Regulation of Na+-K+-ATPase: effect of Mg and Ca ions

The participation of Mg2+ and Ca2+ in complicated mechanisms of Na+, K(+)-ATPase regulation is discussed in the survey. The regulatory actions of Mg2+ on Na+, K(+)-ATPase such as its participation in phosphorylation and dephosphorylation of the enzyme, ADP/ATP-exchange inhibition, cardiac glycosides and vanadate binding with the enzyme, conformational changes induction during ATPase cycle are reviewed in detail. Some current views of mechanisms of above mentioned Mg2+ regulatory effects are discussed. The experimental evidence of Ca2+ immediate influence on the functional activity of Na+, K(+)-ATPase (catalytic, transport and glycoside-binding) are given. It's noted that these effects are based on the conformational changes in the enzyme and also on the phase transition in membrane induced by Ca2+. Unimmediate action of Ca2+ on Na+, K(+)-ATPase is also discussed, especially due to its effect on other membrane systems functionally linked with Na(+)-pump (for instance, due to Na+/Ca(+)-exchanger activation). It's concluded that Mg2+ and Ca2+ as "universal regulators" of the cell effectively influence the functional activity and conformational states of Na+, K(+)-ATPase.     

Role of Na+-K+-ATPase in insulin-induced lactate release by skeletal muscle

Hyperinsulinemia increases lactate release by various organs and tissues. Whereas it has been shown that aerobic glycolysis is linked to Na+-K+-ATPase activity, we hypothesized that stimulation by insulin of skeletal muscle Na+-K+-ATPase is responsible for increased muscle lactate production. To test this hypothesis, we assessed muscle lactate release in healthy volunteers from the [13C]lactate concentration in the effluent dialysates of microdialysis probes inserted into the tibialis anterior muscles on both sides and infused with solutions containing 5 mmol/l [U-13C]glucose. On one side, the microdialysis probe was intermittently infused with the same solution additioned with 2.10(-5) M ouabain. In the basal state, [13C]lactate concentration in the dialysate was not affected by ouabain. During a euglycemic-hyperinsulinemic clamp, [13C]lactate concentration increased by 135% in the dialysate without ouabain, and this stimulation was nearly entirely reversed by ouabain (56% inhibition compared with values in the dialysate collected from the contralateral probe). These data indicate that insulin stimulates muscle lactate release by activating Na+-K+-ATPase in healthy humans.     

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.     

The Na+-K+-ATPase as self-adhesion molecule and hormone receptor

Thanks to the homeostasis of the internal milieu, metazoan cells can enormously simplify their housekeeping efforts and engage instead in differentiation and multiple forms of organization (tissues, organs, systems) that enable them to produce an astonishing diversity of mammals. The stability of the internal milieu despite drastic variations of the external environment (air, fresh or seawater, gastrointestinal fluids, glomerular filtrate, bile) is due to transporting epithelia that can adjust their specific permeability to H(2)O, H(+), Na(+), K(+), Ca(2+), and Cl(-) over several orders of magnitude and exchange substances with the outer milieu with exquisite precision. This exchange is due to the polarized expression of membrane proteins, among them Na(+)-K(+)-ATPase, an oligomeric enzyme that uses chemical energy from ATP molecules to translocate ions across the plasma membrane of epithelial cells. Na(+)-K(+)-ATPase presents two types of asymmetries: the arrangement of its subunits, and its expression in one pole of the epithelial cell ("polarity"). In most epithelia, polarity consists of the expression of Na(+)-K(+)-ATPase towards the intercellular space and arises in part from the interaction of the extracellular segment of the β-subunit with another β-subunit present in a Na(+)-K(+)-ATPase molecule expressed by a neighboring cell. In addition to enabling the Na(+)-K(+)-ATPase to transport ions and water vectorially, this position exposes its receptors to ouabain and analogous cardiotonic steroids, which are present in the internal milieu because these were secreted by endocrine cells.     

Cardiopulmonary bypass reduces atrial Na+-K+-ATPase expression in children

Cardiopulmonary bypass (CPB) may induce serious side effects, potentially leading to myocardial failure. The Na(+)-K(+)-ATPase is a key component for myocardial function. Due to its developmental regulation, results from adult studies cannot be adopted to the situation in childhood. Right atrial myocardium from patients with left-to-right shunts at atrial level (VO, n=8) and those without (NO, n=8) was excised during heart surgery before and after CPB. Na(+)-K(+)-ATPase isoforms ATP1A1 (p=0.008) and ATP1A3 (p=0.038) decreased during CPB, which decrease was restricted to the VO group. This study highlights the importance of the underlying heart defect for susceptibility to the effects of CPB, showing a reduced Na(+)-K(+)-ATPase mRNA expression only in patients with left-to-right shunts on the atrial level. This seemed to be an early molecular event, as apart from one, none of the patients showed heart failure before or after surgery.     

Na+K+-ATP酶抑制引起的细胞凋亡和杂合性细胞死亡

Na^ -K^ -ATP酶也称Na^ 泵或Na^ -K^ 泵,是哺乳类细胞膜进行离子转运的跨膜载体蛋白。其基本作用是维持细胞膜内外Na^ -K^ 电化学梯度的平衡。近来研究表明,Na^ -K^ -ATP酶在细胞死亡中起重要作用,细胞K 缺失导致凋亡,在某些类型的细胞中,同一细胞兼具细胞肿胀、细胞器溶解等坏死特征和染色质凝集、DNA梯带、caspase级联反应等凋亡特征,呈现一种特殊的细胞死亡形式,即杂合性细胞死亡。     

Regulation of cardiac myocyte contractility by phospholemman: Na+/Ca2+ exchange versus Na+ -K+ -ATPase

Phospholemman (PLM) regulates cardiac Na(+)/Ca(2+) exchanger (NCX1) and Na(+)-K(+)-ATPase in cardiac myocytes. PLM, when phosphorylated at Ser(68), disinhibits Na(+)-K(+)-ATPase but inhibits NCX1. PLM regulates cardiac contractility by modulating Na(+)-K(+)-ATPase and/or NCX1. In this study, we first demonstrated that adult mouse cardiac myocytes cultured for 48 h had normal surface membrane areas, t-tubules, and NCX1 and sarco(endo)plasmic reticulum Ca(2+)-ATPase levels, and retained near normal contractility, but alpha(1)-subunit of Na(+)-K(+)-ATPase was slightly decreased. Differences in contractility between myocytes isolated from wild-type (WT) and PLM knockout (KO) hearts were preserved after 48 h of culture. Infection with adenovirus expressing green fluorescent protein (GFP) did not affect contractility at 48 h. When WT PLM was overexpressed in PLM KO myocytes, contractility and cytosolic Ca(2+) concentration ([Ca(2+)](i)) transients reverted back to those observed in cultured WT myocytes. Both Na(+)-K(+)-ATPase current (I(pump)) and Na(+)/Ca(2+) exchange current (I(NaCa)) in PLM KO myocytes rescued with WT PLM were depressed compared with PLM KO myocytes. Overexpressing the PLMS68E mutant (phosphomimetic) in PLM KO myocytes resulted in the suppression of I(NaCa) but had no effect on I(pump). Contractility, [Ca(2+)](i) transient amplitudes, and sarcoplasmic reticulum Ca(2+) contents in PLM KO myocytes overexpressing the PLMS68E mutant were depressed compared with PLM KO myocytes overexpressing GFP. Overexpressing the PLMS68A mutant (mimicking unphosphorylated PLM) in PLM KO myocytes had no effect on I(NaCa) but decreased I(pump). Contractility, [Ca(2+)](i) transient amplitudes, and sarcoplasmic reticulum Ca(2+) contents in PLM KO myocytes overexpressing the S68A mutant were similar to PLM KO myocytes overexpressing GFP. We conclude that at the single-myocyte level, PLM affects cardiac contractility and [Ca(2+)](i) homeostasis primarily by its direct inhibitory effects on Na(+)/Ca(2+) exchange.     

Renal Na+-K+-ATPase in weanling and adult spontaneously hypertensive rats

The interrelationships among plasma renin activity (PRA, ng AI/ml plasma/hr), aldosterone concentration (ng%), and renal Na+-K+-ATPase activity (mumole PO4/mg protein/hr) were studied in 9 weanling normotensive spontaneously hypertensive rats (SHR), 9 adult hypertensive SHR, and 9 weanling and 9 adult normotensive Wistar-Kyoto rats (WKY). All groups were placed on a normal (0.4% sodium) diet. PRA and plasma aldosterone, measured in samples drawn from the ether-anesthetized rat, were higher in weanling SHR (15.2 +/- 2.0, 37 +/- 4.2) than in WKY. PRA measured in samples collected from a separate group of unanesthetized weanling SHR was also greater than in age-matched WKY. In adult SHR, PRA (6.1 +/- 0.9) and plasma aldosterone (20.0 +/- 2.7) were decreased. During the weanling period Na+-K+-ATPase activity in SHR was not only greater than in age-matched WKY but was also increased compared to adult normotensive and hypertensive rats (137 +/- 9 weanling SHR, 89 +/- 7 weanling WKY, 73 +/- 11 adult SHR, 84 +/- 17 adult WKY). Thus, during the weanling period the renin-angiotensin-aldosterone (R-A-A) system and renal Na+-K+-ATPase activity are activated in SHR. The elevation of Na+-K+-ATPase activity may be due to increased aldosterone levels. It was noted, however, that plasma aldosterone was similar in adult WKY and weanling SHR, while Na+-K+-ATPase activity was higher in SHR. These findings involving R-A-A and renal Na+-K+-ATPase activity prior to the elevation of blood pressure suggest that the kidneys may play a role in the initiation of hypertension in SHR.     

Mechanisms underlying increases in rat soleus Na+-K+-ATPase activity by induced contractions.

Acute regulation of the Na(+)-K(+)-ATPase activity in rat soleus muscle was investigated in response to 15 and 90 min of electrically induced contractile activity (500-ms trains at 30 Hz every 1.5 s). Kinetic measurements of Na(+)-K(+)-ATPase activity, assessed by the 3-O-methylfluorescein K(+)-stimulated phosphatase assay (3-O-MFP), were performed on crude homogenates (Hom) and on tissue separated into two membrane fractions, the sarcolemmal/particulate (SLP) and endosomal (En), in both stimulated (Stim) and contralateral control (Con) muscles. Maximal 3-O-MFP activity (V(max), nmol.mg protein(-1).h(-1)) was elevated (P < 0.05) in Stim by 40% and by 53% in Hom and by 37 and 40% in SLP at 15 and 90 min, respectively. The 38% increase (P < 0.05) in the alpha(2)-isoform subunit distribution in SLP at 15 min, as assessed by quantitative immunoblotting, persisted at 90 min, whereas for En a 42% decrease (P < 0.05) was observed only at 15 min. For the alpha(1)-subunit at 15 min, a 27% decrease (P < 0.05) was observed in En, whereas the 13% increase observed in SLP was not significant (P = 0.08). At 90 min, alpha(1) was increased (P < 0.05) by 14% in SLP and by 29% in En. No changes were observed in beta(1)-subunit distribution in En and SLP regardless of time of stimulation. Immunoprecipitation with antiphosphotyrosine antibody and quantitative immunoblotting with alpha(1)- and alpha(2)-antibodies indicated increases (P < 0.05) in tyrosine phosphorylation of 51% in alpha(2) at 15 min only. These results suggest that the increases in V(max) during contractile activity are mediated both by increased phosphorylation and by translocation of the enzyme to the plasma membrane.     

Studies on (Na+-K+)-activated ATPase XXXIV. Phosphatidylserine not essential for (Na+-K+)-ATPase activity

An (Na⁺-K⁺)-ATPase preparation, consisting of NaI-treated microsomes from cattle brain, was incubated with a phosphatidylserine decarboxylase preparation from Escherichia coli. This led to a reduction in the phosphatidylserine content from 10.1 % to less than 0.1%, accompanied by an equimolar formation of phosphatidylethanolamine. Since the (Na⁺-K⁺)-ATPase activity was not reduced, it can be concluded that phosphatidylserine is not essential for the Na⁺-K⁺)-ATPase activity.     

Sarcolemmal cation channels and exchangers modify the increase in intracellular calcium in cardiomyocytes on inhibiting Na+-K+-ATPase

Although inhibition of the sarcolemmal (SL) Na(+)-K(+)-ATPase is known to cause an increase in the intracellular concentration of Ca(2+) ([Ca(2+)](i)) by stimulating the SL Na(+)/Ca(2+) exchanger (NCX), the involvement of other SL sites in inducing this increase in [Ca(2+)](i) is not fully understood. Isolated rat cardiomyocytes were treated with or without different agents that modify Ca(2+) movements by affecting various SL sites and were then exposed to ouabain. Ouabain was observed to increase the basal levels of both [Ca(2+)](i) and intracellular Na(+) concentration ([Na(+)](i)) as well as to augment the KCl-induced increases in both [Ca(2+)](i) and [Na(+)](i) in a concentration-dependent manner. The ouabain-induced changes in [Na(+)](i) and [Ca(2+)](i) were attenuated by treatment with inhibitors of SL Na(+)/H(+) exchanger and SL Na(+) channels. Both the ouabain-induced increase in basal [Ca(2+)](i) and augmentation of the KCl response were markedly decreased when cardiomyocytes were exposed to 0-10 mM Na(+). Inhibitors of SL NCX depressed but decreasing extracellular Na(+) from 105-35 mM augmented the ouabain-induced increase in basal [Ca(2+)](i) and the KCl response. Not only was the increase in [Ca(2+)](i) by ouabain dependent on the extracellular Ca(2+) concentration, but it was also attenuated by inhibitors of SL L-type Ca(2+) channels and store-operated Ca(2+) channels (SOC). Unlike the SL L-type Ca(2+)-channel blocker, the blockers of SL Na(+) channel and SL SOC, when used in combination with SL NCX inhibitor, showed additive effects in reducing the ouabain-induced increase in basal [Ca(2+)](i). These results support the view that in addition to SL NCX, SL L-type Ca(2+) channels and SL SOC may be involved in raising [Ca(2+)](i) on inhibition of the SL Na(+)-K(+)-ATPase by ouabain. Furthermore, both SL Na(+)/H(+) exchanger and Na(+) channels play a critical role in the ouabain-induced Ca(2+) increase in cardiomyocytes.     

Cigarette smoking impairs Na+-K+-ATPase activity in the human coronary microcirculation

The extracellular K(+) concentration ([K(+)](o)) has been proposed to link cardiac metabolism with coronary perfusion and arrhythmogenesis, particularly during ischemia. Several animal studies have also supported K(+) as an EDHF that activates Na(+)-K(+)-ATPase and/or inwardly rectifying K(+) (K(ir)) channels. Therefore, we examined the vascular reactivity of human coronary arterioles (HCAs) to small elevations in [K(+)](o), the influence of risk factors for coronary disease, and the role of K(+) as an EDHF. Changes in the internal diameter of HCAs were recorded with videomicroscopy. Most vessels dilated to increases in [K(+)](o) with a maximal dilation of 55 ± 6% primarily at 12.5-20.0 mM KCl (n = 38, average: 16 ± 1 mM). Ouabain, a Na(+)-K(+)-ATPase inhibitor, alone reduced the dilation, and the addition of Ba(2+), a K(ir) channel blocker, abolished the remaining dilation, whereas neither endothelial denudation nor Ba(2+) alone reduced the dilation. Multivariate analysis revealed that cigarette smoking was the only risk factor associated with impaired dilation to K(+). Ouabain significantly reduced the vasodilation in HCAs from subjects without cigarette smoking but not in those with smoking. Cigarette smoking downregulated the expression of the Na(+)-K(+)-ATPase catalytic α(1)-subunit but not Kir2.1 in the vessels. Ouabain abolished the dilation in endothelium-denuded vessels to a same extent to that with the combination of ouabain and Ba(2+) in endothelium-intact vessels, whereas neither ouabain nor ouabain plus Ba(2+) reduced EDHF-mediated dilations to bradykinin and ADP. A rise in [K(+)](o) dilates HCAs primarily via the activation of Na(+)-K(+)-ATPase in vascular smooth muscle cells with a considerable contribution of K(ir) channels in the endothelium, indicating that [K(+)](o) may modify coronary microvascular resistance in humans. Na(+)-K(+)-ATPase activity is impaired in subjects who smoke, possibly contributing to dysregulation of the coronary microcirculation, excess ischemia, and arrhythmogenesis in those subjects. K(+) does not likely serve as an EDHF in the human coronary arteriolar dilation to bradykinin and ADP.     

Transgenic mice expressing Na+-K+-ATPase in smooth muscle decreases blood pressure

The Na(+)-K(+)-ATPase (NKA) is a transmembrane protein that sets and maintains the electrochemical gradient by extruding three Na(+) in exchange for two K(+). An important physiological role proposed for vascular smooth muscle NKA is the regulation of blood pressure via modulation of vascular smooth muscle contractility (5). To investigate the relations between the level of NKA in smooth muscle and blood pressure, we developed mice carrying a transgene for either the NKA alpha(1)- or alpha(2)-isoform (alpha(1 sm+) or alpha(2 sm+) mice) driven by the smooth muscle-specific alpha-actin promoter SMP8. Interestingly, both alpha-isoforms, the one contained in the transgene and the one not contained, were increased to a similar degree at both protein and mRNA levels. The total alpha-isoform protein was increased from 1.5-fold (alpha(1 sm+) mice) to 7-fold (alpha(2 sm+) mice). The increase in total NKA alpha-isoform protein was accompanied by a 2.5-fold increase in NKA activity in alpha(2 sm+) gastric antrum. Immunocytochemistry of the alpha(1)- and alpha(2)-isoforms in alpha(2 sm+) aortic smooth muscle cells indicated that alpha-isoform distributions were similar to those shown in wild-type cells. alpha(2 sm+) Mice (high expression) were hypotensive (109.9 +/- 1.6 vs. 121.3 +/- 1.4 mmHg; n = 13 and 11, respectively), whereas alpha(1 sm+) mice (low expression) were normotensive (122.7 +/- 2.5 vs. 117.4 +/- 2.3; n = 11 or 12). alpha(2 sm+) Aorta, but not alpha(1 sm+) aorta, relaxed faster from a KCl-induced contraction than wild-type aorta. Our results show that smooth muscle displays unique coordinate expression of the alpha-isoforms. Increasing smooth muscle NKA decreases blood pressure and is dependent on the degree of increased alpha-isoform expression.     

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.