Zeng Qinghua, Zhou Qingwei, Yao Fanrong, O'Rourke Stephen T, Sun Chengwen
Department of Pharmaceutical Sciences, North Dakota State University, Fargo, ND 58105, USA.
J Pharmacol Exp Ther. 2008 Sep;326(3):732-8. doi: 10.1124/jpet.108.140301. Epub 2008 Jun 6.
It has been shown that reactive oxygen species (ROS) are involved in the intracellular signaling response to G-protein coupled receptor stimuli in vascular smooth muscle cells and in neurons. In the present study, we tested the hypothesis that NAD(P)H oxidase-derived ROS are involved endothelin-1 (ET-1)-induced L-type calcium channel activation in isolated cardiac myocytes. ET-1 (10 nM) induced a 2-fold increase in L-type calcium channel open-state probability (NPo). This effect of ET-1 was abolished by the ET(A) receptor antagonist cyclo(D-Trp-D-Asp-Pro-D-Val-Leu) [BQ-123 (1 microM)] but was not altered in the presence of an ET(B) receptor antagonist N-cis-2,6-dimethylpiperidinocarbonyl-b-tBu-Ala-D-Trp(1-methoxycarbonyl)-D-Nle-OH [BQ-788 (1 microM)]. Pretreatment of cells with the ROS scavenger tempol (100 microM), polyethylene glycol-superoxide dismutase (SOD, 25 U/ml), or the NAD(P)H-oxidase inhibitor gp91ds-tat ([H]RKKRRQRRR-CSTRIRRQL[NH(3)]) (5 microM) significantly attenuated ET-1-induced increases in calcium channel NPo. Tempol, SOD, and gp91ds-tat alone had no effect on basal calcium channel activity. In addition, ET-1 significantly increased NAD(P)H oxidase activity and elevated intracellular superoxide levels in cultured cardiac myocytes. The superoxide generator, xanthine-xanthine oxidase (10 mM, 20 mU/ml), also increased calcium channel NPo in cardiac myocytes, mimicking the effect of ET-1. These observations provide the first evidence that ET-1 induces the activation of L-type Ca(2+) channels via stimulation of NAD(P)H-derived superoxide production in cardiac myocytes.
研究表明，活性氧（ROS）参与血管平滑肌细胞和神经元中G蛋白偶联受体刺激后的细胞内信号转导反应。在本研究中，我们验证了一个假说，即NAD(P)H氧化酶衍生的ROS参与了内皮素-1（ET-1）诱导的离体心肌细胞L型钙通道激活。ET-1（10 nM）使L型钙通道开放状态概率（NPo）增加了2倍。ET-1的这种作用被ET(A)受体拮抗剂环（D-色氨酸-D-天冬氨酸-脯氨酸-D-缬氨酸-亮氨酸）[BQ-123（1 microM）]消除，但在存在ET(B)受体拮抗剂N-顺式-2,6-二甲基哌啶羰基-b-叔丁基丙氨酸-D-色氨酸（1-甲氧基羰基）-D-正亮氨酸-OH [BQ-788（1 microM）]时未改变。用ROS清除剂tempol（100 microM）、聚乙二醇超氧化物歧化酶（SOD，25 U/ml）或NAD(P)H氧化酶抑制剂gp91ds-tat（[H]RKKRRQRRR-CSTRIRRQL[NH(3)]）（5 microM）预处理细胞，可显著减弱ET-1诱导的钙通道NPo增加。单独使用tempol、SOD和gp91ds-tat对基础钙通道活性无影响。此外，ET-1显著增加了培养心肌细胞中NAD(P)H氧化酶活性并提高了细胞内超氧化物水平。超氧化物生成剂黄嘌呤-黄嘌呤氧化酶（10 mM，20 mU/ml）也增加了心肌细胞中的钙通道NPo，模拟了ET-1的作用。这些观察结果首次证明，ET-1通过刺激心肌细胞中NAD(P)H衍生的超氧化物产生来诱导L型Ca(2+)通道的激活。
