ELECTROPHARMACOLOGYCAL ANALYSIS OF INTRACELLULAR SIGNALTRANSDUCTIONS OF VASCULAR ENDOTHERIAL CELLS

在培养的血管内皮细胞中，高浓度（>10 μ M）的组胺和ATP产生一个初始的瞬时，随后是细胞内游离Ca^2+浓度（[Ca^2+] i）的持续升高。在目前的实验中，我们在培养的人主动脉内皮细胞中研究了钙离子浓度持续升高的机制，这些细胞中加入了钙离子荧光指示剂fura-2。后一个时相可通过去除细胞外Ca^2+而消除，并可通过将细胞外Cl^-浓度降至40 mM或Cl^-通道阻断剂N-苯基邻氨基苯甲酸（NPA，1 mM）可逆地消除。同时观察了内质网Ca^<2+>-ATP酶的特异性抑制剂毒胡萝卜素和环匹阿尼酸（CPA）对[Ca^<2+]_i的影响。毒胡萝卜素（1-1000 nM）和CPA（0.1-100 μ M）引起[Ca^2+] i的双相变化。细胞外Ca^<2 +>的清除可消除缓慢下降相，低Cl^-和NPA可阻止缓慢下降相的出现，提示组胺、ATP、毒胡萝卜素和CPA是由细胞外Ca^<2+>的Cl^-敏感性进入产生的，细胞外Ca^<2+>的进入是由[Ca^<2+>]_i和/或通过消耗培养的人主动脉内皮细胞中的细胞内Ca^2+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.