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

本研究揭示了体外培养的人主动脉、人脐静脉和牛肺动脉内皮细胞（ETC）中存在C1-通道。产生C1电流是内皮细胞的一个共同特性。以体外培养的主动脉ETC为主，膜片钳法测定膜电流，fura-2荧光法测定细胞内Ca浓度[Ca]i。主要结果如下：1)外用1-10 μ m ATP诱导钙活化K+电流后的向外整流Cl-电流，2)用pc6和pc5的移液管溶液，C1电流在破膜片膜后延迟几分钟形成，3)钙活化的C1-电流被三氟拉嗪和钙调素拮抗剂W7阻断，4)外用10 μ m ATP使[Ca]i以双相方式增加。(5)将[Cl]o从146 mm降低到20 mm，抑制了ATP诱导的[Ca]i的增加。(6)从细胞贴附膜片钳技术记录的[K]o、[C1]o的变化和ATP的应用所产生的漏电流和单通道电流的变化来看，ETC的静息电位比K+离子的逆转电位高出20-40 mV的去极化电位。这表明C1-电流的功能作用是将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