The effect of exercise and beta2‐adrenergic stimulation on glutathionylation and function of the Na,K‐ATPase in human skeletal muscle

Potassium and sodium displacements across the skeletal muscle membrane during exercise may cause fatigue and are in part controlled by the Na,K-ATPase. Regulation of the Na,K-ATPase is therefore important for muscle functioning. We investigated the effect of oxidative stress (glutathionylation) on Na,K-ATPase activity. Ten male subjects performed three bouts of 4-min submaximal exercise followed by intense exercise to exhaustion with and without beta2-adrenergic stimulation with terbutaline. Muscle biopsies were obtained from m. vastus lateralis at rest (Control samples) and at exhaustion. In vitro glutathionylation reduced (P < 0.05) maximal Na,K-ATPase activity in a dose-dependent manner. Na,K-ATPase α subunits, purified by immunoprecipitation and tested by glutathione (GSH) antibodies, had a basal glutathionylation in Control samples and no further glutathionylation with exercise and beta2-adrenergic stimulation. Immunoprecipitation with an anti-GSH antibody and subsequent immunodetection with β1 antibodies showed approximately 20% glutathionylation in Control samples and further glutathionylation after exercise (to 32%) and beta2-adrenergic stimulation (to 38%, P < 0.05). Combining exercise and beta2-adrenergic stimulation raised the β1 glutathionylation to 45% (P < 0.05). In conclusion, both α and β1 subunits of the Na,K-ATPase were glutathionylated in Control samples, which indicates that the maximal Na,K-ATPase activity is overestimated if based on protein density only. β1 subunits are further glutathionylated by exercise and beta2-adrenergic stimulation. Our data suggest that glutathionylation contributes to the complex regulation of Na,K-ATPase function in human skeletal muscle. Glutathionylation of the Na,K-ATPase may explain reductions in maximal Na,K-ATPase activity after exercise, which may be involved in muscle fatigue.