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)

It has recently been appreciated that thrombin induces the retraction of endothelial cells resulting in an alteration of the integrity of the monolayers. We studied thrombin-induced changes in cytosolic calcium concentration (Ca2+i) using microfluorometry of fura-2-loaded single cells, cell topography (scanning electron microscopy), and cytoskeleton (rhodamine phalloidin) in endothelial cells. Thrombin caused an initial and sustained phase of an increase in Ca2+i. Pretreatment with pertussis toxin abolished both phases of Ca2+i response. Sustained phase of thrombin effect required extracellular calcium. Pretreatment of endothelial cells with indomethacin protracted the sustained phase, whereas a lipoxygenase inhibitor, nordihydroguaiaretic acid curtailed it. Thrombin caused a marked retraction of confluent endothelial cells coincident with the sustained phase of Ca2+i response. This was paralleled by the formation of gaps in F-actin distribution at the periphery of the cells. Pretreatment of endothelial cells with nordihydroguaiaretic acid blunted the thrombin-induced cell retraction. Microinjection of various putative messengers into the endothelial cells showed that initial Ca2+ mobilization is not sufficient to account for sustained elevation of Ca2+i. The sustained response required microinjection of phospholipase A2 or co-injection of phospholipase A2 with phosphatidylinositol 4,5-bisphosphate-specific phospholipase C, phosphatidylinositol 1,4,5-trisphosphate, or CaCl2, further implying that thrombin receptor(s) can be coupled to both phospholipases C and A2. Sustained elevation of Ca2+i was a necessary prerequisite for the thrombin-induced changes in endothelial cell topography.
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PMID:Nature of thrombin-induced sustained increase in cytosolic calcium concentration in cultured endothelial cells. 277 5

The thrombin-dependent losses of eicosapentaenoate (EPA) from the various phospholipids of platelets derived from human subjects ingesting a fish lipid concentrate (MaxEPA) were quantitatively assessed and studied in relation to arachidonate (AA). The net loss of AA and EPA from the total phospholipid, phosphatidylcholine (PC) + phosphatidylethanolamine (PE) + phosphatidylserine (PS) + phosphatidylinositol (PI) (loss from phosphatidylinositol minus accumulated phosphatidate), amounted to 44.4 and 7.3 nmol/2 x 10(9) platelets (mean values, n = 4 subjects), respectively, in response to thrombin (2 units/ml). The phosphatidylcholine, phosphatidylethanolamine (including alkenylacyl), phosphatidylserine, and phosphatidylinositol contributed 46, 17, less than 5, and 33%, respectively, of the AA loss; in contrast to these distributions, the corresponding phospholipid contributions to the net loss of EPA were 71, 27, less than 1, and less than 2%, respectively. Furthermore, the inhibition of AA- and EPA-phospholipid degradation by trifluoperazine indicated that almost all of the release of EPA occurs from PC and PE (greater than 95% of total EPA loss) upon thrombin stimulation and is mediated predominantly via phospholipase A2 activity with almost no contribution from PI. Similarities in the molar ratios of AA/EPA in the PC (3.9) or PE (3.7) which were degraded with those in the corresponding phospholipids from resting platelets suggested no marked selectivity by the phospholipase A2 in intact thrombin-stimulated human platelets in the hydrolysis of AA-PC (or AA-PE) versus EPA-PC (or EPA-PE). Quantitation of the newly released free AA and EPA was determined in the presence of BW755C, a dual cyclooxygenase/lipoxygenase inhibitor which was found not to influence the degradation of individual AA- and EPA-containing phospholipids.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Quantitative loss of individual eicosapentaenoyl-relative to arachidonoyl-containing phospholipids in thrombin-stimulated human platelets. 282 97

Whereas numerous studies deal with the effects and metabolism of eicosapentaenoic acid (20:5(n - 3)) in platelets, very few concern docosahexaenoic acid (22:6(n - 3)), although both acids are consumed in equal amounts from most fish fat. The present paper reports the modulation of 22:6(n - 3) oxygenation as well as that of endogenous arachidonic acid (20:4(n - 6)) in 22:6(n - 3)-rich platelets. Like the oxygenation of 20:5(n - 3), the lipoxygenation of 22:6(n - 3) occurred at a low level when incubated alone, but was markedly increased in the presence of 20:4(n - 6), suggesting a similar peroxide tone dependency. 20:5(n - 3) could not replace 20:4(n - 6) in the increasing 22:6(n - 3) lipoxygenation, whereas 22:6(n - 3) shared the potentiating effect of 20:4(n - 6) on both the cyclooxygenation and the lipoxygenation of 20:5(n - 3). On the other hand, 20:5(n - 3), 22:6(n - 3) or 20:5(n - 3) + 22:6(n - 3) enrichment of platelet phospholipids inhibited the formation of cyclooxygenase but not lipoxygenase products from endogenous 20:4(n - 6) in thrombin-stimulated platelets. In doing so, 22:6(n - 3) appeared even more potent than 20:5(n - 3), although it was not liberated after acylation in phospholipids, the opposite of what was observed with 20:5(n - 3). Therefore, it seems that, in contrast to 20:5(n - 3), which may compete with endogenous 20:4(n - 6) at the cyclooxygenase level, 22:6(n - 3) would affect the latter enzyme activity in a different way. We conclude that 20:5(n - 3) and 22:6(n - 3) behave differently and might act synergistically on the inhibition of platelet functions after fish fat intake.
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PMID:Different metabolic behavior of long-chain n-3 polyunsaturated fatty acids in human platelets. 283 37

This study has examined the thrombin-stimulated release of polyunsaturated fatty acids from endothelial glycerolipids. Human umbilical vein endothelial cells were incubated with 1.25 microM [14C]arachidonate or [14C]eicosapentaenoate and then exposed to thrombin in buffered saline plus albumin. After an incorporation period of 0.5 h, the thrombin-stimulated release of the two radiolabeled fatty acids was quite similar. By contrast, after 24 h of fatty acid incorporation, the thrombin-stimulated release of radiolabeled fatty acid from cells incubated with [14C]eicosapentaenoate was only 25-30% of that from cells with [14C]arachidonate. Analysis of cellular glycerolipids indicated that 23 and 72%, respectively, of the incorporated [14C]arachidonate and [14C]eicosapentaenoate had been elongated to 22-carbon fatty acids in 24 h. Both 20- and 22-carbon 14C-labeled fatty acids were released to albumin in the medium in control incubations. Addition of thrombin stimulated the release of [14C]arachidonate and [14C]eicosapentaenoate, but not of their respective elongation products. Furthermore, endothelial cells incorporated exogenous [14C]docosatetraenoate into cellular glycerolipids but did not release it in response to thrombin. Thus, the thrombin-stimulated release of polyunsaturated fatty acids from vascular endothelial cells is highly selective for arachidonate and eicosapentaenoate. These results suggest that the extensive elongation of eicosapentaenoate by these cells serves to remove n - 3 polyunsaturated fatty acids from the pool of cellular acyl groups which are released in response to thrombin and are thus made available for metabolism by cyclooxygenase and lipoxygenase enzymes.
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PMID:Elongation of arachidonic and eicosapentaenoic acids limits their availability for thrombin-stimulated release from the glycerolipids of vascular endothelial cells. 300 85

Receptor-mediated cyclic GMP formation in N1E-115 murine neuroblastoma cells appears to involve oxidative metabolism of arachidonic acid. Evidence in support of this includes the blockade of this response by lipoxygenase inhibitors, e.g., eicosatetraynoic acid (ETYA) or other metabolic perturbants, e.g., methylene blue. It was recently discovered that the lipoxygenase products 15-hydroxyeicosatetraenoic (15-HETE) acid and 12-HETE, like ETYA, were inhibitors of M1 muscarinic receptor-mediated cyclic GMP formation. In the present report, the effects of monoHETEs are explored in more detail, particularly with regard to the function of the muscarinic receptor. Like 12-HETE and 15-HETE (IC50 = 13 and 11 microM, respectively), 5-HETE inhibited the cyclic GMP response to the muscarinic receptor (IC50 = 10 microM). All three of these monoHETEs were shown also to be inhibitors of the cyclic GMP responses to receptors stimulated by carbachol, histamine, thrombin, neurotensin, and bradykinin. 15-HETE was shown to inhibit the muscarinic receptor-mediated response in a complex manner (apparent noncompetitive and uncompetitive components; IC50 = 18 and 2 microM, respectively). 15-HETE did not inhibit either the M1 muscarinic receptor-stimulated release of [3H]inositol phosphates from cellular phospholipids or the M2 muscarinic receptor-mediated inhibition of hormone (prostaglandin E1)-induced AMP formation. It seemed possible that the monoHETEs could enter into biochemical pathways for arachidonate in N1E-115 cells. [3H]Arachidonate and the three [3H]-monoHETEs all rapidly labeled the membrane lipids of intact N1E-115 cells, with each [3H]eicosanoid producing a unique labeling profile. [3H]15-HETE labeling was noteworthy in that 85% of the label found in the phospholipids was in phosphatidylinositol (PI;t1/2 to steady state = 3 min). Exogenous 15-HETE inhibited the labeling of PI by [3H]arachidonate (IC50 = 28 microM) and elevated unesterified [3H]arachidonate levels. Thus, the mechanism of blockade of receptor-mediated cyclic GMP responses by monoHETEs is likely to be more complex than the simple inhibition of cytosolic mechanisms, e.g., generation of a putative second messenger by lipoxygenase, and may involve also alterations of membrane function accompanying the redistributions of esterified arachidonate.
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PMID:Blockade of receptor-mediated cyclic GMP formation by hydroxyeicosatetraenoic acid. 303 24

It has been postulated that the diacylglycerol lipase pathway is a predominant source of the free arachidonic acid which is released from phospholipids upon the exposure of human platelets to thrombin. The amount of released arachidonic acid and other fatty acids in thrombin-stimulated platelets was determined in the presence of BW755C, the cyclooxygenase/lipoxygenase inhibitor, and in relation to phosphatidylinositol degradation and phosphatidic acid formation. A stearic acid:arachidonic acid molar ratio approaching unity would be expected in the free fatty acid fraction if the latter pathway were a major source of released arachidonic acid. Our results indicate that the diacylglycerol lipase pathway contributes a maximum of 3-4 nmol of arachidonic acid/2 X 10(9) platelets or 12-15% of the total arachidonic acid released (25.8 nmol/2 X 10(9) platelets) upon exposure to thrombin (2 units/ml) for 4 min. Trifluoperazine inhibited most of the thrombin-dependent free arachidonic acid release but only 15% of the absolute loss of arachidonic acid from phosphatidylinositol. Therefore, we conclude that the diacylglycerol lipase pathway represents only a minor source of the free arachidonic acid that is released upon thrombin stimulation of human platelets.
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PMID:Diacylglycerol lipase pathway is a minor source of released arachidonic acid in thrombin-stimulated human platelets. 308 Oct 1

Diverse particulate and soluble stimuli trigger two metabolic bursts in mouse peritoneal macrophages important in the inflammatory and/or cytotoxic actions of the cells: release, oxygenation, and further metabolism of arachidonic acid from endogenous phospholipids and reduction of molecular oxygen to reactive intermediates. We tested the hypothesis that the release of arachidonic acid or formation of its metabolites are obligatory intermediate steps in triggering the NADPH oxidase that reduces O2 to O-2. With phorbol diesters as stimuli, the following inhibitors of phospholipase A2 and lipoxygenase suppressed release of H2O2 at nontoxic concentrations (microM range): p-bromophenacyl bromide, quinacrine, eicosatetraenoic acid, nordihydroguaiaretic acid, and phenidone. Indomethacin and acetylsalicylic acid were ineffective. However, the suppressive effect of the first five agents on H2O2 release could be attributed to their suppression of macrophage glucose uptake at the same concentrations, a previously unrecognized effect of these compounds. Further, concanavalin A, wheat germ agglutinin, and thrombin each stimulated abundant arachidonate release without H2O2 release. Finally, noncytolytic concentrations of cycloheximide and/or emetine suppressed arachidonate release without affecting H2O2 secretion triggered either by phorbol esters or zymosan. Release and metabolism of arachidonic acid and secretion of reactive oxygen intermediates appear to be two frequently coincident but mutually independent metabolic pathways in the mouse peritoneal macrophage.
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PMID:Release of arachidonate and reduction of oxygen. Independent metabolic bursts of the mouse peritoneal macrophage. 309 92

Prostaglandin (PG) H synthase and eicosanoid products of arachidonic acid metabolism have been implicated in several steps in the carcinogenic process. This study assessed these parameters using primary cultures of human urothelial cells. To determine the possible presence of permeability barriers to agonist stimulation, incubations were performed with adherent cells in the presence or absence of thioglycolate pretreatment or with cell suspensions. No evidence for permeability barriers was observed. With adherent cells in the absence of thioglycolate, radioimmunoassayable PGE2 was stimulated by epinephrine less than 12-O-tetradecanoylphorbol-13-acetate = thrombin less than bradykinin = A23187 much less than arachidonic acid. Tumor promoters but not non-tumor promoters stimulated PGE2 synthesis. 1-Oleoyl-2-acetylglycerol which like 12-O-tetradecanoylphorbol-13-acetate activates protein kinase C also increased PGE2 synthesis. Cells prelabeled with [14C]arachidonic acid were exposed to agonists and the profile of eicosanoids synthesized was assessed by high performance liquid chromatography. With bradykinin, the pattern of eicosanoids synthesized was 6-keto-PGE1 alpha (12% of total 14C label), thromboxane B2 (0.4%), PGF2 alpha (1.7%), PGE2 (18%), PGD2 (1%), leukotrienes (0.4 to 1%), 12-hydroxy-5,8,10-heptadecatrienoic acid (3%), 15-hydroxy-5,8,11,13-eicosatetraenoic acid (4%), 12-hydroxy-5,8,10,14-eicosatetraenoic acid (0%) and 5-hydroxy-5,8,12,14-eicosatetraenoic acid (2%). Thus, human urothelial cells contain both prostaglandin H synthase and lipoxygenase pathways with the former being more prominent. These pathways may participate in urinary bladder carcinogenesis.
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PMID:Eicosanoid synthesis by cultured human urothelial cells: potential role in bladder cancer. 309 68

The synthetic antioxidants butylated hydroxytoluene (BHT), nordihydroguaiaretic acid and the one-electron donor 1,1'-dimethylferrocene, inhibit cytosolic Ca++ increase, shape change, aggregation and ATP secretion in aspirinated washed human platelets stimulated by thrombin, vasopressin and platelet-activating factor. The antioxidants also inhibit cytosolic Ca++ increase originating from intracellular stores in the presence of EGTA. The effect of phorbol ester (TPA), which promotes platelet aggregation and secretion without raising the cytosolic Ca++, is also antioxidant-sensitive. Since agonist activation of aspirinated platelets does not involve cyclooxygenase or lipoxygenase metabolites, it is suggested that other yet unknown free radical-dependent pathways are involved in the mechanism of platelet activation, both in the protein kinase C-independent events leading to the cytosolic Ca++ increase, and in those, largely protein kinase C-dependent, leading to aggregation and ATP secretion.
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PMID:Inhibition by antioxidants of agonist evoked cytosolic Ca++ increase, ATP secretion and aggregation of aspirinated human platelets. 309 18

Human blood platelet aggregation and the formation of icosanoids were studied in response to triethyl lead chloride (Et3PbCl). Concentrations higher than 75 microM stimulate platelets to aggregate, whereas low concentrations (less than or equal to 20 microM) caused platelet hypersensitivity to aggregating agents such as collagen or arachidonic acid. Incubation of suspensions of washed platelets with Et3PbCl resulted in a stimulated liberation and subsequent metabolism of arachidonic acid. This response was dependent on the concentration of Et3PbCl and the incubation time. Using low concentrations of Et3PbCl and up to 3 h of incubation, the lipoxygenase product 12-hydroxy-5,8,10,14-icosatetraenoic acid was the major metabolite. Under normal conditions, however, stimulation of platelets with collagen, thrombin, or arachidonic acid leads to higher amounts of the cyclooxygenase products 12-hydroxy-5,8,10-heptadecatrienoic acid and thromboxane B2. The aggregation of human platelets induced by Et3PbCl was inhibited by three different drugs: acetylsalicylic acid, forskolin and quinacrine; but only quinacrine could prevent the liberation of arachidonic acid and the appearance of its metabolites. These specific effects of the inhibitors on Et3PbCl-stimulated platelets as well as the differences in the pattern of arachidonic acid metabolites and phosphatidic acid suggest a direct stimulatory action of Et3PbCl on platelet phospholipase A2.
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PMID:Stimulation of arachidonic acid metabolism via phospholipase A2 by triethyl lead. 310 Feb 96


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