RELAXATION BY CROMAKALIM AND PINACIDIL OF ISOLATED SMOOTH-MUSCLE CELLS FROM CANINE CORONARY-ARTERY - MULTIPLE SITES OF ACTION

Dispersed cells were isolated from the canine coronary artery by enzym atic digestion. Their contraction and relaxation were determined by me asuring their length using a video microscaler system. The cells remai ned structurally intact when examined by Trypan blue exclusion and ele ctron microscopy. The cells showed a concentration-dependent contracti on (EC(50): 2.3 +/- 0.36 x 10(-12) M) to phenylephrine. The phenylephr ine-induced contraction of the intact cells was inhibited by cromakali m (IC50: 1.24 +/- 0.27 x 10(-10) M) and pinacidil (IC50: 6.8 +/- 1.89 x 10(-10) M). The sensitivity of the dispersed cells to cromakalim was approximately 3 orders of magnitude larger than that of the muscle st rips (EC(50): 1.94 +/- 0.22 x 10(-7) M). Glibenclamide (a selective in hibitor of the ATP-sensitive K+ channel in pancreatic beta-cells) comp etitively antagonized the cromakalim-induced inhibition of the phenyle phrine-contraction in intact cells (pA(2): 9.12; slope: 1.13) as well as in muscle strips (pA(2): 7.84; slope: 0.95). Permeabilized cells we re made by a brief exposure of the cells to saponin and were suspended in a buffer medium containing 100 mM KCl and 0.18 mu M Ca++. The cell s showed a concentration-dependent contraction to phenylephrine (EC(50 ): 2.2 +/- 0.40 x 10(-12) M) and inositol 1,4,5-triphosphate (EC(50): 5.3 +/- 1.05 x 10(-11) M). These contractions were concentration-depen dently inhibited by cromakalim and pinacidil. The inhibition by cromak alim of the inositol-induced contraction was markedly antagonized by a pamin and, to a lesser extent, by glibenclamide. Thus, it is suggested that cromakalim and pinacidil exert a potent relaxation by acting on multiple sites: the glibenclamide-sensitive K+ channels of the plasma membrane and the intracellular site sensitive to inositol and apamin.