INTERNATIONAL
REVIEW OF CYTOLOGY VOLUME88
ADVISORY EDITORS DONALD G. MURPHY H. W. BEAMS ROBERT G. E. MURRAY HOWARD A. BERN RICHARD NOVICK GARY G . BORISY ANDREAS OKSCHE PIET BORST MURIEL J. ORD BHARAT B. CHATTOO VLADIMIR R. PANTIC STANLEY COHEN W. J. PEACOCK RENE COUTEAUX DARRYL C. REANNEY MARIE A. DIBERARDINO LIONEL I. REBHUN CHARLES J. FLICKINGER JEAN-PAUL REVEL OLUF GAMBORG JOAN SMITH-SONNEBORN M. NELLY GOLARZ DE BOURNE WILFRED STEIN YUKlO HIRAMOTO HEWSON SWIFT YUKINORI HIROTA K. TANAKA K. KUROSUMI DENNIS L. TAYLOR GIUSEPPE MTLLONIG TADASHI UTAKOJI ARNOLD MITTELMAN AUDREY MUGGLETON-HARRIS ROY WIDDUS ALEXANDER YUDIN
INTERNATIONAL
Review of Cytology EDITED BY
G. H. BOURNE
J. F. DANIELLI
St. George’s University School of Medicine
Danielli Associaies Worcester, Massachusetts
Si.
George’s, Grenada Wesr Indies
ASSISTANT EDITOR K. W. JEON Department of Zoology University of Tennessee Knoxville, Tennessee
VOLUME88
1984
ACADEMIC PRESS, Inc. (Hurcourr Bruce Jovunovich, Publishers)
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84858687
9 8 7 6 5 4 3 2 1
Contents CONTRIBUTORS .............................................................
vii
Lysosomal Functions in Cellular Activation: Propagation of the Actions of Hormones and Other Effectors CLARAM. SZEGOAND RICHARDJ. PIETRAS I. Introduction .........................................................
11. Perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Compatibility of Lysosomal Properties with Proposed
1
...........
16
Functions in Activated Cells 1v. Selected Cellular Functions Subjected to Lysosomal Influence . . . . . . . . . . . . . . . . V. Integration . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
70 212 243 246
Neuronal Secretory Systems MONACASTEL,HAROLDGAINER,AND H.-DIETERDELLMANN I. 11. 111.
IV. V. VI. VII. VIII. IX. X.
..........
..........................................
ackaging in Peptidergic Neurosecretory Cells. . . . . . . . . . . . . . . Morphological Aspects of the Formation of Peptidergic Neu Axonal Transport in NeurosecretoIy Cells . . . . . . . . . . . . . . . Morphology of Transport and Release-Peptidergic Neurons . . . . . . . . . . . . . . . . . ........... Molecular Organization of Secretary Vesicles in Neurons Biosynthesis and Biochemical Aspects of Packaging and Transport of Neurotransmitters in Nonpeptidergic Neurons Morphological Aspects of Formation of Nonpeptidergic Secretory Vesicles. . . . . . Developmental Aspects of the Hypothalamic-Neurohypophysial System . . . . . . . . Versatility of Neurosecretory Neurons . . , . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . References .
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INDEX . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CONTENTS OF PREVIOUS VOLUMES AND SUPPLEMENTS . . . . . . . . . . . . . . . . . . . . . . . . . .
V
304 308 318 338 345 366 376 382 40 1 426 438 46 I 465
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Contributors Numbers in parentheses indicate the pages on which the authors’ contributions begin
MONACASTEL(303), Department of Zoology, Institute of Life Sciences, Hebrew University, Jerusalem, Israel H.-DIETER DELLMANN (303), Department of Veterinary Anatomy, College of Veterinary Medicine, Iowa State University, Ames, Iowa 50011 HAROLDGAINER(303), Laboratory of Neurochemistry and Neuroimmunology, National Institute for Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20205 RICHARD J . PIETRAS( l ) , Department of Biology, The Molecular Biology Institute, and the Jonsson Cancer Center, University of California, Los Angeles, California 90024 CLARAM. SZEGO( l ) , Department of Biology, The Molecular Biology Institute, and the Jonsson Cancer Center, University of California, Los Angeles, California 90024
vii
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INTERNATIONAL REVIEW OF CYTOLOGY,VOL 88
Lysosomal Functions in Cellular Activation: Propagation of the Actions of Hormones and Other Effectors CLARAM. SZEGOAND RICHARDJ. PIETRAS Department of Biology, the Molecular Biology Institute, and the Jonsson Cancer Center, University of California, Los Angeles, California I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
..................... ............................ A. Mechanisms of Hormone B. The Relevant Properties of Lysoso 111. Compatibility of Lysosomal Properties with Proposed Agonal Mediating Functions in Activated Cells ........................ A. Generalized Scheme. . . . . . . . . . . . B. Circumstantial Evidence of Covert Membranes . . . . . . . . . . . . . . . ............. C. The “Target” Cell: Occurrence and Functional Implications of Specific Recognition Sites for Given Effectors in the Plasmalemma . . . . . . . . . D. Consequences of Ligand IV. Selected Cellular Functions Subjected A. Cell Death and Some Anomalies of Interpretation . . . . . . . . . . . B. Cell Growth and Proliferation.. .......................... C. Cellular Transformation: Indications for a Lysosomal Role . . . . V. Integration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. The “Uses” of Compartmentation in the Cellular Econ
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Note Added in Proof .....................
1 1 15
16 16 40 70 70 70
72 73 212 212 220 234 243 243 244 246 246 30 1
I. Introduction A. FIRSTPREMISES
If ever there was a universal indicator of cellular activation (or subduction), it is surface membrane destabilization (or stabilization). All eke follows from this primary event. In an orderly succession of coupled reactions, ever widening to encompass all components and phases of cell function, the remotest reaches of 1 Copyright 19x4 hy Acadcniic P r c s . Inc All rlghis 01 reproduction in any lorm rcrcrvcd ISBN 0.12 7644X8-7
2
CLARA M. SZEGO AND RICHARD J . PIETRAS
subcellular organelles are minutely informed of the change in status quo and are enabled to respond appropriately to the triggering stimulus. Such are the coordinate activities that intimately link nucleus and cytoplasm and their respective suborganellar compartments into a functional whole, and, in turn, promote those quantitatively or qualitatively altered metabolic patterns that may result in greater numbers or differentiated types of cells.
I . The Receptor Concept If one is to trace the progression of these activities from the primary event, it is clear that one must start at the outer cell surface where discriminatory capacity resides. Yet, the cell surface is regularly confronted, even bombarded, with a myriad of potential agonists, endogenous and exogenous. To be on the qui vive toward any and all of these would be disastrous, for, without some means of distinguishing between “valid” and “false” triggers, the efficient economy of eukaryotic cells could not have evolved. Thus, it has been a clarifying and unifying concept that the surface of a given cell is equipped with specialized components able to perform this vital discriminatory function through highaffinity but noncovalent, and, accordingly, reversible, interaction with agonists whose molecular conformation is fundamentally complementary. Nature’s infinite catalog of triggers, present and yet to evolve (or to be designed by man), is immediately brought to a manageable size by the receptor concept, first generalized by Paul Ehrlich (1900; Fig. 1). Mutual recognition has, indeed, proved to be the key to selective responses to specific signals delivered by chemical substances to their “target” cells. Discriminatory capacity of given cells toward closely related molecules is often astonishing, for it appears that receptors and effectors have evolved in coordinate fashion (cf. Niall, 1976, 1982; Blundell and Humbel, 1980; Pierce and Parsons, 1980; LeRoith et ul., 1980; Roth et ul., 1982). Moreover, there are exquisite nuances in the recognition phenomenon that permit distinctions to be drawn among agonists, partial agonists, and antagonists within families of closely related molecular species. This is illustrated from examples representing thyroid and steroid hormones, prostaglandins, and certain opiates (Fig. 2 ) . Additional instances occur among relatively less hydrophobic agents. These latter are too familiar to require specific documentation. 2. Signal verws Noise Granting such specificity, it is necessary, first, to define as precisely as possible the distinctions between specific and nonspecific provocations to the surfaces of responsive cells that represent “signal” and “noise,” respectively. Indeed, and seemingly at paradoxical odds with the specific trigger-receptor interaction defined above, there is a collection of evidence that noise and signal may be “read” by the responding cell as generally equivalent stimuli, not only in degree but in kind. Thus, the coarse provocation delivered, for example, by warming of
LYSOSOMAL FUNCTIONS IN CELLULAR ACTIVATION
3
2
FIG. 1 . Diagrammatic representation of the “side-chain’’ theory to illustrate Ehrlich’s concept of specific recognition sites at the cell surface. (1) Complementarity of agonist and receptor. (2) Specific and reversible binding of agonist only to its own receptor. (3) The bound form of the receptor is unavailable for providing negative feedback toward its own biosynthesis. (4) This results in overcorrection by regeneration. (Reprinted by permission, with minor paraphrasing of the text, from the Croonian Lecture, On Immunity with Special Reference to Cell Life, delivered to the Royal Society by Paul Ehrlich, 22 March, 1900; Collected Papers. 1957.)
a cell culture, with or without the further provision of fresh serum and other nutrients in profusion, or even relative anoxia, may have consequences similar in whole or in part to those elicited by the pinpoint signal delivered by a true agonist-even to promoting induction of specific proteins and, perhaps, mito-
AGONISTS AND PARTIAL AGONISTS A. ESTROGENS
& &
HO
Estradiol-17p (active)
...OH
HO
Estradiol-17u (essentially inactive)
HO
C2H5
Estriol (relatively inactive)
Tamoxifen (some agonal effects1
B. ANDROGENS P
CH3 I
~ : '@ H
/
0 Testosterone-17/9 (act we)
0 Testosterone-17a (inactive)
5-Di hydrotestosterone (DHT; intensely active)
/
CI
0
II C-N,
o&
A H 5
C2H5
[A CH3
C ypraterone 178- Jb -diet hy Ic a r ba (antiandrogen with moyl-4-methyl-4-oza-5asome progestational androstan-3-one effects) (inhibits conversion of testosterone - 1 7 p to DHT; has moderate affinity for androgen receptor 1
B,
C. ADRENOCORTICOIDS CHZOH
CHZOH
I
I
0 I! -0eoxycorticosterone
Aldosterone (minerolocorticoids)
VI
D.
0d
0 Cortisol (glucocor ticoid)
=
O
A spirolactone (minerolocorticoid antagonist)
PROGESTATIONAL STEROIDS
OH
Progesterone (active)
Norethinodrel (contraceptive)
Pregnan-3a,20a-diol (inoct ivel (continued) FIG. 2A-D.
See legend on p. 9
AGONISTS AND PARTIAL AGONISTS
ANTAG ONISTS (svnthet ida
E. GIBBERELLINS
Gibberellin A3 (active)
m
Gibberellin A,2
Gibberellin As
(less active; precursor)
(inactive; degradation product 1
F. INVERTEBRATE HORMONES
Hop
OH OH
HO
I OH
HO
0 20-hydroxy Ecdysone (active)
I OH
HO
0
(p')
Ecdysone
('d)
(relatively inactive)
Fluorogibberellin A,2
H3c0m
H,CO
0 ‘H Juvenile Hormone I
Juvenile Hormone II
Juvenile Hormone
m
(active, naturally o c c u r r i n g )
/
o
0
u
“
0
Precocene 2 (natural product from Ageratum houstonianum; induces toxic effect typical of JH excess)
4
0
Synthetic analogs (with greatly enhanced JH -activities)
G. PROSTAGLANDINS O 0 -H
HO
“
‘&OOH
‘0H
HO
OH
PGE, PGFZ, (active; frequently counterpoised)
k
O
H
OH
HO
PGFzp (inactive) (continued) FIG. 2E-G.
See legend on p. 9.
eoo 9,11-Deoxy-7-oxa-prostanoic
acid
AGONISTS AND PARTIAL AGONISTS
ANTAGONISTS (synthetic)'
ti. PROSTACYCLINS
o=rcOO
JTcooH L
9
OH
OH
Prostocyclin PGI, (active)
OH
OH
6-Ketoprostacyclin (inactive)
t
Func t io no I ant o g on ist s
I . THROMBOXANES 0" -"-COOH OH Thromboxone A, (active)
'
*'-COOH OH
Thrombaxane 6, (inactive)
J. THYROID HORMONES I
I
ty2
I
I
I
I
HO ~ O ~ C H - C H , - - C O O H
O H C 0C H --C H 2Q -O -Q -H O -
1
L-3,5,3: 5'-Tetraiodothyronine; throxine;
T4
3.3'-Diiodothyropropionic acid
(ac t ivel N"2
H O b O & C H 2 - C H - C O O lI
I L-3,5,3'-Triiodothyronine; (more active)
T3
L-3,3@,5'-Triiodothyronine (reverse T3 (rT3); naturolly occurring; virtually inactive)
3' Isopropyl,-3,5 dibromothyronine (synthetic analog; more active than T), ~~~
~~
aUnless otherwise noted FIG. 2. Representative examples of agonists, partial agonists, and antagonists: relatively hydrophobic structures
10
CLARA M. SZEGO A N D RICHARD J . PIETRAS
genesis. Some selected examples of this well-known, but rarely integrated, set of observations are presented in Table I and elsewhere in the text. Inspection of Table I reveals that a wide array of “nonspecific” but by no means invariably noxious stimuli may lead in given cells to metabolic and morphologic events generally construed as anabolic and/or developmental. Likewise, it has long becn recognized that serum itself, added in vitrn to surviving cells or tissue explants, especially at times when the former have reached their growth plateau, whether or not confluency has also been achieved, possesses growth-supportive, if not -stirnulatory potential (cf. Eagle, 1965; Temin, 1971; Baker and Humphrey, I97 I ; Holley, 1975). Generally, the latter observations have been construed to mean that the activity of serum reflects its content of specific growth-promoting substances, whether polypeptide or of relatively low molecular weight (cf. Hayashi and Sato, 1976; Gospodarowicz and Moran, 1976), that, in many instances, can be correlated with the presence of certain hormones. In turn, the latter may, in judiciously chosen concentration for the given cell type, substitute altogether for the putative serum components (Bottenstein et al., 1979). On the otlicr hand, there are many investigators who view the contribution of serum and its derivatives as serving generalized nutrient, and thus relatively nonspecific, functions (cf. Rubin, 1975; Balk et al., 1981),especially since the raw serum may, often with impunity, be heat or acid treated (e.g., Fujiwara et al., 1980). Indeed, in at least one contact-inhibited cell line, simple alkalinization promoted the same quality and degree of biochemical responsiveness that was achieved with serum (Ceccarini and Eagle, 1971). Regardless of this sharp divergence in interpretation, it is instructive to note the parallels between the functions of serum in supporting proliferative activity and those of nonspecific stimuli (cf. Table I), for example, when criteria of amino acid incorporation into protein, or that of thymidine into DNA, are applied. But an additional parallel exists. Whether the resultant growth or differentiation is attributable to serum or to some form of nonspecific, not necessarily noxious, stimulus, the response has a further concomitant: induction of lysosoma1 enzymes and/or the organelles themselves (Rose, 1957; Cohn and Benson, 1965; Ahearn et al., 1966; Cohn and Fedorko, 1969; Gordon and Cohn, 1973; Reikvam et a l . , 1975; Wang and Touster, 1976). Such induction, in turn, may be referable to the initial surface phenomenon, upon which attention was focused above. Especially significant in this context is recent work which has demonstrated that intraperitoneal injection of isologous serum leads within 2 minutes to a 3-fold, and by 30 minutes, to a 7-fold increase in microvillar formation and surface microvesiculation of mesothelial cells of mouse omentum (Madison et al., 1979). Similar, but more gradual effects are elicited by as well characterized a protein as bovine serum albumin. Moreover, in quiescent neuroblastorna cells, the surface-perturbing effects of serum can be identified as a virtually immediate depolarization associated with a sharp decline in membrane resistance (Mool-
TABLE I BIOCHEMICAL A N D MORPHOLOGIC CONSEQUENCES OF SELECTED NONSPECIFIC STIMULI: THEBACKGROUND “NOISE” Stimulus
Consequence
Interpretation
Uterus in siru of ovariectomized rats
Estrogenicomimetic effects on acute blood flow, water imbibition at 4 hours
Ligature
Oviduct of diethylstilbestrolprimed chicks
Induction of avidin, highest in immediate region of ligature
Irritation by trauma or stretch, of locally instilled saline serves to attenuate the net influence of estrogen-released biogenic amines on biochemical and mitogenic evidences of uterine stimulation Membrane damage or histamine liberation believed excluded on basis of very limited attempts at blockade by cortisol, CaCI2, or promethazine ip (see, however, Szego, 1972b)
Stretch (10.8%, 18 hours)
Embryonic chicken skeletal myotubules in vitro
Without increase in cell volume, increased accumulation of [3H]AIB, [I4C]amino acids; increased incorporation of latter into general cellular proteins and myosin heavy chains; increased net protein and myosin heavy chains; increased DNA
A. Mechanical lntraluminally applied 0.154 M NaCI
Object
Reference
Szego and Sloan (1961)
Heinonen and Tuohimaa (1976, 1979)
Vandenburgh and Kaufman (1979)
(conrinued)
TABLE I (Continued) Stimulus Stretch (intermittent)
Endocytosis of I-pm latex beads
Object Rat diaphragm incubated in vitro
B-16 mouse melanoma adapted to in vitro cultivation
Consequence
Interpretation
Reference Reeds er al. (1980)
>2-fold increase in “synthesis” of noncollagen protein; increased glucose uptake and lactate output Augmented secretion of neutral proteinase and collagenase
Some indication that in-
Enhancement of axon fonnation (morphologic differentiation); increased cell and nuclear size (morphologic maturation); >lo-fold increase in acetylcholinesterase activity Translocation of unoccupied estrogen receptor to the nuclear fraction Reversible, ligand-independent redistribution of surface receptors for Ig, H2, and Thy-1.2 antigens, some for Con A
Some evidence of participation of microtubules in induction of differentiation, as judged by inhibitory effect of vinblastine
Prasad (1971); Prasad and Vemadakis (1972)
Mechanism undetermined
Cannon and Gorski (1976)
Association of microvilli with cap region suggested activation of underlying mechanoeffector systems
Yahara and Kakimoto-
creased mobilization of energetic resources may be involved Correlation of surface perturbation with “expression of [enzyme] potential.” Best indication in cell line “low in . . . basal proteinase activities”
Sauk and Witkop (1978)
e
N
B. Radiation and chemioeledric X-Irradiation
Mouse neuroblastoma cells in vitro
Hypertonic sucrose
Immature rat uteri in v i m
Hypertonic buffer
Murine lymphocytes and thymocytes
Sameshima (1977)
X-Irradiation (20,000 rad)
Contact-inhibited human glial cells
Electrical excitation; potassium depolarization
Squid giant axon
Diethyl ether; chloroform
Larvae of Trichosia pubescens
Methylene blue
Mouse peritoneal macrophages cultured in Medium 199 Rat liver microsomes
DMSO
~~
~
By 6 hours, augmented microvillar and endocytotic activity; conspicuous alterations in lysosomal structure, and somewhat later, in number; proliferation of Golgi Protein release to external medium
A large and several smaller puffs in the polytene chromosomes of the salivary glands within a few minutes after exposure; maximal at 60-100 minutes; intense incorporation of [3H]uridine (autoradiography), and accumulation of nonhistone proteins (acidic fast-green stain) in the puff region Induction of plasminogen activator secretion Phosphorylation of tyrosyl residues in a 170K protein corresponding to EGF receptor
Origin of autophagic vacuoles from preexisting lysosomes and/or “flattened vacuolar cytoplasmic elements”
Hamberg et al. (1977)
Solubilization of a particular group of proteins in close association with the membrane Increase in gene transcription at puff; potential ef.fect of lipid solvents on permeability of plasma membrane to inducing substances of unspecified nature that are, in turn, translocated to nucleusa
Pant el al. (1978)
“Electrical stimulation of the hexosemonophosphate pathway” Parallels to EGF actions in this and other respects noted
Schnyder and Baggiolini (1980)
Amabis and Janczur (1978); cf. also Wigglesworth (1957); Kroeger (1967)
Rubin and Earp (1983)
~
(continued)
TABLE I (Continues) Stimulus
Object
C. Relative anoxia; prolonged - incubation at 37°C Left ama Coronary artery occlusion
Prolonged (&hour) incubation at 37°C
Immature rat uteri
Incubation for 0.5 to 2 hours
Epithelium of infantile mouse
Several days of primary culture
Rat hepatocytes and hepatoma cells
Consequence
Interpretation
Increased numbers of [3H]thymidine-labeled nuclei and mitoses in left arrial muscle cells at 3-40 days after left ~ e n tricular infarction
“Reactive synthesis” following edema and exaggerated sarcoplasmic and nuclear basophilia. possibly secondary to “mitral [and associated biochemical] insufficiency” Incubation conditions mimic estrogen action and are “dependent upon protein synthesis“ and a permissive temperature
Rurnyantsev and Mirakjan (19681
Relative anoxia; estrogen uptake unaffected
Ljungkvist and Terenius ( 1970)
Profound changes in cytoskeletal composition during cell culture
Franke er 01. (1981a)
Progressive increase in RNA polymerase (RNA-P) activity; enhanced incorporation of [‘4C]glycine into protein. Both effects similar to those of specific estrogen stimulation; effects on RNA-P suppressed at 23°C or by cycloheximide treatmenth In absence of estrogen, mild to advanced autolyiic changes seen by TEM, but undetectable by light microscopy General induction of vimentin filaments and maintenance of production of cytokeratins
Reference
Nicolette et af. (1968); Nicolette (1 969)
“ S e e also comments by Berendes (1972) and Sin (1975) in context of “heat shock” puffs in salivary glands of Drosophih larvae. In the latter case, the puffs were induced by extracts of “mitochondria” prepared after heat shock exposure However. such preparations have long been recognized for their contamination with lysosomes (cf. Vignais and Nachbaur, 1968). bSee Table XXllI and effects of antibiotics on lysosomal structure and function (textl.
LYSOSOMAL FUNCTIONS IN CELLULAR ACTIVATION
15
enaar et a l . , 1979). These phenomena, suggestive of a transient increase in Na+ conductance, were attributed to putative growth-promoting factors in serum because of the minimal electrophysiological response to the addition of depleted media. Thus, surface membrane events associated with the application of serum and/or given proteins appear primary, and clearly precede the biochemical and morphologic consequences. Indeed, as will be documented below, it appears possible to trace a chain of interlocking events which leads in due course to the latter outcome. Accordingly, this background noise, which can, under some circumstances, overwhelm signal, must be kept in mind throughout our attempts to analyze the potential role of lysosomes, through limited recompartmentation of their specialized components, in the amplification and propagation of the initial effects of the primary trigger that destablizes, and thus activates, given cells.
B. OBJECTIVES AND SCOPE On the basis of these considerations, we shall restrict our analysis of the mechanisms by which the activities of endogenous signals in the form of hormones and certain neurotransmitters are intercepted from the extracellular environment, transduced, progressively propagated, and interpreted in the language of that cell capable of perceiving their presence: i.e., having a given number of recognition sites with the appropriate topology on its surface available at the moment, together with a means of coupling the amplified information derived from the ligand-receptor complex to more remote cellular events. On occasion, we shall consider similar circumstances in relation to exogenous effectors, such as drugs, selected carcinogens, and certain regulatory substances that do not fall into the category of hormones. Some instructive parallels appear to emerge from such comparisons, as will be documented below. On the basis of a growing body of evidence, we have proposed that events set in motion by interaction of surface membrane of target cell with specific ligand are associated with regional endocytosis and site-specific modification of lysosoma1 structure and function and are intimately related to the molecular means by which coordinated cell growth and differentiation are achieved in response to tropic hormones (Szego, 1971a,b, 1974, 1975, 1978; Szego et a l . , 1971; Szego and Pietras, 1981). Taking into account the combined properties of lysosomes on the one hand, and the characteristic pleiotropic actions of hormones on the other hand, it has been suggested that primary lysosomes function in selective uptake of the agonist and, in their secondary, covertly labilized form, in its transcytopiasmic migration and in its introduction into the nucleus of the hormoneactivated cell, accompanied by “transformed” and/or diminished receptor and very limited amounts ( ‘‘microquanta’’) of lysosomal constituents. It will be one purpose of the present account to evaluate aspects of this
16
CLARA M. SZEGO AND RICHARD J. PIETRAS
proposal in light of numerous more recent findings. We hope to identify, as far as possible, the individual steps in the staging of such a vectorial pathway and to assess’their potential metabolic consequences in the processing and execution of information delivered by agonist. A further aim of this essay is to determine, from analysis of as wide an array of effectors and target cells as possible, whether such a pattern is generalized or unique to only certain classes of effectors or target cells. Finally, from these and independent data, implications of lysosoma1 function in propagation and coordination of transcellular events will be considered. It is, of course, recognized that in presenting an apparently sequential array of metabolic events one must guard against the bias inherent in the various sensitivities of the several analytical methods themselves. Of even greater concern is the danger of confusing concornirancy with causality, a pitfall that we hope to avoid. Taking these risks advisedly, we hope that identification of serious gaps in understanding, leading, potentially, to stimulation of more definitive work, will compensate for the inevitable prematurity of an integrative effort applied to currently unfolding data. It is hoped that integration of information to be derived from analysis of these interlocking problems will contribute to our growing, but still very incomplete, understanding of the critical molecular events associated with triggering of cellular responses to tropic hormones and other effectors.
11. Perspective
Having put the cart before the horse in presenting some of the complexities of the subject that have prevented facile integration of the many, apparently unrelated, observations into a coherent whole (cf. Szego, 1982), we now address directly the problems in accounting for hormone action that have necessitated the fresh outlook and the change in emphasis that led to the present hypothesis. A. MECHANISMS OF HORMONE ACTION 1. Evolution of Ideas What Is Limiting.? As with any other developmental phase of functional biochemistry, ideas on the primary events in hormone action have generally reflected the prevailing concepts of the time. Attempts to understand hormoneldrug action in physical terms were at first rather sporadic. Efforts of early cell physiologists centered upon modulation of the cell surface and its role in controlling exchanges with the extracellular environment (cf. Meyer, 1899; Overton, 1901; Traube, 1904; Ponder, 1933). This semiquantitative outlook was soon eclipsed by the more stringent formulations of the enzymologists, who, influenced in part by the “p-hypothesis” of Crozier (1926), which formally
LYSOSOMAL FUNCTIONS IN CELLULAR ACTIVATION
17
advanced the proposal that the slowest of an integrated series of presumed enzymic reactions was the overall pacemaker, emphasized potential inhibitory functions of agonists upon the activities of prevailing enzymes to the exclusion of “positive” controls. The latter represented the “unthinkable” in the tight logic of the times (cf. Green, 1941). In wake of the exponential advance of Lipmann (1941) on energy-yielding mechanisms, this limiting view was to give way to the concept of hormonal participation in metabolic reactions as coenzyme, subject to reversible oxidationireduction (cf. Villee and Hagerman, 1953; Ball and Cooper, 1957; Langer and Engel, 1958; Talalay and Williams-Ashman, 1958; and McKerns and Bell, 1960). When, in turn, this phase yielded to the electrifying concept of the operon (Jacob and Monod, 1961), the latter was, virtually immediately, transposed into the hormonal mode: “the” limiting factor was the means of delivering fresh instructions to protein-synthesizing machinery, i.e., transcription of template and/or other forms of RNA (Karlson, 1963; Edelman et af., 1963; Wilson, 1963; Talwar and Segal, 1963; Noteboom and Gorski, 1963; and Ui and Mueller, 1963). When, some time later, it became evident that neither of the latter two views, themselves not mutually exclusive, was wholly adequate to explain all facets of hormone action, and, indeed, when evidence began to accumulate that indicated far greater complexities than had previously been envisioned,’ it became clear that much deeper analysis was required. Somewhere in the course of these developments, there arose the generalization that the steroid and peptide hormones functioned by independent and mutually exclusive mechanisms (Table 11; see Szego, 1978, for review). This dichotomous view, which no longer appears tenable (Szego, 1974, 1975, 1976, 1978; Szego and Pietras, 1981), was, in fact, based upon inadequate premises: that steroid hormones, being fat-soluble, were not appreciably hindered by the lipid bilayer of the plasmalemma. Instead, they readily and indiscriminately entered all cells but were retained, on encountering “cytoplasmic” receptor, exclusively within “target” cells, thereupon to be transferred, by an unknown mechanism, into the nucleus. In contrast, the peptide hormones, because they were thought to be restrained from crossing the plasmalemma by mass and charge, required participation of secondary messenger(s) for propagation of the effects of their initial recognition at the cell surface. As part and parcel of such divergence in mode of operation of steroid and peptide agonists, it was widely and confidently believed (despite cogent evidence to the contrary), that the action of steroid hormones at their cellular targets was, unlike that of their peptidal counterparts, unaccompanied by abrupt alterations in cyclic nucleotide and/or ionic gradients or other indications of membrane perturbation. ‘In part, this impasse was due to the unrestrained enthusiasm with which data obtained in prokaryotes were applied to eukaryotic organisms, often with the added complications arising from the effects of the highly toxic antibiotics used as “specific” inhibitors of protein and RNA synthesis (see Section IV,A).
18
CLARA M. SZEGO AND RICHARD J . PIETRAS
TABLE 11 POSTULAltU DICHOTOMY IN
M o D t b 01.ACTION . OF STEROID AND PtPTlDF
Parameter
Peptidesb
Location of receptor Ccllular entry
Outer plasmalcnima
Nucleotide cyclase activation Nuclear actions
Yes Indirect
No'
HORMON~S<'
Steroidsc Cytosold Freely enter all cells; retained only by "target" ccllsf Non Direct
"Cifutions are m references in Szego (1978), from which this tablc is reprinted, with permission. hBased on reviews of Hechter and Soifer (1971), Pastan (1972), Cuatrccasas KI ul. (1975), Kahn (1975, 1976), and Catt and Dufau (1976). cBased on reviews of Jensen and DeSombre (1Y72), King and Maiawaring (1974). Gorski and Gannon (1976). Buller and O'Mallcy (1976), and O'Malley and Buller (1976). %ee, however, Szego (1974, 1975). Hirsch and Szego (1974). and Pietras and Szego (1975a,b, 1977a). eSee, however, Szego (1974, 1975) and Table IV in Szego (1978). 'See, however, Pietras and Szego (1977a,c, 1978). ~ S c chowever. , Szego and Davis (1967). Szego [1972a,b, 1974 (Discussion, pp. 228ff.), 19751, and Table VII in Szego and Pietras (1981). %c, however, Table IV in Szego (1978) and the discussion in Section 7 thereof.
2 . Present State-ofthe-Art a. Steroid Hormones. We now know that our "blind guides which strain at a gnat and swallow a camel" have misled us: at the very least, the subject is now in tlux. For, contrary to widespread belief, steroid hormones do indeed appear to be recognized, as are other agonists, at the surface membranes selectively of those cells that are thus inherently equipped to respond. Moreover, in course of such recognition, a battery of signals of membrane perturbation is elicited, including, but not limited to (1) abrupt and transitory cyclic nucleotide generation; ( 2 ) alterations in N a + ,K -ATPase and 5'-nucleotidase activities; (3) fluxes in Ca2+ and other ions, with implications for modulation of neural activities and numerous enzymatic and mechanoeffector systems; (4) release of endogenous amines from sequestration, with implications for modulation of the microcirculation in intact organs; ( 5 ) promotion of lectin-mediated hemagglutination; and (6) enhanced aggregation of like cells, and accentuated delivery of enzymes and other components of lysosomes to the cell surface, with potential for remodeling of the latter, as well as diffusion to adjoining cells (see Fig. 3; and Szego and Pietras, 198I , for comprehensive review). It has been inevitable that, as noted above, principal emphasis during the preceding two decades in analysis of steroid hormone action has been upon the molecular mechanisms of transcriptional regulation at the genome. It has not been unattended by faulty interpretations through the application of flawed meth+
LYSOSOMAL FUNCTIONS IN CELLULAR ACTIVATION
19
odology or oversimplification. Nevertheless, this intensive attack upon a problem of such formidable complexity has yielded brilliant achievements (O'Malley et al., 1979; Royal et al., 1979; Baxter et al., 1979; Tata and Smith, 1979), and holds promise of many additional advances. In addition, there is compelling evidence that molecular processes associated with maturation of the primary 30 -
10
-
5-
CELL DIVISION HISTONE AND DNA SYNTHESIS NET PROTEIN SYNTHESIS INCREASED ACTIVITY OF MANY ENZYMES NET RNA INCREASE INCREASED DNA TEMPLATE ACTIVITY PROTEIN LABELING FROM ISOTOPIC AMINO ACIDS NET UPTAKE No', H 2 0 CYCLIC GMP ELEVATION
1 -
0.5-
0.050.1
0.01
-
0.005
LIPID LABELING FROM ISOTOPIC PRECURSORS N E T UPTAKE ALBUMIN AUGMENTED GLUCOSE METABOLISM NET UPTAKE UREA, MONOSACCHARIDES, AMINO ACIDS INCREASE IN RELATIVE AMINO ACID INCORP'N OF PROTEINS WITH RAPID tV2 (e.g., CREATINE KINASE)
i
NET UPTAKE Co2+ ENHANCED RNA POLYMERASE ACTIVITY ENHANCED INTERCELLULAR ADHESION CALCIUM INFLUX REDISTRIBUTION OF CON A-BINDING SITES AT SURFACE RNA LABELING FROM ISOTOPE PRECURSORS ESTROGEN BINDING IN NUCLEUS NUCLEAR TRANSLOCATION OF LYSOSOME-LIKE VESICLES AND ACID HYDROLASES ENHANCED AVAILABILITY OF CATHEPSIN B, ACID PHOSPHATASE, ACID RIBONUCLEASE AND OTHER LYSOSOMAL ENZYMES MICROPINOCYTOTIC VESICULATION OF PLASMA MEMBRANE INCREASE IN NUMBER AND LENGTH OF MICROVILLI HYPEREMIA; INCREASED AMINO ACID, GLUCOSE, NUCLEOSIDE TRANSPORT CYCLIC AMP ELEVATION CALCIUM EFFLUX HISTAMINE RELEASE
I
ESTROGEN BINDING IN CYTOSOL EXTRACTS
0.001
ESTROGEN BINDING IN PLASMA MEMBRANE
FIG. Schematic representation of the time course of responses c uterus to estra Times shown on the logarithmic scale refer to onset of unequivocal change from baseline values in the ovariectomized or immature rat after intravenous administration of, or in vitro exposure to, the hormone. Thus, times indicated are dependent in part upon sensitivities of the various analytic methods applied and also upon the somewhat arbitrary selection of initial time-points for observation in the various experimental protocols. Data were compiled from Szego (1965, 1973, Singhal(1973), Katzenellenbogen et a / . (1979), Szego and Pietras (1981), and Rambo and Szego (1982, 1983).
20
CLARA M. SZEGO AND RICHARD J . PIETRAS
RNA transcript (cf. Darnell, 1979; Royal et al., 1979; Nordstrom et al., 1979; Abelson, 1979; Reiners and Busch, 1980), with its egress from the nuclear compartment (cf. Moore and Hamilton, 1964), and with its translation at the ribosomal level (cf. Cox, 1978; Pennequin et al., 1978; Gehrke et al., 1981a,b) are all subject to modulation by activities attributable, in the final analysis, to the primary metabolic event: hormonal capture at the cellular target. b. Peptide Hormones. Notwithstanding the meager evidence available until very recently for cellular entry of peptide hormones and other macromolecular coordinating substances (cf. Szego, 1974, 1975, 1978), there is now an avalanche of data in support of interiorization of relatively large molecules hitherto believed (cf. de Duve, 1981) to be excluded from the intracellular environment. Selected examples of this general phenomenon are presented in Table 111. Whether entry per se is of significance in the mechanism of action of such effectors is still under (heated) discussion. This problem will be addressed in detail below. An “exploded” view of the apparent time-course of the metabolic events attendant upon the action of thyroid stimulating hormone (TSH) is given in Table IV. Even a cursory inspection of the data presented in Table 1V for a “typical” peptide hormone reveals a striking correspondence to the observations delineating the presumptive sequence of metabolic events at and in its respective target cells for a “typical” steroid hormone, estrogen (cf. Fig. 3). For, in both sets of cases, one can discern the primary phenomenon: recognition and capture of the ligand at the cell surface, associated with unmistakable signs of membrane perturbation secondary to receptor occupancy. There follows, in apparently cascading fashion, a series of metabolic indications of increased substrate availability and utilization that may lay the material and energetic bases for the anabolic events to come. Virtually concomitantly, there is transmitted to the nuclear compartment information appropriate to the activated cellular state-as judged in part by secondary indications of enhanced genic transcription. Whether, in the case of a typical peptide hormone, such information transfer is associated with nuclear entry of hormone-receptor complex per se, as is unequivocal in the case of all steroid hormones thus far examined, is presently uncertain. Nevertheless, the more remote indications of enhanced transcription culminating in increased net RNA content and the signs of augmented translational activity leading to net synthesis of characteristic protein(s), are likewise evident as further consequences of hormonal stimulation, whether by peptide or steroid. And the ultimate endpoint in both cases is the same: growth and/or developmentmaturation. c. Parallels to Other Mitogens. Although the precise metabolic sequence following application of assorted mitogens such as lectins is perhaps less clearly delineated, there is nevertheless a striking degree of parallelism in the overall pattern, a set of observations to which attention was drawn some time ago
TABLE 111 REPRESENTATIVE EXAMPLES OF RECEPTOR-MEDIATED CELLULAR ENTRYOF NONSTEROID HORMONES A N D OTHER MACROMOl.t:CuLES WITH POTENTIAL INTRACELLULAR FUNCTION"." Ligand [marker] ACTH F'ITC-linked, affinitypurified antiserum to ACTH 1 1-24
Unlabeled hormone
Anti-ACTH 1-24
[1251]-; [FITCI[Ferl-
Target cell(s)
Criteria
Reference
Comment
Hypothalamic cells and axons of normal and hypox rats
ICC localization of endogenous presumptive hormone in formalin-fixed, cryostat sections
Watson er al. (1978)
Cultured xanthophores of goldfish, Carassius aureatus L. Rat adrenocortical sections
SEM; TEM, the latter with aid of HRP
Lo er al. (1979)
ICC
Nolin (1980a)
Reticularis and fasciculata, cytoplasm positive; glomerulosa, also in nuclei and on nuclear membrane
Cultured human fibroblasts
Autoradiography (TEM)
Evidence for lysosomal association
Human epitheloid-carcinoma cells (A-431) As above
Autoradiography; fluorescence microscopy TEM
Carpenter and Cohen (1976)"; Gordon et a l . (1978b)" Haigler et al. (1978)" Haigler et al. (1979a,b)
Specific binding sites; confirmation of progressive uptake into MVB
Blockade by ACTH 1-24: no cross-reactivity with pMSH, @endorphin, plipotropin, met- or leu-enkephalin PM internalization via pits and endocytotic vesicles
(continued)
TABLE 111 (Continued) Ligand [marker]
Target cell(s)
Cnteria
Reference
Comment
Epinephrine
W-
Tetrahymem pyriformis
LM; TEM
Csaba er a / . (1980)
Rat hepatocytes
Quantitative autoradiography (TEM)
Barazzone ef a/.(1980a)’
Only limited portion of total binding was nonspecific; < 1% of grains at PM were with coated regions; progressive increases with time in number of grains in deeper cellular sites with preferential coincidence at lysosomes
Granulosa and lutein cells of ovine corpora lutea in vivo; in vitro
Autoradiography (LM, TEM); ICC (LM)
Chen et a / . (1977)
Progressive lysosomal association striking; emphasis on degradation of hormone
Anti-hCG
Fetal rat testis
Childs
Anti-hCGc; anti+subunit of hCG; anti-rat LH
Rat ovaries
ICC localization at PM and at numerous intracellular sites including lysosomal. Goigi, and nuclear ICC (LM): gonadotropin-like immunoreactivity at PM. over granular structures in cytoplasm, and in penand intranuclear sites
Glucagon p511-
13
Gonadotropins [‘251]hCG~-LH
t-f
al. (1978)
Petrusz and Sar (1978): Petrusz (1978)
Model proposes a class of “regulatory lysosomes” that process H and!or R to forms capable of influencing further intracellular functions of the H:R complex
Rat granulosa cell cultures; observations initiated at 2 hours after exposure
Autoradiography (TEM)
Amsterdam et a / . (1 979)
Clusters of grains on PM; progressive accumulation over lysosomes on sustained exposure; appearance of TCAprecipitable and -soluble lZ5I in medium
[ I2SIl.d
Single proximal tubules of rat kidney
Microperfusion and TEM autoradiography
Stacy et a / . (1976)e.”
[ 125]]-/
Cultured human lymphocytes (IM-9)
In virro incubation; quantitative TEM autoradiography; gel exclusion chromatography of internalized and extracellular radioactivity; evidence of specific and nonspecific binding
Barazzone er a / . (1980b)”
Sequential transfer of labeled material from lumen to microvilli, to apical vesicles, to Iysosomes observed with quantitative methods over times ranging from 1 to 60 minutes. Evidence of degradation by 10 minutes Extends and confirms above. Nevertheless, 58-73% of cell-associated radioactivity retained biochemical properties of intact GH after 15-120 minutes of incubation at 37°C
[‘25I]hCG
Growth hormone
w N
(continued)
TABLE 111 (Continued) Ligand [marker] Immunoglobulins [FITCI-X, [125I]-
Target cell(s)
Criteria
Reference
Comment
Ag internalized simultaneously; both Ag and Ab comigrated with lysosomal hydrolases in Percoll gradients Accumulation in GERL vesicles
Hepatoma tissue culture cells
Fluorescence (LM); Percoll gradient analysis
Baumann and Doyle (1980)w; see also Lewis et al. (1974)“; Unanue et al. (1972)w
Cultured lymphocytes and plasma cells from rodent lymph nodes
LM cytochemistry; TEM
Antoine et nl. (1974)
[ IZSI].
Rat hepatocytes; human lymphocytes (Ih4-9)
Gorden er al. (1978a)Y; Carpentier et al. (1978)“
[Rho]-
3T3 fibroblasts
(12511-
Rat hepatocytes; infused liver in vivo
TEM autoradiography; progressive shift toward cellular interior from initial surface orientation Direct visualization by video-intensified fluorescence microscopy in living cells of binding, surface aggregation and intemalization More quantitative analysis by methods initiated in 1978
[HRPI-, [1251]-anti-Ig
Insulin
2
Schlessinger et al. (1978)
Carpentier er al. ( 1 9 7 9 a - ~ ) ~
“No [evidence for] preferential localization [of internalized insulin] to any intracellular organelle” Mobility of fluorescent derivatives bound to presumptive receptor within membrane; saltatory m e tion in intraellular vesicles* Despite previous disclaimers (and 1979a), demonstration of internalized radioactivity in lysosomal (1979a-c) and, possibly (1979b), Golgi (19794 foci
TEM autoradiography
Bergeron er al. (1979)”; Posner et al. (1981a)’; cf. also Schilling et al. (1979)”
Perfused rat liver
SDG centrifugation of subcellular fractions
Desbuquois et al. (1979)”; Ward and Mortimore ( 1980)”
Murine 3T3 cells
TEM
Nicolson (1974)
[FITCI-, [HRPI-Con A
Cultured murine macrophages
TEM; biochemical
Edelson and Cohn (1974)
Con A
Cultured murine macrophages
TEM; biochemical
Goldman and Raz (1975)
[HRPI-PHA; -Rich
Cultured murine drg neurons
TEM cytochemistry
Gonatas et al. (1977); cf. also Nicolson el al. (1976)
[HRPI-WGA
Submandibular glands and scg of rat in vivo
TEM cytochemistry
Harper et al. (1980)
Lectins [Ferl-Ricin
Distinct concentration of grains over Golgi elements in specific fashion and over “lysosome-like vacuoles,” which did not exhibit AcPase activity
Specific accumulation in endocytotic vesicles Increase in pinosome formation but impaired fusion with lysosomes; see, however, Goldman and Raz (1975) Con A-induced vesicles AcPase-positive; treatment led to increase in total enzyme activity of the cells Lectin:HRF?(presumptive) R complexes localized in elernents of GERL’ Lysosomes and cisternae of GERL; HRP-control in lysosomes only (continued)
TABLE I11 (Conrinued) ~
~
~
~~~~
Ligand [marker]
~
Target cell(s)
Cntena
Reference
[ 1251]WGA
Optic tech cells of chicks injected intravitreally
LM autoradiography
Margohs ef a1 (1981)b
[Ferl-WFA; [FITC]., [Rho]-2nd Ab to WFA
Cultured BALB/c 3T3 fibroblasts
Immunofluorescence (LM); TEM
St. John et al. (1980)
Cultured rat pituitary cells
TEM
Hopkins and Gregory (1977)
Cultured rat pituitary cells
Fluorescence microscopy
Hazurn et al. (1980)
Human fibroblasts
TEM
Anderson ef al. (1977)
LHRH [Fa]-LHRH analog
[Rho]-LHRH analog
Lipoproteins [ W ] - : [Ferl-LDLJ
Comment Evidence of receptor-rnediated uptake and anterograde transport of lectin from retinal ganglion cells to axon endings in the optic tectum of the contralateral eye only, much of the lectin found to have escaped lysosomal degradation Localization of label in lysosome-like organelles
Capping over Golgi pole; generally in gonadotropes; disappearance from surface both by sloughing and endocytosis Entry in vesicular form much diminished in absence of Ca2+
Adsorptive endocytosis; progressive concentration of label over lysosomesk
[ Iz5I]LDL’
a2-Macroglobulin ( a 2 M ) a2M.[ IZ5IJTrypsin complexes [Ferl-a,M;
[HRPJ-aZM-Ab [‘*’I]a2M; a2Mcolloidal Au; [Rho J-cx~M
[‘2JI]PNGF [Rho]-
Cultured murine adrenal cells (Y-I)
Biochemical
Faust et al. (1977)
Cultured rat fibroblasts and smooth muscle cells
Biochemical
lnnerarity et al. ( I 980)
Cultured rabbit alveolar macrophages
Biochemical: (‘251 distribution in cell fractions)
Kaplan and Nielsen (1979)”
Cultured Swiss 3T3 fibroblasts; normal rat kidney (NRK-2T) cells
TEM
Willingham et al. (1979); Dickson er af. (1981a.b)“; Maxfield et al. (1981b)
Chick dorsal root ganglia
Biochemical: l2S1 distribution in isolated particulate preparations
Andres ef af. (1977P
Cultured rat pheochromocytoma (PC 12) Cultured embryonal chick sensory and PC12 cells
Emphasis on hydrolysis of LDL in lysosomes to yield cholesterol as a limiting step in steroid biosynthesis Indications of requirement of homology for proper recognition
Intracellular 1251 cosedimented with a lysosomal marker enzyme in SDG Diffuse surface binding followed by clustering in coated regions and vesicular internalization
Specific accumulation in purified nuclei and their membrane fractions
Yankner and Shooter (1979) FPR; video-intensified fluorescence microscopy
Levi et a/. (1980)
Internalized NGF postulated to govern “delayed” effects (continued)
TABLE I11 (Coniinued) Ligand [marker] Parathyroid Hormone ['251]bFTH (1-34)
Polynucleotidesfl.o ['T]tRNA from 15.
coli
3H- or DAPI-labeled DNA:CaPi (HeLa cells) Homologous deacylated 13H]tRNA Daunorubicin:DN A; adriamycin:DNA
Target cell(s)
Criteria
Reference
Comment
Segments of frontal bone from embryonic chick calvaria
TEM autoradiography
Silve ei a/. (1982)
Internalization, especially in osteoblasts, evident, despite concentration of grains primarily at cell surfaces
Murine LIZ10 leukemia cells; human lymphoblastic cells (NC-37); PHAstimulated human peripheral lymphocytes
Cell fractionation and autoradiography (EM); identification of acylated and methylated derivatives
Herrera ei al. (1970); cf. also, Niu ei al. (1968)
Intravenous or intraaortic administration to rats bearing Walker mammary carcinoma Murine Ltk - -Aprt -
Biochemical: [3H]DNA isolation from nuclei of tumors
Watters and Gullino (1971)
Biochemical and fluorescence
Loyter er al. (1982)
L1210 leukemia cells
Biochemical
Gallagher ei al. (1972)
L1210 leukemia cells inoculated into DBAz mice
Loss of fluorescence, dialyzability, and antibiotic propenies of the complex; regained after (intra)lysosomal digestion of DNA
Tmuet et al. (1972, 1974)
from exogenous tRNA present in nuclei, lysosomes, and mitochondria, as well as cytosol; 20% of the polynucleotides recovered functionally intact after 30-minute incubation Intranuclear penetration of up to 0.55'1% of tumor DNA after several days of infusion Extensive cellular entry, but only limited intranuclear penetration Uptake and aminoacylation of exogenous tRNA High endocytotic activity of tumor cells postulated to provide for preferential uptake of the complex; primary reference(s) on the concept of lysosomotropic cancer chemotherapy
Poly(A)-polY(U) (double-stranded complex), synthesized from 3H- or I4C-labeled nucleoside diphosphates [3H]Uridine-RNAq
[’Hl-U-p~ly(A) RNA‘; poly(A) + mRNA (globin) from rabbit reticulocytes; mRNA for myotype creatine kinase
Prolactin (PRL) [HRPI-rabbit Ab-S
Murine ascites tumor cells and human lymphocytes
Autoradiography (LM)
Fenster et al. (1975)
Substantial proportion of the incorporated complex rapidly reaches nuclei and survives “intact” for > 2 hours
Lymphocytes coincubated 1 hour with labeled macrophages Primary chick myoblast cultures
Autoradiography (TEM)
Jonas et al. (1976)
Kinetics of incorporation of exogenous mRNA into polysomes; faithful translation of is0 and heterologous poly(A) -mRNA
Mroczkowski et al. (1980)
Transfer small, except from antigen-stimulated macrophages Unambiguous study of translational controls during development sheds incidental light on entry of exogenously supplied mRNA into cultured cells and its tissue-specificr translation; cf. also Segal et al. (1965)
ICC (LM)
Nolin and Witorsch (1976)
+
Alveolar cells of lactating rat mammary glands‘
Specific staining of apical regions of epithelial cells (remote from blood supply), strongly indicating prior entry of endogenous hormone (continued)
Ligand [marker]
Target cell(s)
Criteria
Reference
[HRPI-rabbit Ab->
Rat sex accessory organs
ICC (LM)
Witorsch and Smith (1977): Witorsch ( I 978)
[ ll'I]oPRL
Rat liver
Biochemical: cell fractionation
losefsberg er a!. (1979)'
Rabbit adipocytes
Uptake (K l o l l M - I ) and binding studies of isolated cells and components
Par1 er al. (1977)'
[Rho]-
Cultured 3T3-4 murine fibroblasts
Video-intensified fluorescence microcopy
Cheng et a/. (1980);cf. also Maxfield er al. (1981a)
['"'IIT,
Rat liver parenchymal cells and their PM vesicles
Rapid centrifugation technique
Rao et a!. (1981)''
Comment Androgen dependency of immunospecific staining. conspicuously at Golgi in ventral prostate and seminal vesicle; staining seen throughout cytoplasm in epididymis and vas deferens; spermatozoa negative Labeled hormone (retaining full rebinding-integrity to fresh membrane preparations) strongly concentrated in Golgi fractions
Uptake of T3,T4 by lipid components 2-5 X that of intact cells, indicating passive diffusion unlikely Surface association, clustering, and vesicular entry of these low MW, relatively hydrophobic hormones paralleled observations by same group on a*M, insulin Evidence for accumulation of hormone against a gradient
Toxins (bacterial) [HRPI-cholera
W
Cultured murine new roblastoma cells
TEM
Joseph et al. (1978. 1979)
Diphtheria
Chinese hamster lung (V79): African green monkey: kidney (VERO) cells
Draper and Simon (1980); Sandvig and Olsnes (1982)
[Rho]-
Murine 3T3 and human W138 fibroblasts in culture
Biochemical: influence of lysosomotropic drugs on metabolic pathways deranged by the toxin As for insulin. above (Schlessinger et al., 1978)
[1251]-, [FITCI-Ab-Tetanus
Cultured rat cerebral brain cells
Biochemical: influence of unlabeled ligand, tetanus antitoxin, and gangliosides on kinetics of cell association of the toxin
Yavin et al. (1981)
Epithelial cells of opened follicles rat and pig thyroid
TEM
Herzog and Miller (1979)
TSH [Cationized Ferl-latex spheres
Keen et al. (1982)
Binding and internalization; predominant association with GERL Evidence for a lysosome-mediated step in the toxicity of endocytosed toxin Vesicular endocytosis similar in both sensitive (human) and insensitive (murine) cells Confirms gangliosidal nature of receptor(s) and speculates on their role as “shuttle” vehicles
TSH-stimulated vesicular endocytosis, preferentially at coated pits, accompanied by lysosomal uptake by 15 minutes; some also at Golgi (continued)
TABLE 111 (Continued) Ligand [marker] Virus Sindbis; vesicular stomatitis
Target cell(s)
Criteria
Reference
Chick cells; MDBK cells
Lack of sensitization of host cells to lysis by Ab + complement
Fan and Sefton (1978)
BHK-21 cells
Biochemical: suppression of productive infection by lysosomotropic arnines ICC at TEM level
Talbot and Vance (1980)
Herpes simplex
Rabbit corneal cells
S e d i k i Forest (SFV) [Rho]-, IFITCI-,
BHK-21 cells
Fluorescence microscopy; E M ; biochemical: infectivity dependent upon low PH
Helenius et 01. (1980a,bp
MDCK cells
Biochemical and morphologic (ICC)
Matlin et al. (1981)
Hansen et al. (1979)
W
N
[35S]-
Fowl plague 13-%]-; also [Fer-20 Ab]-
Comment Adsorptive endocytosis implicated (cf. Dales, 1973; Tardieu et al., 1982) by apparent lack of membrane-fusion mediated entry (as in the case of Sendai ViNS) Data support a lysosomal route of cytoplasmic entry and infectivity Findings, comprising timecourse of subcellular distribution of viral antigen, including into nucleus, support biochemical observations (see refs.) Adsorptive endocytosis into coated pits and vesicles, followed by fusion with (lysosomal) vesicles at low pH As above
also Szego (1974, 1975); Table IV in Szego (1978); as well as Neville and Chang (1978); Petrusz (1978); Gorden eral. (1980a); Goldfine (1981a.b); Pastan and Willingham (1981); King and Cuatrecasas (1981); and Middlebrwk and Kohn (1981). Although persuasive collectively, the data shown in this summary (as well as in a number of additional tables and figures to follow) possess certain inherent limitations. (1) Liberation of isotopic label from agonist so marked may occur with variable degrees of efficiency on exposure to cellular components at surface or intracellularly. (2) Products of limited or more extensive proteoiysis may
or may not retain activity intrinsic to the native material. (3) Confidence in localization of marker at the EM level requires careful statistical analysis, ideally on observations in serial sections. (4)Immunocytochemical criteria, even with rigorous controls, could be recognizing an unspecified fragment of the native material. (5) Resolution of some of the cited procedures is not yet well advanced. (6) Cellular architecture is occasionally inadequately preserved, through faulty fixation (cf. Novikoff, 1980), a problem that gives rise to inappropriate conclusions on significance of subcellular-marker localization. (7) Finally, the relative contributions of nonspecific vs. specific interactions leading to internalization of bound ligand are only rarely assessed, especially as these are further superimposed on the background “noise” (see Table I). bFITC, Fluorescein isotbiocyanate; Hypox, hypophysectomized; SEM, scanning electron microscopy; TEM, transmission electron microscopy; LM, light microscopy; HRP, horseradish peroxidase; PM, plasmalemma; hCG, human chorionic gonadotropin; ICC, immunocytochemistry; H , hormone; R, receptor; LH, luteinizing hormone; MSH, melanocyte stimulating hormone; MVB, multivesicular body; Ab, antibody; Ag, antigen; AcPase, acid phosphatase; PHA, phytohemagglutinin; drg, dorsal root ganglion; scg, superior cervical ganglion; Con A, concanavalin A; WGA, wheat germ agglutinin; WFA, Wistariafloribunda agglutinin; LDL, low-density lipoprotein; VLDL, very-low-density lipoprotein; LHRH, luteinizing hormone-releasing hormone; bPTH (1-34), bovine parathyroid hormone, active segment comprising residues 1-34;GERL, Golgi-endoplasmic reticulum-lysosomal system; FPR, fluorescence photobleaching and recovery; 0, ovine; T,, 3,5,3’-triiodo-~-thyronine; T,. 3,5.3’,5’-tetraiodo-~-thyronine (thyroxin): MDBK cells, Maden-Darby bovine kidney cells; Fer, fernitin; Rho, rhodamine ?Preparations of anti-hCG used were known to cross-react with rat gonadotropins. mainly LH. dSheep. eThis early paper on peptide hormone internalization, using sophisticated ultrastructurallautoradiographiccriteria, was generally discounted because of the presumptive disposal/degradation implications (cf. Table I1 in Szego, 1978). muman. RAb directed against surface membrane glycoproteins hSee Table VIII. However, problems related to vesicular internalization of free rhodamine (cf. Drucker et al., 1982) may render these and similar observations less than unequivocal. ‘See Fig. 6. /Human. LData presented to demonstrate recognition and internalization of free Fer at coated-PM region [cf. Fawcett (1965); Lagunoff and Curran (1972)] occurred at loci independent of presumptive receptors for [Ferl-LDL. ‘Human and murine sources. ‘“Human and rat sources “No attempt is made here to integrate the exponentially growing information on incorporation of foreign DNA into the genome of the recipient.
(continued)
TABLE I11 (Continued) -
"The far-sighted papers and review of Ledoux (see 1965) recognized the likelihood of intracellular introduction of these highly charged macromolecules by processes such as "pinocytosis." However, specific recognition sites were not envisioned, nor have such been identified, as yet. This family of macromolecules is here included primarily for heuristic reasons (cf. Szego, 3975). Comprehensive reviews of the cellular uptake and fate of polynucleotides are now available (e.g., Stebbing 1979). pFrom lactating mammary glands of isogenic rats or from Micrococcus Iysosodeikticus YPresent in autologous macrophages of mice. rPoly(A) mRNA also taken up but not associated with polysomes and thus: untmnslated; mRNA for brain- and liver-type creatine kinase not utilized in translation. .'Directed toward endogenous PRL: special procedures required to eliminate primary antibody-independent direct binding of the rabbit y-globulin to the target cells. 'See N o h (198Ob) for additional cellular targets in the postpartum rat. lSee also Fig. 12. "lodination of ligand performed by the direct chloramine-T method. Recent work has demonstrated that, except when carried out in the presence of substances capable of attenuating the strongly oxidizing effects of the latter, the above procedure results in production of an iodinated peptidal ligand whose association with receptor is irreversible, possibly covalent (Comens ef al., 1982). Iodination with the aid of lactoperoxidase did not lead to similar artifact (Comens et al.. 1982). See also, C . Heinrich (1982). wBoth chlorarnine-T and lactoperoxidase procedures utilized for catalyzing iodination. Since both products were apparently used interchangeably, it was not possible to determine from this repon which product yielded the given results. Wsed modified chloramine-T method of Frazier er al. (1974) with rigorous care in evaluating retention of native character of the peptidal ligand. ~
LYSOSOMAL FUNCTIONS IN CELLULAR ACTIVATION
35
(Szego, 1975; see also Table V). Can such generality (which may, on the basis of limited data already available, extend also to aspects of the actions of certain viruses, carcinogens, and toxins; see below) be a mere evolutionary accident? That does not seem likely. Instead it appears that recognition of ligand associated with plasmalemmal perturbation (or stabilization, in the case of a limited number of agonists such as insulin, prolactin, and antiinflammatory steriods at optimal concentrations), is common to the acute functions of agonists with extremely variegated structures. In turn, this evidence of the exquisite discriminatory capacity of the target cell for selectivity in relating to the potential agonists in its environment is surely attributable to the receptor population available at its dynamic surface at the moment.* In contrast to the extreme specificity of the surface recognition phenomenon, the subsequent pleiotropic effects of the several agonists appear to be more broadly programmed, and thus shared by diverse cell types in qualitatively similar fashion.
3. Unresolved Problems Through our two-decade preoccupation with transcriptional controls exerted by regulatory agents upon their respective target cells, we have, with notable exceptions, neglected to analyze in adequate depth concomitant, and even precedent, activities in the cytoplasm. Moreover, given the extraordinary coordination between nuclear and cytoplasmic responses attributable to the primary perturbation of agonal capture, we have generally failed to seek in systematic fashion, much less to identify, the precise coupling mechanisms by which such manifestly two-way communication is achieved. Above all, we have neglected detailed analysis of the cytostructural correlates of the subcellular compartmentation, which yields the economical, poised system capable of serving as a means of rapid propagation of initial triggering event, as previously sequestered, potential reactants are rendered accessible to various degrees. By the same token, such accessibility may result in metabolic activities that may mask or overwhelm other functions-a source of profound errors of interpretation not yet widely recognized, especially in relation to labeling of metabolic products from isotopic precursors that has been equated, often without adequate foundation, with net “synthesis. ’ ’ 4. Requirements of Any Hypothesis Purporting to Account for Totality of EfSector Action In reviewing the formidable volume of literature that is presented merely in token exemplary form in Figs. 1-3 and Tables 11, IV, and V, above, it is evident *The coexistence, side by side, of cells responsive, as well as unresponsive, to steroid (Szego et a ! . , 1977; Kierszenbaum et a l., 1980; Nazareno er al., 1981) or peptide (Varga er al., 1974) hormones, is a phenomenon that appears attributable, in part, to the turnover of cell surface constituents including macromolecules with recognition properties for the given agonist.
TABLE IV TIMECOURSEOF RESP~NSESOF THYROID GLANDTO TSHQ Time
< 10 seconds < 30 seconds W QI
3-6 minutes 5-15 minutes
Effect TSH,,binding to plasma membrane Enhinced accumulation of CAMP CAMP peak Intense apical surface activity Formation of large bulbous pseudopods which engulf luminal colloid Masses of colloid droplets rapidly filling apices of follicular cells Phagocytosis of colloid in parallel with lysosome redistribution, basal to apical Exocytosis of proteins into follicle lumen immediately on pseudopod formation Enhancement of glucose Q ~ , ac: tivation of the pentose phosphate pathway Depletion of serotonin; increased blood flow Increased uptake of 24Na
System
Bovine thyroid slices Homogenates of bovine thyroid Homogenates of bovine and canine thyroid after TSH in vivo Male hypophysectomized or thyroxin-suppressed rats, in I’ivo
Reference Pastan er af. (1966) Pastan and Katzen (1967) Zor et a/. (1969)
Wollman et al. (1964); Wetzel et al. (1965); Seljelid (1967a-e); Ekholm and Smeds (1966)
Ekholm el a/. (1975) Canine thyroid slices after TSH in vivo
Field er al. (1%3); Dumont and Rocmans (1964)
Male, thyroxin-suppressed rats
Clayton and Szego (1967)
Chicks
Solomon (1961)
20 minutes
1-6 hours
W
2 1 hours
4
24 hours
48 hours
Increased plasma levels of hormonal iodine and of iodide from preformed hormone; iodide organification Increased permeability [I4C]uridine Colloid droplet digestion after lysosomal fusion Onset of increase in water content Increased permeability [14C]amino acids Increased incorporation 3*P into phospholipid Thyroglobulin maturation 15 S, 19 S Increased incorporation [14C]uridine into RNA; increased net RNA (6 hours) Increased cell height, nuclear volume Elevation of water content Increased incorporation isotopic amino acids into protein (earlier effects may be masked by proteolysis) Thyroglobulin synthesis Increased DNA content Increased mitoses ~~
Thyroid venous effluent in dogs
Rosenberg et a!. (1965)
Chicks Male hypophysectomized or thyroxin-suppressed rats in uiuo Chicks Chicks Guinea pigs
Creek (1965) Seljelid (1967b)
Male, thyroxin-suppressed rats Chicks
Cavalieri and Searle (1967) Creek (1965)
Thyroxin-suppressed dogs (thyroid perfusion) Male, thyroxin-suppressed rats Guinea pig thyroid slices after TSH in uivo
Nhve and Dumont (1970a) Clayton and Szego (1967) Raghupathy et al. (1963)
Hypophysectomized rats Guinea pigs Guinea pigs
Pavlovic-Houmac et al. (1967) Ekholm and PantiC (1963) Gedda ( 1960)
Solomon (1961) Klitgaard et al. (1965) Kerkof and Tata (1967)
~
"Reprinted, with minor alterations, by permission from Szego (1975),wherein the crtutions may befound. See also, Freinkel(1964); Field (1968,1975);Spiro (1980);Nitsch and Wollman (1980);Tata (1980);these latter citations refer to present bibliography.
38
CLARA M. SZEGO AND RICHARD J. PIETRAS TABLE V TRANSFORMATION“ TIMECOURSEOF SMALLLYMPHOCYTE Time after mitogen application
Plasma membrane 5 5 minutes
5-40 minutes
Event
PHA binding Activation of surface-membrane Na+ ,K+-ATPase Mitogen capping (including TMV. Con A, anti-H-2) Development of fluorescein- and ionpermeable intercellular junctions Increased influx of isotopically labeled Pi Uridine 3-0-Methyl glucose Increased 32Pi incorporation into phosphatidylinositol Increased influx of K’ Ca2 cu-Aminoisobutyric acid +
Increased levels of CAMP cCMP Integration of levels of the above cyclic nucleotides with [Ca2+ 1 Lysosomal-vacuome system 10 minutes Transforniation antagonism by inhibitor of cathepsin-like protease 20-30 minute5 Iricrcascd uptake of Neutral red S. fyphimurium endotoxin Reduced structural latency of lysosomal 30- 120 minutes hydrolases Breakdown of RNA Increased total acid phosphatasc activity 4-5 hours Enlargement and increased pglycerophosphate permeability Transformation inhibition by lysosomal (and other membrane) "stabilizers"
Reference
Kay (1971); Mendelsohn el ul. (1971) Quastel and Kaplan (1970), Lauf (1975) Loor et al. (1972); Raff and de Petris (1973) Hulser and Petcrs (1971), Sellin et al (1974) Cross and Ord ( 197I ) Peters and Hausen ( 1971 a) Peters and Hausen (197 I b) Fisher and Mueller (1971)
Quaatel et al. (1970) Allwood et al. (197 I ) Mendelsohn et al. (1971); van den Berg and Betel (1971) Smith er ul. (1971), Webb ~t d. ( 1973) Hadden el ul. (1972) Whitfield et al. (1973)
Saito er al. (1973)
Hirschhorn et a/. (1968) Robineaux et ul. (1969) Hirschhorn et al. (1968) Cooper and Rubin (1965) Gillissen and Mecke (1973) Allison and Mallucci (1964a) Hurvitz and Hirachhorn ( I 965)
LYSOSOMAL FUNCTIONS IN CELLULAR ACTIVATION
39
TABLE V (Continued) Time after mitogen application Nuclear < 10 minutes
10 minutes
15 minutes
20 minutes 30-60 minutes 2-4 hours
24-36 hours
General cellular 2 hours 2 hours + 72 hours
Event
Increased AO binding of deoxyribonucleoprotein Perinuclear localization of [ '2sII]anti-HLA2; intranuclear at 24 hours Perinuclear localization of antigen (immunofluorescence); nuclear at 60 minutes Increased activity of histone phosphatase Nuclear localiztion of [3H]PHA Increased histone acetylation Activity of histone kinase Phosphorylation of nucleoproteins Labeling of RNA from [3H]uridine Nonhistone chromatin proteins DNA-dependent RNA polymerase activity (low salt) DNA-dependent RNA polymerase activity (high salt) DNA polymerase activity Thymidine incorporation into DNA
Increased incorporation of [3H]leucine into protein Accentuated RNA metabolism Mitosis
Reference
Killander and Rigler (1965) Lewis et a!. ( 1 974) Coons et a/. (1950)
Cross and Ord ( I 97 1) Stanley et al. (1971) Pogo et a/. (1966) Cross and Ord (1 97 1 ) Kleinsrnith e t a / . (1966) Kay and Cooper (1969) Levy eJ al. (1973) Handmaker and Graef (1970)
Loeb and Agarwal (1971) MacKinney et al. (1962)
Neiman and MacDonnell (1970) Lucas (1971) Nowell (1960)
"Reprinted with minor alterations, by permission, from Szego (1975), wherein all but the following cirarions muy he,fuuad: Quastel and Kaplan (1970); Lauf (1975); the latter are cited presently. See also, Reilly and Ferber (1976); Hume and Weidernann (1980); Udey and Parker (1980); Sidman (1981); Becker et al. (1981).
that a host of metabolic events, grouping themselves into apparent classes, requires integration if a potentially meaningful interpretation of such reaction sequences is to emerge. The list of phenomena that must be accounted for is likewise extensive. viz.: a. Recognition and capture of (hormonal) agonist ( H ) by mutual complementarity of latter with presumptive receptor ( R ) protein intrinsic to the plasmalemma b. Propagation of the primary event: ( 1 ) Adaptive change in surface organization and its relation to signal trans-
40
CLARA M . SZEGO AND RICHARD J . PIETRAS
duction and enhanced exchanges of cellular components with the extracellular fluid. ( 2 ) The H : R entry mechanism. ( 3 ) R activation and/or transformation to a modified, generally diminished structure, R ’ . (4) H:R’ entry into the nuclear compartment concomitantly with changes in numerous metabolic activities in nucleus and cytoplasm, many of which undergo progressive augmentation in rate. (5) Within the nucleus. (a) Interaction of H : R ’ , or possibly each component, individually, or perhaps only one or the other, with acceptor sites, protein or DNA, in chromatin. (b) Modification of the higher order of DNA structure. (c) Access of appropriate polymerase to specific sites destined, by base sequence and localization of modulatory protein factors, for transcription. (d) Processing of primary RNA transcript(s). (e) Emergence of mature RNA product(s) into the cytoplasm; degradation in situ of certain by-products. (6) In the cytoplasm. ( a ) Translation-with all the contributory fuctors required to be present at the appropriate concentrations: the amino acid mix, together with the relevant activating enzymes and tRNA; the ribosomal complement in functionally active state, along with the requisite initiation, elongation, and termination factors; and the specifying programs inherent in mature mRNA. (b) Changes in rates of degradation of preexisting or nascent proteins and other macromolecules. (7) Depletion of surface-oriented R (“down-regulation”), generally in apparent correlation with H levels. ( 8 ) Repletion of R , and “migration” to, and insertion in, the plasmalemma. The above, by no means intended to be inclusive, are indeed a challenging agenda.
B. THERELEVANTPROPERTIES OF LYSOSOMES Cogent evidence has now accumulated that implicates lysosomal functions in the reception, transduction, and propagation of a wide variety of effectors in diverse cell types. The grounds for this proposal (cf. Szego, 1971a,b; Szego ei a l . , 1971) are inherent in the characteristics of lysosomes, a most heterogeneous and pluripotent class of organelles. Therefore, before undertaking detailed con-
LYSOSOMAL FUNCTIONS IN CELLULAR ACTIVATION
41
sideration of the evidence linking the specific properties of lysosomes to the expression of agonal phenomena, it seems appropriate to summarize the unique properties that render this organelle potentially capable of subserving a substantial number of the functions outlined above. More extensive consideration of lysosomal participation in the normal metabolic economy, as well as under pathological conditions, is available elsewhere (Dingle and various co-editors, 1969, 1973-1975; Jacques, 1972; Dean and Barrett, 1976; von Figura et al., 1980; Callahan and Lowden, 1981; Glaumann et a l . , 1981). Lysosomes constitute a major component of the vacuome, a complex system of intracellular vesicles and structures wherein it was proposed that anabolic and catabolic cell functions are segregated (cf. de Duve, 1969). The anabolic segment of the vacuome consists of rough and smooth endoplasmic reticulum (ER), together with the Golgi apparatus. Proteins destined for secretion, as well as those that will ultimately reside in the plasma membrane, Golgi apparatus, or lysosomes, are all found at early stages of their biosynthesis in ER and appear to pass either through or adjacent to the Golgi apparatus during posttranslational maturation, if any. In cells endowed with the capacity to export certain protein products, secretory proteins are packaged in granules for exocytosis (Palade, 1975), whereas certain membrane proteins appear to associate with clathrincoated vesicles for transport to Golgi and/or plasma membranes (Pearse, 1975; Rothman and Fine, 1980). In contrast, lysosomal enzymes are segregated in primary lysosomes (cf. Hasilik and Neufeld, 1980a,b; Sly, 1980). The latter constitute one component of the vacuolar apparatus (de Duve and Wattiaux, 1966), the second major segment of the vacuome, and that which mediates the catabolism of endogenous and exogenous molecules (de Duve, 1969). Extracellular material taken up in course of invagination of the plasma membrane results in the generation of phagosomes or endocytotic vesicles in the cell interior. The latter vesicles then generally fuse with a given fraction of the primary lysosomal population, yielding secondary lysosomes, which are considered sites of catabolic activity. The lysosomal apparatus (cf. de Duve and Wattiaux, 1966; Bainton, 1981) comprises a dynamic system of primary and secondary lysosomes, autophagosomes (containing sequestered intracellular materials), heterophagosomes (containing sequestered extracellular materials), multivesicular bodies (composed of compound vesicles disposed in an amorphous matrix), and residual bodies (containing incompletely degraded materials). Discharge of the contents of either endocytotic vesicles or lysosomal structures into the extracellular space by exocytosis may occur in some instances (de Duve, 1969). 1, Pathways of Uptake of Nutrient and EfSector Substances into the Vacuolar Apparatus Extracellular materials appear to enter cells either by a process of permeation or by incorporation within membrane-limited vacuoles or vesicles. The former pathway involves penetration of a given substance through the plasma membrane
42
CLARA M. SZEGO A N D RICHARD J . PIETRAS
by passive or facilitated diffusion or by active transport (cf. Stein, 1967; Diamond and Wright, 1969; Dietschy, 1978). The alternative pathway is generally termed endocytosis. several varieties of which have been distinguished. However, it is important to note that these two pathways are not mutually exclusive for uptake of a given substance. Several investigators have presented evidence for simultaneous transport of a given molecular species by passive permeation, as well as by specific endocytosis (cf. Thomson, 1978; Brown and Goldstein, 1979; Szego and Pietras, 1981). The several known and putative forms of endocytosis are represented in schematic form in Fig. 4. Two major classes of endocytotic uptake can be distinguished. Phagocylosis occurs mainly in specialized cells and can be defined as the process of ingestion of solids of relatively large size (e.g., erythrocytes, latex spheres, carbon particles) with little concomitant uptake of fluid (Siiverstein et al., 1977). On the other hand, pinucytusis is a more ubiquitous process exhibited by virtually all cells and leads to the interiorization of fluid and solutes, together with small particles (cf. Simson and Spicer, 1973; Sly, 1980). Pinocytosis may be nonselective (i .e., fluidphase) or selective (adsorptive or receptor-mediated) (Jacques, 1969a) and may result in the formation of intracellular vesicles of 300-1000 nm in diameter (macrupinucytusis) or of about 70 nm in diameter (nzierc~,pinocytosis;cf. Allison and Davies, 1974b; Pratten et al., 1980; Sly, 1980). Some micropinocytotic vesicles bear a smooth-surfaced limiting membrane (Palade, 1960), while others, with diameters ranging from 50 to 250 nm, exhibit a filamentous coat on their cytoplasmic surfaces and appear to arise from specialized regions of the plasma membrane termed “coated pits” (cf. Pearse, 1980). The coating of the latter vesicle is a lattice formed by a single protein species, clathrin (Pearse, 1976), that is capable of self-assembly from its constituent subunits into a symmetrical, trimeric cage-like structure without participation of additional proteins (cf. Kirchhausen and Harrison, 1981). A mongrel form of pinocytotic vesicle, recently christened “receptosome” by Willingham and Pastan (1980), is a smooth-surfaced organelle of 150-300 nm in diameter and appears to be formed by interiorization of plasmalemmal coated pits, with presumedly concomitant shedding or exclusion of coat material from the vesicle upon its interiorization (cf. Willingham et a/., 1981a). However, the likelihood of such an “uncoating” process has been discounted recently on the grounds of the extremely rapid time course (of the order of seconds) that would be required (Willingham and Pastan, 1981). Finally, limited data presently available suggest that some cytoplasmic vesicles with average diameters of about 70 nm may constitute elements of a fused chain of branching, permanent or semipermanent invaginations of the plasma membrane (Simionescu et al., 1975; Bundgaard et al., 1979). The latter workers proposed that such a racemose system of vesicles may provide a hitherto unrecognized pathway for intracellular penetration of given solutes.
LYSOSOMAL FUNCTIONS IN CELLULAR ACTIVATION
43
PHAGOCYTOSIS: VACUOLES, dio > l p m [ P o r t i c l e s ]
MACROPINOCYTOSIS: VESICLES, 3 0 0 - 1 0 0 0 nm [Ferritin]
RECEPTOR-MEDIATED MACROPINOCYTOSIS: [ H L A - A n t i g e n ]
I
CP
RECEPTOR-MEDIATED PINOCYTOSIS VIA COATED P I T : 50-250 n m [LDL-cholesterol] RECEPTOR-MEDIATED PINOCYTOSIS VIA COATED PIT 8 'RECEPTOSOME': 1 5 0 - 3 5 0 n r n [ a z - M G ] MICROPINOCYTOSIS
70 nrn
RACEMOSE VESICULATION: 50-200nm; DIFFUSION
U
1
I
I
2
5
15-30
APPROXIMATE T I M E (rnin) FIG. 4. Schematic representation of pathways for the internalization of extracellular agonists, as modified from Geisow (1980). Examples, within brackets. Observations, both descriptive and quantitative, upon which this generalized scheme for the several classifications of endocytotic activity is based, are as follows: phagocytosis (Metchnikoff, 1883); macropinocytosis (Lewis, 1931); micropinocytosis (Palade, 1960; Casley-Smith, 1969; Casley-Smith and Chin, 1971); racemose vesiculation (Bundgaard et a [ . , 1979); and associated receptor-mediated pathways (Anderson et ul., 1977; Pearse, 1980; Willingham and Pastan, 1980; Montesano et d . , 1982). (Cf. also Jacques, 1969a; Allison and Davies, 1974b; Szego, 1978; Herzog, 1981.)
Extracellular materials and surface membrane components internalized by endocytosis appear to be directed along multiple routes in the cell interior (Jacques, 1972; Farquhar, 1981a,b; Herzog, 1981). In secretory cells, observations with electron-dense tracers reveal two major endocytotic pathways: (1) direct route to lysosomes, from which some material is subsequently transferred to the stacked Golgi cisternae; and (2) direct route to the Golgi apparatus (cf.
44
CLARA M. SZEGO AND RICHARD J . PIETRAS
Farquhar, 1981a,b; Herzog, 1981). Factors that appear to regulate the movement of incoming vesicles to lysosomes or to Golgi cisternae include composition, charge, and size of the tracer, as well as the type and physiological state of the given cell (Herzog, 1981). In highly differentiated glandular cells, and probably in most eukaryotic cells (cf. Chapman-Andresen, 1977), this process appears to provide a mechanism to salvage and reutilize membrane components. However, it apparently also provides direct access of agonists, as well as of macromolecules integral to given membranes (e.g., receptors, enzymic moieties), to critical biosynthetic and degradative compartments of the cell (cf. also, Haimes et al., 1981; Goldfischer, 1982). Further discussion on the traffic patterns of incoming materials may be found in Section II,B,S,a and succeeding sections of this article. A clear mechanismic distinction is evident between, on the one hand, phagocytosis and macropinocytosis and, on the other hand, micropinocytosis and coated vesicle formation (Allison, 1973; Szego, 1978; Ockleford and Munn, 1980; Kusiak et al., 1980; Szego and Pietras, 1981). Although the former processes are strongly depressed by inhibitors of glycolysis or oxidative phosphorylation (cf. Allison and Davies, 1974a,b; Ockleford and Munn, 1980), the formation of microvesicles and coated vesicles is found generally not to require direct input of metabolic energy (Casley-Smith, 1969; Nagura and Asai, 1976; Ockleford and Munn, 1980; however, cf. Munthe-Kaas, 1977). Some investigators (Allison and Davies, 1974a,b; Anderson et al., 1977) report that micropinocytosis is inhibited at low temperature, as are other forms of endocytosis, but others find that the formation of smooth and coated microvesicles is not markedly reduced at 4°C (Casley-Smith and Day, 1966; Nagura and Asai, 1976; however, cf. Ockleford and Munn, 1980). Inhibitors of microtubule assembly (e.g., colchicine) generally show little effect on the several classes of endocytosis (Bhisey and Freed, 1971; Ockleford and Munn, 1980; Kusiak et al., 1980). In contrast, cytochalasin B, a drug which depresses actin-based motile processes such as are integral to the function of microfilaments (Wessells et al., 1971), inhibits phagocytosis and macropinocytosis (Allison, 1973; Ockleford and Munn, 1980) but elicits only slight (Munthe-Kaas, 1977) or no (Allison and Davies, 1974a; Nagura and Asai, 1976) inhibition of micropinocytosis involving either smooth or coated vesicles. Thus, the generation of smooth and coated microvesicles from plasma membrane emerges as a process less dependent on metabolic energy supply and subplasmalemmal mechanoeffector systems than other forms of endocytosis. Evidence for the premise that specific binding of certain macromolecules to plasmalemmal receptors must precede uptake into a given endocytotic transport system originated from studies in several areas. These include (1) selective transport of immunoglobulin in the fetal yolk sac and neonatal intestine (Anderson and Spielman, 1971; Wild, 1973); (2) receptor-mediated uptake of yolk
LYSOSOMAL FUNCTIONS IN CELLULAR ACTIVATION
45
protein by the oocyte (Roth and Porter, 1964; Roth et al., 1976); (3) specific entry of transcobalamin 11-vitamin B, complexes into kidney and liver cells and into fibroblasts (Newmark et al., 1970; Pletsch and Coffey, 1971; YoungdahlTurner et al., 1978); (4) selective hepatic clearance of modified plasma proteins (Ashwell and Morell, 1974); and (5) receptor-mediated endocytosis of cholesterol-lipoprotein complexes in fibroblasts (Anderson er al., 1977). A plethora of corresponding observations for hormones and other effectors is likewise available and allows for evaluation of the cogency and generality of this transport process (see succeeding sections). It is clear that one means of triggering regional internalization of the cell surface (i.e., adsorptive or receptor-mediated endocytosis) is the ligand-induced redistribution of integral proteins into patches and clusters in the plasmalemma. The mechanism for such “provoked internalization” (Szego, 1978) is believed to be related to the stress of deformation of the membrane as a consequence of regional clustering of intrinsic ligand-decorated protein, with resultant local change in permeability to some critical factor (e.g., Ca2+), which, in turn, may activate contractile elements in the subplasmalemmal cytoskeleton (Singer, 1975, 1976; Edelman, 1976; Szego and Pietras, 1981). Microfilaments and perhaps other less well-defined mechanoeffector elements are generally considered to play an active role in ligand-induced clustering of surface components (Rutishauser and Edelman, 1978; Singer et al., 1978). Some investigators suggest that microtubules are also involved in this process (Ukena and Berlin, 1972; Albertini and Clark, 1975; Rutishauser and Edelman, 1978) but others dispute this contention (de Petris, 1974; Singer et al., 1978). Although agonists of low molecular weight, viz. triiodothyronine (Cheng et al., 1980) and estradiol-17P (Szego and Pietras, 1981), promote a redistribution of membrane-associated proteins on specific recognition, a related mechanism by which cells endocytose ligand-receptor complexes may be better adapted to the function of agonists that are not multivalent. For example, surface receptors for cholesterol-lipoprotein complexes in fibroblasts appear to be confined largely to coated pit structures, which constitute an estimated 2% of the total surface area of the plasma membrane (Anderson et al., 1977). Endocytosis resulting from interaction of ligand with its specific receptor so localized is presumably not triggered by ligand-induced clustering of membrane receptors, since the latter were aggregated prior to ligand exposure. Such preaggregation of membrane receptor proteins m-ay be a consequence of the proposed continuous flow of plasmalemmal lipid and protein constituents toward coated regions of the surface membrane (Bretscher, 1976; Pearse, 1980). Under such circumstances, the stimulus for membrane deformation leading to coated vesicle formation may be derived from the ligand-receptor interaction per se, from alteration in the selfassociation (Kirchhausen and Harrison, 1981) or conformation of coat protein (i.e., clathrin) molecules, or, in part, from both processes, especially if the
46
CLARA M . SZEGO AND RICHARD J. PIETRAS
clathrin lattice at the inner membrane face is coextensive with receptor aggregates at the external surface (Kanaseki and Kadota, 1969; Ockleford and Munn, 1980). Neither phagosomes nor macro- and micropinocytotic vesicles contain a random complement of cell surface macromolecules (Tsan and Berlin, I97 1 ; Pearse, 1975; Birchmeier er al., 1979; Suzuki and Kono, 1979; Willinger ct a/., 1979). It is not known whether this is attributable to the occurrence of sites on plasma membrane destined for preferential internalization or to the function of some active but undefined molecular exclusion mechanism (cf. Bretscher, 1976; Pearse and Bretscher, 1981). In any event, newly formed endocytotic vesicles exhibit saltatory motion, in course of which certain of their population undergo fusion, predominantly with those organelles of the lysosomal system that are appropriately disposed (cf. Allison, 1973; de Petris, 1977; Muller et a/., 1980a,b), or marked in some as yet undetermined manner. Some micropinocytotic vesicles may also gain access directly or indirectly to other cellular compartments including the nucleus (cf. Szego, 1975; Y.-J. Schneider et al., 1978; Szego and Pietras, 1981), the Golgi apparatus (Bergeron et al., 1979; Willingham and Pastan, 1980), and the opposing plasma membrane (Allison and Davies, l974b). Although the thermal energy needed for niicrovesicular movement appears to be supplied solely by Brownian motion, the rate of this linear translocation process is extremely rapid (Casley-Smith, 1969; Casley-Smith and Chin, 1971; Green and Casley-Smith, 1972; however, cf. Ockleford and Munn, 1980, and Section II,B,S,a). Interestingly, the clathrin lattice of coated microvesicles is apparently partially or totally shed before fusion of its phospholipid bilayer core with a given membrane (Douglas, 1974; Anderson et al., 1977). Energy requirements for the shedding process are not known, but Ockleford and Munn (1980) suggest that activity of a Ca2 -dependent ATPase associated with such vesicles (Blitz et al., 1977) may be contributory. +
2. Composition and Organization of Lysosomes a. Enzymic Constituents. The pivotal biochemical studies of de Duve and colleagues led to the initial characterization of lysosomes as membrane-limited intracellular organelles sequestering acid hydrolases (cf. de Duve and Wattiaux, 1966; de Duve, 1969; Bainton, 1981). More than 70 different enzymes with a wide variety of substrate specificities are now known to occur in lysosomes of one or more cell types (cf. Barrett and Dean, 1976; Barrett and Heath, 1977). The biocatalytic properties of lysosomal hydrolases are well known to vary with assay conditions, such as the type of buffer (Otto, 1971) and the nature of the substrate (Bohley et al., 1971). Nevertheless, these hydrolases generally exhibit optimal activity at acid pH. However, some lysosomal enzymes are active at neutral or alkaline pH (Hugon and Borgers, 1967; Davies et al., 1971; Bainton, 1973; McDonald and Ellis, 1975; Dean and Barrett, 1976; Eeckhout and Vaes,
LYSOSOMAL FUNCTIONS IN CELLULAR ACTIVATION
47
1977; Hagiwara et al., 1980; Britz and Lowther, 1981; Collins and Wells, 1982, 1983), especially in their membrane-bound form (see Table XV and Melloni et al., 1982a,b). In addition, there is recent evidence for the sequestration of nonhydrolytic enzymes in lysosomes (Mraz et al., 1976; Dousset et al., 1979; Griffiths and Lloyd, 1979; Entenmann et al., 1980; Wells et al., 1981; Geller and Winge, 1982; Collins and Wells, 1982, 1983). Most, if not all, lysosomal enzymes, including cathepsin B (Towatari et al., 1979; Seeler and Szego, 1984), are glycoproteins (Dean, 1975a; Dean and Barrett, 1976). Variation in the classes, distribution, and numbers of these charged carbohydrate moieties may contribute to the occurrence of multiple forms of enzymes with very similar substrate specificities (cf. Dean, 1975a). In turn, variation in classes, distribution, and numbers of such moieties may underlie the differential responses of certain hydrolases, e.g., cathepsin B, toward organic and inorganic modulators as a function of cellular origin of the enzyme (cf. Szego et al., 1976). Moreover, constitutive glycosylation of lysosomal enzymes appears to contribute a critical recognition function for channeling these organellar components to appropriate intracellular and/or surface membrane sites of given cells (see Section II,B,4). b. Heterogeneity of Lysosomes. The initial studies of lysosomes by de Duve and co-workers revealed a remarkable heterogeneity of rat liver lysosomes with respect to their density and size (i.e. calculated diameters of 0.25 to 0.8 pm; de Duve, 1969). However, only minor differences were found in the distribution of acid hydrolases of various subcellular fractions separated by differential pelleting. From such findings arose the view, still widely accepted, that, apart from isolated examples of specialized lysosomes in some cell types (e.g., the acrosome of spermatozoa), lysosomes do not exhibit significant biochemical or enzymic heterogeneity. However, subsequent studies of lysosomal populations of rat liver, as well as those of certain other organs and homogeneous cell lines, have revealed significant differences in the relative enrichment of acid hydrolases among subcellular fractions separated by isopycnic or rate-zonal centrifugation (cf. Davies, 1975; Sloane and Bird, 1977; Dobrota et al., 1979; Tanaka, 1979; Knook and Sleyster, 1980; Rome et al., 1979; Radzun et al., 1980). These studies provide evidence of at least two populations of lysosomes with different densities and with qualitatively different enzyme contents, not only in rat liver, wherein cellular heterogeneity may be a contributory factor (Knook and Sleyster, 1980), but also in Chinese hamster ovary fibroblasts (cf. Davies, 1975) and other cultured cells (cf. Milsom and Wynn, 1973), which constitute homogeneous populations. More recent studies using ultrdcytochemical (Uchiyama and von Mayersbach, 1981) and X-ray microanalytical (BAcsy, 1982) techniques tend to confirm the latter findings. The distinct enzymic heterogeneity of lysosomes implies that packaging of enzymes in lysosomes is not a uniform process but a variable function, probably dependent on the rates of synthesis or availability of individual hydrolases at
48
CLARA M. SZEGO AND RICHARD J . PIETRAS
packaging “stations” (cf. Davies, 1975; Dean and Barrett, 1976). Differential rates of synthesis of individual lysosomal hydrolases, a well-characterized response of specific target cells to hormonal stimulation (cf. Table VI), conceivably could result in the formation of primary lysosomes relatively enriched in specific activities of given hydrolases. Alternatively, hydrolase content might be selectively altered at the level of the smooth ER or GERL (see examples in Davies, 1975; Paigen, 1981), regions in which apparent transitory accumulation of lysosomal enzymes has been reported (cf. Brandes and Anton, 1969; Sloane, 1980;and see below). Moreover, as emphasized by Davies (1975), heterogeneity of structural stabilities or degradation rates of lysosomal hydrolases probably prevails in vivo, thereby leading to accentuation of differences in enzyme content (cf. Nemere and Szego, 1981b). Indeed, data on the ontogeny of capacities for synthesis, as well as for degradation, of given lysosomal constituents and even of the assembled organelles themselves (Quintart et al., 1979a; Lodish et al., 1981) TABLE VI REPRtSENTATIVE EXAMP1,ES O F DELAYEDCHANGES ELICITEDB Y AGONISTSIN Ac-TIVITIES O R CONCEN’IRATIONSOF LYSOSOMAL COMPONENTS“ Agonist Pcptide hormones ACTH
Chorionic gonadotropin
Follicle-stimulating hormonc Glucagon Growth hormone lnsulin Luteinizing hormone
Parathyroid hormone Prolactin Kclaxin
THE
TOTAL
Reference
Szab6 e r a / . (1967); Dominguez et a / . (1974); Kostulak (1977); Laychock r / a/. (1977); Mattson and Kowal (1978); Trzcciak et a/. (1979); Mattson and Kowal (1980) Dimino and Reecc ( 1 973); Cajander and Bjersing (1975, 1976); Dimino e t a / . (1977); Elfont ri ( I / . ( 1977) Elkington and Blackshaw (1973, 1974); Zoller and Wcisz ( 1980) Gilder et a/. (1970); Mortimore and Ncely (1975) Steinetz el a / . (1965); Swank (1978); Huhhard and’ Liberti (1981. 1982) Wildenthal (1973); Mortimore and Neely (1975); Hcdly and Dinsdale ( I 979) Elkington and Blackshaw (1973, 1974); Boer ri a/. (1976); Strauss et ti/. (1978); Witkowskd (1979); Zoller and Weisz (1980); cf. also Okazaki et u / . ( 1977) Hara and Nagatsu (1968); Vaes (1969); Eilon and Raisz (1978) Ciiunta ef a/. (1972); 1-ahav ei trl. (1977) Steinetz rt a/. (1965); Manning er u / . (1967); McDonald and Schwabe (1982)
TABLE VI (Continued) Agonist Thyroid hormones”
Thyroid-stimulating hormone
Steroid hormones Androgen
Ecdy steroid Estrogen
Gibberellins Glucocorticoids
Reference Fox (1973); Farooqui et al. (1977); DeMartino and Goldberg (1978); Mandel er a / . (1978); Mori and Cohen (1978); Coates et al. (1978, 1982); DeMartino and Goldberg (1981); Severson and Fletcher (1981) Ekholm and Smeds (1966); Seljelid et al. (1971); Bigazzi and DeGroot (1973); Starling et al. (1978)
de Duve et al. (1962); Lasnitzki et al. (1965); Males and Turkington (1971); Elkington and Blackshaw (1973); Kochakian and Williams (1973); Ban et al. (1974); Elkington and Blackshaw (1974); Iela er al. (1974); Serova and Kerkis (1974); Brandt et al. (1975); Kamble and Mellors (1975); Milone and Rastogi (1976); Fischer and Swain (1978); Moore et al. (1978); Swank (1978); Tenniswood et al. (1978); Blecher and Kirkeby (1979); Koenig et al. (1980a,b); Goldstone et al. (1981); Watson et al. (1981) Radford and Misch (1971); van Pelt-Verkuil (1979) Fishman and Fishman (1944); Harris and Cohen (1951); Beyler and Szego (1954); de Duve er al. (1962); Steplewski and WaroAski (1973); Banon et al. (1964); Watanabe and Fishman (1964); Lasnitzki et al. (1965); Woessner (1969); Smith and Henzl (1969); Schiebler et al. (1970); Platt (1972); Ban et al. (1974); Moulton (1974, 1982); Nozawa et al. (1974); Wolinsky et al. (1974); Serova and Kerkis (1974); Baron and Esterly (1975); Boshier and Katz (1975); Briggs and Briggs (1975); Gustavii (1975); Kamble and Mellors (1975); Katz et al. (1976); Zachariah and Moudgal (1977); Moore et al. (1 978); Jaccard and Cimasoni (1979); Sengupta et al. (1979); Witkowska (1979); Elangovan and Moulton ( 1980); Sloane ( 1980) Gibson and Paleg (1972, 1976); Gonzilez (1978) de Duve et al. (1962); Weissmann and Thomas (1964); Lasnitzki et al. (1965); Nakagawa er al. (1968); Abraham et al. (1969); Caruhelli and Griffin (1970); Bingham et al. (1971); Bowness and Barry (1972); Bourne et al. (1973); Chertow et al. (1973); Kamble and Mellors (1975); Brehier et al. (1977); Kasukabe et al. (1977); Clarke and Wills (1978); Mandel et al. (1978); Moore et al. (1978); Bagwell and Ferguson (1980); MacDonald et al. (1980) (continued)
50
CLARA M. SZEGO AND RICHARD J. PIETRAS TABLE VI (Continued) Agonist
Progecternne
Testosteronc Vitamin D Othcr effectors Chemical carcinogens
Juvenile hormone Phytohemagglutinin Prostaglandin Fzrr Viruses
Vitamin A
Reference Harris and Cohen (1951); Steinetz CI a/. (1965); Manning rt a / . (1967); Moulton (1974, 1982); Serova and Kerkis (1974); Bazer ef a / . (1975): Roberts ~t id. (1976); Sloane and Bird (1977); Hoversland and Weitlauf (1978); Paavola (197X); Elfont e l ol. (1979); Lucas (1979); Witkowska (1979); Elangovan and Moulton (1980); Sloane ( 1980); Tyree ef a / . ( 19x0) McCluer et a / . (19x1) Lerncr (19x0); Davis and Joncs (1982)
Nodes and Reid (1963): Slater and Grecnbaum (196s); Flaks (1970);Pokrovsky el a / . (1972); Schulze (1973); Berg and Christoffersen (1974); Hultherg and Mitelman (1977); Pietras (1978) Bccl and Feir (1977); Koesterer and Feir (1980) Hirschhom ef a/. (1965, 1967): Konig et d.(1973) McClellan t i d.(1977) Allihon and Sandelin (1963); Wolff and Bubel (1964); Allison and Mallucci (1965): Flanagan (1966); Hotham-Iglewski and Ludwig (1966); Allison and Black (1967); Thacore and Wolff (1968); La Placa el ul. (1969); Greenham and Poste (197 I ) ; Postc (1971 h); S y l v h e/ a / . (1974); Lockwood and Shier (1977) Lucy eta!. (1961); Fell and Dinglc (1963); Poste (I97 1a)
<'Evidencefor response to agonist at times greater than 30 ininutes (i.c., generally several hours or days) after addition in vivo or to isolated cells or tissues in virro. Response is generally an increase in total enzyme activity and considered to be attributable to new enzynic synthesis or to delayed labilization of lysosomes. "It is to be notcd that rebound (?) synthesis of rat liver pcroxisomal coniponcnts occurs in response to chronic treatment with thyroid hormones (Just ei a / . , 1982), a process that may bear significant parallels to the present instances.
indicate that the composition of representative populations of primary lysosomes is a variable expression of the unique cellular environment at distinct stages of differentiation. This view is supported by evidence of the relative lack of immunologic reactivity among lysosome-like organelles in rat uterus, lung, or thymus when challenged with an immunoglobulin directed against a high-density lysosoma1 lipoprotein fraction from preputial gland (Szego et 01.. 1977). Superimposed upon these several sources of variability in lysosomal composi-
LYSOSOMAL FUNCTIONS IN CELLULAR ACTIVATION
51
tion is the further diversity imparted by the environmental history of the given organelle, primary vs secondary, the latter in its numerous stages (cf. Jaeken and Thinks-Sempoux, 198I), and thus, the nonuniform proportion of such exogenous materials introduced during the lifetime of the organelle that serve to dilute and otherwise modify its constitutive nature. Accordingly, marked differences in the size and density of lysosomal vesicles are to be anticipated as a product of dynamic organellar functions (e.g., fusion with endocytotic vesicles, autophagy). However, accumulating evidence indicates that the clearcut distinction between lysosomal and nonlysosomal vesicles (e.g., secretory granules, coated vesicles) inherent in the vacuome concept (de Duve, 1969) may require modification. For example, in several cell types, the entire population of potential secretory granules corresponds either to primary lysosomes as such, or to related vesicular structures with which acid hydrolases and secretory proteins are associated (Novikoff et al., 1962; Cohn et al., 1966; Smith and Farquhar, 1966; Palade, 1975; Holtzman et al., 1977; Bansal et al., 1980). The number of reports that describe coated vesicles, which sequester acid phosphatase and apparently function in the capacity of primary lysosomes, is also increasing (cf. Nevorotin, 1980; Pearse and Bretscher, 1981). Such coated vesicles appear to form by pinching off from Golgi cisternae, GERL (Novikoff et al., 1971), or the innermost Golgi lamellae (Hand, 1971). Acid hydrolases other than acid phosphatase have been demonstrated in some coated vesicles (Decker, 1974), but acid hydrolase-containing coated vesicles generally represent a minority of the total population of such organelles (Nevorotin, 1980). In epithelial cells of the rat vas deferens, acid phosphatase-containing coated vesicles may fuse with plasma membrane and thereby discharge hydrolase from the cell (Friend and Farquhar, 1967). Thus, acid hydrolase-sequestering coated vesicles may constitute a separate population of lysosome-related organelles with as yet unknown functional distinctions from those structures bearing a smooth limiting membrane. c. Membrunes of Lysosomes. Only limited data are currently available on the lipid, carbohydrate (Henning and Stoffel, 1973; Thinb-Sempous, 1973; Knecht and Hernandez, 1978), and protein (D. L. Schneider et af., 1978, and citations therein; Yamamoto et ul., 1980) composition of lysosomal membranes. These membranes are characterized by the presence of cholesterol, sphingomyelin, and N-acetylneuraminic acid at levels similar to those found in purified plasma membranes, but in concentrations substantially greater than those of other endomembranes (i.e., endoplasmic reticulum, Golgi). Nevertheless, the “fluidity” of lysosomal membranes is somewhat diminished relative to that of plasma membranes at equivalent temperatures (Ronveaux-Dupal et al., 1979). A major variation from the composition of plasma membranes is the absence or very low activity in lysosomal membranes of enzymes (ThinesSempoux, 1973; Burnside and Schneider, 1982) and certain unidentified polypeptides (Blizquez et al., 1980) that are characteristic of the plasmalemma.
52
CLARA M. SZEGO AND RICHARD J . PIETRAS
Recent studies of lysosomal membrane fractions from rat liver show that 20% (D. L. Schneider et a l . , 1978) to 38% (Yamamoto et al., 1980; cf. also, Avila and Casanova, 1981) of total lysosomal protein is contained in the membrane proper. From 16 to 25 protein and glycoprotein species were detected by these workers by means of gel electrophoresis (cf. also, Blhzquez et al., 1980). Most of these membrane proteins are apparently exposed to the cytoplasm, a conclusion based on studies with impermeant cross-linking reagents and trypsin susceptibility (cf. also Pontremoli et al., 1982). Such exposed proteins, marked by an unusually high content of thiol groups as well as of carbohydrate constituents, may be considered potential intracellular receptors for the recognition of substrates to be degraded and/or of vesicles containing internalized substrate (e.g., D. L. Schneider et a l . , 1978). d. lntralysosomal Orientation of Components. Little is known about the intralysosomal distribution of enzymes and other materials. It was originally proposed that the numerous hydrolases were present within the intact lysosome in a diffusible, active form (de Duve, 1969). However, several workers have suggested that acidic lipoproteins, which constitute a substantial proportion of nonmembranous lysosomal components, may serve as internal matrices, providing sites for the immobilization of hydrolases and other soluble molecules (Shamberger, 1969; Koenig, 1974; Szego, 1975; cf. however, Dean and Barrett, 1976; Horton, 1982). Similarly, the carbohydrate moieties of lysosomal enzymes may control their intralysosomal distribution by influencing hydrophobic and electrostatic interactions with matrix or membrane proteins (Knecht and Hernindez, 1978; Yamamoto et d . , 1980; Adhikari et d . , 1981). There are numerous data indicating that the several lysosomal enzymes differ in the extent to which they are bound to the lysosomal membrane. For example, a-and P-glucosidases can be solubilized only in the presence of detergents that are capable of disrupting membrane organization (Beck and Tappel, 1968; Burton and Lloyd, 1976; Lloyd, 1977; Avila and Casanova, 1981). Similarly, other investigators have found that a major proportion of hepatic (Sloat and Allen, 1969; Dobrota et a l . , 1979) and leukocyte (Avila and Casanova, 1981) acid phosphatase activity is associated with the lysosomal membrane. Pontrernoli et al. (1974) reported that 20% of total proteolytic activity toward hemoglobin at pH 5 is retained by membrane fractions after vigorous freeze-thaw treatments of lysosomal fractions. These authors (cf. also, Pontrernoli et a l . , 1982) further suggested that some portion of proteinase activity may be available at the cytoplasmic surface of lysosomes, since converson of specific substrate could be achieved in the presence of “intact” organelles (cf. also, Tallman et al., 1974, on hydrolysis of gangliosides by “intact” lysosomes from brain). Of possibly greater functional significance is the observation that catalysis by a cathepsin Blike enzyme occurred most rapidly at pH 6.5 when the enzyme was membrane associated, but at pH 4.5 when solubilized (Pontremoli et al., 1974; Horecker et
LYSOSOMAL FUNCTIONS IN CELLULAR ACTIVATION
53
a l . , 1975; Melloni et al., 1982a,b). Tsuda et al. (1974) have also reported a significant elevation of the pH optima of catheptic, sulfatase, and hyaluronidase activities toward neutrality in apparently intact, as compared with disrupted, macrophages. Similar modulation of catalytic properties, including that of inhibition of cannibalistic denaturation (cf. Wu et al., 1981), attributable to immobilization of enzymes by binding to solid supports or to artificial membranes, has been documented (McLaren and Packer, 1970; Katchalski et al., 1971; Kazakova and Orekhovich, 1975; Masters, 1978; Marshall, 1978; Nemat-Gorgani and Wilson, 1980; Ricard et a l . , 1981). Significantly, the pH optimum of catheptic activity may be displaced substantially upward when assayed with naturally occurring proteins, rather than with the synthetic substrates in conventional use (Bohley et a / . , 1971). Thus, it appears that the biocatalytic properties of lysosomal hydrolases may vary with the degree of immobilization of the enzyme (e.g., by binding to lysosomal membrane), as well as with the nature of the substrate (cf. also, Section 11,B,4). Further modulatory functions toward specific lysosomal hydrolase activities are exhibited by nonenzymic “activator” proteins intrinsic to the organelles (Sandhoff and Conzelmann, 1979). 3 . Lysosomal Membrane Stability and Permeability Degradation and processing of macromolecules in secondary lysosomes result in the production of numerous low-molecular-weight compounds such as monosaccharides, amino acids and small peptides, purine and pyrimidine nucleosides, as well as inorganic phosphate and sulfate products (cf. Dean and Barrett, 1976; Reijngoud and Tager, 1977). Clearly, there must exist a means of removal of such products if osmotic swelling and lysis of the organelles are to be prevented. The latency or unavailability of most lysosomal enzymes in intact organelles is usually interpreted as evidence that macromolecular substrates do not readily permeate the lysosomal membrane (however, cf. Koenig, 1974). The results of qualitative studies on the latency or sedimentability of lysosomal enzymes after suspension of “intact” organelles in solutions of various compounds indicate that permeation across the lysosomal membrane decreases with increasing molecular weight (Reijngoud and Tager, 1977). Compounds with molecular weights exceeding 300 are generally excluded. Lysosomes are relatively impermeable to the passive flux of electrolytes and charged compounds at physiological temperatures. However, some selective cation permeation is evident at 0-4”C, with H+ > K > Na+ (Henning, 1975). The results of several recent studies suggest a most provocative new concept, i.e., that lysosomes may function in normal calcium homeostatic mechanisms. High concentrations of calcium subject to regulation by vitamin D, have been detected by analytical electron microscopy in lysosomes of absorptive cells from chick duodenum (Davis et a l . , 1979; Davis and Jones, 1981). Similarly, electron microprobe analysis was used to demonstrate the presence 0; bound calcium in +
54
CLARA M. SZEGO AND RlCHARD J . PIETRAS
dense lysosome-like vesicles of snail neurons (Sugaya and Onozuka, 1978) and in the outer-mantle epithelial cells of a freshwater mollusc (Jones and Davis, 1982). The mechanism by which these lysosome-like organelles sequester and release calcium is not known, but uptake of Ca2 in vitro by structurally related Golgi membrane vesicles from intestine appears to proceed by carrier-mediated and/or H -Ca2 countertransport modes (MacLaughlin et al., 1980; Freedman et al., 1981). In this regard, the potential role of Ca2+-dependent ATPase activity, as identified in primary lysosomes of human leukocytes (Avila and Casanova, 198 I ) , merits further investigation. Since, when isolated, most lysosomal enzymes exhibit an acid pH optimum, Coffey and de Duve (1968) inferred that the intralysosomal pH must be low. Indeed, estimates of intralysosomal pH in situ and in isolated lysosomes range from pH 3.0 to 6.8, using free or immobilized indicator dyes, and from pH 5.3 to 7.3, using weak acid or weak base distribution methods (cf. Reijngoud and Tager, 1977). In all cases, the level of the intralysosomal pH was maintained lower than that of the ambient tluid. More recent studies with a pH-dependent fluorescent probe indicate that the intralysosomal pH in living cells is maintained in the range 4.7-4.8 (Ohkuma and Poole, 1978; Poole and Ohkuma, 1981). Two general mechanisms for regulating the pH difference across lysosomal membranes have been postulated (cf. Reijngoud and Tager, 1977; Ohkuma and Poole, 1978). According to one set of workers, a Donnan-type equilibrium is responsible for maintaining the pH difference across the relatively impermeable lysosomal membrane, due to the presence of nondiffusible, negatively charged groups within lysosomes (i.e., glycoproteins with relatively low isoelectric points, acidic glycolipids, and anionic lipoproteins). The alternative mechanism invokes the existence of an ATP-dependent proton pump in lysosomal membranes (cf. Duncan, 1966, 1967). Certain discrepancies between the results obtained, on the one hand, by Mego (1973), Iritani and Wells (1974), Dell’Antone (1979), D. L. Schneider (l979), Reeves and Reames (1981), and Working and Meizcl ( 1 981), and, on the other hand, those obtained by Henning (1975), Reijngoud and Tager (1977), and Hollemans et ul. (1980), require resolution in future investigations. However, the application of new and more precise techniques to determine the intralysosomal pH in living cells (Ohkuma and Poole, 1978; Poole and Ohkuma, 1981) and recent efforts to characterize the enzymic complement of purified lysosomes (D. L. Schneider, et al., 1978; D. L. Schneider, 1981; Ohkuma etal., 1982) have provided strong evidence for the presence of a lysosomal proton pump driven by Mg2 and ATP. Pending confirmation in future investigations, such a lysosomal H -ATPase may be related to the well-characterized H -ATPase found in secretion granules of adrenal chromaffin cells (Johnson and Scarpa, 1976; Pollard et al., 1979; Geisow, 1982). Variations in the accessibility and catalytic activity of lysosomal enzymes may be effected by agents that modify the molecular architecture of the lysosomal +
+
+
+
+
+
LYSOSOMAL FUNCTlONS IN CELLULAR ACTIVATION
55
membrane or which act intralysosomally to promote osmotic swelling or changes in pH (cf. de Duve et al., 1974; Peters et al., 1972). For example, lysosomes of rat liver are larger and more fragile in the absence of insulin (Dice and Walker, 1978; Pfeifer, 1978), an established promotor of lysosomal membrane integrity (Mortimore et al., 1978). Detailed surveys of the effects of the numerous substances (e.g., retinoids, steroids, bacterial toxins, asbestos, metals, thiols) with capacity to stabilize or labilize lysosomal structure have been presented (cf. Weissmann, 1964; Lucy, 1969; Allison, 1969; Szego, 1975). The balance among agents with labilizing effects vs those that promote lysosomal and other membrane integrity governs the net outcome in relative membrane stability. In general, the effect of a given substance may vary greatly in different tissues (Weissmann et al., 1967) and also may involve either labilization or stabilization, depending on the biochemical character of the membrane and on concentration of the agonist. Some substances that exhibit such dose-dependent, biphasic effects (e.g., cortisol, indomethacin, retinoids, zinc ions) likewise appear to elicit differential effects on the membranes of primary and secondary lysosomes and probably on plasmalemma and other endomembranes as well (cf. Gordon, 1973; Szego, 1972b, 1974, 1975, 1982). In addition, it is important to note that certain agonists shown to be effective in provoking the selective release of lysosomal enzymes from intact cells fail to exhibit such an effect when added to isolated lysosomes (Nemere and Szego, 1981b; Szego, 1982). The effects of agents that reduce the structural latency of lysosomal marker enzymes are often irreversible, but dimethyl sulfoxide, for example, is known to elicit a reversible change in permeability of lysosomal membranes (Misch and Misch, 1975; see Szego, 1975; Dean and Barrett, 1976; Ludwig and Chvapil, 1980, for other examples). The capacity of lysosomes with an acidic interior to accumulate weakly basic substances (e.g., ammonia, chloroquine, acridine) also leads to reduction of catabolic activity (cf. Wibo and Poole, 1974), as will be considered in more detail in Section II,B,5. 4. Synthesis and Processing of Lysosomal Hydrolases and Cellular Pathways of Lysosome Formation The modes of synthesis and processing of lysosomal enzymes and of their subsequent packaging in lysosomes continue to be subjects of intensive research (Figs. 5A and B). Initial studies with inhibitors of protein synthesis suggested that new enzyme synthesis was required for maintenance of long-term increases in lysosomal hydrolase activity in several cell types (cf. Dean and Barrett, 1976). The marked increase in total P-glucuronidase activity of uteri from ovariectomized rodents after treatment with estrogen is a classic example of hormonal induction of lysosomal enzyme synthesis (Fishman and Fishman, 1944). Quantitative studies like these have been confirmed and extended by numerous investigators (cf. Table VI). Formation of lysosomal vesicles in cells is usually cou-
FIG.5 . ( A ) Diagram illustrating the GERL (Golgiiendoplasmic reticulum/lysosomes) theory of Novikoff. Reprinted with permission from Novikoff ef al. (1964). Following is the original caption. Suggested pathways for movement of materials from endoplasmic reticulum and external milieu. Proteins synthesized at the ribosome (upper right) are considered to gain access to rough endoplasmic reticulum (RER) and move through smooth endoplasmic reticulum (SER) from which they may be routed to microbodies (MB) or lysosomes. Four pathways to lysosomes are indicated: (1) via Golgi aaccules (Sac.) to Golgi vesicles (Ves.); (2) via Golgi saccules to Golgi vacuoles (Vac.); (3) directly from endoplasmic reticulum into dense bodies (DB); and (4)from endoplasmic reticulum into areas of cytoplasm isolated as an autophagic vacuole (AV). Endoplasmic reticulum, ribosomes, and a mitochondrion are shown within the forming autophagic vacuole. The polarized nature of the Golgi apparatus (GA) is suggcstcd, with forming face to right and niature facc to left. Golgi vesicles arc shown moving into a pinocytotic or phagocytic vacuole (PV) forming from cell surface and into a multivesicular body (MVB) to which micropinocytosis vacuoles bring protein molecules. Broken arrows indicate possible formation of residual body of dense body variety (DB) from multivesicular bodies or cndocytotic vacuolcs (PV). (B) Schematic representation of potential pathways for the cellular processing and packaging of lysosomal hydrolases. Enzymes are assumed to he synthesized its. on polysomes bound to rough endoplasmic reticulum (RER) and then to he segregated within -.
LYSOSOMAL FUNCTIONS IN CELLULAR ACTIVATION
57
pled in positive fashion with the synthesis of lysosomal enzymes per se (Ahearn et al., 1966; Sloane, 1980). Lysosomal enzymes are generally assumed to be snythesized on polysomes bound to endoplasmic reticulum (ER) and then to be segregated within its cisternae, as observed for secretory proteins (cf. Cohn and Fedorko, 1969; Erickson and Blobel, 1979; Schachter, 1981; Rosenfeld et al., 1982). Secretory proteins are generally synthesized as precursors with small “signal peptides” thought to be important for the secretion of proteins into the cisternal space of the ER (Blobel, 1977). There is considerable evidence that lysosomal enzymes are likewise synthesized as precursors of higher molecular weight (Erickson and Blobel, 1979; J. S. Huang, et al., 1979; Hasilik, 1980; Hasilik and Neufeld, 1980a,b; Sly, 1980; P. C. Heinrich, 1982), but none as yet to implicate directly the supernumerary sequences of the precursor forms in a vectorial transport function. However, by employing a cell-free mRNA translation system, Erickson and Blobel (1979) have found in in vitro mixing experiments that a 46K-M,, core glycosylated form of cathepsin D is formed at the expense of an initial 43K-M, form only in the presence of added microsomal membrane vesicles (cf. also, Rosenfeld et al., 1982). The 46K-M,, but not the 43K-M, form, is resistant to pronase unless the membrane vesicles are disrupted by detergent treatment. Presumably, the 46K-M, form is protected from proteolysis by its segregation in the luminal space of the microsomes and also by its carbohydrate content. Although another putative precursor of cathepsin D with considerably higher molecular weight (i.e., M,, 100K) has been reported (J. S . Huang et al., 1979), its significance, if any, remains unknown (cf. Erickson and Blobel, 1979; Hasilik and Neufeld, 1980a). A second signal that may allow cells to sort out lysosomal enzymes from other proteins in the cisternal space of ER appears to be a specific carbohydrate constituent of these hydrolases (cf. Knecht and Dimond, 1981). Kaplan et al. (1 977) were first to present evidence implicating mannose-6-phosphate or a closely related phosphorylated sugar as a specific recognition marker for the incorporation of lysosomal enzymes into organellar form in fibroblasts and liver cisternae concomitantly with other secretory and membrane-destined proteins. The newly synthesized proteins migrate from RER to smooth ER (SER) or to the vicinity of the Golgi apparatus. In pathway ( I ) , lysosomal enzymes, but not other proteins ( ), bearing a phosphorylated sugar residue (4) are recognized by specific membrane receptors in the ER-Golgi region and membrane encapsulated, resulting in the formation of primary lysosomes. In pathway (2), lysosomal enzymes destined for packaging are released extracellularly along with the secretory proteins, but are then bound by specific plasma membrane receptors, followed by endocytosis and lysosome formation. Pathway (3) represents a modification of the latter proposal wherein lysosomal enzymes are considered to remain firmly bound to membrane receptors during the entire process of exocytosis. followed by subsequent endocytosis. See text for references and additional details, including probable relative quantitative significance of the proposed pathways. These concepts are still in flux.
58
CLARA M. SZEGO AND RICHARD J. PIETRAS (man)p(glcNAc)zasn,
I I I
I t
1 1 +
(A) \
'4
(mon)r(glcNAc)zasn (8)
( S A ~ g o l ~ g l c N A c ) ~ ( m o n ) I ( g l c N A c ) ~ a(C) sn
(glcNAcOzP-)a(man)t(glcNAc)zosn
1
(HO,P-)r(man)t(glcNAc)zasn
(D)
(El
FIG.6. Proposed steps in the processing of lysosomal enzymes. At a certain stage of processing, asparagine-linked oligosaccharides (A) may be converted either into a high-mannose type (B)by accepting additional mannose residues, a complex type (C) by addition of the appropriate sugars, or a phosphorylated type (D).The signals that dictate the type of oligosaccharide finally found in the protein are probably coded for in the primary structure of the polypeptide chain. Compound (D), the suggested precursor of the recognition marker (E). could be formed by transfer of N-acetylglucosamine-l-phosphate from one of its known pyrophosphate derivatives. Reprinted from Hasilik (1980). with permission. See also Sly (1982) and Mellman (1982).
cells (see reviews by Sly, 1980, 1982; Hasilik, 1980; Fig. 6). The asparaginelinked oligosaccharide chains of such lysosomal glycoproteins are generally considered to originate through sequential processing of a common precursor oligosaccharide acquired by the polypeptide in the endoplasmic reticulum. The glycosylated precursor appears to contain an inner core of three branched sugar chains with 6 to 7 mannose residues (Hasilik et al., 1980; Mizuochi et al., 1981). Up to 3 a-N-acetylglucosamine- 1-phosphate groups are later transferred to the carbon-6 hydroxyl of mannose residues in the precursor structure through the action of an N-acetylglucosaminyl phosphotransferase with high affinity toward native lysosomal enzymes (Tabas and Kornfeld, 1980; Reitman and Kornfeld, 198 1). An N-acetylglucosaminyl phosphodiesterase, found to be 'enriched in Golgi fractions, is helieved subsequently to catalyze the release of N-acetylglucosamine residues (Waheed et ul., 1981). The latter hydrolysis promotes exposure of the mannose-6-phosphate residues which appear to be recognized by specific cellular receptors. For example, binding of P-hexosaminidase B to membrane fractions from rat liver is saturable, competitively inhibited by mannose-6-phosphate and obliterated by prior treatment of the enzyme with alkaline phosphatase or endoglycosidase H (Fischer et al., 1980b). Moreover, in liver and fibroblasts, 80% of such enzyme recognition sites occur in membranes of ER, with 7% in Golgi, 5% in lysosomes, 10% in plasma membranes, and less than 2% in nuclei and mitochondria (Fischer et ul., 1980a,b). Most of the recognition sites in ER fractions were found to be occupied by endogenous ligand that could be displaced by mannose-6-phosphate in the presence of detergent, whereas the bulk of the binding sites in plasma membrane and lysosome fractions were unoccupied (Fischer et u l . , 1980b). Thus, efficient routing of
LYSOSOMAL FUNCTIONS IN CELLULAR ACTIVATION
59
lysosomal enzymes, at least in fibroblast and liver cells, appears to be dependent on binding of the modified hydrolases to phosphomannosyl and possibly other sugar-linked recognition sites that occur predominantly in the ER and Golgi. Partial purification and characterization of such putative membrane receptors has been achieved recently (Sahagian et al., 1981; Steiner and Rome, 1982). Kinetic studies on the translocation of lysosomal enzymes (cf. Cohn and Fedorko, 1969; Goldstone and Koenig, 1973; Dean and Barrett, 1976), which tend to confirm earlier histochemical work (cf. Novikoff et al., 1964; de Duve and Wattiaux, 1966; de Duve, 1969), indicate that newly synthesized enzymes migrate from rough ER to smooth ER, to, or to the vicinity of, the Golgi apparatus, and finally to lysosomes. Novikoff et al. (1964) suggested that formation of lysosomes occurs in a special compartment, the GERL (Golgi-ER-lysosomes), interpolated between the ER and trans -Golgi components (cf. Fig. 5A, and Novikoff and Novikoff, 1977). However, an alternative “secretion-recapture” hypothesis proposes that lysosomal enzymes are first exocytosed as modified glycoproteins (i.e., a pathway similar to that of the aforementioned secretory proteins) and then endocytosed as a consequence of interaction with surface membrane recognition sites (Neufeld et al., 1977; stippled arrows at pathway 2, Fig. 5B). Although considerable amounts of lysosomal enzymes are found in the extracellular media of cell preparations (cf. Pietras et al., 1981a), the relative distribution of intracellular and extracellular enzyme activities in fibroblast and liver cells is not profoundly altered by competitive inhibition with mannose-6phosphate of hydrolase uptake via the plasma membrane route, nor by binding of extracellular enzymes to immobilized antibodies presented in the incubation media (e.g., von Figura and Weber, 1978; Sly, 1980; Hasilik and Neufeld, 1980a). Such findings are consistent with the intrucellular packaging hypothesis (e.g., also, Lloyd, 1977). However, to explain the failure of mannose-6-phosphate in the extracellular medium to deplete intracellular enzyme levels, von Figura and Weber (1978) have suggested that enzymes may be delivered to plasma membrane already tightly bound to the putative recognition site and then integrated without dissociation. In support of the latter proposal, von Figura and Voss (1979) have demonstrated that 0.5 to 4.3% of the intracellular activity of four different lysosomal enzymes is available at the surface membrane of fibroblasts for interaction with monospecific antisera. However, association of lysosomal enzymes with the cell surface may not be a general phenomenon, since cultured smooth muscle cells and endothelial cells do not exhibit cell surface-associated lysosomal hydrolases (see von Figura and Voss, 1979; however, cf. Sloane, 1980). Further, it has been reported that about 90% of endogenous, bound lysosomal enzymes and nearly 99% of occupied phosphomannosyl enzyme recognition sites are normally present intracellularly (and predominantly in ER) in fibroblasts (Fischer et al., 1980a) and in liver (Fischer et al., 1980b). Thus, on the above grounds, one might conclude that (1) the predominant
60
CLARA M. SZEGO AND RICHARD J . PIETRAS
route for transport of lysosomal enzymes to lysosomal vesicles occurs intracellularly by vectorial translocation from specialized regions of ER in close proximity to the Golgi apparatus (see Fig. 5A and also dark arrows, fig. 5B), and (2) the transfer of lysosomal enzymes via the plasma membrane, en route to lysosomes, apparently represents a minor pathway in normal (i.e., nontransformed, nonpathologic) cells. Unfortunately, a number of exceptions to the latter generalizations are now documented. For example, Jessup and Dean ( I 982) find that normal macrophages, unlike normal fibroblasts, continuously secrete lysosomal hydrolases and appear to route enzymes to lysosomes via phosphomannosyl enzyme recognition at the cell surface (i.e., as proposed in the secretion-recapture hypothesis). Several extracellular lysosomal hydrolases are also known to be recognized and taken up by a mannose-specific receptor at the surface of macrophages and Kupffer cells (cf. Sly et al., 1981; Tietze et al., 1982) and possibly by a galactosyl-glycoprotein or other incompletely characterized macromolecular receptor in liver (cf. Sly et al., 1981; Owada and Neufeld, 1982). One lysosomal enzyme in liver, P-glucuronidase, is segregated not only in lysosomes but also in endoplasmic reticulum, apparently through the mediation of a specific P-glucuronidase-binding protein (cf. Paigen, 1981). Moreover, even in fibroblasts, two enzymes, acid phosphatase and P-glucocerebrosidase, are suspected to segregate in lysosomes by a pathway which does not require the phosphomannosyl enzyme recognition marker (cf. Sly et al., 1981). Hence, although the phosphomannosyl enzyme recognition system of the ERGolgi may be the major pathway for delivery of most hydrolases to lysosomes in fibroblasts and perhaps in other cells, it is equally apparent that alternative routes, receptors, and destinations for lysosomal enzymes may exist in certain cases. The interrelationships among these several pathways, represented only in part in Figs. 5A and B, remain to be defined. Clearly, the occurrence of extracellular and plasma membrane-associated lysosomal enzymes in certain situations may be a consequence of quantitative limitations of intracellular recognition systems or of tonic membrane recycling mechanisms dependent upon lysosomal enzyme exocytosis (cf. de Duve, 1969; Lloyd, 1977; Pietras et al., 1981a). Inappropriate localization of hydrolytic enzymes has also been noted in certain genetic diseases (cf. Neufeld el a]., 1975). Thus, Wiesmann et al. (1971) observed that fibroblasts from patients with mucolipidosis I1 (I-cell disease) were deficient in several glycosidases and sulfatases, while extracellular fluids exhibited an excess of these enzymes. Such fibroblasts bear normal recognition sites for the lysosomal enzymes, but the affected enzymes lack a phosphomannosyl recognition marker (Hasilik and Neufeld, 1980b). Consequently, these enzymes are apparently not bound and segregated intracellularly in ER or GERL, but proceed instead through the Golgi apparatus where their oligosaccharide chains appear to be processed to the complex type (i.e., N-acetylglucosamine-, galactose-, and sialic acid-containing chains). The resulting resemblance in carbohy-
LYSOSOMAL FUNCTIONS IN CELLULAR ACTIVATION
61
drate structure to that of secretory glycoproteins is presumed to signal the aberrant secretion of such lysosomal hydrolases (cf. Sly, 1980). Following delivery of lysosomal enzymes to vesicles, the polypeptide chains of the enzymes initially persist as larger molecular weight precursors (not to be confused with the very short-lived forms found in ER) but are subsequently processed to the smaller size of those structures that predominate in vivo (Erickson and Blobel, 1979; J. S. Huang et al., 1979; Hasilik and Neufeld, 1980a). Erickson and Blobel (1979) have suggested that the precursor forms may occur largely in primary lysosomes. Diminished forms retaining enzyme activity would then be produced in activated or secondary lysosomes, possibly by the action of an acid thiol proteinase (Frisch and Neufeld, 1981). Hasilik and Neufeld (1980a) have identified precursor, but not processed, chains of lysosomal enzymes with biocatalytic activity in the extracellular media of fibroblasts. Lysosomal enzyme transport and maturation appears further to involve dissociation of enzyme from membrane-localized phosphomannosyl or other related carbohydrate recognition sites (cf. Das and Bishayee, 1980; Burnside and Schneider, 1980) and recycling of the unoccupied binding moiety (Fischer et al., 1980a). P-Glucuronidase has been found to dissociate from specific recognition sites of fibroblast membranes at pH 5.0 (i.e., comparable to intralysosomal pH levels). The functional activities of such sites are fully restored upon return to neutral pH (Fischer et al., 1980a). The latter authors have also calculated that the pool size of such cellular enzyme recognition sites is inadequate to explain observed rates of enzyme incorporation into lysosomes without invoking some mechanism for reutilization of the binding moieties. Hence, it is important to determine whether the proposed rate of recycling of the specialized receptors is sufficiently rapid to achieve the efficient intracellular capture and segregation of lysosomal enzymes that is required during periods of heightened cellular activity (e.g., after serum or hormone exposure; cf. Szego, 1974, 1975, 1978). If not, exocytosis or mislocalization of enzymes may occur (see below).
5. Functions of Lysosomes in the Normal Cellular Economy a. Mobility and Fusion Potential for Cross-Compartment Communication. Under basal conditions, lysosomal structures are often disposed at the cytoplasmic periphery of cells, presumably a strategic position for the interception of incoming endocytotic vesicles (Szego, 1975). Lysosomal membranes can undergo fusion with those of endocytotic vesicles, with those of endoplasmic reticulum undergoing autophagy, with those of the plasma membrane (i.e., during the process of exocytosis), and with those of other components of the vacuolar apparatus (de Duve, 1969; Dean and Barrett, 1976; Y. J . Schneideret al., 1981). However, under basal conditions, lysosomal membranes do not fuse readily with those of other intracellular organelles such as nuclei, mitochondria, or peroxisomes.
62
CLARA M. SZEGO AND RICHARD J. PIETRAS
Indirect evidence suggests that the intracellular movements of lysosomes and related vesicles may be restrained and/or promoted by microtubular or microfilament systems (cf. Allison, 1973; Szego, 1975; Rohlich and Allison, 1976; Hartwig et al., 1977; Oliver and Berlin, 1979; Mori et al., 1981). One of the more intriguing open questions in relation to impending fusion of lysosomes with the plasmalemma and/or newly formed endocytotic vesicles in “activated” cells is the means of access of lysosomes to the subplasmalemmal cell cortex. It has been proposed by Hartwig et al. (1977), on the basis of observations on macrophage spreading, phagocytosis, and phagolysosome formation, that actin-binding protein and myosin, which are concentrated in the cortical cytoplasm under basal conditions, are displaced into the advancing pseudopodia during spreading. This process, for which no unequivocal explanation is yet available, is inferred to promote gelation and compression of actin with resultant exclusion of organelles from the pseudopod. Concomitantly, actin filaments behind the advancing pseudopodia are disaggregated due to local relative depletion of myosin and actin-binding proteins, thus permitting lysosomes to enter the cell periphery and approach the plasmalemma. Membrane-bounded structures that move through the cytoplasm are sometimes observed in close physical, and occasionally chemically specific, contact with cytoskeletal structures such as microtubules (Smith, 1971; Allen, 1975; Burridge and Phillips, 1975; Sherline et al., 1977; Heggeness et al.. 1978; von Figurd et a l . , 1978; Ockleford and Munn, 1980), niicrofilaments (cf. Allison, 1973; Szego, 1975; Moore et at., 1976), and intermediate filaments (cf. Jimbow and Fitzpatrick, 1975; Dentler, 1981; see Table XVIIB, below). Indeed, lysosomes are capable of saltatory, essentially rectilinear, movements that are often blocked by inhibitors of microtubule assembly (Freed and Lebowitz, 1970; Wagner and Rosenberg, 1973; Malawista, 1975; Parr et al., 1978). However, the requirement of an endergonic propulsive system is not clear. Some investigators (e.g., Ockleford and Munn, 1980) have suggested that a Brownian motion-powered ratchet mechanism, permitting vesicle movement in thermodynamically favorable directions, e.g., alongside microtubules or microfilaments, may be a more energy-efficient alternative. The apparent presence of an (acid) ATPase in lysosomes of liver (Nakabayashi and Ikezawa, 1980) and of actin-like material in thyroid lysosome fractions (Dickson er af.,1979), like that associated with chromaffin granules (Meyer and Burger, 1979), indicates the need for further investigation of the sources of motive power for the rapid translocations to which lysosomes, as well as endocytotic vesicles (Willingham and Pastan, 1978), are subject in nietabolically active cells. Unfortunately, attack upon this major problem is hampered by the need to identify constituents requisite to the generation of motion as unequivocally endogenous to the primary organelle. All models thus far proposed suffer from this crucial deficiency. In cells activated by a variety of circumstances (e.g., commitment toward cell division or differentiation), lysosornes are often observed to migrate to the nu-
LYSOSOMAL FUNCTIONS IN CELLULAR ACTIVATION
63
clear region, and with the conspicuous feature of polarity (cf. Szego, 1974, 1975). Such a perinuclear concentration of lysosomes has been identified in lymphocyte transformation (Allison and Mallucci, 1964b; Hirschhorn and Hirschhorn, 1965), liver regeneration (Kent et al., 1965), premitotic cellular reorganization (Robbins and Gonatas, 1964), spermatogenesis (Henley, 1968), and oocyte maturation (Lopata et al., 1977; Ezzell and Szego, 1979; see Section III,D, below, for additional examples). Concomitantly, minute amounts of lysosomal hydrolase activities, normally foreign to the nuclear compartment (Szego, 1974, 1975, 1978; Fiszer-Szafarz and Nadal, 1977; Mak and Wells, 1977), as well as lysosomal antigens (Szego et al., 1977), may be identified within the nuclei of such activated cells (Szego et a l . , 1977). Additional examples of the unique intracellular mobility of lysosomes occur in the vectorial transfer of these organelles and their characteristic hydrolases to the luminal membrane of cells with specialized secretory (cf. Dumont, 1971; Szego et al., 1977) or transport (Pietras et al., 1975) functions. These will be presented in more detail in succeeding sections (cf. also Y.-J. Schneider et al., 1978). Indeed, there has now arisen a limited body of evidence demonstrating the exocytosis of lysosome-like vesicles, as such, from cells activated by a variety of circumstances, both normal (Mishima and Kondo, 1981) and pathologic (Hashimoto er a!., 1977; Paigen, 1979; Greenberg, 1981). Under certain conditions, such as those associated with pigment granule donation (cf. Mottaz and Zelickson, 1967; Klaus, 1969) or the cytolytic function of activated macrophages (Hibbs, 1974, 1976; Bucana et al., 1976), it has been suggested that such exfoliated organelles may be transferred directly to neighboring cells (cf. however, Urban, 1981). The latter process may not be unlike that of the in vifro uptake of mast cell granules by peritoneal macrophages (Lindahl et al., 1979) or even the intracellular acquisition of liposome-encapsulated markers (cf. Weinstein et al., 1977). Apparent organellar infusion to the oocyte from surrounding follicular cells via transitory needle-like bridges has likewise been identified in cinemicrographic analyses of cell preparations carried out in vitro (A. Lopata, J . R. Fonseca, and C. M. Szego, unpublished observations). Although the exchange of cytoplasm through bridges between mammalian cells has long been known (Bendich et a l . , 1967; Espey and Stutts, 1972), the frequency and significance of organellar exchange, especially in relation to heightened intercellular communication in circumstances of hormonal activation (see Section III,D), remain to be investigated in detail. b. Regulation of the Turnover of Cellular Protein and Other Metabolites. Numerous comprehensive reviews of the astounding digestive capacity of lysosomal enzymes in vitro are available (see citations in opening paragraph of this section). It is clear that the enzyme complement of the lysosomal apparatus is more than sufficient to degrade essentially all endogenous and exogenous proteins, carbohydrate moieties, nucleic acids, and complex lipids to products of
64
CLARA M. SZEGO AND RICHARD J . PIETRAS
low molecular weight (cf. Dean and Barrett, 1976). Such concerted catabolic functions are potentially capable of providing important cellular pools of amino acids, monosaccharides, nucleosides, free fatty acids, inorganic ions, and other constituents that are subject to reutilization in situ in maintenance of vital cell functions (Szego, 197 la). Unfortunately, there is at present little information on the cdtabolism in vivo of macromolecules other than protein by normal lysosomes (cf. Burton et al., 1975; Neufeld el al., 1975; Dean and Barrett, 1976; Rome and Crain, 1981). However, considerable evidence has recently accumulated on the pivotal role of lysosomes in protein metabolism in vivu (cf. Dean, 1975b; Goldberg and St. John, 1976; Segal, 1976; Dean, 1980) and will constitute the focus of the remaining discussion in this section. A priori considerations require that maintenance of stable net levels of given cellular protein(s) can be achieved by restraints on degradation as well as synthesis. However, until very recently, there has been intense preoccupation with factors, including hormones, which regulate synthesis, to the exclusion of controls over degradation. This situation has now changed dramatically. Ballard (1977, 1980) has proposed that lysosomal as well as nonlysosomal pathways of protein degradation, albeit with different specificities, contribute to the general turnover of endogenous protein in animal cells (cf. also, Goldberg and Dice, 1974; Goldberg and St. John, 1976; Dice and Walker, 1978; Seglen et al., 1979; Amenta et al., 1978). Short-lived proteins, which account for the low basal level of protein turnover under nutritionally optimal conditions (e.g., usually identified by incorporation of labeled amino acid within 1 hour), are considered to be preferentially degraded by an extralysosomal pathway. In contrast, it is inferred from experiments with 24-hour labeling that the lysosomal pathway predominates in the catabolism of long-lived proteins such as membrane or exogeneously administered proteins. The latter pathway, which is activated in cell culture by starvation for serum or amino acids or by treatment with specific hormones, is sensitive to inhibition by ammonia and other lysosomotropic drugs, while the nonlysosomal component apparently is not. The short-lived proteins represent about 15-2096 of total protein biosynthesis in liver but, due to their rapid breakdown in vivo, constitute only about 0.2% of total intracellular protein (Mortimore and Hutson, 1981). Dean (1980) has emphasized that the apparent distinctions between lysosomal and nonlysosomal pathways of protein turnover are only relative, and do not account for the cumulative evidence on lysosomal participation in basal protein catabolism. Indeed, Dean (1975b) has provided data on the selective uptake of short-lived cytoplasmic proteins by purified rat liver lysosomes in v i m . He argues forcefully that the different rates of degradation of such cytoplasmic proteins may well be determined by their specific rates of entry into lysosomes. Evidence in support of the latter hypothesis has been presented (Stacey and Allfrey, 1977; Marzella et a[., 1980; Russell et al., 1982). One source of such
LYSOSOMAL FUNCTIONS IN CELLULAR ACTIVATION
65
discrepant views is the tacit assumption by most investigators that, to be susceptible to proteolysis by lysosomal hydrolases, a given protein must find its way into the lysosomal compartment (Goldberg and Dice, 1974). For proteins that enter the cell by endocytosis, this route is now well established. However, as noted above, it has been widely assumed that cytosolic proteins do not have access to the lysosomal interior. Accordingly, a “cytosolic” proteolytic system, independent of the lysosomal compartment, is inferred as a mechanism for the degradation, for example, of microinjected proteins (Bigelow et al., 1981; however, cf. Stacey and Allfrey, 1977). Only minimal consideration has been given to the likelihood that even purportedly “mild” intervention in cellular function (e.g., microinjection or endocytosis of foreign proteins) may lead to limited escape of lysosome-sequestered components from structurally labilized organelles (cf. Table I, and Szego, 1974). The possibility that the active site of some lysosomal enzymes, including cathepsins (Pontremoli et al., 1982), may be accessible for interaction with substrate at the cytoplasmic surface of the organelle (see Section II,B,2,d) also requires extension and clarification. A final point, which remains to be addressed, is the premise that the selectivity of protein degradation in vivo may depend, in part, on the differential susceptibility of proteins to given proteinases (Segal, 1976). Hence, the validity and universality of the dual pathway model proposed by Ballard (1977, 1980) and others cannot yet be fully evaluated. Application of lysosomotropic amines and specific proteinase inhibitors has proved useful in efforts to distinguish between the relative contributions of lysosomal and nonlysosomal pathways in cellular protein turnover. Weakly basic substances such as chloroquine and ammonia are well known to reduce the cellular degradation of proteins (Wibo and Poole, 1974), with preferential inhibition of the catabolism of long-lived (i.e., ca. 70% inhibition), as opposed to short-lived (i.e., ca., 45% inhibition), proteins (Seglen et al., 1979). This effect is believed to be a consequence of intralysosomal accumulation of the weak bases. These diffuse readily into lysosomes in their unionized forms, while ionization of the compounds in the acidic interior of the lysosome severely limits their outward movement. Hence, it is estimated that after 2 to 4 hours of incubation, chloroquine is concentrated from a dilute extracellular solution (i.e., 100 p,M) up to 1000 times by lysosomes of fibroblasts (Wibo and Poole, 1974). Some investigators have proposed that the antiproteolytic action of weak bases may result from neutralization of the acid pH within lysosomes to a point at which most “acid” hydrolases would no longer be active. However, others argue that very little drug would be required to achieve such neutralization, and, unless the intralysosomal pH remained low, there would be no further accumulation of weak base within lysosomes (de Duve er al., 1974; Wibo and Poole, 1974). Indeed, recent determinations of intralysosomal pH in living cells with pH-dependent fluorescent probes show that chloroquine increases pH from 4.5 to
66
CLARA M. SZEGO AND RICHARD J . PIETRAS
5.0 only to about 6.0 to 6.5 (Ohkuma and Poole, 1978; Poole and Ohkuma, 1981). A further proposal to explain the inhibitory effect of weak bases on cellular protein degradation is the direct suppression of specific proteinase activities by such compounds. Thus, Wibo and Poole (1974) reported inhibition by chloroquinc administered in vivo of cathepsin B activity, which represents an estimated 12% of total lysosomal protein in liver (cf. Dean and Barrett, 1976). However, activity of the enzyme in vitro was only partially inhibited, with maximal effects of the drug occurring at a concentration of 0.1 M (cf. also, MacGregor et al., 1979). In addition, chloroquine failed to inhibit the activities of other lysosomal proteinases, including cathepsins D, A, and C (Wibo and Polle, 1974). Observations by Ose et al. (1980) have further shown that the extent of inhibition of the degradation of specific exogenous proteins by hepatocytes in the presence of either ammonia or chloroquine is augmented by addition of leupeptin, an inhibitor of cathepsin B (Umezawa and Aoyagi, 1977). In other words, some finite portion of total enzyme activity, presumably lysosomal cathepsin B, beyond that affected by the weak bases, remains subject to inhibition by leupeptin. Unfortunately, the mode(s) of inhibition of protein degradation by lysosomotropic amines remains uncertain. A selected survey of findings shown in Table VII indicates that the influence of these compounds on protein catabolism in lysosomes may be attributable not to one central action but rather to a concerted series of effects in the cell. Although several studies offer evidence for limitation of the entry of protein into the lysosomal compartment after amine exposure (see Table VIl), others emphasize the generalized toxicity of these compounds including, for example, their capacity to inhibit protein synthesis, features that tend to be overlooked by many investigators. Hence, the problem of signal vs noise, especially wherein the noise may constitute cell death in the present context (cf. Visek et af., 1972; Seglen et al., 1979; Houdebine and Djiane, 1980; see also, Table I, and Section IV,A), remains to be addressed in studies utilizing lysosomotropic amines as metabolic probes. New efforts are currently underway to find agents with a capacity for specific inhibition of lysosomal proteolysis but without significant toxic effects upon other cellular functions. A promising approach appears to be the use of a class of microbial peptides with inhibitory activity twoard specific proteinases (Umezawa and Aoyagi, 1977). The peptide inhibitors can sometimes be delivered to cells in free form or, preferably, they may be directed more efficiently and specifically to the lysosome compartment by encapsulation within cationic lipid vesicles (Dean, 1975b; Lasch et al., 1977; Pietras, 1978; Pietras and Szego, 1979b; Pietras et al., 1981a; see Fig. 7). For example, Dean (1975b) entrapped in liposomes pepstatin, an inhibitor of carboxyl proteinases such as cathepsin D. Perfusion of livers that had been prelabeled with [ ''C]valine for several hours with pepstatinloaded liposomes, but not with control liposomes or free pepstatin, elicited
LYSOSOMAL FUNCTIONS I N CELLULAR ACTIVATION
67
TABLE VI1 R t P R E S t N I A IIVE
EXAMPLES <>I-
I N H l H l T O K Y f%I-t.CTS 01'LYSOSOMOTROPIC AhllNES
O N V A R I O U S CL.l.LUI.AK
Cell function inhibited Activity of cathepsin B lntracellular packaging of newly synthesized hydrolases in lysosonies Protein becretion Clustering of occupied cell surface receptors Adsorptive uptake of macromolecules Fusion of coated vesicles with lysosomes Processing or degradation of internalized peptide hormones
Processing or degradation of internalized toxins Recycling of mannose-specific receptors Protein labeling from isotopic amino acid Activities of several lysosomal enzymes involved in phospholipid catabolism Cholesterol biosynthesis Activities of DNA and RNA polymeraxes RNA and DNA labeling from appropriate isotopic precursor Cell spreading Cell division and viability
FUNCTIONS~' Reference
Wibo and Poole (1974) Willcox and Rattray (1979); Hasilik and Neufeld (1980a,b); Gonzalez-Noriega P I a/. ( 1980) Seglen and Reith (1977); Johnson et a / . (1978); Neblock and Berg ( 1982) Maxfield et a/. (1979a) Corden c't al. (1978b); Gliemann and Some ( 1978); Sando et a/. ( 1979) Libby et a/. (1980); Bursztajn and Libby (1981) Carpenter and Cohen (1976); Ascoli and Puett (1978b); King et a / . (1980a); Tsai and Seeman (1981); Posner et a/. (1982a); Smith and Jarett (1982) Draper and Simon (1980); Leppla et a/. (1980); Houslay and Elliott (1981) Tietze et a/. (1980) Seglen ( I 978); Seglen et a/. ( I 979); Houdebine and Dijiane (1980) Kunze et a/. ( I 982) Beynen et u / . (1981) Cohen and Yielding (1965) Schellenberg and Coatney ( I 96 I); Gabourel (1963); Stollar and Levine (1963); Cohen and Yielding (1965); Lancz e t a / . (1971) Seglen and Gordon ( I 979) Gabourel (1963); Lipp (1964); Visek er a / . (1972); Seglen et al. (1979); Firestone et a / . (1979); King e r a / . (1981)
"Drugs presented to the isolated cells or tissues in the several studies included NH4CI, chloroquine, methylamine, butylamine, and triethylamine at concentrations ranging from 1 pM to 10 mM. It is to be noted that the minimum effective dose and requirement for prolonged exposure to an effective concentration of a given drug vary widely. Failure to provide adequate and sustained levels has sometimes resulted in conflicting data (cf. Maxfield et al., 1979a, vs King er a / . , 1981).
inhibition of the activity of cathepsin D and of the rate of catabolism of endogenous protein (Dean, 1975b). Application of free leupeptin or chymostatin, peptide inhibitors of cathepsin B in muscle, also slows the rate of proteolysis in muscle (Libby and Goldberg, 1978, 1980). Although the delivery of competitive inhibitors in liposornes may restrict such agents largely to the lysosomal com-
68
CLARA M. SZEGO AND RICHARD J. PIETRAS
LEUPEPTIN OR OTHER LYSOSOMAL-ENZYME
HYDROLASES
FIG. 7. Diagram of endocytotic uptake of liposome-entrapped leupeptin into the lysusomal apparatus. Upon interaction of liposomes with plasma membrane, uptake by phagocytosis is stimulated. A small portion of entrapped leupeptin may escape into the cytosol compartment at this step. Formation of secondary lysosomes (SL) ensues by fusion of phagosomes (Ph) with primary lysosomes (PL). Consequent hydrolysis of lipoposornal lipids allows for intralysosomal liberation and inhibitory action of leupeptin molecules on cathepsin B . See text and Pietras and Szego (1979b) for further details. RB, Residual body; M, mitochondrion; N, nucleus; G, Golgi apparatus; ER, endoplasmic reticulum. Redrawn from Lasch et a/.. (1977), and reproduced by permission.
partment (Dean, 1975b; Lasch et al., 1977; Pietras and Szego, 1979b; Pietras et al., 1981 a), the method of administration of unencapsulated compounds cannot. Consequently, the peptides given by the latter route are also free to inhibit the activity of extralysosomal proteinases (cf. Libby and Goldberg, 1980). On the assumption that ammonia elicits a relatively selective and complete inhibition of intralysosomal protein degradation, Grinde and Seglen ( 1980) utilized ammonia treatment to aid in discrimination between lysosomal and nonlysosomal effects of microbial peptides given in free form. These workers estimated that free leupeptin inhibited 80-85% of lysosomal and about 14% of extralysosomal protein breakdown in hepatocytes, while free chymostatin suppressed 45% of intralysosomal and 53% of extralysosomal pathways of protein catabolism (Grinde and Seglen, 1980). An unanticipated finding in studies with microbial proteinase inhibitors has been the observation of Seglen and co-workers that inhibition of lysosomal proteolysis also affects the rate of protein synthesis under conditions in which the supply of exogenous amino acids is limited (Seglen et al., 1979; Grinde and Seglen, 1980). However, Amenta and Brocher (1980) have suggested that the reduction in protein synthesis observed by Seglen and co-workers may be an experimental artifact attributable to (1) blockade of lysosomal proteolysis, resulting in loss of an endogenous pool of amino acids (cf. Seglen et al., 1979) and (2) restricted uptake by competitive inhibition of a limited exogenous supply of
LYSOSOMAL FUNCTIONS IN CELLULAR ACTIVATION
69
essential amino acids [i.e., due to the relatively high concentration of one amino acid (e.g., valine) in the external medium (cf. Seglen, 1978)l. Nevertheless, collectively, these results emphasize that lysosomal proteinases may participate in the cellular control of protein synthesis by providing an important rate-limiting pool of amino acids in situ (cf. Harms et al., 1981). This function of the lysosomal pathway becomes especially critical when the supply of extracellular amino acids is restricted (cf. Geiger, 1947). It is clear that one must be alert to lysosomal participation in the bimodal pathways contributing to “mobilization” vs “deposition” of cellular protein components. Thus, the activity of the lysosomal apparatus apears to be especially important in regulating net cell protein accumulation. In turn, control of this process by hormones and other biocatalytic agents, not excluding those present as trace “growth factors” in serum, may be an essential aspect of growth regulation. The latter topic will constitute a central focus for a succeeding section of this contribution.
GENERALIZED SCHEME
TABLE VIIl ACTIONSOF HORMONES:CORRELATIONS PROPERTIES IN THEIRTARGETCELLS”
FOR THE
Characteristics of hormone action to be accounted for ~
~~
LYSOSOMAL
Characteristics of lysosomes that may subserve these functions
~
1. Instantaneous and specific binding to recep-
1. fpecifc binding properties for a variety of
tors at selective target cells
2. Effectiveness in triggering concentrations
2.
3 . Pleiotropic effects; cascade with expanded time-base; indications of enhanced turnover of cellular constituents
3.
4. Migration into nuclear compartment in as-
4.
sociation with a fragment of initial receptor
5 . Triggering of genic derepression; enhancement of synthetic processes; mitogenesis
5.
6 . Tachyphylaris; receptor induction with pro-
6.
longed stimulation
WITH
organic and inorganic substances, soluble and particulate Propensity for reversible fusion with and detachment from other membranes, including the endosome (and thus, the plasmalemma, together with bound agonist) An array of structurally latent hydrolases and matrix proteins, made accessible by graded labilization of the bounding membrane on uptake of agonist Saltatory movement, apparently associated with microtubules; membrane-oriented ATPase activity; nuclear penetration on cellular activation Matricular proteins and hydrolases with potential for reducing affinity toward chromatin of putative repressors, including histones, by preferential interaction or limited proteolysis; RNA processing Depletion on extreme activation; rebound synthesis
BReprinted with minor modifications from Szego (19781, wherein will be found specific docurnentation.
70
CLARA M. SZECO AND RICHARD J . PIETRAS
111. Compatibility of Lysosomal Properties with Proposed Agonal
Mediating Functions in Activated Cells Given the composite properties of lysosomes outlined in the preceding section, together with the cogent evidence that limited lysosomal activation is not incompatible with normal cellular viability, we may now proceed to examine the initial premise: that lysosomes function in the propagation and amplification of the actions of effectors interacting with cell surface receptors. A. GENERALIZED SCHEME
Table Vlll presents a generalized scheme intended to serve as a frame of reference in analysis of this complex and far-reaching postulate. When taken in context with Figs. 1-4 and Tables 11, 1V and V, the unique lysosomal properties invoked in Table Vlll appear to have relevance for promotion and mediation of hormonal effects. However, crucial to the verification of this generalized postulate is cvidence that application of given effectors to their target cclls is indeed associated with acute recompartmentation of a limited proportion of lysosomal enzymes. Such evidence is presented in Table 1X. OF COVERT LAHILIZATION B. CIRCUMSTANTIAL EVIDENCE LYSOSOMAL MEMBRANES
OF
Even a cursory inspection of the data assembled in Table IX reveals a striking pattern common to the acute effects of hormones and a limited selection of other effectors of widely diverse structures: elevation of the proportion of hydrolytic marker enzyme present in cytoplasni and extracellular fluid (unbound) vs latent (structurally inaccessible to substrate until released by detergent or autolytic attack upon the bounding membrane). Such an apparently general phenomenon can hardly be attributed to “leakage” of enzymes following nonspecific cellular “damage,” for this phenomenon is not accompanied by corresponding liberation of marker enzymes from other subcellular compartments, including plasmalemma1 and mitochondria1 (Szego et d . , 1971; Pietras rt d.,1975; Pietras, 1978; Pietras and Szego, 1979b; Nerncrc and Szego, 1981a). Nor is there evidence for concomitant delivery to the extracellular environment of a soluble cytoplasmic enzyme, such a s lactate dehydrogenasc, as an indication of generalized cellular destruction (Pantalonc and Page, 1975; Pietras and Szego, 1979b; Nemerc and Szego, 1981a). Moreover, i t has been establishcd by analytical summation that no net i‘hungi? in total c~ellularlysosomcil hydroluse occurs during the acute phase of’ recornpNrtnirritLitioii of lysosomtrl i~omponentsthat is triggered by specific hornzorw ri>cognitioriat the plcr,smrilenimti (cf. Szego et a l . , 197 I , 1974a,b, 1976; Nemere and Szego, 1981a). The tentative conclusion, therefore, is that
LYSOSOMAL FUNCTIONS IN CELLULAR ACTIVATION
REPRESENTATIVE
TABLE 1X EXAMPLES Ol- ACWTE CHANGES ELICITED B Y AGONIST IN ACCESSIBILI I Y OR R k C O M P A K T M E N T A T I O N Ob LYSOSOMAL. COMPONtNTS"
Agonists Peptide hormones ACTH Antidiuretic hormone Glucagon
Growth Hormone Insulin'1 Luteinizing hormone Parathyroid hormone Thyroid hormones Thyroid-stimulating hormone
Steroid hormones Androgen
Estrogen
Glucocorticoids" Progesterone Vitamin D Other effectors Acetylcholine Catecholamines Chemical carcinogens Cyclic AMP Cyclic GMP
71
Reference
Szego el a / . (1974a); Gorban and Boyd (1977) Pietras e f a/. (1975, 1976) Deter et a / . (1967); Detcr (1971); Vaviinkova and Mosinger (1971); Guder et a/. (1973); Mortimore et a/. (1973) Kasavina et u/. (1976) Vaviinkovi and Mosinger (1 97 I); Mortimore et a / . ( I 973); Grammeltvedt and Berg ( 1 976) Lopata et a/. (1977): Ezzell and Szego ( 1 979) Nemere and Szcgo (1981a,b) Kasavina et a/. ( I 976) Wollman ct a/. (1964); Bitensky et a/. (1974); Dang el a/. ( 1 975); Kasavina et a / . (1 976)
Szego et a/. ( I97 I); Briggs (1973): Szego and S e e k (1973); Szcgo el ul. (1974b); BLrzu and Cupdrencu ( 1 975); Kasavina et a / . (1976); Koenig et a/. (1977); Sergeev et a/. (1978) Hayashi and Fishman (1964): Szego et a/. (1971); Briggs (1973); Szego and Seeler (1973): Szego et a/. (1974b); Pietras and Szego (1975b); Pietras and Szego (1976); Szego el al. (1976); Feldman et a / . (1977); Szego et a / . (1977); Moore ef a / . (1978); Pietras and Szego (1979a,b): Nansel e t a / . (1981a.b); Pietras e r a / . (1981a.b) Goldstein et al. (1976); Kasavina et a/. (1976); Decker et a/. (1978); Moore et a/. (1978) Badenoch-Jones and Baum (1973, 1974); Briggs (1973); Moore e t a / . (1978) de Duve et a/. (1962); Nemere and Szego (1981a.b)
Schneider (1970); Ignarro et a/. (1974a,b): lgnarro ( I 975) Ignarro er a/. (1974a,b); Nansel e t a / . (1981a) Allison and Dingle (1966); Pietras and Szego (1976); Pietras (1978) Szego (1972a); lgnarro (1975); Weissmann et a/. ( 1976) Ignarro et a / . (1 974a); Ignarro (1975) (continued)
CLARA M. SZEGO AND RICHARD J. PIETRAS
12
TABLE IX (Continued) Agonists MelittinC Phorbol ester Phytohemagglutinin Prostaglandins Vitamin A Vitamin E Vitamin K
Reference Weissmann et ul. (1969) Weissniann et a/. (1968) Hirschhorn e t a / . (1968) Weiner and Kaley (1975); Weissmann et a / . (1976) de Duve et ul. (1962); Wenzel and Acosta (1973); Nin,joor et ul. ( I 978) de Duve ef u / . (1962) de Duve et nl. ( I 962)
“Evidence for response to agonist within 30 minutes after presentation in vivo or to isolated cells or tissues in vitro. Responses include extralysosomal redistribution of lysosomal hydrolases, rnovernentlmigration of lysosomes or structural labilizationlstabilizationof isolated organelles challenged in vitro. bh optimal concentrations, this agonist generally elicits stabilization of lysosomal structure. (‘From bee venom.
perturbation of the plasmalemma is somehow rapidly communicated to an intracellular compartment that is poised for release of limited amounts of membranebounded components on signal. How is such selective activation of a portion of the lysosomal population achieved? Once again, cellular architecture may hold the key to the manifest coupling device.
C. THE “TARGET”CELL:OCCURRENCE AND FUNCTIONAL IMPLICATIONS OF SPECIFIC RECOGNITION SITESFOR GIVENEFFECTORS IN THE PLASMALEMMA As outlined above, an earlier generation of cellular physiologists had recognized the dynamic nature of the cell surface and postulated that effectors of a wide variety of chemical composition were capable of modifying the structure, and, hence, the selective permeability of the cell so modulated toward materials critical to the growth/differentiation process(es) to come (see Roberts and Szego, 1953; Hechter, 1955; Levine and Goldstein, 1955; and Szego, 1957, 1975, 1976, for reviews). These considerations bring us full circle, for they focus once again upon the crucial role of the plasmalemma in the discriminatory recognition of those extracellular signals that ultimately elicit changes in the cell as a whole. According, we shall undertake analysis of the chain of metabolic reactions by which such coordinated responsiveness may be achieved. By necessity, it seems appropriate to begin with evidence for the origin, cellular distribution, and turnover of recognition molecules for specific capture of given regulatory ligands.
Surface Recognition of Ligand a. Interaction of Hydrophobic Agonists with Macromolecules Intrinsic to the Plasmalemma. Very recent observations have demonstrated specific mac-
73
LYSOSOMAL FUNCTIONS IN CELLULAR ACTIVATION
romolecular binding sites for steroid hormones and other lipophilic agonists in membranes of selective target cells (see Table X). Such recognition sites for estradiol- 17p have been identified both by affinity binding (Pietras and Szego, 1977, 1979a) and by analytical cell fractionation carried out in conjunction with extremely conservative homogenization techniques (Pietras and Szego, 1979c, 1980; Pietras, 1981). In our laboratory, these observations have been accompanied by partial purification and characterization of estrogen-binding macromolecules, whose striking correspondence to “cytosolic” recognition moieties for estrogen, and, moreover, whose apparent translocation from plasmalemma to nucleus upon binding of hormone are strongly suggestive of receptor function in situ. In addition, depletion of such activity from the cell surface on hormonal occupancy is reminiscent of “down-regulation,” or relative refractoriness of the target cell toward a second course of hormone exposure (Szego, 1974). Accordingly, such findings have raised the question of whether the widely reported predominance of receptors for estrogen as well as other steroids in the cytosol fraction of their target cells might have resulted from inadvertent extraction of native hormone receptors by homogenization procedures that caused extensive damage to cellular structures (cf. Szego and Pietras, 1981). b. Interaction of Peptidal Agonists with Receptor Molecules at the Target Cell Sulface. Documentation of this aspect of peptide hormone action is extensive, in part because of the limiting concept, that once such surface recognition is achieved and “secondary messenger” generated, the peptide hormone, hitherto thought to be excluded from the cell interior by mass and charge, is dispensable (Kahn, 1976; Catt and Dufau, 1976; Fanestil, 1978; Jensen and Gardner, 1980; Kaplan, 1981). Regardless of newer developments on cellular entry of peptide hormones (cf. Table 111) and its potential significance, the point is indisputable that surface recognition of effector is the primary event. The latter premise receives strong support from recent experiments which show that extracellular, but not intracellular, application of peptidal effectors leads to activation of target cells (Philpott and Petersen, 1979; Heumann et al., 1981; Huez et af., 1983). It is the cascading consequences of surface recognition of specific ligands that we need to pursue if we are to unravel the metabolic chain that underlies the more delayed responses. D. CONSEQUENCES OF LICANDCAPTURE 1 . Signals of Sulface Membrane Deformation a. Patterns of Receptor-Mediated Membrane Reorganization: A Universal Response to Ligand Binding. (1) Theoretical. Singer (1975, 1981) has provided a thoughtful analysis of the relatively rapid concomitant of redistribution of binding sites on target cells when occupied by specific ligand (Fig. 8). Crucial to this redistribution is an increase in fluidity, above a generally limiting temperature of 15”C, of the lipid bilayer. The resultant disorder, coupled with a degree of
-
TABLE X REPRESENTATIVE EXAMPLES OF SPECIFIC INTERACTION OF LIPoPHlLlC AGOUISTS\ L I T H EXTKANIICLEAK Mk.Z.1BKANES O K MEMBRANE-BOUUDED COMPARTMENTS Ob TARGtT C E L L S Cell fraction Ligand class Estrogen
Androgen
Plasma membrane Pietras and Szego (1977) LaBella er a/. (l978b) Pietras and Szego (1 979a-c) Hernandez-PCrez et a/. (1979) Dufy et a f . (1980) Nenci et a / . (1980a) Pietras and Szego (1980) Schaeffer er a/. (1980) Ahvisatos er 01. (1981) C.Y. Cheng, ef al. (1981) Nenci er al. (1981) Pietras ez a/. (1 98 1b) Zanker er al. (198 1) Zyzek er a/.(1981 1 Zolman ( I 982) Towle and Sze (1983) Watanabe and Po (1974) Lefebvre et al. (1976) Farnsworth (1977) LaBella et al. (1978a) MacLeod er al. (1979) Campo e t a / . (1982) Koenig et a/. (1982) Towle and Sze (1983)
Microsome-rich
Mitochondria-lysosome
Talwar et a/.(1964) King et a/. ( 1965) Noteboom and Gorski (1965) Laumas el a / . (1970) Blyth et a/. (1971) Little et a/. (1972) Jackson and Chalkley (1974a.b) Sen et al. (1975) Pietras and Szego ( 1 9 7 9 ~ ) Jungblut e r a / . (1980) Parikh et a/. (1980) Pietras and Szego (1980) Pietras er a/. ( I98 1b) Yamada and Miyaji (1982) Taylor and Smith (1982)
Talwar et a/ (1964) King e t a / (1965) Noteboom and Gorski (1965) Laumas er a/ (1970) Hirsch and Szego (1974) Szego (1974) Sen er a/ (1975) Nichoga (1979) Pietras and Szego (1979a-c) Pankh er a/ (1980) Pietras and Szego (1980) Pietras et Q/ (1981b)
Harding and Samuels (1962) Kowarski ef al. (1969) Baulieu et a/. (1971) Mainwaring and Peterken (1 97 1) Farnsworth ( I 972) Robel et a / . (1974) Liao (1977) Maclndoe et d.(1981) Campo et a/. (1982) Yamada and Miyaji (1982)
Baulieu er a/. (1971) Nichoga (1979) MacIndoe et a / . (1981) Campo er al. (1982)
Progesterone
Mineralocorticoid
Glucocorticoid -1 cn
Vitamin D Thyroid hormones
Smith and Ecker (1971) Masui and Market? (1971) Jacobelli et a/. (1974) Cloud and Schuetz (1977) Ishikawa er u/. ( 1 9 7 7 ) ~ ~ Godeau er d . (1978)" Kim er a / . (1980) Wasserman er a / . ( 1 980) Finidori-Lepicard rt a/. (1981) Sadler and Maller (198 I , 1982) Schorderet-Slatkine er a/. (1981) Kostellow et a/. (1982) Williams and Baba (1967) Forte (1972) Oiegovit et a / . (1977) Williams and Baba (1967) Suyemitsu and Terayama ( 1975) . 1978) Koch rr ~ 1 (1977, Harrison er a / . (1979) Fant et a / . ( 1979) Allera er a/. (1980) Koch e l a/. (1981) Towle and Sze (1983)
Pliam and Goldfine (1977) Eckel et d . (1979) Krenning rr a / . (1979, 1981) Maxfield er ui. ( I98 1 a) Rao (1981) Rao e r a / . (1981)
Haukkdmaa and Luukkainen (1974) Haukhamdd et a/. ( I 980) Drangova and Feuer (1980) Yamada and Miyaji (1982)
Egert et a!. (1977)
Fanestil and Edelman (1966) Williams and Baba (1967)
Fanestil and Edelman (1966) Williams and Baba (1967)
Ulrich (1959) De Venuto er a/. (1962) Litwack er a/. (1963) Brunkhorst and Hess (1964) Bottoms and Goetsch (1967) Moms and Barnes (1967) Williams and Baba (1967) Mayewski and Litwack (1969) Beato e t a / . (1971) Ambellan e r a / . (1981) Brumbaugh and Haussler (1973)
De Venuto et a/. (1962) Bellamy (1963) Dingman and Sporn (1965) Morris and Barnes (1 967) Williams and Baba (1967) Beato et a/. (1971)
Brumbaugh and Haussler (1973) Tata (1975) Greif and Sloane (1978) Sterling et a/. (1978)
(continued)
TABLE X (Continued) Cell fraction Ligand class
Plasma membrane
Microsome-rich
Mitochondria-lysosome
Cardiac glycoside
Caldwell and Keynes (1959) Smith er al. (1972) Mills et al. (1974) Eveloff et a[. (1979)
Dutta et al. (1968, 1972) Dutta and Marks (1969) Rogers and Lazdunski (1979)
Vitamin A (retinoid)
Maraini et al. (1977) Sani (1979) Bongiomo et al. (1980) Kitabchi et al. (1980) Kitabchi and Wimalasena (1982) Rao (1974) Smigel and Fleischer (1977) Rao and Mitra (1979) Carsten and Miller (1980, 1981) Shimizu et al. (1982) Shoyab and Todaro (1980) Ashendel and Boutwell (1981)
Bongiomo er al. (1981)
Dutta ef al. (1968, 1972) Dutta and Marks (1969) Pollack et al. (1981) Cook er al. (1982) Lamb and Ogden (1982) Bongiomo et al. (1981)
Rao and Mitra (1979)
Mitra and Rao (1978)
P.M. Blumberg, er al. (1982)
P.M. Blumberg, et al. (1982)
Miller and Miller (1952) Wiest and Heidelberger (1953) Dingle and Barren ( 1969)
Miller and Miller (1952) Wiest and Heidelberger (1953) Allison and Mallucci (1964a) Allison and Dingle (1966) Dingle and Barrett (1969)
Vitamin E Prostaglandin
Phorbol ester Chemical carcinogen
OHowever, cf. Tso et al. (1982).
77
LYSOSOMAL FUNCTIONS IN CELLULAR ACTIVATION
,,
t elevotion to 22% or
Initiolly Random Distribution of Receplors
>
Receptor
d
r,
r,
-b
Enerqy
[?I Cluslerinq Through o Fluid Mtnbrone
Actomyosin - Like System Under Membrane [?]
Endocylorir
FIG. 8 . (a-e) Endocytotic uptake of given ligands on interaction with specific receptors at the cell surface: a theoretical view. From Singer (1973, reproduced by permission, with approved minor modifications.
cross-linking of neighboring receptors by multivalent ligands (cf. also Rutishauser and Edelman, 1978) leading to “patching” and “capping,” results in relative distortion of that region of the cell surface that has become crowded with ligand-occupied recognition macromolecules. There then follows invagination of the affected region, a process that may be enhanced by local concentration of contractile elements in the subcortical cytoplasm. Such events are the prelude to at least certain forms of receptor-mediated endocytosis, a subject of intense current interest that is, consequently, beset with problems of interpretation (see Fig. 4, and Pastan and Willingham, 1981). By no means is the redistribution of proteins intrinsic to the membrane a universal phenomenon prior to entry. For example, those specialized regions comprising clathrin-decorated coated pits bear receptor sites that are preclustered through unknown mechanisms, even before accumulation of ligand. When occupied by specific agonist, the latter sites invaginate without undergoing further redistribution in the plane of the membrane (Willingham and Pastan, 1980). Loss of the clathrin coat of the internalized vesicle then appears to occur very rapidly, with apparent conservation of the polymer. Some of the anomalies that prevent unequivocal interpretation of this sequence may have been resolved (see Note Added in Proof).
78
CLARA M . SZEGO AND RICHARD J . PIETRAS
( 2 ) Observed. (a) Redistribution ojititegrnl mcmbrune proteins mid changes in surfuce architecture. Regardless of the precise nature of the invagination mechanism(s) yet to be elucidated, a striking general pattern of precedent redistribution of membrane oriented macromolecules, generally visualized by appropriate fluorescence or other form of labeling, may be identified in a broad array of cell types as an early response to the recognition of specific effector (Figs. 9 and 10). It is regrettable that an early indication at the EM level of membrane alterations elicited by insulin at the surfaces of fat cells (Barrnett and Ball, 1960; Fig. 11) failed to be recognized for what it was-receptor-mediated surface perturbation, the general prelude to vesicular internalization. Indeed, even more than a decade later, and notwithstanding substantial further evidence of internalization of peptidal effectors already available (reviewed in Szego, 1975, 1978), similar observations for insulin, with appropriate labeling and far better resolution as a result of technical advances (Jarett and Smith, 1976), were dismissed as evidence for exclusion of the agonist from the cell interior. After the latter turning point, the tlood of observations interpreted to the contrary has, as predicted (Szego. 1978), been manifested (see Table 111 and below). It is noteworthy that there are now substantial indications for redistribution of integral surface-membrane proteins in response to steroid and thyroid hormones in cells sensitive to these agonists (cf. Fig. 9A-G), as well as to less hydrophobic effectors in their respective targets (Fig. 9H-S; Fig. 12). A particularly puzzling impediment to the ready interpretation, in the case of the steroid hormones, of the crosslinking mechanism that seems to underlie the macromolecular redistribution phenomenon, is their “univalent” nature (cf. Fig. 2), especially since the range of their longitudinal dimensions is only - 9-1 1 A. However, it should be pointed out that steroid hormones (and related compounds of similar effectiveness, such as diethylstilbestrol), are not strictly univalent (cf. Duaz et (I/. , 1980): the functional-group substituents at positions 3 and 17, minimally, but also at carbons 11, 18, 20, and 21, potentially, render molecular representatives of every vertebrate steroid class at lenst bivalent, and thus capable of eliciting cross-linking. Nevertheless, their usefulness in unsubstituted form as reagents for the visualization of surface-related events at their target cells is rather limited by comparison with, for example, ‘2sI-labeled insulin or other peptide hormone. However, by even the most conservative view, there is little doubt that receptor occupancy at the cell surface by hydrophobic as well as hydrophilic ligands is accompanied by some form of membrane reorganization. Striking morphologic evidence of such a process in swift response to estradiol-17p is now available by the criterion of scanning electron microscopy. Figure 13B-D demonstrates the astonishingly rapid morphologic indications of the action of estrogen on its uterine target. Thus, within 30 seconds of the iv administration of a physiologic concentration of estradiol- 17p to the ovariectomized rat, maintained with minimal receptor occupancy as previously de-
FIG. 9 . Patterns of surface macromolecular patching and capping: receptor-mediated responses of diverse cell types to specific effectors, steroid (A-G) and peptidal (H-S). (A-G) Fluoresceinserum labeled estradiol- 17p (E-BSA-F; a conjugate of 17~-estradiol-6-carboxymethyloxime-bovine albumin-fluorescein isothiocyanate) incubated with isolated human breast cancer cells at 4°C (A), is distributed homogeneously over the cell membrane. Transfer to 37°C promotes aggregation into patches (B,C; same cell photographed in 2 planes), and, finally, migration toward a cell pole where Reprinted from Nenci etal. (1980a), with permission. Although the E-BSA-F capping is evident (D). probe has certain limitations (see text and Szego and Pietras, 19811, the results are strikingly parallel to those elicited by active estrogen, using fluorescein-labeled concanavalin A (F-Con A) as probe. Darkfield ultraviolet fluorescence micrographs of F-Con A binding for 3 minutes at 22°C to endometrial cells isolated from ovariectomized rats. Before exposure to F-Con A, cells were treated for S minutes in vitro with hormone vehicle (E), or with 1 X lo-’ M Eza,the inactive congener (F), or with I X 10-9 M E2P ( G ) .Capping is elicited only by the latter, active hormone. Binding of F-Con A at the cell surface could be blocked by the addition of a S-fold excess of unmodified Con A. No significant alteration in the level of Con A binding to cells resulted from treatments described above (all at p > 0.1; t test). Approximately SO cells in each treatment group were observed in each of three independent experiments. AH, X 1000. Reprinted from Piestras and Szego (1979b), with permission. (H) Random distribution of F-Con A on the surface membrane of normal fibroblasts. (I) Cap formation in normal lymphocytes. Reprinted from Sachs (l974), with permission. (J-M) Darkfield fluorescence micrographs of F-Con A binding to epithelial cells from bullfrog urinary bladder. Cells were incubated in the absence (J, X400; L, X 1000) or presence (K, x 4 0 0 M , X 1000)of 10 mU of arginine vasopressiniml for IS minutes. Approximately 250 cells in each treatment group were observed in each of four to six independent experiments. Reprinted from Pietras (1976), with permission. (N,O) Localization of receptor-bound human chorionic gonadotropin (hCG) in rat granulosa cell cultures by indirect imrnunofluorescence. Incubations with hormone and antibodies were at 37°C. (N) Cells were incubated with hCG for S minutes and then fixed with formaldehyde. Tiny specks of fluorescence (arrowheads) are visible around the entire circumference of an elongated granulosa cell (g). ( 0 )Cells were incubated for 90 minutes with hCG and then, without prior fixation, with anti-hCG. After removal of excess antibodies, cells were incubated for an additional hour. Note large clusters and caps (arrowheads) over granulosa cells. Reprinted from Amsterdam et al. (1980). with permission. (P,Q) Incubation of embryonic chick sensory cells with rhodaminelabeled nerve growth factor (130 ngiml) either for 3 hours at 4°C (P), which revealed diffuse (continued)
FIG. 9H-S. distribution of fluorebcence, or for I hour at 37°C ( Q ) , which was associated' with clustering. Reprinted from Levi er a / . (1980). with permission. (R,S) Patching of insulin-receptor complexes on surfaces of 3T3 fibroblasts, as probed with a rhodamine-laheled insulin dcrivative. Cells were incubated with 50 ng of R-lactalhumin-insulin for 15 minutes and fixed with formaldehyde. (R)and (S) represent fluorescence micrographs of two different cells. Reprinted from Schlessinger et a / . (1978) with permission. 80
.
..
.a
ti-
FIG. IOJ-L. with LDL-ferritin included (time at 3 7 T , 1 minute); X81,OOO. (C) Formation o f a coated vesicle. As the plasma membrane begins to fuse to form the vesicle, some of the LDL-ferritin is excluded from the interior and is left on the surface of the cell (arrow); time at 37"C, 1 minute; XS4.000. (D) A fully formed, coated vesicle that appears to be losing its cytoplasmic coat on one side (arrow); time at 37°C. 2 minutes), X75.000. Reprinted from Anderson PI u/.(1977). with permission. (E-H) Coated and uncoated pits in Swiss 3T3-4 cells. (E,F) Appcarance of coated pits (arrows) in unstained TEM sections tangential to the cell surface. ( G ) A section from an experiment using indirect immunocytocheinical localization of u,-macroglobulin ( a z - M ) with peroxidase-labeled second antibody. The section, which was incubated at 4°C to minimize endocytosis, shows concentration of label in association with coated pit. (H) Similar to (G), but cut more tangentially, apparently masking communication with the outside, and, thus, presumed to depict the bottom of a coated pit. Fixation was carried out before addition of immunologic reagents. Magnification (E-H), X 50,OOO. Reprinted from Willingham e / a/. (1979), with permission. (I) Endocytotic vesicles containing EGF-ferritin (arrows) from human carcinoma A-43 I cells incubated with the conjugate at 4'C and then warnicd for 2 . 5 minutes to 37°C; X66,OOO. Reprinted from Haigler et a/. (197%). with permission. (J,K) Uncoated pinosomes (large arrows), in cells as in (E-H), except for uanyl acetate-lead citrate counterstain. (K) Cut more tangentially than (J). Alignment in membrane area in very flat cells ascribed to crowding due to profusion of cytoskeletal structures (mf, microfilament; f , 10-nni filament; mt, microtubule); XS0,OOO. Reprinted from Willingham ut u / . (197% with permission. (L) Binding of rhodaminc-labeled triiodothyroninc (35 nM) to Swiss 3T3-4 cells in the absence of t 37°C for 30 minutes, followed by washing and fixation in unlabeled hormone. Incubation w buffered formaldehyde. Fluorescence was abolished in the presence of essentially equivalent concentrations of unlabeled hormone, while the rhodaniine-labeled, hornionally inactive thyronine failed to bind. Reprinted from Cheng el a/. (1980). with perinission.
(A-C) The effects of insulin on ultrastructure of rat adipocytes. Electron micrographs Fir,. 1 I . of rat adipocytes incubated for 20 minutes in the absence ( A ) or presence (B,C) of lo5 (C) or 10' (B) IJ.Uof insulin. Many invaginations of the plasma membrane are apparent in (C), as well as small vesicles (in R,C) just underlying the surface (arrows, C). Smooth, membrane-bounded vesicles (v) of 83
(coririnuc.6)
84
CLARA M. SZEGO AND RICHARD J. PIETRAS
FIG. 12. Internalization of Semliki Forest virus by adsorptive endocytosis in coated pits and coated vesicles of BHK-21 cells, from which they ultimately reach the lysosomal apparatus. Reprinted from Helenius er al. (1980b). with permission.
scribed (Szego, 1974), there is substantial increase in numbers of microvillar modifications of cell surfaces, as compared to control preparations given vehicle alone (Fig. 13A; Rambo and Szego, 1982, 1983). In addition, by 1 minute after hormone, corresponding preparations show unmistakable increases in mean length of the microvilli (Rambo and Szego, 1982, 1983). Parallel examples of ultrarapid surface remodeling occur in cellular targets to NGF (Fig. 13E-G) and EGF (Fig. 13H and I). Such unmistakable correspondence between acute responses to steroid hormone in vivo and to peptidal effectors in vitro further strengthens the view that surface recognition at specific receptors is common to both major classes of hormonal effector and is accompanied by common cellular reorganization. It is noteworthy that the response of luminal endometrial cells to E2P is not uniform and that responsive and unresponsive cells may appear side by side (e.g. various sizes are abundant in the cytoplasm; a few lipid droplets (1) are also evident (C). (B,C) are thick sections incubated in parallel, which, although too thick to permit delineation of membranes surrounding the vesicles that crowd the peripheral cytoplasm of the insulin-incubated specimen (B), dramatically differentiate the latter from the control (A), which contains no comparable vesicular structures. Magnifications: (A) X42.000; (B) ~42,000; (C) ~49,000.This rarely acknowledged primary evidence for pinocytotic activity stimulated by a specific hormone in its target cell, and obtained at a time when TEM technology was in an early stage of development, is deeply significant in retrospect. Reprinted from Barmett and Ball (1960), with permission.
FIG. 13. (A-D). Scanning electron micrographs of the luminal surface of uterine endometrium from ovariectomized rats: acute effects of active estrogen. (A) Thirty seconds after iv injection of control vehicle alone. Cell boundaries marked by brush-like protuberances, short microvilli, and, on a preponderant number of cells, a central cilium, are evident. (B-D) Luminal surfaces of endo(continued)
Fio 13E-I.
See legend p p 85 and 87
LYSOSOMAL FUNCTIONS IN CELLULAR ACTIVATION
87
Fig. 13B; cf. also Pietras and Szego, 1979a; Nenci et al., 1980c; Nazareno et al., 1981) (see footnote 2). Similar observations on heterogeneity among FSH-sensitive Sertoli cells have been identified (e.g., Kierszenbaum et al., 1980). Additional work is necessary to establish the nature and properties of such preferentially responsive cells and to determine whether their plasmalemmal receptor complement varies with surface membrane renewal during the cell cycle. (b) Membrane vesiculation. If, indeed, both steroid and peptidal hormones can provoke the surface deformation that is the apparent prelude to cellular entry by some form of endocytosis-a proposal only very recently advanced for both classes of agonist (Szego, 1974, 1975, 1978; Szego and Pietras, 1981) and now also for peptide hormones (cf. Middlebrook and Kohn, 1981; Pastan and Willingham, 1981; King and Cuatrecasas, 1981)-are there likely to be parallels in the cellular entry mechanisms? This is an especially cogent question in relation to the traditional view that the hydrophobic steroid hormones encounter no obstacle in penetrating the lipid bilayer of the plasmalemma (see Giorgi, 1980; Szego and Pietras, 1981, for reviews). At the present writing, only very preliminary indications are available of surface membrane vesiculation associated with steroid hormone capture at the plasmalemma of cellular targets (Fig. 14; cf. also Nenci et al., 1980a). Nevertheless, once again, the apparent parallel to the corresponding phenomenon for peptidal agonists (e.g., Fig. 10) is striking, especially when attention is focused on very early observations in both sets of cases. However, there is an additional consideration that may modify this view. The application of hormone in vivo is presumably accompanied by the primary cellular recognition at the sites of initial presentation: the blood front. Thus, augmentation of surface activity at the luminaf surface, such as is illustrated in Fig. 13B-D and Fig. 14, may be a reflection of primary events at the basolateral borders of the responding cells (cf. also, Casley-Smith and Chin, 197 1). Similar considerations apply to in vitro treatment of epithelial-cell sheets with effectors (cf. Gluck et al. , 1982). However, in the metrium, showing acute effects of exposure to 0.S (LgiIOO g body wt of E2P. (9)Thirdy seconds after E2P. Microvilli have become more numerous. (C) One minute after E2P. Note clustered microvilli, their increased density at the cell borders, absence of central cilium on some cells, and a degree of variation in responsiveness from cell to cell. (D)Seven minutes after E2P. Microvilli occur in dense patches. All, ~ 4 0 0 0 Reprinted . from Rambo and Szego (1983), by permission. (E-G) Scanning electron micrographs of sympathetic neurons from newborn rats deprived of nerve growth factor (NGF) during 5 hours in culture, with (F,G), or without (E), readdition of NGF for 30 seconds. (E) The neuronal surfaces are quite smooth; X2150. (F) Sympathetic neurons after readdition of NGF. Small microvilli appear on > 90% of the cells; X 2150. (G)At 30 seconds after NGF treatment 8-10% of the cells develop very prominent ruffles; x3200. Reprinted from Connolly et al. (1981), with permission. (H,I) Scanning electron micrographs of human A-43 1 carcinoma cells incubated at 37°C in the presence (I) of 100 ngiml of epidermal growth factor (EGF) for 5 minutes. In contrast to the small plicae on the surfaces of control cells (H),cells exposed to EGF exhibit numerous lamellipodia and filopodia (I); X 1600. Reprinted from Chinkers ef a / . (1979), with permission.
88
CLARA M. SZEGO AND RICHARD J. PIETRAS
FIG. 14. Transmission electron micrographs of luminal endometrial cells with and without estrogen treatment. (a) Taken 2 minutes after iv administration of E# (0.1 pgil00 g body wt) to ovariectomized rat. (b) Control preparation that received equivalent volume of vehicle only (cf. Szego and Davis, 1967). To be noted are the frequency of occurrence of mieropinocytotic vesicles (pv) in the cortical cytoplasm of the estrogen-pretreated cell and the lack of such microvesiculation in the control. N, Nucleus; G, Golgi; mv, microvillus; mt, mitochondrion; X 20,400. Reprinted from Szego and Pietras (1981), with permission.
case of isolated, single cells exposed to agonist in v i m , such “rebound” vesiculation may not be a factor (Figs. 1OJ and K and 1 IB and C). Contributing to these uncertainties is the intensified pinocytotic activity elicited by metabolite excess, especially of protein, as earlier noted. Indeed, such a process may promote receptor-unmediated vesicular entry of ligand still in association with serum protein carrier (cf. Siiteri et al. 1982). Only additional kinetic studies conducted at critically early times, possibly using freeze-fracture analyses or, at the least, serial sections with conventional TEM, will permit reconstruction of the sequence of these events with reliability.
LYSOSOMAL FUNCTIONS IN CELLULAR ACTIVATION
89
(c) Proximate signals of membrane perturbation and their attenuation. If vesiculation of the plasmalemma were a direct consequence of the initial perturbation due to ligand recognition, and, further, if such vesiculation represented the early stages of potential cellular entry for a broad range of effectors together with their receptor molecules, it would be of immense significance to determine whether these structural changes, per se, constitute the coupling devices for transduction and/or propagation of the initial triggering signal generated by the binding process. However, such a prospective linkage may be extremely difficult to establish. For, not only is membrane vesiculation a process that occupies a very brief time-span at physiological temperatures (Casley-Smith and Chin, 1971; Szego, 1975; Anderson et a l . , 1977, and Fig. 4), but also it is far from unique as an indicator of membrane perturbation. Additional signs of the latter occur within the same relatively brief time, and are thus difficult to segregate from the entry event per se (Table XI; see also Fig. 3, Table IV and V; Szego and Pietras, 1981; Rasmussen and Goodman, 1977). Although, as the compilation of data in Table XI demonstrates, the observations are varied and occasionally disparate, probably as a result of the numerous cell types and stimuli investigated, as well as the variations in sensitivity of the several criteria applied, the net outcome is instability of the membrane structure-function continuum generated by recognition of ligand/stimulus. In fact, certain of these membrane signals, attributable to perturbation of molecular architecture on receptor occupancy, alone or in concert, may provide coordinated support for the entry mechanism, as well as for amplification and propagation of second-order and still further responses to the effector. However, so rapidly are the several, apparently primary, membrane indicators of receptor occupancy generated that their relative contributions to the resultant cascade are not readily determined. Moreover, the reciprocal interactions of such consequences of perturbation as may be expressed in concentration gradients of cyclic nucleotides, Ca2+ (cf. Fig. 15), calmodulin, and prostaglandins (cf. Table XII) possess predictable influences of a mutually cooperative nature that, through specific feedback mechanisms in situ, may well amplify and even subserve propagation of the initial signal. In turn, these events are closely linked temporally, and probably causally, with alterations in membrane phospholipid composition and turnover (Table XIII). Table XIII, considered together with review of Tables IV and V and Fig. 3, demonstrates the dual features of abrupt phospholipid metabolism associated with receptor occupancy. On the one hand, the phospholipid environment of the receptor domain clearly influences its accessibility to ligand (cf. Michell, 1975), a finding in keeping with the regulation of the activities of many membrane-bound enzymes by their phospholipid surround (cf. Zakim and Vessey, 1980)-not the least of which is the adenylate cyclase system (cf. Levey and Lehotay, 1976; Hebdon et al., 198 1) with its profound implications for effector-mediated activation. Additionally, certain phosphlipids normally present in the lysosomal/mitochondrial fraction of
A. Alterations in cell N a + , K + , or Na+,K+-ATPase Steroid hormones Endometrial microsomes Liver plasma membrane: brain microsomes Prostate slices and membranes Prostate microsomes
E2P ( l o - " M)
Increased Na * .K -ATPase activity
E# (10-5-10-4 M )
Decreased Na ,K -ATPase activity
Testosterone (10-12-10-8
Increased Na+ ,K+-ATPase activity; increased K + content Slightly decreased Na+ ,K+-ATPase activity
Farnsworth (1968, 1972, 1977)
Increased Na influx Decreased Na ,K+ -ATPase Increased Na+ efflux Increased Na+ ,K -ATPase activity Increased ouabain-sensitive K + flux Increased Rb+ uptakeb; increased Na+ .K+ATPase activity Increased Na+ uptake
Moolenaar ef al. (1982) Bamabei er al. (1973) Rogus ef al. (1969) Gavryck ef al. (1975) Clausen and Hansen (1977) Hadden et al. (1972)
M) Testosterone (10-7-10-4 M)
+
+
+
Ritter el al. (1969) [cf. also Karmakar ( 1969)l Heikel and Lathe (1970); Robinson (1970)
Ahmed and Williams-Ashman (1969)
C G
Peptide hormones Human fibroblasts Liver plasma membrane Skeletal muscle
EGF Glucagon Insulin
+
+
+
Lymphocytes Hepatocytes Thyroid gland Amphibian skin and urinary bladder Fibroblasts
TSH Vasopressin
Increased Rh + uptake Increased net Na+ flux Increased ouabain-sensitive Rb + uptake; increased intracellular Na in presence of ouabainc +
Fehlmann and Freychet (1981) [cf. also, Koch and Leffert (1979)l Clayton and Szego (1967) Ussing and Zerahn (1951); Leaf et al. (1958) Mendoza et a[. (198Ob)
Other effectors Hepatocytes Liver plasma membrane Fibroblasts Oocyte Dorsal root ganglion Fibroblasts and kidney cells Diaphragm
Increased ouabain-sensitive Rb uptake; increased Na+ uptake in presence ouabain Decreased Na+ ,K -ATPase activity Increased ouabain-sensitive Rb+ uptake Increased ouabain-sensitive Rb uptake lncreased Na+ uptake Increased Na+ efflux and Rb+ uptake Increased ouabain-sensitive Rb+ uptake; increased Na+ content in presence of ouabain Increased passive K + efflux
EGF
+
Epinephrine FDGFd Melittin 1-Methyl adenine NGF Serum
+
+
T3
Fehlmann er al. (1981) Bamabei er al. (1973) Bourne and Rozengurt (1 976) Rozengurt et a / . ( I 98 I a) Dor6.e (1981) Skaper and Varon ( 198I ) Mendoza et a/. (1980a) Haber and Loeb ( I 982)
B. Alterations in cell Caz+ or CaZ+-ATPase Steroid hormones Endometrial cells
E ~ (1 P x 10-9,441
'0 Myometrium
DES (2 x lO-'M) Progesterone (50 pgiml)
Oocyte
Peptide hormones Aorta Kidney cells Liver Myometrium
Progesterone (1-5 pg/ml)
Angiotensin Calcitonin Glucagon Oxytocin
Increased Ca2+ efflux, followd by increased Ca2+ uptake Increased Ca2+ content Decreased Ca2+ uptake by cells and by mitochondria Increased ATP-dependent Ca2+ binding by microsomes Increased activity of free Ca2+ in cytoplasm Increased Ca2+ efflux
Pietras and Szego (1975a)
Increased Ca2 release Increased cell Ca2+ content Increased Ca2+ efflux Decreased ATP-dependent Ca2+ binding in microsomes; plasma membranes Decreased Ca2 .Mg2 -ATPase activity
Baudouin er al. (1972) Bode (1975) Friedmann ( 1972) Carsten (1974, 1979); Soloff and Sweet ( 1982) Akerman and Wikstrom (1979)
+
+
+
Pietras and Szego (1981) Batra (1973); Batra and Bengtsson (1978) Carsten (1974, 1979) Wasserman el a/. (1980) Kostellow et af. (1980)
(continued)
TABLE XI (Continued) ~~
Stimulus
Cell/tissue preparation Kidney cells
Parathyroid hormone
Embryonic bone cells Anterior pituitary tumor cells
Thyrocalcitonin TRH
Erythrocytes Amphibian urinary bladder
Thyroxine, triiodothyronine Vasopressin
N ic
Other effectors Hepatocyte Muscle fiber PMN leukocyte Neutrophil T lymphocytes PMN leukocyte Kidney cells Slime mold Liver Insect salivary gland
Asialogl ycoprotein
co2 Chemotactic peptide (F.Met-Leu-Phe) Concanavalin A
Cyclic AMP
Response
Reference
Increased Ca" influx and efflux: increased cytoplasmic Ca'+ pool Increased cell Cal- content Increased Gal- efflux
Borle (1970, 1973) Harell e t a / . (1973) Gershengom e? a / . ( 1981)
Increased Ca2- uptake and intracellular Ca' Pool Decreased Ca'- content Increased Ca2 -ATPase activity
Tan and Tashjian (1981)
Increased Ca2+ efflux, followed by increased Ca?- uptake Increased Ca2+ content of granule-rich cells: decreased Gal- content of mitochondriarich cells
Pietras ( 1974)
Decreased Ca2+ uptake and efflux Increased intracellular free Ca2+ Decreased plasma-membrane associated Ca'
Blomhoff et ul. (1982) Lea and Ashley (1978) Hoffstein (1979)
Rebecchi et a/. (1982) Davis et a / . (1982)
+
Increased Ca2+ uptake Decreased plasma membrane Con A binding Increased Ca2 efflux +
Cd7+
+
at site of
Pietras et a!. (1976)
Naccache et a / . (1979) Freedman e t a / . (1975) Hoffstein (1979) Borle (1970, 1973) Chi and Francis (1971) Friedmann (1972) Prince et al. (1972)
Oocyte Insect salivary gland Papillary muscle Rectus abdominus muscle Isolated ventricle Liver mitochondria
Fertilization 5-Hydroxytryptamine 0u ab ain
Increased cytoplasmic free Ca2 Increased Ca2+ uptake and efflux Biphasic changes in Ca2 content Increased Ca2+ uptake
Prostaglandin El
Increased Ca2
+
+
+
efflux
Cuthbertson et a/. (1981) Prince et a/. (1972) Lee er al. (1961) Shanes (1961) Briggs and Holland (1962) Carafoli and Crovetti (1973)
C. Alterations in membrane potential and other electrical parameters Steroid hormones Tuberal hypothalamus Hypothalamus and midbrain Isolated atria
Cortisol (10-2 M ) Dexamethasone (10-
M)
Estradiol ( lop5 M) Estradiol ( l o p 9 M )
Hypothalamus
Estradiol (20 pg iv)
Preoptic-septa1 neurons Anterior pituitary tumor cells Hippocampus Motor nerve terminals Isolated atria
Estradiol Estradiol (10-
w \c
M) lo-* M )
lo-
Estradiol M) Progesterone (10- M ) Testosterone (10-6-10-5
Decreased unit activity Decreased electrical activity
Mandelbrod et al. (1974) Ruf and Steiner (1967)
Increased duration, area of action potential Brief transitory decrease in duration of action potential Decreased unit basal activity; decreased activity preceded by a transient increase in firing rate Decreased single unit activity Increased Ca2+ -dependent action potential
Gimeno et a/. (1963) de Beer and Keizer (1982)
Increased electrical activity Inhibition of depolarization Increased duration of cardiac action potential
Teyler et a / . (1 980) Pennefather et a/. (1980) Gimeno er al. (1963)
Increased electrical activity in diestrous female Lowered discharge frequencies of electroreceptors in accord with their associated electric organ
Teyler et a / . (1980) Meyer and Zakon (1982)
Dufy et a/. (1976)
Kelly et a/. (1976) Dufy e t a / . (1979, 1980)
M) Hippocampus Electric fish, Srernopjgus
Testosterone (10- 1" M ) Sa-DHT (5 K g i g ; 2 weeks)
(continued)
TABLE XI (Cominued) Cell/tissue preparation Peptide hormones Adrenocortical cells Adrenal zona fasciculata Pancreas acinar cell Ileal mucosa Pancreas acinar cell Liver P u
Stimulus
Response
CCK-pancreozymin Glucagon
Depolarization in K +-free mediae Depolarization Depolarization Hyperpolarization Depolarization Hyperpolarization
ACTH Bombesin-nonapeptide
Skeletal muscle Toad urinary bladder, skin adipocyte Ileal mucosa Myometrium Osteoclasts Ileal mucosa Osteoclasts Anterior pituitary cells
Insulin
Hyperpolarization
Neurotensin Oxytocin Parathyroid hormone Substance P Thyrocalcitonin TRH
Hyperpolarization Depolarization Depolarization Hyperpolarization Hyperpolarization Depolarization
Thyroid gland
TSH
Decreased resistance and increased capacitance of luminal membrane Hyperpolarization Depolarization and hyperpolarization Hyperpolarization Decreased resistance of apical membrane Inhibition of spontaneous action potential
Frog skin
Vasopressin
Telost prolactin cells
Catecholamines
Reference
Matthews and Saffran (1968) Lymangrover er a[. ( I 982) Petersen and Philpott (1979) Kachur er al. (1982a) Nishiyama and Petersen (1974) Petersen (1974); Friedmann and Dambach ( 1980) Zierler (1957) Herrera (1965); Andr6 and Crabb6 (1966); K. Cheng et al. (1981a) Kachur et al. (1982b) Woodbury and McIntyre (1954) Mean (1971) Kachur er al. (1982b) Mean (1971) Taraskevich and Douglas (1977); Dufy er a / . (1979, 1980) Williams (1970) Grollman et al. (1977) Pace and Gaitan (1979) Ussing and Zerahn (1951) Els and Helman (1981) Taraskevich and Douglas (1978)
Other effectors Adrenal chromaffin cells Sperm Granulocytes Hepatocytes Necturus gallbladder Parotid acinar cell Echinoderm egg Pancreas islet cell Macrophage Neuroblastoma X glioma Brown fat cell Parotid acinar cell Neuroblastoma X glioma Neuroblastoma
Acetylcholine Acrosome reaction Concanavalin A Cyclic AMP
Epinephrine Fertilization Glucose Latex sphere Morphine Norepinephrine Prostaglandin El Serum
Depolarization Depolarization Triphasic membrane potential change Hyperpolarization Rapid depolarization (reduction of permeability of tight junctions) Hyperpolarization Biphasic membrane potential change Depolarization Hyperpolarization Hyperpolarization Depolarization Hyperpolarization Hyperpolarization Depolarization
Douglas (1968) Schackmann e t a / . (1981) Korchak and Weissmann (1978) Petersen (1974) Duffey et al. (1981) Petersen and Pedersen (1974) Steinhardt et a/. (1971) Dean and Matthews (1970) Kouri et a/. (1980) Traber er a/. (1975) Horwitz et al. (1969) Petersen and Pedersen (1974) Traber et a/. (1975) Moolenaar et a / . (1979)
VI W
“Effects of various stimuli are limited to those reported to begin within 30 minutes. However, in most cases, onset of the response appears to occur within the first 1- 10 minutes following stimulation. bRb+ commonly used as a K + analog or tracer. =Ouabain reduces active efflux of cell Na+ via the proposed Na+-K+ pump. dFibroblast-derived growth factor.
-
300
w c 250-
y 2
BASAL
PERIOD
2 20 a 2 ," 200so
'5 y
150-
Q U
02 ! o o - ~-~ - o= - ~ /5%
L
L
U
TI ME (mid
FIG. 15. Calcium flux at short times after application of appropriate tropic hormone to isolated epithelial cells of toad bladder (A) and rat endometrium (B), respectively. Analyses of calcium uptake and efflux were carried out with the use of 4sCa2+, as described in Pietras and Szego (1975a). Rcprinted from Szego (1975), with permission.
porcine adrenal cortex are capable of activating lysosomal cathepsin D of this tissue manyfold (Watabe and Yago, 1983). Moreover, challenge of target cells with any of a number of effectors promotes the turnover of several relatively minor phospholipids, notably phosphatidylinositol and phosphatidate (cf. Rounds et al., 1980; Table XIII). It is readily apparent that the key enzymes in mobilization of phospholipids and polyunsaturated fatty acids from membrane localization may well be lysosomal. Hence, "spilling of packets of lysosomal enzymes," including phospholipases, at the critical cell surface is anticipated to contribute to the plasmalemmal reorganization resulting from the ligand-induced perturbation. Changes in membrane fluidity and their consequences in integral protein redistribution are thus effective in reinforcing the alterations in mem-
97
LYSOSOMAL FUNCTIONS IN CELLULAR ACTIVATION
brane architecture elicited by limited proteolysis, simultaneously under way (see below). In turn, phospholipid-dependent protein kinases may be activated or suppressed concomitantly, further expanding the intercommunicating web of responses provoked by ligand interception at the cell surface. The resultant shifts in ions critical to further modulation and propagation of the primary recognition event are clearly implicit in these cascading consequences. Activation of protein kinases, with its profound consequences, thus constitutes still another repercussion of the primary ligand challenge. Since delivery of microquanta of lysosomal enzymes, including protein kinase(s) and phosphatase(s), together with additional organellar components at the cell surface is TABLE XI1 IMI~LICAIIONO b PROSTAGLANDINS I N ACU rk ACTIONSOF RtPKtStNTAriVt AGONISIS Agonistiligdnd
Intermediate
Acetylcholine
PGEz .T
Bradykinin and angiotensin 11
PGEz t
EGF Glucagon
GnRH
TSH
Prostacyclm t ; PGE? ; PGF2,, 5 ; and thromboxanc
4
A2
1
Asociated tunctions
Reterence
Deacylation of phosphatidylinositol in iiiouse pancreas in 1,irro Deacylation of a limited pool of arachidonic acid in perfused rabbit kidney, with presuniptive effects o n local hcmodynamics Bone resorption in cultured calvaria Augmented autophagy and protein dcgfiidation in rat liver Capacity to syiithcsizc PGEL precedes first prcovulatory surge of gonadotropins Macrophagc activation Decreased autophagy and protein degration Accumulation of product in ovine endometrium ill v i t w enhanced with approaching estrus Uptake of iodide and its organification in cultured porcine thyroid cells
Banschbach and HokinNeaverson ( 1980)
Schwartzman er u/ (1981)
Tashjian and Levine (1 978) Polonovski et a/. ( 1974)
0,jcda and Campbell ( 1982)
Rourer e / L I / . ( 19x0) Polonovski er ui.( 1974)
J.S. Roberts ('r u / . ( 1976)
98
CLARA M . SZEGO AND RICHARD J . PIETRAS
TABLE XI11 REPRESENTATIVE ESAMPI.HS OF RECIPROCAL EFFECTSBETWEEN MEMBKANELIPIDENVIRONMENT A N D RFCOC~NITION OF SELECTEDEFFECTORS Effector
Reference
A. Influence of the lipid environment on receptor accessibility Acetylcholine Gonzalez-Ros et ul. (1982) Epidermal growth factor Shoyab and Todaro (198 I ) Glucagon Houalay et t i / . (1976) GnRH Hazum P I a / . (1982) Gonadotropins Azhar and Menon (1976); Azhai- el a / . ( I 976) Clark er a / . (1978); McCaleb and Donner (1981); Could cf trl. (1982) Insulin B. Abrupt receptor-mediated generation of alterations in components of membrane lipid domainsa Peptides ACTH" Angiotensin II Glucagon Growth factors Insulin LH LHRH parathyroid hormone Phytomitogens'
TSH Vasopreisin Steroids Aldosterone Glucocorticoids Vitamin D3
Laychock cr u l . (1977); Farese e f a / . (1982a); Farese (1983) Benabe er a / . (1982) Epand et a / . (1981) Habenicht of ul. (1981); Chiba et a/. (1982); Bruni ct cd. (1982) Farese P I a / . ( 198%) Lowitt et trl. (1982) Naor and Yavin (1982) Bidot-L6pez or crl. (1981) Reilly and Ferber (1976); Northoff et ul. (1978); Hasegawa-Saaaki and Sasdki ( I Y X I) Rehecchi ef a/. (19x1, 1983)"; cf. however. Irvine et u/. (1980) Alcniany rr ( I / . (19x1); Monaco (1982); PrpiC cf a/. (1982)
Goodman ei t i / . (1975) Nelson ( 1980) Fontaine t t UI. (1981); Kasmusscn
u l . (1982); Norman
PI
a / . (1982)
"Phospholipid methylation is not considcrcd hcrc. Howcvcr. it may be of particular rclcviincc that cspccially active phosphatidylino~itolkinase with a neutral pH optimum has been identilied i n lysosumal membranes from rat livcr (C'ollins ;ind Wells. 1983). "See also footnote a , Table XIXA. < Scc aiw ' h h l c V . ('See also Tahle IV. iin
likewise an early step in membrane activation by specific ligand, it would seem axiomatic that one or more of these mutually reinforcing, interlocking steps is implicated in the coupling of surface perturbation to secondary responses which, by definition, constitutes transduction of the primary signal. What is important to recognize is that all the components or their immediate precursors are virtually instantly available in a poised system that is not only rapid responsive to stimulatory input without the necessity of intervention ol cumbersome transcriptiunal~rranslationalpathways, but is also capable of close modulation by con-
LYSOSOMAL FUNCTIONS IN CELLULAR ACTIVATION
99
comitantly mobilized members of the cascade and subduction by appropriate membrane-stabilizing substances, endogenous and exogenous (Szego, 1972b, 1975; Smith et al., 1976; Flower and Blackwell, 1979; Russo-Marie etal., 1979; Smolen and Weissmann, 1980; Blackwell et al., 1980; Moore and Hoult, 1980a,b; Franson et al., 1980; Moncada with Vane, 1980; Clarke and Ryan, 1980; Shier, 1980). As noted, in situ mobilization of precursors and synthesis of prostaglandins are processes integral to acute membrane challenge by numerous tropic hormones (Ramwell and Shaw, 1970; see also Table XU). Interestingly, levels of prostaglandins E and F in mouse epidermis are increased by certain tumor-promoting phorbol esters (Ashendel and Boutwell, 1979; Furstenberger and Marks, 1980). Such a pathway has been considered obligatory to eventual prereplicative (Boynton and Whitfield, 1980) and proliferative (Furstenberger and Marks, 1980) steps. It is to be assumed that appropriate controls have been utilized in the above investigations to establish baselines in absence of specific agonist, since these and related findings must be viewed in the context of background noise, as mentioned earlier (see Table I). For example, it is known that radiation injury (Trocha and Catravas, 1980a,b) and lipid perioxides (Hemler er al., 1979; Torinuki et a l . , 1980), agents that activate macrophages (Bonney et al., 1978), are associated with prostaglandin synthesis, as well as with selective release of lysosomal hydrolases. Secondary responses attributable to aspects of the prostaglandin cascade that clearly illustrate the complex feedback effects include modulation of membrane fluidity that appears, in turn, to modify the apparent numbers and or affinities of specific binding sites for given agonist (e.g., prolactin; Dave and Knazek, 1980). Similarly, prostaglandin D, elicited depolarization of isolated neuroblastoma-glioma hybrids, as determined with a cyanine dye (Kondo et al., 1981). Since numerous hormones, both steroid and peptidal, as well as other specialized effectors, also provoke acute changes in membrane potential as one indicator of their perturbational effects in situ (see Table XI), it is of some interest to determine whether such influences on ionic flux are mediated by secondary pathways, not excluding cyclic nucleotide generation, phospholipid turnover, arachidonic acid mobilization, and the prostaglandin cascade. There are numerous additional indications that prostaglandins have the capacity of mimic, if not indeed supplant, the actions of the hormone that, in perturbing the target call surface, leads to their abrupt biosynthesis in situ. For example, prostaglandins E, and E, cause a 5- to 10-fold stimulation of ornithine decarboxylase activity in granulosa cells isolated from porcine ovarian follicles (Osterman and Hammond, 1978), as does luteinizing hormone (and its postulated intracellular surrogate, CAMP). Since phospholipase A, activity released from lysosomal sequestration presides over the initial step in prostaglandin synthesis, these activities are too closely interrelated temporally to be as yet unraveled. Conversely, blockade of prostaglandin synthesis at one or more of its several
I00
CLARA M. SZEGO AND RICHARD J. PIETRAS
control points can be achieved by substanccs with the capacity to stabilize the structure of cell membranes (e.g., steroid and nonsteroid antiinflanimatory agents, indomethacin, and certain tranquilizers, as noted above). It is provocative to consider that members of these latter effector families are also capable of degrees of inhibition of some later phases of agonal stimulation including cell growth and developnicnt (Szego, 1972b, 1975). The conclusion is inescapable, therefore, that acute membrane events contribute to homeostatic mechanisms underlying growth and developnient at stages far removed from application of the initial stimulus. b. Relevance to Trunsdirction of Binding Sigiznl. As may be inferred from the foregoing, the primary information inherent in the (probably mutual) conformational changes that occur on receptor recognition of specific ligand (cf. Kaethner, 1977; Chiang and Koshland, 1979a,b; Smith et d.,1980; Pilch and Czech, 1980; Heidmann and Changeaux, 1980; Roy and Ausiello, 1981; Harmon et ul., 1981) requires amplification by means orders, as well as transduction from biophysical to biochemical terms, if it is to be perceived and effectuated within the cell interior and its several organeller compartments. However, in contrast to the exquisite specificity of interactions at the cell surface, the relatively few propagational mechanisms that have been evolved are of a more qualitatively general nature: concentration gradients of ions, potential substrates, cyclic nucleotides, and the secondary manifestations of these acute fluxes in biocatalytic functions. While these virtually universal correlates of receptor-response coupling have received much emphasis in a wide range of biological phenomena (e.g., Rasmussen and Clayberger, 1979; Rodbell, 1980; Lazo et al., 1981; Levitzki, 1981; Eberle et d., 1981; cf. also, Table XI), the potential contributions of lysosomal functions to such events have only recently come to light. Limited numbers of examples of the latter activities appear focused on surface membrane events themselves, and seem to be integral to the early, but c/eur/y secondury, alterations of membrane architecture related to the spilling of restricted amounts of lysosomal enzymes at the cell surface (Table XIV). Although some of the evidence for these comments is indirect, being based in part on the actions of rclatively specific inhibitors, limited proteolysis associated in many cases with enzymic properties consistent with those of cathepsin B (Table XV) appears implicated. As noted in part from Table XIV, unmasking of latent membrane-associated enzymes (Lacombe et ul., 1977; Richert and Ryan, 1977a,b) is but one such surface alteration that has profound consequences in further propagation of the membrane perturbation by initial ligand.3 The early 3There are cogent indications that a cathepsin B-like enzyme may preexist in given cell membranes (cf. Cuenet et uI.. 1982), possibly as a result of an earlier exocytotic fusion with lysosome(s) at that site, and become unmasked during ligand-actuated surface perturbation, a feature of cell membrane architecture that is greatly exaggerated with intense cellular proliferation. The implications of such functions in cell surface remodeling and in the transduction of the primary agonal signal remain to be investigatcd in depth.
E V I I X N C C OF
TABLE XIV LYSOSOMAL PAK~IICIPATION IN ACUTERtOKGANlZATlON Ok TARGETCELL. PLASMALEMMA INCIDENTAL TO RECEPTOROCCUPANCY INTEKNALIZATION: REPRESENTATlVt EXAMPLES FROM STEROID A N D PEPTIDE CLASSESU
Agonist Estrogen
Target cell Rat endometrial
lsoproterenol
Murine thymocytes
hCGd
Rat ovarian membrane fractions
Parathyroid hormone
Cultured murine bones
AND
Criterion
Mechanism
Con A-mediated hemadsorption
Reorganization of surface architecture. promoting cross-linking and clustering of receptor sites Limited proteolysis
Pietras and Szego (1975b)
Reduction of affinity, without substantial effect upon number of binding sites, apparently through limited proteolysis
Pietras and Szego (1979b)”
Irreversible inhibition of onset of hormoneinduced refractory state by NU-Tosyl-~lysine chloromethyl ketone (TLCK)
Zick et al. (1980); cf. also Feny er a / . (1982)
Limited proteolysis
Richert and Ryan (1977b)
Limited proteolysis (at surface lacunae)
DelaissC et al. (1980)
Cell-cell aggregation (inhibited by leupeptin) Concentration-dependent reduction in specific binding by inhibitors of sulfiydryldependent proteases Selective change in activity ratio CAMP-dPK without effect on cell viability(’ Blockade of activation of adenylate cyclase in presence of TLCK and other low-M, protease inhibitors Inhibitors of lysosomal thiol proteases selectively inhibit resorption of bone explants in culture
Reference
Pietras and Szego (1979b, 1981)
(conrinued)
TABLE XIV (Cnnfinued) Agonist
Target cell
Phytohemagglutinin
Human peripheral lymphocytes
T3
Amphibian intestinal brush border enzymes
TSH
Thyroid-suppressed rats, in vivo
Criterion Inhibition by TLCK of blast transformation without appreciable effects on viability, on a membrane-bound neutral protease. or on other metabolic parameters Coordination of biochemical evidence for enhanced activity of alkaline phosphatasee Electron microscopic stereology
Mechanism
Reference
Limited proteolysis
Grayzel et al. (1975)
Parallel development of enzymic complement during metamorphosis, with peak at 3 and 10 days after T,
Dauqa er al. (1980)
Dose-dependent exocytotic reorganization of apical membrane architecture is a primary event in the thyroid follicular cell surface
Engstrorn and Ericson (1 98 1)
-
8
“See also Tables IX, XI, XII, XIXA,B, XXA, XXIV; Fig 13; and K . Cheng, et al. (1981b). ?3ee also Pietras and Szego (1981) for more extensive compilation of parallels to other steroid hormones. =The significance of this selected example is magnified by several orders when coupled with the sensitivities to limited proteolysis at the membrane locus of both adenylate (Pinkett er al., 1979; Stengel er al., 1980) and guanylate (Lacombe and Hanoune, 1979) nucleotide cyclases, the respective cyclic nucleotide-dependent (Alhanaty and Shaltiel, 1979) and -independent (Kiibler et al., 1982) protein kinases, as well as the phosphodiesterases and their regulators (Pinkett er al., 1979; Loten et al., 1980). This is especially significant in light of the occurrence of both protein kinase activities and the relevant substrates (Kang et al. , 1978; Kiibler er nJ., 1982), as well as phosphodiesterases and their regulators (Smoake el al., 1981). at the outer surfaces of certain cells. Such potentially cascading means of modulation, through amplification and propagation or curtailment of the effect of a primary surface trigger, give but mere hints of the fuller potential of a proteolysis-linked chain of surface as well as intracellular events. It is notable that lysosomes may serve as a reservoir for controlled delivery of protein kinase activity (Collins and Wells, 1982), as well as the source of the protease that serves to modulate these (Sakai etal., 1978; Strewler and Manganiello, 1979; Loten et a!., 1980) and numerous other (cf. Reynolds and Wills, 1974) ectomembrane functions. Moreover, multiple basic residues of the phosphorylated serine act as substrate specificity determinants for given protein kinases (cf. Kemp er al., 1977; Cohen, 19801, a finding concordant also with the substrate requirements for cathepsin B (cf. Table XV). Similarly, such observations are to be considered in context with contributions of lysosomal enzyme delivery to parallel and abrupt alterations in the composition of phospholipid constituents of the plasmalemma that are elicited by agonal recognition, as summarized in part in Tables XI1 and XI11 and text. In the latter context it is intriguing that a membrane-associated proteolytic function is activated by binding of IgE receptors on mast cell membranes before phospholipid methylation, cAMP elevation, Ca*+ -influx, or histamine release are identified (Ishizaka, 1982). dActivation of cAMP in comparable preparations by epinephrine and cholera toxin was similarly inhibited. eA lysosome-sequestered enzyme in intestinal mucosa (Ugolev et n l . , 1979).
104
CLARA M. SZEGO AND RICHARD J. PIETRAS TABLE XV PROIW<TIES 01: CATHEPSIN B (EC 3.4.22. I )
Thiol-dependent endopeptidase MW--25,000 Optimum pH-6.2“; sigrr$cuii/ crctivitv still majiifesred at nerrtrul p H Substrate specificity and functions Naturally occurring proteins with dibasic amino acid residues (e.g., aldolasc. collagcn, glucokinase, proinsulin).” Especially vulnerable to cleavage are residues on the carboxy terminal side of paired dibasic amino acidsc Possesses transpeptidase activity (likc papain)“ Synthetic compounds with dibasic amino acid residues (CBZ-Ala-Arg-Arg-R) Inhibitors Endogenous (tiasucs and sera) Thiol-blocking reagents (TLCK, heavy metals. iodoacetate)<’ Peptide aldehydes (lcupeptin, antipain) Excess [Caz + 1’ Cellular localization Lysosomes-endocytotic vesicles Neoplastic and hormone-activated ccll surfaces Neoplastic and hormone-activated cell nuclei (‘As generally assayed in free solution with synthetic substrates. However, pH optirnum may be displaced subatantially upward with certain native proteins as substrates (Bohley et d.,1971) and when the catheptic activity is in resin-bound or membrane-oriented form (cf. Kazakova and Orekhovich, 1975). “May also act as exopeptidase with certain of these (cf. Barrctt and McDonald, 1980). ‘Note relevance of this highly stringent property for processing of precursor forms of numerous hormones, neuropeptides, and receptor molecules, precisely under the conditions imposed by intcrception of surface signals leading to cellular activation (see also Tables XIV, XXA-C, and XXII). (’Note relevance to protein turnover, such as may he misinterpreted as synthesis in experiments with isotopic amino acids; especially prominent in thc case of leucine (Mycek, 1970; cf. also Szego et al.. 1976). ‘Note potential for inhibition by sulfhydryl interaction with molybdate (cf. Wcathers et al., 1979), the latter serving to stabilize steroid-hormone receptors in “unactivated” form (cf. Szego and Pietras, 1981). fMinimum [Caz + ] required in many tissues; lack of appreciation of this feature contributes to occasional failure to consider an SH-dependent, leupeptin-inhibited enzyme as a potential Ca2 + activated protease (e.g., Rodemann et ul., 1982). especially one that is conspicuously concentrated in the “mitochondrial” fraction as routinely isolated (e.g., Beer et al., 1982).
actions of insulin (Lamer et al. 198 I ; Czech et al., 1981) are also characterized by limited proteolysis at or near the target cell surface, indeed, at the level of the very receptor itself (Fig. 16). An intriguing further development has been the suggestion that the insulin receptor, like its EGF counterpart (Cohen et ul., 1980), has intrinsic protein kinase activity (Roth and Cassell, 1983; Shia and
LYSOSOMAL FUNCTIONS IN CELLULAR ACTIVATION
105
Pilch, 1983), an observation that may nevertheless be consistent with conformational alteration elicited by prior limited proteolysis. Likewise, lectin activation of quiescent lymphocytes is associated with an early stimulation of membranelinked proteolytic activity that is subject to inhibition by leupeptin (Saito et al., 1973). In turn, Shoyab and Todaro (1981) have demonstrated that the affinity of EGF for surface binding to mink lung cells is diminished, without effect on receptor number, by treatment with phospholipase C. Reduction of specific binding of glucagon to liver and fat cells or their membranes under similar conditions has also been observed (Cuatrecasas, 1971; Rodbell et ul., 1971). Thus, localized modification of membrane architecture by limited proteolysis (Table XIV) or by alteration of phospholipid environment (Table XIII) has profound influence upon accessibility of receptor domains. Clearly, selective efflux of limited amounts of lysosomal constituents with the requisite enzymic properties may modulate the further responsiveness of the target cell surface to specific agonists as well as to other ligands and, indeed, to the signaling effects of cell-cell membrane contact. In light of the intense current interest in surface proteolysis as a transducing/coupling signal for eventual mitogenesis (cf. Reich et al., 1975; Noonan, 1978; Pietras et ul., 1981a; Pietras and Szego, 1981), continued emphasis upon the potential significance of recompartmentation to the
I
II
02
1 -Lysosornal protease(s1, Elastase FIG. 16. Generation of three ubiquitous insulin receptors by proteolytic fragmentation of the psubunit. Receptor forms I , 11, and 111 are present in membrane preparations in all tissues studied and can be readily resolved in SDS gels (apparent M,, 350K, 320K, and 290K, respectively). The native (c~p)~-350K receptor in intact adipocytes and plasma membrane preparations can be sequentially converted to the 320 and 290K forms in v i m by lysosomal elastase. Reprinted from Czech et al. (1981), with permission.
106
CLARA M. SZEGO AND RICHARD J. PIETRAS
cell surface of lysosomal proteinases (not to the exclusion of phospholipases, protein kinases, and phosphatases, as well as trace amounts of ions and other components requisite to their catalytic function), appears well warranted. In course of such extension, it would be highly desirable to eliminate the possibility, however remote, that the copurification of agonally elicited proteolytic and/or phosphorylation activity (cf. also, Niedel et al., 1983; Deutsch et al., 1983; Shia and Pilch, 1983) represents contamination with membrane-associated enzymes, rather than functions intrinsic to the receptor or, indeed, effector (e.g., Young, 1979a) as such. 2. Cellular Entry of Hormone-Receptor Complex a. Evaluation of Supporting Evidence. Within the methodologic limitations specified in footnotes to Table 111, there are now overwhelming data in support of receptor-mediated, vesicular internalization of macromolecules at specific target cells, a broadly general phenomenon only very recently acknowledged (cf. de Duve, 1981). The data summarized in Tables I11 and XVI and in Figs. 9 and 10 now permit analysis of some of the features of the entry process, as related to further propagation of the primary signal. b. Mechanism(s). The precise molecular mechanisms that underlie and/or promote such interiorization are presently unknown. Nevertheless, occasional disclaimers apart (e.g., Willingham and Pastan, 1980), there are subwantial indications that components of :he cytoskeleton (Peters, 1956), individually or in synergic fashion, are somehow related to the entry process for a wide variety of agonist-receptor complexes (Singer et a l . , 1978; Oliver and Berlin, 1979; Fig. 10; Table XVIIA,B). These activities are likely to be closely coupled to catalytic functions of Ca2+, calmodulin, and fluxes of cyclic nucleotides (Schreiner and Unanue, 1976; Salisbury et a l . , 1980). Such findings give intriguing glimpses of the numerous potential control points subject to hormonal influence at this significant early step in the reaction sequence triggered by mutual ligand-surfacereceptor recognition (Table XVIIIA,B). In turn, there occurs phosphorylationldephosphorylation of proteins integral to, or closely linked with, the plasmalemma-potentially including cytoskeletal elements themselves, or their decorational components (Table XIXA,B; see also Section IIl,D,2,c). As with other similarly rapid membrane-associated events, including altered phospholipid metabolism and triggering of the prostaglandin cascade, as noted above, these activities possess inherent capacities for transduction and amplification of the primary recognition signal. Moreover, the membrane perturbation so generated and propagated may lead to a certain degree of concomitant entry of heterologous substances through engulfment of regions of the plasmalemma and glycocalyx closely adjoining sites of specific receptor-ligand concentration. Such fortuitous coentry is illustrated by the extensive redistribution (now recognized to be preliminary to the endocytotic process) of both receptor and nonreceptor species of
TIMECOURSE OF CELLULAR REDISTRIBUTION
Substance [marker] ~
Cell
OF
TABLE XVI REPRESENTATIVE MACROMOLECULES INTERNALIZED B Y LIGAND-MEDIATED MECHANISMS
Route
Cellular compartment
Onset time (minutes)"
Reference
~~
Steroid hormone receptorsb [3H]Estradiol-l7pc
Rat uterine cells
In vitro
(22°C)
Peptide hormones [Ferl-, [HRPI-EGF
['251]hCG, -LH
PM G
5; peak, 10 15; peak, 30-60
0.5 10-13 15 25-30 30-60 <5 20 20 0.25-1 1-1.5 1-8
Human epithelioid carcinoma (A-431, KB) cells
In vitro
SVd GlCV GIL MVBe SL
Ovine corpora luteal cells
iv
sv
Rat liver
MVB iv
L
L N
DV [ ~25I]Insulin
Instantaneous
PM SER
sv G
['251]LHRH-
Rat pituitary (gonadotrophs)
iv
s v , cv MVB RER PM GIL SG
1.5-10 3 10-30 30
Pietras and Szego (1977) Pietras er al. (1983)
Haigler et al. (1979b) Cohen er at. (1979) McKanna er al. (1979) Willingham and Pastan (1982)
Chen et al. (1 977)
Desbuquois ef al. (1979,1982)k Goldfine et al. (1981)k Khan et al. (1982)k Pilch et al. (1983)
Pelletier et al. (1982) ~
(continued)
TABLE XVI (Continued)
Substance [marker] Plasma proteins [1251]:, [Ferl-LDL
Cell
Human fibroblasts
Route
In vitro
Cellular compartment
cv sv MVB DB
Asialoglycoproteins [Lac-Ferl-, [HRPI-
a2M (as in Table 111)
Rat liver
iv
sv GERL L
See Table 111
sv GERL L
Onset time (minutesP
Reference
1
2 6 6 0.5-2 5 < 7-15 < I 15 15-30
Anderson er a!. (1977)
Wall et al. (1980); Dunn et al. (1980)
Willingham and Pastan (1980)
X
Immunoglobulins IgG vs. Pz-microglobulin, [Ferl-Anti-lgC IgM (vs cell surface receptor): [FerJ-, [FITCIAnti-IgM
Cultured human fibroblasts Cultured human B lymphoblastoid cells (WiL2)
NCI L Polar capg EC NCI
cv
<15 30
Huet ei a/. (1980)
<5 Salisbury et al. (1980)
> 15
L
Lectin receptorsb [3H]Glucosamine-labeled surface glycoproteins; I25I-membrane proteins isolated by affinity chromatography on WGA-agarose columns
HeLa cells
SVh
< 15
Kramer and Canellakis ( 1979Ik
Noxious agents Lectin [HRPI-PHA, -Ricin
ViNS' Vesicular stomatitis
3 rD
Cultured murine drg neurons
BHK cells
In virro
sv
15
GERL
60
Gonatab et a / . (1977); cf. also, Gonatas er a / . (1975)
LJ
23
Miller and Lenard ( 1 980)
OIt is to be noted that many of the cytochemical methods utilized in the papers cited in this table and in Table 111 suffer from technical limitations; (see footnotes to latter table). Primary among these is that of improper preservation of membrane architecture, leading to artifacts and errors of interpretation (see Novikoff, 1980). Moreover, the onset time specified is dependent upon sensitivities of the several methods and is consequently subject to considerable variation. Generally, times denote association of the label with structure indicated at 3 7 T , following a preliminary exposure to ligand at 0-4°C. Abbreviations, unless otherwise denoted, as in Table 111. CP, Coated pit; CV, coated vesicle, iv, intravenous; NCI, noncoated invaginations; EC, endocytotic channel; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; Fer, fenitin; Lac-Fer, lactosaminated femtin; SG, secretory granule. hConcomitancy of entry with ligand generally implied, but only occasionally demonstrated experimentally. CAssay of cell fractions for specific binding was carried out after fractionation in the absence of ligand. dSmall vesicles (<0.1 km). eMultivesicular bodies (a class of lysosomes). fsecondary lysosomes [occasionally identified as dense bodies (DB)J. Rover Golgi region. hIsolated after WGA challenge of cells, and their contents subjected to SDS-PAGE. 'By the same token, examples of specific cell-surface receptors for interferon, their aggregation when occupied, and indications of the interaction of this class of rnacromoleculaes with intracellular structures such as the cytoskeleton (cf. Chany, 1981), implicate endocytotic entry. This does not as yet appear to have been studied directly. 'By inference, from time course of infectivity inhibition by UV-irradiated virus. *See footnotes to Table 111.
TABLE XVll OF IMPI.ICAIION
'nit CYTOSKEI.ETON" I N POTI:N.I.IAL TKANSLOCATION PHENOMENA
Processiparametcr
Cell type
Anchorage modulation in immune recognition; lcctin cross-linking; intercellular recognition during einbryogenesis "Receptor" redistributionlsestraint in response to specific ligands: Ig
Cholera toxin Interferon Eatradiol- 178 Acetylcholine Intimate association of integral surface membrane protcins at specialized regions of the cell surface6 with actin-, filamin-, and vinculin-containing cytoplasmic stress fibers; myosin< and microtubulcsc conspicuous by their absence
Serum-induced microvilli Macrophage spreading; pinocytosis, phagocytosis" Internalization of gap-junction membranes
A. In surface membrane dynamics Variety Edelman (1976, 1977); Edidin (I98 1 ); Hynes ( 1980)
Variety
Erythrocyte ghosts Embryonic muscle cells Cultured human fibroblasts, prostatic cells; murine tumor and lymphoid cells; brain cells
Exocytosis; thrombin-induced serotonin secretion
Ornental inesothelial cells Macrophages; normal and transformed fibroblasts Rabbit granulosa, human SW- I3 adrenocortical adenocarcinoma, murine B-16 melanoma Cultured human B lyniphoblastoid cells (WiL2) Pancreatic acinar cells; human platelets
Cytoskeletal component: organellar association
Cell type
IgG-mediated endocytosis
Reference
Rutishauser and Edclnian (1978); Nelson et a/. (1982) Sahyoun er nl. ( I98 Ib) Chdny (1981) Puca e t a / . (1981) Privcs et u / . (1982) Heggeness et a/. ( 1977); Ash et d.(1980); Mescher et al. (1981). See also Huct et a/. (1980); Geiger et a / . (1980); Keski-Oja and Yamada (1981); Willingham et a/. (1981b); Weatherbee (1981); Trump et al. (1981); Regnouf et al. (1982); Singer (1982); Mooseker et a / . (1982); Bennett e/ a/. (1982); Bourguignon e/ a / . (1982); Hubbard and Ma (1983) Madison et a/. (1979) Hartwig et a / . (1977); Singer (1979); Trotter (1981) Larsen et al. (1979)
Salisbury et al. (1980) Koike er al. (1980); Jennings ef a/. (1981)
Reference
B. In interactions with subcellular organelles'J Microtubules: synaptic vesiclcs; mitochondria
Spinal cord of lamprey, Petromyzon marmus; rat liver
I10
Smith (1971); Smith et a / . (1975); Bernier-Valentin and Rousaet (1982)
TABLE XVII (Continued) Cytoskeletal component: organellar association
Cell type
Reference
Microtubules: membranebounded vesicles closely and regularly related, but not contiguous; intervening space filled with finely filamentous material Actin, myosin: membranes of isolated chromaffin, insulin granules Intermediate filaments: melanin pigment granules
Purumeciurn caitdatum
Allen (1975)
Bovine adrcnal medulla; rat pancreas
Burridge and Phillips (1975); Howell and Tyhurst (1982)
Human, chick melanocytes
Microfilaments: lysosomes
Rabbit peritoneal PMN leukocytes; muscle Cultured human (MRC-5) fibroblasts
Jimbow and Fitzpatrick (1975); Mayerson and Brumbaugh (198 I ) Moore el u/. (1976); Bond and Somlyo (1982) Rohlich and Allison (1976)
Microfilaments: subplasmalemmal (“pinocytotic”‘?) vesicles Microtubules: secretory granules and membranes of the latter Microtubules: mitochondria (no correlation of latter with actin filaments)
Actin: lysosomes Microtubules: Golgillysosomesh Actin filaments: neurosecretory and other smooth microvesicles‘ in axon terminals and in pituicyteg Vimentin-type microfilaments: nucleus; microtubules Actinin: dense bodicsk Microtubules: mitochondria’ Dyneinimici-otubule: pigmcnt granules”’ Microtubules. niicrofilaments, and electron-dense plasmalemmal “plugs” in staging of cxocytotic vehicle movement
Isolated granule preparations from porcine pituitary
Sherline et a/. (1977)
Cultured normal rat kidney and smooth muscle (AID) cells, human W138 fibroblasts. murine peritoneal macrophages Bovine thyroidg Rat uterus; regenerating liver
Heggeness et a / . (1978)
Neurohypophysial system of the rat in vivo; heavy mcromyosin labeling in virro
Dickson et uI. (1979) Parr (1979); Mori et a / . (I 982); cf. also Seiden (1973) Alonso et d.(1981)
Cultured normal fibroblasts
Ball and Singer (1981)
Chicken gizzard smooth muscle Cultured NRK cells and human fibroblasts Isolated erythropores of the squirrel fish Holocentriis uscensionis Purameciurn tetrurireliu
Geiger et a/. (1981) Ball and Singer (1982) Beckerlc and Porter (1982, 1983) Plattner ef a / . (1982)
~
d’lncluding microfilaments. strcss fibers. and other matricuiar proteins of structural and kinetic properties (cf. Pollard. 1978; Weatherbee, 1981; Poste and Nicolson, 1981; Oliver and Berlin, 1982). (umtinued) Ill
TABLE XVIl (Continued) “In basal regions of microspikes and ruffles (sites of incipient endocytosis), and in regions of cell-cell contact. Note that there is presently no agreement on the association of actin-bearing filaments with the subplasmalemmal regions of coated pits @ro: Salisbury et d . , 1980, 1981; Gamer and Lasek, 1981; eon; Willingham et d . , 1981b). Resolution of this problem probably requires extension of observations to additional cell types and ligands, as well as technical improvements in analytic methods. CSee, however, evidence for plasma membranc-bound tubulin in rat hepatic (ascites) tumor cells (Akcdo et al., 198 I).Also, Braun ef ul. (1978) have provided a thoughtful analysis of circumstances in which myosin may or may not be concentrated in the cortical cytoplasm immediately below sites of capping in lymphocytes. Reevaluation of the long-standing problem of the role of microtubules in capping phenomena indicates that a closc correlation exists between Con A-induced capping and the disassembly of microtubules in human lymphocytes (Oliver et ul.. 1980). On the other hand, critical analysis of related problems in patching and capping leads Bretscher (1981) to the conclusion that microtubules are essentially noncontributory, the prime ”niovers” in membrane flow being the ses of rriicrofilaments (see however, Corps et al., 1982). Compounding such problems are the well known toxic side-effects of microtubuleimicrofilarnent-active “inhibitors.” Additional artifacts may be elicited by common synthetic buffers (Waxman el a‘., 1981). (’It is noteworthy that these functions are in abeyance during mitosis (cf. Berlin el d . , 197Xa). “Scc Rcbhun (1972) for a seminal review of this topic. More recent analyses of dynamic aspects of coated vesicle function relative to cytoskeleton (Ockleford and Munn, 19x0). and of inicrotuhulemembrane interactions in cilia and flagella (Dentler, I98 I)are available. Consideration of potential functions of componcnts of the cytoskeleton in lysosome redistribution. notably in polarized fashion, has bcen provided hy Szego (1975). The latter question has also bcen addressed in Section II,B,6a, above, as well as in Table XVII1A.B. [The potential modulatory role of Ca2 icalmodulin, including reversible phosphorylation of the latter, during cytoskcletal facilitation of organellc movement. is clearly evident (cf. Means et a / ., 1982). A new dimension has been added to this complex problem by the growing recognition that intracellular Caz+ i n intestinal epithelium (Colcinan and Terepka, 1972; Davis rr a / . , 1979; MacImghlin er al., 1980; Davis and Jones, 1981; Jones and Davis, 1981; Freedman e~ u l . , I Y X I ) , in neuronal cells and their derivatives (Sugaya and Onozuka, 1978; Grafstein and Fornian, 1980; Blaustein CI d . , 1980; Shaw and Morris, 1980; Akcrnian and Nicholls, 1981a,b), and in the mitotic 1980), as well as calniodulin (Linden et a/.. 1981; apparatus of sea urchin embryos (Silver el d., Sobue et ul., 1981; Means, 1981), are sequestercd in part in membrane-bounded microvesiclcs that may correspond to members of GERL. The extravesicular redistribu/ion of either component under 1981) bears deep significance for transduction and agonal control (c.g., Table XI; Conn et d., propagation of triggering signals impinging at the cell surface. VAn increasc in level of membrane-associated actin with TSH stimulation, Scc Table XVIIIA. ”Association inferred from inhibition of basal migration of lysosoines and randomization uf Golgi and associaled electron-lucent vesiclcs, 130-4.50 iini, after intraperitoncal administration of colchicine. ‘Including coated vesicles in pituicytes. ’In all axonal structures, actin filaments also frequently appeared anchored to the axoleinma, cspecially at perivascular sites, and were closely associated with thc filamentous material surrounding the microtubules. ‘A form of lysosome. ‘Association persisted after treatment of cclls with virus or cycloheximidc, procedures that elicited rcdistrihution of the intermcdiatc filaments to a perinuclear position. segregated from the niicrotubulca; thc latter remained extended to thc ccll periphery. ”‘Intracellular saltatury transport of pigment blocked by inhibitors of dynein, such as vanadatc and erythro-~-3-(2-hydroxy-nonyl)adeninc (EHNA); mechanism proposed is that of inhibiting the ATPase generating motive force originating in dynein and transduced while the latter is in close association with microtubules. The “cytoplast” as a functional and structural unit has been associated with repeated and unindirectional movement of thc same pigment granule (Porter and McNiven, 1482). I12 +
TABLE XVIII FOR PARTICIPATION OF THE CYTOSKELETON I N FUNCTIONS OF REPRESENTATIVE EI-FECTORS" EVIDENCE Agonist
ACTH
Cell type
Rat adrenal cells
Criteria
Corticosteroid production, (fluorometric)
Corticosteroid production (RIA)
Murine adrenal tumor cells
Steroid production (RIA) and LM
TEM, HVEM, SEM
Observations A. Peptidal agonists When cells were preincubated with four MT-inhibitors or cytochalasin B for 45 minutes, then treated with ACTH, steroidogenesis was inhibited When cells were incubated with anti-MT drugs for 24 hours, steroid synthesis was stimulated to the same degree as with ACTH The effectiveness of four cytochalasins as inhibitors of ACTH responses and as inducers of cell rounding was of the same order by both parameters Complete rounding of cells by 2 hours. Fully rounded cells displayed increased numbers of surface blebs, microvilli, and MT arrays, but fewer MF stress fibers
Conclusions
Reference
The cytoskeleton is involved in steroidogenesis
Crivello and Jefcoate (1 978)
MTs are involved in steroidogenesis
Ray and Strott (1978)
Cytochalasins inhibit the responses to ACTH by interfering with MF function
Hall ( 1 9PO)
Steroidogenic response to ACTH is coupled with alterations in MF organization and with MF-related surface changes
Mattson and Kowal (1980)
(continued)
TABLE XVIll (Continued) Agonist
Cell type
Criteria TEM, SEM
Cultured rat adrenocortical cells
SDS-PAGE
Y-1murine
IF
adrenal and CCL43 rat Leydig tumor cells
Observations At doses greater than 10 kg/ml, cytochalasin B inhibited the steroidogenic response. Stress fibers were lost and MFs were converted to felt-like masses Hormone-specific and concentration-dependent decrease in actin content
Prior to ACTH stimulation of steroidogenesis, tubulin was associated predominantly with cholesterolcontaining granules that were also acid-phosphatase positive. Following hormone treatment, tubulin became organized into MTs
Glucagon
Rat yolk sac
Uptake of macromolecular markers
Rat hepatocytes
Biochemical
Glucagon, epinephrine, and dibutyryl CAMP inhibit pinocytosis; insulin did not. Colchicine and vinblastine were also inhibitory Colchicine and cytochalasins B and D inhibited
Conclusions
Reference
Cytochalasin B disrupts the MF arrangement which promotes steroidogenesis
Mattson and Kowal (1982)
Shape change may be mediated by MF reorganization and alteration of actin content Tubulin may function to sequester cholesterol when steroidogenesis is in abeyance. Lysosomal concentration of tubulin implicit
Cheitlin and Ramachandran (1981)
Clark and Shay (1981)
Cyclic nucleotide regulation of MT function may be involved in the pinocytotic response
Brown and Segal (1977)
Cytoskeleton is involved in the glucagon response at a
Tomomura et a/. (1980)
LM
Glucose, lactate, urea production, [ '4CIAIB transPO*
Gonadotropins FSH
Cultured rat Graafian follicles
CAMP. by modified Gilman method
glucagon-induced increase in glycogenolysis; did not inhibit cAMP formation or protein kinase activity Addition of glucagon, hydrocortisone, and insulin to cell cultures prevented cell detachment due to colchicine treatment; omission of any one hormone negated this effect Colchicine treatment did not alter short-term hormonal effects. but diminished long-term effects on aaminoisobutyric acid transport. Vincristine, but not lumicolchicineh, also altered AIB transport Cytochalasin B or anti-actin serum abolished the cAMP elevation in response to LH and choleragen and inhibited the response to FSH, but failed to affect the response to R E 2 . Colchicine or anti-tubulin serum inhibited the response to FSH and PGE2, but not LH
point after protein kinase activation
Hormones may interfere with colchicine binding to tubulin, or may stabilize MTs
Galivan ( 1981)
MTs are involved in hormone-stimulated AIB transport, but not in glucose, lactate, or urea production
Prentki et al. (1981)
MFs are involved in LH and choleragen stimulation of adenylate cyclase, whereas PGEz stimulation is dependent on MTs and FSH action is dependent on both MTs and MFs
Zor er al. (1978)
~
~
~~
(continued)
TABLE XVIII (Conrinued) Agonist
hCG
Cell type
Criteria
Rat ovarian granulosa cells
LM, TEM, SEM
Cultured Sertoli cells from immature pig testis
PCM
Isolated rat luteal cells
Progesterone production (RIA)
Murine Leydig tumor cells
Binding of [12'I]hCG and progesterone production
Observations Increased intracellular CAMP, followed by cell rounding and loss of MF bundles. PGE,. PGE?, cholera enterotoxin. and dibutyryl CAMP had same effect FSH-stimulated cell rounding was prevented by testosterone; FSH induced MF disorganization: testosterone did not. Cytochalasin B, but not colchicine. mimicked the effect of FSH Cytochalasin B resulted in dose- and time-dependent inhibition of gonadotropin-induced steroidogenesis. Colchicine treatment had no effect Colchicine, vinblastine, and cytochalasin B were weak inhibitors of steroidogenesis, but did not affect hCG binding or degradation
Conclusions
Reference
CAMP-mediated changes in the MF network may be responsible for the FSHinduced shape change.
Lawrence et a / . (1979)
Hormonally induced shape change is mediated by alterations in the MF system. In Senoli cells. FSH and testosterone have antagonistic effects on MF organization
Chevalier and Dufaure (1981)
MFs, but not MTs, may regulate gonadotropin-induced steroid production
Azhar and Menon (1981)
Intact cytoskeleton is not required for hormone binding, but is involved in hormone-stimulated steroidogenesis
Ascoli and Puett (1978a)
LH
Isolated rat interstitial cells
Testosterone production (RIA)
Gn RH
Cultured rat anterior pituitary cells
LH and FSH secretion (RIA)
Glucose increased LH-stimulated testosterone synthesis, which was inhibited by cytochalasin B at conc. of 0.1-50 p M . LH-stimulated testosterone synthesis, in the absence of glucose, was inhibited only by doses of cyto. B P lOI*M Preincubation with colchicine or cytochalasin B inhibited LH and FSH release. but did not affect high K -induced LHiFSH release
Cytochalasin B inhibition of testosterone synthesis may be due to blockade of glucose uptake at lower concentrations of drug and blockade of MF function at higher concentrations
Murono er a / . (1982)
The cytoskeleton may be involved in the formation of the GnRH-receptor complex or in the mobility of this complex, but an intact cytoskeleton is not required for the release of secretory proteins
Khar er al. (1979)
In untreated cells, cytokeratin was located in perinuclear aggregates. Following EGF treatment, the aggregates were lost and cytoplasmic keratin filaments were formed EGF inhibited gastric acid secretion, altered the organization of MFs, and interfered with the surface ruffling characteristic of secreting cells
EGF may directly affect the organization of the intermediate filament system
Keski-Oja et al. (1981)
Modifications of the cytoskeleton may mediate the effect of EGF on gastric acid secretion
GonzLlez e l al. (198 1)
+
Growth factors EGF
Murine embryonic epithelial MMCE cells
IF
Rat parietal cells
TEM and stomach fluid pH
(continued)
TABLE XVIII (Conrinrted) Agonist
NGF
Cell type
Criteria
HeLa and 3T3 cells
IF
Chick embryo sensory ganglia
Tubulin content, by colchicine-binding assay
Mouse brain
SDS-PAGE
Chick brain
TEM
Rate of MT assembly, as monitored by turbidity of tubulin solution
Observations Treatment with EGF caused separation of centrosomes
Tubulin content of sensory ganglia was increased by NGF treatment. In ganglia treated with both NGF and vincristine, tubulin synthesis still occurred but neurite extension was blocked. Treatment with db-CAMP stimulated neurite outgrowth but not tubulin synthesis Coprecipitation with tubulin on addition of NGF to a high-speed supernatant Addition of NGF to partially purified actin induced polymerization into paracrystalline arrays and led to increased myosin ATPase activity NGF interacted with purified tubulin to form large complexes, a process which was inhibited by NaCl or
Conclusions As centrosomes are MT-or-
ganizing centers, EGF may induce centrosomal splitting by causing a reorganization of the MTs NGF and CAMP both stimulate neurite extension, but by different mechanisms
Reference Sherline and Mascardo (1 982a)
Hier
el
a/. (1972)
Interaction with tubulin and neurotubules may promote NGF stimulation of neurite outgrowth NGF may favor actin polymerization in vivo and may regulate the actinomyosin system
Calissano and Cozzari (1974)
Some effects of NGF are mediated by interactions with neurotubules
Levi er ai. (1975)
Calissano el a / . (1978)
Rat pheochromocytoma cells. clone PC 12
IF and autoradiography
Binding of [ t2SI]NGF
LM, TEM
SDS-PAGE
GTP. The formation of the NGF-tubulin complex accelerated the rate of assembly of MTs NGF, which inhibits cell division in this line, was found to accumulate progressively as discrete bodies in the perinuclear region slow"^ NGF receptors were associated with Triton-insoluble extranuclear cytoskeletons, whereas “fast” receptors were not When PC12 cells were grown either with or without NGF for 1 day, colchicine treatment caused a complete depletion of MTs. Cells which had been grown with NGF for 21 days were less susceptible to colchicine disruption of MTs NGF treatment led to increased levels of a HMW phosphoprotein. Further characterization indicated that this was a microtubule-associated protein (MAP), probably MAP1
NGF may regulate cell division via an association with perinuclear actin or tubulin
Marchisio et al. (1980)
Association of a subfraction of NGF receptors with the cytoskeleton may be responsible for NGF-induced changes in surface morphology Treatment with NGF may affect MT properties of PC12 cells
Schechter and Bothwell (1981)
The mechanism by which NGF promotes neurite outgrowth may involve the induction of a HMW MAP
Greene et nl. (1983)
Black and Greene (1982)
(continued)
TABLE XVIII (Conrinired) Agonist
Cell type
Criteria Binding of [ '2iI]NGF
-2!
Human thrombin
Chick, mouse, and human fibroblasts
Incorporation of ['Hjthymidine
Insulin
3T3 cells, embryonic mouse and chick fibroblasts
Incorporation of ['Hlthymidine
Observations When cells were previously incubated with WGA for 30 minutes. NGF bound to a homogeneous receptor population which had slow dissociation kinetics. Also, > 90% of the NGF bound to receptors that were associated with Tnton-insoluble cytoskeletons Colchicine-induced MT depolymerization stimulated DNA synthesis to 75% of the maximum level induced by thrombin. Taxol stabilization of MTs inhibited thrombin stimulation of DNA synthesis by 30% Colchicine inhibited mitogenic stimulation by insulin and by EGF in sparse cultures of mouse or chick, but not 3T3, fibroblasts. Colchicine itself was mitogenic in confluent cultures of embryonic chick fibroblasts
Conclusions A cytoskeletal protein may
Reference Vale and Shooter (1982)
act as an effector molecule in modulating receptor properties
MT depolymerization is involved in growth factor stimulation of cell proliferation
Crossin and Carney (1981)
Effects of MT disruption on hormone-induced cell growth are dependent on the cell type as well as on the density of the cultures
McClain and Edelman ( 1980)
Colchicine treatment produced a time- and temperature-dependent decrease in insulin binding
Isolated rat hepdtocyteS
Glucose oxidation; 2-deoxy-nglucose transport
Isolated rat adipocytes
Biochemical
Cultured Reuber hepatoma (H-35) cells
Tyrosine aminotransferase (TAT)
Colchicine inhibited both basal and insulin-stimulated glucose oxidation and 2-deoxy-~-glucose transport Insulin promoted the assembly of MTs. Insulin-stimulated lipid and glycogen synthesis, but not glucose oxidation, were inhibited by colchicine Cytochalasin B inhibited the induction of TAT by insulin or cortisol, but not by db-CAMP
Colchicine may impair the transport of unoccupied (recycled or newly synthesized) receptors to the plasma membrane Effects of colchicine are due to an action at the plasma membrane
Whittaker et al. (198 1)
Capacity of insulin to “direct” glucose metabolism may be dependent upon MTs
Soifer et al. (1971)
Cytochalasin B may prevent TAT induction by altering the MF network. CAMP may promote MF assembly or stabilize MFs, and thus antagonize the effects of cytochalasin B
Butcher and Perdue (1973)
Cheng and Katsoyannis (1975)
(continued)
TABLE XVIII (Continued) Actmist
Cell type
Criteria
Observations ~
Intrrfcron
PTH
Cultured human fibroblasts
LM. IF
Goldfish xanthophores
LM
Bone and CHO cells
Tubulin content. by colchicine binding assay
~~~
Conclusions
Reference
~~~~
Interferon treatment increased the number of actin filaments per cell. but did not affect MT or IF number: the rate of cell locomotion. amount of membrane ruffling. and saltatory movement of intracellular granules were all decreased Carotenoid-containing SER dispersed in response to MSH and aggregated in response to epinephrine. Cytochalasin B inhibited dispersion and colchicine inhibited aggregation The degree of tubulin polymerization was affected by the temperature of cell incubation, but hormoneinduced changes in morphology were not associated with changes in polymerization. However, MF distribution was altered by hormone treatment
Interferon inhibition of cell proliferation may be mediated by cytoskeletal and plasmalemmal alterations
Pfeffer er a!. (1980)
MFs are involved in pigment dispersion and MTs in pigment aggregation
Winchester et a!. (1976)
Suggests that hormone-induced morphological changes are mediated by MFs, not MTs, in cultured bone cells
Beertsen et al. (1982)
Prolactin
Mammary gland cells from pseudopregnant rabbits
Hybridization with [3H]cDNA probes for pcasein mRNA
Rabbit mammary gland explants
Immunoprecipitation with anticasein serum
Rat liver
Distribution of [ 125Ilprolactin and [125I]insulin in density gradients
Rabbit mammary explants
Biochemical
e
N W
Colchicine prevented the prolactin-induced accentuated transcription of the p-casein gene, but not the enhancement of p-casein mRNA stability Three lysomotropic agents, which inhibit degradation of the prolactin-receptor complex, did not prevent prolactin-induced casein synthesis, whereas five MT-disrupting drugs did By 1 hour after colchicine injection, uptake of PRL into light and intermediate Golgi fractions was inhibited. Vincristine was also inhibitory; lumicolchicine was not. Uptake into heavy Golgi and plasmalemmal fractions was unaffected. Colchicine had a similar, but reduced, effect on insulin uptake Griseofulvin, an anti-MT drug, did not prevent prolactin-induced casein synthesis. Also, ['H]colchicine was found to associate with cellular membranes, especially the plasma and Golgi membranes
MTs are involved in the transfer of the prolactin signal to the p-casein gene
Teyssot and Houdebine (1980)
MTs are involved in the mechanism of prolactin action
Houdebine ( 1980)
Prolactin transfer appears to be MT-dependent; insulin transfer less so. Prolactin and insulin may be transferred in different ways
Posner cc al. (1982h)
MTs are not required for prolactin action. However, colchicine binds to and alters the cellular membranes, thus interfering with the mechanism of prolactin-induced casein synthesis
Houdebine et al. (1982)
TABLE XVIII (Con?imed) Agonist
Cell type
Cntena
Observations
Conclusions
The level of membrane-associated actin was increased by TSH treatment. Also, a lysosomal fraction was found to contain a DNase I inhibitor believed to be actin itself Cytochalasin B inhibited the hydroosmotic response to vasopressin. The drug also increased permeability to Na' , CI - , and urea and induced the formation of large intracellular vacuoles Colchicine treatment inhibited osmotic water flow and the formation of intramembrane particle aggregates in response to vasopressin. but only when the colchicine treatment preceded vasopressin stimulation Mucosal microvilli and terminal web of granular cells were lost following vasopressin treatment
Actin association with lysosomes increases in response to TSH and may be involved in directed movement of lysosomes
Dickson er a / . (1979)
Cytochalasin B may interfere with the coupling of fluid transport and solute movement
Davis er al. (1974)
MTs may be involved in the initiation of vasopressininduced responses
J. Muller et al. (1980)
Suggests that vasopressin alters permeability in part by altering cytoskeletal elements in apical and basal regions
DiBona (1981)
TSHc
Bovine thyroid slices
Density gradient fractionation and PAGE of homogenates
Vasopressid
Toad urinary bladder
TEM, biochemical
Freeze-fracture
Granular cell of toad urinary bladder
LM, TEM
Reference
Miscellaneous Con A
Isolated rat adipocytes
Conversion of [U-'%]glucose to 14C02 was measured
1-Methyladenine (maturationinducing hormone) None
Starfish oocytes, stripped of follicle cells
LM, TEM, SEM
Rat erythrocytes
Biochemical
Acetylcholine
Rat soleus muscle
AcCho receptor sensitivity and electrical potential
The ability of Con A to mimic insulin by stimulating glucose oxidation was not blocked by the disruption of MTs and MFs, but was dependent on Con A valence 1-MA induced the formation of spike-like projections, containing bundles of MFs
Interaction of Con A with insulin receptors does not appear to be dependent upon MTs or MFs
Kahn et al. (1981b)
The MF-containing spikes may play a role in the organization of surface-associated 1-MA receptors
Schroeder (1981)
A considerable proportion of adenylate cyclase activity was associated Triton-extracted cytoskeletons
Suggests that hormonal stimulation of adenylate cyclase may be mediated by the cytoskeleton
Sahyoun et a!. (1981a)
Action of colchicine and cytochalasin B on the AcCho receptor is probably not due to MT/MF disruption
Anwyl and Narahashi (1979)
B. Hydrophobic agonistsn Treatment with either colchicine or cytochalasin B altered the neurophysiological properties of the AcCho receptor, but the effective doses were higher than those required for MT or MF disruption
(continued)
TABLE XVIII (Continued) Agonist
Cell type
a-Bgt
Rat and chick muscle
Adrenalineh
Teleost fish melanophores
Ekdysterone
Cultured Drosophila cells
Estrogen
Rat uterus
Criteria
Observations
Binding of [1251]a-A significant number of receptors was retained on Bgt or TMR-athe cytoskeleton following Bgt Triton extraction Cold treatment together with LM, TEM, and HVEM colchicine failed to prevent adrenaline-stimulated granule aggregation Actin content, asActin increased from 4% of sayed by DNase total protein in untreated cells to 9% in cells exI inhibition posed to ecdysterone for 3 days. Globular and filamentous forms of actin increased at parallel rates during the first 2 days, after which the filamentous form predominated Nuclear uptake of Concentrations of 10 - 5 to [3H]estradiol M cytochalasin B did not inhibit translocation of the estrogen- receptor complex to the nucleus. Vincristine did not affect translocation at early times, but did diminish nuclear uptake after 4 hours of preincubation.
Conclusions
Reference
AcCho receptors are intimately associated with the cytoskeleton
F’rives el a!. (1982)
Pigment granule movement is not MT dependent
Schliwa and Euteneuer (1978)
Actin synthesis and polymerization are stimulated by ecdysterone
Couderc et a / . (1982)
MFs and MTs are not required for nuclear uptake of the estrogen-receptor complex
Gorski and Raker (1973)
Water uptake
Nuclear uptake of [3H]estradiol
-
N
-4
Norepinephrine
Erythrocyte
Binding of [3H]estradiol-receptor (E-R) complex
Ehrlich ascites tumor cells
Depolymerized tubulin, by colchicine-binding assay
S49 lymphoma cells
CAMP, by modified Gilman assay
Colchicine inhibited estradiol-stimulated water uptake Colchicine did not inhibit translocation of the estrogen-receptor complex The E-R complex did not bind to erythrocyte ghosts, but did bind with high affinity, and in a time- and temperature-dependent manner, to Triton-extracted erythrocyte cytoskeletons Desensitization of adenylate cyclase to norepinephrine was accompanied by increased tubulin polymerization; no change in actin filament formation was identified Cytochalasin B enhanced the accumulation of CAMP which followed PGE,, isoproterenol, or cholera toxin treatment
MTs may be involved in the mechanism of estrogen stimulation The effect of colchicine on estrogen-stimulated water uptake is not mediated by inhibition of translocation The extrogen receptor may exist as an integral part of the cytoskeletal network
Fujimoto and Mom11 (1978)
MTs are involved in the desensitization process
Kurokawa et a/. (1980)
MFs may regulate the activity of the adenylate cyclase complex
Insel and Koachman (1982)
Kalimi and Fujimoto (1978)
Puca and Sica (1981)
(continued)
Agonist
Cell type
Criteria
Myeloid leukemic cells, nucleated and enucleated
Refercnce
Observations
Conclusions
Both hormones stimulated cAMP formation in MGI+D-' and M G I - D cells, nucleated, as well as enucleated. Colchicine and vinblastine increased the peak of hormone-induced cAMP in nucleated M G I + D + , but not MGI - D . cells Treatment with PGF,, or FGF resulted in an increase in the rate of initiation of DNA synthesis. Anti-MT drugs had a synergistic effect when added within 8 hours of PGF,,, or FGF addition. but the drugs alone had no effect MFs formed bundles within 3 weeks of androgen withdrawal. MT organization remained unchanged
MTs may be involved in both the normal response to hormonal stimuli and the desensitization process
Simantov el (I/. (1980)
.4n intact cytoskeleton ih not required for initiation of DNA synthesis: in fact. cytoskeletal disruption appears to enhance this process
Otto et 01. ( I 979)
As androgen withdrawal induces androgen insensitivity in these cells, it is suggested that androgen binding alters sensitivity via MFs Thyroid hormones may regulate brain development by promoting MT assembly
Yates cr a/.(1980)
-
PGFZU, FGF
3T3 cells
['HIThymidine incorporation
Testosterone
Cultured Shionogi 115 mouse mammary tumor cells
IF
Thyroid hormones
Rat brain
MT polymerization, assayed by turbidimetry
The degree of in vitro MT polymenzation in brain supernatants from hypothyroid rats was less than that seen in control supernatants from euthyroid rats
Francon cr a / . (1977)
Mouac brain
Triiodothyronine (T,)
Miscellaneous Tertiary arnine local anesthetics
Rat liver parenchymal cells
lurine BALBI3T.3 cells
Tubulin-tyrosine ligase (TTL) activity assayed by [?HH]tyrosineincorporation Uptake of [‘2SI]T3 by cells and by isolated plasma membrane vesicles
Brain levels of TTL. an enzyme involved in a-tubulin metabolism, were reduced in hypothyroid neonates Rate of uptake of T3 into isolated cells was decreased by treatment with colchicine or vinblastine: uptake by vesicles was unaffected
Thyroid hormones may regulate brain development enzymatically via TTL
Lakshmanan et a / . (1981)
MTs are involved in plasmalemmal transport process in situ
Rao ef a / . (1981)
IF
Anesthetics inhibited antibody-induced Ig capping. A similar effect was seen following treatment with cytochalasin B or colchicine
IT and MF regulate the mobility of cell surface receptors
Poste et al. (1975)
“Abbreviations: ACTH. adrenocorticotropin; MT, microtubule; RIA, radioimmunoassay; LM, light microscopy; MF, microfilament; TEM, transmission electron microscopy; SEM, scanning electron microscopy; SDS-PAGE. sodium dodecyl sulfate-polyacrylamide gel electrophoresis; IF, immunofluorescence; FSH. follicle-stimulating hormone; CAMP, cyclic adenosine monophosphate; PGE,, PCE, , PGF,,, prostaglandins; LH, luteinizing hormone; PCM, phasecontrast microscopy; hCG, human chorionic gonadotropin: GnRH, gonadotropin releasing hormone; EGF, epidermal growth factor; NGF, nerve growth factor; WGA, wheat germ agglutinin; db-CAMP, dibutyryl cyclic adenosinc monophosphate; SER, smooth endoplasmic reticulum; MSH, melanocyte stimulating hormone; PTH, parathyroid hormone; CHO, Chinese hamster ovary; TSH, thyroid stimulating hormone; Con A, concanavalin A. “Lumicolchicine is an analog of colchicine that does not bind tubulin nor disrupt MTs.
TABLE XIX CELLULAR T4RGETS COUPLED TO ACONALSIGNALS
EVlIJEliCE 01-PHOSPHORYLAlIOKlDEPHOSPHORYLATIOh IN
Homione
ACTH
Preparation
Purified synaptosomal plasma membranes; also, crude, lysed rnitochondriallsynaptosoma1 membrane fraction from limbic system of rat brain Isolated adrenal cells of fasciculatareticularis type
Kinase
Type
ND
cAMPd ( + )
ND
Phosphatase
Substrate
A. Peptidal agonistsa Unaffected Numerous proteins, including B-SOb
Presumptive cAMPd
Cytosolic 150K M , protein
Outcome
Reference
Dose and structuredependent inhibitory effect on phosphorylation of proteins in SPM fractions on addition of 100 rrM ACTH
Zwien et al. (1976, 1978); Jolles er al. (1981); cf. also, De W i d and Jolles (1982); Oestreicher er al. (1982)
Time and dose dependency of phosphorylation; esp. a cytosol protein of M, 150K was transient, with a stnking decrease after a 15 minute peak, not attributable to change in pool sue, believed due to ACTH-activated protein phosphatase
Podesta et a!. (1979)
Quartered adrenals; isolated fasciculata cells from hypophysectomized rats; also in vivo
cAMPd (+)
ND
ADH
Luminal and contraluminal membranes from bovine collecting ducts
cAMPd (luminal) (+)
ND
Angiotensin I1
Cultured smooth muscle cells from rat mesenteric arteries
cAMPi (+)
ND
cAMPd (+)
Numerous proteins of mitochondrial, microsomal, and high-speed supernatant fractions
Unknown
ND
Myosin light chains
Individual proteins Koroscil and Gallant (1980, both enhanced and diminished in de1981) gree of phosphorylation in all fractions (P-450 not among them). Time-course generally preceded steroidogenesis Schwartz et al. ADH binding stimu( 1974) lates adenylate cyclase in contraluminal membrane, providing CAMP for luminal cAMPd PK; believed to regulate permeability response Increased phosAnderson et al. ( 1981) phorylation peaked at 4 minutes; inhibited by CAMP. Believed to represent net processes of phosphorylationi dephosphorylation concomitant with contraction/ relaxation
TABLE XIX (Corimiued) Hormone Glucagon
Preparation Rat liver in vivo
Kinase
Type
Phosphatasc
Substrate
cAMPd" ( + )
ND
Limited fraction of lysine-rich hist on e
cAMPd'(+)
ND
Membrane proteins of mitochondria, lysosomes. and microsomes. with predominant effect on lysosornes
cAMPd[ ( + )
ND
Protein S6 of the 30 S ribosomal subunit
Outcome Without significant change in specific activity of phosphate pools. increased incorporation of QP into histones Increased uptake of phosphate insufficient to account for degree of enhancement of 32P incorporation into serine residues of the membrane proteins: increased incorporation 3ZP into lipid components Influence on efficiency of translation. possibly of mRNA for given enzymes, inferredd
Reference Langan (1969)
Zahlten er a/. (1972)
Gressner and Wool ( 1976)
cAMPd" (+)
ND
HMG-CoA reducrase (EC I . I . I.34); microsomal reductase kinasee
Isolated hepdtocytes and adipocytes
cAMPdd ( + )
Acetyl-CoA carboxylase (EC 6.4.1.2)
Cultured hepatocytes
(+)
Pyruvate kinase (EC 2.7.1.40) and phosphofructokinase (EC 2.7.1.11)
Increased phosphorylation of the rate-limiting enzyme in cholesterol biosynthesis, without change in [32P]ATP specific activity, resulting in decrease in enzymatic activity Phosphorylation accompanied by decline in enzyme activity consistent with physiological eventsf Phosphorylation of both enzymes enhanced, accounting for inhibitory effect of the hormone in the case of pyruvate kinase, but not, purportedly, for the second enzyme
Beg et al. (1980)
Witters et al. (1979)
Claus er al. (1980)
(continued)
TABLE XIX (Continued) Hormone
Preparation
Kinase
Type
Phosphatase
Substrate
Outcome
Reference
Induction of specific competitive inhibitor of the catalytic subunit of cAMPd PK; inhibitor also curtails activity of Ca2+ dphosphodiesterase Intracellular proteins, including vimentin
Effective feedback modulation, limiting cAMPd PK action as well as restricting CAMP degradation
Tash ez a[. (1979)
Possible dependence of intracellular responses to FSH and shape changes Enhanced phosphorylation may not be directly related to rate-limiting steroidogenesis step in mitochondria per se (Bakker et al., 1981) Substrate is inferred to be similar to ribosomal protein S6
DePhilip and Kierszenbaum (1 982)
Gonadotropins
FSH
LH
Sertoli cell-enriched rat testisg, in vivo
Cultured rat Sertoli cells
cAMPd (+)
Leydig cells isolated from adult rat testis
cAMPd (+)
Rat Leydig tumor cells
ND
14.3K.57K, and 77.6K M, proteins from whole cells that roughly paralleled increases in testosterone production M, 33K
Cooke et al. (1977)
Bakker et al. (1982)
hCG
LH; hCG
Rabbit ovarian fob licles at estrus Corpora lutea of 4-day pseudopregnant rabbits; in vivo Bovine luteal cells
cAMPd (+)
Rat Leydig cells in
cAMPdc (+)
I
Cytosolic proteins ( M , 64K, 84K, 93K, 99K, 115K)
virro
hGG
Porcine Leydig cells in vitro
I1
cAMPdC (+)
Cytosolic proteins M , 21K, 25K, 33K, 37K by 30 seconds (max.): up to 10 additional proteins on longer incubation At least 6 endogenous proteins, notably one of M, 90K
Selective activation of the two isoenzymes appears linked to secondary responses in the diverse cell types Progesterone secretion more closely linked to extent of endogenous protein phosphorylation than CAMP production or PK activation Acute action of the gonadotropins believed to be mediated by one or more of the phosphorylated protein substrates
Hunzicker-Dunn (1981)
Phosphorylation preceded testosterone production; dose-response curves of both were parallel
Gonzalez-Martinez er al. (1982)
Darbon et a!. (1981)
Dufau et al. (1981)
(continued)
TABLE XIX (Contirzued) Hormone Growth factors EGF'
NGF'
Preparation
Kinase
Human epidernioid carcinoma A-431 membranes
cAMPi
A-431 cell-free extracts
Caz--. phospholipid-d
Rat superior cenrical ganglia and chick dorsal root ganglia in organ culture; rat PCI2 cells
cAMPd
(+1
(+)
Type
ND
Phosphatase
Substrate
Outcome
Reference
Endogenous proteins; exogenous (histone, phosvitin, ribonuclease); phosphorylation of tyrosyl rehidues in both endogenous and histone substrates Exogenous histones. protamine Two NGF-responsive nuclear proteins in sympathetic neurons; despite certain similarities. HI and the HMG series excluded. In PCI?, one basic nonhistone iM, 3OK
Phosphorylations of components of M , 170K, 150K. 80K. and 72 5K enhanced by EGFh
Carpenter er a / . (1978, 1979): Ushiro and Cohen ( 1980)
Increase in activity, a response inhibited by a phorbol ester Phosphorylations enhanced
Sahai ef a / . (1982)
Yu et a / . ( 1980)
IGF'
PDGF'
PC12 cells and cell-free extracts Stationary chick embryo fibroblasts
Plasma membranes from human fibroblasts or glial cells
Immunoglobulin Anti-IgE Rat basophilic leukemia cells
cAMPd (+)
M , IOOK Ribosomal protein S6
Phosphorylation greatly decreased Several-fold increase in phosphorylation by 5 minutes. associated with tranbition into G , phase of cell cycle
Tyrosine residues in proteins of M , 175K and 130K
M , 35K @-chain of the receptor for IgE; achain not a substrate
End er d.( 1 982) Haselbacher er a/. (1979)
Ek
Protein phosphorylation only on challenge (and crosslinking) of receptors with antibody. However, the ionophore A23 187 led to phosphorylation of related protein in similar cells (Hempstead er a/. .
el
a/. (1982)
Fewtrell et a / . (1982)
198lY (continued)
TABLE XIX (Continued) Hormone _
_
Insulin
_
Preparation ~
Kinase
Type
Phosphatase
Substrate
Outcome
Reference
~
Sarcolemma membranes from rat hindlimb muscles
cAMPi (+)
Cultured human
(+)
lymphocytes (1M-9; insulin “insensitive”) and rat hepatoma (Fa0 cells; “insulin sensitive”) Murine 3T3 cells
0
ND
Proteolipid M, 15K
95K receptor subunit
p-
Uncertain whether effect elicited on a phosphatase that dephosphorylates inhibitor-1 (thus lifting inhibition of phosphatase- 1) or a direct enhancement of a proposed phosphoprotein phosphatase per se
Insulin enhanced phosphorylation of one of the polypeptide subunits (M,3.6K), with implications for modification of membrane transport functions’ In both cell types, insulin enhanced phosphorylation of this protein in a specific and doserelated fashion at 1 and 15 minutes Rapid, concentration dependent enhancement of protein phosphatase activity, using phosphorylase a as substrate
Walaas el al. (1977. 1981)
Kasuga et al. (1982); see also, Roth and Cassell (1983)
Picton (1982)
Murine 3T3-LI preadipoc ytes
(+)
ND
110K M, subunit of ATP citrate lyase
-w *c
Interferons
Murine L cells
dsRNAd (+)
Cell-sap proteins of M, -60K and a second low M , cornponent; exogenous histones
Insulin (0.2 fl)or isoproterenol (the latter in the presence of methylisobutylxanthine) each stimulated the incorporation of 32P into the lyase -3fold, in a nonadditive manner. The 32P-labeled tryptic fragments were indistinguishable from each other, and from the corresponding preparation stimulated by CAMP alone Increase in phosphorylated products in interferontreated cells suggestive, but properties diverge markedly from those of an initiation factor that contributes to inhibition of protein synthesis'."; see, however, Samuel (1981)
Swergold er al. (1982)
W. K. Roberts et al. (1 976); Ken er al. (1980)
(continued)
TABLE XIX (Conrimred) Hormone Oxytocin
Parathyroid hormone
Preparation Rat mammary myoepithelial cells Rabbit renal cortical tubules in
Kinase
Relaxin
Rat uteri in virro
Somatostatin
Rabbit liver in vivo
Phosphatase
Substrate
Outcome
Reference
Caz--d
Myosin light chain; M , 20K
Enhanced phosphorylation
Olins and Bremel ( 1982)
cAMPd' (+)
Enhanced phosphorylation of proteins M, 150K. 125K, IOOK, and 50K Enhanced phosphorylation of proteins of M , 96K and 62K in membranes from parathyroidectomized dogs 2 CAMP vs those of controls Myosin light chain (MLC; M , 20K)
Suggestive of pattern of CAMP-mediated effects of the hormone
Ausiello et a1 (1976)
vitro
Canine renal brush border membrane vesicles
Type
cAMPd (+)
Exogenous purified synthase D from dog muscle
Hammerman and Phosphorylation with Hruska (1982) concomitant i ~ h i b i tion of Na+-stimulared Pi transport
Diminished phosphorylation associated with decreased kinase activity of MLC Precipitous increase in phosphoprotein phosphatase
Nishikori er 01. (1982)
Curnow et al. (1977)
EC 3.1.3.16
Cytosol from liver, pancreas, and gastric and intestinal epithelial cells, in
(+)‘I
32P-phosphorylated exogenous hiatone
vitro
TRH
GH pituitary cells in vitro
cAMPi (+)
ND
ND (+)
TSH
Thyroid slices from control, hypox., goitrogen-. and TSH-treated rats
cAMPd< (+)
ND
(+)
TRH enhanced phosphorylation of a variety of cellular peptideh. while diminishing the phosphorylation of a 55K M , product. Some overlap with, but primarily divergence from, the patterns elicited by CAMP Exogenous histone
Suggest enhanced phosphoprotcin phosphatase activity could account for triggering of some physiological effects of the hormone on GI tract Results support a mechanism of TRH action that is unlikely to depend on intracellular mediation by CAMP
Reyl and Lewin (1981, 1982)
Evidence of parallel regulation of PK and protein phosphatase activities
Huprikar et a / . ( 1979)
Drust et al. (1982); cf. also Drust and Martin (1982)
(continued)
142 HeLa cells
n z
VIP0
ND
Increased phosphorylation of at least 8 of the 350-odd phosphorylated peptides elicited by TSH; majority of latter cAMPi Phosphorylation of endogenous H I , H3, and HMG-14 enhanced by the hormone; HMG-17 was phosphorylated neither in control nor TSHtreated preparations Exogenous histone
Support CAMP-mediation of TSH action
Lecocq et al. (1 979)
i
ND
9
cAMPdC
ND
z
Calf thyroid slices
(+)
5
cAMPd[
z
Dog thyroid slices
Cooper et al. (1982)
Stimulation of phosphorylation, coupled with known enrichment of 0 reproductive tissues in VIP, and high-affinity binding of VIP in HeLa cells, suggestive o f physiologic relevance
Priero et al.
(1981)
B. Relatively hydrophobic agents Androgen DHTq
Testosterone
Human prostatic PM: microsotnes (it2 vitru)
+'
ND.7
+'
Na ,K + -dependent ATPase
Rat ventral prostate and levator ani muscle
cAMPd
1
ND
ND
+
ND
Alk; increase with 6 ; reversed with androgen
cAMPd
I1
Both phosphvitin and histones served as exogenous substrates for PK, with the former a more sensitive indicator of androgen status; endogenous chromatin proteins also effective ND
Prostatic nuclei and chromatin
Rat dorsolateral prostate after 6-8 months treatment with 0.6 mgiday of the propionate
+
Rapid (s) increases in both processes; presumptive association with functional changes in membrane permeability to AIB; steroid specificity Rapid decrease in enzyme activity after castration restored with hormone in vivo Increased net chromatin protein phosphorylation under androgen influence
Famsworth (1977)
Treatment abolished binding of a photoaffinity probe, 8azido-CAMP, to 3 cytosol proteins ( M , 59K, 51K, and 43K)
Chung er al. (1981)
Fuller et al. (1978); see also Wilson and Ahmed (1977) Ahmed er al. (1979)
(continued)
TABLE XIX tCoj7rit7urd)
Hormone
Preparation
Kindsc
Type
Phoaphala\e
Substrate
Outcome
Reference
hlurine uterus (in ~'I'vo): 4 minutes after ip; redbcAMP sults
Phosphorylation of the relekanr kinase (inactivation)
(c.4MPi)
Dephosphorylation (acnvation) by (a) cAMPd 1')) protein phosphdtase
+
I
Provocative implications for translational controls, esp. of proteins of enzymic, rather than "structural" composition Converse data with progesterone treatment of E2Pprimed rabbit'
Berg (1977, 1978)
Endometrium of ovx rabbit (in
12 of 17 aminoacyl-t-RNA synthetases activated; 5 decreased in activity or unchanged Endogenous proteins: in virro. histone, protamines, phosvitin and casein also served as effective substrates. Lack of significant effects on K , for CAMP, and the obliteration of the changes on cycloheximide administration were suggestive of de novo synthesis of the respective enzymes in response to the steroid hormones
Estrogen E2P
-
vivo)
ND
cAMPd (sl I
I1 cAMPd
Miyazaki er al. ( I 980a)
Tubulin
Ovariectomized rats
Uterine cytosol and nuclei from mice
Adrenocortical hormones Aldosterone Kidneys of adrx rats by 40 minutes of the administration of 5 Kgl100 g body wt ip or sc
+ Partially purified from calf uterus to permit limited characterization
+
Ca2+d; ATP requiring
+
ND
ND
Receptor for E2P
(Nuclear); inferred from NaMoOa inhibition''
Nucleoproreins, especially histonesW
Switching in phosphorylation from p- to a-tubulin under estrogen domination: effect inhibited by progesterone "Receptor" inactivated toward EZPbinding by a nuclear phosphatase may be reactivated by a cytosolic activity requiring ATP". "Receptor" translocated to nucleus in association with E2P-antagonists not subject to dephosphorylation
Joseph et u/. ( 1 982)
Presumed to precede and favor activation of gene expression
Liew ef al. (1973)
Migliaccio and Auricchio (1981); Auricchio ef a/. (1982)
(continued)
TABLE XIX (Continued) Hormone
-
Aldosterone and 0thenX
01 P
Preparation
Kinase
1 p M added to membranes and other preparations from toad bladder
0
Administration ip; cytosol from series",y of relevant targets
-(?)
Type -
NDw
Phosphat ase T
+ (?)
Substrate "Protein D" (M, -49K) of membranes and in cell sap
SCARP-'
Outcome
- Effect of CAMP; parallels inferred on actions of aldosterone and ADH (latter via CAMP) on Na+ and HzO permeability of (apical) membrane In each of 5 target organs, the relevant hormone in vivo at substantial doses reduced the degree of endogenous phosphorylation of a protein of electrophoretic properties "as early as 2 hours" [after E,P or testosterone] Adrx. elicited decrease in the inhibitor activity from muscle supernatant of either control or adrx rats; cortisol tended to restore (permissive?)
Reference Liu and Greengard (1 974)
Liu and Greengard (1976); see also Liu er al. (1981)
-
Cortisol
Muscle supematant fraction
0
Phosphorylase phosphatase inhibitor (cAMPd)
Phosphorylase phosphatase
Green er aE. ( 1980)
Cortisol and othersh
Liver and adipose tissues
cAMPd
RII
-
Liver cytosolic Autophosphorylation protein, M, decreased 54K;properties to regulatory subunit 11 Multiple protein Burst of phosphoprotein labeling bebands in electween 2 and 5 tropherograms from both hours after progesterone applicacytosol and tion to the oocytes 100,OM) g pelin vitro preceded let incorporated 32P. source of germinal vesicle which was prebreakdown (GVBD; an indicasumed to be tor of resumption ATP, preof meiotic maturalabeled with tion) at 6 hours 32P in a preliminary incubation; peak band (40-45K) showed sharp decrease after GVBD Trigger for GVBD Unknown
Liu
et
al. (1981)
-
Progesterone
Xenopus laevis oocytesr
ND
ND
ND
-
-
Inferred from blockade of inhibitor-laa
Belle er al. (1978)
Maller and Krebs (1980); Maller and Sadler (1981) (continued)
TABLE XIX (Catz1IriuedJ Hormone
5 P
Insect hormone Ecdysterone
Plant hormones 2 ,4-Dbb
Preparation
Kinase
Type
Substrate
Outcome
Reference
Ribosomal protein S6
Phosphorylation precedes GVBD
Hanocq-Quertier and Baltus (198 1j: Nielsen er a/. (1982); Kalthoff et a[. (1982) MacDonald et al. ( 1982)
Hamster uterus
ND
ND
Nuclear acid phosphatase activity enhanced by 2 hours after progesterone in vii.0; inhibited by Mo and Vr
By inference. (phosphorylated) estrogen “receptor”
Progesterone treatment resulted in roughly parallel loss of estrogenbinding activity; causal relationship inferred
Murine adipose tissue ( i n viva and in virro)
-
cAMPd
Phosphoprotein phosphatase (+)
Unknown
Data may contribute to understanding of heterophylic effects
Catalan ef a/ (1982)
Mature soybean
+
cAMPd
Data also supported increased phosphate tumover
Basic nonhistone nuclear proteins with low mobilities in acidurea gels
Murray and Key (1978)
+
ND
ND
Nonhistone chromatin proteins
Changes paralleled the reported 2,4-Denhanced RNApolymerase I activity and elevation of RNA synthesis in vivo A protein kinase activity, separated from homologous RNA-polymerase I1 by 0.35 M (NH4)2SO,, postulated as an integral
(G/ycine max-)
hypocotyl
GibberellinA3
Phosphatase
Maize seedlings (in viva)
Jankowski and Kleczkowski (1980)
function of the latter enzyme Vitamin D (1,25(OH)2D3)
Chick intestinal brush border fraction and derived membranes ( i n vivo)
+
-
DRPcc. M , 84K
to DRP properties those of a protein induced by the hormone
Wilson and Lawson (1981)
+
Fraction ND (but not derived membranes), Ca2+d, Mg2 i cAMPd ND
Presumptive induction of PK per se
D3-derivative restored the subnorma1 activity of the 2 PK-isoenzymes; increased level of an inhibitor also seen in the deficient tissues, but inadequate to account for quantitative decline in PK
Rudack-Garcia and Henry (1981)
t
ND
Nonhistone chromatin proteins (NHP)
Within hours of T3, enhanced phosphorylation by endogenous PK in same preparations; no effect DIT or rT3; no change in cytoplasmic activities, as assayed with casein, phosvitin, or histone
Gibson et al. (1975); Kruh and Tichonicky (1976); Taningher et al. (1977)
+
Chick kidney (in vivo)
Thyroid hormone Rat myocardium, T3 liver (in vivo)
ND
(continued)
TABLE XIX (Continued) -Because of the extensive nature of the relevant data on neurotransmitters and the availability of numerous reviews, this class of agonists is not considered here. bACTH also elicited a dose-dependent increase in the formation of phosphatidylinositol biphosphate and inhibited the production of phosphatidic acid (470 and 50% of respective controls at 100 fl hormone). CInferred from parallel results with application of derivatized cAMP or of inhibitor of the relevant phosphodiesterase. dlnferred from concomitant increase in cAMP content elicited by the hormone. PDephosphorylation of the kinase was associated with loss of its activity. Since glucagon promoted a 2-fold increase in activity of the reductase kinase, ifs phosphorylation was inferred to be enhanced by the hormone. fl'aradoxically, insulin, known to elicit enhancement in enzyme activity, which is favored by dephosphorylation, likewise promoted a small but significant increase in 3*P incorporation (Witters, 1981; Brownsey and Denton, 1982), in the latter case, possibly by a CAMP-independent kinase. Analysis of these and parallel events, such as the stimulation of phosphorylation of ribosomal protein 56 by insulin, as well a5 by dibutyryl CAMP,has revealed that the hormone and the cyclic nucleotide effect phosphorylations of different sites within the S6 molecule (Lastick and McConkey, 198I). nDevoid of Leydig cell elements. hSubsequent work by this group (Carpenter and Cohen, 1979; Carpenter et a!. 1981) and others (e.g., Fernandez-Pol, 1981 a.b) has revealed ( I ) that the 170K and 150 K proteins may represent forms of the EGF receptor; (2) that EGF receptor appears to have inherent PK activity; and (3) that autophosphorylation of thc latter in presence of EGF occurs at multiple sites (Gates and King, 1982). These combined data suggest that the EGF binding, kinase, and substrate domains may be integral to a single 170K M , molecule (Cohen et al. 1982; Maciag, 1982). 'Epidermal growth factor; nerve growth factor; insulin-like growth factor; platelet-derived growth factor. JSee review by Winslow and Austen (1982). instructive review on the functions of insulin in promoting both CAMP-dependent and -independent phosphorylation and dephosphorylation of membrane proteins has been provided by Houslay (1981); see also. Walton (1978). Detailed analysis of the composite influences of insulin, somatostatin, and glucocorticoids expressed in net phosphorylationidephosphorylationof the enzymes involved in intermediary metabolism has been presented by Curnow and Lamer (1979); cf. also Le Cam (1982), Garrison and Wagner (1982), and Lamer et al. (1982). 'This topic has now been more extensively treated (see Samuel, 1981). "The several actions of interferons have been compared with those of hormonal effectors (Baglioni and Nilsen. 1981). "Indications are provided by Reyl and Lewin (1982) from reconstitution experiments that the mechanism of enhancement of the phosphatase activity by somatostatin involves its interaction with a regulatory subunit. C'Vasoactive intestinal peptide. PInferred from maximal sensitivity of endogenous kinase when hormone added in presence of phosphodiesterase inhibitors
Y I O - ~M . Phosphorylation is Na -dependent; dephosphorylation. K -dependent. ‘ND. Not dctermined. ‘Variation of protein kinase activities during the estrogen-dominated proliferative. and the progesterone-dominated progestational phases, of the human menstrual cycle were compatible with these Observations (Miyazaki er a/., 1980b). “See text and Table XV for indications that molybdate and related transition elements may inhibit proteolysis, rather than, or in addition to, phosphoprotein hydrolase activity. with comparable outcome in retaining “receptor” in “active” form. I Note that this indirect indication of endogenous nuclear “phosphatase” activity, to which the estrogen-loaded “receptor” is subject, differs from that described by Nielsen er a/. (1977) for glucocorticoids in that, in the latter case, only unbound “receptor“ is affected. The apparent requirement of ATP or a system for its generation to promote ligand interaction with “receptor“ following its stripping in the nucleus, is. as in the case of the earlier observations of Munck and BrinckJohnben (1968) and the Pratt group (lshii e l a/., 1972) for glucocorticoids, only very indirect evidence that the trinucleotide may serve as a phosphate donor in rendering “receptor” capable of binding a given hormone (see also Grody e t a / ., 1982). Although subject to phosphorylation by cAMPd protein kinase from bovine heart (Weigel er (I/. , 198 I ) , the oviduct receptor from laying hens does not appear to serve as a substrate for endogenous (Keller er al., 1976) enzyme as tested by Weigel et u / . (1981). Nevertheless. there are indirect indications from the latter work and elsewhere that the two native progesterone-binding subunits occur in partially phosphorylated form. “Acetylation often predominated, in both heart and kidney, at 40 minutes. \Uterus (E#); prostate and seminal vesicle (testosterone); liver (cortisol); toad bladder (aldosterone); rat tissues unless otherwise indicated; note reducrion in autophosphorjhtion of presumptive RII would promote reassociation with. and curtailment of activity of, the catalytic subunit. ’However. M , of SCARP (Steroid- and CAMP-Regulated Phosphoprotein) 49-54K. -Must be applied externally (see Table IIE in Szego and Pietras, IC.81). Note GVBD in rac oocytes is unaffected by progesterone or estrogen added in v i m . Howevcr. GVBD in rat oocytes, preceded by intense pennuclear accumulation of lysosome-like organelles under LH influence (Ezzell and Szego, 1979), is probably the outcome of proteolytic activity released from the hormone-activated organelles at this focal site (Szego, 1976). In starfish oocytes, on the other hand, reinitiation of meiosis and GVBD is elicited by I-methyladenine produced by the follicular cells; it is notable that simultaneous blockade of GVBD as well as inhibition of lysosomal proteolytic function are achieved by application of ammonia and other weak bases (cf. Table VI) within the hormone-dependent period (Dort5e r t a / ., 1982). Such observations are consistent with liberation of lysosomal protease(s) from the activated organelles in the chain of events set in motion by agonal recognition (see Table VIIIA). In turn, the activation of certain protein kinases by limited proteolysis (Del Grande and Traugh, 1982) bears further relevance to cascading events leading to GVBD. ““Protein phosphatase-1 may be injected. “”2,4-Dichlorophenoxyaceticacid, a synthetic auxin established as a plant growth hormone. <‘<(Vitamin) D-Responsive Protein +
-
-
‘A
+
-
IS2
CLAKA M . SZEGO AND RICHARD 1. PIETKAS
surface polypeptide that may be triggered by a given ligand. including a toxic one: e.g., WGA (Kramer and Canellakis, 1979), Con A (Yahara and Edelman, 1973), and ricin (Gonatas et al., 1975). Indeed, the apparent binding of two agonists of dissimilar structure to one and the same receptor, as evidenced by direct competition, highly unlikely on theoretical grounds, may exemplify such regional overlap on the cell surface that present methods are incapable of resolving. Examples include acetylcholine and rabies virus (Lentz er al., 1982), “transforming growth factors” and EGF (Todaro era!. , 1980),and even insulin and its antibody (Kahn et al., 1981a). Moreover, common internalization of EGF, insulin, and a,-macroglobulin in the same “preclustered” patches on the surface of cultured fibroblasts has been described (Maxfield era/., 1978;cf. also Carpentier et al., 1982). However, it should be noted that what may appear to be cocapping,” may not represent topographically identical, but only generally regional interactions when scrutinized with utmost care (cf. Oliver et al., 1980). Nevertheless, sonic of the above and related observations of interactions among otherwise dissimilar agonists (cf. Katzen et ul., 1981; Sorge and Hilf, 1981) may, in turn, hear upon the kinetics of association of the given ligands with receptors that have been considered to exhibit positive or negative “cooperativity.” As indicated in a related context, the very binding of agonist with the specificity and high affinity requisite to interaction with receptor may promote the mutual conformational change in either or both interacting moieties, termed “adaptive complementarity” (Szego and Pietras, 198I ) . Such a process may actually underlie the frequent reports of “positive” and “negative” cooperativity (cf. De Meyts, 1976)exhibited on ligand recognition (Pietras and Szego, 1980;Tait et ( I / . , 1981; Comens et a/., 1982;Corin and Donncr, 1982). Indeed, Conn and co-workers (1982)make a compelling case for conversion of a pure antagonist to a product with agonal properties by immunologic interaction, rendering the microaggregate capable of cross linking two surface receptor molecules within a critical distance of each other and generating a transmembrane response. Regardless of the ultimate mechanism to be determined, these sporadic examples of haftling complexity simply illustrate the degree to which the surface architecture of a cell may he provoked toward disorder when challenged with a given agonist. c. Time Course. Survey of the literature in Tables 111 and XVI demonstrates that with given ligands, the onset of apparent endocytosis follows by mere seconds their surface binding (cf., also King and Cuatrecasas, 1981). Such rapid entry is consistent with the kinetic characteristics of the endocytotic process, whether receptor-linked/ “mediated” or triggered by less specific surface perturbation (see Szegn, 1978, and Section I1,B for references). Likewise, the participation of such a process is in accord with the chronologic staging of numerous secondary responses of a (target) cell (cf. Fig. 3; Tables I, IV, and V), which occur prior to, and, apparently, independently of, genomic activation (cf., “
LYSOSOMAL FUNCTIONS IN CELLULAR ACTIVATION
153
Szego, 1974, 1975, 1976, 1978; Spelsberg and Halberg, 1981; Szego and Pietras, 1981). While alternate interpretations are not excluded, such concomitancy merely establishes compatibility of a cellular entry process with its putative consequences in the cellular activation chain. It is of paramount importance to note that neither surface display of receptor under “basal” conditions (i.e., prior to ligand interception), nor functional correlates of cellular response (i.e., cellular activation/subduction) are quantitatively invariable throughout the cell cycle. To the contrary, cyclicity is a characteristic feature of binding (e.g., Varga et al., 1974; Cidlowski and Michaels, 1977; Cidlowski and Muldoon, 1978; McNeilly el al., 1980; Lustig et al., 1980; Shimizu et al., 1981) and receptor-mediated endocytosis (e.g., Shimizu et al., 1981) of given ligand. It is also an inherent property of the cell surface, as indicated by variability of basal endocytosis in the absence of specific agonist (Davies and Ross, 1980), a phenomenon subject to circadian changes during the cell cycle (Quintart et al., 1979b) and to more profound alteration by cellular differentiation (cf. Reed et al., 1981). In turn, variation of physical properties of lysosomes (Quintart et al., 1979a) and of individual lysosomal enzymes, both intracellular and as apparently shed into the immediate cellular environment, has been described in hepatocytes (Uchiyama et al., 1981; see also, Twardowski et a l . , 1981). Synchronous turnover of peroxisomal proteins has also been identified (Poole et al., 1969). These and related considerations (see Sections II,B and below) may contribute to clarification of the long-known observation that responsive and unresponsive cell populations, which either “recognize” (bind) or fail to bind agonist, characteristically coexist among “target” cells at a given moment (see footnote 2 ) . The simplest interpretation of these biological facts may be that receptor molecules, like other integral membrane components, are synthesized and intercalated into the cell surface as a distinct function of the given phase of the cell cycle. Indeed, features of receptor “shuttling” (a term coined independently in this context by Y.-J. Schneider et al., 1978, and Szego, 1978), down-regulation (see below), and turnover and rebound (up-regulation; see below) may serve as indicators of the synchronous expression and redistribution of receptor macromolecules. d. Significance of Cellular Entry of Effector:Some Ironic Footnotes. Surely the capacity for intracellular entry of a variety of toxic substances was not evolved merely to facilitate their destructive functions! Instead, it appears that the latter substances have subverted a mechanism that, after the all-important primary recognition at the cell surface, serves the orderly internalization of a wide variety of effectors (see Tables 111 and XVI; Figs. 9 and lo), with legitimate regulatory business in the cellular interior. This irony has not been lost on a number of authors in the context of uptake by cells of toxic lectins (Olsnes and Sandvig, 1981), viruses (Helenius et al., 1980a,b), and many other noxious substances, including bacterial toxins as well as colicins (cf. Neilands, 1979),
154
CLARA M. SZEGO AND RICHARD J . PIETRAS
and promotors of carcinogenesis (Shoyab and Todaro, 1980). Occasionally, there has appeared to be some homology among portions of the primary structures of a number of these generally noxious agents and the “normal” effector(s) appropriate to the given cell. Even certain parallels have been identified between the metabolic actions of given toxins and certain growth factors (cf. Cooper and Hunter, 1981). Whatever the outcome of longer term investigations in this area, the evidence now strongly suggests that specific high affinity receptors for noxious agents coexist on cell surfaces with recognition factors for the “normal” effectors. These convenient facts are being exploited for the design of hybrid molecules capable of being targeted to a given cell type by virtue of specific recognition features of the carrier portion of their compound structure, whether monoclonal antibody (e.g., Youle and Neville, 1980; Blythman etal., 1981) or lectin (Youle and Neville, 1979; Edwards and Thorpe, 1981), while covalently linked to the intracellularly active toxin A-subunit, which is not recognized per se. i n turn, composite structures, of which the binding moiety is hormone, can deliver into the cellular interior the noxious A-chain of ricin (Oeltmann and Heath, 1979), or diphtheria toxin (Miskimins and Shimizu, 1979) of cells bearing externally displayed receptor for hormone, whether, e.g., hCG or insulin, respectively. Alternatively, when the ricin B chain serves as recognition moiety, covalently linked insulin is presented to rat hepatoma cells primarily through specific ricin B receptors, as evidence by competition with galactose and failure of competition with free insulin, the outcome is that of insulin-like action quantitatively different from that elicited by the parent hormone (Roth et ul., 1981). Further potential of hybrid technology has been revealed in the intensely enhanced steroidogenic capacity of ACTH (1-24), covalently linked to tobacco mosaic virus, and presented to isolated rat adrenal cells “preclustered” to the extent of - 150 peptide units per virion (Kriwaczek et ul., 1981). 3. Firsioit of Eiiciocytotic Vesicles with Lysosoint~s While attempting to dissect interlocking signals of membrane perturbation (see Section III,D, I ) , it is also instructive to consider the occasional indications of parallel time course for the cellular redistribution of hormone und receptor, as far as present advances permit (Table XVi). Such concomitancy clearly implicates the continued association of the two constituents in the endocytotic-vesicle sequestered state. Indeed, from presently available evidence, it is likely that the two moieties remain in close association beyond even this phase, although not necessarily in their unaltered forms. Thus, as will be detailed in a later section, there is cogent evidence to indicate that processing of both receptor as well as hormone (and their respective biosynthetic precursors) is a closely regulated lysosomal function with potentially powerful implications in intracellular propagation and amplification of the primary triggering signals. The chance and transient encounters between certain endopinosomes and pe-
LYSOSOMAL FUNCTIONS IN CELLULAR ACTlVATION
I55
ripherally disposed primary lysosomes and/or other components of the vacuolar apparatus, attributable to their inherent capacities for mobility or to their productive association with members of the cellular scaffolding, are likely to lead to fusion of the two classes of organelles under specific conditions. Fusion, defined as the mutual apposition of component parts of the independent surface membranes and their stable intermingling (Lucy, 1978, 1982; Poste and Allison, 1973; Gallaher et al., 1980), would be favored under those circumstances in which rapid (and thus, allosteric or enzymatic) rearrangement of the surface(s) is achieved to states capable of forming relatively strong mutual interactions. The increased degree of disorder, i.e., fluidity, of components of the cell surface on receptor-ligand interaction may carry over into the endocytotic organelle itselfa process that would enhance the opportunities for molecular encounters favorable to the fusion process. Indeed, there are striking parallels between the factors promoting fusion at the cellular and subcellular levels (Poste and Allison, 1973). Significantly, circumstances promoting the likelihood of fusion occur in the presence of lysosomal constituents that are realesed to the organellar surface on “activation” by a variety of stimuli, including the mechanical one of the collision per se. It is far from clear that the selective liberation of given constituents from lysosomes thus labilized, is, in and of itself, sufficient to lead invariably to fusion of whole cells (Poste and Allison, 1973). Nevertheless, there is substantial evidence that given enzymes, including proteinases (e.g., Ahkong et al., 1978; Lalazar and Loyter, 1979), even one with properties corresponding closely to those of cathepsin B (Ahkong et a[., 1980; Allan and Michell, 1979), and characteristic lysosomal phospholipases (cf. Poole et al., 1970; Allan and Michell, 1979), as well as protein kinases that would favor fusion through provision of cross-linking sites (cf. Wells et al., 1981; Collins and Wells, 1982), promote and/or support the mutual interactions of membrane components that lead to fusion in response to a variety of stimuli in endocytotic as well as exocytotic contexts. The rapid fluxes and gradients of Ca2+ that occur concomitantly with the activities set in motion on primary surface recognition of ligand (cf. Table XI) may be expected to modulate fusion-fission functions (cf. Ahkong et al., 1975; Blow et al., 1979). Moreover, exposure of appropriate carbohydrate structures on the organellar surfaces may be required for mutual recognition and prolongation of the initial organellar contact (cf. Amano and Mizuno, 1981). The nature of the vesicular contents may also influence rates and extents of fusion (cf. Amano et al. , 1981). Very recent observations indicate that endocytotic vesicles containing a,-macroglobulin undergo some form of acidification before fusion with lysosomes (Maxfield and Tycko, 1981), a phenomenon that may well govern selection of given organelles for fusion. Clearly, circumstances, including precise timing of the several conditions that favor recognition and fusion of apposed membranes, must be subject to wide local variation. Moreover, random collisions of even highly mobile subcellular
156
CLARA M . SZEGO AND RICHARD J. PIETRAS
organelles do not involve more than a given proportion of their population. Hence, it is highly likely that a substantial number of endocytotic organelles that entrain ligand-receptor escape interaction/fusion with lysosomes. And the relative proportion of such independent vesicles, destined, at least in part, to be concentrated at the polar Golgi region of the nucleus (Szego, 1975; Khan et a l . , I982), may vary with environmental conditions, prevailing or experimentally imposed. Deeper understanding of these complex events, particularly difficult to analyze because of the ultrarapid time scale upon which they occur, should shed light upon numerous open questions such as the detailed intracellular fate of the primary ligand-effector, especially in relation to its further metabolism and its potential penetration into the nuclear compartment. Some of these questions will be considered in a subsequent section. The thoughtful and authoritative essay by Jacques (1981) analyzes the “longlasting overemphasis” that has been placed on the linkage between endocytotic activity and lysosomal function, and concludes that each process should be analyzed independently. Indeed, it is all too clear that there is virtual “kneejerk” association of the rapid appearance of internalized ligand-receptor in the lysosoma1 compartment (Table XVI) with a “purposeful” degradation of the endocytosed material to “inert” products. To conclude that this is the sole outcome of the chain of events initiated by specific surface binding of ligand is to ignore a growing body of evidence demonstrating that not only does a significant proportion of endocytotic vesicles escape lysosomal fusion, but also that the secondary lysosomes undergo perinuclear translocation with what appear to be significant consequences in the interior of that compartment (see section below). 4. Controlled Destabilization in the Staging of Agonal Influence: The Cascading Eflects of Lysosomal Membrane Labilization a. Short Term. (I) At the cell surface. The ways in which limited recompartmentation of lysosomal enzymes may contribute to membrane remodeling on primary recognition of specific ligand at the target cell surface have been summarized above and in Table XIV. As in the case of vesicular fusion, the concerted effects of multiple pathways appear to be involved. Among the changes in surface architecture that may be effected by escaping microquanta of lysosomal enzymes are those elicited by limited proteolysis. The potential for rapid remodeling of preexisting macromolecules by excision of specific segments provides a means of response modulation for which explanations have long been sought. Some surface properties subject to rapid modulation by this and additional mechanisms include conformational changes in macromolecules leading to exposure of previously masked domains, the accessibility of receptor to fresh ligands (down- or up-regulation), the spatial reorientation of receptor to coupling devices, and other physical properties of surface membrane such as fluidity, with consequent shifts in disposition of intrinsic proteins. Additional features of mem-
LYSOSOMAL FUNCTIONS IN CELLULAR ACTIVATION
157
brane architecture subject to acute regulation by protein kinases and phosphatases were outlined in Table XIXA,B. Taken in concert, some of these changes may well underlie the altered permeability to ions and nutrients upon which subsequent metabolic responses depend. Thus, beyond their usefulness as indicators of membrane perturbation and its closely linked consequences (Table XI), the molecular repercussions of ligand binding seen at the cell surface give evidence of regulatory mechanisms of exquisite sensitivity and precision that are imposed at the crucial extracellular interface where discrimination and selectivity of responsiveness begin and where limitations in availability of low M, nutrients for the anabolic processes to come may be subject to attenuation. Implicit in the apparently joint internalization of specifically recognized ligand together with its receptor (cf. Fehlmann et al., 1982) is the loss of the latter from the surface of the affected cell. This phenomenon, long associated in a pharmacologic context with tachyphylaxis, appears in part to explain the transitory diminution of responsiveness to freshly applied ligand (cf. Szego, 1974). Interiorization, withdrawing receptor to a compartment in which, even if it were to dissociate appreciably from agonal binding, it would remain unavailable for interaction with additional macromolecules arriving at the cell surface, does appear to account in part for down-regulation by the usual kinetic analyses (cf. Krupp and Lane, 1982; Green and Olefsky, 1982). However, there are further aspects to apparent receptor loss. Some ligand is shed, possibly still in association with receptor into the extracellular environment, a route not normally accounted for in any balance sheet because of its relatively small contribution to receptor down-regulation and the difficulties in quantitating receptor, as such, except as a sink for specific ligand (labeled for experimental feasibility). However, as a result of numerous data obtained with a wide variety of receptor macromolecules, there is little doubt that net loss of the latter into the lysosomal compartment, into which a substantial proportion of endocytotic vesicles is somehow directed very soon after their cellular entry, generally occurs. The intracellular degradation of receptor macromolecules is inferred in part from their failure to be available for interaction with fresh ligand (following stripping of that remaining associated). In rare cases, in which especially sensitive means of analysis for unmodified receptor are available, such as that for acetylcholine (Libby et a / . , 1980) or certain lectins (Kramer and Canellakis, 1979), loss of macromolecular integrity or appearance of diminished product has permitted the unambiguous conclusion that with advancing time after internalization, there is only limited survival of the unaltered receptor. That the process of receptor attrition is a proteolytic one is likewise unequivocal, especially when analyzed with the aid of those inhibitors of the relevant lysosomal enzyme(s), such as leupeptin at modest concentrations, which appear not to possess inherent toxicity and independent membrane-active properties. The latter are unfavorable sideeffects of many of those highly basic substances that interfere with degradative
158
CLARA M . SZECO AND RICHARD J . PIETRAS
processes in the lysosomal compartment by generating a decrease in its acidity (cf. Table VII). However, there is also growing evidence that ligand may be more extensively degraded than is the case for receptor (Hofmann et al., 1981; Krupp and Lane, 1982; Green and Olefsky, 1982; Bridges et al., 1982; Fehlmann et al., 1982). Nevertheless, the significance of receptor (R), possibly in processed form (R’), in the propagation and amplification of the initial surface recognition event, is yet to be fully evaluated. It has frequently been suggested that intracellular, if not intranuclear functions of receptor may outweigh in significance the modulatory capacity of hormone as such (cf. Jensen and DeSombre, 1972; Hollenberg, 1979; Kahn er al., 1980); De Larminat et al., 1981; Carpentier e f al.. 1981a). The former is relatively conserved and, as recently shown, may be “recycled” to, and reinserted in, the cell surface after a brief interval (see also below). As was noted in Table XIX, phosphorylatiorddephosphorylation likewise contributes to the abrupt reorganization of the surface architecture of cells undergoing functional alterations contingent upon receptor-ligand recognition. Additional contributions to acute remodeling of the surface of the responsive cell are undoubtedly made by selective cleavage of carbohydrate functional groups from glycoprotein and glycosphingolipid components. The requisite enzymatic machinery, as well as certain critical cofactors for each of these processes, is sequestered in the lysosomal compartment (see Section 11,B). Spilling of these and independent lysosomal constituents at the strategic outer surfaces of cells undergoing agonally induced activation is indeed an early indicator of receptor-mediated perturbation with qualitative selectivity based upon intrinsic heterogeneity of those members of the lysosomal population that have fused with H:R-containing endosomes. How is such enzymic exposure (see footnote 3 ) achieved’?There seems much in common between the abrupt surface-delivery and cytoplasmic accessibility of the requisite lysosomal consituents. (2) In the cytoplasm. (a) Accessibility of lysosomul enzyme at the cytosolic-organellar interface. By the several processes already indicated, lysosoma1 constituents possessing the capacity to contribute in cascading fashion to the controlled remodeling of the cell surface also participate in limited destabilization of macromolecules that accompanies early hormone action within the major cellular compartment. Here, we encounter examples of processing of macromolecules, the time-base of which is sufficiently rapid to preclude new synthesis, but which, by finite degrees, is less instantaneous than the circumscribed dismantling at the cell surface (cf. Tables IX,XI). Foremost among the processes responsible is limited proteolysis (Table XXA-C), the extent of which, given the stringcncy of the enzymes involved in those cases in which provisional identification has been achieved, is governed by specific substrate structure and cofaetor availability, for example, trace amounts of Ca2+ and agents that maintain sulfhydryl integrity, both of which favor the activity of cathepsin B (see
TABLE XX REPRESENTATIVE EXAMPLES OF LIMITED PROTEOLYSIS IN RECEPTOR-MEDIATED RESPONSES TO SELECTED EFFECTORS Hormone [marker]
Target
Criteria
Implications
Reference
A. Processing of receptor Epidermal growth factor (EGF) [ z511-
'
Estrogen [3H]Estradiol
Swiss murine 3T3 cells
Gel electrophoresis
EGF bound to receptor may elicit a conformational change in the receptor to expose cleavage sites for lysosomal or other endogenous proteases on the cell surface, resulting in electrophoretic band of lower M , than in cells not exposed to EGF. Propose proteolytic activation of internalized receptor
Fox er al. (1979b)
Rat uterus
Sucrose gradient sedimentation and gel chromatography
Rat uterine cytosol [3H]estradiol receptor was transformed from an 8 S to a 4.5 S sedimenting protein in the presence of a partially purified trypsin-like protease isolated from human uterine cytosol. Hormone bound receptor was more likely to be transformed, suggesting a hormone-induced conformational change in the receptor, exposing new cleavage sites. Process thought to promote nuclear entry of receptor, inactivation of the complex to prevent excessive uterine growth, or to be without physiologic significance
Notides et al. (1973)
(continued)
Hormone [marker]
Criteria
Implications
Calf uterus
Enzyme assays, gel filtration, spectrometnc analysis
Lamb uterus
Gel filtration, DEAEcellulose chromatography
Human myometrium
RIA
Estrogen receptor was transformed from an 8.6 S to 4.5 S sedimenting protein by a Ca'+-activated protease. receptor-transforming factor (RTF). RTF-converted receptor and purified nuclear receptor had similar physical properties (sedimentation coefficient, Stokes radius, M,, and isoelectric point). Proposed that the diminished receptor might diffuse more efficiently across the nuclear envelope to initiate its effects on transcription Isolated nuclear form of estradiol receptor with micrococcal nuclease under mild conditions and found it to be clearly different from Ca2+ -transformed receptor of Puca et al. It had a greater apparent M, and bound to DNA, while the Ca2 -transformed receptor bound to nuclei but not to DNA Three forms of cytosolic estrogen receptor in a low salt medium were characterized by sedimentation coefficients of 9.0, 5.4, and 4.3 S . The presence of diisopropylfluorophosphate (DFP) resulted in a higher yield of 9.0 S vs 4.3 S receptor. Suggest that a protease convert5 the 9.0 S form into the 4.3 S form, which binds twice as much estradiol. as measured by the dextran-charcoal technique. The transformed receptor may thus have more exposed binding sites than its precursor
Target
Reference Puca
PI
a / . (1977)
Rochefort and Andrc? (1978)
Liivgren er al. (1979)
Glucocorticoids (GC) ['HIDexamethasone
["]Triamcinolone acetonide
Glutamate
Rat liver
Gel filtration, DNAcellulose chromatography
Chick thymus
Spectrophotometric analysisiDNA-cellulose chromatograPhY
Rat hippocampal membranes
Kinetic binding studies ? Ca2+ and protease inhibitors, including leupeptin
Incubation of dexamethasone receptor complex (Stokes radius 6.1 nm) with rat liver lysosome extract produced two smaller glucocorticoid-receptor complexes with radii of 3.6 and I .9 nm. This cleavage was not prevented by cathepsin inhibitors antipain, leupeptin, or pepstatin. The 3.6 nm complex was the predominant form found in the liver cell nuclei and bound tightest to DNA-cellulose. Suggest that limited lysosomal proteolysis may enhance interaction of the activated steroid-receptor complex with chromatin, while more extensive proteolysis yields an inactive by-product Ca2+ treatment of cytosol strongly curtailed the stability and DNA-binding capability of glucocorticoid receptors. Ca2 may activate an endogenous protease which cleaves the GC receptor and affects its DNAbinding function. Inhibitor studies suggest the enzyme is an SH-protease. Ca2+ levels in vivo may activate protease activity Ca2+- and SH-dependent proteolytic activity intrinsic to the membranes enhances glutamate binding
Carlstedt-Duke er a/.(1 979); cf. however, Barnett and Litwack ( 1982)
Niray (1981)
+
Baudry and Lynch (1980); Baudry e r a / . (1981)
(continued)
TABLE XX (Corifrnued) Hormone [marker]
Target
Criteria
Implications
Reference ~~
Insulin Unlabeled
Rat adipocytes
Enzyme assays. chromatography
Rat adipocytes
SDS-PAGE
The initial action of insulin may be a proteolytic event at or near the external cell membrane which produces a glycopeptide "mediator" that acts as a second messenger. Treatment of rat adipocytes, without glucose, either with insulin or with low concentrations of trypsin resulted in a rapid activation of glucose oxidation. glycogen synthase, and mitochondrial pyruvate dehydrogenase. Both insulin and trypsin produced species of glycoprotein with similar, but not identical properties The insulin receptor is composed of at least four subunits that are disulfide-linked into a large (M, 350K)complex. The two central a-subunits are identical and have a M, of 125K: each is linked to psubunit (M, 90K) to yield a symmetrical receptor (up),. Incubation of the membrane-bound (a@)*receptor with a lysosomal fraction produced fragments of M, 49K, with unaltered insulin-binding capacity. These arose from the @-subunits, and may be the initial products in the in vivo lysosomal processing of the internalized ( c @ ) ~receptor
~
~~
Lamer ez al. (1981)"
Czech era!. (1981)".'
NAPADP[ I2511
Rat adipocytes
Gel electrophoresis. autoradiography , photoaffinity labeling
Labeled insulin-receptor complex was translocated intracellularly where apparent processing occurred. with generation of a M , 1 ISK component from the M , 125K receptor subunit and disappearance of the M, 330K and 295K receptor species, respectively. Choroquine inhibited these events, suggesting that receptor processing may occur in the lysosomes (see however, Hofmann el a / . , 1981)
Berhanu ct a / . (1982)
Large, asymmetric progesterone receptors in cytosol were converted in the presence of Ca2+ to small globular polypeptides with the same steroid-binding properties of the intact receptor, but lacking nuclear binding capacity. Protease inhibitors prevented cleavage suggesting that Ca2+ may have promoted activation of an endogenous protease Isolated from oviducts of laying hens a Ca?+-activated, sulfhydryl-dependent protease which catalyzed limited proteolysis of progesterone receptor subunits A and B to smaller, hormone-binding fragments. The concentration of receptors in the cell is at a K , close to requirements of this protease, suggesting kinetic feasibility under in wivo conditions. The modified receptor retained hormone binding activity but no longer bound to DNA. Parallels to the RTF of Puca et a/. (1977) were drawn
Sherman et a / . (1978)"
Progesterone
L3H1-
Chick oviduct
Chromatography
Chick oviduct
Chromatography
Vedeckis e? al. (1980a)
(continued)
TABLE XX (Continued) Hormone [marker]
Target
Critena
Enzymic properties
Implications
Reference
B. Processing of hormone ACTH Unlabeled
Pituitary gland
RIA with [3H]corticosterone and ["]CAMP
An endogenous pituitary peptide a7-3X-ACTH has no intrinsic steroidogenic activity but competitively inhibits ACTH-induced corticosterone synthesis, as well as cyclic AMP production in v i m and in viuo. This corticotropin-inhibiting peptide. which may be formed through altered processing of the precursor proopiocortin, results in decrease of active ACTH and simultaneous increase of an ACTH antagonist. Such a process provides for rapid and efficient negative mcdulation of the resultant hormonal effect
Lee et al. ( I 980)
Growth hormone Purified from rat GH3 cells
NGF M , 116K
Porcine costal cartilage
M , 22K-24K by SDS-gel electrophoresis; ["S]sulfate incorporation into product, which had sulfation activity
Heat-sensitive, serinelike protease. cleaving rGH at Arg132-Ile'33. integral to purified rGH
GH may attain sulfation factor activity at peripheral tissues via this proteolytic pathway
Maciag ef al. (1980)
Not known
Spectrophotometric enzyme assay with TAMEd; kinetic analyses on controlled dilution; effects of selected inhibitors
Autocatalyic activation; dissociation of subunits believed excluded
Inactive in concentrated form; upon high dilution, autocatalytic activation to full enzymic activity may serve to prevent expression until NGF reaches its target cell(s) or until it recognizes its physiological substrate
Young (1979a,b); cf. however, Calissano and Levi-Montalcini (1979)
Liver
RIA, gel electrophoresis
Cleavage of circulating PTH 1-84, to generate an active N-terminal fragment selectively taken up by bone, regulated by hypocalcemia. Cleavage may also occur at parathyroid gland
Martin er al. (1979)'
PTH
["'I].
(continued)
TABLE XX iConrinued) Hormone [marker]
Criteria
Target ~
-s
Prolactin Unlabeled
TRH ["IPro-TRH
~~
Partially purified membrane preparation from bovine kidney cortex
RIA, gel electrophoresis, immunoassay
Rat ovarian follicles
lmmunohistochemistry
Not known
Paper chromatography
Enzymic properties
Implications
Reference
Acid pH optimum activity, may be a cathepsm, cleavage between residues 29 and 43
Active N-terminal and inactive C-terminal fragments to those generated in liver and kidney in vhw
Botti el af. (1981)
Evidence that PRL fragments have activity at ovarian follicles; PRL-like immunoreactivity of small < 2 K-M, peptidal molecules in the follicular fluid; active fragments detectable in follicular fluid, granulosa cells, and oocytes; the intrafollicular distribution varies to suggest a bioactive role for them in modulating readiness for ovulation
Nolin (1983)
His-Pro-diketopiperazineis more effective than TRH as an antagonist of ethanol-induced sleep in rats
Prasad et al. (1977)
~
Pyroglutamyl-peptidase converts TRH into His-Pro-diketopiperazine
-
Significance Product [marker]
Site
Criteria
Implications
Biosynthesis
Degradation
Reference
C. Processing of neuroendocrine products Endorphin p511-
-
Human pituitary
Chromatography, electrophoresis
Unlabeled
Rat crude synaptosomal plasma membrane
HPLC, RIA
P-Endorphin
Rat pituitary and brain
4 m
Enkephalin Unlabeled
Porcine hypothalamus
HPLC, TLC, enzyme assay
Cathepsin D purified from human pituitary cleaves human p-endorphin at the L e ~ ~ ~ - P bond, he~* generating y-endorphin Found endogenous peptidase activity, distinct from cathepsin D, to convert P-endorpin to y-endorphin; may have physiological function in endorphin homeostasis P-Endorphin has 6 derivative peptides; C'-fragment, p-end~rphin,-~,,des-histidine derivative (p-endorphin,-,,), and their N-acetyl forms; these are all essentially inactive and are found nonuniformly distributed in the brain, suggesting differential processing by specific enzymes in the individual regions Found a hexapeptide, resembling Met-enkephalin except for extra Arg residue at C-terminus. Proteolytic origin from precursor other than that of P-LPH or p-endorphin strongly implicated
+
Benuck e t a / . (1978); see also Graf et a / . (1979)
+
Burbach
+
4
et a / .
(l980a)
Zakarian and Smyth (1982)
W.-Y. Huang et al. (1979)
TABLE XX (Continues) Significance Product [marker]
Site
Criteria
Implications
Bovine adrenal medulla
Chromatography, RIA
Rabbit brain
Chromatography, enzyme assays
Bovine adrenal medulla
Cell free translation
Bovine adrenal medulla
HPLC, Edman degradation
Rat striatum
TLC, RIA
Isolated a 50K polypeptide. that, when exposed to trypsin and carboxypeptidase B , yielded both Met- and Leu-enkephalin in a ratio of 7:l Found membrane-bound dipeptidyl carboxypeptidase which resembles angiotensin I-converting enzyme but is not active on angiotensin. Inactivates enkephalins through cleavage at Gly-Phe bond cDNA showed extensive hybridization with adrenal poly(A)- RNA to suggest a sequence large enough to be the proenkephalin precursor Isolated 3.6K and 4.9K polypeptides with internal enkephalin sequences bracketed by basic amino acids. May be intermediates of larger proenkephalin, requiring trypsin-like, as well as carboxypeptidase B-like, intervention in processing Inhibitors of known enkephalin hydrolyzing enzymes were used to determine that a dipeptidyl carboxypeptidase ("enkephalinase") is the most active in degrading and inactivating Met-enkephalin. May play key role in regulation since it
Biosynthesis
Degradation
+
Reference Lewis et a / . ( 1 980)
+
Benuck and Marks (1980)
+
Gubler el al. (1981)
+
Stem et al. (1981)
+
Patey et al. (1981)
([3S-3H& Tyr)-Metenkephalin
Rat brain
Electrophoresis, HPLC, TLC
“3 S-3H21Tyr)-Leuenkephalin [ 1251]Met-enkephalin
Bovine synaptic membranes
Chromatography
Adrenal medullary chromaffin granules
Enzyme assays, chromatography
[3H]Met-enkephalin
Monkey brain
Column- and thin-layer chromatography
o* \o
is located close to enkephalinergic nerve terminals Isolated and characterized an endogenous enkephalin aminopeptidase which cleaves the Tyr residue of Met-enkephalin, thus inactivating it; M , 102K, pH optimum 6.5-7.0 Suggest that a neutral endopeptidase similar to that in kidney may play a role in neuropeptide metabolism Detected large amounts of Met- and Leu-enkephalin and large enkephalin-containing peptides in chromaffin granules; also found a unique carboxypeptidase which is unlike carboxypeptidase A, B, or N.f Removes the Arg residue from the C-terminus of Met-enkephalin, resulting in apparent activation Isolated two membrane-bound enzymes that inactivated enkephalin, an exoaminopeptidase, which cleaved Met-enkephalin at the Tyr residue, and a dipeptidyl aminopeptidase that released the dipeptide Tyr-Gly. The exoaminopeptidase was inhibited by bestatin and puromycin, while the dipeptidyl aminopeptidase was inhibited only by metal chelators, suggesting that it may be a metalloenzyme. The actions of these two enzymes may regulate enkephalin levels in the brain
+
Wagner et a/. (1981)
+
Fulcher et a / . (1982)
+
Hook et a/. (1982)
+
Hazato et a/. (1982)
(continued)
TABLE XX (Continued) Significance Product [marker] Unlabeled
Site
Criteria
Implications
Bovine adrenal
Affinity column chromatography, enzyme assays, HPLC
Identified and partially purified a "trypsin-like" enzyme that cleaves the dibasic residues of proenkephalin to produce active Metand Leu-enkephalin. Its optimum pH is consistent with the low intraganular pH of chromaffin granule$ suggesting that proenkephalin activation occurs therein A specific cahoxypeptidase. designated enkephalin convertase, is uniquely localized to the chromaffin granules, which contain enkephalin and precursor peptides. It has a high affinity for the hexapeptides [Mets]- and [Leus]-enkephalin-Ag6 and converts them to their respective active forms. The regional distribution in the brain of apparent enkephalin convertase activity is heterogeneous, with similarities to the distribution of enkephalin and opiate receptors
Enzyme assays, HPLC
Biosynthesis
Degradation
Reference
+
Evangelista et al. (1982)
t
Fricker and Snyder (1982)
Rat brain membrdnes
Electrophoresis, HPLC, spectrofluorometric analysis
Aminopeptidase inactivates Met-enkephalin by cleaving the Tyr'-Gly* bond. P-Endorphin and Argu pendorphin are not cleaved but act as competitive inhibitors. Removal of Tyrl from endorphin decreased inhibitory effects. Suggest that the N-terminal amino group and some residues within sequence region 18-31 are involved in binding to the active site of the aminopeptidase. Endorphins may regulate enkephalin levels at the functional receptor sites through this competitive inhibition
Rat medial basal hypothalamus in vitro
RIA
Rat hypothalamus and anterior pituitary in
RIA, electrophoresis
Decrease in exogenous GnRH levels during incubation with hypothalamic tissue suggests that peptidase(s) in hypothalamus may regulate endogenous GnRH levels Hypothalamus and anterior pituitary exhibited similar degradative enzymic activity (each, M , of 67K and optimal activity at pH 7-8 at 37°C). The neutral pH may indicate non-lysosomal origin (see Section
vitro
+
Hui et al. (1982)
+
Powers and Johnson (1981)
+
Hazum et al. (1981)
113) ~~
(continued)
TABLE XX (Continued) Significance Product [marker] LHRH ['HI-
Oxytocin Unlabeled
Biosynthesis
Site
Criteria
Implications
Bovine anterior pituitary
Chromatography, electrophoresis, enzyme assays
Median eminence, preoptic area
HPLC, RIA
Isolated a neutral endopeptidase which degrades LHRH: CaZ -independent, sulfhydryl-requiring, M , of 83K. Cleaves at TyrS-Gly6and HisZ-Trp" bonds. May play regulatory role in LHRH levels Observed LHRH degradation activity in prepubertal rats to be constant, with a steady increase in total LHRH. During first estrous cycle, the LHRH-degrading activity peaked at 300% increase from prepubertal levels at late proestrus, then decreased to former levels at first estrus. Suggests LHRH-degrading activity has a regulatory role in reproductive cycles
Hypothalamus
Cell free translation. immunoassay
Degradation
+
Honthemke and Bauer (1980)
+
Advis
+
Hypothalamic poly(A) RNA was translated in a wheat germ system to synthesize the composite com+
+
Reference
el
al. (1982)
Schmale and Richter (1980)
mon precursors to OT/NpI* and to AVPINpIIx. It reacted with both anti-OT and anti-Np I , which suggests that oxytocin and its camer molecule neurophysin I derive from a common precursor. In presence of microsomal membranes from dog pancreas, the 16.5K pre-proprecursor was converted to the 15.5K pro-form. Proopiomelanocortin WMC) Unlabeled
e
W 4
Anterior and intermediate pituitary lobes
Differential processing of POMC occurs in anterior and intermediate pituitary lobes. Both sites exhibit initial glycosylation and cleavage, with production of f3-LPH and ACTH. Noteworthy are the paired dibasic amino acid residues at sites of cleavage. However, in intermediate lobe, ACTH is converted to a-MSH and CLIP, while P-LPH is converted to y-LPH and p-endorphin. Possible models (a) specific enzymes aligned in order of secretory pathway, (b) structure of substrate determines order of cleavage rather than disposition of enzymes, (c) more than a single gene for different POMC sequences expressed in the several tissues
-I
Herbert (1981Ih
(continues)
TABLE XX (Confinued) Significance Product [marker]
P
Site
Criteria
['HIPhePOMC ['HI A g POMC ['%]MetPOMC
Rat neurointermediate lobe
Pulse chase, electrophoresis
[3H]Arg- or [3H]PhePOMC
Rat pituitary neurointermediate lobe secretory granules
lmmunoprecipitation
Murine hypothalami
Radioimmunology and HPLC
Prosomatostatin Unlabeled
Implications Canavanine. an analog to Arg. was incorporated into POMC instead of A g . Pulse incubation of modified POMC showed decreased maturationicleavage suggesting that the basic amino acid Arg plays an important role as a cleavage site signal Toad POMC was incubated with granule lysates of POMC converting activity (pH optimum 5.0. cleavage at paired basic amino acids). Converting activity appeared due to a thiol protease, probably distinct from cathepsin Blike activity, because of effective inhibition by pepstatin (as well as leupeptin). Cross-species activity suggests parallel POMC processing pathways
M , 15K precursor selectively converted to somatostatin 1 by endogenous protease(s)
Biosynthesis
Degradation
Reference
+
Crine and Lemieux (1982)
+
Loh and Gainer (1982)
+
Morel ef a / . (1981)
Vasopressin Unlabeled
Bovine neurohypophysis
Molecular sieve fractionation, irnmunoadsorption
Hypothalamus
Cell-free translation, cDNA hybridization
Identified high-molecular-weight forms of proteins which reacted with anti-AVP and anti-neurophysin. When isolated in the absence of inhibitors of serine proteascs, it yielded AVP and Np. This protein may be a biosynthetic precursor to AVP and its neurophysin Provides conclusive proof that AVP and its carrier protein, neurophysin 11, are synthesized via a common precursor. The precursor has a signal peptide followed by AVP. A pair of basic amino acids separating AVP from Np I1 serves as a cleavage site
-I
Nicolas et al. (1980)
+
Land er a!. (1982)
uReview. 1 , a biologically active, photosensitive derivative. bNAPA-DP, B2 (2-nitro-4-azidophenylacety1)-des-PheB =See also Fig. 16. dNU-p-Toluenesulfonyl-L-argininemethyl ester. fReview. fAlthough the chromaffin granules were presumed to be free of mitochondria and lysosomes, the pH optimum of 6.0 for the carboxypeptidase and the trypsinlike substrate specificity are strongly reminiscent of properties of cathepsin B (see Table XV). Moreover, the internal pH of chromaffin granules is, like that of lysosomes, maintained in the acid range by a proton-translocating ATPase in the respective membranes (cf. Geisow, 1982). Thus, the suggestion of an analogy with lysosome-sequestered proteases is not too remote (cf. however, Lindberg et a(., 1982: Mizuno et a/., 1982). SNeurophysin Iioxytocin; neurophysin IIiarginine vasopressin. hReview.
176
CLARA M. SZEGO AND RICHARD J . PlETRAS
Table XV). Additional regulation is imposed by an array of counterpoised extraand intracellular antagonists, such as cymacroglobulins and other endogenous inhibitors of specific proteinases, including those lysosomal. These combined circumstances not only permit fine tuning of effector-responsive activities in the cytoplasm, but also set the stage for the unfolding of later steps in hormone action that may include events in the nuclear compartment. (b) Processing of receptor. The processing of receptor at the target cell level (see Table XXA) has had the conventionally accepted dual interpretations related to the putative divergence in the mechanisms of action of hydrophobic vs hydrophilic agonist (see Table 11). On the one hand, the apparent involvement of limited proteolysis of steroid receptor, as one stage in its “activation” toward chromatin accessibility, has been suggested (Szego, 1969, 1971a, 1974; Puca et al., 1972, 1977; Sherman et al., 1978; Carlstedt-Duke et al., 1979; Miller, 1980). On the other hand, similar, controlled and limited proteolysis of receptor for protein hormone is not yet widely envisioned. instead, intralysosomal degradation to products no longer capable of specific binding of the agonist is almost universally viewed as a means of disposal of “excess” receptor., once it has become detached from surface orientation and entered the lysosornal compartment. Nevertheless, it is becoming increasingly evident that proteolysis of selective receptor is likewise an early correlate of the specific binding of peptidal agonist. Indeed, this phenomenon may have far deeper significance as a regulatory device than merely serving to eliminate “excess” receptor. Limited proteolytic processing of the latter may actually promote its conversion to forms more highly adapted to intracellular functions not yet fully identified. It seems particularly noteworthy that mitogenic agonists have been so closely associated with proteolytic events, often in quite complex, indirect fashion. Thus, plasminogen activation by inherent proteolytic activity is a feature of NGF action upon a nonneural substrate (Orenstein et al., 1978) that may have repercussions at the receptor level. Even more far-reaching implications are evident in the newly described sequence homologies among tonin (a protein isolated from rat submaxillary gland), the NGF y-subunit, EGF-binding protein, and serine proteases (Lazure et d . , 198I ) . Insulin-like effects of dithiothreitol, a powerful promoter of cathepsin B activity (see Table XV), on isolated rat adipocytes, was concomitant with alteration of insulin binding sites to a low-affinity state (Goko et a / . , 1981), a finding that may have circumstantial relevance in the present context. More significantly, Larner and co-workers have identified an insulingenerated “mediator” of insulin action, a low-molecular-weight peptide that is formed in situ by limited proteolysis of a cellular precursor (see Larner et al., 1981, for review). In light of its influence in promoting dephosphorylation of specific proteins, while an accompanying oligopeptide promotes their phosphorylation, this peptide has powerful implications for propagating the multiple further effects of the hormone. Such observations demonstrate how closely inter-
LYSOSOMAL FUNCTIONS IN CELLULAR ACTIVATION
177
woven is the web of cellular regulatory processes that are called into play on target cell-surface recognition of a specific ligand. There may exist numerous further links between proteolysis and phosphorylation/dephosphorylationthat have yet to be identified in reaction chains triggered by ligand capture (cf. also, Table XIV). In either event, invocation of these enzymatic processes permits the poised status quo to be converted rapidly to one of orderly change long before genic processes can be activated toward synthesis of new products. These considerations aside, there exists additional evidence that some fraction of cellular receptors escapes from appreciable degradation in the course of vesicular internalization in association with ligand. Indeed, it seems evident that sparing of receptor, presumably to be reutilized by some form of recycling, is a feature of the metabolic economy of given cells (cf. Kaplan, 1980; Hofmann et al., 1981; Kassis and Gorski, 1981; Anderson etal., 1982; Harford and Ashwell, 1982; Octave et al., 1982). Such observations have been ascribed to the early divergence of the intracellular pathways taken by receptor from those of the agonist, with indications that a class of vesicles that differ from the classical Golgi, lysosome, and plasma membrane fractions may be involved in a form of receptor salvage (Debanne et al., 1982). As indicated above, however, the total destruction of ligand is not a necessary correlate of such findings. (c) Processing of hormone. Except for provision of free cholesterol from its esterified storage form, there has been little indication of hydrolytic enzymic function in steroid hormone biosynthesis. In contrast, there is now very full documentation of limited proteolysis, indeed, by enzymes with the properties of lysosomal cathepsins, for conversion of prohormone to hormone as a distinct step in the provision of peptide agonists at sites of their biosynthesis on environmental demand (Table XXI). The very conditions that elicit secretion of the given hormone are those that mobilize the processing enzymes from organellar sequestration. However, it is not yet known, although occasionally inferred, whether some diminished form of a given peptidal agonist has some unique function as an outcome of its association with the lysosomal proteolytic machinery in the cellular target (cf. Table XXB,C). This possibility has been given only limited consideration (cf. King and Cuatrecasas, 1981), in favor of the idea that, as in the case of their “occupied,” internalized receptors (see above), once peptide agonists have entered the lysosomal compartment, they have sealed their own death warrant: their degradation to ineffective forms is a foregone conclusion. Such a conclusion is clearly premature on the basis that such products of proteolysis, whether limited or more extensive, are yet to be isolated and tested for activity in systematic fashion. Production of acid-soluble catabolites of iodinated hormone is surely an inadequate criterion. Moreover, as will be detailed below, the association with the nuclear compartment of lysosomes containing isotopically labeled peptide hormone gives a tantalizing glimpse of regulatory events in hormonal function yet to be analyzed in molecular terms. Ironically,
TABLE XXI HORMONE FROM PROHORMONE: REPRESENTATIVE EXAMPLES OF POSTTRANSLATIONAL PROTEOLYSIS ASSocl 4 T E D SYSTEMIC DELIVERY AS A FUVCTION OF ENVIRONMENTAL DEM,~NLV.‘~ Precursor ca. 30K pro-ACTH-endorphinc
1 1.8K pro-Calcitonin
9K pro-EGF
Cell type Neurointermediate lobe of dark-adapted toads, Xenopus laevis Murine anterior pituitary cell culture monolayersd Subcellular fractions of rat anterior- and intermediate-lobe cells Human medullary thyroid carcinoma
Mouse submaxillary glands
Product(s)
WITH
Enzymic property
ACTH, P-lipotropin. aMSH
MATUK4TIOV
AVl)/
Reference Loh and Gainer ( 1979)
ACTH, P-lipotropin
Trypsin-like
Hinman and Herbert (1980)
As above, with sequential passage through RER. Golgi, and secretory granules In puise-chase experiments, depletion of the presumptive precursor concomitantly with formation of one whose M , was consistent with that of human CT EGF
NSe
Glembotski (1981)
NSeJ
Jullienne et af. (1980); cf. also Jacobs e t a / . (1981): Bimbaum er af. (1982); Maclntyre et a / . (1982)
Native high M,-EGF (74K) occurs with 2 hormonal peptides of 6K each, in association with a specific “trypsin-like” (cf. Bothwell ef a / ., 1979) arginine esteropeptidase
Frey et a[. (1979); Knauer and Cunningham (1982)
18K pro-Glucagon
Isolated rat islet cells
pro-Insulin
Pancreatic p cells in numerous mammalian species Rat liver lysosomal fractions
-
4
pro-Parathyroid Hormone (PTH)
(pre)pro-Relaxin
M , 3.5K, 4.5K, 13K peptides, the latter of which may be intermediates, reactive with the glucagon antiserum, while another peptide of 10K was an unreactive by-product Insulin and C-peptide
Isolated secretory granules from angler fish islets Parathyroid gland of man and other animals
Insulin and glucagon from the respective pro-hormones PTH conversion from proPTH is accomplished by removal of the N-terminal hexapeptide in the Golgi region
Rat ovarian clone ”bank” of mRNA sequences
cDNA for relaxin demonstrates that the hormone is synthesized as a prepro-molecule, with a large, 105-amino acid connecting peptidei
“Trypsin-like” and “carboxypeptidase-like” activities inferred from model experiments with these enzymes8
Patzelt et al. (1979); cf. also Shields er a / . (1981) and references therein
“Trypsin-like” endopeptidase and a carboxypeptidase B-like exopeptidase in Golgi region Cathepsins B and L, possibly with the further participation of a carboxypeptidase B-like activity Sulfhydryl-dependent “trypsinlike” activity corresponding to cathepsin B8 “Trypsin-like” may correspond to cathepsin B (MacGregor et al., 1979); excess Ca2+ promotes more extensive degradation in situ to products without hormonal activityh NSe
Steiner et a\. (1974); Steiner (1976)c Ansorge et a / . (1977)
Fletcher et al. (1981)
Habener et al. (1977)’, Cohn and MacGregor (1981)’, and the earlier papers from their respective groups cited therein Hudson et al. (1981)
(continued)
TABLE XXI (Contiriued) uThis topic has received extensive review recently (cf. Zimmerman et al., 1980: Dean and Judah, 1980; Ceisow and Smyth. 1980; Herbert er a / . , 1981; Chertow, 1981; and Docherty and Steiner, 1982). bSignals believed to control availability or access of the relevant proteolytic activities to preformed prohormone in cytoplasmic or vesicular sites include amino acids and glucose for insulin (cf. Docherty and Steiner, 1582), and [Car+] for parathyroid hormone (cf. Habener et al., 1977’. Cohn and MacGregor, 1981).‘ It may be anticipated on this basis that changes in prevailing levels of additional metabolites, whose concentrations are responsive to the very hormones that modulate their disposal, will he identified as triggering devices for rendering the appropriate active hormone available on “demand.” See additional examples in the case of neuropeptides in Table XVC. Since cellular recognition of circulating metabolites is clearly a surface event, there is profound significance in the manifest parallels to the processes set in motion by hormonal effectors at their cellular targets (cf. Tables XXA-C and text). Participation of cathepsins released from membranebounded form by surface recognition early in the cellular activation process is a further parallel between ligand-heightened functions at sites of hormone action (Tables IX, XIV, XXA-C). as well as provision of an active biosynthetic product from a poised but not fully active form of a given hormone. Thus, it is striking that the formation of active product(s) from proinsulin, proglucagon, proparathyroid hormone. p- and y-lipotropins (etc.) involves “trypsin-like” cleavage at sites adjoining paired dibasic amino acid residues (cf. Steiner. 1976). Barrett (1977) has remarked on the relevance of such stringency to the known properties of lysosomal cathepsin B (cf. Table XV). This inference has now received strong experimental support in the case of insulin (Ansorge ef a!.. 1977; Fletcher et n l . , 198 I j. In turn, present emphasis on proteolysis is not intended to imply exclusion of additional processes and/or enzymic functions in the biosynthetic maturation of fully active hormone in these or other instances. For example. other covalent modifications of precursors, with further implications for modulation of processing (e.g., Loh and Gainer, 1979), clearly include glycosylation (e.g., thyroglobulin), acetylationideacetylation (e.g. processing of pro-ACM-endorphin), and, potentially. phosphorylationldephosphorylation.That in a number of instances, already noted. lysosomal enzymes are of relevance to these additional processes must likewise be taken into account. Beyond even these modulatory activities, the synthetic leader sequence o f preproparathyroid hormone has been shown to inhibit the processing of a series of preprohormones (Majzoub er a / . , 1980). cSee authoritative review by Eipper and Mains (1980). with comments on the degree of appropriateness of precursor nomenclature. dSimilar results were obtained in murine pituitary tumor cells. AtT-20/Dj6,, except that the latter had a more rapid time course; see, however, Eipper and Mains (1980). <’Not specified. fsequential function of “trypsin-like” and “carhoxypeptidase-B-like” activities has been inferred for proteolytic processing of the corresponding precursor from rat thyroid medullary carcinoma (Jacobs et a / . , 1981). rln isolated secretory granules of anglerfish. Fletcher er a / . (1981), using appropriately specific inhibitors, have identified endogenous thiol-dependent proteinase(s) in conversion of proglucagon and proinsulin into their respective active forms. The properties of cathepsin B. which possesses both trypsin-like and exopeptidase functions (cf. Table XV), correspond closely to those of the secretory granule-associated activity. “A calcium-requirement for cathepsin B in some cells has been indicated; however, excess calcium is inhibitory (Szego et al.. 1976) ‘Review. /Corresponding data are available for bovine arginine vasopressin-neurophysin I1 precursor (see Land et al.. 1982 in Table XXC).
LYSOSOMAL FUNCTIONS IN CELLULAR ACTIVATION
181
the sharp analogy to the relevant chain of events in steroid hormone action appears to have escaped the attention of most workers in the peptidal vineyards (cf. however, Johnson et al., 1980). As earlier noted, the limited number of mechanisms available for propagation of a primary membrane-oriented event are few, and many changes must be rung upon the few by the numerous effectors available. As indicated above, only a short time has elapsed since the rather wary acceptance of the phenomenon of receptor-linked internalization of macromolecular tropic agent in responsive cells (cf. Szego 1974, 1975, 1978). What is now in sharp question, however, is the significance of the entry phenomenon itself. For example, while there is general agreement that internalization is closely linked in many cases to disappearance of receptor from the affected cell, as well as concomitant degradation of the agonist to various degrees short of complete, a large group of investigators has already concluded that internalization of H:R has the exclusive function of catabolizing the components and thus ridding the cell of the “excess” (Table XXII). Indeed, significant numbers of workers assert that entry of ligand is not “required” for execution of its cellular effects. This, despite the impressive numbers of data demonstrating that at least some portion of H , if not R , escapes “undamaged” and becomes associated with the nuclear compartment (see below). And still other workers, conservatively concluding that there may indeed be functional correlates of the entry mechanism not yet identified, acknowledge the potential significance of the latter and thus have a foot in both camps (Table XXII). Firm conclusions are not yet possible. Presently available evidence suggests that processing of hormonelreceptor in the lysosomal compartment (whether membrane-bounded or made functionally accessible to the cytoplasmic milieu) may serve to produce fragments of unique intracellular (intranuclear?) function. Some putative “tertiary messengers” are being proposed for the modulation of the more distal metabolic effects of primary ligands such as insulin (Saltiel et al., 1981; Larner et al., 1981; Goldfine et al., 1982a). Moreover, staging, even of a proteolytic cascade triggered by lysosomal function, is entirely compatible with, for example, available evidence in relation to ATP-dependent proteolysis (cf. Wilkinson and Audhya, 198l), a feature highly relevant to processing of many biologically significant macromolecules to forms with retained, enhanced, or even redirected activity. (d) Indirect influences on other metabolic pathways. The fuller potential of limited proteolysis has only recently been recognized as a regulatory device that permits short-term responses from numerous poised systems (Magnusson et al., 1978; Neurath and Walsh, 1978; Agarwal, 1979a,b). However, retrospective analysis of certain aspects of acute hormone action that have not been adequately explained on the basis of transcriptionally augmented synthesis of new molecules of biocatalytic constituents in the affected cell may be contributory in the present context. For example, increases in energy-yielding reactions, that occur in relatively early response to steroid (see text, Section II,A, Fig. 3, and Table II), as
TABLE XXII RoLt OF LIGAND-RETEMOK INTERNALIZATION"
E S A M P L t S OF SPECULA 1 I O N ON T H E
Aponist ACTH EGF: NGF
Depradation exclusive1yh.c
Carpenter (1979);Maxfield er a / . (1979a.h)
Internalization: degradation'." not "required" for action
Internalization and lysosome fusion contributory to full agonal expression
Surface as n d l as (possible) intracellular functions Nolin (1980a) Fox and Das ( I 979);Fox et Carpenter al. (1979a.h); and Cohen (1979); Thoenen and Barde (1980);Levi er al. (1980): Heumann et a/. (1981); Shimizu er al. (1981); Landreth et a / . (1981): Das (1981):King and Cuatrecasas (1982)
Sacion er a/. (1980)
-
N oc
Internalization linked to turnover and renewal of surface receptor
King et al. (1980a); cf. however, ( 19XOb)
Schneider et al.
Immunoglobulins
(1979,1981) Insulin
Grisolia and Wallace (1976);Gliemann and Sonne (1978):Desbuquois et al. ( 1979); Caqentier er al. (1979a,1981 b); Gorden e t a / . (1980a,b); Kosmakos and Roth (1980);Baldwin et a / . (1980); Kasuga ct a/. (1981); Warren and Doyle (1981):Shimizu et al. (1981)d;Duckworth and Kitabchi (1981)
Fain (1974):Le Cam et a/. (1979):
Hammons and Jarett (1980): Marshall and Olefsky (1980); Felig (1981). Greenberg and Rozengurt (1982)
Goldfine (1981a,b): Varandani et a/. ( 1982)
Goldfine er al. (1981);Kahn er al. (1981a);Shimizu and Shimizu (1981'1: Posner er al. (1981h): Draznin and Trowbridge
(1982)
Posner er al. (1978); Corin and Donner (1981);Marshall et a / . (1981);Green and Olefsky ( 1982): Krupp and Lane ( I 982)
Interferon Gonadotropins
Chen er a / . (1977): Conn cr a / . (1978); Amsterdam et a / . (1980), Rajaniemi ef a/. (1981); Hizuka et a / . (1981)
Branca el a / . (1982) Ascoli (1978, 1982); Ascoli and Puett (1978a,b); Houdebine and Djiane (1980); Segaloff et a / . (I98 1); Segaloff and Ascoli (1981)
Szcgo el a / . (1974a.b); Szego (1974, 1975, 1976, 1978P; Petrusz (1978); Petrusz and Sar (1978); Ezzell and Szego (1979)
Growth hormone
-
w oc
Barazzone et a1 ( I980b)
Prolactin
Rillema (1980)
Releasing hormones
Conn ef a/. ( 198 I )
Houdebine (1982); Posner et a / . (1982~) Hazum et a / . (1980); TixierVidal and Gourdji (1980)
USome of these and other alternatives in this yeasty period have also been considered by Cohen et a/. (1979) and others (King and Cuatrecasas, 1981). hAnd thus, presumably, precedent internalization. [In molecular jargonese, often equated with “down regulation” of receptor; applicable to “excess” H and/or R . (’See, however, possible artifacts in presence of certain incubation buffers (Rennie and Gliemdnn, 1981) and as a result of the use of the direct chloramine Tmethod of iodination ot’ ligand under given experimental conditions (see Comens et a/., 1982). where applicable. See footnotes to Tables 111 and XVI. ‘General reviews, not restricted to gonadotropins.
I84
CLARA M. SZEGO AND RICHARD J. PIETRAS
well as peptide (Field, 1968, 1975; Sterling, 1979a,b; and Table IV) hormones, may reflect minor alterations in primary structure of the relevant enzymes. There is now extensive evidence that limited proteolysis leads to activation of given enzymes, while certain other enzymes are rendered inactive by such modification. Summation of such effects, coupled with those imposed by the well-characterized influences of phosphorylation/dephosphorylation(cf. Chock et ul., 1980; Table XIXA,B), are clearly capable of modulating metabolic pathways in the cytoplasmic milieu. That these pathways are to various degrees subject to hormonal regulation through controlled release of lysosomal enzymes requires extensive investigation. The capacity of many enzymes of the lysosomal compartment to function at neutral, or even alkaline, pH has been documented in Section II,B. Moreover, it may be anticipated that, as a result of early metabolic shifts, a relative decline in buffering capacity is likely to prevail in an activated target cell that is not entirely offset by the rapid increase in microcirculation elicited by the appropriate hormone (Spaziani and Szego, 1958; Szego and Gitin, 1964; Szego, 1965; Clayton and Szego, 1967). The latter, in turn, represents responses to the acute delivery of vasoactive substances sequestered in the lysosomal compartment (e.g., histamine, serotonin), or formed in situ by limited remodeling of peptide precursors (Szego, 1971a). Such “reactive hyperemia,” if prolonged, may have a delayed counterpart: the synthesis in siru of angiogenic factors (Folkman et al., 1971; Auerbach, 1981). Further influences contributing to the net outcome of these shifting metabolic pathways are exerted by given ions, e.g., Caz , calcium-binding proteins such as calrnodulin, and by their endogenous inhibitors and activators (cf. Barrett. 1980; Holzer and Heinrich, 1980), including cyclic nucleotide gradients. It is of crucial significance that Ca2+ as well as calmodulin are sequestered in membrane-bounded, lysosome-like organelles, as are proteases capable of activating CAMP-phosphodiesterase, and thus subject to controlled release on environmental signal. Provision of additional substrate through enhanced permeability of the reorganized cell surface contributes still another facet to the overall patterns of expanding cytoplasmic metabolism under hormonal control. The resultant increase in osmotically active particles, in turn, leads to imbibition of water, a common correlate of early hormone action (see Fig. 3 , Tables 1V and V; and Szego, 1971a). These combined data provide indirect evidence in support of limited dismantling, as well as coordinated and potentially reversible covalent modification, of cellular constituents in early hormone action (cf. Szego, 1971a). Controlled delivery of an array of lysosomal enzymes of unique specificities and wide varieties of function (see Section II,B) at early times, through hormonal intervention in promoting covert organellar destabilization, thus has the predictable consequences, with all the earmarks of cascade. ( 3 ) At mitochondria. Extension of an agonal signal delivered at the cell surface to modulation of energy-yielding reactions is difficult to envision without invoking alterations of mitochondria1 function per se. Indeed, there are cogent reasons +
LYSOSOMAL FUNCTIONS IN CELLULAR ACTIVATION
185
to suggest that extrusion of microquanta of lysosomal hydrolases plays a primary role in altering the structure and function of neighboring organelles, including the mitochondria. Although concerted action of an m a y of lysosomal hydrolases may be involved, the data of Tappel and co-workers (1963) indicate predominance of proteolysis in such effects. While the resultant secondary changes in mitochondrial function are more readily visualized with the massive lysosomal enzyme release associated with the application of toxic or otherwise damaging stimuli, it seems entirely possible that the more subtle changes attributable, in the final analysis, to physiological signals such as hormones, have been overlooked. Thus, although Sterling (1979a,b) and co-workers (1980) have suggested primary and relatively early effects of thyroid hormones on liver mitochondrial functions as reflected in altered P/O ratios, their interpretation has overlooked indirect effects that may well have been elicited through prior lysosomal labilization. The latter seems more likely in view of the relatively long lag period (e.g., 30 minutes between hormone administration and organ excision, followed by the interval required for cell fra~tionation),~ together with the evidence long available for heavy lysosomal contamination of mitochondrial fractions prepared by routine methods from organs in which both groups of organelles are strongly represented. The latter consideration would favor focal and prolonged contact of the mitochonrial membranes with the hydrolases and other components of labilized lysosomes in the isolated state. Accordingly, it seems appropriate to reevaluate the evidence for a primary and direct effect of thyroid hormones on the mitochondrial population in order to take into account these additional factors. Similar considerations may well prevail in the acute energy-mobilization reactions of additional target cells to a wider array of effectors. Indeed, the potential influences of limited concentrations of effector-liberated lysosomal enzymes on the structures and functions of additional cellular organelles, including the cellular scaffolding (cf. Table XVIIIA,B), the Golgi network, even the protein-synthesizing machinery, and the very nuclear compartment itself, have scarcely been addressed. One isolated example is the recent demonstration that the cyclic nucleotide phosphodiesterase activator of rat liver (Sakai et a l . , 1978) and kidney is a lysosomal protease with a pH optimum of 7.5 for that substrate (Strewler and Manganiello, 1979). Such a property could have far-reaching effects in modulating the cyclic nucleotide gradient and associated phosphorylation/dephosphorylation cascade elicited by hormone. These open questions require urgent investigation in light of the growing indications for concerted and cascading responses, with common features of limited hydrolysis of macromolecules, to a wide variety of triggering signals (cf. Szego, 1971a, 1975). (4) At the nuclear membrane: a way-station on the inward route. (a) Perinuclear transfer of activated lysosomes. Although doubly segregated from sur4Translocation of vesicle-oriented triiodothyronine to the perinuclear region (Cheng et d., 1980) and liberation of lysosomal hydrolases (Kasavina et al., 1976) occur well within this interval.
I86
CLARA M. SZEGO AND RICHARD J. PlETRAS
rounding cytoplasmic elements, processes in the nuclear compartment function in such exquisite coordination with all other cellular activities that two-way exchange of information/components across the nuclear envelope has long been a foregone conclusion. Nowhere is this tacit assumption better supported than in the case of what appears to be heightened interaction between the two major cellular compartments under circumstances of cellular activation by appropriate effectors. What is far less certain is the means by which such intimate communication is maintained and, more significantly, adjusted to the metabolic demands upon the cell by agonists recognized at the distant plasnialemmal surface. Without specifically addressing the question of the contribution of the nuclear pore complex in such exchanges, a problem that has occupied numerous gifted investigators (Wischnitzer, 1973; Franke, 1974; Franke et al., 1981b; Harris and Marshall, 1981; cf. also, Maul, 1982), it is the present purpose to analyze the available evidence for a significant role of lysosomal function in mediating FIG. 17. Perinuclear accumulation of organelles with the dimensions and staining properties of lyaosomes at short intervals after application of specific agonists or other substances to given cells in vivo (A-E), or in vitro (F-K). (A-H) Darkfield ultraviolet fluorescence micrographs following vital ataining with acridine orange (AO). (A.B) Nuclear fractions of preputial gland of ovariectomized rats injected intravenously 15 minutes earlier with 0. I p g (B), or 1 pg (A), of cstradiol-17Pi 100 g body wt. To be noted are the (metachromatic) fluorescence of the hormone-mobilized lysosomes and their escaping contents surrounding and adhering to some nuclei, often in the polar fashion illustrated in (B), while other nuclei lack adherent lysosomes (e.g., A), thus demonstrating the variable scnsitivities of cells in the same original preparation (see text). Perinuclcar lysosomes are invariably ahserit from the corresponding vchicle control specimens (not illustrated; see Fig. 1 in Szego and Seeler, 1973). ( A ) x 1000, (B), x630. Reprinted from Szego and Seeler (1973) with permission. (C-E) AO-stained rat oocytes examined immediately after aspiration from follicles explanted at spccificd times during the estrous cycle and cultured in hormone-free medium. (C) Oocyte explanted before the preovulatory gonadotropin surge and cultured in v i m for I hour. Note intact geminal vcsiclc (GV), nucleolus (NI), and surrounding granulosa cells (gc). (D) oocyte explanted during the onset of the gonadotropin surge and cultured as (C). Lysosomes are densely accumulated around GV and depleted from the peripheral cytoplasm. (E) Preparation similar to (D), but culture carried out for 2 hours; meiotic maturation is correspondingly more advanced, as exemplified by loss of GV; clumps of lysosomes are localized in arca formerly occupied occupied by intact GV, and they surround NI (arrow). (C-E) x510. Reprinted from Emell and Szego (1979). with permission. (F-H) AO-stained cells from bullfrog urinary bladdcr epithelium. Prior to staining. cells were treated for 10 minutes in riiro with vehicle control solution (F), nr with 10 mU/ml of arginine vasopressin (G,H); X450. Note that vasopressin also proniotcs redistribution of lysosome-like vesicles to surface membrane. Unpublished data of R.J. Pietras. (1,J) Fluorescencc micrographs of HeLa cells microinjected with highly concentrated (24-40 mg/ml) rhodamine-labeled bovine serum albumin, taken immediately (I), or 6 hours thereafter (J). The latter figure demonstrates the intense concentration of the fluorescent protein at onc pole of the nucleus (J), following fusion with acid phosphatase-containing organelles (not illustrated), presumed to be lysosomes. Reprinted from Stacey and Allfrcy (1977), with permission. (K) Visualization of fluorescent perinuclear granules of F-EGF in human carcinoma A-431 cells. The latter were incubated for 45 minutes at 6°C with 200 ng of the hormone, washed extensively to remove unbound excess, and incubated for 10 minutes at 37°C prior to fixation. Reprinted from Haigler rt 01. (1978), with permission.
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nucleocytoplasmic interactions, especially those integral to the cellular processes accentuated by specific agonists. The prime requisite of such a postulated role for lysosomal organelles and their products is accessibility. In preceding sections of this article, evidence has been marshalled to trace the penetration of the lysosomal compartment by given effectors and the consequential liberation of sequestered constituents in essentially graded proportion to the magnitude of the triggering signal. What is more cogent in the present context is the truly extraordinary phenomenon of perinuclear accumulation of large numbers of lysosomes within seconds to minutes of surface perturbation by ligand binding. In part because of the conspiciously polar concentration of such organellar translocation to which attention had been drawn earlier (Szego, 1975, 1978), the participation of cytoskeletal elements has been inferred (cf. also, Tables XVIIB and XVIIIA,B). What concerns us here is the focalized and intimate nature of the biorganellar interaction and the logical inferences to be drawn therefrom. Thus, as illustrated in Fig. 17 for a small but representative series of target cells, perinuclear aggregation of organelles with the properties of lysosomes is an early and apparently general receptor-mediated response to specific agonists. In addition to these several illustrations of rapid perinuclear accumulation of membrane-bounded vesicles corresponding, at least in part, to lysosomes, on challenge of cells with specific effectors, numerous supplementary data are now available. The phenomenon of peri- andlor supranuclear concentration of peptidal ligand-receptor has been described for antibody (Lewis et al., 1974), nerve growth factor (Andres et al., 1977; Marchisio et al., 1980; Heumann et al., 1981), gonadotropins (Petrusz, 1978, and citations therein; Ordronneau 1979), low-density lipoprotein (Krieger et al., 1979), Wistariajoribunda agglutinin (St. John et al., 1980), insulin (Goldfine, 1981b), and androgen-binding protein (Feldman et al., 198I ) . Quite striking parallels may be seen for hydrophobic effectors (cf. Nenci et a / . , I980b; Pertschuk et a/., 1980; Papamichail et al., 1980; Govindan, 1980; Cheng et al., 1980; Springer and Petrakis, 1981). Although the probes used were of variable degrees of reliability, the overwhelming evidence from these and many other observations supports a channel of nucleocytoplasmic communication served by a vesicular vector with attributes of lysosomes. Moreover, the effectors represent a wide
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spectrum of primary peptide structures which, when taken together with the evidence for their receptor-mediated endocytotic entry (Tables 111 and XVI), and further coupled with an apparently parallel phenomenon for hydrophobic agents (Szego, 1975; Szego and Pietras, 1981; and present Table X), argues strongly for some further role in metabolic regulation by a phenomenon so generalized. In light of these considerations, the disclaimers summarized in portions of Table XXII fade substantially in significance. In part, these considerations raise the question of whether or not breaching of the nuclear envelope in the centripetal direction by lysosomes laden with agonist/receptor in native or processed form is not the normal means by which effectors set the stage for forthcoming transcription. Indeed, as noted earlier, intracellular application of peptide hormones such as NGF (Heumann el al., 1981; Huttner and O’Lague, 1981; cf. however, Claude el al., 1982) or the secretogogues caerulein and bombesin nonapeptide (Philpott and Petersen, 1979) fails to elicit the anticipated mitogenesis and potential conductance changes, respectively, that are observed following extracellular presentation of these substances. Quite parallel observations are reported for the characteristic response of steroid hormone-induced meiotic division in Xenopus laevis oocytes (Baulieu ef al., 1978; cf., however, Tso et al., 1982), a further indication, if more were needed, of the occurrence of primary recognition sites at the outer cell surface for both major classes of effectors (cf. Szego and Pietras, 1981). Thus, it seems that a functional channel of potential nuclear entry requires a surface-triggering event. This is the more significant in light of the fact that application of the primary stimulus directly to isolated lysosomes is not sufficient to activate their functions that have been observed in situ (cf. Lucy, 1970, and references therein; Nemere and Szego, 1981b). The conclusion from these combined observations thus seems inescapable that hydrolases, protein kinases, and phosphatases (among the more than threescore sequestered lysosomal enzymes and their several modulators), as well as numerous additional nonenzymic components yet to be characterized, in escaping from lysosomal membrane-bounded form as a result of the covert labilization inflicted by the prior fusion with endocytotic vesicles, are in a strategic position to produce those covalent modifications in the nuclear envelope that, in such highly localized fashion, may promote its “permeabilization,” and thus lead to expansion of the exchange route across this relative barrier.J (b) Altered accessibility of macromolecules to the nuclear compartment, con5Not to be considered here is the structural evidence of the more drastic outcome of such perinuclear accumulation of lysosome-like organelles, that of germinal vesicle breakdown (cf. Fig. 17) at a crucial stage of meiotic maturation under control of specific hormones (Ezzell and Szego, 1979). Whether accomplished by means of proteolytic activity, or through mechanisms of phosphorylation/dephosphorylation of critical membrane components (see Table XIXA,B), this more distal event may likewise reflect the enzymic potential of the lysosomal apparatus mobilized essentially en masse (cf. Fig. 17D and E) by LH (Ezzell and Szego, 1979), by progesterone, or by 1-methyladenine in their respective target cells (see Table XIXB).
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tingent upon perinuclear association of activated lysosomes. When probed with IgGs that had been raised toward a class of high-density lysosomal lipoproteins and which, in common with all other immunoglobulins, are normally excluded from the nuclear compartment, there is indeed evidence that a cell-specific agonist as, for example, estrogen, promotes the intranuclear transfer of the immunoglobulins (Nazareno et al., 1981; Fig. 18A-D). As will be mentioned in the following section, this experimental approach serves also to illustrate the accessibility of the lysosomal antigen to the nuclear compartment under hormonal control. (c) Implications f o r processing of precursor forms of RNA and enhanced egress of products. By the same token as accessibility in the inward direction, a priori evidence would predict that enhancement of intercompartment communication, once established, is, at least for a limited time until it subsides by reason of attrition of the instigating elements and reestablishment of the status quo ante, a two-way process. In accord with this inference, there is evidence for cell-specific hormonal facilitation of extranuclear egress of various forms of RNA, if not ribonucleoprotein particles. For example, target cells responsive to agonists of such varied structure as thyroid hormones (Tata, 1966), insulin (Schumm and Webb, 1981; Goldfine et al., 1982a,b), as well as estrogen (Moore and Hamilton, 1964; Church and McCarthy, 1970; Thampan and Clark, 1981) all share in this phenomenon. Specific mechanisms to account for such mobilization generally postulate changes in pore function, with or without concomitant or precedent alterations in nuclear matrix and nuclear membrane fluidity (Wunderlich, 1981; cf., however, Agutter and Suckling, 1982). Biochemical requirements for transit have been invoked as a regulatory device, with emphasis upon the apparent participation of phosphorylationldephosphorylation mechanisms (cf. Webb et al., 1981). Activation of enzymes preexisting in the nuclear membrane/pore/matrix complex, including those involved in the processing of precursor forms of RNA (Ciejek er al., 1982), may conceivably regulate such macromolecular traffic. A priori, such hypothetical activation would appear to be secondary to the rapid, receptormediated perinuclear accumulation of some fraction of secondary lysosomal and sublysosomal organelles, extruding microquanta of their sequestered components. While a cause-effect relationship is by no means established, the present, far from complete, evidence is nevertheless consistent with lysosomal participation at this crucial site of cellular function. The infinite detail with which preparation for hormonal augmentation of the biosynthetic pathways yet to come is achieved, in part at least, through preliminary dismantling of an array of structural and functional macromolecules, is astounding. But the events most critical to the attainment of metabolic precision lie within the nuclear compartment itself. And it is upon these that we now focus attention, through analysis of the translocation of materiel peculiar to the lysosomal compartment into the nuclear interior.
F K . I X . Influence of estrogen on accessibility of the nuclear compartment of selective target cells to a fluorescent lysosomal probe. Indirect (A-D) and direct (E-H) immunofluorescence of isolated nuclei (A-D) and cryostat scctions (E-H) of preputial glands of ovariectomized rats in the abscncc (B, E), and presence ( A , C, D, F, H) of estradiol-l7P, 0.1-0.5 pg/IOO g body wt, administered iv 2-15 minutes bcforc tissue excision. (C)Tissue section from animal that has received the relatively inactive congener, estradiol-17a; the latter is similar to the vehicle control (E), in which fluorescence attributable to interaction with immunoglobulin directed against a prcputial gland high-density lipoprotein is restricted to the pinpoint loci characteristic of intact lysosomes. In contrast, immunofluorcsccnce is more generally distributed over nuclei and in a pcrinuclear halo in sections exposed it7 vivo to the lyaosonial mcmhrane labilizing effects of active estrogen (F, G , H);
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( 5 )Intranuclear recompartmentation. The technologies presently available for demonstration of transfer of hormone/receptor into the nuclear interior are still in a primitive state. Nevertheless, highly suggestive evidence, as outlined in preceding sections, has been forthcoming that the steroid and peptide hormones, having set in motion the cell-surface perturbation associated with their specific recognition, find their way, together with diminished forms of their specific receptors, into intracellular compartments, including lysosomal, Golgi, as well as the nuclear. What has been lacking in proposals of a viable mechanism for such “migration” that are in full accord with all the available data is a functional vector. For the moment, all signs point to a system of vesicles that serves the transfer process. And, because of their unique properties, including capacity for saltatory motion and potential for fusion with membranes of other organelles (cf. Table VIII), vesicles conforming to the properties of lysosomes and/or their suborganelles are the prime candidates, with possible “assists” by the microtubule/microfilament system with which these organelles have close associations (cf. Table XVIIB). While direct evidence is meager, the indirect may serve. For, if limited numbers of secondary lysosomes in hormone-activated cells are laden with hormone/receptor and/or their partially (or more extensively) hydrolyzed products, as documented in preceding sections and summarized in Tables XVI, XXII, and Figs. 10-12, analysis of transfer of lysosomal constituents per se, which are readily identifiable by unequivocal methods, may serve a heuristic purpose. Thus, lysosomal marker enzymes, foreign to nuclei under basal conditions, are strikingly evident in hormone-exposed nuclei that have undergone extensive purification for removal of cytoplasmic contamination. Such “ultrapurified” nuclei are significantly marked by lysosomal enzymes within 2 minutes of exposure in vivo to the appropriate hormone, steroid (Szego et al., 1974b, 1976; Fig. 19A and B) or peptide (Szego et al., 1974a; Fig. 19C). Similar results are obtained when nuclei are isolated essentially free of cytoplasmic contamination by nonaqueous methods (Szego et al., 1976). It is notable that enzymes characteristic of the nuclear compartment are entirely unaffected during such short times of hormone treatment in vivo (Szego, 1974). The immunologic probe cited earlier can also provide clues to the centripetal pathway in hormone activated cells. Figure 18E-H reveals, by direct immutransfer of antigen 10 the luminal surfaces of preputial gland tissue is likewise evident in (H). In the isolated nuclei (A-D), immunofluorescence is restricted to the immediate periphery in the absence of hormone (B), while accessibility of antigenlantibody to the nuclear interior occurs in many (but not all; cf. A) nuclei that were exposed in vivo to the hormone. These observations clearly illustrate both lysosomal labilization as well as nuclear membrane permeabilization to macromolecules attributable to the action of estrogen at selective target cells for very brief times. The significance of the apparent ; X560; “capping” of a nucleus (arrow, D) is presently unknown. Magnifications: (A) ~ 8 9 0 (B-D) (E) x580; (F-H) x540. Reprinted from Szego et al. (1977) and Nazareno er al. (1981), with permission.
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FIG. 19. Receptor-mediated lysosomal enzyme transfer into ultrapurified nuclei from hormonal target cells. (A-C) Ultrapurified nuclei, stripped of outer membrane, as well as adventitiously adsorbed cytoplasmic material, from preputial glands of ovariectomized rats (A,B) or adrenal glands of hypophysectomized rats (C) were analyzed for characteristic lysosomal enzymes that are essentially foreign to the nuclear compartment in vehicle-control treated preparations (open bars). In contrast, such enzymic activities are evident in corresponding specimens excised from appropriate animals 2 minutes after iv injection of 0.1 pg of EzPilOO g body wt (stippled bars; A,B) or 5 minutes after iv injection of 10 mU of purified ACTH/lOO g body wt (stippled bars, C).
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nofluorescence in frozen sections, the rapid reorientation of lysosomal antigen from randomly distributed punctate loci to perinuclear, supranuclear, cytoplasmic, and luminal-surface sites, in large measure in diffuse form, on challenge of estrogen-sensitive cells with the active hormone. Still other circumstantial evidence in support of the participation of secondary lysosomes in vectorial transfer toward and, potentially, across the nuclear membrane may be adduced from ultrastructural analysis (Fig. 20). The perinuclear association of lysosomes, which is observed in superficial portions (Fig. 20C), as well as in deep invaginations of the nucleus (Fig. 20A and B), is resistant to the conventional methods of nuclear purification, which include centrifugation through heavy sucrose (Fig. 20D). As illustrated in electron micrographs, it requires stripping of the outer nuclear membrane with very low concentrations of nonionic detergent, followed by extensive washing (cf. Fig. 20E), to yield the ‘‘ultrapurified” nuclear preparation (Fig. 20F) suitable for biochemical analyses such as those presented in Fig. 19A-C. Parenthetically, it should be noted that such intimate association of vesicles with the outer nuclear membrane has had a diametrically opposite interpretation. Thus, several workers have speculated that these structures represent blebbing of the nuclear membrane with the resultant organelles destined to enter the cytoplasm (cf. Wischnitzer, 1963, 1973; Fry, 1976), possibly even by “direct passage . . . across the nuclear membranes” (Scharrer and Wurzelmann, 1969).
FIG. 20. Ultrastructural evidence for estrogen-induced nuclear penetration by lysosomes or thcir suborganellar components. (A-C’) Endometrial cells from ovaricctomized rats injected iv with 0.1 ~*g/100g body wt of estradiol-17p. (A) Portion of a nucleus (N), showing extensive cytoplasmic invagination at 30 minutes after hormone. Intense electron opacity in certain areas is due to deposi-
I94
FIG. 20D-F. tion of acid phosphatase reaction product. Arrow within nucleus refers to latter material, possibly a fragment of a dense-body (DB) lysosome. Several additional multivesicular (mvb) and DB lysosomes are also evident in the perinuclear cytoplasm; X 14,400. (B) As (A), except that it is unstained (continued) I95
FIG.20G-I. for acid phosphatase activity and represents a section exposed in vivo to hormone for 15 minutes; cytoplasmic invagination channel contains several vesicles of sublysosomal dimensions (cf. Fig 4); X60,OOO. (C) At 15 minutes after estradiol, a granular inclusion (inc) with characteristic electrondense lysosomal acid phosphatase reaction product may be noted deep within the nucleoplasm in close proximity to nucleolus (nl). The limiting membrane of a DB lysosome appears in intimate contact with the outer membrane of the nuclear envelope; X 12,000. Latter is shown in greater detail in (C'); X60,OOO. (D-F) Isolated nuclear fractions from preputial gland showing localization of acid phosphatase reaction product in associated lysosomes in preparation from animals injected with 0.1 pg of estrddiol-17@/100g body wt 5 minutes before tissue fixation. (D,E) Tenaciously adherent
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In earlier work, attention was drawn to the prevalence of nuclear inclusions with the dimensions and enzymic markers highly suggestive of lysosomal origin, in cells undergoing intense proliferative activity, whether under normal conditions or in transformed state (cf. Figs. 31 and 32, and Table 3 in Szego, 1975). This interpretation is now echoed in the work of others with estrogen (Padykula et al., 1981) and androgen (Thomas el al., 1981), whereas Bouteille and colleagues (Vagner-Capodano et al., 1982), working with TSH-stimulated thyroid cells, suggest that these subnuclear organelles play some role in ribonucleoprotein transport within the nuclear compartment itself. Clearly, no amount of speculation on the origin, nature, and function of nuclear inclusions serves to elucidate these questions, which are awaiting more definitive methodologies for their solution. There remain some striking anomalies, if not paradoxes, in observations related to these findings. These include the presence of “unoccupied” binding sites for a growing number of effectors within nuclei, rendered apparently free of cytoplasmic contamination during preparative manipulations (cf. however, Vanden Berghe, 1973; Szego et ai., 1974a,b, 1976). Coupled wth such observations is evidence for nuclear translocation of “specific receptor” from cytoplasmic sites under instigation of a variety of provocative stimuli, such as hypertonic sucrose, ethanol, and other drugs (cf. Gorski and Gannon, 1976; Okey et al., 1980) or forms of physical trauma such as cautery (Pertschuk et al., 1980). Indeed, the antecedent to this intranuclear transfer phenomenon, that of perinuclear concentration of labeled probes in vesicular form, is likewise noted on challenge of neuronal (Harper et al., 1980) and HeLa (Kramer and Canellakis, 1979) cells with agglutinins. Still earlier observations have identified striking perinuclear association of vesicles with the colligative properties of lysosomes under conditions of intense proliferative activity, such as occurs in liver regeneration and lymphocyte response to PHA (reviewed in Szego, 1971a,b, 1975; cf. also, Gordon and Rothstein, 1981). And the provisional identification of nuclear inclusions as lysosomal products in other forms of cellular hyperactivity, such as cancer (cf. Szego, 1975), is likewise suggestive of some form of abnormality. Is this centripetal translocation of lysosome-like vesicles, then, evidence lysosomes that survived the shearing force of centrifugation through heavy sucrose (D; X25,200), before removal of outer nuclear membrane and adherent organelles (E; X29,000, one lysosome undergoing dissolution in the process), by procedure of Szego er a!. (1974b,1976). The low-power survey view of a corresponding vehicle control-treated specimen (F) shows nuclei only, some with dense nucleoli, but devoid of lysosomes, and without evidence of acid phosphatase reaction product; X 5800. (G-H) Nuclear inclusions in endometrial cells of estrogen-treated animals. (I) Deposition of acid phosphatase reaction product limited to membrane-like microfibrillar material associated with the inclusion; x60,OOO. (G)Substrate-free control; X60,OOO. (H) Further cytochemical control, boiled before incubation in the complete enzyme-staining medium (note resultant clumped chromatin); X35,OOO. Electron micrographs, courtesy of Dr. Robert E. Smith; reprinted from Szego (1975), with permission.
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of underlying pathology or a response to some untoward stimulus‘? Probably not. Instead, it may be viewed as an exaggerated form of a fundamental cellular activity-the noise in the system (cf. Table I). For example, perinuclear lysosome accumulation has been noted after microinjection of a variety of endogenous and exogenous proteins in HeLa cells (Stacey and Allfrey, 1977), and even after mere cultivation of vascular smooth muscle cells (Fowler and Wolinsky, 1980; cf. also, Rose, 1957; Cohn and Benson, 1965, and Section 1,A,2). Association of lysosomal enzymes with purified hepatic nuclei after fasting/refeeding (Mak and Wells, 1977) may have a similar basis-metabolite glut and the concomitant enhancement of surface pinocytotic activity. Accordingly, it seems possible that these common responses to disparate stimuli represent a channel of surface/cytoplasmic/nuclearcommunication, whether of informational molecules (effectors, including the endogenous or exogenous, in association with their respective internalized receptors, in intact or processed form), or of materiel with potential metabolite functions, which is a normal cellular activity elicited by surface perturbation. Such function may be intensely exaggerated under conditions of profound but “nonspecific” surface provocation.6 By the same token, reduction of endocytotic activity by some form of membrane stabilization, such as exhibited by glucocorticoids in appropriate concentrations (cf. Miller and Melnykovych, 1982), predictably diminishes lysosomal activation (Szego, 1972b) and its accompanying nuclear destabilization (Szego, 1974). ( 6 )Acute altercttions in the intranuclear microenvironment elicited by specific hormone: potential contributions of lysosomal constituents delivered under hormonal instigation. Having considered the nuclear envelope as barrier and demonstrated that it may be breached in controlled degree by lysosomal components mobilized in specific cells by a receptor-mediated process, it becomes necessary to examine the question, what are these potentially destructive elements doing in the nuclear compartment in which their very presence is an a n ~ m a l y ?Some ~ 6An alternative vicw based on relative affinities and concentration gradients has been provided for a related set of observations on thc “shuttling” of certain proteins, synthcsized in the cytoplasm, to the nucleus in Arriorhtr proteus in a thoughtful paper of Goldstein and Ko ( 1981). Thus, these workcrs invoke n o vectorial transport proccss for this system. ’The presence of “endogenous” proteases in isolated nuclei and in chromatin has long been recognized. In most, hut not all (cf. Krueger, 1982) cases, these activities can readily be distinguished from those typical of lysosonies by biochemical critcria and sclcctive inhibitors. However, only rarely has the possibility been considered that the proteolytic degradation common to the experience of many invcstigators attempting to isolate nuclcoproteins in maximum yield might be duc to cxcessive trauma to membranes during cell disruption and consequent mobilization of lysosomes-the noise in thc system. These comments may also have relevance to observations following application of toxic drugs, fur example, the intense acceleration of nuclcar transport, as well as proteolysis of nonhistone components of chromatin, following administration of cyclohcxiniide (Boikov e t a / ., 1982; cf. also, Tablc XXIII). On the other hand, nuclei isolated during liver regeneration reveal substantial increases, in specific lysosomal proteases over control levels (Tsurugi and Ogata, 1980; cf. also Dolhcarc, 1973).
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fresh insight may have emerged from recent investigations. In a number of respects, these new findings conform to some of the predictions that may be made from the foregoing. It is acknowledged that despite the enlightenment achieved by primary advances in microbial genetics, we are still remote from understanding how the compact superhelical structure of DNA, when associated with proteins in higher order nucleosomal array, becomes accessible to appropriate polynierases and related modulatory activities at given times after application of mitogenic or developmental stimuli. This formidable problem is central to further progress. Some very limited aspects of this questions have been addressed by Horton (1982) in recent work. Based upon evidence for abrupt nuclear recompartmentation of lysosomal constituents, including hydrolases, nonhydrolytic enzymes, and nonenzymic matricular components, under hormonal control, systematic analysis was undertaken of the protein complement of purified chromatin and of the nucleoplasm in ultrapurified nuclei from the estrogen-sensitive preputial gland excised from ovariectomized rats 2- 15 minutes after in vivo administration of placebo control solution or a physiological concentration of estradiol- 17p. Attributable to hormone pretreatment and identified by a battery of electrophoretic methods were a number of quantitative alterations, as well as some of a qualitative nature, among histone classes and their variants, among the loosely bound chromatin proteins extractable with 0.35 M sodium chloride, as well as among the residual nonhistone proteins (Horton and Szego, 1984). Within 2 minutes of hormone administration there were observed some significant losses of histones especially of Hl(a), the major H1 subcomponent. Histones of the nucleosomal core were less vulnerable, except for H4, which underwent a relative decline at 2 minutes, followed by a sharp increase at 15 minutes after hormone. Indications were also obtained of limited and highly selective proteolytic processing of certain highly and moderately basic proteins, such as could be elicited by the stringent enzyme, cathepsin B (see Table XV) that is known to be transferred to the chromatin fraction of ultrapurified nuclei of preputial gland under estrogen stimulation (Szego et al., 1976, and Fig. 19B). Changes were also noted at 2 minutes among proteins of the nucleoplasm (Horton and Szego, 1984), a subcompartment in dynamic flux with chromatin and the nuclear matrix, but, more to the immediate point, with the cytoplasm. For the latter is the acknowledged source of many nuclear proteins, and it may be anticipated that certain of these would show striking correlations in electrophoretic behavior illustrative of their similarities in primary sequences. However, even more cogent to the present discussion is the striking parallel between certain chromatin proteins, especially of those loosely bound and of a basic nature (Fig. 21A), with lysosomal proteins extracted from the preputial gland organelles by low-salt buffer (Pig. 21B). It is particularly striking that a virtual mass of material with the electrophoretic properties of the
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10
FIG.2 I . (A) Resolution in a two-dimensional polyacrylarnide gel electrophoresis system [acid:urea:Triton (AUT) in the first dimension, followed by electrophoresis in the presence of sodium dodecyl sulfate (SDS)], of 0.35M NaC1-soluble chromatin proteins (36 +g) from preputial glands of ovariectomized rats. Reference lanes include very-low-M, standards (18 pg), and acid-soluble proteins from a corresponding chromatin preparation (21 pg). (B) AUT-to-SDS gel, as in (A), of 50 pg of lysosomal proteins from preputial glands of ovariectomized rats. Reference lanes include 20 pg of acid- soluble chromatin proteins and a 31-pg aliquot of proteins from the lysosomal fraction. Attention is drawn here and in the text to proteins designated L1, L2, L4, L7, and L8, whose correlates corresponded to those of ceaain 0.35 M NaCI-soluble chromatin proteins (cf. A). Also noted are trace amounts of components at positions corresponding to HMG-1and -2. Reprinted from Horton and Szego (1984), with permission.
core histones, especially H2A, H2B, and H3, or variants thereof, is evident in the lysosomal sap (Horton and Szego, 1984; Fig. 21B). It may be inferred from these and related findings (Horton, 1982) that selective and limited proteolytic processing occurs during even such a short time of hormone exposure. That these events were initiated in viwo is rendered likely by the presence throughout the collection, isolation, and analytical steps, of the proteinase inhibitors, leupeptin and phenylmethylsulfonylfluoride, antagonists of
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FIG. 21B.
the predominant preputial gland proteinase, cathepsin B, as well as of trypsinlike serine proteases. Moreover, the extreme rapidity of these E,P-induced alterations and, in many cases, the changes in relative proportions, rather than net gain or loss, possess implications for redistribution and covalent modification of existing proteins, rather than their synthesis de now, at least initially. Taken together, these observations are strongly suggestive of appreciable reorganization of chromatin structure under hormonal influence at a time well before observations of enhanced genic transcription are generally available. Could limited recompartmentation and specific association of lysosomal constituents with nuclear elements underlie the accessibility of precise genomic sites to the transcriptional machinery? With enzymic recognition of potential substrate an especially critical means of specification, this no longer hypothetical, but still manifestly premature, suggestion (cf. Szego, 1971a,b) urgently rewires extension and verification. Similar considerations have been advanced, in turn, for participation of lysosomal ribonuclease activity in the maturational processing of newly transcribed mRNA (Szego, 1982). Moreover, lysosomal hydrolases, escaping from
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membrane-bounded form on organellar activation, contribute to the increased cytoplasmic supply of precursor molecules destined for the anabolic processes to come. This augmentation arises from both limited demolition of macromolecules in situ, as well as from the modifications, already cited, of surface membrane permeability to amino acids and other low M , substances (cf. also, Szego, 1971a). But is effector-driven invasion of the nuclear compartment by lysosomes and their derivatives, coupled with significant dismantling of cytoplasmic components, sufficient to account for that degree of transcriptional/translationalactivation that leads, ultimately, to cell division? Clearly, there are interposed many additional events in the staging of the mitogenic process. Whether lysosomal activities contribute to further steps in such staging will be considered, to the extent thc data permit, in a later section. b. Longer Term Efects: Action and Rcuction. ( I ) Vesicularflow, including that in course of exocytosis: the lysosomal shuttle. The early signs of cellular activation in which lysosomal function appear to play a critical role have, as noted, further repercussions. One link to the unfolding pattern of cellular events distal to receptor-mediated plasmalemmal vesiculation lies in the behavior of individual vesicles that have been monitored by some form of microscopy. Thus, when viewed in living cells by vital staining and time-lapse photography (Rebhun, 1972) or by cinemicrographic recording with Normarski differential interference optics (Lopata et al., 1977; Willingham and Pastan, 1978), organelles with the several properties of lysosomes undergo saltatory , nonrandom linear movement at 0 or 180” relative to their previous position. This property, so generalized in a variety of cell types, has led to the frequent suggestion that the association of these vesicular organelles with the cytoskeleton (cf. Table XVIIB) may underlie their rapid, ordered translocation. Inherent in this property, also, is the potential for bidirectionality of vesicular distribution-both centripetally toward the pole of the nucleus adjacent to the Golgi network, as well as in the reverse direction, toward the plasmalemma. It is in part this behavior, coupled with propensity for reversible fusion with membranes of other cellular structures (cf. Table VIII), that led to the term “lysosomal shuttle” (Szego, 1978; Y.-J. Schneider et al., 1978). The frequent association of receptor/hormone with the Golgi apparatus (see Tables X and XVI, and text) gains additional significance from consideration of lysosomal mobility together with function of the Golgi region as a crossroads of membrane traffic in secretory (Hand and Oliver, 1981; Fig. 22) and even in nonsecretory (Fig. 5A and B) cells. Although alternative patterns clearly exist (cf. Herzog and Miller, 1979; and Section ILB), the reassociation of secondary lysosomes with the site of their origin as membranebounded organelles may bear some analogy to the return of salmon to the microchemical environment characteristic of the headwaters of their home river. For there is a strong probability that, aside from, or in addition to, the contribution of
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Cis FIG. 22. Diagram showing two possible routes that could be taken by surface membrane to reach patches of surface the stacked Golgi cistemae. Following exocytosis of secretory granules (-), membrane are recovered by endocytosis and fuse with the dilated rims of multiple stacked Golgi cistemae. Membrane recovered by endocytosis can either fuse directly with the cistemae (- - - - - -) or fuse first with lysosomes and then with the Golgi (- . - . -). The available evidence suggests that both routes may be used in different cell types (cf. also Farquhar, 1983). Reprinted from Farquhar (1981b), with permission.
microtubules and their constituent elements to orientation, recognition may be achieved by a degree of correspondence of carbohydrate components among Golgi, plasmalemma, and the membranes of the primary lysosome population of selected cells (reviewed in Szego, 1975). Such reassociation of secondary lysosomes with their presumptive site of origin may underlie the occasional indications for occurrence of receptor macromolecules for a number of effectors, as well as the agonists themselves, in the strategic Golgi region of their target cells (Tables 111 and XVI). The potential significance of this phenomenon for information transfer, its processing and modulation and its expression in metabolic regulation remains to be explored in depth. Inherent in the bidirectionality of vesicular traffic (cf. Steinman et al., 1976; Muller et al., 1980a; Vila-Porcile and Olivier, 1980; Pearse and Bretscher, 1981 ; van Deurs and Nilausen, 1982) are both the well-documented observations of attrition of membrane-oriented receptor consequent to interception of homologous8 ligand, and also the renewal of such receptors in the cell surface by a “rebound” process (up-regulation) as a coupled function. A vector of appropriate mobility and fusion potential is clearly indicated in both cases. Such “percolation” of receptor through the several cellular compartments in course of XTachyphyhxis (down-regulation) of receptor is also frequently encountered in relation to heterologous ligands (cf. Kosmakos and Roth, 1980). This phenomenon may be related to concomitant internalization of adjoining plasmalemmal regions, possibly on exposure to gross concentrations of effector, a situation that provokes excess pinocytotic activity and uptake of extracellular substances by bulk flow. In secretory cells, excessive concentrations of secretagogues are known to be associated with lysosomal hyperactivity (Savion and Selinger, 1981).
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its synthesis, maturation, and packaging could account for the numerous observations, superficially at variance with each other, of the predominant subcellular localization of given receptor, with and without cellular challenge by appropriate ligand. The circulation of surface membrane through vesicular internalization and subsequent insertion of appropriately ‘‘marked” replacements is now well documented and widely appre~iated.~ However, only those aspects of such vesicular flow as are clearly influenced by specific effectors, and also seem to possess the hallmarks of lysosomal participation (other than in relation to the ontogeny of lysosomes per se), will be considered here. For, membrane renewal, together with obvious implications for receptor turnover, are unmistakable correlates of the response-train in cells equipped with recognition devices for specific effectors. Part and parcel of this overall process is the exocytotic delivery of cellular products segregated in vesicular form to the external environment of given cellss-even those not normally considered secretory. For example, microvillar membrane vesicles have been shown to accumulate in media during culture of embryonic chick intestine, a process significantly enhanced by addition of thyroxine (Black et al., 1980). Membrane vesiculation with shedding has been identified in response to immunologic challenge of porcine kidney cells (Paul ef a / ., 1979), while the ionophore A23187 provokes rapid exocytosis of lysosomes per se from rabbit polymorphonuclear leukocytes (Moore et a/., 1979). Granule exocytosis from the luminal surface of toad bladder was extensive within 5 minutes of application of physiologic concentrations of antidiuretic hormone to the serosal surface (Gronowicz ef al., 1980; cf. also Fig. 17F-H). An especially intriguing example of direct participation of vesicles with the several properties of lysosomes in the physiological process of urinary acidification has been furnished by Gluck et a/. (1981). These workers noted that CO, stimulated rapid insertion of such vesicles, with their intrinsic H + pumps, into the luminal membrane of turtle bladder cells, thereby promoting H + secretion across the epithelial sheet. The shedding of cell contents, although in forms not by any means always rigorously identified as vesicular, is probably a normal expression of the vital processes shared by all cells’O-their capacity to “condition” the medium in which they function. It now seems clear from a substantial body of evidence that the exocytotic process, together with surface renewal, is a function closely regulated by the signals set in motion by surface recognition of specific ligands. For example, 9Some sober reservations on the too-ready generalizations on this subject have been voiced by Holtzman and Mercurio (1980), while Novikoff (1980) and others (cf. Tanaka er al., 1980) have drawn attention to the morphologic artifacts that are encountered when insufficient attention has been paid to fixation of preparations for electron microscopy. ‘This property is exaggerated in many cancer cells in which the surface membrane may be relatively freer of the constraints imposed by both exo- and endoskeletal components (Black, 1980; Poste, 1980).
LYSOSOMAL FUNCTIONS IN CELLULAR ACTIVATION
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release of vesicle-bounded hormones from their secretory cells by an exocytotic process triggered by the appropriate tropic agent" is now well established in a substantial number of cases. In many of these, there is circumstantial evidence of lysosomal involvement, in part because of concomitant or closely coupled proteolytic or other means of processing of the precursor to its mature secretory form, presumably within secretory granules. In specific cases, the latter may conform to a type of secondary lysosome (see Table XXI, and Simon and TixierVidal, 1979). This relationship, so strikingly evident in the thyroid gland under TSH control (cf. Engstrom and Ericson, 1981; Miquelis and Simon, 1981, and references therein), is rapidly being extended to the exocytotic delivery of a wide range of cellular products triggered by appropriately recognized effector. In a limited number of cases, parallels between peptidal and steroid hormones as secretory effectors may be identified. Indeed, and perhaps paradoxically, protein-bound steroidal products of secretory cells driven by ACTH (cf. Nussdorfer et al., 1978) and by gonadotropins (Willcox and Alison, 1982, and references therein), respectively, may find their way into the extracellular space through exocytotic delivery of the products of secretory granules. The glucose- or amino acid-triggered release of insulin is a well-known counterpart of such phenomena. Exocytosis of vesicular matrix of chromaffin cells, with conservation of membrane, is a further parallel in cells subject to neuroendocrine control (Winkler, 1977). In turn, secretion into the prostatic lumen, in protein-bound form, of a synthetic estrogen derivative has been described (Forsgren et al., 1979). Moreover, recent evidence of modulation by estrogen and progesterone of LH-RH release in particulate form from hypothalamic "synaptosomes" (Peck et af., 1979) suggests a further parallel in which the effector is steroidal. Likewise, secretory granule release of products accumulated under estrogen dominance at luminal surfaces of endometrial cells is a rapid response to progesterone administration (Fig. 23). A particularly striking example of bidirectional release of a Sertoli cell product, androgen-binding protein, occurs in response both to androgens and FSH. Recent studies have indicated that this well-characterized product gains access to the lumen of the seminiferous tubule at the apical portion of Sertoli cells, while another fraction may be liberated into the blood at the basal aspect (Gunsalus et al., 1980). In either case, transfer by diffusion is unlikely, and the inference is that of a vesicular fusion event preceded by localized remodeling of the relevant membrane surfaces by the processes believed to contribute to fusion (see text above). Pollard et al. (1981) have developed an intriguing chemiosmotic model to encompass the events associated with the exocytotic release of the contents of chromaffin granules. The classical pattern of calcium mediation of stimulus-secretion coupling has been reviewed with significant extensions to membrane conservation by Douglas (198 1). "See footnote a to Table XXI.
FIG. 23. The effects of estrogen and progesterone on exocytosis of secretory granules. ( A ) Urerins horn of ovariectomized cat treated for 14 days with estradiol-17P (E$), followed by 5 minutes of progesterone infusion. Most secretory pranules are of decreased density. Especially to be noted are two granules that are in the process of exocytosis larrows). X 13.500. (B) Exwytosis in an epithelial cell from an animal treated for 14 days with E2P and then infused with progesterone for 5 minutes. Note the membrane irregularity of the other granuks. x32,MOU. (C) Fusion of two granules to release their contents jointly. Animal treated for 14 days with E,P and then infused with progestcrone for 15 minutes. x41 .OOO. Reprinted from Bareither and Verhape (1980). with permission.
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Despite intense current interest in membrane recycling, there is only very limited information on the detailed mechanisms underlying the obviously stringent regulation of this process that permits dynamic equilibrium among the contributing cell compartments (cf. Holtzman, 1981; Farquhar, 1981b; Orci et al., 1981). Lysosomal function in secretory cells has previously been considered in relation primarily to crinophagy and degradation in situ of “excess” product (cf. Smith and Farquhar, 1966). However, there is now growing recognition of the critical contribution of effector-stimulated lysosomal hydrolase release (possibly secondary to calcium influx and microfilament disarray (cf. Savion and Selinger, 19Si), to the localized reorganization of cell surface microarchitecture. The clear relevance of these coupled processes to plasmalemmal fusion with the secretory granule may promote the new experiments required to analyze this phenomenon in depth. Of the several approaches to this complex problem we believe that ligand-induced modulation of vesicular segregation-redistribution holds the most promise for replacement of inference with hard fact. ( 2 ) Promotion of intercellular communication: indications for a further role for lysosomal functions. (a) Modification of the extracellular matrix. The ready exchange of materials between cells and their environment is well established. That cells contribute macromolecular components to their external surfaces, and thus, potentially to the interiors of neighboring cells is likebise axiomatic (cf. Hay, 1981a). Moreover, it has long been recognized that the composition of the extracellular medium in which surviving cells are maintained reflects the metabolic activities of the cells themselves: it is enriched in those products, as well as in some substances presently undefined (cf. Sakiyama, 1980), that promote the transfer of substrates and enzymes in active form, to other cells at physiologic temperatures even if the latter are fixed by glutaraldehyde-suggesting some facilitated means of transfer. Such transfers were not observed when the conditioned medium was boiled; transfer was drastically diminished by treatment of the medium with trypsin or detergent (Sakiyama, 1980). It has already been developed in preceding sections of this article that extrusion of lysosomal enzymes in diffusible form from cells activated by surface recognition of effector is a generalized phenomenon. While analysis of the physiologic effects on the surrounding macromolecular environment has emphasized limited hydrolytic attack, there are also clear implications for the more drastic degradation associated with pathologic processes (Vaes, 1980; Keiser, 1980; Barrett, 1981; Dingle and Saklatvala, 1981; and many others). indeed, surface remodeling by enzymes delivered into the pericellular environment has powerful implications in a wide range of recognition and signal-propagation events (cf. Weiss, 1967), even if the precise nature of the primary recognition is unknown, as in the case of pattern formation and organogenesis (cf. Hay, 1981b). Such factors are the more critical in light of the postulated role of the cellular surface receptor for fibronectin, the universal glycoprotein of basement
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membranes and extracellular matricies, in intercellular behavior (cf. Yamada et al., 1980). Fibronectin is a sensitive and selective substrate for degradation by tissue proteinases (Vartio et al., 1981). Accordingly, its degree of integrity may provide significant modulatory control over cellular interactions with neighboring cells (e.g., Virtanen et al., 1982). However, agonal interactions at cell surfaces are not the exclusive means of provoking delivery of extracellular hydrolytic potential from sequestered form. Indeed, focal contacts between cells also provide the requisite signals. Thus, an apical membrane aminopeptidase, apparently of microsomal origin, appears precisely at the sites of cell-cell contact in cultured kidney epithelial cells (Louvard, 1980; cf. also, Paul et al., 1979). The fact that redistribution of cell surface receptors for lectins was induced in embryonic muscle cells of Xenopus by manipulation to contact (Chow and Poo, 1982) bears significantly upon the likely precedent events-some form of membrane perturbation. Such focal interactions between neighboring cells are essential for induction of gene expression during differentiation of Dictyostelium discoideum (D.D. Blumberg et al., 1982). The precise contributions of this means of cellular communication in normal development and in transformation (cf. Birchmeier, 1981) will be addressed briefly in a later section. The macrophage constitutes a classic case of contact-initiated cellular and transcellular interactions. It is not only a question of activation of the macrophage by a surface contact-mediated event, but also, as anticipated by Metchnikoff (1905) and by Ehrlich (1900), conditioning of the medium in which macrophages are cultured, with repercussions to neighboring cells (cf. Collins, 1979). Release of hydrolases of both neutral and acidic pH optima from mononuclear phagocytes under such circumstances is now very clearly documented (cf. Davies and Bonney, 1980). It is notable that enzymes of the cytosol such as lactate dehydrogenase fail to be secreted under the same provocation, thus indicating selectivity of the response. Moreover, proinflammatory stimuli elicit, while inert particles fail to provoke, hydrolase release (Davies and Bonney , 1980). These events may have far-reaching effects in promotion of angiogenesis (cf. Unanue, 1981), a situation which has certain parallels to early hormone action (cf. Szego, 1971a). It is significant that a complex of hydrolases is secreted by activated macrophages that, in concert, is capable of cutting a wide swath among the components of extracellular matrix (cf. Jones and Scott-Burden, 1979). Moreover, some of these enzymes enhance, while others suppress, the reactivity of neighboring lymphocytes toward mitogenic stimuli (Ryzewska et al., 1981; cf. also, Novogrodsky et a/., 1982). Although numerous unsolved problems exist concerning macrophage interactions with other cells, especially those of tumors (cf. Van Furth, 1977; Unanue, 1981), it is not our intention to consider these here. What is of direct relevance to the thrust of the present contribution is an inquiry into the means of information transmission, and that, in
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turn, beyond the immediate mechanism suggested by cell surface remodeling through selective extrusion of hydrolytic enzymes, with special reference to those of lysosomal origin, into the culture medium. (b) More direct routes of intercellular exchanges. The commonly accepted model of cellular transfer of matkriel, generally as restricted to ions and small molecules, has been the junctional membrane channel (Loewenstein, 1981; Petersen, 1981). This is an appealing concept, in part because in apposed cells, the regions of diminished resistance to electrical communication are subject to modulation in size and frequency by a wide variety of effectors and other environmental signals (see Gilula, 1980, and citations therein). However, the permeability of cell-to-cell membrane channels in mammalian cells is strictly limited by probe size and electronegativity (cf. Flagg-Newton er al., 1979). In light of the growing recognition of the contribution of the plasmalemma per se, in vesiculated form, to entry of macromolecules into affected cells, it might be instructive to examine a corollary: the possibility that macromolecules and, indeed, organelles, may be donated in an active process, through formation of transitory intercellular bridges as a result of effector-provoked membrane instability in a given region. Indeed, needle-like projections from one cell to another have been identified in a limited number of instances in which organellar exchange is a striking common feature. These examples include pigment transfer between neighboring melanocytes and between the latter and keratinocytes in cell cultures of guinea pig skin (Klaus, 1969). This process, which occurs in the absence of detectable extracellular melanin granules and is thus implicitly a cellto-cell transfer event, may have a counterpart in the granulosa-cell-to-ooyte delivery of vesicular organelles with attributes of lysosomes observed by cinemicrographic methods by A. Lopata, J. R. Fonseca, and C. M.Szego (unpublished observations), through such transitory intercellular bridges as have been described by others (Espey and Stutts, 1972; cf. also Bendich et al., 1967). These phenomena may complement the gap-function means of modulation (cf. Gilula et al., 1978) in the cumulus-oocyte complex. Likewise, phase-contrast photomicrographs of mouse peritoneal exudates in short-term culture present numerous similar examples of intercellular bridges, replete with lysosome-like organelles, between macrophages and neighboring lymphocytes (R. M. Ezzell, unpublished observations). While the macrophage may appear to be a “special case” in the immunologic context (cf. Toge er al., 1981), there are now sufficient numbers of examples from additional cell types to indicate that transitory intercellular bridges, through which microscopic vesicles as well as macromolecules may pass within very brief times, may be a more general means of cell-to-cell communication than has been envisioned. Indeed, as noted in a previous section, 70-nm vesicles, followed with the aid of electron microscopic tracers, were found to form on one side of mouse heart endothelial and diaphragm mesothelial cells and, within 3-5 seconds, to fuse with the cell mem-
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brane on the contralateral side (Casley-Smith and Chin, 1971). Although these data relate to vesicular dyanmics in the inrrucellular context, the order of time, the indications for polarity, and, above all, alternate vesiculation and membrane fusion may possess implications at the inlercellular level as well. In consideration of the available evidence for exchange of materials between cells, it is advantageous to recognize the significance of vesicular encapsulation in promoting the efficiency of such transactions. For example, while receptormediated uptake of purified lysosomal enzymes in soluble form may occur to a limited extent, even in genetically deficient cells (reviewed in Sly, 1980; cf. also, Section II,B), it is now possible to visualize direct cell-to-cell transfer of lysosomal constituents. Thus, translocation of lysosomal organelles per se from cytotoxic macrophages into the cytoplasm of tumor cells has been identified by time-lapse cinemicrographic procedures supplemented with immunologic and EM methods (Bucana e t a / . , 1976). In turn, rapid exocytosis of intact lysosomes has been reported from PMN-leukocytes exposed either to micromolar concentrations of the ionophore A23 187 or to niillimolar levels of extracellular calcium (Moore et a/., 1979). Similar observations on exocytosis of intact lysosomes from chicken skeletal muscle after chloroquine treatment in vivo were made by Stauber et al. ( 1 98 1 >.That such extruded organelles may be internalized by fusion with membranes of neighboring cells is clearly a possibility. Thus it is not too far a cry from such observations to the transcellular acquisition of biochemical markers, shed or presented in vesicular form (cf. Doyle et al., 1979; Rando and Bangerter, 1982) between cocultivated cells, even when the two cell classes are heterophylic (e.g., Doyle et ul., 1979), through uptake of vesicles in vivo. The success of liposomes as a means of introducing material into cells gives striking evidence of art imitating nature in this respect (cf. Papahadjopoulos, 1978; Celis et al., 1980). However, even more direct data arc now available, indicating that the lysosomal enzyme P-glucuronidase is acquired by deficient fibroblasts from direct cell-to-cell contact with normal lyphocytes rather than by mere fusion with shed extracellular vesicles (Olsen et al., 1981). Are the foregoing indications for exchanges of lysosomal products between cells subject to regulation by well-characterized effectors? And are such transfers of direct relevance to the functions of the recipient cells'? If so, how? In the remaining sections of this article we provide some suggestions, clearly speculative and requiring rigorous testing, of the potential significance to the life of the cell of the controlled passage of critical macromolecules in vesicular form across its boundaries. First, as to regulation of such passages, a few examples may suffice: Mattson had demonstrated that ACTH elicits in cultured murine adrenocortical tumor cells striking extensions of microvillar processes between neighboring cells. These processes are crowded with lysosomes in orderly longitudinal arrays suggestive of participation of microtubules (Mattson and Kowal, 1982). It will be
LYSOSOMAL FUNCTIONS IN CELLULAR ACTIVATION
21 1
recalled that when cultured cells of murine adrenal and rat Leydig cell tumor lines were treated with ACTH or with CAMP, respectively, the steroidogenic responses were accompanied by redistribution of tubulin, as determined by indirect immunofluorescence, from membrane-bounded granules to organized microtubular form (Clark and Shay, 1981). The granules, which were also enriched in acid phosphatase, had the additional features of appropriate dimensions (0.2-0.6 pm diameter) and sensitivity to structural labilization that were in accord with their probable identity as lysosomes. The potential of organized microtubules for routing of intracellular precursor traffic during steroidogenesis is evident from this and related work (see Table XVIIIA,B). Acquisition of such potential by recompartmentation of the monomeric elements from a reservoir with lysosomal features is indeed an economical means of coupling secondary metabolic responses to an initial receptor-mediated event. However, such a mechanism taken in context with the elegant ultrastructural observations of Mattson and Kowal (1982) is also strongly suggestive of a potential means of signal transmission to that neighboring cell with which the elongated pseudopodium, elicited by progressive microtubule organization (Clark and Shay, 198I ) and further promoted by apparent loss of subplasmalemmal microfilaments (Mattson and Kowal, I982), makes intimate physical contact under hormonal control. Gap junctions formed and, in turn, dispersed under phased hormonal control may also serve to couple the metabolic responses of neighboring cells, e.g., in oocyte maturation (Anderson and Albertini, 1976; Gilula et al., 1978). Direct communication seems indicated in the promotion by peritubular cells, added to Sertoli cell culture, of androgen-binding protein secretion by the latter; conditioned medium alone did not suffice (Hutson and Stocco, 1981; Hutson, 1982), while passage of an LHRH factor from testicular Sertoli to Leydig cells in vitro has been indicated (Sharpe et al., 1981). External passage by cell-cell contact of an EGF receptor may represent a corresponding means of cellular coordination (cf. Das et al., 1981). Indeed, it is implicit in the degree of metabolic cooperation and the coordinated, occasionally sequential (cf. Korach and Lamb, 198I), responsiveness of neighboring cells that information in the form of macromolecules (cf. Meda et al., 1982) and, potentially, vesicles(?) is being exchanged. The partial coupling of the cell cycles to those of neighboring imaginal disc cells may be a further illustration of such phenomena (Adler and MacQueen, 1981) that would bear direct investigation. Cell-cell channel formation in uterine smooth muscle cells is shown to be elicited by liposome-encapsulated mRNA for gap junction protein (Dahl et al., 198 I ) . Other means of manipulation of cellular interactions, both pro and con, include application to the cultured cells of antisera to the proteins shed by mouse mammary carcinoma (Damsky et al., 1981). Increase in size and number of gap junctions between pancreatic p cells during stimulation of insulin secretion (Meda et al., 1979) has been correlated with the insulin content of the respective cells (Meda et al., 1980). These randomly
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selected illustrations serve to indicate the plasticity to a variety of challenges of the membrane barriers intervening between cells. In light of some of the above indications for developmental effects, it is not too surprising that evidence for cell-to-cell passage of vesicular components during embryonic differentiation is already available. Thus, Cunha and co-workers, who have studied extensively the role of tissue interactions in morphogenesis and cytodifferentiation of female urogenital organs, present ultrastructural evidence strongly suggestive of active exocytosis of vesicular material from stromal cells, accompanied by vesicular uptake at the epithelial cell interface, in neonatal mice, with or without estrogen treatment (Cunha and Lung, 1979). In support of the heterotypic transfer of vesicular material critical to epithelial development in the known direction of inductive stimulation (from stroma to epithelium), these workers undertook morphometric analysis of the spatial distribution of such vesicles and found a gradient of the appropriate polarity (Cunha and Lung, 1979). While the nature and source of the vesicular contents are unknown, the findings are indeed provocative and may have their counterparts in additional developmental processes, including application to the role of the androgenresponsive mesenchyme in the induction of prostatic epithelium (Lasnitzki and Mizuno, 1979; cf. also, Cunha et al., 1980). In turn, androgen sensitivity is elicited by induction of receptors to testicular hormones in mesenchyme as a result of epithelial interactions (Heuberger et al., 1982). The reciprocal interaction in the mesenchymally induced formation of androgen sensitivity of epithelium has also been identified (Cunha et at., 1980).
IV. Selected Cellular Functions Subject to Lysosomal Influence A. CELLDEATHAND SOMEANOMALIES OF INTERPRETATION In view of these relatively recent developments, and in consideration also of the profound increase in lysosomal numbers and activities during hormonally regulated tissue remodeling of the more drastic kind, as in the development of cells at expense of the destruction of others in insect metamorphosis (Lockshin and Beaulaton, 1974a,b; van Pelt-Verkuil, 1979), it is instructive to review briefly still earlier indications of profound lysosomal dominance in such developmental processes. The early observation of Hamilton and Teng (1965) on the probable lysosomal source of the Mullerian inhibitory factor that is responsible for destruction of the embryonic female generative tract, a regressive process later recognized to be under the control of fetal androgen in the male genotype, is a case in point (cf. also, Wolff, 1959). More recently, it has been shown that extracts of newborn calf testis enriched in Mullerian regression factor possessed cytotoxicity toward human ovarian cancer cells (Donahoe et ul., 1979). Like-
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wise, the destruction of embryonic epithelial mammary gland buds by the underlying mesenchyme under androgen influence (Kratochwil and Schwartz, 1976) may well have a lysosomal feature not hitherto identified. Thus, cell death under unmitigated hormonal control (cf. Schwartz and Truman, 1982; Moulton, 1982) is a phenomenon with the more familiar ring of the original “suicide-bag’’ concept of lysosomal function. There are as many unresolved questions on the precise molecular mechanisms associated with cell death (cf. Berlin et al., 1978b) as there are on those related to cell growth. The highly specialized subject of natural killer cells and their environmental regulation will not be considered here, although it is tempting to do so, in part because of the crucial role of surface recognition as the primary and discriminatory step in the initiation of a staged effector-mediated process. Moreover, in the immunologic context, there are some striking, but hitherto isolated (cf. Loor, 1981), examples of transfer of “small vesicles” at regions of deep penetration of killer pseudopodes into the target cell cytoplasm (Koren et al., 1973; Adelstein et al., 1976; cf. however, Sanderson and Glauert, 1979). Nevertheless, the biochemical mechanisms thus far identified clearly implicate lysosomal functions in the natural killer cell system (e.g., Roder et al., 1980; Hart, 1981, 1982; Om et al., 1982). Indeed, the recent demonstration of peroxidase activity in lysosomes (Bursztajn and Libby, 1981) may have a bearing on peroxidative mediation of some cidal effects (Hart, 1982; Klebanoff et al., 1982). Modulation of the latter may likewise be a function of relative availability of lysosomal superoxide dismutase (cf. Geller and Winge, 1982). What will be emphasized, instead, are some observations, interpreted in the first flush of enthusiasm for hormonal control at the transcriptional level, which led to the premature conclusion that RNA and protein “synthesis” were obligatory antecedents to cell death. It is instructive to review these ideas in the clear light of historical precedent. In the mid-l940s, White and Dougherty demonstrated a striking involution of lymphoid tissue, together with profound depletion of lymphocytes in the circulation, in rodents treated either with ACTH or with milligram doses of glucocorticoids (reviewed in White, 1949, 1950). Concomitantly, there was a rise in plasma amino acid and elevation in urea nitrogen, suggestive of accentuated catabolism of the proteins so clearly mobilized from the lymphocyte germinal centers. Histologic examination of the latter revealed that the lymphocytes were in all stages of cellular breakdown-from “foam cells” to naked nuclei. Indeed, the lymphoid tissue hypertrophy in adrenalectomized animals was viewed as withdrawal of the tonic mobilizing activity of endogenous adrenocortical function. Somewhat later, Roberts (1953) demonstrated that the effects of adrenocortical hormones on nitrogen mobilization were tissue-specific and biphasic, with inversion of the catabolic dominance at low doses. These latter findings helped to reconcile the earlier data with the puzzling requirement for “permissive” levels
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of adrenocortical hormones in the promotion of growth in young, and in niaintenance of nitrogen equilibrium in mature animals. However, such relatively “gross” observations at the whole animal level were rapidly eclipsed in the wave of enthusiasm for analyses of adrenocortical steroid hormone action at the genomic level. Such was the impact of the operon, a concept that was revolutionizing microbiology. Accordingly, and generally prematurely (see Section II,A), its implications were widely accepted as directly applicable to eukaryotic cells as well. Unfortunately, however, the experimental base was flawed, for highly toxic inhibitors were utilized to “block specific DNA-dependent KNA synthesis” in experiments to determine whether effectors could elicit their costomary metabolic influences either in isolated cells (in which severe membrane damage and death ensued) or in whole animals (ditto). To the astonishment of many, application of the inhibitors themselves, in the absence of putative effectors, elicited, per se, “paradoxical induction” of the very proteins whose synthesis was subject to agonal control. It is by no means outside the scope of this review to consider the evidence for lysosomal participation not only in the programmed consequences of surface receptor interactions with specific effectors, but also in those cases of “paradoxical” induction of given proteins that have been generally viewed as anomalous responses to a variety of relatively toxic substances (Table XXIII). In the case of the latter phenomena, some intriguingly discordant explanations have thus far been put forward (Tomkins et al., 1972; Palmiter and Schimke, 1973; Kenney et al., 1973); none of these has fully explained the observations. The purpose of presentation of the assorted data in Table XXIIl is not to raise a straw man. Indeed, the gross membrane damage and generalized toxicity elicited by these several and many other “specific” inhibitors of protein and nucleic acid “synthesis” have long been recognized [e.g., Waksman et al., 1941; Robinson and Waksman, 1942; Hackmann, 1954; Philips et al., 1960; Gale, 1963; Harris and Sabath, 1964; Revel et al., 1964; Korn et al., 1965; Lippe and Szego, 1965; Szego and Lippe, 1965; Greif et a/., 1965; Laszlo et al., 1966; Soeiro and Amos, 1966; Spaziani and Suddick, 1967; Weinstock, 1970; Miles, 1970; Verbin e t a / ., 1971; Schwartz, 1973 (cf., however, Jones ef al.. 1974); Pater and Mak, 1974; Meller el al., 1974; Sturgess et ul., 1975; Mitranic et al., 1976; and Kellokumpu-Lehtinen and Tuohimaa, 19781. Moreover, the secondary effects of antibiotics upon metabolic functions, including those related to plasmalemmal, Golgi, EK, and mitochondria1 activities, may be attributable to relatively gross primary effects upon lysosomal organelles, accompanied by massive, but sublethal, “spilling” of hydrolase content. It has long been recognized that lysosomes selectively accumulate a wide range of exogenous substances, both organic and inorganic, soluble as well as particulate (cf. Allison, 1968). In keeping with this property, there is found among cultured rat fibroblasts an extraordinary degree of uptake and accumulation of aminoglycoside (Tulkens and Trouet, 1978) and
TABLE XXlII SELECTED EXAMPLES<’ ILLUSTRATING “PARADOXICAL” INDLICTION OF SPECIFIC PROTEINS B Y 1NHlBlTORS OF MACROMOLECULAR SYNTHESIS A N D OTHER TOXIC CHEMICALS APPLIED in VitrO TO ISOLATED CELLS A N D TISSUES Agent
Object
Observations
Interpretation
Reference
Elaborate model of a putative, labile repressor molecule which reversibly inhibits the synthesis of mRNA for TAT
Tomkins et a!. (1966, 1969, 1972)
Blockade of production of postulated repressor(s) which prevent accumulation of stable, active template
Actinomycin D (AMD); 5-fluorouracil; 5-fluorouridine; mitomycin C
Rat hepatoma (HTC) cells
Mithramycin: daunomycin
Rat HTC cells
After withdrawal of steroid hormone inducer, 5 Kliml AMD in a dexamet hasone-preinduced culture containing 5 x 105 HTC cellsiml elicited augmented concentration of tyrosine aminotransferase (TAT; immunoprecipitation) and in its rate of labeling from a [3H]amino acid mixture, without change in enzyme degradation Induction of TAT
Cytosine arabinoside; cycloheximide followed by AMD
Embryonic chick retina in culture
Induction of glutamine synthetase
Unpublished data cited without comment by Martin e? al. (1969) Moscona et al. (1970)
(continued)
TABLE XXIII (Continued) Agent
N m
AMD
AMD
Object
Observations
Immature chick oviduct magnum explants in culture
‘‘Superinduction’’ of
Cultured HTC cells under “full growth conditions” (Eagle’s medium with 4 X the usual concentration of amino acids and vitamins, and with 10% fetal calf serum)
Severely and progressively decreased rate of degradation of prelabeled, “superinduced” TAT in face of striking inhibition of incorporation of [3H] leucine
ovalbumin, conalbumin, and lysozyme on addition of drug, without increase in total mRNA; increased size of polysomes and enhanced rate of elongation
Interpretation
Reference
“Under conditions of inhibi- Palmiter and Schimke (1973) tion of new transcrip tion,” preferential survival of long-lived -As, which are then capable of increased translation on the basis of more favorable competition for rate-limiting initiation and elongation factors Kenney et al. (1973) Effects of AMD believed inconsistent with Tomkins model
AMD
Immature chick oviduct
AMD. ethionine. thio-
Iinmature chick oviduct
acetamide
1.J
4
Induction of avidin by 16 hours after AMD; equivalent to the maximal level elicited by hormone (progesterone): cumulative with mechanical damage (cf. Heinonen and Tuohimaa, 1976, 1979 in Table 1) Induction of ovalbumin and conalbumin synthesis in estrogen-primed and -withdrawn chicks; concomitant elevation of serum progehterone; celiular changes characteristic of hormone administration
Uncertain
Elo et a/. (1975)
Uncertain
Sharma er al. (1976); Sharma and Borek (1977); Sharma (1978)
"A far more extensive series of examples in the bame context for .4MD. as w d as for an array of antibiotics other than the latter, and including, but not limited to. cycloheximide. puromycin, rifampicin. mitomyciii C , mithramycin. and daunomycin, applied in some cases in vivo, was presented by Tomkins and co-workers (1972).
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many other antibiotics. Associated with this process is structural labilization of the bounding membranes of lysosomes as, for example, those of rat liver after exposure in v i m to aflatoxin B1 (Pitout and Schabort, 1973), skeletal muscle after chloramphenicol (Nakahara, 1974), and kidney following gentamicin (Leseur et al., 1975). These events, which may be attenuated by appropriate concentrations of lysosomal membrane stabilizers such as chloroquine (Leseur et a l . . 1973, are accompanied by abrupt release of lysosomal enzymes, as for example, cathepsin D from cultured limb-bone rudiments exposed to cyclohexamide (Hille et ul., 1970). Such processes appear to underlie the accumulation of inflammatory exudates concomitantly with mobilization of leukocytes and release of histamine and acid hydrolases into the peritoneal and pleural cavities of rats given puromycin (Spaziani and Suddick, 1967) or actinomycin (Spaziani and Suddick, 1967; Giri el al., 1975). Moreover, exposure to such antibiotics is accompanied by profound depletion of identifiable lysosomes and impairment of their new formation (StGplewski and Waronski, 1963; Ahearn er al., 1966; Gordon and Cohn, 1973; Lockshin and Beaulaton, 1974b). Thus, it is not surprising that exposure of cells to such powerful antibiotics results in impairment of processcs attributable to the unimpeded function of lysosomes, such as proteolysis of cytosol proteins of organ-cultured heart (Wildenthal and Griffin, 1976), rat liver (Grisolia et a l . , 1977), and rat embryo fibroblasts (Amenta et al., 1978). These observations notwithstanding, some investigators profess to remaining puzzled by the exaggeration by antibiotics, such as cycloheximide, puromycin, and actinomycin D, of membrane-destabilization phenomena (cf. Nickols and Brooker, 1980), and, by the same token, curtailment by inhibitors of RNA and protein synthesis of events related to stabilization of cell membranes (cf. Danon and Assouline, 1978; Tsurufuji et al., 1979; Russo-Marie et al., 1979). Accordingly, and in the face of the profound disruption of normal processes in the presencc of toxic antibiotics, it is not too surprising that “specific inhibitors” of protein and RNA synthesis, whcn presented to isolated cells and tissues that are normally hormone-sensitive, prior to or coticomitantly with the tropic agent, prevent expression of the hormone response (see refs. 1-5 in Szego and Lippe, 1965). Nevertheless, there persists a strong line of investigation, the data from which are interpreted to suggest that a cell destined to be killed by (excess) adrenocortical steroid must ,first (because steroid hormones are still widely presumed to act essentially exclusively at the genomic level) synthesize a specific mRNA that is then translated into a protein essential for the cell death response (reviewed in Hurley, 1978; Wyllic et al., 1980; Gehring, 1980; Bourgeois and Newby, 1980). Such observations are not confined to glucocorticoids. Thus, at concentrations greater than 2 X 10- M , estradiol and active congeners, naturally occurring and synthetic. are growth inhibitory (Sairam and Berman, 1979; Wychc and Noteboom, 1979; Gay and Hilf, 1980), interfere with membrane
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transport functions (Gay and Hilf, 1980), and are also cytotoxic (Gay and Hilf, 1980; Lippman et al., 1976; cf. also, Breslow et a / . , 1979). Moreover, CAMP excess, a condition that is known to be associated with lysosomal hyperactivation (Szego, 1972a, 1975), is similarly damaging to given cells (Gehring and Coffino, 1977), whereas in physiologic concentrations it is growth promoting (cf. Whitfield et al., 1970). That variant cell lines deficient in receptor for the steroid are also resistant to its lytic effects is taken as support for the hypothesis. However, beyond, this, potential deficiencies in the numerous secondary steps in the chain of events leading to cell death have not been demonstrated (cf., however, Gasson and Bourgeois, 1983). There is presently a rich area for investigating in this context possible membrane defects, more specifically, those related to occurrence of reduced numbers or abnormal composition of lysosomes, including problems related to their fusion with endocytotic vesicles bearing hormone-receptor complexes and the failure of requisite processing. Moreover, defects in a possible microtubular translocation mechanism, comprising inadequacies in tubulin assembly or in associated proteins, including nucleotide phosphatase(s), could lead to deficient nuclear transfer of the agonal-macromolecular complex. Nevertheless, none of these potential control points, singly or in concert, can account for the orderly progression of cells through their normal attrition cycles, such as those in the luminal epithelium of the mouse uterus (cf. Finn and Publicover, 1981), in which death of a given proportion of cells occurs at a specific stage of their growth pattern, as programmed by endogenous hormones. Despite these conceptual limitations, there are growing indications that the lysosomal contribution to cell death has not been entirely overlooked. For example, Cidlowski and colleagues have reestablished the link between the early work on glucocorticoid-induced lyphocytolysis and its correlates in protein degradation. The nature of the proteolytic activity rendered accessible and/or induced by glucocorticoids in mediation of cell growth (at low concentrations) or cell death (at excessive levels), is consistent with properties of lysosomal cathepsins (MacDonald et al., 1980; MacDonald and Cidlowski, 1981a,b, 1982; see also, Hopgood et al., 1981; cf. however, Mayer et al., 1982). Moreover, the likelihood of concerted “spilling of packets of lysosomal enzymes” is in accord with the evidence for enhanced RNA degradation in lymphocytes treated with high levels of glucocorticoids and destined for cell death (Cidlowski, 1982). More indirect indications are available for accentuated catabolism of mRNA for collagen in additional tissues exposed to excess glucocorticoid (Rokowski et al., 1981; Oikarinen and Ryhanen, 1981) or to interferon (WCrenne and Bartholeyns, 1977). And, despite assertions that lysosomal activation was not a factor, the dose-dependent increase of an incompletely characterized endonuclease activity in glucocorticoid-treated rat lymphocytes (Wyllie et al., 1980) is compatible with the present indications for a wave of lysosome-generated catabolic activity
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that precedes cell death in response to excessive levels of these hormones. Indeed, concerted proteolytic and endonucleolytic activities, of unspecified intracellular origin, as well as promotion of nuclear lysis (Giddings and Young, 1974), have been identified as effects of substantial levels of glucocorticoids in lymphoid cells. Parenthetically, it should be noted that the biphasic character of the metabolic correlates of these hormones, including their function in the promotion of cell growth at appropriately low concentrations (cf. Whitfield rt of., 1970), has often been overlooked when the emphasis has been placed exclusively on their capacity to provoke cytolysis (e.g., Fig. 1 in Bourgeois and Newby, 1977). In light of the foregoing, it may now be relevant to focus once more upon the cytotoxicity of untoward environmental stimuli and their capacity to provoke graded lysosomal instability (Table I). Indeed, in view of the known responsiveness of the lysosome system to such insults as hyperthermia (cf. Magun, 1981; Keech and Wills, 1981), it seems entirely possible that the cell-cycle synchronizing effects of heat shock may result from coordinated death of cells at those stages of their growth cycle at which their surface membrane-lysosomal membrane continuum is most vulnerable, leaving behind a “stem” population that is heat shock resistant for reasons that require systematic investigation. In this context, the “heat shock proteins” rendered available and, secondarily, induced, in sensitive cells by the thermic stimulus bear a degree of qualitative similarity to those of the lysosomal matrix (cf. Horton, 1982). The dose dependency of lysosomal destabilization in relation to magnitude of the stimulus, whether receptor mediated or “nonspecific,” is a key to further analysis of distinctions between growth and proliferative responses or cellular disaster. As has been outlined above, redistribution of lysosomes and accessibility of their astonishingly varied contents to formerly inacessible sites possess the deepest possible significance for communication of molecular information in course of tissue remodeling during differentiation-from embryonic stages onward, as well as in relation to cell death and the concomitant macromolecular demolition and salvage pathways. In the remaining space, we shall examine some additional selected cellular functions subject to lysosomal influence, with particular reference to patterns of lysosomal distribution and access congruent with the ordered participation of these organelles in cell growth and proliferation.
B . CELLGROWTH AND PROLIFERATION I . Primary Observations on Lysosomal Participation Long before more sophisticated analytical methods became available, there were circumstantial observations linking lysosomal function to preproliferative activities in small lymphocytes. These seminal findings included the now classical demonstration by Allison and Mallucci (1964b), rapidly confirmed by
LYSOSOMAL FUNCTIONS IN CELLULAR ACTIVATION
22 1
Hirschhorn and collaborators ( 1965), that lysosomal membranes become more permeable and that the organelles are strikingly reduced in number at critical times preceding mitosis in those cells stimulated to proliferate by a variety of exogenous stimuli. These stimuli shared the significant property of being recognized by what we have come to learn are surface oriented macromolecules of specific topologic complementarity-receptors, by definition-whose engagement by ligand leads to a staged sequence of metabolic and structural alterations culminating in cell division (see Table V). Some of these early observations, in which lysosomal membrane destabilization was the common denominator, had counterparts in other types of cells characterized by intense proliferative activity, such as hepatocytes during liver regeneration (Adams, 1963; cf. also, Kent et a l . , 1965; Becker and Lane 1965, 1966) and HeLa cells (Robbins and Gonatas, 1964). Agents promoting stability of lysosomal membranes, such as appropriate concentrations of glucocorticoids, were inhibitory, whereas excess vitamin A, which is known to reduce lysosomal membrane integrity, also serves to stimulate mitotic activity of cultured fibroblasts (cf. Allison and Mallucci, 1964b). These and related observations have since been extended and summarized (Barka, 1974; Szego, 1975; Isanin and Yakovlev, 1977; Fiszer-Szafarz and Nadal, 1977). Clusters of lysosomes were especially conspicuous in cultured thyroid epithelial cells undergoing mitotic activity in response to TSH (Nitsch and Wollman 1980). The direct causal relationship, if any, between preproliferative events and enhanced accessibility of lysosomal components to other parts of the cell remains undefined. Nevertheless, the combined observations are “consistent with the view that release of lysosomal materials [in limited amountsI2 into the cytoplasm [and the nucleus]’* acts as a trigger initiating division in a cell that is ready for it, ’’ I3 2 . Search for “the” Transduction Mechanism More recent emphasis on potential means of signal transmission from the cell surface into the still murky cell interior, especially that of the nucleus, has focused on proximate common surface events that appear to serve as early correlates of the mitogenic reaction sequence. These events can be grouped into two major categories that are not in themselves necessarily independent. Indeed, both types of motogenic signals would appear to arise from surface membrane deformation: communication of the structural change in a direct biophysical manner, Fossibly to the cytoskeletal apparatus, on the one hand, and, on the other hand, intervention of a parameter in which some form of metabolic cascade is triggered by cellular accessibility of a key biochemical intermediate in con’2lnwrtions ours. See also Section ITI,D,4,a(5) ’.‘Allison and Mallucci (1964b)
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centrations normally limited by basal membrane status. Circumstantial data are available to implicate augmented lysosomal function in relation to either postulate. Indeed, by the rationale provided in the preceding sections of this article, each of these ostensibly independent parameters may be part of a larger continuum. a. Physical Transduction. There are cogent reasons to implicate the cellular scaffolding in the transmembrane propagation of signals impinging on the cell surface. Not only are these structural elements physically linked or in close contiguity to the cell surface (see Table XVIIA), but they also provide a communicating network anchoring the latter to the nucleus (DuPraw, 1968). What is even more significant, the microtubules and microfilaments constitute a highly poised system capable of rapid rearrangement, pro and con, in response to the events associated with generation of cell surface perturbation (see Tables XI and XVIIIA,B). Accordingly, it is not too surprising that changes, for example, in the polymerization/depolymerization of microtubules, which occur within very brief time intervals after cell surface capture of appropriate ligand, have been implicated in transmembrane control leading to mitogcnesis (cf. McClain and Edelman, 1978). A corollary of this general concept is that centrosomal separation, secondary to Ca2 influx, calmodulin activation, and microfilament contraction, possibly also associated with microtubule depolymerization (cf. Brinkley et al., 1981), is an obligatory antecedent to EGF-induced mitogenesis (Sherline and Mascardo, 1982b). Moreover, microtubule-disrupting drugs enhance the effect of growth factors on DNA synthesis (Otto et d.,1979; Crossin and Carney, 1981). However, additional work has demonstrated that microtubule integrity can be associated with expression of either negative or positive controls on cell growth, depending on the degree of confluence and lineage of thc cells in culture (McClain and Edelman, 1980). By mechanisms not yet identified, the ubiquitous hormone, somatostatin, inhibits both centrosomal separation and nuclear incorporation of labeled thymidine induced by EGF (Mascardo and Sherline, 1982). Moreover, there is growing evidence that the state of centriole ciliation is related to quiescence vs commitment to DNA synthesis and proliferative activity in nonneoplastic vertebrate cells. Thus, resorption, reappearance, and, finally, loss of the central cilium occur in distinct phases in quiescent cells stimulated to mitosis by exogenous factors (Rieder et al., 1979; Tucker et al., 1979; Rambo and Szego, 1983). The initial disappearance of the central cilium is astonishingly rapid in luminal epithelial cells challenged with EZPin vivo (sce Fig. 13A-D), as is its reppearance during the early preproliferative period (Rambo and Szego, 1983). These enigmatic, “precocious” events (cf. Wheatley, 1982) precede by many hours the eventual S phase of cellular activity. The intimate relationship among centriolar separation, the diplosomes with their surrounding amorphous components, and the microtubular system, the latter, in turn, subject to nuclea+
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tion, is now well documented (Wheatley, 1982). However, the implications of such a dynamic network in propagation/amplification of an agonal signal remain elusive. Still, one can speculate that the sharp flux of critical ions, especially calcium, from intra- as well as extracellular origins, including the lysosomal (see refs. in Section II,B and Table XI), the swift accessibility from vesicular, if not lysosomal, sources of a reservoir of tubulin (Clark and Shay, 198 I ) , of calmodulin (e.g., Linden et al., 1981), of protein kinase(s) (Collins and Wells, 1982), and of the counteractive phosphatases and other highly stringent enzymes capable of effecting precise macromolecular modifications (Szego, 1975), can, in concert, elicit the altered intra- and pericellular environment that provokes kinetic activity-especially when reinforced with catalytic concentrations of phospholipids and cyclic nucleotides on membrane activation. It will have become evident in course of the preceding sections of this article that lysosomes or similar cell components may serve as the source of a considerable number of the above factors. Delivered in concert by mechanisms related to cell surface destabilization, they are surely capable of triggering the mechanoeffector reactions in question. b. Biochemical Transduction. Proposals abound on what is limiting in the early commitment of cells to a preproliferative state. Whether the decisive signal is availability of critical ions (Rubin and Koide, 1976; Whitfield et al., 1979). nutrients or growth factors of unspecified nature (cf. Holley, 1975; Barsh and Cunningham, 1977), which are apparently capable of being transferred to quiescent cultures in particulate-free media conditioned by growing cells (cf. Jazwinski et al., 1976; Floros et al., 1978; Meats et al., 1980; see also Smith and Stiles, 1981), or the correct "mix" of cyclic n u c l e o t i d e ~ 'or ~ some or all of these, is still uncertain. These sporadic observations have a common theme: membrane alterations leading to enhanced access of hitherto relatively unavailable substances of potential interactivity . Indeed, the biochemical and the biophysical signals to eventual mitogenesis may not be mutually exclusive, as noted above, but, in fact, different parts of the same elephant. For, underlying these truly varied means of signal transduction leading to growth commitment is the common mechanism of reorganization of the cell surface with concomitant alteration of its permeability. In cells responsive to many hormones, this mechanism is reinforced with an increase in blood flow that occurs as one of the "Tyclic AMP has had a chequered career as a growth regulator, not only pro (e.g., Whitfield et al., 1979; Pawelek, 1979; Rozengurt e r a / . , 1981b) and con (Pastan and Willingham, 1978; Martin and Kowalchyk, 1981). but also deemed irrelevant thereto (Coffey ef a/., 1978; Oleinick er a / . , 1981). Some of these inconsistencies may be resolved by the recognition that the physiological concentrations of the nucleotide (- lop8 M ) elicit lysosomal activation, whereas excess CAMP provokes undue destabilization of lysosomal membranes with resultant escape of toxic concentrations of contents (reviewed in Szego, 1975). Variability of cell type, especially among the transformed lines investigated in this context, is also a clear factor in contribution to these diverse results.
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primary events recognizable across a variety of cell types that are stimulated by mitogens of widely diverse character. The factors that are capable within such short order as seconds to expand the local microcirculation (Szego, 1965) include liberation of local stores of biogenic amines from membrane-bounded organelles akin to lysosomes, and also production of vasoactive peptides from inactive precursors by limited proteolysis in situ (Fig. 24): the very essence of the lysosoma1 cascade. Since surface reorganization, a process generally recognized to possess important but incompletely understood influence in growth control, likewise depends upon limited attack upon preexisting macromolecules by stringent enzymes translocated or unmasked (cf. Table XIV), the potential of such processes for altering cell cycle status will serve as the remaining focus for this section (cf. Cunningham, 1981). Clearly, lysosomal potcntial in this context cannot be overlooked. However, neither can it be emphasized sufficiently that all of these proposals on what is limiting in agonist-induced growth share one
I ESTROGEN
GENERALIZED CYTOSTRUCTURAL REORIENTATION THROUGH RECEPTOR INTERACTION
M ITOCHONDR IAL MEMBRANE(S1
i
PER INUCLEAR MIGRATION INCREASED PERMEAB I L l T Y
RELEASE OF
IE;ERsoAMNA.:
(
RELEASE OF MULTIPLE HYDROLASES
CASCADING OEPOLYMER IZATION
PLETHORA OF SYNTHESIS. . . . . . . .
.-/
ACIDIC INTERMEDIATES
L 1-
. . . . . . . .SYNTHESIS
FIG. 24. Working hypothesis for the propagated effects of estrogen in specific target cells. See text for supporting evidence. The dotted arrows leading to “Synthesis” are used to denote an appreciable time lag, and include reutilization of intermediates by “salvage” pathways as well as by more indirect routes involving accessibility of substrate from extracellular sources. The potential participation of CAMP, as well as othcr second messengers, is incompletely represcnted (see text and Szego, 1972a). Reprinted with minor modifications from Szego (I97 la), with permission.
LYSOSOMAL FUNCTIONS IN CELLULAR ACTIVATION
225
common defect and, thus, a potential fallacy: the post hoc, propter hoc argument. Nothing will overcome this problem except more work. Limited proteolytic cleavage. This topic has been addressed in several contexts in preceding sections of this article. Therefore, a brief recapitulation will serve to highlight some of the potential means by which scission of preexisting macromolecules by highly selective lysosomal proteases, made newly accessible to strategic cellular sites, can contribute to anabolic and proliferative processes in normal cells. It has long been known that treatment of normal cells under mild conditions with exogenous proteinases leads to alterations in cell surface properties (cf. Burger, 1973; Nicholson, 1976; Noonan, 1978) and a level of cell growth usually seen only in transformed or tumor cells. Although the nature of the coupling of the resultant surface membrane reorganization and the staged responses leading to cell growth remains moot (see above section), there are some striking parallels in endogenous proteolytic function in aspects of normal cell growth and differentiation. Thus, protease activities appear to be involved in slime mold development (Fong and Bonner, 1979), in anabolic events in Neurospora crassa (Cohen and Drucker, 19771, in posthatching maturation of brine shrimp (Garesse et ul., 1980) and in that of reticulocytes (Boches and Goldberg, 1982), as well as in myotube development (McElligott et a/., 198 1) and neurogenesis (Krystosek and Seeds, 198 I ) . Indeed, specific activities of lysosonial enzymes, including cathepsin B, are enhanced during myoblast differentiation (McElligott et ul., 1981). Mitogenic stimulation by exogenous proteases such as thrombin (cf. Chen and Buchanan, 1975) has certain properties in common with the actions of several hormonal growth factors. Thus, thronibin-stimulated cell division is associated with its binding to cell surface receptors (Baker et al., 1979) and with their proteolysis (Glenn and Cunningham, 1979). These properties are reminiscent of the endogenous proteolytic activities intrinsic to, or rapidly unmasked in, cell surface membranes as a consequence of specific binding of EGF (Green and Moore, 1980). Proteolytic activities that arc likewise intrinsic to (Orenstein et ( I / . , 1978) or closely associated with (Calissano and Levi-Montalcini, 1979) NGF, as well as with receptor-enriched membrane fractions for acetylcholine (Verdenhalven et u / . , 1982), have been described. Growth restraint by exogenous protease inibitors lends support to the indications already available of protease activity in promotion of cell growth, both “basal” and exponential (cf. Cockle and Dean, 1982). Among thc most cffective of thcse exogenous substances are the microbial substances, leupeptin (Aoyagi cz ul., 1969) and pepstatin (Uniezawa et a / . , 1970), capable of inhibiting rather selectively cathepsins B and D, respectively (Umezawa and Aoyagi, 1977). These antagonists, especially leupeptin, inhibit liver regeneration (Miyamoto et a / ., 1973) and suppress the mitogenic response of thymocytes to
226
CLARA M. SZEGO AND RICHARD J . PIETRAS
phytohemagglutinin (Saito et al., 1973). Moreover, TLCK (tosyllysylcholormethyl ketone) elicits division delay in sea urchin embryos (Penn et al., 1976). TLCK is effective as an inhibitor of trypsin-like (serine-), as well as sulfhydryldependent, proteinases (cf. also, Table XV). As judged by the effects of further specific inhibitors (cf. Fischer et al., 1975), additional degradative enzymes of lysosomal origin, such as P-galactosidase, may likewise be involved in “triggering of growth of cells that are ready for it” (Allison and Mallucci, 1964b). The chromatin-bound proteolytic activity of rabbit thymus that is effectively inhibited by leupeptin, TLCK, and TPCK (Krueger, 1982) has some intriguing potential correlations with the cathepsin B that is transferred to the nuclear compartment in ligand-mediated fashion (see Section III,D,.5 and Fig. 19B). However, the evidence implicating the growth-triggering effects of lysosomal proteinase is by no means based exclusively on the effects of exogenous inhibitors, many of them synthetic. Growth modulation appears also to be exerted by endogenous inhibitors, many of which possess considerable selectivity for proteases of lysosomal character. In addition, a number of such growth antagonists may be formed in situ by the actions of cell surface membrane-bound proteases-thus providing an autoregulatory device both economical and site restricted. Examples of endogenous inhibitors of proteolytic enzymes range from the classic a,-macroglobulin to low-molecular-weight substances in body fluids and in cell extracts that inhibit thiol proteinases (cf. Kopitar et al., 1978) and several cathepsins, including B (Lenney et ul., 1982). It is highly suggestive that NGF is immobilized and rendered inactive as a growth promoter by binding to 1979). Numerous other growth inhibitors, cera2-macroglobulin (Ronne et d., tain of which restrict proteolytic activity (Garesse et al., 1980), have been isolated from such diverse sources as Artemia salina (Garesse er al.. 1980), kidney epithelial cells (Holley et al., 1980), and hepatocytes (McMahon et al., 1982). The latter activity specifically inhibits proliferation of normal, but not malignant, hcpatocytes. An endogenous inhibitor is likewise associated with a calcium-dependent proteinase, selective for a high M , component of neurofilaments, in a cytoskeletal preparation of mammalian spinal cord (Tashiro and Ishizaki, 1982). Moreover, estrogens that are known to enhance liberation of lysosomal catheptic activities from uterus (see below) likewise promote uptake of a,-protease inhibitor and other plasma proteins by this organ (Finlay et al., 198I ) , presumably in course of promoting endocytotic activities of the surface membranes (see above). These combined observations point to significant degree of autoregulation in protease-promoted growth. One anomalous result that appears to militate against participation of lysosomal proteases in some aspects of growth control is the observation that two glycoproteins, whose augmented release into the medium of murine 3T3 cells responding to growth factors has heen identified (Nilson-Hamilton c’t N . , 198 1; Steck et ul., 1982), arc likewise liberated from these cells subjected to agents such as amnionium chloride, meth-
LYSOSOMAL FUNCTIONS IN CELLULAR ACTIVATION
227
ylamine, and chloroquine that increase lysosomal pH, a circumstance believed to suppress intralysosomal hydrolase function (Nilsen-Hamilton et a l . , 198 1). What must be recalled in this context, however, is the structural labilization of lysosoma1 membranes resulting from such expedients, associated with release of sequestered hydrolases to the environment, intra- and extracellular (cf. Table VII), with the consequences predicted by the above data. In closing this section we shall examine the evidence available to implicate enhanced lysosomal cathepsin B accessibility to strategic cellular sites as a normal concomitant of estrogen-induced mitogenesis in the selective target cells responsive to this hormone. Cell death as a consequence of application of excessive levels of this hormone and related substances has already been cited and will not be pursued at this point except parenthetically, to indicate still another unsolved problem: the shedding of degenerating, ‘‘cornified” epithelial cells into the vaginal lumen at fixed intervals during the rodent estrous cycle and also at given times after administration of a single dose of estrogen to the ovariectomized rat. This latter phenomenon still bears the hallmarks of prior lysosomal action, however, to a degree considerably beyond that presently proposed in the control of normal growth. l 5 To analyze the question of staging of mitogenesis in response to steroid and peptide agonists in a meaningful way that is nevertheless economical of space, it seems appropriate to reconsider at this point the data already presented in schematic fashion in relation to estrogen action in Fig. 3, data that, in turn, bear significant parallels to the sequential actions of other mitogenic agents (e.g., TSH, Table IV; PHA, Table V) on their respective target cells. The seemingly smooth sequence from (1) specific ligand interception at the cell surface, through (2) membrane perturbations, (3) augmentation of transport processes, and (4) energy-yielding reactions, together with (5) lysosomal activation, and, more distally, (6) enhanced transcription followed by augmented translational activities, and, ultimately, (7) mitosis in those cells that were at the appropriate stage of receptor availability at their outer membranes (eg., Fig. 3 in Pietras and Szego, 1981), may now be correlated with the data summarized in Table XXIV. Clearly, some observations remain circumstantial. However, from the collective 15The converse view is frequently presented, namely, that growth arrest is the outcome of gross enhancement of macromolecular degradation by lysosomal enzymes (cf. Saha er a / ., 1981; Lockwood and Minassian, 1982). Growth in response to mitogens has long been recognized to be a biphasic phenomenon, with imperceptible changes on the low side of the dose progression, and cell death (cf. Nitsch and Wollman, 1980) in the presence of excessive levels or prolonged exposure. This poorly understood inversion [which may account for the suggestion that thyrotropin (Westermark et al., 1979), as well as ACTH (Masui and Garren, 1971), are growth inhibitory to their respective target cells in virro] is nevertheless compatible with the concepts set forth in the preceding sections of this article on optimal stimulation of metabolic processes with limited lysosomal activation in contrast to attrition at excessive levels. It would be of deep significance if the balance between these counterpoised factors were to be found to contribute to a steady state in tissue mass and composition.
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C L A R A M . SZEGO AND RICHARD J. PIETRAS
TABLE XXIV E V ~ U ~ Nt OCKLPARTICIPATION CX- PRVlLlNASl ACTIVITY IN PKOPALAIION OF AGONALEI-.tbcisc* Agonist Steroid hormones Estrogen
Observation
Cyclic variation of fibrinolytic activity in rat vagina associated with ncutral proteinase Secretion of uterine Ca2+ -activated endopeptidase at proeatrus Protcinases spccific for argininc or lysine bonds enhance EJklicited incorporation of ‘H from UTP into acid-precipitable form in rat uterinc chromatin preparations E2P elicits abrupt extracellular rclease of cathepsin B-like’’ enzyme from uterus E$ stimulates trypsin-like activity associated with utcrine mitochondria-lysosonie fraction E$ promotes rapid transfer of lysosoma1 cathcpsin B-like” enzyme to nucleus in uterus and preputial gland Ca2+ -activated proteinase transforms uterine cytosol rcceptor to lower M , form Lcupeptin and antipain restrict DNA synthesis and fertility in cyclic mice Reduction by proteinase inhibitors TLCK, TPCK, PMSF)< of E2P binding in rat kidney cytosol Ca* -dependent, Icupeptin-sensitive protcinase cleaves cytosol receptor into steroid- and nuclcar-binding domains Inhibition by liposome-entrapped Ieupeptin of E$-stimulated endometrial cell proliferation; partial reduction by leupeptin of hormone bound by intact cells but not by cytosol Lcupcptin and antipain suppress Ezpinduced proliferation of mouse embryo cclls Partial reduction in E,B-binding capacity cif utcrine cytosol after exposure to mitochondria-lysosome lractions +
FACILI IArION O K
Reference
Astrup cf c d . (1967)
Joshi ef a / . (1970) Katz er a/. (1972)
Pietraq and Szcgo (1975h, 1979b); Szego (1975) Katz ef n / (1976)
Szego et al. (1976)
Puca er al. (1977)
Katz ei ul. (1977) Baker and Fancstil (1977); Baker rf al. (197X) Sherman et a[. (197X; cf. also Ratajczak e l d., 1981) Pietras and Szego (1979b, 1981)
Kennedy and Weichsclbaum (1981)
Pino and Sierralta (1981)
LYSOSOMAL FUNCTIONS IN CELLULAR ACTIVATION
229
TABLE XXlV (Continued) Agonist
Observation
Glucocorticoid
Proteinase inhibitors reduce deoxycorticosterone and dexamethasone binding to cytosol extracts Limited proteolysis of glucocorticoid receptor by crude lysosomal preparation from rat liver Increase in rate of protein degradation and in serine proteinases in lymphocytes within 8 hours Proteolytic activity associated with purified glucocorticoid receptor Enhanced synthesis of proteolytic activity with properties of cathepsin D in rat uterus Endogenous Ca2+ -activated. sulfhydryl proteinase elicits partial proteolysis of receptor A- and B-subunits of chick oviduct to hormone-binding, nonDNA-binding fragments Acute extracellular release of cathepsin B-likeh enzyme from rat intestinal epithelial cells
Progesterone
Vitamin D (la,2S-dihydroxycholecal ciferol; active form) Peptide hormones Chorionic gonadotropin Insulin
Oxytocin
Parathyroid hormone Thyroxine
Proteinase inhibitors block activation of adenylate cyclase by hornione in rat ovary Cleavage of a small peptide with potential mediator properties from membrane-enriched fractions of rat adipocytes Inhibition of hormone-induced glucose transport and oxidation by TLCK in rat adipocytes In limbic brain tissue, limited protcolysis of hormone to two peptide fragments with potential biological activity Acute extracellular release of cathepsin B-like” enzyme from rat intestinal epithelial cells Antipain, leupeptin and chymostatin inhibit hornionc-induced synthesis of specific liver enzymes
Reference Baker and Fanestil (1977); Baker et al. (1978) Wrange and Gustafsson (1978); Carlstedt-Duke er a / . (1979) MacDonald et a/. (1980); MacDonald and Cidlowski (1981a) Grandics and Litwack (1982) Elangovan and Moulton ( 1 980) Vedeckis et a/. (1980a,b)
Nemere and Szego (1981a,b)
Richcrt and Ryan (1977b)
Larner et a / . ( 1 979); Seals and Jarett (1980); Seals and Czech ( I 980) Larner et a/. ( I98 I )
Burbach ri u / . (1980b)
Ncmcrc and Szego (1981a,b)
Mori and Cohen (1978)
230
CLARA M. SZEGO AND RICHARD J. PIETRAS TABLE XXlV (Continued) Agonist
Vasopressin
Observation Acute extracellular release of cathepsin B-likeh enzyme at apical surfaces of urinary bladder cells Inhibition of hormone-induced water flow by proteinase inhibitors and by suppression of secretion of cathepsin B-like” enzyme at apical surfaces of bladder cells
Mitogens and carcinogens Dibutyl nitroAcute increase in extracellular release of cathepsin B-like6 enzyme from urisamine nary bladder cells Inhibition by leupeptin of membrane alterations and cell proliferation induced by carcinogcn Dimerhyl benTumorigenesis initiated by the carcinogen and promoted with phorbol zanthracene esters suppressed and/or delayed by proteinase inhibitors EGF Limited proteolysis of photoaffinity-labeled “receptor” in mitochondrialysosome fraction of Swiss mouse 3T3 cclls Lysosomotropic amines inhibit mitogenesis induced by EGF and by insulin in human fibroblastsd Phytohemagglukctin-induced lymphocyte transformatinin tion inhibited by leupeptin Thrombin promotes proteolysis of its cell ‘Ihrombin surface receptor
Reference Pietras er a / . (1975)
Pietras (1976); Pietras er al. ( 1976)
Pietras and .%ego ( 1976); Pietras (1978) Pietras (1978)
Troll er (11. (1975)
Das and Fox (1978)
King et ul. (1981)
Saito ct a!. f 1973) Glenn and Cunningham (1979)
“See also Table XXA-C. W a ? + -dependent, sulfhydryl requiring, leupeptin-sensitive, cleavage at aginine or lysine residue,; see also Table XV. It is also to be noted that in “adequate” concentrations in vivo. leupeptin may be expected (cf. Sutherland and Greenbaum, 1983) to elicit rebound synthesis ofcathepsin B, cunceivably as a result of removal of negative feedback. “Abbreviations: TLCK, Na-p-tosyl-1.-lysine chloromethyl ketone; TPCK, L-I -tosylamido-2-pheriylcthyl chloromcthyl kctone; PMSF, phenylinethane sulfonyl fluoride. “See also Table VII.
data, the implication is unequivocal of ligand-mediated availability of proteinase with properties akin to those of cathepsin B (cf. Table XV) from previously sequestered form. There is likewise evidence in the compiled data for cumulative effects-some early, some late-that would correlate with the staging pattern in
23 1
LYSOSOMAL FUNCTIONS IN CELLULAR ACTIVATION
the unfolding response sequence exemplified in Fig. 3 and in Tables IV and V. The "rebound" synthesis of additional lysosomal activities, including cathepsins, following attrition of the organelles and, presumably, removal of negative feedback on synthesis of lysosomal components (cf. Table VI and Section II,B, above), is likewise implicit in the incomplete summary provided in Table XXIV. Because interpretation of such composite observations as are presented in Table XXIV is limited by their retrospective compilation and their very generalization, it would be appropriate to examine a few specific instances in which enhanced delivery of cathepsin B activity to strategic cellular sites is more directly identified with ligand recognition. Accordingly, and with emphasis on estrogen action, it may be noted from Table XXV that there is a sharp increase in availability of cathepsin B to the outer surfaces of isolated endometrial cells on brief incubation in a serum-free defined medium in the presence of active hormones, whereas the relatively inactive estrogen, estradiol-17cx (see Fig. 2), is inert. Moreover, there is direct correlation between such release and the reorganization of surface architecture (see Pietras and Szego, 1979b, and Table XI), a consequence of E2P action that can be blocked by inhibitors of cathepsin B, such as leupeptin (Fig. 25). Although many steps intervene between ligand recognition and cell division, it is nevertheless significant that the blockade in intra- and extracellular cathepsin B activity that is effected by liposome-delivered leupeptin TABLE XXV FOR 30 MINUTES ON ACTIVITIES OF HYDROLYTIC ENZYMES IN EFFECTOF ESTROGENTREATMENT THE PARTICLE-FREE MEDIAFROM SUSPENSIONS OF ENDOMETRIAL CELLS Extracellular enzyme activity (pmollminlmg cell protein)" Group
Cathepsin B
P-Glucuronidase
Alkaline phosphatase
Control (0.02% ethanol) Estradiol- I 7 a (1 x 10-YM) Estradiol- 17p (1 x 1 0 - 9 ~ ) Estriol (1 x 10-9 M )
43 t 3 (5)
2227 t 30 ( 5 )
10 t 1 ( 5 )
* 42
11 t 2 (3)
45
* 1 (3)
2272
(3)
88 t 1 (5)b
2606 2 48 (5)d
11 t 1 ( 5 )
70
2450 t 102 (3)
10 t 2 (3)
2
2 (3)<
aAfter 30-minute incubation at 2 2 T with or without estrogens, enzyme activities detected in particle-free supernatant fractions from cell suspensions were less than 0.5% of total available cell hydrolase activities; no significant change in ford enzyme contents was found after 30-minute E2P treatment (p > 0.80 vs controls). Values in parentheses represent numbers of separate experiments. Reprinted from Pietras and Szego (l979b), with permission. "Value significantly different from control at p < 0.001. cValue significantly different from control at p < 0.01 dValue significantly different from control at p < 0.05
232
CLARA M . SZEGO AND RICHARD J. PIETRAS
rJ 2501 a
b
1
CONTROL ESTRADIOL- ESTRADIOL-
17a I E p l
178 (E281
.EUPEPTIN
Is
C
LEUPEPTIN 8 E219
250
T
COh1R:)L- (OhlHll. I t -1'cI'l h .ELPEPT.LPO:,OMCS PI.SOMES : POSOMES POSOMES
8
€28
8
w
Frti. 25. (a-c) The influence of free or lipososome-entrapped leupcptin on estrogen-enhanced incorporation of [%]thymidine into acid-prccipitable form and on proliferation of endometrial cellh. Values of [ 'Hlthyinidine incorporation after 20 hours (open bars) and ccll nuriibcrs after 2 days of incuhation (shaded bars) were obtained in 3-4 independent experiments in the presence and absence of 1 x M estrogen, as indicated. Details are given in Pietras and Szego (1Y7Yb), from which this figure is reprinted, by permission.
at nontoxic (cf. also, Bursztajn and Libby, 1981; Ballard, 1982) concentrations, is likewise associated with inhibition of the mitogenic indices shown. Antagonists of proteinases other than cathepsin B were ineffective (Pietras and Szego, 1979b). Further correlations between abrupt availability of extracellular cathepsin B and eventual cell proliferation in the presence of estrogen are available elsewhere (Pietras crt ul., 1981a). The parallels between these data and the impetus toward neoplastic transformation delivered by proteolytic activities to strategic cellular sites are considered briefly in the next section. This etnphasis upon acute delivery of proteinase activity from lysosomes to ligand-stimulated cells is not to be construed to imply that enhanced availability of critical ions, for example, Ca2+, or of cyclic nucleotides, e.g., CAMP, is not contributory to the pathways leading to normal proliferative functions. However, there are some especially significant indications in these latter contexts that require us to focus once again upon lysosonial behavior. Thus, Ca2+, which is required for cathepsin B activity in some cell types, but which, in excess, is inhibitory (see Szego el u l . , 1976; Quinn and Judah, 1978), is now recognized to be sequestered in lysosomes (see Section U , B , and Davis and Jones, 1982). Indeed, the latter data may make it necessary to reevaluate the role of mitochondria as the predominant reservoir of intracellular calcium, for these organelles are notoriously difficult to separate from lysosomes. Moreover, CAMP is a promoter of lysosome labilization and serves to reinforce the activa-
LYSOSOMAL FUNCTIONS IN CELLULAR ACTIVATION
233
tion of lysosomal function mediated by ligand or even to supplant the latter (Szego, 1972a). Although in the preceding sections, protease activities delivered to crucial cellular sites, especially to surface and to the nuclear compartment, at critically early times after agonal recognition, from a portion of the lysosomal population has been emphasized in the propagation and mediation of mitogenic signals (see footnote 15), it is clear that lysosomal activation by itself is a minimum requirement. For one thing, apparently multiple or prolonged encounters with the appropriate agonist are required for full expression of a differentiative or proliferational response (cf. Hopkins, 1980; Shaw and Griffin, 1981; Ramachandran et al., 1981; Segaloff and Ascoli, 1981; Korach and Fox-Davies, 1982). Moreover, a nonmitogenic analog of EGF elicits the early responses in membrane and cytoskeletal structure and function that characterize effects of the hormone itself (Yarden et al., 1982), suggesting either that the early signals are not required or, more likely, not in themselves sufficient for induction of DNA synthesis. Further, an estrogen antagonist such as tamoxifen, itself a weak estrogen, is a highly effective inhibitor of the anabolic and proliferative effects of the hormone in cultured cells, despite the fact that it is capable of promoting estrogen receptor activation and translocation to the nucleus (cf. Evans et al., 1982) and, indeed, depleting the cell of receptor moieties (Martin, 1981; Iacobelli et al., 1982). These findings alone militate against a simplistic view. The common pattern of sequential responses or orderly staging of the effects elicited by agonist (see Fig. 3; Tables IV and V) suggests a far more complex reaction series. Contributing to the staging of such a pattern are cellular entry (Tables 111, XVI, XXII), nuclear penetration of (H), (R)16 or their processed moieties, and, through some form of mutual recognition by specific regions of chromatin, a resultant orderly transition of the compact state of the genome to a transcriptional or replicative mode at given sites specified by nucleotide sequence or associated protein. In preceding sections we have pointed out some potential means by which lysosomal components may contribute to these effects. Unfortunately, the uncertainties are legion. One need not belabor the potential further significance of changes in surface properties of cells in altering the course of growth, differentiation, and interaction with neighboring cells in normal and ectopic sites. It would be of deep significance to learn whether such surface modifications are indeed the direct product of lysosomal enzyme release during action of hormones and other physical and chemical perturbations. These considerations, together with the striking mobility of lysosomal vesicles already emphasized and the indications for inserI6It seems noteworthy that in some tumor cells, specific, unoccupied rcccptor for a given agonist is already present in the nucleus without apparent benefit of hormonal mediation (Geier e r a / . , 1982; Meikle et al., 1982). This observation is reminiscent of the nuclear inclusion phenomenon, which is characteristic not only of cells undergoing intensive proliferation in response to known mitogenic agents, but, likewise, evident in many tumor cells (see Fig. 20G-I).
234
CLARA M. SZEGO AND RICHAKD J. PIETRAS
tion of limited amounts of lysosomal components into the nucleus under conditions of heightened cellular activity, may shed light upon the long-sought coupling mechanism between the surface of the cell and its more central organellar compartments. C. CELLULAR TRANSFORMATION: INDICATIONS FOR
A
LYSOSOMAL ROLE
Evidence for the involvement of acid hydrolases in promoting the growth and metastasis of tumor cells first appeared more than 50 years ago (Carrel and Ebeling, 1928; Purr, 1934). Since that time, there have been numerous additional data in support of a role for lysosomal components in the pathogenesis and expression of malignancy (see reviews by Allison, 1969; Poole, 1973; Pietras et al., 1981a). This section will provide a brief analysis of more recent developments, especially as they appear to reflect exacerbation of those aspects of lysosomal structure and function that have already been implicated in normal proliferative activities. 1 . Lysosomal Alterations in Chemical Carcinogenesis There is considerable biochemical and morphologic evidence that within relatively short times of application, chemical carcinogens and tumor promoters elicit reduction in the structural latency of lysosomal components in target cells. A survey of representative findings is presented in Table XXVI. As is the case with “normal” effectors of mitogenesis (Table X), numerous chemical carcinogens undergo accumulation in lysosome fractions of the affected cells (Table XXVI) where they elicit decreased structural latency of lysosomal hydrolases and, presumably, nonhydrolytic enzymes and other components, as do normal tropic agents (cf. Tablc IX), and redistribution of the organelles to perinuclear and intranuclear sites (Pietras, 1978; cf. Figs. 17-20 for the “normal” counterparts). These events are followed in some instances by increased total lysosomal hydrolase activities after exposure of sensitive cells to chemical carcinogens (Levi et al., 1969; Flaks, 1970), as well as to their normal effectors (Table VI). Collectively, these parallel findings indicate that the initial cellular response to carcinogen may well be analogous to, or convergent with, the process of receptor-mediated endocytosis coupled with lysosome fusion that serves to deliver hormones and other biologically active substances to the cell interior, as has been documented in previous sections of this article. Indeed, tumor cells exhibit not only an exaggerated degree of endo-ipinocytotic activity beyond that seen in normal cells, but also intensification (Liepins, 1982) of the level of vesicular exocytosis or surface shedding characteristic of normal cells (Doljanski, 1982). The molecules so released to the extracellular environment may or may not be represented in the cell surface per se. Conspicuous among those constituents consistently delivered to the extracellular environment of tumor cells are lysoso-
235
LYSOSOMAL FUNCTIONS IN CELLULAR ACTIVATION
TABLE XXVI REPRESENTATIVE EVIDENCE OF INTERACTIONS OF CARCINOGENS AND TUMORPRVMOTORS WITH LYSOSOMAL STKUCT~JRES OF TARGETCELLSO Agonist Carcinogens Dimethylbenzanthracene Methylcholanthrene Dibenzanthracene Benzo(o)pyrene Dimethyl benzanthracene Benzo(a)pyrene
Observation
Reference
Carcinogens taken up and concentrated by lysosomes of HeLa, macrophage and kidney cells after 2-24 hours in viirob
Allison and Mallucci ( I964b)
Partial purification of a kidney lysosome component with strong binding of carcinogen Carcinogen promoted progressive release of hepatic pglucuronidase and glycosidases prior to malignant transformation in vivo; and modified structural properties of “lysolemma” in
Barrett and Dingle ( 1 967)
Carevid and Carevid ( I 982)
virro
Aflatoxin Bl
Diethylnitrosamine
Dibutylnitrosamine
Diethy lstilbestrol
Carcinogen enhanced redistribution of acid DNase from liver lysosomes to supernatant and nuclear fractions; inactive carcinogen, aflatoxin B2. failed to affect lysosomal enzyme recompartmentation Carcinogen promoted structural labilization of galactosidase activity and increased activity of acid DNase in purified nuclei by 24 hours Carcinogen, but not diphenylnitrosamine, promoted extracellular release of cathepsin Blike enzyme from urinary bladder cells within 30 minutes; and redistribution of lysosome-like organelles toward the nucleus and periphery within 1 hour Suspected carcinogen promoted extracellular release of cathepsin Blike enzyme from uterine endometrial cells by 30 minutes
Pitout e t a / . (1971); Pokrovsky er a/. (1972); Pitout and Schabort (1973)
Schulze (1973)
Pietras and Szego (1976); Pietras (1978)
Pietras and Szego ( I 976)
(continued)
236
CLARA M. SZEGO AND RICHARD J . PIETRAS TABLE XXVl (Coritinrtetl) ~
Agonist Ttunor prornotors Croton oil Croton oil, phorbol estcrs
Phorbol iiiyristate acetatc
Observation
Enhanced labilization uf lysosoincs of intact cells Promoted rclcasc of lysosoinal enzymes from particulate fractions irr vitro. in pinportion to tuniospromoting activity it7 vivo; doubled total cathepsin B in 24 houra Stimulated extension of microtubules and 10-nm filaments, with concomitant redistribution of lysosornes by 90 minutes
Reference
Allison ( 1966)
Weissmann ct (I/. (1968); Dolbcare ( 1979)
Phaire-Washington ef nl. ( 1980)
"All intcractions within 24 hours. "Independent evidence of organ sclcctivity and structural spccificity of compoundz, in interaction uf hydi-ucai-hons with lysosotnes presented in Allison (1966).
ma1 hydrolase activities, especially those with degradative functions toward proteins, glycoproteins, phosphoproteins, and polynucleotides (Tables XXVII and XXVIII). Once again, this phenomenon has its counterpart, although in diminished degree, in normal cells undergoing relatively intense proliferative activity in response to specific effectors, as earlier discussed. Extracellular liberation of lysosomal hydrolases, notably cathepsins (e.g., Table XXV), has been implicated in reorganization of the plasmalemmal architecture in the staging of normal cellular responses to their selective agonists (cf. Table XIV). Such a process. whcn duly intensified in degree or extended in time, or possibly in both circumstances combined, may elicit the extremes of surface alterations characteristic of cells stably transformed to the malignant phenotype (Poste, 1976; Kennett et al., 1982). The capacity of lysosomal hydrolases to modify the cell surface and the extracellular matrix is well documented (cf. Poste, 197 la; Bosmann, 1972; Poole, 1973). Moreover, early in the transformation of epithelial cells by the carcinogen, dibutylnitrosamine, clustering of lectin-binding sites at the external surface and changes in the rate of intercellular adhesion correlate closely with variations in the extracellular availability of lysosomal thiol proteinase activity (Pietras and Szego, 1976; Pietras, 1978). In turn, covalent modification by lysosomal hydrolases of the turnover and processing of nucleotides (Fuhge and Otto, 1980; Szego, 1982) and nuclear proteins (Hoiton and Szego, 1984) or their carbohydrate moieties, processes already documented for normal growth, could also contribute substantially in the progression of cells to malignancy, through provoking those changes in critical structural and functional macromolecules that
LYSOSOMAL FUNCTIONS IN CELLULAR ACTIVATION
237
exceed the capacity of normal repair systems and/or lead to genic alterations (cf. Allison and Paton, 1965; Kinsella and Radman, 1980), resulting in expression of otherwise silent genes.
2. Potential Significance of' Cathepsin B-like Thiol Proteinuse in Neoplastic Growth Although much recent work has postulated participation of serine proteinases in the regulation of cell proliferation (cf. Ossowski et al., 1973; Christman et al., 1977; Skriver et ul., 1982), numerous independent studies have failed to provide
Hydrolase activity Acid proteinase(s)
Cathepsin B-like thiol proteinase
Extracellular compartment Extracellular mcdiab Plasma Interstitial fluids Serum Extracellular media" Extracellular fluids'
Acetylglucosaminidase P-Glucuronidase Hyaluronidase
Extracellular media" Serum Serum Interstitial fluid
Elastase Acid phosphatase
Exti-accllular media" Plasma Interstitial space
Acid ribonuclease
Serum Serum
Reference Carrel and Ebeling (1928) Ottoson and SylvCn (1960) SylvCn and Bois-Svensson (1965) Pietras ef a/. (1978, 1979); Huseby and Smith ( 1980) Mort et a / . (1980); Recklies et a/. (1980, 1982); Pietras et a/. ( I 98 1 a); Sloane et a/. ( I 982a) Rinderknecht and Renner (1980); Craf e r ti/. (1981); Orlowski et a / . (1981); Mort P I a / . (1981) Hultbcrg and Mitelman (1977) Pietras ef a/. ( I 979) Boyland of a / . (1955); Nagasue et a / . (1982) Fiszer-Szafarz and Gullino (1970); Fiszer-Szafarz (1981)
Orkin ef a/. (1982) Egbring et a / . (1977) Urban and Unsworth (1977); Schenk and Konrad ( 1980) Nagasur et a/. (1982) Reddi and Holland (1976)
"Malignant tumor samples compared with appropriate controls. Although sevcral reports provide indirect evidence that such elevated lysosomal enzyme activity in the tumor periphery may be due, in part, to the contribution of necrotic tumor cells, invading macrophages, or other host stromal cells in vivo (cf. Weiss, 1978; Dobrossy e t a / ., 1980; Graf et a / . , 1981), recent studies with tumor cell lines maintained in virro indicate that viable malignant cells continue to release acid hydrolase activity extracellularly even in the absence of contaminant host cells (see Fig. 1 in Pietras e t a / . , 1981a; cf. also, Sloane er a / . , 1982a; Recklies ef a / . . 1982). " I n w'tro.
[In vivo, excluding serum.
238
CLARA M. SZEGO AND RICHARD J . PIETRAS
TABLE XXVlll ARNORMAI.ITII:S IN THI. DlSTltIBU~I ION AND/OR ACTIV1TII:S 0 1 . VAIUOUS LYSUSOMAI HYDROIASFS I N TRANSFORM~,I) CELLS:S t L t c r m EXAMPI.I:S" Hydrolasc activity
Celllfissue
Obacrvat ion
Cathcpain B-like enzyme
Gynecologic tu-
Incrcascd activity assnciated with increased invasivcncss Immunofluorescent evidence of enzynie at or near cell surface Redistribution to and enrichment in purified plasma membranes and ultrapurified nuclei Increased activity in cell lines with high metastasis Slightly increased activity Redistribution of activity from lysosomes to microsomes and soluble fractions Increased activity correlated with initiation of metastasis Enrichment of activity in purified plasma membranes
Illorb
Transforincd fibroblasts Cervical carcinoma cells
Melanoma
Cathepsin D
Skin cancer Hepaloma
Acid phosphatasc
Lung carcinoma
Astroc ytoma
Cervical carcinoma cells Astrocytoma
GI ycosidases
Transformed fibroblasts
Sarcoma
Reference Blackwood et ul. (1965)
SylvC11 et nl. ( 1974)
Pietraa and Roberts (1981)
Sloanc et u/ . ( I98 I , I982a)
Shamberger and Rudolph ( 1967) Fiszer-Szafarz (1981)
Dobrossy ef d.(1980)
Knowles er al. (1981)
Pietras and Roberts ( I98 I ) Plasma membrane acid phosphatase showed specificity for phobphotyrosine proteins Increased activities of acet y Iglucosaminidase, glucosidase, galactosidase, mannosidase, acetylgalactosaminidase Increased acetylglucosaminidase
Leis and Kaplan (1982)
Bosmann (1969)
Hultberg and Mitclman (1977)
LYSOSOMAL FUNCTIONS IN CELLULAR ACTIVATION
239
TABLE XXVlII (Continued) Hydrolase activity
Cellitissue
Observation
Polyoma-transformed BHK cells
Hepatoma
Activities of acetylglucosaminidase, acetylgalactosaminidase, galactosidase, and mannosidase exceeded those of quiescent, but nor those of growing, normal cells Variable content of 8 different enzymes in various forms of leukemia Increased acid RNase
Takano et nl. (1971)
Sarcoma Hepatoma
Increased acid DNase
Bhandarkar er al. (1974) Shamberger et al. (1971)
Leukemic cells
Acid RNase and DNase
Reference Thompson et a / . (1978)
Tanaka et a / . (1981); (cf. also Rundell et al., 1974)
UDistribution or specific activity of hydrolase in tumor sample compared with that of appropriate nonmalignant control, unless indicated otherwise.
conclusive evidence for a relation between secretion of serine proteinases and malignancy (cf. Mott et al., 1974; Chen and Buchanan, 1975; Nicolson, 1976; Pearlstein et al., 1976; Rosenthal et at., 1978; Smith et al., 1979; Recklies et al., 1980). Hence, the potential role in malignancy of cathepsin B-like enzyme, which exhibits substantial activity at both acid and alkaline pH (Sylven, 1968; Szego et al., 1976; Pietras et al., 1981a; Recklies et af., 1982), warrants renewed consideration. Indeed, recent quantitative studies provide substantial evidence of a selective and pronounced alteration in the cellular distribution and release of cathepsin B-like thiol proteinase in malignancy (see Table XXVII). The rate of secretion of cathepsin B-like activity in v i m by cells undergoing neoplastic transformation (Pietras and Szego, 1976; Pietras, 1978) and by established malignant cells (Poole et al., 1978; Pietras et at., 1981a) exceeds that by their untransformed counterparts by as much as 50-fold, a level unparalleled by that of several other hydrolases. Especially pronounced alterations in the cellular and extracellular activity of cathepsin B-like enzyme have been found in those variant subpopulations of tumor cells with high rates of proliferation in soft gels (Pietras et ul., 1981a; Pietras and Roberts, 1981) and in those with high metastatic capacity (Sloane et ul., 1982a). The potential importance of variations in the distribution and activities of lysosomal hydrolases in the processes of tumor formation, invasiveness, and metastasis is indicated from the results of studies with inhibitors of enzyme activity, as summarized in Table XXIX. In particular,
240
CLARA M. SZECO AND RICHARD .I.PIETRAS TABLE X X l X
TUMORFOKMAIION ANU Inhibitor
Lcupeptin
Antipain
Ot SPREAD EFFI CTS O F ~ N H I I l I I O l < S
AclU HYDRO[AST ACTIVIIY
Observation
A. Inhibitors of cathepsin M i k e enzymes Blockade of tumorigenesis induced hy polycyclic hydrocarbons in niousc skin Leupeptin-loaded liposomcs reduced cell cathepsin B-like activity and suppressed rnernbrane alterations end growth induced by the carcinogen, dibutylnitrosarnine, in urinary bladder cells Suppression of malignant transforrnation of embryo cclls induced by X rays ir7 b'itro Suppression 0 1 aistcr chi-oinatitl exchange induced by phorbol esters and irradiation Inhibition of blood-borne nictaatasis of ascites hepa!nina cells t o lung Leupeptin-loaded liposoriics selectively rcduccd cell cathepsin B-like enzyme activity and auppreascd proliferation of ncnplnstic cervical epilhelial cells it7 dro Suppression of tumor cell-induced platclct aggregation associated with inhibition ol' cathepsin B-like enzynic activity Suppression of maligniint transformation of embryo cells induced by X rays ill ),i/ro Inhibition (11 etiibryo ccll trmsformotion when inhibitor added 10 iiiinutcs aftcr iiwiid iat ion Inhibit inn of carcinogen-iritltiCcCl clii- liitiow iiiiil ahsrrarioiis i n Chinese hamster cclls without signil'icantly tlccrca\ing their \I ti rvi vii I Inhibition ol c c l l transfi,rmattc,n induced by clicmical carcinogens in rodcni cell cuiturcs
Reference
Hozurni ef ul. (1972) Pietrah (1978)
Kennedy and Little (1978, 1981) Nagasawa and Little (1979) Saito et d.(19x0) Pietras
Honn
P / a/.
c'f
(I9X Ia)
crl. ( 10x2)
Kcnncdy and Littlc ( Ic)7X. 1081) Borck el ol ( 1979)
Kinwlln and Radman 11 9x0)
Kuroki and Drevon ( 1970)
B. Inhihitors of cathepsin D Reduced cathcpsin D i / i i,iw a d rctiirdcd ascite\ toriri;rtion i n tumor-hearing rats
Sacchar<)-I .-l-lactoiic a n d
2-acetatriiilt,-2-dc(~xy~ gluconolac tone
C. Inhihitors of glycosidasrs Reduction 0 1 subcutancow grnwth nt transpl;intahlc tuiiiors
Greenbauiii (1979)
Carr (196.7)
24 1
LYSOSOMAL FlJNCTIONS IN CELLULAR ACTIVATION
TABLE XXIX (Contirirredl Inhibitor
Meth ylprednisolone
Medroxyprogesterone, dexarnethasone. cortisol Indomethacin
Brornodeoxyuridine
Observation
D. Lysosome stabilizing agents Retardation of growth of niairirnary carcinonla and reduction of tissue levels of cathepsin D Blockade of neovascularization and growth o f tumors implanted in rahbit cornea Reduction of numbcrs of tumors induced by dimethylnitrosaminc in rat intestine
E. Miscellaneous agents Reduction of acid and neutral proteinase activities of melanoma cells concomitantly with suppression of tumorigenicity
Rcference
Bagwell and Fergu son ( I 980) Gross et ul. ( 1 % I )
Pollard and Luckert (1981); cf. however, Sloane et ul. (1982b)
Evans and Bosnlann (1 977)
it has been demonstrated that high concentrations of leupeptin and antipain administered in vivo can exert quantitative restraint upon tumorigenesis and metastasis (cf. also, Rossman and Troll, 1980). Although thiol proteinase activity secreted by a variety of human and rodent tumors has indeed been shown to possess enzymic properties similar to those of lysosomal cathepsin B (cf. Table XV), there are, nevertheless, some distinctions. Thus, the tumor enzyme is more stable than is lysosomal cathepsin B from untransformed cells to inactivation above neutral pH and to heat denaturation (Pietras and Roberts, 1981; Recklies et ul., 1982), and exhibits additional behavior indicative of certain alterations in primary structure, such as increased molecular mass and evidence of a more anionic isoenzyme pattern (cf. Mort et d., 1980; Pietras and Roberts, 1981). Hence, as proposed earlier (Pietras et ul., 198la), it appears worthwhile to invesitgate the hypothesis that neoplastic cells produce altered forms or isoenzymes of cathepsin B capable of escape from the usual cellular recognition systems for the intracellular transport and insertion of the enzyme in lysosomes (cf. Fig. 5B; and Sly and Fischer, 1982). Likewise, the activities of such isoenzymes may not be subject to attenuation by naturally occurring inhibitors. Alternatively, the extralysosornal tumor enzymes might be products of a transforming gene and fortuitously share catalytic properties with those of lysosomal cathepsin B. Purification of these cathepsin B-like enzymes and subsequent production of specific antisera will be helpful in addressing such questions. It will be recalled that, by a similar line of reasoning, participation of limited proteolysis was likewise invoked in the triggering andlor propagation of normal
242
CLARA M . SZECO AND RICHARD J . PIETRAS
cell growth: Can one have it both ways? However, it will be recalled that liberation of lysosomal protease, presumably in nutive isoenzyme forms in limited amounts from its organeliar confines at appropriate times in the cell cycle was postulated in preproliferative events under control of effectors unknown. If some is good, more is by no means betterespecially when the lysosomal destabilization event is elicited by uptake of viral genes, toxins, organic and metallo derivatives, and other cellular pollutants. These noxious subtraces gain access to, and are concentrated in, the lysosomal compartment through the intensive endocytotic activity characteristic of rapidly growing cells, and, in due course, penetrate the nuclear envelope by the mechanisms outlined in preceding sections of this article. Under such circumstances, especially with prolonged or persistently repeated exposure to the agonist and/or promoter, proteolytic activities are far from “limited” in degree, or confined to the precise times in the cell cycle during which they can trigger division “in a cell prepared for it” (Allison and Mallucci, 1964b). Moreover, the nuclear matrix, now clearly identified as a significant site of RNA processing in eukaryotic cells (Ciejek et al., 1982), shows evidence of substantial decrease in proportion of high M,polypeptides in livers of rats following low-dose carcinogen intoxication (Clawson and Smuckler, 1982). Thus, and without minimizing the potential contributions of additional lysosomal constituents such as protein kinase(s) and phosphatase(s), or even nonhydrolytic components, loci and mechanisms are clearly available for exaggerated lysosomal proteolytic activity in perversion of normal cell function by carcinogenic agents. A summary of the many cellular alterations leading to progressive acquisition of a malignant phenotype, and that may be attributable, in part, to the action of cathepsin B-like enzyme, is assembled in Fig. 26. Apart from evidence presented in the figure, additional studies indicate that such catheptic activity may also participate in the production of non-complenient-fixing, blocking factors from limited hydrolysis of cytotoxic antitumor antibodies (Dauphinee et a / . , 1974; Keisari and Witz, 1975). Such degradation products could compete successfully for tumor antigens and thereby protect the tumor cell from immune destruction. It is conceivable that aberrant activity of cathepsin B-like enzyme might also initiate a cascade of cellular events by activation or inactivation of growth factors (Smith and van Frank, 1975; King et al., 1981; cf. also, Todaro et al., 1981), regulatory proteins (Fuhge and Otto, 1980; Christopher and Morgan, 198 I ; Bond and Barrett, 1980), and/or structural proteins (cf. Noda et ul., 1981), possibly those of the cytoskeletal system and their modulators (cf. Boschek et al., 1981; Chafouleas el al., 1981). Elucidation of the nature of cathepsin B-like enzyme and its relationship to normal, as well as malignant growth are challenging areas of inquiry. Detailed understanding of the molecular basis of the compartmentation and activity of this enzyme may well prove to be important in defining a new locus for intervention in the pathogenesis of cancer.
243
LYSOSOMAL FUNCTIONS IN CELLULAR ACTIVATION COLLAGEN
LECTIN-MEDI AGGLUTINAT
(t)
CLUSTERING OF LECTIN-BINDING SITES
‘ION
ADHESION GLASS ( FR AGM E N T AT ION OF F IB R ON E C T I N
..
0 ’
Y-DEPENDENT INHIBITION OF GROWTH
FIG. 26. Cellular alterations associated with enhanced activity of cathepsin B-like thiol proteinase. The structural and molecular changes indicated schematically in the figure are representative of those characteristic of the malignant phenotype (cf. Poste, 1976; Nicolson, 1976). The latter include variations in the cellular distribution of cathepsin B-like hydrolase and in its activity toward collagen (Burleigh et al., 1974), proteoglycans (Morrison et a/., 1973), fibronectin (Isemura e t a / ., 1981). chromatin proteins (Szego e t a / . , 1976; Suhar and Marks, 1979), plasma membrane proteins (Seetharam et ul., 1976), hexose carrier molecules (Christopher and Morgan, 1981), cytoskeletal and contractile proteins (Schwartz and Bird, 1977: Quali and Valin, 1981; Noda et al., 1981). and various intracellular proteins (cf. Dean, 1976; Barrett, 1977), including zymogen activation (cf. Smith and van Frank, 1975; Eeckhout and Vaes, 1977; Quinn and Judah, 1978; Coradello et al., 1981: Luetscher et al., 1982; Takashi e t a / ., 1982). Additional features comprise decreased adhesion of cells to glass (SylvCn, 1968), clustering of concanavalin A-binding sites at the cell surface associated with increased lectin-mediated cellular agglutination (Pietras and Szego, 1976: Pietras, 1978), and decreased density-dependent inhibition of cell growth (Pietras, 1978; Pietras et a/., 1981a). Redrawn with minor modifications from Robbins and Nicolson (197% with permission.
V. Integration A. THE “USES”
OF
COMPARTMENTATION I N THE CELLULAR ECONOMY
The present review has sought to address the great gaps in our understanding of the molecular means of propagation and amplification of the initial disturbance generated at the outer cell surface on mutual recognition of receptoreffector that leads to pleiotropic effects culminating in cell division. Having long been alert to compartmentation as a primitive but effective and economical
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CLARA M . SZECO AND RICHARD J . PIETRAS
means of separating potential reactants, we have attempted in the present article to utilize this concept as a framework in analysis of the available but often scattered data on effector-mediated alterations in cellular structure and function. Accordingly, we have placed particular emphasis upon early and controlled attenuation of cellular barriers (or their stabilization) on recognition of specific ligand by membrane-oriented rectptor. Indeed, removal of cytostructural barriers altogether by unduly rigorous homogenization is a means of achieving metabolic chaos (Hill, 1928). However, compartmentation and its corollary, accessibility of critical factors to sites of utilization from which they are relatively restricted under status quo ante, implies a means of regulation of molecular traffic that has generally emphasized the limiting membranes of only three sites: those of the plasmalemma proper, the mitochondrion, and the nuclear envelope, to the essential exclusion of boundaries of other organelles, notably those of the lysosome (cf. Srere and Estabrook, 1978; Herman et a l . , 1980; Sies, 1982). The present analysis has proposed that escape of “microquanta” of individual lysosomal components at strategic cellular sites generates the secondary molecular cascade initiated by that recognition of ligand at the outer cell surface from which all else stems. B. THEINDISPENSABILITY OF LYSOSOMAL FUNCTION
The bases for this broad conclusion have been presented in detail from evaluation of the known properties of lysosomes in context with a wide variety of effector-actuated events, with emphasis on hormone-induced growth and development. However, by virtue of hindsight, one may now identify significant parallels in other contexts. These range from the wound-related shifts in protein metabolism in plants (Ryan, 1980), through immune responses (e.g., Carlo et al., 1981; Ziegler and Unanue, 1982), including, by implication, the functions of interferon in the activation of NK cells (cf. Bloom, 1980), nervous activity, such as storage of neurotransmitter (Schwartz et d . , 1979), axonal transport (Broadwell et u l . , 1980), stimulatory phenomena by nerve trophic factors (Thoenen and Barde, 1980), vision (Regan et a / . , 1980; Hayasaka et a/., 1981), and neurosecretion (Berry, 1981; cf. also Table XXC). Likewise, phenomena associated with reproduction, comprising oocyte maturation (Lopata et al., 1977; Ezzell and Szego, 1979), ovulation (Cajander and Bjersing, 1975), fertilization and related activitics, (Farooqui and Srivastava, 1980; Yamada et al., 1982), implan1979; Moulton, 1982), and even parturition (Gustavii, tation (Sengupta et d., 1975; Schwarz et m l . , 1980; Bryant-Greenwood, 1982) possess demonstrable correlates of limited lysosomal activation. Similarly, absorption and metabolic homeostasis (Jacques, 1969b; Ono, 1979; Christopher and Morgan, 198 I ; DeMartino and Goldberg, 1981), osmoregulation (Moore er id.,1980), as well as local control of the microcirculation (Lewis and Austen, 1981) appear to share
LYSOSOMAL FUNCTIONS IN CELLULAR ACTIVATION
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this common denominator: elicitation of a degradative cascade or its attenuation. Can it be that the dismantling process itself wipes the slate clean, as in insect and amphibian metamorphosis, concomitantly making available the raw materials from which the celliorganism is remodeled or more radically reconstructed’? The participation of lysosomal function in growth processes has been invoked on grounds less heroic, but having, nevertheless, the common element of limited degradation at strategic cellular sites. The common link among these apparently widely dissimilar processes may lie in the pluripotentiality of the lysosome and its heterogeneous components. lndeed, the recent evidence for sequestration in this class of organelles of constitutents above and beyond hydrolases, including, in various cell types, Ca2+ (Davis and Jones, 1982), protein kinase (Collins and Wells, 1982), tubulin (Clark and Shay, 1981), as well as basic proteins with certain correlates of histones and their variants (Horton and Szego, 1984), gives tantalizing glimpses of nnnhydrolytic intervention in the transcompartmental information transfer which this organelle is manifestly or potentially capable of carrying out. For example, it seems altogether feasible to contemplate reorganization of cytoskeletal structure in the anabolic direction, through enhanced availability of limiting components that are relatively inaccessible to sites of their utilization under basal conditions. The translocation of secondary lysosomes to the nuclear region may indeed depend upon their interaction with the cytoskeletal network, if not upon its limited reconstitution, as the covertly labilized organelles deliver, in a selective manner yet to be understood, their complex array of catalytic and structural components. By the same token, the pathway connecting lysosomes to the cell surface (cf. Fig. 22, and Nichols, 1982) renders that structure, as well as the extracellular matrix, amenable to limited remodeling at early intervals after plasmalemmal perturbation. The net outcome of such enhanced transcompartment and transcellular communication may then be a function of the state of the metabolic machinery characteristic of that particular cell, as it is affected by cascading events triggered by structural labilization of lysosomal components. The precise pathways are presently mere conjecture. The wide variety of physiological functions that are now identified with limited recompartmentation of lysosomal constituents indicate that these organelles have successfully adapted to requirements of increasing complexity in eukaryotes (cf. Tumel’yan and Vasil’ev, 1982). Indeed, the strong conservation of this organelle from an allegedly ancestral eukaryotic precursor of plant and animal cells (de Duve, 1980), together with the occurrence of hydrolases characteristic of lysosomes, even in the absence of typical organellar structures, in prokaryotes, strongly suggest the indispensability of some form of limited degradative function in all cells. It is the adaptation of this generalized function to the intracellular implementation of surface-generated signals that distinguishes the lysosome as it has evolved in the higher forms of plant and animal life.
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C. CLOSINGCOMMENTS
Despite the intense preoccupation with the nature and means of communication of mitogenic and other perturbational signals from the cell surface to intracellular compartments that has characterized the literature for the past several years, our ignorance of these problems, both in breadth and in detail, is considerable. It is hoped that provision of some integration among the many, sometimes conflicting, observations, together with identification of areas of particularly gross uncertainty, may contribute to our understanding of the functional relations among the outer cell surface and the nuclear and cytoplasmic Compartments in the coordinated response to tropic hormones, as these agents interact with specific receptors at the cellular boundary. The detailed functions of lysosomes in providing intimate communication among these widely separated, responsive cellular elements appear worthy of continued examination. It is readly acknowledged that the formulation of a conceptual framework for currently developing data is inevitably premature, and surely will require modification with advances to come. Nevertheless, extrapolation from the known permits us to escape from the “grooves” worn by repeated assertions of ostensibly established fact” and to project generalizations that lend themselves to experimental testing. Else what is reason for?
ACKNOWLEDGMENTS The literature survey leading to the preparation of Tables XVIIIA,B and of XXA-C was conducted with the active collaboration of prcdoctoral associates Carol 0. Rambo and Kevin Lee, respectively. We thank Ms. Barbara .I.Seeler for expert assistance in all laboratory phases of this work not previously acknowledged and for logistic support in countless other ways. Ms. Margaret Kowalczyk executed most of the graphic illustrations and Mr. Herman Kabe contributed skilled services in their photographic reproduction. We are grateful to the following colleagues for helpful comments and/or provision of preprints of papers that were in press at the time the literature survey for this contribution was in progress: M. Corcoran, California State University, Northridge; M. Fain, M. J. Horton, E. Levi, C. 0. Rambo, and C. A. West, University of California, Los Angeles; P. Mayerson, University of California, Irvine. Ms. Maureen Gardner and Ms. Jean Sartor provided valued bibliographic support. Ms. Phong Hua and Ms. Mary Seraydarian cheerfully processed the manuscript through its several drafts. Portions of this work were aided by research Grants PCM 80-21829 (NSF), HD 4354, and FX 7009 (USPHS), the Eli Lilly Research Laboratories, and by General Research Funds of the University of California.
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A selection of recent publications provides extension and clarification in several areas covered in the present review. Critical treatment of the process of endocytosis has been provided by Steinman ei al. ( J . CellEiol. 96, 1-27, 1983). Using serial sections, Willingham and Pastan (Proc. Natl. Acad. Sci. U.S.A. 80, 5617-562 I , 1983) have presented evidence that (1) all coated structures participating in endocytosis of concanavalin A by fibroblasts are connected to the cell surface, and (2) endosomes
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containing ligand-receptor coinplexcs appcar to balloon out from membrane adjacent to such coated regions (cf. also, Geure et a / . , Cell 32, 277-287, 1983). Two independent reports provide evidence that tubulin is a major protein constituent of coated vcsiclcs (Pfeffer et a / . , J . Cell Biol. 97, 40-47, 1983; Wiedenmann and Mimms, Binchem. Diophys. Rrs. Cornmun. 115, 3(13-31 1, 1983). In addition, endocytotic vesicles contain an ATP-dependent acidification nicchanism similar to the proton pump charactcrizcd in lysosonics (Yamashiro et ul.. J . Cell Biol. 97, 928-934, 1983). Finally, coated vesicles appear to bear mannose-6-phosphate receptors which face toward the inside of the vesicles (Campbell et u l . , J . Biol. Chem. 258, 2628-2633, 1983). Lienhard (TIDS 8, 125-127, 1983) has reviewed the subject of rapid modulation of pasma niembranc transport rates by hormones that promote vesicular insertion and retrieval of endogenous transport elements. In this regard, TSH is found to stimulate transcytosis of vesicles in thyroid cells (Herzog, J . Cell Riol. 97, 607-617, 1983), while vasopressin elicits the formation of coated pits at the luminal membrane of kidney collecting duct cells [Brown and Orci, Nature ( L u n c h ) 302, 253-25s. 19x31. Duval and colleagues (Biochini. Biuphys. Acfa 737, 409-442, 1983) have reviewed the interactions of steroid hormones with membrane structure and functions. Preliminary studies of Garcia era/. (BiUrhem. Biuphjs. Res. Cummun. 113, 960-966, 1983) suggest that components of chick oviduct progesterone receptor (or possibly minor contaminants in close association) exhibit protein kinase activity. Finally, protcolysis ot f'ibronectin by cathephin D generates regulatory peptides that promote DNA synsthesis in fibroblasts [Humphries and Avad, Nnture (London) 305, 81 1-813, 19831, thus underscoring the evidence already presented of the critical contribution of lysosomal activities at the cell surface, including the extracellular matrix, to modulation of cell function.
INTERNATIONAL REVIEW OF CYTOLOGY. VOL. X8
Neuronal Secretory Systems1 MONACASTEL,*HAROLDGAINER,^
AND
H.-DIETERDELLMANNI.
*Department of Zoologyj Institute of Lge Sciences, Hebrew University of Jerusalem, Jerusalem, Israel; ?Laboratory of Neurochemistry and Neuroimmunology, National Institute for Child Health and Human Development, National Institutes of Health, Bethesda, Maryland; and $Department of Veterinary Anatomy, College of Veterinary Medicine, Iowa State University, Ames, Iowa ....................................... kaging in Peptidergic Neurosecretory Cells. . . . A. The Neurohypophysial Secretory Products, . , , . . . , . , . . . . . . . . B. Separate Hormones and Separate Neurophysins in Separate
....................................... Neurohypophysial Peptides and Neurophysins . D. The Neurosecretory Granulated as a Site of Posttranslational Processing. . . .................... E. Biosynthesis and Functional Ac 111. Morphological Aspects of the Formation of Peptidergic Neurosecretory Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. The Perikaryal Endoplasmic Reticulum . . . . . . . . . . . . . . . . . . . . B. “Colloid Droplets” . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. The Golgi-GERL System.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Additional Peptides in Vasopressin and Oxytocin Neurons . . . . IV. Axonal Transport in Neurosecretory Cells . . . . . . . . . . . . . . . . . . . . . . A. Multiple Components (Waves) of Axonal Tran B. Axonal Transport in the Hypothalamic-Neurohypophysla System _ . . . . . . . . . . . . _ . . . . _ . . . . . . . . . . . . . V. Morphology of Transport and Release-Peptidergic Neurons . . . . . . A. Anterograde Transport of Neurosecretory Granulated Vesicles . B. The Axonal Smooth Endoplasmic Reticu C. Hormone Pools in the Neurohypophysis D. Microvesicles . . . . , . . , . . . . . , . , , , , , , . . . , , , . . . . . . . . . . . . . . E. The Lysosomal System . . . . . . . , . . . . . . VI. Molecular Organization of Secretory Vesicles A. Physical Characteristics of Secretory Vesicles , . . . . . . . . . . . . . . B. The Cholinergic Vesicle . . . . , . . . , . . . . . . . . . . . . . . . . . . . . . . . C. Vesicles in Sympathetic Neurons . . . , , . . . . . . . . . . . . . . . . . . . . D. The Chromaffin Granule.. . . . . . , . , , , . . . . . . . . . . . . . . . . . . . . E. Neurosecrctory Granulated Vesicles . . . . . . . . . . . . . . . . . . . . . . . VII. Biosynthesis and Biochemical Aspects of Packaging and Transport of Neurotransmitters in Nonpeptidergic Neurons. . . . . . . . . . . . . . . . . .. .. ,.. . A. Cholinergic Neurons . , . . , . , . , . . . . . , . . . . . . B. Catecholamine-Containing Cells . , , . , . . . . . . . . . . . . . . . . . . . . . C. Catecholamine Uptake into Chromaffin Vesicles. . . . . . . . . . . . .
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‘Dedicated to Prof. Berta Scharrer with esteem and affection
303 Cupyrighl I984 hy Academic I ’ r w . lnc All right5 of rcproduclm in any lorin roerved
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Morphological Aspects of Formation of Nonpeptidergic Secretory Vesicles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. The Adrcnomedullary Chromaffin Cell . . . . . . . . . . B . Visualization of Biogenic Amines in Neurons. . . . . , . . . . . . . . . C. Immunocytocheinistry of Biogenic Amines . . . . . . . D. Life Cycles of Vesicle5 in Adrenergic Ncurons. . . . . . . . . . . . . . E. Vesicle Life Cycle in an Identified Serotonergic Neuron F. Vesicles in Cholinergic Neurons. . , . . . . . . . . . . . . . . . . . . . . . . . IX. Developmental Aspects of the Hypothalamic-Neurohypophysial System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Magnocellular Hypothalamic Nuclei B. Median Eminence and Neurohypophysis . . . . . . . . . . . . . . . . . . . C. Neurohormones in the Hypothalamus D. Neurohormones in the Ncurohypophy E. Ncurohormones in the Plasma. X. Versatility of Neurosccrctory Ncuro A . Cells and Projections beyond the Hypothalamus. . . . B . Peptidcrgic Synapses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Nonsynaptic Release in the Brain D. In Conclusion.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .........,,....... References . . . . . . . , . . . . . . . . . . . . Note Added in Proof
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“There is no real difference between structure and function; they arc two sides ofthe same coin. If structure does not tell us anything about function, it means we havc not lookcd at it correctly.” (1951) A. SZ.~NT-GYOKGI
I. Introduction “Neurosecretory neurons can be defined as nerve cclls that engage in secretory activity to a degree that greatly surpasses that of conventional neurons, and which is comparable to that of gland cells” (B. Scharrer, 1978). This characterization cnconipasses the views put forward by Speidel (1919) and E. Scharrer ( 1928) in their pioneering studies on neurosecretory cells and, unlike many other attempts at a dcfinition of this type of neuron (for history see Knowles, 1974), has now found wide acceptance. In fact, it was thc glandular nature of the neurosecretory neuron that first caused it to attract the attention of a wide variety of biologists (for reviews on neurosecretion see Berlind, 1977; Mason and Bern, 1977; Yagi and Iwasaki, 1977; Morris e r a / . , 1978; Pickering, 1978; Madrell and Nordmann, 1979; Silverman and Zimnierman, 1983). However, the significance of the neurosecretory ccll today extends far beyond its original concept, for with
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the realization that the “peptidergic neuron” is a major neuronal component in the functioning of the nervous system (Gainer, 1977; Gotto et al., 1979; Hokfelt et al., 1980), it is apparent that it is no longer an isolated phenomenon. The functional significance of the hypothalamic neurohypophysial system (HNS)2 was established over 30 years ago with the aid of classical histochemical methods, that identified the neurosecretory material by its high cysteine content (Bargmann, 1949; Bargmann and Scharrer, 1951; Sloper, 1955). Since that time the HNS has been extensively studied by many other techniques and approaches (for reviews see Sachs et al., 1969; Sachs, 1970; Cross et al., 1975; Mason and Bern, 1977; Morris et a / ., 1978; Cohen et al., 1979; Brownstein et al., 1980). The neurohormones, oxytocin (OT) and vasopressin (VP), are synthesized by magnocellular neurons located in the bilateral supraoptic (SON), paraventricular (PVN), and accessory nuclei of the mammalian hypothalamus (see Fig. I), and are intracellularly transported to the neurohypophysis where they are secreted into the circulatory system. Similar neurosecretory pathways elaborating structurally related peptides are found in the lower vertebrates. Historically the focus has been on the neurohemal location of the nerve terminals in the neurohypophysis, however, within the past decade studies have shown that some of these neurons (particularly in the PVN) project to other regions of the central nervous system (CNS) (see Section X). These include projections to the median eminence of the hypothalamus (DeMey et al., 1975; Dierickx et al., 1976; Silverman, 1976; Zimmerman, 1976), as well as to other diencephalic and mesencephalic subcortical brain areas, medulla oblongata, and spinal cord (Swanson, 1977; Buijs, 1978; Buijs et al., 1978; Sofroniew and Weindl, 1978a, 1981; Swanson and Sawchenko, 1980, 1983). In addition, parvicellular neurons in the suprachiasmatic nucleus (SCN) (Fig. 1) synthesizing VP (but not OT) have been shown by immunocytochemical methods to project to extrahypothalamic areas (Buijs, 1978; Sofroniew and Weindl, 1978b, 1981). Immunoultrastructural studies suggest that at least some extrahypothalamic projections terminate as synaptic-like endings (Buijs and Swaab, 1979; Sterba et al., 1979) (see Section X,B). ’Abbreviations: AChE, acetylcholine esterase; AcPase, acid phosphatase; aSER, axonal smooth endoplasmic reticulum; cDNA, complement~yDNA; ChAt, choline acetyltransferase; CNS, central nervous system; COMT, catechol-o-methyltransferase; cpm, counts per minute; DBH, dopamine-@hydroxylase; DDC, Dopa-decarboxylase; DOPA, dihydroxyphenyl-alanine;dpn, days postnatal; fd, fetal day; GCN, giant cerebral ganglion; GERL, Golgi associated endoplasmic reticulum and lysosomes; HNS, hypothalamic neurohypophysial neurosecretory system; LDCV, large dense-core vesicle; MAO, monoamine oxidase; ME, median eminence; mRNA, messenger RNA; MW, molecular weight; NGV, neurosecretory granulated vesicle; OT, oxytocin; PNMT, phenylethanolamine N methyl transferase; pp. posterior pituitary; PVN, paraventricular nucleus; RER. rough endoplasmic reticulum; RIA, radioimmunoassay; SCN, suprachiasmatic nucleus; SDCV, small dense-core vesicle; SON, supraoptic nucleus; TH, tyrosine hydroxylase; TPPase, thiamine pyrophosphatase; VP, vasopressin.
FIG. 1. Hypothalamus of‘ desert mouse, Acornys cahirinus; light microscopy; preembedding immunocytochemical staining of vibratome scction with antivasopressin as primary antiserum in Sternberger’s pcroxidase-antiperoxidase procedure. (a) Paraventricular nucleus (PVN), supraoptic nucleus (SON), accessory nuclei (Acc), suprachiasmatic nucleus (SCN); note immunoreactive proceqses coursing from PVN and Acc toward ventral hypothalamus, while fine processes from SCN appear to course in dorsal direction. Profusion of periventricular VP-immunoreactive perikarya is characteristic of Acomys cohirinrts. F, Fornix, Op, optic tract. (b) Magnification of two VP-immunoreactive neurons from Acc, illustrating Golgi-like “impregnation.”
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Recently, small VP-imniunoreactive cells were identified in several additional brain locations, not associated with the hypothalamic nuclei (Van Leeuwen and Caffe, 1983; Caffe and Van Leeuwen, 1983) (see Section X,A). It has been argued that the neurons of the HNS are exceptionally useful models for study of the cell biology of secretion (Pickering, 1978, 1981; Gainer et al., 1979), since three major aspects of the secretory process are topographically segregated: biosynthesis and packaging of secretory peptides in the hypothalamic perikarya, transport of secretory vesicles via the axons that traverse the infundibular stalk, and secretion from the nerve terminals in the neurohypophysis. Considerable work has been doen on these three processes in the magnocellular neurons, and perhaps it is not surprising (in view of the “glandular” nature of these cells), that much of the conceptualization has been dominated by the Palade scheme (Palade, 1975) of the secretory process. It is our purpose in this review to critically examine these processes in the magnocellular neurons, and to consider to what extent they are variations of Palade’s model, and whether they display any unique characteristics, particularly in comparison with other types of neurons. Some of the following questions will be addressed in this review. 1 . What is the molecular organization of the secretory vesicle, and how does it relate to the biosynthesis of its contents? 2 . Which intracellular compartments are involved in the formation of the secretory vesicle, and what is the contribution of each compartment? 3. How is the secretory material transported intraaxonally, how is it secreted, and what is the fate of the retrieved membrane’? 4. What is known about the organization of the axonal smooth endoplasmic reticulum (aSER) and related membrane systems and what purpose do they serve‘? 5 . How does the functioning of the magnocellular neurons compare to “nonpeptidergic’ ’ neurons (aminergic and cholinergic)‘? 6. How does the ontogeny of the neurosecretory neuron contribute to an understanding of its function in the adult? 7. In what ways d o the so-called neurohypophysial peptides enlarge their spectrum of neurochemical mediation within the brain, and how does this modify the classical concept of neurosecretion’?
Some of these issues, particularly with regard to nonpeptidergic neurons, have recently been reviewed by Holtzman and his associates (Holtzman, 1977; Holtzman et al., 1977; Holtzman and Mercurio, 1980). Hence, this review will address selective points as they relate by comparison or contrast to definition of peptidergic neurosecretory cells.
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11. Biosynthesis and Packaging of Peptidergic Neurosecretory Cells
Aminergic and cholinergic neurons differ significantly from one another in the organization of their transmitter bisynthesis mechanisms (for instance, catecholamine synthesis involves cytosolic and intravesicular enzymes, whereas acetylcholine is made in the cytosol only), but peptidergic neurosecretory cells differ even more radically in this regard. Whereas catecholamines and acetylcholine are synthesized by enzymes (see Section Vll), and most of the synthesis occurs at nerve terminals, neuropeptides are made by de novo protein synthesis on ribosomes of the rough endoplasmic reticulum (RER) in the cell body (see Section 111). The cell biological implications of this difference are manifold, and the significance of this with regard to the organization of the cell will be considered here and in Section VIII. The best studied, prototypic peptidergic neurosecretory cells are the magnocellular neurons of the HNS that secrete nonapeptides (VP and OT) as well as proteins (neurophysins) into the circulatory system. Hence, this section will deal with the cell biology of this group of neurons, from a biochemical point of view. SECRETORY PRODrJCTS A. THENEUROHYPOPHYSIAL
Ever since their discovery by Du Vigneaud and his colleagues (Du Vigneaud, 19561, the neurohypophysial hormones have been studied in about 40 species belonging to 7 vertebrate classes (Acher, 1978). These hormones, irrespective of the vertebrate class i n which they are found, are all nonapeptides with a hemicystinyl residue at the aminoterininus linked by a disulfide bond to a cysteine at position 6, and a glycinamide at the carboxyl terminus (see Fig. 2 , for OT and VP). Amino acid variations occur only in residues 3, 4, and 8 producing the
different types of neurohypophysial hormones. Comparative studies show that, with the exception of the cyclostomes which appear to produce only arginine vasotocin, each vertebrate class is characterized by two distinct neurohypophysial peptides. Mammals synthesize oxytocin and arginine vasopressin (AVP) ( Lys8 vasopressin in pigs and related species). Nonmammalian tetrapods have iuesotocin (Ile8-oxytocin) and arginine vasotocin (Arg8-oxytocin), whereas fishes produce arginine vasotocin and isotocin (SerJ-Ilex-oxytocin in bony fishes) or a more heterogeneous set of oxytocin-like peptides (in cartilaginous fishes). On the basis of these comparative biochemical data, Acher (1 978) hypothesized that there were two evolutionary lines, an oxytocin line with 3 steps (isotocin, mesotocin, and oxytocin) and a vaspressin line (vasotocin, vasopressin), and that the abrupt change in both peptides in pituitaries of nonmammalian tetrapods versus mammals may be correlated with the emergence of new functions, i.e., lactation and antidiuresis. The biological activities of these diverse molecules have been reviewed (Altura and Altura, 1977; Jard and Bockaert, 1975; Soloff and Pearl-
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VASOPRESSIN
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383&33NH2 OXYTOCIN
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9
Schematic presentation of amino acid sequence in arginine-vasopressin and oxytocin.
mutter, 1979) and conformational studies on OT and VP related to their biological activities have been published (Urry and Walter, 1971; Walter, 1977; see also Cohen et al., 1979). Each of the neurohypophysial hormones is associated in situ with a specific, acidic, low-molecular-weight protein (MW = l0,OOO) with which it is synthesized, packaged in granulated vesicles, intraaxonally transported, and coordinately secreted (see below). These proteins are rich in cysteine (about 16% of the amino acid content linked by disulfide bonds), glycine, and proline residues, and are known collectively as the neurophysins (Acher, 1978; 1979; Breslow, 1979; Cohen et al., 1979; Pickering and Jones, 1979). The neurophysins are also called “carrier proteins” because of their ability to bind the neurohypophysial peptides (maximum binding constant about l o p s M - I ) , and because of their intravesicular location in amounts equimolar to the peptides. Although chemically distinct neurophysins are separately associated in vivo with OT and VP, respectively, this biological compartmentation does not signify a selectivity in binding since either nonapeptide can be bound by either neurophysin in vitro. More than 9 distinct mammalian neurophysins have been completely or partially sequenced. There appears to be a considerable amount of sequence homology between the different neurophysins (see Breslow, 1979; Pickering and Jones, 1979), and in general, the central part of the molecule (residues 10-74) shows the least variation. Positions 38-57 of the molecule, which are virtually invariant, are found in a polar region. Another interesting structural feature of the neurophysins is that there appears to be sequence duplication in the single polypeptide chain (Breslow, 1979; Pickering and Jones, 1979). For example, residues 12-31 have a greater than 50% homology with residues 60-77, in contrast
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MONA CASTEL ET A L
to residues 32-59 in the polypeptide which d o not appear to be duplicated. This observation has led to the proposal that the origin of neurophysin was, in part, via an ancestral gene coding for a 50-60 amino acid peptide that underwent gene duplication and partial fusion to yield the archetypal neurophysin gene. Extensive studies have been done on the in v i m interactions between the nonapeptides and neurophysins, and these have been reviewed (Breslow, 1979; 1979). In summary, these studies indicate that there are potentially Cohen et d., two binding sites on neurophysin for the nonapeptides, a strong binding site for both OT or VP (with a maximum binding constant between 10' and lo6 M - l at 25"C), and a markedly weaker site for VP (at standard salt conditions). The strong binding site is well characterized, and is believed to represent the principal site for the I :1 binding of hormone to neurophysin in situ. Two-thirds of the free energy of binding at the strong binding site resides in the first three amino acids of the nonapeptide, and tripeptides (e.g., methionine-tyrosine-phenylalanine) and have been used as model compounds for binding studies. Only peptides with hydrophobic amino acids in position 2 bind (tyrosine or phenylalanine). A free N-terminus in the peptides is also required for electrostatic interaction with an ionized carboxyl ion (presumably Glu3') in the neurophysin molecule, and the amino acid in position 3 of the peptide has a weak apolar interaction. In contrast to the weaker site, the binding of peptides to the strong binding site is not effected by nitration of TyflY in the neurophysin. The weak binding site binds VP, but not OT under standard salt conditions, however, in 1.4 M LiCl oxytocin will also bind. Nitration of Tyfl9 blocks binding by this site, and it is believed that binding at this site involes the Asp77 (or GIu'~)and T y F of the neurophysin molecule. The physiological significance of the second binding site is not clear, and several possibilities are discussed by Breslow (1979).
B.
SEPARATE
HORMONES AND
SEPARATE NEUROPHYSINS IN SEPARATE CELLS
Earlier studies suggested that OT and VP were made in separate nuclei in the hypothalamus. This is now known not to be the case, and the peptides are found about equally distributed in the SON, the PVN, and the accessory nuclei (Zimmerman and Defendini, 1977; Rhodes et u l . , 1981). It is now generally accepted, largely on the basis of immunohistochemical studies (see Dierickx, 1980), that OT and VP (and their respective neurophysins) are located in separate neurons (Dierickx et a/., 1977; Zimmerman ef al., 1977; Rhodes et a / . , 1981). Physiological studies also support the idea that the peptide hormones are synthesized in and secreted from separate cells. Legros et ul. (1975a,b) have shown in cows that neurophysin II (VP-associated neurophysin) is selectively released during hemorrhage, whereas neurophysin I (OT-associated neurophysin) is selectively secreted during milking and suckling. In the human, VP and OT associated neurophysins are selectively secreted into the blood in response to stimulation by nicotine and estrogen, respectively (Seif and Robinson, 1978).
NEURONAL SECRETORY SYSTEMS
311
Electrophysiological studies in the rat also support this concept. Two distinct cell types in the supraoptic (SON) and paraventricular (PVN) nuclei can be distinguished physiologically; the oxytocin cell which selectively responds to the suckling stimulus with a rapid (2-4 second) burst of impusles causing a release of oxytocin into the blood (i.e., the milk ejection reflex; Wakerley and Lincoln, 1973; Lincoln and Wakerley, 1974), and the vasopressin cell which responds relatively selectively to hemorrhage as a stimulus (Poulain et al., 1977; Wakerly et al., 1975). Both cell types, however, are osmoresponsive (Brimble and Dyball, 1977). Poulain and Wakerley (1982) have recently reviewed the electrophysiology of hypothalamic magnocellular neurons secreting OT and VP. OF C. BIOSYNTHESIS
THE
NEUROHYPOPHYSIAL PEPTIDESAND NEUROPHYSINS
The pioneering work of Howard Sachs and his colleagues (Sachs and Takabatake, 1964; Takabatake and Sachs, 1964; Sachs er al., 1969) generated the concept that the neurohypophysial peptides and neurophysins were synthesized as common precursors (prohormones) on ribosomes in the cell perikaryon and subsequently cleaved enzymatically to their peptide end products. The legacy of experimental data offered by the Sachs group to support this hypothesis is as follows: 1. The synthesis of VP occurs only in the hypothalamus (the site of the magnocellular perikarya) and not in the neurohypophysis (containing axons and terminals of the system). 2. There was a 1- 1.S hour lag period between the administration of ['?3Jcysteine and the appearance of labeled VP in the dog hypothalamus in vivo, and in guinea pig hypothalamic slices in vitro. The protein synthesis inhibitor, puromycin, if applied before the pulse-label, completely inhibited the incorporation of [3sS]cysteine into VP. However, if puromycin was administered after the pulse-label the incorporation of radioactivity into VP was not inhibited. 3. There was parallel synthesis and axonal transport of the peptides and the neurophysins (Fawcett et al., 1968; and see Pickering, 1978, for a review of these studies) suggesting a common precursor. Consistent with this view was the finding of Sachs et al. (1969) that inhibition of neurophysin synthesis by incubation of hypothalamic tissue in analogs of amino acids found in neurophysin but not VP also inhibited the synthesis of VP. The absence of specifically the VPassociated neurophysin in Brattleboro rats, a strain with a genetic deficiency in VP synthesis (Valtin et al., 1974), also supported the hypothesis of a common precursor. Finally, the physicochemical properties of the neurophysins indicated that these proteins were derived from a larger protein (Chaiken et ul., 1975).
The key issue was whether biosynthetic evidence could be obtained for the existence and identity of the putative common precursors of OT and its neu-
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MONA CASTEL ET AL.
rophysin, and VP and its neurophysin. The paradigm for such biosynthetic studies had been established by Steiner and his colleagues (Steiner and Oyer, 1967; Steiner et al.. 1974) in their search for the prohormone of insulin, proinsulin. Since the discovery of proinsulin, several other prohormones have been identified (see Habener and Kronenberg, 1978) including the common precursors of ACTH and P-lipotrophin (Mains et al., 1977; Nakanishi et al., 1978; Freedman and Hawkins, 1980; Koch and Richter, 1980; E. A. Zimmerman et al., 1980). In addition, it is now known that prohormones are not the original de novo synthesized proteins, but that longer proteins (preprohormones) extended at their N-terminals by so-called “signal sequences” are synthesized by the mRNAKER complex (Habener and Kronenberg, 1978; Campbell and Bloebel, 1976; Lingappa et ul., 1978; Rubenstein et al., 1977). The “signal sequence” is inimediately removed enzymatically when the protein is transported into the RER cisternae, and therefore preprohormones can be isolated only in vitro (cell free) translation experiments. Pulse-chase experiments in the hypothalamus of dogs and guinea pigs (Walter er uf., 1977; Mendelson and Walter, 1978) gave some indication of quickly labeled proteins which declined in radioactivity as neurophysin increased in radioactivity, but no identification of a specific precursor protein was possible in these experiments. An experimental approach based on a conceptual model of the peptidergic neuron in which the peptide is first synthesized as a precursor molecule on the KER in the cell perikaryon, and then packaged in secretory vesicles in the Golgi apparatus before axonal transport (the vesicles were presumed to be packaged with prohormones; see Gainer ct ( I / . , 1977a), was used to identil‘y these precursors (Gainer et d., 19773-c; Brownstein and Gainer, 1977; Brownstein r t al., 1977; Gainer and Brownstein, 1978a; for review of this work see Brownstein et u l . , 1980). The rationale of this approach was that if the prohormone were pickaged into secretory vesicles which were then rapidly transported down the axon to the terminal, one might expect to detect the prohormone in the axon and to witness its subsequent conversion to neurophysin and pcptides intraaxonally. This was observed in pulse-chase experiments in the rat, when [‘sSlcysteine was injected near the SON, and a 3sS-labeled 20,000 MW ncurophysin precursor could be idcntificd, which was transported by and converted to neurophysin in the axons of the median eminence (Gainer er a / . , 1977b,c). Similar data were obtained from samples of the SON after short pulses (Gainer et d., 1977~; Gainer and Brownstein, 1978a) and the 3sS-labeled 20,000 MW putative precursor could be iiiirnunoprecipitated by antibodies to neurophysin (Brownstein et t r l . , 1977; Gainer and Brownstein. 1978a). The first direct biosynthetic evidence favoring the common precursor hypothesis was obtained by further analysis of the labeled neurophysin precursors by isoclectric focusing, showing that it was actually comoosed of two proteins, one
NEURONAL SECRETORY SYSTEMS
313
with a pl (isoelectric point) of 5.4, and the other with a pl of 6.1. The latter precursor was absent from the SON of the VP-deficient Brattleboro rat, and hence was identified as the VP-related neurophysin precursor (Brownstein and Gainer, 1977; Gainer and Brownstein, 1978a). Evidence that these precursors for neurophysins also contained the nonapeptides, and were therefore common precursors, was obtained by analysis of tryptic peptides generated from the two precursors (Russell et ul., 1979, 1980a; Brownstein e t a / . , 1977, 1980). The pl 6. I , 20,000 MW precursor for VP and its associated neurophysin (propressophysin) is a glycoprotein, whereas the pi 5.4, 16,000 MW precursor for OT and its associated neurophysin (prooxyphysin) is not (Brownstein et a / ., 1980; Russell et al., 1980b). Tentative models based on this in vivo data for propressophysin and prooxyphysin were suggested, the order of the peptide components being deduced from the analysis of cyanogen bromide cleavage products derived from the precursors (Russell et al., 1981). The presence of a glycopeptide moiety in propressophysin is indicated by the binding of this prohormone (but not prooxyphysin) to concanavalin A affinity columns, and the incorporation of labeled fucose into propressophysin. Furthermore, a small fucosylated glycopeptide is synthesized in the SON and transported to the posterior pituitary of normal rats (Gainer and Brownstein, 1978b; Russell et a / . , 1980b) but not of VP-deficient Brattleboro rats (Russell e t a / . , 1980a). These data are consistent with the identification of the propressophysin as a glycoprotein, and it has recently become apparent from recombinant DNA studies (see below) that a glycopeptide which was discovered in the porcine pituitary and sequenced (Holwerda, 1972a,b) is related to this glycopeptide on propressophysin. Moreover, the validity of these data has now been bourne out by the recent deduced amino acid sequences of the VP and OT preprophomones from the nucleotide sequences of the cloned cDNAs in which they were encoded (Land et ul., 1982, 1983) (see Fig. 3). Several laboratories have shown that mRNA isolated from bovine, mouse, or rat hypothalami could serve as templates in cell-free translation systems to synthesize proteins which were immunoprecipitable by neurophysin antisera, and which were comparable in molecular weight to the in vivo synthesized precursors (Guidice and Chaiken, 1979; Lin et u / . , 1979; Schmale et al. 1979). The most VASOPRESSIN-PREPROHORMONE >*-
0
*
25% SIGNAL
AVP
I]I
NEUROPHYSIN II
I I GLYCOPEPTIDE
OXYTOCIN- PREPROHORMONE
2I
tinu
i5f
SIGNAL
I OT I [ I I
NEUROPHYSIN
I
FIG. 3. S t r u c t u r c s of preprohormoncs for bovine vasopressin (AVP) and oxytocin (OT), adapted from Schrnale er ul. (1983).
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MONA CASTEL ET AL
extensive series of such in vitro translation experiments have been performed by Schmale, Richter, and their colleagues (Schmale et al., 1979; Richter et al., 1980; Schmale and Richter, 1980, 1981a-c; h e l l et al., 1981). These authors have shown the following: 1. Bovine hypothalamic mRNA can be used to synthesize separate common precursors for VP and bovine neurophysin 11, as well as for OT and bovine neurophysin I. 2. Both in vitro synthesized precursors can be converted from preprohormones to prohormones by dog liver microsomes, but only the VP prohormone is glycosylated. 3. The in vitro translated VP preprohormone is 21,000 daltons, is cleaved to a 19,000 dalton prohormone (if glycosylation is prevented by tunicamycin), but the prohormone is 23,000 daltons if glycosylation is allowed to proceed n vitro. 4. The in vitro synthesized O T preprohormone is about 16,500 daltons, and is cleaved to a 15,500 dalton prohormone which is not glycosylated. 5 . Tryptic maps of the VP preprohormone and prohormone indicate that the VP follows the signal sequence and precedes the neurophysin II moiety in the preprohormone. The results of these in vitro translation experiments are in complete agreement with the conclusions deduced from the in vivo studies described above.
‘The most recent advance in the elucidation of the VP precursor has come from recombinant DNA technology. Land et (11. (1982) have recently reported the entire nucleotide sequence of cloned cDNA encoding the bovine VP-neurophysin 11 preprohormone. The aniino acid sequence contains 166 amino acids, the first I9 belonging to the signal sequence. Figure 4 illustrates certain important features of this preprohormone. The order of peptide components in the preprohormone is as deduced from the in vivo and in vitro studies described above. Several important new pieces of information are provided by this data: 1. The cleavage site between AVP and neurophysin 11 contains Gly-Lys-Arg (positions 10- 12), a sequence characteristic of prohormones that will be cleaved and subsequently C-terminally amidated (as in the case of AVP). 2. The cleavage site between neurophysin I1 and the 39 amino acid C-terminal peptide (at position 108) is a single Arg, which is uncharacteristic since pairs of basic amino acids are usually found at such sites. 3. The 39 amino and C-terminal peptide has an amino acid sequence (in positions I 14- 1 16) of Asn-Ala-Thr, characteristic of asparagine-linked glycopeptides. The sequence of this 39 amino acid peptide is identical to peptides previously isolated by Smyth and Massey (1979) and Holwerda ( I 972a,b) from pig, sheep, and bovine pituitaries. Until this recombinant DNA study was done it was not realized that these glycopeptides were part of the VP precursor.
315
NEURONAL SECRETORY SYSTEMS
@ Signalase -1
$ - +2HN
@Amidati 6
1
AVP
-s---s-
4 @ Disulfide Bond Formation
13
Neurophysin II
9
Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Arg-Gly
Ala-
(
7 -S-S-bonds 1
+lo
12
11
Gly-Lys-Arg-
t t Enzymes
@Converting
107 108
104
-Pro-Arg-Arg-Val
1
Arg-
22-97
109 Glycopept&
114
Ala-
116
-Am-Ala-Thr-
-Tyr
COOH
/b
@ Glycosylation FIG. 4. Structure of the bovinc arginine vasopressin-neurophysin I1 precursor. Data obtained from recombinant DNA studies (Land ci a / ., 1982). The arrows (A-E) denote posttranslational cvents occurring at specific sites in the precursor.
Thus, 18 years after its first proposal, the common precursor for VP and neurophysin has been fully identified and characterized. There have been suggestions in the literature for yet larger precursors of AVP and neurophysin (i.e., around 80-140,000 daltons). It is important to note, however, that these reports (Lauber et al., 1979; 1981; Camier et al., 1979; Beguin et id., 1981) do not include in vivo or in vitro biosynthesis or recombinant DNA data. Until such data are available, such putative precursors remain in doubt. The amino acid sequence of the bovine OT-precursor, derived from analysis of the relevant cDNA, was reported within a year of the VP-precursor sequence (Land et al., 1983). As in the latter case, the signal sequence is followed by the nonapeptide hormone which is connected to the neurophysin via Gly-Lys-Arg. An interesting feature is the 197-nucleotide perfect homology in the middle part of the sequences encoding neurophysins I and 11. D. THE NEUROSECRETORY GRANULATED VESICLEAS POSTTRANSLATIONAL PROCESSING
A
SITEOF
Conversion of the putative prohormone to its peptide products intragranularly during axonal transport was suggested by the early work of Sachs (Sachs, 1963a,b; Sachs et al., 1969). He found that secretory granules isolated from the hypothalamus of dogs preinfused with labeled cysteine contained VP of much lower specific activity than secretory granules isolated from the neurohypophysis, and that hypophysial granules contained 5-fold more VP than the hypothalamus-derived granule fraction (Sachs, 1963a). Sachs concluded that the forma-
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MONA CASTEL ET AL
tion of VP occurred between the hypothalamus and the hypophysis. A quantitative autoradiographic study, at the electron microscopic level, performed by Kent and Williams (1974) is particularly relevant here. These authors showed that proteins labeled with [3H]cysteine in the magnocellular neurons were transported to the neural lobe principally within the neurosecretory granulated vesicles (NGV). Since it is now known that these labeled proteins are largely (in the early times after the pulse) in the form of the prohormones (Gainer et a / . , 1977b,c; Gainer and Brownstein, 1978a), it is then reasonable to conclude that the prohormones are packaged into the secretory vesicles that are transported into the axon. Therefore, since conversion of prohormones to nonapeptides and neurophysins occurs primarily in the axons (Gainer et ul., 1977b,c; Gainer and Brownstein, 1978a), the conclusion that the secretory vesicle is the main site of the posttranslational processing events becomes credible. The conversion process need not occur only in the axon. In the presence of the mitotic inhibitor, colchicine, which inhibits the fast axonal transport of the newly synthesized neurosecretory proteins and produces a pile-up of NGVs in the perikaryon (Norstrijm et ul., 1971; Hindelang-Gertner et a l . . 1976; Morris et al., 1978; Dellmann and Sikora-Van Meter, 1982), the conversion of prohormone to nonapeptides and neurophysins can occur in the ccll body as well. Posttranslational cleavage processes in the secretory vesicle have also been proposed for Aplysia neurosecretory cells (Gainer et al., 1982) as well as secretory p cells in the pancreas (Steiner et al., 1974; Fletcher et al., 1980, 1981). Given this hypothesis that the NGV is a major site of posttranslational processing then one should expect to find the processing enzymes within the NGVs. The arrows in Fig. 4 show what the expected processing events would be for the AVP-neurophysin I1 precursor. The first three events, cleavage of the signal peptide by a signalase, disulfide bond formation, and the core glycosylation, appear to occur during translation and are associated with the endoplasmic reticulum ( M . Zimmerman et al., 1980; Freedman and Hawkins, 1980; Koch and Richter, 1980). A second stage of glycosylation occurs in the Golgi apparatus. The enzymatic clcavage of the prohormone (step D in Fig. 4) at the basic amino acid residues appears to occur in the NGVs, as does the amidation (step E) of the C-terminal glycine of AVP. Because of the nature of the cleavages that convert prohormones to hormones in general (see Habener and Kronenberg, 1978; Stcincr et al., 1974; Tager et al., 1979), the effects of trypsin on the neurohypophysial prohormones (Russell et al., 1979, 1980a), and the sequences shown in Fig. 4, the enzymes predicted to be in the NGVs have been referred to as trypsin-like, carboxypeptidase-B-like, and an amidating enzyme. Previous work by North et al. (1977) described a chymotryptic-like enzyme involved in the degradation of neurophysin as being in the neurosecretory vesicles. This enzyme may be involved in cleaving the glycopeptide moiety in the AVP precursor (see Fig. 4). Tager et al. f 1979) have discussed the difficulties in assaying for such prohormone converting enzymes.
NEURONAL SECRETORY SYSTEMS
317
Recent work in a number of systems, aided by the realization that the NGV’s internal microenvironment is acidic (Russell and Holz, 198l), has produced significant progress in the search for prohormone converting enzymes. Such enzyme activities have now been measured and partially characterized in secretory vesicles isolated from anglerfish pancreas islet cells (Fletcher et al., 1980, 1981), neural and intermediate lobes of the rat pituitary (Loh and Gainer, 1982; Loh and Chang, 1982), and bovine posterior pituitary (Chang et al., 1982). The enzyme activities in all of these secretory vesicles are due to unique acid, thiol proteases which specifically cleave at pairs of basic amino acids (e.g., Lys-Arg). Enzyme inhibitor studies indicate these enzymes are distinct from pancreatic trypsin and lysosomal cathepsin B , and they have been referred to as prohormone converting enzymes. Whether they represent a new family of intracellular enzymes specific for prohormone processing is currently under study. VP and OT secretory vesicles (NGVs), when isolated from bovine neural lobes and lysed, are imbued with proopiocortin-converting enzyme activity (Chang et al., 1982). It is certainly interesting that NGVs, which are customarily involved in processing VP- and OT-precursors, should be capable of in vitro cleaving of a prohormone associated with the intermediate lobe. Although at first thought it may be dismissed as highly unlikely that this would occur in vivo, it should be bourne in mind that several derivatives of the proopiocortin precursor have recently been localized by RIA within the HNS, and by immunocytochemistry within the same neurons and even the same secretory vesicles as the neurohypophysial hormones (see Section IILD). E. BIOSYNTHESIS AND FUNCTIONAL ACTIVITY
Prolonged secretion of the neurohypophysial hormones, induced by water deprivation or salt loading of rats, causes depletion of hormone stores in the neural lobe to values of about 10% of normal (Jones and Pickering, 1969). Mice, however, more resistant to osmotic stress, may maintain hormone levels in the face of similar challenges (Castel and Abraham, 1969, 1972; Broadwell et al., 1979). In osmotically stressed rats biosynthesis and axonal transport of hormones is augmented about 5-fold (Gainer et al., 1 9 7 7 ~Morris ; et al., 1978). In addition to this increase in biosynthetic rate, the prohormones are posttranslationally processed at a %fold more rapid rate (Russell et a!. , 1980b). As in other neuronal systems (see Section IV), the rate of axonal transport is unaffected by functional activity, and only the amount of newly synthesized neurosecretory material being transported is increased. The increase in rate of processing of the prohormone is consistent with the need for maintaining an augmented secretion rate, under conditions of hormone depletion. In this way the newly synthesized hormone being transported to the axon terminals would be completely processed to its final products before release. The mechanisms which underlie this increase in processing rate (occurring presumably in the secretory vesicles) are not under-
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MONA CASTEL ET AL
stood at present. Possibly bearing on this issue are recent findings demonstrating the ubiquitous presence of axonal smooth- endoplasmic reticulum (aSER) in ncurosccrctory neurons, and its substantial increase during osmotic stress, particularly when NGVs are scarce (Alonso and Assenmacher, l978b, 1979a,b; Caste1 and Dellmann, 1981; Castel et al., 1981). These authors suggest that aSER may participate in hormone transport, especially during dehydration (see Section V for further discussion of this issue). Several possibilities exist to explain how the information about the state of release in the nerve terminal (in the neural lobe) may be signaled to the perikaryon (in the hypothalamus) in order to regulate the biosynthetic level of the latter. These include (1) the action potential itself, which at the terminal is coupled to secretion, in the perikaryon may be coupled to biosynthesis; (2) information about release from the terminal may be relayed to the perikaryon via retrograde axonal transport (possibly in the form of recovered vesicle membranes after exocytosis); and (3) a direct action of synaptic transmitters (conceivably involving second messengers) from the afferent input, could be the signal for increased hormone biosynthesis. In order to decide which, if any, of these possibilities can account for the relevant regulatory signal, it will be necessary to have an in vitrn model in which these variables can be independently controlled.
111. Morphological Aspects of the Formation of Peptidergic Neurosecretory Vesicles The functional morphology of the magnocellular neurosecretory cells has been dealt with in several admirably comprehensive reviews (see especially Mason and Bern, 1977; Morris el al., 1978). The following selective overview is intended to provide a context for the scrutiny of some unresolved issues, and recent data that might contribute to their elucidation. In this section special attention will be accorded phenomena relating to the rough endoplasmic reticulum (RER), functional polarity of the Golgi system, and immunocytochemical evidence for peptides other than VP and OT in magnocellular neurons of the HNS. Section V deals with morphological correlates of NGV maturation, microvesicles, the lysosmal system, and issues relating to the axonal smooth endoplasmic reticulum (aSER). ENDOPLASMIC RETICUILJM A. T H EPERIKARYAL
In unstressed animals the perikaryal Nissl substance of thc majority of magnocellular neurosecretory cells may be visualized at the ultrastructural level as extensive peripheral stacks of flattened RER (Morris and Dyball, 1974; Tweedle and Hatton, 1976) (Fig. SA). In osmotically stimualted specimens there is a
FIG. 5A. Electron micrograph of mouse supraoptic nucleus; portions of two neurosecretory cells; cytochemical reaction for TPPase (thiamine pyrophosphatase) emphasizes the Golgi system (GO); arrowheads indicate some of thc stacks of rough endoplasmic reticulum. N, Nucleus; HB, herring body; Op, optic tract. (From Caste1 and Dellmann, 1980). x25.000.
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MONA CASTEL ET AL
FIG. 5B. Electron micrograph of neurosecretory cell in supraoptic nucleus of of 2-day waterdeprived rat. Arrows point to dilated and fragmented RER. N. Nucleus. x2900.
preponderance of cells with distended and fragmented RER dispersed throughout the cytoplasm (Zambrano and De Robertis, 1966; Kalimo and Rinne, 1972; Picard et al., 1972; Morris and Dyball, 1974; Tweedle and Hatton, 1976) (Fig. SB). RER dilation is generally taken to denote increased synthetic activity, although extreme cases of vacuolation may indicate exhaustion (Kalimo, 1975). Irrespective of the physiological state, lack of synchrony between cells as regards the state of the RER has been noted, and graded intermediate stages between flattened and dilated cisterns are usually apparent (Morris, 1978; Morris et al., 1978). Ultrastructural three-dimensional visualization of the perikaryal endoplasmic reticulum in neurons of the SON has recently been reported using heavy-metal impregnated thick sections viewed at high voltage (Alonso and Assenmacher, 1979b). In control specimens, the peripherally located ER in 70% of the cells appeared as extensive lamellar structures connected to loosely anastomosed tubules, whereas in water-deprived rats 85% of the cells displayed a highly devel-
NEURONAL SECRETORY SYSTEMS
321
oped tubular network throughout the cytoplasm. Concomitant high resolution autoradiography with [3H]leucine indicated that the tubular elements were the most active sites of protein synthesis. Thus it appears that the lamellar structures represent the flattened stacks of RER seen with conventional transmission electron microscopy in controls, while the tubular network is the three-dimensional equivalent of the apparently fragmented and dispersed RER in stimulated cells. However, despite these ultrastructural indications of heightened neurohorrnone synthesis in water-deprived rats, actual perikaryal levels may be significantly lower than those in controls. In immunocytochemically treated hypothalami of 3-day water-deprived rats, VP immunoreactivity in cell bodies was markedly reduced, concomitant with VP-RIA measurements of total hypothalamus (Epstein et al., 1983). Essentially similar VP-RIA results were reported by Negro-Vilar and Samson ( 1979) for individually punched-out magnocellular nuclei of 7-day water-deprived rats. At the ultrastructural level, the moderately electron-dense fine granular content of the RER has long been taken as an indication of newly synthesized protein, especially since it may be abolished with puromycin treatment (Zambrano and De Robertis, 1966). Broadwell et al. (1979), in an elegant immunoelectron microscopic study of the mouse SON, succeeded in providing conclusive evidence for immunoreactive neurophysin (presumably denoting neurohormone precursor) in the RER, including the cistern of the nuclear envelope (Fig. 6). This was achieved by preembedding immunocytochemistry, and by utilizing antibodies to the “carrier” protein, neurophysin. Despite the anticipated problem of antibody penetration, this technique has yielded results that cannot be obtained with the postembedding procedure. The latter method renders membranes virtually invisible, may be deleterious to antigen preservation, leading to false negative results in perikaryal locations (Zimrnerman, 1976), and may also induce nonspecific staining of putative secretory vesicles (Buijs and Swaab, 1979). The choice of antineurophysin as the primary antibody by Broadwell et al. ( 1979) was probably an additional factor conducive to positive results, for the immunoreactivity of the 10,000 MW protein is greater than that of the associated 1000 MW neuropeptide. It is not surprising, given the technical limitations, that in numerous postembedding studies employing antibodies to vasopressin, oxytocin, and even neurophysin, the only immunoreactive organelles identified unequivocally at the ultrastructural level were secretory granules (Pelletier et al., 1974; Silverrnan and Zimmerman, 1975; Castel and Hochman, 1976; Krisch, 1976, 1980a; Morris et al., 1977; van Leeuwen and Swaab, 1977). Despite the success of Broadwell et al. (1979) with antineurophysin as primary antiserum in the preembedding procedure, the same approach using antivasopressin induces mainly diffuse immunoreactive marking on the outer aspect of perikaryal membrane systems and NGVs and on microtubules, suggesting artifactual diffusion
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MONA CASTEL ET AL.
NEURONAL SECRETORY SYSTEMS
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and absorption of the low-molecular-weight antigen during the protracted staining procedure (Castel, Dellmann, and Sivan, unpublished results). An anastomosing network of smooth ER in the cell body and dendrites of HNS neurosecretory cells has been identified with the cytochemical reaction for glucose-6-phosphatase (Broadwell and Cataldo, 1982), known to mark the ER in other types of cells (Leskes et ul., 1971). Glucose-6-phosphatase activity was not found within organelles that are usually acid phosphatase-positive, nor in organelles with endogenous thiamine pyrophosphatase activity (see Sections III,C and V,B). In axons and terminals only sporadic segments of SER showed glucose-6-phosphatase activity.
B. “COLLOIDDROPLETS” There are indications that intracisternal hormone precursors may also assume the form of large, electron-dense accumulations in dilated portions of RER, commonly known as “colloid droplets” (see Figs. 7A and 7B). In the hypothalamic neurosecretory system of fish, amphibia, birds, and reptiles it has long been assumed that the large colloid droplets ( 1 - 12 p,m) in some perikarya represent neurosecretory material (Scharrer, 1928; Scharrer and Scharrer, 1954, for references; Oksche et al., 1963), this assumption having been based mainly on selective neurosecretory staining with aldehyde fuchsin or pseudoisocyanine. At the untrastructural level the “colloid” has been shown to consist of electrondense material within dilated cisterns of the RER (Murakami, 1963, 1964; Gonzales and Rodriguez, 1980). Gonzales and Rodriguez (1 980) succeeded in unmasking neurophysin immunoreactivity within the “colloid” droplets of the lizard SON and PVN. Routine immunocytochemical procedures with antineurophysin were adequate for demonstration of NGV immunoreactivity, but the “colloid droplets” required pretreatment with urea and trypsin in order to unmask their antigenic sites. As limited tryptic hydrolysis is a popular method for cleavage of several identified prohormones (see Section lI,C), Gonzales and Rodriguez reason that their results “strongly suggest that the secretory droplets are stores of neurophysin precursor and that the tryptic digestion has either triggered its conversion into neurophysin or exposed immunoreactive sites otherwise inaccessible.” FIG. 6. Immunoelectron micrographs of neurosecretory perikarya in mouse supraoptic nucleus; prcembedding immunocytocheniistry, antineurophysin primary antiserum in Sternberger’s peroxidase-antiperoxidase procedure. (a) Note neurophysin-immunoreactivity within cisterns of rough endoplasmic reticulum and (b) within the cistern of the nuclear envelope (NE). (c) Neurophysinimmunoreactivity within cisterns of the Golgi system and on some secretory granules (NGV), but not within GERL (arrow) nor on the putative secretory granule arising from GERL. (d) Immunoreactive secondary lysosome. (From Broadwell and Oliver, 1980.) (a) X27,OOO. (b) X39,OOO. (c) X50,OOO. (d) ~ 3 9 , 0 0 0 .
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FIG.7A. Three large “colloid droplets” in neurosecretory cell of lizard supraoptic nucleus. (From Gonzales and Rodriguez, 1980.)
It would be interesting to know whether it is obligatory for normal hormone secretion that these large droplets undergo further processing or packaging in the Golgi cisterns, or whether they represent a form of Golgi bypass as postulated for similar inclusions in the amphibian pars intermedia (Castel, 1972). The most characteristic enzymes of the Golgi apparatus are those related to the transfer of
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FIG,7B. Electron micrograph showing portion of neurosecretory cell from supraoptic nucleus of water-deprived, colchicine-treated rat. Note (upper) heavily electron-dense “colloid droplets” within ribosome-studded ER, and (lower) accumulation of secretory vesicles (NGVs) of medium electron-density. L, Lipid. X24,650.
oligosaccharides to proteins (glycosyltransferases) (MorrC and Ovtracht, 1977), a step that may be essential for packaging of polypeptides into secretory entities (Eylar, 1965; Gonzales et a / . , 1981). However, the colloid droplets in question seem to be already glycosylated within the RER cisterns, judging by histochemical evidence from many sources (see references in Scharrer and Scharrer, 1954; Oksche et al., 1963; Gonzales and Rodriguez, 1980), and in keeping with the possibility that glycosylation of nascent protein molecules may occur while still attached to ribosomes. Speculation about an alternative route for NGV production, that is, directly from the endoplasmic reticulum and not necessarily via the Golgi apparatus, is not merely of theoretical interest, but might have far-reaching implications regarding local formation of secretory vesicles from smooth endoplasmic reticulum within axons, or even alternative modes of hormone transport and release from this reticulum (see Section V,B). While it is noteworthy that mammals, in contrast to fish, amphibia, birds, and reptiles, do not normally store neurosecretory substances in the form of large intracisternal “colloid droplets,” laboratory rats have been experimentally in-
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duced to do so by intracerebral administration of tunicamycin (Gonzales et al., 1981), an antibiotic that selectively blocks glycosylation of polypeptides (Takatsuki et u l . , 1975; Lehle and Tanner, 1976; Loh and Gainer, 1979). Gonzales et at. (198 1 ) reported that after intracisternal injection of tunicamycin, large “colloid droplets” (1-3 pm) (Fig. 7A) bounded by ribosome-studded membrane, appeared in the SON perikarya of the experimental rats, concomitant with an increase in intensity of neurosecretory staining and neurophysin immunoreactivity in the same location. The filling up and distension of the RER cisterns with neurosecretory material was directly proportional to the dose of tunicamycin, while NGV production by the Golgi complex was curtailed and hormone transport to the neurohypophysis severely rcduced. This differs from the block in transport that follows colchicine administration, when NGV movement but not production is hampered (Norstrom et ul., 197 I ; Dustin ef ul., 1975; HindelangGertner et ul., 1976; Dellmann and Sikora-Van Meter, 1982). Thus it appeared that tunicamycin treatment interfered primarily with the packaging of secretory material. The antibiotic is known to inhbit the N-linked attachment of glycoside to nascent polypeptide chains in the RER (Lehle and Tanner, 1976), and the block in secretory packaging appeared to be somewhere between the RER and the Golgi apparatus. Gonzalez et a/. (1981) concluded that the tunicamycin syndrome is probably based on interference in .glycosylation of the prohormone. This conclusion may be premature for several reasons: (1) apart from the prohormone, many other glycoproteins which are membrane proteins will also be unglycosylated by tunicamycin, and could be the cause of the packaging block; (2) the packaging block seemed to be an overall phenomenon in the magnocellular secretory neurons, but only the VP-prohormone and not the oxytocin prohormone is glycosylated (see Section 11,C); ( 3 ) the cited analogy between naturally occurring and tunicatuycin-induccd “colloid droplets” seems inappropriate, since there is little doubt. about the glycoprotein content of the former. Given these considerations, it is more likely that the block in packaging of hormones caused by tunicamycin is due to unglycosylated vesicle membrane proteins rather than prohormones. “Colloid droplets” within the RER are normally a rare occurrence in mammals. However, a clearly discernible increase in both “colloid droplets” and NGVs may be induced in rat neurosecretory perikarya by a combination of saline drinking, that increases neurohypophysial hormone production, and colchicine treatment that interferes with transport of hormone out of the cell body (Fig. 78).
C. TIIEGOLGI-GERL SYSTEM The prohormones, propressophysin and prooxyphysin (Russell et d., 1979), segregated within the RER cisterns, presumably transfer vectorially to the Golgi system for further processing and packaging into secretory entities, the NGVs. Within the Golgi elements the fate of the neurosecretory material may involve
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condensation by removal of water, modifications of the sugar moieties on the glycopeptide sequence of propressophysin, sulfation, and even partial proteolysis [see Favard (1977), More6 (1977), and Farquhar and Palade (1981) for basic general information regarding functional events in the Golgi apparatus, and Goldfisher ( I 982) for cytochemical aspects]. It is presumably within the Golgi saccules that the prohormones become associated with the proteases destined to effect their eventual cleavage within the NGVs during their passage along the axon (Gainer et a / . , 1977a,b; North et al., 1977). The apparent low activity of these proteases in the Golgi region may indicate that the pH and other conditions are suboptimal at this location in comparison with those in the axon (see Section I1,D for discussion of the posttranslational cleavage of prohormones). Ultrastructural studies reveal several Golgi configurations, generally in perinuclear array, in each neurosecretory cell (Osinchak, 1964; Kalimo, 1971; Picard et al., 1972; Broadwell and Oliver, 1981) (see Fig. 5A). Increase in absolute volume of the Golgi complex has been measured after dehydration (Reinhardt et a/., 1969); while proliferation and fragmentation have been reported following a variety of stresses (Zambrano and De Robertis, 1968; Kalimo and Rinne, 1972; Picard e t a / . , 1972; Fig. 8B). Each Golgi stack is composed of
FIG. 8A. Supraoptic nucleus of normal mouse. Electron micrograph showing the Golgi apparatus and GERL. A putative neurosecretory granulated vesicle (NGV) is forming from GERL. RER, Rough endoplasmic reticulum. (From Broadwell and Oliver, 1980.) X50,OOO.
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FIG.8 8 . Supraoptic nucleus of saline-drinking mouse. Electron micrograph showing the Golgi apparatus and GERL (arrow); neurosecretory granulated vesicles seem to be forming from all cisterns, including GERL. (From Broadwell and Oliver, 1980.) X30,OOO.
3-5 closely apposed cisterns and additional GERL (Golgi associated Endoplasmic Reticulum that gives rise to Lysosomes) elements aligned at a slight distance from the last or innermost Golgi cistern (Osinchak, 1964; Picard ez al., 1972,1978; Kalirno, 1975; Broadwell andoliver, 1981). (Figs. 8A and8B). Most cisterns reveal some degree of fenestration which increases during osmotic stimulation, and is especially obvious in tangential sections of cytochemically stained cells (Fig. 9). It is customary to refer to the convex, cis or condensing face of each Golgi stack versus the concave trans or forming face, but this implication of secretory polarity within the Golgi system may not accurately describe the events in magnocellular neurosecretory cells (see Fig. 10, and discussion below). In the transition zone between the RER and the outermost aspect of the Golgi stack putative transition vesicles may shuttle bank and forth between the two compartments (Palade, 1975), or the vesicles may become incorporated into the
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Golgi stack, which seems likely in the case of neurosecretory cells, judging by the vesicular aspect of the outermost Golgi element (Fig. 8A). Moreover, as the Golgi apparatus continually loses membrane in the form of NGVs, producing about 35 of these per minute under basal conditions (Morris, 1976), membrane components derived from transition vesicles are presumably necessary to replenish the loss. The vesiculation apparent in the outermost Golgi cistern seems to be abolished in a graded manner, for while the following cistern is segmented,
FIG. 9. Supraoptic nucleus of saline-drinking mouse. Thiamine pyrophosphatase activity, normally present in only one or two of the innermost Golgi cisterns (see Fig. 5 ) , appears in almost all Golgi cisterns as well as in CERL-like cisterns during osmotic stress. Enzyme activity also appears on secretory granules (arrows) forming off the various cisterns. (From Broadwell and Oliver, 1980.) X 30.000.
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A
Normal control
Salt treated
PPase
t
[ Granule
8
GERL. AcPase
FIG. 10. Two schematic versions of Golgi apparatus activity in hypothalamic magnocellular neurosecretory cells. (A) According to Broadwell and Oliver (1980) secretory granules and lysosomes arc normally produced only from GERL, while during osmotic strcss secretory granules may be produced from both Golgi and GERL cisterns. (B) According to Picard et al. (1978) the Golgi-GERL system is functionally bipolar at all times, with Golgi proper producing bona fide secrctory granules and GERL producing lysosomes. GERL may haw direct connections with the rough endoplasmic reticulum. See text, Section III,C, for further details. (Adapted from Picard et al.. 1978; and Broadwell and Oliver, 1980.)
the innermost elements of Golgi proper usually demonstrate more continuity (Figs. 8A and 8B). Membrane conversion in a unipolar direction may be indicated (Morr6 and Ovtract, 1977), although a case has also been made for the uniqueness and stability of each Golgi cistern (see Farquhar and Palade, 1981). In HNS perikarya formation of secretory granules is not always strictly parallel with the presumed direction of membrane flow in Golgi-GERL, for all cisterns
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are potentially able to produce NGVs, depending on the physiological state of the animal (Picard e t a / . , 1972, 1978; Broadwell and Oliver, 1981; see Fig. 10). Different degrees of vesiculation, and possibly also differences in membrane thickness as suggested by Kalimo (1971), imbue the Golgi cistern with morphological heterogeneity, while the application of cytochemical and immunocytochemical procedures indicates distinct enzymatic and chemical heterogeneity. Glycoproteins, which have been cytochemically demonstrated throughout both Golgi and GERL components in neurons of the SON (Tasso and Rua, 1975), increase in staining intensity from the outer to the inner face of the Golgi stack (Rambourg et uf., 1969; Thiery, 1969). Prolonged osmication at low pH selectivity impregnatse one or two outer cisterns (Picard et a/., 1972, 1978), while the outermost cistern is also glucose-6-phosphatase reactive (Broadwell and Cataldo, 1982). The intermediate and inner Golgi cisterns are thiamine phyrophosphatase (TPPase) positive (Fig. 5), while GERL elements contain acid hydrolases (Osinchak, 1964; Picard et a/., 1972, 1978; Castel and Dellmann, 1980; Broadwell and Oliver, 1981). TPPase is an enzyme that may be involved in glycosylation in the Golgi apparatus (Hand and Oliver, 1977b), while acid hydrolases are widely implicated in lytic processes throughout the cell (Novikoff, 1961, 1973; Holtzman, 1976; Bainton, 1981; Glaumann et al., 1981; see also Section V,E). As for immunocytochemical heterogeneity, according to the preembedding study by Broadwell et al. (1979) immunoreactive neurophysin was revealed in all Golgi cisterns, but not in GERL (see Fig. 6). However, the authors believe that neurophysin may nevertheless be present in GERL and consider the lack of immunoreactivity as a false-negative result that may be attributed to the impenetrability of the unique GERL membrane to high-molecular-weight antibodies. An analogy is invoked with exocrine gland peroxidase producing cells that failed to reveal diaminobenzidine (DAB) reaction product in GERL, despite the anticipated presence of the enzyme in this location, and the contention is that GERL was inaccessible to DAB by virtue of its impenetrable membrane (Hand and Oliver, 1977a,b). However, as regards neurosecretory cells, other explanations for lack of neurophysin immunoreactivity in GERL may be applicable, and exemplify the current differences of opinion regarding the functional duality of the Golgi system (see Fig. 10). The crux of the matter seems to be whether GERL in neurosecretory cells takes its origin from the Golgi stack (Broadwell and Oliver, 1981), or whether it is a separate system with a different input by direct connections with the RER (Picard et al. 1972, 1978). This point is also important when considering the two types of membrane-bounded packages produced by the Golgi-GERL complex: the NGV containing neurohormone and the primary lysosomes containing hydrolytic enzymes (see also Section V,E). Castel and Dellmann (1980) associate NGV production primarily with TPPase-positive Golgi cisterns in mice. However, they too have found, as stated by
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Kalimo (1971) in regard to PVN cells of rats, that some NGV production may be associated with other cisterns of the Golgi-GERL complex as well. Based on studies in laboratory rats, Picard et al. (1972, 1978) maintain that normally the osmiophilic cisterns at the cis Golgi face are the site of accumulation of dense material that transforms into NGVs, while the intermediary TPPase-positive cisterns may become involved in NGV production following osmotic stress. The AcPase-positive GERL or inner cistern, often separated from Golgi proper by small vesicles, is considered by these investigators to give rise to lysosomes only. Many of these primary lysosomes are small (about 30-50 nm) and electron lucent, with smooth or coated limiting membrane, while others are electron dense, in the size range of NGV (about 100-150 nm) with which they may be confused. Although primary lysosomes are generally of the small electron-lucent variety in gland cells and most other cells (Holtzman, 1976), large electrondense lysosomes are produced by neurons (Novikoff et al., 1971; Sotelo and Palay, 1971). Picard and his colleagues (Fig. 10B) consider the Golgi-GERL complex as bipolar giving rise at one pole to NGVs and at the othcr to two morphologically distinct types of lysosomes, Moreover, they maintain that while the Golgi component receives its input of posttranslational protein via transition vesicles from the peripheral stacks of RER, GERL receives synthesized material by direct connections with local RER, which is in keeping with views held by the Novikoffs and their co-workers (Novikoff and Novikoff, 1977; Novikoff et a / ., 1977). While the entire Golgi-GERL complex is commonly known to hypertrophy and show signs of hypersecretion in dehydrated rats (Reinhardt et al., 1969; Kalimo, 1971; Picard et d.,1972, 1978) and mice (Broadwell and Oliver, 1981), Picard and his associates have observed dissociation between Golgi and GERL activity during the early stages of rehydration, when the Golgi proper seems quiescent, while GERL actively produces both large and small vesicles. This is interpreted as enhanced lysosomal activity by GERL to cope with readjustment of the cell to basal conditions, which presumably also implies degradation of redundant NGVs, possibly by crinophagy (Farquhar, 1971). This interpretation endorses the likelihood that GERL produces lysosomes only and precludes the possibility that the 100- to 150-nm electron-dense vesicles that bud off GERL could be neurohormone-containing secretory vesicles, despite their morphological similarity to classical NGV. Extreme hyperactivity of GERC has been noted in neurosecretory cells of water-loaded, ethanol-anaesthetized rats, when VP secretion is measurably reduced (Castel, unpublished; Fig. 11). Broadwell and Oliver (1980, 1981), working on laboratory mice, present a radically different interpretation of Golgi-GERL functions in neurosecretory cells of the SON (Fig. 10A). They maintain that GERL is the principal site of NGV production and the sole site of lysosome formation, while NGV production by the cisterns of Golgi proper is invoked mainly during osmotic stress. As for primary lysosomes, they presumably recognize as such only the small electron-
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lucent vesicles (40-60 nm) associated with GERL, while all larger electrondense vesicles (100-120 nmj are considered as bona fide NGV. This view is based on the assumption that GERL contains neurohormone precursors, regardless of the fact that immunoreactive neurophysin could not be demonstrated within the lumen. It was for this reason that the authors sought an explanation involving the impenetrability of GERL membrane to antibodies (see Fig. 6). Broadwell and co-workers believe also that GERL takes its origin from Golgi, or at least maintains connections with the innermost Golgi element. This latter premise is strengthened by the situation in hyperosmotically stressed mice where TPPase activity is no longer restricted to the innermost one or two Golgi saccules, but is found in almost all Golgi elements as well as in GERL. This carryover of specific enzymatic activity from Golgi to GERL, plus the increased production of putative NGVs by all cisterns, including GERL, tends to support the neurohormone packaging potentiality of the latter. As for AcPase activity during hyperosmotic stress, this is shown to be cytochemically reduced in GERL (Broadwell and Oliver, 1981), and biochemically decreased in the entire SON (Jongkind and Swaab, 1967; Jongkind, 1969). Thus an inverse relationship exists between neurohormone production and AcPase activity, but it is difficult to conclude whether this indicates that acid hydrolases are unnecessary for routine neurohormone processing, or on the contrary, that their reduction attests to the extent that they have been used up during heightened neurosecretory activity. Thus the precise function of GERL in neurosecretory cells remains equivocal. Putative NGVs of GERL origin may bear coated vesicular attachments identified as primary lysosomes in other cells. What does this imply’?Osinchak (1964) has suggested that at least theoretically this could mean that redundant NGVs are drawn to a lysosomal source of degradation. On the other hand Broadwell and his associates regard such coated vesicular attachments as an indication of the source of the membrane of these putative NGVs, and as a sign of transient association of neurohormone with hydrolases that need not imply degradation. Novikoff and Novikoff (1979) consider “routing of some secretory materials through a hydrolase-rich pathway” quite feasible, and now advocate the view that “the essence of GERL’s function in secretory cells might be the partial hydrolysis of secretory molecules.” However, even if this view is accepted in principle as regards neurosecretory cells, the fact remains that the secretory packages that arise from Golgi proper are probably different in some way from those that take their origin from GERL. This may indicate that neurosecretory cells are producing two types of neurosecretion in one cell (see Section 111,Dj. Species differences may account for some of the contradictory results cited above. While laboratory rats are highly sensitive to hypersomotic stress, rapidly succumb to negative water balance, and readily demonstrate a hyperactive HNS, mice are far more resilient both as regards readapting to positive water balance and as regards preserving the status quo in the HNS (Castel and Abraham, 1969,
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FIG. I IA. Supraoptic nucleus of water-loaded rat, in which vasopressin secretion is depressed. Electron micrographs show apparent hyperactivity of GERL. (A I ) Arrows indicate electron-dense vesicles originating from GEKL (lysosonies'? NGVs'?); note also formation of numerous coated vesicles (primary lysosomes?) from GERL. (A2) Thiamine pyrophosphatase reaction product in some Golgi ckterns. Arrow indicates unusually large electron-dense vesicle (lysosome?) forming from GERI. cistern; compare with nearby putative NGV. (Al) X62,OOO. (A2) X47.000.
1972). These difference are dramatized during stress, but probably exist in normal specimens as well. D. ADDITIONAL PEPTIDES IN VASOPRESSIN AND OXYTOCIN NEURONS
It is becoming increasingly clear that the hypothalamic magnocellular nuclei, particularly thc P V N , produce a host of neuroactive substances in addition to VP and OT and their respective neurophysins (see Table 1 in Swanson and Sawchenko, 1983). In many instances the parvocellular components of the magnocellular nuclei are responsible for the additional substances, but in several instances
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FIG. 1 1B. Synaptoid contact between enkephalin-immunoreactive terminal and pituicyte (P) in rat neural lobe. Extravesicular, electron-dense reaction product surrounds large (“empty”) vesicles. CV, Small, electron-lucent vesicles; L, lipid droplet. Preembedding immunostaining, ultrathin section of vibratome slice. (From Van Leeuwen et al.. 1983.) X37,OOO.
“extra” peptides are claimed to coexist in the same neurons as the neurohypophysial hormones. Joseph and Sternberger (1979) revealed the existence of immunoreactive plipotropin in magnocellular vasopressinergic neurons, by meticulous sequential immunoperoxidase staining of parraffin sections with different chromogens on the same section, without elution. In this instance peptide immunocytochemistry may have presaged the recent biochemical finding that six consecutive residues
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of the glycopolypeptide of propressophysin are homologous to the amino-terminal region of P-lipotropin (Land et a f . , 1982). Further support for the feasibility of finding opioids in the same cells as neurohypophysial hormones may be derived from the claim that an 80,000 dalton protein may be extracted from the neurohypophysis, serving as a giant precursor for VP and various proopiocortinderived peptides (Lauber et al., 1981). Leu-enkephalin was among the opioid peptides identified biochemically in the HNS by Kossier et ul. (1979, 1980). According to electrophysiological data, Clarke et al. (1979) proposed inhibitory regulation of OT release from neurohypophysial terminals by enkephalin. Martin and Voigt ( 198la,b) provided immunocytochemical evidence for met-enkephalin in OT-terminals and leu-enkephalin in VP-terminals, claiming that the opioid peptide is located within the same secretory vesicles as the neurohypophysial principles (Martin et a / ., 1983). Van Leeuwen ( 1982a,b), however, maintains that met-enkephalin is present only in separate neurohypophysial terminals that do not contain either VP or OT. Convincing immunoelectron microscopic evidence has been provided (see Fig. 1 19)that enkephalinergic processes terminate in synaptoid fashion on pituicytes, and it has been proposed that these glial elements could mediate the inhibitory action of enkephalin on VP release (Van Leeuwen et a / . , 1983). An inhibitory action of pituicytes on neurohypophysial hormone release has also been suggested on the basis of a measurably closer engulfment of neurosecretory axons by pituicytes in control rats as compared to water-deprived rats (Tweedly and Hatton, 1980). The fact that Van Leeuwen and his colleagues have found no support for the coexistence of enkephalins and neurohypophysial hormones in the same terminals, while Martin ef a / . (1983) have provided painstaking proof to the contrary, may probably bc attributed to basic differences in immunocytochemical technique. The conclusions of Van Leeuwen et al. derive from yreembrrlding methods applied to vibratome sections, while the results of Martin et a/. (1983) are based on postemheciding procedures performed on epoxy-embedded material. Preembedding immunostaining was carried out on tissue that had undcrgone only aldehyde fixation and buffer washing, whereas postembedding staining was pcrformed on tissues that had been subjected also to alcohol dehydration and epoxy infiltration, as well as the rigors of methanol-benzene and Na-methoxide for removal of embedding nicdium. While such drastic treatnicnts may unmask relevant antigcnic sites, they may also lead to perturbations and relocations within the tissue, which have not even begun to be understood. An illustration in case is the immunostaining of putative VP-synapses in extrahypothalamic sites: Buijs arid Swaab (1979) found that the postembedding procedure induced indiscriminate “inimunorcactivity” in all synaptic profiles, while preembedding staining was more selective and authentic. Thus the contentions of Martin e t a / . ( 1983), relating to the intraneuronal and intravesicular coexistence of en-
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kephalins and neurohypophysial hormones, would benefit from confirmation by preembedding procedures. Using an affinity-purified antiserum to the nonenkephalin part of the dynorphin molecule, it has been demonstrated that dynorphin and VP probably coexist in the same hypothalamic magnocellular neurons of rats (Watson et al., 1982). In VP-deficient Brattleboro rats dynorphin persists in the “empty” magnocellular neurons (but does not occur in oxytocin neurons), indicating that dynorphin is biosynthetically dissociated from VP, despite their colocalization in normal rats. It is interesting that Watson et ul. (1982) could not detect leu-enkephalin in the dynorphin-positive cells of the HNS. Their immunocytochemical strategy involved preembedding staining of frozen serial sections. The colocalization of cholecystokinin and OT-neurophysin has been indicated in some magnocellular HNS neurons, based on immunocytochemical data from consecutive paraffin sections (Vanderhaegen et al., 1981). Oxytocin terminals in the neurohypophysis of normal rats and VP-deficient Brattleboro rats are reported to contain not only immunoreactive cholecystokinin but also immunoreactive met-enkephalin, as demonstrated in Epon-embedded serial sections at both light and electron microscopic levels (Martin er al., 1983). Substance-P immunoreactivity, with the aid of monoclonal antibodies, has been located within cell bodies and processes of the mouse HNS on semithin Epon sections (Stoekel er al., 1982). Colchicine treatment was required to reveal the perikaryal staining. Substance-P immunostaining occurred in some VP (but not OT) magnocellular neurons, but was conspicuously absent from the parvicellular VP-containing cells of the SCN. Renin-like immunoreactivity has been reported in oxytocin cells of the rat PVN and SON (Calza et nl., 1982), and has been morphometrically assessed (Fuxe er al., 1982). The interesting issue relating to the coexistence of angiotensin I1 and VP’in the same magnocellular neurons (Phillips et al., 1979; Kilcoyne et al., 1980) has not been resolved (E. A . Zimmerman et al., 1980). Recently, VP immunoreactivity has been detected in many cells of the locus coerulus (Caffk and Van Leeuwen, 1983) (see Section X,A). As these are likely to be adrenaline-containing cells (Swanson, 1977), colocalization of VP and noradrenaline is indicated, but further work is necessary to verify this initial observation. No attempt is made here to deal exhaustively with the steadily increasing claims for additional neuroactive substances in neurosecretory neurons (see Swanson and Sawchenko, 1983; Silverman and Zimmerman, 1983). Proof of peptide colocalization within neurons rests heavily on immunocytochemical data, and caution regarding the authenticity of these claims is often reiterated. Conflicting reports are not unusual, as for instance the case of the enkephalins referred to above. Speculation is rife about the precise subcellular location, mode of production, functional implications, and evolutionary significance of copep-
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tides in neurosecretory cells. The flood of new information needs to be integrated into current neuropeptide biosynthetic concepts. It has been suggested that all “the bioactive neuropeptides of today arc derived from a very few ancestral protein precursors” (Scharrer, 1982). Whcn more is learned about the precise identity and intracellular distribution of the so-called “extra” peptidcs in neurosecretory cells, it is possible that apparent paradoxcs relating to the Golgi-GERL system (Scction Ill,C), the undefined function of the profuse aSER (Section V,B), and the role of the enigmatic microvesicles (Section V ,D) will be resolved.
IV. Axonal Transport in Neurosecretory Cells Ever since the classic work by Weiss and Hiscoe (1948) who by elegantly simple experiments discovered “axoplasmic flow,” and Droz and LeBlond’s ( 1963) use of radioactive amino acids and autoradiography to demonstrate the phenomenon, this has been an unusually active and productive avenue of research. It was soon recognized that the phenomenon involved more than flowing axoplasm, and also included membrane-bound components which moved orders of magnitude more rapidly (Lasek, 1970) than the 1 mm/day rate described by Weiss and Hiscoe (1948). This phenomenon has been referred to as “axoplasmic transport” and/or “axonal transport.” An extensive review of this area is beyond thc scope of this section, and furthermore is unnecessary since several excellent reviews have already been published (see Lubinska, 1975; Lasek, 1970; Droz, 1975; Ochs, 1975; Grafstein, 1977; Schwartz, 1979; Grafstein and Forman, 1980). We will not deal here with mechanisms of axonal transport, but refer only to those issues which are particularly relevant to neurosecretory cells. A. MULTIPLE COMPONENTS (WAVES) OF AXONALTRANSPORT What makes axonal transport particularly interesting to a cell biologist is that this phenomenon dramatically reveals the organizational complexity of neurons. The simple fact that proteins synthesized in the cell body are transported relatively coherently down the axon in different rate classes (waves) with distinct compositions suggests an organizational segregation of proteins in the neuron with obvious functional implications. The initial recognition that the fast component of axonal transport contained mainly membrane-bounded organelles and that the slow component contained “soluble” proteins (Grafstein, 1967; McEwan and Grafstein, 1968; Lasek, 1970) has led to a more sophisticated analysis of the protein composition of these “waves” (Willard et al., 1974), an identification of organelles in fast transport (Droz, 1975; Schwartz, 1979; Grafstein and Forman, 1980), and an analysis of the network characteristics of the
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“soluble” proteins in slow transport (Hoffman and Lasek, 1975; Lasek and Hoffman, 1976; Lorenz and Willard, 1978; Willard and Hulebak, 1977; Lasek and Black, 1977). Indeed, the slowly transported proteins provide a variety of moelcular candidates for the cytoskeletal system described by Porter and his colleagues (Porter eta!., 1979; Wolosewick and Porter, 1979). The discovery of retrograde axonal transport (see Kristensson and Olsson, 1973; Grafstein, 1977; Grafstein and Forman, 1980, for reviews) immediately suggested a mechanism for the transfer of information from the distant nerve terminals to the synthetic machinery of the neuron in the perikaryon. A brief summary of the components of axonal transport is shown in Table 1 (for details see reviews by Grafstein and Forman, 1980; Lasek and Shelanski, 1981). The fast component is usually classified as having rates exceeding 250 mmiday (maximum rate of about 410 mmiday), and is composed of a wide variety of organelles (see Table 11). Most of the “packaged” material in the cell body (discussed in Sections I1 and 111) which is destined for the axon and terminals is routed via the Golgi (Hammerchlag er al., 1982) and transported in this anterograde component. Calcium ion which seems to be involved in packaging is also transported in this component (Hammerschlag er al., 1975), and has been implicated in the mechanism of transport at the axonal level (Ochs et al., 1977). The molecular composition of the fast transport component is very complex, as one might expect, since it should contain virtually all the membrane proteins found in the axon. Polyacrylamide gel electrophoretic patterns of proteins in the fast component indicate a great similarity between diverse cell types (e.g., motor, sensory, sympathetic nerves) although some differences do exist (e.g., DBH in sympathetic cells, neurophysin in neurosecretory cells, etc.). This overall similarity in electrophoretic patterns is again not surprising since one would expect that many constitutive membrane proteins would be similar in the different cells. The complex composition of fast transport stands in dramatic contrast to the Sca (V) component of slow transport. Only 5 proteins have been detected in this component which moves at 0.3-1.2 mmiday; three proteins (212,000, 160,000, and 68,000 daltons) making up the “neurofilament triplet,” and the 57,000 and 53,000 subunits of tubulin (Hoffman and Lasek, 1975; Lasek and Hoffman, 1976). Lasek and co-workers refer to this component as the microtubule-neurofilament network, noting that although these proteins are not membrane-bound they move as if in a tightly associated network, and may be responsible for maintaining the stable linear form of the axon. The second subcomponent of slow transport, designated Scb by Lasek and Hoffman (1976), is very complex and is believed by these workers to constitute the microtrabecular system of the axon. This component also moves as if it were a unit, and included in it are “soluble” proteins such as enolase, creatinelanase, etc. (see Table 11). Scb moves at the same rate at which axons grow (Lasek and Black 1977) and it is
340
MONA CASTEL ET AL TABLE I COMPONENTS OF AXONALTRANSPORT^
Classification of components
Transport Nature of structural rate organization (e.g., (nm/day) organelles)
Anterograde transport >250 Fast transporl (1)
Intermediate transport (11) (111)
Slow trans-
20-68
4-8
I .5-4
port (IVScb)
(V-Sca)
Retrograde transport
0.3-1.2
70-312
Nature of molecules
Mcmbrane associated proteins; SBH, glycoproteins, lipids, AChE, neurophysin, neurohormones, neurotransmitters, Ca*+ polyamines Mitochondria1 Mitochondria enzymes ATPase Mitochondria, con- Myosin-like acrin binding protein tractile proteins, souble enzymes (MI), MAO, COMT, PNMT, ChAT Microtubules, mi- Actin, clathrin myocrofilarnents, sin-like actin, tumicrotrabeculae bulin, neuron specific enolase, crcatinc kinase, GAD, microtrabcculae proteins Neurofilament trip- Neurofilament prolet and microteins, tubulin tubule network Mitochondria mul- Proteins, glycoprotivesicular and teins, lipids, AchE, DBH (inactive) multilamellar bodies, lysoso68,000 dalton promal-related tein (major protcin), proteins for organelles, SERlike tubules extracellular spaces
Membrane-bound organelles, plasmamembrane, SER, secretory vesicles, lysosoma1 related structures
Inhibitors
Colchicine, vinblastinc etc., 0 2 deprivation, inhibitors of oxidative-phosphorylation, e.g., 2-4DNP. NaCN, IAA ?
?
Colchicine?
Colchicine?
Same as fast transPO*
"The classification of axonal transport components by rate is largely from Lorenz and Willard (1978) although the designation of Sca and Scb is from Lasek and Hoffman (1976). Data in Table I , i n addition to the above papers, were derived from Willard ef a!. (1974). Willard and Huleback (1977), Lasck and Black (1977), Grafstcin and Forman (1980), and Lasek and Shelanski (1981).
NEURONAL SECRETORY SYSTEMS
34 1
noteworthy, in this regard, that actin is a major protein in this component (Willard et al., 1974; Black and Lasek, 1979). Retrograde axonal transport was originally suspected as a phenomenon due to the movement of certain viruses and toxins in peripheral nerves (see Kristensson and Olsson, 1973). The molecular composition of this transport is also complex, and it has been compared to fast (anterograde) transport because of its relatively fast rate (50-70% of fast transport), its membrane-bound nature, similar electrophoretic patterns of proteins, and its inhibition by microtubule disrupting agents (see Grafstein and Forman, 1980). Its characteristics are most convincingly demonstrated by the use of exogenous proteins such as tetanus toxin (TT), nerve growth factor (NGF), and HRP which are taken up at the axon endings by endocytosis and moved retrogradely in membrane-bounded compartments. The HRP may be found in cisternal or tubular organelles (LaVail and LaVail, 1974; LaVail et al., 1980) in the axon, and ends up mainly in the lysosomal compartment of the cell body (Holtzman, 1977). As for endocytotic mechanisms, uptake is either by a high affinity uptake mechanism involving membrane receptors (absorptive pinocytosis) or by a low affinity uptake mechanism (fluid phase pinocytosis) described by Silverstein et al. (1977). The former mechanism characterizes the uptake of NGF and TT, whereas the latter requires high concentrations of the protein in the extracellular fluid and is typical of HRP, ferritin, and serum albumin uptake. Obviously, these mechanisms are used normally in vivo in the absence of exogeneously administered proteins, and recently methods were devised to investigate the retrograde transport of endogenous (to the axon) proteins (see Fink and Gainer, 1980a,b). These studies have shown that the major protein being transported retrogradely in peripheral nerves has a molecular weight of 68,000. It is possible that this protein is serum albumin (Gainer and Fink, 1982) taken up by fluid phase pinocytosis and that this process represents the mechanism by which the neuron samples the external environment in its periphery. Since most of the organelles moving retrogradely are lysosome related (Table I , and Forman et al., 1977) it is likely that most of the imbibed proteins are degraded, but those molecules resistant to degradation by the lysosomal system may be candidates for information transfer from the periphery. TRANSPORT IN THE HYPOTHALAMIC-NEUROHYPOPHYSIAL B . AXONAL SYSTEM Evidence that the neurosecretory proteins and peptides are synthesized in the magnocellular perikarya and are intraaxonally transported to the terminals in the posterior pituitary has been obtained by a wide variety of autoradiographic and biochemical techniques (Norstrom and Sjostrand, 1971a-c; Jones and Pickering, 1970, 1972; Burford and Pickering, 1973; Norstrom et al., 1971; Norstrom, 1975; Kent and Williams, 1974; Flament-Durand et al., 1975; Gainer et al.,
342
MONA C A S l t L El A L
1977b,c). The focus of these studies has been mainly on the transport of neurosecretory materials which occurs in the “fast” component of axonal transport, and which can be blocked by the application of microtubule disrupting agents such as colchicine (Norstriim and Sjiistrand, 1971a; Norstrom et al., 1971; Flament-Durand and Dustin, 1972; Flament-Durand et d.,1975; Dustin rt a / . , 1975; Hindelang-Gertner et a / . , 1976; Gainer et a / . , 1977b,c). Thc ratc of transport of the neurosecretory proteins has been stated as being between 70 and 200 mm/day. However, these values are usually determined by calculating the time of arrival of labeled protein in the neurohypophysis following injection of the radioactive amino acid in the hypothalamus. Because of the short distance between thc hypothalamic neurons and the pituitary in the rat (about 7 mm), the lack of information about the residence time of the labeled protein in the cell body after synthesis, and the low specific activity of [3sS]cysteine used in most of these studies, these calculations are subject to significant error. Some of these difficultics have bccn ovcrcoinc by the use of high spccific activity cysteinc and by comparing the time arrival of [ ?3]cysteine labeled proteins at the half-way point of the axons (in the median eminence) to the time of arrival in the neurohypophysis (Gainer et u l . , 1977b,c). Calculations based on these data are also approximate, and give a value of about 140 mmlday. After an injection of [35SJcysteine in the SON, virtually all the labeled neurosecretory proteins pass through the median eminence (as a broadwave) by 4 hours after injection (see Fink e t a / ., 1981). These labeled proteins and peptides that enter the pituitary can be released by Ca2 -dependent depolarization of the terminals (Gainer et al., 1 9 7 7 ~ Russell ; el al., 1980a; Nordmann and Labouesse, 1981). The rate of transport does not change with enhanced functional activity (Norstrom and Sjiistrand, 1972a,b), but the rate of conversion of the prohormone to the final products does (see Section 11,E). As is the case for neurons in general (see Table I), the hypothalamic magnocellular neurons exhibit more than one rate of axonal transport, with different protein compositions characteristic of each transport wave. Due to the short length of the hypothalamic-neurohypophysial pathway in the rat it is difficult to clearly resolve the separate components of axonal transport as has been done for the visual system (Willard et a l . , 1974) and various peripheral nerves (Lasek and Hoffman, 1976). A procedure especially adapted for analysis of transport waves in short-axon systems is illustrated in Fig. 12 (see Fink et al., 1981). The radioactivity in a given protein band on SDS gels derived from the median eminence (ME), “halfway” from the SON to the posterior pituitary (neurohypophysis), was compared to its radioactivity in the posterior pituitary (PP) in each animal, at a specific time point: ME-cpm/ME-cpm + PP. cpm is a measure of how much of the labeled protein is passing through the midpoint of the axon at any given time relative to the total protein label transported. The ratio is deter+
343
NEURONAL SECRETORY SYSTEMS ESTIMATED TRANSPORT RATE (mm/day)
’200 1
6
3
1
1
-
I
[3sS]Methlohinr &-A
45,000 MW peak
-13,000
MW peak
[ 35 s ] c yete i n e G----o
13.000 MWpsak (neurophysin)
h
1 -
57,000 53.000 45,000 35,000
-
43,000
0
6 12
24
48
72
86
HOURS
FIG. 12. Quantitative evaluation of the axonal transport of the 45,000 MW peak (coruns with actin) and the 13,000 MW peak (coruns with neurophysin)-labeled proteins through the median eminence. The ratio of cpm in the median eminence (ME) to the total transported protein [ME and posterior pituitary (PP)]for each protein band reflects the relative amount of the labeled protein that has arrived at the halfway point in the axon (ME) at various times after injection of the precursor amino acid ([35S]rnethionine or [3sS]cysteine) in the SON, versus the total amount of transported protein. Thus, a ratio of 1:0 indicated that the labeled protein was in the ME but had not yet reached the PP. As the labeled protein enters the P the ratio will fall, and will rise again only when a new “wave” of transport of the labeled protein passes through the ME. The movement of labeled neurophysin is reflected by the 13,000 MW peak after [35S]cysteine injection (open circles, dotted line). Note that multiple waves of transport are visualized after [35Sjmethionine injection. The drawing at the right of the graph is of a typical autoradiograph of an SDS gel containing labeled proteins from the posterior pituitary 7 days after injection of [35S]methionine into the SON. The dark thick bands represent the most heavily labeled protein bands. Modified from Fink er ul. (1981).
mined only from samples in the same animal, and hence, normalizes for interanima1 variation in the injection of the labeled amino acid into the SON, and other variations. A ratio of 1 .O indicates the labeled protein has arrived in the ME, but not yet in the posterior pituitary. For those proteins transported only at rapid rates (e.g., the [35S]cysteine-labeled < 13,000MW protein [neurophysin] in Fig. 12), the ration will rapidly fall to lower values which remain low as the protein continues to accumulate in the posterior pituitary. Proteins transported at more than one rate will show an initial fall in ratio and then an increase and fall in ratio as the next discrete wave passes through the ME region on its way to the pituitary.
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MONA CASTEL ET AL.
In earlier experiments it was found that [3sS]methionine was more efficacious as an amino acid precursor to evaluate general protein transport in the hypothalamo-neurohypophysial system, as opposed to [‘%S]cysteine which gave a selective view of the neurophysins (Gainer and Brownstein, 1978). Figure 12 shows the transport pattern of the 45,000 MW peak (which coruns with actin) and the < I 13,000 MW peak labeled with [3sS]methionine. One can immediately see, particularly for the 45,000 dalton peak, that there are multiple waves (rates) of transport (see upper abscissa in Fig. 12) that correspond reasonably well with those rates found in other neurons (see Table I). From the data in Fig. 12 one could predict that the fast component of the 45,000 dalton labeled peak would arrive in the pituitary virtually completely by 4-12 hours, and the slower components by 8 days. Identification of the 45,000 dalton labeled peak as actin was successful only for the slower waves (see Fink et al., 19811, consistent with the observations of Willard et al. (1979) and Black and Lasek (1979) that actin moves primarily in the Scb (or 1V) component. What the identity of the 45,000 dalton peak in fast transport is remains unknown at present. It could be a protein associated with the neurosecretory granule membrane. Analysis of the < 13,000 dalton, [35S]methionine-labeled protein shows that there is a small protein other than neurophysin which is moving at slow rates (i.e., between 1 and 3 nim/day). The analysis for neurophysin indicates it moves primarily by fast transport (dotted line Fig. 12; see also Fink et al., 1981). The existence of this nonneurophysin small protein which is slowly transported may explain why previous workers, basing their conclusions solely on polyacrylamide gel analysis of labeled proteins transported to the pituitary, believed that there was a slow transport of neurophysin in addition to the fast transported wave (Norstriirn and Sjiistrand, I 97 Ib) . Further resolution of the various rate components and labeled proteins transported to the neurohypophysis is possible by two-dimensional gel electrophoresis and autoradiography (Fig. 13). From Fig. 13 it can be seen that the various rate components are quite heterogeneous and that both tubulin (T) and actin (A) move principally in the slow component, whereas labeled neurophysin (Np) begins to arrive in the pituitary after only 2 hours. This analysis of multiple anterograde transport waves in the H N S indicates a basic similarlity in organization in these specialized (for neurosecretion) neurons and “conventional” sensory-motor neurons. What makes the neurosecretory system unique is that the fast component is so abundant (in quantity of proteins) and that there is an isolatable and identifiable organelle (the ncurosecretory granulated vesicle) found in this component that can be studied in the context of axonal transport experiments. Future studies on the relationships of the transport of these vesicles to the cytoskeletal proteins may provide useful insights into the fundamental mechanisms underlying fast axonal transport.
345
NEURONAL SECRETORY SYSTEMS
A
2 Hours 24 Hours
SDS PAGE
168 Hours
+
-
IEF
+
FIG. 13. Two-dimensional gel patterns of neurohypophysial proteins. (A) Protein distribution wen after staining gel with Coomassie blue. ( B ) Distribution of major labelcd proteins (revealed by autoradiography) 2, 24, and 168 hours after injection of [35S]rnethionine into the SON. Arrows indicate migration positions of tubulin (T), actin (A). neurophysin (Np), and rat seium albumin (RSA). (From Fink PI a / . . 1981.)
V. Morphology of Transport and Release-Peptidergic
Neurons
A. ANTEROCRADE TRANSPORT OF NEUROSECRETORY GRANULATED VESICLES The NGVs produced in the magnocellular hypothalamic perikarya and destined for translocation via the hypophysial stalk to the neurohyophysis proceed
346
MONA CASTEL ET AL
on their way by fast axonal transport (see Section IV,B). The posttranslational processing of the prohormones, possibly initiated already in the Golgi system (Farquhar and Palade, 198l ) , continues intragranularly during transport. Newly formed NGVs are distinguished by a dense granular core that does not occupy the entire vesicular lumen, but is surrounded by an electron-lucent rim, and as maturation proceeds the core gradually expands until it fills the entire vesicle. Unlike aminergic and cholinergic secretory vesicles that accumulate substances from the cytosol during maturation (see Sections V1, VII, VM), peptidergic NGVs appear to contain their full complement of neurohormone precursors and posttranslational processing enzymes by the time they bud off the Golgi cisterns, so that any subtle change in NGV morphology during maturation is generally attributed to intragranular conformational modification at the molecular level, possibly augmented by osmotic and pH changes. (The characteristics of recently arrived versus older NGVs in the neurohypophysis are discussed in relation to preferential hormone release in Section V,C.) In keeping with the notion of “self-sufficiency,” isolated NGVs appear to contain appropriate converting enzymes (see Section II,D), consistent with the recently reported granule-mediated conversions of proinsulin and prosomatostatin (Fletcher et ul., 1980, 1981). It is not known which forces influence the intraaxonal movement of NGVs, although some force-transducing mechanism is anticipated. A transport filament theory has been proposed which associates secretory vesicles with microtubules via ”side bridges” (transport filaments) (Ochs, 1975; McKelvy et a/., 1980). This is claimed to be consistent with reports showing pile-up of NGVs due to interruption of flow caused by alkaloid poisons that bind to microtubules (Dustin et al., 1975; Hindelang-Gertner et al., 1976; Parish et af., 1981; Dellmann and Sikora-Van Meter, 1982). However, this could equally well be related to microtubules as tracks with no physical links involved, as no evidence has yet been presented that shows a consistent association between NGV membrane and microtubular structures. Trabeculae, rather controversial structures, have also been implicated in translocation of secretory granules and other cell organelles (Porter et d.,1979). Using stereo-transmission electron microscopy on polyethylene glycol-embedded and subsequently deembedded neurohypophysis, Kondo et ul. (1981) described an intraaxonal microtrabecula system, quite distinct from microtubules. The system appears to form a lattice in which NGVs are suspended, and the authors suggest that this arrangement is involved in transport of NGVs along the axon. However, due to the unconventional method of tissue preparation it is diffiuclt to define the precise nature of the microtrabeculae, and they may in fact represent collapsed elements of various intraaxonal membrane systems such as the aSER (see Section V,B). Heuser and Kirshner (1980) suggest that microtrabeculae are artifacts produced by agglutination of various filaments on which soluble cytoplasmic components are deposited.
NEURONAL SECRETORY SYSTEMS
347
While the rate of hormone transport in the HNS has proved to be an unalterable parameter, even during osmotic stress, the amount of hormone transported to the neurohypophysis is known to increase (see Section 11,E). In a comparative autoradiographic study of the HNS in several murids, Levy (1978) found that the radioactive grain count was invariably greater in desert mice, in which the HNS is particularly resilient (Castel and Abraham, 1969, 1972). However, this greater amount of 3sS-labeIed neurosecretory material did not arrive in the neurohypophysis any more rapidly than in other murid strains. During osmotic stress, when the HNS is highly stimulated to secrete and synthesize neurohormone, greater numbers of NGVs might be expected along axons in the neurohypophysial stalk. This was indeed shown to be the case in salt-stressed laboratory mice (Broadwell and Oliver, 1980), but in similarly treated laboratory rats fewer than normal NGVs were found along axons in the stalk region (Castel and Gainer, un-
FIG. 14. Mouse neurohypophysis, routine fixation for electron microscopy; membranes delineated by fortuitous lead precipitate during grid staining. Note pleomorphic tubules (some attached to neurosecretory granulated vesicles) which may represent the axonal smooth endoplasmic reticulum (aSER). x 300,000.
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MONA CASTEL E T AL
published observations). It has been proposed that in the dehydrated rat the augmented amount of neurohormone might be conveyed in an axonal compartment other than the N G V s (see Alonso and Assenmacher, 1978b, 1979aj. The possible role of the aSER in neurosecretion will be considered. B. THEAXONALSMOOTH ENUOPI.ASMIC RE-~CULUM Although the present review is intended to deal primarily with secretion phenomena in neurons, and the direct involvement of the aSER in peptidergic secretion has not been proven. this membrane system is nevertheless refered to here in some detail. More than a decade ago Dellmann and Rodriguez (197 1) reported proliferation of “tubular formations” (presumptive aSERj in the proximal stump of the lesioned amphibian HNS, and speculated that these structures might be supplementary vehicles of neurohornione transport, particularly when NGVs are scarce. Adequate characterization of the aSER in neurosecretory neurons his been prevented due to the labile nature of this membrane system, making it difficult to preserve intact either by routine microscopic methods (Fig. 14) or by fractionation and isolation procedures. Similar mcmbrane systems in aminergic and cholinergic neurons have long been given their due, not only as conveyors of membrane constituents and trophic substances, but also for their involvement in storage and transport of neurotransmitters, transmitter constituents, and enzymes associated with transmitter metabolism (see Section VI11). In peripheral neurons it has been recognized that the aSER is a calcium-sequestering compartment (Erogu and Keen, 1977; Duce and Keen, 1978). Interest was revived in the aSER of neurosecretory neurons when methods were devised to visualize its ubiquitous presence in the HNS (Alonso and Assenmacher, 1978b, 1979b; Castel and Dellniann, 1980; Castel er a / . , 1981). With routine electron microscopic procedures aSER-like profiles are only occasionally seen in neurosecretory neurons (Fig. 14). However, both heavy-metal irnpregnation (Alonso and Assenmacher 1978b, 1979b) (Fig. 15) and phosphatase cytochemistry (Castel and Dellman, 1980) (Figs. 16, 17, and 18) stabilize the aSER in neurons of the H N S and facilitate its ultrastructural identification. Droz (I975) introduced heavy-metal impregnated thick sections, viewed at high voltage, as a means for ultrastructural visualization of axonal membrane systems in myelinated and unmyelinated neurons, and combined with high resolution autoradiography demonstrated the central role of the aSER in fast axonal transport (Droz et nl., 1979; Markov er nl., 1976; Rambourg and Droz, 1980). In thick osmieated sections of the neurohypophysis, impregnated with salts of uranium, lead, and copper, extensive aSER has been demonstrated in the laboratory rat (Alonso and Assenmacher, 1978b, 1979a) and in several strains of mice (Castel and Dellmann, 1980, and Fig. 17). Goniornetric tilting confirmed continuity between some NGVs and a SER tubules (Alonso and Assenmacher, 1979a). The
FIG. IS. Mouse neurohypophysis. heavy metal impregnation; 0.5- I .O pm sections viewed at 100 kV, in ordrr to visualize aSER (axunal smooth endoplasmic reticulnni). (A) Above, longitudinal section of neurosecretory axon showing several NGVs, microtubules, mitochondrion (m) and branch-
ing profiles of aSER (arrowheads). Below, portion of axonal swelling replete with NGVs; dcnsc precipitation product between NGVs may denote web of aSER. X70,OOO. (B) Portion of axonal swclling showing clcctron-dcnsc aSER (arrowheads) closly associated with NGVs, mitochondria and lyaoaomea. X60,OOO. (C) Organelles in axonal swelling apparently cnmcshed in web of electrondense aSER. X70.000. Inset: Note close association between NGV and aSER. x30,OOO. (From Castel and Dcllrnann, 19x0.)
350
MONA CASTEL ET AL.
FIG. 16. Mouse neurohypophysis; cytochemical reaction for TPPase (thiamine pyrophosphatase) in order to visualize aSER. Electron micrograph shows axon swelling with clcctron-dense aSER undulating between NGVs. X 100,000.
latter authors maintain that small vesicles (about 30 nm) in terminal axon swellings also originate by budding from the free ends of the aSER tubules (see Fig. 19 and Section V,D for discussion of other roles attributed to small vesicles in neurosecretory terminals). They also suggest that the proliferation of aSER that occurs during water deprivation, when NGVs are scarce while hormone demand is augmented, may indicate that the tubular system is conveyng a nongranular pool of hormone. During short-term rehydration, when the urgent need for water-conserving hormones is lessened, the aSER thickens and accumulates an electron-dense content. Eventually NGVs appear to form from the most dilated portions of this thickened aSER (see Fig. 18 for a graphic interpretation of the views of Alonso and Assenmacher). Three-dimensional visualization of thc aSER throughout the neurosecretory axon is certainly an advantage afforded by viewing thick impregnated sections at high electron microscopic voltage. However, care must be taken with interpretation of the images, for cytological resolution is poor, creating ambiguous relationships between the aSER and other organelles, while the heavy-metal salt method is not selective, impregnating also other organelles and membrane systems, including lysosomal bodies, and even extracellular space. Nevertheless, these drawbacks may be minimized by comparing with images from tissues prepared by other methods.
NEURONAL SECRETORY SYSTEMS
35 1
FIG. 17. Mouse neurohypophysis, cytochemical reaction for TPPase in order to visualize aSER and derivatives. (A) aSER containing dark, coarsely grained TPPase reaction product, and also less electron-dense fine-grained material (arrow) resembling that within NGVs. Arrowhead indicates possible confluence between NGV and aSER. X 106,000. (B) TPPase-positive aSER closely associated with NGVs of various densities, and mitochondrion (m).XY8,OOO. (C) TPPase-positive aSER in neurosecretory terminal; possible continuity between NGV and aSER (arrowhead). X69,OOO. (D) Neurosecretory terminal; TPPase reaction product within small vesicles and tubule. X 103,000. (E) Neurosecretory terminal showing aSER formation associated with mitochondrion. X 92,000. (F) Cistemal element of aSER containing dark reaction product. NGV may be continuous with aSER. X 103,000. (From Caste1 and Dellmann, 1980.)
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MONA CASTEL
r r AL
FIG. 18. Neurohypophysis of saline-drinking mouse. Profuse TPPase-positive aSER, Inset: Arrow indicates apparent continuity between aSER and NGV. X IXO.000.
X 90,000.
NEURONAL SECRETORY SYSTEMS
353
Castel and Dellmann (1980, 198 1) have shown that the aSER in neurosecretory cells may be demonstrated not only by heavy-metal impregnation, but also by a more selective method based on the cytochemical reaction for TPPase (see Figs. 16, 17, and 18). Coarsely grained precipitate of lead phosphate is seen within long stretches of branching and anastomosing aSER, the tubulo-cisternal profiles ranging in width from about 25 to 120 nm. These images are not sporadic, for cross sections of the stalk region reveal TPPase-positive profiles of aSER in virtually every axon. Within axonal swellings and terminals aSER undulates and branches between mitochondria and NGVs, forming characteristic juxtapositions with the former (Fig. 17E) and sometimes appearing confluent with the latter (Figs. 17A, B, C; inset Fig. 18). Associations of this type may indicate involvement of the aSER in the secretory process. Mitochondrial-aSER connections, recently described in a wide variety of neurons by Spacek and Lieberman ( 1980), may foster cooperation between these two calcium-sequestering compartments. Confluence between aSER and NGVs, provided artifact is ruled out, may indicate that NGVs are not entirely “self-sufficient” on leaving the Golgi apparatus and require additional factors from the aSER, or that some NGVs may originate by budding from the aSER just as others take their origin from Golgi cisterns (see Section VIII for dual origin of vesicles in nonpeptidergic neurons). This would be consistent with the suggestion that neurohormone may be present in the aSER. The presence of TPPase activity both within the Golgi cisterns and the aSER may indicate that this enzyme is associated with processing of neurohormone in both locations. It is interesting that osmotic stress leads to a 40% increase of the TPPase activity in the rat HNS (Jongkind and Swaab, 1967; Jongkind, 1969), concomitant with proliferation of both Golgi system (Reinhardt et al., 1969; Kalimo and Rinne, 1972; Picard et ul., 1972) and aSER (Rougon-Rapuzzi rt al., 1978; Alonso and Assenmacher, 1979b; Castel et al., 1981; see also Fig. 18). The “empty” axon dilatations commonly seen in neural lobes of water-deprived specimens prepared by routine electron microscopic methods (Morris et ul., 1978) are seen to be replete with aSER by heavy metal impregnation and by TPPase cytochemistry (Alonso and Assenmacher, 1979b; Castel et al., 1981). It it unfortunate that current immunocytochemical and autoradiographic procedures do not sufficiently preserve the aSER in neurosecretory neurons, since these methods would be appropriate to provide direct evidence regarding the content of this membrane system. The skepticism evoked by claims of neurohormone location within the aSER is due not only to the inconclusive circumstantial evidence raised in support of the idea, but also to current knowledge of the biosynthetic pathway of the neurohypophysial peptides, in which NGVs play a central role both in transport and in prohormone processing (see Sections I1 and IV). Therefore this hypothesis of an alternate vehicle for neurohormone conveyance seems superfluous, unless it could be convincingly demonstrated that such an alternative route would accommodate specific phys-
MONA CASTEL ET AL.
354
A
B
C
Fic;. 19. Diagrammatic presentation of the aSER in neurohypophysial axons during different physiological states. adapted from Alonson and Assenmacher (1979). who consider the possibility that neurohormones may be conveyed in both NGVs and aSER, depending on osmotic conditions. (A) During water deprivation, when secretion of neurohormone is elevated (indicated by multiple arrows), NGVs are scarce but the aSER undergoes extensive development. Numerous small vesicles appear to bud off the free ends of aSER and may convey neurohormone to the axolemma for release. (B) During short-term rehydration, when neurohormone secretion is drastically reduced, neurosecretory material may accumulate within the aSER, inducing dilatation of segments of aSER and budding of NGVs from the aSER. The number of small vesicles budding from the aSER is reduced. (C) Under normal condition5 NGVs are the most prominent conveyors of neurohormone, but the aSER and the small vesicles presumably retain their neurohormone sequestering ability. (Speculations pertaining to the functional capabilities of the aSER and the small vesicles rely on circumstantial evidence only, for no immunocytochemical or biochemical evidence has as yet been presented.)
iological needs not met by NGVs alone (see also Section V , E for aSER and the lysosomal system, and Section 1X,B,2 for aSER during development of the HNS).
c. HORMONEPOOLS IN THE NEUROHYPOPHYSIS Axons from the hypothalamic magnocellular neurons that course through the infundibular stalk terminate in the neurohypophysis among blood vessels and glid pituicytes. About 42% of the volume of the gland is occupied by neurosecretory axons (Nordmann, 1977), comprising undilated portions of axon and
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dilatations that may be divided into swellings and endings. Many profiles of the latter are seen to abut on perivascular basement membranes, demonstrating images associated with neurohormone release. The neurohypophysis has been a model system for studies on the mechanisms of peptidergic secretion at neurohema1 sites (see Dreifuss, 1975; Gratzl et al., 1977, 1980). The most prevalent organelles visualized in the dilatations are NGVs, microvesicles, vacuoles, membrane delimited sacs and tubules of lysosomal nature, elements of the aSER (provided they have survived tissue processing) and mitochondria, Classification of the axon dilatations as preterminal swellings for storage of older NGVs, and endings from which newer NGVs preferentially release their contents, was originally based on the autoradiographic evidence of Heap et al. (1975). These authors showed that when radioactive cysteine was injected into the brain, incorporated into neurosecretory material and transported to the neurohypophysis, silver grains were detected first over nerve endings and only later over nerve swellings. This suggested that the newly formed NGVs arrive first at the endings where they may release their content, while the older NGVs vacate the endings that abut on the perivascular space and move into swellings deeper in the neuropil (Heap et al., 1975; Morris et al., 1978). However, in a recent study on NGV distribution in vasopressin-treated Brattleboro rats (Chapman et al., 1982), it was observed that NGVs initially accumulate randomly in both swellings and endings, and when endings are subsequently emptied by normal hormone release, newly arriving NGVs appear to refill the vacated space. Thus, endings apparently contain and preferentially secrete the new-formed ‘‘readily releasable” pool of hormone. Such schemes of secretory granule movement and distribution may be oversimplified, for they do not take into account the fate of retrieved NGV membrane nor the possible growth and plasticity of the axolemma. A series of studies by Morris and Nordmann and their associates, correlating morphometric and experimental data, have extended the analysis of nerve swellings and nerve endings in relation to the neurohypophysial hormone pools (Nordmann et al., 1979; Lescure and Nordmann, 1980; Morris and Nordmann, 1980; Nordmann and Cazalis, 1981; Nordmann and Labouesse, 1981). The percentage of total volume occupied by NGVs in each axonal compartment of the neurohypophysis was calculated: undilated axons 8.4%, nerve swellings k 30%, nerve endings k 19%. Following in vitro high potassium stimulation of the isolated neurohypophysis, the volume occuped by NGVs in undilated axons and in swellings remained unchanged, while in endings abutting on the basement membrane there was a 51% reduction of the NGV population. Moreover, this reduction was most pronounced in the 200 nm zone nearest the contact area with the basement membrane, where NGV candidates for imminent exocytosis would be expected to be found. Veratridine, added to the incubation medium when the effect of high potassium depolarization had worn off, induced additional hor-
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mone release (presumably by keeping sodium channels open and causing a larger influx of calcium), concomitant with further reduction of thc NGV population in endings only. Thus it appears, at least in vitro, that the “ready relensability” of vesicle-contained neurohormones from the neurohypophysis depends on the nature of the stimulus, the location of the NGVs in the axon (within t 200 nm of the contact zone between nerve ending axolemma and basement membrane), and probably also the age-related propcrties of NGVs (see below). In the intact neural lobe the average diameter of NGVs in nerve endings is smaller (170 nm) than those in nerve swellings (188 nm), and the latter are usually somewhat lcss clectron dense. Several years ago Morris and Cannata ( 1973) hypothesized that “the most likely explanation for granulc swelling and loss of electron-density is osmotic imbibition of fluid into granules. Recently Nordmann rt ul. (1979), by a subcellular fractionation procedure utilizing an isosmotic sucrose-metrizamide gradicnt, isolated two distinct granule populations from the neurohypophysis, both containing vasopressin and oxytocin. The lighter fraction, comprising smaller granules (t 172 nm) was osmotically inactive, whereas the larger granules (t 197 nm) in the heavier fraction were osmotically active and behaved as “perfect osmometers.” This difference in osmotic behavior of the two NGV fractions, which correlated with the differential distribution of larger and smaller NGVs in swellings and endings, respectively, seemed to reflect the postulated aging process. Recently Nordmann and Labouesse (1981) provided further proof for this hypothesis by demonstrating than when [3sS]cysteine was injected into the third ventricle, and the animals sacrificed after various time intervals, the label was at first incorporated into the lighter fraction of newly arrived NGVs within the neurohypophysis, and only after an interval of about 10 days did radioactivity appear also in the heavier fraction of older NGVs. Moreover, when neural lobes in which labeled cysteine had reached homogeneous distribution were stimulated in vitro prior to fractionation, radioactivity decreased only in the lighter fraction. When the number of NGVs has been reduced by dehydration, rehydration results first in an increase of the hormone content of the lighter fraction of newly formed NGVs (Nordmann and Cazalis, 198 I). Chromatofocusing of 1251-labeled granule contents shows certain bands unique to each fraction. The NGVs in the heavier fraction contain small, unidcntificd proteins not present in the newly formed NGVs, and it is speculated that these postmaturational fragments may be physiologically relevant, for instance as activators of lysosomes or as messenger molecules that return retrogradely to the cell body, but this remains a subject for further study (see Sections 11,C and D for discussion of cleavage products of the prohormones). Recent findings by Nordmann and Morris (1983) indicate that aged NGVs contain fewer molecules of hormone and neurophysin, but whether this implies degradation by continuous postmaturational cleavage or leakage of substances out of the NGVs is not clear. In electron micrographs the limiting ”
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membrane of the larger, paler, presumably aged NGVs is very often defective at some point, suggesting that not only content but also membrane properties may be different from those in newly formed NGVs. VP and OT and their respective neurophysins, produced by separate cells in the hypothalamus, are contained in separate axons in the neurohypophysis. However, fractionation procedures do not differentiate between NGVs on the basis of their neurohormone content. Topographically vasopressin-NGVs may be segregated from oxytocin-NGVs by immunocytochemistry using different antibodies on adjacent sections, or sequential immunostaining on the same section with two antisera and visualization with two different chromogens (Swaab et al., 1975; Vandesande and Dierickx, 1975; Aspeslagh et al., 1976; Van Leeuwen and Swaab, 1977). Ultrastructural glycoprotein cytochemistry, using silver proteinate, silver methamine, potassium ferrocyanide, and osmium tetroxide differentiates between vasopressin-NGVs encircled by electron-dense deposits, and negative oxytocin-NGV of homogeneous content and low electron density (Tasso et al., 1976, 1977). This positive reaction of vasopressin-NGVs also correlates with biochemical data attributing a sugar residue to propressophysin but not prooxyphysin (see Section 11,C). Recently a simple method was reported for differentiating electron-dense VP-NGVs from pale OT-NGVs in glutaraldehyde fixed, Epon-embedded neural lobes that had not been osmicated (Alonso et al., 1981).
D. MICROVESICLES Microvesicles within neurohypophysial axons were first reported by Palay (1 955), and have been repeatedly described since in a variety of neurosecretory neurons. However, to this day there is little agreement about their origin, fate, and function. They have been called “synaptic vesicles,” implying that neurosecretory terminals could contain transmitter in addition to neurohormone (Koelle and Geesey, 1961; De Robertis, 1962). The term “synaptoid vesicles,” denoting small electron-lucent vesicles (30-50 nm) typical of neurosecretory endings, was introduced in order to avoid the implication that an additional active substance existed together with neurosecretory material (Scharrer, 1968). However, the coexistence in the same cell of peptide and transmitter or two unrelated peptides is now becoming an established fact (Hokfelt et al., 1980; see Section 111,D). Hence it would not be too surprising if microvesicles were found to contain a transmitter or an “extra” peptide in neurosecretory cells. Ultrastructural studies have suggested that some microvesicles may derive by budding directly from NGVs or via membrane protrusions ( “pseudopodia”) that extend from NGVs (Castel, 1977a; Scharrer and Wurtzelman, 1978; Fig. 20). Such morphological observations are difficult to reconcile with recent results from fractionation studies indicating that the membrane of microvesicles differs
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FIG.20. Irnagcs of neurohypophysial NGVs apparently giving rise to small vesicles. (From Castel, 1977a.) (A and B) X330,OOO. (C) X300,OOO.
radically in protein composition from that of NGV membrane (Torp-Pedersen et al., 1980). It is possible, however, that NGV-derived microvesicles comprise so small a proportion of the heterogeneous microvesicular population, that their intrinsic traits are not expressed in the obligatory averaging of fractionation data. On the other hand Holtzman and Mercurio (1980) have suggested that “the
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vesicles that bud from a given membrane may be selectively constructed so as to differ in composition from the parent membrane. It has been propsoed that microvesicles could ferry small aliquots of neurohormone from NGVs to axolemma for release (Bern and Knowles, 1966) and in insects apparent fragmenting of large NGVs into smaller vesicles has been described (Juberthie and Juberthie, 1974). However, in mammals, immunoreactive neurohormone within microvesicles has not, to our knowledge, been demonstrated in neurohaemal locations (see Section X,B for immunoreactive synaptic vesicles). It has also been hypothesized that during osmotic stress, when NGVs are scarce in the neurohypophysis, microvesicles budding off the aSER could convey neurohormone to sites of release (Alonso and Assenmacher, 1979a; Fig. 19). Although it is feasible that microvesicles in neurosecretory neurons could originate from the aSER, as postulated for aminergic and cholinergic neurons (Section VIII), in the HNS there is no immunocytochemical or other proof that either aSER or microvesicles contain neurohormones. It may be relevant, however, that some microvesicles have a cytochemical marker, TPPase, in common with trans Golgi cisterns and aSER (see Fig. 17D). The most oft-quoted theory regarding microvesicles in neurosecretory neurons relates them to retrieval of membrane from the axolemma following exocytosis of NGV content (Douglas et al., 1971a,b: Nagasawa et al., 1971). However, reports from several laboratories using horseradish peroxidase as a marker for endocytotic activity and membrane retrieval have revealed that only a small proportion of microvesicles imbibe tracer, while macrovesicular structures and various cisterns are responsible for most of the heterophagy and internalization of membrane at neurosecretory terminals (Castel, 1974; Nordmann et al., 1974; Theodosis et al., 1976) (see Fig. 22B). The most significant recent contribution to characterization of neurohypophysial microvesicles has come from the laboratory of Thorn, where it has been shown that in the presence of ATP and Mg2+, pure microvesicular fractions accumulate Ca2+ (Torp-Pedersen et al., 1980). This confirms the cytochemical localization of calcium within microvesicles of neurosecretory endings demonstrated by the pyroantimonate technique and X-ray microanalysis (Stoeckel e t a l . , 1975; Shaw and Morris, 1980; Morris etal., 1981; seeFig. 21). Hence, it has been postulated that microvesicles play an important part in calcium regulation. While the entry of extracellular calcium is an essential trigger factor in stimulus-secretion coupling, it is equally important to regulate excess cytoplasmic calcium by sequestration in mitochondria, aSER, and microvesicles. As microvesicles are probably the most mobile of these calcium-sequestering organelles, they would be able to extrude excess calcium by exocytosis at the axolemma, and this may provide an explanation for the ability of some microvesicles to imbibe extracellular substances, including tracers, especially in stimulated glands. TOT-Pedersen et al. (1980) also conclude, from SDS-polyacrylamide gel ”
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FIG. 21. Neurosecretory ending in rat ncurohypophysis. Calcium is visualized by electron-dense precipitates in microvesicles, tubules (sAER?), and mitochondria. Oxalate-pyroantimonate after calcium-free wash to remove extracellular calcium and calcium entering the cytoplasni during fixation (Shaw and Morris, 1Y80). Electron micrograph kindly supplied by Dr. John Morris. X50,OOO.
electrophoresis data, that the protein content of microvesicle membrane is “very simple and very different” from that of NGV membrane, so that interchange of membrane between these two types of organelle is improbable. Thus retrieval of postexocytotic NGV membrane on a large scale in the form of microvesicles is unlikely according to these authors.
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36 1
The microvesicle population in neurosecretory axons is heterogeneous, both in size range and in distribution, and this may reflect the variety of origins and functions attributed to these organelles. Clustered microvesicles are significantly smaller (? 43 nm) than those dispersed throughout the axoplasm (? 61 nm), and more of the latter imbibe extracellular HRP (Theododis et al., 1977) possibly because greater mobility brings them in more frequent contact with the axolemma. It is possible that a portion of the microvesicles, for instance, the clusters, represents artifactual vesiculation of the aSER that may occur during tissue processing. The changes in size range and distribution of microvesicles in the HNS during fetal and postnatal development probably have a functional basis (Dellmann et ul., 1979; see also Section IX). Some microvesicles are coated, a phenomenon that is probalby more prevalent than generally visualized, for most of the clathrin coating is not preserved during routine fixation, but requires special treatment (Kanaseki and Kadota, 1969). Electron micrographs of the microvesicular fractions prepared by Torp-Pedersen et al. (1980) from bovine neurohypophysis showed that “interspersed between the microvesicles was a background of irregular patches and lattices of hexagons and pentagons compatible with the structure of isolated coats.” Vasopressinergic and oxytdcinergic axons of hypothalamic origin terminate not only in neurohemal regions such as median eminence and neurohypophysis, but also in extrahypothalamic CNS sites where they appear to form synaptic configurations with other neuronal elements (see Section X). Microvesicles ( 2 50 nm) are the most prominent organelles in these synaptic boutons, and only the fact that they are immunoreactive to neurohypophysial hormones distinguishes them from aminergic and cholinergic synaptic vesicles (Buijs and Swaab, 1979; Dogterom and Buijs, 1980). The processes that terminate in vasopressin synapses in the CNS probably do not derive from the same hypothalamic neurons that project to the neurohypophysis. Double-tracer studies indicate that different cells in the PVN project to the neurohypophysis and to the brainstem (Swanson and Sawchenko, 1980). Species, strain, and fixation differences may play a significant role in variation of NGV size. Moreover, within each animal the size of the neurohormonecontaining package differs in each location: k 170-200 nm in the neu90-100 nm in the median eminence rohypophysis (Morris et al., 1978), (Silverman and Zimmerman, 1975), +- 50 nm in synaptic boutons of the CNS (Buijs, 1978). In PVN neuronal perikarya, nascent NGVs are somewhat smaller ( 2 150 nm) than those in neurohemal endings, increase in size presumably being associated with the maturation process during axonal transport. Assuming that similar PVN neurons project to extrahypothalamic sites in the brain and terminate in synaptic boutons, it would be interesting to know how the larger NGVs transform into smaller synpatic vesicles, or whether the same or analogous neurohormone is carried in two different types of vesicle. VP-secreting cells of
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the parvocellular SCN, which project only to CNS sites, also produce NCV that are larger (294 nm) (Van Leeuwen et al., 1978) than vasopressinergic synaptic vesicles. As stated in Section V1 in relation to the molecular organization of secretory vesicles, the factors that determine vesicular size are still unknown.
E. THE LYSOSOMAL SYSTEM By virtue of their glandular nature, neurosecretory cells appear to have a more prominent lysosomal apparatus than other neurons. In cholinergic and aminergic neurons much of the synaptic vesicle membrane and content is locally recycled for use at terminals, without the intervention of extensive degradative mechanisms (see Sections VII and VIII). Although nothing is yet known about secretion or membrane retrieval at peptidergic synapses in the CNS, it is well established that neurosecretory endings in neurohemal regions of the HNS lose hormones into perivascular space and thence into the blood system, while depleted NGV membranes remain to be disposed of intracellularly. In addition, under basal conditions a large population of aging and redundant NGVs needs to be degraded. Concomitant with these secretory-related lysosomal activities are the usual degradative cycles involving all neuronal membranes and organelles, including the aSER, so proliferous in neurosecretory neurons. Thus it is not surprising that under a variety of physiological conditions, including both hydration and dehydration, putative lysosomal structures are readily encountered in both cell bodies and processes. Separate facets of the lysosomal system in neurosecretory cells of the HNS have been dealt with, often extensively, by several different authors. Dellmann (1973) has proposed a functional classification of the various Herring bodies (see also Dellmann and Rodriguez, 1979). Boudier and her associates have provided meticulous accounts of the formation and stereology of autophagic vacuoles (Boudier and Picard, 1976; Boudier, 1978; Boudier et al., 1981). Broadwell and his associates have described the intracellular distribution of organelles with endogenous acid hydrolase activity that also sequester exogenous horseradish peroxidase (Broadwell and Brightman, 1979; Broadwell et al., 1980). Morris and Nordmann (1979) have been concerned with the nature of the signal that initiates lysosomal degradation of aged NGVs. However, despite the extent and variety of available data, a coherent picture of the entire lysosomal system in neurosecretory cells does not emerge. This lack of coherence seems to apply also to other cells, as stated by Glaumann et al. (1981) in a recent review of lysosoma1 mechanisms: “Our knowledge as to the factors that control the traffic of these lysosomal forms and the manner in which they recognize each other and fuse is still restricted.” The task is complicated by the polymorphic and dynamic nature of the lysosomal system, for it is not only acid hydrolase containing structures such as primary and secondary lysosomes that require consideration,
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but also affiliated structures such as prelysosomes (that have not yet acquired their full aliquot of degradative enzymes) and postlysosomes (that have already lost their enzymatic content) (see Bainton, 1981). A wide variety of vesicles, cisterns, tubules, and reticular structures exist in neurosecretory neurons but only a portion of these comprise the lysosomal apparatus (Castel, 1977b; Castel and Dellmann, 1980). Identification of lysosomal structures by their acid hydrolase content has been facilitated by application of lead-based enzyme cytochemical techniques, using a variety of different substrates (Broadwell and Brightman, 1979; Broadwell et al., 1980). In the HNS of laboratory mice these authors have demonstrated selective marking of GERL and small primary lysosomes (40-60 nm) with cytidine 5-monophosphate (CMP) and P-glycerophosphate (PGP) as substrates, while secondary lysosomes were best identified with trimetaphosphate (TMP) and p-nitrocathechol sulfate (for aryl sulfatase). The enzymatic content of some sacs and cisterns reacted with all four substrates. Such cytochemical heterogeneity is to be expected, considering that over 50 different hydrolytic enzymes have been detected in lysosomes of eukaryotes (see Bainton, 1981). As the various lysosomal enzymes seem to be distributed unevenly between different compartments, efforts to mark the entire lysosomal system require the use of multiple cytochemical substrates. As discussed in relation to the Golgi-GERL complex in magnocellular neurons (see Section III,C), AcPase-positive GERL cisterns appear to produce both primary lysosomes and putative NGVs. In a variety of cells it has been shown that the biosynthetic pathway for lysosomal hydrolases, almost all of them glycoproteins, is similar to that for secretory proteins (Neufeld, 1981). Sorting out of lysosomal enzymes from other proteins in a common compartment such as GERL may be accomplished by specific receptors associated with clathrincoated membrane (Goldstein et al., 1979). In neurosecretory cells some of the small coated vesicles (40-60 nm) originating from GERL are indeed considered primary lysosomes (see Broadwell and Oliver, 1981), but it is also possible that electron-dense vesicles (100-200 nm) of GERL origin are anoter type of “virgin” lysosome (Picard et al., 1972, 1978). Diversity of primary lysosomal compartments has recently been described also in crab photoreceptors (Blest et al., 1980) and in exocrine acinar cells (Oliver, 1980). Elongated primary lysosomes, identified by trimetaphosphatase content, appear to arise directly from the ER, so that “in addition to delivery via GERL, there may be another way by which lysosomes receive their enzymes” (Oliver, 1980). Some authors maintain that in neurosecretory cells lysosomal enzymes are conveyed by the aSER (Boudier, 1978; Boudier et al., 1981), which could imply formation of primary lysosomes not only from GERL in the perikaryon but also from aSER along axons and in terminals. The question of aSER conveyance of lysosomal enzymes is equivocal. Castel and Dellmann (1980) have shown that the extensive system of branching and
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anastomosing aSER in neurosecretory neurons is TPPase-positive (a syntehtic rather than a degradative enzyme), and only rarely, in osmotically stressed specimens, are limited tracts of aSER AcPase-positive (see also Section V,B). There has been a tendency to indiscriminately refer to a variety of tubular and vacuolar structures as parts of the aSER, but as aptly stated by Broadwell et u1. (1980), elongated lysosomal sacs and cistersn are probably not genuine aSER, but rather a separate system of discrete organelles. Many of these were shown to sequester endogenous acid hydrolases as well as exogenous HRP imbibed from extracellular space, attesting to at least partial endocytotic origin. This is in line with the evidence of LaVail et al. (1980) from serially sectioned chick ciliary ganglion showing retrograde axonal conveyance of HRP in blunt-ended sacs, not in the branching and anastomosing system of aSER. Images of putative lysosomes with small tails attached, both in neurosecretory (Boudier et a / . , 1981) and in other cells (Novikoff and Shin, 1964, 1978), have been presented as evidence of continuity between the lysosomal compartment and the aSER. but the validity of this assertion remains uncertain. The uptake and fate of exogenous blood-borne markers, such as HRP, exemplifies the process of heterophagy associated with the lysosomal system. About 2 hours after endocytosis at neurohypophysial endings the marker has been conveyed retrogradely within elongated cisterns into the hypophysial stalk, and within 6 hours reaches the perikaryon (Broadwell et a / . , 1980; Price and Fisher, 1978; Theodosis, 1981). Exactly at what point in timc and space these endocytotic organcllcs acquire their lysosomal properties is uncertain. In neurohypophysiai endings AcPase has been detected within macrovesicles appearing identical with secretion-related cndocytotic vesicles that imbibe HRP (Castel and Dellmann, 1981; Fig. 2 2 ) . However, Braodwell rt u l . (1980) maintain that only during subsequent retrograde transport of HRP-sequestering organelles do they fuse with lysosomal elements to form compound structures. Examples of the latter are vacuoles with attached elongated cisterns found in the cell body, particularly following osmotic stre The ultimate fate of the membrane and content 0 1 such organelles remains an enigma; HRP has not been detectcd within Golgi or GERL. The intracellular route of cationized or lectin-bound tracers in neurosecretory cells has not, to our knowledge, been reported to date. Evidcnce of lytic processes per se is rare in neurosecretory perikarya, although primary lysosomes are produced in that location and postlysosomal organelles such as dense bodies are frequently found there. Even during pile-up of NGVs in the perikaryon, following colchicine administration (Boudier and Picard, 1976; Dellmann and Sikora, 198 I ) or anterior hypothalamic Icsions (Carithers et a/. , 1981), thcrc is little evidence of granulolysis either by crinophagy or autophagy in the cell body. According to Boudier (1978) crinophagy (fusion of secretory granules with lysosomes as described by Smith and Farquhar, 1966) is rare throughout the neurosecretory ccll. However, ininiunocytochemical evidence
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FIG.22. Mouse neurohypophysis. Electron micrographs show that (A) acid-phosphatase-pow rive cisterns in neurosecretory endings appear to be the same as those that (B) sequester extracellular horseradish-peroxidase, and are considered to be secretion-related. (From Caste1 and Dellmann, 1981.) (A) X47,OOO. (B) X86,OOO.
provided by Broadwell et ul. (1979) attests to the presence of neurohypophysial hormones within some secondary lysosomes. In contrast to the perikaryon, a variety of autophagic images are readily encountered in axon varicosites in the neurohypophysis of both normal and osmotically stressed specimens (Pilgrim, 1970; Dellmann, 1973; Livingston, 1973; Boudier, 1978, 1979; Boudier et ul., 1981). A scheme of autophagic vacuole (AV) genesis by Boudier (1978) depicts the limiting membrane of AVs originating from dumbbell-shaped profiles that wrap around and segregate structures destined for degradation. Stereology has revealed that the “wrappers” are
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in fact flat, saucer-shaped cisterns, often of polymorphic design (Boudier et al., 1981). These authors favour the view that the flat, wrapper cisterns are derived from the aSER, presumably containing latent hydrolases. However, studies of autophagy in cultured glial cells indicate that preexisting lysosomes may change their shape, flatten out, and thcn wrap around other organelles (Hamberg et a / ., 1977). These two views may be reconciled by the idea that sequestering membranes of AVs in neurons may derive from a system of lysosomal organelles capable of transient attachment and detachment from the aSER (Tsukita and Ishikawa, 1980). Autophagic “wrappers” may also be of endocytotic origin, and Boudier (1979) has suggested, as an alternative possibility, that dumbbellshaped profiles involved in autophagy may be identical with the secretion-associated, endocytotic sickle-shaped organelles described by Theodosis et al. (1976). However, this idea complicates the issue, for sickle-shaped cisterns are also associated with heterophagous activity, such as uptake of markers from extracellular space (Theodosis et. al., 1976; Broadwell et al., 1980). A mingling of heterophagic and autophagic compartments has not, to our knowledge, been reported in other cells. It is difficult at present to integrate the available data on lysosome-related phenomena in neurosecretory cells, particularly as most of the observations are based on morphological studies only, Profiles in electron micrographs represent only fragments of complex systems, and even when enzyme cytochemical methods are used, false negatives may fail to identify structural links in a particular system. Absence of cytochemical reaction may be due to low sensitivity of the procedure employed, or use of an irrelevant substrate. On the other hand, false positive results due to nonenzymatic binding of lead also plague cytochemical methods currently in use (Holtzman et al., 1977). The recent suggestion that lysosomal enzymes may be involved in processing neurosecretory products indicates an additional direction for research (Broadwell and Oliver, 1981).
VI. Molecular Organization of Secretory Vesicles in Neurons It is generaly believed that neurotransmitters and neurohormones are stored in vesicular organelles and secreted at nerve terminals by an exocytotic process (Llinas and Heuser, 1977). Perhaps the most compelling case for this view is the observation that macromolecules stored in these vesicles, such as dopamine-@hydroxylase in catecholamine-containing vesicles and neurophysin in neurohypophysial peptide-containing vesicles, are coordinately secreted with the smaller biologically active molecules. However, even heroic efforts to prove the “vesicular hypothesis” at cholinergic synapses (Heuser et al., 1979) do not satisfy the objections of some of the sceptics (for example, see Marchbanks, 1975; Tauc, 1979). Although secretory mechanisms will be discussed in a later
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section, it follows from the above that studies on the composition of secretory vesicles have played a major role in this issue. In a similar manner such studies serve to focus attention on those biochemical and structural features of the neuron which are necessary for the production of membrane-bounded compartments that store, translocate, and release specific biologically active molecules. A. PHYSICAL CHARACTERISTICS OF SECRETORY VESICLES The physical properties of the vesicles, such as their diameters and electron densities observed by transmission electron microscopy, are as much differentiated properties of specific neurons as are the neurotransmitters that they synthesize and secrete. Table I1 illustrates the large range of vesicle sizes found in neurons (and neurally derived tissues such as the adrenal medulla). The ratio of TABLE I1 PHYSICAL CHARACTERISTICS AND BIOLOGICALLY ACTWEMOLECULECONTENTS OF VARIOUS SECRETORY VESICLESO
Tissue Frog motorneuronb Sympathetic neuron‘ Neurosecretory cell (posterior pituitaIy)d Adrenal medullar
Average Volume of vesicle Volume of vesicle diameter vesicle core membrane (nm) (n~n)~ (nm)3
Percentage total volume of vesicle membrane
50
2.44
X
lo4 3.96
X
lo4
62
75
1.16
X
105
1.08
X
lo5
48
175
2.13 X 106 0.61
X
106
22
340
1.77
X
106
14
X
lo7 2.83
Number of active molecules per vesicle 104 (acetycholine) 1.5 X lo4 (norepinephrine) 8 . 4 X lo4 (vasopressin)
5.9 X lo6 (catechalomine) 1.7 x 104 (enkephalin)
Concentration of molecules in core ( M ) 0.68 0.21 0.07
0.55
oThe volumes were calculated assuming perfect spheres, and a membrane thickness of 7 nm. bDiameter of cholinergic vesicle taken from Heuser et al. (1979) and number of acetylcholine molecules per vesicle from Kuffler and Nicholls (1978). ‘Diameter of large dense-cored vesicle and number of norepinephrine molecules per vesicle from Lagercrantz (1976). “Diameter of neurosecretory granulated vesicle from Nordmann el al. (1979), and number of vasopressin molecules per vesicle from Morris (1976). ‘Diameter of chromaffin granule from Kirschner (1 974). The number of catecholamine molecules per vesicle was calculated from the 0.55 M concentration given in Winkler (1976). See Winkler and Westhead (1980) for more complete analysis of vesicle components.
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adrenal chromaffin granule volume to that of the cholinergic vesicles is more than 300: I , and the chromaffin granule contains about 500 times more catecholamine molecules than acetylcholine molecules present in the cholinergic vesicle. While the electron density of the vesicle cores can be attributed to their macromolecular constituents (Lagercrantz, 1976), virtually nothing is known about the parameters that govern vesicle size. Whether the vesicular dimensions are determined by the specific lipid and protein compositions of their membranes, or by the structural constraints exerted by the contents of the vesicle is not known. Studies of the composition of secretory vesicles are limited by the technical problems of isolating pure granule fractions free of other contaminating membrane components in the cell (see Jones, 1975; Morgan, 1976). Even for the chromaffin granules, where the isolation procedures have been refined for over 20 years, the results have often been contradictory (Winkler, 1976). This problem is even more severe for the smaller vesicles such as the cholinergic vesicles and the dense core vesicles in sympathetic neurons. The contribution of the membrane phase to the total volume in these vesicles is about SO% (Table 11; in the small dense core vesicles in sympathetic nerves the value is 68%; Smith, 1972), and therefore the reliability of the compositional analysis of these vesicles is more critically determined by the purity of the fraction, e.g., contamination of a fraction containing 100 cholinergic vesicles (50 nm in diameter) by one 340 n m vesiculated membrane would contaminate the contents of the vesicle membranes by SO%; and hence even the usual electron microscopic demonstrations of the purity of the isolated fractions would hardly be reassuring. Nevertheless, in spite of these limitations compositional analyses of the vesicles are often useful, particularly with regard to the contents of the core. For example, in the case of the cholinergic vesicle, analysis of the lipid to protein ration gives a value of 3.5 (w/w) and this itself argues against the presence of a protein core (Tashiro and Stadlar, 1978). In contrast, in the chromaffin granule this ratio is about 0.5 (Winkler, 1976). This difference cannot only be attributcd to the larger size of the chomaffin granulc, since the larger dense core vesicles in sympathetic nerves which are closer in size to the cholinergic vesicles are relatively protein rich, i.e., large dense-core vesicles contain 0.78 Fmol lipid-phosphorusirng protein compared to values of 0.45 in chroniaffin granules (Lagercrantz, 1976; Fillenz, 1977) versus 3.8 in cholinergic vesicles (Tashiro and Stadler, 1978). The significance of this point is that these data themselves are inconsistent with the original report of a putative core protein in cholinergic vesicles (i.e., vesiculin; Whittaker et a / . , 1974). Hence, it is not surprising that recent studies have demonstrated “vesiculin” to be a glycosaminoglycan (Stadler and Whittaker, 1978; Tashiro and Stadler, 1978), of about 10,000 in niolecular weight, which relatively selectively binds neutral and acidic amino acids. Cholinergic vesicles isolated from electric organs of Torpedo contain about 0.2 mg of this glycosaminoglycan per mg of protein (Stadler and Whittaker, 1978).
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These authors suggest that the 10,000 MW molecule may in fact be the breakdown product of a higher molecular weight proteoglycan degraded during the isolation procedure.
B. THECHOLINERGIC VESICLE The concentration of acetylcholine in the cholinergic vesicle has been estimated to be from 70 to 80 mM (Dunant et a l . , 1972) to 520 mM (Wagner and Kelley, 1979), the variation in part due to the reasons cited above, Calculations such as that shown in Table 11, based on physiological estimations of the number of acetylcholine molecules per quantum (or vesicle), yield 0.68 M ,a value more consistent with the upper range of concentrations measured biochemically (Wagner and Kelly, 1979; Tashiro and Stadler, 1978). The cholinergic vesicle has also been reported to contain 170 mM ATP and 20 mM GTP (Wagner and Kelly, 1979). A recent review of the stoichiometry of biochemical components in cholinergic vesicles isolated from electric organs of Torpedo and Narcine has been published by Morris (1980). In this review, the author points out that the vesicle diameters measured by electron microscopy are decreased by about 15% (i.e., morphological analyses yields 84-88 nm diameters, whereas physical measurements indicate diameters of about 100 nm in Torpedo). Each Torpedo vesicle contains, according to this analysis, 8.0 X protein, 7.2 X g g of lipid, 19.1 X lo4 molecules of ATP. glycosaminoglycan, 1.35 X The acetylcholine/ATP ration ranges from 5.8 to 7.4, and the cholesterol: phospholipid ratio is 0.42:l. The major phosopholipids in the vesicle are phosphatidylcholine (46.6%), phosphatidylethanolamine (29.5%), phosphatidylserine (l2.6%), phosphatidylinosital (5. I %), and sphingomyelin (5.1%). Lysolecithin (0.4%) and phosphatidic acid (0.6%) are of relatively low concentration. Morris (1980) points out that these vesicle contents are isomolar with the cytosol (see below). Analysis of the protein species in highly purified cholinergic vesicle membranes shows that there are 14 distinct proteins on SDS gels ranging in molecular weight from 20 to 160,000, one of which is actin (Tashiro and Stadler, 1978; Stadler and Tashiro, 1979). Wagner and Kelly (1979) report that there are eight proteins found in synaptic vesicle membranes isolated from Narcine electric organ. Using iodination procedures they conclude that six are on the external (cytoplasmic) face of the membrane, and two on the inner membrane surface. Curiously actin is reported to be on the internal surface. Of these eight proteins, six are specific to the vesicle membrane, whereas two (of which one is actin) are found on other membranes. Stadler and Tashiro (1979) similarly find that actin is common to vesicle membranes and synaptosomal plasma membranes, whereas the other proteins are not. These data differ substantially from the work of Morgan et al. (1973) which showed many common proteins between vesicle
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membranes and plasma membranes. Presumably this difference is due to improvements in the purity of the vesicle preparations. Recent work by Zimmerman and his colleagues (Zimmerman, 1979; Giompres et a / ., 198la-c) indicate that cholinergic vesicles in Torpedo electric-organ may be heterogeneous in size depending on the state of the nerve ending. Stimulation of the electric organ at 0.1 Hz produces a decrease in tissue response and acetylcholine content but no decrease in vesicle number. After 1800 impulses, in 80.5% of the vesicles measured there is a 25% decrease in diameter (Zimmerman and Denston, 1977a). These smaller vesicles contain most of the newly synthesized acetylcholine, with a specific activity ratio in newly synthesized acetylcholine between the smaller and normal sized vesicles of 16.5 (Zimmerman and Denston, 1977b). Vesicle diameters return to control values after 23 hours in vivo (Zimmerman and Denston, 1977a). While these data are usually discussed in the context of the vesicular hypothesis debate (Zimmerman and Whittaker, 1977; Suszkiw et a / . , 1978; Tauc, 1979), the change in vesicular size itself is of some interest. It would be useful to know if the number of acetylcholine molecules per vesicle in the two vesicles differ or are the same. How does the 25% smaller vesicle increase in size after 23 hours? It is more membrane added directly to the vesicle membrane, and if so, from what source? The data of Bennett et al. (1976) indicate that under conditions of intense stimulation neurotransmitter quanta1 sizes may decrease, and therefore newly recycled vesicles may be ready for release before they contain their full complement of neurotransmitter. In a recent series of experiments this group (Giompres et al., 1981a-c) has begun to address some of these issues. Their conclusions are that the stimulationinduced smaller vesicles (about 64 nm) have a lower osmotic pressure than the resting vesicles (91 nm), and that the loading of the small vesicles with acetylcholine is accompanied by osmotic induced rehydration of the vesicles. In this manner, the vesicles return to their 91 nm diameter. These authors (Giompres et al., 1981~)further speculate that the exocytosis of the acetylcholine leads to a stoichiometric exchange with Ca2 (possibly bound to the glycosaminoglycan), resulting in an electrochemical but not osmotic equilbrium (but increased density) in the recycled vesicle. They then propose that the Ca2+ in the vesicle exchanges with acetylcholine in the cytosol via a Ca2 activated ATPase during loading of the vesicle, and that intravesicular ADP exchanges with cytosolic ATP via an attractyloside inhibitable mechanism. Further experimental work is necessary in order to evaluate this interesting proposal. +
+
C. VESICLES IN SYMPATHETIC NEURONS Heterogeneity of vesicle size is characteristic of sympathetic neurons (see also Section VII1,D). Noradrenaline is stored in sympathetic nerves in large (75 nm) and small (45nm) dense-cored vesicles (Geffen and Livett, 1971; Smith, 1972;
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Lagercrantz, 1976; and Fillenz, 1977). The large and small dense-cored vesicles can be separated by density gradient centrifugation since the small vesicles have a lighter buoyant density. Large dense-cored vesicles resemble chromaffin granules in their composition in that they contain, in addition to noradrenaline, also dopamine-P-hydroxylase (DBH), chromagranin A (in trace amounts), cytochrome b s 6 , , MgZ+-stimulated ATPase, ATP, and chromomembrin B (Lagercrantz, 1976). The large dense-cored vesicle (LDCV) differs from the chromaffin granule in its storage capacity for catecholamine (the LDCV contains 110- 130 nrnol/mg protein versus 2500 nmol/mg protein in the chromaffin granule), and in that it contains very little chromagranin A. Hence, DBH is the major protein stored in the LDCV. Another difference is that whereas 80% of the chromaffin granule’s protein (64% of the DBH) is water soluble, only 25% of the LDCV’s protein (8-18% of the DBHO is water soluble (Lagercrantz, 1976; Winkler, 1976; Fillenz, 1977). The small dense-cored vesicles (SDCV) also contain DBH (Bisby et ul., 1973; DePotter and Chubb, 1977). Comparisons of the water-soluble (core) and insoluble (membrane-associated) proteins of the LDCV and chromaffin granule show that a total of 7 soluble and 5 insoluble proteins appear to be common to both vesicles, although quantitative differences in these proteins do exist (Bartlett et ul., 1976; see also Abbs and Phillips, 1980). The distribution patterns of the LDCVs and SDCVs in sympathetic neurons are not homogeneous (see Section VII1,D). LDCVs predominate in the axon (Dahlstrom, 1971, Fillenz, 1977), whereas the SDCVs make up between 80% (in the spleen) and 95% (in the vas deferens and heart) of the total vesicle population in nerve terminals (Fillenz, 1977). The small vesicle population itself may not be homogeneous (Fillenz and Pollard, 1976; DePotter and Chubb, 1977). The relationship between all these vesicles and the release process is not entirely certain. DePotter and Chubb (1977) suggest, on the basis of stimulation experiments with the splenic nerve of the dog, that the two types of SDCVs in the terminal have different origins. One type is believed to be derived from the aSER (see also Tranzer, 1972, 1973; Teichberg and Holtzman, 1973), does not contain DBH and therefore cannot synthesize but may store noradrenaline. This is the major form in the resting terminal. The other type of SDCV, the authors propose, is derived from the recycled membrane of the LDCV after exocytosis during stimulating conditions (see Fig. 25A and B). The evidence for this is that before stimulation the SDCVs have very little DBH, whereas following stimulation the membrane-bounded DBH is quantiatively recovered in the SDCV fraction. Thus, the DBH-containing SDCVs are, in this view, derived from the LDCVs which were originally produced from the perikaryal Golgi complex, and delivered by axonal transport to the terminals. Furthermore, the quantiative distribution of these three types of vesicles in the nerve terminal would then be determined by the state of the activity of the neuron. Proof as to whether this attractive hypothesis is correct requires further work. It is quite likely that LDCVs and SDCVs are both involved in transmitter release in sympathetic nerves (see Geffen and Jar-
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rott, 1977; and Section VI11,D). SDCVs have also been reported to be present in growth cones of cultured sympathetic neurons (Landis, 1978). D. THE CHROMAFFIN GRANULE Ever since the classic analysis of the composition of chromaffin granules by Hillarp (1959), the study of this model secretory vesicle has been an especially active area (for reviews see Kirschner, 1974; Helle and Serck-Hanssen, 1975; Winkler, 1976; Pollard et ul., 1979a). The critical reviews by Winkler (1976) and Winkler and Westhcad ( 1 980) are the most comprehensive accounts of the composition of chromaffin granules. The chromaffin granules contain about 0.55 M catecholamine (72% adrenaline, 27% noradrenaline, and 1% dopamine), about 0.01 M Leu- and Met-enkephalin, 0.13-0.16 M ATP, 0.02 M GTP, 0.03 M CaZ , 0.006 M Mg2 , and 0.03 M ascorbic acid. The water-soluble components of the granule include all the catecholamine, opioid peptides, nucleotides, most of the divalent cations, some mucopolysaccharides (chondroitin sulfate), and 77% of the total granule’s protein. The major soluble protein (40-50%) is a 74-8 1,200 MW acidic protein called chromogranin A. Virtually all this protein is soluble, and whether any of it is membrane-bound is a matter of debate (Winkler, 1976). In contrast, the enzyme DBH represents about 5 % of the total soluble protein. About 64% of the granule’s total DBH is water-soluble, the rest being membrane-bound (see below). DBH is a glycoprotein of about 28,000 MW (composed of 4 subunits of 75,000 MW), and amino acid analysis of the soluble and membrane-bound forms of DBH (the membrane DBH was also referred to as chromomembrin A in the early literature) indicate that they are the same. The catecholamine content is 2.5 Fmol/mg protein, and the catecho1amine;enkephalin:ATPmolar ratio is 1:0.029:0.22. The vesicle membrane proteins include DBH (20% of total membrane pro1976) as do all the teins), which faces the inside of the vesicle (Konig et d., glycoproteins. Approximately 40 individual proteins have been detected that are associated with this membrane, 11 of which face inside the vesicle, and 30 of which can be labeled from the cytoplasmic side by surface labeling methods (Winkler and Westhead, 1980). Among these proteins are a-actinin (97,000 daltons, 4% of total protein), which faces the cytoplasmic side; and actin (2% of total protein) which may be binding to the a-actin on the outside (see below regarding cytoskeletal proteins). Another identified membrane protein facing the cytoplasmic surface is cytochrome hs(,, (molecular weight 21 -28,000, about 10% of total protein), formally referred to in the earlier literature as chromomembrin B. Several enzymes have been localized in the vesicle membrane, phosphatidlylinositol kinase (facing the cytosol), and a Mg2 -activated ATPase (about 10 moleculeslgranule). This ATPase is the basis of the proton pump mechanism (see Section VII) and is composed of a 400,000 MW F, complex +
+
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(facing the cytosol) which hydrolyses ATP, and F,, subunits ((8- 10,000 molecular weight, embedded in the membrane) which are responsible for the proton translocation. The vesicle membrane lipids are composed of phospholipids (2 pmolimg protein) and cholesterol. The molar ration of cholesterol to phospholipid is 0.6. The most distinctive feature of the lipids in this membrane are the high levels of lysolecithin (i.e., 16% of total phospholipids). Surface labeling methods (Winkler and Westhead, 1980) have shown most of the phosphatidylcholine is on the surface facing the granule core, whereas phosphatidylethanolamine faces the cytosol. Phosphatidylserine and phosphatidylinositol (10%) face the cytosolic surface, and provide most of the negative charges characteristic of this granule surface. Several cytoskeletal proteins have been shown to associate with the chromaffin granule membrane. These include actin (Burridge and Phillips, 1975) which may comprise 2% of the membrane protein (Meyer and Burger, 1979a), and a-actininin (Jockusch et al., 1977). Furthermore, Pollard e t a ! . (1979b) have found a 47,000 MW protein, extractable from adrenal medulla, which they call synexin, and which can promote Ca2+ -dependent aggregation of the granules. The authors suggest that this protein may play a role in the fusion process during exocytosis. Morris and Hughes (1979) confirm this action of synexin and its Ca2+ specificity, but argue that its action is not specific for granule membranes and similar results can be obtained with mitochondria, microsomes, and phosphatidlycholine vesicles. These authors state that this lack of specificity argues against a role for synexin in exocytosis (for theory of aggregation see Haynes et al., 1979). Another protein of about 51,000 daltons which binds to chromaffin granules, but which differs from synexin, has been isolated from the plasma membranes of adrenal medulla (Meyer and Burger, 1979b). Recently, small peptides in the 2-3000 molecular weight range were isolated from the chromaffin granule (Roda and Hogue-Angeletti, 1979). These peptides have now been characterized and are Met-enkephalin and Leu-enkephalin (Stern et al., 1979; Winkler and Westhead, 1980). The high concentration of small molecules in the chromaffin granule suggests that they must be organized in an osmotically inactive complex (see discussion in Winkler and Westhead, 1980). Osmometric studies on isolated chromaffin granules show that they contain an osmotically inactive space of about 37% (Morris and Schovanka, 1977; Morris et al., 1977). The molecular organization of the granule core which produces such a situation is again not understood, although a recent publication by Pollard et al. (1979a) sheds some light on this issue. This study utilizes 31Pnuclear magnetic resonance spectroscopy to measure the internal pH and state of ATP in the chromaffin granule. The intragranular pH was found to be 5.7, a value in agreement with previous studies using [ "Tlmethylamine distribution methods (Johnson and Scarpa, 1976; Casey et al., 1977). The
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intragranular ATP does not behave as if it were in free solution, nor does it appear to be associated with Caz+ or Mg2+, but resembles crystalline ATP (although it is known from X-ray studies that there is no crystalline structure in the granule core; see Pollard et al., 1973). The authors concluded, on the basis of pK, values, that the intragranular ATP was in a form with a minimum net negative charge so that it could not function as a counterion for the stored catecholamine, in contrast to previous views on the significance of the 4: 1 ratio of amines to ATP found intragranularly. While these observations do not clarify the nature of the package, they do serve to eliminate one possible mechanism, i.e., electrostatic interaction between ATP and catecholamine. The abidic intragranular pH is consistent with a chemiosmotic model of catecholamine accumulation to be discussed in Section VII, and in addition is close to the in vitro pH optimum (5.5) of DBH activity (Johnson and Scarpa, 1976). This acidic internal pH of secretory vesicles is a widespread phenomenon. The histamine-concentrating granules in rat mast cells are also acidic (Pletscher and DaPrada, 1975), and the serotonin-containing granules of pig platelets have an internal pH of 5.74 (Johnson et ul., 1978). All these vesicles accumulate amines and it has been suggested that this is the primary role of this acidic internal pH, i.e., to provide the necessary pH gradient (see Sections I1 and VII for discussion of other roles for this acidic intravesicular pH).
E. NEUROSECRETORYGRANULATED VESICLES Despite the fact that neurosecretory granulated vesicles (NGVs) (also known as neurosecretory granules, NSGs) have been isolated from neural lobes in reasonably pure form for some time (Barer ef ul., 1963; Ginsburg and Ireland, 1966; Dean and Hope, 1967, 1968; Labella et al., 1963) relatively little information about their composition is available (in comparison to the chromaffin granule). Although these granule preparations are heterogeneous, i.e., they are usually composed of OT-storing and VP-storing granulated vesicles, the separation of these two vesicles from one another by ultracentrifugation methods is problematic (Labella er al., 1963; Dean et al., 1968), and most analyses of composition have been done on mixed fractions. It is now generally agreed that the OT and VP peptide hormones and their vesicles are synthesized in separate neurons in the hypothalamus, and transported in separate axons to the pituitary, where they are stored in and released from separate nerve terminals (see Section 11). The neurohormones are stored in the vesicles in complex with an equimolar amount of a 10,000 molecular weight “carrier protein,” neurophysin, at a concentration of about 0.1 M (Dean and Hope, 1967, 1968; Dreifuss, 1975; Morris, 1976; and Table 11). The soluble proteins are composed primarily of the neurophysins which make up about 50% of the total protein. The granules also contain about 1 nmol ATP/mg of protein (Poisner and Douglas, 1968), which on
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a molar basis is 100 times less than the ATP stored in chromaffin granules (Dreifuss, 1975). The granules contain Ca2+ in a concentration (73 M l m g protein) comparable to that of chromaffin granules (Thorn et al., 1975). Information about the lipid composition of the granules is in a confused state (see discussion in Cohen et al., 1979), but it is known that in contrast to the chromaffin granules, lysolecithin accounts for less than 1% of the total phospholipid (Vilhardt and Holmer, 1972). The concentration of hexosamine in the granules is 28 p.g/mg protein (Thorn et al., 1975), and indirect data from studies on the biosynthesis of the hormones (Brownstein et al., 1980) indicate that a small glycoprotein (10,000 molecular weight) is present in the vasopressin granules (but not the oxytocin granules) in an amount equimolar to the vasopressin. This is consistent with recent evidence (Land et al., 1982; see Section 11) that the vasopressin prohormone is a glycoprotein, and contains a 39 amino acid glycopeptide moiety. Histochemical data (Tasso et al., 1976) from the neural lobe support this contention in that only one group of granulated vesicles reacts for glycoprotein. Several enzymes have been reported to be associated with the NGVs: 3'3'CAMP-dependent kinase (McKelvy , 1975), a membrane-bound adenylate cyclase (Bonne et al., 1977), and a Ca2+-Mg2+ -activated ATPase (Vilhardt and Hope, 1974). In the case of the latter enzyme, the authors, after repeated purifications of the granule fraction, could not discriminate whether the ATPase present was truly a granule enzyme or was due to contamination of the fraction. Recent studies by Russell (1984) using highly purified vesicles and vesicle membranes have shown that a Mg2+-ATPase very similar to that found in the chromaffin granule membrane is also located in the NGV membrane. Since intravesicular conversion of the prohromones has been postulated (see Section 11), one would predict that various processing enzymes should reside within the NGVs. Thus far, a prohormone converting enzyme cleaving at pairs of basic amino acids (Chang et d.,1982; Section 11) and an acidic carboxypeptidase B-like enzyme have been found in these vesicles. A chymotrypsin-like enzyme presumably involved in the degradation of neurophysin has also been found associated with the granule fraction (North et al., 1977). As was mentioned above, the intragranular pH appears to be comparable to that of the chromaffin granule (i.e., around 5.5). The value of this internal pH in the NGV may be related to the fact that the neurohormone-neurophysin complex in vitro (Breslow, 1979; Cohen et aL., 1979; Pickering and Jones, 1979) and in situ in the granule (Morris and Cannata, 1973; Cannata and Morris, 1973) is highly pH dependent and has a pH optimum for binding of about 5.5. Thus, this internal pH would be essential in maintaining the osmotically inactive volume within the NGV core. In this regard, it is interesting to note that several electron microscopic studies of neurosecretory granules in some mammalian species report a crystalline-like core structure (Holmes and Kiernan, 1964; Barer and
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Lederis, 1966; Bargmann and Gaudecker, 1969; Donev, 1970a; Rodriguez, 1971; Boudier and Burlet, 1978).
VII. Biosynthesis and Biochemical Aspects of Packaging and Transport of Neurotransmitters in Nonpeptidergic Neurons A. CHOLINERCIC NEURONS The biosynthesis of acetylcholine is by the enzymatic transfer of the acetyl group of acetyl CoA to the hydroxyl group of choline. The synthetic enzyme is choline acetyltransferase (ChAt, EC 2.3.2.6), which is considered a soluble enzyme in axoplasm, although it has been found both in soluble and membranebound forms (Fonnum, 1967, 1968, 1970; Kuczenski et ul., 1975). The axonal transport of ChAt appears to be slow, between 0.3 and 3 mm/day in mammalian nerves (Jablecki and Brimijoin, 1975; Wooten and Coyle, 1973; Tucek, 1975) consistent with its soluble status, although a small fraction of the ChAt is reported to move at higher rates (Fonnum et al., 1973). Jope (1979) has recently suggested that the membrane-bound form of ChAT may be directly coupled to the high affinity transport of choline from the extracellular space so that acetylation may occur concurrently with transport of the choline into the cell. The low rate of axonal transport of intracellular ChAT stands in contrast to the consistent reports of rapid transport of acetylcholine (Koike et- u l . , 1972; Schafer, 1973; Haggendahl et ul., 1973; Dahlstriim and Heiwall, 1975; Waldclind and Anderson, 1978), and that this acetylcholine transport can be blocked by colchicine or vinblastine (Heiwall et al., 1976; Woodward and Lindstrom, 1977). However, it appears that most of the acetylcholine in the axon is in the soluble form and not transported. It is most likely that the transported form is membrane-bounded (Koike c r a/., 1972), but this rnembrane-bounded acetylcholine can [reely exchange with the soluble pool on the axoplasm. The morphological nature of this acetylcholine-containing membrane compartment is not known, and very few synaptic vesicles may be observed in transit along cholinergic axons (see Section VIII,F for discussion on possible origins of synaptic vesicles). Two candidates for the origins of the synaptic vesicles are the Golgi region in the pcrikaryon and the agranular reticulum within the axon (aSER) (Holtzman, 1977; Holtzman C? al., 1977; Droz, 1975). However, the relative importance of these two sites as sources of new vesicle membranes is unresolved at present (see Section VII1.F; Fig. 26). One unambiguous source is at cholinergic nerve terminals via vesicle membrane retrieval and recycling following exocytosis (Heuser and Keese, 1973). At present the quantitative relationship between the newly synthesized membrane delivered via axonal transport and the recycled membrane
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with regards to vesicle membrane turnover is still unclear. Acetylcholine is synthesized in the axoplasm and transferred into the vesicles in which it is accumulated in high concentration. The degradation of acetylcholine to acetate and choline occurs by the enzyme acetycholinestrease, also called actelycholine acetylhydrolase (AChE, EC 3.1.1.7). AChE can be found in both soluble and membrane-bounded forms (Ross et a l . , 1971), and is found in multiple molecular forms in mammalian peripheral nerves. Although the enzyme is reported to move by both anterograde and retrogade transport (Lubinska and Niemierko, 1971; Ranish and Ochs, 1972) most of it in the axon is stationary (Ranish and Ochs, 1972; Brimijoin and Wiermaa, 1978). Two higher molecular weight forms (10 S and 16 S AChE) appear to be carried by fast axonal transport (DiGiamberadino and Courand, 1978; DiGiamberadino et al., 1979; Festoff and Fernandez, 1978), and the 16 S form of the enzyme seems to account for most of the total (3%) of the sciatic nerve AChE which is transported (Fernandez et all, 1980). The 16 S AChE is sedimentable and appears to be selectively confined to cholinergic motor fibers (Fernandez et al., 1979) whereas the 10 S AChE and 4 S AChE appear to be on the outer axolemmal surface and in extracellular space (Brimijoin, 1972). B . CATECHOLAMINE-CONTAINING CELLS The biosynthesis of catecholamines proceeds through a well-established series of enzymatic reactions (see Blaschko, 1959; Krischner, 1975). Tyrosine, taken up through the plasmalemma, is converted to 1 -dihydroxyphenylanine ( 1DOPA) by the enzyme tyrosine hydroxylase (TH, EC 1.14.16.2). DOPA is then converted to dopamine by DOPA-decarboxylase (DDC, EC 4.1.1.28). DDC is also known as 1-aromatic amino acid decarboxylase due to its relative lack of specificity (i.e, a variety of aromatic amino acids such as tyrosine, tryptophane, phenylalanine, and histidine can be decarboxylated by it). TH and DDC are both considered soluble enzymes located in the cytoplasm, although a fraction of TH may be found absorbed to membranes (Laduron and Belpaire, 1968; Wurzburger and Musacchio, 197 1 ; Lloyd and Kaufman, 1974). Both enzymes require soluble cofactors for activity: tetrahydrobipterin in the case of TH, and pyridoxal phosphate by DDC. The conversion of dopamine to norepinephrine is catalyzed by dopamine-P-hydroxylase (DBH, EC 1.14.17.1) which is found in the storage vesicles (see Section VI). Hence, this reaction, which requires Cu2 , ascorbate, and molecular oxygen (Friedman and Kaufman, 1965), takes place only after doparnine has been transported into the vesicles. Conversion of norepinephrine to epinephrine is catalyzed by the enzyme phenylethanolamine N-methyltransferase (PNMT, EC 2.1.1.28) using S-adenosylmethionine as the methyl donor. Since the PNMT is in the cytosol, storage of epinephrine by the chromaffin granule in the adrenal medulla would require that after norepinephrine is +
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formed in the vesicle it would exit from the vesicle into the cytosol where it is converted to epinephrine by PNMT. The epinephrine would then be taken up into the chromaffin granule for storage (Winkler, 1977). Thus, the uptake process in catecholamine vesicles (see later for discussion of uptake mechanism) plays a critical role in the biosynthesis of catecholamines. Of all the above enzymes, TH is the rate-limiting enzyme in catecholamine biosynthesis. It also is affected by feedback inhibition by all the intermediate and end-products of synthesis which competitively antagonize the binding of the pteridine cofactor to the apoenzyme (Nagatsu et al., 1964; Ikeda et al., 1966; Wurtman et al., 1969). Catecholamine biosynthesis is regulated by neuronal activity and several reviews discuss the mechanisms of long- and short-term regulation (Fillenz, 1977; Winkler, 1977; Vizi, 1979; Costa et al., 1974). The enzymes involved in the degradation of catecholamines are monoamine oxidase (MAO, EC 1.4.3.4) and catechol-o-methyltransferase (COMT, EC 2.1.1.6). The M A 0 action is via oxidative deamination and requires flavin and iron as cofactors (Kopin, 1972; Youdim et al., 1974). COMT action is by o-methylation with S-adenosylmethionine as the methyl donor (Kopin, 1972). MA0 is found on the outer membrane of mitochondria, whereas COMT is found in soluble and plasma membrane-bound forms. Although TH and DDC are considered soluble enzymes, their axonal transport rates are relatively rapid. Only one report identifies TH as being in the slow component (Kopin and Silberstein, 1972), while most others indicate transport rates greater than 100 mm/day (Jarrot and Geffen, 1972; Oesch et ul., 1973; Wooten and Coyle, 1973). In stop-flow experiments, Brimijoin and Wiermsa (1977a) find that TH moves with a maximum velocity of 300 mm/day (modal velocity of 200 mmiday) and suggest that it may move in association with an organelle. Immunocytochemical studies (see Section VII1,C) find that TH is associated with ER, Golgi complex, and microtubules in neuronal perikarya, and with microtubules, dense core granules, and 40- to 80-nm vesicles in axons (Pickel et ul., 1975a, 1976). However, the relationship of these immunocytochemical findings to the axonal transport rates is unclear (see discussion on limitations of current immunocytochemical methods in Section VI1,C). DDC moves more slowly than TH, but still more rapidly than the slow transport. Starkey and Brimijoin (1978)show a maximum transport rate for DDC of 150 mmiday, and find that only 9% is moving at any moment. These authors suggest an intermittent, reversible binding of soluble DDC to rapidly moving organelles is responsible for the apparent fast rate. Anterograde axonally transported DBH moves at a maximum velocity of 430 mm/day (modal velocity is about 300 mm/day), a value consistent with DBH being located in a catecholamine storage vesicle (Brimijoin, 1975; Brimijoin and Wiermaa, 1977b). Norepinephrine, presumably in the same storage vesicle, has the same rate of transport as the DBH (Brimijoin and Wiermaa, 1977b). lmmu-
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nocytochemical data indicate that axonal DBH is located in ER-like membranes and synaptic vesicles (Pickel et at., 1976; Glazer and Joh, 1977). DBH but not norepinephrine moves in the retrograde direction at about 288 mm/day (Brimijoin and Helland, 1976). The dissociation between norepinephrine and DBH movement in the retrograde direction is consistent with the observation that most of this enzyme is nonfunctional (Nagatsu et al., 1976) but can be shown to be immunoreactive (Fillenz et al., 1976; Ziegler et al., 1976). It appears that the retrogradely transported DBH is destined for degradation in the cell body. The degradative enzymes, MA0 and COMT, move at slow rates (3 mm/day) of anterograde axonal transport (Dahlstrom et al., 1969; Wooten and Coyle, 1973). The biogenesis of new vesicles in sympathetic neurons is believed to occur either in the Golgi region of the perkaryon, where large dense core vesicles (LDCVs) are formed, which in turn give rise to the SDCVs (Fillenz, 1977; Holtzman, 1977; DePotter and Chubb, 1977), or directly as SDCVs from the aSER (Hokfelt, 1973; Teichberg and Holtzman, 1973; Holtzman et a / ., 1977). Quatacker and DePotter (1978; also Quatacker, 1981) have attempted to evaluate by histochemical criteria the structures involved in catecholamine storage and transport in sympathetic neurons, and have made interesting observations regarding putative extensions of the Golgi system in the axon, facilitating catecholamine elaboration throughout the neuron (see Section VIII for details). Little is known about the relative quantitative contributions, at any given moment, of the newly synthesized vesicles versus recycled vesicles in the transmitter economy of the sympathetic neurons. Early studies of vesicle turnover suggested a life span of 3-4 weeks (Dahlstrom, 1971). However, recent studies using cycloheximide inhibition of de ROVO protein synthesis or colshicine blockade of axonal transport give half-lives of DBH (a biochemical marker of vesicles) of less than 24 hours (DePotter and Chubb, 1971; Thoenen et al., 1971; Brimijoin, 1972; Molinoff ef al., 1972). These studies on DBH turnover suggest a very important role for new vesicle formation, but the half-life values are probably underestimates due to the unphysiological conditions under which these data are obtained. As might be expected, the most thorough biochemical analysis of secretory vesicle biogenesis has been done for chromaffin granules. Only a brief summary of the conclusions are presented here since an excellent review by Winkler (1977) is available (see also Section VIl1,A). The newly synthesized chromaffin granules assembled in the Golgi region are less dense on sucrose density gradient centrifugation than mature granules, and are lower in ATP and cathecholamine content. These are often referred to as “progranules.” It takes about 24 hours for the progranules to accumulate ATP and catecholamines to their final levels, and to acquire the density of mature chromaffin granules. The ATP and Ca2 appear to be taken up more rapidly than the catecholamines. The ATP and catecholamines are transported via distinct carrier mediated transport systems, but both appear to require a pH gradient across the granule membrane. Pulse-chase label+
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ing studies using [4H]leucine, [3H]fucose, "SO 4' and "PO, all support the contention that the soluble macromolecular content of the granule turns over at a much faster rate than the granule membrane's proteins and lipids, suggesting considerable reutilization of membrane retrieved after exocytosis (see Fig. 7 in Winkler, 1977). Nascent secretory granules are normally stored in the cell for several hours in order to mature and accumulate catecholamine content. However, during periods of stimulation and augmented secretion, various regulatory mechanisms are employed by the cells to speed up maturation of the vesicles (see Winkler, 1977). In this regard, it is interesting that neurosecretory cells, which accumulate secretory products by entirely different mechanisms, also regulate vesicular maturation rates to match variations in secretion rates (see Section 11). C. CATECHOLAMINE UPTAKEINro CJIROMAFFIN VESICLES As discussed above, the uptake and concentration of neurotransmitters in the vesicle are critical properties of the vesicle itself. Little is known about the mechanisms which underly this important phenomenon either in cholinergic neurons or in aminergic vesicles in sympathetic nerves (however, see von Euler and Lishajko, 1969). The chromaffin granule, however, has been extensively studied with regard to this issue for almost 20 years, and recent data have led to some plausible models of the uptake process (see Njus and Radda, 1978, for historical review and models). The uptake process is energized by a M g 2 + activated ATPase (Kirshner, 1962; Banks, 1965) which is located in the vesicle membrane. That the uptake process is primarily a membrane function was indicated by the fact that it can occur in chromaffin granule ghosts (Taugner, 1971; Phillips, 1974; Njus and Radda, 1979; Johnson et a / . , 1979). The first clue that membrane ATPase was involved in an indirect way, as a proton pump, came from observations that catecholamine uptake could be inhibited by uncouplers of oxidative phosphorylation (Bashford et al., 1975, 1976; Casey et a / . , 1976), while thc ATPase activity was stimulated. Since uncouplers are believed to conduct protons across membranes, this suggested that the ATPase was a protonpump, and that their inhibition of the eatecholamine uptake was due to the abolition of the pH gradient across the vesicle membrane (see Section It). Consistent with this interpretation was the observation by Casey et al. (1977) that ATP hydrolysis in the presence of Mg2+ causes the internal pH of the already acidic vesiclc to decrease by 0.3-0.5 units. Thus, the pH gradient across the vesicle membrane was believed to be established by an electrogenic proton pump driven by ATPase in the vesicle membrane. In addition, a catecholamine carrier with saturation kinetics appears to be involved. Catecholamine uptake is inhibited by reserpine, whereas ATP uptake is not, but ATP uptake is specifically inhibited by atractyloside (a blocker of the nucleotide carrier in mitochondria). Given this differential inhibition, separate carriers for ATP and catecholamines are proposed (see Njus and Radda, 1978).
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Recent discussions and experiments (Holz, 1979p Njus and Radda, 1979; Johnson et al., 1979; Johnson and Scarpa, 1979) have focused on the question of whether the proton-catecholamine exchange is driven by the pH gradient, or the electrical potential across the membrane, or both. The current view is that the maximum rate of uptake and accumulation of catecholamines in either intact vesicles or ghosts require both the pH gradient and membrane potential, however, either the pH gradient or the electrogenic membrane potential alone is sufficient for ATP stimulated uptake. In the absence of ATP the membrane potential, as measured by [3HH]tetramethylphenylphosphonium(TPMP+), is around - 70 mV, and the addition of ATP Mg2+ shifts the potential to +50 mV (Holz, 1979), a change of about 120 mV. The proton carrier, carboxylcyanide p-trifluoromethoxyphenyl hydrazone (FCCP), inhibits Mg2 , ATP-induced catecholamine uptake and shifts the membrane potential from +50 to -70 to 90 mV (Holz, 1979; Johnson and Scarpa, 1979). In the absence of ATP, catecholamine uptake may be due to the pH gradient only, whereas in the presence of ATP it is coupled to the entire electrochemical gradient (Holz, 1979). Figure 23 illustrates a model of catecholamine uptake in the chromaffin vesicle. The current data indicate that the amines penetrate and are translocated across the membrane in an uncharged form associated with a carrier (the carrier-amine complex having a negative charge) and are exchanged for protons at the inner membrane surface of the vesicle. A preliminary report of solubilization and reconstitution of the putative catecholamine carrier has recently been published (Maron er al., 1979).
+
+
ATP
Membrane Potential in ATP,Mg2+ Fiti. 2 3 . Cheniiosniotic mechanism of catecholamine uptakc in a chromaffin vesiclc. [Adaptcd from Winkler (19771, Holz (1979) and Johnson and Scarpa (1979).1
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Whether the mechanism described above for the uptake of catecholamines is relevant for the uptake of acetylcholine in synaptic vesicles remains to be determined. It is notable in this regard that a paper citing evidence that an ATPase and a proton-motive force function in the transport of acetylcholine into storage vesicles has recently been published (Toll and Howard, 1980) (see also Section VI,B regarding acetylcholine loading of vesicles). Similar ['Hlmethylamine and [ 14C]SCN distribution experiments have been reported for bovine neurosecretory vesicles (NGVs) (Russell and Holz, 198I). In this case, evidence has been presented to show that the internal pH of the vesicle is 5.8 when the medium pH is 7, and fluctuates little with changes in the external pH. Fluorescence measurements using 9-aminoacridine gave similar results. Recent data on rat NGVs (Scherman and Nordmann, 1982) suggested that these vesicles were less acidic (i.e., around pH 6.6 at an external pH of 7.0). However, the rat vesicle preparation appeared to be significantly more leaky (to sucrose, sorbitol, and cations), and was dominated by Donnan distribution mechanisms. In contrast, the bovine NGV preparation data (Russell and Holz, 1981) are consistent with the chromaffin granule data cited above, i.e., the acidic vesicle interior appears to be due to a Mg*+-ATPase on the vesicle membrane which behaves as an electrogenic proton pump. Biochemical evidence for such an ATPase on the NGV membrane has recently been obtained (Russell, 1984). The proton-pump mechanism in NGV membranes cannot be for the purpose of providing a chemiosmotic mechanism for the uptake of transmitters (as for the chromaffin granule). The apparent value of the acidic interior for the NGV is to provide an acidic microenvironment to allow the prohormone-converting enzymcs, which operate only in acidic ranges, to function (see Section 11). This function may also be required in the chromaffin granule since it has recently been discovered that acidic prohormone-converting enzymes are also located in these vesicles (Loh and Chang, I983), presumably to process proenkephalin (see Section VI).
VIII. Morphological Aspects of Formation of Nonpeptidergic Secretory Vesicles In contrast to peptidergic neurons, exemplified by the magnocellular cells of the HNS, in aminergic and cholinergic neurons the precise role of perikaryal organelles in secretion is ill defined in most instances. As it is believed that most of the transmitter synthesis in nonpeptidergic neurons occurs in axon terminals, these regions have been the most thoroughly investigated for production of secretory packages, while far less attention has been accorded to synthetic activity in the perikaryon. Moreover, it is often impossible to directly correlate events in the nonpeptidergic perikaryon with those in its processes due to exten-
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sive branching of the latter, the distant and diffuse location of the terminals, and the complexity of the nervous system as a whole. However, a coherent sequence of events relating to neuronal catecholamine synthesis may be reconstructed by analogy with the secretory process in the neuronally derived chromaffin cells, compactly concentrated in the adrenal medulla. The chromaffin cell, with no axonal process interposed between synthetic machinery and release site, has facilitated experimentation and subsequent interpretation of the roles of the various cellular compartments involved in the production of epinephrine (see Sections VI and VII for biochemical data; see Winkler; 1977; Winkler and Westhead, 1980, for excellent reviews). CHROMAFFIN CELL A. THEADRENOMEDULLARY In keeping with the Palade (1975) concept of secretion, it is generally agreed that in chromaffin cells the rough endoplasmic reticulum (Abrahams and Holtzman, 1973) and the Golgi complex (Beneczky and Smith, 1972; Holtzman et al., 1973) participate in the initial formation of secretory granules. According to autoradiographic data (Winkler and Smith, 1975) and ultrastructural observations (Beneczky and Smith, 1972) a working hypothesis has been formulated, suggesting that the nascent secretory granules receive their content not only from the Golgi cisterns from which they arise, but also via a shuttle system of coated vesicles (? 75 nm) coming directly from the RER and by-passing the Golgi complex. An earlier study of adrenal medullary cells by Holtzman and Dominitz (1968) described apparently functional RER associations with the Golgi apparatus at both cis and trans aspects, thus also implying dual input into the forming secretory vesicles. In neurosecretory perikarya of the HNS, coated vesicles originating from trans Golgi cisterns or from GERL may also be associated with NGVs (Section III,C), but although morphologically similar to those in chromaffin cells, these coated vesicles may be functionally different and do not indicate Golgi by-pass. Another basic difference is that peptidergic NGVs budding from Golgi cisterns are believed to contain their full aliquot of active substances (prohormones and cleavage enzymes), while the newly formed secretory vesicles in adrenomedullary cells are termed presecretory granules, in keeping with the belief that they contain little or no adrenaline. Even the moderate electron density of these relatively large (100-300 nm) prosecretory granules in glutaraldehyde-fixed tissue is not attributed to cathecholamine content but to the proteinaceous core (chromogranins, DBH) (see Sections VI and VI1,B.) The intense electron density of the larger (k 340 nm), more mature secretory granules indicates the presence of catecholamines accumulated from the cytosol. Autoradiographic studies (Coupland and Kobayashi, 197u) show that newly synthesized macromolecules pass through the Golgi apparatus and are present in the prosecretory granules within 30 minutes after their synthesis, but these gran-
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ules tend to remain in the Golgi region for several hours before they are translocated toward release sites at the plasmalemma. This slowing-down mechanism seems to ensure adequate filling of secretory granules with active substances and prevention of premature discharge of their contents. In aminergic neurons axonal transport is the “slowing-down” niechanisni that allows for maturation of vesicular contents prior to release. Evidence against concomitant synthesis of the membrane of the chromaffin granule and its exportable content is cited in detail by Winkler (1977), who concludes that “membrane synthesis might occur only in cycles at certain periods, whereas secretory proteins are synthesized continuously. This contention is also directly related to the phenomenon of membrane retrieval following release of granule contents, for if membranes are reused their rate of synthesis should be lower than that of the secretory product which is continuously lost, as epinephrine is secreted into the blood circulation. (This argument may not be strictly analogous with the situation in amine-secreting neurons where both the membrane and some of the vesicular content may be rccycled.) On the basis of experimental data from various sources it is suggested that under basal conditions chromaffin granule membrane is reused about 5 secretory cycles, while about 20% of the recycled membrane is destined for incorporation into lysosomes (Winkler, 1977). lt has been suggested that following exocytosis, membrane may be retrieved in the chromaffin cell by coated vesicles (* 100 nm) that bud from the exocytotic pit itself, as shown by both transmission electron microscopy (Benedeczky and Smith, 1972; Nagasawa and Douglas, 1972) and by freeze-etching (Smith et crf., 1972), these vesicles being smaller than the original granule membrane. Nagasawa and Douglas ( 1972) described this phenomenon as ’‘micropinocytosis superimposed on exocytosis,” and confirmed the inward movement of the coated vesicles by uptake of thorium dioxide particles from the extracellular space. These authors seek to generalize this mode of membrane retrieval, extending it also to the neurohypophysis, but experimental data from several sources favor membrane retrieval by larger vacuoles and cisterns in neurohypophysial terminals (see Section V). Judging by uptake of exogenous peroxidase, other forms of membrane retrieval have been recognized in chromaffin cells too, for Holtzman and Dominitz ( 1968) reported electron-dense reaction product in both tubules and cup-shaped structures as well as in vesicles. Winkler ( 1 977) hypothesizes that the retrieved coated vesicles are reused as secretory granule membrane in the Golgi region, where their larger size (t 100 nm) distinguishes them morphologically from other categories of coated vesicles, for instance those that shuttle back and forth from the KEK (? 75 nm). This hypothetical scheme is based on the assumption that the membrane of the retrieval vesicles is similar in composition to that of the chromaffin granules, but this has not yet been proved, despite improved isolation procedures (Pearse, ”
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1976). In the neurohypophysis it has recently been shown that the membrane of two populations of clathrin-coated microvesicles is basically very different from that of the secretory vesicles (Torp-Pedersen et al. 1980), but perhaps drawing too close an analogy between peptidergic neurons and chromaffin cells is not appropriate. It has long been known that elevated adrenomedullary secretion leads to formation of chromaffin granules of reduced size (? 95 nm), a phenomenon that has been confirmed by combined morphometric and biochemical studies (Gagnon et al., 1977). It is a source of speculation whether this is a means of efficiently using up available membrane (the membrane to core ratio is increased), or whether synthesis of core materials is reduced and less available for packaging. Heterogeneity of secretory vesicle size and formation of smaller vesicles for refilling are common phenomena in adrenergic axons, and will be discussed below (see Sections VIII,D and E). In cholingergic synapses, stimulation leads to the appearance of a population of vesicles about 25% smaller in diameter than normal (Zimmermann and Denston, 1977b; see also Sections VII,A and B). In peptidergic neurosecretory neurons newly formed NGVs originating from Golgi cisterns are invariably smaller than mature NGVs, but are not associated with recycling phenomena.. However, in chromaffin granules of Golgi origin, at least part of the membrane involved in their formation is believed to be recycled, as described above. Ultrastructural studies of chromaffin cells, both thin sections and freeze-etch replicas, have provided evidence of secretion by exocytosis (Beneczky and Smith, 1972; Smith et al., 1973; Aunis et al., 1979), confirming biochemical data indicating that the contents of chromaffin granules are secreted without concomitant release of cytoplasmic constituents (Douglas, 1968; Smith and Winkler, 1972). A preexocytotic stage has been described by Aunis et al. (1979), characterized by a distinct area of electron desnity, about 25 nm in width, between secretory granule and plasmalemma, while freeze fracture images indicate connections via particles spanning the space between the two membranes. The authors speculate that the bridging material could represent a lectin-like protein on the outside of the granule membrane attached to a specific site on the inner aspect of the plasmalemma, or possibly an actin and myosin connection. An immunocytochemical study (Aunis et al., 1980) has identified myosin immunoreactivity around exocytotic profiles. Paradoxically, freeze-fracture images of actual fusion stages between granule membrane and plasmalemma revealed a dearth of intramembranous particles, as if these had migrated away from the fusion site (Aunis et u I . , 1979). Similar observations have been made in relation to exocytosis in the neurohypophysis (Theodosis el a / . , 1978). This complies with one of the current models of membrane fusion, postulating an increase in fluidity of lipid constituents, accompanied by lateral displacement of intramembranous particles away from prospective fusion zones (Ahkong et a / .,
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1975; Chi et al., 1976; Friend et al., 1977; Lawson et al., 1977; Orci et al., 1977). A second model of membrane fusion stipulats characteristic configurations of intramembranous particles as essential to recognition of fusion sites (Plattner et al., 1973; Satir et al., 1973; Venzin et al., 1977; Tokunaga et al., 19791, and has been meticulously demonstrated and documented at the cholinergic neuromuscular junction by Heuser et al. (1974, 1979) and Heuser and Reese (1981). It is difficult to decide at present whether both these freeze fracture models for membrane fusion at secretory sites are equally valid, whether they are related to identical or different stages in the fusion process, and to what extent the phenomena described are conditioned by different methods of tissue preparation (for further discussion, see Section VIII,F).
B. VISUALIZATION OF BIOGENIC AMINESIN NEURONS Several histochemical procedures for visualization of biogenic amines have contributed greatly to the understanding of amine transmitter biosynthesis, showing that these active substances in neurons of the central and peripheral nervous systems are most concentrated in axonal varicosities and terminals, are present also in dendrites and to a lesser extent in neuronal perikarya. This distribution reflects the biosynthetic pathway of amine-containing organelles, which derive mainly membrane, protein, and enzymes from the perikaryal synthetic apparatus, while active substances are accumulated and synthesized along neuronal processes (see Section VI1,B). Much of the basic information about intraneuronal amine distribution at the light microscopic level is due to the classic method of Falk and Hillarp that converts biogenic amines to fluorescent derivatives via reaction with aldehydes 1962). Several excellent reviews have dealt with the methodology (Falk et d., (Fuxe et al., 1970; Bjorklund et al, 1972; Heym, 1981), which is generally based on induction of fluorescence by gas phase or aqueous formaldehyde (Hoyte et al., 1979). Recently the use of glyoxylic acid as a simple and rapid fluorescent inducer has been advocated (Furness and Costa, 1975; De la Torre, 1980). Discrimination between different amines is facilitated by use of filters or by quantitative microspectrofluorometry. Various pharmacological procedures may selectively increase or decrease fluorescence of a specific amine (see reviews by Jonsson, 1980, 198I ) . When intraneuronal amine concentrations are low, fluorescence may be enhanced by treatment of the animal or the tissue with exogenous amine, which is then taken up and concentrated by mechanisms typical of aminergic cells (see Section Vl1,C). At the ultrastructural level neurotransmitter amines are stored mainly in small dense-cored vesicles (SDCV, ? 50 nm), also in large dense-cored vesicles (LDCV, ? 100 nm) and in a neuronal tubular reticulum (aSER) (Trdnzer et al., 1969; Tranzer and Richards, 1976). Routine primary fixation with glutaralde-
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hyde and postfixation with osmium tetroxide confer electron density to the aminergic core, probably by reduction of the metallic salt in the fixative by the monoamines. Fixation with osmium tetroxide alone does not induce osmiophilia of the core, a prior condition being cross-linking of the amine by glutaraldehyde (see Richards, 1981, for details). Electron density of SDCVs, derived by fractionation from peripheral nerves and treated with glutaraldehyde and osmium tetroxide, has been meaningfully correlated with biochemically estimated noradrenaline content (Fried et af., 1981). Loss of dense cores and reduction in core size are associated with reduction of adrenaline content in stimulated nerves (Basbaum and Heuser, 1979). As in the case of enhanced fluorescence, the amine accumulating properties of dense-cored vesicles may be exploited cytopharmacologically to increase their electron density by administration of exogenous amines, both natural and false transmitters (Richards and Tranzer, 1970; Thoenen and Tranzer, 1971). The affinity of biogenic amines for chromium salts (the chromaffin reaction of Wood and Barnett, 1964) has been widely exploited for improving their ultrastructural identification (Tranzer and Richards, 1976), although this cytochemical method does not differentiate between noradrenaline, dopamine, and serotonin. Oxidation with potassium permanganate is also a useful technique for ultrastructural demonstration of amines (Hokfelt, 1971), and this as well as several other methods have been critically assessed by Bloom (1 970). Recently a uranaffin reaction was introduced for identification of amine-storing organelles (Richards and Da Prada, !977, 1980; Richards, 1981). Uranyl ions have an extremely high affinity for adenine nucleotides (Richards and Da Prada, 1977), thus the uranaffin reaction exploits the high concentration of ATP in aminergic dense-cored vesicles (see Section VI1,B). Although both chromaffin and uranaffin reactions have proved extremely useful for the study of biogenic amines in peripheral nerves, their applicability to the central nervous system has unfortunately proved limited (see Richards 1981). It is uncertain whether this reflects biochemical differences or whether it is merely a question of hampered accessibility to the more compact brain tissue. Cytopharmacological methods applicable to the central nervous system often involve the use of false transmitters such as 5-OHDA or 6-OHDA, which are readily accumulated by aminergic dense-cored vesicles, selectively increase their electron density and thus facilitate recognition of amine trasnmitters in the brain (Thoenen and Tranzer, 1971; Jonsson, 1981; Richards 1981). Autoradiography of labeled amines is an additional means for identifying amine transmitters (Aghajanian and Bloom, 1969; Descarries and Droz, 1970; see Chan-Palay, 1977). However, unlike the case of the HNS peptidergic neurons, where autoradiography provided valuable information about the time course of NGV biogenesis and axonal transport (see Sections IV and V), in aminergic neurons label is incorporated simultaneously into both neuronal cell bodies and processes, due to the amine-uptake
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properties of the secretory vesicles. In the central nervous system ['HIDOPA incorporation into the neuropil is simultaneous with and invariably greater than that of the cell bodies (Ishii, 1971). Imniunochcmistry is currently a popular technique for localization of biogenic amines.
C. IMMUNOCYTOCIIEMISTRY OF BIOGENIC AMINES fmiiiunocytochemical procedures for identifying biogenic aniines are based mainly on the use of antibodies against specific enzymes that participate in their biosynthesis. The presence of tyrosine hydroxylase (TH), rate-limiting in the synthesis of dopmaine and noradrenaline, and dopamine-B-hydroxylase (DBH) rcsponsiblc for the conversion of dopaminc to noradrcnalinc (SCC Scction VII,B), has been exploited to identify catecholaminergic neurons in the central and peripheral nervous systems by imniunotluorescence (Hokfelt et ul., 1977) and by immunoperoxidase techniques at both light (Pickel et al., I97Sa) and electron microscopic levels (Pickel et ul,, 1975b, 1976; Cimarusti et ul., 1979). These methods are adequate for identifying specific inimunoreactive perikarya, processes, and terminals, but at high electron miscroscopic resolution, immunoreactivity of subcellular structures is difficult to interpret unequivocally, due to diffusion and absorption artifacts that arise during tissue processing. Anti-TH revealed diffuse staining of perikaryal cytoplasm, reaction product adhering also to the endoplasmic reticulum and Golgi membranes, while in axons and dendrites neurotuhulcs wcre intensely immunoreactive. Biochemical analysis of tissue fractions has indeed suggested that TH occurs in both soluble and insoluble forms (see Section VII,B), but it is uncertain whether the diffuse versus the neurotubule-bound immunoperoxidase marking is a valid morphological correlate of these forms. Anti-DBH also has elicited diffuse immunocytochemical staining in perikaryal cytoplasm, and in axons and dendrites on membranes of endoplasmic reticulum (Pickel et ul., 1975b), while a more recent report describes specific immunoperoxidase reaction product on membranes of Golgi cisterns, aSER, and dense-cored vesicles (Cimarusti et al., 1979). Binding of antibodies to organelles engaged in protein synthesis and packaging (RER and Golgi), transport (aSER), and transmitter storage (DCVs) could indeed be of functional significance, but the fact that the reaction product adhered to the cytoplasmic (outer) surface of the organelles in question, probably indicates diffusion of antigen from original intraorganellar sites. With the advent of preembedding immunocytochemistry it is interesting that many neuronal antibodies have been found to elicit intense immunoreactive marking of microtubular structures: anti-TH in noradrenergic and dopaminergic neurons (Pickel et ul., 1975b); anti-tryptophane hydroxylase in serotonergic neurons (Pickel et ul., 1977); anti-substance P in neurons of spinal cord sensory
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ganglia (Chan-Palay and Palay, 1977; Kakudo et al., 1981); anti-LHRH in specific hypothalamic neurons (Kozlowski et al., 1980); anti-vasopressin in magnocellular neurons (Armstrong et al., 1982) (see Fig. 45). To our knowledge there is no presently known functional basis for this ubiquitous binding to microtubular structures, for the antigens involved are in many cases expected to move within vesicular structures. As antigens are liable to diffuse from original locations during protracted immunocytochemical procedures, marking on neurotubules may indicate nonspecific absorption, possibly mediated by glutaraldehyde cross-linking. It is noteworthy that while this possible intraneuronal diffusion and absorption artifact is regrettable for accurate subcellular localization of antigens, it may well provide the basis for the excellent light microscopic Golgi-like images of immunocytochemically treated neurons, in which cell bodies, axons, and even dendrites appear “impregnated” by dense immunoreactive product (for example, Figs. 1 and 44). Using anti-DBH in the unlabeled antibody peroxidase method, Grzanna et al. ( 1 978) demonstrated by “transmitter-specific Golgi images” the impressive three-dimensional organization of noradrenergic neurons throughout the central nervous system. D. LIFECYCLESOF VESICLESI N ADRENERGIC NEURONS Schemes relating to the biogenesis of noradrenaline-sequestering vesicles in neurons are based on a combination of data from fractionation-isolation procedures accompanied by assay of active substances, ligation experiments, and ultrastructural observations (see Geffen and Jarrot, 1977). Probably because correlation between these various experimental approaches is often by extrapolation, almost all that these schemes have in common is the recognition of LDCVs and SDCVs as amine-containing compartments, but the genesis and transformation of the vesicles have been interpreted in a variety of different ways. Many studies relating to the genesis of noradrenergic vesicles have been confined to synaptic varicosities only, and this limitation should be borne in mind when evaluating the various schemes. Formation of LDCVs (80-250 nm) from Golgi cisterns has been described in identified catecholaminergic neurons (Hokfelt, 197 1 ; Ishii, 197 1 ; Machado, 1971; Teichberg and Holtzman, 1973; Richards and Tranzer, 1975; see also Geffen and Jarrot, 1977). It is uncertain whether their origin is Golgi proper or GERL, for all cisterns involved may be AcPase-positive (Quatacker, 198 I ) , making definition of GERL difficult by the usual cytochemical criteria. These LDCVs may be analogous with the prosecretory granules of adrenomedullary chromaffin cells, rich in macromolecular content but poor in catecholamines as well as in catecholamine uptake mechanisms. Thus the electron density of perikaryal LDCVs does not increase appreciably after administration of exogenous catecholamines, nor does it diminish under conditions that deplete transmitter
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content, such as tyramine administration (Klein and Thuresen-Klein, 1974). As axonal LDCVs are imbued with DBH activity and contain noradrenaline, a maturation process is indicated during transport from the perikaryon. The principle of maturation during transport is analogous with that of peptidergic NGVs (see Sections 11,Dand V,C), but while the mechanism in the latter is intravesicular posttranslational cleavage, LDCVs accumulate active substances by uptake and synthesis (Sections VI and VII). Dense-cored vesicles may arise not only from Golgi cisterns but also from aSER (Fillenz, 197 1; Machado, 197 1; Teichberg and Holtzman, 1973; Droz, 1975; Tranzer and Richards, 1976; Quatacker, 1981). Holtzman et al. (1977) interpret this phenomenon as “geometric variation on a common theme,” quoting evidence from immature rat neurons (Steltzer, 1971) and from invertebrate neurons (Lane and Swales, 1976) that elements of the aSEK are directly related to the Golgi apparatus, so that interchange of roles between these two organelles is feasible, depending on the developmental stage and on the physiological state of the animal. According to Richards and Trdnzer (1975), who provided cytochemical evidence that not only LDCVs and SDCVs but also tubular reticulum (aSER) are all amine-containing compartments, LDCVs arise from
A
FIG. 24. Schematic presentation of secretory granule genesis in aminergic neurons. (A) Large dense-cored vesicles (LDCV) may be produced from Golgi (GO) cisterns, and small dense-cored vesicles (SDCV) from aSER in the axon. (B)An alternative possibility is that both LDCVs and SDCVs may be produced from Colgi related tubular formations (aSER) in the perikaryon and also in axonal dilations. N, nucleus (see text for funher discussion; Section VII1,D).
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Golgi cisterns in the perikaryon, while SDCVs arise from tubular reticulum along the axon (Fig. 24A). De Potter and Chubb (1977), from fractionation data on splenic nerve, concluded that there are two categories of SDCVs: those prevalent in resting neurons, arising from tubular reticulum, devoid of DBH, and containing only noradrenaline (Fig. 25A), and others formed during stimulation from recycled membrane subsequent to LDCV exocytosis, containing DBH and capable of accumulating noradrenaline (Fig. 25B) (see also Sections VI and VII). The prominence of the sAER (tubular reticulum) in aminergic secretion has recently been the subject of a detailed cytochemical investigation by Quatacker (1981) (see also Quatacker and De Potter, 1978). In adrenergic neurons of the superior cervical ganglion of the rat the sAER appears to be a direct extension of the perikaryal Gogli system into the axon. This observation was supported by results from a variety of cytochemical procedures: the chromaffin reaction revealed tubular formations with associated LDCVs and SDCVs throughout the neuron, particularly near the inner aspect of the Golgi apparatus and along axons; phosphotungstic acid staining at low pH demonstrated carbohydrate macromolecular content, presumably indicating direct functional relationship with the Golgi apparatus, the chief carbohydrate elaborating system in the perikaryon. The carbohydrate nature of the content was confirmed by the periodic acidthiocarbohydrazide silver proteinate reaction, while the less specific zinc iodide osmium tetroxide staining procedure served to emphasize the morphology of the elements involved, demonstrating unambiguous continuity between tubules of different caliber and between tubules and dense-cored vesicles. As these distinctive tubular formations near the trans aspects of the Golgi apparatus lack acid hydrolase activity, they are probably not analogous with GERL in other secretory cells. Quatacker (1981) suggests that the clusters of tubules and vesicles are cathecolamine-elaborating systems directly related to the Golgi apparatus, and that they are exported into the axon terminals by fast axonal transport (Droz, 1975, 1979; Markov et al., 1976) where they are engaged in local production of cathecholamines. It is not certain whether distant clusters maintain connections with the Golgi elements or whether they become autonomous, but it is clear that they are associated with the Golgi system and do not have any demonstrable connection with the perikaryal RER. Quatacker (1 98 1) indicates that both LDCVs and SDCVs may be produced at the same site from aSER clusters (see Fig. 24B), yet states that their composition need not necessarily be identical. It is also possible that LDCVs actually bud from Golgi proper in the perikaryon, and only later become entangled in webs of sAER. According to other schemes LDCVs that have arisen from GERL in the perikaryon, synthesize and store adrenaline as they move into axon terminals by fast axonal transport. However, there is less unanimity about the manner in which LDCVs transform into SDCVs, and how exocytosis of vesicle contents is ef-
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6
A
A
C
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; ... .....@
.. ...I.... .: .. _.;:.
I.:
sv
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FIG. 25. Various schemes related to sccretory events in aminergic (A-E) and serotoncrgic (F) terminals. ( A ) According to de Potter and Chubb (1977), in rcsting neurons most of the catecholamine-containing SDCVs (filled circles) arc derived from aSER and are poor in DBH content; Colgi derived LDCVs contain both catecholamine and DBH. (B) In stimulated neurons most of the SDCVs (crosses) are derived from recycled LI)CVs, and therefore contain both catecholamine and DBH. (All SDCVs arc morphologically identical, but biochemical proccdures differentiate between the two types.) (Adapted from de Potter and Chubb, 1977.) (C) Both LDCVs and SDCVs may participate in exocytosis of transmitter. SDCVs may arise from recycled LDCV membrane. (D)
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fected (see Fig. 25C-E, adapted from Geffen and Jarrott, 1977). If exocytosis is a function of LDCVs it is uncertain whether it is a step-wise, repetitive process leading to gradual emptying of the large vesicle (Fig. 25C), or whether it is a single event (Fig. 25D). In both cases small vesicles are expected to arise from LDCV membrane, either by budding inward from exocytotic pits (a5 has been proposed in adrenomedullary cells, see Section VIII,A), or by retrieval from the axolemma subsequent to exocytosis of LDCV. Another variation visualizes SDCVs budding directly from LDCVs, or via tubular formations, with subsequent exocytosis being a function of SDCVs only (Fig. 25E). In ligated sciatic nerve, cathecholaminergic terminals contain large elongated dense-cored vesicles in addition to regular LDCVs, SDCVs, and tubular reticulum (Lascar, 1980). It is postulated that the elongated vesicles, which may also be dumbbell shaped, represent an intermediate stage of LDCVs locally transforming into SDCVs. However, despite schemes and speculations it is still far from certain that SDCVs do indeed arise from LDCVs, either directly or indirectly. According to a meticulous study by Basbaum and Heuser (1979) on adrenergic axon varicosities in mouse vas deferens, electrical stimulation reduces the number of SDCVs to less than half, yet the numbers of LDCVs remain stable. Moreover, core depletion was evident only in the small vesicle population. Experiments using HRP as an extracellular tracer indicated that only the SDCVs were engaged in exocytosis. The question arises whether LDCVs are implicated at all in secretion cycles involving aminergic SDCVs, or whether they may perhaps contain neuropeptides (see Hokfelt et a!. , 1980) and thus be intended for other functions governed by different parameters.
E. VESICLELIFE CYCLEIN
AN
IDENTIFIED SEROTONERCIC NEURON
Serotonergic neurons have common features in vertebrate and invertebrate nervous systems, and are basically analogous with the adrenergic prototype. A series of elegant studies by Schwartz and his associates has demonstrated that the giant cerebral ganglion (GCN) of the mollusc, Aplysia, may serve as a model for Exocytosis of LDCVs may be a repeated and step-wise process. SDCVs may originate from recycled LDCV membrane, and both small and large vesicles may participate in exocytosis. (E) It is possible that only SDCVs participate in exocytosis. However, SDCVs may be of dual origin: by fragmentation of LDCVs and by budding from the a SER. (C-E adapted from Geffen and Jarrott, 1977.) (F) Axon varicosity of an identified serotonergic neuron (GCN) in Aplysia (see Section VI11,E). Large Golgi-derived compound vesicles (CV, “vesicle within a vesicle”) accumulate transmitter and become dense-cored vesicles (DCV); these exocytose once in a lifetime, the outer membrane is retrieved as a large, electron-lucent vesicle (LV) while the inner membrane is recycled as small vesicles capable of accumulating serotonin and exocytosing repeatedly. (Adapted from Shkolnik and Schwartz, 1980.)
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genesis and maturation of serotonergic vesicles (Goldman et al., 1976; Ambron et al.. 1980; Shkolnick and Schwartz, 1980). Due to large size and accessibility of GCN, radiolabeled serotonin or glycoprotein may be injected directly into the cell body, The label is then conveyed by fast axonal transport along axon ramifications and into varicosities, facilitating identification of peripheral GCN profiles by electron microsccpic autorddiography, and making it possible to affiliate features in the cell body with those in distant terminals. Thus GCN has provided the rare opportunity of studying in a single neuron a coherent sequence of secretory vesicle formation, a condition that is rarely if ever met in morphological studies of serotonergic neurons in the vertebrate nervous system (Chan-Palay, 1977; Pickel et al., 1977). The model proposed by Shkolnick and Schwartz (1980) indicates that in GCN vesicle genesis commences in the perikaryon by budding from clusters of smooth endoplasmic reticulum adjacent to the Golgi apparatus, possibly analogous to GERL. With routine gIutaraldehyde/OsO, fixation, the perikaryal vesicle (outer diameter, -+ 75 nm) is recognizably compound, containing a smaller electronlucent vesicle, one within the other (see Fig. 2SF, adapted from Shkolnick and Schwartz, 1980). Cytochemical treatment with chromium salts or KMnO,, intended to reveal amine content, darkens the small inner vesicle, rendering its closely applied limiting membrane virtually indiscernible, and producing an apparent “LDCV” with an outer diameter of k7.5 nm. However, this compound vesicle, found in the cell body and along axons, differs from the LDCVs ( 2 95 nm) in terminal varicosities that display core density even with routine glutaraldehyde/OsO, treatment, due to their greater amine content. The electron-lucent compound vesicle is considered the precursor of the larger LDCV that matures within the terminal and stores the amine transmitter, this increase in size being attributed to the greater load of serotonin causing osmotic swelling. In addition to compound vesicles and LDCVs there are also small (+ 53 nm) and large (+ 86 nm) electron-lucent vesicles in the axon varicosities of GCN. The four types of vesicles are distributed heterogeneously, albeit not randomly, among the numerous varicose profiles. According to quantitative analysis of the regional vesicle distribution, Shkolnick and Schwartz (1980) have suggested that each LDCV, once in a life time, releases its amine-core content by exocytosis into the extracellular space (see Fig. 2SF). While the large emptied outer vesicle is retracted, only the small inner vesicle or core participates in actual exocytosis, becoming transiently incorporated into the axolemma. The source of the small vesicle population in GCN may be the recycled inner vesicles of LDCVs, but also possibly derivation by fragmentation of large electron-lucent vesicles or by budding of local aSER. Shkolnick and Schwartz (1980) favour the notion that ‘‘the small electron-lucent vesicles mediate the routine and repeated release of serotonin at active zones,” which is in keeping with the contention of Bashbaum and Heuser (1979) (see Section VII1,D) that mainly small vesicles participate in
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exocytosis. As in other secretory schemes, lysosomes apparently play a significant secretion-related role in GCN. Specific association of neurotransmitter with perikaryal lysosomes indicates that redundant secretory vesicles, including their serotonin content, are engulfed and degraded, probably for recycling purposes (Schwartz et al., 1979). The most interesting speculation of Shkolnick and Schwartz ( 1980) concerns the evolutionary significance of the fact that “two distinct forms of vesicular release . . . both occur in the same nerve terminal.” LDCVs appear to release their serotonin content in a manner reminiscent of glandular secretion (for instance, in neurohypophysial axon endings), without any distinct synaptic specialization. However, small vesicles in GCN mediate transmitter release repeatedly at sites reminiscent of the active zones in cholinergic synapses. A possible evolutionary sequence is indicated in this spectrum of neuronal release possibilities. It is interesting that the neurohypophysial hormones of the HNS may be secreted in a grandular fashion by large NGVs at neurohemal sites, and also in an apparently synaptic manner by small vesicles in the CNS (Buijs, 1978).
F. VESICLESIN CHOLINERGIC NEURONS Within the spectrum of neuronal secretion mechanisms, the cholinergic synaptic terminal is the most highly specialized, both morphologically and electrophysiologically (see reviews by Gray, 1976; Heuser and Reese, 1977; Jones, 1978; Tucek, 1978). The cholinergic synapse has rivetted the attention of neurobiologists for the past three decades, with the emphasis on short-term events such as neural transmission, local transmitter synthesis, and recycling phenomena (see Sections VI,B and VILA). With the advent of the quantum theory of acetylcholine release (Fatt and Katz, 1952; Katz, 1969), supported by the electron microscopic discovery of abundant small, translucent vesicles at synaptic sites (Palay, 1956, 1958), the synaptic vesicle became a prime feature in synaptic events. Although it is assumed that vesicle constituents, and possibly even the vesicles themselves, originate at distant sites in the perikaryon and move by axonal transport toward the terminal, few attempts have been made to render a holistic account of vesicle life history in cholinergic neurons (Holtzmann, 1977; Holtzmann et al., 1977). There has been a tendency to generalize and extrapolate from aminergic and even peptidergic systems, despite biological disparity. Due to lack of an unambiguous morphological marker for cholinergic vesicles, it has not been possible to identify them with certainty outside the synaptic region. Thus, although there is strong circumstantial and experimental evidence for association between transmitter and electron-lucent microvesicles in terminals, it is difficult to make similar assertions about structurally identical vesicles in other regions of the cholinergic neuron. Moreover, there is as yet no definite claim regarding exactly which intraneuronal membrane system gives rise
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RER
A
B
FIG.26. Diagrammatic reprcsentations of thc rolc of the aSER in axonal trampor( and vesicle genesis, according to Droz and his associates (see Section VIII,F for details). (A) The aSER is visualized as originating from the Golgi apparatus (adapted from Droz ri u l . , 1975). (B) The aSER is depicted as an extention of the perikaryal endoplasmic reticulum into the axon (adapted from Ramhourg and Droz, 1980). N, Nucleus; RER, rough cndoplasniic reticulum; GO, Golgi system; GE. GERL; aSER. axonal smooth cndoplasniic reticulum; SV, synaptic vesicles; LY, lysosome.
to virgin cholinergic vesicles and continues to replenish the synaptic,population. Synaptic exocytotic-endocytotic cycles support local economy and reuse of vesicular constituents, but a continuous supply of new elements is required to
replace degraded vesicles. Although a great deal is known about local synaptic events, far less is known about cholinergic vesicle genesis, and it is in this context that we propose a scrutiny of recent work on axonal rnembrdne systems. Thin sections of conventionally fixed tissue viewed in the transmission electron microscope at moderate accelerating voltage reveal a variety of discrete, intraaxonal membranous profiles. Apart from neurotubules and neurofilaments there are blunt-ended tubules, polymorphic cisterns, vacuoles, dense bodies, occasional microvesicles, and mitochondria (Peters rt ul., 1976). However, even the arduous process of serial thin-sectioning often fails to establish relationships between the separate membranoub profiles within axons. Current interest in accurately defining intraaxonal organclles stcms from thc knowledge that fast axonal transport of essential macromolecules, both anterograde and retrograde, is associated mainly with particulate fractions (Grafstein, 1967; Grafstein and Forman, 1980; see also Section 1V ,A). Histochemistry and autoradiography have made important contributions to elucidating the functional morphology of axonal
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transport, and establishing both route and rate of conveyance of neuronal constituents from the perikarya, along axons and into terminals (Droz, 197.5, 1979; Droz etal., 1975; Markov et ul., 1976; Tsukita and Ishikawa, 1976, 1979, 1980; Rambourg and Droz, 1980). Droz, concerned with visualizing in a coherent fashion anatomical compartments involved in axonal transport, introduced heavy-metal impregnation of thick sections (0.2-2.0 k m ) viewed at high electron microscopic voltage, often in combination with autoradiography (Droz, 1975; Droz et ul., 1975). Impregnation methods used for this purpose include treatment of tissue with uranyl acetate followed by copper and lead citrate (Thiery and Rambourg, 1976), ferrocyanide-reduced osmium tetroxide (Karnovsky, 197 I ) , and also a diaminobenzidine, osmium-ferrocyanide sequence (Tsukita and Ishikawa, 1976). Although such procedures d o not “stain” specifically, their selectivity for particular membranes may be enhanced by manipulating the pH of the fixative. At neutral pH neutrotubules and neurofilaments are invisible, while intraneuronal membrane systems such as the axonal smooth endoplasmic reticulum (aSER) may be discerned as highly electron-dense three-dimensional structures. The aSER, the most conspicuous intraaxonal membrane system was revealed as a continuous network of anastomosing longitudinal tubules, extending from perikarya, along axons and into terminals (see Fig. 26 adapted from Droz, 1975; Droz et ul., 197.5; Rambourg and Droz, 1980). The use of a variety of radiolabeled precursor molecules in combination with quantitative autoradiography indicated that the aSER was the most heavily labeled intraaxonal organelle during the first 3-6 hours after a pulse. Only later did radioactivity appear in axolemma, mitochondria, and putative synaptic vesicles. Lacking the vectorial nature of the axon, the time course of the appearance of radioactivity within perikaryal organelles may be more difficult to interpret, beyond the certainty of initial labeling of the RER. According to a stereoscopic study of impregnated tissue, the aSER may be continuous with the perikaryal SER, which in turn arises from the RER (Beaudet and Rambourg, 1979) where most neuronal proteins are synthesized (Fig. 26B). This may imply Golgi bypass, for the nature of the anatomical link between the axonal reticulum and the Golgi apparatus is currently in a state of uncertainty, leaving unaccounted for the route by which glycosylated proteins reach the aSER from Golgi (Rambourg and Droz, 1980). Possibly the small electron-lucent vesicles reported by some authors to bud off Golgi-GERL (see Akert et ul., 1971) may not after all be virgin cholinergic vesicles destined to reach synaptic terminals by fast axonal transport, but rather transition elements conveying glycosylated proteins to the aSER. Recently Hammerschlag et ul. ( 1982) provided evidence from monensin-treated dorsal root ganglia, in which the Golgi system was grossly distorted, that all fast transported proteins (both secretory and membrane) pass through the Golgi apparatus. The earlier concept of Droz (1975) clearly accorded the Golgi system a central
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role in the genesis of the aSER, so that theoretically the aSER could even be regarded as an extension of the Golgi-GERL system into the axon (see Fig. 25A and also Section VII1,D). Such a concept could reconcile the possible dual origin of synaptic vesicles, from Golgi-GERL and from aSER, representing “geometric variation on a common theme” (Holtzmann et ul., 1977). Holtzmann suggests that “the comparative importance of the reticulum and the Golgi apparatus could vary in different neuron typcs or during different periods of neuronal growth and maintenance.” The improved visualization of the aSER by heavy-metal impregnation is based not only on the three-dimensional aspect afforded by thick sections, but also on the fact that the membrane system is stabilized and preserved intact, in contrast to its fragmented aspect after routine processing. Among the functional modifications of the aSER revealed by impregnation are subaxolemmal meshworks of fine-caliber tubules that probably serve as local sources of membrane constituents for the axolemma. In presynaptic regions the aSER transforms into a tangle of irregular tubules from which synaptic vesicles appear to bud (Rambourg and Droz, 1980). Nevertheless, this circumstantial evidence of vesicles budding from aSER tubules remains speculative, for heavy-metal impregnation, despite its cited advantages, reduces electron microscopic resolution and obscures fine structural subtleties, making it difficult to discern whether or not the aSER tubules merely terminate blindly among the synaptic vesicles. The presence of the aSER in presynaptic regions, requiring special procedures to keep it intact, led Gray (1976, 1977) to propose the possibility that synaptic vesicles seen with routine treatment may in fact represent fixation artifacts derived from the highly labile aSER (Fig. 27), and that a quantal release of transmitter might occur by a peristaltic mechanism directly from the aSER. It has been shown that membranes in glutaraldehyde-fixed neurons may indeed be prone to production of artifacts by blebbing and vesiculation (see Reese and Reese, 198I). However, Gray’s “vesicle artifact” theory, relying mainly on morphological concepts, has not as yet met the challenge of the “vesicle hypothesis,” backed by correlative physiological, biochemical and morphological data (see Sections VI and VII,A). At neuromuscular terminals stimulated to hypersecrete with 4-aminopyrine, images of synaptic vesicle exocytosis have been captured by quick freezing and correlated with quantal acetylcholine release (Heuser et al., 1979). Recycling of retrieved vesicle membrane may occur via cisterns (Heuser and Reese, 1977) that superficially resemble profiles of aSER, but in fact probably arise by invagination of the axolemma following intense exocytosis. Prior to exocytosis, synaptic vesicles interact with the presynaptic axolemma at discrete “active zones” (membrane retrieval probably occurs at other sites). Paramembranous densities are associated with “active zones,” assuming characteristic forms in different synapses (bars, spicules, and other variations; see Jones, 1978), but the identity
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B
FIG. 27. Speculative diagrams showing that (A) synaptic vesicles visualized after conventional electron microscopic fixation may in fact represent artifactual fragmentation of the (B) aSER that exists intact in vivo. (Adapted from Gray, 1976, 1977.)
of this electron-dense material is as yet unknown, as is also that found in synaptic
clefts and at postsynaptic sites. Freeze-etching has revealed arrays of large intramembranous particles within the presynaptic membrane at active zones, and these are now thought to embody voltage-sensitive calcium channels (Pumplin et al., 1981). As calcium channels are probably obligatory at all secreting terminals, it is “disappointing” that in peptidergic neurosecretory endings exocytotic sites are exemplified by a dearth of intramembranous particles (Dreifuss et al., 1976). Extensive immunochemical and immunohistochemical studies on cholinergic neurons have been carried out by Whittaker and his associates (Ulmar and Whittaker, 1974a,b; Jones et al., 1981, 1982; Walker et al., 1982). Antigens were derived from the entirely cholinergic electric organ of the ray, Torpedo marmorata, which has been widely used as a model cholinergic system (see Whittaker and Zimmermann, 1976). Antisera were raised in rabbits and guinea pigs to isolated presynaptic plasma membranes and synaptic vesicles, and after extensive absorption and elution procedures (see Walker et al., 1982) yielded purified antibodies to plasma membrane (to a 33,000 MW polypeptide) and to a vesicle-derived glycosaminoglycan ( “vesiculin”; see Section VI,B). The antisera were used for immunofluorescence histochemistry on cryostat sections of Torpedo electric organ and mammalian tissue (rat diaphragm and hippocampus). Kelly and his associates have raised antisera to synaptic vesicles purified from Narcine electric organ (Wagner and Kelly, 1979; Carlson and Kelly, 1980; Hooper et al., 1980; von Wedel et al., 1981). The above-mentioned antisera, derived from components of cholinergic terminals, are useful for marking terminals in both nonmammalian and mammalian species, and may also prove valuable for tracing the life cycle of synaptic vesicles throughout the cholinergic neuron (Jones et al., 1982). At the light microscopic level, within the electric organ immunofluorescence reveals vesicle-related antigen at discrete perikaryal
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sites, particularly clustered near the axon hillock. Along axons there are intensely immunofluorescent spherical bodies (0.25-0.5 km), considerably larger than synaptic vesicles (+ 0.08 Fm), that may represent “transport vacuoles” or possibly fragmented aSER. Ligation experiments that interrupt anterograde flow show build-up of immunofluorescence proximal to the ligature. Stimulation of the electric organ causes the interesting phenomenon of increased immunofluorescence at the terminals, and this has been interpreted by Jones et ul. ( 1982) as increased accessibility of the glycosaminoglycan antigen during exocytosis of transmitter by the synaptic vesicles. The glycosaminoglycan, presumably associated with the inner aspect of the vesicle membrane, is exposed when the vesicles open into extracellular space via the terminal axolemma. This is in keeping with the report by von Wedel el al. (1981) that upon chemical stimulation of the frog cutaneous pectoris muscle, synaptic vesicle antigens are transferred to the presynaptic membrane. The glycosaminoglycan in question is likely tightly bound to vesicle membrane protein, according to a recent report by Carlson and Kelly (1981). Clathrin-coated vesicles havc divcrsc rolcs in all categories of neurons (for example Sections lll,C, V,D, V,E, and VII1,A). In cholinergic synaptic terminals they appear to be associated with retrieval and recycling of membranes (see Heuser and Reese, 1977). Recently, coated vesicles were shown to have a role in intracellular transport of both acetylcholine receptors, which arc intrinsic axolemmal proteins, as well as acetylcholinesterase, a secretory protein (PorterJordan ef ul., 1981). Immunocytochemistry has been used to study the in situ distribution of a ma.jor 180,000 MW protein of coated vesicles in rodent cere1981). lmmunoperoxidase staining at the electron microbellum (Cheng ef d., scopic lcvcl revealcd inimunoreactive coated vesicles in perikarya and terminals. As immunoreactivity was associated not only with the coat encasing the vesicles but also with the cytoplasmic matrix in restricted regions, it is suggested that there may be at least two forms of coated vesicle protein in neurons, and that the soluble cytoplasmic pool is concentrated where it is most needed, that is, near the Golgi apparatus and within synaptic terminals. The immunocytochemical indication of coated vesicle protein along the inner aspect of the presynaptic membrane is of special interest since the coating may imbue particular regions of‘ the axolemma with discriminatory functions pertaining to endocytosed substances. Coated pits and coated vesicles may be associated with receptor-mediated endocytosis (Coldstein el ul., 1979). Monoclonal antibodies to ncuronal antigens pregent a burgeoning new field of research (for reviews see McKay et ul., 1981; Reichardt and Matthew, 1982). Synaptic densities (comprising pre- and postsynaptic membrane and cleft material) purified from mammalian brain and fish electric organ have been used as immunogens (Matthew el al., 1981). Among the monoclonal antibodies that
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developed in the spleens of the immunized mice, two were found directed against a synaptic vesicle protein on the outside of the vesicle membrane. The 65,000 dalton protein, apparently ubiquitous in synaptic vesicle membrane and in restricted regions of the plasmalemma associated with exocytosis, has been identified immunologically in a wide variety of neuronally derived cells throughout the vertebrate phylum. Monoclonal antibodies directed against this protein are not associated with transmitter specificity, but may nevertheless be useful for studying vesicle genesis and neuronal differentiation. It has been found that both the vesicle and the axolemmal antigens appear simultaneously in developing neurons that invade the rat superior cervical ganglion, attesting to the early presence of putative synaptic vesicles during differentiation (K. Greif, in Reichardt and Matthew, 1982). As the determinant for this monoclonal antibody is on the outside of the synaptic vesicle membrane, it effectively binds to intact vesicles, readily identifying them for immunocytochemical purposes, and precipitating them in pure form for biochemical procedures. Several other monoclonal antibodies raised against the synaptic density fraction are specific to mitochondria, postsynaptic densities, and other synaptic components (Reichardt and Matthew, 1982). The advantages of monoclonal antibodies may be manifold in the production of antisera against known antigens that are difficult to purify, and in the discovery of hitherto unrealized common denominators between particular cells, organelles, and systems. Nevertheless, conventional polyclonal antibodies retain their usefulness and even superiority in some experimental procedures such as the production of specific immune lesions. Immunocytochemistry with monoclonal antibodies may also present technical problems, possibley related to reduced avidity of antibody-antigen binding.
IX. Developmental Aspects of the Hypothalamic-Neurohypophysial System During the past decade studies on the morphology and physiology of the hypothalamic-neurohypophysialsystem (HNS) during pre- and postnatal development, using a wide variety of methods, have provided new insights into this system and contributed to a better understanding of its structure and function. The correlation of data on the cytologic differentiation of peptidergic neurosecretory neurons, and those on the onset of neurophysin and hormone biosynthesis and secretion and their further development during prenatal and early postnatal life, has not only confirmed our concepts and expanded our knowledge of the HNS, but has also raised some new and puzzling problems, waiting to be solved through future research.
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A. MAGNOCELLULAR HYPOTHALAMIC NUCLZI 1. Early Origin and Development With ['Hlthymidine autoradiography, the formation of prospective cells of the SON, PVN, and accessory magnocellular groups in the rat can fairly accurately be placed between 13 and 15 fetal days (fd) (Altrnann and Bayer, 1978a,b; Anderson, 1978), with particularly heavy labeling at 14 fd (Ifft, 1972) and near zero labeling at 16 and 17 fd (Anderson, 1978). From a diamond-shaped region of the third ventricle, cells migrate lateroventrally and the first neurons of the SON gather lateral to the optic tract at 16 fd (Altman and Bayer, 1978a,b). At that time they can be identified with certainty in Holtzman rats (Dellmann et ul., 1981), but in Wistar-Lewis and Sprague-Dawley strains this is possible only at 17 fd (Danilova, 1965; Altrnan and Bayer, 1978a,b; Gash et al., 1980; Sladek et ul., 1980) or even at 18 fd (Smiechowska, 1964; Hyyppa, 1969a). The same is essentially true for the PVN which develops in the vicinity of the periventricular origin of its cells. The further pre- and postnatal development of the SON is characterized by more numerous and increasingly larger neurons (Rodeck and Caesar, 1956; Dawson, 1966; Enestrom, 1967; Hyyppa, 1969a; Gash et al., 1980; Sladek et al., 1980; Dellmann et a l . , 1981); at the end of their growth period between 25 and 30 days postnatal (dpn), the perikarya of the SON reach their definitive size (Rodeck and Caesar, 1956; Khatchaturian and Sladek, 1980; Krisch, 1980a; Dellmann et a l . , 1981). In histochemical investigations of the postnatal development of the rat hypothalamus, Pilgrim (1967, 1968) found that reactions for several enzymes also reach typical adult characteristics only between 3 and 4 or (for TPP) even 6 weeks postnatal. In the mouse, precursor neurons of the SON and PVN appear between 11 and 14 fd (Shirnada and Nakamura, 1973; Gracheva and Danilova, 1978; Karim and Sloper, 1981) and the respective nuclei at 13 and 14 fd (Karim and Sloper, 1980). 2. Ultrastructure Ultrastructural investigations in the rat have revealed uniformly undifferentiated cells that cannot be identified as prospective magnocellular supraoptic neurons at 15 fd (Dellrnann et a l . , 1981) or 16 fd (Gash et a l . , 1980; Sladek et a l . , 1980) (Figs. 28 and 29), but a few clearly recognizable neurosecretory neurons have differentiated 1 day later. These neurons contain all cytoplasmic organelles
FIG. 28. Electron micrographs of fetal rat neural lobe. (a) On fetal day 16.5 undifferentiated pituicytes are seen closely apposed to one another. X7250. (b) Portion of pituicyte on fetal day 18, undergoing differentiation and demonstrating active Golgi configurations. N , Nucleus of pituicyte. Neurosecretory axon profiles are seen in upper area of electron micrograph. x 17,400.
FIG.29. (a) This perikaryon from the 16.5 fd rat supraoptic nucleus possesses all the morphological characteristics of a highly active secretory cell. X 13,500. Inset: A typical NGV in the vicinity of the Golgi coniplex of a neurosecretory cell in the 16.5 fd rat supraoptic nucleus. X67,SOO. (b) In this 21 fd supraoptic neuron, the Golgi complex is more extcnsivc and the NGVs more nunieroub and larger than at younger fetal ages. XS4,OOO.
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necessary for the biosynthesis of their specific secretory products (Fig. 28) and their secretory activity is indicated by the presence of occasional NGVs, up to 100 nm in diameter (Gash rt ul., 1980; Sladek et a[., 1980; Dellmann er ul., 1981). The scarcity of NGVs at this time contrasts with the first light microscopic detection of neurophysin-immunoreactive perikarya at 16 fd (Wolf and Trautmann, 1977; Wolf and Sterba, 1978) and 17 fd (Khatchaturian and Sladek, 1980; Sladek et ul., 1980) and VP- and OT-immunoreactive perikarya at 16 fd (Boer et ul., 1980b; Buijs er a l . , 1980b). The positive immunocytochemical reaction is most likely due to the presence of unpackaged secretory material in the RER and Golgi complex (Broadwell rt ul., 1979; Krisch, 1980a) and perhaps also in the aSER (see Section V,B). Subsequent prenatal ultrastructural changes in the rat SON concern primarily the extent and quantity of organelles (Enestrom, 1967; Gash et a/., 1980; Sladek et al., 1980; Dellmann et ul., 1981). The cisternae of the RER become more abundant and the Golgi complex more extensive, and more and increasingly larger NGVs that by 21 fd have increased to sizes comparable with those in adults are present (Fig. 29). Axon profiles, with NGVs, and also dendrites (Sladek et u l . , 1980) become clearly identifiable. Typical axon hillocks apparently do not occur; interestingly, the axons contain abundant elongated profiles of aSER with frequent dilatations whose electron-dense content resembles that of NGVs (Fig. 30) (Dellmann e t a / . , 1979, 1981). While many of these profiles are likely involved in the fast axonal transport of substances needed for growth of the axon, some may participate in neurohormone transport (see Section V,B).
FIG. 30. Neurosecretory axon “hillock” in the supraoptic nucleus of a 21 fd rat, containing many profiles of aSER and putative NGVs arising from aSER-like profiles. X47,OOO.
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FIG. 31. This perikaryon from the supraoptic nucleus of a 12 hour postnatal rat contains an extensive Golgi complex with numerous NGVs of varying electron density in its vicinity. x21,OOO. FIG. 32. At 6 dpn, this supraoptic perikaryon possesses many profiles of rough endoplasmic reticulum and numerous NGVs in the vicinity of the Golgi complex. Also notice lysosomes.
X
17,500.
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The postnatal maturation of the SON, assessed by morphological criteria, takes place within 3 to 4 weeks after birth. During this time span, ultrastructural, immunohistochemical, and immunocytochemical characteristics as well as the synaptic input (see below) reach adult proportions. Physiological data by Sinding et al. (1980a) corroborate these findings by showing that neurophysin and neurohormones in the HNS reach adult levels at 21 to 30 days postnatal (dpn). During postnatal ultrastructural maturation (Dellmann et al., 198l), the number of NGV-containing perikarya increases (Fig. 3 1) as well as the abundance of NGV in both perikarya and processes; the Golgi complexes become larger and multilocular and RER cisterns more elongated and numerous, forming typical aggregates evident at 6 dpn (Fig. 32). The most conspicuous changes occur between 1 and 6 dpn (Priimak and Hajos, 1972; Dellmann et af., 1981), when Khatchaturian and Sladek (1980) reported also the greatest changes in neurophysin immunoreactivity (see below), neuronal size, and number of axonal and dendritic profiles. Adult characteristics are acquired between 25 and 30 dpn (Dellmann et al., 1981; Fig. 33), these findings matching with Krisch’s (1980) observation of immunocytochemically fully mature NGVs between 3 and 4
FIG.33. This supraoptic neuron, at 25 dpn, possesses all the morphologic characteristics of a typical adult pcptidergic neurosecretory neuron. X 8500.
408
MONA CASTEL ET AL.
weeks postnatal, and Khatchaturian and Sladek’s ( I 980) light microscopic studies. At the ultrastructural level, synapses containing only electron-lucent vesicles, occasionally related to profiles of aSER, are first identified in the 17 fd rat (Fig. 34) (Sikora nd Dellmann, 1980a,b) or at later prenatal (Enestrom, 1967; Gash e? al., 1980; Sladek et al., 1980) and even postnatal ages (Priimak and Hajos, 1972). With histofluorescence methods, Khatchaturian and Sladek (1980) demonstrated the first presence of catecholamine fibers in both the SON and PVN at 17 fd, when axons with growth cones were observed at the ultrastructural level (Gash et al., 1980), but it was not until 21 fd that juxtaposition occurred between catecholamine variscosities and magnocellular neurons, also coinciding with the presence of acetylcholinesterase in both magnocellular nuclei (Hyyppa, 1969b,c; Danilova, 197 1). These observations were confirmed at the ultrastructural level in the SON (Gash et al., 1980; Sladek et ul., 1980; Sikora and Dellmann, 1980b), the latter authors observing presynaptic terminals at 20 fd which besides electron-lucent vesicles contain also dense-cored vesicles considered to be characteristic of catecholaminergic terminals (Fig. 35). An increase in density of fluorescent catecholamine fibers continues throughout postnatal stages and reaches adult proportions at 28 dpn (Khatchaturian and Sladek, 1980), while Sikora and Dellmann (1980b) described adult levels of synapses already by 25 dpn. Synaptic maturation occurs at a time when the SON has acquired all other adult characteristics. Interestingly, synapses in the mouse magnocellular nuclei are almost completely absent prior to and immediately after birth, but are present in significant numbers at 5 dpn and increase to adult levels at 24 dpn (Silverman and Goldstein, 1979; Silverman et nl., 1980). Possible reasons for discrepant reports on the synaptic development in the SON have been discussed by Sikora and Dellmann (1980b). Neither the initiation of biosynthetic activity nor the capability of the HNS to respond to physiologic stimuli and thus to secrete is dependent upon the initial presence of synpatic input for the first phenomenon, and upon maturity of the input for the second (Sinding et al., 1980b). Volume and osmoreceptors are apparently functional at birth, but since synaptic contacts are relatively sparse during prenatal and early postnatal development, the question arises whether they are sufficient to mediate responses to physiologic stimuli (e.g., Sinding et a/. , 1980b), or whether nonsynaptic mechanisms are operative at that time, as proposed by Sladek et al. (1980). In the adult rat there is electrophysiological evidence for extensive coordination between magnocellular neurosecretory cells within a nucleus (Poulain and Wakerley, 1982), and the structural basis for this may be direct membrane appositions between neurosecretory elements (Tweedle and Hatton, 1976; Theodosis rt ol., 1981). Hence it is feasible that even limited synaptic input during early stages of development need not impede adequate functioning of the magno-
NEURONAL SECRETORY SYSTEMS
409
34
FIG. 34. Axosomatic synapse in the supraoptic nucleus of a 17 fd rat; notice elcctron-luccnt vesicles and a profile of smooth endoplasrnic reticulum in the anon. (From Sikora nd Dellmann, 1980b.) X77,500. FIG. 35. In thc 20 fd rat supraoptic nucleus, axosomatic synapses are observed for the first time that contain both electron-luccnt and and dcnse-core vesicles. X 43,750.
410
MONA CASTEL ET AL
cellular nuclei. Further investigations of the morphometry of developing synapses and deafferation experiments may provide more clues. The observation of the in v i m differentiation of rat, mouse, and guinea pig magnocellular neurons from undifferentiated or little differentiated cells, to neurons producing NGVs after 9 to 21 days of culture (Benda et al., 1975; Marson and Privat, 1979; Reisert et al., 1980; Jirikowski et al., 1981) is remarkable in that it clearly shows that in vitru techniques may be used for the study of the HNS development, and that no or very little synaptic input (a few synapses actually developed) is necessary for differentiation. The successful cloning of hypothalamic neurons that possess the ultrastructural, biochemical, and histochemical characteristics of peptidergic neurosecretory cells (De Vitry et al., 1974, 1975; Tixier-Vidal and de Vitry, 1976; for review see Tixier-Vidal and de Vitry, 1979) represents the first step of another line of research that promises to yield valuable information on the morphology and function of developing neurosecretory neurons and also the mechanisms that regulate their differentiation. Recently Notter et d.(1981) addressed themselves to the interesting question of the influence of the pituicytes on the maturation of neurosecretory neurons. They cocultured dispersed and subsequently reaggregated hypothalamic cells from newborn rats with dispersed neural lobe cells (age not reported), and observed higher concentrations of VP in the tissue and culture medium than in the hypothalamic reaggregates cultured alone. Since this effect was absent in 19 fd cocultures, they speculate that during a critical time in development, the neurohypophysis (pituicytes?) may either promote survival of the cultured neurons or stimulate their secretory activity. Morphologic evidence indicates extensive synthetic activity of the pituicytes at the time of invasion of the neural lobe anlage (consisting virtually only of pituicytes) by neurosecretory axons (Galabov and Schleiber, 1978a,b; Dellmann and Sikora, 1981). Further investigations at earlier stages of development may contribute to our understanding of the role that pituicytes play in the development of secretory activity of neurosecretory neurons. It has recently been reported that enkephalinergic synpases impinge on pituicytes in the rat neurohypophysis (van Leeuwen, 1982a,b) and it has been suggested that the regulatory effect that this opioid exerts on the release of VP and OT (Iversen et al., 1980) may be via the pituicytes (see Section 111,D). Whether the hypothalamic factor stimulating neurosecretory activity that Pearson et al. (1975) and Schas et al. (1975) detected in the guinea pig hypothalamus between 40 and 55 fd plays a role in the maturation of neurosecretory neurons remains to be investigated. 3. Immunohistochemi~try Earlier reports on prenatal (Benirschke and McKay, 1953; Scharrer, 1954; Rodeck and Caesar, 1956; Kivalo and Talanti, 1957; Barry and Bugnon, 1958; Enemar, 1961; Rinne et al., 1962; Rinne and Kivalo, 1965; Danilova, 1965;
NEURONAL SECRETORY SYSTEMS
41 1
Enestrom, 1967; Bock et af., 1968; Donev, 1970a; Perks and Vizsolyi, 1973) and postnatal (Bargmann, 1949; Dawson, 1953; Diepen et af., 1954; Green and van Breeman, 1955; Rodeck and Caesar, 1956; Amoroso et al., 1958; Rodeck, 1958; Smiechowska, 1964; Danilova, 1965; Bock et al., 1968) occurrence of aldehyde-fuchsin-positiveor Gomori-positive material in a wide variety of species were not only aimed at the detection of neurohormone activity in the HNS, but also at the solution of the long-lasting controversy of whether neurohormone biosynthesis was an exclusive perikaryal function or whether it could also take place in the axon. These studies are now superseded by far more specific and sensitive immunohistochemical and immunocytochemical data which, together with those from radioimmunoassays and bioassays, have provided a clear-cut solution to these problems. In all investigated species (man, rat, mouse, guinea pig, pig, horse, chicken), neurophysin and VP are immunohistochemically detectable in the prenatal hypothalamus (Ellis and Watkins, 1975; Silverman, 1975; Choy and Watkins, 1979; Wolf and Trautmann, 1977; Wolf and Sterba, 1978; Dubois, 1978; Paulin et a f . , 1978; Fellmann et af., 1979; Silverman and Goldstein, 1979; Khatchaturian and Sladek, 1980; Silverman et al., 1980; Buijs ef af., 1980a,b; Sladek ef al., 1980; Fisher et af., 1981). In the rat neurophysin was first detected in the SON at 16 fd (Wolf and Trautman, 1977; Wolf and Sterba, 1978; Fisher et af.,1981) or 17 fd (Khatchaturian and Sladek, 1980), and in the PVN at 17 fd (Khatchaturian and Sladek, 1980), at 18 fd (Choy and Watkins, 1979), and 19 fd (Wolf and Trautman, 1977; Wolf and Sterba, 1978; Fisher et al., 1981), and at the same time VP was detected in these nuclei (Buijs et al., 1980b). The earlier occurrence of neurophysin and neurohormone in the SON has also been reported by Silverman (1975) in the guinea pig and by Ellis and Watkins (1975) in the pig, and was confirmed by Watkins and Choy (1979) and the immunocytochemical observations by Krisch (1980b). OT appears to escape prenatal detection in these nuclei (Buijs et al., 1980b; Fischer et al., 1981) where it was only found by Choy and Watkins (1979) in the rat. In man, however, OT neurons were first detected simultaneously in the SON and PVN at 14 fetal weeks (Paulin et ul., 1978), 3 weeks after the first appearance of neurophysin-positive neurons (Fellman et af., 1979), which seems to indicate that the appearance of the prohormone precedes maturity of its cleaving enzymes system (see Section IUD). These results are corroborated by those of Fisher et af. (198 1) who detect VPneurophysin immunoreactive perikarya in the rat SON at 16 fd and in the PVN at 18 fd; immunoreactivity against OT-neurophysin appeared simultaneously in the SON and the PVN at 18 fd. Buijs et al. (1980a) reported the presence in the 16 fd rat hypothalamus of VP- and OT-containing cells within the ependyrnal lining and projecting into the hypothalamus, and Boer et al. (1980a) likewise described VP-positive cells in contact with the cerebrospinal fluid during late gestation and up to 14 days postnaturn, and supraependymal (intraventricular) VP-positive
412
MONA CASTEL ET AL
nerve fibers up to 16 dpn (see also Buijs et al., 1980b), that were also observed in the infundibular recess of early postnatal rats by Ugrumov and Mitskevich (1981). Within maximally 24 hours after their arrival at the definitive site of the future SON, undifferentiated cells have differentiated into cells with ultrastructural and immunohistochemical characteristics of neurohormone-producing cells. Since Sinding er al. (1980b) were able to detect radioimmunoassayable neorophysin in the fetal rat hypothalamus as early as 13 fd, and in the mouse staining for neurophysin even preceded arrival of the prospective magnocellular neurons at their definitive site (Silverman and Goldstein, 1979), it seems that these cells despite their “undifferentiated” ultrastructural appearance, are definitely capable of neurophysin (prohormone) synthesis that is not detectable with postembedding immunohistochemistry. As in the adult system, the more sensitive preembedding immunohistochemistry and immunocytochernistry is expected to reveal reaction product earlier during development and prior to the NGV stage of the secretory products. In the hands of Krisch (1980a,b), even the postembedding technique has yielded reaction product within presumptive Golgi and RER of magnocellular neurons in the neonate. However, postembedding immunocytochemistry may induce nonspecific staining due to a variety of procedural factors (Buijs and Swaab, 1979). Even though in the mouse, neurophysin-immunoreactive cells were detected as early as 13.5 fd, either in migration from the ependymal wall or in the presumptive SON, it was not until 16.5 fd that neurophysin-positive cells were present within the PVN (Silverman and Goldstein, 1979; Silverman et al., 1980). Despite the discrepancies in the observed first detection of immunoreactive neurons, for which a variety of factors could be responsible (e.g., disparity in determining conception; paraplast versus vibratome sections; different immune sera; etc.), all reports are in agreement that the onset of biosynthetic activity (or differentiation) in the SON precedes that of the PVN and other magnocellular peptidergic cell groups, with quantitative differences persisting throughout the first weeks postnatum (Wolf and Trautman, 1977). This obvious lateral-to-media1 trend in maturation (Fisher et al., 1981) is intriguing, especially since both magnocellular nuclei originate simultaneously from the same area in the developing hypothalamus (Altman and Bayer, 1978a,b), and since they contain both (in the adult) VP- and OT-producing cells. Could this differential development be induced by biosynthesis-stimulating factors such as those identified by Pearson et al. (1975) in the guinea pig hypothalamus, or do the more ventrolateral neurons develop neurites that reach the median eminence earlier than the dorsomedial ones and could thus be susceptible to stimulating influences from the developing vascular network of the glial cells in the median eminence and/or the neural lobe? Clearly, further studies are called for to explain these puzzling phenomena.
NEURONAL SECRETORY SYSTEMS
413
The further light microscopic development of immunoreactivity is characterized by a progressive increase in staining intensity and in the number of positive perikarya and the prenatal appearance of axons and dendrites; these changes reach a plateau between 25 and 28 dpn in the rat, in which 65% of the SON magnocellular perikarya are neurophysin-positive at birth and remain at this level through 28 dpn (Fig. 36) (Khatchaturian and Sladek, 1980). In the guinea pig, the SON at birth contains 95% of the adult population of neurophysinpositive cells, the PVN only 70% or less (Silverman, 1975). 4. Immunocytochemistry The only immunoultrastructural studies on the development of the HNS nuclei are those by Krisch (1980a,b), and they are limited to the postnatal development of the VP-secreting system in the rat. Labeled NGVs are present in both the SON and PVN in the neonate, with more cells reacting in the SON than in the PVN. In addition to intensely and homogeneously stained NGVs (100 to 110 nm), weakly and inhomogeneously labeled NGVs are also present. In some cells, only the Golgi complex is labeled, even though presumptive NGVs are present in the cytoplasm. At the end of the first postnatal week, within the SON larger NGVs (120- 130 nm), labeled to various degrees are present, as well as smaller homogeneously labeled NGVs (70 nm), while in the PVN all NGVs are weakly but evenly stained. At the end of the second week, the staining intensity has increased, but some NGVs in the SON are still inhomogeneously stained; in the PVN there is a noticeable increase in the number of NGVs, and an annular deposit of reaction product is observed in those cells in which the Golgi complex and adjacent RER are labeled. At the end of the third postnatal week, in the PVN the cytochemical reaction is almost that of adult animals. The total absence of reaction product within some NGVs, similar to that observed in mature animals (Morris et al., 1978), or uneven staining of NGVs could be explained by varying proportions of propressophysin that does not react with VP antibodies within these granules, or belated maturation of the cleavage enzymes. The conversion of prohormone may proceed more slowly and irregularly within NGVs of the immature animal, thus accounting for the erratic results. AND NEUROHYPOPHYSIS B. MEDIANEMINENCE 1. Light Microscopic Observations Accounts of the first occurrence of aldehyde-fuchsin-positive or Gomori-positive material in the median eminence (ME) and the neural lobe (NL) are mainly of historical interest (Bargmann, 1949; Benirschke and McKay, 1953; Dawson, 1953, 1966; Scharrer, 1954; Rodeck and Caesar, 1956; Kivalo and Talanti, 1957; Barry, 1961; Rodeck et a l . , 1960; Wurster and Benirschke, 1964;
414
MONA CASTEL ET AL.
.
. '
22 dpc
- **
, 0
.
FIG.36. On~ogcnyof immunocytochemically stained neurophysin neurons in the rat supraoptic nucleus. Light microacopy. (From Khachaturian and Sladek, 1980.) X 163.
NEURONAL SECRETORY SYSTEMS
415
Danilova, 1965; Rinne and Kivalo, 1965; Bock et al., 1968; Fiorini and Wechsler, 1968; Donev, 1970a; Eurenius and Jarskar, 1974). The conclusions that may be drawn from these observations are stainable material is present before birth; its first appearance in the NL precedes that in the hypothalamus (notable exceptions are the descriptions of Barry, 1961; and Bock et al., 1968, of the simultaneous stainability of hypothalamic nuclei and the NL); it occurs first around the capillaries and in the vicinity of the intermediate lobe; its staining intensity increases with increasing age of the animal; and typical Herring bodies appear relatively late during development. From light microscopic immunohistochemical investigations of the developing ME and NL (Silverman, 1975; Wolf and Trautmann, 1977; Paulin et al., 1978; Swaab et al., 1978; Wolf and Sterba, 1978; Burlet et al., 1979; Choy and Watkins, 1979; Silverman and Goldstein, 1979; Watkins and Choy, 1979; Buijs er al., 1980a, b; Krisch, 1980a, b; Silverman et al., 1980; Sladek et al., 1980) it may be concluded that neurophysin-positive axons can be detected in these areas at the time of or approximately 1 day after their first appearance in the hypothalamus, that the staining for neurophysin precedes that for VP and OT, that the distribution of reactive material in the developing NL coincides with that of aldehyde-fuchsin-positive or Gomori-positive material, and that the external zone of the ME contains during late prenatal and early postnatal development a large number of neurophysin or VP-positive axons. In the rat ME, the amount of immunoreactive material is greatest for approximately 1 week postnatal and then decreased to reach near adult proportions at the end of the third (Wolf and Trautmann, 1977; Watkins and Choy, 1979; Krisch, 1980a,b) or fourth postnatal week (Burlet er al., 1979). Silverman (1975), who likewise observed neurophysin and VP-positive axons in the external zone of the guinea pig ME, reported adult levels at 60 fd. There is undoubtedly a striking similarity between these findings and those reported after adrenalectomy (see Bock et al., 1980, for literature), and in view of the sharp increase in the amount of pituitary ACTH reported by Chiappa and Fink (1977) between 5 and 9 dpn in the rat, the CRF nature of the immunoreactive material is likely (Krisch, 1980a). Further correlative investigations on the development of these two systems (neurophysin-VPCRF and ACTH) should also take into account the development of the capillary loops of the hypothalamic-hypophysial portal system and their relationship to the axon terminals. The onset of extrahypothalamic innervation by magnocellular neurons coincides with the first occurrence of neurosecretory axons in the NL; at 17 fd VPpositive fibers are present in the olfactory bulb; at 18 fd they reach the amygdala via the stria terminals, the corpus callosum via the medial septum or the claustrum, and penetrate the ependyma; at 19 fd, the caudal commissure contains VPpositive fibers; at 20 fd, fibers are present in the region of the angular bundle in the ventral hippocampus and in the vicinity of the solitary tract (Buijs er al., 1980b).
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MONA CASTEL ET AL
2. Electron Microscopic. Investigations
The difficulties in handling the immature (fetal) brain for ultrastructural immunocytochemistry are such that it is not surprising that only a few reports deal with that subject in the developing ME and NL. In a study on the postnatal development of the ME, Krisch (1980a,b) describes small (65 nm) neurophysin-labeled NGVs in the external zone of the ME in the neonate. In addition, large labeled NGVs occur after the first week postnatal, with the same labeling characteristics as those in the PVN; this may, despite size differences, indicate a connection between these two areas (Krisch, 1980b). In the internal zone, with advancing age of the animals, more fibers with labeled NGVs of increasing diameter are observed; large axons are present with dense networks labeled by PAP complexes that in nonincubated serial sections are identified as a system of interconnected tubules. Leclerc and Pelletier (1977) have investigated the prenatal rat NL in which they discover small, nonimmunoreactive NGVs (40-90 nm) at 16 fd, and the first neurophysin-positive NGVs (75-1 10 nm) at 18 fd. A compound that crossreacted with OT and VP was present in NGVs (140 nm) in the rat NL at term (Swaab et al., 1977). During the early postnatal development of the NL, the neurosecretory axons resemble those in the ME in that reaction product appears to be detectable within an axoplasmic tubular network (Krisch, 1980a). In addition, homogeneously and moderately labeled NGVs, inhomogeneously labeled NGVs, and NGVs with “frayed” appearance are present; by the end of the third week postnatal, the immunocytochemical characteristics of the NL are almost those of the adult rat. At the ultrastructural level it has been shown that neurosecretory axons at the time of their invasion of the NL contain NGVs, in the mouse (Eurenius and Jarskar, 1974) and in the rat (Fink and Smith, 1971; Leclerc and Pelletier, 1977; Dellmann et al., 1978). The NGVs are first seen shortly prior to or at the time when neurophysin and VP are immunohistochemically detectable. At that time, NGVs are usually less electron-dense than at later stages of development (Dellmann et al., 1978), and subsequently increase in electron density, number, and size. They reach almost adult size in the later stages of development in the mouse (Silverman and Desnoyers, 1976) and guinea pig (Eurenius and Jarskar, 1974), at 19 fetal weeks in human (Okado and Yokota, 1980), and at an undetermined time postnatally in the rat, in which at 10 dpn the NGVs are still significantly smaller than in the adult (Table 111) (Dellmann et al., 1978; Krisch, 1980b). Obviously the size changes of the vesicles are related to the maturation of the hypothalamic-neurohypophysial system, but it is not clear why the neurosecretory cell at an early stage of its development should produce smaller NGVs, nor whether they contain less neurohormone than in the adult. It is also not known whether NGVs that have reached the NL remain the same size until released, or
417
NEURONAL SECRETORY SYSTEMS
TABLE 111 MEANDIAMETER +- SD AND SIZERANGEOF NVG I N NEUROSECRETORY AXONSAT DIFFERENT ACES Age (days) 18 (fetal) 0 1 5 10
Adult
Mean
* SD (nm)
114.9 f 19.1* 138.5 C 26.3" 146.5 2 22.9'* 151.4 23.3 n.s. 153.1 C 35.3* 171.3 30.0
*
*
Range (nm) 85-160 85-190 85-210 100-230 100-240 100-260
*p < 0.001. significantly different from next older group. **p < 0.005, significantly different from neXt older group.
released, or increase in size before release. Since the lowest values of the range of diameters of NGVs remain constant between 18 fd and 1 dpn (Dellmann et al., 1978), it appears that NGV diameters do not change after arrival in the NL, where these vesicles are destined either for release or disposal by lysosomal events (see, however, Section V,C concerning different hormone pools in the adult neurohypohysis) . Several other populations of granulated vesicles of enigmatic significance, that disappear before birth, have been described (see Dellmann et al., 1978), as well as a group of relatively large (70- 125 nm) electron-lucent vesicles that may play a role in membrane retrieval following hormone secretion (Morris et al., 1978). The sudden increase in number of the latter vesicles at 1 dpn could be indicative of increased hormone release, which has indeed been shown to occur in the neonate rat (see below and Sinding et al., 1980a). Although there are indications of hormone release prior to birth, it has not been possible to detect plasma levels in the fetal rat (see below). While large ncurophysin- or VP-immunoreactive dilatations (Herring bodies) of the size found in adults are not observed before birth (Choy and Watkins, 1979), ultrastructural investigations have revealed the presence of neurosecretory axon dilatations at the very time of their invasion of the NL. These Herring bodies, throughout 10 dpn, do not contain large numbers of NGVs or other organelles (Dellmann et al., 1978). Based upon immunocytochemical studies, Krisch ( 1980b) suggested that the nongranular immunoreactive material present within these Herring bodies, and similar ones in the internal zone of the ME, was contained within the tubules of the aSER. Krisch surmised that neurosecretory neurons at that time may not be able to fully fulfill functional demands through the production of sufficient numbers of NGVs, and that additional hormone is transported via the aSER (Dellmann et al., 1978). In the fetal guinea pig there is a clear discrepancy between the level of immunoreactive neurophysin and VP at
418
MONA CASTEL E T AL.
FIG. 37. Neurohypophysis of fetal and neonate rats illustrating the abundance of axonal smooth endoplasmic reticulum (aSER). (a) Fetal day 18; neurosecretory axon with aSER containing electrondense material; vesicles may be NGVs or lysosomes. X35.000. (b) Fetal day 19; aSER with dilatations. X52,OOO. (c) Fctal day 19; apparently twisted aSER with dilatations. X37.000. (d) Fetal
NEURONAL SECRETORY SYSTEMS
419
45 fd and the scarcity of NGVs (Silverman, 1975; Silverman and Desnoyers, 1976), and these authors also hypothesize about an extragranular pool of neurohormone in the neurohypophysis. Anastomosing profiles of aSER containing material of varying electron density are particularly abundant in developing neurosecretory axons (Eurenius and Jarskar, 1974; Dellman et al., 1978; Krisch, 1980a) (Fig. 37). The electron-dense material often appears similar to that within NGVs, while constrictions along segments of aSER and apparent formation of vesicles suggest that this membrane system, at least in the fetus and the neonate, may be involved in conveyance of neurohormone and formation of NGVs. Admittedly, unequivocal evidence for the presence of neurosecretory material within the aSER of peptidergic neurons has not been provided to date (see Section V,B for additional discussion). IN THE HYPOTHALAMUS C. NEUROHORMONES
The application of RIA (radioimmunoassay) for the assessment neurophysin, VP, and OT to the developing HNS has made it possible to establish the first occurrence of neurohormone and gauge its subsequent changes, thus providing valuable information on the maturation of the system. Since the results reported by different authors vary within wide limits, even for the same species, and the standards of measurement vary (per total pituitary, per mg/pituitary, per entire HNS, per body weight, or 100 g/body weight), no attempt will be made here to systematically list exact figures or to reproduce tables and graphs. However, our review of the literature clearly indicates that the first basic data upon which future research can be built are now available. Comparing the immunohistological, immunocytochemical, ultrastructural, light microscopic, and RIA data obtained in various laboratories for one species, for example the rat, it must be emphasized that a precise correlation of these data is virtually impossible. For correlative purposes the most reliable data are undoubtedly those obtained with a variety of methods in a single laboratory (see Sladek ef al., 1980). Discrepancies in reported results are most likely reflections of differences in determining length of pregnancy and fetal age, differences in strain (Sprague-Dawley , Wistar, Wistar-Lewis, Holtzman), extraction procedures, antibodies used, fixation procedures, to name only the most obvious. In the rat, neurophysin is present in the HNS as early as 13 fd, and then increases dramatically at 14 fd; subsequently it decreased to 19 fd, when it starts day 20; appearance of wider, less twisted aSER (asterisks); arrow indicates putative NGV with reticular protrusion. x45,OOO. (e) Day 4 neonate; neurosecretory axons with aSER and NGVs. X4Y.000. (0 Day 7 neonate; apparently vesiculating aSER. X88,OOO. (g) Day 1 neonate; arrow indicates possible formation of NGVs from aSER. X45,OOO. (h) Day 2 neonate; aSER; also elongated, blunt-ended multivesicular body (arrow). X45.000. (i) Day 3 neonate; arrow indicates vesicular structure (NGV? lysosome?) forming from aSER. X 110,000.
420
MONA CASTEL ET AL.
to rise again modestly through 8 dpn (Figs. 38 and 39) (Sinding et al., 1980c,d). VP and OT are absent at 13 fd, but are measured at low levesl at 14 fd (Sinding and Robinson, 1978; Sinding et al., 1980c), 16 fd (Buijs et al., 1980; Boer et al., 1980b), or 17 fd (Gash et al., 1980; Sladek et al., 1980; Swaab, 1980). A sharp increase in VP occurs between 18 and 22 fd, and a modest increase in OT, however, a decrease from 16 to 18 fd in the brain and concomitant increase in the neural lobe was observed by Buijs et al. (1980b). Subsequently OT rose steadily in the brain, while in the neural lobe it decreased to 20 fd, and then also rose (Pitzel et al., 1982). VP decreases at birth (but not significantly; see Sladek et al., 1980), while OT continues to rise. Subsequently VP increases rapidly during the first clays postnatal, while OT increases very slowly through 8 dpn, and then both subsequently increase to reach a plateau between 21 and 30 dpn (Fig. 39) (Sinding et al., 1980~).Sladek et al. (1980), however, reported a significant depletion of OT 2 horus after birth, and Swaab (1980) a decrease in OT concentration between 17 fd and 1 dpn. Neural lobes of fetal (Boer e? al., 1980; Pitzel et al., 1982) and newborn (Pitzel et al., 1982) rats contained approximately 20 times more VP than OT. Interestingly, there is a 350-fold molar excess of neurophysin to OT plus VP at 14 fd; near molar equivalency occurs at 19 fd, primarily due to the high content of VP, and following the sharp rise of VP concentration, a molar deficiency of neurophysin at the time of delivery, with a ratio of 0.12 (Sinding et al., 1980~).
I2
14
I6
18
20
22 days
Q
FIG. 38. Content of vasopressin (VP),neurophysin (NP), and oxytocin (OXY) in fetal rats. Days of gestation and time of delivery (arrow) are indicated (Sinding et al., 1980a).
NEURONAL SECRETORY SYSTEMS
0
42 1
OXYTOCIN VASOPRESSIN NEUROPHYSIN
FIG. 39. Content of oxytocin, vasopressin, and neurophysin in infant rat pituitaries. Levels expressed per 100 g body weight (Sinding et al., 1980b).
Sinding et al. (1980~)were unable to find an explanation for these molar discrepancies, particularly they did not find any evidence for the presence of vasotocin, confirming the findings of Dogterom et al. (1979) and Negro-Vilar et al. (1979). It is conceivable that the observed molar excess of neurophysin is due to the fact that the neurophysin antibodies recognize precursor molecules as well. It is also possible that “neurohypophysial peptides go through several changes of their molecular form which modify the antigenicity of the peptides”; in particular it is feasible that a VP-related neurophysin is present in the developing rat that is not recognized by the antibody used in the RIA or because it was not extracted (Sinding et al., 1980a). This idea is supported by the observation that the neurophysin/OT ratio is 0.83 at birth, i.e., it appears that the neurophysin measured in the HNS at birth is OT-related, as is probably the one in plasma, since plasma VP levels were undetectable postnatally. These data are corroborated by the observation of elevated OT-neurophysin levels and decreasing VP-neurophysin levels during the first few days after birth in man (Robinson et af., 1977). In addition to “regular” OT and VP, Sinding et al. (1980~)identified putative large molecular forms of OT and VP (molecular weight 100,000); “big” VP amounted to more than 90% prior to and less than 40% after 18 fd. Comparing results obtained with immunocytochemical methods and those of RIA, it is obvious that with RI neurophysin appeared considerably earlier during development. This discrepancy is probably accounted for by the technical difficulties encountered with immunocytochemistry of fetal material. It remains a remarkable fact that in the rat, assayable neurophysin was already present in the HNS (at 13 fd) when the first cells of the SON and PVN started to migrate from their site of origin near the ventricle toward their definitive nuclear sites. While
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MONA CASTEL ET AL
the relatively high levels of neurophysin correspond to the migrating period, the decline in neurophysin concentration coincides approximately with the differentiation of the neurosecretory cells and their axonal growth. It is conceivable that during that period most of the protein-synthesizing machinery of the cell is producing growth-related substances rather than secretory proteins, which is resumed once the axons have reached the neurohypophysis. The ability of the devleoping rat HNS to respond to physiologic stimuli was tested by Sinding et al. (1980b) who showed that dehydration in 2 to 30 dpn rats leads to a marked decrease in neurohypophysial VP (26-38%) which is more pronounced after 24 hours of dehydration in younger (2 dpn) rats (26%) than in older ones (7%), confirming earlier findings by Heller and Lederis (1959); at the same time neurophysin also decreased significantly. With the increase in neurohypophysial VP between 8 and 15 pdn, a greater increase in VP plasma levels in response to dehydration was observed, possibly supporting the concept of a readily releasable pool of newly synthesized hormone (Heap et al., 1975; Dreifuss, 1975; see Section lV,C).
D. NEUROHORMONES I N THE NEUROHYPOPHYSIS Despite discrepancies regarding the time of first occurrence of neurohypophysial peptides in the neural lobe, their concentrations and their proportions, there is now general agreement that neurophysin, VP, and OT are detectable in the hypophysis prior to birth. Neurophysin is demonstrated prior to VP and OT, and during development higher pituitary concentrations of VP than of OT are present. Data on the first occurrence of radioimmunoassayable neurophysin are only available for the guinea pig in which it was detected at 20 fd by Burton and Forsling (1972). Neurohypophysial extracts from the pig yielded neurophysin 1, 11, and Ill (minor neurophysin) (Ellis and Watkins, 1975) whose concentration rose from 24% of the total neurophysin content in the fetal (98 fd) pig to 30% in the neonate, and declined to 14% in the adult. This led the authors to speculate on the presence of an as yet to be identified neurohypophysial hormone with which Np 111 may be associated. Hypothalamic extracts that included the hypophysis yielded neurophysin as early as I3 fd in the rat, before neither VP nor OT could be detected, with a marked increase at 14 fd and subsequent decline to 19 fd (Sinding et al., 1980c,d). It would be extremely interesting, in view of the ultrastructural evidence for intense secretory activity of the pituicytes (Galabov and Schiebler, 1978a, b; Dellmann and Sikora, 1979, 1981), to assay the isolated neurophypophysis for the presence of a substance that may be detected by RIA for neurophysin. If such a peptide is present, it would provide an alternative explanation for the 350-fold molar excess of neurophysin over VP plus OT at 14 fd, and its decline to molar equivalency at 19 fd, when the morphologic signs of intense secretory activity of the pituicytes have subsided.
NEURONAL SECRETORY SYSTEMS
423
There have been several reports of argirine vasotocin in the fetal NL of a variety of animals (Burton and Forsling, 1972; Perks and Vizsolyi, 1973, 1974; Pavel, 1974, 1975; Vizsolyi and Perks, 1976b; Skowsky and Fisher, 1973, 1977; Swaab et a/., 1977), but specific RIA for vasotocin could not confirm these findings (Dogterom et a / . , 1979; Negro-Vilar, et al., 1979; Sinding et a/., 1980a; Swaab et a / ., 1979; Lederis, personal communication). At this point it may be concluded that AVT is not produced in substantial amounts in fetal or adult neural lobes. However, since bioassay distinctly revealed “vasotocin activity,” it is possible, that a peptide is present that closely resembles but is not identical to vasotocin (Swaab, 1980). In this connection, Pavel’s (1974, 1975) investigations are of interest reporting the release of a principle with the specific pharmacologic profile of argininevasotocin into the culture medium of human fetal NLs cultured for 43 days. Whether or not the released substance is in fact vasotocin or whether the cells responsible for its synthesis are ependymal cells or pituicytes, is of secondary importance; the concept of the secretory activity of the cells of the fetal NL is of particular interest. An exhaustive correlative study of the fetal neural lobe with a variety of techniques is required to provide solutions to these still unsolved questions. At birth neurophysin levels in the NL are low and are only 0.8% of adult levels (La Rochelle et al., 1977; Sinding e t a / ., 1980a). The concentration remains low for approximately 1 week postnatal and then increases (La Rochelle et a/., 1977) and reaches a plateau at 21 dpn (Sinding et al., 1980a). Radioimmunoassayable VP has been reported to occur in the rat NL as early as 12 fd (Forsling, 1973), thus about 4 days prior to the first occurrence of neurosecretory axons in the NL (Dellmann et a / ., 1978); this again underlines the necessity for a comprehensive correlative study of the developing NL. Other reports set the onset of VP immunoreactivity in the rat NL at between 16 and 18.5 fd with subsequent increase during prenatal development (Roffi, 1958; Pearson et al., 1975; Boer et a / . , 1980a; Buijs et a/., 1980a; Sladek et al., 1980). A slight increase in VP content of the rat NL (NHS) was observed by Sladek et al. (1980) 2 hours postnatal; in contrast, a significant decrease occurs at 1 dpn and is followed by a rapid increase between 1 and 2 dpn, and also between 8 and 15 dpn, and finally a decreases of 33% at 0.5 hours plateau at 21 and 30 dpn (Sinding et a / . , 1980~); postnatal and of 52% at 1 to 2 hours postnatal with a return to prenatal levels at about 1 dpn were reported in the rabbit (Froger and Roffi, 1974; Froger et al., 1976). Hemorrhage causes a 58% decrease in the rabbit neural lobe VP content at term (Froger et al., 1976). These widely varying data could be a reflection of differences in stress situations that the animals were exposed to at varying postnatal times when the NLs were collected (Sladek et al., 1980), and it may well be that environmental stress was greater than that of birth stress. Rat postnatal VP levels are substantially lower than in the adult (Heller, 1947; Dicker and Tyler, 1953a; Roffi, 1958; Heller and Lederis, 1959; Sinding eta/.,
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MONA CASTEL ET AL.
1980a; Sladek et al., 1980). However, near adult levels have been reported at term in the sheep (Alexander et al., 1974a; Vizsolyi and Perks, 1976a,b), the guinea pig (Heller and Lederis, 1959; Fliickiger and Opperschall, 1962), which is probably a reflection of the general maturity of these neonates by other criteria as well. Acomys cahirinus, a desert mouse, has a gestation period of about 7 days longer than that of the laboratory mouse. At 16 fd (Boer et al., 1980b) or 17 fd (Forsling, 1973) OT is first detectable in the rat NL, however, in the HNS it is present as early as 14 fd (Sinding et al., 1980~).After an increase to 22 fd (Buijs et al., 1980b; Sinding et al., 1980c; Sladek el al., 1980), it was observed to remain at low levels to 8 dpn and to increase to 21 dpn when it reached a plateau (Sinding et al., 1980~).However, found that the OT content of the NL decreases by 30% during Boer et al. (1980~) or just before labor, while Sladek et ul. (1980) observed a significant depletion at 2 hours postnatal. A comparison of the available data on neuropeptide concentrations in the developing NL clearly reveals low postnatal levels that are most likely due to the elevated hormone release, and possibly to a relatively low rate of synthesis. Repletion follows the “perinatal stress situation” at different rates for the two hormones: VP increases dramatically within 2 days while OT (and its associated neurophysin; see above) apparently continues to be released (Robinson et al., 1977; Sinding er al., 1980c) and both begin to increase only at approximately 8 dpn (Sinding et al., 1980~). During development, VP predominates over OT in a wide variety of species (Heller and Zaimis, 1949; Dicker and Tyler, 1953a,b; Acher et al., 1956; Heller and Lederis, 1959; Capek and Heller, 1961; Perks and Vizsolyi, 1973; Boer et al., 1980a; Sinding et al., 1980a; Schubertet al., 1981). With increasing postnatal age there is a decrease in the VP/OT ratio that in the rat is close to 1 at 21 dpn (Sinding et al., 1980a) or 30 dpn (Dicker and Tyler, 1953, a,b; Acher ef al., 1956; Heller and Lederis, 1959), but in man already in full-term infants (Heller and Zaimis, 1949; Dicker and Tyler, 1953b). In contrast to the marked increase in the pituitary VP/OT ratio toward the end of gestation in man, reversal and decrease to 0.04 of the hypothalamic VP/OT ratio were observed by Schubert et al., (1981), possibly an indication of increased synthesis and release of OT (see below).
E. NEUROHORMONES IN
THE
PLASMA
The developing HNS undoubtedly synthesizes peptides that are transported to the NL where their concentration increases throughout development. Storage ensues, and the question remains to be answered: when are the hormones first released? In the rat, Sinding et al. (1980~)found plasma neurophysin levels to be higher than adult levels in 2 dpn rats, to decrease subsequently, and to reach adult levels by 8 dpn; in contrast, VP could not be detected throughout 15 dpn
NEURONAL SECRETORY SYSTEMS
425
(Boer et al., 1980a) or even 30 dpn except for a few 8 dpn animals (Sinding et al., 1980a). Since neurophysin plasma levels were high at 2 dpn and remained high for at least 8 days, when they begin to rise in the NL, and since there is no parallel increase in plasma VP, the low content of OT and neurophysin in the NL during the first 8 dpn could possibly be explained by a high secretion rate of both OT and OT-neurophysin in the newborn rat (Sinding et al., 1980~).High levels of plasma OT-neurophysin have been reported by Robinson et al. (1977) in infants for 14 dpn. In the fetal and newborn calf, high plasma neurophysin levels could be detected, decreasing after the first week postnatal (Robinson et al., 1971), probably again a stress-related phenomenon. OT and VP are likewise present at high levels in human umbilical cord blood (Chard et al., 1971, 1977; Chard, 1973; Polin et al., 1977; Swaab et al., 1978) and rise during the last hours of pregnancy and at the time of delivery in man (Chard, 1973; Chard et al., 1977) and sheep (Forsling et al., 1975; Stark, 1977). Glatz et al. (1981), however, do observe a postnatal increase in OT. Even though VP was not detectable in the plasma of control animals, intraperitoneal injection of 5% saline increased plasma VP in 2 to 30 dpn old rats to levels comparable to those obtained in adult animals, while 6 and 24 hours of dehydration had no or little effect (Sinding and Robinson, 1978) but did cause an appreciable decrease in the NL (Heller and Lederis, 1959; Sinding and Robinson, 1978) and in increase in plasma neurophysin and VP in the adult (Seif et al., 1977). The presence of a large quantity of OT in the amniotic fluid would indicate that the fetus is actually releasing this hormone prior to delivery (Sinding et al., 1980c), but Dicker (1966) found that only 7 out of 21 one dpn rat NLs secrete OT when incubated in Locke solution, while all NLs released VP, although appreciably less (1/10) than from adultderived NLs. In contrast, Pitzel et al. (1982) observe the release of both VP and OT from isolated fetal and newborn rat neural lobes. The release of VP was significantly increased at 1 dpn, simultaneously K stimulation caused the release of more than 30% of the neural lobe’s OT content. The greater maturity of the sheep at the time of delivery is likewise expressed in the ability of the HNS to release peptides prior to birth. There appears to be general agreement that plasma VP levels are undetectable in younger but present in older fetuses (Alexander et al., 1971, 1973; Skowsky et al., 1973; Smith et al., 1977; Rurak, 1978; Weitzman et al., 1978). They increase in response to 5% saline injection (Smith et al., 1977; Weitzman et al., 1978), fetal hemorrhage (Alexander et al., 1974b, 1976; Czernichow, 1978; Robillard et al., 1979), hypoxia (Rurak, 1978), or hypertonic saline injection into the mother (Leake et al., 1977). High OT blood levels were reported in the sheep fetus by Skowsky et al. (1973); they rise during the last hour of pregnancy (Forsling et al., 1975; STark, 1977) as they do in man (Chard et al., 1977) where high blood levels were found in umbilical cord blood (Chard et al., 1971, 1977; Swaab et al., 1978). Further evidence for the responsiveness of the fetal HNS to stress is the higher VP plasma level in infants born in deliveries with minor problems (Czer+
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MONA CASTEL ET AL
nichow and Pattin, 1978) and by vaginal delivery rather than those born by caesarian section (Hoppenstein et al., 1968; Chard er al., 1971). Additional evidence for functional volume and osmoreceptor systems has been reported (Ames, 1953; Smith, 1959; Fisher et ol., 1963).
X. Versatility of Neurosecretory Neurons Originally this review set out to describe the hypothalamic-neurohypophysial axis as a model peptidergic system, and to underscore its uniqueness vis-a-vis other neuronal secretory systems. The concept of the neurohypophysial peptides as hormonal messengers dispatched copiously into the blood, conformed with the central tenets of neurosecretion (Scharrer and Scharrer, 1963). In contrast, “conventional” aminergic and cholinergic neurons dispense their neuroactive substances with considerable more economy into limited synaptic spaces. However, it has meanwhile become evident that the so-called neurohypophysial hormones are released not only in a glandular fashion at neurohernal sites (median eminence, neurohypophysis), but also at other locations throughout the CNS, in the manner of neurotransmitters and neuromodulators. Hence, this final section briefly considers the evidence for modes of operation of VP and OT neurons beyond the HNS. BEYOND A. CELLSAND PROJECTIONS
THE
HYPOTHALAMUS
The existence of peptidergic neurosecretory projections within the CNS, beyond the hypothalamus, was presaged by the early work of Barry (1954) and Legait and Legait (1958), using selective neurosecretory stains to identify the neurosecretory material by its high cysteine content. These important findings were neglected for two decades, until verified immunocytochemically and greatly expanded (Weindl and Sofroniew, 1976; Swanson, 1977; Buijs, 1978; Sofroniew and Weindl, 1978a,b), as may be seen from Table IV. In fact, projections containing immunoreactive neurohypophysial peptides may be detected in CNS regions ranging from the olfactory bulb to the spinal cord. The immunocytochemical findings have been endorsed by sensitive rddioimmunoassays for VP and OT in minute circumscribed brain regions (Dogterom et al., 1978; Glick and Brownstein, 1980). There is now ample evidence for CNS vasopressinergic and oxytocinergic systems, apart from the HNS (Buijs et al., 1980a; Nilaver er al., 1980; Swanson and Sawchenko, 1980, 1983; Zimmerman er al., 1980; Sofroniew and Schrell, 1981; De Vries and Buijs, 1983; Silverman and Zimmerman, 1983). However, the precise connections between identified perikarya, projections, and terminal fields of these additional VP and OT networks are currently in a state of confusion. In fact, many a red herring has been pursued in
TABLE IV DISTRIBUTION OF IMMUNOREACTIVE VASOPRESSIN AND OXYTOCIN I N THE RATCENTRAL NERVOUS HYPOTHALAMIC-NEUROHYPOPHYS~AL SYSTEM,OTHERTHANTHE CLASSICAL NEUROSECRETORY SYSTEM^
OT
VP
L. Immunoreactive processes and terminalsb Target area Telencephalon Diagonal band of Broca Lateral septum Medial septum Amygdala: central, anterior, basal, laterial Amygdala: medial Amygdala: cortical Ventral hippocampus
++ ++++ + + +++ + +
+ +
Diencephalon Periventricular posteriodorsal hypothalamus Mediodorsal thalamus Lateral habenula Supramammillary nucleus
+
+
Mesencephalon Substantia nigra Central gray Dorsal raphe nucleus Interpenduncular nucleus Cuneiform nucleus
+ + + +
++
++++ + +
+++ +++ ++++
+
Pons Dorsal parabrachial nucleus Ventral parahrachial nucleus Locus coerulcus Nucleus reticularis parvocellularis Nucleus raphe pontis Central gray Medulla Nucleus of the solitary tract Dora1 nucleus of the vagus Nucleus commissuralis Lateral reticular nucleus Nucleus ambiguus Central gray Nucleus raphe magnus Nucleus raphe obscurus Suhstantia gelatinosa trigemini
+t
+ + + + + ++ ++ ++ + + + + + +
++++ ++++ ++++ +++ +
+ ++ + +
(continued)
428
MONA CASTEL ET AL. TABLE IV (Conrinued) VP Spinal cord Laminae 1-111 Lamina X Intermediolateral nucleus of spinal cord
JJ. Jmmunoreactive cell bodiesC Lateral septumd Bed nucleus of the stria terminalis Dorsomedial hypothalamus Medial amygdaloid nucleus Locus coeruleus
+ + +
OT
+ ++
+
++ ++++ ++ ++ ++++
aPartly adapted from Sofroniew (1980) and Sofroniew and Weindl (1981). Cell body data are from Van Leeuwen and Caffc? (1983) and Caffb and Van Leeuwen (1983). bData on precise cells of origin have been omitted due to current contradictions and uncertainties (see Section X,A). CFollowing colchicine treatment. dPossibly part of diffuse magnocellular system of hypothalamus.
attempts to link up distant projections with neurosecretory cells in the hypothalamus, and published circuitries are being repeatedly revised (for example, septal vassopressinergic innervation, see Buijs, 1978; De Vries et al., 1981; Hoorneman and Buijs, 1982; De Vries and Buijs, 1983). Although a small number of hypothalamic magnocellular neurons may send projections both to the neurohypophysis and to the brain (Swanson and Kuypers, 1980; Zerihun and Harris, 1981), most studies have provided evidence that different cells project to neurohemal sites and to neural targets within the CNS (On0 et al., 1978; Hosoya and Matsushita, 1979; Armstrong et al., 1980; Swanson and Sawchenko, 1980). Immunoreactive magnocellular projections directed toward the neurohypophysis are generally thicker and more varicose than those directed toward extrahypothalamic sites, a phenomenon that is particularly obvious in water-deprived rats (Epstein et al., 1983) (Fig. 40). The basis for this FIG. 40. (A) Vasopressin-immunoreactive processes; part of hypothalamic-neurohypophysial tract. Preembedding immunoperoxidase procedure on vibratome sections; light microscopy. op. Optic tract. (Al) Control rat. (A2) Water-deprived rat; note varicosities (Herring bodies) along axons. X520. (B) Vasopressin-immunorcactive processes in periventricular thalamic region of (B I ) control rat and (B2) water-derpived rat. v, Ventricle. Note that immunoreactive fibers are finer and sparser than those of the hypothalamic-neurohypophysialtract depicted in A. Source of thalamic vasopressin innervation possibly hypothalamic suprachiasmatic nucleus. x432. (C) Vasopressinimmunoreactive processes in lateral septum of (CI) control rat and (C2) water-deprived rat. v, Lateral ventricle. Immunoreactive fibers finer and sparser than those of the hypothalamic-neurohypophysial tract (see A). Source of septal vasopressinergic innervation possibly hypothalamic paraventricular nucleus. x700. (From Epstein er al., 1983.)
. ..
.
B2
*
. '
. : ..
.
,..
i--
..
_ I
FIG. 40A
~
. .
AND
B.
..
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MONA CASTEL Er AL.
FIG. 40C. See legend on p. 428
difference in axon caliber may be that the glandular type of neurosecretion, destined for neurohemal release, is particularly copious, while projections that are expected to terminate synaptically in the brain are sustained by a more economical supply of active substances. In addition to the VP and OT magnocellular perikarya that project either to the neural lobe or to the CNS, there are the parvicellular VP-producing cells of the SCN (Vandesande and Dierickx, 1975) that project only to the brain. This issue has been further compounded by the recent discovery that following colchicine treatment, additional hitherto unrecognized VP-immunoreactive cell bodies may be identified in the stria terminalis, the lateral septum (Van Leeuwen and Caffi, 1983), the dorsomedial hypothalamus, the medial amygdaloid nucleus, and the locus coeruleus (Caffi and Van Leeuwen, 1983) (Table 1V; Fig. 41). These newly described VP-immunoreactive cells are smaller, morphologically distinct,
b
FIG. 41. Immunocytochemical staining with antivasopressin of various cell groups in forebrain of colchicine-treated rat (light microscopy, preembedding procedure, vibratome sections). Note morphological differences between various cell groups. (a) Bed nucleus of the stria terminalis (BST), small multipolar and bipolar cells, far more numerous and quite distinct from the few magnocellular VP-immunoreactive neurons usually found in the BST. (b) Suprachiasmatic nucleus (SCN), particularly heavily stained after colchicine treatment. V, Ventricle; Op, optic tract. (c) Lateral septum (LS). VP-immunoreactive cells may be part of diffuse magnocellular system. (From Van Leeuwen and Caffe, 1983.)
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MONA CASTEL ET AL.
and obviously not part of the diffuse magnocellular system of the hypothalamus, with the possible exception of the septa1 group. Future statements relating to the origin and circuitry of VP projections in the CNS will need to take into account these additional VP-irnmunoreactive perikarya. No "new" OT-immunoreactive cells have come to light following colchicine treatment, which is in line with the observation that ablation of the PVN is sufficient to drastically reduce extrahypothalamic oxytocinergic projections (De Vries and Buijs, 1983). While the anatomically well-defined HNS was amenable to biosynthetic and structure-function studies (see Sections 11-V, IX), the vasopressinergic and oxytocinergic systems in the rest of the brain are difficult to segregate, and pose a new challenge. In order to establish the circuitry of VP and OT projections, a battery of experimental and neuroanatomical techniques needs to be applied, including focal lesions, tracing of individual neurons, and close coordination with specific immunocytochemical staining. Several exellent neuroanatomical studies relating to hypothalamic nuclei have been undertaken in recent years (for
FIG.42. Immunoelectron micrograph; vasopressin-immunoreactive presumptive synapse on unidentified cell in PVN. Arrow indicates possible postsynaptic density. Small vesicles heavily stirrounded by iinmunoreactive material; mitochondrion is not stained. X37,800.
NEURONAL SECRETORY SYSTEMS
433
FIG. 43. Immunoelectron micrograph;vasopressin-positive terminal forming synapse on unidentified dendrite in lateral habenular nucleus of rat; arrow indicates postsynaptic density. Note electronlucent synaptic vesicles; electron-dense reaction product is mostly extravesicular, but some is over synaptic vesicles. (From Buijs and Swaab, 1979.) X60.000.
review see Swanson and Sawchenko, 1983), but considering the cellular heterogeneity of the PVN, the SCN and possibly also the SON, it is not always possible to reach unequivocal conclusions regarding the exact neuropeptide content of individual neurons, the more so as the discovery of copeptides and cotransmitters compounds the issue further (see Section 111,D). It is anticipated, however, that a basic biosynthetic analogy may be drawn between VP- and OT-producing neurons in diverse locations. Independent immunocytochemical observations of extrahypothalamic cells and projections have shown that each neuropeptide (VP
V
V
FIG. 44. Vasopressin-immunoreactive processes appear to contact the cerebral ventricular lumen. Light microscopy, prcembcdding immunopcroxidase procedure, vibratome sections. (a) Mouse hypothalamus; conspicuous immunoreactive dendrites, originating from perikarya in the
NEURONAL SECRETORY SYSTEMS
435
and OT) is invariably accompanied by it respective neurophysin (I1 and I), indicating that posttranslational processing of precursors is probably operative irrespective of whether these neurosecretory cells project to the HNS or the CNS. An interesting line of speculation relates to the possibility that hitherto unrecognized cleavage products of the large precursors, propressophysin and prooxyphysin, may be biologically active in CNS sites beyond the HNS (Brownstein et al., 1980). This would be in line with the diverse neuroactive segments that are cleaved from other brain prohormones, the proopiocortin precursors.
SYNAPSES B. PEPTIDERGIC As current expectations focus on the neurohypophysial peptides as possible neurotransmitters in the CNS, the peptidergic synapse calls for morphological definition. Barry ( I 956) did indeed refer to “de synapses neuroskcretoires” long before the importance of brain neuropeptides was appreciated. At the ultrastructural level, Bargmann et al. (1967) identified neurosecretory synapses terminating on cells of the pars intermedia of the pituitary gland, and defined these as “peptidergic synapses. The electrophysiological concept of recurrent inhibition by neurosecretory neurons (Hayward and Jennings, 1973) implies that peptidergic synapses impinge upon the neurosecretory cells themselves, although this has not been visualized with certainty to date. VP-immunoreactive synapselike profiles may occasionally be seen on unidentified cells of the PVN (Fig. 42). In the CNS putative peptidergic synapses were visualized at the ultrastructural level by Sterba (1974) and Buijs (Buijs and Swaab, 1979). However, Sterba’s oxidation technique for neurophysin (Nauman and Sterba, 1976), although useful when supported by additional evidence (Sterba et d., 1980), is of unaccountable specificity. The immunouitrastructural studies of Buijs (Buijs and Swaab, 1979; Buijs ef al., 1981) provide the best evidence to date for VP and OT synapses (Fig. 43), and this has recently been corroborated with experimental data (Buijs and Van Heerikhuize, 1983). At the light microscopic level terminal fields may be identified tentatively by the coiling and branching of VP and OT fibers among presumptive target cells. Although cell bodies in lateral septum and habenula nucleus appear to be encircled by VP fibers, immunoelectron microscopy reveals that most immu”
paraventricular nucleus (pvn). surround the third ventricle (v); some irnmunoreactive endings may traverse the ependyma (e). X200. (M. Castel and J . Morris, unpublished.) (b) Hypothalamus of heterozygote Brattleboro diabetes insipidus rat; irnmunoreactive dendrite-like processes appear to traverse the ependyma (e) and contact the lumen of the third ventricle (v). X220. Inset is enlargement ( X 1100) of area indicated by arrow (Castel E I ul.. 1982). (c) Rat hypothalamus; immunoreactive beaded axon appears to bifurcate and terminate at ventricular lumen. Foramen of Monroe (fm) connects between lateral and third ventricles. The irnmunoreactive axon originated from a magnocellular neuron in the bed nucleus of the stria terminalis. (Dark blob in center of micrograph is artifact.) X700.
436
MONA CASTEL ET AL.
noreactive synaptic profiles are in fact found on dendrites (Buijs and Swaab, 1979; Buijs et al., 1981). In the medial nucleus of the amygdala some VP terminals form synapses on oligodendrocyte-like cells (Buijs, 1980). However, little progress has been made to date in establishing the identity of the postsynaptic elements of presumptive VP and OT synapses. New stratagems involving combined immunocytochemical and tracer studies are called for. Peptidergic synapses may display pre- and postsynaptic densities and a widening of the synaptic gap (see Fig. 43). According to Buijs, VP- and OT-immunoreactive boutons in the rat brain contain small (about 30 nm) electron-lucent synaptic vesicles surrounded by extravesicular reaction product, and possibly a few reactive dense-cored vesicles of about 100 nm (NGVs in the neural lobe measure about 180-220 nm). Sterba et ul. (1979, 1981), whose evidence is based mainly on submammalian species, maintain that in addition to electronlucent synaptic vesicles, neurophysin-immunoreactive synapses contain a substantial number of “regular-sized” NGVs (180-200 nm); this may be typical of the amphibian and reptilian species investigated by these authors.
RELEASEIN C. NONSYNAFTIC
THE
BRAIN
While it is now evident that the neurohypophysial peptides are not only secreted at neurohemal sites, it is by no means claimed that the only alternative is synaptic transmission in the CNS. According to electrophysiological data, in addition to neurotransmitter roles, VP and OT may function as neuromodulators dispatched into extracellular space in a diffuse manner (Leng and Wiersma, 1981). It has long been known that neurohypophysial peptides may be assayed in the cerebrospinal fluid (Heller et al., 1968; Dogterom et al., 1977; Mens et al., 1982). It has been suggested that direct release of these neurohormones into the cerebral ventricular system occurs only during fetal life, when VP may have a trophic value in the cerebrospinal fluid (Boer et al., 1980b). However, VPimmunoreactive processes presumably contacting the ventricular lumen have been reported in adult animals as well (Brownfield and Kozlowski, 1977; Castel et al., 1982; Kozlowski, 1982) (see Fig. 44). It is not uncommon to see conspicuous VP-immunoreactive dendrites, originating in the PVN and accessory nuclei, directed toward the third ventricle in the hypothalamus (Sofroniew and Glasmann, 1981; Castel et al., 1982), where their function may be either sensory or secretory. The dendrites in question, on passing through subependymal regions on their way toward the third ventricle, are profusely innervated by a variety of afferent synapses (Fig. 45). It has not been possible to establish unequivocally whether the intense VP-immunoreactivity within dendrites of neurosecretory cells (see Fig. 44a and b and Fig. 45) denotes merely artifactual diffusion of reactive substances from lysed secretory vesicles in the cell body, or whether a legitimate dendritic route for transport and release of neuropeptide is indicated (see also Section VIII,C).
FIG. 45. Axodendritic synapses (asterisks) on vasopressin-immunoreactive dendrites in the subependymal region near the paraventricular nucleus of the mouse hypothalamus. Note that electrondense reaction product is on neurotubules and in dendroplasrn (artifact?); mitochondira are unstained. (a) Single axo dendritic bouton (asterisk). (b) Multiple axodendritic boutons (asterisks). (In lower right comer small-caliber axon profile containing irnmunoreactive neurotubules.) X37,800.
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D. IN CONCLUSION Historically VP and OT are designated as neurohypophysial hormones, for the HNS was the first neuronal secretory system in which they were identified in profusion. However, it is now abundantly evident that the spectrum of neurochemical activity of these peptides extends beyond the “parochial” HNS. Multiple projections and venues of release within the CNS vastly increase the variety of target sites available to VP and OT, which in turn is reflected in the wide range of CNS effects that are currently attributed to these neuropeptides (de Wied and Bohus, 1979; de Wied and Versteeg, 1979; Bohus, 1980; Swanson and Mogenson, 1981; Wang et al., 1981; Pittman et a / . , 1982; SzczepanskaSadowska et al., 1982). Moreover, it is feasible, though not yet proven, that specific enzymes could vary the cleavge products of propressophysin and prooxyphysin in different CNS sites. In addition, modifying influences are probably exerted by the different copeptides and cotransmitters within VP and OT neurons. The rich efferent input from other neuronal systems, the feedback relays, and the selective coordination between neurosecretory cells (Poulain and Wakerley, 1982) all contribute to the extended repertoire of the so-called neurohypophysial peptides. The diversity and versatility of the peptidergic neurosecretory cells that produce VP and OT reveal affinities with other neurons in the CNS. However, on the other hand it may well be that “the hallmark of neurosecretory neurons, in contradistinction to conventional neurons, is their capacity for multiple modes of operation” (Scharrer, 1981). ACKNOWLEDGMENTS The research in M.C.’s laboratory has been generously supported by the U.S.A.-Israel Binational Science Foundation, Grants 200/74, 2325/80, and by the Israel Academy of Sciences and Humanities. The technical assistance of Naorni Sivan and the graphic work of Ada Dorman are greatly apprcciatcd. REttKENCES Abbs, M. T., and Phillips, J . G. (1980). Riochim. Biophys. Actu 595, 200.
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NOTL AUUWI N PROOF.Recently several reviews about thc vasopressin and oxytocin precursors were published [Gainer, H. (1983). Prog. Brain Res. 60, 205-215; and 17,cIl, R., Schmale, H . , and
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Richter, D. (1983). Neuroendocrinology 37, 235-2391, and the rat gene for the vasopressin precursor has been characterized [Schmale, H., Heinsohn, S . , and Richter, D. (1983). EMBO J . 2, 763-7671. The self-association constant for the AVP precursor with neurophysin has been determined [Chaiken, I . , Tamaoki, H., Brownstein, M., and Gainer, H. (1983). FEBS Lett. 164, 361-3651 and discussed in the context of prohormone packaging and processing. An updated review on prohormone proceaaing will soon appear [Loh, Y. P., Brownstein, M., and Gainer, H. (1984). Annu. Rev. Neurosci. 7, in press], and cytochrome bS6, has been found in NGVs isolated from bovine neurohypophysis [Duong, L., Fleming, P., and Russell, J . T. (1984). J . B i d . Chem., in press]. A new postembedding immunocytochemical procedure which is superior to preembedding procedures has been used to demonstrate that dynorphin 1-8 is contained specifically within vasopressin NGVs in rat pituitary [Whitnall, M . , Gainer, H . , Cox, B., and Mollineaux, C. (1983). Science 222, 1137- 1 1391. Monoclonal antibodies which react specifically with vasopressin-associated neurophysin or oxytocin-associated neurophysin and their respective precursors, with no cross-reactivity, have been produced [Ben-Barak, Y., Russell, J. T., Ozato, K., and Gainer, H . (1984). Submitted]. These monoclonal antibodies have been used in irnmunocytochemical studies of developing rat brain and have shown that both the oxytocin and vasopressin precursors are expressed early (around fetal day 16), but that only the vasopressin precursor is processed significantly to AVP during fetal life. In contrast, in the rat, the oxytocin precursor processing is delayed until birth [Whitnalll, M., Key, S . , Ben-Barak, Y., Ozato, K . , and Gainer, H . (1984). Submitted.].
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Index
A
Adrenergic neuron, visicle life cycle in, 389-393 Agonists, lipophilic, interaction with extracellular membranes, 74-76
B Biogenic amines immunocytochemistry of, 388-389 visualization in neurons, 386-388 Brain, nonsynaptic release of neurotransmitters in, 436-438
Cytoskeleton, participation in functions of effectors, 113-129
E Effector substances, pathways of uptake by lysosomes, 41-46 Endocytic vesicles, fusion with lysosomes, 154-156 Endoplasmic reticulum perikaryal, peptidergic neurosecretory products and, 318-323 smooth axonal, of peptidergic neurons, 348-354
C Catecholamine(s), uptake into chromaffin vesicles, 380-382 Catecholamine-containing cells, packaging and transport of neurotransmitter in, 377-380 Cholinergic neurons packaging and transport of neurotransmitters in, 376-377 vesicles in, 395-401 Cholinergic vesicle, molecular organization of, 369-370 Chromaffin cell, adrenomedullary, vesicle formation in, 383-386 Chromaffin granule, molecular organization of, 372-374 Chromaffin vesicles, uptake of catecholamines into, 380-382 Colloid droplets, peptidergic neurosecretory products and, 323-327 Compartmentation, uses in cellular economy, 243-244
G Golgi-GERL system, peptidergic neurosecretory products and, 327-334
H Hormones early effects of, 90-95 formation from prohormone, 178- I80 mechanisms of action on lysosomes evolution of ideas, 16-18 hypothesis for totality of effector action, 35-40 present state of art, 18-35 unresolved problems, 35 Hormone-receptor complex, cellular entry of evidence, 106, 107-109 mechanisms, 106, 110-152 significance, 153- I54 time course, 152- 153 46 1
462
INDEX
Hydrolases, lysosomal, synthesis and processing of. 55-61 Hypothalamic-neur.ohypophysialsystem axonal transport i n , 341-345 developmental aspects of, 401 magnocellular hypothalamic nuclei, 402-4 13 median eminence and neurohypophysis 41 3-4 I 9 neurohormones in hypothalamus, 419-422 neurohornmonea in neurohypophysis, 422-424 neurohormones in plasma, 424-426 Hypothalamus cells and projections beyond, 426-435 magnocellular nuclei of early origin and development, 402 immunocytochemistry, 4 I3 immunohistochemistry, 410-41 3 ultrastructure, 402-410 neurohormones in, 419-422
L Ligand-receptor, role of internalization, 182-183 Lysosomal aystcrn, of peptidergic neurons, 362-366 Lysosomea agonists and partial agonists, 4--9 antagonists, 4-9 cellular functions influenced by cell death and anomalies of interpretation, 2 12-220 growth and proliferation, 220-234 transformation, 234-243 compatibility of propertics with proposed agonal mediating functions in activated cells consequcnces of ligand capture, 73-2 I2 evidence of covert labilization of lysosonmal membranes, 70-72 generalized schcme, 70 target cell: occurrence and functional i m plications of specific recognition sites for given effectors in plasmalemma, 72-73 consequences of ligand capture
cellular entry of hormone-receptor complex, 106- I54 controlled destabilization of nicmbrane, 156-212 fusion of endocytic vesicle with lysosome, 154- 156 signals of nicmbrane deformation controlled destabilization of membrane at cell surface, 156- I58 in cytoplasm, 158-202 promotion of intercellular communication, 207-212 vesicular flow and, 202-217 first premises, 1-2 receptor concept, 2 signal versus noise, 2- I5 fusion with endocytic vesicles, 154- 156 indispensability of function of, 244-245 mechanisms of hormone action evolution of ideas, 16-18 hypothesis for totality of effector action, 35-40 present state of art, 18-35 unresolved problems, 35 objectives and scope of review, 15-16 participation in reorganization of target cell plasmalemma, 101-103 relevani properties of, 40-41 composition and organization, 46-53 function in normal cellular economy, 61-69 membrane stability and permeability, 53-55 pathways of uptake of nutrients and effector substances, 41-46 synthesis and processing of hydrolases and pathways of lysosome formation, 55-61
M Median emincnce, neurohypophysis and electron microscopic observations, 416-41 9 light microscopic observations, 413-415 Membrane, see ulso Surface membrane lysosonial, stability and permeability, 53-55 vesiculation, ligand binding and, 87-88 proximate signals of perturbation and their attenuation, 88- 100
463
INDEX Microvesicles, in peptidergic neurons, 357-362
N Neurohypophysis hormone pools in, 354-357 median eminence and electron microscopic observations, 416-4 I9 light microscopic observations, 413-4 I5 neurohormones in, 422-424 peptides and neurophysins of, 311-315 secretory products of, 308-310 Neurons adrenergic, life cycle of vesicles in, 389-393 molecular organization of secretory vesicles in, 366-367 cholinergic vesicle, 369-370 chromaffin granule, 372-374 neurosecretory granulated vesicles, 374-376 physical characteristics of vesicles, 367-369 vesicles in sympathetic neurons, 370-372 serotonergic, vesicle life cycle in, 393-395 visualization of biogenic amines in, 386-388 Neurosecretory cells, see also Nonpeptidergic; Peptidergic axonal transport in hypothalamic-neurohypophysial system. 341-345 multiple components of, 338-341 Neurosecretory granulated vesicles anterograde transport of, 345-348 molecular organization of, 374-376 as site of posttranslational processing, 315-317 Neurosecretory neurons, versatility of cells and projections beyond the hypothalamus, 426-435 nonsynaptic release in brain, 436-438 peptidergic synapses, 435-436 Nonpeptidergic neurons, biosynthesis and biochemical aspects of packaging and transport of neurotransmitters in catecholamine-containing cells, 377-380
catecholamine uptake into chromaffin vesicles, 380-382 cholinergic neurons, 376-377 Nonpeptidergic secretory vesicles, morphological aspects of formation of, 382-383 adrenomedullary chromaffin cell, 383-386 immunocytochemistry of biogenic amines, 388-389 life cycles of vesicles in adrenergic neurons, 389-393 vesicles in cholinergic neurons, 395-401 vesicle life cycle in serotonergic neuron, 393-395 visualization of biogenic amines in neurons, 386-388 Nutrients, pathways of uptake by lysosomes, 41-46 0 Oxytocin neurons, additional peptides in, 334-338
P Peptidergic neurons, morphology of transport and release by anterograde transport of neurosecretory granulated vesicles, 345-348 axonal smooth endoplasmic reticulum, 348-354 hormone pools in neurohypophysis, 354-351 lysosomal system, 362-366 microvesicles, 357-362 Peptidergic neurosecretory cells, biosynthesis and packaging in functional activity and, 317-318 neurohypophysial peptides and neurophysins, 311-315 neurohypophysial secretory products, 308-310 separate hormones in separate cells, 310-311 neurosecretory granulated vesicle as site of posttranslational processing, 3 15-3 17 Peptidergic neurosecretory products, morphological aspects of formation of
464
INDEX
additional peptides in vasopressin and oxytocin cells, 334-338 colloid droplets, 323-327 Golgi-GERL system, 327-334 perikaryal endoplasmic reticulum, 3 18-323 PhosphorylationIdephosphorylation,in cellular targets coupled to agonal signals, 130- I5 I Plasma, neurohormones in, 424-426 Protein(s), membrane, redistribution after ligand binding, 78-87 Proteolysis, in response to selected effectors, 159-175
S Secretory vesicles, physical characteristics of, 367-369
Serotonergic neuron, identified, vesicle life cycle in, 393-395 Surface membrane, signals of deformation relevance to transduction of binding signal, 100-106 response to ligand binding, 73-100 Sympathetic neurons, vesicles in, 370-372 Synapses, peptidergic, 435-436
V Vasopressin neurons, additional peptides and, 334-338
Contents of Recent Volumes and Supplements Volume 70
Volume 73.
*
Cycling Noncycling Cell Transitions in Tissue Aging, Immunological Surveillance, Transformation, and Tumor GrowthSEYMOUR GELFANT The Differentiated State of Normal and Malignant Cells or How to Define a “Normal” Cell in Culture-MiNA J. BISSELL On the Nature of Oncogenic Transformation of C e k A E R A L D L. CHAN Morphological and Biochemical Aspects of Adhesiveness and Dissociation of Cancer Cell+HIDEOHAYASHI A N D YASUJl ISHIMARU The Cells of the Gastric MUCOSa-HtRBERT F. HELANDER Ultrastructure and Biology of Female Gametophyte in Flowering Plants-R. N. KAPILA N D A. K. BHATNACAR
Microtubule-Membrane Interactions in Cilia and Flageb--wILLIAM L. DENTLER The Chloroplast Endoplasmic Reticulum: Structure, Function, and Evolutionary Significance-SARAH P. GIBBS DNA Repair-A. R. LEHMANNAND P. KARRAN Insulin Binding and Glucose Transport-RusSELL HILF, LAURIE K. SORGE, A N D ROGER J. GAY Cell Interactions and the Control of Development in Myxobacteria POpUkitiOnS-DAVlD WHITE Ultrastructure, Chemistry, and Function of the Bacterial Wall-T. J. BEVERIDGE INDEX
INDEX
Volume 73 Volume 71 PrOtOplaStS Of Eukaryotic Alga+MARTHA D. Integration of Oncogenic Viruses in Mammalian BERLINER CCk
INDEX
465
466
CONTENTS OF RECENT VOLUMES AND SUPPLEMENTS
Volume 74
Organization and Expression of Viral Genes in Adenovirus-Transformed Cells-S. J. FLINT The Plasma Membrane as a Regulatory Site in Highly Repeated Sequences in Mammalian GeGrowth and Differentiation of Neunomes-MAxiNE F. SINGER roblastoma Cells-SiscmisD W. DE LAAT Moderately Repetitive DNA in EvolutionROBERTA. BOUCHARD AND PALIL. T. VAN DER SAAG Mechanisms That Regulate the Structural and Structural Attributes of Membranous Organelles in Bacteria--CHARLEs C. REMSEN Functional Architecture of Cell SurfacesJANET M. OLIVERA N D RICHARD D. BERLIN Separated Anterior Pituitary Cells and Their ReGenome Activity and Gene Expression in Avian sponse to Hypophysiotropic HormonesErythroid Ct?lk-KARLEN G . GASARYAN CARL D E N E ~LUC , SWFNNEN, A N D MARIA Morphological and Cytological Aspects of Algal ANDRIES Calcification-MICHAEL A. BOROWITZKAWhat Is the Role of Naturally Produced Electric Naturally Occurring Neuron Death and Its RegCurrent in Vertebrate Regeneration and Healulation by Developing Neural PathwaysiIIg'?-RlcHARD B. BORGENS J. CUNNINGHAM Metabolism of Ethylene by Plants-JoHN TIMOTHY The Brown Fat Cd-JAN NEDERGAARD AND Donns A N D MICHAEL A. HALL INDEX OLOVLINDBERG INDEX
Volume 75
Volume 77
Mitochondria1 Nuclei-TsuNEYosHI KUROIWA Calcium-Binding Proteins and the Molecular Slime Mold Lectins-JAMES R. BARTLES, Basis of Calcium ACtion-LINI>A J. VANELWILLIAMA. FRAZIER,A N D STEVEND. D I K , JOS~PH 0 . ZENtVECUI, DANIEI.R. MARSHAK,AND D. M A R I I NW A ~ E R S V N RostN Lectin-Resistant Cell Surface Variants of Eu- Genetic Predisposition to Cancer in Man: In karyotic Cells-Evt BARAKBRILES V i m Studies-LEv Y K V P ~ L O ICH V Cell Division: Key to Cellular Morphogenesis in Menibrane Flow via the Golgi Apparatus of Higher Plant Ceh-DAvlD G . ROBINSON the Fission Ycast, SrhizosacchnromycesBYRONF. JOHNSON,CODE B. CALLEJA, AND UDO KRISTEN BONGY. Yoo, MICHAEL ZUKER,A N D IAN Cell Membranes in SpongeS-wERNm E. G . MULLER J. MCDONALD Microinjection of Fluorescently Labeled Pro- Plant Movements in the Space Environmentteins into Living Cells, with Emphasis on D A V I D G. HEATHCOTE Cytoskeletal ProteinS-THOMAS E. KREIS Chloroplasts and Chloroplast DNA of AcetahuAND WALTER BIRCHMEIER lurin mediferruneu: Facts and HypothesesANGELALUTTKEAND SlLVANO BONUI-IO Evolutionary Aspects of Cell DifferentiationStructure and Cytochemistry of the Chemical R. A. FLICKINGER Synapses-STEPHEN MANALOV A N D WLADStructure and Function of Postovulatory Follicles (Corpora Lutea) in the Ovaries of NonI M I R OVTSCHAROFF mammalian Vertebrates-SRiNivAs K. SAI- INDEX DAPUR INDtX
Volume 78 Volume 76 Cytological Hybridization to Mammalian ChromoSOmes-ANN s. HENDERSON
Bioenergetics and Kinetics of Microtubule and Actin Filament Assembly-DissassemblyTERRELI. L. HILL AND MARCW. KIKSCHNER
CONTENTS OF RECENT VOLUMES AND SUPPLEMENTS Regulation of the Cell Cycle by SomatomedinsHowARD ROTHSTEIN Epidermal Growth Factor: Mechanisms of ActiOn-MANJUSRI DAS Recent Progress in the Structure, Origin, Composition, and Function of Cortical Granules in Animal Egg-SARDUL S. GURAYA
467
Immunofluorescence Studies on Plant C e l l s 4 . E. JEFFREE,M. M. YEOMAN,A N D D. C. KILPATRICK Biological Interactions Taking Place at a LiquidSolid IntelfaCe-ALEXANDRE ROTHEN INDEX
INDEX
Volume 81 Volume 79 Oxidation of Carbon Monoxide by BacteriaYOUNGM. KIM AND GEORGED. HEGEMAN The Formation, Structure, and Composition of the Mammalian Kinetochore and Ki- Sensory Transduction in Bacterial ChemotaxAND SHIGEAKI i d E R A L D L. HAZELBAUER netochore FiberxoNLY L. RIEDER HARAYAMA Motility during Fertilization4ERALD SCHATThe Functional Significance of Leader and TrailTEN er Sequences in Eukaryotic mRNA&F. E. Functional Organization in the NucleusRONALDHANCWK A N D TENI BOULIKAS BARALLE The Relation of Programmed Cell Death to De- The Fragile X ChrOmOSOme~RANT R. SUTHERLAND velopment and Reproduction: Comparative Psoriasis versus Cancer: Adaptive versus Studies and an Attempt at ClassificationIatrogenic Human Proliferative DiseasesJACQUESBEAULATONAND R~CHARDA. SEYMOUR GELFANT LOCKSHIN Cryofixation: A Tool in Biological Ultrastruc- Cell Junctions in the Seminiferous Tubule and the Excurrent Duct of the Testes: Freezeturd Research-HELMUT PLAITNER A N D Fracture Studies-TosHio NAGANOAND LUISBACHMANN FUMIESUZUKI Stress Protein Formation: Gene Expression and Environmental Interaction with Evolutionary Geometrical Models for Cells in Tissues-HisAO HONDA Significance<. ADAMSA N D R. W. RINNE Growth of Cultured Cells Using Collagen as INDEX Substrate-JASON YANG AND s. NANDI INDtX
Volume 80 DNA Replication Fork Movement Rates in Mammalian Cells-LEON N. KAPP AND ROBERTB. PAINTER Interaction of Virsues with Cell Surface ReceptOrS-MARC TARDIEU, ROCHELLEL. EPSTEIN, AND HOWARD L. WEINER The Molecular Basis of Crown Gall InductionW. P. ROBERTS The Molecular Cytology of Wheat-Rye Hybrids-R. APPELS Bioenergetic and Ultrastructural Changes Associated with Chloroplast Development-A. R. WELLBURN The Biosynthesis of Microbodies (Peroxisomes, Glyoxysomesj H . KINDL
Volume 82 The ExonJntron Structure of Some Mitochondrial Genes and Its Relation to Mitochondria1 Evohtion-HENRY R. MAHLER Marine Food-Borne Dinoflagellate ToxinsDANIELG. BADEN Ultrastructure of the Dinoflagellate AmphieSma-LENITA c. MORRILL AND ALFREDR. LOEBLICH111 The Structure and Function of Annulate Lamellae: Porous Cytoplasmic and Intranuclear Membrane-RICHARD G. KESSEL Morphological Diversity among Members of the
468
CONTENTS OF RECENT VOLUMES AND SUPPLEMENTS
Gastrointestinal Microflora-DWAYNE SAVAGE
C.
INDEX
Cell Surface Receptors: Physical Chemistry and Cellular Regulation-Doucus LAUFFENBURGER AND CHARLESDELIS Kinetics of Inhibition of Transport Systems-R. M. KRUPKAAND R. DEW%
Volume 83
INDEX
Transposable Elements in Yeast-VALERIE MoVolume 85 ROZ WILLIAMSON Techniques to Study Metabolic Changes at the Cellular and Organ LeVel-ROBERT R. DE- Receptors for Insulin and CCK in the Acinar Pancreas: Relationship to Hormone ActionFURlA AND MARYK. DYGERT IRA D. GOLDFINE A N D JOHN A. WILLIAMS Mitochondria1 Form and Function Relationhips in Vivo: Their Potential in Toxicology and The Involvement of the Intracellular Redox State and pH in the Metabolic Control of StimPathology-ROBERT A. SMITH AND MURIEL ulus-Response COUphig-zYGMUND ROTH, J. ORD N A O M l CHAYEN, AND SHABTAY DIKSTEIN Heterogeneity and Territorial Organization of Regulation of DNA Synthesis in Cultured Rat the Nuclear Matrix and Related StructureHepatoma Cek-ROELAND V A N WIJK M. BOUTEILLE,D. BOUWER,AND A. P. Somatic Cell Genetics and Gene Mapping-FASEVE TENKAO Changes in Membrane Properties Associated with Cellular Aging-A. MACIEIRA-COELHOTubulin lsotypes and the Multigene Tubulin Families-N. J. COWANA N D L. DUDLEY Retinal Pigment Epithelium: Proliferation and Differentiation during Development and Re- The Ultrastructure of Plastids in Roots-JEAN M. WHATLEY g e n e r a t i o d L c A G. STROEVAAND VicThe Confined Function Model of the Golgi TOR I. MITASHOV Complex: Center for Ordered Processing of INDEX Biosynthetic Products of the Rough Endoplasmic Reticuhm----ALANM. TARTAKOFF Problems in Water Relations of Plants and Volume 84 Cells-PAUL J . KRAMER Controls to Plastid Divsion-J. V. POSSINGHAM Phagocyte-Pathogenic Microbe InteractionA N T O I N E ~RYTER E AND CHANTALDE AND M. E. LAWRENCE C ASTELLIER Morphology of Transcription at Cellular and Molecular kVek--FRANCINE PUVION- INDEX DUTlLLEUL An Assessment of the Evidence for the Role of Volume 86 Ribonucleoprotein Particles in the Maturation Toward a Dynamic Helical Model for the Influof Eukaryote mRNA-J. T. KNOWLER Degradative Plasmids-J. M. PEMBERTON ence of Microtubules on Wall Patterns in Regulation of Microtubule and Actin Filament P l a n t s 4 L i v E W. LLOYD Assembly-Disassembly by Associated Small Cellular Organization for SteroidogenesisPETERF. HALL and Large Molecules-TERRELL L. HILL Cellular Clocks and Oscillators-R. R. AND MARCW. KIRSCHNER K L E V ~ C ZS,. A. KAUFL-MAN, Long-term Effects of Perinatal Exposure to Sex AND R. M. SHYMKO Steroids and Diethylstilbestrol on the ReMaturation and Fertilization in Starfish productive System of Male MammalsYASUMASA ARAI, TAKAOMORI,YOSHIHIDE OOCyteS-LAURENT MEIJER AND PIERRE SUZUKI,AND HOWARDA. BERN GUERRIER
CONTENTS OF RECENT VOLUMES AND SUPPLEMENTS Cell Biology of Trypanosoma cruzi-wAN-
469
Cultured Vascular Endothelial Cells as a Model System for the Study of Cellular SenesThe Neuronal Organization of the Outer PlexCence-ELuoT M. LEVINE AND STEPHEN ifOrm Layer of the Primate Retina-ANDREW M. MUELLER P. MARIANI Vascular Smooth Muscle Cells for Studies of INDEX Cellular Aging in Virro; an Examination of Changes in Structural Cell L i p i d d L G A 0. BLUMENFELD,ELAINE SCHWARTZ,VERVolume 87 ONICA M. HEARN, AND MARIE J. KRANEFQOL The Modeling AppTOaCh-BARBARA E. Chondrocytes in Aging Research-EDWARD J. WRIGHT MILLERAND STEFFAN GAY Protein Diffusion in Cell Membranes: Some Biological ImpliCatiOnS-MICHAEL Mc- Growth and Differentiation of Isolated Calvarium Cells in a Serum-Free M e d i u w CLOSKEYAND MU-MINGPo0 JAMESK. BURKSAND WILLIAMA. PECK ATPases in Mitotic Spindles-M. M. PRATT Studies of Aging in Cultured Nervous System Nucleolar S t r u c t u r e G u y GOESSENS Tissue-DONALD H. SILBERBERG AND Membrane Heterogeneity in the Mammalian SEUNG U . KIM Spermatozoon-W. V. HOLT Capping and the Cytoskeleton-LILLv Y. W. Aging of Adrenocortical Cells in Culture-PETER J. HORNSBY,MICHAELH. SIMONIAN, BOURGUIGNON A N D GERARD J. A N D GORDON N. GILL BOURGUIGNON s. AMThyroid Cells in CUltUre-FRANCESCO The Muscle Satellite Cell: A ReVieW-DENNIS BESI-IMPIOMBATO AND HAYDENG . COON R. CAMPION Cytology of the Secretion in Mammalian Sweat Permanent Teratocarcinoma-Derived Cell Lines Ghds-KAzUMASA KUROSUMI,S u s u ~ u Stabilized by Transformation with SV40 and SV40tsA Mutant V ~ S ~ W A R R EMAL~ZN SHIBASAKI, AND TOSHlO IT0 MAN, DANIEL I. H. LINZER, FLORENCE INDEX BROWN,ANGELIKA K. TERESKY,MAURICE ROSENSTRAUS, AND ARNOLDJ. LEVINE Supplement 10: Differentiated Cells in Aging Nonreplicating Cultures of Frog Gastric Tubular Research AND CelISqERTRUDE H. BLUMENTHAL DINKARK. KASBEKAR Do Diploid Fibroblasts in Culture Age?-EuINDEX GENE BELL, LOUIS MAREK, STEPHANIE SHER, CHARLOTTE MERRILL, DONALD A N D IAN YOUNG LEVINSTONE, Urinary Track Epithelial Cells Cultured from Supplement 11A: Perspectives in Plant Cell Human Urine-J. S. FELIX AND J. W. and Tissue Culture LITTLEFIELD The Role of Terminal Differentiation in the Cell Proliferation and Growth in Callus CulFinite Culture Lifetime of the Human Epidertures-M. M. YEOMANAND E. FORCHE mal Keratinocyte-JAMES G . RHEINWALD Cell Proliferation and Growth in Suspension Culture-P. J. KING Long-Term Lymphoid Cell CUltUredEORGE F. SMITH, PARVIN JUSTICE, HENRI CytOdiffe~ntiatiOn-RICHARD PHiLLips FRISCHER, LEE KINCHU,AND JAMESKRK Organogenesis in Vitro: Structural, PhysiologiType I1 Alveolar Pneumonocytes in V i f r e cal, and Biochemical Aspects-TREVOR A. THORPE WILLIAMH. J. DOUGLAS, JAMES A. MCATEER,JAMESR. SMITH,A N D WALTER Chromosomal Variation in Plant Tissues in CulR. BRAUNSCHWEIGER ture-M. W. BAYLISS DERLEY DE SOUZA
470
CONTENTS OF RECENT VOLUMES AND SUPPLEMENTS
K. VASII. A N D Clonal Propagation-INuKA VIMLAVASIL Control of Morphogenesis by Inherent and Exogenously Applied Factors in Thin Cell Layers-K. TRANTHANHVAN Androgenetic Haphd5-hVKA K. VASIL Isolation, Characterization, and Utilization of Mutant Cell Lines in Higher Plants-PAL MALICA S U B J t C I INVtX
Supplement 11B: Perspectives in Plant Cell and Tissue Culture Isolation and Culture of PrOtOph$tS-INDRA K . V A S I I . A N D VlMLA VASIL Protoplast Fusion and Somatic Hybridization0n0SCHIEDER A N D INUKA K. VASIL Genetic Modification of Plant Cells Through Uptake of Foreign DNA-€. I. KADOA N D A . KIXINHOFS Nitrogen Fixation and Plant Tissue CultureKENNETHL. GILESAND INDRA K . VASIL Preservation of Gcrmplasm-LuNnsEv A. WITHERS lntraovarian and in Vitro Pollination-M. ZENKTELER Endosperm Culture-B. M. JOHRI,P. S. SRIVASTAVA, AND A. P. RASE The Formation of Secondary Metabolites in Plant Tissuc and Cell Cultures-H. BOHM Embryo Culture-V. RACHAVAN The FUtUre--&ORG MELCHERS
Electrophysiology of Cells and Organelles: Studies with Optical Potentiometric IndicaA N D PHILIP C. tOTS-JEFFREY c. FREEDMAN LAKIS Synthesis and Assembly of Membrane and Organelle PrOtCinS-HARVEY P. LODISH, WILI-IAMA. B R A ~ L ALAN L. L. SCHWAKTZ, GER J . A . M. STROUS, A N D ASHER ZILUERSTEIN The Importance of Adequate Fixation in Preservation of Membrane UltrastructureRONALDB. L u m c , A N D PAUL.N. McMILLAN Liposomes-As Artificial Organelles, Topochemical Matrices, and Therapeutic Carrier Systems-PETER NICHOLLS Drug and Chemical Effects on Membrane TransP O I ' - ~ I L L I A M 0 . BtKNVl INDEX
Supplement 13: Biology of the Rhizobiaceae
The Taxonomy of the [email protected] H. EILKAN Biology of Agrobacrerium rumefacien.c: Plant Interactions-L. W. MOORE A N D D. A . COOKSEY Agrobacterium tumefuciens in Agriculture and Research-FAwzi EL-FIKIA N D K E N N ~ TL.H GlLtS Suppression of, and Recovery from, the Neoplastic State-ROBERT TURGEON Plasmid Studies in Crown Gall TumorigenesisSUBJECT INDEX STEPHtN L. DF.I.I.APORTAA N D RICK L. PESANO Supplement 12: Membrane Research: Classic The Position of Agrobacterium rhizogenesJESSE M. JAYNESA N D GARYA. STROBEL Origins and Current Concepts Recognition in Rhizobium-Legume Symbioses-TERRENCE L. GRAHAM Membrane Events Associated with the Generation of a BhtOCySt-MART!N H. JOHNSON The Rhizobium Bacteroid State-W. D. SUTStructural and Functional Evidence of CooperA N D A. S. CRAIG TON, C. E. PANKHURST, ativity between Membranes and Cell Wall in Exchange of Metabolites and Energy between IMSANUt Legume and Rhizohium-JOHN Bacteria-MANFRED E. BAYER KONPlant Cell Surface Structure and Recognition The Genetics of Rhizobium-ADAM WROSI A N D ANDREW W. B. JOHNSTON Phenomena with Reference to SymbiosesIndigenous Plasmids of Rhizubium-J. DEENPATRICIAS. REISERT ARIEE, P. BOISTARD,FKANCINE CASSEMembranes and Cell Movement: Interactions of DELBART,A. G. ATHERLY,J. 0. BERRY, Membranes with the Proteins of the AND P. RUSSELL Cytoskeleton-JAMES A. WEATHERBEE
CONTENTS OF RECENT VOLUMES AND SUPPLEMENTS
47 1
Nodules Morphogenesis and DifferentiationWILLIAMNEWCOMB Mutants of Rhizobium That Are Altered in Legume Interaction and Nitrogen FixatioL. D. KUYKENDALL The Significance and Application of Rhizobium in AgriCUkUK-HAROLD L. PETERSON A N D THOMASE. LOYNACHAN
Cellular and Molecular Mechanisms of Intracellular Symbiosis in Leishmaniasis-K.-P. CHANG Symbiotic Interaction between Legionella pneumophila and Human Leukocytes-MARCUS A. HORWITZ Plastids-Past, Present, and FU~UR-JEAN M. WHATLEY
INDEX
INDEX
Supplement 14: Intracellular Symbiosis
Supplement 15: Aspects of Cell Regulation
Cellular Factors Which Modulate Hormone Responses: Glucocorticoid Action in PerspectiV+ROBERT w . HARRISON,111 Regulation of Genetic Activity by Thyroid Hormones-A. ABDUKARIMOV The Partitioning of Cytoplasmic Organelles at Cell Divisi0n-C. WILLIAMBIRKY,JR. Cell Cycle MUtant+wILLIAM L. WISS~NCER AND RICHARD J. WANG Formation of Glyoxysomes-J. MICHAELLORD MANN AND LYNNEM. ROBERTS Endonuclear Symbionts in Ciliates-HANS-DIEMitochondria, Cell Surface, and CarTER GORTZ cinogenesis-D. WILKIE,I. H. EVANS,V. Metabolic Interchange in Algae-Invertebrate EGILSSON,E. S. DIALA,AND D. COLLIER SymbiOSiS
Some Eco-evolutionary Aspects of Intracellular Symbiosis-F. J . R. TAYLOR Integration of Bacterial Endosymbionts in Amoebae-KwANG w. JEON Perspective on Algal Endosymbionts in Larger Foraminifera-JOHN J. LEE The Biology of the Xenosome, an Intracellular Symbiont-A. T. SOLW HECKEndosymbionts of Eupbtes-KLAUS
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