Characterization of the vasodilator properties of peroxynitrite on rat pulmonary artery: role of poly (adenosine 5′‐diphosphoribose) synthase

The pulmonary vasculature is constantly exposed to oxygen and reactive oxygen species such as nitric oxide (NO) and superoxide anions which can combine at a near diffusion limited rate, to form the powerful oxidant, peroxynitrite (ONOO − ). When formed in large amounts, ONOO − is thought to contribute to tissue injury and vascular dysfunction seen in diseases such as the acute respiratory distress syndrome (ARDS) and septic shock. Recent studies have shown that ONOO − can cause vasodilatation and at higher concentrations can activate poly (adenosine 5′‐diphosphoribose) synthase (PARS) leading to consumption of nicotinamide adenine dinucleotide (NAD + ) and adenosine 5′‐triphosphate (ATP). As the lung represents a prime site for ONOO − formation, we characterized its effects on pulmonary vascular tone and on endothelial function. In addition, we have assessed the role of PARS in producing the vasoactive properties of ONOO − on pulmonary artery rings. Isolated pulmonary artery rings from rats were mounted in organ baths containing warmed and gassed (95% O 2 : 5% CO 2 ) Krebs buffer. Force was measured with isometric force transducers. After equilibration, ONOO − (10 n M –100 μ M ) was added in a cumulative manner. In separate experiments designed to assess any vasodilator properties of ONOO − , tissues were pre‐contracted with the thromboxane mimetic U46619 (1 μ M ). Once a stable base‐line was achieved, ONOO − was added in a cumulative fashion. ONOO − had no significant effect on resting pulmonary artery tone but caused concentration‐dependent relaxations of pre‐contracted vessels in the range 1 μ M to 100 μ M . In some experiments the effects of freshly prepared ONOO − solutions were compared with those allowed to decay at 4°C for 2 days. In some experiments either vehicle or ONOO − (1, 10 or 100 μ M ) was added for 15 min before U46619 (1 μ M ). Concentration‐response curves to the endothelium‐dependent vasodilator, acetylcholine (10 n M –100 μ M ) were then constructed. In these experiments, ONOO − (1 μ M or 10 μ M ) had no effect on the actions of acetylcholine. However, at the highest concentration tested (100 μ M ), ONOO − increased acetylcholine‐induced relaxations. The vasodilator actions of ONOO − were unaffected by the NO synthase inhibitor, N G ‐nitro‐ L ‐arginine methyl ester ( L ‐NAME; 100 μ M ) or by removal of superoxide anions with superoxide dismutase (SOD) (30 units ml −1 ). However, the relaxations induced by ONOO − were significantly inhibited by the PARS inhibitor, 3‐aminobenzamide (10 μ M ). In contrast to its effects on ONOO − , 3‐aminobenzamide had no effect on the relaxation caused by acetylcholine or sodium nitrite, but actually increased that induced by sodium nitroprusside. These data show that ONOO − causes vasodilatation of rat pulmonary arteries, probably via activation of PARS. Moreover, at concentrations where relaxation was achieved, ONOO − did not affect the ability of pulmonary artery rings to relax to acetylcholine. We propose that ONOO − , but not endothelially derived NO, activates PARS resulting in the rapid depletion of ATP and a consequent reduction in contraction as well as other active processes of vascular smooth muscle. The finding that 3‐aminobenzamide inhibited the actions of ONOO − but not acetylcholine, suggests that NO and ONOO − cause relaxation by independent mechanisms. It has been suggested that ONOO − is responsible for the vascular hyporesponsiveness to constrictor agents seen in experimental sepsis. This observation together with our current finding, that 3‐aminobenzamide inhibits the relaxation induced by ONOO − but not by acetylcholine, suggests that inhibitors of PARS may reduce the persistent hypotension seen in sepsis without affecting the actions of endothelium‐derived NO. Thus, the use of PARS inhibitors may represent a novel therapeutic approach to the treatment of septic shock.