Radical-induced inactivation of kidney Na+,K(+)-ATPase: sensitivity to membrane lipid peroxidation and the protective effect of vitamin E

The Na+,K(+)-ATPase is a membrane-bound, sulfhydryl-containing protein whose activity is critical to maintenance of cell viability. The susceptibility of the enzyme to radical-induced membrane lipid peroxidation was determined following incorporation of a purified Na+,K(+)-ATPase into soybean phosphatidylcholine liposomes. Treatment of liposomes with Fenton's reagent (Fe2+/H2O2) resulted in malondialdehyde formation and total loss of Na+,K(+)-ATPase activity. At 150 microM Fe2+/75 microM H2O2, vitamin E (5 mol%) totally prevented lipid peroxidation but not the loss of enzyme activity. Lipid peroxidation initiated by 25 microM Fe2+/12.5 microM H2O2 led to a loss of Na+,K(+)-ATPase activity, however, vitamin E (1.2 mol%) prevented both malondialdehyde formation and loss of enzyme activity. In the absence of liposomes, there was complete loss of Na+,K(+)-ATPase activity in the presence of 150 microM Fe2+/75 microM H2O2, but little effect by 25 microM Fe2+/12.5 microM H2O2. The activity of the enzyme was also highly sensitive to radicals generated by the reaction of Fe2+ with cumene hydroperoxide, t-butylhydroperoxide, and linoleic acid hydroperoxide. Lipid peroxidation initiated by 150 microM Fe2+/150 microM Fe3+, an oxidant which may be generated by the Fenton's reaction, inactivated the enzyme. In this system, inhibition of malondialdehyde formation by vitamin E prevented loss of Na+,K(+)-ATPase activity. These data demonstrate the susceptibility of the Na+,K(+)-ATPase to radicals produced during lipid peroxidation and indicate that the ability of vitamin E to prevent loss of enzyme activity is highly dependent upon both the nature and the concentration of the initiating and propagating radical species.     

羟基自由基对兔脑微粒体膜脂及膜蛋白的损伤

本文研究了过氧化氢与亚铁离子体系产生的羟基自由基对兔脑微粒体脂质过氧化作用及对膜上(Na~++K~+)-ATP酶活性的影响.结果表明,羟基自由基导致兔脑微粒体脂质过氧化,增加丙二醛的含量.羟基自由基还使微粒体膜巯基数下降,(Na~++K~+)-ATP酶活力受到抑制.阿魏酸钠对抑制微粒体脂质过氧化及对膜巯基和(Na~++K~+)-ATP酶均有保护作用.自旋捕集实验结果进一步证明药物对羟基自由基的猝灭作用.     

Mechanical stretching of alveolar epithelial cells increases Na(+)-K(+)-ATPase activity.

Alveolar epithelial cells effect edema clearance by transporting Na(+) and liquid out of the air spaces. Active Na(+) transport by the basolaterally located Na(+)-K(+)-ATPase is an important contributor to lung edema clearance. Because alveoli undergo cyclic stretch in vivo, we investigated the role of cyclic stretch in the regulation of Na(+)-K(+)-ATPase activity in alveolar epithelial cells. Using the Flexercell Strain Unit, we exposed a cell line of murine lung epithelial cells (MLE-12) to cyclic stretch (30 cycles/min). After 15 min of stretch (10% mean strain), there was no change in Na(+)-K(+)-ATPase activity, as assessed by (86)Rb(+) uptake. By 30 min and after 60 min, Na(+)-K(+)-ATPase activity was significantly increased. When cells were treated with amiloride to block amiloride-sensitive Na(+) entry into cells or when cells were treated with gadolinium to block stretch-activated, nonselective cation channels, there was no stimulation of Na(+)-K(+)-ATPase activity by cyclic stretch. Conversely, cells exposed to Nystatin, which increases Na(+) entry into cells, demonstrated increased Na(+)-K(+)-ATPase activity. The changes in Na(+)-K(+)-ATPase activity were paralleled by increased Na(+)-K(+)-ATPase protein in the basolateral membrane of MLE-12 cells. Thus, in MLE-12 cells, short-term cyclic stretch stimulates Na(+)-K(+)-ATPase activity, most likely by increasing intracellular Na(+) and by recruitment of Na(+)-K(+)-ATPase subunits from intracellular pools to the basolateral membrane.     

Upregulation of apical NHE3 in renal OK cells overexpressing the rodent alpha(1)-subunit of the Na(+) pump

Vectorial Na(+) reabsorption across the proximal tubule is mediated by apical entry of Na(+), primarily via Na(+)/H(+) exchanger isoform 3 (NHE3), and basolateral extrusion via the Na(+) pump (Na(+)-K(+)-ATPase). We hypothesized that regulation of Na(+) reabsorption should involve not only the activity of the basolateral Na(+)-K(+)-ATPase, but also the apical NHE3, in a concerted manner. To generate a cell line that overexpresses Na(+)-K(+)-ATPase, opossum kidney (OK) cells were transfected with the rodent Na(+)-K(+)-ATPase alpha(1)-subunit (pCMV ouabain vector), and native cells were used as a control. The existence of distinct functional classes of Na(+)-K(+)-ATPase in wild-type and transfected cells was confirmed by the inhibition profile of Na(+)-K(+)-ATPase activity by ouabain. In contrast to wild-type cells, transfected cells exhibited two IC(50) values for ouabain: the first value was similar to the IC(50) of control cells, and the second value was 2 log units greater than the first, consistent with the presence of rat and opossum alpha(1)-isozymes. It is shown that transfection of OK cells with Na(+)-K(+)-ATPase increased Na(+)-K(+)-ATPase and NHE3 activities. This was associated with overexpression of the Na(+)-K(+)-ATPase alpha(1)-subunit and NHE3 in transfected OK cells. The abundance of the Na(+)-K(+)-ATPase beta(1)-subunit was slightly lower in transfected OK cells. In conclusion, the increase in expression and function of Na(+)-K(+)-ATPase in cells transfected with the rodent Na(+) pump alpha(1)-subunit cDNA is expected to stimulate apical Na(+) influx into the cells, thereby accounting for the observed stimulation of the apical NHE3 activity.     

Invited review: lung edema clearance: role of Na(+)-K(+)-ATPase.

Acute hypoxemic respiratory failure is a consequence of edema accumulation due to elevation of pulmonary capillary pressures and/or increases in permeability of the alveolocapillary barrier. It has been recognized that lung edema clearance is distinct from edema accumulation and is largely effected by active Na(+) transport out of the alveoli rather than reversal of the Starling forces, which control liquid flux from the pulmonary circulation into the alveolus. The alveolar epithelial Na(+)-K(+)-ATPase has an important role in regulating cell integrity and homeostasis. In the last 15 yr, Na(+)-K(+)-ATPase has been localized to the alveolar epithelium and its contribution to lung edema clearance has been appreciated. The importance of the alveolar epithelial Na(+)-K(+)-ATPase function is reflected in the changes in the lung's ability to clear edema when the Na(+)-K(+)-ATPase is inhibited or increased. An important focus of the ongoing research is the study of the mechanisms of Na(+)-K(+)-ATPase regulation in the alveolar epithelium during lung injury and how to accelerate lung edema clearance by modulating Na(+)-K(+)-ATPase activity.     

Age-associated differential expression of Na(+)-K(+)-ATPase subunit isoforms in skeletal muscles of F-344/BN rats.

Skeletal muscle expresses multiple isoforms of the Na(+)-K(+)-ATPase. Their expression has been shown to be differentially regulated under pathophysiological conditions. In addition, previous studies suggest possible age-dependent alterations in Na(+)-K(+) pump function. The present study tests the hypothesis that advancing age is associated with altered Na(+)-K(+)-ATPase enzyme activity and isoform-specific changes in expression of the enzyme subunits. Red and white gastrocnemius (Gast) as well as soleus muscles of male Fischer 344/Brown Norway (F-344/BN) rats at 6, 18, and 30 mo of age were examined. Na(+)-K(+)-ATPase activity, measured by K(+)-stimulated 3-O-methylfluorescein phosphatase activity, increased by approximately 50% in a mixed Gast homogenate from 30-mo-old compared with 6- and 18-mo-old rats. Advancing age was associated with markedly increased alpha(1)- and beta(1)-subunit, and decreased alpha(2)- and beta(2)-subunit in red and white Gast. In soleus, there were similar changes in expression of alpha(1)- and alpha(2)-subunits, but levels of beta(1)-subunit were unchanged. Functional Na(+)-K(+)-ATPase units, measured by [(3)H]ouabain binding, undergo muscle-type specific changes. In red Gast, high-affinity ouabain-binding sites, which are a measure of alpha(2)-isozyme, increased in 30-mo-old rats despite decreased levels of alpha(2)-subunit. In white Gast, by contrast, decreased levels of alpha(2)-subunit were accompanied by decreased high-affinity ouabain-binding sites. Finally, patterns of expression of the four myosin heavy chain (MHC) isoforms (type I, IIA, IIX, and IIB) in these muscles were similar in the three age groups examined. We conclude that, in the skeletal muscles of F-344/BN rats, advancing age is associated with muscle type-specific alterations in Na(+)-K(+)-ATPase activity and patterns of expression of alpha- and beta-subunit isoforms. These changes apparently occurred without obvious shift in muscle fiber types, since expression of MHC isoforms remained unchanged. Some of the alterations occurred between middle-age (18 mo) and senescence (30 mo), and, therefore, may be attributed to aging of skeletal muscle.     

大鼠单根近端肾小管Na+-K+-ATPase活性测定方法的改进

本研究旨在对Doucet等报道的定量检测大鼠单根近端肾小管Na^＋-K^＋-ATPase活性方法进行改进。取经过Ⅱ型胶原酶消化的大鼠肾脏皮质组织,在体视显微镜下手工分离单根近端肾小管,并测量其长度,经低渗和冻融处理后与[γ-^32P]ATP共同孵育,液闪法检测从[γ-^32P]ATP解离出的^32Pi,采用修正后的公式计算Na^＋-K^＋-ATPase活性。改良法与Doucet等的方法比较,测定单根近端肾小管Na^＋-K^＋-ATPase活性无显著性差异（P〉0．05）。改进后的方法节省试剂,操作简便、省时。     

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.     

Effect of lipid peroxidation on Na+,K(+)-ATPase, 5'-nucleotidase and CNPase in mouse brain myelin

In the presence of H2O2, solutions of Fe2+ were applied to brain homogenate and isolated myelin from adult SWV control mice and the shiverer dysmyelinating mutant mouse as a source of a reactive oxygen species (Fenton reaction). Under these conditions, lipid peroxidation was initiated and measured as thiobarbituric acid-reactive oxidation products (TBAR). This was accompanied by 85% inhibition of myelin-associated Na+,K(+)-ATPase and 25% inhibition of 5'-nucleotidase. In contrast, CNPase activity was not altered. Studies on the shiverer mutant brain revealed that in spite of hypomyelination and prevalence of premature, myelin-like membranes in the homogenate, the myelin-related enzymes reacted as normal enzymes to peroxidation. Differences in the resistance of Na+,K(+)-ATPase to peroxidation in the brain homogenate and myelin suggest that the myelin enzyme is extremely sensitive to reactive oxygen toxicity.     

Role of inwardly rectifying K(+) channels in K(+)-induced cerebral vasodilatation in vivo

We tested whether activation of inwardly rectifying K(+) (Kir) channels, Na(+)-K(+)-ATPase, or nitric oxide synthase (NOS) play a role in K(+)-induced dilatation of the rat basilar artery in vivo. When cerebrospinal fluid [K(+)] was elevated from 3 to 5, 10, 15, 20, and 30 mM, a reproducible concentration-dependent vasodilator response was elicited (change in diameter = 9 +/- 1, 27 +/- 4, 35 +/- 4, 43 +/- 12, and 47 +/- 16%, respectively). Responses to K(+) were inhibited by approximately 50% by the Kir channel inhibitor BaCl(2) (30 and 100 microM). In contrast, neither ouabain (1-100 microM, a Na(+)-K(+)-ATPase inhibitor) nor N(G)-nitro-L-arginine (30 microM, a NOS inhibitor) had any effect on K(+)-induced vasodilatation. These concentrations of K(+) also hyperpolarized smooth muscle in isolated segments of basilar artery, and these hyperpolarizations were virtually abolished by 30 microM BaCl(2). RT-PCR experiments confirmed the presence of mRNA for Kir2.1 in the basilar artery. Thus K(+)-induced dilatation of the basilar artery in vivo appears to partly involve hyperpolarization mediated by Kir channel activity and possibly another mechanism that does not involve hyperpolarization, activation of Na(+)-K(+)-ATPase, or NOS.     

Aldosterone regulation of intestinal Na absorption involves SGK-mediated changes in NHE3 and Na+ pump activity

Aldosterone-induced intestinal Na(+) absorption is mediated by increased activities of apical membrane Na(+)/H(+) exchange (aNHE3) and basolateral membrane Na(+)-K(+)-ATPase (BLM-Na(+)-K(+)-ATPase) activities. Because the processes coordinating these events were not well understood, we investigated human intestinal Caco-2BBE cells where aldosterone increases within 2-4 h of aNHE3 and alpha-subunit of BLM-Na(+)-K(+)-ATPase, but not total abundance of these proteins. Although aldosterone activated Akt2 and serum glucorticoid kinase-1 (SGK-1), the latter through stimulation of phosphatidylinositol 3-kinase (PI3K), only the SGK-1 pathway mediated its effects on Na(+)-K(+)-ATPase. Ouabain inhibition of the early increase in aldosterone-induced Na(+)-K(+)-ATPase activation blocked most of the apical NHE3 insertion, possibly by inhibiting Na(+)-K(+)-ATPase-induced changes in intracellular sodium concentration ([Na](i)). Over the next 6-48 h, further increases in aNHE3 and BLM-Na(+)-K(+)-ATPase activity and total protein expression were observed to be largely mediated by aldosterone-activated SGK-1 pathway. Aldosterone-induced increases in NHE3 mRNA, for instance, could be inhibited by RNA silencing of SGK-1, but not Akt2. Additionally, aldosterone-induced increases in NHE3 promoter activity were blocked by silencing SGK-1 as well as pharmacological inhibition of PI3K. In conclusion, aldosterone-stimulated intestinal Na(+) absorption involves two phases. The first phase involves stimulation of PI3K, which increases SGK-dependent insertion and function of BLM-Na(+)-K(+)-ATPase and subsequent increased membrane insertion of aNHE3. The latter may be caused by Na(+)-K(+)-ATPase-induced changes in [Na] or transcellular Na flux. The second phase involves SGK-dependent increases in total NHE3 and Na(+)-K(+)-ATPase protein expression and activities. The coordination of apical and BLM transporters after aldosterone stimulation is therefore a complex process that requires multiple time- and interdependent cellular processes.     

Regulation of fish gill Na(+)-K(+)-ATPase by selective sulfatide-enriched raft partitioning during seawater adaptation

Na(+)-K(+)-ATPase is arguably the most important enzyme in the animal cell plasma membrane, but the role of the membrane in its regulation is poorly understood. We investigated the relationship between Na(+)-K(+)-ATPase and membrane microdomains or "lipid rafts" enriched in sulfatide (sulfogalactosylceramide/SGC), a glycosphingolipid implicated as a cofactor for this enzyme, in the basolateral membrane of rainbow trout gill epithelium. Our studies demonstrated that when trout adapt to seawater (33 ppt), Na(+)-K(+)-ATPase relocates to these structures. Arylsulfatase-induced desulfation of basolateral membrane SGC prevented this relocation and significantly reduced Na(+)-K(+)-ATPase activity in seawater but not freshwater trout. We contend that Na(+)-K(+)-ATPase partitions into SGC-enriched rafts to help facilitate the up-regulation of its activity during seawater adaptation. We also suggest that differential partitioning of Na(+)-K(+)-ATPase between these novel SGC-enriched regulatory platforms results in two distinct, physiological Na(+) transport modes. In addition, we extend the working definition of cholesterol-dependent raft integrity to structural dependence on the sulfate moiety of SGC in this membrane.     

Effect of increased calcium intake on cardiac and vascular Na(+)-K(+)-ATPase activity in oral contraceptive-treated female Sprague-Dawley rats

The present study aimed at investigating the influence of increased dietary calcium on Na(+)-K(+)-ATPase activity in heart and aorta of female Sprague-Dawley rats treated with oral contraceptive (OC) steroids. Rats were grouped as control (CR), OC-treated and OC+calcium-treated. OC-treated and OC+calcium-treated received a combination of OC steriods (ethinyloestradiol and norgestrel; ig). OC+calcium-treated rats were fed with 2.5% calcium diet, while OC-treated and CR groups were fed on 0.9% calcium diet. The activity of Na(+)-K(+)-ATPase in heart and aorta was significantly lower in OC-treated rats than those in the other groups. OC treatment caused significant increase in plasma glucose and significant decrease in plasma K+ as compared to control group. Decrease in Na(+)-K(+)-ATPase activity and plasma K+ was abrogated by increased calcium intake, while increase in plasma glucose was not normalized by calcium supplementation. Plasma levels of Na+, lipid peroxidation index and ascorbic acid were comparable among the three groups. These results showed that OC treatment could lead to impaired activity of cardiac and vascular Na(+)-K(+)-ATPase, possibly due to reduced plasma K+ level and these effects could be abolished by high calcium diet.     

Ouabain-insensitive acidification by dopamine in renal OK cells: primary control of the Na(+)/H(+) exchanger

The present study was aimed at evaluating the role of D(1)- and D(2)-like receptors and investigating whether inhibition of Na(+) transepithelial flux by dopamine is primarily dependent on inhibition of the apical Na(+)/H(+) exchanger, inhibition of the basolateral Na(+)-K(+)-ATPase, or both. The data presented here show that opossum kidney cells are endowed with D(1)- and D(2)-like receptors, the activation of the former, but not the latter, accompanied by stimulation of adenylyl cyclase (EC(50) = 220 +/- 2 nM), marked intracellular acidification (IC(50) = 58 +/- 2 nM), and attenuation of amphotericin B-induced decreases in short-circuit current (28.6 +/- 4.5% reduction) without affecting intracellular pH recovery after CO(2) removal. These results agree with the view that dopamine, through the activation of D(1)- but not D(2)-like receptors, inhibits both the Na(+)/H(+) exchanger (0.001933 +/- 0.000121 vs. 0.000887 +/- 0.000073 pH unit/s) and Na(+)-K(+)-ATPase without interfering with the Na(+)-independent HCO transporter. It is concluded that dopamine, through the action of D(1)-like receptors, inhibits both the Na(+)/H(+) exchanger and Na(+)-K(+)-ATPase, but its marked acidifying effects result from inhibition of the Na(+)/H(+) exchanger only, without interfering with the Na(+)-independent HCO transporter and Na(+)-K(+)-ATPase.     

Gill Na(+)-K(+)-2Cl(-) cotransporter abundance and location in Atlantic salmon: effects of seawater and smolting

Na(+)-K(+)-2Cl(-) cotransporter abundance and location was examined in the gills of Atlantic salmon (Salmo salar) during seawater acclimation and smolting. Western blots revealed three bands centered at 285, 160, and 120 kDa. The Na(+)-K(+)-2Cl(-) cotransporter was colocalized with Na(+)-K(+)-ATPase to chloride cells on both the primary filament and secondary lamellae. Parr acclimated to 30 parts per thousand seawater had increased gill Na(+)-K(+)-2Cl(-) cotransporter abundance, large and numerous Na(+)-K(+)-2Cl(-) cotransporter immunoreactive chloride cells on the primary filament, and reduced numbers on the secondary lamellae. Gill Na(+)-K(+)-2Cl(-) cotransporter levels were low in presmolts (February) and increased 3.3-fold in smolts (May), coincident with elevated seawater tolerance. Cotransporter levels decreased below presmolt values in postsmolts in freshwater (June). The size and number of immunoreactive chloride cells on the primary filament increased threefold during smolting and decreased in postsmolts. Gill Na(+)-K(+)-ATPase activity and Na(+)-K(+)-2Cl(-) cotransporter abundance increased in parallel during both seawater acclimation and smolting. These data indicate a direct role of the Na(+)-K(+)-2Cl(-) cotransporter in salt secretion by gill chloride cells of teleost fish.     

Postischemic Na(+)-K(+)-ATPase reactivation is delayed in the absence of glycolytic ATP in isolated rat hearts

Normalization of intracellular sodium (Na) after postischemic reperfusion depends on reactivation of the sarcolemmal Na(+)-K(+)-ATPase. To evaluate the requirement of glycolytic ATP for Na(+)-K(+)-ATPase function during postischemic reperfusion, 5-s time-resolution 23Na NMR was performed in isolated perfused rat hearts. During 20 min of ischemia, Na increased approximately twofold. In glucose-reperfused hearts with or without prior preischemic glycogen depletion, Na decreased immediately upon postischemic reperfusion. In glycogen-depleted pyruvate-reperfused hearts, however, the decrease of Na was delayed by approximately 25 s, and application of the pyruvate dehydrogenase (PDH) activator dichloroacetate (DA) did not shorten this delay. After 30 min of reperfusion, Na had almost normalized in all groups and contractile recovery was highest in the DA-treated hearts. In conclusion, some degree of functional coupling of glycolytic ATP and Na(+)-K(+)-ATPase activity exists, but glycolysis is not essential for recovery of Na homeostasis and contractility after prolonged reperfusion. Furthermore, the delayed Na(+)-K(+)-ATPase reactivation observed in pyruvate-reperfused hearts is not due to inhibition of PDH.     

Monoclonal antibody HK4001 completely inhibits K(+)-dependent ATP hydrolysis and H+ transport of hog gastric H+,K(+)-ATPase

A monoclonal antibody (designated as HK4001) was prepared against hog gastric H+,K(+)-ATPase. It dose-dependently inhibited the H+,K(+)-ATPase activity, formation of the K(+)-sensitive phosphoenzyme, and proton uptake into gastric vesicles. The H+,K(+)-ATPase activity was completely inhibited by addition of the antibody at a molar ratio of 1:2 (antibody/catalytic subunit) at pH 7.8. The maximal inhibition decreased with decrease in pH of the medium (7.8 greater than 7.4 greater than 6.2). The Fab fragment obtained by digestion of the antibody with papain was also inhibitory. The antibody did not inhibit the K(+)-dependent p-nitrophenylphosphatase or the labeling of the enzyme with fluorescein isothiocyanate. It inhibited gastric H+,K(+)-ATPase from rabbits and rats, but did not cross-react with related cation-transport ATPases (Na+,K(+)-ATPase or Ca2(+)-ATPase) or H(+)-ATPase in the multivesicular body. From these and related findings, the antibody was suggested to recognize a highly specific site on the cytosolic surface of H+,K(+)-ATPase. The conformation of the epitope was conserved after treatment with Triton X-100, but not sodium dodecyl sulfate. In addition, judging from the stoichiometry of inactivation of H+,K(+)-ATPase by this antibody, the functional unit of H+,K(+)-ATPase was suggested to be a dimer or a tetramer (not a trimer) of the catalytic unit.     

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.