Background: The hemodynamic and anti-ischemic effects of nitroglycerin (GTN) are rapidly blunted as a result of the development of nitrate tolerance. According to the oxidative stress concept, increased GTN-induced vascular superoxide formation will deplete vascular nitric oxide (NO) resulting in the increased formation of the potent oxidant peroxynitrite. It was previously shown that hydralazine decreases vascular reactive oxygen species (ROS) formation in tolerant aorta and normalizes vascular responsiveness to GTN and other NO-dependent vasodilators. The beneficial effects of hydralazine on the efficacy of organic nitrate therapy has been demonstrated by the V-HeFT and A-HeFT studies using a combination of hydralazine and isosorbide dinitrate (ISDN). We here show that these beneficial effects may in part be mediated by the radical scavenging properties of hydralazine.

Methods: The antioxidant effects of hydralazine were tested on ROS formation in different systems using chemiluminescence as well as fluorescence. The peroxynitrite scavenging property was assessed by inhibition of nitration of phenol. Prevention of impairment of NO signaling and GTN bioactivation was determined by measurement of P-VASP and mitochondrial aldehyde dehydrogenase (ALDH-2) activity.

Results: Hydralazine dose-dependently decreased the chemiluminescence signal induced by peroxynitrite from SIN-1, by superoxide from HX/XO, by superoxide from stimulated smooth muscle cells and by ROS from GTN-stimulated mitochondria. Moreover, hydralazine inhibited the peroxynitrite-mediated nitration of phenols as well as proteins in smooth muscle cells in a dose-dependent fashion. Finally, hydralazine normalized impaired NO signaling in tolerant aorta as well as impaired vascular ALDH-2 activity.