Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.4.3 (phospholipase C)
 18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

There is now considerable evidence that a single receptor subtype can couple to multiple effector pathways within a cell. Recently, Kenakin proposed a new concept, termed "agonist-directed trafficking of receptor stimulus", that suggests that agonists may be able to selectively activate a subset of multiple signaling pathways coupled to a single receptor subtype. 5-HT2A and 5-HT2C receptors couple to phospholipase C-(PLC) mediated inositol phosphate (IP) accumulation and PLA2-mediated arachidonic acid (AA) release. Relative efficacies of agonists (referenced to 5-HT) differed depending upon whether IP accumulation or AA release was measured. For the 5-HT2C receptor system, some agonists (e.g. TFMPP) preferentially activated the PLC-IP pathway, whereas others (e.g. LSD) favored PLA2-AA. As expected, EC50's of agonists did not differ between pathways. For the 5-HT2A receptor system, all agonists tested had greater relative efficacy for PLA2-AA than for PLC-IP. In contrast, relative efficacies were not different for 5-HT2A agonists when sequential effects in a pathway were measured (IP accumulation vs. calcium mobilization). These data strongly support the agonist-directed trafficking hypothesis.
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PMID:Pleiotropic behavior of 5-HT2A and 5-HT2C receptor agonists. 992 46

The mitogen-activated protein kinase (MAPK) pathway, classically associated with cell growth and dependent on tyrosine kinases such as MAPK kinase (MEK), can modulate smooth muscle contractility, and our laboratory has tested the hypothesis that 5-HT can activate the MAPK pathway in arterial smooth muscle through activation of a 5-HT2A receptor. Tyrosine kinase inhibitors including genistein and the specific MEK inhibitor PD098059, but not the inactive tyrosine kinase congener daidzein reduced and shifted 5-HT-induced contraction rightward in isolated, endothelium-denuded rat arteries. Activation of a tyrosine kinase/MEK via the 5-HT2A receptor was partially independent of two major signaling pathways typically associated with the 5-HT2A receptor--activation of L-type voltage gated calcium channels and phospholipase C. Western analyses using antibodies directed against tyrosyl-phosphorylated-, activated Erk MAPK, and MEK proteins from cultured aortic smooth muscle cells demonstrated that 5-HT activated MEK and the Erk MAPKs in a time-, concentration-, receptor- and tyrosine kinase-dependent manner. Taken together, these findings provide evidence for a novel pathway of vascular signal transduction--activation of the MAPK pathway--for the 5-HT2A receptor.
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PMID:Activation of the mitogen-activated protein kinase pathway via the 5-HT2A receptor. 992 53

5-Hydroxytryptamine 5-HT2A and 5-HT2C receptors share many properties, including a common ability to stimulate phospholipase C. Traditionally, this activation was thought to be initiated only after agonist binding, in accordance with the ternary complex model of receptor function. Recently, though, the 5-HT2C receptor was shown to deviate from this tenet by spontaneously isomerizing into the active receptor state, thereby activating G proteins in the absence of agonist. To determine if 5-HT2A receptors share this property of constitutive activity, 5-HT2A and 5-HT2C receptor function was evaluated in transiently transfected NIH 3T3 fibroblasts. In 3T3 cells expressing 5-HT2C receptors, agonist-independent phosphatidyl inositol hydrolysis was substantially elevated relative to mock-transfected cells. In contrast, expression of the 5-HT2A receptor at the same density caused only a marginal increase in basal signaling. Control experiments in the current and previous papers establish that basal activity does not reflect contaminating serotonin. In addition, the magnitude of serotonin-induced signaling was the same in cells expressing either receptor, suggesting that the intrinsic ability of the two receptors to couple to G proteins is comparable. These data indicate that the 5-HT2A receptor has a much lower intrinsic ability to spontaneously adopt or maintain the active receptor conformation than does the closely related 5-HT2C receptor.
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PMID:Differences in agonist-independent activity of 5-Ht2A and 5-HT2c receptors revealed by heterologous expression. 993 46

We have previously reported that a triple-helical, collagen-related peptide (CRP; also known as CRP-XL) containing a glycine-proline-hydroxyproline (GPP*) repeat motif and cross-linked through cysteine residues at its N-terminus and C-terminus is a powerful stimulus of platelet aggregation and secretion through the surface receptor glycoprotein VI (GPVI). The activation of platelets is associated with tyrosine phosphorylation of the tyrosine kinase Syk and phospholipase C gamma2 (PLCgamma2). We now report that the non-cross-linked backbone of CRP, monomeric CRP (mCRP), stimulates the tyrosine phosphorylation of Syk and PLCgamma2 in platelets and induces the weak secretion of [3H]5-hydroxytryptamine ([3H]5-HT) and aggregation. The action of mCRP does not seem to be due to spontaneous cross-linking, because alkylation of the cysteine residues leads to an increase in activity. The tripeptide backbone of CRP, GPP*10 (in which P* represents hydroxyproline) also stimulates platelet shape change and the weak tyrosine phosphorylation of Syk and PLCgamma2, but is unable to induce aggregation or secretion. The monomeric peptides partly inhibit the release of [3H]5-HT by CRP, suggesting that they are partial agonists of the collagen receptor GPVI. These results demonstrate that GPP* present as a repeat motif is sufficient to activate the platelet collagen receptor GPVI but that the cross-linking of monomers brings about an increase in activity.
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PMID:Monomeric (glycine-proline-hydroxyproline)10 repeat sequence is a partial agonist of the platelet collagen receptor glycoprotein VI. 1019 Dec 74

Intercellular Ca2+ signaling in intact salivary glands of the blowfly Calliphora erythrocephala was studied by fluorimetric digital imaging combined with microinjection of putative messenger molecules. Iontophoretic injection of D-myo-inositol 1,4, 5-trisphosphate (InsP3) into salivary gland cells evoked regenerative intercellular Ca2+ waves that spread through the impaled cell and several rows of surrounding cells. Ca2+ increases induced by microinjection of Ca2+ ions were confined to the injected cells and their nearest neighbors. Depletion of intracellular Ca2+ stores by thapsigargin pre-treatment did not alter the time course of the Ca2+ increase caused by Ca2+ injection. However, activation of Ca2+ release became clearly evident when Ca2+ was injected in the presence of serotonin (5-HT). Under these conditions, injection of Ca2+ triggered intercellular Ca2+ waves that consecutively passed through >10 cells. The phospholipase C inhibitor U73122 blocked 5-HT-induced Ca2+ increases but did not affect InsP3-dependent Ca2+ spiking and intercellular Ca2+ wave propagation. The results demonstrate that propagation of agonist-evoked Ca2+ waves in the blowfly salivary gland requires supra-basal [InsP3] but does not depend on feedback activation of phospholipase C. We conclude that the intra- and intercellular transmission of these Ca2+ waves is mediated by diffusion of Ca2+ and Ca2+-induced Ca2+ release via the InsP3 receptor channel.
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PMID:The mechanism mediating regenerative intercellular Ca2+ waves in the blowfly salivary gland. 1036 63

This study was designed to investigate the effects of serotonin on changes in intracellular Ca(2+) concentration ([Ca(2+)](i)) in cultured rat heart endothelial cells. Serotonin stimulated a biphasic change in cytosolic Ca(2+) of rat heart endothelial cells: an initial transient increase, which primarily reflects the release of Ca(2+) from internal stores, followed by a slow rise in [Ca(2+)](i) during the incubation with serotonin. Our study also demonstrated that the pattern of the serotonin-induced increase in [Ca(2+)](i) was different from that induced by thrombin in rat heart endothelial cells. In this study, the role of [Ca(2+)](i) on endothelial paracellular barrier function was also investigated. Serotonin induced an increase in endothelial permeability which paralleled the rise in [Ca(2+)](i) and was blocked by the 5-HT(2) receptor antagonist cyproheptadine. Therefore, the serotonin-stimulated increase in cytosolic Ca(2+) and macromolecular permeability was receptor-mediated in rat heart endothelial cells. Further experiments demonstrated that the serotonin-induced increase in [Ca(2+)](i) was inhibited by the phospholipase C inhibitors, neomycin and [6-[[17beta-3-methoxyestra-1,3, 5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122). Experiments involving the rapid depletion of intracellular Ca(2+) stores and Ca(2+)-free medium demonstrated that the biphasic response of endothelial Ca(2+) to serotonin was related to the release of Ca(2+) from intracellular stores and to the influx of extracellular Ca(2+). We also suggest that serotonin-induced changes in [Ca(2+)](i) are related to Ca(2+) channels sensitive to voltage-operated and inorganic Ca(2+) channel blockers.
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PMID:Serotonin-stimulated increase in cytosolic Ca(2+) in cultured rat heart endothelial cells. 1061 20

6-chloro-5-methyl-1-[6-(2-methylpyridin-3-yloxy) pyridin-3-ylcarbamoyl] indoline (SB242,084) is a novel, selective 5-HT(2C) receptor antagonist, but its actions at these sites have been little characterised at the cellular level. We employed a rapid and innovative approach to investigate its functional activity at phospholipase C (PLC)-coupled human 5-HT(2C) receptors expressed in CHO cells. PLC activity was determined as a decrease in the [3H]phosphatidylinositol ([3H]PI) content of cell membranes. Serotonin (5-HT) stimulated [3H]PI depletion (pEC50=8.74), and SB242,084, like mesulergine, completely reversed this action of 5-HT (pK(B)=9.25 and 9.01, respectively). Further, in Schild analysis, SB242,084 behaved as a high affinity competitive antagonist, inducing a parallel, rightward displacement of the 5-HT stimulation isotherm without loss of maximum efficacy. The pA2 of 9.50 was similar to its binding affinity (pKi=9.38). SB242,084 also displayed antagonist properties when PLC activity was examined by conventional determination of [3H]inositol phosphate generation. Employing this parameter, the potency of SB242,084 (pK(B)=9.21) and that of mesulergine (pK(B)=9.06) closely resembled those determined by [3H]PI depletion. In conclusion, determination of [3H]PI depletion constitutes a useful and novel technique to characterise agonist and antagonist properties of ligands at PLC-coupled receptors.
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PMID:An innovative method for rapid characterisation of phospholipase C activity: SB242,084 competitively antagonises 5-HT2C receptor-mediated [3H]phosphatidylinositol depletion. 1068 80

Serotonin (5-HT) stimulates mitogenesis in rat renal mesangial cells through a G protein-coupled 5-HT(2A) receptor. We tested the hypothesis that oxidants might be involved in the signal transduction pathway linking the receptor to extracellular signal-regulated protein kinase (ERK). 5-HT rapidly increased the activity and phosphorylation of ERK. These effects were blocked by the 5-HT(2A) receptor antagonist ketanserin. The peak effect was noted at 5-10 min, and half-maximal stimulation was achieved at 10-30 nM 5-HT. Chemical inhibitor and activator studies supported the involvement of phospholipase C, protein kinase C (PKC), and reactive oxygen species (ROS, i.e., H(2)O(2) and superoxide) generated by an NAD(P)H oxidase-like enzyme in the ERK activation cascade. Mapping studies supported a location for the NAD(P)H oxidase enzyme and the ROS downstream from PKC. Our studies are most consistent with an ERK activation pathway as follows: 5-HT(2A) receptor --> G(q) protein --> phospholipase C --> diacylglycerol --> classical PKC --> NAD(P)H oxidase --> superoxide --> superoxide dismutase --> H(2)O(2) --> mitogen-activated extracellular signal-regulated kinase --> ERK. These studies demonstrate a role for the 5-HT(2A) receptor in rapid, potent, and efficacious activation of ERK in rat renal mesangial cells. They support a role for oxidants in conveying the stimulatory signal from 5-HT, because 1) chemical antioxidants attenuate the 5-HT signal, 2) oxidants and 5-HT selectively activate ERK to a similar degree, 3) 5-HT produces superoxide and H(2)O(2) in these cells, and 4) a specific enzyme [NAD(P)H oxidase] has been implicated as the source of the ROS, which react selectively downstream of classical PKC.
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PMID:5-HT(2A) receptors stimulate mitogen-activated protein kinase via H(2)O(2) generation in rat renal mesangial cells. 1075 Dec 27

The novel benzopyranopyrrolidine and potential antipsychotic, S16924 ((+)-2-[[1-[2-(2,3-dihydrobenzo[ 1,4] dioxin-5-yloxy)-ethyl]-pyrrolidin-3yl]]-1-(4-fluoro-ph enyl)-ethanone), displays marked affinity for serotonin (5-HT)1A, 5-HT2A and dopamine D2 receptors. Herein, we show that it also possesses high affinity for the cloned, INI isoform of h5-HT2c receptors (pKi=8.28) stably expressed in CHO cells. Similarly, clozapine (8.04) was a potent ligand, whereas haloperidol (<6.0) showed low affinity. As demonstrated by fura2-detection, S16924 concentration-dependently abolished (pKb=7.93) the 5HT-induced elevation in intracellular levels of Ca2+ ([Ca2+]i) in a CHO cell line stably expressing the INI isoform of 5-HT2c receptors. Further, as determined by depletion of membrane-bound levels of pre-labelled [3H]phosphatidylinositols ([3H]PI), S16924 concentration-dependently, surmountably and competitively blocked the activation of phospholipase C by 5-HT. This action was expressed with a pA2 of 7.89 according to Schild analysis. Clozapine likewise inhibited 5-HT-induced alterations in [Ca2+]i and [3H]PI levels with pKbs of 7.43 and 7.84, respectively, whereas haloperidol was inactive (<5.0 in each case). Applied alone, S 16924, clozapine and haloperidol modified levels of neither [Ca2+]i nor [3H]PI. In conclusion, in analogy to clozapine, and in contrast to haloperidol, S16924 behaves as a potent and competitive antagonist at h5-HT2c receptors, the blockade of which may contribute to its distinctive functional profile of activity.
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PMID:Antagonist properties of the novel antipsychotic, S16924, at cloned, human serotonin 5-HT2C receptors: a parallel phosphatidylinositol and calcium accumulation comparison with clozapine and haloperidol. 1083 10

Contractile agonists may stimulate mitogenic responses in airway smooth muscle by mechanisms that involve tyrosine kinases. The role of contractile agonist-evoked activation of tyrosine kinases in contractile signaling is not clear. We addressed this issue using cultured rat airway smooth muscle cells. In these cells, serotonin (5-HT, 1 microM) caused contraction (quantitated by a decrease in cell area), which was blocked by the tyrosine kinase inhibitor genistein (40 microM). Genistein and tyrphostin 23 (40 and 10 microM, respectively) significantly decreased 5-HT-evoked peak Ca(2+) responses, and the effect of genistein could be observed in the absence of extracellular Ca(2+). The specific inhibitor of mitogen-activated protein kinase kinase PD-98059 (30 microM) had no significant effect on peak Ca(2+) levels. Western analysis of cell extracts revealed that 5-HT caused a significant increase in tyrosine phosphorylation of proteins with molecular masses of approximately 70 kDa within 10 s of stimulation but no measurable tyrosine phosphorylation of the gamma isoform of phospholipase C (PLC-gamma). Tyrosine phosphorylation was inhibited by genistein. Furthermore, genistein (40 microM) significantly attenuated 5-HT-induced inositol phosphate production. We conclude that in airway smooth muscle contractile agonists acting on G protein-coupled receptors may activate tyrosine kinase(s), which in turn modulate calcium signaling by affecting, directly or indirectly, PLC-beta activity. It is unlikely that PLC-gamma or the mitogen-activated protein kinase pathway is involved in Ca(2+) signaling to 5-HT.
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PMID:Tyrosine kinase-dependent calcium signaling in airway smooth muscle cells. 1083 18


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