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)

CD69 is a signal transducing disulfide-linked homodimer functionally expressed on platelets, CD3bright thymocytes, and activated lymphocytes. In an attempt to investigate early molecular events in CD69-mediated cell activation we studied the relative contribution of phospholipase A2 (PLA2) and phosphatidylinositol-specific phospholipase C-dependent pathways during platelet activation induced by CD69 stimulation. Thromboxane A2 (TXA2) synthetase inhibitor and TXA2R inhibitor R68070 were able to inhibit platelet aggregation induced by CD69 stimulation, indicating that TXA2 was the main mediator of the response. CD69-induced arachidonic acid release and TXA2 production were essentially PLA2 dependent because they could be blocked by the PLA2 inhibitor quinacrine. Inositol 1,3,4-trisphosphate generation was clearly detectable after CD69 cross-linking, but it was completely abrogated by quinacrine and R68070 and therefore secondary to TXA2 release and TXA2R engagement. Finally, direct measurement of enzymatic activity in vitro using radiolabeled phospholipid vesicles showed that CD69 cross-linking resulted in PLA2-dependent arachidonic acid and lysophosphatidylcholine generation from phosphatidylcholine, which was sensitive to quinacrine but not to R68070. By contrast, CD69-induced 1,2-diacylglycerol release from phosphatidylinositol 4,5-bisphosphate was blocked by both inhibitors. These results indicate a preferential involvement of PLA2 in CD69-dependent signal transduction in platelets and provide evidence for the unique role of PLA2-mediated activation pathways in transmembrane receptor signaling.
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PMID:Preferential involvement of a phospholipase A2-dependent pathway in CD69-mediated platelet activation. 131 60

Thromboxane (Tx)A2 has been reported to play an important role in modulating airway contractility under various conditions associated with airways inflammation. To identify its potential role in contributing to airway smooth muscle (ASM) hyperplasia, a characteristic feature of asthmatic airways, the mitogenic effect and mechanism of action of TxA2 were investigated in cultured rabbit ASM cells. The stable TxA2 mimetics, carbocyclic TxA2 (CTA2) and U-46619, elicited dose-dependent (10(-12) to 10(-6) M) increases in ASM cell number and induced acute augmentation of intracellular inositol 1,4,5-trisphosphate accumulation. The latter action was blocked by neomycin, a phospholipase C inhibitor; however, neomycin had no effect on the promitogenic action of the TxA2 mimetics. In contrast, TxA2-induced ASM cell proliferation was inhibited by inhibitors of phospholipase A2 and 5-lipoxygenase, as well as blockade of the leukotriene (LT)D4 receptor. Moreover, in complementary studies, we found that exogenous administration of LTD4 (10(-14) to 10(-6) M) potently induced ASM cell proliferation and that the TxA2 mimetics evoked the enhanced release of endogenous leukotrienes from the cultured ASM cells. Taken together, these observations provide new evidence that 1) TxA2 stimulates ASM cell proliferation; 2) the promitogenic effect of TxA2 is associated with activation of phospholipase A2; and 3) the latter mediates ASM cell proliferation via the release and autocrine mitogenic action of leukotrienes. The findings support a potential role for TxA2 in contributing to the characteristic increase in ASM cell mass obtained in asthma and other chronic airway diseases.
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PMID:Role and mechanism of thromboxane-induced proliferation of cultured airway smooth muscle cells. 144 59

S. aureus alpha-toxin and E. coli hemolysin (Hly) represent two prototypes of pore-forming cytolysins. Both are established virulence factors and have been implicated in the development of septic lung failure. Low doses of these agents cause thromboxane-mediated vasoconstriction and edema formation in isolated perfused rabbit lungs. In a preceding investigation, we observed that alpha-toxin causes overt endothelial cell damage in these lungs, as demonstrable by electron microscopy (Seeger W, Birkemeyer RG, Ermert L, Suttorp N, Bhakdi S, Duncker HR: Lab Invest 63:341, 1990). Here, we present results of a parallel study conducted with E. coli hemolysin. Thromboxane-dependent pulmonary hypertension was suppressed by the addition of acetylsalicylic acid to the perfusion fluid in all cases. Administration of 0.2 hemolytic units (HU; i.e., 20 ng/ml protein) resulted in progressive weight gain after a lag period of 10 to 15 minutes, and 30 minutes after toxin application the gravimetrically determined capillary filtration coefficients (Kfc) were increased greater than 10-fold. Perfusion was terminated when the total lung weight gain surpassed 20 gm. 0.12 HU/ml E. coli hemolysin caused 2- to 3-fold increased capillary filtration coefficients values within 110 minutes, concomitant with intermediate quantities of edema formation (9.7 +/- 2.7 gm). Potassium liberation in the absence of lactate dehydrogenase release occurred in all toxin treated lungs. Electron microscopic examination after perfusion fixation revealed interstitial edema formation in areas remote from the blood-gas exchange barrier. Increased numbers of endothelial plasmalemmal vesicles were visualized at the very onset of edema formation in lungs exposed to 0.2 HU/ml, and after a 110-minute exposure to 0.12 HU/ml of the toxin, but not in lungs exhibiting severe edema (greater than 20 gm weight gain). In contrast to our previous results with alpha-toxin, endothelial cells displayed normal electron density here and were not detached from the fused basal lamina. Hence, although both pore formers provoke severe vascular leakage in our experimental model, the underlying pathways probably divert fundamentally from each other.
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PMID:Induction of severe vascular leakage by low doses of Escherichia coli hemolysin in perfused rabbit lungs. 153 89

We tested the hypothesis that increased systemic vascular resistance in spontaneously hypertensive rats may be secondary to enhanced phospholipase C activity in response to vasoconstrictor stimuli. Activation of phospholipase C by angiotensin II (Ang II), thromboxane A2, arginine vasopressin, and endothelin-1 was compared in cultured glomerular mesangial cells and mesenteric vascular smooth muscle cells taken from 13- to 14-week-old hypertensive and normotensive Wistar-Kyoto rats (blood pressure, 185 +/- 1 versus 135 +/- 2 mm Hg). Phospholipase C was assessed by measuring cytosolic free calcium and by the accumulation of radiolabeled inositol phosphates. Basal cytosolic calcium did not differ between mesangial cells taken from both strains but was greater in smooth muscle cells from hypertensive rats (210.1 +/- 8.2 versus 149.2 +/- 4.7 nM). The responsiveness of cytosolic calcium and inositol phosphate accumulation to Ang II was significantly enhanced in mesangial cells from hypertensive rats (10(-7) M Ang II: peak increase of calcium, 1,266 +/- 181 versus 603 +/- 93 nM; percent increment of inositol phosphates at 1 minute, 266 +/- 26 versus 98 +/- 10%). Vascular smooth muscle cells from hypertensive rats, when compared with normotensive rats, showed a similar augmentation of Ang II-stimulated intracellular calcium and inositol phosphates. Thromboxane A2-induced enhancement of intracellular calcium and inositol phosphate accumulation in vascular smooth muscle cells was also greater in hypertensive animals. However, the responses to vasopressin and endothelin in mesangial or vascular smooth muscle cells did not differ between the normotensive and hypertensive animals. There was no significant difference in Ang II receptor number and affinity between hypertensive- and normotensive-derived mesangial cells. We conclude that genetically increased blood pressure in rats may be secondary to enhanced post-receptor signaling in glomerular mesangial cells activated by Ang II and to enhanced signaling in vascular smooth muscle cells stimulated by either Ang II or thromboxane A2.
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PMID:Phospholipase C responses in cells from spontaneously hypertensive rats. 156 63

The mode of action of E5510, 4-cyano-5,5-bis(4-methoxyphenyl)-4-pentenoic acid, which has very potent anti-platelet activities, was investigated by examining its effects on the biochemical responses in the process of human platelet activation. In a whole-cell system, E5510 inhibited the increased turnover of inositol phospholipids arising from phospholipase C activation, arachidonic acid release from phospholipids by phospholipase A2, mobilization of intracellular free Ca2+, protein kinase C activation, and thromboxane A2 production. In a cell-free system, E5510 inhibited cyclooxygenase activity and cyclic AMP-dependent phosphodiesterase activity in a dose-dependent manner. An elevation of cyclic AMP in platelets was also observed at a relatively high concentration of E5510. It was suggested that receptor-mediated turnover of inositol phospholipids, intracellular Ca2+ increase, arachidonic acid release from phospholipids and protein kinase C activation might be indirectly inhibited by the increased cyclic AMP level in platelets. Thromboxane A2 production in the whole-cell system was very strongly inhibited by E5510, and the IC50 for this effect was 100 times lower than that of direct inhibition of cyclooxygenase in the cell-free system. It was concluded that although the primary mode of action of E5510 is the inhibition of the cyclooxygenase pathway of positive signal transduction in platelets, E5510 has another mode of action by increasing platelet cyclic AMP, which can act as a negative messenger in platelet signal transduction, and these multiple sites of action synergistically antagonize platelet cellular activation.
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PMID:A new anti-platelet drug, E5510, has multiple suppressive sites during receptor-mediated signal transduction in human platelets. 164 15

Elevated eicosanoid biosynthesis characterizes certain forms of human and experimental glomerular proliferative disease. Thromboxane A2 (TxA2) and other prostaglandins (PG) act through specific receptors and mechanisms of intracellular signal transduction in human mesangial cells. We studied the actions of U-46619, a TxA2 mimetic which stimulates mesangial phospholipase C, and of the PGI2 analogue, Iloprost, a potent activator of adenylate cyclase, on proliferation of cultured human mesangial cells. When applied alone to quiescent cells, U-46619 had only weak mitogenic activity, as assessed by [3H]thymidine [( 3H]-TdR) incorporation and cell counts. On the other hand, addition of U-46619 10 minutes prior to stimulation of the cells with 1 to 17% fetal bovine serum (FBS) for 24 hours, potently and dose-dependently inhibited FBS-stimulated [3H]-TdR incorporation. Similarly, U-46619 inhibited the effects of 10 ng/ml platelet-derived growth factor (PDGF), epidermal growth factor or basic fibroblast growth factor on [3H]-TdR incorporation, by 55, 79 and 88%, respectively. The effects of U-46619 were not mimicked by another stimulus of phospholipase C, angiotensin II. Iloprost also inhibited FBS-activated proliferation. Neither eicosanoid inhibited the rise of cytosolic Ca2+ induced by FBS or PDGF. The actions of TxA2 and Iloprost in cultured cells point to multiple functional interactions between eicosanoids and growth factors in the control of mesangial cell proliferation.
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PMID:Regulation of human mesangial cell growth in culture by thromboxane A2 and prostacyclin. 169 33

1. rPDGF stimulates PGE2 release in wild type, but not ras transformed NIH-3T3 cells. 2. Ras transformation blocks PGE2 release by inhibiting phospholipase C activation, IP3 synthesis, and Ca2+ mobilization. 3. rPDGF stimulation of wild type NIH-3T3 cells increases both prostaglandin H synthase (PGHS) mRNA levels and PGHS enzyme levels as measured by immunoblot. However, PGHS gene transcription is not required for PDGF-stimulated PGE2 release. 4. Ras transformed NIH-3T3 cells display elevated basal PGE2 synthesis, and very high levels of both PGHS mRNA and enzyme. rPDGF does not further stimulate PGHS gene transcription. 5. Exogenous PGE2 attenuates rPDGF-stimulated cell proliferation in both wild type and ras transformed cells. 6. These data suggest that increased PGHS gene expression and enhanced basal PGE2 synthesis may be in response to the unregulated growth of ras transformed cells.
Adv Prostaglandin Thromboxane Leukot Res 1991
PMID:Elevated prostaglandin H synthase gene expression in ras-transformed cells. 182 90

Thromboxane (Tx) A2 is a product of cyclooxygenase catalyzed metabolism of arachidonic acid. It is formed via prostaglandin (PG) endoperoxide intermediates (PGG2 and PGH2) by a specific synthase. PGH2 appears to exert the same biologic effects as TxA2. The cDNA for a TxA2 receptor has been cloned from a human placental library. Although pharmacologic and biochemical studies suggest the presence of multiple isoforms, this remains to be confirmed at the molecular level. A hydropathy plot of the deduced amino acid sequence of the available clone suggests that it has 7 transmembrane spanning domains, typical of a G protein linked receptor. Pharmacologic studies imply that Tx receptors in platelets are linked to phospholipase C activation via pertussis toxin insensitive G proteins. Candidates include the 42 kD Gq and the 60 kD Ge. TxA2 acts as an amplifying signal for platelet agonists and the response to this eicosanoid is tightly regulated. Mechanisms include rapid hydrolysis of the agonist to the inactive TxB2, autoinactivation of Tx synthase, rapid homologous TxA2 receptor desensitization due to receptor-G protein uncoupling, coincidental sensitization to counterregulatory Gs linked receptor systems and stimulation of prostacyclin formation by TxA2. Due to its role as an amplification signal in platelet activation, inhibition of Tx synthesis and action is an effective mechanism for preventing platelet-dependent vascular occlusion. Aspirin is of proven efficacy in this regard. Tx synthase inhibitors and antagonists are under clinical investigation.
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PMID:Mechanisms of platelet activation: thromboxane A2 as an amplifying signal for other agonists. 189 57

Thromboxane A2 (TXA2) induces platelet shape change, secretion, and aggregation. Using a novel TXA2/prostaglandin endoperoxide receptor antagonist, [1r-[1 alpha(Z),2 beta,3 beta,5 alpha]]-(+)-7-[5-[[(1,1'- biphenyl)-4-yl]methoxy]-3-hydroxy-2-(1-piperidinyl) cyclopentyl]-4-heptenoic acid hydrochloride (GR32191), we demonstrate that these responses are mediated by at least two receptor-effector systems. GR32191 non-competitively inhibited platelet aggregation to the TXA2 mimetics, (15S)-hydroxy-11,9-(epoxymethano) prostadienoic acid (U46619) and [1S-(1 alpha,2 beta(5Z),3 alpha (1E,-3S), 4 alpha)]-7-[3-(3-hydroxy-4-(p-iodophenoxy)-1-butenyl)7- oxabicyclo[2.2.1]hept-2yl]-5-heptenoic acid by binding irreversibly to a TXA2/prostaglandin endoperoxide receptor. Dissociation of [3H]GR32191 from human platelets demonstrated two specific binding sites, one which was rapidly dissociating and a site to which binding was essentially irreversible. Stimulation by U46619 of platelets incubated with GR32191 and subsequently washed to expose the reversible binding site failed to aggregate or to secrete [3H]5-hydroxy-tryptamine; formation of inositol phosphates and activation of protein kinase C were markedly suppressed. In contrast, platelet shape change and calcium stimulation remained at 90% of control. Furthermore, stimulation of the reversible binding site with U46619 induced aggregation in the presence of ADP, demonstrating its functional importance in amplifying the response to other agonists. These data suggest that TXA2 mediates platelet activation through at least two receptor-effector systems; one linked to phospholipase C activation, resulting in platelet aggregation and secretion and a second site mediating an increase in cytosolic calcium and platelet shape change.
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PMID:The response to thromboxane A2 analogues in human platelets. Discrimination of two binding sites linked to distinct effector systems. 213 29

Monoclonal antibody P256, which is specific for glycoprotein IIb-IIIa complex, was found to induce aggregation of normal platelets in plasma. The mechanism of platelet activation induced by this monoclonal antibody was thoroughly studied. The divalent binding to the IIb-IIIa molecule was necessary for triggering aggregation since Fab' fragments did not induce aggregation as did IgG and F(ab')2 fragments; however, F(ab')2 did not induce the release as did the whole IgG. P256-induced aggregation was accompanied by release of all three granule constituents, namely dense granules, alpha-granules and lysosomes, with parallel kinetics showing half-maximum release 50 s after addition of P256. Thromboxane synthesis was initiated at the same time. Using 32P-prelabeled platelets, no variation in level of [32P]phosphatidylinositol 4,5-bisphosphate could be detected in the first minute after P256 addition, indicating no activation of the calcium-independent phospholipase C specific for polyphosphoinositol phospholipid. P256 induced a calcium mobilization as measured by Indo-1 fluorescence of about the third of that measured in the presence of a thrombin concentration giving the same intensity of aggregation. P256 induced phosphorylation of the myosin light chain p20 and of the main substrate of protein kinase C, p43. Addition of aspirin inhibited almost totally calcium mobilization and partially aggregation, release and protein phosphorylations. By contrast, in the absence of external calcium, although no aggregation could occur, the release reaction was only partially reduced. In this activation, the glycoprotein IIb-IIIa complex thus appears to play a role in modulating platelet response, not only via calcium fluxes but also in activating protein kinase C responsible for p43 phosphorylation.
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PMID:Activation of platelets induced by mAb P256 specific for glycoprotein IIb-IIIa. Possible evidence for a role for IIb-IIIa in membrane signal transduction. 236 45


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