Phospholemman overexpression inhibits Na+-K+-ATPase in adult rat cardiac myocytes: relevance to decreased Na+ pump activity in postinfarction myocytes.

Messenger RNA levels of phospholemman (PLM), a member of the FXYD family of small single-span membrane proteins with putative ion-transport regulatory properties, were increased in postmyocardial infarction (MI) rat myocytes. We tested the hypothesis that the previously observed reduction in Na+-K+-ATPase activity in MI rat myocytes was due to PLM overexpression. In rat hearts harvested 3 and 7 days post-MI, PLM protein expression was increased by two- and fourfold, respectively. To simulate increased PLM expression post-MI, PLM was overexpressed in normal adult rat myocytes by adenovirus-mediated gene transfer. PLM overexpression did not affect the relative level of phosphorylation on serine68 of PLM. Na+-K+-ATPase activity was measured as ouabain-sensitive Na+-K+ pump current (Ip). Compared with control myocytes overexpressing green fluorescent protein alone, Ip measured in myocytes overexpressing PLM was significantly (P < 0.0001) lower at similar membrane voltages, pipette Na+ ([Na+]pip) and extracellular K+ ([K+]o) concentrations. From -70 to +60 mV, neither [Na+]pip nor [K+]o required to attain half-maximal Ip was significantly different between control and PLM myocytes. This phenotype of decreased V(max) without appreciable changes in K(m) for Na+ and K+ in PLM-overexpressed myocytes was similar to that observed in MI rat myocytes. Inhibition of Ip by PLM overexpression was not due to decreased Na+-K+-ATPase expression because there were no changes in either protein or messenger RNA levels of either alpha1- or alpha2-isoforms of Na+-K+-ATPase. In native rat cardiac myocytes, PLM coimmunoprecipitated with alpha-subunits of Na+-K+-ATPase. Inhibition of Na+-K+-ATPase by PLM overexpression, in addition to previously reported decrease in Na+-K+-ATPase expression, may explain altered V(max) but not K(m) of Na+-K+-ATPase in postinfarction rat myocytes.     

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.     

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.     

Changes in the expression of cardiac Na+-K+ ATPase subunits in the UM-X7.1 cardiomyopathic hamster

Previous studies have shown that cardiac Na+ -K+ ATPase activity in the UM-X7.1 hamster strain is decreased at an early stage of genetic cardiomyopathy and remains depressed; however, the mechanism for this decrease is unknown. The objective of the present study was to assess whether changes in the expression of cardiac Na+-K+ ATPase subunits in control and UM-X7.1 cardiomyopathic hamsters are associated with alterations in the enzyme activity. Accordingly, we examined sarcolemmal Na+-K+ ATPase activity as well as protein content and mRNA levels for the alpha1, alpha2, alpha3 and beta1-subunit of the Na+-K+ ATPase in 250-day-old UM-X7.1 and age-matched, control Syrian hamsters; this age corresponds to the severe stage of heart failure in the UM-X7.1 hamster. Na+-K+ ATPase activity in UM-X7.1 hearts was decreased compared to controls (9.0 +/- 0.8 versus 5.6 +/- 0.8 micromol Pi/mg protein/hr). Western blot analysis revealed that the protein content of Na+-K+ ATPase alpha1- and beta1-subunits were increased to 164 +/- 27% and 146 +/- 22% in UM-X7.1 hearts respectively, whereas that of the alpha2- and alpha3-subunits were decreased to 82 +/- 5% and 69 +/- 11% of control values. The results of Northern blot analysis for mRNA levels were consistent with the protein levels; mRNA levels for the alpha1- and beta1-subunits in UM-X7.1 hearts were elevated to 165 +/- 14% and 151 +/- 10%, but the alpha2-subunit was decreased to 60 +/- 8% of the control value. We were unable to detect mRNA for the alpha3-subunit in either UM-X7. 1 or control hearts. These data suggest that the marked depression of Na+-K+ ATPase activity in UM-X7.1 cardiomyopathic hearts may be due to changes in the expression of subunits for this enzyme.     

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.     

Physiological role of the alpha1- and alpha2-isoforms of the Na+-K+-ATPase and biological significance of their cardiac glycoside binding site

An interesting feature of Na+-K+-ATPase is that it contains four isoforms of the catalytic alpha-subunit, each with a tissue-specific distribution. Our laboratory has used gene targeting to define the functional role of the alpha1- and alpha2-isoforms. While knockout mice demonstrated the importance of the alpha1- and alpha2-isoforms for survival, the knockin mice, in which each isoform can be individually inhibited by ouabain and its function determined, demonstrated that both isoforms are regulators of cardiac muscle contractility. Another intriguing aspect of the Na+-K+-ATPase is that it contains a binding site for cardiac glycosides, such as digoxin. Conservation of this site suggests that it may have an in vivo role and that a natural ligand must exist to interact with this site. In fact, cardiac glycoside-like compounds have been observed in mammals. Our recent study demonstrates that the cardiac glycoside binding site of the Na+-K+-ATPase plays a role in the regulation of blood pressure and that it mediates both ouabain-induced and ACTH-induced hypertension in mice. Whereas chronic administration of ouabain or ACTH caused hypertension in wild-type mice, it had no effect on blood pressure in mice with a ouabain-resistant alpha2-isoform of Na+-K+-ATPase. Interestingly, animals with the ouabain-sensitive alpha1-isoform and a ouabain-resistant alpha2-isoform develop ACTH-induced hypertension to a greater extent than wild-type animals. Taken together, these results demonstrate that the cardiac glycoside binding of the Na+-K+-ATPase has a physiological role and suggests a function for a naturally occurring ligand that is stimulated by administration of ACTH.     

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.     

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.     

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 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.     

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.     

Modulation of alpha-ENaC and alpha1-Na+-K+-ATPase by cAMP and dexamethasone in alveolar epithelial cells

cAMP and dexamethasone are known to modulate Na+ transport in epithelial cells. We investigated whether dibutyryl cAMP (DBcAMP) and dexamethasone modulate the mRNA expression of two key elements of the Na+ transport system in isolated rat alveolar epithelial cells: alpha-, beta-, and gamma-subunits of the epithelial Na+ channel (ENaC) and the alpha1- and beta1-subunits of Na+-K+-ATPase. The cells were treated for up to 48 h with DBcAMP or dexamethasone to assess their long-term impact on the steady-state level of ENaC and Na+-K+-ATPase mRNA. DBcAMP induced a twofold transient increase of alpha-ENaC and alpha1-Na+-K+-ATPase mRNA that peaked after 8 h of treatment. It also upregulated beta- and gamma-ENaC mRNA but not beta1-Na+-K+-ATPase mRNA. Dexamethasone augmented alpha-ENaC mRNA expression 4.4-fold in cells treated for 24 h and also upregulated beta- and gamma-ENaC mRNA. There was a 1.6-fold increase at 8 h of beta1-Na+-K+-ATPase mRNA but no significant modulation of alpha1-Na+-K+-ATPase mRNA expression. Because DBcAMP and dexamethasone did not increase the stability of alpha-ENaC mRNA, we cloned 3.2 kb of the 5' sequences flanking the mouse alpha-ENaC gene to study the impact of DBcAMP and dexamethasone on alpha-ENaC promoter activity. The promoter was able to drive basal expression of the chloramphenicol acetyltransferase (CAT) reporter gene in A549 cells. Dexamethasone increased the activity of the promoter by a factor of 5.9. To complete the study, the physiological effects of DBcAMP and dexamethasone were investigated by measuring transepithelial current in treated and control cells. DBcAMP and dexamethasone modulated transepithelial current with a time course reminiscent of the profile observed for alpha-ENaC mRNA expression. DBcAMP had a greater impact on transepithelial current (2.5-fold increase at 8 h) than dexamethasone (1.8-fold increase at 24 h). These results suggest that modulation of alpha-ENaC and Na+-K+-ATPase gene expression is one of the mechanisms that regulates Na+ transport in alveolar epithelial cells.     

Contraction-induced increases in Na+-K+-ATPase mRNA levels in human skeletal muscle are not amplified by activation of additional muscle mass

The present study tested the hypothesis that exercise with a large compared with a small active muscle mass results in a higher contraction-induced increase in Na(+)-K(+)-ATPase mRNA expression due to greater hormonal responses. Furthermore, the relative abundance of Na(+)-K(+)-ATPase subunit alpha(1), alpha(2), alpha(3), alpha(4), beta(1), beta(2), and beta(3) mRNA in human skeletal muscle was investigated. On two occasions, eight subjects performed one-legged knee extension exercise (L) or combined one-legged knee extension and bilateral arm cranking (AL) for 5.00, 4.25, 3.50, 2.75, and 2.00 min separated by 3 min of rest. Leg exercise power output was the same in AL and L, but heart rate at the end of each exercise interval was higher in AL compared with L. One minute after exercise, arm venous blood lactate was higher in AL than in L. A higher level of blood epinephrine and norepinephrine was evident 3 min after exercise in AL compared with L. Nevertheless, none of the exercise-induced increases in alpha(1), alpha(2), beta(1), and beta(3) mRNA expression levels were higher in AL compared with L. The most abundant Na(+)-K(+)-ATPase subunit at the mRNA level was beta(1), which was expressed 3.4 times than alpha(2). Expression of alpha(1), beta(2), and beta(3) was less than 5% of the alpha(2) expression, and no reliable detection of alpha(3) and alpha(4) was possible. In conclusion, activation of additional muscle mass does not result in a higher exercise-induced increase in Na(+)-K(+)-ATPase subunit-specific mRNA.     

Parathyroid hormone-mediated regulation of Na+-K+-ATPase requires ERK-dependent translocation of protein kinase Calpha

Parathyroid hormone (PTH) inhibits Na+-K+-ATPase activity by serine phosphorylation of the alpha1 subunit through protein kinase C (PKC)- and extracellular signal-regulated kinase (ERK)-dependent pathways. Based on previous studies we postulated that PTH regulates sodium pump activity through isoform-specific PKC-dependent activation of ERK. In the present work utilizing opossum kidney cells, a model of renal proximal tubule, PTH stimulated membrane translocation of PKCalpha by 102 +/- 16% and PKCbetaI by 41 +/- 7% but had no effect on PKCbetaII and PKCzeta. Both PKCalpha and PKCbetaI phosphorylated the Na+-K+-ATPase alpha1 subunit in vitro. PTH increased the activity of PKCalpha but not PKCbetaI. Coimmunoprecipitation assays demonstrated that treatment with PTH enhanced the association between Na+-K+-ATPase alpha1 subunit and PKCalpha, whereas the association between Na+-K+-ATPase alpha1 subunit and PKCbetaI remained unchanged. A PKCalpha inhibitory peptide blocked PTH-stimulated serine phosphorylation of the Na+-K+-ATPase alpha1 subunit and inhibition of Na+-K+-ATPase activity. Pharmacologic inhibition of MEK-1 blocked PTH-stimulated translocation of PKCalpha, whereas transfection of constitutively active MEK-1 cDNA induced translocation of PKCalpha and increased phosphorylation of the Na+-K+-ATPase alpha1 subunit. In contrast, PTH-stimulated ERK activation was not inhibited by pretreatment with the PKCalpha inhibitory peptide. Inhibition of PKCalpha expression by siRNA did not inhibit PTH-mediated ERK activation but significantly reduced PTH-mediated phosphorylation of the Na+-K+-ATPase alpha1 subunit. Pharmacologic inhibition of phosphoinositide 3-kinase blocked PTH-stimulated ERK activation, translocation of PKCalpha, and phosphorylation of the Na+-K+-ATPase alpha1 subunit. We conclude that PTH stimulates Na+-K+-ATPase phosphorylation and decreases the activity of Na+-K+-ATPase by ERK-dependent activation of PKCalpha.     

Dopamine increases lung liquid clearance during mechanical ventilation

Short-term mechanical ventilation with high tidal volume (HVT) causes mild to moderate lung injury and impairs active Na+ transport and lung liquid clearance in rats. Dopamine (DA) enhances active Na+ transport in normal rat lungs by increasing Na+-K+-ATPase activity in the alveolar epithelium. We examined whether DA would increase alveolar fluid reabsorption in rats ventilated with HVT for 40 min compared with those ventilated with low tidal volume (LVT) and with nonventilated rats. Similar to previous reports, HVT ventilation decreased alveolar fluid reabsorption by ~50% (P < 0.001). DA increased alveolar fluid reabsorption in nonventilated control rats (by ~60%), LVT ventilated rats (by approximately 55%), and HVT ventilated rats (by ~200%). In parallel studies, DA increased Na+-K+-ATPase activity in cultured rat alveolar epithelial type II cells (ATII). Depolymerization of cellular microtubules by colchicine inhibited the effect of DA on HVT ventilated rats as well as on Na+-K+-ATPase activity in ATII cells. Neither DA nor colchicine affected the short-term Na+-K+-ATPase alpha1- and beta1-subunit mRNA steady-state levels or total alpha1- and beta1-subunit protein abundance in ATII cells. Thus we reason that DA improved alveolar fluid reabsorption in rats ventilated with HVT by upregulating the Na+-K+-ATPase function in alveolar epithelial cells.