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)

We examined the relationship between phosphatidylcholine (PC) hydrolysis, phosphoinositide hydrolysis, and diacylglycerol (DAG) formation in response to muscarinic acetylcholine receptor (mAChR) stimulation in 1321N1 astrocytoma cells. Carbachol increases the release of [3H]choline and [3H]phosphorylcholine ([3H]Pchol) from cells containing [3H]choline-labeled PC. The production of Pchol is rapid and transient, while choline production continues for at least 30 min. mAChR-stimulated release of Pchol is reduced in cells that have been depleted of intracellular Ca2+ stores by ionomycin pretreatment, whereas choline release is unaffected by this pretreatment. Phorbol 12-myristate 13-acetate (PMA) increases the release of choline, but not Pchol, from 1321N1 cells, and down-regulation of protein kinase C blocks the ability of carbachol to stimulate choline production. Taken together, these results suggest that Ca2+ mobilization is involved in mAChR-mediated hydrolysis of PC by a phospholipase C, whereas protein kinase C activation is required for mAChR-stimulated hydrolysis of PC by a phospholipase D. Both carbachol and PMA rapidly increase the formation of [3H]phosphatidic acid ([3H]PA) in cells containing [3H]myristate-labeled PC. [3H]Diacylglycerol ([3H]DAG) levels increase more slowly, suggesting that the predominant pathway for PC hydrolysis is via phospholipase D. When cells are labeled with [3H]myristate and [14C]arachidonate such that there is a much greater 3H/14C ratio in PC compared with the phosphoinositides, the 3H/14C ratio in DAG and PA increases with PMA treatment but decreases in response to carbachol. By analyzing the increase in 3H versus 14C in DAG, we estimate that the DAG that is formed in response to PMA arises largely from PC. Muscarinic receptor activation also causes formation of DAG from PC, but approximately 20% of carbachol-stimulated DAG appears to arise from hydrolysis of the phosphoinositides.
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PMID:Muscarinic receptor activation of phosphatidylcholine hydrolysis. Relationship to phosphoinositide hydrolysis and diacylglycerol metabolism. 254 33

The effect of short-term cholinergic desensitization on muscarinic acetylcholine receptor (mAChR)-mediated activation of phospholipase C was investigated in membranes isolated from the bovine iris sphincter smooth muscle. Membranes prepared from normal or desensitized muscles, prelabeled with either [3H]myo-inositol or 32P from [gamma-32P]ATP, were incubated with a hydrolysis-resistant analogue of GTP, GTP gamma S, or GTP gamma S plus carbachol (CCh), and the production of [3H]myo-inositol 1,4,5-trisphosphate (IP3) and the breakdown of polyphosphoinositides were assessed. In normal membranes, GTP (greater than or equal to 1 mM), GTP gamma S (greater than 10 microM) and GTP gamma S (1 microM) plus CCh (10 microM), but not GDP or GDP beta S, increased phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis and IP3 production. GTP gamma S increased IP3 accumulation in a time- and dose-dependent manner, and CCh, which had no effect on phospholipase C activity in the absence of GTP gamma S, potentiated the effects of GTP gamma S. The effect of CCh plus GTP gamma S on IP3 production was inhibited by atropine, had an absolute requirement for nM amounts of Ca2+ and was not affected by pertussis toxin. At higher concentrations (greater than 1 microM), Ca2+ alone induced PIP2 hydrolysis. Short-term exposure (less than 60 min) of the muscle to CCh (100 microM) did not affect the total number (Bmax) of mAChRs nor their affinity (KD) for [3H]-N-methylscopolamine. Desensitization did, however, result in: (1) a loss of the CCh-high affinity binding state of the sphincter mAChRs in a manner analogous to that produced by GTP gamma S; (2) a loss of the ability of GTP gamma S to affect CCh binding to the receptors; and (3) an attenuation of the GTP gamma S plus CCh-stimulated PIP2 hydrolysis. In conclusion, the data presented suggest that, in the iris smooth muscle, G-proteins are involved in the coupling of mAChRs to phospholipase C and that short-term cholinergic desensitization results in (1) the uncoupling of the receptor-G-protein complex and (2) the attenuation of mAChR-activation of phospholipase C.
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PMID:Muscarinic-agonist and guanine nucleotide stimulation of myo-inositol trisphosphate formation in membranes isolated from bovine iris sphincter smooth muscle: effects of short-term cholinergic desensitization. 255 97

The m1 muscarinic acetylcholine receptor gene was transfected into and stably expressed in A9 L cells. The muscarinic receptor agonist, carbachol, stimulated inositol phosphate generation, arachidonic acid release, and cAMP accumulation in these cells. Carbachol stimulated arachidonic acid and inositol phosphate release with similar potencies, while cAMP generation required a higher concentration. Studies were performed to determine if the carbachol-stimulated cAMP accumulation was due to direct coupling of the m1 muscarinic receptor to adenylate cyclase via a GTP binding protein or mediated by other second messengers. Carbachol failed to stimulate adenylate cyclase activity in A9 L cell membranes, whereas prostaglandin E2 did, suggesting indirect stimulation. The phorbol ester, phorbol 12-myristate 13-acetate (PMA), stimulated arachidonic acid release yet inhibited cAMP accumulation in response to carbachol. PMA also inhibited inositol phosphate release in response to carbachol, suggesting that activation of phospholipase C might be involved in cAMP accumulation. PMA did not inhibit prostaglandin E2-, cholera toxin-, or forskolin-stimulated cAMP accumulation. The phospholipase A2 inhibitor eicosatetraenoic acid and the cyclooxygenase inhibitors indomethacin and naproxen had no effect on carbachol-stimulated cAMP accumulation. Carbachol-stimulated cAMP accumulation was inhibited with TMB-8, an inhibitor of intracellular calcium release, and W7, a calmodulin antagonist. These observations suggest that carbachol-stimulated cAMP accumulation does not occur through direct m1 muscarinic receptor coupling or through the release of arachidonic acid and its metabolites, but is mediated through the activation of phospholipase C. The generation of cytosolic calcium via inositol 1,4,5-trisphosphate and subsequent activation of calmodulin by m1 muscarinic receptor stimulation of phospholipase C appears to generate the accumulation of cAMP.
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PMID:A transfected m1 muscarinic acetylcholine receptor stimulates adenylate cyclase via phosphatidylinositol hydrolysis. 255 56

The regulation of cellular signal transduction and growth by four human muscarinic acetylcholine receptor (mAChR) subtypes has been studied comparatively. The four mAChRs fall into two functional sub-groups, based on their primary effects on second messenger formation; two of the receptors strongly inhibit adenylyl cyclase activity, whereas the other two strongly stimulate PI hydrolysis. Studies on mAChR regulation of two cellular events involved in cellular growth regulation, the transcription of proto-oncogene c-fos and DNA synthesis, indicate that these events are efficiently activated by those mAChRs which couple primarily to phospholipase C.
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PMID:Functional diversity of muscarinic receptor subtypes in cellular signal transduction and growth. 269 17

Islet-activating protein (IAP) was used to investigate the role of the guanosine triphosphate binding proteins Gi and/or Go in muscarinic acetylcholine receptor-mediated responses in neuroblastoma cells (clone N1E-115). Incubation of intact cells for 24 h with 20 ng/ml IAP resulted in inhibition of subsequent IAP catalyzed incorporation of [32P]ADP-ribose into a membrane protein doublet of molecular weight 40,000 (Gi alpha and Go alpha). IAP treatment fully blocked muscarinic receptor-mediated inhibition of cAMP accumulation. Incubation of intact cells with carbachol for 8 h resulted in the concentration dependent loss of membrane muscarinic receptor. Pretreatment of cells with IAP prior to carbachol exposure partially blocked the subsequent decrease in receptor number. Pretreatment of cells with IAP had no effect on the ability of carbachol to stimulate phosphoinositide hydrolysis in neuroblastoma cells. Thus, while the guanosine triphosphate binding proteins Gi and/or Go are involved in coupling the muscarinic receptor to some of the physiological responses in these cells, it is clear that activation of phospholipase C by the muscarinic receptor is a Gi/Go independent response.
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PMID:Modification of neuronal muscarinic receptor-mediated responses by islet-activating protein. 284 Oct 15

The negative inotropic effect of carbachol, as well as phosphoinositide hydrolysis, was measured in atria from neonatal and adult rats. Carbachol increased phosphoinositide hydrolysis and decreased dF/dt of both neonatal and adult atria; however, the neonatal atria showed hypereactivity to carbachol as compared with adult atria. Inhibition of phospholipase C reduced the supersensitivity to carbachol upon contractility in neonatal atria producing values similar to those of the adult atria, suggesting that muscarinic acetylcholine receptor (mAchR) stimulation is secondary to receptor-mediated hydrolysis of phosphoinositides. Pharmacological analysis with mAchR antagonists tends to support the idea that m1 and m2 subtypes are the most important mediators of the response to carbachol in neonatal atria. In adult atria, the effect of carbachol is coupled only to mAchR m2 subtypes.
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PMID:Pharmacological evidence for the existence of different subtypes of muscarinic acetylcholine receptors for phosphoinositide hydrolysis in neonatal versus adult rat atria. 755 85

We used solution hybridization, immunoprecipitation, and immunoblot analyses to examine the developmental expression of chicken m2 (cm2), cm3, and cm4 muscarinic acetylcholine receptor (mAChR) mRNA and protein in embryonic and post-hatched chick heart and retina in order to correlate developmental expression patterns with known physiological events. cm2 is the predominant mAChR subtype expressed in chick heart. cm3 and cm4 protein and mRNA expression is very low in chick heart, and cm3 expression is highest early in development. The decrease in cm3 expression correlates well with the developmental decrease in mAChR-mediated activation of phospholipase C. cm4 is the predominant mAChR subtype expressed in chick retina. The expression of both cm4 protein and mRNA is highest early in development and decreases as development progresses. cm2 and cm3 mAChR are expressed at approximately equivalent levels and have similar patterns of expression. The cm2 and cm3 protein levels increase throughout development, while cm2 and cm3 mRNA levels peak at embryonic day 15 and then decrease after hatching. Our data indicate that the three mAChR subtypes are differentially regulated in chick heart and retina and that the patterns of expression of mAChR may be important in the development and physiology of these tissues.
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PMID:Tissue-specific regulation of muscarinic acetylcholine receptor expression during embryonic development. 765 43

Gq is the heterotrimeric guanine nucleotide-binding protein that activates the beta isoforms of phosphatidyl-inositol-specific phospholipase C (PI-PLC). The Gq alpha-subunit polypeptide (alpha qa) was N-terminally modified by addition of a 9-aa sequence, YPYDVPDYA. Placement of the 9-aa epitope tag at the N terminus allowed expression of functional alpha q polypeptides and selective identification of plasmid-expressed wild-type and mutant G-protein alpha subunits. Mutation of glutamine-209 to leucine in the N-terminally epitope-tagged alpha q (N(epi) alpha qQ209L) inhibited GTPase activity and persistently activated PI-PLC, resulting in high steady-state levels of inositol phosphates. The elevated levels of inositol phosphates resulting from N(epi) alpha qQ209L expression were similar to those obtained with carbachol activation of the M1 muscarinic acetylcholine receptor. The Gq-coupled M1 receptor, which stimulates PI-PLC activity, and phorbol esters, acting via protein kinase C, activate the cytoplasmic mitogen-activated protein kinase in COS cells. However, the constitutive activation of PI-PLC enzymatic activity resulting from expression of GTPase-deficient alpha q was unable to persistently activate this kinase. The results indicate that persistent PI-PLC activation is insufficient to sustain the stimulation of a cytoplasmic serine/threonine protein kinase regulated by Gq-coupled receptor signal-transduction pathways.
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PMID:Epitope-tagged Gq alpha subunits: expression of GTPase-deficient alpha subunits persistently stimulates phosphatidylinositol-specific phospholipase C but not mitogen-activated protein kinase activity regulated by the M1 muscarinic acetylcholine receptor. 768 19

Previous studies have shown that a single type of transmembrane receptor is able to regulate multiple effectors through the activation of heterotrimeric G proteins. For example, the m2 muscarinic acetylcholine receptor (mAChR) expressed in Chinese hamster ovary (CHO) cells inhibits adenylyl cyclase, stimulates phospholipase C-dependent intracellular Ca2+ release, and activates phospholipase A2 through pertussis toxin-sensitive G proteins. However, it is unclear whether multiple effector enzymes can be regulated by one type of heterotrimeric G protein within a single cell. To investigate this question, we constructed a derivative of G alpha i3 (termed G alpha i3 C > S) in which the carboxyl-terminal cysteine residue, the site for pertussis toxin modification, was changed to a serine. Following pertussis toxin treatment of transfected CHO cells expressing the m2 mAChR, we found that the G alpha i3 C > S protein underwent guanine nucleotide exchange in response to the muscarinic agonist carbachol, while the m2 mAChR failed to activate the endogenous G alpha i2 and G alpha i3 proteins. Moreover, coupling of heterotrimeric G proteins containing G alpha i3 C > S to the m2 mAChR resulted in pertussis toxin-resistant inhibition of adenylyl cyclase, stimulation of phospholipase C-induced intracellular Ca2+ release, and phospholipase A2-mediated arachidonic acid release. Therefore, these studies provide conclusive evidence that heterotrimeric G proteins containing just G alpha i3 can regulate multiple effector enzymes within the same cell type.
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PMID:Heterotrimeric G proteins containing G alpha i3 regulate multiple effector enzymes in the same cell. Activation of phospholipases C and A2 and inhibition of adenylyl cyclase. 796 42

The feasibility of using a permeabilized preparation of human SH-SY-5Y neuroblastoma cells for studies of muscarinic acetylcholine receptor (mAChR) sequestration has been evaluated. Exposure of cells permeabilized with digitonin, streptolysin-O, or the alpha-toxin from Staphylococcus aureus to oxotremorine-M (Oxo-M) for 30 min resulted in a 25-30% reduction in the number of cell surface mAChRs, as monitored by the loss of N[3H]methylscopolamine ([3H]NMS) binding sites. The corresponding value for intact cells was 40%. For cells permeabilized with 20 microM digitonin, the Oxo-M-mediated reduction in [3H]NMS binding was time (t1/2 approximately 5 min) and concentration (EC50 approximately 10 microM) dependent and was agonist specific (Oxo-M > bethanechol = arecoline = pilocarpine). In contrast, no reduction in total mAChR number, as monitored by the binding of [3H]quinuclidinyl benzilate, occurred following Oxo-M treatment. The loss of [3H]NMS sites observed in the presence of Oxo-M was unaffected by omission of either ATP or Ca2+, both of which are required for stimulated phosphoinositide hydrolysis, but could be inhibited by the inclusion of guanosine 5'-O-(2-thiodiphosphate). mAChRs sequestered in response to Oxo-M addition were unmasked when the cells were permeabilized in the presence of higher concentrations of digitonin (80 microM).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Sequestration of muscarinic cholinergic receptors in permeabilized neuroblastoma cells. 815 29


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