Nanomolar level of ouabain increases intracellular calcium to produce nitric oxide in rat aortic endothelial cells
Nanomolar level of ouabain increases intracellular calcium to produce nitric oxide in rat aortic endothelial cells
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DOI：
10.1111/j.1440-1681.2004.03995.x
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发表时间：
2004-05-01
影响因子：
2.9
通讯作者：
Takahashi, H
中科院分区：
文献类型：
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作者：
Dong, XH;Komiyama, Y;Takahashi, H
1. 跨细胞膜和/或肌浆网的 [Ca2+](i) 变化调节内皮一氧化氮 (NO) 合酶活性2。在本研究中，我们通过使用 NO 特异性实时传感器连续监测 NO 产生并使用高速波长切换荧光显微成像技术测量 [Ca2+](i) 的变化，研究了哇巴因 (Na+/K+-ATPase 的特异性抑制剂) 对培养的大鼠主动脉内皮细胞 (RAEC) 中 NO 释放和 [Ca2+](i) 运动的影响。以t(1/2)(10μmol/L缓激肽刺激后[Ca2+](i)下降至基础水平的半衰期)作为[Ca2+](i)排出的指标。 3．非常低浓度的哇巴因（10 nmol/L）不会增加NO产生的峰值，但会降低NO释放的衰减，因此，通过缓激肽诱导的最大剂量反应浓度增加整体NO产生。相同剂量的哇巴因影响缓激肽诱导的 [Ca2+](i) 穿过细胞膜和/或肌浆网的运动，其时间过程与 NO 释放的时间过程相似。而且t(1/2)显着提高。 4．用Na+/Ca2+交换抑制剂的无Na+溶液和六水氯化镍预处理RAEC可以防止缓激肽和哇巴因诱导的效应。5．这些使用实时记录的观察结果表明，少量哇巴因通过抑制质膜 Na+/K+-ATP 酶活性和增加细胞内 Na+ 浓度，有助于缓激肽刺激的 NO 产生增加。然后膜去极化，通过正向模式 Na+/Ca2+ 交换，延长 Ca2+ 信号时间，导致缓激肽刺激的 [Ca2+](i) 增加下降。
1. Changes in [Ca2+](i) across the cell membrane and/or the sarcoplasmic reticulum regulate endothelial nitric oxide (NO) synthase activity2. In the present study, we investigated the effect of ouabain, a specific inhibitor of Na+/K+-ATPase, on NO release and [Ca2+](i) movements in cultured rat aortic endothelial cells (RAEC) by monitoring NO production continuously using an NO-specific real-time sensor and by measuring the change in [Ca2+](i) using a fluorescence microscopic imaging technique with high-speed wavelength switching. The t(1/2) (half-time of the decline of [Ca2+](i), to basal levels after stimulation with 10 mumol/L bradykinin) was used as an index of [Ca2+](i) extrusion.3. A very low concentration of ouabain (10 nmol/L) did not increase the peak of NO production, but decreased the decay of NO release and, accordingly, increased integral NO production by the maximal dose-response concentration induced by bradykinin. The same dose of ouabain affected [Ca2+](i) movements across the cell membrane and/or sarcoplasmic reticulum induced by bradykinin with a time-course similar to that of NO release. Moreover, the t(1/2) was significantly increased.4. Pretreatment of RAEC with Na+-free solution, an inhibitor of the Na+/Ca2+ exchanger, and nickel chloride hexahydrate prevented the effects induced by bradykinin and ouabain.5. These observations using real-time recording indicate that a small amount of ouabain contributes to the bradykinin-stimulated increase of NO production through inhibition of plasma membrane Na+/K+-ATPase activity and an increase in intracellular Na+ concentrations. The membrane was then depolarized, leading to a decline in the bradykinin-stimulated increase in [Ca2+](i) by forward mode Na+/Ca2+ exchange to prolong the Ca2+ signal time.