Coordinate regulation of endothelin and adrenomedullin secretion by oxidative stress in endothelial cells

To elucidate the significance of oxidative stress in the modulation of endothelial functions, we examined the effects of H₂O₂ on the expression of two endothelium-derived vasoactive peptides, endothelin (ET) and adrenomedullin (Am), and their interaction. H₂O₂ dose dependently suppressed ET secretion and ET-1 mRNA expression in bovine carotid endothelial cells (ECs). Menadion sodium bisulfate, a redox cycling drug, also decreased ET secretion in a dose-dependent manner. Catalase, a H₂O₂ reductase, and dl-α-tocopherol (vitamin E) significantly inhibited H₂O₂-induced suppression of ET secretion. Downregulation of ET-1 mRNA under oxidative stress was regulated at the transcriptional level. In contrast, H₂O₂ increased Am secretion (and its mRNA expression) accompanied by the augmentation of cAMP production. Am, as well as 8-bromo-cAMP and forskolin decreased ET secretion in a dose-dependent fashion. Furthermore, an anti-Am monoclonal antibody that we developed abolished H₂O₂-induced suppression of ET secretion at 6-24 h after the addition of H₂O₂. H₂O₂ increased the intracellular Ca²⁺ concentration ([Ca²⁺]_i). Moreover, treatment with ionomycin, a Ca²⁺ ionophore, and thapsigargin, an inhibitor of endoplasmic reticulum ATPase, decreased ET secretion dose dependently for 3 h. These results suggest that the production of ET was decreased via activation of the Am-cAMP pathway and by the elevation of [Ca²⁺]_i under oxidative stress. These findings elucidate the coordinate expression of two local vascular hormones, ET and Am, under oxidative stress, which may protect against vascular diseases.