Depressed Na+-K+-ATPase activity in skeletal muscle at fatigue is correlated with increased Na+-K+-ATPase mRNA expression following intense exercise

We investigated whether depressed muscle Na(+)-K(+)-ATPase activity with exercise reflected a loss of Na(+)-K(+)-ATPase units, the time course of its recovery postexercise, and whether this depressed activity was related to increased Na(+)-K(+)-ATPase isoform gene expression. Fifteen subjects performed fatiguing, knee extensor exercise at approximately 40% maximal work output per contraction. A vastus lateralis muscle biopsy was taken at rest, fatigue, 3 h, and 24 h postexercise and analyzed for maximal Na(+)-K(+)-ATPase activity via 3-O-methylfluorescein phosphatase (3-O-MFPase) activity, Na(+)-K(+)-ATPase content via [(3)H]ouabain binding sites, and Na(+)-K(+)-ATPase alpha(1)-, alpha(2)-, alpha(3)-, beta(1)-, beta(2)- and beta(3)-isoform mRNA expression by real-time RT-PCR. Exercise [352 (SD 267) s] did not affect [(3)H]ouabain binding sites but decreased 3-O-MFPase activity by 10.7 (SD 8)% (P < 0.05), which had recovered by 3 h postexercise, without further change at 24 h. Exercise elevated alpha(1)-isoform mRNA by 1.5-fold at fatigue (P < 0.05). This increase was inversely correlated with the percent change in 3-O-MFPase activity from rest to fatigue (%Delta3-O-MFPase(rest-fatigue)) (r = -0.60, P < 0.05). The average postexercise (fatigue, 3 h, 24 h) alpha(1)-isoform mRNA was increased 1.4-fold (P < 0.05) and approached a significant inverse correlation with %Delta3-O-MFPase(rest-fatigue) (r = -0.56, P = 0.08). Exercise elevated alpha(2)-isoform mRNA at fatigue 2.5-fold (P < 0.05), which was inversely correlated with %Delta3-O-MFPase(rest-fatigue) (r = -0.60, P = 0.05). The average postexercise alpha(2)-isoform mRNA was increased 2.2-fold (P < 0.05) and was inversely correlated with the %Delta3-O-MFPase(rest-fatigue) (r = -0.68, P < 0.05). Nonsignificant correlations were found between %Delta3-O-MFPase(rest-fatigue) and other isoforms. Thus acute exercise transiently decreased Na(+)-K(+)-ATPase activity, which was correlated with increased Na(+)-K(+)-ATPase gene expression. This suggests a possible signal-transduction role for depressed muscle Na(+)-K(+)-ATPase activity with exercise.     

Ionic mechanisms of excitation-induced regulation of Na+-K+-ATPase mRNA expression in isolated rat EDL muscle

This study investigated the effects of electrical stimulation on Na+-K+-ATPase isoform mRNA, with the aim to identify factors modulating Na+-K+-ATPase mRNA in isolated rat extensor digitorum longus (EDL) muscle. Interventions designed to mimic exercise-induced increases in intracellular Na+ and Ca2+ contents and membrane depolarization were examined. Muscles were mounted on force transducers and stimulated with 60-Hz 10-s pulse trains producing tetanic contractions three times at 10-min intervals. Ouabain (1.0 mM, 120 min), veratridine (0.1 mM, 30 min), and monensin (0.1 mM, 30 min) were used to increase intracellular Na+ content. High extracellular K+ (13 mM, 60 min) and the Ca2+ ionophore A-23187 (0.02 mM, 30 min) were used to induce membrane depolarization and elevated intracellular Ca2+ content, respectively. Muscles were analyzed for Na+-K+-ATPase alpha1-alpha3 and beta1-beta3 mRNA (real-time RT-PCR). Electrical stimulation had no immediate effect on Na+-K+-ATPase mRNA; however at 3 h after stimulation, it increased alpha1, alpha2, and alpha3 mRNA by 223, 621, and 892%, respectively (P = 0.010), without changing beta mRNA. Ouabain, veratridine, and monensin increased intracellular Na+ content by 769, 724, and 598%, respectively (P = 0.001) but did not increase mRNA of any isoform. High intracellular K+ concentration elevated alpha1 mRNA by 160% (P = 0.021), whereas A-23187 elevated alpha3 mRNA by 123% (P = 0.035) but reduced beta1 mRNA by 76% (P = 0.001). In conclusion, electrical stimulation induced subunit-specific increases in Na+-K+-ATPase mRNA in isolated rat EDL muscle. Furthermore, Na+-K+-ATPase mRNA appears to be regulated by different stimuli, including cellular changes associated with membrane depolarization and increased intracellular Ca2+ content but not increased intracellular Na+ content.     

Muscle Na+-K+-ATPase activity and isoform adaptations to intense interval exercise and training in well-trained athletes.

The Na+ -K+ -ATPase enzyme is vital in skeletal muscle function. We investigated the effects of acute high-intensity interval exercise, before and following high-intensity training (HIT), on muscle Na+ -K+ -ATPase maximal activity, content, and isoform mRNA expression and protein abundance. Twelve endurance-trained athletes were tested at baseline, pretrain, and after 3 wk of HIT (posttrain), which comprised seven sessions of 8 x 5-min interval cycling at 80% peak power output. Vastus lateralis muscle was biopsied at rest (baseline) and both at rest and immediately postexercise during the first (pretrain) and seventh (posttrain) training sessions. Muscle was analyzed for Na+ -K+ -ATPase maximal activity (3-O-MFPase), content ([3H]ouabain binding), isoform mRNA expression (RT-PCR), and protein abundance (Western blotting). All baseline-to-pretrain measures were stable. Pretrain, acute exercise decreased 3-O-MFPase activity [12.7% (SD 5.1), P < 0.05], increased alpha1, alpha2, and alpha3 mRNA expression (1.4-, 2.8-, and 3.4-fold, respectively, P < 0.05) with unchanged beta-isoform mRNA or protein abundance of any isoform. In resting muscle, HIT increased (P < 0.05) 3-O-MFPase activity by 5.5% (SD 2.9), and alpha3 and beta3 mRNA expression by 3.0- and 0.5-fold, respectively, with unchanged Na+ -K+ -ATPase content or isoform protein abundance. Posttrain, the acute exercise induced decline in 3-O-MFPase activity and increase in alpha1 and alpha3 mRNA each persisted (P < 0.05); the postexercise 3-O-MFPase activity was also higher after HIT (P < 0.05). Thus HIT augmented Na+ -K+ -ATPase maximal activity despite unchanged total content and isoform protein abundance. Elevated Na+ -K+ -ATPase activity postexercise may contribute to reduced fatigue after training. The Na+ -K+ -ATPase mRNA response to interval exercise of increased alpha- but not beta-mRNA was largely preserved posttrain, suggesting a functional role of alpha mRNA upregulation.     

Both enalapril and losartan attenuate sarcolemmal Na+-K+-ATPase remodeling in failing rat heart due to myocardial infarction

To investigate the mechanisms underlying the depressed sarcolemmal (SL) Na(+)-K(+)-ATPase activity in congestive heart failure (CHF), different isoforms and gene expression of Na(+)-K(+)-ATPase were examined in the failing left ventricle (LV) at 8 weeks after myocardial infarction (MI). In view of the increased activity of renin-angiotensin system (RAS) in CHF, these parameters were also studied after 5 weeks of treatment with enalapril (10 mg x kg-1 x day-1), an angiotensin-converting enzyme inhibitor, and losartan (20 mg.kg-1.day-1), an angiotensin II type 1 receptor antagonist, starting at 3 weeks after the coronary ligation in rats. The infarcted animals showed LV dysfunction and depressed SL Na(+)-K(+)-ATPase activity. Protein content and mRNA levels for Na(+)-K(+)-ATPase alpha2 isoform were decreased whereas those for Na(+)-K(+)-ATPase alpha3 isoform were increased in the failing LV. On the other hand, no significant changes were observed in protein content or mRNA levels for Na(+)-K(+)-ATPase alpha1 and beta1 isoforms. The treatment of infarcted animals with enalapril or losartan improved LV function and attenuated the depression in Na(+)-K(+)-ATPase alpha2 isoform as well as the increase in alpha3 isoform, at both the protein and mRNA levels; however, combination therapy with enalapril and losartan did not produce any additive effects. These results provide further evidence that CHF due to MI is associated with remodeling of SL membrane and suggest that the blockade of RAS plays an important role in preventing these alterations in the failing heart.     

Muscle Na+-K+-ATPase response during 16 h of heavy intermittent cycle exercise

This study investigated the effects of a 16-h protocol of heavy intermittent exercise on the intrinsic activity and protein and isoform content of skeletal muscle Na(+)-K(+)-ATPase. The protocol consisted of 6 min of exercise performed once per hour at approximately 91% peak aerobic power (Vo(2 peak)) with tissue sampling from vastus lateralis before (B) and immediately after repetitions 1 (R1), 2 (R2), 9 (R9), and 16 (R16). Eleven untrained volunteers with a Vo(2 peak) of 44.3 +/- 2.3 ml x kg(-1) x min(-1) participated in the study. Maximal Na(+)-K(+)-ATPase activity (V(max), in nmol x mg protein(-1) x h(-1)) as measured by the 3-O-methylfluorescein K(+)-stimulated phosphatase assay was reduced (P < 0.05) by approximately 15% with exercise regardless of the number of repetitions performed. In addition, V(max) at R9 and R16 was lower (P < 0.05) than at R1 and R2. Vanadate-facilitated [(3)H]ouabain determination of Na(+)-K(+)-ATPase content (maximum binding capacity, pmol/g wet wt), although unaltered by exercise, increased (P < 0.05) 8.3% by R9 with no further increase observed at R16. Assessment of relative changes in isoform abundance measured at B as determined by quantitative immunoblotting showed a 26% increase (P < 0.05) in the alpha(2)-isoform by R2 and a 29% increase in alpha(3) by R9. At R16, beta(3) was lower (P < 0.05) than at R2 and R9. No changes were observed in alpha(1), beta(1), or beta(2). It is concluded that repeated sessions of heavy exercise, although resulting in increases in the alpha(2)- and alpha(3)-isoforms and decreases in beta(3)-isoform, also result in depression in maximal catalytic activity.     

Dissociation between changes in muscle Na+-K+-ATPase isoform abundance and activity with consecutive days of exercise and recovery

The early plasticity of vastus lateralis Na(+)-K(+)-ATPase to the abrupt onset of prolonged submaximal cycling was studied in 12 untrained participants (Vo(2 peak) 44.8 +/- 2.0 ml x kg(-1) x min(-1), mean +/- SE) using a 6-day protocol (3 days of exercise plus 3 days of recovery). Tissue samples were extracted prior to (Pre) and following exercise (Post) on day 1 (E1) and day 3 (E3) and on each day of recovery (R1, R2, R3) and analyzed for changes in maximal protein (beta(max)) (vanadate-facilitated [(3)H]ouabain binding), alpha- and beta-isoform concentration (quantitative immunoblotting) and maximal Na(+)-K(+)-ATPase activity (V(max)) (3-O-methylfluorescein K(+)-stimulated phosphatase assay). For beta(max) (pmol/g wet wt), an increase (P < 0.05) of 11.8% was observed at R1 compared with E1-Pre (340 +/- 14 vs 304 +/- 17). For the alpha-isoforms alpha(1), alpha(2), and alpha(3), increases (P < 0.05) of 46, 42, and 31% were observed at R1, respectively. For the beta-isoform, beta(1) and beta(2) increased (P < 0.05) by 19 and 28% at R1, whereas beta(3) increased (P < 0.05) by 18% at R2. With the exception of alpha(2) and alpha(3), the increases in the isoforms persisted at R3. Exercise resulted in an average decrease (P < 0.05) in V(max) by 14.3%. No differences were observed in V(max) at E1 - Pre and E3 - Pre or between R1, R2, and R3. It is concluded that 3 days of prolonged exercise is a powerful stimulus for the rapid upregulation of the Na(+)-K(+)-ATPase subunit isoforms. Contrary to our hypothesis, the increase in subunit expression is not accompanied by increases in the maximal catalytic activity.     

Ischemia-reperfusion alters gene expression of Na+-K+ ATPase isoforms in rat heart

The present study investigated whether oxidative stress plays a role in ischemia-reperfusion-induced changes in cardiac gene expression of Na(+)-K(+) ATPase isoforms. The levels of mRNA for Na(+)-K(+) ATPase isoforms were assessed in the isolated rat heart subjected to global ischemia (30 min) followed by reperfusion (60 min) in the presence or absence of superoxide dismutase (5 x 10(4)U/L) plus catalase (7.5 x 10(4)U/L), an antioxidant mixture. The levels of mRNA for the alpha(2), alpha(3), and beta(1) isoforms of Na(+)-K(+) ATPase were significantly reduced in the ischemia-reperfusion hearts, unlike the alpha(1) isoform. Pretreatment with superoxide dismutase+catalase preserved the ischemia-reperfusion-induced changes in alpha(2), alpha(3), and beta(1) isoform mRNA levels of the Na(+)-K(+) ATPase, whereas the alpha(1) mRNA levels were unaffected. In order to test if oxidative stress produced effects similar to those seen with ischemia-reperfusion, hearts were perfused with an oxidant, H(2)O(2) (300 microM), or a free radical generator, xanthine (2mM) plus xanthine oxidase (0.03 U/ml) for 20 min. Perfusion of hearts with H(2)O(2) or xanthine/xanthine oxidase depressed the alpha(2), alpha(3), and beta(1) isoform mRNA levels of the Na(+)-K(+) ATPase, but had lesser effects on alpha(1) mRNA levels. These results indicate that Na(+)-K(+) ATPase isoform gene expression is altered differentially in the ischemia-reperfusion hearts and that antioxidant treatment appears to attenuate these changes. It is suggested that alterations in Na(+)-K(+) ATPase isoform gene expression by ischemia-reperfusion may be mediated by oxidative stress.     

Effect of two different intense training regimens on skeletal muscle ion transport proteins and fatigue development

This study examined the effect of two different intense exercise training regimens on skeletal muscle ion transport systems, performance, and metabolic response to exercise. Thirteen subjects performed either sprint training [ST; 6-s sprints (n = 6)], or speed endurance training [SET; 30-s runs approximately 130% Vo(2 max), n = 7]. Training in the SET group provoked higher (P < 0.05) plasma K(+) levels and muscle lactate/H(+) accumulation. Only in the SET group was the amount of the Na(+)/H(+) exchanger isoform 1 (31%) and Na(+)-K(+)-ATPase isoform alpha(2) (68%) elevated (P < 0.05) after training. Both groups had higher (P < 0.05) levels of Na(+)-K(+)-ATPase beta(1)-isoform and monocarboxylate transporter 1 (MCT1), but no change in MCT4 and Na(+)-K(+)-ATPase alpha(1)-isoform. Both groups had greater (P < 0.05) accumulation of lactate during exhaustive exercise and higher (P < 0.05) rates of muscle lactate decrease after exercise. The ST group improved (P < 0.05) sprint performance, whereas the SET group elevated (P < 0.05) performance during exhaustive continuous treadmill running. Improvement in the Yo-Yo intermittent recovery test was larger (P < 0.05) in the SET than ST group (29% vs. 10%). Only the SET group had a decrease (P < 0.05) in fatigue index during a repeated sprint test. In conclusion, turnover of lactate/H(+) and K(+) in muscle during exercise does affect the adaptations of some but not all related muscle ion transport proteins with training. Adaptations with training do have an effect on the metabolic response to exercise and specific improvement in work capacity.     

Training-induced changes in skeletal muscle Na+-K+ pump number and isoform expression in rats with chronic heart failure.

The mechanisms responsible for the decrements in exercise performance in chronic heart failure (CHF) remain poorly understood, but it has been suggested that sarcolemmal alterations could contribute to the early onset of muscular fatigue. Previously, our laboratory demonstrated that the maximal number of ouabain binding sites (B(max)) is reduced in the skeletal muscle of rats with CHF (Musch TI, Wolfram S, Hageman KS, and Pickar JG. J Appl Physiol 92: 2326-2334, 2002). These reductions may coincide with changes in the Na(+)-K(+)-ATPase isoform (alpha and beta) expression. In the present study, we tested the hypothesis that reductions in B(max) would coincide with alterations in the alpha- and beta-subunit expression of the sarcolemmal Na(+)-K(+)-ATPase of rats with CHF. Moreover, we tested the hypothesis that exercise training would increase B(max) along with producing significant changes in alpha- and beta-subunit expression. Rats underwent a sham operation (sham; n = 10) or a surgically induced myocardial infarction followed by random assignment to either a control (MI; n = 16) or exercise training group (MI-T; n = 16). The MI-T rats performed exercise training (ET) for 6-8 wk. Hemodynamic indexes demonstrated that MI and MI-T rats suffered from severe left ventricular dysfunction and congestive CHF. Maximal oxygen uptake (Vo(2 max)) and endurance capacity (run time to fatigue) were reduced in MI rats compared with sham. B(max) in the soleus and plantaris muscles and the expression of the alpha(2)-isoform of the Na(+)-K(+)-ATPase in the red portion of the gastrocnemius (gastrocnemius(red)) muscle were reduced in MI rats. After ET, Vo(2 max) and run time to fatigue were increased in the MI-T group of rats. This coincided with increases in soleus and plantaris B(max) and the expression of the alpha(2)-isoform in the gastrocnemius(red) muscle. In addition, the expression of the beta(2)-isoform of the gastrocnemius(red) muscle was increased in the MI-T rats compared with their sedentary counterparts. This study demonstrates that CHF-induced alterations in skeletal muscle Na(+)-K(+)-ATPase, including B(max) and isoform expression, can be partially reversed by ET.     

Na+-K+-ATPase properties in rat heart and skeletal muscle 3 mo after coronary artery ligation.

This study was designed to determine whether chronic heart failure (CHF) results in changes in Na(+)-K(+)-ATPase properties in heart and skeletal muscles of different fiber-type composition. Adult rats were randomly assigned to a control (Con; n = 8) or CHF (n = 8) group. CHF was induced by ligation of the left main coronary artery. Examination of Na(+)-K(+)-ATPase activity (means +/- SE) 12 wk after the ligation measured, using the 3-O-methylfluorescein phosphatase assay (3-O-MFPase), indicated higher (P < 0.05) levels in soleus (Sol) (250 +/- 13 vs. 179 +/- 18 nmol.mg protein(-1).h(-1)) and lower (P < 0.05) levels in diaphragm (Dia) (200 +/- 12 vs. 272 +/- 27 nmol.mg protein(-1).h(-1)) and left ventricle (LV) (760 +/- 62 vs. 992 +/- 16 nmol.mg protein(-1).h(-1)) in CHF compared with Con, respectively. Na(+)-K(+)-ATPase protein content, measured by the [(3)H]ouabain binding technique, was higher (P < 0.05) in white gastrocnemius (WG) (166 +/- 12 vs. 135 +/- 7.6 pmol/g wet wt) and lower (P < 0.05) in Sol (193 +/- 20 vs. 260 +/- 8.6 pmol/g wet wt) and LV (159 +/- 10 vs. 221 +/- 10 pmol/g wet wt) in CHF compared with Con, respectively. Isoform content in CHF, measured by Western blot techniques, showed both increases (WG; P < 0.05) and decreases (Sol; P < 0.05) in alpha(1). For alpha(2), only increases [red gastrocnemius (RG), Sol, and Dia; P < 0.05] occurred. The beta(2)-isoform was decreased (LV, Sol, RG, and WG; P < 0.05) in CHF, whereas the beta(1) was both increased (WG and Dia; P < 0.05) and decreased (Sol and LV; P < 0.05). For beta(3), decreases (P < 0.05) in RG were observed in CHF, whereas no differences were found in Sol and WG between CHF and Con. It is concluded that CHF results in alterations in Na(+)-K(+)-ATPase that are muscle specific and property specific. Although decreases in Na(+)-K(+)-ATPase content would appear to explain the lower 3-O-MFPase in the LV, such does not appear to be the case in skeletal muscles where a dissociation between these properties was observed.     

Human nongastric H+-K+-ATPase: transport properties of ATP1al1 assembled with different beta-subunits

To investigate whether nongastric H+-K+-ATPases transport Na+ in exchange for K+ and whether different beta-isoforms influence their transport properties, we compared the functional properties of the catalytic subunit of human nongastric H+-K+-ATPase, ATP1al1 (AL1), and of the Na+-K+-ATPase alpha1-subunit (alpha1) expressed in Xenopus oocytes, with different beta-subunits. Our results show that betaHK and beta1-NK can produce functional AL1/beta complexes at the oocyte cell surface that, in contrast to alpha1/beta1 NK and alpha1/betaHK complexes, exhibit a similar apparent K+ affinity. Similar to Na+-K+-ATPase, AL1/beta complexes are able to decrease intracellular Na+ concentrations in Na+-loaded oocytes, and their K+ transport depends on intra- and extracellular Na+ concentrations. Finally, controlled trypsinolysis reveals that beta-isoforms influence the protease sensitivity of AL1 and alpha1 and that AL1/beta complexes, similar to the Na+-K+-ATPase, can undergo distinct K+-Na+- and ouabain-dependent conformational changes. These results provide new evidence that the human nongastric H+-K+-ATPase interacts with and transports Na+ in exchange for K+ and that beta-isoforms have a distinct effect on the overall structural integrity of AL1 but influence its transport properties less than those of the Na+-K+-ATPase alpha-subunit.     

Influence of chronic and acute spinal cord injury on skeletal muscle Na+-K+-ATPase and phospholemman expression in humans

Na(+)-K(+)-ATPase is an integral membrane protein crucial for the maintenance of ion homeostasis and skeletal muscle contractibility. Skeletal muscle Na(+)-K(+)-ATPase content displays remarkable plasticity in response to long-term increase in physiological demand, such as exercise training. However, the adaptations in Na(+)-K(+)-ATPase function in response to a suddenly decreased and/or habitually low level of physical activity, especially after a spinal cord injury (SCI), are incompletely known. We tested the hypothesis that skeletal muscle content of Na(+)-K(+)-ATPase and the associated regulatory proteins from the FXYD family is altered in SCI patients in a manner dependent on the severity of the spinal cord lesion and postinjury level of physical activity. Three different groups were studied: 1) six subjects with chronic complete cervical SCI, 2) seven subjects with acute, complete cervical SCI, and 3) six subjects with acute, incomplete cervical SCI. The individuals in groups 2 and 3 were studied at months 1, 3, and 12 postinjury, whereas individuals with chronic SCI were compared with an able-bodied control group. Chronic complete SCI was associated with a marked decrease in [(3)H]ouabain binding site concentration in skeletal muscle as well as reduced protein content of the α(1)-, α(2)-, and β(1)-subunit of the Na(+)-K(+)-ATPase. In line with this finding, expression of the Na(+)-K(+)-ATPase α(1)- and α(2)-subunits progressively decreased during the first year after complete but not after incomplete SCI. The expression of the regulatory protein phospholemman (PLM or FXYD1) was attenuated after complete, but not incomplete, cervical SCI. In contrast, FXYD5 was substantially upregulated in patients with complete SCI. In conclusion, the severity of the spinal cord lesion and the level of postinjury physical activity in patients with SCI are important factors controlling the expression of Na(+)-K(+)-ATPase and its regulatory proteins PLM and FXYD5.     

Chronic intermittent hypoxia and incremental cycling exercise independently depress muscle in vitro maximal Na+-K+-ATPase activity in well-trained athletes.

Athletes commonly attempt to enhance performance by training in normoxia but sleeping in hypoxia [live high and train low (LHTL)]. However, chronic hypoxia reduces muscle Na(+)-K(+)-ATPase content, whereas fatiguing contractions reduce Na(+)-K(+)-ATPase activity, which each may impair performance. We examined whether LHTL and intense exercise would decrease muscle Na(+)-K(+)-ATPase activity and whether these effects would be additive and sufficient to impair performance or plasma K(+) regulation. Thirteen subjects were randomly assigned to two fitness-matched groups, LHTL (n = 6) or control (Con, n = 7). LHTL slept at simulated moderate altitude (3,000 m, inspired O(2) fraction = 15.48%) for 23 nights and lived and trained by day under normoxic conditions in Canberra (altitude approximately 600 m). Con lived, trained, and slept in normoxia. A standardized incremental exercise test was conducted before and after LHTL. A vastus lateralis muscle biopsy was taken at rest and after exercise, before and after LHTL or Con, and analyzed for maximal Na(+)-K(+)-ATPase activity [K(+)-stimulated 3-O-methylfluorescein phosphatase (3-O-MFPase)] and Na(+)-K(+)-ATPase content ([(3)H]ouabain binding sites). 3-O-MFPase activity was decreased by -2.9 +/- 2.6% in LHTL (P < 0.05) and was depressed immediately after exercise (P < 0.05) similarly in Con and LHTL (-13.0 +/- 3.2 and -11.8 +/- 1.5%, respectively). Plasma K(+) concentration during exercise was unchanged by LHTL; [(3)H]ouabain binding was unchanged with LHTL or exercise. Peak oxygen consumption was reduced in LHTL (P < 0.05) but not in Con, whereas exercise work was unchanged in either group. Thus LHTL had a minor effect on, and incremental exercise reduced, Na(+)-K(+)-ATPase activity. However, the small LHTL-induced depression of 3-O-MFPase activity was insufficient to adversely affect either K(+) regulation or total work performed.     

Preconditioning attenuates ischemia-reperfusion-induced remodeling of Na+-K+-ATPase in hearts

The aim of this study was to determine whether changes in protein content and/or gene expression of Na+-K+-ATPase subunits underlie its decreased enzyme activity during ischemia and reperfusion. We measured protein and mRNA subunit levels in isolated rat hearts subjected to 30 min of ischemia and 30 min of reperfusion (I/R). The effect of ischemic preconditioning (IP), induced by three cycles of ischemia and reperfusion (10 min each), was also assessed on the molecular changes in Na+-K+-ATPase subunit composition due to I/R. I/R reduced the protein levels of the alpha2-, alpha3-, beta1-, and beta2-isoforms by 71%, 85%, 27%, and 65%, respectively, whereas the alpha1-isoform was decreased by <15%. A similar reduction in mRNA levels also occurred for the isoforms of Na+-K+-ATPase. IP attenuated the reduction in protein levels of Na+-K+-ATPase alpha2-, alpha3-, and beta2-isoforms induced by I/R, without affecting the alpha1- and beta1-isoforms. Furthermore, IP prevented the reduction in mRNA levels of Na+-K+-ATPase alpha2-, alpha3-, and beta1-isoforms following I/R. Similar alterations in protein contents and mRNA levels for the Na+/Ca2+ exchanger were seen due to I/R as well as IP. These findings indicate that remodeling of Na+-K+-ATPase may occur because of I/R injury, and this may partly explain the reduction in enzyme activity in ischemic heart disease. Furthermore, IP may produce beneficial effects by attenuating the remodeling of Na+-K+-ATPase and changes in Na+/Ca2+ exchanger in hearts after I/R.     

Glucose supplements increase human muscle in vitro Na+-K+-ATPase activity during prolonged exercise

Regulation of maximal Na(+)-K(+)-ATPase activity in vastus lateralis muscle was investigated in response to prolonged exercise with (G) and without (NG) oral glucose supplements. Fifteen untrained volunteers (14 males and 1 female) with a peak aerobic power (Vo(2)(peak)) of 44.8 +/- 1.9 ml.kg(-1).min(-1); mean +/- SE cycled at approximately 57% Vo(2)(peak) to fatigue during both NG (artificial sweeteners) and G (6.13 +/- 0.09% glucose) in randomized order. Consumption of beverage began at 30 min and continued every 15 min until fatigue. Time to fatigue was increased (P < 0.05) in G compared with NG (137 +/- 7 vs. 115 +/- 6 min). Maximal Na(+)-K(+)-ATPase activity (V(max)) as measured by the 3-O-methylfluorescein phosphatase assay (nmol.mg(-1).h(-1)) was not different between conditions prior to exercise (85.2 +/- 3.3 or 86.0 +/- 3.9), at 30 min (91.4 +/- 4.7 vs. 91.9 +/- 4.1) and at fatigue (92.8 +/- 4.3 vs. 100 +/- 5.0) but was higher (P < 0.05) in G at 90 min (86.7 +/- 4.2 vs. 109 +/- 4.1). Na(+)-K(+)-ATPase content (beta(max)) measured by the vanadate facilitated [(3)H]ouabain-binding technique (pmol/g wet wt) although elevated (P < 0.05) by exercise (0<30, 90, and fatigue) was not different between NG and G. At 60 and 90 min of exercise, blood glucose was higher (P < 0.05) in G compared with NG. The G condition also resulted in higher (P < 0.05) serum insulin at similar time points to glucose and lower (P < 0.05) plasma epinephrine and norepinephrine at 90 min of exercise and at fatigue. These results suggest that G results in an increase in V(max) by mechanisms that are unclear.     

Modification of sarcolemmal Na+-K+-ATPase and Na+/Ca2+ exchanger expression in heart failure by blockade of renin-angiotensin system

The activities of both sarcolemmal (SL) Na(+)-K(+)-ATPase and Na(+)/Ca(2+) exchanger, which maintain the intracellular cation homeostasis, have been shown to be depressed in heart failure due to myocardial infarction (MI). Because the renin-angiotensin system (RAS) is activated in heart failure, this study tested the hypothesis that attenuation of cardiac SL changes in congestive heart failure (CHF) by angiotensin-converting enzyme (ACE) inhibitors is associated with prevention of alterations in gene expression for SL Na(+)-K(+)-ATPase and Na(+)/Ca(2+) exchanger. CHF in rats due to MI was induced by occluding the coronary artery, and 3 wk later the animals were treated with an ACE inhibitor, imidapril (1 mg.kg(-1).day(-1)), for 4 wk. Heart dysfunction and cardiac hypertrophy in the infarcted animals were associated with depressed SL Na(+)-K(+)-ATPase and Na(+)/Ca(2+) exchange activities. Protein content and mRNA levels for Na(+)/Ca(2+) exchanger as well as Na(+)-K(+)-ATPase alpha(1)-, alpha(2)- and beta(1)-isoforms were depressed, whereas those for alpha(3)-isoform were increased in the failing heart. These changes in SL activities, protein content, and gene expression were attenuated by treating the infarcted animals with imidapril. The beneficial effects of imidapril treatment on heart function and cardiac hypertrophy as well as SL Na(+)-K(+)-ATPase and Na(+)/Ca(2+) exchange activities in the infarcted animals were simulated by enalapril, an ACE inhibitor, and losartan, an angiotensin receptor antagonist. These results suggest that blockade of RAS in CHF improves SL Na(+)-K(+)-ATPase and Na(+)/Ca(2+) exchange activities in the failing heart by preventing changes in gene expression for SL proteins.     

Age-associated differential expression of Na(+)-K(+)-ATPase subunit isoforms in skeletal muscles of F-344/BN rats.

Skeletal muscle expresses multiple isoforms of the Na(+)-K(+)-ATPase. Their expression has been shown to be differentially regulated under pathophysiological conditions. In addition, previous studies suggest possible age-dependent alterations in Na(+)-K(+) pump function. The present study tests the hypothesis that advancing age is associated with altered Na(+)-K(+)-ATPase enzyme activity and isoform-specific changes in expression of the enzyme subunits. Red and white gastrocnemius (Gast) as well as soleus muscles of male Fischer 344/Brown Norway (F-344/BN) rats at 6, 18, and 30 mo of age were examined. Na(+)-K(+)-ATPase activity, measured by K(+)-stimulated 3-O-methylfluorescein phosphatase activity, increased by approximately 50% in a mixed Gast homogenate from 30-mo-old compared with 6- and 18-mo-old rats. Advancing age was associated with markedly increased alpha(1)- and beta(1)-subunit, and decreased alpha(2)- and beta(2)-subunit in red and white Gast. In soleus, there were similar changes in expression of alpha(1)- and alpha(2)-subunits, but levels of beta(1)-subunit were unchanged. Functional Na(+)-K(+)-ATPase units, measured by [(3)H]ouabain binding, undergo muscle-type specific changes. In red Gast, high-affinity ouabain-binding sites, which are a measure of alpha(2)-isozyme, increased in 30-mo-old rats despite decreased levels of alpha(2)-subunit. In white Gast, by contrast, decreased levels of alpha(2)-subunit were accompanied by decreased high-affinity ouabain-binding sites. Finally, patterns of expression of the four myosin heavy chain (MHC) isoforms (type I, IIA, IIX, and IIB) in these muscles were similar in the three age groups examined. We conclude that, in the skeletal muscles of F-344/BN rats, advancing age is associated with muscle type-specific alterations in Na(+)-K(+)-ATPase activity and patterns of expression of alpha- and beta-subunit isoforms. These changes apparently occurred without obvious shift in muscle fiber types, since expression of MHC isoforms remained unchanged. Some of the alterations occurred between middle-age (18 mo) and senescence (30 mo), and, therefore, may be attributed to aging of skeletal muscle.     

Na+-K+-ATPase in rat skeletal muscle: muscle fiber-specific differences in exercise-induced changes in ion affinity and maximal activity

It is unclear whether muscle activity reduces or increases Na(+)-K(+)-ATPase maximal in vitro activity in rat skeletal muscle, and it is not known whether muscle activity changes the Na(+)-K(+)-ATPase ion affinity. The present study uses quantification of ATP hydrolysis to characterize muscle fiber type-specific changes in Na(+)-K(+)-ATPase activity in sarcolemmal membranes and in total membranes obtained from control rats and after 30 min of treadmill running. ATPase activity was measured at Na(+) concentrations of 0-80 mM and K(+) concentrations of 0-10 mM. K(m) and V(max) values were obtained from a Hill plot. K(m) for Na(+) was higher (lower affinity) in total membranes of glycolytic muscle (extensor digitorum longus and white vastus lateralis), when compared with oxidative muscle (red gastrocnemius and soleus). Treadmill running induced a significant decrease in K(m) for Na(+) in total membranes of glycolytic muscle, which abolished the fiber-type difference in Na(+) affinity. K(m) for K(+) (in the presence of Na(+)) was not influenced by running. Running only increased the maximal in vitro activity (V(max)) in total membranes from soleus, whereas V(max) remained constant in the three other muscles tested. In conclusion, muscle activity induces fiber type-specific changes both in Na(+) affinity and maximal in vitro activity of the Na(+)-K(+)-ATPase. The underlying mechanisms may involve translocation of subunits and increased association between PLM units and the alphabeta complex. The changes in Na(+)-K(+)-ATPase ion affinity are expected to influence muscle ion balance during muscle contraction.