MEMBRANE HYPERPOLARIZATION INHIBITS AGONIST-INDUCED SYNTHESIS OF INOSITOL 1,4,5-TRISPHOSPHATE IN RABBIT MESENTERIC-ARTERY

1. Effects of membrane hyperpolarization induced by pinacidil on Ca2+ mobilization induced by noradrenaline (NA) were investigated by measuring intracellular Ca2+ concentration ([Ca2+]i), isometric tension, membrane potential and production of inositol 1,4,5-trisphosphate (IP3) in smooth muscle cells of the rabbit mesenteric artery. 2. Pinacidil (0.1-10-mu-M) concentration dependently hyperpolarized the smooth muscle membrane with a reduction in membrane resistance. Glibenclamide (1-mu-M) blocked the membrane hyperpolarization induced by 1-mu-M-pinacidil. NA (10-mu-M) depolarized the smooth muscle membrane with associated oscillations. Pinacidil (1-mu-m) inhibited this response and glibenclamide (1-mu-M) prevented the action of pinacidil on both the NA-induced events. 3. In thin smooth muscle strips, 10-mu-M-NA produced a large phasic and a subsequent small tonic increase in [Ca2+]i with associated oscillations. These changes in [Ca2+]i seemed to be coincident with phasic, tonic and oscillatory contractions, respectively. Pinacidil (0.1-1-mu-M) inhibited the increases in [Ca2+]i and in tension induced by NA, but not by 128 mM-K+. Glibenclamide inhibited these actions of pinacidil. Pinacidil (1-mu-M) also inhibited the contraction induced by 10-mu-M-NA in strips treated with A23187 (which functionally removes cellular Ca2+ storage sites), suggesting that membrane hyperpolarization inhibits Ca2+ influxes activated by NA. 4. In Ca2+-free solution containing 2 mM-EGTA, NA (10-mu-M) transiently increased [Ca2+]i, tension and synthesis of IP3. Pinacidil (over 0.1-mu-M) inhibited the increases in [Ca2+]i, tension and synthesis of IP3 induced by 10-mu-M-NA in Ca2+-free solution containing 5.9 mM-K+, but not in a similar solution containing 40 or 128 mM-K+. Glibenclamide (1-mu-M) inhibited these actions of pinacidil. These inhibitory actions of pinacidil were still observed in solutions containing low Na+ or low Cl-. These results suggest that pinacidil inhibits NA-induced Ca2+ release from storage sites through an inhibition of IP3 synthesis resulting from its membrane hyperpolarizing action. 5. In beta-escin-treated skinned strips, NA (10-mu-M) or IP3 (20-mu-M) increased Ca2+ in Ca2+-free solution containing 50-mu-M-EGTA and 3-mu-M-guanosine triphosphate (GTP) after brief application of 0.3-mu-M-Ca2+, suggesting Ca2+ is released from intracellular storage sites. Heparin (500-mu-g/ml, an inhibitor of the IP3 receptor), but not pinacidil (1-mu-M) or glibenclamide (1-mu-M), inhibited the Ca2+ release from storage sites induced by NA or IP3. These results suggest that membrane hyperpolarization is essential for the inhibitory action of pinacidil on the NA-induced Ca2+-releasing mechanism. Thus, the membrane hyperpolarization induced by pinacidil negatively controls the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) induced by NA and causes vasodilatation together with a blockade of spike generation in smooth muscle cells of the rabbit mesenteric artery.