Modulation of intracellular calcium by potassium channel openers in vascular muscle.

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 fura 2 (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 lambda 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.