Smooth muscle depolarization and contraction with U46619 in mesenteric arteries is associated with a depression in the subsequent non-nitric oxide, endothelium-dependent hyperpolarization and relaxation (Plane & Garland, 1996). This is in marked contrast to stimulation with α-adenoreceptor agonists, when reproducible nitric oxide-independent hyperpolarization and relaxation is obtained (Garland & McPherson, 1992). The present study addresses the mechanism which may underlie this effect.
Male Wistar rats (200-250 g) were humanely killed by cervical dislocation and exsanguination. Small mesenteric resistance arteries were isolated and mounted in a Mulvany-Halpern myograph for simultaneous measurement of smooth muscle membrane potential and tension charge (Garland & McPherson, 1992). All experiments were done in the presence of L-NAME (100 µM). ACh (30 nM-3 µM) evoked endothelium-dependent and reproducible smooth muscle cell hyperpolarization, increasing the resting potential from -53 ± 1 to -75 ± 2 mV (mean ± S.E.M., n = 5). This hyperpolarization was abolished in the presence of 50 nM apamin. Repeated exposure to ACh was associated with a small but significant depression in the maximum amplitude of hyperpolarization, so that following a third exposure to ACh the potential only increased to -69 ± 3 mV (n = 5; P < 0.05, ANOVA). Stimulation with 1 nM-0.1 µM U46619, followed by washout and subsequent exposure to ACh, was associated with a progressive decline in the ACh-evoked hyperpolarization. After three exposures to U46619, ACh failed to evoke a hyperpolarization (resting potential -52 ± 1 mV; with 3 µM ACh present, -55 ± 1 mV, n = 8). If ACh was applied during ongoing stimulation with U44619, it evoked membrane repolarization with an overshooting hyperpolarization to -62 ± 3 mV. These increases in potential were associated with 86 ± 8 % relaxation (n = 8). By the third exposure, ACh only reversed the depolarization to U46619 to close to the original resting potential (-50 ± 2 mV, n = 8), although relaxation was not altered. Depression in the ACh hyperpolarization was not observed if the arteries were exposed for similar periods to phenylephrine rather than U46619.
These data indicate a progressive inhibition in the ability of ACh to stimulate endothelium-dependent hyperpolarization as a consequence of repeated smooth muscle stimulation with U46619. The fact that the hyperpolarization to ACh could also be blocked with apamin, raises the possibility that U46619 may inhibit SKca channels.
This work was supported by The Wellcome Trust, 039904