Reduced myocardial Na+, K(+)-pump capacity in congestive heart failure following myocardial infarction in rats

We examined changes in expression and function of the cardiac Na+, K(+)-pump in a post-infarction rat model of hypertrophy and congestive heart failure (CHF). Myocardial infarction was induced by ligation of the left coronary artery in Wistar rats and hearts were obtained from animals with CHF and from sham operated rats after 6 weeks. In the CHF group the ratio of heart weight to body weight was 70% greater compared to sham (*P < 0.05) and all left-ventricular end-diastolic pressures (LVEDP) were above 15 mmHg. The expression of the alpha 1- and beta 1-subunits (mRNA and protein) of the Na+, K(+)-pump was not significantly different in CHF and sham. As compared to sham the alpha 2 isoform, mRNA and protein levels were lower in CHF hearts by 25 and 55%, respectively, whereas the alpha 3 isoform mRNA was greater by 120% in CHF. The alpha 3 protein was not detectable in sham but a prominent band was seen in CHF. Cell volume of isolated cardiomyocytes was 30% larger in CHF. Cardiomyocytes containing the Na+ sensitive fluorescent dye SBFI were loaded to an intracellular Na+ concentration ([Na+]i] of about 140 mM in a K(+)- and Mg(2+)-free medium (140 mM Na+, free Ca2+ of 10(-8) M). To avoid back leak of Na+ and to ensure no voltage effects on the Na+, K(+)-pump extracellular Na+ was subsequently removed, and 6 mM Mg2+ was added to the superfusate, The Na+, K(+)-pump was then reactivated by 10 mM Rb+. SBFI fluorescence ratio decreased mono-exponentially with a time constant (tau) of 191 +/- 15 s in sham (n = 8) and 320 +/- 38 s in CHF (n = 9) rats (P < 0.01). These changes in fluorescence indicate that the maximum rate of decline of [Na+]i from 100 to 35 mM was 39% (P < 0.005) slower in CHF compared to sham, whereas maximum pump rate per cell was not significantly altered (9.0 +/- 0.7 fmol/s in sham and 7.1 +/- 0.7 fmol/s in CHF cells). The [Na+]i which caused 50% pump activation (k0.5) was also not altered in CHF (40 mM in both groups). We conclude that the number of Na+, K(+)-pumps per cell was maintained in CHF but an isoform switch of the alpha 3-replacing the alpha 2-isoform occurred. However, maximum Na+, K(+)-pump rate in terms of rate of change of [Na+]i was significantly attenuated in CHF, most likely as a result of increased cell size.     

Inward-directed current generated by the Na+,K+ pump in Na(+)- and K(+)-free medium

In Na(+)- and K(+)-free solution, an inward-directed current can be detected in Xenopus oocytes, which is inhibited by cardiac glycosides and activated by ATP. Therefore, it is assumed to be generated by the Na+,K+ pump. At negative membrane potentials, the pump current increases with more negative potentials and with increasing [H+] in the external medium. This current is not observed when Mg2+ instead of Ba2+ is the only divalent cation present in the bath medium, and it does not depend on whether Na+ or K+ is present internally. At 5 to 10 mM Na+ externally, maximum pump-generated current is obtained while no current can be detected in presence of physiological [Na+]. It is suggested that in low-Na+ and K(+)-free medium the Na+,K+ pump molecule can either form a conductive pathway that is permeable to Ba2+ or protons or operate in its conventional transport mode accepting Ba2+ as a K+ congener. A reversed pump mode or an electrogenic uncoupled Na(+)-efflux mode is excluded.     

Coexisting NPY and NE synergistically regulate renal tubular Na+, K(+)-ATPase activity

The sympathetic renal nerves are of central importance for the regulation of sodium balance. Sodium excretion decreases following renal nerve activation and increases following denervation. These effects have been attributed to norepinephrine (NE) acting on alpha-adrenergic receptors. In the present study, using isolated permeabilized rat renal proximal convoluted tubule (PCT) cells, neuropeptide Y (NPY) was shown to stimulate Na+, K(+)-ATPase activity. This 36-amino acid peptide is a messenger molecule in the sympathetic nervous system which is co-stored with NE and dopamine-beta-hydroxylase (DBH), the NE synthesizing enzyme in the renal nerves. The effect is likely to be mediated via the NPY Y2 receptor, a pertussis toxin (PTX)-sensitive G-protein, and calcium. It is partially antagonized by alpha-adrenergic antagonists, and enhanced by the subthreshold doses of alpha-adrenergic agonists. Our results suggest an important role for this peptide in the regulation of the sodium balance in the kidney.     

Kinetics of Na(+)-dependent conformational changes of rabbit kidney Na+,K(+)-ATPase

The kinetics of Na(+)-dependent partial reactions of the Na+,K(+)-ATPase from rabbit kidney were investigated via the stopped-flow technique, using the fluorescent labels N-(4-sulfobutyl)-4-(4-(p-(dipentylamino)phenyl)butadienyl)py ridinium inner salt (RH421) and 5-iodoacetamidofluorescein (5-IAF). When covalently labeled 5-IAF enzyme is mixed with ATP, the two labels give almost identical kinetic responses. Under the chosen experimental conditions two exponential time functions are necessary to fit the data. The dominant fast phase, 1/tau 1 approximately 155 s-1 for 5-IAF-labeled enzyme and 1/tau 1 approximately 200 s-1 for native enzyme (saturating [ATP] and [Na+], pH 7.4 and 24 degrees C), is attributed to phosphorylation of the enzyme and a subsequent conformational change (E1ATP(Na+)3-->E2P(Na+)3 + ADP). The smaller amplitude slow phase, 1/tau 2 = 30-45 s-1, is attributed to the relaxation of the dephosphorylation/rephosphorylation equilibrium in the absence of K+ ions (E2P<==>E2). The Na+ concentration dependence of 1/tau 1 showed half-saturation at a Na+ concentration of 6-8 mM, with positive cooperatively involved in the occupation of the Na+ binding sites. The apparent dissociation constant of the high-affinity ATP-binding site determined from the ATP concentration dependence of 1/tau 1 was 8.0 (+/- 0.7) microM. It was found that P3-1-(2-nitrophenyl)ethyl ATP, tripropylammonium salt (NPE-caged ATP), at concentrations in the hundreds of micromolar range, significantly decreases the value of 1/tau 1, observed. This, as well as the biexponential nature of the kinetic traces, can account for previously reported discrepancies in the rates of the reactions investigated.     

Modulation of mouse cerebral Na+,K(+)-ATPase activity by oxygen free radicals

There is increasing evidence that oxygen free radicals (OFR) are involved in cerebral ischaemia-reperfusion injury, possibly via a modulation of Na+,K(+)-ATPase activity, one of the major membrane pumps responsible for ionic homeostasis. We measured OFR-mediated modulation of this enzymatic activity and examined the roles of lipid and/or protein alterations. Using mouse brain microsomes exposed to UV-C irradiation, our results show a good correlation between activity inhibition and lipoperoxidation estimated by PUFA loss as well as malondialdehyde production. The protective effect of thiourea (OH scavenger) and the lack of effect noted with DTT (thiol protector) suggest that the functionality of the Na+,K(+)-ATPase is altered by perturbation of membrane integrity rather than by a structural alteration of the protein itself.     

5-HPETE is a potent inhibitor of neuronal Na+, K(+)-ATPase activity

The effects of 1 microM concentrations of arachidonic acid hydroperoxide (HPETES) products of 5-, 12- and 15-lipoxygenase on Na+, K(+)-ATPase activity were investigated in synaptosomal membrane preparations from rat cerebral cortex. 5-HPETE inhibited Na+, K(+)-ATPase activity by up to 67 %. In contrast, 12-HPETE and 15-HPETE did not inhibit Na+, K(+)-ATPase activity. In addition, neither 5-HETE or LTA4 inhibited Na+, K(+)-ATPase activity. Dose-response studies indicated that 5-HPETE was a potent (IC25 = 10(-8) M) inhibitor of Na+, K(+)-ATPase activity. These findings indicate that 5-HPETE inhibits Na+, K(+)-ATPase activity by a mechanism that is dependent on the hydroperoxide position and independent of further metabolism by 5-lipoxygenase. It is proposed that 5-HPETE production by 5-lipoxygenase and subsequent inhibition of neuronal Na+, K(+)-ATPase activity may be a mechansim for modulating synaptic transmission.     

A Glu329-->Gln variant of the alpha-subunit of the rat kidney Na+,K(+)-ATPase can sustain active transport of Na+ and K+ and Na+,K(+)-activated ATP hydrolysis with normal turnover number

An allelic variant of the ouabain-insensitive rat kidney Na+,K(+)-ATPase alpha 1-isoform was identified by chance in a cDNA library. The variant differed from the wild-type rat kidney Na+,K(+)-ATPase by a single G-to-C base substitution in the cDNA, which on amino acid level gave rise to a glutamine in place of the glutamate residue Glu329 previously suggested as a likely donator of oxygen ligands for Na+ and K+ binding. The variant cDNA was transfected into COS-1 cells and the transfectants expanded with success into stable cell lines that were able to grow in the presence of a concentration of ouabain highly cytotoxic to the parental cells containing only the endogenous COS-1 cell Na+,K(+)-ATPase. Under these conditions, the viability of the cells depended on the cation transport mediated by the ouabain-insensitive Glu329-->Gln variant, whose cDNA was shown by polymerase chain reaction amplification to be stably integrated into the COS-1 cell genome. The maximum specific ATP hydrolysis activity of isolated plasma membranes of the Glu329-->Gln variant did not differ significantly from that of plasma membranes containing the wild type. A method was established for measurement of the phosphorylation capacity of the expressed Glu329-->Gln variant and wild-type enzyme, and it was thereby demonstrated that the variant had a turnover number similar if not identical to that of the wild-type.     

Separation and characterization of Na+,K(+)-ATPase containing vesicles

Na+,K(+)-ATPase was reconstituted in vesicles prepared by a dialysis method. Ion-exchange chromatography was used to obtain well characterized fractions from the inhomogeneous vesicle preparation. Lipid and protein content was determined by optical methods during the elution process. It was possible to separate fractions with distinct enzymatic and transport activities. A protocol was set up, which allowed to calculate the average number of 5-IAF labeled ion pumps per vesicle in the different fractions. The dependence of the number of protein molecules per vesicle was studied as function of the initial protein concentration added to the lipid solution before dialysis. The transport activity disappears completely at very low protein concentrations (3.3 micrograms protein per mg lipid). This observation is in favor of the proposal discussed in the literature, that the heterodimer (alpha beta)2 is the transport-active form of the Na+,K(+)-ATPase. The presented method can be applied to all reconstituted vesicle preparations in which the proteins can be labeled quantitatively with a fluorescence dye.     

The Na+/K(+)-pump in rat peritoneal mast cells: some aspects of regulation of activity and cellular function

The mast cell contains potent mediators of inflammation which are released after IgE-directed and non-IgE-directed stimulation of the cell. This highly specialized cell is therefore ascribed a role in the pathogenesis of disease states in which the inflammatory response plays a role for the development of the clinical symptoms. Thus, besides being of interest in basic research, studies of the cellular processes leading to release of inflammatory mediators from the mast cell also have important clinical implications. The aim of the present work has been to document the existence of the Na+/K(+)-pump in rat peritoneal mast cells, to investigate the regulation of the pump activity and to explore whether modulation of the pump activity interferes with the cellular stimulus/secretion coupling mechanism. The Na+/K(+)-pump activity following stimulation of the mast cell was also investigated. The pump activity was assessed as the ouabain-sensitive cellular potassium uptake with 86Rb+ as a tracer for potassium. The histamine release from the mast cell following IgE-directed and non-IgE directed stimulation of the cell was used as a parameter for cellular degranulation. Histamine was measured by spectrofluorometry. The finding of an ouabain-sensitive uptake mechanism in the mast cell documents the presence of a functional Na+/K(+)-pump in this cell. The pump activity is inhibited by lanthanides and by the divalent cations calcium, magnesium, barium and strontium. The pump has a large reserve capacity which probably is caused by a low intracellular concentration of sodium. This enables the pump to respond to changes in the intracellular sodium concentration. The inhibitory effect of di- and trivalent ions on the pump activity is probably a result of the inhibitory effect of these ions on the cellular sodium uptake. The digitalis glycosides, ouabain and digoxin, but not the more lipophilic drug digitoxigenin, increase both IgE-directed and non-IgE-directed histamine release from the mast cell in a calcium-free medium, while there is no effect of digitalis glycosides in a medium containing physiologically relevant concentrations of calcium. The effect of digitalis glycosides on the histamine release is dependent on the drug concentrations used and the time of preincubation. An increase in the intracellular concentration of sodium secondary to inhibition of the Na+/K(+)-pump is the effector mechanism likely to explain the effect of digitalis glycosides on the mast cell histamine release. Increases in intracellular sodium might affect the intracellular concentration of calcium via changes in Na+/Ca(2+)-exchange. IgE-directed and non-IgE-directed stimulation of the mast cell activates the Na+/K(+)-pump. In case of compound 48/80-induced histamine release, the pump is stimulated for at least 2 hr. It is proposed, that the poststimulatory activation of the Na+/K(+)-pump is due to increased cellular sodium uptake associated with the release process. This sodium uptake may occur via Na+/Ca(2+)-exchange, Na+/H(+)-exchange, Na+/K+/2Cl(-)-cotransport or a non-selective ion channel. Besides describing aspects of the function and regulation of the Na+/K(+)-pump in the rat peritoneal mast cells, this thesis points to the potential role of sodium transport mechanisms in mast cell physiology. Pharmacological manipulations of such transport mechanisms might in the future add to the treatment of allergic diseases.     

Electrogenic property of Na+, K(+)-ATPase through computer simulation

A computer simulation of the electrogenic nature of the membrane-bound Na+, K(+)-ATPase is presented. The model involves coupling two simulation systems for passive and active transports, using a minimum of empirical parameters, and studies the contribution of the pump to the membrane potential. The simulation results indicate that electrogenic active transport accelerates the restoration of the resting electrochemical gradients and contributes approximately 0.44-1.1 mV to the resting potential of the membrane, depending on the Na:K coupling ratio. The effect of membrane potential and the physical positioning of the enzyme from the passive transporting channel on the enzyme function is also presented. The validity of the model is checked by comparing our results with reported literature values.     

Captopril ameliorates the decreased Na+,K(+)-ATPase activity in the retina of streptozotocin-induced diabetic rats

PURPOSE: To examine the effect of captopril, an angiotensin-converting enzyme (ACE) inhibitor, on the activity of retinal sodium-potassium ATPase (Na,K-ATPase) and the activity of ACE in the serum and retina of streptozotocin (STZ)-induced diabetic rats. METHODS: Experimental diabetes was induced in male Long-Evans rats by a single intraperitoneal injection of STZ (55 mg/kg body weight). Some groups of normal and diabetic animals were treated with captopril (10 mg/kg per day) added to the drinking water for either a week or a month. After 2 and 4 months of diabetes, the specific activity of retinal total Na,K-ATPase was determined. The components of the activity of Na,K-ATPase caused by the alpha 1 and alpha 3 isoforms were pharmacologically separated by their different sensitivity to ouabain. The activity of ACE in the serum and retina was measured by radioassay using benzoyl-gly-gly-gly as substrate (10(5) cpm, 5 mM). RESULTS: The total Na,K-ATPase activity was decreased significantly after 2 (16%, P < 0.02) and 4 months (15%, P < 0.02) of diabetes. At both time points examined, the activities of the alpha 1-low-ouabain-affinity isoform and the alpha 3-high-ouabain-affinity isoform of retinal Na,K-ATPase were significantly reduced compared to those of age-matched controls (alpha 1, 9% to 14%, P < 0.05; alpha 3, 14% to 19%, P < 0.05 and P < 0.02 respectively). After 1 month of captopril administration, the activities of both Na,K-ATPase isoforms were at control level in 2-month diabetic rats, whereas they were restored only partially in 4-month diabetic rats. In age-matched normal animals, 1 month of captopril treatment did not alter the specific activities of either Na,K-ATPase isoform. One week or 1 month of captopril administration to diabetic rats did not change the activities of retinal Na,K-ATPase isoforms. Serum ACE activity was elevated significantly in both groups of untreated STZ rats (55% and 40%, respectively). One month of captopril administration further increased the ACE levels in 2- and 4-month diabetic rats (101% and 94%, respectively) and also enhanced significantly the serum ACE activity in normal animals (131%) versus the basal values. In contrast, retinal ACE activity was decreased significantly in both groups of untreated STZ rats (approximately 37%). Captopril exerted a significant inhibitory effect on the retinal ACE activity in 2- and 4-month diabetic rats (37% and 31%, respectively) compared to untreated diabetic animals as well as in normal rats (29%). CONCLUSIONS: These data suggest that stimulation of retinal Na,K-ATPase activity in diabetes is most likely one of the mechanisms through which captopril can improve retinal complications. The effect of captopril seems to be related to local effects in the retina. Whether the inhibition of retinal ACE is part of the mechanism of action of captopril requires further study.     

Regulation of Na+,K(+)-ATPase activity by dopamine in cultured rat aortic smooth muscle cells

We investigated the effect of dopamine on Na+,K(+)-ATPase activity in cultured aortic smooth muscle cells. Na+,K(+)- ATPase activity was measured by a coupled enzyme assay. Our results demonstrate that dopamine and dopamine receptor agonists, SKF-38393 (a D1 receptor agonist) and quinpirole (a D2 receptor agonist) produced 62%, 50% and 49% inhibition of Na+,K(+)-ATPase activity in aortic smooth muscle cells, respectively. The combination of the two agonists produced inhibition similar to that of dopamine. Dopamine- and the agonist-induced Na+,K(+)-ATPase inhibition was blocked by selective receptor antagonists. The Na+,K(+)-ATPase inhibition by SKF-38393 but not by quinpirole was abolished by pertussis toxin. Na+,K(+)-ATPase inhibition was also achieved by guanosine triphosphate analog GTP-gamma-S. SKF-38393 but not quinpirole stimulated phosphoinositide hydrolysis rate in rat aortic slices. SKF-38393-induced phosphoinositide hydrolysis stimulation was reversed by SCH-23390, a dopamine D1 receptor antagonist, and attenuated by pertussis toxin. In conclusion, our observations indicate that dopamine and dopamine receptor agonists inhibit Na+,K(+)-ATPase activity through specific vascular receptors. Dopamine D1 receptors are linked to pertussis toxin sensitive-mechanism(s) and a GTP-binding protein appears to be coupled to the enzyme inhibition. Finally, the inhibition of Na+,K(+)-ATPase activity in response to dopamine D1 receptor activation may be mediated by the phospholipase C signaling pathway.     

L-phenylalanine effect on rat brain acetylcholinesterase and Na+,K(+)-ATPase

The effect of different L-phenylalanine (Phe) concentrations (0.1-12.1 mM), on acetylcholinesterase (AChE) and Na+,K(+)-ATPase activities of brain homogenate and pure enzymes, was investigated at 37 degrees C. AChE and Na+,K(+)-ATPase activities were determined according to Ellman G. L., Courtney D., Andres V. and Featherstone R. M. (1961), Biochem. Pharmacol. 7, 88-95 and Bowler K. and Tirri R. (1974), J. Neurochem. 23, 611-613) respectively, after preincubation with Phe. AChE activity in brain homogenate or in pure eel E.electricus enzyme showed a decrease, which reached up to 18% with concentrations of 0.9-12.1 mM. Brain homogenate Na+,K(+)-ATPase activity showed an increase 16-65% with 0.24-0.9 mM of Phe, while an activity increase of 60-65% appeared with 0.9-12.1 mM of Phe. Pure enzyme activity (from porcine cerebral cortex) was not affected by high Phe concentrations, while it was increased by low concentrations. The above results suggest: a) A direct effect of Phe on AChE, b) A direct effect of low Phe concentrations and an indirect effect of high ones on Na+,K(+)-ATPase.     

Effect of methyl isocyanate on rabbit cardiac Na+, K(+)-ATPase

The present study describes the effect of methyl isocyanate (MIC) on rabbit cardiac microsomal Na+, K(+)-ATPase. Addition of MIC in vitro resulted in dose-dependent inhibition of Na+, K(+)-ATPase, Mg(2+)-ATPase and K(+)-activated p-nitrophenyl phosphatase (K(+)-PNPPase). Activation of Na+, K(+)-ATPase by ATP in the presence of MIC showed a decrease in Vmax with no change in Km. Similarly, activation of K+ PNPPase by PNPP in the presence of MIC showed a decrease in Vmax with no change in Km. The circular dichroism spectral studies revealed that MIC interaction with Na+, K(+)-ATPase led to a conformation of the protein wherein the substrates Na+ and K+ were no longer able to bind at the Na(+)- and K(+)-activation sites. The data suggest that the inhibition of Na+, K(+)-ATPase was non-competitive and occurred by interference with the dephosphorylation of the enzyme-phosphoryl complex.     

Inhibition of Na+,K(+)-ATPase by platelet-activating factor in dog submandibular glands

Physiological stimulation of dog submandibular gland has been shown to generate platelet-activating factor (PAF). However, PAF is not released from cells in the tissue. To assess its intracellular activity, the effect of PAF on Na+,K(+)-ATPase was examined in dog submandibular gland cells. PAF inhibited Na+,K(+)-ATPase in membrane preparations, and the inhibitory effect was dependent on the protein concentration in the enzyme preparation. The inhibitory effect of a low concentration of PAF was antagonized by a PAF-receptor antagonist, BN 50,739, but at high concentrations, PAF was not antagonized. Kinetic analysis of PAF inhibition of Na+,K(+)-ATPase suggests that the inhibition of Na+,K(+)-ATPase by PAF is not due to competition by PAF at K(+)- or Na(+)-binding sites on the enzyme, but by complex inhibitory mechanisms. These results suggest that PAF may interact with specific and nonspecific site of action resulting in the inhibition of Na+,K(+)-ATPase. Ouabain increased mucin release from dog submandibular gland cells. Because Na+,K(+)-ATPase and ion exchange pathways are important in the secretory responses of acinar cells, PAF may regulate intracellularly the secretory function of acinar cells by modulating Na+,K(+)-ATPase and ionic homeostasis.     

A putative fourth Na+,K(+)-ATPase alpha-subunit gene is expressed in testis

The Na+,K(+)-ATPase alpha subunit has three known isoforms, alpha 1, alpha 2 and alpha 3, each encoded by a separate gene. This study was undertaken to determine the functional status of a fourth human alpha-like gene, ATP1AL2. Partial genomic sequence analysis revealed regions exhibiting sequence similarity with exons 3-6 of the Na+,K(+)-ATPase alpha isoform genes. ATP1AL2 cDNAs spanning the coding sequence of a novel P-type ATPase alpha subunit were isolated from a rat testis library. The predicted polypeptide is 1028 amino acids long and exhibits 76-78% identity with the rat Na+,K(+)-ATPase alpha 1, alpha 2 and alpha 3 isoforms, indicating that ATP1AL2 may encode a fourth Na+,K(+)-ATPase alpha isoform. A 3.9-kb mRNA is expressed abundantly in human and rat testis.     

Endothelin 1 stimulates Na+,K(+)-ATPase and Na(+)-K(+)-Cl- cotransport through ETA receptors and protein kinase C-dependent pathway in cerebral capillary endothelium

The effect of endothelins (ET-1 and ET-3) on 86Rb+ uptake as a measure of K+ uptake was investigated in cultured rat brain capillary endothelium. ET-1 or ET-3 dose-dependently enhanced K+ uptake (EC50 = 0.60 +/- 0.15 and 21.5 +/- 4.1 nM, respectively), which was inhibited by the selective ETA receptor antagonist BQ 123 (cyclo-D-Trp-D-Asp-Pro-D-Val-Leu). Neither the selective ETB agonists IRL 1620 [N-succinyl-(Glu9,-Ala11,15)-ET-1] and sarafotoxin S6c, nor the ETB receptor antagonist IRL 1038 [(Cys11,Cys15)-ET-1] had any effect on K+ uptake. Ouabain (inhibitor of Na+,K(+)-ATPase) and bumetanide (inhibitor of Na(+)-K(+)-Cl- cotransport) reduced (up to 40% and up to 70%, respectively) the ET-1-stimulated K+ uptake. Complete inhibition was seen with both agents. Phorbol 12-myristate 13-acetate (PMA), activator of protein kinase C (PKC), stimulated Na+,K(+)-ATPase and Na(+)-K(+)-Cl- cotransport. ET-1- but not PMA-stimulated K+ uptake was inhibited by 5-(N-ethyl-N-isopropyl)amiloride (inhibitor of Na+/H+ exchange system), suggesting a linkage of Na+/H+ exchange with ET-1-stimulated Na+,K(+)-ATPase and Na(+)-K(+)-Cl- cotransport activity that is not mediated by PKC.     

Functional analysis and tissue-specific expression of Drosophila Na+,K+-ATPase subunits

We have previously purified and characterized a nervous system-specific glycoprotein antigen from adult Drosophila heads, designated Nervana [nerve antigen (NRV)] and identified two separate genes coding for three different proteins. All three proteins share homology with the beta subunits of Na+,K+-ATPase from various other species. In this study we have isolated a new Drosophila Na+,K+-ATPase alpha subunit cDNA clone (PSalpha; GenBank accession no. AF044974) and demonstrate expression of functional Na+,K+-ATPase activity when PSalpha mRNA is coinjected into Xenopus oocytes along with any of the three different Nrv mRNAs. Western blotting, RNase protection assays, and immunocytochemical staining of adult fly sections indicate that NRV2 is expressed primarily in the nervous system. Staining is most intense in the brain and thoracic ganglia and is most likely associated with neuronal elements. NRV1 is more broadly expressed in muscle and excretory tissue and also shows diffuse distribution in the nervous system. Similar to other species, Drosophila expresses multiple isoforms of Na+,K+-ATPase subunits in a tissue- and cell type-specific pattern. It will now be possible to use the advantages of Drosophila molecular and classical genetics to investigate the phenotypic consequences of altering Na+,K+-ATPase expression in various cell and tissue types.     

Measurement of the total concentration of functional Na+, K(+)-pumps in rumen epithelium

Using the technique of vanadate-facilitated [3H]ouabain binding we have developed a simple and reliable assay for measuring the concentration of [3H]ouabain binding sites in small fresh or frozen biopsies of rumen epithelium papillae. In bovine and ovine rumen epithelium obtained from the cranio-ventral rumen sac the concentration of [3H]ouabain binding sites was 1.6-4.9 nmol g dry wt-1 (n = 32) and 3.7-5.2 nmol g dry wt-1 (n = 6), respectively. When incubated in oxygenated Krebs-Ringer bicarbonate buffer fresh biopsies of rumen epithelium maintained a high K+ and low Na+ content for at least 6 h. Na+ loading of the biopsies induced about 20-fold increase of the Na+, K(+)-pump activity based on measurement of ouabain suppressible net [86Rb+] influx. The ouabain suppressible net influx of [86Rb+] measured in Na+ loaded biopsies showed a close correlation to the [3H]ouabain binding capacity (r = 0.80, P < 0.01) and corresponded to 47 +/- 2% (n = 9) of the theoretical maximum flux rate. The ouabain suppressible net influx of K+ and [86Rb+] were linearly related (r = 0.73; P < 0.001). The net Na+ efflux was 1.21 times the net K+ influx. It is concluded that rumen epithelium has a large capacity for active Na+/K+ transport and that there is agreement between the concentration of [3H]ouabain binding sites in the epithelium and the ouabain suppressible rate of net [86Rb+] influx in Na+ loaded biopsies in spite of some uncertainty about the maximum turnover number of the Na+, K(+)-pump in rumen epithelium.     

Inhibitory effect of several nitric oxide-generating compounds on purified Na+,K(+)-ATPase activity from porcine cerebral cortex

Nitric oxide (NO)-generating compounds (NO donors) such as sodium nitroprusside, S-nitroso-N-acetylpenicillamine, S-nitroso-L-glutathione, 3-morpholino-sydnonimine (SIN-1), (DL)-(E)-ethyl-2-[(E)-hydroxyimino]-5-nitro-5-3-hexenamide, and 1-hydroxy-2-oxo-3-(N-methyl-3-aminopropyl)-3-methyl-1-triazene inhibited the Na+,K(+)-ATPase activity purified from porcine cerebral cortex. NO-reducing or -scavenging agents, such as superoxide dismutase or N-(dithiocarbamate)-N-methyl-D-glucamine sodium salt, L-ascorbic acid; and sulfhydryl (SH) compounds, such as dithiothreitol or the reduced form of glutathione, but not alpha-tocopherol, prevented the inhibition of the enzyme activity by all NO donors except sodium nitroprusside. Enzyme inhibition could also be reversed by these SH compounds, but not by superoxide dismutase, L-ascorbic acid, and alpha-tocopherol. 2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazolin-1-oxyl 3-oxide (PTIO), which is able to scavenge NO radicals and generate nitrogen dioxide radicals (.NO2), potentiated the inhibition of this enzyme activity induced by all NO donors (except SIN-1). PTIO did not potentiate, but rather attenuated, the SIN-1-induced inhibition. SIN-1 has been reported to release both NO and superoxide and thereby to rapidly form peroxynitrite (ONOO-). These potentiated and attenuated inhibitions of the enzyme activity induced by PTIO plus all of the NO donors except sodium nitroprusside were prevented by SH compounds, but not by superoxide dismutase, L-ascorbic acid, and alpha-tocopherol. These results suggest that NO donors may release NO or NO-derived products, presumably .NO2 and ONOO-, and may inhibit the Na+,K(+)-ATPase activity by interacting with a SH group at the active site of the enzyme.