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.     

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.     

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.     

Acute changes in gill Na+-K+-ATPase and creatine kinase in response to salinity changes in the euryhaline teleost, tilapia (Oreochromis mossambicus)

Some freshwater (FW) teleosts are capable of acclimating to seawater (SW) when challenged; however, the related energetic and physiological consequences are still unclear. This study was conducted to examine the changes in expression of gill Na(+)-K(+)-ATPase and creatine kinase (CK) in tilapia (Oreochromis mossambicus) as the acute responses to transfer from FW to SW. After 24 h in 25 ppt SW, gill Na(+)-K(+)-ATPase activities were higher than those of fish in FW. Fish in 35 ppt SW did not increase gill Na(+)-K(+)-ATPase activities until 1.5 h after transfer, and then the activities were not significantly different from those of fish in 25 ppt SW. Compared to FW, the gill CK activities in 35 ppt SW declined within 1.5 h and afterward dramatically elevated at 2 h, as in 25 ppt SW, but the levels in 35 ppt SW were lower than those in 25 ppt SW. The Western blot of muscle-type CK (MM form) was in high association with the salinity change, showing a pattern of changes similar to that in CK activity; however, levels in 35 ppt SW were higher than those in 25 ppt SW. The activity of Na(+)-K(+)-ATPase highly correlated with that of CK in fish gill after transfer from FW to SW, suggesting that phosphocreatine acts as an energy source to meet the osmoregulatory demand during acute transfer.     

Decoupling the Na+-K+-ATPase in vivo: a possible new role in the gills of freshwater fishes

The literature suggests that when Na(+)-K(+)-ATPase has reduced access to its glycosphingolipid cofactor sulfogalactosyl ceramide (SGC), it is converted to a Na(+) uniporter. We recently showed that such segregation can occur within a single membrane when Na(+)-K(+)-ATPase is excluded from membrane microdomains or 'lipid rafts' enriched in SGC (D. Lingwood, G. Harauz, J.S. Ballantyne, J. Biol. Chem. 280, 36545-36550). Specifically we demonstrated that Na(+)-K(+)-ATPase localizes to SGC-enriched rafts in the gill basolateral membrane (BLM) of rainbow trout exposed to seawater (SW) but not freshwater (FW). We therefore proposed that since the freshwater gill Na(+)-K(+)-ATPase was separated from BLM SGC it should also transport Na(+) only, suggesting a new role for the pump in this epithelium. In this paper we discuss the biochemical evidence for SGC-based modulation of transport stoichiometry and highlight how a unique asparagine-lysine substitution in the FW pump isoform and FW gill transport energetics gear the Na(+)-K(+)-ATPase to perform Na(+) uniport.     

Modeling the effect of stretch and plasma membrane tension on Na+-K+-ATPase activity in alveolar epithelial cells

While a number of whole cell mechanical models have been proposed, few, if any, have focused on the relationship among plasma membrane tension, plasma membrane unfolding, and plasma membrane expansion and relaxation via lipid insertion. The goal of this communication is to develop such a model to better understand how plasma membrane tension, which we propose stimulates Na(+)-K(+)-ATPase activity but possibly also causes cell injury, may be generated in alveolar epithelial cells during mechanical ventilation. Assuming basic relationships between plasma membrane unfolding and tension and lipid insertion as the result of tension, we have captured plasma membrane mechanical responses observed in alveolar epithelial cells: fast deformation during fast cyclic stretch, slower, time-dependent deformation via lipid insertion during tonic stretch, and cell recovery after release from stretch. The model estimates plasma membrane tension and predicts Na(+)-K(+)-ATPase activation for a specified cell deformation time course. Model parameters were fit to plasma membrane tension, whole cell capacitance, and plasma membrane area data collected from the literature for osmotically swollen and shrunken cells. Predictions of membrane tension and stretch-stimulated Na(+)-K(+)-ATPase activity were validated with measurements from previous studies. As a proof of concept, we demonstrate experimentally that tonic stretch and consequent plasma membrane recruitment can be exploited to condition cells against subsequent cyclic stretch and hence mitigate stretch-induced responses, including stretch-induced cell death and stretch-induced modulation of Na(+)-K(+)-ATPase activity. Finally, the model was exercised to evaluate plasma membrane tension and potential Na(+)-K(+)-ATPase stimulation for an assortment of traditional and novel ventilation techniques.     

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.     

Tissue ATPase activity and recovery in the freshwater crab Oziotelphusa senex senex exposed to a sublethal concentration of endosulfan for varying periods of time.

1. Na(+)-K+ and Mg(2+)-tissue ATPases of the freshwater crab Oziotelphusa senex senex showed increasing inhibition when exposed to a sublethal concentration (1.86 mg/l = 0.1 of LC50) of endosulfan for 1-30 days. 2. Na(+)-K(+)-ATPase activity in all tissues (thoracic nerve mass, gill, hepatopancreas and claw muscle) was higher than Mg(2+)-ATPase activity. 3. After 30 days exposure tissue Mg(2+)-ATPase was less affected than Na(+)-K(+)-ATPase. 4. Crabs exposed to endosulfan and then returned to uncontaminated water showed greater recovery of Mg(2+)-ATPase than Na(+)-K(+)-ATPase with 90-95% recovery after 1 day exposure and 60-65% recovery after 30 days exposure. 5. Changes in behaviour of the crabs were noted after 7 days exposure to endosulfan with progressive loss of coordination, decreased activity and increased exudation of mucus.     

Hyperoxia reduces plasma membrane fluidity: a mechanism for endothelial cell dysfunction

To evaluate the relative contributions of three possible mechanisms that can be advanced to explain the observation that hyperoxia decreases serotonin uptake by endothelial cells, we examined the effect of high O2 tensions on Na+-K+-ATPase activity, ATP content, and plasma membrane fluidity in cultured endothelial cells. Confluent monolayers of pulmonary artery and aortic endothelial cells were exposed to 95% O2 (hyperoxia) or 20% O2 (controls) in 5% CO2 at 1 ATA for 4-42 h. Exposure to high O2 tensions had no effect on Na+-K+-ATPase activity or ATP content in pulmonary artery or aortic endothelial cells in culture. However, hyperoxia decreased the fluidity of the plasma membrane of pulmonary artery and aortic endothelial cells in culture, and the time course for the decrease in fluidity parallels that of the hyperoxic inhibition of serotonin transport. These results indicate that hyperoxia decreases fluidity in the hydrophobic core of the plasma membranes of cultured endothelial cells. Such decreases in plasma membrane fluidity may be responsible for hyperoxia-induced alterations in membrane function including decreases in transmembrane transport of amines.     

栀子提取物ZG对副流感病毒1型感染后宿主细胞膜的影响

为了探讨栀子提取物ZG抗病毒作用的生物学机制,观察了栀子提取物ZG对副流感病毒1型(PIV-1)感染后宿主细胞膜电位、膜Na -K -ATP酶活性和膜流动性的影响。以氯化乙酰胆碱为阳性对照,采用荧光探针Di-BAC4(3)标记Hep-2细胞膜电位,借助流式细胞仪检测膜电位;定磷法,分光光度计检测Na -K -ATP酶活性;荧光探针NBD-C6-HPC标记细胞膜磷脂,以荧光漂白恢复法和激光扫描共聚焦显微镜检测膜流动性。结果显示:PIV-1感染后宿主细胞膜电位下降,处于超极化状态;膜Na -K -ATP酶活性显著增加,膜流动性显著降低。栀子提取物ZG作用后,对宿主细胞膜的超极化状态没有明显影响;对膜Na -K -ATP酶活性没有明显影响;而对膜流动性则有明显的恢复作用。阳性对照药乙酰胆碱能明显改善病毒感染后膜电位的超极化状态。PIV-1感染后膜电位、Na -K -ATP酶活性和膜流动性等细胞膜能态和功能的改变,可能为病毒感染的生物学机制之一;栀子提取物ZG可能是通过改善细胞膜流动性,维持细胞膜的正常功能来发挥抗病毒感染的作用,而与膜电位和膜Na -K -ATP酶活性等能态来源的环节可能无关。     

Mechanism of acid adaptation of a fish living in a pH 3.5 lake

Despite unfavorable conditions, a single species of fish, Osorezan dace, lives in an extremely acidic lake (pH 3.5) in Osorezan, Aomori, Japan. Physiological studies have established that this fish is able to prevent acidification of its plasma and loss of Na(+). Here we show that these abilities are mainly attributable to the chloride cells of the gill, which are arranged in a follicular structure and contain high concentrations of Na(+)-K(+)-ATPase, carbonic anhydrase II, type 3 Na(+)/H(+) exchanger (NHE3), type 1 Na(+)-HCO(3)(-) cotransporter, and aquaporin-3, all of which are upregulated on acidification. Immunohistochemistry established their chloride cell localization, with NHE3 at the apical surface and the others localized to the basolateral membrane. These results suggest a mechanism by which Osorezan dace adapts to its acidic environment. Most likely, NHE3 on the apical side excretes H(+) in exchange for Na(+), whereas the electrogenic type 1 Na(+)-HCO(3)(-) cotransporter in the basolateral membrane provides HCO(3)(-) for neutralization of plasma using the driving force generated by Na(+)-K(+)-ATPase and carbonic anhydrase II. Increased expression of glutamate dehydrogenase was also observed in various tissues of acid-adapted dace, suggesting a significant role of ammonia and bicarbonate generated by glutamine catabolism.     

Na(+)-K(+)-ATPase is distributed to microvillous and basal membrane of the syncytiotrophoblast in human placenta

Despite its importance for placental function, syncytiotrophoblast Na(+)-K(+)-ATPase has not been studied in detail. We purified syncytiotrophoblast microvillous (MVM) and basal (BM) membranes from full-term human placenta. Western blotting with isoform-specific antibodies demonstrated the presence of the alpha(1)-subunit, but not the alpha(2)- or alpha(3)-subunits, in MVM and BM. Relative density per unit membrane protein in BM was 48 +/- 1% (mean +/- SE, n = 4, P < 0.02) of that in the MVM. The activity of Na(+)-K(+)-ATPase was lower in BM (1.4 +/- 0.14 micromol. mg(-1). min(-1), n = 8, P < 0.02) than in MVM (3.9 +/- 0.25 micromol. mg(-1). min(-1)). Immunocytochemistry confirmed the distribution of Na(+)-K(+)-ATPase to MVM and BM. These findings suggest that the syncytiotrophoblast represents a type of transporting epithelium different from the classical epithelia found in the small intestine and kidney, where Na(+)-K(+)-ATPase is confined to the basolateral membrane only. This unique polarization of the Na(+) pump does not, however, preclude a net transcellular transport of Na(+) to the fetus.     

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.     

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.     

cAMP and sodium transport in the freshwater crayfish, Cherax destructor.

Crayfish in which sodium absorption was maximally stimulated had elevated levels of both cAMP and Na(+)-K(+)-ATPase activity in gill tissue. The concentration of cAMP and activity of Na(+)-K(+)-ATPase in gill tissue were monitored following transfer of crayfish from water containing 125 mmol x l(-1) Na to Na-free media. Both parameters were significantly elevated within 10 min of transfer to Na-free media and [cAMP] peaked between 1 and 2 h before falling transiently to the control level at 3 h. A second peak of [cAMP] and a further rise in Na(+)-K(+)-ATPase activity were evident 6 h after transfer and elevated levels were then maintained. The pattern observed was consistent with the existence of two separate mechanisms for the control of sodium absorption both of which stimulated the activity of Na(+)-K(+)-ATPase via elevation of the intracellular concentration of cAMP. The initial response was very rapid (<10 min) but of brief duration (1-2 h) and this mechanism appeared to be sensitive to changes in external ion levels. The second mechanism exhibited a much longer response time (3-6 h) and duration and was likely to be sensitive to changes in internal ion concentrations.     

FXYD5 modulates Na+ absorption and is increased in cystic fibrosis airway epithelia

FXYD5, also known as dysadherin, belongs to a family of tissue-specific regulators of the Na(+)-K(+)-ATPase. We determined the kinetic effects of FXYD5 on Na(+)-K(+)-ATPase pump activity in stably transfected Madin-Darby canine kidney cells. FXYD5 significantly increased the apparent affinity for Na(+) twofold and decreased the apparent affinity for K(+) by 60% with a twofold increase in V(max) of K(+), a pattern that would increase activity and Na(+) removal from the cell. To test the effect of increased Na(+) uptake on FXYD5 expression, we analyzed Madin-Darby canine kidney cells stably transfected with an inducible vector expressing all three subunits of the epithelial Na(+) channel (ENaC). Na(+)-K(+)-ATPase activity increased sixfold after 48-h ENaC induction, but FXYD5 expression decreased 75%. FXYD5 expression was also decreased in lung epithelia from mice that overexpress ENaC, suggesting that chronic Na(+) absorption by itself downregulates epithelial FXYD5 expression. Patients with cystic fibrosis (CF) display ENaC-mediated hyperabsorption of Na(+) in the airways, accompanied by increased Na(+)-K(+)-ATPase activity. However, FXYD5 was significantly increased in the lungs and nasal epithelium of CF mice as assessed by RT-PCR, immunohistochemistry, and immunoblot analysis (P < 0.001). FXYD5 was also upregulated in nasal scrapings from human CF patients compared with controls (P < 0.02). Treatment of human tracheal epithelial cells with a CFTR inhibitor (I-172) confirmed that loss of CFTR function correlated with increased FXYD5 expression (P < 0.001), which was abrogated by an inhibitor of NF-kappaB. Thus FXYD5 is upregulated in CF epithelia, and this change may exacerbate the Na(+) hyperabsorption and surface liquid dehydration observed in CF airway epithelia.     

A quantitative immunocytochemical study of Na+,K+-ATPase in rat hepatocytes after STZ-induced diabetes and dietary fish oil supplementation.

Because diabetes causes alterations in hepatic membrane fatty acid content, these changes may affect the Na+,K+-ATPase. In this study we documented the effects of streptozotocin (STZ)-induced diabetes on hepatic Na+,K+-ATPase catalytic alpha1-subunit and evaluated whether these changes could be normalized by fish oil supplementation. Two groups of diabetic rats received fish oil or olive oil supplementation. Both groups had a respective control group. We studied the localization of catalytic alpha1-subunit on bile canalicular and basolateral membranes using immunocytochemical methods and confocal laser scanning microscopy, and the Na+, K+-ATPase activity, membrane fluidity, and fatty acid composition on isolated hepatic membranes. A decrease in the alpha1-subunit was observed with diabetes in the bile canalicular membranes, without changes in basolateral membranes. This decrease was partially prevented by dietary fish oil. Diabetes induces significant changes as documented by enzymatic Na+,K+-ATPase activity, membrane fluidity, and fatty acid content, whereas little change in these parameters was observed after a fish oil diet. In conclusion, STZ-induced diabetes appears to modify bile canalicular membrane integrity and dietary fish oil partly prevents the diabetes-induced alterations.