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

2008 ◽  
Vol 294 (4) ◽  
pp. E761-E767 ◽  
Author(s):  
H. J. Green ◽  
T. A. Duhamel ◽  
R. D. Stewart ◽  
A. R. Tupling ◽  
J. Ouyang
Keyword(s):  
Vastus Lateralis ◽  
Abrupt Onset ◽  
Ouabain Binding ◽  
Tissue Samples ◽  
Atpase Subunit ◽  
Average Decrease ◽  
Subunit Expression ◽  
Powerful Stimulus ◽  
Phosphatase Assay ◽  
Quantitative Immunoblotting

The early plasticity of vastus lateralis Na+-K+-ATPase to the abrupt onset of prolonged submaximal cycling was studied in 12 untrained participants (V̇o2 peak 44.8 ± 2.0 ml·kg−1·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 (βmax) (vanadate-facilitated [3H]ouabain binding), α- and β-isoform concentration (quantitative immunoblotting) and maximal Na+-K+-ATPase activity ( Vmax) (3- O-methylfluorescein K+-stimulated phosphatase assay). For βmax (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 α-isoforms α1, α2, and α3, increases ( P < 0.05) of 46, 42, and 31% were observed at R1, respectively. For the β-isoform, β1 and β2 increased ( P < 0.05) by 19 and 28% at R1, whereas β3 increased ( P < 0.05) by 18% at R2. With the exception of α2 and α3, the increases in the isoforms persisted at R3. Exercise resulted in an average decrease ( P < 0.05) in Vmax by 14.3%. No differences were observed in Vmax 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.

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

2007 ◽  
Vol 293 (2) ◽  
pp. E523-E530 ◽  
Author(s):  
H. J. Green ◽  
T. A. Duhamel ◽  
G. P. Holloway ◽  
J. W. Moule ◽  
J. Ouyang ◽  
...  
Keyword(s):  
Aerobic Power ◽  
Intermittent Exercise ◽  
Binding Capacity ◽  
Vastus Lateralis ◽  
Intrinsic Activity ◽  
Cycle Exercise ◽  
Maximum Binding Capacity ◽  
Phosphatase Assay ◽  
Quantitative Immunoblotting

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 ∼91% peak aerobic power (V̇o2 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 V̇o2 peak of 44.3 ± 2.3 ml·kg−1·min−1 participated in the study. Maximal Na+-K+-ATPase activity ( Vmax, in nmol·mg protein−1·h−1) as measured by the 3- O-methylfluorescein K+-stimulated phosphatase assay was reduced ( P < 0.05) by ∼15% with exercise regardless of the number of repetitions performed. In addition, Vmax at R9 and R16 was lower ( P < 0.05) than at R1 and R2. Vanadate-facilitated [3H]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 α2-isoform by R2 and a 29% increase in α3 by R9. At R16, β3 was lower ( P < 0.05) than at R2 and R9. No changes were observed in α1, β1, or β2. It is concluded that repeated sessions of heavy exercise, although resulting in increases in the α2- and α3-isoforms and decreases in β3-isoform, also result in depression in maximal catalytic activity.

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

2007 ◽  
Vol 293 (1) ◽  
pp. R354-R362 ◽  
Author(s):  
H. J. Green ◽  
T. A. Duhamel ◽  
K. P. Foley ◽  
J. Ouyang ◽  
I. C. Smith ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Prolonged Exercise ◽  
Serum Insulin ◽  
Vastus Lateralis ◽  
Oral Glucose ◽  
Vastus Lateralis Muscle ◽  
Ouabain Binding ◽  
Similar Time ◽  
Phosphatase Assay

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 (V̇o2peak) of 44.8 ± 1.9 ml·kg−1·min−1; mean ± SE cycled at ∼57% V̇o2peak 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 (Vmax) 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 (βmax) measured by the vanadate facilitated [3H]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 Vmax by mechanisms that are unclear.

Malleability of human skeletal muscle Na+-K+-ATPase pump with short-term training

2004 ◽  
Vol 97 (1) ◽  
pp. 143-148 ◽  
Author(s):  
H. J. Green ◽  
D. J. Barr ◽  
J. R. Fowles ◽  
S. D. Sandiford ◽  
J. Ouyang
Keyword(s):  
Skeletal Muscle ◽  
Human Skeletal Muscle ◽  
Aerobic Power ◽  
Vastus Lateralis ◽  
Peak Oxygen Consumption ◽  
Rapid Adaptation ◽  
Ouabain Binding ◽  
Short Term ◽  
Power Peak ◽  
Phosphatase Assay

To investigate the hypothesis that short-term submaximal training would result in changes in Na+-K+-ATPase content, activity, and isoform distribution in skeletal muscle, seven healthy, untrained men [peak aerobic power (peak oxygen consumption; V̇o2 peak) = 45.6 ml·kg−1·min−1 (SE 5.4)] cycled for 2 h/day at 60–65% V̇o2 peak for 6 days. Muscle tissue, sampled from the vastus lateralis before training (0 days) and after 3 and 6 days of training and analyzed for Na+-K+-ATPase content, as assessed by the vanadate facilitated [3H]ouabain-binding technique, was increased ( P < 0.05) at 3 days (294 ± 8.6 pmol/g wet wt) and 6 days (308 ± 15 pmol/g wet wt) of training compared with 0 days (272 ± 9.7 pmol/g wet wt). Maximal Na+-K+-ATPase activity as evaluated by the 3- O-methylfluorescein phosphatase assay was increased ( P < 0.05) by 6 days (53.4 ± 5.9 nmol·h−1·mg protein−1) but not by 3 days (35.9 ± 4.5 nmol·h−1·mg protein−1) compared with 0 days (37.8 ± 3.7 nmol·h−1·mg protein−1) of training. Relative isoform distribution, measured by Western blot techniques, indicated increases ( P < 0.05) in α2-content by 3 days and β1-content by 6 days of training. These results indicate that prolonged aerobic exercise represents a potent stimulus for the rapid adaptation of Na+-K+-ATPase content, isoform, and activity characteristics.

Effect of exercise and training on phospholemman phosphorylation in human skeletal muscle

2011 ◽  
Vol 301 (3) ◽  
pp. E456-E466 ◽  
Author(s):  
Boubacar Benziane ◽  
Ulrika Widegren ◽  
Sergej Pirkmajer ◽  
Jan Henriksson ◽  
Nigel K. Stepto ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Human Skeletal Muscle ◽  
Acute Exercise ◽  
Vastus Lateralis ◽  
Exercise Bout ◽  
Short Term ◽  
Atpase Subunit ◽  
Acute Bout ◽  
Subunit Expression ◽  
The Impact

Phospholemman (PLM, FXYD1) is a partner protein and regulator of the Na+-K+-ATPase (Na+-K+ pump). We explored the impact of acute and short-term training exercise on PLM physiology in human skeletal muscle. A group of moderately trained males ( n = 8) performed a 1-h acute bout of exercise by utilizing a one-legged cycling protocol. Muscle biopsies were taken from vastus lateralis at 0 and 63 min (non-exercised leg) and 30 and 60 min (exercised leg). In a group of sedentary males ( n = 9), we determined the effect of a 10-day intense aerobic cycle training on Na+-K+-ATPase subunit expression, PLM phosphorylation, and total PLM expression as well as PLM phosphorylation in response to acute exercise (1 h at ∼72% V̇o2peak). Biopsies were taken at rest, immediately following, and 3 h after an acute exercise bout before and at the conclusion of the 10-day training study. PLM phosphorylation was increased both at Ser63 and Ser68 immediately after acute exercise (75%, P < 0.05, and 30%, P < 0.05, respectively). Short-term training had no adaptive effect on PLM phosphorylation at Ser63 and Ser68, nor was the total amount of PLM altered posttraining. The protein expressions of α1-, α2-,and β1-subunits of Na+-K+-ATPase were increased after training (113%, P < 0.05, 49%, P < 0.05, and 27%, P < 0.05, respectively). Whereas an acute bout of exercise increased the phosphorylation of PKCα/βII on Thr638/641 pre- and posttraining, phosphorylation of PKCζ/λ on Thr403/410 was increased in response to acute exercise only after the 10-day training. In conclusion, we show that only acute exercise, and not short-term training, increases phosphorylation of PLM on Ser63 and Ser68, and data from one-legged cycling indicate that this effect of exercise on PLM phosphorylation is not due to systemic factors. Our results provide evidence that phosphorylation of PLM may play a role in the acute regulation of the Na+-K+-ATPase response to exercise.

scholarly journals β-Subunit overexpression alters the stoicheometry of assembled Na-K-ATPase subunits in MDCK cells

2008 ◽  
Vol 295 (5) ◽  
pp. F1314-F1323 ◽  
Author(s):  
Rebecca J. Clifford ◽  
Jack H. Kaplan
Keyword(s):  
Protein Interactions ◽  
Mdck Cells ◽  
Β Subunit ◽  
Protein Protein Interactions ◽  
Cellular Mechanisms ◽  
Atpase Subunit ◽  
Subunit Expression ◽  
Atpase Subunits ◽  
Confocal Images ◽  
The Individual

In eukaryotic cells, the apparent maintenance of 1:1 stoicheometry between the Na-K-ATPase α- and β-subunits led us to question whether this was alterable and thus if some form of regulation was involved. We have examined the consequences of overexpressing Na-K-ATPase β1-subunits using Madin-Darby canine kidney (MDCK) cells expressing flag-tagged β1-subunits (β1flag) or Myc-tagged β1-subunits (β1myc) under the control of a tetracycline-dependent promoter. The induction of β1flag subunit synthesis in MDCK cells, which increases β1-subunit expression at the plasma membrane by more than twofold, while maintaining stable α1 expression levels, revealed that all mature β1-subunits associate with α1-subunits, and no evidence of “free” β1-subunits was obtained. Consequently, the ratio of assembled β1- to α1-subunits is significantly increased when “extra” β-subunits are expressed. An increased β1/α1 stoicheometry is also observed in cells treated with tunicamycin, suggesting that the protein-protein interactions involved in these complexes are not dependent on glycosylation. Confocal images of cocultured β1myc-expressing and β1flag-expressing MDCK cells show colocalization of β1myc and β1flag subunits at the lateral membranes of neighboring cells, suggesting the occurrence of intercellular interactions between the β-subunits. Immunoprecipitation using MDCK cells constitutively expressing β1myc and tetracycline-regulated β1flag subunits confirmed β-β-subunit interactions. These results demonstrate that the equimolar ratio of assembled β1/α1-subunits of the Na-K-ATPase in kidney cells is not fixed by the inherent properties of the interacting subunits. It is likely that cellular mechanisms are present that regulate the individual Na-K-ATPase subunit abundance.

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

2005 ◽  
Vol 99 (2) ◽  
pp. 656-664 ◽  
Author(s):  
D. J. Barr ◽  
H. J. Green ◽  
D. S. Lounsbury ◽  
J. W. E. Rush ◽  
J. Ouyang
Keyword(s):  
Coronary Artery ◽  
Skeletal Muscles ◽  
Left Main Coronary Artery ◽  
Fiber Type ◽  
Coronary Artery Ligation ◽  
Ouabain Binding ◽  
Artery Ligation ◽  
Adult Rats ◽  
Type Composition ◽  
Phosphatase Assay

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 [3H]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 α1. For α2, only increases [red gastrocnemius (RG), Sol, and Dia; P < 0.05] occurred. The β2-isoform was decreased (LV, Sol, RG, and WG; P < 0.05) in CHF, whereas the β1 was both increased (WG and Dia; P < 0.05) and decreased (Sol and LV; P < 0.05). For β3, 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.

scholarly journals ACTH-induced hypertension is dependent on the ouabain-binding site of the α2-Na+-K+-ATPase subunit

2008 ◽  
Vol 295 (1) ◽  
pp. H273-H280 ◽  
Author(s):  
John N. Lorenz ◽  
Elizabeth L. Loreaux ◽  
Iva Dostanic-Larson ◽  
Valerie Lasko ◽  
J. Renee Schnetzer ◽  
...  
Keyword(s):  
Binding Site ◽  
Systolic Pressure ◽  
Cardiac Performance ◽  
Differential Sensitivity ◽  
Wild Type ◽  
Ouabain Binding ◽  
Atpase Subunit ◽  
Acth Treatment ◽  
Induced Hypertension ◽  
Tail Cuff

ACTH-induced-hypertension is commonly employed as a model of stress-related hypertension, and despite extensive investigation, the mechanisms underlying elevated blood pressure (BP) are not well understood. We have reported that ACTH treatment increases tail-cuff systolic pressure in wild-type mice but not in mutant mice expressing ouabain-resistant α2-Na+-K+-ATPase subunits (α2R/R mice). Since tail-cuff measurements involve restraint stress, the present study used telemetry to distinguish between an effect of ACTH on resting BP vs. an ACTH-enhanced stress response. We also sought to explore the mechanisms underlying ACTH-induced BP changes in mutant α2R/R mice vs. wild-type mice (ouabain-sensitive α2-Na+-K+-ATPase, α2S/S mice). Baseline BP was not different between the two genotypes, but after 5 days of ACTH treatment, BP increased in α2S/S (104.0 ± 2.6 to 117.7 ± 3.0 mmHg) but not in α2R/R mice (108.2 ± 3.2 to 111.5 ± 4.0 mmHg). To test the hypothesis that ACTH hypertension is related to inhibition of α2-Na+-K+-ATPase on vascular smooth muscle by endogenous cardiotonic steroids, we measured BP and regional blood flow. Results suggest a differential sensitivity of renal, mesenteric, and cerebral circulations to ACTH and that the response depends on the ouabain sensitivity of the α2-Na+-K+-ATPase. Baseline cardiac performance was elevated in α2S/S but not α2R/R mice. Overall, the data establish that the α2-Na+-K+-ATPase ouabain-binding site is of central importance in the development of ACTH-induced hypertension. The mechanism appears to be related to alterations in cardiac performance, and perhaps vascular tone in specific circulations, presumably caused by elevated levels of circulating cardiotonic steroids.

Extra- and intracellular water spaces in muscles of man at rest and with dynamic exercise

1982 ◽  
Vol 243 (3) ◽  
pp. R271-R280 ◽  
Author(s):  
G. Sjogaard ◽  
B. Saltin
Keyword(s):  
Water Content ◽  
Vastus Lateralis ◽  
Lactate Concentration ◽  
Total Water Content ◽  
Total Water ◽  
Triceps Brachii ◽  
Bicycle Exercise ◽  
Tissue Samples ◽  
Intracellular Space ◽  
Extracellular Lactate

A method was established to analyze the extracellular water space (H2Oe) in small muscle tissue samples as [3H]inulin distribution space. After initial experiments on rats, the method was applied on 13 men and 6 women. Muscles with different fiber compositions (soleus, S; vastus lateralis, (VL; gastrocnemius, G; triceps brachii, TB) were studied at rest. The total water content was the same for all muscles, 320 (313-330) ml/100 g dry wt. However, differences were demonstrated for H2Oe, with 26-34 ml/100 g dry wt in VL and 38-54 ml/100 g dry wt in S, (P less than 0.05); the values for G and TB were in between those for VL and S. The differences in H2Oe were not related to the fiber composition of the muscles. During 3 x 3 min of intense bicycle exercise demanding about 120% VO2 max (6 men), total water content increased in VL from 313 to 359 ml/100 g dry wt and H2Oe increased from 34 to 60 ml/100 g dry wt (P less than 0.05), In TB, which is relatively inactive during bicycle exercise, no such changes occurred. The calculated intracellular lactate concentration increased in VL from 5.7 to 30.6 mmol/l H2Oi. The extracellular lactate concentration amounted to 13.6 mmol/l H2Oe at the end of exercise. The concentration gradient for lactate of 2 from intra- to extracellular space favored a flux of water to the intracellular space. The relative large increase in H2Oe may then be caused by a hydrostatic rather than an osmotic factor.U

scholarly journals Marinobufagenin enhances cardiac contractility in mice with ouabain-sensitive α1 Na+-K+-ATPase

2009 ◽  
Vol 296 (6) ◽  
pp. H1833-H1839 ◽  
Author(s):  
Arshani N. Wansapura ◽  
Valerie Lasko ◽  
Zijian Xie ◽  
Olga V. Fedorova ◽  
Alexei Y. Bagrov ◽  
...  
Keyword(s):  
Binding Site ◽  
Functional Role ◽  
Cardiac Contractility ◽  
Cardiac Performance ◽  
Wild Type ◽  
Ouabain Binding ◽  
Atpase Subunit ◽  
Cardiac Inotropy ◽  
Rats And Mice

Endogenous Na+ pump inhibitors are thought to play important (patho)physiological roles and occur in two different chemical forms in the mammalian circulation: cardenolides, such as ouabain, and bufadienolides, such as marinobufagenin (MBG). Although all α Na+-K+-ATPase isoforms (α1-4) are sensitive to ouabain in most species, in rats and mice the ubiquitously expressed α1 Na+-K+-ATPase is resistant to ouabain. We have previously shown that selective modification of the putative ouabain binding site of either the α1 or α2 Na+-K+-ATPase subunit in mice substantially alters the cardiotonic influence of exogenously applied cardenolides. To determine whether the ouabain binding site also interacts with MBG and if this interaction plays a functional role, we evaluated cardiovascular function in α1-resistant/α2-resistant (α1R/Rα2R/R), α1-sensitive/α2-resistant (α1S/Sα2R/R), and α1-resistant/α2-sensitive mice (α1R/Rα2S/S, wild type). Cardiovascular indexes were evaluated in vivo by cardiac catheterization at baseline and during graded infusions of MBG. There were no differences in baseline measurements of targeted mice, indicating normal hemodynamics and cardiac function. MBG at 0.025, 0.05, and 0.1 nmol·min−1·g body wt−1 significantly increased cardiac performance to a greater extent in α1S/Sα2R/R compared with α1R/Rα2R/R and wild-type mice. The increase in LVdP/d tmax in α1S/Sα2R/R mice was greater at higher concentrations of MBG compared with both α1R/Rα2R/R and α1R/Rα2S/S mice ( P < 0.05). These results suggest that MBG interacts with the ouabain binding site of the α1 Na+-K+-ATPase subunit and can thereby influence cardiac inotropy.

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