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)

When exogenous ACh is loaded into the cytoplasm of cultured amphibian myocytes and fibroblasts, the cells undergo spontaneous quantal ACh secretion, as detected by the appearance of pulsatile membrane currents in Xenopus myocytes which are manipulated into contact with the cells. These currents resemble in many ways the miniature endplate currents (MEPCs) observed at developing neuromuscular synapses formed on these Xenopus myocytes. Analyses of the frequency, amplitude, and time course of these currents suggests similarity in the cellular mechanisms involved in the packaging and secretion of ACh quanta in fibroblasts, myocytes, and developing neurons. The size of the ACh packets released by the non-neuronal cells were found to be very similar to the size of the neuronal ACh quanta, which are thought to result from the exocytotic release of synaptic vesicles. Moreover, the kinetics with which the ACh packets are discharged from all three cell types are comparable, although the speed of secretion in non-neuronal cells is somewhat slower and more irregular. The spontaneous quantal ACh secretion from neurons and myocytes was decreased by reducing cytosolic Ca2+ level and enhanced by activation of protein kinase C with phorbol ester, but secretion from fibroblasts was unaffected by both treatments. The spontaneous secretion from fibroblasts did show some sensitivity to a rise in cytosolic Ca2+ after treatment with a Ca2+ ionophore. These observations support the hypothesis that the basic machinery for transmitter secretion operating in neurons derive from a more ubiquitous mechanism used for constitutive secretion and membrane trafficking in non-neuronal cells, and neuronal differentiation involves expression of additional unique components for the regulation of the spontaneous quantal secretion.
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PMID:Spontaneous quantal transmitter secretion from myocytes and fibroblasts: comparison with neuronal secretion. 772 32

Acetylcholine (ACh) caused a dose-dependent contraction of gallbladder muscle cells in either a normal (1.9 mM) Ca2+, zero-Ca2+ or 4 mM Sr2+ medium, with a maximal contraction about 21 +/- 1% at 10(-6) M. Pirenzepine, methoctramine and p-fluoro-hexahydro-sila-difenidol (the M1, M2 and M3 antagonist, respectively) alone had no inhibitory effect on ACh-induced contraction in normal Ca2+ medium, which was blocked by the combination of methoctramine and p-F-HHSiD. In the 4 mM Sr2+ medium, methoctramine dose dependently inhibited ACh-induced contraction and shifted the ACh dose-response curve to the right. The contraction induced by ACh was further blocked by 10(-4) M propranolol (phosphatidic acid phosphohydrolase inhibitor that prevents the production of diacylglycerol from phospholipase D activation), 10(-5) M H-7 and chelerythrine (the protein kinase C inhibitors) by 64%, 75% and 77%, respectively. In contrast, in the zero-Ca2+ medium, p-fluoro-hexahydro-sila-difenidol dose-dependently inhibited ACh-induced contraction and shifted the ACh dose-response curve to the right. The action of ACh was further blocked by 10(-6) M U-73122 (phospholipase C inhibitor) and 10(-5) M CGS 9343B (calmodulin antagonist) by 95% and 77%, respectively. In conclusion, ACh contracts the gallbladder muscle by stimulating the M2 and M3 muscarinic receptors. The M2 receptors are linked to Ca2+ influx, activation of phospholipase D and protein kinase C-dependent pathway, whereas the M3 receptors are preferentially associated with the activation of phospholipase C, intracellular Ca2+ release and calmodulin-dependent pathway.
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PMID:Distinct muscarinic receptors and signal transduction pathways in gallbladder muscle. 775 67

Acetylcholine releases calcium from cytoplasmic stores and permits an influx of calcium in salivary acinar cells. The resultant rise in [Ca2+]i causes an increase in potassium permeability which is an important part of the secretory response. We have investigated the effects of 12-O-tetradecanoyl phorbol-13-acetate, a potent activator of protein kinase C, upon this regulation of potassium permeability in superfused pieces of rat submandibular salivary gland. This compound inhibited the initial [Ca2+]O-independent component of the response of acetylcholine but had no effect upon the subsequent [Ca2+]O-dependent phase. This compound does not, therefore, appear to inhibit receptor-regulated calcium influx.
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PMID:The effect of a phorbol ester upon the cholinergic regulation of potassium permeability in the rat submandibular gland. 787 47

1. This study examined the ability of various nitro-vasodilators, 8-bromo cyclic guanosine 3':5' monophosphate (8-BrcGMP) and forskolin to relax rings of rat thoracic aorta pre-contracted with either noradrenaline (0.1 microM) or the protein kinase C activators, phorbol 12,13-dibutyrate (PDB, 0.1 microM) or phorbol 12-myristate 13-acetate (PMA, 0.5 microM). 2. In noradrenaline pre-contracted rings, acetylcholine (10 nM-10 microM), sodium nitroprusside (1 nM-0.5 microM), the calcium ionophore A23187 (10 nM-10 microM) and 8-BrcGMP (10 mM) totally reversed the smooth muscle contraction. In PDB-contracted aortic rings acetylcholine, sodium nitroprusside and 8-BrcGMP-induced relaxation was reduced compared to that in noradrenaline-contracted aortic rings, but A23187 and forskolin-induced relaxations were unaffected. Both acetylcholine and A23187-induced relaxations in PDB-contracted rings were abolished in the presence of the nitric oxide synthesis inhibitor N omega-nitro-L-arginine (NOLA, 100 microM). 3. Acetylcholine and sodium nitroprusside were even less potent in their ability to relax PMA-contracted aortic rings compared with noradrenaline and PDB-contracted rings. A23187-induced relaxation was also inhibited in PMA-contracted rings. 4. These results show that protein kinase C activation reduces the ability of agents which liberate nitric oxide to induce smooth muscle relaxation, and also inhibits the biochemical pathways which are subsequently activated by nitric oxide and lead to vascular smooth muscle relaxation.
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PMID:Phorbol esters impair endothelium-dependent and independent relaxation in rat aortic rings. 792 9

Changes in neocortical immunoreactivity (ir) for muscarinic acetylcholine receptors (mAChRs), protein kinase C gamma (PKC gamma), microtubule-associated protein 2 (MAP-2), and the calcium-binding protein parvalbumin (PARV) induced by the performance of a one-trial passive shock avoidance (PSA) task were studied in young adult male Wistar rats. In experiment I, four groups of animals were formed: three control groups (N, naive; H, habituated but nonshocked; and S, habituated and shocked), and a fully trained group (T, habituated and shocked, followed by a retention trial 24 hr after the footshock). Compared to naive animals, the H, S, and T animals all revealed enhanced cortical ir for mAChRs, PKC gamma, and MAP-2 in discrete subsets of cortical neurons in layers 2, 3, and 5, while no changes were found for PARV. The neurons displaying enhanced levels of ir are of the pyramidal and nonpyramidal cell type and are arranged in a columnar manner. Immunofluorescent double-labeling experiments for mAChR, PKC gamma, and MAP-2 revealed that individual cortical neurons localized within the columns display enhanced ir for all three functionally related proteins. Compared to naive animals, all experimental groups revealed significant increases in the total size of cortical areas showing enhanced ir (H, S, and T over N). A further significant increase is found in animals receiving a footshock over nonshocked animals (S over H, respectively). The retention trial, however, did not induce a further increase (T over S). In some of the animals the patterns appeared to be lateralized, in either the left or right hemisphere. In order to test the role of cholinergic innervation in the induction of enhanced mAChR-ir, unilateral lesions of the nucleus basalis magnocellularis (nbm) were performed in experiment II. Apparently, an intact cholinergic innervation from the nbm is not required for the occurrence of the aforementioned columnar patterns. However, when the enhanced columnar patterns in the sensory areas of the cortex are cholinergically deprived, clear deficits in PSA performance are observed. These results indicate that although ACh is not a prerequisite for the induction of enhanced ir for mAChRs in cortical cells, such neurons demand cholinergic neurotransmission for optimal retention of the shock experience. The alterations in ir for coexpressed mAChR, PKC gamma, and MAP-2 in a discrete subset of cholinoceptive cortical neurons arranged in characteristic patterns most likely represent part of the neuronal substrate involved in functional cortical plasticity related to PSA training.
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PMID:Passive avoidance training induces enhanced levels of immunoreactivity for muscarinic acetylcholine receptor and coexpressed PKC gamma and MAP-2 in rat cortical neurons. 795 Mar 10

The differences of intracellular signalling mechanisms between muscarinic acetylcholine m1 and m3 receptors, which are coupled with polyphosphoinositide turnover, were examined by using m1- and m3-transfected NG108-15 cells. The c-fos mRNA was induced by 1 mM acetylcholine peak at 60 min in both m1 and m3 cells. The c-fos induction in m1 cells was inhibited by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester (BAPTA-AM) and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7), but was not inhibited by prolonged treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA), suggesting that intracellular Ca2+ and calmodulin are involved in the induction. The c-fos induction in m3 cells was inhibited by BAPTA-AM and prolonged treatment with TPA, but was not influenced by W-7, suggesting that protein kinase C is mainly involved in m3-induced c-fos expression. Acetylcholine induced an increase in inositol phosphates and a transient increase in the intracellular concentration of Ca2+ in both m1 and m3 cells. Sustained stimulation of acetylcholine strongly increased the inositol monophosphate content in m3 cells, but that of inositol trisphosphate and inositol diphosphate in m1 cells. These results suggest that the difference between m1- and m3-induced c-fos mRNA induction mechanisms is due to the difference in respective properties in polyphosphoinositide turnover.
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PMID:Distinct induction of c-fos mRNA in NG108-15 cells transfected with muscarinic m1 and m3 receptors. 795 39

1. 5-Hydroxytryptamine (5-HT) has been shown to induce contraction of tracheal smooth muscle. However, the mechanisms of action of 5-HT are not known. We therefore investigated the effects of 5-HT on phospholipase C (PLC)-mediated phosphoinositide (PI) hydrolysis and its regulation in canine cultured tracheal smooth muscle cells (TSMCs) labelled with [3H]-inositol. 5-HT-induced inositol phosphates (IPs) accumulation was time- and dose-dependent with a half-maximal response (EC50) and a maximal response at 0.38 +/- 0.05 and 10 microM, respectively. 2. Ketanserin and mianserin (10 and 100 nM), 5-HT2 receptor antagonists, were equipotent in blocking the 5-HT-induced IPs accumulation with pKB values of 8.46 and 8.21, respectively. In contrast, the dose-response curves of 5-HT-induced IPs accumulation were not shifted until the concentrations of NAN-190 and metoclopramide (5-HT1A and 5-HT3 receptor antagonists, respectively) were increased up to 10 microM. 3. Pretreatment of TSMCs with pertussis toxin or cholera toxin did not inhibit the 5-HT-induced IPs accumulation, but partially inhibited the AlF(4-)-induced IPs response. 4. Stimulation of IPs accumulation by 5-HT required the presence of external Ca2+ and was blocked by EGTA. The addition of Ca2+ (3-620 nM) to digitonin-permeabilized TSMCs directly stimulated IPs accumulation. A further Ca(2+)-dependent increase in IPs accumulation was obtained by inclusion of either guanosine 5'-O-(3-thiotriphoshate) (GTP gamma S) or 5-HT. The combination of GTP gamma S and 5-HT elicited an additive effect on IPs accumulation. 5. Treatment with phorbol 12-myristate 13-acetate (PMA, 1 microM, 30 min) abolished the 5-HT-induced IPs accumulation. The concentrations of PMA that gave a half-maximal and maximal inhibition of 5-HT-induced IPs accumulation were 2.2 +/- 0.4 nM and 1 microM, n = 3, respectively. The protein kinase C (PKC) activator, 4 alpha-phorbol 12,13-didecanoate, at 1 microM, did not influence this response. The inhibitory effect of PMA was reversed by staurosporine, a PKC inhibitor, suggesting that the inhibitory effect of PMA is mediated through the activation of PKC. 6. The site of this inhibition was further investigated by examining the effect of PMA on AlF(4-)-induced IPs accumulation in canine TSMCs. AlF(4-)-stimulated IPs accumulation was inhibited by PMA treatment, suggesting that the effect of PMA is distal to the 5-HT receptor. 7. Acetylcholine-induced IPs accumulation was completely inhibited by atropine, but not affected by ketanserin or mianserin, suggesting that 5-HT-induced IPs accumulation is not due to release of acetylcholine.8. These results demonstrate that 5-HT directly stimulates PLC-mediated PI hydrolysis via a pertussis toxin- and cholera toxin-insensitive GTP binding protein in canine TSMCs and that this coupling process is negatively regulated by PKC. 5-HT2 receptors may be predominantly mediating IPs accumulation and presumably IP-induced Ca2+ release may function as the transducing mechanism for 5-HT stimulated contraction of tracheal smooth muscle.
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PMID:5-Hydroxytryptamine receptor-mediated phosphoinositide hydrolysis in canine cultured tracheal smooth muscle cells. 801 56

The nicotinic acetylcholine receptor (AChR) is a pentameric complex made up of four types of subunits in the stoichiometry alpha 2 beta gamma delta. These subunits have been shown to be differentially phosphorylated by cAMP-dependent protein kinase (PKA) protein kinase C, and a protein tyrosine kinase. A variety of studies have suggested that phosphorylation of the AChR in vitro and in vivo regulates the rate of desensitization of the receptor. In this study we have used site-specific mutagenesis and patch-clamp techniques to examine the role of phosphorylation in the regulation of desensitization of the AChR expressed in Xenopus oocytes Expression of wild-type AChR in Xenopus oocytes results in the constitutive phosphorylation of the AChR on the gamma and delta subunits. This phosphorylation is apparently due to the high basal level of PKA in oocytes since a specific peptide inhibitor of PKA completely eliminated phosphorylation of the AChR by oocyte extracts in vitro. The phosphorylation of the AChR in oocytes was not significantly enhanced by forskolin or cAMP analogs or by coexpression with the catalytic subunit of PKA, suggesting that the basal activity of PKA in oocytes is sufficient to phosphorylate the receptor to a high stoichiometry. Using site-specific mutagenesis, the sites of phosphorylation were determined to be serines 353 and 354 on the gamma subunit and serines 361 and 362 on the delta subunit. To examine the functional properties of wild-type and mutant receptors lacking phosphorylation sites, we used patch-clamp techniques to measure the responses of out-side-out patches to repetitive pulses of ACh using a rapid perfusion system. Wild-type and mutant receptors showed rapid concentration-dependent activation and desensitization to applied agonist. The time constant of desensitization of ensemble mean currents ranged from several hundred milliseconds at low ACh concentrations to 100-200 msec at saturating concentrations. The desensitization time constants for mutant receptors lacking all phosphorylation sites were significantly slower than wild-type phosphorylated receptors at all concentrations of ACh tested. In addition, mutant receptors that had the serine residues changed to glutamate residues in order to mimic the negative charge of the phosphorylated serine residue produced receptors that had desensitization rates approaching those of the wild-type phosphorylated receptor. These results provide further support that phosphorylation of the nicotinic ACh receptor regulates rate of desensitization.
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PMID:Role of phosphorylation in desensitization of acetylcholine receptors expressed in Xenopus oocytes. 802 70

The incorporation of [3H]serine into lipids, water-soluble metabolites and proteins by the human neuroblastoma cell line LA-N-1 exposed to oxotremorine-M, a muscarinic agonist, was investigated. Oxotremorine-M increased the incorporation of this labelled precursor into phosphatidylserine and proteins in a concentration-dependent manner, with the maximal stimulation at 250 microM. This activation was blunted by 100 microM atropine. There were no detectable changes of the radioactivity in the water-soluble metabolites. Acetylcholine, another muscarinic agonist, slightly decreased the serine incorporation into lipids, but did not affect the protein or water-soluble compartments. Several other muscarinic agonists, including 250 microM pilocarpine, 100 microM McN-A-343 and 1 mM carbachol, did not effect these [3H]serine incorporations. Preincubation of cells with 1 mM oxotremorine M, or 1 mM carbachol, or 1 mM McN-A-343, for 4 h prevented the oxotremorine-M-induced increase of serine incorporation. These observations are consistent with the oxotremorine-M action being mediated by muscarinic-receptor occupancy. The G-protein inhibitor guanosine 5'-[beta-thio]diphosphate (1 mM) and the G-protein activators, guanosine 5'-[gamma-thio]triphosphate (100 microM) and A1F3, prevented the oxotremorine stimulation. The muscarinic agonists, 250 microM oxotremorine-M, 1 mM carbamoylcholine and 500 microM acetylcholine, triggered the accumulation of inositol mono- and di-phosphates by cells that had been prelabelled with myo-[3H]inositol, and this phospholipase C activation was blunted by 100 microM atropine. The protein kinase C inhibitor H7 prevented the oxotremorine-M stimulation of serine incorporation. Over-night exposure of LA-N-1 cells to 100 nM phorbol 12-myristate 13-acetate resulted in a decrease of cytosolic protein kinase C activity, and prevented the oxotremorine-M stimulation of serine incorporation. Neither oxotremorine-M nor acetylcholine caused a redistribution of protein kinase C activity between the cytosol and membrane compartments. In addition, oxotremorine-M did not activate phospholipase D of the LA-N-1 cells.
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PMID:Modulation of phosphatidylserine synthesis by a muscarinic receptor occupancy in human neuroblastoma cell line LA-N-1. 817 97

Acetylcholine (ACh) binding to atrial muscarinic receptors activates an inwardly rectifying K+ current (IK[ACh]) via a pertussis toxin-sensitive GTP-binding protein (GK). The muscarinic K+ channel (termed GIRK1) has been cloned, and the nucleotide sequence contains nine consensus sites for protein kinase C (PKC) phosphorylation (16). Dephosphorylation of the muscarinic K+ channel has been implicated in rapid IK[ACh] desensitization in the presence of agonist (13). Staurosporine is a widely used membrane-permeant inhibitor of PKC and other protein kinases (7), including G protein-coupled receptor kinases. We investigated the role of phosphorylation in the regulation of IK[ACh] by examining the effect of a variety of protein kinase inhibitors. Staurosporine produced a rapid and reversible dose-dependent decrease in IK[ACh], activated by either GTP or guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S). Other PKC inhibitors, including calphostin C and K-252b, were without effect on GTP gamma S-activated IK[ACh]. In excised patches of atrial membrane under nonphosphorylating conditions (0 ATP, 1 mM 5'-adenylylimidodiphosphate), staurosporine reversibly reduced muscarinic K+ channel activity without altering single-channel current amplitude. These results suggest that staurosporine inhibits IK[ACh] by a mechanism independent of intracellular protein kinases.
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PMID:Protein kinase-independent inhibition of muscarinic K+ channels by staurosporine. 817 60


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