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Both nitric oxide (NO) and natriuretic peptides produce apoptosis of vascular smooth muscle cells. However, there is evidence that NO induces endothelial cell proliferation, which suggests that there is a difference in the response of endothelial cells to natriuretic peptides. The purpose of this study was to investigate the effect of atrial natriuretic peptide (ANP) on human endothelial cell survival. ANP within the physiologicai concentration ([10.sup.-11] mol/l) induced a 52% increase in the number of human coronary arterial endothelial cells and a 63% increase in human umbilical vein endothelial cells at a low concentration of serum. The increase in cell numbers was blocked by pretreatment with RP8-CPT-cGMP (RP8), a cGMP-dependent protein kinase inhibitor, with wortmannin, an Akt/PKB inhibitor, and with PD-98059, an ERK1/2 inhibitor. In a Transwell migration test, ANP also increased the cell migration, and RP8, wortmannin, and PD-98059 blocked this increase. A wound healing assay was performed to examine the effects of ANP on regeneration in vitro. ANP increased both cell numbers and migration, but the effects were blocked by the above three kinase inhibitors. ANP increased the expression of phospho-Akt and of phospho-ERK1/2 within 1.5 h. These results suggest that ANP can potentiate endothelial regeneration by cGMP-dependent protein kinase stimulation and subsequent Akt and ERK1/2 activations. cGMP-dependent protein kinase; Akt/protein kinase B; extracellular signal-regulated kinase 1/2

To elucidate the significance of oxidative stress in the modulation of endothelial functions, we examined the effects of [H.sub.2][O.sub.2] on the expression of two endothelium-derived vasoactive peptides, endothelin (ET) and adrenomedullin (Am), and their interaction. [H.sub.2][O.sub.2] 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 [H.sub.2][O.sub.2] reductase, and dl-[Alpha]-tocopherol (vitamin E) significantly inhibited [H.sub.2][O.sub.2]-induced suppression of ET secretion. Downregulation of ET-1 mRNA under oxidative stress was regulated at the transcriptional level. In contrast, [H.sub.2][O.sub.2] 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 [H.sub.2][O.sub.2]-induced suppression of ET secretion at 6-24 h after the addition of [H.sub.2][O.sub.2]. [H.sub.2][O.sub.2] increased the intracellular [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.i]). Moreover, treatment with ionomycin, a [Ca.sup.2+] 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 [[[Ca.sup.2+]].sub.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. intracellular [Ca.sup.2+]; hydrogen peroxide; cAMP; nitric oxide; C-type natriuretic peptide