INTRACELLULAR ALKALINIZATION INDUCED BY BRADYKININ SUSTAINS ACTIVATION OF THE CONSTITUTIVE NITRIC-OXIDE SYNTHASE IN ENDOTHELIAL-CELLS

The transient increase in [Ca2+](i) in endothelial cells after stimula tion with bradykinin can account for the initiation but not the sustai ned production of nitric oxide (NO). Therefore, we investigated whethe r this sustained activation of the constitutive NO synthase (cNOS) cou ld be mediated by an increase in pH(i), which is induced by an activat ion of the Na+-H+ exchanger rather than an increase in [Ca2+](i). Cult ured human endothelial cells grown on coverslips were loaded with eith er C.SNAFL-2 or fura 2-AM for fluorometric analysis of either pH, or [ Ca2+](i). NO release was assayed by the ability of effluent from endot helial cells to stimulate purified soluble guanylyl cyclase. The pH de pendence of a microsomal cNOS preparation was determined by assay of L -[H-3]citrulline formation from L-[H-3]arginine. Bradykinin (10 nmol/L ) induced a biphasic change in endothelial pH(i) consisting of an init ial acidification followed by a prolonged alkalinization above resting values. Inhibition of the Na+-H+ exchanger using HOE 694 (10 mu mol/L ) prevented this increase in pH(i). The L-citrulline assay revealed a twofold increase in cNOS activity on increasing pH from 6.7 to 7.4, an optimum at pH 7.5, and a complete abolition of activity at pH 8.6. En dothelial production of NO 15 minutes after starting the infusion of b radykinin was maintained at significantly higher levels in control cel ls compared with cells pretreated with HOE 694. The latter effect cann ot be accounted for by an increase in intracellular Ca2+, since [Ca2+] (i) levels were not significantly different between the two groups. Th ese findings support the hypothesis that alterations in pH(i), indepen dent of changes in [Ca2+](i), represent a second intracellular mechani sm for the regulation of cNOS activity in endothelial cells.