Nitric oxide:: inhibitory effects on endothelial cell calcium signaling, prostaglandin I2 production and nitric oxide synthase expression
Nitric oxide:: inhibitory effects on endothelial cell calcium signaling, prostaglandin I2 production and nitric oxide synthase expression
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DOI：
10.1016/j.cardiores.2003.12.028
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发表时间：
2004-04-01
影响因子：
10.8
通讯作者：
Hayashi, H
中科院分区：
文献类型：
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作者：
Takeuchi, K;Watanabe, H;Hayashi, H
目的：诱导型一氧化氮合酶大量产生的一氧化氮（NO）具有刺激炎症、诱导细胞凋亡等多种有害作用。内皮细胞是NO的主要生理来源，NO的过量产生对内皮细胞功能的影响尚不完全清楚。本研究旨在探讨NO对内皮细胞Ca ~（2+）信号转导和内皮细胞功能的调节作用。方法：原代猪主动脉内皮细胞（PAECs）用于所有这些研究。用Fura-2/AM测定细胞内Ca ~（2+）浓度（[Ca ~（2+）]（i））。采用酶免疫分析法测定前列腺素I-2（PGI（2））和环鸟苷酸（cGMP）的产生，采用Western印迹法测定内皮细胞NO合酶蛋白的表达。结果：缓激肽（BK，10 nM）和毒胡萝卜素（TG，1 μ M）引起[Ca 2 +]（i）大量增加。NO供体NOC 12在100 μ M时分别降低了21%和31%，在300 μ M时降低了60%和55%，在500 μ M时降低了74%和78%。这些影响也观察到与其他NO供体，包括精胺NONOate和NOC 18，并完全防止羧基PTIO（200 μ M），NO清除剂。而8-Bromo-cGMP对BK和TG刺激的Ca ~（2+）反应无影响。在用BK刺激的细胞中观察到PG 12产生增加30倍。在10、100、300和500 μ M时，NOC 12再次分别使该反应降低12%、54%、83%和95%。内皮细胞NO合成酶蛋白水平分别降低了2%，15%，36%和47%，孵育后2，6，12和24小时，与NOC 18，一个NO供体具有长半衰期。结论：NO，当大量产生时，可以独立于环GMP抑制激动剂诱导的Ca 2+反应，减少内皮源性舒张因子（EDRF）的产生并干扰内皮NO合酶蛋白的表达。(C)2004年欧洲心脏病学会。Elsevier B. V.出版，保留所有权利。
Objective: Nitric oxide (NO) produced in large amounts by inducible nitric oxide synthase exerts many harmful effects such as stimulation of inflammation and induction of apoptosis. The effects of excessive NO production on functions of endothelial cells, the major physiological source of NO, are not completely known, The aim of this study was to investigate the role of NO on the regulation of endothelial cell Ca2+ signaling and endothelial cell function. Methods: Primary porcine aortic endothelial cells (PAECs) were used for all these studies. Intracellular Ca2+ concentrations ([Ca2+](i)) were measured using fura-2/AM. Production of prostaglandin I-2 (PGI(2)) and cyclic GMP were assessed using enzyme immunoassays, and endothelial NO synthase protein expression was evaluated by Western blotting. Results: Bradykinin (BK, 10 nM) and thapsigargin (TG, 1 muM) provoked large increases in [Ca2+](i). The NO donor NOC12 reduced these responses, respectively, by 21% and 31% at 100 muM, 60% and 55% at 300 muM, and 74% and 78% at 500 muM. These effects were also observed with other NO donors including spermine NONOate and NOC18, and were completely prevented by carboxy-PTIO (200 muM), an NO scavenger. 8-Bromo-cGMP, however, had no effects on BK- and TG-stirnulated Ca2+ responses. A 30-fold increase in PG12 production was observed in cells stimulated with BK. NOC12 again reduced this response by 12%, 54%, 83% and 95% at 10, 100, 300 and 500 muM, respectively. Endothelial NO synthase protein level was reduced by 2%, 15%, 36 and 47% after 2, 6, 12 and 24 h, respectively, of incubation with NOC18, a NO donor with long half-life. Conclusions: NO, when produced in large amounts, can inhibit agonist-induced Ca2+ responses independently of cyclic GMP, reduce the production of endothelium-derived relaxing factors (EDRFs) and interfere with endothelial NO synthase protein expression. (C) 2004 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.