Chen J, Wang Y, Nakajima T, Iwasawa K, Hikiji H, Sunamoto M, Choi D K, Yoshida Y, Sakaki Y, Toyo-Oka T
Second Department of Internal Medicine, the Department of Oral and Maxillofacial Surgery, and the Human Genome Center, Institute of Medical Science, University of Tokyo, Tokyo 113-0033, Japan.
J Biol Chem. 2000 Sep 15;275(37):28739-49. doi: 10.1074/jbc.M000910200.
The rise in cytosolic Ca(2+) concentration (Ca(2+)(i)) in vascular endothelial cells (ECs) activates the production and release of nitric oxide (NO). NO modifies Ca(2+)(i) homeostasis in many types of nonendothelial cells. However, its effect on endothelial Ca(2+)(i) homeostasis at basal and excited states remains unclear. In the present study, to elucidate the effect of NO on basal Ca(2+)(i), inositol 1,4,5-trisphosphate-induced Ca(2+)(i) release (IICR) was blocked by expressing an antisense against type-1 inositol 1,4,5-trisphosphate receptors or by microinjecting heparin to individual ECs, and the effects of NO that was released by and diffused from adjacent IICR-intact ECs were recorded. After ATP or bradykinin stimulation, IICR-inhibited ECs showed a marked reduction of basal Ca(2+)(i), which was abolished by N(G)-monomethyl-l-arginine monoacetate pretreatment. The reduction disappeared in sparsely seeded ECs. Exogenous NO gas mimicked the effect of ATP or bradykinin to reduce basal Ca(2+)(i). Blocking plasma membrane Ca(2+)-ATPase (PMCA), but not Na(+)-Ca(2+) exchange or sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase, suppressed the reduction, indicating that the reduction resulted from a NO-dependent potentiation of PMCA. To elucidate the effect of NO on elevated Ca(2+)(i), ATP-, bradykinin-, or thapsigargin-evoked Ca(2+)(i) response in the presence and absence of NO production was compared in adjacent IICR-intact ECs. NO was found to potentiate PMCA, which, in turn, greatly attenuated agonist-evoked Ca(2+)(i) elevation. NO also potentiated Ca(2+) influx, which markedly increased the sustained phase of Ca(2+)(i) elevation and possibly NO production. NO did not affect other Ca(2+)(i)-elevating and Ca(2+)(i)-sequestrating components. Thus, NO-dependent potentiation of PMCA is crucial for Ca(2+)(i) homeostasis over a wide Ca(2+)(i) range.
血管内皮细胞（ECs）中胞质钙离子浓度（Ca(2+)(i)）的升高会激活一氧化氮（NO）的产生与释放。NO会改变多种非内皮细胞类型中的Ca(2+)(i)稳态。然而，其在基础状态和兴奋状态下对内皮Ca(2+)(i)稳态的影响仍不清楚。在本研究中，为了阐明NO对基础Ca(2+)(i)的影响，通过表达针对1型肌醇1,4,5-三磷酸受体的反义核酸或向单个ECs显微注射肝素，阻断肌醇1,4,5-三磷酸诱导的Ca(2+)(i)释放（IICR），并记录由相邻IICR完整的ECs释放并扩散的NO的作用。在ATP或缓激肽刺激后，IICR受抑制的ECs显示基础Ca(2+)(i)显著降低，而N(G)-单甲基-L-精氨酸单乙酸盐预处理可消除这种降低。在稀疏接种的ECs中，这种降低消失。外源性NO气体模拟了ATP或缓激肽降低基础Ca(2+)(i)的作用。阻断质膜Ca(2+)-ATP酶（PMCA），而非钠钙交换或肌浆网/内质网Ca(2+)-ATP酶，可抑制这种降低，表明这种降低是由NO依赖性增强PMCA所致。为了阐明NO对升高的Ca(2+)(i)的影响，在相邻IICR完整的ECs中比较了有无NO产生时ATP、缓激肽或毒胡萝卜素诱发的Ca(2+)(i)反应。发现NO增强了PMCA，这反过来又极大地减弱了激动剂诱发的Ca(2+)(i)升高。NO还增强了Ca(2+)内流，这显著增加了Ca(2+)(i)升高的持续阶段并可能增加了NO的产生。NO不影响其他Ca(2+)(i)升高和Ca(2+)(i)螯合成分。因此，NO依赖性增强PMCA对于在广泛的Ca(2+)(i)范围内维持Ca(2+)(i)稳态至关重要。
