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 family of serotonin receptors consists of at least eight distinct subtypes, divided into four classes based on their pharmacological and functional characteristics. Here we report the cloning and expression in Swiss 3T3 cells of the human 5-HT2 and 5-HT1A receptor subtypes. Both genes encode functional receptors for 5-HT, that differ considerably in genomic structure, primary amino acid sequence, pharmacology and signal transduction. The 5-HT1A receptor transfectants displayed a single high affinity site for the agonist [3H](+/-)-8-hydroxy-2-(di-n-propylamino)tetralin HBr ([3H]8-OH-DPAT) and a pharmacological profile specific for the 5-HT1A receptor. In these transfectants, 5-HT mediated a dose-dependent inhibition of forskolin-stimulated cAMP levels. Cells expressing the 5-HT2 receptor exhibited high affinity binding for the antagonist [3H]ketanserin with a 5-HT2 receptor specific pharmacological profile. In these cells 5-HT activated phospholipase C in a dose-dependent manner. The 5-HT2 receptor displayed a genomic organization quite different from the 5-HT1A, 5-HT1B and 5-HT1D receptor subtypes. While these receptors are encoded by one single exon, the 5-HT2 receptor is encoded by three exons separated by two introns. The latter finding adds and additional molecular criterion for receptor classification.
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PMID:Genomic organization, coding sequence and functional expression of human 5-HT2 and 5-HT1A receptor genes. 133 Jun 47

Proliferation of smooth muscle cells in arteries is associated with contractile hypersensitivity to serotonin (5-HT). A possible explanation is that smooth muscle cells express increased numbers of phospholipase C (PLC)-coupled 5-HT receptors (5-HTR), which could mediate contractile and mitogenic signals via phosphatidylinositol turnover. To test this hypothesis, we performed a molecular characterization of 5-HTR subtypes in normal aorta and passaged rat aortic smooth muscle cells (RASM) in culture. Northern blot analysis revealed that growth-arrested cultured cells expressed 5-HT2R mRNA at 50-fold greater levels than aorta. 5-HT1CR mRNA was not detected in either case. 5-HT stimulated intracellular Ca2+ mobilization (fivefold peak increase) and c-fos mRNA induction (10-fold peak increase); both responses were strongly inhibited by selective 5-HT2R antagonists. Specific agonists for the 5-HT1AR, 5-HT1BR, and 5-HT1DR failed to induce c-fos mRNA. Although 5-HT (10 microM) increased [3H]thymidine incorporation (28% relative to 10% calf serum), it was a weak mitogen for cultured RASM based on cell counts. Thus there is high level expression of 5-HT2R mRNA by cultured RASM relative to aorta, and the 5-HT2R appears to be the only 5-HTR subtype mediating early growth signals in these cells. These data suggest that, following arterial injury in vivo, smooth muscle cells may overexpress the 5-HT2R, resulting in 5-HT contractile hypersensitivity and increased responsiveness to other growth factors.
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PMID:5-HT2 receptor mRNA is overexpressed in cultured rat aortic smooth muscle cells relative to normal aorta. 153 23

To assign the GTP-binding protein (G-protein) subtype involved in the signal transduction from exogenous receptors to phospholipase C in the Xenopus oocyte translation system, antisense DNA complementary to rat G-protein alpha-subunit mRNA was designed and injected together with rat brain poly(A)+ RNA. Current response of mRNA-injected oocytes to acetylcholine (ACh) was suppressed dose-dependently by a co-injection of Gil alpha-antisense DNA, but response of the same oocytes to serotonin (5-HT) was not inhibited. In the oocytes co-injected with Go alpha-antisense DNA, the 5-HT response was more effectively suppressed than the ACh response. These results suggest that Go alpha but not Gil alpha intermediates brain 5-HT1C receptor function, and in contrast, muscarinic receptors derived from rat brain utilize Gil alpha rather than Go alpha to activate phospholipase C.
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PMID:Metabotropic responses to acetylcholine and serotonin of Xenopus oocytes injected with rat brain mRNA are transduced by different G-protein subtypes. 154 91

When expressed into Xenopus oocytes, HLK3 K+ channel (Kv1-3) induced a slowly inactivating voltage-dependent K+ current. We have studied the modulation of this K+ current by co-expressing a cloned 5-HT2 receptor together with HLK3 K+ channel protein. Application of 5-HT caused a long-lasting inhibition of the voltage-gated K+ current. This inhibitory modulation was mimicked by intracellular injection of inositol triphosphate or Ca2+, as well as by incubation with phorbol esters or diacylglycerol analogs. Oocytes pretreatment with staurosporine and EGTA fully prevented 5-HT inhibitory action. Elevation of cAMP and cGMP levels into oocytes did not produce any detectable effect on the current recorded in the absence or the presence of 5-HT. These data suggest that the second messengers generated by phospholipase C activation may be important modulators of HLK3 K+ channels in the immune and the central nervous systems.
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PMID:Regulation of a major cloned voltage-gated K+ channel from human T lymphocytes. 160 23

To examine the mechanism of action of antidepressant drugs, we studied the effect of desipramine (DMI) in vitro on agonist-stimulated inositol phosphate formation and inositol phospholipids in rat brain and human platelets. We observed that DMI inhibited thrombin-stimulated 3H-inositol bisphosphate (IP2) and 3H-inositol trisphosphate (IP3) but not 3H-inositol monophosphate (IP1) formation in human platelets. DMI also inhibited norepinephrine (NE) and serotonin (5-HT) stimulated 3H-IP1 formation in rat cerebral cortex. DMI increased levels of all three 3H-inositol phospholipids, 3H-phosphatidyl inositol (PI), 3H-PI-4-phosphate (PIP), and 3H-PI 4,5-bisphosphate (PIP2), in both platelets and rat cortex. The decreased formation of inositol phosphates and increased levels of [3H]-PI, [3H]-PIP, and [3H]-PIP2 by DMI appears to be due to the inhibition of the enzyme phospholipase C rather than its effects on receptors. It is thus possible that interaction of tricyclic antidepressant drugs with the PI-signaling system may be related to their mechanism of action.
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PMID:Effect of desipramine on inositol phosphate formation and inositol phospholipids in rat brain and human platelets. 177 95

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

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 isolated a hamster fibroblast cDNA clone that encodes a serotoninergic receptor whose deduced amino acid sequence displays 94% identity with the rat brain serotonin (5-HT) type 2 receptor. When expressed in Xenopus oocytes, the hamster receptor efficiently couples to the phosphoinositide second messenger system and leads to intracellular Ca2+ mobilization in response to 5-HT. To determine the pharmacological properties of this receptor, and to evaluate the role of phospholipase C (PLC) activation in growth modulation by 5-HT, we have expressed it in hamster fibroblasts. Transfected cells that express 5-HT receptors were selected using a novel method based on coexpression of the Na+/H+ antiporter gene as a selectable marker. After co-transfection of the 5-HT receptor and Na+/H+ antiporter cDNAs in fibroblasts lacking antiporter activity (variants of the CCL39 line), 50% of the clones resistant to an acute acid load express functional receptors. The pharmacological profile of the transfected receptor is consistent with it being of the 5-HT2 subtype, and the extent of 5-HT-stimulated PLC activation in independent clones correlates with their relative level of cRNA expression. In cells in where addition of 5-HT leads to strong activation of PLC, and inhibition of adenylate cyclase via endogenous 5-HT1b receptors, 5-HT alone has little effect on DNA synthesis stimulation. Thus we conclude that activation of the PLC signalling pathway in these cells is not sufficient to trigger G0/G1 to S phase transition. Strong activation of PLC via 5-HT2 receptors does however contribute to the synergy observed between 5-HT (Gi-coupled pathway) and fibroblast growth factor (tyrosine kinase-activated pathway) on DNA synthesis reinitiation in transfected cells.
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PMID:Cloning, functional expression and role in cell growth regulation of a hamster 5-HT2 receptor subtype. 194 95

Electropermeabilized human platelets containing 5-hydroxy[14C]tryptamine ([14C]5-HT) were suspended in a glutamate medium containing ATP and incubated for 10 min with (in various combinations) Ca2+ buffers, phorbol 12-myristate 13-acetate (PMA), guanine nucleotides, and thrombin. Release of [14C]5-HT and beta-thromboglobulin (beta TG) were used to measure secretion from dense and alpha-granules, respectively. Ca2+ alone induced secretion from both granule types; half-maximal effects were seen at a -log [Ca2+ free] (pCa) of 5.5 and maximal secretion at a pCa of 4.5, when approximately 80% of 5-HT and approximately 50% of beta TG were released. Addition of PMA, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), GTP, or thrombin shifted the Ca2+ dose-response curves for secretion of both 5-HT and beta TG to the left and caused small increases in the maximum secretion observed. These results suggested that secretion from alpha-granules, like that from dense granules, is a Ca(2+)-dependent process stimulated by the sequential activation of a G-protein, phospholipase C, and protein kinase C (PKC). However, high concentrations of PMA and GTP gamma S had distinct effects in the absence of Ca2+ (pCa greater than 9); 100 nM PMA released approximately 20% of platelet 5-HT but little beta TG, whereas 100 microM GTP gamma S stimulated secretion of approximately 25% of each. Simultaneous addition of PMA greatly enhanced these effects of GTP gamma S. Phosphorylation of pleckstrin in permeabilized platelets incubated with [gamma-32P]ATP was used as an index of the activation of PKC during secretion. In the absence of Ca2+, 100 nM PMA caused maximal phosphorylation of pleckstrin and 100 microM GTP gamma S was approximately 50% as effective as PMA; neither GTP gamma S nor Ca2+ enhanced the phosphorylation of pleckstrin caused by 100 nM PMA. These results indicate that, although activation of PKC promoted secretion, GTP gamma S exerted additional stimulatory effects on secretion from both dense and alpha-granules that were not mediated by PKC. Measurement of [3H]inositol phosphate formation in permeabilized platelets containing [3H]phosphoinositides showed that GTP gamma S did not stimulate phosphoinositide-specific phospholipase C in the absence of Ca2+. It follows that in permeabilized platelets, GTP gamma S can both stimulate PKC and enhance secretion via G-protein-linked effectors other than this phospholipase.
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PMID:Factors affecting dense and alpha-granule secretion from electropermeabilized human platelets: Ca(2+)-independent actions of phorbol ester and GTP gamma S. 196 91

1. 5-Hydroxytryptamine (5-HT) produced a concentration-dependent increase in the membrane concentration of 1,2-diacylglycerol (DG) in the rabbit isolated basilar artery, but did not stimulate the hydrolysis of membrane phosphoinositide. 2. The 5-HT-induced accumulation of DG could be blocked with the putative phospholipase C inhibitor 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate (NCDC; 70 microM), but not with the protein kinase C inhibitor, 1-(5-isoquinolinesulphonyl)-2-methyl piperazine (H7; 50 microM). 3. Direct stimulation of protein kinase C with phorbol 12,13-dibutyrate (PDBu) produced sustained smooth muscle contraction which was fairly rapid in onset and could be reversed by H7 but not by NCDC. The inactive phorbol, 4 alpha phorbol 12,13-dideceonate, did not produce contraction in the basilar artery. 4. 5-HT-induced contractions (1 nM-100 microM) were blocked or greatly reduced in the presence of the protein kinase inhibitor H7 or polymyxin B, and with the phospholipase C inhibitor, NCDC. The concentrations of these inhibitors which abolished contraction to 5-HT, did not alter smooth muscle contraction produced in response to 30 mM K(+)-physiological salt solution (PSS). 5. These data suggest that DG production and the subsequent activation of PKC forms an important component of the cerebrovascular contractile response to 5-HT. As the DG does not appear to arise from membrane phosphatidylinositol, it appears that 5-HT can stimulate the production of this second messenger in cerebral arteries by a mechanism which is different from peripheral arteries.
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PMID:5-hydroxytryptamine-stimulated accumulation of 1,2-diacylglycerol in the rabbit basilar artery: a role for protein kinase C in smooth muscle contraction. 201 23


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