Quantitative analysis of the interaction between the fluorescent probe eosin and the Na+/K+-ATPase studied through Rb+ occlusion.

We report a study on the effect of the fluorescent probe eosin on some of the reactions involved in the conformational transitions that lead to the occlusion of the K(+)-congener Rb(+) in the Na(+)/K(+)-ATPase. Eosin decreases the equilibrium levels of occluded Rb(+), this effect being fully attributable to a decrease in the apparent affinity of the enzyme for Rb(+) since the capacity for occlusion remains independent of eosin concentration. The results can be quantitatively described by a model that assumes that two molecules of eosin are able to bind to the Na(+)/K(+)-ATPase, both to the Rb(+)-free and to the Rb(+)-occluded enzyme regardless of the degree of cation occlusion. Concerning the effect on the affinity for Rb(+) occlusion, transient state experiments show that eosin reduces the initial velocity of occlusion, and that, like ATP, it increases the velocity of deocclusion of Rb(+). Interactions between eosin and ATP on Rb(+)-release experiments seem to indicate that eosin binds to the low-affinity site of ATP from which it exerts effects that are similar to those of the nucleotide.

Trafficking of Na,K-ATPase fused to enhanced green fluorescent protein is mediated by protein kinase A or C.

Fusion of enhanced green fluorescent protein (EGFP) to the C-terminal of rat Na,K-ATPase a1-subunit is introduced as a novel procedure for visualizing trafficking of Na,K-pumps in living COS-1 renal cells in response to PKA or PKC stimulation. Stable, functional expression of the fluorescent chimera (Na,K-EGFP) was achieved in COS-1 cells using combined puromycin and ouabain selection procedures. Na,K-pump activities were unchanged after fusion with EGFP, both in basal and regulated states. In confocal laser scanning and fluorescence microscopes, the Na,K-EGFP chimera was distributed mainly along the plasma membrane of COS cells. In unstimulated COS cells, Na,K-EGFP was also present in lysosomes and in vesicles en route from the endoplasmic reticulum to the plasma membrane, but it was almost absent from recycling endosomes labelled with fluorescent transferrin. After activation of protein kinase A or C, the density of co-localizing Na,K-EGFP and transferrin vesicles was increased 3-4-fold, while the ouabain-sensitive 86Rb uptake was reduced by 22%. Simultaneous activation of PKA and PKC had additive effects with a 6-fold increase of co-localization and a 38% reduction of 86Rb uptake. Responses of similar magnitude were seen after inhibition of protein phosphatase by okadaic acid. Reduction of the amount of Na,K-ATPase in surface plasma membranes through internalization in recycling endosomes may thus in part explain a decrease in Na,K-pump activity following protein kinase activation or protein phosphatase inhibition.

Uptake of reconstituted Na,K-ATPase vesicles by isolated lymphocytes measured by FACS, confocal microscopy and spectrofluorometry.

Na,K-ATPase (EC 3.6.1.37, Na,K-ATPase) is a fundamental vital membrane transport and receptor system which, after biosynthesis, is exported to the plasma membrane in inside-out vesicles. Na,K-ATPase can be extracted form the natural membrane and inserted into artificially formed phosphatidylcholine vesicles (liposomes). The ultrastructure of the reconstituted vesicles has been fully described. In the present work, the Na,K-ATPase-vesicles were labeled with fluorescent tracers either in their water or membrane phase, incubated with freshly isolated human lymphocytes, and the resulting cellular fluorescence measured with fluorescence activated cell sorting (FACS), confocal microscopy and spectrofluorometry. The FACS data show that all lymphocytes take up Na,K-ATPase-vesicles in a dose- and temperature-dependent fashion. Three-dimensional analysis of the fluorescence by confocal microscopy reveals that the fluorescence is contained within the cells. Quantitative determination by spectrofluorometry indicates that depending on the vesicle/cell ratio, a single lymphocyte takes up 650 to 36,500 vesicles within 30 min at 37 degrees C together with up to about 200,000 renal Na,K-ATPase molecules.

Inhibition of Na,K-ATPase by an endotoxin extracted from Leptospira interrogans: a possible mechanism for the physiopathology of leptospirosis.

Clinical manifestations of leptospirosis include disorders of the electrolytical balance which might be related to inhibition of Na,K-ATPase. Although the physiopathological cellular mechanism of leptospirosis remains unknown, a bacterial endotoxin has been incriminated. Therefore, we evaluated whether a glycolipoprotein fraction extracted from Leptospira interrogans and known to be cytotoxic might inhibit Na,K-ATPase. This glycolipoprotein fraction (GLP) inhibited Na,K-ATPase activity in rabbit kidney epithelial cells as well as Na,K-ATPase purified from rabbit kidney medulla. Inhibition was dose-dependent, and at maximum it almost abolished Na,K-ATPase activity whereas it had no effect on other enzymes. The GLP did not change the apparent affinity of Na,K-ATPase for potassium whereas it increased that for sodium, revealing a mechanism of inhibition different from that of ouabain. Finally, the inhibitory principle present in the GLP preparation was thermostable and was curtailed by the presence of albumin. In conclusion, a glycolipoproteic fraction extracted from Leptospira interrogans contains a specific inhibitor of Na,K-ATPase. This glycolipoproteic fraction which is present in diseased tissues might induce, through this inhibitor, cellular dysfunctions responsible for the symptoms, in particular those associated with electrolytical disorders such as disturbances of renal electrolyte handling, cardiac arrhythmia or diarrhoea.

Mechanisms of brain ion homeostasis during acute and chronic variations of plasma potassium.

Brain and CSF potassium concentrations are well regulated during acute and chronic alterations of plasma potassium. In a previous study, we have shown that during chronic perturbations, regulation is achieved by appropriate adaptation of potassium influx, but that the degree of such adaptation during acute perturbations is much less. To elucidate further potential regulatory mechanisms, rats were rendered acutely or chronically hyper- or hypokalemic (range 2.7-7.6 mM). Measurements were made of brain and CSF water and ion contents to examine whether regulation occurred by modulation of K+ uptake into parenchymal cells. Furthermore, the permeability-surface area products (PSs) of 22Na+ were determined, because changes in K+ efflux fia Na+,K(+)-ATPase on the brain-facing side of the blood-brain barrier might be reflected in modified Na+ permeability. Brain and CSF K+ concentrations and Na PS were all independent of chronic changes in plasma K+ and acute hypokalemia, suggesting that neither modulation of parenchymal K+ uptake nor K+ efflux via the Na+,K(+)-ATPase is involved in extracellular K+ regulation in these conditions. In contrast, Na PSs were increased by 40% (p < 0.05) in acute hyperkalemia. This was accompanied by a slight loss of tissue K+ and water from the intracellular space. These results suggest that increased potassium influx in acute hyperkalemia is compensated by stimulation of K+ efflux via Na+,K(+)-ATPase. A slight degree of overstimulation, as indicated by a net loss of tissue K+, leads us to hypothesize that other factors, apart from the kinetic characteristics of Na+,K(+)-ATPase, may regulate this enzyme at the blood-brain barrier.

A serial study of erythrocyte sodium pump kinetics and sodium content in the puerperium.

OBJECTIVE: Our purpose was to describe the alterations in erythrocyte sodium pump kinetics and sodium content occurring during the puerperium. STUDY DESIGN: Twelve healthy primigravid women were studied serially from late pregnancy until 20 weeks after delivery. Erythrocyte sodium pump rate constant, maximum velocity, and sodium affinity were calculated from the ouabain-sensitive sodium flux measured in whole blood and in erythrocytes in which sodium content had been altered with the ionophore nystatin. The Student t test was used to compare the regression coefficients of the values plotted against log time for specific periods. RESULTS: The sodium pump rate constant, maximum velocity, and sodium affinity were lower 20 weeks after delivery than in late pregnancy (0.339 +/- 0.018 vs 0.399 +/- 0.016/hr, 7.02 +/- 0.08 vs 9.98 +/- 0.078 mmol/kg/hr, 2.65 +/- 0.21 vs 3.16 +/- 0.20 mmol/kg). The decrease in the rate constant commenced after 4 days of the puerperium, whereas the decrease in maximum velocity and Michaelis-Menten constant did not commence until after 2 weeks. Erythrocyte sodium content was greater 20 weeks after delivery than in late pregnancy (4.71 +/- 0.20 vs 4.14 +/- 0.15 mmol/kg cells) and the increase was gradual over the time studied. CONCLUSIONS: After delivery the rate constant of the sodium pump measured in plasma and the erythrocyte sodium content changed before any significant alteration in the maximum velocity of the pump. The return of sodium pump function to the nonpregnant state continues beyond 6 weeks after delivery.

[A kinetic analysis of the action of a synaptosomal factor on Na, K-ATPase]. / Kineticheskii analiz deistviia sinaptosomal'nogo faktora na Na, K-ATFazu.

A synaptosomal factor stimulated by neurotransmitters activates the Na, K-ATPase system effecting the phosphorylating intermediates moving the Na, K-ATPase system in the mode of simultaneous transport of Na+ and K+. This conclusion has been made during the analysis of kinetics of the effect of MgATP complex, free Mg2+ ions and ATP on Na, K-ATPase activity. Unlike the EGTA, the factor under study does not change the number of essential activators (ions of Na+ and K+) of the Na, K-ATPase system at the equimolar ATP and Mg2+ correlation.

Changes in Na,K-ATPase, sodium ion, and glucose transport in isolated enterocytes in an experimental model of malabsorption.

Nippostrongylus brasiliensis infection of the rat resulted, at day 10 of infection, in decreased levels of jejunal enterocyte sodium-potassium-activated adenosine triphosphatase (Na,K-ATPase) and potassium-activated p-nitrophenyl phosphatase (K-pNPPase) activities. Parallel decreases occurred in active sodium efflux from jejunal enterocytes in the presence and absence of actively transported monosaccharides. Ileal enterocyte Na,K-ATPase and K-pNPPase activities were significantly increased, as was active sodium efflux. In contrast to controls, the presence of monosaccharides produced a stimulation of active sodium efflux from ileal enterocytes derived from infected rats. Enzyme and sodium transport changes in the jejunal enterocytes probably reflect cellular immaturity. Functional changes in ileal enterocytes probably represent a compensatory phenomenon.

Isoforms of Na,K-ATPase in Artemia salina: II. Tissue distribution and kinetic characterization.

To characterize the molecular properties conveyed by the isoforms of the alpha subunit of Na,K-ATPase, the two major transepithelial transporting organs in the brine shrimp (Artemia salina), the salt glands and intestines, were isolated in pure form. The alpha isoforms were quantified by ATP-sensitive fluorescein isothiocyanate (FITC) labeling. The salt gland enzyme exhibits only the alpha 1 isoform, whereas the intestinal enzyme exhibits both the alpha 1 and the alpha 2 isoforms. After 32 hours of development, Na,K-ATPase activity [in mumol Pi/mg protein/hr (1 mu)] in whole homogenates was 32 +/- 6 in the salt glands and 12 +/- 3 in the intestinal preparations (mean +/- SEM). The apparent half-maximal activation constants (K1/2) of the salt gland enzyme as compared to the intestinal enzyme were 3.7 +/- 0.6 mM vs. 23.5 +/- 4 mM (P less than 0.01) for Na+, 16.6 +/- 2.2 mM vs. 8.29 +/- 1.5 mM for K+ (P less than 0.01), and 0.87 +/- 0.8 mM vs. 0.79 +/- 1.1 mM for ATP (NS). The apparent Ki's for ouabain inhibition were 1.1 x 10(-4) M vs. 2 x 10(-5) M, respectively. Treatment of whole homogenates with deoxycholic acid (DOC) produced a maximal Na,K-ATPase activation of 46% in the salt gland as compared to 23% in the intestinal enzyme. Similar differences were found with sodium dodecyl sulfate (SDS). The two distinct forms of Na,K-ATPase isolated from the brine shrimp differed markedly in three kinetic parameters as well as in detergent sensitivity.(ABSTRACT TRUNCATED AT 250 WORDS)

The reaction mechanism of Na,K-ATPase: problems regarding the oligomeric/monomeric state and the phosphorylated intermediates of the system.

1. The present overview is concerned with the monomeric/oligomeric structure of Na,K-ATPase and with the number and kinetic competence of the phosphorylated intermediates. 2. The transport system, like many other ion-transporters, seems to be oligomeric in situ. It is, however, uncertain whether the oligomeric structure is of functional importance or not. 3. Phosphorylated intermediates (EP) play a central role in the coupling of ATP-hydrolysis to transport. Occlusion of Na+ takes place concomitantly with EP-formation. 4. Two recent revisionist ideas regarding the phosphorylated intermediates are emphasized: First, there are not two but several different EP-conformations, and second, some of the EP's formed in the presence of Na+ alone are incompetent as intermediates in the Na,K-ATPase reaction. This concept requires a revision of the classical Albers-Post scheme for Na,K-ATPase. 5. It is suggested that the classical notion of two principal conformational states E1 and E2 be abandoned. Instead, one should consider that there are many conformational states of the enzyme protein, partly due to the many different liganded states and partly due to subconformations within each enzyme-ligand complex.