Na+-K+-ATPase in rat skeletal muscle: content, isoform, and activity characteristics.

The purpose of this study was to investigate the hypothesis that muscle Na+-K+-ATPase activity is directly related to Na+-K+-ATPase content and the content of the alpha2-catalytic isoform in muscles of different fiber-type composition. To investigate this hypothesis, tissue was sampled from soleus (Sol), red gastrocnemius (RG), white gastrocnemius (WG), and extensor digitorum longus (EDL) muscles at rest from 38 male Wistar rats weighing 413 +/- 6.0 g (mean +/- SE). Na+-K+-ATPase activity was determined in homogenates (Hom) and isolated crude membranes (CM) by the regenerating ouabain-inhibitable hydrolytic activity assay (ATPase) and the 3-O-methylfluorescein K+-stimulated phosphatase (3-O-MFPase) assay in vitro. In addition, Na+-K+-ATPase content (Bmax) and the distribution of alpha1-, alpha2-, beta1-, and beta2-isoforms were determined by [3H]ouabain binding and Western blot, respectively. For the ATPase assay, differences (P < 0.05) in enzyme activity between muscles were observed in Hom (EDL > WG) and in CM (Sol > EDL = WG). For the 3-O-MFPase assay, differences (P < 0.05) were also found for Hom (Sol > RG = EDL > WG) and CM (Sol = WG > RG). For Bmax, differences in the order of RG = EDL > Sol = WG (P < 0.05) were observed. Isoform distribution was similar between Hom and CM and indicated in CM, a greater density (P < 0.05) of alpha1 in Sol than WG and EDL (P < 0.05), but more equal distribution of alpha2 between muscles. The beta1 was greater (P < 0.05) in Sol and RG, and the beta2 was greater in EDL and WG (P < 0.05). Over all muscles, the correlation (r) between Hom 3-O-MFPase and Bmax was 0.45 (P < 0.05) and between Hom alpha2 and Bmax, 0.59 (P < 0.05). The alpha1 distribution correlated to Hom 3-O-MFPase (r = 0.79, P < 0.05) CM ATPase (r = 0.69, P < 0.005) and CM 3-O-MFPase activity (r = 0.32, P < 0.05). The alpha2 distribution was not correlated with any of the Na+-K+-ATPase activity measurements. The results indicate generally poor relationships between activity and total pump content and alpha2 isoform content of the Na+-K+-ATPase. Several factors, including the type of preparation and the type of assay, appear important in this regard.     

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.     

Reduced activity of muscle Na(+)-K(+)-ATPase after prolonged running in rats.

The purpose of this study was to investigate the hypothesis that Na(+)-K(+)-ATPase activity is reduced in muscle of different fiber composition after a single session of aerobic exercise in rats. In one experiment, untrained female Sprague-Dawley rats (weight 275 +/- 21 g; means +/- SE; n = 30) were run (Run) on a treadmill at 21 m/min and 8% grade until fatigue, or to a maximum of 2 h, which served as control (Con), or performed an additional 45 min of low-intensity exercise at 10 m/min (Run+). In a second experiment, utilizing rats of similar characteristics (weight 258 +/- 18 g; n = 32), Run was followed by passive recovery (Rec). Directly after exercise, rats were anesthetized, and tissue was extracted from Soleus (Sol), red vastus lateralis (RV), white vastus lateralis (WV), and extensor digitorum longus (EDL) and frozen for later analysis. 3-O-methylfluorescein phosphatase activity (3-O-MFPase) was determined as an indicator of Na(+)-K(+)-ATPase activity, and glycogen depletion identified recruitment of each muscle during exercise. 3-O-MFPase was decreased (P < 0.05) at Run+ by an average of 12% from Con in all muscles (P < 0.05). No difference was found between Con and Run. Glycogen was lower (P < 0.05) by 65, 57, 44, and 33% (Sol, EDL, RV, and WV, respectively) at Run, and there was no further depletion during the continued low-intensity exercise period. No differences in Na(+)-K(+)-ATPase activity was observed between Con and Rec. The results of this study indicate that inactivation of Na(+)-K(+)-ATPase can be induced by aerobic exercise in a volume-dependent manner and that the inactivation that occurs is not specific to muscles of different fiber-type composition. Inactivation of Na(+)-K(+)-ATPase suggests intrinsic structural modifications by mechanisms that are unclear.     

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.     

Human neuromuscular fatigue is associated with altered Na+-K+-ATPase activity following isometric exercise.

The purpose of this study was to investigate the hypothesis that reductions in Na+-K+- ATPase activity are associated with neuromuscular fatigue following isometric exercise. In control (Con) and exercised (Ex) legs, force and electromyogram were measured in 14 volunteers [age, 23.4 +/- 0.7 (SE) yr] before and immediately after (PST0), 1 h after (PST1), and 4 h after (PST4) isometric, single-leg extension exercise at ~60% of maximal voluntary contraction for 30 min using a 0.5 duty cycle (5-s contraction, 5-s rest). Tissue was obtained from vastus lateralis muscle before exercise in Con and after exercise in both the Con (PST0) and Ex legs (PST0, PST1, PST4), for the measurements of Na+-K+-ATPase activity, as determined by the 3-O-methylfluorescein phosphatase (3-O-MFPase) assay. Voluntary (maximal voluntary contraction) and elicited (10, 20, 50, 100 Hz) force was reduced 30-55% (P < 0.05) at PST0 and did not recover by PST4. Muscle action potential (M-wave) amplitude and area (measured in the vastus medialis) and 3-O-MFPase activity at PST0-Ex were less than that at PST0-Con (P < 0.05) by 37, 25, and 38%, respectively. M-wave area at PST1-Ex was also less than that at PST1-Con (P < 0.05). Changes in 3-O-MFPase activity correlated to changes in M-wave area across all time points (r = 0.38, P < 0.05, n = 45). These results demonstrate that Na+-K+- ATPase activity is reduced by sustained isometric exercise in humans from that in a matched Con leg and that this reduction in Na+-K+-ATPase activity is associated with loss of excitability as indicated by M-wave alterations.     

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.     

Prolonged exercise to fatigue in humans impairs skeletal muscle Na+-K+-ATPase activity, sarcoplasmic reticulum Ca2+ release, and Ca2+ uptake.

Prolonged exhaustive submaximal exercise in humans induces marked metabolic changes, but little is known about effects on muscle Na+-K+-ATPase activity and sarcoplasmic reticulum Ca2+ regulation. We therefore investigated whether these processes were impaired during cycling exercise at 74.3 +/- 1.2% maximal O2 uptake (mean +/- SE) continued until fatigue in eight healthy subjects (maximal O2 uptake of 3.93 +/- 0.69 l/min). A vastus lateralis muscle biopsy was taken at rest, at 10 and 45 min of exercise, and at fatigue. Muscle was analyzed for in vitro Na+-K+-ATPase activity [maximal K+-stimulated 3-O-methylfluorescein phosphatase (3-O-MFPase) activity], Na+-K+-ATPase content ([3H]ouabain binding sites), sarcoplasmic reticulum Ca2+ release rate induced by 4 chloro-m-cresol, and Ca2+ uptake rate. Cycling time to fatigue was 72.18 +/- 6.46 min. Muscle 3-O-MFPase activity (nmol.min(-1).g protein(-1)) fell from rest by 6.6 +/- 2.1% at 10 min (P <0.05), by 10.7 +/- 2.3% at 45 min (P <0.01), and by 12.6 +/- 1.6% at fatigue (P <0.01), whereas 3[H]ouabain binding site content was unchanged. Ca2+ release (mmol.min(-1).g protein(-1)) declined from rest by 10.0 +/- 3.8% at 45 min (P <0.05) and by 17.9 +/- 4.1% at fatigue (P < 0.01), whereas Ca2+ uptake rate fell from rest by 23.8 +/- 12.2% at fatigue (P=0.05). However, the decline in muscle 3-O-MFPase activity, Ca2+ uptake, and Ca2+ release were variable and not significantly correlated with time to fatigue. Thus prolonged exhaustive exercise impaired each of the maximal in vitro Na+-K+-ATPase activity, Ca2+ release, and Ca2+ uptake rates. This suggests that acutely downregulated muscle Na+, K+, and Ca2+ transport processes may be important factors in fatigue during prolonged exercise in humans.     

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.     

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.     

Endotoxemia stimulates skeletal muscle Na+-K+-ATPase and raises blood lactate under aerobic conditions in humans

We assessed the hypothesis that the epinephrine surge present during sepsis accelerates aerobic glycolysis and lactate production by increasing activity of skeletal muscle Na(+)-K(+)-ATPase. Healthy volunteers received an intravenous bolus of endotoxin or placebo in a randomized order on two different days. Endotoxemia induced a response resembling sepsis. Endotoxemia increased plasma epinephrine to a maximum at t = 2 h of 0.7 +/- 0.1 vs. 0.3 +/- 0.1 nmol/l (P < 0.05, n = 6-7). Endotoxemia reduced plasma K(+) reaching a nadir at t = 5 h of 3.3 +/- 0.1 vs. 3.8 +/- 0.1 mmol/l (P < 0.01, n = 6-7), followed by an increase to placebo level at t = 7-8 h. During the declining plasma K(+), a relative accumulation of K(+) was seen reaching a maximum at t = 6 h of 8.7 +/- 3.8 mmol/leg (P < 0.05). Plasma lactate increased to a maximum at t = 1 h of 2.5 +/- 0.5 vs. 0.9 +/- 0.1 mmol/l (P < 0.05, n = 8) in association with increased release of lactate from the legs. These changes were not associated with hypoperfusion or hypoxia. During the first 24 h after endotoxin infusion, renal K(+) excretion was 27 +/- 7 mmol, i.e., 58% higher than after placebo. Combination of the well-known stimulatory effect of catecholamines on skeletal muscle Na(+)-K(+)-ATPase activity, with the present confirmation of an expected Na(+)-K(+)- ATPase-induced decline in plasma K(+), suggests that the increased lactate release was due to increased Na(+)-K(+)-ATPase activity, supporting our hypothesis. Thus increased lactate levels in acutely and severely ill patients should not be managed only from the point of view that it reflects hypoxia.     

Estradiol-induced expression of N(+)-K(+)-ATPase catalytic isoforms in rat arteries: gender differences in activity mediated by nitric oxide donors

We tested the hypothesis that previously demonstrated gender differences in ACh-induced vascular relaxation could involve diverse Na(+)-K(+)-ATPase functions. We determined Na(+)-K(+)-ATPase by measuring arterial ouabain-sensitive 86Rb uptake in response to ACh. We found a significant increase of Na+ pump activity only in aortic rings from female rats (control 206 +/- 11 vs. 367 +/- 29 nmol 86Rb/K.min(-1).g wt tissue(-1); P < 0.01). Ovariectomy eliminated sex differences in Na(+)-K(+)-ATPase function, and chronic in vivo hormone replacement with 17beta-estradiol restored the ACh effect on Na(+)-K(+)-ATPase. Because ACh acts by enhancing production of NO, we examined whether the NO donor sodium nitroprusside (SNP) mimics the action of ACh on Na(+)-K(+)-ATPase activity. SNP increased ouabain-sensitive 86Rb uptake in denuded female arteries (control 123 +/- 7 vs. 197 +/- 12 nmol 86Rb/K.min(-1).g wt tissue(-1); P < 0.05). Methylene blue (an inhibitor of guanylate cyclase) and KT-5823 (a cGMP-dependent kinase inhibitor) blocked the stimulatory action of SNP. Exposure of female thoracic aorta to the Na+/K+ pump inhibitor ouabain significantly decreased SNP-induced and ACh-mediated relaxation of aortic rings. At the molecular level, Western blot analysis of arterial tissue revealed significant gender differences in the relative abundance of catalytic isoforms of Na(+)-K(+)-ATPase. Female-derived aortas exhibited a greater proportion of alpha2-isoform (44%) compared with male-derived aortas. Furthermore, estradiol upregulated the expression of alpha2 mRNA in male arterial explants. Our results demonstrate that enhancement of ACh-induced relaxation observed in female rats may be in part explained by 1) NO-dependent increased Na(+)-K(+)-ATPase activity in female vascular tissue and 2) greater abundance of Na(+)-K(+)-ATPase alpha2-isoform in females.     

Downregulation of Na+-K+-ATPase pumps in skeletal muscle with training in normobaric hypoxia.

To investigate the effects of training in normoxia vs. training in normobaric hypoxia (fraction of inspired O2 = 20.9 vs. 13.5%, respectively) on the regulation of Na+-K+-ATPase pump concentration in skeletal muscle (vastus lateralis), 9 untrained men, ranging in age from 19 to 25 yr, underwent 8 wk of cycle training. The training consisted of both prolonged and intermittent single leg exercise for both normoxia (N) and hypoxia (H) during a single session (a similar work output for each leg) and was performed 3 times/wk. Na+-K+-ATPase concentration was 326 +/- 17 (SE) pmol/g wet wt before training (Control), increased by 14% with N (371 +/- 18 pmol/g wet wt; P < 0.05), and decreased by 14% with H (282 +/- 20 pmol/g wet wt; P < 0.05). The maximal activity of citrate synthase, selected as a measure of mitochondrial potential, showed greater increases (P < 0.05) with H (1.22 +/- 0.10 mmol x h-1 x g wet wt-1; 70%; P < 0.05) than with N (0.99 +/- 0.10 mmol x h-1 x g wet wt-1; 51%; P < 0.05) compared with pretraining (0.658 +/- 0.09 mmol x h-1 x g wet wt-1). These results demonstrate that normobaric hypoxia induced during exercise training represents a potent stimulus for the upregulation in mitochondrial potential while at the same time promoting a downregulation in Na+-K+-ATPase pump expression. In contrast, normoxic training stimulates increases in both mitochondrial potential and Na+-K+-ATPase concentration.     

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.     

Skeletal muscle ouabain binding sites are reduced in rats with chronic heart failure.

Intrinsic skeletal muscle abnormalities decrease muscular endurance in chronic heart failure (CHF). In CHF patients, the number of skeletal muscle Na(+)-K(+) pumps that have a high affinity for ouabain (i.e., the concentration of [(3)H]ouabain binding sites) is reduced, and this reduction is correlated with peak oxygen uptake. The present investigation determined whether the concentration of skeletal muscle [(3)H]ouabain binding sites found during CHF is related to 1) severity of the disease state, 2) muscle fiber type composition, and/or 3) endurance capacity. Four muscles were chosen that represented slow-twitch oxidative (SO), fast-twitch oxidative glycolytic (FOG), fast-twitch glycolytic (FG), and mixed fiber types. Measurements were obtained 8-10 wk postsurgery in 23 myocardial infarcted (MI) and 18 sham-operated control (sham) rats. Eighteen rats had moderate left ventricular (LV) dysfunction [LV end-diastolic pressure (LVEDP) < 20 mmHg], and five had severe LV dysfunction (LVEDP > 20 mmHg). Rats with severe LV dysfunction had significant pulmonary congestion and were likely in a chronic state of compensated congestive failure as indicated by an approximately twofold increase in both lung and right ventricle weight. Run time to fatigue and maximal oxygen uptake (VO(2 max)) were significantly reduced ( downward arrow39 and downward arrow28%, respectively) in the rats with severe LV dysfunction and correlated with the magnitude of LV dysfunction as indicated by LVEDP (run time: r = 0.60, n = 21, P < 0.01 and VO(2 max): r = 0.93, n = 13, P < 0.01). In addition, run time to fatigue was significantly correlated with VO(2 max) (r = 0.87, n = 15, P < 0.01). The concentration of [(3)H]ouabain binding sites (B(max)) was significantly reduced (21-28%) in the three muscles comprised primarily of oxidative fibers [soleus: 259 +/- 14 vs. 188 +/- 17; plantaris: 295 +/- 17 vs. 229 +/- 18; red portion of gastrocnemius: 326 +/- 17 vs. 260 +/- 14 pmol/g wet tissue wt]. In addition, B(max) was significantly correlated with VO(2 max) (soleus: r = 0.54, n = 15, P < 0.05; plantaris: r = 0.59, n = 15, P < 0.05; red portion of gastrocnemius: r = 0.65, n = 15, P < 0.01). These results suggest that downregulation of Na(+)-K(+) pumps that possess a high affinity for ouabain in oxidative skeletal muscle may play an important role in the exercise intolerance that attends severe LV dysfunction in CHF.     

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.     

Muscle Na-K-pump and fatigue responses to progressive exercise in normoxia and hypoxia

To investigate the effects of hypoxia and incremental exercise on muscle contractility, membrane excitability, and maximal Na(+)-K(+)-ATPase activity, 10 untrained volunteers (age = 20 +/- 0.37 yr and weight = 80.0 +/- 3.54 kg; +/- SE) performed progressive cycle exercise to fatigue on two occasions: while breathing normal room air (Norm; Fi(O(2)) = 0.21) and while breathing a normobaric hypoxic gas mixture (Hypox; Fi(O(2)) = 0.14). Muscle samples extracted from the vastus lateralis before exercise and at fatigue were analyzed for maximal Na(+)-K(+)-ATPase (K(+)-stimulated 3-O-methylfluorescein phosphatase) activity in homogenates. A 32% reduction (P < 0.05) in Na(+)-K(+)-ATPase activity was observed (90.9 +/- 7.6 vs. 62.1 +/- 6.4 nmol.mg protein(-1).h(-1)) in Norm. At fatigue, the reductions in Hypox were not different (81 +/- 5.6 vs. 57.2 +/- 7.5 nmol.mg protein(-1).h(-1)) from Norm. Measurement of quadriceps neuromuscular function, assessed before and after exercise, indicated a generalized reduction (P < 0.05) in maximal voluntary contractile force (MVC) and in force elicited at all frequencies of stimulation (10, 20, 30, 50, and 100 Hz). In general, no differences were observed between Norm and Hypox. The properties of the compound action potential, amplitude, duration, and area, which represent the electromyographic response to a single, supramaximal stimulus, were not altered by exercise or oxygen condition when assessed both during and after the progressive cycle task. Progressive exercise, conducted in Hypox, results in an inhibition of Na(+)-K(+)-ATPase activity and reductions in MVC and force at different frequencies of stimulation; these results are not different from those observed with Norm. These changes occur in the absence of reductions in neuromuscular excitability.     

Differential effects of repetitive activity on sarcoplasmic reticulum responses in rat muscles of different oxidative potential

We investigated the hypothesis that muscles of different oxidative potential would display differences in sarcoplasmic reticulum (SR) Ca2+ handling responses to repetitive contractile activity and recovery. Repetitive activity was induced in two muscles of high oxidative potential, namely, soleus (SOL) and red gastrocnemius (RG), and in white gastrocnemius (WG), a muscle of low oxidative potential, by stimulation in adult male rats. Measurements of SR properties, performed in crude homogenates, were made on control and stimulated muscles at the start of recovery (R0) and at 25 min of recovery (R25). Maximal Ca2+-ATPase activity (Vmax, micromol x g protein(-1) x min(-1)) at R0 was lower in stimulated SOL (105 +/- 9 vs. 135 +/- 7) and RG (269 +/- 22 vs. 317 +/- 26) and higher (P < 0.05) in WG (795 +/- 32 vs. 708 +/- 34). At R25, Vmax remained lower (P < 0.05) in SOL and RG but recovered in WG. Ca2+ uptake, measured at 2,000 nM, was depressed (P < 0.05) in SOL and RG by 34 and 13%, respectively, in stimulated muscles at R0 and remained depressed (P < 0.05) at R25. In contrast, Ca2+ uptake was elevated (P < 0.05) in stimulated WG at R0 by 9% and remained elevated (P < 0.05) at R25. Ca2+ release, unaltered in SOL and RG at both R0 and R25, was increased (P < 0.05) in stimulated WG at both R0 and R25. We conclude that SR Ca2+-handling responses to repetitive contractile activity and recovery are related to the oxidative potential of muscle.     

Inactivation of human muscle Na+-K+-ATPase in vitro during prolonged exercise is increased with hypoxia.

This study investigated the effects of prolonged exercise performed in normoxia (N) and hypoxia (H) on neuromuscular fatigue, membrane excitability, and Na+-K+ -ATPase activity in working muscle. Ten untrained volunteers [peak oxygen consumption (Vo2peak) = 42.1 +/- 2.8 (SE) ml x kg(-1) x min(-1)] performed 90 min of cycling during N (inspired oxygen fraction = 0.21) and during H (inspired oxygen fraction = 0.14) at approximately 50% of normoxic Vo2peak. During N, 3-O-methylfluorescein phosphatase activity (nmol x mg protein(-1) x h(-1)) in vastus lateralis, used as a measure of Na+-K+-ATPase activity, decreased (P < 0.05) by 21% at 30 min of exercise compared with rest (101 +/- 53 vs. 79.6 +/- 4.3) with no further reductions observed at 90 min (72.8 +/- 8.0). During H, similar reductions (P < 0.05) were observed during the first 30 min (90.8 +/- 5.3 vs. 79.0 +/- 6.3) followed by further reductions (P < 0.05) at 90 min (50.5 +/- 3.9). Exercise in N resulted in reductions (P < 0.05) in both quadriceps maximal voluntary contractile force (MVC; 633 +/- 50 vs. 477 +/- 67 N) and force at low frequencies of stimulation, namely 10 Hz (142 +/- 16 vs. 86.7 +/- 10 N) and 20 Hz (283 +/- 32 vs. 236 +/- 31 N). No changes were observed in the amplitude, duration, and area of the muscle compound action potential (M wave). Exercise in H was without additional effect in altering MVC, low-frequency force, and M-wave properties. It is concluded that, although exercise in H resulted in a greater inactivation of Na+-K+-ATPase activity compared with N, neuromuscular fatigue and membrane excitability are not differentially altered.     

Downregulation in muscle Na(+)-K(+)-ATPase following a 21-day expedition to 6,194 m.

To investigate the hypothesis that acclimatization to altitude would result in a downregulation in muscle Na(+)-K(+)-ATPase pump concentration, tissue samples were obtained from the vastus lateralis muscle of six volunteers (5 males and 1 female), ranging in age from 24 to 35 yr, both before and within 3 days after a 21-day expedition to the summit of Mount Denali, Alaska (6,194 m). Na(+)-K(+)-ATPase, measured by the [(3)H]ouabain-binding technique, decreased by 13.8% [348 +/- 12 vs. 300 +/- 7.6 (SE) pmol/g wet wt; P < 0.05]. No changes were found in the maximal activities (mol. kg protein(-1). h(-1)) of the mitochondrial enzymes, succinic dehydrogenase (3.63 +/- 0.20 vs. 3.25 +/- 0.23), citrate synthase (4. 76 +/- 0.44 vs. 4.94 +/- 0.44), and malate dehydrogenase (12.6 +/- 1. 8 vs. 12.7 +/- 1.2). Similarly, the expedition had no effect on any of the histochemical properties examined, namely fiber-type distribution (types I, IIA, IIB, IC, IIC, IIAB), area, capillarization, and succinic dehydrogenase activity. Peak aerobic power (52.3 +/- 2.1 vs. 50.6 +/- 1.9 ml. kg(-1). min(-1)) and body mass (76.9 +/- 3.7 vs. 75.5 +/- 2.9 kg) were also unaffected. We concluded that acclimatization to altitude results in a downregulation in muscle Na(+)-K(+)-ATPase pump concentration, which occurs without changes in oxidative potential and other fiber-type histochemical properties.