Effects of nonintermittent treatment of rabbits with pentaerythritol tetranitrate on vascular reactivity and superoxide production

Abstract

Pentaerythritol tetranitrate is an organic nitrate ester that undergoes metabolization to pentaerythritol, pentaerythritol trinitrate, pentaerythritol dinitrate and pentaerythritol mononitrate. Recent data suggested that pentaerythritol tetranitrate is endowed with vasoprotective activities in experimental atherosclerosis. This study was undertaken to gain insight into the underlying mechanism. The basic mechanism of action of all pentaerythritol nitrates was evaluated by measuring liberation of nitric oxide (NO), stimulation of human soluble guanylate cyclase and vasorelaxation in rabbit aorta. A subsequent in vivo study in New Zealand White rabbits was performed to investigate the effects of a 4 months lasting nonintermittent oral treatment with 6 mg pentaerythritol tetranitrate kg −1 day −1 on vascular superoxide production, endothelium dependent vasorelaxation and vasorelaxation to pentaerythritol tetranitrate itself. The formation rates of NO from the pentaerythritol nitrates (100 μM, n=5) in presence of 5 mM cystein were (in nM min −1): 62.1±3.2 (pentaerythritol tetranitrate), 21.3±0.9 (pentaerythritol trinitrate), 6.4±0.6 (pentaerythritol dinitrate) and 3.2±0.4 (pentaerythritol mononitrate). Similarly, the pD 2 values (−log M) for half-maximal activation of soluble guanylate cyclase decreased from pentaerythritol tetranitrate (3.391±0.09, n=4) to pentaerythritol mononitrate (2.655±0.04, n=3) as did the p D 2 values (in −log M) for half-maximal relaxation of rabbit aortic rings ( n=7) from pentaerythritol tetranitrate (8.3±0.17) to pentaerythritol mononitrate (5.0±0.11). Significant correlations were found between the NO formation rates and the p D 2 values for enzyme stimulation ( r=0.98, P=0.002) and vasorelaxation ( r=0.90, P=0.049) suggesting that these effects of the pentaerythritol nitrates were mediated by NO. The results of the in vivo study showed that aging induces a significant increase of aortic superoxide production (median values, n=10) from 2.45 nM mg −1 min −1 (age 7 months) to 3.39 nM mg −1 min −1 (age 11 months, P<0.01) that was prevented by concurrent treatment with pentaerythritol tetranitrate (2.76 nM mg −1 min −1). In vitro vasorelaxation to pentaerythritol tetranitrate was identical in all groups indicating absence of nitrate tolerance. Endothelium-dependent vasorelaxation was also identical in all groups. These data suggest that oral treatment with pentaerythritol tetranitrate reduces vascular oxidant stress by an NO-dependent pathway, which may contribute to the vasoprotective activity of pentaerythritol tetranitrate in experimental atherosclerosis.