Inositol phosphate production and Ca2+ mobilization in human umbilical-vein endothelial cells stimulated by thrombin and histamine.
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ABSTRACT:
SUBMITTER: Pollock WK
PROVIDER: S-EPMC1135419 | biostudies-other | 1988 Dec
REPOSITORIES: biostudies-other
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Project description:Pure samples of inositol 1,3,4-trisphosphate, inositol 1,3,4,5-tetrakisphosphate and inositol 1,2-cyclic 4,5-trisphosphate were prepared and tested for their ability to mobilize calcium from intracellular stores in a permeabilized Swiss mouse 3T3 cell preparation. In this system inositol 1,4,5-trisphosphate mobilizes Ca2+ with a half-maximal dose of 0.3 microM. Inositol 1,2-cyclic 4,5-trisphosphate mobilized Ca2+ to the same extent with a half-maximal dose of 0.3 microM, whereas inositol 1,3,4-trisphosphate required a half-maximal dose of approx. 9 microM to give the same effect. Inositol 1,3,4,5-tetrakisphosphate was ineffective up to 20 microM and at that concentration did not antagonize the mobilization induced by inositol 1,4,5-trisphosphate. The relevance of these findings to the function of the inositol tris/tetrakis-phosphate pathway is discussed.
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Project description:In several cell types, including hepatocytes, submaximal concentrations of Ins(1,4,5)P3 stimulate an initial rapid mobilization of intracellular Ca2+ stores that is followed by either no further Ca2+ release or very much slower release. Further additions of Ins(1,4,5)P3 then evoke further Ca2+ mobilization. Such 'incremental' responses [Meyer & Stryer (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 3841-3845] could result from all-or-nothing emptying of stores that differ in their sensitivities to Ins(1,4,5)P3 or from partial emptying of stores that are more uniformly sensitive, but unable to release all of their Ca2+ because the response to Ins(1,4,5)P3 rapidly attenuates. By measuring unidirectional 45Ca2+ efflux from intracellular stores stimulated with Ins(1,4,5)P3 under conditions where they continue to sequester 40Ca2+, we provide evidence suggesting that Ins(1,4,5)P3 stimulates all-or-nothing emptying of stores that differ in their sensitivities to Ins(1,4,5)P3, a quantal response pattern.
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Project description:Previous reports demonstrated that the expression of thrombomodulin (TM) in endothelial cells was modulated by various agents. Although TM was down-regulated by endotoxin or cytokines, up-regulation of TM was accomplished when endothelial cells were stimulated with unphysiologically high concentrations of cyclic AMP derivatives or tumour-promoting phorbol esters. We investigated the expression of TM in human umbilical-vein endothelial cells (HUVECs) by physiological substances that can be released into the bloodstream. Histamine (0.1-10 microM, 1-48 h) increased TM activity, TM antigen in cell lysates and TM mRNA levels, but 5-hydroxytryptamine and bradykinin had no effect. Enhancement of TM activity by histamine was completely blocked by the H1-selective antagonist pyrilamine, whereas the H2-antagonist cimetidine had no effect, showing that histamine up-regulates TM activity via H1-receptors on HUVECs. Enhanced TM activity by histamine and the resultant increase in protein C activation might play a role in a feedback regulation for prevention of vascular thrombosis.
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Project description:Most studies of tissue factor (TF) expression in endothelial cells (EC) are performed under stationary culture conditions. The purpose of this study was to determine the influence of mechanical stimuli such as cyclic strain (CS) on the expression of TF in EC exposed to thrombin (Thr). Human umbilical vein endothelial cells (HUVEC) were exposed to 4 U·mL(-1) Thr in the presence or absence of 10% average CS at 60 cycles·min(-1) and then TF expression was measured. TF messenger RNA (mRNA) expression peaked at 2 hours in HUVEC exposed to Thr, but at 4 hours in HUVEC exposed to both Thr + CS. TF expression was inhibited by p38 and extracellular signal-regulated protein kinase (ERK) inhibitors. For both Thr or Thr + CS stimuli, p38 and ERK activity peaked at 5 minutes (p < 0.05). Nuclear factor-kappa B levels remained high in the Thr group but not in the Thr + CS group, while Egr-1 levels were elevated in the Thr + CS group. We demonstrated CS-delayed, Thr-induced TF mRNA expression in HUVEC, which may be modulated by p38 and ERK inhibitors.
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Project description:The causal relationships between cytosolic free-Ca2+ concentration ([Ca2+]i) increases and production of nitric oxide (NO) have been investigated mostly with indirect methods and remain unclear. Here we demonstrate, by direct real-time measurements of [NO] with a porphyrinic microsensor, that Ca2+ entry, but not an increase in [Ca2+]i, is required for triggering of NO production in human endothelial cells. Histamine, ranging from 0.1 to 100 microM, increased both NO production and [Ca2+]i when given in a single dose. However, histamine caused increased NO release but induced progressively smaller [Ca2+]i changes when cumulatively added. In the absence of a transmembrane Ca2+ gradient, no significant NO release was detectable, despite the marked Ca2+ peak induced by histamine. Inhibition of Ca2+ entry by SK&F 96365 abolished histamine-elicited NO production but only reduced the transient [Ca2+]i rise. The suppression of the sustained [Ca2+]i response under these two conditions suggests that NO release was closely associated with Ca2+ entry from the extracellular space. In addition, membrane depolarization, achieved by increasing the extracellular K+ concentration from 5 to 130 mM, reduced both the amplitude of histamine-induced sustained [Ca2+]i elevation and NO production. These results lead us to propose that the availability of numerous Ca2+ ions around the internal side of the plasma membrane would promote the association between nitric oxide synthase and calmodulin, thereby activating the enzyme.
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