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)

Stimulation of the T-cell antigen receptor (TCR), which itself is not a protein-tyrosine kinase (PTK), activates a PTK and phospholipase C (PLC). Using the human T-cell leukemic line Jurkat and normal peripheral blood lymphocytes, we demonstrate that stimulation of the TCR specifically induces the recovery of PLC activity in eluates from anti-phosphotyrosine immunoprecipitates. Stimulation of the human muscarinic receptor, subtype 1, when expressed in Jurkat activates PLC through a guanine nucleotide binding protein but does not induce the recovery of PLC activity in eluates from anti-phosphotyrosine immunoprecipitates. Western blot analysis reveals that PLC-gamma 1 is tyrosine-phosphorylated in response to TCR stimulation. Nearly all of the PLC activity recovered in eluates from anti-phosphotyrosine immunoprecipitates was depleted by anti-PLC-gamma 1 antibodies. Stimulation of the TCR on mutants derived from Jurkat that are defective in TCR-induced PLC activation results in markedly reduced, if any, PLC activity recovered in phosphotyrosine immunoprecipitates and in no detectable PLC-gamma 1 tyrosine phosphorylation. Thus, the TCR functions like PTK growth factor receptors, but through an indirect interaction, to induce tyrosine phosphorylation of PLC-gamma 1. Since other studies have implicated two members of the src family of PTKs in TCR-mediated signal transduction, our findings suggest that the induction of tyrosine phosphorylation of PLC-gamma 1 by a mechanism involving a src-like kinase may be the means by which the TCR regulates PLC activity in T cells.
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PMID:Functional activation of the T-cell antigen receptor induces tyrosine phosphorylation of phospholipase C-gamma 1. 171 1

The effects of norepinephrine (NE), carbachol (CCh), NaF, 3-isobutyl-1-methylxanthine (IBMX), and high K+ concentration (80 mM) depolarization on inositol trisphosphate (IP3) accumulation, cyclic AMP (cAMP) formation, and contraction were investigated in the dilator and sphincter smooth muscles of the sympathetically denervated as well as the normal rabbit eye. (a) In the denervated dilator muscle, NE-stimulated IP3 production and contraction are enhanced. (b) In the sphincter muscle of rabbits that have undergone sympathetic denervation. CCh-stimulated IP3 production and contraction are attenuated. (c) The increase in tension by a maximal effective dose of NaF (209 mM) in the dilator was 12.5 and 18 mg of tension/mg wet weight in normal and denervated tissue, respectively, and in the sphincter was 33.8 and 15.2 mg of tension/mg wet weight in normal and denervated tissue, respectively. NaF had no effect on cAMP formation. (d) Addition of NE had no effect on cAMP formation in both the normal and denervated dilator, whereas basal and IBMX-induced cAMP formation increased. in the denervated sphincter over that of the normal tissue by 15 and 60%, respectively. (e) Isoproterenol (5 microM) increased cAMP formation in the normal and denervated sphincter by 47 and 91%, respectively. (f) Whereas CCh inhibits cAMP formation in the normal sphincter, it lost its inhibitory effect in the sphincter with denervation. (g) IBMX (0.1 mM) attenuated the CCh-stimulated IP3 production and contraction of the sphincter by approximately 30% of their respective controls. (h) High K+ concentration depolarization attenuated contraction in both dilator and sphincter muscles with denervation. These observations suggest that an increase in the level of cAMP in the iris sphincter due to sympathetic denervation could lead to inhibition of phospholipase C (or other target sites, such as phosphorylation of the muscarinic receptor, Gp protein itself, myosin light chain kinase, or the IP3 receptor), IP3 production, and contraction. In conclusion, we suggest that the supersensitivity and subsensitivity observed after surgical sympathetic denervation of the iris dilator and sphincter muscles, respectively, are caused by alterations in the efficiency of coupling, probably through the Gp proteins, between their respective receptors and the breakdown of polyphosphoinositides by phospholipase C. In addition, we propose that the sympathetic nervous system can regulate, through alterations in cAMP levels, the muscarinic stimulation of IP3 accumulation and contraction in the iris sphincter. These findings add further support to the hypothesis that there are reciprocal interactions between the cAMP and IP3-Ca2+ signaling systems and the contractile response in the iris smooth muscle.
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PMID:Effects of surgical sympathetic denervation on myo-inositol trisphosphate production and contraction in the dilator and sphincter smooth muscles of the rabbit iris: evidence for interaction between the cyclic AMP and calcium signaling systems. 171 29

Endothelial cells, either in vivo or freshly isolated, respond when exposed to muscarinic agonists with an increase in cytosolic free calcium concentration ([Ca2+]i) and release of endothelium-derived relaxing factor (EDRF). When placed in culture, however, endothelial cells rapidly lose these responses, which may be related to changes in muscarinic receptor expression. Northern blot analysis of poly(A) + RNA from freshly isolated or cultured bovine aortic endothelial cells was used to address this problem. Through the use of specific cDNA probes complementary to the nonconserved regions of the m1, m2, m3, m4, and m5 muscarinic receptors, mRNA transcripts for the m1 (3.9 kb), m2 (3.8 kb), and m3 (3.1 kb) receptor subtypes were identified in freshly isolated endothelial cells, whereas m1 and m3 transcripts were identified in aortic smooth muscle. In contrast, cultured endothelial cells contained mRNA for only the m2 receptor subtype. Transcripts for the m4 or m5 receptors were not detected in either freshly isolated or cultured endothelial cells. Since m1 and m3 receptor subtypes are coupled to phospholipase C, activation of which is required for EDRF release, these observations may explain the failure of muscarinic agonists to elicit a rise in [Ca2+]i and EDRF release from cultured endothelial cells.
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PMID:Differential muscarinic receptor mRNA expression by freshly isolated and cultured bovine aortic endothelial cells. 173 30

IMR-32 and SK-N-MC cells were found to contain [3H]quinuclidinyl benzilate specific binding sites inhibited by pirenzepine in a manner suggesting the presence of both M1-type and M2-type muscarinic receptor recognition sites. Neither cell had detectable [3H]8-OH-DPAT binding sites. Carbachol stimulated the rate of inositol phospholipid breakdown in IMR-32 and SK-N-MC human neuroblastoma cells with an EC50 value of about 50 microM in both cases. Pirenzepine inhibited the carbachol (100 microM)-stimulated inositol phospholipid breakdown in both cells with Hill slopes of unity and IC50 values of 15 nM (IMR-32) and 12 nM (SK-N-MC). The 5-HT1A receptor agonist 8-OH-DPAT competitively inhibited carbachol-stimulated inositol phospholipid breakdown with pA2 values of 5.78 (IMR-32) and 5.61 (SK-N-MC). These values are consistent with the inhibitory potency of 8-OH-DPAT towards [3H]quinuclidinyl benzilate binding in these cells. The 5-HT agonists 5-MeODMT and buspirone at micromolar concentrations inhibited carbachol-stimulated breakdown in IMR-32 cells. The inhibition by 8-OH-DPAT and 5-MeODMT was not affected by preincubation with (-)alprenolol. 5-HT (10-100 microM) was without effect on either basal or carbachol-stimulated breakdown. It is concluded that IMR-32 and SK-N-MC neuroblastoma cells express muscarinic M1-type but not serotoninergic receptors coupled to phosphoinositide-specific phospholipase C. 8-OH-DPAT acts as a weak antagonist at these muscarinic receptors.
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PMID:Antagonism by 8-hydroxy-2(di-n-propylamino)tetraline and other serotonin agonists of muscarinic M1-type receptors coupled to inositol phospholipid breakdown in human IMR-32 and SK-N-MC neuroblastoma cells. 182 86

Engagement of the TCR initiates at least two transmembrane signaling pathways, the phosphatidylinositol pathway and a tyrosine kinase pathway. The T cell leukemic line Jurkat was used to study the relationship between the number of occupied TCR on the cell surface and the TCR-mediated activation of phosphatidylinositol-specific phospholipase C. We characterized a series of Ti beta-chain transfectants of the Jurkat mutant J.RT3-T3.5, in which surface expression of the TCR is limited by expression of the TCR beta-chain. Calibrated flow cytometry was used to determine the number of binding sites for anti-CD3 mAb on the surface of these cells, which was less than 1.2 x 10(3) to 1.2 x 10(4) sites/cell. In the presence of lithium chloride, the accumulation of inositol phosphates (InsP) in these cell lines in response to saturating concentrations of anti-CD3 mAb was proportional to the calculated surface TCR number. This result was consistent with dose-response studies using anti-CD3 mAb in Jurkat cells, in which ligand concentration, rather than number of binding sites, was limiting. Increase in intracellular free calcium concentration was a sensitive indicator of TCR engagement and correlated with the level of TCR expression, but less closely than did InsP levels. Induction of the early lymphocyte activation marker CD69 by anti-CD3 mAb also correlated with surface expression of TCR. In order to test whether limitation of this signaling pathway by TCR number may be relevant to signal transduction in the wild-type cell, we compared PLC activity in Jurkat cells during soluble anti-CD3 mAb-induced internalization of the TCR and also in response to immobilized mAb. The net accumulation of InsP per min decreased linearly with TCR number during the rapid phase of TCR internalization, confirming the limiting role of TCR number in this system. When internalization was prevented by immobilization of the stimulus, there was no decrease in the net accumulation of InsP per minute over time. In a Jurkat cell line transfected with the heterologous human muscarinic receptor, subtype 1, the InsP response to a muscarinic agonist was unaffected by TCR internalization, indicating that the distal phosphatidylinositol pathway was not affected by prolonged stimulation of the TCR. We conclude that transmembrane signaling through the TCR may be regulated by the number of surface TCR-ligand complexes. This observation has implications for transmembrane signaling in both mature T cells and thymocytes.
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PMID:Signaling via the inositol phospholipid pathway by T cell antigen receptor is limited by receptor number. 182

The ability of muscarinic receptors, present in either the cell surface or sequestered compartments of intact human SK-N-SH neuroblastoma cells, to stimulate phosphoinositide hydrolysis has been examined. When cells were first exposed to carbachol for 1 h at 37 degrees C, approximately 50% of the cell surface receptors became sequestered, and this was accompanied by a comparable reduction in the subsequent ability of muscarinic agonists to stimulate phosphoinositide turnover, as monitored by the release of labeled inositol phosphates at 10 degrees C. At this temperature, muscarinic receptor cycling between the two cell compartments is prevented. Upon warming the carbachol-pretreated cells to 37 degrees C, receptor cycling is reinitiated and stimulated phosphoinositide turnover is fully restored within 5-8 min. When measured at 10 degrees C, the reduction of stimulated phosphoinositide turnover observed following carbachol pretreatment was similar in magnitude for both hydrophilic (carbachol, oxotremorine-M) and lipophilic (arecoline, oxotremorine-2, and L-670,548) agonists. The loss of response for both groups of agonists could be prevented if the incubation temperature was maintained at 37 degrees C, rather than at 10 degrees C. At the latter temperature carbachol pretreatment of SK-N-SH cells reduced the maximum release of inositol phosphates elicited by either carbachol or L-670,548 but not the agonist concentrations required for half-maximal stimulation. Radioligand binding studies, carried out at 10 degrees C, indicate that following receptor sequestration, significantly higher concentrations of carbachol were required to occupy the available muscarinic receptor sites. In contrast the lipophilic full agonist L-670,548 recognized receptors present in control and carbachol-pretreated cells with comparable affinities. Analysis of the inositol lipids present after carbachol pretreatment indicate that only a minimal depletion of the substrates necessary for phospholipase C activation had occurred. The results indicate that the agonist-induced sequestration of muscarinic receptors from the cell surface results in a loss of stimulated phosphoinositide hydrolysis when measured under conditions in which the return of the sequestered receptors to the cell surface is prevented. Thus, only those receptors present at the cell surface are linked to phospholipase C activation.
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PMID:Preferential coupling of cell surface muscarinic receptors to phosphoinositide hydrolysis in human neuroblastoma cells. 184 33

Serotonin 5-HT1A receptors have been reported to be negatively coupled to muscarinic receptor-stimulated phosphoinositide turnover in the rat hippocampus. In the present study, we have investigated further the pharmacological specificity of this negative control and attempted to elucidate the mechanism whereby 5-HT1A receptor activation inhibits the carbachol-stimulated phosphoinositide response in immature or adult rat hippocampal slices. Various 5-HT1A receptor agonists were found to inhibit carbachol (10 microM)-stimulated formation of total inositol phosphates in immature rat hippocampal slices with the following rank order of potency (IC50 values in nM): 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) (11) greater than ipsapirone (20) greater than gepirone (120) greater than RU 24969 (140) greater than buspirone (560) greater than 1-(m-trifluoromethylphenyl)piperazine (1,500) greater than methysergide (5,644); selective 5-HT1B, 5-HT2, and 5-HT3 receptor agonists were inactive. The potency of the 5-HT1A receptor agonists investigated as inhibitors of the carbachol response was well correlated (r = 0.92) with their potency as inhibitors of the forskolin-stimulated adenylate cyclase in guinea pig hippocampal membranes. 8-OH-DPAT (10 microM) fully inhibited the carbachol-stimulated formation of inositol di-, tris-, and tetrakisphosphate but only partially antagonized (-40%) inositol monophosphate production. The effect of 8-OH-DPAT on carbachol-stimulated phosphoinositide turnover was not prevented by addition of tetrodotoxin (1 microM), by prior destruction of serotonergic afferents, by experimental manipulations causing an increase in cyclic AMP levels (addition of 10 microM forskolin), or by changes in membrane potential (increase in K+ concentration or addition of tetraethylammonium). Prior intrahippocampal injection of pertussis toxin also failed to alter the ability of 8-OH-DPAT to inhibit the carbachol response. Carbachol-stimulated phosphoinositide turnover in immature rat hippocampal slices was inhibited by the protein kinase C activators phorbol 12-myristate 13-acetate (10 microM) and arachidonic acid (100 microM). Moreover, the inhibitory effect of 8-OH-DPAT on the carbachol response was blocked by 10 microM quinacrine (a phospholipase A2 inhibitor) but not by BW 755C (100 microM), a cyclooxygenase and lipoxygenase inhibitor. These results collectively suggest that 5-HT1A receptor activation inhibits carbachol-stimulated phosphoinositide turnover by stimulating a phospholipase A2 coupled to 5-HT1A receptors, leading to arachidonic acid release. Arachidonic acid could in turn activate a gamma-protein kinase C with as a consequence an inhibition of carbachol-stimulated phosphoinositide turnover. This inhibition may be the consequence of a phospholipase C phosphorylation and/or a direct effect on the muscarinic receptor.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Potential mechanisms involved in the negative coupling between serotonin 5-HT1A receptors and carbachol-stimulated phosphoinositide turnover in the rat hippocampus. 184 78

We have examined the effects of the muscarinic antagonist atropine, on non-receptor-mediated activation of intracellular calcium (Ca2+i) mobilization events. In dispersed rat parotid acini, atropine significantly lowered basal inositol trisphosphate levels and delayed AlF4(-)-stimulated Ca2+i elevation in a dose-dependent manner. In a cell line transfected with the m1 muscarinic receptor (M1-CHO), atropine significantly lowered (approximately 60%) AlF4- stimulated Ca2+i elevation. These aggregate findings suggest that atropine, acting via the muscarinic receptor, decreases the efficacy of G protein-mediated activation of phospholipase C.
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PMID:Muscarinic receptor antagonist effects in parotid acini and M1-CHO cells: evidence of G protein involvement. 190 23

Many hormones have been shown to activate phospholipase C, which results in the hydrolysis of membrane polyphosphoinositides, such as phosphatidylinositol 4,5-bisphosphate (PIP2). Two second messengers are known to be produced by PIP2 hydrolysis, 1,2-diacylglycerol, an endogenous activator of a family of enzymes called protein kinase C (PKCs), and inositol 1,4,5-trisphosphate, which raises free levels of intracellular Ca2+. Treatment of various cells with 4 beta-phorbol 12-myristate 13-acetate (PMA), a specific exogenous activator of PKCs, causes an enhancement or sensitization of adenylyl cyclase activities. This finding prompted us to examine the effects of direct hormonal activation of PIP2 hydrolysis on the sensitization of adenylyl cyclase. Liao et al. [J. Biol. Chem. 265:11273-11284 (1990)] have shown that P2 purinergic receptor agonists such as ATP and muscarinic receptor agonists such as carbachol stimulate PIP2 hydrolysis in L cells expressing the M5 muscarinic acetylcholine receptor. We investigated the effects of these hormones on adenylyl cyclase and contrasted these effects with the sensitizing effects of PMA. We found that ATP pretreatment of two different types of L cells resulted in a rapid 50-150% sensitization of prostaglandin E1-, epinephrine-, and forskolin-stimulated adenylyl cyclase activity, with an EC50 of 3 microM ATP. This effect was qualitatively similar to that caused by 10 nM PMA. The enhancement of adenylyl cyclase activity was associated with an increase in the Vmax for hormonal stimulation and with a lack of significant effects of ATP on the EC50. The effect was completely eliminated when adenylyl cyclase was assayed in the presence of high free Mg2+ levels (10 mM). Down-regulation of PKCs with long term PMA treatment did not affect the ATP-induced sensitization of adenylyl cyclase, although the PMA-induced sensitization of adenylyl cyclase was eliminated. In contrast to the effects of ATP and PMA, treatment of the cells with carbachol alone had no effect on adenylyl cyclase; however, in combination with nanomolar concentrations of PMA, synergism of the sensitization of adenylyl cyclase was observed. These data indicate that the activation of P2 purinergic receptors by ATP, and possibly activation of M5 muscarinic receptors by carbachol, may be important in the signal transduction pathways leading to the increases in the responsiveness of hormone-stimulated adenylyl cyclase.
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PMID:Sensitization of adenylyl cyclase by P2 purinergic and M5 muscarinic receptor agonists in L cells. 192 86

The effects of culture and membrane potential on Go alpha 39 expression were examined in neonatal rat cardiac myocytes. During six days of culture, the amount of Go alpha 39 in myocytes increased six-fold. The increase in Go alpha 39 appeared to be programmed, since Go alpha 39 of rat hearts also increased in vivo within three days after birth before declining by six days after birth. Furthermore, the age of the rat from which cardiac myocytes were isolated determined the amount of Go alpha 39 that accumulated in cultured cells with myocytes from two day-old rats producing more Go alpha 39 than myocytes from six day-old rats. In addition, agents which alter membrane potential (KCl and bupivacaine) inhibited the accumulation of Go alpha 39 in cultured myocytes. In an attempt to identify the signaling pathway in which cardiac Go alpha 39 is involved, muscarinic receptor-stimulated inositol phosphate production was examined, but was found to be comparable in myocytes that had six-fold differences in Go alpha 39 content. Thus Go alpha 39 does not appear to couple muscarinic receptors to phospholipase C in rat cardiac myocytes.
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PMID:The effect of culture and membrane potential on Go alpha expression in neonatal rat cardiac myocytes. 192 3


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