Analysis of acetylcholine-induced membrane responses in vascular endothelial cells of the guinea-pig mesenteric artery using mefloquine as a gap junction blocker

Abstract

Acetylcholine (ACh)-induced membrane currents were investigated using freshly isolated endothelial layers prepared from the guinea-pig mesenteric artery. Gap junctions were blocked by mefloquine and the whole-cell patch clamp method was applied to individual endothelial cells within each multicellular preparation. While mefloquine effectively blocked the gap junctions, it hyperpolarized the membrane by some 10 mV. As this hyperpolarization was absent when the intracellular Cl -concentration was increased, mefloquine may increase the membrane conductance for Cl -. Besides this minor hyperpolarizing effect, mefloquine did not have serious side effects and ACh could activate a sustained outward current producing a membrane hyperpolarization at concentrations as low as 100 nM. At the beginning of ACh application, the reversal potential of the AChinduced current was around the equilibrium potential for K + indicating that this was a K + current. The reversal potential then gradually became less negative suggesting that other ionic conductances with less negative equilibrium potentials were involved. As the AChinduced outward current was completely blocked by charybdotoxin (CTX, 100 nM), this current seemed to be due to CTX-sensitive K + channels, possibly IKCa channels. After the K + current had been blocked, ACh gradually activated the membrane current which reversed the polarity at around -10 mV, which was most likely due to Ca 2+ -activated nonselective cation channels. These ionic conductances may be responsible for the variety in agonist-induced membrane responses observed in different types of vascular preparations.

Direct activation of Ca2+ and voltage-gated potassium channels of large conductance by anandamide in endothelial cells does not support the presence of endothelial atypical cannabinoid receptor

“…This value is close to those reported earlier for the membrane potential of endothelial cells of the same cell line (Bondarenko et al, 2011a, 2011b; Kamouchi et al, 1999; Sheng et al, 2009) and human umbilical vein endothelial cells (Adams et al, 1989), but rather depolarized compared to the membrane potential values of smooth muscle cells. Values reported for membrane potential of in situ endothelial cells range from −58 mV (Marchenko and Sage, 1993) to −15 to −18 mV (Kohler et al, 2001; Yamamoto and Suzuki, 2010). It is difficult to explain this variability, but it can be due to either specific experimental conditions or interspecies variations.…”