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)

We have investigated synthesis of 3-phosphorylated inositol lipids in growth factor-stimulated Swiss 3T3 cells. Those growth factors tested which act via tyrosine kinase-containing receptors (platelet-derived growth factor (PDGF), insulin growth factor I (IGF-I), epidermal growth factor (EGF), and basic fibroblast growth factor (bFGF)) caused the rapid synthesis of [32P]PtdIns(3,4)P2 and [32P]PtdIns(3,4,5)P3 (PtdIns is phosphatidylinositol) in [32P]P(i)-prelabeled cells and the appearance of an inositol lipid 3-OH kinase in antiphosphotyrosine immunoprecipates. In contrast, those growth factors tested which act via G-protein-coupled receptors (bombesin, vasopressin, prostaglandin E1) were unable to stimulate either of the above responses. Furthermore, while PDGF was able to increase the formation of PtdIns(3,4)P2 and PtdIns(3,4,5)P3 in streptolysin-permeabilized cells, guanosine 5'-3-(thio)triphosphate and guanyl-5'-yl imidodiphosphate were not. These results suggest that Swiss 3T3 cells possess the machinery for tyrosine kinase but not G-protein-mediated activation of PtdIns(4,5)P2 3-OH kinase; a situation which is the inverse to that recently described for human neutrophils. The tyrosine kinase-containing receptors differed markedly in their relative abilities to elevate the levels of [32P] PtdIns(3,4,5)P3 (ranked in the order PDGF greater than or equal to IGF-I greater than EGF greater than bFGF), [32P]Ptd-OH (PDGF greater than EGF greater than bFGF; undetectable for IGF-I), and [32P]PtdIns4P (EGF greater than bFGF greater than PDGF; undetectable for IGF-I) in [32P]P(i)-prelabeled cells. These differences are epitomized by IGF-I, which was the joint most powerful stimulus for [32P] PtdIns(3,4,5)P3 formation, but was unable to stimulate a measurable accumulation of [32P]Ptd-OH (and hence, by deduction, was unable to stimulate phospholipase C). These results indicate that there is a differential ability among the tyrosine kinase-containing receptors present in a single cell to recruit phospholipase C and PtdIns(4,5)P2 3-OH kinase into their signalling complexes and further emphasizes the notion that the rapid synthesis of PtdIns(3,4,5)P3 may be a signalling event.
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PMID:Receptor specificity of growth factor-stimulated synthesis of 3-phosphorylated inositol lipids in Swiss 3T3 cells. 132 11

Recent cloning studies confirm the presence of two subtypes of bombesin (Bn) receptors. In contrast to the gastrin-releasing peptide (GRP)-preferring subtype, which has been widely studied, nothing is known about the cellular mechanisms of the neuromedin B (NMB)-preferring subtype, which occurs widely in the central nervous system and gastrointestinal tissues, partially because of the lack of a cell line with functional receptors. In the present study we have investigated Bn receptors on the rat glioblastoma cell line C-6, reported to contain mRNA of the NMB receptor subtype. Binding of 125I-[D-Tyr0]NMB to these cells was time- and temperature-dependent, saturable, reversible, and only inhibited by Bn receptor agonists or antagonists. For Bn receptor agonists the relative potencies were: NMB (1.7 nM) approximately equal to litorin (3 nM) greater than ranatensin (8 nM) greater than Bn (19 nM) greater than neuromedin C (NMC) (210 nM) greater than GRP (500 nM). These relative affinities were almost identical to those for the NMB receptor subtype on rat oesophageal tissue and for Balb 3T3 cells stably transfected with the NMB receptor subtype. These potencies differed from those for the GRP receptor subtype on rat pancreatic acini [Bn approximately equal to litorin (4 nM) greater than ranatensin, NMC, GRP (15-20 nM) much greater than NMB (351 nM)]. The relative potencies of four different classes of Bn receptor antagonists were compared. Results from C-6 tumour cells agreed closely with those for binding to the NMB receptor subtype on rat oesophageal tissue and in Balb 3T3 cells stably transfected with this receptor, and differed markedly from those for binding to the GRP receptor subtype on rat pancreatic acini. Four Bn receptor antagonists had a higher affinity for the GRP subtype ([D-Phe6]Bn-(6-13)ethyl ester (500 x), [D-Phe6][psi 13-14,Cpa14]Bn- (6-14) (70 x) (where psi 13-14 refers to the replacement of the -CONH- peptide bond between Leu13 and Met14 by -CH2NH2) [psi 13-14,Leu14]Bn, [D-Phe6]Bn-(6-13) propylamide (30 x)] and two had a higher affinity for the NMB subtype on C-6 cells and transfected cells ([D-Pro4,D-Trp7,9,10] substance P-(4-11) (9 x) and [Tyr4,D-Phe12]Bn (18 x)]. In C-6 tumour cells, Bn receptor agonists caused an increase in cytosolic Ca2+ and the generation of inositol phosphates. For both responses, NMB was more than 50-fold more potent than GRP. Neither NMB nor GRP increased cyclic AMP. These results demonstrate that the rat glioblastoma cell line C-6 possesses functional NMB-preferring Bn receptors, and agonist occupation activates phospholipase C, thus increasing cytosolic Ca2+ and inositol phosphate formation. Because the interaction of Bn-related peptides with C-6 cell receptors is identical with that reported in other tissues containing the mRNA for the NMB subtype, this cell line should prove useful in exploring further the cellular basis of action of the peptides that interact with this receptor in the central nervous system and various other tissues.
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PMID:Activation of neuromedin B-preferring bombesin receptors on rat glioblastoma C-6 cells increases cellular Ca2+ and phosphoinositides. 132 46

The protein kinase C (PKC) activator 12-O-tetradecanoylphorbol 13-acetate (TPA) has been shown to potentiate the stimulatory effect of ethanol on the hydrolysis of phosphatidylethanolamine (PtdEtn) in NIH 3T3 fibroblasts. Following an initial 20-min period, the main product of PtdEtn degradation in cells treated with TPA plus ethanol was ethanolamine phosphate. Here, we have examined the regulatory role of PKC and the possible catalytic role of phospholipase C in the formation of ethanolamine phosphate. TPA, bryostatin, and bombesin, direct or indirect activators of PKC, had similar potentiating effects on ethanol-induced formation of [14C]ethanolamine phosphate from [14C]PtdEtn in [14C]ethanolamine-prelabelled NIH 3T3 fibroblasts. At lower concentrations of ethanol (40-80 mM), significant stimulation of ethanolamine phosphate formation required longer treatments (2 h or longer). The combined effects of TPA (100 nM) and ethanol (50-200 mM) on ethanolamine phosphate formation were not inhibited by the PKC inhibitors staurosporine or 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7). In contrast, these inhibitors significantly inhibited TPA-induced formation of ethanolamine, catalyzed by a phospholipase-D-type enzyme. In membranes isolated from TPA+ethanol-treated cells, enhanced formation of ethanolamine phosphate was maintained for at least 20 min. Down-regulation of PKC by prolonged (24-h) treatment of NIH 3T3 fibroblasts by 300 nM TPA enhanced, while overexpression of alpha-PKC in Balb/c fibroblasts diminished, the stimulatory effect of ethanol on the formation of ethanolamine phosphate. Finally, addition of the protein phosphatase inhibitor okadaic acid (2 microM) to fibroblasts inhibited TPA+ethanol-induced formation of ethanolamine phosphate. These results suggest that alpha-PKC-mediated protein phosphorylation may negatively regulate PtdEtn hydrolysis and that the potentiating effect of TPA may result, at least partly, from increased degradation of this PKC isoform.
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PMID:The long-term combined stimulatory effects of ethanol and phorbol ester on phosphatidylethanolamine hydrolysis are mediated by a phospholipase C and prevented by overexpressed alpha-protein kinase C in fibroblasts. 132 80

Treatment of Swiss 3T3 cells with a subsaturating concentration of recombinant Pasteurella multocida toxin (rPMT) markedly potentiated the production of inositol phosphates induced by bombesin, vasopressin, and endothelin but not by platelet-derived growth factor (PDGF) (AA and BB homodimers). Similarly, the neuropeptides but not PDGF caused a shift in the dose-dependent increase in inositol phosphates induced by rPMT. The rate of accumulation of inositol phosphates induced by bombesin was increased 2-fold by rPMT treatment while that of PDGF was unaffected. rPMT treatment also enhanced bombesin-induced inositol(1,4,5)trisphosphate, the direct product of phosphatidylinositol 4,5-bisphosphate hydrolysis. In contrast, treatment of cells with rPMT had no effect on the tyrosine phosphorylation of phospholipase C gamma. Depletion of protein kinase C increased rPMT-induced inositol phosphates in a manner similar to that observed for bombesin but not PDGF. Thus, rPMT selectively potentiates neuropeptide-mediated inositol phosphate production. The action of rPMT on phosphatidylinositol 4,5-bisphosphate hydrolysis persisted in streptolysin O-permeabilized cells. Addition of guanosine 5'-O-(beta-thiodiphosphate) to permeabilized cells markedly reduced rPMT-induced inositol phosphates in a time- and dose-dependent manner. rPMT also increased the sensitivity of phospholipase C for free calcium. Our results strongly suggest that the action of rPMT facilitates the coupling of G protein to phospholipase C.
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PMID:Pasteurella multocida toxin selectively facilitates phosphatidylinositol 4,5-bisphosphate hydrolysis by bombesin, vasopressin, and endothelin. Requirement for a functional G protein. 133 89

The receptor that interacts with the mammalian bombesin-related peptide neuromedin B (NMB) is ubiquitous in the gastrointestinal tract and central nervous system. However, little is known regarding its cellular mechanisms of action. This receptor has been recently cloned, sequenced, and stably transfected into BALB 3T3 fibroblasts, permitting detailed study of the pharmacology and coupled biological activities of this receptor. In the present study, we compare the ability of transfected receptors to alter cell function with that of receptors natively expressed in small numbers by the rat glioblastoma cell line C6. NMB inhibited binding of 125I-[D-Tyro]NMB with high affinity in transfected cells (Ki = 3.08 +/- 0.14 nM) and in C6 cells (Ki = 1.90 +/- 1.10 nM), whereas the bombesin-related agonists gastrin-releasing peptide (GRP) and [D-Phe6, D-Ala11, Leu14]bombesin(6-16) (GRP analogue) had 100- and 300-fold lower affinities, respectively, for NMB receptors in either cell type. For both cell systems, maximal binding was observed between 5 and 15 min at 22 degrees. Both cell types internalized NMB at similar rates, with > 70% of bound ligand being internalized by 60 min at 22 degrees. The nonhydrolyzable guanosine analogue guanosine 5'-(beta,gamma-imido)triphosphate was equipotent in causing a decrease in binding of 125I-[D-Tyro]NMB due to decreased receptor affinity in both cell types, without a change in receptor number, demonstrating that the NMB receptor remained coupled to a guanine nucleotide-binding protein in both native and transfected cells. In both cell systems, NMB increased inositol monophosphate, inositol bisphosphate, and inositol trisphosphate in a time-dependent fashion. Inositol phosphates were increased in a dose-dependent fashion, with similar half-maximal values being obtained for NMB in both cell types (transfected, 1.01 +/- 0.09 nM; C6, 2.09 +/- 0.15 nM) and for the GRP analogue (transfected, 1855 +/- 140 nM; C6, 2129 +/- 250 nM). NMB mobilized intracellular Ca2+ in both cell systems, and the dose-response curves were superimposible (EC50 for transfected, 0.10 +/- 0.08 nM; C6, 0.11 +/- 0.02 nM). These data demonstrate that activation of the receptor for NMB stimulates phospholipase C and increases intracellular Ca2+. These results also demonstrate that transfected and native NMB receptors behave similarly, suggesting that the transfected cell line will be useful in future studies investigating ligand-receptor interactions, as well as in molecular biological studies of the structure-function relationship of the receptor.
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PMID:Neuromedin B receptors retain functional expression when transfected into BALB 3T3 fibroblasts: analysis of binding, kinetics, stoichiometry, modulation by guanine nucleotide-binding proteins, and signal transduction and comparison with natively expressed receptors. 133 12

The proliferation of the bronchial epithelium and tumors associated with this tissue is controlled by various growth factors. The main factor is Gastrin Releasing Peptide (GRP), the human counterpart of the amphibian bombesin. These neuropeptides also act as neuromediators and gut hormones. All peptides of this family share a conserved C terminal sequence which is required for biological activity. The determination of this sequence has provided the basis for the design of specific agonist and antagonist peptides and for the generation of monoclonal antibodies (Mab). GRP interacts with a receptor coupled to a G protein and the signalling process leads to the activation of phospholipase C and kinases, and the mobilization of calcium. GRP promotes the proliferation of foetal and adult bronchial epithelium and of small cell lung cancer (SCLC) cells. GRP is also an autocrine growth factor for some SCLC cell lines. The growth of these lines is reduced in vitro and in vivo by MAb and specific antagonists. Hyperplasia of GRP producing cells has been shown in various lung diseases in adults and children. Pharmacological data on GRP suggest that its antagonists could be used in the treatment of SCLC (in addition to chemotherapy) and of interstitial lung disease. The cloning of the GRP receptor should facilitate the design of specific and potent antagonists of the peptide.
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PMID:[The role of gastrin releasing peptide as a lung growth factor]. 156 25

The mitogenic neuropeptides bombesin and vasopressin markedly increased tyrosine and serine phosphorylation of multiple substrates in quiescent Swiss 3T3 fibroblasts, including two major bands of Mr 90,000 and 115,000. Tyrosine phosphorylation of these proteins was increased as judged by immunoprecipitation of 32Pi-labeled cells and immunoblotting of unlabeled cells with monoclonal antiphosphotyrosine antibodies, elution with phenyl phosphate, and phospho amino acid analysis. Phosphotyrosyl proteins generated by bombesin and vasopressin did not correspond either by apparent molecular weight or by immunological and biochemical criteria to several known tyrosine kinase substrates, including phospholipase C gamma, the microtubule-associated protein 2 kinase, GTPase-activating protein, or phosphatidylinositol kinase. The effect was rapid (within seconds), concentration dependent, and inhibited by specific receptor antagonists for both bombesin and vasopressin. The endothelin-related peptide, vasoactive intestinal contractor, also elicited a rapid and concentration-dependent tyrosine/serine phosphorylation of a similar set of substrates. These results demonstrate that neuropeptides, acting through receptors linked to GTP-binding proteins, stimulate tyrosine phosphorylation of a common set of substrates in quiescent Swiss 3T3 cells and suggest the existence of an additional signal transduction pathway in neuropeptide-induced mitogenesis.
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PMID:Bombesin, vasopressin, and endothelin rapidly stimulate tyrosine phosphorylation in intact Swiss 3T3 cells. 164 10

The murine pancreatic receptor for bombesin and gastrin releasing peptide (GRP) has been characterized. Analysis of the binding of 125I-GRP to membranes indicates a single class of sites (10(-13) mol/mg protein) with Kd of 43 pM. A 70 kDa membrane protein was cross-linked to 125I-GRP by bis(sulfosuccinimidyl) suberate; labeling was blocked by GRP, GRP (14-27), AcGRP(20-27), GRP(18-27), bombesin and ranatensin, was partially blocked by [Leu13 psi (CH2NH)Leu14]bombesin and was unaffected by GRP(21-27) and GRP(1-16). The IC50 values for the competitive displacement of 125I-GRP from intact membranes by these peptides were similar to those obtained by the cross-linking experiments showing that the 70 kDa protein is the GRP receptor. The GRP receptor is G-protein coupled; divalent cations are required for high-affinity binding and nonhydrolyzable GTP analogs decrease receptor affinity. In minced pancreas, GRP caused a dose-dependent increase in inositol phosphates implicating phospholipase C in signal transduction. We suggest that the murine pancreatic receptor for bombesin/GRP is a 70 kDa membrane protein, is associated with a G-protein and stimulates phosphatidylinositol turnover.
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PMID:Characterization of the murine pancreatic receptor for gastrin releasing peptide and bombesin. 165 Sep 53

Mastoparan, a basic tetradecapeptide isolated from wasp venom, is a novel mitogen for Swiss 3T3 cells. This peptide induced DNA synthesis in synergy with insulin in a concentration-dependent manner; half-maximum and maximum responses were achieved at 14 and 17 microM, respectively. Mastoparan also stimulated DNA synthesis in the presence of other growth promoting factors including bombesin, insulin-like growth factor-1, and platelet-derived growth factor. The synergistic mitogenic stimulation by mastoparan can be dissociated from activation of phospholipase C. Mastoparan did not stimulate phosphoinositide breakdown, Ca2+ mobilization or protein kinase C-mediated phosphorylation of a major cellular substrate or transmodulation of the epidermal growth factor receptor. In contrast, mastoparan stimulated arachidonic acid release, prostaglandin E2 production, and enhanced cAMP accumulation in the presence of forskolin. These responses were inhibited by prior treatment with pertussis toxin. Hence, mastoparan stimulates arachidonic acid release via a pertussis toxin-sensitive G protein in Swiss 3T3 cells. Arachidonic acid, like mastoparan, stimulated DNA synthesis in the presence of insulin. The ability of mastoparan to stimulate mitogenesis was reduced by pertussis toxin treatment. These results demonstrate, for the first time, that mastoparan stimulates reinitiation of DNA synthesis in Swiss 3T3 cells and indicate that this peptide may be a useful probe to elucidate signal transduction mechanisms in mitogenesis.
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PMID:Mastoparan, a novel mitogen for Swiss 3T3 cells, stimulates pertussis toxin-sensitive arachidonic acid release without inositol phosphate accumulation. 170 71

The inositol phosphate responses to substance P, bombesin, cholecystokinin, and the muscarinic cholinergic agonist methacholine were examined in the rat pancreatoma cell line AR4-2J. It was found that each agonist produced a distinct temporal pattern of inositol phosphate formation. Furthermore, these different response patterns resulted, at least in part, from different patterns of homologous receptor desensitization. The response to substance P desensitized rapidly and completely within 90 sec. After a 10-15-min refractory period, the response recovered with a t1/2 of approximately 1 hr. The response to methacholine also completely desensitized. However, in this case desensitization developed slowly over the course of 40 min, and no recovery of responsiveness was detected for up to 45 min after the cessation of stimulation. The inositol phosphate responses to bombesin and cholecystokinin were similar to one another and appeared to be composed of two phases. Initially, there was a robust activation of phospholipase C. This initial phase was followed within 20 sec by a second phase of lesser magnitude. For bombesin, attenuation of the initial phase was due to rapid, but only partial, desensitization of the response. Furthermore, the concentration of bombesin required to maintain the second phase of the response was about 100-fold lower than that required to maximally activate the initial phase of the response. These results may indicate multiple mechanisms for the regulation of different phospholipase C-linked receptors in this cell line.
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PMID:Different modes of regulation for receptors activating phospholipase C in the rat pancreatoma cell line AR4-2J. 171 68


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