The mechanism of insulin stimulation of (Na+,K+)-ATPase transport activity in muscle

Since the mechanism underlying the insulin stimulation of (Na+,K+)-ATPase transport activity observed in multiple tissues has remained undetermined, we have examined (Na+,K+)-ATPase transport activity (ouabain-sensitive 86Rb+ uptake) and Na+/H+ exchange transport (amiloride-sensitive 22Na+ influx) in differentiated BC3H-1 cultured myocytes as a model of insulin action in muscle. The active uptake of 86Rb+ was sensitive to physiological insulin concentrations (1 nM), yielding a maximum increase of 60% without any change in 86Rb+ permeability. In order to determine the mechanism of insulin stimulation of (Na+,K+)-ATPase activity, we demonstrated that insulin also stimulates passive 22Na+ influx by Na+/H+ exchange transport (maximal 200% increase) and an 80% increase in intracellular Na+ concentration with an identical time course and dose-response curve as insulin-stimulated (Na+,K+)-ATPase transport activity. Incubation of the cells with high [Na+] (195 mM) significantly potentiated insulin stimulation of ouabain-inhibitable 86Rb+ uptake. The ionophore monensin, which also promotes passive Na+ entry into BC3H-1 cells, mimics the insulin stimulation of ouabain-inhibitable 86Rb+ uptake. In contrast, incubation with amiloride or low [Na+] (10 mM), both of which inhibit Na+/H+ exchange transport, abolished the insulin stimulation of (Na+,K+)-ATPase transport activity. Furthermore, each of these insulin-stimulated transport activities displayed a similar sensitivity to amiloride. These results indicate that insulin stimulates a large increase in Na+/H+ exchange transport and that the resulting Na+ influx increases the intracellular Na+ concentration, thus activating the internal Na+ transport sites of the (Na+,K+)-ATPase. This Na+ influx is, therefore, the mediator of the insulin-induced stimulation of membrane (Na+,K+)-ATPase transport activity classically observed in muscle.     

Angiotensin II modulates the activity of Na+,K+-ATPase in cultured rat astrocytes via the AT1 receptor and protein kinase C-delta activation

In astrocytes the activity of the Na+,K(+)-ATPase pump maintains an inwardly directed electrochemical sodium gradient used by the Na+-dependent transporters and regulates the extracellular K+ concentration essential for neuronal excitability. We show here that incubation of cultured rat astrocytes with angiotensin II (Ang II) modulates Na+,K(+)-ATPase activity, in a dose- and time-dependent manner. Na+,K(+)-ATPase activation was mediated by binding of Ang II to AT1 receptors as it was completely blocked by DuP 753, a specific AT1 receptor subtype antagonist. Stimulation of Na+,K(+)-ATPase activity by Ang II was dependent on protein kinase C (PKC) activation because PKC antagonists abolished the inducing effect of Ang II and the PKC activator phorbol 12-myristate 13-acetate enhanced transporter activity. Ang II stimulated translocation of PKC-delta but not that of other PKC isoforms from the cytosol to the plasma membrane. These results indicate that the activity of Na+,K(+)-ATPase in astrocytes is increased by physiological concentrations of Ang II and that the AT1 receptor subtype mediates the Na+,K(+)-ATPase response to Ang II via PKC-delta activation.     

Further biochemical characterization of an Na+ pump inhibitor purified from human urine

An increase in endogenous Na+,K+-ATPase inhibitor(s) with digitalis-like properties has been reported in chronic renal insufficiency, in Na+-dependent experimental hypertension and in some essential hypertensive patients. The present study specifies some properties and some biochemical characteristics of a semipurified compound from human urine having digitalis-like properties. The urine-derived inhibitor (endalin) inhibits Na+,K+-ATPase activity and [3H]-ouabain binding, and cross-reacts with anti-digoxin antibodies. The inhibitory effect on ATPases of endalin is higher on Na+,K+-ATPase than on Mg2+-ATPase and Ca2+-ATPase. The mechanism of endalin action on highly purified Na+,K+-ATPase was compared to that of ouabain and was similar in that it reversibly inhibited Na+,K+-ATPase activity; it inhibited Na+,K+-ATPase non-competitively with ATP; its inhibitory effect was facilitated by Na+; K+ decreased its inhibitory effect on Na+,K+-ATPase; it competitively inhibited ouabain binding to the enzyme; its binding was maximal in the presence of Mg2+ and Pi; it decreased the Na+ pump activity in human erythrocytes; it reduced serotonin uptake by human platelets; and it was diuretic and natriuretic in rat bioassay. The endalin differed from ouabain in only three aspects: its inhibitory effect was not really specific for Na+,K+-ATPase; its binding to the enzyme was undetectable in the presence of Mg2+ and ATP; it was not kaliuretic in rat bioassay. Endalin is a reversible and partial specific inhibitor of Na+,K+-ATPase, its Na+,K+-ATPase inhibition closely resembles that of ouabain and it could be considered as one of the natriuretic hormones.     

Development of Ion Metabolism in Reaggregated Brain Cell Cultures

Mouse brain cell reaggregates have been used to study changes in sodium- and potassium-dependent ouabain-sensitive adenosine phosphohydrolase (Na+, K+-ATPase) activity and in 86Rb+ uptake and exit during development. Na+, K+-ATPase activity in these cultures has two ouabain-inhibitable components, both of which increased severalfold between day 3 and day 17 in culture. This increase, however, was less than that in developing brain. Little change in either total or extracellular water or in the equilibrium levels of Na+ and K+ occurred during development. The uptake of 86Rb+ measured a 10-min incubation showed only a modest increase during culture, whereas the exit of 86Rb+ from reaggregates preloaded with the tracer increased approximately fourfold. The exit consisted of both K+-independent and K+-stimulated components and the K+-stimulated fraction contributed most of the developmental change. When uptake rates were corrected for the contribution of the developmental changes in exit, these rates were found to increase as well. The 86Rb+ uptake correlated closely with the activity of the Na+,K+-ATPase during development. The pattern of developmental changes in enzyme activity and 86Rb+ uptake and exit suggest that, while little change in the steady-state levels of the ions occurred, the rates of ion movement increase markedly.     

Acute changes in myo-inositol uptake and 22Na+ flux in murine neuroblastoma cells (N1E-115) following insulin

myo-Inositol uptake was investigated in a murine neuroblastoma clone (N1E-115) to determine the effect of altered Na+,K+-ATPase activity. The Na+ ionophore monensin, and veratridine, an alkaloid affecting voltage-dependent Na+ entry, increased acute 22Na+ uptake and 22Na+ efflux from pre-loaded cells, concomitant with enhanced myo-inositol uptake. This effect was also seen following insulin. Insulin-stimulated myo-inositol uptake was inhibited by amiloride, ouabain and pyrithiamine. Amiloride inhibition suggests that activation of Na+/H+ exchange preceding Na+,K+-ATPase activation is involved in insulin stimulation of myo-inositol uptake. Pyrithiamine inhibition is an indication of prior activation of the Na+,K+-ATPase alpha + catalytic subunit by insulin. The results provide evidence that insulin contributes to the maintenance of Na+,K+-ATPase in neuronal tissue.     

Specific effects of spermine on Na+,K+-adenosine triphosphatase

Specific effects of spermine on Na+,K+-ATPase were observed using an enzyme partially purified from rabbit kidney microsomes by extraction with deoxycholate. 1. Spermine competed with K+ for K+-dependent, ouabain-sensitive nitrophenylphosphatase. The K1 for spermine was 0.075 mm in the presence of 1 mM Mg2+ and 5 mM p-nitrophenylphosphate at pH 7.5. 2. spermine activated Na+,K+-ATPase over limited concentration ranges of K+ and Na+ in the presence of 0.05 mM ATP. The spermine concentration required for half maximal activation was 0.055 mM in the presence of 1 mM K+, 10 mM Na+, 1 mM Mg2+, and 0.05 mM ATP. 3. The activation of Na+,K4-ATPase was not due to substitution of spermine for K+, Na+, or Mg2+. 4. When the concentration of K+ or Na+ was extremely low, or in excess, spermine did not activate Na+,K+-ATPase, but inhibited it slightly. 5. Plots of 1/v vs. 1/[ATP] at various concentrations of spermine showed that spermine decreased the Km for ATP without changing the Vmax. 6. Plots of 1/v vs. 1/[ATP] at concentrations of K+ from 0.05 mM to 0.5 mM showed that K+ increased the Km for ATP with increase in the Vmax in the presence of 0.2 mM spermine similarly to that in the absence of spermine. The contradictory effects of spermine on this enzyme system suggest that the K+-dependent monophosphatase activity does not reflect the second half (the dephosphorylation step) of the Na+,K+-ATPase catalytic cycle.     

Cilostazol, a cyclic AMP phosphodiesterase inhibitor, stimulates nitric oxide production and sodium potassium adenosine triphosphatase activity in SH-SY5Y human neuroblastoma cells.

Deficiencies in cellular cyclic AMP (cAMP) and nitric oxide (NO) production are thought to be involved in the pathogenesis of diabetic neuropathy. We used a human neuroblastoma cell line, SH-SY5Y, to investigate the effect of cilostazol, a specific cAMP phosphodiesterase inhibitor, on NO production and Na+, K+-ATPase activity. SH-SY5Y cells were cultured under 5 or 50 mM glucose for 5-6 days, the cells were then exposed to cilostazol or other chemicals and nitrite, cAMP and Na+, K+-ATPase activity were measured. In cells grown in 50 mM glucose, cilostazol was observed to increase significantly both NO production and cellular cAMP accumulation in a time- and dose-dependent manner. Cilostazol also significantly recovered reduced levels of protein kinase A activity (PKA) in 50 mM glucose. Furthermore, a PKA inhibitor, H-89 significantly suppressed the increase in NO production stimulated by cilostazol, suggesting that cilostazol stimulates NO production by activating PKA. Cilostazol did not affect either sorbitol or myo-inositol concentrations. Dexamethasone, which is known to induce inducible NO synthase, had no effect on NO production stimulated by cilostazol, suggesting that cilostazol stimulates NO production catalyzed by neuronal constitutive NO synthase (ncNOS) in SH-SY5Y cells. L-arginine, which is an NO agonist enhanced Na+, K+-ATPase activity in cells grown in 50 mM glucose, NG-nitro-L-arginine methyl ester (L-NAME), which is an NOS inhibitor inhibited basal Na+, K+-ATPase activity in 5 mM glucose and suppressed the increased enzyme activity induced by cilostazol in 50 mM glucose. The above results confirmed our previous observation that NO regulates Na+, K+-ATPase activity in SH-SY5Y cells and suggest that cilostazol increases Na+, K+-ATPase activity, at least in part, by stimulating NO production. The present results also suggest that cilostazol has a beneficial effect on diabetic neuropathy by improving Na+, K+-ATPase activity via directly increasing cAMP and NO production in nerves.     

Potassium as a signal for both proliferation and differentiation of rabbit retinal (Müller) glia growing in cell culture

Retinal glial (Müller) cells were grown from explants of early postnatal rabbit retinae. The resulting monolayers of flat cells were exposed to control media (containing 5.85 mM K+), and to media with enhanced K+ concentrations (10 and 20 mM) or arginine-vasopressin (AVP, 20 micrograms/ml) or epithelial growth factor (EGF, 10 ng/ml). Autoradiographically, protein synthesis was quantified as L-[3H]-lysine incorporation, and DNA synthesis as [3H]-thymidine incorporation. Furthermore, the activity of Na+,K(+)-ATPase was measured radiochemically. Short exposure to either moderately enhanced K+ concentrations (10 mM) or to AVP, stimulated L-[3H]-lysine incorporation into the cells. Long-lasting exposure to either high K+ concentrations (20 mM) or to EGF stimulated [3H]-uptake. The Na+,K(+)-ATPase activity of cell cultures increased with increasing K+ concentration of the media. It is suggested that release of K+ by active neuronal compartments stimulates local protein synthesis of glial cells, resulting in the formation of glial sheaths with active K+ uptake capacity. Strong K+ release may even induce glial proliferation.     

The effects of storage of sarcoplasmic reticulum fragments on the Ca2+, Mg2+-ATPase.

The effects of K+ and Na+ on the Ca2+,Mg2+-ATPase of sarcoplasmic reticulum fragments (SRF) were investigated at 1 mM ATP. There was an alteration of the sensitivity of the ATPase to the monovalent cations during storage of the SRF preparation. The Ca2+, Mg2+-ATPase of freshly prepared SRF was slightly activated by 5-10 mM K+ and Na+. Mg2+-ATPase was inhibited by both the monovalent cations to the same extent, and this response to the ions was independent of the freshness of the preparations. After storage of SRF, however, the Ca2+,Mg2+-ATPase was markedly activated by higher concentrations of K+ and Na+ (0.2-0.3 M). K+ and Na+ reduced the Ca uptake at the steady state in freshly prepared SRF, but did not affect pre-steady state uptake. In the presence of oxalate, the rate of Ca accumulation both in fresh and stored preparations was activated by 0.1-0.2 M K+ and Na+. The Ca2+, mg2+-ATPase with oxalate, so-called "extra ATPase," showed the same response to the ions as did the activity without oxalate during storage.     

Regulation of acetylated tubulin/Na+,K+-ATPase interaction by L-glutamate in non-neural cells: involvement of microtubules

A subpopulation of membrane tubulin consisting mainly of the acetylated isotype is associated with Na+,K+-ATPase and inhibits the enzyme activity. We found recently that treatment of cultured astrocytes with L-glutamate induces dissociation of the acetylated tubulin/Na+,K+-ATPase complex, resulting in increased enzyme activity. We now report occurrence of this phenomenon in non-neural cells. As in the case of astrocytes, the effect of L-glutamate is mediated by its transporters and not by specific receptors. In COS cells, the effect of L-glutamate was reversed by its elimination from culture medium, provided that d-glucose was present. The effect of L-glutamate was not observed when Na+ was replaced by K+ in the incubation medium. The ionophore monensin, in the presence of Na+, had the same effect as L-glutamate. Treatment of cells with taxol prevented the dissociating effect of L-glutamate or monensin. Nocodazole treatment of intact cells or isolated membranes dissociated the acetylated tubulin/Na+,K+-ATPase complex. The dissociating effect of nocodazol does not require Na+. These results indicate a close functional relationship among Na+,K+-ATPase, microtubules, and L-glutamate transporters, and a possible role in cell signaling pathways.     

Inhibition of hamster sperm Na+, K+-ATPase activity by taurine and hypotaurine

Taurine, hypotaurine and the structural analogue, beta-alanine, were tested for their effects on Na+, K+-ATPase activity of crude homogenates prepared from washed cauda epididymal hamster sperm. Preincubation with 0.1-10 mM taurine or hypotaurine inhibited Na+, K+-ATPase in a dose-dependent manner, while beta-alanine had an inhibitory effect only at 10 mM. The results of this study are the first evidence to demonstrate inhibition of Na+, K+-ATPase activity by taurine and hypotaurine and are discussed in relation to the ability of these compounds to sustain hamster sperm motility and fertility.     

Mutant Phe788 --> Leu of the Na+,K+-ATPase is inhibited by micromolar concentrations of potassium and exhibits high Na+-ATPase activity at low sodium concentrations.

Mutant Phe788 --> Leu of the rat kidney Na+,K(+)-ATPase was expressed in COS cells to active-site concentrations between 40 and 60 pmol/mg of membrane protein. Analysis of the functional properties showed that the discrimination between Na+ and K+ on the two sides of the system is severely impaired in the mutant. Micromolar concentrations of K+ inhibited ATP hydrolysis (K(0.5) for inhibition 107 microM for the mutant versus 76 mM for the wild-type at 20 mM Na+), and at 20 mM K+, the molecular turnover number for Na+,K(+)-ATPase activity was reduced to 11% that of the wild-type. This inhibition was counteracted by Na+ in high concentrations, and in the total absence of K+, the mutant catalyzed Na(+)-activated ATP hydrolysis ("Na(+)-ATPase activity") at an extraordinary high rate corresponding to 86% of the maximal Na+,K(+)-ATPase activity. The high Na(+)-ATPase activity was accounted for by an increased rate of K(+)-independent dephosphorylation. Already at 2 mM Na+, the dephosphorylation rate of the mutant was 8-fold higher than that of the wild-type, and the maximal rate of Na(+)-induced dephosphorylation amounted to 61% of the rate of K(+)-induced dephosphorylation. The cause of the inhibitory effect of K+ on ATP hydrolysis in the mutant was an unusual stability of the K(+)-occluded E2(K2) form. Hence, when E2(K2) was formed by K+ binding to unphosphorylated enzyme, the K(0.5) for K+ occlusion was close to 1 microM in the mutant versus 100 microM in the wild-type. In the presence of 100 mM Na+ to compete with K+ binding, the K(0.5) for K+ occlusion was still 100-fold lower in the mutant than in the wild-type. Moreover, relative to the wild-type, the mutant exhibited a 6-7-fold reduced rate of release of occluded K+, a 3-4-fold increased apparent K+ affinity in activation of the pNPPase reaction, a 10-11-fold lower apparent ATP affinity in the Na+,K(+)-ATPase assay with 250 microM K+ present (increased K(+)-ATP antagonism), and an 8-fold reduced apparent ouabain affinity (increased K(+)-ouabain antagonism).     

Effects of L-phenylalanine on acetylcholinesterase, (Na+,K+)-ATPase and Mg2+-ATPase activities in adult rat whole brain and frontal cortex

The effect of different L-phenylalanine (Phe) concentrations (0.12-12.1 mM) on acetylcholinesterase (AChE), (Na+,K+)-ATPase and Mg2+-ATPase activities was investigated in homogenates of adult rat whole brain and frontal cortex at 37 degrees C. AChE, (Na+,K+)-ATPase and Mg2+-ATPase activities were determined after preincubation with Phe. AChE activity in both tissues showed a decrease up to 18% (p<0.01) with Phe. Whole brain Na+,K+-ATPase was stimulated by 30-35% (p<0.01) with high Phe concentrations, while frontal cortex Na+,K+-ATPase was stimulated by 50-55% (p<0.001). Mg2+-ATPase activity was increased only in frontal cortex with high Phe concentrations. It is suggested that: a) The inhibitory effect of Phe on brain AChE is not influenced by developmental factors, while the stimulation of Phe on brain Na+,K+-ATPase is indeed affected; b) The stimulatory effect of Phe on rat whole brain Na+,K+-ATPase is decreased with age; c) Na+,K+-ATPase is selectively more stimulated by high Phe concentrations in frontal cortex than in whole brain homogenate; d) High (toxic) Phe concentrations can affect Mg2+-ATPase activity in frontal cortex, but not in whole brain, thus modulating the amount of intracellular Mg2+.     

Sodium-potassium adenosine triphosphatase activity of human lymphocyte membrane vesicles: kinetic parameters, substrate specificity, and effects of phytohemagglutinin.

We have prepared human blood lymphocyte membrane vesicles of high purity in sufficient quantity for detailed enzyme analysis. This was made possible by the use of plateletpheresis residues, which contain human lymphocytes in amounts equivalent to thousands of milliliters of blood. The substrate specificity and the kinetics of the cofactor and substrate requirements of the human lymphocyte membrane Na+, K+-ATPase activity were characterized. The Na+, K+-ATPase did not hydrolyze ADP, AMP, ITP, UTP, GTP or TTP. The mean ATPase stimulated by optimal concentrations of Na+ and K+ (Na+, K+-ATPase) was 1.5 nmol of P(i) hydrolyzed, microgram protein-1, 30 min-1 (range 0.9-2.1). This activity was completely inhibited by the cardiac glycoside, ouabain. The K(m) for K+ was approximately 1.0 mM and the K(m) for Na+ was approximately 15 mM. Active Na+ and K+ transport and ouabain-sensitive ATP production increase when lymphocytes are stimulated by PHA. Na+, K+-ATPase activity must increase also to transduce energy for the transport of Na+ and K+. Some studies have reported that PHA stimulates the lymphocyte membrane ATPase directly. We did not observe stimulation of the membrane Na+, K+-ATPase when either lymphocytes or lymphocyte membranes were treated with mitogenic concentrations of PHA. Moreover, PHA did not enhance the reaction velocity of the Na+, K+-ATPase when studied at the K(m) for ATP, Na+, K+ OR Mg++, indicating that it does not alter the affinity of the enzyme for its substrate or cofactors. Thus, our data indicate that the increase in ATPase activity does not occur as a direct result of PHA action on the cell membrane.     

Lysophosphatidylcholines containing polyunsaturated fatty acids were found as Na+, K+-ATPase inhibitors in acutely volume-expanded hog

Na+, K+-ATPase inhibitors possessing inhibitory activities against the specific binding of ouabain to Na+, K+-ATPase and 86Rb uptake into hog erythrocytes have been purified from the plasma of acutely saline-infused hog. The purifications were performed by a combination of Amberlite XAD-2 adsorption chromatography and four steps of high-performance liquid chromatography with four different types of columns. Fast atom bombardment (FAB) mass and proton NMR spectrometric studies identified the purified substances as gamma-arachidoyl- [LPCA(gamma), 34%], beta-arachidoyl- [LPCA(beta), 4%], gamma-linoleoyl- (LPCL, 33%), and gamma-oleoyl- (LPCO, 25%) lysophosphatidylcholine, expressed in molar ratio in the plasma. Small amounts of gamma-docosapentaenoyl-, gamma-eicosatrienoyl-, and gamma-palmitoyllysophosphatidylcholine were also detected by both FAB mass and 1H NMR spectrometric studies. Only gamma-acyl-LPC's showed inhibitory activities on Na+,K+-ATPase and ouabain-binding activities. These LPC's were effective at 100 microM levels in attaining 50% inhibition of the enzyme activity. The inhibition of Na+,K+-ATPase activity due to these compounds was always more sensitive than that of both ouabain-binding and 86Rb uptake activities. The ouabain-displacing activity in plasma due to these compounds increased with time during saline infusion. The maximal plasma level was approximately 10 times higher than that in the preinfusion plasma sample. Although these results suggest the gamma-acyl-LPC's with long-chain polyunsaturated fatty acids are not simple competitive inhibitors to Na+, K+ -ATPase, these compounds could be implicated in the pathogenesis of the circulation abnormality through the modulation of membrane enzyme.     

Effects of Organic and Inorganic Lead on Synaptosomal Uptake, Release, and Receptor Binding of Glutamate in Young Rats

Chemical changes in central glutamate neurotransmission were assessed by measuring synaptic membrane receptor binding, the uptake and release by synaptosomes of glutamate in rats treated acutely with tetraethyl lead and chronically with lead acetate. The activity of Na+, K+-ATPase in synaptosomes was measured to correlate with the changes in uptake/release studies. The affinity of receptor binding and uptake systems was significantly reduced although the number of receptor sites and the capacity of uptake systems were increased. The activity of Na+, K+-ATPase was also found to be increased in synaptosomes. The changes were more marked in inorganic lead toxicity, and all three regions studied--cerebral cortex, cerebellum, and brainstem--showed significant alterations.     

Spectrophotometric method for the determination of renal ouabain-sensitive H+,K+-ATPase activity

The aim of this work was to develop a method for renal H+,K+-ATPase measurement based on the previously used Na+,K+-ATPase assay (Beltowski et al.: J Physiol Pharmacol.; 1998, 49: 625-37). ATPase activity was assessed by measuring the amount of inorganic phosphate liberated from ATP by isolated microsomal fraction. Both ouabain-sensitive and ouabain-resistant K+-stimulated and Na+-independent ATPase activity was detected in the renal cortex and medulla. These activities were blocked by 0.2 mM imidazolpyridine derivative, Sch 28080. The method for ouabain-sensitive H+,K+-ATPase assay is characterized by good reproducibility, linearity and recovery. In contrast, the assay for ouabain-resistant H+,K+-ATPase was unsatisfactory, probably due to low activity of this enzyme. Ouabain-sensitive H+,K+-ATPase was stimulated by K+ with Km of 0.26 +/- 0.04 mM and 0.69 +/- 0.11 mM in cortex and medulla, respectively, and was inhibited by ouabain (Ki of 2.9 +/- 0.3 microM in the renal cortex and 1.9 +/- 0.4 microM in the renal medulla) and by Sch 28080 (Ki of 1.8 +/- 0.5 microM and 2.5 +/- 0.9 microM in cortex and medulla, respectively). We found that ouabain-sensitive H+,K+-ATPase accounted for about 12% of total ouabain-sensitive activity in the Na+,K+-ATPase assay. Therefore, we suggest to use Sch 28080 during Na+,K+-ATPase measurement to block H+,K+-ATPase and improve the assay specificity. Leptin administered intraperitoneally (1 mg/kg) decreased renal medullary Na+,K+-ATPase activity by 32.1% at 1 h after injection but had no effect on H+,K+-ATPase activity suggesting that the two renal ouabain-sensitive ATPases are separately regulated.     

Regulation of Na+ transport in brown adipose tissue.

In order to test the hypothesis that Na+, K+-ATPase (Na+,K+-dependent ATPase) is involved in the noradrenaline-mediated stimulation of respiration in brown adipose tissue, the effects of noradrenaline on Na+,K+-ATPase in isolated brown-fat-cell membrane vesicles, and on 22Na+ and K+ (86Rb+) fluxes across the membranes of intact isolated cells, were measured. The ouabain-sensitive fraction of the K+-dependent ATPase activity in the isolated membrane-vesicle preparation was small and was not affected by the presence of noradrenaline in the incubation media. The uptake of 86Rb+ into intact hormone-sensitive cells was inhibited by 80% by ouabain, but it was insensitive to the presence of noradrenaline. 22Na+ uptake and efflux measured in the intact cells were 8 times more rapid than the 86Rb+ fluxes and were unaffected by ouabain. This indicated the presence of a separate, more active, transport system for Na+ than the Na+,K+-ATPase. This is likely to be a Na+/Na+ exchange activity under normal aerobic conditions. However, under anaerobic conditions, or conditions simulating anaerobiosis (2 mM-NaCN), the unidirectional uptake of Na+ increased dramatically, while efflux was unaltered.     

Functional consequences of alterations to Pro328 and Leu332 located in the 4th transmembrane segment of the alpha-subunit of the rat kidney Na+,K(+)-ATPase.

Site-specific mutagenesis was used to analyse the functional roles of the residues Pro328 and Leu332 located in the conserved PEGLL motif of the predicted transmembrane helix M4 in the alpha 1-subunit of the ouabain resistant rat kidney Na+,K(+)-ATPase. cDNAs encoding either of the Na+,K(+)-ATPase mutants Pro328-->Ala and Leu332-->Ala, and wild type, were cloned into the expression vector pMT2 and transfected into COS-1 cells. Ouabain-resistant clones growing in the presence of 10 microM ouabain were isolated, and the Na+,K+, ATP and pH dependencies of the Na+,K(+)-ATPase activity measured in the presence of 10 microM ouabain were analysed. Under these conditions the exogenous expressed Na+,K(+)-ATPase contributed more than 95% of the Na+,K(+)-ATPase activity. The Pro328-->Ala mutant displayed a reduced apparent affinity for Na+ (K0.5 (Na+) 13.04 mM), relative to the wild type (K0.5 (Na+) 7.13 mM). By contrast, the apparent affinity for Na+ displayed by the Leu332-->Ala mutant was increased (K0.5 (Na+) 3.92 mM). Either of the mutants exhibited lower apparent affinity for K+ relative to the wild type (K0.5 (K+) 2.46 mM for Pro328-->Ala and 1.97 mM for Leu332-->Ala, compared with 0.78 mM for wild type). Both mutants exhibited higher apparent affinity for ATP than the wild type (K0.5 (ATP) 0.086 mM for Pro328-->Ala and 0.042 mM for Leu332-->Ala, compared with 0.287 mM for wild type). The influence of pH was in accordance with an acceleration of the E2 (K)-->E1 transition in the mutants relative to the wild type. These data are consistent with a role of Pro328 and Leu332 in the stabilization of the E2 form and of Pro328 in Na+ binding. The possible role of the mutated residues in K+ binding is discussed.     

(Na+ + K+)- and Na+-stimulated Mg2+-dependent ATPase activities in kidney of sea bass (Dicentrarchus labrax L.)

1. Sea bass kidney microsomal preparations contain two Mg2+ dependent ATPase activities: the ouabain-sensitive (Na+ + K+)-ATPase and an ouabain-insensitive Na+-ATPase, requiring different assay conditions. The (Na+ + K+)-ATPase under the optimal conditions of pH 7.0, 100 mM Na+, 25 mM K+, 10 mM Mg2+, 5 mM ATP exhibits an average specific activity (S.A.) of 59 mumol Pi/mg protein per hr whereas the Na+-ATPase under the conditions of pH 6.0, 40 mM Na+, 1.5 mM MgATP, 1 mM ouabain has a maximal S.A. of 13.9 mumol Pi/mg protein per hr. 2. The (Na+ + K+)-ATPase is specifically inhibited by ouabain and vanadate; the Na+-ATPase specifically by ethacrynic acid and preferentially by frusemide; both activities are similarly inhibited by Ca2+. 3. The (Na+ + K+)-ATPase is specific for ATP and Na+, whereas the Na+-ATPase hydrolyzes other substrates in the efficiency order ATP greater than GTP greater than CTP greater than UTP and can be activated also by K+, NH4+ or Li+. 4. Minor differences between the two activities lie in the affinity for Na+, Mg2+, ATP and in the thermosensitivity. 5. The comparison between the two activities and with what has been reported in the literature only partly agree with our findings. It tentatively suggests that on the one hand two separate enzymes exist which are related to Na+ transport and, on the other, a distinct modulation in vivo in different tissues.