Nitric oxide inhibits capacitative Ca2+ entry and enhances endoplasmic reticulum Ca2+ uptake in bovine vascular endothelial cells
Nitric oxide inhibits capacitative Ca2+ entry and enhances endoplasmic reticulum Ca2+ uptake in bovine vascular endothelial cells
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DOI：
10.1113/jphysiol.2001.013258
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发表时间：
2002-02-15
影响因子：
5.5
通讯作者：
Blatter, LA
中科院分区：
文献类型：
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作者：
Dedkova, EN;Blatter, LA
在血管内皮细胞中，胞浆游离钙浓度（[Ca 2 +]（i））的升高引起一氧化氮合酶（NOS）的激活和一氧化氮（NO）的释放。该研究的目的是表征这种细胞类型中[Ca 2 +]（i）和NO产生之间的相互作用。培养的牛血管内皮细胞[Ca ~（2+）]（i）和细胞内NO浓度的同时测定分别用荧光指示剂fura-2和fura-2对CPAE细胞系进行荧光检测，结果显示激动剂诱导的细胞内钙库耗竭后，（capacitative Ca 2 + entry，CCE）是激活内皮型一氧化氮合酶（eNOS）的首选Ca 2+来源。暴露于NO供体硝普钠（SNP）表明，高水平的NO抑制CCE和质膜Ca 2 +-ATP酶的Ca 2+挤出的抑制作用。这种对CCE的抑制作用被膜渗透性cGMP类似物8-溴-cGMP模仿，但被NO清除剂血红蛋白逆转，并被NO敏感的鸟苷酸环化酶ODQ的抑制剂阻止。短暂暴露于SNP降低了ATP诱导的Ca 2+从内质网（ER）释放的峰值，并加速了Ca 2+再摄取到ER中。与SNP长时间孵育导致增强的Ca 2+负载的ER，所揭示的存储内容与ER截留的低亲和力Ca 2+指示剂mag-fura-2的直接测量。结果表明，在血管内皮细胞中，NO合成是在涉及NO依赖性[Ca 2 +]（i）调节的自动调节控制下。通过cGMP依赖性抑制CCE和加速Ca 2+螯合到ER中，NO可以降低[Ca 2 +]（i），因此对其自身的Ca 2+依赖性合成施加自调节负反馈。
In vascular endothelial cells, elevation of cytosolic free calcium concentration ([Ca2+](i)) causes activation of nitric oxide synthase (NOS) and release of nitric oxide (NO). The goal of the study was to characterize the interplay between [Ca2+](i) and NO production in this cell type. Simultaneous measurements of [Ca2+](i) and intracellular NO concentration ([NO](i)) in cultured bovine vascular endothelial cells (CPAE cell line) with the fluorescent indicators fura-2 and DAF-2, respectively, revealed that Ca2+ influx following agonist-induced intracellular Ca2+ store depletion (capacitative Ca2+ entry, CCE) represents the preferential Ca2+ source for the activation of the Ca2+-calmodulin-dependent endothelial NOS (eNOS). Exposure to the NO donor sodium nitroprusside (SNP) showed that high NO levels suppressed CCE and had an inhibitory effect on Ca2+ extrusion by the plasmalemmal Ca2+-ATPase. This inhibitory effect on CCE was mimicked by the membrane-permeant cGMP analogue 8-bromo-cGMP, but was reversed by the NO scavenger haemoglobin and prevented by the inhibitor of the NO-sensitive guanylate cyclase ODQ. Brief exposure to SNP reduced the peak of ATP-induced Ca2+ release from the endoplasmic reticulum (ER) and accelerated Ca2+ reuptake into the ER. Prolonged incubation with SNP resulted in enhanced Ca2+ loading of the ER, as revealed by direct measurements of store content with the ER-entrapped low-affinity Ca2+ indicator mag-fura-2. The results suggest that in vascular endothelial cells, NO synthesis is under autoregulatory control that involves NO-dependent [Ca2+](i) regulation. Via cGMP-dependent inhibition of CCE and acceleration of Ca2+ sequestration into the ER, NO can lower [Ca2+](i) and therefore exert an autoregulatory negative feedback on its own Ca2+-dependent synthesis.