Overproduction of nitric oxide by inducible nitric oxide synthase contributes to the progression of cardiovascular disease. We investigated the effects of azelnidipine and other Ca2+-channel blockers on nitric oxide production by cultured aortic smooth muscle cells isolated from Wistar rats and human umbilical vein endothelial cells (HUVECs), using the Griess reaction and oxyhemoglobin method. Release of lactic dehydrogenase (LDH) was measured to evaluate cell damage, and immunohistochemistry was performed to examine the expression of inducible nitric oxide synthase and nitrotyrosine protein. Azelnidipine and other Ca2+-channel blockers inhibited the release of nitric oxide induced by lipopolysaccharide plus interferon-gamma. Azelnidipine inhibited it most potently among the Ca2+-channel blockers tested (azelnidipine, amlodipine, nifedipine, diltiazem, verapamil, and nicardipine) at a concentration of 10 microM. Longer stimulation with these agents induced the expression of inducible nitric oxide synthase and nitrotyrosine, with an increase of lactic dehydrogenase release, whereas azelnidipine suppressed these changes. In human umbilical vein endothelial cells, azelnidipine enhanced basal nitric oxide production by endothelial nitric oxide synthase. In conclusion, azelnidipine potently inhibited the induction of inducible nitric oxide synthase and then nitric oxide production in vascular smooth muscle cells, while enhancing constitutive nitric oxide production by endothelial cells. Azelnidipine may inhibit nitrotyrosine expression and cell damage caused by overproduction of nitric oxide, suggesting a mechanism for its cardiovascular protective effect.

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SUMMARYAmiodarone (AM) is a potent vasodilator and exhibits diverse cardiovascular protective effects in vivo, but their underlying mechanisms remain unsettled. We investigated the effects of AM and N-desethylamiodarone (DEA), the major metabolite of AM, on endothelial nitric oxide (NO) production using cultured human umbilical vein endothelial cells (HUVECs). The release of NO was evaluated as measured by nitrite, a stable metabolite of NO, using the Griess reaction and also measured directly by a NO-selective electrode. The expression of each nitric oxide synthase (NOS) mRNA was examined by reverse transcriptase-polymerase chain reaction (RT-PCR), and the effects of AM on eNOS mRNA expression were studied by quantitative real-time RT-PCR. AM and DEA(1-30 µM) enhanced NO production in a concentration-dependent manner. DEA was capable of producing more NO than AM. L-NAME, a nonselective NOS inhibitor, EGTA, a Ca 2+ -chelating agent, and nickel, a nonspecific Ca 2+ blocker, all inhibited AM-induced NO production. However, LY294002, an Akt pathway inhibitor and SB202190, a MAP kinase inhibitor, did not significantly suppress the production. In RT-PCR analysis, only eNOS mRNA was detected. Treatment with AM for 4 hours did not show a significant increase in the expression of eNOS mRNA. AM lower than 30 µM did not induce apoptosis, net cell loss, or LDH release from cells. The present study provides the first evidence that therapeutic concentrations of AM and DEA enhance eNOS-mediated NO production without any toxic or apoptotic effects. This mechanism may underlie the cardiovascular protective effects of AM and its metabolite observed in a clinical setting. (Int Heart J 2006; 47: 85-93)

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