Modulation of intracellular calcium by potassium channel openers in vascular muscle

Summary We investigated two putative K⁺ channel openers, pinacidil and BRL34915 (cromakalim), and demonstrated their vasorelaxant effectiveness on rat artery contractions induced by K⁺, tetraethylammonium (TEA), or norepinephrine. The K⁺ channel opener-induced decrease in tension was rapid, even when tension was stimulated by 100 mmol/l K⁺. Measurements of intracellular free Ca⁺⁺ (activity) by ultra-high sensitivity digital imaging microscopy was carried out by briefly loaded fura2 (fluorescence ratio) quantitation in isolated, contracting cells of rat azygos vein. Submicron resolution was achieved by measuring cytoplasmic Ca⁺⁺-sensitive fluorescence at each pixel, and size and intensity of areas with high Ca⁺⁺ concentrations, called hot spots, were determined by a computer-generated, 3 algorithm. Hot spots, which most likely represent the sites of Ca⁺⁺ release and re-uptake by Ca⁺⁺-regulatory organelles, increased in size and intensity upon addition of K⁺ or norepinephrine, reaching an early peak prior to the whole cell average peak in cytoplasmic Ca⁺⁺ activity. Both norepinephrine and K⁺-induced stimulation resulted in Ca⁺⁺ activity increases that were primarily due to Ca⁺⁺ release from storage sites. Reduction of free Ca⁺⁺ activity to resting or lower levels occurred upon addition of pinacidil or cromakalim. Intracellular Ca⁺⁺ decreases due to K⁺ channel openers appeared abruptly beginning at the central portions of the cells, resulting in a pronounced early drop in central Ca⁺⁺ activity while elevated Ca⁺⁺ levels persisted at the periphery. While this late stage residual of peripheral Ca⁺⁺ appears to be a significant step in the vascular muscle relaxant action of both K⁺ channel opener drugs, the level of Ca⁺⁺ at peripheral sites was greater in response to pinacidil than to cromakalim. The results of this study suggest that in addition to increasing K⁺ conductance, pinacidil and cromakalim cause 1) decreased Ca⁺⁺ activity in central regions of the myocytes, and 2) a shift in Ca⁺⁺ distribution to primarily subsarcolemmal sites. These observations lead us to hypothesize separate control of peripheral and central Ca⁺⁺ activity within a vascular muscle cell, with Ca⁺⁺ redistribution that can be altered by vasorelaxants. We suggest that intracellular Ca⁺⁺ redistribution may contribute the membrane potential-independent part of the vasorelaxant action of the K⁺ channel openers.This study was supported by NIH grants HL38537 and HL38645, and Eli Lilly Co. P.E. was supported by the Swiss Foundation of Cardiology and by the SNF 32-029 975.90