Electrical coupling and release of K⁺ from endothelial cells co-mediate ACh-induced smooth muscle hyperpolarization in guinea-pig inner ear artery

The physiological basis of ACh-elicited hyperpolarization in guinea-pig in vitro cochlear spiral modiolar artery (SMA) was investigated by intracellular recording combined with dye labelling of recorded cells and immunocytochemistry. We found the following. (1) The ACh-hyperpolarization was prominent only in cells that had a low resting potential (less negative than -60mV). ACh-hyperpolarization was reversibly blocked by 4-DAMP, charybdotoxin or BAPTA-AM, but not by N^ω-nitro-L-arginine methyl ester, glipizide, indomethacin or 17-octadecynoic acid. (2) Ba²⁺ (100 μm) and ouabain (I μm) each attenuated ACh-hyperpolarization by ∼30% in smooth muscle cells (SMCs) but had only slight or no inhibition in endothelial cells (ECs). A combination of Ba²⁺ and 18β-glycyrrhetinic acid near completely blocked the ACh-hyperpolarization in SMCs. (3) High K⁺ (10 mM) induced a smaller hyperpolarization in ECs than in SMCs, with an amplitude ratio of 0.49: 1. Ba²⁺ blocked the K⁺-induced hyperpolarization by ∼85% in both cell types, whereas ouabain inhibited K⁺ -hyperpolarization differently in SMCs (19%) and ECs (35%) and increased input resistance. 18β-Glycyrrhetinic acid blocked the high K+ -hyperpolarization in ECs only. (4) Weak myoendothelial dye coupling was detected by confocal microscopy in cells recorded with a propidium iodide-containing electrode for longer than 30 min. A sparse plexus of choline acetyltransferase-immunoreactive (ChAT) fibres was observed around the SMA and its up-stream arteries. (5) Evoked excitatory junction potentials (EJP) were partially blocked by 4-DAMP in half of the cells tested. We conclude that ACh-induced hyperpolarization originates from ECs via activation of Ca²⁺ -activated potassium channels, and is independent of the release of NO, cyclo-oxygenase or cytochrome P450 products. ACh-induced hyperpolarization in smooth muscle cells involves two mechanisms: (a) electrical spread of the hyperpolarization from the endothelium, and (b) activation of inward rectifier K⁺ channels (K_ir) and Na⁺ -K⁺ pump current by elevated interstitial K⁺ released from the endothelial cells, these being responsible for about 60% and 40% of the hyperpolarization, respectively. The role ratio of K_ir and pump current activation is at 8: 1 or less.