Characterization of gill Na/K-ATPase activity and ouabain binding in Antarctic and New Zealand nototheniid fishes

The effects of thermal acclimation in two Nototheniid species, the stenothermal Antarctic Trematomous bernacchii and the eurythermal New Zealand Notothenia angustata, were investigated. Serum osmolality, gill Na/K-ATPase activity, sodium pump density and ouabain affinity were determined. Both fish were acclimated at their upper and lower viable thermal temperatures. Warm acclimation (+4 degrees C) of the T. bernacchii significantly decreased their serum osmolality from 550 to 450 mOsm/kg compared to cold-acclimation (-1.5 degrees C) and this was accompanied by a two-fold increase in gill Na/K-ATPase activity. Warm-acclimation (+14 degrees C) of N. angustata did not significantly change their serum osmolality from 330 mOsm/kg or gill Na/K-ATPase activity compared to the cold-acclimated (+4 degrees C) N. angustata. Using [(3)H]ouabain binding techniques, the B(max) and K(d) values of gill Na/K-ATPase enzymes were determined. No difference in the B(max) or K(d) of the warm-acclimated T. bernacchii accounted for the increase in Na/K-ATPase activity. We conclude that the change in gill Na/K-ATPase activity in the warm-acclimated T. bernacchii is not mediated by an increase in the number of enzyme sites and is not reflected in a change in ouabain affinity for Na/K-ATPase.     

The effect of ionizing radiation on the regulation of the Na,K-ATPase activity of the kidney by univalent cations

The effect of ionizing radiation of 0.206 C/kg on the kinetics of activation of rat kidney Na,K-ATPase preparation by Na and K ions was studied as an index of possible qualitative and quantitative changes in the properties of the enzyme. Ionizing radiation was shown not only to increase the enzyme activity but also to change the optimal rate of ATP hydrolysis by Na,K-ATPase and to induce some differences in the shape of the curve for Na,K-ATPase dependence upon Na-sodium//potassium ion ratio in the incubation medium.     

Postradiation changes in the system of active transport of ions in the CNS. Analysis of the cation centers of Na,K-ATPase

A study was made of the influence of ionizing radiation of 0.31 C/kg on the kinetic parameters showing the activity of brain Na, K-ATPase preparation to be a function of ion-regulator concentration. The use of the new method for the analysis of the enzyme cation centers permitted to estimate that whole-body irradiation of rats with the above dose did not cause in vitro a substantial change in the pattern of Na, K-ATPase activation by Na and K ions.     

Postradiation changes in the systems of active ion transport in the CNS. Na,K-ATPase

A study was made of the effects of irradiation with lethal (0.31 C/kg) and superlethal (12.9 C/kg) doses on Na,K-ATPase activity of various membrane formations in rat brain cortex. The results obtained indicated a significant increase in the activity of the enzyme at different times after irradiation.     

Bile pigments and bilirubin UDP-glucuronyl transferase during the metamorphosis of Rana catesbeiana tadpoles

1. Cold-acclimated (1 degree C) goldfish (Carassius auratus) branchial Na/K-ATPase activity was elevated 100% while renal Na/K-ATPase activity was not significantly affected compared with warm-acclimated (20 degrees C) goldfish. 2. Cold-acclimated goldfish branchial and renal Mg-ATPase activity was reduced 18 and 30% on a per mg protein basis, respectively. 3. Renal Na/K-ATPase activity was 4.6- and 1.6-fold greater than gill in cold- and warm-acclimated fish, respectively. 4. The elevated branchial Na/K-ATPase activity and the unchanged renal Na/K-ATPase activity are consistent with the maintenance of the reduced blood ion level in cold-acclimated goldfish.     

Alterations of Na,K-ATPase Isoenzymes in the Rat Diabetic Neuropathy: Protective Effect of Dietary Supplementation with n-3 Fatty Acids

Abstract: Diabetic neuropathy is a degenerative complication of diabetes accompanied by an alteration of nerve conduction velocity (NCV) and Na,K-ATPase activity. The present study in rats was designed first to measure diabetes-induced abnormalities in Na,K-ATPase activity, isoenzyme expression, fatty acid content in sciatic nerve membranes, and NCV and second to assess the preventive ability of a fish oil-rich diet (rich in n-3 fatty acids) on these abnormalities. Diabetes was induced by intravenous streptozotocin injection. Diabetic animals (D) and nondiabetic control animals (C) were fed the standard rat chow either without supplementation or supplemented with either fish oil (DM, CM) or olive oil (DO, CO) at a daily dose of 0.5 g/kg by gavage during 8 weeks. Analysis of the fatty acid composition of purified sciatic nerve membranes from diabetic animals showed a decreased incorporation of C16:1(n-7) fatty acids and arachidonic acids. Fish oil supplementation changed the fatty acid content of sciatic nerve membranes, decreasing C18:2(n-6) fatty acids and preventing the decreases of arachidonic acids and C18:1(n-9) fatty acids. Protein expression of Na,K-ATPase α subunits, Na,K-ATPase activity, and ouabain affinity were assayed in purified sciatic nerve membranes from CO, DO, and DM. Na,K-ATPase activity was significantly lower in sciatic nerve membranes of diabetic rats and significantly restored in diabetic animals that received fish oil supplementation. Diabetes induced a specific decrease of α1- and α3-isoform activity and protein expression in sciatic nerve membranes. Fish oil supplementation restored partial activity and expression to varying degrees depending on the isoenzyme. These effects were associated with a significant beneficial effect on NCV. This study indicates that fish oil has beneficial effects on diabetes-induced alterations in sciatic nerve Na,K-ATPase activity and function.     

Properties of oligomycin-induced occlusion of Na+ by detergent-solubilized Na,K-ATPase from pig kidney or shark rectal gland.

Oligomycin induces occlusion of Na+ in membrane-bound Na,K-ATPase. Here it is shown that Na,K-ATPase from pig kidney or shark rectal gland solubilized in the nonionic detergent C12E8 is capable of occluding Na+ in the presence of oligomycin. The apparent affinity for Na+ is reduced for both enzymes upon solubilization, and there is an increase in the sigmoidicity of binding curves, which indicates a change in the cooperativity between the occluded ions. A high detergent/protein ratio leads to a decreased occlusion capacity. De-occlusion of Na+ by addition of K+ is slow for solubilized Na,K-ATPase, with a rate constant of about 0.1 s-1 at 6 degrees C. Stopped-flow fluorescence experiments with 6-carboxyeosin, which can be used to monitor the E1Na-form in detergent solution, show that the K(+)-induced de-occlusion of Na+ correlates well with the fluorescence decrease which follows the transition from the E1Na-form to the E2-form. There is a marked increase in the rate of fluorescence change at high detergent/protein ratios, indicating that the properties of solubilized enzyme are subject to modification by detergent in other respects than mere solubilization of the membrane-bound enzyme. The temperature dependence of the rate of de-occlusion in the range 2 degrees C to 12 degrees C is changed slightly upon solubilization, with activation energies in the range 20-23 kcal/mol for membrane-bound enzyme, increasing to 26-30 kcal/mol for solubilized enzyme. Titrations of the rate of transition from E1Na to E2K with oligomycin can be interpreted in a model with oligomycin having an apparent dissociation constant of about 2.5 microM for C12E8-solubilized shark Na,K-ATPase and 0.2 microM for solubilized pig kidney Na,K-ATPase.     

Investigation of the effect of phospholipase on Na,K-ATPase activity]

Temperature dependence of bovine brain NA,K-ATPase before and after the short-term treatment of enzyme preparations with phospholipases A, C and D is investigated. Arrhenius plots of the temperature dependence of the reaction rate catalysed by Na,K-ATPase are non-linear, they have an inflection at the region of about 20 degrees C. The treatment of the enzyme with phospholipase A makes the inflection more smooth, phospholipase D shifts the inflection by 4 degrees C to lower temperature and simultaneously activates Na,K-ATPase. Phospholipase C sharply changes the Arrhenius curve and makes it linear. The data obtained are discussed with respect to the role of phospholipids in the formation of membrane bilayer and in the regulation of Na,K-ATPase activity.     

Direct activation of gastric H,K-ATPase by N-terminal protein kinase C phosphorylation. Comparison of the acute regulation mechanisms of H,K-ATPase and Na,K-ATPase

In this study we compared the protein kinase dependent regulation of gastric H,K-ATPase and Na,K-ATPase. The protein kinase A/protein kinase C (PKA/PKC) phosphorylation profile of H,K-ATPase was very similar to the one found in the Na,K-ATPase. PKC phosphorylation was taking place in the N-terminal part of the alpha-subunit with a stoichiometry of approximately 0.6 mol Pi/mole alpha-subunit. PKA phosphorylation was in the C-terminal part and required detergent, as is also found for the Na,K-ATPase. The stoichiometry of PKA-induced phosphorylation was approximately 0.7 mol Pi/mole alpha-subunit. Controlled proteolysis of the N-terminus abolished PKC phosphorylation of native H,K-ATPase. However, after detergent treatment additional C-terminal PKC sites became exposed located at the beginning of the M5M6 hairpin and at the cytoplasmic L89 loop close to the inner face of the plasma membrane. N-terminal PKC phosphorylation of native H,K-ATPase alpha-subunit was found to stimulate the maximal enzyme activity by 40-80% at saturating ATP, depending on pH. Thus, a direct modulation of enzyme activity by PKC phosphorylation could be demonstrated that may be additional to the well-known regulation of acid secretion by recruitment of H,K-ATPase to the apical membranes of the parietal cells. Moreover, a distinct difference in the regulation of H,K-ATPase and Na,K-ATPase is the apparent absence of any small regulatory proteins associated with the H,K-ATPase.     

Regulation of alpha1 Na/K-ATPase expression by cholesterol

We have reported that α1 Na/K-ATPase regulates the trafficking of caveolin-1 and consequently alters cholesterol distribution in the plasma membrane. Here, we report the reciprocal regulation of α1 Na/K-ATPase by cholesterol. Acute exposure of LLC-PK1 cells to methyl β-cyclodextrin led to parallel decreases in cellular cholesterol and the expression of α1 Na/K-ATPase. Cholesterol repletion fully reversed the effect of methyl β-cyclodextrin. Moreover, inhibition of intracellular cholesterol trafficking to the plasma membrane by compound U18666A had the same effect on α1 Na/K-ATPase. Similarly, the expression of α1, but not α2 and α3, Na/K-ATPase was significantly reduced in the target organs of Niemann-Pick type C mice where the intracellular cholesterol trafficking is blocked. Mechanistically, decreases in the plasma membrane cholesterol activated Src kinase and stimulated the endocytosis and degradation of α1 Na/K-ATPase through Src- and ubiquitination-dependent pathways. Thus, the new findings, taken together with what we have already reported, revealed a previously unrecognized feed-forward mechanism by which cells can utilize the Src-dependent interplay among Na/K-ATPase, caveolin-1, and cholesterol to effectively alter the structure and function of the plasma membrane.     

Phosphorylation of phospholemman (FXYD1) by protein kinases A and C modulates distinct Na,K-ATPase isozymes

Phospholemman (FXYD1), mainly expressed in heart and skeletal muscle, is a member of the FXYD protein family, which has been shown to decrease the apparent K(+) and Na(+) affinity of Na,K-ATPase ( Crambert, G., Fuzesi, M., Garty, H., Karlish, S., and Geering, K. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 11476-11481 ). In this study, we use the Xenopus oocyte expression system to study the role of phospholemman phosphorylation by protein kinases A and C in the modulation of different Na,K-ATPase isozymes present in the heart. Phosphorylation of phospholemman by protein kinase A has no effect on the maximal transport activity or on the apparent K(+) affinity of Na,K-ATPase alpha1/beta1 and alpha2/beta1 isozymes but increases their apparent Na(+) affinity, dependent on phospholemman phosphorylation at Ser(68). Phosphorylation of phospholemman by protein kinase C affects neither the maximal transport activity of alpha1/beta1 isozymes nor the K(+) affinity of alpha1/beta1 and alpha2/beta1 isozymes. However, protein kinase C phosphorylation of phospholemman increases the maximal Na,K-pump current of alpha2/beta1 isozymes by an increase in their turnover number. Thus, our results indicate that protein kinase A phosphorylation of phospholemman has similar functional effects on Na,K-ATPase alpha1/beta and alpha2/beta isozymes and increases their apparent Na(+) affinity, whereas protein kinase C phosphorylation of phospholemman modulates the transport activity of Na,K-ATPase alpha2/beta but not of alpha1/beta isozymes. The complex and distinct regulation of Na,K-ATPase isozymes by phosphorylation of phospholemman may be important for the efficient control of heart contractility and excitability.     

Amplification of DNA sequences coding for the Na,K-ATPase alpha-subunit in ouabain-resistant C+ cells.

We have studied the mechanism of cellular resistance to cardiac glycosides in C+ cells. C+ cells were resistant to ouabain and overproduced plasma membrane-bound Na,K-ATPase relative to parental HeLa cells. Overexpression of Na,K-ATPase in C+ cells correlated with increased ATPase mRNA levels and amplification (approximately 100 times) of the ATPase gene. Growth of C+ cells in ouabain-free medium resulted in a marked decline in ATPase mRNA and DNA levels. However, when cells were reexposed to ouabain, they proliferated and ATPase mRNA and DNA sequences were reamplified. Restriction analysis of C+ and other human DNA samples revealed the occurrence of rearrangements in the region of the Na,K-ATPase gene in C+ cells. Furthermore, C+ cells expressed an ATPase mRNA species not found in HeLa cells. These results suggest that amplification of the gene coding for Na,K-ATPase results in overproduction of Na,K-ATPase polypeptides. Amplification of the ATPase gene or the expression of new ATPase mRNA sequences or both may also be responsible for acquisition of the ouabain-resistant phenotype.     

Effect of diazepam on the Na, K-ATPase state in a penicillin--induced hyperactivity focus in the cerebral cortex]

Intramuscular injection of diazepam to rats at doses of 0.01 and 2 mg/kg 25-30 min after penicillin application to the rat brain cortex leads to alteration of periodic appearance of epileptic seizures (ES), to changes in the seizure pattern, and to emergence of periodic acceleration of epileptiform discharges (ED). Injection of diazepam at a dose of 2 mg/kg 20 min before penicillin application results in the reduction of ED latency in the epileptogenic focus and in a decrease in their frequency before seizures as compared to the control animals without diazepam injection. ES appear irregularly, their quantity is markedly reduced while duration is increased. Diazepam injection leads to disappearance of the rat moving reaction during ER and ES. In vivo experiments diazepam (2 mg/kg) does not influence brain cortex Na, K-ATPase of crude synaptosomes. However, diazepam leads to an increase in Na, K-ATPase activity both in the primary and dependent secondary epileptogenic foci. It is suggested that the anticonvulsant action of diazepam may be underlain by its activating effect on Na, K-ATPase of neuronal membranes in the epileptogenic focus.     

Antioxidant SMe1EC2 may attenuate the disbalance of sodium homeostasis in the organism induced by higher intake of cholesterol

The study was focused to the influence of higher intake of cholesterol on properties of the renal Na,K-ATPase, a key system in maintaining the homeostasis of sodium in the organism. Feeding for 4 weeks with cholesterol-enriched food for rats afflicted with hereditary hypertriglyceridemia by itself enhanced the activity of Na,K-ATPase, probably as a consequence of higher number of active enzyme molecules as suggested by 32 % increase of V (max) value. This may be hypothesized as a reason for the increased retention of sodium. Three-week-lasting treatment of animals kept on high cholesterol diet with antioxidant SMe1EC2 in a dose of 10 mg kg(-1) day(-1) normalized the function of renal Na,K-ATPase to the level comparable in hypertriglyceridemic rats fed with the standard diet. Therefore, our results suggest that the antioxidant SMe1EC2 in the applied dose seems to be effective in the attenuation of cholesterol-induced retention of sodium. Treatment for 3 weeks with Fenofibrate in a dose of 100 mg kg(-1) day(-1) reversed the function of renal Na,K-ATPase only slightly.     

Characteristics of ATPase activity in the sarcolemma of longitudinal and circular muscles of the canine ileum

The subcellular fraction enriched in sarcolemmal vesicles was isolated from the longitudinal muscle (LM) and the circular muscle (CM) of the canine ileum by sucrose density gradient centrifugation. Treatment of the LM and CM membranes with sodium dodecylsulfate (0.2 mg/kg protein) led to a 3-fold increase in Na,K-ATPase activity (up to 24 and 39 mumol Pi/mg protein/h, respectively) and to a 90-95% inactivation of Mg-ATPase which was 2 and 8 times (for the CM and the LM, respectively) more active than Na,K-ATPase in the untreated sarcolemma. A specific inhibition of Na,K-ATPase activity by acetylcholine (Ach) and serotonin (ST) was observed which could de blocked in the presence of muscarinic and serotonin receptor antagonists. Sensitivity of the enzyme to ST was more than one order of magnitude higher than to Ach (IC50 = 10(-8) vs 1.2 x 10(-7) M). The inhibition of Na,K-ATPase activity by the neurotransmitters was more pronounced in the LM membranes (30-40%) than in the CM ones (10-20%). These data indicate that cell membranes of the LM and CM differ both in specific ATPase activities and the responsiveness of Na,K-ATPase to the receptor-mediated effects of Ach and ST.     

Regulation of caveolin-1 membrane trafficking by the Na/K-ATPase

Here, we show that the Na/K-ATPase interacts with caveolin-1 (Cav1) and regulates Cav1 trafficking. Graded knockdown of Na/K-ATPase decreases the plasma membrane pool of Cav1, which results in a significant reduction in the number of caveolae on the cell surface. These effects are independent of the pumping function of Na/K-ATPase, and instead depend on interaction between Na/K-ATPase and Cav1 mediated by an N-terminal caveolin-binding motif within the ATPase α1 subunit. Moreover, knockdown of the Na/K-ATPase increases basal levels of active Src and stimulates endocytosis of Cav1 from the plasma membrane. Microtubule-dependent long-range directional trafficking in Na/K-ATPase–depleted cells results in perinuclear accumulation of Cav1-positive vesicles. Finally, Na/K-ATPase knockdown has no effect on processing or exit of Cav1 from the Golgi. Thus, the Na/K-ATPase regulates Cav1 endocytic trafficking and stabilizes the Cav1 plasma membrane pool.     

Possibilities of pharmacologic correction of Na, K-ATPase activity in the spinal cord in botulism

The experiments on white rats have shown that gutimin is capable of reactivating Na, K-ATPase of the synaptosomes of the jugular spinal cord in type C botulinic intoxication. Serotonin prevented Na, K-ATPase activity inhibition only in preclinical period of intoxication. Parmidin injection did not prevent suppression of Na, K-ATPase activity either in preclinical period or in skeletal muscle paresis.     

Role of the transmembrane domain of FXYD7 in structural and functional interactions with Na,K-ATPase

Members of the FXYD family are tissue-specific regulators of the Na,K-ATPase. Here, we have investigated the contribution of amino acids in the transmembrane (TM) domain of FXYD7 to the interaction with Na,K-ATPase. Twenty amino acids of the TM domain were replaced individually by tryptophan, and combined mutations and alanine insertion mutants were constructed. Wild type and mutant FXYD7 were expressed in Xenopus oocytes with Na,K-ATPase. Mutational effects on the stable association with Na,K-ATPase and on the functional regulation of Na,K-ATPase were determined by co-immunoprecipitation and two-electrode voltage clamp techniques, respectively. Most residues important for the structural and functional interaction of FXYD7 are clustered in a face of the TM helix containing the two conserved glycine residues, but others are scattered over two-thirds of the FXYD TM helix. Ile-35, Ile-43, and Ile-44 are only involved in the stable association with Na,K-ATPase. Glu-26, Met-30, and Ile-44 are important for the functional effect and/or the efficient association of FXYD7 with Na,K-ATPase, consistent with the prediction that these amino acids contact TM domain 9 of the alpha subunit (Li, C., Grosdidier, A., Crambert, G., Horisberger, J.-D., Michielin, O., and Geering, K. (2004) J. Biol. Chem. 279, 38895-38902). Several amino acids that are not implicated in the efficient association of FXYD7 with the Na,K-ATPase are specifically involved in the functional effect of FXYD7. Leu-32 and Phe-37 influence the apparent affinity for external K+, whereas Val-28 and Ile-42 are implicated in the apparent affinity for both external K+ and external Na+. These amino acids act in a synergistic way. These results highlight the important structural and functional role of the TM domain of FXYD7 and delineate the determinants that mediate the complex interactions of FXYD7 with Na,K-ATPase.     

Identification of a phospholemman-like protein from shark rectal glands. Evidence for indirect regulation of Na,K-ATPase by protein kinase c via a novel member of the FXYDY family

The Na,K-ATPase provides the driving force for many ion transport processes through control of Na(+) and K(+) concentration gradients across the plasma membranes of animal cells. It is composed of two subunits, alpha and beta. In many tissues, predominantly in kidney, it is associated with a small ancillary component, the gamma-subunit that plays a modulatory role. A novel 15-kDa protein, sharing considerable homology to the gamma-subunit and to phospholemman (PLM) was identified in purified Na,K-ATPase preparations from rectal glands of the shark Squalus acanthias, but was absent in pig kidney preparations. This PLM-like protein from shark (PLMS) was found to be a substrate for both PKA and PKC. Antibodies to the Na, K-ATPase alpha-subunit coimmunoprecipitated PLMS. Purified PLMS also coimmunoprecipitated with the alpha-subunit of pig kidney Na, K-ATPase, indicating specific association with different alpha-isoforms. Finally, PLMS and the alpha-subunit were expressed in stoichiometric amounts in rectal gland membrane preparations. Incubation of membrane bound Na,K-ATPase with non-solubilizing concentrations of C(12)E(8) resulted in functional dissociation of PLMS from Na,K-ATPase and increased the hydrolytic activity. The same effects were observed after PKC phosphorylation of Na,K-ATPase membrane preparations. Thus, PLMS may function as a modulator of shark Na,K-ATPase in a way resembling the phospholamban regulation of the Ca-ATPase.     

The adhesion molecule on glia (AMOG) is a homologue of the beta subunit of the Na,K-ATPase

AMOG (adhesion molecule on glia) is a Ca2(+)-independent adhesion molecule which mediates selective neuron-astrocyte interaction in vitro (Antonicek, H., E. Persohn, and M. Schachner. 1987. J. Cell Biol. 104:1587-1595). Here we report the structure of AMOG and its association with the Na,K-ATPase. The complete cDNA sequence of mouse AMOG revealed 40% amino acid identity with the previously cloned beta subunit of rat brain Na,K-ATPase. Immunoaffinity-purified AMOG and the beta subunit of detergent-purified brain Na,K-ATPase had identical apparent molecular weights, and were immunologically cross-reactive. Immunoaffinity-purified AMOG was associated with a protein of 100,000 Mr. Monoclonal antibodies revealed that this associated protein comprised the alpha 2 (and possibly alpha 3) isoforms of the Na,K-ATPase catalytic subunit, but not alpha 1. The monoclonal AMOG antibody that blocks adhesion was shown to interact with Na,K-ATPase in intact cultured astrocytes by its ability to increase ouabain-inhibitable 86Rb+ uptake. AMOG-mediated adhesion occurred, however, both at 4 degrees C and in the presence of ouabain, an inhibitor of the Na,K-ATPase. Both AMOG and the beta subunit are predicted to be extracellularly exposed glycoproteins with single transmembrane segments, quite different in structure from the Na,K-ATPase alpha subunit or any other ion pump. We hypothesize that AMOG or variants of the beta subunit of the Na,K-ATPase, tightly associated with an alpha subunit, are recognition elements for adhesion that subsequently link cell adhesion with ion transport.