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)

To characterize G-proteins which mediate the signal transduction from ligand stimulated receptor to phospholipase C (PLC), we injected antisense DNAs complementary to Xenopus G(o) alpha or Gi-l alpha to suppress these endogenous G-proteins, together with the mRNAs encoding metabotropic glutamate receptor 1 (mGluR1), 5 (mGluR5) or with M1 type muscarinic receptor into oocytes. Receptor-stimulated chloride current responses were reduced by the suppression of Xenopus G(o) alpha regardless of the types of receptors. However, injection of Gi-1 antisense DNA resulted in the reduction of M1-stimulated responses but not mGluR-stimulated responses. These results suggested that all these receptors could use G(o) alpha, and M1 receptors, but not mGluRs, could also use Gi-1 proteins, to activate PLC in Xenopus oocytes.
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PMID:Inositol phospholipid metabolism in Xenopus oocytes mediated by endogenous G(o) and Gi proteins. 795 59

Expression of certain subtypes of human muscarinic receptors in NIH 3T3 cells provides an agonist-dependent model of cellular transformation by formation of foci in response to carbachol. Although focus formation correlates with the ability of the muscarinic receptors to activate phospholipase C, the actual mitogenic signal transduction pathway is unknown. Through cotransfection experiments and measurement of the activation state of native and epitope-tagged Ras proteins, the contributions of Ras and Ras GTPase-activating protein (Ras-GAP) to muscarinic receptor-dependent transformation were defined. Transforming muscarinic receptors were able to activate Ras, and such activation was required for transformation because focus formation was inhibited by coexpression of either Ras with a dominant-negative mutation or constructs of Ras-GAP that include the catalytic domain. Coexpression of the N-terminal region of GAP or of its isolated SH3 (Src homology 3) domain, but not its SH2 domain, was also sufficient to suppress muscarinic receptor-dependent focus formation. Point mutations at conserved residues in the Ras-GAP SH3 domain reversed its action, leading to an increase in carbachol-dependent transformation. The inhibitory effect of expression of the Ras-GAP SH3 domain occurs proximal to Ras activation and is selective for the mitogenic pathway activated by carbachol, as cellular transformation by either v-Ras or trkA/nerve growth factor is unaffected.
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PMID:Muscarinic receptors transform NIH 3T3 cells through a Ras-dependent signalling pathway inhibited by the Ras-GTPase-activating protein SH3 domain. 796 34

The coupling of muscarinic-cholinergic receptors (mAChR) to adenylate cyclase and phospholipase C (PLC) second messenger systems has been demonstrated in many animal species. However, little is known about this association in the developing human central nervous system. Because of the proposed role of acetylcholine in the regulation of development and differentiation of neural cells, an understanding of these relationships during human fetal development gains importance. We report, in this communication, the coupling of mAChR with PLC in the human fetal brain. This coupling was determined using two independent approaches that relied upon estimating the accumulation of inositol phosphates (IPs) and cytidine diphosphate diacylglycerol (CDP-DAG). Carbachol treatment of brain slices, in the presence of lithium, resulted in the accumulation of IPs. Analysis of the kinetics of this accumulation showed that IP3 and IP2 increased rapidly, reaching a peak or plateau before IP. The results also showed that agonist-stimulated PLC produced two second messengers, IP3 and DAG. The production of DAG was strongly supported by the carbachol-dependent increase of CDP-DAG. The accumulation of IP and CDP-DAG was dependent on agonist concentration. The obtained EC50 values were approximately: carbachol 47 microM; acetylcholine 6 microM; and oxotremorine 25 microM. Unexpectedly, all three agonists demonstrated a similar efficacy. The cholinergic stimulation of inositide hydrolysis appears to be the result of activation of the m1 muscarinic receptor.
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PMID:Muscarinic receptor-dependent activation of phospholipase C in the developing human fetal central nervous system. 798 80

We evaluated the G protein selectivity of chimeric M1 and M2 muscarinic cholinergic receptors in which either the third intracellular (I3) loop or the N-terminal portion of this loop (the I3N peptide) was replaced by the corresponding sequence from the beta 1-adrenergic receptor. The chimeras retained agonist-dependent G protein regulatory activity, but were completely promiscuous among potential G protein targets. When expressed in transfected cells, the chimeric receptors activated adenylyl cyclase, the major target of the beta-adrenergic receptor, and activated phospholipase C via a pertussis toxin-insensitive G protein, presumably a Gq. Gs is not a target of either muscarinic receptor, and Gq is not a cellular target of either the M2 muscarinic or beta-adrenergic receptor. When co-reconstituted into phospholipid vesicles with purified G proteins, the chimeric receptors were completely nonselective among all G proteins tested. They activated Gi, G(o), Gz, and Gs with similar efficiencies. This promiscuity was largely suppressed, both in transfected cells and in reconstituted vesicles, by the additional replacement of the second intracellular (I2) loop of the beta-adrenergic receptor. Such double substitutions created receptors specific for Gs, the target of the beta-adrenergic receptor. These findings suggest that G protein specificity depends on the proper combination of multiple regions on a receptor's cytoplasmic surface. In addition, the promiscuous receptors described here may be useful for regulating novel G proteins whose natural regulators are not yet known.
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PMID:Chimeric muscarinic cholinergic:beta-adrenergic receptors that are functionally promiscuous among G proteins. 803 54

In cultured striatal neurons from embryonic mice, carbachol was found to stimulate the release of arachidonic acid (AA) EC50 = 87 microM) and formation of inositol phosphates (IPs) (EC50 = 54 microM). Both responses were reproduced by muscarinic but not nicotinic agonists, and both exhibited the same pharmacological profile toward four muscarinic antagonists. Furthermore, both responses were insensitive to pertussis toxin, providing additional evidence for the involvement of the same muscarinic receptor(s), most probably of the m1 subtype. Both carbachol-evoked responses were also highly sensitive to the presence of external calcium. The calcium ionophore ionomycin, ineffective alone on AA release, strongly potentiated the carbachol response. In contrast, ionomycin alone stimulated the formation of IPs but did not significantly modify the carbachol response. Protein kinase C activation positively regulated the carbachol-evoked release of AA because this response was markedly potentiated by phorbol 12-myristate 13-acetate (PMA) and was abolished by sphingosine and Ro 31-8220. In contrast, PMA markedly inhibited the carbachol-evoked formation of IPs. The carbachol-evoked release of AA was not mimicked by the combined applications of ionomycin and PMA, which suggests that phospholipase C stimulation alone is not sufficient to trigger AA release. Taken together, these results suggest that the coupling of m1 receptors to a putative phospholipase A2 that is positively regulated by protein kinase C and by calcium is necessary for the carbachol-evoked release of AA.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Muscarinic cholinergic agonists stimulate arachidonic acid release from mouse striatal neurons in primary culture. 818 31

Chinese hamster ovary cells were transfected with both A1 adenosine receptor and muscarinic type 3 acetylcholine receptor cDNAs. The muscarinic receptor agonist carbachol stimulated phospholipase C activity, resulting in Ca2+ mobilization and arachidonate release. N6-Cyclopentyladenosine (CPA), an A1 receptor agonist, did not activate Ca(2+)-related signal transduction systems by itself but instead inhibited cAMP accumulation. In the presence of carbachol, however, the A1 receptor agonist enhanced muscarinic receptor agonist-induced phospholipase C/Ca2+ responses. In addition, the arachidonate release caused by Ca2+ ionophores or thapsigargin was also amplified by CPA, without a change in phospholipase C activity. Thus, CPA augments Ca(2+)-mediated phospholipase A2 activation in addition to and separate from its ability to amplify phospholipase C-mediated Ca2+ mobilization. Because the permissive actions of CPA on phospholipase C and phospholipase A2 activation were each reversed by pertussis toxin treatment, in a manner similar to that of the CPA-induced inhibition of cAMP accumulation, we conclude that a single species of A1 receptor expressed in Chinese hamster ovary cells can couple to multiple signal transduction systems stemming from phospholipase C stimulation, phospholipase A2-mediated and Ca(2+)-dependent arachidonate release, and inhibition of cAMP accumulation. A pertussis toxin-sensitive G protein (or proteins) mediates the permissive actions of the A1 receptor in the stimulation of phospholipase C- and phospholipase A2-mediated arachidonate release.
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PMID:A single species of A1 adenosine receptor expressed in Chinese hamster ovary cells not only inhibits cAMP accumulation but also stimulates phospholipase C and arachidonate release. 819 94

Muscarinic agonist-induced contraction of the ciliary muscle is generally believed to increase aqueous outflow facility and effect accommodation. We used cultured human ciliary muscle cells as a model to study the muscarinic receptor subtype(s) involved in the contractile response of the muscle. Thus, a single cell contraction assay for these muscle cells was developed. And since agonist-induced contraction of smooth muscles is expected to involve the activation of phospholipase C (PLC), we also monitored the PLC activity in these cells. Carbachol caused contraction of the muscle cells in a dose-dependent and time-dependent manner with an estimated EC50 of 1-3 microM. The contractile effect of 100 microM carbachol was antagonized by pretreatment of atropine (1 microM) and 4DAMP (10 nM, antagonist selective for the M1 and M3 receptors) but not by pirenzepine (10 microM, antagonist selective for the M1 receptor), suggesting the involvement of the M3 but not the M1 muscarinic receptor. M3 receptor is also essential for the carbachol-induced PLC activation in the ciliary muscle cells, as indicated by the activity profiles of receptor subtype selective antagonists. For example, the stimulative effect of carbachol (EC50 = 20 microM) was antagonized by pirenzepine (pKi = 6.8), HHSiD (pKi = 7.6), 4DAMP (pKi = 9.5) and methoctramine (pKi < 6). Thus, these results indicate that an M3-like receptor subtype is essential in mediating the muscarinic agonists-induced functional changes, such as PLC activation or muscle contraction, in the ciliary muscle.
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PMID:Characterization of muscarinic receptor involvement in human ciliary muscle cell function. 820 20

The maturation of the cholinergic innervation of the rat cochlea is associated with a transient increase in the muscarinic-receptor activated inositol phosphate synthesis. In order to investigate the mechanisms involved in this transient enhancement of the inositol phosphate response, the binding properties of the cochlear muscarinic receptors were studied during rat cochlear development. Incubating the membranes from 4-day-old, 12-day-old and adult cochleas with [3H]quinuclidinyl benzylate indicates that their respective, mean concentrations of cholinoceptors are 454 +/- 51 (+/- SEM), 39 +/- 2 and 42 +/- 3 fmol/mg of protein. The dissociation constants at equilibrium are 207 +/- 80, 42 +/- 7 and 28 +/- 3 pM for the binding sites of the 4-day-old, 12-day-old and adult cochleas, respectively. Pharmacological characterization of the binding, using selective antagonists, shows that M3 cholinoceptors are expressed in developing and adult cochleas. The data demonstrate that changes in muscarinic receptor affinity and number do not correlate with the previously observed peak of the inositol phosphate metabolism. The transient enhanced inositol phosphate response is therefore not due to changes in cholinoceptors, but probably due to alterations involving the intrinsic activity of the phospholipase C and/or the efficacy of coupling of the transduction system.
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PMID:Characterization of muscarinic binding sites in the adult and developing rat cochlea. 825 26

The functional integrity of the cortical muscarinic receptor (MR)-mediated phosphatidylinositol 4,5-bisphosphate (PIP2)-specific phospholipase C signalling pathway was assessed in Alzheimer disease (AD) and age-matched control subjects. There was no difference in the basal hydrolysis of [3H]-PIP2 to [3H]-inositol phosphates between control and AD membrane preparations. However, muscarinic agonist-stimulated PIP2 hydrolysis was significantly diminished in the AD cases. Diminished agonist-stimulated PIP2 hydrolysis correlated with the loss in high affinity agonist binding (KL/KH ratio) to the M1 muscarinic receptor subtype in the disease. These data further support the hypothesis that muscarinic receptor-mediated signal transduction is altered in AD, and that the defect lies at the level of muscarinic receptor-G protein/effector coupling.
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PMID:Diminished muscarinic receptor-stimulated [3H]-PIP2 hydrolysis in Alzheimer's disease. 825 45

The activation of phospholipase C (PLC) was examined in membranes of rat cerebral cortex simultaneously exposed to monoaminergic receptor and muscarinic receptor agonists after the treatment of membranes with two alkylating agents, N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (100 microM EEDQ) and propylbenzilylcholine (10 nM PrBCM). Treatment of membranes with PrBCM results in a selective inactivation of M3 muscarinic receptors, while treatment with EEDQ results in a relative sparing of M1 muscarinic receptors. Stimulation of PLC by GTP gamma S alone in rat cortical membranes had an apparent EC50 of about 0.4 microM, but in the presence of carbachol (1 mM) was 0.09 microM. Treatment of rat cortical membranes with EEDQ or PrBCM did not modify the concentration-response curves for GTP gamma S alone, but the ability of carbachol (1 mM) to shift the EC50 of GTP gamma S was lost in PrBCM-treated membranes. We have previously shown that dopamine, working through D1-like dopamine receptors, alters the PLC response to carbachol by preventing this shift in the apparent EC50 for GTP gamma S16. When we reproduced these experiments in EEDQ- and PrBCM-treated membranes, only in EEDQ-treated membranes was dopamine able to inhibit the PLC response to carbachol. The results indicate that the post-receptor mechanisms of PLC activation are distinct for the putative M1 as opposed to M3 muscarinic receptors in rat cortical membranes. Further, there appears to be a specific interaction between D1 and M3 receptors.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Modulation of the phospholipase C activity in rat brain cortical membranes by simultaneous activation of distinct monoaminergic and cholinergic muscarinic receptors. 825 72


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