VOLUME 126
SERIES EDITORS Geoffrey H. Bourne James F. Danielli Kwang W. Jeon Martin Friedlander
1949-1988 1949-1984 19671984-
ADVl SORY EDlTORS H. W. Beams Howard A. Bern Dean Bok Gary G. Borisy Bharat B. Chattoo Stanley Cohen Rene Couteaux Marie A. DiBerardino Donald K. Dougall Charles J. Flickinger Nicholas Gillham M. Nelly Golarz De Bourne Elizabeth D. Hay Mark Hogarth Keith E. Mostov Audrey Muggleton-Harris
Andreas Oksche Muriel J. Ord Valdimir R. Pantic M. V. Parthasarathy Lionel 1. Rebhun Jean-Paul Revel L. Evans Roth Jozef St. Schell Hiroh Shibaoka Joan Smith-Sonneborn Wilfred Stein Ralph M. Steinman Hewson Swift Masatoshi Takeichi M. Tazawa Alexander L. Yudin
K.W. Jeon Department of Zoology The University of Tennessee Knoxville, Tennessee
M. Friedlander Jules Stein Eye Institute UCLA School of Medicine Los Angeles, California
VOLUME 126
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9 8 7 6 5 4 3 2 1
CONTENTS
Contributors
............................................................................................................
vii
Melanin-Concentrating Hormone: A General Vertebrate Neuropeptide Bridget I . Baker I. 11. 111 . IV. V. VI . VII .
Introduction ................................................................................................. Anatomical Distribution of MCH in Vertebrates and Invertebrates ............... Structure of MCH and Its cDNA Sequence ......................... ................. Biosynthesis of MCH .................................................................................... Structure-Activity and Molecular Modeling Studies ..................................... Physiology of MCH ...................................................................................... Conclusion: MCH, Past and Future ............................................................... References ...................................................................................................
1 3 14 20 21 30 40 44
Regulation of Synthesis and Transport of Secreted Proteins in Cereal Aleurone Russell L. Jones and John V . Jacobsen I. It. Ill. IV. V. VI . VII .
Introduction ....................................................... ...... Aleurone Tissue ........................................................................................... a-Amylase Genes and Control of Their Expression ...................................... Mechanism of Hormone Action .................................................................... lntracellular Transport and Exocytosis of Secretory Proteins ....................... The Role of Calcium ..................................................................................... Perspective ...................... ............................... References ...................................................................................................
49 49 54 63 70 79 83 84 V
CONTENTS
vi
Multiphasic Uptake Mechanisms in Plants Per Nissen ................... 1. .......................... .. ......................... II. ...................................... Ill. IV. Other Models ................................................... V. VI. .........................
89 91 93 122 126 128 131
Glycosylation in Intestinal Epithelium Douglas J. Taatjes and Jurgen Roth ................................... .... I. .......................................... ~. II. Ill. Methods Employed to Investigate Cellular Glycosylation Reactions .......................................... in Intestine .......................................... IV.
V. VI. VII.
nal Glycosyltransferases and Their Saccharide .......... ............,....................................... Agents on Intestinal Glycosyltran and Glycosylation .................................. ..................................... .. Differentiation and Glycosylation in Intestinal Cell Culture Systems ............. Concluding Remarks ........................... ................................. ................................................... References .,.,,, ..,......,
135 139 147 151 183 185 187 188
Physiological and Pharmacological Regulation of Biological Calcification Daniel C. Williams and Charles A. Frolik .............................................. I Introduction . ............. I I The Vertebrate Skeleton ............................................ Ill Physiological Regulation of Calcification in the Verteb IV Pharmacological Regulators of Calcification ............. ............................................ V. Summary and Conclusions ................ .......................................................... References ._................. Index .........................................................................................................................
195 199 217 246 262 263 293
Numbers in parentheses indicate the pages on which the authors' contributions begin.
Bridget I. Baker (l), School of Biological Sciences, University of Bath, Cleverton Down, Bath BA2 7AY, England Charles A. Frolik (195),Department of Biochemistv Research, Lily Research LaboratoriesJEli Lil& and Company, Indianapolisl Indiana 46285 John V. Jacobsen (49),Division of Plant Industry, CSIRO, Canberra,Australia Russell L. Jones (49),Department of Plant Biology, University of California, Berkelev, Berkeley, California 94720 Per Nissen (89),Department of Biology and Nature Conservation,Agricultural University of Norway, N-I432&NLH, Norway Jurgen Roth (135),lnterdepartrnental Electron MicroscopyJBiocenter, Universily of Basel, CH-4056 Basel, Switzerland Douglas J. Taatjes (135),lnterdepartmntal Electron Microscopy, Biocenter, University of Basel, CH-4056 Basel, Switzerland Daniel C. Williams (195),Bone Biology Research Group, Department of Connective Tissue and Monoclonal Antibody Research, Lily Research Laboratories, Eli Lily and Company, Indianapolis, Indiana 46285
vii
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INTERNATIONAL REVIEW OFCYTOLOGY. VOL. 126
Melanin-Concentrating Hormone: A General Vertebrate Neuropeptide BRIDGETI. BAKER School of Biological Sciences, Universiv of Bath, Cleverton Down, Bath BA2 7AY. England
I. Introduction We owe much of our current appreciation of the endocrine repertoire to comparative studies on lower vertebrates, which have shown that while identical or highly homologous hormones occur in all vertebrate classes, their physiological roles may differ. A hormone may be involved with several diverse functions even within one animal, but the emphasis on a particular function can shift during the course of evolution, so that homologous hormones in fishes and mammals may come to serve different roles in the two classes. It follows that a hormone or neuropeptide exerting an obscure (although not necessarily unimportant) effect in mammals might elicit a strikingly obvious response in lower vertebrates, which is the cause of its discovery. This possibility alone would justify the research of lower vertebrates, since such studies can draw attention to previously unsuspected messenger molecules in mammals. In no tissue is this possibility greater than in the brain and the pituitary gland. The neuropeptide discussed in the present chapter - the melanin-concentrating hormone (MCH) - was discovered because of its striking effect in teleost fish, causing them to become pale in color. Although this pigmentary response appeared to be restricted to bony fishes, it soon became clear that a similar molecule existed in the brains of several vertebrate classes. More recent determination of the structure of MCH from fish, rat, and human shows that the peptide has been highly conserved. It seems to be abundant in the mammalian brain but we still know very little about its physiological role there. On the other hand, several physiological effects of MCH have now been studied in fish. It is the aim of this chapter to review these data and to consider whether this knowledge can assist our search for the potential roles of MCH in mammals. A. DISCOVERY OF MCH There are many examples of physiological events that are regulated by two opposing hormones, an arrangement which gives more rapid and finer control than changing titers of a single hormone. The idea that rapid changes in skin color, seen when lower vertebrates such as amphibians and fishes move between 1
CopyrightQ I991 by Academic Press. Inc. All rights of reproduction in any form reserved.
2
BRIDGET I. BAKER
dark and pale-colored backgrounds, are controlled by two antagonistic hormones seemed uncontentious when it was first proposed (Hogben and Slome, 1931, 1936). it turned out, however, that the evidence from amphibians on which the proposal was based had been misinterpreted. Hogben and Slome ( 1936) observed that both frogs and the clawed toad Xenopus became extremely dark after cautery of the pituitary lobe known as the pars tuberalis. They concluded that this lobe secreted a paling hormone which opposed the darkening effects of the melanocyte-stimulating hormone (MSH) secreted by the intermediate lobe of the pituitary. The pars tuberalis lies closely applied to the floor of the hypothalamus and its destruction can cause damage to the hypothalamo-pituitary tract which restrains the activity of the pars intermedia. Etkin (1962) showed some years later, that such damage results in the hypersecretion of MSH and this is now believed to be the only pituitary hormone regulating color change in amphibia. There is no doubt that rejection of the dual hormone hypothesis for amphibians discouraged credence in a paling or melanin-concentrating hormone when the concept was subsequently applied to bony fish. In this group of animals, however, the evidence was quite different. It was realized early on that some teleosts responded to injections of teleost pituitary gland extract by exhibiting pallor, even though the extract contained MSH (Fries, 1943; Hewer, 1926; Weisel, 1948). The evidence for the existence of a melanin-concentrating hormone was fully documented by Pickford and Atz (1957) but at that time the few attempts to purify the hormone and separate it from MSH were unconvincing (addendum in Pickford and Atz, 1957). Its site of production was another enigma. Kent (1959) showed that MCH bioactivity was most abundant in the neurointennediate lobe of the minnow pituitary gland but the report by Healey (1948) that removal of this lobe did not prevent pallor when minnows were transferred to a pale-colored background suggested that the hormone was associated with the remaining anterior pituitary lobe. Based on his observation that MCH bioactivity could be detected in the catfish hypothalamus, Enami (1955) proposed that it was a neurohypophysial hormone. We know now that this is the correct conclusion and it would, of course, have explained the findings of other workers, but his observations could not be repeated by Kent and were generally ignored, except by Pickford and Atz (1957). interest in the control of color change waned in the 1960s and the existence of a melanin-concentrating hormone did not attract attention again until two decades later, when observations by Baker and Ball (1975) on color changes in the teleost Poeciliu seemed to be explicable only in terms of a dual hormonal control. This reawakened interest in the hormones involved. Subsequent studies by Rance and Baker (1979) and Baker and Rance (1983) showed that MCH bioactivity did indeed exist in the trout hypothalamus but that it was rapidly destroyed when the brain was extracted at pH 5.0; that its concentration in hypothalamic and pituitary tissue changed when fish were switched from white to
MELANIN-CONCENTRATING HORMONE
3
black tanks; and that similar bioactivity existed in the hypothalami of other vertebrates, including lampreys, amphibians, and mammals, suggesting that the peptide might have a widespread distribution. Encouraged by these observations, Kawauchi and co-workers (1983) purified and sequenced the melaninconcentrating hormone from the salmon pituitary gland and showed that it was a heptadecapeptide. Antibodies raised against this sequence were then used by Naito et al. (1985) to reveal the MCH-secreting neurons in the basal hypothalamus of salmonids, from which axons projected not only to the pituitary neural lobe but also up into the brain. Subsequent immunocytochemical studies and radioimmunoassays have confirmed the existence of MCH-like neurons in the hypothalamus of other groups of vertebrates (Section 11).
11. Anatomical Distribution of MCH in Vertebrates and Invertebrates
The distribution of MCH perikarya and fibers has been examined in rather few species but these studies suggest that the organization found in bony fish may differ slightly from that in other vertebrates. The teleost pattern will be discussed first. OF MCH IN TELEOST FISH A. DISTRBUTION
In teleost fish, most of the MCH activity is found in the neurohypophysial lobe where the peptide is stored, ready for release into the blood (Naito et al., 1985; Bird et al., 1989). The relative abundance of MCH in the brain and pituitary depends on a number of factors including the method used to measure the hormone. Bioassays for MCH, which depend on the ability of the peptide to concentrate the melanin granules in isolated scale melanophores, respond poorly to the MCH precursor molecule (Kawazoe et al., 1987a). They also give low values in the presence of MSH, the functional antagonist of MCH on melanophores (Baker, 1988a). This accounts for the apparently low pituitary : hypothalamic ratio of MCH reported both by Enami (1955) and by Rance and Baker (1979) in early studies using scale bioassays. Antibodies against the active peptide also appear to recognize the precursor, so that radioimmunoassays (RIAs) of hypothalamic extracts indicate a higher content of MCH than do bioassays (Kawazoe et al., 1987a; B. I. Baker, unpublished observations). The abundance of immunoreactive MCH (ir-MCH) in the brain and pituitary gland of trout depends on the age of the fish and the color of the tank in which it is kept (Table I), since this affects the rate of hormone synthesis and its release. Changes in the pituitary content of MCH in response to changes of background color have been observed in other species of fish, including the eel Anguilla anguilla (Powell and Baker, 1988), and the grass carp Ctenopharyngodon idellus (Bird and Baker, 1989).
4
BRIDGET I. BAKER TABLE I EFFECTOF REARING TROUTIN BLACKOR WHITE TANKSON MCH CONTENT OF HYPOTHALAMUS AND PITUITARY GLAND"
MCH Contenth
wt
n
tg)
Hypothalamus (Pg)
Pituitary (Pg)
Pituitary/ hypothalamic ratio
4 Months
Black stock B --f B 6 days B + W 6 days White stock W -+ W 6 days W 4 B 6 days
0.8 f 0.06 1.3 f 0.30 0.3 f 0.03 8.1 f 1.9 4.1 f 0.60 8.2 f 0.60
2.3 2.4 2.6 7.6 2.3 2.8
4.7 k 0.6 6.5 + 1.5 0.6 f 0.1' 15.9 f 5.0 2.5 f 0.2"Y 12.0 k 1.4
5.6 5.3 1.8 2.0 0.6 1.6
15 months
Black stock White stock
200
12
200
12
9.2 k 1.4 249.5 ?r 29
231.2 f 25.0 1088.4 f 93.0
25.0 4.0
Trout were reared from the egg stage onwards in off-white- or black-colored tanks (black or white stock). At 4 months, some fish were transferred to clean black or white test tanks and killed after 6 days ( B + B, B -+ W, W -+ W and W + B). Other stock fish were killed at 15 months. "Valuesare means f SEMS. All MCH concentrations in white-reared fish are significantly higher than in their black-reared counterparts. ' p < .01 compared with stock fish.
An early attempt to map the distribution of bioactive MCH within the brain (Baker and Rance, 1983) suggested that the peptide was concentrated in two sites; the majority was located in the ventral hypothalamus near the pituitary stalk, while about 30% was found in the dorsal thalamus, near the posterior commissure. This distribution was clarified by an immunocytochemical study of salmon and trout brains by Naito and co-workers (1985), using antiserum raised against synthetic salmonid MCH (sMCH). They showed that MCH is produced in magnocellular neurons located on the floor of the hypothalamus near the pituitary stalk, in the lateral region of the nucleus lateralis tuberis (NLTpI) (Fig. 1). While most axons are directed to the pituitary, others project dorsally into the brain and are found in the preoptic area and even more abundantly within the pretectal region, either side of the posterior commissure and habenular nuclei (Fig. 2), thus explaining the bioactivity detected in this region. The authors drew attention to the fact that neuroanatomical studies, using tracers such as horseradish peroxidase or [3H]leucineinjected into the eye, have shown this pretectal region receives fibers from the retina and pineal gland, and they proposed that the MCH neurones might receive photosensory information at this site. The
MELANIN-CONCENTRATING HORMONE
5
FIG.1. Sagittal section through the basal hypothalamus and pituitary gland of a trout, immunostained with anti-MCH and counterstained with light green. (A) Parasagittal section at the level of the nucleus lateralis tuberis, pars lateralis (NLTpl), showing numerous MCH perikarya with axons extending ventrally to the pituitary gland and dorsally towards the thalamus. (B) Near mid-sagittal section, showing axons ramifying through the neural tissue (NT) surrounded by the pars intermedia (PI) and extending also into the pars distalis (PD). (LR) Lateral recess of ventricle; (IR) infundibular recess; *, third ventricle. [From Naito et al. (1985). Immunocytochemical identification of melaninconcentrating hormone in the brain and pituitary gland of the teleost fishes Oncorhynchus keta and Salmo gairdneri.]
projection of MCH fibers into the brain implies that the peptide plays a neuromodulatory or neurotransmitter role, apart from its hormonal effect on color change. One may speculate that the release of MCH in the pretectal region could influence the transmission of photic information from the retinal or pineal nerves. The location of MCH immunoreactivity in the trout brain was confirmed by Bird er al. (1989) who showed using fluorescence irnmunostaining on cryostat sections, that MCH fibers also penetrated specific layers of the optic tectum. A comparable distribution of magnocellular MCH perikarya and fibers has been described in the Chinese grass carp, C . ideffus (Bird er af., 1989) and in the molly Poecifia faripinnu (Batten and Baker, 1988). Again, the MCH perikarya are concentrated in the lateral region of the NLT; the majority of fibers supply the pituitary while others project to various regions of the brain - the preoptic area and telencephalon, the pretectal area, lateral hypothalamus, and optic tectum. In all species, MCH perikarya are not totally restricted to the NLT; isolated
6
BRIDGET 1. BAKER
I amprey
trout
dogfish
frog
rat
\*-..
..
... a :
;.: .
,.,.:la:
MELANIN-CONCENTRATING HORMONE
7
cell bodies can sometimes be found near the lateral ventricular recess but their axonal projections have not been traced. The MCH neurons appear to be bi- or multipolar cells, sometimes with branching fibers. It has not been established whether the fibers projecting to the pituitary and the brain arise from the same perikaryon, although this seems likely, judging from the histological picture and from data showing that conditions influencing MCH release from the pituitary can also affect the concentration of MCH in the central regions of the brain (Section VI,B). The data suggest ;hat the availability of MCH for neuromodulatory functions in the brain can be influenced by conditions such as background color, although it does not show whether the release of MCH from the different terminals occurs simultaneously. Most of the axons entering the pituitary gland terminate in the neural lobe but a few penetrate the pars distalis (Naito et al., 1985; Batten and Baker, 1988), coursing near the corticotropes and other cells (Powell and Baker, 1987). No pituitary portal system exists in teleosts but pituitary cells are controlled by hypothalamic neurons which terminate on or near the secretory cells. The presence of MCH fibers in the pars distalis could indicate a potential regulatory effect on one or more pituitary cell types. MCH fibers in the neurohypophysial lobe concentrate near major blood vessels (Naito ef al., 1985) and also, in the case of the eel (Powell and Baker, 1988), terminate on the vascular basement membrane which separates the neural and intermediate lobes. This location permits MCH access to the general circulation and also to cells of the pars intermedia, which accords with the observation that MCH can exert an inhibitory effect on the melanotrope cells (Section VI,A). In some species such as the carp and Poeciliu, in which a basement membrane is less developed, or in the trout in which the fibers can penetrate the basement membrane, the MCH nerve terminals make direct contact with the pars intermedia cells (Batten and Baker, 1988; Powell and Baker, 1987; Naito et al., 1986b). Ultrastructural studies show that MCH is contained within membrane-bound, electron-opaque secretory granules which have a modal width and length of 90 x 120 nm in the carp and eel (Powell and Baker, 1987, 1988); they appear of similar size in Poecilia (Batten and Baker, 1988). In the eel, an increase in the secretory activity of the MCH neurones, caused by adapting the fish to a palecolored background, is accompanied by a decrease in the abundance and pack-
FIG.2. Diagrams of transverse and sagittal sections through the brain of different vertebrates showing the position of the MCH cell bodies (large dots) and their major axonal projections (small dots). Arrows on the sagittal sections indicate the level of the transverse section. Cells and axons are shown on one side only. AVA, area ventralis anterior thalamus; CH, cerebral hemisphere. CP, choroid plexus; LH, lateral hypothalamus; NID,nucleus infundibularis dorsalis; NIL,neurointermediate lobe; NLT, nucleus lateralis tuberis; NPO, preoptic nucleus; NTP, nucleus tuberculi posterioris; NSV, nucleus saccus vasculosus; OT, optic tectum; P, pituitary gland; pc, posterior commissure.
X
BRIDGET 1. BAKER
ing density of the MCH secretory granules at the nerve terminals, together with an increase in the number of synaptoid thickenings on the nerve terminal membrane (Powell and Baker, 1988). In the carp, about 40% of the MCH neurons are also immunostained by antiserum against a-melanocyte stimulating hormone (aMSH) and ultrastructural studies show that both immunoreactivites are located in the same neurosecretory granule (Powell and Baker, 1987). Immunocytochemical localization of other pro-opiomelanocortic (POMC) fragments in the carp brain suggests this immunoreactive a melanocyte-stimulating hormone (iraMSH) cannot be attributed to the coexpression of the POMC precursor in the MCH neurons, and thus does not signal the presence of conventional aMSH (Bird er al., 1989). More recent studies (Section 111) show that the immunostaining is probably attributable to the presence within the MCH precursor sequence of an epitope with minimal resemblance to the C-terminal region of crMSH. Colocalization of these two immunoactivities has not been observed in other teleosts, but occurs in several other vertebrates. Terlou and co-workers (1985) showed that the MCH neurons of trout were immunostained with antiserum raised against methyl-S-glucagon. It seems likely that this antiserum, too, recognizes some epitope within the MCH precursor. Computer sequence comparison (Clustral program) shows that the 15 C-terminal residues of glucagon share three identities and eight similarities with the N-terminal region of prohormone MCH (proMCH) (B. I. Baker, unpublished observations). B. DISTRIBUTION OF MCH IN NONTELEOSTEAN VERTEBRATES
In all nonteleostean vertebrates that have been studied, the MCH perikarya are located more centrally within the hypothalamus and it seems likely that the ventral situation of these neurons in teleosts is associated with their strong axonal projection to the pituitary gland. The hypothalamo-neurohypophysialcomponent is usually much less well developed in nonteleostean vertebrates, while the central projections may be very extensive. In our experience, the MCH neurons are best preserved in tissues that have been fixed by perfusion, and may be poorly preserved in whole brains that have been fixed only by immersion. Thus, even in the small brains of lampreys, good preservation of the cell bodies and their axonal tracts requires that the fixative be injected into the brain ventricles (unpublished observations). Additionally, i.c.v. injection of colchicine enhances the visibility of MCH fibers in mammals. Perfusion and colchicine have not always been used in the studies described below, and it is possible that the distribution of MCH fibers may be more widespread than the current descriptions suggest. I. Cyclostomes
Ln lampreys, MCH neurons can be detected even in young ammocetes (larvae) and become more apparent with increasing age. The cell bodies are lo-
MELANIN-CONCENTRATINGHORMONE
9
cated in a single group near the third ventricle of the hypothalamus, level with the pituitary neurointermediate lobe (Fig. 2). Many neurons are strikingly bipolar, one axon making contact with the ventricular cavity while the other is directed laterally into the brain (Fig. 3). The most prominent axonal tracts are seen in the lateral hypothalamus, projecting backward down the spinal cord and forward toward the optic chiasma and olfactory lobes, and some apparently crossing from side to side above the optic chiasma. Less prominent axonal tracts are directed dorsally toward the region of the habenular nuclei. Immunoreactive MCH fibers are evident in the neurohypophysial lobe of the ammocoete but are less obvious in this location after metamorphosis (unpublished observations). 2 . Elasrnobranchs
The distribution of MCH immunoreactivity has been studied in the dogfish Scyliorhinus canicula by Vallarino and co-workers ( 1989). Immunoreactive MCH cell bodies are found in three hypothalamic sites; the most prominent group is found in the nucleus saccus vasculosus (NSV), where they also react with crMSH antiserum. Fewer irMCH neurons, which apparently do not react with aMSH antiserum, are scattered through the nucleus tuberculus posterioris (NTF’) and the nucleus lateralis tuberis (NLT) (Fig. 2). In addition, some of the secretory cells situated around ventricular spaces within the pituitary gland also reacted positively with the MCH antiserum. No MCH fibers have been detected in the pituitary neural lobe, but they project throughout the posterior hypothalamus, and are found also in the dorsal thalamus and central grey of the mesencephalon.
3. Amphibia In this class of vertebrates, irMCH perikarya may again be located in more than one well defined hypothalamic nucleus (Fig. 2). In Rana ridibunda, the greatest number are found in the preoptic nucleus (NPO),where some make contact with the third ventricle (Andersen el al., 1986). Melanin-concentrating hormone neurons in this area are also immunostained by aMSH antiserum (Andersen el al., 1987). A few MCH perikarya are located in the area ventralis anterior thalami (AVA), lying dorsal to the preoptic nucleus while others, which are not immunostained with aMSH antiserum, occur in the dorsal infundibular nucleus (NID) of the posterior hypothalamus. Scattered nerve fibers are seen in the brain, and a well developed tract of fibers, probably arising from the preoptic region and dorsal infundibular nucleus, supplies the median eminence and the neurohypophysis where numerous fibers terminate around blood sinuses. These two destinations suggest that in R . ridibunda, MCH might serve both as a hormone and as a pituitary regulator. Within the neural lobe, ultrastructural studies show that the hormone is contained within electron-dense granules of about 80-90 nm diameter.
10
BRIDGET 1. BAKER
MELANIN-CONCENTRATING HORMONE
11
A different distribution of MCH cell bodies and fibers has been reported for R. temporaria and Xenopus laevis, in which MCH perikarya have been detected only in the postero-lateral hypothalamus. Fibers from this site project to the midbrain and forebrain bundle, but none were evident in the median eminence or neurohypophysis (Baker, 1988b; Batten et al., 1986). 4. Mammals
Following an early report of MCH-like bioactivity in the rat brain (Baker and Rance, 1983), Naito and co-workers (1988) confirmed that MCH bioactivity and immunoreactivity co-eluted during high-pressure liquid chromatography (HPLC) and thus were probably attributable to the same molecule. They and earlier workers noted, however, that rat MCH (rMCH) behaves as though structurally different from sMCH since it elutes later from HPLC columns, suggesting a more hydrophobic nature (Zamir et al., 1986a; Sekiya et al., 1988). The location of irMCH neurons in the rat brain has been mapped by immunocytochemistry using antiserum against sMCH (Skofitsch et al., 1985; Naito et al., 1988). The perikarya have an extensive distribution through the mid and caudal region of the dorso-lateral hypothalamus (LH) (Figs. 2 and 4A). At their anterior limit they are found dorsal to the paraventricular nuclei, and surround the fornix and the medial forebrain bundle. More caudally, they occur in the subzona incerta region, located above the ventromedial nuclei and medial and dorsal to the optic tract. From these perikarya arises an extensive network of fibers which project widely to most regions of the brain except the cerebellum, and into the spinal cord; a distribution that is confirmed by radioimmunoassays for MCH in dissected regions of the brain and spinal cord (Zamir et al., 1986b; Sekiya et al., 1988). A few MCH fibers project to the median eminence and posterior pituitary. When rat brains are examined by immunocytochemistryat intervals after birth, it is seen that the MCH neurons are poorly developed on postpartum day 1, with sparse granulation apparent in only a few cell bodies. The size of the MCH perikarya and the granulation they contain increases markedly during the first week, so that a greater number become evident. During the next three weeks the neurons increase only slightly in size but become more densely granulated and assume their adult appearance (Fig. 4B). Cell counts in serial sections through the hypothalamus of four rats revealed approximately 8,460 immunostained neurons/hypothalamus on day 4, 19,900 neurons on day 7, 23,960 neurons by day 24 and 22,080 neurons in an adult rat (M. Coles and B. I. Baker, unpublished observations). FIG.3. (A) Transverse section through brain of the lamprey Pefrumyzon mnrinus showing MCH cells with projections both to the ventricle and to the lateral hypothalamus (x 23). (B) Detail of neuronal cell bodies (x 122). LH, lateral hypothalamus; NIL, neurointermediate lobe; V, ventricle of brain.
12
BRIDGET I. BAKER
MELANIN-CONCENTRATING HORMONE
13
In the human, the only other mammal in which the MCH neurons have been located by immunostaining (Pelletier et al., 1987; Bresson et al., 1989), the cell bodies are discernible already in fetuses of 14 weeks (Bresson et al., 1989). In older individuals, the MCH system is said by some workers to be similar in terms of number, morphology, and topography of neurons to that found in the rat (Bresson et al., 1989). But other workers have reported that MCH cells are restricted to the periventricular region (Pelletier er al., 1987). The difference could perhaps be a problem of fixation, as mentioned earlier. As in the rat, axons extend to various regions of the brain, with a few MCH fibers projecting to the median eminence and pituitary stalk. Naito et al. (1988) reported that the MCH cells in rat are of two morphological types. One, located mainly around the zona incerta, is of medium size (15-25 pm diameter) and fusiform in shape; the other type, situated in the lateral hypothalamic and perifornical areas, is described as larger (20-25 pm) and multipolar. Ultrastructural studies show that, as in fish and amphibia, the MCH is contained within golgi-derived elyptical, dense-cored secretory granules whose diameter ranges from 80-110 nm to 90-250 nm, depending on the study (Pelletier et al., 1987; Naito et al., 1988). Nerve terminals of unknown type make synaptic contact with the MCH cell bodies; it is possible that some of these fibers are cholinergic since some MCH neurons in the rat contain acetylcholine esterase (Risold et al., 1989). In addition, MCH fibers make synaptic contact with dendrites of other MCH cells in both the rat (Naito ef al., 1988) and human (Bresson et al., 1989). In the human they are described as forming basketlike endings around both MCH-positive and MCH-negative perikarya. As in several lower vertebrates, the MCH neurons in the rat brain also react with antiserum against aMSH (Naito et al., 1986a; Fellmann et al., 1986). They correspond to the system of hypothalamic aMSH neurons that attracted attention previously because, in contrast to conventional MSH-secreting nerves in the arcuate nucleus, they fail to react with antisera against other POMC-derived peptides such as adrenocorticotropin (ACTH) or P-endorphin (Watson and Akil, 1980; Guy et al., 1980; Kohler et al., 1984; Khachaturian et al., 1986). Other workers subsequently found that the MCH neurons in rat also react with some antisera against human growth hormone-releasing factor (hGRF-37) (Fellmann et al., 1985, 1987) and corticotropin-releasing factor (CRF) (Antoni and Linton,
FIG.4. Immunoreactive MCH cells in the rat brain. (A). Vibrotome section (50 pm) through the hypothalamus of a colchicine-treated rat, showing MCH immunoreactive cells predominantly in the lateral hypothalamus above the fornix (F) and below the medial forebrain bundle (M). At this section thickness, the multipolar nature of the cells is apparent. (*), ventricle (courtesy of N. Naito). (B) Immunoreactive MCH cells (arrowed) in the developing rat. (a) 4 days; (b) 7 days; (c) 24 days; (d) adult, showing axon fibers. Rats were not treated with colchicine and at this section thickness (8 pm) the multipolar nature of the cells is not visible.
14
BRIDGET 1. BAKER
1979: Kawano er al., 1988). The epitopes responsible for this surprising wealth of co-existing immunoreactive molecules have recently been identified as sequences within the MCH precursor, bearing a lesser or greater resemblance to portions of aMSH. hGRF and CRF (Section 111). The observation that MCH and hGRF-37 antisera present a slightly different intracellular immunostaining pattern (Fellmann et al., 1987) might reflect the ability of the antisera to detect their epitopes at different stages of precursor processing. In the human, the MCH neurons are said not to immunoreact with aMSH antiserum but they do stain with antiserum against hGRF-37 (Pelletier et al., 1987; Bresson et al., 1986, 1989). C. INVERTEBRATES MCH-immunoreactive neurons have been described in the locust where they are found in the distal region of the optic lobe, bordering the junction with the ommatidia. and in the cerebral ganglion (Schoofs et al., 1988). The neurons in the optic lobes can also be immunostained by aMSH antiserum.
111. Structure of MCH and Its cDNA Sequence
A. STRUCTURE OF MCH The primary structure of MCH was first determined from purified pituitary extracts of the chum salmon Onchorhynchus keta (Kawauchi et al., 1983) and has since been determined for three further teleost species, the bonito Katsunionus pelamis and the eel Anguilla japonica by amino acid sequencing (Kawauchi, 1989), and the chinook salmon from its cDNA structure (Minth et al., 1989). The hormone is highly conserved in these fish. In all, it is a cyclic heptadecapeptide (Fig. 5) with an identical structure for the two salmonids and bonito and a conservative substitution to Asn' in the eel. More recently, the primary structure of rat MCH has been determined by traditional purification methods (Vaughan et al.. 1989), and that of the human was deduced from its cDNA sequence (Presse er al., 1990). Rat and human MCH are identical (Fig. 5 ) and, in spite of the fact that fish and mammals are separated by approximately 800 million years of evolutionary time, their MCHs are very similar. The central (ring) sequence, between the two cysteines, contains only a conservative mutation, that is, Leu in place of sMCH Val'. The C-terminal sequence also contains a single mutation of glutamine for sMCH GluI6, but the greatest changes involve the N-terminal region where there are two substitutions and two additional terminal residues (Fig. 5 ) . The biological significance of these changes is still uncertain. In teleosts, the ring structure is the most important for melanin concentration and the exocyclic sequences apparently potentiate this bioactivity,
15
MELANIN-CONCENTRATING HORMONE
Primary structure of MCH in different species 5
1
17
14
Eel’
H-~Thr*Met.Arg.Cys.Met.Val*Gly.Arg.Val.Tyr.Arg.Pro.Cys~Trp.Glu.Val*OH I
Salmonids
H-Asp.Thr*Met*Arg.Cys.Met.Val.Gly.Arg.Val.Tyr.Arg.Pro.Cys~Trp.Glu.Val.OH
and Bonito’.* Rat and human3
I
I
H-BSp.~Asp.~L8Y’Arg.Cys.Met.~Gly.Arg.Val.Tyr.Arg.Pro.Cys*Trp.~Val.OH
1
I
FIG. 5. Primary structure of MCH from three fish, the rat and human. Variant amino acids are underlined. I Kawauchi, 1989; * Kawauchi et al., 1983; Minth er al., 1989; Vaughan er al., 1989; Nahon er a/., 1989b; Presse er al., 1990.
perhaps by altering ring conformation and/or enhancing binding to the receptor site (Section V). Rat MCH has about 60% bioactivity compared with sMCH when tested on carp melanophores (unpublished observations) but no bioassay based on mammalian target tissue is yet available. OF TELEOST MCH B. PRECURSOR MOLECULE
Investigation of the molecular size of MCH in the fish hypothalamus initially suggested that, as for other peptides, it must come from a larger precursor molecule (Kawazoe et al., 1987b). This is confirmed by the MCH cDNA structure of two salmonid species, the chum salmon 0. keta (Ono et al., 1988) and the chinook salmon 0. tshawytcha (Minth el al., 1989). The gene for MCH mRNA in 0. keta contains just over 600 base pairs and lacks introns (Takayama et al., 1989). Salmonid fish are tetraploid and have two genes for MCH which are 86% identical. The MCH mRNA, whose cDNA sequences are shown in Fig. 6, each code for a pre-proMCH of 132 amino acids. An initiator MET is followed by a hydrophobic signal sequence and, assuming that signal peptide cleavage occurs after AlaZ4,this is followed by a prohormone of 108 amino acid residues in which MCH occurs at the C terminus (residues 116-132), separated from the rest of the precursor by a classical dibasic amino acid (Arg-Arg) cleavage site. The presence of other dibasic and monobasic amino acids within the precursor suggests that posttranslational processing might liberate additional molecules with potential biological activity. For instance, cleavage at Arglo0 could liberate a 13-amino acid peptide (101-113) which has been termed MCH-gene-related peptide (Mgrp) (Bird et al., 1990). In contrast to the mature MCH, which is identical in the two precursors, the remaining salmonid prohormone is less well conserved. Thus, depending on the species, Mgrp shows one or two amino acid variants (Asp or Gly at position 103 in both salmonids; Asn or Ser at position 107 in 0. keta). The two pre-proMCHs from the same species share 80% (chum) or 81% (chinook) amino acid
CHINOOK ppMCH CHUM p p M C H CHUM cDNA 1
1 10 20 M e t Arg H I S Ser V a l L e u Ser I L E S E R Phe A l a VAL A l a L e u Phe L e u G l u C y s T y r Thr ATG AGA CAC T C T G T C C T C T C C A T C T C C T'lT GCC GTG GCA CTT ? T C T T G GAG T G C T A C ACA
CHUM c D N A 2 CHUM p p M C H CHINOOK ppMCH
ATG AGA GAC T C G G T C C T C T C C G T C A T C TIT G C C T T G GCA CTT TTC T T G GAG T G C T A C ACA M e t A r g A S P S e r V a l L e u S e r V A L ILE Phe A l a L E U A l a L e u Phe L e u G l u C y s T y r Thr
C H I N O O K pp . MCH CHUM p C H CHUM c g N A 1
PRO 30 40 21 P r o S e r THR A l a I l e S E R ILE G l y Lys M e t ASP A s p VAL A l a Leu G l u G l n A s p Thr L e u
CHUM c D N A 2 CHUM p p M C H CHINOOK ppMCH
CCG T C C ATG GCG A T C CCG ATG GGC AAG ATG GAG GAC ACA GCC l T G GAG CAA G A T ACC C T A P r o Ser MET A l a I l e PRO MET G l y Lye M e t G L U A s p THR A l a Leu G l u G l n A s p Thr L e u
CHINOOK ppMCH CHUM p p M C H CHUM c D N A 1
50 ARG 60 A s p Ser L e u L e u SER VAL G l u V a l SER Q L U A S N SER P r o A s p S e r V a l A r g G L Y ARG Ser GAC T C C C T A C T G A G T GTA GAG G T G T C T GAA AAC AGC C C T G A T T C A G T C AGA GGC AGG AGC
CHUM c D N A 2 CHUM p p M C H C H I N O O K ppMCH
GAC T C T C T A C T G AAC GAA GAG GTG G C C G A T AAA AAC C C T G A T T C A G T C AGA AGC GGG AGC A s p Ser Leu L e u A S N Q L U G l u V a l A L A ASP LYS A S N P r o A s p Ser V a l A r g SER G L Y Ser
CHINOOK ppMCH CHUM p p M C H CHUM c D N A 1
61 70 80 Ser Lys I l e V A L L E U L e u A l a A s p Ser G l y L E U T r p MET A s n L e u A s n A r g G l y L e u P r o T C C AAG A T T G T C 7 1 G CTG GCA GAC T C T GGC CTG TGG ATG AAC C T G AAC AGA GGA C T T C C T
CHUM c D N A 2 CHUM p p M C H C H I N O O K ppMCH
T C C AAG A T C A T C GTG T T G GCA GAC T C A GGC ATG TGG AAG AAC C T G AAC AGA GGA ClT C C T Ser Lys I l e ILE V A L L e u A l a A s p Ser G l y MET T r p L Y S A s n Leu A s n A r g G l y L e u P r o
*
t.
L
CCG T C C ACG GCG A T C T C C A T T GGC AAG ATG GAC G A T G T C G C C T T G GAA CAA G A T A C T C T C
* *
*
* ***
*
*
t
*
t
41
**
* *
*
*
* *
*
*
t
*
*
*
*
CHINOOK ppMCH CHUM ppMCH CHUM cDNA 1
81 90 GLN P H E Q r Lys Leu ARQ Ala Ala Ala Ala Gly PRO Asp Arg Ala Leu Thr Leu Asp Arg ARG TTC TAC AAG CTG AGA GCT GCA GCC GCC GGG CCT GAC AGA GCC CTG ACT CTG GAC CGC AGA
CHUM cDNA 2 CHUM ppMCH CHINOOK ppMCH
CTC TAC AAG CTG AAA GCT GCA GCT GCA GGG CTT GAC AGA GCC CTG ACC CTG GAC CGC AGA LEU Tyr Lys Leu LYS Ala Ala Ala Ala Gly LEU Asp Arg Ala Leu Thr Leu Asp Arg ARG
*
h
*
*
*
L A
CHINOOK ppMCH CHUM ppMCH CHUM cDNA 1
Glu Ala GLY Gln Asp Leu SER Pro Ser Ile Ser Ile Val Arq Arq Asp Thr Met Arg Cys GAG GCT GGC CAG GAC CTA AGC CCC AGC ATC TCC ATC GTC AGG AGG GAC ACC ATG AGG TGT
CHUM cDNA 2 CHUM ppMCH CHINOOK ppMCH
GAG GCT GAC CAG GAC CTG AAC CCC AGT ATC TCC ATT GTC AGG AGG GAC ACC ATG AGG TGC Gly Ala ASP Gln Asp Leu ASN Pro Ser Ile Ser Ile Val Arg Arg ASP Thr Met Ara Cys SER L A
CHINOOK ppMCH CHUM ppMCH CHUM cDNA 1
Met Val Gly Arg Val Tyr Arg Pro Cys Trp Glu Val end ATG GTG GGA AGG GTG TAC CGA CCC TGC TGG GAG GTG TAC
CHUM cDNA 2 CHUM ppMCH CHINOOK ppMCH
101
110
*
*
120
*
*
121
*
130
*
*
ATG GTG GGA AGG GTG TAC CGG CCT TGC TGG GAA GTG TAC Met Val Glv Ara Val Tyr Ara Pro CYS Trp Glu Val end
FIG. 6. The nucleotide and amino acid sequence of the two chum salmon (Onochorhynchus keral pre-proMCHs (ppMCH; Ono et al., 1988). Residues which differ in chinook salmon MCH (Minth et al., 1989) are indicated above or below the rows. The predicted signal cleavage is indicated by an arrow. Dibasic, putative cleavage points are indicated by arrowheads. Asterisks show nucleotide mutations. The MCH sequence is underlined.
18
BRIDGET I. BAKER
identities, with 21 of the 26 residue changes in chum salmon and 20 of the 25 changes in chinook salmon involving single nucleotide mutations. Comparing chum with chinook pre-proMCHs reveals a much more striking level of homology (97-99%) between the species than between the two precursors from the same species, indicating that tetraploidy developed before the genus underwent speciation.
C. PRECURSOR MOLECULEOF MAMMALIAN MCH Mammalian MCH shows 76% amino acid identity with the salmonid peptide (excluding the two N-terminal residues of mammalian MCH) but the remaining prohormones (excluding signal) show only 20% identity, the homologous residues tending to be concentrated nearer the C terminus (Fig. 7) (Nahon et al., 1989b; Fellmann et al., 1989). As with fish, several potential proteolytic cleavage sites occur in the precursor so that a number of small peptides could be released. Following the system suggested by Tatemoto and Mutt (198 1 ) for naming neuropeptides, two of these fragments have been named NEI and NGE, viz. pre-proMCH ( I 3 1-144) is neuropeptide-glutamic acid-isoleucine (NEI), and pre-proMCH ( 1 10-128) amide is neuropeptide-glycine-glutamic acid (NGE) (Fig. 7). Fragment NEI is the more highly conserved, showing 30% identity (64% similarity) with salmonid Mgrp. Human pre-proMCH shares 81% identity with the rat precursor (Fig. 7). Both precursors contain 165 residues with the variant amino acids located mainly in the middle region of the molecule. The sequences from residue 114 onwards, which include MCH, NEI and most of the NGE peptide, are identical for the two species (Presse et al., 1990). The structure of the rat proMCH sequence was of considerable interest when it was elucidated because, quite apart from comparison with the teleost MCH precursor. it revealed why the MCH neurons are immunostained by antisera against several other neuropeptides; aMSH, hGRF-37, and CRF (Section 11,4). Workers from the Salk Institute (Nahon et ul., 1989b) showed that rat proMCH has epitopes in common with these other peptides which are recognized by antisera even though the sequence similarities may be very slight. By preabsorbing the antisera with synthetic fragments of rat proMCH, they showed that immunostaining with aMSH antiserum is attributable to the sequence Pro-Ile-NH2 derived from Pro-lle-Gly at the C terminus of peptide NEI. No other sequence similarity between proMCH and aMSH is apparent, and this very slight resemblance between the two molecules emphasizes the inherent pitfalls when interpreting immunostaining results. CRF (24-41) shares six out of a stretch of 18 residues spanning NGE + NEI but immunoabsorption tests suggested that the staining with their CRF antiserum was not attributable to these residues but to
19
MELANIN-CONCENTRATING HORMONE
I
SALMON RAT
1
HUMAN
1
-
Sianal Peptide
I
MRDSVLSVIFALALFLECYTPSMAI * : * ** * * :* MAKMSLSSYMLMLAFSLFSHGILLSASKSIRNVEDDIVFNTFRMGKAF . . .... . . ....* : * ; . . . . : * : : : : : : : : : : : : * * . * : * : : : : : : * : : * : MAKMNLSSYILILTFSLFSQGILLSASKSIRNLDDDMVFNTFRLGKGF
25 48 48
Signal Peptide
SALMON
26
PMGKMEDTALEQDTLDSLLNEEVA----DKNPDSVRSGSSKIIVLADS 69
RAT
49
QKEDTAERSVVAPSLEGYKNDESGFMKDDDDDK'ITKNTGSKQNLVTHGL 96
HUMAN
49
QKEDTAEKSVI APSLEQYKNDESSFMNEEENKVSKNTGSKHNFLNHG~ 96
.* * :*: * . * .. .. .. .. .. .. . * . . * . . . . . . . . . . .* . . * * * * : *
*
*
.. ..
*:
;:::::*:**
MarD
SALMON
70
RAT
97
HUMAN
*
...
v'
G~LNRGLPL---YKLKAAAAFLDRALTLD~E-ADQDLsPs * : * * : * * : : * : * * : * I sI: 1 13 * * :*: PLSLAVKPYLALKGPAVFPAENGVQNTESTQEKREIGDEENSAKFPIG 144 : : : : * : .. . .. ..... .*. . . ....... .. .. .. .. ....... .. .. .. .. ....... .. .. .. .. ......... .. .. 144
NGE
SALMON
114
RAT
145
HUMAN
145
RR--DTMRCMVGRVYRPCWEV
.. .. .. * : : : * : : : : : : : : * . RRDFDMLRCMLGRVYRPCWQV ..................... RRDFDMLRCMLGRVYRPCWQV
NE I
I
132 165 165
MCH FIG.7 Comparison between the pre-proMCHs from chinook salmon (Minth ef al., 1989). rat (Nahon er al., 1989b), and human (Presse et al., 1990).Gaps (-) are introduced to maximize homologies between sequences. (:), Identical amino acids of different species; (*), conservative substitutions; (A),putative proteolytic sites. Putative neuropeptides, NGE and NEI, which might be released from the mammal precursor, and Mgrp (MCHgene-related-peptide) from salmon, are also indicated. ~~
the same epitope recognized by aMSH antiserum, viz. the C-terminal bulky aliphatic amide of NEI. Lastly, the sequence apparently responsible for immunostaining with hGRF antiserum seems to be an 8-residue stretch in NGE, which shares five identical residues with hGRF (30-37). Within hGRF (30-37), residue Ser34corresponds to pre-proMCH SerIz5;it is probably significant that this single residue is not conserved between the human and rat GRF sequences and that antiserum against rGRF does not recognize the MCH precursor.
20
BRIDGET 1. BAKER
D. GENERAL COMMENTS ON MCH PRECURSOR The fact that the MCH neurons of salmonids are not immunostained by aMSH antiserum (Bird ef al., 1989) is probably explained by the sequence difference between the C-terminal region of NEI in rat (Pro-lle-Gly) and its homologue in the salmonid proMCH (residues 1 11-1 13, Ser-He-Val, Fig. 7). The Cterminal amide group, which is derived from the neighboring glycine residue and is a structural requirement for most antisera against aMSH, cannot be formed in salmonids. In the carp, elasmobranchs, and R. ridibundu at least some MCH cell bodies are immunostained with aMSH antiserum and it is possible that posttranslational processing of their proMCH liberates an appropriately amidated C-terminal region. It remains to be explained, nevertheless, why not all of the MCH perikarya in these species react with the aMSH antiserum. It could be that the unreactive MCH neurons produce a slightly different molecular form of proMCH or that posttranslational processing is different in these neurons. For instance, they may lack the relevant amidating enzymes. In the carp, not only the MCH perikarya, but also their nerve terminals in the neurohypophysis differ in whether or not they react with aMSH antiserum (Powell and Baker, 1987). so the distinction between the two neuronal types is unlikely to be simply a question of the rate of posttranslational processing. Many neuropeptides are expressed in nonneuronal tissues such as the gut or gonads, as well as in the central nervous system. The list includes such peptides as vasopressin, oxytocin, POMC. angiotensin 11, cholecystokinin (CCK) and many others. it is therefore interesting that Northern blot analysis, using both polynucleotide and oligonucleotide probes on mRNA extracted from a wide variety of tissues (pituitary gland, adrenal medulla, eye, testis, pyloric caeca, intestine, liver, kidney, and heart), has failed to provide evidence that MCH is expressed outside the hypothalamus (Ono et al., 1988; Minth et al., 1989).
IV. Biosynthesis of MCH The biosynthesis of MCH from its precursor has been studied only in the rainbow trout Uncorhynchus niykiss (formerly Salmo guirdneri; Bird ef al., 1990). In this species, the MCH perikarya are located as a group on the floor of the hypothalamus and radioactively labeled precursors, such as [3sS]cysteine,can easily be injected into their vicinity. If fish are killed at intervals after the injection, the labeled MCH precursor and its products can be extracted from the hypothalamus and pituitary gland, immunoprecipitated and separated by electrophoresis on sodium dodecyl sulfate (SDS) gels. In practice, the injected pool of labeled precursor becomes depleted quite rapidly, presumably due to diffusion or incorporation into other proteins, so that the MCH neurons behave as though they had
MELANIN-CONCENTRATINGHORMONE
21
received a long pulse of labeled precursor. Such studies show that one hr after injection, the label is associated largely with two high-molecular weight proteins (Fig. 8); these then decline in abundance, presumably as a result of conversion to the smaller molecular weight labeled-products which increase progressively in abundance. Melanin-concentrating hormone itself is barely detectable 1 hr after injection but forms a significant peak at 2 hr and is the predominant labeled product within the hypothalamus by 4 hr. The molecular identity of the intermediate-sized precursors has not yet been confirmed. More products are formed than can be accounted for by cleavage at only the dibasic residues in the precursor, and it is possible that some of the single Arg or Lys residues or other amino acids are used as cleavage sites, Separation of the MCH-immunoreactive products on Sephadex G50 reveals a labeled peak in the predicted position of preproMCH (101-132), i.e., MCH plus the preceding fragments (Mgrp, equivalent to rat NEI). This suggests that cleavage may occur first at Arg-Arg (99-100) and subsequently at Arg-Arg (1 14-1 15) but the low abundance of this immediate precursor of MCH implies that it is short-lived and processed rapidly to release mature MCH and the tridecapeptide (Mgrp). Most of the posttranslational cleavage presumably occurs within the secretory granules as they are translocated down the axons to the neurointermediate lobe. Judging from labeling studies, this passage takes 4 hr or more. Separation of MCH from its precursor by gel-permeation on Sephadex G75 shows that proMCH forms less than 1% of the total MCH-like material stored in the neurointermediate lobe. The change in rate of MCH synthesis following transfer of fish from a pale to a dark background and vice versa has been examined in preliminary experiments, making use of the observation that isolated hypothalamic fragments continue to incorporate ['sS]methionine into MCH precursors and process them to mature MCH in v i m (B. I. Baker and D. J. Bird, unpublished observations). Incorporation is doubled within about 7 days after fish are transferred from black- to white-colored tanks, and is enhanced further if fish are also given a daily stress. Incorporation is reduced at a similar rate when white-adapted fish are moved to black tanks. Processing of precursor to mature MCH also continues in vitro but the rate at which this occurs appears constant and unaffected by the rate of precursor synthesis.
V. Structure-Activity and Molecular Modeling Studies A peptide hormone interacts with its receptor in such a way that receptor conformation is changed and a chain of intracellular events, often referred to as signal transduction, elicits the target cell's response. Not all regions of the hormonal molecule interact with the receptor, but they may be important for other
P
MELANIN-CONCENTRATING HORMONE
23
reasons; they may assist interaction with the receptor by enhancing binding to the receptor site, influence the quaternary structure of the hormone, or protect it against degradation in the blood. It is useful to understand the role of the various amino acid residues in the hormone should one wish to modify molecular structure - e.g., by the addition of 1251 or during synthesis of tritiated analogs for binding studies - and still retain biological potency. Some insight into the importance of specific residues can be gained from structure-activity studies in which the bioactive potencies of various hormonal fragments and analogs are compared with the native molecule. Several studies have compared the in vitro effect of fragments and analogs of MCH on melanophores from different teleosts and tetrapods. They have been concerned mainly with the importance of the cyclic configuration of MCH, the effect of the N- and C-terminal exocyclic sequences, and the influence of a few specific residues. ON MELANOPHORES A. MCH HASaMSH-LIKEACTIVITY
The groups headed by Hadley and Hruby in Arizona and by Castrucci in Brazil have contributed much to our understanding in this field. They were the first to report that MCH has no melanin-concentrating effect when tested on tetrapod melanophores, but instead exhibits weak melanin-dispersing activity, being about 600-fold less potent than the melanocyte-stimulating hormone, aMSH, on frog Rana pipiens skin and 300 times less potent when tested on the skin of Anolis carolinensis (Wilkes et al., 1984a). The melanin-dispersing activity of MCH has been confirmed by other workers, although the reported potency of MCH relative to aMSH varies in different studies (Baker et al., 1985a; Ide et al., 1985). When tested on mammalian melanoma cells, sMCH similarly shows aMSH-like effects, stimulating tyrosinase activity and melanin synthesis (Baker et al., 1985a). Several observations suggest that the melanin-concentrating and dispersing activities are elicited by different sequences within the MCH molecule. Thus, when MCH is heated with 0.1 M NaOH (a treatment which racemizes aMSH and potentiates its activity) its aggregating activity is destroyed, while its dispersing activity is either enhanced (Baker et al., 1985a) or not affected (Matsunaga et al., 1989b). Analogs of MCH with truncated N- or C-terminal exocyclic sequences have diminished aMSH-like activity when tested on amphibian melanophores, but their melanin-aggregating effect on Synbranchus skin is not necessarily affected FIG.8. I n vivo incorporation of radioactive methionine and leucine into proMCH by the trout. Labeled amino acids were injected into the NLT, around the MCH perikarya. Trout were killed 1 hr, 2 hr, and 4 hr later, the basal hypothalamus was extracted, and immunoprecipitated MCH-related products were separated by SDS gel electrophoresis. [From Bird et al. (1990)l
24
BRIDGET 1. BAKER
(Hadley et al., 1987; Matsunaga et al., 1989b; Castrucci et al., 1989). Conversely, other MCH analogs in which the size of the ring structure, MCH (5-14), is diminished by shifting Cys5 to positions nearer the C terminus, show a markedly diminished MCH-like activity on Synbranchus skin which is dissociated from their melanin-dispersing activity (Lebl et al., 1988). In the case of the analog [Ala5,Cys'o]-MCH,for example, melanin-dispersing activity on frog skin is the same as for native MCH while its melanin-aggregating activity on eel skin is undetectable; another MCH analog, [AlaS,Cys'O]-MCH (5-17) which also has undetectable aggregating activity on eel skin, has diminished dispersing activity compared with sMCH when tested on Rana skin but is 7-fold more potent than MCH on Anolis skin (Castrucci et al., 1989), emphasizing the different requirements of MSH receptors in different species. This point might be important when interpreting the effects of MCH on teleosts. The tests for melanin dispersion described above used tetrapod melanophores but there is evidence that MCH may also cause melanin dispersion in teleosts. When tested on melanophores of Synhranchus, increasing doses of MCH cause progressive melanin aggregation (lo-" M ) followed by progressive melanin dispersion ( M), producing a bell-shaped dose-response curve (Castrucci et al.. 1987: Hadley et al., 1987). In other systems, a curve of this shape is often attributed to loss of receptor sensitivity at the higher hormone concentrations, but this seems not to be the case here. The melanin-dispersing effect of high peptide concentrations is not seen if calcium is absent from the incubation medium (Oshima et al., 1985; Castrucci et al., 1987) and since calcium is necessary for aMSH to bind to its receptors and cause melanin dispersion (Fujii and Miyashita, 1982; Eberle, 1988), it has been proposed that the melanindispersing effect of MCH results from its interaction with the aMSH receptor. The MCH analog [AlaS,Cys'"]-MCHsimilarly causes darkening of MCH-paled eel skin and requires extracellular calcium to do so (Lebl et al., 1989). The fragment MCH (5-17), which is equipotent with MCH in causing melanin aggregation in Synhranchus skin, does not show "autoantagonism" at high concentrations, suggesting that the MCH ( 1 4 ) sequence is of particular importance for aMSH receptor stimulation in teleost skin (Hadley et al., 1987; Castrucci ef al., 1987, 1989). So far, "autoantagonism"has been reported only for the eel Synbranchus, and only with high concentrations of MCH, but it is possible that MCH exerts some melanin-dispersing action at physiological concentrations in this and other species, since its melanin aggregating potency and that of MCH (1-14) can be enhanced up to 10-fold if tested in medium lacking calcium (Hadley etal., 1988; Visconti ef al., 1989; Castrucci et al., 1989). How MCH interacts with aMSH receptors is so far unexplained; the two hormones show no obvious similarities in their primary structure.
25
MELANIN-CONCENTRATING HORMONE
B. MELANIN-CONCENTRATING ACTIVITY OF MCH The data on the melanin-dispersing effect of MCH and the influence of calcium have been discussed because they have a bearing on the interpretation of work described below. The structure-activity tests with different MCH fragments, using melanophores from different teleost species, have been done in medium containing calcium. They appear to show that the molar potencies of sMCH, MCH (5-17), MCH (1-14), and MCH (5-14) vary in different species (Hadley et af., 1987) (Table 11), which could be interpreted to suggest a difference in structural requirements of their MCH receptors. However, should the melanin-dispersing effect of MCH vary depending on the species, this might contribute to the different aggregating potencies of MCH and some of its fragments. For this and other reasons, interpretation of the various structure-activity studies, done mainly on the eel Synbranchus by Hadley and Castrucci's groups, the Chinese grass carp Ctenopharyngodon by Baker and co-workers, and tilapia by Kawazoe and Kawauchi, is not easy, and only a few general points will be made here. The ring sequence MCH (5-14) is probably the most important for signal transduction. Although removal of the exocyclic sequences (arm structures) causes approximately 100- or 1000-fold loss of potency on eel and carp melanophores, respectively (Table 11), the combined exocyclic sequences, MCH (1-4, 15-17) or MCH (1-4, Aha, 15-17) (where Aha is a spacer molecule) have negligible bioactivity when tested alone (Brown et af., 1990; Baker el al., 1990). Both N - and C-terminal regions apparently serve to enhance the potency of the
TABLE I1 MELANIN-CONCENTRATING POTENCYOF CYCLICsMCH FRAGMENTS ON MELANOPHORES OF DIFFERENTTELEOST SPECIES MCH MCH (5-17) MCH (1-15) MCH (1-14) MCH(1-14) MCH (2-14) MCH (5-14)
Svnbranchus
CaID
Poecilia
TilaDia
100 10 0 h
100
100
looh 10 0 R
-
100 I8od -
-
4d
10 0 h
1O h
-
7R
-
-
Ih
0.lC
-
~
"Valuesare % potency, where MCH is 100%. "Hadley er al. (1987), Castrucci et al. (1987). 'Baker er al. (1990). Wscontin et al. (1989). 'Kawauchi and Kawazoe (1988). Watsunaga et a/.((1989a). PCastrucci et al. (1989). "Kawazoe et al. (1987a).
1'
loo' 100'
1 ow
26
BRIDGET I. BAKER
ring structure, possibly by changing its conformation or by helping to bind the peptide to its receptor site. The individual exocyclic residues vary in importance. Thus, Matsunaga and co-workers (1989a) found that TrpI5 was crucial for interaction with Synbranchus melanophores, MCH (5-1 5) being equipotent with sMCH but MCH (5-14) having only 1% bioactivity. MCH (1-14) also had full potency in some studies on Synbranchus melanophores (Castrucci e f al., 1989) (only 10% potency in other studies; Hadley et af., 1987; Matsunaga et af., 1989a.b) but removal of Asp' reduced potency to 7% that of sMCH, while removal of all four N-terminal residues, to give MCH (5-14), further reduced potency to 1.4%. Rat MCH which has, among other differences, four residue changes in the N-terminal portion (Fig. 5 ) has approximately 60%potency when tested on grass carp (B. I. Baker, unpublished observations). Molecular modeling studies suggest that the exocyclic sequences of sMCH affect the shape of the ring structure; they increase the local conformational flexibility of the two Cys residues and influence interactions between side groups of residues located near the Cys bridge within the ring (Paul et al., 1990). The conformation they induce may improve the interaction of the ring structure with the receptor molecule. It is probable, too, that the side arms enhance potency by helping to bind the peptide to its receptor site. Thus, the bioactivity of [C~S(AC~)'~]-M (9-14) C H is enhanced from
MELANIN-CONCENTRATING HORMONE
27
TABLE 111 POTENCY OF SOME CYCLIC AND LINEARANAL~GS OF SMCH AND SMCH (5-14y Analog sMCH [S-O-Met3. 6]-MCH [Pra3.6]-MCH [Nv~~.~]-MCH [lodinated I-Tyr"]-MCH [NO,-Tyr"]-MCH [DHCH(Arg)4'9.'2]-MCH [NPS-TP'~]-MCH MCH (5-14) [Phe"]-MCH (5-14) [D Ala8]-MCH (5-14) [D Ala8, Phe"]-MCH (5-14)
%Potency 100.0 88.0h, 10.0'.
15.0h 37.0b 0.25b 0.lC 0.1' 100.0' 0.14 0.025d 0.008'9 0.00045d
Linear MCH' [ C ~ S ( A C ~ ) ~ - ' ~ ] - (1-17) MCH
0.90d, ND (< 0.05y
[Cy~(Acm)~.'~]-MCH(5-17)
0.02'9 0.0002d ND (< O . m l ) n 0.03d
[Cy~(Acm)~, I4]-MCH (5-14) [ C ~ S ( A C ~ ) ' ~ ] - M(9-14) CH [Cys(Acm)I4]-MCH (9-17)
" A m , acetamidomethyl; ND, not detectable; Nva, norvaline, h a , propargylglycine. hBaker ef al. (1985a), using the carp scale bioassay. 'Kawazoe er al. (1987a). using the tilapia scale bioassay. dBaker et al. (1990). using the carp scale bioassay. 'Compare with cyclic peptides in Table 11.
1987a). The molecule [Tyr(NO,)'l]-MCH also has drastically reduced bioactivity (Kawazoe et al., 1987a). It seems likely that the introduction of a large iodine atom or NO, group into the ring distorts the overall shape of the molecule. The replacement of Tyr" with Phe, which cannot form hydrogen bonds with Met6 or Cys5, but which would not otherwise affect molecular shape, only reduced the bioactivity of MCH (5-14) about 4-fold (Table 111) (Baker et al., 1990). The importance of the cyclic conformation is suggested also by the reduced activity of linearized MCH molecules, in which disulphide bridge formation is prevented by acetamidomethyl (Acm) groups on the Cys residues (Baker er al., 1985a; Kawazoe er af., 1987a). Linear MCH (5-14) has the same general conformation as cyclic MCH (5-14) in molecular modeling studies, although it can also adopt some conformations not available to the cyclic form (Paul et al., 1990). Bioassays show that the potency of linearized MCH, MCH (5-17), and MCH (5-14) is reduced 100-fold or more relative to the cyclic structures (Table 111), although it is possible that the presence of the relatively bulky Acm side-
MELANIN-CONCENTRATINGHORMONE
29
FIG.9. (continued)
groups might themselves interfere with receptor interaction. As already discussed, contraction of the ring structure, as in [Ala5Cys7]-MCH similarly has drastic effects on the melanin-aggregating effects of MCH, while not necessarily affecting the dispersing effects of the molecule (Lebl et al., 1988; Castrucci et al., 1989). The conclusions to be drawn from all this evidence are that the “message”sequence is contained in the ring structure of MCH, and its potency is enhanced by the presence of the side arm structures; that the correct conformation of this ring is important and may be influenced by the side arms; and that the sequence between Valtoand Cysl4 which is apparently conserved during evolution, might be particularly crucial, although the evidence for this is still tentative and tests with additional analogs are needed for confirmation. The sequences which induce
30
BRIDGET 1. BAKER
melanin dispersion in tetrapods, and probably also in teleosts, involve both exocyclic regions but these apparently interact with receptors for aMSH, not for MCH. Further details can be obtained from the several articles cited here.
VI. Physiology of MCH A. MCH AS A COLOR-CHANGE HORMONEI N FISH
Since MCH was discovered by virtue of its effect on the skin pigment cells in fishes, it is hardly surprising that its role as a physiological regulator of color has received considerable attention. Evidence that the molecule does indeed function as a color-change hormone was initially circumstantial. It depended on observations that the concentration of MCH in the pituitary gland and brain was relatively low in pale-adapted fish (in which hormone release was expected to be intense), but accumulated when such fish were transferred to a dark background (in which it was anticipated that hormone release would be suppressed while synthesis persisted, at least for a while) (Rance and Baker, 1979; Barber et ul., 1987; Powell and Baker, 1988). The development of a solid-phase RIA to measure MCH in the blood confirmed that background color and degree of illumination influence MCH secretion (Kishida et al., 1988; Eberle et al., 1988). Plasma MCH titers in the trout are usually between 50-100 pM in white-adapted fish, 10-20 pM in trout kept on a black background, and about 5 pM in trout kept in the dark for several days. These differences in hormone concentration become evident within two hr or less after fish are moved from one colored background to another. Subsequent studies have concentrated on the environmental conditions which influence the secretion of MCH, and on determining its relative importance for regulating the chromatophores. Several types of chromatophores are present in the skin, each distinguished by the pigment they contain. The pigmentary effects of MCH are not confined to the melanophores; the hormone will also cause aggregation of the red and yellow pigment in the erythrophores and xanthophores, but because of the greater difficulty in observing these cells, they have received relatively little attention (Oshima ef af., 1986; Fujii and Oshima, 1986; Castrucci et al., 1988). The melanophores of all teleost species examined will respond to synthetic sMCH although their sensitivity to the hormone varies considerably, at least in rrifro (Hadley er al., 1987). Some species are extremely sensitive, with an EC,, of about 10 pM (the concentration producing a half-maximal response) (Synbrunchus, the amazonian eel; Hadley ef al., 1988) or 30 pM (Ctenopharyngodon, the Chinese grass carp; Baker et al., 1985a) while trout and tilapia have an EC,, nearer 1 nM (Baker, 1988a; Kawauchi ef al., 1983). There may be technical reasons for these differences, such as problems of hormone penetration to
31
MELANIN-CONCENTRATING HORMONE
the melanophores in v i m , but it is also probable that the importance of MCH for color control varies between species. Physiological (i.e., rapidly adaptive) pigmentary changes in teleost fish can be influenced by several agents, including not only the two color-change hormones (MCH which causes pallor, and its antagonist aMSH which induces darkening), but also neurotransmitters from the autonomic nervous system, involving both melanin-aggregating agents, e.g., noradrenalin (or acetylcholine in some species such as Purusifurus) and putative melanin-dispersing molecules such as ATP (reviewed in Fujii and Oshima, 1986). It would be surprising if the relative importance of these different agents did not vary among species. In flatfish such as the flounder, which can assume a patterned appearance to match a patterned background, it is clear that neuronal control of the melanophores must predominate, with hormones playing relatively little part. This belief is supported by the finding that plasma aMSH titers do not differ in flounders adapted to a dark or a pale background (Fig. 10) (Baker et al., 1984). The involvement of MCH in this species is not known. Most teleosts probably depend on hormones to a greater extent than this and the relative importance of aMSH and MCH may be reflected in the speed with which their concentrations in the blood change when fish move between darkand pale-colored backgrounds. As can be seen in Fig. 10, both the eel and the grass carp show a rapid decline in circulating aMSH titers when they are trans-
500 6oo 400
1
T
-
v
E
2
300
-
200
-
cn
c
t
loo 0
B BW W Carp
B BW W Trout
B BW W Eel
B W Flounder
FIG. 10. Plasma aMSH titers in different fish after adaptation to a black (B) or white (W) background for 18-21 days, and 3-5 hr after transfer from a black to a white background (BW). Vertical bars indicate SEM. The data suggest that species vary in the extent to which they depend on changes in plasma aMSH to achieve physiological color change.
32
BRlDGET 1. BAKER
ferred from black- to white-colored tanks (3-5-fold decrease within about 3 hr, whereas the trout shows only a slow decline in plasma aMSH, which takes days to become apparent (Baker et al., 1984; Rodrigues and Sumpter, 1984). The plasma titers of MCH, on the other hand, change within 2-4 hr when trout are moved between dark and light backgrounds. The rate of MCH-secretion in the eel and carp also appears to alter in response to changes of background color, judging by its concentration in the pituitary gland (Fig. 11) but plasma MCH titers have not yet been measured in these species. In all teleosts examined, MCH and a M S H have mutually antagonistic effects on melanin dispersion, the end result depending on the concentration of either hormone (Wilkes er al., 1984b Baker, 1988a). Competitive assays, in which the effect of aMSH is tested in v i m in the presence of a range of MCH concentrations, suggest that at equimolar titers of the two hormones, within the range of lO-'('-IO-'M, melanophores of the carp, the trout, and the eel show an intermediate state of melanin dispersion (Baker. 1988a). At titers above this range, the effect of one hormone predominates over the other. Thus, aMSH at M and above cannot be antagonized by MCH when tested on the carp; in contrast, it is MCH that has the overriding effect when tested on trout melanophores. This explains the originally puzzling observation that injections of teleost pituitary gland extract, containing both MCH and aMSH, will cause melanin concentra2ooo
. B
a W
1500
r
T
-
M
a
v
E
E*
1000 .
k
500
e*
s2
n "
.
W
WB Eel
W
WB Carp
W
WB Trout
FIG. I I . MCH content of the pituitary gland of fish adapted to a white background (W) for several weeks, and &6 days after transfer from a white to a black background (We). Vertical bars show SEM. Transfer to a black background inhibits the release of MCH and results in its accumulation in both pituitary and hypothalamus.
MELANIN-CONCENTRATING HORMONE
33
tion in some teleost recipients but melanin dispersion in others (Pickford and Atz, 1957). This difference in sensitivity to one or other hormone could depend on the relative numbers of receptors for MCH and aMSH on the melanophores, although receptor numbers on any melanophores, excepting melanoma cells, have yet to be determined. These data imply that fish differ in their use of the two hormones. Some species such as the trout appear to achieve melanin concentration by overriding the effects of aMSH with a rapid release of melanin-aggregating agent(s) such as MCH, while other species such as the carp or eel show a rapid withdrawal of aMSH in a manner akin to amphibians, in which MSH is probably the only color-change hormone. Such a scheme is probably an oversimplification, however, and neglects the role played by nerves. It may be significant that species such as the trout, which are relatively insensitive to MCH and aMSH in vitro, depend also on nervous control to achieve total pallor during adaptation to a pale-colored background. When the melanophores on the caudal fin of trout are denervated by a transverse cut across the fin rays, they fail to show melanin aggregation when the trout is placed on a white background, suggesting that the circulating MCH alone is unable to cause pallor (Baker et al., 1986). This contrasts with some other fish species, such as the molly Poecilia, in which denervated melanophores continue to respond appropriately to changes of background color after denervation (Baker and Ball, 1975; Pickford and Atz, 1957), while MSH secretion remains constant (Ball and Batten, 198 1). The concentration of MCH in the blood of white-adapted trout is usually about 50 pM, more than 20-fold less than that needed to achieve only partial melanin concentration in vitro (Baker, 1988a). These apparent anomalies can be explained by the observation that noradrenalin markedly potentiates the effect of MCH on trout melanophores (Fig. 12) (Green and Baker, 1989). It therefore seems that trout depend on synergy between MCH and the sympathetic nervous system to achieve adaptive pallor. It may be significant that noradrenalin and MCH are not synergistic on carp melanophores which are more sensitive to MCH. Nor-adrenalin is believed to cause melanin aggregation by inhibiting adenyl cyclase via the Gi protein (Eberle, 1988) while MCH has been proposed to act through diacylglycerol from the phosphoinositol pathway (Abrao et al., 1989). Apart from its direct effect on the melanophores, two lines of evidence suggest that MCH also influences pigmentation indirectly, by depressing the release of aMSH from the pars intermedia of the pituitary gland. In one experiment, Baker and co-workers (1986) implanted Alzet minipumps filled with MCH into trout, which were then kept on a black background for several weeks. Such implanted fish exhibited pallor and failed to exhibit the melanogenesis seen in control, black-adapted fish. The histological appearance of the
34
BRIDGET I . BAKER
FIG. 12. Trout scale melanophores, showing synergism between MCH and noradrenalin in achieving melanin aggregation. Trout scales were incubated in virro in solutions containing threshhold doses of ( A ) sMCH ( 3 x It)-''M); ( B ) noradrenalin ( 3 x 10' M); or (C) both agents together. The smaller. paler dots between the melanophores are xanthophores.
pituitary melanotropes, together with the reduced titers of circulating aMSH, suggested that MCH had depressed the secretory activity of the pars intermedia. The failure in melanogenesis can be attributed, therefore, to the depressed release of aMSH, although a direct inhibitory effect on MCH on melanogenesis remains possible. In further studies, trout and eel neurointermediate lobes were incubated for 24 hr in the presence of either rabbit anti-MCH serum or normal rabbit serum (Barber et al.. 1987). The incubated lobes released both MCH and aMSH into the medium but in both species the release of aMSH was significantly enhanced when the MCH was removed by immunoabsorption. This again suggests that MCH, normally released from nerve terminals in the immediate vicinity of the pars intermedia (Secion K A ) , can exert a paracrine effect on the melanotropes and modulate the release of crMSH. Whether MCH acts independently or in synergy with dopamine, the major inhibitor of MSH secretion, is not yet known. The only other vertebrates as yet found capable of responding to MCH by melanin concentration are Holostean fish, which currently are considered to be closely related to the teleosts (Sherbrooke and Hadley, 1988). In other vertebrates, such as amphibians and the reptile Anolis, the peptide causes melanin dispersion rather than concentration, although only at pharmacological doses (Section V,A). It seems likely, therefore, that the use of the peptide as a colorchange hormone is an evolutionary novelty in the teleost branch of vertebrate evolution.
MELANIN-CONCENTRATING HORMONE
35
B. INVOLVEMENT OF MCH IN STRESS-RESPONSE IN FISH Before the characterization of MCH, it was observed that fish kept in darkcolored tanks are more easily stressed, i.e., they develop higher plasma cortisol titers after disturbance, than fish kept in pale-colored tanks (Baker and Rance, 1981). This influence of tank color was initially associated with the enhanced secretory activity of the pars intermedia melanotropes in the black-adapted fish. It seemed possible that either these cells secreted ACTH, the immediate precursor of aMSH, when they became active during adaptation to a black background, or that adrenal activity was stimulated by aMSH itself, or by an N-terminal fragment of the POMC precursor, known to potentiate adrenal responsiveness to ACTH in mammals (Pederson and Brownie, 1980). Subsequent experiments on the isolated trout interrenal gland showed that aMSH will not stimulate cortisol secretion at physiological concentrations (Rance and Baker, 1981). Trout from black tanks were later found to have higher plasma concentrations of ACTH than white-adapted trout; the difference was most marked after stress, suggesting that the hormone is secreted by the corticotropes rather than the melanotropes (Gilham and Baker, 1985). The discovery of MCH and the availability of both the synthetic peptide [Eberle et al., (1986), now available from Peninsula laboratories] and antiserum against it, have made it possible to test the influence of MCH on the hypothalamo-pituitary-interrenal (HPI) axis of trout. It has been shown that MCH has no direct influence on the basal or stimulated secretion of cortisol by trout interrenal tissue in v i m (Green and Baker, 1991) but other experimental results, described below, show that MCH will depress the release both of CRF from the hypothalamus and ACTH from the pituitary. The evidence suggests that MCH plays a modulatory role in the stress response of fish. Isolated trout anterior pituitary lobes secrete ACTH and other POMC-derived peptides when they are incubated in virro, but the amount of hormone released by glands taken from acutely stressed fish is significantly lower if the donors were previously adapted to a white rather than a black background (Baker el al., 1985b). If Alzet minipumps filled with MCH are implanted into black-adapted fish, this too will significantly depress the amount of ACTH released by the pituitary tissue in vifro (Baker ef al., 1986). Possible interpretations are either that MCH depresses the release of corticotropic-releasing factors (CRF) during stress, or that MCH renders the corticotropes less sensitive to the CRF. There is evidence to support both possibilities. Experiments in which trout anterior pituitary lobes were incubated in the presence of synthetic CRF-41 together with different doses of MCH, showed that MCH will depress the response of the corticotropes to CRF in a dose-dependent manner (Baker et af., 1985b). Low doses of MCH, equivalent to those found in the circulation, were effective, although the presence of MCH fibers within the pars dis-
36
BRIDGET 1. BAKER
talk (Section II,A) suggest these could serve as an alternative route of peptide delivery. Further evidence of how MCH can influence the HPI system is provided by a series of experiments done on trout which were reared, from the egg stage until 18 months old, in either black- or pale-colored tanks to achieve the maximum difference between the two groups in their rate of MCH secretion. The values of MCH in the plasma, neurointermediate lobes, ventral hypothalamus (containing the MCH perikarya) and dorsal hypothalamus/thaiamus (containing putative neuromodulatory fibers) are shown in Table IV. In vitro experiments using tissue from these fish showed that the color on which they were reared did not influence either the basal or ACTH-stimulated levels of cortisol secretion from their incubated interrenal tissue, nor the basal corticotropic activity of the pituitary gland. Corticotrophin-releasing bioactivity (b-CRF) was affected, however. To assess the intluence of MCH on b-CRF secretion, hypothalami from these black- or white-reared fish were incubated in vim; the amount of bioactive CRF (possibly a cocktail of several peptides) released into the medium was deterTABLE IV MCH CONTENT OF DORSALA N D VENTRAL HYPOTHALAMI. PITUITARIES, A N D PLASMA OF TROUT REARED IN BLACK-OR WHITE-COLORED TANKS".~ Dorsal Thalamus (pmol)
Ventral Hypothalamus (pmol)
Pituitary (pmol)
Plasma (pmolA)
A. Stock Controls Black -reared White-reared
I .52 f 0.52 4.57 f 0.05
4.0 k 0.05 147.0 f 18.60
1 13 f 24 500 f 72
13f3 49 +lo
B. Transfer and mild stress Black-white White-white
0.38 f 0.05aa 3.86 + 1.05
I .O f 0.09aaa 75.7 f 12.40a
10 C 2aaa 261 f 14aa
54k6 I29 k 25
C. Transfer and injection stress
Black-white White-white
0.33 C O.05aaa 1.81 k 0.19bbb
0.5 f O.05bbb 2 C 0.3bbb 64.7 f 7.60aa 276 f 37.0a
26 C 6 287 f 45
*Fish were reared in black or off-white tanks for 18 months. Group A were killed without further experimentation. Group B and C fish were moved to clean white tanks for I 1 days before autopsy. Group C were injected ip with 1 ml isotonic saline on days 8-1 1 after transfer. MCH was extracted in 0 . 1 molh HCI and assayed by RIA. Molecular weight of sMCH = 2100. "Values are means f SEMS. All values for white-reared fish are very significantly (P < 0.001) higher than for their black-reared counterparts. Comparisons with group A: a, P < 0.05; aa, P < 0.01; aaa, P < 0.001. Comparisons with group B: b. P < 0.05; bb, P < 0.01; bbb, P < 0.001. 'The whole hypothalamus and thalamus, excluding the ventral perikaryal region, were extracted.
37
MELANIN-CONCENTR ATING HORMONE
mined by incubating a control pars distalis in this medium, after first immunoabsorbing the coreleased MCH; measurement of corticotropic secretory products released by the pituitary tissue served as a bioassay for the b-CRF content. Using this protocol, it was shown that hypothalami from black- or white-reared fish released similar basal levels of b-CRF. However, if the hypothalami were incubated in the presence of MCH antiserum in order to immunoneutralize the endogenous MCH, then the amount of b-CRF released by hypothalami from white-reared trout was very significantly increased, while that released from black-reared trout hypothalami was unaltered (Table V). These results show that MCH can depress the release of b-CRF in vitro, and suggest it may exert this effect also in vivo, possibly after release from neuromodulatory MCH fibers in the brain (Green and Baker, 1990). The data suggest also that hypothalami of fish reared on a white background come to contain greater amounts of b-CRF, perhaps because the persistent depressive effect of MCH on their HPI axis leads to a compensatory increase in the amount of b-CRF that is produced. Further studies are required to determine which of the several putative CRFs contained in the fish hypothalamus - CRF-41, vasotocin, or urophysin (Fryer et al., 1985) are affected by MCH. Trout reared on black or white backgrounds were also used to test their relative responsiveness to stress. Fish were transferred from their stock black- or TABLE V IN V/TRO RELEASEOF b-CRF BY HYPOTHALAMI OF TROUTREARED IN BLACKOR WHITE TANKS"
Source of Hypothalamus Experiment 1 None W-reared B-reared Experiment 2 None W-reared B-reared
Incubation Medium n
Hypothalamus
6 6 6
MEM MEM
6 6 6
MEM + a/s MEM + a/s
NPP Release (fmol/PD/30minh~~) Pituitary
MEM + a / s Hth medium + a/s Hth medium + a/s MEM + a/s Hth medium Hth medium
353 f 47 694 f 59f 729 + 32ff 294 f 70ww 1306f200 518+59www
aHypothalami (Hth) from black- (B-) or white- (W-) reared fish were incubated in vitro in 400~1 MEM, with or without the addition of MCH antiserum (a/s). 300~1 of this incubation medium was then added to a pars distalis (PD) from a farm fish; control PDs were incubated in fresh MEM + a/s. When a/s was not added to the incubated hypothalamus (Exp. I), the MCH in hypothalamic medium was immunoabsorbed for 30 min before addition to the test PD. The PDs were incubated for 30 min in the test medium, which was then assayed for NPP as a measure of b-CRF in the hypothalamic medium. "Results are means f SEM. M of NPP = 8500. <Statisticalcomparison used log-transformed data for homogeneity of variance. f, P < 0.05; ff, P < 0.01 compared with control PDs; ww, P < 0.01; www, P < 0.001 compared with incubation medium from W-reared hypothalami.
BRIDGET 1. BAKER
38
W
E
COntrd
0
L
W
B
Control
WW BW Transfer &
WW
BW
Transfer& Mild stress
WW BW Transfer &
B!YLwx Transfer & Injection stress
FIG. 13. Plasma titers of MCH (A) and cortisol (B)in adult trout reared in either off-white tanks ( W I or black tanks ( B ) and subjected to three different treatments I 1 days before autopsy. Control trout were sampled from the stock tanks; some trout were transferred from both stock tanks to bright white tanks (WW and BW) and subjected to mild disturbance for I I days; other trout were transftmed from stock tanks to bright white tanks (WW and BW) for I 1 days and additionally stressed by a daily injection ( I ml saline, ip) for 4 days before autopsy. In each experimental condition, the rise in plasma cortisol is inversely related to plasma MCH.
MELANIN-CONCENTRATING HORMONE
39
pale-colored backgrounds to bright white tanks and subjected to either mild or severe stress to observe the changes in MCH values and cortisol secretion. The results show several interesting features. As far as sensitivity to stress is concerned, fish reared in black tanks always exhibited higher plasma cortisol levels than white-reared fish and, in each condition (Table IVA-C; Fig. 13A-C), there was an inverse relationship between the concentrations of MCH in the plasma and brain and plasma titers of cortisol. The results show also that the secretion of MCH can be enhanced by repeated stress. This has been confirmed in other experiments (Green and Baker, 1991) which show that the stress-induced increment can be suppressed by dexamethasone, suggesting it is normally sensitive to the negative feedback of corticosteroids. A further aspect addressed by these experiments concerns the concentration of MCH within the ascending fibers in the brain, which are presumed to serve a neuromodulatory or neurotransmitter role. It is clear from Table IV that the concentration of MCH available for release in the brain can be influenced by factors such as background color and stress, which regulate the release of MCH from the pituitary. While this does not mean that neuromodulatory MCH is necessarily released within the brain in parallel with hormone release from the pituitary (although this might be the case), it tends to suggest that the neuromodulatory MCH system and the neurohypophysial MCH system are closely connected. The results could support the idea that the MCH fibers projecting to the neurohypophysis and to the brain may arise from the same perikarya. C. EFFECTSOF MCH IN HIGHERVERTEBRATES The available evidence suggests that MCH causes melanin concentration only in teleosts and holosteans (Sherbrooke and Hadley, 1988) and that its use as a color-change hormone is restricted to these fish. With the apparent exception of R . ridibunda, the amount of immunoreactive MCH detected in the neurohypophysis of most vertebrates other than teleosts is trivial: values of 0.6 ng/rat pituitary and 2.7 nghuman pituitary (Sekiya et al., 1988) have been reported, compared with 300-1000 ng/trout pituitary (Table IV). This suggests that MCH is not used as a hormone in most vertebrates. While its presence in the median eminence and posterior pituitary indicate potential functions as a hypophysiotropic or paracrine agent, its most important role is likely to be as a neuromodulator/ neurotransmitter in the central nervous system. A few biological effects of MCH in fish have been investigated in the rat. Baker and co-workers (1985b) found that sMCH depressed the in vitro release of bioactive ACTH from CRF-stimulated rat pituitary fragments but much higher concentrations of MCH were required than were effective on fish pituitaries. These results could not be repeated on dispersed pituitary cells when ACTH was measured by radioimmunoassay (Eberle, 1988). The reason for this discrepancy
40
BRIDGET 1. BAKER
is still unexplained. Synthetic rat MCH has also proved ineffective as a corticotrophin-inhibiting factor on rat pituitary cells; in some experiments, indeed, it exhibited CRF-like effects (Vaughan et al., 1989), or had a tendency to potentiate the effect of CRF (Navarra et al., 1990). Navarra and co-workers have found that rat MCH also failed to influence the release of CRF from the incubated rat hypothalamus. It therefore seems that these effects may be peculiar to teleosts, in which the correlation between MCH and the response to stress is supported by additional lines of evidence (see Section V1,B). High concentrations of sMCH (400pg/kg body weight) will cause a rise in plasma growth hormone when injected intraperitoneally into chlorpromazinetreated rats (Kawauchi et al., 1986). The rise follows a long latent period and reaches a peak 2 hr after injection, implying that the peptide does not act directly on the pituitary cells; the response to GRF, by comparison, occurs within minutes. There have been two unconfirmed reports of responses to MCH administered directly into the rat brain. Thus, Li and co-workers found that MCH reduced plasma luteinizing hormone (Pelletier et al., 1987). Secondly, since MCH will antagonize aMSH on fish melanophores, de Graan and co-workers investigated whether MCH could antagonize aMSH-induced grooming behavior in the rat. They found that MCH (0.4yg sMCH, injected i.c.v.) would partially depress the grooming behavior induced by a novel environment or by 0.3pg aMSH injected a few minutes after the MCH, but did not depress the grooming response induced by ACTH (Eberle, 1988). Zamir et a/. (1986a) observed that the concentration of MCH in the pituitary gland and lateral hypothalamus (the region of the MCH perikarya) was doubled in rats maintained on 2% saline drinking solution for 5 days. By analogy with MCH in fish, and with neuropeptides such as vasopressin in the rat, this increase in MCH concentration suggests a reduced level of peptide release during saline treatment. Zamir and co-workers point out that the lateral hypothalamus has been associated with the regulation of ingestive behavior and that MCH is therefore well situated to exert an effect on eating or drinking.
VII. Conclusion: MCH, Past and Future When salmon MCH was first discovered, it appeared to belong to a new family of hormones and bear no similarity to any other known peptide. Its only apparent resemblance was to the cyclic C terminus of salmon prolactin, which shares five out of a stretch of 12 residues with MCH and which exhibits slight (0.04%)melanin-concentrating activity (Kawauchi et al., 1983). The recent elucidation of the cDNA structures of salmon, rat, and human MCH prohormones has changed our perception of the relationships between MCH and other molecules.
MELANIN-CONCENTRATINGHORMONE
41
FIG. 14. Comparison of the pre-prohormones of Aplysiu peptide A (Pept A), human MCH (hMCH), and human ANP (hANP). Gaps have been introduced to maximize alignment between the residues (indicated by horizontal link-lines). Long arrows show putative sites of signal cleavage; arrow heads mark di- or tri-basic amino acids; single basic amino acids are indicated by vertical bars. The numbers show the percentage of identical amino acids shared by the sequences.
Computer searches of a protein data base for homologies with other sequences (Nahon er al., 1989b,c) revealed similarities between rat pre-proMCH and the precursor for peptide A from the nudibranch Aplysiu. The gene for this peptide is one of probably five highly homologous genes belonging to the Aplysia egg-laying hormone family. These genes are expressed in different tissues (e.g., the abdominal ganglion, bag cells of the abdominal ganglion, and atrial gland) and processed differently to give a variety of end products, including two egg-laying hormones, an acidic peptide, and A and B peptides (Scheller et al., 1983; Mahon et al., 1985). After introducing spaces to permit optimal alignment, Nahon and co-workers (1989b) demonstrated a 24% sequence identity between rat pre-proMCH and the precursor for Aplysia peptide A. Even more striking is the fact that five of the six cleavage sites in the ApEysia precursor match perfectly with mono- or di-basic residues in the rat pro-MCH (Fig. 14). The greatest homology is found in a stretch of 17 residues from rat preproMCH (residues 50-66) which show a 35% identity with Aplysia A peptide. Melanin-concentrating hormone itself becomes aligned with the C-terminal region of the Aplysia prohormone (the acidic peptide homologue) with a 23% identity (29% over a stretch of 17 Aplysia residues). Relatively few gaps needed to be introduced to achieve this level of identity between the precursors and the probability of these accumulated matches occurring by chance was calculated as P = 2.2 x lO-I3. Such an alignment also reveals some homology between the NGE and NEI sequences of rat proMCH and the Aplysiu precursor (19 and 14% identities, respectively). This highly significant resemblance not only implies a common origin for the two prohormones but also suggests there may have been selective pressures during evolution to maintain this similarity, although there is currently no information about the functional importance of the N-terminal region of proMCH. Salmonid pre-proMCH, which shows very little resemblance
32
BRIDGET 1. BAKER 270
256
290
Salmon MCH
AGCTGCAGCTGCAGG---GCTfGACA----GAGCCCTGACCCTGGACCGCA
7 S L mRNA
GGCTGAGGCTGGAGGATCGCTfGAGTCCAGGAGTfCTGGG-CTGTAGTGCG
.. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 50
60
.. .. .. .. .. .. .. .. .. .. .. ..
70
90
80
310 330 Salmon MCH -------GAGAGGCTGACCAGGACCTGAGCCCCAGCATCTCCATfGTCA-------GGAG .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 7 S L mRNA CTATGCCGATCGGGTGTCCGCA--CTAAGTfCG-GCATCAATATGGTGACCTCCCGGGAG 100 110 120 130 140 150
I
350
MCH 370
390
I
Salmon MCH --GGACACCATGAGGTGCATGGTGGGAAGGGTGTACCGGCCTfGCTGGGAAGTGTAGATG
.. .. .. .. .. .. .. .. .. ..
7SL mRNA
.. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. ..
CGGGGGACCACCAGGTTGCCTAA-GGAGGGGTGAACCGGCCCAGGTCGGAAACGGAGCAG 160 170 180 190 200
Fiti. 15. Comparison between the C-terminal region of salmon MCH cDNA and human 7SL RNA. (:I. Identical nucleotides. [From Nahon C r (11. (1989a). cDNA sequence of salmon melaninconcentrating hormone exhibits similarities with 7SL RNA.]
to the mammalian precursor, also share little homology with the Ap[ysia A peptide precursor, suggesting that the selective pressures have not operated along the fish line of evolution. Apart from its similarity to the Aplysia peptide, mammalian pre-proMCH can also be aligned with human pre-proAtrial Natriuretic Peptide (ANP) (Presse et (11.. 1990) (Fig. 14). The greatest resemblance between the two molecules is in the signal region and at the C terminus within the active hormones. Other computer searches (Nahon et al., 1989a) reveal a resemblance between the salmon cDNA MCH sequence and that for human and rat 7SL RNA (Ullu et 01.. 1982, 1984), an essential component of the signal recognition particle which targets secretory proteins to the endoplasmic reticulum during the initial stage of protein translation (Walter and Blobel, 1982; Walter et al., 1984). This resemblance is most striking in the region of MCH itself (Fig. 15), but Nahon et a/. ( I989b) report a significant similarity also between the 7SL RNA and the RNA sequences for Aplysia A peptide, the MCH sequence corresponding to this, and also rhe ANF precursor. As a result of these observations, they speculate that the 7SL gene. which is believed to have appeared very early in evolution (Ullu et ul., 1984). might be ancestral to all these neuropeptides. Finding the biological role of this novel peptide in the brain is rather like looking for a needle in a haystack. The widespread distribution of MCH fibers throughout the brain suggests that it might be involved with a number of independent homeostatic or physiological responses. Unfortunately, neither of the general functions which have been demonstrated for MCH in teleosts - physiological color change and modulating the release of ACTH - have been found to hold for other vertebrates.
MELANIN-CONCENTRATING HORMONE
43
Some putative roles of MCH have been proposed on the basis of similarities between the precursors of MCH and other peptides. A recent suggestion, for instance, is a role in salt or water balance. Thus, Presse and co-workers (1990) draw attention to the fact that products of the Apfysiu Peptide A gene family which are produced in the abdominal ganglion, are concerned with salt and water regulation (Scheller et al., 1984), and with cardiovascular functions (Nambu et ul., 1983). These are also functions of ANF. They therefore speculate that MCH might similarly be concerned with water intake and the control of homeostatic functions. In view of the effect of high salt intake on the MCH content of the rat brain (Zamir er al., 1986a), this idea merits exploration. Such a role has not been considered for fish but it is intriguing to note that cortisol secretion, which is apparently modulated by MCH in fish, is involved in sodium excretion and seawater adaptation in euryhaline teleosts (Doneen, 1976; Folmar and Dickoff, 1980; Masden, 1990). Other approaches to gaining insight into the roles of MCH can be proposed. For instance, using the results from structure-activity studies of MCH, it should be possible to develop a radiolabeled molecule and map the MCH receptors (binding sites) in the brain. Knowledge of these sites, and their relationship with the fibers or receptors of other, better understood, neuropeptides might be informative. Following another line of reasoning, it is possible that the adoption of MCH as a chromatophore-regulatinghormone in teleosts was possible because it originally exerted some function which predisposed or preadapted it for a role in color change, and which selective pressures have emphasized during evolution. Such roles might have persisted in other vertebrates. Candidate functions include the ability of MCH to depress the release of aMSH from the pituitary; or its ability to antagonize the effects of aMSH; or its synergy with catecholamines. Preliminary studies, showing that MCH may indeed oppose behavioral effects of aMSH in the rat brain (i.e., grooming behavior; Section V1,C) suggests this approach might be a profitable one. In this context, comparing the locations of the binding sites of MCH with those of aMSH or noradrenalin in the brain could be revealing. The aim of this chapter has been to summarize our current knowledge of MCH to attract the interest of other vertebrate physiologists. It is hoped that our understanding of the nonpigmentary roles of MCH and its related peptides will be gained more quickly than the acceptance of MCH as a color-change hormone. ACKNOWLEDGMENTS I am very grateful to Dr. N. Naito for providing Figs. 1 and 4A, to Dr. J. Green for Figs. 12 and 13, and to Dr.D. J. Bird for reading the manuscript.
44
BRIDGET I. BAKER
REFERENCES Abrao, M. S., Castrucci. A. M. de L., Hadley, M. E., and Hruby, V. J. (1989). Ahstr., Inr. Symp. Comp.Endocrinol.. 10th. Malaga. Spain. Andersen, A. C., Pelletier. G.. Eberle, A. N., Leroux, P., Jegou, S., and Vaudry, H. (1986). Peptides 7,941-952. Andersen, A. C., Jegou, S., Eberle. A. N., Tonon, M. C., Pelletier, G., and Vaudry, H. (1987).Bruin Res. Bull. 18, 257-259. Antoni, S. I., and Linton. E. A. (1979). Neuroscience 29, 167-174. Baker. B. 1. (1988a). In “Advances in Pigment Cell Research” (J. Y. Bagnara, ed.), pp. 505-515. Alan R. Liss, New York. Baker, B. 1. (1988b). In “The Melanotropins,” Vol. 2, (M. E. Hadley, ed.). pp. 159-173. CRC Press, Boca Raton Florida. Baker. B. I.. and Ball. J. N. (1975).Gen. Con~p.Endocrinol. 25, 147-152. Baker, B. I., and Rance, T. A. (1981). J . Endocrol. 89, 135-140. Baker. B. I., and Rance, T. A. (1983). Gen. Comp.Endocrinol. 50,423431. Baker, B. I., Wilson, J. F.. and Bowley, T. J. (1984).Gen. Comp. Endocrinol. 55, 142-149. Baker, B. I.. Eberle, A. N.. Baumann, J. B., Siegrist, W., and Girard, J. (1985a). Peptides 6, 1125-1 130. Baker, B. I.. Bird. D. J.. and Buckingham, J. C. (l985b).J . Endocrinol. 106, R.548. Baker, B. I.. Bird, D. J.. and Buckingham, I. C. (1986). Gen. Conip. Endocrinol. 6 3 , 6 2 4 9 . Baker, B. I., Brown, D. W., Campbell, M. M.. Kinsman, R. G., Moss, C. A,, Osguthorpe, D. J., Paul, P. K. C.. and White, P. D. (1988).Cht-m. Commun. pp. 1543-1545. Baker, B. I., Kinsman, R. G., Moss, C. A., and Paul, P. K. D. (1991). Peptides (in press). Ball, J. N., and Batten, ?: F. C. (1981). Gen. Comp. Endocrind. 44, 233-248. Barber. L. D.. Baker, B. I., Penny. J. C., and Eberle. A. N. (1987). Gen. Comp. Endocrinol. 65, 79-86. Batten, T. F. C., and Baker, B. 1. (1988). Gen. Comp. Endocrinol. 70, 193-205. Batten, T. F. C.. Powell, K., and Baker, B. I. (1986). Ahsrr., Conf. Eur. Comp. Endocrinol., 13th, Belgrade. Bird, D. J., and Baker B. I. (1989). Neuroscience 28, 245-251. Bird, D. J., Baker, B. I., and Kawauchi, H. (1989). Gen. Comp.Endocrinol. 74,442450. Bird. D. J., Baker. B. I., Eberle. A. N., and Swann, R. W. (1990).J . Neuroendocrinol. 2,309-315. Bresson, J . L., Claverquin. M. C., Fellmann, D., and Bugnon, C. (1986). C. R. Seances Soc. B i d . Ses Fil. 180, 175-183. Bresson. J. L.. Claverquin. M. C., Fellmann, D., and Bugnon, C. (1989).Neurosci. Letr. 102,3943. Brown. D. W., Campbell, M. M.. Kinsman, R. G., Moss, C. A,, Osguthorpe, D. J., Paul, P. K. D., White, P. D., and Baker, B. I. (1990). Biopolymers 29,609422. Castrucci, A. M. de L., Hadley, M. E., Wilkes, B. C.. Zechel. C., and Hruby, V. J. (1987). L(fe Sci. 40, 1845-1852. Castrucci. A. M. de L.. Visconti, M. A., Hadley, M. E., Hruby, V. J., Oshima, N., and Fujii, R. (1988). In “Advances in Pigment Cell Research” (J. Y. Bagnara, ed.), pp. 547-557. Alan R. Liss. New York. Castrucci. A. M. de L.. Lebl. M., Hruby. V. J., Matsunaga, T. 0.. and Hadley. M. E. (1989).Lqe Sci. 45, 1141-1 148. Doneen, B. A. (1976).Gen. Comp. Endocrinol. 28, 3341. Eberle. A. N. ( 1988). “The Melanotropins. Chemistry. Physiology and Mechanisms of Action.” Karger, Basel. Eberle, A. N., Atherton. E., Dryland, A.. and Sheppard, R. C. (1986).J . C. S. Perkin 1. pp, 361-367. Eberle. A. N., Baker, B. I.. Kishida, M.. Baumann. J. B., and Girard, J. (1988). Lqe Sci. 45, I 149-1 154.
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INTERNATIONAL REVIEW OF CYTOLOGY. VOL. 126
Regulation of Synthesis and Transport of Secreted Proteins in Cereal Aleurone RUSSELLL. JONES * AND JOHN v. JACOBSEN+ *Department of Plant Biology, Universityof California,Berkeley,Berkeley, California 94720 ?Divisionof Plant Industry, CSIRO, Canberra, Australia
I. Introduction The cereal aleurone layer has attracted considerable attention from plant cell and molecular biologists because of several unique properties (Akazawa and Hara-Nishimura, 1985; Fincher, 1989). It is one of the few digestive tissues found in plants (Haberlandt, 1884; Jones and Robinson, 1989), and the synthesis and secretion of hydrolytic enzymes by this tissue are controlled primarily by the plant hormones gibberellic acid (GA) and abscisic acid (ABA), and by the calcium ion (Fincher, 1989). Barley grains have also been extensively studied by plant biologists for more than a century because of their importance in malting and in the brewing of beer. Malt diastase (a-amylase) was one of the first enzymes to be isolated and purified'in the early 19th century, and the role of the various barley grain tissues in the synthesis of this enzyme was a source of considerable dispute in the latter part of the 19th century (Haberlandt, 1884). Because the scutellum and aleurone of cereal grains are differentiated as digestive glands, they also attracted particular attention. Green plants are photosynthetic autotrophs; consequently, glands that are specialized for the secretion of digestive enzymes have evolved infrequently. The modified leaves of insectivorous plants (Schnepf, 1975) and the digestive tissues of the embryo (scutellum) and endosperm (aleurone) in cereal grains (Haberlandt, 1884) are notable exceptions.
11. Aleurone Tissue Our knowledge of the aleurone layer and its function has come from the study of several cereal species, predominantly barley. This reflects the importance of germination and endosperm function in the malting process. Although aleurone cells from various cereals appear to have the same basic structure and function and play the same role in germination-related processes, aleurone layers are not all structured the same. Barley has three or four aleurone cell layers, whereas wheat, maize, sorghum, sugar cane, rice, oat, and rye have only one or sometimes two (oat). Also, the control of cellular function does not appear to be the 49 Copyright Q 1991 by Academic Press. Inc. All rights of reproduction in any form reserved.
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RUSSELL L. JONES AND JOHN V. JACOBSEN
REGULATION OF SECRETED PROTEINS IN CEREAL ALEURONE
51
same in all cereals. For example, in wheat aleurone a number of factors including hormones and nitrogenous compounds, in addition to GA (Laidman, 1982), control aleurone function. This article focuses on aleurone cells of barley grains and does not refer to other cereals unless specified. Development of grain tissues, particularly endosperm, has been described previously for wheat (Bhatnagar and Sawhney, 1981; Simmonds and O’Brien, 1981) and barley (Pyliotis, 1977). At the time of fertilization in all cereals, one of the pollen nuclei fuses with two polar nuclei to give a triple fusion nucleus. Subsequent divisions of the nucleus and cellularization give rise to the endosperm. The endosperm rapidly differentiates into two tissues: the inner cells become the large starch- and protein-containing cells of the starchy endosperm (Fig. 1B) and the outer cells give rise to the aleurone layer. The aleurone layer consists of smaller, thick-walled cells (Fig. 1A), which characteristically contain numerous spherical organelles which are the aleurone grains or protein bodies derived from vacuoles (Fig. 2). In mature barley, the aleurone completely surrounds the zygotic tissues. It is three to four cells thick next to the starchy endosperm and only one cell thick (the germ aleurone) next to the embryo (Fig. lA,D). The structure and functions of the cells appear to be the same regardless of their location within the grain (Pogson et al., 1989). Numerous studies of whole germinating grain, de-embryonated grains (halfseeds), and isolated aleurone (Akazawa and Miyata, 1982; Jacobsen, 1983; Ashford and Gubler, 1984; Ho er al., 1987; Jacobsen and Chandler, 1987; MacGregor and MacGregor, 1987; Akazawa et al., 1988; Hill and MacGregor, 1988; Muthukrishnan and Chandra, 1988; Fincher, 1989) show that during germination, or following GA treatment, the activities of a number of enzymes in the aleurone cells increase (Table I). Some of these enzymes are secreted into the aleurone cell walls, whence they find their way into the starchy endosperm (Fig. 1). Many of these enzymes are hydrolytic and degrade the storage polymers of the starch grains, protein bodies, and cell walls, thus providing nutrition for the enlarging and differentiating embryo. The products of this hydrolysis probably enter the embryo through uptake by the epithelial cells of the scutellum (Fig. 1C). These scutellar cells also secrete hydrolytic enzymes into the starchy endosperm during germination.
~
~
FIG. 1. Anatomy of a barley grain. The diagram on the left shows a longitudinal section of a whole grain. The diagrams on the right show (A) enlargements of sections of the aleurone and subaleurone layers, (B) starchy endosperm cells, (C) cells at the interface of the scutellum and the starchy endosperm, and (DI-2) a cross section of whole grain where embryo and endosperm overlap. (Adapted from a drawing by s. I. Wong, provided by Dr. Gamy Fulcher, Plant Research Center, Agriculture Canada, with permission.)
52
RUSSELL L. JONES AND JOHN V. JACOBSEN
Fici. 2. Electronmicrograph o f a barley aleurone protoplast. Note the protein body vacuoles (PB) and extensive ER network. Lipid bodies (oleosomes, 0) and mitochondria (M) are also indicated. (From 1. Zingen-Sell, S. Hillmer. D. G . Robinson. and R . L. Jones, unpublished observations.)
REGULATION OF SECRETED PROTEINS IN CEREAL ALEURONE
53
TABLE I GIBBERELLIN-REGULATED ENZYMES IN CEREAL ALEURONE Secreted
Increased by GA a-Amylase Protease Carboxypeptidase Endoprotease Ribonuclease DNase DNasemNase (1 4 3)-P-Glucanase (1 3) ( 1 + 4)-j3-Glucanase Esterase Acid phosphatases Pentosanase Endoxylanase P-Xy lopyranosidase Arabinofuranosidase a-Glucosidase ( I 4 6)-a-Glucanase Peroxidase Lysophospholipase Catalase Malate dehydrogenase Malate synthase Isocitrate lyase Citrate synthase Phosphorylcholine glyceride transferase Phosphorylcholine cytidyle transferase Polyphenol oxidase Proteases Aleurain ER Ca2+ATPase Tonoplast ATPase
Inhibited by GA Alcohol dehydrogenase Amylase/protease inhibitor
References
Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes No No No No No
Chrispeels and Vamer (1967a) Jacobsen and Vamer (1967) Mikola and Mikola (1986) Koehler and Ho (1988) Chrispeels and Vamer (1967a) Taiz and Starks (1977) Brown and Ho (1986) Jones (1971) Stuart et al. (1986) Jacobsen and Knox (1972) Pollard and Singh (1968); Ashford and Jacobsen (1974) MacLeod and Millar (1962) Taiz and Honigman (1976) Taiz and Honigman (1976) Hardie ( 1975) Hardie (1975) Hardie ( 1975) Jacobsen and Knox ( 1972) Baisted and Stroud (1982) Jones ( I 972) Jones ( 1972) Jones (1972); Doig et al. (1975) Jones (1972); Doig et al. (1975) Jones 1972)
No
Johnson and Kende (1971)
No No No No
No No
Johnson and Kende (1971) Taneja and Sachar ( I 974) Mikola and Mikola (1986) B. C. Holwerda, N. J. Galvin, and J. C. Rogers (unpublished) Bush et al. (1989a) D. S. Bush and R. L. Jones (unpublished)
No No
Noland and Ho (1988) Noland and Ho (1988)
54
RUSSELL L. JONES AND JOHN V. JACOBSEN
111. a-Amylase Genes and Control of Their Expression A. CONTROL OF a-AMYLASE SYNTHESIS
The most extensively studied aleurone enzyme is a-amylase. a-Amylase has a well-defined role in the endosperm during germination; it is made in very large amounts in aleurone; it is easily assayed, purified, and detected on electrophoretic gels; it is very stable; and it consists of a single polypeptide chain. a-Amylase activity increases in germinating barley, and studies have focused on the origin of this enzyme, its role in the germinating grain, the nature of the enzyme itself, and the control of its production (see references listed in Section 11). In some species (barley and wheat) the a-amylase arises predominantly from the aleurone cells, whereas in others (corn, sorghum, and rice) major contributions are made by the scutellum. Gibberellic acid has been shown to cause increased activity of a-amylase and other enzymes in aleurone cells of oat (Naylor, 1966; Chen and Chang, 1972), rice (Tanaka ef al., 1970), wheat (Baulcombe and Buffard, 1983), and barley (Paleg, 1960; Yomo, 1960), and a small and variable response in sorghum (Daiber and Novellie, 1968; Aisien and Palmer, 1983). Although GA does not consistently stimulate enzyme production in corn endosperm above the already high level (Ingle and Hageman, 1965; Harvey and Oaks, 1974; Goldstein and Jennings, 1975), there is evidence that GA may still be the inducer of enzymes (Harvey and Oaks, 1974). These and other studies indicate that GA plays a role in controlling a-amylase in vivo, probably arising in the embryo and controlling a-amylase production in both the aleurone and the scutellum (at least in barley; see Chandler and Mosleth, 1990) during germination (Fig. 1). Much of the work on a-amylase has been done with isolated aleurone layers of barley, particularly from the variety Himalaya. These are prepared from grain which has been de-embryonated and allowed to imbibe water for 3 4 days. The aleurone layers can then be prepared almost free of starch, and with a high degree of aleurone cell integrity. The layers are then usually incubated in liquid media with appropriate additions. Grain is usually selected which has aleurone with an all-or-none response to GA. The intrinsic ability (no hormone added) of aleurone to produce a-amylase varies widely with genotype, growing conditions, grain age, and probably many other factors (Ashford and Gubler (1984); Fincher (1989)]. This is probably a function of variable amounts of GA (and perhaps ABA) present in grain at maturity, but there is little experimental evidence to support this statement. Using isolated aleurone layers of barley, it has been shown that ( I ) a-amylase production is dependent on both GA and Ca'+; (2) most of the enzyme is released from the aleurone cells; (3) ABA antagonizes GA action; (4) a-amylase arises by new synthesis; ( 5 ) the abundance of mRNA for a-amylase increases
REGULATION OF SECRETED PROTEINS IN CEREAL ALEURONE
55
with a-amylase synthesis; and (6) the increase in mRNA is due, at least in part, to an increased rate of transcription of a-amylase genes. These results have been reviewed elsewhere (see references listed in Section 11) and will not be repeated here. However, it is of interest to note the amount of a-amylase synthesized by aleurone cells. Each aleurone layer contains about 100,000cells (Jacobsen et al., 1985) and in 24 hr produces about 45 pg of a-amylase. Thus each cell (weighing about 130 ng) produces about 0.4 ng of a-amylase in 24 hr. This is equivalent to a rate of synthesis of 2 x lo8 molecules per cell per hour. This high rate of production is reflected by other values, namely, that after 12 hr of GA treatment, a-amylase mRNA constitutes about 15-20% of the total translatable mRNA (Higgins et al., 1976; Muthukrishnan et al., 1979) and that a-amylase synthesis constitutes about 30% of total protein synthesis (calculated in Khursheed and Rogers, 1988).
B. a-AMYLASE IS ENCODED IN A MULTIGENE FAMILY 1. Isoforms of a-Amylase
Many studies have recorded the fact that a-amylases from cereal grains consist of a number of isoforms which can be separated by isoelectric focusing (IEF), polyacrylamide gel electrophoresis, or ion exchange chromatography (MacGregor, 1982; MacGregor and MacGregor, 1987; Hill and MacGregor, 1988). The number of isoforms found in the endosperm of barley (synthesized and secreted by the aleurone layer) depends on factors including the separation technique, the variety of barley, and whether one examines germinating whole grain, GA-treated endosperm, or isolated aleurone. Depending on how the isoforms are produced and separated, they fall into two or three isoelectric point (PI) groups. In all cases there are at least two groups, one with PISof about pH 4.6 and the other with PIS of about 5.9. a-Amylase isoform nomenclature is in a confused state and an effort to develop a standardized system is underway (MacGregor and MacGregor, 1987; Sticher and Jones, in press). For the purpose of this article, these groups will be referred to as either the low-PI and high-pI groups or AMY 1 and AMY2, respectively. In some cases there is a third group with PIS of about 6.5 (called AMY3), which are complexes of high-pI isoforms with an inhibitor of a-amylase (MacGregor and MacGregor, 1987). In Himalaya barley about seven major isoforms can be resolved (Jacobsen and Higgins, 1982; Callis and Ho, 1983; Jacobsen et al., 1988), but as many as 12 isoforms of a-amylase can be detected by IEF (Simon and Jones, 1988). Of the four low-PI isoforms that have been resolved, two are posttranslational modifications of the other two (Jacobsen et al., 1988; Aoyagi et al., 1990). At least two major and one minor high-pl isoforms have been resolved. Studies in the early 1980s involving sequencing and proteolytic fingerprinting of purified a-amylases
RUSSELL L. JONES AND JOHN V. JACOBSEN
56
showed that isoforms differed in their amino acid sequences and that these differences were small between isoforms within groups and more extensive between isoforms in different groups (Jacobsen and Higgins, 1982; Callis and Ho, 1983). These results indicated that the diversity of a-amylase isoforms was genetically based, and more recent sequencing studies have confirmed this (Svensson ef a/.. 1985). The isoform groups have been shown to differ in a number of characteristics, including sensitivity to Ca2+,EDTA, sulfhydryl reagents and heavy metals, stability at low pH, and serological characteristics (Jacobsen and Chandler, 1987).
2. ( D N A s and mRNAs A number of cDNA clones for a-amylase have been isolated and sequenced (Table 11). Analyses of low- (Rogers and Milliman, 1984) and high- (Rogers, 1985; Chandler ef a/., 1984 Huang e l af., 1984; Deikman and Jones, 1985) pI clones have verified that there are sequence differences between isofoms and that a high degree of homology exists within groups and less between groups. Base sequence homology within the coding region is 90-95% within groups and about 75% between groups (this figure is about 80% for the mature polypeptides as the signal polypeptides have only about 50% homology). Homology in the noncoding regions (both 3. and 5’) of high- and low-PI isoTABLE 11 ISOLATED A N D SEQUENCED GENOMIC AND CDNA CLONES OF BARLEY a-AMYLASE ~
Low or hieh DI
References
cDNA clones E pM/C pHvl9 96 I03 I68 1-28
Low High High High High High High
Rogers and Milliman (1983) Rogers ( 1985) Chandler er a / . (1984) Huang eta/. (1984) Huang er a / . (1984) Huang er a / . (1984) Diekman and Jones (1985)
Low LOW
Whinier er ol. (1987) Knox er a / . (1987)
High
Know er al. ( 1987)
High High High High High
L. Huiet (unpublished) Khursheed and Rogers (1988) Khursheed and Rogers (1988) Rahmatullah et a / . (1989) Rahmatullah et nl. (1989)
Genomic clones
Amy32b gKamylS5.3 (Sundance) gKAmyl41.117 (Sundance) AmypHv I9 Amy6-4 Amy46 gRAmylS2 gRAmyS6
REGULATION OF SECRETED PROTEINS IN CEREAL ALEURONE
57
forms is only about 25%. These studies also indicated that there were at least five different high-pI isoform sequences. Sequencing full-length clones (Rogers and Milliman, 1984; Rogers, 1985) showed also that there were differences in the masses of the protein. A low-PI isoform contained 438 amino acids and a high-pI isoform 427, indicating a difference of about 1100 Da between high- and low-PI a-amylases. This probably accounts for the difference in mobility between the two noted on SDS polyacrylamide gels (Jacobsen, 1986). Because the low- and high-pl isoform groups appear to migrate as single bands on acrylamide gels, probably all low- and high-pI isoforms differ in length by about 11 amino acids. The a-amylase mRNAs have also been examined by primer extension analysis (Rogers and Milliman, 1984; Rogers, 1985; Chandler et al., 1987; Chandler, 1988; Chandler and Huiet, in press; Chandler and Jacobsen, in press). The results obtained have been variable, but there appear to be two major and one minor low-PI products and at least three major and several minor high-pI products (Fig. 3B). Although the occurrence of multiple bands may have various causes (for discussion see Rogers, 1985; Chandler er al., 1987), this approach indicates that there are two to three low-PI mRNAs and three to five high-pI mRNAs, which is substantially in agreement with the numbers of isoforms detected in protein studies and cDNA sequencing. Although it was not difficult to assign different cDNAs to high- or low-PI isoform groups on the basis of protein sequencing studies (Chandler et al., 1984; Svensson et al., 1987), matching cDNAs and isoforms within groups has been more difficult. To purify and sequence all individual isoforms for comparison with cDNA sequences would be time consuming and laborious. However, a method of expressing barley mRNA in Xenopus laevis oocytes has been developed (Boston et al., 1982), and oocytes injected with a-amylase mRNA, cDNA, and genomic DNA synthesize and secrete a-amylase which is indistinguishable from the protein made in aleurone cells (Simon and Jones, 1988; Aoyagi et al., 1990). Thus any individual full-length nucleotide sequence can now be matched to its isoform(s). 3. Genes
Southern blot analyses have shown that there are about eight a-amylase genes (or pseudogenes) in Himalaya barley and Betzes barley. By using groupspecific clones as probes, these genes have been shown to fall into two classes, about three of them into the low-PI class and five in the high-pI class (Muthukrishnan et al., 1983c; Chandler et al., 1984; Rogers and Milliman, 1984; Rogers, 1985; Khursheed and Rogers, 1988). Again these numbers agree fairly well with those quoted above indicating that probably all of these genes are functional, i.e., they are not pseudogenes. Studies of chromosome addition lines at the DNA and protein levels have shown in Betzes barley that the
58
RUSSELL L. JONESAND JOHN V. JACOBSEN
FIG.3. Comparison of synthesis of high- and low-pl a-amylase isoforms (A) with the abundance of their mRNAs (B). In both experiments. aleurone layers were incubated with GA for different rimes. In A, layers were labeled with [%jmethionine for the last hour of incubation. Polypeptides were extracted. separated by SDS PAGE. and visualized by fluorography. In (B), RNA was isolated from layers at the end of the incubation period. and radiolabeled primer extension products were prepared and separated by electrophoresis (top). Radioactivity in the bands of the fluorograph was quantified by densitornetry (bottom). [(A) is from Nolan and Ho (1988) and (B) from Chandler and Jacobsen (in press).]
REGULATION OF SECRETED PROTEINS IN CEREAL ALEURONE
59
low- and high-pI genes are distributed in two chromosomes, three genes in chromosome 1 and about six in chromosome 6 (Brown and Jacobsen, 1982; Muthukrishnan et al., 1984). The availability of specific cDNA clones has permitted the isolation and sequencing of a number of both low- and high-pI aamylase genes from Himalaya barley. A distinction that appears to be emerging is that the high-pI genes have three introns and the low-PI genes have two (Rogers and Milliman, 1984; Knox et af., 1987; Khursheed and Rogers, 1988; Rahmatullah et al., 1989). Genomic clones have been isolated and sequenced (Table 11) as a prelude to studying the regulation of a-amylase genes by GA and ABA. Sequence comparisons, primarily in the region 5' to the transcription initiation sites, have been made between genes with a view to identifying cis-acting sequences [hormone responsive elements (HREs)] that may be involved in hormonal interaction with the gene and with control of gene expression. Sequence comparisons between barley clones (Table 111) have revealed the presence of a number of highly conserved elements in the promoter regions of a-amylase genes (Huang et al., 1991), but it has not been possible to unequivocally identify HREs from these studies. Studies with promoter-specific clones indicate that there may be a high level of homology between promoters of genes in the same PI group but little homology between groups (Khursheed and Rogers, 1988), which may relate to the differential expression of the genes in different groups in response to hormones described below.
TABLE 111
HIGHLY CONSERVED BARLEYCL-AMYLASE GENEPROMOTER SEQUENCES' Clone
Pyrimidine boxb
-130sequence
-105 sequence
Low PI Amy32b gKAmy155.3
-134 -229INV
TTGCACCTTITCTCGTA CAl'TGCCTiTTGCTTIT
-120 -133
GTAACAGAGTCTGG ATAACAGAGGCCGG
High pl gKAmy141.117 Amy6-4 Amy6-4 Amy46 gRAmy152 gUAmy56 pHv 19
-170 -173 437 -170 -175 -171 -174
ATTCGCCTiTTGAGCTC AATCGCCITTGAGCTC TAAACCCITITGGGGTT AGTCGCCTTTTGAGCTC AATCGCCTlTTGAGCTC AGTCGCCTTITQAGCTC AATCGCCTTITGAGCTC
-140 -143
ATAACAAACTCCGGCCGACATATCCATCG ATAACAAACTCCGGCCGACATATCCACTG
-140 -145 -141 -144
ATAACAAACTCCGGCCGACATATCCATCG ATAACAAACTCCGGCTG ACATATCCACTG ATAACAAACTCCGGCCGACATATCCATCG ATAACAAACTCCGGCCGACATATCCACTG
TATCCATGC TACCCATGC
aAdaptedfrom Huang er al.[ 1990). T h e figures preceding the sequences refer to the numbers of nucleotidesbetween the transcription start site and the fust nucleotide in the sequence.
RUSSELL L. JONES AND JOHN V.JACOBSEN
60
c.
DLFFERENTIAL EXPRESSION OF U - h Y L A S E GENES
Studies of a-amylase gene expression at the protein and RNA levels with probes that are able to distinguish between a-amylase isoforms and RNAs have demonstrated that not only is there a lack of synchrony in isoform synthesis, but also that the order of gene expression may change according to experimental conditions. Using isolated aleurone layers, synthesis of the Iow-pI isoforms can be detected before hormone is added, but synthesis of high-pI isoforms cannot. Following GA addition, the rates of synthesis of both groups increase, that of the low-PI group first. The rate of synthesis of the low-PI isoforms continues to increase and remains high for at least 40 hr, but the rate of synthesis of high-pI isoforms reaches a maximum at about 16 hr and then decreases to a very low value after 30 hr (Fig. 3A) (Jacobsen and Higgins, 1982; Jacobsen, 1983; Ho et ul., 1987; Nolan and Ho, 1988). Using isoform-specific probes and primer extension studies, quantitation of low- and high-pI mRNAs has shown that levels of RNAs correspond fairly well with rates of synthesis of high- and low-PI isoform groups (Fig. 3B) (Huang et al., 1984; Rogers, 1985; Deikman and Jones, 1986; Nolan and Ho, 1988; Chandler and Jacobsen, in press). This demonstrates that differential regulation can be seen at the mRNA level as well as the protein level and that synthesis of the different a-amylase isoform groups is regulated pnmarily by the levels of their mRNAs. Low- and high-pI isoforms are also differentially responsive to GA concentration (Jacobsen and Higgins, 1982; Huang er al.. 1984; Nolan and Ho, 1988; Chandler and Jacobsen, in press). The onset of hormone-initiated synthesis of low-PI isoforms and their mRNAs and their time of maximum accumulation are both initiated by a lower GA concentration (about 1/10) than for high-pI isoforms. Thus, the two isoform groups are differentially regulated both in time and by hormone dose, although these two phenomena may be related. For example, the fundamental difference between the two gene groups may be sensitivity to GA, and the low-PI isoforms may be expressed earlier because the hormone concentration threshold for low-PI genes may be reached earlier as the GA level increases gradually within the cells. Using other experimental conditions, results have vaned from the above (Chandler and Jacobsen, in press). In germinating grains, high-pI isoforms appear first and then disappear after about three days of germination, whereas the low-pl isoforms appear at about two days and steadily accumulate up to eight days. This sequence of events is matched by changes in RNA levels. Similar results have been obtained using de-embryonated seeds (Callis and Ho, 1983). In aleurone protoplasts, the relative levels of mRNAs and proteins are different yet again: the high- and low-PI isoforms and their mRNAs appear simultaneously (Chandler and Jacobsen, in press). There is no simple explanation for this vari-
REGULATION OF SECRETED PROTEINS IN CEREAL ALEURONE
61
ation in time course of appearance of a-amylase mRNAs and proteins, and the results highlight the complexity of the GA response and the need to take care in extrapolating results from one experimental system to another. The action of Ca2+in aleurone has received considerable attention because its effect on a-amylase production is one of its few actions in plants which is defined in molecular terms. Because all a-amylase isofonns are CaZ+metalloproteins (Fischer and Stein, 1960; Bush et af., 1989b), it seems logical that the requirement for Ca2+can be explained in part by a role for CaZ+in conferring activity and in stabilizing enzyme structure (Bush et al., 1989b). Calcium may also regulate a-amylase synthesis and secretion at other levels including gene transcription, mRNA synthesis, processing and transport, or on the synthesis and intracellular transport of the secreted protein. The role of Ca2+in the synthesis of barley a-amylase is discussed in Section VI of this review, but it is appropriate here to record that Ca2+ does not affect the levels of a-amylase mRNA (Deikman and Jones, 1985, 1986) and therefore does not affect gene transcription, mRNA processing, or transport. GA seems to be the sole regulator of mRNA accumulation in these cells. Regulation of isoforms within groups has also been examined at the protein and RNA levels. Studies at the enzyme level (Jacobsen and Higgins, 1982; Jones and Carbonell, 1984) have shown that one of the low-PI isoforms (a-amylase 2) is produced without addition of GA and Ca2+(but still responds to them), and it is produced before all other isofonns. The other low-PI isofonn (a-amylase 1) requires GA and Ca2+for its production. In the presence of GA and Ca2+, a-amylase 2 accumulates to a much higher level than a-amylase 1. All of the high-pI isoforms appear to accumulate simultaneously and to be similarly responsive to GA, although they also accumulate to different extents. In general, RNA studies support the enzyme studies with minor differences. Primer extension studies (Rogers and Milliman, 1984; Chandler and Jacobsen, in press) have provided evidence that mRNAs within PI groups differ in the extent of their accumulation with time in different GA concentrations (Fig. 3). The changes within groups are largely coordinate with respect to time and GA response. Inhibition of individual a-amylase isoforms by ABA can be coordinate or differential depending on how the experiment is done. Added at the same time as GA, ABA inhibits all isoforms about equally (Nolan ef af.,1987) and the levels of rnRNAs are reduced similarly (Nolan and Ho, 1988). Added some time after GA, ABA inhibits the synthesis of high-pI isoforms more strongly than low-PI isoforms. This difference is reflected in the mRNA levels for low- and high-pI isoforms (Nolan et al., 1987). Thus ABA, like GA, regulates synthesis of isoforms by regulating mRNA levels, and this regulation can be differential. What do these differences in regulation mean and how do they occur? There is good evidence that at least part of the action of GA and ABA relates to gene
62
RUSSELL L. JONES AND JOHN V. JACOBSEN
transcription rate. One interpretation of differential regulation consistent with current concepts of the mechanism for gene regulation is that the strength and timing of a-amylase gene expression is a function of the interaction of cis- and trans-acting factors, and differences in these factors could account for differential regulation. Southern blot studies using promoter-specific probes show that there is a high degree of sequence conservation in the promoters of six or seven high-pI genes, little between three (or four) low-PI genes, and little between low- and high-pI genes (Khursheed and Rogers, 1988). Such studies do not necessarily tell us anything about very short base sequences of the sort we might expect to constitute hormone-responsive elements, but many aspects of these results are consistent with the differences in isoform gene expression described in the preceding paragraph. This lends support to the contention that differential gene regulation is, at least in part, due to differences in the cis- acting elements which would dictate the strength of interaction with trans-acting factors and thus the rate of gene transcription. Differences in rates of gene transcription could account for the differences in abundance of the a-amylase isoform mRNAs. mRNA stability may also be involved in a-amylase gene expression. The abundance of an mRNA is a function not only of its rate of synthesis but also its rate of degradation. Therefore, differential gene expression may also be a function of mRNA stability. Reliable turnover rates for a-amylase mRNA have not been reported, and there is no evidence for differential mRNA stabilities or for control of mRNA stabilities by GA or ABA. Measuring mRNA turnover rates is not an easy task, and this line of research has taken a backseat to the easier-towork-with and more fashionable option of transcriptional control. There is evidence that animal hormones can stabilize mRNA as well as regulate gene transcription (Guyette et al., 1979; McKnight and Palmiter, 1979; Shapiro, 1982; Brock and Shapiro, 1983), demonstrating that hormonal control of gene expression can occur posttranscriptionally. There is sufficient sequence diversity between a-amylase isoforms to make differential rates of degradation of a-amylase mRNA an option. Figure 3B shows that the level of high-pI mRNA increases and then decreases rapidly while that of low-p1 mRNA continues to increase. Such differential behavior could be explained, at least in part, by differential mRNA stabilities. In particular, the reversal of accumulation of high-pI mRNAs could be explained by the appearance of high-PI-specific GA-induced nucleases. Cases of such nucleases in aleurone cells have been reported (Chrispeels and Varner, 1967a; Brown and Ho, 1987; Brown ef al., 1988). On the other hand, the differential behavior could also be explained by transcriptional differences. One possibility is that GA may lead to differential gene stability, perhaps by induction of high-pI gene-specific DNAse. DNAse induction by GA is known to occur in aleurone cells (Taiz and Starks, 1977; Brown and Ho, 1986; Kalinska et al., 1986).
REGULATION OF SECRETED PROTEINS IN CEREAL ALEURONE
63
IV. Mechanism of Hormone Action
A. HORMONE BRCEFTION Despite the considerable effort devoted to understanding the control of aamylase gene expression by GA and ABA in aleurone cells, there is remarkably little concept of how the initial steps in the process occur. Perhaps this is due, at least in part, to the tendency in molecular studies of plant hormone action to approach the problem in a reverse order beginning at the organhissue or protein level and progressing backward through RNA and DNA and then to hormone perception. The wave of study of hormonal control of a-amylase gene expression in aleurone cells is currently at the DNA level. Presumably, it will not be long before there will be a much greater effort to understand the events involving hormone perception, signal propagation, and finally, regulation of gene transcription and other events leading to altered patterns of gene expression. There have been only two attempts to detect hormone binding molecules in aleurone cells. Both in vivo (barley) (Keith et al., 1980) and'in vitro (wheat) (Jelsema et al., 1977) studies have detected hormone binding, but these studies have not yet led to the identification of hormone receptors or to an explanation of the processes of hormone perception. It would appear that new approaches are required if progress is to be made in this area. A number of such new approaches have been initiated by Hooley and colleagues (1990) who have demonstrated that aleurone cell protoplasts from oat make a-amylase when incubated with GA immobilized on inert beads. If GA does not enter the cell, the intriguing implications are that GA perception occurs at the cell surface and that the hormone does not have to enter the cytoplasm to regulate gene expression. It may also mean that if GA receptors exist, they may be integral membrane proteins. If this is true, our current concept of GA action would require considerable modification. Another hopeful approach involves the use of anti-idiotypic antibodies. An anti-idiotypic antibody is an antibody to the idiotype or antigen-binding region of another antibody and, as such, may mimic some of the configuration of the antigen against which the first antibody was raised. If the antigen was a hormone, then the anti-idiotypic antibody may recognize the hormone receptor (Strossberg and Schreiber, 1984). Anti-idiotypes have proven to be useful as receptor probes in animal (Farid and Lo, 1985) and plant (Pain ef al., 1988) systems, An anti-idiotypic antiserum raised against antibodies to GA has been found to block GA action in oat aleurone protoplasts (Hooley et al., 1990). This research is very encouraging and perhaps will lead to the production of probes to localize or purify GA receptor(s) in aleurone cells or to clone the gene(s) for receptor protein(s). Because antibodies are not thought to enter intact cells, and
64
RUSSELL L.JONES AND JOHN V. JACOBSEN
because the anti-idiotypic antiserum agglutinated the oat protoplasts, these results also indicate that factors which are involved in GA action exist on the cell surface, thus supporting the observation that immobilized GA can induce ctamylase synthesis. However, anti-idiotypic antibodies may also recognize GA uptake or metabolizing proteins so any moiety recognized by the probe is not necessarily a receptor. Other approaches, such as photoaffinity labeling and the use of immobilized GA in affinity chromatography, have not yet offered much hope of progress. This is due mainly to lack of specificity of binding to these probes (Hooley et ul., 1990). No studies of ABA perception in aleurone cells have yet been reported even though ail of the above approaches are as applicable to the study of ABA action as they are to GA. In summary, the study of hormone perception in aleurone cells is in its infancy. Despite indications that GA perception may occur at the cell surface and may involve an integral membrane GA receptor protein (Hooley et al., 1990), there is no indication yet of how the initial hormone recognition event is translated into an altered pattern of gene expression or other cellular states. B. H O R M O N E ~ E NINTERACTION E In keeping with current concepts of gene regulation (Maniatis et af., 1987; Johnson and McKnight, 1989). it is thought that a-amylase genes are regulated by a hormone-dependent interaction between short base sequences (GA responsive element or GARE) in the promoter (cis-acting) and factors which are probably DNA binding proteins (trans-acting factors). The binding proteins would arise by GA-dependent new synthesis or they may be pre-existing proteins that undergo hormone-dependent activation or relocation. The cis- and trans-acting factors in combination with RNA polymerase, perhaps with the direct participation of GA, would control gene transcription. One or more interactions between these factors in the promoter would be necessary to promote efficient gene transcription. The current wave of activity among research groups working on aamylase gene regulation is to identify the cis- and trans-acting elements. It is possible also that ABA inhibits a-amylase synthesis by causing interaction of ABA-specific trans-acting factors and ABA-responsive elements (ABAREs) in the gene promoter. I
Cis-acting Elements
The classical approach to identifying cis-acting elements has been to perform functional analyses of a promoter deletion series. Progressively shortened promoter fragments are fused to a reporter gene, and the ability of the fragments to initiate reporter gene transcription is assayed in an expression system. Transient expression of such chimaeric plant genes in plant protoplast systems has been
REGULATION OF SECRETEDPROTEINS IN CEREAL ALEURONE
65
obtained in a number of cases (Howard et al., 1987; Marcotte et al., 1989; Roussell et al., 1988), and similar systems for the analysis of a-amylase promoters have been devised. Promoter analyses have been made for a low-PI gene from wheat expressed in oat aleurone protoplasts (Huttly and Baulcombe, 1989) and for a high-pI gene from barley expressed in barley aleurone protoplasts (Jacobsen and Close, in press). GA-responsive zones were located between positions -289 and -164 of the wheat gene and -174 and 4 1 in the barley gene. The major functional zone delineated in the barley gene promoter (see pHv19, Table 111) contains all of the highly conserved sequences previously described (pyrimidine, -1 30, -105, and TATA boxes) (Huang et af.,1991; Jacobsen and Close, 1990), but the functional zone of the wheat a-amylase gene promoter contains only a pyrimidine box, the other sequences being located much closer to the transcription initiation site (Huttly and Baulcombe, 1989). Thus, of these highly conserved sequences, only the pyrimidine box appears to be still a candidate for a GARE. In support of this, a rice gene promoter sequence bound by a GA-related trans-acting factor from rice (see Section IV,B,2) contains two pyrimidine boxes (Ou-Lee et al., 1988). Pyrimidine boxes occur in the promoters of other GA-regulated genes, but the other conserved sequences of a-amylase genes (-130, -105) do not (Huang er al., 1991). Therefore, if there is a sequence which is common to all GA-regulated genes, a unique GARE, the pyrimidine box would appear to be the most likely candidate. It may be too simplistic to expect cis-acting elements to be short base sequences alone. The steroid-responsive elements identified in animal cells are all 13mer or 1Smer palindromes (some imperfect; Beato, 1989) suggesting that these regions are recognized by dimeric receptor proteirdhormone complexes with the receptor molecules being joined back-to-back so that each recognizes one half of the palindrome. The occurrence of palindromes may also indicate the possibility that stem and loop structures are formed and that these may be part of the gene regulating complex. Palindromes have been found in regulatory elements of several plant genes, for example, in the heat shock response elements of heat shock genes and in the ABAREs of the genes for Em protein of wheat and the rab genes of rice (see Table IV). Ou-Lee et af. (1988) have identified a palindromic sequence associated with the pyrimidine box in a binding-protein-protected zone of a rice gene promoter, and Table IV shows that all of the barley a-amylase gene promoters examined so far contain pyrimidine-rich sequences. However, the palindromes are not all the same, and if these sequences are involved in gene regulation, it is likely to be the conformation of the DNA rather than the base sequence that would be the important feature. Table IV also shows some examples of the same thing in other GA-regulated genes from wheat, rice, and barley. In the near future, GAREs will be further defined by functional analysis and promoter deletion studies, by footprinting, and by site-directed mutagenesis.
RUSSELL L. JONES AND JOHN V. JACOBSEN
66
TABLE IV PYRIMIDINE BOXESA N D PALlNDROMiCSEQUENCESIN PROMOTER REGIONS OF GA- A N D ABA-REGULATED GENES IN ALEURONE CELLSa Sequence location
Clones
Pyrimidine box/palidromeh
Barley a-amylase genomic clones Low pl
Arny32b
Amy32b
-
- 129
G A-T G-C A-T CC'ITITCTCGTAAC - GGTATCC
- 205
G-C A-T C -T C-G C-G A-T T-A e C - G GA - TAGTCGTATCTTTTCC - GAATT A -T
- 197
gKAmy155.3
+I:::
CATTIT - AGGCTGGT
High PI
gKAmy 141.117 AmypHV 19 Amy6-4 Amy46 gRAmy 152 gRAmy56
G-C
-
165 10 - 174
At 2
C C m - AC
Other genomic clones
Xniy2/54 (wheat, low PI) (Huttly cf al., 1988)
-
143
b CC-CGT
A-T G-C A-T C -G A-G A-T - ATCCATGC
REGULATION OF SECRETEDPROTEINS IN CEREAL ALEURONE
67
TABLE IV (continued) Clones
OSamy-a (rice a-amylase) (Ou-Lee era/., 1988)
Sequence location
- 166
Pyrimidine boxlpalidrome” T-A T-A T-A T T C-G T-A C T m T A T C -TAG
-
A T-A T T Aleurain (barley) (Whittier et al., 1987)
OSg 2 (rice) (Huang et al., 1990)
- 425
T -A TGCTCTT - ACAT
- 323
C-G T-A AT -CTCA -C-T
-238
C-G G-C G G T -A GGACACG - CAGCAG
- 182
c-G G-C G-C T-A CCGTACG - CCGCC
Promoter regions of ABA-regulated genes
EM protein (Marcotte et a/., 1989)
rab A (rice) (Yamaguchi-Shinozaki el a/., 1990)
“Exceptwhere noted, clones are referenced in Table 11. ”Palindromes are depicted as stem structures.
There has been no success reported yet in locating ABAREs in a-amylase promoters. However, in both the studies of promoter function described above (Huttly and Baulcombe, 1989; Jacobsen and Close, in press), deletions to at least the 5 ’ margin of the GA-responsive zone were still ABA responsive and in the case of the barley gene, all deletions to -1 18 retained some ABA responsiveness, indicating that the ABARE does not exert its effect on transcription at a site upstream from the GARE but at the same site or downstream from it.
68
RUSSELL L. JONES AND JOHN V. JACOBSEN
2 . Trans-actingFactors The trans-acting factors from barley have not been characterized, but a factor has been isolated from rice aleurone cells that binds to the promoter of an apparently low-pl rice a-amylase gene promoter (Ou-Lee et al., 1988). The factor was obtained only from GA-treated aleurone. In exonuclease 111 protection experiments, the factor protected an 80 base pair region which contained a sequence from -143 to -164, including two pyrimidine-rich sequences, a potential stem and loop structure, and an imperfect tandem repeat, all overlapping in the 22 base pair sequence. Functional analysis of this promoter has not been reported, nor is it clear whether this factor binds GA (Ou-Lee et a f . ,1988). The possibility that hormone-dependent synthesis of a trans-acting factor may be part of the signal propogation pathway for GA is consistent with experiments that indicate that both continued protein synthesis and GA are required for synthesis of a-amylase mRNA (Muthukrishnan et al., 1983a,b). The simplest interpretation of these results is that the trans-acting factor is a GA-induced protein. If this were so, then the possibility of GA control of genes for DNA binding factors arises. However, all of the above results have alternative interpretations. For example, a requirement for continuous protein synthesis may also be consistent with a need for continued synthesis of a short-lived receptor, as is the case for steroids (see Fig. 4). Many questions remain to be answered in this area of hormone research.
C . THESTEROID MODEL Because of similarities in structure and biosynthesis of steroids and GAS, it is thought that the mechanism of GA action may resemble that of the steroid hormones. The action of steroids has been the subject of numerous studies, and although many aspects are still obscure, there is a good general understanding of the broad features. Our knowledge of GA action is embryonic in comparison, but there is enough information to begin testing the validity of the hypothesis that GA acts like the steroids. Figure 4 shows a recent model of steroid action. The receptors (solid black figures) are proteins with short half-lives of 4 hr, and all forms of the receptors, either activated [with estrogen, (E)] or nonactivated, occur mainly in the nucleus (Greene and Press, 1986). Steroid molecules pass through the plasma membrane and are ferried through the cytoplasm by lowaffinity carrier molecules into the nucleus, where they combine with receptor molecules giving rise to an activated (probably dimeric) steroid-receptor complex. In combination with RNA polymerase, the complex binds to specific sites on the chromatin and influences gene transcription by interacting with palindromic steroid responsive elements in the gene promoter. A number of these elements have been identified and sequenced (Beato, 1989).
REGULATION OF SECRETED PROTEINS IN CEREAL ALEURONE
I1
Nucleus
Cytoplasm YA
69
I/
ACTIVATION
\I
P&T TRANSLATIONAL MODIFICATICW
FIG.4. Diagram representing the principal features of estrogen action in animal cells. (After Greene and Press, 1986.)
The actions of steroids and GA clearly have a lot in common. Both hormones induce the synthesis of a number of proteins, some of which are secreted. Protein synthesis is regulated by mRNA levels which, in turn, are regulated by increased rates of gene transcription, at least in part. Regulation of transcription appears to involve HREs in the gene promoters. However, at this point, the similarities appear to end. Soluble GA-binding proteins with properties of specific GA receptor molecules have been detected in various plant tissues in which the hormonal response involves cell elongation (Venis, 1985; Stoddart, 1986; Srivastava, 1987; Romanov, 1989), but proteins with similar receptor-like properties have not been reported in aleurone cells. In fact, it appears that perception is a cell surface phenomenon (Section IV,A). There is reason to think that in aleurone cells, the mechanism of GA action may differ from that of steroids in animal tissues. If the perception of GA in aleurone cells is a cell surface phenomenon, then the signal propagation pathway leading to regulation of gene expression for GA must also be different from steroids. It is obviously premature to discuss such processes, but a number of pathways by which cell surface receptors give rise to
70
RUSSELL L. JONES AND JOHN V.IACOBSEN
intracellular messengers are known in animal cells and no doubt will become of greater interest to GA physiologists if the surface receptor concept withstands the test of time.
V. Intracellular Transport and Exocytosis of Secretory Proteins A.
ROUTEOF SECRETION
It is now generally accepted that hydrolytic enzymes are secreted from the barley aleurone layer along the constitutive pathway (Jones and Robinson, 1989). This route of secretion is characterized by the absence of mechanisms for concentrating the secretory product in the cytosol, in contrast to secretion along the regulated pathway, where secretory products accumulate in the cytosol in condensing vacuoles which release their products across the plasma membrane in response to an appropriate signal (Farquhar, 1985; Gebhart and Ruddon, 1986). Ultrastructural studies of the aleurone layer coupled with autoradiography (Chen and Jones, 1974a), histochemistry (Ashford and Jacobsen, 1974; Pyliotis et al., 1979; Jones, 1987), or immunocytochemistry (Gubler et al., 1986, 1987; Zingen-Sell er al., 1990) show that the endoplasmic reticulum (ER) and Colgi apparatus contain secretory proteins, but condensing vacuoles are not apparent in these preparations (Fig. 2). Pulse-chase labeling experiments with barley aleurone layers confirm that secretory proteins do not accumulate in the cytosol and that turnover of secretory vesicles i s rapid. Thus, labeled secreted proteins accumulate in the incubation medium within 10-15 min ofexposurc of aleurone layers lo labeled amino acids (Chen and Jones. 1974b). Evidence for the rapid turnover of secretory vesicles in secretory plant cells comes from the detailed morphometric analyses of Steer and others reviewed by Steer (1988). By measuring the production of Golgi vesicles under conditions of steady-state secretion, Steer ( 1988) calculated that an area of membrane equal to that of the entire surface area of the cell is produced every 10 min in pollen tubes, root cap cells, and epidermal cells. Another distinctive feature of constitutive secretion is the close coupling between the synthesis and the export of secretory proteins (Gebhart and Ruddon, 1986). In the barley aleurone the rate of secretion of a-amylase is very closely coupled to its rate of synthesis (Chrispeels and Vamer, 1967b Higgins et a/., 1976; Jones er al., 1987). and it has proven difficult to experimentally separate the processes of enzyme synthesis and secretion in this system. The calcium ion (Moll and Jones, 1982) and the sodium ionophore monensin (Akazawa and Hara-Nishimura, 1985; Mitsui er al., 1985; Heupke and Robinson, 1985: Melroy and Jones, 1986) block a-amylase secretion from barley aleurone and rice scutellum tissues without significantly affecting a-amylase synthesis. In the
REGULATION OF SECRETED PROTEINS IN CEREAL ALEURONE
71
case of monensin-treated barley aleurone, a-amylase accumulates to high levels within the Golgi apparatus because monensin preferentially inhibits intracellular transport of this enzyme (Heupke and Robinson, 1985; Melroy and Jones, 1986). Withdrawal of calcium or addition of monensin preferentially inhibits the secretion of the AMY2 amylase isoforms in barley aleurone (Jones and Jacobsen, 1983; Melroy and Jones, 1986) and R-form isoforms in rice scutellum (Mitsui et al., 1985). These results led to the proposal that the two isoform groups may be secreted along different pathways, one involving the transport of AMY2 and Rforms of amylase via the Golgi apparatus along a monensin- and calcium-sensitive pathway, and the other the secretion of AMYl and S-forms along a monensin-insensitive pathway bypassing the Golgi apparatus (Akazawa and Hara-Nishimura, 1985). But immunocytochemical localization of AMY 1 and AMY2 in barley aleurone using antibodies raised against these two isoform groups shows that both groups of a-amylase are secreted to the cell exterior via the Golgi apparatus (Zingen-Sell et al., 1990). Differences in the activities and stabilities of AMYl and AMY2 at low calcium concentrations (Bush et a[., 1989b) may provide another explanation for the preferential effects of calcium withdrawal and monensin treatment on the two groups of cereal a-amylase. Whereas AMY 1 isoforms retain activity and are stable at Ca2+concentrations below 1 pM,AMY2 isoforms are inactivated and are unstable at concentrations of Ca2+below 1 pM. We speculate that monensin and low external Ca2+concentrations lower the Ca2+concentration in the endomembrane of the aleurone cell. This speculation is supported by two observations. First, we have shown that GA and Ca2+are required to maintain elevated Ca2+levels in the endomembrane system of the aleurone for the synthesis of active amylase (Bush et al., 1989a,b), and second, that monensin could affect the permeability of the Golgi apparatus membranes to ions (Bush et al., 1988). Monensin could therefore affect the synthesis of AMY2 isoforms by dissipating the Ca2+gradient across the endomembranes of the aleurone cell, whereas calcium withdrawal would directly affect CaZ+availability. Whereas membranes of secretory vesicles and the plasma membrane form the barriers that must be crossed by animal cell secretory proteins, secretory proteins of plant cells must also traverse cell walls before they reach target substrates. It is well documented that plant cell walls have a limited porosity to macromolecules (Carpita et a[., 1979; Fincher, 1989), and the movement of hydrolytic enzymes through the cell wall of the barley aleurone provides evidence that plant cell walls can function as a barrier to the release of secreted proteins (Ashford and Jacobsen, 1974; Pyliotis et al., 1979; Jones, 1987). The cell wall of the aleurone layer is a two-layered structure composed of a thin layer approximately 60-nm wide adjacent to the plasma membrane and a thick wall matrix up to 5-pm wide (Taiz and Jones, 1970, 1973). Following
72
RUSSELL L. JONES AND JOHN V.JACOBSEN
treatment with GA. the cell wall matrix is digested, whereas the inner wall layer, also referred to as the resistant wall, remains intact (Taiz and Jones, 1970). Electron microscopy shows the resistant wall to be fibrous, but the matrix of the wall lacks structural detail at the electron microscope level. Investigations of the chemistry of the aleurone cell wall show that the matrix consists primarily of arabinoxylan (85%) and cellulose (8%) (McNeil et al., 1975) organized as a cellulose-reinforced gel (Fincher and Stone, 1986), but little is known about the chemistry of the resistant wall layer (Fincher, 1989). Histochemical studies of the aleurone layer provide the best evidence that the cell wall matrix functions as a barrier to the release of enzymes (Ashford and Jacobsen, 1974; Pyliotis et al., 1979; Jones, 1987; Benjavongkulchai and Spencer, 1989). A preformed extracellular acid phosphatase can be localized by histochemical methods in aleurone cells that have not been exposed to GA (Ashford and Jacobsen, 1974; Pyliotis et al., 1979; Jones, 1987). This enzyme activity is located between the plasma membrane and the resistant wall layer and in the interstices of the resistant wall, but is absent from the cell wall matrix (Jones, 1987). Following treatment with GA this preformed phosphatase as well as newly synthesized enzymes such as a-amylase (Gubler et al., 1987) are released from the aleurone layer along channels that are digested in the matrix of the wall. Digestion of the cell wall matrix becomes extensive with increasing duration of GA treatment, but the resistant wall is not degraded. These observations show that the undigested cell wall matrix is impermeable to enzymes, whereas the inner wall layer is relatively freely permeable. It is presumed that the digestion of the cell wall matrix is catalyzed by xylanases produced by the aleurone layer in response to GA (Taiz and Honigman, 1976; Fincher, 1989). From the foregoing, it is clear that in plants the transport of secretory proteins from sites of synthesis to target substrates involves both secretion and diffusion. It is also clear that the process of diffusion through the cell wall can be regulated by enzymes that make the cell wall permeable to proteins. The existence of a large pool of enzymes diffusing through the cell wall complicates studies of the process of intracellular transport and secretion in plants. It is frequently difficult to distinguish experimentally the transport of newly synthesized proteins in the cytoplasm from that in the cell wall (Vamer and Mense, 1972; Moll and Jones, 1982). For example, Varner and Mense (1972) argued that the principal role of CaL+and other ions in stimulating the secretion of a-amylase from aleurone layers was via effects on the diffusion of enzymes through the cell wall. Experiments by Moll and Jones (1982). on the other hand, suggested that the role of Ca2+in enzyme secretion was at a metabolically dependent step. Only recent work with wall-less aleurone protoplasts provided convincing evidence that the effects of Ca'+ were not limited to its role in facilitating enzyme diffusion through the cell wall (Bush et al., 1986).
REGULATION OF SECRETED PROTEINS IN CEREAL ALEURONE
73
B. SIGNAL SEQUENCES AND TRANSLOCATION INTO THE ENDOPLASMIC RETICULUM(ER) LUMEN Molecular cloning of cDNAs encoding proteins synthesized by the ER of barley and wheat aleurone cells shows that their derived amino acid sequences possess signal peptides (Jones and Robinson, 1989). Among these proteins are secreted hydrolases, such as a-amylase, P-glucanase, and cysteine protease, and nonsecreted proteins, such as aleurain, that may be transported to intracellular compartments (Holwerda er al., 1990). The signal peptides of plant-secreted proteins are similar to the signal sequences of other eukaryote-secreted proteins. Von Heijne (1985) formulated rules for the physical properties of amino acids in signal peptides after studying more than 100 signal sequences. These rules stipulate that the N-terminus of the peptide consists of a short region of one or two positively charged amino acids, a core of about seven hydrophobic amino acids, and a carboxyl terminus of a minimum of five small and neutral amino acids. The derived amino acid sequences of proteins synthesized on the ER of aleurone cells fit the rules of von Heijne almost precisely. There is now convincing evidence that the signal peptide of plant secretory proteins functions to direct these proteins to the lumen of the ER. First, when mRNAs for secreted proteins are translated in virro, the resulting proteins have molecular masses that are 1500-2000 Da larger than the corresponding secreted forms of the proteins (Okita er al., 1979; Boston er al., 1982; Higgins et al., 1982). Second, the addition of microsomal membranes derived from dog pancreas (Boston er al., 1982; Higgins et al., 1982) or from barley aleurone layers (L. Sticher and R. L. Jones, unpublished observations) to an in v i m protein synthesizing system containing barley a-amylase mRNA can bring about the cleavage of a 1.5- to 2.0-kDa peptide from cereal a-amylase. Adding membranes isolated from barley aleurone also results in the cotranslational modification of other plant-secreted proteins synthesized in virro. For example, barley microsoma1 membranes are as efficient as dog pancreas microsomal membranes in the in virro processing of tomato proteinase inhibitor I (K. Osteryoung, L. Sticher, A. Bennett, and R. L. Jones, unpublished observations). There have been other reports of the efficient cotranslational processing of secreted proteins by microsoma1 membranes isolated from plants. Microsomes from wheat germ (Prehn er al., 1987) and immature kernels of Zea mays (Torrent er al., 1986; Campos er al., 1988) will cotranslationally modify plant secretory proteins, and the membranes from wheat germ will also support the cotranslational modification of animal secretory proteins in v i m . That there is considerable homology between the mechanisms for the synthesis and translocation of proteins among the kingdoms is indicated by experiments showing that plant secretory proteins can be synthesized and secreted
14
RUSSELL L. JONES AND JOHN V. JACOBSEN
from animal, fungal. and bacterial cells. This is particularly well demonstrated with respect to the cereal a-amylases where DNAs encoding them have been expressed in Saccharomyces ceresisiae (Rothstein et al., 1987). Xenopus faevis oocytes (Aoyagi et al., 1990), and Escherichia coli (Gatenby et al., 1986). The cereal a-amylases synthesized in these heterologous systems were secreted in the catalytically active form.
C. POSTTRANSLATIONAL PROTEIN MODIFICATION Proteins translocated into the ER of aleurone cells are posttranslationally processed in a variety of ways. Metalloenzymes, such as a-amylases, bind Ca2+; proteases, such as aleurain and cysteine endoproteinase are proteolytically processed from a proenzyme; other enzymes, including aleurain, p-glucanase and a nuclease are glycosylated; and a-amylases are posttranslationally modified by a lowering of their isoelectric points. The binding of metals to proteins may be among the first posttranslational modifications that occur in the ER. In the case of the a-amylase isoforms of barley aleurone it has been shown that the binding of at least one atom of calcium is essential for the activity and the stability of the enzyme (Bush er at., 1989b). We propose that the binding of Ca2+to barley a-amylase occurs as the protein is translocated into the lumen of the ER. Our proposal is based on two observations. First, analysis of the secondary structure of barley a-amylase (Fig. 5) (Bush et al., 1989b) using Chou-Fassman rules (Chou and Fassman, 1978) shows many similarities with that of porcine pancreatic amylase (PPA) determined crystallographically (Buisson et al., 1987). In particular, the Ca2+ binding region in both barley amylase and PPA is near the amino terminus of the protein. probably at Asn-92, Asp-138, and Asp-149 of barley amylase (Buisson ef al., 1987; Bush et al., 1989b). Thus, it is possible that Ca2+binds to the amino terminal region of the a-amylase molecule before synthesis of the enzyme is complete. Second, isoforms of a-amylase that are known to be precursor forms and which are catalytically active (Jacobsen et al., 1988; Aoyagi el al., 1990) are the predominant form of amylase in the ER of the aleurone cell (L. Sticher and R. L. Jones, unpublished observations). Because the precursor forms of a-amylase in the ER are active, they must bind Ca2+in the ER. Although nothing is known about the mechanism of Ca2+binding to a-amylase in vivo, we speculate that the process does not occur spontaneously. This speculation is based on the observation that when affinity-purified a-amylase from barley is denatured by removal of Ca*+,enzymatic activity cannot be restored by the addition of Ca2+(Bush et al., 1989b). The reduced enzymatic activity of denatured a-amylase is not a result of changes in the primary structure of this protein. Rather, Ca2+removal alters only the secondary or tertiary struc-
75
REGULATION OF SECRETED PROTEINS IN CEREAL ALEURONE
I:
AMY2
(I
2I-
H
0-I-2-
P
AMY1
H
11
-I
-2
PPA
-71 -2
AMYI, AMY2 0 PPA
I
0
'
'
150
190
50 1
50
I
100
I
150
200
250
I
200
250
300 I
300
490
350 I
350
I
I
400
450 I
450
'
5~ I
500
AMINO ACID RESIDUE
FIG.5. A comparison of hydrophobicity (H) and predicted a-helix ( a , open horizontal bars) and P-sheet (P, closed horizontal bars) regions of AMY1 and AMY2 with hydrophobicity and known ahelix and P-sheet regions of porcine pancreatic a-amylase (PPA) (Buisson el al., 1987). AMY1 and AMY2 were aligned with PPA by an 18-amino acid residue offset with respect to PPA. The location of the three structural domains of PPA (A and C, light regions of hydropathy plot, and B, dark regions of hydropathy plot) are shown with our prediction of the corresponding regions in AMY. Disulfide bridges in PPA (C-C) and cysteine residues in AMY (C) where potential disulfide bridges could occur are also indicated. The four amino acid residues known to participate in CaZ+binding in PPA (N,D,D,H) are indicated with our prediction of the corresponding residues in AMY (N,D,D). Amino acid sequence numbers for AMYI, AMY2, and PPA start with the mature peptide and do not include the signal sequence. (Reproduced from Bush ef al., 1989b.)
ture of this enzyme (Bush et al., 1989b). We propose that the folding of secretory proteins in plants is similar to the process in animal cells in that it is catalyzed by proteins such as binding protein (BiP), a resident ER protein involved in assembly of secretory proteins (Pelham, 1989; Sambrook, 1990). Our proposal is based on the observation that a BiP-like protein can be detected in the ER of barley aleurone cells using antibodies raised against yeast BiP (D. S. Bush and R. L. Jones, unpublished observations).
76
RUSSELL L. JONES A N P JOHN V. JACOBSEN
After Ca2+binding, the a-amylases of barley are further posttranslationally modified. This modification leads to a lowering of the p1 of the a-amylase isoforms without causing a measurable change in protein mass, and it occurs in the endomembrane system (Jacobsen et ui., 1988; Aoyagi et a/., 1990; L. Sticher and R. L. Jones, unpublished observations). Several types of experiments have established that AMY 1 and AMY2 isoforms of barley are posttranslationally modified. The existence of precursor forms of a-amylase was initially inferred from experiments with barley aleurone protoplasts that showed that fewer isoforms were present in the incubation medium than were found in protoplast homogenates (Fig. 6) (Jacobsen et d.,1985). Experimental proof of the existence of precursor forms of AMY came from radiolabeling experiments using aleurone layers and protoplasts from Himalaya barley. Jacobsen et al. (1988) showed that radiolabel from [3'S]methionine was first incorporated into isoforms of AMY 1 with PIS of 4.90 and 4.64, and then chased into isoforms with PIS of 4.72 and 4.56 that were subsequently secreted. Experiments with the barley cultivar Betzes that secretes only one AMY 1 isoform having a pl of 4.72 but whose cytosol contains AMY 1 with PIS of 4.90 and 4.72 provided evidence that isoform pl4.9 was a precursor of PI 4.72. By inference, isoform PI 4.64 in the Himalaya cultivar is a precursor of a-amylase PI 4.56 (Jacobsen et al., 1988).
Frc. 6 . IEF of proteins from homogenates and incubation media of Xenopus oocytes injected with RNA and DNA. a-Amylase in incubation media of oocytes injected with poly(A+)RNA (lane 3 ) . pSP.M/C (lane 4). pSP. 1-28 (lanes 5 and 6), and pSP.E (lane 7), and homogenate of oocytes injected with pSP.E (lane 8). Native a-amylase containing AMY 1, AMY2, and AMY3 isoforms was used as a standard (lanes I and 2). Protein was delected by fluorography (lane I), enzyme activity (lanes 2-5. 7, and 8),and protein immunoblotting (lane 6). Incubation media or homogenates of oocytes injected with RNA (lane 3) or DNA (lanes 4-8) were collected after 2 4 days of incubation and 25 pI were loaded per lane. The positions of the a-amylase isoforms synthesized by oocytes injecred with plasmids are indicated by arrows (top to bottom: PI 4.7, 4.85, 6.0, 6.1). (Reproduced from Aoyagi e/ ol.. 1990).
REGULATION OF SECRETED PROTEINS JN CEREAL ALEURONE
77
Perhaps the most convincing proof that multiple forms of barley amylases arise by a posttranslational lowering of the PI comes from work with X . laevis oocytes. Aoyagi et al. (1990) have shown that when recombinant plasmids containing barley a-amylase cDNAs are injected into the nucleus of Xenopus oocytes, two enzymatically active forms of a-amylase are secreted into the incubation medium. When a recombinant plasmid containing cDNA clone E encoding AMY 1 isoforms of Himalaya barley (Rogers and Milliman, 1984) is injected into the oocyte nucleus, two isoforms with PIS of 4.9 and 4.72 that are indistinguishable from native AMY 1 in PI and enzyme activity are secreted into the incubation medium (Fig. 6). Similarly, when recombinant plasmids containing clone pM/C encoding AMY2 Himalaya isoforms (Rogers, 1985) are injected into oocytes, two isoforms with PIS 6.13 and 6.03 are synthesized and secreted (Fig. 6). Because two catalytically active isoforms are synthesized by oocytes programmed with a-amylase cDNA clones, we can conclude that two posttranslational modifications of the primary translation product occur in the oocyte. These modifications are the binding of Ca2+to confer catalytic activity to the aamylase molecule, and a modification that alters enzyme charge (Fig. 6). The nature of the posttranslational modification that lowers a-amylase PI without significantly changing enzyme mass is not understood nor is it known what function this change serves. We have shown that posttranslational protein phosphorylation, sulfation, and fatty acid acylation are not involved in this process. We speculate that the deamidation or transamidation of Gln may be involved in this posttranslational modification (Wold, 1985). Human salivary and pancreatic a-amylases are known to be posttranslationally modified by deamidation that causes a lowering of isoform PI without significantly affecting the molecular weights of these enzymes (Karn er al., 1974). Recently we isolated an enzyme activity from barley aleurone layers that will lower the PI of AMY 1 isoforms in v i m (L. Sticher and R. L. Jones, unpublished observations). This enzyme activity is located in the endomembrane system of aleurone cells, and the enzyme from barley will also lower the PI of human salivary a-amylase in vitro. It is unlikely that this posttranslational modification of barley a-amylase is involved in targeting of the protein to the cell exterior. We base this conclusion on the observation that the lowering of the PI of AMY 1 isoforms in barley is not essential for the secretion of the protein from aleurone cells or X . laevis oocytes. The precursor forms of AMY accumulate in the incubation medium albeit at lower levels than the product forms of the enzyme (Jones et al., 1987; Jacobsen et pl., 1988). Furthermore, when oocytes are injected with plasmids containing cDNAs encoding AMY 1 and AMY2, precursor and product forms of a-amylase are synthesized and secreted in approximately equal quantities. These observations support the hypothesis that secretion of a-amylase from the barley aleurone layer occurs by bulk flow along the default pathway (Jones and Robinson, 1989; Chrispeels and Tague, 1991).
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Many of the hydrolases synthesized by the aleurone layer are also glycoproteins. A mixed linkage P-glucanase (Fincher et a[., 1986) and nuclease (Brown and Ho, 1987). both of which are secreted proteins, contain a single asparaginelinked, high-mannose oligosaccharide, whereas the thiol protease aleurain, a nonsecreted protein, has two oligosaccharide side chains, one high-mannose chain, and one complex oligosaccharide (Holwerda ef al., 1990). The subcellular sites of aleurain and nuclease glycosylation have not been investigated in detail, and nothing is known about the roles of oligosaccharide side chains in the targeting of these proteins to the appropriate subcellular compartment. In addition to being a glycoprotein, aleurain is synthesized as a proenzyme (Holwerda et a/., 1990). Aleurain is a thiol protein that is similar to cathepsin H, a rat liver lysosomal thiol protease (Rogers et al., 1985; Whittier er al., 1987). Based on its cDNA sequence, aleurain is synthesized on the ER as a 37-kD polypeptide and subsequent glycosylation results in the synthesis of a glycoprotein of 42 kD. This 42-kD proenzyme is further processed to the 32-kD mature form by a process that is thought, based on similarities in their predicted amino acid sequences (Holwerda et al., 1990), to be analogous to the processing of rat liver cathepsin H (Nishimura and Kato, 1987). By aligning the predicted amino acid sequence of aleurain with the sequence of mature cathepsin H, Holwerda and colleagues propose that proaleurain is processed to its mature form by cleavage of an amino-terminal pro segment. Aleurain has been localized by immunocytochemical methods to compartments in barley aleurone cells that are similar in structure to microbodies (Holwerda et al., 1990). Microbodies containing enzymes of the glyoxylate cycle (glyoxysomes) have been identified in the aleurone cell (Jones, 1972), but it would be difficult to reconcile the presence of aleurain in glyoxysomes since aleurain is targeted to the ER by a signal peptide (Rogers et al., 1985). It is widely accepted that glyoxysomal enzymes are synthesized on free ribosomes and are imported into the glyoxysome posttranslationally by targeting signals that are present at the C-terminus of these proteins (Could et af., 1989). On the other hand, it is possible that the organelles identified as microbodies to which aleurain has been immunolocalized are indeed distinct lysosomal compartments in the aleurone cell. Germinating barley grains also contain a broad spectrum of protease activities that are synthesized and secreted from the aleurone layer (Jacobsen and Vamer, 1967; Sundblom and Mikola, 1972; Rogers et al.. 1985; Hammerton and Ho, 1986; Koehler and Ho, 1988) and scutellum (Mikola and Mikola, 1986). The posttranslational processing of these enzymes is only beginning to be investigated, but preliminary data show that these proteases are also synthesized as proenzymes (S. Koehler and T.-H. D. Ho, personal communication). The processing of carboxypeptidase I that is secreted from barley scutellum involves the excision of a 55-amino acid polypeptide from a primary translation
REGULATION OF SECRETED PROTEINS IN CEREAL ALEURONE
79
product (Doan and Fincher, 1988). Carboxypeptidases from barley possess two chains termed A and B, which in carboxypeptidase I consist of 266 and 148 amino acid residues, respectively, and the 55-amino acid polypeptide that is excised from the proenzyme links the A and B chains (Doan and Fincher, 1988). The precise mechanism of proenzyme processing is not understood, nor is it known where in the cereal grain these steps occur. Because carboxypeptidase activity is highest in the starchy endosperm, Doan and Fincher (1988) speculated that processing of the proenzyme to the active mature enzyme might occur after secretion of the enzyme into the endosperm.
VI. The Role of Calcium A. BARLEYAMYLASES ARE CALCIUM METALLOPROTEINS The requirement for calcium in the synthesis of barley a-amylase has been known for more than 50 years. The first experiments with aleurone layers isolated from barley endosperm showed that Ca", but not other divalent cations, was required at rnillimolar concentrations, in addition to GA, for the accumulation of a-amylase activity in the incubation medium (Chrispeels and Vamer, 1967b). Chrispeels and Vamer (1967b) proposed that Ca2+played a role in stabilizing secreted a-amylase molecules. The effect of calcium on the stability of barley a-amylase was studied in more detail by Jacobsen et al. (1970). They showed that the AMY1 and AMY2 isoforms of barley exhibited different stabilities in the presence of EDTA and proposed that AMY2 isoforms were calciumcontaining metalloproteins like a-amylases from animals, bacteria, and fungi, whereas AMY 1 isoforms were not calcium-containing enzymes, or that the different groups of barley a-amylase differ in their affinities for calcium. Bush et al. (1989b) have studied the calcium binding properties of affinity purified barley a-amylases in detail. They showed that the binding of calcium to barley a-amylase is essential for the catalytic activity and stability of all isoforms. There is a stoichiometric relationship between Ca2+binding and a-amylase activity, and maximum activity of both isoform groups is reached when one atom of Ca2+is bound per molecule of a-amylase. Although AMY 1 and AMY2 isoforms bind the same amount of Ca2+,the affinities of these two isoform groups for Ca2+differ by an order of magnitude. The concentration of Ca2+required for half-maximal activity of AMY2 is 3 p.~%!but for AMY 1 it is only 300 nM (Fig. 7). Differences in the affinities of barley a-amylases for Ca2+help explain the effect of added Ca2+on the formation of barley a-amylase in vivo as well as the differential effects of EDTA on the activity of barley a-amylase in vitro (Jacobsen et al., 1970). Thus, when barley aleurone layers are incubated in a
80
RUSSELL L. JONES AND JOHN V. JACOBSEN
1 :c 0.2
1
I
I
5
4
3
2
Co2+CONCENTRATION ($a) FIG. 7. The effect of Ca” concentration on the enzymatic activity of two isofoms of AMY and a double reciprocal plot of these data (inset). AMY I and AMY2 were incubated in solutionscontaining 10 mM KCI, 50 mM HEPES, pH 7.0, 100 phf CaCI,, and sufficient BAPTA to set the Caz*concentration beiween pCa 6.5 and 4. Amylase activity was measured after 4 hr of incubation by the AMY 1; AMY2 (Reproduced from Bush er al., 1989b). standard method. (0).
(a),
m,
medium containing no added Ca2+(actual external Ca2+= 10 only AMY 1 isofoms accumulate in the incubation medium, in contrast to layers incubated in 20 mM Ca2+which accumulate both AMY 1 and AMY2 to approximately equal levels (Jacobsen et al., 1970; Jones and Jacobsen, 1983). Calcium binding also protects barley a-amylase from proteolytic degradation. Chymotrypsin will rapidly degrade barley a-amylase that has been inactivated by incubation at pCa 7, whereas a-amylase incubated at pCa 4 is not attacked by this protease (Bush et al., 1989b). Bush et al. (1989b) proposed that the susceptibility of barley a-amylase to proteolytic degradation reflected a structural change in the a-amylase molecule following Ca2+removal. This proposal was supported by circular dichroism measurements and by the altered antigenic properties of the inactivated a-amylase molecule. The structural changes in Ca2+-depletedbarley a-amylase are different from those occurring in a-amylase isolated from other groups of organisms. a-Amylases from animals and bacteria can be reactivated by the addition of Ca2+to the inactivated enzyme in virro (Vallee et a/., 1959), but barley a-amylases cannot be reactivated after Ca2+depletion in vitm (Bush er al., 1989b). We speculate that the binding of Ca2+to barley a-amylase is catalyzed by enzymes such as BiP located in the lumen of the ER (Pelham, 1989; D. S. Bush and R. L. Jones, unpublished observations).
REGULATION OF SECRETED PROTEINS IN CEREAL ALEURONE
B.
81
INTRACELLULAR CA2+LEVELS AND CA2+TRANSPORT IN ALEURONE CELLS
The synthesis of catalytically active a-amylase by barley aleurone cells requires that Ca2+is present in the ER at a concentration of at least 3 pA4 (Bush et al., 1989a). The Ca2+content of the cytosol and ER of barley aleurone was studied using the fluorescent Ca2+indicator indo-1 (Bush and Jones, 1987, 1988; Bush et al., 1989a). Aleurone protoplasts were loaded with the free acid of indo-1 at pH 4.5 (Bush and Jones, 1987). Under these conditions indo-1 enters the cytosol and ER of the aleurone cell. Using the fluorescent ratio technique (Grynkiewicz et at., 1985), the cytosolic level of Ca2+was estimated to be = 200 nM in protoplasts incubated in GA plus 10 pM Ca2+,and = 350 nM in protoplasts incubated in GA plus 20 nM Ca2+(Bush and Jones, 1988). The levels of cytosolic calcium are homeostatically maintained over extended periods (up to 36 hr of incubation of protoplasts in GA in the presence or absence of Ca2+(Bush and Jones, 1988). Calcium in the ER was estimated by measuring fluorescence from membrane fractions isolated and purified from barley aleurone protoplasts loaded with indo-1 at pH 4.5 (Bush et al., 1989a). Calcium was present in the lumen of the ER at a concentration of at least 3 pM. This value represents only an approximation of the Ca2+concentration of the ER since indo-1 is only useful for quantitating CaZ+levels up to about 3 pkf (Grynkiewicz et al., 1985). By measuring the uptake of 45Ca2+into isolated ER membrane fractions, however, we have estimated the Ca2+concentration of the ER lumen to be greater than 1 mM (D. S. Bush and R. L. Jones, unpublished observations). We have shown that high Ca2+concentrations in the lumen of the aleurone cell ER play a role in the maintenance of a-amylase activity (Bush et al., 1989b). Endoplasmic reticulum vesicles containing active a-amylase were isolated from barley aleurone cells treated with GA and 20 mM Ca2+and incubated in solutions of pCa 4 or 7 in the presence and absence of the Ca2+ionophores A23 187 or ionomycin. &Amylase activity in the lumen of the ER was reduced only when ER vesicles were incubated in low Ca2+(pCa 7) in the presence of Ca2+ ionophores. When A23 187 or ionomycin was added to membranes incubated in pCa 4, the activity of amylase in the ER was unchanged relative to control, untreated membranes (Bush et al., 1989b). The maintenance of high lumenal ER Ca2+concentrations is brought about by a Ca2+transporter located on the ER membrane (Bush et al., 1989a). This transporter has the properties of a Ca-ATPase and is similar to the Ca2+transporter located on the ER of other plant cells (Buckhout, 1984; Bush and Sze, 1986; Lew et al., 1986). The K,,, of the aleurone ER Ca2+transporter for Ca2+is 0.5 yM, a value consistent with a role for this pump in transporting Ca2+from the cytosol into the ER (Bush et al., 1989a). An unique feature of the activity of the ER Ca2+pump in barley aleurone is its regulation by GA and ABA (Bush et al., 1989a; Bush and Jones, 1990). The ER
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RUSSELL L. JONES AND JOHN V.JACOBSEN
isolated from GA-treated aleurone layers contains higher levels of CaZ+ATPase activity relative to layers incubated in the absence of GA. There is a lag of about 4-6 hr following GA addition before the Ca2+transport activity of the ER increases, and the stimulatory effect of GA plateaus after about 18-20 hr of GA treatment (Bush er a]., 1989a). The time course of the GA-induced increase in Ca2+transport is almost identical to the time course of GA-induced a-amylase secretion. The effect of GA is not to increase the amount of ER membrane. The amount of ER determined by the activity of cytochrome c reductase (CCR) increases by about 40% after 18 hr of incubation in GA, whereas the activity of the Ca2+ transporter increases by more than tenfold (Bush ef al., 1989a). ABA, on the other hand, causes a marked suppression of the activity of the ER Ca2+ATPase without affecting the level of the ER marker enzyme CCR (D. S. Bush and R. L. Jones, unpublished observations). It is significant that the effects of GA and ABA on Ca" transport activity in the ER of barley aleurone parallel the effects of these two hormones on a-amylase synthesis. Cytosolic Ca2+is homeostatically maintained in GA-treated aleurone layers, indicating that as Ca" is transported into the lumen of the ER to support the synthesis of active and stable a-amylase molecules, a separate transport mechanism must operate to transport Ca2+into the cytosol. The cell exterior and the protein body vacuoles (aleurone grains) are two potential sources of Ca2+for the maintenance of cytosolic Ca" homeostasis in the aleurone cell. Experimental evidence indicates that in whole grain, either vacuole or starchy endosperm could supply the cytosol and ER of the aleurone cell with sufficient Ca2+to sustain the synthesis and secretion of a-amylase. Calcium is present in aleurone and starchy endosperm tissue of barley at about 1-5 mM (Stewart ef al., 1988; D. S. Bush and R. L. Jones, unpublished observations). In the aleurone cell most of the calcium is present in the protein body vacuole as an insoluble complex with phytic acid, and Ca" as well as other inorganic ions are released from phytic acid during germination or following GA treatment (Eastwood and Laidman, 1971; Gabard and Jones, 1986). The relative contributions of vacuolar or endosperm Ca" to cytosolic Ca'* homeostasis in the aleurone layer have not been established. However, the dependence of a-amylase synthesis by isolated aleurone layers on added Ca" indicates that Ca2+transport across the plasma membrane plays an important role in this process. Although a role for Ca" in the synthesis of mature a-amylase molecules has been established, it is not known whether Ca2+plays other regulatory roles in the barley aleurone cell. Like other higher plants, calmodulin genes are present in barley (Zielinski. 1987), and the gene is expressed in barley aleurone tissue (R. L. Jones, unpublished observations). A role for Ca-calmodulin in regulating the synthesis of hydrolases or in the secretion process has not been established. Although high external concentrations of Ca2+are required for the accumulation
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of active a-amylase in barley aleurone, the accumulation of a-amylase mRNAs is not dependent on high external Ca2+concentrations (Deikman and Jones, 1985, 1986). Thus the levels of mRNA for AMY 1 and AMY2 isofoms are increased by the presence of GA in the incubation medium but not by the presence or absence of Ca2+. There is widespread belief that Ca2+affects the secretory process in plants (Poovaiah and Reddy, 1987; Steer, 1988), but a direct effect on Ca2+on this process has not been well documented. Moll and Jones (1982) argued for a role of Ca2+in the secretion process in barley aleurone based on a kinetic study. They showed that the withdrawal of Ca2+from the medium surrounding isolated aleurone layers in a flow-through system caused a-amylase secretion to stop within a minute and that secretion resumed within a few minutes when the Ca2+was replaced (Moll and Jones, 1982). Vamer and Mense (1972) used a similar experimental approach to study the effects of Ca2+in isolated barley aleurone layers, but they concluded that the effect of Ca2+and other divalent cations was to facilitate movement of a-amylase molecules through the cell wall. Clearly, a more accurate description of the role of Ca2+in the secretory response in plants awaits advances that will permit a direct examination of the process of intracellular transport and secretion in reconstituted systems similar to those developed for the study of these processes in animal cells.
VII. Perspective Although molecular cloning has lead to a detailed understanding of the structure of some of the a-amylase genes in barley aleurone and their protein products, we lack a clear understanding of how these genes are regulated. The gap is conceptual. Plant biologists have yet to identify the entity in cells with which plant hormones interact leading to altered gene expression. Does GA recognize a receptor on the plasma membrane, as the experiments of Hooley et al. ( 1 990) suggest, or does GA bind to a receptor in the cytosol or nucleus? If the receptor for GA is located at the cell surface, what is the nature of the signal transduction pathway? If the GA receptor is cytoplasmic or nuclear, what is the sequence of events leading to a-amylase gene expression? Does a GA-receptor complex interact directly with the a-amylase gene promoter or is the sequence of events more complex? Establishing the precise cellular site of GA interaction with the cell and the nature of a presumed second messenger should be immediate goals in achieving an understanding of the molecular details of hormone action in the aleurone layer. The aleurone cell, one of the few digestive glands in plants, is an excellent experimental model for studies of transport processes. The mechanisms that regulate the intracellular transport and secretion of hydrolytic enzymes from the
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barley aleurone cell as well as the mechanisms involved in the targeting of proteins to subcellular compartments and the identity of these compartments need more detailed study. For example, is the cathepsin H-like protease aleurain targeted to a lysosomal compartment in aleurone cells? If so, what is the nature of this compartment? Is the protein body vacuole the analog of the lysosome in plants, or do bona fide lysosomes exist in barley aleurone cells? The role of Ca2+in the secretory process also needs to be resolved. Does Ca2+ function in the ER primarily to achieve the synthesis of active and stable proteins as shown by Bush et al. (1989b), or does ER Ca2+play a more central role in the secretion process (Sambrook, 1990)? The ER of the aleurone cell with its attendant, hormone-regulated Ca” pumps may be an ideal experimental system in which to test the hypothesis that ER Ca2+plays a crucial role in the secretion process. ACKNOWLEDGMENTS The help of Eleanor Crump in the preparation of this manuscript is gratefully acknowledged. Thanks also to Drs. David Baulcombe, Geoff Fincher, David Ho, Richard Hooky, Sandy MacGregor, John Mundy, Ray Rodriguez, and John Rogers for providing manuscripts of unpublished material during the preparation of the manuscript. This work was supported in part by grants from the National Science Foundation to RLJ.
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Gubler, F., Jacobsen, J. V., and Ashford, A. E. (1986). Plunru 168,447452. Gubler, F., Ashford. A. E.. Jacobsen, J. V. (1987). Planfa 172, 155-161. Guyette, W. A,. Matusik, R. J., and Rosen, J. M. (1979). Cell 17, 1013-1023. Haberlandt, G . (1884). “Physiologische F’tlanzenanatomie.” Engelmann. Leipzig. Hammerton, R., and Ho, T.-H. D. (1986). Plunr Physiol. 80,692-697. Hardie. D. G. (197.5). Phytochemisrry 14, 1719-1722. Harvey, B. M. R.. and Oaks, A. (1974). Plunr Physiol. 53,453457. Heupke. H.-J., and Robinson, D. G. (1985). Eur. J . Cell Biol. 39,265-272. Higgins, T. J. V.. Zwar, J. A., and Jacobsen, J. V. (1976). Nuture (London) 260, 166-169. Higgins, T. J. V.. Jacobsen, J. V., and Zwar, J. A. (1982). Plunr Mol. Biol. 1, 191-215. Hill, R. D., and MacGregor, A. W. (1988). Ad,.. Cereul Sci. Techno/. 7, 217-261. Ho. T.-H. D., Nolan. R. C., Lin, L., Brodl, M. R., and Brown, P. H. (1987). I n ”Molecular Biology of Plant Growth Control” (J. E. Fox and M. Jacobs, eds.), pp. 3 5 4 9 . Alan R. Liss, New York. Holwerda, 8.C., Galvin, N. J., and Rogers, J. C. (1990). Submitted. Hooky, R., Beale. M. H.. Smith. S. J., and MacMillan, J. (1990). In “Plant Growth Substances 1988” (R. P. Pharis and S. B. Rood, eds.), pp. 145-153. Springer-Verlag.Berlin. In press. Howard, E. A,. Walker. J. C., Dennis, E. S., and Peacock, W. I. (1987). Plunru 170,535-540. Huang, J. K., Swegle, M., Dandekar, A., and Muthukrishnan. S. (1984). J . Mol. Appl. Genet. 2, 579-588. Huang, N.. Sutliff. T. D.. Litts, J. C., and Rodriguez, R. L. (1991). Plum Mol. Biol. (in press). Hurtly, A.,and Baulcombe,D. C. (1989). EMBOJ. 8, 1907-1913. Huttly. A. K., Martiensen. R. A,, and Baulcombe, D. C. (1988). Mol. Gen. Genet. 214,232-240. Ingle. J., and Hageman. R. H. (1965). Plunr Physiol. 40,672675. Jacobsen. J. V. (1983). In “The Biochemistry and Physiology of Gibberellins” (A. Crozier, ed.), pp. 159-187. Praeger. New York. Jacobsen, J. V. ( I 986). Plum Physiol. 80, 35CL359. Jacobsen. J. V.. and Chandler, P. M. (1987). I n “Plant Hormones and Their Role in Plant Growth and Development” (R. P. Pharis. ed.), pp. 164-1 93. Nijhoff, Dordrecht, Netherlands. Jacobsen, J. V., and Close. T. (1990). Plant Mol. B i d . (in press). Jacobsen. J. V., and Higgins, T. J. V. (1982). Plant Ph-wiol. 70, 1647-1653. Jacobsen, J. V., and Knox, R. B. (1972).I n “Plant Growth Substances” (D. J. Cam, ed.), pp. 344-351. Springer-Verlag. Berlin. Jacobsen. J. V.. and Vamer, J. E. (1967). Plunr Physiol. 42, 1596-1600. Jacobsen. J. V., Scandalios, J. G . and Vamer, J. E. (1970). Plant Physiol. 24,367-371. Jacobsen, J. V., Zwar, J. A,. and Chandler, P. M. (1985). Planfa 163,430-438. Jacobsen. J. V., Bush. D. S.. Sticher, L., and Jones, R. L. (1988). Plant Physiol. 88, 1168-1 174. lelsema, C. L., Ruddat. M., M o m , J. D., and Williamson, F. A. (1977). Plant Cell Physiol. 18, 1009- 1019. Johnson. K. D., and Kende, H. (1971). Pro(.. Nurl. Acud. Sci. U S A . 68,329&3293. Johnson, P. F., and McKnight, S. L. (1989). Annu. Rev. Biochem. 58,799-839. Jones, R.L. (1971). PIunr Physiol. 47,412416. Jones. R. L. (1972). Plunru 103.95-109. Jones, R . L. (1987). Protoplusmu 138,7348. Jones. R. L., and Carbonell, J. (1984). Plunr Physiol. 76, 213-218. Jones. R. L., and Jacobsen. J. V. (1983). Plunru 158, 1-9. Jones. R. L.. and Robinson. D. G. (1989). New Phyrol. 111, 567-597. Jones, R. L., Bush, D. S.. Sticher, L., Simon, P., and Jacobsen, J. V. (1987). I n “Plant Membranes: Structure, Function, Biogenesis” (C. Leaver and H. Sze, eds.), pp. 325-340. Alan R. Liss, New York. Kalinska. A.. Chandra, G. R.,and Muthukrishnan, S. (1986). J . Biol.Chem. 261, 11393-11397.
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INTERNATIONAL REVIEW OF CYTOLOGY. VOL. 126
Multiphasic Uptake Mechanisms in Plants PERNISSEN Department of Biology and Nature Conservation,Agricultural University of Norway, N-1432 As-NLH,Noway
I. Introduction Plants have evolved remarkable but still poorly understood mechanisms for the uptake of nutrients from the soil. In response to an environment (soil solution) in which the concentration of nutrients may vary between exceedingly wide limits, inorganic ions and other solutes appear to be taken up in such a way as to minimize the energy required. Uptake of each solute is mediated by a single structure, presumably a membrane channel, which undergoes a series of allor-none transitions as the external solute concentration is increased. Uptake via the lower phases requires energy and has carrierlike characteristics whereas uptake via the higher phases is by facilitated diffusion and has channellike characteristics. The phenomenon of concentration-dependent discontinuities is well documented and has been shown to involve a separate transition site, at least for inorganic ions, but otherwise remains an enigma in molecular terns and may involve a hitherto unrecognized type of interaction between solutes and membrane components. The study of mechanisms of ion transport through plant cell membranes has been reviewed in this series by Epstein (1973). Through extensive studies, especially of concentration dependencies on the uptake of inorganic ions by plant roots and other tissues, Epstein and his co-workers have developed much of the methodology and concepts for what may be termed the carrier-kinetic approach to the study of transport. This article, which expands on a previous review of uptake mechanisms in plant roots (Nissen, 1991), will mainly be concerned with the influx of solutes across the plasmalemma, the plasma membrane of plant cells. Specifically, it will focus on the number and nature of the uptake mechanism(s), especially as deduced from studies of concentration dependencies and specificities. Similar studies of the activity of a plasmalemma-bound ATPase will also be reviewed. Transport across other plant membranes has been much less studied and will only be briefly considered. Because of the all-important role of plant roots in the uptake of nutrients from the soil, uptake of inorganic ions by roots has been extensively studied over the years. Such studies will form the basis of the present account, but uptake of amino acids, sugars, and other organic compounds will also be considered, as will solute uptake in other plant tissues, single cells, pro89 Copyright 0 1991 by Academic Ress, Inc. All righrs of reproduction in any form reserved.
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toplasts, vacuoles, and plastids. In addition, patterns for long-term accumulation of nutrient elements in intact plants will be considered briefly, as will concentration dependencies for uptake into lower plants and bacteria. The experimental conditions must satisfy certain requirements if influx across the plasmalemma is to be accurately determined (JensCn et al., 1987). In the usual radiotracer experiments in which plant tissue is allowed to take up a radioactively labeled ion or molecule from a solution, short (10 min) uptake periods and still shorter ( 5 min) desorption with cold, unlabeled solutions should preferentially be used. In order to measure only the solutes that have been taken up across the plasmalemma, solutes in the intercellular spaces and cell walls (free space) must be deducted or removed. Water suffices to remove anions in the free space but radioactively labeled cations bound to negatively charged sites must be exchanged by unlabeled cations, e.g., unlabeled uptake solutions. At low solute concentrations, rate-limitation by diffusion in the unstirred layer surrounding the plant tissue may possibly interfere with fluxes and with transport measurements. This limitation, which will produce deviations from Michaelis-Menten kinetics (Winne, 1973), can be alleviated by stirring, shaking, or vigorous aeration of the uptake solution (Polle and Jenny, 1971). Depletion of the investigated solute should also be avoided by the use of large solution volume to tissue ratios and short uptake periods, or should be corrected for. At high external concentrations it may be necessary to correct for ion activities, especially for di- and trivalent ions (Evangelou and Wagner, 1987) and for ion mixtures. It should also be checked that the tracer is appropriate, e.g., that "Rb' can be used as a tracer for K', something which is not always the case (Jacoby and Nissen, 1977). There is still, presumably due to incomplete consideration of the evidence at hand, no consensus on some basic and seemingly simple questions. Is uptake in plants mediated by a single mechanism, or are there two or more independent mechanisms for each solute? Is there also free diffusion across the plasmalemma? Is the relationship between uptake rate and external solute concentration continuous or discontinuous? The failure to rigorously address such questions is hampering the study and understanding of the molecular basis for solute transport across plant membranes. In this article it will be shown that data are available for a resolution of these questions and for an unequivocal evaluation and comparison of the various kinetic models. Specifically it will be shown that the relation between the rate of solute uptake and the external solute concentration can, in the majority of instances, be represented by a single, multiphasic curve or isotherm. Other evidence for and characteristics of multiphasic uptake mechanisms in plants will also be reviewed. Taking the earlier article (Epstein, 1973) as the point of departure for the present article seems appropriate. Epstein's overview of the competence of higher
MULTIPHASIC UPTAKE MECHANISMS M PLANTS
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plants in ion transport, of the sites of ion transport, and of experimental materials and methods, remains relevant and recommendable. In this article such information will be restricted to the discussion immediately at hand. The concept of multiphasic uptake mechanisms differs fundamentally from that of two independent mechanisms developed by Epstein and co-workers, and it may also be useful for this reason to consult the earlier article. A series of questions and answers in that article will, furthermore, be reconsidered in the light of present information and evidence.
11. Dual Model
In view of its wide acceptance and historicai importance, a brief presentation of the dual model of uptake seems in order. In a study of K+ uptake by barley roots, Epstein et al. (1963) represented the uptake as the sum of two MichaelisMenten terms, a high-affinity (low K,) term and a low-affinity (high K,) term. However, a closer examination of uptake in the high concentration range (above 1 mM) revealed a heterogeneous or “bumpy” isotherm in this range for C1(Elzam et a1.,1964) as well as for K+and Na+ (Epstein and Rains, 1965) (Fig. 1). This isotherm was taken as evidence for a spectrum of active sites with slightly
‘Ot I
I
I
I
I
10
20
30
40
50
K (mM1 FIG.1. (-), Uptake of K+ by barley roots; (---), maximal rate of uptake (VmJ by the high-affinity mechanism. (From Epstein and Rains, 1965.)
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different affinities in the low-affinity mechanism (Mechanism 2). The highaffinity mechanism (Mechanism 1) was considered to only have a single site. Although it will be shown later that the data of Epstein and co-workers cannot be resolved into two kinetically meaningful isotherms, cases of truly dual (or triple) uptake mechanisms do exist in higher plants. The isotherm for uptake of choline sulfate (the sulfate ester of choline) by barley roots could be resolved into three independent isotherms, each following Michaelis-Menten kinetics and spanning the entire range of l O - ' O - 10-I M (Nissen, 1974b; see Fig. 2 which shows the two upper isotherms). A pattern similar to that in Fig. 2 was also found for uptake of the analog 2-aminoethyl sulfate and for uptake of choline sulfate by leaf slices of barley. There was no indication of a third mechanism in leaf slices at very low concentrations. Competition experiments with a number of analogs of choline sulfate yielded, in agreement with these results, continuously curvilinear Dixon plots (Nissen, 1974b), in contrast to the multiphasic plots usually obtained (cf. Figs. 7-1 I , 24). These results are for constitutive uptake of choline sulfate. Bacteria-mediated uptake of choline sulfate by plants (Nissen, 1968, 1971a, 1973e; Bakkerud and Nissen, 1980) is outside the scope of this article.
-
-1.0
I
10'~
lo-'
lo-3
I
I
lo-'
Choline sulfate /MI FIG. 2. Uptake of choline sulfate by barley roots. Lines a and b, isotherms for mechanisms a and b. (From Nissen, l974b.)
MULTIPHASIC UPTAKE MECHANISMS IN PLANTS
93
111. Multiphasic Model
A. INITIAL EXPERIMENTS WITH SULFATE AND GLUCOSE
In a study of sulfate uptake by roots and leaf slices of barley (Nissen, 1971b), M, the uptake into both tissues followed Michaelis-Menten kinetics from lowest concentration used, to somewhere above M where there was an abrupt transition from the lower phase to a higher phase as shown for roots in Fig. 3. At a somewhat higher concentration, transition to still another phase occurred. Each phase followed Michaelis-Menten kinetics as revealed by straight lines in a Lineweaver-Burk plot or in an Eadie-Hofstee plot (Fig. 4), a plot in which any deviation from linearity is more reliably detected. The transition between phases 1 and 2 was accompanied by a jump, i.e., the isotherm was noncontiguous in this range, whereas the transition between phases 2 and 3 was discontinuous but contiguous. In a series of overlapping experiments, root uptake was found to be mediated by a total of eight phases up to 2.5 x lo-' M, the highest concentration used. This was confirmed in two comprehensive but less detailed experiments (Fig. 5). There were five phases for leaf uptake in this range (Fig. 5). The following conclusions were drawn regarding the kinetics of the uptake mechanism (Nissen, 1971b): 1. The uptake of sulfate can be described by a single, multiphasic isotherm. 2. The phases are separated by sharply defined inflection points. 3. Each phase obeys Michaelis-Menten kinetics. 4. The kinetic constants increase in a fairly regular manner, the increases in both V,, and K, being small at low to intermediate concentrations of sulfate. 5. Only one phase functions at any one concentration. The last conclusion is evident from the good agreement with Michaelis-Menten kinetics and from the finding that extrapolation and subtraction of any one phase
10.~
10' M SUlFATE
FIG.3. Uptake of sulfate by barley roots. (From Nissen, 1971b.)
PER NISSEN
94 V
600
400
200
FIG.4. Eadie-Hofstee plot of the data in Fig. 3. Linear segments, multiphasic interpretation; curvilinear isotherm. dual + diffusion interpretation. (From Nissen and Nissen, 1983.)
from the adjacent phases result in meaningless Lineweaver-Burk plots, often with negative values for V,,,. For uptake of glucose and 3-0-methyl glucose by potato slices in the range - 10.' M ,Linask and Laties (1973) independently arrived at similar concluI
I
I
I
1
'
10.5 10.4 10.3 10-2 10. M SULFA JE FIG.5. Uptake of sulfate by roots and leaf slices of barley. Transition points are connected by
straight lines. (From Nissen. 1971b.)
MULTIPHASIC UPTAKE MECHANISMS IN PLANTS
I
I
I
5
1
10
I
lo2
I
10’
I
lo4
95
I
lo5
GLUCOSE CONCENTRATION Ip M/ FIG. 6. Uptake of D-glucose by potato slices. (-), Theoretical curves calculated from Lineweaver-Burk plots of data (0); (---), extension of theoretical curves without all-or-none transitions. (From Linask and Laties, 1973.)
sions. Each of the four phases (Fig. 6) followed Michaelis-Menten kinetics as shown by linear Lineweaver-Burk, Eadie-Hofstee, and Hill plots. The in-parallel operation of four carriers was excluded since this would have caused these plots to be continuously curvilinear. B.
SEPARATE UFTAKE AND TRANSITION SITES
The phenomenon of discontinuous phase transitions was investigated in a series of competition experiments (Vange et a1.,1974a,b). In the experiment shown in Fig. 7, barley roots were allowed to take up [35S]sulfatefrom a 10-5M solution in the presence of various concentrations of unlabeled sulfate or tungstate. The large tungstate ion competitively inhibited uptake of [35S]sulfate,but as expected, more weakly than sulfate itself. The addition of sulfate or tungstate also caused transition from phase 1 to phase 2 at 2.9 x or 3.0 x lo-’ M ,respectively. Experiments with sulfite, molybdate, and chromate gave similar results (Vange et al., 1974b).These divalent anions were all equally effective in causing transitions for sulfate uptake but varied widely in their ability to compete for the uptake site. Particularly clear-cut results were obtained in experiments with phosphate and pyrophosphate. At pH 5.8, the pH of the unbuffered uptake solutions, phosphate exists mainly as the monovalent H,PO,- ion. This ion neither inhibited sulfate uptake nor caused transitions, even at a 1000-fold excess (Vange et al., 1974b). At pH 8.15, where phosphate exists mainly as the divalent HPO,,- ion,
PER NISSEN
96
0.5
0 Inhibitor
(M)
FIG.7. Dixon plot for uptake of ["S]sulfate M K,"SO,) by barley roots as a function of K,SO, or Na,WO, added. N o buffer added, pH 5.8. Uptake expressed as nmol (g fresh weight) ' (20 min)-'.Transition points indicated by arrows. (From Vange e t a / . , 1974a.)
phosphate was, however, as effective as sulfate in causing transitions in the up(Fig. 8). But the divalent phosphate ion did not compete for take of [ ~5S]sulfate the uptake site (the three lines for [35S]sulfateuptake in the presence of phosphate are parallel with the .r axis). This was confirmed in a more detailed experiment (Fig. 9) where it is evident that divalent phosphate caused a transition but had no direct effect on uptake. These findings were not caused, somehow, by the high pH. Similar results were obtained with pyrophosphate, both at pH 3.0 (Fig. 10) where it exists mainly as the divalent anion, and at pH 6.9 (Vange et al., 1974b) where it is mainly trivalent. In reciprocal experiments, sulfate had no effect on the multiphasic uptake of monovalent phosphate but caused phase transitions at high pH, i.e., from solutions where the divalent phosphate ion predominates (Vange eta/., 1974b). The results of these experiments, summarized in Table I and Fig. 11, constitute good evidence that there must be separate uptake sites and transition sites in multiphasic uptake of sulfate and phosphate. Only divalent sulfate analogs (including sulfite) competitively inhibited sulfate uptake, whereas all small divalent anions appeared to be equally effective in causing phase transitions. Monovalent phosphate had no effect on sulfate uptake. Reciprocally, sulfate caused transitions in uptake of divalent phosphate but had no effect on the
MULTIPHASIC UPTAKE MECHANISMS IN PLANTS
m 0 Z.SxIO-*
I
I
lo-’
2.5 x 10’
Inhibitor
97
J
(MI
M K2?S04) by barley roots as a function of FIG. 8. Dixon plot for uptake of [3SS]sulfate K,SO, (0)or KH,PO, (0)added. Tris-HCI buffer, pH 8.15. Uptake expressed and transition points indicated as in Fig. 7. (From Vange er al., 1974a.)
I
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pH 8.3
I
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o
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o 0
000
0.20
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I
Phosphate,
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_
M
M K,’%O,) by barley roots as a function of FIG.9. Dixon plot for uptake of [3sS]sulfate ( K,HPO, added. Tris-HC1 buffer, pH 8.3. Uptake expressed as in Fig. 7. (From Vange et al., 1974b.)
PER NISSEN
98
3.06
4 0.04
i0.02
0
lo-*
0 4x10”
Inhibitor, M FIG. 10. Dixon plot for uptake of [’5S]sulfate M K2-”S0,) by barley roots as a function of K,SO, or Na,P,O, added. pH adjusted to 3.0 with HCI. Uptake expressed and transition points indicated as in Fig. 7. The data for pyrophosphate are expanded (right ordinate) for clarity. Phase numbers (14)are indicated. (From Vange el ol., 1974b.)
-I
Interaction with:
V
Uptake site and transition site
C
.....................
~
C
C’
0
a
a
Transition site
No interaction
. Inhibitor FIG. 1 1 . Interaction with uptake site and transition site. Schematic Dixon plot. (From Vange el d..1974b.) See text for details.
MULTIPHASIC UPTAKE MECHANISMS IN PLANTS TABLE I SULFATE AND PHOSPHATE UPTAKE: INTERACTION OF IONS WITH UPTAKE AND M
Solute
Interacting anion Divalent sulfate analogs H,PO;
’SSO 4 2-
HP02-
H,P20,*m20,3~ ~ 3 2 ~ 0 , ~ 3 2 ~ 0 ~ 2 -
so42so,*-
DH 5.8 5.8 8.15 3.0 6.9 5.8 8.15
99 S E TRANSITIONS~
Inhibition ofuDtake
+
Effect on transitions
+
-
-
-
+ + +
-
-From Vange er a[.(1974b).
monovalent species. As indicated in the schematic Dixon plot (Fig. 1l), interaction with the transition site but not with the uptake site resulted in higher than expected uptake rates (b’ and c’ vs. b and c, see also Figs. 8 and 10). The reason for this is not clear. Data for uptake of phosphate by pea plastids (Emes and Traska, 1987) also indicate separate uptake and transition sites. Phosphoglyceric acid inhibited uptake but was without effect on the transitions between the upper phases (Nissen, 1991).
c.
UPTAKE OF INORGANIC IONS
Additional experiments as well as extensive reanalyses of previously published (and some unpublished) data have been carried out to determine to what extent the above findings for sulfate and phosphate uptake also apply to other plants and tissues and to other solutes. It should be noted, first of all, that multiphasic sulfate uptake has been found not only in multicellular root and leaf tissue but also in the unicellular green alga Chlorella (Biedlingmaier and Schmidt, 1989). Uptake was triphasic in the range - 10.’ M , with each phase having Hill coefficients of 1.0 (Fig. 12), i.e., following Michaelis-Menten kinetics. Detailed and wide-range experiments on phosphate uptake by corn roots at temperatures ranging from 5 to 30°C (Nandi ef al., 1987) revealed six (or seven) phases. Five phases are apparent in the range 3 x - 7.5 x lo-*M (Fig. 13). Phase 3 in this figure may possibly encompass two phases as indicated by subsequent experiments (Nandi ef ol., 1987). Phase 1 extended down to 2 x -4 x lo-’ M where there was a transition, in part in the form of a jump, to a sigmoidal phase which extended down to M ,the lowest concentration used (Nandi et al., 1987). Phases 1-3 obeyed Michaelis-Menten kinetics whereas phases 4 and 5 could be represented by curvilinear Lineweaver-Burk plots (Fig. 14). No phys-
0
1
2
3
4
log 5 I m M l FIG. 12. Hill plot for sulfate uptake by Ch/ore//afisca.Phase numbers given by Roman numerals. (From Biedlingmaier and Schmidt, 1989.) See text for details.
FIG. 13. Uptake of phosphate by corn roots. Heavy lines, Multiphasic interpretation; thin lines, single + diffusion interpretation (5 and 10°C) and dual + diffusion interpretation (25 and 30°C). (From Nandi era/.. 1987.)
MUL'IIPHASIC UPTAKE MECHANISMS IN PLANTS
101
0
Lf
L
I
2(
15
1/S ( k m o l
m-3)-'
FIG.14. Lineweaver-Burk plots for part of the data in Fig. 13. Extension of phases 4 and 5 to the Ilv axis is stippled (---), as is the extension of phase 4 to phase 3. Thin lines represent best fit of "continuous"models, see Fig. 13. (From Nandi et al., 1987.)
iological significance is claimed for the hyperbolae fitted to these data, but the hyperbolic curves do provide good fits and, by extrapolation, estimates of V-. Reanalyses of published data for phosphate uptake give similar multiphasic patterns, not only for roots, but also for leaves, and for Chforeffu(Fig. 15). The data were originally taken to represent uptake by two or three independent mechanisms, each following Michaelis-Menten or diffusion kinetics. However,
102
PER NlSSEN I
I
1
I
I
0
-
t
0
-
1
I
I
2
1
3
4
-
corn roots
1
:
~
c3 0
5 2
1
2
1
@ I
10.6
,
I
10-5
corn roots clover roots
2
1
?I
2
3
2
1
@ 01
10-7
corn roots
2
D
0
I
5
3
,
3
,
4
5
{
wheat roots
1
barley roots
{
Elodea leaves
i
Chlorella
I
I
I
I
10-4
10-3
10-2
10-1
Phosphate (kmol
m-3)
FIG. 15. Total concentration range and range of individual phases for experiments on phosphate 1, Ranges within which transitions occurred. Data: 1. experiment in Fig. 13; 2, Carter uptake. and Lathwell (1967): 3. Weigl (1968); 4, Edwards (1968); 5 . Edwards (1970): 6, Barber (1972); 7. Griinsfelder (1971): 8. Jeanjean et a/. (1970). See Nissen (1973b) for reanalysis of Data 5 , and ) reanalysis of Data 2-4 and 6-8. (From Nandi ct of.. 19x7.) Nissen ( 1 9 7 3 ~ for
(hv
these and other data have upon reanalysis been shown to be equally well or better represented by multiphasic isotherms (Nissen, 1973b,c). Data for uptake of other inorganic ions have also been shown (Nissen, 1973a,b.d, 1974a, 1977, 1980, 1987, 1991; Nissen and Nissen, 1983) to be equally or better represented by multiphasic isotherms than by the original, in most cases dual, interpretation. A case in point are the data in Fig. 1 for uptake of K' by barley roots. These data can be precisely represented by a single isotherm with two Michaelis-Menten phases (phases 2 and 3 in Fig. 1 in Nissen, 1973d) and three phases with curvilinear Lineweaver-Burk plots (Fig. 16) of the type found for phosphate uptake by corn roots (cf. Fig. 14). These phases are obtained without subtraction of the contribution of a Mechanism 1 (stippled line in Fig. 1). Subtraction of such a term results in meaningless or, at least, noninterpretable kinetics. Data for uptake of K' and Cl- by roots of tall wheatgrass (Elzam and Epstein, 1969) can also be represented by multiphasic isotherms (Fig. 17). The striking similarities between Figs. 16 and 17 and the equally striking similarities between the patterns for a cation and an anion (Fig. 17) indicate that the present interpretation and phase assignment are indeed correct. Further confirmation of this is provided by the finding that data for uptake of Na' (Epstein and Rains, 1965) and C1- (Elzam et al., 1964) by barley roots also yield similar multiphasic isotherms (Nissen, 1987).
MULTIPHASIC UPTAKE MECHANISMS IN PLANTS l/v I I
103
I
FIG.16. Lineweaver-Burk plot for uptake of K+by barley roots in the range 8-50 M.Data esMultiphasic interpretation; (-), dual interpretation; (---), extrapolatimated from Fig. 1. (-), tions. (From Nissen, 1987.)
0.10 .
I
I
l/v
70.08 I
-c
-
f
0 0
I
I
100 1/S (kmol m-3
20(
I-'
FIG. 17. Lineweaver-Burk plots for uptake of K+ and CI- by roots of tall wheatgrass in the range 5-50 mM. Data estimated from Figs. 7 and 2, respectively, in Elzam and Epstein (1969). (---), Extrapolations. (From Nissen, 1987.)
104
PER NISSEN
Data for uptake of boron, either as the tetrahedral B(0H); ion (Bowen, 1969) or as the nonionized B(OH), molecule (Nissen, 1973d), by leaf tissue of sugarcane (Bowen, 19681, can be represented by multiphasic isotherms with three Michaelis-Menten phases at low to intermediate concentrations (Fig. 54 in Nissen, 1973d) and two phases with curvilinear Lineweaver-Burk plots at high concentrations (Fig. 18). Boron uptake by barley roots was characterized by complex but precise multiphasic isotherms, with the phases separated in part by marked and reproducible jumps (Bowen and Nissen, 1977). Nitrate uptake by dwarf bean plants was also multiphasic, as shown by depletion experiments (Breteler and Nissen, 1982). At low external nitrate concentrations there were four uptake phases which were separated by sharp
A ( 0.5 mot m-3 CaCI21
__-___----
I
I
50
10
l/S (kmol m-3)-1
FIG. 18. Lineweaver-Burk plots for uptake of boron by leaf tissue of sugarcane in the range I W 2 mM. Uptake in the presence of 0.5 mM CaCI,, originally published by Bowen (1968); uptake in the presence of 5 mM CaCI2, unpublished data by the same author. Thick lines, multiphasic interpretation; thin lines. single + diffusion model: (---), extrapolations. (From Nissen, 1987.)
MULTIPHASIC UF'TAKE MECHANISMS IN PLANTS
105
S-l t d& ymd" 1
0
0'02
0.04
0.06
t3 i NO;
0
2
4
FIG. 19. Lineweaver-Burk plot for net nitrate uptake by dwarf bean plants calculated from depletion experiments starting at 0.4 mM nitrate (upper abscissa, phases A, B, and C) and 5 mM nitrate (lower abscissa, phases C and D). The mean uptake rate is given in arbitrary units and approximated 40 pnol hrl g-' in phase C. (From Breteler and Nissen, 1982.)
transitions at about 45, 80, and 480 pM (Fig. 19). Uptake of nitrate was essentially concentration independent in the range of phase C (80480 pW. The physiological significance of this phase could be to maintain a constant provision of nitrate in a concentration range occurring in natural and agricultural ecosystems.
D. UPTAKE
OF AMINOACIDS
Uptake of arginine, lysine, methionine, and proline by barley roots could be precisely represented by multiphasic isotherms (Soldal and Nissen, 1977, 1978), as shown for lysine in the range - lo-' M in Fig. 20. When the range was exM), the number of phases for lysine uptake increased tended (lo-' - 6.3 x from 4 to 5 (Soldal and Nissen, 1978). The data in Fig. 20 have been shown by an F-test to be significantly better represented by the multiphasic model than by
1 06
PER NISSEN
2.0 u
r 0
c
n
3
1.5
I0 - 5
lo*
10-3
L-Lysine, M FIG. 20. Uptake of L-lysine by barley roots. Transition points indicated by arrows. (From Soldal and Nissen, 1977.)
the dual model or other models yielding continuous isotherms (Soldal and Nissen, 1977; see also Nissen, 1987). Results consistent with the multiphasic model have also been obtained by other workers. Multiphasic isotherms have been found for uptake of aaminoisobutyric acid (AIB) (Shtarkshall and Reinhold, 1974) and arginine, aspartic acid, glutamic acid, glycine, and lysine (Lien and Rognes, 1977) by barley leaf tissue. Uptake of AIB by squash hypocotyls was represented by four phases in the range 10 ’ - 10 I M (Hancock, 1975). Uptake of AIB by barley leaf tissue was suggested to be mediated by a multiplicity of carriers, with each carrier being “switched off outside its range (Shtarkshall and Reinhold, 1974: Reinhold and Kaplan, 1984). However, as noted (Nissen, 1971b) for multiphasic sulfate uptake, it is difficult to perceive how a series of parallel structures could become operative and be shut off precisely at the inflection points. Transport into cultured tobacco cells has also been interpreted as multiphasic (2 or more phases) for cysteine (Harrington and Smith, 1977), leucine (Blackman and McDaniel, 1978, 1980 McDaniel er al., 1981), and lysine (Harrington and Henke, 1981). An Eadie-Hofstee plot for leucine uptake (Fig. 2 1 ) shows that the transitions may be accompanied by marked jumps. Other data include biphasic uptake of AIB by leaf discs of broadbean (Desphegel and Delrot. 1983) and triphasic loading of leucine into the phloem of soybean (Servaites e t a / . , 1979).
MULTIPHASICUPTAKE MECHANISMS IN PLANTS I
I
I
I
I
1
I
107 I
FIG. 21. Eadie-Hofstee plot for uptake of L-leucine by tobacco cells. Concentration ranges for phases in mM.(From McDaniel et al., 1981.) (x), growing phase cells; (0). stationary phase cells.
In line with the above findings for sulfate (Fig. 12), uptake of the p-amino acid taurine (2-aminoethanesulfonate) in Chlorellu was also precisely triphasic in the range M (Fig. 22). Leucine uptake by protoplasts from Vincu (Suzuki, 1981) and pea (Dureja et ul., 1984) has been reported to be multiphasic. Examination of the data for pea reveals a curvilinear Lineweaver-Burk plot for the upper phase (Fig. 23). Such kinetics are also found (P. Nissen, unpublished observations) for data of Theodoropoulos and Roubelakis-Angelakis ( 1989) for arginine uptake by protoplasts from grapevine leaves. In contrast to the situation for sulfate, competition experiments contraindicate the existence of separate uptake and transition sites for amino acids. In the experiment in Fig. 24, barley roots were allowed to take up [3H]lysinefrom a M solution in the presence of various concentrations of unlabeled arginine or lysine. Arginine was a better competitive inhibitor of [3H]lysineuptake than lysine itself. It was also more effective in causing transition to a higher phase. The transition occurred at virtually the same degree of saturation of the uptake site [(substrate-site complex + inhibitor-site complex)/total sites; see Soldal and Nissen (1978)], viz. 0.71 for lysine and 0.73 for arginine. For inhibition of
PER NISSEN
108 500
I
I
V
LOO
30C
i
20c
1oc
C
0
0 05
0.10
0 15
-
v/s FIG. 22. Eadie-Hofstee plot for uptake of taurine by Chlorellu fusru. Uptake is expressed as amol cell-' (30 r n i n ) - l , taurine concentration is in pM. (From Biedlingmaier and Schmidt, 1987.)
[3H]methionineuptake by unlabeled methionine, S-ethylcysteine, or S-methylcysteine, transition to a higher phase also occurred at about the same degree of saturation of the uptake site (Soldal and Nissen, 1978).
E. U P ~ A KOF E SUGARS The data of Linask and Laties (1973) for uptake of glucose and 3-0-methyl glucose by potato slices (see Fig. 6) remain the most detailed and precise for sugar uptake. These data cannot be satisfactorily represented by any of the continuous models (Nissen and Nissen, 1983; Nissen, 1987). Uptake of 3-0-methyl glucose by carrot root slices (Reinhold and Eshhar, 1968), leaf discs of geranium (Carlier. 1975), and squash hypocotyls (Hancock, 1975) is also multiphasic or,
MULTIPHASIC UPTAKE MECHANISMS IN PLANTS
-
2-
I
I
I
I
I
I
109
I
.-K
a)
c
2 a 1-
10-4
l/S (kmol m-3)-’ I
10-2 L- leucine (kmol m-3)
lo-’
10-~
FIG.23. Uptake of L-leucine by pea protoplasts in the range 0.05-100 mM. Transitions indicated by arrows. Inset, Lineweaver-Burk plot for uptake in the range 2-100 mM. Data kindly provided by Dr. I. Dureja. (From Nissen, 1987.)
0.10
1
D
, L-arginine
/ 0.05
/
I
L-I y sine
2
I
0
I
lo-‘
1
2.104 Inhibitor. M
M )by barley roots as a function of lysine or FIG.24. Dixon plot for uptake of [3H]~-lysine arginine added. Transition points indicated by arrows. (From Soldal and Nissen, 1977.)
110
PER NISSEN
at least, consistent with such kinetics. Biphasic kinetics are indicated for phloem loading of sucrose in leaves of sugar beet (Sovonick et al., 1974). Komor and Tanner (1974, 1975) have proposed that hexose uptake in Chlorella is mediated by a protein carrier which may exist in a protonated and a deprotonated state. The protonated form has a high affinity for the sugar and is responsible for active uptake; the low-affinity form mediates facilitated diffusion. This will give dual uptake kinetics, and the contribution of the low-affinity term should increase with increasing pH. Data of Komor and Tanner (1975) for uptake of 6-deoxyglucose have been shown, however, to be significantly (p < .01) better represented by multiphasic than by dual isotherms (Nissen, 1976). Other models have also been offered for these and similar data (Reinhold and Kaplan, 1984), and an unequivocal resolution of the kinetics of sugar uptake will, for many systems, have to await more and better data.
F. UPTAKEOF OTHERCOMFQUNDS Uptake of the cytokinin benzyladenine by tuber slices of Jerusalem artichoke in the range M could be represented by isotherms with four phases (Fig. 25). The fit to multiphasic kinetics was significantly better than the fit to cooperative kinetics (4 or 6 subunits) or to the sum of four independent Michaelis-Menten terms (Minocha and Nissen, 1982). Uptake of the auxins 2,4dichlorophenoxyacetic acid (2,4-D) and indoleacetic acid (IAA) in Jerusalem artichoke and potato slices was approximately proportional to the external concentration. The apparently linear relationships could in some experiments be resolved into precisely multiphasic patterns (Minocha and Nissen, 1985b). The pH dependence of uptake was consistent with facilitated diffusion of the undissociated acids. In contrast to the multiphasic patterns for the cytokinin and the auxins, uptake of abscisic acid (ABA) in these tissues was directly proportional to the external concentration over a wide range (4 x lo-'" - lo4 M ) and was in all probability due to simple diffusion of undissociated ABA across the plasmalemma (Minocha and Nissen, 1985a). Uptake of the alkaloid lupanine by epidermal cell layers of Lupinus polyphyllus can in the range 5 x - 2.5 x lo-*M be represented by four phases with curvilinear Lineweaver-Burk plots (P. Nissen; data estimated from Wink and Mende, 1987). Uptake of lupanine by mesophyll tissue (Wink and Mende, 1987) and by protoplasts from cell suspension cultures (Mende and Wink, 1987) also followed multiphasic kinetics.
G. Low vs. HIGHEXTERNAL SOLUTE CONCENTRATIONS In addition to the obvious differences in rates and kinetic constants, uptake of solutes from solutions of low and high concentrations (by high and low
MULTIPHASIC UPTAKE MECHANISMS IN PLANTS
I
I
I
10-8
0-7
10-6
I
s
0-5
111
1
0-4
(M)
FIG. 25. Uptake of benzyladenine by Jerusalem artichoke tuber slices. Transition points indicated by arrows. Significance of fit to adjacent phases (tested against a single phase): *, 0.01
phases) differs also in other respects. Sulfate and K' uptake by barley roots was severely inhibited by 2,4-dinitrophenol at low solute concentrations but was only slightly inhibited at high concentrations (Nissen, 1971b, 1980). This was taken to indicate that uptake from low external solute concentrations (against an electrochemical gradient) is active, i.e., requires metabolic energy, but that the process becomes gradually less active and more similar to facilitated diffusion as the external solute concentration is raised and the electrochemical gradient is diminished or reversed (Nissen, 1980). Whether such decrease in energy requirement is gradual or in step with the transitions to higher phases is not known. A high K' status severely reduced K' uptake by barley roots via the lower phases but had much less effect on uptake via the higher phases (Fig. 26), indicating that there was little allosteric regulation (see also Glass, 1976) at high external K' concentrations. Similar conclusions regarding energy requirements and regulation have been arrived at for K' uptake by wheat seedlings (Erdei et al., 1984). In necessarily vague terms it was proposed (Nissen, 1980) that the uptake site or structure is "tight at low external ion concentrations (low Vmax,low K,, active uptake, allosteric regulation), and "loose"at high concentrations (high V,,,, high
112
PER NISSEN
FIG.26. Uptake of K' by low K (seedlings grown in 0.5 mM CaSO,) and high K (seedlings grown in 0.5 mM CaSO, + 5 mM KCI) barley roots. Two separate but identical experiments. Transition points indicated by arrows. Significance of fit to adjacent phases indicated as in Fig. 25. (From Nissen. 1980.)
K,,,, facilitated diffusion, no regulation). A model (Fig. 37) consistent with this proposal is discussed later. BETWEEN KM AND VMAX H. RELATIONSHIP
As noted above, phases for sulfate uptake were stated to have regularly increasing kinetic constants (Nissen, 1971b). Only recently (Nissen, 1991) was a relationship between K , and V,,, sought. The points for log K, vs. log V,,, lie on a straight line for the first five phases for sulfate uptake by barley roots (Fig. 27). The coefficient (b) for this line was, on the average, 2.44, i.e., a 275-fold increase in K, was required for a 10-fold increase in V,,,. The slope decreased for the higher phases, possibly approaching direct proportionality between the two kinetic constants. Five instead of 8 phases were observed for leaf slices, and the decrease in slope at high sulfate concentrations was less pronounced. Although the kinetic constants for the various phases did not agree in the two root experi-
MULTIPHASIC UPTAKE MECHANISMS IN PLANTS
I
V,(prnol
10
100
113
1000
. g dry wt-'h-')
FIG. 27. Plots of log K,,,vs. log V,, for multiphasic sulfate uptake by roots and leaf slices of barley (experiments in Fig. 5). Data from Tables I and 2 in Nissen (1971b). Coefficient (b) for straight lines for indicated phases calculated by least squares. (From Nissen, 1991.)
ments, the points for both experiments lie on approximately the same line, i.e., the relationship between K,,, and V,,, holds. The good fits in Fig. 27 indicate that sulfate uptake is indeed multiphasic. The points would not have lain so precisely on such lines if the multiphasic isotherms were misinterpreted for multiple mechanisms. Additional results for uptake of inorganic ions by barley roots are shown in Fig. 28. The lines for sulfate and phosphate are similar but with much lower V,, values for sulfate uptake. The lines for uptake of K' and Cl- are virtually identical, indicating a very strong similarity between the two fluxes. Uptake of Na' was somewhat lower with SO,*- as the counterion than with C1-, but the lines are otherwise very similar. Multiphasic uptake of amino acids by barley roots also yields linear or approximately linear plots of log K,,, vs. log V,,,,, with a relatively steep slope for arginine (Fig. 29). Data for uptake of amino acids by leaf slices of barley (Lien and Rognes, 1977) yield very similar results.
114
PER NISSEN
---
0.01
0.I
I
, , ,V
10
100
lo
(pmo1.g fresh w t - ' )
FIG.28. Plots of log K, vs. log V,,, for multiphasic uptake by barley roots. A: Sulfate and phosphate. values for 20-mrn uptake (data from Table 2 in Vange e?al., 1974b). B: CI-and K', values for 4-hr uptake (reanalysis of data in Hiatt. 1968: data from Table 18 in Nissen, 1973d). C: Na', with Sodzor CI as the counterion, values for I-hr uptake (reanalysis of data in Rains and Epstein, 1967; data from Table I 1 in Nissen, 1973d). Otherwise as for Fig. 27. (From Nissen, 1991.)
I. K+-STIMULATED ATPASEACTIVITY Membrane-bound ATPases have been implicated in the transport of ions in plants (Hodges, 1973, 1976). Particularly close correlations exist between a K+stimulated ATPase and uptake of K' across the plasmalemma (Fisher et al., 1970; Leonard and Hodges, 1973; Leonard and Hotchkiss, 1976). Although later work has shown that the coupling between H' and K' transport may only be indirect (Serrano, 1989), the close correlations do indicate a relationship between the H+-ATPaseand K+ transport. Data of Leonard and Hodges (1973) for K'-stimulation of an ATPase associated with plasmalemma from oat roots were originally represented by a continuously curvilinear isotherm and discussed in terms of negative cooperativity. However, the data can be significantly better represented by multiphasic than by cooperative kinetics (Nissen. 1977). Data for K+(86Rb)uptake by corn roots and for K+ stimulation of an ATPase also from corn roots (Leonard and Hotchkiss, 1976) can be represented by remarkably similar triphasic isotherms (Fig. 30). Not only did the transitions occur at virtually identical concentrations, the K,,
FIG. 29. Plots of log K,,,vs. log V,,, for multiphasic amino acid uptake by barley roots. Data from Table 1 in Soldal and Nissen (1978). Otherwise as for Fig. 27. (From Nissen, 1991.)
.+A -r
1 -
r
T
f P
Y
-? Y
?
0,
K+- stimulated ATPase .. ._
5
-8
-1 I
10.~
I
10.~
I
10.~
I
lo-*
I
10-
KCI (M) FIG. 30. K+(86Rb)uptake by corn roots and K' stimulation of plasmalemma ATPase from corn roots. Data of Leonard and Hotchkiss (1976). Transition points indicated by arrows. (From Nissen, 1977.)
116
PER NISSEN
values for uptake and stimulation were, within experimental error, identical for the two lower phases. For K+ stimulation of plasmalemma ATPase from oat roots, Hlvarstein and Nissen (198 1 ) found multiphasic kinetics with the phases separated by jumps (Fig. 3 1). J. TRANSPORT IN BACTERIA Multiphasic uptake mechanisms are clearly present in lower plants, as shown by precisely multiphasic isotherms for uptake of inorganic and organic solutes by the unicellular green alga Chiorella and by yeast cells (Figs. 12 and 22 and examples in Nissen, 1973~).Detailed and wide-range concentration dependence
I/v
0.05
50
-- 0 2
9
n
F
B
-
\
n -
g
-
0.10-
I
-
200
/
2
.-; 0.15-
150
100
;;*y'& n.s
0
*)
).'
(
005-
I
I
I
I
MULTIPHASIC UPTAKE MECHANISMS IN PLANTS
117
data for uptake in nonplant systems appear to be relatively scarce, but there is evidence for the existence of multiphasic uptake mechanisms also in bacteria. Uptake of glucose in the range 2.5 x - 4.0 x lo-' M by an oligotrophic marine bacterium could be represented by four phases separated by sharp transitions at about 1.6 x and 2 x M (Nissen et al., 1984), as shown in Fig. 32 for the two lower transitions. The data in Fig. 32 were found to be better represented by multiphasic isotherms than by models (single + diffusion, dual, triple, cooperative) giving continuous isotherms. In a wider range the marked transition between phases 3 and 4 (not shown) would clearly make the continuous models untenable. Amino acid uptake by bacteria may also be multiphasic. Data for uptake of arginine, lysine, and ornithine in Escherichia coli (Celis et al., 1973) can be represented by multiphasic isotherms (P. Nissen, unpublished analyses). For arginine and lysine, the fit to the multiphasic model was significantly better than the fit to the dual model used by the authors, or to other models giving continuous isotherms. Data for uptake of glutamic acid, alanine, and valine in Mycobacterium smegmatis (Yabu, 1970) are also consistent with a multiphasic interpretation (P. Nissen, unpublished analyses).
-
13-
8
0
0
2 ID-
En
0
0
05-
0 -
indicated by ar-
118
PER NISSEN
K. QUESTIONS AND ANSWERS REVISITED The questions in this section have been taken verbatim from the previous review (Epstein, 1973). With the proviso that dual mechanisms should be replaced by multiphasic mechanisms, most of the questions remain appropriate. Epstein's detailed answers, provided here mostly in conclusion form, should be consulted in any closer study. The information in these answers is updated and, where necessary, corrected. It is now abundantly clear that multiphasic uptake isotherms do reflect membrane events. Earlier suggestions that the complexities in the isotherms reflect artifacts of various kinds can, therefore, be dealt with very briefly. 1 . Is the Pattern Universal in Plant Cells? Epstein lists experiments with nongreen and green tissues, fibrous roots and tuberous storage tissue, lower and higher plants, and with a majority of the essential mineral elements, and concludes that "the dual pattern of ion transport is indeed common, possibly quite general in mature plant tissue." This conclusion remains valid, but, as noted above, for multiphasic rather than for dual pattern(s). Multiphasic uptake mechanisms appear also to be quite general for sugars, amino acids, and other organic compounds. However, constitutive uptake of choline sulfate is mediated by truly dual (or triple) mechanisms (see Fig. 2).
2 . 1s the Appearance of a Type-I Mechanism due to Activities of Microorganisms Associated with the Roots? Epstein concludes that "bacteria thus do not normally contribute significantly to the measured values of amounts of ions absorbed by nonsterile plant tissue." Many experiments have now been camed out under sterile or semisterile conditions, and it is clear that multiphasic uptake patterns are not due to microorganisms associated with plant cells or tissues. The precisely multiphasic isotherms obtained in many experiments preclude, furthermore, a significant contribution by a microbial component.
3. Is the Complexity Observed due to the Presence, in the Plant Materials Used, of Se\*eral Tvpes of Tissues and Cells? Epstein concludes that "the dual pattern is a characteristic of ion transport at the level of the individual cell." From multiphasic patterns found for uptake into protoplasts and plastids, it is evident that the discontinuities in the isotherms do indeed reflect membrane events. Additional evidence for this is provided by the discontinuities, including jumps, in isotherms for K+ stimulation of a plasmalemma-bound ATPase (Figs. 30 and 3 1).
MULTIPHASIC UPTAKE MECHANISMS IN PLANTS
119
4 . Is the Dual Patrern an Artifact due fo Use of Low-Salt Tissue?
Epstein concludes that “there is no evidence that endogenous concentrations of some element change the nature of the mechanisms of absorption of that element.” Although quantitatively different, multiphasic isotherms are qualitatively similar in low- and high-salt roots (Nissen, 1980) (Fig. 26). Of particular note is that the concentrations at which the transitions for K’ uptake occur are not affected by the endogenous concentration of K’. 5 . Do the Two Types of Absorption Mechanisms Operate in Parallel or in Series? The models referred to are shown in Fig. 33. In the parallel model both uptake mechanisms (1 and 2) were assumed to be located at the plasmalemma. In the series model the high-affinity mechanism (1) was taken to be at the plasmalemma and the low-affinity mechanism (2) at the tonoplast. Both models are based on misinterpretations of experimental results. As evidenced above, there are no dual uptake mechanisms and hence no parallel model. The purported dissection (Epstein, 1973) of total rates of uptake into two components representing Mechanisms 1 and 2 by uptake of Na’ in the presence of increasing concentrations of K+ and vice versa can, furthermore, be shown by Dixon plots to yield multiphasic rather than dual kinetics (Nissen, 1973d). For uptake across the tonoplast to become rate-limiting in the range of Mechanism 2, a diffusive component has to exist across the plasmalemma at external salt concentrations above 5 x M (Laties, 1969). In support of Parallel model (Epstein et al.)
,plasmalemma
f
cytoplasm
\
Multiple phase Single phase
Series model (Laties et a\.)
Linear or exponential
1-9
+LI
Single phase
- - - 3 Diffusion
d Active uptake 6+II)Muttiphasic Multiphasic series model (Nissen and Laties) FIG.33. Localization of uptake mechanisms in vacuolated plant cells. Mechanisms 1 and 2 here denoted I and 11, respectively. (From Nissen, 1973b.)
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PER NISSEN
this, uptake into the nonvacuolated cells of corn root tips was interpreted as being linear in the high concentration range, whereas uptake into the vacuolated cells of proximal root sections showed a bumpy isotherm in this range (Torii and Laties, 1966; Liittge and Laties, 1966). Reanalyses of these data show, however, multiphasic isotherms for both tissues (Nissen, 1973a,b,d) (Fig. 34). A multiphasic series model (Fig. 33; see also Nissen, 1973b) in which there is a single, multiphasic mechanism at the plasmalemma and, probably, a similar mechanism at the tonoplast, at least at high concentrations, seems consistent with all experimental findings. This model was also adopted by Laties (1975). Epstein’s conclusions that “the plasmalemma is not permeable to inorganic ions to any marked degree” and that “(the plasmalemma) is negotiated by these (inorganic) ions appreciably only via transport mechanisms, not by diffusion” agree with other conclusions (Nissen, 1973a) and are fully borne out by analyses (Nissen, 1987, 1989) of data taken to represent uptake via one or two Michaelis-Menten mechanisms and a diffusion term, i.e., models 1 and 3 in Table 11. 6 . Does the Dual Pattern of Transport Reflect the Operation of a Single Transport Mechanism?
The single transport mechanism refers to analyses of Thellier (1970), Nissen (1971b), and Gerson and Poole (1971) denying the existence of dual mechanisms of ion transport. The electrokinetic formalism of Thellier ( 1970) abandons the concept of carriers and is inadequate to account for the complex kinetics actually observed (Epstein, 1973; Nissen, 1973a). The unary interpretation of Gerson and Poole (1971) is also inadequate as noted in Section IV,D.
+-
I
I 05r
lo-‘
10-3
CaCI,
CI
lo-2 eq/l
FIG.34. Uptake of chloride by tips and proximal sections of corn roots. Data from Fig. 3 in Torii and Laties (1966). (From Nissen, 1973a.)
MULTIPHASIC UPTAKE MECHANISMS IN PLANTS
121
Epstein notes that an examination of the data of Nissen (1 97 1b) for sulfate uptake reveals excellent agreement with previous experiments and that the facts are as observed before. This is correct; it is the interpretation that differs. Contrary to a further statement (Epstein, 1973), the features of the sulfate uptake isotherm reflect the operation of a single transport system rather than dual systems. Reasons for this were given in the original paper (see also Section 111,A). In support of the duality of uptake isotherms, Epstein (1973) reviews the “dissection” (Welch and Epstein, 1968) of isotherms into two components and concludes that “the data seem incompatible with any but an additive or parallel scheme of two mechanisms.” As noted above, however, Dixon plots of these data reveal multiphasic kinetics (Nissen, 1973d). In its latest version, the dual model is taken to not necessarily imply the functioning of two separate uptake mechanisms, only a marked duality in the isotherm (Epstein, 1976). However, except for choline sulfate (Fig. 2), there is no duality in uptake isotherms (Figs. 5,6, 13, 20,23,25, 26, and 34). Any duality is only apparent and is caused, in part, by the practice of using different concentration scales at low and high solute concentrations. The fact that phase 2 may have lower kinetic constants than phase 1 (e.g., Fig. 34; see also Nissen, 1974a) may also give the impression of a plateau in this region. The straight-line relationship between K, and V,,, for the different phases (Figs. 27-29) is conclusive evidence against any duality in these isotherms. The continued use of terms such as “Mechanism 1” and “Mechanism 2 is highly misleading and should be abandoned.
7. Is There Evidence for the Operation of Dual Mechanisms from Long-Term Experiments with Growing Plants? From a review of various uptake and growth experiments lasting from a few hours to many weeks, Epstein concludes that the duality of ion absorption mechanisms is not a laboratory curiosity but reflects the realities of mineral plant nutrition in nature. The extent to which long-term accumulation of nutrients reflects short-term uptake kinetics is not clear. However, there are many examples of multiphasic accumulation patterns. In a 14-day experiment with excised cotton roots, Johanson and Joham (197 1) recognized a discontinuous relationship between the calcium concentrations in the growth medium and in the roots. These data are represented as multiphasic in Fig. 35, essentially according to the original interpretation. In experiments (Nissen et al., 1980) with seedlings of rice, soybean, and orange grown for 20 to 125 days in nutrient solutions with up to 16 different concentrations of NH,’, H,PO,-, K’, Ca2+,Mg2+,or Zn2+,the relationship between tissue concentration of an element and external concentration of the corresponding nutrient ion was invariably multiphasic, with phases separated by sharp breaks or jumps. In the example in Fig. 36, the phase pattern for
PER NISSEN
122 i
.-cr, 0)
.*.
2-0.5 -
...T
2
u
c 0
3
CI,
0
-1.5l
I
10-5
I
10-4
J
10-3
FIG. 35. Concenmtion of calcium in cotton roots. Excised roots of cotton were grown for 14 days in nutrient solutions containing indicated average concentrations of calcium. Data from Table 2 in Johanson and Joham (1971). Significance symbols as in Fig. 25. (From Nissen et d.,1980.)
the concentration of phosphorus in roots and tops of rice plants differed, for unknown reasons, markedly at low external phosphate concentrations.
IV. Other Models Subsequent to the proposal of the dual and the multiphasic model, several other models have also been proposed to account for the complex uptake isotherms in plants. The most widely used models are shown in Table I1 in order
I
1
I
1
0
5 4
L lo-?
I
, IO-~
1
IO-~
I
IO-~
Phosphate (MI FIG.36. Concentration of phosphorus in roots and tops of rice plants grown for 25 days in nutrient solutions of indicated phosphate concentrations. Transitions indicated by arrows or as jumps. Significance symbols as in Fig. 25. (From Nissen et ol.. 1980.)
MULTIPHASIC UPTAKE MECHANISMS IN PLANTS
123
TABLE I1 KINETIC MODELSFOR SOLUTE UFTAKE IN PLANTS'
Model
Formula
1. Single + diffusion
Vmax
v = -+
v,, s v,
5. Multiphasic
Kochian and Lucas (1982)
s
"= +I Epstein er al. (1963) %,+S
4. Cooperative
References kD S
K,+S
2. Dual 3. Dual + diffusion
S
V,," y
=
Km2+S
sI + vm s + k,
-
K m I + S Km2+S Not given v=
v,, s (for phase n ) Kmn S +
S
Borstlap ( 1983) Hodges (1973) Nissen (197 1b); Linask and Laties (1973)
=Seetext for further description of models. From Nissen (1986). S, Solute concentration; v , uptake rate; V , ,maximal uptake rate; K, ,Michaelis constant; K, ,diffusion constant.
of increasing complexity. (The models will be discussed in approximately chronological order.) Models 1-3 predict the existence of multiple uptake mechanisms, whereas models 4 and 5 predict a single mechanism for uptake of a particular solute. The multiple models (1-3) and the cooperative model predict a continuous relation between uptake rate and external solute concentration, whereas the multiphasic model (5) predicts a discontinuous relation.
A. COOPERATIVE MODEL The proposal by Hodges (1973) that the kinetics of ion uptake and ATPase activity in plants are due to the cooperative interaction of enzyme subunits is attractive in that it would explain these kinetics in terms of known mechanisms. The consideration of cooperative kinetics for uptake of sugars and amino acids has also been urged (Reinhold and Kaplan, 1984). However, there appears to be no good evidence for this model in plant uptake. On the contrary, the fitting of cooperative models (with up to 6 subunits) resulted only in very gradual bends rather than the sharp transitions actually observed (Nissen, 1977; Hivarstein and Nissen, 1981; see also Fig. 31; Minocha and Nissen, 1982). The occurrence of jumps in isotherms and the demonstration that such noncontiguities can be produced in a predictable and reproducible manner by interaction with the transition site (see Figs. 8-10) constitute conclusive evidence against the cooperative model and other models yielding continuous isotherms.
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B. SLNGLE + DIFFUSION MODEL This model, which also may be termed saturable + linear, has been proposed for uptake of alkali cations by barley roots (Waisel, 1962) and for uptake of K' by roots of barley and rye grass (Glass and Dunlop, 1978). The latter data were subsequently shown to be precisely represented by multiphasic isotherms (Nissen, 1980). The model has become quite widely accepted in recent years since its reintroduction by Kochian and Lucas (1982) for uptake of K' by corn roots. It was later also proposed for uptake of C1- (Kochian et af., 1985). The underlying data (Kochian and Lucas, 1982, 1983, 1985; Kochian et al., 1985) cannot be satisfactorily explained by this simple model (Nissen, 1989):
M CaSO, 10-3M KCI), the model fails to account for uptake at low external Rb+ concentrations. The kinetics remain complex after supposedly complete inhibition of the "saturable" component by the sulfhydryl reagent N-ethyl maleimide. The model gives an unacceptably poor fit for influx into protoplasts, at least as represented by the authors. Unacceptably low probabilities were obtained by a test for runs of positive and negative deviations in experiments with different accompanying ions. In the case of detailed and precise data, subtraction of a "linear" component does not yield saturation kinetics.
1. For roots grown under high-salt conditions (in solution of 2 x 10.'
+5x
2. 3. 4. 5.
In short, there is no linear component in these data, and the use of inhibitors to selectively inhibit a saturable component can be shown to be invalid (see also Reinhold and Kaplan, 1984). In contrast, the data may be well represented by a single, multiphasic mechanism for each ion. Other data for K+ uptake by corn roots are also well represented by multiphasic isotherms (Nissen. 1991). Extensive analyses (Borstlap, 1983; Nandi et al., 1987; Nissen, 1987) have shown that the single + diffusion model is inadequate also for other solutes. This is exemplified in Fig. 13 where the thin lines for 5 and 10°C represent the best fit of this model to data for phosphate uptake by corn roots. C. DUAL+ DIFFUSION MODEL
Borstlap (198 la,b; 1983) has claimed that the concept of multiphasic uptake mechanisms in plants is invalid and that there is hardly any evidence that the discontinuities in uptake isotherms are not due to experimental error in the data. He has also suggested that many isotherms may be described by the sum of two saturable terms added to a linear term. These claims and suggestions have been examined in considerable detail and depth (Nissen and Nissen, 1983: Nissen, 1987) and have been found to result from incomplete and, in part, faulty consid-
MULTIPHASIC UPTAKE MECHANISMS IN PLANTS
125
eration of the evidence at hand. In discounting the evidence for discontinuities, no mention was made of the finding of separate uptake and transition sites and the use of this finding to produce jumps in a reproducible manner (see Figs. 8-10). A study of Borstlap’s claims and the answers to these claims should prove instructive and should obviate the need for any further mention. The suggestion that a dual + diffusion model can account for the kinetics of uptake can, similarly, be shown to be invalid in the case of detailed and precise data, e.g., for phosphate uptake by corn roots (Fig. 13). D. PSEUDODUAL MODELS Various models have been put forward to account for the complex kinetics of ion uptake by a single mechanism giving pseudodual kinetics. All appear to be unsatisfactory. The unary interpretation of Gerson and Poole (1971) involves a charged ion-carrier complex but is restricted to anions. A model involving an interaction between processes occurring in an unstirred boundary layer and the negatively charged cell wall (Dalton, 1974) is restricted to cations. Grunwaldt et al. (1979) have proposed that the complex kinetics can arise simply because the plant tissues are multilayered. However, multiphasic uptake kinetics have also been found in unicellular organisms, in isolated cells of higher plants, in protoplasts, and in plastids. Furthermore, multiphasic kinetics for ATPase stimulation have been found in a plasmalemma-enriched membrane fraction (see Figs. 30 and 3 1). Random binding of solute and driver ions to cotransport carrier systems can yield dual isotherms (Sanders, 1986). However, none of the pseudodual models can fully account for the highly complex and discontinuous kinetics actually observed.
E. “DISCONTINUOUS” MODELS
In addition to the multiphasic model, other models have also attempted to account for the discontinuities in uptake isotherms. Bange (1979) proposed that transport of solutes (both in bound and in a free form) through a channel with oscillating binding sites would give rise to inflections in the isotherm. The model is at variance with present information on the structure and functioning of ion channels in microbial and mammalian systems. It also seem questionable if a system of oscillating sites could give truly multiphasic kinetics. Sabater (1982) proposed a mechanism in which the carrier has two binding sites for the solute. The first site binds n - 1 solute ions or molecules, allowing an additional ion or molecule to become bound to and transported by the second site. Both sites can exist in several states with different affinities for the solute. A mathematical formulation of this mechanism, with the proper coefficients, can, for high values of n, yield approximately multiphasic isotherms. However, the model cannot
126
PER NISSEN
account for all the complexities in uptake isotherms. Thus, for a similar model developed by W. D. Stein (personal communication) to give “stepped“curves, it has been shown (P. Nissen, unpublished analyses) that it cannot account for the data for phosphate uptake by corn roots in Fig. 13. Phases 1 and 2 in this experiment cover a wider concentration range than can be accommodated by the model, resulting in local peaks and dips in the isotherm rather than “stepped curves.
V. Molecular Basis of Multiphasic Uptake Little is known about the molecular structure and functioning of uptake mechanisms in plants, but the kinetic information outlined in this article may be combined with information from other systems to give a tentative picture of how inorganic ions and organic solutes are transported into plant cells. A. CARRIERS vs. CHANNELS
Transport of solutes across cell membranes has usually been considered to be mediated by two different mechanisms or structures; by carriers (taken to be mobile proteins), and by channels (transmembrane proteins with a central hydrophilic channel). The characteristics of the two mechanisms have also been taken to differ markedly: Carrier-mediated transport has low rates, high specificities, and can occur against an electrochemical gradient requiring energy. Channel-mediated transport has higher rates and lower specificities, cannot occur against an electrochemical gradient, and requires less energy. For multiphasic uptake it was strongly suggested that only one mechanism or transport structure functions at all solute concentrations (Nissen, 197lb). It was further suggested that the various phases must reflect different conformations of this single structure. The finding of precise relations between log K,,,and log V,,,, (Figs. 27-29) confirms that only a single structure is involved. Although uptake is mediated by a single structure, the characteristics of uptake differ markedly at various external solute concentrations. Uptake at low concentrations (by the lower phases) is dominated by carrier-like properties whereas uptake at high concentrations (by the higher phases) has dominantly channel-like properties. The conclusion that a single structure, albeit in different conformations, may have both carrier-like and channel-like properties is in line with current thinking on the structure and functioning of carriers and ion channels. A carrier need not be a mobile protein but may be a channel with a binding site, or uptake site in the present terminology, which is alternatively exposed via microscopic conformational changes to the two sides of the membrane (see also Lauger, 1984, 1985; Schauf, 1987). Such a channel will show carrier-like prop-
T
127
erties when the rate of translocation is limited by the rate of interconversion between the two states. Figure 37A illustrates how a channel might function at low external solute concentrations. There is tight binding (low K,) of the solute to the outward-facing uptake site and a correspondingly low rate of interconversion. At higher solute concentrations (higher phases), binding to the uptake is weaker and the rate of interconversion between the two states of the uptake site is increased. With faster interconversion, the properties of the channel approach those of a channel with a fixed barrier (Lauger, 1984). Figure 37B illustrates channel-like transport at high external solute concentrations. Uptake at intermediate solute concentrations is proposed to be mediated by stages (phases) intermediate between carriers and channels, i.e., intermediate between Figs. 37A and 37B. For a channel in the camer mode, i.e., at low external solute concentrations, transport against an electrochemical gradient can occur when the uptake site is transiently exposed to the other side of the membrane by input of an external energy source (Lauger, 1984). The properties of the channel are, furthermore, such that no free diffusion of solute occurs, even at high external concentrations. B. TRANSITION SITE According to the proposed model, the transitions observed in multiphasic ion uptake are caused by the interaction of solute ions with a site which differs from that for uptake. Changes in the transition site appear, in turn, to cause conformational changes in the adjacent uptake site, resulting in changes in affinity (K,) and hence also in V,,,. The transition site appears to be hydrophilic and to be located on the outside of the plasmalemma (Nissen, 1980; Breteler and Nissen, 1982). However, it is not clear whether it is a proteinaceous site exterior to the channel or whether it is constituted of the head groups of lipids surrounding the trans-membrane protein (Fig. 37). The temperature independence of the transitions favors the former assignment, whereas the apparently large number of sites required to cause the sharp transitions, if these are due to cooperative interactions, would favor the latter. C. CONCEPTUAL PROBLEMS Truly discontinuous changes are not believed to occur in biological systems (Koshland, 1987), and it may be asked whether the transitions in uptake isotherms are discontinuous or whether they merely reflect highly cooperative interactions. Available evidence, including the existence of jumps, indicates that the transitions are indeed discontinuous. There is no consistent indication of any gradual transitions in the uptake isotherms that have been examined. The absence of gradual transitions is especially clear in the case of curvilinear
128
PER NISSEN
Lineweaver-Burk plots where the phases may intersect almost at a right angle (Fig. 14). In multiphasic uptake it thus appears that we either have (i) a hitherto unrecognized type of interaction in which solutes interact with membranes to give truly discontinuous transitions, or (ii) interactions yielding much higher cooperativities than any previously known for membrane transport. For the latter alternative, calculated using binomial coefficients, at least 1000 sites would be required to confine the transitions to sufficiently narrow concentration ranges (Nissen, 1980). A possibly related problem is the need for some kind of control to ensure that the concentration-dependent phase transitions are not normally accompanied by jumps (Nissen, 1973d). The uptake site, i.e., the binding site in the channel (Fig. 37), and, presumably, also the transition site, must assume at least as many different conformations as the observed number of phases, i.e., 5-8. How this is achieved and how the conformation of the channel is controlled so as to cause transport to be less carrier-like and more channel-like with increasing external solute concentrations is yet unexplained by the present model.
VI. Concluding Remarks The main conclusion of this article can be simply stated: Solute uptake in plants is mediated by multiphasic mechanisms, at least in the great majority of cases. There is, furthermore, strong kinetic evidence that uptake of a particular solute occurs by a single structure, presumably a membrane channel, having carrier-like properties at low external solute concentrations and channel-like properties at high concentrations. With the recognition that this entity may exist in several different concentration-dependent states, it seems that the widely held assumption that carriers and channels represent separate entities should be reexamined. The multiphasic model has been extensively tested and validated over 20 years. The model remains, as far as it can be determined at the present, fully satisfactory to account for the complex concentration dependencies of solute uptake in plants. This does not, of course, mean that the testing and the challenging of the multiphasic model should cease. It means, however, that full cognizance should be taken of the extensive body of evidence in favor of, or consistent with, the multiphasic concept. Anything less will only result in a continuation of the present situation in which a number of clearly unsatisfactory models are being proposed and championed. The present needlessly confused situation should be replaced by one in which the efforts are directed toward solving real problems instead of imaginary ones created by inadequacies in the design, analysis, and/or interpretation of experiments. The following statement by two leading workers in the field seems, unfortunately, to typify the present situation: "It is hard to avoid the conclusion that fur-
MULTIPHASIC UPTAKE MECHANISMS IN PLANTS
out
out
.
in
--.. in
-
out o===
129
71.
in
..out
o====o==-.
e=== _co in
FIG.37. Hypothetical and highly simplified structure for transport of solutes across a plant membrane. (Illustrated for transport of K+.) (A) Low external concentration: Canier mode. Solute strongly bound to uptake site. Rate of "flipping" low (short arrow) and limiting. Energy input for active transport indicated. (B) High external concentration: Channel mode. Solute weakly bound to uptake site. Rate of "flipping" high (long arrow) and nonlimiting. Conversion from canier mode to channel mode, or to any of the intermediate stages, occurs through interaction of the solute with an exterior transition site (- - -). U, Uptake site; T,transition site; R. regulatory site. (From Nissen, 1991.)
130
PER NISSEN
ther kinetic analysis of intact tissues is not the way towards an understanding of K+ transport. There is an aura of almost medieval sophistry surrounding the matter, with unresolved disputes about how many different carriers or carrier states exist' (Luttge and Clarkson, 1989). Similar opinions may, presumably, be held for the transport of other solutes. Such opinions may reflect a lack of information and/or understanding of the issues involved, and a clarification seems in order. To know the number of carriers or mechanisms for uptake of a particular solute is clearly of fundamental importance for understanding the transport of that solute. This question should not be allowed to remain unresolved because of disputes in the past. It behooves the worker in this field to face the issue squarely: Is there or is there not more than one mechanism for transport of the solute under study? As shown in this article, data and evidence have long been available for the unequivocal resolution of this question, at least for the major inorganic ions. Uptake of nutrient ions by plant roots can be precisely represented by multiphasic isotherms, i.e., the multiphasic model is satisfactory. Other models fail to account for the sharp breaks in the isotherms and do, in the case of detailed and precise data, give unacceptably poor fits. Uptake of organic solutes from the soil is of less importance than uptake of inorganic ions and has, consequently, received less attention. However, uptake of amino acids can be better represented by the multiphasic model than by other models. Most data for sugar uptake by plant cells and tissues are insufficiently detailed and/or precise to differentiate between the various models. Better data, analysis, and discussion of already available evidence are needed for uptake of sugars and, in part, also for amino acids. It would also be of considerable interest to see to what extent multiphasic uptake mechanisms are present in bacteria and other organisms. The carrier-kinetic approach has been, and continues to be, indispensable to the study of solute uptake. Only by this approach can the number of uptake mechanisms be resolved. The question of whether or not there is also free diffusion across the plasmalemma can, similarly, only be resolved by this approach. Proper use of the carrier-kinetic approach is also essential for determination of specificities, etc. The fact that several different kinetic models have been proposed for uptake of the same solute, often in the same tissue, does not reflect on the validity or usefulness of the carrier-kinetic approach, only on its practitioners. Prerequisites for the correct resolution of the kinetics are: 1. Concentration-dependence data should cover a sufficiently wide range and
should be sufficiently detailed and precise for meaningful analyses to be carried out. 2. Resolution of isotherms and comparison of models should be done with all due statistical rigor. (This is, incidentally, the antithesis of the sophistry referred to above.)
MULTIPHASIC UPTAKE MECHANISMS IN PLANTS
131
3. All relevant information should be taken into account, not just the result of a particular experiment. Kinetic models have all too often been proposed on the basis of one, or a few, often unsatisfactory, data sets. Influx across the vacuolar membrane (tonoplast) may be similar to that across the plasmalemma. Uptake of C1- by vacuoles isolated from mesophyll protoplasts of barley leaves (Martinoia et al., 1986) can be better represented by 1-3 phases of a multiphasic isotherm than by the single + diffusion model used by the authors (P. Nissen, unpublished analyses; original data kindly provided by Dr. E. Martinoia). However, much more work will be needed to establish the number and nature of mechanisms for transport into the vacuole. Patch-clamp studies on plant membranes (Hedrich et al., 1987; Satter and Moran, 1988) may provide important information on the multistate channel proposed in Fig. 37. Single-channel studies of conductance as a function of ion concentration should, especially when done in conjunction with influx studies, reveal whether or not a channel can undergo the postulated transitions. Isotherms for uptake of widely different solutes, e.g., K ' and C1-, are strikingly similar, maybe more so than would be expected from electroneutrality of total fluxes. Can the identical patterns for uptake of K' and CI- result from a strong coupling of the two fluxes or do they mean that a single mechanism is, somehow, taking up both cations and anions? The answer to this question may bear directly on the structure and functioning of the channel(s). The reason for the deviations from Michaelis-Menten kinetics in many of the higher phases should also be sought. The nature of the transitions remains an enigma. Discontinuous transitions have mainly been found for uptake in higher plants, and there is little to go on regarding the molecular basis of this phenomenon. A membrane-bound ATPase exhibiting discontinuous transitions and organelles and similar simple systems seem best suited for further studies of concentration-dependent discontinuities.
ACKNOWLEDGMENTS I thank Professor L. Reinhold, Dr. H. Breteler, and Dr. R. H. Potter for their helpful comments on the manuscript.
REFERENCES Bakkerud, K.G., and Nissen, P. (1980). Biochim. Biophys. Acru 600, 205-21 1. Bange, G. G. I. (1979). Z. Pflanzenphysiol. 91,75-78. Barber, D. A. (1972). New Phytol. 71,255-262. Biedlingmaier, S . , and Schmidt, A. (1987). Physiol. Plunr. 70,688-696. Biedlingmaier, S., and Schmidt, A. (1989). Z. Nuturforsch. 44C, 495-503. Blackman, M. S., and McDaniel, C. N. (1978). Plant Scz. Lett. 13,27-34. Blackman, M. S., and McDaniel, C. N. (1980). Plant Physiol. 66,261-266.
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[email protected](20), 1-53. Nissen, P. (1974a). Annu. Rev. Plant Physiol. 2553-79. Nissen, P. (1974b). Physiol. Plant. 30, 307-316. Nissen, P. (1976). Physiol. Plant. 36,321-324. Nissen, P. ( 1977). Physiol. Plant. 40,205-2 14. Nissen, P. (1980). Physiol. Plant. 48, 193-200. Nissen, P. (1986). Acra Hortic. 178, 21-28. Nissen, P. (1987). Plant Cell Environ. 10,475485. Nissen, P. (1989). Plant Physiol. 89,231-237. Nissen, P. (1991). In “Plant Roots: The Hidden Half’ (Y. Waisel, A. Eshel, and U. Katkafi, eds.), pp. 483-502. Dekker, New York. Nissen, P., and Nissen, 0. (1983). Physiol. Plant. 57,47-56. Nissen, P., Fageria, N. K., Rayar, A. J., Hassan, M. M., and van Hai, T. (1980). Physiol. Plant. 49, 222-240. and Jenny, H. (1971). Physiol. Plant. 25,219-224. Polk, E. 0.. Rains, D. W., and Epstein, E. (1967). Plant Physiol. 42,3 14-3 18. Reinhold, L., and Eshhar, Z. (1968). Plant Physiol. 43, 1023-1030.
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INTERNATIONALREVIEW OFCYTOLOGY, VOL. 126
Glycosylation in Intestinal Epithelium DOUGLAS J. TAATJES* AND JURGEN Row? Interdepartmental Electron Microscopy, Biocenter, University of Basel, CH-4056 Basel, Switzerland
I. Introduction A great deal of interest has been focused in the last few decades on elucidating the mechanisms and cellular locations of glycosylation reactions. This interest has arisen as the result of many divergent disciplines converging on a central, fundamental question: Where and how are sugar residues added on to proteins and lipids during their journey from the sites of synthesis to delivery at final destinations? This question is of practical concern to biochemists, cell biologists, virologists, and cancer biologists, to name a few, and has emerged from the realization that the sugar moieties of glycoconjugates are involved in a myriad of events related to recognition phenomena. This is especially true of oligosaccharide chains present on plasma membrane glycoconjugates. The first version of the fluid mosaic model of the plasma membrane published in 1972 (Singer and Nicolson, 1972) depicting a lipid bilayer containing integral and peripheral proteins, neglected the existence of the sugar constituents of these molecules. Indeed, almost all plasma membrane proteins are glycoproteins. Given the knowledge that the sugar moieties of glycoconjugates are found on the extracellular side of plasma membranes, it is quite easy to envision their involvement in recognition of another cell, bacterium, or virus (Rademacher et a!., 1988). This idea took on added significance with reports from the 1960s that cancer cell plasma membranes were different from those of normal cells with respect to their oligosaccharide composition (Aub et al., 1963, 1965a,b; Burger and Goldberg, 1967). Although time and much subsequent research have revealed the oversimplification of this notion of malignancy and metastasis, it nevertheless certainly generated a flurry of activity aimed at understanding glycosylation. Perhaps of equal importance was the finding that many pathogenic organisms bind to and gain entry to cells through recognition of sugar molecules present at the cell surface (Paulson, 1985; Kocourek, 1986; Sharon and Lis, 1989). Regardless of the driving forces, the result has been an understanding in detail of some aspects of the process of glycosylation.
*Present Address: Department of Pathology, University of Vermont, Burlington, Vermont 05405 'Present Address: Department of Cell and Molecular Pathology, Institute of Pathology,University of Zurich, CH-8091 Zurich, Switzerland 135 Copyright 0 1991 hy Academic Press.Inc. All rights of reproduction in any form reserved.
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The intestinal epithelium represents a good model system in which to study the glycosylation process. From a simplistic viewpoint, the intestinal epithelium is composed of two basic epithelial cell types; the absorptive enterocyte and the mucus-producing goblet cell (Figs. 1 and 2). These two cell types differ both in form and function, yet are derived from a singular precursor cell (Leblond and Messier, 1958; Cheng and Leblond, 1974). This affords the opportunity to study glycosylation in neighboring but distinct cells. From a historical perspective, the intestine was chosen as one of the earliest systems for the cytochemical investigation of glycosylation. Indeed, in two classic papers investigating intestinal epithelial cells, Neutra and Leblond (1966a,b) provided the first demonstration that complex sugars are added to glycoconjugates in the Golgi apparatus. They injected tritiated hexoses (['H]glucose and [3H]galactose)into rats, and found that autoradiographic grains were first localized over the Golgi apparatus cisternal stack. Radioactivity was then followed into the mucus droplets of the goblet cell and into the plasma membrane of absorptive cells. Further, classical histochemical staining techniques, applied to electron microscopically visualize periodate-reactive carbohydrates (Rambourg er al., 1969), demonstrated reaction in the Golgi apparatus and plasma membrane of intestinal epithelial cells, as previously reported for acid mucopolysaccharides using colloidal iron staining (Revel, 1964; Wetzel er al., 1966; Berlin, 1967). Likewise, .the basic morphology and physiology of the intestinal tract provide an ideal system in which to study glycosylation as it relates to development and differentiation (Fig. 3). The harsh environment of the intestinal lumen results in continuous loss of epithelial cells through sloughing into the lumen. Gastrointestinal epithelial renewal ensues through the processes of cell proliferation, migration, and differentiation (Eastwood, 1977). This renewal occurs in discrete proliferative zones along the gastrointestinal tract. In the small intestine, this proliferative zone is restricted to the base of the crypts, whereas in the large intestine it is less restrictive, occurring in the basal two thirds of the crypt. The definitive autoradiographic studies of Leblond and co-workers have established that in both small (Cheng, 1974; Cheng and Leblond, 1974) and large intestine (Chang and Leblond, 1971), a ring of undifferentiated stem cells situated in the crypts divide to produce daughter cells committed to differentiate into FIG. I . Low-power electron micrograph showing the surface epithelium from the chick duodenum. In this and all subsequent micrographs, the tissue was lightly fixed in aldehydes (without postfixation in osmium tetroxide), followed by low temperature dehydration and embedding in Lowicryl K4M. Thus, membrane delineation is different from that seen in routinely processed tissues. The surface lining the intestinal lumen is composed mainly of two epithelial cell types: the mucus-producing goblet cell interspersed among the absorptive enterocyte. Both cell types possess an elaborate apical plasma membrane called the brush border (arrowheads), which is covered by a layer of mucus rich in glycoconjugates. (md) Mucus droplets; ( n l ) goblet cell nucleus; (n2) absorptive cell nucleus; (n3) nucleus of migrating lymphoid cell. X 1400. Bar = 6.7 l m .
138
DOUGLAS J. TAATJES AND JURGEN ROTH
B r u s h Border
. Multivesicular Body
Apical Cytoplasmic Vesicles
Mucus Droplets ,
Lysosome
Golgi Apparatus Golgi
Apparatus
W FIG.2. Schematic drawing illustrating the morphology of the two basic epithelial cell types lining the intestinal tract. Compare this drawing with the electron micrograph presented in Fig. 1. Of particular imponance with respect to glycosylation are the Golgi apparatus, apical and basolateral plasma membrane domains, absorptive cell apical cytoplasmic vesicles, and goblet cell mucus droplets.
absorptive (columnar), goblet, and endocrine cells. The mechanisms guiding proliferation and migration are not fully understood (Potten and Loeffler, 1987). however, migration time from crypt to surface epithelium is known to take 2-3 days in the rodent. Thus, in a longitudinal section along the crypt-to-surface axis, cells in various degrees of differentiation may be observed, providing a unique in viw system in which to investigate differentiation-related glycosylation events. In this review we will attempt to assimilate the data available from biochemical and cytochemical studies to present a picture of glycosylation reactions in the intestinal epithelium. Very few generalizations will be attempted given the
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variability of the oligosaccharide structures of glycans in different segments of the gastrointestinal tract, as well as between animal species. Moreover, the very fascinating discipline of cancer-related changes in intestinal glycosylation will not be presented here (Boland and Kim, 1984; Bresalier et al., 1985; Kim, 1989a,b). A review by Weiser and co-workers, focusing on intestinal cell membranes (but also discussing glycosylation) has been published in this series (Weiser et al., 1986). 11. Overview of Glycosylation
A brief overview of what is known concerning glycosylation reactions is in order for the understanding of material presented in the rest of this paper. It is not in the realm of this review to present in great detail a treatise on glycosylation reactions in general. Interested readers may consult several excellent papers on this subject published in the last decade (Hanover and Lennarz, 1981; Hubbard and Ivatt, 1981; Kornfeld and Kornfeld, 1985; Hirschberg and Snider, 1987; Roth, 1987a). Most of the work investigating glycosylation has focused on the glycosylation of proteins; not as much is known concerning the glycosylation of lipids. For this reason, the vast majority of this article will focus on glycosylation of intestinal proteins, with only infrequent reference to glycolipids. Glycoproteins can be conveniently divided into two main classes based on the nature of the covalent linkage between the amino acid in a peptide and the sugar residue of an oligosaccharide. One class of glycoproteins are characterized by oligosaccharide chains linked N-glycosidically from the sugar N-acetylglucosamine to the arnide nitrogen of asparagine in the peptide (Fig. 4). A certain restriction seems to be placed on the asparagine residue to be glycosylated; it must be part of the sequence -Asn-X-Sermr (where X can be any amino acid except proline or aspartic acid). Interestingly, even when asparagine is found in the correct sequence it is not always glycosylated, suggesting that accessibility of the sequence may also be a factor. The asparagine-linked (N-linked) oligosaccharides can further be divided into three subclasses depending upon the extent of trimming and elongation reactions: (1) high mannose-type oligosaccharides which contain only the sugars mannose and N-acetylglucosamine (Fig. 4C); (2) complex-type oligosaccharides which contain the sugars galactose, fucose, and sialic acid in addition to mannose and N-acetylglucosamine (Fig. 4B); and (3) hybridtype oligosaccharides which contain both high mannose- and complex-type units (Fig. 4D). Although the three subclasses of N-linked oligosaccharides display a wide variety of structural conformations, they nevertheless all share the common core structure Manal-3(Manal-6)Manp1-4GlcNAc pl-4GlcNAc-Asn'. 'Abbreviations: Fuc, fucose; Gal, galactose; GalNAc, N-acetylgalactosamine; Glc, glucose; GlcNAc, N-acetylglucosamine; Man, mannose; NeuSAc, neuraminic (sialic) acid; GDP-Man, guanosine 5'-diphosphate mannose; UDP-Gal, uridine 5 '-diphosphate galactose; UDP-GlcNAc, uridine 5 '-diphosphate N-acetylglucosamine.
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The second major class of glycoproteins are the so-called 0- or mucin-type glycoproteins and comprise, among others, oligosaccharides attached via an 0glycosidic linkage from the sugar N-acetylgalactosamine to the hydroxyl group of the amino acids serine or threonine in the peptide (Fig. 5). A third, and novel type of glycoprotein has recently been described in which N-acetylglucosamine is 0-glycosidically attached to the peptide (Torres and Hart, 1984). This appears to be a most interesting type of glycoprotein since its synthesis most likely occurs outside of the normal cellular glycosylation pathway. Detailing this type of glycoprotein is beyond the scope of this review; however, this subject has recently been discussed by Hart and co-workers (Hart et al., 1988, 1989). OF N-GLYCOSIDICALLY LINKEDOLIGOSACCHARIDES A. ASSEMBLY
The mechanisms of N-linked glycosylation reactions have been worked out in great detail (Komfeld and Komfeld, 1985; Roth, 1987a) and will only be briefly reviewed here. The classical reactions themselves are restricted to two intracellular compartments, the rough endoplasmic reticulum and the Golgi apparatus. The assembly of N-glycosidically linked oligosaccharide chains follows a unique pathway involving a lipid-linked intermediate. The lipid employed is dolichol phosphate, to which sugars are added in a stepwise manner, with the first seven sugars (two N-acetylglucosamine and five mannose residues) derived from the nucleotide sugars UDP-GlcNAc and GDP-Man, while the next seven sugars are donated from the lipid intermediates Dolichol-P-Man and DolicholP-Glc (Hirschberg and Snider, 1987). The resulting lipid-linked oligosaccharide consists of the structure Glc,Man,GlcNAc,-P-P-Dolichol (Fig. 4A), and is found in the lumen of the rough endoplasmic reticulum. Glycosylation of a growing peptide chain occurs via the en bloc transfer of the oligosaccharide chain from the lipid to an asparagine residue as the peptide is sequestered into the lumen of the rough endoplasmic reticulum. Processing of the oligosaccharide chain seems to begin immediately after its transfer to the peptide. The FIG.3. Light micrographs of semithin sections (1 pm) from rat proximal colon illustrating the basic organization of the intestinal mucosa. (a) The mucosa of the large intestine is composed of the surface epithelium lining the intestinal lumen, and the goblet cell-rich crypt region. Beneath the basement membrane of the surface epithelial cells is the lamina propria (Ip). (b) Higher magnification of the boxed region from (a), demonstrating the organization of the surface epithelium. The section had been incubated with the Lirnaxflavus lectin (specific for sialic acid residues), followed by fetuin-gold complex and silver amplification. All structures containing sialic acid therefore appear black in these photos. Note the intense staining of goblet cell mucus (md), brush border (arrowheads), Golgi apparatus (arrows), and cells in the lamina propria. In contrast, note the lack of nuclear staining (n). A goblet cell in the process of releasing mucus is denoted by an asterisk. x160 (a); x725 (b). Bars = 100 pm (a) and 13 pm (b). (b) (Reproduced with permission from Taatjes and Roth, 1988.)
DOUGLAS J. TAATJES AND fiRGEN ROTH
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A I
@
A I A
@ A A
A
I 0 I
e I
1
e\Jo
a
e I
I 0 I
I
I
0
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m
m
I
m I
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I
I I
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-hm-
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e
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e
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rn
rn
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FIG.4. Schematic representation of typical N-glycosidically linked oligosaccharides. (A) lipidlinked oligosaccharide precursor; (B) complex-type oligosaccharide; (C) high mannose-type hybrid-type oligosaccharide. See Section I1 for details. oligosaccharide: and (D)
extent of processing is dictated by whether the oligosaccharide chain on the glycoprotein is destined to become a high mannose- or complex-type. In the case of the high mannose-type, processing merely involves the removal of the three glucose residues, as well as perhaps a few mannose residues. In contrast, the pro-
GLYCOSYLATION IN INTESTINAL EPITHELIUM
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I
I -Serr/Thr
-
A
Sialic Acid
FIG.5. Schematic representation of typical O-glycosidically linked oligosaccharide. See Section I1 for details.
cessing of complex-type oligosaccharides is more extensive and entails the removal of the three glucose residues and all but three of the mannose residues. These trimming events are carried out by the enzymes glucosidase I, glucosidase 11, and ER mannosidases (Kornfeld and Kornfeld, 1985; Moremen and Touster, 1988). Following these trimming reactions, the glycoprotein is transported via vesicles to the Golgi apparatus where further mannose residues are removed (Moremen and Touster, 1988). Complex-type oligosaccharides are then completed in the Golgi apparatus by the addition of N-acetylglucosamine and the terminal sugars galactose, fucose, and sialic acid.
B. ASSEMBLYOF O-GLYCOSIDICALLY LINKED OLIGOSACCHARIDES
In contrast to the abundant information available concerning the synthesis of asparagine-linked oligosaccharides, less is known regarding the steps involved in the synthesis of O-glycosidically linked oligosaccharides. The synthesis of the oligosaccharide chain seems to be a late posttranslational event, appears to occur by a sequence of classical glycosyl transfer reactions, and does not involve an oligosaccharide-lipid intermediate (Hanover and Lennarz, 1981). The initial event consists of the transfer of N-acetylgalactosamine from UDPGalNAc to serine or threonine in a polypeptide. There seems to be no requirement for a specific amino acid sequence surrounding the serine or threonine, as is necessary for the asparagine in N-glycosidically linked oligosaccharides. However, 0-glycosyl residues are often clustered in regions rich in the amino acid proline, suggesting that a possible signal required for O-glycosylation is contained in the secondary or tertiary structure of the polypeptide chain (Hanover and Lennarz, 1981). The precise cellular location of the onset of O-glycosylation has been difficult to ascertain. Depending upon the methodology employed, various investigators have implicated the rough endoplasmic reticulum (Strous, 1979) or the Golgi apparatus (Hagopian ef al., 1968; Kim et al., 1971; Hanover et af., 1980;
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DOUGLAS J. TAATJES AND JURGEN ROTH
FIG.6. Demonstration of Helixpomario lectin-gold binding sites in sections from chick duodenum. Gold particle label is detectable in goblet cell mucus (md) and Golgi apparatus (arrows). but not in the rough endoplasmic reticulum (rer). At higher magnification (b), label in the Golgi apparatus is seen to be restricted to cis and trans (arrowheads) regions. X 5,500 (a); X 18.000 (b). Bars = 1.8 pm (a) and 0.6 pm (b). (Reproduced from the J. Cell B i d . . 1984,98, 399-406 by permission o f the Rockefeller University Press.)
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Roth, 1984; Deschuyteneer et al., 1988; Tooze et al., 1988) as the site of addition of N-acetylgalactosamine to the peptide chain. These conflicting results are most probably due to the different methodologies employed, or to cell type variability in glycosylation reactions (Section IV,B,2). The majority of the evidence, though, points to the Golgi apparatus (or a pre-Golgi apparatus compartment; see below) as the site of initiation of 0-glycosylation. Elhammer and Kornfeld ( 1984) investigated the subcellular distribution of the initiating polypeptide : N-acetylgalactosaminyltransferase as well as the subsequently acting UDP-Gal : GalNAc-P- 1,3 galactosyltransferase by fractionating the total microsomal membranes of mouse lymphoma BW5147 cells on linear sucrose gradients. They found that the two transferases were present in membranes of different densities. The galactosyltransferase codistributed with the “classical” galactosyltransferase involved in asparagine-linked glycosylation and was shown to reside in trans-Golgi apparatus cisternae of HeLa cells (Roth and Berger, 1982), whereas the polypeptide : N-acetylgalactosaminyltransferase distributed in a fraction intermediate between those containing galactosyltransferase activity and glucosidases I and 11, the latter indicative of the endoplasmic reticulum (Lucocq et al., 1986). These results were interpreted to suggest that the polypeptide : N-acetylgalactosaminyltransferase most likely was derived from membranes representative of the cis-Golgi apparatus, and was contained in a separate Golgi compartment from the galactosyltransferase. In a carefully controlled complimentary biochemical approach, Abeijon and Hirschberg (1987) reported that in rat liver the polypeptide : N-acetylgalactosaminyltransferase activity (using apomucin as an exogenous acceptor) was highly enriched in membranes derived from the Golgi apparatus compared to those derived from the rough and smooth endoplasmic reticulum. Moreover, they found that vesicles prepared from the Golgi apparatus were able to translocate UDP-GalNAc into their lumen in an in vitro assay, at rates 4-6-fold higher than those from rough and smooth endoplasmic reticulum. These results demonstrated that at least in rat liver, all the cellular machinery necessary for the initiation of 0-glycosylation was located within the Golgi apparatus. An independent cytochemical investigation has also implicated the cis-Golgi apparatus as the site for the onset of 0-glycosylation. Roth (1984) used a Helix pomatia lectin-gold complex in a postembedding study as a probe for terminal nonreducing N-acetylgalactosamineresidues. In goblet cells of chick and rat intestine he found label in the cis and trans regions of the Golgi apparatus, but not in the endoplasmic reticulum (Figs. 6 and 7). He interpreted these findings to indicate that the initial transfer of N-acetylgalactosamine to the peptide occurs in the cis region of the Golgi apparatus, while the staining present in the trans region of the Golgi apparatus was representative of terminal N-acetylgalactosamine residues added on to the oligosaccharide chain. These results were corroborated and expanded upon by Deschuyteneer er al., (1988). They used
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FIG.7 . Demonstration of H . pomuria lectin-gold binding sites in ultrathin section from rat colon. Gold particle label is found over cis-cistemae and dilated trans-cistemae (arrowheads) of goblet cell Golgi apparatus. as well as in mucus droplets (md). Note lack of staining over cisternae of rough endoplasmic reticulum (rer). X 11,500. Bar = 0.9 pm. (Adapted from the J. Cell B i d . , 1984, 98, 399-406 by permission of the Rockefeller University Press.)
immuno- and cytochemical means to investigate the site of addition of N-acetylgalactosamine to the peptide chain of porcine submaxillary gland mucin. Employing an antibody which recognized only the deglycosylated form of mucin (apomucin). they found that immunoreactivity was localized throughout the rough endoplasmic reticulum (including the nuclear envelope) of mucous cells. In contrast, staining with a H. pomaria lectin-gold complex was found in the Golgi apparatus and mucus droplets, but not in the rough endoplasmic reticulum, confirming the results obtained by Roth (1984) for intestinal goblet cells. Interestingly, by treating thin sections with a cocktail of glycosidases, Deschuyteneer et al., (1988) were able to detect immunoreactivity with the antibody raised against apomucin in the cis-cistemae of the mucous cell Golgi apparatus. These results provided very strong further support for the contention that the onset of 0-glycosylation is a posttranslational event occurring most likely in cis-cistemae of the Golgi apparatus. Very recently, a third compartment has been implicated as the site of the onset of 0-glycosylation. Tooze and co-workers ( 1988) investigated steps in
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the 0-glycosylation of the El glycoprotein of coronavirus MHV-A59 using a combination of pulse-labeling and morphological techniques. They found that N-acetylgalactosamine was added to the E l protein about 10 min after synthesis, followed about 10 min later by the addition of galactose and sialic acid, producing the mature oligosaccharide. They then took advantage of temperature-sensitive steps in the exocytic transport system of virus release (Saraste and Kuismanen, 1984) to block the movement of the glycosylated El protein out of different compartments. Interestingly, they were able to identify a smooth-membraned compartment situated between the endoplasmic reticulum and the cis-Golgi apparatus where the El glycoprotein was found to already possess N-acetylgalactosamine, but not galactose or sialic acid. They interpreted these results to indicate that the site of addition of the core N-acetylgalactosamine is this smooth membrane compartment, which most likely represents a budding compartment situated between transitional elements of the rough endoplasmic reticulum and the cis side of the Golgi apparatus. However, it should be borne in mind that these results were obtained from virally infected cells, which may or may not behave as normal cells with respect to glycosylation reactions. Nevertheless, in light of these findings, the results of Elhammer and Komfeld (1984) discussed above could also be interpreted to suggest that the polypeptide : N-acetylgalactosaminyltransferase is housed within this preGolgi apparatus budding compartment, rather than within cis-Golgi apparatus cisternae. It seems likely, though, that the definitive answer defining where the onset of 0-linked glycosylation occurs awaits the immunocytochemical localization of the polypeptide : N-acetylgalactosaminyltransferase. Regardless, subsequent 0-linked glycosylation reactions are known to occur in the Golgi apparatus.
111. Methods Employed to Investigate Cellular Glycosylation Reactions in Intestine Investigations into intestinal glycosylation have mainly drawn upon biochemical and morphological techniques for the demonstration of enzymes (glycosyltransferases) involved in glycosylation reactions, as well as the carbohydrate products resulting from the enzymatic action. These techniques are not specific to the study of intestinal tissues, but rather have been used to study glycosylation in general. The unique application of these methods to intestinal tissues stems mainly from the following three areas: (1) separating activities from crypt and villus portions of the small intestine; (2) separating activities from Golgi apparatus membranes and plasma membranes; and (3) investigating glycosylation changes in different intestinal regions.
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A. BIOCHEMICAL METHODS Two main biochemical tacks have been followed in the investigation of intestinal glycosylation. The first was to assay for the activity of specific glycosyltransferases, followed by subsequent purification. These assays were usually employed with the goal of measuring and comparing activities of various glycosyltransferases in the different intestinal segments, as well as crypt versus villus. Moreover, such methods were also extensively used to compare changes in different glycosyltransferase activities during development (Section IV,A,2). The main drawbacks of this method were the possible contamination of cellular fragments which could lead to erroneous conclusions. as well as the inability to identify which cells in the mixture actually contained the glycosyltransferase of interest (Section IV,A, I ) . The second most popular method has been to measure the incorporation of radiolabeled sugars into glycoproteins and glycolipids and following their transport to final destinations (Section IV,A,3). B. MORPHOLOGICAL METHODS As noted in the Introduction, classical morphological and cytochemical methods have been extensively applied to studies of intestinal glycosylation. These studies will not be mentioned again here so that we may focus on more recent types of cytochemical investigations. However, it should be borne in mind that although we now have available more sensitive and specific probes, as well as refined cytochemical techniques, the results obtained have dramatically supported the original conclusions drawn from the earlier studies. Investigations employing lectins have proven to be of particular importance for the study of intestinal glycosylation (Etzler and Branstrator, 1974). Lectins are carbohydrate-binding proteins of nonimmune origin that agglutinate cells and precipitate glycoconjugates (Goldstein and Poretz, 1986). When coupled to an appropriate marker (Fig. 8) they can be used to demonstrate the cellular occurrence and distribution of specific sugar residues (Roth, 1978, 1987b). In intestinal studies they have been used to investigate Golgi apparatus glycosylation events, to determine the pattern of sugar residue expression at the plasma membrane of epithelial cells along the intestinal tract, and to monitor changes in the glycosylation pattern of epithelial cells during development and neoplastic transformation. For convenience, a list of lectins (with their saccharide specificities) mentioned in this article is presented in Table I. A major advance for the field of glycosylation investigation in general (however, also applicable to intestinal studies; Sections IV,B,2; IV,C) was the development of sensitive, yet routine methods for electron microscopic immunocytochemistry. The first was the introduction of colloidal gold particles as an electron dense marker for immunocytochemistry (Faulk and Taylor, 1971). This
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GLYCOPROTEIN - G O L D
\
LECTIN
- GOLD
"\ LECTIN
GLYCOPROTEINS
FIG.8. Schematic representation of lectin-gold techniques for the detection of sugar residues on thin sections from tissues embedded in Lowicryl K4M. Lectins can be applied directly complexed with particles of colloidal gold (left side of figure), or in a two-step cytochemical affinity technique employing an unlabeled lectin followed by a glycoprotein-gold complex (right side of figure). A detailed description of these procedures can be found in Roth er al. (1988a) and Roth (1989).
was followed by the introduction of the protein A-gold technique (Roth et al., 1978) for the postembedding localization of antigenic sites (Fig. 9). The virtues of this method have been detailed in many reviews (Roth, 1983a, 1986, 1989; Bendayan, 1984) and will not be enumerated here. It suffices to say that the protein A-gold technique in conjunction with the introduction of the low temperature embedding methods employing Lowicryl K4M (Carlemalm et al., 1982) provided the means necessary for the precise immunolocalization of the relatively scarce glycosyltransferases. Of course, of equal importance was the production and availability of highly specific anti-glycosyltransferase antibodies (Roth and Berger, 1982; Weinstein et al., 1982; Shaper et al., 1985; Ulrich et al., 1986). The combination of these innovations provided immediate dividends
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PROTEIN A
6 ,
-
GOLD
J
\
L ; N , T I G E N : h
n
\A n,
ANTIBODIES
TISSUE SECTION FIG.9. Schematic representation of the protein A-gold technique for antigen localization on thin sections from tissues embedded in Lowicryl K4M. This is a typical two-step technique in which sections are first incubated with an antibody, followed by protein A-gold complex which binds to the Fc portion of the antibody. As illustrated on the right side of the figure, multiple antigens can be detected on the same section by employing colloidal gold particles of different sizes. Details of this technique can be found in Roth et a/. (1978) and Roth (1983a. 1989). TABLE I SACCHARIDE-BINDING SPECIFICITES OF VARIOUSLECTINS Lectin
Canar,alioensfformis Lens c~ilinaris
Common name
Abbreviation
Pisum sarivrtm Triticum wlgure
Jack bean Lentil Pea Wheat germ
Con A LCL PSL WGA
Dolichos hiflorus
Horse gram
DBL
He1i.r pomatia
Edible snail
HPL
G l w i n e ma.\
soybean
SBL GSL I
Griffonia simpiicifolia I-B, .Arachis hypogaea Ricinus communis I Ricinus rommunis II Durura stramonium
Lorus terragonolobus U1e.r europaeus Lima.\ flaws Samhucus nigra L. Maackia amurensis
-
Peanut Castor bean Thorn apple Asparagus pea Gorse seed Slug Elderberry -
PNL RCL I RCL I I DSL LTL UEL I LFL SNL I MAL
Nominal specificity" aMan > aGIc > GlcNAc aMan > aGlc > GlcNAc aMan > aGlc = GlcNAc GlcNAc ( ~ I P G l c N A c ) ,>~ , PGlcNAc > NeuSAc GalNAc al.3GalNAc >> aGalNAc GalNAc al.3GalNAc > aGalNAc aGalNAc = PGalNAc aGal>> aGalNAc CalPl,3GalNAc > a and PGal pGal> aGal>> GalNAc P and a G a l > GalNAc GalPl,4GlcNAc = GlcNAc (PI,4GlcNAc),., a-L-Fuc a-L-Fuc aNeuSAc > aNeuSGc NeuSAc a2,6Gal/GalNAc NeuSAc a2,3Cal
uAbbreviations: Man, mannose; Glc, glucose; GlcNAc, N-acetylglucosamine; GalNAc, N-acetylgalactosamine; Gal, galactose; Fuc, fucose; NeuSAc, sialic acid.
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with the publication of the first immunocytochemical localization of a glycosyltransferase at the electron microscopic level (Roth and Berger, 1982). This paper provided a key piece of evidence which led to the subcompartmentation model of the Golgi apparatus, which will be discussed in detail in Section IV,B,lb. At the same time, the opportunity had now arisen to explore the subcellular distribution of glycosyltransferases at the electron microscopic level in intestinal epithelial cells.
IV. Distribution of Intestinal Glycosyltransferases and Their Saccharide Products A.
STUDIES ON
WHOLE TISSUE
1 . Measurement of Glyco.$yltransferase Activities in Adult Animals As mentioned earlier, many of the studies concerning glycosyltransferases in intestinal cells sought to compare activities in the crypt with those in the villus, or to compare glycosyltransferases among various segments of the intestinal tract. Weiser (1 973a,b) compared the glycosyltransferase activity of mature cells in the villus with immature cells of the crypt by measuring the incorporation of radiolabeled monosaccharides into surface membrane glycoproteins, and using this as a measure of the corresponding glycosyltransferase activity. He employed a separation method based on citrate and EDTA to dissociate cells, resulting in epithelial cell fractions which defined a gradient of cells from villus tips to crypts. His results demonstrated that the levels of N-acetylgalactosaminyltransferase, galactosyltransferase, and fucosyltransferase were approximately 10-fold greater on crypt as compared to villus cells, whereas sialyltransferase activity was higher on villus cells. In a subsequent study, however, Weiser and co-workers (1978) not only separated crypt from villus cells, but also prepared membrane fractions, and reported that the basolateral plasma membrane of villus cells was rich in galactosyltransferase activity. This discrepancy with their previous results (Weiser, 1973a,b) was explained as resulting from the presence of glycosidases on the microvilli of intact villus cells in the earlier study which had interfered with the detection of glycosyltransferases on the lateral plasma membrane. However, an alternative explanation was proposed by Lau and Carlson (1981). They found that nucleotide pyrophosphatase, an enzyme that interferes with glycosyltransferase assays, is particularly enriched in intestinal mucosa (especially at the villus tips). By assuring inactivation of this enzyme, they determined that the activity of two galactosyltransferases (one acting on asparagine-linked and the other acting on 0-linked oligosaccharides) displayed essentially identical activities on both crypt and villus cells. They
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advocated exercising caution when interpreting the measurement of intestinal glycosyltransferase activities without recognizing the potential influence of nucleotide pyrophosphatase. In subsequent studies, Weiser’s group (Weiser el a/., 1987; Wilson el al., 1987) took precautions against nucleotide pyrophosphatase activity and analyzed the rat intestinal distribution of two different galactosyltransferases; one acting on N-linked oligosaccharides and the other acting on 0-linked (mucintype) oligosaccharides (this enzyme may be identical to that investigated by Lau and Carlson mentioned above). They were still able to detect both crypt : villus differences as well as differences among intestinal segments for both enzymes. The galactosyltransferase acting on 0-linked oligosaccharides showed increased activity in the duodenum and distal ileum of the small intestine, and the cecum and proximal colon of the large intestine (Wilson eta/. , 1987). These areas of increased activity corresponded to areas of increased mucus production. Moreover, within the duodenum this galactosyltransferase showed a moderately increased activity in cells from the crypt region as compared to those of the villus; however, no such difference was detectable in the jejunum or ileum. The galactosyltransferase acting on N-linked oligosaccharides displayed highest activities in the terminal ileum, cecum, and proximal colon, with lesser amounts detected in the jejunum and duodenum (Weiser et af., 1987). Although they could not demonstrate a difference in total homogenate galactosyltransferase activity between crypt and villus cells, they found that assays for cell surface galactosyltransferase revealed an elevation in the crypts (Section IV,C). Kim and co-workers (1975) devised a planar sectioning technique utilizing a mounted razor blade to cut frozen sections for the separation of crypt from villus cells. Upon homogenization the sections were assayed for glycosyltransferase activity. The results showed that sialyltransferase activity was enriched in crypt cells. whereas galactosyltransferase activity was approximately equal in both regions (Kim ef al.. 1975). These results are in contrast to those from Weiser’s group mentioned above. The discrepancy may have arisen from the different methodologies employed by the two groups for the separation of cell populations or from glycosyltransferase activity variation among intestinal sections. Nevertheless, both separation techniques suffer from the questionable purity of the fractions. Indeed, the importance of the purity of intestinal fractions cannot be overstated, since elements other than intestinal epithelial cells have recently been shown to be the major, if not the only source for sialyltransferases in rat small intestine (Paulson et af., 1989). Glycosyltransferase activities were also shown to vary from the proximal to the distal regions of the rat small intestine (Morita et al., 1986). Specifically, activities for two galactosyltransferases (acting on N- and 0-linked oligosaccharides), two sialyltransferases (acting on N- and 0-linked oligosaccharides), fucosyltransferase and N-acetylgalactosaminyltransferasewere consistently found
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to be higher in distal regions of the small intestine compared with proximal regions. These results were corroborated by the carbohydrate analysis of brush border membranes in proximal and distal small intestine. Interestingly, both sialyltransferase enzymes displayed the lowest activity of all the glycosyltransferases assayed. In an immunocytochemical investigation (Section IV,C) we found that the distribution of the a2,6-sialyltransferase was regionalized within the rat intestine (Taatjes et al., 1988a). Although abundant staining was detectable in all regions of the large intestine, no labeling was detectable in any portion of the small intestine from the same animals. These results were corroborated by direct measurement of enzymatic activity for the a2,6- and a2,3-sialyltransferases. In this case, the activity of these two sialyltransferases (both acting on N-linked oligosaccharides) was undetectable in rat small intestine. These results would appear to conflict with those of Morita et al. (1986) mentioned above, as well as those of Van Halbeek er al. (1983) who reported the presence of sialic acid a2,3 linked to galactose in much glycoproteins from rat small intestine. The apparent discrepancy may be explained in part by the fact that we examined sialyltransferase activity only for the mucosal surface of the intestine scraped from the intestinal wall. Indeed, in a subsequent investigation, Paulson et al. (1989) found that homogenates of the intestinal wall itself contain substantial levels of this sialyltransferase in the small intestine. Moreover, in contrast to the results of Morita er al. (1986) they found that the activity for a sialyltransferase (adding sialic acid in an a2,3 position to galactose in 0-linked oligosaccharides) actually decreased in the mucosa from proximal to distal small intestine. Ironically, no activity was detectable in the ileal mucosa, whereas substantial activity was measurable in the ileum wall. Thus, results obtained from intestinal homogenates or segments are not directly comparable. This also indicates that these sialyltransferase enzymes are differentially expressed within different regions of the small intestine, each having specialized functions, yet identical cell types. These results have recently been further supported by in siru lectin-binding studies (Section IV,A,6). The situation is further complicated by the acceptor substrates used for the measurement of glycosyltransferase activity. For instance, according to the “one-enzyme one-linkage’’ hypothesis (Hagopian and Eylar, 1968) at least a dozen different sialyltransferases must exist in order to form the known linkages of sialic acid to penultimate sugars. Thus, fetuin, which is quite often employed as an acceptor substrate for sialyltransferase activity, contains both N- and 0linked carbohydrate groups which are acceptors for at least four different sialyltransferases (Kim er al., 1975; Weinstein et af., 1982; Green et al., 1988). This may explain in part the sometimes variable results reported using different techniques with respect to glycosyltransferase distribution within the intestine. Earlier studies demonstrating glycosyltransferase activity based upon known acceptor substrates may have actually been measuring the activity of several
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different enzymes. However, due to antibody specificity, immunological-based techniques are assaying for the presence of one highly linkage-specific glycosyltransferase. Therefore, these limitations should be borne in mind when interpreting results from different investigators employing different techniques. Much less information is available concerning the glycosyltransferase activity in the large intestine. Freeman et al. (1978) have demonstrated that in adult rat large intestine both gaiactosyltransferase and sialyltransferase activities were significantly greater in proximal than in distal colonic mucosa. We have reported (Taatjes e f al.. 1988a) enzymatic activity for two sialyltransferases acting on asparagine-linked oligosaccharides in mucosal scrapings from rat large intestine (Section 1V.C; Table IV).
2 , Measurement of Glvcosyltransferase Activity during Development The activity of several glycosyltransferases has been found to vary during postnatal development in both rat small and large intestine. Sialyltransferase activity was present in increased levels during the suckling period, and decreased 5-fold during the subsequent weaning and adult periods (Chu and Walker, 1986). On the other hand, fucosyltransferase activity was very low during suckling phase, rapidly increased during weaning, and reached adult levels by 5 weeks of age (Chu and Walker, 1986). The activities of two galactosyltransferases, the UDP-Gal : GlcNAc(P1-4)galactosyltransferase, and UDP-Gal : GalNAc(p 1 -3)-galactosyltransferase have also been found to be under developmental regulation (Ozaki et al., 1989). Both glycosyltransferases demonstrated a marked elevation in activity after the weaning period and into adulthood in all regions of the rat small intestine. All of these results considered together demonstrate that activities for galactosyltransferase, N-acetylgalactosaminyltransferase and fucosyltransferase all increase during postnatal development of rat small intestine. On the other hand, sialyltransferase activity declines during the same developmental period. These data correlate well with known changes in terminal glycosylation of microvillar proteins during postnatal development of rat small intestine (Sections IV,A,4 and 6). The activity of galactosyltransferase has been measured in fetal and postnatal rat large intestine (LaMont and Ventola, 1978). The activity in fetal homogenates increased 4-to 7-fold between 18 and 22 days, the last 4 days of gestation. The enzyme activity then gradually increased postnatally, reaching adult levels by day 15. Determination of the autoradiographic incorporation of [3H]galactose into fetal large intestine glycoconjugates correlated well with the increase in galactosyltransferase activity during this period (Rampal et al., 1978). Interestingly, these autoradiographic studies revealed the selective incorporation of [3H]galactoseinto goblet cells but not into absorptive cells. These results therefore suggest that the maturation of fetal rat large intestine during the
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last 4 days of gestation is accompanied by the appearance of goblet cells and enhanced mucus synthesis.
3. Analysis of Sugar Content of Membrane Glycoproteins in Adult Animals Two basic biochemical methods have traditionally been applied in order to determine the carbohydrate composition of intestinal membrane glycoproteins. The first method requires the administration of radiolabeled sugar precursors into the lumen of the intestine, followed by purification and analysis of the incorporation of the sugars into membrane glycoproteins. The second method employs the binding of labeled lectins to intestinal plasma membrane fractions. In an early study, Kim and Perdomo (1974) traced the incorporation of [14C]glucosamineinto the membranes of intestinal cells. They prepared three membrane fractions, consisting of smooth, rough, and brush border membranes. They observed incorporation first into smooth membranes, followed after a lag period by entrance into rough and brush border fractions. Aside from the peculiar late entrance into a rough membrane fraction, these results trace the transit of ['4C]glucosamine-containingglycoconjugates from the Golgi apparatus to the brush border. A further purification to distinguish apical (brush border) membranes from basolateral was not attempted. Quaroni and co-workers (Quaroni et al., 1980; Herscovics et al., 1980) took these studies further by separating Golgi apparatus, apical, and basolateral membranes, both in crypt and villus cells. They measured the incorporation of ~ - [ 5 , 6 3H]fucose and ~-[2-~H]mannose into intestinal membrane glycoproteins following an intraperitoneal injection of these radiolabeled sugars. The incorporation of mannose was roughly equal in crypt and villus cells, whereas fucose incorporation was elevated in the differentiated villus cells (Quaroni et al., 1980). Fucosylated glycoproteins were originally detected in the Golgi apparatus and basolateral membranes, followed by redistribution into villus membranes after 3 4 hr. In contrast, most mannose-labeled glycoproteins remained in the Golgi and basolateral membrane fractions. They interpreted their results to indicate that fucosylated glycoproteins represent a special class of membrane components that appear with differentiation (absent in undifferentiated crypt cells) and are specifically localized to the luminal portion of the intestinal cell plasma membrane. In an accompanying paper, Herscovics et al. (1980) used similar techniques to demonstrate that high-mannose oligosaccharides were the precursors of complex oligosaccharides. Moreover, they provided evidence that luminal membranes of both crypt and villus cells were greatly enriched in complex oligosaccharides as compared with basolateral plasma membranes, but no qualitative changes were found to occur during cellular differentiation. Thus, their results suggested that intestinal epithelial cells were polarized with respect to plasma membrane glycoconjugate oligosaccharide composition (Section IV,A,5).
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The second biochemical method, utilizing lectin binding to isolated cell plasma membranes, is a more recent innovation, and has found widespread use for the comparison of membrane glycoproteins among intestinal segments as well as during maturation. Kim and co-workers (Morita et a [ . , 1986) examined the reactivity of brush border membrane components with lectins, and the binding of brush border membrane-associated enzymes to Ricinus communis lectin I (RCL I) and wheat germ agglutinin (WGA) in segments from the proximal and distal small intestine. In addition they analyzed the carbohydrate composition of the brush border membranes. Their results indicated that although brush border membrane glycoproteins from distal portions of the small intestine contained more complete oligosaccharide side chains, the glycoprotein profile on SDS gels was less complex than in proximal small intestine. Specifically, more WGA and succinylated-WGA-binding glycoproteins were present on brush border membranes from proximal compared to distal segments. However, the binding of RCL I to brush border membranes was two times higher in the distal as compared to proximal small intestine. Overall sugar content was higher in distal small intestine brush border membranes, reflected mainly by elevated galactose and sialic acid content. The content of N-acetylglucosamine appeared equal in the two intestinal segments. These results suggest that the carbohydrate content of brush border membranes changes with the progression of the gastrointestinal tract, with more distal regions of the small intestine containing more completed oligosaccharide chains. 4 . Analysis of Sugar Content of Membrane Glycoproteins During Development
Similar biochemical methods to those just mentioned above have also been used extensively to investigate changes in the glycosylation pattern of microvillar proteins in postnatal intestine. Mahmood and Torres-Pinedo ( 1 983) incubated microvillar membrane preparations from postnatal rats with radiolabeled lectins to determine the carbohydrate profile of membrane glycoconjugates. They found that the microvillus membrane of suckling rats (from birth to about 2 weeks of age) was rich in glycopeptides containing binding sites for peanut lectin (PNL) in sialyl-substituted form. During the weaning phase (14-21 days postnatal), the membranes lost about half of these binding sites, accompanied by decreased sialic acid content and increased content of glycopeptides containing unsubstituted binding sites for soybean lectin (SBL) and RCL I. Perhaps the most important result from this study was the finding that the sialic acid content of microvillar plasma membrane drastically decreases from the suckling to the weaning period. Indeed, in a subsequent paper (Torres-Pinedo and Mahmood, 1984) they found that this decrease in microvillar plasma membrane sialic acid content was accompanied by a dramatic rise in fucose content. They observed that the binding of '2sl-labeled WGA to neuraminidase-sensitive sites in the microvillar membrane decreased markedly from early suckling to weaning ages.
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At the same time, the binding of Iz5I-labeledUlex europaeus lectin I (UEL I) to microvillar membranes showed an opposite increase from suckling to weaning periods. This developmentally related shift from sialylation to fucosylation was found for both glycoproteins and glycolipids of the microvillar membrane, suggesting that it is a general phenomenon for membrane constituents. They postulated that such a dramatic shift from a strongly acidic to a more neutral microvillar glycocalyx could relate to the physiological changes occumng in the intestine concomitant with development. These studies were followed up by examining the carbohydrate profile of individual microvillar membrane proteins during postnatal development of the rat small intestine. Srivastava et al. (1987) found that the terminal glycosylation of several microvillar glycoproteins of >90,000 Da (most likely hydrolases) does not reach complete maturation until after weaning, although their content within the membrane has reached adult levels by this time. Moreover, several of these glycoproteins were fully sialylated during the suckling period, whereas addition of N-acetylgalactosamine and fucose continued well into the weaning period. Buller el al. (1990) took such investigations one step further by examining the glycosylation of a known glycoprotein, lactase-phlorizin hydrolase, during development of the rat small intestine. Lectin binding to the enzyme immunoprecipitated from microvillus membranes revealed the presence of both N- and 0linked oligosaccharide chains containing mannose and galactose, which did not vary throughout development. In contrast, the content of fucose and sialic acid was developmentally regulated; sialic acid was present at weaning and declined through adulthood, whereas fucose was not detectable until rats were 20 days of age. Thus, by examining a single enzyme, it was established that the core N- and 0-linked oligosaccharide structures of this microvillar hydrolase remain constant during development, whereas alteration in terminal glycosylation occurs with a shift from sialic acid at suckling to fucose in adulthood. The above studies taken together show that a definite change in glycosylation occurs on specific microvillar membrane glycoproteins during the postnatal developmental period. In a related study (Jaswal et al., 1988), the content of sialic acid and fucose in enterocytes was measured in crypt and villus cells from suckling and adult animals. In suckling animals, no change was found in the sialic acid content of enterocytes during progression from crypt to villus. In contrast, the sialic acid content decreased precipitously from crypt to villus in adult animals. The fucose content of enterocytes from suckling animals was greater in the crypts than in the villus, whereas in adult animals fucose content was much greater in the villus.
5 . Cytochemical Detection of Lectin Binding to Intestinal Cells in Situ Much cytochemical data based upon lectin binding studies have contributed to the understanding of intestinal glycosylation patterns. In the absence of
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glycosyltransferase enzymatic measurements, lectin-binding sites can be taken as indicative of specific glycosyltransferase activity. The earliest studies used lectins conjugated to fluorescent dyes in a direct labeling technique, usually on frozen or paraffin sections. Such investigations demonstrated differences in lectin-binding patterns to intestinal epithelial cells in the various segments of the intestinal tract. For instance, Etzler and Branstrator (1974) examined the binding of FITC-conjugated lectins from Dolichos biflorus (DBL), Lotus terragonolobus (LTL), Ricinus communis I, and Triticum vulgare (WGA) to the various regions from rat small intestine. They observed differences in the binding of the lectins to both the epithelial cell plasma membranes, as well as to the goblet cell mucus. With respect to plasma membrane staining, LTL, RCL I, and WGA bound to the microvillar portion of the epithelial cells lining the crypts and villi in the proximal regions of the small intestine. This pattern of staining was altered along the first 15 cm of the small intestine, such that distal to this point the apical surfaces of only those epithelial cells in the crypts and at the base of the villi reacted with LTL and RCL I, while WGA stained the apical surfaces of cells lining the villi. In the distal small intestine, LTL, RCL I, and WGA stained the cell surfaces of only those epithelial cells at the base of the villi and in the crypts. DBL did not stain the epithelial cell surface in any portion of the small intestine. With respect to staining of the mucus content of goblet cells, WGA and DBL stained the goblet cells in proximal portions of the intestine, whereas in middle and distal regions all four lectins were found to stain goblet cell mucus. These results suggested that the content of complex carbohydrates in goblet cell mucus increases from proximal to distal regions of the small intestine. In a preembedding peroxidase study, Ovtscharoff and Ichev (1984) showed that in rat small intestine (middle regions), the pea and soybean lectins stained the microvillar membrane from epithelial cells in the crypts and lower villus more intensely than those in the upper villus and lumen. Essner et al. (1978) also used several lectins conjugated to FITC to investigate the lectin-binding pattern to cryostat sections from portions of the descending colon of the rat. Besides reactivity in goblet cell mucus and plasma membrane, they identified cytoplasmic staining which they attributed to the Golgi apparatus. Binding sites for the lectins from Glycine max (SBL) and Dolichos hiflorus were observed in goblet cell mucus, apical and basolateral plasma membranes, and in the apical cytoplasm, indicating the presence of terminal nonreducing N-acetylgalactosaminyl residues at these sites. WGA, RCL I, UEL I, and concanavalin A (Con A) all stained the cytoplasm of epithelial cells, but did not, or only weakly, stain mucus droplets and plasma membranes. Gorelick et al. (1982) examined lectin-binding patterns in the plasma membrane and goblet cell mucus of epithelial cells in the various regions of guinea pig large intestine. Staining of the brush border with the various rhodamine-labeled lectins tended to be heterogeneous across the regions of the large intestine.
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In general, though, Con A, WGA, RCL 11, and PNL stained the brush border of the right colon, whereas RCL I and SBL stained the transverse colon intensely. Limulin reacted with the brush border only in the left colon. Staining of the goblet cell mucus was even more variable. Gorelick et al. (1982) not only examined the lectin binding to the goblet cell mucus in the various regions of the large intestine, they also separated the intestinal segments according to crypt regions: basal, middle and apical. Again, the staining patterns varied not only among the large intestinal segments, but also within crypt region of individual segments. Noted exceptions were LTL and PNL which did not stain the goblet cell mucus in any area examined. Moreover, goblet cells in the transverse and left portions of the large intestine tended to react more intensely with the various lectins employed, suggesting a maturation of goblet cell mucus along the large intestine. The paper by Gorelick et al. (1982) also served to usher in the modem approach to investigating intestinal lectin-binding sites; namely, the postembedding application of colloidal gold-labeled lectins to ultrathin sections. They prepared a complex of colloidal gold particles with RCL I1 and applied this to sections from intestine embedded in Epon-Araldite. They were able to demonstrate binding of this complex to goblet cell mucus, apical plasma membrane, apical cytoplasmic vesicles, and Golgi apparatus. Since this time, numerous papers have been published utilizing both colloidal gold-labeled lectins and peroxidase-labeled lectins at the light and electron microscopic level to investigate intestinal glycosylation patterns. The main,benefit of such studies has been the increase in resolution over fluorescence studies obtainable with these methods. Many of these studies were interested in investigating the role of the Golgi apparatus in glycosylation, and their results will be described in Section IV,B,3. In the current section we will detail the results of these studies as they relate to plasma membrane and mucous glycoconjugates. Helixpomatiu lectin (HPL) binding has been observed (Fig. 10) in the mucus and apical plasma membrane of chick duodenum (Roth, 1984), rat duodenum (Ellinger and Pavelka, 1985), and rat jejunum (Murata er al., 1986). RCL I binding has been reported in the mucus and apical and basolateral plasma membranes of chick (Roth, 1983b) and rat duodenum (Ellinger and Pavelka, 1985), and the basolateral plasma membrane of rat proximal colon epithelial cells (Roth et al., 1988a). Pavelka and Ellinger (1989b) have shown binding of Erythrina cristagalli lectin (ECL) to apical and basolateral plasma membranes and goblet cell mucus in rat duodenum, while Egea et al. (1989) have reported identical results by using Daturu srramonium lectin (DSL). Lotus terragonolobus lectin (LTL) binding to the apical plasma membrane and goblet cell mucus has been described in chick duodenum (Roth, 1983b), while UEL I binding to apical plasma membrane and goblet cell mucus has been reported for rat duodenum (Ellinger and Pavelka, 1988a). Binding of sialic acid-specific lectins has also been documented in intestinal cells. Roth et ul. (1984) found binding of
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the Limaxflavus lectin (LFL) to the apical plasma membrane and goblet cell mucus in rat distal colon. Similar results were also found in rat proximal colon (Taatjes and Roth, 1988). Recently, staining with Maackia amurensis lectin (MAL) has been reported in the apical and basolateral plasma membranes and goblet cell mucus in pig colon (Sata el al., 1989). Furthermore, we have recently observed staining with Sambucus nigra L. lectin (SNL I) complexed with colloidal gold particles (Taatjes et al., 1988b) in mucus droplets, but not in the plasma membrane of rat jejunal epithelial cells (Taatjes and Roth, 1990; Section IV,A,6). The main message resulting from all of these studies is that lectins recognizing complex carbohydrate structures bind to plasma membranes and goblet cell mucus in both small and large intestine from various species.
6 . CyfochemicalDetection of Lectin Binding to Intestinal Cells in Situ During Development and Diferentiation As described in Section IV,A,2, the activities of several glycosyltransferases are altered during intestinal cell development. Such alterations are also reflected in the modification of lectin binding to epithelial cells that occurs during postnatal development. In the rat small intestine, Etzler and Branstrator (1979) found developmental changes in the binding of RCL I, LTL, and WGA. RCL I stained the brush border of epithelial cells as early as 1 hr after birth. The staining became patchy at the cell surface over the next few days, reacting uniformly with the surface 5-14 days after birth. By 19-24 days postnatal, the epithelial cell surface began to lose its ability to react with RCL I, and by 30 days postnatal, the cell surfaces were no longer stained with RCL I. The onset of LTL staining was a much later event, commencing between 11 and 19 days after birth. By 28 days after birth, regional differences were apparent with respect to LTL binding to intestinal cell surfaces; brush borders of cells lining the villi in the distal portion of the small intestine were no longer bound by LTL. Wheat germ agglutinin (WGA) stained the brush border of epithelial cells from 1 hr after birth, until about postnatal day 19 when cells lining the villi were no longer stained with this lectin. We have recently investigated the binding of sialic acid-specific and fucosespecific lectins to developing rat small intestinal cells (Taatjes and Roth, 1990). In line with the results detailed in Sections IV,A,2 and 3 concerning developmental-related changes in sialyltransferase and fucosyltransferase activities, as FIG. 10. Low-power electron micrograph demonstrating H . pomatiu lectin-gold binding sites in chick duodenum. In the center of the micrograph a prominent goblet cell is displayed, with intense gold particle labeling present in the goblet cell mucus (asterisk), in the Golgi apparatus, and in the apical plasma membrane (arrowheads). Label is also observable in the apical plasma membrane of adjacent absorptive cells (arrowheads). X 1,710. Bar = 5 p n . (Reproduced from the J . Cell B i d . 1984.98, 399406 by permission of the Rockefeller University Press.)
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well as the lectin-binding results of Etzler and Branstrator (1974), we found that binding of SNL I, LFL, and UEL I to intestinal cells changed with postnatal development. SNL I (Fig. lla,b) and LFL stained the brush border and mucus droplets in animals during the suckling phase. During weaning (day 23) we found that individual epithelial cells were no longer stained with SNL I (Fig. llc,d and Fig. 12) and LFL. By adulthood, staining with these two sialic acidspecific lectins was restricted to goblet cell mucus and cells in the lamina propria and submucosa (Fig. 1 le,f). In contrast, binding of fucose-specific UEL I was restricted to goblet cell mucus during the suckling phase, but by day 23 postnatal appeared in the brush border of some epithelial cells. In adults, intense staining with UEL I was found in goblet cell mucus and in the brush border of epithelial cells. All of these results taken together support the premise that during postnatal development of rat small intestine, a progressive change from sialylation to fucosylation of brush border glycoconjugates occurs. Caldero et al. (1988) have performed a detailed investigation of changes in glycoconjugate composition of the rat colonic mucosa during development. They used a battery of eight fluorescein-conjugated lectins recognizing a variety of sugar residues. Their results demonstrated that each lectin showed a unique developmental staining pattern, including differences between the various regions of the colon. In all cases, the adult pattern of staining was achieved 25-30 days after birth. Differentiation-related changes in intestinal cell glycosylation patterns have been described in adult animals during cell migration from crypts to the villus or lumen. Some of these were already described above (Section IV,A,S; Etzler and Branstrator, 1974). We have investigated the localization of LFL binding sites in the plasma membrane of rat colonic epithelial cells during differentiation (Taatjes and Roth, 1988). We found that in the crypt regions, goblet and absorptive cell precursors were stained along their entire plasma membrane (Fig. 13); that is, both apical and basolateral plasma membranes were stained. However, when cells reached the zone of migration (Eastwood, 1977) the staining with LFL became restricted to the apical plasma membrane (Fig. 13). This polarized staining remained a feature of fully mature epithelial cells (both absorptive and goblet) located at the intestinal lumen. These results suggest that a feature of
FIG.1 1 . Light micrographs illustrating the detection of SNL I-gold binding sites in epithelial cells during postnatal development of rat jejunum. At postnatal day 1 (a,b), staining is present in the epithelium along the apical plasma membrane (arrowheads)and in the goblet cell mucus (asterisk). By postnatal day 23 (c,d), individual cells in the epithelium are not stained by the SNL I-gold complex (arrows). In adult animals (e,9, the apical plasma membrane (arrowheads)of all epithelial cells is not stained by SNL I-gold complex, whereas goblet cell mucus (asterisks) and the plasma membrane of cells in the lamina propria (Ip) are intensely stained. a,c,e, Bright-field micrographs; b,d,f, correspondingphase-contrast images. lp, lamina propria. X 368 (a-9. Bar = 5 pm.
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fully differentiated colonic epithelial cells is the polarization of the plasma membrane with respect to the distribution of sialic acid residues on membrane glycoconjugates. A similar phenomenon was apparent in the small intestine, although fully differentiated small intestinal epithelial cells display very sparse LFL binding sites. B. GOLGIAPPARATUS In an early study, Kim and co-workers (1 97 1) investigated the subcellular distribution of the then called “multienzyme system” of glycosyltransferases in rat small intestinal mucosal scrapings. They determined that the polypeptide : Nacetylgalactosaminyltransferase, galactosyltransferase, N-acetylglucosaminyltransferase, and N-acetylgalactosaminyltransferase were enriched in a smooth microsome fraction. This was the first detailed report of the localization of glycosyltransferases in intestinal tissue, and quite accurately determined them to be located in a fraction most likely representing Golgi apparatus membranes. In a subsequent investigation, Kim and Perdomo (1974) investigated the intestinal membrane distribution of five glycosyltransferases: two galactosyltransferases (acting on N- and 0-linked oligosaccharides), sialyltransferase, fucosyltransferase, and N-acetylgalactosaminyltransferase.They found that all five enzymes were enriched in a smooth membrane fraction (Golgi apparatus), with only background amounts detected in a rough membrane fraction and a brush border membrane fraction. More recently the techniques of immuno- and lectin cytochemistry have helped to unravel the pattern of glycosylation reactions in the intestinal cell Golgi apparatus. However, before we begin to detail these intestinal studies, it will be helpful to briefly review the concept of general Golgi apparatus glycosylation as formulated by the assimilation of data from several different techniques.
1. Subcompartmentation Model of the Golgi Apparatus Several recent reviews have considered this topic in detail and the interested reader should refer to them for more information (Dunphy and Rothman, 1985; Farquhar, 1985; Kornfeld and Kornfeld, 1985; Roth, 1987a; Roth and Taatjes, 1989). Briefly, this model proposes that the Golgi apparatus cisternal stack is FIG. 12. Demonstration of SNL I-gold binding sites in the jejunum of sections from postnatal day 23 rat. The apical plasma membrane (brush border) of adjacent epithelial cells is shown. Large gold particles (14-nm diameter) indicative of SNL I binding sites are restricted to the cell on the right. To rule out possible processing artifacts, this section was also stained with RCL I/asialofetuin-gold (small gold particles; 10-nm diameter). As can be seen, both cells are RCL I positive, indicating that loss of binding sites is specific for SNL I. The lateral plasma membrane (arrowheads) separating the two cells contains binding sites for both lectins. X 66,000. Bar = 0.15 pm.
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FIG. 13. Detection of sialic acid residues with the Limo.rf7avus lectin/fetuin-gold technique at the plasma membrane of rat colonic absorptive cells. in differentiated cells at the surface epithelium (a). gold particle label is restricted to the apical plasma membrane (arrowheads). Note the lack of staining in the basolateral plasma membrane (asterisks). In contrast, the basolateral plasma membrane of undifferentiated absorptive cells from the crypts region (b) is intensely stained for sialic acid residues (arrowheads). n, nuclei of absorptive cells. X 18,000 (a); X 55,000 (b). Bars = 0.6 p n ( a ) and 0.2 p n (b). (Reproduced with permission from Taatjes and Roth, 1988.)
GLYCOSYLATIONIN INTESTINAL EPITHELIUM
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functionally subcompartmentalized with respect to the steps involved in the processing of oligosaccharide side chains of glycoconjugates. Although based upon the maturation of N-linked oligosaccharides of glycoproteins, support for this model has also been presented for the case of 0-linked oligosaccharides of glycoproteins. Inherent in this concept is the premise that glycosyltransferases which act early in the pathway are preferentially located in cis-cistemae of the Golgi apparatus, whereas those acting at intermediate steps are located in middle cisternae, and those acting at terminal steps are housed in trans-cisternae. This compartmentation, or spatial separation, would allow the enzymes to act upon an oligosaccharide chain in an “assembly line” progression, without risk of interfering with the action of one another. This model is thus very attractive biochemically, and indeed has received much experimental support. For instance, in cell fractionation studies utilizing analytical sucrose gradients, activities for earlier and later acting oligosaccharide-processing enzymes were detected in distinct Golgi apparatus fractions (Dunphy et al., 1981; Dunphy and Rothman, 1983; Goldberg and Komfeld, 1983). More direct evidence, however, has been provided by numerous investigations analyzing the in situ cytochemical detection of various sugar residues with lectins (Pavelka, 1987; Roth er al., 1988b) and by the immunolocalization of a few glycosyltransferases (Roth and Berger, 1982; Dunphy er al., 1985; Roth et al., 1985a). Indeed, the first direct demonstration of Golgi apparatus subcompartments was provided by the immunocytochemical localization of galactosyltransferase by Roth and Berger (1982). They found that galactosyltransferase immunoreactivity colocalized with thiamine pyrophosphatase activity in one or two trans-Golgi apparatus cisternae in HeLa cells. This result indicated that the Golgi apparatus contained at least two compartments with respect to glycosylation reactions: cis (defined as galactosyltransferase negative) and trans (defined as galactosyltransferase positive). The number of identifiable subcompartments increased to three with the localization of N-acetylglucosaminyltransferase I to middle cisternae of the Golgi apparatus stack (Dunphy et al., 1985). Finally, the most distally acting glycosyltransferase, sialyltransferase, was detected in two trans-cisternae and a complex trans-tubular network continuous with these cisternae in rat hepatocytes (Roth er al., 1985a). Interestingly, sialyltransferase immunoreactivity was found in portions of the Golgi apparatus stack which also contained cytochemically demonstrable thiamine pyrophosphatase activity, a classical trans-Golgi marker, or acid phosphatase (CMPase) activity, a classical marker for the GERL element of the Golgi apparatus. These results suggested that in hepatocytes the Golgi apparatus is composed of three subcompartments with respect to glycosylation reactions: cis, so far delineated by what it does not contain; middle, containing N-acetylglucosaminyltransferase I; and trans, containing sialyltransferase. We include the trans-tubular network (Rambourg and Clermont, 1990), or trans-Golgi network (Griffiths and Simons, 1986) as part of the trans-Golgi apparatus since
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functionally it is involved in sialylation as are trans-cisternae, and structurally it is continuous with trans-Golgi cisternae (Roth et al., 1985a; Taatjes and Roth, 1986). This view differs from that of Griffiths and Simons (1986), who regard the trans-Golgi network as perhaps a fourth Golgi subcompartment, separate from trans-cistemae. Moreover, other investigators have proposed that galactosyltransferase is housed in trans-Golgi apparatus cisternae, whereas sialyltransferase is separated and housed in the more distally located trans-Golgi network (Berger and Hesford, 1985; Thorens and Vassalli, 1986; Berger et al., 1987). Several pieces of evidence contest this view. First, we have shown that sialyltransferase is not only localized in the trans-Golgi network of hepatocytes, but also quite clearly in two trans-cistemae of the Golgi apparatus stack (e.g., Fig. 3 in Roth ef al., 1985a). Second, by double-labeling immunofluorescence we found an identical codistribution of galactosyltransferase and sialyltransferase irnmunoreactivity in cultured rat hepatocytes (Taatjes el al., 1987). Third, Geuze and co-workers ( 1985) found that galactosyltransferase was detectable in the trans-Golgi network, in addition to trans-cisternae in hepatoma cells and liver hepatocytes. Fourth, galactose residues detected with RCL I, were found in the trans-cisternae and trans-Golgi network of hepatocytes (Lucocq et al., 1987). Clearly, the ability to resolve more and more Golgi apparatus subcompartments will come with the introduction of more Golgi apparatus-specific antibodies. Of certain importance for the previous discussion will be the simultaneous immunocytochemical demonstration of galactosyltransferase and sialyltransferase in the same Golgi apparatus at the electron microscopic level. Due to the constraints placed upon imrnunocytochemical investigations by antibody cross-reactivity with other animal species, this experiment has not proven possible. Moreover, cell-specific variability with respect to the organization of Goigi apparatus subcompartments may have been a factor in the above described discrepancies.
2 . Immunoc:\.tochemicalLocalization of Glycosyltransferases in Golgi Apparatus of Intestinal Epithelial Cells To this date, only two glycosyltransferases have been immunocytochemically detected in the Golgi apparatus of intestinal cells; yet, their localization has yielded most interesting results. After having reported on the localization of sialyltransferase in hepatocytes, we sought to expand on these findings by performing similar localizations on intestinal cells. When we examined the Golgi apparatus distribution of sialyltransferase in goblet cells from the rat colon, we observed the expected result (Fig. 14). Namely, immunoreactivity was restricted to transGolgi apparatus cisternae (Roth et al., 1986). Likewise, sialic acid residues, as detected with LFL, were localized to trans-cisternae. However, quite surprisingly when we examined neighboring absorptive cells a quite different pattern of labeling emerged: the entire Golgi apparatus cisternal stack (with the exception of the fenestrated first cis-cistema) was labeled (Figs. 14 and 15a). In a fashion similar
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169
FIG.14. Immunocytochemical localization of sialyltransferasein surface epithelial cells from rat proximal colon. Gold particle label is restricted to trans-Golgi apparatus cistemae in a goblet cell (gc), whereas in a neighboring absorptive cell (ac) label is detectable throughout the Golgi apparatus cistemal stack (with the exception of the fenestrated first cis-cistema). Label is also present in the goblet cell mucus droplets (md) and in the lateral plasma membrane separating the two cells (arrowheads). X 25,500. Bar = 0.4 pm. (Reproduced with permission from Roth er al., 1986.)
to that observed in goblet cells, the distribution of sialic acid residues in absorptive cells was found to mirror that of the sialyltransferase enzyme (Fig. 15b). Quantitative evaluation of the distribution of sialyltransferaseimmunolabel in the Golgi apparatus of absorptive versus goblet cells confirmed the differential label
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FIG. 15. Gold particle label for sialyltransferase (a) and sialic acid residues (b) is distributed throughout the absorptive cell Golgi apparatus cistemal stack (with the exception of the fenestrated first cis-cistema) from rat proximal colon. X 63,000 (a): X 66,500 (b). Bar = 0.16 pm (a) and 0.15 pm (b). (Reproduced with permission from Roth PI of., 1986.)
171
GLYCOSYLATION IN INTESTINAL EPITHELIUM TABLE I1 QUANTIFICATION OF IMMUNOLABEL FOR SIALYLTRANSFERASE IN RAT PROXIMAL COLONIC EPITHELIAL CELLS? Absorptive cell (n = 26) Golgi apparatus cistema”
Gold particle/pm 0.21 +/2.55 +/2.34 +/3.42 +/3.63 +/4.14 +/6.20 +/-
0.08 0.18 0.22 0.33 0.24 0.28 0.41
Total length (p) 115.5 134.3 138.4 132.1 126.8 124.1 125.7
Goblet cell ( n = 23) Gold particle/pm
Total length (pm)
0.45 +I- 0.24 0.16 +/- 0.08 0.15 +/- 0.08 0.16 +/- 0.09 0.18 +/- 0.09 0.18 +/-0.10 0.43 +/- 0.27 4.50 +/- 0.26 6.64 +/- 0.33
148.5 148.5 154.3 154.3 151.7 148.5 147.3 147.9 148.5
“From Roth er nl. (1986). ”Cistema 1 designates the fenestrated first cis-cisterna and the following numbers the subsequent cistemae toward the trans side of the Golgi apparatus.
observed on micrographs (Table 11). Moreover, it was apparent that although the labeling was diffuse throughout the Golgi apparatus of absorptive cells (with the exception of the fenestrated first cis-cistema), the labeling intensity increased gradually from the cis to the trans side. This was the first demonstration of an apparent lack of subcompartmentation for a glycosyltransferase within the Golgi apparatus cistemal stack. In the same study, we found that another terminal glycosyltransferase, the blood group A N-acetylgalactosaminyltransferase,was distributed in strikingly different patterns in the Golgi apparatus of absorptive versus goblet cells from human intestine, restricted to trans-cisternae in goblet cell Golgi apparatus, and diffusely localized throughout the absorptive cell Golgi apparatus (Fig. 16). In accordance with the matching labeling in the Golgi apparatus for sialyltransferase and sialic acid residues in rat intestine, the distribution of blood group A substance (detected with a monoclonal antibody) mirrored that of the blood group A N-acetylgalactosaminyltransferasein both absorptive and goblet cells. These results were then confirmed and extended in further studies of human intestinal cells (Roth er al., 1987, 1988~).Thus, for both N-linked oligosaccharide processing (sialyltransferase),as well as 0-linked (blood group A N-acetylgalactosaminyltransferase) a terminal glycosyltransferase was not distributed in the Golgi apparatus cisternal stack as would be predicted by the subcompartmentation model. The implications of these findings for the elaboration of oligosaccharide side chains of glycoconjugates in intestinal absorptive cells are not clear. Although the subcompartmentation of glycosyltransferases would serve to prevent competing reactions which could alter the normal processing of oligosac-
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FIG.16. Immunocytochemical localization of blood group A al.3-N-acetylgalactosaminyltransferase in absorptive cell Golgi apparatus from human ileum. Label is present throughout the cistemal stack and trans-tubular network of the Golgi apparatus. Note the complexity of the structures at the trans side of the Golgi apparatus. X 48,000.Bar = 0.2 pm. (Reproduced with permission from Roth tv a/., 1986.)
charides, it is not clear that normal processing requires such subcompartmentation. Other mechanisms such as the differential expression of the levels of two competing glycosyltransferases could favor one terminal glycosylation pattern over another. Besides, it is not known if the consequences of having oligosaccharides with one type of terminal structure would have functional significance over another for most proteins. It should be emphasized, though, that more insight into these questions awaits the immunolocalization of other glycosyltransferases in a variety of cell types. Indirect support for these results has emerged from recent lectin-binding investigations. Diffuse labeling throughout the Golgi apparatus cisternal stack has been observed with RCL I in mouse epididymal cells (Yokoyama et al., 1980) and rat absorptive intestinal cells (Pavelka and Ellinger, 1986). Hedman er al. ( 1986) observed label with LFL throughout the cisternal stack with the excep-
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tion of one cis-cisterna in 3T3 cells. Similarly, Roth and co-workers (Lee et al., 1989) found that LFL labeled the entire Golgi apparatus cisternal stack in CHO cells. Moreover, in the same study (Lee et al., 1989) CHO cells were transfected with a cDNA coding for the P-galactoside a2,6-sialyltransferase. This enzyme competed with the endogenous P-galactoside a2,3-sialyltransferase for the termination of oligosaccharide chains. This competition was assessed by the binding of SNL I (specific for NeuSAc a2,6-Gal/GalNAc sequences) to sections from wild type and transfected cells. While SNL I did not stain wild type CHO cells, the Golgi apparatus of transfected cells was labeled throughout the entire cisternal stack. Thus, in both wild type and transfected cells, sialic acid residues were not restricted to trans-cisternae of the Golgi apparatus. Such lectin-binding studies identify any glycoconjugate in the Golgi apparatus carrying the required sugar residues. Although the nature and extent of recycling of glycoconjugates from the plasma membrane through the Golgi apparatus remains controversial (Farquhar, 1985; see Snider and Rogers, 1985, 1986; Neefjes et al., 1988; Reichner et al., 1988, for disparaging views) such recycling could at least in part explain the presence of complex-type oligosaccharide chains in the middle and cis regions of the Golgi apparatus cisternal stack. For this reason, we feel that it is most important to determine the intra-Golgi apparatus distribution of a particular glycosyltransferase before surmising that the pattern of glycoconjugate localization represents the site of glycosyltransferase activity.
3. Demonstration of Lectin-Binding Sites in lntestinal Cell Golgi Apparatus
In contrast to the relatively few investigations detailing the localization of glycosyltransferases within the intestinal cell Golgi apparatus, many studies have employed lectins for the demonstration of sugar residues therein (Pavelka, 1987). Preembedding methods employing peroxidase-conjugated lectins, as well as postembedding methods employing colloidal gold-labeled lectins and glycoproteins have been used. Although the methods and animal species investigated may differ among the various investigators, the lectin-binding patterns to intestinal goblet and absorptive cell Golgi apparatus may be summarized as in Table 111. As can be seen from the table, the interpretations of lectin-binding studies by various investigators tend to overlap, but also display variability. Such discrepancies may result from species variability, variability among intestinal segments as well as crypt versus villus regions, and methodology (preversus postembedding, tissue fixation and processing, probe preparation, etc.). Moreover, probably of equal importance is the very subjective nature of the interpretation of lectin labeling patterns within the Golgi apparatus. It may be rather easy to distinguish between cis- and trans-sides of the Golgi apparatus cisternal stack, yet what defines where the cis region ends and middle begins, or where middle ends and trans begins? How many cisternae compose the desig-
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TABLE 111 LECTINBINDING TO GOLGIAPPARATUS IN INTESTWAL EPITHELIAL CELLS" Goblet cell Lectin ConA SBL PSL LCL HPL
cis nd
LFL UEL I O+,
Absorptive cell cis middle trans
+
+I-
-
+
+
-
nd
nd
nd
nd nd
nd nd
nd nd
+
+I-
-
-
+
+I+I-
+/-
nd
+
nd
nd
t
nd nd
nd nd
nd nd
-
+
+/-
t
GSL I RCL I
middle trans nd nd
+
-
+
+
+
-
+I-
+
-
+
-
-
-
-
-
+
-
-
nd
nd
nd
+
+I-
+
t
-
+I-
+ +
References Pavelka and Ellinger (1985) Tsuyama et al. (1986) Pavelka and Ellinger (1989a) Pavelka and Ellinger (1989a) Murata et a / .(1986) Pavelka and Ellinger (1985); Ellinger and Pavelka (1988b) Roth ( 1984) Ellinger and Pavelka (1988b) Pavelka and Ellinger (1985, 1989b) Tsuyama ef a / . (1986) Roth et al. (1986) Ellinger and Pavelka (1988a)
Staining present; -, not detected; nd, not determined.
nated cis, middle, and trans regions of the stack? In the absence of specific markers, these borders seem to be arbitrarily defined by individual investigators. The situation is further complicated by cell type variability. For instance, some cell types such as hepatocytes may contain Golgi apparatus with as few as three cistemae, whereas the Golgi apparatus of goblet cells may possess up to 20 cisternae. The number of cistemae within a given cell may also vary depending upon the functional condition of the cell. Finally, the plane of section must be considered when interpreting the number of cistemae within, as well as the orientation of the Golgi stack. This may necessitate examining serial sections in order to exclude the possibility of missing a particular region of the cisternal stack in a given section. For instance, Orci et al. (1986) performed a serial sectioning analysis of the transport of horseradish peroxidase from the cell surface to the Golgi apparatus in insulin-secreting B cells. Their results showed quite convincingly that what appeared to be a cis- or trans-cisterna in a random section could always be traced to a position in the Golgi stack intermediate (i.e., middle cistemae) between the cis and trans poles. Taking all of these points into consideration, and assuming some subjectivity on our part, we propose the following scheme for the localization of sugar residues within the intestinal cell Golgi apparatus. In absorptive cells, mannose/glucose residues are restricted to cis and middle portions of the cisternal stack (Pavelka and Ellinger, 1985, 1989a); N-acetylgalactosamine residues are concentrated in cis and trans regions (Pavelka and Ellinger, 1985; Ellinger and Pavelka, 1988b); galactose residues to
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trans- and variably middle cistemae (Pavelka and Ellinger, 1985); fucose residues to middle/trans regions (Ellinger and Pavelka, 1988a); and sialic acid residues diffuse throughout the stack, but concentrated in trans-cistemae (Roth et al., 1986). In goblet cells, mannose/glucose residues are restricted to cis/middle portions of the stack (Tsuyama et al., 1986); N-acetylgalactosamineresidues to cis and trans regions (Roth, 1984); galactose residues to middle/trans portions of the stack (Pavelka and Ellinger, 1985, 1989b); fucose residues to trans-cisternae (Ellinger and Pavelka, 1988a); sialic acid residues to trans-cisternae (Roth er af., 1986); sialic acid a2,3-linked and a2,6-linked to galactose concentrated in trans-cisternae (Sata et al., 1989; Taatjes and Roth, 1990). In a single study, Ellinger and Pavelka ( 1988b) have reported that a-galactose residues as detected with GrifSoonia simplicifolia isolectin I-B4, are restricted to cis- cisternae in intestinal goblet cells. C. POST-GOLGI APPARATUS DISTRIBUTION OF GLYCOSYLTRANSFERASES: FACTOR ARTIFACT? It is well established that glycosyltransferases exist outside of their usual location as Golgi apparatus integral membrane proteins; specifically in cellular plasma membranes and in soluble form in a number of secretions, predominantly milk and colostrum (Andrews, 1970; Barker er al., 1972; Paulson er al., 1977), and serum (Hudgin and Schachter, 1971; Fujita-Yamaguchi and Yoshida, 1981; Kaplan el al., 1983). Immunocytochemical methods have indicated the presence of galactosyltransferase (Pestalozzi et a f . , 1982; Davis et af., 1984; Roth et al., 1985b; Shaper er al., 1985; Bayna er al., 1988), N-acetylgalactosaminyltransferase (Balsam0 er al., 1986), blood group A N-acetylgalactosaminyltransferase (Roth et al., 1987, 1988c), and sialyltransferase (Roth er al., 1986; Taatjes and Roth, 1988; Taatjes et al., 1988a) at the plasma membrane of many cell types. A detailed discussion of cell surface glycosyltransferases is beyond the scope of this review. However, interested readers should consult several excellent reviews of this area (Pierce er al., 1980; Strous, 1986; Shur, 1989). In this section we will focus on the evidence pertaining to the presence of glycosyltransferases outside of the Golgi apparatus in intestinal cells. Such evidence has been presented from three types of experiments: (1) measurement of glycosyltransferase activities in plasma membrane fractions; (2) autoradiographic detection of glycosyltransferase activity in plasma membranes; and (3) in situ immunocytochemical localization of glycosyltransferases. Many studies have reported the presence of glycosyltransferase activity in the plasma membranes of intestinal epithelial cells, and these results were already presented in Sections IV,A, 1 and 2. Briefly, activities for galactosyltransferase and sialyltransferase have been detected on the apical and basolateral plasma membranes of intestinal epithelial cells (Weiser, 1973a,b; Weiser er al., 1978).
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Using a different methodology, Bennett ei al. (1987) have reported on the existence of an active sialyltransferase at the microvillar surface of rat intestinal absorptive cells. They injected CMP-['Hlsialic acid into the intestinal lumen, followed by visualization of autoradiographic products at the light microscopic level. Injection period was restricted to 5 min to ensure that reaction product reflected cell surface phenomena and not activity from the Golgi apparatus. They observed a moderate autoradiographic reaction at the microvillar surface of small intestinal absorptive cells, yet found no reaction at the luminal surface of epithelial cells from gallbladder, ciliary body, and iris. Likewise, the injection of UDP-['H]galactose resulted in no reaction at the cell surface of all these cells, including intestinal absorptive cells. They attributed these results to reflect the presence of a sialyltransferase capable of sialylating endogenous acceptors at the luminal surface of small intestinal absorptive cells. In light of immunocytochemical results to be discussed below, it would have been of interest if Bennett and co-workers had examined reaction in the large intestine as well. As mentioned previously, several immunocytochemical investigations at both the light and electron microscopic levels have reported the presence of immunoreactivity for glycosyltransferases at the plasma membrane, as well as other post-Golgi apparatus sites of intestinal epithelial cells (Pestalozzi et al., 1982; Roth et al., 1985b. 1986, 1987, 1988~;Taatjes and Roth, 1988; Taatjes er al., 1988a). Berger and co-workers (Pestalozzi ei al., 1982), observed at the light microscopic level label with an affinity-purified galactosyltransferase antibody at the apical, but not basolateral plasma membrane of human jejunal enterocytes. Roth ei al., (1985b) performed a similar investigation using postembedding protein A-gold immunocytochemistry at the electron microscopic level. An affinity-purified antibody against human milk galactosyltransferase was applied to thin sections from human duodenum embedded in Lowicryl K4M. Intense gold particle label was observed at the apical (brush border), as well as basolateral plasma membrane of enterocytes. The intensity of label decreased on the lateral plasma membrane as it approached the basal membrane. Staining was completely abolished by preabsorption of the antibody with purified galactosyltransferase antigen. However, the validity of these results as representing true cell surface, or ecto-galactosyltransferase has recently been challenged. First, Boyle ef al. ( 1986) using analytical subcellular fractionation techniques, reported that galactosyltransferase activity was confined to the Golgi apparatus fraction in human jejunal biopsy homogenates, with no significant amount detectable in the brush border membrane fraction. They postulated that the staining observed by Roth et al. (1985b) was probably due to contaminating immunoglobulins present in the milk used as the source of the galactosyltransferase antigen. An alternative explanation for their inability to detect significant amounts of galactosyltransferase activity in their plasma membrane fraction could have resulted from failure to block endogenous intestinal nucleotide py-
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rophosphatase. As reported by Lau and Carlson (1981) (and discussed in Section IV,A, 1), when measuring glycosyltransferase activities in tissues rich in nucleotide pyrophosphatase activity (such as intestinal mucosa), precautions must be taken, including inclusion of EDTA and soybean trypsin inhibitor, to ensure that glycosyltransferase degradation does not occur. A second, and perhaps more troublesome critique, has been the recent revelation that polyclonal antibodies raised against glycoproteins may contain clones directed against carbohydrate epitopes of the antigen (Feizi and Childs, 1987). Human milk galactosyltransferase possesses blood group-related carbohydrate structures as part of its oligosaccharide constituency. Indeed, Feizi and co-workers (Childs et al., 1986) have reported immunochemical data demonstrating that the affinity-purified antibody raised against the human milk galactosyltransferase used in the above-mentioned studies (Pestalozzi et al., 1982; Roth et al., 1985b) contains a minor population of antibodies directed against the blood group-related carbohydrate moiety of the enzyme. When used in immunofluorescence experiments, these antibodies against carbohydrate epitopes of galactosyltransferase intensely stained the brush border of intestinal epithelial cells (Childs et al., 1986). This staining could be abolished by preabsorbing the antibodies with blood group substances, suggesting that the staining did not reflect galactosyltransferase immunoreactivity at the brush border, but rather that of blood group substances. How do these results relate to those published by Roth et al. (1985b) described above? Perhaps a reevaluation is necessary, employing a galactosyltransferase antibody preabsorbed with blood group carbohydrate structures. Alternatively, antibodies could be raised against a deglycosylated form of the enzyme and used for immunocytochemistry.However, it is not clear what effect removal of the carbohydrate moieties would have on the folding and three dimensional conformation of the enzyme. Resulting antibodies could potentially recognize antigenic structures not present on the molecule in situ. Perhaps the best method for resolving this discrepancy would be to utilize the recent successful cloning of several galactosyltransferases (Narimatsu et al., 1986; Shaper et al., 1986, 1988; Masri et al., 1988; Nakazawa et al., 1988; D’Agostaro et al., 1989; Masibay and Qasba, 1989) to produce polypeptide epitope-purified antibodies (Taatjes et al., 1988a) recognizing only the protein portion of the enzyme as related below. During our studies on the subcompartmentation of sialyltransferase in the Golgi apparatus of intestinal epithelial cells, we noted predominant staining over a variety of post-Golgi apparatus structures, including plasma membrane and mucus droplets (Roth et al., 1986). In view of the findings of Childs et al. (1986) concerning the contamination of galactosyltransferase antisera with carbohydrate-directed antibodies noted above, we sought to determine if the immunolabeling we observed for sialyltransferase outside of the Golgi apparatus represented true sialyltransferase enzyme, or rather was due to nonspecific
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cross-reaction with carbohydrate antigens. For this purpose we took advantage of the recent cloning of the gene for this particular sialyltransferase (Weinstein er al., 1987) to prepare polypeptide epitope-purified polyclonal sialyltransferase antibodies (Fig. 17) by adsorption to a recombinant P-galactosidase-sialyltransferase fusion protein produced in Escherichia coli (Taatjes et al., 1988a). Because the fusion protein is nonglycosylated, the resulting purified antibodies recognize only protein epitopes of the sialyltransferase. Using these antibodies for immunoelectron microscopy, we observed immunoreactivity to the sialyltransferase polypeptide in several post-Golgi apparatus structures, in addition to the Golgi apparatus, in both absorptive and goblet cells from the rat colon (Taatjes et al., 1988a). In absorptive cells, labeling was found in the apical and basolateral plasma membranes, lysosomes, and multivesicular bodies, and at the
FIG. 17. Characterization of a P-galactosidase-sialyltransferase fusion protein epitope purified antibody ( h STI). SDS-polyacrylamide gels of rat liver Golgi apparatus (lanes 2 , 4 , 6 ) and purified Galpl.4ClcNAc a-2.6 sialyltransferase (lanes I . 3, 5 ) were stained by Coomassie blue (lanes I and 2) or processed as Immun-blot with fusion protein epitope-purified antibody (lanes 3 and 4) or by antibody mock-purified with P-galactosidase without fused sialyltransferase (h GTI I; lanes 5 and 6). The h ST1 antibody recognizes both the purified and Golgi apparatus forms of sialyltransferase, while the h GT11 control antibody shows only a background level of staining. (Reproduced with permission from Weinstein ef a/., 1987.)
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limiting membrane of apical cytoplasmic vesicles (Fig. 18). In goblet cells (Fig. 19a), label was detected in the apical and basolateral plasma membranes and in mucus droplets (both in the lumen and at the limiting membrane). Surprisingly, label was undetectable (including the Golgi apparatus) in all regions of the small intestine from the same animals (Fig. 19b), as presented in Table IV, and previously detailed in Section IV,A, 1.
FIG.18. Immunocytochemical localization of sialyltransferase with P-galactosidase-sialyltransferase fusion protein epitope-purified antibody in an absorptive cell from rat proximal colon. Sialyltransferase immunoreactivity is detectable in the apical plasma membrane (asterisk) and along the inner aspect of the limiting membrane of apical cytoplasmic vesicles (arrowheads). X 50,000. Bar = 0.2 pn. (Reproduced with permission from Taatjes et al., 1988a.)
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GLYCOSYLATION IN INTESTINAL EPITHELIUM TABLE IV DISTRIBUTION OF SIALYLTFWNSFERASES IN RAT INTESTINE' Intestinal segment Duodenum Jejunum Ileum Colon
Sialyltransferase activityb GalPl,3(4)GlcNAc a2.3-ST' GalPl,4GlcNAc a2.6-STd 0 0 0 12
0 0 0 14
"From Taatjes et al. ( 1988a). hActivity expressed as picomole of [14C]Neu5Actransferred per milligram of protein/hour for both sialyltransferases. 'A value of 0 indicated activity not detected with limit of detection at 10 pmol of ['4C]Neu5Ac/ milligram of proteinhour. dA value of 0 indicates activity not detected with limit of detection at 1 pmol of [14C]Neu5Ac/ milligram of protein/hour.
Thus, the powerful combination of molecular cloning and immunocytochemistry provided very strong support to the contention that glycosyltransferases are also housed in cellular locations distal to the Golgi apparatus. However, the question must be asked whether the post-Golgi apparatus localizations of sialyltransferase in rat intestine are functionally significant. If we consider first the label present in the mucus droplets, it is possible that this luminal sialyltransferase continues its function in the sialylation of glycoproteins. Recently, Paulson and co-workers (Colley et al., 1989) investigated the conversion of membrane-bound Golgi apparatus sialyltransferase to a secretory form of the protein. By replacing the NH,-terminal signal anchor with the cleavable signal peptide from y-interferon, and transfecting CHO cells with this sialyltransferase expression vector, they were able to show that this construct was secreted from the cell with a half time of 2-3 hr. Most importantly, this secreted form of sialyltransferase contained the catalytic portion of the enzyme and was enzymatically active. By analogy, the sialyltransferase located in the mucus may represent a form of the enzyme rendered soluble by cleavage of the NH,-terminal signal anchor by an endogenous protease in the trans region of the Golgi apparatus. Indeed, precedence for such a situation has been documented for the conFIG. 19. Immunocytochemical demonstration of sialyltransferase in goblet cells using P-galactosidase-sialyltransferase fusion protein epitope-purified antibody. (a) Gold particle label indicative of sialyltransferase immunoreactivity is found in trans-cistemae of the Golgi apparatus (facing the mucus droplets), in the mucus droplet lumen (md), and along their limiting membrane (arrowheads), and along the lateral plasma membrane (arrows) of goblet cells from rat proximal colon. In contrast, immunoreactivity for sialyltransferase is undetectable in both the Golgi apparatus and mucus droplets (md) of goblet cells from rat jejunum (b). X 50.000 (a and b). Bar = 0.2 pm. (Reproduced with permission from Taatjes et al., 1988a.)
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version of the blood group A N-acetylgalactosaminyltransferasefrom a membrane-associated to a nonmembrane-associated form in the trans-tubular network of human intestinal goblet cells (Roth et al., 1988~).In this case, active enzyme has been directly demonstrated in the mucin released from the goblet cells (Omtoft et al., 1987). Of course, even if the enzyme is active, continued glycosylation would require the transport of the appropriate nucleotide sugar from the cytoplasm into the lumen by a nucleotide sugar antiport protein (Hirschberg and Snider, 1987). Thus, whether or not the sialyltransferase plays a functional role in the mucus is still an open question. Once it is released with the mucin into the lumen of the gut, it most likely has no catalytic activity due to lack of substrates, and is unlikely to play any role in the physical properties of the much since it would be such a minor component of the total protein (estimated at less than 0.0001% by activity). The demonstration of sialyltransferase at the apical and basolateral plasma membrane of intestinal cells provides further evidence for the existence of ecto-glycosyltransferases. The unambiguous existence of cell surface glycosyltransferases has been difficult to establish since Roseman ( 1970) first proposed their role in cell recognition and adhesion. However, in recent years Shur and co-workers, in a series of elegant studies, have succeeded in demonstrating the role of cell surface galactosyltransferase in such diverse functions as fertilization, preimplantation embryonic development, implantation, mesenchymal cell migration on substrates, and growth control in normal, neoplastic, and metastatic cells (Shur, 1989). Presently, it is not clear if ecto-sialyltransferase plays a functional role in intestinal cells or if its occurrence simply reflects its less restricted distribution in the post-Golgi apparatus membranes of these cells. In this respect, it would be of interest to determine if other glycosyltransferases in these cells have similar or different distributions, since similar distributions would favor the view that their existence on the cell surface is a consequence of an alteration in the underlying mechanism which would restrict their subcellular localization to the Golgi apparatus. Indeed, this view is supported by the immunocytochemical localization of the blood group A N-acetylgalactosaminyltransferase in both the apical and basolateral plasma membrane of human intestinal epithelial cells (Roth et al., 1987). On the other hand, Lopez ez a/. (1989) have reported that at least in F9 embryonal carcinoma cells the levels of cell surface (ecto-galactosyltransferase) and Golgi apparatus galactosyltransferase change relative to one another during cell differentiation, suggesting that these functionally and distinct pools of galactosyltransferase are independently and differentially regulated. This would indicate that we should not necessarily think of ecto-glycosyltransferases as representing nonspecific vesicular transport of the Golgi apparatus form of the enzyme to the plasma membrane, but rather as an independently regulated entity of its own.
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V. Effects of Exogenous Agents on Intestinal Glycosyltransferase Activity and Glycosylation A. HORMONES In adult rats, a continuous subcutaneous administration of testosterone for fourteen days resulted in qualitative and quantitative changes in the glycosphingolipid composition of rat small intestinal mucosa (Dahiya et al., 1989). These changes were accompanied by increases in the enzymatic activities of CMP-Nacetylneuraminic acid : lactosyfceramide sialyltransferase and UDP-galactose : lactosylceramide galactosyltransferase. The authors proposed that testosterone induced the activities of the two glycosyltransferases to increase, resulting in changes in intestinal mucosa glycosphingolipid composition. In a similar investigation, Dudeja et al. (1988) analyzed the activities of the same two glycosyltransferases reported above, in Golgi apparatus membranes, in response to subcutaneous administration of the synthetic glucocorticoid dexamethasone. They found that the activities of both glycosyltransferases were elevated in response to dexamethasone administration. They speculated that the increase in galactosyltransferase activity may have resulted from an increased membrane fluidity caused by the dexamethasone. However, they could not attribute the increase in sialyltransferase activity to the same cause. Several investigations have been aimed at examining the effect of hormone administration on glycosylation activity in developing intestine. As described in Sections 1V,A,2 and 3, the postnatal development of rat small intestine is characterized by a decrease in sialyltransferase activity, with a concomitant increase in fucosyltransferase activity. A postnatal injection of cortisone caused precocious changes in the activities of sialyltransferase and fucosyltransferase in the mucosal fractions from 2-week-old rats (Chu and Walker, 1986). Specifically, cortisone administration resulted in a 50% decrease in sialyltransferase activity and an 8-fold increase in fucosyltransferase activity as compared to control animals. Likewise, glycosidic-bound sialic acid content was significantly decreased, while glycosidic-bound fucose content significantly increased in the hormone-treated animals. Walker and co-workers (Ozaki et al., 1989) have also shown that cortisone injection into suckling rats causes a precocious increase in the activities of two developmentally regulated galactosyltransferases:the UDPwas increased 2.7-fold and the UDPGal : GlcNAc (~1-4)-galactosyltransferase Gal : GalNAc(~1-3)-galactosyltransferaseactivity was increased 1.8-fold. In an earlier study, Mahmood and Torres-Pinedo (1985) injected suckling rats with cortisone, thyroxine, epidermal growth factor, or insulin and measured the effect on the intestinal microvillar membrane content of sialic acid and fucose, as well as subsequent lectin binding. Cortisone treatment was found to lower sialic acid content and raise fucose content of microvillar membranes, as well as
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increase the incorporation of ['Hlfucose into these membranes. These results were also reflected in the binding of '251-labeledlectins to purified microvillar membrane preparations; cortisone administration decreased the binding of WGA to microvillar membranes, while increasing the binding of UEL I and PNL. Thyroxine treatment had a similar effect as cortisone on membranous fucose content and UEL I binding, but did not alter the incorporation of ['Hlfucose into membranes or the sialic acid content of membranes. Epidermal growth factor and insulin did not affect any of these parameters. Thus, these results demonstrated that only cortisone administration to suckling rats induced precocious changes in sialic acid and fucose content of microvillar membranes normally associated with postnatal intestinal development. Kolinska e? al. (1988) examined the effect of hydrocortisone administration on sialyltransferase activity in the crypts versus villus of 10-day-old rat small intestine. They found that the decrease in sialyltransferase activity induced by hydrocortisone administration occurred mainly in the crypt cells. B. DRUGSAND OTHERNoxlOuS STIMULI
Treatment of rats with the microtubule-disrupting drug colchicine or with turpentine results in an increase in the serum level of sialyltransferase activity (Mookerjea et al.. 1977; Kaplan ez al., 1983). Ratnam et al. (1987) speculated that some of this increase in serum sialyltransferase may result from secretion from the small intestine. They injected rats with colchicine and then 4 hr later measured the activity of the a2,6-sialyltransferase in the homogenates from jejunal slices. They found that secretion of soluble sialyltransferase into the medium was elevated in the animals treated with colchicine, as compared to control animals. A similar increase in intestinal and serum sialyltransferase activity has also been shown to be induced by inflammation caused by a standardized 25% body surface area thermal injury in rats (Chu e? al., 1988). These results thus suggested that intestinal sialyltransferase may form part of the acute phase response to inflammation. However, we believe that this sialyltransferase originates from cells of the lamina propria, and not from intestinal epithelial cells. As pointed out in Sections IV,A,l and IV,C, immunoreactivity and enzymatic activity for the a2,6-sialyltransferase were undetectable in rat small intestinal mucosa. Colchicine is normally used as a depolymerizing agent for microtubules to study cellular processes which may be microtubule-dependent. Indeed, Hugon e?al. (1987) performed such a study on mouse jejunal epithelial cells to investigate the role of microtubules in the migration of glycoproteins from the Golgi apparatus to the apical and basolateral plasma membranes. They examined by autoradiography the incorporation of [3H]fucose into glycoconjugates in explants of mouse jejunum cultured in a medium containing colchicine. They
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found that colchicine inhibited the labeling of the brush border by 67%, while labeling of the basolateral plasma membrane increased 114%. Similar results were also obtained with the microtubule-disrupting drug nocodazole. These results suggested that some glycoproteins destined for the apical plasma membrane may be rerouted to the basolateral plasma membrane in the presence of colchicine and thereby suggests a role for microtubules in the transport of glycoconjugates from the Golgi apparatus to the apical plasma membrane in polarized intestinal absorptive cells. Similar effects of colchicine on glycoprotein migration were reported earlier for human jejunal biopsies in culture (Blok et af.,1981) and for rat small intestine (Ellinger et af., 1983). The effect of polyamine deficiency on Golgi apparatus membranes and galactosyltransferase activity in mouse small intestinal epithelial cells was studied by Sakamaki et al. (1989). They produced polyamine-deficient cells by injecting two inhibitors of polyamine synthesis, ethylglyoxal bis(guany1hydrazone) and a-difluoromethylornithine into mice. Polyamine deficiency produced swelling of the Golgi apparatus membranes (demonstrated by electron microscopy) accompanied by a decrease (to approximately 55% of the control value) in galactosyltransferase activity. These results suggested that galactosyltransferase activity is diminished in swollen Golgi apparatus membranes. Umesaki and Ohara (1989) investigated in detail treatments which lead to an increase in GDP-fucose : asialo GH,a(1-2)fucosyltransferase activity in rat small intestinal mucosa. The increase in this particular fucosyltransferase activity was manifested by alteration in the neutral glycolipids of the microvillar plasma membrane. Factors shown to cause an increase in fucosyltransferase activity were microbial contamination of germ-free mice, weaning (see Section IV,A,2), intraperitoneal injection of the protein synthesis inhibitors cycloheximide or emetine (although repeated injection of cycloheximide every 2 hr resulted in a repression of fucosyltransferase activity), injection of a soluble fraction from a small intestinal homogenate, and mechanical injury to the intestinal mucosa. They also analyzed the composition of the glycolipids in mucosal fractions after such treatments and found an increase in their fucose content. Finally, by separating crypt from villus cells, they found that fucosyltransferase activity was increased in villus cells as compared to crypt cells. They attributed these findings to indicate that the increase in fucosyltransferase activity in response to various stimuli is preferentially localized to the postmitotic epithelial cells located on the villus.
VI. Differentiation and Glycosylation in Intestinal Cell Culture Systems Although the morphology and physiology of the intestinal tract are quite amenable for studying differentiation events (Section I), pitfalls of using such an
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organ system in biochemical studies are numerous, i.e., obtaining pure cell populations, experimental manipulation of cells, and difficulty in administering exogenous agents, to name a few. For these reasons, alternative experimental systems such as intestinal organ (Quaroni, 1985) and cell culture (Rousset, 1986) have been introduced. The recent introduction of several stable cell lines derived from intestinal epithelial cells has made this a particularly fruitful avenue of research. The oligosaccharide composition of cell surface glycopeptides was investigated in confluent and subconfluent cultures of the rat small intestinal epithelial cell line IEC-6 by measuring the incorporation of D-[2-3H] mannose and by glycopeptide sensitivity to various oligosaccharide processing enzymes (Sasak et al., 1982). They found that confluent cells contained a much higher proportion of complex oligosaccharides in glycopeptides of the plasma membrane than did subconfluent cells. Only minor differences were observed between total mannose-labeled glycopeptides from confluent and subconfluent cultures, suggesting that the cell surface changes were mainly due to differences in biosynthesis of the carbohydrate moieties and not to the formation of different glycoproteins. Moreover, this alteration in oligosaccharide composition of cell surface glycopeptides was shown to be dependent upon cell density and not on the growth rate of the cells. Interestingly, Sasak et al. (1982) also were able to draw a correlation between degree of cell adhesion to the substratum and cell surface oligosaccharides: confluent cultures containing cell surface glycopeptides with complex-type oligosaccharide structures were more adherent than their subconfluent counterparts displaying more high mannose-type oligosaccharides. Several recent reports have documented the relationship between cell differentiation and the extent of processing of N-linked oligosaccharides in the human colon cancer cell line HT-29 (Trugnan et a / . , 1987; Ogier-Denis et al., 1988, 1989). HT-29 cells remain undifferentiated in media containing glucose, but undergo differentiation when glucose is removed from the media. Trugnan er al. ( 1987) examined the biosynthesis of sucrase-isomaltase, a microvillar membrane protein taken as a marker for differentiated intestinal epithelial cells in v i w , in both differentiated and undifferentiated HT-29 cells. In contrast to the normal processing and expression of this enzyme at the cell surface in differentiated HT-29 cells, in undifferentiated cells no enzyme was detectable at the plasma membrane. They showed that the failure to detect membrane expression was not due to lack of synthesis, but rather to abnormal posttranslational processing. Indeed, as compared to the enzyme synthesized and expressed in differentiated HT-29 cells, sucrase-isomaltase produced in the undifferentiated cells displayed ( I ) an impairment of the conversion from high mannose to complex form of the enzyme; (2) abnormal complex form glycosylation; and (3) rapid intracellular degradation of both high mannose-type and complex-type enzymes. In a subsequent paper (Ogier-Denis et al., 1988), this group investigated
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whether the impairment in glycosylation noted in undifferentiated HT-29 cells was specific for sucrase-isomaltase, or a general glycosylation defect. They found that there is an overall defect in the processing of N-linked oligosaccharides (Section 11) which is manifested by alterations in three processing steps: ( 1) incorporation of ~-[2-~H]mannose into glycoproteins; (2) conversion of high-mannose chains to complex-type N-linked glycans; and (3) trimming of high-mannose chains at the level of conversion of Man,,-GlcNAc,-Asn to Man,-GlcNAc,-Asn. This particular trimming reaction was elaborated on in another report (Ogier-Denis et al., 1989), where it was suggested that it may represent an important regulatory point in the conversion of undifferentiated to differentiated cells. These results, therefore, suggest that there is an impairment in the conversion of high mannose forms of N-linked oligosaccharides into complex-type in undifferentiated HT-29 cells. Future investigations on these cultured intestinal cells should help to unravel in more detail the cellular mechanisms involved in terminal differentiation as it relates to glycosylation.
VII. Concluding Remarks Although much effort has been directed toward elucidating glycosylation mechanisms and patterns in intestinal cells, unequivocal answers have not been forthcoming. This lack of emergence of a unifying concept underlying intestinal cell glycosylation may be the result of many divergent factors. Conflicting results concerning the activities of glycosyltransferasesin intestinal homogenates almost certainly results from variation in methodologies; i.e., (1) mucosal scrapings representing mostly epithelial cells versus homogenates containing submucosa and lamina propria; (2) failure to allow for endogenous intestinal enzymes which could potentially degrade glycosyltransferases; (3) variation in acceptor substrates employed, resulting in the measurement of different glycosyltransferases within the same class; and (4)different techniques for the separation of crypt versus villus epithelial cells. Similar technique-related problems could explain the variation in expression of intestinal carbohydrates. However, this is more likely due to the inherent variability in glycosylation expressed in a given cell type. Many detailed investigations have revealed a marked degree of glycoconjugate heterogeneity, not only among similar cell types from different species (Holthofer, 1983; Schulte and Spicer, 1983a,b; Spicer er al., 1987), but also among supposedly homogeneous cell populations within a given organ (Watanbe er al., 1981; Spicer et al., 1981; LeHir er al., 1982; Roth er al., 1983; Brown et al., 1985; Roth and Taatjes, 1985; Roth er al., 1988b; Taatjes et al., 1988b). Such heterogeneity may reflect blood group specificities, environmental or genetic variation, differentiation state of the cell, or pathological influences. However, a rapidly emerging concept suggests that the glycoconjugate repertoire displayed
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by a given cell reflects its endogenous expression of glycosyltransferases. This concept has recently been discussed in detail by Paulson (1989; Paulson et al., 1989) and by Rademacher et al. (1988), and will not be elaborated on here. Given the role played by terminal oligosaccharide structures in cell-cell recognition phenomena (Section I), the expression of glycosyltransferases would appear to occupy a key position in the posttranslational processing of glycoconjugates and thus influence cellular functions. Does this then mean that the carbohydrate portion of all types of glycoconjugates is important for their biological functioning? Certainly this is not the case for all glycoconjugates, and is an important area of concern in biotechnology. The importance of glycosylation in intestinal systems is mostly unknown at this point, although the well-documented shift from sialylation to fucosylation during rat postnatal development (Sections IV,A,2,4, and 6) has been attributed to represent the change in physiological functioning of the intestine during the weaning phase (Torres-Pinedo and Mahmood, 1984). It seems probable that the application of cDNA probes for various glycosyltransferases to intestinal systems (Paulson er a/., 1989) as well as the development of chimeric and transgenic mice (Gordon, 1989; Trahair et a/., 1989) will provide exciting opportunities in the future for the investigation of the importance of intestinal glycosylation in a myriad of functions. ACKNOWLEDGMENTS The original research described in this paper has received generous continual support from the Swiss National Science Foundation. We would like to thank Daniel Wey, Michele von Turkovitch, and Linda Barcornb for preparing the figures and photographs.
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INTERNATIONALREVIEW OF CYTOLOGY, VOL. 126
Physiological and Pharmacological Regulation of Biological Calcification DANIEL c . WILLIAMS*
AND CHARLES A. FROLIK~
*Bone Biology Research Group, Department of Connective Tissue and Monoclonal Antibody Research, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285 f Department of Biochemistry Research, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285
I. Introduction Calcification is essential to both the physiology and structural integrity of the human body. It is important for growth and maintenance of the skeletal system as well as for mineral homeostasis and several specialized functions. Understanding the mechanism by which calcium deposition and mobilization occurs in the skeletal system and being able to regulate it has important medical connotations. Osteoporosis, a disease characterized by low bone mass and increased bone fragility, affects over twenty-four million people in the United States and is recognized as one of the major concerns of modern medicine. The direct medical costs associated with osteoporosis in the United States alone in 1986 were estimated to exceed 5 billion dollars (Philips et al., 1988). In addition, millions of individuals are affected by improperly healing bone fractures, orthopedic replacement surgery, and ectopic calcification (e.g., kidney stones and calcified atherosclerotic plaques). Add to this the potential involvement of abnormal bone modeling in the pathology of osteoarthritis (over 15 million people) and rheumatoid arthritis (over 2 million people) (Lawrence et al., 1989) and it is clear that the medical implications of calcification disorders are enormous. In a broader sense, the deposition of calcium salts is a widespread phenomenon in biology. It occurs in living organisms ranging phylogenetically from bacteria to higher plants and animals. As a result, biomineralization processes can have profound effects on the environment we live in - modulating the chemistry of the oceans, the shape and composition of the land masses, and the survival of many biological species. The literature relating to the regulation of calcification, particularly in the vertebrate skeleton, has increased dramatically in the last two decades. As a practical matter, this article is limited primarily to regulation of calcification processes in vertebrate bone. Because of the large literature base, representative papers have been cited rather than providing a comprehensive list of references. In many cases, the most recent citations have been chosen so that the readers can 195 Copyright Q 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
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refer to those papers for a more complete survey of the literature in a particular topic area. A. FUNCTION OF BIOLOGICAL CALCIFICATION Calcification is used in biological systems for a number of purposes. Mineralization of the connective tissue matrix of the vertebral skeleton provides it with the rigidity necessary to support the body. Most of this structural support comes from the cortical (compact) bone which comprises 80% of the skeletal mass. In addition, the skeletal framework provides attachment sites for muscles and acts as levers in movement. The hard skeletal infrastructure also provides the sensitive soft tissues of the body with a significant degree of protection against injury. In a similar manner, the calcified egg shells common to avian and reptilian species serve to protect embryos during development. Calcium salts deposited in the teeth, tusks, horns, and antlers provide these organs with the rigidity and strength necessary to serve as tools and weapons. These basic functions of the vertebrate endoskeleton, in many cases, hold true as well for the calcified shells and exoskeletal elements of invertebrates. Calcified tissues are also involved in a number of specialized functions such as sound transmission (as illustrated by the otoliths of the middle ear) and maintenance of balance (e.g., the utoconia of the inner ear). The calcified skeleton is furthermore the major reservoir for ionic calcium, a key cation in physiological regulation of many body functions, and therefore an ion whose concentration in the body fluids and tissues is carefully regulated. The trabecular (cancellous) bone, constituting only 20% of the skeletal mass but more than 60% of the bone surface due to its spongelike macroscopic structure, is metabolically more active than cortical bone and therefore provides a greater contribution to this mineral homeostasis (Jee, 1983; Martin ct al., 1988). Additionally, bone serves as a maternal reservoir for skeletal salts used in both fetal and neonate development. In higher plants, calcification is most often viewed as a mechanism for removing metabolic products from the cellular milieu (Amott and Pautard, 1970; Smith, 1982), but also may function as a storage site for ionic material (Amott and Pautard, 1970) and in intracellular pH balance (Smith, 1982).
B. MECHANISMS OF CALCIFICATION I . Nudeution Theory
These are two general theories proposed for the mechanism of initiation of calcification in biological tissues. One concept, the nucleation theory (Glimcher, 1987, 1989), maintains that, in a biological system, there must be a highly
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ordered nucleation site present that places the substrates for the crystallization process in the correct spatial conformation necessary for the proper interactions to occur to allow crystal formation. The biological nucleator in bone is believed to be the collagen fibril. Indeed, under certain conditions, initial calcium phosphate crystals do appear to be deposited in the hole-zone region of these fibrils (Berthet-Colominaset al., 1978; Glimcher, 1989). While collagen may be a necessary component for formation of these crystals, the noncollagenous proteins appear to be required for control of the process. The nature and function of these proteins will be discussed in a later section.
2. Matrix Vesicle Theory The second theory concerning the initiation of calcification in biological systems involves matrix vesicles (Wuthier, 1988; Anderson, 1989). Matrix vesicles are small membrane-bound compartments believed to be primarily formed by the pinching off of the plasma membrane (Cecil and Anderson, 1978; Ralphs and Ali, 1986; Palumbo, 1986) and are found in hypertrophic cartilage (Anderson, 1969), primitive woven bone early in fetal osteogenesis (Bernard and Pease, 1969), and in some bone repair processes (Schenk et al., 1970), but rarely occur in more mature bone (Landis et al., 1977). The interior of these vesicles are enriched in specific enzymes, proteins, and lipids, many of which appear to be involved in maintaining a high intravesicular calcium and phosphate concentration (Peress et al., 1974; Bernard, 1979; Majeska et al., 1979). Matrix vesicles are postulated to function in the calcification process by: (1) releasing enzymes that remove inhibitors of crystal growth (for example, pyrophosphate and proteoglycans); (2) increasing calcium and phosphate concentrations to adequate levels for calcification to occur; (3) maintaining a protective environment for the formation of the more soluble octacalcium phosphate prior to its conversion to hydroxyapatite; and (4) providing for site-specific deposition of seed crystals into the hole zones of the collagen fibrils. While matrix vesicles may play an important role in initiating calcification (Ali, 1976; Hsu and Anderson, 1978; Wuthier, 1982), once it has begun, they no longer appear to be needed, and disappear. Opponents of the matrix vesicleinduced calcification theory point out that electron micrographs seem to indicate, as discussed above, that calcification first occurs in the hole zone of the collagen fibril, while the matrix vesicle theory has the initial crystals forming separately from the collagen fibrils. It is possible that in mature bone, calcification is initiated by heterogeneous nucleation while in cartilage and early embryonic bone, where there is no clear relationship between collagen fibrils and calcium phosphate crystals (Glimcher, 1989), matrix vesicles control the mineral formation process (Eanes, 1989).
DANEL C. WILLIAMS AND CHARLES A. FROLIK
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DIVERSITY OF BIOLOGICAL CALCIFICATION
1. Chemical Species and Crystal Forms About two-thirds of all biogenic minerals are salts of calcium with a diversified anion composition that includes salts of carbonates, phosphates, citrates, sulfates, and oxalates (Lowenstam, 1981). As a general rule, phosphate salts tend to be most common in vertebrate calcified tissues, carbonates in invertebrate tissues, and oxalates in tissues of higher plants. However, as with all “general rules” in biology, there are many exceptions, particularly when individual tissues are considered (e.g., both the otoconia of the vertebrate inner ear and the cystoliths of higher plants are carbonates). Lowenstam ( 1981) notes that two fundamentally different biomineralization processes occur in nature. In the more primitive systems, such as for certain bacteria and green and brown algae, mineralization occurs by bulk intracellular or extracellular crystal formation in the absence of a preformed organic matrix. This process is far less controlled than those associated with an organic matrix, and the mineral phase is similar to that produced by precipitation from inorganic solutions. In the second, more advanced form of biomineralization, an organism produces an organic matrix which serves as a framework for mineral deposition and crystal growth. In this latter case, the mineral type, crystal orientation, and microarchitecture are under genetic control.
2. Biological Diversity While the emphasis in this review is on the regulation of calcification in the vertebrate skeleton, an appreciation of the diverse species that utilize calcification and how they regulate this process is useful for understanding the common mechanisms employed in biomineralization throughout the biological kingdom. Calcification, even in “simple” unicellular organisms, is often a precisely regulated procedure that may result in the deposition of calcium salts in elaborately sculptured crystalline arrays (Pautard, 1970). Examples of some of the most highly regulated cellular calcification processes occur among microorganisms. The control of crystal shape by the coccolithophorid algae, where the organic matrix is laid down within the Golgi vesicles prior to calcification to form highly sculptured, interconnecting elements, is an elegant example of the role of the organic matrix providing a framework for mineral deposition and growth (Outka and Williams, 1971; Williams, 1974; van der Wal el al., 1983). Even in relatively primitive bacterial mineralizing systems, cellular regulation of calcification is evident. The biosynthesis of hydroxyapatite requires that calcium and phosphate be delivered to the site of membrane-associated nucleation, and that protons formed during mineralization be removed in order to maintain appropriate pH conditions. During calcification of the membrane of the oral bacterium Bacter-
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ionema matruchotii, bacteriorhodopsin serves this role, being an active proton pump whose action is facilitated by calcifiable proteolipids (Swain and Boyan, 1988; Swain et al., 1989). Despite the fact that calcification occurs throughout the plant kingdom, in comparison to what we know about animal calcification, relatively little is known about its regulation. There is, however, general agreement that mineral deposition in plants is a highly regulated function (Amott and Pautard, 1970; Smith, 1982). While deposits of calcium salts can be found in the plant cell wall, more typically they are located intracellularly, occurring predominantly in leaves and stems, but also found in roots, flowers, fruits, and seeds. The shape of calcium deposits and their location in a given species appears to be under genetic control. The diversity of mineralization among invertebrate animals, as discussed in three recent books (Leadbeater and Riding, 1986; Simkiss and Wilbur, 1989; Lowenstam and Weiner, 1989), is enormous. Nevertheless, clearly, there are common factors that tie the calcification process in these various species together. For example, there is a reliance on organic material to nucleate crystals, and to direct and control mineral growth (Weiner, 1986; Wheeler et al., 1988). Also, an important role for active ion transport mechanisms and hormonal regulation is evident in some invertebrate mineralizing systems (Cameron, 1989). Weiner (1986) views the phylogenetically diverse mineralizing tissues as part of a continuum, although the end members may differ markedly in the degree of control they exert over crystal growth.
11. The Vertebrate Skeleton A. BIOCHEMISTRY OF MINERALIZED TISSUES Healthy bone consists of an extracellular matrix that is spatially arranged in a highly organized pattern and is embedded with crystals of hydroxyapatite. While this hydroxyapatite is the major constituent of bone, accounting for 60-70% of the dry weight of cortical bone, there are well over 200 other macromolecules (Delmas et al., 1984) that have important roles in maintenance of bone structure and function. Of these molecules, the collagens play a major role in formation of the bone matrix, while the noncollagenous proteins are believed to regulate calcification and control matrix synthesis and degradation through actions on osteoclasts and osteoblasts. In this section, a brief review of the various components of bone will be presented. While emphasis will be placed on true bone and not mineralized cartilage, many of the molecules found in bone either occur or have related components in cartilage.
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1. Mineral Phase Although hydroxyapatite, Ca,,(PO,),(OH),, was first described as being the mineral component of bone over 60 years ago (DeJong, 1926; Roseberry et al.. 193I), the exact chemical composition and spatial arrangements of minerals in bone is still poorly understood. The mineral phase is not pure hydroxyapatite but contains small portions of carbonate, citrate, sodium, and magnesium as well as other trace metals and elements. Also, bone mineral is in a state of continual flux and the “natural” ions can be replaced by other ions that may be in the local environment at the time that mineralization is taking place. This may lead to unwanted side effects, such as those which occur due to the substitution of lead for calcium in lead intoxication, or to the incorporation of strontium-90 into bone after exposure to nuclear fallout. On the other hand, ion substitution may also have desirable properties. For example, in the prevention of dental caries, fluoride treatment leads to substitution of the fluoride ion for the hydroxyl ion with the subsequent formation of a less soluble fluoroapatite. During the process of mineralization there is still some question as to what is the initial physical state of calciumiphosphate in bone (for reviews see Eanes. 1985; R. G. G. Russell et al., 1986; Glimcher, 1987). Using in vitro and synthetic models to simulate the multistep process of mineralization, several precursors have been postulated to occur during the formation of hydroxyapatite. These include octacalcium phosphate [Ca,H,(PO,), 5H20] (Brown, 1966; Eanes and Meyer, 1977), amorphous calcium phosphate [Ca,(PO,),.,,(HPO,),, , XH,O] (Eanes er al., 19651, and dicalcium phosphate dihydrate [CaHPO, 2H,O] (Francis and Webb, 197 1). Whether these transient intermediates actually do occur in viw is still a matter of debate. 2 . Collagens The word “collagen” is derived from the Greek words kolla and genes, which are literally translated as meaning to produce glue. Collagen is indeed the “glue” produced by cells to provide the coherent structural element that forms the mature tissue. In addition. in developing tissue, collagen appears to have a directive role in morphogenesis. The basic unit of collagen (called tropocollagen) consists of three polypeptide chains wound around each other in a helical motif. There are currently 20 different types of chains known which are associated together in various ratios to form 11 types of collagen (for review see Miller and Gay, 1987). Type I collagen, and small amounts of type V collagen, account for approximately 90% of the proteins in bone. However, while type I cartilage is the predominate form of collagen found in many connective tissues, only in bone is it physiologically involved in the mineralization process. Therefore, the involvement of type I collagen in calcification is most likely not due to a characteristic
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of the collagen backbone itself but probably occurs because of a posttranslationa1 modification of the collagen fiber (Mechanic et al., 1985) and/or to the presence or absence of other factors in the local bone environment (Termine et al., 1981b; Boyan, 1985).
3. Noncollagenous Proteins (NCP) While NCP represent only 10-15% of the organic phase of bone, their role in maintaining bone structure is essential (for reviews see Termine, 1988; Veis, 1988; Heinegard and Oldberg, 1989; Boskey, 1989a). For the purpose of this article, emphasis will be primarily placed on the involvement of the NCP in calcification. The NCP are believed to be associated with both the initiation of mineralization (through removal of inhibitors of nucleation and/or modification of the sites of nucleation) and, once crystallization has begun, with the regulation of the final shape and orientation of the crystal, and restricting the mineralization process within the proper boundaries. Table I lists the broad categories under which the NCP may be grouped. It should be remembered that many of the proteins may fall under more than one of the categories. Thus, osteopontin is not only a phosphoprotein but is also a glycoprotein. In the next few paragraphs, a brief description of the various classes of proteins that are found in bone will be presented along with examples of specific proteins in each class. a. Proteoglycans. The proteoglycans are proteins that contain one or more polysaccharide (glycosaminoglycan or GAG) sidechains (Scott, 1988) with chondroitin sulfate, dermatin sulfate, keratin sulfate, and sometimes heparin sulfate being found most often. The proteoglycans are thought to inhibit the process of mineralization (Howell et al., 1969; Cuervo et al., 1973; Boskey, 1989b), possibly through their ability to bind calcium, thus lowering the effective calcium concentration at the calcification site (Hunter, 1987). Classified according to their size, the very large proteoglycans are found only in calcified cartilage and make up 5-10% of the tissue weight. Their function appears to aid in tissue hydration (Campo, 1988), giving the cartilage resilience and providing a low friction joint surface, as well as to maintain matrix organization and regulate mineralization (Buckwalter et al., 1987). The large and small proteoglycans represent 4 1 0 % of the NCP in bone. In addition to being present in bone, they have a wide tissue distribution, occurring in most connective tissues. Three of these proteoglycans [PG I (also called PG-SI or biglycan), PG I1 (also named PG-SII or decorin), and fibromodulin] have extensive sequence homology (Fisher et al., 1989; Heinegard and Oldberg, 1989). They have been shown to bind to both collagen I and I1 (Hedbom and Heinegard, 1989) and are thought to modulate collagen fibril formation (Scott, 1988), thereby effecting mineral organization and calcification.
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TABLE I NONCOLLAGENOUSPROTEINS OF BONE
Class
Proposed Role
Examples PG-I (PG-Si. biglycan) PG-I1 (PG-Sii, decorin) Fibromodulin
Proteoglycans
Regulate matrix organization and mineral imtion
Clycoproteins
Control extracellularcalcium Osteopontin (BSPI) Regulate crystal growth and shape Bone sialoprotein (BSPII) Provide cell adhesion to bone surface
Phosphoproteins
Control extracellularmatrix formation and mineralization
Osteonectin
y-Carboxyglutamicacidcontaining proteins
Regulate crystal growth Recruit osreoclasts
Osteocalcin (Bone GLA protein) Matrix GLA Protein
Proteolipids
Initiation of crystal formation Ionophores
Enzymes
Formation of mineral phase Bone resorption
Alkaline phosphatase Acid Phosphatase Proteases Collagenase Carbonic Anhydrase
Growth factors
Control of cellular proliferation
Transforming Growth Factor+ (TGFP) lnsulinlike Growth Factor-I,11 (IGF-I, IGF-11) Epidermal Growth Factor (EGF) Fibroblast Growth Factor (FGF) Platelet-DerivedGrowth Factor ( PDGF) Cytokines
b. Glycopr-oteinsand Phosphoproteins. It is difficult to separate the glycoproteins and the phosphoproteins into two different classes since many of the glycoproteins isolated from bone also appear to be phosphorylated. In general, these proteins are highly anionic and therefore bind both hydroxyapatite and calcium. As a result, they are able to either retard or promote mineral deposition on hydroxyapatite, depending on concentration, physical form, and secondary structure (Renugopalakrishnanet al., 1986). Bone sialoprotein (BSPII) was one of the first noncollagenous bone glycoproteins described (Andrews er al., 1967). While originally thought to be specific to bone, Northern blot analysis now is able to detect low levels of mRNA for BSPI1 in highly localized sites in other tissues as well (Fisher ef al., 1990). Bone sialoprotein is a 59,000 MW glycoprotein that contains a high content of sialic acid (Oldberg et al., 1988a; Fisher er al., 1990), and a high glutamic acid and
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phosphorylated serine content which appear to be responsible for its ability to bind calcium (Heinegard and Oldberg, 1989) and hydroxyapatite (Franzen and Heinegard, 1985; Fisher er al., 1987). The primary structure also includes an Arg-Gly-Asp cell-binding sequence typical of cell-adhesion peptides, enabling the protein to bind to osteosarcoma cells (Oldberg et al., 1988b). These characteristics may be partly responsible for the ability of osteoblasts to adhere to bone surfaces recently exposed by resorption. A second phosphorylated glycoprotein, osteopontin (also called BSPI or 2ar), shows many similarities to bone sialoprotein. While it has a lower carbohydrate content, it is highly phosphorylated on serine and contains the Arg-Gly-Asp sequence (Franzen and Heinegard, 1985; Oldberg et al., 1986). Like bone sialoprotein, osteopontin is not specific for bone but has also been detected in a number of other tissues (Nomura et al., 1988; Senger et al., 1989), and has been demonstrated to be synthesized in virro by several epithelial and fibroblast cell lines (Smith et al., 1987), as well as by osteoblastlike cells (Yoon et al., 1987; Mark et al., 1987). The presence of the cell-binding tripeptide sequence and the protein’s localization in bone at the clear zone where osteoclasts are binding to mineral (Heinegard and Oldberg, 1989), has led investigators to speculate that it may be involved in the binding of osteoclast precursor cells to the mineralized matrix of bone. In support of this concept is the observation that osteoclasts contain a high concentration of the vitronectin receptor, a member of the integrin family of receptors that recognizes the Arg-Gly-Asp sequence (Horton, 1988). A third member of this class, osteonectin (also called SPARC, BM-40, or 44K albumin-binding protein) is a 32,000 MW phosphorylated glycoprotein first isolated and characterized by Termine and co-workers in 1981 (Termine et al., 1981a,b; for review see Tracy et al., 1988). While it represents the most abundant noncollagenous protein in mineralized matrix (23% of the noncollagenous proteins) it is also widely distributed in nonmineralized tissues (Wasi et d., 1984; Young et a/., 1986; Holland et al., 1987) and platelets (Stenner er al., 1986). Osteonectin displays a high affinity for calcium, hydroxyapatite, and type I collagen (Termine et a1.,1981a,b; Romberg et al., 1985, 1986; Engel et al., 1987; Domenicucci er al., 1988). Examination of the primary sequence of human (Villarreal et al., 1989) and bovine (Bolander et al., 1988) bone osteonectin indicates several possible calcium binding domains in the amino terminal and carboxy terminal portions of the peptide similar to those observed in other intracellular calcium-binding proteins such as calmodulin (Persechini et al., 1989). Osteonectin may function in a general manner in controlling extracellular matrix formation. It has been shown to increase the binding of hydroxyapatite to collagen and to increase collagenlinked crystal formation from metastable solutions (Termine et al., 1981b). On the other hand, osteonectin at very low concentrations (lo-’ M) also inhibits hydroxyapatite crystal growth in vitro (Romberg ef al., 1985, 1986) and therefore could function to prevent mineralization of newly synthesized osteoid.
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c. yCarhoiyglutamic Acid-Containing Proteins. Another general class of proteins found in bone are those that contain the amino acid y-carboxyglutamic acid. To date, there are two bone-related proteins that fall into this category. The first, osteocalcin (bone Gla protein), represents 10-20% of the NCP in bone (Hauschka et al., 1975; Price er al., 1976a, 1982; for reviews see Lian and Gundberg, 1988; Price, 1988; Hauschka et al., 1989). It is a 5800 MW protein that contains three residues of y-carboxyglutamic acid (Price et al., 1976b) which are formed posnranslationally by a vitamin K-dependent carboxylation reaction. Osteocalcin mRNA is detected only in bone (Yoon et al., 1988: Fraser and Price, 1988) and appears to be synthesized exclusively in the osteoblast (Nishimoto and Price, 1980). It is expressed relatively late in bone development (Otawara and Price, 1986),appearing shortly after the initial deposition of bone mineral (Price el al., 198la; Hauschka e f al., 1983).The synthesisof osteocalcin is under the control of many of the factors that are known to affect bone, including vitamin D (Price and Baukol, 1980; Lian et al., 1985; Spiess ef al., 1986), transforming growth factor-p (TGFP) (Noda, 1989), and parathyroid hormone (PTH) (Noda et al.. 1988a). The protein has been detected in serum where levels appear to correlate with bone turnover, often being used as a diagnostic for bone diseases (Price et al., 1980; Delmas et al., 1983; Slovik et al., 1984)reflecting mainly, if not exclusively, bone formation (Price etal., 1981b; Brown etal., 1984; Riggs etaf., 1986). As with many of the NCPs studied to date, osteocalcin binds both to hydroxyapatite (Poser and Price, 1979) and to calcium (Svard et al., 1986). However, the protein does not appear to be involved in the initial deposition of bone mineral (Boskey et al., 1985) but instead may be involved in inhibition of hydroxyapatite crystal growth (Price et al., 1982; Romberg et al., 1986). This might occur through the binding of osteocalcin to various lipids, making them unavailable for their proposed role in calcification (Gendreau et al., 1989). However, osteocalcin does not appear to be absolutely necessary for normal mineralization, since administration of warfarin to rats (which inhibits osteocalcin synthesis and blocks y-carboxylation) still results in normal bone formation (Hauschka and Reid, 1978; Price et al., 1982). In addition to a proposed regulatory role in mineralization, osteocalcin may also recruit osteoclasts, resulting in an increase in bone resorption (Malone et al., 1982; Lian e f al., 1984; Glowacki and Lian, 1987). Finally, recent evidence indicates that this protein is able to inhibit the activation of prothrombin in vitro, thereby perhaps serving as a natural anticoagulant within the Haversian system or on the endosteal surfaces (Gendreau ef al., 1989). A second y-carboxyglutamic acid-containing protein, matrix Gla protein, has been isolated from bone, dentin, and cartilage (Price et al., 1983). Amino acid sequence analysis shows it to be a peptide of 84 amino acids (10,612 kDa) with five y-carboxyglutamic acid moieties (Price et a/., 1987; Kiefer et al., 1988).
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The C-terminal domain has a 20% homology with osteocalcin (Kiefer et al., 1988). It represents approximately 2% of the NCP and accounts for 10-20% of the Gla-containing protein in adult bone (Otawara and Price 1986). While the protein occurs in calcified tissues, it is also found in noncalcified cartilage (Hale et al., 1988) and mRNA levels for matrix Gla protein are 10-fold higher in lung and heart and 5-fold higher in kidney than in bone, although these tissues do not accumulate much of the protein (Fraser and Price, 1988). As with osteocalcin, 1,25-dihydroxyvitamin D3[1,25-(OH),-D,] stimulates expression of the protein (Fraser et al., 1988). Matrix Gla protein is tightly associated with the organic phase of bone and cartilage (Price et al., 1983) and appears before the onset of mineralization (Otawara and Price, 1986). However, because of its widespread occurrence, its function does not appear to be specific for calcified tissues. It has recently been postulated that matrix Gla protein may be involved in inhibition of the mineralization process and that it is synthesized in those soft tissues that are susceptible to ectopic calcification (Fraser and Price, 1988). d. Proteolipids. Another class of proteins, the proteolipids, also appears to be intimately involved in the calcification process. The proteolipids are hydrophobic membrane proteins which tend to be complexed with various acidic phospholipids, the most common being phosphatidylserine. They are able to cause hydroxyapatite deposition both in vitro (Ennever et al., 1978) and in vivo (Raggio et al., 1986). The proteolipids are found in high concentrations in matrix vesicles (Boyan-Salyers et al., 1978) where they may act as ionophores, serving to export protons and import calcium and phosphate into the vesicle. (Sapirstein and Rounds, 1983; Swain and Boyan, 1988). They are also involved in formation of calcium phosphate-phospholipid complexes (Cotmore et al., 1971; Boskey and Posner, 1976), perhaps functioning to adjust the conformation of the phosphatidylserine to allow it to interact with the ions. These complexes are believed to be involved in the initial formation of hydroxyapatite crystals (Posner, 1985). While the phospholipids, as indicated above, are often found complexed with proteins, they also occur by themselves in close association with the mineral phase (Shapiro et al., 1966) and are enriched in matrix vesicles (Majeska et al., 1979) where they are involved in formation of the lipid bilayer membrane necessary for forming the internal environment of the vesicle. Phospholipid metabolism is influenced by 1,25-(OH),-D,, which increases phosphatidylserine levels in human osteoblastlike cells (Haining et al., 1988), and in UMR-106 rat osteosarcoma cells (Matsumoto et al., 1985). It has therefore been postulated that at least part of the effects of 1,25-(OH),-D3 on bone may be to increase the components necessary for formation of calcium phosphate-phospholipid complexes and, secondarily, hydroxyapatite formation.
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e. Enpmes. Of the enzymes involved in the calcification process, only alkaline phosphatase has been studied extensively. The alkaline phosphatases are glycoproteins that are attached to the cell membrane via interaction with phosphatidylinositol (Low and Zilversmit, 1980). There are several isozymes of alkafine phosphatase that have been separated into three classes (liver, kidney, bone; placental; intestine) based on immune cross reactivity (McKenna et al., 1979). Recently, using specific antibodies, it has become possible to specifically detect bone alkaline phosphatase in mixtures of the other isozymes (Hill and Wolfert, 1989). In general, the enzymes are metalloproteins containing zinc and magnesium with broad substrate specificities and alkaline pH optima. The possible role of alkaline phosphatase in mineralization is supported by a number of observations. First, it was demonstrated quite early that the enzyme is able to cause precipitation of calcium phosphate in solutions of calcium salts and phosphate esters (Robison, 1923). Second, alkaline phosphatase concentration increases at the site of mineralization in bone and cartilage (Martland and Robison, 1924; Follis, 1949: Sandhu and Jande, 1982) and is present at high concentrations in matrix vesicles (Ali et al., 1970; Majeska and Wuthier, 1975). Finally. inhibitors of alkaline phosphatase will also block matrix vesicle mineral ion uptake (Register and Wuthier, 1984). It must be kept in mind, however, that alkaline phosphatases are widely distributed in the body, including locations in tissues that do not calcify. Therefore, although these enzymes may have a role in the calcification process in bone and cartilage, they must also have functions not associated with mineralization. A number of mechanisms have been proposed to explain the participation of an alkaline phosphatase in formation of the mineral phase. First, the enzyme may be involved in removal of phosphorylated inhibitors of crystallization (i.e., pyrophosphate and ATP) (Fleisch and Neuman, 1961; Fleisch and Bisaz, 1962). Second, during the hydrolysis of the phosphorylated inhibitors, inorganic phosphate becomes available for use as a substrate in the crystallization process. However, the levels of phosphorylated substrates found in cartilage extracellular fluid are considered to be too low to supply the necessary phosphate concentration needed at the nucleation site (Wuthier and Register, 1985). Alkaline phosphatase has also been implicated in the transport of inorganic phosphate across the cell membrane (Petit-Clerc and Plante, 1981; Letellier e l al., 1982), though this role has been questioned, since selective removal of alkaline phosphatase from renal tubule brush border membranes actually stimulates phosphate transport (Yusufi et al.. 1983). However, alkaline phosphatase may play a modulatory role as part of a multiprotein membrane complex, perhaps involving phosphorin, a proteolipid postulated to be involved in phosphate transport (Kessler and Vaughn, 1984). In addition to its possible function in controlling phosphate concentrations, the enzyme also has calcium binding properties (Vittur and
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deBemard, 1973) and Ca-ATPase activity (Haussler et al., 1970) although these properties may be due to other components that copurify with the enzyme. Finally, alkaline phosphatase has been reported to have phosphotyrosyl protein phosphatase activity (Swamp et af.,1981). While selective removal of a phosphate group from tyrosine may be involved in controlling many of the responses of growth factor stimulation, the role of alkaline phosphatase in this pathway is still uncertain. In support of this concept, levels of the enzyme rise at confluency when cell growth slows, growth factors are able to stimulate alkaline phosphatase activity (Wuthier and Register, 1985) and factors that suppress alkaline phosphatase activity are able to stimulate cellular proliferation (Carpenter, 1981). B. MODELING AND REMODELING IN SKELETAL TISSUES
In the face of a burgeoning literature that has emphasized the physiology and metabolism of individual cellular components of the skeletal system (e.g., osteoblast, osteoclast, chondrocyte, etc.), Frost and others have emphasized a holistic approach to understanding the biogenesis and maintenance of calcified tissue in the vertebrate skeleton (Frost, 1988, 1989a-d). In order to understand how cells function to form skeletal tissues, and how these tissues interact to form and maintain a functioning skeletal system, it is necessary to realize that the processes which occur during skeletal growth and development, and those that are responsible for skeletal maintenance, often involve the same cellular players. However, these cells may function by a different set of regulatory rules under different physiological conditions (Jee, 1983; Eriksen, 1986; Frost, 1988; Burr and Martin, 1989). Bone modeling is a term used to describe the processes involved in bone growth and changes in bone shape (some investigators, e.g., Frost, 1988, prefer to consider growth and modeling as mechanistically separate). Bone formation and resorption are not “in balance” either locally or systemically during the process of modeling, and both formation and resorption may be occurring simultaneously in different regions of the same bone. While modeling is primarily associated with the immature, growing skeleton, changes in bone shape can also occur in the mature skeleton in response to appropriate stimuli (Frost, 1988). On the other hand, bone remodeling refers to the processes by which bone is maintained in nongrowing tissue. Resorption and formation occur sequentially in a local region of the skeleton and are quantitatively in balance. This equilibrium between bone formation and resorption is often referred to as “coupling” (Frost, 1963; Martin et al., 1988). Errors in the coupling of bone formation and resorption during remodeling can lead to skeletal disease (Eriksen, 1986; Marcus, 1987; Burr and Martin, 1989).
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C. CELLSOF BONE 1. Osteoblasts
Osteoblasts are derived from noncirculating mesenchymal cells called osteoprogenitors. These progenitor cells are found in the soft connective tissue of the periosteum, endosteum, and Haversian canals of bone, and among the stromal cells in the trabecular bone marrow adjacent to the bone (J. Russell, et al., 1986; for review see Marks and Popoff, 1988). Four differentiation stages can be defined morphologically in the osteoblast lineage-the preosteoblast, the osteoblast, the lining cell, and the osteocyte. These cells are recognized by their morphology, by their position in bone, and by their proliferative capacity. Under well-defined conditions within an organized tissue environment, preosteoblasts can be recognized by morphological criteria such as their localization in soft tissues adjacent to bone, their nuclear size and morphology, and their glycogen and alkaline phosphatase content (Scott, 1967; Roberts et al., 1982). Efforts are also underway to define these cells by antigenic characteristics (Nijweide and Mulder, 1986; Nijweide el al., 1988; Nakano el al.,l989; Peny et al., 1990). These endeavors are particularly important in order to better understand the properties of osteogenic cells in culture where their characteristic morphology and tissue relationships may not be evident. Osteoblasts are observed histologically as cuboidal cells lying on the bone surface (Figs. 1 and 2). They are morphologically and functionally polarized cells with abundant rough endoplasmic reticulum and a prominent Golgi region, i.e., characteristics of a secretory cell. Their main product, type I collagen, is assembled extracellularly to form the primary organic constituent of osteoid, the matrix which is mineralized to form the rigid structural elements of bone. The FIG.1. Light micrograph of a section through the metaphysis of a rat long bone showing the classic morphology of osteoblasts (Ob) on the bone surface that are actively secreting bone matrix. The cells are cuboidal to columnar in shape with an acentric nucleus and a prominent central secretory region. Also shown in this section are osteocytes (0)located within the bone matrix, and an osteoblastic transition cell (T) which is located just below the surface layer of osteoblasts. This cell is an osteoblast that is being surrounded with bone matrix and becoming an osteocyte. Bar = 10 pm. FIG.2. Transmission electron micrograph of a demineralized bone preparation showing a section of an osteoblastic (Ob) transition cell in canine rib bone. This cell lies at the surface of the bone matrix (BM), and has been almost completely surrounded by loose collagen fibrils. It has extended a cellular process through the canalicular canal (C) in the bone matrix which allows it to form contacts with osteocytes, and also maintains contact with the overlying osteoblasts by close cellular contacts (arrows) that form gap junctions between the cells. Bar = 1 pm. FIG.3. Light micrograph of a section through a rat tibia1 metaphysis. A large, multinucleate osteoclast (arrow) can be seen on the surface of a bone aabecula. Bar = 10 pm. FIG.4. Transmission electron micrograph showing a large multinucleate osteoclast (Oc) forming a resorption lacuna (RL) between itself and the surface of the bone matrix (BM). The numerous cellular processes extending into the resorption lacuna form the osteoclastic ruffled border. Bar = l pm.
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shape and density of the mineralized matrix varies depending on its location within a bone and on the location of the bone within the skeletal framework. The regulatory steps that determine these characteristics are poorly understood. In adult mammalian bone, osteoblasts lay down collagen fibrils in a parallel manner producing a lamellar arrangement. Individual lamellae are typically 3-7 pm thick and appear to be stacked together to form the bony tissue. The collagen fibers in adjacent lamellae are not parallel with each other and the lamellae are separated by interlamellar “cement.” In embryonic bone, fracture healing, or certain disease states, osteoblasts produce a less organized woven bone in which the collagen fibers are nonparallel and irregularly arranged. Cultured osteoblastic cells have been derived from a variety of species and have proven to be valuable tools in understanding osteoblastic function (e.g., Peck et al., 1964: Wong and Cohn, 1975; Kodama et al., 1981; Williams et al., 1980; Partridge ef al., 1981; Robey and Termine, 1985; McCarthy ef al., 1988). Both primary and longer term cultures of cells obtained from normal tissues have been employed. However, information acquired from primary cultures must generally be considered as being derived from a heterogeneous mix of bone cells, whereas, in long-term cultures, there frequently appears to be a change of phenotypic expression associated with extended culture which can serve to complicate interpretation of data (e.g., Aubin et al., 1988). In addition, a number of the osteoblastic cell lines used as models for bone metabolism studies have been derived from osteosarcomas [e.g., UMR (Partridge et al., 1981) and ROS (Majeska er al., 1980) cell lines]. The extension of information from these cancer cells to normal tissue metabolism must be viewed cautiously. Typical phenotypic markers used to identify osteoblastic cells include type I collagen synthesis, production of alkaline phosphatase, and response to the calciotropic hormone FTH (G. A. Rodan, er at., 1989). Mineralization in vim may also be considered to be an important phenotypic marker (Williams ef al., 1980; Sudo er al., 1983). lntegrens (cell surface receptors for a range of cell and extracellular matrix proteins) on osteoblasts do not seem to differ radically from other stromal or fibroblastic cells (Horton and Davies, 1989). Several animal models have been used to investigate the effects of mechanical loading in modulating bone formation and osteoblast activity. Suppression of weight bearing or complete inactivity will lead to loss of bone, whereas physical activity increases relative levels of bone mass (Schoutens et al., 1989). Experiments applying dynamic loads to functionally isolated bones of skeletally mature roosters also indicate that removal of load bearing results in reduced bone mass, while cyclic loading prevents resorption and causes bone formation (Rubin and Lanyon, 1984). A number of studies, performed on animals in real or simulated space flight conditions, have demonstrated that osteopenia can result from exposure to microgravity. The mechanism of this osteopenia appears to be a transient inhibition of osteoblastic function (Morey and Baylink, 1978;
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Wronski and Morey, 1983; Jee et al., 1983; Simmons et al., 1983) in the presence of unchanged levels of bone resorption (Wronski and Morey, 1983; Jee et al., 1983; Cam and Adachi, 1983; Bikle et al., 1987; Vico et al., 1988). Taken from another view, Pead et al., (1988) have demonstrated that a single short period of dynamic loading is capable of transforming quiescent adult periosteal cells on the bone surface to active bone-forming cells. Studies with bones in culture have shown that intermittent compressive force, applied to mouse bone rudiments, stimulates matrix mineralization and inhibits osteoclastic invasion and resorption of mineralized bone (Burger et al., 1989). Osteoblastic cells in culture will respond to cyclical mechanical deformation by activation of both prostaglandin synthesis and CAMPand phosphodiesterase pathways, by increases in cell division rates, by changes in cell orientation on the culture substrate, and by release of bone resorbing agents (Buckley et al., 1988; Sandy et al., 1989a,b). Bone cells will also respond to electrical stimulation with an enhanced osteogenesis. This response has been used clinically to treat problem fractures (Haupt, 1984). Similarly, a number of recent studies in animal models suggest that electrical (or electromagnetic) stimulation of osteogenesis might find utility in the treatment of osteoporosis. The effects of electrical stimulation in these models appears to be associated with increased bone formation which may or may not involve effects on bone resorption (e.g., Brighton et al., 1988, 1989; Rubin et al., 1989). Pulsed electrical stimulation has also been shown to stimulate DNA synthesis in cultured osteoblastic cells (Ozawa et al., 1989). Finally, a study of the effects of surface charge on beads injected intrafemorally into the medullary cavity of chicken hatchlings indicated that new osteogenesis was found to be preferentially associated with positively charged beads. These positively charged beads at neutral pH created a negative counterionic environment, thus making the finding consistent with the observation that osteogenesis is associated with the cathodes of surgically implanted electrodes (Krukowski et al., 1988). In addition to their role in bone formation per se, osteoblasts (and/or bone lining cells) play a crucial role in bone resorption (see discussions in Rodan and Martin, 1981; McSheehy and Chambers, 1986a; Kahn and Partridge, 1987; Martin et al., 1988). Osteoblasts, but not osteoclasts, have receptors to several resorption-promotingagents, and osteoblasts release factors capable of stimulating bone resorption. Several osteoblast products have been identified which may serve to mediate both the short and long-term osteoblast-osteoclast interaction. These include granulocyte-macrophage colony-stimulating factor (GM-CSF) (Shiina-Ishimi et al., 1986; Felix et al., 1988; Horowitz et al., 1989a,b), prostaglandins (Nolan et al., 1983; MacDonald et al., 1984; Feyen et al., 1984), interleukins (Feyen et al., 1989), TGFP (Centrella and Canalis, 1987; Robey et al., 1987), and a number of less well-characterized factors (e.g., McSheehy and
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Chambers, 1986b; Abe et al., 1988; Sandy et al., 1989b). Osteocalcin, synthesized exclusively by osteoblasts, may be involved in osteoclast recruitment and/or differentiation (Malone ef al., 1982; Lian er al., 1984; Glowacki et al., 1989). In addition, osteoblasts produce a neutral collagenase capable of rernoving the nonmineralized osteoid from the bone surface so the osteoclast can resorb the mineralized bone (Chambers and Fuller, 1985). Finally, the osteoblasts produce plasminogen activator, an enzyme hypothesized to be involved in the activation of collagenase, as well as inhibitors capable of blocking both collagenase and plasminogen activator activity (Kahn and Partridge, 1987; Martin et al., 1988). Osteoblasts also may regulate osteoclast access to the bone surface through changes in cell shape (Rodan and Martin, 1981). Changes in osteoblast cell shape have been observed by several groups in response to either PTH or prostaglandin E, (PGE,) (Jones and Boyde, 1976; Miller et al., 1976; Jones and Ness, 1977; Shen et al., 1986). Ali et al. (1990) found, however, that osteoblasts from 2-week-old rats, growing on a natural substrate, change shape in response to PTH but not PGE,, though both agents are known to induce bone resorption in neonate calvarial culture. These observations, they suggest, indicate that osteoblast shape change may not be essential for bone resorption.
2 . Bone Lining Cells Bone lining cells are thin, flat elongated cells containing few organelles (Luk et al., 1974a,b; Miller et al., 1980; Bowman and Miller, 1986). These cells cover the bone surface (Figs. 5 and 6), appearing to be metabolically inactive with respect to bone formation or resorption, and are often joined to each other by gap junctions. While viewed by many as “inactive” osteoblasts, Miller and Jee (1987) have proposed that the lining cell represents a separate phenotype. Evidence exists for an estrogen-induced transition between the lining cell and osteoblast in avian medullary bone induction (Bowman and Miller, 1986). However, relatively little is known about the function and regulation of lining cells because they are found primarily in nongrowing adult bone, while the vast majority of cell biology studies are carried out in fetal tissues where these cells do not play a significant role (Nijweide et al.. 1986). It has been speculated that they may have one or more of the following functions-progenitors for osteoblasts, selective barriers for mineral exchange between bone and extracellular fluid, regulation of crystal growth, regulation of hemopoiesis, and/or physical barriers to osteoclastic bone resorption (Rodan and Martin, 1981; Miller and Jee, 1987; Marks and Popoff, 1988). 3. Osteorytes
Osteocytes are derived from osteoblasts. They lie within lacunar spaces embedded within the mineralized bone (Figs. 1, 5, and 7). Osteocytic lacunae
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are connected to each other, and to cells on the bone surface, by a complex canalicular lattice (Figs. 2, 5, and 7). This canalicular network allows the osteocytes to communicate with each other and the bone surface via thin cytoplasmic processes which form gap junctions with other cells. The exact function of the osteocyte is not known. Some investigators maintain that the osteocyte can both form and resorb bone (Belanger, 1969; Marks and Popoff, 1988), although this opinion is controversial (Sissons el al., 1984, 1990; Mercer and Crenshaw, 1985). An alternative view is that the osteocyte may be involved in sensing mechanical force on bone and relaying this information to the bone surface where resorptive or formation responses can be initiated (Martin et aZ.,1988). Osteocytes, as well as periosteal cells, will respond to mechanical loading (strain) by rapid changes in glucose-6-phosphatedehydrogenase (G6PD) activity, but not glyceraldehyde-3-phosphate dehydrogenase or aldolase activity, and by a slower (24 hr) increase in RNA synthesis ([3H]uridine uptake). While the functional significance of these changes is not currently known, the increase in the number of osteocytes demonstrating G6PD activity has been shown to be proportional to the mechanical strain applied to the bone, and related to increased bone formation at the bone surface (Skeny er al., 1989). 4. Osteoclasts
Osteoclasts are large multinucleated cells, located on the bone surface, which resorb mineralized bone and cartilage (Figs. 3 and 4). Active osteoclasts have two unique, specialized cell surface regions, a ruffled border, and a clear zone. The ruffled border is a highly folded region of the plasma membrane adjacent to the resorbing bone that has prominent cytoplasmic vacuoles (primary and secondary lysosomes) associated with it. The clear zone is an actin-rich region of the cell cytoplasm next to the ruffled border region and is thought to be involved in cell-substrate adhesion (Martin et al., 1988; Marks and Popoff, 1988). Though for many years osteoclasts and osteoblasts were thought to be derived from a common progenitor cell population, osteoclasts are now generally regarded to be derived from hemopoietic cells. A variety of in vivo and in vitro models have been used to support this hemopoietic origin. Studies using osteopetrotic rodents have proven to be particularly informative. Osteopetrosis is an inherited bone disease in which defective bone resorption results in excessive accumulation of bone throughout the skeleton. Walker (1975) first showed that osteopetrosis in rodents could be reversed by transplantation of hemopoietic tissue. In vitro, osteoclastlike cells can be formed in a variety of marrow cultures (MacDonald et al., 1987; Takahashi el a/., 1988a-c) as well as from circulating mouse blood leukocytes (Helfrich et al., 1989) and spleen cells (Takahashi el al., 1988b; Udagawa et al., 1989). Akatsu et al. (1989) have shown that prostaglandin (especially PGE, and PGE,) can stimulate the formation of osteoclastlike cells from mouse bone marrow cultures, apparently through a
DANIEL C. WILLIAMS A N D CHARLES A . FROLIK
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CAMP-associated mechanism. PTH and 1,25-(OH),-D, can also induce the formation of osteoclastlike cells in these cultures (MacDonald er d., 1987; Takahashi et al., 1988~).In several of these in v i m models, osteoclast formation can be inhibited by calcitonin (e.g., McDonald er al., 1987). The formation of osteoclasts in marrow preparations and in spleen cell preparations is enhanced by the presence of osteoblastic or marrow stromal cells (Takahashi er al., 1988b; Wdagawa et al., 1989). Many workers propose that the osteoclast shares a common origin with phagocytic monocytes and macrophage. The work of Schneider and Relfson ( 1988), however, suggests that osteoclast precursors co-isolate with granulocyte colony-forming cells (G-CFC) but not macrophage colony-forming cells (M-CFC). Granulocyte-macrophage-colony forming cells (GM-CFC) populations were intermediate in their ability to reverse osteopetrosis in rats. These data suggest that the immediate precursors to osteoclasts and monocyte/macrophage are not the same, but do not necessarily indicate that the granulocyte lineage is the source of the osteoclast precursor. A more detailed discussion of the history of osteoclast lineage can be found in the reviews of Nijweide et al. (1986) and Marks and Popoff (1988). Osteoclasts induced to form in mouse marrow preparations are typically characterized by being multinucleate, and containing tartrate-resistant acid phosphatase. Some studies also evaluate the ability of the cells to resorb bone and look for induction of calcitonin receptors in association with osteoclast differentiation (Hattersley and Chambers, 1989; Taylor er al., 1989). Other studies look for evidence of immunologic relationships between cells formed in culture and osteoclasts from bone (Kukita and Roodman, 1989; Kukita ef al., 1989). In addition, integrens, which on mature osteoclasts differ from other hemopoietic cell types (i.e., large amounts of vitronectin receptor and collagen type I receptor), perhaps reflecting their specialized role in bone (Horton and Davies, 1989), may be useful in characterizing osteoclastic cells derived from marrow cultures. Bone resorption occurs in a specialized environment that is under the control of the osteoclast. This cell attaches to the bone surface to form an enclosed space, the resorption lacuna (Fig. 4), which becomes acidified (e.g., Fallon, FIG.5. Light micrograph showing the thin, flat nuclei (arrows) of the cells (lining cells) that line the medullary cavity of a quail long bone diaphysis. Also shown are osteocytes (Oc) and the network of canaliculae that connect the osteocytes to each other and the cells on the bone surface. Bar = 10
w.
FIG. 6. Transmission electron micrograph of a section through lining cells (LC) on the surface of trabecular bone in dog rib. The lining cells form a continuous cellular boundary separating the bone surface from the cells in the marrow cavity. Bar = 1 pn. FIG.7. Transmission electron micrograph of an osteocyte (0)in dog rib bone. The osteocyte is located in a lacunar space that is surrounded by bone matrix. Several thin cellular processes can be seen projecting into canalicular canals (C) that form pathways for cell-cell contact through the dense bone matrix. Bar = 1 pm.
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DANIEL C. WILLIAMS AND CHARLES A. FROLIK
1984; Baron et al., 1985) through the action of a proton pump located in the apical membrane (Anderson et al., 1986; Baron, 1989; Blair et al., 1989). Ultrastructural and immunocytochemical evidence suggests a role for cytoplasmic microfilaments in forming the tight membrane-cytoplasmic adhesion needed to maintain the acidic environment in the resorption lacunae (King and Holtrop, 1975; Turksen et al., 1988; Lakkakorpi et al., 1989). The attachment of the osteoclast and formation of the resorption lacuna defines the morphological and metabolic polarity of the active osteoclast (Baron, 1989). The apical pole of the cell is the region attached to the extracellular matrix, and the apical membrane forms the clear zone and ruffled border. Lysosomal enzymes are vectorially transported to the apical end of the cell in association with mannose-6-phosphate receptors (Baron er al., 1988). Upon fusion with the apical cell membrane and exposure to the acid extracellular environment of the resorption lacuna, the enzymes dissociate from the mannose-6-phosphate receptor and are released into the resorption lacuna. Osteoclastic acidification of the resorption lacuna is stimulated by PTH and inhibited by calcitonin (Baron et al., 1985; Hunter el al., 1988). The acidification of this space is mediated through the action of a proton pump located in the ruffled border membrane adjacent to the resorption lacuna (Baron ef af., 1985; Baron, 1989). The identity of the proton pump is not clear, and evidence for a H+-K+-ATPasepump (Baron et al., 1985; Tuukkanen and Vaananen, 1986; Anderson et al., 1986), a Na+-H+-antiporter(Hall and Chambers, 1990b), and an electrogenic H+-ATPase pump (Blair et ul., 1989; Baron, 1989) have been reported. A significant body of evidence suggests that carbonic anhydrase may also play a role in osteoclast acidification and bone resorption. Carbonic anhydrase is the enzyme responsible for the reversable hydration/dehydration reaction converting carbon dioxide to carbonic acid. Carbonic anhydrase has been localized histochemically to the apical region of the cell (Anderson et al., 1982; Cao and Gay, 1982), and inhibitors of carbonic anhydrase inhibit the acidification process (Hunter et a/., 1988). Carbonic anhydrase levels in osteoclasts can be modulated by FTH (Hall and Kenny, 1985; Silverton eta/. , 1987), and its cellular localization regulated by calcitonin (Anderson ef al., 1982; Cao and Gay, 1982). Thus the H' ions produced as a result of the action of carbonic anhydrase in the apical domain of the osteoclast are transported into the resorption lacuna via the proton pump. Evidence for a complementary CI--HCO,- anion transport system to remove carbonate ions from the cells, and thus balance the intracellular pH, has also recently been reported (Teti et al., 1989; Hall and Chambers, 1989; Klein-Nulend and Raisz, 1989). The cellular localization of this transport system has not been defined, but is assumed to be in the basolateral domain (Baron, 1989). Baron (1989) suggests that the basolateral cell membrane is also enriched with respect to sodium pumps (Na+,K*-ATPase)which may supply the primary force for proton transport, though others suggest an apical localization
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for this ATPase activity (Akisaka and Gay, 1986). A calcium pump (Ca-ATPase) has been shown to be localized on the marrow (basal) face of the osteoclast and may be associated with removing excess calcium derived from resorbed bone out of the intracellular environment (Akisaka et al., 1988). The osteoclast can thus be viewed as a highly organized enzymatic factory in which hydrolytic enzymes are synthesized in the endoplasmic reticuludGolgi apparatus and transported in secretory vesicles (primary lysosomes) to the apical domain of the cell where they are released into a closed, acidic resorption lacuna formed by the tight adhesion of the osteoclast to the bone surface. The products of bone resorption in the resorption lacuna are taken up by the osteoclast by the process of endocytosis (phagocytosis).The osteoclast contains a variety of acidification and ion transport cellular machinery, localized in different functional domains of the cell.
111. Physiological Regulation of CaIeification in the Vertebrate SkeIeton
In the skeletal system, there are many factors that affect the initiation and rate of calcification. These biological regulators include collagen (BerthetColominas et al., 1979; Glimcher, 1989) pyrophosphates (Fleisch and Bisaz, 1962). proteoglycans (Chen et al., 1984), osteocalcin (Menanteau et al., 1982; Boskey et al., 1985), phosphoproteins (Fujisawa et al., 1987), and osteonectin (Menanteau et al., 1982; Romberg er al., 1986). It is clear that anything that increases or decreases the extracellular matrix concentration of these proposed regulators could affect the calcification process. Indeed, several hormones and growth factors are able to modulate the levels of these inhibitors and stimulators of crystal growth (Tables I1 and 111). In this section, these physiological regulators will briefly be discussed. While the focus of this article is on the cellular and biochemical processes directly involved in the formation and maintainence of calcified tissues, it is clear that associated mechanisms, such as the absorption, excretion, and transport of calcium and phosphate, also play an important role in the calcification process. A. ENDOCRINE FACTORS
1 . Parathyroid Hormone (PTH) There are several hormones that act on bone in order to maintain normal blood calcium homeostasis. Included in this group is PTH, a hormone secreted from the parathyroid gland in response to low serum calcium. In addition to its actions on bone, PTH accelerates calcium absorption in the intestine and kidney. In bone, PTH interacts primarily with the osteoblast and chondrocyte which
DANIEL C. WILLIAMS AND CHARLES A. FROLIK
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TABLE I1 ENDOCRINE REGULATORS OF BONEMETABOLISM Hormone
Proposed Role
Parathyroid hormone (PTH) Increase bone formation and resorption
Interaction with Other Factors Increases release of IGF-1, IGF-11, and GMCSF Modulates activity of TGFP. EGE and IL- I Increases levels of 1,25-(OH),-D, Effects inhibited by estrogen and testosterone
Calcitonin
Decrease bone resorption and increase bone formation
Increases levels of I ,25-(OH),-D, Effects enhanced by testosterone Secretion modulated by estrogen
Vitamin D
Regulation of calcification process
Decreases PTH levels Increases EGF receptor number Increases TGFP secretion Increases activity of IGF Modulates response to gonadal steroids Receptors regulated by retinoic acid and glucoconicoids Estrogen increases 1.25-(OH)Z-D, production
Vitamin A (retinoids)
Alter extracellular matrix synthesis Modulates PTH secretion and effects Controls I .25-(OH)2-D,activity and receptor levels Increases growth hormone production
Estrogens
Suppression of bone turnover
Modulates PTH and calcitonin Increases I .2S-(OH)z-D, production Effects modulated by 1,25-(OH),-D, Increases synthesis and secretion of IGF-I and TGFP Increases release of growth hormone
Androgens
Maintenance of bone mass Increase in bone formation
Inhibits PTH response Increases growth hormone and IGF-I production
Growth hormone
Increase in bone formation
Increases synthesis of IGF-I Production increased by retinoids, estrogen and androgens
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TABLE I11 OF BONEMETABOLISM AUTOCRIN~~ARACRUE REGULATORS Growth Factor
Proposed Role
Interaction with Other Factors
IGF
Increase bone matrix production Increase osteoprogenitorcell proliferation
Secretion regulated by growth hormone, estrogen, F'TH, 1,25-(OH)2-DS, ~-miC@Obulin, and glucocorticoids Decreases secretion of growth hormone Regulated 1,25-(OH),-D, levels Synergizeswith EGF, FGF
TGFP
Regulates embryonic formation of cartilage and bone Modulates extracellular matrix production Modulates bone resorption
Activity increased by FGF, IL- I , 1,25-(OH),-DS,estrogen and decreased by calcitonin Modulates effects of FGF, EFG, IL-1, and I,25-(OH),-D3
Bone inductive proteins (BMPs, OF, osteogenin)
Induction of mesenchymd cell differentiation
OIF synergizes with TGFP Osteogenin activity modulated by PDGF and vitamin D BMP activity regulated by cytokines
EGF (TGFa)
Increase osteoblast/osteoclast precursor pool
Receptors modulated by TGFP, FTH Synergizes with IL-1
FGF
Increase osteoprogenitorcell proliferation
Increases TGFP levels Potentiated by TGFP, IGF
PDGF
Stimulatesosteoblast proliferation Stimulatesbone resorption
Effects modulated by IGF-I
Cytokines (IL-1, IL-3, GMCSF, TNF)
Regulation of matrix producing cells Modulation of bone resorption
Effects modulated by TGFP, FGF, 1,25-(OH),-D3 Regulates actions of EGF, TGFa, PTHrP Interacts with estrogen GMCSF induced by PTH
have been shown to possess high affinity receptors for the hormone (Silve et al., 1982; Pliam et al., 1982; Newman el al., 1989; Enomoto et al., 1989). The receptor is a glycoprotein (80,000 kDa with a 59,000 kDa polypeptide backbone) and is localized on the cell surface plasma membrane (Shigeno er al., 1988a,b). Parathyroid hormone will induce receptor down-regulation (Yamamoto er al., 1988; Abou-Samra et al., 1989b) with expression of the receptor being under the control of inhibitory guanyl nucleotide regulatory proteins (Abou-
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DANIEL C. WILLIAMS AND CHARLES A. FROLIK
Samra et al., 1989b). In rat long bones, PTH receptors have been localized to the osteoblast and a preosteoblastlike cell with little or no binding to the osteoclast (Rouleau et al., 1988). This supports the view that PTH acts indirectly on the osteoclast through interaction with the osteoblast, causing release of a soluble factor which then acts on the osteoclast to increase bone resorption (Rodan and Martin, 1981; McSheehy and Chambers, 1986a,b; Perry ef al., 1989). While the primary action of PTH on bone is the stimulation of resorption, (for review see Martin ef al., 1985), effects on bone formation have also been noticed. In viw, administration of physiological amounts of PTH results in gain of bone (Podbesek et al., 1983; Slovik et a[., 1986) due to increases in both cortical and trabecular bone mass (Hock ef al., 1988a). Subcutaneous injections of PTH into rats over a period of 12 days causes an increase in total bone calcium, dry weight, and bone-forming surfaces. These effects were not dependent on bone resorption (Hock et al.,l989a). In v i m , PTH can stimulate the proliferation of chick calvaria cells in culture (Farley ef al., 1988a) and human trabecular bone cells, cultured at high cell density, but has no effect on human skin fibroblasts (MacDonald et al., 1986a). The effect of PTH on bone formation may be through an increase in both the number and activity of osteoblasts, resulting in an increase in the extent of bone-forming surfaces and in the rate of mineral and matrix apposition. Parathyroid hormone causes an increase in the steady state levels of osteocalcin mRNA (Noda et al., 1988a), stimulates amino acid transport in neonatal mouse calvaria (Yee, 1988; Hall and Yee, 1989), DNA synthesis in fetal rat calvaria (Canalis et al., 1989a) and in embryonic chicken chondrocytes (Schluter et al., 1989) and, with transient treatment, increases type I collagen synthesis (Canalis et al., 1989a). The mechanism whereby PTH causes these anabolic effects is not known. They do not appear to be mediated by prostaglandins (Gera et af., 1987) or CAMP (Schluter et al., 1989). Recently, the involvement of the insulinlike growth factors (IGFs) has been suggested. Parathyroid hormone will stimulate the release of IGF-I and IGF-I1 from neonatal mouse calvaria (Linkhart and Mohan, 1989), and from fetal rat calvaria (Canalis et af., 1989a), and will stimulate the synthesis of IGF-I in osteoblastlike cells (McCarthy ef al., 1989a). Also, the effect of PTH on collagen synthesis in fetal rat calvaria is blocked by IGF-I antibodies, although the antibodies will not inhibit the mitogenic effect of PTH (Canalis et al., 1989a). Finally, infusion of IGF-I or PTH alone into the hind limb of a rat will not affect the rate of trabecular bone apposition but, when given together, will cause a significant rate increase (Spencer ef af., 1989). Therefore, at least some of the effects of PTH may arise from an autocrine mechanism through the release of the IGFs or a potentiation of their effects. In addition to IGF, PTH is known to interact with several other growth factors and hormones. In osteoblastlike cells, PTH modulates the activity of TGFP and enhances its cellular binding (Centrella ef al., 1988). The opposite effect has
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been shown for epidermal growth factor (EGF), where PTH decreases both the number of EGF receptors and the responsiveness of the MC3T3-El osteoblastlike cell line to EGF (Ohta et al., 1989). Parathyroid hormone synergizes with interleukin-1 (IL-1) in stimulating bone resorption both in v i m and in vivo (Dewhirst et al., 1987; Sat0 et al., 1989) and induces the secretion of GM-CSF from bone cells (Weir et al., 1989; Horowitz et al., 1989a). Since GM-CSF is able to increase the number of osteoclasts (MacDonald et al., 1986b; Horowitz et al., 1989a), this factor may be the osteoblast-derivedcomponent that mediates PTH-stimulated effects on osteoclast and bone resorption. The effects of PTH on bone resorption are further modulated by the steroid hormones. Estrogen inhibits PTH stimulated bone resorption and PGE, release in the neonatal mouse calvaria (Pilbeam et al., 1989) and PTH-stimulated adenyl cyclase activity in primary cultures of calvaria and trabecular bone cells (Emst et al., 1989), and in SaOS-2 human osteosarcoma cells but not in ROS 17/2.8 rat osteosarcoma cells (Fukayama and Tashjian, 1989a). Testosterone and dihydrotestosterone also inhibit the cAMP response to PTH in SaOS-2 cells (Fukayama and Tashjian, 1989b). The net effect of these actions would be to decrease bone resorption. Finally, PTH plays a role in the metabolism of vitamin D by increasing the 25hydroxyvitamin D,- la-hydroxylase activity in the renal cortex of the kidney, thereby controlling the levels of 1,25-(OH),-D,, the active metabolite of vitamin D (Siege1etal., 1987). Parathyroid hormone may have a dual pathway for generating changes in the osteoblast. It was known quite early that PTH can activate adenyl cyclase and increase cellular levels of cAMP (Chase and Aurbach, 1970). More recently, it has been demonstrated that PTH is also able to stimulate the inositol phosphate pathway in calvarial osteoblastlike cells (Somjen et al., 1987; Farndale et al., 1988; Dunlay and Hruska, 1990). In this pathway, hydrolysis of phosphatidylinositol-4,5-bisphosphateby phospholipase C releases inositol 1,4,5-trisphosphate (which mediates the release of intracellular calcium) and diacylglycerol (which activates protein kinase C) (Berridge and Irvine, 1984). Indeed, both protein kinase C (Somjen et d., 1987; Abou-Samra et al., 1989a) and calcium mobilization (Lowik et al., 1985; Somjen et al., 1987; Civitelli et al., 1989b; Schluter er al., 1989) have been implicated in the PTH response. While it is tempting to speculate that the bone-resorbing effects of PTH may be mediated primarily through the cAMP pathway (Civitelli et al., 1989a), while its anabolic effects occur through its interactions with the phosphoinositide pathway (Somjen et al., 1987; Dunlay and Hruska, 1990), certain PTH analogs that are able to stimulate resorption without increasing cAMP concentrations (Lowik et al., 1985) indicate that it may not be as simple as this. A second protein with PTH-like properties has been found in several different tumor cells (Rodan et al., 1983; Moseley et al., 1987). The first 13 N-terminal amino acid residues of this PTH-related peptide (PTHrP) shares a 62%
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DANIEL C. WILLIAMS AND CHARLES A. FROLIK
homology with PTH while the remaining sequence is markedly different (Suva et al., 1987). Parathyroid hormone-related peptide binds to the same receptor as PTH (Horiuchi et al., 1987; Abou-Samra er al., 1989c; Klein-Nulend et al., 1990). Its effects on bone and kidney are similar to those seen for PTH (Horiuchi et al.. 1987; Klein-Nulend et al., 1990), although it is perhaps slightly less potent (Fukayama et al., 1988; Hock et al., 1989b). Like parathyroid hormone, PTHrP modulates the effects of locally produced growth factors. For example, in osteoblast enriched cultures, PTHrP, while having no effect by itself, enhances the effects of TGFP on DNA and collagen synthesis and matrix turnover (Centrella er al., 1989a). The normal physiological function of PTHrP is unknown. Its presence in tumors (Moseley et al., 1987), and the fact that it produces hypercalcemia in vivo (Horiuchi et al., 1987) has lead to the suggestion that it may be responsible for the hypercalcemia observed in malignancy. 2 . Calcitonin Calcitonin, secreted from the C-cells of the thyroid gland, acts as an antagonist to PTH and decreases serum calcium levels (for reviews see Fischer and Born, 1987; Breimer et al., 1988). It is a 32-amino acid peptide that is the product of a gene complex that produces either calcitonin or calcitonin gene-related peptide (Fischer and Born, 1987; Breimer et al., 1988). The major action of calcitonin is to decrease bone resorption and to enhance kidney production of 1,25(OH),-D,. In modulating resorption, calcitonin appears to act directly on the osteoclast. Calcitonin receptors have been detected on this cell (Warshawsky et al., 1980; Rao et al., 1981; Nicholson et al., 1986) and the hormone abolishes cytoplasmic mobility and spreading of isolated osteoclasts in culture (Chambers and Magnus, 1982; Chambers et al., 1986; Nicholson et al., 1987). The effects of calcitonin may be partly mediated through the production of CAMP(Murad et al., 1970; Heersche ef al., 1974; Rodan and Rodan, 1974), although calcium may also play a modulatory role (Malgaroli et al., 1989). In addition to its effects on inhibition of resorption, calcitonin will also stimulate growth and maturation of embryonic chick cartilage in vitro (Burch, 1984) and bone formation in vivo (Weiss et al., 1981). Although calcitonin is not currently believed to have a significant etiological role in the development of osteoporosis (McDermott and Kidd, 1987; Prince et al., 1989), it has been used as a therapeutic agent in osteoporosis to increase trabecular bone volume and bone mineral content (Gruber et al., 1984; Mazzuoli et al., 1985; Civitelli et al., 1988). These anabolic effects of calcitonin appear to be a result of a direct interaction with the osteoblast. High affinity calcitonin receptors have been detected on a bone stem cell line (Eilon et al., 1983), and on osteoblastlike cells (Findlay and Martin, 1986). Calcitonin is able to stimulate the differentiation of an osteoblastlike cell line, MC3T3-E 1, and the production of alkaline phosphatase
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(It0 et al., 1987). These anabolic effects occur at a similar dose level that causes an inhibition of bone resorption (Farley et al., 1988b). Although not extensively investigated, calcitonin does appear to interact with other factors. Testosterone enhances the hypocalcemic effect of calcitonin in castrated rats (Ogata et al., 1970), while estrogen has been reported to modulate calcitonin secretory capacity, with lower calcitonin levels found in the postmenopausal woman (Reginster er al., 1989). However, other investigators have not observed a direct effect of estrogen on calcitonin secretion and postulate that any observed variations may be due to the effects of estrogen on bone metabolism (Body et al., 1989). In the mouse marrow culture system, 1,25(OH),-D, stimulates the formation of calcitonin-responsive cells and the appearance of calcitonin receptors (Takahashi et al., 1988a; Hattersley and Chambers, 1989). In return, calcitonin is able to act on the kidney to increase the production of 1,25-(OH),-D, (Rasmussen et al., 1972). These higher levels of 1,25-(OH),D, are then able to stimulate intestinal absorption of calcium, thus providing additional motivation for the decrease of bone resorption.
3. Vitamin D Vitamin D and its metabolites, 1,25-(OH),-D3 and 24,25-(OH),-D,, are another group of factors that interact with bone and are involved with calcium homeostasis (for reviews see DeLuca, 1988; Reichel et al., 1989). Specific, high affinity receptors for 1,25-(OH),-D3 have been detected in bone (Kream et al., 1977; Chen et a [ . , 1979), being localized on the osteoprogenitor cells and the osteoblast and osteocyte (Narbaitz et al., 1983; Boivin et al., 1987). The number of receptors in bone decrease with age (Horst et al., 1990). This decrease correlates with a decrease in the response of the osteoblast to 1,25-(OH),-D, (Chen et al., 1986) and is therefore perhaps partly responsible for the age-related changes observed in bone. Although the major site for the synthesis of 1,25-(OH),-D, is in the kidney, human bone cells have been shown to contain both 1- and 24hydroxylase activity (Howard et al., 1981), and evidence exists for the production of 1,25-(OH),-D, in bone cells (Lohnes and Jones, 1987; Sempere er al., 1989). 1,25-(OH),-D, is thought to play an important role in the calcification process by modulating the levels of a number of the substrates and regulators of mineralization. Thus, in osteoblastlike cell lines, 1,25-(OH),-D, has been reported to increase phosphatidylserine levels (Matsumoto et al., 1985; Haining et al., 1988), a constituent of the calcium-phospholipid-phosphate complex formed during the crystal formation process, and to reduce the synthesis and increase the degradation of proteoglycans (Takeuchi et al., 1989), thereby removing a potential inhibitor of mineralization. The synthesis of another component of the calcification process, type I collagen, has been reported to be both stimulated and inhibited by vitamin D. In certain systems employing human bone cells
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(Beresford et al., 1986), or a human osteosarcoma cell line (Franceschi et al., 1988), 1,25-(OH)?-D,enhanced type I collagen synthesis. In other systems that utilized bone cells from rat sources, 1,25-(OH),-D, inhibited type I collagen production (Rowe and Kream, 1982) through the inhibition of the transcription of the type I collagen gene (Harrison et a[., 1989). This regulation was found to be bone-specific, occurring in bone cells but not in fibroblasts (Lichtler et al., 1989). Whether this variation is due to a difference in species or to other factors remains to be investigated. The osteocalcin gene also contains a vitamin Dresponsive promoter region (Lian et al., 1989; Demay et al., 1989) that is controlled by the vitamin D receptor (McDonnell et al., 1989). Binding of the receptor to this promoter causes an increase in the transcription of the gene (Yoon et af., 1988) leading to an increase in the synthesis of osteocalcin (Price and Baukol, 1980; Pan and Price, 1984; Lian et al., 1985). Matrix Gla protein synthesis is likewise stimulated by 1,25-(OH),-D, (Fraser ef al., 1988). It has also been postulated that some of the effects of vitamin D on mineralization may be indirect through an increase in the absorption of calcium and phosphorus from the intestine (Underwood and DeLuca. 1984). Yet another mechanism for the involvement of vitamin D in calcification of bone would be through an increase in the number or activity of differentiated osteoblast cells capable of producing the extracellular matrix required for the formation of crystals. While 1,25-(OH),-D, has been shown to inhibit bone cell proliferation, it increases alkaline phosphatase activity (Majeska and Rodan, 1982; Beresford et al., 1986), a marker for the differentiated osteoblast. In v i m , 1,25-(OH),-D, prevents the dedifferentiation of fetal rat calvarial cells, maintaining the expression of alkaline phosphatase (Fritsch et al., 1985). In vivo, in dogs, the vitamin reportedly increases the activity but not the number of osteoblast cells in bone (Malluche et al.. 1986). In contrast, dihydrotachysterol, an analog of vitamin D, supposedly acts on bone through the activation of an osteoprogenitor cell population with a subsequent increase in the number of osteoblasts (Tabuchi et al., 1989). In chondrocytes, 1,25-(OH),-D, has an effect on the matrix vesicle, causing a greater increase in alkaline phosphatase activity in the vesicle compared to the plasma membrane (Schwartz et al., 1988a; Boyan et al., 1989). Matrix vesicles from growth cartilage chondrocytes display a stimulation of not only alkaline phosphatase activity but also 5’-nucleotidase, phospholipase A2, and sodium/potassium ATPase activity upon treatment with 1,25(OH),-D, (Boyan et al., 1988; Schwartz ef al., 1988b). Other metabolites of vitamin D, in particular 24,25-(OH),-D,, have been suggested to have an effect on bone formation and mineralization (Omoy el al., 1978; Tam et al., 1986). This idea has, however, been questioned by data indicating that 24,24-difluoro-25-(0H),-D3, an analog that could not be metabolized to the 24,25-dihydroxy compound, was still able to enhance mineralization (Tanaka et al., 1979). In contrast to 1,25-(OH),-D,, the 24.25-dihydroxy
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metabolite has no effect on phospholipid metabolism (Haining et al., 1988), on stimulation of alkaline phosphatase activity in human bone cells (Beresford er al., 1986), or on osteocalcin synthesis (Lian et al., 1985). In the matrix vesicle, the response to 24,25-(OH),-D, depends on the stage of chondrocyte differentiation. While it does not have much effect on growth zone cartilage matrix vesicles, it is able to stimulate alkaline phosphatase activity and inhibit phospholipase A, activity in the resting zone matrix vesicle (Schwartz et al., 1988a,b; Boyan ef al., 1988). A second natural metabolite of vitamin D, 1,25-(OH),-D326,23-lactone also appears to have unique properties in bone. It is able to inhibit 1,25-(OH),-D3-induced bone resorption while it has no effect on PTH-induced resorption (Kiyoki et al., 1985). This response is thought to occur through an inhibition of the induction of osteoclastlike cells by 1,25-(OH),-D3 (Ishizuka et a[., 1988a). The lactone metabolite is also able to increase alkaline phosphatase activity and collagen synthesis in osteoblastlike cells (Kiyoki et al., 1985; Ishizuka et al., 1988b), and to stimulate bone formation as evidenced by an increase in matrix production and mineral uptake in vivo during ectopic bone formation (Shima et al., 1990). Vitamin D interacts with a number of other factors to modulate its own response or the response of these factors. For example, 1,25-(OH),-D, blocks transcription of the PTH gene (J. Russell et al., 1986), thereby decreasing levels of pre-proPTH mRNA both in v i m (Silver et al., 1985) and in vivo (NavehMany et al., 1989). In addition, it blocks the synthesis and secretion of PTH from the parathyroid cell in culture (Cantley et al., 1985; Chan et al., 1986) as well as from the parathyroid gland in vivo (Delmez et al., (1989). Vitamin D metabolism has been reported to be regulated by the gonadal steroids. At low levels of estrogen, 25-hydroxyvitamin D is metabolized predominantly to 24,25(OH),-D, in the premenopausal woman, but at higher estrogen concentrations, the formation of 1,25-(OH),-D, is favored (Buchanan er al., 1986a). In postmenopausal women, estrogen increases the levels of circulating biologicallyactive free 1,25-(OH),-D, (Cheema et al., 1989). 1,25-(OH),-D3 also has been reported to selectively affect the biological response of skeletal tissues to the gonadal steroids (Somjen er al., 1989a). Finally, estrogen blocks the 1,25-(OH),D,-stimulated increase in the secretion of the IGFs from the rat osteosarcoma cell line, UMR-106 (Gray er al., 1989a). Receptors for 1,25-(OH),-D3 are regulated by a number of factors, including retinoic acid and glucocorticoids. In two different rat osteosarcoma cell lines, the receptor number is increased by retinoic acid treatment while in normal rat bone derived cells it is decreased (Petkovich et a1.,1984; Lee et al., 1988). This difference in response to retinoic acid is also observed when mouse is compared to rat. 1,25-(OH),-D, receptor number increases after retinoic acid treatment in mouse osteoblasts but decreases in rat osteoblasts (Chen and Feldman, 1985). This is in contrast to glucocorticoids which decrease the number of receptors in the mouse osteoblast and increase it
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in the rat osteoblast (Chen er al., 1983). These changes in receptor levels parallel the observed response to 1,25-(OH),-D, in induction of the 25-hydroxyvitamin D-24-hydroxylase (Chen and Feldman, 1985) and in the modulation of procollagen mRNA levels (Kim and Chen, 1989). In addition to changes in its own receptors, 1,25-(OH),-D, can increase the number of receptors for EGF on osteoblasts (Petkovich et al., 1987). It also increases the secretion of TGFP-like activity from osteoblasts (Petkovich et al., 1987) and enhances the activity of IGF in stimulating alkaline phosphatase activity and collagen synthesis (Kurose et al., 1989). This interaction with various growth factors may help to explain some of the effects of vitamin D on bone growth and differentiation. In addition to its effects on bone formation and mineralization, vitamin D also has both short- and long-term effects on the resorption process. In the short term, l.25-(OH),-D3 has been shown to increase the release of calcium from fetal rat long bone in culture (Raisz et al., 1972). This presumably occurs through the stimulation of osteoblasts to release a factor that, in turn, stimulates the osteoclast (McSheehy and Chambers, 1987; Key et af., 1988). Its long-term effects on resorption appear to be through an increase in the number of osteoclasts (Holtrop er al., 198 I ; Roodman et al., 1985). 4 . Vitamin A (Retinoids)
The retinoids also appear to be involved in regulation of bone formation and resorption. In general, retinoid excess causes a decrease in osteoblast activity resulting in a decrease in formation, while a deficiency results in an increase in the number of osteoblasts and the production of new bone. Retinoids can act directly on bone-forming cells to alter extracellular matrix synthesis. Retinol specifically inhibits collagen synthesis in embryonic chick and neonatal mouse calvaria (Dickson and Walls, 1985) and in a rat osteosarcoma (ROS) cell line (Nishimoto et al., 1987). In ROS 17/2.8 cells, retinoic acid inhibits both alkaline phosphatase production and PTH-stimulated adenyl cyclase without an effect on proliferation (Imai et al., 1988). In addition, retinoid toxicity may result in increased bone resorption (Fell and Mellanby, 1952; Hough et al., 1988). In cartilage, chondroitin sulfate proteoglycan synthesis is inhibited by retinol (Solursh and Meier, 1973). Likewise, retinoic acid stimulates the loss of proteoglycans from adult bovine articular cartilage with the larger proteoglycan being lost at a greater rate than the small proteoglycan (Campbell and Handley, 1987). Finally, retinoic acid rapidly reduces cartilage matrix synthesis in chick sternal chondrocytes by selectively changing the normal pattern of gene expression from synthesis of chondrocyte specific proteoglycan and collagen II to the production of fibronectin and collagen I11 (Horton et al., 1987). The retinoids have been implicated in the normal development of mammalian bone. While specific intracellular cytosolic binding proteins for retinol or retinoic acid could not be detected in UMR-106 osteoblastlike cells in culture
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(Oreffo et al., 1985), retinoic acid receptor mRNA is expressed in the calcifying fronts of developing mouse finger bones (Noji et al., 1989). In embryonic and fetal rat calvaria bone cells, retinoic acid causes increases in both FTH-stimulated adenyl cyclase and alkaline phosphatase activity and in type I procollagen mRNA, all considered to be markers of a more differentiated state (Ng et al., 1988; Heath et al., 1989). As indicated in the discussion on vitamin D, the retinoids are able to modulate 1,25-(OH),-D,- receptor levels. Retinol and retinoic acid have also been reported to stimulate the activity of 25-hydroxyvitamin D 1-hydroxylase in primary cultures of chick kidney cells (Trechsel and Fleisch, 1981) and to enhance the activity of 1,25-(OH),-D, in the stimulation of osteocalcin in ROS 17/23 cells (Nishimoto et al., 1987). In addition to its effects on vitamin D metabolism and response, retinoic acid is able to control growth hormone production in pituitary cells in vitro, acting synergistically with thyroid hormone and glucocorticoids, (Bedo et al., 1989), and selectively increasing mRNA levels for the hormone (Morita et al., 1989). Retinoids are also able to stimulate the release of PTH from the parathyroid gland (Chertow et al., 1977), and testosterone production in rat Leydig cells (Chaudhary et al., 1989). 5 . Steroid Hormones
The steroid hormones, estrogen and testosterone, also appear to have significant effects on bone turnover and mineralization. The effects of estrogen are most clearly demonstrated in those situations where hormone levels are decreased by ovariectomy in the rat and by bilateral oophrectomy or menopause in the human. In the rat, removal of the ovaries results in enhanced bone turnover with increases in both the rates of bone apposition and removal (Wronski et al., 1985, 1988; Turner et al., 1987a). Estrogen treatment suppresses this turnover with declines in formation and resorption (Cruess and Hong, 1979; Turner et al., 1987a; Wronski et al., 1988). In the human, accelerated bone loss resulting in low bone mass and osteoporosis is found in postmenopausal women (Riggs et al., 1982; Nilas and Christiansen, 1987) as well as in the patient who has undergone bilateral oophrectomy (Aitken et al., 1973; Lindsay et al., 1980). Again, estrogen replacement therapy helps to prevent this loss (Meema et al., 1975; Lindsay et al., 1976, 1980). For a long time, the effects of estrogen on bone were considered to be indirect effects. This concept was supported by the fact that estrogen receptors, considered to be essential for the action of estrogen, could not be detected in bone. More recently, however, osteoblasts have been shown to contain estrogen receptors and estrogen-receptor mRNA (Komm et al., 1988; Eriksen et al., 1988; Kaplan et a[., 1988). In support of this data, autoradiographic evidence indicates receptors for estrogen in the periosteum, perichondrium, chondrocytes, and chondroblasts of adult baboon (Aufdemorte et al., 1988). Receptors have
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likewise been observed in articular chondrocytes from both rabbit (Rosner ef al., 1982) and dog (Young and Stack, 1982), but not in epiphyseal chondrocytes from these two species (Kan et al., 1984). Finally, nuclear binding of estradiol has been detected in several different normal human osteoblastlike cell strains, suggesting that the action of estrogen on bone can occur through the classical receptor-mediated mechanism observed in other estrogen-responsive tissues (Colvard et al., 1989a). Additional evidence indicating the direct involvement of estrogen in bone formation comes from in vitro experiments. In neonate rat calvaria cells, estrogen increases cell proliferation and the steady state levels of type I procollagen mRNA while it decreases PTH-stimulated adenyl cyclase activity (Emst et al., 1988, 1989). In UMR- 106 osteosarcoma cells, estrogen decreases proliferation and increases alkaline phosphatase activity (Gray er al., 1987). Avian bone responds to estrogen treatment by inducing synthesis of proteoglycan (Hunter and Schraer, 1983) and of a calcium-binding glycoprotein with alkaline phosphatase activity (Stagni er al., 1980). in the diaphyseal bone of weanling rats, females show an increase in creatine kinase activity after estrogen treatment, while males do not respond to estrogen (although they will respond to androgens) (Somjen rt al., 1989b). Also, differentiation and mineralization are increased by estrogen and progesterone in the matrix-induced endochondral bone formation assay (Bumett and Reddi, 1983). Finally, in an in vivo experiment, local infusion of estradiol into rat bone inhibited bone resorption and stimulated bone formation with no effect on uterine or body weight (TakanoYamamoto and Rodan, 1990). The action of estrogen in cartilage appears to be dependent on the system being studied. While it has been suggested that proliferation and proteoglycan synthesis in bovine articular chondrocytes are not under the control of physiological levels of estrogen (Mackintosh and Mason, 1988), in rabbit cartilage, estrogen has been reported to both suppress proteoglycan synthesis and decrease proteoglycan turnover in vivo (Rosner et al., 1979), and to increase proteoglycan synthesis in vitro (Corvol et al., 1987). Although some of this difference may simply be due to an in vivo versus an in virro response, a portion of it could also be due to the age and sex of the animal from which the cells were derived. In fetal rabbit chondrocytes, estrogen will stimulate proteoglycan synthesis, while in chondrocytes from 5-30 day old animals no response to estrogen was detected. In older animals, the stimulation returned but it was less in the male compared to the female (Corvol er al., 1987). Finally, in the epiphyseal cartilage from weanling rats, estrogen stimulated creatine kinase activity and DNA synthesis (Somjen et al., 1989b). Originally, the effects of estrogen on bone resorption were thought to be indirect. Estrogen was not able to inhibit bone resorption either in fetal rat long bones (Caputo et al., 1976) or in neonatal mouse vertebral bones in culture
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(Stewart and Stern, 1987). Instead, the effects of estrogen were thought to be mediated through an interaction with PTH and/or calcitonin. To get the observed decrease in resorption with estrogen administration requires either a decrease in PTH or an increase in calcitonin levels, or a change in tissue response to these factors. In support of this concept, it has been reported that there is an increased sensitivity to PTH in bone from ovariectomized rats (Orimo et al., 1972). In contrast, however, parathyroidectomized and ovariectomized rats have been reported to respond to estrogens with a conservation of bone to the same extent as sham-operated controls (Goulding and Gold, 1989). Similarly, the involvement of PTH in the in vivo response to estrogen in humans is not clear. While estrogen has been reported to increase PTH secretion from parathyroid tissue from bovine (Greenberg et al., 1987) or human (Duarte et al., 1988) sources, it does not change plasma levels of the hormone in postmenopausal women (Selby et al., 1985). Similarly, in normal premenopausal women, PTH levels remain constant during the menstrual cycle in spite of sharply fluctuating estrogen levels, although increased estrone levels might be associated with reduced circulating PTH (Buchanan et al., 1986b). In fact, early effects of estrogen on bone resorption in postmenopausal women have been reported to be independent of PTH, while later effects are only variably associated with increased PTH (Stock et al., 1989a). More recently, direct effects of estrogen on bone resorption have been observed. In neonatal mouse calvaria, estrogen will inhibit PTH-stimulated PGE, release and PTH-stimulated bone resorption (Atkins et al., 1972; Pilbeam et al., 1989), while in a human, but not a rat osteosarcoma cell line, it will inhibit PTH-stimulated CAMPproduction (Fukayama and Tashjian, 1989a). The mediation of the effects of estrogen on bone resorption through calcitonin have not been as thoroughly studied. In vitro, estrogen will stimulate the secretion of calcitonin from rat thyroid C cells (Greenberg et al., 1986). Also, some in vivo studies indicate that estrogen treatment of postmenopausal women will give rise to a sharp increase in plasma calcitonin (Whitehead er al., 1981; Stevenson et al., 1983; Civitelli et al., 1988). However, other in vivo studies have not demonstrated this effect (Selby et al., 1985; Body et al., 1989). Again, thyroidectomized rats show the same response to estrogen as sham-operated controls, implying the effects of the steroid hormones are independent of calcitonin (Goulding and Gold, 1989). Therefore, the involvement of calcitonin in mediating the effects of estrogens on bone resorption is still open to debate. Besides PTH and calcitonin, estrogens interact with a number of other factors involved in bone metabolism and calcification. The effects of estrogen on vitamin D metabolism and the possible control by 1,25-(OH),-D, of the estrogen response in rat long bone have already been discussed. Estrogens have also been shown to interact with several growth factors in bone and part of the skeletal effects of estrogen may be mediated via regulation of these factors. In rat calvaria cells, estrogen will increase the steady state level of IGF-I mRNA (Emst et
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al., 1989) and stimulate the release of IGF-binding proteins (Schmid et al., 1989a). while UMR-106 osteoblastlike cells respond to estrogen with an increase in the secretion of IGF-I and I1 (Gray et al., 1989a). In bone, TGFP may also be under the control of estrogen. In rat and human osteosarcoma cells, the level of TGFP mRNA is increased by estrogen (Komm et al., 1988), which is translated into an increased production of the growth factor itself (Gray et al., 1989b). There is conflicting data on the role of IL-1 in mediating the action of estrogen on bone resorption. In one study, treatment of postmenopausal women with estrogen did not affect the spontaneous release of IL-I from peripheral monocytes in vitro (Stock et al., 1989b). in a second study, the levels of IL-1 in osteoporotic and nonosteoporotic women correlated inversely with vertebral mineral density. Treatment with estrogen/progesterone caused a significant decrease in interleukin activity (Pacifici et al., 1989). Additional studies will have to be done before a conclusion can be reached concerning the role of cytokines in the action of estrogen on bone. Finally, estradiol has been reported to amplify the neuroendocrine regulation of pulsatile growth hormone release with decreased circulating estradiol concentrations being correlated with decreased growth hormone levels (Ho et al., 1987). Indeed, the growth hormone response to levodopa is depressed in postmenopausal osteoporosis (Rico et ul.,l979). Estrogen administration to the postmenopausal woman results in enhanced growth hormone secretion and increased serum concentrations of growth hormone (Duurama et al., 1984; Dawson-Hughes et al., 1986). In addition to estrogens, the androgens, in particular testosterone and dihydrotestosterone, are involved in bone growth and mineralization. Their role best becomes evident in those situations where a deficiency or excess of the hormone exists. In rats, castration (Wink and Felts, 1980; Verhas et al., 1986) or administration of an antiandrogen (Feldmann et al., 1989) leads to reduced bone mass, as does hypogonadism in the human male (Finkelstein et al., 1989). In the female, androgen excess can cause increased trabecular bone density (Buchanan et al., 1988a) and can maintain normal bone mass even in the presence of undetectable estrogen levels (Dixon et al., 1989). Adrenal androgens have been suggested to have a role in maintaining bone mass in normal postmenopausal women (Devogelaer et al., 1987). In the young, normal woman, the androgens and estrogens appear to function as independent and additive determinants of peak trabecular bone density (Buchanan ef al., 1988b). Some studies have demonstrated a positive correlation between serum testosterone or dehydroepiandrosterone sulfate levels and bone density in aged men (Foresta et al., 1984) and women (Brody et al., 1981; Wild et al., 1987; Deutsch et ul.. 1987; Steinberg et al., 1989), while other studies have not been able to make this association (Meier et a[., 1987). Androgens, like estrogens, interact with their target tissues through a receptormediated mechanism. Indeed, receptors for testosterone have been detected in
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human osteoblastlike cells, with cells from male and female sources containing similar receptor concentrations (Colvard et al., I989a,b). Human fetal epiphyseal chondrocytes have also been shown to contain high affinity binding sites for dihydrotestosterone (Carrascosa et al., 1990). In many tissues that respond to androgens, dihydrotestosterone is considered to be the compound that actually generates the biological response. Whether this androgen is the active metabolite in bone is still an unanswered question. Testosterone can be converted to dihydrotestosterone in bone (Vittek et al., 1974; Schweikert et al., 1980) and in cartilage (Audi et al., 1984), with no differences being observed in osteoporotic bone versus normal bone, or in male verses female. While the possibility exists that the androgens can act in bone by being first converted to estrogen (Frisch et al., 1980), the presence of receptors in bone seem to indicate that at least some of the actions of the androgens are through a direct interaction with the bone cells. Androgen treatment appears to result in an increase in bone formation. Administration of testosterone will produce an increase in skeletal growth both in the experimental animal (Jansson et al., 1983) and in man (Rosenfeld, 1986). In castrated mice, testosterone restores total bone protein in the femoral diaphyses to normal (Broulik et al., 1976). Similarly, direct injection of testosterone into the tibia1 epiphyseal growth plate of castrated rats causes a significant increase in the growth plate width when compared to control (Ren et al., 1989). In vitro, dihydrotestosterone enhances the proliferation of mouse and human osteoblastlike cells and increases the number of cells that produce alkaline phosphatase (Kasperk et al., 1989), although no effect on collagen or DNA synthesis was noted in fetal rat calvaria cultures (Canalis and Raisz, 1978). Using the bone-matrix-induced endochondral bone formation model, androgens markedly elevate the levels of alkaline phosphatase and calcium in the implant and cause an induction of mineralization (Kapur and Reddi, 1989). As is observed for estrogen, fetal rabbit epiphyseal articular chondrocytes respond to testosterone with an increase in proteoglycan synthesis, while chondrocytes from 5-30 day old animals lose their response, which is regained in older animals (Corvol et al., 1987). Testosterone deficiency in males is also associated with increased bone resorption (Stepan et al., 1989). Testosterone and dihydrotestosterone are believed to have no direct effect on resorption in fetal rat long bone in culture (Caputo et al., 1976). Therefore, its effects on inhibiting bone resorption appear to be through PTH and calcitonin. Evidence that supports this concept indicates that testosterone will inhibit the PTH-induced release of calcium from neonatal mouse calvaria (Atkins and Peacock, 1975) and the PTH-stimulated increase in CAMPin human osteosarcoma cells (Fukayama and Tashjian, 1989b). Although testosterone has no effect on the release of calcitonin from rat thyroid C cells in culture (Greenberg et al., 1986), decreased calcitonin levels were found in hypogonadal, osteoporotic young men and testosterone therapy resulted in a
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return to normal values (Foresta et al., 1987). It has also been postulated that an enhanced calcitonin response observed in normal men compared to women may be partially due to the higher testosterone levels found in men (GarciaAmeijeiras et al., 1987). Androgens may also mediate some of their effects on bone through increases in serum levels of IGF-I (Parker et al., 1984; Jasper, 1985; Rosenfeld and Furlanetto, 1985). Part of this increase may be through increased secretion of growth hormone (Parker et al., 1984; Craft and Underwood, 1984; Copeland et al., 1984; Mauras er al., 1987, 1989) which in turn stimulates IGF-I production (Green et a!., 1985; Isaksson er a/., 1985; Nilsson et at., 1986a). However, testosterone can also stimulate growth in the absence of growth hormone (Young et al., 1989), perhaps by directly regulating levels of IGF-I (Wilson, 1986). 6 . Growth Hormone
Before leaving the endocrine factors that regulate skeletal growth and mineralization, the actions of growth hormone on bone should be considered. It has been known for some time that growth hormone is involved in skeletal growth (Raben, 1958). Since many of the effects of growth hormone may be mediated through the IGFs, the interaction of these two factors will be deferred to the discussion of the IGFs. There is evidence, however, that growth hormone may also have direct effects on bone. For example, when continuous intravenous infusion of growth hormone to mutant growth hormone-deficient rats is compared to the infusion of IGF-I, greater bone growth is observed for the growth hormonetreated rats, implying that the two factors may differ in their mechanism of actions (Skottner er al., 1989). While it has been reported that growth hormone may act on the skeleton without changes occurring in the circulating level of IGF-I (Young er al., 1989), this may simply be due to the hormone causing local stimulation of IGF-I, which may not be observable systemically (Green ef al., 1985; Isaksson er al., 1985; Flint and Gardner, 1989). The direct interaction of growth hormone with bone is supported by the detection of specific binding sites for the protein on cultured rabbit chondrocytes (Eden et al.. 1983). Similarly, in young rabbits (20-50 days old), growth hormone receptors were localized on reserve and proliferative chondrocytes in the tibia1 growth plate, while in older animals, where the growth plate was closed. no receptors could be detected (Bernard et al., 1988). Also, treatment of mouse condylar cartilage cells in vitro with growth hormone results in stimulation of cell proliferation, differentiation, and extracellular mineralization (Maor er al., 1989). With fetal mouse calvaria osteoblastlike cells, growth hormone appears to preferentially act on the more differentiated cell resulting in an increase i n the number of osteoblasts (Slootweg er al., 1988). In addition to its interaction with the IGFs, growth hormone secretion may be regulated by several other factors that interact with bone, including retinoids,
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androgens, and estrogens. Since these factors have already been discussed, the reader is referred to those sections in this article that cover these interactions. B. PARACRINE/AUTOCRINEFACTORS 1. lnsulinlike Growth Factors (IGFs)
In addition to the hormonal factors that are produced elsewhere in the body and camed to bone via the circulatory system, there are numerous factors that are now known to be produced in the local bone environment that may play major roles in regulation of bone cell growth and differentiation and, ultimately, the calcification process. Included in this category are the IGFs. Because the IGFs occur in the serum at high concentrations, it was originally thought that the bone was responding to these circulating factors (Salmon and Daughaday, 1957). However, available evidence now seems to indicate that it is probably the locally produced IGFs that are important in stimulating bone growth and mineralization (Skottner et al., 1987). For example, a chronically elevated serum concentration of IGF-I in transgenic mice resulted in an increase in body weight without an apparent effect on skeletal growth (Mathews et a[., 1988). Similarly, infusion of IGF-I into diabetic rats caused no change in bone growth (Carlsson et al., 1989), while infusion into mutant growth hormone-deficient dwarf rats caused smaller changes in bone growth than that observed for growth hormone by itself (Skottner et al., 1989). Studies with hypophysectomized rats are not as clear. Several studies indicate that infusion of IGF-I or IGF-I1 into these animals results in increases in tibial epiphyseal plate width and bone growth similar to that observed with growth hormone (Schoenle et al., 1982; Guler et al., 1988; Shaar et al., 1989) while other studies demonstrate that although infused IGF-I and IGF-I1 are able to increase the width of the tibial epiphysis, growth hormone showed greater changes, again implying that endogenously produced IGFs are more effective than exogenously supplied material (Schoenle et al., 1985). The importance of the IGFs in bone is supported by the occurrence of these factors and their receptors and binding proteins in the bone environment. Skeletal tissues are a rich source of both IGF-I and IGF-I1 (Frolik et al., 1988; Mohan et al., 1988). That these factors are being produced in bone rather than being picked up from the serum is indicated by experiments demonstrating the synthesis of IGF-I in cultured fetal rat calvaria or tibia (Stracke et al., 1984; Canalis et al., 1988a), in embryonic chick pelvic cartilage (Burch et al., 1986) and in osteosarcoma cells (Gray et al., 1989a). Similarly, IGF-I1 is a product of human osteoblastlike cells in culture (Wergedal et al., 1986; Gray et al., 1989a). Using labeled growth factor or immunohistochemical staining techniques, IGF-I has been localized to chondrocytes in the proliferative and hypertrophic zones of porcine (Simon and Cooke, 1988), rat (Nilsson et al., 1986a; Hansson et al.,
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1988), and bovine (Trippel et al., 1986) epiphyseal growth plate cartilage. Activity, however, was not observed in the reserve zone. During rat embryogenesis, the IGF-I1 gene is expressed in chondrocytes and declines substantially prior to ossification (Stylianopoulou et al., 1988). Even though IGF is produced in bone and is present at high concentrations, in order for it to have an effect on bone formation, the cells involved in the actual process need to have the proper receptors. Indeed, specific, high affinity receptors for both IGF-I and IGF-I1 have been detected on osteoblastlike cells from the newborn mouse and embryonic chick calvaria (Mohan et al.. 1989a) and from fetal rat calvaria (Bennett et al., 1984; Cantrell et al., 1989; Centrella et al., 1990). Bovine (Watanabe et al., 1985) and rabbit (Jansen et al., 1989) articular chondrocytes and rat costal chondrocytes (Schalch et al., 1986; Sesseins et al., 1987) also contain IGF-I and I1 receptors. Interestingly, there are 2-6 times more IGF-I receptors on chondrocytes from the bovine growth plate than on articular chondrocytes (Trippel et al., 1983) which is not unanticipated if IGF-I is involved in skeletal growth. In agreement with the immunohistochemical staining experiments, chondrocytes from the growth zone had a higher concentration of IGF-I receptors than those from the resting or hypertrophic zones (Makower et al., 1989a). Insulinlike growth factor-binding proteins, that apparently function to modulate the activity and availability of the IGFs, have been described in a number of tissues (for review see Ooi and Herington, 1988) with bone being no exception. Conditioned medium from rat osteoblastlike cells (Schmid et al., 1989a,b) and from human osteosarcoma cells (Mohan er al.. 1989b). as well as from human bone extracts (Frolik and Black, 1989), contains IGF-binding protein activity that appears to be different from activity found in other tissues. IGF-binding proteins are also secreted by both epiphyseal and articular chondrocytes from rabbit (Froger-Gaillard er al., 1989). chick (Burch et al., 1990), and human (Hill et al., 1989) sources. While the exact function of these binding proteins is still unknown, there is one report that the human amniotic fluid 25-kDa binding protein will inhibit IGF-I stimulated growth of chick embryo pelvic cartilage (Burch et al., 1990). implying that the binding proteins may serve to down regulate the activity of the IGFs. The IGFs have been postulated to have a role in the calcification process directly through increasing bone matrix production by the differentiated osteoblast, and indirectly by increasing the osteoprogenitor cell population and enhancing their differentiation into mature osteoblasts (Guenther et al., 1982; Schmid et al., 1984; Hock et al., 1988b). Thus, IGF-I will stimulate DNA synthesis in rat calvaria (Canalis, 1980; Schmid et al., 1984) and in osteoblastlike cells (Kurose et al., 1989). Similarly, IGF-I1 stimulates DNA synthesis in both rat and chick calvaria cells and in human bone cells (Canalis and Raisz, 1979a; Schmid er al., 1983; Farley e t a / . , 1986). In the osteoblast, the IGFs will stimu-
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late collagen synthesis (Canalis et al., 1977; Canalis, 1980; Linkhart et a/., 1986; Hock el a/., 1988b) by increasing mRNA levels for type I collagen and decreasing collagen degradation (McCarthy et a / . , 1989b; Schmid et al., 1 9 8 9 ~ ) . Insulinlike growth factor-I1 has also been shown to stimulate protein phosphorylation in the chick calvaria (Lau et al., 1988a). As discussed in an earlier section, both of these effects can play a major role in the modulation of the calcification process. In cartilage, besides having a possible role in the in v i m proliferation of the chondroblast (Makower et al., 1989b), the IGFs appear to play a major role in the maintenance of the fully differentiated chondrocyte, especially in regard to the synthesis of proteoglycans (Stevens et al., 1981; Guenther et al., 1982; Hiraki et a/., 1985; McQuillan e t a / . , 1986). As with the collagens, under certain conditions, the IGFs not only increase the synthesis of the proteoglycans, but also decrease their degradation (Tyler, 1989). Under other conditions, they help to maintain an equilibrium between proteoglycan biosynthesis and catabolism (Luyten et al., 1988). The IGF stimulation of proteoglycan synthesis occurs principally in the proliferative zone chondrocyte (Makower et al., 1988; Trippel et al., 1989) resulting in an increase in the size of the proteoglycan monomer (Makower et al., 1988). As already mentioned, the action of growth hormone on bone appears to be intimately intertwined with the IGFs. Treatment with growth hormone in vivo causes increased levels of IGF-I mRNA in rat rib growth plates (Isgaard et a/., 1988a,b), and in rat calvaria (McCarthy et al., 1989a), and of IGF-I protein (Stracke et al., 1984; Orlowski and Chernausek, 1988). The action of growth hormone in stimulation of both long bone growth in the hypophysectomized rat (Russell and Spencer, 1985; Isgaard et a/., 1986) and of isolated osteoblastlike cells in culture is blocked by IGF-I antibodies (Schlechter el al., 1986; Ernst and Froesch, 1988). In vitro, growth hormone stimulates the effects of IGF-I on extracellular matrix synthesis (Smith et al., 1989) and the release of IGF-binding protein (Schmid et al., 1989a,b). A current hypothesis for the mode of action of growth hormone and IGF in cartilage is that growth hormone causes differentiation of a precursor cell (the prechondrocyte)to a mature cell that is more responsive to the IGFs, while the IGFs interact with the cell at a later stage in development causing an increase in number or activity of the differentiated cell (Zezulak and Green, 1982; Nilsson et al., 1986a; Lindahl et a/., 1987). Finally, in perhaps a feedback type control loop, IGF-I has been reported to suppress the expression of the growth hormone gene and the secretion of the protein from the rat pituitary cell (Yamashita et a/., 1987; Namba et al., 1989). Growth hormone is not the only biological factor that controls IGF action. Glucocorticoids have been shown to inhibit the growth hormone induction of IGF-I mRNA levels in bone. This effect has been postulated to be a mechanism of action that can explain the observed growth retardation caused by glucocorti-
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coids (Luo and Murphy, 1989; McCarthy et al., 1990). By themselves, glucocorticoids have been reported to increase the concentration of IGF-I receptors in rat osteoblasttike cells (Bennett et al., 1984) and to inhibit the synthesis of IGF-I (McCarthy et a1.,1990). On the other hand, while glucocorticoids by themselves inhibit collagen synthesis in bone cell populations, they are able to enhance the responsiveness of the cell to the anabolic effects of IGF-I. Therefore, calvaria treated with cortisol and IGF-I showed a marked increase in collagen synthesis compared to cells treated with cortisol alone (Kream et al., 1990). Other factors that increase the concentration of IGF include estrogen (Gray e f al., 1989a; Schmid et al.. 1989a; Emst et al., 1989), 1,25-(OH),-D, (Gray et al., 1989a), PTH (McCarthy et al., 1989a; Linkhart and Mohan, 1989). and P,-microglobulin (Centrella et al., 1989b). Insulinlike growth factor-] has also been postulated to be involved in regulating serum levels of 1,25-(OH),-D, (Gray, 1987; Halloran and Spencer, 1988). Finally, fibroblast growth factor (FGF) and EGF have been reported to act synergistically with IGF-I in the stimulation of DNA synthesis in cultured rabbit costal chondrocytes (Hiraki ef al., 1987). 2 . Transforming Gronith Factors (TGFs) Transforming growth factor P(TGFf.3) is a second growth factor that appears to be intimately involved in modulating the cells that participate in the calcification process. Transforming growth factor f3 exists as a family of proteins (Roberts and Spom, 1988), many of which are found in cells of the skeletal system. From bone, it was originally isolated as cartilage inducing factor A and B (Seyedin et al., 1985) which were later shown to be identical to TGFP-1 (Seyedin et ol., 1986) and TGFP-2 (Seyedin ef ai., 1987). Primary chick embryo chondrocytes have been shown to contain TGFP-3 and -4 (Jakowlew er al.,
1988a,b) while the bone morphogenetic proteins also share sequence identity with the TGFs (Wozney cr al., 1988). The TGFs are thought to be involved in embryonic formation of cartilage and bone, controlling both endochondral and intramembranous ossification (Heine et al., 1987; Pelton et al., 1989).
Employing a model where endochondral bone is generated by implantation of demineralized bone matrix into the muscle of rats, TGFP was localized to the chondrocytes of the calcifying cartilage and to the osteoblasts and mineralized bone matrix. The concentration of TGFP was highest when cartilage was being replaced by bone, again suggesting a role for TGFP in the ossification process (Carrington et al., 1988). That the TGFs are actually being produced in bone has been shown by a number of investigators (Centrella and Canalis, 1985, 1987; Robey et al., 1987; Guenther et al., 1988; Sandberg et al., 1988; Pelton et af., 1989) and specific, high affinity receptors for TGFP have been detected on osteoblastlike cells (Segarini et al., 1987; Robey er al., 1987). In addition to the conventional receptors, TGFP also binds to a high molecular weight proteoglycan component that is found on primary fibroblasts, osteoblasts and chondro-
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blasts but not on epithelial, endothelial or lymphoid-derived cells (Segarini et al., 1989). The function of this binding component is unknown. It does not appear to play a role in any of the known responses to the TGFs. Finally, in other systems, the TGFs have been shown to be produced in a latent form and require activation prior to the generation of their response (Roberts and Sporn, 1988). Similarly, in bone matrix and conditioned medium from calvaria organ cultures, the TGFP is found associated with a high molecular weight glycoprotein that masks the TGFP activity (Jennings and Mohan, 1990; Pfeilschifter ef al., 1990). While the biological mechanism for the activation of this latent TGFP is not clear, in the skeleton, osteoclasts have been reported to be capable of performing this function (Oreffo et al., 1989). The effects of TGFP on bone cell function and mineralization seem to depend on the species being studied, the state of differentiation of the target cell, and the experimental conditions employed. In vivo, TGFP is able to increase periosteal woven bone formation in rats (Noda and Camilliere, 1989). In vitro, it is able to both stimulate (Centrella et al., 1986; Datta et al., 1989; Jennings et al., 1988; Hock et al., 1990) and inhibit osteoblast cell proliferation (Noda and Rodan, 1986, 1987; Elford et al., 1987; Pfeilschifter et al., 1987; Guenther et al., 1988). It is also able to regulate extracellular matrix production. TGFP stimulates expression of the type I collagen gene (Centrella et al., 1987a; Noda and Rodan, 1987), possibly acting through a nuclear factor binding site in the a2(I) collagen promoter (Rossi et al., 1988), as well as the osteonectin (Noda and Rodan, 1987) and osteopontin (Noda et al., 1988b) genes. The response of alkaline phosphatase to TGFP varies, with the activity being stimulated in ROS17/2.8 cells (Noda and Rodan, 1987; Pfeilschifter et al., 1987) and inhibited in MC3T3-El and primary rat osteoblastlike cells (Noda and Rodan, 1986; Elford et al., 1987; Rosen el al., 1988; Wrana et al., 1988; Ibbotson et al., 1989). Finally, the expression of the fibronectin and the osteocalcin genes are decreased by TGFP (Noda and Rodan, 1987; Noda, 1989). These effects on cell function are independent of the effects of TGFP on cell proliferation (Centrella et al., 1987a; Wrana et al., 1988; Ibbotson et al., 1989). In cultured fetal rat calvaria, using histomorphometry and autoradiography, TGFP was shown to have mitogenic effects on all cell zones of the pericranial periosteum with an increase in the number of osteoblasts and a decrease in the number of osteoclasts. The study concluded that the effects of TGFP on matrix protein synthesis was partly due to increased cell proliferation and partly to increases in bone cell differentiation and function (Hock et al., 1990). TGFP also changes the morphological appearance of the cell (Noda and Rodan, 1986; Pfeilschifter et al., 1987; Rosen et al., 1988). This modified behavior of the cell may be due to a change in binding specificity of the integrins that are expressed by the cell after TGFP treatment (Heino and Massague, 1989).
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While TGFP is able to act on the osteoblastlike cell, a significant amount of evidence exists to indicate that it also has a role in cartilage. TGFP mRNA has been shown to be expressed in cartilage during murine embryogenesis (Pelton et al., 1989) with the highest concentration occurring in the growth plate (Sandberg et al., 1988). Immunohistochemical staining techniques support the involvement of TGFP- 1 in cartilage, detecting the factor intracellularly in the chondrocyte and extracellularly in the cartilage matrix (Thompson et al., 1989). TGFP was initially isolated from bone based on its ability to cause differentiation of the neonatal rat muscle mesenchymal cell into chondrocytes and to stimulate production of cartilage-specific proteoglycans and type I1 collagen (Seyedin er d., 1985). In addition to the stimulation of proteoglycan synthesis in cartilage (O'Keefe er al., 1988a; Redini et al., 1988), TGFP inhibits both the degradation of newly synthesized proteoglycans (Morales and Roberts, 1988) and the IL-1stimulated release of proteoglycans (Andrews et al., 1989). Finally, TGFP is able to stimulate DNA synthesis in chick epiphyseal chondrocytes (O'Keefe et al., 1988b). However, TGFP also can inhibit the phenotypic expression of cultured chondroblasts, resulting in a decrease in the production of type I1 collagen and cartilage proteoglycan (Rosen ef a!., 1988) and an inhibition of in vitro cartilage-induced mineralization (Kato et al., 1988). In addition to its effects on bone and cartilage formation, TGFP also has effects on bone resorption. In neonatal mouse calvaria in culture, TGFP stimulates resorption, perhaps through a PGE,-related mechanism (Tashjian et al., 1985). Transforming growth factor-p is able to stimulate the synthesis and release of PGE, from MC3T3-El cells (Sumitani ef a!., 1989) and from fetal rat calvaria (Centrella ef a/., 1986). On the other hand, in cultured fetal rat long bone, TGFP causes a decrease in resorption (Pfeilschifterer af., 1988). This effect of TGFP may be due to inhibition of the proliferation of the osteoclast precursor (Chenu er a/., 1988). Transforming growth factor-P may also have effects on connective tissue degradation. It has been reported to inhibit the IL-I-stimulated production of neutral protease activity by rabbit articular chondrocytes (Chandrasekhar and Harvey, 1988) and to stimulate the synthesis of the collagenase inhibitor tissue inhibitor of metalloprotease (TIMP) and of PAI- I , a plasminogen activator inhibitor, while decreasing collagenase production by normal fetal rat calvaria bone cells thereby suppressing matrix degradation and removal (Overall er al., 1989). The role of TGFP in the regulation of bone growth and calcification may be modified by the action of other agents (Centrella et al., 1987b). Factors that will increase TGFP activity in bone include FGF (Noda and Vogel, 1989), 1,25(OH),-D, (Petkovich er al., 19871, IL-1 (Pfeilschifter and Mundy, 1987), and estrogen (Komm el al.. 1988; Gray et al., 1989b) while calcitonin will decrease TGFP activity (Pfeilschifter and Mundy, 1987). Parathyroid hormone has been reported to increase TGFP activity in the mouse or rat calvaria (Pfeilschifter and Mundy, 1987) but to decrease it in a rat osteosarcoma cell line (Noda and Vogel,
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1989). These factors not only modify the levels of TGFP, but can also regulate its effects. For example, FTH will enhance the binding of TGFP to fetal rat calvaria osteoblastlike cells in culture and decrease the response of the cells to TGFP (Centrella et al., 1988). In contrast, in the same cells, PTH-related peptide has been reported to enhance the effects of TGFP on DNA and collagen synthesis (Centrella et al., 1989a). In rabbit articular chondrocytes grown in soft agar, TGFP has no effect on DNA synthesis unless grown in the presence of EGF (Skantze et al., 1985). While some factors are able to control the activity of TGFP, the converse is true for other factors whose actions in bone are modulated by TGFP. For example, TGFP potentiates the effects of FGF on bone cell proliferation (Hiraki et al., 1988; Globus et al., 1988; Iwamoto et al., 1989). Similarly, chondrocytes, grown in the presence of TGFP and FGF, will express the proteoglycans specific for a differentiated chondrocyte (Inoue et al., 1989). In rat long bone, resorption stimulated by IL-1 or 1,25-(OH),-D3 is inhibited by TGFP (Pfeilschifter et af., 1988). Other effects of TGFP on IL-1 activity have already been described. Interestingly, while short term treatment of MC3T3-E1 cells with TGFP causes an increase in the number of EGF receptors and decreases the rate of DNA synthesis, longer term treatment decreases the number of receptors with an increase in DNA synthesis (Uneno et al., 1989). It should be apparent, from this discussion, that the effects of TGFP on bone involve a complicated interaction with a wide range of other bone regulatory molecules and that the observed response depends on the differentiated state of the bone cell at the time the factor is in the local environment of that cell.
3. Bone tnductive Proteins Another group of factors, the bone inductive proteins, appear to be related to the TGFs in structure but not necessarily in function. They are postulated to be responsible for the differentiation of mesenchymal cells into osteoprogenitor cells, while other factors are involved in the proliferation of these cells (Urist et al., 1983). These proteins were originally discovered, and have been characterized, using the ectopic bone formation assay where extracts from demineralized bone induce new bone when implanted at an ectopic site (Urist, 1965). The sequence of events that occurs during this process are:.(l) the migration and attachment of mesenchymal cells into the implant and their proliferation; (2) by days 5-7, the appearance of chondroblasts and formation of a cartilaginous template: (3) at days 10-14, hypertrophy of the cartilage and vascularization of the extracellular matrix; and (4) removal of the cartilage and replacement with bone, complete with marrow, by day 21 (Reddi and Huggins, 1972; Firschein and Urist, 1972; Reddi, 1981). There are a number of proteins that appear to initiate some of, or all, of these events. Bone inductive proteins that have been purified and at least partially sequenced include the bone morphogenetic proteins (BMPs), the osteoinductive factor (OIF), and osteogenin.
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Bone morphogenetic protein, purified from bovine bone matrix, is an acidic protein of approximately 18.5 kDa with a pl of 4.9-5.1 (Urist et al., 1984; Bessho et al., 1989). Besides being found in bone matrix, it also occurs in dentine, enamel, and cementum (Kawai and Urist, 1989), as well as in different osteosarcomas (Hanamura et al., 1980; Bauer and Urist, 1981; Takaoka et al., 1989). Three different human BMPs have been cloned and complete amino acid sequence obtained (Wozney et al., 1988). Two of the proteins, BMP-2A and BMP-3. are members of the TGFP family, with 34 to 38% sequence homology to TGFP-1 and TGFP-2, while the third, BMP-1, is a unique protein. Initial reports indicated that these proteins, by themselves, will form cartilage when placed ectopically, but the cartilage will not be replaced by bone. However, more recent reports indicate that purified recombinant human BMP-2A is able to induce bone formation by itself in the rat ectopic bone formation assay (Wang et al., 1990). Although TGFP is related to these proteins, subcutaneous injection of TGFP by itself results only in formation of granulation tissue with no appearance of cartilage or bone (Roberts et al., 1986). A fourth member of this group, OIF, has also been isolated from bovine bone and has been shown to be a glycosylated protein of 22-28 kDa with a protein backbone of 12 kDa (Bentz et al., 1989). Although purified OIF is able to produce ectopic bone, its activity is substantially increased by the coadministration of TGFP-1 or TGFP-2. The complete OIF amino acid sequence shows no homology with other reported proteins, including those with osteoinductive activity (Bentz et al., 1990). Finally, osteogenin has also been found in bovine bone matrix (Luyten ef al., 1989) and in rat tooth (Katz and Reddi, 1988). It is a 22-kDa protein whose partial amino acid sequence shows homology to BMP-3 (Luyten et al., 1989). However, unlike BMP-3, it is able to initiate both cartilage and bone formation in vivo and its activity is enhanced by type 1 collagen (Muthukumaran et al.. 1988). In vitro, osteogenin is able to stimulate the expression of the osteogenic phenotype in periosteal cells and osteoblasts as measured by increased alkaline phosphatase activity, collagen synthesis, and PTH-stimulated adenyl cyclase activity, and of the chondrogenic phenotype of chondroblasts as measured by increased production of sulfated proteoglycans. The effects seem to be somewhat specific, as NIH 3T3 fibroblasts do not respond to the factor (Vukicevic et al., 1989). In addition to possible interactions with TGFP, some of the bone inductive proteins may be dependent on vitamin D, since it has been reported that bone matrix from vitamin D-deficient rats have reduced levels of osteogenin (Sampath et al., 1984). The ectopic bone forming activity of osteogenin is enhanced by platelet-derived growth factor (PDGF) while EGF, FGF, and insulin have no effect (Howes ef al., 1988). A role of IL-1 in ectopic bone formation, and perhaps fracture healing, has also been suggested by experiments that show greater amounts of heterotopic ossification induced by BMP in the presence of this cytokine (Mahy and Urist, 1988). Also antiinterleukin antibodies are able to
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abolish the activity of BMP (Mahy and Urist, 1988). Another cytokine, tumor necrosis factor-a, is able to inhibit ectopic bone formation, presumably by preventing mesenchymal cells from differentiating into chondrocytes (Yoshikawa et al., 1988). It would therefore appear that these bone inductive factors may be important for the initial phases of differentiation, after which other factors are necessary to carry out the entire osteogenic process to the final conclusion of the generation of mature bone. 4. Epidermal Growth Factor (EGF)
EGF is another growth factor shown to affect the skeletal system and mineralization. Specific, high affinity receptors for EGF have been found on human osteosarcoma cells (Shupnik and Tashjian, 1981, 1982) and on newborn murine calvaria cells (Shupnik et al., 1980; Ng et al., 1983). These EGF receptors were localized to preosteoclastlike cells, that were found in close association with osteoclasts in the regions of bone or cartilage where matrix was being actively resorbed, and to preosteoblastlike cells in the metaphysis. Mature osteoblasts and osteoclasts themselves were not labeled. (Martineau-Doizeet al., 1988). The number of EGF receptors are modulated by TGFP (Uneno et af., 1989), PDGF (Shupnik et al., 1982), PTH (Ohta et al., 1989), and by agents that increase protein kinase C activity (Borst and Catherwood, 1989). One of the possible roles for EGF in bone is to increase the osteoblast/osteoclastprecursor pool. Evidence in support of this role demonstrates that EGF receptors occur on a population of immature cells that were sequentially released from the proliferative zone of newborn mouse calvaria by enzymatic digestion (VandePol et al., 1989). These same cells also responded to EGF by an increase in proliferation. As observed for TGFP, the response to EGF depends on the conditions of the experiment. Epidermal growth factor has been reported to either have no effect (Shupnik et al., 1980; Shupnik and Tashjian, 1981) or to stimulate (Canalis and Raisz, 1979b; Hiramatsu et al., 1982; Ng et al., 1983; Partridge et af., 1985) DNA synthesis in different in vitro bone systems. In addition to its possible effects on DNA synthesis, EGF is able to stimulate cartilage proteoglycan synthesis (Makower et al., 1989b; Osborn et al., 1989), inhibit type I collagen synthesis in MC3T3-El cells (Hiramatsu et al., 1982; Hata et af., 1984) and in fetal rat calvaria (Canalis and Raisz, 1979b; Canalis, 1983), and inhibit alkaline phosphatase activity (Canalis, 1983; Ibbotson et al., 1986). It also has a differential effect on prostaglandin synthesis, causing stimulation (Shupnik and Tashjian, 1981, 1982; Feyen et al., 1984), no effect (Shupnik and Tashjian, 1982), or an inhibition (Partridge et al., 1985). In general, EGF may have a function in bone formation partly through increasing the number of early preosteoblasts and, at the same time, blocking the more differentiated function of the later preosteoblastlike cell and/or the mature osteoblast. This hypothesis is supported by experiments with the UMR-106 cell where EGF and TGFa block
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a differentiated function of the osteoblastlike cell, that of the PTH induction of adenyl cyclase activity (Gutiemez et al., 1987). A dual role in controlling the process of bone mineralization is emphasized in experiments with fetal rat calvaria cells grown under conditions where bone nodules are able to form. In this situation, EGF can inhibit or stimulate nodule formation, depending upon the time and duration of exposure to the growth factor (Antosz et a1.,1989). This has also been shown in in vivo experiments where short-term administration of physiological levels of EGF causes a transient reduction of endosteal matrix apposition and mineralization, while longer term treatment resulted in an increase in periosteal bone formation (Marie et al., 1990). Again, the effects of EGF on bone resorption are dependent on the system being btudied. Epidermal growth factor and TGFa (a member of the EGF family; for review see Gill et al., 1987)are able to raise the systemic plasma calcium levels in intact mice (Tashjian et al., 1986). In vifro, in neonatal mouse calvaria, these factors stimulate bone resorption (Tashjian and Levine, 1978). most likely through a PGE2-mediatedmechanism (Stem et al., 1985; Ibbotson er af., 1986 Tashjian et al., 1988). However, in fetal rat long bone, while EGF and TGFa are still able to increase bone resorption (Raisz et al., 1980; Ibbotson et al., 1983, 1985, 1986), it does not appear to be through the prostaglandin pathway (Raisz et a[., 1980; Stem et ul., 1985), but rather through a mechanism that results in an increase in the number of osteoclasts (Takahashi et al., 1986). Interestingly, the response in fetal rat long bone could be made prostaglandin-dependent by blocking DNA synthesis (Lorenzo et a/., 1986), or by treatment with IL-1 (Lorenzo et at., 1988). Interleukin-1 also acts synergistically with TGFa in fetal rat calvaria to increase prostaglandin synthesis and inhibit collagen synthesis (Hurley et al., 1989). 5. Fibroblast Gniwrh Factor (FGFI
Fibroblast growth factor (FGF) has been postulated to play a role in bone formation by increasing the number of osteoprogenitor cells that are then available for differentiation to mature bone forming cells capable of synthesizing matrix proteins (Canalis et al., 1987, 1988b). Fibroblast growth factor is able to stimulate the formation of bonelike mineralized nodules in an in vitro bone marrow cell system, presumably through enhanced proliferation and differentiation of an osteoprogenitor cell population (Noff et al., 1989). Factors that have characteristics similar to FGF have been found in bone extracts (Hauschka et al., 1986) and both acidic FGF and basic FGF are produced by cultured fetal bone cells ( G l o b u s et al., 1989). Because t h e FGFs are able to bind to heparin (Gospodarowicz et al., 1984), and since the osteoblasts produce heparin sulfate proteoglycans (Beresford et ul., 1987), it is thought that, once produced, the FGFs bind to the extracellular mamx proteoglycans (Globus et af., 1989). They could then be released and activated by heparinaselike enzymes produced by cells in the immediate environment (Baird and Ling, 1987).
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In addition to their proposed role in the stimulation of precursor cell division, these factors have been shown, in vitro, to increase proliferation in rat osteoblastlike cells (Canalis and Raisz, 1980; Rodan et al., 1987; Globus et al., 1988; Noda and Vogel, 1989; Shen et al., 1989), being more mitogenic for bone cell populations with fewer osteoblastlike characteristics (McCarthy et al., 19894. It also decreases the expression of the osteoblast phenotype, causing diminished levels of alkaline phosphatase, type I collagen, osteocalcin, and PTH responsive-adenyl cyclase activity (Canalis and Raisz, 1980; S . B. Rodan et al., 1989; Noda and Vogel, 1989; McCarthy et al., 1989c; Shen er al., 1989), although it enhances osteopontin ( S . B. Rodan et al., 1989) and, in bovine bone cells, osteocalcin (Globus er al., 1988). In cartilage, FGF stimulates proliferation of chondrocytes in vitro (Kato and Gospodarowicz, 1985) and in vivo (Cuevas e f al., 1988), resulting in increases in the formation of extracellular matrix. In addition to its effects on cell division, FGF is also able to inhibit the terminal differentiation of rabbit growth-plate chondrocytes to hypertrophic cells which results in a decline in cartilage calcification (Kato and Iwamoto, 1990). Part of the effects of FGF on bone may be through enhancement of expression of the TGFP gene (Noda and Vogel, 1989) which, in turn, is able to potentiate the mitogenic effects of FGF (Globus er al., 1988). Insulinlike growth factor-I also is able to enhance the mitogenic effect of FGF on osteoblastlike cells (Rodan et al., 1987). Finally, there is a report that FGF is able to cause a prostaglandin-dependent stimulation of bone resorption in fetal rat long bones (Shen et al., 1989) although the physiological importance of this observation is still to be determined. 6 . Platelet-derived Growth Factor (PDGF)
Although PDGF, or closely related proteins, have been isolated from bone matrix (Hauschka et al., 1986), the role of PDGF in bone has not been extensively studied. While PDGF was not detected in the conditioned medium of normal fetal rat bone cultures (Centrella and Canalis, 1985), it has been shown to be synthesized by human osteosarcoma cells (Heldin et a1.,1986), and by normal human adult bone-derived cells (Graves et al., 1989). Platelet-derived growth factor receptors have been detected on osteoblasts (Centrella et al., 1 9 8 9 ~ ) which respond to the factor with increases in DNA synthesis (Canalis, 1981; Graves et al., 1989; Centrella et al., 1989c; Canalis et a[., 1989b) and general protein synthesis (Centrella et al., 1989c; Canalis et al., 1989b). In addition to its effects on osteoblasts, PDGF also stimulates collagen degradation and bone resorption (Tashjian et al., 1982; Canalis er al., 1989b), perhaps due partly to an increase in prostaglandin synthesis (Tashjian et al., 1982; Habenicht et al., 1985), and partly to an increase in collagenase production (Bauer er al., 1985). These effects of PDGF on collagen degradation are prevented by IGF-I, while the two factors show additive effects on DNA synthesis (Canalis et al., 1989b).
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7. Cytokines The role of the cytokines in bone formation and resorption is just now becoming appreciated. Interleukin- 1 is known to stimulate the proliferation of osteoblastlike cells (Rifas et al., 1984; Gowen el al., 1985a,b; Canalis, 1986; Smith et al., 1987). At the same time, it may also inhibit the differentiation of bone- or cartilage-forming cells, as suggested by decreases in the synthesis of type I collagen (Gallagher et al., 1985; Canalis, 1986; Smith et al., 1987), and proteoglycan (Tyler, 1985), as well as osteocalcin and alkaline phosphatase (Gallagher et al., 1985). A decrease in functional matrix-producing cells predicts that the calcification process would be diminished. Indeed IL-1 has been reported to prevent the formation of mineralized bone nodules by fetal rat osteoblasts in vitm (Stashenko et al., 1987). However, one group has reported an association between an increase in IL-I production by circulating monocytes and bone formation rates (Pacifici et al., 1987). In addition to its possible role in bone formation, IL-1 also stimulates bone resorption in vitro (Gowen et al., 1983; Thomson et af., 1986). Indeed, human osteoclast activating factor has been shown to be identical to IL-lp (Dewhirst et a/., 1985). This effect of IL-I on resorption is apparently mediated through the osteoblast (Thompson et al., 1986). While IL-I induces production of prostaglandins (Sato et al., 1986a,b; Bandara et a1.,1989), its effects on resorption are not caused only through this increase in prostaglandin synthesis. This is evidenced by experiments demonstrating that stimulation of resorption is only partially blocked (Sato et d., 1986a) or unaffected (Gowen and Mundy, 1986) by indomethacin. Interleukin- 1 may also stimulate resorption through activation of matrix degrading enzymes (Krakauer et al., 1985) such as neutral proteinases (Bandara et al., 19891, collagenase (Lin et al., 19881, and phospholipase A2 (Lyons-Giordano et al., 1989) and/or through the enhanced production of macrophage-colony stimulating factor (M-CSF), which may then potentiate resorption through the recruitment of osteoclasts (Sato et al., 1986b). The factor does not appear to act on the osteoblast through a calcium-mediated pathway (Pacifici ec al., 1988). Interleukin-1 will also stimulate resorption in vivo (Sabatini et al., 1988). When administered subcutaneously directly over the calvaria, the early effects of IL-I, which were independent of prostaglandin synthesis, resulted in increased bone resorption while the longer term effects, that appeared to be dependent on prostaglandin production, showed osteoclast-mediated resorption with the resorbed bone replaced by increased amounts of new bone (Boyce et al., 1989). These effects of IL-1 on resorption could also be observed when the factor was membrane-bound, implying that the resorption that occurs in inflammatory diseases such as rheumatoid arthritis and peridontal disease could be due to macrophage membrane-bound IL- 1 (Sabatini et al., 1988). As with the other factors described, IL-I interacts with a number of other regulators of bone metabolism. Several factors have been shown to modulate
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the effects of IL-1 in bone. For example, TGFP opposes the actions of IL-1 on enhanced proteoglycan synthesis and degradation in cartilage and thus may have a protective effect in inflammatory diseases (Andrews et al., 1989). On the other hand, FGF will enhance the IL-1-stimulated production of neutral proteases by chondrocytes (Phadke, 1987) perhaps through an increase in the number of IL-1 receptors in these cells (Chandrasekhar and Harvey, 1989). Finally, 1,25-(OH),-D, will increase the production of IL-1 in macrophage cells. It has therefore been postulated that some of the effects of vitamin D on bone may actually be mediated through the immune system (Hodler et al., 1985). The actions of other factors in bone are, in turn, modified by IL- 1. For example, the resorptive response to TGFa or to EGF is enhanced by IL-1 while it inhibits the mitogenic response to these factors (Lorenzo et af.,1988). Finally, IL-1 acts synergistically with TGFa in fetal rat calvaria to increase prostaglandin synthesis and inhibit collagen synthesis (Hurley et al., 1989), and with PTHrP to stimulate bone resorption both in virro and in vivo (Sato et al., 1989). As indicated earlier, the interaction of IL-1 with estrogen is not clear. In one report, treatment of postmenopausal women with estrogen did not affect the spontaneous release of IL-1 from the peripheral monocytes and it was concluded that the action of estrogen on bone resorption was not mediated by effects on IL-1 production (Stock et al., 1989b). In a second report, estrogen administration caused a significant decrease in IL-1 activity in blood from postmenopausal women, and the opposite conclusion was reached; the inhibition of bone loss by estrogen may, at least in part, be mediated through a decrease in IL-1 (Pacifici er af.,1989). Additional experiments are needed to determine the role of IL-1 in the response to estrogen. Other cytokines are also able to regulate osteoblast activity and, therefore, calcification. GM-CSF is produced by neonatal mouse calvaria and by calvarial osteoblastlike cells (Felix et al., 1988). This production is induced by PTH (Horowitz et al., 1989a,b; Weir er af., 1989). Because GM-CSF is able to induce baboon bone marrow cultures to produce cells with osteoclastlike features (MacDonald et al., 1986b), it has been postulated to be the factor, produced by osteoblasts in response to PTH, that in turn stimulates the osteoclast (Weir et al., 1989). However, in fetal mouse long bone cultures, GM-CSF decreases the development of osteoclasts (Lorenzo et al., 1987). The reasons for the difference in these two systems still needs to be investigated. In addition to its proposed role on osteoclast development, GM-CSF is able to modulate human trabecular osteoblastlike cells, stimulating their proliferation, but antagonizing their differentiated function (Evans et al., 1989). Similar to GM-CSF, IL-3 is able to induce the formation of osteoclastlike cells in bone marrow cultures (Barton and Mayer, 1989). The response to both of these factors is suppressed by TGFP (Sing et al., 1988). Finally, tumor necrosis factor (TNF) is able to stimulate bone resorption in nude mice (Johnson er al., 1989), perhaps through the stimulation of prostaglandin (Tashjian et af., 1987; Gowen et af., 1988) or elastase (Redini et
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al., 1988) synthesis. Tumor necrosis factor has also been reported to inhibit chondrocyte differentiation in the process of ectopic bone formation in mice (Yoshikawa et al., 1988). In vifro,TNF is able to stimulate bone resorption in cultures of fetal rat long bones and to inhibit collagen synthesis in calvaria (Bertolini et a/.. 1986), and proteoglycan synthesis in cartilage explants (Saklatvala, 1986). Like several other factors that stimulate bone resorption, the effects of TNF on the osteoclast appear to be mediated through the osteoblast (Thomson et al.. 1987). Besides stimulating the osteoblast to produce an osteoclast activating factor, TNF is also able to increase bone cell proliferation resulting in an increased number of osteoblastlike cells, although, at the same time, decreasing their differentiated phenotype (Canalis, 1987; Gowen et a/., 1988). Osteoblasts are not only able to respond to TNF but also synthesize this factor in response to IL- 1 or GM-CSF stimulation (Gowen et al., 1990). The role of the immune system and its cytokines in normal bone formation and resorption still needs further experimentation in order to be defined. However, it is not too difficult to expand the effects observed to date and postulate the involvement of these factors in various inflammatory diseases where increased bone resorption occurs.
iV. Pharmacological Regulators of Calcification In this section a structurally diverse group of compounds will be discussed that can be loosely classified as pharmacological regulators of calcification. Using either in vitro or in viva models, these agents have been shown to affect some facet of bone metabolism. In a few cases, the mechanism of action for a compound in bone has been evaluated experimentally. In other examples, the mode of action i s surmised based on the agent’s known biochemical mechanism i n different biological systems. In many instances, however, agents can be shown to act on bone, but t h e biochemical mechanism is unknown. Pharmacological agents which affect bone metabolism may prove not only to be useful therapeutically, but may also be valuable tools in elucidating the details of the calcification process. This survey of pharmacological effectors of calcification in skeletal tissues is not meant to be exhaustive, but rather to provide the reader with a sampling of active agents. The inclusion of particular agents in specific categories (e.g., enzyme inhibitors) is primarily for the convenience of discussion. The reader should recognize that many of these pharmacological agents have multiple sites of action in biological systems. The category in which agents are included may represent only one of the mechanisms by which they can modulate the calcification process.
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A. ENZYME INHIBITORS
I. Carbonic Anhydrase Inhibitors As discussed earlier in this article, carbonic anhydrase I1 plays an important role in osteoclastic bone resorption. Several inhibitors of this enzyme [(acetazolamide, ethoxzolamide, 5-(3-hydroxybenzoyl)2-thiophenesulfonamide (HTS)] have been used to block bone resorption either in vitro or in vivo (e.g., Kenny, 1985; Raisz et al., 1988). One difficulty with the therapeutic use of carbonic anhydrase inhibitors is associated with the wide spread involvement of the bone isozyme in the physiology of other tissues (Pierce and Waite, 1987; Raisz et a[., 1988). An approach to overcoming the problem of selectivity has been to target an inhibitor of the enzyme, e.g., acetazolamide, to bone, by coupling it to a bone-seeking agent such as tetracycline (Pierce and Waite, 1987).
2. Collagenase Inhibitors Collagenases of osteoblastic origin are believed to be important in the process of bone remodeling (e.g., Kahn and Partridge, 1987). Natural inhibitors of collagenases have been characterized from several skeletal tissues (Sellers and Reynolds, 1977; Sellers et al., 1980; Sakamoto et al., 1981; Cawston et al., 1981, 1983; Nagayama et aZ., 1984). However, the role of these enzymes in osteoclastic-mediated bone resorption is not well defined, and collagen breakdown in the acidic resorption lacuna may be mediated through a cysteine proteinase or other lysosomal enzymes, rather than collagenases (Delaisse et al., 1987). C1- 1 [N-(3-N-benzyloxycarbonyIamino- 1-R-carboxypropy1)-L-leucyl-0methyl-L-tyrosine N-methylamide] is reported to be a specific and potent inhibitor of tissue collagenases (Delaisse et af., 1985). In cultured calvaria, a complex system in which both osteoblasts and osteoclasts play a role in the resorption process, C1-1 inhibited PTH-induced resorption of bone matrix (Delaisse et al., 1985). It was, however, ineffective in blocking resorption by isolated chick osteoclasts cultured on a dentin surface, whereas inhibitors of cysteine proteinases were effective inhibitors in this model (Delaisse et al., 1987). 3. Phosphodiesterase Inhibitors
A number of important physiological regulators of bone metabolism, including PTH, calcitonin, and prostaglandins, utilize cAMP as an intracellular second messenger. The intracellular concentration of cAMP is dependent both on the rate of formation via adenyl cyclase and on the rate of degradation via phosphodiesterase (PDE). It is therefore not surprising that PDE inhibitors affect bone metabolism in a complex manner. The PDE inhibitor, theophyllin, retards the fall in serum calcium that follows acute parathyroidectomy in rats, and causes
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elevation of serum calcium in rats parathyroidectomized for 4 days (Wells and Lloyd, 1967), thus implicating PDE in the mechanism of action of PTH and the regulation of blood calcium. In short-term organ culture (less than 24 hr), the PDE inhibitors, theophylline and 3-isobutyl-methylxanthine(IBMX) can inhibit mineral and lysosomal enzyme release from unstimulated or PTH-stimulated mouse calvaria (Lerner, 1980), whereas the nonxanthine PDE inhibitors rolipram (an imidazole) or Ro 20-1724 (a pyrrolidone) did not reduce PTH-stimulated mineral release (Lerner et al., 1986). In longer term cultures (24-120 hr), PDE inhibitors of several types (e.g., xanthine, aminophyllin, theophylline, IBMX, and the two nonxanthine PDE inhibitors, Ro 20-1724 and rolipram) will stimulate the release of calcium, phosphate, and lysosomal enzymes from cultured calvaria (Herrman-Erlee and van der Meer, 1974; h e y et al., 1976; Lemer et al., 1986). This effect is not observed if endogenous prostaglandin production is inhibited, implying that PDE inhibition of resorption is only effective in the presence of stimulators of adenylate cyclase (Ransjo et al., 1988). Chambers and Ali (1983) reported that theophyllin and IBMX increased the activity of prostaglandins and calcitonin in inhibiting the motility of isolated osteoclasts. Similarly, Allan et al., (1986) indicated that IBMX enhanced the activity of PTH, prostaglandins, and calcitonin in stimulating tissue-type plasminogen-activator activity in different strains of the UMR osteosarcoma cell line. Finally, Robin and Ambrus (1982) evaluated two imidazoquinazolinone PDE inhibitors and found that they were able to prevent heparin-induced osteoporosis. The cardiotonic agents amrinone and milrinone have positive ionotropic and vasodilatory effects. Their mechanism of action in the heart may be related to PDE inhibitory effects on alterations in calcium balance (Schneeweiss, 1986). In this regard, amrinone will stimulate bone resorption when given alone, and inhibit bone resorption induced in cultured bone by PTH, 1,25-(OH),-D,, and PGE, (Krieger and Stern, 1982). More recently, the cardiotonic agent milrinone has also been shown to stimulate bone resorption in neonate mouse calvaria organ culture and, at the same time, to inhibit collagen synthesis (Krieger et al., 1987, 1988). Milrinone, but not amrinone, has structural homology to thyroxine (T4) (Davis et al., 1987), which is a well documented agent that stimulates bone turnover both in vivo and in vitro. In addition, thyroid hormones have been shown to have PDE-inhibitory effects (Marcus, 1975). However, milrinone is not effective in competing for thyroid hormone binding sites in bone cultures, thus suggesting that this is not its probable mechanism of action in bone (Krieger et al., 1988). 4 . Prostaglandin Synthesis Inhibitors
Prostaglandins have diverse effects on bone. Their production by osteoblasts in response to a variety of bone-stimulating agents indicates that they may play a key role in mediating the activity of several different physiological regulators of
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bone metabolism. Agents which interfere with prostaglandin synthesis have been used both to discern the mechanism of prostaglandin action on bone, and to evaluate how they might be used in treating diseases of the skeletal system. The effects of prostaglandins, and of the compounds that inhibit their synthesis on bone metabolism are complex. The most consistent observations show that the nonsteroidal antiinflammatory drugs (NSAIDS) that inhibit the cyclooxygenase pathway of prostaglandin synthesis will also inhibit prostaglandin-mediated bone resorption both in v i m (Sandberg et al., 1977; Tashjian and Levine, 1978; Katz et al., 1983) and in vivo (Williams et al., 1985, 1988; Jee et al., 1988; Thompson and Rodan, 1988; Li et al., 1989; Hayward et af., 1989). In addition, cyclooxygenase inhibitors have been shown to modulate osteoblast function with both inhibition and stimulation of DNA synthesis and alkaline phosphatase being reported in v i m (Khokher and Dandona, 1988; Fujimori et al., 1989), and bone formation in vivo (Jee et af., 1988; Li el al., 1989). Nonsteroidal antiinflammatory drugs have been used to prevent the induction of ectopic bone in animal models (Nilsson et af., 1986b). They have also been employed therapeutically to reduce ectopic bone formation following orthopedic surgery (Ritter and Sieber, 1985; Elmstedt et al., 1985), and to diminish alveolar bone loss associated with periodontal disease (Williams er al., 1989). AND ANTAGONISTS B. ESTROGEN AGONISTS
Significant interest has recently been focused on the modulation of bone metabolism by a group of compounds that elicit mixed agonist/antagonist actions on estrogen target tissues (e.g., tamoxifen, clomiphene), and are commonly referred to as antiestrogens (Jordan, 1984). This interest in regard to the biology of bone has been stimulated by recent reports that these compounds may mimic the activity of estrogen in preventing the development of osteopenia in ovariectomized rats (Beall et al., 1984; Jordan et af., 1987; Turner et al., 1987b, 1988), and neurectomized rats (Wakley et al., 1988), and may inhibit bone resorption in v i m (Stewart and Stem, 1986). Feldmann and co-workers (1989), on the other hand, found that antiestrogens caused osteopenic changes in intact rats which were similar to those associated with surgical castration, though one of the compounds tested, tamoxifen, did show some indication of agonist effects at the highest dose when given to ovariectomized rats. Kusuhara and Ishida (1986) evaluated the effect of tamoxifen on capons in which medullary bone had been induced by treatment with estrogens and androgens for 40 days. They found that tamoxifen treatment during the last 10 days of this period appeared to antagonize the effects of the estrogenlandrogen treatment and prevented osteoblast formation and increased osteoclast number. Turken et al. (1989) have reported, in preliminary studies evaluating the therapeutic potential of this class of compounds, that in women being treated with tamox-
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ifen for breast cancer, the compound may reduce bone loss associated with menopause. C. IMMUNE MODULATORS Numerous reports in recent years have demonstrated that the immune/hemopoietic system is closely associated with the generation of osteoclasts, and that cytokine-mediated responses can be observed in both bone-forming and resorbing models. This has led a number of investigators to study the effects of immune modulators, such as cyclosporin A (CsA), on bone metabolism. ?he cyclosporins are potent immunosuppressive agents (Bore], 1989; Kahan, 1989) that also have effects on bone. CsA inhibits bone resorption induced by a variety of stimulating agents (e.g., PTH, IL- 1, PGE,, 1.25-(OH),-D3, lipopolysacchandes or thrombin) in both long bone and calvarial organ culture (Skjotd et al., 1985; Stewart et a/., 1986; Klaushofer et al., 1987; Stewart and Stem, 1988, 1989a,b; Sasagawa et al., 1989). It also antagonizes IL-1-mediated stimulation of PGE, synthesis and inhibition of osteocalcin synthesis in human osteoblastlike cell cultures (Skjodt et a/., 1985). An immunosuppressive analog of CsA, cyclosporin G (CsG), was also effective in inhibiting bone resorption (Stewart and Stern, 1989a), whereas nonimmunosuppressive analogs (cyclosporin F, cyclosporin H) or a weakly suppressive analog (cyclosporin D) were ineffective in blocking resorption in tdtm (Stewart and Stem, 1988, 1989a). Moreover, CsA, given concomitantly with calcitonin, delayed or eliminated the escape phenomenon which is typical of calcitonin inhibition of bone resorption in v i m (Stewart and Stem, 1989b). Sasagawa et al. (1989) examined additional classes of immunosuppressants (lobenzarit, traxanox, mizoribine) in addition to CsA, and found that they too inhibited resorption stimulated by a variety of factors. CsA and mizoribine also inhibited basal resorption in the calvarial cultures. The effects of CsA in iivo are less clear. The compound administered to either male or oophorectomized female rats results in severe osteopenia associated with enhanced bone remodeling (Movsowitz et al., 1988, 1989; Schlosberg et al., 1989). Withdrawal of CsA results in a partial reversal of the osteopenia (Schlosberg et ul., 1989). Orcel et a/. (1989) evaluated the effect of CsA in weanling rats and, in agreement with the studies of Movsowitz and co-workers, found histomorphometric evidence for an increase in bone formation. In contrast to the studies of Movsowitz et al., however, Orcel and co-workers reported that treated rats had reduced bone resorption. These apparent differences can be partially resolved in that, at comparable doses and treatment time, both groups saw increased parameters of bone formation. and either no change, or reduced bone resorption. It was only at higher doses or longer treatment time that Movsowitz and co-workers noted increased resorption.
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D. ION FLUXEFFECTORS A number of agents that affect the passage of ions in and out of cells have been shown to have a direct effect on bone. While there is some controversy in the literature about the effects of calcium ionophores in bone, a majority of investigators have reported that the calcium ionophores (A23 187 and/or ionomycin) stimulate bone resorption (e.g., Dziak and Stem, 1975; Lorenzo and Raisz, 1981; Rabadjija et al., 1987). This stimulated resorption was found to be associated with prostaglandin synthesis (Rabadjija et al., 1987). The calcium antagonists, verapamil and diltiazem, inhibit bone resorption stimulated in vitro by PTH or vitamin D compounds (Herrmann-Erlee et al., 1977; Lerner and Gustafson, 1982; Ly et al., 1985). Guggino et al. (1988, 1989) identified a phenylalkamine (verapami1)-sensitive calcium channel in osteoblastlike osteosarcoma cells that could be stimulated by the dihydropyridine calcium agonist BAY K 8644. Desmethoxyverapamil inhibits PTH-stimulated calcium uptake, osteocalcin synthesis, and bone resorption, while BAY K 8644, on the other hand, stimulates osteocalcin synthesis and bone resorption. Dietrich and Duffield (1979) have shown that verapamil inhibits the synthesis of collagen and noncollagen proteins in vitro in skeletal tissue. Indirect effects of calcium antagonists have also been demonstrated, particularly with respect to modulation of PTH levels, although both increases and decreases in PTH levels have been reported (e.g., Fox, 1988; Seely et al., 1989). Using a variety of pharmacological agents including ouabain, monovalent ionophores, vanadate and dichlorobenzamil, a role for sodium, sodium-potassium and sodium-calcium exchanges in bone resorption has been suggested using in vitro organ culture bone resorption experiments (Krieger and Tashjian, 1980, 1981, 1982, 1983; Krieger and Kim, 1988). The proton pump mechanism in the osteoclast ruffled border likely involves a maleimide-inhibited kidney-type electrogenic proton pump (Baron, 1989; Blair et al., 1989). Omeprazole, an inhibitor of the hydrogen-potassium ATPase also inhibits resorption (Tuukkanen and Vaananen, 1986), suggesting a role for this proton pump as well. Disulfonic stilbene inhibitors of chloride-bicarbonate exchange have been used to inhibit bone resorption both in organ culture (Klein-Nulend and Raisz, 1989) and with isolated osteoclasts (Hall and Chambers, 1989), indicating that anion exchange, presumably associated with the need to regulate intracellular pH in the acid-secreting osteoclast, plays an important role in bone resorption. As might be expected, ion flux i n osteoblasts has also been implicated in the mineralization process (Anderson et al., 1984a) and the effects of hormones and metabolic inhibitors on, these fluxes have been described in osteoblastlike cells (e.g., Anderson et al., 1984b; Reid et al., 1988).
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E. OTHERORGANIC AGENTS I . Bisphosphoncites An extensive literature on the effects of bisphosphonates (formerly referred to as diphosphonates) on biological calcification and calcium metabolism has developed over the past twenty years (for review see Fleisch, 1989). The initial interest in these compounds was based on the suggestion that pyrophosphates and other condensed phosphates may have a role in regulating the physiological formation and dissolution of calcium phosphate crystals (Fleisch et al., 1966; Fleisch. 1989; Blumenthal, 1989). Bisphosphonates are stable analogs of pyrophosphates in which the P-0-P group is replaced with P-C-P. These compounds, while retaining a high affinity for calcified tissues, are resistant to enzymatic hydrolysis. A variety of bisphosphonates have been synthesized by changing the structure of the carbon side chains or by esterification of the phosphate groups, resulting in a biologically diverse group of compounds (Fleisch, 1989; Papapoulos et al., 1989). Bisphosphonates have been shown to inhibit the process of calcification in \Yvo in both skeletal and ectopically mineralizing tissues (Fleisch et al., 1970; Casey et al., 1972; Fraser et al., 1972; Schenk et al., 1973; Levy et al., 1985). This effect is presumably associated with their ability to inhibit crystal growth in solution, rather than being associated with specific cellular effects (Fleisch, 1989). The bisphosphonates are also potent inhibitors of bone resorption both in vifro and in i*iw(e.g., Fleisch et a/., 1969; Russell et al., 1970; Reynolds et al., 1972; Ohya et al., 1985; Reid et al., 1986; Schenk et al., 1986; Wronski et al., 1989). apparently by a variety of mechanisms. Some bisphosphonates may possess a selective toxicity for phagocytic cells such as macrophages and osteoclasts (Boonekamp et al., 1986; Flanagan and Chambers, 1989), while others may inhibit the proliferation and differentiation of macrophage and osteoclastlike cell precursors from bone marrow (Cecchini et al., 1987; Hughes et af., 1989). The mechanism of action of the newer clinically tested bisphosphonates, however, appears to involve absorption of the compound onto the calcified bone matrix. The bisphosphonate may then either inhibit the action of the mature osteoclast, or, at lower concentrations, prevent attachment of the osteoclast and its precursors to the matrix, thus suppressing resorption and phenotypic expression of the mature osteoclast (Boonekamp et al., 1986; Lowik et ul., 1988; Papapoulos et al., 1989). Inhibition of the osteoclast may not be due simply to cytotoxicity since several bisphosphonates are effective in inhibiting bone resorption by mature osteoclasts at concentrations that are not cytotoxic to the cells (Sato and Grasser, 1990). Because of their selective action on mineralized tissues, their amenity to chemical manipulation and the resultant functional diversity, bisphosphonates
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have been evaluated in a variety of clinical disorders. Their main use is in patients with Paget’s disease (Adami et al., 1986; Thiebaud et al., 1987; Atkins et af., 1987; O’Donoghue and Hosking, 1987; Rico et al., 1988). They have also been evaluated in osteoporosis treatment (Valkema et al., 1989; Mallette et al., 1989; Hodsman, 1989), and in treatment of hypocalcemia associated with malignancy (Thiebaud et al., 1986; Coleman et al., 1988; Morton and Howell, 1988), as well as other disorders (Fleisch, 1989). 2 . Thiazide Diuretics
The primary therapeutic utility of diuretics is to lower blood pressure through the enhanced excretion of sodium and chloride. However, these compounds also effect the renal processing of other ions such as calcium. The effects on calcium metabolism differ depending on the type of compound administered (Stier and Itskovitz, 1986). Among the different diuretics, thiazides have received the greatest attention with respect to therapeutic utility in modifying calcifying processes, being suggested for use in both renal stone disease and osteoporosis. In the urinary tract, thiazides are claimed to prevent the formation of calcium stones (e.g., Yendt and Cohanim, 1978; Brocks et al., 1983) although the positive effects of thiazides on stone formation are not universally accepted (Wolf ef al., 1983; Churchill and Taylor, 1985). In bone, thiazide administration results in higher levels of mineral content in treated compared to untreated individuals (e.g., Wasnich et al., 1983). Estrogen therapy, in combination with thiazides, increases bone mineral content even further (Wasnich et al., 1986). However, not all studies agree with this conclusion (Adland-Davenport et al., 1985). Stier and Itskovitz (1986) have recently reviewed the role of diuretics in calcium metabolism, and point out that different diuretics may either increase or decrease calcium excretion by affecting the physiological mechanisms that influence renal handling of calcium. Long-term administration of thiazides causes a reduction in calcium excretion which results in reducing hypercalcuria, thereby preventing renal stone formation (Stier and Itskovitz, 1986). However, thiazides also increase urinary levels of crystallization inhibitors such as pyrophosphate, magnesium, and zinc, and these also may contribute to thiazide effects on renal stone formation (Brandes et al., 1982). It appears that the primary action of thiazides on skeletal metabolism and as a treatment for osteoporosis is associated with its action on renal tubule calcium readsorption, although their final effects are likely the result of interrelated forces including renal, hormonal, and nonhormonal factors. These aspects of thiazide action need further elucidation (Chestnut, 1983; Stier and Itskovitz, 1986). Other classes of diuretics may likewise affect mineral metabolism in complex ways. Amiloride (a structurally distinct diuretic which appears to block sodium channels), for instance, has an additive effect with thiazides in reducing calcium clearance, but also may have direct effects
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on bone as indicated by its ability to inhibit basal resorption in organ culture (Krieger and Kim, 1988). 3. Anticonvulsants Anticonvulsant drugs, such as diphenylhydantoin (phenytoin) or phenobarbitol, have been reported to have complex effects on bone metabolism, resulting in a clinical instance of osteomalacia (Dziak er al., 1988; Takeshita et at., 1989). While the impact of these compounds in patients can in part be explained by their effect on vitamin D metabolism (Hahn, 1980), the compounds also have direct effects on in virro bone metabolism, including inhibition of bone resorption (Somerman et a f . , 1986) and modulation of osteoblast metabolism (Dziak et al., 1988). In patient populations, osteopenia associated with anticonvulsant therapy is accompanied by high bone turnover ( ie . , increased formation and resorption) e.g., Barden er al., 1982; Takeshita et al., 1989). The interpretation of these data is complicated, however, by the heterogeneous nature of the patient population and accompanying drug therapy regimes the subjects were given, as well as possible differential effects on compact versus trabecular bone (Barden et al., 1982). The effects of these drugs in vivo may involve many indirect effects on bone metabolism. Diphenylhydantoin has, for instance, been shown to inhibit calcium-mediated release of calcitonin from thyroid C cells. This latter effect can be reversed by calcium channel activators (Cooper et a!., 1988). 4 . Flavanoids
Flavanoids have been shown to have a variety of effects on bone related systems. Ipriflavone and several analogs have been shown to inhibit the release of calcium from both unstimulated and PTH-stimulated cultured bones (Tsuda et al., 1986). These compounds can also increase secretory levels of calcitonin in the presence of subeffective doses of estrogen (Yamazaki et al., 1986) and moderate the effect on bone of experimentally induced diabetes, glucocorticoid therapy, and vitamin Dkalcium nutritional deficiency (Shino et al., 1988; Yamazaki et al., 1986; Takenaka et al., 1986). In human studies of relatively short-term (6 month) and limited patient numbers, there appeared to be a positive effect of ipriflavone on bone mineral content in the lumbar spine and distal radius (Agnusdei et al., 1989). Catechin, another flavanoid, can stabilize collagen molecules so that they are less susceptible to collagenase digestion (Kuttan et al., 1981; Pontz et al., 1982) and also can inhibit bone resorption induced in cultured calvaria by PTH, PGE,, or retinoic acid (Delaisse et al., 1986). Other flavanoids have been shown to decrease plasma calcium and calcitonin when injected into rats in short-term (2.5 hr) studies (Saija et al., 1988).
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5. Levamisole
Levamisole is an antihelmentic drug with multiple sites of action in bone. As an antihelmentic agent, levamisole (L-tetramisole) is active while its stereoisorner, dexamisole (D-tetramisole) is inactive. Levamisole and an analog, R823 1 (bromotetramisole), have been reported to inhibit the action of bone-associated alkaline phosphatase in a stereospecific manner (Reynolds and Dew, 1977; Lerner and Granstrom, 1984), though the stereospecificity of this reaction has been questioned with regard to chick bone alkaline phosphatase (Tenenbaum, 1987). Levamisole, dexamisole, and their bromotetramisole analogs have also been shown to inhibit calcium release (bone resorption) and reduce lysosomal enzyme release and lactate production in cultured mouse calvarial bones in a nonstereospecific manner (Lemer and Granstrom, 1984). Levamisole has been shown to affect bone matrix apposition and mineral apposition in vivo, effects which have been attributed to its action on alkaline phosphatase (Garba and Marie, 1986). In that levamisole also has immune-modulatory activity, some of its activity on bone may be mediated through effects on the immune system (Sasagawa et al., 1989).
6 . Imidazole Imidazole and the tetramizoles are structurally related and have similar effects on bone (Lemer and Granstrom, 1984). Imidazole has been shown to inhibit bone resorption both in vitro and in vivo (Wells and Lloyd, 1968; Heersche and Gaillard, 1969; Avery et al., 1971; Heersche and Jez, 1981). It will also increase mineral uptake by cultured calvaria (Heersche and Gaillard, 1969), improve osteomalacia induced by low dietary levels of vitamin D and phosphorus in rats (Roudier and Martin, 1984), and promote calcification in bisphosphonateinduced ricketic growth plates (Eguchi ef al., 1989). Because imidazole has calcium binding activity and its action on ricketic growth plate is reversed by diltiazem, a calcium antagonist, it is possible that its action on skeletal tissue is mediated through an influence on intracellular calcium levels (Lerner and Granstrom, 1984; Eguchi et al., 1989). Alternatively (or in addition), Chambers and Ali (1983) noted that imidazole reduced the effectiveness of prostaglandins and calcitonin in inhibiting motility and shape change in isolated osteoclasts, suggesting that this action was associated with irnidazole stimulation of PDE activity.
7 . Promethazine and Related Compounds Promethazine hydrochloride, a phenothiazine derivative, has been reported to be a depressor of the central nervous system (sedative), an H, (histamine) receptor antagonist, an inhibitor of prostaglandin synthesis, and a potent
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inhibitor of macrophage function (Goldhaber and Rabadjija. 1982). It inhibits PTH-induced bone resorption in calvarial culture at concentrations which do not inhibit osteoid formation (Goldhaber and Rabadjija, 1982). The authors attributed these effects to inhibition of macrophage function. In a subsequent report, Goldhaber and Rabadjija (1983) compared the effects of several H, and H2 receptor antagonists. In this study, the HI antagonists inhibited resorption in calvarial cultures, whereas the H, antagonists did not. Promethazine was found to be the most potent of the H I antagonists tested. However, since histamine agonists (histamine, 2-methyl histamine, 2-pyridylethylamine, and betazole) and a histidine decarboxylase inhibitor [4-(4-imidazolyl)-3-amino-2-butanone] failed to stimulate bone resorption, and histamine was ineffective in reversing the promethazine effect, the authors concluded that promethazine was not acting through H, receptor antagonism. They suggested that the mechanism of action of the H I receptor antagonists was dependent on a membrane stabilizing effect. Tyan (1986) evaluated the effects of promethazine in vivo in aged mice and found that the compound appeared to enhance net bone deposition in femoral shafts. In a second report (Tyan and Blahd, 1986), promethazine was compared to chlorpheneramine (an HI blocker) and trifluoperazine (a phenothiazine without H, blocking activity) and it was found that promethazine, but not the other compounds tested, inhibited bone loss in aging mice. Komoda and co-workers (1985). on the other hand, reported that the phenothiazine derivatives chlorpromazine, trifluoperazine, and perphenazine inhibited aspects of bone formation (e.g., alkaline phosphatase, collagen synthesis, bone cell proliferation) in different in vivo and in vitro models, whereas prornethazine had little effect on these parameters. 8. Thiophene Carbo.qlic Acids Thiophene carboxylic acid and its derivatives have been shown to have antiresorptive effects in several bone models. The hypocalcemic and hypophosphatemic effects of 2-thiophene carboxylic acid in intact, parathyroidectornized and thyroparathyroidectomized rats were initially described by Lloyd ef af. (1969). It was subsequently shown to inhibit both basal and PTH-induced bone resorption in calvarial cultures (Fang et a / . , 1971). Two derivatives, benzo[b]thiophene-2-carboxylic acid (BL5583) and dibenzothiophene-4-carboxylic acid (BL5593) were shown to be effective in reducing heparin-induced osteopenia in mice (Robin at al., 1980). Benzo[b]thiophene-2-carboxylic acid was also evaluated in vitro and found to inhibit basal, PTH-, and A23 187- (a calcium ionophore) induced bone resorption and to decrease calcium uptake by cultured calvarial cell populations, but did not affect CAMP levels in these cells (Robin et al., 1984). Benzo[b]thiophene-2-carboxylic acid (thionapthene-2-car-
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boxylic acid) was found to be the most potent of a series of thiophene carboxylic acid derivatives tested by Raisz et al. (1985)for their effects in inhibiting bone resorption in long bone cultures, and its lysine salt was shown to be effective in lowering serum calcium concentrations in rats bearing hypercalcemic Leydig cell tumors (Decker et al., 1989). 9. WR-2721
WR-2721 [S-2(3-aminopropylamino)ethylphosphorothioic acid] is a thiophosphate analog of cystamine that has been used to protect certain normal tissues against the lethal effects of ionizing radiation and akylating agents (Yuhas et al., 1980).It causes hypocalcemia in animals and humans (Glover et al., 1983;Attie et al., 1985),inhibits PTH secretion (Glover et al., 1983),increases urinary calcium excretion (Hirschel-Scholz et al., 1985), and inhibits osteoclastic bone resorption both in vitro and in vivo (Attie et al.; 1985;Shaker et al., 1989).Attie et al. (1985)concluded that the predominant effect in producing hypocalcemia was associated with the compound’s antiresorptive effect in bone. 10. HeparinlProtamine Heparin is best known therapeutically for its anticoagulant activity. However, it has also been used experimentally to enhance the resorptive activity of agents that stimulate bone resorption (Goldhaber, 1965),and to induce osteopenia in animal models (Thompson, 1973;Ambrus et al., 1978;Matzsch et al., 1986). These models have, in turn, been used to evaluate a variety of compounds for their effects on osteoporosis (e.g., Robin et al., 1980, 1983). Heparin has also been implicated as a causal factor in human osteoporosis (De Swiet et al., 1983;Mazanec and Grisanti, 1989),although the mechanism by which heparin causes osteopenia is not known. It is able to increase both collagenase activity in vitro in mouse bone explants (Sakamoto et a f . , 1973), and resorption of experimentally implanted bone particles (Glowacki, 1983). Crisp et al. (1986) have shown that some heparin preparations could “blunt” the activity of calcitonin in inhibiting bone resorption in vitro, even though heparin alone inhibited resorption in the same model. Recent studies have attempted to better define the activity of heparin based on size characteristics, anticoagulant activity of heparin fragments, and the interactions of heparin with other bone-regulating agents. Cochran (1987)and Cochran and Abernathy (1988)examined glycosaminoglycan effects in mouse calvarial organ culture and found that hyaluronic acid and dermatan sulfate stimulated resorption in the absence of added stimuli, while heparin, heparin fragments with differing anticoagulant activity, dextran, and dextran sulfate potentiated the action of PTH in stimulating resorption but failed to stimulate resorption by themselves. When these glycosaminoglycans were combined with IL-1 p, the effects on resorption differed
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from those seen with PTH. Heparin and hyaluronic acid partially inhibited resorption stimulated by IL- I , and the decreasing inhibition of the heparin fragments corresponded to their decreasing anticoagulant activity. These in vitro results point out the potential complexity associated with heparin effects on the skeletal system. Protamine is a basic polypeptide used therapeutically to antagonize the anticoagulant activity of heparin. Because of this fact, it was evaluated for its effects in patients with hypercalcemia associated with malignancy (Anderson et al., 1967), and has been shown to be a potent hypocalcemic agent in animal models (Johnston et al., 1970 Potts et al., 1984). It is able to inhibit bone resorption both in vitro in cultured calvaria (Johnston et al., 1970) and in vivo in association with subcutaneously implanted bone particles (Glowacki, 1983). The data from both intact and surgically modified animals are consistent with a primary site of protamine’s hypocalcemic action being on bone tissue (Johnston et al., 1970 Potts et al., 1984).
11. Glucocorticoids The effects of glucocorticoids on bone are complex and often contradictory, particularly in comparing in vitro and in vivo data. In vivo, both direct and indirect effects of glucocorticoids on mineral metabolism have been documented. Bone formation in vivo appears to be retarded, whereas bone resorption is reported to be either unaffected or enhanced, with the net effect of glucocorticoid treatment being consistently associated with bone loss both in animal models and in clinical human experience (e.g., Als et al., 1985; Ortoft and Oxlund, 1988; Goulding and Gold, 1988; Lukert and Raisz, 1990; Bockman and Weinerman, 1990). in vitro effects, on the other hand, include impaired bone resorptive activity with isolated osteoclasts (Tobias and Chambers, 1989), inhibition of plasminogen activator activity stimulated by bone resorbing hormones (Hamilton et al., 1985), and stimulation of bone formation in cultured osteoblasts (Bellows et d., 1987). Depending on the experimental conditions, glucocorticoids can either inhibit or enhance bone resorption in organ culture (e.g., Raisz et al., 1972; Reid et al., 1986). One explanation for these conflicting results may be the tendency for glucocorticoids to exhibit biphasic concentration responses in some in vitro systems (e.g., Wong, 1979; Anderson et al., 1984b). The literature on glucocorticoid effects on calcified tissues is far too extensive to review in depth here; however, two very recent reviews that emphasize clinical aspects of the problem are available (Lukert and Raisz, 1990; Bockman and Weineman, 1990). From a therapeutic perspective, one relatively new glucocorticoid, deflazacort, holds promise of reduced adverse effects on the skeletal system (Gennari et al., 1984; Balsan et al., 1987).
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EFFECTORS OF CALCInCATION
I . Fluoride The use of fluoride in treating metabolic bone diseases such as osteoporosis has been extensively studied, but remains controversial. Fluoride therapy results in increased trabecular bone mass in the axial (vertebral) skeleton of osteopenic patients. This effect is generally found to be associated with decreased vertebral fracture rates (Heaney et al., 1989; Farley et al., 1989). However, concern persists about the quality of the bone laid down and the effect of therapy on fracture rates in the appendicular skeleton. Published data are in disagreement as to the effects of fluoride therapy on appendicular bone. Some studies indicate no significant enhanced risk of fracture with therapy (Riggs er al., 1987; Mamelle et al., 1988; Heaney et al., 1989), whereas others have suggested that fluoride treatment is associated with reduced cortical bone density (e.g., Hodsman and Drost, 1989; Riggs er al., 1990), and increased fracture incidence in the appendicular bones (Schnitzler and Salmon, 1985; Hedlund and Gallagher, 1989; Schnitzler e f al., 1990; Riggs et al., 1990). Some undesirable side effects (e.g., gastrointestinal and rheumatic) associated with fluoride therapy may be related to pharmacokinetic problems associated with the pulsatile administration of fastrelease fluoride preparations that may periodically increase fluoride concentrations to toxic levels (Pak er al., 1986a,b;Turner et aL1989). Slow-release preparations may therefore prove useful (Pak et al., 1986a,b; Pak, 1989). However, significant amounts of fluoride are incorporated into the bone crystalline structure, which can then act as a slow-release reservoir of fluoride, thus effectively increasing the length of treatment (Baud et al., 1988; Grynpas and Cheng, 1988). Since this may mediate some of the adverse skeletal effects, it is not clear that slow-release formulations will eliminate problems of appendicular fracture. Published data consistently demonstrate that fluoride has a dramatic effect on the process of calcification that may be related both to its direct effects on the crystallization processes, as well as to its effects on the cellular aspects of bone formation. Fluoride has been shown to directly stimulate proliferation and alkaline phosphatase activity in bone forming cells (Farley et al., 1983), which may be mediated through the effects of fluoride in inhibiting phosphotyrosyl protein phosphatase and prolonging the stimulatory response of growth factors whose action is mediated through tyrosyl protein phosphorylation (Lundy et af., 1988; Lau et al., 1989). Fluoride also has been shown to modify the crystallinity and reduce the solubility of bone mineral (Baud er al., 1988; Grynpas and Cheng, 1988). These changes in crystalline structure may account for the increases in microhardness and compressive strength associated with fluoridic bone (Baud et al., 1988). High doses of fluoride (60 mg/kg body weight in rats) were
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demonstrated to be toxic to osteoclasts (Lindskog et al., 1989), but whether this has physiological significance for an osteoclast resorbing fluoridic bone is not clear. Reduced solubility of bone crystal (and/or selective toxicity for osteoclam) could result in decreases in bone resorption (Grynpas and Cheng, 1988) with enhanced bone volume, but reduced repair of microfracture in bone via remodeling processes, and subsequently to stress fracture of bone. Alternatively, Schnitzler et al. ( 1990) suggest that enhanced bone erosion associated with fluoride therapy accompanied by replacement with poorly mineralized osteoid may be the cause of increased incidence of stress fractures.
2. Vanadate Several investigators have demonstrated that vanadate will inhibit bone resorption in mouse calvarial organ culture (e.g., Krieger and Tashjian, 1983), and stimulate DNA, alkaline phosphatase, and collagen synthesis in calvarial cell and organ cultures (Canalis, 1985; Lau et al., 1988b). Moreover, Yamaguchi cr al. (1989b) have recently demonstrated that oral administration of vanadate to weanling rats results in increases in DNA content and alkaline phosphatase activity in femoral diaphyses, while acid phosphatase activity and bone calcium content is not altered. Krieger and Tashjian (1983) conclude that the action of vanadate in stimulating bone resorption is through its effect on Na/K transport and the inhibition of Na,K-ATPase, whereas, Lau et a f .(1988b) suggest that the effects of vanadate in the calvaria may be due to its ability to prolong and/or potentiate the action of mitogenic agents by inhibiting the activity of phosphotyrosy1 protein phosphatase. 3. Aluminrtm
Aluminum accumulation in the skeleton has been connected with the development of a vitamin D-resistant form of osteomalacia, and may be associated with impaired osteoblast numbers and/or rates of matrix synthesis and mineralization (Sedman e t a / . , 1987; Hodsman et al., 1988). Alternatively, aluminum binding to hydroxyapatite crystals may interfere with nucleation and crystal growth (Blumenthal, 1985). The factors causing the development of this syndrome have not been completely defined, but, in animal models, appear to be linked with the vitamin D/PTH status of the subject (Hodsman et al., 1988). Aluminum toxicity has also been implicated in the development of aplastic bone disease in dialysis patients. This disease differs from pure osteomalacia in the absence of a marked osteoid excess. While aluminum-associated aplastic bone disease has been less studied than osteomalacia associated with aluminum toxicity, it does appear to involve impaired osteoblast function (Parisien et al., 1988).
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4. Gallium
Gallium salts have been used therapeutically as an antitumor agent and to reduce hypercalcemia and bone turnover associated with certain malignant tumors (e.g., Warrell et al., 1984, 1987; Warrell and Bockman, 1989). Gallium inhibits bone resorption in vitro in organ culture (Warrell et al., 1984), as well as in cultures of isolated osteoclasts (Hall and Chambers, 1990a), and short term treatment of rats in vivo results in increases in bone calcium and phosphorus content (Repo et al., 1988). In chronically treated rats, both acid phosphatase and alkaline phosphatase levels were increased in calvaria (Coumot-Witmer et al., 1987). Like aluminum, gallium interacts with the mineral as well as the cellular components of bone (Bockman et al., 1986; Blumenthal et al., 1989; Hall and Chambers, 1990a), and this interaction may partially explain the in vivo effects of gallium. 5 . Gold
Complexes of gold are used as treatment for rheumatoid arthritis (Klaushofer et al., 1989). Several groups have reported that these complexes will also inhibit bone resorption in vitro (Goldhaber et al., 1978; Katz and Gray, 1986; Vargas et al., 1987; Klaushofer et al., 1989). These studies that might help to explain the possible mechanism of gold action on bone resorption differ in the models used (e.g., mouse vs. rat, long bone vs. calvaria), and in their results. Klaushofer et al. (1989) reported that, depending on concentration, gold salts can either stimulate or inhibit bone resorption in calvarial organ cultures, with stimulation at lower concentrations (3 x 10-'-3 x M) mediated through effects on prostaglandin production. They suggest that the inhibitory effect of high concentrations of auranofin, the gold compound used in their studies, may be due to cytotoxic effects. Katz and Gray (1986), however, found that while auranofin inhibited collagen synthesis at doses comparable to those inhibiting bone resorption in mouse calvaria, DNA and protein synthesis and lysosomal enzyme release were not significantly affected. Vargas et al., using fetal rat long bone cultures, found that both auranofin and gold sodium thiomalate (GST) inhibited resorption. The effects of auranofin were irreversable and accompanied by inhibition of ['Hlthymidine and ['Hlproline incorporation into bone, whereas GST effects on resorption were reversible and it did not effect DNA or protein synthesis. Both auranofin and GST decreased lysosomal (P-glucuronidase) release from bones.
6. Zinc Zinc is an essential trace element. It is a cofactor for many enzymes, including a number that participate in the processes of transcription and translation (Prasad, 1983). Several investigators have shown a correlation between tissue and body fluid levels of zinc and osteoporosis (e.g., Atik, 1983; Herzberg et al.,
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1990). Zinc deficiency has also been shown to be associated with retardation of ectopic bone formation and calcification in response to implantation of demineralized bone matrix. Evidence indicates that zinc may play an active role in bone metabolism (Calhoun er a/., 1975). However, an alternative explanation for the zinc effects in the ectopic bone implant model relate to impaired vascularization at the site of implantation (Belanger et al., 1977). Yamaguchi and co-workers have studied the effects of zinc on bone metabolism using both in vitro and in v i w animal models (e.g., Yamaguchi and Inamoto, 1986; Yamaguchi and Matsui, 1989; Yamaguchi er af., 1982a,b, 1987, 1988, 1989a,b). In these studies, short termbow dose administration of zinc in weanling rats increased parameters associated with bone formation (increased femur dry weight, DNA content, alkaline phosphatase activity), whereas longer term administration or higher doses decreased formation parameters and increased resorption parameters (femur acid phosphatase activity) (Yamaguchi et al., 1982a,b). Zinc was found to potentiate the bone formation effects of 1,25-(OH),-D,, but not the effects of PTH or calcitonin (Yamaguchi and Inamoto, 1986; Yamaguchi et al., 1989a). The actions of zinc in bone were attributed to its effects in stimulating protein synthesis and aminoacyl-tRNA synthetase (Yamaguchi et al., 1987, 1988). In tissue culture, the positive effects of zinc could be reversed by chelation with dipicolinate, which Yamaguchi and Matsui (1989) suggest supports a role for endogenous zinc in regulating bone protein synthesis.
V. Summary and Conclusions Biological calcification is a highly regulated process which occurs in diverse species of microorganisms, plants, and animals. Calcification provides tissues with structural rigidity to function in support and protection, supplies the organism with a reservoir for physiologically important ions, and also serves in a variety of specialized functions. In the vertebrate skeleton, hydroxyapatite crystals are laid down on a backbone of type I collagen, with the process being controlled by a wide range of noncollagenous proteins present in the local surroundings. In bone, cells of the osteoblast lineage are responsible for the synthesis of the bone matrix and many of these regulatory proteins. Osteoclasts, on the other hand, are continually resorbing bone to both produce changes in bone shape and maintain skeletal integrity, and to establish the ionic environment needed by the organism. The proliferation, differentiation, and activity of these cells is regulated by a number of growth factors and hormones. While much has already been discovered over the past few years about the involvement of various regulators in the process of mineralization, the identification and functional characterization of these factors remains an area of intense investigation.
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As with any complex, biological system that is in a finely tuned equilibrium under normal conditions, problems can occur. An imbalance in the processes of formation and resorption can lead to calcification disorders, and the resultant diseases of the skeletal system have a major impact on human health. A number of pharmacological agents have been, and are being, investigated for their therapeutic potential to correct these defects. While much has been learned recently concerning mineralization and methods of therapeutic intervention in the process, research in this area is only in its infancy. The next few years should bring exciting advancements in this field that will most likely give a better insight into how calcification is initiated and controlled and how to best deal with abnormalities in the process.
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Index
A Abscisic acid cereal aleurone and, 49 a-amylase genes, 54,59,61-62 calcium, 81-82 mechanism of hormone action, 6 3 4 4 , 6647 uptake mechanisms in plants and, I10 Abscisic acid-responsive element, cereal aleurone and, 64-65.67 N-Acetylgalactosamine, intestinal epithelium and, 141, 157 Golgi apparatus, 174-175 oligosaccharides, 143, 145-147 N-Acetylglucosamine, intestinal epithelium and, 139,141,143,156 Acid phosphatase, calcification and, 215,261 Acidification, calcification and, 2 15-2 17 Actin, calcification and, 213 Adaptation, melanin-concentrating hormone and, 7,33,35 Adenyl cyclase, calcification and, 221,226228, 240,242-243 Adrenocorticotropin (ACTH), melaninconcentrating hormone and, 13.35-36, 3940 Affinity calcification and, 219,223,234,241 glycosylation in intestinal epithelium and, 176177 uptake mechanisms in plants and, 91-92, 119, 125 Aleurone, regulation of secreted proteins in, see Cereal aleurone 293
Alkaline phosphatase, calcification and biochemistry, 2 0 6 2 0 7 cells of bone, 209-210 endocrine factors, 226228,231 pharmacological regulation, 249,255, 259-26 1 physiological regulation, 237,240-241, 243-244 Aluminum, calcification and, 260-261 Amiloride, calcification and, 253 Amino acids calcification and, 204,22&222,240 cereal aleurone and a-amylase genes, 5&57 protein transport, 70.73.78-79 glycosylation in intestinal epithelium and, 139,141,143 melanin-concentrating hormone and, 14-1 8, 21,23 uptake mechanisms in plants and, 89, 105-109, 113,123 a-Aminoisobutyric acid, uptake mechanisms in plants and, 106 Amphibians, melanin-concentrating hormone and, 1-3 anatomical distribution, 9-1 1, 13 physiology, 33-34 structure-activity studies, 23 Amrinone, calcification and, 248 a-Amylase, cereal aleurone and, 49,83 calcium, 79-83 genes, 54-62 mechanism of hormone action, 6 3 4 8 protein transport, 70-74.7677 Androgen, calcification and, 230-23 I , 233,249
294
INDEX
Anguilla, melanin-concentrating hormone and,
3, 14 Anolis, melanin-concentrating hormone and, 23-24.34 Anti-idiotypic antibodies, cereal aleurone and. 63-64 Antibodies calcification and, 206,220,235,240 cereal aleurone and, 63-64,7 1,75 glycosylation in intestinal epithelium and. 146, 149. 154, 168, 176-178 melanin-concentrating hormone and, 3 Anticonvulsants, calcification and, 254 Antiestrogens, calcification and. 249 Antigens calcification and, 209 cereal aleurone and. 63 intestinal epithelium and, 149, 176-178 Apical region, calcification and, 2 16-2 17 A p ~ w i umelanin-concentrating . hormone and, 41-43 Arginine. uptake mechanisms in plants and. 107 ATP. melanin-concenaating hormone and, 3 1 ATPase calcification and, 216-217,224,260 cereal aleurone and. 8 1-82 uptake mechanisms in plants and, 89, 123, 125, 131 multiphasic model, 114-116. I18 Atrial natriuretic factor. melaninconcenaating hormone and, 4 2 4 3 Autoantagonism, melanin-concentrating hormone and, 24 Autoradiography calcification and, 227,237 glycosylation in intestinal epithelium and, 137, 154. 175-176, 184 Auxins, uptake mechanisms in plants and, 110
B Bacteria, uptake mechanisms in plants and, 90, 116-118, 192 Barley cereal aleurone and, 83-84 aleurone tissue, 49-52 a-amylase genes, 54-57,59,61 calcium. 79-83
mechanism of hormone action, 6 3 , 6 5 4 8 protein transport, 7&7 I , 73-79 uptake mechanisms in plants and, 124.13 I dual model, 9 1-92 multiphasic model, 93-98. 102-107. 111-1 13 Basement membrane, melanin-concentrating hormone and, 7 Bean, uptake mechanisms in plants and, 104 Benzyladenine, uptake mechanisms in plants and, 110 Binding protein, cereal aleurone and, 75 Biological calcification, see Calcification Bisphosphonates, calcification and, 252-253 Bone, see Calcification Bone inductive proteins, calcification and, 239-24 I Bone lining cells, calcification and, 21 2 Bone marrow, calcification and, 213.215.223, 242,245,252 Bone morphogenetic proteins, calcification and, 239-24 1 Bone sialoprotein, calcification and. 202-203 Boron, uptake mechanisms in plants and, 104 Brain, melanin-concentrating hormone and, 1,3, 4243 anatomical distribution, 3-5,7-8, 11, 13 physiology, 30.37.39-40
C Calcification, 195-196,262-263 biochemistry, 199 r-carboxyglutamic acid, 2 W 2 0 5 collagens, 200-201 enzymes, 206-207 mineral phase, 200 noncollagenous proteins, 201-203 proteolipids, 205 cells ofbone, 208 bone lining cells, 21 2 osteoblasts, 209-2 I2 osteoclasts, 2 13-2 I7 osteocytes, 2 12-2 13 diversity, 198-199 function, 196 matrix vesicle theory. 197 modeling, 207
INDEX nucleation theory, 196-197 pharmacologicalregulation, 246 enzyme inhibitors, 247-249 estrogen, 249-250 immune modulators, 250 inorganic effectors, 259-262 ion flux effectors, 251 organic agents, 252-258 physiological regulation, 2 17-2 19 bone inductive proteins, 239-24 I calcitonin, 222-223 cytokines, 244-246 endocrine factors, 2 18 epidermal growth factor, 24 1-242 fibroblast growth factor, 242-243 growth hormone, 232-233 insulinlike growth factors, 233-236 parathyroid hormone, 217,219-222 platelet-derived growth factor, 243 steroid hormones, 227-232 transforminggrowth factors, 236-239 vitamin A, 226-227 vitamin D, 223-226 Calcitonin, calcification and cells of bone, 215-216 pharmacological regulation, 247-248,257,261 physiological regulation, 222-223.229, 231-232.238 Calcium cereal aleurone and, 49.84 a-amylase genes, 54,56,61 metal loproteins, 79-80 protein transport, 71-72,74,76-77 transport, 81-83 melanin-concentratinghormone and, 2 4 2 5 Calcium phosphate, calcification and, 197,200, 205-206,252 Calmodulin calcification and, 203 cereal aleurone and, 82 Cancer cells calcification and, 210 glycosylation in intestinal epithelium and, 135,186 Carbohydrate calcification and, 203 glycosylation in intestinal epithelium and, 137,147-148,186-188 distribution, 153,155-158, 161
295
post-Golgi apparatus distribution, 177-178 Carbonate, calcification and, 198,200 Carbonic anhydrase, calcification and, 216 inhibition, 247 T-Carboxyglutamic acid, calcification and, 204-205 Carriers, uptake mechanisms in plants and, 126-128 Cartilage, calcification and biochemistry, 199-200,205-206 endocrine factors, 222,224-227 physiological regulation, 235-236,238-24 1, 244-245 Catalysis, glycosylation in intestinal epithelium and, 182 Cathespin H, cereal aleurone and, 78.84 cDNA, see ComplementaryDNA Cellulose, cereal aleurone and, 72 Central nervous system, melanin-concentrating hormone and, 10,39 Cereal aleurone, regulation of secreted proteins in, 49,83-84 aleurone tissue, 49-53 a-amylase genes expression, 60-62 multigene family, 55-59 synthesis, 54-55 calcium metalloproteins, 79-80 transport, 8 1-83 hormone action genes, 64-68 perception, 63-64 steroid model, 68-70 protein transport, 7 4 7 9 ER lumen, 73-74 route, 70-72 Channels, uptake mechanisms in plants and, 126-128,131 Chloride calcification and, 253 uptake mechanisms in plants and, 91,102, 113,124 CHO cells, glycosylation in intestinal epithelium and, 173,181 Choline sulfate, uptake mechanisms in plants and, 92 Chondrocytes,calcification and, 217,220, 224-225
296
INDEX
cytokines. 245-246 endocrine factors. 217,220,224-225.232 physiological regulation, 233-236.238-239, 243 steroid hormones. 227-228.23 I Chorellu, uptake mechanisms in plants and, 99-101, 107. 110, 116 Chromatin. cereal aleurone and. 68 Chromatophores. melanin-concentrating hormone and, 30,43 Chromosomes,cereal aleurone and, 57.59 Chymotrypsin,cereal aleurone and, 80 Cis-acting elements, cereal aleurone and, 59.62, 64-67 Citrate. calcification and, 198.200 Clones calcification and. 240 cereal aleurone and, 83 a-amylase genes, 5657.59 mechanism of hormone action, 63.6-7 protein transport. 73,77 glycosylation in intestinal epithelium and, 177. 181 Colchicine glycosylation in intestinal epithelium and, I84185 melanin-concentratinghormone and. 8 Collagen. calcification and, 197,262 biochemistry, 200-203 cells of bone, 209-210 endocrine factors, 220, 222-226,231 pharmacological regulation, 248.25 1,254, 260-261 physiological regulation, 2 17. 235-237, 239-245 Collagenase.calcification and, 21 2,238, 243-244,254,251 Collagenase inhibition. calcification and, 247 Colloidal gold, glycosylation in intestinal epitheliumand, 148. 159. 161 Colon, glycosylation and. 158-159. 163, 168, 171, 178 Color change, melanin-concentratinghormone and, 1-2.30.39.43 Competition. uptake mechanisms in plants and, 92.95. 107 Complementary DNA cereal aleurone and a-amylase genes. 56-57.59 protein transpon, 73.77-78
glycosylation in intestinal epithelium and, 173, 188 melanin-concentratinghormone and, 14-17, 40.42 Concanavalin A, glycosylation in intestinal epithelium and, 158-159 Cooperative kinetics, uptake mechanisms in plants and. 114, 127-128 Cooperative model, uptake mechanisms in plants and, 123 Corn, uptake mechanisms in plants and, 102. 114, 120, 124-126 Corticosteroids, melanin-concentratinghormone and, 39 Corticotropin-releasingfactor anatomical distribution, 13-14, 18 physiology, 35-37.39-40 Conisol calcification and, 236, 25 I melanin-concentratinghormone and, 35-36, 38-39,43 Cortisone, glycosylation in intestinal epithelium and, 183-184 Creatine kinase, calcification and, 228 Crystallization, calcification and, 197-198.262 biochemistry,201,206 cells of bone, 212 pharmacological regulation, 252-253,259 physiological regulation, 2 17,223-224 Crenophupngodon, melanin-concentrating hormone and, 3,5,25,30 Cyclic AMP,calcification and cells of bone, 2 I 1.215 endocrine factors, 220-222,231 pharmacologicalregulation, 247,256 Cycloheximide,glycosylation in intestinal epithelium and, 185 Cyclooxygenase,calcification and, 249 Cyclosporin, calcification and, 250 Cyclostomes, melanin-concentratinghormone and, 8-9 Cysteine, uptake mechanisms in plants and, 106 Cytochemistry,glycosylation in intestinal epithelium and, 138. 148, 157, 165 Cytochrome c reductase, cereal aleurone and, 82 Cytokines, calcification and, 230,24&241, 244-246.250 Cytokinin, uptake mechanisms in plants and, I10 Cytoplasm. cereal aleurone and, 63. 68
INDEX Cytosol, cereal aleurone and, 70.81-83
D Daruru srramonium lectin (DSL), intestinal
epithelium and, 159 Depletion, uptake mechanisms in plants and, 104 Dexamethasone, glycosylation in intestinal epithelium and, 183 Diacylglycerol, calcification and, 221 Dicalcium phosphate dihydrate, calcification and, 200 2,4-Dichlorophenoxyaceticacid, uptake mechanisms in plants and, 110 Differentiation, glycosylation in intestinal epithelium and, 185-187 Diffusion, uptake mechanisms in plants and, 89, 101,124-125 Dihydrotachysterol, calcification and, 224 Dihydrotestosterone, calcification and, 22 I , 230-23 1 2,4-Dinitrophenol, uptake mechanisms in plants and, 1 1 1 Discontinuous models, uptake mechanisms in plants and, 125-126 Dixon plot, uptake mechanisms in plants and, 99 DNA calcification and cells of bone, 2 11 endocrine factors, 220,222.23 1 pharmacological regulation, 249,260-261 physiological regulation, 234,236, 238-239,241-243 cereal aleurone and, 57,63-65,68,74 DNase, cereal aleurone and, 62 Dolichos biflorus lectin (DBL), intestinal epithelium and, 158 Dual and diffusion model, uptake mechanisms in plantsand, 124-125
E Eadie-Hofstee plot, uptake mechanisms in plants and, 93-95 EDTA, cereal aleurone and, 79 Elasmobranchs, melanin-concentrating hormone and, 9.20 Electron microscopy
297
cereal aleurone and, 72 glycosylation in intestinal epithelium and, 137, 148, 151,159,168, 176 Electrophoresis cereal aleurone and, 54 melanin-concentrating hormone and, 20 Emetine, glycosylation in intestinal epithelium and, 185 Endocrine factors, calcification and, 217-219 calcitonin, 222-223 growth hormone, 232-233 parathyroid hormone, 217,219-222 steroid hormones, 227-232 vitamin A, 226-227 vitamin D, 223-226 Endocytosis, calcification and, 217 Endoplasmic reticulum calcification and, 217 cereal aleurone and, 70.73-75.8042.84 glycosylation in intestinal epithelium and, 143, 145, 147 melanin-concentrating hormone and, 42 Endosperm, cereal aleurone and, 79.82 aleurone tissue, 49,5 1 a-amylase genes, 54-55 Energy, uptake mechanisms in plants and, 89,111 Enterocytes, glycosylation in intestinal epithelium and, 137, 157 Environment melanin-concentrating hormone and, 30 uptake mechanisms in plants and, 89 Enzymes calcification and, 197 biochemistry, 206-207 cells of bone, 212,217 inhibitiors, 247-249 pharmacological regulation, 252,255,261 physiological regulation, 241-242 cereal aleurone and, 49.80.83 aleurone tissue, 5 1.53 a-amylase genes, 54,61 protein transport, 70-72.74-75.77-78 melanin-concentrating hormone and, 20 uptake mechanisms in plants and, 123 Epidermal cells, uptake mechanisms in plants and, 110 Epidermal growth factor, calcification and, 221, 226,236-239-242.245 Epithelium calcification and, 237
298
INDEX
glycosylation in, see Glycosylation in intestinal epithelium Epitopes glycosylation in intestinal epithelium and. 177-1 78 melanin-concentratinghormone and, 8, 14, 18-19 Ervthrrno cristugalli lectin (ECL), intestinal epithelium and, 159 Escherichia coli
cereal aleurone and, 74 glycosylation in intestinal epithelium and, I78 uptake mechanisms in plants and, 117 Estradiol, calcification and, 228,230 Estrogen. calcification and cells of bone. 212 endocrine factors, 223.225.227-231,233 pharmacological regulation, 249-250,254 physiological regulation. 236.245 Evolution melanin-concentratinghormone and. I , 14, 29,34.41-43 uptake mechanisms in plants and, 89 Extracellular matrix. calcification and endocrine factors, 2 17,224 physiological regulation, 235,237,239, 242-243
F Fibroblast growth factor, calcification and, 236, 238-240,242-245 Fibroblasts,calcification and, 203.2 10,220, 224.236.240 Fibronectin. calcification and, 237 Fish, see also Teleosts melanin-concentratinghormone and, 1-2.43 biosynthesis. 20-2 1 color-change hormone, 3 6 3 4 stress respone, 35-40 structure, 14, 18 Flavanoids. calcification and, 254 Fluorescence cereal aleurone and. 8 1 glycosylation in intestinal epithelium and, 158- I59 melanin-concentratinghormone and, 5 Fluoride, calcification and, 200.251, 259-260 Fucose, intestinal epithelium and, 143, 175, 188 distribution, 155-157. 161, 163 exogenous agents, I X3-I85
Fucosyltransferase, intestinal epithelium and, 151, 157, 161,165. 185 G G-binding protein, calcification and, 219 Galactose, intestinal epithelium and, 143, 147 distribution of glycosyltransferases. 1 5 6 157 Golgi apparatus, 174-176 investigation methods, 153-154 Gdactosyltransferase. intestinal epithelium and, I45 distribution, 151-152, 154 exogenous agents, 183, 185 Golgi apparatus, 165, 168 post-Golgi apparatus distribution, 175-177, 182 Gallium, calcification and, 261 Genes, cereal aleurone and differential expression, 6 0 6 2 mechanism of hormone action, 64-68 multigene family, 55-59 synthesis. 54-55 Genetics, calcification and, 198-199 Genomes, cereal aleurone and, 56-57.59.66 Genotype, cereal aleurone and, 54 Gibberellic acid, cereal aleurone and, 49.5 I , 83 a-amylase genes, 5 4 5 5 , 5 9 4 2 calcium, 79.81-83 mechanism of hormone action, 6 3 4 , 6 6 7 0 protein transport, 7 1-72 Gibberellic acid-responsive element, cereal aleurone and. 64-65.67 Glucagon, melanin-concentratinghormone and. 8 Glucocorticoids, calcification and, 225,227, 235-236.254.258 Glucose intestinal epithelium and, 142-143, 174-175, 186 uptake mechanisms in plants and, 94-95, 108 Glucose-&phosphate dehydrogenase, calcification and, 21 3 Glutamic acid, calcification and, 202 Glycogen, calcification and, 209,228 Glycolipids, intestinal epithelium and, 148, 157, 185
Glycopeptides, intestinal epithelium and, 156, 186 Glycoprotein calcification and, 201-203,206,219,237
INDEX cereal aleurone and, 78 glycosylation in intestinal epithelium and, 135,139,141 distribution, 151, 153, 155-157 exogenous agents, 185 Golgi apparatus, 173 investigation methods, 148 oligosaccharides, 143, 147 post-Golgi apparatus distribution, 177, 181 Glycosaminoglycans,calcification and, 257 Glycosylation calcification and, 240 cereal aleurone and, 78 Glycosylation in intestinal epithelium, 135-141, 187-1 88 differentiation, 185-187 distribution of gl ycosyltransferases adult animals, 151-154 development, 154-155 lectin binding, 157-166 sugar content, 155-157 drugs, 184-185 Golgi apparatus, 165 lectin-binding sites, 173-175 localization, 168-173 subcompartmentalization,165, 167-1 68 hormones, 183-184 investigation methods, 147 biochemical, 148 morphological, 148-15 1 oligosaccharides N-glycosidically linked, 141-143 0-glycosidically linked, 143-147 post-Golgi apparatus distribution, 175-182 Glyoxsomes, cereal aleurone and, 78 Goblet cells, glycosylation and, 137-138, 182 distribution, 154-155,158-159, 161 Golgi apparatus, 168-169,17&175 ol igosaccharides, 145- 146 Gold, calcification and, 261 Gold sodium thiomalate, calcification and, 261 Golgi apparatus calcification and, 198,209,217 cereal aleurone and, 7&71 glycosylation in intestinal epithelium and, 137 distribution, 155, 158-159 exogenous agents, 183-1 85 investigation methods, 147-148.15 1 lectin-binding sites, 173-2 75 localization, 168-173 oligosaccharides, 141, 143, 145-147 post-Golgi apparatus, 175-182
299
subcompartmentalization,165,167-1 68 Granculocyte colony-formingcells, calcification and, 215 Granculocyte-macrophage-colonyforming cells, calcification and, 215 Granculocyte-macrophagecolony-stimulating factor, calcification and, 21 1,221,245-246 Growth factors, calcification and, 262 pharmacologicalregulation, 259 physiological regulation, 217,220,222,226 steroid hormones, 229-230 Growth hormone, calcification and, 227,230, 232-233,235
H Helixpomatia, intestinal epithelium and, 145-146 Helixpomatia lectin (HPL), intestinal epithelium and, 159 Hemopoietic cells, calcificationand, 213,215 Heparin, calcificationand, 242,257-258 Histamine, calcification and, 256 Homology cereal aleurone and, 56,59,73 melanin-concentratinghormone and, 1, 18, 20,4142 Hormone-responsiveelements,cereal aleurone and, 59.62 Hormones, see also specific hormone calcification and, 199,217,253,258,262 cereal aleurone and, 49,5 1,82-83 genes, 64-68 perception, 63-64 steroid model, 68-70 glycosylation in intestinal epithelium and, 183-184 Human growth hormone-releasingfactor, melanin-concentratinghormone and, 13-14,18-19 Hydroxyapatite,calcification and, 197-198.262 biochemistry, 199-200,202-205 pharmacologicalregulation, 260 Hypercalcemia,calcification and, 221,257-258, 26 1 Hypertrophy,calcification and, 234,239 Hypocalcemia,calcification and, 223,253, 256-258 Hypothalamo-pituitary-interrenalaxis, melaninconcentrating hormone and, 35-37 Hypothalamus,melanin-concentratinghormone
300
INDEX
and, 2-3,40 anatomical distribution. 3-5.7-9, biosynthesis, 2 C 2 1 structure, 15, 20
1I , 13
I Imidazole. calcification and, 255 Immune modulators, calcification and, 250 Immune system, calcification and, 255 Immunocytochemical localization, glycosylation in intestinal epithelium and, 147, 151, 175, 177, 182 Golgi apparatus, 167-173 Immunocytochemistry cereal aleurone and. 70 glycosylation in intestinal epithelium and. 148, 1.53, 165, 175, 181 melanin-concentrating hormone and, 3-4.8, I1 Immunofluorescence, gl ycosylation in intestinal epithelium and, 168, 177 Immunoglobulin, glycosylation in intestinal epithelium and, 176 Immunoreactive melanin-concentrating hormone, 3.9. 1 1. 14, 39 Immunoreactive a-melonocyte-stimulating hormone, 8 Immunoreactivity glycosylation in intestinal epithelium and. 146,167, 176 melanin-concentrating hormone and, 8.21 lndoleacetic acid. uptake mechanisms in plants and, 110 Inflammatory diseases, calcification and, 244-246 Inhibition calcification and, I97 biochemistry, 201,204-206,210-212,215 cytokines, 245-246 endocrine factors, 217,219,221-226 enzymes, 247-249 pharmacological regulation, 250-261 physiological regulation. 235-239, 241-243 steroid hormones, 228-229,23 I cereal aleurone and, 53,55.61,64,71 glycosylation in intestinal epithelium and. 177, 185 melanin-concentrating hormone and, 7,34
uptake mechanisms in plants and, 96-99, 107, 111, 124 Insulin, calcification and, 240 Insulinlke growth factors, calcification and endocrine factors, 220,225-226,229-230, 232 physiological regulation, 233-236.243 Integral membrane proteins, intestinal epithelium and, 175 Interleukin- 1, calcification and endocrine factors, 221,230,238-239 physiological regulation, 240,242,244-248 Intestinal epithelium, glycosylation in, see Glycosylation in intestinal epithelium Invertebrates, melanin-concentrating hormone and, 14 Ion-carrier complex, uptake mechanisms in plants and, 125 Ion channels, uptake mechanisms in plants and, 125 Ion flux effectors, calcification and, 25 1 Ion transport, calcification and, 199 3-Isobutyl-methylxanthine(IBMX), calcification and, 248 Isoelectric focusing, cereal aleurone and, 55 Isoelectric point (PI), cereal aleurone and a-amylase genes, 55,57,59-60,62 mechanism of hormone action, 65-66 protein transport, 76-77 Isoforms, cereal aleurone and a-amylase genes, 55-56,58,6&62 calcium, 79-82 protein transport, 7 1,7&77 Isotherm, uptake mechanisms in plants and, 122-127, 130-131 dual model, 91-92 multipha.icmodel.93, 113-114, 116-121 amino acids, 105-106 benzyladenine, 110 inorganic ions, 102, 104
K Kidney, calcification and, 217.221-222.227
K,, uptake mechanisms in plants and, 91,93, 111-114, 126-127
L Lacuna. calcification and, 212,215-217
NDEX Lectin, glycosylation in intestinal epithelium and, 145 distribution of glycosyltransferases. 155-1 63 exogenous agents, 183-184 Golgi apparatus, 165,167,172-175 investigation methods, 148, 150, 153 Leucine, uptake mechanisms in plants and, 106-107 Levamisole, calcification and, 255 Leydig cells, calcification and, 227,257 Light microscopy, glycosylation in intestinal epithelium and, 159, 176 Limaxflavus lectin (LFL), intestinal epithelium and, 161,163, 165, 167, 172-173 Lineweaver-Burk plot, uptake mechanisms in plants and, 93-95,99, ilU, 128 amino acids, 107 inorganic ions, 102-104 Lipid intestinal epithelium and, 135, 139, 141, 143 uptake mechanisms in plants and, 127 Localization, glycosylation in intestinal epitheliumand, 147, 151, 175, 177, 182 Golgi apparatus, 167-173 Lotus rerragonolobus lectin (LTL), intestinal epithelium and, 158-159, 161 Lupinus polyphyllus, uptake mechanisms in plants and, 110 Luteinizing hormone, melanin-concentrating hormone and, 40 Lysine, uptake mechanisms in plants and, 105-107 Lysosomes, cereal aleurone and, 84
M Maackia amurensis lectin (MAL), intestinal epithelium and, 161 Macrophage colony-forming cells, calcification and, 215,252 Macrophage-colony stimulating factor, calcification and, 244 Macrophages, calcification and, 21 1,215,221, 245-246.256 Mammals, melanin-concentrating hormone and, 3,23,35,42 anatomical distribution, 8, 11-14 structure, 14-15, 18-20 Mannose glycosylation in intestinal epithelium and,
30 1
157, 165 intestinal epithelium and, 139, 141-143, 155, 186-1 87 Mannose-&phosphate receptor, calcification and, 216 Mapping, melanin-concentrating hormone and, 11
Matrix vesicle, calcification and, 197,205-206, 224-225 Melanin-concentrating hormone, 1,4@43 anatomical distribution, 3.8 in amphibians, 9-1 I in cyclostomes, 8-9 in elasmobranchs, 9 in invertebrates, 14 in mammals, 11-14 in teleost fish, 3-8 biosynthesis, 20-21 discovery, 1-3 physiology color-change hormone in fish, 30-34 higher vertebrates, 3 9 4 0 stress-response in fish, 35-39 precursor molecule of mammalian MCH, 18-19 of teleost MCH, 15-18 structure, 1 4 1 5 , 2 0 structure-activity studies, 2 1-23 melanin-concentrating activity, 25-30 aMSH-like activity, 23-24 Melanin-concentrating hormone-gene-related peptide, 15, 18,21 Melanocyte-stimulating hormone, 2-3 a-Melanocyte-stimulating hormone, 43 distribution, 8-9, 13-14 physiology, 3 I-36,40 structure, 18-20 structure-activity studies, 23-24,30 Melanophores, 3, 15 physiology, 30-33,40 structure-activity studies, 23-26 Mesenchyme, calcification and, 209.238-239, 24 1 Messenger RNA calcification and biochemistry, 202,204-205 endocrine factors, 220,225-230 physiological regulation, 235,238 cereal aleurone and, 73,83 a-amylase genes, 5455,57,60-62 mechanism of hormone action, 68-69
302
INDEX
melanin-concentratinghormone and, 15,20
MET.melanin-concentratinghormone and, 15 Metalloproteins. cereal aleurone and, 79-80 Michaelis-Menten kinetics, uptake mechanisms in plants and, 90. I3 1 dual model, 92 multiphasic model, 93.95,99. 101, 104 Microtubules, glycosylation in intestinal epithelium and. 184-185 Milrinone. calcification and, 248 Mineralization. calcification and. 198-199, 262-263 biochemistry. 200-207 cells of bone, 209-21 2 epidermal growth factor, 241-242 pharmacological regulation, 25 I. 260 physiological regulation. 223-224.226-228, 230-233 transforming growth factors. 237-238 Mitogen, calcification and, 220,237,243 Monensin, cereal aleurone and, 70-7 I Monocytes,calcification and, 215,230,244245 Morphogenesis,calcification and. 200,236 Morphology calcification and, 209,237 glycosylation in intestinal epithelium and, 147-151, I85 melanin-concentratinghormone and, 13 Mucin. intestinal epithelium and, 146, 153. 182 Multienzyme system. intestinal epithelium and. 165 Multiphasic uptake mechanisms in plants, see Uptake mechanisms in plants Mutagenesis. cereal aleurone and, 65 Mutation, melanin-concentratinghormone and, 14. 18
N NEI. melanin-concentratinghormone and, 18-20,4 1 Neurohypophysis,melaninconcentrating hormone and, 7-9.20.39 Neuromodulators,melanin-concentrating hormone and, 5.7.37.39 Neuropeptides,see Melaninconcentrating hormone Neurotransmitters,melanin-concentrating hormone and, 5.3 I , 39
NGE, melanin-concentratinghormone and, 18-19.41 Nitrate, uptake mechanisms in plants and, 1w105 Nocodazoie, glycosylation in intestinal epithelium and, I85 Noncollagenousproteins, calcification and, 197, 201-207.262 Nonsteroidal antiinflammatorydrugs (NSAIDS), calcification and, 249 Nucleation theory. calcification and, 196-197 Nucleotide pyrophosphate, intestinal epithelium and, 151-152, 176-177 Nucleotides cereal aleurone and, 57 glycosylation in intestinal epithelium and, 141, 182 Nucleus lateralis tuberis, melanin-concentrating hormone and, 4-5.9 Nutrition, cereal aleurone and, 5 I
0 Oats. uptake mechanisms in plants and, 114, 116 Oligosaccharides cereal aleurone and, 78 glycosylation in intestinal epithelium and differentiation, 186-1 87 Golgi apparatus. 167, 171 OnchorhJnchus.melanin-concentrating hormone and, 14-15.20 Osteoblasts, calcification and, 209-2 12,262 biochemistry, 199,204 endocrine factors, 2 17,220-228.230-232 pharmacologicalregulation, 247-25 1.258, 260 physiological regulation, 234,236-239, 24 1-246 Osteocalcin, calcification and biochemistry. 204-205 cells of bone, 2 I2 endocrine factors, 217,220,224,227 pharmacologicalregulation, 250-25 I physiological regulation, 237,243,250-251 Osteocalcium phosphate, calcification and, 197, 200 Osteoclasts. calcification and, 2 10-2 17,262 biochemistry, 199,204
INDEX endocrine factors, 220-221.225-226 organic agents, 252,255,257-258 pharmacologicalregulation, 247-248,250,
260 physiological regulation, 237.241-242,
244-246
303
Peptides calcificationand, 203-204 cereal aleurone and, 73,78 glycosylation in intestinal epithelium and,
139,141,145-146,181 melanin-concentratinghormone and, 15, 18,
Osteocytes, calcification and, 209.212-213.223 Osteogenesis, calcification and, 197.211,241 Osteogenin, calcification and, 239-240 Osteoinductive factor, calcification and,
239-240 Osteomalacia, calcification and, 260 Osteonectin, calcification and, 203,217,237 Osteopenia, calcification and, 210,250,254,
257,259 Osteopetrosis, calcification and, 213,215 Osteopontin, calcification and, 201,203,237,
243,249 Osteoporosis, calcification and, 195 pharmacologicalregulation, 253,257,259,
261 physiological regulation, 222,227,230-231 Osteoprogenitors,calcification and, 209,
223-224.242 Osteosarcomacells, calcification and, 210 biochemistry,203,205 endocrine factors, 221,225-231 pharmacologicalregulation, 248,251 physiological regulation, 233-234.238, 240-24 I , 243 Oxalates, calcification and, 198
P Palindromes, cereal aleurone and, 654% Pancreas, cereal aleurone and, 73 Pancreatica-amylase, cereal aleurone and, 77 Parathyroid hormone, calcification and biochemistry, 204 cellsofbone,2lO,212,215-216 endocrine factors, 217-222.226-229.231 inorganic effectors, 260,262 pharmacologicalregulation, 247-248.251,
21.40-43 Perichondrium,calcification and, 227 Periosteal cells, calcification and, 21 1,
227.237 pH, uptake mechanisms in plants and, 95-96, 110
Phagocytes, calcification and, 215 Pharmacologicalregulation, calcification and,
246-25 1,263 inorganic effectors,259-262 organic agents, 252-258 Phenotype, calcification and, 210,212 pharmacologicalregulation, 252 physiological regulation, 238,240,243,246 Phosphate calcification and, 197-198 biochemistry,205,207 pharmacologicalregulation, 248,252 physiological regulation, 217,223 uptake mechanisms in plants and, 124-126 multiphasic model, 95-97.99,102,113,122 Phosphatidylserine,calcification and, 205,223 Phosphodiesterase,calcification and, 21 1,255 inhibitors, 247-248 PhospholipaseA,, calcification and, 225,244 Phospholipase C, calcification and, 221 Phospholipids,calcification and, 205,223 Phosphoprotein,calcification and, 201-202,217 Phosphorus calcification and, 224,255,261 uptake mechanisms in plants and, 122 Phosphorylation,calcification and, 202-203,
206,259
Pituitary calcification and, 227,235-236 melanin-concentratinghormone and, 1-3,43 anatomical dismbution, 3-5,7-9,I I , 13 biosynthesis, 20 physiology, 30,32-37,3940 254,256-258 structure, 14 physiological regulation,238,240,242-243.245 Plants, uptake mechanisms in, see Uptake Parathyroid hormone-relatedpeptide, mechanisms in plants calcification and, 221-222,239,245 Plasma, melanin-concentratinghormone and, Peanut lectin (PNL), intestinal epithelium and,
156,159,184
31-32,36,3840
304
INDEX
Plasma membrane calcificationand. 197. 213. 219. 224 cereal aleurone and, 68.70-7 I. 82-83 glycosylation and, 135. 137. 147-148 differentiation, I86 distribution of glycosyltransferases. 15 I. 155-156 exogenous agents, 184-1 85 Golgi apparatus. 173 lectin binding, 158-159, 161. 163, 165 post-Golgi apparatus distribution. 175-176. 178-179, 182 uptake mechanisms in plants and, 89 Plasmalemma,uptake mechanisms in plants and, 89-90. 130- 1 3 1 molecular basis, 127 multiphasicmodel. 110, 114. 116, 118-120 Plasmids. cereal aleurone and. 77 Plasminogen activator, calcification and. 2 12. 238.24X. 258 Plastids, uptake mechanisms in plants and, 90, 99. 118. 125 Platelet-derivedgrowth factor. calcification and, 240,243 foecilia. melanin-concentratinghormone and, 2. 5.7.33 Polyamine, glycosylation in intestinal epithelium and. 185 Polypeptides calcification and. 200.2 19 cereal aleurone and, 54.56.78-79 glycosylation in intestinal epithelium and, 143. 145. 147. 165, 177-178 POMC. melanin-concentratinghormone and, 8. 13.20,35 Porcine pancreatic amylase. cereal aleurone and. 79 Posttranslational modifications. cereal aleurone and, 55. 7 6 7 1 Potassium calcification and, 2 16.25 I. 260 uptake mechanisms in plants and, 90, 124, 130-131 dual model, 9 I multiphasicmodel. 102, I l l . 114-116. 118-119,121 Pre-prohormonemelanin-concentrating hormone. IS, 18-19.41 Preosteoblasts. calcification and, 209 Procollagen. calcification and, 226-228
Proenzymes, cereal aleurone and, 78-79 Progesterone,calcification and, 228,230 Prohormone melanin-concentratinghormone, 4 1 anatomical distribution, 8 biosynthesis, 2 I structure, 20 Prohormones, melanin-concentratinghormone and, 15, 18.4W1 Prolactin, melaninconcentratinghormone and, 40 Promethazine, calcification and, 255-256 Prostaglandin,calcification and cellsofbone,211-213 endocrine factors, 220,229 pharmacological regulation, 247.25 I , 254 physiological regulation, 238,241-245 synthesis inhibitors, 248-249,255 Proteases, cereal aleurone and. 74.78,80,84 Protein, see also specific protein calcification and, 197,262 biochemistry, 200-207 endocrine factors, 226,23@-232 pharmacological regulation, 259-262 physiological regulation, 234,236,243 glycosylation in intestinal epithelium and, 135, 139. 147-148, 185 distribution of glycosyltransferases, 154, 156157, 172 post-Golgi apparatus distribution, 175, 177-178, 182 melanin-concentratinghormone and, 20-21, 4142 regulation in cereal aleurone. see Cereal aleurone uptake mechanisms in plants and, 1 10, 126127 Protein kinase C , calcification and. 221,241 Protein-A gold technique. glycosylation in intestinal epithelium and. 149, 178 Proteoglycans,calcification and biochemistry, 201-202 endocrine factors, 2 17,223,226,228.23 I physiological regulation, 235-236.238-242, 245-246 Proteolipids,calcification and, 199,205 Proteolysis cereal aleurone and, 74,80 melanin-concentratinghormone and, 18 Prothrombin,calcification and, 204 Protoplasts cereal aleurone and. 6345,72,76,81
305
INDEX uptake mechanisms in plants and, 89-90, 124-125 multiphasic model, 107, 110, 118 Pseudo-dual model, uptake mechanisms in plants and, 125 Pyrimidine, cereal aleurone and, 65-68 Pyrophosphate calcification and, 217,252-253 uptake mechanisms in plants and, 96
R Radioimmunoassays,melanin-concentrating hormone and, 3.1 1,39 Rana, melanin-concentratinghormone and, 24 Rana pipiens. melanin-concentratinghormone and, 23 Rana ridibunda, melanin-concentrating hormone and, 9.20.39 Rana temporaria, melanin-concentrating hormone and, 11 Rat growth hormone-releasingfactor, melaninconcentrating hormone and, 19 Rat melanin-concentratinghormone, 11 Rat osteosarcoma cells, calcification and, 226-221,229,238 Recombination,cereal aleurone and, 77 Retinoic acid, calcification and, 225-227 Retinoids, calcification and, 226-227.232 Retinol, calcification and, 226-227 Rheumatoid arthritis, calcification and, 195, 244,26 1 Rice, uptake mechanisms in plants and, 122 Ricinus communis lectin I (RCL I), glycosylation in intestinal epithelium and, 156,158-159,161,168,172 Ricinus communis lectin I1 (RCL 11). glycosylation in intestinal epithelium and, 159 RNA calcification and, 213 cereal aleurone and, 60,63 melanin-concentratinghormone and, 42 RNA polymerase, cereal aleurone and, 64.68 Rough endoplasmic reticulum, glycosylation in intestinal epithelium and, 141, 143, 145-146 Rubidium, uptake mechanisms in plants and, 90, 124
S Saccharide, glycosylation in intestinal epithelium and, 140 distribution, 151-157 Golgi apparatus, 165-175 lectin binding, 157-166 post-Golgi apparatus distribution, 175-182 Salivary a-amylase, cereal aleurone and, 77 Salmon, melanin-concentratinghormone and, 3-4, 14-15.18 Salmonid melanin-concentratinghormone, 40, 42 anatomical distribution,4, 11 physiology, 3 9 4 0 structure, 14-15 structure-activity studies, 23.25-28 Sambucus nigra L. lectin (SNL I), intestinal epitheliumand, 161, 163,173 Scutellum,cereal aleurone and, 49.5 1,54,71, 78 Second messengers, cereal aleurone and, 83 Secreted proteins in cereal aleurone, see Cereal aleurone Selective pressures, melanin-concentrating hormone and, 4 1 4 3 Sequences calcification and, 204 cereal aleurone and a-amylase genes, 56-57,59,62 mechanism of hormone action, 64-68 melanin-concentratinghormone and, 3.4 1 4 2 structure, 14-20 structure-activity studies, 23,26,29 Sialic acid calcification and, 202 intestinal epithelium and, 143, 147, 176, 183 distribution, 156-157, 159, 161, 163, 165 Golgi apparatus, 168-169, 171 Sialyltransferase,intestinal epithelium and distribution, 151-154, 161 exogenous agents, 183-184 Golgi apparatus, 165,167-169,171,173 post-Golgi apparatus distribution, 175-178, 181-182 Signal transduction cereal aleurone and, 83 melanin-concentratinghormone and, 25 Skeleton, vertebrate, calcification in, see Calcification
306
LNDEX
Sodium calcification and, 216,251,253,260 uptake mechanisms in plants and, 91, 102. 113. 119 Soybean lectin (SBL),intestinal epithelium and, 156,158-159 Spinal cord, melanin-concentratinghormone and, 9. I I Spleen, calcification and, 2 I3,2 15 Starch, cereal aleurone and, 5 I , 54,79,82 Steroids calcification and, 221.225,227-232 cereal aleurone and, 65,68-70 Stress, melanin-concentratinghormone and, 3540 Structure-activity studies, melaninconcentrating hormone and, 2 1-30 Subcompanmentalization,glycosylation in intestinal epithelium and. 165, 167-168, 171-172, 177 Sucrose. uptake mechanisms in plants and, 110 sugar glycosylation in intestinal epithelium and, 135. 137,139. 141,143, 148 distribution of glycosyltransferases, 153, 155-157, 163 Golgi apparatus, 167, 173 uptake mechanisms in plants and, 108, I10 Sulfate calcificationand, 198,201.226 uptake mechanisms in plants and, 93,95-98, 112-113,121 aminoacids, 1 W 1 0 7 inorganic ions, 99-100 Synbranchrcr, melanin-concentratinghormone and. 23-24.26.30
T Tamoxifen, calcification and, 249 Teleosts. melanin-concentratinghormone and, 1-2.42-43 anatomical distribution, 3-8 physiology. 30.33-34.39-40 structure. 14-18 structure-activity studies, 23-25.30 Testosterone calcification and. 221,223,227.230-232 glycosylation in intestinal epithelium and, 183 Theophylline,calcification and, 247-248
Thiamine pyrophosphatase,intestinal epithelium and, 167 Thiazide diuretics, calcification and, 253-254 Thiophene carboxylic acids, calcification and, 256-257 TIMP, calcification and, 238 Tonoplasts, uptake mechanisms in plants and, 119-120,131 Transcription calcification and. 224,261 cereal aleurone and a-amylase genes, 55.61-62 mechanism of hormone action, 63-65, 67-69 Transforming growth factor-a, calcification and, 241-242,245 Transforming growth factor+, calcification and biochemistry, 204 cells of bone, 21 I endocrine factors, 220,222,226,230 physiological regulation, 236241,245 Transforming growth factors, calcification and, 236239 Trans-Golgi network, intestinal epithelium and, 145, 168 Translation, melanin-concentratinghormone and, 42 Translocation cereal aleurone and, 73-74 melanin-concentratinghormone and, 2 1 Tropocollagen,calcification and, 200 Tumor necrosis factor, calcification and, 241, 245-246
U Ulex europaeus lectin I (UEL I), intestinal epitheliumand. 157-159, 163, 184 Uptake mechanisms in plants, 89-91. 122-123. 128, 130-131 cooperative model, 123 discontinuous models, 125-1 26 dual and diffusion model, 124-125 dual model, 9 1-92 molecular basis, 126-129 carriers, 126-1 27 channels, 126-127 transition site, 127 multiphasic model. 1 18-1 22, 128, 130 amino acids, 105-109
INDEX initial experiments, 93-95 inorganic ions, 99-105 K,, 112-1 15 potassium, 114-1 16 solute concentrations, 110-1 12 sugars, 108,110 transition sites, 95-99 transport in bacteria, 116-117 VmaX,112-115 pseudo-dual models, 125 single and diffusion model, 124
V Vacuoles calcification and, 2 13 cereal aleurone and, 5 I , 70,82 uptake mechanisms in plants and, 90, 120, 131 Vanadate, calcification and, 260 Vertebrate skeleton, calcification in, see Calcification Vertebrates, melanin-concentrating hormone and, see Melanin-concentrating hormone Vesicles calcification and, 197-198.205.217 glycosylationin intestinal epithelium and, 143, 145,159,179 Vitamin A, calcification and, 226-227 Vitamin D, calcification and biochemistry, 204
307
endocrine factors, 221,223-227 pharmacological regulation, 25 1,254,260, 262 physiological regulation, 236,238-240.245 Vitronectin, calcification and, 203,215 V,,,,,, uptake mechanisms in plants and, 101, 111-114,121,126-127
W Warfarin, calcificationand, 204 Wheat, cereal aleurone and, 49,5 1 a-amylase genes, 54 mechanism of hormone action, 6 3 , 6 5 4 6 Wheat germ agglutinin (WGA), glycosylation in intestinal epithelium and, 156,158-159, 161, 184 WR-2721,calcification and, 257
X Xenopus, melanin-concentrating hormone and, 2 Xenopus laevis cereal aleurone and, 57,74,77 melanin-concentrating hormone and, 11
Z
Zinc, calcificationand, 206,261-262
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