Neurotensin effect on Na+, K+-ATPase is CNS area- and membrane-dependent and involves high affinity NT1 receptor

We have previously shown that peptide neurotensin inhibits cerebral cortex synaptosomal membrane Na⁺, K⁺-ATPase, an effect fully prevented by blockade of neurotensin NT₁ receptor by antagonist SR 48692. The work was extended to analyze neurotensin effect on Na⁺, K⁺-ATPase activity present in other synaptosomal membranes and in CNS myelin and mitochondrial fractions. Results indicated that, besides inhibiting cerebral cortex synaptosomal membrane Na⁺, K⁺-ATPase, neurotensin likewise decreased enzyme activity in homologous striatal membranes as well as in a commercial preparation obtained from porcine cerebral cortex. However, the peptide failed to alter either Na⁺, K⁺-ATPase activity in cerebellar synaptosomal and myelin membranes or ATPase activity in mitochondrial preparations. Whenever an effect was recorded with the peptide, it was blocked by antagonist SR 48692, indicating the involvement of the high affinity neurotensin receptor (NT₁), as well as supporting the contention that, through inhibition of ion transport at synaptic membrane level, neurotensin plays a regulatory role in neurotransmission.     

Ouabain sensitivity of Na+,K+-ATPase in neuronal membranes]

Na+, K(+)-ATPase preparations of the rat and bovine brain and kidney were studied for ouabain sensitivity. Differences in apparent affinities to inhibitor of alpha(+)- and alpha-isozymes of Na+, K(+)-ATPase catalytic subunit were detected only in rat tissues but not in bovine ones. It is concluded that glycoside-sensitive and glycoside-resistant enzymic forms are not fully identical to alpha(+)- and alpha-subunit forms of Na+, K(+)-ATPase.     

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

A computer simulation of the electrogenic nature of the membrane-boundNa⁺, K⁺-ATPase is presented. The model involves coupling twosimulation systems for passive and active transports, usinga minimum of empirical parameters, and studies the contributionof the pump to the membrane potential. The simulation resultsindicate that electrogenic active transport accelerates therestoration of the resting electrochemical gradients and contributes0.44–1.1 mV to the resting potential of the membrane,depending on the Na:K coupling ratio. The effect of membranepotential and the physical positioning of the enzyme from thepassive transporting channel on the enzyme function is alsopresented. The validity of the model is checked by comparingour results with reported literature values.     

Palytoxin Acts on Na+,K+-ATPase but not Nongastric H+,K+-ATPase

Palytoxin (PTX) opens a pathway for ions to pass through Na,K-ATPase. We investigate here whether PTX also acts on nongastric H,K-ATPases. The following combinations of cRNA were expressed in Xenopus laevis oocytes: Bufo marinus bladder H,K-ATPase α₂- and Na,K-ATPase β₂-subunits; Bufo Na,K-ATPase α₁- and Na,K-ATPase β₂-subunits; and Bufo Na,K-ATPase β₂-subunit alone. The response to PTX was measured after blocking endogenous Xenopus Na,K-ATPase with 10 μm ouabain. Functional expression was confirmed by measuring ⁸⁶Rb uptake. PTX (5 nm) produced a large increase of membrane conductance in oocytes expressing Bufo Na,K-ATPase, but no significant increase occurred in oocytes expressing Bufo H,K-ATPase or in those injected with Bufo β₂-subunit alone. Expression of the following combinations of cDNA was investigated in HeLa cells: rat colonic H,K-ATPase α₁-subunit and Na,K-ATPase β₁-subunit; rat Na,K-ATPase α₂-subunit and Na,K-ATPase β₂-subunit; and rat Na,K-ATPase β₁- or Na,K-ATPase β₂-subunit alone. Measurement of increases in ⁸⁶Rb uptake confirmed that both rat Na,K and H,K pumps were functional in HeLa cells expressing rat colonic HKα₁/NKβ₁ and NKα₂/NKβ₂. Whole-cell patch-clamp measurements in HeLa cells expressing rat colonic HKα₁/NKβ₁ exposed to 100 nm PTX showed no significant increase of membrane current, and there was no membrane conductance increase in HeLa cells transfected with rat NKβ₁- or rat NKβ₂-subunit alone. However, in HeLa cells expressing rat NKα₂/NKβ₂, outward current was observed after pump activation by 20 mm K⁺ and a large membrane conductance increase occurred after 100 nm PTX. We conclude that nongastric H,K-ATPases are not sensitive to PTX when expressed in these cells, whereas PTX does act on Na,K-ATPase.     

Effect of glycosphingolipids on erythrocyte Na+,K+-ATPase activity

The total fractions of gangliosides and cerebrosides isolated from the tissue of human brain were studied for their effect on the Na+, K+-ATPase activity of native erythrocytes and their membranes. It is shown that gangliosides depending on time of their preincubation with the enzyme preparation and concentration produce both the activating and inhibiting action and cerebrosides--only the inhibiting one. Gangliosides inhibit the transport ATPase activity noncompetitively with respect to ATP and Na+ and competitively--to K+, cerebrosides inhibit it noncompetitively with respect to all ATPase activators.     

Na+,K+ -ATPase activity characteristics in human colon adenocarcinoma

To evaluate the enzyme functional changes the Na+,K+-ATPase activity in membrane fraction of human colorectal adenocarcinoma at II and III cancer stages (according to TNM classification) of varying degrees of differentiation has been investigated. The decrease of the Na+,K+-ATPase activity in comparison with conditionally normal tissue of macroscopically unchanged mucosa was revealed in the tumor membrane preparations. Such changes of the Na+,K+-ATPase activity were higher at low differentiation grade and were less pronounced in moderately and highly differentiated adenocarcinomas. At the same time the changes in Na+,K+-ATPase activity have not been revealed between tumor membrane preparations at studied cancer stages when the degree of differentiation was not taken into account. It is supposed that Na+,K+-ATPase functional specificity occurs in colorectal adenocarcinomas and it is associated with tumor differentiation.     

Structural insights into the high affinity binding of cardiotonic steroids to the Na+,K+-ATPase

The Na+,K+-ATPase belongs to the P-ATPase family, whose characteristic property is the formation of a phosphorylated intermediate. The enzyme is also a defined target for cardiotonic steroids which inhibit its functional activity and initiate intracellular signaling. Here we describe the 4.6 ? resolution crystal structure of the pig kidney Na+,K+-ATPase in its phosphorylated form stabilized by high affinity binding of the cardiotonic steroid ouabain. The steroid binds to a site formed at transmembrane segments αM1-αM6, plugging the ion pathway from the extracellular side. This structure differs from the previously reported low affinity complex with potassium. Most importantly, the A domain has rotated in response to phosphorylation and αM1-2 move towards the ouabain molecule, providing for high affinity interactions and closing the ion pathway from the extracellular side. The observed re-arrangements of the Na+,K+-ATPase stabilized by cardiotonic steroids may affect protein-protein interactions within the intracellular signal transduction networks.     

The beta-subunits of Na+,K+-ATPase and gastric H+,K+-ATPase have a high preference for their own alpha-subunit and affect the K+ affinity of these enzymes

The alpha- and beta-subunits of Na+,K+-ATPase and H+,K+-ATPase were expressed in Sf9 cells in different combinations. Immunoprecipitation of the alpha-subunits resulted in coprecipitation of the accompanying beta-subunit independent of the type of beta-subunit. This indicates cross-assembly of the subunits of the different ATPases. The hybrid ATPase with the catalytic subunit of Na+,K+-ATPase and the beta-subunit of H+,K+-ATPase (NaKalphaHKbeta) showed an ATPase activity, which was only 12 +/- 4% of the activity of the Na+,K+-ATPase with its own beta-subunit. Likewise, the complementary hybrid ATPase with the catalytic subunit of H+,K+-ATPase and the beta-subunit of Na+,K+-ATPase (HKalphaNaKbeta) showed an ATPase activity which was 9 +/- 2% of that of the recombinant H+,K+-ATPase. In addition, the apparent K+ affinity of hybrid NaKalphaHKbeta was decreased, while the apparent K+ affinity of the opposite hybrid HKalphaNaKbeta was increased. The hybrid NaKalphaHKbeta could be phosphorylated by ATP to a level of 21 +/- 7% of that of Na+,K+-ATPase. These values, together with the ATPase activity gave turnover numbers for NaKalphabeta and NaKalphaHKbeta of 8800 +/- 310 min-1 and 4800 +/- 160 min-1, respectively. Measurements of phosphorylation of the HKalphaNaKbeta and HKalphabeta enzymes are consistent with a higher turnover of the former. These findings suggest a role of the beta-subunit in the catalytic turnover. In conclusion, although both Na+,K+-ATPase and H+,K+-ATPase have a high preference for their own beta-subunit, they can function with the beta-subunit of the other enzyme, in which case the K+ affinity and turnover number are modified.     

Arrestins and spinophilin competitively regulate Na+,K+-ATPase trafficking through association with a large cytoplasmic loop of the Na+,K+-ATPase

The activity and trafficking of the Na(+),K(+)-ATPase are regulated by several hormones, including dopamine, vasopressin, and adrenergic hormones through the action of G-protein-coupled receptors (GPCRs). Arrestins, GPCR kinases (GRKs), 14-3-3 proteins, and spinophilin interact with GPCRs and modulate the duration and magnitude of receptor signaling. We have found that arrestin 2 and 3, GRK 2 and 3, 14-3-3 epsilon, and spinophilin directly associate with the Na(+),K(+)-ATPase and that the associations with arrestins, GRKs, or 14-3-3 epsilon are blocked in the presence of spinophilin. In COS cells that overexpressed arrestin, the Na(+),K(+)-ATPase was redistributed to intracellular compartments. This effect was not seen in mock-transfected cells or in cells expressing spinophilin. Furthermore, expression of spinophilin appeared to slow, whereas overexpression of beta-arrestins accelerated internalization of the Na(+),K(+)-ATPase endocytosis. We also find that GRKs phosphorylate the Na(+),K(+)-ATPase in vitro on its large cytoplasmic loop. Taken together, it appears that association with arrestins, GRKs, 14-3-3 epsilon, and spinophilin may be important modulators of Na(+),K(+)-ATPase trafficking.     

Na+,K+-ATPase activity in cultured C6 glioma cells

Rat C₆ glioma cells were cultured for 4 days in MEM medium supplemented with 10% bovine serum and Na⁺,K⁺-ATPase activity was determined in homogenates of harvested cells. Approximately 50% of enzyme activity was attained at 1.5 mM K⁺ and the maximum (2.76±0.13 mol Pi/h/mg protein) at 5 mM K⁺. The specific activity of Na⁺,K⁺-ATPase was not influenced by freezing the homogenates or cell suspensions before the enzyme assay. Ten minutes' exposure of glioma cells to 10^–4 or 10^–5 M noradrenaline (NA) remained without any effect on NA⁺,K⁺-ATPase activity. Neither did the presence of NA in the incubation medium, during the enzyme assay, influence the enzyme activity. The nonresponsiveness of Na⁺,K⁺-ATPase of C₆ glioma cells to NA is consistent with the assumption that (+) form of the enzyme may be preferentially sensitive to noradrenaline. Na⁺,K⁺-ATPase was inhibited in a dose-dependent manner by vanadate and 50% inhibition was achieved at 2×10^–7 M concentration. In spite of the fact that Na⁺,K⁺-ATPase of glioma cells was not responsive to NA, the latter could at least partially reverse vanadate-induced inhibition of the enzyme. Although the present results concern transformed glial cells, they suggest the possibility that inhibition of glial Na⁺,K⁺-ATPase may contribute to the previously reported inhibition by vanadate of Na⁺,K⁺-ATPase of the whole brain tissue.     

Time-dependent effect of leptin on renal Na+,K+-ATPase activity

Leptin, secreted by adipose tissue, is involved in the pathogenesis of arterial hypertension, however, the mechanisms through which leptin increases blood pressure are incompletely elucidated. We investigated the effect of leptin, administered for different time periods, on renal Na(+),K(+)-ATPase activity in the rat. Leptin was infused under anesthesia into the abdominal aorta proximally to the renal arteries for 0.5-3 h. Leptin administered at doses of 1 and 10 microg/min per kg for 30 min decreased the Na(+),K(+)-ATPase activity in the renal medulla. This effect disappeared when the hormone was infused for > or =1 h. Leptin infused for 3 h increased the Na(+),K(+)-ATPase activity in the renal cortex and medulla. The stimulatory effect was abolished by a specific inhibitor of Janus kinases (JAKs), tyrphostin AG490, as well as by an NAD(P)H oxidase inhibitor, apocynin. Leptin increased urinary excretion of hydrogen peroxide (H(2)O(2)) between 2 and 3 h of infusion. The effect of leptin on renal Na(+),K(+)-ATPase and urinary H(2)O(2) was augmented by a superoxide dismutase mimetic, tempol, and was abolished by catalase. In addition, infusion of H(2)O(2) for 30 min increased the Na(+),K(+)-ATPase activity. Inhibitors of extracellular signal regulated kinases (ERKs), PD98059 or U0126, prevented Na(+),K(+)-ATPase stimulation by leptin and H(2)O(2). These data indicate that leptin, by acting directly within the kidney, has a delayed stimulatory effect on Na(+),K(+)-ATPase, mediated by JAKs, H(2)O(2) and ERKs. This mechanism may contribute to the abnormal renal Na(+) handling in diseases associated with chronic hyperleptinemia such as diabetes and obesity.     

Guanidinium derivatives act as high affinity antagonists of Na+ ions in occlusion sites of Na+,K(+)-ATPase.

We have screened various alkyl- and arylguanidinium derivatives as possible competitors of Na+ or Rb+ for the cation sites on renal Na+,K(+)-ATPase. Alkyl-monoguanidinium or alkylbisguanidinium (BisG) compounds (chain lengths of C3 to C10) competitively inhibit the occlusion of Rb+ and Na+ with an order of affinities C10 greater than C8 greater than C6 greater than C4 greater than C3. BisG compounds are approximately twice as effective as the equivalent alkylmonoguanidinium compounds. In media of high ionic strength, affinities of tens of micromolar are observed, e.g. 26 microM for BisG 8. m-(mXBG)- and p-xylylenebisguanidinium were synthesized and were found to compete with Rb+ or Na+ with intrinsic affinities of 7.7 and 8.2 microM, respectively. The hydrophobicity rather than the degree of proximity of the guanidinium groups in all BisG compounds appears to determine the binding affinity. A systematic search has been made of conditions in occlusion assays for which the inhibitor affinities are highest. When the pH is raised from 7.0 to 8.5, a 5-fold increase in affinity is observed, suggesting that the guanidinium derivatives compete with protons at sites of pKa approximately 7.5. Replacing Tris-HCl with choline chloride-containing media raised apparent affinities approximately 2-fold. All guanidinium derivatives stabilize the E1 conformation of fluorescein-labeled Na+,K(+)-ATPase, acting as competitive Na+ analogues. In media containing only 1 mM Tris-HCl, pH 8.55, very high affinities were observed for binding to the fluorescein-labeled enzyme (e.g. 0.08 microM for mXBG). In very low ionic strength medium, the inhibition was still competitive with Rb+ ions. However, there was also evidence for nonspecific adsorption to the membranes. The following findings show that mXBG, a typical guanidinium derivative, behaves as a Na(+)-like antagonist. (a) It inhibits Na+,K(+)-ATPase activity, competing strongly with Na+ but only weakly with K+ ions. (b) It inhibits phosphorylation from ATP, competing with Na+ ions. (c) Like Na+ ions, it blocks phosphorylation from inorganic phosphate. Based on these results, we propose that the guanidinium group binds to a relatively wide vestibule at the cytoplasmic surface; but, unlike Na+ or K+ ions, it cannot pass into a narrower region of the cation transport path within the membrane. Therefore, it blocks the occlusion and active transport of cations. In the future, high affinity guanidinium derivatives may serve the purpose of locating cation-binding domains of the pump protein after being converted to reactive affinity or photoaffinity covalent labels.     

Interaction of N-terminal peptide analogues of the Na+,K+-ATPase with membranes

The Na⁺,K⁺-ATPase, which is present in the plasma membrane of all animal cells, plays a crucial role in maintaining the Na⁺ and K⁺ electrochemical potential gradients across the membrane. Recent studies have suggested that the N-terminus of the protein's catalytic α-subunit is involved in an electrostatic interaction with the surrounding membrane, which controls the protein's conformational equilibrium. However, because the N-terminus could not yet be resolved in any X-ray crystal structures, little information about this interaction is so far available. In measurements utilising poly-l-lysine as a model of the protein's lysine-rich N-terminus and using lipid vesicles of defined composition, here we have identified the most likely origin of the interaction as one between positively charged lysine residues of the N-terminus and negatively charged headgroups of phospholipids (notably phosphatidylserine) in the surrounding membrane. Furthermore, to isolate which segments of the N-terminus could be involved in membrane binding, we chemically synthesized N-terminal fragments of various lengths. Based on a combination of results from RH421 UV/visible absorbance measurements and solid-state ³¹P and ²H NMR using these N-terminal fragments as well as MD simulations it appears that the membrane interaction arises from lysine residues prior to the conserved LKKE motif of the N-terminus. The MD simulations indicate that the strength of the interaction varies significantly between different enzyme conformations.     

Glucagon stimulation of hepatic Na+, K+-ATPase

Summary In the perfused rat liver administration of glucagon was shown to result in a transiently increased uptake of K⁺, indicating the possible involvement of the Na⁺, K⁺-ATPase. Direct measurement of the activity of Na⁺, K⁺-ATPase revealed a two-fold stimulation of the enzyme by glucagon. The effect of glucagon on the activity of the enzyme was immediate. Simultaneously with the increase in the activity of the Na⁺, K⁺-ATPase, the activity of Mg²⁺-ATPase decreased. In order to evaluate whether the activation of the Na⁺, K⁺-ATPase by glucagon is related to the metabolic effects of the hormone, experimental conditions known to interfere with the activity of the enzyme were employed and glucagon stimulation of Ca²⁺-efflux, mitochondrial metabolism and gluconeogenesis were measured. K⁺-free perfusate, high K⁺ perfusate or ouabain interfered to varying degrees with the glucagon stimulation of these responses. The combination of K⁺-free perfusate and ouabain almost completely abolished the glucagon stimulation of all three parameters. These results demonstrate the glucagon stimulation of Na⁺, K⁺-ATPase and raise the possibility that the activation of the enzyme by glucagon might be a necessary link for the manifestation of its metabolic effects.     

Electrogenic ion transport by Na+,K+-ATPase

Experimental data on the ion electrogenic transport by Na+,K+-ATPase available in the literature are analyzed. Special attention is paid to the measurements of unsteady-state electric currents initiated by alternating voltage or rapid introduction of the substrate. In the final part, a physical model of the Na+,K+-ATPase functioning is discussed. According to this model, active transport is carried out by opening and closing of the access channels used for the sodium and potassium exchange between solutions on either side of the membrane. The model explains most of the experimental data, although some details (the channel size, rates of individual transport steps) need further refinement.     

Salt,Na+,K+-ATPase and hypertension

Chronic hypertension is characterized by a persistent increase in vascular tone. Sodium-rich diets promote hypertension; however, the underlying molecular mechanisms are not fully understood. Variations in the sodium content of the diet, through hormonal mediators such as dopamine and angiotensin II, modulate renal tubule Na⁺,K⁺-ATPase activity. Stimulation of Na⁺,K⁺-ATPase activity increases sodium transport across the renal proximal tubule epithelia, promoting Na⁺ retention, whereas inhibited Na⁺,K⁺-ATPase activity decreases sodium transport, and thereby natriuresis. Diets rich in sodium also enhance the release of adrenal endogenous ouabain-like compounds (OLC), which inhibit Na⁺,K⁺-ATPase activity, resulting in increased intracellular Na⁺ and Ca²⁺ concentrations in vascular smooth muscle cells, thus increasing the vascular tone, with a corresponding increase in blood pressure. The mechanisms by which these homeostatic processes are integrated in response to salt intake are complex and not completely elucidated. However, recent scientific findings provide new insights that may offer additional avenues to further explore molecular mechanisms related to normal physiology and pathophysiology of various forms of hypertension (i.e. salt-induced). Consequently, new strategies for the development of improved therapeutics and medical management of hypertension are anticipated.     

Increase of Na+ gradient-dependent L-glutamate and L-aspartate transport in high K+ dog erythrocytes associated with high activity of (Na+, K+)-ATPase

As reported previously, some dogs possess red cells characterized by low Na+, high K+ concentrations, and high activity of (Na+, K+)-ATPase, although normal dog red cells contain low K+, high Na+, and lack (Na+, K+)-ATPase. Furthermore, these red cells show increased activities of L-glutamate and L-aspartate transport, resulting in high accumulations of such amino acids in their cells. The present study demonstrated: (i) Na+ gradient-dependent L-glutamate and L-aspartate transport in the high K+ and low K+ red cells were dominated by a saturable component obeying Michaelis-Menten kinetics. Although no difference of the Km values was observed between the high K+ and low K+ cells, the Vmax values for both amino acids' transport in the high K+ cells were about three times those of low ones. (ii) L- and D-aspartate, but not D-glutamate, competitively inhibited L-glutamate transport in both types of the cells. (iii) Ouabain decreased the uptake of the amino acids in the high K+ dog red cells, whereas it was not effective on those in the low K+ cells. (iv) The ATP-treated high K+ cells [(K+]i not equal to [K+]o, [Na+]i greater than [Na+]o) showed a marked decrease of both amino acids' uptake rate, which was almost the same as that of the low K+ cells. (v) Valinomycin stimulated the amino acids' transport in both of the high K+ and the ATP-treated low K+ cells [( K+]i greater than [K+]o, [Na+]o), suggesting that the transport system of L-glutamate and L-aspartate in both types of the cells might be electrogenic. These results indicate that the increased transport activity in the high K+ dog red cells was a secondary consequence of the Na+ concentration gradient created by (Na+, K+)-ATPase.     

Stimulation of rat renal Na+, K+-ATPase activity by thyroid hormones

The effect of thyroid hormones (T4, T3 and reverse T3) on rat renal Na+,K+-ATPase activity was investigated by a cytochemical technique. T3 caused stimulation of Na+,K+-ATPase activity in the renal medulla but not in the renal cortex. There was a peak in enzyme activity after cultured renal segments had been exposed to T3 for 11 min and this time of maximal stimulation did not vary with the concentration of T3. A rectilinear response in Na+,K+-ATPase activity was observed over T3 concentration range 10 pmol l-1 to 100 nmol l-1; at higher T3 concentrations, Na+,K+-ATPase activity was inhibited. The enzyme response was totally blocked by specific T3 antiserum. Addition of T4 and reverse T3 (100 fmol l-1 -1 mmol l-1) failed to stimulate Na+,K+-ATPase activity in any part of the kidney. Plasma (neat and diluted 1:10) stimulated the enzyme in parallel with the dose response curve and the stimulatory effect was abolished by prior addition of specific T3 antiserum.     

Captopril inhibits ouabain-sensitive Na+/K+-ATPase

Captopril has been reported to inhibit ouabain-sensitive Na+/K+-ATPase activity in erythrocyte membrane fragments. We investigated the effect of captopril on two physiological measures of Na+/K+ pump activity: 22Na+ efflux from human erythrocytes and K+-induced relaxation of rat tail artery segments. Captopril inhibited 22Na+ efflux from erythrocytes in a concentration-dependent fashion, with 50% inhibition of total 22Na+ efflux at a concentration of 4.8 X 10(-3) M. The inhibition produced by captopril (5 X 10(-3) M) and ouabain (10(-4) M) was not greater than that produced by ouabain alone (65.3 vs. 66.9%, respectively), and captopril inhibited 50% of ouabain-sensitive 22Na+ efflux at a concentration of 2.0 X 10(-3) M. Inhibition by captopril of ouabain-sensitive 22Na efflux was not explained by changes in intracellular sodium concentration, inhibition of angiotensin-converting enzyme or a sulfhydryl effect. Utilizing rat tail arteries pre-contracted with norepinephrine (NE) or serotonin (5HT) in K+-free solutions, we demonstrated dose-related inhibition of K+-induced relaxation by captopril (10(-6) to 10(-4) M). Concentrations above 10(-4) M did not significantly inhibit K+-induced relaxation but did decrease contractile responses to NE, although not to 5HT. Inhibition of K+-induced relaxation by captopril was not affected by saralasin, teprotide or indomethacin. We conclude that captopril can inhibit membrane Na+/K+-ATPase in intact red blood cells and vascular smooth muscle cells. The mechanism of pump suppression is uncertain, but inhibition of ATPase should be considered when high concentrations of captopril are employed in physiological studies.