Cytoskeleton and molecular mechanisms in neurotransmitter release by neurosecretory cells

European Joun~al of Pharmacology, - Mok,cular Pharmacok~gy Section, 225 (1992) 83-1fl4

83

~) 1992 Elsevier Science Publishers B.V. All rights resel-:ed 0922-4i06/92/$1/5.00

EJPMOL 90268

Review

Cytoskeleton and molecular mechanisms in neurotransmitter release by neurosecretory cells J . - M . T r i f a r d , M . L . V i t a l e a n d A. R o d r l g u e z D e l C a s t i l l o Secretot?," Process Research Program, Department of Pharmacology, Unirersity of Ottawa, Ottawa, Ontario KIH 8M5, Canada

Received i5 November i991. accet~ted i9 November i991

The process of exocytosis is a fascinating interplay between secretory vesicles and cellular components. Secretory vesicles are true organelles which not only store and protect neurotransm;tters from inactivation but also provide the cell with efficient carriers of material for export. Different types of secretory vesicles are described and their membrane components compared. Associations of several cytoplasmic proteins and cytoskeletai components with secretory vesicles and the importance of such associations iv the mechanism of secretion are discussed. A description of possible sites of action for Ca 2 * as well as possible roles for calmodulin, G-proteins and protein kinase C in secretion are aiso presented. Important aspects of the cytosketeton of neurosecretory cells are discussed. The cytoskeleton undergoes dynamic changes as a result of cell :,timulation. These changes (i.e. actin filament disassembly) ~hich are a prelude to exocytosis, play a central role in secretion. Moreover, advanced electrophysiological techniques which allow the study of secretory vesicle-plasma membrane fusion i~ real-time resolution and at the level of the single secretory vesicle, have also provided a better understanding of the secretory process. Neurotransmitter release; Neurosecretory cells; Cytoskeleton; Cytoplasmic proteins; Ca -~+; Calmodulin; G-proteins; Protein kinase C

1. Introduction As early as 1905, Scott advanced the hypothesis that neurons were something more than a system of conducting paths. T h e r e were true secreting cells which act upon one another and upon cells of other tissues by passage of a chemical substance from the first to the second cell (Scott, 1905). Since this early observation, our knowledge about the secretory functions of neurons, as well as other secretory cells, has moved forward quickly, because of the development of powerful electrophysiological, histochemical, electromicroscopic, biochewlcal and molecular biology techniques. Moreover, it has become clear in recent years that secretion by cells can take two forms, constitutive and regulated (Tartakoff et al., 1978; G u m b i n e r and Kelly, t982; Kelly, 19851. Constitutive secretion is the form of secretion which is unregulated and closely follows the rate of synthesis of secretory products. This form of secretion occurs in almost all cell types including lymphocytes, liver, yeast cells, etc. (Buckley and Kelly,

Correspondence to: Dr. J.-M. Trifard. Department of Pharmacology, University of Ottawa. 451 Smyth Road, Ottawa Kltt 8M5, Canada. Tel. (613) 787-6557; Fax (613) 731-8949.

i985; Kelly, 1985). The other form of secretion is highly regulated and is characteristic of acarons as well as endocrine and exocrine cells. Cells displaying regulatory s e c r e t o w pathways store their secretory products in m e m b r a n e bound secretory vesicles or granules (Trifar& 1977; Trifar6 and Poisner, 19821. An exception among endocrine tissues are the steroid-secreting cells (adrenal cortex, ovary, testis). Secretory vesicles are not present '~n these tissues and secretion is constitutive since release of steroids is coupled to their synthesis (Trifar6, 1977). Granule storage not only allows secretory tissues to store large amounts of secretory, material in a relatively small volume, but also protects this material from intracellu!ar degradation and provides a very efficient means for transporting and releasing fixed amounts (quanta) of secretory, substances (Trifar6, 1977; Trifar6 and Poisner, i982). Secretory cells displaying secretory vesicles have been named 'paraneurons', a term which embraces cells generally and traditionally not considered as neurons, and yet should be regarded as 'relatives' of ,leurons on the basis of their strw:ture and function (fig. 1). The chromaffin ceil of the adrenal medulla has shown itself to be one of the most useful systems for the study of development and fuc, ctional aspects of paraneurons (Trifar6, 1982). Chromaffin cells derive embriologically from the

84

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cytoskeleton of the neurosecretory cell (Trifaro, t989b, 1990a; Vitale et al., 199!a). This review seeks to unify published observations concerning cellular and molecdlar events involved in neurosecrction.

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neural crest sharing a common origin with the sympathetic neurons and common subcellular features with many neuro-endocrinc cells (Trifar6, 1982, 1984). Early observations on this secretory cell have been the starting point of many important studies on the cellular and molecular mechanisms involved in the secretions of numerous secretory systems. Consequently, findings on chromaffin cells are frequently mentioned in this review (Trifar6, 1982, 1984). Regulatory secretion is lnediated by exocytosis, a mechanism whereby the membrane of the secretory vesicles fuses with the plasma membranes allowing the escape of the contents of the vesicle to the cell extcrior. Regulatory secretion is triggered by an increase in intracellular Ca 2+. ltowever, despite the fact that the role of Ca 2+ secretion was observed many years ago (Houssay and Molinelli, 1928; Hatwey and Macintosh, i940; Douglas and Rubin, 1961; Douglas, 1968), the exact mechanisms in which Ca 2+ is involved in the secretory process are still poorly understood. One attractive hypothesis is that the action of Ca -,+ in secretion is mediated by calmodu!in (Trifar6 et al., 1985a; Trifar6 and Fournier, 1987; Trifar6 and Kenigsberg, 1987), an intracellular Ca 2+binding protein ubiquitous to the cucaryotcs. Another attractive hypothesis for the role of Ca -,+ in secretion is the regulation by this ion of dynamic changes in the

all-or-none

phenomenon

Work on chromaffin cells has been crucial in the demonstration of the release of secretory products by exocytosis (Douglas, i974; Viveros, 1974; Trifar6, 1977). In 1957, De Robertis and Vaz Ferreira first suggested that secretion was by exocytosis (reverse pinocytosis), a mechanism whereby the membrane of the secretory granules fuses with the plasmalemma allowing the escape of the contents of the granule to the cell exterior. Moreover. chemical studies carried out in adrenal medulla have provided biochemical evidence in favour of release by exocytosis (Douglas, 1974; Viveros, 1974; Trifar& 1977). These studies have shown that catecholamines were released during stimulation along with other secretory granule components. In the chromaffin cell there is a concomitant release of ATP, dopamine /3-hydroxylase, chromogranin A and other neuropeptides along with catecholamines (Banks and Helle, 1965; Douglas et al., 1965; Viveros et al., 1968; Lastowecka and Trifar6, 1974). These soluble substances are quantitatively . recovered in the e ' ';:t;e n t escaping from the stimulated glands. Furthermore, .~abcellular fractionation studies carried out on the stimulated medulla have shown that the entire soluble content of the granule "- discharged to the cell exterior, thus demonstrating ~:_:.' exocytosis is an all-or-none re!ease process (Viveros et ai., 1969; Trifar6. 1977). Moreover, other biochemical studies have demonstrated ;hat granule membrane components are retained within the cells after exocytosis (Trifar6 et al., 1967; Poisner et al., 1967; Vivmos et al., 1969) (fig. 2), and although large molecules (for example, dopamine ,8-hydro~lase) leave the cell during exocytosis, no cytoplasmic marker enzymes such as lactic dehydrogenase, are detected in the effluents escaping from stimulated tissues (Schneider et al., 19967; Matthews et al., 1973; Lastowecka and Trifar6, 1974). This indicates that the vital feature of the neurosecretory cell, that is the membranous isolation of the cytosol from the extraceltular environment, is maintained during exocytosis, a~zvcl" ........ ,n pieces of evidence aiso il~dicate ,1u,,~ . . . . ~,,,.ha release of neurotransmitter from the sympathetic neuron is also by exocytosis. In order to show that the noradrenergic vesicle was the site of the release of noradrenaline, it became necessary to rule out the cytosol as the origin of the noradrenaline found in the effluents from organs during adrenergic nerve stimulation. One way to test this, was to induce the accumulation of noradrenaline in the

~5 with vesicle cytosol. This was achieved by m~,.,_,~.,lng " *....... storage, tk-Jr example by using reserpine. This treatm e n t , w h e n a c c o m p a n i e d by the use of m o n o a m i n e oxidase inhibitors, resulted in the accumulation of ip.tact n o r a d r e n a l i n e in the cytoplasm (Malmfors. 1965; H~iggendal and Malmfors, !969). U n d e r these conditions, sympathetic nerves can take up and store noradrenaline. This type of cytoso! storage can be d e m o n strated by a s m o o t h distribution of fluorescence in the n e u r o n (Malmfors, 1965), by subcellular fractionation studies, which have shown that most o f the nora d r e n a l i n e taken up is not s e d i m e n t a b l e (Iversen et al., 1965; Lundborg, 1967; Potter, 1967) and by autoradiography, which shows the cytoplasm of the n e u r o n and not the vesicles b e c a m e labelled (Taxi, 1969). W h e n the sympathetic nerves were stimulated u n d e r these conditions, no n o r a d r e n a l i n e was r e l e a s e d into the extracellular space (Van O r d e n et al., 1967; H~iggendal and Malmfors, 1969; F a r n e b o and H a m b e r g e r . 1970). T h e r e f o r e , it was c o n c l u d e d f r o m these pharmacological e x p e r i m e n t s , that w h e n a m i n e metabolism and vesicle p u m p w e r e inhibited - - conditions leading to an accumulation of n o r a d r e n a l i n e in extravesicular sites

CONTROL CE'_L

--- no release of amine was observed upon nerve stimulation. E x p e r i m e n t s will] false transmiiterf, also ' u l e out release from extragranular s~ort:s, in addition to n o r a d r e n a l i n e , many pherlylethylamine derivatives can be taken up, stored in vesicles, become hydroxylated and be r e l e a s e d upon nerve stimulation (Muschot! and Maitre, 1963; Muscho!!. 1972). Inhibition of vesicle r e t e n t i o n (i.e. after reserpine) makes the false transmitters unavailable for release (Muscholt, 1972)~ Ano t h e r piece of evidence in favour o f release from vesicles is that (with the exception o f d o p a m i n e and ce-methyldopamine) the output of non-/3-hydroD,lated amines (tyramine, a-methyltyramine, phenylcthyiamine, a m p h e t a m i n e , etc.) is not increased by nerve stimulation. However, the output of their /3-hydroxylated derivative is increased (Musacchio e t a ! . , 1965; Muscholl, 1972). T h e s e results suggest that a substance to be r e l e a s e d from the adrenergic terminal must be stored in a vesicle and this implies that the vesicles are directly involved in the n e u r o t r a n s m i t t e r release mechanism. Biochemical e x p e r i m e n t s on sympathetic neurons also indicated that release was by exocytosis. In these e x p e r i m e n t s d o p a m i n e / 3 - h y d r o x y t a s e (DBH) and

ALL OR NONE

RELEASE

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RELEASE

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> Fig. 2. Diagrammatic representation of all-or-none and partial release. Upper l:)',;': Hypnfl3elical distribution of filled, erupt}, and partiail~ empty storage vesicles within a chromaffin cell. Middle row: Vesicles after homogenization: notice that some vesicles have been disrupted as the result of mechanical stress. Lower row: Dopamine/3-hydrox3,lase( ) and catecho~amines(. . . . . . ) after isopycniccentrifugafion of |arge granular fraction through a linear sucrose density gradient; denser re~ion of gradient is It, left-hand side af each graph. Although amine secretion by all-or-none release of soluble content for each vesicle only decreases dense vesicle peak with a corresponding increase in empty vesicle fraction (lighter dopamine ,61-hydroxylase peak), partial release induces appearance of new enzyme and amine peaks of les.~er buoyancy with an enzyme-to-amine ratio that continuously increases as vesicles become more depleted a!3d lighter. Number of new peaks should he a function of amount of soluble material released each time a vesicle thses vdth the plasma memb,.ane: relative size of each peak x~ill be determined b~ probability of filled, empty and partially empty vesicles to participate in secretion and by magnitude of depiction of chromaffin celt. (Taken from Viveros, 1974.) Published results clearly indicate that stimulation induces 'all-or-none" release and "partial" release is never obse~,ed (Poisner et al., 1967: Trifard et a]., 1967: Viveros. 197.4k

86 chromogranin A were found together with noradrenaline in effluents escaping from the perfused spleen, suggesting that the adrenergic vesicle was involved in the release process (DePotter et al., 1969; Smith et al., 1970; Weinshilboum et al., 1971; Cubeddu et al., 1974a, b). Additional experiments also demonstrated that DBH was released into the incubation medium from the vas deferens during stimulation of the hypogastric nerve (Weinshilboum et al., 1971). All these experiments indicate that dopamine/3-hydro~iase and ehromogranin A were released from nerves and, therefore, from the adrenergic vesicles since these two proteins are components of secretory vesicles. Electrophysiological studies have also provided evidence for exocytosis as a mechanism of release from sympathetic neurons. Excitatory spontaneous action potentials (ESJP~) have been detected by intracellular recording in smooth muscle cells innervated by sympathetic nerves (Ho!man, 1970; Burnstock and Costa, 1975). In the vas deferens, the frequency of the E S J P s increased following stimulation of the hypogastric nerve and decreased following administration of reserpine (Holman, 1970). In the dog retractor penis muscle, the ESJP~ were abolished by denervation (Holman, 1970). These observations suggested that the transmitters, were released in packages. It was not possible to conclude, however, whether such packages were of uniform size (quantum). This might have been due to some of the following reasons: (1) The relationship between varicosities and smooth muscle is extremely variable. The cleft width between the two structures varies from 10 nm in the rat va~ deferens to 100 nm in the tenia coli of the guinea pig (Burnstock and Costa, 1975). (2) There is more than one type of vesicle in the varicosity. (3) There is a possibility of the transmitter being released from successive varicosities of the same adrenergic nerve. (4) There is electrical coupling of neighbouring muscle cells (Burnstock and Costa, 1975). In spite of all these anatgmica! and morphological ddferences, the evidence accumulated so far seems to be in favour of the quantum release of noradrenaline rather than molecule by molecule release. Studies on the uptake, storage and release of amino acid transmitters have also provided strong evidence for exocytosis as the mechanism of release (Burger et al., 1989; Nichols, 1989). It has been demonstrated with rapid freezing techniques at the neuromuscular junction that cholinergic vesicles fuse with the plasma membranes before the onset of postsynaptic signals, and when synaptic vesicle recycling was completely inhibited, the incorporation of vesicle membranes into presynaptic plasmalemma could be demonstrated by immunoc~ctochemieal methods using antibodies against vesicle protein antigens (Ceccarelli et al., 1988). In spite of this evidence, some investigators have suggested that the neurotransmitter acetyleholine was not

released by exocytosis of secretory vesicles but through pores at the level of the presynaptic membranes (Israel et al., !989). It has been suggested that the membrane protein which is responsible for the transport of acetylcholine to the extracellular space is the mediatophore, a protein which has now been identified as a subunit of the proton pamp ATPase. According to these investigators, C a 2+ will induce an opening in the mediatophore allowing the release of a quantum of acetylcholine (Israel et al., 1989).

3. Storage vesicles Biochemical findings in correlation with morphological observations have demonstrated the existence of at least three types of vesicles in neurons: large dense-core vesicles (LDCV) of 750-1000 A, small dense-core vesicles (SDCV) of 400-500 ,~ and small electrontranslucent vesicles of 400-500 A. (Grillo, 1966; Tranzer et ~1., 1969; Bisby and Fillenz, 1971; H6kfelt, 1973). In the adrenergic neuron of the sympathetic system, histochemical methods have demonstrated that in varicosities, both types of dense-core vesicles contain catecholamines (Fillenz, 1971) and that the small translucent vesicles can take up and store amines (Tranzer et al., 1969; Tranzer, 1973). In contrast to the morphological observations on the varicosities, the non-terminal axons of the sympathetic system showed only the large type of vesicles (Smith, 1972a, b). The percentage of each vesicle type was not the same in ~i .-.erve terminals examined. In fact, LDCV represented 26-25% of the vesicle population found in splenic nerve terminals, whereas they constituted only 4 - 5 % of tbe vesicle popula|;3n in t~rminals of the adrenergic nerve innervating the vas de r ,ens, iris, and heart (H6kfelt, 1969; Bisby and Fillenz, 1971; Tranzer, 1973). These observations on the distribution of LDCV and SDCV were reflected in differences in subceUular distribution of noradrenaline. In the vas deferens, only a low density peak of SDCV was observed, whereas in the spleen a clear bimodal distribution of noradrenaline was obtained (Bisby and Fillenz, 1971). Cholinergic neurons and neurons storing amino acid transmitters seem to contain small electrontranslucent vesicles as well as LDCV. Small electrontranslucent vesicles are thought to store classical neurotransmitters whereas evidence seems to indicate that peptide neurotransmitters are stored in LDCVs (DeCamilli and Jahn, 1990). Here again, LCDVs may also contain amines (Fillenz, 1971). In the chromaffin cells and in other neurosecretory cells, secretory granules seem to be related to the neuronal LDCVs. However, immunohistochemistry work with antibodies directed to small synaptic vesicle antigens (p38) has indicated the presence of a population of a small electrontranslucent vesicles in these

~7

cells (Navone et al., 1986, 1989). Moreover, small synaptic vesicle antigens have been identified in electrontranslucent vesicles of non-neuronal cells transfected with genes coding for these vesicle antigers (Johnston et al., 1989; Leube et al., 1989). Although the type of secretory vesicles described above differs in their soluble contents (different neurohormones or neurotransmitters), some intravesicular components (i.e. chromogranins) are expressed in different secretory tissues. Thus, a variety of neuroendocrine cells contains chromgranin A, B or C, in addition to the neurohormone they stored (Lloyd and Wilson, t983; Fisher~Calbric et al., t985; Lassman et al., 1986). Although present in different neuroendocrine cells, the relative proportion of chromogranins varies in vesicles from different secretory tissues (Lassman et al., 1986). Chromogranins are also present in LDCV of neurons and this wilt suggest that the regulated control for neuropeptide secretion must be very similar in these two types of secretory cells. It is known that vesicles which have been depleted by drug treatment (i.e. reserpine) from one or more soluble secretory component, are still capable of exocytosis (Cubeddu et al., 1974a, b; Thoa et al., 1975; Trifar6 and Cubeddu, t979). This would suggest that vesicle membranes contain specific sites necessary for their transpo~-t to release sites and for the fusion of secretory vesicle membranes with the plasma membranes. Although surf?co vesicle components seem to play a role in vesicle fu ~etion, there is, at the oresent time, a disagreement as to what extent the three vesicle types express similar components. This difference of opinion arose from the use of different approaches (biochemical or immunocytochemical) in the study of the distribution of vesicle membrane components. Although there seems to be a general consensus that secretory vesicles contain a protein pump ATPase in their membranes, there is no generai agreement on the presence of other membrane components such as synaptophysin (p38), 65 kDa calmodulin-binding protein (p65) and transmembrane glycoprotein (SV2) in all vesicles. Those groups of investigators suggesting the presence of the above antigens only iv electrontranslucent vesicles, have used this hypothesis as the possible explanation for the differential release observed between classical neurotransmitters and neuropeptides during co-transmission (see below).

4. C o - t r a n s m i s s i o n and differential release

As stated above, the regulated pathway of secretion involves the release of vesicular contents by exocytosis. The regulated pathway in neurons involves at least two types of secretory vesicles - - large and small synaptic vesicles (SSV). The SSV are mainly involved in the storage and release of classical neurotransmitters

(acetylcholine, noradrenalive, glutamate, scrotonin, GABA, etc.* and 1hc LDCV are mainly involved with the storage and release of peptides, although they also store classical neurotransmitters. SSV'; seem to undergo exocytosis/endocytosis at nerve terminals (Cecarelli et al., 1973; Heuser and Reesc, 1973). This local circulation of membrane material between vesicular and plasma membrane components is taking place, according to some investigators, via coated vesicles formation at an area distant from the release sites (Heuser, 1989). However, other schools of thought have suggested that it is the same vesicle membrane which is retrieved after exocytosis (Trifar6 and Poisner, 1982). This latter mechanism will allow conservation of membrane active vesicle antigens (i.e. p38, SV,). It is, however, possible that during intense ne~we stimulation, a condition accompanied by a large number of vesicle membrane incorporation into plasmalemma, excess surface membrane is retrieved via coated vesicles with formation of 'large endosomal' intermediates (Heuser and Reese, 1973). Whatever the real mechanism of exocytosis/membrane retrieval is, it is understood that SSVs originate locally at the level of the terminal, since these vesicles seem to be absent from axons with only LDCV being found. It has also been suggested that in view of the fact that in adrenergic ne~es, SSVs and LDCVs shared proteins necessary for amine uptake (proton pump ATPase) (Cidon and Sihra, 1989) and synthesis (dopamine /3-hydroxylase) (Trifar6 and Cubeddu, 1979), LDCVs are precursors of SSVs, with SSVs being fo,qned after exocytosis of LDCVs (Smith, 1972a, b; DePotter and Chubb, 1977; Boarder, 1989). In neurosecretory cells, LDCVs seem to contain surface antigens, such as 65 kDa ealmodnlin-binding protein (Fournier and Trifar6, 1988a; Tifar6 e t a l . . I989a), which arc also present in SSVs. This would suggest a relationship between small and large vesicles in neurosecretou cells. However, recent observations on neurons suggest that peptide-containing vesicles (LDCVs) release their content at sites distant to the synaptic cleft (Thureson-Klein et ai., 1988) and this argues against precursor-product relationship between the two vesicles (De Camilli and Jahn, 1990). In agreement with this latter observation are experimental demonstrations that differential release of classical neurotransmitters and neuropeptides from neurons can ~e achieved by applying different frequencies of stimulavion (Andersson et al., 1982; Lundberg and H6kfelt, 1986). Classical neurotransmitters seem to be released at tow frequencies of stimulation, whereas high frequencies are needed for the discharge of neuropeptides (Lynch, 1980; Andersson e t a l . , 1982; Lundberg and H6kfelt, 1986). Moreover, a-latrotoxin, a toxin from black widow spider venom (Meldolesi et al., 1986), can induce massive exccytosis of acetylcholine (SSVs) without an~ appreciable change in the number of pep-

8s tide-containing LDCVs in the same nerve terminals (Matteoli et at., 1988). The differential release between classical neurotransmitters and neuropeptides could be the results of: (a) different Ca 2+ sensitivity of the two vesicular pools, (b) differential localization of Ca 2+ channels (i.e. Ca 2+ channels present only at the synaptic cleft and therefore requiring intense stimulation to build up intrace!lular Ca z+ at terminal areas where LDCV are situated), or (c) different types of vesicle-cytoskeleton interaction (i.e. synapsin 1 interacts only with small vesicles, see below). Regardless of the molecular mechanisms involved in the differential release of neuronal secretory material, it is clear that neurons, although containing a cocktail of neurotransmitters and co-transmitters, are capable of changing the ratio of peptide/transmitter released according to different physiological situations.

5. Cellular and molecular events in exocytosis

A large body of information on cellular and molecular events in secretion has been obtained from studies on non-neuronal secretory cells (Trifar6, 1977; Trifar6 and Poisner, 1982). These have been, in part, due to the fact that nerve terminais are not always accessible to the application of techniques directed to the study of secretion and isolated synaptosomes have not been, in some cases, suitable preparations. Furthermore, it has not yet been established whether evidence obtained from non-neuronal cells can be extended to neurons. Chromaffin cells are among those cells, often classified as non-neuronal, from which a large body of information has been collected (Trifar6, 1982, 1990b). Chromaffin cells of the adrenal medulla should be considered as neuronal cells since they derive embryologically from the neural crest, sharing a common origin and common subcellular features (secretory vesicles) with sympathetic neurons (Trifar6, 1982, 1984). Finally, it is not clear whether the molecular events involved in the release of the SSV and LCDV contents are similar, since it has been shown that SSVs and LDCVs have different associated proteins (i.e. synapsin 1).

5.1. The picotaf role of

Ct;I 2+

In 1928 Houssay and Molinelli first suggested the importance of Ca x+ in catecholamine secretion. Later on, Harvey and Macintosh (1940) demonstrated that extracellular Ca ,-+ was also required for acetylcholine release. However, it was Douglas and his colleagues who conceived the role of Ca 2+ in the secretory process as a general one and who gave the name of a 'stimulus-secretion coupling' to the events involved in the secretory process because of the similarity with the

'excitation-contraction coupling" in muscle (Douglas and Rubin, 1961; Douglas 1968). The arrival of the action pntential to the nerve terminal or to the secretory, pole of a neurosecretory cell triggers the influx of extracellular Ca 2+ through specific Ca 2+ channels (slow Ca 2+ channels) (Augustine et al., 1987; Smith and Augustine, 1988). The increase in intracellular Ca 2+ seems to trigger a series of events leading to the extrusion of secretory material (Smith and Augustine, 1988). Under certain experimental conditions, release can be triggered in the absence of extracellular Ca 2+ (Lastowecka and Trifar6, 1974; Adam-Vizi and Ligeti, 1984). However, in this case, the rise of intracellular Ca =+ is due to its release from one or more intracellular pools (Aguirre et al., 1977; Meldolesi et al., 1988). These pools might be contained in cellular organelles (calciosomes) which are shown by conventional EM techniques to be similar to smooth endoplasmic reticulum (SER), but that can be distinguished from SER on the basis of their composition and immunocytochemistry (Meldolesi et al., 1988; Volpe et al., 1988). Moreover, experiments on neurons (Meldolesi et al., 1988; Thayer et al., 1988) and secretory cells (Burgoyne et al., 1989a) preloaded with fura-2 (a fluorescent Ca 2+ indicator) and stimulated with either bradykinin/angiotensin (agents that cause formation of IP3) or caffeine have shown the presence of two specially d~slinct pools that give rise to intracellular Ca 2+ (Meldolesi et al., t988; Burgoyne et al., 1989a). As discussed above, neuropeptides might be released from neurons at presynaptic terminal areas distinct from active zones (presynapt~c areas of clustered SSVs containing classical neurotransmitters). On the basis of fura-2 experiments on squid axons and of EM freeze-fracture studies, it has been sugge=ted that Ca 2+ channeL, are confined to areas of plasma membranes situated at ~i,c active zones. This would suggest that the Ca 2+ concentration reached at active zones during stimulation-evoked Ca 2+ influx is several-fold greater (100 ixM or more) than at other areas of the terminal (Smith and Augustine, 1988). If this were the case, the target(s) for Ca 2+ during exocytosis in these areas would not require a high affinity for Ca 2+. On the other hand, the release of LDCVs' contents might require the presence of high-affinity Ca 2÷ receptors. Several possible sites of action for Ca 2+ in secretion are discussed below.

5.2. hltegral membrane proteins and proteins which bind to cesicles (Jig. 3) 5.2.l. Synaptophysin (p38) This protein is a major component of the membrane of synaptic vesicles (Jahn and Maycox, 1980; Jahn et al., 1985; Wiedenmann and Franke, 1985; Navone et

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Fig. 3. Schematic representation of membrane c ,mponents of the chromaffin granuie. CAT: catecholamines: CM: calmodulin: 65CMBP: 65 kDa calmodulin-binding protein (p65); CAL: caldesmon: G: low molecular weight G-protein; SV,: glycoproteim D~H: dopamine 3-hyd;o~,lase; ~-A: a-actinin; F-A: F-actin; S: synaptophysin (p38) and cyt.bsm: cytochrome.

associated with the SSVs containing ncurotransmitters and is not associated wilh neuropeptides containing LDCVs (Naw)ne et al., 1986, 1988; De Camilti and Navone, 1087; De Camil!i and Jahn, 19901. Tl.is observation has been recently challcnged since work from ether laboratories has shown the presence of synaptophysin in chromaffin granules as well as in other neurosecretory granules (Fournier and Trifard, 1988a; Lowe et al., i988; Oberndorf et ai., 1988; Schil!ing and Gratzl, 1988; Fournier et al., 1989; Trifar6, t990b). Synaptophysin is also present in PC~2 cells and in a variety of neoplasms (Wiedenmann and Franke, t985; Lowe et al., 1988). Moreover, recent experiments have shown that contrary to what was found with 65-CMBP (p65, synaptophysin was not detected in plasma membranes (Fournier and Trifard, 1988b; Trifar6 et al., !98%). Although the presence of this protein ha, not been demonstrated in exocrine gland secretory granules, it is now clear that. in additkm to classic synaptic vesicles, the protein is found in secretory vesicles of many, if not alI, neurosecretory tissues.

5.2.Z Glycoprotehl SV~ al., 1986, 1988; Rehm et al., 1986; De Camilli and Navone, 1987). Synaptophysin has now been characterized by different laboretories and it has been shown to be a glycoprotein with apparent molecular weight of 38,000 as determined by electrophoresis. However, electrophoresis carried out under non-reducing conditions shows an apparent m,qecular weight of 76,000 (Jahn and Maycox, 1980). Tae protein is an integral membrane component, requiring Triton X-100 buffer for extraction. Synaptophysin i~ able to bind Ca ~'+ (Rehm etal., 1986) and, in additic,n, it has been shown that native synaptophysin is a hexaraer with a topology very. similar to channel proteins. When it is introduced into lipid bilayers it shows voltage-dependent channel activity (Thomas et al,, 1988). Three independent groups of investigators have recently published the cloning and sequences of eDNAs encoding synaptophysin (Buckley et ai., 1987; Leube et al., 1987; Sudhof et al., 1987). These studies have shown that the protein has 397 amino acids with a membrane topology showing four hydrophobic transmembrane domait~s with both amino and carboxyl terminals oriented to the cytoplasm. The Ca2+-binding site is in the cytoplasmic domain. Recent phosphorylation studies seem to indicate that synaptophysin is a substrate for tyrosine kinase (Pang et al., 1988). Interestingly enough, tyrosine kinase present in one of two forms of p60 ..... has been found associated with chromaffin vesicles (Parson and Crentz, 1986). Immunocytochemical studies have revealed that synaptophysin is present in all synapses of the mammalian nervous system (De Camiili and Navone, 1987; Navone ct al., 1988). Moreover, it has been suggested that, in neuronal cells, the protein is

Another component common to several secreto~' vesicles is a transmembrane glycoprotein (SV 2) of 100,000 molecular weight (Buckiey and Kelly, 1985). This protein, originally described in synaptie vesicles, has also been found to be present in secreto~3' granules of endocrine tissues incluc,: "g the adrenal medulla, pituitary and endocrine pancreas (Buckley and Kelly, 1985). SV~ is also present in secreto~ vesicles of established cell lines such as PC12 (a line derived from rat pheochromocytoma), GH 3 (a line derived from mouse anterior pituitary) and H I T (insulin secreting line). These different cell types show SV~ proteins of different molecular masses. This i:; due to differences in the composition of their carbohydrate moieties (Buckiey and Kelly, 1985).

5.2.3. 65 kDa cahnodulh>bindh~g protehl (65-CMBP, p65) Calmodulin has been shown to bind to several secretory vesicles such as synaptic vesicles (Moskowitz et al., 19S3), neurohypophyseai granules (Olsen et al., 1983L platelet oe-granules (Grinstein and Furuya, 1982), pancreatic islet secretoQ' granules (Watkins and White, 1985) and chromaffin granules (Geisow et al., 1982; Hikita et al., 1984; Bader et al., 1985). Moreover, recent work demonstrated the presence of a 65,090 molecular weight protein in chromaffin vesicle membranes which binds calmodulin with high affinity (Hikita et al., 1984; Bader et al., 1985). Although several calmodulin-binding proteins have been detected in membranes of other sccreto~' vesicles (Grinstein and Furuya, 1982; Moskowitz et at.. 1983; Watkins and Wi~ite, 1985), no attempts to find a common calmod-

90 ulin-binding protein in these vesicles were made until very. recently (Bader eta!., 1985; Fournier and Trifar6, 1988a). Interestingly, it has been found that several secretory tissues show cross-reactivity to a monoclonal antibody prepared against a 65,000 molecular weight rat brain synaptie vesicle antigen (p65). In these studies, it was not determined if the antigens responsible for the cross-reactivity of other neurosecretory tissues were vesicle antigens (Matthew et al., 1981). Morever, in these studies, no attempt was made to determine the function of the p65 synaptic vesicle surface antigen. Work has continued quite independently in different laboratories using either monoclonal antibodies against p65 (Floor and Leeman, 1985; De Camilli and Navone, 1987; Obata et al., 1987; Lowe et al., 1988) or the 65,000 molecular weight calmodulin-binding protein (Bader et al., 1985; Trifar6 and Fournier, 1987). In most instances, the investigators involved in these studies were not aware that the p65 and the 65,000 molecular weight secretory vesicle calmodulin-binding protein were identical (Trifar6 and Fournier, 1987; Fournier and Trifar6, 1988a). The development of a radioimmunoassay using antibodies against p65 also allowed the demonstration of the presence of the antigen in anterior and posterior pituitaries and in cell lines AtT20 , GH 3 and PC~2 (Matthew et al., 1981; Lowe et al., 1988). In sodium dodew1 sulphate (SDS)-polyacrylamide gels of synaptic plasma membrane and of brain homogenate i'roteins, both antibodies were found to bind to a single protein with a molecular weight of 65,000 (Matthew et al., 1981). It was also demonstrated using this technique, that the antigen was highly conserved through the vertebrae phylogeny since it was found to be present in sharks, amphibia, birds and different mammal brains (Matthew et al., 198!). The same antibodies were also able to precipitate 80-95% of the labelled noradrenaline present in homogenates of PCI2 cells and adrenal medulla previously incubated with radioactive catecholamines (Matthew et al., 1981; Lowe et al., 1988). Additional work has demonstratea the presence of the 65-CMBP in chromaffin, synaptic and neurohypophyseal secretory granules (Fournier and Trifar6, 1988a). The protein was extracted from each vesicle membrane type with Triton X-100 and subsequently purified by calmodulin affinity chromatography. lmmunoblot tests of the secretory vesicle 65CMBP using monoclonal antibodies against p65 demonstrated the immunological identity of the calmodulin-binding proteins isolated from three types of secreto~, vesicle membranes (Fournier and Trifar6, 1988a).

5.2.4. Cytoskeleton proteins Recent experiments seem to suggest that the cell cytoskeleton determines the positioning of secretory vesicles in nerve terminals and in other secretory cells

(Burgoy.ne, 1990; Trifar6, 1990). It is also known that actin microfilaments not only interact with the inner surface of the plasma membrane but also with the cytoplasmic surface of secretory, vesicles (see below) through specific anchorage proteins (a-actinin, fodrin, synapsin I). A great deal of information on the interaction bctween cytoskeletal proteins and secretory vesicles comes from studies on chromaffin granules (Trifar6 et al., 1982, 1985a, b, 1989b; Trifar6, 1990b). Published observations have indicated the association of fodrin with chromaffin granule surfaces (Aunis and Perrin, 1984) and in another set of experiments, aactinin was found to be a component of chromaffin granule membranes (Trifar6 et al., 1982, 1984, 1985a, b; Bader and Aunis, 1983). When cultured chromaffin cells were stained with antibodies against muscle aactinin~ a granular staining pattern was observed in the cytoplasm and neurites, suggesting an association of a-actinin with chromaffin vesicles (Trifar6 et al., 1982, 1984, 1985a, b). SDS-polyacrylamide gel electrophoresis of chromaffin vesicle membranes showed the presence of a protein component that co-migrated with purified c~-actinin (Trifar6 et al., 1982, 1984; Bader and Aunis, 1983). In addition, an a-actinin-like protein was extracted from purified chromaffin vesicles by using conditions known to extract a-actinin from myofibrilar Z lines. The extracted protein of molecular mass 97 kDa and isoelectric point 6.4 was recognized by aactinin antibodies as seen by immunodiffusion and immunoblotting (Bader and Aunis, 1983; Trifar6 et al., 1984). The results obtained with pronase digestion of intact and broken granules suggested a localization on the cytoplasmic surface of the granules of both actin and a-actinin (Bader and Aunis, 1983). The presence of two different actin-binding proteins (a-actinin and fodrin) in vesicle membranes may indicate the existence of v-..~ different types of actin-secretory granule association, with each binding protein involved in one sff~dhc type of interaction (Trifar6 et at., 1984, 1985b). Chromaffin vesicle membranes contain a significant number of actin nuclei which are able to promote actin polymerization and formation of membrane-bound actin filaments (Wilkins and Lin, 1981). Vesicle membranes pre-incubated with a-actinin antibodies showed a reduced number of binding sites, an observation which indicates the absence of actin nuclei in those pre-treated membranes (Trifar6 et al., 1985b). Therefore, a-actinin molecules are either the nuclei themselves or they stabilize the actin nuclei, probably by anchoring these nuclei to the granule membrane. Caldesmon is another protein found associated with chromaffin vesicles (Burgoyne et al., 1986). Caldesmon is a calmodutin-dependent actin-binding protein which, at low Ca 2+ concentrations (10 -v M), binds and crosslinks actin filaments (Burgoyne et al., 1986). The binding of caldesmon to actin filaments is inhibited in the

91 presence of micromolar concentrations of Ca 2 +. Under these conditions, caldesmon interacts reversibly with chromaffin vesicle membranes (Burgoyne et al., t986). This flip-flop regulation of caldesmon may be important for secretory vesicle function during the changes observed in intracellular Ca 2+ levels upon stimulation. Synapsin I is another protein present in synaptic terminals which is associated with synaptic vesicles (De Camilli et al., 1983; Huttner et al., 1983; De Camilli and Grecngard, 1986). Synapsin I is a phosphoprotein which ~s a substrate for cAMP kinase A an~- calmodu!in-dependent protein kinase II (De Camilli and Greengard, 1986). Synapsin I has also been shown to bin0 to spectrin (Baines and Bennet, 1985) and actin (Bahier and Greengard, 1987) with the ability to bundle aetin microfilaments (Bahler and Greengard, 1987; Benfenati et al., 1990). It has been suggested that synapsin ! might serve as an anchor between synaptic vesicles and the cytoskeleton (De Camilli and Greengard, 1986). The affinity of synapsin I for synaptic vesicles is decreased by phosphorylation of the protein (De CamiUi and Greengard, 1986) and it is also known that when neurotransmitter release is triggered, it is accompanied by a reversible phosphorylation of synapsin I (De Camilli and Greengard, 1986). Therefore, synapsin I phosphorylation will result in the release of synaptic vesicles from their anchorage site on the cytoskeleton, thus a~lowing the vesicles to move to the active synaptic zones ~see below).

5.Z5. Ca ~-~-dependent bhMing proteb~s 5.2.5.1. Calmodufin. This t~rotein has been shown to bind with high affinity and in a Ca:+-dependent manner to several secretory vesicles. Among these vesicles are platelet e-granules (Grinstein and Furuya, 1982), synaptic vesicles (Moskowitz ct al., 1983), chromaffin vesicles (Geisow et al., 1982; Hikita et al., 1984; Bader et al., 1985) and posterior pituitary secretory granules (Olsen et al., 1983). Moreover, the association constants (K D) described for calmoduli~ interaction with each secretory vesicle (Kt~ = 49 nM platetets, c~granules; K D = 10 riM, synaptic vesicles; K D = 2.4 riM, posterior pituitary secretory' granules, K D = 9.8 nM chromaffin granules) are surprisingly similar considering the different conditions used to measure binding constants in each granule preparation. The high affinity Ca2+-dependent calmodulin binding involves the 65-CMBP (p65) described above (Bader et al., 1985; Trifar6 and Fournier, 1987). The fact that the 65-CMBP is present in secretory cells from several tissues and that calmodulin binds to this protein with high affinity and in a CaZ+-dependent manner may suggest an important role for calmodulin in the control of exocytosis (Trifar6 and Fournier, 1987; Fournier and Trifar6, 1988b) (see below).

5.2.5.Z Caleactin. This is another CaZ--depen dent binding protein that belongs to the family of annexins (Burgoyne and Gelsow, t989; Burgoync, 1990). Calpactin has the ability to bind reversibiy to chromaffin vesicles in the presence of micromolar Ca 2÷ concentrations (Burgoyne and Cheek, 1987; Burgoyne, 1988; Drust and Creutz, t988a, b). In vitro experiments indicated that caloactin induced secretory vesicle aggregation in the presence of Ca :~ (Drust and Creutz, 1988a) and that the aggregation of the vesicles is followed by membrane fusion if arachidonic acid is present (Drust and Creutz, 1988a). 5.2.6. GTP-binding proteins 5:udies on permeabilized secretory. cells have suggested the involvement of GTP-binding proteins in exocytosis (Knight et al., 1989; Gomperts, 1990). Hydrolysis-resistance GTP analogues stimulate exocytosis in permeabilized ceils, even in the absence of C a : (Knight et al., 1989; Penner and Neher, 1989; Gomperts, 1990). Low molecular weight GTP-binding proteins were found to co-purify with chromaffin vesicles and synaptic vesicles (Burgoyne and Morgan, t989; Doucet et at., 1989; Ngsee et al., 1990). Moreover, low molecular weight GTP-binding proteins have been shown to be involved in the constitutive secretion of yeast (Bourne, 1988). These proteins are related to the ras protein oncogenes (Bourne. 1988). It is unknown if the yeast G-proteins are related to the low molecular weight GTP-binding proteins found in secretory vesicles (Doucet et al., 1989). 5.3. Cahnodulin in secretion 5.3.1. Efj>ct qf cahnodulin antay, onis~s The assumption that calmodulir~ is involved in the regulation of numerous Ca2*-dependent cellular processes has led to the application of pharmacological agents to modify. C a : ' - m e d i a t e d functions. A wide range of chemical and related substances such as phenothiazines, butyrophenones, naphthalene sulphonamides, vinca-alkaloids, local anaesthetics, calmidazolium and compound 48/80, have been found to antagonise calmodulin (Hidaka et al., 1981; Weiss et at., 1982). With the use of these antagonists, calmodulin has been implicated in secretion for a number of cell systems including the adrenal medulla (Kenigsberg et a!., 1982; Sasakawa et at., 1983; Wada et at., 1983), endocrine in pancreas (Schubart et at., 1980), anterior pituitary. (Corm et al., 1981; Fleckman et al., 1981; Sand et al., t983), platelets (Nishikawa et al., 1980; White and Raynor, 1980), mast cells (Douglas and Nemeth, 1982), nerve terminals (De Lorenzo, 1982), adienat cortex (Balla et at., 1982). teukocytes (Etfrynck, 1979) and paramecium cells (Garofalo et al., 1983). In view of the wide range of unrelated pharmacological

92

agents that inhibit eahnodulin, the selectivity of this inhibition is questionable. However, there exists a general correlation between the hydrophobicities of various agents and their potencies as inhibitors of calmodulin (Laporte et at., 1980; Landry et al., 198!). Moreover, it was not surprising to find that other Ca2+-bind ing proteins which exhibit sequence homology with calmodulin were found to bind to these antagonists, nor was it surprising to find that these agents were capable of antagonising calmodulin-independent enzyme systems such as protei~ kinase C (Schatzman et al., 1983; Saitoh and Dobkins, 1986). Consequently, it is apparent that these antagonists cannot be applied as a sole detection of calmodulin-dependent functions. Trifluoperazine (TFP) and N-(6-aminohexyl)-5chloro-l-naphthalene sulphonamide (W7) have been the two most widely used agents to test for the involvement of calmodulin in secretion. However, there is a great discrepancy in the findings and interpretation of the results obtained with these compounds on several secretory systems by different laboratories. Some experiments have shown an enhancement of hormone and neurotransmitter release by TFP, others, on the contrary, have shown inhibition of secretion by TFP. This latter effect was interpreted in some cases as due to inhibition of Ca 2+ entry (Sasakawa et al., 1983; Wada et al., 1983) and, in others, to an intracellular inhibition (Conn et al., 1981; Kenigsberg et al., 1982) which was most probably at the calmodulin level (Kenigsberg et at., 1982). These differences are in part due to dissimilar experimental conditions, especially with regard to different TFP concentrations and periods of incubation. From the data obtained in experiments performed by Clapman and Neher (1984), and taking into consideration the above limitations, it has now become clear that most of the effects described for TFP are concentration-dependent and can also be observed in the same secretory' system. Low concentrations of TFP ( < 1 /xM) can inhibit the activation of some receptors a n d / o r secretion by acting on an intra~ cellular site (Kenigsberg et al., 1982; Clapman ano Neher, 1984). TFP concentrations ranging between 100 nM and 1 /zM alter acetylcholine-induced currents (Clapman and Neher, 1984). Thus, it appears that TFP is approximately 10 times more potent as an inhibitor of receptor-mediated than depolarization (high K +)-induced release. Concentrations of TFP of about 10-20 /xM are able to produce a Ca 2+ channel blockade, and therefore reduce Ca 2+ entry (Clapman and Neher, 1984). Higher concentrations of TFP ( > 25 ixM) produced a detergent-like effect, accompanied by massive release of hormones (Kenigsberg and Trifar6, unpublished observations). Therefore, it is now clear that TFP, in addition to other effects (i.e. Ca 2+ channel blockade), blocks secretion by acting at an intracellular site which is distal to Ca 2+ entry (Corm et al., 1981;

Kenigsberg et al., 1982; Clapman and Neher, 1984) suggesting indirectly a rote for calmodulin in secretion. In support of such suggestions our in vitro experiments demonstrated that the binding of labelled calmodulin to secretory granule membranes is inhibited by TFP (Bader et al., 1985).

5.3.2. Effect of calmoduiin antibodies The use of anti-calmodulin agents on secretory systems has provided circumstantial evidence for a calmodulin role in secretion. More evidence for the calmodulin involvement can be obtained through the use of more highly selective probes such as calmodulin antibodies (Steinhart and Alderton, t982; Kenigsberg and Trifar,5, 1985; Momayezi et al., 1987). Using a preparation of isolated cortex obtained from paramecium cells, Momayezi et al. (t987) were able to demonstrate that calmodulin antibodies inhibit exocytosis in this system. Moreover, exocytosis was also blocked when the paramecium cortical preparation was exposed to antibodies against catcineurin, a Ca 2+ ca!modulin-dependent protein phosphatase (Momayezi et al., 1987). An in vitro system developed in sea urchin eggs also showed that calmodulin antibodies could inhibit secretory-granule membrane interactions. It was found in this model that preincubation of cortical granules and plasma membranes with calmodulin antibodies prevented the fusion of these ~ ' o components (Steinhardt and Alderton, 1982). In a set of experiments performed by Kenigsberg and Trifar6 (1985), it was demonstrated that the delivery ~f monospecific antibodies directly into the cytoplasm of cultured chromaffin ceils by the erythrocyte ghost-mediated microinjection technique (Kenigsberg and Trifald, !985; Trifar6 and Sc,,ard, 1987) inhibited catecholamine release in response :~ Aimulation by different secretagogues without affecting other cellular processes such as the high affinity amine uptake system. Although the results obtained in the experiments using calmodulin antibodies strongly suggest a role for calmoO~lin in secretion, they cannot distinguish with a degree of certainty which calmodulin-dependent process(es) i s / a r e of significance to stimulus-secretion coupling. However, recently published experiments on Paramecium tetraurelia have provided some evidence in favour of a specific calmodulin-dependent process (Momayezi et al., 1987). In these experiments, parameciums were microinjected with antibodies against calcineurin and these produced inhibition of stimulus-induced exocytosis. On the other hand, microinjection of alkaline phosphatase into these paramecium cells induced and exocytotic discharge. These experiments suggest, for the first time, that at least in this secretory system, a calmodulin-dependent protein dephosphorylation step seems to be involved in e×ocytosis.

93

5.3..3. Calmodulin in slirnuhts-secrelion coupling The process of secretion is one of the Ca2+-mediated events in which calmodulin seems to be involved. How calmodulin might be involved in secretion remains to be determined. However, the presence of calmodulin-binding proteins in secretory- vesicles may suggest that ealmodulin is involved either with the transport of vesicles to release sites or with the process of interaction between vesicles and plasma membrane during exoeytosis. Alternatively, calmodulin might be involved in the control of cell viscosity and consequently in vesicle mobility or with the process of retrieval of vesicle membranes after exocytosis. In this regard, high affinity binding sites for calmodulin have been found in coated vesicles (Moskowitz et al.~ 1982). Calmodulin can also be involved in regulation of cytoskeleton proteins by a flip-flop mechanism (Sobue et al., 1983). There is a possible regulation through caldesmon phosphorylation, and in this regard, phosphorylation of caldesmon through a Ca 2+ calmodulindependent mechanism would produce the removal of caldesmon cross-linking from actin filaments. Another possibility is the regulation of the interaction of secretory vesicle anchoraging proteins such as c~-actinin and synapsin 1 (Trifar6 et al., 1982, 1984, Bader and Aunis. 1983; De Camilli and Greengard, 1986). The phospborylation of some of these proteins by Ca 2 + calmodulin-dependent kinases would detach these proteins from secretory vesicles with a consequent increase in their mobility. In o~her words, a flip-flop interaction will take place in the secretory cells: under resting conditions (low Ca2*), these anchorage proteins would interact with the vesicles whereas during stimulation (high Ca 2+) these proteins would bind to calmodulin. In connection with this mechanism, it has been shown that the injection of calmodulin kinase It into the squid giant synapse potentiates neurotransmitter release in response to stimulation (Llinas et ai., 1985). Fodrin is another protein that interacts with ealmodulin and actin presumably in a flip-flop manner. Perrin and Aunis (1985) have shown, using immunofluoreseence techniques, that in the resting chrcmaffin cell, fodrin is mainly localised in the ectoplasmic region under the plasma membrane, showing a continuous ring-like fluorescence when cells are treated with a~;tibodies against fodrin. Stimulation of the cells produces a rearrangement of fodrin in patches. Treatment of the cells with trifluoperazine blocks not only normal release but also fodrin patch formation (Perrin and Aunis, 1985). Moreover, work from the same laboratory has shown that treatment of detergent-permeabilized cells with fodrin antibodies also blocks hormone release in v,.sponse to an increased Ca 2÷ concentration (Perrin et al., t987). Finally. calmodu!in may also be involved in secretory- vesicle-plasma membrane fusion during exocytosis. In addition to the experiments de-

scribed involving paramecium cc(!s, other fusion experiments carried out with plasma membranes and anterior pituitary secretory granules (f)raznin eL at.. 1986) or pancreatic granules (Watkins and Cooperstein, 1983) have indicated the requirement of calmodulin for fusion processes. TFP has also been shown to preven! the interaction of secretory vesicles and plasma r,.lembrahe in intact secretory cells (Burgoyne et al., 1982). Moreover, experiments described above, carried out on paramecium cortical preparations, seem to suggest thal an activation of the Ca 2~ calmodulin-dependent calcineurin system may be involved in the fusion process~ and that this process may be activated through the depho, phorytation of the 65 kDa protein (Momayezi et al., 1987). Interestingly enough, ob;ervations from our laboratory have shown, as described in the previous section, the presence of a similar 65 kDa caimodulinbinding protein in secretory vesicles and plasma membranes of chromaflin cells and these would Iead one to speculate that this 65 kDa protein might be involved in the fusi(~n process (Fournier and Trifard. 1988a, b). Whether the 65 kDa protein described by us and the 65 kDa described in the paramecium studies are the same proteins remains to be determined.

5.4. Dynamic changes in cortical actin filame,,~t networks as preh~de to c:rocrtos#, Secretion is the process ~hich requires the movement of secretoD ' vesicles towards the plasma membrane, the fusion of vesicles with the plasma membrane and the subsequent extrusion of the secretory vesicle contents to the celt exterior (Trifard, 1977: Trifard and Poisner. i982). Consequently. it has been proposed that the process of secretion might be mediated by contractile elements associated with the secretory vesicles or present clsc,~here in the secretory cells (Trifar6, 1978; Trifar6 et at.. 1985a, b). The association of some of these proteins with secreto~' vesicles suggests that contractile proteins and their reguIatory counterparts might play a role in the cellular transport of vesicles a n d / o r in other steps involved in secretion (Trifard et al.. 1985a. b). One possibility is that the cytosketeton proteins and their associated regulatory protei~:s might be involved in the eontroi of cell viscosity and therefore in secretory vesicle mobihty (fig. 4). In this particular case. actin would control secretory cell viscosity through the formation of the mesh of microfilaments which could be cross-linked by fodrin (Perrin and Aunts. t985), caldesmon (Burgoyne e* al.. 1986) and secretory vesicle c~-actinin ('Irifard et aI.. t985a, b) and in the i:erve terminals by synapsin 1 (De Camilli and Greengard, 1986). In this regard, it has been shown that adrenal medullary secretory granules will induce actin polymerization and gel formation in vitro, effects blocked by raising the concentration of Ca 2" in the

94 PM

Hi

~

M

j

'ii!i Fig. 4. A schematic representation of a possible mechanism in which cytoskeleton proteins may play ~ role in chromaffin cell secretion. (A) Under resting conditions actin ( ) controls chromaffin cell cytosol viscosity through the formation of a mesh of microfilaments thi~t are cross-linked and stabilized by (i) fodrin (11) and by (ii) a-actinin (*). The secretory, granule membranes also contain a calmodulin-biuding protein (e). A similar calmodulin-binding protein is also present in plasma membranes. At the C a 2+ concentration found in the resting cells, calmodulin (x) and scinderin (#) are not activated and there is a large percentage of non-filamentous, nonphosphorylated myosin (1). (B) When the chromaffin cell is stimulated, Ca2+ enters the cell and produces (i) a dissociation of actin from fodrin, (ii) patching of fodrin along the plane of the plasma membrane (mm In), and (iii) activation of scinderin with a consequent capping and shortening of the acfin microfilaments. Ca2~ does not affect the binding of actin to granule membrane a-actinin. As a result of both i and iii, the cytosol viscosity decreases, allowing the movement of granules towards the pta:;ma membrane releasing sites. Whether an actin-myosm interaction (sliding mechanism) is also involved in granule movement remains to be determined• "li~eintracellular Ca2+ concentration reached during stimulation is sufficient to activate ca[modulin-dependent processes, including the binding of calmodulin to granule membranes ~md plasma membranes (fusion) and the phosphorylation of myosin-like chains, a condition required for myosin activation and bipolar filament formation (2). (Modified from Trifar6 and Fournier, 1987.)

medium (Fowler and PolLard, 1982; Cheek et al., 1986). Work from our laboratory, as well as others, has demonstrated that filamentous actin is merely Localized in the cortical surface of the chromaffin cells (L~e and Trifard, 1981; Trifar6 et al., 1984, 1989b; Cheek and Burgoyne, 1986). Wc have ab;o suggested that cortical

F-actin acts as a barrier to the secretory • granules impeding their contact with the plasma m e m b r a n e (Trifar6 et al., 1982). Chromaffin granules contain aactinin (Trifar6 et al., 1982; Bader and Aunis, 1983) and fodrin (Perrin and Aunis, 1985) and synaptic vesicles contain synapsin I (De Camilli and Greengard, 1986), these are anchorage proteins which mediate filamentous actin association with the secretory vesicles (see above). In the case of chromaffin cells, the stimulation produces disassembly of actin networks and removal of the barrier (Cheek and Burgoyne, 1986; Burgoyne et al., 1989b; Trifar6 et al., 1989b). This interpretation is based on the follewing evidence. Cytochemical experiments with rhodamine-labelled phalloidin and actin antibodies indicated that, in resting chromaffin cells, a filamentous actin network is visualized as a strong cortical fluorescent ring (Lee and Trifar6, 1981; C h e e k and Burgoyne, 1986, 1987; Trifar6 et al., 1989b). Cholinergic receptor stimulation produces a fragmentation of the fluorescent ring leaving cell cortical areas devoid of fluorescence (Cheek and Burgoyne, 1986, 1987; Trifar6 et al., 1989b). These changes are accompanied by the decrease in F-actin associated with the concomitant increase in G-actin as evaluated by D N A s e I inhibition assay (Cheek and Burgoyne, 1986; Trifar6 et al., 1989b). These changes are also accompanied by a decrease in the amount of F-actin recovered with cytoskeletons p r e p a r e d from stimulated cells (Burgoyne et al., 1989b; Trifar6, 1990b). F-actin network disassembly has also b e e n observed in mast cells upon stimulation (Koffer et al., 1990) and in depolarized (high K +) synaptosomes (Bernstein and Banburg, 1985). The existence of actin-binding proteins that regulate the dynamics of actin networks (Yin and Stosse!, I)79; Craig and Pollard, 1982; Stossel et al., !985; Mae!:_,.¢ a c t al., 1989; Rodriguez Del Castillo et al., 1990) strongly suggests a role for these proteins in the disassembly of actin filaments triggered by cell stimulation. Gelsolin is an actin filament c a p p i n g / severing protein found in many cells including chromaffin cells and extracellular fluids (Yin and Stossel, 1979; Yin et al., 1981; Stossel et al., 1985; Trifar6 et al., 1985b; Bader et al., 1986). Previous work from our laboratory described the presence in chromaffin cells of another actin-binding protein that can be eluted by E G T A containing buffers from actin D N A s e affinity. columns along with gelsolin (Bader et al., 1986). Recently we have isolated and given the n a m e of 'scindefin' to this new protein (Rodrlguez De! Castillo et al., 1990). Scinderin is a 79 kDa cytosolic protein that shortens actin filament length provided Ca z+ is present in the medium (Rodrfguez Del Castillo et al., 1990). Further work from our laboratory has demonstrated different tissue expressions for scinderin and gelsolin. Scinderin seems to be expressed in neuronal, endocrine and exocrine tissues (Tchakarov et al., 1991))

95 systems in which secretion is the main function. Immunocytochemical studies show that in chromaffin cells, scinderin has a diffused cytoplasmic and a more dense sub-plasmalemmal distribution (Rodr~guez Del Castillo et al., 1990). Instead, gelsolin only showed a diffused cytoplasmic distribution (Rodriguez Del Castillo et al., 1990). Immunocytochemical and biochemical experiments from our laboratory have clearly demonstrated that chromaffin cell stimulation produces a concomitant cortical surface distribution of scinderin and F-actin disassembly (Vitale et al., 1991a). The cellular distribution of gelsolin is not affected by cell stimulation. These dynamic changes (scinderin redistribution and F-actin disassembly) precede exocytosis and, as the latter event, require the presence of extracellular Ca 2+ (Vitale et al., 1991a). Stinmlationinduced redistribution of scinderin and F-actin disassembly would produce sub-plasmalemmal areas of decreased cytoplasmic viscosity and high secretory vesicle mobility (fig. 4). Therefore, only secretagogues which induce Ca 2+ entry are able to redistribute sub-plasmalemmal scinderin and produce disassembly of F-actin networks leaving cytoptasmic areas devoid of these two proteins. Published experiments with anti-Drill have indicated that exocytosis pits are preferentially present in plasma membrane areas devoid of F-actin (Vitale et al., 1991a).

5.5. Modulation of release 5.5.1. GTP-binding proteins It is generally accepted that G'l-P-binding proteins (G-proteins) transduce ho_ mona!, neurotransmitter and sensory signals across plasma membranes (Gilman, 1987). More recently, G-proteins have also been implicated in the regulation of ion channels (Brown and Birnbaumer, 1990) and in the vectorial transport of membranes in the secretory pathway (Bourne, 1988). In this regard, reports from several laboratories have described G-proteins associated with secretory. vesicles. These are either pertussis toxin substrates (Toutant et al., 1987) of molecular weight ranging from 39 to 41 kDa or of small molecular weight ras-like GTP-binding proteins (Burgoyne and Morgan, 1989; Doucet et al., 1989; Ngsee et al., 1990). Although the exact role ef G-proteins (large and small) in exocytosis is unknown, there is published evidence suggesting their involvement in exocytosis (Gomperts, 1990). The injection of H-ras protein into mast cells resulted in degranulation (Bar-Sagi and Gomperts, 1988). A rho C-protein, also a ras protein, is the substrate for ADP-ribosylation of the botulinum toxin C3 and probably plays a role in actin neurofilament assembly (Chardin et al., 1989). In this regard, a botulin toxin substrate also co-purifies with synaptic vesicles (Ngsee et al., 1990). Whether this

substrate is the rho C-protein earlier described, remains to be demonstrated. From work on yeast G-proteins, Bourne (1988) has proposed that GTP-binding proteins regulate constitutive vesicle transport through the cell. Moreover, in mammalian cells, the work of Gomperts" group has demonstrated that GTP and Ca 2~ are necessary for exocytosis from permeabilized neutrophils and mast cells (Barrowman et al., 1986; Cockcrofi et al., I987). Their results suggested the involvement of a novel GTP-binding protein (Ge) since GTPyS, a non-hydrolyzable GTP analogue, stimulates exocytosis even in the absence of Ca e* (Barrowman et ai., 1986). In platelets, Ca2+-induced release of serotonin is also potentiated by GTPTS (Haslam and Davison, 1984). In chromaffin cells, early reports have indicated that G T P y S inhibits secretion (Knight and Baker, 1985; Knight et al., 1989). However, newly published information also indicates that non-hydrolyzabte GTP analogues stimulate catecholamine secretion (Bader et al., 1989). In conclusion, evidence is emerging in favour of the role of GTP-binding proteins as modulators of exocytosis. However, it remains to be determined whether G-proteins act in secretion through signal transduction and generation of second messengers or, alternatively. through direct action on secretory vesicle function as suggested for yeast.

5.5.2. Proteh7 kinase C Since its discovery by Nishizuka and co-workers (Takai et al., I979), protein kinase C (PKC) has been shown to be an important element in many biological processes (Nishizuka, 1984; Hum,g, 1989; Rana and Hokin, t990). PKC is an enzyme whose activity depends on Ca :+ and its lipid environment, particularly, phosphatidylserine. Diacylgiycerol, a product of the PIPz cascade, also increases the affinity of the en~Tme for Ca :+. This physiological messenger is produced in many t3'pes of cells in response to synaptic or hormonal stimulation (Nishizuka, 1986). PKC can be stimulated by phorbol esters (Castagna et al., 1982) and these tumour promoters have been a valuable tool in the study of PKC role in cell function. Under resting conditions, the inactive enzyme is mainly cytoplasmic. However, upon stimulation, PKC is transtocated to the membranes where it can associate with phosphatidylserine and diacylglycerol. A series of substrates for PKC have been identified including cytoskeletal and cytoskeletal-associated proteins such as actin, myosin light chain, troponin, caldesmom talin, filamin, neurofilament subunits (Katoh et al., 1983; Naka et al., 1983; Nishizuka et al., 1983; Kawamoto and Hidaka, t984; Werth and Pastan, 1984; Lichtfield and Ball, 1986- Ohta et at., 1987; Sihag et al.. 1988: Georges et al., 1989): acetylcholine (Safran et al., 1987), insulin

% (3acobs et al., I983) and growth factor (Chochet et al., i984) receptors: enzymes such as tyrosine hydroxylase (Albert et al., 1984), guanylate cyciase (Zwillcr et a!., 1984) and glycogen synthase (Ahmed et al., 1984) and membrane proteins such as Na + / H + exchanger (Burns and Rozengurt, 1981) and Na + channel proteins (Costa and Catterall, 1985). PKC is highly concentrated in the brain (Kuo et al., 1980; Kikawa et al., 1982) suggesting an important role for this enzyme in the regulation of neuronal fimction (Kaczmarek, 1987). Activation of PKC by phorbol esters enhances the release of acetylcholine, noradrenaline, serotonin and 7-aminobutyric acid from synaptosomes and brain slices in response to secretagogues or electrical stimulation (Allgaier et al., 1987, 1988; Feuerstein et al., 1987; Vcrsteeg and Florijn, 1987; Versteeg and Ulenkate, 1987; Bartmann et al., 1989; Dekker et al., 1991). Inhibition of PKC activity by polymixin B or staurosporine partially diminishes stimulated neurotransmitter release and blocks the effect of phorbol esters. In nervous tissue, two major substrates for PKC are GAP-43 (B-50, neuromodulin) (Aloyo et al., 1983; Alexander et al., 1987), a major component of the motile growth-cone (Skene et al., 1986), and p87 (Albert et al., 1986; Patel and Kligman, 1987). It has also been shown that phosphorylation of GAP-43 (B-50, neuromodulin) correlates with neurotransmitter release (Dekker et al., 1989a, 1990a, b). Furthermore, antibody raised against GAP-43 inhibits noradrenaline release from rat synaptosomes (Dekker et al., 1989b). In adrenal chromaffin cells, stimulation of PKC by phorbol esters does not induce neurotransmitter release (Brocklehurst and Pollard, 1985; Pocotte et al., 1985; Bader et al., 1989; Bittner and Holz, 1990). However, enzyme activation does potentiate release in response to a physiological stimulus (Brocklehurst and Pollard, 1985; Pocotte et al., 1985; TerBush et al., 1988; Bader et al., 1989; Bitmer and Holz, 1990). There is experimental evidence that PKC may modulate ion transport across the plasma membrane. PKC has been shown to activate the Ca-'+-dependent ATPase (Limas, 1980), to increase Ca 2+ currents in neurons (Kaczmarek, 1987) and to potentiate K+-induced Ca z+ influx (Wakade et al., 1986). It has then been suggested that PKC may reguk~.te exocytosis through modulation of Ca 2+ influx through voltage-dependent Ca 2+ channels (Wakade et al., 1986; Kaczmarek, 1987) and N a + / H + exchange (Negishi et al., 1990). Although modulation of Ca ,-+ channel activity may be an important aspect of PKC action, experiments on ceils rendered 'leaky' by either electric fields or detergents have demonstrated a potentiation by the enzyme ef the Ca2~-dependent secretkm. The fact that, as in this case, PKC activation makes exocytosis nlachinery more sensitive to Ca 2' , strongly suggests other sites of action for PKC.

Work on several tissues and systems has shown that several cytoskeletal proteins are targets for PKC and that targets for PKC such as tJ,ar-,.J arc bound to neuronal membrane cytoskeleton (Moss et al., 1990). It is quite possible that the abi!ity of PKC to phosphorylate cytoskeletal or cytoskeletal-regulating proteins might be critical for neuronal regulation° In this regard, phorbol esters produce dramatic alterations in chroma~fin cell morphology, mainly through an effect on microfilament rearrangement (Grant and Aunis, 1990; Vitale et al., !991b). A short exposure (1 min) to phorbol esters is enough to produce cortical actin filament disassembly in chromaffin cells, a change that primes the cells for exocytosis (Vitale et al., 1991b). It is then possible that this effect of PKC on cortical actin filament networks is the mechanism by which the enzyme potentiates secretion.

5.6. Secretopy cesicle-p&sma membra,Te fitsion During exocytosis, the secretory vesicle membrane fuses with the target plasma membrane and, subsequently, the vesicle releases its soluble contents to the cell exterior. Current concepts on membrane fusion have derived from studies on artificial phospholipid systems (Wilschut and Hoechstra, 1984; Finkelstein et al., t986), from work on the mechanism of action of fusion proteins in the fusion of viral envelopes to membranes of host cells (Stegman et al., 1989) and especially from experiments with quic!:-e~eeze electrov_ microscopy on neurons (Heuser and Reese, !981) and secretory ceils (Chandler and Heuser, 1980; Schmidt et al., 1983). These latter experiments have shown that secretory vesicles frozen at early stages of exocytosis a~c connected :'; the extracellular space by narrow fusion pores. More recently, using the patch-clamp technique, it has been possible to detect exocytosis of single vesicles or group of vesicles (Neher and Marty, 1982; Fernfindez et al., 1984). In these experiments, the electrical capacitance of the plasma membrane is monitored and, as single vesicles are fused with the plasma membrane, stepwise increases in capacitance are observed (Neher and Marty, 1982; Fernfindez et al., 1984; Almers, 1990). Furthermore, giant secretory vesicles are found in mast cells of the mutant mouse, 'beige' mouse (Poon et aI., 1981). Patch-clamp experiments on these cells have shown that each giant vesicle generates, at the time of fusion, a brief current of 200-300 picosiemens (pS) (Breckenridge and Almers, 1987; Spruce et at., 1990). From this conductance value, it can be calculated that the initial diameter of the pore is in the order of 2 nm (Ahners, 1990). The time of exocytosis, as measured from the delay between arrival of the stimulus and the first sign of exocytosis (i.e. capacitance jump), varies from milliseconds in fast vesicles containing classical neurotransmitters (i.e. ncu-

97 romuscular junction) to seconds (i.e. mast cells). The difference in delay times could be the result of interw , t l o n , in each case, of different cytosolic messengers (modulators) or to the removal of different structural barriers (i.e. actin networks, see above). Nevertheless, vesicle-plasma m e m b r a n e fusion is too fast to be explained by a lipid-lipid interaction between membranes (Almers, 1990). Alternatively, specific proteins might be involved in m e m b r a n e fusion. Work on the interaction between virus and host cells has indicated that a fusion protein in the virus envelope should be inserted into the lip,id bilayer of the host cell m e m b r a n e (Stegman et al., 1989). However, this process is much slower than the delay at a fast synapse. Therefore, it has been suggested that fast release requires the pro-assembled fusion protein expanding both fusion membranes (AImers, 1990; Alme~s and Tse, 1990). In this case, the fusion pore protein would be formed by two halves, one in each membrane, and the m e m b r a n e topology of these halves should be such that, at the arrival of the action potential, they would simply dock with each o t h e r (Breckenridge and Almers, 1987a; Thomas et al., t988). T h e r e are several candidates for fusion proteins. Synexin, a protein first discovered in chromaffin cells (Creutz et al., 1978) has been reported to cause Ca 2+d e p e n d e n t aggregation of chromaffin granules and to form Ca 2+ channels in artificial !ipid bilayers (Pollard et al., 1988). Synaptophysin, a sec~. ,tory vesicle protein (see above), is another candidate which forms channels in lipid bilayers with voltage-sensitive conductances in the order of 150 pS (Thomas et al., 19b18). Moreover, a secretory vesicle docking protein has also been purified from chromaffin cell plasma m e m b r a n e s (Meyer and Burger, 1979). Intracellular delivery, into chromaffin cells of antibodies against this secretory vesicle-binding protein impaired the release of catecholamines and the increases ~n m e m b r a n e capacitance observed during exocytosis (Schweizer et al., 1989). In conclusion, new advanced electrophysiolcgical techniques have made possible the study of fusion in exocytosis with real-time resolution and at the level of a single secretory vesicle. However, further studies arc necessary to identify, the molecular components involved in the fusion process.

6. Final remarks The process of exocytosis is a fascinating interplay between cellular components and the secretory' vesicles. These organelles provide the cell with an efficient carrier of material for export. In the vesicle, the secretory material is protected from destruction and it can reach high concentrations. The vesicle also allowed the release of fixed (quantum) amounts of material to the cell exterior. Our knowledge of the membrane compo-

nents of the secreto~, vesicles has increased rapidly in recent years. Thu:;, i::Ic:'ae~2?n::. ~:f.vgc,:e~y .~_ey~!.e~: with calmodulin, GTP-binding proteins, cymskci.z:al components, etc., have been described. This, together with a better understanding of the cytoskeleton network and its dynamics, has made it possible to begin to understand the function of the exocytotic machinery. There is no doubt that exocytosis is an all-or-none p h e n o m e n o n and that Ca ?... plays a pivotal role in secretion, However, the site, or more probably sites, of action for Ca 2+ in exocytosis remain to be elucidated. Ca ?+ is required for second messenger activity (i.e. calmodulin, PKC), for the control of eytoskeleton dynamics (i.e. actin filament disassembly) and probably for the vesicle-plasma m e m b r a n e fusion process and these could possibly be the sites of action for Ca 2 + in secretion. Advanced electrophysiological techniques (i.e. patch-clamp, capacitance measurements), which allowed the study of fusion in real-time resolution and at the level of a single secretory vesicle, have also provided a better understanding of secretion, Moreover, the studies on the dynamic changes in the cortical actin filament network brought about by cell stimulation should continue since we believe that they are of crucial importance in the understanding of secretion at molecular level. Finally, v,e have no doubts that if the study of exocytosis (using a multi-disciplinary approach) continues at the present pace, a clear picture of the molecular mechanisms governing secretion will soon emerge.

Acknowledgements We arc graleful to Mrs. S. Dunn for typing the manuscript. Research performed in the authors" taboratoD, was supported by grants from the Medicat Research Council of Canada.

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