ELECTROPHARMACOLOGYCAL ANALYSIS OF INTRACELLULAR SIGNALTRANSDUCTIONS OF VASCULAR ENDOTHERIAL CELLS

在培养的血管内皮细胞中，高浓度的组胺和ATP （>0 μ m）会产生初始瞬态，随后细胞内游离Ca^<2+>浓度持续升高（[Ca^<2+>]_i）。在本实验中，我们在装载荧光Ca^<2+>指示物fura-2的培养人主动脉内皮细胞中研究了后者持续升高的机制。后一阶段通过去除细胞外Ca^<2+>来消除，并通过将细胞外Cl^-浓度降低到40 mM或通过Cl^-通道阻滞剂n -苯氰苯甲酸（NPA,1 mM）可逆地消除。研究了内质网Ca^<2+>- atp酶特异性抑制剂thapsigargin和cyclopiazonic acid （CPA）对[Ca^<2+>]_i的影响。Thapsigargin （1 ~ 1000 nM）和CPA （0.1 ~ 100 nM）使[Ca^<2+>]_i发生双相变化。缓慢下降期通过去除细胞外Ca^<2+>而消除，并通过低Cl^-条件和NPA加以阻止。这些结果表明，在组胺、ATP、thapsigargin和CPA的作用下，[Ca^<2+ b>] i的持续升高是由[Ca^<2+>] i的升高和/或培养的人主动脉内皮细胞中细胞内Ca^<2+>储存的消耗激活的细胞外Ca^<2+>的Cl^-敏感进入和/或消耗产生的。

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.