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)

The membrane fraction prepared from rat brain was incubated with 0.5 mM N-ethylmaleimide (NEM) for 10 min. 3H-labelled agonist binding to muscarinic, A1-adenosine, opiate and alpha 2-adrenergic receptors was markedly inhibited by this NEM treatment of membranes, which interfered with the subsequent ADP-ribosylation of endogenous G-proteins by pertussis toxin. This indicated that the toxin target cysteine residues of the G-protein were modified by NEM. The NEM-induced inhibition of agonist bindings was mostly reversed by reconstitution of the alpha-subunits of purified Gi or Go into the membranes. The NEM-induced inhibition, together with the reversal by the G alpha reconstitution, was due to changes in the relative number of high- to low-affinity receptors solely without change in the total (high- plus low-affinity) receptor number. Thus, in NEM-treated membranes endogenous G-proteins become uncoupled from receptors, which were coupled to either Gi alpha or Go alpha. Reconstitution of NEM pre-treated membranes showed that Go acted in preference to Gi in interaction with muscarinic receptors and vice versa in interaction with three other types of receptor. The possible involvement of Go in mediating phospholipase C activation and Gi in mediating adenylate cyclase inhibition is discussed.
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PMID:Selective coupling of purified alpha-subunits of pertussis toxin-substrate GTP-binding proteins to endogenous receptors in rat brain membranes treated with N-ethylmaleimide. 217 93

Cholera toxin (CT) inhibited the in vitro growth of three of four human small-cell lung carcinoma (SCLC) cell lines with a 50% inhibitory concentration of 27-242 ng/ml. Loss of surface membrane ruffling and the capacity of [Tyr4]-bombesin, vasopressin, and fetal calf serum to stimulate increases in intracellular free calcium clearly preceded effects on cellular metabolic activity and cell growth. 125I-[Tyr4]-bombesin binding was unaffected by CT treatment but [Tyr4]-bombesin stimulated phospholipase C activity was decreased in membranes from CT-treated SCLC cells. CT stimulated a rapid but transient increase in intracellular cyclic AMP ([cAMP]i) in SCLC. The effects of CT on susceptible SCLC were not reproduced by elevations of [cAMP]i induced by forskolin or cyclic AMP analogues. GM1 ganglioside, the cellular binding site for CT, was highly expressed in the CT-sensitive but not the CT-resistant SCLC cell lines. In contrast, expression of guanine nucleotide binding protein substrates for ADP-ribosylation by CT was similar. These data demonstrate the existence of a CT-sensitive growth inhibitory pathway in SCLC-bearing GM1 ganglioside. Addition of CT results in decreased responsiveness to several mitogenic stimuli. These results suggest novel therapeutic approaches to human SCLC.
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PMID:Cholera toxin inhibits signal transduction by several mitogens and the in vitro growth of human small-cell lung cancer. 217 11

P2-purinergic receptor agonists (UTP) and formylated peptide receptor agonists (FMLP) were found to be equally efficacious in eliciting rapid 6-7-fold increases in inositol polyphosphate accumulation in differentiated HL-60 granulocytes. The activation of this response by either agonist was substantially but incompletely inhibited in cells treated with pertussis toxin. Thus, in cells containing only 1-10% of the control level of non-ADP-ribosylated Gi-2/3, UTP induced rapid 2-fold increases in inositol polyphosphate accumulation whereas smaller 50% increases were observed in FMLP-stimulated cells. Washed membranes prepared from control and toxin-treated HL-60 cells were used to characterize this toxin-insensitive activation of phospholipase C further. The agonist-independent stimulation of phospholipase C by either millimolar Ca2+ or the nonhydrolyzable GTP analog guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) was only modestly attenuated by toxin treatment. There was a 70-80% decrease in the rate and extent of phospholipase C activity stimulated by GTP per se in the absence of receptor agonists. The rate and extent of FMLP-induced potentiation of GTP-dependent phospholipase C activity were also inhibited by greater than 80% in toxin-treated membranes. Conversely, the potency and efficacy characterizing UTP-induced potentiation of GTP-dependent phospholipase C activity were only modestly attenuated (less than 20% inhibition). The results indicate that P2-purinergic receptors (and perhaps other Ca2(+)-mobilizing receptors) activate both pertussis toxin-sensitive and toxin-insensitive pathways for phospholipase C regulation in phagocytic leukocytes.
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PMID:Pertussis toxin produces differential inhibitory effects on basal, P2-purinergic, and chemotactic peptide-stimulated inositol phospholipid breakdown in HL-60 cells and HL-60 cell membranes. 220 20

In voltage-clamped Xenopus oocytes injected with embryonic guinea pig mRNA, effective concentrations of extracellular ATP elicited an inward fluctuating current. This current, carried by Cl-ions, was mainly dependent upon liberation of Ca2+ ions from stores as demonstrated by experiments using intracellular EGTA loading and TMB-8 superfusion. Neomycin inhibited these fluctuating currents indicating that the transplanted purinoceptor is linked to phospholipase C activity and triggers Ins(1,4,5)P3 formation. Ins(1,4,5)P3 production evoked by external ATP was clearly demonstrated by directly measuring the water-soluble Ins(1,4,5)P3 level in injected oocytes. Finally, it is suggested that the ATP effect was mediated by a Ca2+ release from Ins(1,4,5)P3 sensitive pools since heparin blocked the ATP responsiveness. The acquired purinoceptor may be made apparent to a P2 subtype since ATP and ADP were equipotent in eliciting Cl- current while AMP and Adenosine were ineffective in injected oocytes.
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PMID:Ins(1,4,5)P3 formation and fluctuating chloride current response induced by external ATP in Xenopus oocytes injected with embryonic guinea pig brain mRNA. 226 56

1. Stimulated inositolphospholipid turnover has been proposed as a signal-transducing mechanism in many cell types. It appears to be initiated by stimulation of hydrolysis of inositolphospholipid by a phospholipase C. 2. In human endometrial fibroblasts, estradiol was observed to cause sequential enhancement of [32P]phosphate incorporation into phosphatidic acid (PA) and phosphatidylinositol (PI), indicating an accelerating effect of estradiol on inositolphospholipid turnover. Specific 32P-radioactivity in the gamma-phosphate of ATP was increased in response to estradiol. Estrone or estriol were without any effects. 3. To investigate possible mechanisms by which estradiol activates a phospholipase C enzyme in the fibroblasts, the plasma membrane fraction isolated from the fibroblasts was exposed to estradiol in the presence of guanosine triphosphate (GTP) to detect inositol trisphosphate (IP3) production. The IP3 production was Ca2+ dependent, a dependency not affected by estradiol. 4. However, ATP decreased the Ca2+ concentration required for IP3 production in a dose-dependent manner; adenosine diphosphate (ADP), cytidine triphosphate (CTP) showed no effects. 5. These findings from cell and cell-free systems might suggest that estradiol stimulates a phospholipase C, as a result of enhancement of intracellular ATP synthesis, but not as a result of a direct effect on the enzyme molecule or direct activation of receptor-phospholipase C unit. 6. This may give us new insight into estrogen-stimulated cellular phenomenon through some mechanisms other than that classically associated with the action of estrogen.
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PMID:Phospholipase C in human endometrial fibroblasts and its regulation by estrogens. 228 72

Anti-human platelet p24/CD9 (p24/monoclonal antibody 7) causes the activation of platelets and in the presence of calcium induces platelet aggregation. Our studies suggest that platelet response to this antibody is mediated at least in part by the pertussis toxin-sensitive guanine nucleotide-binding proteins (G proteins) that stimulate phosphoinositide hydrolysis and inhibit adenylate cyclase. Prior exposure of saponin-treated platelets to anti-p24/CD9 inhibited the [32P] ADP-ribosylation of the alpha 41 protein by pertussis toxin. Platelet aggregation induced by this antibody is preceded by and/or accompanied by accelerated phosphatidylinositol turnover, the generation of inositol phosphates and diacylglycerol (DAG), calcium mobilization, and protein phosphorylation. The production of inositol phosphate(s) was measurable within 15 s of either anti-p24/CD9 or thrombin addition. Within 10 s of antibody addition (10 micrograms/ml), the level of DAG was 200% over that of the control and similar to that observed with 2 units/ml thrombin (201% over that of the control). Therefore, as it appears to be true for thrombin, platelet response upon binding of anti-p24/CD9 is primarily mediated by the activation of phospholipase C. When platelets pretreated with aspirin (200 microM) and apyrase (1 mg/ml) were subsequently exposed to anti-p24/CD9, aggregation still occurred. This indicates that neither secreted ADP nor thromboxane generation is required for this aggregation response. Using indo-1 and ratio cytofluorometry, we observed that an increase in platelet cytosolic calcium is a relatively early event and occurs in either the presence or absence of calcium in the external media. Phosphorylation studies of platelet proteins showed that anti-p24/CD9 binding to platelets caused increased phosphorylation of four proteins with apparent molecular masses of 50,000, 47,000, 36,000, and 20,000 daltons. These studies suggest that platelet activation mediated by the surface protein p24/CD9 is mainly through the stimulation of a phospholipase C, the activation of which is responsible for the generation of second messengers inositol trisphosphate and DAG.
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PMID:The activation of human platelets mediated by anti-human platelet p24/CD9 monoclonal antibodies. 230 80

The CD9 molecule is a 24 kDa surface-membrane glycoprotein present on platelets and a variety of haematopoetic and non-haematopoetic tissues. In the present study we utilized specific inhibitors of thromboxane A2 (TxA2) formation (aspirin), protein kinase C [H-7 [1-(5-isoquinolinesulphonyl)-2-methylpiperazine]] and autocrine stimulation by secreted ADP (apyrase) to modify platelet activation by a monoclonal antibody ALB-6 to the CD9 antigen. This activation is only partially inhibited by aspirin alone but, in combination with either H-7 or apyrase, more than 50% inhibition of platelet aggregation and secretion was observed. This combination of inhibitors was also required to inhibit effectively the phosphorylation of myosin light chain and the 47 kDa substrate of protein kinase C. Intracellular Ca2+ flux monitored by the fluorescent dye fura-2 showed that this was almost completely mediated by the aspirin-sensitive TxA2 pathway. We suggest that the aspirin-insensitive pathway is primarily mediated by phospholipase C formation of diacylglycerol to activate protein kinase C. The inhibition by apyrase suggests a strong dependency on autocrine stimulation by secreted ADP to fully activate both phospholipase C and express fibrinogen-binding sites mediating platelet aggregation. This alternate pathway of phospholipase C activation by ALB-6 may be mediated by cytoplasmic alkalinization [monitored by SNARF-1 (5'(6')-carboxy-10-bismethylamino-3-hydroxy-spiro-[7H- benzo[c]xanthine-1',7(3H)-isobenzofuran]-3'-one) fluorescence of the dye]. Both activation pathways are dependent on intact antibodies, since F(ab')2 fragments of SYB-1, a monoclonal antibody against the CD9 antigen with activation characteristics identical with those of ALB-6, do not elicit activation. Besides thrombin, collagen is another physiological agonist shown to induce aspirin-insensitive activation. Similarities to ALB-6 in collagen sensitivity to apyrase in combination with aspirin inhibitors were noted with respect to aggregation and secretion, as well as a complete block of Ca2+ flux by aspirin. However, it is unlikely that collagen activation is mediated by the CD9 antigen, since SYB-1 F(ab')2 fragments had no effect on collagen activation and aspirin also completely blocked the alkalinization response to collagen, in contrast with ALB-6.
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PMID:Stimulus-response coupling in human platelets activated by monoclonal antibodies to the CD9 antigen, a 24 kDa surface-membrane glycoprotein. 231 2

alpha-Thrombin and phorbol 12,13-dibutyrate stimulated the mono(ADP-ribosyl)ation of a 42-kDa cytosolic protein of human platelets. This effect was mediated by protein kinase C activation and was inhibited by protein kinase C inhibitor staurosporine. It also was prevented by prostacyclin, which is known to inhibit the phospholipase C-induced formation of 1,2-diacylglycerol, which is one of the endogenous activators of protein kinase C. On sodium dodecyl sulfate/polyacrylamide gel electrophoresis, the 42-kDa protein that is ADP-ribosylated by alpha-thrombin was clearly distinct from the alpha subunits of membrane-bound inhibitory and stimulatory guanine nucleotide-binding regulatory proteins, respectively Gi alpha and Gs alpha; the 47-kDa protein that is phophorylated by protein kinase C in platelets; and the 39-kDa protein that has been shown to be endogenously ADP-ribosylated by agents that release nitric oxide. This information shows that agonist-induced activation of protein kinase leads to the ADP-ribosylation of a specific protein. This covalent modification might have a functional role in platelet activation.
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PMID:Agonist-induced ADP-ribosylation of a cytosolic protein in human platelets. 233 84

The characterization of P2 gamma purinoceptors on vascular endothelial cells has progressed rapidly since their existence was first demonstrated in 1983. They transduce the actions of extracellular ATP and ADP--endothelium-dependent relaxation, prostacyclin synthesis, endothelial cell mitogenesis--which play a vital role in the interaction between platelets (a rich source of extracellular adenine nucleotides) and the vessel wall. Release of prostacyclin limits the extent of intravascular platelet aggregation following vascular damage and platelet stimulation, while the mitogenic effect may accelerate the repair of a lesion. P2 gamma receptors on endothelial cells are coupled to a phospholipase C by a GTP-binding protein. Jean-Marie Boeynaems and Jeremy Pearson explain how the increases in cytoplasmic Ca2+ and diacylglycerol resulting from this initial event mediate several further effects. In particular, activation of a Ca2(+)-sensitive phospholipase A2 explains the increased synthesis of prostacyclin, while the phosphorylation of several proteins by calmodulin-dependent kinases modulates other endothelial cell functions.
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PMID:P2 purinoceptors on vascular endothelial cells: physiological significance and transduction mechanisms. 240 10

At least two signal-generating systems are involved in the actions of various hormonal factors in human platelets--the adenylate cyclase system and the phosphoinositide-metabolizing pathway. The formation of cyclic AMP (cAMP) by the adenylate cyclase system--consisting of the catalyst itself, the Ns and Ni proteins, and various hormone receptors--is stimulated by prostaglandins and adenosine, and is inhibited by alpha 2-adrenergic agonists, ADP, vasopressin, platelet-activating factor, and thrombin. On the other hand, the formation of inositol trisphosphate and diacylglycerol by the phosphoinositide-metabolizing pathway is stimulated by some of the latter agents, particularly by thrombin. There are apparently several mutual interactions between these two signal-generating systems. On the one hand, increases in the level of cAMP inhibit the formation of inositol phosphates and diacylglycerol. It is presently unclear whether this inhibitory effect of cAMP is due to a direct action at the phospholipase C itself or to an indirect mechanism, for example, a depletion of the substrate of the enzyme. On the other hand, protein kinase C, which is activated by diacylglycerol, largely interferes with the adenylate cyclase system. This kinase, when activated by diacylglycerol or phorbol esters, apparently phosphorylates the guanine nucleotide-binding alpha-subunit of Ni, which results in an impairment or loss of the inhibitory hormonal signal transduction to the adenylate cyclase. Thus, available evidence indicates that the two signal-generating systems present in platelet membranes are not completely separated, and furthermore suggests that they may even be causally related to each other.
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PMID:Interactions between the hormone-sensitive adenylate cyclase system and the phosphoinositide-metabolizing pathway in human platelets. 243 28


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