Modifications induced by general anesthetics on Na+/K+ ATPase obtained from human placenta

Previously it was demonstrated that thiopental in vivo anesthesia didn't affect the Na+/K(+)-ATPase activity of syncythiotrophoblast plasma membrane, while affecting other enzymatic activity. The aim of the present work was to investigate if this lack of effect of thiopental on the Na+/K+ ATPase activity might be due to its specificity of action on definite membrane proteins or if the binding sites of the anesthetic to this enzyme might be masked within the membrane. Temperature dependence of the Na+/K(+)-ATPase activity and of a spin label paramagnetic maleimide derivative (MSL,2,2,6,6-tetramethylpiperidin-1-oxyl-4-maleimide), which shows a selective binding to the reduced sulfhydryl groups of proteins were investigated. This report shows that a Na+/K(+)-ATPase membranous preparation obtained from placental tissue is strongly inhibited by thiopental.     

Kinetic studies on Na+/K+-ATPase and inhibition of Na+/K+-ATPase by ATP

Na+/K+-ATPase (EC 3.6.1.3) is an important membrane-bound enzyme. In this paper, kinetic studies on Na+/K+-ATPase were carried out under mimetic physiological conditions. By using microcalorimeter, a thermokinetic method was employed for the first time. Compared with other methods, it provided accurate measurements of not only thermodynamic data (deltarHm) but also the kinetic data (Km and Vmax). At 310.15K and pH 7.4, the molar reaction enthalpy (deltarHm) was measured as -40.514 +/- 0.9kJmol(-1). The Michaelis constant (Km) was determined to be 0.479 +/- 0.020 mM and consistent with literature data. The reliability of the thermokinetic method was further confirmed by colorimetric studies. Furthermore, a simple and reliable kinetic procedure was presented for ascertaining the true substrate for Na+/K+-ATPase and determining the effect of free ATP. Results showed that the MgATP complex was the real substrate with a Km value of about 0.5mM and free ATP was a competitive inhibitor with a Ki value of 0.253 mM.     

Activation of the Na+/K+-ATPase by interleukin-2

Activated B61.SF.1 and CTLL-2 T lymphocyte clones which are strictly dependent on interleukin-2 (IL-2) for growth were used to study the activation of Na+/K+-ATPase. 50% of [3H]thymidine maximal incorporation was obtained when the extracellular concentration of Na+ or K+ was reduced to 50 or 2 mM, respectively. 'Quiescent' CTL clones stimulated with IL-2 showed an increase of 48-380% in ouabain-sensitive 86Rb uptake. Furthermore, this stimulation was completely inhibited by a monoclonal antibody PC.61 directed at the IL-2 receptor. The activation of the pump was dependent on the dose of IL-2, took place at the same doses of IL-2 that were required to stimulate cell proliferation and was linear for at least 30 min.     

Ouabain-induced Internalization and Lysosomal Degradation of the Na+/K+-ATPase

Internalization of the Na⁺/K⁺-ATPase (the Na⁺ pump) has been studied in the human lung carcinoma cell line H1299 that expresses YFP-tagged α1 from its normal genomic localization. Both real-time imaging and surface biotinylation have demonstrated internalization of α1 induced by ≥100 nm ouabain which occurs in a time scale of hours. Unlike previous studies in other systems, the ouabain-induced internalization was insensitive to Src or PI3K inhibitors. Accumulation of α1 in the cells could be augmented by inhibition of lysosomal degradation but not by proteosomal inhibitors. In agreement, the internalized α1 could be colocalized with the lysosomal marker LAMP1 but not with Golgi or nuclear markers. In principle, internalization could be triggered by a conformational change of the ouabain-bound Na⁺/K⁺-ATPase molecule or more generally by the disruption of cation homeostasis (Na⁺, K⁺, Ca²⁺) due to the partial inhibition of active Na⁺ and K⁺ transport. Overexpression of ouabain-insensitive rat α1 failed to inhibit internalization of human α1 expressed in the same cells. In addition, incubating cells in a K⁺-free medium did not induce internalization of the pump or affect the response to ouabain. Thus, internalization is not the result of changes in the cellular cation balance but is likely to be triggered by a conformational change of the protein itself. In physiological conditions, internalization may serve to eliminate pumps that have been blocked by endogenous ouabain or other cardiac glycosides. This mechanism may be required due to the very slow dissociation of the ouabain·Na⁺/K⁺-ATPase complex.     

Binding of manganese ions to the Na+/K+-ATPase during phosphorylation by ATP

The aim of the present work was to study the Mg2+-Na+/K+-ATPase interaction that was proposed to lead to the formation of a stable Mg-enzyme complex during phosphorylation from ATP. Instead of Mg we used Mn, which can replace Mg as essential activator of Na+/K+-ATPase activity. The amounts of steady-state Mn bound to the enzyme were estimated at 0 degree C on the basis of the 54Mn remaining in the effluent after passing the reaction mixture through a cation exchange resin column. As a function of the MnCl2 concentration, the amount of Mn retained by the enzyme in the absence and presence of ATP showed a saturable and a linear component; the slope of the linear component was the same in both instances (0.016 nmol/mg per microM). The ATP-dependent Mn binding could be adjusted to a hyperbolic function with a Km of 0.76 microM. The ratio [ATP-dependent E-Mn]/[E-P] measured at 5 microM MnCl2 and 5 microM ATP was not different from 1.0, both in native (Mn-E2-P) as well as in a chymotrypsin treated enzyme (Mn-E1-P). When the Mn.E-P complex was allowed to react with KCl (E2-P form) or ADP (E1-P form), the enzyme was dephosphorylated and simultaneously lost the strongly bound Mn in such a way that the ratio [ATP-dependent E-Mn]/[E-P] remained 1:1. These results show the existence of strongly bound Mn ions to Na+/K+-ATPase during phosphorylation by ATP. That binding is (i) of high affinity for Mn, (ii) probably on a single site, and (iii) with a stoichiometry Mn-Pi of 1:1.     

The effect of cytoplasmic K+ on the activity of the Na+/K(+)-ATPase.

Experiments with the reconstituted (Na+ + K+)-ATPase show that besides the ATP-dependent cytoplasmic Na(+)-K+ competition for Na+ activation there is a high affinity inhibitory effect of cytoplasmic K+. In contrast to the high affinity K+ inhibition seen with the unsided preparation at a low ATP especially at a low temperature, the high affinity inhibition by cytoplasmic K+ does not disappear when the ATP concentration an-or the temperature is increased. The high affinity inhibition by cytoplasmic K+ is also observed with Cs+, Li+ or K+ as the extracellular cation, but the fractional inhibition is much less pronounced than with Na+ as the extracellular cation. The results suggest that either there are two populations of enzyme, one with the normal ATP dependent cytoplasmic Na(+)-K+ competition, and another which due to the preparative procedure has lost this ATP sensitivity. Or that the normal enzyme has two pathways for the transition from E2-P to E1ATP. One on which the enzyme with the translocated ion binds cytoplasmic K+ with a high affinity but not ATP, and another on which ATP is bound but not K+. A kinetic model which can accommodate this is suggested.     

Primary structure of the Neurospora plasma membrane H+-ATPase deduced from the gene sequence. Homology to Na+/K+-, Ca2+-, and K+-ATPase

The gene for the Neurospora crassa plasma membrane H+-ATPase has been cloned and sequenced. The gene encodes for a protein of 920 amino acids with a molecular weight of 100,002. The coding region is interrupted by four introns: three near the amino terminus and one near the carboxyl terminus. The deduced amino acid sequence of the N. crassa plasma membrane H+-ATPase exhibits 75% homology to the amino acid sequence of the Saccharomyces cerevisiae plasma membrane H+-ATPase. Also, an amino acid comparison with the Na+/K+-ATPase from sheep kidney, Ca2+-ATPase from rabbit muscle, and K+-ATPase from Escherichia coli reveals that certain regions are highly conserved and suggest that these regions may serve essential functions which are common to the various cation-motive ATPases. This observation suggests that the phosphorylatable, cation-motive ATPases may function via a similar energy transduction mechanism.     

Ozone alteration of transport of cations and the Na+/K+-ATPase in human erythrocytes.

We have purified glutaminase 65-fold from cow brain; the final specific activity is 24 μmol/min/mg. The enzyme is stable between pH 7.5 and 9.0 and has maximal activity at pH 8.8. It requires P_i for activity. The dependence of activity on P_i concentration is sigmoidal; 50 mmP_i gives half-maximal velocity at pH 8.8. At 0.2 mP_i, pH 8.8, the dependence of activity on glutamine concentration is hyperbolic; the observed K_Gln was 30 mm. Increasing P_i concentrations increase the apparent V_m and decrease the apparent K_Gln. NH₄⁺ does not inhibit at concentrations up to 0.1 m. Glutamic acid inhibits competitively with respect to glutamine; at 0.2 mP_i pH 8.8, K_Gln was 30 mm and K_Glu was 19 mm. The results are consistent with a model in which NH₄⁺ is released irreversibly from the enzyme-substrate complex and is the first product released. The activity of glutaminase appears to be independent of the nature of the buffer with which it is equilibrated before being assayed.     

Localization of the Na+/K+-ATPase and of an amiloride sensitive Na+ uptake on thyroid epithelial cells

The Na+/K+-ATPase was localized using purified specific antibodies, on the basolateral membranes of rat thyroid epithelial cells and of cultured porcine thyroid cells, by immunofluorescence and immunoelectron microscopy. No staining was observed on the apical membranes. When cultured cells formed monolayers, with their apical pole in contact with the culture medium, 22Na+ uptake was inhibited by amiloride. Inhibition was dependent upon extracellular Na+ concentration, half maximal inhibition was obtained with 0.7 microM amiloride in the presence of 5 mM Na+. Ouabain was ineffective on Na+ uptake into intact monolayers. A brief treatment of the monolayers with ethyleneglycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) opened the tight junctions and allowed the access of ouabain to the basal pole of the cells. In this condition ouabain increased Na+ uptake. When cells were reorganized into follicle-like structures, with their basal pole in contact with the culture medium, Na+ uptake was not modified by amiloride but was increased by ouabain. We conclude that in thyroid cells, the Na+/K+-ATPase is present on the basolateral domain of the plasma membrane whereas an amiloride sensitive sodium uptake occurs at the apical surface.     

Regulation of Glial Na+/K+-ATPase by Serotonin: Identification of Participating Receptors

The purpose of the present study was the characterization of the receptors participating in the regulatory mechanism of glial Na⁺/K⁺-ATPase by serotonin (5-HT) in rat brain. The activity of the Na⁺ pump was measured in four brain regions after incubation with various concentrations of serotoninergic agonists or antagonists. A concentration-dependent increase in enzyme activity was observed with the 5-HT_1A agonist R (+)-2-dipropylamino-8-hydroxy-1,2,3, 4-tetrahydronaphthalene hydrobromide (8-OH-DPAT) in homogenates or in glial membrane enriched fractions from cerebral cortex and in hippocampus. Spiperone, a 5-HT_1A antagonist, completely inhibited the response to 8-OH-DPAT but had no effect on Na⁺/K⁺-ATPase activity in cerebellum where LSD, a 5-HT₆ agonist, elicited a dose-dependent response similar to that of 5-HT. In brainstem, a lack of reponse to 5-HT and other agonists was confirmed. Altogether, these results show that serotonin modulates glial Na⁺/K⁺-ATPase activity in the brain, apparently not through only one type of 5-HT receptor. It seems that the receptor system involved is different according to the brain region. In cerebral cortex, the response seems to be mediated by 5-HT_1A as well as in hippocampus but not in cerebellum where 5-HT₆ appears as the receptor system involved.     

Na+/K+-ATPase inhibition during cardiac myocyte swelling: Involvement of intracellular pH and Ca2+

Previous studies in chick embryo cardiac myocytes have shown that the inhibition of Na⁺/K⁺-ATPase with ouabain induces cell shrinkage in an isosmotic environment (290 mOsm). The same inhibition produces an enhanced RVD (regulatory volume decrease) in hyposmotic conditions (100 mOsm). It is also known that submitting chick embryo cardiomyocytes to a hyperosmotic solution induces shrinkage and a concurrent intracellular alkalization. The objective of this study was to evaluate the involvement of intracellular pH (pH_i), intracellular Ca²⁺ ([Ca²⁺]_i) and Na⁺/K⁺-ATPase inhibition during hyposmotic swelling. Changes in intracellular pH and Ca²⁺ were monitored using BCECF and fura-2, respectively. The addition of ouabain (100 M) under both isosmotic and hyposmotic stimuli resulted in a large increase in [Ca²⁺]_i (200%). A decrease in pH_i (from 7.3 ± 0.09 to 6.4 ± 0.08, n = 6; p < 0.05) was only observed when ouabain was applied during hyposmotic swelling. This acidification was prevented by the removal of extracellular Ca²⁺. Inhibition of Na⁺/H²⁺ exchange with amiloride (1 mM) had no effect on the ouabain-induced acidification. Preventing the mitochondrial accumulation of Ca²⁺ using CCCP (10 M) resulted in a blockade of the progressive acidification normally induced by ouabain. The inhibition of mitochondrial membrane K⁺/H⁺ exchange with DCCD (1 mM) also completely prevented the acidification. Our results suggest that intracellular acidification upon cell swelling is mediated by an initial Ca²⁺ influx via Na⁺/Ca²⁺ exchange, which under hyposmotic conditions activates the K⁺ and Ca²⁺ mitochondrial exchange systems (K⁺/H⁺ and Ca²⁺/H⁺).Deceased     

Identification of cisplatin-binding sites on the large cytoplasmic loop of the Na+/K+-ATPase

Cisplatin is the most widely used chemotherapeutic drug for the treatment of various types of cancer; however, its administration brings also numerous side effects. It was demonstrated that cisplatin can inhibit the Na⁺/K⁺-ATPase (NKA), which can explain a large part of the adverse effects. In this study, we have identified five cysteinyl residues (C452, C456, C457, C577, and C656) as the cisplatin binding sites on the cytoplasmic loop connecting transmembrane helices 4 and 5 (C45), using site-directed mutagenesis and mass spectrometry experiments. The identified residues are known to be susceptible to glutathionylation indicating their involvement in a common regulatory mechanism.     

Effects of Oligomycin on Transient Currents Carried by Na+ Translocation of Bufo Na+/K+-ATPase Expressed in Xenopus Oocytes

Na(+)/K(+)-ATPase (NKA) exports 3Na(+) and imports 2K(+) at the expense of the hydrolysis of 1ATP under physiological conditions. In the absence of K(+), it can mediate electroneutral Na(+)/Na(+) exchange. In the electroneutral Na(+)/Na(+) exchange mode, NKA produces a transient current containing fast, medium and slow components in response to a sudden voltage step. These three components of the transient current demonstrate the sequential release of Na(+) ions from three binding sites. Our data from oocytes provide further experimental support for the existence of these components. Oligomycin is an NKA inhibitor that favors the 2Na(+)-occluded state without affecting the conformational state of the NKA. We studied the effects of oligomycin on both K(+)-activated currents and transient currents in wild-type Bufo NKA and a mutant form of Bufo NKA, NKA: G813A. Oligomycin blocked almost all of the K(+)-activated current, although the three components of the transient current showed different sensitivities to oligomycin. The oligomycin-inhibited charge movement measured using a P/4 protocol had a rate coefficient similar to the medium transient component. The fast component of the transient current elicited by a short voltage step also showed sensitivity to oligomycin. However, the slow component was not totally inhibited by oligomycin. Our results indicate that the second and third sodium ions might be released to the extracellular medium by a mechanism that is not shared by the first sodium ion.     

Investigation of subunit interactions by radiation inactivation: the case of Na+/K+-ATPase.

The target size of Na+/K+-ATPase has been determined by radiation inactivation. To interpret the results, we have performed Monte Carlo simulations of the inactivation process. This seems to be a general method for the interpretation of such studies. The simulation revealed that radiation inactivation can distinguish between monoprotomeric and multiprotomeric models of enzyme action only if the measured reaction requires the actual co-operation of, rather than the mere co-existence of, different protomeres.     

ATP-binding is stabilized by a stacking interaction within the binding site of Na+/K+ -ATPase

Site-directed mutagenesis was applied to modify phenylalanines (Phe(475)Trp, Phe(548)Tyr, and both) to generate mutants on the basis of molecular modeling of the ATP-binding domain of Na(+)/K(+)-ATPase, in order to characterize the forces that stabilize ATP in its binding pocket. Each of the mutants was examined by Raman difference spectroscopy, i.e., as a difference between the spectrum of the domain with and without bound ATP. It was shown that Phe(475) plays a key role in stabilizing ATP-binding by a stacking interaction. Phe(548) co-stabilizes ATP on the opposite site of the binding pocket and its type of interaction with ATP-binding differs from that of Phe(475).     

Energy metabolism of reticulocytes: Two different sources of energy for Na+K+-ATPase activity

Total energy production in rabbit reticulocytes amounted to 136·52 ± 6·50μmol ATP h^?1ml^?1 of reticulocytes: 88·3 per cent was provided by oxidative phosphorylation, whereas only 11·7 per cent by aerobic glycolysis. Na⁺K⁺-ATPase accounted for 23 per cent, i.e. 27·65 ± 2·55μmol ATP h^?1ml^?1 of reticulocytes, in the overall energy consumption in reticulocytes of rabbits. Under basal conditions ATP for Na⁺K⁺-ATPase activity was derived exclusively from oxidative phosphorylation. However, when the activity of Na⁺K⁺-ATPase was increased due to the stimulation of adenylate cyclase by (?)-isoprenaline, the additional energy required was provided by aerobic glycolysis. These results indicate that two different compartments, one cytosolic and the other mitochondrial, provide energy for Na⁺K⁺-ATPase activity in reticulocytes.     

Inhibition of Na+/K+-ATPase and Mg2+-ATPase by metal ions and prevention and recovery of inhibited activities by chelators

Kinetics and inhibition of Na⁺/K⁺-ATPase and Mg²⁺-ATPase activity from rat synaptic plasma membrane (SPM), by separate and simultaneous exposure to transition (Cu²⁺, Zn²⁺, Fe²⁺ and.Co²⁺) and heavy metals (Hg²⁺and Pb²⁺) ions were studied. All investigated metals produced a larger maximum inhibition of Na⁺/K⁺-ATPase than Mg²⁺-ATPase activity. The free concentrations of the key species (inhibitor, MgATP^{2 ?} , MeATP^{2 ?} ) in the medium assay were calculated and discussed. Simultaneous exposure to the combinations Cu²⁺/Fe²⁺ or Hg²⁺/Pb²⁺caused additive inhibition, while Cu²⁺/Zn²⁺ or Fe²⁺/Zn²⁺ inhibited Na⁺/K⁺-ATPase activity synergistically (i.e., greater than the sum metal-induced inhibition assayed separately). Simultaneous exposure to Cu²⁺/Fe²⁺ or Cu²⁺/Zn²⁺ inhibited Mg²⁺-ATPase activity synergistically, while Hg²⁺/Pb²⁺ or Fe²⁺/Zn²⁺ induced antagonistic inhibition of this enzyme. Kinetic analysis showed that all investigated metals inhibited Na⁺/K⁺-ATPase activity by reducing the maximum velocities (V_max) rather than the apparent affinity (K_m) for substrate MgATP^2-, implying the noncompetitive nature of the inhibition. The incomplete inhibition of Mg²⁺-ATPase activity by Zn²⁺, Fe²⁺ and Co²⁺ as well as kinetic analysis indicated two distinct Mg²⁺-ATPase subtypes activated in the presence of low and high MgATP^{2 ?} concentration. EDTA, L-cysteine and gluthathione (GSH) prevented metal ion-induced inhibition of Na⁺/K⁺-ATPase with various potencies. Furthermore, these ligands also reversed Na⁺/K⁺-ATPase activity inhibited by transition metals in a concentration-dependent manner, but a recovery effect by any ligand on Hg²⁺-induced inhibition was not obtained.