Reduced volume but increased training intensity elevates muscle Na+-K+ pump alpha1-subunit and NHE1 expression as well as short-term work capacity in humans

The present study examined muscle adaptations and alterations in work capacity in endurance-trained runners after a change from endurance to sprint training. Fifteen runners were assigned to either a sprint training (ST, n = 8) or a control (CON, n = 7) group. ST replaced their normal training by 30-s sprint runs three to four times a week, whereas CON continued the endurance training (approximately 45 km/wk). After the 4-wk sprint period, the expression of the muscle Na+-K+ pump alpha1-subunit and Na+/H+-exchanger isoform 1 was 29 and 30% higher (P < 0.05), respectively. Furthermore, plasma K+ concentration was reduced (P < 0.05) during repeated intense running. In ST, performance in a 30-s sprint test, Yo-Yo intermittent recovery test, and two supramaximal exhaustive runs was improved (P < 0.05) by 7, 19, 27, and 19%, respectively, after the sprint training period, whereas pulmonary maximum oxygen uptake and 10-k time were unchanged. No changes in CON were observed. The present data suggest a role of the Na+-K+ pump in the control of K+ homeostasis and in the development of fatigue during repeated high-intensity exercise. Furthermore, performance during intense exercise can be improved and endurance performance maintained even with a reduction in training volume if the intensity of training is very high.     

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.     

FXYD proteins reverse inhibition of the Na+-K+ pump mediated by glutathionylation of its beta1 subunit

The seven members of the FXYD protein family associate with the Na(+)-K(+) pump and modulate its activity. We investigated whether conserved cysteines in FXYD proteins are susceptible to glutathionylation and whether such reactivity affects Na(+)-K(+) pump function in cardiac myocytes and Xenopus oocytes. Glutathionylation was detected by immunoblotting streptavidin precipitate from biotin-GSH loaded cells or by a GSH antibody. Incubation of myocytes with recombinant FXYD proteins resulted in competitive displacement of native FXYD1. Myocyte and Xenopus oocyte pump currents were measured with whole-cell and two-electrode voltage clamp techniques, respectively. Native FXYD1 in myocytes and FXYD1 expressed in oocytes were susceptible to glutathionylation. Mutagenesis identified the specific cysteine in the cytoplasmic terminal that was reactive. Its reactivity was dependent on flanking basic amino acids. We have reported that Na(+)-K(+) pump β(1) subunit glutathionylation induced by oxidative signals causes pump inhibition in a previous study. In the present study, we found that β(1) subunit glutathionylation and pump inhibition could be reversed by exposing myocytes to exogenous wild-type FXYD3. A cysteine-free FXYD3 derivative had no effect. Similar results were obtained with wild-type and mutant FXYD proteins expressed in oocytes. Glutathionylation of the β(1) subunit was increased in myocardium from FXYD1(-/-) mice. In conclusion, there is a dependence of Na(+)-K(+) pump regulation on reactivity of two specifically identified cysteines on separate components of the multimeric Na(+)-K(+) pump complex. By facilitating deglutathionylation of the β(1) subunit, FXYD proteins reverse oxidative inhibition of the Na(+)-K(+) pump and play a dynamic role in its regulation.     

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.     

Mechanisms of Na+-K+ pump regulation in cardiac myocytes during hyposmolar swelling

We have previously demonstrated that the sarcolemmalNa⁺-K⁺pump current(I_p) in cardiacmyocytes is stimulated by cell swelling induced by exposure tohyposmolar solutions. However, the underlying mechanism has not beenexamined. Because cell swelling activates stretch-sensitive ionchannels and intracellular messenger pathways, we examined their rolein mediating I_pstimulation during exposure of rabbit ventricular myocytes to ahyposmolar solution.I_p was measuredby the whole cell patch-clamp technique. Swelling-induced pumpstimulation altered the voltage dependence ofI_p. Pumpstimulation persisted in the absence of extracellularNa⁺ and under conditions designedto minimize changes in intracellular Ca²⁺, excluding an indirectinfluence on I_pmediated via fluxes through stretch-activated channels. Pumpstimulation was protein kinase C independent. The tyrosine kinaseinhibitor tyrphostin A25, the phosphatidylinositol 3-kinase inhibitorLY-294002, and the protein phosphatase-1 and -2A inhibitor okadaic acidabolished I_pstimulation. Our findings suggest that swelling-induced pumpstimulation involves the activation of tyrosine kinase,phosphatidylinositol 3-kinase, and a serine/threonine proteinphosphatase. Activation of this messenger cascade maycause activation by the dephosphorylation of pump units.     

Aldosterone regulation of intestinal Na absorption involves SGK-mediated changes in NHE3 and Na+ pump activity

Aldosterone-induced intestinal Na(+) absorption is mediated by increased activities of apical membrane Na(+)/H(+) exchange (aNHE3) and basolateral membrane Na(+)-K(+)-ATPase (BLM-Na(+)-K(+)-ATPase) activities. Because the processes coordinating these events were not well understood, we investigated human intestinal Caco-2BBE cells where aldosterone increases within 2-4 h of aNHE3 and alpha-subunit of BLM-Na(+)-K(+)-ATPase, but not total abundance of these proteins. Although aldosterone activated Akt2 and serum glucorticoid kinase-1 (SGK-1), the latter through stimulation of phosphatidylinositol 3-kinase (PI3K), only the SGK-1 pathway mediated its effects on Na(+)-K(+)-ATPase. Ouabain inhibition of the early increase in aldosterone-induced Na(+)-K(+)-ATPase activation blocked most of the apical NHE3 insertion, possibly by inhibiting Na(+)-K(+)-ATPase-induced changes in intracellular sodium concentration ([Na](i)). Over the next 6-48 h, further increases in aNHE3 and BLM-Na(+)-K(+)-ATPase activity and total protein expression were observed to be largely mediated by aldosterone-activated SGK-1 pathway. Aldosterone-induced increases in NHE3 mRNA, for instance, could be inhibited by RNA silencing of SGK-1, but not Akt2. Additionally, aldosterone-induced increases in NHE3 promoter activity were blocked by silencing SGK-1 as well as pharmacological inhibition of PI3K. In conclusion, aldosterone-stimulated intestinal Na(+) absorption involves two phases. The first phase involves stimulation of PI3K, which increases SGK-dependent insertion and function of BLM-Na(+)-K(+)-ATPase and subsequent increased membrane insertion of aNHE3. The latter may be caused by Na(+)-K(+)-ATPase-induced changes in [Na] or transcellular Na flux. The second phase involves SGK-dependent increases in total NHE3 and Na(+)-K(+)-ATPase protein expression and activities. The coordination of apical and BLM transporters after aldosterone stimulation is therefore a complex process that requires multiple time- and interdependent cellular processes.     

Exercise-induced translocation of Na(+)-K(+) pump subunits to the plasma membrane in human skeletal muscle

Six human subjects performed one-legged knee extensor exercise (90 +/- 4 W) until fatigue (exercise time 4.6 +/- 0.8 min). Needle biopsies were obtained from vastus lateralis muscle before and immediately after exercise. Production of giant sarcolemmal vesicles from the biopsy material was used as a membrane purification procedure, and Na(+)-K(+) pump alpha- and beta-subunits were quantified by Western blotting. Exercise significantly increased (P < 0.05) the vesicular membrane content of the alpha(2)-, total alpha-, and beta(1)-subunits by 70 +/- 29, 35 +/- 10, and 26 +/- 5%, respectively. The membrane content of alpha(1) was not changed by exercise, and the densities of subunits in muscle homogenates were unchanged. The ratio of vesicular to crude muscle homogenate content of the alpha(2)-, total alpha-, and beta(1)-subunits was elevated during exercise by 67 +/- 33 (P < 0.05), 23 +/- 6 (P < 0.05), and 40 +/- 14% (P = 0.06), respectively. It is concluded that translocation of subunits is an important mechanism involved in the short time upregulation of the Na(+)-K(+) pumps in association with human muscle activity.     

K+/Na+ antagonism at cytoplasmic sites of Na+-K+-ATPase: a tissue-specific mechanism of sodium pump regulation

Tissue-distinct interactions of theNa⁺-K⁺-ATPasewith Na⁺ andK⁺, independent ofisoform-specific properties, were reported previously (A. G. Therien,N. B. Nestor, W. J. Ball, and R. Blostein. J. Biol.Chem. 271: 7104-7112, 1996). In this paper, wedescribe a detailed analysis of tissue-specific kinetics particularlyrelevant to regulation of pump activity by intracellularK⁺, namelyK⁺ inhibition at cytoplasmicNa⁺ sites. Our results show thatthe order of susceptibilities of ₁ pumps of various rat tissuestoK⁺/Na⁺antagonism, represented by the ratio of the apparent affinity forNa⁺ binding at cytoplasmicactivation sites in the absence ofK⁺ to the affinity constant forK⁺ as a competitive inhibitor ofNa⁺ binding at cytoplasmic sites,is red blood cell < axolemma  rat₁-transfected HeLa cells < small intestine < kidney < heart. In addition, we havecarried out an extensive analysis of the kinetics ofK⁺ binding and occlusion to thecytoplasmic cation binding site and find that, for most tissues, thereis a relationship between the rate ofK⁺ binding/occlusion and theapparent affinity for K⁺ as acompetitive inhibitor of Na⁺activation, the order for both parameters being heart  kidney > small intestine  rat₁-transfected HeLa cells. Thenotion that modulations in cytoplasmicK⁺/Na⁺antagonism are a potential mode of pump regulation is underscored byevidence of its reversibility. Thus the relatively highK⁺/Na⁺antagonism characteristic of kidney pumps was reduced when rat kidneymicrosomal membranes were fused into the dog red blood cell.

     

Interleukin-1 beta inhibits Na+-K+ ATPase activity and protein expression in cardiac myocytes

Recent studies have shown that heart diseases are always accompanied with high levels of IL-1beta and a decrease in Na+-K+ ATPase concentrations. This work studies the involvement of the cytokine in the observed changes in the pump. Rats were injected intraperitoneally with 400 mg of IL-1beta and 4 h later, the heart was isolated and a crude homogenate of the right and left ventricles was prepared and tested for Na+-K+ ATPase activity and protein expression. IL-1beta inhibited by around 70% the activity of the ATPase in the left and right ventricles. This inhibition of the pump was ascribed to a decrease in its protein expression as demonstrated by western blot analysis. A dose and time response study conducted on isolated cardiac myocytes confirmed the inhibitory role of the cytokine on the ATPase and showed that IL-1beta exerts its maximal down-regulatory effect at 2 h and at a dose of 20 ng/ml. The cytokine caused also an up-regulation of the NaKCl2 cotransporter. Both MEK and p38MAPK were shown to be involved in the signaling pathway activated by the cytokine. It can be concluded that the decrease in the Na+-K+ ATPase concentration observed in heart diseases is a consequence of the accompanying high levels of IL-1beta, and may be responsible for the different symptoms that accompany cardiac ischemia.     

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.     

Role for protein phosphatase 2A in the regulation of Calu-3 epithelial Na+-K+-2Cl-, type 1 co-transport function

Activity of Na+-K+-2Cl- co-transport (NKCC1) in epithelia is thought to be highly regulated through phosphorylation and dephosphorylation of the transporter. Previous functional studies from this laboratory suggested a role for protein phosphatase 2A (PP2A) as a serine/threonine protein phosphatase involved in the regulation of mammalian tracheal epithelial NKCC1. We expand on these studies to characterize serine/threonine protein phosphatase(s) necessary for regulation of NKCC1 function and the interaction of the phosphatase(s) with proteins associated with NKCC1. NKCC1 activity was measured as bumetanide-sensitive 86Rb uptake or basolateral to apical 86Rb flux in primary cultures of human tracheal epithelial cells or in Calu-3 airway epithelial cells grown on Transwell filter inserts. Preincubation with 0.1 nm okadaic acid, a PP2A > phosphatase 1 (PP1) inhibitor, increased NKCC1 activity 3.5-fold in human tracheal epithelial cells and 4.1-fold in Calu-3 cells. Calyculin, a PP1 > PP2A inhibitor, did not alter NKCC1 activity or percent bumetanide-sensitive flux. The effect of OA was dose-dependent with an IC50 of 0.4 nm. The alpha1-adrenergic agonist methoxamine increased NKCC1 activity and transiently increased PP2A activity 3.8-fold but did not alter PP1 activity. OA augmented methoxamine-dependent stimulation of NKCC1 activity. PP1, PP2A, and PP2C but not PP2B were detected in lysates from Calu-3 cells by immunoblot analysis. PP1 was not detected in immunoprecipitates of NKCC1 and vice versa. PP2A co-immunoprecipitated with NKCC1 and protein kinase C-delta (PKC-delta) and was pulled down by a recombinant N terminus of NKCC1 consisting of amino acids 1-286. One novel finding is co-precipitation of STE20-related proline-alanine-rich kinase, a regulatory kinase for NKCC1, with PP2A and PKC-delta. The results suggest a model of actin serving as a scaffold for binding and association of PKC-delta, PP2A, and STE20-related proline-alanine-rich kinase. The role of the complex of serine/threonine protein kinases and a protein phosphatase is probably the maintenance of optimal phosphorylation of NKCC1 coincident with its physiological function in epithelial absorption and secretion.