Analysis of endothelial ionic channels involved in vascular smooth muscle function

为了研究参与所谓的EDHF（内皮源性超极化因子）现象的内皮离子通道，使用膜片钳方法检查分离的内皮细胞的近腔膜。从豚鼠肠系膜动脉中分离出一片内皮细胞，并将其置于玻璃盖片上，使近腔表面朝上。将两个贴片电极施加在两个相邻的内皮细胞上，这两个相邻的内皮细胞应该通过缝隙连接彼此等电位耦合。在一个电极中使膜片膜破裂，并在电流钳模式下监测膜电位。另一个电极用于细胞贴附膜片钳模式，并且使用通过第一个电极记录的膜电位来控制膜片膜电位。静息膜电位通常低于-10 mV，这些细胞似乎已经失去了细胞内的K ^+浓度。在150 mM-K ^+溶液中孵育10 - 15 min，以恢复细胞内高浓度的K ^+，在2 pS或7 pS通道后加入ACh（3 μ M）。这些通道的反转电位接近K ^+的平衡电位。这些通道与非选择性阳离子通道不同，后者也被ACh激活，似乎是负责ACh诱导超极化的K ^+通道。

To investigate endothelial ionic channels involved in the so-called EDHF (Endothelium-Derived Hyperpolarizing Factor) phenomenon, abluminal membranes of isolated endothelial cells were examined using the patch clamp method. A sheet of endothelial cells was isolated from a guinea-pig mesenteric artery and placed on a glass cover slip with the abluminal surface up. Two patch electrodes were applied on two adjacent endothelial cells which should be isopotential coupling to each other electrically with gap junctions. The patch membrane was ruptured in one electrode and the membrane potential was monitored in the current clamp mode. The other electrode was used in the cell-attached patch-clamp mode and the patch membrane potential was controlled using the membrane potential recorded through the first electrode. The resting membrane potential was usually less negative than -10 mV and these cells seemed to have lost intracellular K^+ concentration. Incubation in a 150 mM-K^+ solution for 10-15 min was employed to recover the high intracellular K^+ concentration and ACh (3μM) was applied after 2 pS or 7 pS channels. These channels had reversal potentials close to the equilibrium potential of K^+. These channels were distinct from the nonselective cation channels which were also activated by ACh and seemed to be K^+ channels responsible to the ACh-induced hyperpolarization.