Estradiol-induced expression of N(+)-K(+)-ATPase catalytic isoforms in rat arteries: gender differences in activity mediated by nitric oxide donors

We tested the hypothesis that previously demonstrated gender differences in ACh-induced vascular relaxation could involve diverse Na(+)-K(+)-ATPase functions. We determined Na(+)-K(+)-ATPase by measuring arterial ouabain-sensitive 86Rb uptake in response to ACh. We found a significant increase of Na+ pump activity only in aortic rings from female rats (control 206 +/- 11 vs. 367 +/- 29 nmol 86Rb/K.min(-1).g wt tissue(-1); P < 0.01). Ovariectomy eliminated sex differences in Na(+)-K(+)-ATPase function, and chronic in vivo hormone replacement with 17beta-estradiol restored the ACh effect on Na(+)-K(+)-ATPase. Because ACh acts by enhancing production of NO, we examined whether the NO donor sodium nitroprusside (SNP) mimics the action of ACh on Na(+)-K(+)-ATPase activity. SNP increased ouabain-sensitive 86Rb uptake in denuded female arteries (control 123 +/- 7 vs. 197 +/- 12 nmol 86Rb/K.min(-1).g wt tissue(-1); P < 0.05). Methylene blue (an inhibitor of guanylate cyclase) and KT-5823 (a cGMP-dependent kinase inhibitor) blocked the stimulatory action of SNP. Exposure of female thoracic aorta to the Na+/K+ pump inhibitor ouabain significantly decreased SNP-induced and ACh-mediated relaxation of aortic rings. At the molecular level, Western blot analysis of arterial tissue revealed significant gender differences in the relative abundance of catalytic isoforms of Na(+)-K(+)-ATPase. Female-derived aortas exhibited a greater proportion of alpha2-isoform (44%) compared with male-derived aortas. Furthermore, estradiol upregulated the expression of alpha2 mRNA in male arterial explants. Our results demonstrate that enhancement of ACh-induced relaxation observed in female rats may be in part explained by 1) NO-dependent increased Na(+)-K(+)-ATPase activity in female vascular tissue and 2) greater abundance of Na(+)-K(+)-ATPase alpha2-isoform in females.     

Regulation of renal Na(+),K(+)-ATPase and ouabain-sensitive H(+),K(+)-ATPase by the cyclic AMP-protein kinase A signal transduction pathway

We investigated the effect of the cyclic AMP-protein kinase A (PKA) signalling pathway on renal Na(+),K(+)-ATPase and ouabain-sensitive H(+),K(+)-ATPase. Male Wistar rats were anaesthetized and catheter was inserted through the femoral artery into the abdominal aorta proximally to the renal arteries for infusion of the investigated substances. Na(+),K(+)-ATPase activity was measured in the presence of Sch 28080 to block ouabain-sensitive H(+),K(+)-ATPase and improve specificity of the assay. Dibutyryl-cyclic AMP (db-cAMP) administered at a dose of 10(-7) mol/kg per min and 10(-6) mol/kg per min increased Na(+),K(+)-ATPase activity in the renal cortex by 34% and 42%, respectively, and decreased it in the renal medulla by 30% and 44%, respectively. db-cAMP infused at 10(-6) mol/kg per min increased the activity of cortical ouabain-sensitive H(+),K(+)-ATPase by 33%, and medullary ouabain-sensitive H(+),K(+)-ATPase by 30%. All the effects of db-cAMP were abolished by a specific inhibitor of protein kinase A, KT 5720. The stimulatory effect on ouabain-sensitive H(+),K(+)-ATPase and on cortical Na(+),K(+)-ATPase was also abolished by brefeldin A which inhibits the insertion of proteins into the plasma membranes, whereas the inhibitory effect on medullary Na(+),K(+)-ATPase was partially attenuated by 17-octadecynoic acid, an inhibitor of cytochrome p450-dependent arachidonate metabolism. We conclude that the cAMP-PKA pathway stimulates Na(+),K(+)-ATPase in the renal cortex as well as ouabain-sensitive H(+),K(+)-ATPase in the cortex and medulla by a mechanism requiring insertion of proteins into the plasma membrane. In contrast, medullary Na(+),K(+)-ATPase is inhibited by cAMP through a mechanism involving cytochrome p450-dependent arachidonate metabolites.     

Chimeric domain analysis of the compatibility between H(+), K(+)-ATPase and Na(+),K(+)-ATPase beta-subunits for the functional expression of gastric H(+),K(+)-ATPase.

Gastric H(+),K(+)-ATPase consists of alpha-subunit with 10 transmembrane domains and beta-subunit with a single transmembrane domain. We constructed cDNAs encoding chimeric beta-subunits between the gastric H(+),K(+)-ATPase and Na(+),K(+)-ATPase beta-subunits and co-transfected them with the H(+),K(+)-ATPase alpha-subunit cDNA in HEK-293 cells. A chimeric beta-subunit that consists of the cytoplasmic plus transmembrane domains of Na(+),K(+)-ATPase beta-subunit and the ectodomain of H(+),K(+)-ATPase beta-subunit assembled with the H(+),K(+)-ATPase alpha-subunit and expressed the K(+)-ATPase activity. Therefore, the whole cytoplasmic and transmembrane domains of H(+),K(+)-ATPase beta-subunit were replaced by those of Na(+),K(+)-ATPase beta-subunit without losing the enzyme activity. However, most parts of the ectodomain of H(+),K(+)-ATPase beta-subunit were not replaced by the corresponding domains of Na(+), K(+)-ATPase beta-subunit. Interestingly, the extracellular segment between Cys(152) and Cys(178), which contains the second disulfide bond, was exchangeable between H(+),K(+)-ATPase and Na(+), K(+)-ATPase, preserving the K(+)-ATPase activity intact. Furthermore, the K(+)-ATPase activity was preserved when the N-terminal first 4 amino acids ((67)DPYT(70)) in the ectodomain of H(+),K(+)-ATPase beta-subunit were replaced by the corresponding amino acids ((63)SDFE(66)) of Na(+),K(+)-ATPase beta-subunit. The ATPase activity was abolished, however, when 4 amino acids ((76)QLKS(79)) in the ectodomain of H(+),K(+)-ATPase beta-subunit were replaced by the counterpart ((72)RVAP(75)) of Na(+),K(+)-ATPase beta-subunit, indicating that this region is the most N-terminal one that discriminates the H(+),K(+)-ATPase beta-subunit from that of Na(+), K(+)-ATPase.     

Spectrophotometric assay of renal ouabain-resistant Na(+)-ATPase and its regulation by leptin and dietary-induced obesity

Apart from Na(+),K(+)-ATPase, a second sodium pump, Na(+)-stimulated, K(+)-independent ATPase (Na(+)-ATPase) is expressed in proximal convoluted tubule of the mammalian kidney. The aim of this study was to develop a method of Na(+)-ATPase assay based on the method previously used by us to measure Na(+),K(+)-ATPase activity. The ATPase activity was assayed as the amount of inorganic phosphate liberated from ATP by isolated microsomal fraction. Na(+)-ATPase activity was calculated as the difference between the activities measured in the presence and in the absence of 50 mM NaCl. Na(+)-ATPase activity was detected in the renal cortex (3.5 +/- 0.2 mumol phosphate/h per mg protein), but not in the renal medulla. Na(+)-ATPase was not inhibited by ouabain or an H(+),K(+)-ATPase inhibitor, Sch 28080, but was almost completely blocked by 2 mM furosemide. Leptin administered intraperitoneally (1 mg/kg) decreased the Na(+),K(+)-ATPase activity in the renal medulla at 0.5 and 1 h by 22.1% and 27.1%, respectively, but had no effect on Na(+)-ATPase in the renal cortex. Chronic hyperleptinemia induced by repeated subcutaneous leptin injections (0.25 mg/kg twice daily for 7 days) increased cortical Na(+),K(+)-ATPase, medullary Na(+),K(+)-ATPase and cortical Na(+)-ATPase by 32.4%, 84.2% and 62.9%, respectively. In rats with dietary-induced obesity, the Na(+),K(+)- ATPase activity was higher in the renal cortex and medulla by 19.7% and 34.3%, respectively, but Na(+)-ATPase was not different from control. These data indicate that both renal Na(+)-dependent ATPases are separately regulated and that up-regulation of Na(+)-ATPase may contribute to Na(+) retention and arterial hypertension induced by chronic hyperleptinemia.     

Leptin decreases renal medullary Na(+), K(+)-ATPase activity through phosphatidylinositol 3-kinase dependent mechanism.

We examined the effect of leptin on renal function and renal Na(+),K(+)-ATPase and ouabain-sensitive H(+),K(+)-ATPase activities in the rat. Leptin was infused under general anaesthesia into the abdominal aorta proximally to the renal arteries. Leptin infused at doses of 1 and 10 microg/kg/min increased urine output by 40% and 140%, respectively. Urinary Na(+) excretion increased in rats receiving leptin at doses of 0.1, 1, and 10 microg/kg/min by 57.6%, 124.2% and 163.6%, respectively. Leptin had no effect on creatinine clearance, potassium excretion and phosphate excretion. Na(+),K(+)-ATPase activity in the renal medulla of rats treated with 1 and 10 microg/kg/min leptin was lower than in control animals by 25.5% and 33.2%, respectively. In contrast, cortical Na(+),K(+)-ATPase as well as either cortical or medullary ouabain-sensitive H(+),K(+)-ATPase activities did not differ between leptin-treated and control animals. The effect of leptin on Na(+),K(+)-ATPase activity was abolished by actin depolymerizing agents, cytochalazin D and latrunculin B, and by phosphatidylinositol 3-kinase (PI3K) inhibitors, wortmannin and LY294002. These results indicate that: 1). natriuretic effect of leptin is mediated, at least in part, by decrease in renal medullary Na(+),K(+)-ATPase activity, 2). inhibition of medullary Na(+),K(+)-ATPase by leptin is mediated by PI3K and requires integrity of actin cytoskeleton.     

Nitric oxide -- superoxide cooperation in the regulation of renal Na(+),K(+)-ATPase

The aim of this study was to investigate whether endogenous superoxide anion is involved in the regulation of renal Na(+),K(+)-ATPase and ouabain-sensitive H(+),K(+)-ATPase activities. The study was performed in male Wistar rats. Compounds modulating superoxide anion concentration were infused under general anaesthesia into the abdominal aorta proximally to the renal arteries. The activity of ATPases was assayed in isolated microsomal fraction. We found that infusion of a superoxide anion-generating mixture, xanthine oxidase (1 mU/min per kg) + hypoxanthine (0.2 mumol/min per kg), increased the medullary Na(+),K(+)-ATPase activity by 49.5% but had no effect on cortical Na(+),K(+)-ATPase and either cortical or medullary ouabain-sensitive H(+),K(+)-ATPase. This effect was reproduced by elevating endogenous superoxide anion with a superoxide dismutase inhibitor, diethylthiocarbamate. In contrast, a superoxide dismutase mimetic, TEMPOL, decreased the medullary Na(+),K(+)-ATPase activity. The inhibitory effect of TEMPOL was abolished by inhibitors of nitric oxide synthase (L-NAME), soluble guanylate cyclase (ODQ) and protein kinase G (KT5823). The stimulatory effect of diethylthiocarbamate was not observed in animals pretreated with a synthetic cGMP analogue, 8-bromo-cGMP. An inhibitor of NAD(P)H oxidase, apocynin (1 mumol/min per kg), decreased the Na(+),K(+)-ATPase activity in the renal medulla and its effect was prevented by L-NAME, ODQ or KT5823. In contrast, a xanthine oxidase inhibitor, oxypurinol, administered at the same dose was without effect. These data suggest that NAD(P)H oxidase-derived superoxide anion increases Na(+),K(+)-ATPase activity in the renal medulla by reducing the availability of NO. Excessive intrarenal generation of superoxide anion may upregulate medullary Na(+),K(+)-ATPase leading to sodium retention and blood pressure elevation.     

Analysis of Na+,K+-ATPase motion and incorporation into the plasma membrane in response to G protein-coupled receptor signals in living cells

Dopamine (DA) increases Na(+),K(+)-ATPase activity in lung alveolar epithelial cells. This effect is associated with an increase in Na(+),K(+)-ATPase molecules within the plasma membrane (). Analysis of Na(+),K(+)-ATPase motion was performed in real-time in alveolar cells stably expressing Na(+),K(+)-ATPase molecules carrying a fluorescent tag (green fluorescent protein) in the alpha-subunit. The data demonstrate a distinct (random walk) pattern of basal movement of Na(+),K(+)-ATPase-containing vesicles in nontreated cells. DA increased the directional movement (by 3.5 fold) of the vesicles and an increase in their velocity (by 25%) that consequently promoted the incorporation of vesicles into the plasma membrane. The movement of Na(+),K(+)-ATPase-containing vesicles and incorporation into the plasma membrane were microtubule dependent, and disruption of this network perturbed vesicle motion toward the plasma membrane and prevented the increase in the Na(+),K(+)-ATPase activity induced by DA. Thus, recruitment of new Na(+),K(+)-ATPase molecules into the plasma membrane appears to be a major mechanism by which dopamine increases total cell Na(+),K(+)-ATPase activity.     

Differential regulation of Na(+),K(+)-ATPase and the Na(+)-coupled glucose transporter in hypertensive rat kidney

Several Na(+) transporters are functionally abnormal in the hypertensive rat. Here, we examined the effects of a high-salt load on renal Na(+),K(+)-ATPase and the sodium-coupled glucose transporter (SGLT1) in Dahl salt-resistant (DR) and salt-sensitive (DS) rats. The protein levels of Na(+),K(+)-ATPase and SGLT1 in the DS rat were the same as those in the DR rat, and were not affected by the high-salt load. In the DS rat, a high-salt load decreased Na(+),K(+)-ATPase activity, and this decrease coincided with a decrease in the apparent Mechaelis constant (K(m)) for ATP, but not with a change of maximum velocity (V(max)). On the contrary, a high-salt load increased SGLT1 activity in the DS rat, which coincided with an increase in the V(max) for alpha-methyl glucopyranoside. The protein level of phosphorylated tyrosine residues in Na(+),K(+)-ATPase was decreased by the high-salt load in the DS rat. The amount of phosphorylated serine was not affected by the high-salt load in DR rats, and could not be detected in DS rats. On the other hand, the amount of phosphorylated serine residues in SGLT1 was increased by the high-salt load. However, the phosphorylated tyrosine was the same for all samples. Therefore, we concluded that the high-salt load changes the protein kinase levels in DS rats, and that the regulation of Na(+),K(+)-ATPase and SGLT1 activity occurs via protein phosphorylation.     

Pseudobranch and gill Na(+), K(+)-ATPase activity in juvenile chinook salmon,Oncorhynchus tshawytscha: developmental changes and effects of growth hormone,cortisol and seawater transfer

The teleost pseudobranch is a gill-like structure often fused to the anterior of the opercular cavity. Pseudobranch cells are mitochondria rich and have high levels of Na(+), K(+)-ATPase activity. In this study, pseudobranch Na(+), K(+)-ATPase activity in juvenile chinook salmon (Oncorhynchus tshawytscha) was compared to gill Na(+), K(+)-ATPase activity, a known marker of parr-smolt transformation, in three experiments. In two stocks of New Zealand chinook salmon, pseudobranch Na(+), K(+)-ATPase activity was found to significantly increase during development. At these times gill Na(+), K(+)-ATPase activity was also elevated. Pseudobranch Na(+), K(+)-ATPase activity did not increase 10 days after transfer from fresh water to 34 ppt seawater, a treatment that resulted in a twofold increase in gill Na(+), K(+)-ATPase activity. Cortisol (50 microg/g) and ovine growth hormone (5 microg/g) implants had no effect on pseudobranch Na(+), K(+)-ATPase activity in underyearling chinook salmon, while gill Na(+), K(+)-ATPase activity was stimulated by each hormone. In yearling chinook salmon, only cortisol stimulated pseudobranch Na(+), K(+)-ATPase activity 14 days post-implantation. It was concluded that the pseudobranch differs from the gill in terms of the regulation of Na(+), K(+)-ATPase activity and a role during adaptation to seawater is likely to be limited.     

Chimeras of X+, K+-ATPases. The M1-M6 region of Na+, K+-ATPase is required for Na+-activated ATPase activity, whereas the M7-M10 region of H+, K+-ATPase is involved in K+ de-occlusion

In this study we reveal regions of Na(+),K(+)-ATPase and H(+),K(+)-ATPase that are involved in cation selectivity. A chimeric enzyme in which transmembrane hairpin M5-M6 of H(+),K(+)-ATPase was replaced by that of Na(+),K(+)-ATPase was phosphorylated in the absence of Na(+) and showed no K(+)-dependent reactions. Next, the part originating from Na(+),K(+)-ATPase was gradually increased in the N-terminal direction. We demonstrate that chimera HN16, containing the transmembrane segments one to six and intermediate loops of Na(+),K(+)-ATPase, harbors the amino acids responsible for Na(+) specificity. Compared with Na(+),K(+)-ATPase, this chimera displayed a similar apparent Na(+) affinity, a lower apparent K(+) affinity, a higher apparent ATP affinity, and a lower apparent vanadate affinity in the ATPase reaction. This indicates that the E(2)K form of this chimera is less stable than that of Na(+),K(+)-ATPase, suggesting that it, like H(+),K(+)-ATPase, de-occludes K(+) ions very rapidly. Comparison of the structures of these chimeras with those of the parent enzymes suggests that the C-terminal 187 amino acids and the beta-subunit are involved in K(+) occlusion. Accordingly, chimera HN16 is not only a chimeric enzyme in structure, but also in function. On one hand it possesses the Na(+)-stimulated ATPase reaction of Na(+),K(+)-ATPase, while on the other hand it has the K(+) occlusion properties of H(+),K(+)-ATPase.     

Simultaneous phosphorylation of Ser11 and Ser18 in the alpha-subunit promotes the recruitment of Na(+),K(+)-ATPase molecules to the plasma membrane

Renal sodium homeostasis is a major determinant of blood pressure and is regulated by several natriuretic and antinatriuretic hormones. These hormones, acting through intracellular second messengers, either activate or inhibit proximal tubule Na(+),K(+)-ATPase. We have shown previously that phorbol ester (PMA) stimulation of endogenous PKC leads to activation of Na(+),K(+)-ATPase in cultured proximal tubule cells (OK cells) expressing the rodent Na(+), K(+)-ATPase alpha-subunit. We have now demonstrated that the treatment with PMA leads to an increased amount of Na(+),K(+)-ATPase molecules in the plasmalemma, which is proportional to the increased enzyme activity. Colchicine, dinitrophenol, and potassium cyanide prevented the PMA-dependent stimulation of activity without affecting the increased level of phosphorylation of the Na(+), K(+)-ATPase alpha-subunit. This suggests that phosphorylation does not directly stimulate Na(+),K(+)-ATPase activity; instead, phosphorylation may be the triggering mechanism for recruitment of Na(+),K(+)-ATPase molecules to the plasma membrane. Transfected cells expressing either an S11A or S18A mutant had the same basal Na(+),K(+)-ATPase activity as cells expressing the wild-type rodent alpha-subunit, but PMA stimulation of Na(+),K(+)-ATPase activity was completely abolished in either mutant. PMA treatment led to phosphorylation of the alpha-subunit by stimulation of PKC-beta, and the extent of this phosphorylation was greatly reduced in the S11A and S18A mutants. These results indicate that both Ser11 and Ser18 of the alpha-subunit are essential for PMA stimulation of Na(+), K(+)-ATPase activity, and that these amino acids are phosphorylated during this process. The results presented here support the hypothesis that PMA regulation of Na(+),K(+)-ATPase is the result of an increased number of Na(+),K(+)-ATPase molecules in the plasma membrane.     

ERK1/2 mediates insulin stimulation of Na(+),K(+)-ATPase by phosphorylation of the alpha-subunit in human skeletal muscle cells

Insulin stimulates Na(+),K(+)-ATPase activity and induces translocation of Na(+),K(+)-ATPase molecules to the plasma membrane in skeletal muscle. We determined the molecular mechanism by which insulin regulates Na(+),K(+)-ATPase in differentiated primary human skeletal muscle cells (HSMCs). Insulin action on Na(+),K(+)-ATPase was dependent on ERK1/2 in HSMCs. Sequence analysis of Na(+),K(+)-ATPase alpha-subunits revealed several potential ERK phosphorylation sites. Insulin increased ouabain-sensitive (86)Rb(+) uptake and [(3)H]ouabain binding in intact cells. Insulin also increased phosphorylation and plasma membrane content of the Na(+),K(+)-ATPase alpha(1)- and alpha(2)-subunits. Insulin-stimulated Na(+),K(+)-ATPase activation, phosphorylation, and translocation of alpha-subunits to the plasma membrane were abolished by 20 microm PD98059, which is an inhibitor of MEK1/2, an upstream kinase of ERK1/2. Furthermore, inhibitors of phosphatidylinositol 3-kinase (100 nm wortmannin) and protein kinase C (10 microm GF109203X) had similar effects. Notably, insulin-stimulated ERK1/2 phosphorylation was abolished by wortmannin and GF109203X in HSMCs. Insulin also stimulated phosphorylation of alpha(1)- and alpha(2)-subunits on Thr-Pro amino acid motifs, which form specific ERK substrates. Furthermore, recombinant ERK1 and -2 kinases were able to phosphorylate alpha-subunit of purified human Na(+),K(+)-ATPase in vitro. In conclusion, insulin stimulates Na(+),K(+)-ATPase activity and translocation to plasma membrane in HSMCs via phosphorylation of the alpha-subunits by ERK1/2 mitogen-activated protein kinase.     

Activation of AMP-activated protein kinase stimulates Na+,K+-ATPase activity in skeletal muscle cells

Contraction stimulates Na(+),K(+)-ATPase and AMP-activated protein kinase (AMPK) activity in skeletal muscle. Whether AMPK activation affects Na(+),K(+)-ATPase activity in skeletal muscle remains to be determined. Short term stimulation of rat L6 myotubes with the AMPK activator 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR), activates AMPK and promotes translocation of the Na(+),K(+)-ATPase α(1)-subunit to the plasma membrane and increases Na(+),K(+)-ATPase activity as assessed by ouabain-sensitive (86)Rb(+) uptake. Cyanide-induced artificial anoxia, as well as a direct AMPK activator (A-769662) also increase AMPK phosphorylation and Na(+),K(+)-ATPase activity. Thus, different stimuli that target AMPK concomitantly increase Na(+),K(+)-ATPase activity. The effect of AICAR on Na(+),K(+)-ATPase in L6 myotubes was attenuated by Compound C, an AMPK inhibitor, as well as siRNA-mediated AMPK silencing. The effects of AICAR on Na(+),K(+)-ATPase were completely abolished in cultured primary mouse muscle cells lacking AMPK α-subunits. AMPK stimulation leads to Na(+),K(+)-ATPase α(1)-subunit dephosphorylation at Ser(18), which may prevent endocytosis of the sodium pump. AICAR stimulation leads to methylation and dephosphorylation of the catalytic subunit of the protein phosphatase (PP) 2A in L6 myotubes. Moreover, AICAR-triggered dephosphorylation of the Na(+),K(+)-ATPase was prevented in L6 myotubes deficient in PP2A-specific protein phosphatase methylesterase-1 (PME-1), indicating a role for the PP2A·PME-1 complex in AMPK-mediated regulation of Na(+),K(+)-ATPase. Thus contrary to the common paradigm, we report AMPK-dependent activation of an energy-consuming ion pumping process. This activation may be a potential mechanism by which exercise and metabolic stress activate the sodium pump in skeletal muscle.     

Failure to up-regulate gill Na+,K+-ATPase alpha-subunit isoform alpha1b may limit seawater tolerance of land-locked Arctic char (Salvelinus alpinus)

Many populations of Arctic char (Salvelinus alpinus) are land-locked, physically separated from the ocean by natural barriers and unable to migrate to sea like anadromous populations. Previous studies which experimentally transferred land-locked Arctic char to seawater report high mortality rates due to osmoregulatory failure and an inability to up-regulate gill Na(+),K(+)-ATPase activity. This study examined the mRNA expression of two recently discovered alpha-subunit isoforms of gill Na(+)K(+)-ATPase (alpha1a and alpha1b) during seawater exposure of land-locked Arctic char. mRNA levels of these gill Na(+),K(+)-ATPasealpha-subunit isoforms were compared to Na(+),K(+)-ATPase activity and protein levels and related to osmoregulatory performance. Land-locked Arctic char were unable to regulate plasma osmolality following seawater exposure. Seawater exposure did not induce an increase in gill Na(+),K(+)-ATPase activity or protein levels. Na(+),K(+)-ATPase isoform alpha1a mRNA quickly decreased upon exposure to seawater, while isoform alpha1b levels were unchanged. These results suggest the inability of land-locked Arctic char to acclimate to seawater is due a failure to up-regulate gill Na(+),K(+)-ATPase activity which may be due to their inability to increase Na(+),K(+)-ATPase alpha1b mRNA expression.     

AS160 associates with the Na+,K+-ATPase and mediates the adenosine monophosphate-stimulated protein kinase-dependent regulation of sodium pump surface expression

The Na(+),K(+)-ATPase is the major active transport protein found in the plasma membranes of most epithelial cell types. The regulation of Na(+),K(+)-ATPase activity involves a variety of mechanisms, including regulated endocytosis and recycling. Our efforts to identify novel Na(+),K(+)-ATPase binding partners revealed a direct association between the Na(+),K(+)-ATPase and AS160, a Rab-GTPase-activating protein. In COS cells, coexpression of AS160 and Na(+),K(+)-ATPase led to the intracellular retention of the sodium pump. We find that AS160 interacts with the large cytoplasmic NP domain of the α-subunit of the Na(+),K(+)-ATPase. Inhibition of the activity of the adenosine monophosphate-stimulated protein kinase (AMPK) in Madin-Darby canine kidney cells through treatment with Compound C induces Na(+),K(+)-ATPase endocytosis. This effect of Compound C is prevented through the short hairpin RNA-mediated knockdown of AS160, demonstrating that AMPK and AS160 participate in a common pathway to modulate the cell surface expression of the Na(+),K(+)-ATPase.     

Clathrin-mediated endocytosis of Na+,K+-ATPase in response to parathyroid hormone requires ERK-dependent phosphorylation of Ser-11 within the alpha1-subunit

Parathyroid hormone (PTH) inhibits Na(+),K(+)-ATPase activity through protein kinase C- (PKC) and extracellular signal-regulated kinase- (ERK) dependent pathways and increases serine phosphorylation of the alpha(1)-subunit. To determine whether specific serine phosphorylation sites within the Na(+),K(+)-ATPase alpha(1)-subunit are involved in the Na(+),K(+)-ATPase responses to PTH, we examined the effect of PTH in opossum kidney cells stably transfected with wild type rat Na(+),K(+)-ATPase alpha(1)-subunit (WT), serine 11 to alanine mutant alpha(1)-subunit (S11A), or serine 18 to alanine mutant alpha(1)-subunit (S18A). PTH increased phosphorylation and endocytosis of the Na(+),K(+)-ATPase alpha(1)-subunit into clathrin-coated vesicles in cells transfected with WT and S18A rat Na(+),K(+)-ATPase alpha(1)-subunits. PTH did not increase the level of phosphorylation or stimulate translocation of Na(+),K(+)-ATPase alpha(1)-subunits into clathrin-coated vesicles in cells transfected with the S11A mutant. PTH inhibited ouabain-sensitive (86)Rb uptake and Na(+),K(+)-ATPase activity (ouabain-sensitive ATP hydrolysis) in WT- and S18A-transfected opossum kidney cells but not in S11A-transfected cells. Pretreatment of the cells with the PKC inhibitors and ERK inhibitor blocked PTH inhibition of (86)Rb uptake, Na(+),K(+)-ATPase activity, alpha(1)-subunit phosphorylation, and endocytosis in WT and S18A cells. Consistent with the notion that ERK phosphorylates Na(+),K(+)-ATPase alpha(1)-subunit, ERK was shown to be capable of causing phosphorylation of Na(+),K(+)-ATPase alpha(1)-subunit immunoprecipitated from WT and S18A but not from S11A-transfected cells. These results suggest that PTH regulates Na(+),K(+)-ATPase by PKC and ERK-dependent alpha(1)-subunit phosphorylation and that the phosphorylation requires the expression of a serine at the 11 position of the Na(+),K(+)-ATPase alpha(1)-subunit.     

Effect of the pyridoindole antioxidant stobadine on the cardiac Na(+),K(+)-ATPase in rats with streptozotocin-induced diabetes

In the present study we examined the effect of dietary supplementation with the pyridoindole antioxidant stobadine on functional properties of the cardiac Na(+),K(+)-ATPase in diabetic rats. Diabetes lasting sixteen weeks which was induced by a single i.v. dose of streptozotocin (55 mg x kg(-1)) was followed by decrease in the enzyme activity. Evaluation of kinetic parameters revealed a statistically significant decrease in the maximum velocity (Vmax) (32% for ATP-activation, 33% for Na(+)-activation), indicating a diabetes-induced diminution of the number of active enzyme molecules in cardiac sarcolemma. The ATP-binding properties of the enzyme were not affected by diabetes as suggested by statistically insignificant changes in the value of Michaelis-Menten constant, K(M (ATP)). On the other hand, the affinity to sodium decreased as suggested by 54% increase in the K(M (Na+)) value. This impairment in the affinity of the Na(+)-binding site together with decreased number of active Na(+),K(+)-ATPase molecules are probably responsible for the deteriorated enzyme function in hearts of diabetic animals. Administration of stobadine to diabetic rats dramatically improved the function of cardiac Na(+),K(+)-ATPase with regard to Na(+)-handling, as documented by statistically significant elevation of Vmax by 66 and 47% decrease in K(M (Na+)). Our data suggest that stobadine may prevent the diabetes-induced deterioration of cardiac Na(+),K(+)-ATPase, thus enabling to preserve its normal function in regulation of intracellular homeostasis of Na(+) and K(+) ions.     

Gill Na(+), K(+)-ATPase in a series of hyper-regulating gammarid amphipods: enzyme characterisation and the effects of salinity acclimation

The enzyme Na(+), K(+)-ATPase was investigated in the gills of selected hyper-regulating gammarid amphipods. Gill Na(+), K(+)-ATPase was characterised with respect to the main cation and co-factor concentrations for the freshwater amphipod Gammarus pulex. The optimum cation and co-factor concentrations for maximal gill Na(+), K(+)-ATPase activity in G. pulex were 100mM Na(+), 15mM K(+), 15mM Mg(2+) and 5mM ATP, at pH 7.2. The effects of salinity acclimation on gill Na(+), K(+)-ATPase activity and haemolymph sodium concentrations was investigated in selected gammarid amphipods from different salinity environments. Maximal enzyme activity occurred in all gammarids when acclimated to the most dilute media. This maximal activity coincided with the largest sodium gradient between the haemolymph and the external media. As the haemolymph/medium sodium gradient decreased, a concomitant reduction in gill Na(+), K(+)-ATPase activity occurred. This implicates the involvement of gill Na(+), K(+)-ATPase in the active uptake of sodium from dilute media in hyper-regulating gammarids.     

Diet deficient in alpha-linolenic acid alters fatty acid composition and enzymatic properties of Na+, K+-ATPase isoenzymes of brain membranes in the adult rat

The effects of dietary (n-6)/(n-3) polyunsaturated fatty acid balance on fatty acid composition, ouabain inhibition, and Na(+) dependence of Na(+), K(+)-ATPase isoenzymes of whole brain membranes were studied in 60-day-old rats fed over two generations a diet either devoid of alpha-linolenic acid [18:3(n-3)] (sunflower oil diet) or rich in 18:3(n-3) (soybean oil diet). In the brain membranes, the sunflower oil diet led to a dramatic decrease in docosahexaenoic acid [22:6(n-3)] membrane content. The activities of Na(+), K(+)-ATPase isoenzymes were discriminated on the basis of their differential affinities for ouabain and their sensitivity to sodium concentration. The ouabain titration curve of Na(+), K(+)-ATPase activity displayed three inhibitory processes with markedly different affinity [i.e., low (alpha1), high (alpha2), and very high (alpha3)] for brain membranes of rats fed the sunflower oil diet, whereas the brain membranes of rats fed the soybean oil diet exhibited only two inhibitory processes, low (alpha1) and high (alpha2' = alpha2 + alpha3). Regardless of the diet, on the basis of the Na(+) dependence of Na(+), K(+)-ATPase activity, three isoenzymes were found: alpha1 form displaying an affinity 1.5- to 2-fold higher that of than alpha2 and 3-fold higher that of alpha3. In rats fed the sunflower oil diet, alpha2 isoenzyme exhibited higher affinity for sodium (Ka = 8.8 mmol/L) than that of rats fed the soybean oil diet (Ka = 11.7 mmol/L). These results suggest that the membrane lipid environment modulates the functional properties of Na(+), K(+)-ATPase isoenzymes of high ouabain affinity (alpha2).     

Folic Acid Prevents Behavioral Impairment and Na+,K+-ATPase Inhibition Caused by Neonatal Hypoxia–Ischemia

Folic acid plays an important role in neuroplasticity and acts as a neuroprotective agent, as observed in experimental brain ischemia studies. The aim of this study was to investigate the effects of folic acid on locomotor activity, aversive memory and Na(+),K(+)-ATPase activity in the frontal cortex and striatum in animals subjected to neonatal hypoxia-ischemia (HI). Wistar rats of both sexes at postnatal day 7 underwent HI procedure and were treated with intraperitoneal injections of folic acid (0.011 μmol/g body weight) once a day, until the 30th postnatal day. Starting on the day after, behavioral assessment was run in the open field and in the inhibitory avoidance task. Animals were sacrificed by decapitation 24 h after testing and striatum and frontal cortex were dissected out for Na(+),K(+)-ATPase activity analysis. Results show anxiogenic effect in the open field and an impairment of aversive memory in the inhibitory avoidance test in HI rats; folic acid treatment prevented both behavioral effects. A decreased Na(+),K(+)-ATPase activity in striatum, both ipsilateral and contralateral to ischemia, was identified after HI; a total recovery was observed in animals treated with folic acid. A partial recovery of Na(+),K(+)-ATPase activity was yet seen in frontal cortex of HI animals receiving folic acid supplementation. Presented results support that folic acid treatment prevents memory deficit and anxiety-like behavior, as well as prevents Na(+),K(+)-ATPase inhibition in the striatum and frontal cortex caused by neonatal hypoxia-ischemia.