Analysis of endothelial ionic channels involved in vascular smooth muscle function

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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.