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)

Productive interaction between receptors and G proteins involves multiple intracellular receptor domains, but the role of individual receptor amino acids in directing the selection of specific signaling pathways has not yet been identified. Sequence alignment of several G protein-coupled receptors identified a highly conserved threonine residue in the i2 loop of the 5-hydroxytryptamine 1A (5-HT1A) receptor that is a putative protein kinase C phosphorylation consensus site and is located in a predicted amphipathic alpha-helical domain. To examine the role of this conserved threonine residue in 5-HT1A receptor coupling to Gi/Go proteins, this residue was mutated to alanine (T149A mutant). Wild-type and mutant 5-HT1A receptors were stably transfected into both Ltk- and GH4C1 cells to investigate receptor coupling to multiple signaling pathways. In both cell lines, the T149A mutant displayed similar agonist affinities as the wild-type receptor. In Ltk- cells, the T149A 5-HT1A receptor inhibited cAMP accumulation by 30% compared with wild-type (83%). A 2.6-fold increase in intracellular calcium (due to phospholipase C-mediated calcium mobilization) was observed for the wild-type receptor upon the addition of 100 nM 5-HT; whereas the T149A 5-HT1A receptor failed to mediate a calcium mobilization response at equivalent receptor levels to wild-type. When transfected in GH4C1 cells, the T149A receptor mutant fully inhibited basal cAMP and partially inhibited Gs-stimulated cAMP accumulation compared with wild-type receptor (57 +/- 14% versus 86 +/- 2%). In contrast, the T149A 5-HT1A receptor mutant failed to block the influx of calcium induced by calcium channel agonist (+/-)-Bay K8644, whereas the wild-type 5-HT1A receptor inhibited the calcium influx by 40%. Thus, the Thr149 residue is directly involved in G protein coupling to calcium mobilization (mediated by betagamma subunits of Gi2) and to inhibition of calcium channel activation (mediated by betagamma subunits of Go) but plays a minor role in coupling to alpha1-mediated inhibition of cAMP accumulation. The conserved i2 loop threonine may serve as a G protein contact site to direct the signaling specificity of multiple receptors.
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PMID:A conserved threonine residue in the second intracellular loop of the 5-hydroxytryptamine 1A receptor directs signaling specificity. 922 26

To determine the mechanisms of receptor-dependent Ca2+ sensitization in airway smooth muscle, canine tracheal smooth muscle (CTSM) was permeabilized with alpha-toxin or beta-escin. Although the effects of 5-hydroxytryptamine (100 microM), histamine (100 microM), and the thromboxane A2 analogue U-46619 (100 microM) were negligible, carbachol (100 microM) and endothelin-1 (ET-1, 1 microM) evoked additional contractions of 47.0 +/- 5.90% and 25.0 +/- 5.37% (n = 6) at pCa 6.7 with GTP (3 microM) (normalized to the maximum contraction at pCa 4.5) in alpha-toxin-permeabilized CTSM. GDP-beta-S (1 mM) reversed the carbachol and ET-1 responses completely. GTP-gamma-S (30 microM) and 4 beta-phorbol 12,13-dibutyrate (PDBu, 3 microM) increased the Ca2+ sensitivity (median effective pCa) of contraction by 1.8- and 4.4-fold, respectively (n = 4-11, P < 0.05). The effects of saturating concentrations of GTP-gamma-S and PDBu were additive. A synthetic peptide (T2) corresponding to the actin-binding site of calponin caused a dose-dependent contraction of beta-escin permeabilized CTSM, with the peak effect (25 +/- 4%, n = 4) at 1200 microM, PDBu (3 microM) caused contraction of the T2 peptide-treated CTSM. In conclusion, Ca2+ sensitization of CTSM depends on receptor type and is mediated by G proteins and protein kinase C whose effects are additive, with a partial contribution by calponin.
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PMID:Receptor-dependent G protein-mediated Ca2+ sensitization in canine airway smooth muscle. 923 49

The potent vasoconstrictor substances, 5-hydroxytryptamine (5-HT), angiotensin II (A II), and bradykinin bind to G-protein coupled receptors and activate phospholipase C-beta. Using the Fura-2 technique and microfluorometry we found that all three agonists induce a transient increase in intracellular calcium ([Ca2+]i) by releasing stored calcium in human renal artery smooth muscle cells. Using binding of [3H]-phorbol dibutyrate (PDBu) to quantify membrane-associated protein kinase C (PKC) we also showed that 5-HT, A II and bradykinin induced a rapid but transient translocation of protein kinase C (PKC) to the plasma membrane. The time-course of the rise in [Ca2+]i was similar to that of the increase in [3H]-PDBu binding, suggesting transient activation of the calcium dependent alpha-isoform of PKC. Following prolonged pre-treatment with tetradecanoyl phorbol acetate (100 nmol L-1), which down-regulates PKC-alpha and delta, the angiotensin-induced PKC translocation was lost. 5-HT, A II or bradykinin were unable to increase cell proliferation or act as a co-mitogens with platelet-derived growth factor in human vascular smooth muscle cells. Thus, transient increases in [Ca2+]i or PKC activity by a vasoconstrictor agent are insufficient to cause vascular smooth muscle proliferation.
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PMID:5-Hydroxytryptamine, angiotensin and bradykinin transiently increase intracellular calcium concentrations and PKC-alpha activity, but do not induce mitogenesis in human vascular smooth muscle cells. 924 83

Serotonin (5-hydroxytryptamine; 5-HT) 5-HT2A and 5-HT2C receptors belong to the class of phosphoinositide-specific phospholipase C (PLC)-linked receptors. Conditions were established for measuring 5-HT2A-linked and 5-HT2C-linked PLC activity in membranes prepared from previously frozen rat frontal cortex and caudate. In the presence of Ca2+ (300 nM) and GTPgammaS (1 microM), 5-HT increased PLC activity in caudate membranes. Pharmacological analysis using the selective 5-HT2A antagonist, spiperone, and the nonselective 5-HT(2A/2C) antagonist, mianserin, demonstrated that over half of the 5-HT-stimulated PLC activity was due to stimulation of 5-HT2C receptors as opposed to 5-HT2A receptors. Radioligand binding assays with [3H]RP 62203 and [3H]mesulergine were used to quantify 5-HT2A and 5-HT2C sites, respectively, in caudate. From these data, the Bmax for caudate 5-HT2A sites and 5-HT2C sites was 165.4 +/- 9.7 fmol/mg of protein and 49.7 +/- 3.3 fmol/mg of protein, respectively. In contrast to that in caudate, PLC activity in frontal cortex was stimulated by 5-HT in a manner that was inhibited by the 5-HT2A-selective antagonists, spiperone and ketanserin. Taken together, the results indicate that 5-HT2A- and 5-HT2C-linked PLC activity can be discerned in brain regions possessing both receptor subtypes using membranes prepared from previously frozen tissue. More importantly, significant 5-HT2C-mediated phosphoinositide hydrolysis was observed in caudate, despite the relatively low density of 5-HT2C sites. The significance of these observations with respect to the physiological function of 5-HT2C receptors is discussed.
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PMID:The serotonin 5-HT2C receptor is a prominent serotonin receptor in basal ganglia: evidence from functional studies on serotonin-mediated phosphoinositide hydrolysis. 932 73

Neuropeptide Y (NPY) significantly potentiates the constrictor actions of noradrenaline and ATP on blood vessels via a pertussis toxin (PTX)-sensitive mechanism involving Gi/o (alpha beta gamma) protein subunits (Gi/o, GTP-binding proteins sensitive to PTX). In Chinese hamster ovary K1 (CHO K1) cells expressing specific receptors for these neurotransmitters, stimulation of Gi/o protein-coupled receptors for NPY and other neurotransmitters can augment the Gq/11-coupled (Gq/11, GTP-binding proteins insensitive to PTX) alpha 1B adrenoceptor- or ATP receptor-induced arachidonic acid (AA) release and inositol phosphate (IP) production (early events which may precede vasoconstriction). In this study, we have assessed the role of G beta gamma subunits in the synergistic interaction between Gi/o- (NPY Y1, 5-hydroxytryptamine 5-HT1B, adenosine A1) and Gq/11- [ATP P2Y2 (P2U)]-coupled receptors on AA release by using the specific abilities of regions of the beta-adrenergic receptor kinase (beta ARK1 residues 495-689) and the transducin alpha subunit to associate with G-protein beta gamma subunit dimers and to act as G beta gamma subunit scavengers. Transient expression of beta ARK1(495-689) in CHO K1 cells heterologously expressing NPY Y1 receptors had no significant effect on the PTX-insensitive ability of ATP to stimulate AA release. Stimulation of NPY Y1 receptors (as well as the endogenous 5-hydroxytryptamine 5-HT1B receptor and the transiently expressed human adenosine A1 receptor) resulted in a PTX-sensitive augmentation of ATP-stimulated AA release, which was inhibited by expression of both G beta gamma subunit scavengers. Expression of beta ARK1(495-689) similarly inhibited NPY Y1 receptor augmentation of ATP-stimulated IP production (a measure of phospholipase C activity), a step thought to precede the NPY Y1 receptor-augmented protein kinase C-dependent AA release previously observed in these cells. These experiments demonstrate that G beta gamma subunits, as inhibited by two different G beta gamma scavengers, significantly contribute to the synergistic interaction between NPY Y1 Gi/o- and Gq/11-coupled receptor activity, and are required for the augmentation of IP production and AA release observed in this model cell system.
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PMID:Role of G-protein beta gamma subunits in the augmentation of P2Y2 (P2U)receptor-stimulated responses by neuropeptide Y Y1 Gi/o-coupled receptors. 935 46

1. Extracellular adenosine triphosphate (ATP) is mitogenic for vascular smooth muscle cells (VSMC) and stimulates several events that are important for cell proliferation: DNA synthesis, protein synthesis, increase of cell number, immediate early genes, cell-cycle progression, and tyrosine phosphorylation. 2. Receptor characterization indicates mitogenic effects of both P2U and P2Y receptors. The P2X receptor is lost in cultured VSMC and is not involved. Several related biological substances such as UTP, ITP, GTP, AP4A, ADP, and UDP are also mitogenic. 3. Signal transduction is mediated via Gq-proteins, phospholipase C beta, phospholipase D, diacyl glycerol, protein kinase C alpha, delta, Raf-1, MEK, and MAPK. 4. ATP acts synergistically with polypeptide growth factors (PDGF, bFGF, IGF-1, EGF, insulin) and growth factors acting via G-protein-coupled receptors (noradrenaline, neuropeptide Y, 5-hydroxytryptamine, angiotensin II, endothelin-1). 5. The mitogenic effects have been demonstrated in rat, porcine, and bovine VSMC and cells from human coronary arteries, aorta, and subcutaneous arteries and veins. 6. The trophic effects on VSMC and the abundant sources for extracellular ATP in the vessel wall make a pathophysiological role probable in the development of atherosclerosis, neointima-formation after angioplasty, and possibly hypertension.
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PMID:Extracellular ATP: a growth factor for vascular smooth muscle cells. 959 70

Previous studies have established that dopamine (DA) can stimulate phosphoinositide (PI) metabolism in the CNS and in the periphery. The present study summarizes our attempt to find a cell line that expresses this dopaminergic system. We describe that the stable clonal HN33.11 cell line, established by fusion of mouse hippocampal cells with neuroblastoma cells (N18TG2) that originate from A/J mouse, natively expresses the D1 DA receptor system that couples to PI hydrolysis. In this cell line, 500 microM DA or SKF38393 produced 43 and 75% increases in inositol phosphate (IP) accumulations, respectively. In contrast, noradrenaline or 5-hydroxytryptamine did not affect IP accumulations. The formation of IP that was stimulated by DA or SKF38393 was selectively blocked by the D1 DA receptor antagonist SCH23390 with IC50 values of 13 and 16 microM. This response was not mediated by the D1A DA receptor and was cyclic AMP-independent, as HN33.11 cells did not express this receptor, and DA or SKF38393 was unable to stimulate the formation of cyclic AMP. In Ca2+-free/100 microM EGTA medium, basal IP level was reduced by 31.5%, but SKF38393-stimulated PI hydrolysis was not affected. SKF38393-stimulated IP accumulation was also not affected by pertussis toxin (PTX) treatment (200 ng/ml), suggesting that this dopaminergic response is mediated by PTX-insensitive G proteins. Co-immunoprecipitation studies indicated that in membranes of HN33.11 cells, D1-like binding sites are coupled to G alpha(q) protein. Blockade of SKF38393-induced PI hydrolysis with antiserum against phospholipase C (PLC) isozymes, performed in permeabilized cells, as well as co-immunoprecipitation studies implicate PLCbeta3 and PLCbeta4 in this dopaminergically mediated PI hydrolysis cascade. The results indicate that HN33.11 cells express a D1-like DA receptor that couples to PLCbeta3/4 via G alpha(q) protein. These cells may therefore be a useful model system for investigating this receptor system.
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PMID:Characterization of the phosphoinositide-linked dopamine receptor in a mouse hippocampal-neuroblastoma hybrid cell line. 979 18

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

The effect of 5-hydroxytryptamine (5-HT) on phospholipase C (PLC)-mediated phosphoinositide (PI) hydrolysis and intracellular Ca2+ ([Ca2+]i) changes was investigated in canine cultured aorta smooth muscle cells (ASMCs). 5-HT-stimulated inositol phosphate (IP) accumulation was time and concentration dependent with a half-maximal response (pEC50) and a maximal response at 6.4 and 10 microM, n = 6, respectively. Stimulation of ASMCs by 5-HT produced an initial transient peak followed by a sustained, concentration-dependent elevation in [Ca+]i. The half-maximal response (pEC50) values of 5-HT for the peak and sustained plateau were 7.1 and 6.9, respectively. Ketanserin and mianserin (1 and 3 nM), 5-HT2A antagonists, were equipotent and had high affinity in antagonising the 5-HT-induced IP accumulation and [Ca2+]i change with pK(B) values of 8.6-9.1 and 8.6-9.4, respectively. In contrast, the concentration-effect curves of 5-HT-induced IP and [Ca2+]i responses were not shifted until the concentrations of NAN-190 and metoctopramide (5-HT1A and 5-HT3 receptor antagonists, respectively) were increased to as high as 1 microM with pK(B) values of 5.7-6.3 and 6.1-6.6, respectively, indicating that the 5-HT receptor-mediated responses had low affinity for these antagonists. Pre-treatment of ASMCs with pertussis toxin (100 ng/mL, 24 h) caused a significant inhibition of 5-HT-induced IP accumulation and [Ca2+]i change in ASMCs. Depletion of external Ca2+ or removal of Ca2+ by addition of EGTA led to a significant attenuation of IP accumulation and [Ca2+]i change induced by 5-HT. Influx of external Ca2+ was required for the 5-HT-induced responses, because Ca2+-channel blockers--verapamil, nifedipine and Ni2+--partly inhibited the 5-HT-induced IP accumulation and Ca2+ mobilisation. The sustained elevation of [Ca2+]i response to 5-HT was dependent on the presence of external Ca2+. Removal of external Ca2+ by addition of 5 mM EGTA during the sustained phase caused a rapid decline in [Ca2+]i to lower than the resting level. The sustained elevation of [Ca2+]i could then be evoked by addition of 1.8 mM Ca2+ in the continued presence of 5-HT. These results demonstrate that 5-HT directly stimulates PLC-mediated PI hydrolysis and Ca2+ mobilisation, at least in part, through a pertussis toxin-sensitive G protein in canine ASMCs. 5-HT2A receptors may be predominantly mediating IP accumulation, and subsequently IP-induced Ca2+ mobilisation may function as the transducing mechanism for 5-HT-stimulated contraction of aorta smooth muscle.
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PMID:5-Hydroxytryptamine-induced phosphoinositide hydrolysis and Ca2+ mobilisation in canine cultured aorta smooth muscle cells. 1037 10

The effects of concomitant P1-receptor stimulation on peak intracellular Ca2+ release by extracellular adenosine 5'-triphosphate (ATP) and 5-hydroxytryptamine (5-HT) were investigated in cultured airway smooth-muscle (ASM) cells. The results show that peak Ca2+ release to ATP is enhanced by preincubation with adenosine (ADO) and with the specific A3 receptor agonist 1-Deoxy-1-(6-([(3-iodophenyl)methyl] amino)-9H-purin-9-yl)-N-methyl-beta-D-ribofuranuronamide (1B-MECA). The response to 5-HT, a smooth-muscle contractile agonist, was also enhanced after preincubation with ADO. Further measurements showed that this enhancement of the response to ATP was dependent on extracellular calcium because it was abolished by the removal of Ca2+ from the extracellular fluid and by incubation with the calcium channel blocker nifedipine. In addition, there was no difference between the levels of total inositol phosphates measured in the presence of ATP alone or of ADO + ATP. AACOCF3, a specific blocker of phospholipase A2, decreased the peak Ca2+ response to ATP and abolished the enhanced response to ATP and 5-HT produced by ADO. We conclude that stimulation of P1 and P2 receptors in ASM cells activates not only phospholipase C but also phospholipase A2. The enhancement of ATP-induced and 5-HT-induced Ca2+ release is due to Ca2+ influx from the extracellular fluid through a Ca2+ channel presumably modulated by arachidonic acid. These data show that endogenous ADO may modulate airway hyperresponsiveness by enhancing the ASM response to contractile agonists.
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PMID:Mechanisms of the potentiation by adenosine of adenosine triphosphate-induced calcium release in tracheal smooth-muscle cells. 1038 90


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