Biochimica et Biophysica Acta, 1071 ( 1991 ) 174-21)2 © 1991 Elsevier Science Publishers B.V. 0304-4157/91/$03.50 ADONIS 03(}441579100059Z
174
Review
BBAREV 85381
Control of exocytosis in adrenal chromaffin cells R o b e r t D. B u r g o y n e Department of Physiology, University of Lit'erpool, Lil'erpool (U. K. ) (Received 8 January 1991)
Contents I,
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
174
I!.
Technical approaches for the study of exocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
176
III.
Receptor activation and exocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
177
IV.
The control of exocytosis by second messengers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
178
A. Calcium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Cyclic nucleotides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
178 184
V.
The cytoskeleton, granule movement and exocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
185
VI.
The exocytotic mechanism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Intracellular requirements for exocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Effect of toxins on exocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Phospholipase A2 and arachidonic acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Metailoendoproteinases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Calmodulin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. Phosphorylation, dephosphorylation and exocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G. Protein kinase C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . H. Guanine nucleotide binding proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I. Plasma membrane proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . J. Secretory vesicle proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . K. Involvement of annexins in exocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . L. Cytosolic proteins in exocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
187 187 188 189 190 1911 190 191 192 193 194 194 197
Vll.
Conclusions
Acknowledgements
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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction Over the past few years, considerable progress has been made in understanding the organisation of the secretory pathway and the molecular basis of vesicular transport. A number of proteins involved in vesicle budding, transport and the intracellular fusions that
Correspondence: R.D. Burgoyne, Department of Physiology, University of Liverpool, PO Box 147, Liverpool L69 3BX, U.K.
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occur during transport from the endoplasmic reticulum to the Golgi and between the stacks of the Golgi have been identified from studies on yeast secretory mutants and in vitro preparations from mammalian cells [20,21,84,141,234,235,274]. The regulation of the final step of the secretory pathway, exocytotic fusion of constitutive or regulated secretory vesicles, has been studied in detail for many cell types but still little is known about the fundamental mechanisms involved in exocytosis. One of the reasons for this is that it has been difficult to establish in vitro fusion systems be-
175
Fig. 1. Electron micrographs of isolated bovine adrenal chromaffin cells. The cortical region of cytoplasm from which most secretory granules are excluded can be seen in the higher power micro~raph in B. Scale bars: 1 ~m in A; 200 nm in B.
ACh
tween secretory vesicles and plasma membrane that would allow identification of the required components. In addition, in the study of regulated secretion, experimental approaches aimed at understanding exocytosis are complicated by the fact that the exocytotic process is regulated by several interacting second messenger systems. Thus, in these cells, the exocytotic machinery is overlaid by additional components whose function is regulatory rather than essential. This means that manipulations of cells that modify secretion may not necessarily do so directly at the level of the exocytotic machinery. Amongst those cells in which regulated exocytosis has been examined is the bovine adrenal chromaffin cell (Fig. 1). For a variety of reasons this cell has been intensively studied by those interested in endocrine mechanisms, neuronal function and the basic cell biology of the secretory process [46,162,280]. The secretory vesicle of the chromaffin cell, the chromaffin granule, can readily be prepared in a highly purified form and has been studied by many laboratories. Bovine adrenal chromaffin cells can be isolated in large quantities to provide a relatively homogenous cell population for the study of secretion by a variety of technical approaches including morphological, electrophysiological and biochemical analyses. For those interested in neuronal function, chromaffin cells possess the advantage that they are derived during embryogenesis from the same precursors as sympathetic neurons and possess some properties in common with neurons such as the possession of voltage-dependent Na ÷ and Ca-"÷ channels and a variety of 'neuronal-specific' proteins. In this article I will review current knowledge on the control of exocytosis in chromaffin cells and discuss progress that is being made in identifying the components involved in
ACh
a
Ca"
Fig. 2. General scheme showing cholinergic stimulation and the control of exocytosis in bovine adrenal chromaffin cells. The sources of calcium include entry from outside and release from two separate internal stores. The cytoskeletal barrier at the cell cortex is disassembled in response to calcium or activation of protein kinase C. The exocytotic machinery is shown as a filled box. Following exocytosis vesicle membrane is recovered by endocytosis through coated vesicles. Abbreviations: pic, phospholipase C; pkc, protein kinase C; ACh, acetyicholine; m, muscarinic receptor; n, nicotinic receptor.
176 the exocytotic process in these cells. I will deal almost exclusively with work on chromaffin cells of bovine origin since chromaffin cells of this species have been most studied and significant species differences between chromaffin cells do exist. I will, however, at times mention work on exocytosis in other secretory cell types where study of those cells has illuminated mechanisms that may also be important in bovine adrenal chromaffin cells and indicate where aspects of the control mechanisms described may be peculiar to chromaffin cells rather than being general to all secretory cells. The major physiological stimulus for exocytosis in chromaffin cells is a rise in [Ca 2+]i (Fig. 2). Ca 2+ is sufficient to activate exocytosis in permeabilized cells and Ca2+-dependent exocytosis is known to be modulated by several intracellular factors. The mechanisms by which calcium activates exocytosis and the way in which this is modulated still remains to be fully elucidated. In this review I will discuss the nature of the Ca 2+ signal, the role of other second messengers and the factors that regulate or may be components of the Ca2÷-dependent exocytotic mechanism. II. Technical approaches for the study of exocytosis A number of technical approaches have been developed that allow detailed examination of the control of exocytosis in chromaffin cells. Changes in [Ca2+]i have been studied in single cells and the use of video-imaging techniques has allowed the spatial characteristics of the Ca 2÷ signal to be investigated. Complementary to these studies have been attempts to develop assays for exocytosis in single cells. Membrane capacitance measurement with the whole cell patch-clamp technique [200,213] has allowed direct determination of the extent of exocytotic membrane fusion and this can be combined with measurement of [Ca2+]i [2121. The whole cell patch clamp approach suffers from the disadvantage that the internally dialysed cells do not respond to agonists. A co-culture technique has been developed that allows the localization of [Ca2+]i and exocytosis of ATP to be monitored in single chromaffin cells [78]. This technique is based on the fact that ATP, which is stored at high levels in the secretory granules of chromaffin cells, elicits a rise in [Ca z÷ ]i in surrounding fibroblasts by acting on ATP receptors. This assay allows spatial aspects of exocytosis to be examined. Exocytosis can also be visualised in single ceils by staining intact ceils with antisera against secretory granule components such as dopamine-/3-hydroxylase that appear on the cell surface following exocytotic granule fusion [78,210,211,220]. This technique does not allow the time course of exocytosis in single cells to be monitored and is only semi-quantitative. A sensitive voltametric technique for the assay of catecholamine
release from single chromaffin cells has recently been described and it was claimed that the technique is sufficiently sensitive to detect catecholamine release from a single chromaffin granule [171]. This technique will probably be very useful for the determination of the kinetics of exocytosis under various experimental conditions. Direct information on the factors controlling exocytosis has come from the use of techniques that allow access to the intracellular sites of exocytosis and thus the ability to directly manipulate intracellular factors that may control exocytosis [8,159,176]. This approach was pioneered by Baker and Knight [19,155] who permeabilized chromaffin cells by electric discharge (electro-permeabilization). Subsequently, chromaffin cells have been permeabilized by treatment with saponin [42], digitonin [106,279], staphylococcal alpha toxin [17], or streptolysin O [251]. The plasma membrane lesions generated by the permeabilization methods vary with those due to saponin, digitonin and streptolysin O being large enough to allow proteins to slowly enter or leave the cells. Cells permeabilized with alpha toxin [8] or by electro-permeabilization have much smaller lesions (approx. 2-4 nm [155]) that only allow passage of molecules of up to around 1000 daltons. Permeabilized cells allow contr,al of the intracellular conditions and have resulted in the demonstration that in chromaffin cells, Ca 2+ is sufficient to activate exocytr~sis. The concentration of Ca 2÷ required to activate exocytosis is similar with all permeabilization techniques (10 ~M for maximal response) except for the alpha toxin method in which much higher Ca 2÷ levels are required. The reason for the different results with alpha toxin-permeabilized cells has not been established but it could be due to a limitation in the rate of diffusion of Ca2+-buffers into these cells through the small pores formed by the toxin. In the case of saponin- and digitonin-permeabilized cells, convincing electron microscopical evidence has shown that the cells release catecholamine by exocytosis [41,242]. It has been suggested from electron microscopical observations that cells permeabilized with digitonin have lesions large enough for chromaffin granules to be lost from the cells [128]. This conclusion may have resulted from the extreme fragility of digitoninpermeabilized cells when processed for electron microscopy. Others have shown that intact granules are not released from these cells in the presence or absence of Ca 2÷ [137] and that the morphology of the cells is very similar to intact cells [242]. In recent years experimental work has increasingly been carried out on chromaffin cells maintained in culture for varying periods of time. Two notes of caution must be made about the use of such cells. One is that chromaffin cells do change functionally during time in culture (e.g., Ref. 195). In some laboratories
177 chromaffin cells also begin to change morphologically and grow processes after several days in culture. In addition, freshly isolated cell p r e p a r a t i o n s contain a relatively small p e r c e n t a g e of c o n t a m i n a t i n g cells which can easily be r e d u c e d by differential plating [273a] but these c o n t a m i n a t i n g cells divide in culture and can exert major distorting effects on biochemical m e a s u r e ments on o l d e r cultures. It is t h e r e f o r e essential that chromaffin ceils should be used after only short culture periods before these changes occur and where possible the results confirmed with freshly isolated cells. Ill.
Receptor
activation
and
exocytosis
I reviewed exocytosis in chromaffin cells in a B B A Review on B i o m e m b r a n e s in 1984 [46]. At that time the only r e c e p t o r clearly linked to stimulation of the exocytosis of catecholamine in bovine chromaffin cells was the nicotinic cholinergic receptor. Since then it has become clear that activation of a n u m b e r of o t h e r receptors on chromaffin cells will stimulate catecholamine secretion and that activation of others modulates nicotine-induced secretion (Table I). R e c e p t o r activation in chromaffin cells e i t h e r results in depolarisation and o p e n i n g of v o l t a g e - d e p e n d e n t channels, direct activation of phospholipase C with subsequent generation of the Ca 2 +-mobilising signal inositol 1,4,5trisphosphate (Ins(1,4,5)P 3) [28] and in some cases generation of Ins(1,4,5)P 3 and o p e n i n g of so far uncharacterised non-voltage d e p e n d e n t Ca 2+ channels. Mus-
carinic, angiotensin II, bradykinin, histamine, prostaglandin E2, V I P and A T P receptors are all linked to Ins(1,4,5)P 3 p r o d u c t i o n (Table 1). Muscarinic stimu!ation has also b e e n shown to raise the levels of inositol t e t r a k i s p h o s p h a t e [235a]. Nicotinic stimulation and depolarisation with high K + leads to lns(1,4,5)P 3 production t h r o u g h CaZ+-dependent activation of phospholipase C [108,109,195] and in addition nicotinic stimulation results in an increase in the levels of inositol p e n t a k i s p h o s p h a t e [238] the significance of which is unclear. The efficacy of the various r e c e p t o r agonists in stimulating secretion is very variable with nicotinic stimulation giving the biggest response° In fact, a p a r t from nicotinic agonists only histamine and G A B A produce a substantial secretory response (Fig. 3). T h e various receptors that stimulate exocytosis appear to be linked to changes in intracellular calcium concentration ([Ca2+]i) due to either Ca 2+ entry or release of Ca :+ from internal stores following production of Ins(1,4,5)P 3. T h e extent of secretion of catecholamine in response to various agonists is not correlated with the peak rise in [Ca2+]i obtained (Fig. 3). However, secretion in all cases is abolished by removal of external Ca 2+ (Fig. 4 and Ref. 206). This demonstrates in a simple fashion that it is Ca 2+ entry and not release of Ca 2+ from internal stores that activates exocytosis in chromaffin cells. Secretion in response to G A B A , which is not shown in Figs. 3 and 4, is also d e p e n d e n t on external Ca 2+ and Ca 2+ entry occurs following depo-
TABLE I Receptors present on borine adrenal chromaffin cells
The effect of a rar~ge of receptor agonists on [Ca" ~-]i and the levels of lns(l,4,5)P3 and whether they stimulate Ca-"+ entry is shown. The effect of the agonists on basal secretion and secretion in response to nicotinic stimulation is indicated. +, stimulation; - , inhibition: 0, ,to effect. Receptor
[Ca-"÷ ]i r i s e
Ins(1,4,5)P 3 production
Ca-' ÷ entry
Secretion basal
Ref. nicotine
Angiotensin II ATP ANP Bradykinin Chromostatin
'~ + + 9 + 0
? ++ ++ "~ ++ '~
'~ + + '~ + 0
+ + + 0 + -
Dopamine D a
9
'~
+
9
Dopamine D z Endothelin GABA A GABA a
9 + ++ '~
~ '~ 9 '~
9 ++ o
0 + ++ -
9 9
H i s t a m i n e H~
+ + + +
+ + +
+ ~.
+ + +
9
IGFI Muscarinic Nicotinic Opioid
9 +
'~ +
'~ 0
0 0
+ 0
+ + +
+
+ + +
+ + + +
"
0
'~
0
-
PGE2
+ 9 9 0
++ 9 ,~ ++
+ '~ 9 9
0/+ 0 0 0
+
Adenosine
Somatostatin Substance P VIP
0 0 "~ + '~ 9
-
82 45,206,221,255,282 151,239 2O7 206,209a.221 118a.247 12 30,175,252 226 67,102,146 67 68a,201,202,206,221,255,273 93,94 71,113,206 168 137a,145,189a,199,221a 173,186,231 173,231 277
178
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300
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174
Review
BBAREV 85381
Control of exocytosis in adrenal chromaffin cells R o b e r t D. B u r g o y n e Department of Physiology, University of Lit'erpool, Lil'erpool (U. K. ) (Received 8 January 1991)
Contents I,
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
174
I!.
Technical approaches for the study of exocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
176
III.
Receptor activation and exocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
177
IV.
The control of exocytosis by second messengers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
178
A. Calcium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Cyclic nucleotides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
178 184
V.
The cytoskeleton, granule movement and exocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
185
VI.
The exocytotic mechanism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Intracellular requirements for exocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Effect of toxins on exocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Phospholipase A2 and arachidonic acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Metailoendoproteinases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Calmodulin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. Phosphorylation, dephosphorylation and exocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G. Protein kinase C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . H. Guanine nucleotide binding proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I. Plasma membrane proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . J. Secretory vesicle proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . K. Involvement of annexins in exocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . L. Cytosolic proteins in exocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
187 187 188 189 190 1911 190 191 192 193 194 194 197
Vll.
Conclusions
Acknowledgements
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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction Over the past few years, considerable progress has been made in understanding the organisation of the secretory pathway and the molecular basis of vesicular transport. A number of proteins involved in vesicle budding, transport and the intracellular fusions that
Correspondence: R.D. Burgoyne, Department of Physiology, University of Liverpool, PO Box 147, Liverpool L69 3BX, U.K.
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.
.
.
,
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,
198
,
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.
,
! 98
198
occur during transport from the endoplasmic reticulum to the Golgi and between the stacks of the Golgi have been identified from studies on yeast secretory mutants and in vitro preparations from mammalian cells [20,21,84,141,234,235,274]. The regulation of the final step of the secretory pathway, exocytotic fusion of constitutive or regulated secretory vesicles, has been studied in detail for many cell types but still little is known about the fundamental mechanisms involved in exocytosis. One of the reasons for this is that it has been difficult to establish in vitro fusion systems be-
175
Fig. 1. Electron micrographs of isolated bovine adrenal chromaffin cells. The cortical region of cytoplasm from which most secretory granules are excluded can be seen in the higher power micro~raph in B. Scale bars: 1 ~m in A; 200 nm in B.
ACh
tween secretory vesicles and plasma membrane that would allow identification of the required components. In addition, in the study of regulated secretion, experimental approaches aimed at understanding exocytosis are complicated by the fact that the exocytotic process is regulated by several interacting second messenger systems. Thus, in these cells, the exocytotic machinery is overlaid by additional components whose function is regulatory rather than essential. This means that manipulations of cells that modify secretion may not necessarily do so directly at the level of the exocytotic machinery. Amongst those cells in which regulated exocytosis has been examined is the bovine adrenal chromaffin cell (Fig. 1). For a variety of reasons this cell has been intensively studied by those interested in endocrine mechanisms, neuronal function and the basic cell biology of the secretory process [46,162,280]. The secretory vesicle of the chromaffin cell, the chromaffin granule, can readily be prepared in a highly purified form and has been studied by many laboratories. Bovine adrenal chromaffin cells can be isolated in large quantities to provide a relatively homogenous cell population for the study of secretion by a variety of technical approaches including morphological, electrophysiological and biochemical analyses. For those interested in neuronal function, chromaffin cells possess the advantage that they are derived during embryogenesis from the same precursors as sympathetic neurons and possess some properties in common with neurons such as the possession of voltage-dependent Na ÷ and Ca-"÷ channels and a variety of 'neuronal-specific' proteins. In this article I will review current knowledge on the control of exocytosis in chromaffin cells and discuss progress that is being made in identifying the components involved in
ACh
a
Ca"
Fig. 2. General scheme showing cholinergic stimulation and the control of exocytosis in bovine adrenal chromaffin cells. The sources of calcium include entry from outside and release from two separate internal stores. The cytoskeletal barrier at the cell cortex is disassembled in response to calcium or activation of protein kinase C. The exocytotic machinery is shown as a filled box. Following exocytosis vesicle membrane is recovered by endocytosis through coated vesicles. Abbreviations: pic, phospholipase C; pkc, protein kinase C; ACh, acetyicholine; m, muscarinic receptor; n, nicotinic receptor.
176 the exocytotic process in these cells. I will deal almost exclusively with work on chromaffin cells of bovine origin since chromaffin cells of this species have been most studied and significant species differences between chromaffin cells do exist. I will, however, at times mention work on exocytosis in other secretory cell types where study of those cells has illuminated mechanisms that may also be important in bovine adrenal chromaffin cells and indicate where aspects of the control mechanisms described may be peculiar to chromaffin cells rather than being general to all secretory cells. The major physiological stimulus for exocytosis in chromaffin cells is a rise in [Ca 2+]i (Fig. 2). Ca 2+ is sufficient to activate exocytosis in permeabilized cells and Ca2+-dependent exocytosis is known to be modulated by several intracellular factors. The mechanisms by which calcium activates exocytosis and the way in which this is modulated still remains to be fully elucidated. In this review I will discuss the nature of the Ca 2+ signal, the role of other second messengers and the factors that regulate or may be components of the Ca2÷-dependent exocytotic mechanism. II. Technical approaches for the study of exocytosis A number of technical approaches have been developed that allow detailed examination of the control of exocytosis in chromaffin cells. Changes in [Ca2+]i have been studied in single cells and the use of video-imaging techniques has allowed the spatial characteristics of the Ca 2÷ signal to be investigated. Complementary to these studies have been attempts to develop assays for exocytosis in single cells. Membrane capacitance measurement with the whole cell patch-clamp technique [200,213] has allowed direct determination of the extent of exocytotic membrane fusion and this can be combined with measurement of [Ca2+]i [2121. The whole cell patch clamp approach suffers from the disadvantage that the internally dialysed cells do not respond to agonists. A co-culture technique has been developed that allows the localization of [Ca2+]i and exocytosis of ATP to be monitored in single chromaffin cells [78]. This technique is based on the fact that ATP, which is stored at high levels in the secretory granules of chromaffin cells, elicits a rise in [Ca z÷ ]i in surrounding fibroblasts by acting on ATP receptors. This assay allows spatial aspects of exocytosis to be examined. Exocytosis can also be visualised in single ceils by staining intact ceils with antisera against secretory granule components such as dopamine-/3-hydroxylase that appear on the cell surface following exocytotic granule fusion [78,210,211,220]. This technique does not allow the time course of exocytosis in single cells to be monitored and is only semi-quantitative. A sensitive voltametric technique for the assay of catecholamine
release from single chromaffin cells has recently been described and it was claimed that the technique is sufficiently sensitive to detect catecholamine release from a single chromaffin granule [171]. This technique will probably be very useful for the determination of the kinetics of exocytosis under various experimental conditions. Direct information on the factors controlling exocytosis has come from the use of techniques that allow access to the intracellular sites of exocytosis and thus the ability to directly manipulate intracellular factors that may control exocytosis [8,159,176]. This approach was pioneered by Baker and Knight [19,155] who permeabilized chromaffin cells by electric discharge (electro-permeabilization). Subsequently, chromaffin cells have been permeabilized by treatment with saponin [42], digitonin [106,279], staphylococcal alpha toxin [17], or streptolysin O [251]. The plasma membrane lesions generated by the permeabilization methods vary with those due to saponin, digitonin and streptolysin O being large enough to allow proteins to slowly enter or leave the cells. Cells permeabilized with alpha toxin [8] or by electro-permeabilization have much smaller lesions (approx. 2-4 nm [155]) that only allow passage of molecules of up to around 1000 daltons. Permeabilized cells allow contr,al of the intracellular conditions and have resulted in the demonstration that in chromaffin cells, Ca 2+ is sufficient to activate exocytr~sis. The concentration of Ca 2÷ required to activate exocytosis is similar with all permeabilization techniques (10 ~M for maximal response) except for the alpha toxin method in which much higher Ca 2÷ levels are required. The reason for the different results with alpha toxin-permeabilized cells has not been established but it could be due to a limitation in the rate of diffusion of Ca2+-buffers into these cells through the small pores formed by the toxin. In the case of saponin- and digitonin-permeabilized cells, convincing electron microscopical evidence has shown that the cells release catecholamine by exocytosis [41,242]. It has been suggested from electron microscopical observations that cells permeabilized with digitonin have lesions large enough for chromaffin granules to be lost from the cells [128]. This conclusion may have resulted from the extreme fragility of digitoninpermeabilized cells when processed for electron microscopy. Others have shown that intact granules are not released from these cells in the presence or absence of Ca 2÷ [137] and that the morphology of the cells is very similar to intact cells [242]. In recent years experimental work has increasingly been carried out on chromaffin cells maintained in culture for varying periods of time. Two notes of caution must be made about the use of such cells. One is that chromaffin cells do change functionally during time in culture (e.g., Ref. 195). In some laboratories
177 chromaffin cells also begin to change morphologically and grow processes after several days in culture. In addition, freshly isolated cell p r e p a r a t i o n s contain a relatively small p e r c e n t a g e of c o n t a m i n a t i n g cells which can easily be r e d u c e d by differential plating [273a] but these c o n t a m i n a t i n g cells divide in culture and can exert major distorting effects on biochemical m e a s u r e ments on o l d e r cultures. It is t h e r e f o r e essential that chromaffin ceils should be used after only short culture periods before these changes occur and where possible the results confirmed with freshly isolated cells. Ill.
Receptor
activation
and
exocytosis
I reviewed exocytosis in chromaffin cells in a B B A Review on B i o m e m b r a n e s in 1984 [46]. At that time the only r e c e p t o r clearly linked to stimulation of the exocytosis of catecholamine in bovine chromaffin cells was the nicotinic cholinergic receptor. Since then it has become clear that activation of a n u m b e r of o t h e r receptors on chromaffin cells will stimulate catecholamine secretion and that activation of others modulates nicotine-induced secretion (Table I). R e c e p t o r activation in chromaffin cells e i t h e r results in depolarisation and o p e n i n g of v o l t a g e - d e p e n d e n t channels, direct activation of phospholipase C with subsequent generation of the Ca 2 +-mobilising signal inositol 1,4,5trisphosphate (Ins(1,4,5)P 3) [28] and in some cases generation of Ins(1,4,5)P 3 and o p e n i n g of so far uncharacterised non-voltage d e p e n d e n t Ca 2+ channels. Mus-
carinic, angiotensin II, bradykinin, histamine, prostaglandin E2, V I P and A T P receptors are all linked to Ins(1,4,5)P 3 p r o d u c t i o n (Table 1). Muscarinic stimu!ation has also b e e n shown to raise the levels of inositol t e t r a k i s p h o s p h a t e [235a]. Nicotinic stimulation and depolarisation with high K + leads to lns(1,4,5)P 3 production t h r o u g h CaZ+-dependent activation of phospholipase C [108,109,195] and in addition nicotinic stimulation results in an increase in the levels of inositol p e n t a k i s p h o s p h a t e [238] the significance of which is unclear. The efficacy of the various r e c e p t o r agonists in stimulating secretion is very variable with nicotinic stimulation giving the biggest response° In fact, a p a r t from nicotinic agonists only histamine and G A B A produce a substantial secretory response (Fig. 3). T h e various receptors that stimulate exocytosis appear to be linked to changes in intracellular calcium concentration ([Ca2+]i) due to either Ca 2+ entry or release of Ca :+ from internal stores following production of Ins(1,4,5)P 3. T h e extent of secretion of catecholamine in response to various agonists is not correlated with the peak rise in [Ca2+]i obtained (Fig. 3). However, secretion in all cases is abolished by removal of external Ca 2+ (Fig. 4 and Ref. 206). This demonstrates in a simple fashion that it is Ca 2+ entry and not release of Ca 2+ from internal stores that activates exocytosis in chromaffin cells. Secretion in response to G A B A , which is not shown in Figs. 3 and 4, is also d e p e n d e n t on external Ca 2+ and Ca 2+ entry occurs following depo-
TABLE I Receptors present on borine adrenal chromaffin cells
The effect of a rar~ge of receptor agonists on [Ca" ~-]i and the levels of lns(l,4,5)P3 and whether they stimulate Ca-"+ entry is shown. The effect of the agonists on basal secretion and secretion in response to nicotinic stimulation is indicated. +, stimulation; - , inhibition: 0, ,to effect. Receptor
[Ca-"÷ ]i r i s e
Ins(1,4,5)P 3 production
Ca-' ÷ entry
Secretion basal
Ref. nicotine
Angiotensin II ATP ANP Bradykinin Chromostatin
'~ + + 9 + 0
? ++ ++ "~ ++ '~
'~ + + '~ + 0
+ + + 0 + -
Dopamine D a
9
'~
+
9
Dopamine D z Endothelin GABA A GABA a
9 + ++ '~
~ '~ 9 '~
9 ++ o
0 + ++ -
9 9
H i s t a m i n e H~
+ + + +
+ + +
+ ~.
+ + +
9
IGFI Muscarinic Nicotinic Opioid
9 +
'~ +
'~ 0
0 0
+ 0
+ + +
+
+ + +
+ + + +
"
0
'~
0
-
PGE2
+ 9 9 0
++ 9 ,~ ++
+ '~ 9 9
0/+ 0 0 0
+
Adenosine
Somatostatin Substance P VIP
0 0 "~ + '~ 9
-
82 45,206,221,255,282 151,239 2O7 206,209a.221 118a.247 12 30,175,252 226 67,102,146 67 68a,201,202,206,221,255,273 93,94 71,113,206 168 137a,145,189a,199,221a 173,186,231 173,231 277
178
700
16 x x
600
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0 0
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4
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bas meth ang
bk
caff
K÷
hist
x. \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ x x \ \ \ \
0
500
C '--
400
O
300
L.
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C '"
200
I11 0 (3=
100
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