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.     

Effects of phorbol esters, diglyceride, and cholinergic agonists on the subcellular distribution of protein kinase C in intact or digitonin-permeabilized adrenal chromaffin cells

Phorbol esters which activate protein kinase C increased the percentage of membrane-bound protein kinase C activity in bovine adrenal chromaffin cells from less than 10 to 20-50% within 30 min. Permeabilization of chromaffin cells with digitonin in the absence of Ca2+ and phorbol esters caused virtually 100% of the protein kinase C activity to leave the cells within 1 h, which is consistent with protein kinase C being soluble and cytosolic. However, if cells were incubated for 15-30 min with 12-O-tetradecanoylphorbol-13-acetate (TPA) prior to permeabilization, 50-60% of the protein kinase C activity exited from the cells within 1 h of permeabilization. In cells not incubated with phorbol ester, permeabilization in the presence of 1-10 microM Ca2+ also decreased the rate at which protein kinase C exited from the cells. The slower release of protein kinase C caused by prior incubation of the cells with TPA or because of the presence of micromolar Ca2+ in permeabilized cells was associated with increased membrane-bound protein kinase C. The effects of TPA and permeabilization in the presence of micromolar Ca2+ were approximately additive. Active phorbol esters had different abilities to cause retention of protein kinase C in digitonin-treated cells. Dioctanoylglycerol, which activates protein kinase C in vitro and enhanced Ca2+-dependent secretion from permeabilized chromaffin cells similarly to TPA, also increased membrane-bound protein kinase C in intact cells, but had no effect on the retention of protein kinase C in permeabilized cells in the presence or absence of Ca2+. The different abilities of protein kinase C activators to cause retention of protein kinase C in subsequently permeabilized cells suggest differences in the reversibility of the binding. The mixed nicotinic-muscarinic agonist carbachol and the nicotinic agonist 1,1-dimethyl-4-phenylpiperazinium, but not the muscarinic agonist muscarine, caused 3-10% of the total protein kinase C activity to become membrane-bound within 3 min in intact chromaffin cells. Thus, nicotinic stimulation of chromaffin cells may rapidly activate protein kinase C.     

Catecholamine secretion from digitonin-treated PC12 cells. Effects of Ca2+, ATP, and protein kinase C activators

PC12 cells, a cloned rat pheochromocytoma cell line, were treated with digitonin to render the plasma membrane permeable to ions and proteins. At a cell density of 2-6 X 10(5) cells/cm2, incubation with 7.5 microM digitonin permitted a Ca2+-dependent release of 25-40% of the catecholamine within 18 min in the presence of 10 microM Ca2+. Half-maximal secretion occurred at 0.5-1 microM Ca2+. PC12 cultures at lower cell densities were more sensitive to digitonin and gave more variable results. Secretion in the presence of digitonin and Ca2+ began after a 2-min lag and continued for up to 30 min. When cells were treated for 3 min in digitonin and then stimulated with Ca2+ in the absence of digitonin, secretion occurred in the same manner but without the initial lag. Optimal secretion from PC12 cells was also dependent upon the presence of Mg2+ and ATP. Permeabilized PC12 cells exhibited a slow time-dependent loss of secretory responsiveness which was correlated with the release of a cytosolic marker, lactate dehydrogenase (134 kDa). This suggests that digitonin permeabilization allows soluble constituents necessary for secretion to leave the cell in addition to allowing Ca2+ and ATP access into the cell interior. Ca2+-dependent secretion was completely inhibited by exposure of digitonin-permeabilized cells to 100 micrograms/ml trypsin (27 kDa), whereas secretion was only slightly inhibited by trypsin exposure prior to digitonin treatment. Thus, an intracellular, trypsin-sensitive protein is probably involved in secretion. The data also indicate that the same population of digitonin-treated cells which responded to Ca2+ was permeable to a 27-kDa protein. 1,2-Dioctanoylglycerol and phorbol esters which activate protein kinase C enhanced the Ca2+-dependent and Ca2+-independent secretion in digitonin-permeabilized PC12 cells. Thus, protein kinase C appears to be involved in the regulation of catecholamine secretion from permeabilized PC12 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.     

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.     

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.     

A role for soluble NSF attachment proteins (SNAPs) in regulated exocytosis in adrenal 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. Such secretory run-down can be retarded by cytosolic fractions, thus providing an assay for proteins potentially involved in the exocytotic process. We have used this assay to investigate the role of N-ethylmaleimide-sensitive fusion protein (NSF) and soluble NSF attachment proteins (SNAPs) in regulated exocytosis. Recombinant alpha- and gamma-SNAP stimulated Ca(2+)-dependent exocytosis, although recombinant NSF was ineffective, despite the fact that NSF and alpha-SNAP leak from the permeabilized cells with similar time courses. However, around one third of cellular NSF was found to be present in a non-cytosolic form and so it is possible that this is sufficient for exocytosis and that exogenous SNAPs stimulate the exocytotic mechanism by acting on the leakage-insensitive NSF. The stimulatory effect of alpha-SNAP displayed a biphasic dose-response curve and was maximal at 20 micrograms/ml. The effect of alpha-SNAP was Ca(2+)- and MgATP-dependent and was inhibited by N-ethylmaleimide and botulinum A neurotoxin, indicating a bona fide action on the exocytotic mechanism. Furthermore, Ca2+ concentrations which trigger catecholamine secretion acted to prevent the leakage of NSF and alpha-SNAP from permeabilized cells. These findings provide functional evidence for a role of SNAPs in regulated exocytosis in chromaffin cells.     

Calmodulin is involved in catecholamine secretion from digitonin-permeabilized bovine adrenal medullary chromaffin cells.

The role of calmodulin in exocytotic secretion was studied using digitonin-permeabilized bovine adrenal medullary chromaffin cells. Addition of calmodulin to the permeabilized cells increased Ca(2+)-dependent norepinephrine release in a dose-dependent manner. Unlike calmodulin, addition of caldesmon, actin or bovine serum albumin did not increase the release. Calmodulin increased the release at Ca2+ concentrations of more than 10(-6) M and its effect increased with increase in Mg2+ concentration. Th release of norepinephrine enhanced by calmodulin was inhibited by tetanus toxin, which specifically inhibits exocytotic secretion. These results indicate directly that calmodulin plays an important role in exocytotic secretion from chromaffin cells.     

Synergistic actions of Ca2+ and the phorbol ester TPA on K+-induced catecholamine release from bovine adrenal chromaffin cells

Enhancement of Ca2+-dependent high K+-evoked catecholamine secretion was observed after pretreatment of cultured bovine adrenal chromaffin cells with the phorbol ester 4B-phorbol 12-myristate 13-acetate (TPA) in the absence of added extracellular Ca2+. This effect of TPA was not reproduced when the secretagogues acetylcholine, nicotine, or veratrine were substituted for high K+. The implications of these results are discussed in relation to the role of protein kinase C in stimulus-secretion coupling in the chromaffin cell.     

Barium and Calcium Stimulate Secretion from Digitonin-Permeabilized Bovine Adrenal Chromaffin Cells by Similar Pathways

We compared the characteristics of secretion stimulated by EGTA-buffered Ba(2+)- and Ca(2+)-containing solutions in digitonin-permeabilized bovine adrenal chromaffin cells. Half-maximal secretion occurred at approximately 100 microM Ba2+ or 1 microM Ca2+. Ba(2+)-stimulated release was not due to release of sequestered intracellular Ca2+ because at a constant free Ba2+ concentration, increasing unbound EGTA did not diminish the extent of release due to Ba2+. The maximal extents of Ba(2+)- and Ca(2+)-dependent secretion in the absence of MgATP were identical. MgATP enhanced Ba(2+)-induced secretion to a lesser extent than Ca(2+)-induced secretion. Half-maximal concentrations of Ba2+ and Ca2+, when added together to cells, yielded approximately additive amounts of secretion. Maximal concentrations of Ba2+ and Ca2+ when added together to cells for 2 or 15 min were not additive. Tetanus toxin inhibited Ba(2+)- and Ca(2+)-dependent secretion to a similar extent. Ba2+, unlike Ca2+, did not activate polyphosphoinositide-specific phospholipase C. These data indicate that (1) Ba2+ directly stimulates exocytosis, (2) Ba(2+)-induced secretion is stimulated to a lesser extent than Ca(2+)-dependent secretion by MgATP, (3) Ba2+ and Ca2+ use similar pathways to trigger exocytosis, and (4) exocytosis from permeabilized cells does not require activation of polyphosphoinositide-specific phospholipase C.     

Plasma Membrane and Chromaffin Granule Characteristics in Digitonin-Treated Chromaffin Cells

Digitonin permeabilizes the plasma membranes of bovine chromaffin cells to Ca2+, ATP, and proteins and allows micromolar Ca2+ in the medium to stimulate directly catecholamine secretion. In the present study the effects of digitonin (20 microM) on the plasma membrane and on intracellular chromaffin granules were further characterized. Cells with surface membrane labeled with [3H]galactosyl moieties retained label during incubation with digitonin. The inability of digitonin-treated cells to shrink in hyperosmotic solutions of various compositions indicated that tetrasaccharides and smaller molecules freely entered the cells. ATP stimulated [3H]norepinephrine uptake into digitonin-treated chromaffin cells fivefold. The stimulated [3H]norepinephrine uptake was inhibited by 1 microM reserpine, 30 microM NH4+, or 1 microM carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). The data indicate that [3H]norepinephrine was taken up into the intracellular storage granules by the ATP-induced H+ electrochemical gradient across the granule membrane. Reduction of the medium osmolality from 310 mOs to 100 mOs was required to release approximately 50% of the catecholamine from chromaffin granules with digitonin-treated chromaffin cells which indicates a similar osmotic stability to that in intact cells. Chromaffin granules in vitro lost catecholamine when the digitonin concentration was 3 microM or greater. Catecholamine released into the medium by micromolar Ca2+ from digitonin-treated chromaffin cells that had subsequently been washed free of digitonin could not be pelleted in the centrifuge and was not accompanied by release of membrane-bound dopamine-beta-hydroxylase. The studies demonstrate that 20 microM of digitonin caused profound changes in the chromaffin cell plasma membrane permeability but had little effect on intracellular chromaffin granule stability and function. It is likely that the intracellular chromaffin granules were not directly exposed to significant concentrations of digitonin. Furthermore, the data indicate that during catecholamine release induced by micromolar Ca2+, the granule membrane was retained by the cells and that catecholamine release did not result from release of intact granules into the extracellular medium.     

Loss and Ca2+-Dependent Retention of Scinderin in Digitonin-Permeabilized Chromaffin Cells: Correlation with Ca2+-Evoked Catecholamine Release

Exposure of chromaffin cells to digitonin causes the loss of many cytosolic proteins. Here we report that scinderin (a Ca(2+)-dependent actin-filament-severing protein), but not gelsolin, is among the proteins that leak out from digitonin-permeabilized cells. Chromaffin cells that were exposed to increasing concentrations (15-40 microM) of digitonin for 5 min released scinderin into the medium. One-minute treatment with 20 microM digitonin was enough to detect scinderin in the medium, and scinderin leakage levelled off after 10 min of permeabilization. Elevation of free Ca2+ concentration in the permeabilizing medium produced a dose-dependent retention of scinderin. Results were confirmed by immunofluorescence microscopy of digitonin-permeabilized cells. Subcellular fractionation of permeabilized cells showed that scinderin leakage was mainly from the cytoplasm (80%); the remaining scinderin (20%) was from the microsomal fraction. Other Ca(2+)-binding proteins released by digitonin and also retained by Ca2+ were calmodulin, protein kinase C, and calcineurins A and B. Scinderin leakage was parallel to the loss of the chromaffin cell secretory response. Permeabilization in the presence of increasing free Ca2+ concentrations produced a concomitant enhancement in the subsequent Ca(2+)-dependent catecholamine release. The experiments suggest that: (1) scinderin is an intracellular target for Ca2+, (2) permeabilization of chromaffin cells with digitonin in the presence of micromolar Ca2+ concentrations retained Ca(2+)-binding proteins including scinderin, and (3) the retention of these proteins may be related to the increase in the subsequent Ca(2+)-dependent catecholamine release observed in permeabilized chromaffin cells.     

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.     

Evidence that the inositol phospholipids are necessary for exocytosis. Loss of inositol phospholipids and inhibition of secretion in permeabilized cells caused by a bacterial phospholipase C and removal of ATP.

We directly manipulated the levels of PtdIns, PtdInsP and PtdInsP2 in digitonin-treated adrenal chromaffin cells with a bacterial phospholipase C (PLC) from Bacillus thuringiensis and by removal of ATP. The PtdIns-PLC acted intracellularly to cause a large decrease in [3H]inositol- or [32P]phosphate-labelled PtdIns, but did not directly hydrolyse PtdInsP or PtdInsP2. [3H]PtdInsP and [3H]PtdInsP2 levels declined markedly, probably because of the action of phosphatases in the absence of synthesis. Removal of ATP also caused marked decreases in [3H]PtdInsP and [3H]PtdInsP2. The decrease in polyphosphoinositide levels by PtdIns-PLC treatment or ATP removal was reflected by the inhibition of the production of inositol phosphates upon subsequent activation of the endogenous PLC by Ca2(+)-dependent catecholamine secretion from permeabilized cells was strongly inhibited by PtdIns-PLC treatment and by ATP removal. Ca2(+)-dependent secretion was similarly correlated with the sum of PtdInsP and PtdInsP2 when the level of these lipids was changed by either manipulation. PtdIns-PLC inhibited only the ATP-dependent component of secretion and did not affect ATP-dependent secretion. Both PtdIns-PLC and ATP removal inhibited the late slow phase of secretion, but had little effect on the initial rapid phase. Although we found a tight correlation between polyphosphoinositide levels and secretion, endogenous phospholipase C activity (stimulated by Ca2+, guanine nucleotides and related agents) was not correlated with secretion. Additional experiments indicated that neither the products of the PtdIns-PLC reaction (diacylglycerol and InsP1) nor the inability to generate products by subsequent activation of the endogenous PLC is likely to account for the inhibition of secretion. Incubation of permeabilized cells with neomycin in the absence of ATP maintained the level of polyphosphoinositides and more than doubled subsequent Ca2(+)-dependent secretion. The data suggest that: (1) Ca2(+)-dependent secretion has a requirement for the presence of inositol phospholipids; (2) the enhancement of secretion by ATP results in part from increased polyphosphoinositide levels; and (3) the role for inositol phospholipids in secretion revealed in these experiments is independent of their being substrates for the generation of diacylglycerol and InsP3.     

Guanine nucleotide effects on catecholamine secretion from digitonin-permeabilized adrenal chromaffin cells

The nonhydrolyzable GTP analogue guanosine 5'-(beta, gamma-imido)triphosphate (GMP-PNP) produced an ATP-dependent but Ca2+-independent stimulation of [3H]norepinephrine release from permeabilized chromaffin cells. This stimulation of secretion was 25-35% of the secretion induced by 10 microM Ca2+. A similar Ca2+-independent stimulation was produced by other non-hydrolyzable GTP analogues. No effect was seen with a variety of other nucleotides, including GTP. The GMP-PNP effect was specifically inhibited by low concentrations of guanine nucleotides. Addition of cAMP did not mimic the Ca2+-independent GMP-PNP effect, but did slightly enhance Ca2+-dependent secretion. Pretreatment with pertussis toxin had no effect on Ca2+-dependent secretion or on the GMP-PNP effect. There was no detectable diglyceride or inositol phosphate produced during GMP-PNP treatment, and addition of diglyceride and inositol trisphosphate did not induce secretion. Guanosine 5'-(beta-thio)diphosphate (GDP-beta-S), in addition to its ability to inhibit the GMP-PNP effect, partially inhibited Ca2+-dependent secretion. At 10 microM free Ca2+, the effects of GMP-PNP and Ca2+ were nonadditive. In fact, secretion in the presence of both GMP-PNP and 10 microM Ca2+ was slightly less than secretion due to Ca2+ alone. These data suggest that a guanine nucleotide-dependent process interacts in some way with one or more components of the normal Ca2+-dependent secretory pathway. However, it may not be an intrinsic part of the mechanism underlying Ca2+-dependent secretion.     

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)     

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.     

The phorbol ester TPA enhances A23187--but not carbachol- and high K+-induced catecholamine secretion from cultured bovine adrenal chromaffin cells

The effect of the phorbol ester TPA on catecholamine secretion was studied in cultured bovine adrenal chromaffin cells. The pretreatment of chromaffin cells with TPA caused the enhancement of catecholamine secretion induced by the calcium ionophore, A23187. By contrast, neither carbachol- nor high K+-induced secretion was changed by TPA pretreatment. These results support the concept that protein kinase C plays an important role as a factor transducing the Ca2+ signal to the exocytotic process of catecholamine secretion in bovine adrenal chromaffin cells.     

Evaluation of the role of inositol trisphosphate in IgE-dependent exocytosis.

A close correlation exists between inhibition by 12-O-tetradecanoylphorbol 13-acetate (TPA) of inositol trisphosphate (InsP3) formation and the rise in internal Ca2+ concentrations in IgE-stimulated rat basophilic leukemia (RBL-2H3) cells. Inhibition of both processes is dose-dependent, with half-maximal and maximal inhibition occurring at 1.5 and 10 ng of TPA/ml respectively. At a similar range of concentrations TPA does not inhibit, but rather enhances, IgE-dependent secretion. When added to antigen-activated cells. EGTA immediately abrogates secretion and stimulates InsP3 production. In contrast, EGTA has only a small inhibitory effect on IgE-induced secretion from TPA-activated cells. In antigen-activated cells, EGTA partially inhibits InsP1 formation, suggesting that, unlike InsP3, InsP1 may in part be formed directly from phosphatidylinositol in a Ca2(-)-dependent manner. Together, these findings suggest that under physiological conditions the stimulated formation of InsP3 is insufficient for triggering secretion, and that Ca2+ influx is essential. Moreover, InsP3 formation is not obligatory for IgE-mediated exocytosis, provided that the cells are activated by TPA. Secretion from TPA-activated cells, which is independent of InsP3 formation and the rise in internal Ca2+, does not require the presence of external Ca2+, implying that the presence of external Ca2+ during IgE-induced secretion is required for producing the Ca2+ signal and not for exocytosis per se.     

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.