A sensitive in vitro functional assay to detect K+ — channel-dependent vasodilators

Abstract

This study describes a sensitive in vitro relaxation assay using isolated rabbit mesenteric artery to detect the activity of a vasodilator as a K⁺-channel activator. Thus, comparison of several known K⁺-channel activators was made with other vasodilators known to work via various cellular mechanisms. The vasodilators used were minoxidil sulfate (MNXS; 5 μM), BRL-34915 (cromakalim, 0.1 μM), nicorandil (10 μM), pinacidil (1 μM), diazoxide (100 μM), sodium nitroprusside (10 μM), forskolin (1 μM), D600 (0.5 and 10 μM), hydralazine (10 μM), and viprostal (PGE1 analog, 5 μM). The concentrations chosen were equipotent to produce greater than 80% relaxation of the maximal norepinephrine (NE) (5 μM) contraction. At these concentrations, MNXS, cromakalim, pinacidil, nicorandil, and diazoxide were found to be ineffective in producing relaxation of 80 mM K⁺-contractions. Subsequently, pretreatment of tissues with 20 mM K⁺ before NE contraction was found to attenuate relaxation significantly by these agents, but had no effect on the relaxations by forskolin or D600. These initial criteria helped to establish cromakalim, pinacidil, nicorandil, and diazoxide as compounds acting similarly to MNXS as K⁺-channel-dependent. In another set of experiments, the effects of tetraethylammonium (TEA) (10 mM), Ba²⁺ (0.5 mM), and glyburide (1 μM) as K⁺-channel blockers were examined. Again it was found that these blockers had the most inhibitory effect on the class of compounds identified as K⁺ channel activators. Additionally, it was found that these K⁺-channel activators were without any significant effect on the NE-sensitive intracellular Ca²⁺ release as studied by contraction in a Ca²⁺-free solution. Thus, this series of functional criteria clearly show that the profile of these K⁺-channel activators is distinctly different from the vasodilators working via other mechanisms such as cyclic AMP (cAMP) (forskolin), cyclic GMP (cCMP) (nitroprusside), and Ca²⁺ antagonists (D600). It is suggested that appropriately defined, systematic functional studies, such as the one described here, can provide a sensitive and reproducible vascular model to discover and delineate the role of pharmacologically relevant mechanisms for vasodilation.