Proteolytic processing of chromogranin A in cultured chromaffin cells

The prohormone chromogranin A is the major soluble component of secretory granules in chromaffin cells of adrenal medulla and in many other different endocrine cell types. The proteolytic processing of chromogranin A was studied in cultured bovine chromaffin cells using [35S]methionine to label proteins and a specific antibody to immunoprecipitate the native protein and its breakdown products. In resting cells, it was found that the degradation of chromogranin A is a slow process, since no degradation was observed after a 40 h incubation with radiolabelled methionine. Stimulation of cells with a single pulse or with successive pulses of nicotine did not significantly enhance the degree of proteolytic processing of chromogranin A. As it has recently been shown (Simon, J.P., Bader, M.F. and Aunis, D. Biochem. J. (1989) 260, 915-922) that protein kinase C may be involved in the regulation of chromogranin A synthesis, the possibility that prohormone processing may also be controlled by protein kinase C was examined using the activator of protein kinase C, 12-O-tetradecanoylphorbol 13-acetate (TPA). However, incubation of cells with TPA did not significantly modify chromogranin A processing, indicating that biosynthesis and proteolytic processing of chromogranin A are two distinctly regulated mechanisms. Glucocorticoids are known to exert regulatory control of chromaffin cell metabolism; however, incubation of cells with dexamethasone did not alter slow chromogranin A processing. Stimulation of labelled cells rapidly released newly synthesized chromogranin A into external medium. In addition, released chromogranin A was found to be actively processed into its 60 kDa and 43 kDa breakdown products. This extracellular proteolytic degradation mechanism may be of importance with regard to the function of chromogranin A as a prohormone.     

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.     

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.     

Phorbol ester stimulates catecholamine synthesis in isolated bovine adrenal medullary cells

In isolated bovine adrenal medullary cells, the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA), an activator of protein kinase C, stimulated [14C]catecholamine synthesis from [14C]tyrosine, but not from [14C]DOPA. This stimulatory effect of TPA on [14C]catecholamine synthesis was not dependent upon extracellular Ca2+, and TPA did not affect the uptake of 45Ca2+ or the release of catecholamine by the cells. TPA also did not affect the intracellular cyclic AMP (cAMP) level. 4 alpha-Phorbol 12, 13-didecanoate, which is not an activator of protein kinase C, did not stimulate the synthesis of [14C]catecholamine from [14C]tyrosine. The stimulatory effect of TPA on [14C]catecholamine synthesis was additive with that of carbamylcholine, but not with that of dibutyryl cAMP (DB-cAMP). From these results, it was suggested that protein kinase C is involved in the regulation of tyrosine hydroxylase activity and that this regulatory mechanism might be similar to that involving cAMP.     

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.     

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.     

Muscarinic receptor-linked elevation of cAMP in SH-SY5Y neuroblastoma cells is mediated by Ca2+ and protein kinase C.

The mechanisms of muscarinic receptor-linked increase in cAMP accumulation in SH-SY5Y human neuroblastoma cells has been investigated. The dose-response relations of carbachol-induced cAMP synthesis and carbachol-induced rise in intracellular free Ca2+ were similar. The stimulated cAMP synthesis was inhibited by about 50% when cells were entrapped with the Ca2+ chelator BAPTA or in the presence of the protein kinase C (PKC) inhibitor staurosporine. Production of cAMP could be induced also by the Ca2+ ionophore, ionomycin and by TPA, an activator of PKC. When added together TPA and ionomycin had a synergistic effect. When cAMP synthesis was activated with cholera toxin, PGE1 or PGE1 + pertussis toxin carbachol stimulated cAMP production to the same extent as in control cells. Ca2+ and protein kinase C thus seem to be the mediators of muscarinic-receptor linked cAMP synthesis by a direct action on adenylate cyclase.     

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.     

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.     

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.     

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.     

Differential effects of protein kinase C activators on carbamylcholine- and high K+-induced rises in intracellular free calcium concentration in cultured adrenal chromaffin cells

Pretreatment of adrenal chromaffin cells with protein kinase C activators, i.e. 12-O-tetradecanoyl phorbol-13-acetate (TPA) and 1-oleoyl 2-acetyl glycerol (OAG), partially inhibited carbamylcholine (CCh)-induced rise in intracellular free Ca2+ concentration ([Ca2+]i). The apparent IC50 values of TPA and OAG were 3 nM and 25 microM, respectively. The effect of TPA on the CCh-induced rise in [Ca2+]i was overcome by pretreatment of the cells with a protein kinase C inhibitor, 1-(5-isoquinidinesulfonyl)-2-methylpiperazine hydrochloride (H-7). In contrast, KCl-induced rise in [Ca2+]i was not affected by pretreating the cells with TPA or OAG. An inactive phorbol ester, 4 alpha-phorbol 12,13-didecanoate failed to affect the CCh-induced rise in [Ca2+]i. CCh-induced 45Ca2+ uptake was also partially inhibited by pretreatment of the cells with TPA or OAG, but KCl-induced 45Ca2+ uptake was not affected by these pretreatments. These results indicate that protein kinase C activation causes an uncoupling of signal transduction between the nicotinic receptors and Ca2+ channels.     

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.     

Opposing effects of calcium entry and phorbol esters on fusion of chick muscle cells

Studies utilizing cultured muscle cells have shown that myoblast fusion requires extracellular Ca2+ and involves transient coordinated changes in cell membrane topography and cytoskeletal organization. However, neither the mechanisms by which Ca2+ influences these changes nor its cellular sites of action are known. We have investigated the effects of Ca2+ channel modulators and phorbol esters on fusion of embryonic chick myoblasts in culture. Myoblast fusion was inhibited by the Ca2+ channel blockers D600 and nitrendipine and stimulated by the Ca2+ channel activator Bay K 8644. We have obtained evidence that the tumor promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibits fusion through activation of protein kinase C. Myoblasts prevented from fusing by Ca2+ channel blockers or TPA display a distinctive elongated morphology that is characteristic of cells prevented from fusion by Ca2+ deprivation. The inhibition of fusion by D600 and TPA is significantly diminished in the presence of the Ca2+ ionophore A23187. TPA arrest of myoblast fusion was found to be accompanied by an increase in phosphorylation of the 20-kDa light chain of cytoplasmic myosin in a dose- and time-dependent manner. The effects of TPA on myoblast fusion and phosphorylation of myosin light chain were mimicked by the cell permeant diacylglycerol sn-1,2-dioctanoylglycerol, a potent activator of protein kinase C. The present results suggest that activators of protein kinase C block fusion by interfering with a Ca2+ signal transduction pathway and that this interference may be associated with a protein kinase C catalyzed inhibitory phosphorylation of myosin light chain.     

Calcium-dependence of chromogranin A-catecholamine interaction

Major components of the secretory organelle of bovine adrenal medullary cells, the chromaffin vesicles, are the acidic protein chromogranin A, catecholamines and Ca2+. The binding of Ca2+ to chromogranin A has been established. To study the interaction between chromogranin A and catecholamines and its dependence on Ca2+ we immobilized chromogranin A to a newly raised monoclonal antibody. It is shown that chromogranin A can bind (i) about 0.5 mol catecholamines per mol in a non-calcium-dependent manner and (ii) about 5 mol per mol in the presence of calcium. These results further support the notion that chromogranin A may act as a secretory granule-condensing protein.     

Enhancement of Ca2+-induced catecholamine release by the phorbol ester TPA in digitonin-permeabilized cultured bovine adrenal chromaffin cells

The phorbol ester, 4 beta-phorbol 12-myristate acetate (TPA), increased the extent of catecholamine release induced by Ca2+, without affecting the basal release response in digitonin-permeabilized chromaffin cells. This finding is consistent with the hypothesis that protein kinase C has a role to play in stimulus-secretion coupling in the bovine adrenal medullary chromaffin cell.     

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.     

Muscarinic receptor-mediated increase in cytoplasmic free Ca2+ in isolated bovine adrenal medullary cells. Effects of TMB-8 and phorbol ester TPA

The change in cytoplasmic free calcium, [Ca2+]i in isolated bovine adrenal medullary cells during stimulation by acetylcholine (ACh) in Ca2+-free incubation medium was measured using the fluorescent Ca2+ indicator quin2. ACh (1-100 microM) caused an increase in [Ca2+]i by mobilization of Ca2+ from the intracellular pool. Nicotine (10 microM) did not increase [Ca2+]i in the absence of extracellular Ca2+. Pretreatment of the cells with atropine (10 microM) completely inhibited ACh-induced increase in [Ca2+]i, whereas pretreatment with hexamethonium (100 microM) did not. The intracellular Ca2+ antagonist 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), inhibited ACh-induced increase in [Ca2+]i. The activator of protein kinase C 12-O-tetradecanoylphorbol-13-acetate (TPA), but not its 'inactive' analog 4 alpha-phorbol-12,13-didecanoate (PDD), also inhibited ACh-induced increase in [Ca2+]i. These findings suggest that in bovine adrenal medullary cells, stimulation of muscarinic ACh receptor causes an increase in [Ca2+]i by mobilizing Ca2+ from the intracellular pool and that protein kinase C is involved in 'termination' or 'down regulation' of this response.     

Control of bovine placental progestin synthesis: calcium dependent steroidogenesis is modulated at the site of the cholesterol side chain cleavage enzyme

We have previously reported that progesterone synthesis in the bovine placenta is regulated by Ca2+ dependent and cyclic nucleotide independent mechanism. In studies conducted to further define the role of Ca2+ in the synthesis of progestins in bovine placental tissue, it was found that both protein kinase C (PKC), as determined by phosphorylation, and cytochrome P-450 side chain cleavage, as determined by Western blot analysis, were detectable in the steroidogenetically active portion of the placentome. To determine the site of action of PKC, fetal cotyledon cells were incubated in media containing 25-hydroxycholesterol in the absence or or presence of 10 ng/ml 12-O-tetradecanoyl-phorbol-13-acetate (TPA). It was found that TPA significantly (P less than 0.05) increased the conversion of the exogenous cholesterol analog to progesterone. To determine if the TPA could act synergistically with calcium activators, fetal cotyledon cells were incubated with either methyl isobutyl xanthine (MIX), an activator of intracellular calcium, or the calcium ionophore, A23187, which increases extracellular calcium influx, or both of these agents, in the presence or absence of TPA. It was found that TPA synergistically increased the conversion of sterol to progestins induced by submaximal concentrations of either MIX or A23187. In the presence of both compounds, TPA induced an even more dramatic increase in progestin synthesis. In experiments in which cyanoketone, an agent that inhibits the conversion of pregnenolone to progesterone, was added, TPA addition resulted in increased pregnenolone production, indicating that side chain cleavage of cholesterol is the site of action. The data, therefore, suggest that: (a) Ca2+ affects mechanisms regulating placental steroidogenesis; (2) one locus of Ca2+ is the cholesterol side chain cleavage reaction; and (3) PKC found in this tissue has a role in the Ca activated progestin production.     

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.