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Hoffmann, R. A wiki for the life sciences where authorship matters. Nature Genetics (2008)
 
 
 
 
 

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 stimulation with bradykinin can account for the initiation but not the sustained production of nitric oxide (NO). Therefore, we investigated whether this sustained activation of the constitutive NO synthase (cNOS) could be mediated by an increase in pHi, which is induced by an activation of the Na(+)-H+ exchanger rather than an increase in [Ca2+]i. Cultured human endothelial cells grown on coverslips were loaded with either C.SNAFL-2 or fura 2-AM for fluorometric analysis of either pHi or [Ca2+]i. NO release was assayed by the ability of effluent from endothelial cells to stimulate purified soluble guanylyl cyclase. The pH dependence of a microsomal cNOS preparation was determined by assay of L-[3H]citrulline formation from L-[3H]arginine. Bradykinin (10 nmol/L) induced a biphasic change in endothelial pHi consisting of an initial acidification followed by a prolonged alkalinization above resting values. Inhibition of the Na(+)-H+ exchanger using HOE 694 (10 mumol/L) prevented this increase in pHi. 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. Endothelial production of NO 15 minutes after starting the infusion of bradykinin was maintained at significantly higher levels in control cells compared with cells pretreated with HOE 694. The latter effect cannot be accounted for by an increase in intracellular Ca2+, since [Ca2+]i levels were not significantly different between the two groups.(ABSTRACT TRUNCATED AT 250 WORDS)[1]

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