EC:2.7.11.13 - FACTA Search

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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)

The suppressive action of carbachol (CCh) on the Ca2+ current (ICa) in smooth muscle cells of the guinea-pig urinary bladder was investigated using the whole-cell patch clamp technique. Bath application of 10 microM CCh reduced the amplitude of ICa by 92 +/- 3.8% (n = 9). Adding 1 microM atropine to the bath completely blocked the action of CCh, indicating that the suppressive action of CCh on ICa is mediated by the activation of muscarinic receptors. Intracellular perfusion of the non-hydrolysable GTP analogue, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S; 200 microM) mimicked the effects of CCh. Sustained suppression of ICa was observed when GTP gamma S was present in the cytoplasm. Intracellular perfusion of inositol 1,4,5-trisphosphate (InsP 3; 20 microM) also suppressed ICa; its effect was not sustained but transient. The protein kinase C activator, phorbol 12,13-dibutyrate (PDBu), however, could not mimic the effects of CCh on ICa. When intracellular Ca2+ was strongly buffered by the Ca2+ chelator EGTA (20 mM) in the patch pipette, the sustained suppression of ICa was abolished. Inclusion of 3 mg/ml heparin, a blocker of InsP3-induced Ca2+ release, in the patch pipette reduced the degree of sustained ICa suppression by 43.2 +/- 1.9% (n = 7). Adding thapsigargin (TG), a sarcoplasmic reticulum Ca2+-ATPase inhibitor, to a wash solution reduced the recovery of ICa by about 50%, suggesting that approximately half of the ICa suppression induced by CCh is due to Ca2+ release from TG-sensitive internal Ca2+ stores. From these results it appears that CCh suppresses ICa via two independent mechanisms: (1) Ca(2+)-mediated inactivation of the Ca2+ channel, which is caused by Ca2+ release from InsP3- and TG-sensitive internal stores, and (2) a GTP-binding protein-mediated mechanism, which requires intracellular Ca2+.
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PMID:Muscarinic suppression of Ca2+ current in smooth muscle cells of the guinea-pig urinary bladder. 757 97

The phosphorylation state of cp20, a low molecular weight membrane-associated GTP-binding protein, was previously shown to increase two- to threefold 24 h after associative conditioning. Here, cp20 is shown to be phosphorylated by protein kinase C (PKC) in vitro. Pronounced differences in activity were observed with the three major isoforms of PKC, whereas casein kinase, calcium/calmodulin-dependent protein kinase II, and cyclic AMP-dependent protein kinase produced no detectable phosphorylation of cp20. Phosphorylation of cp20 had no effect on its GTPase or GTP-binding activity but caused a translocation of cp20 from cytosol to the nuclei/mitochondrial particulate fraction. These results suggest that the increase in phosphorylation of cp20 after conditioning may be due to PKC.
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PMID:Phosphorylation of the conditioning-associated GTP-binding protein cp20 by protein kinase C. 759 25

We studied the involvement of protein kinase C (PKC) and a small GTP-binding protein (G-protein), rho, in receptor-mediated Ca2+ sensitization of the contractile apparatus of smooth muscle of guinea pig vas deferens. In beta-escin-permeabilized smooth muscle strips, norepinephrine (NE) in the presence of GTP caused further contraction of the preparations at a constant Ca2+ level (Ca2+ sensitization). Prazosin and GDP beta S, a nonhydrolyzable GDP analogue, inhibited NE-induced Ca2+ sensitization, indicating an alpha-1 adrenoceptor/G-protein mediated response. GTP alone (> 10 microM) and GTP gamma S, a non-hydrolyzable GTP analogue, also induced Ca2+ sensitization. Pretreatment of preparations with C3 exoenzyme of Clostridium botulinum, which is known to ADP-ribosylate rho family proteins, with NAD resulted in complete inhibition of NE- and GTP (GTP gamma S)-induced Ca2+ sensitization. AIF4-, which activates heterotrimeric G-, but not small G-protein also induced Ca2+ sensitization. Interestingly, AIF4(-)-induced Ca2+ sensitization was inhibited by not only GDP beta S but also C3-treatment, suggesting that activation of heterotrimeric GTP-binding protein precedes activation of rho protein. On the other hand, phorbol 12,13-dibutyrate, like NE, also induced Ca2+ sensitization. The sensitization was inhibited by PKC(19-31), a PKC inhibitor peptide. However, PKC(19-31) did not have any effect on NE- or AIF4(-)-induced Ca2+ sensitization.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Involvement of heterotrimeric GTP-binding protein and rho protein, but not protein kinase C, in agonist-induced Ca2+ sensitization of skinned muscle of guinea pig vas deferens. 761 45

Phosphatidylcholine-specific phospholipase D (PLD) is an important signalling phospholipase in mammalian cells. Recently, PLD activity has been shown to be positively regulated by the GTP-binding protein ARF (ADP-ribosylating factor). In the present work, we document the presence of a factor negatively regulating PLD activity in bovine brain cytosol. The inhibitory factor is characterized as a large protein or a complex of proteins with a molecular mass higher than 300 kDa. Using permeabilized and pre-permeabilized HL-60 cells depleted of their cytosol, we demonstrate that the inhibitor acts on GTP[S]-stimulated PLD activity. This effect is immediate, persistent and dose dependent for GTP[S]-stimulated PLD. Different possibilities for a mechanism of action of the inhibitory factor on the regulation of GTP binding of ARF were investigated. This inhibitory factor is not the guanine-dissociating inhibitor (GDI) for the small G-binding proteins Rho (Rho-GDI), reported to be a PLD inhibitor, since specific antibodies against this protein did not recognize a protein in the peak containing the inhibitory factor for PLD activity. Furthermore, the inhibitory factor does not prevent the binding of GTP[S] to ARF in the presence of HL-60 membranes. This excludes its possible role as an inhibitor of an ARF/guanine exchange factor. The inhibitory factor not only inhibits a pathway of PLD through GTP[S] activation in particular of the small GTP-binding protein, ARF, but it also inhibits PLD activated via either protein kinase C (PKC) or tyrosine kinase activation. The inhibitory factor also decreases PLC activity and this effect seems to be secondary to the inhibition of PLD activity. We discuss a mechanism of action of the inhibitor on PLD and the importance of this enzyme activity for membrane traffic.
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PMID:A soluble protein negatively regulates phospholipase D activity. Partial purification and characterization. 762 81

The mechanisms of alpha 1-adrenoceptor agonist-mediated sensitization of the contractile apparatus of smooth muscle to Ca2+ were studied in beta-escin-permeabilized thoracic arterial smooth muscle of rabbit. Addition of norepinephrine (10 microM) plus guanosine 5'-triphosphate (GTP, 50 microM) significantly enhanced Ca2+ sensitivity as compared with the addition of 0.3 microM Ca2+ alone (pCa6.5). In beta-escin-skinned smooth muscle of chloroethylclonidine-treated tissues, the enhancement of Ca(2+)-contraction produced by norepinephrine or clonidine was completely inhibited by guanosine 5'-O-(beta-thiodiphosphate) (GDP beta-S, 1 mM). In addition, Clostridium botulinum C3, which inactivates low molecular weight GTP-binding protein families, abolished norepinephrine- or clonidine-induced Ca(2+)-sensitization, but did not affect clonidine-induced translocation of protein kinase C to the membrane. The norepinephrine-enhanced Ca2+ sensitivity was partially reversed by a pretreatment with a selective myosin light chain kinase inhibitor (8R*, 9S*, 11S*)-(-)-9-hydroxy-9-methoxycarbonyl-8-methyl-14-n- propoxy-2,3,9,10-tetrahydro-8,11-epoxy,1H,8H,11H-2,7b,11a- triazadibenzo[a,g]cycloocta[cde]trinden-1-one (KT5926, 500 nM), but those of clonidine and in the chloroethylclonidine-treated tissues norepinephrine were not. These results suggest that Ca(2+)-sensitization produced by the activation of the alpha 1-adrenoceptor subtypes is linked via a low molecular weight GTP-binding protein (Rho), and the regulations of phosphorylation in contractile elements.
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PMID:Involvement of botulinum C3-sensitive GTP-binding proteins in alpha 1-adrenoceptor subtypes mediating Ca(2+)-sensitization. 766 21

1. The mouse AtT-20/D16-16 anterior pituitary tumour cell line was used as a model system for the study of protein kinase C (PKC)-mediated enhancement of calcium- and guanine nucleotide-evoked adrenocorticotrophin (ACTH) secretion. 2. A profile of the PKC isozymes present in AtT-20 cells was obtained by Western blotting analysis and it was found that AtT-20 cells express the alpha, beta, epsilon and zeta isoforms of PKC. 3. PKC isozymes were activated by the use of substances reported to activate particular isoforms of the enzyme. The effects of these substances were investigated in both intact and electrically-permeabilized cells. Phorbol 12-myristate 13-acetate (PMA, EC50 = 1 +/- 0.05 nM, which activates all isozymes of PKC, except the zeta isozyme), thymeleatoxin (TMX, EC50 = 10 +/- 0.5 nM, which activates the alpha, beta and gamma isozymes) and 12-deoxyphorbol 13-phenylacetate 20-acetate (dPPA, EC50 = 3 +/- 0.5 nM, a beta 1-selective isozyme activator) all stimulated ACTH secretion from intact cells in a concentration-dependent manner. Maximal TMX stimulated ACTH secretion was of a similar degree to that obtained in response to PMA but maximal dPPA-stimulated ACTH secretion was only 60-70% of that obtained in response to PMA or TMX. 4. Calcium stimulated ACTH secretion from electrically-permeabilized cells over the concentration-range of 100 nM to 10 microM. PMA (100 nM), TMX (100 nM) but not dPPA (100 nM) enhanced the amount of ACTH secreted at every concentration of calcium investigated. PMA (100 nM) and TMX (100 nM)significantly enhanced ACTH secretion in the effective absence of calcium (i.e. where the free calcium concentration is nM).5. GTP-gamma-S stimulated ACTH secretion from permeabilized cells in a concentration-dependent manner with a threshold of 1 micro M. PMA (100 nM), TMX (100 nM) but not dPPA (100 nM) increased the amount of ACTH secretion evoked by every concentration of GTP-gamma-S investigated.6. The PKC inhibitor, chelerythrine chloride (10 micro M), blocked the PMA (100 nM)-evoked enhancement of calcium- and GTP-micro-S-stimulated ACTH secretion but did not significantly alter calcium- or GTP-micro-S-evoked secretion itself.7. The present paper indicates that AtT-20 cells express multiple isoforms of PKC and that these act at different sites in the secretory pathway for ACTH secretion. The alpha and epsilon isozymes of PKC can act distal to calcium entry to modulate the ability of increased cytosolic calcium concentrations to stimulate ACTH secretion. This site of action is either at the level of, or at some stage distal to, a GTP-binding protein which mediates the effects of calcium upon ACTH secretion. The beta isozyme of PKC may act ata stage early in the secretory pathway to regulate the cytosolic calcium concentration.
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PMID:Involvement of multiple protein kinase C isozymes in the ACTH secretory pathway of AtT-20 cells. 767 Jul 32

1. Effects of cholinergic agonists on the cultured chick cochlear ganglion (CG) neurone were examined using the whole-cell patch-clamp method. 2. Acetylcholine (ACh, 0.1-100 microM) and its non-hydrolysable form, carbamylcholine (CCh, 0.1-300 microM), suppressed the outward current. The CCh-sensitive current was activated at membrane potentials more positive than -70 mV. 3. The CCh-sensitive current slowly activated after step depolarization with a time constant from 20 to 150 ms. The activation time constant decreased monotonically with depolarization of the membrane. 4. The reversal potential of CCh-sensitive current changed as a function of the external K+ concentration (-79, -65 and -44 mV in 5, 10 and 25 mM, respectively) and was approximately equal to the potassium equilibrium potential (-89, -71 and -48 mV in 5, 10 and 25 mM, respectively). The CCh-sensitive current is concluded to be K+ selective. 5. The CCh-sensitive current showed a sigmoid log dose vs. response relationship with an apparent dissociation constant (KD) of 1.4 microM and a Hill coefficient of 1.0. When ACh was applied, an apparent KD of 1.8 microM and a Hill coefficient of 1.0 was measured. 6. The suppression of K+ current by CCh was blocked by atropine (3 microM) and pirenzepine (3 microM), suggesting that the current is mediated by an M1 muscarinic receptor. 7. The CCh suppression of the K+ current was enhanced by GTP-gamma-S (0.1 mM), suggesting that a GTP-binding protein is involved. 8. The CCh suppression of the K+ current was mimicked by protein kinase C activators, 1-oleoyl-2-acetyl-sn-glycerol (OAG, 100 microM), phorbol dibutyrate (PDBu, 2 microM) and phorbol 12-myristate 13-acetate (PMA, 1 microM). The protein kinase inhibitor, staurosporine (0.2 microM) applied internally blocked the CCh suppression of the K+ current which suggests an involvement of protein kinase C.
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PMID:Suppression of the slow K+ current by cholinergic agonists in cultured chick cochlear ganglion neurones. 769 17

The role of mitogen-activated protein (MAP) kinase in the release of arachidonic acid was examined in a mutated mast cell (RBL-2H3(m1)) line that expressed both native Fc epsilon R1 and the G protein-coupled muscarinic m1 receptor. Stimulation of these cells with Ag, carbachol, Ca(2+)-ionophore, or thapsigargin resulted in the phosphorylation of Raf1, MEK1, p42mapk MAP kinase, and the recently cloned cytosolic phospholipase A2 (PLA2) and increased activities of both MAP kinase and PLA2, as well as release of arachidonic acid. Because this cascade of reactions was inhibited by guanosine 5'-(2-thiodiphosphate), it appeared to be dependent on a GTP-binding protein(s). These reactions, however, were not dependent on protein kinase C; the cascade was totally resistant to the actions of a selective protein kinase C inhibitor, Ro31-7549, whereas release of the secretory granule marker, hexosaminidase, was blocked by this agent. Differences between the stimulatory pathways for release of arachidonic acid and hexosaminidase were evident also from the effects of the kinase inhibitor, quercetin. The above cascade of reactions, including release of arachidonic acid, was inhibited by 50% with approximately 5 microM quercetin, whereas secretion was inhibited only at higher concentrations of inhibitor. Moreover, inhibition of the activation of MAP kinase and release of arachidonic acid were closely correlated. This and previous findings suggested that release of arachidonic acid was attributable to the regulation of cytosolic PLA2 by MAP kinase (for activation of PLA2) and Ca2+ (for association of PLA2 with the membrane), whereas release of hexosaminidase was regulated primarily by Ca2+ and protein kinase C.
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PMID:Activation of the mitogen-activated protein kinase/cytosolic phospholipase A2 pathway in a rat mast cell line. Indications of different pathways for release of arachidonic acid and secretory granules. 773 Jun 40

Inositol 2,4,5-trisphosphate irreversibly activated capacitative calcium entry in Xenopus oocytes, whereas guanosine thiotriphosphate (GTP[S]) and AIF4- only activated capacitative calcium entry transiently. Both GTP[S] and AIF4- inhibited capacitative calcium entry activated by thapsigargin pretreatment, but guanosine thiodiphosphate (GDP[S]), inositol 2,4,5-trisphosphate and dibutyryl cyclic GMP did not affect capacitative calcium entry. This suggests the involvement of heterotrimeric GTP-binding proteins in the regulation of capacitative calcium entry. Activation of protein kinase C or cyclic-AMP-dependent protein kinase had profound effects on capacitative calcium entry, which were consistent with the hypothesis that the effects of GTP[S] and AIF4- on capacitative calcium entry may be mediated via heterotrimeric GTP-binding protein stimulation of kinases. Further evidence for this hypothesis was derived from the result that the effects of GTP[S] on calcium entry could be inhibited by the application of the protein kinase inhibitor staurosporine.
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PMID:G-protein regulation of capacitative calcium entry may be mediated by protein kinases A and C in Xenopus oocytes. 774 94

It has been reported that glucocorticoid modifies phosphoinositide (PI) hydrolysis stimulated by vasoactive agents in vascular smooth muscle cells. In the present study, we investigated the point at which glucocorticoid affects vasopressin-induced PI hydrolysis in primary cultured rat aortic smooth muscle cells. The pretreatment with dexamethasone significantly amplified the formation of inositol trisphosphate (IP3) induced by vasopressin in a dose-dependent manner in a range of 1 pM to 10 nM. The effect of dexamethasone was dependent on the time of pretreatment up to 8 h. Dexamethasone had little effect on the number of vasopressin receptor and its affinity to vasopressin. The pretreatment with dexamethasone also amplified the formation of IP3 induced by NaF, a GTP-binding protein activator, or angiotensin II. 12-O-Tetradecanoylphorbol-13-acetate, a protein kinase C (PKC)-activating phorbol ester, significantly reduced the dexamethasone-induced enhancement of IP3 formation stimulated by vasopressin, angiotensin II or NaF 4 alpha-Phorbol-12, 13-didecanoate, a PKC-nonactivating phorbol ester, had little effect on the enhancement by dexamethasone. These results strongly suggest that glucocorticoid amplifies vasopressin-induced PI hydrolysis at a point downstream from GTP-binding protein in primary cultured rat aortic smooth muscle cells, and that the activation of PKC has a negative feedback effect on the amplification by glucocorticoid of vasopressin-induced PI hydrolysis.
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PMID:Glucocorticoid amplifies vasopressin-induced phosphoinositide hydrolysis in aortic smooth muscle cells. 776 86

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