Molecular and Electropharmacological Analysis of Capacitative Ca^<2+> Entry channels of Vascular Endothelial Cells

在培养的人主动脉内皮细胞（HAECs）中，组胺（1-100 μ M）引起[Ca^2+] i的双相反应。我们采用全细胞电流记录结合细胞内Ca^<2+>浓度（[Ca^<2+]_i）荧光测量的方法来研究内皮细胞中C1敏感性Ca^<2+内流的机制。用制霉菌素穿孔膜片钳技术测定单个内皮细胞的膜电流。组胺诱导的Ca^2+内流可通过降低细胞外Cl^-浓度或Cl^-通道阻断剂可逆地抑制。组胺激活一个外向电流，随后在-50 mV时产生一个持续的内向电流。在正常台氏液和含30 ε lm Cl^-的内液中，初始外向电流的逆转电位比-60 mV更负，持续内向电流的逆转电位约为-30 mV。通过微管施加的流体流也引起了增加。 ...更多信息 [Ca^<2+>]_i增加，且与流速和细胞外Ca^<2+>浓度有关。液流诱导的[Ca^<2+>] i增加伴随着-53 mV的内向电流的激活。通过降低外部Cl^-浓度，液流诱导的电流的反转电位向正电位移动。从组胺和液流的作用结果我们可以得出结论，不仅组胺诱导的内向电流，而且液流诱导的电流也主要由Cl^-携带。磺酰脲类药物通过阻断ATP敏感性钾通道（KATP），抑制囊性纤维化跨膜调节因子（CFTR）的Cl^-通道、容积敏感性Cl ^-通道和Ca^2+激活的Cl^-通道，从而抑制内皮细胞和心肌细胞的Ca ^2+内流。格列本脲（10-500 μ M）对组胺诱导的[Ca^2+] i的初始瞬时升高几乎没有影响，但以浓度依赖性方式抑制随后的[Ca^2+] i的持续升高。IC 50值为151.8 μ M，希尔系数为1.9。格列本脲也能抑制ATP（100 μ M）和cyclopiazonic酸（10 μ M）引起的[Ca^<2+>]_i的持续升高。格列本脲抑制组胺诱导的Cl^-电流，IC 50值为12 μ M。100 μ M格列本脲几乎完全消除组胺诱导的Cl^-电流，但[Ca^2+]持续增加，我只是被部分压制了。因此，格列本脲诱导的Ca^2+内流抑制并不是由于抑制Cl^-电流而可能引起的膜电位变化，而是由于对Ca^2+内流途径的直接影响。结果表明，格列本脲对Ca^2+内流通路的干扰不依赖于对Cl^-电流的抑制，而对Cl^-通道的激活可能与血管内皮细胞Ca^2+内流通路的激活在功能上是偶联的。少

In cultured human aortic endothelial cells (HAECs) histamine (1-100 muM) produced a biphasic response in [Ca^<2+>]_i. We conducted whole-cell current recording combined with fluorescence measurement of intracellular Ca^<2+> concentration ([Ca^<2+>]_i) in order to investigate the mechanism underlying the C1-sensitive Ca^<2+> entry in endothelial cells. Membrane currents from single endothelial cells were measured using nystatin-perforated patch clamp technique. The histamine-induced Ca^<2+> entry is inhibited reversibly either by decreasing the extracellular concentration of Cl^- or by Cl^- channel blockers. Histamine activated an outward current, followed by a sustained inward current at -50 mV.The reversal potential was more negative than -60 mV for the initial outward current and approximately -30 mV for the sustained inward current with the normal Tyrode solution and the internal solution containing 30 epsilonlm Cl^-. The fluid stream applied through a micro tube also induced an inc … More rease in [Ca^<2+>]_i, which was dependent on both the flow rate and the extracellular Ca^<2+> concentration. The fluid stream-induced increase in [Ca^<2+>]_i was accompanied by the activation of an inward current at -53 mV.The reversal potential of the fluid stream-induced current shifted to positive potentials by reducing the external Cl^- concentration. Results from the effects of histamine and a fluid-stream we concluded that not only the histamine-induced inward current but also the fluid stream-induced current is carried mainly by Cl^-. And Cl^- current plays a crucial role in modulating the Ca^<2+>_i influx by altering the membrane potential of endothelial cells.It is well known that sulfonylureas, which block ATP-sensitive K^<2+> channels (KATP), inhibit cystic fibrosis transmembrane regulator (CFTR) Cl^- channels, volume-sensitive Cl^- channels and Ca^<2+> activated Cl^- channels in epithelial and cardiac cells. Glibenclamide (10-500 muM) has little effect on the initial transient increase in [Ca^<2+>]_i induced by histamine but inhibited the following sustained increase in [Ca^<2+>]_i in a concentration-dependent manner. The IC50 value was 151.8 muM and the Hill's coefficient was 1.9. The sustained increase in [Ca^<2+>]_i induced by ATP (100 muM) and cyclopiazonic acid (10 muM) were also suppressed by glibenclamide, Under the patch-clampcondition, however, glibenclamide suppressed the histamine-induced Cl^- current with an IC50 value of 12 muM.Histamine-induced Cl^- current was almost completely abolished by 100 muM glibenclaminde but the sustainedincrease in [Ca^<2+>]_i was only partially suppressed. Thus, the glibenclamide-induced inhibition of the Ca^<2+> entry was not due to possible changes of membrane potential by suppression of Cl^- current, but due to direct effects on the Ca^<2+> influx pathway. The present results indicate that glibenclamide disturbs the Ca^<2+> influx pathway independent of the inhibition of Cl^- current, However, the activation of Cl^- channels might be functionally coupled with Ca^<2+> influx pathway in vascular endothelial cells. Less