Oxidative stress (glutathionylation) and Na,K-ATPase acvity in rat skeletal muscle

Abstract

Background: Changes in ion distribution across skeletal muscle membranes during muscle activity affect excitability and ay impair force development. These changes are counteracted by the Na,K-ATPase. Regulation of the Na,K-ATPase is herefore important for skeletal muscle function. The present study investigated the presence of oxidative stress glutathionylation) on the Na,K-ATPase in rat skeletal muscle membranes. Rsults: Immunoprecipitation with an anti-glutathione antibody and subsequent immunodetection of Na,K-ATPase protein ubunits demonstrated 9.061.3% and 4.1 61.0% glutathionylation of the a isoforms in oxidative and glycolytic skeletal uscle, respectively. In oxidative muscle, 20.066.1% of the b1 units were glutathionylated, whereas 14.862.8% of the b2- ubunits appear to be glutathionylated in glycolytic muscle. Treatment with the reducing agent dithiothreitol (DTT, 1 mM) ncreased the in vitro maximal Na,K-ATPase activity by 19% (P,0.05) in membranes from glycolytic muscle. Oxidized lutathione (GSSG, 0-10 mM) increased the in vitro glutathionylation level detected with antibodies, and decreased the n vitro maximal Na,K-ATPase activity in a dose-dependent manner, and with a larger effect in oxidative compared to lycolytic skeletal muscle. Conclusion: This study demonstrates the existence of basal glutathionylation of both the a and the b units of rat skeletal uscle Na,K-ATPase. In addition, the study suggests a negative correlation between glutathionylation levels and maximal a,K-ATPase activity. Perspective: Glutathionylation likely contributes to the complex regulation of Na,K-ATPase function in skeletal muscle. specially, glutathionylation induced by oxidative stress may have a role in Na,K-ATPase regulation during prolonged uscle activity.