Effects of increased intracellular Cl- concentration on membrane responses to acetylcholine in the isolated endothelium of guinea pig mesenteric arteries

Abstract

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 IKCa and SKCa 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.