Sarcolemmal Na(+)-K(+)-ATPase: inactivation by neutrophil-derived free radicals and oxidants.
One of the targets of free radicals and neutrophil-derived oxidants that is known to be generated during ischemic-reperfusion injury of the myocardium is the sarcolemma. We therefore examined the susceptibility of sarcolemmal Na(+)-K(+)-ATPase and ouabain binding sites to O2-., H2O2,.OH, HOCl, NH2Cl, and stimulated neutrophils. O2-. generated from xanthine oxidase action on xanthine had no significant effect on Na(+)-K(+)-ATPase activity. The inhibition of Na(+)-K(+)-ATPase activity and ouabain binding by H2O2 was dependent on concentration and the time of incubation. H2O2 (10 mM) inhibited 80% of Na(+)-K(+)-ATPase activity at 90 min..OH generated by Fenton's reagent (200 microM Fe2+ + 5 mM H2O2) significantly decreased maximum binding of ouabain (43.06 +/- 1.45 to 31.96 +/- 2.37 pmol/mg) and was significantly protected by 5 mM mannitol (P less than 0.05). The dissociation constant of ouabain binding was unaffected by Fenton's reagent or H2O2. In contrast, lower concentrations of HOCl, NH2Cl, or PMA-stimulated human neutrophils (4 X 10(6) cells/ml) had significant inhibitory effects on Na(+)-K(+)-ATPase activity. We conclude that O-2. per se is not damaging to sarcolemmal Na(+)-K(+)-ATPase activity. The formation of H2O2 and the more destructive .OH or HOCl and NH2Cl disrupt sarcolemmal function by inhibiting Na(+)-K(+)-ATPase activity and destroying ouabain binding sites.
Kukreja
R C
RC
Department of Medicine, Medical College of Virginia, Richmond 23298.
Weaver
A B
AB
Hess
M L
ML
D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D004285:Dogs; D004789:Enzyme Activation; D005609:Free Radicals; D006861:Hydrogen Peroxide / Q000494:pharmacology; D006878:Hydroxides / Q000494:pharmacology; D006997:Hypochlorous Acid / Q000494:pharmacology; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism*; D010042:Ouabain / Q000037:antagonists & inhibitors; D010100:Oxygen / Q000378:metabolism / Q000502:physiology*; D012508:Sarcolemma / Q000201:enzymology* / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D005609:Free Radicals; D006878:Hydroxides; D000643:Ammonium Chloride; D010042:Ouabain; D006997:Hypochlorous Acid; D006861:Hydrogen Peroxide; D000254:Sodium-Potassium-Exchanging ATPase; D013755:Tetradecanoylphorbol Acetate; D010100:Oxygen