FORMATION OF BIOLOGICALLY-ACTIVE AUTACOIDS IS REGULATED BY CALCIUM INFLUX IN ENDOTHELIAL-CELLS
FORMATION OF BIOLOGICALLY-ACTIVE AUTACOIDS IS REGULATED BY CALCIUM INFLUX IN ENDOTHELIAL-CELLS
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DOI：
10.1161/01.atv.14.11.1821
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发表时间：
1994-11-01
期刊：
影响因子：
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通讯作者：
WEBER, PC
中科院分区：
文献类型：
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作者：
KRUSE, HJ;GRUNBERG, B;WEBER, PC
用受体介导的钙内流阻断剂SK&F 96365评价钙内流对生物活性内皮前列环素和内皮衍生松弛因子形成的影响。SK&F 96365抑制组胺刺激的钙离子进入人脐静脉内皮细胞，但不抑制组胺刺激的钙离子从细胞内钙离子排出，这是通过荧光分光光度法检测Fura-2负载细胞内钙离子浓度的变化确定的。SK&F 96365以剂量依赖的方式抑制组胺诱导的内皮细胞合成6-酮-前列腺素F-1α和血栓素B-2。为了评价前列环素和EDRF对血小板内皮形成的功能意义，在内皮细胞与血小板的共同孵育实验中，分析了两种血小板蛋白Rap1B和50kD血管扩张剂刺激的磷酸蛋白(VASP)依赖的cAMP和cGMP的磷酸化。组胺刺激内皮细胞释放的类胡萝卜素可引起血小板RAP1B和VASP的磷酸化，消炎痛或N-G-单甲基-L-精氨酸只部分抑制这种作用，但SK&F 96365几乎完全抑制这种作用。伴随的血栓素A(2)内皮细胞释放对蛋白激酶C和钙依赖的血小板蛋白的磷酸化没有影响。结果表明，SK&F 96365阻断受体介导的钙内流可显著减少血管内皮细胞释放具有生物活性的前列环素和内源性内分泌因子。因此，在受体刺激的内皮细胞内前列环素和EDRF的形成和释放中，钙离子内流而不是钙离子释放起着关键作用。
The blocker of receptor-mediated calcium entry SK&F 96365 was used to evaluate the contribution of calcium influx to the formation of biologically active endothelial prostanoids and endothelium-derived relaxing factor (EDRF). SK&F 96365 inhibited histamine-stimulated calcium entry into human umbilical vein endothelial cells but not its discharge from intracellular stores as determined spectrofluorometrically by changes of intracellular calcium concentration in fura-2-loaded cells. Concordantly, SK&F 96365 inhibited histamine-induced endothelial synthesis of 6-keto-prostaglandin F-1 alpha and thromboxane B-2 in a dose-dependent manner. To assess the functional significance of endothelial formation of prostacyclin and EDRF to platelets, the cAMP- and cGMP-dependent phosphorylation of two platelet proteins, rap1B and a 50-kD vasodilator-stimulated phosphoprotein (VASP), was analyzed in coincubation experiments of endothelial cells with platelets. Autacoids released by histamine-stimulated endothelial cells caused the phosphorylation of rap1B and VASP in platelets, which was only partly inhibited by either indomethacin or N-G-monomethyl-L-arginine but was almost completely suppressed by SK&F 96365. The concomitant endothelial release of thromboxane A(2) had no effect on protein kinase C- and calcium-dependent phosphorylation of platelet proteins. The results demonstrate that blockade of receptor-mediated calcium entry by SK&F 96365 markedly reduced the release of biologically active prostacyclin and EDRF from endothelial cells. Thus, calcium influx but not calcium release from intracellular stores plays a critical role in the receptor-stimulated formation and liberation of prostacyclin and EDRF in endothelial cells.