Maximal Na⁺-K⁺-ATPase activity is upregulated in association with muscle activity.

Importance of the Na -K pump. The ion gradients across muscle membranes undergo pronounced perturbations during intense muscle activity. These activity-induced changes in ion distribution affect excitability and may lead to impairment of force development (muscle fatigue). The Na -K -ATPase (Na -K pump) counteracts changes in ion gradients due to muscle activity. Regulation of the Na -K -ATPase is therefore important for muscle function, and especially the regulation in relation to muscle activity is crucial. It is generally accepted that the Na -K pump is upregulated during muscle activity. The underlying mechanisms include electrical stimulation and hormones such as insulin, catecholamines, insulin-like growth factor 1, and calcitonins (6). However, due to conflicting results it is unclear whether the maximal Na -K -ATPase activity is increased or decreased in association with muscle activity. A review paper concluded that the maximal in vitro Na -K pump activity is reduced (pump inactivation) after muscle activity (17). In contrast, it has recently been reported that ion affinity and maximal Na -K -ATPase activity are increased in association with muscle activity (13). It is argued below that the former paper is based on an inappropriate method for ATPase quantification. The 3-O-MFPase method. This method is based on K-dependent phosphatase activity using the artificial substrate 3-Omethylfluorescein phosphate. The K-stimulated phosphatase method does not allow measurements in the presence of Na , thus changes in Na affinity during muscle activity cannot be detected (10). In addition, the dose-response curve is steep and bell shaped; small exercise-induced changes in affinity could therefore influence the results. Ion fluxes in intact cells. The best method to maximal quantify pump activity in intact tissue is to measure the fluxes of radiolabeled Na and Rb (representing K ). However, this technique is difficult to use for measurements of the kinetic parameters Km and Vmax, as it is difficult to change intracellular Na in intact muscle cells. ATPase assay based on Pi release. Methods for direct ATPase quantification have been difficult to adapt to muscle. One problem is the extremely high background of other ATPases (especially the Ca -ATPase). However, the method has recently been improved. Pi release from ATP hydrolysis can be quantified with the use of P-ATP or the malachitebased Biomol Green reagent (14, 20). The advantage is that most Ca -ATPase activity is removed by homogenization and spinning, and the background activity is further reduced by using EGTA-containing solutions. The activity is then measured in the purified membranes obtained with ultra-spinning. The recovery of Na -K -ATPase activity after membrane fractionation is 0.5. The remaining background activity is quantified in samples without Na , and the Na -dependent response is quantified in samples with varying Na concentrations. This method allows calculation of Km for Na (Na affinity) and Vmax. Reduced maximal in vitro Na -K -ATPase activity after muscle activity? A number of papers have consistently reported a reduced maximal pump activity after exercise, a phenomenon usually referred to as inactivation of Na -K pumps. The exercise protocols included submaximal cycling in humans (18), fatiguing knee extensor exercise of short duration (9, 18), high-intensity interval cycling (2), submaximal cycling (12, 15), incremental exercise in humans (1, 21), and prolonged treadmill running in rats (8) In contrast, one study using high-frequency stimulation of rat fast-twitch muscle found unchanged 3-O-MPFase activity (11), and one study using electrical stimulation in rats reported an increased Na -K ATPase activity after contractions (22). These studies used the 3-O-MFPase method to quantify maximal pump activity. A review paper listing these and other papers concluded that the maximal in vitro Na -K pump activity is reduced after exhaustive muscle activity (17). It has been suggested that the reduction is partly (one-third) due to reactive oxygen species (18). Ion flux measurements. A study with electrical stimulation of isolated rat muscle and quantification of Rb influx found that excitation can induce an increase in ion fluxes, probably mediated by an increased intracellular Na affinity of the pump (5). On the basis of ion substitution experiments, the authors concluded that excitation-associated activation of the Na -K pump can be elicited by the action potentials per se and may not depend on a Na influx. Although the mechanism is unknown, such experiments demonstrate the existence of additional stimulatory mechanisms. Effects of exercise investigated with direct measurements of ATP utilization. Studies with purified membranes from oxidative and glycolytic muscle have shown that Km for Na was significantly higher in glycolytic muscle compared with oxidative muscle due to different distributions of Na -K pump and -isoforms (14). Treadmill running significantly reduced the Km for Na in membranes from glycolytic muscle (13), which implies that the maximal pump activity at physiological intracellular Na concentrations ( 15 mM) is increased after muscle exercise. Address for reprint requests and other correspondence: C. Juel, Dept. of Biology, Univ. of Copenhagen, August Krogh Bldg., Universitetsparken 13, DK-2100, Copenhagen, Denmark (e-mail: [email protected]). J Appl Physiol 112: 2121–2123, 2012; doi:10.1152/japplphysiol.01421.2011. Perspectives