Direct nitric oxide release from nipradilol in human coronary arterial smooth muscle cells observed with fluorescent NO probe and NO-electrode

Abstract

Background: Nipradilol (3,4-dihydro-8-[2-hydroxy-3-isopropyl-amino]propoxy-3-nitroxy-2- H-1-benzopyran), a potent non-selective β-adrenoceptor antagonist, has been shown to increase NO production. The mechanisms are up-regulation of nitric oxide synthase (NOS) and direct release of NO from nipradilol. The process of direct NO release from nipradilol requires a reductase, such as glutathione S-transferase (GST) in some cells but non-enzymatic NO release was reported in pig coronary arteries. Direct NO release from nipradilol in human coronary arteries has not been examined yet, though this information is of importance. Purpose: To demonstrate direct NO release from nipradilol in human coronary arterial smooth muscle cells (HCASMC) by using a fluorescent NO probe (DAF-2) and an NO-electrode. Methods and results: HCASMC were loaded with DAF-2 and images of fluorescence (515 nm) were obtained under excitation at 488 nm through an intensified CCD with an inverted phase-contrast microscope. Concomitantly, NO was measured using an NO-electrode (0.2 mm o.d.; 501, Inter Medical Co. Ltd., Nagoya, Japan) after addition of various concentrations of nipradilol (1, 5 or 10 μM) with or without ethacrynic acid (GST inhibitor). The cells showed no fluorescence at baseline, but intense fluorescence appeared at 30 min after addition of 10 μM nipradilol. The intensities of fluorescence at 30 min in the control, nipradilol and nipradilol with ethacrynic acid groups were 98 ± 6, 163 ± 10 and 128 ± 6% of the baseline level, respectively. Ethacrynic acid itself did not affect the fluorescence. Continuous measurements of NO by the electrode showed the NO generation peaked at about 30 min, remained at the same level till about 45 min and then gradually declined. Nipradilol did not produce NO at all in the absence of cells. The dose-dependency study of NO release from nipradilol showed 45 ± 12, 72 ± 24 and 157 ± 23 nM, respectively, at 1, 5 and 10 μM nipradilol. All experiments were performed under conditions where endogenous formation of NO was inhibited by an NOS inhibitor (10 −4 M N G-monomethyl- l-arginine ( l-NMMA)). Conclusion: Nipradilol can release NO in the presence of human coronary arterial smooth muscle cells and the denitration reaction catalyzed by a reductase such as glutathione S-transferase contributes substantially to NO release from nipradilol.