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:3.4.21.5 (thrombin)
33,306 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The present study has examined the role of vitamin E, a natural lipid antioxidant, in the production of diacylglycerol (DAG) and phosphatidic acid (PA) in thrombin-stimulated human endothelial cells. Cells were labelled with [3H]myristate and the incorporation and distribution of [3H]myristate into cellular lipids was not affected by vitamin E. However, in response to thrombin stimulation, considerably more PA and less DAG were formed in cells enriched with vitamin E. The time-course of thrombin stimulation indicated that vitamin E attenuated the accumulation of sustained DAG levels with a concomitant increase in PA. Direct determination of DAG mass further confirmed that vitamin E suppresses the accumulation of DAG induced by thrombin. In the presence of ethanol, the formation of [3H]phosphatidylethanol (PEt) in [3H]myristate-labelled cells stimulated by thrombin was unaffected by vitamin E enrichment. DL-Propranolol, a PA phosphohydrolase inhibitor, caused an accumulation of PA, without affecting DAG formation in either vitamin E-treated and untreated cells. This indicated that the increase in PA and decrease in DAG in vitamin E-treated cells was not due to a stimulation of phospholipase D or an inhibition of PA phosphohydrolase. Determination of inositol phosphates formation in response to thrombin showed that the change of DAG levels elicited by vitamin E was independent of phospholipase C-induced hydrolysis of inositol phospholipids. In contrast, analysis of DAG kinase activity revealed that vitamin E enrichment enhanced the activity of the enzyme in both basal and thrombin-stimulated cells. Taken together, these data indicated that vitamin E caused an increased conversion of DAG to PA by activating DAG kinase activity without causing any change in the activities of phospholipase D, PA phosphohydrolase or phospholipase C.
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PMID:Vitamin E suppresses diacylglycerol (DAG) level in thrombin-stimulated endothelial cells through an increase of DAG kinase activity. 818 Feb 45

Studies on electropermeabilized human platelets indicated that any two of three distinct factors must be present for marked secretion of dense or alpha-granule constituents to occur. These factors are Ca2+, activation of protein kinase C (PKC) and activation of an unidentified GTP-binding protein ('GE'). Thus, in the absence of Ca2+, phorbol ester and GTP[S] acted synergistically to promote secretion, whereas in the presence of Ca2+, either activation of PKC or addition of GTP[S] was sufficient. In all cases, secretion correlated with the activation of phospholipase D (PLD), as detected by the formation of [3H]phosphatidic acid (PA) in the absence of ethanol or of [3H]phosphatidylethanol (PEt) in the presence of ethanol. Secretion did not correlate with phospholipase C (PLC) activity or with the accumulation of 1,2-diacylglycerol (DAG), both of which required Ca2+ and were inhibited by phorbol ester. Ethanol partially inhibited secretion in the absence of Ca2+. BAPTA, a known inhibitor of Ca(2+)-independent secretion in permeabilized cells, caused parallel inhibitions of secretion and PLD activity. GTP[S] enhanced PKC activity, as indicated by pleckstrin phosphorylation, apparently by stimulating the formation of PA in the absence of Ca2+, as well as of DAG in the presence of Ca2+. PA and stable analogues, including PEt, stimulated the Ca(2+)-independent phosphorylation of pleckstrin and other proteins in platelet supernatant fraction. The results suggest that PA formed by activation of PLD may mediate secretion from permeabilized platelets by PKC-dependent and independent mechanisms. However, in intact platelets stimulated by thrombin, PLD accounted for only 10-20% of the total PA formed and can only play a major role in secretion if this PA fraction is distinct from that formed by the combined actions of PLC and DAG kinase.
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PMID:Evidence that activation of phospholipase D can mediate secretion from permeabilized platelets. 820 83

alpha-Thrombin induced a change in the cell morphology of IIC9 fibroblasts from a semiround to an elongated form, accompanied by an increase in stress fibers. Incubation of the cells with phospholipase D (PLD) from Streptomyces chromofuscus and exogenous phosphatidic acid (PA) caused similar morphological changes, whereas platelet-derived growth factor (PDGF) and phorbol 12-myristate 13-acetate (PMA) induced different changes, e.g., disruption of stress fibers and cell rounding. alpha-Thrombin, PDGF, and exogenous PLD increased PA by 20-40%, and PMA produced a smaller increase. alpha-Thrombin and exogenous PLD produced rapid increases in the amount of filamentous actin (F-actin) that were sustained for at least 60 min. However, PDGF produced a transient increase of F-actin at 1 min and PMA caused no significant change. Dioctanoylglycerol was ineffective except at 50 micrograms/ml. Phospholipase C from Bacillus cereus, which increased diacylglycerol (DAG) but not PA, did not change F-actin content. Down-regulation of protein kinase C (PKC) did not block actin polymerization induced by alpha-thrombin. H-7 was also ineffective. Exogenous PA activated actin polymerization with a significant effect at 0.01 microgram/ml and a maximal increase at 1 microgram/ml. No other phospholipids tested, including polyphosphoinositides, significantly activated actin polymerization. PDGF partially inhibited PA-induced actin polymerization after an initial increase at 1 min. PMA completely or largely blocked actin polymerization induced by PA or PLD. These results show that PC-derived PA, but not DAG or PKC, activates actin polymerization in IIC9 fibroblasts, and indicate that PDGF and PMA have inhibitory effects on PA-induced actin polymerization.
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PMID:Activation of actin polymerization by phosphatidic acid derived from phosphatidylcholine in IIC9 fibroblasts. 827 97

The aim is to summarize briefly the evidence for the existence and possible functions of receptor-mediated activity of phospholipases C and D in the myocardium. Muscarinic, alpha 1-adrenergic, angiotensin II, endothelin-1, thrombin, adenine nucleotide and opioid peptide receptors are all linked through GTP-binding proteins to phospholipase C which hydrolyses phosphatidylinositol 4,5-bisphosphate (PIP2) in the myocardium. Events that are not linked to receptors, such as mechanical loading (stretching) of cardiomyocytes, can also activate phospholipase C. The high capacity for resynthesis of PIP2 maintains the pool of PIP2, even during maximal activation of phospholipase C. Activation of phospholipase C by endothelin-1, alpha 1-adrenoceptor and angiotensin II, is subject to different rates of homologous desensitization. Protein kinase C is probably not involved in the desensitization of the response to endothelin-1. One of the products of the hydrolysis of PIP2, inositol 1,4,5-trisphosphate (IP3), releases Ca2+ from the sarcoplasmic reticulum. This intracellular response seems to be causally related to positive inotropy. The phosphorylated product of IP3, inositol 1,3,4,5-tetrakisphosphate (IP4), is believed to play a role in the handling of intracellular Ca2+, as well as in the inotropic response; however, its formation is controversial. At present the oscillations in the level of intracellular Ca2+ underlying, for example, the positive inotropy induced by alpha 1-adrenoceptors or endothelin are not clearly identified. The other product of phospholipase C, 1,2-diacylglycerol, activates Ca(2+)-dependent protein kinase C and potentially controls a wide array of cellular functions such as ion transport, myofibrillar Ca2+ sensitivity, "cross-talk" between phospholipases C and D, gene expression, protein synthesis and hypertrophic cell growth. Alterations in the fatty acid composition, particularly the polyunsaturated fatty acids, modify the phosphoinositide response induced by hormones. Cultured cardiomyocytes, incubated in sera containing the fatty acids 18:2n-6 or 20:5n-3, but not 18:0 and 18:1n-9, show a decrease in the phospholipase C responses mediated by alpha 1-adrenoceptors. The fatty acid composition of myocardial phosphatidyl inositol 4-monophosphate (PIP) and PIP2 differs from that of phosphatidylinositol, which indicates that phosphatidylinositol kinases have a certain substrate specificity or have access to localized phosphatidylinositol molecules. The estimation of the level of stimulated 1,2-diacylglycerol is complicated by the contribution of the activity of receptor-mediated phospholipase D. The identification of the molecular species of 1,2-diacylglycerol is crucial in establishing the roles and the sources of 1,2-diacylglycerol. The fatty acids covalently bound in the membrane phospholipids may also influence phospholipases C and D.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Receptor-mediated signalling pathways acting through hydrolysis of membrane phospholipids in cardiomyocytes. 840 19

Regulation of phospholipase D (PLD) activity was investigated in cultured monolayers of bovine pulmonary artery endothelial cells (BPAECs). Agonists such as bradykinin, histamine, vasopressin, alpha-thrombin, and adenosine triphosphate (ATP) stimulated up to 15-fold accumulation of phosphatidylethanol (PEt) in the presence of ethanol through PLD-catalyzed phosphatidyltransferase activity. To examine mechanisms of PLD regulation, we investigated the role of protein kinase C (PKC) and Ca2+ fluxes in agonist-induced PLD activation. The PKC activator 12-O-tetradecanoylphorbol-13-acetate (TPA, 100 nmol/L) produced up to a 25-fold increase in PEt formation in a time- and dose-dependent manner. PEt production was also stimulated by other cell-permeant PKC activators such as 1,2 dioctanoylglycerol and 1-oleyl-2-acetylglycerol, whereas inactive phorbol derivatives 4-alpha-phorbol-12,13-didecanoate and 4-beta-phorbol showed no effect. The effect of TPA on PEt accumulation was inhibited by the PKC inhibitors staurosporine (5 mumol/L, 95% inhibition) and sphingosine (10 mumol/L, 50% inhibition). TPA-induced PEt accumulation was almost completely abolished (> 95% inhibition) by PKC down-regulation accomplished by long-term treatment with 100 nmol/L TPA. In contrast, bradykinin- or ATP-induced phosphorus 32-labeled PA and [32P]-labeled PEt formation was only partially blocked (70% inhibition) by either staurosporine (10 mumol/L) or PKC down-regulation, suggesting that part of agonist-stimulated PLD activity may occur in the absence of PKC activation. An increase in Cai2+ appears to be involved in agonist-induced PLD activation as bradykinin-, ATP-, or Ca2+ ionophore-induced [32P]. PEt production was attenuated by either depletion of extra-cellular Ca2+ with EGTA or chelation of intracellular Ca2+ by BAPTA. TPA-mediated PEt accumulation was not affected by EGTA treatment, whereas BAPTA reduced TPA-mediated PEt formation by 50%. These results suggest that direct PKC activation is a potent stimulus for PLD activity and that the major pathway for agonist-induced PLD activation involves PKC activation and is dependent on an increase in intracellular Ca2+. Further, these studies suggest that agonist-induced PLD activation may also involve a PKC-independent mechanism.
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PMID:Agonist-induced activation of phospholipase D in bovine pulmonary artery endothelial cells: regulation by protein kinase C and calcium. 843 44

1. The comparative study of the effect of bradykinin (BK) in young and old IMR-90 human fibroblasts shows that old cells are characterized by a reduced increase in 1,2-diacylglycerol (1,2-DAG) generation upon stimulation after short-term treatment and a significant higher increase after long-term agonist treatment. BK-induced activation of phospholipase D (PLD), the major enzyme involved in sustained 1,2-DAG generation, was 2.5-fold higher in old cells, strongly suggesting that it is involved in the potentiated increase of 1,2-DAG formation. The increased activation of PLD by BK in old cells was specific, since in parallel experiments the effect of thrombin was not significantly different in young and old cells. PLD activity in old cells was only reduced by down-regulation of protein kinase C (PKC) activity, in contrast to what was observed in young cells where it was completely abolished. This indicates that the enzyme activity in old cells was partially PKC-independent. BK was also able to increase the release of [14C]ethanolamine, a water-soluble product of hydrolysis of phosphatidylethanolamine (PtdEtn), through PLD activation in young and old cells. The BK effect was significantly higher in old cells and, very likely, PKC-independent, since phorbol 12-myristate 13-acetate failed to induce PtdEtn hydrolysis. 2. The present results indicate that the PLD/1,2-DAG pathway is specifically potentiated by BK in old fibroblasts, demonstrating that the formation of positive effectors of PKC activation is not necessarily decreased in cellular senescence. It remains to be established whether the increased generation of DAG upon BK stimulation plays any role in the altered PKC signalling pathway which characterizes old fibroblasts.
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PMID:Potentiated bradykinin-induced increase of 1,2-diacylglycerol generation and phospholipase D activity in human senescent fibroblasts. 855 23

We examined the effect of thrombin on phosphatidylcholine-hydrolyzing phospholipase D activity in osteoblast-like MC3T3-E1 cells. Thrombin stimulated the formation of choline dose dependently in the range between 0.01 and 1 U/ml, but not the phosphocholine formation. Diisopropylfluorophosphate (DFP)- inactivated thrombin had little effect on the choline formation. The combined effects of thrombin and 12-O-tetradecanoylphorbol-13-acetate, a protein kinase C-activating phorbol ester, on the choline formation were additive. Staurosporine, an inhibitor of protein kinases, had little effect on the thrombin-induced formation of choline. Combined addition of thrombin and NaF, an activator of heterotrimeric GTP-binding protein, did not stimulate the formation of choline further. Pertussis toxin had little effect on the thrombin-induced formation of choline. Thrombin stimulated Ca2+ influx from extracellular space time and dose dependently. The depletion of extracellular Ca2+ by EGTA exclusively reduced the thrombin-induced choline formation. Thrombin had only a slight effect on phosphoinositide-hydrolyzing phospholipase C activity. Thrombin induced diacylglycerol formation and DNA synthesis, and increased the number of MC3T3-E1 cells, but DFP-inactivated thrombin did not. Thrombin suppressed both basal and fetal calf serum-induced alkaline phosphatase activity in these cells. Propranolol, an inhibitor of phosphatidic acid phosphohydrolase, inhibited both the thrombin-induced diacylglycerol formation and DNA synthesis. These results suggest that thrombin stimulates phosphatidylcholine-hydrolyzing phospholipase D due to self-induced Ca2+ influx independently of protein kinase C activation in osteoblast-like cells and that its proliferative effect depends on phospholipase D activation.
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PMID:Thrombin induces proliferation of osteoblast-like cells through phosphatidylcholine hydrolysis. 864 17

Mitogenic signalling mechanisms emerged as novel targets for tumor chemotherapy. Current strategies for pharmacological interventions are briefly discussed. Phospholipid analogues are treated in greater detail. It is shown here that this new class of antitumor agents acts as inhibitors of mitogenic signal transduction. The common target of all phospholipid analogues studied so far is the phosphatidylinositol (PI)-specific phospholipase C (PLC). This results in an attenuated formation of inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). The reduction in IP3-levels leads to a depressed release of Ca2+ from internal stores, and the reduced formation of DAG interferes with the growth factor-induced activation of protein-kinase C (PKC). In addition to the effect on PI-specific PLC, most phospholipid analogues inhibit PKC directly by interacting with the regulatory domain of the enzyme. This effect, however, is not observed with all phospholipid analogues. Some potent growth inhibitory representatives from this group like hexadecylphosphoserine or hexadecylphosphonoserine do not affect PKC in cell-free extracts. It is concluded, therefore, that the direct inhibition of PKC is not required for the growth-inhibitory activity of these agents. The ability of phospholipid analogues to interact with PKC was also not found to be correlated the occurrence of unwanted side effects. Phospholipid analogues have also been found to act as inhibitors of phospholipase D (PLD). However, in this case the correlation to the growth inhibitory potency of various phospholipid analogues was less clear, so that the contribution of the PLD inhibition to the growth inhibitory effect of these agents still remains to be established. The inhibition of the thrombin-induced rise in cytosolic free Ca2+ by phospholipid analogues is reversible by washing the cells in phospholipid-free medium. These findings suggest that phospholipid analogues do not cause persistent membrane damage and may act as cytostatic rather than cytotoxic agents.
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PMID:Cellular signalling as a target in cancer chemotherapy. Phospholipid analogues as inhibitors of mitogenic signal transduction. 886 57

Besides its role as a putative second messenger releasing Ca2+ from intracellular stores, sphingosine-1-phosphate (SPP) has recently been identified as an extracellularly acting ligand activating a high affinity G protein-coupled membrane receptor in various cell types. Since SPP can be released from activated platelets, we examined in the present study whether endothelial cells express receptors for SPP and related sphingolipids. In bovine aortic endothelial cells loaded with fura-2, addition of SPP caused a rapid and transient increase in intracellular Ca2+ concentration ([Ca2+]i), amounting to maximally about 230 nM. Removal of extracellular Ca2+ revealed that SPP-induced [Ca2+]i elevations were due to both release of Ca2+ from intracellular stores and influx of extracellular Ca2+. Pretreatment of the cells with pertussis toxin inhibited the SPP-induced increase in [Ca2+]i by 83%, in line with the previously reported involvement of G proteins of the Gi/o family in SPP signalling in other cell types. In contrast to other [Ca2+]i-elevating agonists, e.g., ATP and bradykinin, SPP did not activate phospholipase C in bovine aortic endothelial cells, suggesting the involvement of a novel, unidentified signalling pathway in SPP-induced release of intracellular Ca2+. Furthermore, SPP also did not cause activation of either phospholipase D or A2. Out of various related sphingolipids studied, only sphingosylphosphorylcholine (SPPC) induced a similar maximal increase in [Ca2+]i as SPP, and its effect was also fully pertussis toxin-sensitive. However, the potencies of the two sphingolipids to increase [Ca2+]i differed by more than two orders of magnitude, with the EC50 values being 0.8 nM and 260 nM for SPP and SPPC, respectively. These results identify SPP and SPPC as novel and potent endothelial agonists, inducing calcium signalling by activation of a Gi/o protein-coupled receptor(s). Given the recently reported release of SPP from thrombin-activated platelets, SPP may represent a novel mediator of platelet-endothelial cell interactions.
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PMID:Calcium signalling by G protein-coupled sphingolipid receptors in bovine aortic endothelial cells. 889 40

In this paper we demonstrate for the first time a mitogen-induced activation of a nuclear acting phosphatidylcholine-phospholipase D (PLD) which is mediated, at least in part, by the translocation of RhoA to the nucleus. Addition of alpha-thrombin to quiescent IIC9 cells results in an increase in PLD activity in IIC9 nuclei. This is indicated by an increase in the alpha-thrombin-induced production of nuclear phosphatidylethanol in quiescent cells incubated in the presence of ethanol as well as an increase in PLD activity in isolated nuclei. Consistent with our previous report (Wright, T. M., Willenberger, S., and Raben, D. M. (1992) Biochem. J. 285, 395-400), the presence of ethanol decreases the alpha-thrombin-induced production of phosphatidic acid without affecting the induced increase in nuclear diglyceride, indicating that the increase in nuclear PLD activity is responsible for the effect on phosphatidic acid, but not that on diglyceride. Our data further demonstrate that RhoA mediates the activation of nuclear PLD. RhoA translocates to the nucleus in response to alpha-thrombin. Additionally, PLD activity in nuclei isolated from alpha-thrombin-treated cells is reduced in a concentration-dependent fashion by incubation with RhoGDI and restored by the addition of prenylated RhoA in the presence of guanosine 5'-3-O-(thio)triphosphate. Western blot analysis indicates that this RhoGDI treatment results in the extraction of RhoA from the nuclear envelope. These data support a role for a RhoA-mediated activation of PLD in our recently described hypothesis, which proposes that a signal transduction cascade exists in the nuclear envelope and represents a novel signal transduction cascade that we have termed NEST (nuclear envelope signal transduction).
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PMID:Nuclear translocation of RhoA mediates the mitogen-induced activation of phospholipase D involved in nuclear envelope signal transduction. 903 May 50


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