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)

Addition of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) to intact Chinese hamster lung fibroblasts (CCL39) depolarized by high K+ concentrations results in activation of phosphoinositide-specific phospholipase C (PLC) (at GTP gamma S concentrations greater than 0.1 mM), inhibition of adenylate cyclase (between 10 microM and 0.5 mM), and activation of adenylate cyclase (above 0.5 mM). Since GTP gamma S-induced activation of PLC is dramatically enhanced upon receptor-mediated stimulation of PLC by alpha-thrombin, we conclude that in depolarized CCL39 cells GTP gamma S directly activates various guanine nucleotide-binding regulatory proteins (G proteins) coupled to PLC (Gp(s)) and to adenylate cyclase (Gi and Gs). Pretreatment of cells with pertussis toxin strongly inhibits GTP gamma S-induced activation of PLC and inhibition of adenylate cyclase. GTP gamma S cannot be replaced by other nucleotides, except by guanosine 5'-O-(2-thiodiphosphate) (GDP beta S), which mimics after a lag period of 15-20 min all the effects of GTP gamma S, with the same concentration dependence and the same sensitivity to pertussis toxin. We suggest that GDP beta S is converted in cells into GTP beta S, which acts as GTP gamma S. Since cell viability is not affected by a transient depolarization, these observations provide a simple method to examine long-term effects of G protein activation on DNA synthesis. We show that a transient exposure of G0-arrested CCL39 cells to GTP gamma S or GDP beta S under depolarizing conditions is not sufficient by itself to induce a significant mitogenic response, but markedly potentiates the mitogenic action of fibroblast growth factor, a mitogen known to activate a receptor-tyrosine kinase. The potentiating effect is maximal after 60 min of pretreatment with 2 mM GTP gamma S. GDP beta S is equally efficient but only after a lag period of 15-20 min. Mitogenic effects of both guanine nucleotide analogs are suppressed by pertussis toxin. Since the activation of G proteins by GTP gamma S under these conditions vanishes after a few hours, we conclude that a transient activation of G proteins facilitates the transition G0----G1 in CCL39 cells, whereas tyrosine kinase-induced signals are sufficient to mediate the progression into S phase.
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PMID:Guanosine 5'-O-(3-thiotriphosphate) and guanosine 5'-O-(2-thiodiphosphate) activate G proteins and potentiate fibroblast growth factor-induced DNA synthesis in hamster fibroblasts. 216 8

In stimulated human platelets dense-granule secretion in response to the 'weak agonists' ADP, adrenaline, platelet activating factor and low concentrations of thrombin as well as Ca2+ mobilisation in response to thrombin are enhanced by a Na+/H+ exchanger. In the present study the role of this antiport in collagen stimulated human platelets was examined. While stimulation of platelets loaded with the fluorescent intracellular pH-sensitive dye, bis-carboxyethyl-5-(6)-carboxyfluorescein (BCECF) with thrombin resulted in the activation of the Na+/H+ exchanger, activation of this antiport did not occur in collagen-stimulated platelets. The lack of antiport activity in response to collagen using BCECF-loaded platelets correlated with the lack of any functional role of the antiport in collagen stimulated platelets. In the presence of a Na+/H+ exchange inhibitor, ethylisopropylamiloride, neither collagen-induced platelet aggregation or dense-granule secretion was affected. Furthermore, while the removal of extracellular Na+ (Na+ext), a condition that also prevents activation of the antiport, inhibited dense-granule secretion in response to a low concentration of thrombin, collagen-induced secretion was potentiated. This potentiatory effect could not be attributed to changes in either the membrane potential or in collagen-induced phospholipase C or protein kinase C activity. The present results indicate that in contrast to the 'weak agonists' (1) collagen-induced platelet activation does not require activation of the Na+/H+ exchanger and (2) Na+ext per se is an inhibitor of collagen-induced secretion.
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PMID:Stimulation of human platelets by collagen occurs by a Na+/H+ exchanger independent mechanism. 216 91

The responses to alpha- and gamma-thrombin were studied in normal and Bernard-Soulier platelets labelled with [32P]phosphate, to investigate the relationship between thrombin binding to the platelet membrane glycoprotein Ib (GPIb) and thrombin-induced platelet activation. For this purpose we conducted parallel studies of the kinetics of platelet aggregation, granule secretion, hydrolysis of polyphosphoinositides, formation of phosphatidic acid, phosphorylation of the myosin light chain (p20) and of the 43 kDa protein (p43), and thromboxane B2 formation. Like alpha-thrombin, gamma-thrombin activated control platelets via all the above metabolic responses, but only after a prolonged lag. In Bernard-Soulier platelets, alpha-thrombin induced polyphosphoinositide hydrolysis and phosphatidic acid formation, p20 and p43 phosphorylation, thromboxane B2 formation, secretion and to a lesser extent aggregation, but only after a prolonged lag. The metabolic responses of Bernard-Soulier platelets to gamma-thrombin were very similar to those of control platelets. We have previously showed that GPIb which is not present in Bernard-Soulier platelets binds alpha- but not gamma-thrombin. The present results indicate that thrombin binding to GPIb is not directly coupled either with the activation of phospholipase C specific to polyphosphoinositides, or with the activation of protein kinase C and phospholipase A2. However, thrombin binding to GPIb appears to promote an early mechanism which accelerates all the platelet responses.
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PMID:The common pathway for alpha- and gamma-thrombin-induced platelet activation is independent of GPIb: a study of Bernard-Soulier platelets. 216 23

Rabbit platelet-rich plasma was incubated with [32P]orthophosphate, after which the platelets were washed, further incubated in the absence or presence of verapamil and subsequently stimulated with PAF-acether or thrombin. In the absence of verapamil, a rapid increase in radioactivity in phosphatidic acid was observed in platelets stimulated with PAF-acether or thrombin. This was inhibited by verapamil over the concentration range 10(-7) to 10(-4) M, at which concentration the rise in phosphatidic acid was completely abolished. In unstimulated platelets, 10(-4) M verapamil induced an increase in radioactivity in polyphosphoinositides but not significantly in phosphatidylinositol. When these verapamil-treated platelets were stimulated with PAF-acether or thrombin, there was a rapid, sustained loss of the additional radioactivity induced in the polyphosphoinositides by verapamil. Polyphosphoinositide radioactivity remained unchanged in platelets stimulated in the absence of verapamil. Verapamil may stimulate formation of a separate pool of polyphosphoinositide which is susceptible to agonist-induced phospholipase C, and failure to re-synthesize this polyphosphoinositide could result from inhibition of phosphatidic acid synthesis.
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PMID:Verapamil inhibits phosphatidic acid formation and modifies phosphoinositide metabolism in stimulated platelets. 217 46

In order to evaluate the role of phosphoinositide turnover in growth factor action, we expressed human M1 muscarinic acetylcholine (Hm1) receptors in Chinese hamster lung fibroblasts (CCL39 cell line). In the transfected cells (39M1-81 clone), but not in wild type fibroblasts, the muscarinic agonist carbachol induced a release of inositol phosphates as strong as alpha-thrombin, a very potent growth factor and activator of phosphoinositide-specific phospholipase C (PLC) in this cell system. In contrast to thrombin, carbachol-stimulated PLC activity was not inhibited by pertussis toxin treatment of cells. At concentrations that elicited a comparable initial rate of inositol phosphate release (10 nM for thrombin and 0.1 mM for carbachol), both agents gave rise to an identical calcium signal and equally stimulated Na+/H+ exchange and the transcription of the early genes c-jun, c-fos, and c-myc. Surprisingly, however, carbachol is not a mitogen for 39M1-81 cells, and even if tested in association with insulin or fibroblast growth factor, its effects on cell proliferation remained weak when compared with thrombin. Also, the muscarinic agonist did not stimulate soft agar colony forming capacity and did not prevent growth arrest in Go upon serum deprivation of cycling 39M1-81 cells. The failure of carbachol to induce cell proliferation could not be attributed to rapid and complete desensitization of Hm1 receptors nor to the activation of inhibitory pathways like adenylyl cyclase stimulation. We conclude that strong and persistent activation of phosphoinositide turnover elicits early biochemical events generally associated with mitogenesis, but is not sufficient to stimulate or maintain continuous cell proliferation. On the basis of our results, we postulate that thrombin mitogenesis depends critically on signaling events different from phosphoinositide turnover, possibly the stimulation of a receptor tyrosine kinase or a Gi protein-activated tyrosine kinase.
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PMID:Strong and persistent activation of inositol lipid breakdown induces early mitogenic events but not Go to S phase progression in hamster fibroblasts. Comparison of thrombin and carbachol action in cells expressing M1 muscarinic acetylcholine receptors. 217 13

alpha-Thrombin, gamma-thrombin, and platelet-activating factor each stimulated the mobilization of intracellular Ca2+ stores in aspirin-treated human platelets. This was followed by desensitization of the receptors, as shown by the return of the Ca2+ level to basal values and by the fact that a subsequent addition of a second different agonist, but not the same agonist, could again elicit a response. Epinephrine, acting on alpha 2-adrenergic receptors, was by itself ineffective at mobilizing Ca2+ stores. However, when added after the thrombin-induced response, epinephrine could evoke a considerable release of Ca2+ from cellular stores. This appeared to be due to epinephrine recoupling thrombin receptors to phospholipase C. In support of this, epinephrine was able to induce the formation of inositol triphosphate when added after the response to thrombin had also become desensitized. Alone, epinephrine was without effect. Pre-activation of protein kinase C with the phorbol ester abolished these effects of epinephrine, suggesting that epinephrine was working by activating a protein which could be inactivated by phosphorylation. Our current work is to characterize this protein that may be a member of the Gi, GTP-binding protein family.
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PMID:Regulation of hormone-induced Ca2+ mobilization in the human platelets. 219 Aug 17

Since protein kinase C (PKC) plays an important role in the control of platelet biological responses, we investigated whether it can be involved in the enhanced platelet reactivity to thrombin which is observed in spontaneously hypertensive rats (SHR) in comparison to that observed in controls (WKY). PKC activity was determined by measuring the phosphorylation of P47 protein (the endogenous substrate of PKC in the platelet). Mean effective concentration (EC50) values for phorbol ester and synthetic diacylglycerol (direct activators of PKC) were similar in SHR and WKY, thus revealing similar intrinsic activity of PKC in both rat strains. EC50 values for thrombin were approximately 30% lower in SHR v WKY. Enhanced PKC activity in SHR is likely the result of an increased diacylglycerol formation and release of Ca2+ from intracellular pools, consequent to an increased thrombin-induced phospholipase C activity.
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PMID:Protein kinase C and platelet reactivity in spontaneously hypertensive rats. 226 Nov 54

Trypsin causes rapid activation of intact platelets that mimics many actions of thrombin, including the stimulation of phospholipase C (PLC). We have examined the effects of thrombin and trypsin on PLC in a platelet membrane preparation using exogenous [3H]-phosphatidylinositol 4,5-bisphosphate (PIP2) as substrate. Trypsin induced PIP2 breakdown, which was maximal at 20 micrograms/ml, but was reduced at higher concentrations. alpha- and gamma-Thrombins also stimulated PLC-induced hydrolysis of PIP2 in membranes. This effect was inhibited by leupeptin. Exogenous [3H]phosphatidylinositol 4-monophosphate (PIP) was hydrolyzed in response to both thrombin and trypsin in the same ratio as PIP2. Activation of membrane-bound PLC persisted after removal of thrombin and trypsin. The hydrolysis of [3H]phosphatidylinositol was not activated by alpha-thrombin and trypsin. We examined the question of whether calpain was involved in the observed PLC activation by thrombin and trypsin. Although dibucaine activated a Ca2(+)-dependent protease as judged by the hydrolysis of actin-binding protein and by the activation of phosphoprotein phosphatases, it failed to stimulate the generation of phosphatidic acid in 32P-prelabeled platelets. Moreover, when PLC was assayed in the membranes, the addition of Ca2(+)-activated neutral proteinases did not increase the rate of hydrolysis of either PIP or PIP2. Our results show that proteases such as trypsin and thrombin are able to stimulate membrane-bound PLC, but this activation does not seem to be related to calpain.
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PMID:Persistent activation of platelet membrane phospholipase C by proteolytic action of trypsin and thrombin. 229 26

In a previous paper (Rath, H. M., Doyle, G. A. R., and Silbert, D. F. (1989) J. Biol. Chem. 264, 13387-13390), we reported a selection for the isolation of Chinese hamster lung fibroblasts (CCL39) defective in thrombin-induced mitogenesis. One mutant, D1-6b, had decreased production of inositol phosphates when challenged with activators of phosphatidylinositol turnover and extracts of this mutant showed a marked decrease in phospholipase C (PLC) activity toward phosphatidylinositol. In the current studies, the PLC activities of wild type CCL39 and D1-6b cytosolic extracts are further characterized. Wild type cytosol had at least two phosphatidylinositol-specific PLC isoenzymes, which could be separated by anion exchange chromatography and behaved differently in thermal inactivation studies. Since gel filtration of PLC activity in wild type extracts gave Mr values similar to that of previously characterized PLCs (140,000-200,000), immunoblots with antibodies to bovine brain isoenzymes were used to show that the PLC activities obtained by anion exchange chromatography were PLC-delta and PLC-gamma. Immunoblots with mutant D1-6b cytosol confirmed the presence of the PLC-gamma but showed no detectable PLC-delta. This activity in the mutant extracts eluted at the same conductivity on anion exchange columns and had the same kinetics of thermal inactivation as the PLC-gamma found in the wild type extracts. PLC-gamma from mutant extracts was active in assays containing phospholipid detergent mixed micelles but not in assays utilizing phospholipid vesicles, in sharp contrast to PLC-gamma from CCL39 extracts, which was active under either condition. Thus, the phosphatidylinositol-specific phospholipase C activity of mutant D1-6b is diminished both by the loss of PLC-delta and by the compromised behavior of PLC-gamma.
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PMID:Characterization of phosphatidylinositol-specific phospholipase C defects associated with thrombin-induced mitogenesis. 230 41

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


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