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)

Platelet aggregation and histamine release were evaluated in normal and IgE pretreated human platelets exposed in vitro to IgE, anti-IgE and thrombin. The response of platelets from atopic donors directly stimulated with anti-IgE was also evaluated. Histamine release was measured by fluorimetric analysis of histamine content in platelets and in supernatants. The morphology of platelets exposed to immunological and non-immunological stimuli was recorded using an electron microscope. A detectable amount of histamine was measured in quiescent platelets. Their exposure to varying concentrations of thrombin produces a progressive aggregation which runs parallel to histamine release. The effects were significantly enhanced in platelets pretreated with IgE. Incubation of normal platelets with increasing concentrations of IgE myeloma protein, or with anti-human IgE antibody was ineffective on both aggregation and histamine release. However, incubation of platelets passively sensitized with IgE-myeloma protein with different concentrations of anti-human IgE antibody produces a concentration-dependent increase both in aggregation and histamine release. The same effects were obtained using platelets from atopic donors directly stimulated with anti-IgE. The electron microscopic pattern of platelet aggregation induced by thrombin was indistinguishable from that evoked by anti-IgE in IgE pretreated platelets. Loratadine, a non-sedative H1-receptor blocker, significantly abated platelet aggregation and histamine release induced by anti-IgE in IgE pretreated platelets.
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PMID:Platelet aggregation and histamine release by immunological stimuli. 752 32

Effects of vasoactive agonists on endothelial permeability was assessed by measurement of transendothelial electrical resistance (TEER) of human umbilical vein endothelial cells (HUVECs) grown on porous polycarbonate supports. Because of the low values of TEER obtained in this preparation (< 5 omega cm2) a design of an Ussing type recording chamber was chosen that provided for a homogeneous electric field across the monolayer and for proper correction of series resistances. Precision current pulses and appropriate rates of sampling and averaging of the voltage signal allowed for measurement of < 0.1 omega resistance changes of the endothelium on top of a 21 omega series resistance of the support and bathing fluid layers. Histamine (10 microM) and thrombin (10 U/ml) induced an abrupt and substantial decrease of TEER, bradykinin (1 microM) was less effective, PAF (380 nM) and LTC4 (1 microM) had no effect. TEER was also reduced by the calcium ionophore A-23187 (10 microM). The technique allows for measurements of TEER in low resistance monolayer cultures with high precision and time resolution. The results obtained extend previous observations in providing quantitative data on the increase of permeability of HUVECs in response to vasoactive agonists.
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PMID:High precision measurement of electrical resistance across endothelial cell monolayers. 766 75

1. The regulation of histamine-induced [3H]-inositol phosphate formation was studied in human cultured umbilical vein endothelial cells (HUVEC). 2. Histamine (EC50 4.8 microM) produced a 12.7 fold increase in [3H]-inositol phosphate formation over basal levels. Prior exposure to 0.1 mM histamine (2 h) produced a 78% reduction in the response to subsequent histamine (0.1 mM) challenge. The IC50 for this histamine-induced desensitization was 0.9 microM. 3. The inositol phosphate response to histamine (0.1 mM) was inhibited by phorbol dibutyrate (IC50 40 nM; maximal reduction 64%). This effect was antagonized by both staurosporine (100 nM) and Ro 31-8220 (10 microM). However, the histamine-induced desensitization of the H1-receptor-mediated inositol phosphate response was insensitive to the protein kinase inhibitors, staurosporine, Ro 31-8220, K252a and KN62. 4. Prior exposure to sodium nitroprusside (100 microM), forskolin (10 microM) or dibutyryl cyclic AMP (1 mM) had no effect upon histamine-induced [3H]-inositol phosphate formation. 5. NaF (20 mM) and thrombin (EC50 0.4 u ml-1) also induced inositol phosphate formation in HUVEC. Histamine pretreatment (0.1 mM, 10-120 min) failed to modify the inositol phosphate response to a subsequent NaF or thrombin challenge. 6. We conclude that the desensitization of histamine H1-receptor-mediated [3H]-inositol phosphate formation occurs at the level of the receptor and involves a mechanism independent of activation of protein kinase A, G, or C, or calcium calmodulin-dependent protein kinase II.
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PMID:Agonist-induced desensitization of histamine H1 receptor-mediated inositol phospholipid hydrolysis in human umbilical vein endothelial cells. 785 73

We examined the contribution of actin-myosin contraction to the modulation of human umbilical vein endothelial cell focal adhesion caused by histamine and thrombin. Focal adhesion was measured as the electrical resistance across a cultured monolayer grown on a microelectrode. Actin-myosin contraction was measured as isometric tension of cultured monolayers grown on a collagen gel. Histamine immediately decreased electrical resistance but returned to basal levels within 3-5 min. Histamine did not increase isometric tension. Thrombin also immediately decreased electrical resistance, but, however, resistance did not return to basal levels for 40-60 min. Thrombin also increased isometric tension, ML-7, an inhibitor of myosin light chain kinase, prevented increases in myosin light chain phosphorylation and increases in tension development in cells exposed to thrombin. ML-7 did not prevent a decline in electrical resistance in cells exposed to thrombin. Instead, ML-7 restored the electrical resistance to basal levels in a shorter period of time (20 min) than cells exposed to thrombin alone. Also, histamine subsequently increased electrical resistance to above basal levels, and thrombin initiated an increase in resistance during the time of peak tension development. Hence, histamine and thrombin modulate endothelial cell focal adhesion through centripetal and centrifugal forces.
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PMID:Histamine and thrombin modulate endothelial focal adhesion through centripetal and centrifugal forces. 861 24

Histamine and thrombin increase myosin light-chain kinase-mediated phosphorylation of myosin light chain (MLC) in human umbilical vein endothelial cells (HUVEC). The increase in MLC phosphorylation caused by thrombin persists longer (330 min) than the increase caused by histamine (<5 min), although both increase cell calcium similarly. We hypothesized that some of the longer duration of the increase in MLC phosphorylation caused by thrombin was because of inhibition of myosin dephosphorylation by thrombin. Calyculin A, an inhibitor of type 1 and 2A protein phosphatases, caused a time-dependent increase in MLC phosphorylation in unstimulated HUVEC. As thrombin-stimulated phosphorylation approached its peak at 15 min, calyculin A caused progressively less of an increase in MLC phosphorylation in thrombin-stimulated HUVEC, and no increase at the peak of thrombin stimulation. In HUVEC in which cell calcium was maintained at 600 nM, thrombin increased MLC phosphorylation above the level caused by increased calcium alone at a time coinciding with the peak of thrombin stimulation. However, when phosphatase activity was already inhibited with calyculin A, thrombin did not further increase MLC phosphorylation in cells in which calcium was maintained at 600 nM calcium. Thrombin increases MLC phosphorylation in HUVEC not only by increasing cell calcium but also by inhibiting calyculin A-sensitive dephosphorylation of MLC.
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PMID:Thrombin inhibits myosin light chain dephosphorylation in endothelial cells. 912 83

There is growing evidence that proteinuric hypertension of pregnancy (preeclampsia) is associated with endothelial dysfunction. The aim of this study was to evaluate the effects of serum from preeclamptic patients on basal and agonist-stimulated prostacyclin production by human umbilical vein endothelial cells (HUVEC) in culture and to compare these to the effects of serum from normal pregnant and nonpregnant women. During a 24 h incubation of HUVEC with 20% of preeclampsia serum, baseline prostacyclin output was significantly (P < 0.01) increased over the control groups. However, this response was attenuated by extending the exposure to 72 h. Histamine, thrombin and the calcium ionophore, A23187, all acutely increased prostacyclin production, but the increase relative to baseline levels was greatest in HUVEC preincubated for 24 h in normal serum transiently promotes prostacyclin production in HUVEC derived from normal pregnancies, preeclampsia serum transiently promotes prostacyclin production in HUVEC derived from normal pregnancies, and 2) the relative increase in response to agonists is reduced by preeclampsia serum, compared to normal pregnancy sera.
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PMID:Effects of serum from preeclamptic women on prostacyclin production by human endothelial cells. 936 Jan 80

Interleukin (IL)-8, a C-X-C chemokine, activates integrin-mediated adhesion of neutrophils. Presentation of IL-8 on the endothelial cell surface may promote leukocyte extravasation. We found that cultured human microvascular endothelial cells from the intestine (HIMEC) and from nasal polyps (PMEC), but not human umbilical vein endothelial cells (HUVEC), contained IL-8 in intracellular granules that coexpressed von Willebrand factor (vWf ). This observation was corroborated by the immunohistochemical observation of double-positive granules (IL-8(+)vWf+) in vessels of small and large intestine, nasal mucosa, and skin, whereas umbilical cords revealed no endothelial IL-8. After treatment of HIMEC or PMEC with histamine or thrombin, a dramatic increase in supernatant IL-8 concentration was observed within 3 min, whereas no increase in IL-8 was detected in supernatants of identically treated HUVEC cultures. Histamine or thrombin treatment also caused IL-8-containing granules to rapidly disappear from HIMEC. In HUVEC, IL-8-containing granules were inducible by treatment with recombinant human IL-1beta for 24 h; additional histamine treatment doubled IL-8 secretion from HUVEC in the same rapid manner observed for mucosal EC. These data suggested that IL-8 prestored in microvascular endothelial cells may provide a rapid pathway for specific activation of neutrophil adhesion at sites of acute inflammation.
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PMID:Rapid secretion of prestored interleukin 8 from Weibel-Palade bodies of microvascular endothelial cells. 980 86

In the present study, we differentiated between short- and long-term effects of vasoactive compounds on human endothelial permeability in an in vitro model. Histamine induced a rapid and transient (<3 minutes) decrease in barrier function, as evidenced by a decreased transendothelial electrical resistance and an increased passage of 22Na ions. This increase in permeability was inhibited completely by chelation of intracellular calcium ions by BAPTA-AM and inhibition of calmodulin activity and myosin light chain (MLC) phosphorylation. The presence of serum factors prolonged the barrier dysfunction induced by histamine. Thrombin by itself induced a prolonged barrier dysfunction (>30 minutes) as evidenced by an increased passage of peroxidase and 40 kDa dextran. It was dependent only partially on calcium ions and calmodulin. The protein tyrosine kinase inhibitors genistein and herbimycin A, but not the inactive analogue daidzein, inhibited to a large extent the increase in permeability induced by thrombin. Genistein and BAPTA-AM inhibited the thrombin-induced permeability in an additive way, causing together an almost complete prevention of the thrombin-induced increase in permeability. Inhibition of protein tyrosine kinase was accompanied by a decrease in MLC phosphorylation and a reduction in the extent of F-actin fiber and focal attachment formation. Inhibition of RhoA by C3 transferase toxin reduced both the thrombin-induced barrier dysfunction and MLC phosphorylation. Genistein and C3 transferase toxin did not elevate the cellular cAMP levels. No evidence was found for a significant role of protein kinase C in the thrombin-induced increase in permeability or in the accompanying MLC phosphorylation. These data indicate that in endothelial cell monolayers that respond to histamine in a physiological way, thrombin induces a prolonged increase in permeability by "calcium sensitization," which involves protein tyrosine phosphorylation and RhoA activation.
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PMID:Transient and prolonged increase in endothelial permeability induced by histamine and thrombin: role of protein kinases, calcium, and RhoA. 983 6

Primary cultures of human cerebral microvascular endothelial cells (HCMEC) were loaded with fura-2. The intracellular free Ca2+ concentration ([Ca2+]i) was measured by digital imaging microscopy. Agonists ATP (100 micro), thrombin (10 units/ml), and histamine (25 microM) induced a transient [Ca2+]i increase. Histamine (100 microM) induced a biphasic [Ca2+]i increase with an initial [Ca2+]i peak followed by a [Ca2+]i plateau. The [Ca2+]i plateau was blocked by the receptor-operated Ca2+ channel (ROC) blockers SK&F 96365 and NCDC, indicating a contribution by Ca2+ influx through ROC to the [Ca2+]i plateau. However, this [Ca2+]i plateau was not blocked by the voltage-gated Ca2+ channel (VGC) blocker diltiazem (DTZ). Depolarization with 80K+ or application of the VGC agonist BAY K 8644 did not alter the resting [Ca2+]i; but 80K+ reduced the histamine (100 microM) induced [Ca2+]i plateau. These results show that HCMEC are devoid of functional VGC. Thus the membrane potential (Em) regulates Ca2+ entry mainly by enhancing the electrochemical Ca2+ gradient, such that hyperpolarization increases while depolarization decreases [Ca2+]i. Blockade of sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) by CPA increased [Ca2+]i. This effect was dependent on extracellular Ca2+ and reduced by iberiotoxin (IBTX) blockade of Ca2+-activated K+ channels (Kca), suggesting a role for Kca in regulating Ca2+ influx. Ca2+ is the principal activator of endothelial nitric oxide synthase (eNOS), which stimulates cyclic GMP production. The final result that the eNOS inhibitor L-NAME enhanced the histamine (100 microM) induced [Ca2+]i plateau suggests a negative feedback loop (via cGMP) of endothelial NO on its own synthesis in the regulation of endothelial [Ca2+]i signal.
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PMID:Agonist-stimulated calcium entry in primary cultures of human cerebral microvascular endothelial cells. 1032 49

Although known for its role in hemostasis, there is a growing body of evidence that thrombin can induce leukocyte recruitment and contribute to the inflammatory response. An in vitro parallel-plate flow chamber was used to systematically examine thrombin-induced neutrophil interactions with human endothelium. Stimulation of endothelial cells with thrombin (1 U/ml) resulted in an immediate, P-selectin-dependent increase in neutrophil rolling and adhesion that was comparable in magnitude to optimal levels of histamine (the classical inducer of P-selectin). However, thrombin, but not histamine, induced a delayed (4 h) E-selectin-dependent rolling similar to that of tumor necrosis factor-alpha, suggesting that thrombin has the unique ability to recruit neutrophils by an early P-selectin and a delayed E-selectin pathway. Surprisingly, inhibition of E-selectin expression with the general protein synthesis inhibitor cycloheximide induced P-selectin expression 4 h after thrombin stimulation. Cycloheximide and thrombin (4 h) induced sufficient P-selectin-dependent rolling to recruit as many neutrophils as were recruited with 4 h of stimulation with thrombin alone. Histamine in the presence of cycloheximide or cycloheximide alone did not evoke the P-selectin response at 4 h, suggesting that this was not due to direct cycloheximide induction of P-selectin. Treatment of endothelium with tumor necrosis factor-alpha (an E-selectin inducer) and cycloheximide also eliminated E-selectin expression but, much like thrombin, induced P-selectin expression and neutrophil recruitment. In conclusion, inhibition of E-selectin via protein synthesis inhibition activates the protein synthesis-independent pathway of P-selectin expression to support adequate leukocyte recruitment.
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PMID:Translational inhibition of E-selectin expression stimulates P-selectin-dependent neutrophil recruitment. 1074 18


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