ELECTROPHARMACOLOGYCAL ANALYSIS OF INTRACELLULAR SIGNALTRANSDUCTIONS OF VASCULAR ENDOTHERIAL CELLS

在培养的血管内皮细胞中，组胺和三磷酸腺苷在高浓度(&gt；10um)时产生一过性的细胞内游离钙离子浓度的持续升高。在本实验中，我们用钙荧光指示剂Fura-2在培养的人主动脉内皮细胞中研究了后者持续升高的机制。后者可通过去除胞外钙离子而消除，并可通过将胞外氯离子浓度降至40 mM或用氯离子通道阻断剂N-苯基邻氨基苯甲酸(NPA，1 mM)可逆地消除。同时观察内质网钙离子的特异性阻断剂thapsigargin和环哌酸(CPA)对[Ca^&lt；2&gt；-ATPase]_i的影响。山茶素(1-1000 nM)和CPA(0.1-100微米)引起[Ca^&lt；2&gt；]_i的双相变化。缓慢下降的阶段被细胞外钙的去除消除，并被低氯条件和NPA阻止。这些结果表明，组胺、ATP、thapsigarin和CPA引起的[Ca^&lt；2&gt；]_i的持续升高是由于[Ca^&lt；2&gt；2&gt；_i和/或通过耗尽培养的人主动脉内皮细胞内的钙储备。

In cultured vascular endothelial cells, histamine and ATP at high concentrations (>10 muM) produce an initial transient followed by sustained elevation in intracellular free Ca^<2+> concentration ([Ca^<2+>]_i). In the present experiments, mechanisms responsible for the latter sustained elevation were studied in cultured human aortic endothelial cells loaded with the fluorescent Ca^<2+> indicator fura-2. The latter phase was eliminated by removal of extracellular Ca^<2+>, and was reversibly abolished by reduction of extracellular Cl^- concentration to 40 mM or by the Cl^- channel blocker N-phenylanthranilic acid (NPA,1 mM). Effects of thapsigargin and cyclopiazonic acid (CPA), specific inhibitors of endoplasmic reticulum Ca^<2+>-ATPase, on [Ca^<2+>]_i were also examined. Thapsigargin (1-1000 nM) and CPA (0.1-100 muM) produced a biphasic change in [Ca^<2+>]_i. The slow declining phase was eliminated by removal of extracellular Ca^<2+> and was prevented by the low Cl^- condition and NPA.These results suggest that the sustained elevation of [Ca^<2+>]_i in response to histamine, ATP,thapsigargin and CPA is produced by the Cl^--sensitive entry of extracellular Ca^<2+> activated by the rise in [Ca^<2+>]_i and/or by the depletion of intracellular Ca^<2+> stores in cultured human aortic endothelial cells.