Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.13 (protein kinase C)
 49,245 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Nicotinic stimulation and high K+ depolarization of bovine chromaffin cells cause disassembly of cortical filamentous actin networks. Previous work from our laboratory has demonstrated that disassembly of actin filaments is Ca(2+)-dependent, precedes exocytosis and occurs in cortical areas of low cytoplasmic viscosity which are the sites of exocytosis. It has also been suggested that protein kinase C is involved in catecholamine secretion from chromaffin cells. Therefore, the possibility that protein kinase C activation might be implicated in cortical filamentous actin disassembly was investigated. Here we report that phorbol myristate acetate, a protein kinase C activator, causes cortical filamentous actin disassembly. Short-term phorbol ester treatment does not alter the morphology of chromaffin cells; however, 1 h after phorbol ester exposure an increase in cell flattening and membrane ruffling is observed. Phorbol ester-induced cortical filamentous actin disassembly is inhibited by protein kinase C activity inhibitors, is independent of extracellular Ca2+ and has a slower time course than that induced by either nicotinic receptor stimulation or K(+)-depolarization. Phorbol ester effects are likely to be mediated by activation of protein kinase C and not by any changes in intracellular Ca2+ levels, as indicated by measurements of Ca2+ transients. Pretreatment of chromaffin cells with phorbol myristate acetate increases the initial rate of nicotine-evoked catecholamine release. Nicotine-induced cortical actin filament disassembly and catecholamine secretion are partially (29-40%) inhibited by pretreatment of cells with either calphostin C, staurosporine or sphingosine. The results suggest that protein kinase C may be involved in the reorganization of the cortical actin filament network priming the cells for release by removing a barrier to secretory granule mobility. However, its role in exocytosis is modulatory but not essential.
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PMID:Protein kinase C activation by phorbol esters induces chromaffin cell cortical filamentous actin disassembly and increases the initial rate of exocytosis in response to nicotinic receptor stimulation. 128 30

Previous results from our laboratory suggest that long-term treatment of primary cultured bovine adrenal medullary (BAM) chromaffin cells with nicotine or phorbol 12-myristate 13-acetate, either of which directly activates protein kinase C (PKC), increases the mRNA levels encoding catecholamine-synthesizing enzymes and proenkephalin. In the present study, we have examined the effects of nicotine on BAM cell PKC activity with special emphasis on long-term effects. Nicotine increased particulate PKC activity in a concentration-dependent manner when measured using in vitro enzyme assay with histone as the substrate. This effect is mediated through nicotinic cholinergic receptors, because 1,1-dimethylphenylpiperazinium, a nicotinic agonist, had a similar effect. In addition, chlorisondamine, a specific nicotine-receptor blocking drug, antagonized the effect of nicotine. Nicotine also increased specific [3H]phorbol 12,13-dibutyrate ([3H]PdBu) binding within 1 min, the effect of which was maximal between 3 and 12 min. This effect was reversed by chlorisondamine similarly after 12 min and after 18 h of nicotine treatment, indicating that continual nicotinic-receptor occupancy is required for persistent PKC activation. Compared to PKC activation, the onset of nicotine-stimulated diacylglycerol production was slow, and it was observed after 12 min of incubation with nicotine. The diacylglycerol levels, specific [3H]PdBu binding, and PKC activity remained significantly elevated for at least 18 h with continuous nicotine incubation. Furthermore, nicotine increased the PKC immunoreactivity of a particulate protein with a molecular mass of 82 kDa in the western blot. These results suggest that nicotinic-receptor activation increases PKC activity and immunoreactivity in BAM cells.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Long-term activation of protein kinase C by nicotine in bovine adrenal chromaffin cells. 156 Feb 24

The purpose of the present study was to determine the molecular mechanism of stimulatory actions of ACh and vasoactive intestinal polypeptide (VIP) by determining the role of various second messengers in the neurohumoral secretion. Toward such a goal, we measured cAMP, cGMP, protein kinase (PKC) activity, 3H-inositol triphosphate (3H-IP3), and 45Ca uptake in the adrenal medulla subjected to various treatments. Stimulation of splanchnic nerve endings increased 45Ca uptake, cAMP content, 3H-IP3, and PKC activity in the adrenal medulla. If muscarinic receptors of chromaffin cells were selectively activated by perfusion with muscarine, 3H-IP3 content and PKC activity were enhanced. Nicotine, on the other hand, increased only 45Ca uptake without affecting any other second messenger. Perfusion with VIP increased PKC activity and cAMP and 3H-IP3 content. None of the procedures affected cGMP content. Interplay among various second messengers was further investigated by studying interactions of nicotinic, muscarinic, and VIP-ergic receptors in modulation of catecholamine (CA) secretion and by using agents known to activate specific second messengers (e.g., forskolin, phorbol esters). Our results show that muscarine, VIP, and phorbol ester facilitated nicotine-evoked secretion by increasing PKC activity, and it was associated with an additional increase in 45Ca accumulation. On the other hand, secretion evoked by nicotine as well as muscarine was facilitated by forskolin without additional increase in 45Ca accumulation. A novel feature of the study is that ACh and VIP activate three types of receptors on chromaffin cells to stimulate and mutually facilitate the secretion of CA by generating various second messengers.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Cross-communication between acetylcholine and VIP in controlling catecholamine secretion by affecting cAMP, inositol triphosphate, protein kinase C, and calcium in rat adrenal medulla. 255 6

The uptake and release of catecholamines was investigated in the isolated perfused adrenal gland of the rat after preloading the preparation with [3H]norepinephrine, and the effects of various agents were examined on the stimulation-evoked secretion of catecholamines and total tritium. Large quantities of tritium were found in the adrenal medulla after either intravenous injection of [3H]norepinephrine to the rat, or perfusion of the isolated adrenal gland with Krebs-bicarbonate solution containing [3H]norepinephrine. The retention of the tritium was inhibited 90% by desipramine. Acute treatment with guanethidine and chronic treatment with 6-hydroxydopamine abolished the secretion of tritium without affecting the secretion of catecholamines evoked at 1 Hz. Nicotine, muscarine and acetylcholine enhanced the secretion of catecholamines but not tritium, whereas tyramine and ephedrine enhanced the secretion of tritium but not catecholamines. It is concluded that chromaffin cells do not possess the norepinephrine uptake mechanism and that the uptake of [3H]norepinephrine occurs mainly in sympathetic nerve terminals present in the adrenal gland and the surrounding blood vessels (adrenal and renal veins). The differential localization of [3H]norepinephrine and catecholamines allowed us to test the effects of a variety of pharmacological agents that alter neurotransmitter release by acting on receptors on the neuronal membrane, acting on sodium and potassium channels, or acting to alter the intracellular concentrations of adenosine 3',5'-cyclic monophosphate and protein kinase C. Transmural stimulation (1 Hz for a total of 300 pulses) markedly enhanced the release of catecholamines and tritium which was blocked by tetrodotoxin (sodium channel-blocker) and potentiated by tetraethylammonium and gallamine (potassium channel-blockers). Phentolamine, an alpha adrenergic blocking agent which acts on both alpha-1 and alpha-2 receptors, caused a 3- to 4-fold facilitation of the tritium secretion while inhibiting catecholamine secretion by 45%. [Met]enkephalin almost completely inhibited the evoked-secretion of tritium but had very little effect on the secretion of catecholamines. Forskolin inhibited the tritium secretion by 80% but produced more than a 2-fold facilitation of catecholamine secretion. Phorbol 12,13-dibutyrate caused facilitation of evoked secretion of both catecholamines and tritium. A combination of phorbol ester and forskolin had a synergistic effect on stimulation-evoked secretion of catecholamines, whereas phorbol ester partially reversed the inhibitory effects of forskolin on the tritium secretion.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Simultaneous secretion of catecholamines from the adrenal medulla and of [3H]norepinephrine from sympathetic nerves from a single test preparation: different effects of agents on the secretion. 376 30

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.
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PMID:Muscarinic receptor-mediated increase in cytoplasmic free Ca2+ in isolated bovine adrenal medullary cells. Effects of TMB-8 and phorbol ester TPA. 404 96

It has been demonstrated that filamentous actin (F-A) is mainly localized in the cortical surface of the chromaffin cell. This F-A network acts as a barrier to the chromaffin granules impeding their contact with the plasma membrane. Stimulation of chromaffin cells with either nicotine or a depolarizing concentration of K+ induces the disassembly of cortical F-A in focal areas underneath the plasma membrane. Sites of exocytosis are localised to these areas with low concentration of F-A. The cortical surface of the chromaffin cell also contains scinderin, a Ca(2+)-dependent actin filament-severing protein recently isolated in our laboratory. Nicotine and high K+ stimulation also induce redistribution of cortical scinderin. Both nicotine and high K(+)-induced scinderin redistribution and F-A disassembly are Ca(2+)-dependent events which seem to precede neurotransmitter secretion. A possible target for protein kinase C in the modulation of secretion is the cortical F-A network. Treatment of chromaffin cells with phorbol esters prior to secretion induced scinderin redistribution, F-A disassembly and enhanced the initial rate of subsequent nicotine-evoked catecholamine release. The present results strongly indicate the involvement of the cortical cytoskeleton in the regulation of neurotransmitter release.
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PMID:Scinderin and chromaffin cell actin network dynamics during neurotransmitter release. 790 66

Annexin 2 phosphorylated in vitro by protein kinase C has been shown to restore partially catecholamine secretion in streptolysin O-permeabilized chromaffin cells depleted of their protein kinase C activity. This result suggested a phosphorylation of annexin 2 in stimulated cells. Nicotine stimulation induced an increase of 32P incorporation in annexin 2 heavy chain concomitant with catecholamine release. This incorporation results from phosphorylation by protein kinase C because (a) serine was the only phosphorylated residue, (b) 32P incorporation was inhibited by the protein kinase inhibitors H7, GF 109203X, and staurosporine, and (c) activators of this enzyme, 12-O-tetradecanoylphorbol 13-acetate and 1,2-dioctanoylglycerate, increased the incorporation of radioactivity. The phosphorylated heavy chain had an electrophoretic mobility lower than that of the unmodified one, thus allowing determination of the fraction of phosphorylated protein. In the resting state, a significant fraction of annexin 2 heavy chain was phosphorylated, and nicotine stimulation resulted in an activation of both phosphorylation and dephosphorylation. Phosphorylation was largely increased in the presence of okadaic acid, indicating the involvement of type 1 and 2A phosphatases.
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PMID:Phosphorylation by protein kinase C of annexin 2 in chromaffin cells stimulated by nicotine. 908 46

The alpha4beta2 nicotinic acetylcholine receptors (nAChRs), a major subtype in the brain, have been shown to be modulated by chronic treatment with nicotine. In this study, the regulation of recombinant human alpha4beta2 nAChR subtype by (-)-nicotine and other cholinergic channel modulators was studied using human embryonic kidney 293 cells stably expressing this subunit combination. The treatment of transfected cells with (-)-nicotine and other activator ligands, including (-)-cytisine, 1,1-dimethyl-4-phenylpiperazinium, (S)-3-methyl-5-(1-methyl-2-pyrrolidinyl)isoxazole, and (+/-)-epibatidine, resulted in concentration-dependent increases in the levels of alpha4beta2 nAChRs. The increase in [3H]cytisine binding sites was initiated by low concentrations of (-)-nicotine (<100 nM); was maximal at 10 microM (15-fold), rapid (t0.5 = 4.0 +/- 0.5 hr), and totally reversible (t0.5 = 11.7 +/- 0.1 hr); and occurred with no change in ligand binding affinity. Antagonists, including dihydro-beta-erythroidine, d-tubocurarine, and methyllycaconitine, also elicited significant increases in receptor levels. A good correlation was observed between the Ki values for binding inhibition and the EC50 values for receptor up-regulation. Treatment of cells with mecamylamine, a noncompetitive antagonist, did not change receptor levels or alter (-)-nicotine-evoked up-regulation. (-)-Nicotine-evoked up-regulation was blocked by cycloheximide, suggesting a role for protein synthesis. Treatment of cells with (-)-nicotine or dihydro-beta-erythroidine differentially modulated the efficacy of acetylcholine to activate cation efflux. Both 6-beta-[beta'(piperidino)propionyl]forskolin and phorbol-12-myristate-13-acetate increased [3H]cytisine binding sites and nAChR function and enhanced the effects of chronic (-)-nicotine treatment in a synergistic manner. These results collectively demonstrate that human alpha4beta2 nAChRs can be differentially up-regulated by chronic treatment with nAChR ligands and activation of protein kinase A- and protein kinase C-dependent mechanisms.
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PMID:Regulation of human alpha4beta2 neuronal nicotinic acetylcholine receptors by cholinergic channel ligands and second messenger pathways. 928 15

In canine cerebral artery strips contracted with prostaglandin F2alpha, transmural electrical stimulation (5 Hz for 40 s) produced a relaxation which was abolished by tetrodotoxin. The neurogenic response was inhibited moderately by [S]-5-isoquinolinesulfonic acid,4-[2-[(5-isoquinolinyl-sulfonyl)methylamino]-3-oxo-(4-phenyl-1-piperazinyl)-propyl] phenyl ester (KN62), an inhibitor of Ca2+ /calmodulin-dependent protein kinase II, which however did not alter or only slightly reduced the relaxant response to electrical nerve stimulation in canine coronary arterial strips that is mediated via beta-adrenoceptors stimulated by norepinephrine. Nicotine-induced relaxation, mediated by nitric oxide (NO) derived from perivascular nerves, was also attenuated by KN62, whereas the response to exogenous NO was unaffected. The nicotine-induced increase in the cyclic GMP content in cerebral arteries was depressed by KN62. The neurogenic relaxation was not influenced by phorbol 12-myristate 13-acetate, an activator of protein kinase C. 8-Bromo-cyclic GMP and 8-bromo-cyclic AMP did not significantly alter the response to nerve stimulation. It is concluded that the phosphorylation pathway involving Ca2+/calmodulin-dependent protein kinase II, but not other protein kinases so far tested, appears to be involved in the function of vasodilator nerves innervating the cerebral artery.
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PMID:Effect of Ca2+/calmodulin-dependent protein kinase II inhibitors on the neurogenic cerebroarterial relaxation. 952 7

FGF-2, a mitogenic/neurotrophic protein, controls the development and plasticity of many types of neural cells. In neural crest-derived adrenal pheochromatocytes, induction of FGF-2 coincides with the establishment of functional innervation and is reproduced in vitro by stimulating acetylcholine receptors (AChR). The mechanisms by which AChR activate the FGF-2 gene were examined in cultured bovine adrenal medullary chromaffin (BAMC) cells in which AChR induce expression and nuclear accumulation of growth-promoting FGF-2 and FGF-2 receptors. Carbachol or nicotine increased expression of transfected FGF-2 gene promoter-luciferase constructs and were more potent than the muscarinic agonist ABMCB. Deletion analysis has identified a unique -555/-512 bp element that confers AChR stimulation and basal activity to the downstream FGF-2 promoter, and a separate protein kinase C/cAMP-responsive sequence (-625/-555 bp). Stimulation of AChR increased in vitro formation of protein complexes with the AChR-responsive element which were not displaced by target oligonucleotides for common trans-activators. Southwestern analysis identified 50-55, 125, 140 and 170 kDa proteins that interact with the AChR-responsive element in a manner stimulated by AChR. Nicotine increased tyrosine phosphorylation of cytoplasmic and nuclear proteins, including 50-55 kDa promoter-binding factors. Activation of the FGF-2 promoter was reduced by genistein. Thus, nicotinic AChR activate the FGF-2 gene via a new signaling mechanism separate from the cAMP/PKC pathways. It utilizes tyrosine phosphorylation and interaction of trans-activating factors with a novel cis-acting element. It offers a new pathway through which trans-synaptic signals may control neural development and plasticity.
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PMID:Increased tyrosine phosphorylation and novel cis-acting element mediate activation of the fibroblast growth factor-2 (FGF-2) gene by nicotinic acetylcholine receptor. New mechanism for trans-synaptic regulation of cellular development and plasticity. 958 40


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