Studies on the Effect of Insulin-Like Growth Factor-I on Catecholamine Secretion from Chromaffin Cells

Chromaffin cells cultured in serum-free medium secreted a smaller percentage of their catecholamine stores in response to stimulation by high K+ (55 mM) than did cells cultured in serum-containing medium. Addition of insulin-like growth factor-I (IGF-I) to serum-free medium restored high K(+)-stimulated catecholamine secretion to the levels seen in serum-treated cultures. In contrast, addition of IGF-I to serum-containing medium had little effect on catecholamine secretion. These results suggest that serum contains IGF-I or another factor that maintains the secretory responsiveness of chromaffin cells. IGF-I not only enhanced high K(+)-stimulated catecholamine secretion, but also augmented secretion elicited by the nicotinic agonist dimethyl-phenylpiperazinium, the dihydropyridine agonist Bay K 8644, and Ba2+. IGF-I did not affect the dependence of catecholamine secretion on extracellular Ca2+ concentration nor did it affect the time course of secretion. Experiments using 45Ca2+ demonstrated that IGF-I treatment enhanced Ca2+ uptake into the cells. When cells were permeabilized by treatment with digitonin, Ca2(+)-dependent catecholamine secretion was slightly, but consistently, greater from IGF-I-treated cells than from untreated cells. Our results suggest that IGF-I may enhance catecholamine secretion partly by increasing Ca2+ entry into the cells and partly by affecting a step distal to Ca2+ entry.     

Regulation of Guanosine Triphosphate Cyclohydrolase and Tetrahydrobiopterin Levels and the Role of the Cofactor in Tyrosine Hydroxylation in Primary Cultures of Adrenomedullary Chromaffin Cells

Selective modification of the tetrahydrobiopterin levels in cultured chromaffin cells were followed by changes in the rate of tyrosine hydroxylation. Addition of sepiapterin, an intermediate on the salvage pathway for tetrahydrobiopterin synthesis, rapidly increased intracellular levels of tetrahydrobiopterin and elevated the rate of tyrosine hydroxylation in the intact cell. Tyrosine hydroxylation was also enhanced when tetrahydrobiopterin was directly added to the incubation medium of intact cells. When the cultured chromaffin cells were treated for 72 h with N-acetylserotonin, an inhibitor of sepiapterin reductase, tetrahydrobiopterin content and the rate of tyrosine hydroxylation were decreased. Addition of sepiapterin or N-acetylserotonin had no consistent effect on total extractable tyrosine hydroxylase activity or on catecholamine content in the cultured chromaffin cells. Three-day treatment of chromaffin cell cultures with compounds that increase levels of cyclic AMP (forskolin, cholera toxin, theophylline, dibutyryl- and 8-bromo cyclic AMP) increased total extractable tyrosine hydroxylase activity and GTP-cyclohydrolase, the rate-limiting enzyme in the biosynthesis of tetrahydrobiopterin. Tetrahydrobiopterin levels and intact cell tyrosine hydroxylation were markedly increased after 8-bromo cyclic AMP. The increase in GTP-cyclohydrolase and tetrahydrobiopterin induced by 8-bromo cyclic AMP was blocked by the protein synthesis inhibitor cycloheximide. Agents that deplete cellular catecholamines (reserpine, tetrabenazine, and brocresine) increased both total tyrosine hydroxylase and GTP-cyclohydrolase activities, although treating the cultures with reserpine or tetrabenazine resulted in no change in cellular levels of cyclic AMP. Brocresine and tetrabenazine increased tetrahydrobiopterin levels, but the addition of reserpine to the cultures decreased catecholamine and tetrahydrobiopterin content and resulted in a decreased rate of intact cell tyrosine hydroxylation in spite of the increased activity of the total extractable enzyme. These data indicate that in cultured chromaffin cells GTP-cyclohydrolase activity like tyrosine hydroxylase activity is regulated by both cyclic AMP-dependent and cyclic AMP-independent mechanisms and that the intracellular level of tetrahydrobiopterin is one of the many factors that control the rate of tyrosine hydroxylation.     

Induction of Gene Expression of the Catecholamine-Synthesizing Enzymes by Insulin-Like Growth Factor-I

Abstract: The effects of insulin-like growth factor-I (IGF-I) on gene expression and the activities of the three enzymes specific for catecholamine biosynthesis, tyrosine hydroxylase (TH), dopamine β-hydroxylase (DBH), and phenylethanolamine N -methyltransferase (PNMT), were determined in bovine adrenomedullary chromaffin cells primary cultured in serum-free medium. The mRNA level of TH was maximally elevated in the presence of IGF-I by 3.1 ± 0.4-fold after 48 h, DBH by 5.1 ± 0.3-fold in 24 h, and PNMT by 2.8 ± 0.5-fold in 72 h. In addition, the activity of TH was increased by 77%, DBH by 70%, and PNMT by 23% in IGF-I-exposed cultures. In the absence of the growth factor, the mRNA levels of TH and DBH were decreased to 45 ± 10% and 35 ± 12% of the time-zero control within 48 h while PNMT mRNA was decreased to 82 ± 5% only after 72 h. When the cells were cotreated with the protein tyrosine kinase inhibitor genistein, DBH induction by IGF-I was suppressed, confirming that the effect is mediated by tyrosine kinase. Cotreatment with the protein kinase A (PKA) inhibitor H89 caused complete reversal of the IGF-I-induced DBH increase and the effects of IGF-I treatment and PKA activation by forskolin were not additive, suggesting that PKA is involved in the signaling initiated by IGF-I in these cells.     

Cholinergic Receptor-Mediated Phosphorylation and Activation of Tyrosine Hydroxylase in Cultured Bovine Adrenal Chromaffin Cells

We have identified a 56-kilodalton protein in cultured bovine adrenal chromaffin cells that is phosphorylated when catecholamine secretion is stimulated. Immunodetection on Western blots from both one- and two-dimensional polyacrylamide gels indicated that this protein was tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis. Two-dimensional polyacrylamide gel electrophoresis of proteins from unstimulated cells revealed small amounts of phosphorylated protein with a molecular weight of 56K and pI values of 6.37 and 6.27 which were subunits of tyrosine hydroxylase. Nicotinic stimulation of chromaffin cells caused the phosphorylation of three proteins of 56 kilodaltons with pI values of approximately 6.37, 6.27, and 6.15 which were tyrosine hydroxylase. The immunochemical analysis also revealed that there was unphosphorylated tyrosine hydroxylase 56 kilodaltons with a pI of 6.5 which may have decreased on nicotinic stimulation. The phosphorylation of tyrosine hydroxylase was associated with an increase in in situ conversion of [3H]tyrosine to [3H]dihydroxyphenylalanine ([3H]DOPA). Muscarinic stimulation also caused phosphorylation of tyrosine hydroxylase, but to a smaller extent than did nicotinic stimulation. The secretagogues, elevated K+ and Ba2+, stimulated phosphorylation of tyrosine hydroxylase and [3H]DOPA production. The effects of nicotinic stimulation and elevated K+ on tyrosine hydroxylase phosphorylation and [3H]DOPA production were Ca2+-dependent. Nicotinic agonists also raised cyclic AMP levels in chromaffin cells after 2 min. Dibutyryl cyclic AMP and forskolin, which have little effect on catecholamine secretion, also caused phosphorylation of tyrosine hydroxylase. These stimulators of cyclic AMP-dependent processes caused the appearance of two phosphorylated subunits of tyrosine hydroxylase with pI values of 6.37 and 6.27. There was also a small amount of phosphorylated subunit with a pI of 6.15. Both agents stimulated [3H]DOPA production. The experiments indicate that tyrosine hydroxylase is phosphorylated and activated when chromaffin cells are stimulated to secrete. The data suggest that the earliest phosphorylation of tyrosine hydroxylase induced by a nicotinic agonist occurs through stimulation of a Ca2+-dependent protein kinase. After 2 min phosphorylation by a cyclic AMP-dependent protein kinase may also occur. Phosphorylation of tyrosine hydroxylase is associated with an increase in in situ tyrosine hydroxylase activity.     

Insulin-Like Growth Factor-I-Enhanced Secretion Is Abolished in Protein Kinase C-Deficient Chromaffin Cells

Abstract: Previous studies have demonstrated that bovine chromaffin cells cultured in medium with 10 nM insulin-like growth factor-I (IGF-I) secrete about twofold more catecholamine when exposed to secretory stimuli than do cells cultured without IGF-I. The purpose of this study was to determine whether protein kinase C (PKC) is involved in the effect of IGF-I on secretion from these cells. PKC was down-regulated in the cells by 16–18 h of treatment with β-phorbol didecanoate (β-PDD; 100 nM). Such treatment had no effect on high-K⁺-stimulated secretion from cells cultured without IGF-I; however, secretion from cells cultured with IGF-I was reduced to a level comparable to that in cells cultured without the peptide. The inactive isomer, α-PDD (100 nM), had no effect on secretion from untreated or IGF-I-treated chromaffin cells. The effect of β-PDD was time and concentration dependent, with 100 nM β-PDD producing a maximal effect in 8–10 h. In situ PKC activity measured in permeabilized cells treated with PMA (300 nM) was decreased by～40% by 10 h and was reduced to almost basal levels by 18 h. Immunoblotting experiments demonstrated that both α-and ε-PKC were lost from the cells with time courses similar to that seen in the in situ PKC assay. Overnight treatment with the PKC inhibitor H7 (100 μM) prevented the enhanced secretion normally seen in IGF-l-treated cells, whereas HA1004 had no effect. High-K⁺-stimulated ⁴⁵Ca²⁺ uptake in IGF-I-treated cells was attenuated by long-term treatment with β-PDD (200 nM) or H7 (100 μM). Together these observations suggest that PKC is required for IGF-I-enhanced secretion from chromaffin cells.     

Effects of substance P on the long-term regulation of tyrosine hydroxylase activity and catecholamine levels in cultured adrenal chromaffin cells

Acetylcholine, released from splanchnic nerve terminals innervating adrenal chromaffin cells, is known to increase synthesis of adrenal tyrosine hydroxylase, the rate-limiting enzyme in catecholamine synthesis. The neuropeptide substance P is also present in the splanchnic nerve innervating the adrenal medulla, and this study examined whether substance P has any long-term effects on tyrosine hydroxylase activity and catecholamine levels in cultures of adult bovine adrenal chromaffin cells. When cultures were incubated for 3 days with substance P and carbachol, a cholinergic agonist, substance P (10(-6) M, and greater) completely inhibited the increase in tyrosine hydroxylase activity normally induced by carbachol. Long-term stimulation with carbachol also depleted endogenous catecholamines from the cells and substance P prevented this carbachol-induced depletion of catecholamine content. Substance P by itself, in the absence of carbachol, had only a slight effect on tyrosine hydroxylase activity. 8-Bromoadenosine 3':5'-cyclic monophosphate, an analogue of adenosine 3':5'-cyclic monophosphate, also increases tyrosine hydroxylase activity in chromaffin cells; however, substance P had no effect on the increase in tyrosine hydroxylase activity induced by this analogue. These results indicate that substance P's effects are relatively specific for the carbachol-induced increased in tyrosine hydroxylase activity and that the primary site of action of substance P is not a site common to the mechanism of tyrosine hydroxylase induction by carbachol and 8-bromoadenosine 3':5'-cyclic monophosphate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential Effects of Nerve Growth Factor and Ciliary Neuronotrophic Factor on Catecholamine Storage and Catecholamine Synthesizing Enzymes of Cultured Rat Chromaffin Cells

The effects of nerve growth factor (NGF) and ciliary neuronotrophic factor (CNTF) on catecholamine content and in vitro activities of tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT) were studied in adrenal chromaffin cells cultured from 8-day-old rats. Both NGF and CNTF enhanced chromaffin cell survival and partially prevented losses of adrenaline during the 4-day culture period in a dose-dependent manner. CNTF was more potent, although cellular levels of adrenaline and noradrenaline were not maintained. NGF did not add to the effect of CNTF. The effect of CNTF on catecholamine storage was not accompanied by changes in the activities of TH and PNMT. In contrast, NGF induced TH but not PNMT activity. These data indicate differences between the mechanisms by which NGF and CNTF affect adrenal chromaffin cells.     

Bovine Chromaffin Cells Have Insulin-Like Growth Factor-I (IGF-I) Receptors: IGF-I Enhances Catecholamine Secretion

The binding of 125I-insulin-like growth factor-I (125I-IGF-I) to bovine chromaffin cells was measured. Chromaffin cell cultures contained 111,000 +/- 40,000 IGF-I binding sites/cell. These sites bound IGF-I with a KD of 1.1 +/- 0.3 nM and had a much lower affinity for insulin. Cross-linking studies showed that 125I-IGF-I bound to a protein that had an Mr of approximately 125,000, similar to the Mr of the alpha subunit of the IGF-I receptor in other tissues. Cells cultured with IGF-I (10 nM) for 4 days exhibited an almost twofold increase in high K+-evoked catecholamine secretion. Insulin was much less potent than IGF-I in enhancing catecholamine secretion. These data indicate that binding of IGF-I to its receptors on chromaffin cells can modulate the function of these cells.     

Restoration of Catecholamine Content of Previously Depleted Adrenal Medulla In Vitro: Importance of Synthesis in Maintaining the Catecholamine Stores

The functional integrity of adrenal chromaffin storage vesicles was studied in the perfused rat adrenal gland subjected to intense exocytosis. Continuous perfusion with 55 mM K+-Krebs solution produced a large and uninterrupted secretion of catecholamines. Total amounts secreted within 45 min were 4.66 micrograms and represented almost 30% of the total tissue catecholamine content. If perfusion with excess K+ was extended to 90 min, the secretion increased further to 5.76 micrograms. Despite such a large secretory response, the catecholamine content of the K+-stimulated adrenal medulla was comparable to that of unstimulated control, suggesting an enhanced resynthesis to maintain the normal levels. Pretreatment of rats with alpha-methyl-p-tyrosine, and including this agent in the perfusion medium during stimulation with K+, caused a marked reduction in catecholamine content. The degree of depletion depended on the extent of stimulation with K+ (45% in 45 min and 60% in 90 min). Although depleted catecholamine stores did not show spontaneous recovery in 2 h, inclusion of tyrosine, L-3,4-dihydroxyphenylalanine or dopamine (but not epinephrine or norepinephrine) completely restored the catecholamine content of previously depleted adrenal medulla. Repletion achieved by tyrosine was time dependent (evident in 30 min and maximum in 2 h) and blocked by alpha-methyl-p-tyrosine but not by calcium deprivation. The ratio of epinephrine to norepinephrine remained constant during various stages of the experiment, suggesting both types of vesicles were equally affected by different treatments. The secretory response (10 Hz for 30 s) was unaffected even though tissue catecholamine stores were significantly depleted (50%). In summary, we have demonstrated that catecholamine content of the isolated perfused adrenal gland can be reduced by stimulation of exocytotic secretion in the presence of tyrosine hydroxylase inhibitor. Since the depleted stores can be fully refilled by synthesis of catecholamines from its precursors, it is suggested that chromaffin vesicles may be reutilized for the purpose of synthesis, storage, and secretion of adrenal medullary hormones.     

Arachidonic acid stimulates phosphoinositide metabolism and catecholamine release from bovine adrenal chromaffin cells

Arachidonic acid (AA) evoked a dose-dependent increase in the accumulation of inositol phosphates in cultured bovine adrenal chromaffin cells, and this effect was specific for AA. AA also induced a rise in [Ca2+]i, but this rise was markedly reduced by removal of extracellular Ca2+. AA-induced accumulation of inositol phosphates was absolutely dependent on extracellular Ca2+, and nicardipine and nifedine partially reduced it but verapamil had no effect. Moreover, AA dose-dependently stimulated catecholamine release from chromaffin cells in the presence of ouabain, and this effect was specific for AA. AA-induced catecholamine release in the presence of ouabain was also inhibited by nicardipine and nifedipine but not by verapamil. Furthermore, the phospholipase C inhibitor neomycin inhibited the release. These results taken together suggest that AA stimulates catecholamine release in the presence of ouabain by stimulation of phosphoinositide metabolism in a Ca2(+)-dependent manner.     

Effects of Hypoxia on the Catecholamine Release, Ca2+ Uptake, and Cytosolic Free Ca2+ Concentration in Cultured Bovine Adrenal Chromaffin Cells

The purpose of the present study is to clarify the effects of hypoxia on catecholamine release and its mechanism of action. For this purpose, using cultured bovine adrenal chromaffin cells, we examined the effects of hypoxia on high (55 mM) K(+)-induced increases in catecholamine release, in cytosolic free Ca2+ concentration ([Ca2+]i), and in 45Ca2+ uptake. Experiments were carried out in media preequilibrated with a gas mixture of either 21% O2/79% N2 (control) or 100% N2 (hypoxia). High K(+)-induced catecholamine release was inhibited by hypoxia to approximately 40% of the control value, but on reoxygenation the release returned to control levels. Hypoxia had little effect on ATP concentrations in the cells. In the hypoxic medium, [Ca2+]i (measured using fura-2) gradually increased and reached a plateau of approximately 1.0 microM at 30 min, whereas the level was constant in the control medium (approximately 200 nM). High K(+)-induced increases in [Ca2+]i were inhibited by hypoxia to approximately 30% of the control value. In the cells permeabilized by digitonin, catecholamine release induced by Ca2+ was unaffected by hypoxia. Hypoxia had little effect on basal 45Ca2+ uptake into the cells, but high K(+)-induced 45Ca2+ uptake was inhibited by hypoxia. These results suggest that hypoxia inhibits high K(+)-induced catecholamine release and that this inhibition is mainly the result of the inhibition of high K(+)-induced increases in [Ca2+]i subsequent to the inhibition of Ca2+ influx through voltage-dependent Ca2+ channels.     

Effects of changes in osmolality on the stability and function of cultured chromaffin cells and the possible role of osmotic forces in exocytosis

Recent evidence indicates that osmotic forces may play a role in exocytosis. To examine this possibility and to investigate the osmotic properties of storage granules within cells, we investigated the effects of changes of osmolality on stability and function of cultured bovine chromaffin cells. Cell volume measurements indicated that the cells behaved as osmometers and that the intracellular osmolality rapidly equilibrated with the osmolality of the extracellular medium. Hyperosmotic solutions strongly inhibited nicotinic agonist-stimulated secretion but did not alter nicotinic agonist-stimulated Ca(2+) uptake. Hyperosmotic solutions also strongly inhibited elevated potassium- stimulated secretion but only weakly inhibited elevated K(+)-stimulated Ca(2+) uptake. Thus, hyperosmotic solutions inhibited secretion at a step after calcium entry. Cells exposed to 165 mOs(1) solutions did not lyse and retained their capacity to store and secrete catecholamine upon stimulation. Significant intracellular lysis of chromaffin granules occurred within cells exposed to lower osmolalities. In contrast, 75 percent of the catecholamine was released from granules from cultured cells or from fresh adrenal medulla incubated in vitro at 210 mOs. The data provide evidence for a role for osmotic forces in exocytosis and suggest that if osmotic stress of the granule occurs during exocytosis, then water influx into chromaffin granules increases granule volume by at least 70 percent. The results also indicate that the osmotic properties of the granules are altered upon homogenization and subcellular fractionation of the cells.     

Phosphorylation of tyrosine hydroxylase by cGMP-dependent protein kinase in intact bovine chromaffin cells.

The phosphorylation of the enzyme tyrosine hydroxylase by the cGMP pathway was investigated in chromaffin cells from the bovine adrenal medulla. The nitric oxide donor, sodium nitroprusside, and the natriuretic peptide, C-type natriuretic peptide, which are able to increase cGMP levels and cGMP-dependent protein kinase activity, produced significant increases in the phosphorylation level of tyrosine hydroxylase in a time- and concentration-dependent manner. The pretreatment of the cells with the soluble guanylyl cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one blocked the effect of sodium nitroprusside. This result indicates that cGMP production by this enzyme mediated this effect. Experiments performed with a cGMP-dependent protein kinase inhibitor, the Rp-isomer of 8-(4-chlorophenylthio)-cyclic guanosine monophosphorothioate, which blocked the effects of both sodium nitroprusside and C-type natriuretic peptide, demonstrated that the phosphorylation increases evoked by both compounds were mediated by the activation of cGMP-dependent protein kinase. In cells incubated with the adenylyl cyclase activator, forskolin, an increase in the phosphorylation level of the tyrosine hydroxylase was also found. When cells were treated simultaneously with forskolin and sodium nitroprusside or C-type natriuretic peptide, an additive effect on tyrosine hydroxylase phosphorylation was not observed. This suggests that cAMP- and cGMP-dependent protein kinases may phosphorylate the same amino acid residues in the enzyme. Western blot analysis of soluble extracts from chromaffin cells detected specific immunoreactivity for two different commercial antibodies raised against cGMP-dependent protein kinase (both Ialpha and Ibeta isoforms). Electrophoretic mobility correlates with that of purified PKG Ialpha. Because the phosphorylation of the tyrosine hydroxylase correlates with increases in its enzymatic activity and thus with augmentation in the cell capacity to synthesize catecholamines, our results indicate that a cGMP-based second messenger pathway participates in catecholamine biosynthesis regulation in chromaffin cells, a mechanism which may be widespread in other catecholamine-synthesizing cells.     

Depolarization-stimulated catecholamine biosynthesis: involvement of protein kinases and tyrosine hydroxylase phosphorylation sites in situ

Depolarizing stimuli increase catecholamine (CA) biosynthesis, tyrosine hydroxylase (TH) activity, and TH phosphorylation at Ser19, Ser31, and Ser40 in a Ca(2+)-dependent manner. However, the identities of the protein kinases that phosphorylate TH under depolarizing conditions are not known. Furthermore, although increases in Ser31 or Ser40 phosphorylation increase TH activity in vitro, the relative influence of phosphorylation at these sites on CA biosynthesis under depolarizing conditions is not known. We investigated the participation of extracellular signal-regulated protein kinase (ERK) and cAMP-dependent protein kinase (PKA) in elevated K(+)-stimulated TH phosphorylation in PC12 cells using an ERK pathway inhibitor, PD98059, and PKA-deficient PC12 cells (A126-B1). In the same paradigm, we measured CA biosynthesis. TH phosphorylation stoichiometry (PS) was determined by quantitative blot-immunolabeling using site- and phosphorylation state-specific antibodies. Treatment with elevated K(+) (+ 58 mM) for 5 min increased TH PS at each site in a Ca(2+)-dependent manner. Pretreatment with PD98059 prevented elevated K(+)-stimulated increases in ERK phosphorylation and Ser31 PS. In A126-B1 cells, Ser40 PS was not significantly increased by forskolin, and elevated K(+)-stimulated Ser40 PS was three- to five-fold less than that in PC12 cells. In both cell lines, CA biosynthesis was increased 1.5-fold after treatment with elevated K(+) and was prevented by pretreatment with PD98059. These results suggest that ERK phosphorylates TH at Ser31 and that PKA phosphorylates TH at Ser40 under depolarizing conditions. They also suggest that the increases in CA biosynthesis under depolarizing conditions are associated with the ERK-mediated increases in Ser31 PS.     

Activation of Tyrosine Hydroxylase in PC12 Cells by the Cyclic GMP and Cyclic AMP Second Messenger Systems

Tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis, is subject to regulation by a variety of agents. Previous workers have found that cyclic AMP-dependent protein kinase and calcium-stimulated protein kinases activate tyrosine hydroxylase. We wanted to determine whether cyclic GMP might also be involved in the regulation of tyrosine hydroxylase activity. We found that treatment of rat PC12 cells with sodium nitroprusside (an activator of guanylate cyclase), 8-bromocyclic GMP, forskolin (an activator of adenylate cyclase), and 8-bromocyclic AMP all produced an increase in tyrosine hydroxylase activity measured in vitro or an increased conversion of [14C]tyrosine to labeled catecholamine in situ. Sodium nitroprusside also increased the relative synthesis of cyclic GMP in these cells. In the presence of MgATP, both cyclic GMP and cyclic AMP increased tyrosine hydroxylase activity in PC12 cell extracts. The heat-stable cyclic AMP-dependent protein kinase inhibitor failed to attenuate the activation produced in the presence of cyclic GMP. It eliminated the activation produced in the presence of cyclic AMP. Sodium nitroprusside also increased tyrosine hydroxylase activity in vitro in rat corpus striatal synaptosomes and bovine adrenal chromaffin cells. In all cases, the cyclic AMP-dependent activation of tyrosine hydroxylase was greater than that of the cyclic GMP-dependent second messenger system. These results indicate that both cyclic GMP and cyclic AMP and their cognate protein kinases activate tyrosine hydroxylase activity in PC12 cells.     

Differential control of tyrosine hydroxylase activation and catecholamine secretion by voltage-operated Ca2+ channels in bovine chromaffin cells

Contributions of L-, N-, and P/Q-type voltage-operated Ca2+ channels to two responses of bovine adrenal chromaffin cells have been studied using the nonreceptor stimulus K+ depolarization. Tyrosine hydroxylase activity and catecholamine secretion were both increased by K+ over a similar concentration range and in a Ca(2+)-dependent manner. At a submaximal concentration of 20 mM K+, tyrosine hydroxylase activation was reduced by nitrendipine but unaffected individually by (+/-)-Bay K 8644, omega-conotoxin GVIA, omega-agatoxin IVA, and omega-conotoxin MVIIC. It was fully blocked by combined inhibition of L-, N-, and P/Q-type channels. With a maximal concentration of 50 mM K+, tyrosine hydroxylase activation was unaffected by nitrendipine as well as by each of the other drugs on its own; however, it was reduced by 71 % by combined inhibition of L-, N-, and P/Q-type channels. In contrast, catecholamine secretion with both 20 and 50 mM K+ was enhanced by (+/-)-Bay K 8644, partially inhibited by nitrendipine and omega-conotoxin MVIIC, and completely blocked by a combination of antagonists for L-, N-, and P/Q-type channels. The results show that Ca2+ entry through voltage-operated Ca2+ channels can differentially regulate distinct chromaffin cell responses and that this is an intrinsic property of the mechanisms by which Ca2+ entry activates these responses. It is not dependent on the parallel activation of other signaling events by receptors.     

Different contributions of voltage-sensitive Ca2+ channels to histamine-induced catecholamine release and tyrosine hydroxylase activation in bovine adrenal chromaffin cells.

Histamine stimulates catecholamine release and tyrosine hydroxylase activity in a Ca(2+)-dependent manner in bovine adrenal chromaffin cells. The role of voltage-sensitive Ca2+ channels in these two responses has been investigated. Using an EC50 concentration of histamine, 1 microM, catecholamine release was enhanced by (+/-)BayK8644, and partially inhibited by nitrendipine and omega-agatoxin IVA, blockers of L- and P/Q-type Ca2+ channels. omega-Conotoxin GVIA gave small and variable inhibitory effects. With a maximal histamine concentration, 10 microM, similar results were obtained except that now omega-conotoxin GVIA reliably reduced release. In contrast, neither (+/-)BayK8644 nor any of the individual Ca2+ channel antagonists had any significant effect on tyrosine hydroxylase (TOH) activation induced by either an EC50 or a maximal concentration of histamine. When high concentrations of nitrendipine, omega-conotoxin GVIA and omega-agatoxin IVA were combined with omega-conotoxin MVIIC (a non-selective blocker of N, P and Q channels) to block voltage-sensitive Ca2+ channels in these cells, release induced by K+ depolarization was completely blocked. Release caused by histamine, however, was substantially reduced but not abolished. The combination of antagonists also only partially inhibited TOH activation by histamine. The results show that the G protein-coupled receptor agonist histamine activates several different types of voltage-sensitive Ca2+ channels in chromaffin cells to mediate its cellular effects. Histamine may also activate additional pathways for Ca2+ entry. The results also suggest that the manner by which Ca2+ controls release and TOH activation once it has entered chromaffin cells through these channels are different.     

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.     

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.