EC:2.7.11.13 - FACTA Search

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)

1. The present study employed a [(35)S]-GTPgammaS binding protocol in conjunction with immunoprecipitation (IP) of the Galpha subunits to investigate the desensitization of G(q/11)-coupled receptors at the level of the G-protein activation. Membranes from SH-SY5Y cells expressing the recombinant human alpha(1B)-adrenoceptor (alpha(1B)-AR) (and endogenously expressing the M(3) muscarinic acetylcholine receptor (M(3)-AChR)) exhibited G(q/11) activation in a concentration-dependent manner in response to noradrenaline or methacholine. 2. Pre-treatment of intact cells with agonist prior to membrane preparation and use in the [(35)S]-GTPgammaS IP assay demonstrated that both receptors were homologously desensitized by pre-treatment with agonist since the G(q/11) activation in response to a secondary challenge with agonist was markedly reduced. Stimulation of alpha(1B)-AR was effective at heterologously desensitizing the M(3)-AChR. The PKC inhibitor, Ro-31-8220 (10 microM) was ineffective at preventing the agonist-mediated receptor desensitization. 3. [(32)P]P(i)-labelled cells allowed the detection of increases in receptor phosphorylation. Phorbol 12,13 dibutyrate (PDBu) (1 microM) was effective at producing a Ro-31-8220 (10 microM)-sensitive, detectable increase in alpha(1B)-AR but not M(3)-AChR phosphorylation. Noradrenaline (30 microM) stimulated alpha(1B)-AR phosphorylation, which could be partially inhibited by Ro-31-8220 (10 microM). The phosphorylation of M(3)-AChR was increased by methacholine (100 microM) incubation and this effect appeared to be insensitive to Ro-31-8220 (10 microM). 4. These findings demonstrate that [(35)S]-GTPgammaS-Galpha-subunit IP can be used to estimate receptor desensitization as a decline in receptor-G-protein coupling. Both the alpha(1B)-AR and M(3)-AChR undergo rapid homologous desensitization that is associated with an increase in receptor phosphorylation. The heterologous desensitization of M(3)-AChR produced by alpha(1B)-AR stimulation is not associated with a detectable increase in M(3)-AChR phosphorylation, suggesting that receptor phosphorylation is not necessarily a prerequisite for desensitization.
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PMID:Homologous and heterologous uncoupling of muscarinic M(3) and alpha(1B) adrenoceptors to Galpha(q/11) in SH-SY5Y human neuroblastoma cells. 1156 43

alpha(1a)-Adrenergic receptors (ARs) couple to phosphoinositide hydrolysis, adenylyl cyclase, and mitogen-activated protein kinase (MAPK) pathways. However, the interaction among these signaling pathways in activating extracellular signal-regulated kinase 1/2 (ERK1/2) is not well understood. We investigated the coupling of alpha(1a)-ARs to ERK1/2 in Chinese hamster ovary (CHO)-K1 cells stably transfected with mouse alpha(1a)-ARs, as well as the interaction between ERK1/2 and norepinephrine-induced cAMP accumulation. alpha(1a)-AR activation by norepinephrine increased the cytosolic Ca(2+) concentration and phosphorylated ERK1/2 in a time- and concentration-dependent manner. ERK1/2 phosphorylation was blocked by the MAPK kinase 1/2 inhibitor 2'-amino-3'-methoxyflavone (PD 98059) and the alpha(1)-AR antagonist prazosin. A transient elevation in intracellular Ca(2+) was required for the phosphorylation of ERK1/2; however, activation of protein kinase C did not seem to be required for ERK1/2 phosphorylation. Norepinephrine also stimulated cAMP accumulation in transfected CHO-K1 cells in a concentration-dependent manner via alpha(1a)-ARs, which was blocked by the Ca(2+) chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. Norepinephrine-induced ERK1/2 phosphorylation was inhibited by the adenylyl cyclase activator forskolin and was enhanced by the adenylyl cyclase inhibitor 9-(tetrahydro-2-furanyl)-9H-purine-6-amine (SQ 22536) and the protein kinase A inhibitor 4-cyano-3-methylisoquinoline. In conclusion, in transfected CHO-K1 cells, alpha(1a)-AR activation activates both phospholipase C and adenylyl cyclase-mediated signaling pathways. alpha(1a)-AR-mediated ERK1/2 phosphorylation was dependent on a rise in intracellular Ca(2+), and this pathway was reciprocally regulated by the concomitant activation of adenylyl cyclase, which inhibits ERK1/2 phosphorylation. Thus, alpha(1a)-AR stimulation of cAMP production may play an important role in regulating ERK1/2 phosphorylation in cell lines and native tissues.
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PMID:Tonic inhibitory role for cAMP in alpha(1a)-adrenergic receptor coupling to extracellular signal-regulated kinases 1/2. 1223 58

1. The aim of the present study was to investigate noradrenaline (NA)-induced regulation of alpha1-adrenoceptor (AR) mRNA expression in human embryonic kidney (HEK) 293 cells stably expressing cloned alpha1-AR subtypes with similar receptor densities. Stable transfection was performed by calcium phosphate precipitation. Receptor expression was detected by radioligand binding assay. The mRNA expression was measured by RNase protection assay. 2. alpha1-Adrenoceptor subtype mRNA respond in distinct ways following prolonged exposure to NA. The mRNA level of the alpha 1A-AR subtype was unchanged, the mRNA level of the alpha 1B-AR subtype was increased and the mRNA level of the alpha 1D-AR subtype declined time dependently. The protein kinase C (PKC) inhibitor calphostin C or RO 31-8220 abolished the NA-induced downregulation of alpha 1D-AR mRNA. Phorbol myristate acetate (PMA), a PKC activator, similarly repressed the effects of NA on alpha 1D-AR. However, calphostin C, RO 31-8220 or PMA had no effect on the induction of alpha 1B-AR mRNA by NA. The Ca2+-ATPase inhibitor thapsigargin or the calcium chelator 1,2-bis-(o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid tetra (acetoxymethyl) ester (BAPTA/AM) had no effect on the repression of alpha 1D-AR mRNA, but did inhibit the induction of alpha 1B-AR mRNA by NA. Noradrenaline significantly decelerated the degradation of alpha 1B-AR mRNA, but had no effect on the degradation of alpha 1D-AR mRNA. 3. Thus, the mRNA expression of three alpha1-AR subtypes in HEK293 cells is differentially regulated through distinct signal transduction pathways under sustained NA stimulation. The upregulation of alpha 1B-AR mRNA is via the Ca2+ pathway, whereas the downregulation of alpha 1D-AR mRNA is via the PKC pathway.
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PMID:Changes in mRNA expression induced by sustained noradrenaline stimulation are different for alpha1-adrenoceptor subtypes in HEK293 cells. 1239 Feb 96

Chronic surgical denervation of the rat vas deferens leads to an enhanced contractile response of the tissue to norepinephrine in vitro. Norepinephrine produces a higher rate of protein kinase C translocation to the particulate fraction of denervated tissues as compared with the paired, control vas deferens. Diacylglycerol generation in response to norepinephrine and contractile responses to phorbol diacetate were not altered by chronic denervation of the vas deferens. However, the contractile response to norepinephrine in these tissues was less susceptible to the inhibitory effects of the calcium channel blocker nifedipine. A potential role of protein kinase C in sensitizing the contractile apparatus to mobilized calcium in denervation supersensitivity is discussed.
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PMID:A role for protein kinase C in the supersensitivity of the rat vas deferens following chronic surgical denervation. 1244 1

Noradrenaline-stimulated phosphoinositide breakdown in cultured glia was found to be mediated by alpha(1A)-adrenoceptors. The alpha(1A)-selective agonist A61603 was as effective as noradrenaline in eliciting 3H-inositol phosphate (IP) accumulation but was approximately 50-fold more potent. In addition, the use of selective antagonists revealed a clear rank order of potency in the ability of these drugs to reverse the effect of noradrenaline on phosphoinositide breakdown: RS17053 (alpha(1A)-selective) >>AH11110A (alpha(1B)-selective)>BMY7378 (alpha(1D)-selective). Pre-treatment of cultured glia with the protein phosphatase inhibitor okadaic acid resulted in a concentration- and time-dependent reduction in noradrenaline-evoked 3H-IP accumulation. This effect was mimicked by, but was not additive with, a phorbol ester, was reversed by protein kinase C (PKC) inhibitors and was not evident in cells which had been PKC depleted. The ability of cell extracts to dephosphorylate radiolabelled glycogen phosphorylase revealed the presence of the phosphatases PP1 and PP2A in almost equal abundance. Okadaic acid pre-treatment of intact cultures elicited a marked reduction in total phosphatase activity, particularly that mediated by PP2A. We also determined the effect of okadaic acid pre-treatment on PKC and cyclic AMP-dependent protein kinase (PKA) activities in these cells. PKC and PKA activities in cell extracts were assessed by determining the incorporation of 32P into histone and kemptide, respectively. Okadaic acid elicited increases in both Ca(2+)-dependent and Ca(2+)-independent PKC activity; in addition, increases in both initial and total PKA activities were also recorded. The effect of okadaic acid on noradrenaline-stimulated 3H-IP accumulation were not, however, mimicked by either forskolin or 8-bromo-cyclic AMP, suggesting that this event is not regulated by PKA. Our data point to roles for both PKC and PP2A in the regulation of alpha(1A)-adrenoceptor-linked phosphoinositide metabolism in cultured cortical glia.
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PMID:Regulation of alpha(1)-adrenoceptor-linked phosphoinositide metabolism in cultured glia: involvement of protein phosphatases and kinases. 1261 15

Norepinephrine (NE) stimulates phospholipase D (PLD) activity and cell proliferation in vascular smooth muscle cells (VSMCs). The objective of this study was to determine the contribution of PKC-zeta to NE-induced PLD activation and cell proliferation in VSMCs. PLD activity was measured by the formation of [3H]phosphatidylethanol in VSMCs labeled with [3H]oleic acid and exposed to ethanol. A high basal PLD activity was detected, and NE increased PLD activity over basal by 70%. This increase was abolished by the broad-range PKC inhibitor Ro 31-8220 (1 micromol/L, 30 minutes) and myristoylated PKC-zeta pseudosubstrate peptide inhibitor (25 micromol/L, 1 hour). Transfection of VSMCs with PKC-zeta antisense, but not sense, oligonucleotides, which reduced PKC-zeta protein level and basal PLD activity, caused a 92% decrease in NE-induced PLD activation. NE-induced increase in PLD activity was also reduced by 61% in cells transfected with kinase-deficient FLAG-T410A-PKC-zeta plasmid but not in those transfected with wild-type PKC-zeta. NE increased immunoprecipitable PKC-zeta activity and phosphorylation, reaching a maximum at 2 and 5 minutes, respectively. NE-induced increase in PKC-zeta activity was inhibited by Ro 31-8220 and by the pseudosubstrate inhibitor. Treatment of VSMCs for 48 hours with PKC-zeta antisense, but not sense, oligonucleotides also inhibited basal and NE-stimulated cell proliferation by 54% and 57%, respectively, as measured by [3H]thymidine incorporation. The inhibitor of PLD activity n-butanol, but not its inactive analog tert-butanol, also reduced the basal and blocked NE-induced cell proliferation. These data suggest that PKC-zeta mediates PLD activation and cell proliferation elicited by NE in rabbit VSMCs.
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PMID:PKC-zeta mediates norepinephrine-induced phospholipase D activation and cell proliferation in VSMC. 1262 98

The effect of exercise training (9 weeks of running) on norepinephrine-induced inhibition of insulin secretion was examined in rat islets. Insulin secretions from islets in the presence of glucose (> or =5.5 mmol/L) were significantly lower in trained (TR) than in control rats (CR). Norepinephrine inhibited 5.5 mmol/L glucose-stimulated insulin secretions and cyclic adenosine monophosphate (cAMP) contents in a dose-dependent manner in CR. Norepinephrine (10 micromol/L)-induced inhibition of insulin secretion was reversed by the blockade of the alpha(2)-adrenergic receptor in CR, but not in TR. Exercise training substantially shifted the dose-dependent curve for clonidine-induced inhibition of insulin secretions and that of cAMP contents to the right. Exercise training did not alter the density of the alpha(2)-adrenergic receptor either per islet or per protein of islet crude membrane. However, exercise training significantly reduced the protein expression of G alpha i-2 without change in G alpha i-2 mRNA. In CR but not in TR, norepinephrine significantly inhibited insulin secretions elicited by a combination of high glucose, a protein kinase C activator, and an adenylate cyclase activator under Ca(2+)-free conditions. Thus, exercise training appears to provoke a decreased expression of G alpha i-2 protein. This, at least in part, results in loss of the inhibitory effect of norepinephrine either on cAMP content or on insulin secretion at the post-calcium events in stimulus-secretion coupling, which, in turn, leads to the blunted inhibitory effects of norepinephrine on insulin secretion.
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PMID:Desensitization of the inhibitory effect of norepinephrine on insulin secretion from pancreatic islets of exercise-trained rats. 1553 96

Incubation of rat pancreatic islets for 4-6 h with 100 micromol/l fatty acid-free BSA induced a 3- to 10-fold enhancement of insulin release to a subsequent challenge with 16.7 mmol/l glucose, without changing the typical biphasic pattern of the response. A similar enhancement was observed with other stimuli, such as leucine, depolarizing concentrations of KCl and tolbutamide, pointing to a general phenomenon and common mechanism for the augmentation. Norepinephrine completely blocked the stimulated response. The protein kinase C (PKC) inhibitor Ro 31-8220, which acts at the ATP-binding site and inhibits all PKC isoforms, strongly inhibited the enhancement of a subsequent glucose challenge when present during the BSA pretreatment period. In contrast, Go 6976, an inhibitor of conventional PKC isoforms, was without effect, even at the high concentration of 1 micromol/l. Preincubation with calphostin C, which competes for the diacylglycerol (DAG)-binding site, therefore inhibiting conventional, novel, and PKC isoforms of the PKD type, completely abolished the enhancing effect of the BSA but did not affect secretion in islets treated with 10 micromol/l fatty acid-free BSA. We conclude that the remarkable enhancement of insulin release is due to a change in glucose signaling and activation of a novel PKC isoform or a DAG-binding protein.
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PMID:Massive augmentation of stimulated insulin secretion induced by fatty acid-free BSA in rat pancreatic islets. 1556 45

We determined the contribution of the Rho family of low molecular GTP-binding proteins to phorbol ester-induced contraction in swine pulmonary artery smooth muscle. In Ca2+-free medium containing 1 mM EGTA, 12-deoxyphorbol 13-isobutyrate (DPB, 1 microM), a protein kinase C (PKC) activator, elicited sustained contractions, which were not inhibited by treatment with verapamil, a voltage-dependent Ca2+ channel antagonist, and Y27632, a Rho-associated kinase inhibitor. Immunoblot analysis showed three PKC isoforms (alpha, epsilon, and zeta) and two Rho GTPases (RhoA and Cdc42) in both cytosolic and the membrane fractions from quiescent strips. DPB (1 microM) significantly induced PKCalpha and epsilon to translocate from the cytosolic to the membrane fraction in Ca2+-free medium. DPB also elicited the translocation of Cdc42, but not RhoA to the membrane fraction. Similarly, in the experiment for measurement of Rho GTPase activity by pull-down assay, DPB (1 microM) significantly increased the activity of Cdc42 in Ca2+-free medium. Norepinephrine (NE, 10 microM) stimulated the redistribution of RhoA from the cytosolic to the membrane fraction in swine pulmonary artery smooth muscle. In contrast, NE did not alter the subcellular distributions of Cdc42 and the PKC isoforms. These results indicate that phorbol ester evokes PKC-mediated Ca2+-independent contraction via a Rho GTPase pathway, especially Cdc42, in smooth muscle from swine pulmonary arteries.
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PMID:Cdc42 contributes to phorbol ester-induced Ca2+-independent contraction of pulmonary artery smooth muscle. 1614 65

Properties of repetitive firing, including spike adaptation, are considered to play an essential role in controlling neural excitability in the central nervous system. Noradrenaline is one of major neurotranmitters that modulate repetitive firing in the cerebral cortex. Although activation of beta-adrenoceptors increases firing frequency similarly to noradrenaline, it is still controversial whether alpha(1)-adrenoceptor activation influences repetitive firing. In the present study, we examined the effects of adrenoceptor agonists on firing properties and the intracellular mechanism for alpha(1)-adrenoceptor-dependent modulation of firing in pyramidal neurons of rat cerebral cortex. In agreement with previous reports, bath application of 100microM isoproterenol, a beta-adrenoceptor agonist, increased firing frequency in response to a long intracellular depolarizing current injection. Phenylephrine (100microM), an alpha(1)-adrenoceptor agonist, also increased firing rate, which was inhibited by 100microM prazosin, an alpha1-adrenoceptor antagonist. The extent of increment in firing rate is comparable to that induced by isoproterenol. Furthermore, phenylephrine's effects on firing properties were mimicked by 2-5microM phorbol ester, a protein kinase C (PKC) activator, and pre-application of 10microM chelerythrine, a PKC inhibitor, prevented phenylephrine-induced facilitation of repetitive firing. These results suggest that phenylephrine has a facilitatory effect on repetitive firing through PKC activation.
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PMID:Activation of alpha1-adrenoceptors increases firing frequency through protein kinase C in pyramidal neurons of rat visual cortex. 1806 48

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