Abstract
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.
Highlights
Ion gradients across the muscle membrane undergo pronounced perturbations during intense muscle contractions
Similar experiments with a(all) antibodies detected 23% glutathionylation in the control samples and no significant effects of exercise and terbutaline plus exercise. This is the first study to investigate the effect of glutathionylation on Na,K-ATPase activity in human skeletal muscle
ATPase activity, (2) that a basal glutathionylation is present in samples taken at rest, and (3) that exercise and beta2-adrenergic stimulation increase the glutathionylation of the Na,K-ATPase b1 subunit, whereas the glutathionylation level is constant for the other subunits
Summary
Ion gradients across the muscle membrane undergo pronounced perturbations during intense muscle contractions. These activity-induced changes in ion distribution affect muscle excitability and may lead to impairment of force development (muscle fatigue). The Na,K-ATPase (=Na,K-pump) counteracts the rundown of transmembrane gradients of Na+ and K+. Regulation of the Na,KATPase is important for muscle functioning. It is generally accepted that the Na,K-ATPase is upregulated during muscle activity by a multifactorial process that includes sensitivity to hormones and elevated intracellular Na+ concentrations. Purinergic stimulation may be involved (Walas and Juel 2012; Juel et al 2014)
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