Annexin 2 promotes the formation of lipid microdomains required for calcium-regulated exocytosis of dense-core vesicles

Annexin 2 is a calcium-dependent phospholipid-binding protein that has been implicated in a number of membrane-related events, including regulated exocytosis. In chromaffin cells, we previously reported that catecholamine secretion requires the translocation and formation of the annexin 2 tetramer near the exocytotic sites. Here, to obtain direct evidence for a role of annexin 2 in exocytosis, we modified its expression level in chromaffin cells by using the Semliki Forest virus expression system. Using a real-time assay for individual cells, we found that the reduction of cytosolic annexin 2, and the consequent decrease of annexin 2 tetramer at the cell periphery, strongly inhibited exocytosis, most likely at an early stage before membrane fusion. Secretion also was severely impaired in cells expressing a chimera that sequestered annexin 2 into cytosolic aggregates. Moreover, we demonstrate that secretagogue-evoked stimulation triggers the formation of lipid rafts in the plasma membrane, essential for exocytosis, and which can be attributed to the annexin 2 tetramer. We propose that annexin 2 acts as a calcium-dependent promoter of lipid microdomains required for structural and spatial organization of the exocytotic machinery.     

S100A10‐Mediated Translocation of Annexin‐A2 to SNARE Proteins in Adrenergic Chromaffin Cells Undergoing Exocytosis

In neuroendocrine cells, annexin‐A2 is implicated as a promoter of monosialotetrahexosylganglioside (GM1)‐containing lipid microdomains that are required for calcium‐regulated exocytosis. As soluble N‐ethylmaleimide‐sensitive factor attachment protein receptors (SNAREs) require a specific lipid environment to mediate granule docking and fusion, we investigated whether annexin‐A2‐induced lipid microdomains might be linked to the SNAREs present at the plasma membrane. Stimulation of adrenergic chromaffin cells induces the translocation of cytosolic annexin‐A2 to the plasma membrane, where it colocalizes with SNAP‐25 and S100A10. Cross‐linking experiments performed in stimulated chromaffin cells indicate that annexin‐A2 directly interacts with S100A10 to form a tetramer at the plasma membrane. Here, we demonstrate that S100A10 can interact with vesicle‐associated membrane protein 2 (VAMP2) and show that VAMP2 is present at the plasma membrane in resting adrenergic chromaffin cells. Tetanus toxin that cleaves VAMP2 solubilizes S100A10 from the plasma membrane and inhibits the translocation of annexin‐A2 to the plasma membrane. Immunogold labelling of plasma membrane sheets combined with spatial point pattern analysis confirmed that S100A10 is present in VAMP2 microdomains at the plasma membrane and that annexin‐A2 is observed close to S100A10 and to syntaxin in stimulated chromaffin cells. In addition, these results showed that the formation of phosphatidylinositol (4,5)‐bisphosphate (PIP₂) microdomains colocalized with S100A10 in the vicinity of docked granules, suggesting a functional interplay between annexin‐A2‐mediated lipid microdomains and SNAREs during exocytosis.     

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.     

Annexin II in exocytosis: catecholamine secretion requires the translocation of p36 to the subplasmalemmal region in chromaffin cells

Annexin II is a Ca(2+)-dependent membrane-binding protein present in a wide variety of cells and tissues. Within cells, annexin II is found either as a 36-kD monomer (p36) or as a heterotetrameric complex (p90) coupled with the S-100-related protein, p11. Annexin II has been suggested to be involved in exocytosis as it can restore the secretory responsiveness of permeabilized chromaffin cells. By quantitative confocal immunofluorescence, immunoreplica analysis and immunoprecipitation, we show here the translocation of p36 from the cytosol to a subplasmalemmal Triton X-100 insoluble fraction in chromaffin cells following nicotinic stimulation. A synthetic peptide corresponding to the NH2-terminal domain of p36 which contains the phosphorylation sites was microinjected into individual chromaffin cells and catecholamine secretion was monitored by amperometry. This peptide blocked completely the nicotine-induced recruitment of p36 to the cell periphery and strongly inhibited exocytosis evoked by either nicotine or high K+. The light chain of annexin II, p11, was selectively expressed by adrenergic chromaffin cells, and was only present in the subplasmalemmal Triton X-100 insoluble protein fraction of both resting and stimulated cells. p11 can modify the Ca(2+)- and/or the phospholipid-binding properties of p36. We found that loss Ca2+ was required to stimulate the translocation of p36 and to trigger exocytosis in adrenergic chromaffin cells. Our findings suggest that the translocation of p36 to the subplasmalemmal region is an essential event in regulated exocytosis and support the idea that the presence of p11 in adrenergic cells may confer a higher Ca2+ affinity to the exocytotic pathway in these cells.     

Control of exocytosis from adrenal chromaffin cells

1. Calcium-dependent exocytosis of catecholamines from intact and digitonin-permeabilized bovine adrenal chromaffin cells was investigated. 2. 45Ca2+ uptake and secretion induced by nicotinic stimulation or depolarization in intact cells were closely correlated. The results provide strong support for Ca2+ entry being the trigger for exocytosis. 3. Experiments in which the H+ electrochemical gradient across the intracellular secretory granule (chromaffin granule) membrane was altered indicated that the gradient does not play an important role in exocytosis. 4. Ca2+ entry into the cells is associated with activation of phospholiphase C and a rapid translocation of protein kinase C to membranes. 5. The plasma membrane of chromaffin cells was rendered permeable to Ca2+, ATP, and proteins by the detergent digitonin without disruption of the intracellular secretory granules. In this system in which the intracellular milieu can be controlled, micromolar Ca2+ directly stimulated catecholamine secretion. 6. Treatment of the cells with phorbol esters and diglyceride, which activate protein kinase C, enhanced phosphorylation and subsequent Ca2+-dependent secretion in digitonin-treated cells. 7. Phorbol ester-induced secretion could be specifically inhibited by trypsin. The experiments indicate that protein kinase C modulates but is not necessary for Ca2+-dependent secretion.     

Ultrastructural demonstration of exocytosis in intact and saponin-permeabilized cultured bovine chromaffin cells

Exocytosis is the release of intracellular vesicular contents directly to the cell exterior after fusion of the vesicular and plasma membranes. It is generally accepted as the process by which transmitters and hormones are released from neurons and neurosecretory cells. There is overwhelming biochemical evidence that exocytosis is the mechanism by which catecholamines are released from adrenal chromaffin cells. With the exception of the hamster, however, there is little ultrastructural evidence to support such a mechanism. We have used a modified in vitro tannic-acid method to visualize exocytosis by transmission electron microscopy in intact and saponin-permeabilized bovine chromaffin cells. When cells are exposed to tannic-acid-containing medium, the content of vesicles involved in exocytosis is coagulated in situ as the vesicle opens to the exterior. Numerous exocytotic profiles were observed. The exposed vesicle contents appeared more granular than those of vesicles in the cell interior. Tannic acid also made the plasma membrane more distinct. Small holes were apparent in the plasma membrane of saponin-treated cells, with little disruption of underlying cytoplasmic structure. Furthermore, when these cells were stimulated with calcium, exocytosis was evident only at regions of intact plasma membrane, not at the holes. Parallel measurements of secretion showed no secretion in the presence of tannic acid. Pretreatment with tannic acid prevented subsequent secretion by intact cells and markedly reduced that of permeabilized cells, indicating a probable change in the nature of the plasma membrane.(ABSTRACT TRUNCATED AT 250 WORDS)     

Exocytosis in Single Chromaffin Cells: Regulation by a Secretory Granule-Associated Go Protein

1. Besides having a role in signal transduction, trimeric G proteins may also be involved in membrane trafficking events. In chromaffin cells, Go has beenfound associated with the membrane of secretory granules. Here we examined the role of Go in regulated exocytosis using pressure microinjection combined with amperometric measurement of catecholamine secretion from individual chromaffin cells.2. Microinjection of GTPS and mastoparan strongly inhibits the amperometric response to either nicotine or high K⁺.3. The presence of mastoparan in the cell incubation medium had no effect on K⁺-evoked secretion, suggesting that mastoparan blocks the exocytotic machinery through an intracellular target protein not located just beneath the plasma membrane.4.Microinjection of anti-Go antibodies potentiates by more than 50% the K⁺-evoked secretion, whereas anti-Gi_1/2 antibodies have no effect.5. Thus an inhibitory Go protein, probably associated with secretory granules, controls exocytosis in chromaffin cells. The intracellular proteins controlling organelle-associated G proteins are currently unknown. The neuronal cytosolicprotein GAP-43 stimulates Go in purified chromaffin granule membranes and inhibits exocytosis in permeabilized cells. We show here that microinjection of a synthetic peptide corresponding to the domain of GAP-43 that interacts with Go inhibits secretion. We suggest that GAP-43 or a related cytosolic protein controls the exocytotic priming step in chromaffin cells by stimulating a granule-associated Go protein.     

Activation of annexin 7 by protein kinase C in vitro and in vivo

Annexin 7, a Ca(2+)/GTP-activated membrane fusion protein, is preferentially phosphorylated in intact chromaffin cells, and the levels of annexin 7 phosphorylation increase quantitatively in proportion to the extent of catecholamine secretion. Consistently, various protein kinase C inhibitors proportionately reduce both secretion and phosphorylation of annexin 7 in these cells. In vitro, annexin 7 is quantitatively phosphorylated by protein kinase C to a mole ratio of 2.0, and phosphorylation is extraordinarily sensitive to variables such as pH, calcium, phospholipid, phorbol ester, and annexin 7 concentration. Phosphorylation of annexin 7 by protein kinase C significantly potentiates the ability of the protein to fuse phospholipid vesicles and lowers the half-maximal concentration of calcium needed for this fusion process. Furthermore, other protein kinases, including cAMP-dependent protein kinase, cGMP-dependent protein kinase, and protein-tyrosine kinase pp60(c-)(src), also label annexin 7 with high efficiency but do not have this effect on membrane fusion. In the case of pp60(c-)(src), we note that this kinase, if anything, modestly suppresses the membrane fusion activity of annexin 7. These results thus lead us to hypothesize that annexin 7 may be a positive mediator for protein kinase C action in the exocytotic membrane fusion reaction in chromaffin cells.     

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.     

Exocytosis of catecholamine (CA)-containing and CA-free granules in chromaffin cells

Recent evidence suggests that endocytosis in neuroendocrine cells and neurons can be tightly coupled to exocytosis, allowing rapid retrieval from the plasma membrane of fused vesicles for future use. This can be a much faster mechanism for membrane recycling than classical clathrin-mediated endocytosis. During a fast exo-endocytotic cycle, the vesicle membrane does not fully collapse into the plasma membrane; nevertheless, it releases the vesicular contents through the fusion pore. Once the vesicle is depleted of transmitter, its membrane is recovered without renouncing its identity. In this report, we show that chromaffin cells contain catecholamine-free granules that retain their ability to fuse with the plasma membrane. These catecholamine-free granules represent 7% of the total population of fused vesicles, but they contributed to 47% of the fusion events when the cells were treated with reserpine for several hours. We propose that rat chromaffin granules that transiently fuse with the plasma membrane preserve their exocytotic machinery, allowing another round of exocytosis.     

A role for diacylglycerol in annexin A7-mediated fusion of lung lamellar bodies

Lung surfactant secretion in alveolar type II cells occurs following lamellar body fusion with plasma membrane. Annexin A7 is a Ca2+-dependent membrane-binding protein that is postulated to promote membrane fusion during exocytosis in some cell types including type II cells. Since annexin A7 preferably binds to lamellar body membranes, we postulated that specific lipids could modify the mode of annexin A7 interaction with membranes and its membrane fusion activity. Initial studies with phospholipid vesicles containing phosphatidylserine and other lipids showed that certain lipids affected protein interaction with vesicle membranes as determined by change in protein tryptophan fluorescence, protein interaction with trans membranes, and by protein sensitivity to limited proteolysis. The presence of signaling lipids, diacylglycerol or phosphatidylinositol-4,5-bisphosphate, as minor components also modified the lipid vesicle effect on these characteristics and membrane fusion activity of annexin A7. In vitro incubation of lamellar bodies with diacylglycerol or phosphatidylinositol-4,5-bisphosphate caused their enrichment with either lipid, and increased the annexin A7 and Ca2+-mediated fusion of lamellar bodies. Treatment of isolated lung lamellar bodies with phosphatidylinositol- or phosphatidylcholine phospholipase C to increase diacylglycerol, without or with preincubation with phosphatidylinositol-4,5-bisphosphate, augmented the fusion activity of annexin A7. Thus, increased diacylglycerol in lamellar bodies following cell stimulation with secretagogues may enhance membrane fusion activity of annexin A7.     

Secretion of annexin A3 from ovarian cancer cells and its association with platinum resistance in ovarian cancer patients

Early detection of resistance to platinum-based therapy is critical for improving the treatment of ovarian cancers. We have previously found that increased expression of annexin A3 is a mechanism for platinum resistance in ovarian cancer cells. Here we demonstrate that annexin A3 can be detected in the culture medium of ovarian cancer cells, particularly these cells that express high levels of annexin A3. Levels of annexin A3 were then determined in sera from ovarian cancer patients using an enzyme-linked immunosorbent assay. Compared with those from normal donors, sera from ovarian cancer patients contain significantly higher levels of annexin A3. Furthermore, serum levels of annexin A3 were significantly higher in platinum-resistant patients than in platinum-sensitive patients. To gain insight into the mechanism of secretion, the ovarian cancer cell lines were examined using both transmission electron microscopy and immunoelectron microscopy. Compared with parent cells, there are significantly more vesicles in the cytoplasm of ovarian cancer cells that express high levels of annexin A3, and at least some vesicles are annexin A3-positive. Moreover, some vesicles appear to be fused with the cell membrane, suggesting that annexin A3 secretion may be associated with exocytosis and the release of exosomes. This is supported by our observation that ovarian cancer cells expressing higher levels of annexin A3 released increased numbers of exosomes. Furthermore, annexin A3 can be detected in exosomes released from cisplatin-resistant cells (SKOV3/Cis) by immunoblotting and immunoelectron microscopy.     

Cofilin activation during Ca(2+)-triggered secretion from adrenal chromaffin cells

Cofilin is one of the major actin depolymerizing proteins in eukaryotic cells and involved in many membrane modulating activities, such as cell growth and motility. Here we examined whether cofilin is activated upon Ca(2+) regulated noradrenalin secretion from bovine adrenal chromaffin cells. We found that triggering exocytosis by nicotine causes a dephosphorylation and thereby activation of cofilin. Furthermore, in permeabilized chromaffin cells the addition of Ca(2+) alone is sufficient to trigger both, regulated exocytosis and cofilin activation. This is consistent with cofilin activation being required for actin reorganization during exocytosis.     

Cholesterol regulates membrane binding and aggregation by annexin 2 at submicromolar Ca(2+) concentration

Annexin 2 is a member of the annexin family which has been implicated in calcium-regulated exocytosis. This contention is largely based on Ca(2+)-dependent binding of the protein to anionic phospholipids. However, annexin 2 was shown to be associated with chromaffin granules in the presence of EGTA. A fraction of this bound annexin 2 was released by methyl-beta-cyclodextrin, a reagent which depletes cholesterol from membranes. Restoration of the cholesterol content of chromaffin granule membranes with cholesterol/methyl-beta-cyclodextrin complexes restored the Ca(2+)-independent binding of annexin 2. The binding of both, monomeric and tetrameric forms of annexin 2 was also tested on liposomes of different composition. In the absence of Ca(2+), annexin 2, especially in its tetrameric form, bound to liposomes containing phosphatidylserine, and the addition of cholesterol to these liposomes increased the binding. Consistent with this observation, liposomes containing phosphatidylserine and cholesterol were aggregated by the tetrameric form of annexin 2 at submicromolar Ca(2+) concentrations. These results indicate that the lipid composition of membranes, and especially their cholesterol content, is important in the control of the subcellular localization of annexin 2 in resting cells, at low Ca(2+) concentration. Annexin 2 might be associated with membrane domains enriched in phosphatidylserine and cholesterol.     

Participation of annexins in Ca2+-regulated secretion of catecholamines

Secretion of catecholamines by adrenal medulla chromaffin cells occurs after their stimulation by nicotine or depolarization of plasma membrane. Adrenal medulla secrets mostly noradrenaline and adrenaline, both having pleyotropic action in the organism. Central role in regulation of exocytosis of catecholamines play calcium ions. Their intracellular concentration increases as a cell response to stimulus and creates signal to start secretion. Moreover, annexins are known to participate in regulation of biological membrane dynamics during intracellular transport processes, however their participation in secretion is less established then in endocytosis. Among twelve annexin subfamilies (AnxA1-A11 i A13) expressed in mammalian organisms only involvement of AnxA2 and AnxA6 in endocytosis is well documented. Some data suggests that annexins may play important functions also in Ca2+-regulated catecholamine secretion.     

Identification of a key domain in annexin and 14-3-3 proteins that stimulate calcium-dependent exocytosis in permeabilized adrenal chromaffin cells

Calcium-dependent secretion in digitonin-permeabilized adrenal chromaffin cells is stimulated by exogenous annexin II and 14-3-3 proteins. These proteins share a conserved domain that has been suggested to be involved in specific protein-protein interactions. We examined whether this domain was involved in secretion by using a synthetic peptide (P16) of sequence KGDYQKALLYLCGGDD corresponding to the C-terminus of annexin II. P16, but not truncated peptides, prevented the stimulation of secretion by 14-3-3 proteins and produced a partial inhibition of control secretion. These data suggest that the shared annexin/14-3-3 domain is important in the mechanisms controlling Ca²⁺-dependent secretion and may play a key role in protein-protein interactions during exocytosis.     

Regulation of neuroendocrine exocytosis by the ARF6 GTPase-activating protein GIT1

Neuroendocrine cells release hormones and neuropeptides by exocytosis, a highly regulated process in which secretory granules fuse with the plasma membrane to release their contents in response to a calcium trigger. Using chromaffin and PC12 cells, we have recently described that the granule-associated GTPase ARF6 plays a crucial role in exocytosis by activating phospholipase D1 at the plasma membrane and, presumably, promoting the fusion reaction between the two membrane bilayers. ARF6 is activated by the nucleotide exchange factor ARNO following docking of granules to the plasma membrane. We show here that GIT1, a GTPase-activating protein stimulating GTP hydrolysis on ARF6, is the second molecular partner that turns over the GDP/GTP cycle of ARF6 during cell stimulation. Western blot and immunofluorescence experiments indicated that GIT1 is cytosolic in resting cells but is recruited to the plasma membrane in stimulated cells, where it co-localizes with ARF6 at the granule docking sites. Over-expression of wild-type GIT1 inhibits growth hormone secretion from PC12 cells; this inhibitory effect was not observed in cells expressing a GIT1 mutant impaired in its ARF-GTPase-activating protein (GAP) activity or in cells expressing other ARF6-GAPs. Conversely reduction of GIT1 by RNA interference increased the exocytotic activity. Using a real time assay for individual chromaffin cells, we found that microinjection of GIT1 strongly reduced the number of exocytotic events. These results provide the first evidence that GIT1 plays a function in calcium-regulated exocytosis in neuroendocrine cells. We propose that GIT1 represents part of the pathway that inactivates ARF6-dependent reactions and thereby negatively regulates and/or terminates exocytotic release.     

Possible involvement of actin and myosin in Ca2+ transport through the plasma membrane of chromaffin cells

The exocytosis of catecholamines by chromaffin cells following stimulation (e.g. by acetylcholine) is accompanied by a rise in the level of intracellular free Ca2+. Actually, secretion can be induced merely by making the cells leaky to Ca2+ from the external medium. We have recently demonstrated that secretion can be increased by the introduction of DNase-I, the F-actin depolymerizing agent, or of heavy meromyosin, the enzymatically active fragment of myosin. Suspecting that these changes might be associated with a higher intracellular level of Ca2+, we now have measured the influx of 45Ca2+ into chromaffin cells which have undergone fusion with DNase-I- or with heavy meromyosin-loaded liposomes. In both cases, a marked increase in Ca2+ uptake has been observed, which could be abolished by Co2+ ions (a Ca2+ channel blocker), suggesting an intimate involvement of the cellular actomyosin system in the process of Ca2+ ions transport through the Ca2+ channels of the plasma membrane.     

The participation of annexin II (calpactin I) in calcium-evoked exocytosis requires protein kinase C

Permeabilized adrenal chromaffin cells secrete catecholamines by exocytosis in response to micromolar calcium concentrations. Recently, we have demonstrated that chromaffin cells permeabilized with digitonin progressively lose their capacity to secrete due to the release of certain cytosolic proteins essential for exocytosis (Sarafian T., D. Aunis, and M. F. Bader. 1987. J. Biol. Chem. 34:16671-16676). Here we show that one of the released proteins is calpactin I, a calcium-dependent phospholipid-binding protein known to promote in vitro aggregation of chromaffin granules at physiological micromolar calcium levels. The addition of calpactin I into digitonin- or streptolysin-O-permeabilized chromaffin cells with reduced secretory capacity as a result of the leakage of cytosolic proteins partially restores the calcium-dependent secretory activity. This effect is specific of calpactin I since other annexins (p32, p37, p67) do not stimulate secretion at similar or higher concentrations. Calpactin I requires the presence of Mg-ATP, suggesting that a phosphorylating step may regulate the activity of calpactin. Calpactin is unable to restore the secretory activity in cells which have completely lost their cytosolic protein kinase C or in cells having their protein kinase C inhibited by sphingosine or downregulated by long-term incubation with TPA. In contrast, calpactin I prephosphorylated in vitro by purified protein kinase C is able to reconstitute secretion in cells depleted of their protein kinase C activity. This stimulatory effect is also observed with thiophosphorylated calpactin I which is resistant to cellular phosphatases or with phosphorylated calpactin I introduced into cells in the presence of microcystin, a phosphatase inhibitor. These results suggest that calpactin I is involved in the exocytotic machinery by a mechanism which requires phosphorylation by protein kinase C.     

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.