Smooth muscle membrane potential modulates endothelium‐dependent relaxation of rat basilar artery via myo‐endothelial gap junctions

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Abstract
The release of endothelium-derived relaxing factors, such as nitric oxide (NO), is dependent on an increase in intracellular calcium levels ([Ca2+]i) within endothelial cells. Endothelial cell membrane potential plays a critical role in the regulation of [Ca2+]i in that calcium influx from the extracellular space is dependent on membrane hyperpolarization. In this study, the effect of inhibition of vascular smooth muscle delayed rectifier K+ (KDR) channels by 4-aminopyridine (4-AP) on endothelium-dependent relaxation of rat basilar artery to acetylcholine (ACh) was assessed. ACh-evoked endothelium-dependent relaxations were inhibited by N-(Ω)-nitro-l-arginine (l-NNA) or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), confirming a role for NO and guanylyl cyclase. 4-AP (300 μm) also suppressed ACh-induced relaxation, with the maximal response reduced from ≈92 to ≈33 % (n = 11; P < 0.01). However, relaxations in response to exogenous NO, applied in the form of authentic NO, sodium nitroprusside or diethylamineNONOate (DEANONOate), were not affected by 4-AP treatment (n = 3-11). These data are not consistent with the view that 4-AP-sensitive KDR channels are mediators of vascular hyperpolarization and relaxation in response to endothelium-derived NO. Inhibition of ACh-evoked relaxation by 4-AP was reversed by pinacidil (0.5-1 μm; n = 5) or 18β-glycyrrhetinic acid (18βGA; 5 μm; n = 5), indicating that depolarization and electrical coupling of the smooth muscle to the endothelium were involved. 4-AP caused depolarization of both endothelial and vascular smooth muscle cells of isolated segments of basilar artery (mean change 11 ± 1 and 9 ± 2 mV, respectively; n = 15). Significantly, 18βGA almost completely prevented the depolarization of endothelial cells (n = 6), but not smooth muscle cells (n = 6) by 4-AP. ACh-induced hyperpolarization of endothelium and smooth muscle cells was also reduced by 4-AP, but this inhibition was not observed in the combined presence of 4-AP and 18βGA. These data indicate that 4-AP can induce an indirect inhibition of endothelium-dependent relaxation in the rat basilar artery by electrical coupling of smooth muscle membrane depolarization to the endothelium via myo-endothelial gap junctions.