Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.4.3 (phospholipase C)
18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The purpose of this study was to visualize muscarinic receptors and their distribution on cardiomyocytes and to examine the effects of muscarinic cholinergic receptor (mACh-R) stimulation with carbachol on phosphatidylcholine hydrolysis. Cardiomyocytes were prepared as primary culture from 7-day-old chick embryo hearts. Cardiomyocytes, grown on cover slips, were labelled with BODIPY PZ, a fluorescent analog of the muscarinic receptor antagonist pirenzepine, and examined with a laser scanning confocal microscope, mACh-R clusters were visualized and were fairly homogeneous in size with diameters ranging from 0.5 to 1.0 micron. The number of receptor clusters per cell was 83.5 +/- 6.8 (mean +/- SEM) and clusters were found at the periphery of the cell. Cardiomyocytes, grown as a monolayer in dishes, were treated with the 10(-4) M carbachol, a mACh-R agonist, and the effects on phosphatidylcholine hydrolysis were ascertained in cells preincubated with [methyl-3H]choline for 18 h. Cells were washed, lysed, and subjected to thin-layer chromatography to separate [3H]choline in various metabolites of phosphatidylcholine. Carbachol significantly (p < 0.05) increased intracellular free choline and decreased cellular phospholipid consistent with phosphatidylcholine hydrolysis. Carbachol increased the amount of [3H]choline that effluxed out of the cardiomyocyte into the medium. Carbachol-induced choline efflux was not prevented by pretreatment with n-butanol, a phospholipase D inhibitor, suggesting that other lipases such as phospholipase C are the major enzyme involved in phosphatidylcholine hydrolysis. Pertussis toxin prevented carbachol-induced choline efflux and the changes in intracellular free choline and phospholipid. An action of carbachol through G proteins was supported by the ability of pertussis toxin to antagonize the carbachol-induced reduction in cAMP generation from isoproterenol. In summary, mACh-Rs, visualized in living cardiomyocytes, were peripheral to the nucleus. Phosphatidylcholine hydrolysis induced by mACh-R stimulation may be a signal transduction pathway for mACh-R in the cardiomyocyte, operating through inhibitory G proteins sensitive to pertussis toxin.
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PMID:Visualization of muscarinic cholinergic receptors on chick cardiomyocytes and their involvement in phosphatidylcholine hydrolysis. 925 Mar 60

Carbachol can stimulate insulin release in RINm5F cells by a mechanism that does not involve the elevation of cytosolic free Ca2+ concentrations or the activation of conventional protein kinase Cs (Mol Pharmacol 47:863-870, 1995). Thus, a novel signal transduction pathway links the muscarinic activation of the cells to increased insulin secretion. The question arises as to whether the pathway results from a novel receptor, different from the five established muscarinic receptors, or whether a "normal" receptor in the RINm5F cell activates a novel pathway. To distinguish between these two possibilities, the muscarinic receptors in the RINm5F cell were identified. Using polymerase chain reaction, combined with subcloning and DNA sequencing techniques, the cDNAs that encode the established M3 and M4 receptors were identified. The cDNAs for the Ml, M2, and M5 receptors were not found. Pharmacological studies showed a rank order of potency for muscarinic receptor subtype antagonists to inhibit carbachol-induced insulin release (half-maximal inhibitory concentration [pIC50] values given in parentheses): atropine (nonselective, 9.0) > 4-diphenyl-acetoxy-N-methyl piperidine methiodide (M3/M1, 8.6) > para-fluoro-hexahydrosiladiphenidol (M3, 8.1) > hexahydrosiladiphenidol (M3, 8.0) > tropicamide (M4, 6.4) > pirenzepine (M1, 6.1) > methoctramine (M2, 5.9). This antagonist profile suggests that it is the M3 receptor that mediates carbachol-induced insulin release. In this case, the novel signaling involved in the unusual carbachol response would not be due to a novel receptor but to the well-characterized M3 receptor. It appears, therefore, that the novel portion of the signaling pathway lies downstream of the M3 receptor and may consist of products of phosphatidylinositol hydrolysis, other than inositol triphosphate and diacylglycerol, resulting from the activation of phospholipase C. While a contributory role of the M4 receptor cannot be ruled out, there is no evidence in its favor other than its presence in the cell.
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PMID:Identification of muscarinic receptor subtypes in RINm5F cells by means of polymerase chain reaction, subcloning, and DNA sequencing. 928 41

In ileal absorptive cells, carbachol inhibits NaCl absorption and its component brush border Na+/H+ exchanger, acting via basolateral membrane receptors. This carbachol effect involves (i) activation of brush border phosphatidylinositol 4,5-bisphosphate-specific phospholipase C (PLC) activity and brush border but not basolateral membrane translocation of PLC-gamma1 (Khurana, S., Kreydiyyeh, S., Aronzon, A., Hoogerwerf, W. A., Rhee, S. G., Donowitz, M., and Cohen, M. E. (1996) Biochem. J. 313, 509-518); and (ii) brush border tyrosine kinase(s) because mucosal but not serosal addition of the tyrosine kinase inhibitor genistein prevents the carbachol-induced inhibition of NaCl absorption and brush border Na+/H+ exchange. In the present work we identify a pool of villin (a brush border actin-binding protein) in the microvillus membrane fraction of rabbit ileum; this pool of villin is tyrosine-phosphorylated and associates with brush border membrane PLC-gamma1. Villin is present both in the Triton X-100-soluble and -insoluble fractions of the brush border. The Triton X-100-soluble pool is approximately 4-fold smaller than the brush border pool of villin that is present in the Triton X-100-insoluble fraction. Only the villin present in the Triton X-100-soluble fraction of ileal villus brush border associates with PLC-gamma1 and is tyrosine-phosphorylated. Carbachol increases the tyrosine phosphorylation of villin rapidly (as early as 30 s) and transiently. Carbachol also increases the amount of tyrosine-phosphorylated villin that associates with PLC-gamma1. These studies demonstrate that carbachol effects on NaCl absorption are accompanied by an increase in brush border PLC-gamma1 association with villin and an increase in tyrosine phosphorylation of villin. To study the role of cytoskeletal rearrangement in carbachol-induced inhibition of NaCl absorption, we used the F-actin stabilizing drug jasplakinolide. Jasplakinolide prevents the carbachol inhibition of ileal NaCl absorption. This suggests that F-actin severing is necessary for carbachol to inhibit ileal villus NaCl absorption. Since villin is known to sever actin, these studies suggest a role for villin in the signaling cascade that begins at the basolateral membrane with carbachol binding to its receptor and ends at the apical membrane in inhibition of NaCl absorption.
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PMID:Ileal microvillar protein villin is tyrosine-phosphorylated and associates with PLC-gamma1. Role of cytoskeletal rearrangement in the carbachol-induced inhibition of ileal NaCl absorption. 937 90

Muscarinic-cholinergic signals in brain are mediated in part through the hydrolysis of phosphoinositides (PtdIns) by phospholipase C (PLC). To test the hypothesis that muscarinic PtdIns signals change during aging, membranes were prepared from the cerebral cortex and hippocampus of young (4-6 months old), middle aged (8-10 months old) and senescent (24-26 months old) Fisher 344 rats. Carbachol dose-dependently increased [3H]-PtdIns hydrolysis in both brain regions in all three age groups, however, in senescent rats the maximal response was decreased to 69.26 +/- 4.33% (p < 0.01) in cortex and to 48.29 +/- 2.55% (p < 0.01) in hippocampus of young rat values. In contrast to the decrease in carbachol-stimulated phosphoinositide hydrolysis, calcium-stimulated phosphoinositide hydrolysis was not altered. GTP gamma S also dose-dependently increased [3H]-PtdIns hydrolysis in membranes from all three age groups through G-protein-PLC activation. Similar to carbachol, GTP gamma S-activated [3H]-PtdIns hydrolysis was reduced approximately 40% in senescent rats membranes. Muscarinic receptor (mAChR) density, as determined by [3H]-QNB binding decreased slightly in cortical membranes, but not in hippocampal membranes. These data suggest that muscarinic stimulated [3H]-PtdIns responses are decreased in senescent brain primarily due to an uncoupling of the receptor-G-protein and/or G-protein-PLC link, although decreases in receptor density may also contribute to reduced muscarinic [3H]-PtdIns signaling.
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PMID:Uncoupling of muscarinic cholinergic phosphoinositide signals in senescent cerebral cortical and hippocampal membranes. 946 Jul 9

Oxidative stress appears to contribute to neuronal dysfunction associated with Alzheimer's disease and other CNS neurodegenerative disorders. This investigation examined if oxidative stress might contribute to impairments in cholinergic receptor-linked signaling systems and if intracellular glutathione levels modulated responses to oxidative stress. To do this the activation of the AP-1 and NF-kappaB transcription factors and of the phosphoinositide second-messenger system was measured in human neuroblastoma SH-SY5Y cells after exposure to the oxidants H2O2 or diamide, with or without prior depletion of cellular glutathione. H2O2 concentration-dependently inhibited carbachol-stimulated AP-1 activation and this inhibition was potentiated in glutathione-depleted cells. Carbachol-stimulated NF-kappaB activation was unaffected by H2O2 unless glutathione was depleted, in which case there was a H2O2 concentration-dependent inhibition. Glutathione depletion also potentiated the inhibition by H2O2 of carbachol- or G-protein (NaF)-stimulated phosphoinositide hydrolysis, whereas phospholipase C activated by the calcium ionophore ionomycin was not inhibited. The thiol-oxidizing agent diamide also inhibited phosphoinositide hydrolysis stimulated by carbachol or NaF, and glutathione depletion potentiated the diamide concentration-dependent inhibition. Unlike H2O2, diamide also inhibited ionomycin-stimulated phosphoinositide hydrolysis. Activation of both AP-1 and NF-kappaB stimulated by carbachol was inhibited by diamide, and glutathione depletion potentiated the inhibitory effects of diamide. Thus, diamide inhibited a wider range of signaling processes than did H2O2, but glutathione depletion increased the susceptibility of phosphoinositide hydrolysis and of transcription factor activation to inhibition by both H2O2 and diamide. These results demonstrate that the vulnerability of signaling systems to oxidative stress is influenced by intracellular glutathione levels, indicating that cell-selective susceptibility to inhibition of signal transduction systems by oxidative stress can arise from cellular variations in antioxidant capacity.
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PMID:Glutathione depletion exacerbates impairment by oxidative stress of phosphoinositide hydrolysis, AP-1, and NF-kappaB activation by cholinergic stimulation. 947 71

We observed that AP-3, an antagonist of metabotropic glutamate receptors, reduced carbachol-induced hydrolysis of phospholipids in hippocampal slices. This inhibition could be explained in different ways, e.g.: 1) AP-3 acts also as antagonist of muscarinic receptors mediating the hydrolysis of phospholipids induced by carbachol, 2) Carbachol induces the release of glutamate which, by activating metabotropic glutamate receptors, leads to additional hydrolysis of phospholipids. The aim of this work was to test these possibilities. It is shown that AP-3 reduces carbachol-induced hydrolysis of phospholipids in hippocampal slices but not in cerebellar neurons at 10-14 days of culture, when these cells are not able to induce hydrolysis of phospholipids following activation of metabotropic glutamate receptors. It is also shown that carbachol induces a release of [3H]aspartate in hippocampal slices. The results reported suggest that the hydrolysis of phospholipids induced by carbachol in hippocampal slices would have two components. One part would be due to direct activation by carbachol of muscarinic receptors associated to activation of phospholipase C. This part would not be inhibited by AP-3. The second part would be due to subsequent release of glutamate and activation of metabotropic glutamate receptors. This part would be inhibited by AP-3.
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PMID:Carbachol-induced hydrolysis of phospholipids in hippocampal slices may be mediated in part by subsequent activation of metabotropic glutamate receptors. 958 Mar 90

1. The purpose of the present study was to explore the different mechanisms of [Ca2+]i oscillations induced by high concentrations of either carbachol (CCh) or extracellular Ca2+ ([Ca2+]o). First, we compared the oscillations induced by CCh at concentrations of 100-300 micromol/L and [Ca2+]o (5 mmol/L) in the single rat ventricular myocyte. Second, we studied CCh- and [Ca2+]o-induced [Ca2+]i oscillations following either interference with the production of inositol trisphosphate (IP3), reductions in cytosolic Ca2+ ([Ca2+]i), inhibition of Ca2+ influx and Na+-Ca2+ exchange or depletion of Ca2+ from its intracellular store. 2. The [Ca2+]i oscillations induced by CCh were frequent and were superimposed on [Ca2+]i transients in electrically stimulated cells, whereas those induced by high [Ca2+]o were occasional and occurred in quiescent cells and between [Ca2+]i transients in electrically stimulated cells. In both cases, [Ca2+]i oscillations were preceded by an increase in resting levels of [Ca2+]i. 3. Carbachol-induced [Ca2+]i oscillations were accompanied by an increase in amplitude and prolongation of the time of decline to 80% of the peak of the [Ca2+]i transient, while high [Ca2+]o-induced [Ca2+]i oscillations were the opposite. 4. A reduction of [Ca2+]o to 0.1 mmol/L and treatment with Ni2+ or ryanodine or 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid AM (BAPTA-AM) abolished the [Ca2+]i oscillations induced by both CCh and high [Ca2+]o. 5. The calcium channel blockers verapamil and nifedipine and inhibitors of phospholipase C (neomycin and U-73122) abolished the [Ca2+]i oscillations induced by CCh; Li+ accelerated the onset of the [Ca2+]i oscillations induced by CCh. 6. These observations suggest that the mechanisms responsible for the [Ca2+]i oscillations induced by CCh and high [Ca2+]o are different from each other. Other than an increase in extracellular Ca2+ influx as a mechanism common for both CCh- and high [Ca2+]o-induced [Ca2+]i oscillations, the CCh-induced [Ca2+]i oscillations involve influx of Ca2+ via L-type Ca2+ channels, Na+-Ca2+ exchange, mobilization of intracellular Ca2+ and IP3 production.
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PMID:Different mechanisms for [Ca2+]i oscillations induced by carbachol and high concentrations of [Ca2+]o in the rat ventricular myocyte. 959 May 79

Carbachol-stimulated insulin release in the RINm5F cell is associated with elevation of the cytosolic Ca2+ concentration ([Ca2+]i) through mobilization of Ca2+ from thapsigargin-sensitive intracellular stores and with the generation of diacylglycerol (DAG). Thus carbachol activates phospholipase C, and this was thought to be the means by which it stimulates insulin secretion. However, when the elevation of [Ca2+]i was blocked by thapsigargin, the effect of carbachol to stimulate insulin release was unchanged. Thus the effect of carbachol to increase [Ca2+]i was dissociated from the stimulation of release. When the role of protein kinase C (PKC) was examined, carbachol-stimulated insulin release was found to be unaffected by phorbol ester-induced downregulation of PKC, using 12-O-tetradecanoylphorbol-13-acetate (TPA), and by the PKC inhibitors staurosporine, bisindolylmaleimide, and 1-O-hexadecyl-2-O-methylglycerol (AMG-C16). These treatments abolished the stimulation of release by TPA. Thus the carbachol activation of PKC appeared also to be dissociated from the stimulation of insulin release. However, when the activation of several different PKC isozymes was studied, an atypical PKC isozyme, zeta, was found to be translocated by carbachol. By Western blotting analysis, carbachol selectively translocated the conventional PKC isozymes alpha and beta (the activation of which is dependent on Ca2+ and DAG) from the cytosol to the membrane. Carbachol also translocated the atypical PKC isozyme zeta, which is insensitive to Ca2+, DAG, and phorbol esters. The PKC inhibitors staurosporine, bisindolylmaleimide, and AMG-C16 blocked the stimulated translocation of PKC-alpha and -beta, but not that of PKC-zeta. Prolonged treatment of the cells with TPA downregulated PKC-alpha and -beta, but not PKC-zeta. Under all these conditions, carbachol-stimulated insulin release was unaffected. However, a pseudosubstrate peptide inhibitor specific for PKC-zeta inhibited the translocation of PKC-zeta and 70% of the carbachol-stimulated insulin secretion. The data indicate that carbachol-stimulated insulin release in RINm5F cells is mediated to a large degree by the activation of the atypical PKC isozyme zeta.
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PMID:Atypical protein kinase C isozyme zeta mediates carbachol-stimulated insulin secretion in RINm5F cells. 960 67

1. Whole-cell patch-clamp recordings were made from smooth muscle cells isolated from the longitudinal muscle layer of guinea-pig ileum. Carbachol (acting at muscarinic receptors) or histamine (acting at H1 histamine receptors) suppressed Ca2+ channel current. The effect of either agonist had an initial transient component followed by a sustained component. 2. Wortmannin inhibited transient and sustained components of carbachol-induced Ca2+ channel current suppression: half-effective inhibitory concentrations (IC50) were 1.1 microM and 0.6 microM for the two components respectively. Wortmannin also inhibited the transient phase of carbachol-induced cationic current (IC50 1.6 microM) and Ca2+-dependent K+-current (IC50 1.7 microM). Wortmannin did not appear to produce any direct block of cationic channels or Ca2+ channels. 3. Intracellular application of the phospholipase inhibitor D609 (tricyclodecan-9-ylxanthogenate) inhibited transient and sustained components of histamine action on the Ca2+ channel current: the IC50 was about 130 microM for both components. Carbachol action on Ca2+ channels was also inhibited by D609. D609 had no significant direct blocking effect on Ca2+ channels, cationic channels activated by carbachol, or Ca2+-activated K+-current in response to flash-photolysis of caged-inositol 1,4,5-trisphosphate. 4. Micromolar concentrations of wortmannin and D609 are inhibitors of both components of spasmogen-induced Ca2+ channel suppression. The data suggest that both components are mediated by a common, or similar, signal transduction element which is a phospholipase C (PLC) or phospholipase D (PLD) isoform.
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PMID:Inhibitors of spasmogen-induced Ca2+ channel suppression in smooth muscle cells from small intestine. 983

The involvement of protein kinase C (PKC) and protein kinase A (PKA) in cholinergic signalling in CHO cells expressing the M3 subtype of the muscarinic acetylcholine receptor was examined. Muscarinic signalling was assessed by measuring carbachol-induced activation of phospholipase C (PLC), arachidonic acid release, and calcium mobilisation. Carbachol activation of PLC was not altered by inhibition of PKC with chelerythrine chloride, bisindolylmaleimide or chronic treatment with phorbol myristate acetate (PMA). Activation of PKC by acute treatment with PMA was similarly without effect. In contrast, inhibition of PKC blocked carbachol stimulation of arachidonic acid release. Likewise, PKC inhibition resulted in a decreased ability of carbachol to mobilise calcium, whereas PKC activation potentiated calcium mobilisation. Inhibition of PKA with H89 or Rp-cAMP did not alter the ability of carbachol to activate PLC. Similarly, PKA activation with Sp-cAMP or forskolin had no effect on PLC stimulation by carbachol. Carbachol-mediated release of arachidonic acid was decreased by H89 but only slightly increased by forskolin. Forskolin also increased calcium mobilisation by carbachol. These results suggest a function for PKC and PKA in M3 stimulation of arachidonic acid release and calcium mobilisation but not in PLC activation.
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PMID:Involvement of protein kinase C and protein kinase A in the muscarinic receptor signalling pathways mediating phospholipase C activation, arachidonic acid release and calcium mobilisation. 1035 92


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