Kruse H J, Grünberg B, Siess W, Weber P C
Institut für Prophylaxe und Epidemiologie der Kreislaufkrankheifen, University of Munich, FRG.
Arterioscler Thromb. 1994 Nov;14(11):1821-8. doi: 10.1161/01.atv.14.11.1821.
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 F1 alpha and thromboxane B2 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 NG-monomethyl-L-arginine but was almost completely suppressed by SK&F 96365. The concomitant endothelial release of thromboxane A2 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.
使用受体介导的钙内流阻滞剂SK&F 96365来评估钙内流对生物活性内皮前列腺素和内皮衍生舒张因子（EDRF）形成的作用。SK&F 96365抑制组胺刺激的钙进入人脐静脉内皮细胞，但不抑制其从细胞内储存库释放，这是通过用fura-2负载细胞中细胞内钙浓度的变化进行荧光分光光度法测定的。与此一致，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F1α和血栓素B2。为了评估内皮细胞形成前列环素和EDRF对血小板的功能意义，在内皮细胞与血小板的共孵育实验中分析了两种血小板蛋白rap1B和一种50-kD血管舒张刺激磷蛋白（VASP）的cAMP和cGMP依赖性磷酸化。组胺刺激的内皮细胞释放的自分泌物质导致血小板中rap1B和VASP的磷酸化，吲哚美辛或NG-单甲基-L-精氨酸仅部分抑制该磷酸化，但SK&F 96365几乎完全抑制该磷酸化。内皮细胞同时释放的血栓素A2对血小板蛋白的蛋白激酶C和钙依赖性磷酸化没有影响。结果表明，SK&F 96365阻断受体介导的钙内流显著减少了内皮细胞释放生物活性前列环素和EDRF。因此，钙内流而非细胞内储存库的钙释放在内皮细胞中受体刺激的前列环素和EDRF的形成和释放中起关键作用。
