The reactive nitrogen species peroxynitrite is a potent inhibitor of renal Na-K-ATPase activity

Abstract

Reifenberger MS, Arnett KL, Gatto C, Milanick MA. The reactive nitrogen species peroxynitrite is a potent inhibitor of renal Na-K-ATPase activity. Am J Physiol Renal Physiol 295: F1191-F1198, 2008. First published August 13, 2008; doi:10.1152/ajprenal.90296.2008Peroxynitrite is a reactive nitrogen species produced when nitric oxide and superoxide react. In vivo studies suggest that reactive oxygen species and, perhaps, peroxynitrite can influence Na-K-ATPase function. However, the direct effects of peroxynitrite on Na-K-ATPase function remain unknown. We show that a single bolus addition of peroxynitrite inhibited purified renal Na-K-ATPase activity, with IC 50 of 107 Ϯ 9 M. To mimic cellular/physiological production of peroxynitrite, a syringe pump was used to slowly release (ϳ0.85 M/s) peroxynitrite. The inhibition of Na-K-ATPase activity induced by this treatment was similar to that induced by a single bolus addition of equal cumulative concentration. Peroxynitrite produced 3-nitrotyrosine residues on the ␣, ␤, and FXYD subunits of the Na pump. Interestingly, the flavonoid epicatechin, which prevented tyrosine nitration, was unable to blunt peroxynitrite-induced ATPase inhibition, suggesting that tyrosine nitration is not required for inhibition. Peroxynitrite led to a decrease in iodoacetamidofluorescein labeling, implying that cysteine modifications were induced. Glutathione was unable to reverse ATPase inhibition. The presence of Na ϩ and low MgATP during peroxynitrite treatment increased the IC 50 to 145 Ϯ 10 M, while the presence of K ϩ and low MgATP increased the IC50 to 255 Ϯ 13 M. This result suggests that the EPNa conformation of the pump is slightly more sensitive to peroxynitrite than the E(K) conformation. Taken together, these results show that peroxynitrite is a potent inhibitor of Na-K-ATPase activity and that peroxynitrite can induce amino acid modifications to the pump. P-type ATPase; reactive oxygen species; nitrotyrosine; nitric oxide THE CONTRIBUTION OF ELEVATED production of reactive oxygen and nitrogen species (ROS and RNS, respectively) to cell dysfunction in a variety of disease states via induction of oxidative damage to cell macromolecules, such as lipids, DNA, and proteins, is increasingly recognized (1, 17). Peroxynitrite is a damaging RNS that is produced in vivo when nitric oxide (NO) and superoxide react (for review see Refs. 17 and 21). The reaction rate between NO and superoxide is nearly diffusion limited; it is three-to fourfold higher than the reaction rate between superoxide and SOD (17, 18) when superoxide is less than its K m . Thus, even if the NO concentration is as low as the SOD concentration, peroxynitrite is likely to form, even in the presence of SOD.Peroxynitrite has a short (Ͻ1-s) half-life. It can react with tyrosine residues, generating 3-nitrotyrosine, which is stable. 3-Nitrotyrosine levels are elevated in a number of conditions, including cardiovascular diseases, hyperglycemia, and renal ischemia-reperfusion injuries (17, 25).The effect of peroxynitrite on ...

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