Characterization of hormone and protein release from alpha-toxin-permeabilized chromaffin cells in primary culture

Addition of Staphylococcus aureus alpha-toxin to adult bovine chromaffin cells maintained in primary culture causes permeabilization of cell membrane as shown by the release of intracellular 86Rb+. The alpha-toxin does not provoke a spontaneous release of either catecholamines or chromogranin A, a protein marker of the secretory granule, showing the integrity of the secretory vesicle membrane. However the addition of micromolar free Ca2+ concentration induced the co-release of noradrenaline and chromogranin A. In alpha-toxin-treated cells, the released chromogranin A could not be sedimented and lactate dehydrogenase was still associated within cells, which provides direct evidence that secretory product is liberated by exocytosis. By contrast, permeabilization of cells with digitonin caused a Ca2+-dependent but also a Ca2+-independent release of secretory product, a dramatic loss of lactate dehydrogenase, as well as release of secretory product in a sedimentable form. Ca2+-dependent exocytosis from alpha-toxin-permeabilized cells required Mg2+-ATP and did not occur in the presence of other nucleotides. Thus alpha-toxin is a convenient tool to permeabilize chromaffin cells, and has the advantage of keeping intracellular structures, specifically the exocytotic machinery, intact.     

Interaction of protein kinase C with chromaffin granule membranes: effects of Ca2+, phorbol esters and temperature reveal differences in the properties of the association and dissociation events

Interaction of protein kinase C with chromaffin granule membranes has been studied as a means of investigating the translocation of protein kinase C from cytosol to intracellular membrane surfaces, which is believed to occur during secretion. Protein kinase C in an adrenal medullary soluble fraction was found to bind reversibly to granule membranes in a Ca2+-dependent fashion. Association and dissociation events were sensitive to Ca2+ concentrations in the low micromolar range, and the Ca2+ sensitivity of both processes was increased when the membranes had been preincubated with the protein kinase C-activating phorbol ester, 4 beta-phorbol 12-myristate 13-acetate (TPA). Binding of protein kinase C to granule membranes occurred at 0 and 37 degrees C, irrespective of whether the membranes had been preincubated with TPA. However, dissociation of protein kinase C from granule membranes that had been preincubated with TPA occurred only at 37 degrees C and not at 0 degree C, even though dissociation of the enzyme from membranes which had not been preincubated with TPA would occur at both 37 and 0 degrees C. These effects of TPA were not reproduced by 4 alpha-phorbol 12,13-didecanoate (4 alpha PDD), a phorbol ester which does not activate protein kinase C. Soluble protein kinase C activity also associated with chromaffin granules in a Ca2+-dependent manner in an adrenal medullary homogenate, indicating that granules can compete with other intracellular membranes for the binding of protein kinase C. Results obtained with this model system differ from other systems where the interaction of protein kinase C with plasma membranes has been studied and have general implications for studies performed on the translocation of protein kinase C in intact cells and for the role of protein kinase C in stimulus-secretion coupling in the chromaffin cell.     

Effects of Osmolality and Ionic Strength on Secretion from Adrenal Chromaffin Cells Permeabilized with Digitonin

Hyperosmotic solutions inhibit exocytosis of catecholamine from adrenal chromaffin cells at a step after Ca2+ entry into the cells. The possibility that the inhibition resulted from an inability of shrunken secretory granules to undergo exocytosis was investigated in cells with plasma membranes permeabilized by digitonin. The osmoticants and salts used in this study rapidly equilibrated across the plasma membrane and bathed the intracellular organelles. When sucrose was the osmoticant, secretion was not significantly inhibited unless the osmolality was raised above 1,000 mOs. When the osmolality was raised with the tetrasaccharide stachyose or a low-molecular-weight maltodextrin fraction (average size a tetrasaccharide), one-half maximal inhibition occurred at 900-1,000 mOs. Prior treatment of permeabilized cells with Ca2+ in hyperosmotic solution did not result in enhanced secretion when cells were restored to normal osmolality. Increased concentrations of potassium glutamate or sodium isethionate were more potent than carbohydrate in inhibiting secretion. Half-maximal inhibition occurred at 600-700 mOs or when the ionic strength was approximately doubled. The inhibition by elevated potassium glutamate also occurred when the osmolality was kept constant with sucrose. Increasing the ionic strength did not alter the Ca2+ sensitivity of the secretory response. Reducing the ionic strength by substituting sucrose for salt reduced the Ca2+ concentration required for half-maximal stimulated secretion from approximately 1.2 microM to 0.5 microM. Chromaffin granules, the secretory granules, are known to shrink in hyperosmotic solution. The experiments indicate that shrunken chromaffin granules can undergo exocytosis and suggest that in intact cells elevated ionic strength rather than chromaffin granule shrinkage contributes to the inhibition of secretion by hyperosmotic solutions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ca2+ influx causes rapid translocation of protein kinase C to membranes. Studies of the effects of secretagogues in adrenal chromaffin cells

In bovine adrenal chromaffin cells nicotinic stimulation or a depolarizing concentration of K+ caused a rapid, transient translocation to membranes of as much as 14% of the total cellular protein kinase C activity. The quantitative relationship between membrane-bound protein kinase C and Ca2+-dependent secretion was determined in cells rendered leaky by digitonin treatment. Intact cells were incubated with various concentrations of 12-O-tetradecanoylphorbol-13-acetate (TPA) to activate and cause translocation of protein kinase C to membrane before permeabilization in the presence of Ca2+. For the same amount of membrane-bound protein kinase C, a similar degree of enhancement of Ca2+-dependent secretion occurred in cells incubated for 1 or 30 min with TPA. Translocation of as little as 2-3% of the cellular protein kinase C to the membrane enhanced Ca2+-dependent secretion by 25-30%. Muscarinic agonists caused a 5% increase in membrane-bound protein kinase C at 2 s which rapidly reversed. Nicotinic and muscarinic receptor-mediated increases in membrane-bound protein kinase C were additive at 10 s and synergistic at 3 min. Muscarinic stimulation enhanced nicotinic receptor-dependent secretion. Prior incubation with TPA caused a similar enhancement of nicotinic-mediated secretion. The data indicate that protein kinase C which is translocated within seconds of stimulation of the cells with a nicotinic agonist or elevated K+ probably enhances the secretory response immediately or soon after exocytosis begins. In addition, the muscarinic receptor-mediated enhancement of nicotinic receptor-stimulated secretion may be due to newly activated protein kinase C.     

Bovine chromaffin granule membranes undergo Ca(2+)-regulated exocytosis in frog oocytes

We have devised a new method that permits the investigation of exogenous secretory vesicle function using frog oocytes and bovine chromaffin granules, the secretory vesicles from adrenal chromaffin cells. Highly purified chromaffin granule membranes were injected into Xenopus laevis oocytes. Exocytosis was detected by the appearance of dopamine-beta-hydroxylase of the chromaffin granule membrane in the oocyte plasma membrane. The appearance of dopamine-beta-hydroxylase on the oocyte surface was strongly Ca(2+)-dependent and was stimulated by coinjection of the chromaffin granule membranes with InsP3 or Ca2+/EGTA buffer (18 microM free Ca2+) or by incubation of the injected oocytes in medium containing the Ca2+ ionophore ionomycin. Similar experiments were performed with a subcellular fraction from cultured chromaffin cells enriched with [3H]norepinephrine-containing chromaffin granules. Because the release of [3H]norepinephrine was strongly correlated with the appearance of dopamine-beta-hydroxylase on the oocyte surface, it is likely that intact chromaffin granules and chromaffin granule membranes undergo exocytosis in the oocyte. Thus, the secretory vesicle membrane without normal vesicle contents is competent to undergo the sequence of events leading to exocytosis. Furthermore, the interchangeability of mammalian and amphibian components suggests substantial biochemical conservation of the regulated exocytotic pathway during the evolutionary progression from amphibians to mammals.     

Evidence that the ability to respond to a calcium stimulus in exocytosis is determined by the secretory granule membrane: Comparison of exocytosis of injected bovine chromaffin granule membranes and endogenous cortical granules inXenopus laevis oocytes

Summary 1. To understand better the mechanisms which govern the sensitivity of secretory vesicles to a calcium stimulus, we compared the abilities of injected chromaffin granule membranes and of endogenous cortical granules to undergo exocytosis inXenopus laevis oocytes and eggs in response to cytosolic Ca²⁺. Exocytosis of chromaffin granule membranes was detected by the appearance of dopamine--hydroxylase of the chromaffin granule membrane in the oocyte or egg plasma membrane. Cortical granule exocytosis was detected by release of cortical granule lectin, a soluble constituent of cortical granules, from individual cells.2. Injected chromaffin granule membranes undergo exocytosis equally well in frog oocytes and eggs in response to a rise in cytosolic Ca²⁺ induced by incubation with ionomycin.3. Elevated Ca²⁺ triggered cortical granule exocytosis in eggs but not in oocytes.4. Injected chromaffin granule membranes do not contribute factors to the oocyte that allow calcium-dependent exocytosis of the endogenous cortical granules.5. Protein kinase C activation by phorbol esters stimulates cortical granule exocytosis in bothXenopus laevis oocytes andX. laevis eggs (Bement, W. M., and Capco, D. G.,J. Cell Biol. 108, 885–892, 1989). Activation of protein kinase C by phorbol ester also stimulated chromaffin granule membrane exocytosis in oocytes, indicating that although cortical granules and chromaffin granule membranes differ in calcium responsiveness, PKC activation is an effective secretory stimulus for both.6. These results suggest that structural or biochemical characteristics of the chromaffin granule membrane result in its ability to respond to a Ca²⁺ stimulus. In the oocytes, cortical granule components necessary for Ca²⁺-dependent exocytosis may be missing, nonfunctional, or unable to couple to the Ca²⁺ stimulus and downstream events.     

A pleckstrin homology domain specific for phosphatidylinositol 4, 5-bisphosphate (PtdIns-4,5-P2) and fused to green fluorescent protein identifies plasma membrane PtdIns-4,5-P2 as being important in exocytosis

Kinetically distinct steps can be distinguished in the secretory response from neuroendocrine cells with slow ATP-dependent priming steps preceding the triggering of exocytosis by Ca(2+). One of these priming steps involves the maintenance of phosphatidylinositol 4, 5-bisphosphate (PtdIns-4,5-P(2)) through lipid kinases and is responsible for at least 70% of the ATP-dependent secretion observed in digitonin-permeabilized chromaffin cells. PtdIns-4,5-P(2) is usually thought to reside on the plasma membrane. However, because phosphatidylinositol 4-kinase is an integral chromaffin granule membrane protein, PtdIns-4,5-P(2) important in exocytosis may reside on the chromaffin granule membrane. In the present study we have investigated the localization of PtdIns-4,5-P(2) that is involved in exocytosis by transiently expressing in chromaffin cells a pleckstrin homology (PH) domain that specifically binds PtdIns-4, 5-P(2) and is fused to green fluorescent protein (GFP). The PH-GFP protein predominantly associated with the plasma membrane in chromaffin cells without any detectable association with chromaffin granules. Rhodamine-neomycin, which also binds to PtdIns-4,5-P(2), showed a similar subcellular localization. The transiently expressed PH-GFP inhibited exocytosis as measured by both biochemical and electrophysiological techniques. The results indicate that the inhibition was at a step after Ca(2+) entry and suggest that plasma membrane PtdIns-4,5-P(2) is important for exocytosis. Expression of PH-GFP also reduced calcium currents, raising the possibility that PtdIns-4,5-P(2) in some manner alters calcium channel function in chromaffin cells.     

Protein phosphorylation and secretion in digitonin-permeabilized adrenal chromaffin cells. Effects of micromolar Ca2+, phorbol esters, and diacylglycerol

The effects of phorbol esters, dioctanoylglycerol (DiC8), and micromolar Ca2+ on protein phosphorylation and catecholamine secretion in digitonin-treated chromaffin cells were investigated. [gamma-32P]ATP was used as a substrate for phosphorylation in the permeabilized cells. 12-O-Tetradecanoylphorbol-13-acetate (TPA) enhanced Ca2+-dependent catecholamine secretion from digitonin-permeabilized cells. The enhancement required MgATP. Only those phorbol esters which activate protein kinase C in vitro enhanced both catecholamine secretion and protein phosphorylation. DiC8, which activates protein kinase C in vitro and mimics phorbol ester effects in situ, also enhanced both catecholamine secretion and protein phosphorylation. Preincubation of intact cells with TPA or DiC8 was necessary for maximal effects on both catecholamine secretion and protein phosphorylation in subsequently digitonin-treated chromaffin cells. The TPA-induced enhancement of protein phosphorylation was almost entirely Ca2+-independent, whereas DiC8-induced enhancement of protein phosphorylation was mainly Ca2+-dependent. Micromolar Ca2+ alone also enhanced the phosphorylation of a large number of proteins. Most of the proteins phosphorylated in response to TPA or potentiated by DiC8 in combination with Ca2+ were also phosphorylated by micromolar Ca2+ in the absence of exogenous protein kinase C activators. In intact cells, 1,1-dimethyl-4-phenylpiperazinium (DMPP) induced Ca2+-dependent phosphorylation of at least 17 proteins which were detected by two-dimensional gel electrophoresis. All of the proteins phosphorylated upon incubation with 1,1-dimethyl-4-phenylpiperazinium were phosphorylated upon incubation with micromolar Ca2+ in digitonin-treated cells. These results demonstrate that TPA- or DiC8-enhanced Ca2+-dependent catecholamine secretion is associated with enhanced protein phosphorylation which is probably mediated by protein kinase C and that activation of protein kinase C modulates catecholamine secretion from digitonin-treated chromaffin cells.     

Relationship between arachidonic acid release and Ca2(+)-dependent exocytosis in digitonin-permeabilized bovine adrenal chromaffin cells.

The relationship between Ca2(+)-dependent arachidonic acid release and exocytosis from digitonin-permeabilized bovine adrenal chromaffin cells was investigated. The phospholipase A2 inhibitors mepacrine, nordihydroguaiaretic acid and indomethacin had no effect on either arachidonic acid release or secretion. The phospholipase A2 activator melittin had no effect on secretion. The specific diacylglycerol lipase inhibitor RG80267 had no effect on secretion, but decreased basal arachidonic acid release to such an extent that the level of arachidonic acid in treated cells in response to 10 microM-Ca2+ was equivalent to that of control cells in the absence of Ca2+. Staurosporine, a protein kinase C inhibitor, was found to abolish Ca2(+)-dependent arachidonic acid release completely, but had only a slight inhibitory effect on Ca2(+)-dependent secretion. It is concluded that arachidonic acid is not essential for Ca2(+)-dependent exocytosis in adrenal chromaffin cells.     

Activation of protein kinase C is not required for exocytosis from bovine adrenal chromaffin cells. The effects of protein kinase C(19-31), Ca/CaM kinase II(291-317), and staurosporine

We examined whether protein kinase C activation plays a modulatory or an obligatory role in exocytosis of catecholamines from chromaffin cells by using PKC(19-31) (a protein kinase C pseudosubstrate inhibitory peptide), Ca/CaM kinase II(291-317) (a calmodulin-binding peptide), and staurosporine. In permeabilized cells, PKC (19-31) inhibited the phorbol ester-mediated enhancement of Ca2(+)-dependent secretion as much as 90% but had no effect on Ca2(+)-dependent secretion in the absence of phorbol ester. The inhibition of the phorbol ester-induced enhancement of secretion by PKC (19-31) was correlated closely with the ability of the peptide to inhibit in situ phorbol ester-stimulated protein kinase C activity. PKC(19-31) also blocked 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced phosphorylation of numerous endogenous proteins in permeabilized cells but had no effect on Ca2(+)-stimulated phosphorylation of tyrosine hydroxylase. Ca/CaM kinase II(291-317), derived from the calmodulin binding region of Ca/calmodulin kinase II, had no effect on Ca2(+)-dependent secretion in the presence or absence of phorbol ester. The peptide completely blocked the Ca2(+)-dependent increase in tyrosine hydroxylase phosphorylation but had no effect on TPA-induced phosphorylation of endogenous proteins in permeabilized cells. To determine whether a long-lived protein kinase C substrate might be required for secretion, the lipophilic protein kinase inhibitor, staurosporine, was added to intact cells for 30 min before permeabilizing and measuring secretion. Staurosporine strongly inhibited the phorbol ester-mediated enhancement of Ca2(+)-dependent secretion. It caused a small inhibition of Ca2(+)-dependent secretion in the absence of phorbol ester which could not be readily attributed to inhibition of protein kinase C. Staurosporine also inhibited the phorbol ester-mediated enhancement of elevated K(+)-induced secretion from intact cells while it enhanced 45Ca2+ uptake. Staurosporine inhibited to a small extent secretion stimulated by elevated K+ in the absence of TPA. The data indicate that activation of protein kinase C is modulatory but not obligatory in the exocytotoxic pathway.     

Phosphorylation of a chromaffin granule-binding protein in stimulated chromaffin cells

A procedure was devised to determine whether in the stimulated chromaffin cell phosphate is incorporated into specific proteins ("chromobindins") that bind to chromaffin granule membranes in a Ca2+-dependent manner. Cells were preincubated with 32P-labeled orthophosphate, then challenged with secretory stimuli. A postmicrosomal supernatant fraction was prepared from the cells and incubated with unlabeled chromaffin granule membranes in the presence of 5 mM Ca2+. Proteins that bound to the membranes were isolated by centrifugation and examined for 32P content by electrophoresis and autoradiography. Stimulation by carbamylcholine, nicotine, 56 mM K+, or 2 mM Ba2+ led to the incorporation of 32P into a 37-kDa protein that had previously been characterized as a substrate for protein kinase C in vitro (chromobindin 9, or CB9; Summers, T. A., and Creutz, C. E. (1985) J. Biol. Chem. 260, 2437-2443). Incorporation of 32P into this protein was dependent on extracellular Ca2+ and followed a time course that paralleled secretion of catecholamines, returning to base-line levels after 30 min, when secretion terminated. 32P was also incorporated into a 58-kDa protein that may be tyrosine hydroxylase and into an unidentified 28-kDa protein in response to cell stimulation, but neither of these proteins bound to granule membranes in a Ca2+-dependent manner. Treatment of cells with phorbol 12,13-dibutyrate, an activator of protein kinase C, led to 32P incorporation into the 37-kDa protein that was only 30% of the level obtained with nicotinic stimulation, suggesting that additional kinases may be involved in phosphorylating this protein in the stimulated cell.     

Biochemical characterization of the inhibitory effect of CsA on cytolytic T lymphocyte effector functions

We have examined the effects of cyclosporine A (CsA) on a number of CTL effector functions. CsA partially inhibited the CTL-mediated lysis of Ag-bearing target cells. Both target cell- and anti-TCR mAb-induced granule exocytosis were markedly inhibited by CsA. In addition, marked inhibition of PMA and calcium ionophore (A23187) induced granule exocytosis was produced by CsA suggesting that the inhibitory effects of CsA on granule exocytosis involve biochemical events after protein kinase C activation and increases in intracellular free Ca2+. CsA had no inhibitory effects on TCR-mediated phosphatidylinositol metabolism. The inhibitory effects of CsA were not mediated by the cAMP-dependent protein kinase inhibitory pathway and no effect of CsA on the Ca2+-induced binding of calmodulin to calmodulin-binding proteins could be demonstrated. CsA was also a potent inhibitor of IgE receptor-mediated exocytosis in rat basophil leukemia cells. CsA had no effect on receptor-mediated phosphatidylinositol hydrolysis; 400 ng/ml CsA resulted in a 90% inhibition of serotonin release but had no effect on phosphatidylinositol hydrolysis. These results indicate that CsA may inhibit some common event in Ca2+-dependent secretory cells. Taken together, these results suggest that CsA does not inhibit signal transduction but rather interferes with the biochemical events in the later stages of Ca2+-dependent reactions that follow the binding of calmodulin to cytoskeletal or cytoplasmic calmodulin binding proteins.     

Effects of trypsin on secretion stimulated by micromolar Ca2+ and phorbol ester in digitonin-permeabilized adrenal chromaffin cells

1. Catecholamine secretion from digitonin-treated chromaffin cells is stimulated directly by micromolar Ca2+ in the medium. The permeabilized cells are leaky to proteins. 2. In this study trypsin (30-50 micrograms/ml) added to cells after digitonin treatment completely inhibited subsequent Ca2+-dependent catecholamine secretion. The same concentrations of trypsin did not inhibit secretion from permeabilized cells if trypsin was present only prior to cell permeabilization. 3. The data indicate that trypsin entered digitonin-treated chromaffin cells which were capable of undergoing secretion and that an intracellular, trypsin-sensitive protein is involved in secretion. Chymotrypsin was less potent but had effects similar to those of trypsin. 4. The enhancement of Ca2+-dependent secretion from permeabilized chromaffin cells induced by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) was inhibited by trypsin added simultaneously with Ca2+ to permeabilized cells at concentrations (3-10 micrograms/ml) which had little or no effect on Ca2+-dependent secretion from cells untreated with TPA. Ca2+-dependent secretion in TPA-treated cells was reduced by trypsin only to the level that would have occurred in cells not treated with TPA. Trypsin reduced the large TPA-induced increment of membrane-bound protein kinase C.     

Widespread association of annexin II with intracellular membrane systems and involvement of annexin II in granule--granule fusion in addition to granule--plasma membrane fusion in anterior pituitary cells

The subcellular localization in anterior pituitary secretory cells of annexin II, one of the Ca2+-dependent phospholipid-binding proteins, was examined by immunohistochemistry and immunoelectron microscopy. Annexin II was associated with the plasma membrane, the membranes of secretory granules and cytoplasmic organelles, such as rough endoplasmic reticulum, mitochondria and vesicles, and with the nuclear envelope. Annexin II was frequently detected at the contact sites of secretory granules with other granules and with the plasma membrane. The anterior pituitary and adrenal medulla were treated with Clostridium perfringens enterotoxin, which induces Ca2+ influx, and examined under an electron microscope. The anterior pituitary cells showed multigranular exocytosis, i.e. multiple fusions of secretory granules with each other and with the plasma membrane, but adrenal chromaffin cells, which lack annexin II on the granule membranes, never showed granule--granule fusion and only single granule exocytosis. From these results, we conclude that, in anterior pituitary secretory cells, annexin II is involved in granule--granule fusion in addition to granule--plasma membrane fusion. © 1998 Chapman & Hall     

Introduction of Macromolecules into Bovine Adrenal Medullary Chromaffin Cells and Rat Pheochromocytoma Cells (PC12) by Permeabilization with Streptolysin O: Inhibitory Effect of Tetanus Toxin on Catecholamine Secretion

Conditions are described for controlled plasma membrane permeabilization of rat pheochromocytoma cells (PC12) and cultured bovine adrenal chromaffin cells by streptolysin O (SLO). The transmembrane pores created by SLO invoke rapid efflux of intracellular 86Rb+ and ATP, and also permit passive diffusion of proteins, including immunoglobulins, into the cells. SLO-permeabilized PC12 cells release [3H]dopamine in response to micromolar concentrations of free Ca2+. Permeabilized adrenal chromaffin cells present a similar exocytotic response to Ca2+ in the presence of Mg2+/ATP. Permeabilized PC12 cells accumulate antibodies against synaptophysin and calmodulin, but neither antibody reduces the Ca2+-dependent secretory response. Reduced tetanus toxin, although ineffective when applied to intact chromaffin cells, inhibits Ca2+-induced exocytosis by both types of permeabilized cells studied. Omission of dithiothreitol, toxin inactivation by boiling, or preincubation with neutralizing antibodies abolishes the inhibitory effect. The data indicate that plasma membrane permeabilization by streptolysin O is a useful tool to probe and define cellular components that are involved in the final steps of exocytosis.     

Sodium-dependent calcium efflux from adrenal chromaffin cells following exocytosis. Possible role of secretory vesicle membranes.

Although cytosolic Ca2+ transients are known to influence the magnitude and duration of hormone and neurotransmitter release, the processes regulating the decay of such transients after cell stimulation are not well understood. Na(+)-dependent Ca2+ efflux across the secretory vesicle membrane, following its incorporation into the plasma membrane, may play a significant role in Ca2+ efflux after stimulation of secretion. We have measured an enhanced 45Ca2+ efflux from cultured bovine adrenal chromaffin cells following cell stimulation with depolarizing medium (75 mM K+) or nicotine (10 microM). Such stimulation also causes Ca2+ uptake via voltage-gated Ca2+ channels and secretion of catecholamines. Na+ replacement with any of several substitutes (N-methyl-glucamine, Li+, choline, or sucrose) during cell stimulation inhibited the enhanced 45Ca2+ efflux, indicating and Na(+)-dependent Ca2+ efflux process. Na+ deprivation did not inhibit 45Ca2+ uptake or catecholamine secretion evoked by elevated K+. Suppression of exocytotic incorporation of secretory vesicle membranes into the plasma membrane with hypertonic medium (620 mOsm) or by lowering temperature to 12 degrees C inhibited K(+)-stimulated 45Ca2+ efflux in Na(+)-containing medium but did not inhibit the stimulated 45Ca2+ uptake. Enhancement of exocytotic secretion with pertussis toxin resulted in an enhanced 45Ca2+ efflux without affecting calcium uptake. The combined results suggest that Na(+)-dependent Ca2+ efflux across secretory vesicle membranes, following their incorporation into the plasma membrane during exocytosis, plays a significant role in regulating calcium efflux and the decay of cytosolic Ca2+ in adrenal chromaffin cells and possibly in related secretory cells.     

Synhibin: a new calcium-dependent membrane-binding protein that inhibits synexin-induced chromaffin granule aggregation and fusion

We report the isolation and purification of synhibin, a new M_r 68000 protein, which inhibits synexin. Synexin mediates Ca²⁺-dependent chromaffin granule aggregation and fusion, processes perhaps important during exocytosis. Our data indicate that synhibin action involves competition with synexin for a site on the chromaffin granule membrane involved in membrane contact. Synhibin may thus be an important intracellular regulator of synexin action during secretion.     

Calcium-binding proteins and secretion

The Ca ion plays a central role in the control of the regulated pathway of exocytotic secretion in eukaryote cells. Most secretagogues either directly or indirectly raise cytosolic free Ca levels which in turn affects granule biogenesis, contractile events, gel/sol transition in intracellular matrix and membrane fusion events occurring at exocytosis. Many of these responses are mediated by Ca-binding proteins among which calmodulin and protein kinase C have received prominent attention. Studies of the nature and inter-relationship of proteins which undergo Ca-dependent association with intracellular membranes in secretory tissue reveal that there may be further Ca-binding proteins in these cells which act as intracellular transducers of the Ca signal during secretion.     

Arachidonic Acid Release and Catecholamine Secretion from Digitonin-Treated Chromaffin Cells: Effects of Micromolar Calcium, Phorbol Ester, and Protein Alkylating Agents

The relationship between catecholamine secretion and arachidonic acid release from digitonin-treated chromaffin cells was investigated. Digitonin renders permeable the plasma membranes of bovine adrenal chromaffin cells to Ca2+, ATP, and proteins. Digitonin-treated cells undergo exocytosis of catecholamine in response to micromolar Ca2+ in the medium. The addition of micromolar Ca2+ to digitonin-treated chromaffin cells that had been prelabeled with [3H]arachidonic acid caused a marked increase in the release of [3H]arachidonic acid. The time course of [3H]arachidonic acid release paralleled catecholamine secretion. Although [3H]arachidonic acid release and exocytosis were both activated by free Ca2+ in the micromolar range, the activation of [3H]arachidonic acid release occurred at Ca2+ concentrations slightly lower than those required to activate exocytosis. Pretreatment of the chromaffin cells with N-ethylmaleimide (NEM) or p-bromophenacyl bromide (BPB) resulted in dose-dependent inhibition of 10 microM Ca2+-stimulated [3H]arachidonic acid release and exocytosis. The IC50 of NEM for both [3H]arachidonic acid release and exocytosis was 40 microM. The IC50 of BPB for both events was 25 microM. High concentrations (5-20 mM) of Mg2+ caused inhibition of catecholamine secretion without altering [3H]arachidonic acid release. A phorbol ester that activates protein kinase C, 12-O-tetradecanoylphorbol-13-acetate (TPA), caused enhancement of both [3H]arachidonic acid release and exocytosis. The findings demonstrate that [3H]arachidonic acid release is stimulated during catecholamine secretion from digitonin-treated chromaffin cells and they are consistent with a role for phospholipase A2 in exocytosis from chromaffin cells. Furthermore the data suggest that protein kinase C can modulate both arachidonic acid release and exocytosis.     

Identification of Exo2 as the catalytic subunit of protein kinase A reveals a role for cyclic AMP in Ca(2+)-dependent exocytosis in chromaffin cells.

Digitonin-permeabilized chromaffin cells secrete catecholamines by exocytosis in response to micromolar Ca2+ concentrations, but lose the ability to secrete in response to Ca2+ as the cells lose soluble proteins through the plasma membrane pores. We have previously shown [Morgan and Burgoyne (1992) Nature, 355, 833-836] that cytosol can retard this loss of secretory competence and that two distinct stimulatory activities (Exo1 and Exo2) are present in cytosol. Here we report that Exo2 behaved as a single peak of activity through purification on hydroxyapatite, ammonium sulfate precipitation and gel filtration and the activity correlated with a single polypeptide of approximately 44 kDa on SDS gels. Protein sequencing of this band revealed it to be the catalytic subunit of cyclic AMP-dependent protein kinase (PKA). Both cyclic AMP and the commercially available catalytic subunit of PKA stimulated exocytosis in a dose-dependent manner which was absolutely dependent on the presence of micromolar Ca2+. These data show that PKA (Exo2) regulates Ca(2+)-dependent exocytosis in bovine adrenal chromaffin cells.