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

¹Department of Medical Pharmacology and Physiology, School of Medicine, and Dalton Cardiovascular Research Center, University of Missouri, Columbia, Missouri; and ²Division of Biomedical Sciences, Department of Biological Sciences, Illinois State University, Normal, Illinois

Submitted 7 May 2008 ; accepted in final form 11 August 2008

Peroxynitrite 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 IC₅₀ of 107 ± 9 µM. To mimic cellular/physiological production of peroxynitrite, a syringe pump was used to slowly release (0.85 µM/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 , β, 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⁺ and low MgATP during peroxynitrite treatment increased the IC₅₀ to 145 ± 10 µM, while the presence of K⁺ and low MgATP increased the IC₅₀ to 255 ± 13 µM. 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

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