Adrenomedullin and nitric oxide inhibit human endothelial cell apoptosis via a cyclic GMP-independent mechanism

Adrenomedullin, which was discovered as a vasodilating peptide, has been re ported to be produced in various organs, in which adrenomedullin regulates not only vascular tone but also cell proliferation and differentiation in a n autocrine/paracrine manner. We evaluated the effect of adrenomedullin on endothelial cell apoptosis. Human umbilical vein endothelial cells underwen t apoptosis when cultured in serum-free medium. Treatment with adrenomedull in reduced the number of cells with pyknotic nuclei (Hoechst 33258 staining ) and inhibited cell death (dimethylthiazo-diphenyltetrazolium bromide assa y) in a dose-dependent manner. The administration of adrenomedullin did not alter the expression levels of Bcl-2 family proteins. Experiments with ana logs of cAMP or a cAMP-elevating agonist demonstrated that elevation of the intracellular cAMP concentration does not mediate the antiapoptotic effect of adrenomedullin. The coadministration of N-nitro-L-arginine methyl ester (2 mmol/L), an inhibitor of nitric oxide synthase, abrogated the effect of adrenomedullin. Lower doses of sodium nitroprusside (1 to 10 mu mol/L), a nitric oxide donor, mimicked the antiapoptotic effect of adrenomedullin. Th e antiapoptotic effect of sodium nitroprusside was not attenuated by the in hibition of soluble guanylyl cyclase with 1 mu mol/L oxadiazolo-quinoxalin- 1-one nor could apoptosis be inhibited by the incubation of human umbilical vein endothelial cells with 1 mmol/L 8-bromo-cGMP, a cell-permeant cGMP an alog. These results indicate that adrenomedullin and nitric oxide inhibit e ndothelial cell apoptosis via a cGMP-independent mechanism.