Regulation of excitability of smooth muscle by endothelium in cerebral arterioles -Intercellular humoral and electrical signal transduction-

在本研究项目中，我利用从大鼠脑软膜分离的小动脉，通过细胞与戴尔的体液和电通讯，研究内皮(EC)对平滑肌细胞(SMC)的调节。1.采用穿孔式全细胞膜片钳技术，在电压钳模式下对系膜细胞施加超极化电压阶跃，引起通过系膜细胞膜的内向电流和通过内皮细胞膜的外向电流(缝隙连接电流)，该电流被甘草次酸(α-α-GA)减弱。这一结果表明，SMC和EC之间存在电耦合。2.为探讨体液因素的作用，在α-GA阻断交界性电流的条件下，观察了缓激肽对内皮素-1(ET-1)诱发的振荡钙激活氯电流(I_&lt；Cl(Ca)&gt；)的影响。结果表明，缓激肽可将一氧化氮释放到细胞外间隙，并抑制ET-1诱导的血管内皮细胞内钙离子。3.相邻的SMC之间存在缝隙连接。结点电流随时间呈下降趋势。这种现象是电压依赖的，并且对于正、负结电压是对称的。另一方面，SMC和EC之间的结电流表现出不对称的电压依赖性。间隙电导可以根据测量的宏观电流、电极产生的串联电阻以及周围单元之间的间隙电阻应并联的假设来估计。在适当浓度的缝隙连接抑制剂条件下，也可以计算单通道电导。所有这些技术都可能分析具有完整结构的微血管细胞之间的各种类型的通讯。

In the present research project, I attended to investigate regulation of smooth muscle cells (SMCs) by endothelium (EC) via cell-to-dell humoral and electrical communication, using arterioles dissected from the rat cerebral pial membrane. 1. By means of the perforated whole-cell patch-clamp technique, a hyperpolarizing voltage step applied to SMC in voltage-clamp mode elicited the inward current through the SMC membrane as well as the outward current through EC membrane (gap-junctional current), which was attenuated by α-glycyrrhetinic acid (α-GA). This result suggests the existence of electrical coupling between SMC and EC. 2. To investigate the role of humoral factors, I examined the effect of bradykinin on endothelin-1 (ET-1)-evoked oscillatory Ca2+-activated Cl- currents (I_<Cl(Ca)>) under the condition where junctional current was abolished by α-GA. As a result, bradykinin was shown to release nitric oxide to an extracellular space, and to inhibit an ET-1-evoked I_<Cl(Ca)> in smooth muscle cells. 3. Gap junction was demonstrated to exist between adjacent SMCs. The junctional current declined time-dependently. This phenomenon was voltage-dependent, and symmetrical for the positive and negative junctional voltages. On the other hand, the junctional current between SMC and EC exerted asymmetrical voltage-dependency. Gap-conductance could be estimated according to the macroscopic current measured, series resistance created by the electrodes, and assumption that gap-resistance among the surrounded cells should be connected in parallel. Single-channel conductance could also be calculated under the condition where a gap-junction inhibitor was performed at a modest concentration. All of these techniques are likely to enable to analyze various types of communication between cells of microvasculature with intact structure.