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)上，组胺(1-100微米)对[Ca^&lt；2+&gt；]_i产生双相反应。我们采用全细胞电流记录结合细胞内[Ca^&lt；2+&gt；]_i的荧光测量，以探讨C1敏感的[Ca^&lt；2+&gt；]_i进入内皮细胞的机制。用制霉菌素穿孔膜片钳技术测定单个内皮细胞的膜电流。组胺诱导的钙内流可通过降低胞外氯离子浓度或被氯离子通道阻滞剂可逆地抑制。组胺激活外向电流，然后在-50 mV产生持续的内向电流。在正常的Tyrode溶液和含有30 epsilonlm Cl^-的内液中，初始外向电流的反转电位大于-60 mV，持续的内向电流的反转电位约为-30 mV。通过微管施加的流体也诱导了Inc.…胞外[Ca~(2+)]_i的降低幅度更大，且与流速和胞外Ca~(2+)浓度有关。流体流诱导的[Ca~(2+)]_i增加伴随着-53 mV的内向电流的激活，随着外加氯离子浓度的降低，流体流诱导电流的反转电位向正电位移动。结果从组胺和流体流的作用来看，不仅组胺诱导的内向电流主要由氯离子携带，流体流诱导的电流也主要由氯离子携带。而氯离子电流通过改变内皮细胞的膜电位，在调节钙离子内流中起着至关重要的作用。众所周知，磺脲类药物阻断三磷酸腺苷敏感的钾通道(KATP)，抑制囊性纤维化跨膜调节因子(CFTR)氯离子通道、容量敏感性氯离子通道和激活的氯离子通道。格列本脲(10-500微米)对组胺引起的短暂[Ca~(2+)]_i升高无明显影响，但对随后持续升高的[Ca~(2+)]_i有浓度依赖性抑制作用。IC50值为151.8微米，希尔系数为1.90。在膜片钳条件下，格列本脲也能抑制由ATP(100微米)和环匹安酸(10微米)引起的[Ca^&lt；2+&gt；]_i的持续升高，但格列本脲可抑制组胺诱导的氯离子电流，IC50值为12微米。100微米的格列本脲几乎完全消除组胺诱导的氯离子电流，但对[Ca^&lt；2+&gt；]_i的持续升高仅有部分抑制作用。因此，格列本脲抑制Ca~(2+)-Gt~(2+)内流不是通过抑制Cl~+电流而改变膜电位，而是直接影响Ca~(2+)~(2+)~(2+)内流途径。以上结果表明，格列本脲对血管内皮细胞Ca~(2+)内流途径的干扰不依赖于Cl~-电流的抑制，而可能与血管内皮细胞的Ca~(2+)~(2+)内流途径发生功能上的偶联。较少

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