ADVANCES IN LIPOBIOLOGY Volume 7 • 1996
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ADVANCES IN LIPOBIOLOGY Editor: RICHARD W. GROSS Department of Bioorganic Chemistry and Molecular Pharmacology Washington University School of Medicine St Louis, Missouri
VOLUME 1 • 1996
UQIJ) JAI PRESS INC. Greenwich, Connecticut
London, England
Copyright © 1996 byJAI PRESS INC. 55 Old Post Road, No. 2 Greenwich, Connecticut 06836 JAI PRESS LTD. 38 Tavistock Street Covent Garden London, WC2E 7PB England All rights reserved. No part of this publication may be reproduced, stored on a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, filming, recording, or otherwise, without prior permission in writing from the publisher. ISBN: 1-55938-635-5 Manufactured in the United States of America
CONTENTS
LIST OF CONTRIBUTORS
ix
PREFACE Richard W. Cross
xiii
REGULATION OF MAMMALIAN CTP: PHOSPHOCHOLINECYTIDYLYLTRANSFERASE Rosemary B. Cornell
1
INCORPORATION AND TURNOVER OF FATTY ACIDS IN ESCHERICHIA CO/./MEMBRANE PHOSPHOLIPIDS Charles O. Rock and Suzanne Jackowski
39
A BRANCHED METABOLIC PATHWAY IN ANIMAL CELLS CONVERTS 2-MONOACYLGLYCEROL INTO sn-1 -STEAROYL-2-ARACHIDONOYL PHOSPHATIDYLINOSITOL AND OTHER PHOSPHOGLYCERIDES John A. Glomset
61
PROPERTIES AND REGULATION OF MAMMALIAN NONPANCREATIC PHOSPHOLIPASE A2 ENZYMES Christina C. Leslie
101
THE FATE OF PLATELET-ACTIVATING FACTOR: PAF ACETYLHYDROLASES FROM PLASMA AND TISSUES Tada-atsu Imaizumi, Diana M. Stafforini, Yoshiji Yamada, Guy A. Zimmerman, Thomas M. Mclntyre, and Stephen M. Prescott
141
BIOSYNTHESIS OF PLASMALOGENS IN MAMMALIAN CELLS AND THEIR ACCELERATED CATABOLISM DURING CELLULAR ACTIVATION David A. Ford and Richard W. Gross
163
vi PLASMALOGENS, NITROXIDE FREE RADICALS, AND ISCHEMIA-REPERFUSION INJURY IN THE HEART Richard Schuiz PHOSPHOLIPID HYDROLYSIS IN PANCREATIC ISLET BETA CELLS AND THE REGULATION OF INSULIN SECRETION John Turk, Richard W. Gross, and Sasanka Ramanadham THE ROLE OF PAF IN REPRODUCTIVE BIOLOGY Hisashi Narahara, Rene A. Frenkel, and John M. Johnston SPHINGOLIPIDS AS REGULATORS OF CELLULAR GROWTH, DIFFERENTIATION, and BEHAVIOR Alfred H. Merrill, Jr., Dennis C. Liotta, and Ronald T. Riley PHOSPHATIDYLSERINE DYNAMICS AND MEMBRANE BIOGENESIS Pamela J. Trotter and Dennis R. Voelker DIACYLGLYCEROL METABOLISM IN CELLULAR MEMBRANES Rosalind A. Coleman and Steven H. Zeisel PHOSPHATIDYLINOSITOL 4-KINASES IN SACCHAROMYCES CEREVISIAE George M. Carman, Rosa J. Buxeda, and Joseph T. Nickels, Jr. PHOSPHOINOSITIDE METABOLISM IN MYOCARDIAL TISSUE Robert A. Wolf ROLE OF ARACHIDONATE IN MONOCYTE/MACROPHAGE FUNCTION Michelle R. Lennartz and James B. Lefkowith
INDEX
CONTENTS
193
215
241
273
299
337
367
387
429
463
LIST OF CONTRIBUTORS
Rosa J. Buxeda
Department of Food Science Rutgers University New Brunswick, New Jersey
George M. Carman
Department of Food Science Rutgers University New Brunswick, New Jersey
Rosalind A. Coleman
Department of Pediatrics Duke University Medical Center Durham, North Carolina
Rosemary Cornell
Department of Chemistry Simon Fraser University Burnaby, British Columbia, Canada
David A. Ford
Department of Internal Medicine Washington University School of Medicine St. Louis, Missouri
Rene A. Frenkel
Department of Biochemistry University of Texas Southwestern Medical School Dallas, Texas
John A. Clomset
Howard Hughes Medical Institute Research Laboratories University of Washington Seattle, Washington
Richard W. Gross
Department of Bioorganic Chemistry and Molecular Pharmacology Washington University School of Medicine St. Louis, Missouri
LIST OF CONTRIBUTORS Tada-atsu Imaizumi
Department of Internal Medicine and Biochemistry University of Utah School of Medicine Salt Lake City, Utah
Suzanne jackowski
Department of Biochemistry St. Jude Children's Research Hospital Memphis, Tennessee
John M. Johnston
Department of Biochemistry University of Texas Southwestern Medical School Dallas, Texas
James B. Lefkowith
Department of Medicine Washington University School of Medicine St. Louis, Missouri
Michelle
Departments of Physiology and Cell Biology Albany Medical College Albany, New York
Lennartz
Christina C. Leslie
Department of Pediatrics National Jewish Center Denver, Colorado
Dennis C. Liotta
Department of Chemistry Emory University Atlanta, Georgia
Thomas M. Mclntyre
Department of Internal Medicine and Biochemistry University of Utah School of Medicine Salt Lake City, Utah
Alfred H. Merrill, Jr
Department of Biochemistry Emory University School of Medicine Rollins Research Center Atlanta, Georgia
Hisashi Narhara
Department of Biochemistry University of Texas Southwestern Medical School Dallas, Texas
List of Contributors
IX
Joseph T. Nickels, Jr.
Department of Food Science Rutgers University New Brunswick, New Jersey
Norman S. Radin
Nephrology Division University of Michigan Medical Center Ann Arbor, Michigan
Sasanka Ramanadham
Washington University School of Medicine St. Louis, Missouri
Ronald T. Riley
Toxicology and Mycotoxins Research Unit/USDA-ARS Athens, Georgia
Charles O. Rock
Department of Biochemistry St. Jude Children's Research Hospital Memphis, Tennessee
Richard Schuiz
Departments of Pediatrics and Pharmacology University of Alberta Edmonton, Alberta, Cananda
Diana M. Stafforini
Department of Internal Medicine University of Utah School of Medicine Salt Lake City, Utah
Pamela J. Trotter
Department of Medicine National Jewish Center Denver, Colorado
John Turk
Washington University School of Medicine St. Louis, Missouri
Dennis Voelker
Department of Medicine National Jewish Center Denver, Colorado
Robert A, Wolf
Department of Medicine Washington University School of Medicine St. Louis, Missouri
LIST OF CONTRIBUTORS Yoshiji Yamada
Department of Internal Medicine and Biochemistry University of Utah School of Medicine Salt Lake City, Utah
Steven H. Zeisel
Department of Pediatrics Duke University Medical Center Durham, North Carolina
Guy A. Zimmerman
Department of Internal Medicine and Biochemistry University of Utah School of Medicine Salt Lake City, Utah
PREFACE
The last decade has witnessed explosive advances in our understanding of the role of membranes, lipid second messengers, and lipid metabolism in the molecular mechanisms regulating cell growth, differentiation, and ligand-regulated cellular activation. In large part, these insights have been facilitated by the exploitation of new methodologies including genetic engineering (molecular biology), analytical instrumentation (biophysical chemistry), and computer technology (molecular modeling and drug design). While the application of these methodologies to their parent disciplines has resulted in the easily detectable emergence of new concepts and principles, their central role in the growth of lipobiology has been less evident, especially to students embarking on new scientific careers. Accordingly, it was envisaged that a series of cohesive didactic discussions identifying rapidly-evolving scientific arenas in lipobiology would serve to illustrate the dynamic nature of the field and encourage students to participate in this rapidly-evolving discipline focused at the interface of lipid chemistry and biology. This series was created to provide a forum for leading scientists in the field of lipobiology to: (1) broadly interpret the potential significance of recent findings in the area of lipid structure and function, (2) identify what does (and what does not) constitute "proof of concept," and (3) provide a critical foundation for evaluation of experimental results and strategies in these rapidly evolving arenas. While some historical information has been presented, the purpose of this series is to provide a medium for discussion of emerging concepts by experts in the field.
xii
PREFACE
It is my sincere hope that the extensive efforts by the contributors in this edition to illustrate the recent growth in the scope of investigations in lipid research will be recognized by the next generation of scientists and encourage them to fulfill the promise of our conjoint expectations. Richard W. Gross Editor
REGULATION OF MAMMALIAN CTP: PHOSPHOCHOLINECYTIDYLYLTRANSFERASE
Rosemary B. Cornell
I. ROLE OF CT IN PC SYNTHESIS A. CT Catalyzes a Rate-Limiting Reaction B. Evidence for Other Rate-Limiting Steps 11. ROLE OF CT IN CONTROL OF LIPID SECOND MESSENGER CONCENTRATIONS A. CT Controls the PC Metabolic Cycle B. CT Attenuates the Agonist-Induced DAG Signal III. CT IS A REGULATED ENZYME A. Posttranslational Regulation B. Pretranslational Regulation IV. INTRACELLULAR DISTRIBUTION AND TRANSLOCATION BETWEEN SITES A. Distributionof CT as Assessed by Cell Fractionation B. Activity of the Cytosolic and Membrane Forms C. Interconversion of Soluble and Membrane-Bound CT D. Physiological Relevance ofthe Translocation Model E. Identity ofthe Membrane to Which CT Binds F. Two Forms of Soluble CT G. Nontranslocatable Membrane-Bound CT Advances in Lipobiology Volume 1, pages 1-38. Copyright © 1996 by JAI Press Inc. AH rights of reproduction in any form reserved. ISBN: 1-55938-635-5 1
2 2 4 4 4 5 5 6 6 7 7 7 8 10 11 12 14
2
ROSEMARY B. CORNELL
V
VI.
VII.
VIII.
IX.
REGULATION OF CT ACTIVITY BY LIPIDS/TV r/r/?0 A. Classification of Lipid Modulators B. Critical Features ofthe Activating Membrane REGULATION OF CT ACTIVITY BY PHOSPHORYLATION A. Effects ofManipuiation of Phosphorylation Conditions B. cAMP and Phorbol Esters do not Affect CT Phosphorylation C. CT is a Phosphoenzyme D. Effects ofOkadaic Acid on CT E. Effects of Cholecystokinin F. Effects of PhospholipaseC G. Effects of Oleic Acid STRUCTURE OF MAMMALIAN CT A. Molecular Mass and Subunit Structure B. Amino Acid Sequence and Sequence Homologies C. Phosphorylation Sites D. Secondary and Tertiary Structural Predictions E. Membrane-Binding Domain F. Dimerization Domain G. ModelofCT Interaction With Membranes MECHANISM OF THE REGULATION OF ACTIVITY/iVr/FO A. Regulation Induced Solely by Changes in the Lipid Composition B. Regulation Induced Solely by Changes in Phosphorylation C. Regulation Involving Changes in Both Lipid Composition and Phosphorylation D. Interrelationship ofthe Various CT Conformations FUTURE DIRECTIONS ACKNOWLEDGMENTS REFERENCES
14 15 16 17 17 18 19 19 20 20 21 21 21 22 . 23 24 24 26 26 27 27 28 29 30 31 32 32
I. ROLE OF CT IN PC SYNTHESIS CTPrphosphocholine cytidylyltransferase (EC. 2.7.7.15, CT) is an important regulatory enzyme in phosphatidylcholine (PC)^ metabolism. It catalyzes the transfer of a cytidyl group from CTP to phosphocholine to form CDPcholine, the head group carrier molecule. CDPcholine is subsequently attacked by diacylglycerol (DAG), releasing CMP and forming PC. This pathway for forming PC is the dominant pathway in all animal cells (see Figure 1). A. CT Catalyzes a Rate-Limiting Reaction The ratio ofthe metabolite concentrations in a pathway can indicate the slowest step. The pool sizes of the choline containing metabolites in the CDPcholine pathway have been analyzed in liver (Sundler et al., 1972), lung (Post et a l , 1984), skeletal muscle cells (Sleight and Kent, 1980), HeLa cells (Vance et a l , 1980; Wang
Regulation of Mammalian CTP
GPC choline /
phosphocholine
lysoPC ■FA^ f
y
\
PLD
CDPcholine-^ )PLC "PA
©
y
Figure 1. CT is activated by at least three products of PC catabolism. The evidence for activation by PA is based on in vitro effects only.
et al., 1993a), and several other cell lines (Cornell and Goldfme, 1983; Sleight and Kent, 1983a; Tessner et al., 1991). The ratio of phosphocholine to CDPcholine ranges from 10 (Post et al., 1984) to 150 (Wang et al., 1993a). This indicates a bottle-neck at the CT catalyzed reaction. Secondly, ^H-choline pulse-chase studies have directly indicated that the conversion of phosphocholine to CDP-choline is the slowest step in the pathway (Vance et al., 1980; Pritchard and Vance, 1981; Post et al., 1982; Cornell and Goldfine, 1983). In addition there are several examples of changes in the rate of PC synthesis which correlate with changes in the relative ratio of phosphocholineiCDPcholine or changes in the turnover rate of phosphocholine. For example, fatty acid stimulation of PC synthesis in HeLa cells caused a decrease in phosphocholine and an increase in CDP-choline such that the ratio decreased from 150 to 12 (Wang et al., 1993a). Phospholipase C (PLC) treatment of chick myoblasts stimulated PC synthesis and caused a 60% decrease in phosphocholine and a 2.5-fold increase in CDPcholine (Sleight and Kent, 1980). PC synthesis was elevated in lung from prematurely bom rats due to activation of CT. The phosphocholine pool size decreased at least fourfold (Possmayer et al., 1981; Weinhold et al., 1982). Inhibitors of cholesterol synthesis inhibited PC synthesis in L^ myoblasts and led to an increase in the ratio of phosphocholineiCDPcholine, and an increase in the turnover of phosphocholine (Comell and Goldfme, 1983). Poliovirus infection (Vance et al., 1980) or phorbol ester (Paddon and Vance, 1980) stimulated PC synthesis twofold in HeLa cells and increased the phosphocholine turnover rate twofold.
4
ROSEMARY B. CORNELL B. Evidence for Other Rate-Limiting Steps
The reaction catalyzed by CT may not be the rate-limiting step under every condition. The concentration of DAG, the substrate for the terminal reaction, can be the rate-limiting factor. PC synthesis was inhibited when hepatocytes were treated with cAMP analogues, however there was no effect on CT activity. Rather, the DAG content of cellular membranes decreased probably due to inhibition of fatty acid production (Jamil et al., 1992). Replenishment of the DAG restored the PC synthesis rate in a direct concentration-dependent manner. Secondly, PC synthesis was stimulated only threefold when CT was over-expressed in COS cells, although the amount of the active form of CT increased nearly 20-fold. The CDPcholine concentration increased 12-fold, indicative of a bottleneck at the terminal step. Increasing the supply of DAG stimulated PC synthesis fourfold (Walkeyetal., 1994). The choline kinase catalyzed step can also be rate limiting. This conclusion is based on a change in the specific activity of choline kinase that correlates with the change in the PC synthesis rate (e.g., Warden and Friedkin, 1985), or a change in the ratio of cholineiphosphocholine in a direction opposite to that of choline flux. Regulation of PC synthesis by choline kinase or the supply of choline has been recently reviewed (Tijburg et al., 1989; Kent et al., 1991).
li. ROLE OF CT IN CONTROL OF LIPID SECOND MESSENGER CONCENTRATIONS A. CT Controls the PC Metaboh'c Cycle
PC is the source of DAG production via phospholipase D and phosphatidic acid phosphatase in response to bombesin, epinephrine, vasopressin, cholecystokinin, and other agonists (Billah and Anthes, 1990). In these pathways, phosphatidylinositol bisphosphate- (PIP2) specific PLC generates the first wave of DAG production, followed by more sustained production of DAG via the hydrolysis of PC. PC is also the immediate precursor to DAG via PC-specific PLC in response to interleukins 1 or 3, tumor necrosis factor a, interferon-a, and colony stimulating factor-1 (Liscovitch, 1992). In these latter signal transduction pathways no PIP2 hydrolysis occurs; rather PC seems to be the sole generator of DAG. PC is also a source of arachidonic acid via PLA2. Protein kinase C (PKC) appears to be both required for and activated by the sustained production of second messengers from PC. For the long-term effects of PKC activity, a prolonged production of DAG would be needed. If enhanced PC hydrolysis were to continue for several hours without any stimulation of PC synthesis this would likely lead to a fatal reduction in membrane PC content. However, in every system investigated the stimulation of PC catabolism results in an acceleration of synthesis (e.g.. Sleight and Kent, 1980; Guy and Murray, 1982; Lacal, 1990). This and other data (Terce et al., 1991; Tijburg et a l .
Regulation of Mammalian CTP
5
1991; Walkey et al, 1994) provide strong evidence for a tightly controlled PG metabolic cycle in which synthesis and degradation are closely coupled (Pelech and Vance, 1989; Tronchere et al., 1994). Stimulation of PC synthesis usually involves activation of CT. Three of the products of degradation of PC are known activators of CT: fatty acids, DAG, and PA (Figure 1). In this way CT plays a regulatory role in the maintenance of PC homeostasis in cells activated by a wide variety of agonists. B. CT Attenuates the Agonist-Induced DAG Signal
CT may also be involved in attenuation of the DAG signal. DAG can be metabolized by lipase to produce fatty acids, by kinase to produce PA, by acyltransferase to produce triacylglycerol, or by phosphotransferases to produce PE or PC. The importance of the phosphotransferase pathway in the metabolism of DAG has been illuminated recently. DAG accumulates in liver of rats in which PC synthesis has been inhibited by deprivation of choline (Blusztajn and Zeisel, 1989). Similarly, in pancreatic acini, inhibition of PC synthesis by cholecystokinin resulted in increased DAG levels, suggesting that the major clearance route for DAG in these cells is via incorporation into PC (Matozaki et al., 1991). Using labeled long-chain DAG species introduced into 3T3 fibroblasts by liposome fusion, Florin-Christensen et al. (1992) found that the predominant metabolic destiny of [1-18:0, 2-20:4]DAG was PC. The DAGs appeared to be incorporated intact into PC, that is, without first being degraded to the free fatty acid. Increases in [DAG] stimulate DAG kinase or lipase by substrate level control, whereas increases in [DAG] may stimulate the synthesis of PC at the substrate level and/or by allosteric activation of CT. DAG has been shown to activate CT both in vitro (Cornell and Vance, 1987a,b; Cornell, 1991b) and in vivo (Rosenberg et al., 1987; Kolesnick and Hemer, 1990; Slack et al., 1991). Thus increases in [DAG] would accelerate the formation of CDPcholine, the rate-limiting substrate in the pathway, and thus the rate of the CPT-catalyzed reaction, which converts DAG to PC.
III. CT IS A REGULATED ENZYME The activity of CT is regulated by many factors. The addition to cultured cells of phorbol esters (Pelech et al., 1984a), phospholipases (Sleight and Kent, 1980), fatty acids (Pelech et al., 1984b), diacylglycerol (Utal et al., 1991), calcium ionophore (Sanghera and Vance, 1989a), and CSF-1 (Tessner et al., 1991) all lead to stimulation of CT activity. Glucocorticoid or estrogen treatment also activates CT in developing lung (Possmayer et al,, 1981; Chu and Rooney, 1985; Rooney et al., 1990; Xu et al., 1990). Treatment of cells with okadaic acid (Hatch et al., 1992), cholesterol synthesis inhibitors (Cornell and Goldfine, 1983), cholecystokinin (Matozaki et al., 1991), alkyl phosphocholine (Geilen et al., 1992), and transfection with H-ras (Teegarden et al., 1990) inhibit CT activity. Are these agents acting via
6
ROSEMARY B. CORNELL
common or distinct mechanisms? There is abundant evidence for posttranslational regulation of CT activity, and evidence for pretranslational control is also emerging. A.
Posttranslational Regulation
Many of the regulatory molecules alter CT activity very rapidly after addition to cells and require no new synthesis of CT to exert their effect. Moreover, fatty acids, DAG, phospholipase C, alkyl phosphocholine, and anionic phospholipids can modulate CT in vitro, suggesting a direct mechanism of action. Rapid alteration of CT activity has been proposed to be mediated by changes in the phosphorylation state of the enzyme, by changes in the lipid composition of the membrane or lipid particle with which CT associates, or by a combination of the two. In most, but not all, cases the change in CT activity is associated with changes in its intracellular location: the cytosol (or soluble inactive form) vs. membrane (active form). The mechanism of posttranslational regulation of CT activity is discussed in Section VIII. B. Pretranslational Regulation
Evidence for control of CT activity by alteration in the level of CT mRNA has recently been presented. In most cells examined thus far the CT message exists as a dominant -^5 kb species. In some, but not all, cells a ~1.5 kb species accompanies the larger mRNA on Northern blots. Tessner et al. (1991) measured an increase in a ~5 kb mRNA for CT, identified by hybridization with a CT cDNA probe, in a macrophage cell line after treatment with CSF-1. This mitogen elevated sequentially the CT mRNA level, CT activity, and PC synthesis. It did not change the intracellular distribution of CT activity. The turnover time of the CT mRNA was decreased by CSF-1 treatment. Thus the increase in CT activity is at least in part due to stabilization of the CT message. The analyses did not rule out an effect on transcription of the CT gene, nor posttranslational effects. Aside from showing that the CT message levels can be regulated, this work also showed that CT is a target for a factor that stimulates cell cycling. This finding emphasizes the role of CT in controlling the PC metabolic cycle which is critical in growth factor signaling to the nucleus. The regeneration of rat liver after partial hepatectomy is accompanied by increased PC synthesis rates per cell (Houweling et al., 1993). The amount of CT protein, quantitated via immunoprecipitation with anti-CT antibodies, and the amount of CT mRNA, quantitated with a rat CT cDNA, increased two to threefold. Whether the increase in CT mRNA was due to increased transcription, or posttranscriptional changes was not investigated. These examples point out that pretranslational control of CT may be more important than was previously thought. In developing lung (Cassola et al., 1981; Rooney et al., 1986), and in other systems (Choy, 1982; Lim et al., 1983; Weinhold et al., 1991) increases in total cellular CT activity have been observed, rather than
Regulation of Mammalian CTP
7
simply a redistribution of existing CT between the soluble and membrane bound forms. Increased CT expression is a potential explanation. The level of the 1.5 kb CT mRNA increased in cultured explants of fetal lung along with other markers of lung maturation, such as fatty acid synthase and surfactant proteins A, B, and C (Fraslon and Batenburg, 1993). The CT gene has been isolated only from yeast. Regulatory elements in the yeast 5' untranslated region have not been identified. Clues to the mechanisms of transcriptional control of mammalian CT expression await the isolation and sequencing of a mammalian CT gene.
IV. INTRACELLULAR DISTRIBUTION AND TRANSLOCATION BETWEEN SITES A. Distribution of CT as Assessed by Cell Fractionation When primary tissues or cells are homogenized and separated into soluble and particulate fractions by differential centrifugation, CT is found distributed predominantly in the soluble, or cytosolic, fraction but with significant amounts in various particulate fractions. For example, in adult rat liver (Schneider, 1963) or rabbit skeletal muscle (Cornell and MacLennan, 1985) -90% is cytosolic, with the remainder in the nuclear, mitochondrial, and microsomal fractions. In adult rat lung 60-70% is cytosolic (Feldman et al., 1990). In cultured cells CT is again found in both cytosolic and particulate fractions, typically with 60-70% cytosolic fractions. In cultured cells the subcellular distribution of CT activity has also been examined by digitonin permeabilization (MacKall et al., 1979). This procedure divides the activity into soluble (digitonin-released) and particulate fractions (cell-associated). The results closely resemble those obtained by homogenization and centrifugation (Pelech et al., 1984b; Cornell and Vance, 1987a; Yao et al., 1990; Wang et al., 1993a). These studies of CT distribution were based on assay of the enzyme's activity under optimal conditions. More recently, with the availability of CT-specific antibodies, immunotitration of CT protein in the various subcellular fractions has been carried out (Weinhold et al., 1991; Utal et al., 1991), confirming that the majority of CT is in the cytosol. In COS cells that overexpress CT 40-fold, the distribution of CT is the same as in nontransfected COS cells (Walkey et al., 1994). This suggests that the equilibrium distribution is a function of its intrinsic membrane partition coefficient, rather than, e.g., a membrane-bound saturable receptor. B. Activity of the Cytosolic and Membrane Forms To measure the intracellular distribution of CT using an activity assay, it is necessary to add saturating amounts of lipid vesicles to the cytosolic fraction. Typically sonicated vesicles composed of egg PC/oleic acid (1/1 M/M) are used. In the absence of vesicles the activity in the cytosol is very low. The activity of the soluble form was completely eliminated by extracting the residual lipids with
8
ROSEMARY B. CORNELL
isopropyl ether (Feldman et al., 1981). Enzyme activity was restored upon adding back the extracted lipids. The membrane-associated form of CT, on the other hand, is not stimulated or is only slightly stimulated by exogenous lipids. (The activity associated with the low-speed, 1,000 x g pellet is often stimulated three to fourfold by lipid vesicles, due to contamination of the soluble form in this fraction. CT activity in washed microsomes is lipid-independent.) The observation that cytosolic CT is generally inactive but can be activated ~ 10-fold by exogenous lipids generated the idea that lipids are primary regulators of CT activity, and that the cytosolic, or soluble form, of CT acts as an inactive reserve of enzyme that can be recruited to the membrane when there is increased demand for PC synthesis. C. Interconverslon of Soluble and Membrane-Bound CT
As mentioned in section III. A. above, many agents which stimulate PC synthesis also change the subcellular distribution of CT without changing the overall level of CT in the cell. This amphitropism, or the ability to interconvert between an inactive soluble and active membrane-bound form, places CT in the same class as PKC, DAG kinase, arachidonate 5-lipoxygenase, and cellular phospholipases. A list of agents that stimulate CT translocation is given in Table 1. The first documentation of translocation between cytosol and membranes was in chick myoblasts in response to PLC. Sleight and Kent (1980) showed that the threefold stimulation of PC synthesis by PLC treatment correlated with a -threefold increase in the CT activity in cell homogenates when assayed in the absence of exogenous lipid. When assayed in the presence of a crude lipid preparation there was no difference between control and PLC-treated cells. The cytosolic activity (assayed in the presence of lipid) decreased twofold and the membrane associated activity increased threefold. Thus in control cells most of the CT was cytosolic (78%) and inactive, whereas in PLC-treated cells, most of the CT was membranebound (70%) and active. The authors concluded that the rate of PC synthesis and CT activity are determined by the amount of membrane-bound CT. They suggested that CT translocation was signaled by the deficiency of PC content in the membrane (Sleight and Kent, 1980, 1983b). PLC-induced translocation was readily reversed by withdrawal of the lipase from the medium (Wright et al, 1985). Subsequently, similar translocation phenomena were described using fatty acids as the effectors. Oleic acid (added to cells as sodium oleate) stimulates PC synthesis 3—20-fold, depending on the cell and mode of administration (Pelech et al., 1983a, 1984b; Cornell and Vance, 1987a; Burkhardt et al, 1988; Weinhold et al., 1991; Terce et al., 1991). Oleate induced a dramatic redistribution of CT activity and CT protein, such that virtually all the CT became membrane-bound (Pelech et al, 1984b; Houweling et al., 1993b; Wang et al., 1993). At the oleate concentrations used to effect maximal translocation, the microsomal membranes were highly enriched with free oleic acid (15 mole percent; Cornell and Vance, 1987a). The membrane translocation induced by oleate could be readily reversed by a high
Regulation of Mammalian
CTP
Table 1, Translocation Correlations CT PC Phosphorylation Activity Synthesis References
Effector
is
E
Lipid A
PLC Fatty acid Phorbol ester^ DAG Choline deficiency Alkyl Pchol^ Okadaic acid Ca ionophore'^
i i i i i
t t t t t
DAGT pel fatty acidt DAGt DAGt
i i —
?ci
I
i
t t
i i
alkylPCholT
9
i i
i i i
t
7
t
t
1
—
?
t 9
T
t t t
t t t t
1-4 5-9 10-12 2,9,12,13 14,15 16 17 18
Notes: ^Watkins and Kent (1990) reported no effect on CT translocation in HeLa cells. ''Boggs et al. (1995) reported CT translocation from cytosol to membranes in a macrophage cell line treated with alkylphosphocholines. Posse de Chaves et al. (1995) reported an inhibition of PC synthesis in sympathetic neurons treated with alkylphosphocholine, but no effect on CT. '^The opposite effects on CT activity and PC synthesis in pancreatic acinar cells were reported by Matosoki etal.(1991). Ej. = soluble CT; E^ = membrane-bound CT; ? = not measured; — = no change. References: I have referenced either the original discovery and/or particularly thorough or insightful studies. 1. Sleight, R. & Kent, C. (1980). J. Biol. Chem. 255, 10644-10650. 2. Slack, B., et al. (1991). J. Biol. Chem. 266, 24503-24508. 3. Watkins, J. & Kent, C. (1990). J. Biol. Chem. 266, 21113-21117. 4. Jamil, H., et al. (1993). Biochem. J. 291,419-427. 5. Houweling, M., et al. (1993b), submitted. 6. Pelech, S., et al. (1983a). J. Biol. Chem. 258, 6782-6788. 7. Wang, Y., et al. (1993). J. Biol. Chem. 268, 8. Weinhold, R, et al. (1984). J. Biol. Chem. 259, 10315-10321. 9. Cornell, R. & Vance, D. (1987a,b). Biochim. Biophys. Acta 919, 26-36; 919, 37-48. 10. Pelech, S., et al. (1984a). Biochim. Biophys. Acta 795,447-451. 11. Kolesnick, R. (1987). J. Biol. Chem. 262, 14525-14530. 12. Utal, A., etal. (1991). J. Biol. Chem. 266, 24084-24091. 13. Kolesnick, R. & Hemer, M. (1990). J. Biol. Chem. 265, 1090-10904. 14. Jamil, H., et al. (1990). J. Biol. Chem. 265,4332-4339. 15. Weinhold, RA., Charles, L., & Feldman, D. (1994). Biochim. Biophys. Acta 1210, 335-347. 16. Geilen, C , et al. (1992). J. Biol. Chem. 267, 6719-6724. 17. Hatch, G., et al. (1992). J. Biol. Chem. 267, 15751-15758. 18. Sanghera, J. & Vance, D. (1989a). Biochim. Biophys. Acta 1003, 284-292.
concentration of BSA (Weinhold et al, 1984; Cornell and Vance, 1987a,b), which effectively extracts the fatty acids from the cell membranes. This suggests that the presence of the fatty acid in the membrane is essential for recognition of the membrane by CT. The fatty acid induction of membrane binding occurred within five minutes of the addition to cells (Terce et al., 1991; Houweling et al., 1993b), and the dissociation from the membrane within one minute after addition of BSA (Wang et al, 1993a). This rapid association/dissociation-activation/inactivation is
10
ROSEMARY B. CORNELL
compatible with a model where the membrane lipid directly associates with and activates CT. In support of this idea, cytosolic CT (Feldman et al., 1985; Cornell and Vance, 1987b) and pure CT (Cornell, 1991a,b) bound to and were activated by pure lipid vesicles containing oleic acid or other lipid activators. Thus, no membrane-bound protein receptor or any other soluble proteins are needed for CT-membrane binding. The stimulation of PC synthesis and CT activity by phorbol esters and DAG can also be accounted for by the translocation of CT from cytosol to membrane. The addition of TPA to HeLa cells stimulated PC synthesis threefold (Paddon and Vance, 1980). CT activity in the homogenate, assayed without exogenous lipid, increased two to threefold; the activity in the cytosol decreased threefold, and the activity in the membrane increased threefold (Pelech et al., 1984b; Utal et al., 1991). Twofold increases in membrane-bound CT were associated with TPA treatment of GH3 pituitary cells (Kolesnick and Hemer, 1990). Soluble, short-chain DAG (diCg) increased membrane-bound CT and PC synthesis twofold (Utal et al., 1991). The increase in membrane-bound CT was matched by the loss of soluble enzyme. Agents which stimulate the transfer of CT from membrane to cytosol are associated with a decrease in CT activity and PC synthesis. Okadaic acid, an inhibitor of type 1 and 2a cellular phosphatases, is the most thoroughly studied effector of this type (Hatch et al., 1991). The translocation to cytosol can be reversed or overridden by fatty acids (Hatch et al., 1992). While all of the agents listed in Table 1 which increase the amount of membrane CT could have influence by changing the lipid composition of the membrane, there is no data suggesting a change in lipid composition is involved in the mechanism of action of okadaic acid. Rather, there is an increase in the phosphorylation state of CT. In summary, these examinations of the reversible membrane interactions of CT reveal that: (a) cytosolic CT is an inactive reserve of the enzyme which can be recruited to the membrane when there is an increased demand for PC synthesis; (b) translocation can be triggered solely by changes in the lipid composition of the membrane; and (c) translocation between compartments is the major mechanism for regulation of CT in most mammalian cells. As more is learned about CT regulatory mechanisms, these conclusions may be modified. D.
Physiological Relevance of the Translocation Model
The studies cited above involved cultured cells treated with often non-physiological concentrations of effectors. There are at least two in vivo models which support CT translocation. First, in developing lung and liver of the rat there is an increase in CT activity and membrane CT content at birth (Weinhold et al., 1973, 1984; Pelech et al., 1983c). In rat lung the increase in membrane CT correlated with an increase in the free fatty acid content of the cell (Weinhold et al., 1984). At birth there is a need for increased PC synthesis for surfactant production in the lung. Second, choline deprivation of rats leads to increased CT associated with mem-
Regulation of Mammalian CTP
11
branes and a loss of cytosolic CT (Yao et al., 1990). (The translocated CT is potentially active, yet there is no increase in PC synthesis due to limitation of choline.) E. Identity of the Membrane to Which CT Binds
For many years it was presumed that the active form of CT was associated with the endoplasmic reticulum (ER), since that is the accepted site of PC synthesis. Certainly, the next enzyme in the CDP-choline pathway, cholinephosphotransferase, is a constituent of the ER, and is very often used as a marker for this organelle. It was also assumed that agents that promoted CT-membrane interaction promoted CT binding to the ER. If CT is an ER enzyme then one would expect to find most of the membrane-associated activity in the microsomal pellet. However, in most cells the majority of the particulate CT activity actually sediments at < 1,000 X g. The first thorough examination of the membrane localization of CT was done using Krebs II ascites cells after PLC treatment. Terce et al., (1988) fractionated the 1,000 X g supernatant on a Percoll density gradient which separated plasma membrane, golgi, mitochondria, and ER. CT activity colocalized with two ER marker enzymes, but not with any of the other membrane fractions. The 1,000 x g pellet contained 15% of the total cellular CT activity, but PLC treatment did not increase the percent in this fraction, whereas CT activity in the ER fraction was elevated twofold by PLC treatment. Vance and Vance (1988) found CT associated with the ER and Golgi. The CT-specific activity in rat liver Golgi, isolated by three different methods, was as high or higher than that of the ER. Nuclease-treated whole homogenates of PLC-treated CHO cells were fractionated on Percoll gradients of various concentrations (Morand and Kent, 1989). CT activity fractionated differently from two ER enzyme markers. A separate sucrose density gradient separated the Golgi marker enzyme from CT activity. Differential centrifugation revealed that 80% of the total particulate CT from control, PLC, or N-monomethyl PE-enriched CHO cells sedimented at only 65 x g x 5 minutes. This fraction contained nuclei, identified by microscopy, and other organelles which were separated by Percoll gradients. CT activity colocalized with the nuclei. Wang et al. (1993b) enucleated untreated L cells to determine if CT in its soluble form would partition with a cytosolic enzyme (LDH) or with the karyoplasts (nuclei plus some cytosol and plasma membrane). Ninety-seven percent of the CT, but only 38% of the LDH, activity was recovered with the karyoplasts. This result suggests a nuclear location for both the soluble and membrane-bound form of CT in CHO cells. Immunofluorescence analysis of fixed cells has corroborated these biochemical analyses. A fluorescent antipeptide antibody directed against the N-terminus of CT revealed staining of the nuclear envelope. CHO that had been treated with PLC (Watkins and Kent, 1992), or HeLa cells that had been treated with fatty acid (Wang et al., 1993b) were analyzed. The antibody, although affinity purified, cross-reacted
12
ROSEMARY B. CORNELL
with a 50 kDa soluble protein, which could have accounted for the nuclear staining. However, immunofluorescence with this antibody on a temperature sensitive CT mutant of CHO cells showed very weak fluorescence compared to wild type CHO. Expression of a rat liver CT cDNA in the CT mutant cells reinstated the nuclear fluorescence. Nuclear staining with the fluorescent antibody was obtained for Hep G2, NIH 3T3, L cells, and rat liver slices. None of these cells contained proteins other than CT that cross-reacted with the anti-CT antibody (Wang et al., 1993a). These studies support a model where CT resides in the nucleoplasm in its inactive form, and translocates to the inner nuclear membrane for activation. There are two putative nuclear targeting signals in mammalian CT (residues 12—16 and 248-254). Fusion of residues 8—28 of rat liver CT onto the N-terminus of P-galactosidase was sufficient to target the chimera to the nucleus of stably transfected CHO cells. Deletion of residues 12-16 resulted in the immunolocalization of CT to both the cytoplasm and nucleus (Wang et al., 1995). These data strongly suggest a nuclear localization for wild-type CT. If CT were a nuclear enzyme, why is it released into the cytosol after homogenization of cells, or why is it released from the cell ghost by digitonin treatment? CT, a large but elongated molecule (Weinhold et al., 1989), may be able to pass unimpeded through the nuclear pores. Why does a significant proportion of the membrane-bound CT activity colocalize on density gradients with the ER and Golgi? It could be that after homogenization nuclear membrane fragments may be released from the nucleus, and would have densities overlapping with ER and/or Golgi. The physiological significance of a nuclear location for CT is not readily apparent. One possibility relates to the recent reports of nuclear PC cycle and DAG production accompanying a proliferative response (Divecha et al., 1991; Banfic et al., 1993). DAG is one of the primary candidates for the activation of CT translocation to membranes. CT may be located in the nucleus to respond to fluctuations in the DAG concentrations that indirectly regulate DNA replication. If choline kinase is cytosolic, cholinephosphotransferase is ER-bound, and CT is nuclear, the three-step CDP-choline pathway involves three separate cellular compartments. Choline, phosphocholine, and CDP-choline are all water soluble so that there is no metabolite diffusion barrier presented by catalysts bound to different organelles. The rationale for a nuclear localization is made more complex by the finding that the nuclear localization mutant (lacking the N-terminal targeting sequence) was none-the-less able to support PC synthesis in CT-deficient CHO-58 cells (Wang et al., 1995). Evidence of metabolite channeling suggests that the enzymes of the CDPcholine pathway from the plasma membrane choline transporter to CPT are a physically-linked functional unit. Exogenous phosphocholine or CDPcholine that is introduced into C6 glioma cells via electropermeabilization do not compete with labeled choline for incorporation into PC (George et al., 1989, 1991). Labeled phosphocholine and labeled CDPcholine introduced in the same way also do not incorporate into PC. These results suggest that endogenous metabolite pools are
Regulation of Mammalian CTP
13
not in rapid exchange with exogenous pools, a characteristic of organized complexes. This hypothesis requires further testing using other cells and methods of introducing metabolites. F. Two Forms of Soluble CT
In some, but not all, cells there are two forms of soluble CT: an inactive (i.e., lipid-dependent) low molecular weight form—the L-form—^and an active high molecular weight form—the H-form (Stem et al., 1976; Choy et al., 1977, 1979). The L-form is a '^90 kDa homodimer that does not contain bound lipid, whereas the H-form is a ^300 kDa complex of CT homodimer plus -250 phospholipid molecules. These forms were separated by density differences and characterized from lung tissue, alveolar type II cells, A549 cells, and Hep G2 cells (Weinhold et al, 1989). Colocalization of [^H]-PI and the H form of CT on glycerol density gradients suggested that the H form contains PI, and may contain other anionic lipids as well. The H-form is not stable. In crude fractions it can be dissociated to the L-form by simply incubating at 37° C for 15 minutes. It may be that a high concentration of lipid is required to stabilize the H-form. The L-form can be converted into the H-form by addition of lipid vesicles composed of PI, PG, or PC/oleic acid, but not by PC vesicles (Weinhold et al., 1989). In adult rat lung soluble CT is almost exclusively in the H-form, while in fetuses CT is solely in the L-form (Feldman et al., 1980). In lung, the ratio of H- to L-form increases with premature birth (Weinhold et al., 1981). Treatment of Hep G2 cells with oleic acid increased both microsomal and soluble H-form CT (Weinhold et al, 1991), with no decrease in the amount of L-form. Glucocorticoids increase CT activity predominantly in the cytosolic fraction of lung (Chu and Rooney, 1985; Possmayer et al., 1981; Rooney et al., 1986). The stimulation is mediated by phospholipids (Chu and Rooney, 1985; Xu et al., 1990), and is likely accompanied by an increase in the H:L ratio, although an examination of changes in this ratio has not been reported. Based on these observations, a regulatory mechanism involving conversion between H- and L-forms of CT was proposed (Weinhold et al., 1989). The interrelationship of the H-, L-, and membrane forms of CT was analyzed (Feldman et al., 1990). The two soluble forms, as well as the membrane-bound form, are immunochemically the same and are interconvertible. Triton, at sub-solubilizing concentrations, extracted an H-form lipid-CT aggregate from microsomes. The authors suggested that the cytosolic H-form originates via fragmentation of the membrane as a CT-phospholipid aggregate. Further experiments suggested that H-form must dissociate to the L-form prior to membrane binding (Feldman et al., 1990). In effect the CT replaces one lipid environment for another. These results may appear to be at odds with the membrane translocation/activation model for CT regulation. However, both models support the notion that CT is a lipid requiring enzyme. The form of the activating lipid may be micellar or membranous, depending on the cell. Relevant to this point, purified rat liver CT
14
ROSEMARY B. CORNELL
binds to and is activated by well characterized and Triton-lipid micelles (Cornell, 199 la). Since the associations of H-form CT and lipid were probed in cell extracts, it is difficult to know if the lipid-CT aggregate was produced as a consequence of the cell disruption, or whether it was formed prior to cell disruption. The tissue specific differences in the abundance of H- vs. L-forms could be due to differences in the amount or composition of cytosolic lipid. For example, removal of phospholipid from the alveolar spaces prior to homogenization of lung reduced the fraction of cytosolic CT in the H-form (Feldman et al., 1980), suggesting that the L-form CT in the homogenate bound to (in this case) extracellular phospholipid, when present. This concern with cell disruption artifacts applies also to digitonin released cytosol, since the detergent itself could solubilize some membrane lipid creating a micelle to which CT could bind. During the process of isolating the soluble L-form of CT it became clear that this form is not very soluble. Detergents were required at an early stage of the purification to avoid aggregation. What stabilizes L-form CT in cells? Are chaperonins involved? CT may be synthesized on cytosolic ribosomes and may associate with a chaperonin until recruited to membranes. If CT is targeted after synthesis to the nucleus, does it do so in complex with another protein(s)? One candidate protein has been suggested, a 112 kDa protein from rat liver (Feldman and Weinhold, 1993). A CT-horse radish peroxidase conjugate bound to a partially purified form of the 112 kDa protein that was immobilized on nitrocellulose. The binding was competed by pure CT and affmity-purified antibodies against the 112 kDa protein. Both CT and the 112 kDa species co-eluted through two rounds of DEAE Sepharose chromatography. It would be informative to determine if this protein colocalizes intracellularly with CT, using the immunocytochemical approach (Wang et al., 1993b). Chemical cross-linking might also help evaluate the association of these proteins in vivo. G.
Nontranslocatable Membrane-Bound CT
Every cell or tissue examined has some membrane-bound CT in the basal condition. This membrane pool is presumably responsible for the basal rate of PC synthesis. This form of CT may differ from the translocatable form in its mode of membrane association, or the translocated CT may mix with and become indistinguishable from the pre-existing membrane-bound CT. A hint that there may be a nontranslocatable pool comes from the observations that albumin extraction of fatty acids from microsomes of oleic acid treated cells did not remove all of the bound CT (Cornell and Vance, 1987a; Terce et al., 1992). The remaining activity corresponded to the basal membrane activity (Terce et al., 1992). These results might imply that the basal membrane pool is insensitive to the translocation promotor, oleic acid. On the other hand, albumin extraction of fatty acid from untreated microsomes released at least 50% of the CT activity (Weinhold et al., 1984),
Regulation of Mammalian CTP
15
suggesting that the CT membrane association in unstimulated cells is maintained by lipid activators in those membranes.
V. REGULATION OF CT ACTIVITY BY LIPIDS/NV/r/fO The soluble form of CT can bind to phospholipid vesicles of certain compositions, which activate the enzyme via increases in the V^^,^. K^ values forCTP and phosphocholine are not affected (Weinhold et al., 1986). The lipid specificity for activation of CT has been investigated by many laboratories using different sources of CT and different methods to prepare the lipid suspensions. A. Classification of Lipid Modulators
Analysis of CT lipid specificity using crude extracts is problematic for two major reasons, (a) The added lipids risk lipolysis. This explanation is likely for early results suggest that lyso PE and oleoyl CoA activate CT (Fiscus and Schneider, 1966; Choy and Vance, 1978). Fatty acid, generated by lipases in the crude extracts, is likely the actual stimulator, (b) The added lipids might bind to other lipid binding proteins changing their effective concentrations. On the other hand, the potential drawback of a purified system is that if proteins or other factors are involved in modulating the lipid specificity of activation, these interactions will be missed. In spite of these shortcomings, lipid specificity studies using either crude (Choy and Vance, 1978; Feldman et al., 1978, 1980; Choy et al, 1979) or pure enzyme (Weinhold et al, 1986; Feldman and Weinhold, 1987; Cornell, 1991a,b) have confirmed that there are at least two classes of lipid activators. Class I is composed of anionic phospholipids. Phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidic acid (PA), phosphatidylglycerol (PG), and cardiolipin all activate CT. Class 11 is composed of neutral lipids with small polar head groups, such as mono and diacylglycerol, and oleyl alcohol. These lipids must be intercalated into phospholipid bilayers in order to activate CT (Weinhold et al., 1986; Cornell and Vance, 1987b; Cornell, 1991a,b). Fatty acids are also potent activators when presented as a component of a PC vesicle. Fatty acids resemble class I activators by having the negative charge, but resemble class II activators by having a small polar group. Purified rat liver CT is activated equally well by palmitic as by oleic acid in vesicles composed of egg PC: fatty acid (2/1). Activation requires chain lengths longer than 12 carbons for both phospholipid and fatty acid, and also requires that the lipids be in the liquid crystalline phase (Cornell, 1991b). Cationic lipids and amphiphiles with long alkyl chains (e.g., sphingosine, oleyl amine) are inhibitors, and antagonize the action of the anionic lipids (Sohal and Cornell, 1990). Alkylphosphocholine also inhibits CT (Geilen et al., 1992). Zwitterionic phospholipids, i.e., PC, sphingomyelin, PE, and also triacylglycerol have negligible effects.
16
ROSEMARY B. CORNELL
The association of CT with membranes can best be described as the partitioning of an amphiphile (CT) between a polar and nonpolar solvent. The relative affinity for different lipid vesicles would best be described by comparing the membrane partition coefficients. These values have not been reported because a rapid, simple, and quantitative assay for CT binding to lipid vesicles has yet to be developed. B. Critical Features of the Activating Membrane
Only a few studies have been published to date which analyze the chemistry of the lipid-CT interaction using the purified rat liver enzyme and pure lipids. CT purified from rat liver cytosol and from baculovirus expression systems has very low activity without exogenous lipids. The purification protocol involves addition of large concentrations of egg PC-oleic acid vesicles to achieve the initial fractionation. Traces of this activating lipid contaminate the purified preparation. Removal of this lipid by ion exchange chromatography results in total inactivation. The activity can be completely restored by rapid addition of lipid vesicles (Cornell, 1991b). These studies with pure CT confirm the idea that lipids are necessary and sufficient for activation. There are two major conclusions that can be drawn from these studies. First, the membrane negative charge density/surface potential is influential in activation of CT. The purified enzyme can be activated by anionic lipids when presented in micellar (Cornell, 1991a) or vesicular form (Feldman and Weinhold, 1987; Sohal and Cornell, 1990; Cornell, 1991b). The potency of the activating lipid in Triton micelles is related to its negative charge, and not its chemical structure; thus CL (charge = "2) activates fully at 10 mole percent whereas PG, PS, or PI (charge = ~1) activate fully at 20 mole percent. Sphingosine and other cationic lipids antagonize the activation by anionic lipids (Sohal and Cornell, 1990). CT binds to the anionic micelles or vesicles, as shown by coelution of micelles or vesicles with CT on a gel filtration column (Cornell, 1991a). The negative surface charge could initiate the binding of CT to the vesicle as the first step in activation. In sonicated vesicles, PI stimulates CT when it is present at concentrations as low as ~10 mole percent, concentrations that are near physiological (Feldman and Weinhold, 1987). The second conclusion is that defects in lipid packing promote CT binding/activation. Anionic lipids, by virtue of charge repulsion of neighboring molecules, perturb the lipid head group packing, and increase the curvature of the vesicle. Small unilamellar vesicles are much more effective activators than planar multilamellar vesicles of the same composition (Feldman et al., 1978; Cornell, 1991b). Packing defects are also generated by lipids with small uncharged head groups such as mono and diacylglycerol and oleyl alcohol, all of which activate CT (Cornell and Vance, 1987b; Cornell, 1991b). One vision of the role of lipids in promoting CT interactions is to facilitate the intercalation of a portion of CT into a nonpolar
Regulation of Mammalian CTP
17
environment. In support of an intercalation model, CT is anomalously activated by liposomes composed of PG/PC (1 /3) at the gel -> liquid crystalline phase transition temperature. Lipid packing is the most disordered at the gel/fluid phase boundaries that exist at the phase transition. In this state the lipid vesicle would present the least barrier against penetration by peptides. The interaction between CT and membranes, whether promoted by anionic (class I) or neutral (class II) activators, would involve both electrostatic and hydrophobic interactions. For example, binding of CT to oleic acid-enriched membranes was only partially blocked by high ionic strength medium (Cornell and Vance, 1987a). Although class II activators have no associated charge, the association of CT with these vesicles could involve electrostatic interactions with phosphate groups. Additional studies on the kinetics of CT binding to lipid vesicles are needed to unravel the order and degree of cooperation between the electrostatic and hydrophobic parameters of the CT-lipid interaction. Milligram quantities of pure CT have recently been prepared using the baculovirus expression system (MacDonald and Kent, 1993; Luche et al., 1993). Thus mechanistic studies on CT interaction with lipids are now very feasible. Do the results of these in vitro analyses using purified components concur with the information obtained using cell culture models? In cell culture fatty acids and DAG are the two effector lipids most often associated with CT activation? A role for anionic phospholipids in vivo has not been rigorously demonstrated. There is a ~ twofold increase in PG content in lung during the latter stages of development which may contribute to the increase in CT activity and the amount of H-form at birth (Feldman et al., 1978). Jamil et al. (1993) examined the effects of PLC and PLA2 on CT translocation in hepatocytes to determine if there was a unifying principle involved in the signal for CT translocation to membranes. The translocation of CT did not correlate in each case with changes in the concentrations of PC or DAG. They did notice a tendency of lipid activators to prefer nonbilayer packing arrangements, whereas lipids that decrease CT binding to membranes prefer a bilayer organization. Thus DAG, oleic acid, PE or mono-methyl PE, and oleyl alcohol promote the Hjj phase, while PC or dimethyl PE promote the lamellar phase. Decreases in PC content and increases in DAG or oleic acid correlate with increased CT translocation. They proposed that CT senses even small changes in the ratio of nonbilayer/bilayer lipids. The physiological rationale for such a regulatory mechanism is obvious. Increased CT activity would result in increased production of the major bilayer stabilizer, PC. The small percent of nonbilayer forming lipid generated would not be sufficient to lyse the membranes. More likely, it would create small localized packing instabilities which would generate optimal sites for CT to bind and intercalate.
18
ROSEMARY B. CORNELL
VI. REGULATION OF CT ACTIVITY BY PHOSPHORYLATION A.
Effects of Manipulation of Phosphorylation Conditions
Several early studies suggested that CT might be regulated by phosphorylation. Incubation of cultured hepatocytes with cAMP analogs or aminophylline, a cAMP phosphodiesterase inhibitor (Pelech et al, 1981) was reported to inhibit PC synthesis and CT activity. The inhibitory effects of cAMP on PC synthesis were more pronounced after one hour (Pelech et al., 1981; Jamil et al., 1992). In lung type II cells, on the other hand, the opposite effect of cAMP analogs was seen on PC synthesis (Niles and Makarski, 1979). It was suggested that the protein kinase A phosphorylation system participated in the regulation of CT. In a more recent analysis, cholera toxin raised the cAMP content of cultured hepatocytes more than 50-fold but had very little effect on PC synthesis after one hour (Watkins et al., 1992). Incubation of cytosol from rat liver with Mg ATP or NaF, a protein phosphatase inhibitor, resulted in an inhibition of CT activity that could be reversed by protein kinase inhibitors (Pelech and Vance, 1982). Similarly, CT from fetal rabbit lung was inhibited by incubation with Mg ATP (Radika and Possmayer, 1985). The discovery that phorbol esters stimulate PC synthesis (Wertz and Mueller, 1978; Kinzel et al, 1979), CT activity, and translocation (Paddon and Vance, 1980; Pelech et al, 1984a) raised the hypothesis that PKC might be involved in the phosphorylation control of CT. The stimulation of PC synthesis by PLC (Kent, 1979) also suggested the potential for PKC involvement. However, subsequent studies (see below) showed that the mechanisms for the inhibition of PC synthesis by cAMP and the stimulation of PC synthesis by phorbol esters did not involve changes in the phosphorylation state of CT. B. cAMP and Phorbol Esters do not Affect CT Phosphorylation
Jamil et al. (1992) showed that cAMP analogs and glucagon had no effect on the •^^P incorporation into CT, which was analyzed by immunoprecipitation with an affinity purified ^ntipeptide antibody. The cAMP analogs also did not affect CT distribution or activity. Rather, the inhibition of PC synthesis was due to a decrease in the membrane supply of DAG for PC synthesis. The effects on PC synthesis were reversed by incubation with oleic acid, which increased the DAG concentration. Jamil et al. (1992) proposed that the decrease in DAG content was the consequence of inhibition of acetyl Co A carboxylase and fatty acid synthesis by protein kinase A (PKA). These results are consistent with earlier observations that fatty acids can override the effects of cAMP analogs (Pelech et al., 1983b). In agreement with Jamil et al. (1992), Kolesnick (1990) showed that short chain soluble DAGs could reverse the inhibitory effect on PC synthesis of cholera toxin, an adenylate cyclase activator, implying that DAG can overcome the effects of phosphorylation by PKA. Finally, Sanghera and Vance (1989b) reported that PKA phosphorylated purified rat liver
Regulation of Mammalian CTP
19
CT in vitro, but since only 0.2 mol P per mole enzyme was incorporated on serine residues, it was later concluded that this was a nonspecific effect. The closest resemblance to a PKA site in the rat liver sequence is threonine 63. Similarly, the effect of phorbol esters appears to be mediated by DAG rather than changes in CT phosphorylation. Purified PKC had no effect on CT activity when added to a partially purified preparation of rat liver CT (Cook and Vance, 1985), suggesting that CT was not controlled by the PKC pathway. Watkins and Kent (1990) found that phorbol ester (TPA) treatment of HeLa cells did not change the overall level of phosphorylation of CT. But since CT was phosphorylated on numerous sites they concluded that the sensitivity of their analysis was not sufficient to detect a change in the phosphorylation at one site. Utal et al. (1991) confirmed the lack of a measurable effect of phorbol ester on CT phosphorylation. On the other hand the content of DAG increased twofold in TPA-treated HeLa cells (Utal et al., 1991). The kinetics of the changes in DAG content, CT translocation, and PC synthesis suggested a cause-effect relationship. Incubation of the cells with short-chain DAG stimulated PC synthesis and CT translocation twofold. Utal et al. (1991) suggested that the effect of TPA on CT activity was via an increase in the DAG content, which stimulates CT translocation. DAG would be generated by TPA activation of PKC, which activates PC specific phospholipases that generate DAG (Liscovitch, 1992). C. CT is a Phosphoenzyme CT is polyphosphorylated. CT has been immunoprecipitated from hepatocytes labeled with ^^P-phosphate. After further purification by electrophoresis the 42 kDa CT band was digested and the peptides separated on 2D maps. There were at least 12 (Watkins et al, 1992) or 8 (Hatch et al, 1992) phosphopeptides. Different peptide isolation protocols were used by each laboratory. While some of the peptide spots may represent different fragments containing the same phosphorylated residue, it is likely that there are several distinct sites that are phosphorylated in vivo. In HeLa cells, immunoprecipitated CT was phosphorylated on serine residues only (Watkins and Kent, 1990). On SDS gels, two or sometimes three phosphorylated species of cytosolic CT, detected with anti-CT antibodies, can be resolved: the 42 kDa band and slightly slower migrating forms. Two distinct -^^P-labeled species from CHO cytosol were analyzed for phosphopeptides (Watkins and Kent, 1991). The peptide maps of the slower and faster migrating ^^P-labeled forms were quite different, although some of the spots were the same. The slower migrating form had more phosphopeptides than the faster migrating form. The slower migrating forms are more highly phosphorylated (Wang and Kent, 1995b). The phosphorylation state of CT varies during the cell cycle. CT was immunoprecipitated from a macrophage cell line after release from G^ arrest with colony stimulating factor-1. The phosphorylation state was assessed by resolution of three electrophoretic forms. The slower migrating phosphorylated forms were prominent
20
ROSEMARY B. CORNELL
in the arrested state; the faster hypophosphorylated form dominated early Gl. The phosphorylated forms accumulated during the progression through Gl to S phase (Jackowski, 1994). D.
Effects of Okadaic Acid on CT
A role for phosphorylation control of the activity and intracellular distribution of CT has emerged in studies with okadaic acid. Okadaic acid is a specific inhibitor ofcellular protein phosphatases 1 and 2a. Preincubation of rat liver postmitochondrial supernatant with okadaic acid for one hour inhibited CT activity, decreased the amount of membrane CT, and increased the amount of soluble CT (Hatch et al., 1990). Redistribution of CT from membranes to cytosol also occurred when hepatocytes were treated with the phosphatase inhibitor. PC synthesis was inhibited -twofold after two hours (Hatch et al., 1991,1992). Okadaic acid treatment caused an increase in the amount of ^^P associated with immunoprecipitated CT. Peptide maps of control and okadaic acid treated cytosolic samples showed similar phosphorylated peptides, but the ^^P associated with several of the peptides was increased by okadaic acid (Hatch et al., 1992). Oleic acid treatment of hepatocytes led to increases in DAG and reversed all the effects of okadaic acid. These results suggested that okadaic acid inhibited dephosphorylation of CT, which decreased the stability of the membrane-bound form, resulting in increased soluble CT. Oleic acid, probably acting via DAG, counteracted the effects of phosphorylation by stabilizing the membrane-bound form. E. Effects of Cholecystokinin
The effects of this hormone imply a role for Ca^"^ calmodulin kinase in the regulation of CT. Cholecystokinin (CCK) stimulates pancreatic acinar cells to secrete digestive enzymes. It also inhibits PC synthesis, and this effect may be related to the generation of DAG, a second messenger in stimulus-secretion coupling (Matozaki et al., 1991). CCK inhibited CT activity (both soluble and particulate, assayed in the absence of lipid vesicles), and the degree of inhibition increased when phosphatase inhibitors were added to the extracts for enzyme activity analysis. The inhibition of CT was prevented by a calmodulin antagonist (W-7), suggesting that the Ca^"^-calmodulin activated protein kinase may participate in the inactivation of CT (Matozaki, 1991). The phosphorylation state of CT was reduced by CCK treatment. Moreover CT proteolytic processing was stimulated by this hormone, producing a -30 kDa non-phosphorylated fragment (Groblewski etal., 1995). F. Effects of Phospholipase C
PLC stimulation of PC synthesis is associated with translocation of soluble CT to membranes. Watkins and Kent (1991) analyzed the phosphorylation state of CT
Regulation of Mammalian
CTP
21
and changes in phosphorylation accompanying the PLC-induced translocation by immunoprecipitation after in vivo ^^P-labeling CHO cells. The soluble enzyme in control cells was multiply phosphorylated, and the membrane-bound CT in PLCtreated cells was nonphosphorylated implying that translocation to membranes was associated with dephosphorylation. If the cells were pretreated with okadaic acid, the effects of PLC on PC synthesis and membrane binding were diminished. In agreement with the studies of Hatch et al. (1992), okadaic acid increased the phosphorylation state of the soluble form of the enzyme. The authors concluded that dephosphorylation was a prerequisite for membrane binding. Houweling et al. (1993b) also observed that PLC-induced translocation of soluble CT to membranes in hepatocytes was accompanied by dephosphorylation. However, in vitro soluble CT associated with membranes from PLC-treated rat hepatocytes without significant dephosphorylation. This would suggest that the phosphorylated enzyme can bind membranes and is subsequently dephosphorylated. G. Effects of Oleic Acid A time-course of the effects of oleic acid on ^^P-labeled CT in the cytosol and membrane fractions of hepatocytes indicated that CT translocated to the membranes in phosphorylated form and was subsequently (within one hour) dephosphorylated (Houweling et al., 1993b). In HeLa cells oleic acid-induced translocation also correlated with CT dephosphorylation within 15 minutes (Wang et al., 1993a). Extraction of the oleic acid from the cell membranes by treatment of the cells with BSA resulted in very rapid release (<1 minute) of CT from the membranes. The released form was not phosphorylated initially, but became fully phosphorylated within 30 minutes, as assessed by migration shifts on SDS gels. The studies with okadaic acid, oleic acid, and PLC indicate that CT is in dynamic equilibrium between a cytosolic phosphorylated form and a membrane bound dephosphorylated form. The exact role of phosphorylation changes and the relative significance of the role of phosphorylation vs. lipid signals in the control of translocation needs additional clarification. In the final section I present some ideas as to how these signals are coordinated.
VII. STRUCTURE OF MAMMALIAN CT The purification of rat liver CT (Weinhold et al., 1986; Feldman and Weinhold, 1987) ushered in a new era of research on CT regulation. The protocol involves three major purification steps: (a) affinity for PC/oleic acid vesicles (the vesicles with bound protein are precipitated with acid and resuspended in octylglucoside (25-fold purification)); (b) DEAL Sepharose chromatography (fourfold); and (c) hydroxylapatite chromatography (20-fold). A second HA chromatography step removes a --36 kDa contaminant protein (Feldman and Weinhold, 1987). Alternatively a Mono-Q column has been substituted for the second HA column to obtain
22
ROSEMARY B. CORNELL
nearly homogeneous enzyme (Sanghera and Vance, 1989b). The enzyme elutes in the final elution step bound to a Triton micelle. The yields are reasonable (10-25%), but since CT is so scarce in rat liver, only -100 jug pure enzyme can be obtained from 10 livers. Overexpression in Sf9 insect cells using baculovirus has resulted in CT becoming the major protein in these cells. The Weinhold method has been adapted with success to obtain milligrams of pure CT (MacDonald and Kent, 1993; Lucheetal., 1993). A. Molecular Mass and Subunit Structure
The molecular mass (Mj.) of rat liver CT, based on the cDNA-derived protein sequence, is 41,720 Da (Kalmar et al., 1990). On SDS gels CT migrates with an apparent molecular mass of-42 kDa. By gel filtration, purified CT complexed with the Triton micelle is -240 kDa. This result originally suggested that native CT is a tetramer of the 42 kDa subunit. Further studies revealed the gel filtration behavior to be anomalous. The molecular mass of the two cytosolic forms from lung was estimated using standardized glycerol density gradients and gel filtration to obtain sedimentation coefficients and stokes radii (Weinhold et al., 1989). The M^. for the L-form was 97,690 Da, and the M^ for the H-form was 284,000 Da. The H-form was proposed to be a lipoprotein micelle of the CT dimer (-90 kDa) plus phospholipids (-250 molecules/micelle). Chemical cross-linking of the purified rat liver CT with four different reagents yielded a 84 kDa band, which was resolved after reduction in the second dimension into the 42 kDa species (Cornell, 1989). Thus CT is a homodimer of a 42 kDa polypeptide. The cross-linking study indicated that the dimer is present when bound to a Triton micelle or to a phospholipid vesicle. The interactions of the dimer are very strong. Disulfide bonds do not appear to be involved. Some dimer persists after boiling samples in 8% SDS and 2% p-mercaptoethanol for 5 minutes, and SDS PAGE. B. Amino Acid Sequence and Sequence Homologies
The amino acid sequence of rat liver CT was deducedfi-omthe cDNA sequence (Kalmar et al., 1990; MacDonald and Kent, 1993). The rat liver enzyme is 367 amino acids long. It is a hydrophilic protein; the longest continuous stretch of nonpolar residues is five. Thus CT has poor potential for spanning the membrane as an a-helix. There are no consensus motifs for covalent attachment of lipids. There is a 11-mer motif repeated three fimes in tandem between residues 256-288. This motif falls within a predicted amphipathic a-helix (see Figure 2). The C-terminal 50 residues are very rich in serine and proline residues, and contain the motif SPSSSP twice and also the motif SPSPS. There are two potential nuclear localization signals; ^^RKRRK and ^'^^KVKKKVK. A central domain of the rat liver CT shows striking homologies to a similarly located domain in yeast CT and to Bacillus subtilis glycerol phosphate cytidylyltransferase (catalyzes the reaction CTP + G3P -> CDPglycerol + PP). The yeast
Regulation of Mammalian CTP Nuclear Localization
^''
.catalytic domain
23
'\
lipid-binding • ; Ilmer
] N h ^
phosphorylation •.
'
-f^y -235 I ^ S T ! : 315 ! ■ ■ ■ ■ ■ ■ ■ ■ — I I 1 1 l|il l| conserved central domain Helix-1 Helix-2
367' I
Figure 2. Domain structure of mammalian CT.
and rat CT sequences share 64% identity between residues 74—234 of the rat and 100-262 of the yeast CT. The same region of the rat CT shares 33% identity with the B. subtilis CT (Mauel et al., 1991). The strong homologies within this region argue for its role as the active site. The yeast and rat CTs are only weakly homologous outside this conserved central domain. Mammalian, yeast, and the B. subtilis CT all contain a motif, HXGH, where X = serine, tryptophan, or leucine, which is shared with class I amino acyl tRNA synthetases. In the crystal structures of two tRNA synthetases, this motif participates in binding ATR The analogous motif in the cytidylyltransferases has been proposed as a CTP binding site (Bork et al., 1995). In support of this notion, a mutation of the glycine of the HXGH motif to serine in the rat liver CT caused a 25-fold increase in the K^^ for CTP (Veitch and Cornell, 1996). Mammalian CTs from mouse (Rutherford et al., 1993), a human erythroleukemic cell line (Kamar et al., 1994), CHO cells (Sweitzer and Kent, 1993; Hogan et al., 1995) and have recently been cloned and sequenced. The mouse clone has only three conservative changes from the rat. The human clone has 14 amino acid changes; all but two are clustered at the extreme N- or C-terminus, The CHO clone is similarly 98% identical to the rat. The high homology between these four mammalian CTs suggest only one cDNA isoform has been isolated. C. Phosphorylation Sites
Rat liver CT was purified from the baculovirus expression system after labeling in vivo with ^^P. Sequencing of phosphopeptides revealed that all of the 16 serines within the C-terminal 53 amino acids were phosphorylated to some extent. This same C-terminal region has been identified as the sole domain of phosphorylation (domain P) by analysis of deletion mutants expressed in COS cells (Cornell et al., 1995) or CHO cells (Wang and Kent, 1995a). CT phosphorylation in vitro was catalyzed by casein kinase II, cell division control 2 kinase (cdc2 kinase), protein kinase C, and glycogen synthase kinase-3 (GSK-3), but not by MAP kinase. All kinase targets were localized to the C-terminal domain P, except for protein kinase C, which phosphorylated sites throughout the protein (Cornell et al., 1995). The stoichiometry of phosphorylation catalyzed by any of these kinases was low in this
24
ROSEMARY B. CORNELL
study (< 0.2 mol/mol), because the purified enzyme was in a phosphorylated form, and no effects on enzyme activity were detected. D. Secondary and Tertiary Structural Predictions
Using the Gamier et al. (1978) and Chou and Fasman (1978) algorithms, a model of CT secondary structure emerged. The N-terminal two thirds (to residue 235) alternates between short a and P segments linked by turns, suggestive of a tightly folded globular domain. This domain contains the putative catalytic site. Residues 236-316 are predicted to be entirely a helical with one turn between 294-297. We refer to these domains as helix-1 and helix-2 (see Figure 2). Helix-1 contains the 11-mer repeat. Both helices are strikingly amphipathic. Helix-1 has a highly charged polar face with 16 positive and 13 negative charges, and the potential for numerous helix-stabilizing salt bridges. The polar face of helix-2 is very basic (net charge = +4). C-terminal to the long helical domain is a nonstructured domain containing eight prolines within 29 residues. Thus we proposed that CT consists of a globular head domain linked to a C-terminal extended tail (Craig et al., 1993). The prediction of the bipartite tertiary structure of CT has been confirmed by limited proteolysis. Chymotrypsin and other proteases digest CT progressively from the C-terminus producing relatively resistant fragments that have been mapped to the N-terminal two thirds; i.e., the proposed tightly folded globular domain (Craig et al., 1993). The C-terminus is more protease-sensitive suggesting it is in an extended conformation. Three functional domains are proposed to correlate with three structural regions, (a) The catalytic domain (conserved domain) resides within the N-terminal globular region (residues 74-235); (b) a membrane-binding domain is composed of the first long helix (residues 236-293); and (c) a phosphorylation domain is located in the C-terminal unstructured 50 amino acids. E. Membrane-Binding Domain
What kind of structure would mediate a membrane interaction that is electrostatic and hydrophobic, reversible, lipid specific, and sensitive to the phosphorylation state of the enzyme? We proposed that the amphipathic a-helical region would accommodate most of these properties (Kalmar et al, 1990). We envisioned, drawing on proposals for the interaction of apolipoproteins with lipid particles, that the amphipathic helix would lie on the surface of the bilayer with its helix axis parallel to the membrane surface, its hydrophobic face penetrating into the bilayer core, and its polar face interacting with water, phospholipid head groups, or other domains of CT in the aqueous phase (see Figure 3). A role for the amphipathic helix in membrane binding was demonstrated in membrane binding studies of proteolytic fragments of CT. Partial chymotrypsin digestion of CT produced five fragments between 39 and 26 kDa, all lacking the
Regulation of Mammalian CTP
25
C-terminus, but containing the N-terminus, as assessed by reactivity with antipeptide antibodies against N- and C-terminal CT peptides. The 26, 28, and 30 kDa fragments lack the amphipathic helix domain, and did not bind to phospholipid vesicles containing class I or class II activators. However the 35 kDa fragment (which contains helix-1) and the 39 kDa fragment (which contains both helix 1 and 2) behaved much like undigested CT and did bind to these membranes (Craig et al., 1994). These experiments indicate a correlation between the presence of the amphipathic helix-1 and membrane binding. Additional experiments suggested that chymotryptic sites within and adjacent to the amphipathic helix-1 are less accessible to protease when CT is bound to activating lipid vesicles. The simplest explanation is that the helix becomes buried with the vesicle bilayer upon CT binding (Craig et al, 1993). Evidence that the putative helical domain is in fact a-helical has emerged from circular dichroism analysis of peptides derived from the sequence of helix-1. A 33-mer comprised of the three 11-mer repeats was largely random coil in dilute aqueous buffer but was 60-70% helical in the presence of anionic phospholipid small unilamellar vesicles (Johnson and Cornell, 1994). The peptide contains one tryptophan in the center of the nonpolar face. The fluorescence E^^^^ of this tryptophan blue-shifted in the presence of anionic vesicles, indicative of a change to a more nonpolar environment. In the presence of anionic vesicles the trp fluorescence was shielded from quenching by iodide, an aqueous phase quencher. The trp fluorescence was quenched 60% in the presence of anionic vesicles containing 50 mole percent 9,10-dibromo-PC, a lipid phase quencher. No changes in the degree of a-helicity, wavelength of the trp E^^^, or iodide quenching of the trp fluorescence were observed in the presence of vesicles composed of PC alone, PC-sphingosine, or PC-DAG (Johnson and Cornell, 1994). These results suggest that the amphipathy of this peptide is induced selectively by anionic phospholipid vesicles, that the peptide binds to these vesicles in an a-helical conformation, and that binding involves intercalation into the hydrophobic core. The 11-mer repeat is either not involved or is only a portion of the domain required for interaction with membranes containing class II lipid activators. Studies with alternative peptides are underway in an attempt to delineate the domain that interacts with these lipids. At this time the molecular basis for the lipid specificity is far from understood. Evidence from cDNA deletion analysis also implicates the amphipathic helix domain in membrane binding. Expression of CT mutants truncated at amino acids 228 or 236 resulted in a reduced association of CT with membranes of COS or CHO cells, respectively (Cornell et al., 1995; Wang and Kent, 1995b). F. Dimerization Domain A suggestion that one or both of the amphipathic a-helices could mediate the dimerization of the 42 kDa subunit in the manner of a coiled-coil (Kalmar et al.,
26
ROSEMARY B. CORNELL
1990) now appears to be wrong. Chymotrypsin fragments of CT lacking the C-terminus including all of the amphipathic helix still form tight dimers that can be detected on SDS gels. These dimer bands (56-60 kDa) are seen only in connection with the 28-30 kDa bands, and react with antipeptide antibodies against the N-terminal peptide and a peptide from the conserved central domain, but do not react with anti-C-terminal peptide antibodies (Craig et al., 1993). Thus the N-terminal domain is sufficient for dimerization. The amphipathic helix may also participate in dimerization, but it is not required. Since there is evidence that the amphipathic helix-1 is the lipid binding domain, and since CT binds membranes as a dimer, it is reasonable that a site outside the amphipathic helix would mediate the interactions of the dimer. G. Model of CT Interaction With Membranes
A model of CT interacting with membranes is shown in Figure 3. The enzyme is shown as a dimer in a parallel arrangement (head to head; tail to tail). We show the amphipathic helices of the two monomers interacting, although there is no direct evidence for this. Helix-2 is not binding to the membrane, in keeping with proteolysis data showing it is not needed for binding to lipid vesicles. The Nterminal 230 amino acids are portrayed as a large globular domain.
C-termlnus
Figure 3. A model for the structure of CT and its interaction with the lipid bilayer.
Regulation of Mammalian CTP
17
VIII. MECHANISM OF THE REGULATION OF ACTIVITY IN VIVO A.
Regulation Induced Solely by Changes in the Lipid Composition
Phorbol esters represent the best example of an effector that activates CT in the absence of any detectable changes in the phosphorylation state. Two laboratories have reported no change in ^^P association with CT after immunoprecipitation from HeLa cells cultured in the presence or absence of TPA (Watkins and Kent, 1990; Utal et al, 1991). Utal found that after 1 hour incubation with 100 nM TPA, the level of CT phosphoenzyme in the particulate fraction was increased. This suggests that phosphorylated CT bound to the membranes. Both groups found evidence for stimulation of the CT reaction, however Watkins and Kent (1990) did not observe enhanced membrane binding, in contrast to the findings of others (Pelech et al., 1984a; Utal et al., 1991). Utal et al. (1991) obtained a twofold increase in DAG in TPA-treated cells which preceded the binding of CT to membranes. DAG was directly tested as an activator using soluble, di Cg, and was found to stimulate CT activity and translocation (Utal, 1991; Kolesnick and Hemer, 1990). The available data would suggest that the activation of CT by phorbol esters is mediated by increases in membrane DAG concentration alone. When neutrophils were treated with the chemotactic peptide fMLP or fMLP plus cytochalasin, phospholipase D activation led to the rapid accumulation of PA or PA plus DAG, respectively (Tronchere et al., 1995). CT translocation to the cell membranes was correlated with DAG, but not PA production. Phorbol ester treatment of neutrophils stimulated CT translocation which correlated kinetically with the production of DAG from PC, but not from phosphatidylinositol bis phosphate hydrolysis (Tronchere et al., 1995). Regulation of CT activity can be accomplished in vitro without any change in the phosphorylation state. For example purified CT is activated by pure phospholipid vesicles of the appropriate composition. There are no protein phosphatases in this system. But this does not eliminate an important role for changes in phosphorylation in vivo. For one thing the precise phosphorylation state of purified rat liver CT is unknown. Fractionation may result in specific loss of phosphorylated forms. Assuming for the moment that it is at least partially phosphorylated, then in the model system phosphorylated enzyme interchanges between an active (E^) and inactive (E2) conformation. The lipid vesicles stabilize the acfive form, and thus shift the equilibrium from E2 ^ Ej (see Figure 4). If the pure enzyme were completely dephosphorylated, much lower concentrations of lipid activator might be required to favor Ej. The lipid activators and CT phosphorylation would have opposite effects on the membrane/aqueous partition coefficient. Thus the relative strength of the phosphorylafion vs. lipid activator signals would influence the
28
ROSEMARY B. CORNELL
intracellular distribution between soluble and membrane-bound states. In vivo the concentration of lipid activators may never reach more than a few mole percent. B. Regulation Induced Solely by Changes in Phosphorylation
At this time the experimental evidence for regulation solely by phosphorylation is relatively meager. Okadaic acid caused an increase in the degree of phosphorylation of CT, which correlated with decreased activity and membrane binding (Watkins and Kent, 1991; Hatch et al., 1992). The magnitude of this shift in distribution was small compared to the large effects of fatty acids or PLC, which clearly alter both the lipid content and phosphorylation state of CT. Treatment of hepatocytes with okadaic acid did not change the free fatty acid content, but it did result in lower DAG content compared to control hepatocytes incubated identically except without okadaic acid (Hatch et al., 1992). The PC content was not measured. When oleate was added with the okadaic acid the relative decline in the DAG content was blocked, as was CT translocation to the cytosol. These results might suggest that, in addition to CT phosphorylation changes, diacylglycerol was participating in the regulation of CT translocation. However, since the DAG content did not decrease over the 2 hour okadaic acid treatment, yet the PC synthesis rate declined and CT was released from the membrane during this time, the authors concluded that DAG was not limiting PC synthesis (Hatch, 1992). Further studies on the role of DAG concentrations in the okadaic acid induced inhibition of CT activity are necessary before it can be concluded that changes in CT phosphorylation alone are sufficient to shift the E^ ^ E2 equilibrium. C. Regulation Involving Changes in Both Lipid Composition and Phosphorylation
PLC and fatty acids are two effectors that have now been demonstrated to activate CT via changes in both the lipid composition and the phosphorylation state. The lipid signal associated with PLC action was originally attributed to a decrease in the relative amount of PC (Sleight and Kent, 1980, 1983b), since PC was the primary phospholipid degraded by PLC. However, decreases in mass were not detected because of the rapidly induced stimulation of PC synthesis (Kent, 1979). Slack et al. (1991) also were unable to detect decreases in PC content due to PLC treatment, except at very high concentrations (0.5 units/ml). CT activation is maximal at 0.1 unit/ml. PLC treatment generated two to threefold increases in DAG content (Sleight and Kent, 1980; Slack et al., 1991; Jamil et al., 1993). Thus the lipid alteration that triggers CT translocation may be either an elevation of DAG content, or a combination of an increase in DAG and a decrease in PC. PLC treatment also affects the phosphorylation state. In CHO cells (Watkins and Kent, 1991) and rat hepatocytes (Houweling et al., 1994) the activation of CT by membrane translocation was associated with a decrease in the phosphorylation state of immunoprecipitated CT. Reversal of the PLC treatment resulted in increased
Regulation of Mammalian CTP
29
soluble CT which was phosphorylated. The translocation and dephosphorylation of CT, which was also evident from gel migration shifts, was blocked by pretreatment of CHO cells with okadaic acid. This implied that dephosphorylation might be a prerequisite for CT binding to membranes (Watkins and Kent, 1991). Another explanation of these results is that the phosphorylation of CT counteracted the stabilization of the membrane form by the PLC modification of membrane lipids, which shifted the equilibrium towards the soluble form. In vitro, soluble enzyme did bind in its phosphorylated form to membranes from PLC-treated cells (Houweling et al, 1994), suggesting that the lipid signal (DAG or PC) can overpower the phosphorylation signal if an excess of membranes is present. Fatty acid treatment of cultured cells leads to large increases in the membrane content of fatty acid. The increase in membrane CT directly correlates with the increase in membrane fatty acid content (Weinhold et al., 1984; Cornell and Vance, 1987a). The rapid reversibility of the membrane association of CT by albumin also suggests that membrane free fatty acids are responsible for the CT binding. In hepatocytes incubated with oleate plus 1% BSA the uptake of the fatty acid was slower such that free fatty acids did not accumulate; rather, DAG accumulated under these conditions (Hatch et al., 1992). As with PLC treatment, oleate induction of CT translocation to membranes correlated with a reduction in ^^P-labeling of CT (Wang et al, 1993a; Houweling et al., 1993). A time course of the change in ^^P associated with soluble and membrane CT, after addition of oleate to the cells, indicated that the enzyme translocated to membranes in the phosphorylated form and was subsequently dephosphorylated (Houweling et al., 1994). The lipid signal in this case which promoted membrane binding was stronger than the phosphorylation signal which stabilized the soluble form. Dephosphorylation accompanied the long-term, stable association of CT with the membrane, but was not required for the initial binding. Furthermore, the translocated, phosphorylated enzyme was active. This study illuminated the role of membrane-bound phosphatases in the control of the phosphorylation state of CT, and demonstrated that CT can be membrane-bound, phosphorylated, and active (Ej(L)(P) of Figure 4). Wang et al. (1993a) analyzed the change in phosphorylation after BSA reversal of the oleate-induced membrane binding. CT was released from the membranes in less than one minute after BSA addition; it was released in a dephosphorylated form that was subsequently (within 16 minutes) phosphorylated by cytosolic kinases. The released soluble enzyme was initially partially active. Its activity declined in correlation with increased phosphorylation, as assessed by gel migration shifts. D. Interrelationship of the Various CT Conformations In the scheme depicted in Figure 4,1 have tried to integrate the effects of lipid activators and phosphorylation into a coherent scheme which treats both modifications as stabilizers of two rapidly interchanging conformations of CT. Lipids
30
ROSEMARY B. CORNELL
^E1(L)(P)^=:^E2(L)(P) E1(L)
E2(P)
Figure 4. Pathways for interconversion of CT forms.
stabilize Ej and phosphorylation stabilizes £2- Lipids (L) refers to a lipid aggregate, either in membrane form or the micellar organization of the cytosolic H-form. The binding of CT to this aggregate depends on the presence of activator lipids v^ithin the aggregate which destabilize bilayer packing. Phosphorylation (P) refers to phosphoryl modification of critical sites that decrease the membrane partition coefficient. Phosphorylation at some sites may not influence the activity or intracellular distribution. In fact, Houweling et al. (1994) discovered some new phosphopeptides in the membrane-bound CT from oleic acid treated hepatocytes that were not present in the soluble CT, suggesting that phosphorylation of CT at some sites occurs while membrane bound. Ei(L) is the most abundant, stable membrane-bound active form, and E2(P) is the most abundant soluble phosphorylated form. The most likely pathways for their interconversion are diagrammed in Figure 4. The interconversion between these forms can proceed via phosphorylation/dephosphorylation in the cytosol (or nucleoplasm) by soluble kinases or phosphatases (lower route in Figure 4), or by phosphorylation/dephosphorylation on the membrane (upper route in Figure 4). E2 is a transient form which results from dephosphorylation prior to membrane binding. Its formation from E2(P) was blocked in CHO cells treated with okadaic acid prior to PLC treatment. Ej is another intermediate soluble form. This is the active, but lipid-free* and dephosphorylated form released by BSA treatment of oleic acid enriched cells (Wang et al., 1993a). E2(L)(P) is the transient form that binds membranes after fatty acid enrichment (Houweling et al., 1994). It is rapidly converted to Ej(L)(P) and more slowly dephosphorylated to Ej(L). Ej(L)(P) accumulates in cells treated with okadaic acid plus oleate. Okadaic acid prevents dephosphorylation so the active enzyme remains as Ej(L)(P) (Hatch et al., 1992). Ej(L)(P) is also the form which is bound to membranes in phorbol ester treated cells, where no change in the phosphorylation state was detected. Whether the enzyme that is modified by both phosphorylation and lipids is in the active (Ej) or inactive (E2) conformation depends on the relative strengths of these signals, that
Wang et al. showed that this form was membrane-free. It may have not been lipid-free, if released as the H-form (e.g., Feldman et al., 1990).
Regulation of Mamma Han CTP
31
is, the nature of the activating Hpid and its mole fraction in the membrane and the occupation of the several critical phosphorylation sites. It is noteworthy that those effectors that induce the greatest redistribution of CT in the cell affect both phosphorylation and lipid composition (e.g., oleic acid and PLC). The scheme presented in Figure 4 is based on quite limited information on the relationship between the two effectors of CT activity. It is presented as a guide to integrate our thinking and as a model to be tested. It will likely be modified and refined as we learn more about the regulation of CT.
IX. FUTURE DIRECTIONS Future research will concentrate on unraveling the molecular basis for the regulation of CT activity and expression. Identification of the important functional domains of CT and elucidation of their structure is required before we can ask many important questions, such as: How does lipid binding or phosphorylation at certain sites change the conformation of the catalytic domain? What is the molecular basis for the lipid selectivity in the membrane interaction? What is the role of dimerization in the function of this enzyme? Transcriptional control of CT expression has never been demonstrated. Sequencing mammalian CT genes would give clues to how its transcription is regulated. Identification of the kinases and phosphatases that directly or indirectly control CT activity, as well as the lipid species that regulate CT in vivo, will be important steps in determining how CT integrates into the cellular metabolic network, and how CT participates in signal transduction pathways.
ACKNOWLEDGMENTS The research in the author's laboratory was funded by the Natural Science and Engineering Research Council and the British Columbia Health Care Research Foundation. I am grateful to Dr. Dennis Vance, Dr. Claudia Kent, Dr. Martin Houweling, Joanne Johnson, and Dallas Veitch for helpful comments on this manuscript.
REFERENCES Banfic, H,, Zizak, M., Divecha, N., & Irvine, R.F. (1993). Nuclear diacylglycerol is increased during cell proliferation in vivo. Biochem. J. 290, 633-636. Billah, M.M. & Anthes, J.C. (1990). The regulation and cellular functions of phosphatidylcholine. Biochem. J, 269, 281-291. Blusztajn, J.K. & Zeisel, S.H. (1989). l,2-5«-Diacylglycerol accumulates in choline-deficient liver: A possible mechanism of hepatic carcinogenesis via alteration in protein kinase C activity? FEBS Lett.243, 267-270. Boggs, K., Rock, C, & Jackowski, S. (1995). Lysophosphatidylcholine and 1-0-octadecyl- 2-0-methylrac-glycero-3-phosphocholine inhibit the CDP-choline pathway of phosphatidylcholine synthesis at the CTP: Phosphocholine cytidylyltransferase step. J. Biol. Chem. 270, 13, 7757-7764.
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Bork, P., Holm, L., Koonin, E., & Sander, C. (1995). The cytidylyltransferase superfamily: Identification of the nucleotide-binding site and prediction. Proteins: Struct. Func. Genet. 22, 259-266. Burkhardt, R., Von Wichert, P., Batenburg, J., & Van Golde, L. (1988). Fatty acids stimulate PC synthesis and cytidylyltransferase activity in type II pneumocytes. Biochem J. 254, 495-500. Casola, P.O., Chan, F., MacDonald, P.M., Ryan, S., McMurray, W.C, & Possmayer, F. (1981). Coordinate increases in the enzyme activities responsible for phosphatidylglycerol synthesis and CTP: Phosphocholine cytidylyltransferase activity in developing rat lung. Biochem. Biophys. Res. Comm. 96, 120^1215. Choy, RC, Lim, RH., & Vance, D.E. (1977). Purification and characterization of CTP: Phosphocholine cytidylyltransferase from ratsliver cytosol. J. Biol. Chem. 252, 7673-7677. Choy, RC. & Vance, D.E. (1978). Lipid requirements for activation of CTP: Phosphocholine cytidylyltransferase from rat liver. J. Biol. Chem. 253, 5163—5167. Choy, RC, Farren, S.B., & Vance, D.E. (1979). Lipid requirements for the aggregation of CTP: Phosphocholine cyltidylyltransferase in rat liver cytosol. Canad. J. Biochem. 57, 605-612. Choy, RC. (1982). Control of PC biosynthesis in myopathic hamster heart. J. Biol. Chem. 257, 10928-10933. Chou, P. & Fasman, G. (1978). Empirical predictions of protein conformation. Annu. Rev. Biochem. 47,251-276. Chu, A.J. & Rooney, S.A. (1985). Stimulation of cholinephosphate cytidylyltransferase activity by estrogen in fetal rabbit lung is mediated by phospholipids. Biochim. Biophys. Acta 834,346-356. Cook, H.W. & Vance, D.E. (1985). Evaluation of possible mechanisms of phorbol ester stimulation of phosphatidylcholine synthesis in HeLa cells. Can. J. Biochem. Cell Biol. 63, 145-151. Cornell, R.B. (1989). Chemical cross-linking reveals a dimeric structure for CTP:phosphocholine cytidylyltransferase. J. Biol. Chem. 264, 9077-9082. Cornell, R.B. (1991a). Regulation of CTP:phosphocholine cytidylyltransferase by lipids. 1. Negative surface charge dependence for activation. Biochemistry 30, 5873-5880. Cornell, R.B. (1991b). Regulation of CTP:phosphocholine cytidylyltransferase by lipids. 2. Surface curvature, acyl chain length, and lipid-phase dependence for activation. Biochemistry 30, 5881— 5888. Cornell, R.B. & Goldfine, H. (1983). The coordination of sterol and phospholipid synthesis in cultured myogenic cells: Effect of cholesterol synthesis inhibition on the synthesis of phosphatidylcholine. Biochim. Biophys. Acta 750, 504-520. Cornell, R.B., Kalmar, G., Kay, R., Johnson, M., Sanghera, J., & Pelech, S. (1995). Functions of the C-terminal domain of CTP:phosphocholine cytidylyltransferase. Biochem. J. 310, 699-708. Cornell, R.B. & MacLennan, D.H. (1985). The capacity of the sarcoplasmic reticulum for phospholipid synthesis: A developmental study. Biochim. Biophys. Acta 835, 567-576. Cornell, R.B. & Vance, D.E. (1987a). Translocation of CTP:phosphocholine cytidylyltransferase from cytosol to membranes in HeLa cells: Stimulation by fatty acid, fatty alcohol, mono- and diacylglycerol. Biochim. Biophys. Acta 919,26-36. Cornell, R.B. & Vance, D.E. (1987b). Binding of CTP:phosphocholine cytidylyltransferase to large unilamellar vesicles. Biochim. Biophys. Acta 919, 37—48. Craig, L., Johnson, J.E., & Cornell, R.B. (1994). Identification of the membrane-binding domain of rat liver CTP:phosphocholine cytidylyltransferase using chymotrypsin proteolysis. J. Biol. Chem. 269,3311-3317. Divecha, N., Banfic, H., & Irvine, R.R (1991). The polyphosphoinositide cycle exists in the nuclei of Swiss 3T3 cells under the control of a receptor for IGF-1 in the plasma membrane, and stimulation of the cycle increases nuclear diacylglycerol and induces translocation of protein kinase C to the nucleus. EMBO J. 10, 3207-3214. Feldman, D.A., Kovac, C.R., Dranginis, P.L., & Weinhold, P. A. (1978). The role of phosphatidylglycerol in the activation of CTP:phosphocholine cytidylyltransferase from rat lung. J. Biol. Chem. 253, 4980-4986.
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Feldman, D.A., Dietrich, J.W., & Weinhold, RA. (1980). Comparison of the phosphoHpid requirements and molecular form of CTP:phosphocholine cytidylyltransferase from rat lung, kidney, brain and liver. Biochim. Biophys. Acta 620, 603-611. Feldman, D.A., Brubaker, P.G., & Weinhold, P.A. (1981). Activation of CTPiphosphocholine cytidylyltransferase in rat lung by fatty acids. Biochim. Biophys. Acta 665, 53-59. Feldman, D.A., Rounsifer, M.E., & Weinhold, RA. (1985). The stimulation and binding of CTP:phosphorylcholine cytidylyltransferase by phosphatidylcholine-oleic acid vesicles. Biochim. Biophys. Acta 833,429-437. Feldman, D.A. & Weinhold, P.A. (1987). CTP:phosphorylcholine cytidylyltransferase from rat liver: Isolation and characterization of the catalytic subunit. J. Biol. Chem. 262, 9075-9081. Feldman, D.A., Rounsifer, M.E., Charles, L., & Weinhold, RA. (1990). CTPiphosphocholine cytidylyltransferase in rat lung: Relationship between cytosolic and membrane forms. Biochim. Biophys. Acta 1045, 49-57. Feldman, D.A. & Weinhold, P. A. (1993). Identification of a protein complex between choline-phosphate cytidylyltransferase and a 112-kDa protein in rat liver. J. Biol. Chem. 268, 3127—3135. Fiscus, W.G. & Schneider, WC. (1966). The role of phospholipids in stimulating phosphorylcholine cytidyltransferase. J. Biol. Chem. 241, 3324-3330. Florin-Christensen, J., Florin-Christensen, M., Delfmo, J. Stegmann, T, & Rasmussen, H. (1992). Metabolic fate of plasma membrane diacylglycerols in NIH 3T3 fibroblasts. J. Biol. Chem. 267, 14783-14789. Fraslon, C. & Batenburg, J.J. (1993). Pre-translational regulation of lipid synthesizing enzymes and surfactant proteins in fetal rat lung explant culture. FEBS Lett. 325, 285-290. Gamier, J. Osguthorpe, D., & Robson, B. (1978). Analysis of the accuracy and implications of simple methods for predicting the secondary structure of globular proteins. J. Mol. Biol. 120, 97-120. Geilen, C.C., Wieder, T, & Reutter, W (1992). Hexadecylphosphocholine inhibits translocation of CTPrcholine-phosphate cytidylyltransferase in Madin-Darby canine kidney cells. J. Biol. Chem. 267,6719-^724. George, T.R, Morash, S.C., Cook, H.W., Byers, D.M., Palmer, F.B.S.C, & Spence, M.W. (1989). Phosphatidylcholine biosynthesis in cultured glioma cells: Evidence for channeling of intermediates. Biochim. Biophys. Acta 1004, 283-291. George, T.R, Cook, H.W., Byers, D.M., Palmer, F.B.S.C, & Spence, M.W. (1991). Channeling of intermediates in the CDP-choline pathway of phosphatidylcholine biosynthesis in cultured glioma cells is dependent on intracellular Ca^"^. J. Biol. Chem. 266, 1241^12423. Groblewski, G., Wang, Y., Ernst, S., Kent, C, & Williams, J. (1995). Cholecystokinin stimulates the down-regulation of CTP: Phosphocholine cytidylyltransferase in pancreatic acinar cells. J. Biol. Chem. 270, 1437-1442. Guy, G.R. & Murray, A.W. (1982). Tumor promoter stimulation of phosphatidylcholine turnover in HeLa cells. Cancer Res. 42, 1980-1985. Hatch, G., Tsukitani, Y., & Vance, D.E. (1991). The protein phosphatase inhibitor, okadaic acid, inhibits PC synthesis in isolated rat hepatocytes. Biochim. Biophys. Acta 1081, 25-32. Hatch, G.M., Lam, T.-S., Tsukitani, Y, & Vance, D.E. (1990). Effect of NaF and okadaic acid on the subcellular distribution of CTP:phosphocholine cytidylyltransferase activity in rat liver. Biochim. Biophys. Acta 1042, 374-379. Hatch, G.M., Jamil, H., Utal, A.K., & Vance, D.E. (1992). On the mechanism of the okadaic acid-induced inhibition of phophatidylcholine biosynthesis in isolated rat hepatocytes. J. Biol. Chem. 267, 15751-15758. Hogan, M., Zimmerman, L.J., Wang, J., Kuliszewski, M., Lui, J., & Post, M. (1994). Increased expression of CTP: Phosphocholine cytidylyltransferase in maturing type II cells. Amen J. Physiol. 267, L25-35.
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Houweling, M., Tijburg, L.B.M., Vaartjes, W.J., Batenburg, J.J., Kalmar, G.B., Cornell, R.B., & van Golde, L.M.G. (1994). Evidence that CTPxholine-phosphate cytidylyltransferase is regulated at a pretranslational level in rat liver after partial hepatectomy. Eur. J. Biochem. 214, 927-933. Houweling, M., Jamil, H., Hatch, G., & Vance, D.E. (1994). Dephosphorylation of CTPxholine-phosphate cytidylyltransferase is not required for binding to membranes. J. Biol. Chem. 269, 75447551. Jackowski, S. (1994). Coordination of membrane phospholipid synthesis with the cell cycle. J. Biol. Chem. 269, 3858-3867. Jamil, H., Yao, Z., & Vance, D.E. (1990). Feedback regulation of CTP:phosphocholine cytidylyltransferase translocation between cytosol and endoplasmic reticulum by phosphatidylcholine. J. Biol. Chem. 265, 4332-4339. Jamil, H., Utal, A.K., & Vance, D.E. (1992). Evidence that cAMP-induced inhibition of phosphatidylcholine biosynthesis is caused by a decrease in cellular diacylglycerol levels in cultured rat hepatocytes. J. Biol. Chem. 267, 1752-1760. Jamil, H., Hatch, G.M., & Vance, D.E. (1993). Evidence that binding of CTP:phosphocholine cytidylyltransferase to membranes in rat hepatocytes is modulated by the ratio of bilayer- to non-bilayerforming lipids. Biochem. J. 291, 419-427. Johnson, J.E., Kalmar, G., Sohal, P, Walkey, C, Yamashita, S., & Cornell, R.B. (1992). Comparison of the lipid regulation of yeast and rat CTPiphosphocholine cytidylyltransferase expressed in COS cells. Biochem. J. 285, 815-820. Johnson, J.E. & Cornell, R.B. (1994). A membrane-binding amphipathic a-helix derived from CTPiphosphocholine cytidylyltransferase. Biochemistry 33,4327-4335. Kalmar, G.B., Kay, R.J., Lachance, A., Aebersold, R., & Cornell, R.B. (1990). Cloning and expression of rat liver CTPiphosphocholine cytidylyltransferase: An amphipathic protein that controls phosphatidylcholine synthesis. Proc. Natl. Acad. Sci. USA, 87, 6029-6033. Kalmar, G., Kay, R., LaChance, A., & Cornell, R. (1994). Primary structure and expression of a human CTP: Phosphocholine cytidylyltransferase. Biochimica et Biophysica Acta 1219, 328-334. Kent, C. (1979). Stimulation of phospholipid metabolism in embryonic muscle cells treated with phospholipase C. Proc. Natl. Acad. Sci. USA, 76, 4474-4478. Kent, C, Carman, G.M., Spence, M.W., & Dowhan, W. (1991). Regulation of eukaryotic phospholipid metabolism. FASEB J. 5, 2258-2266. Kinzel, V, Kreibich, G., Hecker, E., & Suss, R. (1979). Stimulation of choline incorporation in cell cultures by phorbol derivatives and its correlation with their irritant and tumor-promoting activity. Cancer Res. 39, 2743-2750. Kolesnick, R.N. (1990). 1,2-Diacylglycerols overcome cyclic AMP-mediated inhibition of phophatidylcholine synthesis in GH3 pituitary cells. Biochem. J. 267, 17-22. Kolsesnick, R.N. & Hemer, M.R. (1990). Physiological 1,2-diacylglycerol levels induce protein kinase C-independent translocation of a regulatory enzyme. J. Biol. Chem. 265, 10900-10904. Lacal, J.C. (1990). Diacylglycerol production in Xenopus laevis oocytes after microinjection of p2l'^^ proteins is a consequence of activation of phosphatidylcholine metabolism. Mol. Cell. Biol. 10, 333-340. Lim, PH., Pritchard, PH., Paddon, H.B., & Vance, D.E. (1983). Stimulation of hepatic PC synthesis in rats fed a high cholesterol and cholate diet correlates with translocation of CTPiphosphocholine cytidylyltransferase from cytosol to microsomes. Biochim. Biophys. Acta 753, 74-52. Liscovitch, M. (1992). Cross-talk among multiple signal-activated phopholipases. Trends Biochem. Sci. 17,393-399. Luche, M.M., Rock, CO., & Jackowski, S. (1993). Expression of rat CTPiphosphocholine cytidylyltransferase in insect cells using a baculovirus vector. Arch. Biochem. Biophys. 301, 114-118. MacDonald, J. & Kent, C. (1993). Baculovirus mediated expression of rat liver CTPiphosphocholine cytidylyltransferase. Prot. Express. Purif 4, 1-7.
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MacDonald, J., and Kent, C. (1994). Identification of phosphorylation sites in rat liver CTP: Phosphocholine cytidylyltransferase. J. Biol. Chem. 269, 10529-10537. MacKall, J. Meredith, M., & Lane, M.D. (1979). A mild procedure for the rapid release of cytoplasmic enzymes from cultured animal cells. Anal. Biochem. 95, 270-274. Matozaki, T., Sakamoto, C , Nishisaki, H., Suzuki, T., Wada, K., Matsuda, K., Nakano, O., Konda, Y., Nagao, M., & Kasuga, M. (1991). Cholecystokinin inhibits phosphatidylcholine synthesis via a Ca ^-calmodulin-dependent pathway in isolated rat pancreatic acini: A possible mechanism for diacylglycerol accumulation. J. Biol. Chem. 266, 22246-22253. Mauel, C , Young, M., & Karamata, D. (1991). Genes concerned with synthesis of poly(glycerol phosphate), the essential teichoic acid in Bacillus subtilis strain 168, are organized in two divergent transcription units. J. Gen. Micro. 137, 929-941. Morand, J.N. & Kent, C. (1989). Localization of the membrane-associated CTP:phosphocholine cytidylyltransferase in Chinese hamster ovary cells with an altered membrane composition. J. Biol. Chem. 264, 13785-13792. Niles, R.M. & Makarski, J.S. (1979). Regulation of phosphatidylcholine metabolism by cyclic AMP in a model alveolar type 2 cell line. J. Biol. Chem. 254, 4324-4326. Paddon, H.B. & Vance, D.E. (1980). Tetradecanoyl-phorbol acetate stimulates phosphatidylcholine biosynthesis in HeLa cells by an increase in the rate of the reaction catalyzed by CTP:phosphocholine cytidylyltransferase. Biochim. Biophys. Acta 620, 636-640. Pelech, S.L., Pritchard, P.H., & Vance, D.E. (1981). cAMP analogues inhibit phosphatidylcholine biosynthesis in cultured rat hepatocytes. J. Biol. Chem. 256, 8283-8286. Pelech, S.L. & Vance, D.E. (1982). Regulation of rat liver cytosolic CTP:phosphocholine cytidylyltransferase by phosphorylation and dephosphorylation. J. Biol. Chem. 257, 14198-14202. Pelech, S.L., Pritchard, P.H., Brindley, D.N., & Vance, D.E. (1983a). Fatty acids promote translocation of CTP:phosphocholine cytidylyltransferase to the endoplasmic reticulum and stimulate rat hepatic phosphatidylcholine synthesis. J. Biol. Chem. 258, 6782-6788. Pelech, S.L., Pritchard, RH., Brindley, D.N., & Vance, D.E. (1983b). Fatty acids reverse the cyclic AMP inhibition of triacylglyerol and phosphatidylcholine synthesis in rat hepatocytes. Biochem. J. 216, 129-136. Pelech, S., Power, E., & Vance, D.E. (1983c). Activities of the phosphatidylcholine biosynthetic enzymes in rat liver during development. Can. J. Biochem. Cell Biol. 61, 1147-1151. Pelech, S.L., Paddon, H.B., & Vance, D.E. (1984a). Phorbol esters stimulate phosphatidylcholine biosynthesis by translocation of CTP:phosphocholine cytidylyltransferase from cytosol to microsomes. Biochim. Biophys. Acta 795,447-451. Pelech, S.L., Cook, H.W., Paddon, H.B., & Vance, D.E. (1984b). Membrane-bound CTP:phosphocholine cytidylyltransferase regulates the rate of phosphatidylcholine synthesis in HeLa cells treated with unsaturated fatty acids. Biochim. Biophys. Acta 795,433-440. Pelech, S.L. & Vance, D.E. (1989). Signal transduction via phosphatidylcholine cycles. Trends Biochem. Sci. 14, 28-30. Posse de Chaves, B., Vance, D., Campenot, R., & Vance, J. (1995). Alkylphosphocholines inhibit choline uptake and phosphatidylcholine biosynthesis in rat sympatic neurons and impair axonal extention. Biochem J. 312,411-^17. Possmayer, F., Cassola, P., Chan, F., MacDonald, P., Ormseth, M., Wong, T, Harding, P., & Tokmakjian, S. (1981). Hormonal induction of pulmonary maturation in the rabbit fetus. Biochim. Biophys. Acta 664, 10-21. Post, M., Batenburg, J.J., Schuurmans, E.A.J.M., & van Golde, L.M.G. (1982). The rate-limiting step in the biosynthesis of phosphatidylcholine by alveolar type II cells from adult rat lung. Biochim. Biophys. Acta 712, 390-394. Post, M., Batenburg, J.J., Smith, B.T., & van Golde, L.M.G. (1984). Pool sizes of precursors for phosphatidylcholine formation in adult rat lung type II cells. Biochim. Biophys. Acta 795, 552-557.
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Pritchard, P.H. & Vance, D.E. (1981). Choline metabolism and phosphatidylcholine biosynthesis in cultured rat hepatocytes. Biochem. i. 194, 261—267. Radika, K. & Possmayer, F. (1985). Inhibition of fetal pulmonary choline-phosphate cytidylyltransferase under conditions favouring protein phosphorylation. Biochem. J. 232, 833-840. Rooney, S.A., Dynia, D.W., Smart, D.A., Chu, A.J., Ingleson, L.D., Wilson, CM., & Gross, I. (1986). Glucocorticoid stimulation of choline-phosphate cytidylyltransferase activity in fetal rat lung: Receptor-response relationships. Biochim. Biophys. Acta 888, 208-216. Rooney, S.A., Smart, D.A., Weinhold, P.A., & Feldman, D.A. (1990). Dexamethasone increases the activity but not the amount of choline-phosphate cytidylyltransferase in fetal rat lung. Biochim. Biophys. Acta 1044,385-389. Rosenberg, I.L., Smart, D.A., Gilfillan, A.M., & Rooney, S.A. (1987). Effect of l-oleoyl-2-acetylglycerol and other lipids on phosphatidylcholine synthesis and cholinephosphate cytidylyltransferase activity in cultured type II pneumocytes. Biochim. Biophys. Acta 921,473-480. Rutherford, M., Rock, C, Jenkins, N., Gilbert. D., Tessner, T., Copeland, N., & Jackowski, S. (1993). The gene for murine CTP: Phosphocholine cytidylyltransferase (Ctpct) is located on mouse chromosome 16. Genomics 18, 698-701. Sanghera, J.S. & Vance, D.E. (1989a). Stimulation of CTP:phosphocholine cytidylyltransferase and phosphatidylcholine synthesis by calcium in rat hepatodytes. Biochim. Biophys. Acta 1003, 284-292. Sanghera, J.S. & Vance, D.E. (1989b). CTP:phosphocholine cytidylyltransferase is a substrate for cAMP-dependent protein kinase in vitro. J. Biol. Chem. 264, 1215-1223. Schneider, W.C. (1963). Intracellular distribution of enzymes XIII: Enzymatic synthesis of dCDPcholine and lecithin in rat liver. J. Biol. Chem. 238, 3572-3577. Slack, B.E., Breu, J., & Wurtman, R.J. (1991). Production of diacylglycerol by exogenous phospholipase C stimulates CTP:phosphocholine cytidylyltransferase activity and phosphatidylcholine synthesis in human neuroblastoma cells. J. Biol. Chem. 266, 24503-24508. Sleight, R. & Kent, C. (1980). Regulation of phosphatidylcholine biosynthesis in cultured chick embryonic muscle treated with phospholipase C.J. Biol. Chem. 255, 10644—10650. Sleight, R. & Kent, C. (1983a). Regulation of phosphatidylcholine biosynthesis in mammalian cells. 1. Effects of phospholipase C treatment on phosphatidylcholine metabolism in Chinese hamster ovary cells and LM mouse fibroblasts. J. Biol. Chem. 258, 824—830. Sleight, R. & Kent, C. (1983b). Regulation of phosphatidylcholine biosynthesis in mammalian cells. 1. Effects of phospholipase C treatment on the activity and subcellular distribution of CTP: phosphocholine cytidylyltransferase in Chinese hamster ovary and LM cell lines. J. Biol. Chem. 258, 831-835. Sohal, P.S. & Cornell, R.B. (1990). Sphingosine inhibits the activity of rat liver CTP:phosphocholine cytidylyltransferase. J. Biol. Chem. 265, 11746-11750. Stem, W., Kovac, C, & Weinhold, P.A. (1976). Activities and properties of CTP:phosphocholine cytidylyltransferase in adult and fetal rat lung. Biochim. Biophys. Acta 441,280-293. Sundler, R., Arvidson, & Akesson, B. (1972). Pathways for the incorporation of choline into rat liver phosphatidylcholine in vivo. Biochim. Biophys. Acta 280, 559-568. Sweitzer, T. & Kent, C. (1993). Genbank accession # L13244. Sweitzer, T., & Kent, C. (1994). Expression of wild-type and mutant rat liver CTP: Phosphocholine cytidylyltransferase in a cytidylyltransferase-deficient Chinese hamster ovary cell line. Arch. Biochem. Biophys. 311,107-116. Teegarden, D., Taparowsky, E.J., & Kent, C. (1990). Altered phosphatidylcholine metabolism in C3H10T(l/2) cells transfected with the Harvey-ra5 oncogene. J. Biol. Chem. 265, 6042-6047. Terce, P., Record, M., Ribbes, G., Chap, H., & Douste-Blazy, L. (1988). Intracellular processing of cytidylyltransferase in Krebs II cells during stimulation of phosphatidylcholine synthesis: Evidence that a plasma membrane modification promotes enzyme translocation specifically to the endoplasmic reticulum. J. Biol. Chem. 263, 3142-3149.
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Terce, R, Record, M., Tronchere, H., Ribbes, G., & Chap, H. (1991). Cytidylyltransferase translocation onto endoplasmic reticulum and increased de novo synthesis without phosphatidylcholine accumulation in Krebs-II ascites cells. Biochim. Biophys. Acta 1084, 69-77. Terce, F., Record, M., Tronchere, H., Ribbes, G., & Chap, H. (1992). Reversible translocation of cytidylyltransferase between cytosol and endoplasmic reticulum occurs within minutes in whole cells. Biochem. J. 282, 333-338. Tesan, M., Anceschi, M.M., & Bleasdale, J.E. (1985). Regulation of CTP:phosphocholine cytidylyltransferase activity in type II pneumonocytes. Biochem. J. 232, 705-713. Tessner, T.G., Rock, CO., Kalmar, G.B., Cornell, R.B., & Jackowski, S. (1991). Colony-stimulating factor 1 regulates CTP:phosphocholine cytidylyltransferase mRNA levels. J. Biol. Chem. 266, 16261-16264. Tijburg, L.B.M., Geelen, M.J.H., & van Golde, L.M.G. (1989). Regulation of the biosynthesis of triacylglycerol, phosphatidylcholine and phosphatidylethanolamine in the liver. Biochim. Biophys. Acta 1004, 1-19. Tijburg, L.B.M., Nishimaki-Mogami, T., & Vance, D.E. (1991). Evidence that the rate of phosphatidylcholine catabolism is regulated in cultured rat hepatocytes. Biochim. Biophys. Acta 1085, 167-177. Tronchere, H., Record, M., Terce, R, & Chap, H. (1994). Phosphatidylcholine cycle and regulation of phosphatidycholine biosynthesis by enzyme translocation. Biochimica et Biophysica Acta 1212, 137-151. Tronchere, H., Planat, V., Record, M., Terce, R, Ribbes, G., & Chap, H. (1995). Phosphatidylcholine turnover in activated human neutrophils. J. Biol. Chem. 270, 13138-13146. Utal, A.K., Jamil, H., & Vance, D.E. (1991). Diacylglycerol signals the translocation of CTP: phosphocholine cytidylyltransferase in HeLa cells treated with TPA. J. Biol. Chem. 266, 24084-24091. Vance, D.E., Trip, E.M., & Paddon, H.B. (1980). Poliovirus increases phosphatidylcholine biosynthesis in HeLa cells by stimulation of the rate-limiting reaction catalyzed by CTP:phosphocholine cytidylyltransferase. J. Biol. Chem. 255, 1064-1069. Vance, J.E. & Vance, D.E. (1988). Does rat liver golgi have the capacity to synthesize phospholipids for lipoprotein secretion? J. Biol. Chem. 263, 5898-5909. Veitch, D., & Cornell, R. (1996). Substitution of serine for glycine-91 in the HXGH motif of CTP: Phosphocholine cytidylyltransferase implicates this motif in CTP binding (submitted). Walkey, C , Kalmar, G., & Cornell, R.B. (1993). Over-expression of CTP:phosphocholine cytidylyltransferase accelerates PC synthesis and degradation. J. Biol. Chem. 269, 5742-5749. Wang, Y., MacDonald, J.I.S., & Kent, C. (1993a). Regulation of CTP:phosphocholine cytidylyltransferase in HeLa cells: Effect of oleate on phosphorylation and intracellular localization. J. Biol. Chem. 268, 5512-5518. Wang, Y., Sweitzer, T.D., Weinhold, RA., & Kent, C. (1993b). Nuclear localization of soluble CTP: Phosphocholine cytidylyltransferase. J. Biol. Chem. 268, 5899-5904. Wang, Y, & Kent, C. (1995). Identification of an inhibitory domain of CTP: Phosphocholine cytidylyltransferase. J. Biol. Chem. 270, 18948-18952. Wang, Y, MacDonald, J., & Kent, C. (1995). Identification of the nuclear localization signal of rat liver CTP: Phosphocholine cytidyltransferase. J. Biol. Chem. 270, 354-360. Wang, Y, & Kent, C. (1995). Effects of altered phosphorylation site on the properties of CTP phosphocholine cytidylyltransferase. J. Biol. Chem. 240, 17843-17849. Warden, C. & Friedkin, M. (1985). Regulation of choline kinase activity and phosphatidylcholine synthesis by mitogenic growth factors. J. Biol. Chem. 250, 6006-6011. Watkins, J.D. & Kent, C. (1990) Phosphorylation of CTP:phosphocholine cytidylyltransferase in vivo: Lack of effect of phorbol ester treatment in HeLa cells. J. Biol. Chem. 265, 2190-2197. Watkins, J.D. & Kent, C. (1991). Regulation of CTP:phosphocholine cytidylyltransferase activity and subcellular location by phosphorylation in Chinese hamster ovary cells: The effect of phospholipase C treatment. J. Biol. Chem. 266, 21113-21117.
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Watkins, J.D. & Kent, C. (1992). Immunolocalization of membrane-associated CTP:phosphocholine cytidylyltransferase in phosphatidylcholine-deficient Chinese hamster ovary cells. J. Biol. Chem. 267, 5686-5692. Watkins, J.D., Wang, Y, & Kent, C. (1992). Regulation of CTP:phosphocholine cytidylyltransferase activity and phosphorylation in rat hepatocytes: Lack of effect of elevated cAMP levels. Arch. Biochem. Biophys. 292, 360-367. Weinhold, P.A., Skinner, R., & Saunders, R. (1973). Activity and properties of choline kinase, cholinephosphate cytidylyltransferase, and cholinephosphotransferase during liver development in the rat. Biochim. Biophys. Acta 326,43-51. Weinhold, P.A., Feldman, D.A., Quade, M.M., Miller, J.C, & Brooks, R.L. (1981). Evidence for a regulatory role of CTPrphosphocholine cytidylyltransferase in the synthesis of phosphatidylcholine in fetal lung following premature birth. Biochim. Biophys. Acta 665, 134-144. Weinhold, RA., Rounsifer, M.E., Williams, S.E., Brubaker, P C , & Feldman, D.A. (1984). CTP:phosphocholine cytidylyltransferase in rat lung. J. Biol. Chem. 259, 10315-10321. Weinhold, PA., Rounsifer, M.E., & Feldman, D.A. (1986). The purification and characterization of CTPiphosphocholine cytidylyltransferase from rat liver. J. Biol. Chem. 261, 5104-5110. Weinhold, PA., Rounsifer, M.E., Charles, L., & Feldman, D.A. (1989). Characterization of cytosolic forms of CTP:phosphocholine cytidylyltransferase in lung, isolated alveolar type II cells, A549 cell and Hep G2 cells. Biochim. Biophys. Acta 1006, 299-310. Weinhold, PA., Charles, L., Rounsifer, M.E., & Feldman, D.A. (1991). Control of phosphatidylcholine synthesis in Hep G2 cell. J. Biol. Chem. 266, 6093-6100. Weinhold, PA., Charles, L., & Feldman, D. (1994). Regulation of CTP: Phosphocholine cytidylyltransferase in HepG2 cells: Effect of choline depletion on phosphorylation, translocation and phosphatidylcholine levels. Biochimica et Biophysica Acta 1210, 335-347. Wertz, PW & Mueller, G. (1978). Rapid stimulation of phsopholipid metabolism by tumor-promoting phorbol esters. Cancer Res. 38, 2900-2904. Wright, PS., Morand, J., & Kent, C. (1985). Regulation of PC biosynthesis in CHO cells by reversible membrane association of CTP: phosphocholine cytidylyltransferase. J. Biol. Chem. 260, 79197926. Xu, Z.-x., Smart, D. A., & Rooney, S. A. (1990). Glucocorticoid induction of fatty-acid synthase mediates the stimulatory effect of the hormone on choline-phosphate cytidylyltransferase activity in fetal rat lung. Biochim. Biophys. Acta 1044, 70-76. Yang, W., Boggs, K., & Jackowski, S. (1995). The association of lipid activators with the amphipathic helical domain of CTP: Phosphocholine cytidylyltransferase accelerates catalysis by increasing the affinity of the enzyme for CTP J. Biol. Chem. 270(41), 23951-23958. Yao, Z., Jamil, H., & Vance, D.E. (1990). Choline deficiency causes translocation of CTP:phosphocholine cytidylyltransferase from cytosol to endoplasmic reticulum in rat liver. J. Biol. Chem. 265, 4326-4331.
INCORPORATION AND TURNOVER OF FATTY ACIDS IN ESCHERICHIA COiL/MEMBRANE PHOSPHOLIPIDS
Charles O. Rock and Suzanne Jackowski
I. II. III. IV. V
INTRODUCTION 39 THREE ACYLTRANSFERASES 40 TURNOVER OF PHOSPHOLIPID ACYL MOIETIES 45 UPTAKE AND METABOLISM OF FATTY ACIDS AND PHOSPHOLIPIDS . 48 FUTURE PROSPECTS 54 ACKNOWLEDGMENTS 54 REFERENCES 55
I. INTRODUCTION The study of lipid metabolism in Escherichia coli continues to make significant contributions to our understanding of membrane phospholipid synthesis and remodeling. The ability to manipulate the genotype of this organism allows scientists to directly address the physiological roles of the phospholipids. The lipid compo-
Advances in Lipobiology Volume 1, pages 39-59. Copyright © 1996 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-635-5 39
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CHARLES O. ROCK and SUZANNE JACKOWSKI
sition of E. coli is relatively simple and although the majority of the enzymes involved in their biosynthesis and turnover are known, research over the last 10 years has uncovered novel pathways, significant new enzymes, and unexpected interrelationships linking lipid metabolism with posttranslational protein modification, oligosaccharide biosynthesis, and transcriptional control. The wealth of information on the physiology, genetics, and biochemistry of E. coli enables the integration of the metabolic origin and fate of lipids into the overall physiology of the organism. Practical advantages of the system include: (a) complete control over the conditions of cell growth; (b) large quantities of cells for biochemical analysis; and (c) molecular biology techniques to regulate the levels of expression of the enzymes of interest. Consequently, most of the information available on bacterial lipid metabolism is based on studies of £. coli and this organism will be the sole focus of this review. However, considering the diversity of procaryotic lifestyles and lipid structures, it would be incorrect to consider E. coli a typical microorganism. There are several recent excellent reviews covering phospholipid biosynthesis and function (Vanden Boom and Cronan, 1989; Raetz and Dowhan, 1990; Jackowski et al., 1991), fatty acid P-oxidation (Nunn, 1986), lipid A metabolism (Raetz, 1987, 1990) and the enzymes and regulation of fatty acid biosynthesis (Magnuson et al., 1993). Therefore, this review will focus on the metabolism of phospholipid acyl moieties including their initial incorporation into phospholipid, metabolic turnover, resynthesis via the acylation of lysophospholipids, and the assimilation of exogenous fatty acids into membrane phospholipids.
II. THREE ACYLTRANSFERASES The 5«-glycerol-3-phosphate acyltransferase catalyzes the first step in the biosynthesis of membrane phospholipids from fatty acid precursors (Figure 1). The indispensable role of this enzyme was recognized with the isolation ofplsB mutants which possess an 5«-glycerol-3-phosphate acyltransferase (PlsB) that is inactive in vivo under normal growth conditions due to an elevated Km for glycerol-3-phosphate (Bell, 1975). PlsB is capable of transferring both acyl-acyl carrier protein (ACP) and acyl-CoA derivatives to glycerol-3-phosphate (Green et al., 1981; Rock et al., 1981). The plsB gene has been cloned, sequenced and the properties of the expressed protein have been extensively investigated by Bell and coworkers (Larson et al., 1980; Lightner et al., 1980; Green et al., 1981, 1983; Lightner et al., 1983). Primarily saturated fatty acids are transferred to the 1-position of ^w-glycerol-3-phosphate in vivo, and PlsB has a lower Km for saturated thioesters than for unsaturated thioesters (Green et al., 1981). When PlsB is overproduced, the cells form ordered tubular arrays of the enzyme (Wilkison et al., 1986). The formation of these tubular arrays is dependent on the presence of heat shock proteins, since they do not form in htpR mutants (Wilkison and Bell, 1988). The dimer structure of PlsB is consistent with kinetic analysis of purified PlsB that shows substrate
FattK/Ac/cfs/n Escherichia coli
41 0 II R2-C-S-C0A
0 II R^-C-S-CoA
or
or
0
0 Ri-C-S-ACP 0 II 0-- C - R ,
HjC-OH HO-CH 1 H g C - O - PO-3
Rg-C-S-ACP
PIsB
HO-CH 1 H 2 C - 0 - -PO-3
PIsC
0 II 0 HX-O-C-R, II ^ R2-C-O-CH H2C-O-PO3
Figure 1, Formation of phosphatidic acid. Two discrete acyltransferases are required for the formation of phosphatidic acid. PIsB catalyzes the formation of 1 -acyl-sr?-glycerol-3-phosphate utilizing either acyl-ACP or acyl-CoA thioesters as acyl donors. This enzyme is most active with 16:0 and 18:1 A l l thioesters and exhibits a low activity towards 16:1 A9 thioesters. PIsC catalyzes the synthesis of phosphatidic acid from 1-acyl-SA7-glycero-3-phosphate and prefers 16:1A9 and 18:1 A l l as opposed to 16:0 thioesters. The substrate specificity of the two acyltransferases contributes to the observed asymmetric distribution of fatty acid found in E co//phospholipids.
cooperativity with respect to glycerol-3-phosphate (Scheideler and Bell, 1991), and examination of the tubular arrays by electron microscopy reveals a repeating unit consistent with a dimer (Wilkison et al., 1992). The freeze fracture analysis of the tubules shows only a limited contact of PlsB with the membrane with the major portion of the protein forming a large globular domain facing the cytoplasm. Consistent with this configuration, analysis of the predicted PlsB protein sequence fails to reveal any obvious transmembrane segments. PlsB is the first step in the conversion of soluble intermediates to membrane phospholipid, and is ideally located to control the amount of phospholipid synthesized. However, the available data indicate that in cells growing in minimal medium, membrane phospholipid synthesis is regulated primarily at a step early in fatty acid biosynthesis (for review, see Magnuson et al, 1993). Acyl-ACP molecules are the products of de novo biosynthesis and the substrates for the PlsB acyltransferase. Since the size of the acyl-ACP pool is very small, the acyl-group composition of this pool is a major determinant of the acyl-group distribution among the phospholipids during normal growth where the formation of membrane is determined by the supply of fatty acids. Overexpression of the PlsB protein does not cause overproduction of membrane phospholipids, and inactivation of the acyltransferase activity by reduction of the available 5«-glycerol-3-phosphate in the plsB mutant results in accumulation of acyl-ACPs, supporting the view that a normal level of PlsB protein is more than enough to catalyze the incorporation of all of the products of fatty acid biosynthesis into membrane phospholipid and that the biochemical substrate selectivity of the enzyme becomes important when the size of the acyl-ACP pool increases to the point where choices are possible.
42
CHARLES O. ROCK and SUZANNE JACKOWSKI
A different situation exists in cells growing in the presence of exogenous fatty acids. In this case, the synthesis of endogenous fatty acids is suppressed and it is likely that PlsB regulates phospholipid synthesis by determining the rate of glycerol-3-phosphate acylation with exogenous fatty acids that are presented to the enzyme as acyl-CoA derivatives. Cells grown in the presence of large amounts of exogenous fatty acids do not overproduce phospholipid products suggesting that PlsB limits membrane formation under such circumstances. The exit of fatty acids from plsB strains (Cooper et al., 1987) is one means of regulating the supply of substrates to the acyltransferase and points to PlsB as a slow step in the pathway. Incorporation of exogenous fatty acids into phospholipids via PlsB is negatively regulated by amino acid starvation (Nunn and Cronan, 1974) which stimulates the levels of ppGpp, an intracellular alarmone. Although a link between the shutdown of protein synthesis and concomitant shutdown of membrane synthesis is provided by this scenario, which is known as the "stringent response", the specific target enzymes inactivated by ppGpp are not yet definitively identified. Biochemical experiments with enzyme preparations suggest that PlsB activity is inhibited by ppGpp but the effect is critically dependent on the Mg^"*" and protein concentrations in the assay (Lucking and Goldfine, 1975). Inducible plasmids that can elevate the alarmone levels in the absence of the global side effects associated with amino acid starvation have recently been constructed and provide a means to address the role of PlsB, among other targets, in the control of phospholipid synthesis during the stringent response. The second acyltransferase in membrane phospholipid synthesis is the 1-acyl5«-glycerol-3-phosphate acyltransferase (Figure 1). This acyltransferase (PlsC) is immediately downstream of PlsB in the pathway of phosphatidic acid synthesis and is also capable of utilizing either acyl-ACPs or acyl-CoAs as the acyl donors (Rock et al., 1981). Presumably the PlsC has a higher affinity for unsaturated fatty acyl-derivatives since these are preferentially found in the sn-2 position ofE. coli phospholipids. Previous to the discovery of PlsC, experiments with crude inner membrane fractions demonstrated that the native distribution of fatty acids on the glycerol-phosphate backbone could be reconstructed in vitro when acyl-ACPs were used as substrate rather than acyl-CoAs (Rock et al., 1981). The recent important advance in this area comes from the isolation of l-acyl-5«-glycerol-3-phosphate acyltransferase mutants {plsC) and the cloning of the structural gene for this protein (Coleman, 1990, 1992). PlsC is an essential protein in E. coli. ^htnplsC mutants are grown at the permissive temperature, approximately 20% of the phospholipid is l-acyl-5«-glycerol-3-phosphate, and following the shift to the nonpermissive temperatures, l-acyl-5«-glycerol-3-phosphate accumulates to 30% of the total phospholipid and cell growth ceases. PlsC has a molecular weight of 27.5 kDa and analysis of the predicted amino acid sequence reveals three short hydrophobic domains that may anchor it to the inner membrane. IhQplsC gene is the same as the parF gene in S. typhimurium which encodes an essential 25 kDa protein
Fatty Acids in Escherichia coW
43
(Luttinger et al., 1991). Experiments can now be designed to address the structure and function of the PlsC acyltransferase in lysophospholipid metabolism. PlsC is completely dependent on PlsB for substrate and cannot acylate sn-glycerol-3-phosphate. Turnover of the sn2 fatty acids of E. coli phospholipids is not evident and therefore the acylated product of the PlsC protein is probably stable and not subject to remodeling schemes. At first glance, the possibilities for regulation by PlsC of the quantity of phospholipid seem limited, but it may be interesting to examine the extent of regulation of fatty acid composition under conditions of growth on exogenous fatty acid where acyl-CoAs substitute for acyl-ACPs as substrates for PlsC. The third acyltransferase involved in phospholipid metabolism is 2-acylglycerophosphoethanolamine (2-acyl-GPE) acyltransferase. This acyltransferase functions as a heterodimer consisting of a membrane associated catalytic subunit and ACP. The acyltransferase has been purified, mutants isolated, and the structural gene {aas) has been mapped and cloned (Cooper et al., 1989; Hsu et al., 1991; Rock et al., 1993). The Aas protein was originally thought to possess a molecular weight of 27 kDa based on gel electrophoresis of the purified protein preparation (Cooper et al., 1989); however, more recent results show that the DNA sequence of the aas clone codes for an 80.6 kDa protein and specific expression of the aas gene yields an 81 kDa protein in agreement with the predicted product of the aas gene. The 27 kDa protein is an artifact due either to proteolytic degradation of Aas during purification or to the mistaken identification of a major protein contaminant as Aas. Although Aas is tightly associated with the inner membrane, like PlsB, analysis of the predicted amino acid sequence does not reveal obvious transmembrane segments. The catalytic cycle of Aas is shown in Figure 2. The first step is the activation of the fatty acid by ATP and the formation of a putative acyl-AMP intermediate coupled to the release of pyrophosphate. The fatty acid is then transferred to the terminal sulfliydryl of the 4'-phosphopantetheine prosthetic group of ACP. The acyl-ACP intermediate remains bound to the enzyme during the normal catalytic cycle, but can be released from the enzyme by the addition of high salt concentrations to the assay. The ability of high salt concentrations to dissociate the bound ACP subunit from the acyltransferase protein is the basis for the acyl-ACP synthetase activity exhibited by this enzyme that led to its initial characterization (Ray and Cronan, 1976). The acyl-ACP synthetase activity of Aas has proved to be very useful in the preparation of acyl-ACP intermediates for use as substrates in the characterization of other acyltransferases (Rock and Garwin, 1979). Under physiological conditions the acyl moiety is then transferred from acyl-ACP to the 1-position of 2-acyl-GPE to form phosphatidylethanolamine (PtdEtn). It is important to understand that the reaction catalyzed in vivo is the 2-acyl-GPE acyltransferase reaction and that the enzyme does not release acyl-ACPs that can be utilized by the enzymes of fatty acid biosynthesis or by other acyltransferases. Despite the similarity in the reactions that they catalyze, there are no obvious stretches of primary sequence that are common to all three enzymes. In general, all
44
CHARLES O. ROCK and SUZANNE JACKOWSKl PtdEtn V
Enz
Enz
' +
ACPSH■
ACPSH
ATP ♦ Fatty Acid
y
2-Acyl-GPE
Enz + Acyl-ACP
Enz Acyl-ACP
AMP + PPi
Figure 2. Reaction mechanism for 2-acyl-GPE acyltransferase. The first step in the reaction is the activation of fatty acid with ATP to form an acyl-adenylate intermediate. The fatty acid is then transferred to the sulfhydryl of the bound ACP subunit forming acyl-ACP. The acyl moiety is then transferred to the 1-position of 2-acyi-GPE regenerating ACP. The acyl-ACP intermediate normally remains bound to the enzyme; however, in the presence of high salt concentrations in the \n vitro assay, acyl-ACP dissociates from Aas and acyl-ACP accumulates. This partial reaction of the acyltransferase accounts for the acyl-ACP synthetase activity of Aas.
three acyltransferases are basic enzymes with isoelectric pHs of 10.2, Aas; 8.6, PlsB; and 10.4, PlsC. The three proteins also differ in their molecular weights. PlsB is a 91.3 kDa protein, Aas is a 80.6 kDa protein whereas PlsC is a 27.5 kD protein. Coleman (1992) notes that there are nine conserved amino acids over a 94 residue stretch of LpxA, PlsB, and PlsC. Even this low degree of homology is not evident when the Aas primary sequence is compared to the other acyltransferases. The significance of this region of homology is unclear, but it is tempting to speculate that it may represent the acyl-ACP binding region of these proteins (Coleman, 1992). The definition of a consensus ACP binding pocket would be of broad significance since a large number of enzymes use ACP thioesters as substrates. Future work should be directed toward site-directed mutagenesis of the acyltransferases aimed at defining the site of ACP interaction with these enzymes. Aas seems the most suitable candidate for this work because the enzyme binds ACP with the highest affinity and Aas is not essential for cell viability allowing the facile characterization of catalytically compromised mutants. Are there any additional components of the acyltransferase systems? Based on the available data, it seems unlikely. However, there remain some unexplained data that may provide a surprise. Most notable is the function of thep/^Xgene. TheplsB mutants are phenotypically recognized as glycerol-phosphate auxotrophs; however, an additional gene tQvmedplsXis required in the genetic background in order
Fatty Acids in Escherichia coli
45
forplsB mutants to exhibit glycerol-3-phosphate auxotrophy (Larson et al., 1984). In other words, cells with a defective PlsB do not have perturbed phospholipid synthesis unless they are also plsX mutants. It is not known whether plsX is a gain-of-function or a loss-of-function mutant. Recently, the plsX locus has been localized within a cluster of lipid biosynthetic genes in the 24 min region of the chromosome (Oh and Larson, 1992). The gene order in this region is plsX-fabHfabD-fabG-acpP. It seems unlikely that PlsX is another glycerol-3-phosphate acyltransferase, but may be important to regulating PlsB. Support for this concept is derived from the experiments of Scheideler and Bell (1991) who show that solubilization of PlsB results in the conversion of PlsB from an acyltransferase with a low Km for glycerol-3-phosphate to a high Km enzyme reminiscent of the enzymatic activity found in membranes prepared fromplsB mutants. One hypothesis that warrants investigation is that PlsX is a cofactor or regulatory subunit of PlsB (Scheideler and Bell, 1991). The acquisition of the p/^X clone provides the molecular tools to determine the function of the PlsX protein in modulating the glycerolphosphate acyltransferase system.
III. TURNOVER OF PHOSPHOLIPID ACYL MOIETIES The only established metabolic cycle for the turnover of membrane phospholipid acyl moieties is the 2-acyl-GPE cycle (Figure 3). This cycle involves the removal of 1-position acyl moieties from PtdEtn and their replacement by 2-acyl-GPE acyltransferase (Rock, 1984). The rate of acyl group turnover is low, amounting to only 3-5% of the PtdEtn pool per generation. Thus, detection of acyl group turnover in pulse-chase metabolic labeling experiments is technically challenging due to the large amount of PtdEtn in the cell (ca. 10^ molecules per cell). Although this low rate of turnover may seem insignificant, it may play an essential metabolic role when considered in light of the synthesis of membrane protein components that are three to four orders of magnitude less abundant. A second approach to detecting phospholipid fatty acid turnover is to look for lysophospholipid substrates and enzymes that reacylate endogenous lysophospholipids (Rock, 1984). In these experiments 2-acyl-GPE and I-acylglycerol-3-phosphate were the only lysophospholipids detected in E. coli membranes. These data corroborate the existence of the three acyltransferases described above (Figures 1 and 2), but do not reveal the presence of other lysophospholipid intermediates that may indicate the operation of additional acyl group turnover cycles. A complete picture of the destination of the 1-position acyl moieties is not available, but the amine-linked fatty acid on the amino terminus of the major outer membrane lipoprotein is one significant fate (Jackowski and Rock, 1986). Although the transfer of 1-position fatty acids in PtdEtn to lipoprotein can be directly demonstrated in vivo (Jackowski and Rock, 1986), the transacylases responsible for lipoprotein acylation do not have an absolute specificity for PtdEtn since the lipoprotein is acylated and correctly processed inpss mutants that lack PtdEtn (Gupta et al., 1991). In this case, the fatty
46
CHARLES O. ROCK and SUZANNE JACKOWSKI
0 II
O II
L(p)-Etn\
pIdA Protein / Acylationl
P^^^^" K ^^^y
0 /wwv^oH ACP, ATP
L ® - Etn 2-Acyl-GPE PtdGro pIdB O
"
^
II
O II
O II
OH
rOH
L®-J-, Acyl-PtdGro 0 ^ pOH HO-I
'{
HO-
[email protected],n Qp^
>
©-Etn
Figure 3. Metabolism of 2-acyl-GPE. 2-Acyl-GPE is generated by the action of phospholipase Ai or by transfer of the 1-position acyl moiety to the amino terminus of the lipoprotein. 2-Acyl-GPE can be converted to PtdEtn by 2-acyl-GPE acyltransferase/acyl-ACP synthetase (Aas). An alternate fate of 2-acyl GPE is metabolism via the pldB gene product which either hydrolyzes the lypsophospholipid to fatty acid and GPE or transfers the acyl group to the polar head group of phosphatidylglycerol (PtdGro). Adapted from Hsu et al. (1991).
acids are derived from phosphatidylglycerol. Membrane phospholipids are also the source of fatty acids attached to membrane proteins of Mycoplasma capricolum (Dahl and Dahl, 1984), indicating that protein acylation is a driving force for fatty acid turnover in membrane phospholipids and is a widespread phenomenon. The contribution of phospholipases to membrane phospholipid turnover is undefined. The best-studied phospholipase is phospholipase A^ that is located in the outer membrane. The protein is very stable and has been purified to homogeneity. Mutants lacking phospholipase Aj activity have been isolated (pldA), and XhQpldA gene has been cloned and sequenced (Ohki et al., 1972; Abe et al., 1974; Nishijima et al, 1977; Homma et al, 1984b). Interestingly, cells that lack PldA do not have a phenotype and cells that overexpress the PldA possesses increased phospholipase
Fatty Acids in Escherichia coli
47
activity in vitro, but do not exhibit an obvious phenotype (Homma et al., 1984a). The effect of the presence or absence of this enzyme on 1-position turnover in PtdEtn has not been, but should be, examined to determine the contribution of this enzyme to the turnover rate. However, PldA appears to be present in a latent form in vivo (Homma et al., 1984a). The reason for the inactivity of the enzyme in intact cells is unknown, but the activity can be revealed when the cell envelope formation is perturbed. For example, mutants (envC) defective in an inner membrane protein (Klein et al., 1991) form chains and accumulate 2-acyl-GPE, presumably due to the activation of PldA (Michel et al., 1977; Michel and Starka, 1979). Activation of PldA by membrane perturbants is important to the ability of polymorphonuclear leukocytes to kill E. coli (for review, see Elsbach et al., 1990). Although it is clear that the presence of PldA in the outer membrane determines the sensitivity of E. coli to polymixin B (Elsbach et al., 1985) or cytotoxicity when ingested by leukocytes (Wright et al, 1990), the importance of this latent enzyme to bacterial physiology remains a mystery. E. coli also possess a lysophospholipase activity that plays a role in preventing the accumulation of lysophospholipids in vivo (Figure 3). The gene for this enzyme (pldB) has been cloned and sequenced (Homma et al., 1983). ThtpldB gene is found as an operon withpldA (Kobayashi et al, 1985a,b) and the PldB protein, an inner membrane component, has been purified and characterized (Karasawa et al., 1985). PldB hydrolyzes 2-acyl-GPE more readily that the 1 -acyl isomer and does not attack diacylphospholipids. PldB also transfers the fatty acid from the 2-position of 2-acyl-GPE to the polar headgroup of phosphatidylglycerol forming acyl phosphatidylglycerol (Nishijima et al., 1978; Karasawa et al., 1985). These two activities of PldB (Figure 3) work in concert to ensure that membrane-damaging lysophospholipids do not accumulate in vivo. In aas mutants, 2-acyl-GPE does not accumulate even though the activity of 2-acyl-GPE acyltransferase cannot be detected (Hsu et al., 1991). Instead, a new phospholipid identified as acylphosphatidylglycerol appears indicating that the PldB pathway for 2-acylGPE metabolism is significant in the absence of the acyltransferase/synthetase activity. Mutants that either lack or overexpress PldB activity do not have a discemable phenotype (Kobayashi et al., 1984). The accumulation of 2-acyl-GPE in aas pldB double mutants confirms the role of PldB in acylphosphatidylglycerol synthesis and 2-acyl-GPE degradation. However, 2-acyl-GPE does not accumulate to high levels in these double mutants pointing to yet another pathway for the removal of 2-acyl-GPE. A membrane-associated transacylase activity has been characterized that converts two 2-acyl-GPEs to PtdEtn and GPE (Homma and Nojima, 1982a,b; Homma et al., 1987), and there is a second lysophospholipase that cleaves 1-acyl isomers localized in the soluble fraction (Doi and Nojima, 1975). The role of these two enzymes in membrane phospholipid turnover awaits the isolation of mutants and clones that can be used to manipulate the intracellular levels of these enzymes. The fact that acylphosphatidylglycerol accumulates in aas mutants suggests that reacylation of 2-acyl-GPE by Aas is the predominant pathway for the metabolism of 2-acyl-GPE.
CHARLES O. ROCK and SUZANNE JACKOWSKI
48
IV. UPTAKE AND METABOLISM OF FATTY ACIDS AND PHOSPHOLIPIDS Our working hypothesis on the mechanisms for fatty acid uptake and metabolism in E. coli is shown in Figure 4. Fatty acids are presented to the cell as mixed micelles with detergents and are then bound to the outer membrane FadL protein which
2-Acyl-GPE Acyltransferase"1[ACP,
Glycerol-P Acyltransferase acyl-CoA • /J-oxidatlon
Figure 4. Pathways for the incorporation of exogenous fatty acids into phospholipid. Exogenous fatty acids transit the outer membrane via interaction with the FadL protein. The fatty acids then diffuse to the cytoplasmic aspect of the inner membrane where they are available as substrates for one of the two synthetases. Acyl-CoA synthetase (FadD) converts fatty acids to their corresponding acyl-CoA derivatives. Acyl-CoAs are released from the enzyme and are then used as substrates for the p-oxidation system or by the sn-glycerol-3-phosphate acyltransferase system (PIsB + PisC) to form phosphatidic acid. 2-Acyl-GPE acyltransferase/acyl-ACP synthetase activates the fatty acid for transfer of 2-acyl-lysophospholipids via the formation of an enzyme-bound acyl-ACP intermediate. The acyl-ACP intermediate remains associated with Aas and is not available to the enzymes of fatty acid biosynthesis or to the PIsB/PisC acyltransferase system.
Fatty Acids in Escherichia coli
49
facilitates the translocation of the fatty acid into the cell. The fatty acid then transits the periplasmic space and diffuses through the inner membrane. Acyl moieties that are presented on the inner surface of the inner membrane are available to react with one of two synthetases found in E. coli. The quantitatively most important synthetase is acyl-CoA synthetase (FadD) which ligates fatty acids to CoA. These acyl-CoAs are either used as a carbon source via the P-oxidation system or are incorporated into phospholipid via the glycerol-3-phosphate acyltransferase system. An alternate metabolic destination for intracellular fatty acids is the acyl-ACP synthetase/2-acyl-GPE acyltransferase (Aas). Aas is an inner membrane-associated enzyme that exists as a heterodimer between a catalytic subunit and ACP. Unlike FadD, the fatty acids activated by Aas are not released by the catalytic subunit, and are therefore not available as a potential substrate for other enzymes in the cell. FadL activity is critical for the uptake of long-chain fatty acids, but is not required for the uptake of medium- and short-chain acids. ThcfadL gene was first recognized as a mutation required for the p-oxidation of long-chain fatty acids (Nunn and Simons, 1978). ThefadL gene was originally thought to encode an inner membrane protein required for the concentrative uptake of fatty acids (Maloy et al., 1981; Ginsburgh et al., 1984; Nunn et al., 1986). However, thefindingthat strains resistant to bacteriophage T2 are also defective in fatty acid utilization (Morona and Henning, 1986; Black, 1988), coupled with the reevaluation of the localization and properties of FadL (Black et al., 1987), led to the conclusion that FadL is actually located in the outer membrane. ThQ fadL gene was cloned (Black et al., 1985) and its DNA sequence (Black, 1991) predicts a protein of 48.8 kDa containing a signal sequence and signal peptidase cleavage site consistent with its outer membrane localization. In addition, FadL exhibits heat-modifiable electrophoretic mobility and the predicted amino acid sequence of FadL is similar to other heat-modifiable outer membrane proteins (Black, 1991). FadL binds 16:0 and 18:1 fatty acids with high affinity (ca. 10~^ M). Binding of 14:0 is nearly an order of magnitude weaker and the association of 10:0 with FadL is not detected (Black, 1990). These binding studies are consistent with the physiological experiments with fadL mutants that show a functional y^JZ gene product is required for the uptake of long-chain, but not short-chain, fatty acids (Nunn and Simons, 1978). Linker insertion mutagenesis experiments point to the amino terminal portion of FadL as important for fatty acid binding activity, whereas mutations in the DNA sequence corresponding to the carboxyl terminal domain of the protein retain fatty acid binding activity, but are unable to facilitate the transfer of fatty acids across the outer membrane (Kumar and Black, 1991). Although a functional FadL protein is required for optimal growth of strains on fatty acids greater than twelve carbons long, fatty acid uptake appears independent of FadL in other experiments. For example, FadL activity is not required for the efficient N-myristolylation of foreign proteins expressed in E. coli (Knoll and Gordon, 1993). We attribute the difference between these two results to the differences in the amount of fatty acid that must enter the cell to give a maximal response in these two different assay systems. Growth on fatty acid as a sole carbon
CHARLES O. ROCK and SUZANNE JACKOWSKI
50
Domain I Consensus
SGXXGXPKG
Aas
368
VVHSi
FadD
210
AMLTI
VV L K B ^ AMVTI
FAA1
273
RATACS
274
Aas
504
FadD
350
i
Domain II
FAA1
449
RatACS
452
Aas
590
FadD
432
V V S I N V g M A A K P TV LVSVNP^D I DHS S I |S T T I L D M-A N F E L R T A G C C L S L IGDWTA
LR I
Domain III FAA1
532
RatACS
535
YD LH FK LH
VRFDEQgFVQHOG AVMDEE iRpiV GEWEAN Ml GKWLPN
Figure 5. Domain structure of fatty acid activating synthetases. The primary protein sequence of Aas (Jaci
source necessitates the uptake of much larger amounts of fatty acid from the medium compared to that required for the N-myristolation of proteins. Thus, it appears that 14:0 crosses the outer membrane at a measurable rate, but that this rate is unable to support the rapid growth of the bacteria on 14:0 as a carbon and energy source. The fact that short-chain fatty acids (<12:0) are rapidly metabolized by the cell in the absence of FadL protein suggests that the outer membrane is not a barrier to these acyl moieties. It is not known whether metabolic energy is coupled to the release of fatty acids from FadL. It would be interesting to test whether FadL function is coupled to periplasmic energy transducing molecules such as TolA or TonB. The characterization of fatty acid transport has been hampered by confusion in the field over what constitutes a transport assay. The standard approach to transport measurements dictates that the intracellular metabolism of the solute being investigated must be blocked or a non-metabolizable analogue should be employed in order to ensure that the transport activity measurement is independent of incorporation into cellular constituents. Alternatively, very short time points on the order
Faffy/Ac/ds/n Escherichia coli
51
of seconds should be taken to determine the linear range of the transport assay that represents first-order kinetics. To date, these criteria have not been met in the assays to measure fatty acid transport and, therefore, these experiments primarily determine the rate of incorporation of exogenous fatty acids into membrane phospholipids that results from the transport of fatty acids to the cytoplasmic aspect of the inner membrane. This point was made in the initial experiments on fatty acid uptake from Overath's laboratory which showed that phospholipids were the primary products in the uptake assay and not the accumulation of intracellular fatty acids (Klein et al., 1971). Incorporation experiments with both fadD or fadL mutants showed that accumulation of phospholipid was blocked in both cases and led to the conclusion that both of these proteins were actively involved in the movement of fatty acid from the medium into the cell. Our laboratory has also investigated this point and found that the inhibition of phospholipid synthesis and P-oxidation using plsBfadE double mutants prevents the time-dependent accumulation of fatty acids into the cell (Jackowski and Rock, unpublished observations). In our experiments, the only process detected was an initial, rapid, and saturable binding of fatty acids to the cell. Isolated FadL protein binds fatty acids and is most likely responsible for the rapid saturable binding (Black, 1990). Thus, the evidence argues that acyl-CoA synthetase is not necessary for the transport of fatty acids and is therefore not a component of the fatty acid transport process despite the fact that exogenous fatty acids fail to accumulate in fadD mutants. Acyl-CoA synthetase (FadD) is a key enzyme in the metabolism of exogenous fatty acids. The importance of this enzyme was first recognized from the isolation of mutants lacking acyl-CoA synthetase activity (fadD) which were unable to incorporate exogenous fatty acids into phospholipids or utilize fatty acids as a carbon source (Overath et al., 1969). The biochemical and physiological properties of fadD mutants lead to the conclusion that there is only a single acyl-CoA synthetase in E. coli. Purification of FadD yielded a single protein of apparent subunit molecular weight of 47 kDa (Kameda and Nunn, 1981). FadD is a soluble protein, although about 10% of the FadD activity is found associated with the membrane fraction. Since FadD can be extracted by washing the membrane pellet with buffer (Kameda and Nunn, 1981), FadD should be classified as a soluble or extrinsic membrane protein. The location of FadD in the cytosol is not consistent with a role in the vectorial transport of fatty acid across the inner membrane. The recent cloning and sequencing ofiYiQfadD gene has significantly advanced our knowledge of the structure of FadD (Black et al., 1992). The DNA sequence predicts a protein of 62 kDa and the amino terminal sequence of the purified FadD matches the predicted protein sequence. The expression of yeast acyl-CoA synthetase is capable of complementingyaJD mutants and enabling the cells to undertake both phospholipid synthesis and growth on fatty acids as a sole carbon source (Knoll and Gordon, 1993), thereby illustrating that heterologous, soluble acyl-CoA synthetases can functionally substitute for FadD. The acyl-CoA synthetase obviously
52
CHARLES O. ROCK and SUZANNE JACKOWSKI
is a high-capacity enzyme system that channels fatty acids to both phospholipid synthesis and |3-oxidation. Acyl-ACP synthetase/2-acyl-GPE acyltransferase (Aas) is a second route for the assimilation of exogenous fatty acids (Rock and Jackowski, 1985). In this pathway, fatty acids are activated and transferred to the 4'-phosphopantetheine prosthetic group of a tightly bound AGP subunit. The enzyme-bound acyl-ACP then reacts with 2-acyl-GPE to form PtdEtn. It is important to note that the acyl-ACPs formed by this enzyme are not released and therefore are not available to other enzymes in fatty acid biosynthesis that react with acyl-ACP. The absolute requirement for acyl-ACP derivatives in the acylation of lipid A, coupled with the inability to introduce exogenous fatty acids into the fatty acid biosynthetic pathway, accounts for the observation that mutants in total fatty acid synthesis cannot be rescued by feeding them a mixture of saturated and unsaturated fatty acids. The acyl-ACP synthetase reaction only operates in vitro where the acyl-ACP product is dissociated from the Aas protein by the inclusion of high concentrations of salt in the assay (Rock and Cronan, 1979). Saturated fatty acids are the preferred substrates for Aas although unsaturated fatty acids are esterified to AGP at significant rates (Rock and Garwin, 1979). Fatty acids with chain-lengths below eight carbons are not substrates for Aas. In contrast to FadD, Aas exclusively fractionates with the membrane fraction and detergents are required to solubilize Aas (Rock and Cronan, 1979; Cooper et al., 1989). Purified Aas binds AGP with high affinity. The AGP subunit can be stripped from the catalytic subunit with high salt and the acyltransferase activity is reconstituted by the re-addition of AGP. The analysis of aas mutants has yielded the most insight into the function of this enzyme in phospholipid metabolism (Hsu et al., 1991). Mutants were selected based on the absence of acyl-ACP synthetase activity; however, all aas mutants lack both acyl-ACP synthetase and 2-acyl-GPE acyltransferase activities in vitro. In aas mutants, there is no detectable acyl-ACP synthetase-dependent incorporation of exogenous fatty acids into PtdEtn or the major outer membrane lipoprotein and the cells lack lysophospholipid uptake and acylation. Aas is not necessary for fatty acid transport per se since FadD-dependent phospholipid synthesis and p-oxidation are not affected in aas mutants. Lipoprotein acylation by phospholipids synthesized by the FadD pathway is not altered in aas mutants showing that Aas is not directly involved in lipoprotein biogenesis. The aas mutants have an altered membrane phospholipid composition and accumulate both 2-acyl-GPE and acylphosphatidylglycerol. Acylphosphatidylglycerol is the product of lysophospholipase L2 (the pldB gene product) and is absent in aaspldB double mutants where the 2-acyl-GPE correspondingly accumulates to higher levels than in aas mutants. The biochemical, physiological, and genetic analysis of aas mutants support the conclusion that 2-acyl-GPE acyltransferase and acyl-ACP synthetase are two activities of the same protein and confirm that this enzyme system participates in membrane phospholipid turnover and governs the acyl-CoA-independent incorporation of exogenous fatty acid and lysophospholipids into the membrane.
Faffy/Ac/ds/n Escherichia coli
53
FadD and Aas have similarities in their primary structure that reflect their common biochemical mechanism. These structural comparisons are possible due to the recent cloning and sequencing ofihQfadD (Black et al., 1992) and aas genes (Jackowski et al., 1993). There are three regions with a high degree of similarity between the two synthetases (Figure 5). Domain I is a region that has the characteristic signature for enzymes that operate via the formation of an acyl-adenylate intermediate. This region of similarity between synthetases is referred to as the AMP binding domain and has the consensus sequence of GXXGXPKG. This structural motif is found in over 20 synthetases in the protein structure data base. The functions of Domains II and III are less well established and the structural similarity in these two domains are not found in synthetases. Domains II and III are also found in the long-chain acy 1-Co A synthetases from yeast (Duroino et al, 1992) and rat brain (Suzuki et al., 1990; Fujino and Yamamoto, 1992). The functional significance of Domains II and III are unknown. Since Aas does not transfer fatty acids to CoA (Ray and Cronan, 1976), and Domains II and III are not found in acetyl-CoA synthetases (Eggen et al., 1991), it seems that these structural motifs are related to the binding of long-chain acyl-AMP rather that the association of a CoA derivative with the enzymes. The precise characterization of the function of this common region between these two proteins awaits dissection by site-directed mutagenesis. The prospect of identifying additional players in the fatty acid transport pathway has tantalized investigators for years. Specific fatty acid translocases exist in mammalian cells. Two examples are the 4,4'-diisothiocyanostilbene-2,2'-disulfonatesensitive translocase of adipose tissue (Schwieterman et al., 1988) and the sodiumdependent liver transporter (Stremmel, 1987). However, inner membrane fatty acid translocators have not been identified genetically or biochemically in E. coli. Furthermore, attempts to find fatty acid binding proteins in the periplasmic compartment that would facilitate the movement of the hydrophobic fatty acids between the outer and inner membrane have not been found. Most of these negative experiments have not been published; however, Mangroo and Gerber (1992) undertook a systematic photo-affmity labeling study to determine the number and properties of fatty acid binding proteins in E. coli. These investigators show the cross-linking of ll-m-diazirinophenoxy-[ll-^H]undecanoate to FadL, but do not detect fatty acid binding proteins associated with the inner membrane or periplasm. The problem of how a fatty acid is translocated across the periplasmic space may be more apparent than real since the inner and outer membranes of E. coli are actually attached to one another at numerous places and FadL may function at these zones of adhesion to deliver exogenous fatty acids directly to the inner membrane. Fatty acid movement is bidirectional. When fatty acid utilization at the glycerophosphate acyltransferase step is blocked using plsB mutants, fatty acids derived from de novo biosynthesis are excreted from the cell (Cooper et al., 1989). It is not know whether fatty acid efflux requires the activity of FadL. At present, it seems unlikely that there are additional proteins required for fatty acid uptake other than those shown in Figure 4.
54
CHARLES O. ROCK and SUZANNE JACKOWSKI
The pathway and mechanisms for the uptake of exogenous phospholipids are less well studied. Intact phospholipids are not taken up by E. coli except by strains that are impaired in outer membrane assembly (Jones and Osbom, 1977). Phospholipid vesicles can be fused to these "deep rough" mutants and incorporated into the membrane (Lai and Wu, 1980). In these experiments there is extensive hydrolysis of the phospholipids and the data do not rule out the possibility that phospholipids are incorporated into the membrane via hydrolysis to lysophospholipids and fatty acids followed by resynthesis. The ability of deep rough strains to incorporate lysophospholipids was established in a careful study using double-labeled substrate, and l-acylglycerol-3-phosphate was shown to be incorporated intact into plsB deep rough mutants (Mclntyre and Bell, 1978). In contrast, 2-acyl-lysophospholipids are taken up and acylated by E. coli with native outer membrane structures (Homma et al., 1981; Hsu et al, 1989). However, the efficiency of extracellular lysophospholipid uptake is low and accounts for approximately 2.5% of the normal phospholipid biosynthetic rate. Analysis ofaas mutants shows that the 2-acyl-GPE acyltransferase pathway is the only mechanism for the incorporation of exogenous lysophospholipids into the membrane. Mutants defective in membrane phospholipid synthesis cannot be rescued by exogenous lysophospholipids, even in the presence of increased expression of the aas gene product. At present, it seems that diffusion is the primary mechanism by which lysophospholipids gain access to Aas; however, it would be interesting to determine if FadL is required for the movement of the lysophospholipid across the outer membrane.
V. FUTURE PROSPECTS The combination of biochemistry, genetics, and molecular biology have proven to be powerful tools in dissecting the metabolism of membrane phospholipids in E. coli. These approaches have led to the detailed understanding of the metabolic pathways for membrane phospholipid synthesis and remodeling. However, the regulatory aspects of phospholipid turnover and the role of the acyltransferases in determining the rate of membrane phospholipid formation are important unresolved problems that should lend themselves to the molecular tools that have become available in the last few years. Most importantly, our knowledge of how membrane phospholipid formation and fatty acid metabolism is integrated into the global control of other structural components (e.g., protein synthesis, ribosomal RNA synthesis, and DNA replication) is meager and the most exciting and fruitful advances in understanding lipid metabolism will be found in this area.
ACKNOWLEDGMENTS This work was supported by grant GM34496 from the National Institutes of General Medical Sciences, Cancer Center (CORE) Support Grant CA21765 from the National Cancer Institute, and the American Lebanese Syrian Associated Charities.
Fatty Acids in Escherichia coli
55 REFERENCES
Abe, M., Okamoto, N., Doi, O., & Nojima, S. (1974). Genetic mapping of the locus for detergent-resistant phospholipase A (pldA) in Escherichia coli K-12. J. Bacteriol. 119, 543-546. Bell, R.M. (1975). Mutants of Escherichia coli defective in membrane phospholipid synthesis: Properties of wild type and Km defective 5«-glycerol-3-phosphate acyltransferase activities. J. Biol. Chem. 250, 7147-7152. Black, P.N., Kianian, S.F., DiRusso, C.C, & Nunn, W.D. (1985). Long-chain fatty acid transport in Escherichia coli. Cloning, mapping, and expression of the fadL gene. J. Biol. Chem. 260, 1780-1789. Black, RN., Said, B., Ghosn, C.R., Beach, J.V., & Nunn, W.D. (1987). Purification and characterization of an outer membrane-bound protein involved in long-chain fatty acid transport in Escherichia coli. J. Biol. Chem. 262, 1412-1419. Black, P.N. (1988). The fadL gene product of Escherichia coli is an outer membrane protein required for uptake of long-chain fatty acids and involved in sensitivity to bacteriophage T2. J. Bacteriol. 170,2850-2854. Black, P.N. (1990). Characterization of FadL-specific fatty acid binding in Escherichia coli. Biochim. Biophys. Acta 1046,97-105. Black, P.N. (1991). Primary sequence of the Escherichia coli fadL gene encoding an outer membrane protein required for long-chain fatty acid transport. J. Bacteriol. 173, 435-442. Black, P.N., DiRusso, C.C, Metzger, A.K., & Heimert, T.L. (1992). Cloning, sequencing, and expression of the fadD gene of Escherichia coli encoding acyl coenzyme A synthetase. J. Biol. Chem. 267, 25513-25520. Coleman, J. (1990). Characterization of Escherichia coli cells deficient in l-acyl-5«-glycerol-3-phosphate acyltransferase activity. J. Biol. Chem. 265, 17215-17221. Coleman, J. (1992). Characterization of the Escherichia coli gene for 1-acyl-5«-glycerol-3-phosphate acyltransferase (plsQ. Mol. Gen. Genet. 232, 295-303. Cooper, C.L., Jackowski, S., & Rock, CO. (1987). Fatty acid metabolism in 5w-glycerol-3-phosphate acyltransferase (plsB) mutants. J. Bacteriol. 169, 605-611. Cooper, C.L., Hsu, L., Jackowski, S., & Rock, CO. (1989). 2-Acylglycerolphospho-ethanolamine acyltransferase/acyl-acyl carrier protein synthetase is a membrane-associated acyl carrier protein binding protein. J. Biol. Chem. 264, 7384-7389. Dahl, CE. & Dahl, J.S. (1984). Phospholipids as acyl donors to membrane proteins of Mycoplasma capricolum. J. Biol. Chem. 259, 10771-10776. Doi, O. & Nojima, S. (1975). Lysophospholipase of Escherichia coli. J. Biol. Chem. 250, 5208-5214. Duroino, R.J., Knoll, L.J., & Gordon, J.I. (1992). Isolation of a Saccharomyces cerevisiae long-chain fatty acyl:CoA synthetase gene {FAAl) and assessment of its role in protein N-myristoylation. J. Cell Biol. 117,515-529. Eggen, R.I.L., Geerling, A.C.M., Boshoven, A.B.P., &de Vos, W.M. (1991). Cloning, sequence analysis, and functional expression of the acetyl coenzyme A synthetase genefromMethanothrix soehngenii m Escherichia coli. J. Bacteriol. 173, 6383-6389. Elsbach, P., Weiss, J., & Kao, L. (1985). The role of intramembrane Ca ^ in the hydrolysis of the phospholipids of Escherichia coli by Ca ^-dependent phospholipases. J. Biol. Chem. 260, 16181622. Elsbach, R, Weiss, J., Wright, G., Forst, S., van den Bergh, C.J., & Verheij, H.M. (1990). Regulation and role of phospholipases in host-bacteria interaction. Prog. Clin. Biol. Res. 349, 1-9. Fujino, T. & Yamamoto, T. (1992). Cloning and functional expression of a novel long-chain acyl-CoA synthetase expressed in brain. J. Biochem. Ill, 197-203. Ginsburgh, C.L., Black, P.N., & Nunn, W.D. (1984). Transport of long chain fatty acids in Escherichia coli. Identification of a membrane protein associated with the fadL gene. J. Biol. Chem. 259, 8437-8443.
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Green, P.R., Merrill, A.H., Jr., & Bell, R.M. (1981). Membrane phospholipid synthesis in Escherichia coli: Purification, reconstitution, and characterization of 5«-glycerol-3-phosphate acyltransferase. J. Biol. Chem. 256, 11151-11159. Green, P.R., Vanaman, T.C., Modrich, R, & Bell, R.M. (1983). Partial NH2- and COOH-terminal sequence and cyanogen bromide peptide analysis of Escherichia coli 5«-glycerol-3-phosphate acyltransferase. J. Biol. Chem. 258, 10862-10866. Gupta, S.D., Dowhan, W., & Wu, H.C. (1991). Phosphatidylethanolamine is not essential for the N-acylation of apolipoprotein in Escherichia coli. J. Biol. Chem. 266, 9983—9986. Homma, H., Nishijima, M., Kobayashi, T., Okuyama, H., & Nojima, S. (1981). Incorporation and metabolism of 2-acyl lysophospholipids by Escherichia coli. Biochim. Biophys. Acta 663, 1-13. Homma, H., Kobayashi, T., Ito, Y., Kudo, I., Inque, K., Ikeda, H., Sekiguchi, M., & Nojima, S. (1983). Identification and cloning of the gene coding for lysophospholipase L2 ofE. coli K-12. J. Biochem. 94,2079-2081. Homma, H., Chiba, N., Kobayashi, T., Kudo, I., Inque, K., Ikeda, H., Sekiguchi, M., & Nojima, S. (1984a). Characteristics of detergent-resistant phospholipase A overproduced in E. coli cells bearing its cloned structural gene. J. Biochem. 96, 1645-1653. Homma, H., Kobayashi, T., Chiba, N., Karasawa, K., Mizushima, H., Kudo, I., Inque, K., Ikeda, H., Sekiguchi, M., & Nojima, S. (1984b). The DNA sequence encoding/?/
Faffy/\c/c/s//I Escherichia coli
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cerevisiae acyl-CoA synthetase, and its utilization by S. cerevisiae myristoyl-CoA:protein Nmyristoyltransferase. J. Biol. Chem. 268, 4281-^290. Kobayashi, T., Homma, H., Natori, Y., Kudo, I., Inque, K., & Nojima, S. (1984), Isolation of two kinds of £. coli K-12 mutants for lysophospholipase L2: One with an elevated level of the enzyme and the other defective in it. J. Biochem. 96, 137-145. Kobayashi, T., Kudo, I., Homma, H., Karasawa, K., Inoue, K., Ikeda, H., & Nojima, S. (1985a). Gene organization of pldA and pldB the structural genes for detergent-resistant phospholipase A and lysophospholipase L2 of Escherichia coli. J. Biochem. 98, 1007-1016. Kobayashi, T., Kudo, I., Karasawa, K., Mizushima, H., Inoue, K., & Nojima, S. (1985b). Nucleotide sequence of the pldB gene and characteristics of deduced amino acid sequence of lysophospholipase L2 in Escherichia coli. J. Biochem. 98, 1017-1025. Kumar, G.B. & Black, P.N. (1991). Linker mutagenesis of a bacterial fatty acid transport protein. Identification of domains with functional importance. J. Biol. Chem. 266, 1348-1353. Lai, J.-S. & Wu, H.C. (1980). Incorporation of acylmoieties of phospholipids into murein lipoprotein in intact cells by Escherichia coli by phospholipid vesicle fusion. J. Bacteriol. 144, 451^53. Larson, T.J., Lightner, V.A., Green, P.R., Modrich, P., & Bell, R.M. (1980). Membrane phospholipid synthesis in Escherichia coli: Identification of the 5«-glycerol-3-phosphate acyltransferase polypeptide as the plsB gene product. J. Biol. Chem. 255, 9412-9426. Larson, T.J., Ludtke, D.N., & Bell, R.M. (1984). 5«-Glycerol-3-phosphate auxotrophy of plsB strains of Escherichia coli: Evidence that a second mutation,/7/5A', is required. J. Bacteriol. 160,711-717. Lightner, V.A., Larson, T.J., Tailleur, R, Kantor, G.D., Raetz, C.R.H., Bell, R.M., & Modrich, R (1980). Membrane phospholipid synthesis in Escherichia coli: Cloning of a structural gene (plsB) of the 5«-glycerol-3-phosphate acyltransferase. J. Biol. Chem. 255, 9413-9420. Lightner, V.A., Bell, R.M., & Modrich, P. (1983). The DNA sequences encoding jo/^^ and dgk loci of Escherichia coli. J. Biol. Chem. 258, 10856-10861. Lucking, D.R. & Goldfine, H. (1975). The involvement of guanosine 5-diphosphate-3-diphosphate in the regulation of phospholipid biosynthesis in Escherichia coli. Lack of ppGpp inhibition of acyltransfer from acyl-ACP to 5«-glycerol-3-phosphate. J. Biol. Chem. 250, 4911-4917. Luttinger, A.L., Springer, A.L., & Schmid, M.B. (1991). A cluster of genes that affects nucleoid segregation in Salmonella typhimurium. New Biologist 3, 687-697. Magnuson, K., Jackowski, S., Rock, CO., & Cronan, J.E., Jr. (1993). Regulation of fatty acid biosynthesis in Escherichia coli. Microbiol. Rev. in press. Maloy, S.R., Ginsburgh, C.L., Simons, R.W., & Nunn, W.D. (1981). Transport of long and medium chain fatty acids by Escherichia coli K12. J. Biol. Chem. 256, 3735-3742. Mangroo, D. & Gerber, G.E. (1992). Photoaffinity labeling of fatty acid-binding proteins involved in long chain fatty acid transport in Escherichia coli. J. Biol. Chem. 267, 17095-17101. Mclntyre, T.M. & Bell, R.M. (1978). Escherichia coli mutants defective in membrane phospholipid synthesis: Binding and metabolism of 1-oleoylglycerol 3-phosphate by d^plsB deep rough mutant. J. Bacteriol. 135,215-226. Michel, G., De Savino, D., & Starka, J. (1977). Phospholipid composition and phenotypic correction of an e«vC mutant of Escherichia coli. J. Bacteriol. 129, 145-150. Michel, G. & Starka, J. (1979). Phospholipase A activity with integrated phospholipid vesicles in intact cells of an envelope mutant of Escherichia coli. FEBS Lett. 108, 261-265. Morona, R. & Henning, U. (1986). New locus {ttr) in Escherichia coli K-12 affecting sensitivity to bacteriophage T2 and growth on oleate as the sole carbon source. J. Bacteriol. 168, 534-540. Nishijima, M., Nakaike, S., Tamori, Y., &, Nojima, S. (1977). Detergent-resistant phospholipase A of Escherichia coli K-12 purification and properties. Eur. J. Biochem. 73, 115-124. Nishijima, M., Sa-eki, T., Tamori, Y, Doi, O., & Nojima, S. (1978). Synthesis of acyl phosphatidylglycerol from phosphatidylglycerol in Escherichia coli K-12. Biochim. Biophys. Acta 528, 107-118.
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Nunn, W.D. (1986). A molecular view of fatty acid catabolism in Escherichia coli. Microbiol. Rev. 50, 179-192. Nunn, W.D., Colbum, R.W., & Black, RN. (1986). Transport of long-chain fatty acids in Escherichia coli. Evidence for role offadL gene product as long-chain fatty acid receptor. J. Biol. Chem. 261, 167-171. Nunn, W.D. & Cronan, J.E., Jr. (1974). re/Gene control of lipid synthesis in Escherichia coli. Evidence for eliminating fatty acid synthesis as the sole regulatory site. J. Biol. Chem. 249, 3994-3996. Nunn, W.D. & Simons, R.W (1978). Transport of long-chain fatty acids by Escherichia coli: Mapping and characterization of mutants in the fadL gene. Proc. Natl. Acad. Sci. USA 75, 3377—3381. Oh, W. & Larson, T.J. (1992). Physical location of genes in the me{ams)-rpmF-plsX-fab region of the Escherichia coli K-12 chromosome. J. Bacteriol. 174, 7873-7874. Ohki, M., Doi, O., & Nojima, S. (1972). MuXdinioiEscherichia coli K-12 deficient for detergent-resistant phospholipase A. J. Bacteriol. 110, 864—869. Overath, P., Pauli, G., & Schairer, H.U. (1969). Fatty acid degradation in Escherichia coli: An inducible acyl-CoA synthetase, the mapping of o/^f-mutations, and the isolation of regulatory mutants. Eur. J. Biochem. 7, 559-574. Raetz, C.R.H. (1987). Structure and biosynthesis of lipid A in Escherichia coli. In: Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology (Neidhardt, F.C., Ingraham, J.L., Low, K.B., Magasanik, B., Schaechter, M., & Umbarger, H., eds.), pp. 498-503, American Society for Microbiology, Washington, D.C. Raetz, C.R.H. (1990). Biochemistry of endotoxins. Ann. Rev. Biochem. 59, 12^170. Raetz, C.R.H. & Dowhan, W (1990). Biosynthesis and function of phospholipids in Escherichia coli. J. Biol. Chem. 265, 1235-1238. Ray, T.K. & Cronan, J.E., Jr. (1976). Activation of long chain fatty acids with acyl carrier protein: Demonstration of a new enzyme, acyl-acyl carrier protein synthetase, in Escherichia coli. Proc. Natl. Acad. Sci. USA 73,4374-4378. Rock, CO., Goelz, S.E., & Cronan, J.E., Jr. (1981). Phospholipid synthesis in Escherichia coli. Characteristics of fatty acid transfer from acyl-acyl carrier protein to 5«-glycerol-3-phosphate. J. Biol. Chem. 256, 736-742. Rock, CO. (1984). Turnover of fatty acids in the 1-position of phosphatidylethanolamine m Escherichia coli. J. Biol. Chem. 259, 6188-6194. Rock, CO. & Cronan, J.E., Jr. (1979). Solubilization, purification, and salt activation of acyl-acyl carrier protein synthetase from Escherichia coli. J. Biol. Chem. 254, 7116-7122. Rock, CO. & Garwin, J.L. (1979). Preparative enzymatic synthesis and hydrophobic chromatography of acyl-acyl carrier protein. J. Biol. Chem. 254, 7123-7128. Rock, CO. & Jackowski, S. (1985). Pathways for the incorporation of exogenous fatty acids into phosphatidylethanolamine in Escherichia coli. J. Biol. Chem. 260, 12720-12724. Scheideler, M.A. & Bell, R.M. (1991). Characterization of active and latent forms of the membrane-associated 5«-glycerol-3-phosphate acyltransferase of Escherichia coli. J. Biol. Chem. 266, 14321— 14327. Schwieterman, W, Sorrentino, D., Potter, B.J., R&, J., Kiang, C.-L., Stump, D., & Berk, RD. (1988). Uptake of oleate by isolated rat adipocytes is mediated by a 40-kDa plasma membrane fatty acid binding protein closely related to that in liver and gut. Proc. Natl. Acad. Sci. USA 85, 359-363. Stremmel, W. (1987). Translocation of fatty acids across the basolateral rat liver plasma membrane is driven by an active potential-sensitive sodium-dependent transport system. J. Biol, Chem. 262, 6284-6289. Suzuki, H., Kawarabayasi, Y, Kondo, J., Abe, T, Nishikawa, K., Kimura, S., Hashimoto, T., & Yamamoto, T. (1990). Structure and regulation of rat long-chain acyl-CoA synthetase. J. Biol. Chem. 265, 8681-8685. Vanden Boom, T. & Cronan, J.E., Jr. (1989). Genetics and regulation of bacterial lipid metabolism. Annu. Rev. Biochem. 43, 317-343.
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Wilkison, W.O., Walsh, J.P., Corless, J.M, & Bell, R.M. (1986). Crystalline arrays of the Escherichia coli 5«-glycerol-3-phosphate acyltransferase, an integral membrane protein. J. Biol. Chem. 261, 9951-9958. Wilkison, W.O., Bell, R.M., Taylor, K.A., & Costello, M.J. (1992). Structural characterization of ordered arrays of 5«-glycerol-3-phosphate acyltransferase from Escherichia coli. J. Bacteriol. 174, 66086616. Wilkison, W.O. & Bell, R.M. (1988). 5«-glycerol-3-phosphate acyltransferase tubule formation is dependent upon heat shock proteins (hptR). J. Biol. Chem. 263, 14505-14510. Wright, G.C., Weis, J., Kim, K.S., Verheij, H., & Elsbach, R (1990). Bacterial phospholipid hydrolysis enhances the destruction of Escherichia coli ingested by rabbit neutrophils. Role of cellular and extracellular phosphohpases. J. Clin. Invest. 85, 1925-1935. Yamakawa, A., Nishizawa, M., Fujiwara, K.T., Kawai, S., Kawasaki, H., Suzuki, K., & Takenawa, T. (1991). Molecular cloning and sequencing of cDNA encoding the phosphatidylinositol kinase from rat brain. J. Biol. Chem. 265, 17580-17583.
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A BRANCHED METABOLIC PATHWAY IN ANIMAL CELLS CONVERTS 2-MONOACYLGLYCEROL INTO sn-1 -STEAROYL-2-ARACHIDONOYL PHOSPHATIDYLINOSITOL AND OTHER PHOSPHOGLYCERIDES
John A. Glomset
ABSTRACT I. INTRODUCTION 11. EVIDENCE FOR 2-MG INCORPORATION INTO PHOSPHOGLYCERIDES A. Swiss 3T3 Cells in Culture Incorporate 2-Arachidonoyl MG into 5«-l-Stearoyl-2-Arachidonoyl Species of PI and other Phosphoglycerides B. Swiss 3T3 Cells also Convert 2-oleoyl MG into Phosphoglycerides . . . . C. A Possible Role for an Arachidonoyl-specific DG Kinase in the 2-MG Incorporation Pathway
Advances in Lipobiology Volume 1, pages 61—100. Copyright © 1996 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN; 1-55938-635-5 61
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III. UNANSWERED QUESTIONS A. What is the Source of 2-MG/« Kivo? B. What Kinases Normally Convert 2-MG to 2-acyl LysoPA? C. What is the Donor in the Unesterified CoA-Dependent, Stearoyl-Specific Transacylase Reaction? D. What is the Basis of the Preferential Incorporation of 2-Arachidonoyl MG int0 5«-l-Stearoyl-2-ArachidonoylPI? E. Does the 2-MG Incorporation Pathway Play a Role in the PI Cycle? . . . F. How do Swiss 3T3 Cells Convert 2-MG into PE, PS, and PC? G. How does the 2-MG Incorporation Pathway Relate to other Pathways of Phosphoglyceride Biosynthesis? H. Do the sn-1 -Stearoyl-2-acyl Phosphoglycerides Formed by the 2-MG Incorporation Pathway Play a Special Role in Cell Membranes? . . IV. CONCLUSION ACKNOWLEDGMENTS ABBREVIATIONS REFERENCES
73 73 75 76 77 77 79 84 86 90 91 91 92
ABSTRACT Swiss 3T3 cells in culture have recently been shown to incorporate exogenous,[^H]glycerol-labeled 2-monoacylglycerol into phosphoglycerides. The pathways that form these phosphoglycerides have yet to be characterized in detail, but a monoacylglycerol kinase and a stearoyl-specific transacylase seem to act successively to initiate one of the major pathways that are involved. This pathway is of special interest because its products appear to include 5«-l-stearoyl-2-arachidonoyl phosphatidylinositol and 5/2-l-stearoyl-2-acyl species of phosphatidylethanolamine and phosphatidylserine. These phosphoglyceride species are key components of animal cell membranes that seem not to be formed by the classical pathways that synthesize phosphoglycerides de novo. Their precise function remains to be determined, but phosphoinositides, phosphatidylethanolamine, and phosphatidylserine are known to be present in the cytoplasmic leaflet of the plasma membrane where they contribute to mechanisms of stimulus transduction and serve as binding sites for many intracellular proteins. Therefore, the 5«-l-stearoyl-2-acyl phosphoglycerides that the 2monoacylglycerol incorporation pathway forms may have an important influence on intracellular events.
1. INTRODUCTION The phosphoglyceride composition of mammalian cell membranes is remarkably complex. Several classes of phosphoglycerides are present that differ in headgroup content; some of these classes consist of up to three subclasses that have either an sn-1,2-diacyl structure, an sn-1 -alkyl-2-acyl structure, or an sn-1 -alk-1 '-enyl-2-acyl structure; and some subclasses consist of as many as 20 separate molecular species
Phosphoglyceride Biosynthesis from 2-Monoacylglycerol
63
that differ in content of specific esterified fatty acyl groups or ether-linked hydrocarbon groups. The potential for variation is such that as many as 200 separate kinds of phosphoglycerides may be present in a single cell membrane (Myher et al., 1989). This raises the possibility that the membrane phosphoglyceride bilayer may encode much more information than is commonly supposed. The fact that some classes of phosphoglycerides have a high potential for providing information has been appreciated for several years. For example, inositol-containing phosphoglycerides and choline-containing phosphoglycerides are known to play important roles in stimulus transduction (Berridge 1987, 1993; Exton, 1994; Billah and Anthes, 1990; Bishop et al., 1992), and both inositol-containing phosphoglycerides and serine-containing phosphoglycerides have been shown to function as anchorage sites for peripheral membrane proteins (Geisow et al., 1987; Klee, 1988; Isenberg, 1991). However, the possibility that different phosphoglyceride subclasses and molecular species also may have specific functions largely remains to be explored. All that is known at present is that 5«-l-alkyl2-arachidonoyl glycero-3-phosphocholine can serve as a precursor of platelet activating factor (reviewed in Snyder et al., 1992) and that other phosphoglycerides that contain an s«-2-arachidonyl group can serve as sources of the arachidonic acid that is used in eicosanoid production. One reason for believing that additional phosphoglyceride molecular species may have specific functions is that the distribution of molecular species among different classes of phosphoglycerides appears to be regulated. For example, analyses of several animal cells and tissues have shown that molecular species that contain a palmitoyl group or an oleoyl group in the snA position and an oleoyl group or a linoleoyl group in the sn-2 position generally predominate in phosphatidylcholine (PC), whereas the 6:«-l-stearoyl-2-arachidonoyl species usually comprises 50% or more of the phosphatidylinositol (PI) and 5«-l-stearoyl-2-acyl species account for about 90% of the phosphatidylserine (PS) (Myher et al., 1989; Mahadevappa and Holub, 1982; Nakagawa et al., 1985; Takamura et al, 1990; Lee and Hajra, 1991; Aveldano et al., 1992). Furthermore, some tissues contain a characteristic distribution of phosphoglyceride molecular species that appears to have been conserved during evolution (Crawford et al., 1977). How might one investigate the potential functions of individual molecular species, given the probability that all membrane phosphoglycerides generally contribute to the structure of the lipid bilayer and the possibility that many phosphoglycerides may function interchangeably? One way might be to focus attention on metabolic pathways that form specific molecular species. If key enzymes in these pathways could be identified and the genes that encode them could be cloned, enzyme knock-out experiments might be done to demonstrate special membrane requirements. Moreover, the same type of approach might yield valuable information about membrane biogenesis. Unfortunately, relatively little information about the pathways that form different species of phosphoglycerides is currently available. The best-known pathways
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JOHN A. GLOMSET
convert glycerol-3-phosphate into diacyl phosphoglycerides de novo (Kent, 1995). They form molecular species, such as the 5«-l-palmitoyl-2-oleoyl species and the 5«-l,2-dioleoyl species, that are typically found in PC (Hill et al, 1968; Arvidson, 1968; Trewhella and Collins, 1969; Holub and Kuksis, 1971). In contrast, pathways that are less well understood form other molecular species, such as the snAstearoyl-2-arachidonoyl species of PI and the ^«-l-stearoyl-2-acyl species of PS. It has been proposed that pathways form 5«-l-stearoyl-2-arachidonoyl species by successively deacylating and reacylating phosphoglycerides that have been formed de novo (for reviews, see Lands and Crawford, 1976, Holub and Kuksis, 1978; MacDonald and Sprecher, 1991; and Snyder et al., 1992). For example, after 5«-l-palmitoyl-2-oleoyl PI is formed de novo a series of four successive reactions of deacylation and reacylation might conceivably convert it into 5«-l-stearoyl-2arachidonoyl PI (Fig. 1). In support of this possibility, cell labeling experiments with radioactive phosphate and radioactive glycerol have demonstrated rapid labeling of monoenoic and dienoic molecular.species of phosphoglycerides, but a much slower labeling of tetraenoic species (Holub and Kuksis, 1978). Moreover, pulse-chase experiments with radioactive glycerol have demonstrated delayed labeling of 5«-l-stearoyl-2acyl species of PC, PE, PI, and PS (Glomset et al., to be published); and separate acylCoA-dependent acyltransferase activities have been reported to catalyze the s/7-1-palmitoyl-2-oleoyl PI >. PHOSPHOLIPASE Ag arachldonoyi CoA S . ^
sn-1-palmitoyl lysoPI -^^
^
oleic acid
ACYLTRANSFERASE 2
x:
stearoyi CoA ' X
sn-1-palmitoyl-2-arachiclonoyl PI
^ y^—s/>2-arachidonoyl lysoPI -^--^
^ ^^ palmitic acid
ACYLTRANSFERASE1 ^ ^ s/>1-stearoyl-2-arachidonoyl PI Figure /. Potential reactions of deacylation and reacylation that might "remodel" PI. Reactions such as those shown here have been postulated to convert species of PI formed de novo into the snA -stearoyl-2-arachidonoyl species. However, the reaction sequence implies a degree of enzyme fatty acyl group specificity that has yet to be convincingly demonstrated.
Phosphoglyceride Biosynthesis from 2-Monoacylglycerol
65
transfer of arachidonoyl and stearoyl groups to lysophosphoglycerides (Lands and Crawford, 1976). Nevertheless, many questions about this proposed mechanism of phospholipid "remodeling" remain to be answered. Precisely which intracellular phospholipases are involved, and what controls their activity? What is the relation between the relatively nonspecific, acyl CoA-dependent acyltransferases that use lysophosphoglycerides as substrates (Sanjanwala et al., 1988; Sanjanwala et al., 1989) and the apparently highly specific arachidonoyl and stearoyl transacylases that have recently been described (Waku, 1992; Snyder et al., 1992; Masuzawa et al., 1989; Darnell and Saltiel, 1991; Simpson et al., 1991; Itabe et al., 1992)? What mechanisms cause the distribution of 5«-l-stearoyl-2-acyl and 5/7-l-acyl-2-arachidonoyl species to differ so strikingly among different phosphoglyceride classes? What is the relation between the proposed reactions of deacylation and reacylation and the biological activity of animal cells? To complicate matters further, a metabolic pathway in Swiss 3T3 cells has recently been shown to incorporate 2-monoacylglycerol (2-MG) preferentially into several molecular species of phosphoglycerides including 5«-l-stearoyl-2-arachidonoyl PI (Simpson et al., 1991), and this has raised a new set of questions concerning the pathways that form these molecular species. The aim of this chapter is to summarize the existing evidence for the 2-MG incorporation pathway, to discuss some current unanswered questions concerning the pathway, and to present a tentative framework of ideas that may serve as a target for future research.
IL EVIDENCE FOR 2-MG INCORPORATION INTO PHOSPHOGLYCERIDES A. Swiss 3T3 Cells in Culture Incorporate 2-Arachidonoyl MG into sn-1-Stearoyl-2-Arachidonoyl Species of PI and other Phosphoglycerides
Swiss 3T3 cells are useful models because they can be maintained in culture in a quiescent state, but replicate readily in response to agonists such as platelet-derived growth factor (PDGF). Therefore, their ability to metabolize phosphoglycerides can be studied under conditions that reflect membrane turnover in the steady state, under conditions that accompany agonist-induced phosphoglyceride degradation and resynthesis, or under conditions that are associated with cell cycle-dependent membrane biogenesis. Furthermore, many investigators use Swiss 3T3 cells for studies of different cell functions, so the potential of this model system to provide integrated information is likely to increase. To examine mechanisms that might form 5«-l-stearoyl-2-acyl and 5'«-l-acyl-2arachidonoyl phosphoglycerides in quiescent Swiss 3T3 cells, pulse-chase experiments were done with radioactive glycerol (Glomset et al., to be published). The cells were incubated for 1 h with the labeled precursor. Then a large excess of unlabeled glycerol was added, and the incubation was continued for an additional
JOHN A. GLOMSET
66
24 h. The results of these experiments provided evidence that the cells initially formed labeled phosphoglycerides that contained a palmitoyl group, an oleoyl group, or a myristoyl group in the sn-1 position and an oleoyl group or a linoleoyl group in the sn-2 position. In contrast, during the chase they primarily formed sn-1 -stearoyl-2-acyl- and sn-1 -acyl-2-arachidonoyl phosphoglycerides. By the end of the chase, the content of labeled 5«-l-stearoyl-2-acyl species was particularly high in PS (about 80% of the total labeled PS), whereas the highest relative content of 5'«-l-acyl-2-arachidonoyl species was found in PI (about 80% of the total PI that became labeled). This suggested that the pathways that form 5/7-l-stearoyl-2-acyland 5«-l-acyl-2-arachidonoyl phosphoglycerides in Swiss 3T3 cells might resemble those that form the corresponding phosphoglycerides in other animal cells. To test the possibility that a 2-MG incorporation pathway might be involved, Simpson et al. (1991) incubated quiescent Swiss 3T3 cells for 1 h in the presence of [^H]-glycerol-labeled 2-arachidonoyl MG and included a large excess of unlabeled unesterified glycerol in the culture medium in order to dilute any labeled unesterified glycerol that might be released by hydrolysis. These experiments demonstrated rapid labeling of several cell lipids including PI, phosphatidylethanolamine (PE), PC, diacylglycerol (DG), and triacylglycerol (TG). In addition, a
PI CDP-DG PA Figure 2, Percentage distribution of radioactivity In lipids from quiescent Swiss 373 cells that were labeled for 1 h with either [ HJglycerol or [ H]glycerol-labeled 2-arachidonoyl MG. The data are expressed as percentage distribution of radioactivity among the indicated lipid classes. They represent the means ± standard deviations of four replicates within a single experiment (from Simpson et al., 1991, with permission). Abbreviations used are: PI (phosphatidylcholine), CDP-DG (cytidine diphosphoryldlacylglycerol), PA (phosphatidic acid), DG (diacylglycerol), TG (triacylglycerol), PC (phosphatidylcholine), PE (phosphatidylethanolamine), PS (phosphatidylserlne).
Phosphoglyceride Biosynthesis from
67
2-Monoacylglycerol
comparison of the results with those of parallel incubation experiments using unesterified [^H]-glycerol provided evidence for preferential incorporation of the radioactive 2-arachidonoyl MG into both PI and PE (Fig. 2). Very little PS became labeled in the 1 h cell incubation experiments with radioactive 2-arachidonoyl MG or radioactive unesterified glycerol, but, chase-associated labeling of PS was observed when cells were incubated for 1 h with radioactive 2-arachidonoyl MG and then incubated for an additional 24 h in unlabeled medium (Fig.3). Furthermore, comparable results were obtained in pulse-chase experiments with radioactive unesterified glycerol (Glomset et al., to be published). The chase-associated labeling of PS was not surprising because animal cells are known to form PS indirectly by exchanging serine for the choline of PC or the ethanolamine of PE (Vance, 1991).
medium change
PC PE PI PS
Incubation Time (h) Pulse
Chase
Figure 3. Time course of radioactive 2-arachidonoyl MG incorporation into lipids of quiescent Swiss 3T3 cells. The cells were incubated with the radioactive precursor for 0, 0.5, or 1 h in the presence of a 7000-fold excess of unlabeled glycerol. Then the medium on some of the cells that had been incubated for 1 h was changed to medium containing no radioactive precursor, and the cells were incubated for a further 24 hours. The data represent the means ± standard deviations of four replicates within a single experiment (modified from Simpson et al., 1991, with permission).
68
JOHN A. GLOMSET
To test the possibility that the cells might have incorporated radioactive 2-arachidonoyl MG into specific molecular species of phosphoglycerides, phosphoglycerides that had become labeled during pulse-chase experiments were converted into acetylated DGs and then analyzed by reverse phase high performance liquid chromatography (HPLC). The results of separate analyses of the acetylated DGs derived from PI, PE, and PS revealed that 70 to 90% of the radioactivity in each of these phosphoglyceride classes was present in the 5«-l-stearoyl-2-arachidonoyl species. In contrast, this species contributed only about 40% of the total radioactivity in PC and DG (Simpson et al, 1991). This raised the possibility that separate 2-MG incorporation pathways might have been involved. To identify potential intermediates in these pathways, cell lipids that had become labeled during the first few seconds of an incubation experiment with radioactive 2-arachidonoyl MG were analyzed by thin layer chromatography. The results demonstrated very early labeling of DG and lysophosphatidic acid (lysoPA). Furthermore, subsequent incubation experiments with cell-free systems revealed that the cell membranes contained both an acylCoA-dependent acyltransferase that could convert 2-arachidonoyl MG to DG and one or more MG kinases that could convert 2-arachidonoyl MG into >s«-2-arachidonoyl lysoPA (Simpson et al., 1991). These results suggested that each type of enzyme might be initiating a separate 2-MG incorporation pathway. To identify further steps in a possible 2-MG kinase-initiated pathway, the cell membranes were incubated with ^^P-labeled 5«-2-arachidonoyl lysoPA. The results of these experiments provided evidence that the membranes contained both an unesterified coenzyme A (CoA)-dependent transacylase that could selectively convert this substrate into 5'«-l-stearoyl-2-arachidonoyl PA and a relatively nonspecific acyltransferase that could convert ^^P-labeled 5/2-2-arachidonoyl lysoPA into a mixture of 5«-l-palmitoyl-2-arachidonoyl PA and 5«-l-stearoyl-2-arachidonoyl PA. To determine whether the stearoyl-specific transacylase reaction could promote the incorporation of ^^P-labeled 5«-2-arachidonoyl lysoPA into PI, cell membranes were incubated with this substrate in the presence or absence of unesterified CoA, cytidine triphosphate (CTP), and free inositol. The results demonstrated unesterified CoA-dependent labeling of both cytidine diphosphoryldiacylglycerol (CDPDG) and PI (Table 1). Furthermore, subsequent structural analysis of the PI showed that the 5n-l-stearoyl-2-arachidonoyl species accounted for almost all of the recovered radioactivity (Simpson et al., 1991). B. Swiss 3T3 Cells also Convert 2-oleoyl MG into Phosphoglycerides To examine the cells' ability to incorporate other species of 2-MG into phosphoglycerides, the cells were incubated with [^H]-glycerol-labeled 2-oleoyl MG (Glomset et al, to be published). As in the earlier experiments with radioactive 2-arachidonoyl MG, the cells incorporated the radioactive 2-MG into PI, PE, PC, DG, and TG during a 1 h pulse, and formed labeled PS during a subsequent 24 h >
Phosphoglyceride Table h
Biosynthesis from 2-Monoacylglycerol
69
Production of PI From sn-2-Arachidonoyl lysoPA by Swiss 3T3 Cell Membranes in the Presence of CTP and Myoinositol Without unesterified Co//
Cofactors added
PA
CDP-DG
With unesterified CoA
PI
PA
CDP-DG
134.6 14.4 125.9 7.5
1.3 81.8 2.8 54.8
PI
pmol/mg protein None CTP Inositol CTP and inositol
19.0 4.3 22.6 2.1
0.0 8.6 0.0 6.1
0.6 5.1 0.3 7.5
0.5 37.4 0.3 54.9
Notes: ^^P-labeied 5/7-2-arachidonoyl lysoPA was incubated with the membranes of Swiss 3T3 cells at 37° C in the presence of magnesium acetate; the PAphosphohydrolase inhibitors, NaF and Na3V04; and various cofactors. The incubation periods were 5 min for those with unesterified CoA and 20 min for those without unesterified CoA (from Simpson et al., 1991, with permission).
chase. Furthermore, PI and PE appeared to be labeled preferentially; analysis of the lipids that were labeled at early time points revealed that radioactivity was present in 5«-l-stearoyl-2-oleoyl species of PI, PE, PC, PS, and DG; and cell-free incubation experiments demonstrated that cell membranes could convert ^^P-labeled sn-2-oleoyl lysoPA into 5«-l-stearoyl-2-oleoyl species of PA and PI. These results provided evidence that 2-MG incorporation pathways in Swiss 3T3 cells convert both 2-arachidonoyl MG and 2-oleoyl MG into phosphoglycerides. Additional results of the incubation experiments with radioactive 2-oleoyl MG provided further important information. For example, during the 1 h pulse-incubation experiments with this substrate, the cells formed about five-fold less PI than they had formed in the corresponding experiments with labeled 2-arachidonoyl MG. Furthermore, only about 40% of the labeled PI consisted of the ^w-l-stearoyl2-oleoyl species, while another 40% consisted of the 5«-l,2-dioleoyl species. By comparison, in the cell-incubation experiments with radioactive 2-arachidonoyl MG almost 90% of the labeled PI consisted of the 5'«-l-stearoyl-2-arachidonoyl species, and diarachidonoyl species of PI were not detected. These results indicate that the 2-MG incorporation pathway forms 5«-l-stearoyl-2-arachidonoyl PI in preference to 5'«-l-stearoyl-2-oleoyl PL The molecular basis for this preference remains to be determined, but the fact that Swiss 3T3 cells incorporate radioactive 2-oleoyl MG into both 5«-l-stearoyl2-oleoyl- and 5'«-l,2-dioleoyl species of PI may depend on a hitherto undescribed, membrane-associated transacylase activity. In support of this possibility, cell-free incubation experiments showed that the membranes of Swiss 3T3 cells contain at least two different unesterified CoA-dependent transacylases that can catalyze the conversion of ^^P-labeled 5«-2-oleoyl lysoPA into PA, the stearoyl-specific transacylase that was mentioned earlier and a second transacylase that catalyzes the
70
JOHN A. GLOMSET
formation of 5«-l,2-dioleoyl PA. Furthermore, the two transacylases may normally compete for sn-2-o\Qoy\ lysoPA on an essentially equal basis because the cell incubation experiments with radioactive 2-oleoyl MG showed that comparable amounts of radioactivity accumulated in ^«-l-stearoyl-2-oleoyl- and sn-l,2'dioleoyl species not only of PI, but also of PE and PC. A final important result of the cell-incubation experiments with radioactive 2-oleoyl MG provided evidence that the 2-MG incorporation pathway is coupled to a pathway that preferentially remodels PI. Both the the 5'«-l-stearoyl-2-oleoyl PI and the ^-z?-1,2-dioleoyl PI that accumulated during the 1 h cell incubation with radioactive 2-oleoyl MG disappeared rapidly during the subsequent chase and were selectively replaced by the corresponding 5«-l-stearoyl-2-arachidonoyl- and sn-loleoyl-2-arachidonoyl species. On the other hand, 5«-l-stearoyl-2-oleoyl- and 5/2-1,2,-dioleoyl species of other phosphoglycerides were replaced more slowly. The mechanism of the PI "remodeling" reaction was not investigated, but the successive action of a phospholipase A2 and an arachidonoyl-specific transacylase may have been involved. An enzyme that can transfer arachidonoyl groups from PC to lysoPI has been described (Irvine and Dawson, 1979). C. A Possible Role for an Arachidonoyl-specific DC Kinase in the 2-MG Incorporation Pathway
In the course of an investigation of the intracellular location of the MG kinaseinitiated pathway, cell homogenates were subfractionated by centrifugation on Percoll gradients and the distribution of the stearoyl-specific transacylase was compared with the distributions of CTPiPA cytidyl transferase, PI synthase, and several different marker enzymes for subcellular organelles (W. Thomas and J.A. Glomset, unpublished experiments). The results of these experiments provided evidence that the stearoyl-specific transacylase, CTP:PA cytidyl transferase, and PI synthase activities were all located in the endoplasmic reticulum. Furthermore, an arachidonoyl-specific DG kinase appeared to be located there as well (Fig. 4).
Figure 4, Distribution of enzymes among subcellular organelles in a Swiss 3T3 cell homogenate. Quiescent Swiss 3T3 cells were homogenized in a buffer containing 0.25 M sucrose. Then a post-nuclear supernatant was prepared and centrifuged in a Percoll gradient. Part A shows the distribution of several organelle marker enzymes (N-acetyl glucosaminidase, lysosomes; citrate synthase, mitochondria; NADPH cyt. C reductase, endoplasmic reticulum; galactosyl transferase, golgi; and NaVK"^ ATPase, plasma membrane). Part B shows the distribution of two key enzymes of PI biosynthesis: CTP:PA cytidyl transferase, and PI synthase. Part C shows the distribution of the unesterified CoA-dependent, stearoyl-specific transacylase, an acylCoA:2-acyl lysoPA acyltransferase, and the membrane-associated arachidonoyl DG kinase (W. Thomas and J.A. Glomset, unpublished data).
71
Phosphoglyceride Biosynthesis from 2-Monoacylglycerol 40%
30% 1
20% H
10% 1
c 9 c o
JUVo A
o
B
CTP. PA cylidyl liansJerase
.....^...
PI synthase
20% -
2o c o 0) Q_
'
10%-
n% -
1—1
1—1
1
1—1
1—1
V
^
1
1—1
\
1—r-
I
■
^
'
JUVo " ■
20% -
~c^
Acyllransferase
----a---
StearoyI transacylase
--"•--
ArachidonoyI DG kinase /
A
rv
10% -
\\
V
k-ci 0 ° ^ ^ F*=^:
2
J-
4
1
1
6
1
1
8
.
1
10
.
1
12
fraction
14
16
18
20
72
JOHN A. GLOMSET
This DG kinase was discovered in Swiss 3T3 cells by MacDonald and colleagues, who provided evidence that it plays a role in the PI cycle (MacDonald et al, 1988a, 1988b). A similar enzyme has recently been purified from bovine testis membranes (Walsh et al., 1994). It shows an approximately 18-fold preference for sn-\-acyl2-arachidonoyl DG over 5«-l-acyl-2-oleoyl DG and distinguishes between sn-\acyl-2-arachidonoyl DG and 5«-l-alk-r-enyl-2-arachidonoyl DG (Walsh et al., 1994;Lemaitreetal., 1990). The finding that both the arachidonoyl DG kinase and the stearoyl-specific transacylase are located in the endoplasmic reticulum raises the possibility that the two enzymes may be contributing to a common pathway that forms 5«-l-stearoyl2-arachidonoyl PI in that organelle. Thus, if cells normally convert several different species of 2-MG to the corresponding species of 5«-l-stearoyl-2-acyl PA, as the cell-incubation experiments with radioactive 2-arachidonoyl MG and 2-oleoyl MG have suggested, a PAphosphohydrolase activity might subsequently convert much of the PA to a mixture of 5«-l-stearoyl-2-acyl species of DG. One role of the arachidonoyl-specific DG kinase might then be to salvage 5«-l-stearoyl-2-arachidonoyl DG from this mixture by selectively reconverting it to ^«-l-stearoyl-2-
^ . .^
acyl CoA -dependent acyltransferase
2-MG —
-sn-1-acyl-2-acylDG
2-MG kinase acyl CoA-dependent acyltransferase
sn-2-acy\ lysoPA
-sn-1-acyl-2-acyl PA
unesterified CoA-dependent stearoyi transacylase PA phosphohydrolase
sn-1-stearoyl-2-acyiPA sn-1-stearoyl-2-acyl CDPDG
sn-1-stearoyl-2-acyl PI
sn-1-stearoyl-2-acyl DG
[3] arachidonoyl DG kinase
s/v1-stearoyl-2-arachidonoyl PA
sn-1-stearoyl-2-oleoyl PI phosphoiipase Ag j
5^1 .stearoyl-2-arachidonoyl CDPDG
s/^1-stearoyllysoPI arachidonoyl transacylase
sn-1-stearoyl-2-arachiclonoyl PI
s/>1-stearoyl-2-arachiclonoyl PI
5n-1-stearoyl-2-arachldonoyl PI
Figure 5. Three different branches of the 2-MG incorporation pathway may form sn-1-stearoyl-2-arachidonoyl PI. Broken lines between sn-1-stearoyl-2-acyl PI and SA7-1-stearoyl-2-oleoyl- or sn-1-stearoyl-2-arachidonoyl PI are meant to indicate that the latter two compounds are components of a mixture of Pis that are formed by the 2-MG incorporation pathway.
Phosphoglyceride Biosynthesis from 2-Monoacylglycerol
73
arachidonoyl PA. A salvage mechanism of this type might promote the formation of 5«-l-stearoyl-2-arachidonoyl PI by increasing the amount of substrate available for reaction with the CTP:PA cytidyltransferase and the PI synthase. Moreover, it might partially account for the fact that considerably more PI became labeled in the cell incubation experiments with radioactive 2-arachidonoyl MG than in the corresponding experiments with radioactive 2-oleoyl MG. In summary, the results that have been obtained to date demonstrate that Swiss 3T3 cells readily convert 2-MG into several phosphoglycerides, suggest that an MG kinase-initiated pathway may be involved, and raise the possibility that this pathway may have at least two branches including one that forms 5«-l-stearoyl-2acyl phosphoglycerides and one that forms 5«-l,2-dioleoyl phosphoglycerides. Furthermore, it appears that the stearoyl-specific branch of the 2-MG-incorporation pathway may form 5«-l-stearoyl-2-arachidonoyl PI by at least three different mechanisms: (1) It may successively convert 2-arachidonoyl MG to 5«-2-arachidonoyl lysoPA and the corresponding 5«-l-stearoyl-2"arachidonoyl species of PA, CDPDG, and PI (Simpson et al, 1991); (2) it may convert other 2-acyl MGs to the corresponding 5«-l-stearoyl-2-acyl Pis by the same set of reactions and then "remodel" these Pis to the 5«-l-stearoyl-2-arachidonoyl species; and (3) it may convert a mixture of 5/2-l-stearoyl-2-acyl PAs to a corresponding mixture of DGs and then salvage sn-1 -stearoyl-2-arachidonoyl DG from this mixture by selectively reconverting this species of DG to PA (Fig. 5).
III. UNANSWERED QUESTIONS It should be evident at this point that many questions about the 2-MG incorporation pathway remain to be answered. Information about the pathway has largely been derived from studies of quiescent Swiss 3T3 cells and a recent study of intestinal epithelial cells (Lehner and Kuksis, 1992), and it remains to be demonstrated that 2-MG incorporation pathways in other animal cells function in the same way. In addition, available evidence about the mechanisms that contribute to the 2-MG incorporation pathway in Swiss 3T3 cells mainly relates to the formation of 5/7-1-stearoyl-2-arachidonoyl PI, and this evidence is incomplete. The branches of the pathway that convert 2-MG into PE, PC, and PS have yet to be characterized; very little is known about the regulation of the pathway or its relation to other pathways of phosphoglyceride synthesis; and the intracellular fate and function of the phosphoglyceride molecular species that are formed remain to be clarified. Some questions that are related to these issues are discussed below. A. What is the Source of 2-MG In Wvo? Several different mechanisms might generate 2-MG in vivo, including the following (see also Fig. 6):
74
JOHN A. GLOMSET
1. Phospholipase C-dependent pathways that hydrolyze phosphatidylinositol4,5-bisphosphate (PIP2) or PC and PE might generate 5'«-l,2-DG, and a DO lipase might subsequently hydrolyze this DO to 2-MG. 2. A phospholipase D-dependent pathway might hydrolyze PC and PE to form PA, a phosphohydrolase might convert the PA to sn-1,2-DG, and a DG lipase might hydrolyze the DG to 2-MG. 3. Lysosomal phospholipase Aj and C activities or a lysosomal phospholipase C and a neutral lipase activity might act together to degrade endocytosed membrane or lipoprotein phospholipids to 2-MG (Huterer and Wheirett, 1984; Irvine et al., 1978; Matsuzawa and Hostetler, 1980; Imanaka et al., 1985). 4. A cytoplasmic lipase might hydrolyze intracellular TG to 2-MG (Cook and Spence, 1985). 5. An sn-1,2-DG transacylase, such as the one described by Lehner and Kuksis (1993), might generate 2-MG. 6. Lipoprotein lipase or hepatic lipase might generate 2-MG extracellularly by hydrolyzing plasma lipoprotein TG, or the latter might be hydrolyzed intracellularly by a lysosomal lipase. The relative importance of these potential mechanisms remains to be determined and may well differ as a function of cell type and metabolic state. In the case of
Possible sources of 2-monoacylglycerol membrane phosphoglycerides
\
/
phospholipase C . DG lipase
0^"^°!!^^^^°, * ''^ + DG P'°n'^1°J7f°'''' lipase Cell DG
I
—lipoprotein lipase ^ ^ \
lipoprotein
/
DGjransacylase
hepatic lipase lysosomal --^ "TG lipase"
j '
lysosomal phospholipases Al and C
I
y ^
neutral lipase ^
Cell TG
lysosomal phospholipase C + "TG lipase"
\
phospholipid inclusions in lysosomes figure 6. Potential sources of 2-MG for the 2-MG incorporation pathway.
Phosphoglyceride Biosynthesis from 2-Monoacylglycerol
75
Swiss 3T3 cells, all that is known at present is that quiescent cells that are incubated for 1 h with radioactive glycerol form labeled MG, including arachidonoyl MG, and that the amount of labeled MG remains relatively constant during a subsequent 24 h chase (Glomset et al., to be published). Furthermore, cells that have been prelabeled with radioactive glycerol for 24 h and then stimulated with PDGF geherate MG, including arachidonoyl MG, during the first hour of cell cycle traverse (Habenicht et al, 1981; Glomset, 1990). Thus, quiescent Swiss 3T3 cells clearly generate MG both under steady-state conditions and in response to a potent mitogenic stimulus. B. What Kinases Normally Convert 2-MG to 2-acyl LysoPA?
Once 2-MG is formed or becomes available to cells, it may have several immediate fates. For example, a lipase may hydrolyze it; an acylCoA-dependent acyltransferase may convert it to DG; one or more kinases may convert it to 5«-2-acyl lysoPA; or it may transfer from the cells into the extracellular fluid. More information is needed about these possibilities and the factors that determine their relative importance in cells. For example, the fatty acyl group specificity of the acylCoA:2-MG acyltransferase has not been determined, and it is uncertain how many different MG kinase activities are present. Indeed, it remains to be demonstrated that MG-specific kinases exist, though the partial purification of a putative MG kinase has been claimed (Shim et al., 1989). Instead, some DG kinases have been shown to have significant MG kinase activity. For example, Kanoh and colleagues (1986) showed that a DG kinase from pig brain could phosphorylate MG and that the kinase reacted preferentially with the 2-isomer. Other investigators have obtained generally similar results with other enzymes (Chen et al., 1993). A potential problem with regard to the preference of kinases for 2-MG as opposed to 1- or 3-MGs is that MGs tend to isomerize rapidly. For example, Kanoh et al.(1986) tested the reactivity of their enzyme toward 1 -MG and found evidence for some phosphorylation of this substrate. However, analysis of the lysoPA that was formed showed that about 70% of it was the 5«-2-acyl isomer. Therefore, the isomeric preference of MG kinases must be analyzed with careful attention to possible acyl chain migradon. Available evidence with regard to Swiss 3T3 cells indicates that they contain at least three different enzymes that can phosphorylate DG: the previously mentioned arachidonoyl-specific DG kinase, which is present exclusively in membranes (MacDonald et al., 1988b), and two nonspecific DG kinases, which are mainly present in the cytosol (Thomas and Glomset, to be published). Furthermore, recent studies of the two soluble enzymes have indicated that they both have MG kinase activity (Thomas and Glomset, to be published). Whether additional MG kinases are present remains to be demonstrated.
76
JOHN A. GLOMSET C. What is the Donor in the Unesterified CoA-dependent, Stearoy I-Specific Transacylase Reaction?
As mentioned earlier, the membranes of Swiss 3T3 cells contain an unesterified CoA-dependent, stearoyl-specific enzyme that can catalyze the conversion of 5^2-2-arachidonoyl lysoPA or sn-2-o\eoy\ lysoPA into the corresponding sn-\stearoyl-2-acyl species of PA; and it appears that this enzyme may contribute to the 2-MG incorporation pathway. However, much more information about the enzyme is needed because its properties have yet to be characterized and its functional significance is far from clear. Fortunately, bovine testis membranes contain relatively high amounts of a similar enzyme, and attempts to purify this enzyme and characterize its properties are underway. Initial studies showed that the enzyme can be solubilized in buffer containing the neutral detergent, Triton X-100 and that the solubilized enzyme functions as a transacylase when incubated with unesterified Co A and mixed micelles containing Triton X-100 and donor and acceptor phospholipids (Itabe et al., 1992). More recent studies of a partially purified enzyme preparation have provided evidence that it can use 5«-l-stearoyl-2-acyl species of PA or PI as stearoyl donors and sn-l-acyl species of lysoPA or lysoPI as stearoyl acceptors in a stearoyl-specific transacylase reaction, but can also use added acylCoA as a donor in a relatively nonspecific acyltransferase reaction (Hollenback and Glomset, to be published). These findings raise important questions about the enzyme's normal function in vivo. Does the enzyme act primarily as a stearoyl-specific transacylase or as a relatively nonspecific acyltransferase? If the enzyme acts as a stearoyl-specific transacylase, does it form stearoylCoA as a reaction intermediate? If it forms stearoylCoA as a reaction intermediate, how does it distinguish this stearoylCoA from "exogenous" acylCoAs that are present in the cytosol? One way to approach these questions might be to do experiments in vitro in the presence or absence of the cytosolic acylCoA binding protein described by Knudsen and his colleagues (Rosendal et al., 1992; Rasmussen et al., 1993). Because this protein appears to regulate the access of acylCoA to enzymes of lipid biosynthesis, it might limit the ability of the partially purified transacylase to use added acylCoA as an acyl donor. On the other hand, it might not prevent the transacylase from transferring stearoyl groups from donor phospholipids to acceptor lysophospholipids via an "endogenously" formed acylCoA intermediate. If the enzyme acts primarily as a stearoyl-specific transacylase, what substrates does it interact with in vivo? One way to address this question might be to use an in vitro assay system that is designed to mimic conditions presumed to exist in intact cells. The partially purified enzyme could be reconstituted into well-characterized, unilamellar vesicles containing a mixture of potential donor phosphoglycerides. Then radioactive, acceptor 5«-2-acyl lysophosphoglycerides could be supplied in the presence of unesterified CoA. A vesicle assay system of this type might provide important information about the relative accessibility of different membrane-
Phosphoglyceride Biosynthesis from 2-Monoacylglycerol
77
associated substrates to the enzyme's active site and might also reveal requirements for additional factors and enzymes in the 2-MG incorporation pathway. D. What is the Basis of the Preferential Incorporation of 2-Arachidonoyl MG into sn-1-Stearoyl-2-Arachidonoyl PI?
As mentioned earlier, considerably more PI became labeled in Swiss 3T3 cell-incubation experiments with radioactive 2-arachidonoyl MG than in corresponding experiments with radioactive 2-oleoyl MG. Furthermore, in the former experiments a higher proportion of the labeled PI consisted of the 5«-l-stearoyl-2acyl species. This selectivity might conceivably depend on the substrate specificities of the enzymes that successively convert 2-MG into 5«-2-acyl lysoPA, 5'«-l-stearoyl-2-acyl PA, 5«-l-stearoyl-2-acyl CDPDG, and 5'«-l-stearoyl-2-acyl PI. But little evidence for arachidonoyl specificity has been obtained so far for the relevant enzymes of 3T3 cells or other tissues (Simpson et al., 1991; Itabe et al., 1992; Bishop and Strickland, 1976; Murthy and Agranoff, 1982). If further studies of these enzymes also provide little evidence for arachidonoyl specificity, then other possibilities will have to be considered. One such possibility is that the 2-MG incorporation pathway may be coupled to an arachidonoyl DG kinase-dependent mechanism that preferentially converts sn-1 -acyl-2-arachidonoylglycerol into sn-1 -acy 1-2-arachidonoyl PA (see also pages 72-73). However, this is highly speculative. Swiss 3T3 cells contain an arachidonoyl DG kinase that might contribute to such a mechanism, but direct evidence that this DG kinase plays a role in the 2-MG incorporation pathway remains to be obtained. An approach involving selective knock-out of the enzyme's activity in intact cells would probably yield the most convincing results, but in the absence of specific enzyme inhibitors this will have to await the accumulation of information about the enzyme's molecular biology. If future experiments demonstrate that the arachidonoyl DG kinase does indeed play a role in the 2-MG incorporation pathway, additional work will be required to identify the factors that regulate its activity and channel the PA that it forms toward the CTP:PA cytidyltransferase instead of toward the PA phosphohydrolase (Fig. 5). E. Does the 2-MG Incorporation Pathway Play a Role in the PI Cycle?
According to the conventional view of the PI cycle, agonists interact with receptors on the cell surface and thereby promote the hydrolysis of PIP2 to DG and inositol trisphosphate, which act as second messengers. Then a DG kinase reaction initiates the resynthesis of phosphoinositides by converting the DG to PA (Kanoh et al., 1990). Furthermore, it has sometimes been assumed that both the degradation and the resynthesis of phosphoinositides occur on the plasma membrane. However, recent results have suggested that the PI cycle may be considerably more complicated than originally supposed. For example, consider the following:
78
JOHN A. GLOMSET
1. It's now clear that the DG that transiently accumulates in response to agonists may be derived not only from the hydrolysis of PIP2, but also from the hydrolysis of PC and possibly also PE (see, for example, Bishop et al., 1992). Therefore, it is necessary to think not only in terms of a PI cycle, but also in terms of potential PC and PE cycles. 2. Evidence has been accumulating that the hydrolysis of PIP2 may occur not only on the plasma membrane, but also in the nucleus (Irvine and Divecha, 1992). Therefore, the question of the location of the reactions that resynthesize phosphoinositides following the hydrolysis of PIP2 may need to be reexamined. 3. As mentioned previously, animal cells have been shown to contain several DG kinase activities including nonspecific, soluble forms and an arachidonoyl-specific, membrane-associated form. Therefore, the possibility that the different DG kinases may have different functions must be considered. 4. DG kinase reactions are not the only potential sources of PA in agonist-induced cell response systems because a phospholipase D that hydrolyzes PC to PA also can be activated (Exton, 1994). 5. Furthermore, not all of the DG that is formed in response to agonists may subsequently be phosphorylated because a DG lipase activity may hydrolyze much of the DG to 2-MG (Prescott and Majerus, 1983; Habenicht et al., 1981;Glomset, 1990). 6. With regard to the enzymes that promote the conversion of PA into phosphoinositides, a PI synthase activity has been detected in pituitary cell plasma membranes (Imai and Gershengorn, 1987; for review, see also Monaco and Gershengom, 1992). Nevertheless, several investigators have failed to find evidence for the presence of PI synthase or PA:CTP cytidyl transferase activities in the plasma membranes of other cells including hepatocytes (Jelsema and Morre, 1978; Lundberg and Jergil, 1988), rat glioma and murine neuroblastoma cells (Morris et al., 1990), astrocytoma cells (Sillence and Downes, 1993), Chinese hamster ovary (CHO) cells (Helms et al., 1991), and Swiss 3T3 cells (Fig. 4). Instead, these enzymes appear to be located in the endoplasmic reticulum and other compartments (see Sillence and Downes, 1993). Therefore, compartments other than the plasma membrane may play a key role in replacing the phosphoinositides (and other phosphoglycerides) that are degraded in response to agonists. To the extent that the endoplasmic reticulum participates in phosphoinositide resynthesis, both transfer of substrates to this organelle and transfer of products from it would presumably be required. With respect to the former possibility, mechanisms that transfer DG or PA between intracellular membranes might conceivably be involved; and a search for specific DG- or PA-transfer proteins is warranted even though little or no evidence for such proteins currently exists. However, an alternate possibility is that both the
Phosphoglyceride Biosynthesis from 2-Monoacylglycerol
79
agonist-induced hydrolysis of PIP2 to DG and the agonist-induced hydrolysis of PC to DG or PA might be coupled to mechanisms that separately generate sn-l -acyland sn-2-dicy\ species of lysoPA and that the relatively hydrophilic lysoPAs might subsequently transfer among intracellular membranes (Fig. 7). Thus, a DG lipase/MG kinase-dependent mechanism or a PA phospholipase A^-dependent mechanism (Higgs and Glomset, 1994, 1996) might generate sn-2-?icy\ lysoPAs including 5«-2-arachidonoyl lysoPA, and a phospholipase A2 might hydrolyze PA to ^«-l-acyl lysoPAs including 5«-l-stearoyl lysoPA. The 5/2-2-acyl lysoPAs and ^«-l-acyl lysoPAs might then transfer to the endoplasmic reticulum and react with enzymes of the 2-MG incorporation pathway or other membrane-associated enzymes to generate sn-1,2-diacyl PAs for use in the resynthesis of phosphoglycerides including phosphoinositides. In support of these possibilities, animal tissues have been found to contain significant amounts of lysoPA (Das and Hajra, 1989); studies of human platelets have shown that they generate both arachidonoyl lysoPA and stearoyl lysoPA in response to thrombin (Mauco et al., 1978; Gerrard and Robinson, 1989); the latter study provided evidence that one mechanism caused the accumulation of arachidonoyl lysoPA while a second, phospholipase A2-dependent mechanism caused the accumulation of stearoyl lysoPA; and studies in two different laboratories demonstrated that lysoPA transfers readily between intracellular membranes and the endoplasmic reticulum (Haldar and Lipfert, 1990; Vancura et al., 1991; Das etal., 1992). However, one problem with this hypothesis is that thrombin-stimulated platelets may represent a special case. Indeed, Eichholtz et al. (1993) have recently reported that thrombin-stimulated human platelets release lysoPA into the medium, and it appears that lysoPA released from platelets in vivo may subsequently act as a local mediator of cell proliferation (Moolenaar et al., 1992). Therefore, it would be advisable to determine whether other agonist-induced response systems also generate sn-2-2icy\- and sn-1 -acyl lysoPAs before attempting to examine the hypothesis in more detail. F. How do Swiss 3T3 Cells Convert 2-MG into RE, PS, and PC?
As mentioned earlier, incubation experiments with quiescent Swiss 3T3 cells have shown that these cells convert radioactive 2-arachidonoyl MG into snAstearoyl-2-arachidonoyl species of PI, PE, PS, PC, and DG. However, these species account for a significantly larger proportion of the recovered radioactivity in PI, PS and PE than they do in PC and DG. The basis for this difference is not known, but the stearoyl-specific, unesterified CoA-dependent branch of the 2-MG incorporation pathway may conceivably form much of the PI, PE, and PS, while both this branch of the pathway and branches that have other specificities may form PC (Fig. 8). The fact that Swiss 3T3 cells preferentially incorporate 2-arachidonoyl MG and 2-oleoyl MG into the corresponding 5n-l-stearoyl-2-acyl species of PS is of
PC
PIP,
2-MG
J sn -2-acyl lyso PA
PC Phospholipase D
+
-
I
I DG kinase I
+ PA
PA ph osph ohydrola se /
I
A Phospholipase A,
sn -1 -acyl lyso PA
I I
I
plasma membrane or nuclear membrane
I I
I I
I
Transfer through the cytosol
I I I
I
I
I
I
t
t
sn -2-acyl lyso PA
sn -1-acyl lyso PA Transacylases and/or
endoplasmic reticulum
sn -1,2-diacyl PA
J
/PI
l\
PC, PE, PS
Figure7. Potential sources of sn-1 -acyl and sn-2-acyl IysoPA in agonist-stimulated cells. According to this postulated reaction sequence, both PIP2 and PC may be indirect sources of sn-1-acyl and sn-2-acyl IysoPA in agonist-stimulated cells. The two forms of IysoPA may then transfer to the endoplasmic reticulum and react with transacylases and/or acyltransferases to form sn-1 -stearoyl-2-acyl PA. Finally, the latter may be converted to PI by the reactions shown in Figure 5.
82
JOHN A. GLOMSET
_
2-MG
acyl CoA -dependent acyltransferase
-sn-1-acyl-2-acyl DG
PC ?
2-MG kinase 1 '
SA7-2-acyl lysoPA
acyl CoA-dependent acyltransferase
-SAh1-acyl-2-acyl PA —>
PC ?
free CoA-dependent stearoyi transacylase PA phosphohydrolase
sn-1-stearoyl-2-acyl PA
-sn-1-stearoyl-2-acyl DG
PE
-PS ?
r\ PI PI PI Figure 8. Potential reaction sequences that might lead to the conversion of 2-MG into PE, PS, and PC.
considerable interest because 5«-l-stearoyl-2-acyl species typically account for about 90% of the PS found in animal cells, as mentioned earlier. The mechanisms that cause sn-1 -stearoyl-2-acyl species of PS to accumulate have yet to be identified, but two separate head group exchange reactions are known to form PS in animal cells. A reaction catalyzed by PS synthase 1 exchanges serine for the choline of PC or the ethanolamine of PE, whereas a reaction catalyzed by PS synthase 2 selectively exchanges serine for the ethanolamine of PE (Kuge et al., 1986a, 1986b). Because 5«-l -stearoyl-2-acyl species normally make up a relatively low proportion of the PC of animal cells, some special mechanism or combination of mechanisms must account for the pronounced accumulation of these species in PS. In principle, one might imagine several different possibilities: 1. One or both of the head group exchange enzymes might conceivably show a preference for ^«-l-stearoyl-2-acyl precursors. 2. The head group exchange enzymes might show no such preference, but be located in close proximity to enzymes in a pathway, such as the 2-MG incorporation pathway, that preferentially forms sn-1 -stearoyl-2-acyl precursors. 3. 5«-l-Stearoyl-2-acyl species of PS might not be formed preferentially, but might accumulate selectively because they are metabolized more slowly than other species. 4. A combination of these possibilities might be involved, as suggested in Fig. 9. Several investigators have provided evidence that the PS formed by the PS synthase 1 reaction may subsequently be decarboxylated to PE in mitochondria (see, for example, Voelker, 1990). Indeed, Nishijima and colleagues have shown that mutant CHO cells that have a lethal, temperature-sensitive defect in PS
Phosphoglyceride
Biosynthesis from
83
2-Monoacylglycerol
Plasma membrane
18:0-X PS PS synthase 2
e.r. cJomain (2)
18:0-X PE
18:1-18:1 PS e.r domain (1)
I PS synthase 1
2-MG pathway 18:1-18:1 PE
T
2-MG
18:1-18:1 PC
t Glycerol-3-P pathway
Glycerol-3-P
Figure 9. Postulated role of the 2-MG incorporation pathway in the biosynthesis of PS. PS synthase 1 appears to contribute to the biosynthetic pathway that forms ethanolannine in animal cells. Its role is to convert PC into PS, which can subsequently be decarboxylated by an enzyme in mitochondria. In contrast, PS synthase 2 is postulated to form the sn-l-stearoyl-2-acyl species of PS that accumulate in membranes and serve as binding sites for peripheral membrane proteins. Note that PS synthase 1 and PS synthase 2 are postulated to be located in two separate domains of the endoplasmic reticulum (e.r.). No evidence for or against this possibility exists at present, though evidence that the endoplasmic reticulum may contain subregions has recently been reviewed (Sitia and Medolesi, 1992).
synthase 1 can be rescued by adding PE to the medium (Kuge et al, 1986b). Therefore, it is conceivable that PS synthase 1 may normally convert sn-\-pd\mitoyl-2-oleoyl and 5«-l ,2-dioleoyl species of PC to PS, and that most of this PS may subsequently be metabolized to the corresponding species of PE. In contrast, the cell incubation experiments with radioactive 2-MG that were described earlier in this chapter have suggested that the stearoyl-specific branch of the 2-MG incorporation pathway may selectively form sn-1 -stearoyl-2-acyl species of both PE and PS. This raises the possibility that the 2-MG-incorporation pathway may be coupled to the PS synthase 2 reaction and that the PS formed by this reaction may accumulate in cell membranes. The mutant CHO cells that Nishijima and colleagues have isolated might provide a powerful means of testing these possibili-
84
JOHN A. GLOMSET
ties. If the cells could be grown at the nonpermissive temperature to inactivate PS synthase 1 under conditions associated with full activity of PS synthase 2, it might be possible to do cell incubation experiments with radioactive serine, glycerol, or 2-MG and obtain evidence related to the proposed role of the 2-MG incorporation pathway. In addition, it might be possible to do incubation experiments with cell membrane preparations to examine the enzyme's specificity directly. It would clearly be of interest to determine whether PS synthase 2 reacts with sn-1 -stearoy 12-acyl PEs in preference to ^«-l-palmitoyl-2-acyl- or 5«-l-oleoyl-2-acyl PEs. Furthermore, because most animal membranes contain both PE {sn-\,2- diacylglycero-3-phosphoethanolamine) and plasmenylethanolamine (^w-l-alk-l'-enyl2-acylglycero-3-phosphoethanolamine), but contain little or no plasmenylserine (Horrocks, 1972), it would be of considerable interest to determine whether PS synthase 2 discriminates against ethanolamine-containing phosphoglycerides that have an ether-linked hydrocarbon chain in the 5«-l-position. G.
How does the 2-MG Incorporation Pathway Relate to other Pathways of Phosphoglyceride Biosynthesis?
Important questions remain to be answered about the relation between the 2-MG incorporation pathway and the metabolic pathways that either form phosphoglycerides de novo or "remodel" these phosphoglycerides into other molecular species. For example, the stearoyl-specific branch of the 2-MG incorporation pathway is of special interest because it forms 5«-l-stearoyl-2-arachidonoyl phosphoglycerides that appear not to be formed de novo, but accumulate selectively in animal cell membranes and may fill specific membrane requirements (see below). But if arachidonoyl groups and stearoyl groups are poorly incorporated into phosphoglycerides de novo, how are they introduced into the substrates that are used in the 2-MG incorporation pathway? One possibility is that relatively nonspecific acyltransferase reactions may be involved. For example, some of the PC that is formed de novo might be hydrolyzed by phospholipase Aj or phospholipase A2 reactions to generate a mixture of lysoPCs. Then these lysoPCs might react with relatively nonspecific acyltransferases to form a mixture of PCs including sn-\stearoyl-2-acyl and 5«-l-acyl-2-arachidonoyl species (Fig. 10). Finally, further metabolism of the PCs might generate stearoyl or arachidonoyl group-containing substrates that could react with acyl chain-specific enzymes in the 2-MG incorporation pathway. In this way pathways that show relatively little acyl chain specificity might be coupled to a pathway that shows a high degree of acyl chain specificity to generate special phosphoglyceride products. Pulse-chase experiments with radioactive stearate might be used to evaluate the validity of this type of reaction sequence. If an adequate chase could be effected, one might expect to find a rapid initial incorporation of stearoyl groups into PC, as already demonstrated by Woldseth et al. (1993), followed by a slower transfer of these groups to PI, PE, and PS. A similar approach has already been used to follow
Phosphoglyceride Biosynthesis from 2-Monoacyiglycerol
85
Glycerol-3-phosphate diacyl phosphoglyceride biosynthesis de novo ▼ R6:0-18:l1 PCs 18:1-18:1 16:0-18:2
phospholipase A-i or Ag
sn-2-acyl lyso PC or sn-1-acyl lyso PC incorporation of 18:0 or 20:4 by relatively nonspecific, acylCoA-dependent, acyltransferases
PCs phospholipases ± DG/MG kinases
sn-2-20:4 lyso PA
sn-1-18:0lysoPA
I
\ transacylase-dependent pathways 18:0- and 20:4-containing species of PA, PI, PE, and PS Figure 10, Potential relation between the pathway that forms PC de novo, the mechanisms of deacylation and reacylation that "remodel" some of this PC, and subsequent reactions that may form stearoyi- and/or arachidonoyi group—containing species of other phosphoglycerides. A pathway that transfers arachidonoyi groups directly from PC to lysoPE or lysoplasmenylethanolamine has been described, but is not shown.
86
JOHN A. GLOMSET
the initial incorporation of arachidonoyl groups into PC and subsequent transfer of these groups to other phosphoglycerides (Blank et al, 1992; Hall et al., 1987; Chilton and Murphy, 1986; Colard et al., 1984; Spinedi et al, 1990). Additional questions concern the relation between the branches of the 2-MG incorporation pathway that form ^A2-l-stearoyl-2-arachidonoyl PI and the de novo pathway that forms other species of PI: Do the same two CTP:PAcytidyl transferase and PI synthase enzymes contribute to both pathways, or are different enzyme isoforms involved? Are the two pathways spatially segregated within separate subdomains of the endoplasmic reticulum? Are they separately regulated? Are the molecular species of PI that the two pathways form distributed to different intracellular membranes or membrane domains? Do the different molecular species of PI have different functions? Answers to these questions are likely to be difficult to obtain. To determine whether different isoforms of the CTP:PAcytidyltransferase and PI synthase exist, purification of both enzymes will be required and molecular biological studies may be required as well. To determine whether the 2-MG incorporation pathway and the de novo pathway are spatially segregated, high titer antibodies to key enzymes will have to be prepared so that the precise location of the enzymes in the endoplasmic reticulum can be examined by immunoelectron microscopy. To determine whether the two pathways are regulated separately, conditions will have to be developed that allow the content of PI in endoplasmic reticulum membranes to be systematically altered. To determine whether the molecular species of PI formed by the two pathways are distributed differently within cells, cell homogenates will have to be fractionated using methods that yield relatively pure organelles, and the distribufion of PI molecular species among the organelles will have to be analyzed by reverse phase high-performance liquid chromatography. To determine whether the different PI molecular species have different functions, enzyme knock-out experiments will probably have to be done, as mentioned previously. H.
Do the sn-1 -Stearoyl-2-acyl Phosphoglycerides Formed by the 2-MG Incorporation Pathv^ay Play a Special Role in Cell Membranes?
The physiological role of the 2-MG incorporation pathway is unclear. The pathway's role may simply be to recycle 2-MG that has been formed by the hydrolysis of phosphoglycerides or TG. On the other hand, it may have additional functions related to the special molecular species of phosphoglycerides that it forms. For example, there is good reason to believe that the 5«-l-stearoyl-2-acyl phosphoglycerides that the pathway forms may be of special functional importance. To begin with, those products of the pathway that contain the highest proportions ofsn-1 -stearoyl-2-acyl species, i.e., PI, PE, and PS, are known to be asymmetrically distributed across the plasma membrane lipid bilayer (Butikofer et al, 1990) and may be similarly distributed across the lipid bilayer of certain intracellular membranes as well. This asymmetric distribution depends in part on an ATP-dependent
Phosphoglyceride Biosynthesis from 2-Monoacylglycerol
87
translocase that is present in these membranes (Devaux, 1992). The enzyme can transfer PS and PE from the extracellular face to the cytoplasmic face of the plasma membrane lipid bilayer and may act similarly to transfer PS and PE from the lumenal face to the cytoplasmic face of secretory granule membranes. The net effect of the asymmetric distribution of PI and PS is to create a special, negatively charged interface between the membrane and the cytosol. This interface is clearly an important attachment site for intracellular proteins. Many soluble proteins have been shown to bind noncovalently to anionic phosphoglycerides, and evidence has been obtained that some of these proteins also affect the structure of the lipid bilayer (see below). The presence of 5«-l-stearoyl groups in the anionic phosphoglycerides, as opposed to that of ^w-l-palmitoyl- or sn-\-o\eoy\ groups, would be expected to increase intermolecular van der Waals interactions within the lipid bilayer. Therefore, it is possible that the role of stearoyl groups may be to increase the stability of attachment sites for peripheral membrane proteins. Experiments designed to test this possibility are needed because studies of the binding of these proteins that have been done to date have focused on the effects of different phospholipid head groups, not different diacylglycerol structures. The information that has been obtained in these studies can be summarized as follows: 1. Some soluble intracellular proteins bind preferentially to PIP and PIP2, others also bind to additional anionic phosphoglycerides including PS, and still others bind to PS in response to an increase in the concentration of intracellular Ca^" (Table 2). 2. Different types of phosphoglyceride-binding domains seem to be involved. Several of the proteins that bind to PIP and PIP2 contain the consensus sequences, KxxxKxKK and/or KxxxxKxRR (Yu et al., 1992). On the other hand, some proteins that bind to PS in the presence of Ca^"*", including several isoforms of protein kinase C and rabphilin, contain a phosphoglyceride-binding domain of about 40 amino acids, referred to as a "C2" domain (recently reviewed by Hug and Sarre, 1993); and annexins contain a special "core" structure that consists of three or more copies of the following Ca"^"^- binding motif: KGxGT-(38 residues)-E/D (Bewley et al., 1993). 3. When proteins bind to anionic phosphoglycerides, they may influence the structure of the lipid bilayer in several ways. Some proteins, including vinculin, vimentin, synapsin 1, and spectrin have been shown to penetrate into the hydrophobic core of the lipid bilayer (Niggli et al., 1986; Benfenati et al., 1989; Johnson et al., 1991). Other proteins, including the profilin of platelets (Goldschmidt-Clermont et al., 1990), C2 domain-containing isoforms of protein kinase C, and annexins (Bazzi and Nelsestuen, 1991), have been shown to bind multiple phosphoglycerides and induce clustering of these phosphoglycerides. Annexins V and VII have been shown to form
JOHN A. GLOMSET
88
Table 2, Some Soluble Intracellular Proteins that Bind to Anionic Phospholipids Proteins that Bind Specifically to PIP and/or PIP2 profilin gelsolin villin severin cofilin destrin gCap39 a-actinin: striated muscle protein 4.1 m-calpain
(Goldschmidt-Clermont et al., 1990; Machesky et al., 1990) (Janmey and Stossel, 1989; Janmey et al., 1987) (Janmey and Matsudaira, 1988) (Yinetal., 1990) (Moriyama et al., 1992; Yonezawa et al., 1991; Yonezawa et al., 1990) (Yonezawa et al., 1990) (Yuetal., 1990) (Fukami et al., 1992) (Anderson and Marchesi, 1985) (Saidoetal., 1992)
Proteins that Bind to PS and/or Other Anionic Phosphoglycerides myosin I caldesmon synapsin I neuromodulin vinculin vimentin tubulin spectrin protein 4.1 MAP2 Ras GTPase-activating protein myelin basic protein
(Doberstein and Pollard, 1992; Zot et al., 1992; Hayden et al., 1990; Adams and Pollard, 1989) (Czurylo et al., 1993; Vorotnikov et al., 1992;) (Benfenati et al., 1989) (Houbreetal., 1991) (Niggli et al., 1990; Niggli et al., 1986; Ito et al., 1983) (Perides et al., 1987; Perides et al., 1986) (Caron and Berlin, 1987) (Maksymiw et al., 1987; Bonnet and Begard, 1984; Mombers et al., 1980) (Cohen et al., 1988; Sato and Ohnishi, 1983) (Surridge and Bums, 1992; Yamauchi and Purich, 1987) (Tsaietal., 1991; Serth etal., 1991) (ter Beest and Hoekstra, 1993; Sankaram et al., 1991)
Proteins that Show Ca^^-Mediated Binding to Anionic Phospholipids Annexin I Annexin II Annexin IV Annexin V
Annexin VI Annexin VII Annexin XI scinderin adseverin calcineurin protein kinase C* rabphilin Note:
(Schlaepfer and Haigler, 1987) (Thiel et al., 1991; Johnsson and Weber, 1990; Glenney et al., 1987; Glenney, 1986) (Edwards and Crumpton, 1991) (Boustead et al., 1993; Concha et al., 1992; Tait and Gibson, 1992; Andree et al., 1992; Meers et al., 1991; Rojas et al., 1990; Schlaepfer et al., 1987) (Bazzi and Nelsestuen, 1992; Bazzi and Nelsestuen, 1991) (Pollard et al., 1988; Pollard et al., 1990; Rojas and Pollard, 1987; Hong et al., 1982; Hong et al., 1981) (Tokumitsu et al., 1992) (Rodriguez del Castillo et al., 1992) (Maekawa and Sakai, 1990) (Politino and King, 1990; Huang et al., 1989; Politino and King, 1987) (Bazzi et al., 1992; Bazzi and Nelsestuen, 1990; Bazzi and Nelsestuen, 1988) (Yamaguchi et al, 1993)
*A mixture of type A isoforms was used. Other isoforms show different activation characteristics (for example, see Nakanishi et al., 1993; Kochs et al., 1993) and may conceivably show different phosphoglyceride-binding characteristics as well.
Phosphoglyceride
Biosynthesis from
89
2-Monoacylglycerol
# 0 0 Ca+
Ca++
Ca++
Ca+
Ca+
Annexin V Figure 11. Schematic view of a section through an annexin V molecule and a segment of a unilamellar phosphoglyceride vesicle that contains PS. The serine head groups of the PS molecules are shown as filled circles. The annexin V molecule is shown in a helix cylinder diagram that is roughly based on data reviewed by Creutz (1992). Interhelical loops, depicted as projecting from the convex surface of the annexin V molecule, contain carbonyl- and carboxyl groups that can partially coordinate Ca"^"*" ions. Negatively charged PS carboxyl groups that project from the surface of the phosphoglyceride vesicle can also bind Ca"^"^ ions. When Ca"^"^ ions are added to a mixture of annexin V molecules and phosphoglyceride vesicles, they are thought to bind concommitantly to the protein- and vesicle surfaces. Several coordination sites are involved for each Ca"^"^ ion though only two such sites, depicted by broken lines, are shown. The figure suggests that the resulting protein-Ca'^'^-PS bridges act together to distort the shape of the vesicles.
voltage-gated Ca"^"^ channels (Bums et al, 1989; Rojas et al., 1990); and annexin V, has been shown to distort the shape of large unilamellar phosphoglyceride vesicles (Andree et al., 1992). The latter effect probably depends on the structure of the annexin V molecules. Studies of the crystal structures of annexin V and other annexins have revealed the presence of several repeats of five alpha helices w^ound into a right-handed superhelix. These repeats are all arranged along a surface that forms one convex side of the molecule (Huber et al., 1992). This surface contains the Ca"^"^- binding sites and the phosphoglyceride-binding sites as well (Jost et al., 1992); the bound
90
JOHN A. GLOMSET
Ca"^"^ ions are thought to form a bridge between the protein and the negatively charged phosphoglycerides. When molecules of annexin V bind to phosphoglyceride bilayers, they form trimers or higher aggregates (Zaks and Creutz, 1991; Newman et al, 1991); and large phosphoglyceride vesicles that have aggregates of annexin V bound to them assume "bizarre, sharply edged shapes" (Andree et al., 1992). The convex, phosphoglyceride-binding surfaces of the aggregated Annexin V molecules apparently distort the shape of the vesicles by promoting the formation of planar facets (Fig. 11). These findings suggest a possible approach for future experimentation. The effects of annexin-dependent distortion on large unilamellar vesicles might be measured as a combined function of phosphoglyceride class and molecular species content. Bazzi and Nelsestuen (1992) have already shown that the presence of PE in vesicles can profoundly affect the annexin-dependent clustering of PS. Therefore, it might be informative to use vesicles containing specific molecular species of phosphoglycerides to examine the potential stabilizing effects of ^«-l-stearoyl groups. Indeed,.a similar approach might be used to examine the potential effects of 5«-2-arachidonoyl groups in phosphoglycerides. Recent molecular modeling calculations have suggested that the 5«-l-stearoyl-2-arachidonoyl backbone structure of some phosphoglycerides may promote the formation of tightly packed, highly regular arrays in monolayers (Applegate and Glomset, 1991).
IV. CONCLUSION The combined results of experiments with intact cells, cell-free systems, and a solubilized, stearoyl-specific transacylase have provided evidence that a branched metabolic pathway in animal cells incorporates 2-MG into several classes of phosphoglycerides. The pathway is of interest for several reasons: 1. One of its branches selectively incorporates stearoyl groups into the sn-lacyl position of phosphoglycerides, while another branch forms dioleoyl species. 2. The products of the stearoyl-specific branch of the 2-MG incorporation pathway include 5«-I-stearoyl-2-arachidonoyl PI and may also include 5'«-l-stearoyl-2-acyl species of PE and PS. 3. All three classes of phosphoglyceride may be key components of the cytoplasmic leaflet of the lipid bilayer in plasma membranes and some intracellular membranes. 4. Both inositol-containing phosphoglycerides and PS are known to be anchorage sites for peripheral membrane proteins. Because work on the pathway began only recently, many questions about it have yet to be answered. Some of these questions were discussed in this chapter, but
Phosphoglyceride Biosynthesis from 2-Monoacylglycerol
91
additional questions also warrant attention. For example, what is the quantitative significance of the pathway as a source of cell phosphoglycerides? Is the pathway modified in different cells and tissues to suit special membrane requirements? Did development of the pathway during the course of evolution coincide with the development of new membrane functions? It is important that questions like these be addressed in view of the possibility that the 2-MG incorporation pathway may play a key role in programming the interface between cell membranes and the cytoplasm. With regard to this possibility, evidence is accumulating that phosphoglycerides on the cytoplasmic face of the lipid bilayer may not only anchor proteins to the membrane, but also in some cases activate them. This suggests that the phosphoglyceride surface of membranes may be an important allosteric modifier of protein function. In other words, when proteins bind to mukiple, negatively charged phosphoglycerides or insert into the phosphoglyceride bilayer, critical changes in protein conformation may result. If both the acyl chains and the head groups of phosphoglycerides influence these changes, then pathways, such as the 2-MG incorporation pathway, that may control the distribution of phosphoglyceride molecular species within membranes take on added significance. Further studies of these pathways and their effects on the membrane lipid bilayer are likely to be required if we are to understand the principles that govern the formation and function of animal cell membranes.
ACKNOWLEDGMENTS Research in the author's laboratory described in this chapter was supported by funds from the Howard Hughes Medical Institute and by United States Public Health Service Grant RR-00166.
ABBREVIATIONS PC PI PIP PIP2 PS PDGF MG DG TG PE PA ATP GTP CTP
phosphatidylcholine phosphatidylinositol phosphatidylinositol-4-phosphate phosphatidylinositol-4,5-bisphosphate phosphatidylserine platelet-derived growth factor monoacylglycerol diacylglycerol triacylglycerol phosphatidylethanolamine phosphatidic acid adenosine triphosphate guanosine triphosphate cytidine triphosphate
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CoA CDP-DG CHO Cyt
coenzyme A cytidine diphosphoryldiacylglycerol Chinese Hamster Ovary cytochrome
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PROPERTIES AND REGULATION OF MAMMALIAN NONPANCREATIC PHOSPHOLIPASE A2 ENZYMES
Christina C. Leslie
ABSTRACT 102 I. INTRODUCTION 102 II. PROPERTIES AND FUNCTION OF MAMMALIAN PLA2 ENZYMES . . . 103 A. Secreted PLA2 Enzymes 103 B. Role of Group IIPLA2 in Arachidonic Acid Release 105 C. Cytosolic PLA2 Enzymes 107 D. Calcium-Dependent, Arachidonic Acid-Specific Cytosolic PLA2 Enzymes . 107 E. Calcium-Independent Cytosolic PLA2 Enzymes Ill III. PLA2 REGULATION 113 A. Transcriptional Regulation of PLA2 113 B. Regulationof PLA2 by Phosphorylation 115 C. Regulation ofPLA2 by Calcium 119 D. Regulation ofPLA2 by ATP 123 E. Regulation of PLA2 by G-Proteins 125 ACKNOWLEDGMENTS 127 REFERENCES 127
Advances in Lipobiology Volume 1, pages 101-139. Copyright © 1996 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-635-5 101
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CHRISTINA C.LESLIE
ABSTRACT Protein purification and molecular cloning approaches have led to the identification of a variety of structurally distinct phospholipase A2 (PLA2) enzymes in mammalian cells. PLA2 enzymes, which degrade phospholipid to free fatty acid and lysophospholipid, function in the processes of membrane repair, digestion of dietary lipid, microbial degradation, and the generation of both extracellular and intracellular lipid mediators and second messengers. The immediate products of PLA2 and their metabolites, the eicosanoids and platelet activating factor, have been implicated as mediators in many diverse tissue and cell functions. In the past decade, mammalian PLA2 enzymes have been identified that play important roles in the pathogenesis of a variety of inflammatory diseases and in signal transduction. This includes low molecular weight secreted PLA2 enzymes, that are structurally similar to the PLA2 enzymes in snake venoms, as well as a heterogeneous group of higher molecular weight, cytosolic PLA2 enzymes that exhibit unique substrate specificities. Current evidence suggests that these PLA2 enzymes are differentially regulated, thus providing alternative pathways for the highly controlled process of phospholipid degradation. In this review the properties, function, and the regulation of mammalian nonpancreatic PLA2 enzymes will be described.
I. INTRODUCTION Mammalian phospholipases A2 (PLA2) are a diverse group of acylhydrolases that function in the catabolism of phospholipid to produce sn-2 fatty acid and lysophospholipid (Waite, 1987; Mukherjee, 1990; Wong and Dennis, 1990). The processes of digestion, membrane repair, microbial degradation and the production of bioactive lipid mediators all involve phospholipid degradation by PLA2. These enzymes play an important role in normal homeostasis and cell function, however, uncontrolled production and activation of PLA2, leading to excess amounts of fatty acid and lysophospholipid, has been implicated in the pathogenesis of various diseases (Vadas and Pruzanski, 1986). It is now well recognized that activation of intracellular phospholipases, including phospholipases C, D, and A2, is important for the generation of a variety of lipid mediators that play a role in stimulus-response coupling (Dennis et al, 1991). Because of the role of PLA2 enzymes in the production of inflammatory mediators and potential involvement in the pathogenesis of diseases, they are important pharmacological targets (Chang et al, 1987; Dennis, 1987; Mayer and Marshall, 1993). In this review the properties, function, and regulation of mammalian PLA2 enzymes will be described with particular emphasis on the nonpancreatic PLA2 enzymes and their role in arachidonic acid mobilization. The immediate products of PLA2 action, namely fatty acids and lysophospholipids, are themselves implicated as important regulatory molecules. Lysophospholipids are active surfactants and at high enough concentrations act as detergents
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and disrupt biological membranes leading to cell lysis (Weltzien, 1979; Stafford and Dennis, 1988). Their production as a result of PLA2 activation during cardiac ischemia is implicated in the electrophysiologic abnormalities of ischemic myocardium (Corr et al., 1984). At sublytic concentrations, lysophospholipids are thought to play a role in membrane fusion (Weltzien, 1979). More recently, lysophosphatidylcholine has been shown to potentiate antigen-stimulated secretion in mouse mast cells (Marquardt and Walker, 1991), and promote T lymphocyte activation induced by diacylglycerol and calcium (Asaoka et al., 1992). Both unsaturated fatty acids and lysophospholipids have been shown to regulate protein kinase C (PKC) activity (McPhail et al., 1984; Murakami et al., 1986; Oishi et al., 1988; Yoshida et al., 1992). A role for specific eicosanoids as mediators in a variety of cell processes such as ion transport, mitogenesis, and differentiation has also emerged and has recently been reviewed (Bonventre, 1992). The products of PLA2, arachidonic acid, and lysophospholipid are also precursors of potent lipid mediators, the eicosanoids and platelet-activating factor (PAF), that as a group can mediate all phases of the inflammatory response (Larsen and Henson, 1983; Feuerstein and Hallenbeck, 1987; Samuelsson et al., 1987; Smith, 1989; Prescott et al., 1990). These lipid mediators act in autocrine or paracrine fashion through specific cell surface receptors.
II. PROPERTIES AND FUNCTION OF MAMMALIAN PLA2 ENZYMES A. Secreted PLA2 Enzymes
It has become clear that most mammalian cells contain multiple PLA2 enzymes including the more well characterized low molecular weight secreted forms, as well as cytosolic and membrane associated intracellular PLA2 enzymes (Ross et al., 1985; Errasfa, 1991; Miyake and Gross, 1992; Murakami et al, 1992) (Table 1). The secreted PLA2 enzymes are low molecular weight (14 kDa), calcium-dependent enzymes that share characteristics with the PLA2 enzymes found in snake venoms (Waite, 1987; Mayer and Marshall, 1993). They do not exhibit any fatty acyl specificity but prefer phosphatidylethanolamine (PE) over phosphatidylcholine (PC) as phospholipid substrate. The low molecular weight secreted PLA2 enzymes have been classified into two groups based on the configuration of the disulfide bonds (Heinrikson et al., 1977). They contain seven disulfide bonds which renders them stable to heat and acid but sensitive to reducing agents. Pancreatic PLA2 and PLA2 enzymes of snake venom from Elapidae (cobras) and Hydrophidae (sea snakes), are group I enzymes and contain characteristic half cysteines at positions 11 and 77. The group IIPLA2 enzymes contain an extended C-terminus which ends in a half cysteine that links to a unique half cysteine at position 50. Mammalian group IIPLA2 enzymes have been found in a variety of cell types and inflammatory exudates.
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CHRISTINA C.LESLIE Table 1. Mammalian PLA2 Enzymes
Type Secreted Pancreatic Nonpancreatic
MW
Ca'' requirement
14kDa
+
14kDa
+
85kDa
+
30kDa
+
40kDa
—
Homology
Regulation
Substrate preference
snake venom PLA2 (group I) snake venom PLA2 (group II)
posttranslational
PE>PC
transcriptional
PE>PC
Nonsecreted PKC/GAP/PLCy/ transcriptional p65 CaLB domain posttranslational 9 14-3-3 proteins 7
ATP
sn-2 20:4 plasmalogens sn-2 20:4 plasmalogens sn-2 20:4
The group I pancreatic PLA2, which is produced as an inactive zymogen form by the pancreas, is activated by proteolysis after secretion and fimctions to digest dietary phospholipid (Waite, 1987). Group I type PLA2 enzymes have also been detected in a variety of other tissues such as lung, stomach, spleen, and serum where their fiinction is unknown (Nishijima et al, 1983; Seilhamer et al., 1986; Waite, 1987; Tojo et al., 1988; Sakata et al., 1989; Yasuda et al, 1990). Recent studies have demonstrated the presence of high affinity binding sites of 200 kDa on a variety of cells including vascular smooth muscle cells, endothelial cells, chondrocytes, synovial cells, and gastric mucosal cells that are specifically recognized by the active group I PLA2 enzyme but not by the group I proenzyme nor by group II PLA2 (Arita et al., 1991; Hanasaki and Arita, 1992). Binding of these sites by group I PLA2 was found to induce DNA synthesis in 3T3 fibroblasts at concentrations of PLA2 that did not yield measurable phospholipid hydrolysis (Arita et al., 1991). Downregulation of the binding sites was observed after long term exposure to group I PLA2 and to cAMP elevating agents and glucocorticoids. These are intriguing findings and suggest a more diverse function for group I PLA2 than previously thought. There has been intense interest in elucidating the structural and ftinctional properties of mammalian nonpancreatic PLA2 enzymes because of their potential roles in inflammatory disease processes and in signal transduction. In diseases such as septic shock and rheumatoid arthritis, PLA2 increases both locally in inflammatory exudates and in serum, and is thought to play a role in the pathogenesis of these diseases (Vadas and Hay, 1983; Pruzanski et al., 1985, 1988; Vadas et al., 1985, 1988). Extracellular PLA2 fi'om synovial fluid has been purified and found to be an acid-stable, 14 kDa cationic protein that requires calcium for activity and prefers phospholipid substrates in the form of Escherichia coli membranes (Vadas et al..
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1985; Kara et al, 1988; Kramer et al, 1989). Cloning of the PLA2 revealed the presence of a signal peptide characteristic of secreted proteins, and sequence homology characteristic of group IIPLA2 enzymes (Kramer et al., 1989; Seilhamer et al., 1989). The group II enzymes exhibit a pi of 10.5 and contain basic amino acids near the N-terminus which is characteristic of PLA2 enzymes that can degrade the phospholipids of E. coli that have been killed by bactericidal permeability increasing protein (BPI) (Forst et al., 1986; Wright et al., 1990b). APLA2 identical to the synovial fluid enzyme has also been described in platelets and is rapidly secreted in response to thrombin stimulation (Kramer et al., 1989). PLA2 enzymes with structural similarities to group IIPLA2, based on partial amino acid analysis, have been purified from peritoneal exudate and a variety of cells and tissues suggesting they are broadly distributed (Verger et al., 1982; Forst et al., 1986; Hayakawa et al., 1988a,b; Ono et al, 1988; Aarsman et al., 1989; Wright et al, 1990b). The recently determined crystal structure of the type II synovial fluid PLA2 revealed identical features to other extracellular group I and II PLA2 enzymes suggesting they share a common catalytic mechanism (Scott et al., 1991; Wery et al., 1991). Amino acids involved in the catalytic site and calcium-binding loop are conserved in the synovial fluid enzyme. The group II PLA2 in inflammatory exudates may be derived from serum or produced locally by cells such as platelets, chondrocytes, synovial fibroblasts, and hepatocytes, which have all been shown to produce the enzyme. It has recently been suggested that the group II PLA2 is an acute phase protein since IL-6 can induce PLA2 synthesis in hepatoma cells, and the PLA2 gene contains an apparent IL-6 responsive element in the 5'-flanking region similar to other acute phase protein genes (Crowl et al., 1991). This data along with the established role of the group II PLA2 in degrading microorganisms implicates a role for this enzyme in host defense mechanisms (Elsbach and Weiss, 1988; Wright et al, 1990a). Group II type PLA2 with properties of an integral membrane protein has also been purified from spleen (Ono et al, 1988). The cloned spleen enzyme was found to contain a signal sequence typical of secretory proteins which raises the interesting question of the mechanism for its membrane association (Ishizaki et al., 1989). From Northern analysis the spleen PLA2 mRNA was distributed in a variety of tissues but enriched in ileal mucosa. A similar enzyme with characteristics of a peripheral membrane protein has been purified from platelet membranes and liver mitochondria (Hayakawa et al., 1988a; Aarsman et al, 1989). Thefiinctionof these membrane associated forms of PLA2 is not known but they have been postulated to be involved in membrane repair and/or arachidonic acid release. B. Role of Croup 11 PLA2 in Arachidonic Acid Release Whether the group II PLA2 is involved in the mobilization of intracellular arachidonic acid has been controversial. Arguments used to suggest that the group II secreted PLA2 is not involved in intracellular mobilization of arachidonic acid
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CHRISTINA C.LESLIE
are: (a) its lack of fatty acid specificity, (b) its inactivation by reducing agents and hence probable inactivation by the reducing environment of the cytosol, and (c) its requirement for concentrations of calcium higher than that found intracellularly. However, the group II PLA2 can hydrolyze arachidonic acid and it could be targeted to a cellular membrane location where it may not be susceptible to reduction. In addition there could be mechanisms operative that modulate the affinity of the enzyme for calcium. In two studies, the group IIPLA2 has been overexpressed in cells to specifically address whether it could mediate enhanced arachidonic acid release. When the group IIPLA2 was overexpressed 100-fold in Chinese hamster ovary (CHO) cells, stimulation with ATP, thrombin, or A23187 did not enhance arachidonic acid release compared to untransfected cells (Lin et al., 1992b). In contrast, in CHO cells overexpressing the 85 kDa cytosolic PLA2 these stimuli greatly enhanced arachidonic acid release. Overexpression of the type IIPLA2 in a fibroblast line resulted in increased cytosolic and membrane associated PLA2 activity and when the cells were stimulated with TPA or aluminum flouride, a small increase (twofold) in arachidonic acid release resulted (Pemas et al., 1991). It is possible that the large amount of PLA2 that was secreted by these cells played a role in the arachidonic acid release in response to TPA or aluminum flouride (discussed below). Recent results by Inoue's group has provided the most definitive evidence that the group II PLA2 can mediate eicosanoid production when fiinctioning as an ectoenzyme. Their data supports an interesting mechanism whereby the group II PLA2, produced by endothelial cells in response to tumor necrosis factor (TNF), becomes associated with the cell surface heparan sulfate proteoglycan where it can hydrolyze membrane phospholipid (Murakami et al., 1993). PGI2 production by TNF-treated endothelial cells could be partially blocked when antibody to the group IIPLA2 was added to the culture medium, and PLA2 activity could be released from the cell surface by addition of excess heparin to the medium. Also, the addition of exogenous group II PLA2 to TNF-treated endothelial cells augmented PGI2 production, which could be blocked by heparin, or antibody to the heparin-binding domain of group II PLA2. In other cell types, exogenous group II PLA2 has been shown to augment eicosanoid production but only when the cells have been exposed to stimulants such as A23187 or antigen (Hara et al., 1991; Murakami et al., 1991). Addition of group IIPLA2 to unstimulated cells did not induce eicosanoid production. Augmentation of eicosanoid production by group II PLA2 depended on the type of cell stimulus, since there was no increased PGI2 production when group II PLA2 was added to thrombin stimulated cells (Murakami et al., 1993). These data, along with the observation that injection of group II PLA2 into tissues promotes inflammation (Bomalaski et al., 1991), support the concept that the high levels of PL A2 found in inflammatory exudates are responsible in part for enhanced production of inflammatory lipid mediators that could play a role in exacerbating inflammation. Although the mechanism by which the group II PLA2 on the cell surface mediates arachidonic acid release is not known, exposure of cells to certain stimulants may
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modify the membrane allowing access of the PLA2 on the cell surface to arachidonoyl-containing phospholipids. It was suggested that A23187 may affect lipid packing or increase the distribution of certain phospholipids on the outer leaflet of the plasma membrane (Hara et al., 1991). It has been documented that cell stimulation results in movement of phospholipids normally on the inner leaflet of the plasma membrane, such as phosphatidylserine, to the outer leaflet (Bevers et al., 1983; Bratton et al., 1992; Bratton, 1993). This process has similarities to the mechanisms involved in PLA2-mediated degradation of ingested bacteria by neutrophils (Elsbach and Weiss, 1988). The neutrophil PLA2 will not degrade bacterial membrane phospholipid unless the bacterial membrane has been perturbed or destabilized by the action of BPI. It is postulated that as a result of BPI action, the flip-flop of phospholipids from the inner leaflet of the outer bacterial membrane occurs creating bilayer regions, and this, along with mobilization of calcium, may contribute to activation of the PLA2 enzymes. The PLA2 enzymes implicated in the hydrolysis of bacterial membrane phospholipid include the bacterial PLA2, a neutrophil granule PLA2, and PLA2 present in the extracellular inflammatory exudate that can be translocated into the phagolysosome during the pmcess of phagocytosis (Wright et al., 1990b). The PLA2 enzymes in the neutrophil granule and in inflammatory exudate have been purified and have properties of group II PLA2 (Forst et al., 1986; Wright et al, 1990a). C. Cytosolic PLA2 Enzymes
Prior to the mid 1980s, the prevailing dogma was that PLA2 enzymes were in general low molecular weight, calcium-dependent enzymes that did not exhibit any substrate specificity. Since that time, novel cytosolic PLA2 enzymes have been identified that are highly substrate specific, such as the PLA2 enzymes selective for 5«-2 arachidonic acid and for plasmalogens, and include forms that function independent of calcium. These cytosolic PLA2 enzymes are a heterogeneous group of higher molecular weight (30-85 kDa) proteins that were identified primarily because specific phospholipid substrates, rather than E. coli membranes or disaturated phospholipids, were used for their characterization and subsequent purification. Although two of these cytosolic enzymes are classified as calcium-dependent enzymes, they do not exhibit an absolute requirement for calcium for catalytic activity as described for the secreted PLA2 enzymes. In addition, all the higher molecular weight phospholipases exhibit varying degrees of lysophospholipase activity. The structure of the active site(s) and the mechanism of catalysis for these enzymes is for the most part not known. D. Calcium-Dependent, Arachidonic Acid-Specific Cytosolic PLA2 Enzymes
Since arachidonic acid is normally found esterified in the sn-l position of membrane phospholipid, and the availability of free arachidonic acid is considered
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to be a rate-limiting step in eicosanoid production, the important role of PLA2 in mediating arachidonic acid mobilization and eicosanoid production has long been recognized. In certain systems it has also been demonstrated that cell stimulation can lead to the selective release of arachidonic acid, implicating a PLA2 that exhibits preference for arachidonic acid (Irvine, 1982). The importance of PLA2 activation in the synthesis of another lipid mediator of inflammation, PAF has also been recognized. In 1979, the structure of PAF, l-O-alkyl-2-acetyl-glycerophosphocholine was elucidated and subsequent studies have demonstrated that it is synthesized by many cell types including neutrophils, macrophages, platelets, endothelial cells and basophils (Benveniste et al., 1979; Blank et al., 1979; Demopoulos et al, 1979). PAF is a potent inflammatory mediator that acts through specific cell surface receptors, and can increase vascular permeability, activate neutrophils, macrophages and platelets, and induce smooth muscle contraction (Hanahan, 1986; Snyder, 1989; Prescott et al., 1990). Analysis of the phospholipid composition of cells that synthesize PAF revealed a high proportion of the 1-0-alkyl-analogue of PC, which was found to be enriched in arachidonic acid compared to diacyl-linked PC (Mueller et al.,-1982, 1984; Nakagawa et al., 1985). Based on several studies, a scheme for the coordinate production of PAF and the eicosanoids has been proposed and postulated to be initiated by PLA2-mediated hydrolysis of arachidonic acid from 1-0-alkyl-linked PC to yield precursors of both the eicosanoids and PAF (Swendsen et al., 1983; Albert and Snyder, 1984; Chilton et al., 1984; Chilton and Connell, 1988; Chilton, 1989). The involvement of an arachidonoylspecific PLA2 in this pathway was suggested by studies showing that neutrophils obtained from essential fatty acid deficient animals, or differentiated HL60 cells depleted of arachidonic acid, failed to synthesize PAF in response to A23187 (Ramesha and Pickett, 1986; Suga et al, 1990). In the HL60 cell system, A23187 stimulation of cells that had been supplemented with arachidonic acid could synthesize PAF, which was accompanied by a decrease in arachidonic acid from alkyl-arachidonoyl-PC (Suga et al., 1990). Recently, an alternate route for the synthesis of PAF has been proposed which involves transfer of arachidonic acid from alkyl-arachidonoyl-PC to a lyso phospholipid acceptor by a coenzyme A-independent transacylase (Uemura et al, 1991; Venable et al, 1991). This reaction is calcium-independent, selective for arachidonic acid, and does not appear to involve a free fatty acid intermediate (Sugiura et al., 1987, 1990; Uemura et al., 1991; Venable et al., 1991; Winkler et al, 1991, 1992). Additional studies have suggested that the preferred lysophospholipid acceptor for the transferred arachidonic acid is lyso PE plasmalogen, which has been shown to be made in sufficient quantities in stimulated neutrophils to drive this reaction (Nieto et al., 1991). From these studies—primarily using homogenized cell preparations—a. scheme for the production of lyso PAF has been proposed. The first step in this proposed pathway is the stimulus-induced activation of PLA2 that selectively hydrolyzes l-alkenyl-2-arachidonoyl-PE forming free arachidonic acid and lyso PE plasmalogen. The lyso PE plasmalogen serves as an acceptor for
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arachidonic acid which is transferred from alkyl-arachidonoyl-PC by a coenzyme A-independent transacylase. The lyso PAF formed can then be acetylated to PAF or reacylated to PC. Studies using stimulated neutrophils and mast cells have documented that PE plasmalogen is the major source of free arachidonic acid that is mobilized in response to cell stimulation (Chilton and Connell, 1988; Tessner et al, 1990; Nakamura et al, 1991; Fonteh and Chilton, 1992). Whether PLA2 selectively hydrolyzes PE plasmalogen or can also directly hydrolyze alkyl-arachidonoyl-PC in stimulated cells remains to be determined. Regardless, a variety of studies have implicated involvement of an arachidonic acid selective PLA2. One of the earliest reports describing a PLA2 activity that exhibited specificity for arachidonic acid was a study identifying a calcium-dependent PLA2 activity in macrophage homogenates that had an alkaline pH optimum and exhibited a 30-fold greater affinity for PC containing sn-l arachidonic acid compared to sn-2 oleic acid (Flesch et al., 1985). Shortly thereafter a cytosolic, calcium-dependent PLA2 with an alkaline pH optimum was identified in human neutrophils that preferred PC containing sn-2 arachidonic acid but did not exhibit any specificity for either an snA acyl- or alkyl-linked PC substrates (Alonso et al., 1986). This PLA2 activity increased in the cytosol after stimulation of the cells with a calcium-ionophore suggesting that the increased activity was due to stable modification of the enzyme. These studies also demonstrated that the presence of calcium during homogenization greatly reduced recovery of the cytosolic PLA2 activity which is now know to be due to the calcium-dependent membrane association of this enzyme. This cytosolic, arachidonoyl-specific PLA2 has now been purified and cloned revealing that it does not exhibit any homology to the low molecular weight secreted forms of PLA2 (Leslie etal., 1988; Clark etal., 1990,1991;DiezandMong, 1990;Gronich etal., 1990; Kim etal., 1991; Kramer etal., 1991; Leslie, 1991; Sharp etal., 1991; Wijkander and Sundler, 1991). The PLA2 cDNA encodes a protein of 85 kDa, although the enzyme migrates at 100-110 kDa by SDS-PAGE for reasons that are not understood, but has been suggested to be due to a high proline content. By radiation inactivation, the size of this PLA2 has been estimated to be 77—80 kDa (Tremblay et al., 1992). The PLA2 appears to be present as a single gene and not a member of a closely related gene family. The purified enzyme exhibits a biphasic calcium dose response curve exhibiting significant activity at concentrations of calcium found intracellularly (100 nM-1 uM), followed by a plateau and another burst of activity from 0.1-5 mM calcium. The reason for the increased activity at the higher concentrations of calcium is not known. The PLA2 contains a Ca^"^dependent phospholipid binding domain (CaLB domain) that shares homology to a similar domain on PKC, GTPase activating protein (GAP), the synaptic vesicle protein, p65, and phospholipase Cy (Clark et al., 1991; Sharp et al., 1991). One of the strongest arguments implicating the 85 kDa PLA2 in the mobilization of intracellular arachidonic acid is its selective hydrolysis of sn-2 arachidonic acid. Previous studies demonstrated that the PLA2 purified from RAW 264.7 cells preferentially hydrolyzed sn-2 arachidonic acid compared to linoleic or oleic acids
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from PC but did not exhibit specificity for sn-2 arachidonoyl-containing PC or PE nor for 1 -acyl or 1 -alkyl-linked PC (Leslie et al, 1988). However, this study utilized defined phospholipid substrates and it was possible that differences in physical properties of the substrate could in part have contributed to the specificity observed. This substrate specificity has recently been confirmed using an in vitro system composed of small quantities of phospholipid substrates added to dimyristoylphosphatidyl methanol vesicles, thus allowing activities to be measured under conditions where the physical properties of the substrate are not a factor (Diez et al., 1992). In a study using macrophage membranes as a substrate, it was demonstrated that upon calcium-dependent translocation of the PLA2 to membranes, radiolabeled arachidonic was preferentially hydrolyzed compared to radiolabeled oleic acid (Channon and Leslie, 1990). It has also been shown that recombinant 85 kDa PLA2 hydrolyzes 3—5 times greater amounts of arachidonic acid than oleic acid from natural membranes despite a threefold greater amount of sn-2 oleic acid than arachidonic acid in the membranes (Clark et al., 1991). Taking into account the amounts of arachidonic acid and oleic acid in the membrane this represents about a 20-fold selectivity for arachidonic acid. In contrast, the synovial fluid PLA2 shows no specificity for sn-2 fatty acid but prefers PE over PC. An unusual property of the 85 kDa PLA2, which is not shared by the secreted PLA2 enzymes but is exhibited by other cytosolic PLA2 enzymes (described below), is that it exhibits lysophospholipase activity (Leslie, 1991). Both PLA2 and lysophospholipase activities copurify, and recombinant 85 kDa PLA2 exhibits lysophopholipase activity (Leslie, unpublished data), verifying that a single protein exhibits dual activities. When assayed against sonicated dispersions of palmitoyllyso-PC or palmitoyl-arachidonoyl-PC, the purified 85 kDa PLA2 exhibits higher lysophospholipase activity than PLA2 activity. However, the physiological relevance of the lysophospholipase activity is not clear particularly since levels of lysophospholipid substrate above the critical micellar concentration are required for activity and such levels would not be expected to be achieved in the cell without deleterious effects. However, it is possible that local concentrations of lysophopholipids high enough for enzyme activity could be achieved. The lysophopholipase activity may represent an efficient mechanism to regulate levels of lysophopholipids formed from PLA2 action on membrane phospholipid. However, sn-1 alkyl-linked lysophopholipids, the precursors of PAF, would not be susceptible to hydrolysis and therefore available for acetylation. The level of complexity and diverse mechanisms that are potentially involved in the regulation of arachidonic acid mobilization is further substantiated by the identification and recent cloning of another arachidonic acid selective PLA2 origincily purified from sheep platelets (Loeb and Gross, 1986). This PLA2 is a cytosolic 30 kDa dimeric protein that can be activated by concentrations of calcium found intracellularly (300-800 nM) and shows 100-fold selectivity for plasmalogen substrate (l-hexadecenyl-2-oleoyl-PC) compared to l-palmitoyl-2-oleoyl-PC. Both purification and cloning revealed the existence of several isoforms of this
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PLA2. Cloning of the PLA2 demonstrated a 73% homology to a member of the 14-3-3 protein family (Zupan et al., 1992). The 14-3-3 dimeric proteins, which exist as multiple isoforms, are widely distributed but are enriched in neuronal tissue (Aitken et al, 1992). Although the function of the isoforms is not well understood, forms have been described that appear to regulate enzyme activities including PKC, aromatic hydoxylases, and N-acetyltransferase. The recombinant 30 kDa PLA2 was shown to catalyze cleavage ofsn-2 fatty acid by forming a covalently-linked acyl enzyme intermediate (Zupan et al., 1992). The transesterification was selective for arachidonic acid and was stimulated by calcium, but calcium was not absolutely required. This mechanism of catalysis is distinct from the mechanism used by secreted group II PLA2, which involves activation of an adjacent H2O molecule (Waite, 1987). The 30 kDa PLA2 exhibits lysophospholipase activity and can potentially function as a transacylase as well as a PLA2. The multiple enzyme activities exhibited by the higher molecular weight cytosolic PLA2 enzymes suggests they may play multiple roles in cells involving diverse mechanisms of arachidonic acid mobilization as well as regulation of lysophospholipid levels. E. Calcium-Independent Cytosolic PLA2 Enzymes
Certain tissues are highly enriched in a specific PLA2 enzyme such as myocardium in which greater than 95% of the PLA2 activity is a calcium independent, cytosolic PLA2. The purified mocardial PLA2 is a 40 kDa protein with an acidic pH optimum that preferentially hydrolyzes arachidonoyl-containing plasmalogen substrates and is the first calcium-independent PLA2 purified that is regio-specific for sn-2 fatty acid (Hazen et al., 1990). The purified enzyme exhibits very high specific activity in the range of 200 jumole/min/mg. Like the 30 kDa and 85 kDa PLA2 enzymes, it also has lysophospholipase activity, but the activity is low relative to PLA2 activity. In addition, under physiologically relevant conditions, in which a low level of lysophospholipid was incorporated into a PC bilayer, the enzyme did not exhibit lysophospholipase activity. Consequently, this PLA2 probably functions primarily as a PLA2 in vivo. A high degree of purification was necessary for this PLA2 and was achieved by the use of ATP-affinity chromatography. ATP was later found to activate and stabilize the crude cytosolic PLA2 and may play an important role in regulating this enzyme (discussed below). The myocardial sarcolemma membrane is the major site for increased phospholipid hydrolysis during ischemia (Miyazaki et al., 1990; Gross, 1992). This membrane contains primarily the plasmalogen species of phospholipid which are enriched in sn-2 arachidonic acid. Since the myocardial calcium-independent PLA2 exhibits selectivity for plasmalogens containing sn-l arachidonic acid, it is a likely candidate involved in mediating the phospholipid breakdown during ischemia. As previously discussed, PE plasmalogen is highly enriched in arachidonic acid and has been shown to be the major source of arachidonic acid in certain stimulated cells, although the specific PLA2 involved in its breakdown has not been identified. Consequently, considering the
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substrate specificity of the myocardial enzyme for sn-2 arachidonoyl-containing plasmalogens, it would be of interest to determine if a similar calcium-independent PLA2 may play a more ubiquitous role in mediating arachidonic acid release in other cells. The cellular and tissue distribution of the 40 kDa calcium-independent myocardial PLA2 is not known, although a PLA2 activity with identical characteristics has recently been characterized in vascular smooth muscle cells (Miyake and Gross, 1992). Another calcium independent phospholipase has been purified from heart cytosol, and this enzyme exhibits both PLAj and PLA2 activities (Cao et al., 1987). The purified phospholipase B was shown to be a calcium-independent enzyme with a pH optimum of 9 and a molecular weight of 140 kDa by gel filtration, but was found to be composed of identical 14 kDa subunits. Neither lysophospholipids nor neutral lipids could be hydrolyzed by this enzyme. This enzyme hydrolyzed acyl groups from both PC and PE, with preference for substrates containing sn-2 arachidonic acid. The ability of the enzyme to hydrolyze plasmalogen substrates was not tested. Compared to the 40 kDa plasmalogen-selective PLA2, the PLA2 activity of the phospholipase B was several orders of magnitude lower. Although not a cytosolic enzyme, a calcium-independent PLA2 from intestine, that exhibits lysophospholipase activity, will be described since its molecular weight and dual enzyme activities suggests similarity to other high molecular weight PLA2 enzymes. It has been purified from guinea pig intestinal brush border membrane and is postulated to play a role in intestinal phospholipid digestion (Gassama-Diagne et al., 1989). The phospholipase has characteristics of an ectoenzyme since it can be recovered from membrane vesicles by papain treatment or Triton-X-100 solubilization. This is characteristic of other intestinal brush border membrane hydrolases which have a short hydrophobic tail that anchors the protein in the membrane, and a glycosylated, globular domain containing the catalytic site. Consequently, this phospholipase may not play a role in hydrolyzing cellular phospholipids but rather exogenous phospholipids. Consistent with this hypothesis is the observation that this phospholipase is only active against substrates dispersed in mixed micelles with detergents. A similar enzyme has also been detected in rabbit and rat. The purified phospholipase is a 97 kDa protein with a specific activity of 22 jimol/min/mg and hydrolyzes linoleic acid from PC and PE with similar activity. The substrate specificity of the purified guinea pig phospholipase has recently been studied in detail showing that it can hydrolyze neutral glycerides as well as phospholipids and hence can be classified as a broad specificity glycerol ester lipase (Gassama-Diagne et al., 1992). Because of some similarities with the 85 kDa cytosolic PLA2, the ability of the guinea pig 97 kDa enzyme to hydrolyze 1stearoyl-2-arachidonoyl-PC was tested under conditions used to assay the 85 kDa PLA2 (in the absence of detergents). It was unable to hydrolyze the arachidonoylcontaining substrate either in the presence or absence of calcium and demonstrates that the two enzymes are fimctionally distinct.
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III. PLA2 REGULATION There have been many mechanisms proposed for regulating PLA2 activities. In this review more recent developments on the role of calcium, G-proteins, specific ligands such as ATP, phosphorylation, and transcriptional regulation will be discussed with particular emphasis on studies in which attempts have been made to characterize the specific PLA2 enzymes involved. There are many studies in which the regulation of arachidonic acid release or eicosanoid production has been investigated, however, in most cases it is not known which PLA2 enzyme has been affected, and could actually represent effects on multiple forms of PLA2. A. Transcriptional Regulation of PLA2
The group II secreted PLA2 was the first form of PLA2 demonstrated to be subject to transcriptional regulation, which has been studied primarily in cultured cell systems, but has also been verified to occur in vivo. Early studies had demonstrated that the inflammatory cytokines, IL-1, and TNF could enhance the secretion of PLA2 and stimulate prostaglandin production from a variety of cells (Chang et al., 1986; Oilman et al., 1988; Godfrey et al., 1988; Pfeilschifter et al., 1989). Subsequently, IL-1 treatment of rabbit chondrocytes for 20 hours was found to enhance cellular levels of PLA2 activity and arachidonic acid, which correlated with an increase in mRNA levels of group IIPLA2 (Kerr et al., 1989). A coordinate increase in PLA2 activity, prostaglandin production, and mRNA levels to group IIPLA2 has also been demonstrated in IL-1 treated mesangial cells. These increases were evident at six hours after IL-1 exposure and continued to increase up to 24 hours (Nakazato et al., 1991). Similarly, treatment of mesangial cells with IL-1 plus forskolin induced an increase in group II PLA2 between 8 and 24 hours after cell stimulation, and almost 90% of the induced PLA2 was secreted (Schalkwijk et al., 1991). In smooth muscle cells, agents that increase intracellular cAMP (forskolin, dibutyryl cAMP, isobutylmethylxanthine), as well as, IL-1, TNF, and lipopolysaccharide (LPS) increased the level of group II PLA2 mRNA, and the amount of secreted group II enzyme, but did not affect the level of group IPLA2 (Nakano et al., 1990b). IL-1, TNF, and LPS did not increase cAMP levels and acted synergistically with forskolin indicating they regulate PLA2 levels by different mechanisms. In rat astrocytes, two different pathways leading to increased group II PLA2 gene expression have also been described (Oka and Arita, 1991). Unlike smooth muscle cells, astrocytes do not respond to forskolin alone but it greatly enhances PLA2 expression in response to TNF. In contrast, LPS-induced PLA2 expression in astrocytes is not affected by forskolin but appears to involve activation of PKC. Consequently, the mechanisms involved in induction of group II PLA2 gene expression appear cell type specific. Increased gene expression of group II PLA2 has also been verified to occur in vivo in brains of LPS-treated rats, and in serum and tissues of rats suffering from
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endotoxic shock. Notably, the enhanced gene expression of group II enzyme in these rats can be inhibited by dexamethasome (Nakano and Arita, 1990; Oka and Arita, 1991). In cultured cell systems, the increased synthesis of group II PLA2, elevation in mRNA levels, and enhanced prostaglandin production induced by cytokines or LPS can also be inhibited by dexamethasone (Nakano et al., 1990a; Oka and Arita, 1991; Schalkwijk et al., 1991). The ability of dexamethasone to inhibit group IIPLA2 synthesis and gene expressioh depends on the tissue as well as the stimulus. In astrocytes, dexamethasone has no effect on TNF-induced synthesis or expression of PLA2, but in smooth muscle cells, it inhibits TNFinduced synthesis and secretion of PLA2, without affecting TNF-induced PLA2 expression (Nakano et al., 1990a; Oka and Arita, 1991). Consequently, glucocorticoids can potentially exert effects on regulation of the group II PLA2 at transcriptional or posttranscriptional levels. Two growth factors, platelet-derived growth factor (PDGFP) and transforming growth factor P (TGPP), when preincubated with mesangial cells, have been shown to suppress cytokine induced gene expression of group IIPLA2 (Muhl et al., 1991; Schalkwijk et al., 1992a). These and potentially other factors may play a role in attenuating pathological effects of overproduction of this extracellular PLA2 and the resulting sequelae. An intriguing finding in studies using PDGFp and TGFP was that they can potentiate cytokine-stimulated prostaglandin production despite the complete downregulation of the group IIPLA2 (Pfeilschifter et al., 1990). These results have now been clarified by the finding that cytokines such as IL-1, TGpp, and TNFa can induce an increase in the synthesis of the 85 kDa cytosolic PLA2. IL-1 or TGFp stimulation of mesangial cells, and TNFa stimulation of HEp-2 cells, have been shown to result in a twofold increase in PLA2 activity with characteristics of the 85 kDa enzyme (Goppelt-Struebe and Rehfeldt, 1992; Lin et al., 1992a; Schalkwijk et al., 1992b). The increase in PLA2 activity is apparent beginning eight hours after cell stimulation and is inhibited by actinomycin D or cycloheximide. In mesangial cells similar doses of IL-1 P induced an increase in the levels of both secreted group II PLA2 and the cytosolic 85 kDa PLA2, and both of the PLA2 enzymes appeared to contribute to prostaglandin production (Schalkwijk et al, 1992b). In contrast, TGFp induced an increase in only the cytosolic PLA2, which was. accompanied by an increase in PGE2 production, implicating a role for the cytosolic PLA2 in prostaglandin production in mesangial cells (Schalkwijk et al., 1992b). In other cell types, such as synovial fibroblasts and WI-38 cells, IL-1 induces an increase only in the cytosolic 85 kDa PLA2 (Hulkower et al., 1992; Lin et al., 1992a). In IL-1 sfimulated WI-38 cells, the increased PLA2 acfivity was identified as the 85 kDa PLA2 by Western blotting (Lin et al., 1992a). The increase in 85 kDa PLA2 protein was evident by eight hours and continued to increase up to 24 hours which correlated with the time course of increased prostaglandin production and cellular PLA2 activity. Importantly, dexamethasone was shown to inhibit both PGE2 production and increased levels of the cytosolic PLA2 in the IL-1 treated WI-38 cells (Lin et al. 1992a). In contrast, neither IL-1 nor dexamethasone had an effect on
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levels of either the constitutive or induced-form of cyclooxygenase. These results strongly implicate the cytosolic PLA2 in prostaglandin production in WI-38 cells stimulated with IL-1. Treatment of monocytes with macrophage colony stimulating factor (M-CSF) has recently been shown to induce a biphasic increase in PLA2 activity measured in cell-free homogenates that correlates with a biphasic increase in mRNA levels of the 85 kDa PLA2 (Nakamura et al, 1992). The first increase in mRNA occurred 15-30 minutes after stimulation, which declined by three hours and was followed by a second increase in mRNA between 24-48 hours, which remained elevated for at least 94 hours. The second phase of PLA2 mRNA induction was clearly associated with an increase in PLA2 protein. The increase in mRNA was in part attributed to an increase in mRNA stability, however, transcriptional rates of the CPLA2 gene could not be measured. These results suggest that increases in PLA2 activity in cell-free homogenates that occur after short-term stimulation (15-30 minutes) can potentially be due to increased gene expression and/or posttranslational modification of the PLA2 (discussed below), depending on the cell type and the stimulus used. Since cytokines can stimulate induction of at least two forms of PLA2, depending on the cell type and expression of both forms can be inhibited by dexamethasone, analysis of both enzymes is necessary in determining the mechanisms involved in prostaglandin production. In addition, with the recent identification of an upregulated form of cyclooxygenase (Kujubu et al., 1991; O'Banion et al., 1991; Fletcher et al., 1992; Kujubu and Herschman, 1992), all these enzymes can potentially contribute to increased prostaglandin production in stimulated cells. Moreover, the ability of dexamethasone to inhibit expression of at least two forms of PLA2, and prostaglandin production, provides at least one mechanism to explain the antiinflammatory effect of glucocorticoids. The observation that dexamethasone inhibits PLA2 expression also challenges the previously held hypothesis that glucocorticoids inhibit prostaglandin production through increased synthesis of the purported PLA2 inhibitory protein, lipocortin (Flower and Blackwell, 1979; Hirata et al., 1980). B. Regulation of PLA2 by Phosphorylation
There have been numerous studies in a variety of systems implicating phosphorylation events in regulating PLA2 activity. In many cell systems, activation of PKC has been shown to play a role in activation of PLA2. The PKC activator, phorbol myristate acetate (PMA), can directly stimulate arachidonic acid release or eicosanoid production in cells such as macrophages and MDCK cells (Bonney et al., 1980; Beaudry et al., 1982; Daniel et al., 1984; Ohuchi et al., 1985; Emilsson and Sundler, 1986;Pfannkucheetal., 1986; Parker etal., 1987;Chaoetal., 1990). Since PMA has been shown not to induce an increase in intracellular calcium in macrophages, but can stimulate eicosanoid production, it suggests that PKC can directly
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activate PLA2 without an increase in intracellular calcium in certain cell types (Chao et al., 1992). In other cells types PMA acts synergistically with calcium ionophore, or augments the effect of other stimuli, to promote arachidonic acid release (Halenda et al, 1985; Craven et al, 1987; Mclntyre et al., 1987; MargoHs et al., 1988; Slivka and Insel, 1988; Weiss and Insel, 1991). It has also been shown that prolonged exposure to PMA, which downregulates PKC, can suppress arachidonic acid release in response to certain stimuli (Wijkander and Sundler, 1989; Chao et al., 1990; Godson et al, 1990; Chao et al., 1992). Specific isoforms of PKC namely a, p, and, 8 have been suggested to be involved in regulating arachidonic acid release or prostaglandin production in MDCK cells, Kupffer cells, and mesangial cells, respectively (Duyster et al., 1983; Godson et al., 1990; Huwiler et al., 1991). Using another approach, studies have shown that the addition of specific protein kinases to cell homogenates can modulate PLA2 activity. In macrophage lysates and in lysed synaptosomes addition of cAMP-dependent protein kinase has been shown to potentiate PLA2 activity (Wightman et al, 1982; Piomelli and Greengard, 1991). In the synaptosome system addition of casein kinase II was also shown to enhance PLA2 activity, whereas, addition of calcium/calmodulin-dependent PKC completely abolished PLA2 activity (Piomelli and Greengard, 1991). In addition to a potential role of these serine/threonine kinases on PLA2 regulation, a role for tyrosine kinase activation has also been suggested. Inhibition of prostaglandin production has been observed with the tyrosine kinase inhibitors genistein and tyrphostin in mouse peritoneal macrophages stimulated with zymosan, PMA, or A23187, and with genistein in PAF-stimulated, LPS-primed P388Dj macrophages (Glaser et al., 1990, 1993). In macrophages, concentrations of tyrphostin or genistein that inhibited prostaglandin production also inhibited stimulus-induced protein tyrosine phosphorylation (Glaser et al., 1993). Similarly, a correlation between inhibition of protein tyrosine phosphorylation and arachidonic acid release, but with the tyrosine kinase inhibitor herbimycin, has been demonstrated in LPS-stimulated RAW 264.7 macrophages (Weinstein et al., 1991). Although a role for protein phosphorylation in regulating PLA2 activity is strongly suggested by these studies, the specific PLA2 enzyme involved was not identified, nor could it be determined whether phosphorylation of the PLA2 itself, or a regulatory protein, was involved. A direct phosphorylation of PLA2 is suggested in studies showing that cell stimulation can result in an increase in PLA2 activity in cell lysates indicating that a stable modification of the enzyme had occurred. Stimulation of mesangial cells with vasopressin, PMA, or EGF has been shown to result in a 2—3-fold increase in cytosolic PLA2 activity that is retained after partial purification of the PLA2 (Gronich et al., 1988; Bonventre et al., 1990). In contrast, A23187 stimulation did not result in an increase in PLA2 activity in cell lysates of mesangial or HER 14 cells (Bonventre et al., 1990; Goldberg et al., 1990). However, in other cell types such as neutrophils, smooth muscle cells, and macrophages (Leslie, unpublished observation), A23187 stimulation does result in a stable increase in PLA2 activity
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in cell lysates (Alonso et al, 1986; Balsinde et al, 1988; Chakraborti et al., 1991). In smooth muscle cells, PLA2 activation by A23187 was either PKC-dependent or independent depending on whether high or low doses of A23187 were used, respectively (Chakraborti et al, 1991). The vasopressin and PMA-induced increase in PLA2 activity in mesangial cells was suppressed by prolonged exposure to PMA implicating a role for PKC activation, however, EGF-induced PLA2 activity was independent of PKC (Gronich et al, 1988; Bonventre et al, 1990). In nontransformed cells such as mesangial cells that have a low number of EGF receptors/cell, EGF does not activate phospholipase C nor result in an increase in cytosolic calcium (Bonventre et al., 1990). However, EGF stimulation of mesangial cells results in increased PLA2 activity in cell lysates but intact cells treated with EGF do not release arachidonic acid or eicosanoids unless PMA or calcium ionophore is also added. Based on these observations it has been suggested that despite a stable modification of the PLA2, an increase in intracellular calcium, or PKC activation, are required for full PLA2 activation to promote arachidonic acid release in EGF stimulated mesangial cells (Bonventre et al., 1990). Characterization of the EGF and PMA stimulated PLA2 in NEF and mesangial cells revealed that it had characteristics similar to the 85 kDa, arachidonic acid-specific PLA2, including chromatographic properties, specificity for arachidonoyl-containing phospholipid substrates, and calcium-dependent membrane association (Gronich et al., 1988; Spaargaren et al., 1992). Phosphorylation of the 85 kDa cytosolic PLA2 in intact cells has now been demonstrated using specific antibody. Stimulation of rat 1A cells with the mitogens PDGF or EGF induced a 2-4-fold increase in phosphorylation of the PLA2 and the enzyme was found to be phosphorylated only on serine residues (Lin et al., 1992b). Similarly, when a CHO cell line overexpressing the 85 kDa PLA2 was stimulated with ATP, thrombin, or PMA, a twofold increase in phosphorylation of the PLA2 on serine was observed that correlated with enhanced arachidonic acid release (Lin et al., 1992b). Treatment of the transfected CHO cells with these stimuli was shown to induce a shift in the molecular weight of the PLA2 to a more slowly migrating species that was reversed upon treatment with phosphatase. ATP, thrombin, and PMA induced a shift in 100% of the PLA2 suggesting stoichiometric phosphorylation. Of interest, the calcium ionophore A23187 induced a shift in molecular weight of 50% of the PLA2 protein suggesting that calcium mobilization activates a kinase that phosphorylates the 85 kDa PLA2 in these cells (Lin et al., 1992b). A correlation between phosphorylation of the PLA2 and enhanced arachidonic acid release was provided by showing that the induction of the gel shift of the PLA2 and stimulusinduced arachidonic acid release could be prevented with the protein kinase inhibitor staurosporine (Lin et al., 1992b). In addition, ATP stimulation of the CHO cells overexpressing the PLA2 induced a twofold increase in PLA2 activity in cell lysates that could be reduced to control levels by phosphatase treatment. This study provided convincing evidence that the 85 kDa PLA2 can mediate hormonallyinduced arachidonic acid release, and that phosphorylation of the PLA2 plays a role
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in its activation. It should be noted that in most cell systems where agonist-induced increases in PLA2 activity in cell lysates have been measured, generally 2—3-fold increases in activity have been observed, whereas larger increases in arachidonic acid from the stimulated cells are evident. It is apparent that the PLA2 from unstimulated cells exhibits significant inherent activity when assayed in vitro in the presence of calcium, and phosphorylation appears to increase the specific activity by approximately 2—3-fold. In another study, PM A stimulation of mouse peritoneal macrophages labeled with ^^P- was found to result in a 1.6-fold increase in activity of partially purified PLA2 that correlated with increased ^^P-labeling of a 100 kDa protein on SDS-polyacrylamide gels (Wijkander and Sundler, 1992b). Phosphatase treatment of the partially purified macrophage preparation resulted in a decrease in both PLA2 activity and the amount of ^^P-label in the 100 kDa band suggesting phosphorylation contributed to the increase in activity of a PLA2 with characteristics similar to the 85 kDa PLA2. When the calcium requirement of the PLA2 from unstimulated and PMA treated cells was compared, no differences were observed suggesting that phosphorylation did not affect the calcium requirement. However, this experiment used a defined arachidonoyl-containing PC substrate and it is possible that phosphorylation may affect the affinity of the enzyme for calcium if assayed using natural membranes as substrate. Studies have recently been carried out to identify the kinases that may be involved in phosphorylating the 85 kDa PLA2. A cytosolic PLA2 with properties characteristic of the 85 kDa, archidonoyl-specific PLA2 was purified from the J774 macrophage cell line and was shown to be phosphorylated by PKC in vitro to a stoichiometry of 0.5 mol phosphate/mol enzyme (Wijkander and Sundler, 1991). However, this phosphorylation did not result in an increase in enzyme activity. Recent studies have demonstrated that the 85 kDa PLA2 can be phosphorylated by MAP kinase in vitro resulting in an increase in PLA2 activity (Lin et al., 1993; Nemenoff et al., 1993). MAP kinase is a threonine/serine kinase that can be activated in many cells types by stimuli such as PMA, growth factors, and agonists acting through G-protein coupled receptors (Cobb et al., 1991; Thomas, 1992). MAP kinase requires phosphorylation on threonine and tyrosine for activation. The 85 kDa PLA2 contains one MAP kinase consensus phosphorylation sequence, Pro-Leu-Ser-Pro (Clark et al., 1991; Sharp et al., 1991). This serine is at position 505 in the PLA2 sequence. In one study, the 85 kDa PLA2 was shown to be phosphorylated in vitro by PKC and by p42 MAP kinase each resulting in 0.3 mol ^^P/mol of PLA2, and a 40% and 80% increase in PLA2 activity, respectively (Nemenoff et al., 1993). Another form of MAP kinase, p54 did not phosphorylate the PLA2. In vitro phosphorylation of fusion proteins of different regions of the PLA2, in combination with two dimensional phosphopeptide mapping, revealed that PKC and MAP kinase phosphorylated different sites on the PLA2. The fragment phosphorylated by MAP kinase did contain the MAP kinase consensus site. In an attempt to relate these observations to PLA2 activation in the cell, it was found that
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stimuli that induce arachidonic acid release in mesangial cells by PKC-dependent or PKC-independent pathways all activated MAP kinase. Although this data is consistent with a role for MAP kinase activation in the activation of the PLA2, no evidence was presented to show phosphorylation of the PLA2 in vivo on the site shown to be phosphorylated by MAP kinase in vitro. Additional evidence has been provided suggesting that the 85 kDa PLA2 is phosphorylated by MAP kinase in vivo (Lin et al, 1993). In this study, PKC, MAP kinase, and protein kinase A were shown to phosphorylate the 85 kDa PLA2 in vitro but only MAP kinase, which phosphorylated the PLA2 on serine residues, induced an increase in PLA2 activity by 2.8-fold. In addition phosphorylation by MAP kinase, but not the other kinases, induced a mobility shift of the PLA2 on SDSpolyacrylamide gels as described for the PLA2 in cells stimulated with agents that induce arachidonic acid release. All the PLA2 shifted to the slower migrating species when phosphorylated by MAP kinase in vitro suggesting stoichiometric phosphorylation. Strong evidence was presented indicating that MAP kinase does phosphorylate the PLA2 on serine-505. A mutated form of the PLA2, in which serine-505 was replaced by alanine, was not phosphorylated by MAP kinase in vitro. In addition CHO cells overexpressing the mutant form of the PLA2 released much less arachidonic acid when stimulated, compared to cells overexpressing the wild type PLA2, and the PLA2 in cells expressing the mutant form of the enzyme did not exhibit a gel shift to a slower migrating species. Comparison of phosphopeptide maps of the PLA2 from TPA treated cells to the PLA2 phosphorylated in vitro revealed that similar sites were phosphorylated, and that these sites were absent on the mutated form of the PLA2 derived from TPA stimulated cells. These data strongly suggest that MAP kinase phosphorylates and activates the 85 kDa PLA2 resulting in arachidonic acid release in the simulated CHO cells. It is also possible that other kinases phosphorylate the 85 kDa PLA2 in vivo since additional phosphopeptides, as well as those phosphorylated by MAP kinase, were observed on the 2-dimensional maps of the PLA2 from TPA-stimulated CHO cells. A role for PKC in directly phosphorylating the PLA2 in vivo and modulating PLA2 activity is not clear at this time. Only one of the three studies demonstrating phosphorylation of the PLA2 in vitro by PKC showed a small increase in PLA2 activity. However, it is possible that phosphorylation of the PLA2 on certain sites has functional consequences other than affecting enzyme activity. Of all the PLA2 enzymes described only the 85 kDa cytosolic PLA2 has been shown to be regulated by phosphorylation. C. Regulation of PLA2 by Calcium
The low molecular weight, extracellular PLA2 enzymes exhibit an absolute requirement for calcium for catalytic activity and other cations will not substitute (Verheij et al., 1974). Structural analysis has shown that calcium binds to the active site and is involved in the catalytic mechanism (Dennis, 1983; Scott et al., 1990;
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Jain et al, 1991). Maximal activity of the secreted synovial fluid PLA2 enzyme is achieved at mM concentrations of calcium, which is consistent with these enzymes functioning extracellularly (Hara et al., 1988; Kramer et al., 1989). However, a side-by-side comparison of the calcium requirement of recombinant synovial fluid PLA2 and partially purified 85 kDa PLA2 surprisingly showed that these enzymes exhibited identical calcium dose response curves (Marshall and McCarte-Roshak, 1992). When low concentrations of calcium were adjusted using calcium/EGTA buffers, both enzymes exhibited the biphasic dose response curve previously observed for the 85 kDa PLA2. However, neither enzyme exhibited significant enzyme activity at concentrations of calcium below 10 juM when EGTA buffers were not used. The presence of EGTA itself was found to modulate enzyme activity at low levels of calcium. It is possible that the EGTA is acting to chelate other metals that are inhibiting the PLA2 enzymes at low levels of calcium. Alternatively, it suggests that for these PLA2 enzymes to function intracellularly in a calciumdependent manner, mechanisms to modulate their aflfinity for calcium would be required. As previously suggested (Krause et al, 1991; Lathrop and Biltonen, 1992), one possibility is that the affinity of these PLA2 enzymes for calcium may be affected by the presence of other divalent cations such as magnesium. For the 85 kDa PLA2, phosphorylation may modulate the affinity of the enzyme for calcium. The composition of the substrate may also play a role in influencing the affinity of the 85 kDa PLA2 for calcium. It has previously been shown that when anionic phospholipids are codispersed with the arachidonoyl-containing PC substrate, the activity of the 85 kDa PLA2 at low levels of calcium is greatly enhanced (Leslie and Channon, 1990). In contrast to the secreted PLA2 enzymes, the 85 kDa and 30 kDa cytosolic PLA2 enzymes do not exhibit an absolute requirement for calcium. For these cytosolic enzymes it has been shown that their calcium requirement can be overcome when assayed in the presence of molar concentration of NaCl together with excess calcium chelators (Zupan et al., 1991; Wijkander and Sundler, 1992a). A variety of salts were effective. Neither venom nor pancreatic PLA2 enzymes exhibited activity in the presence of high salt without calcium. Detailed kinetic analysis of the 85 kDa PLA2 has also provided evidence for the mechanism by which calcium regulates this enzyme. The purified 85 kDa PLA2 has been shown to exhibit unusual kinetic properties when assayed against sn-2 arachidonoyl-containing PC vesicles (Leslie, 1991). The reaction progress ceases abruptly despite only limited substrate hydrolysis, and addition of more enzyme, but not substrate, could reinitiate catalysis. Since both substrate depletion and end product inhibition were ruled out, the data appeared consistent with enzyme inactivation. However, in a more recent study it was found that readdition of buffer containing salt or albumin could reinitiate catalysis indicating the PLA2 was not inactive (Ghomashchi et al., 1992). Additional data suggested that the PLA2 becomes trapped on phospholipid vesicles as product accumulates in the vesicle, and that trapping is independent of catalysis. Interestingly, trapping was dependent on the nature of the fatty acid product and
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was highly selective for arachidonic acid. Both trapping and activity of the PLA2 required a similar concentration of calcium, but other divalent cations (Sr^"^ > Ba^"^» Mg^^) could substitute for calcium, suggesting that calcium is required for interfacial binding rather than as a cofactor for enzyme activity. Consistent with this hypothesis is the observation that the 85 kDa PLA2 can be induced to bind biological membranes in a calcium-dependent manner. When cells are homogenized in the presence of calcium chelators or at levels of calcium found intracellularly in unstimulated cells (100 nM), the PLA2 is found almost exclusively in the 100,000 g supernatant (Channon and Leslie, 1990; Diez and Mong, 1990). When homogenized in the presence of higher concentrations of calcium, that would occur intracellularly in response to calcium mobilizing agonists (>200 nM), the enzyme quantitatively associates with the particulate fraction. The membrane associated enzyme could be removed from the membrane with calcium chelators (Channon and Leslie, 1990). This process has been verified using recombinant PLA2 which bound to natural membrane vesicles at concentration of calcium > 300 nM, and it was additionally shown that a fragment of the PLA2 containing the CaLB domain associated with natural membrane vesicles in the presence of calcium (Clark et al., 1991). A recent study has shown that the 85 kDa PLA2 together with ^^Ca^"*" and phospholipid vesicles can form a stable ternary complex and the results suggested a single binding site for Ca^"^ on the PLA2 (Wijkander and Sundler, 1992a). These data suggest a potential regulatory mechanism whereby increases in intracellular calcium can induce PLA2 association with membrane phospholipid substrate. However, it is also possible that other mechanisms are involved in regulating the membrane association of the 85 kDa PLA2 such as phosphorylation. There are cell types such as macrophages where PMA stimulation can induce an increase in PLA2 activity and arachidonic acid release without an apparent increase in intracellular calcium (Chao et al., 1992). This suggests that phosphorylation may increase the affinity of the PLA2 for calcium thus allowing for membrane association at resting levels of calcium. The mechanisms involved in regulating the membrane association of the 85 kDa PLA2 in response to cell stimulation are poorly understood. There have not been any studies that have actually demonstrated the translocation of the 85 kDa PLA2 in response to cell stimulation by immunological methods that verify an increase in the amount of PLA2 protein on cell membrane. In studies using monoc3^es or macrophages, agents the activate PKC have been shown to induce a decrease in cytosolic PLA2 activity accompanied by an increase in PLA2 activity in the 100,000 g particulate fraction (Schonhardt and Ferber, 1987; Rehfeldt et al., 1991; Shibata et al., 1992). The PLA2 assayed in these studies had characteristics of the 85 kDa enzyme. However, studies investigating translocation by measuring PLA2 activity are potentially complicated since the stimuli can induce an increase in the specific activity of the enzyme. In addition, to study the stimulus-induced translocation process by probing subcellular fractions it is important to control for the levels of calcium present during homogenization since the PLA2 can be induced to bind to
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membrane at very low levels of calcium (>200 nM), which is in the range of calcium that potentially contaminates unchelated buffers. Conversly, the PLA2, once induced to associate by cell stimulation, can be dissociated from the membrane if the cells are homogenized in the presence of excess calcium chelators (Leslie, unpublished observation). However, it is possible that binding of the PLA2 to membrane in some cells in response to certain stimuli may result in a stable association that is calcium independent as described for PKC. Recent results with the 85 kDa PLA2, PKC, PLCy and the p65 synaptic vesiclespecific protein, showing that they all bind to Triton-insoluble cytoskeleton elements, may begin to shed light on the mechanisms involved in their association with the cellular particulatefi-action(Wolf and Sahyoun, 1986; Jaken et al, 1989; Hyatt et al, 1990; Mochly-Rosen et al, 1990, 1991; Grondin et al, 1991; Kiley et al., 1991; McBride et al, 1991; Vaziri and Downes, 1992; Akiba et al., 1993). The first report suggesting the involvement of the cytoskeleton in PLA2 activation showed that cytochalasin B, an inhibitor of actin polymerization, could inhibit collagen-induced arachidonic acid liberation (Nakano et al., 1989). Recently, thrombin stimulation of rabbit platelets has been shown to result in about a twofold increase in PLA2 activity in the Triton-insoluble residue (Akiba et al., 1993). This PLA2 had characteristics of the 85 kDa PLA2 including immunoreactivity, preference for phospholipid substrates containing arachidonic acid, and a calcium requirement characteristic of this enzyme. Interestingly, to obtain the Triton-insoluble pellet containing the PLA2, the cells were homogenized in the presence of excess calcium chelators suggesting the association of the PLA2 to this material was not simply a calcium-dependent binding. It was suggested that the cytosolic PLA2 may be induced to associate with the cytoskeleton as a result of thrombin stimulation. However, it is also possible that posttranslational modification of the PLA2, such as phosphorylation, resulted in an increase in activity of the PLA2 that had been associated with this fraction in unstimulated cells. Experiments to demonstrate that the increase in PLA2 activity represented an increase in PLA2 protein were not presented. However, the results do suggest an interaction between the 85 kDa PLA2 and cytoskeletal elements. Although the involvement of the CaLB domain in this interaction was not addressed, recent data has provided evidence that the homologous C2 domain in PKC and p65 mediates binding of these proteins to cytoskeletal elements. Several studies have now demonstrated that in stimulated cells, PKC isoforms are targeted to specific intracellular sites including cytoskeletal elements (Wolf and Sahyoun, 1986; Jaken et al., 1989; Hyatt et al., 1990; Mochly-Rosen et al., 1990, 1991; Kiley et al, 1991). Specific intracellular proteins in the Triton insoluble fraction have been identified that bind PKC and have been called receptors for activated C-kinase (RACKs) (Mochly-Rosen et al., 1991). Direct protein-protein interaction is involved in PKC binding to RACKs and the binding requires phosphatidylserine and diacylglycerol. Recent experiments were conducted to identify the region on PKC and p65 that may be involved in binding the RACKs (Mochly-Rosen et al, 1992). A fragment of p65 synaptic vesicle protein
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containing the region homologous to the PKC C2 domain was found to bind to RACKS and was dependent on phospholipid and calcium. However, a smaller fragment of p65 that still included the C2 domains was found to bind RACKs in a calcium and phospholipid independent manner suggesting that amino acids outside the C2 domain are important for calcium and phospholipid dependent binding. Evidence was presented suggesting that the p65 fragment and PKC bind to the same site on RACKs, and that binding occurs through the C2 domain, which is the only homologous region shared by p65 and PKC. It has previously been suggested that the CaLB region on the 85 kDa PLA2 that shares homology to the C2 region of PKC and p65 mediates binding of the PLA2 to membrane lipid (Clark et al, 1991). However, in light of this recent work with PKC and p65, there may be an interaction of the PLA2 with specific proteins in the particulate fraction. We do know that calcium-induced PL A2 association with the particulate fraction results in hydrolysis of membrane phospholipid verifying association with phospholipid, however, it is possible that a binding protein is also involved. It should also be noted that other enzymes in the eicosanoid biosynthetic pathway—^5 and 12 lipoxygenases—^have been shown to exhibit calcium-induced membrane association (Rouzer and Kargman, 1988; Wong et al., 1988; Baba et al., 1989; Kargman et al., 1991). Five-lipoxygenase has also been demonstrated to translocate to membranes in cells stimulated with physiological agents (Kargman et al., 1991; Wong et al., 1992; Malaviya et al., 1993). The primary structure of 5-lipoxygenase does not contain a region homologous to the C2 domain (Yamamoto, 1992). The translocation of 5-lipoxygenase can be inhibited by specific indole and quinoline leukotriene synthesis inhibitors which have been shown to bind to an 18 kDa membrane protein called 5-lipoxygenase activating protein (Miller et al., 1990; Rouzer et al., 1990; Kargman et al, 1991). Transfection studies have demonstrated that both 5-lipoxygenase and the activating protein are required for leukotriene biosynthesis (Dixon et al., 1990). The activating protein is thought to mediate translocation of 5-lipoxygenase to the membrane, however, the mechanism is not understood. Direct binding of 5-lipoxygenase to the activating protein has not been demonstrated. Since PLA2 activation is required to provide the substrate, arachidonic acid, for the 5-lipoxygenase, understanding the relationship and subcellular distribution of specific PLA2 enzymes and the 5-lipoxygenase is an intriguing problem yet to be clarified. D.
Regulation of PLA2 by ATP
A unique property of the 40 kDa, calcium-independent PLA2 from myocardium is its ability to bind to ATP affinity columns, which suggested possible regulation of the PLA2 by this ligand. Numerous lines of investigation have provided evidence that ATP regulates both rabbit and human calcium-independent PLA2 which exists as a high molecular weight, cytosolic complex that is composed of the 40 kDa catalytic subunit and a high molecular weight regulatory protein (Hazen and Gross,
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1990; Hazen et al., 1991). ATP was found to protect the complex in the cytosol from thermal denaturation and to enhance PLA2 activity, but had no effect on the purified 40 kDa catalytic subunit. When the purified 40 kDa catalytic subunit was reconstituted with cytosols depleted of PLA2, ATP was found to stimulate PLA2 activity. Nonhydrolyzable analogues of ATP could exert effects similar to ATP suggesting that a phosphorylation event by cytosolic kinases was not involved. The activation also did not appear to be G-protein mediated since it involved cytosolic components, was independent of calcium, and ATP was more effective than GTP. Analysis of the nucleotides present in myorcardial tissue demonstrated that ATP was the only nucleotide present at a concentration sufficient to activate the PLA2. Based on these observations, it was suggested that changes that occur in ATP levels during ischemia reperfiision may play a role in regulating PLA2 during these processes. In a rabbit model of myocardial ischemia, it has been found that activation of a plasmalogen-selective, calcium-independent PLA2 is an early event (< 5 minutes) that precedes the cellular damage (Ford et al., 1991). Interestingly, the large increase (10-fold) in PLA2* activity that occurred during the first five minutes of ischemia occurred in the membrane, whereas cytosolic activity remained unchanged (Ford et al., 1991; Hazen et al., 1991). During reperfiision after ischemia the activity of the PLA2 rapidly returned to baseline levels. The increase in PLA2 activity was not a result of increased protein synthesis since it occurred in hearts pretreated with cycloheximide and actinomycin D. Characterizafion of the membrane associated PLA2 revealed that it had properties of an integral membrane protein with a pH optimum of 8.5, compared to 6.5 for the cytosolic calcium-independent PLA2. The membrane PLA2 was calcium-independent and was selective for plasmalogen substrates. The large increase in membrane associated PLA2 activity was not accompanied by a concomitant decrease in the cytosolic PLA2 suggesting that the cytosolic form did not translocate to the membrane. Experiments similar to those used to show translocation of the 85 kDa PLA2 did not demonstrate any translocation of the calcium-independent PLA2. Proposed mechanisms for activation of the membrane associated cardiac PLA2 include: (a) activation of a latent membrane associated enzyme, (b) translocation and activation of a latent cytosolic enzyme, or (c) translocation of a cytosolic regulatory protein. PLA2 activity in heart tissue of humans suffering from end-stage ischemic heart disease has recently been characterized (Hazen and Gross, 1992). Most of the PLA2 activity (98%) in the human myocardium was found to be calcium-independent and plasmalogen selective. However, unlike normal myocardium from other mammalian species, most of the activity was membrane associated (65%). With the exception of pH optima, which was 8.5 for the microsomal enzyme and 7.0 for the cytosolic enzyme, both enzymes were similar and shown to be inhibited by covalent modification of an essential thiol group and sensitive to mechanism-based inhibition with bromoenol lactone. The effect of ATP on the membrane associated PLA2 activity was not determined. It was suggested that the large amount of PLA2 in the membrane fraction may be a characteristic of diseased tissue.
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E. Regulation of PLA2 by G-Proteins
A role for G-proteins in the regulation of intracellular phospholipases A2, C, and D has been well documented as discussed in a review (Cockcroft, 1992). The regulation of PLA2 activation by G-proteins can be indirect as a result of G-protein linked activation of PLC, which results in PKC activation and Ca^"^ mobilization, both of which can affect PLA2 activity. However, many studies primarily using permeabilized cells have demonstrated that GTPyS stimulation of arachidonic acid release can be dissociated from PLC activation suggesting a direct link between G-proteins and PLA2 (Burch et al., 1986; Burch and Axelrod, 1987; Nakashima et al., 1987; Kaya et al, 1989; Teitelbaum, 1990; Portilla et al, 1992; Stasi et al, 1992). Studies using neutrophil or platelet membrane preparations have also demonstrated activation of PLA2 activity by GTPyS suggesting a direct activation (Silk et al., 1989; Ando et al., 1992). Using a different approach in which dominant negative Gaj subunit constructs were transfected into CHO cells, it was shown that ATP or thrombin stimulation of arachidonic acid release was inhibited in the transfected cells, but activation of PLC was unaffected (Gupta et al., 1990). It has also been shown that overexpression of a GTPase deficient Gai2 subunit in CHO cells attenuates thrombin-induced arachidonic acid release (Lowndes et al., 1993). These studies, as well as other reports documenting that stimulated arachidonic acid release is inhibited by pertussis toxin, implicate the pertussis sensitive Gj or G^ classes of G-proteins (Bokoch and Gilman, 1984; Okajima and Ui, 1984; Murayama and Ui, 1985; Nakamura and Ui, 1985; Jelsema, 1987; Murayama et al., 1990). In addition to a potential role of the a subunit, modulation of PLA2 activation by PY G-protein subunits has been suggested in a study showing they stimulate PLA2 activity in rod outer segments that have been depleted of transducin, the major G-protein in rod outer segments (Jelsema and Axelrod, 1987). There is now substantial evidence in several cell systems that activation of PLA2 and PLC can be mediated by distinct G-proteins (Burch et al., 1986; Portilla et al, 1992). In certain cell types, primarily hemopoietic cells, PLC activation has also been shown to be regulated by a pertussis toxin sensitive G-protein and (iy subunits appear to be the moieties involved (Martin, 1989; De Vivo and Gershengom, 1990; Fain, 1990; Camps et al., 1992). PLCP2 and PLCI33, and to a lesser extent PLCp^ and PLC6j, have been shown to be activated by Py subunits in a membrane-free reconstitution system (Park et al., 1993). In other cell types PLC activation through pertussis toxin insensitive G-proteins of the Gq class has been documented and the a subunit has been shown to be the stimulatory moiety (Blank et al., 1991; Smrcka et al., 1991; Taylor et al., 1991; Waldo et al., 1991; Wu et al., 1992). Although the studies discussed above clearly establish G-protein linked activation of PLA2, the identity of the specific PLA2 enzyme(s) targeted were not determined. The 85 kDa cytosolic PLA2 is a likely candidate, however, this can not be assumed. Reconstitution experiments have demonstrated that exogenously supplied group II type PLA2 from Naja naja venom or group I type PLA2 from
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porcine pancreas, to permeabilized rat basophilic leukemia cells, can be synergistically activated by a combination of GTPyS and IgE at levels of free calcium between 10~^ and 10"^ M (Narasimhan et al., 1990). Prior to permeabilization and addition of the exogenous PLA2, the cells had been treated with p-bromoacetophenone to inhibit cellular PLA2. These are intriguing results and it was proposed that the G-protein, either directly or indirectly, may act to increase the affinity of these low molecular weight PLA2 enzymes for calcium. A recent study has provided evidence suggesting that the 85 kDa PLA2 can be activated by GTPyS (Xing and Mattera, 1992). Using electropermeabilized HL60 cells it was shown that Mg^"^ATP was required for GTPyS stimulation of arachidonic acid release which occurred maximally at 1 |LIM Ca^"^. Several lines of evidence showed that the requirement for ATP was not for interaction with purinergic receptors and it was postulated that ATP was necessary for phosphorylation events that were required for GTPyS/Ca^"^ stimulation of arachidonic acid release. Consistent with this hypothesis was the observation that staurosporine, a kinase inhibitor, completely suppressed GTPyS/Mg^"^ATP stimulation of arachidonic acid release in the permeabilized cells. Short term preincubation of the cells with PMA slightly stimulated the GTPyS/Mg^"^ATP stimulation of arachidonic acid release that was partially inhibited by staurosporine. In cell free homogenates of the HL60 cells, Ca^"^ itself could stimulate arachidonic acid release and the response could be increased twofold with GTPyS, but addition of ATP had no effect in contrast to the electropermeabilized cells. The stimulation of arachidonic acid release from the membrane fraction by Ca^^ and GTPyS required the presence of cytosol which was found to contain the 85 kDa PLA2. Similarly, a requirement for cytosol and membrane for GTPyS activation of PLD and PLC has been demonstrated and suggests that cytosolic forms of all these phospholipases can bind to membrane and be activated in a G-protein dependent manner (Olson et al., 1991; Thomas et al., 1991). In the experiments using HL60 homogenates, pretreatment of the cytosol and membrane fraction with antibody to the 85 kDa PLA2 attenuated PLA2 activation by Ca^"" and GTPyS. The lack of ATP requirement in the homogenates, compared to permeabilized cells, was postulated to mean that normally the PLA2 is under inhibitory constraint that can be relieved by phosphorylation, as reflected by the ATP requirement in permeabilized cells, or relieved after N2 cavitation. The 85 kDa PLA2 was verified to be phosphorylated in the electropermeabilized cells and phosphorylation could be enhanced by either Ca^"^ or PMA. This study provides evidence that the 85 kDa PLA2 can be coupled to G-proteins. Although a direct link between G-proteins and the 85 kDa PLA2 is postulated in this study, the evidence is circumstantial. Now that purified forms of PLA2 enzymes are available, reconstitution experiments can be carried out using specific G-protein subunits to better define these interactions and the components required. Now that various forms of PLA2 have been purified, the tools will be available to identify the PLA2 enzymes involved in mediating phospholipid hydrolysis under specific conditions. Based on current information there are several structurally
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diverse PLA2 enzymes that can potentially mediate arachidonic acid release. Given the unique properties of these PLA2 enzymes, they appear to be differentially regulated thus providing for alternative pathways for phospholipid breakdown and generation of lipid mediators. Differences in subcellular distribution and substrate specificities of the various PLA2 enzymes may also allow for the hydrolysis of specific phospholipid pools. The generation of products of PLA2 action at specific subcellular sites may be important in determining their subsequent metabolic fate with respect to conversion to specific lipid mediators, and hence the biological responses that are elicited.
ACKNOWLEDGMENTS This work was supported by National Institutes of Health grant HL34303. The help of Brenda Sebem is greatly appreciated.
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THE FATE OF PLATELET-ACTIVATING FACTOR: PAF ACETYLHYDROLASES FROM PLASMA AND TISSUES
Tada-atsu Imaizumi, Diana M. Stafforini, Yoshiji Yamada, Guy A. Zimmerman, Thomas M. Mclntyre, and Stephen M. Prescott
I. II. III. IV.
ABSTRACT INTRODUCTION PATHOLOGICAL AND PHYSIOLOGICAL ACTIONS OF PAF PAF ACTS BY BINDING TO A RECEPTOR SYNTHESIS OF PAF A. Enzymatic Mechanisms B. Formation of Oxidatively Fragmented Phospholipids That Are Structurally Similar to PAF
Advances in Lipobiology Volume 1, pages 141-162. Copyright © 1996 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-635-5 141
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V. DEGRADATION OF PAF AND OXIDIZED PHOSPHOLIPIDS BYTHEPAFACETYLHYDROLASE A. Biochemical Characteristics of the PAF Acetylhydrolase(s) B. Properties ofthe Plasma Form of the PAF Acetylhydrolase C. The PAF Acetylhydrolase From Human Plasma Prevents Oxidative Modification of LDL D. Population Studies of PAF Acetylhydrolase Activity in the Plasma of Normal Human Subjects and Animals E. Genetics ofthe Plasma PAF Acetylhydrolase F. The Plasma PAF Acetylhydrolase in Disease G. Intracellular PAF Acetylhydrolase ACKNOWLEDGMENTS REFERENCES
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ABSTRACT Platelet-activating factor (PAF) likely mediates a variety of physiological and pathological events. There is abundant evidence that the concentration of PAF in blood or tissues is influenced by its rate of degradation. Two forms ofthe degradative enzyme, PAF acetylhydrolase, have been purified and studied in detail. Changes in the activity of the plasma enzyme have been observed in human diseases, physiological responses, and in animal models, suggesting that it may be a key step. The plasma PAF acetylhydrolase has several interesting properties including marked substrate specificity and association with lipoproteins. Studies that define the molecular basis for these properties and elucidate the role ofthe enzyme in physiological processes should be forthcoming, and will provide insight into the function of PAR
I. INTRODUCTION Platelet-activating factor (PAF, l-0-alkyl-2-acetyl-5«-glycero-3-phosphocholine) is a potent phospholipid autacoid that has diverse actions. The structure of this compound v^as elucidated by two different groups: one seeking to characterize a factor in the blood of rabbits undergoing anaphylactic shock, and the other trying to identify an endogenous compound that lowered blood pressure (reviewed in Hanahan, 1986; Snyder, 1987; Prescott et al, 1990; Venable et al., 1993a). The trivial name, PAF, is incomplete as it refers to only the earliest recognized effect. However, this name has found general acceptance while others have not. PAF acts by binding to a specific receptor, which has recently been characterized in detail following the cloning of its cDNA, and its expression in heterologous cells. The synthesis of PAF can occur through one of two described synthetic pathways, and the synthetic process is tightly regulated. PAF is degraded by PAF acetylhydrolase, which catalyzes the hydrolysis ofthe esterified acetate at the sn-2 position.
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In this chapter we will review how PAF is made and has its effects, and will concentrate on the PAF acetylhydrolase, which is an unusual phospholipase that catalyzes the hydrolysis of PAF and its analogs. In particular, we will analyze the evidence that this enzyme may have a major role in limiting the actions of PAF and, conversely, that a decrease in its activity may unmask PAF bioactivity.
II. PATHOLOGICAL AND PHYSIOLOGICAL ACTIONS OF PAF PAF has multiple physiological and pathological actions. It probably is a mediator of physiological inflammation, may facilitate normal hemostasis, and likely contributes to several stages of events in reproduction. Moreover, there is evidence that it is one of the pathological mediators in asthma and other allergic responses, vascular damage including ischemia and reperfusion injury, various forms of shock (particularly endotoxic shock), and other syndromes in which there is a marked inflammatory response (e.g., the adult respiratory distress syndrome and inflammatory bowel disease). The evidence that PAF participates both in normal responses and diseases was reviewed recently (Yue et al., 1991; Zimmerman et al, 1992) and will only be summarized here. The in vitro effects of PAF include activation of platelets and leukocytes including neutrophils, monocytes, and macrophages, which support a role in thrombosis and inflammation. Many other cells and tissue respond as well—^there is glycogenolysis in perfused livers, growth in smooth muscle cells, and activation of neural cells. Intravenous infusions of PAF into animals result in a marked increase in vascular permeability, adhesion of leukocytes to endothelium, decreased cardiac output, hypotension, shock, and death. Selective administration in vivo or to isolated tissues has been shown to cause contraction of uterine muscle, bronchoconstriction, and ulcers in the gastrointestinal tract. PAF has been found in the plasma of patients with sepsis, and in blood and tissues of animal models of disease (Yue et al., 1991; Zimmerman et al., 1992). Finally, one of the main lines of evidence supporting a role for PAF in pathological events is that pharmacological agents that antagonize its binding to its receptor have been found to attenuate certain diseases where PAF is the suspected mediator (Yue et al., 1991; Zimmerman et al., 1992). The effects of PAF on cells and tissues are concentration-dependent, and the earliest response (threshold) is usually between 10~^^ to 10"^ M, although effects at lower concentrations have been described. The concentration of PAF in blood or tissues depends on the difference between the rates of synthesis and degradation. Most of the cells that are capable of synthesizing PAF do not do so under basal conditions, but only when they are activated. One mechanism for pathological effects, then, would be the inappropriate activation of the synthetic pathways. Conversely, PAF is rapidly degraded by the actions of the PAF acetylhydrolase, and a decreased activity of this enzyme could allow the accumulation of concentrations of PAF that would provoke a pathological response.
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A variety of cells (e.g., neutrophils, macrophages, and endothelial cells) synthesize PAF when stimulated and which of them is the source of PAF observed in sepsis and other disease circumstances is not known. We have shown that PAF is synthesized by activated endothelial cells but is not secreted (Prescott et al., 1984; Mclntyre et al, 1985). However, the PAF made by monocytes and macrophages is secreted, as is a portion of the PAF synthesized by neutrophils (Elstad et al., 1988). Although endothelial cells do not secrete PAF, it is expressed on the surface of the cells where it serves as a component of the signal for the binding of neutrophils to endothelial cells (Zimmerman etal., 1990;Lorantetal., 1991,1993). This probably is a homeostatic response since the adhesion of leukocytes to the endothelium is the first step in physiological inflammation. However, inappropriate or excessive expression of the adhesion signal followed by the attraction and activation of large numbers of leukocytes could result in further vascular damage due to the secretion of proteases and oxygen radicals by the leukocytes.
III. PAF ACTS BY BINDING TO A RECEPTOR PAF exerts its effects through a receptor that has been well characterized pharmacologically, and many potent antagonists have been developed. Most of those described are competitive antagonists although they usually share no structural homology with PAF. The competitive nature of their actions is potentially a drawback since PAF may often be expressed at a surface and/or in a restricted environment. If so, the effective concentration could be extremely high and overcome the blockade by a competitive antagonist—even a potent one. Several groups have isolated cDNAs encoding the receptor (Gibson et al., 1991; Honda et al., 1991; Ye et al, 1991; Kunz et al., 1992; Seyfried et al., 1992), and have shown that it is a member of the family of receptors linked to G-proteins, as suggested previously by pharmacological and biochemical studies. The receptor is linked to turnover of phosphatidylinositol, changes in intracellular calcium, activation of protein kinase C, and synthesis of eicosanoids (Shukla, 1992). The known receptor is clearly linked to a G-protein, although which one is not yet known. Additionally, there is phosphorylation on tyrosine residues in unknown proteins in response to PAF (Dhar et al., 1990; Sha'afi et al, 1991; Chao et al., 1992; Tripathi et al., 1992). Although there are pharmacological data to support the existence of two receptors (Hwang, 1988), the molecular studies so far have revealed only one (see cloning references above). PAF receptors in intracellular membranes have been described, but it is not known whether these are spare receptors, or surface receptors in a stage of cycling, or whether they have a specific intracellular function. Stewart et al. concluded that PAF fiinctions as an intracellular messenger since they found that antagonists of the receptor blocked the synthesis of prostaglandins in response to agonists other than PAF (Stewart et al., 1989, 1990).
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IV. SYNTHESIS OF PAF A.
Enzymatic Mechanisms
The actions of PAF are dictated by its concentration in blood and tissues. In general, the concentration is very low—^usually unmeasurable—^and the reason for this is thought to be that cells do not make PAF, or at least not much, constitutively. PAF can be synthesized by two pathways (Figure 1). The first pathway, termed remodeling, begins with the hydrolysis of arachidonate from the sn-2 position of a membrane phospholipid. This reaction probably is catalyzed by an arachidonatespecific phospholipase A2 (PLA2) since several groups have found that PAF synthesis and arachidonic acid release are closely coupled. Wykle and colleagues proposed that the PLA2 hydrolyzes arachidonate from 1-(9-alkyl-2-arachidonoyl5«-glycero-3-phosphocholine generating 1 -6)-alkyl-2-lyso-5A2-glycero-3-phosphocholine (lyso-PAF) and free arachidonic acid (Chilton et al., 1984). An alternative mechanism for the generation of lyso-PAF has come from recent studies in which it appears that the first step is hydrolysis of arachidonate-containing plasmalogen phosphatidylethanolamine (PE) by a PLA2 (Nieto et al., 1991; Snyder et al., 1991; Venable et al., 1991). The lyso-PE product stimulates the transfer of arachidonate from the PAF precursor to the lyso-PE, simultaneously forming lyso-PAF. The latter reaction is catalyzed by a coenzyme A-independent transacylase, the activity of which does not seem to change upon cell stimulation (Venable et al, 1993b). Once lyso-PAF has been produced via one or the other mechanism it then is acetylated in a reaction catalyzed by acetyl coenzyme A:lyso-PAF acetyltransferase, using acetyl coenzyme A as a donor, to form PAF. This enzyme is activated, probably by phosphorylation, when the cell is stimulated (Alonso et al., 1982; Lee et al., 1982; Nieto et al., 1988; Holland et al., 1992). The second route to PAF synthesis, the de novo pathway, begins with l-0-alkyl-5«-glycero-3-phosphate followed by incorporation of an acetate, and then by removal of the phosphate and its replacement with a phosphorylcholine. The enzymes in this pathway appear to be constitutively active, and it has been proposed that there is continuous production of a small amount of PAF via this route (Lee et al., 1986; Blank et al., 1988). B. Formation of Oxidatively Fragmented Phospholipids That Are Structurally Similar to PAF
The polyunsaturated fatty acids in membrane phospholipids are susceptible to free-radical oxidation, which has been shown to occur in some pathologic conditions. These include reperfusion following ischemia, the adult respiratory distress syndrome, and chronic inflammation. We found that oxidation of synthetic phospholipids could fragment the unsaturated fatty acid while it was still esterified. This results in structural analogs of PAF (Figure 2), and we and others have shown that they can act through its receptor to reproduce the effects of PAF (Smiley et al, 1991). Tokumura and co-workers have identified such compounds in extracts of
146
TADA-ATSU IMAIZUMI ETAL. Remodeling
De Novo
H2C-O-CH2R
H2C-O-CH2R
I
I
20:4-CH
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HjC-O-CHsCHR
I OH-CH
-..,^
H2C.(P)-eth \
I
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H2C-O-CH2R O
I
H3C-C-O-CH
HO-CH H2C-(p)-chollne
HaC-®
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Phosphohydrolase
H2C-O-CH2R O H3C-C-O-CH H2C-(g)-chollne
Cholinephosphotransferase
H2C-O-CH2R H3C-C-O-CH H2C-OH
Figure 1. Synthesis of PAF can occur via two pathways. Remodeling pathway (on left). Most PAF is thought to be made by this route, particularly in cells participating in the inflammatory response. This pathway begins with a PLA2 reaction, which may act on 1-0-aIkyl-2-arachidonoyl glycerophosphocholine (right side of left panel) to yield lyso-PAF and free arachidonic acid. The lyso-PAF is converted to PAF by transfer of acetate acetyl coenzyme A by a specific acetyltransferase. Alternatively, the PLA2 may act on plasmalogen phosphatidylethanolamine, and the lyso-phosphatidylethanolamine then serves as an acceptor for arachidonate in a transacylation reaction from the PAF precursor, which yields lyso-PAF (left hand side of left panel). This mechanism fits with many observations on the flux of arachidonic acid. Irrespective of the subsequent steps, the PLA2 step is absolutely required for initiation of PAF synthesis by this pathway—a situation we have termed conditional. Once the lyso-PAF is generated the acetyltransferase reaction becomes limiting, and we have termed this a modulating step. De novo pathway (right side). In this pathway 1 -O-alkyl-sn-glycero3-phosphate is acetylated by a different acetyltransferase, and then l-O-alkyl-2-acetyl-SA7-glycero-3-phosphate phosphohydrolase acts to yield 1-0-alkyl-2-acetyl-sn-glycerol. This Is a substrate that can be converted to PAF by a dithiothreitol-insensitive CDP-cholinephosphotransferase. This pathway seems to act constitutively, being regulated only by the availability of substrate, and may continuously produce a small amount of PAF for some physiological role(s). (Reprinted by permission from J. Lipid Res. 14, 691-702, 1993.)
Metabolism of Platelet-Activating
i
L i-
147
Factor
PC Free Radical ► Oxidation
rr =0
^ 0
1 O-PC
+Fragments
0 Synthetic
6-PC
>=0
Enzymes
Figure 2, Generation of PAF-like oxidized phospholipids contrasted with the synthesis of PAR Phospholipids with bioactivity like that of PAF can result from the oxidation of polyunsaturated fatty acids at the sn-2 position of choline phosphoglycerides (upper panel). Fragmentation of the acyl chain yields compounds that are sufficiently similar in structure to PAF to allow them to bind to the PAF receptor. This response has been observed in model phospholipids, cultured cells, and brain tissue, and may represent an important mechanism by which PAF-like bioactivity can be generated in an unregulated fashion.
brain (Tokumura et al., 1989), and have carried out detailed structural studies (Tanaka et al., 1993). We found that similar compounds are produced when endothelial cells are exposed to oxidants, and that they are released as vesicles (or blebs) from the plasma membrane (Patel et al., 1992). We have show^n that these bioactive lipids are generated v^hen lov^ density lipoprotein particles are exposed to oxidants, and that one of their effects is to stimulate the growth of vascular muscle cells (Heery et al., 1995). This may be particularly relevant since it is thought that oxidation of the lipids in LDL is a crucial step in atherosclerosis (Steinberg et al., 1989a; Witztum and Steinberg, 1991). Since these compounds are made by a free-radical reaction, rather than by regulated enzymatic steps, they could be
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generated in much larger amounts than PAF and at inappropriate times and places. If they escape the usual regulatory controls that govern PAF synthesis and are produced in excess, then degradation via the PAF acetylhydrolase might be crucial in protecting against pathological effects.
V. DEGRADATION OF PAF AND OXIDIZED PHOSPHOLIPIDS BY THE PAF ACETYLHYDROLASE Regardless of the pathway by which PAF is synthesized or the mechanism by which analogs such as the oxidized phospholipids are generated, both are degraded to inactive products by a phospholipase that is specific for PAF and closely related lipids, PAF acetylhydrolase (Figure 3). There are at least two forms of this enzyme: a secreted form present in mammalian plasma (Wardlow et al., 1986; Stafforini et al., 1987a,b), and an intracellular form that is found in various blood cells and tissues (Blank et al, 1981; Stafforini et al, 1993). Although they catalyze the same reaction and have the same substrate specificity, the plasma and intracellular forms of the PAF acetylhydrolase are different proteins as judged by a variety of criteria (see below). The degradation of PAF and related phospholipids by these isoenzymes may be a crucial step that regulates inflammation. A.
Biochemical Characteristics of the PAF Acietylhydrolase(s)
The defining characteristics of this enzyme's activity are that it does not require calcium and is specific for short acyl groups at the sn-2 position of the substrate phospholipid (Blank et al., 1981; Stafforini et al, 1987b). There is no measurable activity when the acyl chain is longer than six carbons. For the plasma enzyme, this trait is essential if the enzyme is to circulate in an active form—^if typical phospholipids were substrates the enzyme would continuously hydrolyze the phospholipids of lipoproteins and cell membranes. The 1 -0-acyl and 3-phosphoethanolamine analogs of PAF, and phospholipids containing oxidatively-fragmented residues at sn-2 also are substrates (Stafforini et al., 1987b; Stremler et al., 1991). It is possible that the enzymatic activity evolved to catalyze the hydrolysis of this last group of compounds which, in addition to receptor-mediated actions, might have a variety of toxic effects on cells. Nonetheless, the recent findings that phospholipids other than PAF are substrates makes the traditional name incorrect, but we have retained it here for consistency with previous work. The PAF acetylhydrolase is a PLA2 with specificity for short acyl chains. The activities in plasma and in cells have identical substrate specificity, but studies of the molecular weight, chemical inhibition, protease inactivation, and antibody recognition have shown them to be distinct proteins (Stafforini et al., 1991, 1993). The plasma protein is resistant to proteolysis, and is unaffected by reagents that derivatize sulfhydryl or histidyl residues, and sodium fluoride. In contrast, the enzyme in spleen, liver and leukocytes all are inhibited, at least partially, by these treatments. Also, the enzyme
Metabolism of Platelet-Activating
Factor
149
A. r-AlkyI
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PAF Acetyihydrolase
-O-CO-CH3
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Oxidized Phospholipids
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0-C0-[CHj3^CH0
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Lyso Phospholipids -1Oxldized Fatty Acids
Figure 3. Degradation of PAF and oxidized phospholipids. Panel A: The degradation of PAF occurs by hydrolysis of the acetyl residue in a reaction catalyzed by a specific phospholipase (PAF acetyihydrolase) which does not require calcium and is specific for short residues at sn-2. In particular, phospholipids with the usual fatty acids are not utilized as substrates. This enzyme also catalyzes the hydrolysis of phospholipids with oxidatively fragmented fatty acids (panel B), an action that may protect against oxidation of low density lipoprotein. Related enzymes with the same substrate specificity are present in tissues.
from erythrocytes, which is inhibited by histidine and cysteine modification, is sensitive to proteolysis and NaF (Stafforini et al, 1993). There may be two or more intracellular enzymes; for example, the neutrophil and erythrocyte enzymes migrated at the same rate during electrophoresis in polyacrylamide gels, while the liver enzyme displayed a higher mass/charge ratio (Stafforini et al., 1991). B.
Properties of the Plasma Form of the PAF Acetyihydrolase
The plasma PAF acetyihydrolase has an apparent mass of 43,500 Da and is tightly associated with low density lipoprotein (LDL) and high density lipoprotein (HDL) (Stafforini et al., 1987a,b). Under optimal conditions the enzyme exhibits identical catalytic properties irrespective of which particle it is associated with. However, in plasma that contains concentrations of PAF described in vivo (10"^ M or lower), the HDL-associated enzyme does not degrade PAF at appreciable rates, while the LDL-associated enzyme does (Stafforini et al., 1989). It is not yet clear why the activity differs in the two types of particles when substrate is limiting, but it may be that the PAF partitions into the particles at a different rate, and that this step limits the access of the enzyme to the substrate. The plasma PAF acetyihydrolase is synthesized and secreted by both macrophages (Stafforini et al., 1990; Narahara et
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al., 1993) and hepatocytes (Carter et al., 1988; Satoh et al, 1991b). In each case, it appears that secretion of the enzyme occurs independently of the secretion of lipoprotein particles, and that the acetylhydrolase then associates with either nascent lipoproteins secreted by the cells (in the absence of serum) or with mature lipoproteins if serum is included in the culture medium. Macrophages also may play a role in the local regulation of PAF levels since the precursor monocytes do not produce the enzyme. However, upon differentiation to macrophages they begin to produce and secrete PAF acetylhydrolase (Elstad et al., 1989). Secretion of the PAF acetylhydrolase by hepatocytes is suppressed by estrogen (Carter et al., 1988; Satoh et al., 1993), which may account for the dramatic fall in the plasma activity that has been observed late in gestation, and which has been proposed to be important in the initiation of labor (see chapter 9, "The Role of PAF in Reproductive Biology"). C. The PAF Acetylhydrolase From Human Plasma Prevents Oxidative Modification of LDL
Modification of LDL is an early event in atherogenesis, and the modified LDL particles are thought to be produced from native particles by oxidation that is somehow carried out by cells in the arterial wall, including endothelial cells, monocyte and macrophages, and smooth muscle cells (Steinberg et al, 1989; Witztum and Steinberg, 1991). Blood monocytes invade the vascular wall and become macrophages, which take up modified LDL through their scavenger receptor to become foam cells (Brown and Goldstein, 1983; Witztum and Steinberg, 1991). This results in the accumulation of cells loaded with cholesterol esters, which is recognized histologically as a fatty streak. The molecular events that result in oxidation of LDL by vascular cells have not been determined. However, a number of biochemical changes have been identified in LDL particles that have been oxidized. One of the most important is the modification of apolipoprotein B-lOO, since this is what results in recognition by the scavenger receptor. The modified particles have an increased electrophoretic mobility due to a change in the charge of the protein (Mahley et al., 1979; Fogelman et al., 1980; Brown and Goldstein, 1983; Gonen et al, 1983), and there is peroxidation of their lipids (Parthasarathy et al, 1989; Steinberg et al., 1989; Lenz et al., 1990; McNally et al., 1990; Rankin et al., 1991). A central role for the latter change is suggested by experiments in which oxidized lipids were added in vitro and shown to directly derivatize apolipoprotein B-100 (Steinbrecher, 1987). Also, antioxidants such as butylated hydroxytoluene and probucol slow atherogenesis in animal models (Parthasarathy et al., 1986; Kita et al, 1987; Carew et al., 1987; Bjorkhem et al., 1991; Reaven et al., 1992). Thus, generation of lipid peroxides may be important in the modification of LDL and the subsequent development of atherosclerosis.
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We showed that the PAF acetylhydrolase catalyzes the hydrolysis of oxidativelyfragmented phospholipids (Stremler et al., 1991). During modification of LDL, polyunsaturated fatty acids in phospholipids (predominantly choline phosphoglycerides) are oxidized, and then are hydrolyzed to yield lysophospholipid and the oxidized fatty acid and/or their fragmentation products (Steinbrecher et al., 1984). The fragments of lipids that have undergone peroxidation are thought to covalently bind to apolipoprotein B-lOO, which causes increased electrophoretic mobility and changes receptor recognition (Steinbrecher, 1987). In this model, the hydrolysis of oxidized phospholipid is required for LDL to be modified. However, we found that hydrolysis of the oxidized phospholipids resulted in the release of water-soluble products, presumably reactive fragments, and that the net effect was to protect apolipoprotein B-lOO from modification (Stafforini et al., 1992). Thus, catalysis of the degradation of oxidatively-fragmented phospholipids by the PAF acetylhydrolase prevents modification of LDL. If this also occurs in vivo, it would indicate that this enzyme is a crucial defense against atherosclerosis. D.
Population Studies of PAF Acetylhydrolase Activity in the Plasma of Normal Human Subjects and Animals
The PAF acetylhydrolase activity in serum is low in human newborns, and it increases linearly with respect to the natural logarithm of the age from birth to 6 weeks (Caplan et al., 1990). In adults, plasma PAF acetylhydrolase activity increases gradually with advancing age (Satoh et al., 199la). It is lower in pre-menopausal women than in men (Farr et al., 1986; Satoh et al., 1991a), however, over 50 years of age the difference between men and women lessens (Satoh et al., 1991 a). In women, the PAF acetylhydrolase activity changes during the menstrual cycle and it is negatively correlated with plasma estrogen level (Miyaura et al., 1991). PAF acetylhydrolase activity decreases in maternal plasma during the latter stages of pregnancy in humans (Johnston, 1989) and rabbits (Maki et al., 1988), and administration of estrogen to rats decreases the plasma activity (Pritchard, 1987; Miyaura et al., 1991). These observations taken together suggest that estrogen decreases the activity in plasma, and that the closer values in older men and women are due to a loss of the suppressive effect of estrogen. Estrogen probably acts by decreasing the secretion of PAF acetylhydrolase by the liver cells, as described above. In an experiment reported by Miyaura et al. (1991), administration of glucocorticoids increased the level of the PAF acetylhydrolase in the plasma of rabbits and reversed the suppressive actions of estrogen. This suggests that a portion of the antiinflammatory actions of steroids may be due to an increase in this enzyme that catalyzes the removal of inflammatory lipids. It will be interesting to elucidate the mechanism for the effects of estrogen and glucocorticoids on the synthesis of PAF acetylhydrolase by hepatocytes once a cDNA probe and antibodies are available.
1 52
TADA-ATSU IMAIZUMI ET AL. E. Genetics of the Plasma PAF Acetylhydrolase
Based on measurement of the enzymatic activity in plasma samples from a population of subjects, Miwa et al. (1988) reported that about 4% of Japanese children and adults are markedly deficient in the plasma PAF acetylhydrolase. These investigators also were able to study five families, and concluded that the mode of transmission was autosomal recessive. They found that deficiency of the PAF acetylhydrolase occurred more frequently in asthmatic children with severe symptoms than in normal children, suggesting that it plays a role in limiting inflammatory and allergic responses. However, there were subjects with low activity of plasma PAF acetylhydrolase who did not have a defined phenotype. There have been no reports of deficiency of this enzyme in other ethnic and racial groups, although the prevalence of 4% in Japan has been confirmed by another group in the northern part of the country (Imaizumi and Satoh, unpublished observation). Importantly, these workers found that deficient subjects examined serially were always low, a result that excludes some trivial causes for the observation. We have tested plasma samples from over 1,000 subjects in the United States and have not found a case of marked deficiency (unpublished results). The molecular basis for the deficiency observed in Japan is unknown since the gene has not yet been identified. However, the possibility of an inhibitor was excluded by mixing studies. If true deficiency is common in some populations, it will provide an experiment of nature to assess the function, if any, of this enzyme in human physiology and disease. F. The Plasma PAF Acetylhydrolase In Disease
The activity of PAF acetylhydrolase in plasma has been reported to be increased in patients with atherosclerotic diseases such as peripheral vascular disease (Ostermann et al., 1987), ischemic stroke (Satoh et al., 1988), and familial HDL deficiency (Tangier disease) (Pritchard et al., 1985). Based on the analogy to inflammatory conditions, we propose that this represents a protective response to some signal generated during atherogenesis. Conversely, it is possible that the increased activity contributes to the onset or progression of atherosclerosis. We conclude that this is less likely because the activity of plasma PAF acetylhydrolase is also increased in insulin-dependent diabetes mellitus (Hofmann et al., 1989), and habitual cigarette smokers (Imaizumi et al., 1990), implying that the increased activity is a result rather than cause in these disorders. However, the observations that the enzyme activity is increased in patients with atherosclerosis, while it is decreased by estrogen, which is associated with a decreased risk of atherosclerosis, are consistent with the counterhypothesis that the enzyme is atherogenic rather than protective. Further experimentation will be necessary to resolve this point. It is also reported that PAF acetylhydrolase activity is increased in plasma from patients with essential hypertension (Satoh et al., 1989) and spontaneously hypertensive rats (Blank et al..
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Table 1. Changes of PAF Acetylhydrolase Activity in Physiological and Pathological Conditions Plasma type PAF-AH activity
I. Plasma Human physiological newborn aging sex pregnancy pathological peripheral vascular disease ischemic stroke myocardial infarction familial HDL deficiency (Tangier Disease) essential hypertension insulin-dependent diabetes mellitus chronic cholestasis
Reference
low Caplanetal., 1990 Satohetal., 1991a increase premenopausal women < men Farretal., 1986; Satohetal., 1991a Johnston, 1989 decrease
primary glomerulonephritis rheumatoid arthritis noninflammatory arthritides habitual cigarette smokers systemic lupus erythematosus septic shock neonatal necrotizing enterocolitis coronary bypass operation child asthma cigarette smoke extract LDL oxidation by Cu Experimental animal models estrogen administration (rat) pregnancy (rabbit) dexamethasone administration (rat) corticosterone implantation (lizard) chronic stress (lizard) gastric ulcer by water-immersion stress (rat) necrotizing enterocolitis (rat) spontaneous hypertensive rat (rat) lupus mouse (mouse) lupus mouse, moribund (mouse) peritoneal LPS administration (guinea pig)
increased increased increased increased increased increased increased normalized after liver transplantation increased increased increased increased decreased decreased decreased decrease increased incidence of deficiency decrease decrease decrease decrease increase increase increase increase decreased increased decreased increased increased in peritoneal supernatant
Ostermann et al., 1987 Satohetal., 1988 Ostermann et al., 1988 Pritchard et al., 1985 Satohetal., 1989 Hofmann et al., 1989 Meade etal., 1991 Latrouetal., 1992 Dulioust et al., 1992 Dulioust et al., 1992 Imaizumi etal., 1990 Tettaetal., 1990 Taylor etal., 1992 Caplanetal., 1990 Stephens et al., 1992 Miwaetal., 1988 Miyaura et al., 1992 Stafforini et al., 1992 Pritchard, 1987; Miyaura et al., 1991 Makietal., 1988 Miyaura et al., 1991 Lenihan et al., 1985 Lenihan et al., 1985 Fujimura et al, 1989 Caplanetal, 1988 Blank etal, 1983 Zhao etal, 1992 Zhao etal, 1992 Karasawa et al, 1992
{continued)
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TADA-ATSU IMAIZUMI ETAL.
Table 1. (Continued) II. Cell Human in vivo RBC aging ischemic stroke (RBC) Experimental animal models lupus mouse, moribund (liver, kidney, lung, spleen)
Intracellular type PAF-AH activity
Reference
decrease decreased
Yoshida et al., 1994 Yoshidaetal., 1992; Yoshida et al., 1993
increased
Zhao etal., 1992
1983). PAF has a hypotensive activity, and there is a possibility that excessive levels of PAF acetylhydrolase could increase blood pressure by removing the hypotensive signal. We believe this is unlikely for two reasons. First there is normally a great excess of PAF acetylhydrolase activity in plasma, and there are no data to suggest that the Tj/2 of PAF would be shorter with increased levels. In fact, we have found that this is not the case (unpublished data). Second, Satoh et al. (1989) found increased levels of PAF acetylhydrolase activity in the plasma of patients with hypertension, but the level increased with the length of time that the patient had hypertension; again suggesting that it was a result rather than a cause. In related experiments, investigators have examined the role of cigarette smoking on the accumulation of PAF or PAF-like lipids in blood and on the enzyme activity. Imaizumi et al. (1991) showed that PAF-like lipids accumulate in the plasma upon acute inhalation of cigarette smoke. Miyaura et al. (1992) found a substance in cigarette smoke extract that inhibits the PAF acetylhydrolase, and we have found that the activity in plasma LDL is lost upon artificial oxidation with Cu^"^ (Stafforini et al., 1992). We postulate that the unidentified compound from cigarette smoke inhibits the PAF acetylhydrolase, which then may lead to the increased levels of PAF or related compounds due to a decreased rate of hydrolysis. This could accelerate vascular damage by the proinflammatory actions of PAF, and could account for some of the deleterious effects of smoking. Alterations of the plasma PAF acetylhydrolase activity have been described in various inflammatory conditions. The enzyme activity in serum is increased in patients with primary glomerulonephritis (Latrou et al., 1992), while that in plasma from patients with systemic lupus erythematosis {Tetta et al., 1990) is decreased. The activity in plasma from lupus mice is also decreased, but when they are moribund, activity increases (Zhao et al., 1992). The PAF acetylhydrolase activity is high in serum from patients with cholestasis caused by liver diseases such as sclerosing cholangitis, advanced primary biliary cirrhosis, or cholangiocarcinoma, but was shown to normalize after successful liver transplantation (Meade et al., 1991). PAF acetylhydrolase activity is decreased in plasma from patients with septic
Metabolism of Platelet-Activating Factor
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shock (Taylor et al., 1992), but it is increased in the exudate in the peritoneal cavity of guinea pigs with experimental endotoxic shock following intraperitoneal administration of lipopolysaccharide (Karasawa et al., 1992). The level of PAF acetylhydrolase activity has been implicated in gastrointestinal disorders as it is decreased in patients with neonatal necrotizing enterocolitis (Caplan et al., 1990) and rats with experimental necrotizing enterocolitis (Caplan et al., 1988). Serum PAF acetylhydrolase activity is increased in patients with rheumatoid and other forms of arthritis (Dulioust et al., 1992). Thus, both increased and decreased enzyme activity have been reported in plasma from patients or animals with various inflammatory conditions (Table 1). This suggests that PAF acetylhydrolase activity may be affected by multiple factors which mediate the inflammatory response, and the response may be complex, perhaps increased during some stages and decreased during others. In support of this, some work suggests that PAF itself, after being generated in pathological inflammation, induces increased synthesis and secretion of the plasma PAF acetylhydrolase activity by the liver. Moreover, we have shown that the secretion of plasma type PAF acetylhydrolase from normal macrophages increases during differentiation (Elstad et al., 1989), and the same has been shown in HL-60 cells (Narahara et al., 1993). We have shown that interferon decreases this response (unpublished observations) and Narahara et al. (1993) found that endotoxin inhibited the secretion of PAF acetylhydrolase by HL-60 cells. Administration of dexamethasone, a potent antiinflammatory steroid hormone, to rats increases the activity in plasma (Miyaura et al., 1991), and this response can be mimicked by treatment of cultured HL-60 cells with dexamethasone. Implantation of corticosterone or chronic stress also increases the activity in the plasma of lizards (Lenihan et al., 1985). These findings may partially account for the mechanisms by which plasma PAF acetylhydrolase activity is altered in pathologic situations such as inflammation. G.
Intracellular PAF Acetylhydrolase
We recently reported the purification and characterization of a PAF acetylhydrolase from human erythrocytes, which is clearly a distinct protein from the plasma form (Stafforini et al., 1993). This should rapidly lead to reagents that will allow us and others to test the role of this enzyme. For example, it is puzzling that erythrocytes have such high levels of this enzyme since they do not synthesize PAF, nor do they take up and hydrolyze exogenous PAF at an appreciable rate (Stafforini et al., 1993). We have proposed that the enzyme is present in these cells predominantly as a an antioxidant defense since erythrocytes are particularly prone to oxidative damage as they have high concentrations of oxygen and iron. Conversely, the PAF acetylhydrolase in erythrocytes might contribute to the hydrolysis of PAF if the cells were lysed at the site of inflammation and released their PAF acetylhydrolase. Yoshida et al. (1992a, 1993) showed that PAF acetylhydrolase activity in
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red blood cell cytosol and membrane are decreased in patients with cerebral thrombosis and that the activity was correlated with erythrocytes deformability. They also found that the activity decreases with aging of erythrocytes in vivo (Yoshida et al., 1994). The PAF acetylhydrolase activities in kidney, liver, lung, and spleen have reported to be increased in moribund lupus mice (Zhao et al., 1992). There currently is no other information on changes of the intracellular form of PAF acetylhydrolase activity that have been correlated with physiologic or pathologic events. It will be intriguing to determine if it plays a role in defense against oxidative damage, or has other actions. Since the acceptance of this manuscript, the cDNA for human PAF acetylhydrolase was cloned (Tjoelker, L.W., et al. (1995). Nature 374, 549-553.). The recombinant plasma PAF acetylhydrolase inhibits the inflammatory effects of PAF in in vivo and in vitro experimental models. The cDNAs for three subunits of bovine brain PAF acetylhydrolase were also cloned, and the cDNA for one of the subunits is the bovine homolog of human gene which is causative for Miller-Dieker lissencephaly, a human cerebral malformation (Hattori, M., et al. (1994). Nature 370, 216-218; (1994) J. Biol. Chem. 269, 23150-23155; (1995) J. Biol. Chem. 270,31345-31352.).
ACKNOWLEDGMENTS We are grateful for the technical assistance of Susan Cowley, Donnelle Benson, Bart Tarbett, and Linda Wilcox in the projects from our laboratories. Drs. Kay Stremler and Mark Elstad made important contributions to these studies. The work has been supported by the Nora Eccles Treadwell Foundation and the George and Dolores Dore Eccles Foundation, and by grants from the National Institutes of Health and the American Heart Association. Dr. Stafforini is the recipient of a Minority Scientist Development Award from the American Heart Association and Drs. Prescott and Zimmerman were Established Investigators during much of the work reviewed here.
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BIOSYNTHESIS OF PLASMALOGENS IN MAMMALIAN CELLS AND THEIR ACCELERATED CATABOLISM DURING CELLULAR ACTIVATION
David A. Ford and Richard W. Gross
ABSTRACT I. INTRODUCTION II. PHOSPHOLIPID STRUCTURE ill. PLASMALOGEN BIOSYNTHESIS A. DeTVovo Biosynthesis of Alkyl Ether Glycerophospholipids B. De Novo Synthesis of the Vinyl Ether Bond of Plasmenylcholine C. PlasmenylchoHne Biosynthesis D. Plasmalogen Catabolism During Cellular Perturbation E. Plasmalogen Catabolism in Myocardium . F. Plasmalogen Catabolism in Smooth Muscle Cells G. Plasmalogen Catabolism in Neutrophils IV. CONCLUSION ACKNOWLEDGMENT REFERENCES
Advances in Lipobiology Volume 1, pages 163-191. Copyright © 1996 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-635-5 163
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164
DAVID A. FORD and RICHARD W. GROSS
ABSTRACT Plasmalogens are specialized phospholipids which have a unique conformation, possess distinct molecular dynamics, and serve as the major endogenous phospholipid storage depot for arachidonic acid in many mammalian cells. Recently, several novel intracellular phospholipases A2 have been identified which selectively hydrolyze plasmalogen substrate. Quantitative analysis of phospholipid molecular species in several cell types demonstrate that plasmalogen molecular species containing arachidonic acid are selectively hydrolyzed during cell stimulation and serve as the major source of arachidonic acid mass released during cellular activation. Collectively, these results underscore the importance of plasmalogen hydrolysis as a major mechanism for the release of eicosanoid metabolites during agonist stimulation. Accordingly, this chapter will review recent insights on the de novo synthesis of plasmalogens, address the molecular mechanisms responsible for the enrichment of arachidonic acid in plasmalogen molecular species and,finally,will focus on the mechanisms which mediate accelerated plasmalogen catabolism and the release of arachidonic acid during cellular activation.
I. INTRODUCTION Biological membranes are critical cellular constituents w^hich serve a multiplicity of distinct functional roles in cellular metabolism and physiology. First and foremost, biological membranes constitute the permeability barrier that provides the appropriate physical interface for the sequestration of critical enzymes and metabolites v^hich allows each organism to propagate and reproduce. Without the barrier function of the membrane, life itself would be impossible. Second, biological membranes are the storage depot for many of the chemical precursors utilized in the generation of lipid second messengers (e.g., eicosanoids, diglycerides) which are synthesized during cellular stimulation through the activation of intracellular phospholipases. Third, biological membranes are dynamic, interactive matrices that facilitate the appropriate interactions of multiple transmembrane proteins which, in turn regulate cellular function and adaptive responses. To accomplish these multiple functions, biological membranes have undergone an enormous amount of chemical specialization yielding an astonishing diversity of the individual molecular constituents present in specific membrane compartments. The functional diversity and scope of adaptive membrane responses results, in part, from the presence of thousands of distinct chemical moieties in biological membranes including polar lipids, nonpolar lipids, and proteins interdigitated in a complex dynamic matrix which allows each cell to fulfill its biological destiny. Through the appropriate juxtaposition of unique combinations of these distinct chemical entities within a membrane bilayer, nature has created a mechanism where critical stereoelectronic relationships, membrane physical properties, and membrane dynamics can be
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precisely tailored to facilitate each cell's adaptation to its metabolic history, current external milieu and genetically pre-destined function. The purpose of this chapter is to examine the biology, chemistry, and physiologic significance of one specialized phospholipid subclass in mammalian cell membranes, the plasmalogens. Plasmalogens are phospholipid membrane constituents which possess a distinct covalent structure, adopt a unique molecular conformation, possess specific molecular dynamics, represent the major phospholipid storage depot of arachidonic acid in many cell types and are the target of certain phospholipases activated during cellular stimulation.
11. PHOSPHOLIPID STRUCTURE Mammalian membranes are comprised of a diverse array of polar lipids which (for the most part) are glycerol-based and typically contain two aliphatic chains covalently linked to the sn-l and sn-2 positions of the glycerol backbone. The sn-3 position contains a polar head group covalently attached to the glycerol backbone through a phosphodiester linkage. Variations in the chemical nature of the polar head group (e.g., choline, ethanolamine, inositol, serine) give rise to the structural diversity known as phospholipid classes (e.g., phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, etc.). Variations in the polar head group produce substantial changes in the surface charge characteristics of the membrane and in critical stereoelectronic relationships within the membrane bilayer. Each phospholipid class can contain (in theory) three subclasses representing variations in the nature of the covalent attachment of the sn-\ aliphatic linkage to the glycerol backbone. Phospholipid subclasses include diacyl phospholipids (ester linkages at both the sn-\ and sn-2 positions), alkyl ether phospholipids (an alkyl ether linkage at the sn-\ position and an ester linkage at the sn-2 position) and plasmalogen molecular species (a vinyl ether linkage at the sn-1 carbon and an ester linkage at the sn-2 position) as shown in Figure 1. In most mammalian membranes, diacyl phospholipids are far more abundant than either alkyl ether- or vinyl ether-containing phospholipids. Furthermore, phospholipids with either alkyl ether or vinyl ether constituents occur predominantly within the choline and ethanolamine glycerophospholipid classes. Each cell type and subcellular membrane compartment has a distinct and highly regulated complement of specific phospholipid classes and subclasses. For example, myocardial cells have a high content of plasmenylcholine (plasmalogens with a choline polar head group) while hepatocytes contain only diminutive amounts of plasmenylcholine. This diversity in mammalian phospholipids is further amplified by a vast array of different aliphatic constituents linked to the sn-\ and sn-2 carbon of the glycerol backbone which vary in their chain length and the degree and position of their olefinic carbons. Common aliphatic constituents typically include saturated fatty acids, predominantly found at the sn-\ position (palmitic and stearic acids) and
166
DAVID A. FORD and RICHARD W. GROSS Rj
Phospholipid Subclass
H H rO-Ri
-C=C-R '
Plasmalogen
O II
I-O-C-R2 O
H H
-c-C-Ri' I I H H
Alkyl Ether
i-O-p-o-X O
II -C-Ri'
Diacyl
Figure / . Phospholipid subclasses. Phospholipid subclasses are distinguished by the nature of covalent bond connecting the sn-^ aliphatic constituent to the glycerol backbone. The aliphatic constituent of plasmalogen, alkyl ether and diacyl subclasses of phospholipids contains a vinyl ether, alkyl ether, or ester bond at the sn-^ position, respectively. The abbreviations used are R2, long chain aliphatic constituent (e.g., C19H31), X, polar head group (e.g., choline) Ri', aliphatic chain.
unsaturated fatty acids, predominantly present at the sn-2 carbon (oleic, linoleic and arachidonic acids). The combined diversity present in the multiplicity of phospholipid classes (~ 10 different polar head groups), subclasses (three different covalent linkages at the sn-1 carbon), and individual molecular species (20 different fatty acid combinations at the sn-l and sn-2 carbons) gives rise to hundreds of specific chemical entities which collectively comprise the phospholipids of biological membranes. The complexity inherent in the membrane is further demonstrated by the presence of specific nonpolar lipids in individual membrane compartments (e.g., cholesterol in the plasma membrane) and individual protein constituents, which are each present in specific subcellular membrane compartments. The evolutionary design of these multiple constituents to facilitate cellular adaptation to diverse perturbations underscores the importance of these specialized features in the organization, surface charge characteristics, molecular dynamics, conformation, and physical properties of the membrane bilayer. Although the dramatic differences precipitated by changes in the physical characteristics of the membrane bilayer elicited by alterations in phospholipid class composition (i.e., changes in the polar head group) or changes in individual molecular species (i.e., alterations in the sn-l and. ^«-2 constituents) are well characterized, the significance of subclass specialization in biologic membranes has just begun to be appreciated. The structural difference between plasmalogen and diacyl phospholipids (i.e., the replacement of an ester linkage with a vinyl ether linkage), however subtle, results in a substantially different molecular geometry of plasmalogen molecular species in comparison to their diacyl phospholipid coun-
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167
terparts (a consequence of the presence of two sp^ carbon atoms in the proximal portion of the sn-l aliphatic chain) and resultant changes in lipid packing and molecular dynamics in membrane bilayers comprised of plasmalogens (Han and Gross, 1991).
III. PLASMALOGEN BIOSYNTHESIS Although the de novo biosynthetic pathways responsible for alkyl ether glycerophospholipid synthesis, as well as for the synthesis of the ethanolamine plasmalogens (plasmenylethanolamine) have been known for many years, the biosynthetic pathway for plasmenylcholine remained enigmatic until recently. The oxidation of plasmanylethanolamine to plasmenylethanolamine is catalyzed by a mixed-function oxidase and represents the only known mechanism for the introduction of a vinyl ether linkage into mammalian phospholipids. Thus, while it was generally believed that the direct metabolic precursor of plasmenylcholine is plasmenylethanolamine, the biochemical mechanisms responsible for this conversion were unknown. The transformation of plasmenylethanolamine to plasmenylcholine could result from either polar head group remodeling or from the sequential iV-methylation of the ethanolamine head group of plasmenylethanolamine. This section will first review the biosynthesis of plasmenylethanolamine and subsequently focus on recent insights into the de novo pathways of plasmenylcholine biosynthesis. A.
De Novo Biosynthesis of Alkyl Ether Glycerophospholipids
The biosynthetic mechanism responsible for the generation of the ether bond in ether-containing glycerophospholipids was described independently by Hajra and Snyder et al. over two decades ago (Hajra, 1970; Snyder et al., 1971). The critical reaction in the introduction of the ether bond was demonstrated to result from an exchange between a fatty alcohol (e.g., palmitoyl alcohol) and the acyl moiety of monoacyl dihydroxyacetone phosphate, resulting in the synthesis of monoalkyl dihydroxyacetone phosphate (Hajra, 1970; Lumb et al., 1971) (Figure 2). Both substrates in this reaction are present in substantial quantities in eukaryotic cells. Monoacyl dihydroxyacetone phosphate is produced by the acylation of the glycolytic intermediate, dihydroxyacetone, and fatty alcohol is synthesized by reduction of fatty acid (Rizzo et al., 1987). The next step in alkyl ether glycerophospholipid de novo biosynthesis is the reduction of the alkyl dihydroxyacetone phosphate by an NADPH-dependent alkyl dihydroxyacetone phosphate reductase (Figure 2). The product of this reaction, l-O-slkyl-snglycero-3-phosphate, is then sequentially acylated at the sn-2 carbon and subsequently dephosphorylated at the sn-3 carbon. Dephosphorylation is mediated by a microsomal phosphatase that requires magnesium ion. The resultant alkyl ether diglyceride, l-(9-alkyl-2-acyl-5A2-glycerol, represents a branch point intermediate in the biosynthesis of polar and nonpolar ether lipids since the alkyl-acyl glycerol
DAVID A. FORD and RICHARD W. GROSS
168
HjC-O-C-R, 0=C
O
I
II
HjC-0-P-OH
r R, CH2CH2OH
A
O II
R,C-OH ^
H2C-O-CH2CH2R,
o=i
o
H,C-0-P-OH
NADP
'NADPH
J
HjC-O-CHjCHjR, HO-CH
O
H,C
0-P-OH
RjC-SCoA-^ CoASH
O
^
H2C-O-CH2CH2R,.
RjC - 0 - C H 0 I II HjC-0-P-OH O® H2O ->^| O e II 0-P-OH ^ I OH
;:i
O
H,C-0-CH2CH2R,
RjC - 0 - C H HjC - O H CDP-Ch^
CDP-Etn ^ *
O
H2C-O-CH2CH2RV
O 11
RjC-O-CH
O
H,C - 0 - P - 0 - C H 2 C H 2 N ( C H 3 ) 3
l^RiCSCoA
')_ CMP^
CMPV^
H^C-O-CHjCHjR, ' 1
R2C - 0 - C H
?
?•
1
O
H^C-O-P-O-CHjCHj^Hj
HjC-0-CH2CH2R, R2C - O - C H O I II H,C-0-C-R,
O^
Figure 2. The de novo biosynthetic pathway for plasmanylethanolamine. Monoacyl dihydroxyacetone phosphate and fatty alcohol are the precursors of the alkyl ether phospholipids. Fatty alcohol exchange with the fatty acyl moiety of monoacyl dihydroxyacetone phosphate is catalyzed by alkyl dihydroxyacetone phosphate synthase and is the first committed step in the biosynthesis of ether containing lipids. Through the sequential reduction and acylation at the sn-2 carbon, alkyl ether phosphatidic acid is synthesized. Dephosphorylation of alkyl ether phosphatidic acid generates 1-0-alkyl-2-acyl-sn-glycerol which can be utilized by amino alcohol phosphotransferases or diradyl glycerol acyl transferase for the synthesis of either choline and ethanolamine alkyl-ether glycerophospholipids or alkyl-ether triradyl glycerols, respectively.
Plasma Iogen Metabolism
169
can either be subsequently acylated (by a diglyceride acyl transferase) to generate a triglyceride, phosphorylated to generate phosphatidic acid, or utilized as a cosubstrate for condensation with CDP-choline or CDP-ethanolamine to generate plasmanylcholine or plasmanylethanolamine (Figure 2). B. De Novo Synthesis of the Vinyl Ether Bond of Plasmenylcholine
The only known enzymic mechanism for the introduction of a vinyl ether linkage into phospholipids utilizes a pyridine nucleotide-dependent mixed-function oxidase which has an absolute specificity for desaturation of l-O-alkyl-2-acyl-GPE (plasmanylethanolamine) to generate the corresponding l-O-alk-r-enyl-2-acylGPE (plasmenylethanolamine) (Schmid et al, 1972; Wykle et al., 1972). This mixed-function oxidase is inhibited by cyanide, but not by carbon monoxide, and requires molecular oxygen, reduced pyridine nucleotide (NADH or NADPH), and cytochrome b5. These characteristics are similar to those of the fatty acid desaturase that catalyzes the synthesis of monoenoic fatty acids. Multiple l-O-alkyl-2-acylGPE molecular species can be utilized by the mixed-function oxidase (Blank et al, 1986). However, neither 3-0-alkyl-2-acyl-5«-glycero-l-phosphorylethanolamine, 1 -0-alkyl-GPE, 1 -0-alkyl-2-acyl-5«-glycero-3-phosphoryl(A^-dimethyl)ethanolamine, nor, most importantly, plasmanylcholine is utilized by this mixed-function oxidase (Schmid et al., 1972; Wykle et al., 1972; Paltauf and Holasek, 1973; Blank et al., 1986). No alkyl ether desaturase activities have been demonstrated which can catalyze the desaturation of plasmanylcholine to plasmenylcholine despite numerous attempts to demonstrate this activity in broken cell preparations, intact cells or intact tissue. Accordingly, the only pathway for introduction of the vinyl ether linkage into cellular lipids is by oxidation of plasmanylethanolamine to plasmenylethanolamine. Thus, it is generally accepted that plasmenylcholine must be generated from plasmenylethanolamine. C. Plasmenylcholine Biosynthesis
Three pathways which could potentially generate plasmenylcholine from plasmenylethanolamine have been deduced predominantly from broken cell experiments as well as from a limited number of studies with intact cells and intact tissue. Pathway I involves the condensation of CDP-choline with l-O-alk-r-enyl-2-acyl5«-glycerol and was initially suggested by Kiyasu and Kennedy (1960). The key intermediate in this pathway, 1 -0-alk-1 '-enyl-2-acyl-5«-glycerol, was subsequently identified as an endogenous constituent of myocardium (Ford and Gross, 1988; Ford et al., 1992). The strategy employed by Pathway I is the exploitation of polar head group remodeling (initiated by either phospholipase C directly or by the sequential actions of phospholipase D coupled with phosphatidate phosphohydrolase) to facilitate the introduction of the vinyl ether linkage into plasmenylcholine. The subsequent condensation of l-0-alk-r-enyl-2-acyl-5«-glycerol with CDPcholine catalyzed by choline phosphotransferase results in the synthesis of plas-
CMP Ethanolamine Phosphotransferase
CDP - Ethanolamine CDP - Choline
Phosphorylethanolamine Phosphorylcholine
Choline Phosphotransferase
CMP
II
O "2-0-0-^
O
j - O - C=C-R, C O 0
II
R,-C-0
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"
■ - O - P - G - CCH2CH2N(CH3)3 h A
J
CDP-Choline^ O
|-0-C=C-R,
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II
R,-C-0
L-0-P-0-CH2CH2NH3
{
OH O e >■ O-P-OH I
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O
.-T'
—7^
OH
r-0-C=C-Ri
II
R2-C-O
O
L-O-P-OH
R2CSC0A
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3 S-adenosyl methionine
-C=C-R, O
3 S-adenosyt homocysteine
k0-C-R2
I-O-C-R2
N - methyl transferase
o II
pO-C=C-R, O ■>
II
II
®
'-0-P-0-CH2CH2N(CH3), 0^
«
l-O-P-O-CHjCHjNHa O^
Figure 3. Proposed pathways for de novo plasmenylcholine biosynthesis. Three pathways have been proposed for the de novo biosynthesis of plasmenylcholine which each utilize plasmenylethanolamine as the precursor of the vinyl ether aliphatic group of plasmenylcholine. Through Pathway I, plasmenylcholine is synthesized by polar head group remodeling of plasmenylethanolamine which is initiated by phospholipase C (or a phospholipase C equivalent) catalyzed hydrolysis of plasmenylethanolamine. Pathway II is initiated by phospholipase A2 catalyzed hydrolysis of plasmenylethanolamine and through remodeling of the sn-2 and sn-3 constituents generates l-0-alk-V-enyl-2-acyl-SA7-glycerol which condenses with CDP choline resulting in the biosynthesis of plasmenylcholine. Pathway III is mediated by the sequential N-methylationoftheethanolamine head group of plasmenylethanolamine. 170
Plasmalogen Metabolism
171
menylcholine. Pathway II was initially proposed by Wykle and co-workers (Wykle and Schremmer, 1974; Wykle and Snyder, 1976) and is initiated by phospholipase A2-mediated hydrolysis of plasmenylethanolamine, resulting in the generation of lysoplasmenylethanolamine. The subsequent hydrolysis of the ethanolamine head group catalyzed by lysophospholipase D can generate plasmalogenic lysophosphatidic acid. Plasmalogenic lysophosphatidic acid can then be sequentially acylated and dephosphorylated resulting in l-0-alk-r-enyl-2-acyl-5«-glycerol production. Plasmenylcholine can be subsequently synthesized by choline phosphotransferasemediated condensation of l-0-alk-r-enyl-2-acyl-5«-glycerol with CDP choline. Thus, Pathway I and Pathway II share common final steps in the generation of plasmenylcholine (i.e., phosphatidate phosphohydrolase and choline phosphotransferase) and utilize the same key intermediate (l-0-alk-r-enyl-2-acyl-5«glycerol) which is generated by different enzymatic pathways. Pathway III utilizes the sequential 7V-methylation of the ethanolamine moiety of plasmenylethanolamine mediated by one or more A^-methyl transferases to introduce the vinyl ether linkage into plasmenylcholine (Mogelson and Sobel, 1981). Through sequential A^-methylations utilizing S-adenosylmethionine as the methyl donor, monomethyl plasmenylethanolamine, dimethyl plasmenylethanolamine and,finally,plasmenylcholine are produced. The evidence supporting each of these pathways will be briefly summarized and their quantitative importance, mechanisms of regulation, and some of the directions of future research in this area will be discussed on the following pages. The discovery in 1985 of a neutral-active phospholipase C in myocardium that utilizes choline and ethanolamine glycerophospholipids (including plasmalogen molecular species) (Wolf and Gross, 1985) led to the hypothesis that plasmenylcholine biosynthesis in myocardium is initiated by phospholipase C-mediated hydrolysis of plasmenylethanolamine. The direct generation of the key intermediate, l-0-alk-r-enyl-2-acyl-^«-glycerol, from plasmenylethanolamine in conjunction with the known ability of choline phosphotransferase to utilize l-0-alk-r-enyl-2-acyl-5«-glycerol to generate plasmenylcholine would provide a direct route to the synthesis of plasmenylcholine molecular species through polar head-group remodeling (e.g., Pathway I, Figure 3). Initial experimental support for this pathway included the demonstration of l-0-alk-r-enyl-2-acyl-5«-glycerol as an endogenous lipid in rabbit myocardium and the demonstration that endogenous microsomal l-0-alk-r-enyl-2-acyl-5«-glycerol is selectively utilized by myocardial microsomal choline phosphotransferase (Ford and Gross, 1988). The selectivity of myocardial choline phosphotransferase for the synthesis of plasmenylcholine was demonstrated in studies in which the water soluble metabolite, CDP-choline, was added to rabbit myocardial microsomes and the utilization of each endogenous microsomal l,2-diradyl-5«-glycerol by microsomal choline phosphotransferase was quantified. These experiments demonstrated that plasmenylcholine and phosphatidylcholine molecular species were synthesized at similar rates (corresponding to their tissue distribution in rabbit myocardial microsomal lipids), despite the fact
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DAVID A. FORD and RICHARD W. GROSS
that over a 20-fold molar excess of l,2-diacyl-5«-glycerol as compared to 1-0-alk1 '-enyl-2-acyl-5«-glycerol was present in myocardial microsomes (Ford and Gross, 1988). Furthermore, these studies also documented the metabolic significance of this pathway in maintaining myocardial plasmenylcholine levels, since the rate of plasmenylcholine synthesis by choline phosphotransferase utilizing endogenous l-0-alk-r-enyl-2-acyl-5«-glycerol and physiologic concentrations of CDPcholine was comparable to the rate of plasmenylcholine catabolism in intact tissue (Ford and Gross, 1988). Taken together, these studies demonstrated the presence of the key intermediate of Pathway I (e.g., l-0-alk-r-enyl-2-acyl-^«-glycerol), the selectivity of the choline phosphotransferase for alk-r-enyl-2-acyl diradyl glycerol, and the metabolic capacity of myocardial choline phosphotransferase to catalyze the major portion of plasmenylcholine biosynthesis in intact tissues utilizing endogenous concentrations of l-0-alk-r-enyl-2-acyl-5«-glycerol and CDP-choline. To further identify the salient kinetic characteristics resulting in the molecular species distribution of choline and ethanolamine plasmalogens in mammalian cells, the substrate selectivity and kinetic profile of myocardial ethanolamine phosphotransferase were characterized. Quantification of the initial rates of CDPethanolamine condensation with l-(9-alk-r-enyl-2-acyl-5«-glycerol (AAG) compared to l,2-diacyl-^«-glycerol (DAG) catalyzed by ethanolamine phosphotransferase demonstrated a 16-fold selectivity for utilization of vinyl ether diradyl glycerol in comparison to diacyl diradyl glycerol (Ford et al., 1992). Furthermore, direct competition experiments employing mixed micelles comprised of equimolar amounts of AAG and DAG (containing palmitic acid at the sn-l position and arachidonic acid at the sn-2 position) demonstrated a 20:1 ratio of plasmenylethanolamine production compared to phosphatidylethanolamine production. Additionally, incubation of rabbit myocardial microsomes with CDPethanolamine utilizing endogenously produced diradyl glycerols as substrate resulted in the robust production of 16:0-20:4 plasmenylethanolamine, 18:1—20:4 plasmenylethanolamine, and 18:0-20:4 plasmenylethanolamine with only diminutive amounts of phosphatidylethanolamine synthesized. Thus, these results demonstrate the substantial substrate selectivity of mammalian ethanolamine phosphotransferase for plasmenylethanolamine synthesis in direct comparison to utilization of DAG substrate for phosphatidylethanolamine synthesis, and underscore the importance of the ethanolamine phosphotransferase reaction in determining the subclass distribution of endogenous ethanolamine glycerophospholipids in mammalian cells. Comparisons of the molecular species selectivity of rabbit myocardial microsomal ethanolamine phosphotransferase demonstrated that its reaction velocity was predominantly determined by the subclass distribution of diradyl glycerols and not the molecular species distribution since utilization of l-0-alk-l-enyl-2-acyl-5«glycerol containing either oleic or arachidonic acid by the ethanolamine phosphotransferase proceeded with similar velocities. Similarly, all molecular species
Plasmalogen Metabolism
1 73
of l-0-alkyl-2-acyl-5«-glycerols were utilized with similar reaction velocities. Additional experiments demonstrated a modest selectivity for utilization of diacyl glycerophospholipid molecular species containing arachidonic compared to oleic acid at the sn-2 position. However, this selectivity was dwarfed by the fact that the reaction velocity utilizing any molecular species of AAG was over 20-fold greater than that manifest by the best diacyl glycerol substrate. Collectively, these results demonstrated that the primary determinant of rabbit myocardial ethanolamine phosphotransferase substrate selectivity is the covalent nature of the sn-l aliphatic group of diradyl glycerol acceptors. Based upon these observations, several biochemical correlates concerning the subclass and molecular species distribution of plasmalogen and diacyl phospholipids in mammalian cells can be made. First, the dramatic selectivity of myocardial ethanolamine phosphotransferase for the synthesis of plasmenylethanolamines (in comparison to phosphatidylethanolamines) identifies one biochemical mechanism responsible for the abundance of the plasmalogen subclass in ethanolamine glycerophospholipids. Furthermore, identification of the substantial enrichment of arachidonic acid in l-0-alk-r-enyl-2-acyl-5«-glycerol molecular species in conjunction with the selectivity of ethanolamine phosphotransferase for l-0-alk-r-enyl-2-acyl-5«-glycerol demonstrates one mechanism contributing to the high content of arachidonic acid in plasmenylethanolamine molecular species. Thus, the subclass distribution of plasmalogens in ethanolamine glycerophospholipid pools results, in large part, from the preferred utilization of 1-0-alkr-enyl-2-acyl-5'«-glycerol moieties by ethanolamine phosphotransferase and the enrichment of arachidonic acid in vinyl ether containing diradyl glycerols. The selective synthesis of arachidonylated l-0-alk-r-enyl-2-acyl-5«-glycerol could occur either through selective phospholipase C- or D-mediated cleavage of plasmalogens containing arachidonic acid at the sn-2 position. Based on these studies, the biochemical mechanisms responsible for plasmenylcholine biosynthesis have been refined as depicted in Scheme I. The essential features inherent in this scheme include: (a) the initial generation of the vinyl ether linkage in plasmenylethanolamine by de novo synthesis catalyzed by alkyl ether desaturase; (b) the subsequent shuttling of vinyl ether equivalents carried by l-6)-alk-r-enyl-2-acyl-5«-glycerols; and (c) the de novo synthesis of plasmenylcholine or the regeneration of plasmenylethanolamine by their respective amino alcohol phosphotransferases each utilizing a common l-0-alk-r-enyl-2-acyl-5«glycerol intermediate. There are several important physiologic correlates which are implicit in this scheme. First, the content of vinyl ethers present in choline and ethanolamine glycerophospholipid classes is determined, at least in part, by the substrate selectivity of each amino alcohol phosphotransferase for 1 -0-alk-1 '-enyl2-acyl-5n-glycerol versus l,2-diacyl-5«-glycerol. This explains why the fractional percentage of phospholipids containing vinyl ethers in the ethanolamine glycerophospholipid pool is substantially higher than that in the choline glycerophospholipid pool (i.e., the selectivity of the ethanolamine phosphotransferase greatly
174
DAVID A. FORD and RICHARD W. GROSS iPLASMANYLETHANOLAMirJEl
IPLASMENYLETHANOLAMINEI
Scheme 1. The plasmenylethanolamine: 1-0-alk-r-enyl-2-acyi-sA7-glycerol cycle: Regulation of de novo biosynthesis of plasmenylcholine by 1-0-alk-1'-enyl-2-acylsn-glycerol. The abbreviations used are: EPT, ethanolamine phosphotransferase; Etn, ethanolamine; PA, phosphatidic acid; PAP, phosphatidic phosphohydrolase; PEtn, phosphorylethanolamine; PLC, phospholipase C; PLD, phospholipase D.
exceeds that of the choline phosphotransferase). Secondly, we point out that the enrichment of endogenous l-0-alk-r-enyl-2-acyl-5«-glycerol molecular species in arachidonic acid contributes to the enrichment of both the plasmenylethanolamine and plasmenylcholine pools in arachidonic acid, since neither choline phosphotransferase nor ethanolamine phosphotransferase possesses an inherent ability, at least in vitro, to discriminate between diradyl molecular species containing oleic acid or arachidonic acid at the sn-2 position. Since the molecular species of l-(9-alk-r-enyl-2-acyl-5«-glycerol are similar to the molecular species distribution of plasmenylethanolamine in rabbit membranes, these results suggest that plasmenylethanolamine is the predominant substrate for the generation of 1 -O-alkr-enyl-2-acyl-5w-glycerol in this system by phospholipase C or phospholipase D. The selective utilization of plasmenylethanolamine by myocardial phospholipase C or phospholipase D would thereby facilitate the flux of vinyl ether linkages into the choline glycerophospholipid pool and the generation of plasmenylcholine enriched in arachidonic acid. Recently, pulse-chase radiolabeling techniques have been utilized with precursors of the sn-l, sn-2 and sn-3 functionalities of plasmenylethanolamine and plasmenylcholine to quantitatively compare the rates of de novo synthesis to the rates of turnover of the sn-2 aliphatic chain and the sn-3 polar head group in intact contracting myocardium. Through this approach, the relative magnitudes of de novo synthesis versus remodeling can be ascertained. Since utilization of fatty alcohol is an obligatory reaction in the de novo synthetic pathway, de novo
Plasmalogen Metabolism
175
plasmalogen biosynthesis was assessed by pulse-chase radiolabeling of Langendorff perfused rabbit hearts with l-[-^H]-hexadecanoL Pulse-chase radiolabeling of perfused rabbit hearts with I -[^H]-hexadecanol resuhed in the rapid and progressive incorporation of radiolabel into plasmenylethanolamine molecular species (e.g., after 0.5 hours of radiolabeling, 10% of l-[-'H]-hexadecanol incorporated into ethanolamine glycerophospholipid was in plasmenylethanolamine, and after 1.5 hours, 21% was in plasmenylethanolamine), with no detectable radiolabeling of plasmenylcholine until three hours after the pulse (Figure 4). The temporal course of the appearance of plasmanylethanolamine, plasmenylethanolamine, and plas-
Plasmanyicholine
Plasmanylethanolamine
10 15 PERFUSION INTERVAL (Hours) BEGINNING OF CHASE INTERVAL BEGINNING OF PULSE LABELING
Figure4, Thetemporalcourseof1-[^H]-pa(mitoyl alcohol {1-[^H]-hexadecanol) into myocardial choline and ethanolamine glycerophospholipid pools. Rabbit hearts were perfused in a recirculating Langendorff mode with modified Krebs-Henseleit buffer containing 1-[^H]-palmitoyl alcohol for selected intervals. After a 1.5 hour radiolabeling interval the hearts were perfused with unlabeled palmitoyi alcohol (20 \xN\). At the indicated times, perfusions were terminated by freeze-clamping. Choline and ethanolamine glycerophospholipids were purified by straight phase HPLC and incorporation of radiolabel into ether lipids was determined by the generation of fatty aldehyde after exposure to acid fumes as well as by reverse phase HPLC separation of the molecular species oi each glycerophospholipid.
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DAVID A. FORD and RICHARD W. GROSS
menylcholine is consistent with precursor-product relationships between both plasmanylethanolamine and plasmenylethanolamine as well as between plasmenylethanolamine and plasmenylcholine in intact tissue. Analysis of the molecular species of plasmenylcholine and plasmenylethanolamine formed in the l-[^H]-hexadecanol pulse-chase radiolabeling experiments demonstrated that the predominant plasmenylethanolamine molecular species synthesized were 16:0-20:4 and 16:0-18:1, whereas the predominant plasmenylcholine molecular species synthesized was 16:0-20:4. Thus, the pulse-chase labeling profiles of individual molecular species suggest that arachidonoylated molecular species of plasmenylethanolamine are the preferred precursors for shuttling into plasmenylcholine by the selective phospholipase C- or phospholipase D-mediated hydrolysis of plasmenylethanolamine molecular species. These results are entirely consistent with the aforementioned in vitro studies demonstrating the selective synthesis of arachidonoylated plasmenylethanolamine molecular species by ethanolamine phosphotransferase and identifying the predominant endogenous molecular species of l-0-alk-r-enyl-2-acyl-5«-glycerol in myocardium as 1-0hexadec-r-enyl-2-eicosatetra-5',8',ir,14'-enoyl-5«-glycerol (Ford et al., 1992). Thus, plasmenylethanolamine molecular species enriched in arachidonic acid are both synthesized and degraded more rapidly than their monounsaturated counterparts. To substantiate the importance of polar head group remodeling and to further exclude the participation of an alkyl ether desaturase which could utilize plasmanylcholine, additional studies utilizing l-(9-[^H]-alkyl-GPC (^H-lysoPAF) were performed. Although ^H-lysoPAF was rapidly incorporated into plasmanylcholine through acylation, no detectable plasmenylcholine or plasmenylethanolamine was synthesized even after extended perftision intervals. Thus, even in intact tissue, plasmanylcholine is not a direct precursor of plasmenylcholine. To assess the relative rates of polar head group remodeling, sn-2 remodeling of plasmalogens, and de novo vinyl ether biosynthesis, comparisons of the flux of either choline, ethanolamine (polar head group remodeling), arachidonic acid (sn-2 remodeling), or hexadecanol into plasmenylcholine and plasmenylethanolamine were performed. Remarkably, the rate of polar head group remodeling of plasmalogens was over 300-fold more rapid than the rate ofde novo plasmalogen synthesis in intact, ftinctioning myocardium (Table 1). Furthermore, sn-2 remodeling of plasmalogens occurs at rates over 100-fold greater than that ofde novo plasmalogen biosynthesis (Table 1). Additionally, sn-2 group remodeling of plasmalogen molecular species likely contributes to their enrichment in arachidonic acid, since the rate of incorporation of arachidonic acid into plasmenylcholine and plasmenylethanolamine was threefold greater than the rate of incorporation of oleic acid. In contrast, rates of incorporation of oleic acid were more rapid into phosphatidylcholine and phosphatidylethanolamine in comparison to their plasmalogen counterparts.
Plasmalogen Metabolism
1 77
Table 1. Incorporation Rates of Polar Head Group and Aliphatic Precursors of Diacyl and Plasmalogen Glycerophospholipids in Isolated Perfused Rabbit Hearts Precursors (nmol/g^^,h) Product Plasmenylcholine Phosphatidylcholine Plasmenylethanolamine Phosphatidylethanolamine Note:
Hexadecanol
Choline
<0.01
21 131 — —
0.09
Ethanolamine — — 31 139
Arachidonic acid Oleic acid 35 113 12 16
11 93 4 19
Rabbit hearts were perfused in a recirculating Langendorff mode with modified Krebs-Henseleit buffer containing either l-pH]-hexadecanol, pH] choline, or [-^H] ethanolamine for 1.5 hours or alternatively with modified Krebs-Henseleit buffer containing either [^H] oleic acid or [^H] arachidonic acid for 20 minutes. The flux of precursors into each lipid pool was calculated from the specific activity of radiolabel in each lipid pool and the incorporation of radiolabel into the designated products.
Thus, independent results utilizing a variety of methodologies employing both broken cells and intact tissue have elucidated the pathways for plasmenylcholine biosynthesis and the biochemical mechanisms responsible for the enrichment of plasmalogen molecular species in arachidonic acid. These studies underscore the quantitative importance of remodeling in comparison to de novo synthesis and the inherent selectivity of the salient enzymic machinery for remodeling, which is critical in facilitating the rapid hydrolysis and resynthesis of specific phospholipid constituents in mammalian cells. Conceptually, Pathway I and Pathway II are similar since they both (ultimately) require plasmenylethanolamine polar head group remodeling and employ the common intermediate, l-(9-alk-r-enyl-2-acyl-5«-glycerol, for plasmenylcholine biosynthesis. However, Pathway II involves a multi-step process for the remodeling of the sn-2 and sn-?> constituents of plasmenylethanolamine which is initiated by a plasmalogen-selectivephospholipase A2 (Figure 3). Pathway II was first envisioned nearly 20 years ago, based on the discovery of a lysophospholipase D activity which demonstrated selectivity for ether-linked lipids (Wykle and Schremmer, 1974; Wykle and Snyder, 1976). Pathway II is initiated by phospholipase A2-mediated hydrolysis of plasmenylethanolamine followed by hydrolysis of the sn-2> polar head group of the resultant lysoplasmenylethanolamine by lysophospholipase D to generate lysophosphatidic acid containing a vinyl ether linkage at the sn-1 position. After acylation and dephosphorylation, the resultant l-(9-alk-r-enyl-2-acyl-5«glycerol, the intermediate which represents the convergence point of Pathways I and II, could be utilized by choline phosphotransferase resulting in the synthesis of plasmenylcholine. Thus, in this scheme the shuttling of vinyl ether moieties from plasmenylethanolamine into the choline glycerophospholipid pool is initiated by
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phospholipase A2 and requires multiple sequential reactions, while Pathway I generates the key intermediate directly. Although Pathway II was based on broken cell studies measuring lysophospholipase D activity (Wykle and Schremmer, 1974; Wykle and Synder, 1976), recent studies demonstrating the existence of plasmalogen-selective phospholipase A2 and the rapid disappearance of lysoplasmalogen have supported the presence of Pathway II. Further support for Pathway II as an enzymatic mechanism responsible for plasmenylcholine biosynthesis has been gained through studies utilizing [1-^H]-1O-alk-l'-enyl-GPE to label plasmalogen pools in intact HL-60 cells (Blank et al., 1993). In these studies, [l-^H]-l-0-alk-r-enyl-GPE was initially incorporated into plasmenylethanolamine molecular species which predominantly contain 20:3 fatty acid at the sn-2 carbon. Subsequently, radiolabel appeared in plasmenylcholine molecular species predominantly containing 16:0 at the sn-2 carbon. The disparity of radiolabeled individual molecular species of plasmenylcholine and plasmenylethanolamine was interpreted to support the role of phospholipase A2 in Pathway 11. Additionally, since both plasmenylethanolamine and plasmenylcholine contained molecular species with 16:0 aliphatic chains at the sn-2 carbon, it was concluded that at least some plasmenylcholine biosynthesis is initiated by phospholipase C (or phospholipase D, or their equivalents) (e.g.. Pathway I) or by direct base exchange (e.g.. Pathway III). The third pathway for plasmenylcholine biosynthesis (Figure 3) involves the A^-methylation of plasmenylethanolamine. Support for this pathway was obtained through demonstration that myocardium contains enzymes which catalyze the 7V-methylation of plasmenylethanolamine to plasmenylcholine (Mogelson and Sobel, 1981). It is not known if more than one enzyme is required for the complete A^-methylation of plasmenylethanolamine as in the case of phosphatidylethanolamine (Higgins, 1981) or if the enzyme(s) catalyzing the A^-methylation of plasmenylethanolamine are identical to those previously characterized for phosphatidylethanolamine. However, it seems unlikely that this pathway contributes substantively to plasmenylcholine biosynthesis since: (a) the measured biosynthetic capacity of this pathway is relatively low (Mogelson and Sobel, 1981) as compared to the metabolic capacity of enzymes responsible for plasmenylcholine catabolism; (b) myocardial phospholipase D activity is diminutive (Schmid et al., 1983); and (c) at each concentration of S-adenosyl methionine studied phosphatidylethanolamine was A^-methylated more rapidly than plasmenylethanolamine (Mogelson and Sobel, 1981) and A^-methylation of phosphatidylethanolamine is a quantitatively minor component of phosphatidylcholine synthesis. However, A^methylation of plasmenylethanolamine may be of importance under some pathophysiologic conditions in which accelerated A^-methylation has been demonstrated (e.g., Horrocks et al., 1986a,b).
Plasmalogen Metabolism D.
179
Plasmalogen Catabolism During Cellular Perturbation
The identification of the unique conformational motif of plasmalogen molecular species (Han and Gross, 1990), their enrichment in arachidonic acid (Chilton et al., 1984; Gross, 1984; Ford and Gross, 1989a), their accelerated turnover during cellular stimulation (Ford and Gross, 1989a; Tessner et al., 1990), and the identification of plasmalogen-selective phospholipases A2 each serve to underscore the importance of plasmalogen molecular species in cellular activation. This portion of the chapter will focus on salient aspects of plasmalogen catabolism including the turnover of plasmalogen molecular species in activated cells, recognition of plasmalogens by intracellular phospholipases, and the importance of plasmalogens as storage depots for the arachidonic acid mass released during cellular activation in several representative cell types including cardiac myocytes, smooth muscle cells, and neutrophils. E. Plasmalogen Catabolism in Myocardium
The demonstration that plasmalogens are localized in specific subcellular loci (i.e., sarcolemma and sarcoplasmic reticulum. Gross, 1984; Gross, 1985) suggested their importance as critical components essential for the appropriate physiologic function of myocardium. It has long been appreciated that ischemia is accompanied by accelerated phospholipid metabolism (Boime et al., 1970) which results in the accumulation of amphiphilic metabolites (e.g., lysophospholipids and arachidonic acid) as well as the release of oxygenated arachidonate metabolites from ischemic myocardium subjected to reperfusion (Hsueh et al., 1977). Since sarcolemmal plasmalogens are enriched in arachidonic acid (Gross, 1984), and since myocardium contains a plasmalogen-selective phospholipase A2, sarcolemmal plasmalogens have been implicated as a source of the arachidonic acid released during myocardial ischemia. Recently, evidence demonstrating the importance of accelerated sarcolemmal plasmalogen catabolism in ischemic myocardium has been accrued utilizing quantitative electron microscopic autoradiography of isolated myocytes subjected to metabolic deprivation. Sarcolemmal phospholipids were the predominant target of accelerated catabolism during metabolic deprivation as assessed by the selective incorporation of arachidonic acid into sarcolemmal phospholipids resulting from deacylation-reacylation cycling in reversibly injured cardiac myocytes (Miyazaki et al., 1990). These observations in tissue culture systems have been substantiated in intact tissue studies which demonstrated the activation of microsomal calcium-independent plasmalogen-selective phospholipase A2 during brief (i.e., reversible) intervals of myocardial ischemia. Activation of this phospholipase A2 is reversible upon reperfusion (Ford et al., 1991; Hazen et al., 1991a) and the temporal course of alterations in phospholipase A2 activation precisely parallels the temporal course of alterations in glycolytic flux. The physiological significance of accelerated plasmalogen catabolism during myocardial
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ischemia is underscored by the enrichment of arachidonoylated molecular species of plasmalogens in the sarcolemma of cardiac muscle cells and the fact that arachidonic acid has profound effects on ion channel function. Accordingly, the accelerated hydrolysis of sarcolemmal phospholipids results in alterations in the physicochemical properties of the membrane due to accumulation of amphiphilic constituents which are tightly coupled with the function of critical transmembrane proteins such as ion channels (Lenaz, 1987; Kim and Clapham, 1989; Alvermann et al., 1992). Thus, the selective hydrolysis of sarcolemmal plasmalogens and the consequent release of arachidonic acid during myocardial ischemia results in a sarcolemmal microenvironment enriched in arachidonic acid and lysophospholipids which would have profound effects on K"^ channel function, ligand-receptor coupling, and ion pump function. Accordingly, the attenuation of accelerated plasmalogen turnover and arachidonic acid release mediated by the activation of calcium-independent phospholipase A2 in ischemic myocardium represents an important target for pharmacologic intervention in the treatment of electrophysiologic dysfunction and myocytic cellular necrosis during ischemia. Although numerous studies suggested the importance of accelerated phospholipid metabolism during ischemic injury, previous attempts to document activation of myocardial phospholipase A2 during ischemia have been unsuccessful utilizing diacyl phospholipid substrates (Das et al., 1986; Bentham et al., 1987). Recently, the synthesis of plasmalogen substrates have been instrumental in the identification of the activation of phospholipase A2 This ischemia-activated phospholipase A2 is membrane-associated, calcium-independent and preferentially cleaves plasmalogen substrate (Ford et al., 1991; Hazen et al., 1991a). Remarkably, membrane-associated, calcium-independent, plasmalogen-selective phospholipase A2 activity was demonstrated to increase over fivefold during two minutes of global ischemia, was nearly maximally activated (>eightfold) after only five minutes of ischemia, and remained activated throughout a one hour ischemic interval (Figure 5). Activation of plasmalogen-selective phospholipase A2 was shown to be rapidly reversible after reperfiision of ischemic tissue (Figure 5). Furthermore, the activation of plasmalogen-selective phospholipase A2 occurred concordantly with activation of anaerobic metabolism (as assessed by myocardial lactate levels). Thus, activation of calcium-independent phospholipase A2 is one of the earliest measurable biochemical manifestations of acute myocardial ischemia, occurs prior to irreversible myocardial ischemic injury (as determined by the absence of electron microscopic evidence of cellular damage at these early time points), and is entirely reversible with reperfiision. Furthermore, the membrane-associated calcium-independent phospholipase A2 is specifically inhibited by the mechanism-based inhibitor, (£)-6-(bromomethylene)tetrahydro-3-( 1 -naphthalenyl)- 2H-pyran-2-one. Accelerated plasmalogen catabolism during myocardial ischemia can also occur by the acfivation of myocardial phospholipases C or D. The possibility that l-(9-alk-r-enyl-2-acyl-5«-glycerol accumulation during myocardial ischemia occurs through phospholipase C activation is suggested by the demonstration that
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Experimental Interval (min) Figure 5. The concordant activation of membrane-associated phospholipase A2 activity and glycolysis during myocardial ischemia and their reversibility during reperfusion. Ischemic and reperfused rabbit hearts were initially perfused for a 10 minute equilibration interval before a 0-60 minute experimental period. After the initial 10 minute pre-equilibration interval, hearts were rendered ischemic for 2, 5, or 60 minutes (—), or rendered ischemic for five minutes and subsequently reperfused for an additional 10 minutes (i.e., 15 minute data points), or reperfused for an additional 55 minutes (i.e., 60 minute data points) ( ). Phospholipase A2 activity was assessed by incubating microsomes (8 ^g) from either control, ischemic, or reperfused rabbit hearts with 16:0, [ H] 20:4 plasmenylcholine (•) in the presence of 4 m M EGTA as previously described (Hazen et al. 1991). Tissue lactic acid content (n) was quantitated spectrophotometrically. In control hearts phospholipase A2 activity as well as lactate production were not elevated.
myocardium contains a neutral active phospholipase C that catalyzes the hydrolysis of plasmalogen molecular species whose activity is regulated by an endogenous inhibitor (Wolf and Gross, 1985). The demonstration that l-O-alk-l'-enyl-l-acylsn-g\ycQTo\ accumulates during brief myocardial ischemia (Ford and Gross, 1989b) (two and fivefold following 20 and 60 minutes of global ischemia, respectively) illustrates the likelihood of accelerated plasmalogen polar head group turnover during the ischemic interval (Figure 6). In sharp contrast, diacyl glycerol content decreases during myocardial ischemia (Ford and Gross, 1989b). The discordant changes in l-0-alk-r-enyl-2-acyl-5«-glycerol and diacyl glycerol content in ischemic myocardium likely reflects the disparate rates of metabolic clearance of each diradyl glycerol molecular subclass. Since 1 -(9-alk-1 '-enyl-2-acyl-5«-glycerol is a poor substrate for both diglyceride kinase and diglyceride lipase which function to remove diacyl glycerol, the potential for accumulation of r-O-alk-enyl-2-acylglycerol is substantially greater than that of diacyl glycerol (Ford and Gross, 1990a).
DAVID A. FORD and RICHARD W. GROSS
182
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Figure 6. The accumulation of 1-0-alk-r-enyI-2-acyl-sn-gIycerol in perfused and ischemic rabbit myocardium. Rabbit hearts were either perfused normally or rendered ischemic for the indicated times. Rabbit myocardial 1-0-alk-1'-enyl-2-acyl-sn-glycerol was quantitated after purification as previously described (Ford and Gross, 1988). Values represent the mean ± SEM for six determinations.
It should be recognized that l-0-alk-r-enyl-2-acyl-5«-glycerol content was observed to increase prior to arachidonic acid accumulation (i.e., arachidonic acid accumulation was not detected until 60 minutes of global ischemia) (Ford and Gross, 1989b). The only l-0-alk-r-enyl-2-acyl-5«-glycerol molecular species that was detected in both control and ischemic rabbit hearts was l-O-hexadec-T-enyl2-acyl-5«-glycerol. Since this molecular species is enriched in plasmenylcholine, but only present in moderate amounts in plasmenylethanolamine, these findings suggested that l-0-alk-r-enyl-2-acyl-5«-glycerol production was mediated by phospholipase C-catalyzed hydrolysis of plasmenylcholine. Alternatively, it should be recognized that another potential source of l-O-alk-T-enyl-l-acyl-^w-glycerol is via the selective hydrolysis of specific molecular species of plasmenylethanolamine containing a 16:0 vinyl ether at the sn-\ carbon by phospholipase C or its equivalent (i.e., sequential phospholipase D and phosphatidate phosphohydrolase activities). The temporal course of 1 -O-alk-1 '-enyl-2-acyl-5n-glycerol accumulation is similar to that of the increase in intracellular free calcium during myocardial ischemia. Accordingly, 1 -0-alk-1 '-enyl-2-acyl-5'«-glycerol and calcium could synergistically
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183
activate specific myocardial protein kinase C isozymes during the ischemic event. It is of interest that l-(9-alk-r-enyl-2-acyl-5«-glycerol may induce the specific phosphorylation of critical myocardial proteins since it is a potent activator of myocardial protein kinase C which possesses an obligatory requirement for physiologic increments in free calcium concentration (Ford and Gross, 1990b). In contrast, myocardial protein kinase C activity activated by diacyl glycerol is partially calcium-independent (Ford and Gross, 1990b). The calcium requirement for the activation of individual protein kinase C isozymes by l-O-alk-r-enyl-2acyl-5«-glycerol was further elucidated after the demonstration that the purified P isozyme of protein kinase C is activated by diacyl glycerol in the absence of free calcium while l-0-alk-r-enyl-2-acyl-5«-glycerol-mediated activation of the P isozyme has an absolute requirement for physiologic increments in free calcium (Ford et al., 1989). In sharp contrast, activation of the purified a isozyme of protein kinase C by both l-0-alk-r-enyl-2-acyl-5«-glycerol and diacyl glycerol requires physiologic increments in calcium ion. Taken together, these results demonstrate that accelerated plasmalogen polar head group hydrolysis results in the generation of a plasmalogen diradyl glycerol, l-0-alk-r-enyl-2-acyl-5«-glycerol, that potentially mediates alterations in myocardial performance through the activation of distinct isozymes of myocardial protein kinase C which, in turn, phosphorylates specific myocardial proteins. F. Plasmalogen Catabolism in Smooth Muscle Cells
The regulation of phospholipid catabolism in smooth muscle cells has been the focus of intense investigation since phospholipid-derived second messengers including eicosanoids, diglycerides, and platelet activating factor have profound effects on vascular smooth muscle contractility. In fact, the production of specific eicosanoids in the vascular bed represents a major mechanism responsible for the regulation of vascular tone and the appropriate distribution of blood flow to specific organs to fulfill each tissue's hemodynamic requirement. Accordingly, the mechanisms responsible for the release of arachidonic acid from vascular smooth muscle cells, as well as other cells in the vascular bed (e.g., endothelial cells), have been extensively investigated. The goal of many of these studies is to identify the source of released arachidonic acid, the phospholipase(s) mediating release with each agonist (i.e., the subcellular compartment targeted for hydrolysis and the individual molecular species which are hydrolyzed) and to identify the physiologic importance of the released metabolites in the vasculature. Much of the work in the field of arachidonic acid release in activated smooth muscle cells has focused on inositol phospholipids which are rapidly hydrolyzed after stimulation with many agonists (e.g., vasopressin, angiotensin). However, the majority of arachidonic acid mass released in stimulated smooth muscle cells likely results from phospholipase A2-mediated cleavage of choline and ethanolamine glycerophospholipids. Several lines of evidence support this conclusion. First,
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DAVID A. FORD and RICHARD W. GROSS
plasmenylethanolamine is the major endogenous phospholipid storage depot of arachidonic acid in rabbit aortic intimal smooth muscle. In fact, 80% of the arachidonic acid present in aortic smooth muscle cell phospholipid is sequestered in plasmenylethanolamine molecular species (Ford and Gross, 1989a). Second, vasopressin stimulation of cells results in major losses in arachidonic acid content in choline and ethanolamine glycerophospholipids. Utilizing aortic rings prelabeled with [^H] arachidonic acid the specific activities of the choline and inositol glycerophospholipid pools were similar while ethanolamine glycerophospholipid had a specific activity of only 20% of that present in the choline and inositol glycerophospholipid pools. Despite the marked disparity in the specific activities of these three phospholipid classes after the prelabeling interval employed, angiotensin II stimulation resulted in nearly identical fractional losses (35-41%) of [^H] arachidonic acid from aortic smooth muscle cell choline, ethanolamine, and inositol glycerophospholipid classes demonstrating that plasmenylethanolamine, due to its high arachidonic acid content and low specific activity, was the major source of released arachidonic acid mass in this system. Reverse phase HPLC confirmed that over 60% of the arachidonic acid released from ethanolamine glycerophospholipids during angiotensin II stimulation originated from plasmenylethanolamine molecular species (Ford and Gross, 1989a). The demonstration that arachidonic acid was selectively released from vascular smooth muscle cell plasmenylethanolamine pools suggested that vascular smooth muscle contained plasmalogen-selective phospholipase(s) A2 which facilitated the release of arachidonic acid from specific pools. To clarify the salient kinetic characteristics of the phospholipase(s) A2 in smooth muscle cells, in vitro analyses of their calcium requirements, substrate selectivities, and kinetic properties were performed. Three separate and distinct phospholipase A2 activities are present in vascular smooth muscle including: (a) a cytosolic calcium-independent phospholipase A2 that is activated by nucleotide di- and triphosphates; (b) a cytosolic calcium-dependent phospholipase A2 which is activated by physiologic increments in calcium ion concentration; and (c) a microsomal calcium-independent phospholipase A2 which is highly selective for plasmenylcholine substrate (Miyake and Gross, 1992). Traditionally, the importance of a given enzyme or protein responsible for a specific physiologic or pathophysiologic effect after cellular perturbation has been clarified through utilization of inhibitors which possess substantial specificity for the reactions or processes under consideration. In the case of phospholipases, the assignment of the role of individual polypeptides in the release of arachidonic acid after agonist stimulation represents a critical issue both for the understanding of smooth muscle cell biochemistry as well as identification of the mechanisms of potential therapeutic effects. Our ability to dissect critical biochemical events leading to the release of arachidonic acid after smooth muscle cell activation has been hampered by the relatively poor selectivity of existing pharmacologic agents for the inhibition of specific types of phospholipase A2. In many cases, utilization
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of nonspecific inhibitors has produced ambiguous results which has led to substantial confusion regarding the importance of a given type of phospholipase in mediating arachidonic acid release during a specific agonist-mediated event. Accordingly, the identification of inhibitors which possess selectivity for each specific type of intracellular phospholipase would be of great utility in identifying the contributions of each of the individual phospholipases A2 in the release of arachidonic acid. Recognizing that one suicide inhibitor previously developed by Katzenellenbogen and co-workers (Daniels et al., 1983; Daniels and Katzenellenbogen, 1986), contained a vinyl ether linkage (a chemical moiety which was specifically recognized by calcium-independent phospholipases) and an oxyester linkage which was potentially susceptible to hydrolysis by calcium-independent phospholipase A2, Hazen et al. (1991b) identified (F)-6-(bromomethylene)tetrahydro-3-(l-naphthalenyl)-2H -pyran-2-one (HELSS) as a specific mechanism-based inhibitor which possessed over a 1,000-fold selectivity for inhibition of calciumindependent phospholipase A2 in comparison to calcium-dependent phospholipase A2. Since HELSS did not contain a charged functionality, it was reasonable to expect that it could freely diffuse through cellular membranes. The thoracic aortic cell line, A10 smooth muscle cells, expresses vasopressin receptors of the VI subtype, whose stimulation results in the selective release of arachidonic acid. Accordingly, we exploited the specificity inherent in mechanism-based inhibition to identify the intracellular phospholipase(s) responsible for arachidonic acid release during vasopressin stimulation of aortic smooth muscle cells. Prelabeling of smooth muscle A10 cells with [^H]-arachidonic acid followed by treatment with 1 )LiM arginine vasopressin for five minutes resulted in the release of approximately 4% of total [^H] arachidonic acid in cellular phospholipids (Lehman et al., 1993). The only metabolite released by these cells was [^H]-arachidonic acid without demonstrable lipoxygenase or cyclooxygenase metabolites observed (i.e., these smooth muscle cells do not contain substantial quantities of eicosanoid oxidative enzymes). Remarkably, treatment of smooth muscle A10 cells with only 1 JLIM HELSS resulted in the inhibition of over one-half of pH] arachidonic acid release, and treatment with 5 JLIM HELSS resulted in the inhibition of over two-thirds of [^H]-arachidonic acid release. Thus, the majority of arachidonic acid release by arginine vasopressin was attributed to calcium-independent phospholipase A2. Since arachidonic acid attenuates the rate of myosin light chain dephosphorylation and is associated with increased smooth muscle cell contractility, it seems likely that calcium-independent phospholipase A2 is an important modulator of the increase in contractile force in smooth muscle mediated by arginine vasopressin stimulation. G. Plasmalogen Catabolism in Neutrophils Accelerated plasmalogen catabolism in activated neutrophils plays a major role in the production of eicosanoids as well as platelet acfivating factor during the
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inflammatory response. The importance of accelerated ether lipid catabolism, arachidonic acid release, and platelet activating factor biosynthesis in neutrophils can easily be demonstrated simply by recognizing that 66% of the arachidonic acid in the choline glycerophospholipid pool is present in alkyl ether choline glycerophospholipids (i.e., the precursor of platelet activating factor) and 71% of arachidonic acid in the ethanolamine glycerophospholipid pool is present in the plasmalogen subclass (Chilton and Connell, 1988). Initial investigations predominantly focused on the concomitant production of arachidonic acid and platelet activating factor utilizing calcium ionophore- or zymosan-stimulated neutrophils (Chilton et al., 1984). Since platelet activating factor production is initiated by the hydrolysis of arachidonic acid from alkyl-acyl-glycerophosphoryl choline, it was envisaged that a single enzymatic reaction (i.e., phospholipase A2) could result in the initiation of two classes of lipid mediators (i.e., eicosanoids and platelet activating factor). The concept that platelet activating factor and eicosanoid production are initiated through phospholipase A2-mediated hydrolysis of alkyl-acylGPC was further supported by experiments demonstrating similar specific activities in alkyl-acyl-GPC, leukotriene B4, and 20-hydroxy-leukotriene B4 in stimulated neutrophils that were prelabeled with [^H]-arachidonic acid (Chilton, 1989). Since phospholipase A2 likely mediates the release of arachidonic acid and the initiation of platelet activating factor production in stimulated neutrophils, substantial effort has been directed toward identifying the mechanism of phospholipase A2 activation in neutrophils. One hypothesis that has been given considerable attention is that the activation of phospholipase A2 in stimulated neutrophils is mediated by the activation of protein kinase C through diradyl glycerols produced after agonist stimulation. This hypothesis was first supported by the demonstration that neutrophils stimulated with the chemotactic peptide,flVILP,accumulate l-(9-alkyl-2-acyl^«-glycerol and l,2-diacyl-5«-glycerol (Rider et al., 1988). The production of 1-0-alkyl-2-acyl-5«-glycerol in stimulated neutrophils is mediated by phospholipase D-catalyzed hydrolysis of alkyl-acyl-GPC followed by phosphatidate phosphohydrolase-mediated hydrolysis of the resultant phosphatidic acid (Chabot et al., 1992; Strum et al, 1993). Diradyl glycerol-mediated regulation of neutrophil phospholipase A2 has been supported by the demonstration that 1,2-diacyl-5«-glycerol and l-(9-alkyl-2-acyl-5/7-glycerol not only prime neutrophil respiratory bursts for stimulation by fMLP, but l,2-diacyl-5«-glycerol and l-(9-alkyl-2-acyl also prime neutrophils for arachidonic acid release during fMLP stimulation (Bauldry et al, 1988). Of further interest is the observation that only l,2-diacyl-5'«-glycerol and not l-0-alkyl-2-acyl-5«-glycerol primes neutrophil production of LTB4 (Bauldry et al., 1991). Taken together, it is likely that diradyl glycerols modulate neutrophil phospholipase A2 and possibly lipoxygenase activities. Several recent studies have elucidated the role of plasmalogen catabolism in neutrophil arachidonic acid release and platelet activating factor production. The
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observation that plasmenylethanolamine catabolism resulted in lysoplasmenylethanolamine accumulation in stimulated neutrophils (Tessner et al., 1990) led to the discovery of a novel enzymatic pathway for platelet activating factor biosynthesis in neutrophils which is initiated by phospholipase A2-mediated hydrolysis of plasmenylethanolamine (Nieto et al, 1991; Uemura et al., 1991). Via this pathway, the subsequent transacylation of lysoplasmenylethanolamine with arachidonic acid from l-(9-alkyl-2-arachidonoyl-GPC generates lyso-platelet activating factor. Lyso-platelet activating factor is then readily acetylated by a reaction mediated by acetyl CoA transferase or a CoA-independent transacetylation pathway (Lee et al., 1986) resulting in the synthesis of platelet activating factor. Accelerated plasmalogen catabolism in activated neutrophils also results in the accumulation of an acetylated plasmenylethanolamine species (Tessner and Wykle, 1987). Although this plasmalogenic platelet activating factor analog is chemically similar to platelet activating factor, it does not share the biological activities of platelet activating factor and it has not yet been assigned a biological activity. The biosynthetic pathway for plasmalogenic platelet activating factor resembles that of platelet activating factor, since lysoplasmenylethanolamine can be converted to its acetylated platelet activating factor analog by either a CoA-independent transacetylase activity utilizing the acetate of platelet activating factor as the donor or via an acetyl transferase pathway (Lee et al., 1992). Collectively, multiple studies have underscored the importance of the hydrolysis of arachidonoylated plasmenylethanolamine in activated neutrophils catalyzed by phospholipase A2 resulting in the generation not only of arachidonic acid and its bioactive oxygenated products, but also the initiation of the synthesis of other bioactive lipid second messengers. Since these lipid second messenger molecules likely are key elements in inflammatory responses, specific inhibitors would be of great utility in attenuating the accumulation of these biomolecules. Furthermore, since the hydrolysis of plasmenylethanolamine is an initial step in the cascade in the synthesis of these molecules, the identification of inhibitors that selectively inhibit plasmenylethanolamine hydrolysis by phospholipase A2 represent a particularly attractive pharmaceutical target.
IV. CONCLUSION The last decade has witnessed remarkable advances in our understanding of the structure and function of ether phospholipids. The observation that plasmalogens represent the predominant phospholipid constituents of the sarcolemmal membrane of myocardium has underscored the importance of plasmalogen constituents in membrane function. The enrichment of arachidonic acid in plasmalogen molecular species in conjunction with the demonstration of the activation of plasmalogen-selective phospholipases A2 has now provided unambiguous evidence delineating the
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role of the plasmalogen subclass as a reservoir of eicosanoids released during cellular activation. Concomitant with the rapid expansion of our knowledge on plasmalogen conformation, catabolism and the role of plasmalogens in cellular function, the current concepts of rapid polar head group turnover in the de novo synthesis of plasmenylcholine have evolved. It is important to realize that these insights on plasmalogen structure, function, and metabolism do not circumscribe the entire plasmalogen dynamic but, rather, provide the initial insights into the molecular mechanism and covalent structures which underlie nature's own specialized system integrating form and function.
ACKNOWLEDGMENT This research was supported by NIH grant HL42665.
REFERENCES Alvermann, G., Ford, D.A., Friedrich, M., Gross, R.W., Han, X., Hirche, H., Zupan, L,A., & Benndorf, K. (1992). Lysoplasmenylcholine (LPC) decreases Ca-current (I^a) in isolated guinea pig heart cells. Pflugers Arch. 420, R80. Bauldry, S.A., Wykle, R.L., & Bass, D,A. (1988). Phospholipase A2 activation in human neutrophils: Differential actions of diacylglycerols and alkylacylglycerols in priming cells for stimulation by A^-formyl-met-leu-phe. J. Biol. Chem. 263, 16787-16795. Bauldry, S.A., Wykle, R.L., & Bass, D.A. (1991). Differential actions of diacyl- and alkylacylglycerols in priming phospholipase A2, 5-lipoxygenase and acetyltransferase activation in human neutrophils. Biochim. Biophys. Acta 1084, 178-184. Bentham, J.M., Higgins, A. J., & Woodward, B. (1987). The effects of ischemia, lysophosphatidylcholine and palmitoylcamitine on rat heart phospholipase A2 activity. Basic Res. Cardiol. 82, 127—135. Blank, M.L., Lee, T.C., Cress, E.A., Fitzgerald, V., & Snyder, F. (1986). Plasmalogen biosynthesis in Madin-Darby canine kidney cells: Selectivity in the acylation of l-alkyl-2-lyso-5n-glycerol-3phosphoethanolamine and the subsequent desaturation step. Arch. Biochem. Biophys. 251,55-60. Blank, M.L., Fitzgerald, V., Lee, T.C., & Snyder, F. (1993). Evidence for biosynthesis of plasmenylcholine from plasmenylethanolamine in HL-60 cells. Biochim. Biophys. Acta 1166, 309-312. Boime, I., Smith, E.E., & Hunter, F.E. (1970). The role of fatty acids in mitochondrial changes during liver ischemia. Arch. Biochem. Biophys. 139, 425-443. Chabot, M.C., McPhail, L.C., Wykle, R.L., Kennedy, D.A., & McCall, C.E. (1992). Comparison of diglyceride production from choline-containing phosphoglycerides in human neutrophils stimulated with iV-formylmethionyl-leucylphenylalanine, ionophore A23187 or phorbol 12-myristate 13-acetate. Biochem. J. 286, 693-699. Chilton, F.H., Ellis, J.M., Olson, S.C, & Wykle, R.L. (1984). l-0-Alkyl-2-arachidonoyl-5«-glycero-3phosphocholine: A common source of platelet activating factor and arachidonate in human polymorphonuclear leukocj^es. J. Biol. Chem. 259, 12014-12019. Chilton, F.H. & Connell, T.R. (1988). 1-Ether-linked phosphoglycerides: Major endogenous sources of arachidonate in the human neutrophil. J. Biol. Chem. 263, 5260-5265. Chilton, F.H. (1989). Potential phospholipid source(s) of arachidonate used for the synthesis of leukotrienes by the human neutrophil. Biochem. J. 258, 327-333.
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Daniels, S.B., Cooney, E., Sofia, M.J., Chakravarty, P,K., & Katzenellenbogen, J.A. (1983). Haloenol lactones. Potent enzyme-activated irreversible inhibitors for alpha-chymotrypsin. J. Biol. Chem. 258, 15046-15053. Daniels, S.B. & Katzenellenbogen, J.A. (1986). Haloenol lactones: Studies on the mechanism of inactivation of alpha-chymotrypsin. Biochemistry 25, 1436-1444. Das, D.K., Engelman, R.M., Rousou, J.A., Breyer, R.H., Otani, H., & Lemeshow, S. (1986). Role of membrane phospholipids in myocardial injury induced by ischemia and reperfusion. Am. J. Physiol. 251, H71-H79. Ford, D.A. & Gross, R.W. (1988). Identification of endogenous l-0-alk-r-enyl-2-acyl-5«-glycerol in myocardium and its effective utilization by choline phosphotransferase. J. Biol. Chem. 263, 264^2650. Ford, D.A. & Gross, R.W. (1989a). Plasmenylethanolamine is the major storage depot for arachidonic acid in rabbit vascular smooth muscle and is rapidly hydrolyzed after angiotensin II stimulation. Proc. Natl. Acad. Sci. USA 86, 3479-3483. Ford, D.A. & Gross, R.W. (1989b). Differential accumulation of diacyl and plasmalogenic diglycerides during myocardial ischemia. Circ. Res. 64, 173—177. Ford, D.A., Miyake, R., Glaser, P.E., & Gross, R.W. (1989). Activation of protein kinase C by naturally occurring ether-linked diglycerides. J. Biol. Chem. 264, 13818-13824. Ford, D.A. & Gross, R.W. (1990a). Differential metabolism of diradyl glycerol molecular subclasses and molecular species by rabbit brain diglyceride kinase. J. Biol. Chem. 265, 12280-12286. Ford, D.A. & Gross, R.W. (1990b). Activation of myocardial protein kinase C by plasmalogenic diglycerides. Am. J. Physiol. 258, C30-C36. Ford, D.A., Hazen, S.L., SafTitz, J.E., & Gross, R.W. (1991). The rapid and reversible activation of a calcium-independent plasmalogen-selective phospholipase A2 during myocardial ischemia. J. Clin. Invest. 88,331-335. Ford, D.A., Rosenbloom, K.B., & Gross, R.W, (1992). The primary determinant of rabbit myocardial ethanolamine phosphotransferase substrate selectivity in the covalent nature of the sn-\ aliphatic group of diradyl glycerol acceptors. J. Biol. Chem. 267, 11222-11228. Gross, R.W. (1984). High plasmalogen and arachidonic acid content of canine myocardial sarcolemma: A fast atom bombardment mass spectroscopic and gas chromatography-mass spectroscopic characterization. Biochemistry 23, 158-165. Gross, R.W. (1985). Identification of plasmalogen as the major phospholipid constituent of cardiac sarcoplasmic reticulum. Biochemistry 24, 1662-1668. Hajra, A.K. (1970). Acyl dihydroxyacetone phosphate: Precursor of alkyl ethers. Biochem. Biophys. Res. Commun. 39, 1037-1044. Han, X. & Gross, R.W. (1990). Plasmenylcholine and phosphatidylcholine membrane bilayers possess distinct conformational motifs. Biochemistry 29, 4992-4996. Han, X. & Gross, R.W (1991). Modulation of cardiac membrane fluidity by amphiphilic compounds and their role in the pathophysiology of myocardial infarction. In: Drug and Anesthetic Effects on Membrane Structure and Function (Aloia, R.C., Curtain, C.C., & Gordon, L.M., eds.), pp. 225-243. Wiley-Liss, New York. Hazen, S.L., Ford, D.A., & Gross, R.W. (1991a). Activation of a membrane-associated phospholipase A2 during rabbit myocardial ischemia which is highly selective for plasmalogen substrate. J. Biol. Chem. 266, 5629-5633. Hazen, S.L., Zupan, L.A., Weiss, R.H., Getman, D.R, & Gross, R.W (1991b). Suicide inhibition of canine myocardial cytosolic calcium-independent phospholipase A2. J. Biol. Chem. 266, 7227— 7232. Higgins, J.A. (1981). Biogenesis of endoplasmic reticulum phosphatidylcholine. Translocation of intermediates across the membrane bilayer during methylation of phosphatidylethanolamine. Biochim. Biophys. Acta 640, 1-15.
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Horrocks, L.A., Harder, H.W., Mozzi, R., Goracci, G., Francescangeli, E., Porcellati, S., & Nenci, G.G. (1986a). Receptor-mediated degradation of choline plasmalogens and glycerophospholipid methylation: Anew hypothesis. In: Enzymes of Lipid Metabolism II (Frey, S.Z.L., Dreyfus, H., Massarelli, R., & Gatt, S.), pp. 707-711. Plenum, New York. Horrocks, L.A., Yeo, Y.K., Harder, H.W., Mozzi, R., & Goracci, G. (1986b). Choline plasmalogens glycerophospholipid methylation; and receptor-mediated activation of adenylate cyclase. Advances in Cyclic Nucleotide and Protein Phosphorylation Research. 20, 263-292, Raven, New York. Hsueh, W., Isakson, P.C, & Needleman, P. (1977). Hormone selective lipase activation in the isolated rabbit heart. Prostaglandins 13,1073-1091. Kim, D. & Clapham, D.E. (1989). Potassium channels in cardiac cells activated by arachidonic acid and phospholipids. Science 244, 1174-1176. Kiyasu, J.Y. & Kennedy, E.P. (1960). The enzymatic synthesis of plasmalogens. J. Biol. Chem. 235, 2590-2594. Lee, T., Malone, B., & Snyder, F. (1986). A new de novo pathway for the formation of l-alkyl-2-acetyl5«-glycerols, precursors of platelet activating factor. J. Biol. Chem. 261, 5373-5377. Lee, T.-C, Uemura, Y, & Snyder, F. (1992). A novel CoA-independent transacetylase produces the ethanolamine plasmalogen and acyl analogs of platelet-activating factor (PAF) with PAF as the acetate donor in HL-60 cells. J. Biol. Chem. 267, 19992-20001. Lehman, J.J., Brown, K.A., Ramanadham, S., Turk, J., & Gross, R.W. (1993), Arachidonic acid release from aortic smooth muscle cells induced by [Arg ]vasopressin is largely mediated by calcium-independent phospholipaseA2. J. Biol. Chem. 268, 20713-20716. Lenaz, G. (1987). Lipid fluidity and membrane protein dynamics. Bioscience Reports 7, 823-837. Lumb, R.H. & Synder, F. (1971). Arapid isotopic methods for assessing the biosynthesis of ether linkages in glycerolipids of complex systems. Biochim. Biophys. Acta 244, 217—221. Miyake, R. & Gross, R.W. (1992). Multiple phospholipase A2 activities in canine vascular smooth muscle. Biochim. Biophys. Acta 1165, 167—176. Miyazaki, Y, Gross, R.W., Sobel, B.E., & Saffitz, J.E. (1990). Selective turnover of sarcolemmal phospholipids with lethal cardiac myocytes injury. Am. J. Physiol. 259, C325-C331. Mogelson, S. & Sobel, B. E. (1981). Ethanolamine plasmalogen methylation by rabbit myocardial membranes. Biochim. Biophys. Acta 666, 205-211. Nieto, M.L., Venable, M.E., Bauldry, S.A., Greene, D.G., Kennedy, M., Bass, D.A., & Wykle, R.L. (1991). Evidence that hydrolysis of ethanolamine plasmalogens triggers synthesis of plateletactivating factor via a transacylation reaction. J. Biol. Chem. 266, 18699-18706. Paltauf, F. & Holasek, A. (1973). Enzymatic synthesis of plasmalogens. J. Biol. Chem. 248,1609-1615. Rider, L.G., Dougherty, R.W., & Niedel, J.E. (1988). Phorbol diesters and dioctanoylglycerol stimulate accumulation of both diacylglycerols and alkylacylglycerols in human neutrophils. J. Immunology 140, 200-207. Rizzo, W.B, Craft, D.A., Dammann, A.L., & Phillips, M.W. (1987). Fatty alcohol metabolism in cultured human fibroblasts. J. Biol. Chem. 262, 17412-17419. Schmid, H.H.O., Muramatsu, T., & Su, K.L. (1972). On the nonconversion of alkyl acyl choline phosphatides to the corresponding plasmalogens in myelinating rat brain. Biochim. Biophys. Acta 270,317-323. Schmid, RC, Reddy, RV, Natarajan, V., & Schmid, H.H.O. (1983). Metabolism of N-acylethanolamine phospholipids by a mammalian phosphodiesterase of the phospholipase D type. J. Biol. Chem. 258,9302-9306. Snyder, F., Blank, M.L., & Wykle, R.L. (1971). The enzymic synthesis of ethanolamine plasmalogens. J. Biol. Chem. 246, 3639-3645.
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Strum, J.C., Nixon, A.B., Daniel, L.W., & Wykle, R.L. (1993). Evaluation of phospholipase C and D activity in stimulated human neutrophils using a phosphono analog of choline phosphoglyceride. Biochim. Biophys. Acta 1169,25-29. Tessner, T.G. & Wykle, R.L. (1987). Stimulated neutrophils produce an ethanolamine plasmalogen analog of platelet-activating factor. J. Biol. Chem. 262, 12660-12664. Tessner, T.G., Greene, D.G., & Wykle, R.L. (1990). Selective deacylation of arachidonate-containing ethanolamine-linked phosphoglycerides in stimulated human neutrophils. J. Biol. Chem. 265, 21032-21038. Uemura, Y., Lee, T.C., & Snyder, F. (1991). A coenzyme A-independent transacylase is linked to the formation of platelet-activating factor (PAF) by generating the lyso-PAF intermediate in the remodeling pathway. J. Biol. Chem. 266, 8268-8272. Wolf, R.A. & Gross, R.W. (1985). Identification of neutral active phospholipase C which hydrolyzes choline glycerophospholipids and plasmalogen selective phospholipase A2 in canine myocardium. J. Biol. Chem. 260, 7295-7303. Wykle, R.L., Blank, M.L., Malone, B., & Snyder, F. (1972). Evidence for a mixed function oxidase in the biosynthesis of ethanolamine plasmalogens from l-alkyl-2-acyl-5«-glycero-3-phosphorylethanolamine. J. Biol. Chem. 247, 5442-5447. Wykle, R.L. & Snyder, F. (1976). Microsomal enzymes involved in the metabolism of ether-linked glycerolipids and their precursors in mammals. In: Enzymes of Biological Membranes (Martonosi, A., ed.), pp. 87-117. Plenum, New York. Wykle, R.L. & Schremmer, J.M. (1974). A lysophospholipase D pathway in the metabolism of ether-linked lipids in brain microsomes. J. Biol. Chem. 249, 1742-1746.
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PLASMALOGENS, NITROXIDE FREE RADICALS, AND ISCHEMIA-REPERFUSION INJURY IN THE HEART
Richard Schuiz
ABSTRACT 194 I. INTRODUCTION 194 II. UNIQUE ROLE OF PLASMALOGENS IN THE HEART . 196 A. Prevalence 196 B. Arachidonic Acid Content 197 C. Phospholipase(s) A2 Activity 197 D. Effectsof Free Fatty Acids and Eicosanoids on the Heart 198 E. Detrimental Actions of Lysophospholipids 199 III. FREE RADICALS AND ISCHEMIA-REPERFUSION INJURY 201 A. Susceptibility of Plasmalogens to Free Radical Attack 202 B. Questions Regarding the Generation of Oxygen-Derived Free Radicals . 205 C. Potential Role of Nitroxide Free Radicals in Reperfusion Injury 206 D. Role of Peroxynitrite 207
Advances in Lipobiology Volume 1, pages 193-214. Copyright © 1996 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-635-5 193
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ACKNOWLEDGMENTS NOTE ADDED IN PROOF REFERENCES
209 209 209
ABSTRACT Myocardial ischemia and reperfusion injury results in major disturbances in membrane phospholipid homeostasis. The physicochemical properties of the phospholipid microenvironment in the cell membrane affects the function of membrane-bound proteins, ion channels and signal transducers. Alterations in the composition of the phospholipids will result in a disturbance in cellular function. The extent of the changes to the membrane phospholipids may determine whether damage to the myocardium is reversible or irreversible. The myocardium is unique compared to other tissues as it contains a high content of plasmalogens (alkyl-1-enyl phospholipids) in the sarcolemma and sarcoplasmic reticulum, a large proportion of which contain arachidonic acid esterified in the sn-2 position. Two primary mechanisms contributing to changes in the phospholipid environment during myocardial ischemia and reperfusion injury include activation of phospholipases and attack by oxygenderived free radicals. During ischemia there is a rapid activation of Ca^'^-independent phospholipase A2 activity in the myocardium which is plasmalogen-selective. Upon reperfusion of ischemic myocardium, a burst of oxygen-derived free radicals is released which may preferentially attack plasmalogens. The exact nature of the free radicals is disputed, however, nitric oxide released from the endothelial cells of the coronary vasculature and the myocardium could combine with superoxide anion to form peroxynitrite, a potent delivery form of the highly reactive hydroxyl radical. This review will outline the role of these two mechanisms in myocardial ischemia and reperfusion injury and how they may affect myocardial plasmalogens.
I. INTRODUCTION Ischemic heart disease remains one of the leading causes of death in North America (Mickelson et al., 1990). During ischemia (a reduction in blood flow and, thus, deprivation of oxygen and nutrient supply to a portion of the myocardium), a number of pathophysiological changes occur which alter the physicochemical properties of the myocardial phospholipids. The extent of alterations to the phospholipid environment may determine whether the ensuing damage to the ischemic region is either reversible or irreversible. The restoration of blood flow and the reintroduction of oxygen to the previously ischemic myocardium results, paradoxically, in an enhanced deterioration of the phospholipid pool (Das et al., 1986; Otani et al., 1989; Davies et al., 1992). The phospholipid bilayer is the basis for the integrity of the myocyte since it forms the barrier across which the ionic gradients of the cell membrane are formed. Furthermore, the activity of membrane associated pumps, ion channels, and cellular transducers is dependent upon the nature of the phospholipid microenvironment
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195
O II r— OCH=CHR R-C-0 -\ L O-P-X alkyl 1- enyl 2- acyl (plasmalogen)
O II rR-C-0 -\
0 « 0-C-R
O i pOR R-C-0 - J
Lo-p-x 1,2- diacyl
L_ O-P-X 1-alkyl 2-acyl ether
P-X: phosphocholine, phosphoethanolamine R: aliphatic chain Figure 1. Glycerophospholipids.
surrounding these proteins (Sandermann, 1978; Bennett et al., 1980; Corr et al, 1984; Spector and Yorek, 1985; Han and Gross, 1991). The sarcolemma and sarcoplasmic reticulum of cardiomyocytes are highly enriched in plasmalogens (up to 45% of sarcolemmal phospholipids in human, canine, bovine, and rabbit hearts) and phospholipids with a vinyl ether (alkyl-1-enyl) in the sn-l position of the glycerol backbone (Figure 1). The first double bond of the sn-\ acyl chain has the CIS configuration. The plasmalogens are also highly enriched with unsaturated fatty acids, and in particular, with arachidonic acid, in the sn-2 position (Gross, 1984, 1985). Although the function of plasmalogens in the heart is only beginning to be unraveled, it is now apparent from recent studies that the plasmalogens may play a key role in the phospholipid-mediated alterations which occur during ischemia and ischemia-reperfusion injury (Snyder et al., 1981; Kako, 1986; Hazen et al., 1990; Ford etal., 1991; Hazen etal., 1991a; Daviesetal., 1992; Gross, 1992; Hazen and Gross, 1992). Earlier studies of ischemia or ischemia followed by reperfusion in the heart have shown a variety of changes to the myocardial phospholipids including: (a) accelerated cataboHsm of phospholipids (Sobel et al, 1978; Chien et al., 1981; Shaikh and Downar, 1981; Das et al, 1986; Otani et al, 1989), (b) accumulation of lysophospholipids with arrhythmogenic effects on the myocardium (Sobel et al, 1978; Snyder et al, 1981; Clarkson and Ten Eick, 1983; Akita et al, 1986; Liu et al, 1991), and (c) enhanced release of free fatty acids, including arachidonic acid, which may then be metabolized to prostaglandins and leukotrienes or participate in free radical propagating chain reactions (Van der Vusse et al, 1982; Chien et al, 1984; Karmazyn, 1986; Kuzuya et al, 1987; Sen et al, 1988; Tada et al, 1988). These reports, however, have only measured overall changes in the content of glycerophospholipids or their metabolites and have not specifically addressed
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changes in the plasmalogens, which are now recognized to be the most abundant subclass of phospholipid in the myocardial cell membrane. Activation of phospholipases and attack by oxygen-derived free radicals are the two primary mechanisms acting during ischemia and reperfusion injury to alter the composition of the phospholipids in the cell membrane. During ischemia there is a rapid activation of a plasmalogen-selective phospholipase A2 activity in the myocardium which accounts for the vast majority of overall phospholipase A2 activity observed (Ford et al, 1991; Hazen et al., 1991a; Hazen and Gross, 1992) and which can lead to altered cell membrane properties which can cause dysfunction and even myocyte death. Upon reperfusion, further damage ensues due to the release of oxygen-derived free radicals in the heart which attack numerous cellular targets including the phospholipid membranes. Plasmalogens are more susceptible than diacyl phospholipids to degradation by free radicals (Zoeller and Raetz, 1986; Morand et al., 1988). The precise nature of the free radicals is uncertain, however, most agree that superoxide anion (O2*) and the ultimate production of hydroxyl (OH*) free radicals are involved. It has been suggested that peroxynitrite (0N00~*), an adduct of superoxide anion and nitric oxide (NO) could mediate free radical injury (Beckmann et al., 1990; Beckmann, 1991). The conditions during myocardial reperfusion may be ideal for the release of both superoxide anion and NO. The possible role of peroxynitrite in mediating phospholipid degradation in the ischemic and reperfused heart has not been studied to date. This brief review will present ideas concerning the role of phospholipids in myocardial ischemia and reperfusion injury, including: (a) the unique composition of myocardial phospholipid in terms of its plasmalogen content, (b) the importance of myocardial phospholipids in maintaining cellular homeostasis and how they may be altered during ischemia and reperfiision injury, (c) the role of activation of phospholipase(s) A2 during ischemia, and (d) the possible contribution of oxygenderived free radicals, including myocardial-derived NO, to degradation of phospholipids during reperftision of the ischemic zone. Our knowledge of the role of myocardial plasmalogens is in its infancy, however, recent literature has suggested that this new focus may bring some advances in our understanding of myocardial ischemia and reperfiision injury.
II. UNIQUE ROLE OF PLASMALOGENS IN THE HEART A.
Prevalence
Plasmalogens were first discovered in 1924 by Feulgen and Voit and were recognized to contain a unique chemical moiety as part of their lipid structure since they formed an aldehyde intermediate during acid hydrolysis of a variety of fresh tissue slices for histological processing (see Rapport and Norton, 1962). The chemical structure of the plasmalogens remained elusive until the late 1950s, when the precise structure of the alkyl-1-enyl linkage of the ethanolamine plasmalogens
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Table 1, Species Differences in Myocardial Plasmalogen Content % Total phospholipid Rat Cow Pig Rabbit Dog* Human
12" 40^ 9
31" 45' 32«
% Phosphatidylcholine 1^ 39C 36^ 39' 47f ca. 40*
% Phosphatidylethanolamine 9
ca. 56^
Notes: *Sarcolemma. References: ^Rapport & Norton, 1962; ^Schulz et al., 1993; '^Pugh et al., 1977; ''Shaikh & Downar, 1981; ^Osani & Sakagami, 1979; ^Gross, 1984; ^Horrocks, 1972.
was discovered primarily in the laboratories of Rapport and Marinetti, Gray, Klenk and Debuch (see Snyder, 1991). The tissue specific distribution of the plasmalogens is notable: nervous tissue, white blood cells, testes, uterus, ovaries, kidney, erythrocytes, bone marrow, spleen, platelets, and lipoproteins have considerable plasmalogen content (up to 20% of total phospholipid content), whereas, in contrast, liver contains 3% or less (Rapport and Norton, 1962). Most striking, however, is their marked enrichment in the phospholipids of the heart: in the dog, rabbit, and human heart they account for over 30% of total phospholipid (Table 1). There is, however, a striking species difference since the rat heart contains only 12% plasmalogen. The heart is also unique in that it contains significant quantities of plasmenylcholine, whereas most other tissues contain only very small quantities of this phospholipid. B. Arachidonic Acid Content
Plasmalogens are highly enriched in arachidonic acid esterified in the sn-2 position of the glycerol backbone. In canine sarcolemma, over 40% of plasmenylcholine molecular species and 86% of plasmenylethanolamine molecular species contain arachidonic acid (Scherrer and Gross, 1989). In the rat heart we have shown that 28% of total arachidonic acid found in phosphatidylcholine and -ethanolamine fractions is found esterified to the plasmalogens (Schulz et al., 1993). The plasmalogens in the heart could be a major source of this fatty acid which is a substrate for eicosanoid synthesis during myocardial ischemic injury. C. Phospholipase(s) A2 Activity
Although it is well known that myocardial ischemia is accompanied by a selective release of arachidonic acid (Chien et al., 1984), previous attempts to measure phospholipase A2 activity using conventional diacyl phospholipid substrates in the presence of Ca^"^ failed to detect any increase in phospholipase A2 activity (Das et
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al, 1986). It is now evident that the majority of myocardial phospholipase A2 activity is Ca^"^-independent and selective for plasmalogen substrate (for review see Gross, 1992). The use of an inhibitor of the Ca^"*"-independent phospholipase A2, (E)-6-(bromomethylene)-3-( 1 -napthalenyl)-2//-tetrahydropyran-2-one, with 1,000-fold selectivity over Ca^"^-dependent forms of the enzyme, has been described (Hazen et al., 1991b) and has proven very useful in studying Ca^'^-independent phospholipase A2 activity. There are apparently both membrane associated (Hazen et al, 1991a) and cytosolic (Hazen and Gross, 1991a) forms of the enzyme, the latter of which has been purified to homogeneity (Hazen et al., 1990). The inhibitor is equally effective in blocking the activity of both forms of the enzyme as measured in the ischemic human heart (Hazen and Gross, 1992). Within five minutes of ischemia there is maximal activation of a Ca^"^-independent phospholipase A2 associated with the membrane. This is accompanied by a significant decrease in the cytosolic activity of this enzyme during the ischemic period. The increase in microsomal activity is greater than the loss of cytosolic activity, suggesting that a simple translocation of enzyme is not occurring (Ford et al, 1991). The membrane-bound form accounts for 90% of total plasmalogen specific phospholipase A2 activity in the ischemic rabbit heart, whereas in ischemic human myocardium about 35% of total plasmalogen-specific phospholipase A2 activity is found in the cytosol (Hazen and Gross, 1992). The cytosolic enzyme is stimulated by ATP (Hazen and Gross, 1991a,b). Although bulk ATP concentrations decline within 15 minutes of severe ischemia, it is yet unknown whether other ligands and modes of interaction of the catalytic complex with its microenviroment (phosphorylation, spatial translocation, etc.) may account for continued phospholipase A2 activity during longer periods of ischemia (Gross, 1992). However, during reperfiision, there is a rapid repletion of ATP stores which could then reactivate this e'lzyme. Phospholipid metabolism by action of phospholipase(s) A2 results in the formation of two products, fi"ee fatty acids and lysophospholipids, each with potential detrimental actions on myocardial function. There is a loss of arachidonic acid from the ischemic heart after prolonged ischemia in vivo (Chien et al., 1984) or after depletion of ATP from cultured myocytes in vitro (Chien et al., 1985; Sen et al., 1988). The recent discovery of a myocardial plasmalogen-selective phospholipase A2 activity which is rapidly activated during myocardial ischemia and which preferentially hydrolyzes plasmalogen species containing arachidonic acid in the sn-2 acyl chain (Hazen et al., 1990) suggests that plasmalogens may be a major source of this fatty acid precursor of leukotriene, prostaglandin, and hydroperoxyeicosatetraenoic acid production. D.
Effects of Free Fatty Acids and Eicosanoids on the Heart
Although the capacity of the cardiac myocyte to metabolize arachidonic acid is low (Lucchesi and Mullane, 1986), the arachidonic acid made available by phos-
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pholipase action could be metabolized by cells of the coronary vasculature (Gerritsen and Printz, 1981; Piper etal., 1983) or by the adherent leukocytes associated with later stages of reperfusion injury (Mullane et al., 1984). Whether the eicosanoids could exacerbate myocardial injury, either through actions on coronary flow, thrombus formation, or by direct effects on the myocardium (Karmazyn, 1986) is unknown. Other fatty acids released from the phospholipid pool could have direct actions on ion channels in the cardiac myocyte. Huang et al. (1992) have shown that long-chain fatty acids (greater than 12 carbons in chain length), both saturated and unsaturated, stimulate voltage-dependent Ca^'*"-currents in isolated ventricular myocytes. This occurred at low concentrations of free fatty acids (3—30 |LiM), required the presence of a free carboxyl group, and could not be prevented by inhibitors of eicosanoid production, fatty acid oxidation, or by inhibition of protein kinase A or C. Although oleic acid was the most potent, arachidonic acid was also effective. Some effect of arachidonic acid through its metabolism to prostaglandin or leukotrienes could not, however, be discounted. Whether the release of arachidonic acid and other free fatty acids is significant to the development of irreversible myocardial dysfunction during ischemia-reperfiision injury or merely indicative of the imbalance in phospholipid metabolism must be ascertained. E. Detrimental Actions of Lysophospholipids The evidence regarding the detrimental role of lyosphospholipids in the heart is more compelling. Lysophospholipids are amphiphilic compounds with a molecular geometry which disrupts membrane properties such that the activity of membrane bound proteins is altered (Karli et al., 1979; Briggs and Lefkowitz, 1980). The lysophospholipids have deleterious electrophysiological effects in a number of cardiac tissues including isolated dog (Corr et al., 1979) and sheep Purkinje fibers (Amsdorf and Sawicki, 1981) and in isolated cat papillary muscle (Clarkson and Ten Eick, 1983). Ischemia in the heart results in a rapid increase in the content of lysophospholipids (Sobel et al, 1978; Shaikh and Downar, 1981; Corr et al, 1982; Corr et al., 1989). Early studies documented an acute rise in lysophosphatidylcholine and lysophosphatidylethanolamine within 10 minutes of the onset of ischemia (Sobel etal., 1978; Shaikh and Downar, 1981; Corr etal., 1982). However, these early studies only reported bulk changes in lysophospholipids and did not measure the contribution of lysoplasmalogens. Indeed, acidic extraction procedures which hydrolyze the alkyl-1-enyl bond of plasmalogens may have caused erroneously high levels of lysophospholipids. However, careful extraction and analysis under nonacidic conditions resulted in overall lower levels of lysophospholipids, but still documented an increase in lysophospholipids within 10 minutes of the onset of ischemia (Shaikh and Downar, 1981). Fewer studies have documented the changes in lysophospholipids during ischemia and reperfusion. In porcine hearts subjected to two hours of left anterior
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descending coronary artery occlusion followed by 15 minutes of reperfusion in situ, an accumulation of lysophosphatidylcholine and a decrease in phosphatidylcholine and phosphatidylethanolamine content was seen (Das et al, 1986). In the Langendorff perfused rat heart, using a 30 minute ischemia period followed by 30 minutes of reperfusion, the enhancement in lysophosphatidylcholine content was similar to that seen after a 60 minute period of ischemia. The increase in lysophosphatidylcholine during ischemia was not, however, accompanied by a loss of phosphatidylcholine content, as was the case after reperfusion (Otani et al., 1989). These studies did not specifically address the role of plasmalogens in contributing to changes in phospholipid membrane during ischemia and reperfusion. We therefore developed a two stage high performance liquid chromatography method to determine changes in lysoplasmenylcholine and lysoplasmenylethanolamine in isolated working rat hearts (Davies et al., 1992). Hearts were perfused with Krebs buffer or with modified Krebs buffer, containing 1.2 mM palmitate bound to 3% bovine serum albumin, to give a free fatty acid concentration which mimics that which is seen in blood after myocardial infarction (Opie, 1975). In hearts perfused with either buffer, there was no significant accumulation of lysoplasmalogens after 30 minutes of ischemia. However, fatty acid perftised hearts showed a significant increase in lysoplasmenylethanolamine, but not lysoplasmenylcholine, content following 30 minutes of reperfusion. Since fatty acid perfused hearts, but not hearts perftised with glucose alone, show a significantly depressed recovery of function following ischemia, the accumulation of lysoplasmalogen in these hearts suggests a potential detrimental action of lysoplasmalogen on myocardial ftinction. A similar increase in lysoplasmenylethanolamine was observed following reperfusion in fatty acid perftised rabbit hearts, indicating that the phenomenon is not species specific. The mechanism by which lysoplasmalogens increase in the myocardium following ischemia and reperftision injury is unclear. This could include one or many of the following: activation of plasmalogen-selective phospholipase(s) A2, free radical attack on plasmalogens, or diminished activities of enzymes which remove potentially harmful lysoplasmalogens. If a plasmalogen specific phospholipase A2 activity is involved, the question is open as to whether the enzyme shows a substrate preference for ethanolamine over choline containing plasmalogens. The cell has mechanisms to remove harmftil lysophospholipids which possess membrane disruptive properties (Gross and Sobel, 1982). Lysophospholipase and lysophospholipase-transacylase activity is abundant in rabbit myocardium (Gross and Sobel, 1983; Corr et al., 1984). These enzyme activities are inhibited by long-chain acyl carnitine and acyl CoA, metabolites which accumulate during ischemia (Idell-Wanger et al., 1978) and which, particularly in our model, are known to accumulate when using relevant high fatty acid concentrations in the perftisate (Lopaschuk et al., 1988). Furthermore, lysoplasmalogens are not hydrolyzed by these lysophospholipases but must be either transacylated or reacylated (Gross, 1992). To date no lysoplasmalogenase or lysophospholipase C has been identified which could remove these lysoplasmalogens. Thus, it is more likely that
Plasmalogens, NO, and the Ischemic-Reperfused Heart
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lysoplasmalogen could accumulate as there are mechanisms whereby other lysophospholipids can be removed in the ischemic myocardium. Model membranes composed of plasmalogens show markedly different molecular dynamics than comparable membranes consisting of diacyl phospholipids (Boggsetal. 1981;Lohneretal., 1984;Paketal., 1987). These studies suggest that the higher plasmalogen content in excitable tissues such as the heart provide a unique microenviroment providing for optimal transmembrane protein function. Perturbations in this phospholipid microenvironment in plasmalogen-rich membranes such as the myocardium are likely to cause even larger derangements in membrane dynamics than in diacylphospholipid membranes (Han and Gross, 1991). Using binary mixtures of choline glycerophospholipids and lysophospholipids as model membranes they showed that: (a) lysoplasmenylcholine caused a four to sixfold greater perturbation of membrane dynamics (decrease in steadystate anisotropy) in bilayers consisting of plasmenylcholine than the effects of lysophosphatidylcholine on phosphatidylcholine bilayers, (b) lysoplasmenylcholine showed greater potency than lysophosphatidylcholine in altering dynamics of phosphatidylcholine bilayers, and, most intriguingly, (c) bilayers of plasmenylcholine were much more susceptible to perturbation by amphiphiles such as lysoplasmenylcholine, lysophosphatidylcholine, or long-chain acylcamitines than were matrices composed of phosphatidylcholine. This suggests that small changes in lysoplasmalogen accumulation in a plasmalogen-rich environment could cause dramatic changes in membrane dynamics and the function of membrane associated proteins. As the myocardial phospholipids are primarily made up of plasmalogens, accumulation of lysoplasmalogens after reperfusion following ischemia could have dramatic consequences on functional recovery of the heart.
III. FREE RADICALS AND ISCHEMIA-REPERFUSION INJURY A distinction must be made with processes which lead to ischemic injury and those which result by reintroduction of oxygen and nutrient flow to an area which was previously ischemic (reperfusion injury). Although deprivation of oxygen and nutrients by diminishing blood flow to a region of the heart will eventually lead to irreversible cell damage, short periods of ischemia can be well tolerated. A concept of myocardial injury arises in which cells progress from reversible cellular injury (during the ischemic period, depending upon its duration and severity) to permanent damage (necrosis). Longer periods of ischemia followed by the reintroduction of blood flow and oxygen supply will result in a proportionately larger area of cell necrosis to an extent not observable during the ischemic period itself (Reimer et al., 1977). This enhanced myocardial injury is attributed to the release of oxygenderived free radicals which are released in an explosive burst during the first seconds to minutes of reperfusion.
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The release of oxygen-derived free radicals during reperfiision will damage the integrity of myocardial phospholipid membranes. This damage is not limited to the lipid components of the cell as DNA and proteins are also susceptible to alteration in structure by free radical attack. However, the membrane phospholipids are sensitive targets offreeradical induced damage, specifically in the unsaturated fatty acyl chains, and in terms of free radical propagating mechanisms this may provide for the acceleration of free radical mediated injury. This could lead to peroxidation and deacylation of membrane phospholipids and result in the release of lysophospholipids, as well as lipid peroxides. These changes in the phospholipid environment of membrane-associated proteins could result in a major alteration in the function of these proteins, disrupting cellular homeostasis and leading to the entry of Ca^"^ and finally irreversible changes in myocyte function. The changes in membrane phospholipids caused by free radical production during reperfusion could exacerbate alterations which take place during ischemia, including those changes induced by the activation of myocardial-specific phospholipase A2 activity. A. Susceptibility of Plasmalogens to Free Radical Attack Considering the aforementioned changes in myocardial phospholipase activity, free radical attack represents an alternative or additional mechanism for the alterations to the phospholipid environment, accumulation of lysophospholipids and lysoplasmalogens, and release of free fatty acids, including arachidonic acid. The vinyl-ether moiety of plasmalogens is susceptible to free radical attack by singlet oxygen on the carbon-carbon double bond (Frimer, 1979), resulting in 2-acyl lysophosphatidylcholine and its oxidation products in plasmenylcholine bilayers (Scherrer and Gross, 1989). Using an oxygen free radical generating system, cleavage of the alkyl-1-enyl bond has been demonstrated, both in rat brain homogenates (Yavin and Gatt, 1972) and in ram spermatozoa (Jones and Mann, 1976), the latter being associated with a loss of function. The high degree of unsaturation in the sn-2 fatty acyl chain is another site of free radical attack in plasmalogens, leading to lipid peroxidation, autocatalysis, and the resultant release of free fatty acids (Corr et al., 1984). The molecular configuration of the plasmalogen has also been hypothesized to increase the susceptibility of the sn-2 fatty acid to free radical attack (Zoeller and Raetz, 1986; Morand et al, 1988). An inhibitor of lipid peroxidation, U74006F (Braughler et al., 1987), belonging to a group of compounds collectively referred to as lazaroids, has been found to be beneficial in attenuating myocardial ischemia and reperftision injury in stunned and reperfused canine myocardium (Holzgrefe et al., 1990) and in isolated nonworking rabbit hearts (Carrea et al., 1992). Using our model of ischemia and reperfusion injury in isolated working rat hearts we have found this compound to be beneficial in preserving myocardial function and phospholipid integrity. Spontaneously beating, isolated working rat hearts were perfused using a modified Krebs buffer (containing 1.2 mM palmitate buffer bound to 3% bovine serum
203
Plasmalogens, NO, and the Ischemic-Reperfused Heart
albumin) for 15 minutes as aerobically working hearts, followed by 30 minutes of global no-flow ischemia, and then reperfiised aerobically for a further 30 minute period as previously described (Davies et al., 1992). U74006F (20 |LIM) was added to the perfusion buffer 10 minutes before the onset of ischemia. Additional groups consisted of ischemic hearts, which were not reperfused, and a group of aerobic working hearts which were perfused for a total of 45 minutes. All hearts were frozen at the end of the perfusion period. We found that U74006F significantly improved the functional recovery of the isolated working hearts during the reperfiision period (Figure 2). Analysis of lysoplasmenylcholine and lysoplasmenylethanoline content of the heart tissue (Davies et al., 1992) revealed no changes in their content at the end of ischemia. Upon reperfusion of the untreated hearts, however, there was a significant accumulation of lysoplasmenylethanolamine and lysoplasmenylcholine content. The increase in lysoplasmalogen content of the heart seen after reperfusion was prevented by U74006F (Figure 3). Although we do not know the precise mechanism by which U74006F acts, these results suggest that inhibition of free radical production may prevent accumulation of potentially harmful lysoplasmalogens during the reperfusion period. Interestingly, a related lazaroid antioxidant 30 MIN ISCHEMIiAL
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Figure 2. Protective action of U74006F (20 ^iM), a lipid peroxidation inhibitor, on recovery of function of isolated working rat hearts subjected to ischemia and reperfu> sion injury. Control (n = 9) and U74006F (n = 10) hearts were subjected to 15 minutes of aerobic perfusion followed by 30 minutes of global, no-flow ischemia and 30 minutes of aerobic reperfusion. * p < 0.05 (Student's unpaired t-test) compared with control group.
204
RICHARD SCHULZ 1600 n nn AEROBIC EZl END OF ISCHEMIA ■ i REPERFUSED FOLLOWING ISCHEMIA REPERFUSED FOLLOWING ISCHEMIA+U74006F
CHOLINE
ETHANOLAMINE
Figure 3. Prevention of lysoplasmalogen accumulation by U74006F (20 |LIM) in isolated working rat hearts subjected to ischemia and reperfusion injury, n = 6-13 hearts per group. * p < 0.05 (ANOVA followed by Fisher's least significant difference test for individual comparisons) compared with aerobic and reperfused following ischemia + U74006F groups (for lysoplasmenylcholine) and aerobic, end of Ischemia, and reperfused following ischemia + U74006F groups (for lysoplasmenylethanolamine).
U74500A has been shown to inhibit malondialdehyde formation in low density lipoprotein oxidized by a free radical generating system and the increase in systolic Ca^"^ in isolated cardiomyocytes exposed to oxidized lipoprotein (Liu et al., 1993). The production of oxygen-derived free radicals (superoxide anion, hydroxyl radical, etc.) in the first seconds to minutes of reperfusion is considered to be crucial to the process of myocardial reperfusion injury. Evidence for their formation has been provided in studies which have measured these species directly by the use of electron paramagnetic spin resonance spectroscopy or indirectly using spin trapping agents (Kramer et al, 1987; Garlick et al., 1987; Zweier et al, 1987, 1989; BoUi et al., 1988; Henry et al, 1990). The exact nature of these radicals, the mechanisms of their formation, and how they are transformed into highly reactive species such as the hydroxyl radical are disputed, however, most agree that superoxide anion and hydroxyl free radical are made and that their formation during the first seconds to minutes of reperfusion are integral to the extent of damage which is caused (Cohen, 1989). This discussion is not intended to review the previous literature regarding free radical injury but will address the possibility that NO, derived from a variety of cellular sources in heart tissue, may play a distinctive role
Plasmalogens, NO, and the Ischemic-Reperfused Heart
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in ischemia-reperfusion injury by combining with superoxide anion to form peroxynitrite (0N00~ ), a stable delivery form of free hydroxyl radicals. B. Questions Regarding the Generation of Oxygen-Derived Free Radicals
Three key features of oxygen-derived free radical mediated injury are: (a) the extremely rapid time course, with the greatest concentration of free radicals being formed within the first seconds to minutes of reperfusion, (b) the potential sources and cell types involved in free radical production, and (c) how superoxide anion and hydrogen peroxide produced intracellularly, with their limited cellular toxicity, leave the cell and form highly reactive hydroxyl radicals at or near their target site which may be a number of cell diameters distant from the cell of production. Where could the free radicals be made? Suggested sources of free radical production include the endothelium, myocytes, and leukocytes. The accumulation of neutrophils in the myocardium is, however, a late response in myocardial reperfusion injury and likely extends the period of injury over the first 24 hours following reperfusion. Furthermore, numerous in vitro studies of ischemic hearts using aqueous buffers show the release of oxygen-derived free radicals and myocardial injury following reperfusion in the absence of circulafing cells. Organelles such as mitochondria and the endoplasmic reticulum, which are targets for free radical-mediated damage, may also contribute to the production of hydrogen peroxide and superoxide anion. However, the species attributed to causing the majority of free radical-mediated damage, the hydroxyl radical, is so reactive that at most it can only proceed a few molecular diameters before it is consumed. The question arises, in regards to leukocyte or endothelium-mediated free radical production, as to how superoxide anion and hydrogen peroxide may be generated in the vascular space and cross a number of cell diameters to then be transformed into hydroxyl radicals to act at a distant target deep within the myocardium. Since a large protein such as superoxide dismutase, which does not readily enter cells, provides protection from myocardial injury if administered with catalase only one minute before the onset of reperfusion (Przyklenk and Kloner, 1989), an "antioxidant paradox" (Gutteridge and Halliwell, 1990) has been suggested. In other words, how do extracellular antioxidants, which either by virtue of their inaccessibility into the intracellular space, or their rapid onset of action in a time frame where they have not yet entered the intracellular space in sufficient concentration to be effective there, protect the myocardium from intracellular injury by oxygen-derived free radicals? The issues in myocardial free radical induced injury which remain unanswered are: which free radicals are excreted into the extracellular space; how are they able to diffuse unreacted across cells to their target sites; and how are they then able to form hydroxyl radicals or other strong oxidants which are understood to mediate the bulk of free radical induced cellular damage? Under conventional theory, superoxide anion is thought to react with hydrogen peroxide to yield hydroxyl free radicals (the Haber-Weiss reaction. Equation 1).
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RICHARD SCHULZ
This reaction progresses very slowly to form hydroxyl radicals unless catalyzed by Fe^"*" (described thus as the Fenton reaction). The free level of Fe^"^ is, however,
H2O2 + O-* -> O2 + OH- + OH*
(1)
strictly controlled in the cell and it is normally completely bound in the form of ferritin, such that free levels are negligible. It is questionable whether there is a sufficient concentration of free Fe*^"^ present to catalyze the Haber-Weiss reaction. Furthermore, the protective action of desferrioxamine, usually described as an iron chelator, in various experimental models, has not satisfactorily accounted for all of its beneficial effects (Gutteridge and Halliwell, 1990). Previous reports have indicated that desferrioxamine acts to scavenge hydroxyl radicals in an action distinct from its iron-chelating properties (Hoe et al., 1982; Maruyama et al., 1991). Beckmann et al. (1990) have shown that desferrioxamine, in an action completely independent of its iron-chelating ability, is a potent and competitive inhibitor of peroxynitrite-initiated oxidation. How are sufficient quantities of superoxide anion transformed into highly reactive hydroxyl radicals, without necessitating a requirement for "free" Fe^"^ for catalysis, in a very short period of time, to cause free radical-induced reperfusion injury? C. Potential Role of Nitroxide Free Radicals in Reperfusion Injury Nitric oxide, which best accounts for the biological activity of endothelium-derived relaxing factor (EDRF) (Furchgott and Zawadzki, 1980; Ignarro et al., 1987; Palmer et al., 1987), is synthesized by the vascular endothelium from L-arginine (Palmer et al., 1988) by the enzyme NO synthase (Equation 2). Nitric oxide causes vasorelaxation by stimulation of soluble guanylate cyclase in vascular smooth muscle (Rapoport and Murad, 1983). It is a short-lived, yet potent, signaling O2 + L-arginine -^ NO + L-citruUine
(2)
molecule with a half-life of less than 10 seconds in most biological systems (Gryglewski et al., 1986), breaking down in plasma to nitrate by reaction with oxyhemoglobin (Kosaka et al., 1979). It is also one of the important endogenous factors which regulate perfusion through the coronary circulation. Stimulation of isolated hearts with agonists which provoke the release of EDRF from vascular endothelium (Furchgott and Vanhoutte, 1989) results in a decrease in coronary perfusion pressure which can be blocked by hemoglobin and by NO synthase inhibitors (Amezcua et al., 1988, 1989; Kelm and Schrader, 1990). The rapid changes in coronary flow and fluid shear stress between systole and diastole are possibly the major physiological stimuli of NO release in the coronary circulation
Plasmalogens, NO, and the Ischemic-Reperfused Heart
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as flow-dependent release of EDRF has been demonstrated in the peripheral vasculature (Rubanyi et al, 1986; Pohl et al, 1988). Flow-dependent dilatation in canine epicardial arteries after reactive hyperaemia is attenuated after endothelium removal (Inoue et al., 1988) or in the presence of the NO synthase inhibitor L-nitroarginine methyl ester (Kostic and Schrader, 1992). Moreover, atherosclerotic coronary arteries are shown to have a diminished flow dependent dilatation (Drexler et al., 1989) and diminished basal and stimulated release of NO in response to vasodilator agonists (Chester et al, 1990). D.
Role of Peroxynitrite
Recently it has been proposed that peroxynitrite (ONOO"), a product of the combination of superoxide anion and NO (Equation 3), could explain the damage implicated to be caused by superoxide anion (Beckmann et al, 1990; Gutteridge and Halliwell, 1990). At physiological pH, peroxynitrite is sufficiently stable to diffuse over several cell diameters to critical cellular targets before decomposing upon protonation to yield strong oxidants including nitronium ion (NO2) and an intermediate with hydroxyl radical character (Koppenol et al., 1992), the species generally ascribed to mediating oxygen radical-induced toxicity (Equation 4). Peroxynitrite also initiates lipid peroxidation and reacts directly with sulhydryl groups at a 1,000-fold greater rate than hydrogen peroxide does at pH 7.4 (Radi et al., 1991). O"* + NO -^ ONOO"
(3)
ONOO" + H^ -^ NOJ + OH"
(4)
How is peroxynitrite formed? The change in shear stress caused by the reintroduction of flow is likely the key stimulus for a burst of NO which is released during reperfusion. Furthermore, as NO is synthesized from oxygen and L-arginine, the reintroduction of oxygen should drive the formation of NO rightwards (Equation 2). The quick kinetics of the release of NO from endothelium as well as its short half-life in biological millieu (Palmer et al., 1987) coincide with the findings that reperfusion damage occurs within the first minute of reintroducing flow to the ischemic zone. The vascular endothelium is one of the cell types providing a source of NO to drive the formation of peroxynitrite (Equation 3). The maximally sfimulated rate of endothelial NO production has been calculated to be 8 jiM/min (Beckmann, 1991). However, NO production in the heart is not limited only to the coronary vascular endothelium. We have shown that both the endocardium and cardiac myocytes express Ca^'^-dependent NO synthase activity (Schulz et al., 1991, 1992) and could act as additional sources for the formation of NO. It is possible that during reperfusion, mechanisms which increase intracellular Ca^^
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RICHARD SCHULZ
levels would stimulate Ca^"^-dependent NO synthase in the endothelium, endocardium, and cardiac myocytes. NO could then combine with superoxide anion released upon reperfusion to yield peroxynitrite which acts as a transmembrane and transcellular delivery form for the highly toxic hydroxyl radical. In support of this finding an unidentified nitrogen-centered radical has been detected during postischemic reperfusion in isolated rat hearts (Zweier et al, 1987,1989). Since the rate of peroxynitrite formation is dependent upon the product of superoxide anion and NO concentrations, it will increase 100-fold for every 10-fold increase in the concentration of superoxide anion and NO (Beckmann et al., 1990). In a model of reoxygenation injury in the hypoxic piglet undergoing cardiopulmonary bypass, it has now been shown that the NO synthase inhibitor N^-nitro-Larginine methyl ester, or the combination of mercaptopropionyl glycine (a hydroxyl radical scavenger) with catalase, provided similar and nearly complete protection against myocardial injury, which was accompanied by a reduction in the level of NO decomposition products in the plasma (Matheis et al., 1992). Furthermore, the increase in the level of conjugated dienes (a measure of lipid peroxidation) in endocardial biopsy samples seen after reoxygenation injury was prevented with either drug treatment. It is possible that an excess short-term burst of NO during the first seconds to minutes of reperfiision is first initiated with the shear stress-mediated release of NO fi"om the endothelium and then progresses to include the release of NO from the myocardium by the Ca^^ dependent NO synthase, as the wavefi'ont of Ca^"^ entry which extends during the reperfusion phase advances deeper into heart tissue. In combination with superoxide anion to form peroxynitrite, NO may contribute to myocardial injury through free radical mediated attack on myocardial phospholipids. Whether the production of peroxynitrite and subsequent formation of hydroxyl radicals could specifically account for changes in plasmalogens (i.e., lysoplasmalogen accumulation or release of free fatty acids) is currently under investigation in my laboratory. These results must be contrasted with the finding that NO donors have cardioprotective effects in a feline model of ischemia-reperfiision injury (Siegfried et al., 1992). Lower doses of NO released by such drugs may have beneficial actions in protecting hearts through coronary vasodilatation, as well as by preventing thrombosis and neutrophil aggregation. A diminished basal release of NO from the endothelium of coronary arteries subjected to ischemia and reperfusion injury has been shown (Ma et al., 1993). If there is a significant loss in the capacity of the coronary endothelium to make NO as one of the physiological mediators of coronary vasodilator tone, replacement with small quantities of NO provided by a NO donor through the vascular space will help to ensure coronary perfusion. However, the continuous low output basal release of NO from the endothelium is very distinctfi-oma burst of excessive NO release in pathophysiological quantities throughout the myocardium during the first seconds of reperfusion. Plasmalogens play a unique role in myocardial cell function and are likely an essential feature of the distinctive electrophysiological properties of this excitable
Plasmalogens, NO, and the Ischemic-Reperfused Heart
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tissue. During myocardial ischemia and reperflision injury, the plasmalogens are a target for both the actions of plasmalogen-specific phospholipases and oxygenderived free radicals. The by-products of plasmalogen degradation, lysoplasmalogens and free fatty acids, including the predominant constituent, arachidonic acid, have actions consistent with further derangements in myocardial cell function. An excessive burst of NO released during reperfusion from the coronary endothelium and myocardium could act to enhance injury through the production of peroxynitrite and hydroxyl radicals, causing further damage to the phospholipid environment. Future therapies effective for the prevention of ischemia and reperfusion injury may include inhibitors of the plasmalogen-selective phospholipase(s) A2 and NO synthase.
ACKNOWLEDGMENTS I gratefully thank Ken Strynadka and Donna Panas for their excellent technical assistance and Lorraine Hirsch for typing the manuscript. U74006F was a generous gift from Upjohn. This work was supported by a grant from the Heart and Stroke Foundation of Alberta. The author is a Scholar of the Alberta Heritage Foundation for Medical Research and the Medical Research Council of Canada.
NOTE ADDED IN PROOF We have shown the two inhibitors of NO synthase, N^-nitro-L-arginine methyl ester (L-NAME) and N^-monomethyl-L-arginine (L-NMMA) protect isolated working rabbit hearts from ischemia-reperfusion injury (Schulz and Wambolt, 1995). Moreover, reperfusion of isolated rat hearts subjected to global ischemia resulted in the acute release of ONOO~ in the coronary effluent which peaked within 30 seconds of reperfusion. The release of 0N00~ was abolished by L-NMMA and was accompanied by the improved recovery of mechanical function. The protective action of L-NMMA showed a marked and bell-shaped concentration-dependence (Yasmin and Schulz, 1995).
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Amsdorf, M.F. & Sawicki, G.J. (1981). The effects of lysophosphatidylcholine, a toxic metabolite of ischemia, on the components of cardiac excitability in sheep Purkinje fibers. Circ. Res. 49, 16-30. Beckmann, J.S. (1991). The double-edged role of nitric oxide in brain function and superoxide-mediated injury. J. Develop. Physiol. 15, 53-59. Beckmann, J.S., Beckman. T.W., Chen, J., Marshall, P.A., & Freeman, B.A. (1990). Apparent hydroxyl radical production by peroxynitrite: Implications for endothelial injury from nitric oxide and superoxide. Proc. Natl. Acad. Sci. USA 87, 1620-1624. Bennett, J.P., McGill, K.A., & Warren, G.B. (1980). In: Carriers and Membrane Transport Proteins (Bronner, F. & Kleinzeller, A., eds.), pp. 127-164. Academic Press, New York. Boggs, J.M., Stamp, D., Hughes, D.W., & Deber, CM. (1981). Influence of ether linkage on the lamellar to hexagonal phase transition of ethanolamine phospholipids. Biochemistry 20, 5728-5735. Bolli, R., Patel, B.S., Jeroudi, M.O., Lai, E.K., & McCay, RB. (1988). Demonstration of free radical generation in "stunned" myocardium of intact dogs with the use of the spin trap a-phenyl n-tert-butyl nitrone. J. Clin. Invest. 82, 476-485. Braughler, J.M., Pregenzer, J.F., Chase, R.L., Duncan, L.A., Jacobsen, E.J., & McCall, J.M. (1987). Novel 21-amino-substituted steroids as potent inhibitors of iron-dependent lipid peroxidation. J. Biol. Chem. 262, 10438-10440. Briggs, M.M. & Lefkowitz, R.J. (1980). Parallel modulation of catecholamine activation of adenylate cyclase and formation of the high-affmity agonist-receptor complex in turkey erythrocyte membranes by temperature and cis-vaccenic acid. Biochemistry 19, 4461-^466. Carrea, F.R, Lesnefsky, E.J., Kaiser, D.G., & Horwitz, L.D. (1992). The lazaroid U74006F, a 21 -aminosteroid inhibitor of lipid peroxidation, attenuates myocardial injury from ischemia and reperfusion J. Cardiovasc. Pharmacol. 20, 230-235. Chester, A.H., O'Neil, G.S., Moncada, S., Tadjkarimi, S., & Yacoub, M.H. (1990). Low basal and stimulated release of nitric oxide in atherosclerotic epicardial coronary arteries. Lancet 336, 897-900. Chien, K.R., Han, A., Sen, A., Buja, CM., & Willerson, J.T. (1984). Accumulation of unesterified arachidonic acid in ischemic canine myocardium. Circ. Res. 54, 313—322. Chien, K.R., Reeves, J.R, Buja, L.M., Bonte, F., Parkey, R.W., & Willerson, J.T. (1981). Phospholipid alterations in canine ischemic myocardium. Temporal and topographical correlations with Tc-99PPi accumulation and an in vitro sarcolemmal Ca ^ permeability defect. Circ. Res. 48, 711-719. Chien, K.R., Sen, A., Reynolds, R., Chang, A., Kim, Y, Gunn, M.D., Buja, L.M., & Willerson, J.T. (1985). Release of arachidonate from membrane phopholipids in cultured neonatal rat myocardial cells during adenosine triphosphate depletion. J. Clin. Invest. 75, 1770-1780. Clarkson, C.W. & Ten Eick, R.E. (1983). On the mechanism of lysophosphatidylcholine-induced depolarization of cat ventricular myocardium. Circ. Res. 52, 543-556. Cohen, M.V. (1989). Free radicals in ischemic and reperfusionmyocardial injury: Is this the time for clinical trials? Ann. Internal Med. 111,918-931. Corr, RB., Cain, M.E., Witkowski, F.X., Price, D.A., & Sobel, B.E. (1979). Potential arrhythmogenic electrophysiological derangements in canine Purkinje fibers induced by lysophosphoglycerides. Circ. Res. 44, 822-832. Corr, RB., Creer, M.H., Yamada, K.A., Saffitz, J.E., & Sobel, B.E. (1989). Prophylaxis of early ventricular fibrillation by inhibition of acylcamitine accumulation. J. Clin. Invest. 83, 927-936. Corr, P.B., Gross, R.W., & Sobel, B.E. (1984). Amphipathic metabolites and membrane dysfunction in ischemic myocardium. Circ. Res. 55, 135-154. Corr, RB., Snyder, D.W., Lee, B.I., Gross, R.W, Keim, C.R., & Sobel, B.E. (1982). Pathophysiological concentrations of lysophosphatides and the slow response. Am. J. Physiol. 243, H187-H195. Das, D.K., Engelman, R.M., Rousou, J.A., Breyer, R.H., Otani, H., & Lemeshow, S. (1986). Role of membrane phospholipids in myocardial injury induced by ischemia and reperfusion. Am. J. Physiol. 251, H71-H79.
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Davies, N.J., Schulz, R., Olley, P.M., Strynadka, K.D., Panas, D.L., & Lopaschuk, G.D. (1992). Lysoplasmenylethanolamine accumulation in ischemic/reperfiised isolated fatty acid-perfused hearts. Circ. Res. 70, 1161-1168. Drexler, H., Zeiher, A.M., Wollshlager, H., Meinertz, T., Just, H., & Bonzel, T., (1989). Flow-dependent coronary artery dilatation in humans. Circulation 80, 466-474. Ford, D.A., Hazen, S.L., Saffitz, J.E., & Gross, R.W. (1991). The rapid and reversible activation of a calcium-independent plasmalogen-selective phospholipase A2 during myocardial ischemia. J. Clin. Invest. 88, 331-335. Frimer, A. (1979). Reaction of singlet oxygen with olefins. Question of mechanism. Chem. Reviews 79, 359-386. Furchgott, R.F. & Vanhoutte, P.M. (1989). Endothelium-derived relaxing and contracting factors. FASEB J. 3, 2007-2018. Furchgott, R.F. & Zawadzki, J.V. (1980). The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature 288, 373-376. GarUck, P.B., Davies, M.J., Hearse, D.J., & Slater, T.F. (1987). Direct detection offi*eeradicals in the reperfiised rat heart using electron spin resonance spectroscopy. Circ. Res. 61, 757-760. Gerritsen, M.E. & Printz, M.P. (1981). Sites of prostaglandin synthesis in the bovine heart and isolated bovine coronary microvessels. Circ. Res. 49, 1152-1163. Gross, R.W. (1984). High plasmalogen and arachidonic acid content of canine myocardial sarcolemma: A fast atom bombardment mass spectroscopic and gas chromatography-mass spectroscopic characterization. Biochemistry 23, 158-165. Gross, R.W. (1985). Identification of plasmalogen as the major phospholipid constituent of cardiac sarcoplasmic reticulum. Biochemistry 24, 1662-1668. Gross, R.W. (1992). Myocardial phospholipases A2 and their membrane substrates. Trends Cardiovasc. Med. 2, 115-121. Gross, R.W. & Sobel, B.E. (1982). Lysophosphatidylcholine metabolism in the rabbit heart. J. Biol. Chem. 257, 6702-6708. Gross, R.W. & Sobel, B.E. (1983). Rabbit myocardial cytosolic lysophospholipase. J. Biol. Chem. 258, 5221-5226. Gryglewski, R.J., Moncada, S., & Palmer, R.M.J. (1986). Bioassay of prostacyclin and endotheliumderived relaxing factor (EDRF) from porcine aortic endothelial cells. Br. J. Pharmacol. 87, 685-694. Gutteridge, J.M.C. & Halliwell, B. (1990). Reoxygenation injury and antioxidant protection: A tale of two paradoxes. Arch. Biochem. Biophys. 283, 223—226. Han, H. & Gross, R.W. (1991). Alterations in membrane dynamics elicited by amphiphilic compounds are augmented in plasmenylcholine bilayers. Biochem. Biophys. Acta 1069, 37-45. Hazen, S.L. & Gross, R.W. (1991a). ATP-dependent regulation of rabbit myocardial cytosolic calciumindependent phospholipase A2. J. Biol. Chem. 266, 14526-14534. Hazen, S.L. & Gross, R.W. (1991b). Human myocardial cytosolic Ca ^- independent phospholipase A2 is modulated by ATR Biochem. J. 280, 581-587. Hazen, S.L. & Gross, R.W. (1992). Identification and characterization of human myocardial phospholipases A2fi*omtransplant recipients suffering from end stage ischemic heart disease. Circ. Res. 70, 486-495. Hazen, S.L., Ford, D.A., & Gross, R.W. (1991a). Activation of a membrane-associated phospholipase A2 during rabbit myocardial ischemia which is highly selective for plasmalogen substrate. J. Biol. Chem. 266, 5629-5633. Hazen, S.L., Stuppy, R.J., & Gross, R.W. (1990). Purification and characterization of canine myocardial cytosolic phospholipase A2: A calcium independent phospholipase with absolute sn-2 regiospecificity for diradyl glycerophospholipids. J. Biol. Chem. 265, 10622-10630. Hazen, S.L., Zupan, L.A., Weiss, R.H., Getman, D.R, & Gross, R.W. (1991b). Suicide inhibition of canine myocardial cytosolic calcium-independent phospholipase A2. J. Biol. Chem. 266, 7227-7232.
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Henry, T.D., Archer, S.L., Nelson, D., Weir, E.K., & From, A.H.L.(1990). Enhanced chemiluminescence as a measure of oxygen-derived free radical generation during ischemia and reperfusion. Circ. Res. 67, 1453-1461. Hoe, S., Rowley, D.A., & Halliwell, B. (1982). Reaction of ferrioxamine and desferrioxamine with the hydroxyl radical. Chem. Biol. Interact. 41, 75-81. Holzgrefe, H.H., Buchanan, L.V., & Gibson, J.K. (1990). Effects of U74006F, a novel inhibitor of lipid peroxidation, in stunned reperftised canine myocardium, J. Cardiovasc. Pharmacol. 15, 239-248. Horrocks, L.A. (1972). Content, composition, and metabolism of mammalian and avian lipids that contain ether groups. In: Ether Lipids, Chemistry and Biology (Snyder, P., ed.), pp. 177-272. Academic Press, New York. Huang, J.M.-C, Xian, H., & Bacaner, M. (1992). Long-chain fatty acids activate calcium channels in ventricular myocytes. Proc. Natl. Acad. Sci. USA 89, 6452-6456. Idell-Wenger, J.A., Grotyohann, L.W., & Neely, J.R. (1978). Coenzyme A and carnitine distribution m normal and ischemic hearts. J. Biol. Chem. 253, 4310-4318. Ignarro, L.J., Buga, G.M., Wood, K.S., Byms, R.E., & Chaudhuri, G. (1987). Endothelium-derived relaxing factor produced and released from artery and vein is nitric oxide. Proc. Natl. Acad. Sci. USA 84, 9265-9269. Inoue, T., Tomoike, H., Hisano, K., & Nakamura, M. (1988). Endothelium determines flow-dependent dilation of the epicardial coronary artery in dogs. J. Am. Coll. Cardiol. 11, 187-191. Jones, R. & Mann, T. (1976). Lipid peroxides in spermatozoa.Formation, role of plasmalogen, and physiological significance. Proc. Roy. Soc. Lond. B 193, 317-333. Kako, K.J. (1986). Membrane phospholipids and plasmalogens in the ischemic myocardium. Can. J. Cardiol. 2, 184-194. Karli, J.N., Karikas, G.A., Hatzipavlou, RK., Levis, G.M., & Moulopoulos, S.N. (1979). The inhibition of Na and K stimulated ATPase activity of rabbit and dog heart sarcolemma by lysophosphatidylcholine. Life Sci. 24, 1865^1875. Karmazyn, M. (1986). Contribution of prostaglandins to reperfusion-induced ventricular failure in isolated rat hearts. Am. J. Physiol. 251, H133-H140. Kehn, M. & Schrader, J. (1990). Control of coronary vascular tone by nitric oxide. Circ. Res. 66,1561-1575. Koppenol, WH., Moreno, J.J., Pryor, W.A., Ischiropoulos, H., & Beckman, J.S. (1992). Peroxynitrite, a cloaked oxidant formed by nitric oxide and superoxide. Chem. Res. Toxicol. 5, 834—842. Kosaka, H., Imaizumi, K., Imai, K., & Tyuma, I. (1979). Stoichiometry of the reaction of oxyhemoglobin with nitrite. Biochim. Biophys. Acta 581, 184-188. Kostic, M.M. & Schrader, J. (1992). Role of nitric oxide in reactive hyperemia of the guinea pig heart. Circ. Res. 70,20^212. Kramer, J.H., Arroyo, CM., Dickens, B.F., & Weglicki, WB. (1987). Spin-trapping evidence that graded myocardial ischemia alters post-ischemic superoxide production. Free Radic. Biol. Med. 3, 153-159. Kuzuya, T., Hoshida, S., Nishida, M., Kim, Y, Kamada, T., & Tada, M. (1987). Increased production of arachidonate metabolites in an occlusion-reperfusion model of canine myocardial infarction. Cardiovasc. Res. 21, 551-558. Liu, E., Goldhaber, J.I., & Weiss, J.N. (1991). Effects of lysophosphatidylcholine on electrophysiological properties and excitation-contraction coupling in isolated guinea pig ventricular myocytes. J. Clin. Invest. 88, 1819-1832. Liu, K., Massaeli, H., & Pierce, G.N. (1993). The action of oxidized low density lipoprotein on calcium transients in isolated rabbit cardiomyocytes. J. Biol. Chem. 268, 4145-4151. Lohner, K., Hermetter, A., & Paltauf, F. (1984). Phase behaviour of ethanolamine plasmalogen. Chem. Phys. Lipids 34, 163-170. Lopaschuk, G.D., Wall, S.R., Olley, P.M., & Davies, N.J. (1988). Etomoxir, a carnitine palmitoyltransferase I inhibitor, protects hearts from fatty acid-induced ischemic injury independent of changes in long chain acylcamitine. Circ. Res. 63, 1036-1043.
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Lucchesi, B.R. & Mullane, K.V. (1986). Leukocytes and ischemia-induced myocardial injury. Ann. Rev. Pharmacol. Toxicol. 26, 201-224. Ma, X., Weyrich, A.S., Lefer, D.J., & Lefer, A.M. (1993). Diminished basal nitric oxide release after myocardial ischemia and reperfusion promotes neutrophil adherence to coronary endothelium. Circ. Res. 72,403-412. Maruyama, M., Pieper, G.M., Kalyanaraman, B., Hallaway, P.E., Hedlund, B.E., & Gross, G.J. (1991). Effects of hydroxyethyl starch conjugated deferoxamine on myocardial functional recovery following coronary occlusion and reperfusion in dogs. J. Cardiovasc. Pharmacol. 17, 166-175. Matheis, G., Sherman, M.R, Buckberg, G.D., Haybron, D.M., Young, H.H., & Ignarro, L.J. (1992). Role of L-arginine-nitric oxide pathway in myocardial reoxygenation injury. Am. J. Physiol. 262, H616-H620. Mickelson, J.K., Simpson, P.J., & Lucchesi, B.R. (1990). Ischemic heart disease: Pathophysiology and pharmacologic management. In: Cardiovascular Pharmacology (Antonaccio, M., ed.), pp. 127164. Raven Press New York. Morand, O.H., Zoeller, R.A., & Raetz,'C.R.H. (1988). Disappearance of plasmalogens from membranes of animal cells subjected to photosensitized oxidation. J. Biol. Chem. 263, 11597-11606. Mullane, K.M., Read, N., Salmon, J.A., & Moncada, S. (1984). Role of leukocytes in acute myocardial infarction in anesthetized dogs: Relationship to myocardial salvage by anti-inflammatory drugs. J. Pharmacol. Exp. Then 228, 510-522. Opie, L.H. (1975). Metabolism of free fatty acids, glucose and catecholamines in acute myocardial infarction. Am. J. Cardiol. 36, 938-953. Osani, A. & Sakagami, T. (1979). Compositions of diacyl-, alkenyl-acyl-, and alkyl-acyl-glycerylphosphorylcholine and -ethanolamine in male and female rabbit hearts. J. Biochem. 85, 1453-1459. Otani, H., Prasad, M.R., Jones, R.M., & Das, D.K. (1989). Mechanism of membrane phospholipid degradation in ischemic-reperfused rat hearts. Am. J. Physiol. 257, H252-H258. Pak, J.H., Bork, V.R, Norberg, R.E., Creer, M.H., Wolf, R. A., & Gross, R.W. (1987). Disparate molecular dynamics of plasmenylcholine and phosphatidylcholine bilayers. Biochemistry 26, 4824-4830. Palmer, R.M.J., Ashton, D.S., & Moncada, S. (1988). Vascular endothelial cells synthesize nitric oxide from L-arginine. Nature 333, 664-666. Palmer, R.M.J., Ferrige, A.G., & Moncada, S. (1987). Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature 327, 524—526. Piper, P.J., Letts, L.G., & Galton, S.A. (1983). Generation of a leukotriene-like substance from porcine vascular and other tissues. Prostaglandins 25, 591—599. Pohl, U., Busse, R., Kuon, E., & Bassenge, E. (1988). Pulsatile perfusion stimulates the release of endothelial autocoids. J. App. Cardiol. 1, 215-235. Przyklenk, K. & Kloner, R.A. (1989). "Reperfusion injury" by oxygen-derived free radicals? Effect of superoxide dismutase plus catalase, given at the time of reperfusion, on myocardial infarct size, contractile function, coronary microvasculature and regional myocardial blood flow. Circ. Res. 64, 86-96. Pugh, E.L., Kates, M., & Hanahan, D.J. (1977). Characterization of the alkyl ether species of phosphatidylcholine in bovine heart. J. Lipid Res. 18, 710-716. Radi, R., Beckman, J.S., Bush, K.M., & Freeman, B.A. (1991). Peroxynitrite oxidation of sulfhydryls. The cytotoxic potential of superoxide and nitric oxide. J. Biol. Chem. 266, 4244-4250. Rapport, M.M. & Norton, W.T. (1962). Chemistry of the lipids. Ann. Rev. Biochem. 31, 103-138. Reimer, K., Lowe, J., Rasmussen, M., & Jennings, R. (1977). The "wavefront phenomenon" of ischemic cell death I: Myocardial infarct size versus duration of coronary occlusion in dogs. Circulation 56, 78^794. Rapoport, R.M. & Murad, F. (1983). Agonist induced endothelium-dependent relaxation in rat thoracic aorta may be mediated through cyclic GMP. Circ. Res. 52, 352-357. Rubanyi, G.M., Romero, J.C, & Vanhoutte, P.M. (1986). Flow-induced release of endothelium-derived relaxing factor. Am. J. Physiol. 250, H1145-H1149.
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Sandermann, Jr., H. (1978). Regulation of membrane enzymes by lipids. Biochim. Biophys. Acta 515, 20^237. Scherrer, L.A. & Gross, R.W. (1989). Subcellular distribution,molecular dynamics and catabolism of plasmalogens in myocardium. Mol. Cell. Biochem. 88, 97-105. Schulz, R., Nava, E., & Moncada, S. (1992). Induction and potential biological relevance of a Ca ^-independent nitric oxide synthase in the myocardium. Br. J. Pharmacol. 105, 575-580. Schulz, R., Smith, J.A., Lewis, M.J., & Moncada, S. (1991). Nitric oxide synthase in cultured endocardial cells of the pig. Br. J. Pharmacol. 104, 21-24. Schulz, R., Strynadka, K.D., Panas, D.L., Olley, RM., & Lopaschuk, G.D. (1993). Analysis of myocardial plasmalogen and diacyl-phospholipids and their arachidonic acid content using high performance liquid chromatography. Anal. Biochem. 213, 140-146. Schulz, R., & Wambolt, R. (1995). Inhibition of nitric oxide synthesis protects the isolated working rabbit heart from ischaemia-reperfusion injury. Cardiovasc. Res. 30, 432-439. Sen, A., Miller, J.C, Reynolds, R., Willerson, J.T., Buja, L.M., & Chien, K.R. (1988). Inhibition of the release of arachidonic acid prevents the development of sarcolemmal membrane defects. J. Clin. Invest. 82, 1333-1338. Shaikh, N.A. & Downar, E. (1981). Time course of changes in porcine myocardial phospholipid levels during ischemia: A reassessment of the lysolipid hypothesis. Circ. Res. 49, 316-325. Siegfried, M.R., Erhardt, J., Rider, T., Ma, X-L., & Lefer, A.M. (1992). Cardioprotection and attenuation of endothelial dysfunction by organic nitric oxide donors in myocardial ischemia-reperfusion. J. Pharmacol. Exp. Ther. 260, 668-675. Snyder, D.W., Crafford, W.A. Jr., Glashow, J.L., Rankin, D., & Sobel, B.E. (1981). Lysophosphoglycerides in ischemic myocardium effluents and potentiation of their arrhythmogenic effects. Am. J. Physiol. 241, H700-H707. Snyder, F. (1991). Metabolism, regulation, and function of ether-linked glycerolipids and their bioactive species. In: Biochemistry of Lipids, Lipoproteins and Membranes (Vance, D.E. & Vance, J., eds.), pp. 241-268. Elsevier, Amsterdam. Sobel, B.E., Corr, RB., Robison, A.K., Goldstein, R.A., Witkowski, F.X., & Klein, M.S. (1978). Accumulation of lysophosphoglycerides with arrhythmogenic properties in ischemic myocardium. J. Clin. Invest. 62, 546-553. Spector, A.A. & Yorek, M.A. (1985). Membrane lipid composition and cellular function. J. Lipid Res. 26,1015-1035. Tada, M., Kuzuya, T., Hoshida, S., & Nishida, M. (1988). Arachidonate metabolism in myocardial ischemia and reperfusion. J. Mol. Cell. Cardiol. 20, 135-143. Van der Vusse, G.J., Roeman, T.H.M., Prinzen, F.W., Coumans, W.A., & Reneman, R.S. (1982). Uptake and tissue content of fatty acids in dog myocardium under normoxic and ischemic conditions. Circ. Res. 50, 538-546. Yasmin, W., & Schulz, R. (1995). Detection of peroxynitrite after ischemia-reperfusion in isolated hearts. Circulation 92,1-563 (abstract). Yavin, E. & Gatt, S. (1972). Oxygen-dependent cleavage of the vinyl-ether linkage of plasmalogens. 1. Cleavage by rat brain supernatant. 2. Identification of the low molecular weight active component and the reaction mechanism. Eur. J. Biochem. 25, 431-446. Zoeller, R.A. & Raetz, C.R.H. (1986). Isolation of animal cell mutants deficient in plasmalogen biosynthesis and peroxisome assembly. Proc. Natl. Acad. Sci. USA 83, 5170-5174. Zweier, J.L., Flaherty, J.T., & Weisfeldt, M.L. (1987). Direct measurement of free radical generation following reperfusion of ischemic myocardium. Proc. Natl. Acad. Sci. USA 84, 1404—1407. Zweier, J.L., Kuppasamy, R, Williams, R., Raybum, B.K., Smith, D., Weisfeldt, M.L., & Flaherty, J. (1989). Measurement and characterization of postischemic free radical generation in the isolated perfused heart. J. Biol. Chem. 264, 18890-18895.
PHOSPHOLIPID HYDROLYSIS IN PANCREATIC ISLET BETA CELLS AND THE REGULATION OF INSULIN SECRETION
John Turk, Richard W. Gross, and Sasanka Ramanadham
I. 11. III. IV V
ABSTRACT INTRODUCTION PANCREATIC ISLET BETA CELL D-GLUCOSE RECOGNITION IONIC EVENTS IN GLUCOSE-INDUCED INSULIN SECRETION NONESTERIFIED ARACHIDONIC ACID AS A SECOND MESSENGER IN PANCREATIC ISLET BETA CELLS CONTROL OF ARACHIDONATE HYDROLYSIS FROM ISLET MEMBRANE PHOSPHOLIPIDS A BETA CELL ATP-STIMULATED, Ca^^-INDEPENDENT PHOSPHOLIPASE A2 ENZYME
Advances in Lipobiology Volume 1, pages 215-239. Copyright © 1996 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-635-5
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VII. HYDROLYSIS OF ARACHIDONATE FROM BETA CELL MEMBRANE PHOSPHOLIPIDS, REGULATION OF THE BETA CELL CYTOSOLIC CALCIUM ION CONCENTRATION, AND INSULIN SECRETION VIII. ARACHIDONATE-CONTAINING PLASMALOGEN MOLECULAR SPECIES IN PANCREATIC ISLETS IX. ASCI-PLA2 AS A COMPONENT OF THE BETA CELL D-GLUCOSE SENSOR APPARATUS X. ARACHIDONATE METABOLISM IN ISLET BETA CELLS ACKNOWLEDGMENTS REFERENCES
227 229 232 233 235 235
ABSTRACT The p-cells of pancreatic islets maintain the blood D-glucose concentration within a narrow range by secreting insulin in response to rising D-glucose concentrations in the fluid bathing them. D-glucose must be transported into the p-cell and metabolized there in order to induce insulin secretion. Signals derived from the metabolism of D-glucose within p-cells increase the membrane permeability to Ca^^ and induce a rise in the cytosolic [Ca^"^], which is a critical signal in the induction of insulin secretion. Upon stimulation with secretagogues including D-glucose, phospholipid hydrolytic events occur in p-cells and result in the accumulation of a variety of phospholipid-derived mediators including nonesterified arachidonic acid and arachidonate metabolites. A major fraction of the D-glucose-induced hydrolysis of arachidonate from P-cell membrane phospholipids is independent of Ca^"^ influx and occurs in Ca^"^-free medium, but D-glucose-induced eicosanoid release from islets does not occur when glucose metabolism is prevented by compounds which inhibit the phosphorylation of D-glucose and its entry into glycolysis. Sufficient nonesterified arachidonate accumulates in D-glucose-stimulated islets to achieve an increment in the P-cell concentration of at least 30-70 |aM, and such concentrations of arachidonate induce a rise in the p-cell cytosolic [Ca^"^] and amplify depolarization-induced insulin secretion. The D-glucose-induced hydrolysis of arachidonate from p-cell membrane phospholipids is mediated by a phospholipase A2 enzyme whose activity is independent of Ca^^ and stimulated by ATP and which prefers plasmalogen substrates with sn-2 arachidonoyl residues. Selective inhibition of this islet ATP-stimulated, Ca^'^-independent (ASCI)-phospholipase A2 (PLA2) with a haloenol lactone suicide substrate which is sterically similar to plasmalogens suppresses the D-glucoseinduced hydrolysis of arachidonate from P-cell phospholipids, the rise in P-cell cytosolic [Ca^"^], and insulin secretion. Islets contain substantial amounts of plasmenylethanolamine molecular species bearing arachidonoyl residues in the sn-2 position, and these molecules are hydrolyzed more rapidly than diacyl-phospholipid substrates by islet ASCI-PLA2 in vitro and also undergo hydrolysis in intact, secretagogue-stimulated islets. Islet ASCI-PLA2 appears to constitute an important component of the P-cell D-glucose sensor apparatus.
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!• INTRODUCTION Pancreatic islet p-cells respond to increased D-glucose concentrations in the fluid bathing them by secreting insulin, which suppresses hepatic glucose production, accelerates glucose uptake into skeletal muscle and adipose tissue, and lowers the blood glucose concentration (1). In insulin-dependent diabetes mellitus (IDDM), hyperglycemia results from a loss of P-cell mass and the resultant inability to secrete insulin (1). In contrast, in the more prevalent disorder noninsulin dependent diabetes mellitus (NIDDM), most of the P-cell mass is retained, and P-cells can be induced to secrete insulin by nonglucose secretagogues such as arginine (1). The insulin secretory response to D-glucose is diminished, however, as is the potentiation by D-glucose of secretion induced by nonglucose secretagogues (1). The attenuated response to D-glucose by P-cells in NIDDM has prompted intensive investigation of how P-cells recognize D-glucose and couple alterations in glucose concentration to insulin secretion. In many types of cells, transduction of extracellular signals to responses such as secretion involves phospholipid hydrolysis and accumulation of phospholipidderived mediators including arachidonic acid and its metabolites (2), and stimulation of isolated islets with insulin secretagogues also induces accumulation of these mediators (3-5). This chapter attempts to relate the accumulation and action of nonesterified arachidonate and its metabolites to other signaling events that are known to occur in secretagogue-stimulated islet P-cells.
II. PANCREATIC ISLET BETA CELL D-GLUCOSE RECOGNITION Many endocrine cells recognize their secretagogues via plasma membrane receptors, and insulin secretion is modulated by receptor agonists including the neurotransmitter acetylcholine, which acts on P-cells via a muscarinic receptor (6). Insulin secretion induced by D-glucose, however, does not appear to be initiated by plasma membrane D-glucose receptors but depends on glucose metabolism within the P-cell (7,8). Both D-glucose transport into P-cells and the rate-limiting enzymatic step in its entry into glycolysis are mediated by proteins whose tissue distribution is limited to cells with glucose sensor functions, including p-cells and hepatocytes. The P-cell/hepatocyte facilitative glucose transporter (GLUT2) exhibits a high apparent dissociation constant (K^ about 15 mM) for glucose relative to facilitative glucose transporter isoforms expressed in other cells (9). This property of the GLUT2 transporter causes the rate of glucose entry into the P-cell to be proportional to the extracellular glucose concentration over the range of 4-17 mM in which the insulin secretory rate rises with glucose concentration (10). The conversion of D-glucose to the first intermediate in glycolysis (glucose-6-phosphate) is catalyzed in p-cells and in hepatocytes by the enzyme glucokinase, which exhibits a K^ for glucose of about 5 mM (11). In other tissues this reaction is
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catalyzed by hexokinases with a K^ for glucose of less than 1 mM. The lower affinity of glucokinase for glucose relative to other hexokinases causes the entry of glucose into glycolysis in P-cells to rise with glucose concentrations in the range of^lTmM. The requirement that D-glucose be metabolized within p-cells in order to induce insulin secretion is reflected by the fact that mannoheptulose, which prevents the phosphorylation of glucose and its entry into glycolysis, inhibits glucose-induced insulin secretion from isolated islets (7,8). The rate of glycolytic utilization of various carbohydrates by islets correlates well with their ability to induce insulin secretion (7,8). Carbohydrates other than D-glucose which are also metabolized by the glycolytic pathway in P-cells, such as mannose and glyceraldehyde, also induce insulin secretion, but carbohydrates which are not metabolized via glycolysis in islets, such as L-glucose and 3-0-methylglucose, fail to induce insulin secretion.
III. IONIC EVENTS IN GLUCOSE-INDUCED INSULIN SECRETION A second event that is required for glucose-induced insulin secretion is Ca^"^ entry from the extracellular space (12). Glucose-induced insulin secretion from isolated islets does not occur in Ca^'^-free medium and is attenuated by pharmacologic inhibition of Ca^"^ channels (3,12). The metabolism of glucose and the entry of Ca^"^ into P-cells have been related by an attractive model involving coordinated regulation of membrane channels for K"^ and for Ca^"^. Electrically excitable cells including P-cells have negative resting membrane potentials because they have far more open channels selective for K"^ than for other ions and because intracellular [K"^] (ca. 155 mM) exceeds extracellular [K"^] (ca. 4 mM) (13,14). A large fraction of P-cell plasma membrane K"^ permeability is mediated by a K'^-selective channel (K^yp) whose activity is inhibited by ATP (14,15) generated by metabolism of glucose (16,17). The P-cell K^yp channel is also the target of the hypoglycemic sulfonylureas, which inhibit channel activity, and of diazoxide, which maintains the channel in an open state (18). P-cell K^^p channel closure reduces the membrane K"^ conductance and induces a rise in membrane potential, which activates voltageoperated Ca^"*"-channels. Calcium ion then enters from the extracellular space down its concentration gradient, and p-cell cytosolic [Ca^"^] rises (19,20), which is thought to be a critical signal in glucose-induced insulin secretion. The question to which the remainder of this chapter is directed is how secretagogue-mduced phospholipid hydrolytic events in P-cells might relate to this model of insulin secretion.
IV. NONESTERIFIED ARACHIDONIC ACID AS A SECOND MESSENGER IN PANCREATIC ISLET BETA CELLS Isolated pancreatic islets contain substantial amounts of arachidonic acid esterified in membrane phospholipids, and arachidonate accounts for 30% of the total
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glycerolipid fatty acyl mass (21). Stimulation of islets with glucose induces hydrolysis of membrane phospholipids and the accumulation of nonesterified arachidonic acid in amounts sufficient to achieve an incremental change in the cellular concentration of 35—70 jiM, as measured by stable isotope dilution mass spectrometry (Figure 1) (22,23). The majority of the liberated arachdionate remains within the islet, and no glucose-induced liberation of the fatty acid palmitate is observed in islets (23). In contrast, the oxygenated arachidonate metabolite PGE2 is released into the perifusion medium of glucose-stimulated islets, and such eicosanoid release serves as a readily measured marker event for glucose-induced hydrolysis of arachidonate from islet membrane phospholipids (23). By following this marker event, hydrolysis of arachidonate from islet membrane phospholipids is observed within 2 minutes of stimulation with glucose and continues with a time course roughly parallel to that of glucose-induced insulin secretion (22,23). The amount of nonesterified, unmetabolized arachidonate which accumulates in glucose-stimulated islets (22,23) is substantially greater than that of the most abundant of the arachidonate metabolites in islets, which accumulate to concentrations no greater than 2.5 |LIM (24). This quantitative predominance of unmetabolized, nonesterified arachidonate raises the question of whether arachidonic acid itself may play some messenger function in islets that does not require its conversion to an oxygenated metabolite. It is possible that participation in the regulation of Ca^"^ gating by arachidonate represents such a second messenger role. Low micromolar concentrations of arachidonic acid amplify Ca^"^ entry through N-methyl-D-aspartate (NMDA)-operated Ca^"^ channels in cerebellar cells (25) and induce a rise in cytosolic [Ca^"^] in anterior pituitary cells (26) and in the GH3 clonal pituitary cell line (27). In GH3 cells, patch-clamping observations indicate that arachidonic acid shifts the activation curve of voltage-operated Ca^"^ channels to more negative potentials on the voltage axis (27). In the presence of arachidonate, a measurable Ca^"^ current flows through these channels at potentials only slightly above the resting membrane potential and at which there is no Ca^"^ current in the absence of arachidonate (27). Arachidonate therefore amplifies voltage-dependent Ca^"^ entry at small degrees of membrane depolarization. Such an effect of arachidonate in P-cells could constitute a mechanism to amplify the insulin secretory response to small changes in glucose concentration and in membrane potential. It seems likely that such amplification mechanisms must operate in vivo because in the normal subject the blood D-glucose concentration changes very little, but insulin secretion still occurs. Arachidonate (5-30 [iM) has been found to amplify the insulin secretory response of isolated islets to depolarizing concentrations of KCl by shifting the concentration-response curve to the left on the [KCl] axis (23) and therefore to lower transmembrane potentials (Figure 2). In the presence of arachidonic acid, measurable increments in insulin secretion occur at KCl concentrations which do not induce secretion in the absence of arachidonate, but neither basal nor maximal depolarization-induced insulin secretion is affected (23). That this effect of arachi-
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3 mM Glucose 28 mM Glucose
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Time (min) Figure 1. Glucose-induced accumulation of nonesterified arachidonic acid in isolated pancreatic islets demonstrated by stable isotope dilution gas chromatographymass spectrometry. The upper portion of the figure illustrates GC-MS tracings of the deuterium-labeled internal standard arachidonate and endogenous arachidonate derived from isolated pancreatic islets (upper two tracings) or incubation medium containing no islets (lower two tracings) after extraction, conversion to the pentafluorobenzyl ester derivative, reverse phase HPLC purification, and GC-MS analysis. The lower portion of the figure illustrates the Influence of stimulation with 28 m M D-glucose on the islet content of nonesterified arachidonate measured in this way. These figures have been modified with permission from Wolf et al. (1986). J. Biol. Chem. 261,3501-3511. 220
Phospholipid Hydrolysis and Regulation of Insulin Secretion A.) Amplification of KCI-lnduced Insulin Secretion by Arachldonate
221
B.) Arachldonate Concentratlon-Dependence of Amplification of Secretion
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Figure 2. Amplification of depolarization-induced insulin secretion from isolated pancreatic islets by arachidonic acid. In the left panel, 30 islets per condition were incubated under static conditions in medium containing 3 m M D-glucose and the indicated concentration of KCI with (closed squares) or without (open squares) arachidonic acid (20 jiM) for 30 minutes at 37° C, and the insulin content of the supernatant was then measured by radioimmunoassay. In the right panel, similar experiments were performed but the KCI concentration was fixed at 20 m M , and the arachidonate concentration was varied as indicated on the horizontal axis. These figures have been modified with permission from Wolf et al. (1991). Biochemistry 30, 6372-6379.
donate might be attributable to enhanced Ca^"^ entry is suggested by findings with single p-cells prepared by fluorescence-activated cell sorting (FAGS) and loaded with the Ca^"^-chelating fluorescent indicator Fura-2 (28). Arachidonate induces a rise in p-cell cytosolic [Ca^"^] (Figure 3) that is concentration-dependent (1-20 juM), readily reversible on washout, abolished upon removal of extracellular Ca^"^, and blunted by the Ca^'^-channel blocker nifedipine (28). The effect appears to be exerted by arachidonate itself rather than by a metabolite because it is unaffected by inhibitors of arachidonate oxygenases such as BW755C and indomethacin (28). These observations suggest that glucose-induced accumulation of nonesterified arachidonate could play a role in amplifying Ca^"^ entry into P-cells and in insulin secretion.
JOHN TURK, RICHARD W. GROSS, and SASANKA RAMANADHAM
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Phospholipid Hydrolysis and Regulation of Insulin Secretion
223
Figure 3, Arachldonic acid-induced rise in the cytosolic calcium ion concentration of single pancreatic islet p-cells. Single islet p-cells prepared by fluorescence-activated cell sorting were attached by glass cover slips, loaded with Fura-2, perifused in a thermostatic cell, and monitored microfluorimetrically in a PTI DeltaScan instrument fitted with a Nikon microscope with excitation at 340 or 380 nm and emission measurements at 500-530 nm. Arachidonic acid (AA) was introduced at varied concentrations at the time indicated and subsequently washed out with bovine serum albumin-containing medium. The ratio of fluorescence intensity observed at an excitation wavelength of 340 nm divided by that at 380 nm is proportional to the cytosolic [Ca '^]. The upper portion of the figure (panels A through F) illustrates data from individual cells, and the lower portion of the figure (panel G) illustrates mean data from 14 cells per condition. This figure has been reproduced with permission from Ramanadham etal. (1992). Biochem. Biophys. Res. Commun. 184, 647-653.
A.) Insulin Secretion
3Q/0Ca
17Q/0Ca
B.) Eicosanoid Release
3G/0Ca
17G/0Ca
Figure 4, Influence of extracellular calcium ion concentration on glucose-induced insulin secretion and eicosanoid release from isolated pancreatic islets. Fifty islets per condition were incubated under static conditions for 30 minutes at 37° C in medium containing 3 mM or 17 mM D-glucose and 2.5 mM Ca^"^ (3G or 1 7 G) or in medium containing 3 or 17 mM D-glucose and no added Ca^"^ (3G/0Ca or 17G/0Ca). At the end of that period supernatant was removed and the content of insulin (A) or PGE2 (B) was measured by radioimmunoassay or enzyme immunoassay, respectively. This figure has been modified with permission from Wolf et al. (1991). Biochemistry 30, 6372-6379.
JOHN TURK, RICHARD W. GROSS, and SASANKA RAMANADHAM
224
V. CONTROL OF ARACHIDONATE HYDROLYSIS FROM ISLET MEMBRANE PHOSPHOLIPIDS Most recognized phospholipases which catalyze hydrolysis of arachidonate from phospholipids either require Ca^"^ for activity or are activated by rising cytosolic [Ca^"^] (29). Indeed, other phospholipid hydrolytic events induced in islets by glucose, such as phosphoinositide hydrolysis, are attributable to glucose-induced Ca^"^ influx (30). If glucose-induced arachidonate hydrolysis from islet membrane phospholipids were triggered solely by Ca^"^ influx, arachidonate accumulation might occur too late in the sequence of biochemical responses to glucose to influence Ca^"^ entry. In fact, a major fraction of glucose-induced release of arachidonate from islet membrane phospholipids is independent of Ca^"^ influx because it occurs in Ca^'^-free medium (Figure 4) and in the presence of the Ca^^-channel blockers verapamil and nifedipine (23,31). A. Carbohydrate-Induced Insulin Release
B. Carbohydrate-Induced Eicosanoid Release
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Figures. Carbohydrate insulin secretagogues must be metabolized to induce insulin secretion and eicosanoid release from isolated pancreatic islets. Isolated islets (150 per condition) were perifused for 15 minutes at 37 C with medium containing 3 mM D-glucose; 17 mM D-glucose; 17 mM D-glucose plus 17 mM mannoheptulose; 3 mM D-glucose plus 17 mM D-glyceraldehyde; or 3 mM D-glucose plus 17 mM 3-O-methylglucose. Perifusion effluent was collected, and the content of insulin (A) and PGE2 (B) was measured by radioimmunoassay or by enzyme immunoassay, respectively. This figure has been reproduced with permission from Turk et al. (1992). Biochim. Biophys. Acta 1125,280-291.
Phospholipid Hydrolysis and Regulation of Insulin Secretion
225
D-glucose fails to induce eicosanoid release from islets, however, unless it is metabolized (31). Inhibition of the phosphorylation of D-glucose and its entry into glycolysis with mannoheptulose totally prevents D-glucose-induced eicosanoid release (Figure 5) (31). Other carbohydrate fuel secretagogues which are metabolized via glycolysis, such as D-mannose and D-glyceraldehyde, also induce eicosanoid release from islets, but carbohydrates which are not metabolized and do not induce insulin secretion, such as L-glucose and 3-0-methylglucose, fail to induce eicosanoid release (31). Glycolytic metabolism appears to be required for this effect, because lactate, which is readily oxidized by the P-cell mitochondrion but which does not induce insulin secretion, fails to induce eicosanoid release from islets (31). A signal derived from glycolytic metabolism of glucose therefore appears to be required for the induction of hydrolysis of arachidonate from membrane phospholipids, just as it is for insulin secretion. This is also the case for glucose-induced closure of K^^p channels in P-cells (16), and in that case the metabolic signal is thought to be ATP (15).
VI. A BETA CELL ATP-STIMULATED, C A ^ ^ - I N D E P E N D E N T PHOSPHOLIPASE A2 ENZYME A novel phospholipase A2 activity recently identified in myocardium (32-36) has several properties which make it an attractive candidate for mediating a component of the glucose-induced hydrolysis of arachidonate from islet membrane phospholipids. The enzyme has no requirement for Ca^"^ and is fully active in EGTAcontaining medium (32-36). Interestingly, the enzyme is activated by physiological concentrations of ATP, and this activation involves interaction of a 40 kDa catalytic subunit with a 350 kDa regulatory unit which is the ATP-sensing component of the complex (36). The enzyme hydrolyzes phospholipids containing either choline or ethanolamine headgroups, prefers sn-2 arachidonoyl residues over other fatty acyl substituents, and prefers a vinyl ether (plasmalogen) linkage in the sn-\ position over an acyl linkage (32—36). The enzyme is also irreversibly inactivated by a haloenol lactone suicide substrate (HELSS), which is sterically similar to plasmalogens and which selectively inhibits the ATP-stimulated Ca^"^-independent (ASCI)phospholipase A2 (PLA2) at concentrations which have no influence on the activities of Ca^'^-dependent phospholipases A2 from a variety of sources (35). The chemical identity of HELSS is (E)-6-(bromomethylene)tetrahydro-3-(lnaphthalenyl)-2H-pyran-2-one] (35). The fact that ASCI-PLA2 is stimulated by ATP and does not require Ca^"^ has prompted a search for a similar enzymatic activity in isolated islets. In experiments with synthetic phospholipid substrates containing radiolabeled fatty acids in the sn-l position, acyl or alkenyl (plasmenyl) linkages in the snA position, and choline or ethanolamine head groups, rat islets have been found to contain substantial amounts of a Ca^'^-independent PLA2 activity in both cytosolic and membranous subcellular fractions which catalyzes hydrolysis of the radiolabeled fatty acid from
226
JOHN TURK, RICHARD W. GROSS, and SASANKA R A M A N A D H A M Islet
ASCI-PLA2
Substrate
Preference
D
Cytosol
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Membranes
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Figure 6. Phospholipid substrate preference of a pancreatic islet calcium ion-independent PLA2 activity. Cytosol or membranous fractions prepared from isolated pancreatic islets were incubated with synthetic phospholipid substrates in medium containing no added calcium ion and 10 m M EGTA. PLA2 activity was monitored as release of radiolabeled fatty acids from the sn-2 position of the synthetic substrates and expressed as a specific enzymatic activity after calculations involving the amount of fatty acid released, the specific radioactivity of the substrate, and the measured protein content of the incubation mixture. All substrates contained a saturated 16 carbon chain in the sn-1 position (16:0). The notation " a " denotes an acyl linkage to a palmitic acid residue in the sn-1 position. The notation " p " denotes a plasmenyl (vinyl ether) linkage to a palmitic aldehyde residue in the sn-1 position. In individual phospholipid substrates, the radiolabeled fatty acid residue in the sn-2 position was palmitate (16:0), oleate (18:1), linoleate (18:2), or arachldonate (20:4). The phospholipid substrate head-group was either choline (PC) or ethanolamine (PE). This figure has been constructed with permission from data in Gross et al. (1993). Biochemistry 32, 327-336.
these substrates (37). The amounts of Ca^'*'-independent PLA2 activity in islets in fact exceed the amounts of Ca^'^-dependent PLA2 activity. The islet Ca^+-independent activity prefers plasmenyl over acyl linkages in the sn-l position, prefers arachidonoyl over palmitoyl residues in the sn-2 position, and catalyzes hydrolysis of substrates with choline or ethanolamine headgroups (Figure 6). The rat islet cytosolic Ca^"*"-independent PLA2 activity is augmented several-fold by ATP (0.1—
Phospholipid Hydrolysis and Regulation of Insulin Secretion
227
Figure 7, Stimulation by ATP of islet cytosolic calcium ion-independent PLA2 activity. Calcium ion-independent PLA2 activity was measured as in Figure 6 in islet cytosolic (open symbols) or membranous (closed symbols) subcellular fractions in incubation medium containing no added calcium ion, 10 m M EGTA, and either no ATP or ATP at a concentration between 10 i i M and 10 m M . Phospholipid substrates were 16:0p/18:1-PC (panel A, circles), 16:0a/20:4-PE (panel B, squares), or 16:0a/20:4-PC (panel C, triangles), where the notation follows the conventions described in the legend of Figure 6. This figure has been reproduced with permission from Gross et al. (1993). Biochemistry 32, 327-336.
10 mM) (Figure 7) and is inactivated by low micromolar concentrations of the suicide substrate HELSS (Figure 8). Both isolated human islets and clonal insulinsecreting RIN-m5f cells also express a similar Ca^'^-independent PLA2 activity (37). Although the human islet Ca^'^-independent PLA2 activity is also stimulated by ATP, the RIN cell activity is not, a finding of interest in view of the fact that REN cells do not secrete insulin in response to glucose (38).
VII. HYDROLYSIS OF ARACHIDONATE FROM BETA CELL MEMBRANE PHOSPHOLIPIDS, REGULATION OF THE BETA CELL CYTOSOLIC CALCIUM ION CONCENTRATION, AND INSULIN SECRETION The ASCI-PLA2 suicide substrate HELSS has been used in experiments with intact islets to examine the role of ASCI-PLA2 in glucose-induced eicosanoid release and in insulin secretion. HELSS has been found to inhibit glucose-induced eicosanoid
228
JOHN TURK, RICHARD W. GROSS, and SASANKA R A M A N A D H A M
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Figure 8, Inhibition of islet calcium ion-independent PLA2 activity with a selective HELSS. Calcium ion-independent PLA2 activity was measured as in Figure 6 in islet cytosolic (open symbols) or membranous (closed symbols) subcellular fractions in incubation medium containing no added calcium ion and 10 m M EGTA. The HELSS was either absent from the incubation medium or present at a concentration between 10 nM and 10 ^iM. The chemical identity of HELSS is (E)-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one]. Phospholipid substrates were 16:0p/18:1PC (panel A, circles), 16:0a/20:4-PE (panel B, squares), or 16:0a/20:4-PC (panel C, triangles), where the notation follows the conventions described in the legend of Figure 6. This figure has been reproduced with permission from Gross et al. (1993). Biochemistry 32, 327-336.
release from islets in a concentration- and time-dependent manner without affecting basal eicosanoid release (Figure 9) (39). This suggests that ASCI-PLA2 mediates glucose-induced hydrolysis of arachidonate from islet membrane phospholipids. With a similar concentration-dependence and time course, HELSS also suppresses glucose-induced insulin secretion (Figure 9) from islets (39), suggesting that glucose-induced hydrolysis of arachidonate from membrane phospholipids participates in the signal transduction pathways leading to insulin secretion. At these concentrations, HELSS influences neither islet oxidation of [^"^CJ-glucose to [^'^C]02 nor carbachol-induced inositol trisphosphate accumulation in islets (39). With FACS-purified, Fura-2-loaded P-cells, HELSS suppresses the glucose-induced rise in cytosolic [Ca^"^] by 65% but does not influence the rise in P-cell cytosolic [Ca^"^] induced by depolarization with KCl, by stimulation with carbachol, or by exogenous arachidonate (Figure 10) (39). These observations suggest
Phospholipid Hydrolysis and Regulation of Insulin Secretion
A.) HELSS on PQE2 relesM
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Figures. Inhibition of glucose-induced insulin secretion and eicosanoid release from pancreatic islets by a selective suicide substrate of the islet ATP-stimulated, Ca^'^-independent PLA2 enzyme. Isolated pancreatic islets were perifused, and the contents of insulin and PGE2 in the effluent were measured as in Figure 5. In panels A (PGE2 release) and B (insulin secretion), these parameters were measured in islets that had been pre-treated with 25 |iM HELSS (open symbols) or with vehicle (closed symbols) for 30 minutes at 37° C and then perifused for 15 minutes with medium containing glucose at the concentrations indicated on the horizontal axis. In panel C, islets were perifused with 17 m M D-glucose after pretreatment with HELSS at the concentration indicated on the horizontal axis. In panel D, islets were perifused with 17 m M D-glucose after pretreatment with 25 |LIM HELSS for the period indicated on the horizontal axis. This figure has been modified with permission from Ramanadham et al. (1993). Biochemistry 32, 337-346.
that the glucose-induced hydrolysis of arachidonate from islet membrane phospholipids participates in the rise in P-cell cytosolic [Ca^"^] induced by glucose.
Vni. ARACHIDONATE-CONTAINING PLASMALOGEN MOLECULAR SPECIES IN PANCREATIC ISLETS To determine whether islets contain endogenous plasmalogen phospholipids bearing sn-l arachidonoyl residues which might serve as substrates for ASCI-PLA2,
230
JOHN TURK, RICHARD W. GROSS, and SASANKA R A M A N A D H A M A.
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Figure 10. Inhibition of the glucose-induced rise in p-cell cytosolic calcium ion concentration by a selective suicide substrate of the islet ATP-stimulated, Ca^'^-independent PLA2 enzyme. Single pancreatic islet p-cells were prepared, loaded with Fura-2, and monitored fluorimetrically as described in Figure 3. Increasing values of the parameter displayed on the vertical axis correspond to increasing values of cytosolic [Ca^"^]. Cells were pre-treated for 30 minutes at 37° C with 3 |aM HELSS (panels B and D) or with vehicle (panels A and C). Cells were perifused with medium containing 3 m M D-glucose and monitored continuously. In panels A and B, arachidonate (AA) was added at a concentration of 15 |aM and then washed out (W) with medium containing bovine serum albumin at the times indicated by the arrows. In panels C and D, the medium D-glucose concentration was increased to 1 7 m M (G) and then washed out (W) with medium containing 3 m M D-glucose at the times indicated by the arrows. This figure has been reproduced with permission from Ramanadham et al. (1993). Biochemistry 32, 337-346.
islet phospholipids have been separated into head-group classes by normal phase HPLC (21). Each head-group class has then been digested with B. cereus phospholipase C to yield the diglycerides, which have then been converted to acetate derivatives and analyzed by TLC under conditions where alkenylacyl-, alkylacyl-, and diacyl-acetylglycerols are clearly separated (21). These analyses have revealed that islet ethanolamine-phospholipids contain substantial amounts of alkenylacyldiglyceride, reflecting the presence of plasmenylethanolamine molecular species in islet membranes.
Phospholipid Hydrolysis and Regula tion of Insulin Secretion
2 31
To determine whether these plasmenylethanolamine species contain arachidonate, islets have been prelabeled with [^H]-arachidonate, and their phospholipids have been extracted and analyzed by sequential normal phase and then reverse phase HPLC under conditions where individual molecular species of head-group classes of phospholipids are resolved (21). Such analyses have revealed that there are six major arachidonate-containing molecular species of islet ethanolaminephospholipids. Three of these peaks have been found to be acid-labile, which is characteristic of plasmalogens, and the remaining three peaks to be acid-stable. The molecular identities of these peaks have been established by acid methanolysis and gas chromatography-mass spectrometry (GC-MS) in the positive ion-methane chemical ionization (PCI) mode. Acid methanolysis releases fatty aldehyde residues from the sn-\ position of plasmalogens as dimethylacetal (DMA) derivatives, which yield a strong (M-31) ion on PCI-MS analysis. Acid methanolysis releases fatty acyl residues from phospholipids as fatty acid methyl ester (FAME) derivatives, which yield a strong protonated molecular ion (M+1) on PCI-MS analysis. Individual molecular species of FAME and DMA derivatives are clearly separated on GC analysis. Application of these procedures to the islet ethanolamine phospholipid molecular species isolated by reverse phase HPLC has revealed that the three acid-labile peaks each contain an arachidonate residue and a fatty aldehyde residue (21). The fatty aldehyde residues in these three peaks are palmitic aldehyde, oleic aldehyde, and stearic aldehyde, respectively. Similar analyses of the acid-stable islet ethanolamine-phospholipid peaks have revealed that these materials each contain two fatty acyl residues, one of which is arachidonate. The identities of the other residues are palmitate, oleate, and stearate, respectively. The arachidonate content of these islet phospholipids has been quantitated after saponification in the presence of deuterium-labeled arachidonate as an internal standard, conversion of the liberated fatty acids to pentafluorobenzyl ester derivatives, and stable isotope dilution GC-MS measurements in the negative ion-(methane) chemical ionization mode (21). These measurements have revealed that arachidonate constitutes 30% of the total fatty acyl mass of islet glycerolipids, which is among the highest reported for any tissue. Of that amount, 30% resides in islet ethanolamine-phospholipids, 44% of which is contained in the three plasmenylethanolamine molecular species mentioned above (21). The rat islet content of plasmenylethanolamine is virtually identical to that reported for rat cerebral cortical neurons (40), which, like other electrically active tissues such as myocardium (41,42), contain substantially greater amounts of plasmalogens than do electrically inactive tissues such as liver (43,44). Incubation of endogenous islet ethanolamine-phospholipid molecular species isolated by sequential normal phase and then reverse phase HPLC with islet cytosolic ASCI-PLA2 activity has revealed that plasmenylethanolamine substrates are hydrolyzed substantially more rapidly (1.86-10.30 pmol/mg-min) than is the corresponding diacyl species (1-stearoyl, 2-arachidonoyl)-phosphatidylethanolamine (0.04 pmol/mg-min) (21). This is similar to the substrate preference observed
232
JOHN TURK, RICHARD W. GROSS, and SASANKA RAMANADHAM
with synthetic phospholipids both for myocardial (32-36) and islet (37) cytosolic ASCI-PLA2. Islet ASCI-PLA2-catalyzed hydrolysis of endogenous plasmenylethanolamine species in vitro has been found to be inhibited by the ASCIPLA2 suicide substrate and plasmalogen analog HELSS (21), as previously observed for ASCI-PLA2-catalyzed hydrolysis of synthetic phospholipid substrates (37). Prolonged perifusion of isolated islets with secretagogues has been found to result in a multiphasic temporal pattern of insulin secretion and an oscillatory, multiphasic release of eicosanoids reflecting hydrolysis of arachidonate from islet phospholipids (21). All of the temporal phases of both insulin secretion and eicosanoid release are inhibited to a similar degree by the ASCI-PLA2 suicide substrate HELSS (21). The periodicity of the oscillations in eicosanoid release under these conditions is similar to that recently reported for the oscillatory rates of glycolytic metabolism of D-glucose in islets (45) and may reflect cycles of ASCI-PLA2 activation induced by oscillations in the ATP/ADP ratio (46). Under these conditions, prolonged perifusion with secretagogues has been found to induce a decline in the arachidonoyl content of islet plasmenylethanolamine species of 5.9 pmol/islet and a decline in the arachidonoyl content of phosphatidylethanolamine species of 2.1 pmol/islet, suggesting that plasmenylethanolamine species undergo hydrolysis in the secretagogue-stimulated islet (21). Collectively, these observations suggest that ASCI-PLA2-catalyzed hydrolysis of arachidonate from endogenous plasmenylethanolamine molecular species is an intermediary biochemical event in the induction of insulin secretion.
IX. ASCI-PLA2 AS A COMPONENT OF THE BETA CELL D-GLUCOSE SENSOR APPARATUS A model (47) which incorporates the observations described above into existing knowledge of the early events in glucose-induced insulin secretion is that glucose enters the P-cell via the GLUT2 transporter, is phosphorylated by glucokinase, and enters glycolysis. ATP generated in the cytosol then interacts with two targets, the K^yp channel and the ATP-sensing unit of ASCI-PLA2. The effect of ATP on the K^yp channel is to induce closure of a fraction of the channel population, which results in membrane depolarization. For small increments in glucose concentration, this effect may be insufficient to raise membrane potential from the resting level of about—70 mV (13) to the-40 mV level required for activation of voltage-operated Ca^'^-channels (27). Interaction of ATP with the ATP-sensing unit of ASCI-PLA2 enzyme, however, activates the phospholipase, which catalyzes hydrolysis of arachidonate from membrane phospholipids including plasmenylethanolamine molecular species. Nonesterified arachidonate then accumulates in the membrane and alters the properties of voltage-operated Ca^'*"-channels to enhance their activity at small degrees of membrane depolarization in the range of-60 mV to -40 mV
Phospholipid Hydrolysis and Regulation of Insulin Secretion
233
(27). This results in amplification of Ca^"^ entry from the extracellular space, a rise in the P-cell cytosolic [Ca^"^], and the induction of insulin exocytosis. Although this model fits several observations discussed above, it obviously oversimplifies actual P-cell physiology. Complexities not incorporated include the fact that: 1. Factors in addition to ATP, including ADP (48,49), and possibly others (50), influence P-cell K^jp channel activity, and this may also be true for ASCIPLA2. 2. Both glucose metabolism (51,52) and high concentrations of arachidonate (53—55) stimulate insulin secretion by mechanisms in addition to promoting Ca^"^ influx into p-cells. 3. As P-cell [Ca^"^] rises, Ca^"^-dependent PLA2 (56-60) may be activated and further amplify arachidonate release. 4. PLA2 action also yields lysophospholipids which may promote insulin secretion (61-63). 5. A fraction of the arachidonate in glucose-stimulated islets is converted to metabolites which may influence secretion (64-69), as discussed below.
X. ARACHIDONATE METABOLISM IN ISLET BETA CELLS Of the nonesterified arachdionate which accumulates in glucose-stimulated islets, a fraction is converted to cyclooxygenase products, which may negatively modulate insulin secretion (8), and to 12-lipoxygenase products, which may promote insulin secretion (64—69). The most abundant of the arachidonate metabolites produced by islets is the 12-lipoxygenase product 12-hydroxy-(5,8,10,14)-eicosatetraenoic acid (12-HETE), which is formed from its 12-hydroperoxy precursor (12-HPETE). Both rat (65-67) and human (68) islets synthesize 12-HETE from endogenous arachidonate as the quantitatively predominant arachidonate metabolite. Islet 12-HETE consists exclusively of the S-stereochemical isomer with no detectable R-enantiomer (67), and this is consistent with an origin of this compound from lipoxygenase catalysis. D-glucose and other fuel secretagogues stimulate accumulation of 12-HETE in islets to concentrations up to 2.5 |LIM (66,69), and several structurally distinct pharmacologic inhibitors of the 12-lipoxygenase suppress both glucoseinduced insulin secretion and islet 12-HETE production with similar concentrationdependence (65-68). Attempts to reverse this inhibition with exogenous 12-lipoxygenase products in experiments with islets from adult animals have been disappointing (24,67), but 12-HPETE stimulates insulin secretion from neonatal pancreatic islet cells (65). Recent observations with P-cells from adult rat islets suggest that the arachidonate 12-lipoxygenase and its products may play a role in p-cell function. An antibody directed against neutrophil isozyme of the arachidonate 12-lipoxygenase and an antibody directed against the arachidonate 5-lipoxygenase have been used
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to probe the cellular distribution of these enzymes in immunocytochemical studies using a biotin-avidin-peroxidase detection system (70). In sections of whole pancreas, 12-lipoxygenase antigen is detected only within islets and not in pancreatic exocrine tissue, and no 5-lipoxygenase antigen is detected in islets. Immunoblotting of extracts from isolated islets reveals that a single protein band is recognized by the anti-12-lipoxygenase antibody and that this protein co-migrates (Mj. = 72 kDa) with authentic neutrophil 12-lipoxygenase on SDS-PAGE analysis (70). Dispersed cells from isolated islets of adult rats have been separated into virtually homogeneous populations of (3-cells and into a-cell-enriched populations by FACS. Only FACS-purified P-cells and not a-cell-enriched populations contain 12-lipoxygenase antigen, and the FACS-purified p-cells contain no detectable 5-lipoxygenase antigen (70). In addition, only the P-cells and not the a-cell-enriched populations convert exogenous arachidonate to the 12-lipoxygenase product 12-HETE, the identity of which has been verified by HPLC analysis and by mass spectrometry in both electron impact and negative ion chemical ionization modes (70). Neither P-cells nor a-cell-enriched populations convert arachidonate to the 5-lipoxygenase product 5-HETE (70). These observations indicate that pancreatic islet P-cells selectively express an arachidonate 12-lipoxygenase enzyme and suggest that this enzyme may play some unique role in P-cell function. At least two questions are raised by these observations. If 12-lipoxygenase products participate in insulin secretion, why have exogenous 12-lipoxygenase products been relatively ineffective in influencing insulin secretion, and with what intracellular targets might 12-lipoxgenase products interact in the P-cell? With respect to the former question, the observation that P-cells express the neutrophil isozyme of the 12-lipoxygenase may have some bearing. At least two isozymes of the 12-lipoxygenase exist, which are distinguishable by antigenic determinants and substrate specificity (71). The platelet isozyme accepts arachidonic acid as substrate but is virtually inactive with linoleic acid. In contrast, the neutrophil isozyme catalyzes oxygenation of linoleic acid as well as arachidonic acid. The linoleate oxygenation product is 13-hydroperoxy-octadecadienoic acid, which is then reduced to the corresponding 13-hydroxy compound. The possibility that islets may synthesize these products is suggested by the observations that linoleic acid is roughly one half as abundant as arachidonic acid in islet phospholipids (72), and that the concentration of nonesterified linoleate also rises in glucose-stimulated islets (23). One of these linoleate products may exert an influence on insulin secretion that previously has been difficult to demonstrate with the corresponding products from arachidonate. It is also possible that certain more complex 12-lipoxygenase products may prove to have such activity (73) or that glycerolipids modified by the action of the 12-lipoxygenase may have a role in insulin secretion (74). With respect to potential targets of 12-lipoxygenase products within p-cells, it is of interest that these products act as second messengers of neurotransmitters in the organism Aplysia California. The neurotransmitters histamine and the peptide
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FMRFamide induce changes in membrane potential in sensory neurons of this organism that patch-clamping observations indicate is mediated by a direct action of a metabolite of 12-HPETE on plasma membrane K"^-channels (75). Certain 12-HPETE metabolites close such channels and hyperpolarize the cells (76), but other 12-HPETE metabolites depolarize Aplysia neurons (76). The prospect that 12-lipoxygenase products might modulate P-cell K"^-channel activity is an intriguing one in view of recent skepticism that ATP is the sole modulator of the activity of the p-cell K^-^p channel (50).
ACKNOWLEDGMENTS These studies were supported by NIH grants DK-34388, DK-01553, andHL-34839. Superb technical assistance was provided by Alan Bohrer, Mary Mueller, Kelly Kruszka, and Lori Zupan.
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6. Garcia, M.-G., Hermans, M.P., & Henquin, J.-C. (1988). Glucose-, Ca -, and concentration dependence of acetylcholine stimulation of insulin release and ionic fluxes in mouse islets. Biochem. J. 254,211-218. 7. Ashcroft, S.J.H. (1980). Glucoreceptor mechanisms and the control of insulin release and biosynthesis. Diabetologia 18, 5-15. 8. Meglasson, M.D. & Matschinsky, F.M. (1986). Pancreatic islet glucose metabolism and regulation of insulin secretion. Diabetes/Metabolism Rev. 2, 163—214. 9. Garvey, W.T. (1992). Glucose transport and NIDDM. Diabetes Care 15, 396-417. 10. Ashcroft, S.J.H., Bassett, J.M., & Randle, P.J. (1971). Insulin secretion mechanisms and glucose metabolism in islets. Diabetes 21, 538-545. 11. Matschinksy, F.M. (1990). Glucokinase as glucose sensor and metabolic signal generator in pancreatic beta cells and hepatocytes. Diabetes 39, 647-652. 12. Wollheim, C.B. & Sharp, G.W. (1981). Regulation of insulin release by calcium. Physiol. Rev. 61, 914-973. 13. Hille, B. (1984). In: Ionic Channels of Excitable Membranes. Sinauer Associates, Sunderland, Massachusetts. 14. Cook, D.L., Satin, L.S., Ashford, M.L.J., & Hales, C.N. (1988). ATP-sensitive K"" channels in pancreatic beta cells. Spare channel hypothesis. Diabetes 37, 495-498. 15. Cook, D.L. & Hales, C.N. (1984). Intracellular ATP directly blocks K"^ channels in pancreatic beta cells. Nature 311, 271-273.
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16. Ashcroft, F.M., Harrison, D.E., & Ashcroft, S.J.H. (1984). Glucose induces closure of single K"^ channels in pancreatic beta cells. Nature 213, 446-448. 17. Rorsman, P. & Trube, G. Glucose dependent K channels in pancreatic beta cells are regulated by intracellular ATP. Pflugers Arch. 405, 305-309. 18. Trube, G., Rorsman, P., & Ohno-Shosaku, T. (1986). Opposite effects of tolbutamide and diazoxide on the ATP-dependent K^ channel in mouse pancreatic beta cells. Pflugers Arch. 407,493-499. 19. Arkhammar, P, Nilsson, T, Rorsman, P, & Berggren, P-0. (1987). Inhibition of ATP-regulated K"*^ channels precedes depolarization-induced increase in cytoplasmic free Ca^"^ concentration in pancreatic beta cells. J. Biol. Chem. 262, 5448-5454. 20. Gylfe, E. (1988). Glucose-induced early changes in cytoplasmic calcium of pancreatic beta cells studied with time-sharing dual-wavelength fluorometry. J. Biol. Chem. 262, 5044-5048. 21. Ramanadham, S., Bohrer, A., Mueller, M., Jett, P., Gross, R.W., & Turk, J. (1993). Mass spectrometric identification and quantitation of arachidonate-containing phospholipids in pancreatic islets: Prominence of plamenylethanolamine molecular species. Biochemistry 32,5339-5351. 22. Wolf, B.A., Turk, J., Sherman, W.R., & McDaniel, M.L. (1986). Intracellular Ca^"" mobilization by arachidonic acid. Comparison with myo-inositol 1,4,5-trisphosphate in isolated pancreatic islets. J. Biol. Chem. 261, 3501-3511. 23. Wolf, B. A., Pasquale, S.M., & Turk, J. (1991). Free fatty acid accumulation in secretagogue-stimulated pancreatic islets and effects of arachidonate on depolarization-induced insulin secretion. Biochemistry 30,'6371-^379. 24. Turk, J., Colca, J.R., McDaniel, M.L. (1985). Arachdionic acid metabolism in isolated pancreatic islets III. Effects of exogenous lipoxygenase products and inhibitors on insulin secretion. Biochim. Biophys. Acta 834,23-36. 25. Miller, B., Sarantis, M., Traynelis, S.F., & Attwell, D. (1992). Potentiation of NMDA receptor currents by arachidonic acid. Namre 355, 722-725. 26. Knepel, W., Schofl, C , & Gotz, D.M. (1988). Arachidonic acid elevates cytosolic free calcium concentration in rat anterior pituitary cells. Naunyn-Schmieberg's Arch. Pharmacol. 338,303—309. 27. Vacher, P., McKenzie, J., & Dufy, B. (1989). Arachidonic acid affects membrane ionic conductances of GH3 pituitary cells. Am. J. Physol. 257, E203-E211. 28. Ramanadham, S., Gross, R., & Turk, J. (1992). Arachidonic acid elevates the cytosolic calcium concentration in individual pancreatic islet beta cells. Biochem. Biophys. Res. Commun. 184, 647-653. 29. Bonventre, J.V. (1992). Phospholipase A2 and signal transduction. J. Am. Soc. Nephrol. 3,128-150. 30. Biden, T.J., Peter-Riesch, B., Schlegel, W, & WoUheim, C.B. (1987). Ca^'^-mediated generation of inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate in pancreatic islets. Studies with K , glucose, and carbamylcholine. J. Biol. Chem. 262, 2567-3571. 31. Turk, J., Mueller, M., Bohrer, A., & Ramanadham, S. (1992). Arachidonic acid metabolism in isolated pancreatic islets VI. Carbohydrate insulin secretagogues must be metabolized to induce eicosanoid release. Biochim. Biophys. Acta 1125,280-291. 32. Wolf, R.A. & Gross, R.W. (1985). Identification of neutral active phospholipase C which hydrolyzes choline glycerophospholipids and plasmalogen selective phospholipase A2 in canine myocardium. J. Biol. Chem. 260, 7295-7303. 33. Hazen, S.L., Stuppy, R.J., & Gross, R.W. (1990). Purification and characterization of canine myocardial cytosolic phospholipase A2. A calcium-independent phospholipase with absolute sn-2 regiospecificity fordiradyl glycerophopholipids. J. Biol. Chem. 265, 10622-10630. 34. Hazen, S.L., Ford, D.A., & Gross, R.W. (1991). Activation of a membrane-associated phospholipase A2 during rabbit myocardial ischemia which is highly selective for plasmalogen substrate. J. Biol. Chem. 266, 5629-5633.
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35. Hazen, S.L., Zupan, L.A., Weiss, R.H., Getman, D.R, & Gross, R.W. (1991). Suicide inhibition of myocardial cytosolic Ca "^-independent phospholipase A2. Mechanism-based discrimination between Ca """-dependent and independent phospholipases A2. J. Biol. Chem. 255, 7227-7232. 36. Hazen, S.L. & Gross, R.W. (1991). ATP-dependent regulation of rabbit myocardial cytosolic calcium-independent phospholipase A2. J. Biol. Chem. 266, 14526-14534. 37. Gross, R.W., Ramanadham, S., Kruszka, K.K., Han, X., & Turk, J.(1993). Rat and human 2+ pancreatic islet cells contain a Ca -independent phospholipase A2 activity selective for hydrolysis of arachidonate which is stimulated by ATP and is specifically localized to islet p-cells. Biochemistry 32, 327-336. 38. Praz, G.A., Halban, RA., Wollheim, C.B., Blondel, B., Strauss, A.J., & Renold, A.E. (1983). Regulation of immunoreactive insulin release from a rat cell line (RINm5f). Biochem. J. 210, 345-352. 39. Ramanadham, S., Gross, R.W, Han, X., & Turk, J. (1993). Inhibition of arachidonate release suppresses both insulin secretion and the rise in P-cell cytosolic Ca ^ concentration. Biochemistry 32,337-346. 40. Freysz, L., Bieth, C , Sensenbreener, M., Jacob, M., & Mandel, P. (1968). Quantitative distribution of phospholipids in neutrons and glial cells isolated from rat cerebral cortex. J. Neurochem. 15, 307-313. 41. Gross, R.W. (1984). High plasmalogen and arachidonic acid content of canine myocardial sarcolemma: A fast atom bombardment mass spectroscopic and gas chromatography-mass spectroscopic characterization. Biochemistry 23, 158-165. 42. Gross, R.W. (1985). Identification ofplasmalogen as the major phospholipid constituent of cardiac sarcoplasmic reticulum. Biochemistry 24, 1662-1668. 43. Dawson, R.M.C., Hemington, N., & Davenport, J.B. (1962). Improvements in the method of determining individual phospholipids in a complex mixture by successive chemical hydrolyses. Biochem. J. 84,497-501. 44. Scott, T.W., Setchell, B.P., & Bassett, J.M. Characterization and metabolism of ovine foetal lipids. Biochem. J. 104, 1040^1047. 45. Chou, H.-F., Berman, N., & Ipp, E. (1992). Oscillations of lactate released from islets of Langerhans: Evidence for oscillatory glycolysis in p-cells. Am. J. Physiol. 262, E800-E805. 46. Corkey, B.C., Tomheim, K., Deeney, J.T., Glennon, M.C., Parker, J.C., Matschinsky, P.M., Ruderman, N.B., & Prentki, M. (1988). Linked oscillations of free Ca^"" and the ATP/ADP ratio in permeabilized RINm5F insulinoma cells supplemented with a glycolyzing cell-free muscle extract. J. Biol. Chem. 263, 4254^258. 47. Turk, J., Gross, R.W., & Ramanadham, S. (1993). Amplification of insulin secretion by lipid messengers. Diabetes 42, 367-374. 48. Kakei, M., Kelley, R.P, Ashcroft, S.J.H., & Ashcroft, P.M. (1986). The ATP-sensitivity of K^ channels in rat pancreatic B-cells is modulated by ADP. FEBS Lett. 208, 63-66. 49. Misler, S., Falke, L.C., GiUis, K., & McDaniel, M.L. (1986). A metabolite regulated potassium channel in rat pancreatic B cells. Proc. Natl. Acad. Sci. USA 83, 7119-7123. 50. Ghosh, A., Ronner, P, Cheong, E., Khalid, P, & Matschinksy, F.M. (1991). The role of ATP and free ADP in metabolic coupling during fuel-stimulated insulin release from islet beta cells in the isolated perfused rat pancreas. J. Biol. Chem. 266, 22887-22892. 51. Gembal, M., Gilon, P., & Henquin, J.C. (1992). Evidence that glucose can control insulin release independently from its action on ATP-sensitive K channels in mouse B cells. J. Clin. Invest. 89, 1288-1295. 52. Sato, Y, Aizawa, T., Komatsu, M., Okada, N., & Yamada T. (1992). Dual functional role of membrane depolarization/Ca "^ influx in rat pancreatic B cells. Diabetes 41, 438-443.
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53. Metz, S.A., Draznin, B., Sussman, K.E., & Leitner, J.W. (1987). Unmasking of arachidonate-induced insulin release by removal of extracellular Ca ^. Arachidonic acid mobilizes cellular Ca ^ in rat islets of Langerhans. Biochem. Biophys. Res. Commun. 142, 251-258. 54. Metz, S.A. (1988). Exogenous arachidonic acid promotes insulin release from intact or permeabilized rat islets by dual mechanisms. Putative activation of Ca ^ mobilization and protein kinase C. Diabetes 37, 1455-1469. 55. Band, A.M., Jones, P.M., & Howell, S.L. (1992). Arachidonic acid-induced insulin secretion from rat islets of Langerhans. J. Molec. Endocinol. 8, 95-101. 56. Laychock, S.G. (1982). Phospholipase A2 activity in pancreatic islets is Ca -dependent and stimulated by glucose. Cell Calcium 3, 43—54. 57. Mathias, P.C.F., Best, L., & Malaisse, W.J. (1985). Stimulation by glucose and carbamylcholine of phospholipase A2 in pancreatic islets. Diabetes Res 2, 267-270. 58. Metz, S., Holmes, D., Robertson, R.P., Leitner, W., & Draznin, B. (1991). Gene expression of type I phospholipase A2 in pancreatic beta cells. FEBS Lett. 295, 110-112. 59. Metz, S.A. (1991). The pancreatic islet as Rubik's cube. Is phospholipid hydrolysis a piece of the puzzle? Diabetes 40, 1565-1573. 60. Konrad, R.J., Jolly, Y.C., Major, C , & Wolf, B.A. (1992). Inhibition of phospholipase A2 and insulin secretion in pancreatic islets. Biochim. Biophys. Acta 1135, 215-220. 61. Konrad, R.J., Johhy, C , Major, C , & Wolf, B.A. (1992). Carbachol stimulation of phospholipase A2 and insulin secretion in pancreatic islets. Biochem. J. 287, 283-290. 62. Metz, S.A. (1986). Ether-linked lysophospholipids initiate insulin secretion. Lysophospholipids may mediate effects of phospholipase A2 activation on hormone release. Diabetes 35, 808—817. 63. Metz, S.A. & Dunlop, M. (1990). Sodium fluoride unmasks the accumulation of lysophosphatidylcholine in intact pancreatic islet cells. Biochem. Biophys. Res. Commun. 167, 61-66. 64. Robertson, R.P. (1988). Eicosanoids as pluripotential modulators of pancreatic islet function. Diabetes 37, 367-370. 65. Metz, S., VanRollins, M., Strife, R., Fujimoto, W, & Robertson, R.R (1983). Lipoxygenase pathway in islet endocrine cells. Oxidative metabolism of arachidonic acid promotes insulin release. J. Clin. Invest. 71,1191-1205. 66. Turk, J., Colca, J., Kotagal, N., & McDaniel, M. (1984). Arachdionic acid metabolism in isolated pancreatic islets II. The influence of glucose and of inhibitors of arachidonate metabolism on insulin secretion and metabolite synthesis. Biochim. Biophys. Acta 794, 125—136. 67. Turk, J., Wolf, B.A., Easom, R.A., Hughes, J., & McDaniel, M. (1989). Arachidonic acid metabolism in isolated pancreatic islets V. The enantiomeric composition of 12-hydroxy(5,8,10,14)-eicosatetraenoic acid indicates synthesis by a 12-lipoxygeanse rather than a monooxygenase. Biochim. Biophys. Acta 1001, 16-24. 68. Turk, J., Hughes, J.H., Easom, R.A., Wolf, B.A., Scharp, D.W., Lacy, RE., & McDaniel, M.L. (1988). Arachidonic acid metabolism and insulin secretion by isolated human pancreatic islets. Diabetes 37, 992-996. 69. Metz, S.A. (1985). Glucose increases the synthesis of lipoxygenase-mediated metabolites of arachidonic acid in intact rat islets. Proc. Natl. Acad. Sci. 82, 198-202. 70. Shannon, V.R., Ramanadham, S., Turk, J., & Holtzman, M.J. (1992). Selective expression of an arachidonate 12-lipoxygenase by pancreatic islet beta cells. Am. J. Physiol. 263, E908-E912. 71. Takahashi, Y., Ueda, N., & Yamamoto, S. (1988). Two immunologically and catalytically distinct arachidonate 12-lipoxygenases of bovine platelets and leukocytes. Arch. Biochem. Biophys. 266, 613-621. 72. Turk, J., Wolf, B.A., Lefkowith, J.B., Stump, W T , & McDaniel, M.L. (1986). Glucose-induced phospholipid hydrolysis in isolated pancreatic islets: Quantitative effects on the phospholipid content of arachidonate and other fatty acids. Biochim. Biophys. Acta 879, 399-409.
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73. Pace-Asciak, Martin, J.M., Corey, E.J., & Su, W.-G. (1985). Endogenous release of hepoxilin A3 from isolated perifused pancreatic islets of Langerhans. Biochem. Biophys. Res. Commun. 128, 942-946. 74. Laychock, S.G. (1985). Effects of hydroxyeicosatetraenoic acids on fatty acid esterification in phospholipids and insulin secretion in pancreatic islets. Endocrinology 117, 1011-1014. 75. Buttner, N., Siegelbaum, S.A., & Volterra, A. (1989). Direct modulation of Aplysa S-K"*" channels by a 12-lipoxygenase metabolite of arachidonic acid. Nature 342, 553-555. 76. Piomelli, D. (1991). Metabolism of arachidonic acid in nervous system of marine mollusk Aplysia califomica. Am. J. Physiol. 260, R844^R848.
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THE ROLE OF PAF IN REPRODUCTIVE BIOLOGY
Hisashi Narahara, Rene A. Frenkel, and John M. Johnston
ABSTRACT I. INTRODUCTION II. THE ROLE OF PAF IN THE EARLY STAGES OF PREGNANCY A. The Role of PAF in Sperm Function and Metabolism B. The Role ofPAF in Ovulation C. The Role of PAF in the Endometrium and Implantation III. THE ROLE OF PAF IN FETAL LUNG MATURATION AND PARTURITION A. ThePresenceof PAF in Amniotic Fluid B. PAF Metabolism and its Role in Fetal Lung Maturation C. PAF Receptors in Type II Pneumonocytes D. Effectsof PAF on Lung Glycogen Metabolism E. Effect of PAF on the Secretion of Surfactant by Type II Pneumonocytes F. PAF and Surfactant Replacement Preparations
Advances in Lipobiology Volume 1, pages 241-27L Copyright © 1996 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-635-5 241
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G. PAF Metabolism in Fetal Membranes 250 H. Relationship Between Fetal Lung Maturation and the Stimulationof Fetal Membranes 252 I. PAF Metabolism in the Maternal Compartment 255 J. Plasma PAF-AH Activity During Pregnancy 255 K. The Role of the Macrophage in PAF Metabolism 256 L. Hormonal Regulation of PAF-AH Activity 257 M. Presence of PAF Receptors in Human Myometrium and the Effect of PAF and a Receptor Antagonist on Myometrial Contraction and Parturition 259 N. Effectof a PAF Receptor Antagonist on the Time of Delivery in Rats . . .260 O. Role of Estrogens in the Timing of Parturition 260 IV. PAF AND PERINATAL COMPLICATIONS 261 A. PAF and Intrauterine Infections 261 B. PAF and Cigarette Smoking 262 C. Pregnancy Induced Hypertension/Preeclampsia 263 D. PAF and Necrotizing Enterocolitis 263 ACKNOWLEDGMENTS 264 REFERENCES 265
ABSTRACT Platelet-activating factor (PAF) has been shown to play an important role in both fetal lung maturation and in parturition and it has been demonstrated that part of the PAF present in the amniotic fluid is associated with surfactant and, therefore, of lung origin. PAF receptors are present in type II pneumonocytes and the addition of the autacoid promotes both glycogenolysis and increased surfactant secretion from these cells. Moreover, type II pneumonocytes and a human amnion-derived cell line (WISH) have the capability of metabolizing PAF to ethanolamine phospholipids. It has also been established that PAF will cause intracellular Ca^"^ mobilization and promote myosin phosphorylation and increased contractility in myometrial strips. Increased PAF biosynthesis in fetal tissues takes place during the latter stages of pregnancy, simultaneously with a decrease in the activity of PAF-acetylhydrolase (PAF-AH) in maternal plasma. The activity of PAF-AH is decreased by the administration of estrogens in vivo and is increased by glucocorticoids and progestins. A number of agents lower the secretion of PAF-AH by decidual macrophages, including 1,25(OH)2D3, bacterial lipopolysaccharides, and some cytokines (ILl, TNF-a). This inhibitory action may result in higher levels of PAF and increased myometrial contractility as a consequence of infectious processes. PAF causes a profound intestinal necrosis when administered to rats by intravenous injection. Pretreatment with dexamethasone protects against this disease. The presence of PAF-AH in human milk may explain the protective effect of mother's milk against necrotizing enterocohtis.
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I. INTRODUCTION Platelet-activating factor (l-(9-alkyl-2-acetyl-5«-glycero-3-phosphocholine, PAF) has been shown to be involved in a number of diseases including asthma, endotoxic shock, inflammation, diabetes, acute allergic reactions, thrombosis, and ischemic bowel necrosis (Snyder, 1990; Hanahan, 1986). Furthermore, it is clear that PAF may be one of the most potent lipid mediators thus far described, having both pathological and physiological effects at concentrations as low as 10~^^ to 10"^^ M. The role of PAF in a number of these pathological conditions has been previously reviewed (Prescott et al., 1990). In terms of the physiological actions of PAF, one of the major areas in which a role for PAF has been established is that of reproductive biology. PAF has been implicated throughout reproduction, ranging from its secretion by the periovulatory follicles (Alexander et al., 1990) and its role in the enhancement of the acrosome reaction of sperm (Ricker et al., 1989; Kuzan et al., 1990) to the role of PAF in human fetal lung maturation and the relation of this phenomenon to the initiation and maintenance of parturition (Johnston et al., 1992). The latter topic will be emphasized in this review.
II. THE ROLE OF PAF IN THE EARLY STAGES OF PREGNANCY A. The Role of PAF in Sperm Function and Metaboh'sm
The first report on the presence of PAF in rabbit spermatozoa was published in 1988 (Kumar et al, 1988). Subsequently, numerous studies have been published on the role of PAF in male reproduction. Several groups of investigators, in particular Minhas and colleagues, have suggested that PAF facilitates sperm motility (Ricker et al., 1989). PAF is present in human spermatozoa, but not found in seminal fluid. An explanation for this finding was recently provided by the fact that PAF-acetylhydrolase (PAF-AH), the enzyme that inactivates PAF, is present in human seminal plasma (Letendre et al., 1992). PAF addition to human semen samples has also been reported to enhance the sperm penetration into the zone-free hamster oocyte (Minhas, 1993). B. The Role of PAF in Ovulation
The involvement of PAF in ovulation was recently suggested (Abisogun et al., 1989). It was reported that the local administration of a PAF receptor antagonist inhibited follicle rupture in rats which were previously stimulated with human chorionic gonadotrophin. The inhibition was reversed by the simultaneous administration of PAF. Further support for a role for PAF in ovulation was provided by Li et al. (1991) who also demonstrated that PAF was involved in the rupture of follicles during gonadotropin-induced ovulation and that PAF action was independent from the known effect of eicosanoids.
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HISASHI NARAHARA, RENE A. FRENKEL, and JOHN M. JOHNSTON C. The Role of PAF in the Endometrium and Implantation
PAF has been detected in uterine tissue of rats (Yasuda et al, 1986), rabbits (Angle et al, 1988a), and humans (Alecozay et al, 1989). Support of the contention that it is indeed the endometrium that is involved in PAF metabolism is the observation that the PAF concentration in rabbit endometrium was significantly higher than that found in the myometrium of the same species. It has not been clearly established whether the increase in endometrial PAF is due to an increased synthesis or a decreased breakdown in this tissue. It has been suggested that the capacity to synthesize PAF is relatively high in human endometrial tissue as judged by lyso-PAF-acetyltransferase activity (Nonogaki et al., 1989). The presence of 1-alkyl-2-acetyl-5'«-glycerol:cytidine diphosphate-choline choline phosphotransferase (a reflection of the de novo pathway for PAF biosynthesis) has also been demonstrated in the microsomal fraction obtained from endometrial tissue. The presence of PAF receptors in the rat uterus, as well as the presence of the PAF receptor in a purified endometrial membrane preparation, have recently been demonstrated (Kudolo and Harper, 1990). PAF has also been reported to induce a dose-dependent decidua like reaction in the pseudopregnant rat (Acker et al., 1989). One of the aspects of PAF in the area of reproduction that has received major attention is that relating the autacoid to implantation. O'Neill and colleagues (for review see O'Neill, 1989) were the first to demonstrate that PAF was secreted by the preimplantation embryo and that the secretion was related to the viability of the embryos. We observed that the PAF concentration in the culture media was related to the age of the embryo and to its developmental stage (Punjabi et al., 1990). In addition, PAF production was higher in the embryo culture media obtained from clinical pregnancies compared to preclinical pregnancies. Similar findings have also been reported by Nakatsuka et al. (1992). Several years ago, Ryan et al. (1990) reported that the supplementation of culture media with PAF in the in vitro fertilization procedure in the mouse increased the rate of embryo implantation without altering congenital abnormalities. More recently, O'Neill et al. (1992) reported that the pregnancy rate was significantly increased by the addition of PAF to human IVF culture media. This group also suggested that PAF was responsible for the mild maternal thrombocytopenia that occurs in response to fertilization. PAF has also been suggested as the agent which induces the release of "early pregnancy factor" from spleen cells (Clarke et al., 1990). Based on these findings, it has been concluded that PAF produced by the developing embryo has a functional role in the phenomenon of implantation. Whether or not the small amounts of PAF produced by the embryo are directly responsible for the maternal thrombocytopenia is not so clear. As an alternative, it has been suggested that the embryonic production of PAF results in the activation of the uterine endometrium to produce PAF, thereby further amplifying the signal of embryo PAF production (Angle and Johnston, 1990).
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III. THE ROLE OF PAF IN FETAL LUNG MATURATION AND PARTURITION The possible relationship between maturation of the fetus and the initiation of parturition was suggested many years ago in several species by Hippocrates, William Harvey, and Spiegelberg. As stated by William Harvey in the 1600s: "the assistance of the foetus is chiefly required in the birth, is evident not in birds only, which due to their own industry without help of the parents breakup the shell, but also in other animals" (cited by Bleasdale and Di Renzo, 1989). It is cl^ar, however, that this symbiotic relationship may also exist for PAF at an early stage of pregnancy, namely that of implantation, as previously discussed. A. The Presence of PAF in Amniotic Fluid
The communication from the fetus to the maternal compartment is most likely mediated via the amniotic fluid and amnion tissue. This tissue is morphologically attractive for the transduction of the signal(s) to the chorion laeve and decidua and ultimately to the myometrium and cervix, because (a) it is metabolically active with respect to O2 consumption and contains an abundance of lipid droplets (the importance of lipids in parturition will be discussed); (b) it contains a large surface area due to the abundance of microvilli, numerous intracellular junctions, and cytoplasmic filaments to accept a signal; and (c) amnion tissue has a high activity of various enzymes involved in PAF and eicosanoid biosynthesis, some of which are increased at term (Johnston et al., 1992). It has been demonstrated that the increased arachidonic acid that was present in the amniotic fluid during labor was derived primarily from two glycerophospholipids of fetal membranes, namely diacylphosphatidylethanolamine and phosphatidylinositol (Okita et al., 1982). A model system based on the enzyme activities and their substrate specificity was proposed to explain the selective release of arachidonic acid from these two glycerophospholipids. A central role for Ca^"^ in the regulation of the release of this polyunsaturated fatty acid was also established (Bleasdale and Johnston, 1984). Similar findings have been reported by Olsen et al. (1983). It was proposed that PAF could have a role in parturition, since this autacoid is generally associated with an increase in intracellular Ca^"^ (Snyder, 1985). PAF was identified and characterized in the amniotic fluid obtained from women in active labor (Billah and Johnston, 1983), but only a trace amount of PAF was detected in the amniotic fluid obtained from women at term and not in labor. These observations were confirmed by Nishihira and colleagues (1984). PAF was also shown to cause a stimulation of prostaglandin E2 (PGE2) formation in amniotic tissue (Billah et al., 1985; Morris et al., 1992) and in anmion cells in culture fBleasdale, 1989).
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HISASHI NARAHARA, RENE A. FRENKEL, and JOHN M. JOHNSTON B. PAF Metabolism and its Role in Fetal Lung Maturation
Although amniotic tissue contains the enzymes and substrates that are required for the synthesis of PAF, we were unable to demonstrate a significant release of PAF into the incubation medium (Billah and Johnston, 1983). Similar findings have been reported in endothelial cells (Elstad et al., 1989b). Amniotic fluid, at term, is enriched with a surface-active agent, a lipoprotein complex that is synthesized by the type II pneumonocytes of the fetus and transported by fetal breathing to the amniotic fluid in the form of lamellar bodies. One of the precursors of PAF, namely alkyl-acyl-glycero-phosphocholine (GPC), has been shown to be present in surfactant (Kumar et al., 1985). When the distribution of PAF between the lamellar bodies and the supernatant fractions was determined in amniotic fluid obtained from women in labor at term, approximately 44% of the PAF was found to be associated with the lamellar body-enriched fraction (Billah and Johnston, 1983). Approximately 50% of lysoPAF and over 90% of the alkyl-acyl-GPC was associated with the lamellar body fraction found in amniotic fluid and the concentration of alkylacyl-GPC was of the order of 10"* times higher than that of PAF. A similar ratio was found in amnion tissue (Ban et al., 1986). The possibility that PAF found in amniotic fluid originated, in part, from the lung was an attractive working hypothesis, not only from the standpoint that the fetal lung may participate in providing one of the signals involved in parturition, but also because PAF may have a direct function in fetal lung maturation. It has been suggested that fetal lung glycogen can serve as a precursor of the surfactant glycerophospholipids (Maniscalco et al., 1978; Bourbon et al., 1982) and a role for PAF in hepatic glycogenolysis has been proposed by Hanahan (1986). The concentration of PAF and its lipid precursors was determined in lung and liver tissue of fetal rabbits throughout gestation (Hoffman et al., 1986a). It was found that the concentration of PAF in fetal rabbit lung increased some threefold between day 21 and day 31 of gestation, whereas that in liver did not change. A similar increase in PAF concentration was found in human fetal lung tissue in organ culture (Hoffman etal., 1986b). The activities of some of the enzymes involved in PAF biosynthesis were also assayed in lung tissue throughout gestation. A Ca^'^-independent phospholipase A2 (PLA2) present in fetal rabbit lung (Angle et al., 1988b). The activity of lysoPAF:acetyl-coenzyme A (CoA) acetyltransferase increased threefold between the 21st and 24th day of gestation and remained elevated until birth in fetal rabbit lung (Hoffman et al, 1986a) and human fetal lung explants placed in organ culture (Hoffman et al., 1986b). The activity of the specific choline phosphotransferase involved in the de novo pathway for PAF biosynthesis also increased (Hoffman et al, 1988a). It was concluded that fetal lung had the capacity for the synthesis of PAF and that the elevated enzymatic activity could account for the increase in PAF in the lung which occurs during late gestation.
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C. PAF Receptors in Type II Pneumonocytes
The presence of the PAF receptor in a number of cells and its characterization have been due primarily to the investigations of Hwang and Wang (1990). The PAF receptor was recently cloned by Honda et al. (1991) from a guinea pig lung DNA library and expressed in Xenopus oocytes. Subsequently, PAF receptor DNAs have been cloned from an HL-60 granulocyte cDNA library (Ye et al., 1991), from a U-937 myeloid cell expression library (Kunz et al, 1992), and human leukocytes (Nakamura et al, 1991). We have determined the presence of PAF receptors employing purified type II pneumonocytes (Eguchi et al, 1993). A single class of binding sites was found, with a dissociation constant of 0.46 nM and it was computed that approximately 3,000 specific PAF binding sites were present per type II pneumonocyte. The specific binding was blocked by prior incubation of the type II cells with the potent PAF receptor antagonist WEB-2086. D.
Effects of PAF on Lung Glycogen Metabolism
During fetal development the type II pneumonocytes undergo a truly remarkable alteration: from cells very rich in stored carbohydrates in the form of glycogen (a characteristic noted by Claude Bernard in 1859), the pneumonocytes undergo a marked metabolic change and synthesize the lipids required for the production and secretion of lung surfactant. This striking metabolic transformation takes place late in gestation, when the lung must be prepared for secreting surfactant into the alveoli in order to prevent alveolar collapse once the neonate initiates breathing. In view of the known glycogenolytic effect of PAF in liver (Buxton et al., 1984), it was considered of crucial importance to establish whether PAF would promote glycogen breakdown in fetal lung. Since glucose-6-phosphatase activity is not present in lung to any significant degree, the expected products of accelerated glycogenolysis and glycolysis are pyruvate and lactate. It is, however, necessary to employ fetal tissue to test this effect, since adult lung has a very low glycogen content. The role of PAF in glycogen mobilization in fetal lung tissue was directly assessed (Hoffman et al., 1988b). PAF was injected into 24-day fetal rabbits in utero, and it was found that the fetal pulmonary and hepatic glycogen concentrations were significantly reduced and the lactate concentrations in fetal lung, liver, and plasma were increased. When either SRI-63-441 (PAF receptor antagonist) or the PAF antagonist plus PAF were injected, no effect on glycogen breakdown or lactate formation was found compared with the controls. Similarly, the injection of the enantiomer of the natural occurring PAF, sn-l PAF, did not alter the glycogen or lactate content of fetal lung, liver, or plasma. Additional information about the effects of PAF in fetal glycogen metabolism was obtained by direct infusion of PAF
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HISASHI NARAHARA, RENE A. FRENKEL, and JOHN M. JOHNSTON
into tlie rat vitellin vein (Bourbon et al., 1991). In the PAF-treated group, glycogen content was reduced up to 19%, as compared with controls. E. Effect of PAF on the Secretion of Surfactant by Type II Pneumonocytes
PAF also stimulates surfactant release by type II pneumoncytes (Johnston et al., 1987; Kumar and Hanahan, 1987). In our study, type II pneumonocytes were labeled with [^H]palmitic acid and the influence of PAF on surfactant secretion was examined. PAF caused a dose-dependent and significant (p < 0.01) increase of surfactant secretion. When PAF at a concentration of 5 x 10"^ M was added, disaturated phosphatidylcholine secretion was stimulated some 3.5- to 5-fold (Eguchi et al., 1993). When methylcarbamyl-PAF (a stable derivative of PAF) was used as a PAF-receptor agonist, a similar effect was observed, but a 10-fold higher concentration was required to obtain a stimulation similar to that caused by PAF. The PAF-stimulated surfactant secretion was inhibited by the PAF-receptor antagonist WEB-2086. On the basis of these findings, it has been postulated that PAF can funcfion as a potent stimulator of surfactant secretion. /. Synthesis of Ether Clycerophosoholipids Pneumonocytes
From Hexadecanol by Type II
Principally due to the woric of Snyder and colleagues (Snyder, 1989) as well as Hajra (1983), the metabolic sequence that leads to biosynthesis of ether lipids has been well established. In view of the fact that the enzymes required for the de novo pathway of PAF biosynthesis are present in lung (Johnston & Maki, 1989), and considering the potential impact of PAF in the metabolism of this organ, studies were conducted with isolated rat type II pneumonocytes. The interest in establishing the presence of this biosynthetic pathway in lung acquired particular significance since a long-chain fatty alcohol, hexadecanol, is one of the constituents added as a spreading agent to a synthetic surfactant-replacement preparation (Long and Sanders, 1988). These preparations are presently in use for the treatment of respiratory distress syndrome (RDS) in the newborn (see below). Isolated type II pneumonocytes (Frenkel, Narahara, Eguchi and Johnston, 1993) were incubated for various times with l-[-^H]hexadecanol. The results of one such experiment are depicted in Figure 1. As can be seen, there was a rapid transfer of label from hexadecanol to species that correspond to ethers of phosphafidylcholine, phosphatidylethanolamine, and to some form of neutral lipid. Detailed analyses of the individual peaks further established these conclusions. In summary, in the studies employing purified adult rat type II cells, we have demonstrated: (a) the presence of a PAF receptor, (b) that PAF will simulate surfactant release, and (c) that these cells contain the necessary activities to convert long-chain alcohols into ether lipids. In addition, we have shown that PAF is converted to alkyl-acyl-GPC and alkyl-acyl-GPE (Eguchi et al., 1993).
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Figure 1. Radioactivity scan of lipid extract from type 11 pneumonocytes incubated with [1-^H]hexadecanol, for one hour. Total lipids extracted from the cells were separated by thin layer chromatography on silica gel and scanned for radioactivity.
F. PAF and Surfactant Replacement Preparations
During the last few years, great strides have been made in the treatment of premature infants suffering from RDS by the use of surfactant-replacement preparations. It has become increasingly evident that this type of therapy is extremely beneficial for those infants bom with inadequate amounts of pulmonary surfactant. Several preparations obtained from natural sources have become available for clinical use (Avery and Merritt, 1991). In addition, a chemically defined, totally synthetic preparation consisting of pure dipalmitoylphosphatidylcholine has been in use for some time in which hexadecanol was added as a spreading agent (Long and Sanders, 1988). In view of the occurrence of PAF in the lamellar bodies obtained from amniotic fluid, it was considered likely that the surfactant preparations obtained from natural sources could contain detectable amounts of PAF which could have some effects when administered to the newborn. For these reasons, the PAF content of several surfactant-replacement preparations was determined (Moya et al., 1993). The results of the analyses are shown in Table 1. As can be observed.
250
HISASHI NARAHARA, RENE A. FRENKEL, and JOHN M. JOHNSTON Table 1. PAP Content of Surfactant Preparations Type
Exosurf Survanta Infasurf Curosurf
PAF Content (pmol PAF/ml)
0 36 ± 7 218 ± 1 8 * ^ 107+14*''
Notes: Values are mearI ± SD of three separate determinations. *P < 0.0001 vs. Exosurf;"' p < 0.001 vs. Survanta; * p < 0.01 vs. Curosurf.
all the preparations from natural sources analyzed, whether from whole lung tissue or from lavage, contained variable amounts of PAF. As expected, the totally synthetic preparation was devoid of PAF. It should be noted, however, that the synthetic surfactant suspension contains 0.15% hexadecanol; this represents 50 l^imoles in the usual dosage of 8 ml administered to infants affected by RDS. In view of the capacity of type II cells to synthesize ethers, as discussed in an earlier section, the addition of a long-chain alcohol might result in the production of increased amounts of phospholipid ethers, the most potent being PAF. The role of PAF in fetal lung maturation can be summarized as follows: It is suggested that the last major organ system to develop in the fetus, namely the lung, produces and secretes surfactant with its associated PAF into the amniotic fluid. The amniotic fluid PAF interacts with amniotic membranes to stimulate the production and release of PGE2. PAF may act autocatalytically in amnion to cause an increase in PAF synthesis by increasing the intracellular Ca^"^, which in turn activates lysoPAF:acetylCo-Aacetyltransferase (see below). A similar autocatalytic synthesis of PAF has been shown to occur in neutrophils (Doebber and Wu, 1987). G. PAF Metabolism in Fetal Membranes
We have demonstrated that 1-0-alkyl- and l-O-alkenyl-2-acyl-GPE (ethanolamine plasmalogen) increase in amnion from early pregnancy (13 to 17 weeks) to term (Johnston and Maki, 1989). The relative amount of ethanolamine plasmalogens was similar to the diacyl-GPE content in those amnions obtained early in pregnancy but was two times higher than the diacyl species at term. As mentioned earlier, PAF associated with lamellar bodies that originate in fetal lung may stimulate the synthesis of PAF by amnion. The enzymes and substrates required for the synthesis of PAF have been demonstrated in this tissue (Ban et al., 1986). If PAF is converted to ethanolamine plasmalogens, these compounds could be a reflection of increased concentrations of PAF in amnion. To test this postulate, WISH cells (a tumor cell line derived from human amnion) were incubated with PAF labeled with ^H in the alkyl side chain (Frenkel and Johnston, 1992). Analysis
PAF in Reproductive
Biology
251 IV
AfkyI Lyso GPC -^
II Alkyl Acyl GPC
► Alkyl Acyl GP
CMP IX CDP-ChoHno CDF^elhanolomine Alkyl Lyso GP
Alkyl G Alkenyl Acyl GPE (ethanolamlne pla3mak>g€n) Vill
1-Lyso 2-Acyl GPE Figure 2, Possible pathways for the conversion of alkyl-acyl-GPC Into ethanolamlne plasmalogen. The following reactions are indicated: Rxl: phosphollpase C; Rxll: phospholipase D; Rxlll: phosphatidate phosphohydrolase; RxlV: phosphollpase A2; RxV: CDP-choline:dlradylglycerol chollnephosphotransferase; RxVI: CDP-ethanolamlne:dlradyl-glycerol ethanolamlnephosphotransferase; RxVII: alkyl-acyl-GPE desaturase (plasmalogen synthase); RxVIII: plasmalogenase; RxlX: lysophospholipase D; RxX:alkyl-lysoGP phosphohydrolase (postulated); RxXI: alkyl-glycerohacyl-CoAacyltransferase.
of the different glycerophospholipid fractions isolated from the cells have resulted in establishing that PAF is converted into ethanolamine phospholipids, including a high proportion of ethanolamine plasmalogens. Similar conversions of PAF into ethanolamine glycerophospholipids have been reported by Wykle and colleagues (Strum et al., 1992). The exact sequence of events that takes place during this metabolic transformation has not been elucidated, but a number of possible pathways are depicted (Figure 2). The conversion of the alkyl group of PAF into the alkenyl group of the ethanolamine plasmalogens provides a mechanism by which the stable 1-alkyl bond may be metabolized. The resulting alkenyl bond of the phosphatidylethanolamine is rapidly metabolized in most tissues by the enzyme
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HISASHI NARAHARA, RENE A. FRENKEL, and JOHN M. JOHNSTON
plasmalogenase (Warner and Lands, 1961) with the release of a long-chain aldehyde. The only other pathway for the cleavage of the ether bond is by a pteridinedependent enzyme (Tietz et al., 1964) and the activity of this enzyme is thought to be quite low in most tissues. A role for alkenyl-lyso-GPE in the transacylation with alkyl-acyl-GPC has been postulated. Since the transacylase is rather specific for arachidonate, this mechanism provides an explanation for the increased enrichment with arachidonate of the ethanolamine plasmalogen fraction that we observed in human amnion tissue late in gestation (Okita et al., 1982). These interrelationships will be further discussed in a subsequent section. H.
Relationship Between Fetal Lung Maturation and the Stimulation of Fetal Membranes
We have previously suggested a role for PAF in stimulating glycogen breakdown and surfactant biosynthesis and secretion during fetal lung maturation (Johnston et al., 1992). The finding that a significant amount of the PAF in amniofic fluid is associated with the surfactant was the basis for the suggestion that fetal lung maturation and the initiation of parturition may have certain interactions. A second communication between fetal lung maturation and the initiation of parturition is suggested by the relationships presented in Figure 3. In addition to PAF, a significant amount of alkyl-acyl-GPC is also present in lamellar bodies (Billah and Johnston, 1983). Previously, Kumar et al. (1985) demonstrated the presence of alkyl-acyl-GPC in the surfactant prepared from dog lung. When PAF metabolism was examined in lung type II pneumonocytes, we could not detect the synthesis of either the plasmalogen of phosphatidylcholine or -ethanolamine, although alkyl-acyl-GPC and -GPE were produced. When a similar incubation was carried out with [^H]alkyl-PAF and amnion-derived WISH cells, a significant amount of radioactivity was incorporated into the alkenyl (plasmalogen) species of ethanolamine (Frenkel and Johnston, 1992), as discussed. It would thus appear that type II pneumonocytes have a limited capacity for plasmalogen synthesis (Eguchi et al., 1993). Thisfindingwould explain the presence of alkyl-acyl-GPC in the surfactant (Billah and Johnston, 1983). Rustow et al. (1992) have observed a high ether content in the phospholipids isolated from type II cells. The alkyl-acyl-GPC secreted along with surfactant into the amniotic fluid may serve as a precursor of PAF by its conversion to lysoPAF through the action of a PLA2. The resulting lyso derivative can be incorporated into PAF after acetylation by the enzyme lysoPAF:acetyl-CoA acetyltransferase. Both lysoPAF and alkylacyl-GPC are known to be present in amnion tissue. In addition, the PAF can be converted to alkenyl-acyl-GPE in amnion derived WISH cells as has been discussed. Previously, we had demonstrated the presence of a PLA2 in amnion tissues that preferentially cleaved diacyl-GPEs containing an arachidonoyl species in the sn-l position (Okazaki et al., 1981b). The activity of this enzyme increased in amnion tissue during the last third of pregnancy (Okazaki et al., 1981b). We
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Table 2. Substrate Specific:ity of the PLA2 Present in Amn ion Microsomes nmoles >: mirf X mg~ protein Substrates l-Acyl-2-[''^]C-arachidonyl-GPE l-Alkenyl-2-[-^^P]-arachidonoyl-GPE Note:
Ca^^ (+)
Ca'U-)
25.7 31.4
9.3 12.2
Assay mixture contained Tris HCl (30 mM, pH 8.5), 0.02% BSA, 50 fig amnion microsomal protein and substrate (10 nmol) in a total volume of 0.5 ml. When Ca^"^ was added it was 2 mM.
reported that the arachidonate utilized for eicosanoid formation in amnion tissue during early labor was derived primarily from diacyl-GPE and phosphatidylinositol (Okita et al., 1982). Although almost 50% of the arachidonate in amnion tissue was present in the alkenyl-acyl-GPE, no decrease in the arachidonate content of the alkenyl-acyl-GPE species was found during early labor. The substrate specificity of the PLA2 activity was, therefore, examined to see if only the diacyl-GPE was a substrate for this enzyme. Diacyl-GPE containing [^"^CJarachidonate in the sn-2 position, and l-alkenyl-2-acyl-GPE were compared as substrates employing the microsomal fraction prepared from amnion tissue (Ban and Johnston, unpublished observations). The results are shown in Table 2. The validity of the assay system was checked employing the PLA2 from Crotalus adamanteus which preferentially cleaves the diacyl-GPE (Waku and Nakazawa, 1972). As can be seen, [^"^CJarachidonate was released from both substrates were cleaved to a similar degree. It was, therefore, concluded that the specificity of the PLA2 in amnion microsomes could not account for the fact that the arachidonate was not lost from the plasmalogen species during early labor. Dennis and colleagues (Jarvis et al., 1984) have demonstrated the presence of an active lysophospholipase in this tissue and we have confirmed these findings (Ban and Johnston, unpublished observations). The formation of a 1 -alkenyl-2-lyso-GPE which is not cleaved to the extent of the acyl-lyso-GPE may have a marked stimulatory effect on the synthesis of PAF by this tissue. As has been previously discussed, l-alkenyl-2-lyso-GPE will increase lysoPAF formation by acting as an arachidonoyl acceptor in the transacylase reaction, the donor being alkyl-arachidonoyl-GPC (Uemura et al., 1991; Strum et al., 1992). We have recently demonstrated the presence of the transacylase activity in amnion derived WISH cells (Toyoshima, Frenkel, and Johnston, 1994). The resulting lysoPAF could rapidly be converted to PAF by lysoPAF:acetyl-Co-Aacetyltransferase. The latter enzyme has been well characterized in amnion tissue (Ban et al., 1986). This series of reactions could account for the enrichment of arachidonate in the l-alkenyl-2-acyl-GPE fraction. The PLA2 present in this tissue could regenerate the 1-alkenyl-lyso-GPE with the liberation of arachidonic acid which could be utilized for eicosanoid
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HISASHI NARAHARA, RENE A. FRENKEL, and JOHN M. JOHNSTON
synthesis in this tissue (Okazaki et al, 1981b). The proposed mechanism for stimulation of PAF formation by 1-alkenyl-lyso-GPE in amnion tissue and its relation to fetal lung is illustrated in Figure 3. Thus, in addition to a direct relationship between fetal lung maturation and parturition involving PAF, a second mechanism of action of PAF is proposed in which the alkyl-acyl-GPC secreted in association with the surfactant from the fetal lung can also serve as a precursor of PAF by two routes: (a) by the action of a PLA2 with the release of free arachidonic acid and lysoPAF and (b) by participating in the transacylation reaction with alkenyl-lyso-GPE as an acceptor, also with the formation of lysoPAF. Several years ago Lopez Bemal et al. (1989) reported that the addition of surfactant to cultured amnion cells stimulated prostaglandin E2 formation by these cells. They reported that the stimulation was not due to the
AMNIOTIC FLUID
AMNION CELL
Figure 3. Relationships between PAF, lyso-PAF, and alkyl-arachidonoyl-GPC (AAGPC) in the amniotic fluid and the amnion cells. PAR lyso-PAF, and AA-GPC are found In the amniotic fluid, partially in association with the surfactant-containing lamellar bodies. Cleavage of PE-plasmalogen (PE-P'gen) by a PLA2 will result in the release of arachidonic acid (AA) which can be converted to prostaglandin E2 by these cells. The other product of AA-GPC cleavage is lysoPAF. The latter can also be formed via a transacylase reaction in which the lyso PE-P'gen produced from PE-P'gen acts as acceptor of an arachidoyi residue from AA-GPC. LysoPAF formed by either mechanism can be converted rapidly to PAF by the lysoPAF:acetyl-CoA acetyltransferase which is present In this tissue (Ban et al., 1986). Thus, two powerful autacoids, PAF and prostaglandin E2, are generated in a closely interrelated manner.
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presence of PAF in the surfactant. An alternate explanation would be that alkylacyl-GPC, known to be present in the surfactant found in amniotic fluid (Billah and Johnston, 1983), serves as a precursor of PAF by the mechanism outlined above. I. PAF Metabolism in the Maternal Compartment
As discussed, the increase in the biosynthesis of PAF was suggested as the mechanism which altered fetal lung maturation and the initiation of parturition. The PAF concentration may also be modified by a change in the activity of the enzyme that inactivates PAF, PAF-AH. There are at least two isozymic forms of PAF-AH (Snyder, 1990): one form is present in the intracellular compartment of most cells and the second is found in the plasma. The plasma PAF-AH activity is increased during a "stress" reaction in the lizard (Lenihan et al., 1985), during bovine platelet aggregation (Suzuki et al., 1988), in patients with ischemic cerebrovascular disease (Satoh et al., 1988), in the rabbit neonate (Maki et al., 1988), in insulin-dependent diabetes mellitus (Hofman et al., 1989), in the spontaneous hypertensive rat (Blank et al., 1979), and in hypertensive human males (Satoh et al., 1989; Snyder, 1990). It is decreased in the plasma of asthmatic children (Miwa, 1988), and in maternal plasma during the second half of pregnancy in the rabbit (Maki et al., 1988), human (Johnston, 1989; Johnston et al., 1990), and rat (Yasuda and Johnston, 1992). The activity of PAF-AH was first described several years ago (Farr et al., 1978) as the acid-labile factor in rabbit serum that is associated with anaphylaxis. Farr et al. (1983) reported that this enzyme was, indeed, the "acid labile factor" present in human plasma and demonstrated that the activity was associated with low density lipoprotein (LDL) fractions. The plasma enzyme has been characterized and partially purified by Stafforini et al. (1987b). J. Plasma PAF-AH Activity During Pregnancy
Inactivation of PAF prior to contact with the myometrium would be of critical importance in order to maintain the quiescent state of the uterus throughout most of pregnancy. This would be advantageous since PAF is one of the most potent stimulators of myometrial contraction (Nishihira et al., 1984). In consideration of these findings, we assayed the specific activity of the plasma PAF-AH in rabbit plasma throughout pregnancy. Prior to insemination, the activity was approximately 130 nmol x min~^ x ml"^ plasma (Maki et al., 1988). The enzyme activity decreased to a minimum value of 10 nmol x min~^ x ml"^ plasma between day 23 and day 29 of gestation and rapidly returned to the nonpregnant levels following delivery. We have considered that the decidua vera may be the site of interaction between fetal PAF and maternal plasma PAF-AH due to its abundant blood supply. The plasma PAF-AH in the decidua may, therefore, inactivate PAF produced in the fetal compartments, thus preventing PAF from reaching the myometrium throughout most of pregnancy. Late in pregnancy, however, the PAF might escape inactivation in the decidua due to the decrease of the maternal plasma PAF-AH activity.
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HISASHI NARAHARA, RENE A. FRENKEL, and JOHN M. JOHNSTON K. The Role of the Macrophage in PAF Metabolism
Macrophages may also be a source of PAF-AH in the decidua. The activity of PAF-AH has been implicated in the regulation of PAF secretion by various macrophage preparations (Prescott et al., 1990). It has been reported that PAF production in macrophages diminished during differentiation due, in part, to the known increased secretion of PAF-AH activity. The PAF-AH secreted by various macrophage preparations has been shown to be the plasma type isozyme and it has also been reported that its secretion increased dramatically (260-fold) during the differentiation of human peripheral monocytes into macrophages (Elstad et al., 1989a). Prior to these observations, it was generally assumed that the tissue origin of the plasma PAF-AH activity was the liver, since this enzyme is present in plasma in association with the lipoprotein fraction (Farr et al., 1983). The observation that various macrophage preparations also secrete PAF-AH of the plasma type prompted us to examine the secretion of this enzyme by the macrophage population known to be present in the decidua vera (Narahara et al., 1993a). PAF-AH secretion was observed in dispersed human decidual cells and the secreted enzyme was the plasma type isozyme. As previously discussed, our interest in decidual tissue in relation to PAF metabolism and its role in parturition was focused on the function of plasma PAF-AH activity in this tissue due to its blood supply. Macrophages comprise approximately 20% of the human decidual cell population as judged by the presence of a specific antigen. It was observed that the amounts of PAF-AH activity secreted by decidual cells into the culture medium correlated positively with the percentage of macrophages in the population. Moreover, pretreatment of the cells with CD 14 monoclonal antibody and complement specifically decreased the PAF-AH secretion by these cells. We have also isolated a relatively pure population of human decidual macrophages by antibody labeling, flow cytometry, and cell sorting. PAF-AH activity secreted by decidual cells could be accounted for by that secreted by the macrophage population. Based on these observations, it was concluded that decidual macrophages were the major cell type that produces and secretes PAF-AH. Furthermore, it is clear that decidual macrophages secrete PAF-AH which has all the characteristics of the plasma type enzyme. The mechanisms involved in the regulation of the PAF-AH secretion by decidual macrophages in the tissue are yet to be elucidated. We have previously reported that the injection of nonpregnant rats (Yasuda and Johnston, 1992) with dexamethasone or medroxyprogesterone causes an increase, and estrogen causes a decrease, in plasma PAF-AH activity. We have suggested that the hormonal regulation of plasma PAF-AH activity in the pregnant rat may be, in part, via the secretion of PAF-AH by macrophages (Yasuda et al., 1993). The importance of decidual macrophage activation has been recently stressed (MacDonald et al., 1991). For example, decidual tissue produces interleukin 1 (IL-1) (Romero etal., 1989) as well as tumor necrosis factor (TNF) (Jattela et al., 1988) both of which have been shown to be
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secreted by the decidual macrophages (Vince et al., 1989). The relationships of endotoxins and cytokines with PAF have been recognized in a number of pathophysiological conditions (for review see Braquet et al., 1987). We have found that bacterial lipopolysaccharide (LPS) inhibits the PAF-AH secretion by human peripheral monocytes/macrophages and decidual macrophages. The observation that LPS decreases PAF-AH secretion by these cells may provide further evidence as to why gram-negative infections may be involved in premature delivery, as will be discussed in a subsequent section. L. Hormonal Regulation of PAF-AH Activity
The dramatic change in steroid hormones that occurs during the last half of pregnancy led us to examine the role of various hormones on the plasma PAF-AH activity. In an attempt to show that the plasma PAF-AH activity closely paralleled the plasma lipoprotein concentration in the rabbit, Pritchard reported that the activity of PAF-AH in rat plasma was decreased following the injection of 17aethynylestradiol (Pritchard, 1987). When we administered 17a-ethynylestradiol to both adult female and male rats for three days, the plasma PAF-AH activity decreased to approximately one-fifth that of the vehicle-injected group. Dexamethasone administration, on the other hand, resulted in a three-fold increase in plasma PAF-AH activity (Miyaura et al., 1991). This increase in PAF-AH activity following dexamethasone treatment may account, at least in part, for the known antiinflammatory properties of this steroid. It was demonstrated that medroxyprogesterone administration to adult rats caused an increase in the plasma PAF-AH activity (Yasuda and Johnston, 1992). The decrease in plasma PAF-AH activity by the administration of various estrogens was also determined. 17a-Ethynylestradiol was the most potent estrogen tested, although estriol, estradiol, and estrone were also active in that order. Similar changes in the plasma PAF-AH activity caused by dexamethasone, estrogen, or medroxyprogesterone administration have also been shown in pregnant rats (Yasuda and Johnston, unpublished observations). The observation that estrogens decrease and progestins increase plasma PAF-AH activity may have an important implication in parturition. As is well documented, the ratio of estrogens to progestins increases dramatically during the latter stages of pregnancy in most species (Challis and Olson, 1988). We have recently reported (Johnston et al., 1992; Narahara et al., 1993b) that various macrophage preparations also respond to hormonal treatment. Increased secretion of PAF-AH by rat alveolar and spleen macrophages in primary culture was observed when either progesterone or medroxyprogesterone was added to the media. Dexamethasone addition also caused a significant increase in the secretion of PAF-AH. Estrogen addition to the media was without effect. Tarbet et al. (1991) have reported that dexamethasone treatment of HepG-2 cells resulted in an increased secretion of PAF-AH, while estrogen treatment decreased this secretion. Further support for a role of PAF in the initiation of parturition and the relationship of PAF to estrogens was provided by a
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series of experiments in which 17a-ethynylestradiol (2.5 mg/kg) was administered to pregnant rats for three days starting on the 17th day of gestation. The plasma PAF-AH activity decreased from 52 nmoles x min~^ x ml"' on day 17 to 10 nmoles X min~^ X ml"^ on day 20. All of the estrogen-treated animals delivered by day 20 (Yasuda and Johnston, unpublished observations). This finding provided further support for the concept of the involvement of PAF in the events of parturition and the role of estrogen in this process. It has been reported that 1,25-dihydroxyvitamin D3 [l,25"(OH)2D3] is increased in maternal plasma throughout pregnancy (Kumar et al., 1979). l,25-(OH)2D3 has also been detected in increased amounts in the amniotic fluid at term, while 24,25-(OH)2D3 was undetectable (Lazebnik et al., 1983). The origin of 1,25(OH)2D3 in the amniotic fluid is thought to be the fetal kidney (Bouillon, 1990). Weisman et al. (1979) have reported that 1,25-(OH)2D3 is synthesized in the human decidual tissue and suggested that this tissue is also a possible source of the hormone in the amniotic fluid. A central role for [Ca^"^]! in the initiation and maintenance of parturition has been discussed. Although l,25-(OH)2D3 produced in the fetal and maternal compartments is thought to serve as a major regulator of Ca^"^ metabolism and homeostasis (Weisman et al., 1979; Lazebnik etal., 1983; Bouillon, 1990), its physiological role in parturition and its mechanism of action are largely unknown. The l,25-(OH)2D3 was shown to stimulate prostaglandin E2 production in cultured human amnion cells (Casey et al, 1986).
0
0.16
0.8
4
20
100
1,25~(0H)2D3 concentrations (nmol/L) Figure 4. Effect of various concentrations of 1,25-(OH)2D3 on PAF-AH secretion by decidual macrophages following treatment for six days. The values are reported as the mean ± standard deviation.
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We have recently investigated the effect of l,25-(OH)2D3 on the metabolism of PAF in human decidual cells. l,25-(OH)2D3 inhibited PAF-AH secretion by a decidual macrophage population in a dose dependent manner (Figure 4) (Narahara and Johnston, 1993a). The half-maximal inhibition (IC50) was obtained at 2.4 ± 1.3 nM (n = 6). An inhibition of the enzyme secretion was observed within the range of the reported plasma concentrations during pregnancy (Kumar et al., 1979). It is suggested, therefore, that the active metabolite of vitamin D3 may elevate the concentration of PAF in the human decidua due to its inhibitory effect on the PAF-AH secretion by decidual macrophages. The increase in the local concentration of PAF might stimulate the production of prostaglandin E2 and also result in a direct stimulation of myometrial contraction (Tetta et al., 1986; Zhu et al., 1992). Thus, in parallel with the increase in PAF synthesis in tissues of fetal origin (lung and kidney) that occurs late in gestation, there is a decrease in the capacity to inactivate PAF catalyzed by PAF-AH in maternal plasma. The tissue origin of this enzyme may be, in part, the decidual macrophage population. It is suggested that the secretioii of PAF-AH by the macrophages in decidual tissue, combined with the high circulating plasma PAF-AH in the blood supply of this tissue contributes to the resting state of the myometrium that occurs during most of pregnancy by preventing PAF from reaching this tissue. The decrease in the maternal plasma PAF-AH activity that occurs late in gestation, due to either the surge in estrogen production, the decrease in progesterone, or both, may result in an increase in PAF. The proposed mechanism by which this occurs would be by a decrease in plasma PAF-AH activity and a possible decrease in the secretion of PAF-AH by decidual macrophages. M. Presence of PAF Receptors in Human Myometrium and the Effect of PAF and a Receptor Antagonist on Myometrial Contraction and Parturition
Maintenance of a quiescent uterus until the appropriate time, followed by initiation of myometrial reactivity, is required for a successful pregnancy. Nishihira and colleagues (1984) have shown that PAF (either an authentic sample or that purified from amniotic fluid obtained from women in labor) could initiate and propagate contractions of the rat uterus. Similar results have been reported for guinea pig (Montrucchio et al., 1986) and human (Tetta et al., 1986) myometrial strips. We have also demonstrated that PAF will contract myometrial strips at concentrations as low as 10"^^ M to 10"^^ M (Zhu et al., 1992). The force of spontaneous contractions and the contractile force in response to PAF were determined by quantification of the area of active force during a period prior to PAF and after PAF addition. PAF induced a three-fold increase in contractile activity. A PAF receptor in the plasma membrane fraction prepared from human myometrium has been identified and a single class of PAF receptors was established. The binding was inhibited when the membranes were preincubated with the PAF receptor antagonist, L-659,989 (kindly supplied by Merck, Sharp and Dohme, Rahway, NJ).
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HISASHI NARAHARA, RENE A. FRENKEL, and JOHN M. JOHNSTON
The effect of PAF on [Ca^"^]} was examined in cultured myometrial cells (Zhu et al., 1992). The [Ca^^^Jj was quantified by observing the fluorescence of fura-2/AM. PAF caused an almost three-fold increase in the [Ca^'^]^ of these cells (from 112 to 301 nM). The increase in [Ca^"^]! was transient, with maximal values observed after 12—15 seconds, returning to basal levels by one minute. Similarly, treatment of human myometrial cells with PAF (10"^^ M) for 15 seconds, resulted in a 13-fold increase in myosin light-chain phosphorylation. The observations that PAF receptors are present in human myometrium, that this potent autacoid will increase [Ca^"^]!, and that myosin light-chain phosphorylation is increased dramatically following the addition of PAF, provide additional evidence for a role for PAF in the events of parturition. N.
Effect of a PAF Receptor Antagonist on the Time of Delivery in Rats
To evaluate the effect of PAF on parturition, the PAF receptor antagonist L659,989 was administered to 17-day timed pregnant rats and the effects on labor and delivery monitored (Zhu et al., 1991). The duration of gestation was not altered by administration of the antagonist, but the duration of parturition was increased, in a concentration-dependent manner, from two- to five-fold. The toxicity was minimal, as no change in morbidity or mortality was noted. We suggest that the PAF antagonist interferes with the normal events of parturition and that the site of action of the antagonist is the myometrium. A possible reason for the prolongation of delivery time, but not of the length of gestation, is the fact that the PAF antagonist may not cross the placenta and therefore would not affect the fetal component of PAF action. O.
Role of Estrogens in the Timing of Parturition
Recently, we have reported that the injection of 17a-ethynylestradiol caused a decrease in the plasma PAF-AH activity of nonpregnant and pregnant rats (Miyaura et al., 1991; Yasuda and Johnston, 1992; Yasuda et al., 1996). If PAF plays a role in the initiation and maintenance of parturition as has been suggested, the administration of estrogen might be expected to influence the timing of parturition by altering the PAF-AH activity. In this study, pregnant rats were injected with 17a-ethynylestradiol on day 17 for three days. The plasma activity in this group decreased from approximately 50 nmol x min~^ x ml"^ plasma on day 14 to 10 nmol X min~^ X m\~^ on day 19 or 21. The vehicle-treated animals decreased to approximately 40 nmol x min~^ x m\~\ The estrogen treated rats delivered 24 hours prior to the control animals. Although numerous effects of estrogens can be invoked on the timing of parturition, one mechanism may be the effect of this hormone on the PAF-AH activity and PAF concentration.
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IV. PAF AND PERINATAL COMPLICATIONS A.
PAF and Intrauterine Infections
Gram-negative organisms elicit an inflammatory reaction which is largely induced by one of their cell wall constituents, endotoxin (lipopolysaccharide, LPS). The onset of preterm labor caused by bacterial infections can be considered as a pathophysiologic counterpart to other endotoxin-induced reactions, including endotoxic shock, and thus as a host mediated response. Bacterial endotoxins induce the release of a vast array of host mediators, including TNF-a, interleukins, interferons, colony stimulating factors, arachidonate metabolites (eicosanoids), and PAF (Braquet et al., 1987). It has been proposed that the host mediators released from decidua by endotoxins may play an important role in the onset of labor associated with infection (MacDonald et al., 1991; Romero et al., 1991; Gibbs et al., 1992). As previously described, PAF is present in the amniotic fluid of women at term and in labor, but only present in trace quantities in women at term and not in labor (Billah and Johnston, 1983). PAF can also be detected in increased amounts in the amniotic fluid of patients with preterm labor and premature rupture of membranes (Hoffman et al., 1990). In view of these observations, it is postulated that a key mediator, PAF, is elevated at the fetal-maternal interface in response to bacterial infections which lead to preterm labor or premature rupture of membranes. The inactivation of PAF produced in the fetal and maternal compartments prior to its contact with the myometrium would be of considerable importance in the prevention of myometrial contraction (Nishihira et al., 1984; Zhu et al., 1992). As previously discussed, decidua may be the tissue site of the PAF-inactivation. PAF produced in the fetal and maternal compartments would be inactivated by both plasma PAF-AH supplied from maternal blood flow and the enzyme secreted by decidual macrophages, thus preventing PAF from reaching the myometrium. We have recently investigated the effects of LPS and cytokines on the PAF-AH secretion by decidual macrophages (Narahara and Johnston, 1993a). It was demonstrated that LPS inhibited the secretion of PAF-AH by these cells. Therefore, the decrease in PAF-AH production by decidual macrophages may be related directly to the inflammatory responses caused by various LPS. As previously discussed, LPS stimulates the monocyte/macrophage system to induce the release of cytokines such as TNF-a, IL-la, and IL-ip (Hinshaw, 1990). We tested the possibility that these cytokines might mediate the inhibitory effect of LPS on the PAF-AH secretion by decidual macrophages. The LPS-induced inhibition was partially, but significantly, reversed by IL-1 receptor antagonist or by neutralizing antibodies against IL-la, IL-1 (3, or TNF-a, which is consistent with such a mechanism. The findings are consistent with the concept that TNF-a, IL-la, and IL-ip might participate in the LPS-induced inhibition of PAF-AH secretion by decidual macrophages. All three of these cytokines decreased the PAF-AH secretion by these cells. It has recently been reported that IL-1 receptor antagonist, a naturally occurring one, is
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HISASHI NARAHARA, RENE A. FRENKEL, and JOHN M. JOHNSTON
physiologically present in human amniotic fluid (Romero et al., 1992). Therefore, the complete reversibility also suggests the possible modulation of PAF metabolism by the IL-1 receptor antagonist in the intrauterine tissue: the antagonist might decrease PAF concentration by antagonizing the action of IL-1, a cytokine that not only stimulates PAF production (Braquet et al., 1987), but also inhibits PAF-AH secretion. Based on these findings, it is suggested that PAF is involved in the pathogenesis of preterm labor or premature rupture of membranes caused by endotoxins and the subsequent activation of cytokine networks. B. PAF and Cigarette Smoking
It has been suggested in epidemiologic studies that maternal smoking in pregnancy is associated with a significant increase in preterm labor, premature rupture of membranes, and premature delivery (Underwood et al., 1967; Meyer and Tonascia, 1977; Harger et al., 1990; Haas et al, 1991). However, the precise biochemical mechanisms that associate cigarette smoking with these complications of labor are largely unknown. It has been proposed that cigarette smoking may result in the disruption of the endothelial lining, activate platelets, and increase their adherence to the damaged endometrium (Hawkins, 1972). The bioactive mediators released from the lesion may amplify the cascades of thrombosis, atherogenesis, and vasoconstriction, resulting in perivascular tissue damage (Ross, 1986). The mediators may include growth factors, coagulation factors, cytokines, eicosanoids, and PAF, since these bioactive molecules have been considered important in the cellular interactions among platelets, endothelial cells, and leukocytes such as neutrophils and monocytes/macrophages (Braquet et al., 1987; Hinshaw, 1990). A cigarette smoke extract has been shown to oxidize LDL and the oxidized form is preferentially taken up by macrophages (Yokode et al, 1988). Recently, cigarette smoking has been reported to be linked to an increased production of PAF or similar lipid(s) associated with plasma lipoproteins (Imaizumi et al., 1991). These observations are of particular importance since oxidized phospholipids associated with LDL have been found to be substrates for plasma PAF-AH (Stremler et al, 1989) and the enzyme is associated with LDL (Stafforini et al., 1987a). Thus, the capacity of PAF-AH to degrade both PAF and oxidized molecules suggests that this enzyme may regulate, at least in part, the mechanism underlying the pathogenesis related to cigarette smoking. We have previously reported that a cigarette smoke extract (CSE) inhibited plasma PAF-AH activity (Miyaura and Johnston, 1992). Therefore, it is suggested that PAF levels may be increased in plasma by smoking via at least a dual mechanism, in which smoking not only stimulates the production of PAF or oxidized phospholipids in LDL but also inhibits plasma PAF-AH activity. We have demonstrated that CSE inhibited the secretion of PAF-AH by decidual macrophages and peripheral blood monocytes/macrophages (Narahara and Johnston, 1993b). The capacity of these cells to secrete the enzyme was extremely sensitive to CSE compared to the direct inhibitory effect of CSE on the plasma
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enzyme activity. This could result in an elevated concentration of PAF which may play a critical role in the stimulation of uterine endothelium and myometrium. Moreover, the uncontrolled myometrial stimulation by PAF may contribute to the incidence of preterm labor or premature rupture of membranes. C. Pregnancy Induced Hypertension/Preeclampsia
Since PAF is known to be a potent hypotensive agent (Snyder, 1990) and normal pregnancy is thought to be associated with lower responsiveness of the blood vessels to various vasopressor agents (Gant, 1989), we have postulated that physiological levels of PAF that are needed to maintain the responsiveness may be decreased in patients with pregnancy induced hypertension/preeclampsia. It was found that the plasma PAF-AH activity in preeclamptic patients at 32 weeks of gestation was approximately 1.6 times higher than that in the gestationally matched controls (Maki et al., 1993). It is suggested that the higher enzyme activity in patients with this complication may lead to a lower PAF concentration compared to the normotensive group, resulting in a relatively higher sensitivity to various pressor agents. Benedetto et al. (1989), however, have reported a reduced serum inhibition of PAF in patients with preeclampsia, which may cause an increase in PAF in these patients. On the other hand, Kahn et al. (1992) reported no difference in the plasma enzyme activity between patients with pregnancy induced hypertension and normotensive pregnant women. The difference in the selection of patients' groups may be a possible explanation for the discrepancy, in which patients with the mild type disease may have normal (Kahn et al., 1992) or higher (Maki et al., 1983) activity of PAF-AH, while groups with the severe form may have lower enzyme activity (Benedetto et al., 1989). It is evident that future studies are necessary in order to address the role of PAF in vasomotor activity during pregnancy. D. /.
PAF and Necrotizing Enterocolitis
PAF and its Role in the Pathogenesis of Necrotizing
Enterocolitis
Acute necrotizing enterocolitis (NEC) is a highly lethal disease of the gastrointestinal tract in the newborn infant and it is seen primarily in low birth weight infants (Kliegman and Fanaroff, 1984). It has been suggested that PAF, along with endotoxin and TNF-a, plays an important role in the development of NEC of the newborn (Gonzalez-Crussi and Hsueh, 1983; Caplan et al., 1990). Patients with this disease were shown to have higher plasma PAF concentrations and to have lower plasma PAF-AH activity. We have investigated the role of PAF in the pathogenesis of NEC and the mechanism involved in the protective effect of breast feeding in the prevention of this disease (see below). The administration of PAF via the descending aorta induced lesions with the pathological characteristics of NEC (Furukawa et al., 1993a). The induction was dependent on the site of the
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HISASHI NARAHARA, RENE A. FRENKEL, and JOHN M. JOHNSTON
injection and dose of PAF administered. The injection at a site lower than the branch of the inferior mesenteric artery was less effective. A dose of 0.35 jag PAF in a 200 g rat caused the NEC-like disease. When plasma PAF-AH activity was lowered by pretreatment with 17a-ethynylestradiol, the lesion was observed at a dose as little as 0.175 |ig/rat. Pretreatment of rats with therapeutic levels of dexamethasone resulted in an increase in the plasma PAF-AH activity two- to three-fold above controls and protected against the formation of the lesion. Although the relationship between elevated plasma PAF-AH activity and the protection against NEC has not been presently established, it would appear that the increase in PAF-AH may play a role in the modification of this disease. Indeed, several reports have appeared in which the administration of glucocorticoids for treatment of other conditions such as RDS has resulted in a decreased incidence of NEC (Morales et al., 1986; Halac etal., 1990). 2.
PAF-Acetylhydrolase in Milk
The beneficial effects of breast feeding compared to formula feeding in the prevention of NEC in the human has been reported (Lucas and Cole, 1990). Recently, we have demonstrated that PAF-AH activity is present in human milk (Furukawa et al., 1993b). It was demonstrated that this activity is not due to lipoprotein lipase, bile salt-stimulated lipase, or Ca^'^-dependent PLA2. Inhibitor studies revealed that the enzyme in human milk was of the plasma type. The enzyme activity was stable at pH 4 at 37°C, and it was concluded that the enzyme may not be degraded in the gastrointestinal tract of the neonate. Cow's milk contained very low PAF-AH activity, which might explain the observation that raw cow's milk was not a satisfactory substitute for breast milk (Park et al., 1983). It has also been suggested that the white cells in milk may play an important role in the prevention of NEC (Pitt et al., 1977). Macrophages isolated from human milk secreted PAF-AH activity of the plasma type into the culture medium. The presence of PAF-AH in human milk and the demonstration that this enzyme could be secreted by the macrophage population in human milk may explain, in part, the beneficial effects of nursing on the prevention of the development of NEC. We have emphasized a role for PAF in fetal lung maturation, the initiation and maintenance of parturition, and in certain complications associated with a premature delivery. Although PAF is known to be the most potent lipid mediator and its importance in reproductive biology is well documented, it is our view that the events cannot be attributed solely to PAF. In all likelihood a number of autacoids participate in these essential processes.
ACKNOWLEDGMENTS This work was supported, in part, by NIK grants HD13912 and HDll 149, as well as grants from the Chilton Foundation and The Robert A. Welch Foundation, Houston, TX. The authors appreciate the editorial assistance of Ms. Dolly Tutton.
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265 REFERENCES
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SPHINGOLIPIDS AS REGULATORS OF CELLULAR GROWTH, DIFFERENTIATION, AND BEHAVIOR
Alfred H. Merrill, Jr., Dennis C. Liotta, and Ronald T. Riley
ABSTRACT 274 I. INTRODUCTION 274 II. BASIC PARADIGMS FOR THE ROLE OF SPHINGOLIPIDS IN THE REGULATION OF CELL GROWTH, DIFFERENTIATION, AND BEHAVIOR 277 A. Regulationof Cell Growth by Sphingolipids 277 B. Regulation of Protein Kinases and Protein Phosphatases by Sphingolipids . . 279 C. Inhibitors of Long-Chain Base Biosynthesis as the Mechanism for the Toxicity and Carcinogenicity of Fumonisins 282 D. Hypotheses for Growth Regulation by Sphingolipids 284 III. APPROACHES TO STUDYING THE FUNCTIONS OF SPHINGOLIPIDS . 285 A. Additionof Exogenous Sphingolipids and Sphingolipid Analogues . . . 285
Advances in Lipobiology Volume 1, pages 273-298. Copyright © 1996 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-635-5 273
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B. Effects of Adding Exogenous Agents That Bind or Chemically Modify Sphingolipids 287 C. Studies With Metabolic Inhibitors 287 D. Genetic Approaches to Altering Sphingolipid Biosynthesis 288 IV. CRITERIA FOR EVALUATING THE RESULTS OF STUDIES OF SPHINGOLIPIDS 288 V. PERSPECTIVES FOR FUTURE RESEARCH ON SPHINGOLIPIDS . . . . 290 A. Opportunities for the Discovery of Diseases Caused by Aberrant Sphingolipid Metabolism 290 B. New Strategies for Preventing and Treating Disease Based on an Understandingofthe Activities of Sphingolipids 290 ACKNOWLEDGMENTS 291 REFERENCES 291
ABSTRACT Sphingolipids have been suggested to participate in a wide range of cellular processes that are important to growth and differentiation, by functioning as ligands for proteins of the extracellular matrix and cell-cell communication, modulators of the activity of growth factor receptors, lipid second messengers in the action of cytokines and other factors, and as potent activators and inhibitors of cellular regulatory systems such as protein kinase C (as well as other protein kinases and phosphatases), phospholipase C and D, phosphatidic acid phosphatase, and other proteins. For the most part, these roles have been suggested by studies in which specific sphingolipids (e.g., gangliosides, ceramides, sphingosine, and sphingosine 1-phosphate, among others) have been added to cells or enzyme preparations to elicit a response; therefore, the direct involvement of the compounds as regulators of most of these systems in vivo remains to be proven. Nonetheless, a clearer picture is beginning to emerge for three cases, namely: the regulation of the epidermal growth factor receptor by ganglioside GM3, the participation of a sphingomyelin/ceramide cycle in the action of tumor necrosis factor and a number of other agonists, and the disruption of free long-chain (sphingoid) base metabolism as the mechanism of action of fumonisins and related mycotoxins. These provide models for additional studies of the roles of sphingolipids in the regulation of cellular growth, differentiation, neoplastic transformation, and diverse biological functions.
I. INTRODUCTION Since their first description by Thudichum in 1884, sphingolipids have been one of the more intriguing classes of biomolecules. They encompass a diverse family of compounds with the backbone lipid domain termed a "ceramide" (a long-chain, sphingoid base with an amide-linked fatty acid) and a highly water-soluble headgroup (e.g., phosphorylcholine, simple to complex carbohydrates, or polypep-
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tides that are thereby anchored to membranes). Sphingolipids may well be the most structurally diverse class of lipids in eukaryotic organisms, with hundreds of subclasses varying in their headgroups, each of which is present in multiple forms due to heterogeneity in the long-chain (sphingoid) bases and long-chain fatty acids of the ceramide backbone (Barenholz and Thompson, 1980; Kanfer and Hakomori, 1983; Sweeley, 1991; Bell et al, 1993). Such structural diversity implies that these compounds have complex cell functions, and this idea is further supported by changes in sphingolipids during cell growth and differentiation, embryogenesis and development, and in cancer and other diseases (Hakomori, 1981) (for specific references and recent reviews see Bell et al., 1993). Thus, many of the proposed functions for sphingolipids (see examples in Figure 1) are associated with the ability of cells to interact with their environment. The physical characteristics of sphingolipids (both the nature of the headgroups and the hydrophobic domains) probably have widespread effects on membrane structure and dynamics (i.e., membrane internalization, sorting, recycling, and turnover). However, there are many cases in which glycolipids associate specifically with proteins; prominent among these interactions are sphingolipid binding to extracellular matrix proteins and receptors (as well as with bacterial toxins and antibodies). Sphingolipids have been recently found to be covalently attached to some proteins as lipid anchors, and to also affect the membrane behavior of an analogous group of proteins anchored via phosphatidylinositol glycan groups. Sphingolipids have joined the list of membrane lipids that may serve as intracellular second messengers upon hydrolysis to substitutents that affect cell regulatory systems. Application of this paradigm to sphingolipids began with the discovery that sphingosine (Hannun et al., 1986) is a potent inhibitor of protein kinase C (PKC). Since this discovery, micromolar concentrations of sphingosine have been found to affect many systems and additional sphingolipids have been found to be plausible candidates for lipid mediators; these include ceramides, ceramide 1 -phosphate, sphingosine 1-phosphate, lysosphingolipids, and N-methyl-sphingosines. Other, more structural, roles for sphingolipids are their contribution to the permeability barrier of skin (Holleran et al., 1993), where ceramides are a major repository for essential fatty acids and an important target in essential fatty acid deficiencies (Wertz and Downing, 1989). Sphingomyelin affects not only the physical properties of membranes and lipoproteins, but also the metabolism of other lipids, especially phosphatidylcholine and cholesterol (Slotte et al., 1990; Gupta andRudney, 1991). Experts in "sphingolipidology" are, therefore, in the perplexing position of having to unravel how these molecules function in some of the most intricate biological processes. Their counterparts—scientists interested in particular biological events—^are often bewildered by the complexity of the sphingolipids. Are there any straightforward ways to uncover the true biological functions of the sphingolipids and then to proceed to understand how defects in these processes lead to disease? And, are these approaches accessible to one not trained in the preparation
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Sphingolipid
,0-Glu-Gal-NAM
{ Ganglioside CMS
Inhibition of EGF Receptor kinase Inhibition of Cell Growth
,0-Glu-Gal I Stimulation of Cell Growth Lactosylceramide OH
OH ,CH 20POCH2CH2N(CH3)3
,NH
Sphingomyelin OH
Ceramide OH
Sphingosine
I Inhibition of colon carcinogenesis 1 Interaction with Cholesterol *- Source of Ceramide via Sphingomyelinase Activation of Protein Phosphatase(s) Inhibition of Cell Growth Induction of Apoptosis, Differentiation Mediator of Cellular Responses to: TNF, ILl &Gamma Interferon Inhibition of Protein kinase C, Na+/K+ ATPase Phosphatidic Add Phosphatase Activation of Phospholipase C & D, EGF Receptor Phosphorylation, Protein kinase(s) Inhibition of Cell Transformation, ODC Induction, Tumor Invasion/Metastasis Stimulation /Inhibition of Growth/EHfferentiation Activation of Phospholipase D Release of Intracellular Caldimi Stimulation of Cell Growth
Sphingosine l-phosphate Figure 1, Structures of representative sphlngolipids and examples of biological activities that are associated with these compounds in vitro or in vivo. For simplicity, variations in the long-chain base or fatty acid backbones have not been shown. The abbreviations are: Glu, glucose; Gal, galactose; N A M , N-acetylneuraminic acid, or sialic acid; TNF, tumor nectosis factor; I L l , Interleukin 1 -beta; EGF, epidermal growth factor; and ODC, ornithine decarboxylase. For more details about these activities, see the text.
and handling of sphingolipids? There are, of course, no easy answers to these questions. But there are a number of emerging concepts that are helpful in framing new hypotheses about sphingolipids, and recently developed tools that can be useful in testing them. This chapter will summarize many of these points, as well as
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propose basic criteria that should be used to evaluate research that attempts to relate sphingolipids to biological phenomena. As part of this, we will describe the recent discovery of a family of compounds that cause diseases of plants and animals via disruption of sphingolipid metabolism.
IL BASIC PARADIGMS FOR THE ROLE OF SPHINGOLIPIDS IN THE REGULATION OF CELL GROWTH, DIFFERENTIATION, AND BEHAVIOR The initial evidence that complex sphingolipids may be involved in the regulation of growth and differentiation was that glycolipid profiles change with the cell cycle, as cells become confluent, and with transformation or differentiation (reviewed in Ogura and Sweeley, 1992; Zeller and Marchase, 1992). While suggestive, this does not establish causality. A. /.
Regulation of Cell Growth by Sphingolipids
Gangliosides and Growth Factor Receptors
The next step toward establishing a more concrete relationship has usually been to add sphingolipids to cells and determine how this affects growth. Gangliosides Gj^3 or Gj^j inhibit growth through extension of the Gj phase of the cell cycle (Laine and Hakomori, 1973; Keenan et al., 1975) and make cells refractory to stimulation by fibroblast growth factor (Bremer et al., 1984). Bremer et al. (1984) noted that Gj^3 and G^^ inhibit PDGF binding and PDGF-induced protein phosphorylation in 3T3 cells, and suggested that inhibition of cell growth could be achieved both by interference with growth factor binding and with the cellular response. Addition of Gj^3 to A431 cells, which have a high level of epidermal growth factor (EGF) receptors, inhibited growth and studies with KB and A431 cells found that Gj^3 inhibited EGF-induced phosphorylation (on tyrosine only) without inhibition of EGF binding (Bremer et al., 1986). Weis and Davis (1990) used a mutant Chinese hamster ovary (CHO) cell line that has a conditional defect in ganglioside biosynthesis and stable cell lines expressing the EGF receptor to show that variation of the physiological levels of gangliosides (primarily Gj^3) modulated signal transduction by the EGF receptor. There are several lines of evidence in favor of these effects of gangliosides being mediated via direct binding to the receptor. Affinity purification of the EGF receptor from a subclone of A431 cells that is only weakly responsive to EGF (A5S cells show weak growth stimulation by EGF) yielded Gj^3 (Hanai et al., 1988b); whereas, Gj^3 did not co-purify with the EGF receptor of a subclone (A5I) that exhibits growth inhibition in response to EGF, although more polar gangliosides were obtained. Other studies have used the proteins that bind sphingolipids (such as antibodies or the beta subunit of cholera toxin, which binds ganglioside Gj^j) to compete with
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the binding of glycolipids to their cellular "receptor(s)." The beta subunit is mitogenic for rat thymocytes and quiescent 3T3 cells, and potentiates the response of the latter to EGF, PDGF, and insulin (Spiegel and Fishman, 1987). There is a rapid increase in intracellular Ca^^ that is sensitive to the extracellular calcium concentration, which suggests that the increase does not arise from intracellular stores (Spiegel and Panagiotopoulos, 1988). In rapidly growing 3T3 cells and ^^'-transformed 3T3 cells, however, the beta subunit is inhibitory; therefore, gangliosides have been described as bimodal regulators of cell growth (Spiegel and Fishman, 1987; Buckley et al., 1990; Hakomori, 1990). Another approach has been to remove gangliosides by treating cells with enzymes that cleave the headgroup, such as endoglycoceramidase or neuraminidase (Ogura and Sweeley, 1992), which is growth stimulatory. The latter treatment may have a physiological counterpart because a Gj^3 sialidase activity is found in the culture medium of human fibroblasts (Usuki et al., 1988a,b). To assess the importance of extracellular sialidase(s) to cell growth, fibroblasts were incubated with a sialidase inhibitor (2-deoxy-2,3-dehydro-N-acetylneuraminic acid), which reversibly inhibited growth and the turnover of labeled Gj^3 (Usuki et al, 1988a). Thus, it appears that Gj^3 turnover to lactosylceramide is more active for growing cells—to release them from the growth inhibitory effects of the intact ganglioside— but that the sialidase activity is low at confluence (and G,^3 turnover is reduced) to contribute to the downregulation of growth factor receptor(s). 2. Modified Gangliosides and Growth Factor Receptors
A number of modified gangliosides have also been implicated as bioactive species. LysoGj^3 (which lacks the amide-linked fatty acid) inhibits the receptor kinase, and has been found in small amounts in the cells (Hanai et al., 1988b). De-N-acetyl-Gf^3 (with loss of the N-acetyl group from the sialic acid) strongly enhances the EGF receptor kinase activity (and was growth stimulatory) without altering the binding of EGF; this compound was also found in small amounts in cells (Hanai et al., 1988a). On the basis of these findings, Hanai et al. (1988a,b) have suggested that gangliosides and their N-deacetylated derivatives (i.e., lacking an N-acetyl group from the sugar or a fatty acid from ceramide) may represent a mechanism for stimulating and inhibiting growth. However, other investigations by Song et al. (1991) indicate that the effects of the deacylated analogues on EGF receptor phosphorylation may be artifactual because they only occur in vitro. They confirm that Gj^3 does affect labeled thymidine incorporation into DNA in vivo. 3.
Lactosylceramide and Growth
Lactosylceramide, which is formed upon removal of the sialic acid from G^^, increases DNA synthesis in fibroblasts (Ogura and Sweeley, 1992), and glucosylceramide stimulates liver growth and DNA synthesis in liver (Datta and Radin, 1988).
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Other Metabolites of Complex Sphingolipids Implicated in Growth
Ceramide is growth stimulatory in Swiss 3T3 cells (Olivera et al., 1992) and inhibitory in HL-60 cells (Okazaki et al., 1989, 1990) and yeast (Fishbein et al., 1993). Ceramide can also activate the mitogen-activated protein kinase in HL-60 cells (Raines et al., 1993); in other cases, it induces programmed cell death (Obeid et al., 1993). Long-chain bases stimulate DNA synthesis and growth at low concentrations, but are inhibitory at higher levels (Zhang et al., 1990; Stevens et al., 1990a). Sphingosine 1-phosphate (Zhang etal, 1991), sphingosine 1-phosphorylcholine (Desai et al., 1993), and N,N-dimethylsphingosine (Felding-Habermann et al, 1990) also affect growth. B. Regulation of Protein Kinases and Protein Phosphatases by Sphingolipids
The idea that components of sphingolipids might have biological activity gained serious attention with the discovery that sphingosine inhibits PKC in vitro and in intact cells (Hannun et al, 1986; Merrill et al., 1986; Wilson et al., 1986). Sphingosine inhibits PKC competitively with diacylglycerol, phorbol dibutyrate, and Ca^"*", and blocks activation by unsaturated fatty acids and other lipids (Hannun et al., 1986; Wilson et al., 1986; Oishi et al., 1988; El Touny et al., 1990); therefore, it would appear that sphingosine could serve as a natural antagonist to the lipid activators of this enzyme. Even though studies to date have not proven that long-chain bases are involved in the downregulation of PKC in vivo, these findings raised the possibility that cells might utilize hydrolysis products of sphingolipids as "lipid second messengers." Subsequent investigations have found that 100 different cellular systems are affected by long-chain bases, and a large number of additional compounds (sphingosine 1-phosphate, N-methylsphingosines, ceramides, lysosphingolipids, etc.) have been discovered to have potent biological activities. This indicates that the sphingolipid signaling pathways may be as diverse as the glycerolipid second messengers (or more so). 7. Sphingosine and Cell Growth
Long-chain bases cause growth inhibition and cytotoxicity for CHO cells (Merrill et al., 1989; Stevens et al., 1990a), growth stimulation for Swiss 3T3 cells (Zhang et al., 1990), and reversal of the growth inhibition caused by PKC activation in vascular smooth muscle cells (Weiss et al., 1991). While some of these effects may be due to PKC, long-chain bases affect many additional processes that are involved in growth regulation. Sphingosine activates a phosphatidylethanolaminespecific phospholipase D (Kiss et al., 1991) and phospholipase C delta (Pawelczyk and Lowenstein, 1992). Long-chain bases are also potent inhibitors of phosphatidic acid phosphohydrolase (Jamal et al., 1991; MuUmann et al., 1991; Aridor-Piterman et al., 1992), which removes the phosphatidic acid produced after phospholipase D
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A. H. MERRILL, JR., D. C. LIOTTA, and R. T. RILEY
activation. Based on studies with human neutrophils, it appears that phosphatidic acid phosphatase may be the most sensitive target of sphingosine action (i.e., about 10-fold more potent as an inhibitor of phosphatidic acid phosphohydrolase than as an inhibitor of PKC) (Perry et al., 1992). The mechanism of inhibition has recently been analyzed using phosphatidic acid hydrolase(s) purified from yeast (Wu et al., 1993). All together, these findings suggest that long-chain bases both stimulate the formation of phosphatidic acid and its dephosphorylation. The net effect would be accumulation of phosphatidic acid, which is often regarded as a mitogenic glycerolipid. The effects of long-chain bases on lipid metabolism may be far-reaching because Pinkerton et al. (1993) have reported that sphingosine reduced 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in CHO cells. Long-chain bases have also been reported to affect several ion transport systems. Of particular interest is the finding that sphingosine and sphingosylphosphorylcholine induce calcium release from intracellular stores in smooth muscle cells permeabilized with saponin (Ghosh et al., 1990). Sphingosine and sphingosine 1-phosphate additionally stimulate increases in intracellular calcium in Swiss 3T3 cells (Zhang et al., 1991), and these observations have led to the hypothesis that a sphingosine metabolite—possibly sphingosine 1-phosphate—^might act as a regulator of calcium mobilization. Spiegel and co-workers have shown that sphingosine and sphingosine 1-phosphate stimulate proliferation of quiescent Swiss 3T3 fibroblasts (Zhang et al, 1990) with increases in phosphatidic acid levels (Desai et al., 1992). Other recent work has found sphingosine 1-phosphate to inhibit cell motility and phagokinesis of B16 melanoma cells (Sadahira et al., 1992). Sphingosine has also been shown to activate some protein kinases. Davis and co-workers (Faucher et al., 1988) demonstrated that sphingosine increases the phosphorylation at threonine 669 (the MAP kinase site) of the EGF receptor through a pathway that appears to be independent of PKC. Sphingosine increases the activity of the cytoplasmic tyrosine kinase domain of the EGF receptor to equal or greater than that of the ligand-activated holo EGF receptor (Wedegaertner and Gill, 1989). The phosphorylation of threonine 669 may be due to the formation of ceramide and activation of a ceramide activated kinase (Goldkom et al., 1991); nonetheless, sphingosine and N-methylated long-chain bases also directly induce phosphorylation on threonine 669 (Goldkom et al., 1991). N-methyl-sphingosines inhibit both in vitro and in vivo growth of tumor cells in nude mice (Endo et al., 1991) and inhibit metastasis (Okoshi et al., 1991). Sphingosine induces dephosphorylation of the retinoblastoma gene product (Chao et al., 1992), a nuclear phosphoprotein that is thought to function as a tumor suppressor. As noted by the authors, the phosphorylated forms of retinoblastoma protein are observed in the S and G2/M phases of the cells cycle, whereas the less phosphorylated forms are found in GQ/GJ. Therefore, sphingosine may modulate phosphorylation/dephosphorylation of retinoblastoma protein and progression through the cell cycle. Pushkareva et al. (1992) have reported that D-ery^/zro-sphingosine also induces the phosphorylation of a number of cytosolic proteins in Jurkat
Bioactive Sphingolipids
2 81
T cells. There appear to be several sphingosine-activated protein kinases because the concentration dependence on sphingosine varied among the endogenous substrates and there were differences in utilization of ATP and GTR The erythro-sphinganines were less active than sphingosine, and threo-sphinganinQ was not active. Virus replication, as another type of growth, is also affected by sphingosine, which inhibits the induction of DNA polymerase and DNase activities in EpsteinBarr virus infected cells treated with phorbol esters and n-butyrate (Nutter et al., 1987). Phorbol esters alter the sphingosine levels in Epstein-Barr virus transformed B lymphocytes (Miccheli et al., 1991). Interestingly, Van Veldhoven et al. (1992) have found that ceramide, but not sphingosine, is increased in HIV-infected cells. For some of the enzymes that are affected, but require high concentrations of sphingosine, the effects are probably not relevant to the regulation of these enzymes in vivo. In these cases, the enzymes may be sensitive to disruption of the surface charge of the membrane by any positively charged molecule, or to the coincidental binding of sphingolipids to hydrophobic regions of the polypeptide. 2. Ceramides, as Part of a Sphingomyelin/Ceramide and Differentiation
Cycle, in Cell Growth
Hannun and co-workers discovered (Okazaki et al., 1989, 1990) that treatment of HL-60 cells with 1-alpha, 25-dihydroxyvitamin D3 to induce differentiation resulted in hydrolysis of a significant portion of the cellular sphingomyelin to ceramide and phosphorylcholine. The maximal response occurred after approximately two hours, and the amounts of sphingomyelin and ceramide returned to basal levels by four hours. The sphingomyelin turnover was related to activation of a neutral sphingomyelinase. Ceramide formation appeared to be part of the cellular response to this agonist because an effect was also seen if short-chain ceramides were added instead of the agonist. On the basis of these observations, they proposed that cells possess sphingomyelin/ceramide cycle(s) that serve as a lipid second messenger system (Hannun, 1994) that function in cell cycle arrest (Jayader et al., 1995). Sphingomyelin/ceramide cycles appear to be involved in the action of at least three other agonists: tumor necrosis factor (TNF) (Kim et al., 1991; Mathias et al., 1991), gamma-interferon (Kim et al., 1991), and interleukin 1 (Ballou et al., 1992; Mathias etal, 1993). Ceramide has been found to activate a serine/threonine protein phosphatase (Dobrowsky and Hannun, 1992). The characteristics of the ceramide-activated protein phosphatase (C APP) are similar to the subgroup 2 A protein phosphatases (i.e., it is cation independent and sensitive to inhibition by okadaic acid). CAPP is activated by a variety of ceramides with different hydrophobic moieties; however, studies with N-acyl-phenylaminoalcohol analogues indicate that it is stereospecific (Bielawska et al., 1992). Kolesnick and co-workers have shown that ceramide also activates protein kinase(s) (Mathias et al., 1991; Kolesnick and Golde, 1994). These studies arose from investigation of the mechanism of sphingosine activation of the epidermal
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A. H. MERRILL, JR., D. C. LIOTTA, and R. T. RILEY
growth factor receptor (Goldkom et al, 1991), and found that ceramide was able to induce phosphorylation of the receptor; therefore, at the concentrations of sphingosine that caused activation of the receptor, Goldkom et al. (1991) they hypothesized that it is actually this metabolite of sphingosine that is active. Subsequent studies (Mathias et al., 1991) utilizing a polypeptide substrate (representing amino acids 663-681 of the EOF receptor), found a Mg^'^-dependent, ceramide-activated kinase activity in the membrane fraction from A431 cells. Activation of this kinase activity has been demonstrated in a cell-free system (Dressier et al, 1992) in which postnuclear supernatants from HL-60 cells were treated with TNF to induce sphingomyelin turnover to ceramide, and could be mimicked by adding exogenous sphingomyelinase to the preparation instead of TNF. Ceramide has also been found to activate the mitogen-activated protein kinase in HL-60 cells (Raines et al., 1993). From these studies, it appears that a sphingomyelin/ceramide cycle meets the main criteria of a second messenger system: ceramide is formed in response to the agonist, and its formation occurs in the correct time frame and in amounts necessary to activate the next steps of the pathway, in this case protein kinase(s) and protein phosphatase(s), and ceramides alone can lead to the biological response. Some structural specificity is seen in the ceramides, and inhibitors (i.e., okadaic acid, which inhibits protein phosphatases) block the biological response. In these studies of ceramide, the authors have noted that sphingosine was not formed in response to the agonists; nonetheless, ceramide can be converted to additional bioactive products in other cells. Sphingomyelin turnover in isolated plasma membranes from rat liver yields both ceramide and sphingosine (Slife et al., 1989) and growth factors trigger sphingolipid turnover to ceramide, sphingosine and sphingosine 1-phosphate (Su et al., 1994; Coroneos et al., 1995). Cells can also produce ceramide 1-phosphate via a ceramide kinase (Bajjalieh et al., 1989; Kolesnick and Hemer, 1991). Little is known about the biological activity (if any) of ceramide phosphates; however, some bacteria and the brown recluse spider produce a sphingomyelinase D (which forms ceramide phosphate) and injection of this enzyme causes a prolonged inflammatory response (Rees et al., 1984), which indicate that these molecules may have profound biological activities in vivo. C.
Inhibitors of Long-Chain Base Biosynthesis as the Mechanism for the Toxicity and Carcinogenicity of Fumonisins
While searching for agents that increase the amounts of free sphingosine via stimulation of sphingolipid turnover, we have discovered a family of toxins that cause long-chain bases to accumulate due to disruption of de novo sphingolipid biosynthesis (Wang et al., 1991). These toxins are produced by Fusarium moniliforme (Sheldon) and related molds, which are the most prevalent molds on com, sorghum, and a number of other grains throughout the world. Consumption oi K
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moniliforme-conteimimtQd corn causes several diseases of agricultural concern (e.g., equine leukoencephalomalacia and porcine pulmonary edema) (Kriek et al., 1981; Marasas et al., 1988; Ross et al., 1990) and has been correlated with human esophageal cancer (Lin et al., 1980; Yang, 1980; Marasas, 1982). In studies with laboratory rats, it is clear that the toxins are hepatotoxic and hepatocarcinogenic (Marasas et al., 1984; Gelderblom et al., 1988, 1991). The causative agents are thought to be a group of mycotoxins termed fumonisins (Bezuidenhout et al., 1988) because the feeding of purified fumonisins have been able to reproduce the diseases associated with consumption of contaminated com or culture materials (Norred, 1993). Because the fumonisins bear a remarkable structural similarity to sphinganine, we hypothesized that they might act via disruption of sphingolipid metabolism. Fumonisins were found (Wang et al., 1991) to inhibit the incorporation of [^"^CJserine into [^"^CJsphingosine by hepatocytes, with an IC5Q of approximately 0.1 \xM, and the effects were selective because there was no reduction in the radiolabeling or mass of fatty acids, phosphatidylserine, phosphatidylethanolamine, or phosphatidylcholine. The primary target of the fumonisins is ceramide synthase, which is inhibited in vitro and in vivo, as shown by the accumulation of sphinganine by 110-fold (i.e., to 1.50 ± 0.02 nmol/dish) when hepatocytes were treated with fumonisin Bj for four days (Wang et al., 1991). To determine if these in vitro observations were relevant in vivo, the amounts of sphingosine, sphinganine, and total sphingolipids were measured using serum from ponies that had been given fumonisin-contaminated feed. There was a large increase in sphinganine and sphingosine, and a reduction in the amounts of complex sphingolipids in serum (Wang et al., 1992). Similar findings have been obtained subsequently in feeding studies with pigs, rats, chickens, and turkeys (reviewed in Merrill et al., 1993). Additional studies have related the accumulation of sphinganine to the inhibition of cell growth, and ultimately cells death, using LLC-PKl cells (Norred et al., 1992; Yoo et al., 1992). Given the known cytotoxicity of free long-chain bases (Stevens et al., 1990a), it is likely that the large increase in cellular sphinganine contributes to cell death. More recent studies (Schroeder et al., 1993) have shown that fumonisins cause sphinganine accumulation in Swiss 3T3 cells, and that this is responsible for an increase in DNA synthesis and cell division in these cells when incubated with fumonisin Bj (Schroeder et al., 1994). This fumonisin- (and sphinganine-) induced mitogenicity closely matches the known growth stimulation by sphingosine in Swiss 3T3 cells (Zhang et al, 1990). Therefore, these findings suggest that the carcinogenicity of the fumonisins may be mediated via disruption of sphingolipid biosynthesis because a key event in the action of many carcinogens is the induction of cell growth.
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A. H. MERRILL, JR., D. C. LIOTTA, and R. T. RILEY D.
Hypotheses for Growth Regulation by Sphingoh'pids
Why and how do cells utilize gangliosides and products of ganglioside turnover to modulate the behavior of receptors? The utilization of gangliosides to modulate receptor function enables cells to fine tune their responsiveness to external stimuli. For example, the release of sialidase by fibroblasts cultured at low density (Usuki et al., 1988a,b) prevents G^^ accumulation and, thereby, would cause minimal inhibition of the EGF receptor. As the cells become dense (in crude analogy to the final steps in wound healing), it is no longer desirable for the cells to be responsive to growth factors, so sialidase activity decreases and Gj^3 levels rise. Modulation of growth factor receptor function by glycolipids also provides a mechanism for coordinating several receptors (including receptors on neighboring cells), or for linking them to other cell cycle-related events at the cell surface, such as the interaction of the plasma membrane with the extracellular matrix and/or other cells. In this way, one might envision that glycolipid turnover would both make cells responsive to growth factors and break some of the contacts with the extracellular matrix that are needed for the changes in cell morphology during cell division. The hydrolysis of sphingolipids liberates other bioactive molecules that could function as ligands for other proteins at the cell surface and/or at intracellular sites. By conventional usage, if the cleavage products are formed rapidly and activate or inhibit ion channels, protein kinases or phosphatases, etc., they would be described as second messengers. However, there is no reason to assume a priori that the release of bioactive sphingolipids must be rapid because many of the processes that are associated with sphingolipids (growth and differentiation) progress slowly. The responses might occur over longer times to coordinate many aspects of cellular metabolism because the products of sphingolipid turnover affect many different targets—spanning the growth factor receptors, ceramide-activated protein kinases and phosphatases, sphingosine stimulation/inhibition of phospholipases, phosphatidic acid phosphatase, PKC, the EGF receptor kinase, etc.—^highlighting just a few of the systems that may be affected by this class of molecules, as was illustrated in Figure 1. Furthermore, the effects of the sphingolipid metabolites (ceramide, sphingosine, etc.) can interact (Gomez-Munoz et al., 1995). Figure 2 presents a hypothetical model of the ways that bioactive sphingolipids may arise from both biosynthetic reactions and from complex sphingolipid turnover, appear in multiple intracellular sites, and affect cell responses. Long-chain bases can move among membranes (Merrill et al, unpublished observations); therefore, sphinganine, that is an intermediate of the de novo biosynthetic pathway, or sphingosine released during lysosomal catabolism of sphingolipids (or in response to agonist-induced sphingolipid turnover), could affect targets in multiple cellular compartments regardless of their initial site of formation. More hydrophobic sphingolipids (e.g., ceramides) might be transported by intracellular membrane movement and/or lipid transport proteins (Hoekstra and Kok, 1992).
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Agonists
I Endosomes Cer- SM and
Cer
I Cellular responses | -^i^^Cer, SM and GSL
^
f^ SaP
'^y^^ Serine + Endoplasmic Palmitoyl-CoA reticidum
I N-MeSo 1
SoP
^ ^ . - ^
SM and GSL
So^^SoLysosomes
Figure 2, A hypothetical scheme depicting how complex sphingolipids might function in cell regulation. The abbreviations are: Sa, sphinganine; Cer, ceramide and dihydroceramide; SM, sphingomyelin; GSL, glycosphingolipid; SMase, sphingomyelinase; LacCer, lactosylceramide; GM3, ganglioside CMS; SO, sphingosine; SoP, sphingosine 1-phosphate; SaP, sphinganine 1-phosphate; and N-MeSo, N-methyl- and N,N-dimethylsphingosine. The thin lines represent metabolic reactions of these molecules; the bold lines illustrate the molecules that have been reported to have biological activities. For more details about these events, see the text.
This model is mainly speculative because no studies to date have provided a step-by-step change in these sphingolipids or in these targets. Nonetheless, there is a provocative symmetry to the systems that are affected by sphingolipids as illustrated by the inhibition of growth by Gj^3 complemented by the stimulatory effects of lactosylceramide, ceramide, sphingosine, and sphingosine 1-phosphate. If these are merely coincidental, then this is surely one of the most amazing coincidences in biochemistry. If not, then there is clearly a lot of work to be done to unravel the enigmas.
III. APPROACHES TO STUDYING THE FUNCTIONS OF SPHINGOLIPIDS A. Addition of Exogenous Sphingolipids and Sphingolipid Analogues
There are a number of general principles that underlie the utilization of bioactive lipids that may not be appreciated by investigators unaccustomed to studying lipids.
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The first consideration, of course, is the source of the compounds because the fatty acid and long-chain base composition of a given sphingoHpid varies considerably from tissue to tissue, and sometimes among animals of different ages (Kanfer and Hakomori, 1983). The purity of the compounds should be checked, both because quality control can vary among suppliers and because standard procedures such as drying may lead to decomposition, especially for free long-chain bases. Few chemically synthesized sphingolipids are available commercially and, when available, may contain natural and unnatural stereoisomers that differ in biological activity. Sphingolipids are sometimes finicky to handle. Gangliosides and other glycolipids with highly polar headgroups, as well as sphingosine 1-phosphate and many lysosphingolipids, are not readily extracted by organic solvents and, therefore, may be underestimated if all fractions are not analyzed. Some sphingolipids exhibit varying solubilities depending on the pH, the presence of other ions, the purity of the sample, and other conditions. Several strategies are used to deliver sphingolipids to cells. For the more hydrophobic sphingolipids (ceramides, sphingomyelin, glucosylceramide, etc.), it is necessary to dissolve them in water-miscible solvents (DMSO or alcohol) or detergents, and to add them in small volumes to minimize the effects of the solvent. Even so, this approach can give erratic results because the sphingolipids may aggregate upon dilution of the organic solvent, and become unavailable for cellular uptake. It is not uncommon for sphingolipids to precipitate upon addition to culture media; therefore, they will be lost if the media is filter-sterilized after adding the sphingolipid. Precipitafion can occur even with relatively water-soluble compounds such as long-chain bases. However, these compounds present an additional complication, which is that they may be so readily taken up by cells that the levels become high enough to disrupt the membranes. This is usually avoided by either adding them in low concentrations (in which case, repetitive addifion may be necessary if they are removed by metabolism), or after first preparing the albumin complex to facilitate solubility and to provide a "time release" delivery vehicle (Hannun et al., 1991). Delivery may be facilitated by delivering the sphingolipid to cells as part of liposomes or lipoprotein complexes, or by the use of analogues with shorter alkyl chains (for example, N-acetylsphingosine is often used in place of the very water-insoluble ceramides with longer fatty acids). When incorporating sphingolipids into liposomes and other particles, care should be taken to mix the other components thoroughly because most sphingolipids have saturated alkyl sidechains, which gives them high phase transition temperatures (Merrill and Nichols, 1985). In some cases (such as for gangliosides), sphingolipids form water-soluble micelles that are relatively easy to administer to cells (see examples cited earlier in this review). Another approach, if it is not necessary for the sphingolipid to be taken up by the cells (for example, for studies of binding by extracellular matrix proteins), is to bind sphingolipids to thin-layer chromatography
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(TLC) plates or the wells of microtiter plates and determine binding to the immobilized ligand. Dose-response relationships are much more complicated for hydrophobic ligands than for simple, water-soluble compounds. The relevant concentration is not the amount that has been added to the cells, but rather, the amount that partitions into cellular membranes (Hannun et al., 1991). This is most frequently noted when comparing the results of experiments with different numbers of cells, or in some instances, when comparing cells that differ greatly in the amounts of membranes and other lipids (Robertson et al., 1989). To compare the results among experiments, it is often helpful to express the results as the mole percentage of the added lipid versus the cellular lipids. The results can also be affected by the binding to serum proteins and lipoproteins. Other factors that warrant concern are whether the exogenously added sphingolipids undergo metabolism. If so (and this is often the case for added sphingolipids) (Trinchera et al., 1990; Ladenson et al., 1993), the response may be due to a metabolite rather than to the added sphingolipid per se. B. Effects of Adding Exogenous Agents That Bind or Chemically Modify Sphingolipids
One approach to studies of sphingolipids has been to add proteins that bind or hydrolyze the compound of interest. For example, the beta-subunit of cholera toxin binds ganglioside Gj^j (Buckley et al., 1990). A similar approach can be used with antibodies to glycolipids. It has also been useful to add extracellular neuraminidase to cleave gangliosides (Ogura and Sweeley, 1992), endoclycoceramidase (Slife et al., 1989), and sphingomyelinases (Okazaki et al, 1990; Kolesnick, 1991; Olivera etal., 1992). C. Studies With Metabolic Inhibitors
Metabolic inhibitors are now available for many of the early steps of sphingolipid metabolism, and these can also be used to explore the roles of sphingolipids in biological processes. The first enzyme of de novo sphingolipid biosynthesis is inhibited by cycloserine (Sundaram and Lev, 1984; HoUeran et al., 1990), haloalanines (Medlock and Merrill, 1988), and sphingofiingins (Zweerink et al., 1992) and ISPl (Myake et al., 1995). Although haloalanines are not specific for serine palmitoyltransferase, this enzyme appears to be one of the more sensitive targets of this compound (Medlock and Merrill, 1988). The acylation of long-chain bases by ceramide synthases can be inhibited by fumonisins (Wang et al., 1991), as has been described earlier in this review, and australifungin (Mandala et al., 1995). It should be borne in mind that these inhibitors not only blocks inhibition of complex sphingolipid formation, but also causes sphinganine to accumulate in cells. Radin's group has prepared a series of ceramidelike compounds that can be used to inhibit the initial step of glycolipid biosynthesis (Vunnam and Radin, 1980;
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Inokuchi and Radin, 1987), the most active of which is D-threo-1-pheny\-2-dQcanoylamino-3-moq3holine-1 -propanol (PDMP). At high concentrations, this compound also blocks sphingomyelin synthesis (Rosenwald et al, 1992). PDMP is relatively non-toxic in vivo, although administration to mice causes poor growth of kidneys and liver, with the kidney being the more sensitive organ (Shukla et al., 1991). Studies with Madin-Darby canine kidney cells (Shayman et al., 1991) found that PDMP causes ceramide accumulation and increases in free sphingosine. In addition to these systems, it warrants mention that sphingosine and related analogues are inhibitory for the de novo pathway for making sphingolipids (Van Echten etal, 1990). It would be useful to have selective inhibitors of sphingolipid turnover. Usuki et al. (1988a) used 2-deoxy-2,3-dehydro-N-acetylneuraminic acid to inhibit the sialidase activity of fibroblasts; copper II and ganglioside GM3 has been reported to inhibit the neutral sphingomyelinase (Lister et al., 1993). Sphingosine kinase in platelets and some tissues is inhibited by the threo-isomQV sphinganine (Buehrer and Bell, 1992). D. Genetic Approaches to Altering Sphingolipid Biosynthesis
Unfortunately, there have been few reports of the use of modem genetic approaches to studying sphingolipid metabolism and function. This is likely to change because mutants have been isolated for the initial enzyme ofde novo sphingolipid biosynthesis (serine palmitoyltransferase) in yeast (selected by auxotrophy for exogenously provided long-chain bases) (Dickson et al., 1990) and CHO cells (isolated as a temperature-sensitive activity) (Hanada et al., 1990, 1992).
IV. CRITERIA FOR EVALUATING THE RESULTS OF STUDIES OF SPHINGOLIPIDS The physical properties of lipids make it difficult to prove that a given molecule is responsible for the regulation of a biological function. As already discussed, the addition of lipids to cells (and their removal) is far from simple; furthermore, analyses of the endogenous amounts in whole cells are unlikely to give a true picture of the concentrations in the relevant intracellular compartments. When these factors are considered in the context of the complex biological events that have been presumed to be regulated in some way by sphingolipids (e.g., growth and differentiation), it is not surprising that so many fundamental questions remain unanswered. To be regarded as regulatory for a given process, a number of criteria must be met. 1. The sphingolipid(s) must cause biological effects when added to cells, preferably with structural specificity and, when possible, with supporting
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3.
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5.
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evidence that it is the molecule/7er se—not a metabolite^hat accounts for the biological activity. The sphingolipid(s) must be present in cells and change appropriately under the conditions that control the relevant biological response of the cells. For example, in the case of growth regulation of gangliosides, the compounds should increase and decrease in amounts concomitant with the growth status of the cell. The changes in sphingolipid amount and type should occur within a time frame consistent with its proposed biological function. Traditionally, compounds are described as second messengers only when the response is rapid. Disruption of the formation and/or removal of the bioactive specie(s) in vivo by antagonists, metabolic inhibitors, exogenously added enzymes, or genetic means should cause predictable changes in the cellular behaviors thought to be regulated by the sphingolipid. It should be possible t^-identify the downstream targets of the bioregulatory sphingolipids (i.e., protein kinases, protein phosphatases, etc.) and their activity should be closely tied to the levels of the putative sphingolipid mediators.
As described relatively briefly in this review, there appear to be three experimental systems where information is accumulating in each of these categories: the modulation of growth via effects of gangliosides on growth factor receptors; the mediation of the action of TNF via a sphingomyelin/ceramide cycle; and the pathophysiology of the diseases caused by fumonisins via disruption of the normal pathways of sphingolipid metabolism. While these are useful models for future studies, it should not be assumed that the only modes of action of sphingolipids would be via the classical signal transduction paradigms. Sphingolipids may have equally important effects on cell regulation by monitoring subtle properties of the cell surface and adjusting the behavior of the cells accordingly. It is also possible that bioactive "mediators" such as sphingosine might exert some of their regulatory effects by influencing the charge of the membrane surface, rather than by more restrictive lipid-protein interactions. Field effects can be very powerful in regulating the properties of membrane proteins and other biological processes, but they would not be defined as specific according to the current definition (i.e., the effects might be mimicked by other stereoisomers and compounds with very different structures as long as they could interact with the membranes in a similar way). In this context, it warrants comment that long-chain (sphingoid) bases are the only cationic lipids in mammalian cell membranes; therefore, nature may have capitalized on this fact to regulate many membrane-mediated events via a common mechanism.
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V. PERSPECTIVES FOR FUTURE RESEARCH O N SPHINGOLIPIDS A. Opportunities for the Discovery of Diseases Caused by Aberrant Sphingolipid Metabolism
Genetic defects in sphingolipid catabolism have long been known to cause a wide range of diseases (Sweeley, 1991). The pathology of these diseases has usually been attributed to cell disruption by the accumulating sphingolipids; however, it is also possible that some of these molecules, or others that are formed by side pathways (such as the accumulation of psychosine), contribute to the abnormal cell behavior by affecting these regulatory pathways (Hannun and Bell, 1987). Furthermore, recent findings with fumonisins have shown that these mycotoxins exert their toxicity, and perhaps carcinogenicity, through disruption of sphingolipid biosynthesis (Wang et al, 1991; Merrill et al, 1993). Other recent studies that may provide examples of genetic defects in sphingolipid biosynthesis include the reports by Boiron-Sargueil et al. (1992) that Trembler mice have very low levels of ceramide synthase, and by Goldin et al. (1992) that fibroblasts from patients with Niemann Pick's disease type C have unusually high amounts of sphinganine (which usually only appears in significant amounts as an intermediate of sphingolipid synthesis). Kendler and Dawson (1992) have found that hypoxic injury to rat oligodendrocytes causes ceramide to accumulate in the endoplasmic reticulum and Wright et al. (1996) have demonstrated that a tumor cell line is resistant to aptosis has a defect in sphingomyelinase activation. B. New Strategies for Preventing and Treating Disease Based on an Understanding of the Activities of Sphingolipids
As such bioactive molecules, sphingolipid present opportunities for the development of new types of dietary or pharmacologic agents to control aberrant cell behaviors. For example, sphingosine and other long-chain bases might be antitumor agents due to their inhibition of PKC (Hannun et al, 1986). Topical application of sphingosine blocked the induction of ornithine decarboxylase by phorbol esters in mouse skin (Gupta et al., 1988; Enkvetchakul et al, 1989), which is one biochemical marker of tumor promotion. But sphingosine did not reduce the number of tumors in a longer term study (Enkvetchakul et al., 1992). Since skin is already rich in sphingosine (Wertz and Downing, 1989) this might have diminished the impact of exogenous sphingosine addition. In a cell culture model of transformation (mouse C3H10T1/2 cells) (Borek et al., 1991), sphingosine and sphinganine reduced cell transformation in response to gamma irradiation and phorbol esters (PMA). Furthermore, Dillehay et al. (1994) have found that feeding sphingomyelin to mice treated with N,N-dimethylhydrazine (DMH) reduced the number of aberrant colonic crypts in short term studies and decreased the tumor incidence by approximately one-third. Long-chain bases
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have also been found to induce differentiation and, when used in combination with other agents that induce differentiation, result in cells with a more fully differentiated phenotype (Stevens et al., 1989, 1990b; Yung et al., 1992). Sphingosines, N-methyl sphingosines, and sphingosine 1-phosphate reduce tumor growth and metastasis (Sadahira et al., 1992). All together, these findings suggest that longchain bases still offer promise in the prevention and/or treatment of cancer. Based on the diverse signal transduction pathways affected by sphingolipids, one can envision that these compounds—or more likely new compounds based on the major pharmacophors of the bioactive sphingolipids—^will prove to be useful in the prevention and treatment of a wide range of diseases.
ACKNOWLEDGMENTS The authors thank the past and current students, postdoctoral fellows, and technicians who have been part of our investigations and to the many collaborators that have contributed substantially to our thinking about these systems, particularly R.M. Bell, D.F. Birt, C. Borek, G.A. Carman, D.L. Dillehay Y.A. Hannun, R.N. Kolesnick, J.D. Lambeth, W.R Norred, K. Sandhoff, S. Spiegel, D.E. Vance, G. Van Echten, and K.A. Voss. Thanks are also due Mrs. Winnie Scherer for help with the preparation of this review. The work by the authors has been supported primarily by NIH grants GM33369 and GM46368, USDA/ARS funds and USDA-NRI Competitive grants 91-37204-6684 and 92-022509, and funds from the National Dairy Council and the American Institute for Cancer Research.
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Inokuchi, J. & Radin, N.S. (1987). Preparation of the active isomer of l-phenyl-2-decanoylamino-3morpholino-l-propanol, inhibitor of murine glucocerebroside synthetase. J. Lipid Res. 28, 565— 571. Jamal, H., Martin, A., Gomez-Munoz, A., & Brindley, D.N. (1991). Plasma membrane fractions from rat liver contain a phosphatidate phosphohydrolase distinct from that in the endoplasmic reticulum and cytosol. J. Biol. Chem. 266, 2988-2996. Kanfer, J.N. & Hakomori, S.-I. (eds.) (1983). In: Sphingolipid Biochemistry: The Handbook of Lipid Research, Vol. 3, p. 485, Plenum Press, New York. Keenan, T.W., Schmid, E., Franke, W.W., & Wiegandt, H. (1975). Exogenous glycosphingolipids suppress growth rate of transformed and untransformed 3T3 mouse cells. Exp. Cell. Res. 92, 259-270. Kendler, A. & Dawson, G. (1992). Hypoxic injury to oligodendrocytes: Reversible inhibition of ATP-dependent transport of ceramide from the endoplasmic reticulum to the Golgi, J. Neurosci. Res. 31, 205-211. Kim, M.-Y., Linardic, C , Obeid, L., & Hannun, Y.A. (1991). Identification of sphingomyelin turnover as an effector mechanism for the action of tumor necrosis factor alpha and gamma-interferon. J. Biol. Chem. 266, 484-489. Kiss, Z., Crilly, K., & Chattopadhyay, J. (1991). Ethanol potentiates the stimulatory effects of phorbol ester, sphingosine and 4-hydroxynonenal on the hydrolysis of phosphatidylethanolamine in NIH 3T3 cells. Eur. J. Biochem. 197, 785-790. Kolesnick, R.N. (1991). Sphingomyelin and derivatives as cellular signals. Prog Lipid Res. 30, 1-38. Kolesnick, R.N. & Hemer, M.R. (1991). Characterization of a ceramide kinase activity from human leukemia (HL-60) cells. Separation from diacylglycerol kinase activity. J. Biol. Chem. 265, 18803-18808. Kriek, N.RJ., Kellerman, T.S., & Marasas, W.F.O. (1981). A comparative study of the toxicity of Fusarium verticillioides (= E moniliforme) to horses, primates, pigs, sheep and rats. Onderstepoort J. Vet. Res. 48, 129-131. Ladenson, R.C., Monsey, J.D., Allin, J., & Silbert, D.F. (1993). Utilization of exogenously supplied sphigosine analogues for sphigolipid biosynthesis in Chinese hamster ovary and mouse LM cell fibroblasts. J. Biol. Chem. 268, 7650-7659. Laine, R.A. & Hakomori, S.-I. (1973). Incorporation of exogenous glycosphingolipids in plasma membranes of cultured hamster cells and concurrent change of growth behavior. Biochem. Biophys. Res. Comm. 54, 1039-1045. Lin, M., Lu, S., Ji, C , Wang, Y., Wang, M., Cheng, S., & Tian, G. (1980). Experimental studies on the carcinogenicity of fungus-contaminated food from Linxian County. In: Genetic and Environmental Factors in Experimental and Human Cancer (Gelboin, H.V., ed.), pp. 139-148. Japan Sci. Soc. Press, Tokyo. Lister, M.D., Crawford-Redick, C.L., & Loomis, C.R. (1993). Characterization of the neutral pH-optimum sphingomyelinase from rat brain: Inhibition by copper II and ganglioside GM3. Biochim. Biophys. Acta 1165, 314^320. Marasas, W.F.O. (1982). Mycotoxicological investigations on com produced in oesophageal cancer areas in Transkei. In: Cancer of the Oesophagus (Pfeiffer, C.J., ed.). Vol. 1, pp. 29-40, CRC Press, Boca Raton. Marasas, W.F.O., Kellerman, T.S., Gelderblom, WC.A., Coetzer, J.A.W., Thiel, RG., & van der Lugt, J.J. (1988). Leukoencephalomalacia in a horse induced by frimonsin Bi isolated from Fusarium moniliforme. Onderstepoort J. Vet. Res. 55, 197-203. Marasas, W.F.O., Kriek, N.P.J., Fincham, J.E., & van Rensburg, S.J. (1984). Primary liver cancer and oesophageal basal cell hyperplasia in rats caused by Fusarium moniliforme. Int. J. Cancer 34, 383-387. Mathias, S., Dressier, K.A., & Kolesnick, R.N. (1991). Characterization of a ceramide-activated protein kinase: Stimulation by tumor necrosis factor alpha. Proc. Natl. Acad. Sci. USA 88,10009-10013.
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PHOSPHATIDYLSERINE DYNAMICS AND MEMBRANE BIOGENESIS
Pamela J. Trotter and Dennis R. Voelker
ABSTRACT : 300 I. INTRODUCTION 300 11. PHOSPHATIDYLSERINE STRUCTURE AND SYNTHESIS 301 A. Structure 301 B. Cytidine Diphosphate-Diacylglycerol Dependent Reactions 301 C. Cytidine Diphosphate-Diacylglycerol Independent Synthesis 303 D. Subcellular Localization of Eukaryotic Phosphatidylserine Synthase . . . 304 E. CouplingofPtdSer Synthesis to Calcium Sequestration 305 III. PHOSPHATIDYLSERINE METABOLISM AND INTERORGANELLE TRANSPORT 309 A. Phosphatidylserine Decarboxylase 309 B. Interorganelle Cooperation in Phosphatidylethanolamine Formation . . . 310 C. Phosphatidylserine Translocation From Endoplasmic Reticulum to Mitochondria 311 D. Mitochondrial Import of Exogenous Phosphatidylserine 320 E. Mitochondrial Export of Phosphatidylethanolamine 320 IV. INTRAMEMBRANE TRANSPORT OF PHOSPHATIDYLSERINE 321 A. Translocation within Intracellular Membranes 322
Advances in Lipobiology Volume 1, pages 299-335. Copyright © 1996 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-635-5 299
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B. Translocations Within the Plasma Membrane CONCLUDING REMARKS ABBREVIATIONS ACKNOWLEDGMENTS REFERENCES
323 328 329 329 329
ABSTRACT Phosphatidylserine (PtdSer) is a lipid of relatively low abundance, yet its anionic character, dynamic role in phospholipid biosynthesis, and activity in a number of diverse cellular processes makes it a vital component of biological membranes. PtdSer is synthesized by a cytidine diphosphate-diacylglycerol-dependent mechanism in bacteria and yeast, and a cytidine diphosphate-diacylglycerol-independent, Ca^"^-dependent reaction in mammalian cells. A number of mechanisms for the coupling of Ca^"^ transport and PtdSer synthesis can be proposed. Following inter-organelle transport between the endoplasmic reticulum and mitochondria, PtdSer can be decarboxylated to form phosphatidylethanolamine. Recent investigation of this interorganelle cooperation in intact and permeabilized cells and isolated organelles has provided new information about the energetics of the process and mechanisms of transport. Regulation of the distribution of PtdSer within the leaflets of both intracellular and plasma membranes has also been the focus of recent investigations and provided substantial new information about transbilayer lipid movement and its ATP and membrane protein dependence. This article will review recent findings about the synthesis and metabolism of PtdSer that have helped illuminate some of the mechanisms of membrane assembly.
L INTRODUCTION Phosphatidylserine (PtdSer) is a minor but readily detectable phospholipid present in the membranes of mammalian, yeast, and bacterial cells. This lipid contributes anionic character to eukaryotic membranes and also functions as an important intermediate in the synthesis of phosphatidylethanolamine (PtdEtn) in both prokaryotes and eukaryotes. In addition, PtdSer can function as an important cofactor for protein kinase C (PKC) and enzymes involved in the blood clotting cascade, and may also serve as an external cell surface signal for the clearance of effete blood cells. Recent evidence has revealed that PtdSer metabolism within eukaryotic cells encompasses a dynamic range of processes involved in its synthesis and interorganelle transport. Interest in this lipid as an indicator for intramembrane assembly processes, intermembrane lipid transport phenomena and interorganelle cooperation in membrane biogenesis is growling. This article will summarize some of the salient findings about the enzymology and metabolism of PtdSer and elaborate on several of the more recent observations that have engendered new enthusiasm for examining the intracellular metabolism of this lipid.
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II. PHOSPHATIDYLSERINE STRUCTURE AND SYNTHESIS A.
Structure
The presence of an a-amino nitrogen compound as a component of hydrolysis products of the cephalin fraction of brain tissue (a mixture of lipids containing nitrogen and phosphorus) was first demonstrated by Mac Arthur in 1914 (Mac Arthur, 1914). However, it was not until 1948 that Folch, utilizing data on cleavage products and reactivity of the purified compound, proposed a chemical structure for PtdSer (Folch, 1948). The positions of the phosphoserine moiety and the fatty acids on the glycerol backbone, however, remained unclear. In 1955, Baer and Maurakas successfully synthesized PtdSer by phosphorylation of a, P-distearin followed by esterification with L-serine to yield L-a-(distearoyl)-phosphatidyl-Lserine (Baer and Maurakas, 1955). Based on the comparison of the optical properties of the synthetic PtdSer and that which was isolated from cephalin and catalytically hydrogenated, the structure of naturally-occurring PtdSer was established to be of the a conformation. Thus, L-serine is esterified to the phosphate moiety of 1,2 diacyl-5«-glycero-3-phosphate. B. Cytidlne Diphosphate-Diacylglycerol Dependent Reactions
PtdSer can be synthesized either by cytidine diphosphate (CDP)-diacylglycerol dependent or independent (see below) reactions. The CDP-diacylglycerol dependent reaction was first elucidated in Escherichia coli as a logical consequence of the seminal observation by Kennedy and Weiss (1956) that cytidine nucleotide derivatives played an essential role in the synthesis of phosphatidylcholine (PtdCho). Kanfer and Kennedy (1962) provided the first direct evidence for CDP-diacylglycerol dependent PtdSer synthesis by demonstrating that an enzyme preparation from E. coli catalyzed incorporation of L-serine into PtdSer in a CDP-dipalmitin dependent reaction. In this PtdSer synthase reaction, the phosphatidyl group is transferred to the hydroxyl function of the L-serine acceptor concomitant with cleavage of the phosphodiester bond in CDP-diacylglycerol to yield PtdSer and cytidine monophosphate (CMP). The reaction is utilized by both bacteria and yeast (Steiner and Lester, 1972), but is not used in mammalian cells. The phosphatidylserine synthase (PSS) gene has been cloned, sequenced and overexpressed in both E. coli and Saccharomyces cerevisiae. Bacterial mutants in the PSS gene were first isolated using [^H]-serine suicide or in situ colony autoradiography techniques (Ohta et al., 1974; Raetz, 1976). These E. coli mutants exhibit temperature sensitive PtdSer synthesis and conditional lethality. At the permissive temperature PtdSer synthase activity is present, and PtdEtn (which is made by decarboxylation of PtdSer) is found at the normal level of-80% of total phospholipid. Incubation at nonpermissive temperatures results in loss of PSS activity, a drop in PtdEtn levels to 35% of total phospholipid, increased accumulation of phosphatidylglycerol (by 40%) andbisphosphatidylglycerol (fourfold), and
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cessation of cell division. The E. coli PSS gene was isolated based on its ability to correct the phenotype of a PSS mutant (Raetz et al, 1977). Overexpression of the E. coli gene leading to a 6- to 15-fold increase in PSS activity did not affect the phospholipid composition, indicating that the enzyme is not rate-limiting for PtdEtn synthesis in this organism. DeChavigny et al. (1991) have constructed null mutants by disrupting the PSS gene with a marker gene for kanamycin resistance (pss::kan). These strains require the presence of a plasmid encoding the PSS gene for routine growth. Under growth conditions that permit plasmid loss, the cells stop dividing when the PtdEtn level decreases to 30% of total phospholipid. Growth of both the temperature-sensitive and null mutants in the presence of Ca^"^ and Mg^"^ suppresses the deleterious effects of PtdEtn loss (Dowhan, 1992) . When the null mutants are grown in medium containing high concentrations of divalent cations, enzyme activity remains undetectable, PtdEtn content falls to an undetectable level, and phosphatidylglycerol and bisphosphatidylglycerol accumulate (DeChavigny et al, 1991). It has been suggested, based on these data, that PtdEtn serves as an uncharged structural component of the membrane and that the divalent cations nonspecifically counteract the negative charge of the membranes that lack PtdEtn. In addition, the divalent cations may act to facilitate formation of nonbilayer structures needed for membrane fusion events, which are normally generated by PtdEtn (Dowhan, 1992). Yeast mutants defective in PSS were originally isolated as choline or ethanolamine auxotrophs and given the designation cho 1 (Atkinson et al., 1980). The reason that mutants in PSS are choline auxotrophs is a consequence of the multiple metabolic pathways that produce PtdEtn and PtdCho in yeast. In the absence of ethanolamine and choline, yeast synthesize PtdCho from PtdSer first by decarboxylation to form PtdEtn, and then methylation of this latter product to form PtdCho. In the absence of PSS activity, yeast therefore require ethanolamine or choline to make PtdCho. The PtdSer synthase enzyme has been purified to homogeneity and characterized (Bae-Lee and Carman, 1984). The yeast PSS gene was cloned by genetic complementation of cho 1 strains (Letts et al., 1983; Nikawa et al., 1987). Constructs of the PSS gene disrupted with a selectable nutritional marker gene were used to create null mutations in the PSS gene. Such mutations are lethal unless the cells are supplemented with ethanolamine, mono- or di-methylethanolamine or choline (Bailis et al, 1987; Hikiji et al., 1988). The null mutants fail to make PtdSer and provide clear evidence that this phospholipid is dispensable in yeast. Growth of the PSS null mutants in the presence of choline revealed that the cells synthesize low but significant levels of PtdEtn, suggesting the presence of a pathway for PtdEtn formation in yeast that bypasses the PtdSer precursor (Hikiji et al., 1988). The synthesis of PtdEtn in such strains may be via the degradation of sphingolipids (Stoffel et al., 1968; Atkinson, 1984). The activity of yeast PSS can be regulated transcriptionally and posttranslationally via phosphorylation by cyclic-AMP dependent protein kinase (Kinney and Carman, 1988) in response to changing levels of choline and inositol (BaiHs et al., 1992).
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C. Cytidine Diphosphate-Diacylglycerol Independent Synthesis
Shortly after the description of the enzymology of PtdSer synthesis in E. coli, it became apparent that the mechanism for synthesis of this Hpid in mammahan cells was fundamentally different. In 1959, Hubscher (Hubscher et al., 1959) initially described a Ca^"^ dependent incorporation of radiolabeled L-serine into phospholipids by membranes derived from rat liver. Further characterization by Hubscher (1962) showed that the enzyme was primarily located in the microsomal (endoplasmic reticulum (ER)) fraction. The enzyme activity was reported to require millimolar concentrations of Ca^"^, have a K^ for L-serine of approximately 0.5 mM, a pH optimum between 8 and 9, and did not require ATP or cytidine nucleotides or their derivatives for catalysis. Later investigation of this reaction revealed that not only serine, but also choline and ethanolamine could be incorporated into phospholipid in Ca^"^ dependent reactions that were also independent of the presence of cytidine nucleotides (Borkenhagen et al., 1961; Kanfer, 1972; Bjerve, 1973a). Kanfer (1972) reported that incubation of particulate fractions prelabeled with radioactive serine, choline, or ethanolamine with the corresponding unlabeled amino alcohols resulted in the liberation of radioactivity into the aqueous phase. Thus, the reaction was that of an exchange of free serine, choline or ethanolamine for these same moieties present in lipids. The generic term base exchange became a convenient way to describe the collective enzyme activities. The base exchange pathway provides the major route for PtdSer biosynthesis in mammalian cells, while synthesis of PtdCho and PtdEtn via these enzyme reactions is considered to be negligible (Kanfer, 1980). In marked contrast to PtdSer, PtdCho and PtdEtn are principally synthesized by other reaction schemes, the most notable of which respectively utilize CDP-choline or CDP-ethanolamine, and diacylglycerol as substrates. Based on data from competitive inhibition experiments, Bjerve (1973b) was the first to suggest that the base-exchange reactions of L-serine, choline, and ethanolamine are not catalyzed by a single enzyme. From pulse-chase data, Bjerve (1973b) proposed that the base-exchange reactions could be divided into two activities. Subsequently, successful solubilization and chromatographic resolution of base exchange activity (Taki and Kanfer, 1978) provided evidence that two enzymes were involved. One enzyme exchanges L-serine, choline, and ethanolamine and one exchanges L-serine and ethanolamine. Kuge et al. (1986) have proposed that the enzymes be referred to as PtdSer synthase I and PtdSer synthase II, respectively. The PtdSer synthase II activity has been purified to homogeneity (Suzuki and Kanfer, 1985) but sequence information and isolation of the cDNA have yet to be reported. Somatic cell mutants defective in PtdSer synthesis have been isolated using either in situ autoradiography for ATP-independent incorporation of [^Hjcholine into lipid (Kuge et al., 1985) or screening for ethanolamine auxotrophy (Voelker and Frazier, 1986). Mutant cell lines isolated by either technique are PtdSer auxotrophs and
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appear to be defective in the enzyme that exchanges serine, choline, and ethanolamine. Chinese hamster ovary cell (CHO) mutants defective in choline exchange activity (Kuge et al., 1985) also had a 50% decrease in both L-serine and ethanolamine exchange activity, and markedly reduced biosynthesis of PtdSer. However, despite the evident defect in choline-exchange, the cells continued to exhibit normal levels of PtdCho synthesis. The authors concluded that the choline-exchange enzyme primarily functions in whole cells to synthesize PtdSer by L-serine exchange, and that its contribution to total PtdCho biosynthesis in CHO cells is insignificant. Kuge et al. (1991) were able to isolate a cDNA clone that complemented the defect in the mammalian PtdSer synthase I and the DNA sequence of this clone has been determined. Voelker and Frazier (1986) have isolated a mutant from CHO cells that requires exogenous ethanolamine or PtdSer for growth. The mutant has markedly depressed levels of incorporation of L-serine into PtdSer, and in the absence of ethanolamine a significant decrease in cellular PtdSer was observed. The defect was demonstrated to be primarily in PtdCho-dependent PtdSer synthesis, although PtdEtn-dependent synthesis was also reduced (-20%). These findings support the possibility that at least one of the mammalian PSS enzymes is capable of utilizing both PtdCho and PtdEtn as a donor, while the other is specific for PtdEtn. D. Subcellular Localization of Eukaryotic Phosphatidylserine Synthase The initial localization of mammalian PtdSer synthase was to the microsomal membrane fraction (Hiibscher, 1962). Marker enzyme analysis of the subcellular fractions indicated that the major site of serine incorporation into lipid was the ER with some activity present in fractions enriched with the Golgi apparatus (Jelsema and Morre, 1978; Vance and Vance, 1988). There is also an association of enzyme activity with the nuclear fraction that is likely a consequence of the tight association of some elements of the ER with the nuclear membrane. Recently, a subcellular fraction expressing high levels of PtdSer synthase and enriched in glucose 6-phosphatase but markedly reduced in other characteristic ER marker enzymes, such as CTP:phosphocholine cytidyltransferase and NADPH cytochrome c reductase, has been identified by Vance (1990). This membrane fraction has PSS specific activities that are two to three times the levels found for typical microsomal fractions. A provocative observation about this membrane fraction is that it co-sediments with mitochondria prepared from liver by routine methods. In contrast, density gradient fractionation using self-forming PercoU gradients allows for the separation of this population of membranes enriched in PtdSer synthase away from mitochondria. In rat liver this membrane fraction, denoted fraction X by Vance (1990) and referred to as MAM (for mitochondria associated membrane) in this chapter, has recently been shown to express a novel PtdEtn methyltransferase enzyme that has until now gone unrecognized (Cui et al., 1993).
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There is currently much interest in the MAM fraction and speculation that this membrane may play a specialized role in the synthesis of PtdSer that is destined for transport to the mitochondria. The exact physical interaction between MAM and the mitochondria remains to be resolved, but there is clear evidence from electron microscopy that elements of the ER can often be found in close association with the mitochondria (Lewis and Tata, 1973). Whether these associations are a consequence of structural linkage or random chance distribution has not been determined. As will be discussed later, there is some biochemical evidence to suggest that specific physical associations between MAM and the mitochondria exist. In S. cerevisiae the pattern of subcellular distribution of the PSS enzyme has been complicated to resolve. Initial localization studies suggested that the mitochondrial outer membrane was the likely membrane where the enzyme resided (Kuchler et al., 1986). However, subcellular fi-actionation in yeast is not simple, and there is pronounced difficulty in obtaining relatively pure preparations of ER. The most recent data provide evidence that the yeast PtdSer synthase is associated with a specialized membrane fraction that may have some characteristics of the ER but which has a propensity to sediment with the mitochondria (Zinser et al, 1991). As in the case of mammalian cells, there is provocative evidence for the existence of a specialized organellar system which may be a transitionary membrane between the ER and the mitochondria. While the evidence for this membrane system in both yeast and animal cells is solid, it is also scant. Additional morphological and biochemical studies are needed to provide more information to characterize this population of membranes in eukaryotes. E. Coupling of PtdSer Synthesis to Calcium Sequestration
One curious aspect of the enzymology of mammalian PtdSer synthesis has been the requirement for high levels of Ca^"^ (10 mM) necessary to drive the reaction (Hiibscher, 1962). These levels are essentially five orders of magnitude higher than the Ca^"^ levels present in the cytosol of mammalian cells. The disparity between the cytosolic levels of Ca^"*" and those necessary to effect catalysis has recently been resolved using permeabilized animal cells (Voelker, 1990). In permeabilized CHOKl cells the synthesis of PtdSer was shown to require ATP at 400 nM Ca^^ (levels that are close to those found in most resting cells). The requirement for ATP was essentially absolute but could be circumvented by the addition of 1 mM Ca^"^. The interrelationship between Ca^"^ and ATP requirements for PtdSer synthesis was made clear by examining the coupling between PtdSer synthesis and Ca^"^ sequestration using several inhibitors that either directly or indirectly affect the activity of the microsomal Ca^"^-ATPase (see Figure 1). At physiological intracellular Ca^"^ levels, the ATP requirement for PtdSer synthesis is essentially absolute. However, if the Ca^"^ is chelated with excess [ethylenebis (oxyethylenenitrilo)] tetraacetic acid (EGTA), PtdSer synthesis is completely
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2+ EGTA (t-Bu)2HQ Thapslgarginf lonomycin
ENDOPLASMIC RETICULUM
Ser
i^ATP
Cs I
2+
▼ RdSer
ATPJ, EXPORT TO OTHERl
|-j RESTRICTED TRANSPORT (
Figure 1, Biochemical events in the synthesis and metabolism of PtdSer in eukaryotic cells. PtdSer is synthesized via a reaction that is coupled to ATP dependent Ca^"^ sequestration in the ER or mitochondria associated membrane. The sequestration of Ca "^ can be inhibited in vitro by chelators (EGTA), inhibitors of the Ca "^ ATPase (2,5-di(ferf)butyl hydroquinone, (t-Bu)2HQ), and thapsigargin), and Ca^"^ ionophores (ionomycin). Nascent PtdSer is transported to the mitochondrial outer membrane by reactions requiring ATP, and by a mechanism that is restricted to autologous mitochondria. Translocation of PtdSer from the outer to the inner mitochondrial membrane is ATP independent but is inhibited by adriamycin and likely occurs at inner membrane-outer membrane contact sites. At the inner membrane the PtdSer is decarboxylated to form PtdEtn v^hlch is efficiently exported to other cellular organelles. In yeast the process is believed to be similar except that PtdSer synthesis is independent of Ca "^ sequestration and utilizes CDP-diacylglycerol and Ser as substrates.
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arrested. A second agent that arrests the ATP-dependent synthesis of PtdSer is di-tertiary butyl hydroquinone, a compound that has marked specificity for the microsomal Ca^"^ sequestering ATPase (Moore et al., 1987; Kass et al., 1989). Thapsigargin, an agent that is more specific for the ATPase (Thastrup et al, 1990), also efficiently arrests PtdSer synthesis. A third class of agent that inhibits ATP-dependent PtdSer synthesis is ionomycin which functions to effectively cause Ca^"^ that is pumped into the lumenal compartment of the ER and closely related membranes to leak back out (Liu and Hermann, 1978). Collectively these results indicate that the synthesis of PtdSer is coupled to ATP-dependent Ca^"^ sequestration phenomena mediated by the thapsigargin sensitive Ca^"^ ATPase (Thastrup et al., 1990). The ATPase can concentrate Ca^"^ in the lumen of the ER to levels in the millimolar range (Moore et al., 1975; Brattin et al., 1982). The findings with permeabilized cells have been corroborated using microsomal membranes derived from rat liver (Voelker, unpublished observations). The realization that PtdSer synthesis is coupled to Ca^"^ sequestration by the ER (and probably closely related membranes such as MAM that are also involved in PtdSer synthesis) raises several basic questions about this phenomenon regarding the topics of the regulation of the coupling, the topology of the processes, and the mechanisms of catalysis by PtdSer synthase. Although the data are reasonably good to indicate that concentration of Ca^"^ in the lumen of the ER can drive PtdSer synthesis, it is not clear if there is any coordination of the action of the Ca^"^ ATPase and PtdSer synthase. It would be interesting to determine if PtdSer depleted membranes have enhanced rates of Ca^"^ transport or if modulation of Ca^^ transport rates affects the initial rates of PtdSer synthesis. In addition, the stoichiometry of Ca^^ transport and PtdSer formation remain unknown parameters that may supply insight into the relationship between the two processes. Studies with permeabilized cells in vitro and intact cells in vivo, however, provide a clear connection between Ca^"^ sequestration and the rates of PtdSer formation. The studies with intact cells also highlight the fact that mobilization of ER Ca^"^ stores by agents such as inositol-tris phosphate leads to a clear decrease in the rates of PtdSer synthesis (Pelassy et al., 1992). Whether this situation also serves a role in transiently altering membrane composition for some ulterior regulatory purpose is unclear. Although PKC isozymes require PtdSer for activation (Bell and Bums, 1991), it would seem that the decrement in PtdSer synthesis would be too small to aher kinase activity. Nonetheless, additional work is required to discern if the reduction in PtdSer synthesis that accompanies Ca^"^ mobilization is merely a bystander effect or has other consequences. 7. Mechanisms of Coupling
Work by Bell and colleagues (Bell et al., 1981) on the topology of lipid synthetic enzymes in the ER provided evidence that the mammalian PtdSer synthase enzymes possess peptide sequences that are important for catalysis on the cytosolic face of the membrane. The topology experiments used the criteria of proteolytic inactiva-
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tion of catalysis to infer structural information. While such studies are an excellent first approximation of domain organization in the absence of specific structural information, the interpretation of the results must be tempered with caution. The coupling of Ca^"*" sequestration within the lumen of the ER to the activity of PtdSer synthase raises critical questions about the topography of the enzyme and its mechanism of catalysis, as outlined in Figure 2. If Ca^"^ is required at high concentrations at the active site of the enzyme in addition to serine, a simple model would suggest that the active site of PtdSer synthase may reside within the lumen of the ER (Figure 2a). Such a model for enzyme structure and function would therefore imply that serine, as well as Ca^"^, must be imported into the lumen of the ER to accomplish catalysis. For the collective base exchange activities that also include synthesis of PtdEtn and PtdCho, this model further implies transport of ethanolamine and choline into the lumen of the ER. A less simplistic model might invoke a two site mechanism whereby lumenal Ca^"^ induces structural changes in the enzyme that effect catalysis on the cytosolic surface of the membrane. This model obviates the need for secondary transport systems for serine, choline, and ethanolamine. Clearly, structural information about the enzyme with regard to soluble substrate binding sites, lipid substrate binding sites and Ca^^ binding sites coupled with topological studies defining the location of peptide domains are necessary to provide evidence for such a model. A more sophisticated model for catalysis incorporates the chemical driving force of sequestered Ca^^ into the reaction mechanism (Figure 2b). In a resting cell, the ratio of Ca^"^ in the lumen of the ER to that in the cytosol is 10"^ (Moore et al., 1975; Brattin et al., 1982). Such a Ca^^ gradient corresponds to a chemical potential of 5.7 kcal/mole or 0.74 ATP equivalents. Chemiosmotic theory clearly predicts that such Ca^"^ gradients should be adequate to accomplish the formation of PtdSer by cleavage and reformation of a phosphomonoester bond (Harold, 1986). The energetics for catalysis could be acquired either by a coupling factor or directly incorporated into the PtdSer synthase enzyme if it also functioned as a transport channel that could harness the energy of Ca^"^ transit down a concentration gradient. A somewhat less intuitive model that incorporates features of the chemiosmotic model and the other models is that the PtdSer synthase functions as an export channel for Ca^"^ leak out of the ER and that the local concentration of Ca^^ at the cytosolic domain of the enzyme is sufficient to effect catalysis (Figure 2c). In principal the Ca^"^ concentration within the putative export channel, or at a site adjacent to the channel, would effectively be the same as that within the lumen of the ER. The availability of the cDNA for PtdSer synthase I (Kuge et al., 1991) and the Ca^^ sequestering ATPase should provide the biochemical raw material to begin to address some of the above issues by direct experimentation. Reconstitution experiments with recombinant PtdSer synthase should help to better define the topology of the enzyme and its relationship with Ca^"^ transport phenomena.
Phosphatidylserine
309
Metabolism and Translocation
ER LUMEN
Ca
r^ mmmmmmmmmmm
ATPase
mmmmmm^m
Ca 2+
Figure 2. Models for coupling between PtdSer synthase and the Ca sequestering ATPase of the ER. (a) Ca^"^ and serine may interact with the enzyme active site at the lumenal face of the ER. Alternatively high lumenal Ca^"^ may induce structural changes in the enzyme that facilitate catalysis at the cytosolic surface, (b) The driving force for the condensation of serine with the phosphatidyl moiety derived from PtdCho or .2+ down a concentration .entrdLiun gradient giduieiii (Ca v^d PtdEtn may come from the movement of Ca^"^ 2+ cytosol alO^). (c) Local concentration of Ca^"^ within a protein channel lumen:Ca^"^ may effect catalysis on the cytosolic surface and at the same time function as an export route for the ion.
III. PHOSPHATIDYLSERINE METABOLISM AND INTERORGANELLE TRANSPORT A.
Phosphatidylserine Decarboxylase
The enzyme catalyzing the conversion of PtdSer to PtdEtn, PtdSer decarboxylase, v^as first identified in bacteria (Kanfer and Kennedy, 1962). Consequently the bacterial enzyme is the most studied and best understood. Of particular note is the finding that the bacterial decarboxylase is a pyruvoyl enzyme (Sartre and Kennedy, 1978). The pyruvate moiety arises from an autoproteolytic processing of a precursor enzyme into a large and small subunit (Li and Dowhan, 1990). The large subunit has a molecular mass of 35 kDa and the small subunit has a mass of 7 kDa and contains an amino terminal pyruvoyl moiety. The carbonyl function of the pyruvate forms a Schiff's base with the amino group of PtdSer and functions in essentially the same manner as pyridoxal prosthetic groups in effecting the elimination of the CO2 moiety in amino acid decarboxylases (van Poelje and Snell, 1990). Eukaryotic PtdSer decarboxylases have been localized to the mitochondria (Dennis and Kennedy, 1972; Van Golde et al, 1974; Kuchler et al., 1986). Subfractionation of mitochondria and proteolytic treatment of intact mitochondria
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PAMELA J. TROTTER and DENNIS R. VOELKER
and mitoplasts has been used to localize the enzyme to the inner mitochondrial membrane (Van Golde et al., 1974; Zborowski et al., 1983; Kuchler et al, 1986). Further sub fractionation of the mitochondria from rat liver has led to the suggestion that the decarboxylase may be enriched at contact sites between the inner and outer mitochondrial membranes (Ardail et al., 1991). However, Hovius et al. (1992) were not able to find direct evidence that the decarboxylase is located at contact sites. A partial cDNA sequence has been obtained for the PtdSer decarboxylase from mammalian cells, but this construct does not contain the critical 5' region that encodes important amino acid residues necessary for mitochondrial targeting of the newly translated protein. Two groups have independently cloned and sequenced the yeast gene for the decarboxylase (Trotter et al., 1993; Clancey et al., 1993). The deduced amino acid sequence of the yeast enzyme shows 28% position identity with the bacterial enzyme and 43% position identity with the predicted partial sequence from the mammalian cDNA. The site of the large-small subunit cleavage is preserved across all three species, indicating that the yeast and mammalian enzymes, like their bacterial counterpart, are pyruvoyl enzymes. B. Interorganeile Cooperation in Phosphatidylethanolamine Formation
The existence of multiple pathways for the synthesis of PtdEtn has long been recognized. One pathway originally described by Kennedy and Weiss (1956) utilizes CDP-ethanolamine and diacylglycerol as co-substrates in the reaction catalyzed by ethanolamine phosphotransferase. This latter reaction appears to occur primarily in the ER and requires an extracellular supply of free ethanolamine. The second pathway for PtdEtn formation is the decarboxylation of PtdSer described above that occurs at the inner mitochondrial membrane. Initially most of the interest of biochemists focused on the microsomal ethanolamine phosphotransferase in mammalian cells. However, the observation that the decarboxylase enzyme can actually play a major role in PtdEtn synthesis in some mammalian cells (Voelker, 1984), and the realization that the segregated subcellular localization of PtdSer synthase and PtdSer decarboxylase could allow one to use PtdSer metabolism as an indicator reaction for the interorganeile transport of phospholipid (Voelker, 1984, 1985), has sparked an increasing number of investigators to focus on the decarboxylase enzyme and its dynamic position in metabolism. The utility of the decarboxylase enzyme as an indicator of lipid transport processes in yeast has also been appreciated (Simbeni et al., 1990). Since PtdEtn is an abundant component of virtually all cell membranes, its biosynthesis from a PtdSer precursor has important implications for interorganeile lipid movement. As stated above, PtdSer is synthesized primarily in the ER and/or MAM, while the decarboxylation of this lipid occurs at the inner mitochondrial membrane. Thus in the absence of exogenous ethanolamine, all cellular organelles must derive their PtdEtn via the following steps: (a) PtdSer synthesis in the ER or MAM, (b) PtdSer transport from its site of synthesis to the mitochondria, (c) PtdSer
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import from the outer to the inner mitochondrial membrane, (d) decarboxylation of PtdSer at the inner mitochondrial membrane, (e) export of nascent PtdEtn from the inner mitochondrial membrane to the outer membrane, and (f) transport of PtdEtn from the outer mitochondrial membrane to other cellular organelles such as the ER, Golgi apparatus, plasma membrane, and lysosomes. The following sections will highlight investigations of the above processes tracing the metabolism of PtdSer and PtdEtn. The main events in this metabolic journey taken by PtdSer are outlined in Figure 1. C. Phosphatidylserine Translocation From Endoplasmic Reticulum to Mitochondria 1. Intact Cells
The synthesis and translocation of PtdSer from the ER to the mitochondria has been examined in intact mammalian cells by examining the metabolism of radiolabeled serine (Voelker, 1984, 1985). By following the incorporation of the serine precursor first into PtdSer and subsequently into PtdEtn, one measures discrete events taking place at the ER (and/or MAM) and the inner mitochondrial membrane, respectively. Pulse chase experiments in mammalian cells using [^H]serine reveal that a significant fraction of nascent PtdSer turns over to form PtdEtn. When a combination of NaN3 plus NaF are added at the end of the pulse labeling period, the conversion of PtdSer to PtdEtn is arrested indicating that ATP is required for some step in the process. Since the PtdSer decarboxylase enzyme has no requirement for ATP and is not inhibited by NaN3 and NaF, the results suggest that the inhibitors are acting at a transport step. Comparative analysis of subcellular fractions derived from metabolically poisoned and control cells shows that PtdSer accumulates in the microsomal fraction of the poisoned cells. This observation demonstrates that ATP depletion blocks the translocation of newly made PtdSer between its site of synthesis and the inner mitochondrial membrane. In addition to metabolic poisons that block ATP formation, inhibitors of protein synthesis also alter PtdEtn formation from a PtdSer precursor (Voelker, 1985). Cycloheximide is the most effective, essentially mimicking the results obtained with NaN3 plus NaF treatment of intact cells. The precise mechanism by which cycloheximide works to alter the translocation dependent decarboxylation of PtdSer remains unknown but suggests that there may be some interrelationship between protein and lipid import into mitochondria. These effects appear specific for cycloheximide, however, since inhibition of protein synthesis with puromycin only marginally decreases PtdEtn formation from PtdSer. Pulse-chase experiments examining PtdSer metabolism to PtdEtn have also been performed in yeast cells and provided evidence that ATP may also be required at a putative translocation step (Gnamusch et al, 1992). However, the results obtained with yeast do not provide as clear a definition of the ATP requirement, because the inhibition of PtdEtn formation is not as dramatic as the result obtained with
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mammalian cells. Furthermore, these experiments do not provide evidence for the accumulation of PtdSer in an ER or MAM fraction at the expense of PtdEtn formation in the mitochondria. Despite these shortcomings, a general picture is emerging to suggest that yeast and mammalian cells may use mechanistically similar processes to transport newly synthesized PtdSer to the mitochondria. This latter idea is important as the yeast system provides powerful investigative advantages over mammalian cells with respect to genetic experimentation. The complementation of biochemical experiments with a genetic approach to the investigation of lipid translocation holds promise for more rapidly advancing our understanding of the processes. The initial finding that PtdSer translocation processes could be arrested by ATP depletion at the level of the whole cell (Voelker, 1985) did not fit with the (then prevailing) general hypothesis that phospholipid exchange/transfer proteins were essential components in intracellular lipid transport. In vitro, lipid transfer proteins exhibit no ATP dependence (Wirtz and Gadella, 1990). Consequently the results of the ATP depletion experiments raised questions about the activity of the nonspecific lipid transfer protein (this was the protein implicated by in vitro studies to be involved in PtdSer transport) in mammalian cells under the conditions of the experiment. Measurement of nonspecific lipid transfer protein activity revealed that treatment of cells with NaNj plus NaF failed to alter the transfer of PtdSer subsequently measured in extracts from these cells. Independent examination of this result using cell lines with greatly diminished levels of nonspecific transfer protein gave results consistent with the conclusion that PtdSer translocation to the mitochondria is independent of the activity of this protein (Van Heusden et al., 1990). Collectively, the results obtained with intact cells set the stage for reconstitution of the ATP dependent translocation process in vitro. 2, Isolated Organelles The initial system used for reconstituting PtdSer translocation consisted of rat liver microsomes, utilized as a source of PtdSer synthase and as the donor membrane population, and rat liver mitochondria, which functioned as the acceptor membranes that contained the decarboxylase enzyme (Voelker, 1989b; Vance, 1990). The subcellular fractions from rat liver were chosen since they are reasonably well characterized and can be isolated in large quantities. Ptd[^H]Ser was first synthesized in the microsomal membranes using the resident PtdSer synthase. The radiolabeled microsomes were subsequently washed free of the [^H]serine precursor and Ca^"^ cofactor by centrifugation and incubated with mitochondria. The Ptd[^H]Ser synthesized in the microsomal compartment was readily transferred to the mitochondria and decarboxylated to form PtdEtn. Analysis of the latency of cytochrome c oxidase provided clear evidence that mitochondrial integrity was preserved during incubation with microsomes. The results were most consistent with a collision-based transfer of the newly synthesized Ptd[^H]Ser present in the
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microsomes to the mitochondria (presumably the outer aspect of the outer membrane). These early studies established other important criteria of the transport process with respect to the properties of the donor and acceptor compartments. Microsomes were completely dispensable as the donors. Liposomes of mixed lipid composition containing radiolabeled PtdSer functioned as well as microsomes, implying that elements critical for mitochondrial uptake of PtdSer resided principally with the mitochondria. The uptake process was saturable and dependent upon the absolute concentration of PtdSer in the donor membranes. Further examination of the decarboxylase part of the reaction sequence using detergent solubilization established that the rate limiting step in the overall sequence of events preceded the action of the decarboxylase at the inner membrane. This observation indicated that the rate limiting step in the reconstituted system was either at the level of PtdSer transfer to the outer membrane or between the outer and inner membrane. Since mitochondria fail to accumulate significant levels of PtdSer in the outer membrane in vivo (Colbeau et al., 1971; Jelsema and Morre, 1978), the results favor transfer between the donor and acceptor membrane as the rate limiting step. Of particular importance in studies using reconstitution of transport with isolated organelles was the finding that the process was independent of mitochondrial membrane potential, and the addition of ATP and cytosol. This result contrasts sharply with studies of reconstituted protein transport among isolated organelles which in crude preparations exhibit an absolute dependence upon ATP and cytosol (Balch, 1989). The results are consistent with those observed in intact cells which implied that the cytosolic nonspecific lipid transfer protein was not an obligatory component of the lipid transfer process (Voelker, 1985; Van Heusden et al., 1990). Despite the fact that the nonspecific lipid transfer protein is not required for PtdSer translocation, it can accelerate the rate of translocation between microsomes or liposomes and mitochondria in the in vitro assay (Voelker, 1989b; Hovius et al., 1992; Simbeni et al., 1993). This result further implies that the rate limiting step in the reconstituted translocation assay is the transfer of lipid from the donor membranes (either microsomes or liposomes) to the outer mitochondrial membrane. Effective enhancement of the PtdSer translocation from prelabeled microsomes to mitochondria requires purified protein at relatively high concentrations. The above sequence of events is true for yeast cells as well as mammalian cells (Simbeni et al., 1993). PtdSer generated in preparations of yeast mitochondria in vitro is also imported to the mitochondrial inner membrane and decarboxylated to form PtdEtn (Simbeni et al., 1990). Reassessment of the components of the mitochondrial membrane preparations in yeast suggests that they are contaminated by a MAM-like fraction that harbors the PtdSer synthase (Vance, 1990; Zinser et al., 1991). As described for mammalian cells (Voelker, 1989b), the import process for PtdSer between the outer and inner membrane did not require a membrane potential or ATP (Simbeni etal., 1993).
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The pool of PtdSer (formed either in intact cells or subcellular fractions) that is transferred to the mitochondria may not be homogenous. Pulse chase experiments with intact cells indicate that PtdSer turnover to form PtdEtn can proceed with a t j/2 as long as seven hours (Voelker, 1984). This turnover time (which is proportional to an Intermembrane transit time) seems inordinately long in comparison with values obtained for other phospholipids and sphingolipids. These estimates of PtdSer turnover time assume a single homogenous pool of the lipid. This assumption, however, is clearly an over-simplification of the system. Bjerve, using hepatocytes, provided evidence for multiple pools of PtdSer within living cells (Bjerve, 1985). Intuitively this idea is logical, since most organelle membranes contain PtdSer and the entire pool cannot be destined for decarboxylation immediately after synthesis. Using double labeling protocols, Bjerve has argued that newly synthesized PtdSer is more likely to be decarboxylated than older pools of the lipid. The "older" pools may simply represent PtdSer that has been targeted to organelles other than mitochondria and therefore recycling may be required before the lipid is available (if at all) for decarboxylation. Vance has reached similar conclusions using isolated organelles and the reconstituted system for the synthesis, translocation, and decarboxylation of PtdSer (Vance, 1990). 3.
Permeabilized Cells
There has remained a significant disparity between reconstituted PtdSer translocation with isolated organelles (mitochondria, microsomes, and MAM) and events with the intact cell regarding the energy dependence of the translocation process. While depletion of ATP in intact cells arrests PtdSer translocation to the mitochondria (Voelker, 1985), in vitro reconstitution using isolated organelles has not been shown to require ATP (Voelker, 1989b; Vance, 1990; Ardail et al., 1991; Hovius et al., 1992; Simbeni et al., 1993). Studies with permeabilized cells constitute an approach to the problem that possesses experimental advantages of both the intact cell and isolated organelles. The dramatic progress made in dissecting interorganelle protein transport in permeabilized cells (Balch, 1989) attests to the validity and power of this experimental approach. A key attribute of permeabilized cells is that they retain elements of structural organization critical to cellular functions that are lost upon the classical preparation of subcellular organelles. The initial studies of PtdSer transport to the mitochondria in permeabilized cells demonstrated that PtdSer synthesized in intact cells could subsequently be transported to the mitochondria after the cells were permeabilized (Voelker, 1989a). These studies also provided strong evidence that the translocation of PtdSer to the mitochondria required ATP, corroborating the results obtained with intact cells. Refinement of the permeabilized cell system revealed a number of important properties about PtdSer synthesis and its translocation to the mitochondria (Voelker, 1990, 1991, 1993). As described earlier in the chapter (see section II. E), the synthesis of PtdSer in permeabilized cells was completely dependent upon the addition of ATP when the cells were maintained at physiological levels of intracel-
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lular Ca^"^. This ATP requirement is now known to be necessary to drive Ca^"^ sequestration by the ER or MAM (Voelker, 1990). The elevated lumenal concentration of Ca^"^ functions by presently unknown mechanisms to effect catalysis by PtdSer synthase (see Figure 1). The synthesis of PtdSer is relatively stable in permeabilized cell preparations and can be linear for periods up to two hours in the presence of ATP and a regenerating system. The relative durability of this system has made it available to a number of experimental manipulations that have proved useful in sorting out some of the steps in the transport process. 4.
Phosphatidylserine Translocation Is Independent of Synthesis
One important problem that the permeabilized cells were used to address was the role of active lipid synthesis in PtdSer transport from the ER or MAM to the mitochondria. The coupling of PtdSer synthase to Ca^"^ sequestration provided a convenient means to examine this question since agents that arrested Ca^"^ sequestration could be used to completely inhibit the coupled synthesis of PtdSer. In such experiments the PtdSer pool was pulse labeled with a [^H]serine precursor for 40 minutes and then further incorporation was arrested by the addition of EGTA which chelates Ca^"^ and thereby inhibits sequestration of this ion by the Ca^"^ ATPase. Under these "pulse-arrest" conditions, the nascent PtdSer made during the pulse labeling phase of the incubation continues, during the arrest period, to be translocated to the mitochondria and imported to the inner membrane where it is decarboxylated to form PtdEtn. These experiments provided clear evidence demonstrating that translocation of PtdSer to the mitochondria is an event that does not require continuous synthesis of the lipid. This same issue was addressed in experiments that utilized a different means of inhibiting the PtdSer synthase. Ethanolamine is known to compete for serine incorporation into lipid catalyzed by both PtdSer synthases I and II. Consequently, permeabilized cells can be pulse labeled with [^H]serine, and then the incorporation of radiolabel into PtdSer can be efficiently arrested by the addition of 5 mM ethanolamine which competes with serine as a substrate for the enzyme. The results of "pulse-arrest" experiments with ethanolamine as an inhibitor are identical to those employing EGTA, such that PtdSer translocation to the mitochondria proceeds in the absence of continuing synthesis of this lipid. Thus, these "pulse-arrest" studies provide unambiguous evidence that PtdSer translocation is not driven by the synthesis of this lipid. The relationship between the newly synthesized pool of PtdSer made in permeabilized cells (Voelker, 1990) and the "old and new" pools of PtdSer made in intact cells (Bjerve, 1985) is not entirely clear. The pulse-arrest experiments indicate that PtdSer made during the pulse period is readily transferred during the arrest period. The simple interpretation is that the permeabilized cells do not contain PtdSer in compartments that operationally correspond to the "old pool" of PtdSer observed in intact cells. If, as hypothesized above, the true difference between old and new pools of PtdSer is subcellular location, then this result might be explained by a
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failure of the permeabilized cells to export the bulk of the newly synthesized PtdSer to organelles other than the mitochondria. 5.
Phosphatidylserine Translocation Requires ATP
Another aspect of PtdSer translocation examined using permeabilized cells is the ATP dependence of the process (Voelker, 1989a, 1990). By establishing that the continual synthesis of PtdSer was not required for transport to the mitochondria, it was next possible to use ATP depletion experiments to examine their effect upon the transport process. For these experiments, permeabilized cells were pulse labeled with ^H-serine for 40 minutes in the presence of ATP and then incorporation of the precursor into PtdSer was arrested by addition of apyrase (a phosphohydrolase that sequentially converts ATP to ADP and AMP). The depletion of ATP not only arrested PtdSer synthesis but also prevented its transport dependent conversion to PtdEtn. This result clearly established that ATP was required for a step between synthesis and decarboxylation. Analysis of the above result, in conjunction with results obtained with isolated organelles that indicated PtdSer translocation between the outer and inner mitochondrial membrane was ATP independent (Voelker, 1989b; Vance, 1990; Ardail et al., 1991; Hovius et al., 1992; Simbeni et al., 1993) thus place the ATP requirement between the site of synthesis and the outer mitochondrial membrane (see Figure 1). The fact that the nonhydrolyzable ATP analogue, AMP-P(NH)P, fails to support the translocation of PtdSer to the mitochondria (Voelker, 1989a) indicates that ATP is cleaved as part of the transport mechanism. Exactly how ATP is utilized in the transport of PtdSer is not known. Likely mechanisms for ATP consumption include vesicle formation and transport, organelle movement (along cytoskeletal elements) and membrane fusion events. Although the exact transport mechanism remains undefined, some of its characteristics have been determined. Unlike protein transport out of the ER, PtdSer transport does not require GTP or cytosol (Voelker, 1990). Addition of cytosol is actually slightly inhibitory to PtdSer transport. The nucleotide analogue, GTPyS, which inhibits protein transport out of the ER, is without effect on PtdSer transport to the mitochondria. The above observations indicate that the mechanisms governing PtdSer movement between the ER or MAM and mitochondria are fundamentally different from those involved in protein export out of the ER. The translocation of PtdSer to the mitochondria in permeabilized cells is also insensitive to dilution (Voelker, 1990). To measure the effects of dilution, the permeabilized cells were subjected to labeling using the pulse-arrest technique with EGTA. Subsequent to EGTA addition, the cells were diluted 45-fold under conditions favorable for PtdSer transport. The transport of PtdSer in diluted samples was virtually identical to that found in undiluted samples. These results suggested that there was not free diffusion of the translocation intermediate through the soluble compartment and that there may be some tight physical association between donor compartments (ER or MAM) and the mitochondria.
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6. Phosphatidylserine Translocation Is Restricted to Autologous Mitochondria
The mechanism of PtdSer translocation was examined in further detail using permeabilized cells whose structural organization had been further disassembled by shearing through a 27 gauge needle (Voelker, 1993). The shearing process disrupts the residual cell structure present in permeabilized cells. In such preparations, one typically observes intact nuclei and fine granular debris that is characteristic of cells homogenized by various methods. The disrupted permeabilized cells exhibit the major properties for PtdSer synthesis and transport to the mitochondria that are found in their nondisrupted counterparts. Specifically, the synthesis of PtdSer is ATP dependent and coupled to Ca^"^ sequestration, and the translocation step remains sensitive to ATP depletion. In addition, the latency of cytochrome c oxidase remains high. Preparations of disrupted permeabilized cells provide the tools to determine if there is a unique relationship between the donor and acceptor compartments for PtdSer transport (Voelker, 1993). In these experiments, schematically outlined in Figure 3, a group of cells that form the donor pool were treated with 10 mM hydroxylamine to irreversibly inactivate the PtdSer decarboxylase. These donors were subsequently permeabilized and disrupted by shearing. Next, the donor population was pulse labeled with [^HJserine in the presence of ATP for 40 minutes. Following the labeling period, further incorporation of [^H]serine was arrested by the inclusion of EGTA. The above series of manipulations thus yields disrupted permeabilized cells that contain newly made radiolabeled PtdSer. These cells are unable to decarboxylate the PtdSer because they were previously poisoned with hydroxylamine. A second population of cells, denoted as acceptors, is not treated with hydroxylamine but is permeabilized and disrupted and mixed with the prelabeled donor cell extract. Experimentally this system is poised to measure the translocation of PtdSer from the donor preparation to the acceptor cell population. No PtdSer decarboxylation can occur in the donor mitochondria as a consequence of the hydroxylamine poisoning and no PtdSer synthesis can occur in the acceptors as a result of the EGTA arrest. Because the cells have been sheared, the major structural organization is lost enabling organelles from donor and acceptor compartments to freely intermix. Under these conditions the result obtained is striking —none of the PtdSer made in the donors is transferred to the heterologous acceptor mitochondria. This result indicates that translocation of PtdSer is a process that is restricted to autologous mitochondrial acceptors. Several control experiments were performed to rule out trivial explanations of the above data. In particular, the hydroxylamine poisoning of the donors was carried out under conditions where there is no carry over of the inhibitor to the final incubation where donor and acceptor cell extracts are mixed. In addition, examination of the concentration dependence of hydroxylamine inhibition indicates that the decarboxylation reaction, but not the translocation reaction, is affected by the
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ACCEPTORS
DONORS
X-NH,OH Permeabilize Shear
Pulse label with ^H-Serine for 40 min Arrest further RdSer synthesis with EGTA. Mix & Measure Translocation Dependent Decarboxylation of PtdSer Figure 3. Experimental outline for measuring PtdSer translocation to heterologous mitochondria. CHO-K1 cells were divided into two groups: one of which was sham treated (ACCEPTORS) and the other of which was treated with 10 mM NH2OH to inhibit PtdSer decarboxylase (DONORS). Both groups were subsequently permeabilized and sheared. The donor group was radiolabeled with pHJserine for 40 minutes to pulse label the PtdSer pool, and further PtdSer synthesis was arrested by the addition of EGTA. The donor and acceptor cells were then mixed and the translocation-dependent decarboxylation of PtdSer measured. The change in stippling of the mitochondria after NH2OH treatment indicates that these organelles have been rendered incompetent to decarboxylate PtdSer. MT, Mitochondria; NUC, nucleus; ER, endoplasmic reticulum. (Reprinted with permission from the Journal of Biological Chemistry.)
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inhibitor. The above results imply that there is a physical channeling of PtdSer between the ER or MAM and its corresponding mitochondria. This finding strongly supports the idea that there is a physical connection between the ER and mitochondria which functions as a corridor for ATP dependent PtdSer transport. While one can find electron micrographs that depict zones of close apposifion of mitochondria and ER (Lewis and Tata, 1973), it has not been clear whether these are a consequence of discrete structural interactions or whether they are of functional significance. The restricted transport of PtdSer constitutes a strong argument for interorganelle connections with defined transport fiinctions. 7.
Role of Mitochondrial
Membrane Contact Sites
Hypothetically, the import of PtdSer into the mitochondria might be expected to follow routes similar or identical to those taken by proteins as they enter the same organelle. However, experiments using isolated organelles have largely failed to provide any evidence favoring coordinate import of protein and lipid. While it is clear that factors governing protein import, such as membrane potential and matrix ATP content, are not required for PtdSer import (Voelker, 1989b; Vance, 1990; Ardail et al., 1991; Hovius et al., 1992; Simbeni et al., 1993), it is still uncertain whether the physical structures of the mitochondria through which proteins pass is also the same through which PtdSer passes. The key organelle domain appears to be the contact sites between the inner and outer mitochondrial membrane. Ardail et al. (1991) have reported that newly imported PtdSer is concentrated in these membrane contact sites and Hovius et al. (1992) have provided data that dinitrophenol mediated decreases of membrane contact sites cause decreases in the initial rate but not the extent of PtdSer import. Contact sites have also been implicated as the routes for PtdSer import into yeast mitochondria (Simbeni et al., 1993) In permeabilized cells the antitumor drug adriamycin has also been used to investigate the process of PtdSer import into mitochondria (Voelker, 1991). Adriamycin has a high affinity for bisphosphatidylglycerol which is known to be concentrated in the inner mitochondrial membrane. One effect of adriamycin is to disrupt contact sites between the mitochondrial membranes (Nicolay et al., 1984) and this in part explains some of the effects of the drug as an inhibitor of protein import into the mitochondria (Eilers et al., 1989). When permeabilized cells are treated with adriamycin, the import of newly made PtdSer into mitochondria is rapidly and dramatically arrested. The IC50 of adriamycin was 150 |iM, a value compatible with the drug concentration that similarly affects the import of some proteins into the mitochondria (Eilers et al., 1989). The site of action of the drug was examined using the PtdSer analogue l-acyl-2C^-NBD-PtdSer (NBD-PtdSer) (Voelker, 1990). The NBD-PtdSer analogue nonspecifically partitions into organelle membranes including the mitochondrial outer membrane (Sleight and Pagano, 1984; Pagano and Sleight, 1985). The partitioning property thus enables NBD-PtdSer to be loaded into the mitochondrial outer membrane and the kinetics of import and decarboxylation can be followed by
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measuring the formation of NBD-PtdEtn. Adriamycin blocks the synthesis of NBD-PtdEtn but this drug does not specifically inhibit the decarboxylase enzyme. These results provide strong evidence that adriamycin is blocking the movement of the NBD-PtdSer from the outer to the inner mitochondrial membrane (see Figure 1). It is likely, although not proven, that the inhibition of PtdSer transport by adriamycin is a consequence of the disruption of contact sites between the mitochondrial membranes. D. Mitochondrial Import of Exogenous Phosphatidylserine
While the above discussions of interorganelle PtdSer import into the mitochondria have centered around the pathway taken by newly synthesized PtdSer, there is another aspect of transport of this lipid for which some data have accumulated, and that is the transport of exogenous PtdSer. This is a multifaceted topic that includes elements of intra- and intermembrane transport. The intramembrane aspect is discussed later in this chapter. Studies with mammalian cells defective in PtdSer synthase I demonstrated that they were PtdSer auxotrophs (Kuge et al, 1986; Voelker and Frazier, 1986). The PtdSer was not only taken up by the cell but was also translocated to the mitochondria and decarboxylated. Use of long-chain PtdSer analogues Ci2NBDPtdSer suggests that the routing of this lipid to the mitochondria may occur in part via the Golgi apparatus (Kobayashi and Arakawa, 1991). Acritical issue to be gleaned from these studies is that pathways clearly exist for transporting significant quantities of lipid from the extracellular space and cell surface to the inner mitochondrial membrane. More experimentation examining transport of exogenous PtdSer will be necessary to determine if this lipid enters some common recycling pool within the cell or if it is specifically targeted to the mitochondria after its uptake from the media. E. Mitochondrial Export of Phosphatidylethanolamine
Analysis of the amount of decarboxylation of PtdSer provides clear evidence that this pathway can meet the requirement of several cell types for the synthesis of PtdEtn that is necessary for cell growth and division (Voelker, 1984). Implicit in this finding is the conclusion that the mitochondria play an active role in lipid synthesis and export to other organelles. Evidence for the mitochondria acting as a lipid export organelle has also been demonstrated directly in subcellular fractionation experiments that show that PtdEtn formed at the mitochondrial inner membrane translocates to the ER-derived microsomal fraction (Voelker, 1985). More compelling evidence has been provided by Vance who measured the appearance of PtdEtn, formed by the decarboxylation reaction within the mitochondria, at the outer aspect of the plasma membrane (Vance et al., 1991). The kinetics of PtdEtn transport from the mitochondria to the cell surface are quite rapid and are comparable to the rates observed for PtdEtn formed in the ER by the CDP-ethanolamine dependent pathway. Notably, the PtdEtn made within the mitochondria is
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transported to the plasma membrane by a route that is insensitive to the effects of brefeldin A (Vance et al., 1991). Brefeldin A causes disassembly of the Golgi apparatus and concomitantly blocks protein transport from the ER to other cellular organelles (Lippincott-Schwartz et al., 1989). The contrasting effects of brefeldin A on PtdEtn and protein transport indicate that movement of this lipid to the plasma membrane can readily occur via mechanisms that are different from those required for protein translocation. In addition to being used for membrane synthesis, the PtdEtn synthesized in the mitochondria is also used to supply PtdEtn and PtdCho (synthesized by methylation of PtdEtn) for lipoprotein assembly (Vance and Vance, 1986). The export of PtdEtn from the mitochondria is not unique to mammalian cells. In the absence of exogenous choline and ethanolamine, yeast cells synthesize PtdEtn in the mitochondria and this lipid must be exported to other membranes (Kuchler et al., 1986; Carman and Henry, 1989). Collectively there is unambiguous evidence that the mitochondria can play a major role in PtdEtn export in eukaryotes, but currently very little is known about the biochemical mechanisms governing these transport processes and this should prove an exciting research topic in the near future.
IV. INTRAMEMBRANE TRANSPORT OF PHOSPHATIDYLSERINE In addition to interorganelle transport, movement of phospholipids within biological membranes adds another dimension of complexity to the problems of cellular lipid synthesis and distribution. The localization of PtdSer within the bilayers of the cell is a critical parameter which must be controlled. There is a wealth of evidence that PtdSer is distributed asymmetrically in several subcellular membranes. This asymmetrical localization of PtdSer within membranes is important to such diverse functions as membrane attachment of Ca^"^ binding proteins that mediate cytoskeletal rearrangements during secretion and cell motility (Geisow and Walker, 1986; Klee, 1988), regulation of PKC activation (Newton and Koshland, 1990; Bell and Bums, 1991), enhancement of the hemostatic process (Bevers et al., 1983), and the interaction of blood cells with the reticuloendothelial system (Schlegel and Williamson, 1987). Mechanisms must therefore exist by which the asymmetric distribution of PtdSer within membranes is formed and maintained. Spontaneous translocation of phospholipids across model membranes is an extremely slow process with half-times on the order of several hours (Kornberg and McConnell, 1971), yet the transbilayer diffusion of some phospholipids would need to be much more efficient to maintain the correct distributions within biological membranes. Investigation into the mechanism whereby PtdSer is translocated within intracellular and plasma membranes has provided evidence that these processes are mediated by specific proteins within the bilayer.
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PAMELA J. TROTTER and DENNIS R. VOELKER A. Translocation within Intracellular Membranes
/.
Endoplasmic
Reticulum
The ER is the major locus of cellular lipid synthesis (Colbeau et al., 1971; Van Golde et al, 1974; Jelsema and Morre, 1978; Bell et al., 1981; Vance & Vance, 1988) and several studies have focused on the capacity of this membrane to distribute lipids between its cytosolic and lumenal faces. Herrman et al. (1990) have examined the transbilayer movement of spin-labeled PtdSer analogues introduced to the outside of rat liver microsomal vesicles. The kinetics of movement to the inside were determined by using a "back exchange" technique. A spin-labeled phospholipid analogue suspension was introduced to the outer surface of the microsomes, and the kinetics of spin-label translocation to the inner leaflet were determined by subsequent extraction of the remaining label from the outer leaflet at reduced temperature by bovine serum albumin. The ti/2 of outside-inside translocation was calculated to be 20 minutes, at which point -45% of the labeled molecules had moved to the inside of the vesicles. These data clearly indicate that the translocation process occurs more rapidly than could be explained by the spontaneous "flip-flop" found in model membranes, and is most likely mediated by a membrane protein. Treatment of the microsomes with the sulfhydryl modifying reagent N-ethylmaleimide (NEM) significantly inhibited lipid translocation and also implicates a protein carrier in the process. In addition, evidence was obtained for saturability and broad specificity (including PtdSer, as well as PtdCho, PtdEtn, sphingolipids, and lysophospholipids) of the process. These data were very similar to earlier reports by Bishop and Bell (1985) and Backer and Dawidowicz (1987) which indicated a role for a membrane "flippase" in the transbilayer movement of PtdCho within the ER. 2.
Chromaffin
Granule
The membrane of the chromaffin granule has also been the subject of lipid translocation experiments. Secretion of epinephrine and norepinephrine stored in chromaffin granules in the adrenal gland is thought to be mediated by annexin proteins, via Ca^"*"-dependent binding to PtdSer (Geisow and Walker, 1986). For this to occur, the PtdSer must be localized to the cytoplasmic monolayer of the chromaffin granule membrane. Zachowski et al. (1989) have studied transmembrane movement of spin-labeled phospholipid analogues in chromaffin granules from bovine adrenal glands. The translocation activity in the granules acted to move PtdSer from the lumenal to the cytoplasmic face of the bilayer. The translocation reaction was ATP-dependent and sensitive to the sulfhydryl reagent NEM. Chromaffin granules possess only two detectable ATPase activities, referred to as I and II, and the authors suggest, based on the level of NEM sensitivity, that PtdSer translocation is mediated by ATPase II. The ATPase, purified from chromaffin granules by Moriyama et al. (1991), is specifically activated by PtdSer while other
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phospholipids including PtdCho, PtdEtn, Ptdlns, and lysophospholipids are without effect. This is the only evidence, thus far, for such a specific, ATP-dependent phospholipid translocase in an intracellular organelle. B. Translocations Within the Plasma Membrane /.
Platelets
The maintenance of PtdSer asymmetry at the plasma membrane has been studied primarily in platelets and red blood cells (RBC). In the platelet almost all the PtdSer is localized to the inner leaflet of the membrane. Bevers et al. (1983) have demonstrated that activation of platelets by thrombin and collagen results in a significant reorganization of PtdSer to the outer leaflet. This translocation of PtdSer to the outer layer is considered to be of major importance in hemostasis. Exposure of PtdSer at the outer monolayer of the platelets results in acceleration of the clotting process by increasing the formation of coagulation factor Xa, and thereby enhancing the conversion of prothrombin to thrombin. Sune et al. (1987) have studied the mechanism of inward transbilayer movement of platelet phospholipids by introducing spin-labeled analogues to the external monolayer, and monitoring translocation by following the spontaneous reduction of the probe molecules due to cytosolic reducing agents. These studies demonstrated that whereas the tj 2 of translocation for PtdCho was -^40 minutes, it was less than seven minutes for PtdSer. In addition, the inward translocation of aminophospholipoids, but not PtdCho, was notably inhibited after aging of the platelets, which results in ATP depletion. Maintenance of bilayer asymmetry in platelets appears to be mediated by a membrane protein since asymmetry was lost upon treatment with the sulfhydrylmodifying reagents pyridyldithioethylamine and diamide (Bevers et al., 1983, 1989). Subsequent treatment of the platelets with dithiothreitol (DTT) reversed the effects of the sulfhydryl reagents, resulting in translocation of the PtdSer back to the inner leaflet of the bilayer (Bevers et al., 1989). However, treatment of the platelets with proteases prior to addition of DTT blocked the reversibility of the PtdSer exposure to the outer leaflet. Data such as these lead to the conclusion that an ATP-dependent translocating enzyme specific for aminophospholipids is likely responsible for the inward movement of PtdSer in platelet membranes. 2.
Phosphatidylserine Externalization as a Clearance Signal
The loss of the asymmetrical localization of PtdSer within the plasma membrane of blood cells is hypothesized to play a role in the clearance of aged cells from the circulation. In RBC, PtdSer is localized exclusively to the inner leaflet of the plasma membrane. However, RBC that have PtdSer exposed in the outer leaflet are cleared from the circulation by the liver and spleen (Schroit et al, 1985). Actual demonstration of increased PtdSer in the outer leaflet of aged RBC membranes, isolated by differential centrifugation, has not been successful (Devaux, 1992), suggesting that the required change may be very small and the population may be so rapidly
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removed that it is not recoverable. In sickled RBC, however, it has been effectively demonstrated that PtdSer is significantly enriched in the outer leaflet of the membrane (Lubin et al, 1981). This property of sickled RBC accounts for increased recognition and adherence by monocytes, and perhaps decreased survival time in the circulation (Schwartz et al., 1985). Further, Schlegel and colleagues (McEvoy et al., 1986; Schlegel and Williamson, 1987) have demonstrated that exposure of PtdSer in the plasma membrane of RBC leads to increased binding and phagocytosis by macrophages. These investigators have proposed that such loss of lipid asymmetry may provide a general signal for the removal of many types of blood cells from the circulation by the reticuloendothelial system. Indeed, Fadok et al. (1992) have studied the changes in PtdSer transmembrane distribution during apoptosis of lymphocytes. These studies, utilizing an extracellular derivatization technique, demonstrated an increase in PtdSer in the outer leaflet of plasma membranes from apoptotic cells. Concomitant with this externalization of PtdSer, the cells became recognizable by macrophages and were readily phagocytosed. Phagocytosis of the apoptotic lymphocytes was blocked by liposomes containing PtdSer, glycerophosphoserine or phosphoserine, but not by other anionic phospholipids. Taken together, these data infer that in sickly or aged blood cells, the aminophospholipid asymmetry is gradually lost, and exposure of PtdSer in the outer half of the plasma membrane serves as an important signal for removal from circulation. 3.
Red Blood Cells
The mechanism by which the membrane asymmetry in RBC is generated and maintained has received much attention and may have important implications for similar processes occurring in other cell types (Siegneuret and Devaux, 1984; Tilley etal., 1986; Devaux, 1992; Martin and Pagano, 1987). Spin-labeled or radiolabeled analogues of PtdSer, PtdEtn, and PtdCho were introduced to the outer leaflet of the membrane of RBC, and it was observed that, while the PtdCho remained primarily in the outer leaflet, the aminophospholipids PtdSer and PtdEtn were rapidly translocated to the inner leaflet. The translocation process of aminophospholipids required ATP and was accompanied by a notable change in RBC shape. Daleke and Heustis (1985, 1989) utilized this change in cell shape to further characterize aminophospholipid translocation and distribution in the plasma membrane. Using ^"^C-dilauryl PtdSer, which spontaneously transfers from liposomes to the RBC membrane, they described dramatic cell shape changes that are a consequence of the transbilayer localization of the phospholipids. Upon addition of the exogenous PtdSer and its transfer to the outer monolayer of RBC, the cells take on a spiny, echinocytic morphology. The extent of this shape change is dose dependent and is likely due to the excess lipid in the outer leaflet of the membrane. After 10 to 20 minutes incubation, the cells rapidly return to their discoid shape, coincident with translocation of the PtdSer to the inner leaflet of the RBC membrane. As translocation of the PtdSer to the inner monolayer continues, the cells eventually acquire
Phosphatidylserine Metabolism and Translocation
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an invaginated or stomatocytic morphology. This latter change is thought to result from excess PtdSer in the inner leaflet of the membrane, and is blocked by treatment with sulfhydryl modifying agents and ATP depletion. Similar experiments with ^"^C-dilauroylphosphatidylcholine (PtdCho) showed the initial echinocytic shape change followed by a very slow return to discoid shape, but no development of the stomatocytic morphology. Most of the added PtdCho (>95%) remained in the outer monolayer and was extractable by extracellular acceptor vesicles, whereas most of the PtdSer (-^75%) became unextractable. Longer incubation (>15 hours) of stomatocytic cells containing exogenously added PtdSer with acceptor vesicles results in the gradual return to a discoid shape, indicating that PtdSer translocated to the inner leaflet can be slowly moved outward (see below) to reduce the excess in the inner leaflet. These studies provide direct evidence that the transbilayer movement and distribution of PtdSer influences the shape of RBC. In addition, since the reaction is dependent upon Mg^'^-ATP, is inhibited by protein modifying reagents, and does not act on PtdCho, the translocation was concluded to be effected by an ATP-dependent aminophospholipid translocase. Devaux and colleagues (Zachowski et al., 1986; Morrot et al., 1989) have investigated the kinetics and specificity of the putative aminophospholipid translocating enzyme. Initial velocities of outer-inner leaflet movement were measured using spin-labeled analogues of PtdSer, PtdEtn, and PtdCho, and saturability of the process was observed. Assuming Michaelis-Menten kinetics, the maximum rates of PtdSer and PtdEtn translocation were similar and the apparent K^ values obtained had a ratio (K^PS/K^PE) of 1:34. In contrast to the aminophospholipids, translocation rates of PtdCho failed to show saturability. N-ethylmaleimide inhibited translocation of both PtdSer and PtdEtn, but PtdCho movement was unaffected. Changing the hydrophobic component of PtdSer from diacylglycerol to ceramide completely abolished the rapid translocation from the outer to inner monolayer thereby demonstrating chemical specificity. In addition, lysoPtdSer was only weakly transported, indicating the need for an acyl group at the sn-2 position of the glycerol moiety. These data indicate that the enzyme translocates glycerol based aminophospholipids from the outer to the inner monolayer, and displays the highest affinity for PtdSer. More recent studies have demonstrated that the movement of phospholipids across the plasma membrane is not merely from the outer to inner monolayer, but is a bidirectional process. Daleke and Heustis (1989) showed that the stomatocytic morphology of RBC acquired after translocation of exogenous PtdSer to the inner leaflet of the membrane could be reversed to a discoid morphology upon treatment of the RBC with extracellular acceptor vesicles. These data indicated that the PtdSer in the inner leaflet was translocated back to the outer leaflet for extraction by the acceptor vesicles. In more direct experiments, Bitbol and Devaux (1988) exposed RBC to spin-labeled phospholipid analogues and allowed them to be translocated to the inner leaflet where the nitroxide radical was spontaneously reduced. Outward movement was subsequently monitored by examining the susceptibility of the
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probe to extraction from the membrane by bovine serum albumin and oxidation by ferricyanide. The translocation of PtdSer and PtdEtn from inner to outer leaflet was reported to be 3-^-fold more rapid than for PtdCho. The authors suggest that the previously proposed inward aminophospholipid transporter may also be responsible for outward movement. Schroit and colleagues (Connor et al., 1992) have reported similar experiments utilizing fluorescent analogues of PtdSer, PtdEtn, and PtdCho. Like inward translocation, the outward movement was sensitive to sulfhydryl oxidizing reagents, ATP and Mg^"*". In contrast to the results of Bitbol and Devaux (1988), these authors noted that the kinetics of outward movement of the three analogues was similar, and the extent of translocation reflected the normal membrane asymmetrical distribution in the outer leaflet (PtdCho > PtdEtn > PtdSer). They hypothesized that outward movement of phospholipids across the RBC membrane is mediated by a membrane protein with no head group specificity. Lipid distribution in the membrane, then, would be a cumulative consequence of continuous, slow transport of all lipids from the inner to the outer leaflet, while the specific translocase enzyme catalyzes rapid movement of the aminophospholipids from the outer to inner leaflet. The basis for the contradictory results obtained by these two groups is likely related to the use of different probes and methodology. Clearly, more investigation into the mechanism of inward-outward translocation of phospholipids in the plasma membrane is needed. 4. Aminophospholipid
Transporters
Putative aminophospholipid transporter proteins have been identified by several groups. Using an iodinated derivative of the thiol reagent pyridyldithioethanolamine (PDA), which reversibly inhibits PtdSer translocation, Connor and Schroit (1988) identified a 31 kDa protein that was preferentially labeled by the *^^I-PDA molecule and comigrated with RBC integral membrane protein band 7. A similar result was obtained utilizing photoactivatable ^^^I-N3-PtdSer, which labeled a 32 kDa protein (Connor and Schroit, 1988). The specific crosslinking of these two agents to a similarly-sized protein led to the suggestion that it plays a role in PtdSer transbilayer movement. In addition, the crosslinked probe was immunoprecipitable with antibodies to Rh proteins, which prompted the suggestion that Rh proteins may be involved in aminophospholipid translocation (Connor et al, 1992). In a recent report, however, Daleke et al. (1992) demonstrated that in Rh'''*^ RBC that lack Rh proteins, ^^^I-PDA still crosslinks proteins of 28-32 kDa. This indicates that the 32 kDa protein is not part of the Rh family. While the exact identity of this 32 kDa protein is in question, the crosslinking results with PtdSer analogues clearly make it a credible candidate for the aminophospholipid translocase. Connor and Schroit (1990) further investigated whether other sites on the 32 kDa protein were important for transport activity, using other sulfhydryl reagents. The investigators observed that PDA-inhibition of aminophospholipid translocation was not reversed by endogenous glutathione. However, inhibition by diamide, an agent known to crosslink the RBC cytoskeletal spectrin, was reversed by endo-
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genous glutathione and the depletion of endogenous glutathione also inhibited PtdSer translocation. These authors suggested, as have others (Kumar et al, 1990), that there may be a protein at the endofacial surface, perhaps a component of the cytoskeleton, which is also involved in maintenance of the asymmetrical distribution of PtdSer in the plasma membrane. Other groups, however, dispute a significant role for cytoskeleton in plasma membrane lipid asymmetry. Calvez et al. (1988) found that the asymmetrical phospholipid distribution in RBC vesicles was maintained, despite being almost entirely devoid of cytoskeletal proteins. Gudi et al. (1990) showed that heat-treatment of RBC, which alters the interaction between spectrins in the cytoskeleton, had no effect on lipid asymmetry. Further, Pradhan et al. (1991) demonstrated that the pattern of cytoskeletal/phospholipid interactions, as assessed by crosslinking with a photoactivatable phospholipid analogue, were identical in RBC ghosts whether the lipids were distributed symmetrically or asymmetrically. Thus, it seems that despite indirect data suggesting a role for cytoskeleton in lipid asymmetry, direct experimentation does not support such a mechanism. Morrot et al. (1990) and Daleke et al. (1992) have characterized another putative aminophospholipid translocase. Using a method similar to that used to isolate ATPases from chromaffin granules (Moriyama et al., 1991) and clathrin-coated vesicles (Xie et al., 1988), a Mg-ATPase-enriched fraction from RBC was utilized to isolate a vanadate-sensitive 120 kDa Mg^"^-ATPase that required PtdSer for optimal activity. A role for this protein in aminophospholipid translocation is supported by its requirement for PtdSer, Mg^"^, and its inhibition by Ca^"^, vanadate and sulfhydryl reagents (Morrot et al., 1990; Daleke et al., 1992). Proof that this protein mediates bilayer translocation of aminophospholipids will require complete purification and reconstitution. Thus, despite the proposal of such candidate proteins, the actual identity of the aminophospholipid translocase remains ambiguous. 5.
Fibroblasts
Although transbilayer movement of PtdSer has been studied primarily in blood cells and platelets, as mentioned above, asymmetry of PtdSer distribution is found in the plasma membrane of many cell types. Martin and Pagano (1987) have investigated the process of PtdSer and PtdEtn translocation across the plasma membrane of cultured fibroblasts. Exogenous fluorescently labeled (1-palmitoyl2-C^-NBD) PtdSer was added to the plasma membrane by incubating the cells with liposomes for 30 minutes at 2- C. Subsequent incubation of the cells at 7- C resulted in fluorescent labeling of intracellular membranes by a process which reportedly did not include endocytosis, but was a consequence of transbilayer movement. The translocation of the C^-NBD-PtdSer was reversibly inhibited by reducing the level of cellular ATP. Translocation was also blocked by the sulfhydryl reagent NEM, and by the PtdSer analogue glycerophosphoserine. The translocation process was also demonstrated to be stereospecific for the L-isomer of C^ NBD-PtdSer. Kobayashi and Arakawa (1991) have also examined transport of PtdSer in fibroblasts,
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but used an NBD analogue with a longer acyl chain in the sn-2 position, (1-palmitoyl-2-C,2-NBD)PtdSer. This PtdSer analog was incorporated and transported primarily to the Golgi as well as the mitochondria. Based on the kinetics of transport at different temperatures, the requirement for ATP and inhibition by NEM and structural analogues, the authors concluded that the Cj2-NBD-PtdSer was transported to the Golgi via translocation from the outer to inner leaflet of the membrane followed by a nonvesicular transport mechanism through the cytoplasm to the Golgi. Collectively, the above studies indicate that transbilayer movement of PtdSer in fibroblasts is mediated by an ATP-dependent plasma membrane protein analogous to the aminophospholipid translocated in RBC. Intramembrane transport and distribution of PtdSer is clearly of vital importance in phospholipid biosynthesis, blood cell senescence, and the activity of various plasma membrane proteins. Many questions, however, remain unanswered. What is the identity of the ER phospholipid "flippase," how does it function and how is it regulated? Which candidate plasma membrane aminophospholipid translocase enzyme is responsible for generating and maintaining phospholipid asymmetry in RBC? Is the same protein involved in this process in other cell types? What is the mechanism of phospholipid translocation? How are inner-outer and outer—inner translocations carried out and regulated? Does the same protein mediate transport in both directions? Further purification and reconstitution of these proteins as well as isolation of the genes encoding them will be necessary to address these questions.
V. CONCLUDING REMARKS We now have a solid understanding of the enzymology and the gene structure of PtdSer synthase from bacteria and yeast. A number of critical issues remain to be investigated for this enzyme, including structure-function analyses of the mature proteins, that will help provide a clear understanding of the mechanisms of catalysis. Efforts to achieve crystallization of the enzymes and resolve their structure also remain important goals. Information about the primary structure of the mammalian PtdSer synthase I is also available and should provide the basis of new studies to learn more about higher order structure, function, topology, and coupling of this enzyme to Ca^"^ sequestration. The bacterial PtdSer decarboxylase has been studied in detail especially with regard to its posttranslational processing. In contrast, although primary structural information has recently become available for the yeast and mammalian enzymes, little is known about the processing events that lead to assembly of the mature protein in the mitochondrial inner membrane and this should be an area of important research efforts in the future. A general picture of the biochemical events in the translocation of PtdSer from the MAM or ER to the mitochondria is emerging in both the higher and lower eukaryotes. Future efforts must now be directed at more precise physical disassembly and reconstitution of individual steps in the translocation process to better identify the biochemical details. Specifically the cytological and biochemical
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intermediates need to be elucidated as do the reactions that consume ATP. As more information is obtained from the yeast system, it is likely that genetic strategies for addressing the interorganelle translocation problem will become evident and provide powerful approaches to identify specific genes involved in the process. The general details of the transbilayer movement of PtdSer in the plasma membrane of higher eukaryotes are now well established. Purification of the proteins involved remains a very difficult and important problem. The use of the PtdSer stimulated ATPase assays constitutes a rational approach to this effort. However, the reconstitution of functional transport remains as the critical test that must be applied to all candidate proteins. The biochemical and cytological events accompanying the synthesis and metabolism of PtdSer are numerous and it is clear that each one of these events can reveal important new information about the structure, function, or mechanism of assembly of cell membranes. It seems likely that interest in this phospholipid will continue to expand as it provides clues about the complex processes of membrane biogenesis.
ABBREVIATIONS ADP, adenosine diphosphate; AMP, adenosine monophosphate; AMP-P(NH)P, adenyl-5'-yl imidodiphosphate; ATP, adenosine triphosphate; CDP, cytidine diphosphate; CHO, Chinese hamster ovary cells; CMP, cytidine monophosphate; DTT, dithiothreitol; EGTA, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid; ER, endoplasmic reticulum; GTP, guanosine triphosphate; GTPyS, guanosine 5'-0-(3thiotriphosphate); MAM, mitochondria associated membrane; C^NBD, 1-acyl2[N-(6-[7-nitrobenz-2-oxa-l,3-diazo-4-yl)]aminocaproyl)]; NEM, N-ethylmaleimide; PDA, pyridyldithioethanolamine; PKC protein kinase C; PSS, phosphatidylserine synthase; PtdCho, phosphatidylcholine; PtdEtn, phosphatidylethanolamine; Ptdlns, phosphatidylinositol; PtdSer, phosphatidylserine; RBC, red blood cells.
ACKNOWLEDGMENTS The authors thanks Ms. Peggy Hammond for excellent secretarial assistance. This work was supported by NIH grant GM32453.
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Sleight, R.G. & Pagano, R.E. (1984). Transport of a fluorescent phosphatidylcholine analogue from the plasma membrane to the Golgi apparatus. J. Cell Biol. 99, 742-751. Steiner, M.R. & Lester, R.L. (1972). In vitro studies of phospholipid biosynthesis in Saccharomyces cerevisiae. Biochim. Biophys. Acta 26, 222-243. Stoffel, W., Sticht, G., & Le Kim, D. (1968). Degradation in vitro of dihydrosphingosine and dihydrosphingosine phosphate to palmitaldehyde and ethanolamine phosphate. Hoppe-Seyeler's Z. Physiol. Chem. 349, 1745-1748. Sune, A., Bette-Bobillo, P., Bienveniie, A., Fellmann, P., & Devaux, P.F. (1987). Selective outside-inside translocation of aminophospholipids in human platelets. Biochem. 26, 2972-2978. Suzuki, T.T. & Kanfer, J.N. (1985). Purification and properties of an ethanolamine-serine base exchange enzyme of rat brain microsomes. J. Biol. Chem. 260, 1394-1399. Taki, T. & Kanfer, J.N. (1978). A phospholipid serine base exchange enzyme. Biochim. Biophys. Acta 528,309-317. Thastrup, O., Cullen, P.J., Drobak, B., Hanley, M.R., & Dawson, A.P. (1990). Thapsigargin, a tumor promoter, discharges intracellular Ca stores by specific inhibition of the endoplasmic reticulum Ca^'^-ATPase. Proc. Natl. Acad. Sci. USA 87, 2466-2470. Tilley, L., Criber, S., Roelofsen, B., Op de Kamp, J.A.F., & van Deenen, L.L.M. (1986). ATP-dependent translocation of amino phospholipids across the human erythrocyte membrane. FEBS Lett. 194, 21-27. Trotter, P.J., Pedretti, J., & Voelker, D.R. (1993). Phosphatidylserine decarboxylase from Saccharomyces cerevisiae: Isolation of mutants, cloning of the gene and creation of a null allele. J. Biol. Chem. 268,21416-21424. Van Golde, L.M.G., Raben, J., Batenburg, J.J., Fleischer, B., Zambrano, F., & Fleischer, S. (1974). Biosynthesis of lipids in Golgi complex and other subcellular fractions from rat liver. Biochim. Biophys. Acta 360, 179-192. Van Heusden, G.RH., Bos, K., Raetz, C.R.H., & Wirtz, K.W.A. (1990). Chinese hamster ovary cells deficient in peroxisomes lack the nonspecific lipid transfer protein (sterol carrier protein 2). J. Biol. Chem. 265, 4105-4110. van Poelje, RD. & Snell, E.E. (1990). Pyruvoyl-dependent enzymes. Ann. Rev. Biochem. 59, 29-59. Vance, D.E. & Vance, J.E. (1986). Specific pools of phospholipids are used for lipoprotein secretion by cultured rat hepatocytes. J. Biol. Chem. 261, 4486-4491. Vance, J.E. (1990). Phospholipid synthesis in a membrane fraction associated with mitochondria. J. Biol. Chem. 265, 7248-7256. Vance, J.E., Aasman, E.J., & Szarka, R. (1991). Brefeldin A does not inhibit the movement of phosphatidylethanolamine from its site of synthesis to the cell surface. J. Biol. Chem. 266, 8241-8247. Vance, J.E. & Vance, D.E. (1988). Does rat liver Golgi have the capacity to synthesize phospholipids for lipoprotein secretion? J. Biol. Chem. 263, 5898-5909. Voelker, D.R. (1984). Phosphatidylserine functions as the major precursor of phosphatidylethanolamine in cultured BHK 21 cells. Proc. Natl. Acad. Sci. USA 81, 2669-2673. Voelker, D.R. (1985). Disruption of phosphatidylserine translocation to the mitochondria in baby hamster kidney cells. J. Biol. Chem. 260, 14671-14676. Voelker, D.R. (1989a). Phosphatidylserine translocation to the mitochondrion is an ATP dependent process in permeabilized animal cells. Proc. Natl. Acad. Sci. USA 86, 9921—9925. Voelker, D.R. (1989b). Reconstitution of phosphatidylserine import into rat liver mitochondria. J. Biol. Chem. 264, 8019-8025. Voelker, D.R. (1990). Characterization of phosphatidylserine synthesis and translocation in permeabilized animal cells. J. Biol. Chem. 265, 14340-14346. Voelker, D.R. (1991). Adriamycin disrupts phosphatidylserine import into the mitochondria of permeabilized CHO-Kl cells. J. Biol. Chem. 266, 12185-12188.
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Voelker, D.R. (1993). The ATP-dependent translocation of phosphatidylserine to the mitochondria is a process that is restricted to the autologous organelle. J. Biol. Chem. 268, 7069-7074. Voelker, D.R. & Frazier, J.L. (1986). Isolation and characterization of a Chinese hamster ovary cell line requiring ethanolamine or phosphatidylserine for growth and exhibiting defective phosphatidylserine synthase activity. J. Biol. Chem. 261, 1002-1008. Wirtz, K.W.A. & Gadella, T.W.J. (1990). Properties and modes of action of specific and nonspecific phospholipid transfer proteins. Experientia 46, 592-599. Xie, X.-S., Stone, D.K., & Racker, E. (1988). Proton pump of clathrin-coated vesicles. Meth. Enzymol. 157,634-646. Zachowski, A., Favre, E., Cribier, S., Herve, P., & Devaux, P.F. (1986). Outside-inside translocation of aminophospholipids in the human erythrocyte membrane is mediated by a specific enzyme. Biochemistry 25, 2585-2590. Zachowski, A., Henry, J.P., & Devaux, P.F. (1989). Control of transmembrane lipid asymmetry in chromaffin granules by an ATP-dependent protein. Nature 340, 75-76. Zborowski, J., Dygas, A., & Wojtczak, L. (1983). Phosphatidylserine decarboxylase is located on the external side of the inner mitochondrial membrane. FEBS Lett. 157, 179-182. Zinser, E., Sperka-Gottlieb, C.D.M., Fasch, E.V., Kohlwein, S.D., Paltaufif, F., & Daum, G. (1991). Phospholipid synthesis and lipid composition of subcellular membranes in the unicellular eukaryote Saccharomyces cerevisiae. J. Bacteriol. 173, 2026-2034.
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DIACYLGLYCEROL METABOLISM IN CELLULAR MEMBRANES
Rosalind A. Coleman and Steven H. Zeisel
I. INTRODUCTION 338 II. DIACYLGLYCEROL IS A SUBSTRATE FOR TRIACYLGLYCEROL AND COMPLEX LIPID SYNTHESIS 338 III. DIACYLGLYCEROL ACTIVATES PROTEIN KINASE C 340 A. Nuclear Protein Kinase C 342 B. Regulation of Protein Kinase C by Sphingolipids 343 IV. SOURCES OF DIACYLGLYCEROL 343 A. Diacylglycerol From De Novo Synthesis 344 B. Diacylglycerol Arises via Recycling of Cellular Glycerolipids 346 C. Source of Nuclear Diacylglycerol 346 D. Phosphatidic Acid Phosphohydrolase 2 347 E. Triacylglycerol Lipase 347 V. DIACYLGLYCEROL SIGNAL ATTENUATION AND RECYCLING . . . . 348 A. Diacylglycerol Kinase 348 B. Diacylglycerol Lipase 348 VI. CELLULAR DIACYLGLYCEROL CONTENT AND PROTEIN KINASE C 350 VII. MOVEMENT OF DIACYLGLYCEROL WITHIN CELLS 351
Advances in Lipobiology Volume 1, pages 337-366. Copyright © 1996 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-635-5 337
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ROSALIND A. COLEMAN and STEVEN H. ZEISEL
VIII. MOLECULAR SPECIES OF DIACYLGLYCEROL AND DIRADYLGLYCEROL A. Molecular Species of Diaradylglycerols and Protein Kinase C Activation B. Diacylglycerol Individuality and Removal C. Diacylglycerol Individuality and Free Fatty Acids Formed IX. THE FATE OF MEDIUM CHAIN TRIACYLGLYCEROLS X. IMPORTANT AREAS FOR FUTURE INVESTIGATION REFERENCES
353 353 355 355 .356 356 357
I. INTRODUCTION The synthesis, degradation, and transport of 5«-l,2-diacylglycerol (DAG) within cells has become a critical focus of study because DAG plays a central role in membrane synthesis, energy storage, and signal transduction (Figure 1). Quantitatively, the most important phospholipids (phosphatidylcholine, phosphatidylethanolamine, and, indirectly, phosphatidylserine) are all derived from DAG. It is also the precursor of triacylglycerol, the major storage form of energy in all cells. DAG is an important intracellular second messenger, mediating the actions of numerous cytokines, drugs, and hormones by activating protein kinase C (PKC). Other roles for DAG have been suggested. It promotes actin nucleation in Dictyostelium discoideum (Shariff and Luna, 1992), regulates enzymes such as cytidylyltransferase (Utal et al., 1991) and monoacylglycerol acyltransferase (Coleman, 1992), and increases the potential for membrane fusion (Siegel et al., 1989). In this review of DAG metabolism, we do not discuss details of enzymology that have been well-covered elsewhere (Bell and Coleman, 1983; Bishop and Bell, 1988; Hjelmstad and Bell, 1991a), nor do we discuss the intracellular trafficking of individual phospholipids (Voelker, 1991), or the metabolism of glycerolipids in bacteria, plants, and yeast. The metabolism of DAG includes the use of DAG as a substrate for the synthesis of complex glycerolipids, as well as the recycling or hydrolysis of DAG. We will examine DAG formation, use, and metabolism from these viewpoints.
II. DIACYLGLYCEROL IS A SUBSTRATE FOR TRIACYLGLYCEROL AND COMPLEX LIPID SYNTHESIS Three synthetic enzymes (diacylglycerol acyltransferase, cholinephosphotransferase, and ethanolaminephosphotransferase) in the endoplasmic reticulum (ER) incorporate DAG into triacylglycerol, phosphatidylcholine, and phosphatidylethanolamine. Studies of the regulation of these enzymes in mammalian cells have been hampered by the lack of purified enzymes or cloned cDNAs. The specific activity of diacylglycerol acyltransferase, the enzyme unique to triacylglycerol
Diacy Iglycerol
Metabolism
339 Attenuation/ Recyciing
de novo Synthesis PHOSPHATIDYLINOSITOL PHOSPHATIDYLGLYCEROL PHOSPHATIDYLSERINE CARDIOLIPIN
GLYCEROL 3-P Acyl-CoA A ^ CoA-*-^
CDP-DIACYLGLYCEROL ,
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GLYCEROL + FATTY ACID
A ^ ' CERAMIOE ---PHOSPHATIDICACID
Phosphatidylcholine -
sn-2-MONOACYLGLYCEROL Sphingomyelin
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"
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Attenuation/ Products CDP-choline CMP PHOSPHATIDYLCHOLINE' CDP-ethanolamine CMP
PHOSPHATIDYLETHANOLAMINE
PHOSPHATIDYLCHOLINE TRIACYLGLYCEROL
TRIACYLGLYCEROL
Figure / . Enzymes of diacylglycerol metabolism in mammals and their subcellular locations. The enzymes that catalyze these reactions are: 1, sr7-glycerol-3-phosphate acyltransferase (EC 2.3.1.15); 2, lysophosphatidic acid acyltransferase (EC 2.3.1.0); 3, phosphatidic acid phosphohydrolase 1 ; 4, sphingomyelin synthase; 5, monoacylglycerol acyltransferase; 6, diacylglycerol kinase; 7, diacylglycerol lipase; 8, diacylg l y c e r o l c h o l i n e p h o s p h o t r a n s f e r a s e (EC 2 . 7 . 8 . 2 ) ; 9, d i a c y l g l y c e r o l ethanolaminephosphotransferase (EC 2.7.8.1); 10, diacylglycerol acyltransferase (EC 2.3.1.20); 11, triacylglycerol lipase; 12, phospholipase D; 13, phosphatidic acid phosphohydrolase 2; 14, phospholipase C. Subcellular locations are indicated by the following superscripts: a, plasma membrane; b, endoplasmic reticulum; c cytosol; d, mitochondrial membrane; e, nuclear membrane; f, Golgi; g, lysosomes.
synthesis, varies by as much as 100-fold in different tissues and cultured cells, during differentiation of 3T3-L1 adipocytes (Coleman et al., 1978) and perinatal liver (Coleman and Haynes, 1983), and under conditions of physiological stress such as diabetes (Schoonderwoerd et al., 1990; Mostafa et al.,1993) and hepatic regeneration (Tijburg et al., 1991). These data suggest that regulation of triacylglycerol synthesis is related to the amount of diacylglycerol acyltransferase activity. Recent studies have also indicated that phosphorylation activates diacylglycerol acyltransferase in guinea pig parotid gland (Soling et al., 1989a,b) and in hamster fibroblasts (Maziere et al, 1986), but that phosphorylation inactivates the activity in rat liver (Haagsman et al, 1982) and adipose tissue (Rodriguez et al., 1992).
340
ROSALIND A. COLEMAN and STEVEN H. ZEISEL
Studies in permeabilized rat hepatocytes suggest that the same pool of microsomal DAG is used by cholinephosphotransferase and diacylglycerol acyltransferase. The amount of phosphatidylcholine synthesized appears to be controlled by the availability of cytidine diphosphate- (CDP) choline (Stals et al, 1992), whereas the amount of triacylglycerol that is synthesized is limited by the activity of diacylglycerol acyltransferase (Mayorek et al., 1989). This view implies that a relative lack of diacylglycerol acyltransferase activity might result in the accumulation of DAG in the ER or other membranes. CDP-choline is synthesized by the CTP: phosphocholine cytidylyltransferase, an ambipathic enzyme that, like PKC and PAPase 1, is active in its membrane-associated form (Vance, 1990). Recently, Kent and colleagues localized rat liver CTP:phosphocholine cytidylyltransferase in the nucleus (Wang et al., 1993,1995). It translocates from cytosol to microsomes when it is dephosphorylated, when oleic acid concentration is high, and when the membrane content of phosphatidylcholine decreases (Vance, 1990). Thus, under these conditions, an increase in the availability of CDP-choline would enhance the utilization of DAG substrates by diacylglycerol cholinephosphotransferase. In heart, cholinephosphotransferase has a greater apparent affinity for plasmenyl-diglycerides than for DAGs; synthesis of plasmenylcholine and phosphatidylcholine were equal despite the fact that DAG is present at more than 10-times the mass of endogenous ^A2-l-0-alk-r-enyl-2-acylglycerol (Ford and Gross, 1988). The cDNAs of yeast diacylglycerol choline- and ethanolamine-phosphotransferases have been cloned (Hjelmstad and Bell, 1991 b), but neither enzyme has been purified from any source. Both choline- and ethanolamine-phosphotransferases are predominantly microsomal activities but some cholinephsphotransferase activity may be present in Golgi (Vance and Vance, 1988), thereby providing a potential product for the DAG that has been synthesized by the sphingomyelin synthase (see below). Synergistic activation of PKC by DAG and lysophospholipids has also been described (Sasaki et al., 1993). Few studies have focused on ethanolaminephosphotransferase, its regulation, or its role in DAG metabolism. Phosphatidylethanolamine is highly enriched in polyunsaturated fatty acids, and it has been suggested that the ethanolaminephosphotransferase prefers DAG substrates that contain C22:6, C20:4, and C22:5 fatty acids (Masuzawa et al., 1986). In rabbit heart, ethanolaminephosphotransferase is 16 fimes more selective for 5«-renyl-2-acylglycerol compared to 5«-l,2-diacylglycerol, and use of endogenous substrates was greater for the plasmenyldiglycerides of which 75% contained arachidonic acid at the sn-2 position (Ford et al., 1992).
Hi. DIACYLGLYCEROL ACTIVATES PROTEIN KINASE C PKC is a major second messenger whose activation triggers a wide variety of cellular responses including photoreceptor function (Kapoor et al., 1987; Ilincheta
Diacylglycerol
Metabolism
PKC isotvpe
341
Structure
a, P, Y 6, e, r\, e
C1 t,X
ines ' ^ phosphatidylserines
Eaa^Hg^lLCl^
diacylglycerol ^^^Jru,di! ^ regulatory domain ^ ' (mimics substrate)
site
catalytic site C4>ATP binding site Figure 2, Isotypes of protein kinase C and their activation. The isotypes of protein kinase C (PKC) share some sequence homology. All have a common ATP binding site (C3) and catalytic site (C4). Some isotypes also have a calcium binding site (C2), and/or a lipid binding site (CI). Normally, the catalytic site is occupied by a region of the enzyme that resembles the substrate (pseudosubstrate). When the concentration of intracellular calcium increases, PKC becomes more closely associated with membranes that contain phosphatidylserine, making it possible for DAG to bind to PKC. When this occurs, the conformation of PKC changes, exposing the catalytic site and thereby activating it.
and Giusto, 1992), neurotransmission (Shearman et al., 1991), T-cell activation (Berry and Nishizuka, 1990), platelet activation (Kajikawa et al., 1989), cell proliferation (Berridge et al., 1985), oocyte fertilization (Berridge et al, 1988), insulin response (Christensen et al, 1987; Farese, 1988; Metz, 1988), vasopressin response (Bondy and Gainer, 1988; Brown and Chen, 1990), receptor response to muscarinic hormones (el Fakahany et al, 1988), and the response to prolactin in liver (Buckley et al., 1988). PKC may be involved in thousands of additional essential cell functions (Nishizuka, 1992). At least 10 isoforms of PKC exist, each encoded by a distinct gene (Figure 2) (Nishizuka, 1992). PKCa, pi/2, and y are Ca^'^-dependent. PKC 5, 81/2, ^ 6, and Tj lack the calcium binding C2-domain of PKC and are not, therefore, calciumdependent (Stabel and Parker, 1991). Calcium acts by increasing the tightness w^ith
342
ROSALIND A. COLEMAN and STEVEN H. ZEISEL
which susceptible PKC isoforms associate with membranes, thereby increasing membrane occupancy. This facilitates DAG binding to the cysteine-rich sequences of the CI domain of PKC. These CI domains contain tightly bound zmc ions. Two such sequences are present on all PKCs except for the C^ isoform which has only one (Crabos et al., 1992; Rando and Kishi, 1992). The C^ isoform may (Crabos et al., 1992) or may not (Nakanishi and Exton, 1992; Nishizuka, 1992) be capable of binding DAG. When DAG binds to PKC, the complex approaches the membrane more closely, thereby placing the kinase in a pocket of negatively charged phosphatidylserine head groups, into which additional Ca^"^ is attracted. Thus, DAG increases the affinity of PKC for calcium. Normally PKC is folded so that an endogenous "pseudosubstrate" region on the protein is bound to the catalytic site and kinase activity is inhibited. The binding of DAG and Ca^"^ induces flexion at a hinge region of PKC, causing the pseudosubstrate to withdraw and unblocking the PKC catalytic site (Figure 2) (Stabel and Parker, 1991). Although most cellular DAG arises via de novo synthesis by the glycerol-3-P pathway, it is believed that PKC is activated only by the DAG that is released from membrane phosphatidylinositol-bisphosphate (PI-P2) and phosphatidylcholine after these phospholipids are hydrolyzed by receptor-mediated activation of phospholipases. Hydrolysis of PI-P2 by phospholipase C also releases inositol-1,4,5-trisphosphate (Ins-1,4,5-P3) which interacts with receptors on the ER to release calcium. The resulting increase in cytosolic calcium makes more calcium available for binding to the Ca^"^ dependent-PKC isoforms (Berridge and Taylor, 1988). Free fatty acids can also activate PKC. In the absence of DAG, oleate preferentially activates "soluble" rather than membrane-bound PKC (Touny et al., 1990). In the presence of DAG, fatty acids act synergistically to increase PKC activation (Yoshida et al, 1992). These observations may be related to differences in the isoforms of PKC present in the experimental systems used. The time period during which PKC remains activated depends on the continued presence of DAG (Nishizuka, 1992). In a number of cell types, a prolonged phase of agonist-induced DAG production continues after the hydrolysis of PI-P2. The acyl groups present in this late-phase DAG are consistent with its formation from phosphatidylcholine (Nishizuka, 1992). Phosphatidylcholine hydrolysis is catalyzed either by a specific phospholipase C (releasing DAG and phosphocholine) or by phospholipase D plus phosphatidic acidphosphohydrolase (releasing, sequentially, choline plus phosphatidic acid and then DAG plus inorganic phosphate) (Huang et al., 1992). Thus, phosphatidylcholine hydrolysis acts to sustain the DAG message that was initially transmitted via the hydrolysis of PI-P2. A.
Nuclear Protein Kinase C
The nuclear membrane is a second site for PKC activation, and different isoforms may be present in different tissues. Incubation of glioblastoma cell lines with phorbol-myristyl-acetate, which acts at the DAG-binding site of PKC, results in
Diacylglycerol Metabolism
343
the translocation of 48% and 33% of cytosolic PKC-a to the nuclear and plasma membranes, respectively, whereas PKC-y is not translocated (Misra-Press et al, 1992). In brain, both PKC-a and -y are associated with the nuclear membrane (Buchner et al., 1992). During liver regeneration, nuclear PKC-a decreases and -5 increases (Alessenko et al., 1992), and in leukemia HL60 cells, PKC-P II, but not -a, associates with the nuclei after treatment with bryostatin (Hocevar and Fields, 1991). Activated PKC phosphorylates a nuclear Ins-1,4,5-P3 receptor and thereby increases Ca^"^ release by Ins-1,4,5-P3 (Matter et al., 1993). Among other proteins phosphorylated by PKC are lamin B2 (Matter et al., 1993) and an 80 kDa protein (Steinschneider et al., 1991). Phosphorylation of the nucleolar protein B23 (Beckmann et al., 1992) indicates that PKC has access to the interior of the nucleus as well as to the nuclear membrane. B. Regulation of Protein Kinase C by Sphingolipids
The PKC phosphorylation cascade can be terminated by messengers produced by sphingomyelin hydrolysis (Hannun et al., 1986, 1991). More recent reviews of sphingomyelin signaling (Hannun, 1994; Jayadev et al., 1995). Many of the agonists that induce PI-P2 hydrolysis activate sphingomyelinase in the plasma membrane (Slife et al., 1989) and release phosphocholine and ceramide (Merrill and Jones, 1990; Merrill, 1992). Ceramide promotes cell differentiation (Merrill, 1992), induces apontosis (Obeid et al., 1993), and is a competitive inhibitor of DAG kinase (Younes et al., 1992). Cleavage of ceramide releases sphingosine, a potent inhibitor of PKC. Sphingosine acts by blocking DAG-mediated activation of PKC, perhaps by binding at or near the CI domain of PKC (Merrill and Stevens, 1989). Sphingosine also inhibits phospholipases A2 and D, (Franson et al., 1992) and PAPase (Mullmann et al., 1991).
IV. SOURCES OF DIACYLGLYCEROL The cellular content of DAG depends on the relative rates of DAG formation, utilization, and degradation. DAG is formed in two ways, by de novo synthesis from glycerol-3-P, dihydroxyacetone-P, monoacylglycerol, and the phosphatidylcholine-related synthesis of sphingomyelin, and from the recycling of intracellular glycerolipids (Figure 1). Apart from the possible entry into cells of synthetic medium-chain DAGs (see below), there is no evidence that natural DAGs enter cells directly. Although the relative contribution of recycling is not known, it is likely that most DAG originates from de novo synthesis. Synthesis would be especially prominent in cells whose rapid rates of division require extensive biogenesis of new membranes, and in cells—such as hepatocytes, intestinal mucosa cells, adipocytes, and mammary cells—that synthesize large amounts of triacylglycerol.
344
ROSALIND A. COLEMAN and STEVEN H. ZEISEL A.
Diacylglycerol From De Novo Synthesis
The glycerol-3-P pathway, first described by Kennedy and his collaborators (Bell and Coleman, 1983), provides the major route for the de novo synthesis of DAG in most cells. Changes in the DAG content of the ER must primarily be the result of a balance between the flux of intermediates through the glycerol-3-P pathway and the utilization of DAG by the activities that synthesize triacylglycerol, phosphatidylcholine, and phosphatidylethanolamine. The committed step of the synthetic pathway is the acylation of glycerol 3-P by the glycerol-3-P acyltransferase. Independent glycerol-3-P acyltransferase isoenzymes are present in microsomes and mitochondria (Bell and Coleman, 1983; Hjelmstad and Bell, 1991a). Neither isoenzyme has been purified. Rat liver mitochondrial glycerol-P acyltransferase (Vancura and Haldar, 1994) and the recombinant murine enzyme (Yet et al., 1995) have recently been purified. The mitochondrial isoenzyme appears to have been cloned, and its mRNA abundance is regulated by fasting and refeeding (Shin et al., 1991). The microsomal glycerol-3-P acyltransferase activity increases 70-fold during 3T3-L1 adipocyte differentiation (Coleman et al., 1978) and in perinatal rat liver (Coleman and Haynes, 1983), suggesting that it plays a major role in glycerolipid synthesis in these tissues. The second pathway enzyme, the lysophosphatidic acid acyltransferase, is also located in both microsomes and mitochondria and acylates 5'«-l-acyl,2-lyso-glycerol-3-P to form phosphatidic acid. The activity increases 60-fold during adipocyte differentiation (Coleman et al., 1978) and eightfold in perinatal rat liver (Coleman and Haynes, 1983). Lysophosphatidic acid acyltransferases have recently been cloned from several plant species (Brown et al., 1994). In the acylation of the alternative triose-P, the dihydroxyacetone-P, the resulting products must first be reduced to l-acyl,2-lyso-glycerol-3-P or to phosphatidic acid before DAG can be synthesized (Bell and Coleman, 1983; Hjelmstad and Bell, 1991a). Little information is available on the regulation of any of these activities. The final step in the synthesis of DAG is catalyzed by phosphatidic acid phosphohydrolase (PAPase). At least two PAPase isoenzymes are present in cells. PAPase 1, which is distinguished by its sensitivity to A^-ethylmaleimide and its location in the ER, functions in the glycerol-3-P pathway to catalyze the release of inorganic phosphate and DAG from phosphatidic acid. PAPase 1 activity is ambipathic and moves from cytosol to the ER when oleate is added to the media of cultured cells (Walton and Possmayer, 1986; Day and Yeaman,1992). Although work in permeabilized cells suggests that PAPase does not regulate this pathway (Mayorek and Bar-Tana, 1985; Stals et al., 1992), other work supports the role of PAPase 1 in regulating the flux of phosphatidic acid towards DAG (and subsequently triacylglycerol, phosphatidylcholine, and phosphatidylethanolamine synthesis) or towards the synthesis of the anionic phospholipids, phosphatidylglycerol, phosphatidylinositol, and cardiolipin via CDP-DAG (Brindley, 1984). Okadiac acid prevents translocation of PAPase 1 to the ER, suggesting that phosphorylation is involved (Gomez-Munoz et al., 1992). Because the soluble form
Diacylglycerol Metabolism
345
of the activity is stimulated by phosphatidyiethanolamine and inhibited by the anionic phospholipids (Humble and Berglund, 1991), membrane composition might alter the flux between the synthesis of the anionic phospholipids and the synthesis of phosphatidylcholine and phosphatidyiethanolamine. Since PAPase 1 is not present in mitochondria, any phosphatidic acid synthesized by the mitochondrial glycerol-3-P acyltransferase and lysophosphatidic acid acyltransferase must either be used as a substrate for the synthesis of the anionic phospholipids or be transported to the ER. Phosphatidic acid appears to be an intracellular second messenger (Breittmayer et al., 1991; Metz and Dunlop, 1991; Cano et al, 1992; Purkiss and Boarder, 1992); thus, its synthesis and transport is likely to be closely regulated. The monoacylglycerol pathway provides the major route for DAG synthesis in intestinal mucosa and, thus, for the synthesis of chylomicra-triacylglycerol (Polheim et al., 1973). This pathway is also prominent in liver during selected physiological periods characterized by high rates of lipolysis and/or P-oxidation (Coleman, 1992). Monoacylglycerol acyltransferase (MGAT), the enzyme that characterizes the monoacylglycerol pathway, stereospecifically synthesizes 5«-l ,2DAG (Coleman et al., 1986) from 5«-2-monoacylglycerol and long-chain fatty acyl-CoA. The 5«-2-monoacylglycerol substrate is a competitive inhibitor of glycerol-3-P acyltransferase (Polheim et al., 1973; Coleman, 1988), suggesting that when large amounts of monoacylglycerol are present in microsomal membranes, the monoacylglycerol pathway of DAG synthesis would predominate. In liver, partial lysosomal hydrolysis of lipoprotein remnant-glycerolipids may provide the main source of the monoacylglycerol substrate, since as much as 90% of the product of acid DAG lipase is monoacylglycerol (Xia and Coleman, 1992). Experiments performed in tetrahymena have provided direct evidence for the lysosomal route (Arai et al., 1987). A second route may involve the hydrolysis of triacylglycerol from storage droplets (Wiggins and Gibbons, 1992). MGAT can use xenobiotic carboxylic acids to acylate 5«-2-monoacylglycerol (Dodds, 1991); some of these DAG analogues can activate PKC. Little information is available concerning the regulation of MGAT in cells other than liver and intestinal mucosa. In Balb/c 3T3 cells, however, platelet derived growth factor (PDGF) appears to stimulate the incorporation of labeled fatty acids into DAG via the monoacylglycerol pathway (Hata et al., 1989). The synthesis of 5«-l-C18:0,2-C20:4 phosphatidylinositol from 5«-2-C20:4-glycerol apparently proceeds via the acylation of 2-acylglycerol-P followed by action of phosphatidic phosphohydrolase 1 rather than by an initial acylation to DAG (Simpson et al., 1991). DAG is also a product of the formation of sphingomyelin from phosphatidylcholine and ceramide catalyzed by phosphatidylcholine xeramide choline phosphotransferase (Marggraf and Kanfer, 1984; Merrill and Jones, 1990; Koval and Pagano, 1991).
346
ROSALIND A. COLEMAN and STEVEN H. ZEISEL B. Diacylglycerol Arises via Recycling of Cellular Glycerolipids
/.
Phospholipase C
Numerous PI-P2 specific phospholipases C exist, including proteins varying in size from 70 to 154 kDa, designated P through y (Meldrum et al., 1991). All share two highly conserved domains which form the catalytic region. It is believed that specific receptors couple to specific phosphatidylinositol-phospholipase C isotypes (Meldrum et al., 1991). Agonist-mediated activation is regulated by two G-protein subunits in liver plasma membrane: a 43 kDa a subunit and a 35 kDa P subunit (Exton et al., 1992). Immunological studies show that the a subunit is closely related to the Gq class of G-proteins (Stemweis and Smrcka, 1992). The muscarinic receptor, for example, operates via this mechanism after it interacts with its ligand acetylcholine (Exton, 1990; Qian and Drewes, 1990). 2.
Phospholipase D and Phosphatidylcholine-Specific
Phospholipase C
Though phospholipase C-mediated hydrolysis of PI-P2 was once thought to be the sole mechanism for DAG production, the hydrolysis of phosphatidylcholine appears to sustain the production of DAG during the PKC signal-transduction cascade. Phosphatidylcholine, the major substrate for phospholipase D, is cleaved to form phosphatidic acid and choline (Billah et al., 1989). Conversion of phosphatidic acid to DAG is then catalyzed by PAPase 2 (see below). Some agonists trigger phospholipase D-mediated phosphatidylcholine hydrolysis via the activation of PKC (Nishizuka, 1992) in a G-protein mechanism coupled to the receptor (Garland, 1992). The ability of PKC to activate phospholipase D may be mediated by a separate catalytic site that does not involve an ATP-dependent phosphorylation (Conricode et al., 1992). Inhibitors of PKC, and down-regulation of PKC attenuate phosphatidylcholine hydrolysis. Phospholipases D and A2 (which releases fatty acid and 1 -acyl, 2-lysophosphatidylcholine) are stimulated by intracellular calcium, thereby modulating phosphatidylcholine hydrolysis. Also, it is likely that the activation of tyrosine kinases is directly linked to the hydrolysis of phosphatidylcholine (Exton, 1990). This linkage may explain why the stimulation of phosphatidylcholine hydrolysis by epidermal growth factor and PDGF elevates cellular DAG for hours even though PI-P2 hydrolysis is not triggered. In a similar manner to that previously described for PI-P2-specific phospholipase C, specific receptors appear to be linked to activation of specific phosphatidylcholine-phospholipases C (Exton, 1990). GTP analogues and Mg^"^ stimulate phosphatidylcholine hydrolysis in rat liver plasma membranes, indicating that a G-protein may mediate this phospholipase C activation (Exton et al., 1992). C. Source of Nuclear Diacylglycerol
The DAG responsible for the translocation of PKC from cytosol to the nuclear membrane is generated via phospholipase C-mediated PIP and PIP2 hydrolysis.
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possibly occurring within the nucleus itself (Manzoli et al, 1988; Divecha et al., 1991; Cocco et al, 1992; Martelli et al, 1992). IGF-I stimulation of Swiss 3T3 fibroblasts results in a decrease of PI-P and PI-P2 mass and an increase in nuclear DAG that precedes PKC translocation (Divecha et al., 1991; Martelli et al., 1992). Phosphatidylinositol turnover occurs via the nuclear P isozyme of phosphoinositidase C; the cytosolic phosphoinositidase C is unaffected (Martelli et al., 1992). Stimuli for PKC translocation (and thus, presumably, stimuli for DAG production) include IL-4 for human monocytes (Arruda and Ho, 1992), and ACTH for rat adrenal (Lehoux et al., 1991). Both DAG kinase and phospholipase C activities have been identified in the internal matrix of the nucleus (Payrastre et al., 1992). D.
Phosphatidic Acid Phosphohydrolase 2
In addition to the PAPase 1 that is part of the glycerol-3-P pathway of triacylglycerol, phosphatidylcholine, and phosphatidylethanolamine synthesis, a second PAPase is located in the plasma membrane (Jamal and et al., 1991; Day and Yeaman, 1992). This 7V-ethylmaleimide-resistant PAPase 2 catalyzes the release of DAG from the phosphatidic acid that arises from phospholipase D hydrolysis of phosphatidylcholine. The two PAPases from rat liver were partially purified by gel filtration and anion-exchange chromatography (Day and Yeaman, 1992). The plasma membrane PAPase has an apparent M(r) of 240,000 and the ER form has an apparent M(r) of 540,000. In neutrophils, sphingosine inhibits PAPase hydrolysis of alkyl, acyl-phosphatidic acid (Mullmann et al., 1991). The alkyl, acyl-PAPase, alkyl, acetyl-PAPase, and PAPase 2 are probably different proteins (Lee et al., 1988). When 0.1 mM oleate is added to rat liver plasma membranes, [^H]choline is released from phosphatidyl[^H]choline and [^"^CJDAG is released from [^^CJphosphatidic acid (Siddiqui and Exton, 1992). These results are consistent with the stimulation of phospholipase D and PAPase 2 by oleate. The data suggest that unsaturated fatty acids may regulate DAG production in the plasma membrane (Siddiqui and Exton, 1992). Involvement of a G-protein in this process is supported by a report that cells transformed with the Hsi-ras oncogene increase DAG mass without a concomitant rise in inositol phosphates (Lacal et al., 1987). PAPase 2 also plays a role in the production of DAG from a special group of phosphatidic acids that contain polyunsaturated fatty acids. These are synthesized by lysophosphatidic acid acyltransferase in response to activation by L-1 (Bursten et al., 1991). This route of DAG formation in mesangial cells may be regulated by a pertussis-sensitive G-protein, independent of the phosphatidylinositol cycle and phospholipase C (Bursten and Harris, 1991). E. Triacylglycerol Lipase
Apart from the hormone-sensitive lipase of adipocytes and adrenal cells, the lysosomal acid triacylglycerol lipase/cholesterol esterase, and several car-
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ROSALIND A. COLEMAN and STEVEN H. ZEISEL
boxyesterases (Mentlein et al, 1987), intracellular triacylglycerol lipases have not been purified. The hormone-sensitive lipase, as well as lipoprotein lipase, and pancreatic lipase hydrolyze both the sn-l- and 3-positions; it is not known whether other cellular triacylglycerol lipases are also DAG lipases. In rat heart the predominant triacylglycerol lipase is lysosomal with a 4.8 pH optimum, whereas the predominant DAG and monoacylglycerol lipases are microsomal and have neutral pH optima (Stam et al., 1986). These data suggest that, in heart, DAG might be released from lysosomes and transported to the ER for further metabolism. How the DAG would be specifically routed to the ER remains unclear. In fetal rat lung, the distribution of triacylglycerol lipase and DAG lipase is similar, but in adult rat lung, DAG lipase is prominent in the cytosol (Brooks and Weinhold, 1986). In primary cultures of bovine chromaffin cells, however, RG 80267, an inhibitor of DAG lipase, had little effect on the pH 4.3 plasma membrane triacylglycerol lipase, suggesting that the two activities might be separable (Galatioto and Zahler, 1993). Carbichol stimulates triacylglycerol lipase activity threefold and increases labeled DAG content, indicating that hormonally-mediated triacylglycerol hydrolysis might be a DAG source (Hundley and Rubin, 1992).
V. DIACYLGLYCEROL SIGNAL ATTENUATION AND RECYCLING A.
Diacylglycerol Kinase
Diacylglycerol kinase regulates the metabolism of diacylglycerol released in agonist-stimulated cells. The major purified form of the enzyme is cytosolic and may become membrane-associated after phosphorylation by PKC in a calcium-dependent manner (Kanoh et al., 1990; Schaap et al., 1990; Sakane et al, 1991). In addition, there are several kinase subspecies immunologically distinct from the major enzyme (Kanoh et al, 1990; Yada et al., 1990). These isozymes exhibit specificity for DAG species that differ in saturation of acyl-groups (Stathopoulos et al., 1990), and different tissues may express different isozymes (Lemaitre et al., 1990). B. Diacylglycerol Lipase
DAG lipase activities have been reported in ER, cytosol, and lysomes. The cytosolic DAG lipase from rat liver is stimulated two- to threefold by snA{3)- and 2-monooleylglycerol ethers (Xia and Coleman, 1992). A pi 5.2 carboxyesterase purified from rat liver microsomes has DAG lipase activity and also hydrolyzes 12-0-tetradecanoyl phorbol 13-acetate and acylcarnitines (Mentlein, 1986).
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Vl. CELLULAR DIACYLGLYCEROL CONTENT AND PROTEIN KINASE C The normal content of DAG in unstimulated cultured cells is 0.2 to 4 nmol/100 nmol phospholipid (nmol percent) (Table I). Typically, the increase in membrane DAG content after phospholipase C is stimulated is between 150 and 300%. Many studies have overestimated the cellular content of DAG because the enzymatic assay (Preiss et al., 1986) does not distinguish between 1,2-diacylglycerol and l-alkyl,2-acylglycerol. Thus, in most cases, the changes listed in Table I are really those of diradylglycerol rather than DAG. Few studies in stimulated cells have both quantified DAG and have also shown that PKC is simultaneously activated. In most studies, PKC activation is used to infer an increase in cellular DAG content. The discovery of PKC isoforms that do not require DAG for activation (Nakanishi and Exton, 1992) indicates that this assumption may not always be justified. Additionally, an increase in cellular DAG content is not invariably associated with PKC activation. Lack of concordance between cellular DAG concentration and PKC activation was reported in rat-6 PKC-3 fibroblasts that overexpress the 1 isozyme of PKC (Pai et al., 1991), and in a-thrombin-stimulated fibroblasts (Leach et al., 1991). Either the DAG was sequestered in a membrane compartment that is uninvolved in PKC activation or the presence of inhibitors of PKC counteracted the effect of the increased DAG. Similar lack of concordance between the cellular DAG concentration and PKC activity and location was observed in postnatal rat liver (Xia and Coleman, 1992), although the changes in cellular DAG concentrations were equal to those that have been associated with profound alterations in PKC-mediated events (Wright et al., 1988). Hepatic DAG content increases dramatically from 600 nmoles per liver in control fed animals to 15,000 nmoles per liver in choline-deficient rats (daCosta et al., 1993). This prolonged elevation of DAG and activation of PKC in choline deficient rat liver is now known to last more than 27 weeks (da Costa et al., 1995). However, most of the DAG is sequestered in triacylglycerol-rich lipid droplets and only a small portion of the DAG is present in cell membranes (150% of control DAG levels) where it is presumably capable of activating PKC. In cells such as liver and adipocytes, the amount of DAG produced in response to hormone-activated phosphatidylcholine and PI-P2 hydrolysis is small compared to the amount of DAG routinely produced by the hydrolysis of intracellular droplet triacylglycerol, and, in liver, the lysosomal hydrolysis of triacylglycerol that has entered the cell with chylomicron and VLDL remnants. It is likely that the glycerol-3-P pathway provides the major source of DAG in most cells. The problem is intensified, however, in cells that are rapidly dividing or that specialize in triacylglycerol synthesis, such as adipocytes, hepatocytes, hepatic lipocytes (Ito cells), intestinal mucosa cells, and mammary cells. Furthermore, when serum fatty acids rise, cells store them temporarily in triacylglycerol, synthesized via the glycerol-3-P pathway.
ROSALIND A. COLEMAN and STEVEN H. ZEISEL
350
Table 1. Cell Diradylglycerol^ Content: Selected Examples Cell/Fraction/Tissue Cell platelets
Basal DRG
Stimulated DRG
Stimulant
Reference
0.3
thrombin
hepatocytes NRX cells
0.1 nmol/10^ platelet 3.6 nmol% 0.47 nmol%
11 1.26
A431
0.38 nmol%
1.2
Arg-vasopressin 1 Preissetal., 1986 K-ras Preissetal., 1986 transformed time in culture Van Veldhoven and Bell, 1988 serum deprived Paietal., 1991 Thompson et al., 1990 fMetLeuPhe Tyagietal., 1989 fMetLeuPhe
rat-6 fibroblast human neutrophils human neutrophils
1.2nmol% 4nmol/10^cells 10-18 nmol/10^ cells human neutrophils 10-18 nmol/10^ cells 180nmol/10^ NIH 3T3 cells cells 260nmol/10^ Swiss 3T3 cells cells IIC9 hamster fibroblast 0.23 nmol% 0.20 nmol% human fibroblasts
Fraction liver plasma membrane 8.9 nmol/mg protein 12-18 nmol/mg lung microsomes protein 0.1 nmol/mg Swiss 3T3 nuclei protein 6.0 g/mg protein heart microsomes Tissue rat liver postnatal day 1 1.8nmol% 0.7 nmol% days 8-30 4.6 nmol/mg adult protein 140 nmol/g wet rat lung wt fetal 400 nmol/g wet adult wt
0.35 20 1.6-fold increase 6-8-fold increase 500 370
Zymosan
Tyagietal., 1989
bradykinin
Fuetal., 1992
EOF
Cook and Wakelam, 1992 Wright etal, 1988 Van Veldhoven and Bell, 1988
0.59 (early) 0.24
a-thrombin time in culture
12.9
6 wk choline deficient
0.2
Preissetal., 1986
IGF-1
daCostaetal., 1993 Rustow and Kunze, 1987 Divecha et al., 1991
—
Ford etal., 1992
— —
Xiaetal., 1993 Xiaetal., 1993 daCostaetal., 1993
10.1
6 wk choline deficient
—
Brooks and Weinhold, 1986
—
Brooks and Weinhold, 1986
Notes: ^Many of the studies did not distinguish between DAG, l-alkyl,2-acylglycerol, and 1-vinyl-ether, 2-acylglycerol. ''l-Alkyl,2-acylglycerol content was 4.0-9.5 nmol/10^ ceils.
DiacyIglycerol Metabolism
3 51
Hepatic DAG concentrations are altered under a variety of conditions. Rats fed soybean protein, for example, decrease both triacylglycerol and DAG content in liver (Ide et al, 1992b) and soybean phospholipid reduces liver microsomal DAG (Ide et al., 1992a). In partially hepatectomized rats, DAG content is reported to increase (Bocckino et al., 1989) or to remain unchanged (Houweling et al., 1992). DAG is elevated in vivo in ra^-transformed liver from neonatal transgenic mice (Wilkison et al., 1989). NIH 3T3 cells transformed with Ha-ra^ , Ki-ra^, v-src, or \-fms oncogenes contain elevated DAG levels as well as tonically activated and partial down-regulated PKC (Wolfman et al., 1987). These elevated DAG levels are produced by activation of phosphatidylcholine hydrolysis (Price et al., 1989; Diaz-Laviada et al., 1990). In erft^-transformed fibroblasts DAG accumulates because its removal by DAG kinase is slowed (Kato et al., 1989).
VII. MOVEMENT OF DIACYLGLYCEROL WITHIN CELLS The existence of the numerous sources and fates of DAG make it necessary to reconcile several disparate findings by answering the following questions: 1. Do separate pools of DAG exist, one that activates PKC, the other destined for synthesis of triacylglycerol and phospholipid? At present, this question remains unresolved. Data have been presented to support the hypothesis that only a small fraction of the endogenous microsomal DAG is actually available for phosphatidylcholine synthesis (Rustow and Kunze, 1987). It has been suggested that glycerolipid intermediates are channeled between the glycerolipid synthetic enzymes in a multienzyme complex (Rustow and Kunze, 1987). This idea is supported by the finding that choline, but not phosphocholine or CDP-choline, can be incorporated into phosphatidylcholine in glioma cells permeabilized by electroporation (George et al., 1989). Although evidence for channeled intermediates was not found in HeLa cells permeabilized with digitonin, it is possible that the detergent disrupted the postulated multienzyme complex (Vance, 1990). 2. Is the DAG that is synthesized in the ER, lysosomes, and Golgi restricted from moving to the plasma and nuclear membranes that are involved in PKC-related signaling? Existing data strongly indicate that DAG moves freely within and between membranes. In egg phosphatidylcholine bilayers and small unilamellar vesicles, NMR spectra with [13C]-l,2-dilauralglycerol showed two carbonyl resonances, consistent with the presence of 1,2-dilaural glycerol on both the outer and inner leaflets of the vesicle bilayer (Hamilton et al, 1991). At 38° C only a single resonance was observed, suggesting equilibration of the two monolayer D AGs. The rate constant was approximately 60 s~' with a tj/2 of 10 ms, indicating very rapid transbilayer movement (Hamilton et al., 1991). DAG is able to rapidly traverse natural membrane bilayers and artificial membranes (Pagano, 1988). In in vivo studies, the use of fluorescent phosphatidic acid has provided evidence for the rapid movement of DAG from plasma membrane to
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ROSALIND A. COLEMAN and STEVEN H. ZEISEL
the ER. When Chinese hamster fibroblasts are incubated with C^-NBD-PA liposomes at 2° C, phosphate is released, presumably by PAPase 2. The resulting C^-NBD-DAG rapidly crosses the bilayer and translocates to intracellular membranes that include mitochondria, ER, and the nuclear envelope (Pagano and Longmuir, 1985). When cells are prelabeled with P32, the resulting C^-NBD-PA becomes labeled, indicating phosphorylation by DAG kinase. Since movement of C^-NBD-DAG between small unilamellar vesicles is slow (tj/2 = 168 minutes at 5° C), this study suggests that cellular DAG moves between membranes by facilitated translocation. Further, the C^-NBD-DAG is metabolized to phosphatidylcholine and triacylglycerol when cells are subsequently warmed to 37° C (Pagano et al., 1983), indicating that the DAG transported to the ER is available to the DGAT and CPT activities. These data argue against channeling of glycerol-3-P metabolites in the synthetic pathway. Further work using [^"^CjDAGs that are incorporated into NIH Swiss 3T3 fibroblasts via liposome fusion shows that the incorporation of the labeled DAG into specific phospholipids depends strongly on the DAG acyl-composition (Florin-Christensen et al, 1992). Although triacylglycerol and phosphatidylcholine are major products with both l-C18:0,2-C14:0-glycerol and 1-C18:0, 2-C20:4-glycerol, little of the disaturated compound is incorporated into phosphatidic acid and phosphatidylinositol, whereas phosphatidic acid and phosphatidylinositol are significant products from the 1-CI8:0,2-C20:4-glycerol substrate. The authors of this study concluded that attenuation of a DAG signal originating at the plasma membrane includes both the use of the DAG or its immediate metabolite, phosphatidic acid, in the synthesis of complex glycerolipids as well as in the lipolytic breakdown of DAG to monoacylglycerol and fatty acid. Incubation with the DAG kinase inhibitor R59022 or the DAG lipase inhibitor RG 80267 alters the amount of label that is incorporated into phosphatidic acid (and phosphatidylinositol) or into monoacylglycerol and fatty acid, respectively, but has little effect on the amount of label incorporated into triacylglycerol and phosphatidylcholine. This study indicates that synthesis of complex lipids is not merely a function of the amount of DAG available. In a cell-free system, DAG can also move from transitional elements of the ER to the Golgi; this transfer is stimulated by ATP (Moreau and Morre, 1991). 3. Current data suggest that DAG activates PKC only at the plasma and nuclear membranes. What prevents PKC activation at the ER, or the lysosomal and Golgi membranes? If DAG is sequestered, does the presence of PKC inhibitors outweigh the effect of DAG on PKC activation, or are appropriate PKC isoforms and substrates absent from these other membranes? PKC activity has been measured in a mitochondrial fraction from mouse kidney cortex; there, a 43 kDa mitochondrial protein is phosphorylated in the presence of DAG and calcium (Boneh and Tenenhouse, 1988). PKC has been localized to presynaptic vesicles in presynaptic vesicles of axon terminals (Wood et al., 1986; Kose et al, 1988). 4. After DAG is produced in plasma and nuclear membranes, where is the DAG signal attenuated? Is the DAG metabolized within its membrane of origin, allowing
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transport of a metabolite, such as phosphatidic acid, to another membrane, or does the transport process itself function to attenuate the signal? If DAG itself is transferred, how is the transfer accomplished? Since the nuclear membrane is itself an extension of the ER, how is the DAG released from the hydrolysis of PI-P2 kept separate from the DAG synthesized by the glycerol-3-P pathway via PAPase 1? Golgi DAG might be transferred to the plasma membrane and then recycled to the ER via budding of membrane vesicles, direct membrane connections, or lipid carrying proteins. Of potential interest is the identification of a soluble DAG-transfer protein in yeast mitochondrial intermembrane space (Muller and Bandlow, 1989). To test the hypothesis that DAG released at the plasma membrane might be recycled back to PI-P2 within the plasma membrane, the activities of the enzymes needed were measured in purified plasma membrane from cultured neural cells (Morris et al., 1990). Only DAG kinase and PIP kinase were found in the plasma membrane. CTPiphosphate cytidylyltransferase and CMP-phosphate:inositol phosphatidyltransferase were located only in a microsomal fraction enriched in ER markers (Morris et al, 1990). Thus, resynthesis of PI-P2 must require transport of DAG or phosphatidic acid to the ER.
VIII. MOLECULAR SPECIES OF DIACYLGLYCEROL AND DIRADYLGLYCEROL The fatty acid species in phosphatidylcholine differ from those in PI-P2; thus, the diradylglycerols (DRGs) generated from each will differ. PI-P2 consists predominantly of the ester-linked 1-stearoyl, 2-arachidonoyl (18:0/20:4) species (Holmsen et al., 1992). In contrast, phosphatidylcholine contains both ester-linked 1,2-diacylglycerol (DAG) species, and ether- or vinyl-linked species (1-alkyl, 2-acyl-glycerols (AAG)). Within each of these DRG species in phosphatidylcholine, the lengths and degree of unsaturation of the radyl chains differ considerably. Thus, the DRG species available in cells may vary significantly depending on the dietary intake of fatty acid species as well as upon whether they have been generated from phosphatidylcholine or from PI-P2. This individuality of DRG molecular species determines packing density, metabolic fate, and difference in substrate presentation. In rat sciatic nerve (Zhu and Eichberg, 1990) and rat brain (LeeandHajra, 1991)the 18:0/20:4 and the 16:0/18:1 species of DRG predominate. Rat retina DAGs(Stinsonetal., 1991) contain mainly 18:0/20:4,18:0/22:6n-3 and 16:0/20:4 species. A. Molecular Species of Diaradylglycerols and Protein Kinase C Activation The differences in DRG species released from membrane phospholipids may have important consequences for PKC activation. It is possible that subspecies of DRG are recognized by special PKC isoforms that act in different membranes. This
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ROSALIND A. COLEMAN and STEVEN H. ZEISEL
could provide a mechanism that maintains signal specificity when specific agonists appear to use common signal transduction pathways. The subclasses of DRG (DAG, AAG,and vinyl-A AG) may differ in their ability to activate PKC (Cabot and Jaken, 1984; Dawson and Cook, 1987; Heymans et al, 1987; Daniel et al., 1988; Bass et al, 1989; Ford et al., 1989). DAG is the best studied activator of PKC. Although AAG was thought to be incapable of activating PKC (Cabot and Jaken, 1984), recent studies indicate that naturally occurring species of AAG can be activators (Dawson and Cook, 1987; Ford and Gross, 1988; Ford et al., 1989). Activation may require the high concentrations of free calcium that are only found in stimulated cells (Ford et al., 1989). Compared to the corresponding DAG, the presence of an alkyl group in the sn-1 -position diminishes the molecule's ability to activate PKC (Heymans et al., 1987), and AAGs that contain short-chain fatty acids appear to be better activators than those with longer-chain fatty acids (Bass et al, 1989). l-O-alkyl-2-acetyl analogues of AAG inhibit PKC activation by DAG (Daniel et al., 1988). These acetyl analogues may differ from other AAGs in biologic activity and their biologic activity may depend on their conversion to l-alkyl,2-acetyl-glycerophosphocholine (platelet activating factor) (Rider et al., 1988). Another problem in ascertaining the role of AAGs is the fact that the structural requirements for interaction with PKC have been primarily studied in detergent/lipid micelles. The assumption that the relevant recognition sites are similar in detergent/lipid micelles and in natural phospholipid bilayers may not be valid (Bonser, FEBS Lett., 1988). In lipid-micelle studies, most of the specificity of PKC for DAG lies near the hydrophilic glycerol backbone (Ganong et al., 1986; Rando, 1988). DAG analogues are less potent activators of PKC if they lack either the free hydroxyl group at the sn-3> position or an ester moiety at the sn-\ position (Molleyres and Rando, 1988). Acyl-chain hydrophobicity is also important for PKC activation (Molleyres and Rando, 1988). Most studies in cultured cells have used the 8:0/8:0 DAG species which is modestly soluble in aqueous solutions and can enter cells (Rider et al., 1988). The 10:0/10:0 species of DAG also enters cells, but longer-chain species do not (Go et al., 1987). The activity of DAG depends strongly on the length of the carbon chain; in lymphocytes, dihexanoylglycerol is a more potent PKC activator than dibutyrylglycerol or diacetylglycerol (Kerr et al., 1987). Of course, in vivo, DAGs with acyl-chains shorter than 16 carbons are not likely to exist. DAG analogues containing hydrophilic polyether-containing ester groups are not active. Diolein, dilinolein, diarachidonin, 1-stearoyl-2-oleoylglycerol, and l-stearoyl-2-linoleoylglycerol are similar in their ability to activate PKC. In contrast, dipalmitin and distearin have almost no ability to activate PKC (Kishimoto et al., 1980). This difference is probably not due to a difference in solubility because adding Triton X-100 does not change the efficacy of these DAGs. Saturation of 1-acyl group is not as important as unsaturation at the ^«-2-position.
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355
The effects of different DAG species and PKC activation in intact cells have been rarely studied. Rat cardiomyocytes grown in medium supplemented with docosahexanoic acid (22:6 n-3) form a docosahexanoic acid-enriched DAG after a-1 adrenoceptor stimulation (Hrelia et al., 1992). The docosahexanoic acid-enriched DAG stimulates a more persistent translocation of PKC from cytosol to plasma membrane, compared to that observed in control unsupplemented cells. In controls, PKC activity in the particulate fraction peaks 30 seconds after stimulation and returns to normal by 60 seconds. In docosahexanoic acid-supplemented cells, PKC activity in the particulate fraction peaks at 30 seconds, but remains associated with membrane for 10 minutes. B. Diacylglycerol Individuality and Removal
Most of the thought as to how DAG molecular species might influence signal transduction has concentrated on the molecular interaction between DAG and PKC. However, accumulation of DAG in cells depends not only on its rate of production but also on its rate of removal. Removal may occur by phosphorylation, hydrolysis, and incorporation into complex glycerolipids. Thus, not only do DAG molecular species vary in binding affinity to PKC, their concentrations within cells may vary if the enzymes catalyzing DAG removal exhibit specificity for particular molecular species of DAG. Examples of enzyme specificity include a DAG kinase that prefers l-stearoyl,2-arachidonyl-glycerol (MacDonald et al., 1988), and the fact that thrombin stimulation of platelets generates more than seven species of DAG, but platelet DAG kinase phosphorylates only one of them (1-18:0,2-20:4 n-6) (Holmsen et al., 1992). This marked specificity observed in vivo is not always observed with in vitro detergent experiments. Isozymes of DAG kinase differ in activity depending on the detergent content of the extraction buffers. In the presence of 1 mM deoxycholate, DAG kinase phosphorylates diC 18:1-glycerol about three times faster than diC 10:0-glycerol. In the absence of deoxycholate, no specificity is noted (Holmsen et al., 1992). The distinct specificity for certain DAG species observed in vivo may actually reflect a net balance between the opposing DAG kinase and PAPase reactions or enhanced activity of DAG lipase for certain DAG species. C.
Diacylglycerol Individuality and Free Fatty Acids Fornned
Some fatty acids greatly potentiate the DAG-dependent activation of PKC (Seifert et al., 1988; Shinomura et al., 1991) as enhancers rather than second messengers, since the presence of DAG is absolutely required (Yoshida et al., 1992). Most studies have been performed using PKC a, p, and y (Shinomura et al, 1991; Yoshida et al., 1992). Saturated fatty acids of 14—20 carbons are inactive and the structural requirements for the free fatty acids include unsaturated fatty acids of 16-18 carbons with trans double bonds, and of 14—20 carbons with cis double bonds. The presence of a carboxyl group is critical; methyl arachidonate is inactive. The ability to potentiate activation increases with chain length. Though fatty acids
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ROSALIND A. COLEMAN and STEVEN H. ZEISEL
can inhibit DAG lipase, in platelets the rate of disappearance of DAG is not increased by treatment with fatty acids (Yoshida et al., 1992).
IX. THE FATE OF MEDIUM CHAIN TRIACYLGLYCEROLS Synthetic medium-chain triacylglycerols (MCT) are used medically as diet supplements. Because the triacylglycerol is hydrolyzed at the sn-^ and 1-positions by gastric and pancreatic lipases, some water-soluble DAGs may be released in the intestinal lumen and could activate PKC in intestinal mucosa cells. MCT itself can be absorbed intact into intestinal mucosa in the absence of lipases (Swift et al., 1990) and taken up into mucosal cells [^"^CJDAG added into gut loops (Morotomi and Weinstein, 1991). Recent data shows effects of dietary MCT that might be mediated by PKC, including increased proliferation of small intestinal cells (Takase and Goda, 1990), consistent with PKC's role as a mitogen. Unlike long-chain triacylglycerol, MCT fails to suppress sucrase activity and increases jejunal alkaline phosphatase activity (Takase and Goda, 1990). In premature infants fed MCT, gastric lipase activity decreases 87%, compared with long-chain triacylglycerol formula (Hamosh et al., 1989). Dietary MCT also alters several enzyme activities in liver. Although MCT-derived DAGs might reach the liver after reincorporation into chylomicra (Swift et al., 1990), a more likely route involves the PKC-mediated secretion of cytokines released from activated gastrointestinal cells. In rats, dietary MCT increases the secretion of cholecystokinin as much as 17-fold more than does feeding long-chain triacylglycerol (Douglas et al., 1990). The MCT-associated increase in hepatic fatty acid synthase and malic enzyme activities cannot entirely be explained by an increase in insulin (Takase and Goda, 1990). Long-term MCT feeding in humans has also been associated with micronodular cirrhosis (Partin et al., 1974), and choline-deficient rats that accumulate DAG in liver have a greatly increased incidence of spontaneous hepatocarcinoma and of atypical hepatic foci that express fetal enzymes (90% versus 0% in controls) (daCosta et al., 1993). A detailed description of the pathology found in these rats has been recently published (daCosta et al., 1995).
X. IMPORTANT AREAS FOR FUTURE INVESTIGATION Advances in understanding DAG metabolism and its several roles will be greatly aided by the ability to study purified enzymes of synthesis and metabolism and the ability to transfect their cDNAs into cells. The movement of DAG between cell membranes requires further study, as does the possible channeling of DAG within enzyme pathways. Discovery of selective inhibitors of DAG production and attenuation or use will allow an improved understanding of DAG regulation and PKC activation.
Diacylglycerol Metabolism
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Muller, G. & Bandlow, W. (1989). An amphitropic cAMP-binding protein in yeast mitochondria. 3. Membrane release requires both Ca ^^ -dependent phosphorylation of the cAMP-binding protein and a phospholipid-activated mitochondrial phospholipase. Biochemistry 28, 9974-9981. Mullmann, T.J., Siegel, M.I., Egan, R.W., & Billah, M.M. (1991). Sphingosine inhibits phosphatidate phosphohydrolase in human neutrophils by a protein kinase C-independent mechanism. J. Biol. Chem. 266, 2013-2016. Nakanishi, H. & Exton, J.H. (1992). Purification and characterization of the zeta isoform of protein kinase C from bovine kidney. J. Biol. Chem. 267, 16347-16354. Nishizuka, Y. (1992). Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C. Science 258, 607-614. Obeid, L.M., Linardic, CM., Karolak, L.A., & Hannun, Y.A. (1993). Programmed cell death induced by ceramide. Science 259, 1769-1771. Pagano, R.E. (1988). What is the fate of diacylglycerol produced at the Golgi apparatus? Trends Biochem. Sci. 13,202-205. Pagano, R.E. & Longmuir, K.J. (1985). Phosphorylation, transbilayer movement and facilitated intracellular transport of diacylglycerols are involved in the uptake of a fluorescent analog of phosphatidic acid by culture fibroblasts. J. Biol. Chem. 260, 1909-1916. Pagano, R.E., Longmuir, K.J., & Martin, O.C. (1983). Intracellular translocation and metabolism of a fluorescent phosphatidic acid analogue in cultured fibroblasts. J. Biol. Chem. 258, 2034-2040. Pai, J.K., Pachter, J.A.,.Weinstein, I.B., & Bishop, W.R. (1991). Overexpression of protein kinase C beta 1 enhances phospholipase D activity and diacylglycerol formation in phorbol ester-stimulated rat fibroblasts. Proc. Natl. Acad. Sci. USA 88, 598-602. Partin, J.S., Partin, J.C, Schubert, W.K., & McAdams, A.J. (1974). Liver ultrastructure in abetalipoproteinemia: Evaluation of micronodular cirrhosis. Gastroenterology 67, 107-118. Payrastre, B., Nievers, M., Boonstra, J., Breton, M., Verkleij, A. J., & Van, B.e.H.RM. (1992). A differential location of phosphoinositide kinases, diacylglycerol kinase, and phospholipase C in the nuclear matrix. J. Biol. Chem. 267, 5078-5084. Polheim, D., David, J.S.K., Schultz, P.M., Wylie, M.B., & Johnston, J.M. (1973). Regulation of triglyceride biosynthesis in adipose and intestinal tissue. J. Lipid Res. 14,415-421. Preiss, J., Loomis, C.R., Bishop, W.R., Stein, R., Niedel, J.E., & Bell, R.M. (1986). Quantitative measurement of 5n-l,2-diacylglycerols present in platelets, hepatocytes, and ras- and sis-transformed normal rat kidney cells. J. Biol. Chem. 261, 8597-8600. Price, B.D., Morris, J.D., Marshall, C.J., & Hall, A. (1989). Stimulation of phosphatidylcholine hydrolysis, diacylglycerol release, and arachidonic acid production by oncogenic ras is a consequence of protein kinase C activation. J. Biol. Chem. 264, 16638-16643. Purkiss, J.R. & Boarder, M.R. (1992). Stimulation of phosphatidate synthesis in endothelial cells in response to P2-receptor activation. Evidence for phospholipase C and phospholipase D involvement, phosphatidate and diacylglycerol interconversion and the role of protein kinase C. Biochem J. 287, 31-36. Qian, Z. & Drewes, L.R. (1990). A novel mechanism for acetylcholine to generate diacylglycerol in brain. J. Biol. Chem. 265, 3607-3610. Rando, R.R. (1988). Regulation of protein kinase C activity by lipids. FASEB J. 2, 2348-2355. Rando, R. R. & Kishi, Y. (1992). Structural basis of protein kinase C activation by diacylglycerols and tumor promoters. Biochemistry 31, 2211-2218. Rider, L.G., Dougherty, R.W., & Niedel, J.E. (1988). Phorbol diesters and dioctanoylglycerol stimulate accumulation of both diacylglycerols and alkylacylglycerols in human neutrophils. J. Immunol. 140, 200-207. Rodriguez, M.A., Dias, C , & Lau, T.E. (1992). Reversible ATP-dependent inactivation of adipose diacylglycerol acyltransferase. Lipids 27, 577-581. Rustow, B. & Kunze, D. (1987). Further evidence for the existence of different diacylglycerol pools of the phosphatidylcholine synthesis in microsomes. Biochim. Biophys, Acta 921, 552-558.
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PHOSPHATIDYLINOSITOL 4-KINASES IN SACCHAROMYCES CEREVISIAE
George M. Carman, Rosa J. Buxeda, and Joseph T. Nickels, Jr.
ABSTRACT I. INTRODUCTION II. PURIFICATION OF PI 4-KINASES A. Purification of45kDa PI 4-Kinase B. Purification of 55 kDa PI 4-Kinase C. Purification of 125 kDa PI 4-Kinase III. PROPERTIES OF PI 4-KINASES A. Physiochemical Properties B. Enzymological Properties IV. KINETIC PROPERTIES OF PI 4-KINASES A. Role ofTritonX-100 in Catalysis B. Dependenceof PI4-KinasesonPIand ATP V. REGULATION OF PI 4-KINASE ACTIVITIES BY PHOSPHORYLATION
Advances in Lipobiology Volume 1, pages 367-385. Copyright © 1996 by JAI Press Inc. Allrightsof reproduction in any form reserved. ISBN: 1-55938-635-5 367
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VI. REGULATION OF PI 4-KINASE ACTIVITIES BY NUCLEOTIDES VII. PI 3-KINASE VIII. CONCLUSIONS ACKNOWLEDGMENTS REFERENCES
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ABSTRACT Phosphatidylinositol 4-kinase from the yeast Saccharomyces cerevisiae catalyzes the formation of phosphatidylinositol 4-phosphate and ADP from phosphatidylinositol and ATP. Phosphatidylinositol 4-kinase catalyzes the first phosphorylation reaction in the reaction sequence phosphatidylinositol -^ phosphatidylinositol 4-phosphate -^ phosphatidylinositol 4,5-bisphosphate. This phosphorylation sequence in S. cerevisiae is regulated by glucose and sterol. Phosphatidylinositol 4,5-bisphosphate appears to play an essential role in cell proliferation of S. cerevisiae. Since phosphatidylinositol 4-kinase catalyzes the first step in the phosphorylation sequence of phosphatidylinositol, the enzyme should play a major role in phosphoinositide synthesis and cell growth in S. cerevisiae. Two membrane-associated (45 kDa and 55 kDa) forms and one cytosolic-associated (125 kDa) form of phosphatidylinositol 4-kinase have been purified and characterized from S. cerevisiae. The membraneassociated phosphatidylinositol 4-kinases differ with respect to their physiochemical, enzymological, and kinetic properties. The binding and catalytic steps of the reactions catalyzed by the membrane-associated phosphatidylinositol 4-kinases toward phosphatidylinositol has been defined through meaningful kinetic analyses using Triton X-100/PI-mixed micelles. Detailed kinetic analyses of the inhibition of membraneassociated phosphatidylinositol 4-kinases by nucleotides has led to insights on the regulation of phosphoinositide synthesis in response to glucose.
I. INTRODUCTION In the yeast Saccharomyces cerevisiae, phosphatidylinositol (PI) accounts for 10-40% of the total membrane phospholipids depending on strain and culture condition (Henry, 1982). PI is the phospholipid precursor to the phosphoinositides PI 4-phosphate (PIP) (Lester and Steiner, 1968; Patton and Lester, 1992), PI 4,5-bisphosphate (PIP2) (Lester and Steiner, 1968; Patton and Lester, 1992), PI 3-phosphate (Auger et al., 1989), and inositol-containing sphingolipids (Becker and Lester, 1980; Patton and Lester, 1991) (Figure 1). PI also serves as an anchor for plasma membrane glycoproteins (Conzelmann et al., 1992) and is required for the synthesis of cell wall glycans (Hanson and Lester, 1982; Hanson, 1984). A number of studies have shown that PI is essential for the growth and metabolism of S. cerevisiae. When inositol auxotrophic {inol) mutants are deprived of inositol (the water-soluble precursor of PI), cells undergo changes in the metabolism of lipids,
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CDP-diacylglycerol Inositol h> CMP
I' Phosphatidylinositol^
h
ATP ADP
Sphingolipids
Phosphatidylinositol 3-P
Phosphatidylinositol 4-P
t
ATP ADP
Phosphatidylinositol 4,5-P2 Figure 1, Biosynthesis of phosphoinositides in 5. cerevisiae.
carbohydrates, proteins, and nucleic acids, resulting in a loss of viability (Henry et al., 1977; Becker and Lester, 1980). Cells carrying a disrupted copy of the PIS gene encoding the enzyme responsible for PI synthesis (i.e., PI synthase) do not synthesize PI and fail to grow (Nikawa et al., 1987). PIP2 (Uno et al., 1988) and inositol-containing sphingolipids (Pinto et al., 1992) have been shown to play essential roles in cell viability. Thus, inositol-containing lipids are essential for the growth of 5. cerevisiae. In higher eucaryotic organisms, the phosphorylation of PI to PIP and PIP2 and their subsequent phospholipase C-mediated hydrolysis are involved with several cellular responses to hormones, growth factors, and neurotransmitters (Berridge, 1987; Downes and Macphee, 1990). The hydrolysis products of PIP2, namely diacylglycerol and inositol trisphosphate, activate protein kinase C and increase cytosolic calcium levels, respectively (Berridge, 1987; Downes and Macphee, 1990). Due to its tractable molecular genetic system, 5. cerevisiae is being used as a model eucaryote to study phosphoinositide metabolism and signal transduction. The synthesis of PIP and PIP2 in S. cerevisiae is stimulated by glucose (Talwalkar and Lester, 1973; Kaibuchi et al, 1986) and ergosterol (Dahl and Dahl, 1985; Dahl et al., 1987). Talwalkar and Lester (1973) were the first to show that the addition of glucose to glucose-starved cells results in increases in PIP and PIP2 whereas glucose starvation results in the opposite effect. Kaibuchi et al. (1986) have shown
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that the addition of glucose to glucose-starved cells results in the generation of inositol trisphosphate and calcium mobilization; these results have suggested that a phospholipase C-mediated hydrolysis of PIP2 occurs in S. cerevisiae. However, recent studies by Hawkins et al. (1993) have shown that glucose-stimulated cells do not produce inositol trisphosphate, but instead produce glycerophosphoinositol, glycerophosphoinositol 4-phosphate, and glycerophosphoinositol 4,5-bisphosphate. Therefore, these results have suggested that glucose does not stimulate a phospholipase C-mediated hydrolysis of phosphoinositides, but instead stimulates specific phospholipases A or B (Hawkins et al, 1993). Angus and Lester (1972) were the first to suggest this effect. Thus, the PI signaling pathway of higher eukaryotes involving phospholipase C has not been firmly established in S. cerevisiae. The glucose effect on the levels of PIP and PIP2 has been attributed to cellular levels of ATP and ADP (Talwalkar and Lester, 1973; Patton and Lester, 1992). In addition, studies using mutants defective in the RASIcAMV pathway have led to conflicting conclusions whether the glucose effect on phosphoinositide synthesis is mediated by the RASIcPMV pathway (Kaibuchi et al, 1986; Kato et al., 1989; Frascotti et al., 1990; Hawkins et al., 1993). Thus, the mechanism by which glucose regulates the synthesis of PIP and PIP2 in S. cerevisiae is also unclear. PI 4-kinase catalyzes the formation of PIP and ADP from PI and ATP (Colodzin and Kennedy, 1965). Because PI 4-kinase catalyzes the committed step in the synthesis PIP and PIP2, it is widely anticipated that the regulafion of PI 4-kinase activity plays an important role in phosphoinositide synthesis and cell growth in eukaryotic organisms (Michell, 1992). A great deal is known about the biochemistry of PI 4-kinase from S. cerevisiae. PI 3-kinase and PIP kinase, the enzymes that catalyzes the formation of PI 3-phosphate and PIP2, respecfively, have been identified from S. cerevisiae (Auger et al, 1989; Kato et al., 1989; Uno et al., 1988). However, little is known about the biochemistry of these enzymes. This chapter will primarily focus on the biochemistry and regulation of PI 4-kinases from S. cerevisiae.
IL PURIFICATION OF PI 4-KINASES PI 4-kinase activity is associated with the membrane and cytosolic fractions of 5. cerevisiae (Talwalkar and Lester, 1974; McKenzie and Carman, 1982; Auger et al., 1989). Membrane-associated PI 4-kinase activity is associated with secretory vesicles destined for the plasma membrane (Kinney and Carman, 1990) and the reaction product PIP is localized in the plasma membrane (Patton and Lester, 1992). The purification of membrane-associated PI 4-kinase has proven to be cumbersome owing to the general difficulty of purifying membrane-associated enzymes and, in particular, the lability of enzyme activity after solubilization with detergents (McKenzie and Carman, 1983; Belunis et al., 1988). The cytosolic-associated PI 4-kinase has also been difficult to purify due to its apparent association with heat
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shock proteins (Flanagan and Thomer, 1992). Despite these difficulties, procedures have been devised for the purification of two membrane-associated forms (45 and 55 kDa) (Belunis et al., 1988; Buxeda et al., 1991; Nickels et al., 1992) and one cytosolic-associated form (125 kDa) (Flanagan and Thomer, 1992) of PI 4-kinase. A.
Purification of 45 kDa PI 4-Kinase
The 45 kDa form of PI 4-kinase has been purified from the microsomal fraction of 5. cerevisiae (Buxeda et al., 1991). The purification procedure includes Triton X-100 solubilization of microsomal membranes, DE-52 (DEAE-cellulose) chromatography, hydroxylapatite chromatography, and fast protein liquid chromatography with Mono Q. The purification is conducted in the presence of Triton X-100 to prevent protein aggregation. This procedure results in the isolation of a nearly homogeneous preparation of the 45 kDa PI 4-kinase (Belunis et al., 1988). The identity of the 45 kDa protein as PI 4-kinase has been confirmed by the measurement of enzyme activity directly from sodium dodecyl sulfate-polyacrylamide gel slices (Buxeda et al., 1991). Overall, the 45 kDa PI 4-kinase is purified 8,300-fold over the cell extract to a final specific activity of 4 |Limol/min/mg with an activity yield of 12% (Buxeda et al., 1991). PI 4-kinase activity is extremely labile after solubilization with Triton X-100 and up through the hydroxylapatite chromatography step (Carman et al., 1992). The success of the purificafion of the 45 kDa PI 4-kinase also appears to be strain dependent (Carman et al., 1992). The procedure that is used for the purification of the 45 kDa PI 4-kinase is adapted from a previously published procedure for the purification a 35 kDa PI 4-kinase (Belunis et al., 1988). The procedure for the 35 kDa PI 4-kinase includes an octyl-Sepharose chromatography step and a second Mono Q chromatography step (Belunis et al., 1988). The octyl-Sepharose chromatography step is particularly time consuming and results in the proteolytic degradation of the enzyme. Immunoblot analysis using affinity purified antibodies raised against the 45 kDa PI 4-kinase has shown that the 35 kDa PI 4-kinase is a proteolysis product of the 45 kDa enzyme (Buxeda et al., 1991). B. Purification of 55 kDa PI 4-Kinase
Preliminary studies have shown that two forms of membrane-associated PI 4-kinase exist in S. cerevisiae (McKenzie, 1982). This is based on the observation that PI 4-kinase has two pH optima in microsomal membranes. The 55 kDa form of PI 4-kinase has been subsequently purified to near homogeneity from the microsomal fraction of S. cerevisiae (Nickels et al., 1992). The procedure includes solubilization of microsomal membranes with Triton X-100 followed by chromatography with DE-52, hydroxylapatite I, Q-Sepharose, Mono Q, and hydroxylapatite II in the presence of Triton X-100. The 55 kDa protein has been confirmed to be a PI 4-kinase by a combination of native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis and measurement of activity from gel slices (Nickels et
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al, 1992). Overall, the 55 kDa PI 4-kinase is purified 10,166-fold over the cell extract to a final specific activity of 5 |Limol/min/mg with an activity yield of 6.5% (Nickels et al, 1992). The 55 kDa PI 4-kinase can be separated from the 45 kDa form of the enzyme by DE-52 chromatography. Forty-five kDa PI 4-kinase elutes from the DE-52 column with 0.1 M NaCl (McKenzie and Carman, 1983; Belunis et al., 1988) whereas the 55 kDa PI 4-kinase elutes from the column with 0.3 M NaCl (Nickels et al., 1992). Owing to the lability of both forms of PI 4-kinase during DE-52 chromatography, both enzymes are step-eluted from the column with 0.3 M NaCl. The two forms of the enzyme are then separated on the first hydroxylapatite column (Nickels et al., 1992). C. Purification of 125 kDa PI 4-Kinase
The 125 kDa form of PI 4-kinase has been purified 25,000-fold from the cytosolic fraction of S. cerevisiae (Flanagan and Thomer, 1992). The purification scheme includes ammonium sulfate fractionation of the cytosol followed by chromatography with S-Sepharose, phosphocellulose, threonine-agarose, and fast protein liquid chromatography with Mono Q and Mono S (Flanagan and Thorner, 1992). The 125 kDa PI 4-kinase is purified to a final specific activity of 4.3 |Limol/min/mg with an activity yield of 12% (Flanagan and Thomer, 1992). The 125 kDa protein that is purified by the above procedure has been identified as a PI 4-kinase by measuring activity from sodium dodecyl sulfate-polyacrylamide gel slices (Flanagan and Thomer, 1992). The initial stages of the purification of the 125 kDa PI 4-kinase is hampered due to the presence of very abundant heat shock proteins, Hsc82 and Hsp82, present in the cytosolic fraction of wild-type cells (Flanagan and Thomer, 1992). This problem has been alleviated by using a combination of molecular genetics to constmct a null mutation in HSC82, altered growth conditions to minimize expression from the inducible HSP82 gene, and high ionic strength fractionation conditions to remove residual Hsp82. The purified preparations of the 45, 55, and 125 kDa forms of PI 4-kinase have very similar final specific activities (Table 1). To obtain pure preparations of enzyme, the 125 kDa PI 4-kinase requires 2.5- to 3-fold greater purification than the purifications needed for the 45 and 55 kDa forms of the enzyme (Table 1). This raises the suggestion that, in vivo, the 125 kDa cytosolic form of PI 4-kinase is less abundant than the membrane-associated forms of the enzyme. The 45,55, and 125 kDa forms of PI 4-kinase phosphorylate the D-4 ring position of PI to form PIP (Buxeda et al., 1991; Flanagan and Thomer, 1992; Nickels et al., 1992). The product of the reactions catalyzed by all three forms of the enzyme has been confirmed to be PIP by thin-layer chromatography and by analyzing the water-soluble methylamine hydrolysis product of PIP by high-performance liquid chromatography (Buxeda et al., 1991; Flanagan and Thomer, 1992; Nickels et al., 1992). 5. cerevisiae does contain a PI 3-kinase activity which catalyzes the formation of PI 3-phosphate (Auger et al., 1989).
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III. PROPERTIES OF PI 4-KINASES The physiochemical and enzymological properties of the purified PI 4-kinases are summarized in Table 1. A.
Physiochemical Properties
In addition to having different subunit molecular masses, the 45 and 55 kDa forms of PI 4-kinase differ with respect to their physiochemical properties (Nickels et al., 1992). The amino acid compositions of the 45 and 55 kDa forms of PI 4-kinase differ as well as their proteolysis patterns after treatment with TPCK-trypsin and V8 protease. The isoelectric points of the 45 and 55 kDa enzymes are 6.53 and 5.02, respectively. Similar information for the 125 kDa PI 4-kinase has not been reported. The 45,55, and 125 kDa forms of PI 4-kinase do not appear to be immunologically related. The affinity purified antibodies to the 45 kDa PI 4-kinase do not cross-react with the pure 55 kDa PI 4-kinase (Nickels et al., 1992) and do not detect the 55 and 125 kDa PI 4-kinases in cell extracts (Buxeda et al., 1991). B. Enzymological Properties
All three forms of PI 4-kinase require Mg^"^ ions and Triton X-100 for maximum activity at their pH optimum (Belunis et al., 1988; Buxeda et al., 1991; Flanagan Table 1, Enzymological Properties of Purified PI 4-Kinases PI 4-kinase Property Subcellular localization Purification (-fold) Specific activity Isoelectric point pH optimum Cofactor dependence Mg^^ Mn^" Triton X-100:PI dependence Reaction mechanism Activation energy Inhibition by Ca ^ Inhibition by thioreactive agents
45 kDa
55 kDa^
Microsomes 8,300 4 ^mol/min/mg 6.53 8.0
Microsomes 10,166 5 fimol/min/mg 5.02 7.0
Cytosol 25,000 4.3 f^mol/min/mg NR^ 7.5
27 mM
10 mM 0.5 mM 16:1 Sequential 12.1 kcal/mol IC5o=5.6mM Yes
10 mM
64:1 Sequential 31.5kcal/mol IC5o=10mM Yes
Notes: ^Data taken from Belunis et al. (1988) and Buxeda et al. (1991). ''Data taken from Nickels et al. (1992). '^Data taken from Flanagan and Thomer (1992). •^NR, not reported.
125 kDa""
15.6:1 NR NR NR NR
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G. M. CARMAN, R. J. BUXEDA, and J. T. NICKELS, JR.
and Thomer, 1992; Nickels et al., 1992). However, these activity requirements differ among the three PI 4-kinases. The Mg^"^ ion dependence of the 55 kDa PI 4-kinase can be substituted by Mn^"^ ions. The 45 and 55 kDa forms of PI 4-kinase also differ with respect to their activation energies and sensitivity to inhibition by Ca^^ ions. Both membrane-associated forms of the enzyme are inhibited by thioreactive agents suggesting that a sulfhydryl group is required for catalysis. The reaction mechanism for the 45 and 55 kDa forms of PI 4-kinase is sequential, where the enzyme binds to PI before ATP and PIP is the first product released in the reaction sequence (Belunis et al., 1988; Buxeda et al., 1991; Nickels et al., 1992). This mechanism is based on the results of detailed kinetic analyses of each enzyme (see below) and the ability of each enzyme to catalyze a series of isotopic exchange reactions between substrates and products (Belunis et al, 1988; Buxeda et al., 1991; Nickels et al., 1992). The reaction mechanism for the 125 kDa enzyme has not been reported. The differences in the enzymological properties of the PI 4-kinases could be exploited to differentially measure the relative activities of each enzyme in cell extracts or membrane preparations.
IV. KINETIC PROPERTIES OF PI 4-KINASES The kinetic analyses of purified PI 4-kinases from various eukaryotes are difficult to interpret due to different ways the substrate PI is delivered (e.g,. sonicated vesicles and detergent micelles) to the assay systems (Downes and Macphee, 1990). Kinetic analyses of enzymes using phospholipid substrates are best performed using detergent/phospholipid-mixed micelle systems (Dennis, 1983; Hannun et al., 1985; Walsh and Bell, 1986; Scheideler and Bell, 1989; Bae-Lee and Carman, 1990; Lin and Carman, 1990). The mixed micelle system is well defined and may provide an environment that mimics the physiological surface of the membrane (Dennis, 1983; Hannun etal, 1985). A.
Role of Triton X-100 in Catalysis
The addition of Triton X-100 to the assay systems for the 45 and 55 kDa forms of PI 4-kinase resuhs in the stimulation of PI 4-kinase activity to a maximum (molar ratios of Triton X-100:PI of 64:1 and 16:1, respectively), followed by an apparent inhibition of activity at higher Triton X-100 concentrafions (Belunis et al., 1988; Buxeda et al., 1991; Nickels et al., 1992). These results are typical of "surface dilution" kinetics (Deems et al., 1975) commonly observed for purified phospholipid biosynthetic enzymes (Dowhan et al., 1974; Carman and Dowhan, 1979; Fischl and Carman, 1983; Bae-Lee and Carman, 1984; Dutt and Dowhan, 1985; Kelley and Carman, 1987; Lin and Carman, 1990) which use Triton X-100/phospholipid-mixed micelle substrates. The function of Triton X-100 in the assay systems for PI 4-kinase is to form a uniform mixed micelle with the phospholipid substrate PI (Buxeda et al., 1991). The Triton X-100 micelle serves as a catalytically
PI 4-Kinases in Yeast
375
inert matrix in which PI is dispersed preventing high local concentration of substrate at the active site (Buxeda et al., 1991). The apparent inhibition of the PI 4-kinase activities by high Triton X-100 concentrations (i.e., molar ratios of Triton X-100:PI above the maximum) is simply due to the association of the enzyme with the micelle (Buxeda et al., 1991; Nickels et al., 1992) and a dilution of PI at the surface of the micelle (Deems et al., 1975). Thus, Triton X-100 does not inhibit PI 4-kinase activity. Dennis and co-workers (Deems et al., 1975; Roberts et al., 1977; Hendrickson and Dennis, 1984) have developed the "surface dilution" model to explain the kinetic behavior of enzymes toward their phospholipid substrates at the interface of Triton X-100 micelles. According to the surface dilution model (Deems et al., 1975; Roberts et al, 1977; Hendrickson & Dennis, 1984), the enzyme associates with the Triton X-100 micelle surface followed by the formation of an enzyme-substrate complex, catalysis, and release of products. The enzyme may specifically bind to its substrate in the micelle surface or nonspecifically bind to the micelle surface (Deems et al., 1975; Roberts et al., 1977; Hendrickson and Dennis, 1984). The kinetic equation for this model is given in Equation 1.
^r^JAXB)
(1)
The term Amay be defined as the sum of the molar concentrations of Triton X-100 plus phospholipid or the molar concentration of phospholipid. The term B is the mol fraction of phospholipid substrate in the mixed micelle. The V^^^ is the true ^max ^^ ^^ infinite mol fi"action of phospholipid substrate, K^ is the dissociation constant for the mixed micelle binding site, and K^ is the interfacial Michaelis constant (expressed in surface concentration). The dependence of the 45 kDa (Buxeda et al., 1991) and 55 kDa (Nickels et al., 1992) forms of PI 4-kinase on PI in Triton X-100/PI-mixed micelles has been examined in detail according to the surface dilution model. An example of the kinetic data that is obtained from such an analysis is shown in Figure 2. In the example shown, 45 kDa PI 4-kinase activity is measured as a function of the molar (bulk) concentration of PI at several set surface concentrations (in mol %) of PI. Forty five kDa PI 4-kinase activity is dependent on both the bulk and surface concentrations of PI below 0.1 mM. At concentrations above 0.1 mM, activity is independent of the bulk concentration of PI but dependent only on the surface concentration of PI. By constructing a number of replots of the kinetic data and performing the appropriate calculations, kinetic constants can be obtained (Deems et al., 1975). Kinetic constants for the 45 kDa (Buxeda et al., 1991) and 55 kDa (Nickels et al., 1992) forms of PI 4-kinase have been obtained using this type of analysis and are summarized in Table 2. Based on the dissociations constants (K^ values) for the micelle and PI, the 45 and 55 kDa PI 4-kinases have more affmity for the micelle surface when their substrate PI is present. Physical data for the
G. M. CARMAN, R. J. BUXEDA, and ). T. NICKELS, JR.
376
Surface PI, Mol % 1.57 J
4
^
W
Q I
Bulk PI, mM Figure 2, Activity of 45 kDa PI 4-kinase toward PI in mixed micelles with Triton X-100. Forty-five kDa PI 4-kinase was measured as a function of the bulk (mM) concentration of PI at the indicated set surface (mol%) concentrations of PI. The figure was redrawn from the data in Buxeda et al. (1991).
Table 2. Kinetic Constants of Purified PI 4-Kinases PI 4-kinase Kinetic constant K^ (micelle) ^,(PI) ^.(PI) ^,„(MgATP) A:,(MgADP)ATP ^,(MgADP)pi ^,(APS)ATP
^/(CTPW ^/(CTP)p, Notes:
45kDa^ 118 mM 0.26 mM 0.4 mol % 0.5 mM 0.14 mM 1.3 mM 0.22 mM Nf NI
55kDa^ 11.8 mM 0.035 mM 1.7 mol % 0.36 mM 0.25 mM 0.9 mM 0.27 mM 1.5mM 4.0mM
^Data taken from Buxeda et al. (1993) and Buxeda et al. (1991). •'Data taken from Buxeda et al. (1993) and Nickels et al. (1992). ^ I , not inhibitory.
PI 4-Kinases in Yeast
377
association of both enzymes to Triton X-100/PI-mixed micelles has been obtained by glycerol gradient centrifugation experiments (Buxeda et al., 1991; Nickels et al., 1992). The 55-kDa PI 4-kinase has a 7-fold greater affinity (reflected in the K^ value for PI) for the Triton X-100 micelle surface when compared to the affinity for the micelle of the 45 kDa PI 4-kinase (Buxeda et al, 1991). However, once bound to PI in the micelle surface, the 45 kDa PI 4-kinase has a 4.7-fold greater catalytic efficiency (reflected in the K^ values) for PI than does the 55 kDa PI 4-kinase. The differences in the binding and catalytic properties of the 45 and 55 kDa forms of PI 4-kinase raise the suggestion that these enzymes may be regulated differentially in vivo. B. Dependence of PI 4-Kinases on PI and ATP
The PI 4-kinases catalyze reactions involving two substrates and two products (Bi Bi reaction). The concentration of one substrate affects the concentration dependence of the enzyme on the other substrate (Buxeda et al., 1991; Nickels et al., 1992). Thus, to obtain meaningful kinetic constants for enzymes like PI 4-kinase, experiments to examine the dependence of the enzyme on one substrate must be performed at several fixed concentrations of the second substrate (Segel, 1975). This detailed kinetic analysis is complicated by the fact that the 45 kDa (Buxeda et al., 1991) and 55 kDa (Nickels et al., 1992) forms of PI 4-kinase are dependent on both the bulk and surface concentrations of PI below a bulk PI concentration of 0.1 mM (see Figure 2) and 0.15 mM, respectively. This fact made the initial kinetic studies of the 45 kDa (35 kDa) PI 4-kinase difficult to interpret (Belunis et al., 1988; Buxeda et al, 1991). Therefore, in the kinetic experiments to examine the dependence of the 45 kDa (Buxeda et al., 1991) and 55 kDa (Nickels et al., 1992) PI 4-kinases on PI and ATP, the PI 4-kinases are measured such that their activities are dependent only on the surface concentration of PI. Under these conditions, the PI 4-kinase activities follow saturation with respect to one substrate and several fixed concentrations of the other substrate (Buxeda et al, 1991; Nickels et al, 1992). The family of lines resulfing from double-reciprocal plots of these data are consistent with both enzymes catalyzing sequential reaction mechanisms (Buxeda et al., 1991; Nickels et al, 1992). Furthermore, these detailed kinetic analyses have resulted in meaningful kinetic constants for each enzyme which are summarized in Table 2. The K^ values for PI for each PI 4-kinase were the same as that obtained through the kinetic analyses using the surface dilution model described above. The K^ values for ATP for both PI 4-kinases are similar. The kinetic constants for PI and ATP determined for the 45 and 55 kDa forms of PI 4-kinase can not be compared with the kinetic constants determined for the 125 kDa form of the enzyme. In the kinetic studies for the 125 kDa PI 4-kinase, the enzyme dependence on one substrate has been measured at only one concentration of the second substrate (Flanagan and Thomer, 1992). Thus, under these conditions only apparent kinetic constants have been obtained. Moreover, these experiments have
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G. M. CARMAN, R. J. BUXEDA, and J. T. NICKELS, JR.
been conducted using only one surface concentration of PI (i.e., at a constant molar ratio of Triton X-100:PI) (Flanagan and Thomer, 1992). Whether or not the 125 kDa PI 4-kinase activity is dependent on both the bulk and surface concentrations of PI has not been addressed. For the same reasons, it is difficult to compare the kinetic constants of the 45 and 55 kDa forms of PI 4-kinase with the kinetic constants determined for purified PI 4-kinases from higher eukaryotic cells.
V. REGULATION OF PI 4-KINASE ACTIVITIES BY PHOSPHORYLATION It is unclear whether the RAS/cAMP pathway plays a role in the glucose-induced changes in phosphoinositide synthesis in S. cerevisiae. Using S. cerevisiae mutants {rasl, ras2, bcyl, rasl ras2 bcyl, and RAS2'^^^^^) defective in the RAS/cAM? pathway, Kaibuchi et al. (1986) have carried out studies that led to the conclusion that /L4*S-encoded proteins regulated PIP and PIP2 synthesis. On the other hand, studies by Kato et al. (1989) using similar mutants (cyri, bey I, ras2, and rasl ras2 bcyl) have led to the conclusion that cAMP levels affect phosphoinositide synthesis. Their studies, using crude membrane preparations, raised the suggestion that PI kinase activity is activated by cAMP-dependent protein kinase phosphorylation which correlates with increases in the synthesis of PIP. Contrary to both of these reports, the studies of Holland et al. (1988), using cell extracts derived from wild-type cells, have suggested that PI kinase activity is inactivated by cAMP-dependent protein kinase phosphorylation. There are a number of factors that could account for these conflicting conclusions. Kato et al. (1989) and Holland et al. (1988) used different strains, growth conditions, and enzyme assay conditions in their studies. S. cerevisiae cells have two very different types of PI kinase activities. In addition to the PI 4-kinase activities, S. cerevisiae also has a PI 3-kinase (Auger et al., 1989). In the studies of Kato et al. (1989) and Holland et al. (1988) PI kinase activity is measured in the presence of Triton X-100. As discussed above, the PI 4-kinase activities in 5. cerevisiae are dependent on Triton X-100 (Belunis et al., 1988; Buxeda et al., 1991; Nickels et al., 1992). The PI 3-kinase activity in S. cerevisiae is inactivated by Triton X-100 (Auger et al., 1989). Thus, the activity that was being measured in the studies of Kato et al. (1989) and Holland et al. (1988) is most likely that of PI 4-kinase. The membrane-associated 45 and 55 kDa PI 4-kinases and the cytosolic-associated 125 kDa PI 4-kinase have very different enzymological properties (Belunis et al., 1988; Buxeda et al., 1991; Flanagan and Thomer, 1992; Nickels et al, 1992). Because Kato et al. (1989) and Holland et al. (1988) were using different enzyme assay conditions, it is very difficult to know which form of the PI 4-kinases is affected. The availability of purified preparations of the 45 and 5 5 kDa forms of PI 4-kinase has permitted experiments to determine if these PI 4-kinases are phosphorylated and regulated by cAMP-dependent protein kinase (Buxeda et al., 1993). The results of these studies have shown that cAMP-dependent protein kinase has absolutely
PI 4-Kinases in Yeast
379
no effect on the activities of 45 and 55 kDa PI 4-kinases. Furthermore, cAMPdependent protein kinase does not catalyze the incorporation of the y-phosphate from ATP into the 45 and 55 kDa PI 4-kinases (Buxeda et al., 1993). The lack of regulation of the 45 and 55 kDa PI 4-kinases by cAMP-dependent protein kinase phosphorylation in vivo is supported by the findings of Frascotti et al. (1990). Using cdc25 mutants, these workers have demonstrated that the glucose effect on phosphoinositide metabolism is not mediated through the RAS/cAMP pathway. The cdc25 mutants are defective in the CDC25 gene product which activates the RASIQAM? pathway (Broek et al. , 1987). Thus while the activity of membraneassociated PI 4-kinases may have been affected in crude systems which favor cAMP-dependent protein kinase activity (Holland et al, 1988; Kato et al., 1989), the effects observed are most likely indirect. It is not known whether the 125 kDa form of PI 4-kinase is regulated by cAMP-dependent protein kinase.
VI. REGULATION OF PI 4-KINASE ACTIVITIES BY NUCLEOTIDES The 45 and 55 kDa PI 4-kinases are inhibited to varying degrees by adenosine derivatives and nucleotides when activities are measured using an ATP concentration near the K^ values for the enzymes (Buxeda et al., 1993). Using saturating concentrations of ATP masks the inhibitory effects of adenosine derivatives and nucleotides on PI 4-kinase activity (Belunis et al., 1988; Buxeda et al., 1993). The inhibitory compounds include a- and P-adenosine, AdoMet, AdoHcy, AMP, c AMP, and ADP. The inhibitory effects of adenosine nucleotides increase with the number of phosphate groups attached to adenosine (Buxeda et al., 1993). The most potent adenosine nucleotide inhibitor of the 45 and 55 kDa PI 4-kinases is ADP, the nucleotide product of the PI 4-kinase reaction (Buxeda et al., 1993). Detailed kinetic analyses (taking into account the kinetic considerations discussed above) on the inhibition of the 45 and 55 kDa PI 4-kinases by ADP have been performed (Buxeda et al., 1993). The results of these kinetic analyses have shown that ADP is a competitive inhibitor of both PI4-kinases with respect to ATP and a noncompetitive inhibitor with respect to PI (Buxeda et al., 1993). The kinetic constants for ADP have been determined and are summarized in Table 2. ADP is not a substrate for either one of the PI 4-kinase reactions and thus, the inhibition of the enzymes by ADP is the result of a dead-end complex (Buxeda et al., 1993). Both membraneassociated PI 4-kinases are also inhibited competitively by the ADP analogue APS (adenosine 5'-phosphosulfate) and thus the phosphorous in the P position can be substituted by a sulfur. In a preliminary experiment, the 125 kDa PI 4-kinase has been shown to be inhibited by ADP (Flanagan and Thomer, 1992). However, this inhibition can not be compared with that described for the membrane-associated forms of PI 4-kinase because this experiment was conducted in the presence of a saturating concentration of ATP and defined kinetic analyses have not been performed.
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G. M. CARMAN, R. J. BUXEDA, and J. T. NICKELS, jR.
The inhibition of the 45 and 55 kDa PI 4-kinase activities by ADP appears to be more than just simple product inhibition. Based on the cellular concentrations of ATP and ADP and the kinetic constants for these nucleotides (Table 2), an argument has been made for the regulation of PI 4-kinase activity by ATP and ADP in vivo (Buxeda et al., 1993). The K^ values both enzymes have for ATP are about 6-fold lower than the cellular concentration of ATP (Ozier-Kalogeropoulos et al., 1991). The K. values for ADP (with respect to ATP) of both enzymes are 1.5- to 2-fold lower than the K^ values both enzymes have for ATP. Furthermore, these K- values for ADP are 1.6- to 2.8-fold lower than the cellular concentration of ADP (Talwalkar and Lester, 1973; Ozier-Kalogeropoulos et al., 1991). Although the K. values for ADP are below the cellular level of ADP, the cellular levels of ATP and PI (Patton and Lester, 1991) are well above the K^ values for these substrates. Thus, the reaction in cells grown with glucose would be driven toward the synthesis of PIP. However, upon glucose starvation the cellular ATP levels drop about 4-fold while the cellular ADP levels increase about 2-fold (Talwalkar and Lester, 1973). This would bring the cellular ATP levels near the K^ values for ATP and the cellular ADP levels about 3- to 6-fold higher than the K. values for ADP. Therefore, changes in the cellular levels of ATP and ADP brought about by glucose starvation could result in a significant effect on the synthesis of PIP through the inhibition of PI 4-kinase activity. The 55 kDa PI 4-kinase is also inhibited by the mono-, di-, and triphosphorylated derivatives of cytidine, thymidine, uridine, and guanosine with CTP being the most potent inhibitor (Buxeda et al., 1993). In contrast, the 45 kDa PI 4-kinase is not affected by these nucleotides (Buxeda et al., 1993). A detailed kinetic analysis of the inhibition of the 55 kDa PI 4-kinase by CTP has shown that CTP is a mixed-type of inhibitor with respect to ATP and a noncompetitive inhibitor with respect to PI (Buxeda et al., 1993). The K. values for CTP are presented in Table 2. CTP does not serve as a phosphate donor for the reaction (Buxeda et al., 1993). The K- value for CTP is 4-fold higher than the K^ value for MgATP and 7-fold higher than the cellular concentration of CTP. Thus, CTP would not be expected to regulate 55 kDa PI 4-kinase activity in vivo. However, if the cellular concentration of CTP were to rise near the K- value, CTP could play a regulatory role on 55 kDa PI 4-kinase activity. The inhibition of the 55 kDa PI 4-kinase by CTP and ADP is additive (Buxeda et al., 1993). It is unclear whether or not CTP binds to the ATP binding site and/or a different site on the enzyme (Buxeda et al., 1993). CTP plays an important role in phospholipid biosynthesis in S. cerevisiae (Carman and Henry, 1989). This nucleotide is the precursor of the liponucleotide CDP-diacylglycerol, the source of the phosphatidyl moiety of all phospholipids synthesized by the primary' (phosphatidylethanolamine methylation) phospholipid biosynthetic pathway (Carman and Henry, 1989). CTP is also a substrate for two reactions in the auxiliary (CDP-ethanolamine- and CDP-choline-based) phospholipid pathway (Carman and Henry, 1989). It is known that the biosynthesis of PI, the substrate of 55 kDa PI 4-kinase, is coordinately regulated with the biosyn-
PI 4-Kinases in Yeast
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thesis of phosphatidylcholine through the primary and auxiliary pathways by genetic and biochemical mechanisms (Carman and Henry, 1989). It may be that CTP plays a role in this coordinate regulation.
VIL PI3-KINASE PI 3-kinase catalyzes the phosphorylation of the D-3 ring position of PI to form PI 3-phosphate (reviewed in Carpenter and Cantley, 1990). It is now known that PI 3-phosphate as well as PIP exists in S. cerevisiae (Auger et al., 1989). Indeed, S. cerevisiae does have PI 3-kinase activity which is associated with the membrane and cytosolic fractions of the cell (Auger et al., 1989). In mammalian cells PI 3-kinase exists as a heterodimer consisting of a 110 kDa catalytic subunit and an 85 kDa regulatory subunit (Carpenter and Cantley, 1990). The gene encoding for the 110 kDa catalytic subunit has been recently isolated from bovine brain (Hiles et al., 1992). The results of a computer search indicated that the cDNA sequence of the bovine brain gene for the 110 kDa subunit shares significant sequence similarity with the VPS34 gene of 5. cerevisiae (Hiles et al., 1992). The VPS34 gene product is essential for sorting of soluble hydrolases to the lysosome-like vacuole of S. cerevisiae (Herman and Emr, 1990). Through a series of molecular genetic and biochemical studies, Schu et al. (1993) have shown that the VPS34 gene product is the PI 3-kinase of S. cerevisiae. Thus, PI 3-kinase in S. cerevisiae plays a role in the regulation of vacuolar protein sorting. Most of the PI 3-kinase in S. cerevisiae is membrane-associated (Schu et al., 1993). This membrane-association is mediated by a protein kinase (Stack et al., 1993), which is encoded by the VPS15 gene (Herman et al., 1991). Like the PI 3-kinase, the Vpsl5 protein kinase is essential for the sorting of soluble hydrolases to the vacuole (Herman et al., 1991). Furthermore, the activity of PI 3-kinase is dependent on Vpsl5 protein fiinction (Stack et al., 1993). PI 3-kinases have been purified from mammalian cells (Walker et al, 1988). However, the enzyme has yet to be purified from S. cerevisiae and its biochemical and kinetic properties have not been addressed.
VIII. CONCLUSIONS In higher eukaryotes the synthesis of PIP and PIP2 and their subsequent hydrolysis to diacylglycerol and inositol trisphosphate play an important role in cell signaling in response to a plethora of agonists (Berridge, 1987; Downes and Macphee, 1990). The only agonists shown to have an effect on PIP and PIP2 synthesis in S. cerevisiae are glucose (Talwalkar and Lester, 1973; Kaibuchi et al., 1986) and ergosterol (Dahl and Dahl, 1985; Dahl et al., 1987). It is unclear whether or not S. cerevisiae uses the PI signaling pathway of higher eukaryotes involving the phospholipase Cmediated production of diacylglycerol and inositol trisphosphate. Yet, many of the enzymes in the PI signaling pathway, including PI 4-kinase (Belunis et al., 1988;
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Buxeda et al, 1991; Flanagan and Thomer, 1992; Nickels et al., 1992) and protein kinase C (Ogita et al., 1990), have been purified from S. cerevisiae. It is also unclear whether or not the RASIcAM? pathway plays a role in the glucose-induced changes in PIP and PIP2 synthesis. The kinetic analyses performed on the inhibition of the 45 and 55 kDa forms of PI 4-kinase by ADP (Buxeda et al., 1993) support the conclusion (Talwalkar and Lester, 1973) that the regulation of phosphoinositide synthesis by glucose is mediated by the cellular levels of ATP and ADP through the regulation of PI 4-kinase activity. The phosphorylation studies performed on the 45 and 55 kDa forms of PI 4-kinase (Buxeda et al., 1993) do not support the conclusion (Kato et al., 1989) that the regulation of phosphoinositide synthesis is mediated by the RASIcAMV pathway through the cAMP-dependent protein kinase phosphorylation of the membrane-associated PI 4-kinases. A great deal has been learned about the biochemical regulation of the PI 4-kinases of S. cerevisiae through the purification of these enzymes. A molecular genetic approach has led to an understanding of the function of PI 3-kinase in S. cerevisiae. Clearly, the cloning of the structural genes encoding for the PI 4-kinases will be required to obtain a greater understanding of the genetic regulation of these enzymes and their roles in signal transduction.
ACKNOWLEDGMENTS This work was supported by United States Public Health Service Grant GM-35655 from the National Institutes of Health, New Jersey State funds, and the Charles and Johanna Busch Memorial Fund. We acknowledge the helpful discussions with Dr. Edward A. Dennis concerning the kinetic analyses of the membrane-associated forms of PI 4-kinase using the surface dilution kinetic models.
REFERENCES Angus, W.W. & Lester, R.L. (1972). Turnover of inositol and phosphorus containing lipids in Saccharomyces cerevisiae: Extracellular accumulation of glycerophosphorylinositol derived from phosphatidylinositol. Arch. Biochem. Biophys. 151,483-495. Auger, K.R., Carpenter, C.L., Cantley, L.C., & Varticovski, L. (1989). Phosphatidylinositol 3-kinase and its novel product, phosphatidylinositol 3-phosphate, are present in Saccharomyces cerevisiae. J. Biol. Chem. 264, 20181-20184. Bae-Lee, M. & Carman, G.M. (1984). Phosphatidylserine synthesis in Saccharomyces cerevisiae. Purification and characterization of membrane-associated phosphatidylserine synthase. J. Biol. Chem. 259, 10857-10862. Bae-Lee, M. & Carman, G.M. (1990). Regulation of yeast phosphatidylserine synthase and phosphatidylinositol synthase activities by phospholipids in Triton X-100/phospholipid mixed micelles. J. Biol. Chem. 265, 7221-7226. Becker, G.W. & Lester, R.L. (1980). Biosynthesis of phosphoinositol-containing sphingolipids from phosphatidylinositol by a membrane preparation from Saccharomyces cerevisiae. J. Bacteriol. 142, 747-754. Belunis, C.J., Bae-Lee, M., Kelley, M.J., & Carman, G.M. (1988). Purification and characterization of phosphatidylinositol kinase from Saccharomyces cerevisiae. J. Biol. Chem. 263, 18897-18903.
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Berridge, MJ. (1987). Inositol lipids and cell proliferation. Biochim. Biophys. Acta 907, 33-45. Broek, D., Toda, T., Michaeli, T., Levin, L., Birchmeier, C , Zoller, M., Powers, S., and Wigler, M. (1987). The S. cerevisiae CDC25 gene product regulates the RAS/adeny\ate cyclase pathway. Cell 48, 78^799. Buxeda, R.J., Nickels, J.T., and Carman, G.M. (1993). Regulation of the 45- and 55-kDa forms of phosphatidylinositol 4-kinase from the yeast Saccharomyces cerevisiae by nucleotides. J. Biol. Chem. 268, 624^-^255. Buxeda, R.J., Nickels, J.T. Jr., Belunis, C.J., and Carman, G.M. (1991). Phosphatidylinositol 4-kinase from Saccharomyces cerevisiae. Kinetic analysis using Triton X-100/phosphatidylinositol-mixed micelles. J. Biol. Chem. 266, 13859-13865. Carman, G.M., Belunis, C.J., and Nickels, J.T. (1992). Phosphatidylinositol 4-kinase from yeast. Methods Enzymol. 209, 183-189. Carman, G.M. & Dowhan, W. (1979). Phosphatidylserine synthase from Escherichia coli. The role of Triton X-100 in catalysis. J. Biol. Chem. 254, 8391-8397. Carman, G.M. & Henry, S.A. (1989). Phospholipid biosynthesis in yeast. Annu. Rev. Biochem. 58, 635-669. Carpenter, C.L. & Cantley, L.C. (1990). Phosphoinositide kinases. Biochemistry 29, 11147-11156. Colodzin, M. & Kennedy, E.P. (1965). Biosynthesis of diphosphoinositide in brain. J. Biol. Chem. 240, 3771-3780. Conzelmann, A., Puoti, A., Lester, R.L., and Desponds, C. (1992). Two different types of lipid moieties are present in glycophosphoinositol-anchored membrane proteins of Saccharomyces cerevisiae. EMBO J. 11,457-466. Dahl, C , Biemann, H.-P, and Dahl, J. (1987). A protein kinase antigenically related to pp60v-src possibly involved in yeast cell cycle control: Positive in vivo regulation by sterol. Proc. Natl. Acad. Sci. USA 84, 4012-4016. Dahl, J.S. & Dahl, C.E. (1985). Stimulation of cell proliferation and polyphosphoinositide metabolism in Saccharomyces cerevisiae GL7 by ergosterol. Biochem. Biophys. Res. Commun. 133,844—850. Deems, R.A., Eaton, B.R., and Dennis, E.A. (1975). Kinetic analysis of phospholipase A2 activity toward mixed micelles and its implications for the study of lipolytic enzymes. J. Biol. Chem. 250, 9013-9020. Dennis, E.A. (1983). Phospholipases. In: The Enzymes (Boyer, P.D., ed.) vol. 16, pp. 307-353; Academic Press, New York. Dowhan, W., Wickner, W.T., and Kennedy, E.P. (1974). Purification and properties of phosphatidylserine decarboxylase from Escherichia coli. J. Biol. Chem. 249, 3079-3084. Downes, C.P. & Macphee, C.H. (1990). /w>^o-Inositol metabolites as cellular signals. Eur. J. Biochem. 193, 1-18. Dutt, A. and Dowhan, W. (1985). Purification and characterization of a membrane-associated phosphatidylserine synthase from Bacillus licheniformis. Biochemistry 24, 1073—1079. Fischl, A.S. & Carman, G.M. (1983). Phosphatidylinositol biosynthesis in Saccharomyces cerevisiae: Purification and properties of microsome-associated phosphatidylinositol synthase. J. Bacteriol. 154,30^311. Flanagan, C.A. & Thomer, J. (1992). Purification and characterization of a soluble phosphatidylinositol 4-kinase from the yeast Saccharomyces cerevisiae. J. Biol. Chem. 267, 24117-24125. Frascotti, G., Baroni, D., and Martegani, E. (1990). The glucose-induced polyphosphoinositides turnover in Saccharomyces cerevisiae is not dependent on the CDC25-RAS mediated signal transduction pathway. FEBS Lett. 274, 19-22. Hannun, Y.A., Loomis, C.R., and Bell, R.M. (1985). Activation of protein kinase C by Triton X-100 mixed micelles containing diacylglycerol and phosphatidylserine. J. Biol. Chem. 260, 1003910043. Hanson, B. (1984). Role of inositol-containing sphingolipids in Saccharomyces cerevisiae during inositol starvation. J. Bacteriol. 159, 837-842.
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Hanson, B.A. & Lester, R.L. (1982). Effect of inositol starvation on the in vitro synthesis of mannan and N-acetylglucosamine-pyrophosphoryldolichol in Saccharomyces cerevisiae. J. Bacteriol. 151, 334^342. Hawkins, P.T., Stephens, L.R., and Piggott, J.R. (1993). Analysis of inositol metabolites produced by Saccharomyces cerevisiae in response to glucose stimulation. J. Biol. Chem. 268, 3374-3383. Hendrickson, H.S. & Dennis, E.A. (1984). Kinetic analysis of the dual phosphlipid model for phospholipase A2 action. J. Biol. Chem. 259, 5734^5739. Henry, S.A. (1982). Membrane lipids of yeast: Biochemical and genetic studies. In: The Molecular Biology of the Yeast Saccharomyces: Metabolism and Gene Expression (Strathem, J.N., Jones, E.W., & Broach, J.R., eds.), pp. 101-158; Cold Spring Harbor Laboratory, Cold Spring Harbor. Henry, S.A., Atkinson, K.D., Kolat, A.J., and Culbertson, M.R. (1977). Growth and metabolism of '\nos\\.o\-si2iTWQd Saccharomyces cerevisiae. J. Bacteriol. 130, 472-484. Herman, P.K. & Emr, S.D. (1990). Characterization of VPS34, a gene required for vacuolar protein sorting and vacuole segregation in Saccharomyces cerevisiae. Mol. Cell. Biol. 10, 6742-6754. Herman, P.K., Stack, J.H., DeModena, J.A., and Emr, S.D. (1991). A novel protein kinase homolog essential for protein sorting to the yeast lysosome-like vacuole. Cell 64, 425—437. Hiles, LD., Otsu, M., Volinia, S., Fry, M.J., Gout, I., Dhand, R., Panayotou, G., Ruiz-Larrea, P., Thompson, A., Totty, N.F., Hsuan, J.J., Courtneidge, S.A., Parker, P.J., and Waterfield, M.D. (1992). Phosphatidylinositol 3-kinase: Structure and expression of the 110 kd catalytic subunit. Cell 70, 419-429. Holland, K.M., Homann, M.J., Belunis, C.J., and Carman, G.M. (1988). Regulation of phosphatidylinositol kinase activity in Saccharomyces cerevisiae. J. Bacteriol. 170, 828—833. Kaibuchi, K., Miyajima, A., Arai, K.-L, and Matsumoto, K. (1986). Possible involvement of Z^.^^encoded proteins in glucose-induced inositolphospholipid turnover in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 83, 8172-8176. Kato, H., Uno, L, Ishikawa, T, and Takenawa, T. (1989). Activation of phosphatidylinositol kinase and phosphatidylinositol-4-phosphate kinase by cAMP in Saccharomyces cerevisiae. J. Biol. Chem. 264,3116-3121. Kelley, M.J. & Carman, G.M. (1987). Purification and characterization of CDPdiacylglycerol synthase from Saccharomyces cerevisiae. J. Biol. Chem. 262, 14563—14570. Kinney, A.J. & Carman, G.M. (1990). Enzymes of phosphoinositide synthesis in secretory vesicles destined for the plasma membrane in Saccharomyces cerevisiae. J. Bacteriol. 172, 4115—4117. Lester, R.L. «fe Steiner, M.R. (1968). The occurrence of diphosphoinositide and triphosphoinositide in Saccharomyces cerevisiae. J. Biol. Chem. 243, 4889-4893. Lin, Y.-P. & Carman, G.M. (1990). Kinetic analysis of yeast phosphatidate phosphatase toward Triton X-100/phosphatidate mixed micelles. J. Biol. Chem. 265, 166-170. McKenzie, M.A. (1982). Partial purification and properties of membrane-associated phosphatidylinositol kinase from Saccharomyces cerevisiae. M.S. thesis, Rutgers University, New Brunswick, NJ. McKenzie, M.A. & Carman, G.M. (1982). Solubilization of membrane-associated phosphatidylinositol kinase from Saccharomyces cerevisiae. J. Food Biochem. 6, 77—86. McKenzie, M.A. & Carman, G.M. (1983). Membrane-associated phosphatidylinositol kinase from Saccharomyces cerevisiae. J. Bacteriol. 156, 421—423. Michell, R.H. (1992). Inositol lipids in cellular signalling mechanisms. Trends Biochem. Sci. 17, 274-276. Nickels, J.T., Buxeda, R.J., and Carman, G.M. (1992). Purification, characterization, and kinetic analysis of a 55-kDa form of phosphatidylinositol 4-kinase from Saccharomyces cerevisiae. J. Biol. Chem. 267, 16297-16304. Nikawa, J., Kodaki, T, and Yamashita, S. (1987). Primary structure and disruption of the phosphatidylinositol synthase gene oiSaccharomyces cerevisiae. J. Biol. Chem. 262, 4876-4881.
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Ogita, K., Miyamoto, S., Koide, H., Iwai, T., Oka, M., Ando, K., Kishimoto, A., Ikeda, K., Fukami, Y, and Nishizuka, Y. (1990). Protein kinase C in Saccharomyces cerevisiae: Comparison with the mammaUan enzyme. Proc. Natl. Acad. Sci. USA 87, 5011-5015. Ozier-Kalogeropoulos, O., Fasiolo, F., Adeline, M.-T., Collin, J., and Lacroute, F. (1991). Cloning, sequencing and characterization of the Saccharomyces cerevisiae URA 7 gene encoding CTP synthetase. Mol. Gen. Genet. 231, 7—16. Patton, J.L. & Lester, R.L. (1991). The phosphoinositol sphingolipids of Saccharomyces cerevisiae are highly localized in the plasma membrane. J. Bacteriol. 173, 3101-3108. Patton, J.L. & Lester, R.L. (1992). Phosphatidylinositol phosphate, phosphatidylinositol bisphosphate, and the phosphatidylinositol sphingolipids are found in the plasma membrane and stimulate the plasma membrane H+-ATPase of Saccharomyces cerevisiae. Arch. Biochem. Biophys. 292, 70-76. Pinto, W.J., Srinivasan, B., Shepherd, S., Schmidt, A., Dickson, R.C., and Lester, R.L. (1992). Sphingolipid long-chain-base auxotrophs of Saccharomyces cerevisiae: Genetics, physiology, and a method for their selection. J. Bacteriol. 174, 2565-2574. Roberts, M.F., Deems, R.A., and Dennis, E.A. (1977). Dual role of interfacial phospholipid in phospholipase A2 catalysis. Proc. Natl. Acad. Sci. USA 74, 1950-1954. Scheideler, M.A. & Bell, R.M. (1989). Phospholipid dependence of homogeneous, reconstituted sn-glycerol-3-phosphate acyltransferase of Escherichia coli. J. Biol. Chem. 264, 12455-12461. Schu, RV., Takegawa, K., Fry, M.J., Stack, J.H., Waterfield, M.D., and Emr, S.D. (1993). Phosphatidylinositol 3-kinase encoded by yeast VPS34 gene essential for protein sorting. Science 260, 88-91. Segel, LH. (1975). Enzyme Kinetics. Behavior and Analysis of Rapid Equilibrium and Steady-State Enzyme Systems. John Wiley and Sons, New York. Stack, J.H., Herman, P.K., Schu, P.V., and Emr, S.D. (1993). Amembrane-associated complex containing the Vpsl5 protein kinase and the Vps34 PI 3-kinase is essential for protein sorting to the yeast lysosome-like vacuole. EMBO J. 12, 2195-2204. Talwalkar, R.T. & Lester, R.L. (1973). The response of diphosphoinositide and triphosphoinositide to perturbations of the adenylate energy charge in cells of Saccharomyces cerevisiae. Biochim. Biophys. Acta 306,412-421. Talwalkar, R.T. & Lester, R.L. (1974). Synthesis of diphosphoinositide by a soluble fraction of Saccharomyces cerevisiae. Biochim. Biophys. Acta 360, 306-311. Uno, L, Fukami, K., Kato, H., Takenawa, T., and Ishikawa, T. (1988). Essential role for phosphatidylinositol 4,5-bisphosphate in yeast cell proliferation. Nature 333, 188-190. Walker, D.H., Dougherty, N., and Pike, L.J. (1988). Purification and characterization of a phosphatidylinositol kinase from A431 cells. Biochemistry 27, 6504-6511. Walsh, J.P. & Bell, R.M. (1986). sn-1, 2-diacylglycerol kinase of Escherichia coli. Mixed micellar analysis of the phospholipid cofactor requirement and divalent cation dependence. J. Biol. Chem. 261,6239-6247.
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PHOSPHOINOSITIDE METABOLISM IN MYOCARDIAL TISSUE
Robert A. Wolf
ABSTRACT I. INTRODUCTION 11. DE NOVO SYNTHESIS AND PHOSPHORYLATION OF PHOSPHOINOSITIDES IN CARDIAC TISSUE III. QUANTIFICATION OF PHOSPHOINOSITIDE-DERIVED SECOND MESSENGERS IV. MOLECULAR HETEROGENEITY OF SOLUBLE PHOSPHOLIPASE C IN CARDIAC TISSUE V. MEMBRANE-ASSOCIATED PHOSPHOLIPASE C IN CARDIAC TISSUE VI. MECHANISMS FOR REGULATION OF MYOCARDIAL PHOSPHOLIPASE C A. G-Proteins as Regulators of Myocardial PhospholipaseC B. Protein Kinases as Regulators of Myocardial PhospholipaseC C. Regulationof MyocyticPhosphohpaseCby Phosphatidic Acid
Advances in Lipobiology Volume 1, pages 387-428. Copyright © 1996 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-635-5 387
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VII. HYPOXIA, ISCHEMIA, AND REPERFUSION AS DETERMINANTS OF MYOCARDIAL METABOLISM OF PHOSPHOINOSITIDES VIII. IONIZED CALCIUM CONCENTRATION AS A DETERMINANT OF PHOSPHOLIPASEC ACTIVITY IX. FUTURE DIRECTIONS IN THE INVESTIGATION OF MYOCARDIAL METABOLISM OF PHOSPHOINOSITIDES ACKNOWLEDGMENTS REFERENCES
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ABSTRACT Hydrolysis of phosphoinositides by phospholipase C (PLC) plays an important role in signal transduction by a variety of receptors that are expressed in the adult mammalian heart. Hydrolysis of myocardial phosphoinositides is correlated with changes in inotropic function and electrophysiologic properties of cardiac myocytes as well as altered expression of myocardial genes. There is substantial evidence that metabolism of phosphoinositides is altered in ischemic and reperfused myocardium. Thus, an understanding of phosphoinositide metabolism in cardiac tissue will provide important insights into the physiology of the normal and the diseased heart. Cardiovascular investigators face significant challenges in delineating the biochemical events that regulate PLC activity in cardiac myocytes. To characterize myocytic metabolism of phosphoinositides, it is necessary to separate cardiac myocytes from nonmyocytic cells under conditions that preserve the integrity of the myocyte. There are significant methodological problems associated with the quantification of phosphoinositide-derived second messengers in cardiac myocytes. Concepts for regulation of myocardial PLC must consider the molecular heterogeneity of isoenzymes of PLC that are expressed in the adult mammalian heart. Each of these issues will be discussed in terms of future strategies for elucidating mechanisms for regulation of myocardial PLC.
I. INTRODUCTION There is substantial evidence that phosphoinositide metabolism plays an important role in signal transduction in myocardial tissue. A variety of agonists stimulate phosphodiesteric hydrolysis of phosphoinositides by myocardial phospholipase C (PLC), including a^-adrenergic agonists, muscarinic agonists, thrombin, endothelin, and angiotensin II (Brown et al., 1985; Baker and Singer, 1988; Jones et al., 1989; Vigne et a l , 1989). Myocardial inotropic function, electrophysiologic function, gene expression, and contractile protein synthesis are altered subsequent to hydrolysis of myocardial phosphoinositides by PLC (Shubeita et al., 1990; Endoh et al., 1991; Steinberg et al., 1991). Abnormal contractile function, sarcolemmal dysfunction, and myocardial hypertrophy are among the most important
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consequences of cardiovascular disease. Thus an understanding of phosphoinositide metabolism in cardiac tissue will provide important insights into the physiology of normal and diseased myocardium. Cardiovascular investigators face significant challenges in understanding the biochemical events that couple receptors to the hydrolysis of myocardial phosphoinositides. Ventricular myocardium contains multiple cell populations that may contribute to the metabolism of phosphoinositides. To characterize myocytic metabolism of phosphoinositides, it is necessary to separate biologically intact cardiac myocytes from nonmyocytic cells that inhabit cardiac tissue. Since differentiated adult myocytes do not proliferate in culture, myocytic metabolism of phosphoinositides must be investigated in primary cultures of myocytes that are prepared by enzymatic and mechanical disaggregation of ventricular myocardium (Heathers et al., 1987). There are several experimental problems associated with primary cultures of ventricular myocytes. Proteases used to isolate myocytes may modify the extracellular domains of receptors that are coupled to myocytic PLC. Long-term experiments using isolated myocytes are difficult to perform because prolonged culture of myocytes can result in loss of viability of myocytes or overgrowth by nonmyocytic cells such as cardiac fibroblasts. To reduce contamination by nonmyocytic cells, adult cardiac myocytes can be purified by the use of density gradients (Heathers et al., 1987) or neonatal myocytes can be irradiated to prevent overgrowth by contaminating cell populations (Steinberg et al, 1991). Some of the receptors and the enzymes involved in phosphoinositide metabolism are expressed in low abundance in cardiac myocytes, making it difficult to characterize the sequence of biochemical events that leads to production of phosphoinositide-derived second messengers. Livestigators must also overcome the inherent problems in quantifying second messenger production in small populations of cardiac myocytes. Biosynthesis of phosphoinositides differs from most glycerophospholipids in that the polar head group of phosphatidylinositol (PI) can undergo sequential phosphorylation at the 4 hydroxy and 5 hydroxy groups to produce phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) as shown in Figure I. Inositol 1,4,5-trisphosphate is produced in cardiac myocytes that have been stimulated by agonists coupled to myocardial PLC (Figure 2), indicating that PIP2 is a physiologic substrate for PLC in cardiac tissue (Steinberg et al., 1989). A second messenger function has been proposed for inositol 1,4,5trisphosphate in cardiac myocytes, based on the ability of this hydrophilic product to mobilize calcium from intracellular organelles in cardiac myocytes. The sarcoplasmic reticulum is presumed to be the site of inositol 1,4,5-trisphosphate-induced calcium release, although there is some controversy regarding the magnitude and the location of the calcium pool that is mobilized by this second messenger (Vites and Pappano, 1990; Borgatta et al., 1991). The other product of phosphodiesteric hydrolysis of PIP2 is 1,2-diacylglycerol (Figure 2). 1,2-Diacylglycerol is believed to play a second messenger function by regulation of myocytic protein kinase C (PKC) activity (Takai et al., 1979). 1,2-Diacylglycerol is hydrophobic and is likely
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ROBERT A. WOLF Sarcolemma
T PI
PIP,
T-Tubule
T-Tubule 4
ATP
ADP
ATP
ADP
29kDa PI Transfer Protein
CMP Inositol
n
PI \
^
PP|
CDP-
CTP
,V y p.
KJ Longitudinal Junctional Sarcoplasmic Reticulum
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Figure 1. Biosynthesis of phosphoinositides in cardiac membranes. De novo synthesis of phosphatidylinositol (PI) in the longitudinal sarcoplasmic reticulum is accomplished by reaction of CTP w i t h phosphatidic acid (PA) to form cytidine diphosphodiacylglycerol (CDP-DAG) followed by reaction with inositol to form PI. PI undergoes sequential phosphorylation to form phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) by phosphoinosltide kinases associated with the cytoplasmic leaflet of the sarcolemma. A 29 kDa PI transfer protein expressed in the cytosol of rabbit myocardium can shuttle PI from the sarcoplasmic reticulum to the sarcolemma (see text for details).
to remain in the membrane where phosphoinositides undergo hydrolysis by PLC. Production of 1,2-diacylglycerol is believed to cause translocation of PKC to sites of PLC activation resulting in the selective phosphorylation of myocardial proteins (Takai et al., 1979). At the present time it is not clear whether the selective phosphorylation of myocardial proteins is mediated by substrate specificity of PKC or by compartmentalization of phosphoinosltide hydrolysis and PKC substrates in adjacent membrane sites. Mammalian tissues express several distinct isoenzymes of PLC that are regulated by independent mechanisms. It is important to consider expression and compartmentalization of myocardial isoenzymes of PLC in order to understand the molecular details of coupling of PLC to myocardial receptors. In this chapter, I will summarize recent data concerning the compartmentalization and the expression of critical enzymes involved in myocardial metabolism of phosphoinositides. I will review the biosynthesis of myocardial substrates of PLC and the subsequent hydrolysis of these substrates by myocardial PLC. Based on these data, I will evaluate current concepts of regulation of myocardial PLC with particular attention to the influences of ischemia, reperfiision, and intracellular calcium concentration on myocardial PLC.
Myocardial
Phosphoinositides
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PI
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Figure 2, Second messenger production by myocardial phospholipase C (PLC). Hydrolysis of phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP), and phosphatidyl inositol 4,5-bisphosphate (PIP2) by PLC will produce inositol phosphates (represented by IPx) and diacylglycerol (DAG). Identification of 1(1,4,5)P3 in stimulated myocytes confirms that PIP2 is a physiologic substrate for myocardial PLC. The positional isomers for inositol phosphate species are shown in parentheses. 1(1,4,5)P3 can undergo phosphorylation by a kinase expressed in cardiac myocytes to form l(1,3,4,5)P4.
11. DE NOVO SYNTHESIS AND PHOSPHORYLATION OF PHOSPHOINOSITIDES IN CARDIAC TISSUE Several groups have reported that cardiac membranes contain the necessary substrates and enzymes to synthesize polyphosphoinositides. Varsanyi et al. reported that microsomes prepared from rabbit myocardium were capable of incorporating ^^P from [y^^P]-ATP into PIP and PIP2 (Varsanyi et al., 1986). Phosphorylation of endogenous phosphoinositides was documented in a low density fraction of microsomes that contained muscarinic receptors, but not in a high density fraction enriched for cardiac dyads (Varsanyi et al., 1986). These authors interpreted these data to indicate that biosynthesis of PIP and PIP2 was enriched in nonjunctional portions of the sarcolemma of rabbit myocardium. Quist and co-workers (1989) have characterized phosphoinositide kinases in low density, intermediate density, and high density microsomes prepared from canine myocardium. This group reported that phosphorylation of endogenous PI and PIP was enriched in low density microsomes that were enriched in ouabain-sensitive Na, K-ATPase, suggesting a predominance of phosphoinositide kinases in the sarcolemma. Quist and co-workets also reported that the phosphorylation reaction had substantial latency to treatment with alamethicin. Alamethicin is a nonspecific
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ionophor that increases access of substrates to enzymes located in the luminal leaflet of sealed membrane vesicles. PI kinase activity has 71% latency to treatment with alamethicin in a tightly sealed, asymmetrically oriented (82% right side-in) preparation of canine sarcolemmal vesicles (Wolf, 1992). These data are consistent with asymmetric distribution of phosphoinositide kinases in the sarcolemma, with enrichment of these enzymes in the cytoplasmic leaflet of the membrane. De novo synthesis and phosphorylation of phosphoinositides has been characterized in rabbit myocardial membranes enriched for sarcolemma, free (longitudinal) sarcoplasmic reticulum, or junctional sarcoplasmic reticulum (Wolf, 1990). Biosynthesis of phosphatidylinositol was quantified by incorporation of [^H]-m>'oinositol into PI in the presence of MgCl2 and CTP (see Figure 1). De novo synthesis of PI was highly enriched in preparations of free (longitudinal) sarcoplasmic reticulum as compared to junctional sarcoplasmic reticulum and sarcolemma prepared from rabbit myocardium (see Figure 3 A). These data are consistent with data from noncardiac tissues which indicate that de novo synthesis of PI occurs principally in internal membrane compartments of mammalian cells (Brophy et al., 1978). De novo synthesis of PI was also documented in junctional sarcoplasmic reticulum (Wolf, 1990). The rate ofde novo synthesis of PI in sarcolemma-enriched membranes was substantially less than that for internal membrane fractions (Figure 3). While these data establish that longitudinal sarcoplasmic reticulum contain the necessary hydrophobic substrates and the necessary enzymes for synthesis of PI, these experiments do not establish whether synthesis of PI is limited by availability of substrates or availability of biosynthetic enzymes. PI kinase activity (see Figure 3B) was characterized in these membrane preparations using exogenous PI as substrate in the presence of Triton X-100 to optimize the kinase reaction rate (Wolf, 1990). In contrast to de novo synthesis of PI, the phosphorylation of PI was highly enriched in sarcolemma compared to junctional sarcoplasmic reticulum and free (longitudinal) sarcoplasmic reticulum. Similar results were obtained when phosphorylation of endogenous PI and PIP was assayed in these membrane preparations (Wolf, 1990). These data indicate that de novo synthesis and phosphorylation of phosphoinositides are highly compartmentalized in discontinuous membrane sites in cardiac tissue. This fact has important implications for radioisotopic studies of phosphoinositide metabolism in cardiac tissue (see below). Compartmentalization of PIP2 synthesis and surface membrane receptors in the sarcolemmal membrane is consistent with receptor-coupled hydrolysis of PIP2 in this membrane site. Although biosynthesis of phosphoinositides is highly compartmentalized in cardiac tissue, there is evidence for metabolic exchange between de novo synthesized PI and pools of PI that can undergo phosphorylation to PIP and PIP2. PI synthesized de novo in the sarcoplasmic reticulum can be transferred to sarcolemmal membranes by a 29 kDa PI transfer protein that is expressed in the cytosol of rabbit myocardium (Wolf, 1990). Meij and Lamers (1989) have shown that myocytes prepared from neonatal rat hearts can incorporate exogenous [^HJ-w^^-inosi-
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JSR
FSR
Figure 3. De novo synthesis and phosphorylation of phosphatidylinositol (PI) by subcellular fractions of rabbit myocardium. De novo synthesis of PI (A) and phosphorylation of PI (B) were characterized in sarcolemma (SL), junctional sarcoplasmic reticulum (JSR), and free or longitudinal sarcoplasmic reticulum (FSR) prepared from rabbit myocardium. De novo synthesis and phosphorylation of PI occur principally in discontinuous membrane sites. Reproduced with permission of the American Physiological Society and with permission of the author (Wolf, 1990).
tol into PI, PIP, and PIP2. Thus [^H]-m>^o-inositol incorporated into myocytic PI can eventually undergo phosphorylation to form radiolabeled polyphosphoinositides. Intermembrane transfer of PI may serve as one mechanism to overcome compartmentalization of phosphoinositide synthesis and phosphorylation in cardiac tissue (Wolf, 1990). The kinetics of this process have not been carefully examined and rigorous criteria for isotopic equilibrium during metabolic labeling of cardiac myocytes or myocardial tissue with [•^H]-Aw>^o-inositol have not been established. Myocardial synthesis, transfer, and phosphorylation of phosphoinositides are summarized in Figure 1.
III. QUANTIFICATION OF PHOSPHOINOSITIDE-DERIVED SECOND MESSENGERS Numerous investigators have labeled isolated myocytes or cardiac tissue by incubation with [^H]-mjo-inositol to assess activation of PLC during stimulation of myocardial receptors. In these experimental systems, activation of myocardial PLC is quantified by the appearance of radioisotope in different species of inositol
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phosphates after separation of inositol phosphates by anion exchange chromatography or by high performance Hquid chromatography. In view of the extensive compartmentaHzation of phosphoinositide synthesis and phosphorylation in cardiac tissue (Varsanyi et al, 1986; Quist et al., 1989; Wolf, 1990) these data should be interpreted with caution since there may be a variable relationship between mass and incorporation of radioisotope into inositol phosphates. If isotopic equilibrium has not been achieved during the labeling interval, then an increase in radiolabeled inositol phosphates could occur as a consequence of altered synthesis, transfer, or phosphorylation of substrates for myocardial PLC. Some investigators have attempted to overcome the issue of isotopic disequilibrium by directly measuring the mass of phosphoinositide-derived second messengers in isolated myocytes or myocardial tissue. Three approaches have been utilized to assess the mass of inositol phosphates in cardiac tissue. Heathers and co-workers (1989b) developed a gas-liquid chromatographic method for quantification of inositol phosphates in isolated adult cardiac myocytes. This method was based on the extraction of myocytic inositol phosphates followed by separation of inositol phosphate species on anion exchange resins. The salts used to elute inositol phosphate species from the anion exchange resin were removed, inositol phosphates were enzymatically dephosphorylated, and the resulting mjo-inositol was derivatized and separated by gas-liquid chromatography and quantified by a flame-ionization detector (Heathers et al., 1989b). This method was used to characterize mass of inositol phosphates in adult myocytes stimulated with a,-adrenergic agonists (Heathers et al., 1989a), and was limited by the loss of inositol phosphates during the desalting step and by the limited sensitivity of the flameionization detector for quantification of derivatized inositol. Subsequently, DaTorre et al. (1990) modified the desalting step and resorted to gas-liquid chromatography-mass spectrometry to increase both the overall recovery and the sensitivity of inositol phosphate assay in isolated myocytes. This method has been used to characterize the time course of production of various species of inositol phosphates in stimulated adult myocytes (Kurz et al., 1993). The gas-liquid chromatographymass spectrometry method can detect as little as 200 fmol of derivatized myo-inositol and permits the simultaneous assay of different inositol phosphates from a single biological sample. Thus this method overcomes the issue of isotopic disequilibrium and provides adequate sensitivity to quantify mass of inositol phosphates in isolated myocytes (DaTorre et al, 1990; Kurz et al., 1993). However, this technique is labor-intensive and requires the use of equipment that is not widely available to cardiovascular investigators. An additional limitation of this method is that it cannot distinguish inositol 1,4,5-trisphosphate from inositol 1,3,4-trisphosphate. In view of the fact that a second messenger function has been identified for inositol 1,4,5-trisphosphate but not for its isomer (Berridge, 1987), it is desirable to selectively assay positional isomers of inositol trisphosphate. This could be accomplished by high performance liquid chromatographic separation of inositol phosphates (Steinberg et al., 1989) prior to gas-liquid chromatography-mass spectrometry.
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Inositol 1,4,5-trisphosphate mass can also be quantified by the use of a competitive protein binding assay (Mouton et al., 1991; Kurz et al., 1993). This proteinbinding assay can be performed with equipment that is widely available and has been used to quantify inositol 1,4,5-trisphosphate mass in stimulated cardiac myocytes (Kurz et al., 1993). This assay is selective for inositol 1,4,5-trisphosphate which is uniquely derived by phosphodiesteric hydrolysis of PIP2 by PLC. However, this assay cannot be used to detect inositol phosphates derived by hydrolysis of PI and PIP or by dephosphorylation of other inositol phosphates (see Figure 2). There are limited data on the quantification of mass of 1,2-diacylglycerol in cardiac tissue. Okumura and co-workers have used thin-layer chromatography and flame-ionization detection to quantify the mass of 1,2-diacylglycerol in rat hearts and in canine hearts (Okumura et al., 1988; Kawai et al., 1990). Alternatively, diglyceride kinase has been utilized to quantify 1,2-diglyceride production in isolated cardiac membranes (Wolf, 1992). It should be noted that the mass of 1,2-diglycerides cannot be specifically related to activation of phosphoinositidespecific PLC since this second messenger can also be generated by phosphodiesteric hydrolysis of choline glycerophospholipids by a distinct myocardial PLC (Wolf and Gross, 1985), by activation of myocardial phospholipase D (Lindmar et al., 1988) and the subsequent dephosphorylation of phosphatidic acid, or by acylation or deacylation of neutral lipids. Thus from the standpoint of characterizing metabolism of phosphoinositides, measurement of mass of 1,2diglycerides is a somewhat ambiguous experimental endpoint. Some investigators have characterized the fatty acid constituents of 1,2-diglycerides to infer the phospholipid source of this second messenger (Ford and Gross, 1989), but this approach has not yet been applied extensively to cardiac tissue. In general, the overall increase in mass of 1,2-diglycerides that has been observed during stimulation of myocardial receptors has been modest. However, the mass of 1,2-diglyceride has been quantified in tissue extracts and it is conceivable that compartmentalization of phosphoinositide hydrolysis could lead to substantial increases in the mass of 1,2-diglycerides in specific membrane sites within selected cell populations of cardiac tissue. Translocation of isoenzymes of PKC has been used as a surrogate marker for the local production of 1,2-diglycerides during activation of PLC in cardiac myocytes (Mochly-Rosen et al., 1990). Distinct isoenzymes of PKC are translocated to the sarcolemma and to myofibrils in cardiac myocytes that have been stimulated with norepinephrine (Mochly-Rosen et al., 1990). Since norepinephrine induces hydrolysis of phosphoinositides in this cell population (Steinberg et al., 1991), these data are consistent with receptor-coupled hydrolysis of phosphoinositides in the sarcolemma. Receptor-mediated translocation of a distinct isoenzyme of PKC to the myofibril has not been explained yet, but this observation raises the interesting possibility that nonsarcolemmal phosphoinositides may be hydrolyzed during stimulation of sarcolemma-associated receptors (Mochly-Rosen et al., 1990).
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ROBERT A. WOLF
IV. MOLECULAR HETEROGENEITY OF SOLUBLE PHOSPHOLIPASE C IN CARDIAC TISSUE Early evidence for molecular heterogeneity of myocardial PLC was based on the separation of myocardial PLC into multiple peaks of enzyme activity during chromatography on anion exchange resins (Low and Weglicki, 1983; Wolf, 1989). Further evidence for heterogeneity of myocardial PLC was based on differential sensitivity of chromatographically separated subforms of PLC to stimulation by acidic phospholipid (Wolf, 1989) or differential substrate specificity of soluble and membrane-associated PLC isolated from cardiac tissue (Schwertz et al., 1987b). These experiments did not establish the molecular basis for heterogeneity of myocardial PLC since chromatographic or functional heterogeneity could have been a consequence of distinct isoenzymes of PLC, posttranslational modification of a single enzyme (i.e., proteolytic cleavage or phosphorylation by kinases), or a consequence of complex formation between PLC and multiple myocardial proteins. There is now convincing evidence that mammalian tissues express multiple isoenzymes of PLC that represent the products of separate genes (Rhee and Choi, 1992). cDNA encoding multiple isoenzymes of mammalian PLC have been sequenced (Rhee and Choi, 1992). Based on the amino acid sequence, each of these isoenzymes can be assigned to one of three families of PLC (Figure 4). There are M-qPL-HY-i-SSHNTYL—Q
SS-EY—L—GCRCvELD-WH5 ■Y-
PLC-Pl N
I I I II I I I
PLC-yl N
11 I 11 |l
PLC-61 N LSRIYP-G-R-DSSNY-P—W—G-Q-mVALNFQT
Figure 4, Linear display of three types of mammalian phospholipase C (PLC) representing'three different families (PLCp, PLCy, and PLCs) of isoenzymes of PLC. Amino acids that are invariant (long vertical lines) or conservatively changed (short vertical lines) in all sequenced forms of PLC are located primarily in either the X domain or the Y domain of each family of Isoenzymes. Two highly conserved domains are further Identified by brackets and by the single letter amino acid code for the conserved sequences. The domains of PLCyi that share sequence homology with the src-protooncogene are represented by SH2 and SH3. Reproduced with permission of the American Society for Biochemistry and Molecular Biology and with permission of the authors (Rhee and Choi, 1992).
Myocardial Phosphoinositides
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two domains of limited sequence homology (the X and Y domains) that are shared by members of different families of PLC (Rhee and Choi, 1992). Based on deletion studies, the X and Y domains of PLC are important to catalytic activity while the nonhomologous domains are believed to facilitate unique mechanisms for regulation of each family of isoenzymes (Kriz et al., 1990). Within a given family of PLC isoenzymes, subtypes of PLC share limited sequence homology throughout the entire length of the gene product. Isoenzymes representing each of the three families (PLCp, PLC , and PLCg) are abundantly expressed in bovine brain, and much of our current knowledge on the regulation of PLC is based on characterization of PLC originally purified from this tissue (Ryu et al., 1987b). I will briefly discuss the structural and functional characteristics of each of the three families of PLC prior to discussing myocardial expression of isoenzymes of PLC. In general, the schemes that have been described for regulation of these isoenzymes have not yet been validated in cardiac tissues or cardiac myocytes. The PLCp group of isoenzymes migrates on SDS-polyacrylamide gels with M^. of approximately 140-150 kDa. One member of this family of isoenzymes, PLCpj, was originally purified from bovine brain (Ryu et al., 1987a) and is regulated by the a subunit of a pertussis-toxin resistant G-protein (G ; see Blank et al., 1991). The cDNA encoding related isoenzymes of PLC (PLCQ2 and PLCgj) has been cloned from promyelocytes and fibroblasts (Kriz et al., 1990). There is evidence that Gj^, a member of the G family of a subunits of G-proteins, selectively regulates PLCQ2 (Rhee and Choi, 1992). Interaction between the G family of G-proteins and members of the PLCQ family of isoenzymes is believed to be mediated by the highly charged carboxy-terminal region that is unique to members of the PLCp family (Rhee and Choi, 1992). PLCp, is a substrate for PKC, and phosphorylation of this isoenzyme by PKC has been suggested as a mechanism for negative feedback regulation of phosphoinositide metabolism (Rhee and Choi, 1992). The PLC^ family of phospholipases migrates with Mj. 145 kDa on SDS-polyacrylamide gels and is distinguished by three domains that share limited sequence homology with the regulatory region of the src proto-oncogene (Meisenhelder et al., 1989). Transfection and deletion studies have demonstrated that PLC j is a substrate for phosphorylation by tyrosine kinase-related receptors, and that the ^rc-like domains of PLC j are essential for phosphorylation of this isoenzyme in response to receptor stimulation (Kriz et al., 1990). Growth factors that stimulate phosphoinositide hydrolysis cause the redistribution of PLC , from cytosol to membranes and cause the selective phosphorylation of tyrosine and serine residues of PLC^j (Meisenhelder et al., 1989). There is substantial evidence that phosphorylation of PLC J is a critical step in regulation of this isoenzyme (Rhee and Choi, 1992). In general, phosphorylation of PLC^, by tyrosine kinase is associated with increased hydrolysis of phosphoinositides, while phosphorylation of PLC j by protein kinase A may prevent phosphorylation of PLC j by tyrosine kinase and inhibit receptor-coupled hydrolysis of phosphoinositides (Park et al., 1992).
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ROBERT A. WOLF
The PLCg group of isoenzymes is characterized by a smaller molecular mass (M^. of 85-87 kDa) than PLCQ and PLC . PLCgj was originally isolated from bovine brain and cDNAs for structurally-related isoenzymes have been identified for brain and fibroblasts (Kriz et al., 1990). The role of PLCg in transmembrane signaling has yet to be defined (Rhee and Choi, 1992). Consistent with the lack of a highly charged carboxy-terminal domain and the lack of 5rc-like domains, PLCgj is not regulated by G and is not a substrate for tyrosine kinase (Rhee and Choi, 1992). However, there is intriguing evidence that PLCg plays an important physiologic role in cardiovascular tissue. There have been numerous reports that phosphoinositide metabolism is altered in cardiac myocytes, cardiac fibroblasts, and in vascular smooth muscle cells derived from spontaneously hypertensive (SHR) strains of rats (Millanvoye et al., 1988; Kato et al., 1992). There is a temporal relationship between the expression of PLCg in vascular tissues and the onset of hypertension that can be correlated with enhanced calcium sensitivity of vascular PLCg in SHR rats (Kato et al., 1992). Yagisawa et al. (1991) have reported that vascular smooth muscle cells isolated from SHR rats express mRNA that encodes point mutations in the X domain of PLCgj. The physiologic role of PLCg in cardiovascular tissue is under active investigation. Data based on the application of immunochemical and molecular biological techniques have confirmed that multiple isoenzymes of PLC are expressed in adult myocardium. Suh and co-workers (1988a) used a tandem radioimmunoassay to detect immunoreactive PLC^ and PLCg in soluble extracts of bovine myocardium. These investigators were unable to detect immunoreactive PLCp in myocardial extract and reported that the concentration of PLC^ in extracts of bovine myocardium was less than 10% of the concentration of this isoenzyme in extracts of bovine brain (Suh et al., 1988a). The concentration of PLCg in myocardial extract was similar to that detected in noncardiac tissues (Suh et al, 1988a). Immunoblotting has been utilized to characterize the expression of isoenzymes of PLC in canine myocardium (Wolf, 1992). Soluble PLC activity was characterized by DEAE-cellulose chromatography followed by immunoblotting with monoclonal antibodies and with polyclonal antibodies prepared against isoenzymes of PLC that had been purified from bovine brain. As shown in Figure 5, DEAEcellulose chromatography separated myocardial PLC into two major peaks of enzymatic activity. Monoclonal antibodies to bovine PLC^j recognized a 145 kDa protein that comigrated with the second peak of PLC activity. The mobility of this myocardial protein on SDS-polyacrylamide gels was similar to that of PLC^j from bovine brain (data not shown). The first peak of myocardial PLC activity contained an 85 kDa protein that comigrated with bovine PLCgj on SDS-polyacrylamide gels (see outside lanes of Figure 5B) and was recognized by polyclonal antibodies to bovine PLCgj (see Figure 5B). This protein reacted very weakly with monoclonal antibodies to bovine PLCgj (see Figure 5A). The discrepancy in immunoblotting of canine PLC with monoclonal and with polyclonal antibodies to PLCgj may be a consequence of interspecies variability in the amino acid sequence and the
Myocardial Phosphoinositides
399
antigenicity of this isoenzyme (Suh et al, 1988b). Consistent with this hypothesis, extracts of bovine myocardium contained an 85 kDa form of PLC that reacted strongly with monoclonal antibodies to bovine PLCgj and that comigrated with canine PLCg on DEAE-cellulose (Wolf, 1992). The first peak of PLC activity eluted during DEAE-cellulose chromatography of canine cytosol also contained a 68 kDa protein that was recognized by polyclonal antibodies to PLCg (Figure 5B). This protein corresponds in molecular mass to PLC^ which is antigenically related to
40-1
A
-^PLCv
^
t Bovine
Bovine PLCd
Figure 5, Immunoblot analysis of PLC5 and PLCy expressed in canine myocardial cytosol. Canine myocardial phospholipase C (PLC) was characterized by DEAE-cellulose chromatography and by immunoblot analysis using monoclonal antibodies to PLCsi (A) polyclonal antibodies to PLC51 (B), and monoclonal antibodies to PLCyi. Samples of bovine PLCs were included in immunoblots for canine PLC5 to show the electrophoretic position of this isoenzyme. Reproduced with permission of the American Physiological Society and with permission of the author (Wolf, 1992).
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ROBERT A. WOLF
PLCg and may be derived by proteolytic cleavage of the 85 kDa form of PLCg (Taylor et al, 1992). Consistent with the radioimmunoassay data reported by Suh et al. (1988a), immunoreactive PLCp could not be detected by conventional immunoblotting of soluble extracts of either canine or bovine myocardium in spite of the fact that polyclonal antibodies to PLCpj were able to detect this isoenzyme in extracts of brain from both species (Wolf, 1992). However, many investigators have speculated that a G-protein-coupled isoenzyme of PLC is expressed in cardiac tissue (Jones et al., 1988; Steinberg etal, 1989; Renard and Poggioli, 1990). To further characterize myocardial expression of PLCg, selected fractions from DEAE-cellulose chromatography of myocardial PLC were characterized by an amplified immunoblotting technique (Figure 6).
t Rat Brain PLCfl
Rat Brain PLCfl
Figure 6. Amplified immunoblot analysis of PLCp expressed in canine myocardial cytosol. Canine myocardial phospholipase C (PLC) was characterized by DEAE-cellulose chromatography and by an amplified immunoblot technique to demonstrate myocardial expression of low concentrations of PLCp using a polyclonal antibody to PLCpi. Samples of rat brain PLCp were included in the immunoblot to show the electrophoretic position of this isoenzyme.
Myocardial
Phosphoinositides
401
This amplified detection system uses biotinylated secondary antibodies and streptavidin-alkaline phosphatase complex to provide a 20-fold increase in sensitivity for detection of antigens compared to conventional immunoblotting with alkaline phosphatase-conjugated secondary antibodies (Warren et al., 1989). Amplified immunoblotting detected a 150 kDa myocardial protein that was recognized by polyclonal antibodies to PLCpj and that comigrated with PLC^j extracted from rat brain on SDS-polyacrylamide gels (Figure 6). This myocardial protein was partially resolved from myocardial PLC on DEAE-cellulose, consistent with previous reports on the chromatographic behavior of PLCp^ (Ryu et al., 1987b). Chromatography and amplified immunoblotting of cytosol from bovine myocardium also demonstrated a 150 kDa protein that could only be detected using the amplified detection system (data not shown). When combined with the radioimmunoassay data reported by Suh and co-workers (1988a), these data are consistent with the expression of a 145 kDa form of PLC and an 85 kDa form of PLCg in extracts of canine myocardium and bovine myocardium. It is likely that a small quantity of a 150 kDa form of PLCg is also expressed in adult myocardium of both species. Homma and co-workers (1989) have utilized Northern blotting to characterize expression of isoenzymes of PLC in cardiac and in noncardiac tissues derived from adult rats using cDNA probes isolated from a rat brain library. These investigators demonstrated that adult rat myocardium contained a prominent 6.5 kb transcript that was recognized by a probe to PLC^j and a weak signal for a 3.5 kb transcript that was recognized by a probe to PLCgp Consistent with our immunochemical data on canine and bovine myocardium, short-term autoradiography failed to demonstrate a signal for mRNA to PLCQJ in rat myocardium but trace amounts of mRNA to PLCQJ could be detected in rat myocardium after prolonged autoradiography of Northern blots (Homma et al., 1989). In general, immunochemical and molecular biological data are consistent with expression of PLC and PLC§ in various species of adult myocardium. These techniques also indicate that very small amounts of PLCp are also expressed in the adult heart. Myocardial expression of multiple isoenzymes of PLC has important implications for mechanisms of transmembrane signaling in the adult heart. PLCo is regulated by G-proteins that do not regulate either PLC^ or PLCg. Tyrosine kinases that regulate PLC^ apparently do not regulate either PLCg or PLCg (Rhee and Choi, 1992). Thus myocardial expression of PLCp, PLC^, and PLCg may provide for diversification in mechanisms for signal transduction by myocardial PLC. It is tempting to speculate that the mechanisms that regulate noncardiac isoenzymes of PLC also regulate phosphoinositide metabolism in cardiac myocytes. However the data concerning myocardial expression of isoenzymes of PLC are derived from tissue extracts that may contain contributions from a variety of nonmyocytic cells (e.g., vascular smooth muscle cells, endothelial cells, cardiac fibroblasts) that also inhabit ventricular myocardium. There is a need to systematically characterize expression and regulation of isoenzymes of PLC in purified populations of cardiac
402
ROBERT A. WOLF
myocytes. Preliminary experiments have confirmed that an 85 kDa form of PLCg and a 145 kDa form of PLC^ are expressed in a highly purified population of myocytes isolated from adult rabbit myocardium (Wolf, unpublished observation). It should be noted that neonatal or embryonic myocytes have frequently been used to characterize myocytic metabolism of phosphoinositides (Jones et al., 1989; Steinberg et al., 1989, 1991). Kato and co-workers (1992) have recently demonstrated that expression of PLCg and PLC^ is developmentally regulated in aortic tissue indicating that metabolism of phosphoinositides may differ in neonatal and adult cardiovascular tissues. Thus expression and regulation of isoenzymes of PLC need to be specifically characterized in both adult and neonatal cardiac myocytes.
V. MEMBRANE-ASSOCIATED PHOSPHOLIPASE C IN CARDIAC TISSUE Numerous investigators have reported that cardiac membranes contain endogenous PLC activity (Schwertz et al., 1987b; Wolf, 1989; Edes and Kranias, 1990). The identity of membrane-associated PLC and its relationship to soluble PLC in cardiac tissue has been the source of some controversy. Based on the enzymatic properties of myocardial PLC, some investigators have reported that membrane-associated PLC is either similar to soluble PLC (Edes and Kranias, 1990) or distinct from soluble PLC (Schwertz et al, 1987b). In vitro activity of PLC is determined not only by the identity of the PLC isoenzyme, but also by the lipid milieu of the substrate as well as the buffering conditions of the assay (Ryu et al., 1987b). Thus, identification of membrane-associated PLC by enzymatic properties alone is likely to yield ambiguous results. Availability of immunochemical reagents to selectively identify isoenzymes of PLC has facilitated the identification of membrane-associated PLC in cardiac (Wolf, 1992) and in noncardiac (Lee et al., 1987) tissues. Immunoblotting has been used to characterize PLC activity associated with a highly enriched preparation of sarcolemmal vesicles prepared from canine myocardium (Wolf, 1992). This sarcolemmal preparation was substantially free of contamination by nonsarcolemmal membranes and was highly enriched for phosphoinositide kinases (Wolf, 1992). Sarcolemmal PLC was solubilized with Triton X-100 and was characterized by anion exchange chromatography on DEAE-cellulose (Figure 7). The majority of solubilized PLC activity comigrated with cytosolic PLCg on DEAE-cellulose (compare Figures 5,6, and 7). Partially purified sarcolemmal PLC contained immunoreactive PLCg (85 kDa band in Figure 7, lane 3) while immunoblotting for PLCp (Figure 7, lane 1) and PLC^ (Figure 7, lane 2) was negative. These data contrast with analysis of membrane-associated PLC activity in bovine brain, which is predominantly a 140 kDa form of PLCp (Lee et al., 1987). The association of PLCg with a myocardial site of PIP2 synthesis suggests that this isoenzyme participates in the generation of PIP2-derived second messengers in cardiac tissue. The mechanism(s) for association of PLCg with the sarcolemma and the mechanism(s) for regulation of this isoenzyme have not yet been described.
+
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z
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200
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- 1 l6.5\ pLC - 80
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/
0
0 10
30
50
70
Fraction Figure 7. lmmunoblot analysis of phospholipase C (PLC) associated with a highly enriched preparation of canine sarcolemma. PLC activity was solubilized from canine sarcolemma and characterized by DEAE-cellulose chromatography. Partially purified sarcolemmal PLC (shown by the bracket) was characterized by immunoblot analysis using polyclonal antibodies to PLCpi (lane 11, monoclonal antibodies to PLC,] (lane 2), and polyclonal antibodies to PLCsi (lane 3). Sarcolemmal PLC activity correlated with an 85 kDa form of immunoreactive PLCs (lane 3). The position of authentic isoenzymes of PLC and the position of molecular weight markers is shown next to lane 3. Reproduced with permission of the American Physiological Society and with permission of the author (Wolf, 1992).
404
ROBERT A. WOLF
However, it is interesting to note that transfection of COS-1 cells with cDNA to PLC52 results in a selective increase in membrane-associated PLC activity (Meldrum et al., 1991). Thus members of the PLCg family of isoenzymes are associated with membranes in both cardiac and noncardiac tissues. There are several important caveats to the characterization of PLC in subcellular fractions of cardiac tissue. There is evidence that the canine sarcolemmal preparation used in this study (Wolf, 1992) contains membranes derived from endothelial cells (Tomlins et al., 1986). Thus PLCg cannot be unambiguously assigned to the sarcolemmal membrane of cardiac myocytes. Furthermore, translocation or inactivation of PLC could have occurred during membrane isolation or during extraction of sarcolemmal PLC. Thus additional isoenzymes may be associated with the sarcolemma that were not detected in this study. At the present time it is not possible to assign numerical subscripts for the identification of myocardial isoenzymes of PLC because there are insufficient data to determine which subtype of each family is expressed in cardiac tissue. While immunochemical reagents can clearly distinguish isoenzymes from different families of PLC (Suh et al., 1988a), there has not been a systematic investigation into the ability of these reagents to distinguish different subtypes within a family. Subtype identification will require the sequencing of homogeneous preparations of myocardial PLC or the use of molecular biological techniques to sequence cDNA to myocardial PLC. Based on available data on the regulation of PLCp^, PLCp2, PLC J, and PLC 2 (Rhee and Choi, 1992) it seems likely that a given model for regulation of PLC activity can be generalized within a family of isoenzymes, although distinct mediators (e.g., distinct G-proteins for each subtype of PLCp) may independently regulate each subtype within a family.
VI. MECHANISMS FOR REGULATION OF MYOCARDIAL PHOSPHOLIPASE C At the present time, there are no data that demonstrate coupling of a specific isoenzyme of PLC to a specific receptor in cardiac myocytes. Mechanisms for regulation of myocardial PLC must be inferred from our knowledge of myocardial expression of isoenzymes of PLC and from evidence derived from noncardiac tissues. Based on myocardial expression of PLCp and PLC^, G-proteins and protein kinases are possible mediators for regulation of myocardial PLC activity. Potential mechanisms for regulation of myocardial PLCg have not yet been characterized. A. G-Proteins as Regulators of Myocardial Phosphollpase C
G-proteins have a heterotrimeric structure consisting of a, p, and y subunits. The a subunit contains a high affinity binding site for guanine nucleotides (GDP and GTP). In the resting state, the a subunit binds GDP and is complexed with the P and y subunits. When an appropriate receptor is activated, it interacts with the
Myocardial Phosphoinositides
405
G-protein, causing the a subunit to release GDP, bind GTP, and dissociate from the PY heterodimer (Hepler and Gilman, 1992). The activated a subunit then interacts with an effector molecule, such as PLCpj, to stimulate the production of intracellular second messengers. In some cases, the Py heterodimer may interact with an effector molecule, such as PLCp3 (Park et al., 1993). The activated a subunit returns to the resting state when the bound GTP is hydrolyzed by an intrinsic GTPase activity to yield GDP. In some cases, a subunits possess cysteine residues that can undergo covalent modification by pertussis toxin that will prevent conversion to the active form, thus uncoupling second messenger production from receptor activation (Hepler and Gilman, 1992). From an experimental point of view, G-protein-dependent signal transduction can be identified by demonstrating a GTP requirement for second messenger production or by demonstrating that second messenger production can be inhibited by pretreatment with pertussis toxin. Although there have been isolated reports that activation of myocardial PLC can be inhibited by pertussis toxin, most investigators have reported that receptorstimulated hydrolysis of phosphoinositides is resistant to pertussis toxin in adult myocardium (Masters et al., 1985; Schmitz et al, 1987), indicating that receptorinduced activation of myocardial PLC is mediated either by pertussis toxin-resistant G-proteins or by G-protein-independent mechanisms. There is preliminary evidence that a 150 kDa form of PLCp is expressed in adult myocardium (see Figure 6) and transcripts to both PLCo and G have been detected in adult myocardium (Homma et al., 1989; Strathmann and Simon, 1990). There is a preliminary report that G may participate in transmembrane signaling in isolated myocytes (LaMorte et al., 1992). If PLCoj and G are both expressed in cardiac myocytes, these proteins could provide a mechanism for coupling cell surface receptors to phosphoinositide metabolism by a G-protein-dependent, pertussis toxin-resistant mechanism. It is important to emphasize that expression of PLC Q has not yet been demonstrated in isolated myocytes, and there are currently no data to confirm the coupling of a specific isoenzyme of PLC to a specific G-protein in cardiac myocytes. Several investigators have characterized the effects of GTPyS, a nonhydrolyzable analogue of GTP, on phosphoinositide metabolism in cardiac membranes and in isolated cardiac myocytes. Jones et al. (1988) have shown that GTPyS can stimulate the production of radiolabeled inositol phosphates in permeabilized chick myocytes and that the effect of GTPyS on inositol phosphates is at least additive to the effect of thrombin (Jones et al., 1989) in this cell preparation. Steinberg et al. (1989) have characterized G-protein-dependent production of radiolabeled inositol phosphates in membranes prepared from isolated neonatal rat myocytes that had been prelabeled during 72 hour incubations with [^H]-w70-inositol. They demonstrated that GTPyS stimulated the production of radiolabeled inositol phosphates, and that GTPyS augmented the ability of norepinephrine to stimulate production of radiolabeled inositol phosphates. Renard and Poggioli (1990) have characterized the effects of GTPyS and GDPpS on the hydrolysis of radiolabeled phosphoinositides in membranes prepared from adult rat myocardium that had been prelabeled with
ROBERT A. WOLF
406
400
300H
300
200 H
200 h
100
-Log (Ca2+)M
Figure 8. GTPyS-induced accumulation oi [ H]-inositol phosphates in cardiac membranes. Rat hearts were perfused with buffer containing myo-[ H]-inositol and crude membranes were prepared by homogenization of radiolabeled myocardium. Production of radiolabeled inositol phosphates was characterized in EGTA-buffered CaCb in the absence of GTPyS (•), or in the presence of 0.1 \xhA GTPyS (A) or 10 |aM GTPyS (▼). Data are expressed as a percent of control values observed in 100 nM ionized Ca "^ in the absence of GTPyS. Data identified by (A) are significantly different from control (P< 0.05). Reproduced with permission of Academic Press and with permission of the authors (Renard and Poggioli, 1990).
perfusate containing [•^H]-w>'o-inositol. These investigators characterized the production of radiolabeled inositol phosphates as a function of ionized calcium concentration in the presence or absence of GTPyS and reported that GTPyS increased the production of radiolabeled inositol trisphosphate at physiologic concentrations of ionized calcium (Figure 8). They also reported that the effect of GTPyS w^as blocked by GDPf3S (Renard and Poggioli, 1990). The antagonistic effect of GDPpS is an important criterion for participation of a G-protein in inositol phosphate metabolism since some investigators have reported that nucleotide triphosphates can nonspecifically activate PLC (Rock and Jackowski, 1987). In general, experimental evidence supports the hypothesis that G-proteins participate in the regulation of myocardial phosphoinositide metabolism by a mechanism that is resistant to pertussis toxin. Myocardial expression of PLCp and G suggests that myocardial G-protein(s) may directly regulate myocardial PLC based on reconstitution experiments that have been performed on PLC3 and G prepared from noncardiac sources (Blank et al., 1991). It should be noted that all of the available data on regulation of myocardial PLC by G-proteins is based on production of radiolabeled inositol phosphates in experimental systems that have not been shown to be in isotopic equilibrium. In view of the fact that some of the enzymes involved in biosynthesis of polyphosphoinositides are regulated by G-proteins
Myocardial Phosphoinositides
407
(Smith and Chang, 1989), care must be exercised in the interpretation of these data. There is little doubt that G-proteins modify phosphoinositide metabolism in cardiac membranes and in some preparations of cardiac myocytes, but activation of enzymes other than PLC may contribute to the transfer of radiolabeled precursors to inositol phosphates. Measurement of GTPyS-dependent hydrolysis of exogenous [^H]-PIP2 or direct measurement of the mass of inositol phosphates would be helpful to confirm regulation of myocardial PLC by myocardial G-proteins. As described above, it has been relatively difficult to demonstrate myocardial expression of PLCg and there are currently no data that demonstrate expression of PLCp in cardiac myocytes. Based on immunochemical data (Suh et al., 1988a; Wolf, 1992) and Northern blotting data (Homma et al., 1989) from myocardial extracts, it is likely that PLCoj is expressed in low abundance if it is expressed at all in cardiac myocytes. It is conceivable that cardiac myocytes express additional isoenzymes of PLC that are regulated by distinct G-proteins. In this regard, Im and co-workers (1992) have described a novel G-protein (Gj^) that is coupled to a,-adrenergic receptors and interacts with a PLC expressed in rat liver to stimulate hydrolysis of PIP2. Based on rholecular mass and functional properties of Gj^, this G-protein appears to be distinct from G (Im et al., 1992). Although the PLC regulated by Gj^ has not yet been identified, this G-protein does not regulate in vitro activity of PLCpj, PLCyi, or PLC51 (Im et al., 1992). In a preliminary report, Das and co-workers (1992) have identified a Gj^-like protein in extracts of bovine myocardium, but coupling of G^ to myocardial PLC or to myocardial a ^-adrenergic receptors has not been reported. Blank and co-workers (1992) have recently described a PLC activity expressed in cytosol of liver tissue that is regulated by a heterodimer of Py subunits of G-proteins. Based on chromatographic and immunochemical characteristics, the PLC regulated by heterodimers of Py is distinct from PLCpi, PLC^i, and PLCg, (Blank et al., 1992). Recent data indicate that PLCp3 may be regulated by heterodimers of Py (Park et al., 1993). These data raise the interesting possibility that signaling systems distinct from PLCQJ and G may couple GTP-dependent hydrolysis of phosphoinositides to cell surface receptors. Thus characterization of G-protein-dependent mechanisms for activation of myocytic PLC should include a search for isoenzymes of PLC other than PLCoj. B. Protein Kinases as Regulators of Myocardial Phospholipase C
As described above, there is evidence that PLCpj is negatively regulated by PKC, while PLC^j is negatively regulated by protein kinase A and positively regulated by tyrosine kinase (Rhee and Choi, 1992). Phosphorylation and regulation of myocytic isoenzymes of PLC by endogenous protein kinases has not yet been demonstrated under physiologic conditions. However, there is evidence that transmembrane signaling by myocytic PLC is regulated by PKC. Meij and Lamers (1989) have demonstrated that aj-adrenergic receptor-dependent production of radiolabeled inositol phosphates can be attenuated by pretreatment of isolated
408
ROBERT A. WOLF
-205 -116.5 -80 -49,5
Figure 9. Phosphorylation of PLCyi by myocardial protein kinase A. Purified PLCyi (3 |Lig) was incubated with [y ^P]-ATP in the presence (lane 1) or absence (lane 2) of catalytic subunit of protein kinase A that had been purified from bovine myocardium. Incorporation of ^^P into PLCyi was confirmed by autoradiography. The position of molecular weight markers is shown next to lane 2.
neonatal rat myocytes with the PKC agonist, phorbol 12-myristate 13-acetate. Phosphorylation of myocytic proteins by PKC was not characterized in this study, but the authors speculated that impaired production of radiolabeled inositol phosphates was secondary to phosphorylation of a j-adrenergic receptors by PKC (Meij and Lamers, 1989). An alternative explanation is that myocytic PLC was phosphorylated by PKC, leading to inhibition of receptor-coupled hydrolysis of phosphoinositides. The receptors for both platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) contain endogenous tyrosine kinase activity that can catalyze the transfer of phosphate groups to tyrosine residues of PLC^j during stimulation with the corresponding growth factors (Rhee and Choi, 1992), leading to synthesis of DNA and cell proliferation. Phosphorylation of PLC^j by myocytic tyrosine kinase has not been confirmed, although PLC^ is expressed in this cell population (see above). EGF can stimulate DNA synthesis in myocytes isolated from adult rat myocardium, presumably by activating the EGF-receptor-associated tyrosine kinase (Claycomb and Moses, 1988). The intracellular substrates for myocytic tyrosine kinase have not yet been identified. Phosphorylation of PLC^j by protein kinase A can inhibit phosphorylation of this isoenzyme by tyrosine kinase and decrease receptor-coupled hydrolysis of phosphoinositides in some mammalian cells (Rhee and Choi, 1992). Although phospho-
Myocardial Phosphoinositides
409
rylation of PLC^j by myocytic protein kinase A has not been demonstrated under physiologic conditions, this isoenzyme is clearly a substrate for phosphorylation by the catalytic subunit of protein kinase A that is expressed in bovine myocardium (Figure 9). C. Regulation of Myocytic Phospholipase C by Phosphatidic Acid
A variety of agonists have been shown to stimulate an increase in phosphatidic acid in mammalian cells (Bocckino et al., 1987; Rubin, 1988). The precise mechanisms for receptor-coupled production of phosphatidic acid may vary depending on the cell type, although stimulation of phospholipase D or stimulation of PLC and subsequent phosphorylation of 1,2-diglycerides have been identified during receptor stimulation (Bocckino et al., 1987; Rubin, 1988). Exposure to exogenous phosphatidic acid, exogenous lysophosphatidic acid, or exogenous phospholipase D can induce many of the same biologic responses that can be induced during receptor stimulation of mammalian cells, including the stimulation of DNA synthesis and cell proliferation, the mobilization of intracellular stores of calcium, and the expression of genes related to mitogenesis (Knauss et al., 1990). Exposure to exogenous phosphatidic acid is temporally associated with activation of phosphoinositide metabolism in mammalian cells, and the biologic response to phosphatidic acid resembles the response to stimulation of cell surface receptors that are coupled to intracellular PLC. Based on these observations, some investigators have proposed that phosphatidic acid may initiate or sustain phosphoinositide hydrolysis under physiologic conditions (Jackowski and Rock, 1989). The effects of phosphatidic acid on phosphoinositide metabolism have recently been characterized in isolated myocytes prepared from adult rabbit myocardium (Kurz et al., 1993). Mass of myocytic inositol phosphates was quantified by the use of the competitive protein binding assay for inositol 1,4,5-trisphosphate and by the use of the gas-liquid chromatography-mass spectrometry method for quantification of inositol phosphate species. Phosphatidic acid induced a rapid and transient increase in the mass of inositol 1,4,5-trisphosphate in adult cardiac myocytes (Figure 10). A four- to fivefold increase in the mass of this second messenger was observed within 30 seconds of exposure to 1.0 juM phosphatidic acid. The increase in myocytic inositol 1,4,5-trisphosphate was a function of phosphatidic acid concentration, with the concentration required for 50% maximal stimulation (EC5Q) being only 4.4 x 10"^ M (Figure 11). The time course of production of other inositol phosphate species in response to phosphatidic acid is shown in Figure 12. In general, these data demonstrate a sequential increase in the mass of inositol trisphosphate, inositol tetrakisphosphate, inositol bisphosphate, and inositol monophosphate after exposure to phosphatidic acid. There have been reports that phosphatidic acid can act as a calcium ionophore in some preparations (Salmon and Honeyman, 1980). To exclude a calcium ionophore effect as the mechanism for altered phosphoinositide metabolism in myocytes, the
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1—I—I—r 100 120 Time (seconds] Figure 10, Time course of production of inositol 1,4,5-trisphosphate (l[1,4,5]P3) in response to phosphatidic acid (1 |iM) in myocytes prepared from ventricular myocardium of adult rabbits. Myocytes were exposed to 1 (iM phosphatidic acid at 37° C and mass of l(1,4,5)P3 was quantified by a specific protein binding assay. Values significantly different from control values are indicated (* P < 0.05, **P < 0.01). Reproduced with permission of the American Heart Association and with permission of the authors (Kurzetal., 1993).
myocyte experiments were repeated in extracellular buffers that lacked calcium salts and in buffers that contained 1.0 mM EGTA. Exclusion of calcium from extracellular buffer did not attenuate the inositol 1,4,5-trisphosphate response in cardiac myocytes, indicating that extracellular calcium was not essential to the phosphatidic acid effect (Kurz et al., 1993). There is controversy regarding the relative importance of phosphatidic acid and lysophosphatidic acid as signal transducers in mammalian tissues. This controversy is due, in part, to the fact that some preparations of phosphatidic acid contain substantial contamination (up to 11 %) by lysophosphatidic acid (Jalink et al., 1992; Knauss et al., 1990). Jalink and co-workers have concluded that lysophosphatidic acid, but not phosphatidic acid, is a potent calcium mobilizing stimulus in human fibroblasts (Jalink et al, 1992). These investigators were unable to elicit a calcium response in fibroblasts using chromatographically purified phosphatidic acid that had been solubilized with bovine serum albumin (Jalink et al., 1992). Hashizume et al. (1992) have recently noted that bovine serum albumin can inhibit responsive-
Myocardial
Phosphoinositides
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Phosphatidic Acid [M] • Figure 11, Accumulation of inositol 1,4,5-trisphosphate (l[1,4,5]P3) as a function of concentration of phosphatidic acid. Myocytes prepared from adult rabbit myocardium were exposed to various concentrations of phosphatidic acid for 20 seconds and mass of I(1,4,5)P3 was quantified by a specific protein binding assay. The dose-response curve, expressed as a percent of the maximum response, is shown in the inset. Values significantly different from control are indicated ( P < 0.05, P < 0.01). Reproduced with permission of the American Heart Association and with permission of the authors (Kurzetal., 1993).
ness to chromatographically purified phosphatidic acid. These investigators demonstrated that liposomes of chromatographically purified long-chain and mediumchain phosphatidic acid, prepared in the absence of bovine serum albumin, can stimulate PLC in rabbit platelets and induce platelet aggregation whereas the corresponding lysophosphatides were less potent agonists in this preparation (Hashizume et al., 1992; Sato et al., 1992). Control experiments demonstrated that the increase in inositol 1,4,5-trisphosphate observed in phosphatidic acid-stimulated myocytes was not due to contaminating lysophosphatidic acid (Kurz et al., 1993). However, lysophosphatidic acid has not been systemically characterized as an agonist of phosphoinositide metabolism in cardiac myocytes. The biochemical mechanism of the phosphatidic acid effect has not yet been determined. Phosphatidic acid can stimulate in vitro activity of both membrane-associated and soluble PLC activity in platelets (Jackowski and Rock, 1989), suggesting that phosphatidic acid may interact with PLC. Alternatively, phosphatidic acid has unique physical-chemical properties that may alter the lipid milieu of PLC
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160 i c
140 ■
V
o o.
120-
o>
E 100-
IPl
0}
o
E
o.
80 •
IP2
60 40 20
IP3 IP4 45 60 Time [seconds]
120
Figure 12. Production of inositol monophosphate (IP1, n), inositol bisphosphate (IP2, ♦), inositol trisphosphate (IP3, • ) , and inositol tetrakisphosphate (IP4, o) after stimulation of adult cardiac myocytes with phosphatidic acid. Adult ventricular myocytes were stimulated with 1 |LIM phosphatidic acid and the mass of IP1, IP2, IP3, and IP4 were quantified using anion exchange chromatography followed by gas-liquid chromatography-m^ass spectrometry. Values significantly different from control are indicated ( P < 0.05, P < 0.01). Reproduced with permission of the American Heart Association and with permission of the authors (Kurz et al., 1993).
substrates to make them more susceptible to hydrolysis by PLC. When diluted into lipid bilayers containing phosphatidylcholine or phosphatidylethanolamine, phosphatidic acid can undergo lateral phase separation in response to an increase in calcium ions or exposure to certain cytosolic proteins (Bazzi and Nelsestuen, 1991). Furthermore, phosphatidic acid binds calcium with high affmity and an increase in calcium concentration or a decrease in pH can induce the formation of nonlamellar structures by phosphatidic acid (Farren et al, 1983). There has also been a report that phosphatidic acid may induce the selective phosphorylation of myocardial proteins by a unique myocardial protein kinase (Bocckino et al., 1991). Stimulation of PLC by lysophosphatidic acid in fibroblasts is augmented by GTPyS, suggesting the participation of a G-protein in this reaction (van Corven et al, 1989). There is evidence that lysophosphatidic acid acts at an extracellular site to stimulate PLC activity in fibroblasts (Jalink et al., 1992), whereas uptake of phosphatidic acid appears to be a critical event in modulation of PLC activity in platelets (Hashizume et al., 1992; Sato et al., 1992). Thus it is conceivable that lysophosphatidic acid and
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phosphatidic acid regulate PLC activity by distinct mechanisms. At the present time, it is not clear which of these mechanisms, if any, mediate the effect of phosphatidic acid on phosphoinositide metabolism in cardiac myocytes.
VII. HYPOXIA, ISCHEMIA, AND REPERFUSION AS DETERMINANTS OF MYOCARDIAL METABOLISM OF PHOSPHOINOSITIDES Numerous investigators have characterized the effects of hypoxia, ischemia, and reperfusion on myocardial metabolism of phosphoinositides. These studies have been motivated, in part, by the observation that calcium metabolism and phosphoinositide metabolism are simultaneously altered during ischemia and reperfusion, suggesting that a cause-effect relationship may exist between calcium overload and activation of myocytic PLC. Phosphoinositide-derived second messengers may increase concentration of ionized calcium in cardiac myocytes, or alternatively calcium-dependent PLC may become activated during calcium overload in cardiac myocytes. In addition, there is substantial evidence that myocardial tissue has increased sensitivity to stimulation of a ^-adrenergic receptors during ischemia and reperfusion (Heathers et al., 1988). Since myocardial aj-adrenergic receptors are coupled to the production of phosphoinositide-derived second messengers (Brown et al., 1985), increased biological sensitivity to a^adrenergic stimulation has motivated a search for altered coupling of myocytic PLC under pathophysiologic conditions. A variety of techniques and preparations have been used to characterize the effects of hypoxia, ischemia, and reperfusion on myocardial metabolism of phosphoinositides. In 1987, Schwertz and colleagues characterized the effects of global ischemia on PI mass and on PLC activity in isolated, perfused adult rat hearts. PLC activity in cytosol and in crude membranes was characterized by hydrolysis of exogenous [^H]-PI in the presence of 1.0 mM ionized calcium at pH 5.5 (Schwertz et al., 1987a). These investigators reported that mass of myocardial PI increased and that activity of soluble PLC decreased within 30 minutes of ischemia. These investigators interpreted these data to indicate that ischemia induced a decrease in phosphodiesteric hydrolysis of PI, leading to an increase in mass of myocardial PI, although they noted that additional mechanisms may have contributed to the increase in myocardial PI (Schwertz et al., 1987a). Using this assay system, these investigators detected minimal activity of PLC in cardiac membranes and did not detect any changes in membrane-associated PLC activity during ischemia. In a more recent study, this group characterized PLC activity in cytosol and in crude membranes prepared from myocardial biopsies taken during normal oxygen level perfusion, global ischemia, and reperfusion of isolated adult rat hearts (Schwertz and Halverson, 1992). In this study, Schwertz and Halverson characterized hydrolysis of exogenous [^H]-PIP and [^H]-PIP2 at an ionized calcium concentration of approximately 30 |LIM, pH 6.8. These assay conditions permitted the detection of
ROBERT A. WOLF
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I Membrane ^Cytosol DHomogenate
30
2 20 c o o 10
c CO
0
O-IO
m^ J
ryn
I
I
Q_ I
CL TT -20
-30
10 30 60 Time of Ischemia (min)
Figure 13, Cytosollc and membrane-associated phospholipase C (PLC) in ischemic rat myocardium. Sequential biopsies were obtained from the left ventricle of isolated, perfused rat hearts during global ischemia. Cytosolic and crude membrane fractions were prepared by homogenization of myocardial biopsies and PLC activity was assayed in each fraction and in total homogenate. Data significantly different from control are indicated ( P < 0.05). Reproduced with permission of Springer-Verlag Publishers and with permission of the authors (Schwertz and Halverson, 1992).
membrane-associated PLC activity that was not detected in the previous study (Schwertz et al., 1987a). Within five minutes of onset of ischemia, there was a slight increase in membrane-associated PLC activity, and a slight decrease in soluble PLC activity. After 10 minutes of ischemia, membrane-associated PLC activity decreased significantly and soluble PLC activity increased significantly compared to control activity (Figure 13). The increase in soluble PLC activity and the decrease in membrane-associated activity was sustained after 30 and 60 minutes of global ischemia (Schwertz and Halverson, 1992). Thus, using different assay techniques, these investigators reached opposite conclusions regarding the effects of ischemia on soluble PLC activity in the isolated rat heart. These discrepant results might be
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resolved by considering the enzymatic properties of isoenzymes of PLC that are expressed in adult myocardium. Enzymatic activity of PLC is optimized using PI as substrate at ionized calcium concentrations above 1.0 mM (Ryu et al., 1987a; Kriz et al., 1990). In contrast, activity of PLCg is optimized using PIP2 as substrate at ionized calcium concentrations between 10 [iM and 100 jiM (Ryu et al., 1987a; Kriz et al., 1990). Thus the assay conditions used by Schwertz et al. in their 1987 study probably optimized detection of PLC while the more recent study used assay conditions that were optimal for PLCg. The enhanced ability to detect membraneassociated activity in the second study is consistent with data showing that PLCg is present in both cytosol and in membranes prepared from myocardium, while PLC is preferentially distributed in cytosol (see above). While these data indicate that ischemia can differentially affect soluble and membrane-associated PLC activity, it is not possible to determine if the effect of ischemia is attributable to translocation of PLC or a modification of the enzymatic properties of myocardial PLC. As suggested by the authors, immunoblotting of isoenzymes of PLC in soluble and in membranous compartments could be used to address the issue of translocation of myocardial. PLC during ischemia (Schwertz and Halverson, 1992). In their study, Schwertz and Halverson (1992) characterized the effects of reperfusion on soluble and membrane-associated PLC activity after 40 minutes of global ischemia. They detected a significant increase in soluble PLC activity in reperftised myocardium, compared to both control and ischemic myocardium (Schwertz and Halverson, 1992). Membrane-associated PLC activity also increased significantly during reperfiision when compared to activity in membranes prepared from nonischemic myocardium. This increase in membrane-associated PLC was particularly striking when compared to the decrease in activity found in membranes isolated from myocardium after 30 to 60 minutes of ischemia, indicating that profound changes occurred in membrane-associated PLC when ischemic myocardium was reperfiised. For the reason described above, further studies will be necessary to delineate the mechanisms for these changes in membrane-associated and soluble PLC during reperfusion of myocardium. Since both soluble and membrane-associated activities increased during reperfusion, it seems unlikely that translocation is the only mechanism for the observed changes in PLC activity. Otani and co-workers (1988) have characterized the effects of ischemia and reperfusion on the incorporation of radiolabeled precursors into phosphoinositides in isolated, perfiised rat hearts. Hearts were prelabeled by brief (45 minutes) perfusion with [^E]-myo-inosito\ prior to 30 minutes of global ischemia and 30 minutes of reperfusion with buffer containing LiCl (to inhibit dephosphorylation of inositol monophosphate). Data from reperftised hearts were compared to data from prelabeled hearts that were perfused with normoxic buffer for 60 minutes. Compared to control hearts, 30 minutes of ischemia caused a decrease in the incorporation of [^H]-m>;(9-inositol into PIP and PIP2, but did not significantly alter labeling of PI. During reperfusion, incorporation of radioisotope into PIP and PIP2 increased and approached the level of radioisotopic incorporation that was achieved
ROBERT A. WOLF
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in control hearts. There was enhanced incorporation of [^H]-m>^o-inositol into inositol phosphates during reperfusion of ischemic myocardium compared to control hearts. The mass of myocardial PI was stable during 30 minutes of ischemia (in contrast to the results reported by Schwertz et al., 1987a) but tended to decrease slightly during reperfusion. If perfusion with [^H]-w;;o-inositol was delayed until the reperfusion interval, there was enhanced incorporation of radioisotope into PI, PIP, and PIP2 in reperfused hearts compared to nonischemic control hearts. The authors interpreted these data to be consistent with inhibition of PLC during ischemia and stimulation of PLC during reperfusion, although they noted that reactions proximal to phosphodiesteric hydrolysis of phosphoinositides by PLC could have mediated the observed changes in incorporation of radioisotope (Otani et al., 1988). They also presented data on the incorporation of ['"^CJ-arachidonic acid and [^H]-glycerol into phosphoinositides during ischemia and reperfusion. These data were generally supportive of the hypothesis that metabolic turnover of phosphoinositides is impaired after 30 minutes of ischemia and stimulated during reperfusion. Interpretation of the data from this study is subject to the limitations
_140 1 130 1120 S110
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aioo 90 BO ' 70 ^- 60 50
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30 (min)
igatjon Figure 14, Mass of 1,2-diacylglycerol (1,2-DG) in ischemic (•) and nonischemic (o) myocardium after coronary ligation in the absence (—) or in the presence (---) of prazosin. Data are expressed as a percent of control values and values significantly different from control are indicated (**P < 0.01 compared to 0 minutes, *P < 0.01 compared to nonischemic myocardium, P < 0.01 compared to absence of prazosin). Reproduced with permission of Kluwer Academic Publishers and with permission of the authors (Kawai et al., 1990).
Myocardial Phosphoinositides
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that the precise enzymatic steps altered by ischemia and reperfiision can not be identified and altered phosphoinositide metabolism can not be localized to a particular cell population within the myocardium. Several investigators have characterized the effects of ischemia and reperfusion on the mass of phosphoinositide-derived second messengers in ischemic and reperfused myocardium. Kawai et al. (1990) quantified 1,2-diglycerides in ischemic and nonischemic zones of the left ventricle following occlusion of the left anterior descending coronary artery in anesthetized dogs. Five minutes after occlusion of the artery, 1,2-diglycerides were increased in both the ischemic and nonischemic zones, although the increase was more significant in the ischemic zone (Figure 14). After 30 minutes of coronary artery occlusion, the mass of 1,2diglycerides decreased relative to control values, especially in the ischemic zone. The initial increase and the later decrease in 1,2-diglycerides in the ischemic zone was markedly attenuated by prazosin. 1,2-Diglycerides were also quantified in nonischemic and postischemic zones during reperfusion of the occluded artery (Figure 15). These data revealed a transient increase in 1,2-diglycerides during
**
^160 i 1 50 " 1 40 30 S 1 20 5 1 10 = 1 00 90 80
^::i«
0 2 5
t
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30 (min)
reperfusion Figure 15, Mass of 1,2-diacylglycerol (1,2-DG) in ischemic (•) and nonischemic (o) myocardium after reperfusion in the absence (—) or in the presence (—) of prazosin. Values shown are expressed as percent of value at time 0 minutes. Values significantly different from control are indicated ( P < 0.05 compared to 0 minutes, P < 0.01 compared to 0 minutes, *^ P < 0.01 compared to nonischemic myocardium, '^"'^ P < 0.01 compared to absence of prazosin). Reproduced with permission of Kluwer Academic Publishers and with permission of the authors (Kawai et al., 1990).
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reperflision that was especially significant in the postischemic zone. The reperfusion-induced increase in 1,2-diglycerides was also attenuated by prazosin. These data implicate a j-adrenergic receptors as mediators of altered 1,2-diglyceride metabolism in ischemic and reperfused myocardium. Ford and Gross (1989) have characterized diacyl and ether-linked species of 1,2-diglycerides in isolated perfused rabbit hearts. They observed a time-dependent decrease in 1,2-diacylglycerides in normoxic and in ischemic rabbit hearts. Based on the fatty acid constituents of myocardial 1,2-diacylglycerides, these investigators concluded that the majority of 1,2-diacylglycerides present in normoxic and in ischemic rabbit hearts were derived from lipid pools other than phosphoinositides. Similarly, Chien et al. (1984) reported a time-dependent decrease in 1,2diglycerides in both ischemic and nonischemic zones of canine myocardium following the occlusion of the left anterior descending coronary artery. Mouton and colleagues (1991) have quantified inositol 1,4,5-trisphosphate mass by the competitive binding protein assay in isolated adult rat hearts subjected to ischemic arrest followed by reperflision. These investigators documented a significant decrease in mass of inositol 1,4,5-trisphosphate after 20 minutes of ischemic arrest followed by a significant increase during reperfusion. The increase in mass of inositol 1,4,5-trisphosphate that occurred during reperfusion was prevented by the PLC inhibitor, neomycin. These data were therefore consistent with activation of myocardial PLC during reperfusion, although the role of a j-adrenergic receptors during reperfusion was not characterized in this study. Two groups have characterized the effects of hypoxia on transmembrane signaling by PLC in purified populations of cardiac myocytes. Heathers et al. (1989a) characterized the effects of hypoxia on the coupling of a j-adrenergic receptors to myocytic metabolism of phosphoinositides. These investigators utilized gas-liquid chromatography to characterize inositol phosphate species in myocytes prepared from canine myocardium. They reported that norepinephrine stimulated a sequential increase in inositol trisphosphate, inositol tetrakisphosphate, inositol bisphosphate, and inositol monophosphate in canine myocytes. Hypoxia caused a sixfold reduction in the concentration of norepinephrine required to achieve 50% maximal stimulation of inositol trisphosphate production, and a 100-fold reduction in the threshold concentration of norepinephrine required to elicit an inositol trisphosphate response. These authors concluded that hypoxia increased a j-adrenergic responsiveness of PLC in cardiac myocytes. Steinberg and Alter (1991) have reported preliminary data on the effects of hypoxia on the incorporation of [^H]myo-inosiio\ into inositol phosphates in neonatal rat myocytes during stimulation with a variety of agonists that are coupled to PLC. They reported that hypoxia increased the incorporation of radioisotope into inositol phosphates in response to stimulation with norepinephrine, thrombin, and carbachol without affecting the production of cAMP in response to stimulation by isoproterenol. They interpreted these data to indicate that hypoxia enhances coupling of myocytic receptors to PLC, but does not alter coupling of receptors to adenylyl cyclase.
Myocardial Phosphoinositides
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Thus a variety of preparations have been used to document altered metabolism of phosphoinositides during hypoxia, ischemia, and reperfusion. It is important to consider the unique properties of each of these experimental preparations as well as the limitations of the methods used by each investigator. The experiments performed by Schwertz et al, Otani et al, and Mouton et al. were all performed on isolated perfused rat hearts. While these ex vivo preparations were denervated, it is still possible that neurotransmitters could have influenced the observed changes in phosphoinositide metabolism during ischemia and early reperfusion. Schomig and colleagues (1984) have documented profound increases in catecholamines, including norepinephrine, in coronary effluent collected during early reperfusion of isolated rat hearts. These data are consistent with a dramatic increase in interstitial concentrations of catecholamines during myocardial ischemia, with redistribution and washout of catecholamines during reperfusion. In view of the fact that a^-adrenergic receptors are coupled to production of phosphoinositide-derived second messengers in cardiac myocytes, myocardial metabolism of norepinephrine may be an important determinant of phosphoinositide metabolism in ischemic and reperfused myocardium. The data reported by Kawai et al. (1990) may have been influenced by neural traffic in the autonomic nervous system since this group characterized second messenger production during in vivo experiments performed on anesthetized dogs. Since each of these investigators characterized phosphoinositide metabolism in tissue extracts, there is ambiguity regarding the cell population that was responsible for the observed changes during ischemia and reperfusion. The data reported by Heathers et al. and Steinberg and Alter were derived from isolated myocytes. Myocyte preparations permit localization of phosphoinositide metabolism to this cell population and exclude the influence of neurotransmitters and metabolites that accumulate in ischemic and reperfused myocardium. It is also important to recognize that some of these investigators directly measured the mass of phosphoinositide-derived second messengers, while others relied on the incorporation of radioactive precursors to assess phosphoinositide metabolism. It is difficult to synthesize data from multiple experimental preparations into a single model that can explain modulation of phosphoinositide metabolism during ischemia and reperfusion. However, there are some general trends that can be observed in these data. Further work is necessary to characterize myocardial metabolism of phosphoinositides during early ischemia, but available data indicate that 30 minutes or more of ischemia results in decreased metabolism of myocardial phosphoinositides and decreased myocardial content of phosphoinositide-derived second messengers. Schwertz and Halverson (1992) reported that in vitro activity of membrane-associated PLC increased after five minutes of ischemia, but subsequently decreased during more prolonged ischemia. The decrease in membraneassociated PLC activity after 30 minutes of ischemia was not altered by pretreatment with prazosin suggesting that changes in membrane-associated PLC activity were independent of aj-adrenergic receptors. It is tempting to speculate
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that these changes in membrane-associated PLC activity contributed to the observed changes in metabolism of phosphoinositides by ischemic myocardium. During early reperfusion, there is increased metabolic turnover of myocardial phosphoinositides (Otani et al, 1988) and increased mass of phosphoinositide-derived second messengers in myocardial extracts (Kawai et al., 1990; Mouton et al, 1991). These results correlate with an apparent increase in in vitro activity of membrane-associated PLC and soluble PLC in homogenates of reperfused myocardium (Schwertz and Halverson, 1992). The available data are therefore consistent with accelerated hydrolysis of phosphoinositides by myocardial PLC during reperfusion. The mechanism(s) of increased hydrolysis of phosphoinositides by PLC in reperfused myocardium has not been determined. Some investigators have examined the role of aj-adrenergic receptors in mediating this effect. Otani et al. (1988) reported that aj-adrenergic blockade did not alter the accelerated incorporation of [^H]-m7o-inositol into inositol phosphates during reperfusion, while Kawai et al. (1990) reported that prazosin markedly attenuated the increase in 1,2-diglycerides that occurred in reperfused myocardium. It should be noted that these two groups were investigating different experimental endpoints in two different experimental preparations. Redistribution of neurotransmitters, alterations in receptor coupling, alterations in intracellular calcium concentration, and alterations in PLC activity may conspire to modulate myocardial metabolism of phosphoinositides in a time-dependent fashion during early reperfusion. Thus, independent investigators may reach different conclusions about this nonequilibrium state as a function of experimental protocol.
VIII. IONIZED CALCIUM CONCENTRATION AS A DETERMINANT OF PHOSPHOLIPASE C ACTIVITY Depolarization of cardiac myocytes during the action potential results in an increase of intracellular ionized calcium from a diastolic concentration of approximately 100—300 nM to a peak systolic concentration of approximately 800 nM (Peeters et al., 1987). In vitro activity of PLC increases significantly when ionized calcium concentration is increased from 100 nM to 1,000 nM (Ryu et al., 1987b), causing some investigators to speculate that myocytic PLC activity may be determined, in part, by physiologic changes in intracellular calcium concentration. Electrical stimulation, increased extracellular potassium concentration, and sodium channel activation have all been used to characterize the effects of membrane depolarization and intracellular calcium concentration on myocardial metabolism of phosphoinositides. Poggioli et al. (1986) reported that rapid electrical stimulation of isolated rat hearts caused an increase in the incorporation of [^H]-mj;o-inositol into inositol phosphates and concluded that membrane depolarization results in activation of myocardial PLC. In contrast, Otani et al. (1986) reported that electrical stimulation did not alter the incorporation of radioisotope into inositol phosphates after brief (15 minutes) incubation of rat papillary muscle with [-^HJ-mj^o-inositol. Depolari-
Myocardial Phosphoinositides
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zation of myocardial tissue with 40 mM KCl enhances receptor-activated hydrolysis of phosphoinositides and depolarization with 100 mM KCl has been reported to directly stimulate hydrolysis of myocardial phosphoinositides by PLC (Kaku et al., 1991). Intracellular calcium concentration was not monitored in these studies, so it is not possible to determine if these potassium-induced changes in phosphoinositide metabolism correlated with changes in intracellular ionized calcium concentration. McDonough et al. (1988) have shown that activation of sodium channels increases ionized intracellular calcium concentration in embryonic chick heart myocytes coincident with an increase in [^H]-m>^(9-inositol labeled mositol phosphates. Extracellular calcium and sodium were both required to stimulate phosphoinositide metabolism in response to sodium channel activation, suggesting that the Na/Ca exchange mechanism may mediate the activation of PLC. Otani et al. (1988) reported that the increase in phosphoinositide metabolism that accompanies reperfusion can be inhibited by reperfiision with calcium-free perfusate and concluded that calcium influx is critical to enhanced turnover of phosphoinositides in the reperfused heart. Taken together, these data indicate that experimental conditions that increase intracellular ionized calcium concentration or depolarize the sarcolemma may enhance coupling of PLC to myocardial receptors or may directly stimulate the hydrolysis of myocardial phosphoinositides by PLC. However, there is some controversy regarding the systolic increase in intracellular calcium as a determinant of myocytic PLC activity. This issue should be considered by investigators working with isolated myocytes since neonatal myocytes have spontaneous action potentials and calcium transients in culture, whereas adult myocytes are quiescent unless electrically stimulated. Electrical field stimulation of isolated adult myocytes has not been characterized extensively as a determinant of phosphoinositide metabolism.
IX. FUTURE DIRECTIONS IN THE INVESTIGATION OF MYOCARDIAL METABOLISM OF PHOSPHOINOSITIDES There is convincing evidence for molecular diversity of PLC in cardiac tissue, suggesting that there are diverse mechanisms for regulation of myocardial metabolism of phosphoinositides. There is a need to further define the expression of isoenzymes of PLC in purified populations of cardiac myocytes. There is preliminary evidence that an 85 kDa form of PLCg and a 145 kDa form of PLC^ are expressed in a purified population of ventricular myocytes prepared from adult rabbit myocardium (Wolf, unpublished observation). However, these data are based on immunochemical identification of PLC and it is not clear whether these immunochemical reagents can distinguish different subtypes of PLC within a family. Although functional differences among subtypes within a family have not yet been demonstrated, sequence analysis has demonstrated interesting differences in the primary structure of subtypes belonging to the PLCg family of isoenzymes. PLC52 contains six consecutive glutamic acid residues (residues 440-445) that are lacking
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in PLCg, (Suh et al., 1988b; Meldrum et al., 1991). Because of the relatively low abundance of these proteins in cardiac tissue, it is likely that a molecular biological approach will be necessary to characterize the expression of subtypes of PLCg in myocardial tissue and in cardiac myocytes. Association of PLC§ with sarcolemmal sites of PIP2 synthesis (Wolf, 1992) and the recent discovery of mutant forms of PLCgj in vascular tissues of the SHR rat (Yagisawa et al., 1991) suggest that this family of PLC may play an important physiologic role in cardiovascular tissues. It is therefore important to determine how this family of isoenzymes of PLC is regulated and to determine the mechanisms for association of PLCg with biological membranes. The expression of members of the PLCo family in cardiac myocytes remains an unsettled issue. Many of the receptors that are coupled to myocardial PLC contain seven membrane-spanning domains. This structural feature is shared by receptors that are coupled to second messenger production by membrane-associated G-proteins (Hepler and Oilman, 1992). Thus it is likely that G-protein-coupled forms of PLC, such as PLCgj, are expressed in cardiac myocytes. However, recent data demonstrate that isoenzymes distinct from PLCp, are coupled to G-proteins (Blank et al., 1992; Im et al., 1992), indicating that the search for G-protein-dependent PLC should not be limited to a search for PLCQJ in the cardiac myocyte. There is substantial evidence that phosphorylation of PLC^j by tyrosine kinase plays an important role in mediating the proliferative response to growth factors in certain cell types. Although some investigators have reported that adult cardiac myocytes can be stimulated to synthesize DNA in response to growth factors (Claycomb and Moses, 1988), tyrosine phosphorylation of PLC^ has not been documented in cardiac myocytes. The fact that PLC is a substrate for in vitro phosphorylation by myocardial protein kinase A could provide a mechanism for "cross talk" between transmembrane signaling mechanisms that generate cAMP and inositol phosphates in cardiac myocytes. Thus phosphorylation of myocytic isoenzymes of PLC, including PLC^, deserves further investigation as a mechanism for regulation of phosphoinositide metabolism. Stimulation of PLC by phosphatidic acid could provide a novel mechanism for initiating or sustaining second messenger production in cardiac myocytes (Kurz et al, 1993). Exogenous phosphatidic acid and phospholipase D can induce slow action potentials in potassium-depolarized atrial myocytes (Knabb et al., 1984). Exogenous phospholipase D also induces a significant increase in contractility in neonatal rat myocytes coincident with an increase in myocytic phosphatidic acid (Burt et al., 1984). These effects were previously attributed to altered Na/Ca exchange or to a calcium ionophoric effect of phosphatidic acid (Burt et al., 1984). However, production of phosphoinositide-derived second messengers may also have contributed to the altered electrical and mechanical function observed in response to phosphatidic acid. A physiologic function for phosphatidic acid-mediated phosphoinositide hydrolysis presumes that myocytic phosphatidic acid content is modulated under
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physiologic conditions. Potential mechanisms for an increase in myocytic phosphatidic acid include activation of endogenous phospholipase D, phosphorylation of endogenous 1,2-diacylglycerol, and exposure to exogenous phospholipase D. There is evidence that muscarinic receptors are coupled to the production of phosphatidic acid in myocardium by hydrolysis of choline glycerophospholipids by an endogenous phospholipase D (Lindmar et al., 1988). Human plasma and human neutrophils contain endogenous phospholipase D that is selective for inositol phospholipids (Cockcroft, 1984). In view of the fact that neutrophils invade myocardium during the early hours following coronary artery occlusion, these inflammatory cells could be a source for exogenous phospholipase D during myocardial infarction. At the present time, a physiologic role for phosphatidic acid in cardiac myocytes remains speculative. Future efforts will be directed to characterizing mechanisms for modulation of phosphatidic acid in adult myocytes. There is evidence that ischemia and reperfusion significantly alter phosphoinositide metabolism in myocardial tissue. These changes in phosphoinositide metabolism are temporally related to changes in in vitro activity of PLC in membranes and in cytosol derived from homogenates of myocardial tissue. Translocation, proteolysis, and phosphorylation of myocardial PLC have been proposed as potential mediators of the observed changes in phosphoinositide metabolism in the ischemic and in the reperfused heart (Schwertz and Halverson, 1992). Monoclonal antibodies to isoenzymes of PLC are now commercially available, and these reagents could be used to characterize each of these proposed mechanisms for modulation of myocardial PLC. Ischemia and reperfusion-induced changes in the lipid milieu of substrates of PLC should also be considered as a potential determinant of membrane-associated PLC activity. Much of our current knowledge regarding myocytic metabolism of phosphoinositides is based on radioisotopic experiments performed on neonatal rat myocytes and embryonic chick myocytes. These experimental preparations have provided important insights into phosphoinositide metabolism in cardiac tissue. However, developmental changes in membrane structure (Wibo et al., 1991), developmental regulation of expression of isoenzymes of PLC (Kato et al., 1992), and interspecies differences in phosphoinositide metabolism (Endoh et al., 1991) may limit the applicability of these data to the adult human heart. In view of the extensive compartmentalization of synthesis of phosphoinositides in cardiac tissue, it is desirable to combine direct measurement of inositol phosphate mass with radioisotopic data to characterize physiologic mechanisms for regulation of myocytic PLC. Efforts to overcome these experimental difficulties are justified by the opportunity to further understand mechanisms that regulate electrophysiologic function, mechanical function, and gene expression in the normal and in the diseased heart.
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ACKNOWLEDGMENTS The author gratefully acknowledges the useful comments on this manuscript provided by Sharon Boyce, Mike Leonas, Kathryn Yamada, and Richard Gross. Excellent secretarial assistance was provided by Barbara Donnelly. The work was supported by Grant-in-Aid 900746 from the American Heart Association and by NIH Grant HL 17646, SCOR in Coronary and Vascular Diseases.
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ROLE OF ARACHIDONATE IN MONOCYTE/MACROPHAGE FUNCTION
Michelle R. Lennartz and James B. Lefkowith
ABSTRACT 430 I. INTRODUCTION 430 II. PHOSPHOLIPASE AND ARACHIDONATE RELEASE 431 A. Monocytic PLA2S 432 B. Regulation of PLA2 Activity 433 C. Substrates and Products 436 III. MONOCYTE/MACROPHAGE ARACHIDONATE METABOLISM 439 A. Stimuli for Arachidonate Release 439 B. Macrophage Subsets and Eicosanoid Profiles 440 C. Regulation of Arachidonate Metabolism 441 D. Regulatory Role for Eicosanoids 442 IV. ARACHIDONATE AND MONOCYTE/MACROPHAGE TRAFFICKING . 443 A. Dietary PUFA Manipulation and Inflammation 443 B. EFA Deficiency and Resident Macrophages 444 C. EFA Deficiency and Chronic Inflammation 445
Advances in Lipobiology Volume 1, pages 429-462. Copyright © 1996 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-635-5 429
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D. Arachidonate and Monocyte/Macrophage Migration V INVOLVEMENT OF ARACHIDONATE IN MONOCYTE/MACROPHAGE FUNCTION A. Monocyte/Macrophage Adherence B. Phagocytosis of IgG-Opsonized Targets C. Fusionof Intracellular Vesicles VI. CONCLUDING COMMENTS ACKNOWLEDGMENTS REFERENCES
446 447 448 448 552 554 554 554
ABSTRACT Monocytes/macrophages are a major cellular source of arachidonate. This fatty acid is of considerable biological importance since it serves as the cyclooxygenase and lipoxygenase substrate for production of the eicosanoid inflammatory mediators. It can be released from cells via phospholipase A2(PLA2) hydrolysis of membrane phospholipids. This chapter summarizes the current literature regarding the identification, characterization, and regulation of monocyte/macrophage PLAzs. Additionally, it discusses differential metabolism of the released arachidonate as a function of (a) the stimulus for release, (b) the source and activation state of the macrophages, and (c) the regulation of the cyclooxygenase and lipoxygenase metabolic pathways. Finally, the evidence supporting a role for arachidonate as an autocrine mediator of monocyte/macrophage function is presented. The effects of arachidonate depletion on macrophage trafficking are described in the essential fatty acid deficient mouse and are related to in vitro studies demonstrating an arachidonate requirement for monocyte and macrophage adherence. Additionally, the involvement of arachidonate as an intracellular second messenger is discussed in the context of phagocytosis and membrane fusion.
I. INTRODUCTION Monocytes/macrophages play an important role in a panoply of immune responses. Monocytes constitutively populate tissues and become resident macrophages, cells which may play an important role in immune surveillance and may impart the immunologic identity to tissues. Additionally, monocytes are elicited into tissues during inflammation. These cells, which become elicited macrophages, perform various functions in situ related to inflammation such as phagocytosis, cytokine production, antigen presentation, etc. One important function related to inflammation that monocytes/macrophages subserve is the regulated release and metabolism of arachidonate. This polyunsaturated fatty acid (PUPA) is liberated in response to a variety of stimuli and is metabolized to the prostaglandin (PG) and leukotriene (LT) inflammatory mediators. These arachidonate metabolites (known collectively as the eicosanoids) me-
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diate a variety of the immunologic events occurring during inflammation. Over the past several years significant new insights into the release mechanisms and metabolism of arachidonate have been made. In addition to its role in eicosanoid generation, arachidonate may be an autocrine modulator of monocyte/macrophage function. Recent evidence suggests that arachidonate per se is required for phagocytosis (Lennartz and Brown, 1991; Lennartz et al., 1993), adherence (Lefkowith et al., 1991, 1992) and superoxide generation (Sakata et al., 1987). Additionally, experiments using the strategy of dietary manipulation of fatty acids (in particular, essential fatty acid (EFA) deprivation) have shown that arachidonate may play a key role in monocyte/macrophage traffficking both constitutively and in the contest of inflammation. Collectively, these studies suggest a critical role for arachidonate in intracellular signal transduction in these phagocytes. This review will attempt to summarize the latest developments concerning how monocytes/macrophages control the release and metabolism of arachidonate and the role that this fatty acid plays in monocyte/macrophage function. We will specifically discuss recent advances in the area of phospholipase structure/function, focusing on phospholipases of the A2 class, which appear to be the principal enzymes responsible for arachidonate release. We will also review new developments in understanding how arachidonate metabolism is regulated by monocytes/macrophages. We will further consider the evidence that arachidonate plays an important role in monocyte/macrophage trafficking and finally consider data which suggest that arachidonate may be an autocrine regulator of monocyte/macrophage function.
II. PHOSPHOLIPASE AND ARACHIDONATE RELEASE Arachidonate can be released from membrane phospholipids by at least two pathways, directly via phospholipase A2 (PLA2) or by the sequential action of phospholipase C (PLC) and diglyceride lipase (Dennis, 1990). Although PLC/diglyceride lipase pathways account for release of arachidonate in several non-monocytic cell types (e.g., Mauco et al., 1984; Zahler et al., 1986; Nakashima et al., 1988), and a diacylglycerol lipase activity has been reported in cytosolic fractions from rat alveolar macrophages (Errasfa, 1992), the association of PLA2 activity with numerous stimulus-response systems in monocytic cells supports its role as a major participant in regulated release of arachidonate in this cell type (Chang etal., 1986; Godfrey etal., 1988; Lennartz and Brown, 1991; Errasfa, 1992; Lefkowith et al., 1992). The best characterized PLA2 enzymes are secreted, Ca^"^-dependent enzymes, often of relatively low molecular weight (12-18 kDa) and containing up to seven disulfide bonds. The enzymes are generally classified as type I or type II based on the location of the cysteine residues in their primary structure and the pattern of disulfide cross-linking associated with that structure (for reviews see, Waite, 1990;
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Ward and Pattabiraman, 1990). Although this classification may provide a useful tool for structure/function analysis of the secreted lipases, some of the more recently described enzymes, particularly the higher molecular weight, cell-associated (LesHe et al, 1988; Clark et al, 1990; Diez and Mong, 1990; Kramer et al, 1991; Wijkander and Sundler, 1991), and Ca^"^-independent (Hazen et al., 1990; Lennartz et al., 1993) PLA2S share few of the characteristics of their secreted counterparts (with the obvious exception of the enzymatic activity). By virtue of their cellular association, the higher molecular weight PLA2S probably play a role in such cellular functions as membrane turnover and remodeling, destruction of intracellular bacteria, and release of arachidonate for generation of inflammatory mediators (Pruzanski and Vadas, 1990). In addition, recent evidence that arachidonate activates protein kinase C (PKC) (Naor et al., 1988; Khan et al., 1992) and respiratory burst (Sakata et al., 1987; Steinbeck et al, 1991), opens ion channels (Kim and Clapham, 1989; Ordway et al., 1989), participates in membrane fusion (Meers et al., 1988; Fry et al., 1991) and releases [Ca^"^]j from intracellular stores (Chow and Jondal, 1990) or from the cell (Randriamampita and Trautmann, 1990) suggests that this fatty acid may also function as a second messenger for signal transduction. As such, the characterization of monocyte/macrophage PLA2S (including enzyme structure, mechanisms of activation/deactivation, identification of second messengers, and the targets of those messengers), would be invaluable for our understanding of the mechanisms of signal transduction in general and, more specifically, how information about the environment is translated into the biochemical and physiological changes necessary for the specialized functions of these immune cells. A. Monocytic PLA2S
In the 1970s, Mason and co-workers (1972) reported that arachidonate constitutes 20% of the fatty acids associated with monocyte phospholipids, suggesting that this cell type may be a major cellular source of arachidonate. Release of arachidonate from monocytes and macrophages by phagocytic targets (yeast, IgG, and complement-opsonized particles), phorbol esters, and calcium ionophores demonstrated that the esterified arachidonate could be mobilized for the production of inflammatory mediators. Indeed, extensive studies have been done characterizing the differences in arachidonate metabolism upon stimulation with different compounds (Pawlowski et al., 1983; Aderem et al., 1986). Identification of arachidonate-releasing PLA2S from homogenates of rat alveolar (Franson and Waite, 1973) and mouse peritoneal macrophages (Wightman et al., 1981) provided the mechanism for arachidonate release. Since these early studies, much progress has been made in the purification and characterization of monocyte/macrophage PLA2S (Table 1). Current efforts are aimed at elucidating the signaling mechanisms for receptor-mediated release of arachidonate and the subsequent action of arachidonate on cellular processes. Several of these systems will be discussed in detail below.
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Table 1. Purified Macrophage Phospholipases A2S Substrate specificity Ca sn-2 Fraction Size (kDa) (EC^Q) Headgroup Fatty acid
Cloned
Reference
P388Di membrane 18 2 mM PC, PE 16:0,20:4 No Ulevitchetal., 1988; (mouse) Glaseretal., 1990 RAW cytosol 60-70 1 mM PE>PC 20:4»18:1, No Leslie etal, 1988 264.7 - 18:2 (mouse) U937 cytosol 56 300 nM PE>PC>PI 2 0 : 4 » 16:0 No Diez and Mong, 1990 U937* cytosol 100-110 400 nM ND 20:4>18:2> Yes Clark etal., 1990; 18:1»16:0 Krameretal, 1991 J774* cytosol 100 700 nM PC, PE, PI 20:4>18:1, Yes Wijkander and Sundler, (mouse) 18:2 1991 Notes:
*Sequence analysis and cloning has determined that these two enzymes are identical. They have been designated cytosolic PLAj (cPLAj) (Clark et al., 1991; Wijkander and Sundler, 1991). ND, Not determined.
B. Regulation of PLA2 Activity 7. Mechanisms of Activation
As can be seen from Table 1, the cellular PLA2S are generally of higher molecular weight than the secreted forms, are of cytosolic origin, and require Ca^"^ for maximal activity. Their soluble nature presents an apparent paradox when one considers that the substrate is a membrane phospholipid. While it is clear that monocyte activation by both particulate (Pawlowski et al., 1983; Aderem and Cohn, 1988; Lennartz et al., 1993) and soluble (Pawlowski et al., 1983; Aderem and Cohn, 1988) stimuli generate free arachidonate, the mechanism of activation of unclear. Does "activation" involve an increase in enzyme activity toward a given substrate, i.e., enhanced catalytic activity, or is the stimulated release of product a measure of the extent to which the enzyme is translocated to the membrane, in which case the intrinsic enzyme activity is unchanged? The answer may be a combination of the two. 2.
PLA2 Translocation
Cloning of the U937 enzyme (CPLA2) has revealed a number of interesting features (Clark et al., 1991: Sharp et al., 1991). First, the deduced amino acid structure has no homology with the lower molecular weight, secreted PLA2S and thus codes for a novel enzyme. Second, the amino terminus of the clone exhibits extensive homology with PKC, a protein which translocates to membranes in response to increases in intracellular free calcium ([Ca^^Jj). Consistent with this finding, translocation of the CPLA2 N-terminal peptide to cellular membranes in
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MICHELLE R. LENNARTZ and JAMES B. LEFKOWITH
the presence of physiological concentrations of Ca^"^ has been demonstrated (Clark et al., 1991). Similar results showing Ca^'^-dependent translocation of PLA2 activity from soluble to particulate cell fractions have been obtained using cellular homogenates from RAW 264.7 macrophages (Channon and Leslie, 1990) and Kupffer cells (Krause et al., 1991). Finally, phospholipids containing arachidonate are preferentially hydrolyzed over those containing other unsaturated sn-2 fatty acids, providing further evidence for the role of CPLA2 in generation of inflammatory mediators in intact cells. The fact that translocation parallels the increase in arachidonate release suggests that activation of CPLA2 resides, at least partially, in the increased association of the enzyme with its membrane substrate. It further suggests that if the calcium requirement is at the level of enzyme-substrate association, the enzymes themselves may not require calcium for catalytic activity (Channon and Leslie, 1990). This is in direct contrast to the secreted PLA2, in which calcium participates as a cofactor in phospholipid hydrolysis (Scott et al., 1990). Recent studies describing Ca^^-independent PLA2 in canine myocardium (Hazen et al, 1990) and human monocytes (Lennartz et al., 1993) support the hypothesis that catalysis does not necessarily require calcium. The canine enzyme has been purified and found to be of lower molecular weight (40 kDa) than its Ca^'^-dependent counterparts. Thus, it is tempting to speculate that it may share catalytic homology with the Ca^^-dependent enzymes but lacks the PKC homology region that confers Ca^"^-sensitivity. A comparison of the sequences of the Ca^"*"-dependent and -independent PLA2 will answer this question and may provide additional insight into alternative mechanisms for PLA2-membrane association and clues as to the pathway(s) for Ca^^independent phospholipid hydrolysis. 3.
Catalytic
Activation
Catalytic activation of enzymes often involves phosphorylation. Rhee and coworkers pioneered the study of receptor-activated phospholipases with their work involving epidermal growth factor (EGF)-stimulated activation of PLC. They demonstrated that ligation of EGF receptors causes rapid phosphorylation of PLC-y that correlated with an increase in phosphatidylinositol turnover and membrane association (Todderud et al., 1990). Further investigations measuring cytosol and membrane-associated PLC activity from control and growth factor treated fibroblasts revealed an increase in PLC-y specific activity in both fractions upon incubation with EGF (Wahl et al., 1992); additional studies revealed that tyrosine phosphorylation of PLC-y enhanced its catalytic activity (Nishibe et al., 1990). Although phosphorylation-dependent activation of PLC has been well documented for the EGF system, similar evidence concerning PLA2 activation is relatively recent. Several investigators have reported PLA2 phosphorylation in cellular homogenates and a phosphorylation-dependent increase in substrate turnover, but the effect of PLA2 phosphorylation on membrane association was not assessed (Lin et al..
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1992; Xing and Mattera, 1992). Additionally, recent reports from two laboratories demonstrate that human CPLA2 can be phosphorylated in vitro by PKC, microtubule-associated protein kinase, or mitogen-activated protein kinase, and that phosphorylation enhances enzyme activity (Lin et al., 1993; Nemenoff et al, 1993). These results demonstrate that, like PLC, PLA2 activity can be modulated by phosphorylation. It should be noted, however, that the pattern of phosphorylation differs between the two phospholipase classes; PLC activation requires tyrosine phosphorylation (Nishibe et al., 1990), and PLA2 activity is modulated by phosphorylation of serine 505 (Lin et al., 1993). The effect of this modification on PLA2 membrane association has yet to be determined. Regulation of proteins such as PKC and adenyl cyclase occurs at the level of multi subunit complexes containing regulatory and catalytic subunits. In these systems, stimulus-dependent dissociation of the regulatory subunit causes enzyme activation. There is evidence that similar mechanisms may regulate some of the cytosolic PLA2S. For example, the soluble sheep platelet PLA2 is apparently a Ca^'^-dependent enzyme, a requirement that can be circumvented by the addition of high concentrations of salt to the purified enzyme (Zupan et al., 1991), thus generating a Ca^'^-independent lipase. In contrast, similar treatment of crude cytosol does not elicit Ca^'^-independent substrate hydrolysis, suggesting that the cytosolic enzyme is complexed with other protein(s) that effectively mask its Ca^"^-independent activity. These "regulatory" elements are lost during purification, revealing the Ca^"^-independent nature of the enzyme. Thus, the sheep platelet PLA2 activity may be modulated in a manner similar to PKC and adenyl cyclase, in which the regulatory subunit inhibits substrate catalysis. In contrast, activation of Ca^'^-independent PLA2S may occur by a novel variation on this subunit theme. Hazen et al. (1990) reported that the Ca^''"-independent PLA2 from canine myocardium binds ATP. Characterization of the ATP-lipase interaction revealed the existence of a 400 kDa enzyme species that hydrolyzes plasmenylphosphatidylcholine and interacts with ATP. ATP acts to stabilize the enzyme against denaturation and enhances the initial rate of catalysis as determined by kinetic studies (Hazen and Gross, 1991 a,b). However, the purified PLA2 exhibits a molecular weight of 40 kDa and is insensitive to ATP, suggesting the existence of a regulatory element responsible for the ATP effects. This regulatory component was recovered in a high molecular weight fraction upon gel filtration; its activity was confirmed by addback studies in which this fraction was able to restore ATP-responsive activity to the purified enzyme (Hazen and Gross, 1991a,b). The results of these studies demonstrate the existence of PLA2 regulatory proteins which modulate Ca^"^-independent enzyme activity. However, unlike the PKC and adenyl cyclase complexes in which the regulatory subunit represses the catal3^ic component, the enzymatic activity of the Ca^'^-independent PLA2S is apparently protected by association with the high molecular weight protein(s).
436 4.
MICHELLE R. LENNARTZ and JAMES B. LEFKOWITH Regulation by G-Proteins
Although G-protein mediated activation of PLA2 has been described in several cell types, the mechanism(s) responsible for the enhanced catalysis have yet to be elucidated. Treatment of platelet membranes, prelabeled with [^H]-arachidonate, with G-protein activators (AIF^ or GTPyS) or inhibitors (GDPpS) caused respective increases or decreases in arachidonate liberation (Silk et al., 1989). As the membranes used for these experiments were washed free of cytosol, regulation of arachidonate release appears to be a function of the phospholipase/G protein interaction rather than a translocation-mediated event. Although stimulated arachidonate release was not significantly affected by the PLC inhibitor neomycin in these studies, further characterization is necessary to establish that the enzyme activity of interest is indeed a PLA2. The putative interaction of PLA2 with G-proteins can be disrupted by treatment of platelet membranes with anti-G-protein antibodies or by incubation with pertussis toxin, a compound known to inhibit some classes of G-proteins (Murayama et al, 1990). Similar studies, utilizing neutrophils and neutrophil-like differentiated HL-60 cells stimulated with the chemotactic factor f-met-leu-phe, provide evidence for G-protein regulation of PLA2S in phagocytes (Cockroft and Stutchfield, 1989) and further suggest that the G-protein of interest bears a Gj a subunit (Bokoch and Gilman, 1984). Unfortunately, little work has been done evaluating the role G-proteins play in monocyte PLA2 activation. C. Substrates and Products
2-Arachidonyl phosphatidylcholine is commonly used as a substrate for in vitro PLA2 assays and is often presented to the enzyme as a micelle or vesicle. Enzymes assayed by this method exhibit little or no specificity towards the phospholipid headgroup (Table 1). However, the physical characteristics of the substrate micelles or vesicles present in the in vitro assay varies with the composition of the phospholipids and may or may not approximate cellular membranes, making it difficult to generalize the results obtained in vitro to intact cells. In cases where a known stimulus causes release of arachidonate from intact cells, the substrate of the activated enzyme can be identified using cells endogenously labeled with ^H-arachidonate. For example, stimulation of human neutrophils with a variety of agents results in release of ^H-arachidonate from phosphatidylcholine (PC) and phosphatidylinositol (PI) (Cockroft and Stutchfield, 1989). Similarly, thrombin stimulation of platelets decreases PC and Pl-associated ^H-arachidonate (Bills et al., 1976). The use of radiolabeled cells as PLA2 substrates has the advantage of maintaining membrane composition and integrity, thus providing the PLA2 with a physiological substrate. However, the results obtained from such studies may be influenced by the conditions used to radiolabel the cells as the incorporation of ^H arachidonate into the different phospholipid classes is a function of labeling time and arachidonate concentration (Sugiura et al., 1984; Chilton, 1991). This issue has been discussed in detail in a recent review in which
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437
the phospholipid sources of the arachidonate, released from human neutrophils upon stimulation by ionophore, were identified. -^H-arachidonate was liberated predominantly from PC in neutrophils radiolabeled with ^H-arachidonate; in unlabeled cells, the same stimulus caused a depletion in arachidonate from phosphatidyl ethanolamine (PE) as measured by gas chromatography/mass spectrometry (GC/MS) (Chilton, 1991). We have used a similar method to demonstrate arachidonate depletion from PE during antibody-mediated phagocytosis (Lennartz et al., 1993) and to characterize the fatty acid alterations associated with EFA deprivation in mice (Lefkowith et al, 1985). Taken together, these studies indicate that in vitro PLA2S exhibit little head group specificity. In intact cells, PC is the apparent substrate for PLA2 action when assayed in radiolabeled cells, whereas measurements in unlabeled cells demonstrate selective arachidonate depletion from PE, suggesting that PE is the physiological substrate for PLA2 action in intact cells. For cellular PLA2S this would make teleologic sense since PE is concentrated in the inner leaflet and PC on the outer leaflet of the plasma membrane. In contrast to the relative nonselectivity of PLA2S for the phospholipid headgroup, these enzymes preferentially hydrolyze arachidonate over other sn-l fatty acids (Table I) in in vitro assays, a finding that underscores its importance as a mediator of cellular response. Data from our laboratory suggests that stimulation of human monocytes results in very specific release of arachidonate. These studies were performed on unlabeled peripheral blood monocytes exposed to phorbol dibutyrate (PDBu), a tumor promoting phorbol ester, the calcium ionophore A23187, or IgG-opsonized glass beads. After stimulation, the lipids were extracted from the reaction mixture by the method of Thigh and Dyer and the free fatty acids were derivatized and analyzed by GC. This procedure allowed us to identify all the fatty acids released during monocyte stimulation with various agonists. The results indicate that arachidonate is virtually the only fatty acid released by monocytes upon activation with either specific (IgG-opsonized beads) or non-specific (PDBu) stimuli (Figure 1 and Lennartz et al., 1993). The release of arachidonate in response to phagocytosis of IgG-opsonized targets is consistent with activation of a receptor-associated PL. Receptors for EGF (Teitelbaum, 1990), IgE (Narasimhan et al., 1990), and histamine (Murayama et al, 1990) have been shown to be coupled to arachidonate-generating PLA2 via G-proteins. In phagocytes, Suzuki et al. (1982) have shown a specific enhancement of PLA2 activity upon binding of IgG2b immunoglobulins to FcyR in a mouse macrophage-like cell line. However, we were surprised to find that PDBu (and the calcium ionophore A23187) also liberated arachidonate to the exclusion of other free fatty acids. As these treatments have the more global effects of activating PKC and Ca^"*'-dependent enzymes, respectively, we expected that they would activate several phospholipases and generate a pattern of free fatty acid release reflecting the composition of unsaturated fatty acids in the membrane phospholipids. The selective arachidonate release suggests that either the cellular phospholipases are
438
MICHELLE R. LENNARTZ and JAMES B. LEFKOWITH
IT BBSA BIgG m PDBu ■ BIgG + BPB \1 □ \m_BIgG + BEL No change
16:0
16:1
18:0
18:1
18:2
20:4
Fatty Acid Figure 1, Release of fatty acids from monocytes. Monocytes (4 x 10^/ml) were incubated with PDBu (100 ng/ml) or glass beads opsonized with bovine serum albumin (BBSA) or IgG (BIgG) for 60 minutes (37^ C). To assess the ability of p-bromophenacyl bromide (BPB) and bromoenol lactone (BEL) to inhibit AA release, monocytes were incubated with 10 ^iM BPB (30 minutes, 0^ C) or 100 |LIM BEL (60 minutes, 23- C) prior to addition of BIgG. Control incubations contained no additions. The free fatty acid pool was resolved by 2-D TLC and analyzed by GC (Lefkowith et al., 1987). Results are presented as Stimulated/Control (ratio of 1 = no change). Standard lipid nomenclature is used to identify the free fatty acids, the first number referring to the number of carbons in the fatty acid, the second designating the number of carbon-carbon double bonds. Only AA (20:4) was increased in the free fatty acid pool in response to either BIgG or PDBu. No significant changes were found in any other fatty acids. BBSA had no effect on the quantity or character of the free fatty pool. Data are reported as average ± SEM; BBSA (n = 7), BIgG (n = 9), PDBu (n = 2), BPB or BEL + BIgG (n = 3). The average concentration of free AA in resting cells was 118 ± 21 pmol/10^ cells (n = 7). Taken from Lennartz et al. (1993).
arachidonate specific or that this fatty acid is uniquely accessible to cellular phospholipase. The fact that many purified phospholipases are active—^albeit to a lesser extent—towards substrates bearing fatty acids other than arachidonate (Waite, 1987; Glaser et al., 1990), suggests that the arachidonate specificity may lie in the characteristics of the membrane rather than the phospholipase. Having reviewed the literature with respect to monocyte/macrophage PLA2S —their characterization, structure, potential mechanisms of regulation, and substrates—^we will turn our attention to their arachidonate product, its metabolism and regulation, its involvement in cellular trafficking, and finally to its potential role as a second messenger in monocj^e/macrophage function.
Arachidonate
in Macrophage Function
439
III. MONOCYTE/MACROPHAGE ARACHIDONATE METABOLISM A. Stimuli for Arachidonate Release Monocytes/macrophages contain substantial amounts of arachidonate within their phospholipids, which they release and metabolize in response to a variety of stimuli. In general, these cells have the capacity to produce both cyclooxygenase metabolites and 5-lipoxygenase metabolites of arachidonate (Lefkowith and Schreiner, 1987) in addition to a variety of hydroxyeicosatetraenoic acids (HETEs), notably 5-, 12-, and 15-HETEs (Baiter et al., 1989). The capacity of these cells to produce specific metabolites of arachidonate is a highly regulated process and depends on the activating stimulus as well as the cell state. Stimuli which are known to cause release of arachidonate can be classified broadly into two categories: soluble (including lipopolysaccharide, LPS phorbol esters) or particulate (zymosan, immune complexes). Soluble stimuli induce the formation of cyclooxygenase products by macrophages, whereas particulate stimuli induce the formation of both cyclooxygenase and 5-lipoxygenase products (Humes et al., 1982). The initial conception was that there were two pools of arachidonate within cells that have different accessibility to the metabolizing enzymes. This would explain these differences in metabolic profile between different stimulating agents (Humes et al., 1982). It was subsequently appreciated that this disparity was more likely due to differences in alterations in intracellular Ca^"^ between these two classes of stimuli. By providing a subthreshold amount of Ca^"^ ionophore along with a soluble stimulus (e.g., phorbol esters or LPS), macrophages were ''primed" for leukotriene production and synthesized both cyclooxygenase and 5-lipoxygenase products rather than just cyclooxygenase products (Tripp et al., 1985b; Aderem and Cohn, 1988). Thus, the explanation behind the differences in eicosanoid profiles between classes of agonists most likely relates to the relative thresholds of activation for Ca^"^ for the phospholipase and 5-lipoxygenase (Aderem and Cohn, 1988). Soluble agonists appear to be able to induce the release of arachidonate without liberating sufficient Ca^"^ to activate the 5-lipoxygenase. It still may be the case that there is compartmentalization of arachidonate release and metabolism within the cells and tight coupling between these processes. Evidence for such an hypothesis is derived from investigations which have shown substantial discrepancies between the metabolism of exogenously vs. endogenously supplied arachidonate by macrophages (Baiter et al, 1989). Moreover, arachidonate release by monocytes/macrophages can be two orders of magnitude greater than eicosanoid production (Lefkowith et al., 1992). How arachidonate release is coupled to metabolism remains to be elucidated. In addition to the above noted stimuli, adherence is also a stimulus for the release and metabolism of arachidonate acid in monocytes/macrophages (Lefkowith et al., 1992). Interestingly, this stimulus leads to the production of only cyclooxygenase
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MICHELLE R. LENNARTZ and JAMES B. LEFKOWITH
metabolites and is thus similar to soluble stimuli (Lefkowith et al, 1992). The lack of lipoxygenase products during adherence most likely relates to a lack of sufficient Ca^"^ influx with simple adherence to activate the 5-lipoxygenase. B. Macrophage Subsets and Eicosanoid Profiles Macrophages appear to differ in the profiles of eicosanoids that they are capable of producing depending on their source and state of activation. Resident macrophages seem to be among the most prodigious producers of eicosanoids, both in terms of total amounts and product profiles. Resident murine peritoneal macrophages, for example, produce PGI, PGE, and thromboxane (Tx) via the cyclooxygenase pathway in response to zymosan with the former being the predominant product (Lefkowith et al, 1987). These cells produce substantial amounts of LTC but only modest amounts of LTB (Lefkowith et al, 1987). Interestingly murine pulmonary macrophages (which are a type of resident macrophage) appear to be more capable of producing products of the 5-lipoxygenase pathway relative to resident peritoneal macrophages, and exhibit a lesser capacity to produce cyclooxygenase products (Rouzer et al, 1982). Moreover, pulmonary macrophages produce substantial amounts of LTB (Baiter et al, 1989). The genesis of this heterogeneity of arachidonate metabolic capacity in the resident macrophages of differing tissues remains to be clarified. In contrast to resident cells, macrophages elicited into the peritoneum by an inflammatory stimulus (i.e., elicited macrophages), exhibit a much more limited capacity to release and metabolize arachidonate (Humes et al., 1980; Tripp et al., 1985a, 1986a). Their production of PGI, PGE, and LTC is much less than that of resident macrophages, although their production of Tx is relatively preserved. By examining the conversion of the various substrates of the arachidonic acid cascade, it is evident that these cells contain less cyclooxygenase, 5-lipoxygenase, prostacyclin synthase, PGH-PGE isomerase than resident macrophages (Tripp et al., 1985b). Interesting, Tx synthase is highly conserved and highlights the important role that Tx may play in inflammation (see below). These differences between elicited and resident macrophages appear attributable to the ontogeny of the cells. Blood monocytes are very similar to elicited macrophages in terms of their arachidonate metabolism (both quantitatively and qualitative) (Tripp et al., 1986a). Moreover, the shift in arachidonate metabolic capability of peritoneal macrophages during intraperitoneal inflammation coincides with the influx of blood monocytes and can be attenuated by monocyte depletion by irradiation (Tripp et al, 1986a). It has thus been suggested that elicited macrophages are immediately derived fi-om blood monocytes and consequently manifest a similar arachidonate metabolic phenotype. The origin of the resident macrophage and the genesis of its marked capacity to metabolize arachidonate, however, is unclear.
Arachidonate in Macrophage Function C.
441
Regulation of Arachidonate Metabolism
Over the past two years, it has become apparent that there are at least two isoforms of cyclooxygenase. One, often termed cyclooxygenase-1, is a constitutive enzyme expressed by multiple tissues. This was the original enzyme purified from sheep seminal vesicle and eventually cloned (Merlie et al, 1988). It became apparent, however, that changes in cyclooxygenase activity did not always correlate with changes in the mRNA for this enzyme (Linn et al., 1989). It was subsequently appreciated that there was another cyclooxygenase mRNA expressed of slightly largely size (Hla and Neilson, 1992). This latter species is often referred to as cyclooxygenase-2. Cyclooxygenase-2 is approximately 60% identical to cyclooxygenase-1, belongs to the family of immediate-early response genes (Kujubu et al., 1991), and is preferentially induced by inflammatory stimuli, although cyclooxygenase-1 can be regulated to a certain extent (Hla and Neilson, 1992). The regulation of cyclooxygenase appears to be a dynamic process in monocytes/macrophages. Turnover of the enzyme in culture is on the order of hours (Hoffman et al., 1992). Cyclooxygenase activity is also induced in these cells in response to inflammatory stimuli both in vitro and in vivo. Monocytes exhibit an upregulated cyclooxygenase in vitro in response to phorbol esters and LPS (Fu et al., 1990; Hla and Neilson, 1992). Resident peritoneal macrophages upregulate their cyclooxygenase in vivo in response to systemic endotoxin (Masferrer et al., 1990). This upregulation of cyclooxygenase is largely due to the marked induction of cyclooxygenase-2 (Hla and Neilson, 1992). Cytokines may also regulate cyclooxygenase expression. CSF-1 (which may play a role in monocyte differentiation into macrophages) has been shown to upregulate cyclooxygenase activity in vitro (Orlandi et al., 1989). Adherence per se may also have the capacity to regulate cyclooxygenase expression. Adherence itself can upregulate the expression of various monocyte mediators and proto-oncogenes, and possibly the inducible form of cyclooxygenase (Juliano and Haskill, 1993). Macrophage cyclooxygenase may be tonically regulated in vivo by glucocorticoids (Masferrer et al., 1990). Macrophages from adrenalectomized mice exhibit a marked increase in cyclooxygenase relative to normal macrophages presumably due to an induction of cyclooxygenase-2. This increase in cyclooxygenase is reversed by supplementation of adrenalectomized animals with glucocorticoids. Interestingly, no change in renal cyclooxygenase occurs in adrenalectomized animals suggesting that most of the renal cyclooxygenase is of the constitutive type (i.e., cyclooxygenase-1). In addition, dexamethasone can regulate the induction of cyclooxygenase-2 in macrophages in response to inflammatory stimuli (Masferrer et al., 1990). LPS in vivo induces an increase in peritoneal macrophage cyclooxygenase expression. Dexamethasone appears to inhibit this upregulation probably via an inhibition of the expression of cyclooxygenase-2. This action of corticosteroids may be part of their antiinflammatory potential.
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MICHELLE R. LENNARTZ and JAMES B. LEFKOWITH
Whether other stimuH can down-regulate cyclooxygenase expression in monocytes/macrophages is uncertain. A decrease in cyclooxygenase expression (cyclooxygenase-1) in endothelial cells with acidic FGF has been observed (Hla and Maciag, 1991). The relevance of these data for monocytes/macrophages is uncertain. Additionally, interleukin-4 can down-regulate prostaglandin production in monocytes, although the enzymatic site of regulation is not clear (i.e., whether the decrease is due to a decrease in phospholipase activity, cyclooxygenase activity, or both; Hart et al, 1989). It has also been noted that cultured resident macrophages down-regulate their cyclooxygenase over time, but the mechanism underlying this effect, and its physiologic relevance, is unclear (Tripp et al., 1986a). The regulation of the 5-lipoxygenase pathway in monocytes/macrophages is less well delineated. This enzymatic step involves two proteins: the 5-lipoxygenase and the 5-lipoxygenase activating protein (termed FLAP) (Dixon et al., 1990). The 5-lipoxygenase is translocated to the membrane and is activated via its association with FLAP by an increase in intracellular Ca^"^ (Dixon et al., 1990). It appears that the regulation of this pathway is less dynamic than that of the cyclooxygenase pathway. In experiments were cyclooxygenase is upregulated by inflammatory stimuli, no change in 5-lipoxygenase activity has been seen (Masferrer et al., 1990). Moreover, turnover of these proteins in monocytes in vitro is much less rapid than that of cyclooxygenase (Hoffman et al., 1992). It is clear, however, that there are vast differences in the capacity of blood monocytes and tissue macrophages to produce products of the 5-lipoxygenase pathway. Blood monocytes express very little 5-lipoxygenase protein and make little, if any, leukotrienes (Pueringer et al., 1992). Nonetheless, tissue macrophages, such as alveolar macrophages which are presumably derived from blood monocytes, contain markedly more 5-lipoxygenase and make substantial amounts of leukotrienes (Pueringer et al., 1992). What accounts for this difference and its mechanism are undetermined; however, in vitro systems of monocyte/macrophage development using HL-60 cells suggest that both the 5-lipoxygenase and FLAP can be up-regulated during differentiation (Bennett etal., 1993). D.
Regulatory Role for Eicosanolds
Eicosanoids serve as autocrine/paracrine mediators of inflammatory phenomena. With respect to their potential autocrine role in monocytes/macrophages, most studies have focused on PGE as a negative feedback modulator of macrophage function. PGE has been shown to down-regulate a variety of macrophage functions including la expression, interleukin-1 production, and cytolytic capacity (Taffet and Russell, 1981; Snyder etal., 1982; Kunkel and Chensue, 1985; Tripp etal., 1986b). The mechanism underlying this effect is most likely mediated via an increase in cAMP (Snyder et al., 1982). Whether these observations represent in vitro phenomena or occur in vivo is less clear.
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PGE may not be the only prostaglandin with an autoregulatory role as both PGE and PGI have been found to act as negative modulators of la expression in vitro (Tripp et al., 1986b), with PGI being substantially more potent than PGE. Moreover, only PGI appears to be produced in sufficient quantities by macrophages to produce this effect (Tripp et al, 1986b). Thus PGI may be the more relevant autoregulator of la expression. Of note, although macrophages also produce substantial amounts of Tx and LTC, neither of these eicosanoids apparently exert an autoregulatory role on la expression at relevant physiologic concentrations (Tripp et al., 1986b). Eicosanoids clearly mediate a panoply of inflammatory phenomena in a paracrine fashion, a subject which goes beyond the scope of this review. It is worthwhile, however, to consider the role of macrophage-derived Tx in inflammation. As noted above, the ability to produce Tx is conserved in inflammatory macrophages despite the down-regulated production of the other cyclooxygenase metabolites. This conservation of Tx synthetic capacity suggests an important role for this eicosanoid in inflammation. In support of this contention, inhibition of Tx production during infection with Listeria monocytogenes was subsequently found to potentiate mortality from the infection, an effect which could be reversed by administration of a stable Tx analogue or other vasoconstrictor (Tripp et al., 1987). These data suggest an important regulatory role for the vasoconstrictor/platelet aggregatory properties of this eicosanoid in inflammation. Conjecturally, Tx generated by macrophages during the inflammatory response elicited by Listeria isolates the focus of infection, prevents dissemination of bacteria, and aids the cellular immune response.
IV. ARACHIDONATE AND MONOCYTE/MACROPHAGE TRAFFICKING A.
Dietary PUFA Manipulation and Inflammation
One way in which the role of arachidonate in inflammation in vivo has been assessed has been to use dietary PUFA manipulation. There are two basic manipulations which have been examined in detail: EFA deficiency in which animals are deprived of (co-6) PUFAs, or dietary (co-3) PUFA enrichment. With the former, tissue (co-6) PUFA are depleted (i.e., linoleate and arachidonate) and (co-9) fatty acids accumulate, notably 18:1 (co-9) (i.e., oleate) and 20:3(co-9) (often termed Mead acid) (Lefkowith et al., 1985; Lefkowith, 1988). With dietary (co-3) fatty acid supplementation, tissue arachidonate is replaced with 20:5(co-3) (Lefkowith et al., 1990a). Studies to date show that dietary PUFA modulation can ameliorate murine lupus glomerulonephritis (Hurd et al., 1981; Prickett et al., 1981; Kelley et al, 1985; Robinson et al., 1985), nephrotoxic nephritis (Schreiner et al, 1989), hydronephrosis (Spaethe et al, 1988), interstitial nephritis (Harris et al., 1990), glomerulosclerosis (Diamond et al, 1989), myocardial infarction (Freed et al., 1989), several variants of immune-mediated diabetes (Wright et al., 1988; Lefkowith et al., 1990b), collagen-induced arthritis (Leslie et al., 1985), a model of acute respiratory
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distress syndrome (Morganroth et al., 1989), and experimental acute and chronic inflammation (Bonta et al., 1974, 1977; Lefkowith et al., 1988; Lefkowith et al., 1990a). The mechanisms underlying the protective effect of dietary PUFA manipulation have been the subject of substantial scrutiny. Although no single mechanism may explain the immunologic effects of dietary PUFA modulation, it appears that one common element underlying the antiinflammatory effects of dietary PUFA manipulation, EFA deficiency in particular, is a unique ability to alter monocyte/macrophage trafficking both constitutively as well as in the context of inflammation. B. EFA Deficiency and Resident Macrophages
EFA deficiency has been shown to have the unique capacity to deplete populations of resident macrophages (Lefkowith and Schriener, 1987; Lefkowith, 1988; Lefkowith et al., 1988). In the rat kidney both the population of resident glomerular macrophages, as well as interstitial macrophages, are diminished by EFA deficiency (Lefkowith and Schreiner, 1987; Lefkowith et al., 1988), in mice, EFA deficiency depletes only the resident peritoneal macrophage population (Lefkowith, 1988). Fatty acid supplementation of EFA-deficient animals, moreover, shows that this decrease in the resident macrophage population may be a specific function of (co-6) fatty acids (Lefkowith and Schreiner, 1987). Administration of linoleic acid (18:2(co-6)), but not linolenic acid (18:3(co-3)), to EFA-deficient rats reverses the decrease in resident glomerular macrophages, presumably due to its conversion into arachidonate (20:4(co-6)). These data suggest that arachidonate may be the important variable in the population of the kidney with resident macrophages, and that 20:5(co-3), which is produced from 18:3(co-3), cannot substitute for arachidonate in this regard. However, dietary enrichment of (co-3) PUFA does not appear to lead to a decrease in resident peritoneal macrophages in mice, as does EFA deprivation, suggesting the opposite, that 20:5(co-3) may be an adequate substitute for arachidonate (Lefkowith et al., 1990a). Whether this difference in mouse peritoneal and rat glomerular macrophages represents a species or tissue difference is unclear. This depletion of resident macrophages with EFA deprivation is also accompanied by a decrease in tissue eicosanoid production (Lefkowith and Schreiner, 1987; Lefkowith, 1988; Lefkowith et al., 1988). Although the simple assumption is that this decrease is due to a depletion of arachidonate, arachidonate levels in tissue even with EFA deficiency are on the order of nmol/mg protein whereas eicosanoid production is on the order of pmol/mg protein (Lefkowith and Schreiner, 1987). Moreover, this decrease in eicosanoid production is seen only with certain agonists (Lefkowith and Schreiner, 1987). The decrease in eicosanoid production seen with EFA deficiency, however, is reversed by (co-6) fatty acid supplementation of EFA-deficient animals concomitantly with the repopulation of tissue with macrophages. Additionally, a decrease in tissue eicosanoid production, specifically LTB4, can be induced by macrophage depletion (Lefkowith et al., 1988). These data
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suggest that EFA deficiency may secondarily exert effects on eicosanoid production via its effects on macrophage migration and the population of tissues with resident macrophages. C. EFA Deficiency and Chronic Inflammation
In addition to the effects of the deficiency state on resident macrophage populations, it has been shown that EFA deficiency can profoundly affect the influx of monocytes/macrophages into a focus of inflammation. EFA deficiency inhibits the influx of macrophages into tissues in glomerulonephritis (Schreiner et al., 1989), interstitial nephritis (Harris et al., 1990), autoimmune diabetes (Wright et al., 1988), and glomerulosclerosis (Diamond et al., 1992). This inhibited influx of macrophages can secondarily exert profound effects on the extent of the metabolic and functional alterations which accompany the inflammatory response. The inhibition of macrophage invasion in the context of renal inflammation markedly attenuates the tissue eicosanoid production which, in turn, may alter the regulation of such local events as blood flow and filtration function (Schreiner et al., 1989). Moreover, the inhibited invasion of macrophages can attenuate local production of macrophage-derived cytokines (which are derived from these cells) and the accompanying proliferation of mesenchymal cells which may lead to local scarring and irreversible loss of tissue function (Diamond and Pesek, 1991; Diamond et al.,^ 1992). Data from these in vivo studies in models of chronic inflammation suggest that the effects of dietary PUFA manipulation may be more a function of a lack of arachidonate as opposed to the presence of an alternative eicosanoid precursor, such as 20:3(co-9) or 20:5(co-3). In autoimmune diabetes, a model of inflammation in which macrophages appear to play a pivotal role, it was possible to show that the antiinflammatory effect of EFA deficiency directly correlated with the depletion of arachidonate as opposed to the presence of 20:3(co-9) (Lefkowith et al., 1990b). These results suggest that the lack of arachidonate (or a metabolite) is the critical variable in mediating the effects of dietary PUFA manipulation in monocyte/macrophage-mediated inflammation as opposed to the presence of an alternative eicosanoid precursor (or metabolite therefrom). This result is distinctly different from the situation in acute inflammation in which the antiinflammatory effect of dietary PUFA manipulation appears to be a function of the accumulation of the alternative eicosanoid precursor. In acute inflammation, LTB plays a substantial role as a chemoattractant for neutrophils (Leflcowith et al., 1988). With dietary PUFA manipulation, 20:3(co-9) or 20:5(co-3) is substituted for arachidonate. These two alternative substrates for the 5-lipoxygenase form LTAs (i.e., LTA3 or UrA5) which are poor substrates for the LTA hydrolase; their presence prevents the metabolism of arachidonate to LTB4 (Evans et al, 1985; Nathaniel et al, 1985). The resultant decrease in UTB levels leads to the diminished influx of neutrophils in acute inflammation, a condition that can be
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MICHELLE R. LENNARTZ and JAMES B. LEFKOWITH
reproduced with 5-lipoxygenase blockade (Lefkowith, 1988; Lefkowith et al., 1990a). The antinflammatory effects of dietary PUFA manipulation in acute inflammatory are directly proportional to the inhibitory efficacy of the respective LTAs formed. That is, EFA deficiency is more effective in decreasing acute inflammation than ((0-3) fatty acid supplementation because LTA3 is a better inhibitor of LTA hydrolase than LTA5 (Lefkowith, 1988; Lefkowith et al., 1990a). D. Arachidonate and Monocyte/Macrophage Migration The mechanism underlying the inhibition of macrophage trafficking with EFA deprivation is not entirely clear. Two basic (and nonmutually exclusive) possibilities have been pursued. One is that there is a lipid chemoattractant, the production of which is inhibited by dietary PUFA manipulation. A second possibility is that monocyte/macrophage function may be altered by changes in membrane fatty acids. Initial presumptions regarding the effects of dietary PUFA manipulation and monocyte/macrophage trafficking were that a decrease in LTB4 or PAF production, or both, was the underlying mechanism. These lipid mediators can be chemoattractants for monocytes/macrophages and induce adherence of these cells to the endothelium although they tend to be less potent in this regard than for neutrophils (Smith et al., 1980; Tonnesen et al, 1989; Rovin et al., 1990). Moreover, the production of both of these autacoids is inhibited by dietary PUFA modulation (Ramesha and Pickett, 1986). Investigations into this possibility in the context of glomerulonephritis, however, have failed to show a convincing role for LTB4 or PAF in mediating macrophage migration (Rovin et al., 1990; Nagamatsu et al., 1992). Nonetheless, studies do suggest that a noncyclooxygenase non-5-lipoxygenase metabolite (such as a P450-derived metabolite) may play a role in monocyte/macrophage trafficking (Nagamatsu et al., 1992). Efforts to isolate a lipid monocyte/macrophage chemoattractant from inflamed glomeruli have also suggested the possibility of a lipid-mediator of monocyte/macrophage trafficking, the production of which is inhibited by EFA deficiency; however, identification of this factor has proved elusive to date (Rovin et al, 1990). As an alternative possibility, it has been considered that dietary PUFA modulation may alter monocyte/macrophage membrane fatty acid composition, and thereby, macrophage function. Data supporting this contention early on were derived from experiments in which macrophages were enriched with the abnormal saturated fatty acid 19:0 (Mahoney et al., 1977). These cells exhibited a relatively global defect in function; both pinocytosis and phagocytosis were impaired. These defects in function were attributed to the altered membrane fluidity that occurred with the enrichment of membrane with saturated fatty acid. Similar investigations on EFA-deficient macrophages did show that these cells were functionally deficient, however, they manifested much more selective defects in their function. Specifically, EFA-deficient macrophages exhibited intact zymosan phagocytosis but impaired receptor-mediated pinocytosis (Lefkowith et al., 1987). These data suggest
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that the altered function induced by manipulation of arachidonate by EFA deprivation might be more specific than a gross alteration in membrane physical properties. Of particular relevance to macrophage migration, it was subsequently shown that EFA deficiency markedly impaired the capacity of resident murine macrophages to spread and adhere in vitro (Lefkowith et al., 1991). This characteristic of EFA-deficient macrophages may contribute to the impaired macrophage emigration seen both constitutively and in the context of inflammation in whole animals. However, the decrease in adherence did not appear to be a simple function of the alteration of membrane fatty acid composition, in that elicited EFA-deficient macrophages (which were found to be equally EFA-deficient to resident EFA-deficient cells) were normally adherent. Thus, the explanation of the defective adherence did not appear to be an alteration in membrane fluidity. The defective macrophage adherence observed with EFA deficiency also did not appear to be due to an alteration in the expression of cell surface adherence molecules (Lefkowith et al., 1991). The presence of cell surface adherence molecules such as Fc receptors or the P2 integrins was unaffected by EFA deficiency. Moreover, an adherence defect could not be induced in normal cells pharmacologically with cyclooxygenase/lipoxygenase blockade or a PAF receptor antagonist. In contrast, phospholipase inhibition was found to induce a spreading and adherence defect in resident macrophages similar to that seen with EFA deficiency, suggesting that decreased levels of PLA2 product(s) (such as arachidonate) may modulate macrophage/substratum interactions. The implications of this finding will be discussed in detail below. In sum, the data derived from dietary PUFA manipulation suggests that arachidonate plays an integral role in monocyte/macrophage trafficking. The simple assumption that this effect is due to alterations in membrane physical properties such as fluidity does not appear to be the case. Rather it appears that arachidonate may be an important autocrine regulator of monocyte/macrophage adherence.
V. INVOLVEMENT OF ARACHIDONATE IN MONOCYTE/MACROPHAGE FUNCTION The fact that monocytes/macrophages contain substantial amounts of arachidonate within their membrane and that they express several enzyme activities for the release and metabolism of this fatty acid, raises the question of whether any of the released arachidonate is utilized intracellularly as a second messenger for signal transduction. Precedence for its role in intracellular signaling is derived from work demonstrating that arachidonate activates PKC (Naor et al., 1988; Khan et al., 1992) and respiratory burst (Sakataetal., 1987; Steinbeck etal., 1991), opens ion channels (Kim and Clapham, 1989; Ordway et al., 1989), participates in membrane fusion (Meers et al., 1988; Fry et al., 1991), and release of [Ca^"^]! from intracellular stores (Chow and Jondal, 1990) or from the cell (Randriamampita and Trautmann, 1990). Thus, a question of interest is: Does the arachidonate, released upon mono-
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MICHELLE R. LENNARTZ and JAMES B. LEFKOWITH
cyte/macrophage stimulation, provide eicosanoid mediators for paracrine action or does it also have an autocrine function? Work from our group and others suggests that, although arachidonate-derived eicosanoid mediators perform an essential paracrine function in inflammation, arachidonate per se acts as a second messenger in monocyte/macrophage signal transduction. Data supporting this hypothesis has been generated using three different model systems: adherence (Lefkowith et al, 1991; Lefkowith et al., 1992), phagocytosis of IgG-opsonized targets (Lennartz and Brown, 1991; Lennartz et al., 1993), and fusion of intracellular vesicles (Mayorga et al., 1993); the results from these studies are presented here. A. Monocyte/Macrophage Adherence
The inability of EFA-deficient resident macrophages to adhere in vitro raised the possibility that their arachidonate deficiency and adherence defects were related. To study this hypothesis, we attempted to reproduce the EFA-deficient phenotype in normal macrophages. Phospholipase inhibition of resident mouse macrophages caused a decrease in adherence that directly correlated with the inhibition of arachidonate release, suggesting a role for this fatty acid in cell/substratum interactions (Lefkowith et al., 1991). Adding back exogenous fatty acids to cells after phospholipase inhibition demonstrated that normal adherence was reconstituted with arachidonate or 20:5(co-3). These results suggest that arachidonate may be an intracellular mediator of leukocyte adherence. It is also noteworthy that 20:5(co-3) could substitute for arachidonate with respect to resident murine macrophage adherence in vitro. This finding may relate to the inability of dietary enrichment for this fatty acid to deplete resident peritoneal macrophages in mice like EFA deficiency (Lefkowith et al., 1990a). A potential role for arachidonate in human monocyte adherence is suggested by other studies (Lefkowith et al., 1992). Like murine resident macrophages, human monocyte adherence is decreased by phospholipase inhibition and restored by the readdition of arachidonate. However, in contrast to the murine system, the arachidonate precursor 20:3((o-6) can substitute for arachidonate with respect to restoring human monocyte adherence after phospholipase blockade, possibly due to its rapid conversion into arachidonate. Interestingly, the phospholipase activity coincident with adherence in human monocytes appears not to require a change in intracellular Ca^"*" (Lefkowith et al., 1992). The phospholipase activity which accompanies adherence may thus be different from the Ca^"^-dependent CPLA2 recently purified from a human monocytic cell line (Clark et al, 1990; Diez and Mong, 1990). B. Phagocytosis of IgG-Opsonized Targets
It has been known for some time that phagocytosis of yeast and IgG-opsonized particles by monocytes/macrophages is accompanied by release of arachidonate. Several groups have analyzed the fate of this arachidonate and found its metabolism to be dependent on the stimulus for arachidonate release as detailed above. We asked
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the question: Is the arachidonate, released during IgG-mediated phagocytosis, necessary for particle ingestion, or is it a byproduct of that process? (Previous studies demonstrated that yeast phagocytosis can occur in the absence of arachidonate release; Aderem et al, 1984). Our approach was to determine the affect of inhibition of arachidonate release on phagocytosis by human monocytes. Monocytes were labeled with ^H-arachidonate and subsequently treated with a variety of PLA2 inhibitors, including bromophenacyl bromide (BPB), manoalide, quinacrine, and the bromoenol lactone, (E)-6-(bromomethylene)tetrahydro-3-(lnaphthalenyl)-2H-pyran-2-one (BEL) to determine their affects on the release of ^H-arachidonate and phagocytosis. All the drugs inhibited the release of ^H-arachidonate associated with ingestion of IgG-opsonized erythrocytes (EIgG) in a manner that closely paralleled the decrease in phagocytosis itself (Lennartz and Brown, 1991; Lennartz et al., 1993). Given the disadvantages associated with utilizing radiolabeled cells (i.e., differential labeling of, and release from, intracellular pools) we repeated the inhibition experiments using unlabeled monocytes and analyzed the free fatty acids released by sequential thin layer/gas chromatography (TLC/GC). We determined that arachidonate was the only fatty acid released during phagocytosis and that its release could be abrogated by treatment of the cells with BPB or BEL (Figure 1 and Lennartz et al., 1993), thus confirming the results with radiolabeled monocytes. To ascertain whether the decrease in arachidonate release was related to the alteration in phagocytic function, we examined the ability of exogenous arachidonate or arachidonate analogues, to overcome the effects of BPB or BEL (Figure 2). Arachidonate was able to partially restore phagocytosis to cells treated with either drug (BPB recovery: 69%, n = 21; BEL; 47%, n = 9; Lennartz and Brown, 1991; Lennartz et al., 1993). Interestingly, of the three analogues tested, only 20:3 (co-6), the immediate precursor to arachidonate, supported recovery. The observation that 20:3(co-6), but not its isomer, 20:3(co-9), could restore function to inhibited monocytes may be due to its rapid metabolism into arachidonate, a pathway not available to the co-9 isomer. These findings suggest that the released arachidonate has a very specific function in the phagocytic signaling pathway that cannot be mimicked by closely related fatty acids. We also studied the impact of cyclooxygenase or lipoxygenase inhibitors on IgG-mediated ingestion and found that inhibition of arachidonate metabolism through these pathways does not alter phagocytosis, supporting a role for arachidonate per se as a second messenger in our system. The conclusions from these experiments are (a) arachidonic acid is the only fatty acid released in significant amounts by monocytes during IgG-mediated phagocytosis, (b) its liberation is required for phagocytosis and (c) its metabolism is not. In an effort to assess the role of arachidonate in phagocytosis without using pharmacological drugs, we quantified the level of IgG-mediated phagocytosis in EFA-deficient resident macrophages. As detailed above, EFA-deficient macrophages are deficient in cell-associated arachidonate (Lefkowith and Schreiner, 1987). In spite of the depletion of arachidonate, these cells do ingest zymosan
MICHELLE R. LENNARTZ and JAMES B. LEFKOWITH
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Figure 2. Recovery of BPB-inhibited phagocytosis by exogenously added fatty acids. Varying concentrations of fatty acids were added to fresh monocytes in the presence of 5 i^M BPB. Ingestion of IgG-opsonized sheep red blood cells was assessed after 30 minutes 37-C. Details in Lennartz and Brown, 1991.
normally (Lefkowith and Schreiner, 1987), a finding consistent Wiih previous work demonstrating that zymosan phagocytosis does not require arachidonate release (Aderem et al., 1884; Lefkovv^ith and Schreiner, 1987). In response to zymosan, EFA-deficient phagocytes release the arachidonate analogue, 20:3(co-9) rather than arachidonate. However, the levels of unsaturated fatty acids released by both normal and EFA-deficient cells in response to zymosan are similar, indicating that the arachidonate-releasing phospholipase activity is normal in the EFA-deficient cells. Thus, they are a good model for studying the involvement of arachidonate in IgG-mediated phagocytosis. Comparative measurements of IgG-mediated phagocytosis revealed that EFA-deficient macrophages were incapable of ingesting EIgG targets (phagocytic indices: 45 ± 15, Control; 2 ± 2, EFA-deficient; n = 4,/? < .001; Lennartz, unpublished observations), providing supporting evidence that release of arachidonate is critical for this type of phagocytosis. We next concentrated our efforts on characterizing the phospholipase activated during phagocytosis (which we refer to as the phagocytic phospholipase, or pPL). Analysis of the arachidonate content of the various membrane phospholipids by sequential TLC/GC revealed that arachidonate liberation as a free fatty acid is balanced by a loss in arachidonate from phosphatidylethanolamine (Lennartz et al., 1993). The simplest explanation for these results is that PE is the in vivo substrate for pPL, a result consistent with release of arachidonate from PE in stimulated neutrophils (Chilton, 1991).
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Reports in the literature have demonstrated that IgG-dependent phagocytosis in mouse macrophages occurs in the apparent absence of Ca^"^ (McNeil et al., 1986; Di Virgilio et al., 1988; Greenberg et al., 1991). However, most of the described PLA2S require Ca^"^ for maximal activity. Thus, we tested the effects of calcium depletion on phagocytosis and the release of arachidonate. Monocytes were incubated in media containing 1.5 mM CaCl2 and 1.5 mM MgC^ (control) or 1 mM EGTA and 2 mM MgCl2 (depleted), a treatment that removes Ca^^ from the cytosol and depletes intracellular stores. Ca^^ depletion ([Ca^'^lj < 2 nM) had no significant effect on either phagocytosis or arachidonate release (Lennartz et al., 1993), suggesting that pPL requires little or no Ca^"^ for activity and provides a potential physiological role for the newly identified Ca^^-independent PLA2 (Hazen et al., 1990). Having characterized pPL as a Ca^"^-independent enzyme capable of releasing arachidonate from PE, we used an in vitro assay to determine the pH optimum (9), the subcellular localization (40% membrane, 60% cytosol), and to begin purification studies. We presume pPL is a PLA2 based on the inhibition seen with PLA2 inhibitors. This hypothesis is supported by our preliminary purification data. The enzyme elutes as a single peak upon sequential ion exchange and hydrophobic chromatography. Furthermore, the in vitro enzyme activity recovered from human monocyte cytosol is inhibited by BEL, the mechanism-based inhibitor of the Ca^'^-independent PLA2 purified from canine myocardium (Hazen et al., 1991), supporting our conclusions that pPL is a Ca^'^-independent enzyme. Of note is that this in vitro activity is not modulated by BPB, a finding that was not surprising in light of its activity towards low molecular weight, Ca^'^-requiring PLA2S (Lister et al., 1989) but was inconsistent with our findings that BPB inhibits release of arachidonate and phagocytosis in monocytes (Figure 1, Lennartz and Brown, 1991). Although we do not have an explanation for this apparent paradox, our current findings suggest that phagocytosis requires arachidonate derived from two sources, a phospholipase inhibited by the extracellular PLA2 inhibitor, BPB and one sensitive to the intracellular, Ca-independent PLA2 modulator, BEL. In summary, we have shown that IgG-mediated phagocytosis is associated with the release of arachidonate and a corresponding decrease in PE-associated arachidonate. Liberation of arachidonate occurs in the apparent absence of intracellular Ca^"*" and can be modulated by the Ca^"*"-independent PLA2 inhibitor, BEL. Inhibition of arachidonate release by any of several PLA2 inhibitors results in a corresponding decrease in phagocytosis, an effect that can be reversed by the addition of arachidonate, but not other closely related fatty acids. Additionally, inhibition of the major metabolic pathways for conversion of arachidonate to bioactive eicosanoids has no effect on phagocytosis. Taken together, these results provide evidence that arachidonate per se plays an essential role in the signal transduction pathway that culminates in ingestion of antibody-opsonized targets. Results from our studies on fusion of intracellular vesicles provide clues as to the function of the released arachidonate.
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MICHELLE R. LENNARTZ and JAMES B. LEFKOWITH
C. Fusion of Intracellular Vesicles Intracellular trafficking is required for cellular homeostasis. This includes transport of newly synthesized proteins through the endoplasmic reticulum and Golgi for deposition into intracellular organelles (e.g., lysosomes), membranes, or for transport out of the cell. Similarly, endocytic pathways function in the uptake of nutrients. These processes require multiple fusion events for the formation of the transport vesicles and proper delivery of their contents within the cell or to the extracellular milieu. Since Rothman et al. first reported vesicular transport between Golgi stacks (Rothman et al., 1984a,b), tremendous progress has been made in identifying the proteins involved in the fusion events required for such trafficking. Recent reviews list membrane receptors, cytosolic attachment proteins, and G-proteins as essential components of the "fusion" (Gruenberg and Clague, 1992; Rothman, 1992). However, the actual mechanism of membrane fusion has yet to be elucidated. Recent studies by Stahl and co-workers suggest that PLA2 activity, specifically the arachidonate product of this enzyme, is required for membrane fusion (Mayorga et al., 1993). Their studies involve isolation of endocytic vesicles from the mouse macrophage-like J774 cell line and assessment of the effects of various treatments on the fusibility of the vesicles (experimental protocol detailed in Diaz et al., 1988). Treatment of the endosomes with a variety of PLA2 inhibitors including manoalide, quinacrine, and BEL generated nonfusible vesicles, a condition that could be reversed by the addition of arachidonate but not lysophosphatides, the other product of PLA2-directed phospholipid hydrolysis. PUFA analogues were much less effective than arachidonate at mediating fusion of treated vesicles. Confirmation of the biochemical fusion results was done by electron microscopy, using endosomes loaded with different size gold particles. In this assay, fusion was detected by the presence of more than one size gold particle in a membrane-bound structure (Figure 3a). By this criteria, BEL-treated vesicles formed large aggregates of discrete vesicles each containing a single-size gold particle (Figure 3b). No mixing of the gold particles was seen unless exogenous arachidonate was added to the incubation (Figure 3c). The conclusion from these studies is that araphidonate, derived via PLA2 action, plays a crucial role in membrane fusion of intracellular vesicles, but does not participate in the aggregation of those membranes prior to fusion. These results are consistent with reports that PLA2 is necessary for fusion in the transport of proteins from the endoplasmic reticulum to the Golgi (Slomiany et al., 1992) and between stacks of the Golgi (Tagaya et al., 1993), and that arachidonate is required for fusion of artificial membranes (Meers et al., 1988; Fry et al., 1991). In contrast to results from these other groups, our data suggest that endosome fusion is mediated by a Ca^"*"-independent PLA2. This conclusion is based on the observations that (a) fusion occurs in the presence of 0.5 mM EGTA (Colombo et al., 1992) and (b) fusion is inhibited by BEL, a drug known to selectively inhibit Ca^'^-independent PLA2S (Hazen et al., 1991) and suggests that fusion between different classes of membranes may be mediated by different
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Figure 3. Effects of PLA2 inhibitors and arachidonate on endosome fusion assessed by electron microscopy. Endosomes loaded with mannosylated BSA-coated 5, 10, and 20 nm colloidal gold particles were incubated in fusion buffer with no additions (a), in the presence of 200 pM BEL (b) and in the presence of 20 pM BEL plus 100 pM arachidonate ( c ) .Large arrows point to vesicles containing more than one size of colloidal gold particles. Small arrows in panel (c) show tubular connections between vesicles. Bar = 100 nm. Taken from Mayorga et al. (submitted).
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MICHELLE R. LENNARTZ and JAMES B. LEFKOWITH
PLA2S. Taken together with the results from our phagocytosis work, we propose that some of the arachidonate released during phagocytosis is required for the membrane fusion events essential to phagosome formation. In summary, this body of work provides convincing evidence that arachidonate functions as an autocrine mediator of monocyte/macrophage homeostasis. Although its precise mode of action has yet to be elucidated, the resuhs of the above studies strongly support its role as a mediator of membrane fusion. This is consistent with several reports in the literature (Meers et al., 1988; Fry et al., 1991; Creutz, 1992) and provides a credible explanation for the observed correlation between arachidonate release and phagocytosis. As phagocytosis requires mobilization of large amounts of membrane, local regulated release of arachidonate (by a receptoractivated PLA2?) may direct insertion of membrane at the region of target binding for the formation of pseudopods and subsequent phagosomes. Its role in adherence is less obvious. There are at least two possible ways in which arachidonate might mediate cell adherence. One possibility is that arachidonate, as an amphiphile, might alter membrane properties and, consequently, the function of membrane proteins. Arachidonate could alter the binding characteristics of cell surface adherence receptors within the membrane. A lipid-receptor interaction has been shown for isolated vitronectin receptors (Conforti et al., 1990). In these studies, the lipid environment of the receptor altered both its avidity and substrate specificity. A lipid integrin-modulating factor derived from human neutrophils has also recently been described (Hermanowski-Vosatha et al., 1992). This factor is believed to be an allosteric activator of leukocyte integrins, and the data suggest that it may be a polyunsaturated fatty acid. A second possibility is that the arachidonate may act as a second messenger activating other cell events. Of particular relevance may be the observation that arachidonate may activate PKC (Klucis and Polya, 1987; Sekiguchi et al., 1987; Naor et al., 1988 Shearman et al., 1989).
VI. CONCLUDING COMMENTS In this chapter we have attempted to review the literature regarding the role of PLA2, arachidonate, and arachidonate-derived metabolites on monocyte/macrophage function. In recent years, much progress has been made in the purification, characterization, and cloning of monocytic PLA2S, advances which are providing valuable structural information as well as insights into the mechanisms of catalysis. However, even as the number of systems in which PLA2 and/or arachidonate play a role is increasing, the rules governing PLA2 action in whole cells remain elusive. Current efforts are directed towards understanding the mechanisms of enzyme regulation (modulation by G-proteins, protein kinases, and intracellular translocation) and the effects of both arachidonate and its metabolites on cellular processes. We believe that advances in these areas will reveal novel pathways of second messenger action that are based on the unique hydrophobic properties of both the enzyme and its products. We further believe that the information to be gained from
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such studies will be invaluable to our understanding of immune function in general and, more specifically, to the development of more effective treatments for chronic inflammatory disease and atherosclerosis.
ACKNOWLEDGMENTS The authors wish to acknowledge Drs. Luis Mayorga, Maria Isabel Colombo, and Philip Stahl for the contribution of their vesicle fusion data to this chapter and Drs. Eric Brown and Colombo for critical reading of the manuscript.
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Smith, M., Ford, H.A., & Bray, M.A. (1980). Leukotriene B: A potential mediator of inflammation. J. Pharm. Pharmacol. 32, 517-518. Snyder, D.S., Beller, D.I., & Unanue, E.R. (1982). Prostaglandins modulate macrophage la expression. Nature 299, 163-165. Spaethe, S.M., Freed, M., DeSchryver, K., Lefkowith, J.B., & Needleman, P. (1988). Essential fatty acid deficiency reduces the inflammatory cell invasion in rabbit hydronephrosis resulting in suppression of the exaggerated eicosanoid production. J. Pharmacol. Esp. Then 245, 1088-1094. Steinbeck, M.J., Hegg, G.G., & Kamovsky, M.J. (1991). Arachidonate activation of the neutrophil NADPH-oxidase. J. Biol. Chem. 266, 16336-16342. Sugiura, T., Katayama, O., Fukui, J., Nakagawa, Y., & Waku, K. (1984). Mobilization of arachidonic acid between diacyl and ether phospholipids in rabbit alveolar macrophages. FEBS Lett. 165, 273-276. Suzuki, T., Saito-Taki, T., Sadasivan, R., & Nitta, T. (1982). Biochemical signal transmitted by Fc receptors: Phospholipase A2 activity of the Fc2b receptor of murine macrophage cell line P388Di. Proc. Natl. Acad. Sci. USA 79, 591-595. TafFet, S.M. & Russell, S.W. (1981). Macrophage-mediated tumor cell killing: Regulation of expression of cytolytic activity by prostaglandin E. J. Immunol. 126, 424—427. Tagaya, M., Henomatsu, N., Yoshimori, T., Yamamoto, A., Tashiro, Y, & Fukui, T. (1993). Correlation between phospholipase A2 activity and intra-golgi protein transport reconstituted in a cell free system. FEBS Lett. 324, 201-204. Teitelbaum, I. (1990). The epidermal growth factor receptor is coupled to a phospholipase A2-specific pertussis toxin-inhibitable guanine nucleotide-binding regulatory protein in cultured rat inner medullary collecting tubule cells. J. Biol. Chem. 265,4218-4222. Todderud, G., Wahl, M.L, Rhee, S.G., & Carpenter, G. (1990). Stimulation of phospholipase C-1 membrane association by epidermal growth factor. Science 249, 296-298. Tonnesen, M.G., Anderson, D.C., Springer, T.A., Knedler, A., Avdi, N., & Henson, RM. (1989). Adherence of neutrophils to cultured human microvascular endothelial cells: Stimulation by chemotactic peptides and lipid mediators and dependence upon Mac-1, LFA-1, p 150,95 glycoprotein family. J. Clin. Invest. 83, 637-646. Tripp, C.S., Leaby, K.M., & Needleman, P.N. (1985a). Thromboxane synthase is preferentially conserved in activated mouse peritoneal macrophages. J. Clin. Invest. 76, 898-901. Tripp, C.S., Mahoney, M., & Needleman, P. (1985b). Calcium ionophore enables soluble agonists to stimulate macrophage 5-lipoxygenase. J. Biol. Chem. 260, 5895-5898. Tripp, C.S., Needleman, P., & Unanue, E.R. (1987). Indomethacin in vivo increases the sensitivity to Listeria infection in mice: A possible role for macrophage thromboxane A2 synthesis. J. Clin. Invest. 79, 399-403. Tripp, C.S., Unanue, E.R., & Needleman, P. (1986a). Monocyte migration explains the changes in macrophage arachidonate metabolism during the immune response. Proc. Nad. Acad. Sci. USA 83, 9655-9659. Tripp, C.S., Wyche, A., Unanue, E.R., & Needleman, P. (1986b). The functional significance of the regulation of macrophage la expression by endogenous arachidonate metabolites in vitro. J. Immunol. 137,3915-3920. Ulevitch, R.J., Watanabe, Y, Sano, M., Lister, M.D., Deems, R.A., & Dennis, E.A. (1988). Solubilization, purification and characterization of a membrane-bound phospholipase A2 from the P388Dj macrophage-like cell line. J. Biol. Chem. 263, 3079-3085. Wahl, M.L, Jones, G.A., Nishibe, S., Rhee, S.G., & Carpenter, G. (1992). Growth factor stimulation of phospholipase C-51 activity. J. Biol. Chem. 267, 10447-10456. Waite, M. (1987). The Phospholipases. Plenum, New York. Waite, M. (1990). Phospholipases, enzymes that share a substrate class. In: Biochemistry, Molecular Biology, and Physiology of Phospholipase A2 and Its Regulatory Factors (Mukherjee, A.B., ed.), pp. 1—22, Plenum, New York.
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INDEX
A23187, 116-117 Acyl-ACP synthetase (Aas), 49, 5253,54 Acyl-CoA synthetase (FadD), 49-53 {see also ''Escherichia coli,..") Adriamycin, 319-320 Alamethicin, 391-92 Alkyl-acyl-GPC (AA-GPC), 252-255 Amniotic fluid, PAF in, 245-246 {see also'^VkV.,:-) C A ' ' , 245 Angiotensin, 183-184 Anion exchange chromatography, 394 Anionic phospholipids, 15-17 {see also "Mammalian CTP...") Annexin V, 87-90 {see also "2MG...") Arachidonate, role of in monocyte/ macrophage function, 429462 abstract, 430 concluding remarks, 454-455 introduction, 430-431 eicosanoids, 430-431 phagocytosis, 431 involvement of, 447-454 adherence, 448 as autocrine mediator of monocyte/macrophage homeostasis, 454 463
BEL, 451-453 fusion of intracellular vesicles, 452-454 phagocytic phospholipase (pPL),450 phagocytosis of IgG-opsonized targets, 448-451 and PKC, 454 as second messenger in signal transduction, 448, 449 metabolism, 439-443 adherence, 439-440 cyclooxygenase-1 and -2, 441 eicosanoids, regulatory role for, 442-443 FLAP, 442 HETEs, 439 macrophage subsets and eicosanoid profiles, 440 particulate, 439 PGE, 442-443 PGI, 443 regulation of, 441-442 soluble stimuli, 439 stimuli for arachidonate release, 439-440 Tx, 443 release of, phospholipase and, 431-438 2-arachidonyl phosphatidylcholine, 436
464
ATP-lipase interaction, 435 catalytic activation of enzymes, 434-435 G-proteins, 436 inflammatory mediators, 432 phagocytosis, 437 phosphatidyl ethanolamine (PE) PLA2 translocation, 433-434 PLA2S, monocytic, 431-436 PLC/diglyceride lipase pathways, 431 purified macrophage phospholipases A2S, 433 regulation of PLA2 activity, 433-436 serine phosphorylation, 435 substrates and products, 436438 tyrosine phosphorylation, 435 trafficking, 443-447 dietary PUFA manipulation and inflammation, 443-444 EFA deficiency, 443-446 inflammation, chronic, and EFA deficiency, 445-446 LTAs and LTB, 445-446 macrophages, resident, EFA deficiency and, 444-445 Mead acid, 443 migration, 446-447 Arachidonate hydrolysis, 219-225 Arachidonate metabolites, 261, 430431 {see also "Arachidonate...") Arachidonic acid, 105-107, 125-127, 174-177, 179-187, 202, 21539 in amniotic fluid, 245 Ca^', 219 in heart, 197-99 nonesterified as second messenger in, 218-223
INDEX
Atherosclerosis, 150-151, 152-155 ATP, regulation of PLA2 by, 123124 in heart, 198 in phosphatidylserine, 305-320 BEL, 451-453 (5^e a/50 "Arachidonate. .") Brefeldin A, 321 Ca^', 245, 258, 260 CAPP, 281 CCK, 20 {see also "Cholecystokinin") Ceramide, 274, 279-282 -activated protein phosphatase (CAPP), 281 Chaperonins, 14 Cholecystokinin (CCK), effects of on CT, 20 Cholinephosphotransferase, 11-12 Chromaffin granule membrane, 322323 Cigarette smoking, PAF acetylhydrolase activity and, 152-154, 262-263 Colony stimulating factors, 261 CT, role of in PC synthesis, 2-4 {see also "Mammalian CTP...) Cyclooxygenase-1 and -2, 441 Cytochalasin B, 122 DAG, 2-38, 337-366 {see also "Mammalian CTP..." and "Diacylglycerol metabolism...") changes in content of, 18-19 PC as generator of, 4 signal, 5 DEAE-cellulose (DE-52) chromatography, 371, 372, 398-403 DEAE Sepharose chromatography, 14, 21 Desferrioxamine, 206
Index
Dexamethasone, 114-115, 155,441 and PAF-AH activity, 257-258 Diabetes mellitus, insulin-dependent (IDDM), 217 noninsulin dependent (NIDDM), 217 Diacylglycerol (DAG) metabolism in cellular membranes, 337-366 cellular content and protein kinase C, 349-351 future investigation, areas for, 356 introduction, 338 roles of, 338 as second messenger, 338 molecular species of and diradylglycerol, 353-356 AAGs, 354 diacylglycerol individuality and free fatty acids formed, 355356 diacylglycerol individuality and removal, 355 and protein kinase C activation, 353 movement of within cells, 351-353 phosphatidic acid, 351-353 protein kinase C, activation of by, 340-343 isotypes of, 341 nuclear, 342-343 sphingolipids, regulation of by, 343 signal attenuation and recycling, 348 kinase, 348 lipase, 348 sources of, 343-348 from de novo synthesis, 344-345 glycerol-3-P pathway, 344-345 MOAT, 345 of nuclear diacylglycerol, 346347 PAPase, 344, 347
465
phosphatidic acid phosphohydrolase 2, 347 phosphatidylcholine-specific phospholipase C, 346 phospholipase C, 346 phospholipase D, 346 from recycling of cellular glycerolipids, 346 triacylglycerol lipase, 347-348 as substrate for triacylglycerol and complex lipid synthesis, 338-340 CDP-choline, 340 enzymes of in mammals, 339 triacylglycerols, medium chain (MCT), fate of, 356 Dietary MCT, 356 Digitonin permeabilization, 7 Diradylglycerol (DRGs), 353-356 {see also "Diacylglycerol...") Di-tertiary butyl hydroquinone, 307 Dithiothreitol (DTT), 323 DRGs, 353-356 {see also "Diacylglycerol...") "Early pregnancy factor," 244 EDRF, 206 EGF, 117 {see also "Epidermal growth factor...") EGTA, 120,305-307,315,317 Eicosanoids, 261, 429-462 {see also "Arachidonate...'^ PGE, 442-443 PGI, 443 regulatory role for, 442-443 Electron microscopy, 452-453 Electron paramagnetic spin resonance spectroscopy, 204 Endometrium and implantation in pregnancy, role of PAP in, 244 Endothelium-derived relaxing factor (EDRF), 206
466
Endotoxin, 261 Enzymes, catalytic activation of, 434-435 Epidermal growth factor (EGF) receptors, 211-11% and myocardial phospholipase C, 408 Epstein-Barr virus, 281 Escherichia coli membrane phospholipids, incorporation and turnover of fatty acids in, 39-59 acyltransferases, three, 40-45 Aas, 43-44 acyl-ACP, 43 2-acyl-GPE, 43 amino acid starvation, 42 phosphatidic acid, formation of, 41 PlsB, 40-44 PlsC, 42-44 plsX gene, 44-45 ppGpp, 42 "stringent response," 42 fatty acid uptake and metabolism, 48-54 Aas, 49, 52-53, 54 domain structure, 50 of exogenous phospholipids, 54 FadD (acyl-CoA synthetase), 49-53 FadL, 49-51 future prospects, 54 introduction, 39-40 phospholipid acyl moieties, turnover of, 45-47 aas pldB dovibXt mutants, 47 2-acyl-GPE cycle, 45-47 PldA, 46-47 PldB, 47 PtdEtn, 45 Estrogen, 151 role of in timing of parturition, 260
INDEX
FACS, 221, 223 Familial HDL deficiency (Tangier disease), 152 Fast protein liquid chromatography, 371,372 Flame-ionization detection, 395 FLAP, 447 Fluorescence-activated cell sorting (FACS), 221, 223 Fumonisins, 282-283, 287, 290 G-proteins as regulators of myocardial phospholipase C, 404413 activation of PLA2 by, 436 Gamma-interferon, 281 Gangliosides, 277-278 Gas chromatography/mass spectrometry (GC/MS), 437 Gas-liquid chromatography, 394, 409,418 -mass spectrometry, 394 Glucocorticoids, 114 GLUT2, 217, 232-233 GTP(gamma)S, 405-407 HDL deficiency (Tangier disease), 152 HELSS, 225-232 Heparin, 106 HETEs, 439 Hexadecanol, 248-250 High performance liquid chromatography (HPLC), 68, 230231,372,394-395 Hydroxylapatite chromatography, 21, 371, 372 Hypertension, PAF acetylhydrolase activity and, 154 IDDM (insulin dependent diabetes mellitus), 217 Immunoblotting, 398-404
Index
Insulin secretion, phospholipid hydrolysis and regulation of, 215-239 (5^^ afao "Phospholipid hydrolysis...") Interferon, 155, 261 Interleukins, 261 interleukin 1, 281 lonomycin, 307 Ischemia, 145, 152-155, 179-183 and plasmalogens and nitroxidefree radicals, 193-214 (see also "Plasmalogens...") Lactosylceramide, 278 Lazaroids, 202-204 LDL, oxidative modification of, 150-151 (^e^flfao "Plateletactivating factor...") Lipid activators, 15-17 Lipid metabolism in Escherichia coli, 39-59 (see also ''Escherichia coll..") Lysophosphatidic acid, 410-413 LysophosphoUpids, detrimental actions of to heart, 199-201 {see also "Plasmalogens...") Macrophages, 256-257, 262 arachidonate, role of in function of, 429-462 {see also "Arachidonate...") cyclooxygenase, 441 Mammalian CTP, regulation of, 138 future directions, 31 intracellular distribution and translocation between sites, 7-15 amphitropism, 8 cell fractionation, 7 chaperonins, 14 cholinephosphotransferase, 1112
467
cytosolic and membrane forms, activity of, 7-8 digitonin permeabilization, 7 H-form, 13-14, 22 interconversion of soluble and membrane-bound, 8-10 L-form, 13-14, 22 membrane to which CT binds, identity of, 11-13 nontranslocatable membranebound CT, 14-15 okadaic acid, 10 oleic acid, role of, 8-10, 14 physiological relevance of translocation model, 10-11 soluble CT, two forms of, 13-14 translocation correlations, 9 lipid second messenger concentrations, role of in control of, 4-5 DAG signal, 5 PC metabolic cycle, 4-5 lipids in vitro, regulation of activity by, 15-17 {see also ".. .regulation by lipids...") PC synthesis, role of CT in, 2-4 rate-limiting reaction, 2-3 phosphorylation, regulation of CT activity by, 18-21 cAMP analogs, 18-19 cholecystokinin, effects of, 20 DAG content, 18-19 manipulation of conditions, effects of, 18 okadaic acid, effects of, 20,28,30 oleic acid, effects of, 21, 30 phorbol esters, 18-19 as phosphoenzyme, 19-20 phospholipase C, effects of, 2021 as regulated enzyme, 5-7 posttranslational regulation, 6 pretranslational regulation, 6-7
468
INDEX
regulation of activity in vivo, mechanism of, 27-31 dephosphorylation, 28-29 Ei(L) and E2(P), 30 interrelationship of various conformations, 29-31 lipid composition, changes in, 27-29 32p^ 29
phorbol esters, 27 phosphorylation, changes in, 28-29 in vitro, 27 regulation by lipids in vitro, 15-17 activating membrane, critical features of, 16-17 anionic phospholipids, 15-17 modulators, classification of, 15-16 neutral lipids, 15 PLC and PLA2, 17 structure, 21-26 amino acid sequence and sequence homologies, 22-23 amphipathic domain, 24-25, 26 catalytic domain, 24 dimerization domain, 25-26 helix-1 and helix-2, 24 interaction of CT with membranes, model of, 26 kinase targets, 23 membrane-binding domain, 2425 model of CT interaction with membranes, 26 molecular mass and subunit, 22 phosphorylation domain, 24 phosphorylation sites, 23-24 predictions, secondary and tertiary, 24 putative helical domain, 25
Mammalian nonpancreatic phospholipase A2 enzymes, 101139 {see also "Phospholipase A 2...") Mannoheptulose, 218, 225 MCT, 356 {see also "Diacylglycerol...") Mead acid, 443 2-MG incorporation into phosphoglycerides, 61-99 abbreviations, 91-92 abstract, 62 conclusion, 90-91 importance of, potential, 91 pathway, reasons for interest in, 90 evidence for, 65-73 arachidonoyl-specific DG kinase, role for, 70 PDGF, 65 stearoyl-specific transacylase, 70 Swiss 3T3 cells, 65-75 introduction, 62-65 classes, 62-63 complexity, 62-63 functions, specific, 63 pathways little known, 63-64 "remodeling," 63-64, 70 questions, unanswered, 73-90 annexin V, 87-89 2-arachidonoyl MG, preferential incorporation of into sn1 -stearoyl-2-arachidonoyl PI, 77 ATP-dependent translocase, role of, 86-87 branches of pathway, 73 C2 domain, 87 cell membranes, role in of snAstearoyl-2-acyl phosphoglycerides, 86-90 de novo pathway, 84-86 DG kinases, 77-79
Index
donor in stearoyl-specific transacylase reaction, 76-77 evolutionary development of pathway, 91 kinases converting 2-MG to 2acyl lysoPA, 75 membrane requirements, 91 PE, PS and PC, conversion of 2-MG into, 79-84 phosphoglyceride biosynthesis, pathways of, relation to, 8486 physiological role of 2-MG pathway, 86-90 PI cycle, role of pathway in, 7779 PI synthase activity, 78 PIP2, hydrolysis of, 78-79 significance, quantitative, of pathway as source of cell phosphoglycerides, 91 source in vivo, 73-75 thrombin-stimulated platelets, 79 MGAT, 345 Michaelis-Menten kinetics, 325 Miller-Dieker Ussencephaly, 156 Mono Q chromatography, 371, 372 Monocyte/macrophage function, role of arachidonate in, 429462 (see also "Arachidonate...") Myocardial ischemia, 124 Myocardial tissue, phosphoinositide metaboUsm in, 387-428 (see also "Phosphoinositide...") Necrosis, 201 Necrotizing enterocolitis (NEC), PAF and, 263-264 Neomycin, 418 NIDDM (noninsulin dependent diabetes mellitus), 217
469
Niemann Pick's disease type C, 290 Nitroxide free radicals, 201-209 (see also "Plasmalogens...") NO synthase, 204-205, 207-209 inhibitors, 209 Norepinephrine, 395, 418-419 Northern blotting, 401, 407 Octyl-Sepharose chromatography, 371 Okadaic acid, 10, 21, 28, 30, 344 effects of on CT, 20 Oleic acid, 8-10, 14, 20, 30 effects of on CT, 21 and plasmalogens in heart, 199 ONOO", 207-208, 209 Ovulation, role of PAF in, 243 PAF, 108-109, Ul-162 (see also "Platelet-activating factor...") PAF, role of in reproductive biology, 241-271 abstract, 242 fetal lung maturation and parturition, role of in, 245-260 alkyl-acyl-GPC (AA-GPC), 252-255 Ca'', 245, 258, 260 in amniotic fluid, 245-246 estrogens, role of in timing of parturition, 260 ethanolamine plasmalogens, 250-252 fetal lung maturation and stimulation of fetal membranes, relationship between, 252255 in fetal membranes, 250-252 hexadecanol, 248 hormonal regulation of PAFAH activity, 257-259 human myometrium, receptors in, 259-260
470
IL-la and IL-ljS, 261-262 L-659, 189,259-260 LPS, 261 lung glycogen metabolism, effects on, 247-248 macrophage, role of in PAF metabolism, 256-257, 262 in maternal compartment, 255 metabolism of, 246, 255 and myometrial contraction, 255, 259-260, 261 FAF-AH, 255-257, 262, 264 PAF-AH activity during pregnancy, 255-260 prostaglandin E2, 254, 259 receptor antagonist, effect of PAF receptor on delivery time in rats, 260 receptor antagonist, effect of on myometrial contraction and parturition, 259-260 respiratory distress syndrome (RDS), 248 and surfactant replacement preparations, 249-250 TNF-a, 261 type II pneumonocytes, PAF receptors in, 247, 249 type II pneumonocytes, surfactant secreted by, 248 WEB-2086, 248 WISH cells, 250-252 introduction, 243 in parturition, 245-260 and perinatal complications, 261264 -acetylhydrolase in milk, 264 and cigarette smoking, 262-263 endotoxin, 261 intrauterine infections, 261-262 hypertension/preeclampsia, 263 necrotizing enterocolitis, 263264
INDEX
pregnancy, early stages of, role in, 243-244 autacoid, relating of to implantation, 244 "early pregnancy factor" from spleen cells, 244 in endometrium and implantation, 244 in ovulation, 243 in sperm function and metabolism, 243 PAF-AH, 255-257, 262 PAPase, 344, 347 PDGF, 65, 408 and myocardial phospholipase C, 408 PDMP, 288 PE, 437 Peroxynitrite (ONOO"), 207-209 Pertussis toxin, 405, 436 PGE, 442-443 PGI, 443 Phorbol esters (PMA), 18-19, 27, 290 Phorbol myristate acetate (PMA), 115-119 Phosphatidic acid, regulation of myocytic phospholipase C by, 409-413 Phosphatidyl ethanolamine (PE), 437 Phosphatidylethanolamine, 299-335 {see also "Phosphatidylserine. ..") Phosphatidylinositol (PI)4-kinases in Saccharomyces cerevisiae, 367-385 abstract, 368 cell proliferation, role in, 368 conclusions, 381-382 introduction, 368-370 functions of, 368-369 kinetic properties of, 374-378
Index
mixed micelle system, 374, 376 PI and ATP, dependence of on, 377-378 "surface dilution" kinetics, 374375 Triton X-100, role of in catalysis, 374-377 nucleotides, regulation of by, 379381 ATP and ADP, 379-380 CTP as potent inhibitor, 380381 phosphorylation, regulation of by, 378-379 PI 3-kinase, 381 properties of, 373-374 enzymological, 373-374 physiochemical, 373 purification of, 370-372 cumbersome, 370-371 of45kDaPI4-kinase, 371 of 55 kDa PI 4-kinase, 371-372 of 125 kDa PI 4-kinase, 372 regulation of activities of by nucleotides, 379-381 regulation of activities of by phosphorylation, 378-379 Phosphatidylserine (PtdSer) dynamics and membrane biogenesis, 299-335 abbreviations, 329 abstract, 300 concluding remarks, 328-329 interorganelle transport, metabolism of and, 309-321 adriamycin, 319-320 ATP requirement, 311-320 brefeldin A, 321 cooperation in phosphatidylethanolamine formation, 310-311 cycloheximide, 311 EGTA,317
471
exogenous phosphatidylserine, mitochondrial import of, 320 mitochondrial membrane contact sites, role of, 319-320 organelles, isolated, 312-314 permeabilized cells, 314-315 phosphatidylethanolamine, mitochondrial export of, 320-321 phosphatidylserine decarboxylase, 309-310 "pulse-arrest" studies, 315 PH] serine, 317-319 translocation of as independent of synthesis, 315-316 translocation of from endoplasmic reticulum to mitochondria, 311-320 translocation requirement for ATP, 316 translocation restricted to autologous mitochondria, 317319 in yeast cells, 311-312 intramembrane transport of, 321328 aminophospholipid transporters, 326 "back-exchange" technique, use of, 322 chromaffin granule, 322-323 dithiothreitol (DTT, 323 endoplasmic reticulum, 322 externalization of as clearance signal, 323-324 fibroblasts, 327-328 importance of, 328 N-ethylmaleimide (NEM), 322 PDA, 326 platelets, 323 questions, unanswered, 328 in red blood cells, 324-326
472
in sickled red blood cells, 323324 translocation within intracellular membranes, 322-323 translocation within plasma membrane, 323-328 introduction, 300 and protein kinase C (PKC), 300 PtdEtn, 300-302 metabolism of and interorganelle transport, 309-321 (see also ".. .interorganelle transport...") structure and synthesis, 301-309 ATP requirement, 305-320 biochemical events in, 306 calcium sequestration, coupling of with, 305-309 cytidine diphosphatediacylglycerol dependent reactions, 301-302 cytidine diphosphatediacylglycerol independent synthesis, 303-304 di-tertiary butyl hydroquinone, 307 EGTA, 305-307, 315 eukaryotic phosphatidylserine synthase, subcellular localization of, 304-305 ionomycin, 307 MAM, 305,307,310 mechanisms of coupling, 307309 PSS gene, 301-302, 305 thapsigargin, 307 Phosphoinositide metabolism in myocardial tissue, 387-428 abstract, 388 calcium concentration, ionized, as determinant of phospholipase C activity, 420-421
INDEX
de novo synthesis and phosphorylation of, 391-393 alamethicin, 391-392 future directions, 421-423 hypoxia as determinant of myocardial metabolism of phosphoinositides, 413-420 neomycin, 418 norepinephrine, 418-419 prazosin, 419-420 introduction, 388-391 biosynthesis of, 390 challenges in study of, 389 importance of, 388-389 second messenger function, 389, 391 ischemia as determinant of myocardial metabolism 420 of phosphoinositides, 413420 neomycin, 418 norepinephrine, 418-419 prazosin, 419-420 membrane-associated phospholipase C, 402-404 molecular heterogeneity of soluble phospholipase C in cardiac tissue, 396-402 G-protein, 397, 401-402 immunoblotting, 398-401, 402 src proto-oncogene, 397-398 transmembrane signaling in heart, 401-402 phospholipase C, myocardial, mechanisms for regulation of, 404-413 biochemical, 411-412 G-proteins, 404-407 GTPyS, 405-407, 412 lysophosphatidic acid, 410-413 myocytic protein kinase A, 409 pertussis toxin, 405 phosphatidic acid, 409-413
Index
PKC, 405-406 PLC^, 405-407 protein kinases, 407-409 quantification of phosphoinositide-derived second messengers, 393-395 gas-liquid chromatographic method, 394 gas-liquid chromatographymass spectrometry, 394 norepinephrine, 395 protein binding assay, 395 radioisotope, 393-394 reperfusion as determinant of myocardial metabolism of phosphoinositides, 413-420 neomycin, 418 norepinephrine, 418-419 prazosin, 419-420 Phospholipase A2 enzymes (PLA2), mammalian nonpancreatic, properties and regulation of, 101-139 abstract, 102 function of, 102 ATP-stimulated, Ca^^independent, 225-227, 229 introduction, 102-103 properties and function of, 103112 in arachidonic acid release, 105107 bactericidal permeability increasing protein (BPI), 105, 107 calcium-dependent, arachidonic acid-specific cytosolic, 107111 calcium-independent cytosolic, 111-112 cytosolic, 107 in eicosanoid production, 108, 115-116
473
40kDaprotein. 111 group I type, 104 group II type, 105-107 heparin, 106 lysophospholipase activity, 110 myocardial sarcolemma membrane, 111 97kDa protein, 112 PAF, 108-109 PE plasmalogen, 108-109 secreted, 103-105 spleen, 105 30kDaPLA2, 111 tumor necrosis factor (TNF), 106 regulation, 113-127 A23187, 116-117 by ATP, 123-124 by calcium, 119-123 cytochalasin B, 122 cytoskeleton elements, 122 dexamethasone, 114-115 EGF, 117 EGTA, 120 85kDaPLA2, 118-119, 120-123, 126 by G-proteins, 125-127 glucocorticoids, 114 5-lipoxygenase, 123 M-CSF, 115 MAP kinase, 118-119 myocardial ischemia, 124 ' ' P , 118 PDGF^, 114 pertussis toxin, 125 phorbol myristate acetate (PMA), 115-119 by phosphorylation, 115-119 RACKS, 122-123 serine/threonine kinases, 116 staurosporine, 126 TGF^, 114 transcriptional, 113-115
474
transducin, 125 tyrosine kinase activation, 116 vasopressin, 116-117 Phospholipase C (PLC), 346 effects of on CT, 20-21 Phospholipid hydrolysis in pancreatic islet beta cells and regulation of insulin secretion, 215-239 abstract, 216 arachidonate hydrolysis, control of from islet membrane phospholipids, 219-225 secretagogues, 224-225 arachidonate metabolism in islet beta cells, 233-235 FACS-purified p cells, 234 12-HETE, 233-235 arachidonic acid, nonesterified, as second messenger in, 218223 Ca'", 219 fluorescence-activated cell sorting (FACS), 221, 223 PGE2, 219 ASCI-PLA2 as component of beta cell D-glucose sensor apparatus, 232-233 complexities, 233 GLUT2, 232-233 beta cell cytosolic calcium ion concentration, regulation of, 227-229 D-glucose recognition, 217-218 acetylcholine, 217 and eicosanoid release, 225 GLUT2, 217 mannoheptulose, 218, 225 hydrolysis of arachidonate from beta cell membrane phospholipids, 227-229 ASCI-PLA2, 227-228 HELSS, 227-229
INDEX
insulin secretion, 227-229 introduction, 217 diabetes mellitus, insulindependent (IDDM), 217 D-glucose concentrations, 217 noninsulin dependent diabetes mellitus (NIDDM), 217 ionic events in glucose-induced insulin secretion, 218 K', 218 iS-cell KATP channel, 218 phospholipase A2 enzyme, ATPstimulated and Ca^^independent, 225-227 ASCI-PLA2, 225 HELSS, 225-227 plasmalogen molecular species in, arachidonate-containing, 229-232 ASCI-PLA2, 231-232 HPLC, 230-231 oleate, 231 palmitate, 231 plasmenylethanolamine, 231232 stearate, 231 Phospholipid structure, 165-167 {see also "Plasmalogens...") PLA2, 101-139 (see also "Phospholipase A2...") Plasmalogens, nitroxide free radicals, and ischemiareperfusion injury in heart, 193-214 abstract, 194 free radicals, 201-209 "antioxidant paradox," 205 desferrioxamine, 206 Fenton reaction, 206 ferritin, 206 Haber-Weiss reaction, 206 hydroxyl radical, 204 lazaroids, 202
Index
necrosis, 201 nitroxide, potential role of, 206207 NO, 204-205, 207-209 ONOO", 207-209 oxygen-derived, generation of, 205-206 peroxynitrite, role of, 207-209 susceptibility of to attack by, 202-205 U74006F, 202 introduction, 194-196 glycerophospholipids, 195 ischemic heart disease as leading cause of death, 194 phospholipase A 2 activity, 196 plasmalogens in heart, unique role of, 196-201 arachidonic acid content, 197 ATP, 198 eicosanoids and free fatty acids, effects of on heart, 198-199 lysophospholipids, 199-201 phospholipase A2 activity, 197198 prevalence, 196-197 Plasmalogens in mammalian cells and their metabolism, 163191 abstract, 164 biosynthesis of, 167-187 alkyl ether glycerophospholipids, de novo biosynthesis of, 167-169 angiotensin, 183-184 arachidonic acid, 179-187 catabolism of during cellular perturbation, 179 catabolism of in myocardium, 179-183 catabolism in neutrophils, 185187
475
catabolism in smooth muscle cells, 183-185 K^ channel function, 180 lysoplasmenylethanolamine, 187 phospholipase A2, 180, 181-187 phospholipases C and D, 180-182 of plasmenylcholine, 169-178 protein kinase C, 183, 186 pulse-chase radiolabeling techniques, 174-176 sarcolemmal phospholipids, 179-183 vasopressin, 183-185 vinyl ether bond of plasmenylcholine, de novo synthesis of, 169 conclusion, 187-188 definition, 165 introduction, 164-165 biological membranes, roles of, 164 phospholipid structure, 165-167 aliphatic constituents, 165-166 subclasses, three, 165-166 Platelet-activating factor (PAF), fate of, 141-162 abstract, 142 acetylhydrolase, PAF, 148-156 {see also ".. .degradation of...") degradation of by PAF acetylhydrolase, 148-156 apolipoprotein B-lOO, 151 atherosclerosis, 150-151, 152155 biochemical characteristics of, 148-149 changes of activity of in physiological and pathological conditions, 153-154 in cigarette smokers, 152-154 dexamethasone, 155 diabetes mellitus, insulindependent, 152
476
in erythrocytes, 155-156 estrogen, 151, 152 forms, two, 148 in gastrointestinal disorders, 155 genetics of plasma form, 152 hypertension, 152-154 in inflammatory conditions, 154 interferon, 155 intracellular, 155-156 LDL, oxidative modification of, 150-151 Miller-Dieker lissencephaly, 156 plasma form in disease, 152-155 plasma form of, properties of, 149-150, 152 population studies of activity in human and animal plasma, 151 cDNA, cloning of human PAF acetylhydrolase, 156 introduction, 142-143 pathological and physiological actions of, 143-144 in vitro effects, 143 in v/vo effects, 143 plasma PAF acetylhydrolase, 149150 {seealso'' .. .degradation of...") receptor, binding to, 144 competitive antagonists, 144 synthesis of, 145-148 enzymatic mechanisms, 145 concentration in blood and tissues, 145 and ischemia, 145 lyso-PAF, 145-146 de novo pathway, 145-146 PE, hydrolysis of, 145-146 phospholipids, oxidatively fragmented, similar to PAF, 145-148 remodeling, 145-146
INDEX
Platelet-derived growth factor (PDGF), 65, 408 and myocardial phospholipase C, 408 Platelets, PtdSer and, 323 PLC, 20-21 PLC^, 405-407 PMA, 18-19, 27, 290 Prazosin, 419-420 Preeclampsia, pregnancy-induced, 263 Pregnancy, role of PAF in, 243-244 {see also''PAF..:") Prostaglandin E2, 254, 259 Protein binding assay, 395 Protein kinase C: diacylglycerol, activation of by, 340-343 diradylglycerol (DRGs), 353-356 isotypes of, 341 nuclear, 342-343 and phosphatidylserine, 300 sphingolipids, regulation of by, 279-282, 343 sphingosine as inhibitor of, 275 Protein kinases as regulators of myocardial phospholipase C, 407-409 PtdSer, 299-335 {see also "Phosphatidylserine...") Pulse-chase radiolabeling, 174-175, 303,311 RACKS, 122-123 RDS, 248, 249-250 Receptors for activated C-kinase (RACKS), 122-123 Remodeling, 145-146 Reproductive biology, role of PAF in,24Uni {see also''PAF, role of...") Respiratory distress syndrome (RDS), 248, 249-250
Index
Saccharomyces cerevisiae, phosphatidylinositol 4-kinase in, 367-385 {see also "Phosphatidylinositol...") SDS-PAGE analysis, 234 Serine/threonine protein phosphatase, 281 Sperm function and metabolism, role of PAF in, 243 Sphinganine, 284 and Niemann Pick's disease type C,290 Sphingolipids as regulators of cellular growth, differentiation, and behavior, 273-298 abstract, 274 approaches to study of functions of, 285-288 addition of exogenous sphingolipids and sphingolipid analogues, 285-287 exogenous agents that bind or chemically modify, effects of adding, 287 genetic approaches to altering biosynthesis of, 288 metabolic inhibitors, 287-288 PDMP, 288 criteria for evaluating studies' results, 288-289 future research on, 290-291 {see also ".. .perspectives for...") introduction, TIA-211 biological activities, examples of, 276 ceramide, 274, 279 protein kinase C, inhibitor of, 275 skin, permeability of, 275 sphingosine, 275 structural diversity, 275
477
paradigms for, basic, 277-285 CAPP, 281 for cell growth, 277-279 ceramide, 274, 279-282 ceramide-activated protein phosphatase (CAPP), 281 ceramides in cell growth and differentiation, 281-282 epidermal growth factor (EGF) receptors, 277 Epstein-Barr virus, 281 fumonisins, 282-283, 287, 290 gamma-interferon, 281 gangliosides, 277-278 growth regulation by, hypotheses for, 284-285 interleukin 1, 281 lactosylceramide, growth and, 278 metabolites, other, 279 phosphatidic acid, 280 protein kinases and protein phosphatases, regulation of, 279-282 saponin, 280 sphinganine, 284 sphingosine and cell growth, 279-281 thin-layer chromatography (TLC), 286-287 threonine 669, 280 tumor necrosis factor (TNF), 281,282 virus replication, 281 perspectives for future research on, 290-291 disease, opportunities for discovery of, 290 preventing and treating disease, new strategies for, 290-291 protein kinase C, regulation of by, 343 studies, criteria for evaluation of, 288-289
INDEX
478
Sphingosine, 275 and cell growth, 279-281 Stable isotope dilution mass spectrometry, 219 Staurosporine, 126 Surfactant replacement preparation, 249-250 Swiss 3T3 cells, 65-75 {see also "2MG...") conversion of 2-MG by into PE, PS, and PC, 79-84
THF-a, 261 TLC, 68, 286-287 TNF, 106,281,282 Transducin, 125 Triacylglycerols, medium chain (MCT), 356 {see also "Diacylglycerol...") Triton X-100, 354, 368, 371, 374-378, 392, 402 Tumor necrosis factor (TNF), 106, 281,282
Tangier disease, 152 TGF (beta), 114 Thapsigargin, 307 Thin layer chromatography (TLC), 68, 286-287, 372, 395 Thin-layer/gas chromatography (TLC/GC), 449 Threonine, 19 threonine 669, 280
U74006F, 202-204 van der Waals interactions, 87 Vasopressin, 116-117, 183-185, 341 WISH cells, 250-252 Zymosan, 429-462 {see also "Arachidonate...")
Advances In Neural Science Edited by Sudarshan Malhotra, Department of Zoology, University of Alberta Volume 1,1993, 228 pp. ISBN 1-55938-356-9
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Edited by Sudarshan Malhotra, University of Alberta and G.D. Das, Purdue University CONTENTS: Preface, Sudarshan K. Malhotra. Voltage-Independent Calcium Channels in Neurons, Judith A. Strong. Physiological Aspects of Presynaptic Inhibition, Harold L. Atwood. Neural Transplants: Their Growth and Differentiation Potentials, Gopal D. Das,. A Reevaluation of the Role of Glia in Central Nen/ous System Regeneration, Samuel David. Molecular Aspects of CNS Injury Response: Changes in the Cytoskeletal Gene Expression After Axotomy, Monica M. Oblinger and Susanna A. Mikucki. Neuroendocrine Aspects of Neural Transplantation, David E. Scott and Wutian Wu. Parallel Roles of Astrocytes and Fibroblasts: An Old Concept Revisited, Theodor K. Shnitka and Sudarshan K. Malhotra.The Evolution of Myelin, Betty I. Roots. Subject Index.
Volume 2,1995,244 pp. ISBN 1-55938-625-8
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CONTENTS: Preface, Sudarshan K. Malhotra. Phosphorylation of Neurofilament Proteins, Michael G. Sacher, Eric S. Athlan and Walter W. Mushynski. Neuronal Development in Embryos of the Mollusc, Helisoma trivolvis; Multiple Roles of Serotonin, Jeffrey I. Goldberg. Opioid Growth Factor and Retinal Morphogenesis, Ian S. Zagon, Tomoki Isayama and Patricia J. McLaughlin. The Molecular Bases of Nerve Regeneration, Joanne K. Daniloff and Laura G. Remsen. Trophic Actions of Gonadal Steroids on Neuronal Functioning Normalcy and Following Injury, Kathleen A. Kujawa and Kathryn J. Jones. Are Epigenetic Factors Involved in the Normal Expression of Neuronal Phenotypes During Spinal Development?, Eric Philippe and Raymond Marchand. Plasticity of Descending Spinal Pathways in Developing Mammals, George F. Martin, Ganesh T. Ghooray, Xian Ming Wang and Xiao MingXu. Development of the Mammalian Auditory Hindbrain, Frank H. Willard. Subject Index.
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Volume 2A, Rhodopsin and G-Protein Linked Receptors 1996,227 pp. ISBN 1-55938-659-2
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CONTENTS; Preface, A.G. Lee. Rhodopsin Structure and Function, Burton J. Litman and Drake C. Mitchell. Characterization of the Primary Photochemical Events in Bacteriorhodopsin and Rhodopsin, Jeffrey A. Stuart and Robert R. Birge. Light-Induced-Protein Interactions on the Rod Photoreceptor Disc Membrane, Klaus Peter Hofmann and Martin Heck. Microbial Sensory Rhodopsins, John L. Spudich and David N. lacks. Alpha-Adrenergic Receptors, David B. Bylund.
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A Multi-Volume Work Edited by E. Edward Bittar, Department of Physiology, University of Wisconsin, Madison and Neville Bittar, Department of l\/ledicine. University of Wisconsin, t\/ladison Volume 1A, Bioethlcs 1994,208 pp. ISBN 1-55938-801-3
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CONTENTS: Preface, E Edward Bittar and Neville Bittar. The Emergence of Secular Bioethics, H.r. Engeltiardt. Meaning of the Quality of Life, MattiHayry. Bioethlcs in Health Policy: What Methodology, Daniel Wikler. Randomized Clinical Trials: Ethical Considerations, Robert J. Levine. The Use and Abuse of Animals in Research, Bernard E. Rolin. Systems Bioethics: AIDS, Reproduction, and the Pandemic Destroying Population Groups, Colleen Clements. Death and Permission to Die, Rem B. Edwards and Glen C. Graber. Euthanasia, Helga Kuhsy. Rational Suicide in the Ederly and the Right to Die, David Clark and Laurel Burton. Medicine and the Environment, Michael McCally, John Last and Eric Chivian. Volume I B , Evolutionary Biology 1994,371 pp. ISBN 1-55938-802-1
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CONTENTS: Preface, E. Edward Bittar and Neville Bittar. The Plasma Membrane, S. Malhotra and T.K. Shnitka. The Transport of Macromolecules Across the Nuclear Envelope, N. Pokrywka, David Goldfarb, M. Zillmann, and A. DeSilvia. Chromosomes, Chromatin, and the Regulation of Transcription,
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CONTENTS: Preface, E. Edward Bittar and Nevelle Bittar. The Lysosome: Its Role in the Biology of the Cell and Organism, Brian Storrie. The Golgi Complex, Alan M. Tartakoff and Jerrold R. Turner. The Peroxisome, Colin Masters and Denis Crane.The Mitochondrion, David Drake Tyler. Lysosomal Storage Diseases, Grazia M. S. Mancini and Frans W. Verheijen. Peroxisomal Disorders, Ronald J. A. Wanders and Joseph M.Tager. Molecules in Living Cells, David S. Goodsell. Extracellular Matrix, D. W. L. Hukins, S. A. Weston, M. J.Humphries, and A. J. Freemont Posttranslatlonal Processing of Collagens, Kari I. Kivirikko. Cellular and Molecular Aspects of Selected Collagen Diseases, Rajendra Raghow. Index. Volume 4, Cell Chemistry and Physiology: Part I
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CONTENTS: Preface, E. Edward Bittar and Neville Bittar. Proteins: An Introduction, Nonria M. Allewell, Vince J. Licata,and Xiaoling Yuan. How Enzymes Woric, Gary L Nelsesfuen.Substrate Utilization in Mammalian Cells, Gerd. van der Vusse and Robert S. Reneman. The Organization of Metabolic Pathways In Vivo, G. Rickey Welch. Enzyme Kinetics In Vitro and In Vivo: Michaelis-Menten Revisited, MichaelA. Savageau. The Basis of Enzymatic Adaptation, Kenneth B. Storey and Stephen P.J. Brooks. Our Aqueous Heritage: Evidence for Vicinal Water in Cells, W. Drost-Hansen and J. Lin Singleton. Our Aqueous Heritage: Role of Vicinal Water in Cells, W. Drost-Hansen and J. Lin Singeton. Intracellular Water and the Regulation of Cell Volume and pH, Harold G. Hempling. Protein Synthesis and Regulation in Eukaryotes, Suresh I.S. Rattan. The Role of Glycosylation in Cell Regulation, Elizabeth F. Hounsell. Why Are Proteins Methylated?, Steven Clarke. ADP-Ribosylation Reactions, Colin K. Pearson. Modification of Proteins by Prenyl Groups, Michael H. Gelb. Lipobiology, David A. Ford and Richard W. Gross. Index. Volume 4, Cell Chemistry and Physiology: Part II
1996,400 pp. ISBN 1-55938-806-4
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CONTENTS: Cellular ATP, David Harris. Purines, Charies H. V. Hoyle and Geoffrey Bumstock and Dr. Charies Hoyle. The Role of Multiple Isozymes in the Regulation of Cyclic Nucleotide Syn-
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CONTENTS: Part I: Primary Ion Pumps, Jens P. Andersen. Facilitative Glucose Transport, Charles F. Burant. Ion-Coupled Cotransport, Rose M. Johnstone and John McCormlck. The Sodium-Calcium Exchanger and Calcium Pumps, Emanuel Strehler. The Na+-H+ Exchanger, C.Frelln and P. VIgne. Oxidase Control of Plasma Membrane Proton Transport, F. L Crane, I. L Sun, R.A. Crowe and H. Low. Cell Volume Regulation, D. Haussinger. Part II: Some Aspects of Medical Imaging, H.K. Huang. NMR Studies of Cell Metabolism In Vivo, Rainer dailies and Kevin M. Brindle. Calorimetric Techniques, Ingemar Wadso. Heat Dissipation and Metabolism in Isolated Mammalian Cells, Richard B. Kemp. Volume 4, Cell Chemistry and Physiology: Part IV 1996,M85pp. $128.50 ISBN 1-55938-808-0 CONTENTS: Part I: The Cytoskeleton-Microtubules and Microfilaments: A Biological Perspective, S. K. Malhotra and T K. Shnitka. Actin Polymerization: Regulation by Divalent Metal Ion and Nucleotide Binding, ATP Hydrolysis and Actin Binding Proteins, Marie-France Cahier and Dominique Pantaloni. Myosins, D. D. LorimerandP. de Lanerolle. Cell Motility, Sutherland Km Mciver and Alan Weeds. Mitochondrial Oxidations and ATP Synthesis in Muscle, D.M. Turnbull and H. S. A. Sherratt. Regulation and Activity of Smooth Muscle, Lloyd Barr.The Cellular and Molecular Basis of Skeletal and Cardiac Muscle Contraction, M. Peckham. Muscle Fatigue, EricHultman, andL. L.Spriet. Skeletal Muscle Disorders, M. A. Johnson, K.M.D. Bushby, LV.B. Anderson and John B. Harris. Part II: Principles of Medical Cryobiology: The Freezing of Living Cells, Tissues and Organs, Peter Mazur. Clinical Applications of Cryobiology, J. H. Southard. Malignant Hyperthermia, C. Johnson and S. Kotamraju. Heat Shock Proteins, Donald Jurivich. Cell Culture, Mary Taub. Index.
