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.     

Preconditioning attenuates ischemia-reperfusion-induced remodeling of Na+-K+-ATPase in hearts

The aim of this study was to determine whether changes in protein content and/or gene expression of Na+-K+-ATPase subunits underlie its decreased enzyme activity during ischemia and reperfusion. We measured protein and mRNA subunit levels in isolated rat hearts subjected to 30 min of ischemia and 30 min of reperfusion (I/R). The effect of ischemic preconditioning (IP), induced by three cycles of ischemia and reperfusion (10 min each), was also assessed on the molecular changes in Na+-K+-ATPase subunit composition due to I/R. I/R reduced the protein levels of the alpha2-, alpha3-, beta1-, and beta2-isoforms by 71%, 85%, 27%, and 65%, respectively, whereas the alpha1-isoform was decreased by <15%. A similar reduction in mRNA levels also occurred for the isoforms of Na+-K+-ATPase. IP attenuated the reduction in protein levels of Na+-K+-ATPase alpha2-, alpha3-, and beta2-isoforms induced by I/R, without affecting the alpha1- and beta1-isoforms. Furthermore, IP prevented the reduction in mRNA levels of Na+-K+-ATPase alpha2-, alpha3-, and beta1-isoforms following I/R. Similar alterations in protein contents and mRNA levels for the Na+/Ca2+ exchanger were seen due to I/R as well as IP. These findings indicate that remodeling of Na+-K+-ATPase may occur because of I/R injury, and this may partly explain the reduction in enzyme activity in ischemic heart disease. Furthermore, IP may produce beneficial effects by attenuating the remodeling of Na+-K+-ATPase and changes in Na+/Ca2+ exchanger in hearts after I/R.     

温度和盐度对吉富品系尼罗罗非鱼幼鱼鳃Na+-K+-ATPase活力的联合效应

试验采用中心组合设计(central composite face-centered design,CCF)和响应曲面法(response surface methodology,RSM)研究了温度(12—34℃)和盐度(0—26)两因素对体长为(4.36±0.105)cm,体重为(2.45±0.153)g的吉富品系尼罗罗非鱼(GIFT Nile tilapia,Oreochromis niloticus;简称吉富罗非鱼)幼鱼鳃Na+-K+-ATPase活力的联合效应。结果表明:(1)温度和盐度的一次效应和二次效应对Na+-K+-ATPase活力影响极显著(P<0.01),温度和盐度的互作效应不显著(P>0.05);(2)经响应曲面法分析,随着温度和盐度的增大,Na+-K+-ATPase活力呈先减小后增大的趋势;(3)建立了Na+-K+-ATPase活力与温度、盐度间关系的模型方程(R2=0.9829,Pred.R2=0.8550,P<0.01),并可用于预测吉富罗非鱼幼鱼鳃Na+-K+-ATPase的活力;(4)优化结果显示,温度为24.15℃,盐度为11.75时,Na+-K+-ATPase活力最小为0.62μmol无机磷.mg-1蛋白.h-1,满意度函数值高达0.961。Na+-K+-ATPase活力可以作为检测罗非鱼生长性能的指标,其活力较低时,一般反映了鱼体生存环境适宜,生长代谢旺盛,消耗于渗透调节的能量较少。     

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.     

Increases in transepithelial vectorial Na+ transport facilitates Na+-dependent L-DOPA transport in renal OK cells

The present study evaluated the hypothesis of whether increases in vectorial Na+ transport translate into facilitation of Na+-dependent L-DOPA uptake in cultured renal epithelial tubular cells. Increases in vectorial Na+ transport were obtained in opossum kidney (OK) cells engineered to overexpress Na+-K+-ATPase after transfection of wild type OK cells with the rodent Na+-K+-ATPase alpha1 subunit. The most impressive differences between wild type and transfected OK cells are that the latter overexpressed Na+-K+-ATPase accompanied by an increased activity of the transporter. Non-linear analysis of the saturation curve for l-DOPA uptake revealed a Vmax value (in nmol mg protein/6 min) of 62 and 80 in wild type and transfected cells, respectively. The uptake of a non-saturating concentration (0.25 microM) of [14C]-L-DOPA in OK-WT cells was not affected by Na+ removal, whereas in OK-alpha1 cells accumulation of [14C]-L-DOPA was clearly dependent on the presence of extracellular Na+. When Na+ was replaced by choline, the inhibitory profile of neutral l-amino acids, but not of basic and acidic amino acids, upon [14C]-L-DOPA uptake in both cell types, was significantly greater than that observed in the presence of extracellular Na+. It is concluded that enhanced ability of OK cells overexpressing Na+-K+-ATPase to translocate Na+ from the apical to the basal cell side correlates positively with their ability to accumulate L-DOPA, which is in agreement with the role of Na+ in taking up the precursor of renal dopamine.     

三疣梭子蟹Na+/H+-exchanger基因克隆鉴定及在盐度胁迫下的表达分析

为研究Na+/H+-exchanger基因在三疣梭子蟹（Portunus trituberculatus）盐度胁迫过程中的功能作用,克隆了三疣梭子蟹Na+/H+-exchanger基因并进行表达分析。结果显示,Na+/H+-exchanger基因（GenBank:KU519329）全长4233 bp,5和3非编码区（UTR）长分别为519和753 bp,开放阅读框（ORF）长2961 bp。编码986个氨基酸,预测蛋白质分子量和等电点分别为110.8 kD和7.42,具有信号肽和典型的Na+/H+-exchanger蛋白结构域,含12个跨膜螺旋;三疣梭子蟹Na+/H+-exchanger基因与普通滨蟹（Carcinus maenas）同源性最高,达到87.2%,系统进化分析也显示该序列与普通滨蟹聚为一支;表达分析显示,三疣梭子蟹Na+/H+-exchanger基因在鳃中表达量最高;在低盐（盐度5、10和20）胁迫过程中,Na+/H+-exchanger基因在0-12h上调表达明显,在24-168h间表达量呈下降趋势;在高盐（盐度50）胁迫初期（0-12h）,该基因表达量相对稳定,之后（24-168h）显著下调表达。研究表明低盐显著诱导Na+/H+-exchanger基因的高表达,推测三疣梭子蟹Na+/H+-exchanger基因在低盐环境下发挥重要的渗透调节功能。     

Gill Na+-K+-ATPase activity correlates with basolateral membrane lipid composition in seawater- but not freshwater-acclimated Arctic char (Salvelinus alpinus)

The successful migration of euryhaline teleost fish from freshwater to seawater requires the upregulation of gill Na+-K+-ATPase, an ion transport enzyme located in the basolateral membrane (BLM) of gill chloride cells. Following 39 days of seawater exposure, Arctic char had similar plasma sodium and chloride levels as individuals maintained in freshwater, indicating they had successfully acclimated to seawater. This acclimation was associated with an eightfold increase in gill Na+-K+-ATPase activity but only a threefold increase in gill Na+-K+-ATPase protein number, suggesting that other mechanisms may also modulate gill Na+-K+-ATPase activity. We therefore investigated the influence of membrane composition on Na+-K+-ATPase activity by examining the phospholipid, fatty acid, and cholesterol composition of the gill BLM from freshwater- and seawater-acclimated Arctic char. Mean gill BLM cholesterol content was significantly lower ( approximately 22%) in seawater-acclimated char. Gill Na+-K+-ATPase activity in individual seawater Arctic char was negatively correlated with BLM cholesterol content and positively correlated with %phosphatidylethanolamine and overall %18:2n6 (linoleic acid) content of the BLM, suggesting gill Na+-K+-ATPase activity of seawater-acclimated char may be modulated by the lipid composition of the BLM and may be especially sensitive to those parameters known to influence membrane fluidity. Na+-K+-ATPase activity of individual freshwater Arctic char was not correlated to any membrane lipid parameter measured, suggesting that different lipid-protein interactions may exist for char living in each environment.     

Developmental and thyroid hormone regulation of two molecular forms of Na+-K+-ATPase in brain

In the brain there are two isozymes of Na+-K+-ATPase differing in their catalytic subunits: alpha, indistinguishable from the kidney form of alpha, and alpha +, found in axolemma. The time course of the increase in each alpha during development was described by quantitating the abundance of each form, studied in unpurified membranes resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with specific antibodies and with fluorescein 5'-isothiocyanate. Both the alpha and alpha + subunits, quantitated with antibodies, increased 10-fold in abundance from 18 days gestation to 20 days of age, with alpha + increasing more rapidly than alpha early in development. A 10-fold increase in enzyme activity was also observed during this period. Using fluorescein 5'-isothiocyanate to quantitate the two alpha subunits, a similar increase in alpha + was observed with less of an increase in alpha. The ratio of alpha + to alpha increased from 0.75 at 18 days gestation to 3 at 3 days of age remaining at this ratio to 20 days of age. The possibility that thyroid hormone, a known regulator of brain Na+-K+-ATPase during development, differentially regulated the two forms was tested using 15-day-old hypothyroid rats. The abundance of both forms of alpha was similarly decreased: alpha + to 69% and alpha to 48% of control values. Na+-K+-ATPase activity was 70% of control. We conclude that both alpha and alpha + abundance increase in the brain during pre-and neonatal development and that the increase in both alpha subunits is regulated, directly or indirectly, by thyroid hormones.     

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.     

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.     

A conserved gene encoding the 57-kDa subunit of the yeast vacuolar H+-ATPase

The peripheral (catalytic) sector of vacuolar H+-ATPases contains five different polypeptides denoted as subunits A-E in order of decreasing molecular masses from 72 to 33 kDa. The gene encoding subunit B (57 kDa) of yeast vacuolar H+-ATPase was cloned on a 5-kilobase pair genomic DNA fragment and sequenced. Four open reading frames were identified in the sequenced DNA. One of them encodes a protein of 504 amino acids with a calculated Mr of 56,557. Hydropathy plot revealed no apparent transmembrane segments. Southern analysis demonstrated that a single gene encodes this polypeptide in the yeast genome. The amino acid sequence exhibits extensive identity with the homologous protein from the plant Arabidopsis (77%). This polypeptide also contains regions of homology with the alpha subunits of H+-ATPases from mitochondria, chloroplasts, and bacteria. However, less similarity was detected when it was compared with the beta subunits of those enzymes. The implication of these phenomena on the evolution of proton pumps is discussed.     

Salinity dependent Na+-K+ATPase activity in gills of the euryhaline crab Chasmagnathus granulata

The occurrence and response of Na+-K+ATPase specific activity to environmental salinity changes were studied in gill extracts of all of the gills of the euryhaline crab Chasmagnathus granulata from Mar Chiquita coastal lagoon (Buenos Aires Province, Argentina). All of the gills exhibited a salinity dependent Na+-K+ATPase activity, although the pattern of response to environmental salinity was different among gills. As described in other euryhaline crabs highest Na+-K+ATPase specific activity was found in posterior gills (6 to 8), which, with exception of gill 6, increased upon acclimation to reduced salinity. However, a high increase of activity also occurred in anterior gills (1 to 5) in diluted media. Furthermore, both short and long term differential changes of Na+-K+ATPase activity occurred among the gills after the transfer of crabs to reduced salinity. The fact that variations of Na+-K+ATPase activity in the gills were concomitant with the transition from osmoconformity to ionoregulation suggests that this enzyme is a component of the branchial ionoregulatory mechanisms at the biochemical level in this crab.     

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.     

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.