The reactive nitrogen species peroxynitrite is a potent inhibitor of renal Na-K-ATPase activity.
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 microM. To mimic cellular/physiological production of peroxynitrite, a syringe pump was used to slowly release (approximately 0.85 microM/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 alpha, beta, 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 microM, while the presence of K+ and low MgATP increased the IC50 to 255+/-13 microM. 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.
Reifenberger
Matthew S
MS
Department of Medical Pharmacology and Physiology, School of Medicine, and Dalton Cardiovascular Research Center, University of Missouri, 134 Research Park Rd., Columbia, MO 65211, USA.
Arnett
Krista L
KL
Gatto
Craig
C
Milanick
Mark A
MA
D000818:Animals; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D066298:In Vitro Techniques; D007668:Kidney / Q000201:enzymology*; D010042:Ouabain / Q000494:pharmacology; D030421:Peroxynitrous Acid / Q000494:pharmacology*; D011487:Protein Conformation; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism
D004791:Enzyme Inhibitors; D030421:Peroxynitrous Acid; D014443:Tyrosine; D010042:Ouabain; D000254:Sodium-Potassium-Exchanging ATPase