Effects of Increased Intracellular Cl⁻ Concentration on Membrane Responses to Acetylcholine in the Isolated Endothelium of Guinea Pig Mesenteric Arteries

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ACh-induced membrane responses in vascular endothelial cells that have been reported vary between preparations from a sustained hyperpolarization to a transient hyperpolarization followed by a depolarization; the reason for this variation is unknown. Using the perforated whole-cell clamp technique, we investigated ACh-induced membrane currents in freshly isolated endothelial layers having a resting membrane potential of less negative than −10 mV. A group of cells was electrically isolated using a wide-bore micropipette, and their membrane potential was well controlled. ACh activated K⁺ and Cl⁻ currents simultaneously. The K⁺ current was blocked by a combination of charybdotoxin and apamin and appears to result from the opening of IK_Ca and SK_Ca channels. The Cl⁻ current was partially blocked by tamoxifen, niflumic acid, or DIDS and appears to be produced by Ca²⁺-activated Cl⁻ channels. When the pipettes contained 20 mM Cl⁻, the ACh-induced K⁺ conductance started decreasing during a 1-min application of ACh while the Cl⁻ conductance continued, making the ACh-induced hyperpolarization sustained. When the pipettes contained 150 mM Cl⁻, both conductances started decreasing during a 1-min application of ACh, making the ACh-induced hyperpolarization small and transient. [Cl⁻]_i is very likely modified by experimental procedures such as the cell isolation and the intracellular dialysis with the pipette solution. Such a variability in [Cl⁻]_i may be one of the reasons for the variations in the ACh-induced membrane response.