Electrophysiological study of existence and regulation of chloride current in endothelial cells

本研究揭示了培养的人主动脉、人脐静脉和牛肺动脉内皮细胞(ETC)中存在C_1通道。C_1-电流的产生可以看作是内皮细胞的共同属性。采用膜片钳技术测定膜电流，Fura-2荧光法测定细胞内钙离子浓度。主要结果如下：1)外加1~10um的三磷酸腺苷诱发外向整流性氯电流，继之以钙激活的K+电流；2)用PCA 6和PCA 5的移液管溶液，在膜破裂后几分钟内出现延迟的C1电流；3)三氟拉嗪和W7，钙调素拮抗剂阻断钙激活的氯电流；5)将[Cl]o从146 mM降至20 mM，抑制了ATP引起的[Ca]i升高的紧张期。6)从[K]o、[c1]o变化所记录的泄漏电流和单通道电流的变化和应用ATP的膜片钳技术来看，ETC的静息电位比K+离子的反转电位多20-40 mV。这表明，C_1-电流的作用是使静息膜电位稳定在去极化范围内。

The present study has disclosed the existence of C1- channels in cultured endothelial cells (ETC) from human aorta, human umbilical vein as well as bovine pulmonary artery. The generation of C1- current can be regarded as a common property of the endothelial cells. We used mainly cultured human aortic ETC and measured membrane currents by patch clamp technique and intracellular Ca concentration [Ca]i by fura-2 fluorometry. Major results were : 1) External application of 1-10 uM ATP induced outward-rectifying Cl- currents succeeding to Ca-activated K+ current, 2) With pipette solution of pCa 6 and pCa 5, C1 currents developed with a delay of a few minuts after breaking the patch membrane, 3) The Ca-activated C1- currents were blocked by trifluoperazine and W7, calmodulin antagonists, 4) [Ca]i increased in a bipasic manner by external application of 10 uM ATP, 5) The tonic phase of the ATP- induced [Ca]i increase was suppressed by decreasing [Cl]o from 146 to 20 mM.6) From the change of the leakage current and single channel currents recorded by cell-attached patch clamp technique produced by the change in [K]o, [C1]o and an application of ATP, the resting potential of ETC is at 20-40 mV more depolarized potentials from the reversal potential of K+ ions. It suggests that the functional role of the C1- currents is the stabilization of the resting membrane potential to the depolarized range