Assembly of the alpha-subunit of Torpedo californica Na+/K(+)-ATPase with its pre-existing beta-subunit in Xenopus oocytes

The alpha- and beta-subunits of Torpedo californica Na+/K(+)-ATPase were expressed in turn in single oocytes by alternately microinjecting the specific mRNAs for the alpha- and beta-subunits. The mRNA first injected was degraded prior to the injection of the second mRNA by injecting the antisense oligonucleotide specific for the first mRNA. The pre-existing beta-subunit, which had been synthesized by injecting mRNA for the beta-subunit, could assemble with the alpha-subunit expressed later in the single oocytes and the resulting alpha beta complex acquired both ouabain-binding and Na+/K(+)-ATPase activities. On the other hand, formation of the alpha beta complex was not detected when the alpha-subunit was expressed first, followed by the beta-subunit. These data suggest that the beta-subunit acts as a receptor or a stabilizer for the alpha-subunit upon the biogenesis of Na+/K(+)-ATPase.     

Preferential reduction of Na+/K+ ATPase alpha3 by 17beta-estradiol influences contraction frequency in rat uteri

One beta1 and two alpha (alpha1 and alpha3) isoforms of Na+/K+-ATPase exist in rat uteri. Previous immunocytochemistry studies have suggested that the alpha3 isoform may be involved in calcium regulation indirectly. Estrogens are known to both modulate Na+/K+-ATPase activities in non-uterine tissues and suppress spontaneous uterine contractions in rats. Thus the purpose of this study was to examine the correlation between estrogens-modulated uterine contraction and the expression of Na+/K+-ATPase alpha3 isoform in rats. After 1-, 2-, and 4- day treatments with 17beta-estradiol (E2, 5 microg/ml/kg, s.c., daily), the diameter of uterine horn was measured. The contraction force of uterine strips was measured by standard muscle bath apparatus. The protein abundance and enzyme activity of Na+/K+-ATPase in rat uteri were measured by Western blot analysis and ATPase assay, respectively. One day of E2 decreased both contraction frequency and alpha3-protein expression without the change in uterine diameter, enzyme activity or other isoforms. Two days of E2 reduced contraction frequency, the enzyme activity, as well as alpha3- and beta1- protein abundance but increased alpha1-protein and uterine diameter. Four days of E2 elicited similar effects as two days of E2, but did not affect alpha1-protein abundance. In conclusion, E2 elicits differential effects on isoform expression. After 1-day treatment with 17beta-estradiol, the decrease in the expression of alpha3 and beta1 without a change in Na+/K+-ATPase activity suggests that some isoform other than beta1 exist in rat uteri. The positive correlation between the reduction of alpha3-and the decrease of contraction frequency suggests the involvement of alpha3 isoform in uterine oscillation.     

Expression of multiple Na+,K(+)-ATPase genes reveals a gradient of isoforms along the nonpigmented ciliary epithelium: functional implications in aqueous humor secretion.

The Na+,K(+)-ATPase alpha 1, alpha 2, and alpha 3 subunit isoforms have been shown to be differentially expressed in the nonpigmented (NPE) and pigmented (PE) cells of the ocular ciliary epithelium (CE) (Martin-Vasallo et al., J. Cell. Physiol., 141:243-252, 1989; Ghosh et al., J. Biol. Chem., 265:2935-2940, 1990). In this study we analyzed and compared the pattern of expression of the multiple Na+,K(+)-ATPase alpha (alpha 1, alpha 2, alpha 3) subunit genes with the pattern of expression of the Na+,K(+)-ATPase beta (beta 1, beta 2) subunit genes along the bovine CE. We have selected three regions in the CE, referred to as 1) the anterior region of the pars plicata, near the iris; 2) the middle region of the pars plicata; and 3) the posterior region of the pars plana, near the ora serrata. Using isoform-specific cDNA probes and antibodies for the Na+,K(+)-ATPase alpha 1, alpha 2, alpha 3, beta 1, and beta 2 subunits on Northern and Western blot analysis, we found that mRNA and polypeptides are expressed in all three CE regions with different abundance. The pattern of expression of alpha and beta isoforms detected along the NPE cell layers suggests a gradient of alpha 1, alpha 2, alpha 3, beta 1, and beta 2 mRNAs and polypeptides that correlates with decreasing Na+,K(+)-ATPase activity from the most anterior region at the pars plicata towards the posterior region at the ora serrata. We also found marked differences in the pattern of immunolocalization of Na+,K(+)-ATPase alpha 1, alpha 2, alpha 3, beta 1, and beta 2 subunit isoforms in different regions of the CE. In the anterior region, NPE cells stained intensely at the basal lateral membrane with specific monoclonal and polyclonal antibodies for each of the alpha (alpha 1, alpha 2, alpha 3) and beta (beta 1, beta 2) Na,K-ATPase isoforms. In the middle and posterior regions of the CE, NPE cells showed lower or absent levels of staining with alpha 1, alpha 2, alpha 3, and beta 1 antibodies, although staining with beta 2 was abundant. In contrast, PE cells throughout the CE were stained at the basal lateral membrane by antibodies to alpha 1 and beta 1, while no staining signals were detected with the rest of the antibodies (i.e. alpha 2, alpha 3, and beta 2). Our results support the conclusion that the three alpha and two beta isoforms of the Na+,K(+)-ATPase are differentially expressed in the two cell layers that make up the CE.(ABSTRACT TRUNCATED AT 250 WORDS)     

Na+/K(+)-ATPase regulation by neurotransmitters.

A long period of experimental work has led to the conclusion that Na+/K(+)-ATPase is the enzymatic version of the Na+/K+ pump. This enzymatic system is in charge of various important cell functions. Among them cationic equilibrium and recovering of resting membrane potential in neurons is relevant. A tetrameric ensemble of peptides conform the system known as alpha and beta subunits. The alpha subunit is subdivided in alpha 1, alpha 2 and alpha 3, according to different location and properties. Regulatory factors intrinsic to the Na+/K(+)-ATPase system are: ATP, Na+ and Mg2+ concentrations inside the cell, and K+ outside. The enzyme activity is also regulated by extrinsic factors like some hormones (insulin and thyroxine). Induction of gene expression or post-translational modifications of the preexisting pool of the enzyme are the basic mechanisms of regulation proposed. Other extrinsic factors that seem to regulate the enzyme activity are some neurotransmitters. Among them the most extensively studied are catecholamines, mainly norepinephrine (NE) and lately serotonin (5-HT). The mechanism suggested for NE activation of the enzyme seems to involve specific receptors or a non-specific chelating action related to the catechol group that would relieve the inhibition by divalent cations. Another possibility is that NE removes an endogenous inhibitory factor present in the cytoplasm. The Na+/K(+)-ATPase is activated also by 5-HT. In vivo pharmacological and nutriological manipulations of brain 5-HT are accompanied by parallel responses of Na+/K(+)-ATPase activity. Serotonin agonists do activate the enzyme and antagonists neutralize the activation. In vitro there is a different dose dependent activation, according to the brain region. The mechanism involved seems to implicate a specific receptor system. Serotonin-Na+/K(+)-ATPase interaction in the rat brain is probably of functional relevance because it disappears in amygdaloid kindling. Also it seems to influence the ionic regulation of the pigment transport mechanism in crayfish photoreceptors. In relation to other neurotransmitters, a weak response to histamine was observed with acetylcholine, GABA and glutamic acid, the results were negative.     

The effect of micromolar Ca2+ on the activities of the different Na+/K(+)-ATPase isozymes in the rat myometrium.

In the present work we show the existence of two Na+/K(+)-ATPase isozymes in rat myometrial microsomes and suggest that they have different Ca2+ sensitivities. The catalytic subunits (alpha 1, alpha 2) of Na+/K(+)-ATPase were labelled by fluorescein-isothiocyanate and separated by SDS gel electrophoresis. The two isozyme Ca2(+)-sensitivities were studied by comparing the kinetics of Ca2+, strophantidin, ouabain and N-ethylmaleimide inhibitions. Our results indicate that the activity of the high ouabain-sensitive part (alpha 2 type) of Na+/K(+)-ATPase enzyme could only be inhibited by micromolar Ca2+. Furthermore, treatment of the microsomal preparation with 1mM N-ethylmaleimide selectively inactivated the high Ca2+ sensitive isoform of myometrial Na+/K(+)-ATPase.     

Insulin induces translocation of the alpha 2 and beta 1 subunits of the Na+/K(+)-ATPase from intracellular compartments to the plasma membrane in mammalian skeletal muscle.

Unlike glucose transport, where translocation of the insulin-responsive glucose transporter (GLUT4) from an intracellular compartment to the plasma membrane is the principal mechanism underlying insulin stimulation, no consensus exists presently for the mechanism by which insulin activates the Na+/K(+)-ATPase. We have investigated (i) the subunit isoforms expressed and (ii) the effect of insulin on the subcellular distribution of the alpha beta isoforms of the Na+/K(+)-ATPase in plasma membranes (PM) and internal membranes (IM) from rat skeletal muscle. Western blot analysis, using isoform-specific antibodies to the various subunits of the Na+/K(+)-ATPase, revealed that skeletal muscle PM contains the alpha 1 and alpha 2 catalytic subunits and the beta 1 and beta 2 subunits of the Na+ pump. Skeletal muscle IM were enriched in alpha 2, beta 1, and beta 2; alpha 1 was barely detectable in this fraction. After insulin treatment, alpha 2 content in the PM increased, with a parallel decrease in its abundance in the IM pool; insulin did not have any effect on alpha 1 isoform amount or subcellular distribution. The beta 1 subunit, but not beta 2, was also elevated in the PM after insulin treatment, but this increase originated from a sucrose gradient fraction different from that of the alpha 2 subunit. Our findings suggest that insulin induces an isoform-specific translocation of Na+ pump subunits from different intracellular sources to the PM and that the hormone-responsive enzyme in rat skeletal muscle is an alpha 2:beta 1 dimer.     

Effect of streptozotocin-induced diabetes on rat liver Na+/K+-ATPase.

Na+/K+-ATPase during diabetes may be regulated by synthesis of its alpha and beta subunits and by changes in membrane fluidity and lipid composition. As these mechanisms were unknown in liver, we studied in rats the effect of streptozotocin-induced diabetes on liver Na+/K+-ATPase. We then evaluated whether fish oil treatment prevented the diabetes-induced changes. Diabetes mellitus induced an increased Na+/K+-ATPase activity and an enhanced expression of the beta1 subunit; there was no change in the amount of the alpha1 and beta3 isoenzymes. Biphasic ouabain inhibition curves were obtained for diabetic groups indicating the presence of low and high affinity sites. No alpha2 and alpha3 isoenzymes could be detected. Diabetes mellitus led to a decrease in membrane fluidity and a change in membrane lipid composition. The diabetes-induced changes are not prevented by fish oil treatment. The results suggest that the increase of Na+/K+-ATPase activity can be associated with the enhanced expression of the beta1 subunit in the diabetic state, but cannot be attributed to changes in membrane fluidity as typically this enzyme will increase in response to an enhancement of membrane fluidity. The presence of a high-affinity site for ouabain (IC50 = 10-7 M) could be explained by the presence of (alphabeta)2 diprotomeric structure of Na+/K+-ATPase or an as yet unknown alpha subunit isoform that may exist in diabetes mellitus. These stimulations might be related, in part, to the modification of fatty acid content during diabetes.     

Assembly of a hybrid from the alpha subunit of Na+/K(+)-ATPase and the beta subunit of H+/K(+)-ATPase.

Messenger RNA for the alpha subunit of Torpedo californica Na+/K(+)-ATPase was injected into Xenopus oocytes together with that of the beta subunit of rabbit H+/K(+)-ATPase. The Na+/K(+)-ATPase alpha subunit was assembled in the microsomal membranes with the H+/K(+)-ATPase beta subunit, and became resistant to trypsin. These results suggest that the H+/K(+)-ATPase beta subunit facilitates the stable assembly of the Na+/K(+)-ATPase alpha subunit in microsomes.     

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.     

Aldosterone-mediated regulation of Na+, K(+)-ATPase gene expression in adult and neonatal rat cardiocytes.

By altering the Na+/K+ electrochemical gradient, Na+,K(+)-ATPase activity profoundly influences cardiac cell excitability and contractility. The recent finding of mineralocorticoid hormone receptors in the heart implies that Na+,K(+)-ATPase gene expression, and hence cardiac function, is regulated by aldosterone, a corticosteroid hormone associated with certain forms of hypertension and classically involved in regulating Na+,K(+)-ATPase gene expression and transepithelial Na+ transport in tissues such as the kidney. The regulation by aldosterone of the major cardiac Na+,K(+)-ATPase isoform genes, alpha-1 and beta-1, were studied in adult and neonatal rat ventricular cardiocytes grown in defined serum-free media. In both cell types, aldosterone-induced a rapid and sustained 3-fold induction in alpha-1 mRNA accumulation within 6 h. beta-1 mRNA was similarly induced. alpha-1 mRNA induction occurred over the physiological range with an EC50 of 1-2 nM, consistent with binding of aldosterone to the high affinity mineralocorticoid hormone receptor. In adult cardiocytes, this was associated with a 36% increase in alpha subunit protein accumulation and an increase in Na(+)-K(+)-ATPase transport activity. Aldosterone did not alter the 3-h half-life of alpha-1 mRNA, indicating an induction of alpha-1 mRNA synthesis. Aldosterone-dependent alpha-1 mRNA accumulation was not blocked by the protein synthesis inhibitor cycloheximide, whereas amiloride inhibited both an aldosterone-dependent increase in intracellular Na+ [Na+]i) and alpha-1 mRNA accumulation. This demonstrates that aldosterone directly stimulates Na+,K(+)-ATPase alpha-1 subunit mRNA synthesis and protein accumulation in cardiac cells throughout development and suggests that the heart is a mineralocorticoid-responsive organ. An early increase in [Na+]i may be a proximal event in the mediation of the hormone effect.     

Activity-Dependent Regulation of Na+,K+-ATPase α Isoform mRNA Expression In Vivo

To investigate the functional role of the different Na+, K(+)-ATPase alpha (catalytic) subunit isoforms in neuronal cells, we used quantitative in situ hybridization with riboprobes specific for alpha 1, alpha 2, and alpha 3 isoforms to measure the level of alpha isoform-specific expression in the neuroendocrine cells of the supraoptic (SON) and paraventricular (PVN) nuclei of rat hypothalamus. A prolonged increase in electrical activity of these cells, achieved by 5 days of salt treatment, increased the amount of alpha 1 isoform mRNA in the SON and PVN by 50%. Levels of alpha 1 mRNA in other brain regions and levels of alpha 2 and alpha 3 mRNAs were not affected by salt treatment. We conclude that the alpha 1 isoform Na+, K(+)-ATPase may be specifically adapted to pump out Na+, which enters the cells through voltage-gated channels during neuronal depolarization.     

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.     

Blocking of Na+/K+ transport by the MgPO4 complex analogue Co(NH3)4PO4 leaves the Na+/Na(+)-exchange reaction of the sodium pump unaltered and shifts its high-affinity ATP-binding site to a Na(+)-like form.

Inactivation of Na+/K(+)-ATPase activity by the MgPO4 complex analogue Co(NH3)4PO4 leads, in everted red blood cell vesicles, to the parallel inactivation of 22Na+/K+ flux and 86Rb/Rb+ exchange, but leaves the 22Na+/Na(+)-exchange activity and the uncoupled ATP-supported 22Na+ transport unaffected. Furthermore, inactivation of purified Na+/K(+)-ATPase by Co(NH3)4PO4 leads to a parallel decrease of the capacity of the [3H]ouabain receptor site, when binding was studied by the Mg2+/Pi-supported pathway (ouabain-enzyme complex II) but the capacity of the ouabain receptor site was unaltered, when the Na+/Mg2+/ATP-supported pathway (ouabain-enzyme complex I) was used. No change in the dissociation constants of either ouabain receptor complex was observed following inactivation of Na+/K(+)-ATPase. When eosin was used as a marker for the high-affinity ATP-binding site of the E1 conformation, formation of stable E'2.Co(NH3)4PO4 complex led to a shift in the high-affinity ATP-binding site towards the sodium form. This led to an increase in the dissociation constant of the enzyme complex with K+, from 1.4 mM with the unmodified enzyme to 280 mM with the Co(NH3)4PO4-inactivated enzyme. It was concluded, that the effects of Co(NH3)4PO4 on the partial activities of the sodium pump are difficult to reconcile with an alpha, beta-protomeric enzyme working according the Albers-Post scheme. The data are consistent with an alpha 2, beta 2 diprotomeric enzyme of interacting catalytic subunits working with a modified version of the Albers-Post model.     

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.     

Sodium transport systems in human chondrocytes. I. Morphological and functional expression of the Na+,K(+)-ATPase alpha and beta subunit isoforms in healthy and arthritic chondrocytes.

The chondrocyte is the cell responsible for the maintenance of the articular cartilage matrix. The negative charges of proteoglycans of the matrix draw cations, principally Na+, into the matrix to balance the negative charge distribution. The Na+,K(+)-ATPase is the plasma membrane enzyme that maintains the intracellular Na+ and K+ concentrations. The enzyme is composed of an alpha and a beta subunit, so far, 4 alpha and 3 beta isoforms have been identified in mammals. Chondrocytes are sensitive to their ionic and osmotic environment and are capable of adaptive responses to ionic environmental perturbations particularly changes to extracellular [Na+]. In this article we show that human fetal and adult chondrocytes express three alpha (alpha 1, alpha 2 and the neural form of alpha 3) and the three beta isoforms (beta 1, beta 2 and beta 3) of the Na+,K(+)-ATPase. The presence of multiple Na+,K(+)-ATPase isoforms in the plasma membrane of chondrocytes suggests a variety of kinetic properties that reflects a cartilage specific and very fine specialization in order to maintain the Na+/K+ gradients. Changes in the ionic and osmotic environment of chondrocytes occur in osteoarthritis and rheumatoid arthritis as result of tissue hydration and proteoglycan loss leading to a fall in tissue Na+ and K+ content. Although the expression levels and cellular distribution of the proteins tested do not vary, we detect changes in p-nitrophenylphosphatase activity "in situ" between control and pathological samples. This change in the sodium pump enzymatic activity suggests that the chondrocyte responds to these cationic environmental changes with a variation of the active isozyme types present in the plasma membrane.     

Vacuolar-type H(+)-ATPase and Na+, K(+)-ATPase expression in gills of Atlantic salmon (Salmo salar) during isolated and combined exposure to hyperoxia and hypercapnia in fresh water

Changes in branchial vacuolar-type H(+)-ATPase B-subunit mRNA and Na+, K(+)-ATPase alpha- and beta-subunit mRNA and ATP hydrolytic activity were examined in smolting Atlantic salmon exposed to hyperoxic and/or hypercapnic fresh water. Pre-smolts, smolts, and post-smolts were exposed for 1 to 4 days to hyperoxia (100% O2) and/or hypercapnia (2% CO2). Exposure to hypercapnic water for 4 days consistently decreased gill vacuolar-type H(+)-ATPase B-subunit mRNA levels. Salmon exposed to hyperoxia had either decreased or unchanged levels of gill B-subunit mRNA. Combined hyperoxia + hypercapnia decreased B-subunit mRNA levels, although not to the same degree as hypercapnic treatment alone. Hyperoxia generally increased Na+, K(+)-ATPase alpha- and beta-subunit mRNA levels, whereas hypercapnia reduced mRNA levels in presmolts (beta) and smolts (alpha and beta). Despite these changes in mRNA levels, whole tissue Na+, K(+)-ATPase activity was generally unaffected by the experimental treatments. We suggest that the reduced expression of branchial vacuolar-type H(+)-ATPase B-subunit mRNA observed during internal hypercapnic acidosis may lead to reduction of functional V-type H(+)-ATPase abundance as a compensatory response in order to minimise intracellular HCO3- formation in epithelial cells.     

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.