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.     

Evidence That Antioxidants Prevent the Inhibition of Na+,K+-ATPase Activity Induced by Octanoic Acid in Rat Cerebral Cortex in Vitro

The objective of the present study was to investigate the in vitro effects of octanoic acid, which accumulates in medium-chain acyl-CoA dehydrogenase (MCAD) deficiency and in Reye syndrome, on key enzyme activities of energy metabolism in the cerebral cortex of young rats. The activities of the respiratory chain complexes I–IV, creatine kinase, and Na⁺, K⁺-ATPase were evaluated. Octanoic acid did not alter the electron transport chain and creatine kinase activities, but, in contrast, significantly inhibited Na⁺, K⁺-ATPase activity both in synaptic plasma membranes and in homogenates prepared from cerebral cortex. Furthermore, decanoic acid, which is also increased in MCAD deficiency, and oleic acid strongly reduced Na⁺, K⁺-ATPase activity, whereas palmitic acid had no effect. We also examined the effects of incubating glutathione and trolox (-tocopherol) alone or with octanoic acid on Na⁺, K⁺-ATPase activity. Tested compounds did not affect Na⁺, K⁺-ATPase activity by itself, but prevented the inhibitory effect of octanoic acid. These results suggest that inhibition of Na⁺, K⁺-ATPase activity by octanoic acid is possibly mediated by oxidation of essential groups of the enzyme. Considering that Na⁺, K⁺-ATPase is critical for normal brain function, it is feasible that the significant inhibition of this enzyme activity by octanoate and also by decanoate may be related to the neurological dysfunction found in patients affected by MCAD deficiency and Reye syndrome.     

The inhibitory activity of a brain extract on synaptosomal Na+,K+-ATPase is sensitive to carboxypeptidase A and to chelating agents

In the present study some properties of an inhibitory extract of synaptosomal membrane Na⁺,K⁺-ATPase were investigated. This extract (peak II) was prepared by gel filtration in Sephadex G-50 of a soluble fraction of the rat cerebral cortex. Ultrafiltration of peak II through Amicon membranes indicated that the inhibitor has a low MW (<1000). The inhibitory activity was not modified by heating in neutral pH at 95°C for 20 min but it was destroyed by charring in acid pH at 200°C for 120 min. The inhibitory activity decreased by incubation of peak II with carboxypeptidase A. These findings suggest that the factor responsible for the inhibition of Na⁺,K⁺-ATPase activity is probably a polypeptide. On the other hand, the inhibition was reverted by the chelators EDTA and EGTA, indicating the participation of an ionic compound as well. The increase of Mg²⁺ concentration during the enzyme assay did not increase the inhibition, indicating that the ion involved might not be vanadate. It is suggested that both a polypeptide and an ionic compound coparticipate in the inhibitory effect of peak II on Na⁺,K⁺-ATPase activity.     

Vanadate inhibition of rat cerebral cortex Na+, K+-ATPase during postnatal development

The Na⁺, K⁺-ATPase activity and its response to vanadate inhibition was investigated in cerebral cortex homogenates of 7-, 12- and 18-day-old rats. The enzyme was inhibited by vanadate in a dose-dependent manner in all these age groups. Furthermore, there was a different sensitivity towards vanadate during postnatal development; the concentration of V⁺⁵ needed for 50% inhibiton of Na⁺, K⁺-ATPase was 1.1×10^–6M, 2×10^–7M and 4.4×10^–7M for 7-, 12- and 18-day-old rats, respectively. It is suggested that the different sensitivity of Na⁺, K⁺-ATPase towards vanadate inhibition during postnatal development might be due to age-dependent changes in the ratio of various cell types.Special Issue dedicated to Dr. O. H. Lowry.     

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.     

An endogenous factor which interacts with synaptosomal membrane Na+, K+-ATPase activation by K+

In previous papers, the isolation of brain soluble fractions able to modify neuronal Na⁺, K⁺-ATPase activity has been described. One of those fractions-peak I-stimulates membrane Na⁺, K⁺-ATPase while another-peak II-inhibits this enzyme activity, and has other ouabain-like properties. In the present study, synaptosomal membrane Na⁺, K⁺-ATPase was analyzed under several experimental conditions, using ATP orp-nitrophenylphosphate (p-NPP) as substrate, in the absence and presence of cerebral cortex peak II. Peak II inhibited K⁺-p-NPPase activity in a concentration dependent manner. Double reciprocal plots indicated that peak II uncompetitively inhibits K⁺-p-NPPase activity regarding substrate, Mg²⁺ and K⁺ concentration. Peak II failed to block the known K⁺-p-NPPase stimulation caused by ATP plus Na⁺. At various K⁺ concentrations, percentage K⁺-p-NPPase inhibition by peak II was similar regardless of the ATP plus Na⁺ presence, indicating lack of correlation with enzyme phosphorylation. Na⁺, K⁺-ATPase activity was decreased by peak II depending on K⁺ concentration. It is postulated that the inhibitory factor(s) present in peak II interfere(s) with enzyme activation by K⁺.     

Modification of heart sarcolemmal Na+/K+-ATPase activity during development of the calcium paradox

This study examined the status of sarcolemmal Na⁺/K⁺-ATPase activity in rat heart under conditions of Ca²⁺-paradox to explore the existence of a relationship between changes in Na⁺/K⁺-pump function and myocardial Na⁺ as well as K⁺ content. One min of reperfusion with Ca²⁺ after 5 min of Ca²⁺-free perfusion reduced Na⁺/K⁺-ATPase activity in the isolated heart by 53% while Mg²⁺-ATPase, another sarcolemmal bound enzyme, retained 74% of its control activity. These changes in sarcolemmal ATPase activities were dependent on the duration and Ca²⁺ concentration of the initial perfusion and subsequent reperfusion periods; however, the Na⁺/K⁺-ATPase activity was consistently more depressed than Mg²⁺-ATPase activity under all conditions. The depression in both enzyme activities was associated with a reduction in V_max without any changes in K_m values. Low Na⁺ perfusion and hypothermia, which protect the isolated heart from the Ca²⁺-paradox, also prevented reperfusion-induced enzyme alterations. A significant relationship emerged upon comparison of the changes in myocardial Na⁺ and K⁺ content to Na⁺/K⁺-ATPase activity under identical conditions. At least 60% of the control enzyme activity was necessary to maintain normal cation gradients. Depression of the Na⁺/K⁺-ATPase activity by 60-65% resulted in a marked increase and decrease in intracellular Na⁺ and K⁺ content, respectively. These results suggest that changes in myocardial Na⁺ and K⁺ content during Ca²⁺-paradox are related to activity of the Na⁺/K⁺-pump; the impaired Na⁺/K⁺-ATPase activity may lead to augmentation of Ca²⁺-overload via an enhancement of the Na⁺/Ca²⁺-exchange system.     

Hypoxic preconditioning induces neuroprotection against transient global ischemia in adult rats via preserving the activity of Na(+)/K(+)-ATPase

We demonstrated previously that 30 min of hypoxic preconditioning (HPC) applied 1 day before 10 min of transient global cerebral ischemia (tGCI) reduced neuronal loss in the hippocampal CA1 subregion in adult rats. The aim of the present study was to investigate the role of Na⁺/K⁺-ATPase and protein kinase Mζ (PKMζ) in the protective effect of HPC against tGCI in adult rats. We found that the activity of Na⁺/K⁺-ATPase decreased in the hippocampal CA1 subregion after 10 min of tGCI. This effect was not seen after 30 min of HPC in adult rats. Corresponding to the changes in Na⁺/K⁺-ATPase activity, the surface expression of Na⁺/K⁺-ATPase α1 subunit increased after HPC. Furthermore, HPC dramatically reduced the number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells in the hippocampal CA1 subregion after tGCI. However, neither PKMζ nor phosphorylation of PKMζ was changed after tGCI or HPC. The results of the present study are consistent with the hypothesis that both enhanced recovery of Na⁺/K⁺-ATPase activity due to preserved the protein levels of Na⁺/K⁺-ATPase α1 subunit and reduced DNA fragmentation after tGCI contribute to the protection afforded by HPC. However, PKMζ activation does not appear to play a role in this neuroprotection.     

Inhibition of Rat Brain Na+, K+-ATPase Activity Induced by Homocysteine Is Probably Mediated by Oxidative Stress

The objective of the present study was to investigate the effects of preincubation of hippocampus homogenates in the presence of homocysteine or methionine on Na⁺, K⁺-ATPase and Mg²⁺-ATPase activities in synaptic membranes of rats. Homocysteine significantly inhibited Na⁺, K⁺-ATPase activity, whereas methionine had no effect. Mg²⁺-ATPase activity was not altered by the metabolites. We also evaluated the effect of incubating glutathione, cysteine, dithiothreitol, trolox, superoxide dismutase and GM1 ganglioside alone or incubation with homocysteine on Na⁺, K⁺-ATPase activity. Tested compounds did not alter Na⁺, K⁺-ATPase and Mg²⁺-ATPase activities, but except for trolox, prevented the inhibitory effect of homocysteine on Na⁺, K⁺-ATPase activity. These results suggest that inhibition of this enzyme activity by homocysteine is possibly mediated by free radicals and may contribute to the neurological dysfunction found in homocystinuric patients.     

The interaction between calcium and the activation of Na+, K+-ATPase by noradrenaline

Summary The interaction of noradrenaline, various cation chelators and calcium on Na⁺, K⁺-ATPase from rat cerebral cortex plasma membranes was studied. It was shown that chelation of inhibitory cations by EGTA, EDTA and dipyridyl activated Na⁺, K⁺-ATPase to the same extent as noradrenaline but at higher concentrations; increasing concentrations of EGTA depressed the activation by noradrenaline; calcium in the form of a calcium-EGTA buffer depressed Na⁺, K⁺-ATPase at physiological concentrations; the inhibition of Na⁺, K⁺-ATPase by calcium is dependent on the magnesium concentration in the assay and the inhibition by calcium was partially reversed by noradrenaline.     

Inhibition of Na+,K+-ATPase Activity from Rat Hippocampus by Proline

Na⁺,K⁺-ATPase and Mg²⁺-ATPase activities were determined in the synaptic plasma membranes from hippocampus of rats subjected to chronic and acute proline administration. Na⁺,K⁺-ATPase activity was significantly reduced in chronic and acute treatment by 33% and 40%, respectively. Mg²⁺-ATPase activity was not altered by any treatment. In another set of experiments, synaptic plasma membranes were prepared from hippocampus and incubated with proline or glutamate at final concentrations ranging from 0.2 to 2.0 mM. Na⁺,K⁺-ATPase, but not Mg²⁺-ATPase was inhibited (30%) by the two amino acids. In addition, competition between proline and glutamate for the enzyme activity was observed, suggesting a common binding site for these amino acids. Considering that Na⁺,K⁺-ATPase activity is critical for normal brain function, the results of the present study showing a marked inhibition of this enzyme by proline may be associated with the neurological dysfunction found in patients affected by type II hyperprolinemia.     

Resveratrol prevents oxidative stress and inhibition of Na(+)K(+)-ATPase activity induced by transient global cerebral ischemia in rats

Increased oxidative stress and energy metabolism deficit have been regarded as an important underlying cause for neuronal damage induced by cerebral ischemia/reperfusion (I/R) injury. In this study, we investigated the oxidative mechanisms underlying the neuroprotective effects of resveratrol, a potent polyphenol antioxidant found in grapes, on structural and biochemical abnormalities in rats subjected to global cerebral ischemia. Experimental model of transient global cerebral ischemia was induced in Wistar rats by the four vessel occlusion method for 10 min and followed by different periods of reperfusion. Nissl and fluoro jade C stained indicated extensive neuronal death at 7 days after I/R. These findings were preceded by a rapid increase in the generation of reactive oxygen species (ROS), nitric oxide (NO), lipid peroxidation, as well as by a decrease in Na⁺K⁺-ATPase activity and disrupted antioxidant defenses (enzymatic and non-enzymatic) in hippocampus and cortex. Administrating resveratrol 7 days prior to ischemia by intraperitoneal injections (30 mg/kg) significantly attenuated neuronal death in both studied structures, as well as decreased the generation of ROS, lipid peroxidation and NO content. Furthermore, resveratrol brought antioxidant and Na⁺K⁺-ATPase activity in cortex and hippocampus back to normal levels. These results support that resveratrol could be used as a preventive, or therapeutic, agent in global cerebral ischemia and suggest that scavenging of ROS contributes, at least in part, to resveratrol-induced neuroprotection.     

Dual activity of the H1-H2 domain of the (Na(+)+K+)-ATPase

(Na⁺+K⁺)-ATPase is a target receptor of digitalis (cardiac glycoside) drugs. It has been demonstrated that the H1-H2 domain of the α-subunit of the (Na⁺+K⁺)-ATPase is one of the digitalis drug interaction sites of the enzyme. Despite the extensive studies of the inhibitory effect of digitalis on the (Na⁺+K⁺)-ATPase, the functional property of the H1-H2 domain of the enzyme and its role in regulating enzyme activity is not completely understood. Here we report a surprise finding: instead of inhibiting the enzyme, binding of a specific monoclonal antibody SSA78 to the H1-H2 domain of the (Na⁺+K⁺)-ATPase elevates the catalytic activity of the enzyme. In the presence of low concentration of ouabain, monoclonal antibody SSA78 significantly protects enzyme function against ouabain-induced inhibition. However, higher concentration of ouabain completely inactivates the (Na⁺+K⁺)-ATPase even in the presence of SSA78. These results suggest that the H1-H2 domain of the (Na⁺+K⁺)-ATPase is capable of regulating enzyme function in two distinct ways for both ouabain-sensitive and -resistant forms of the enzyme: it increases the activity of the (Na⁺+K⁺)-ATPase during its interaction with an activator; it also participates in the mechanism of digitalis or ouabain-induced inhibition of the enzyme. Understanding the dual activity of the H1-H2 domain will help better understand the structure-function relationships of the (Na⁺+K⁺)-ATPase and the biological processes mediated by the enzyme.     

In search of synaptosomal Na+, K+-ATPase regulators

The arrival of the nerve impulse to the nerve endings leads to a series of events involving the entry of sodium and the exit of potassium. Restoration of ionic equilibria of sodium and potassium through the membrane is carried out by the sodium/potassium pump, that is the enzyme Na⁺,K⁺-ATPase. This is a particle-bound enzyme that concentrates in the nerve ending or synaptosomal membranes. The activity of Na⁺,K⁺-ATPase is essential for the maintenance of numerous reactions, as demonstrated in the isolated synaptosomes. This lends interest to the knowledge of the possible regulatory mechanisms of Na⁺,K⁺-ATPase activity in the synaptic region. The aim of this review is to summarize the results obtained in the author's laboratory, that refer to the effect of neurotransmitters and endogenous substances on Na⁺,K⁺-ATPase activity. Mention is also made of results in the field obtained in other laboratories. Evidence showing that brain Na⁺,K⁺-ATPase activity may be modified by certain neurotransmitters and insulin have been presented. The type of change produced by noradrenaline, dopamine, and serotonin on synaptosomal membrane Na⁺,K⁺-ATPase was found to depend on the presence or absence of a soluble brain fraction. The soluble brain fraction itself was able to stimulate or inhibit the enzyme, an effect that was dependent in turn on the time elapsed between preparation and use of the fraction. The filtration of soluble brain fraction through Sephadex G-50 allowed the separation of two active subfractions: peaks I and II. Peak I increased Na⁺,K⁺- and Mg²⁺-ATPases, and peak II inhibited Na⁺,K⁺-ATPase. Other membrane enzymes such as acetylcholinesterase and 5′-nucleotidase were unchanged by peaks I or II. In normotensive anesthetized rats, water and sodium excretion were not modified by peak I but were increased by peak II, thus resembling ouabain effects.³H-ouabain binding was unchanged by peak I but decreased by peak II in some areas of the CNS assayed by quantitative autoradiography and in synaptosomal membranes assayed by a filtration technique. The effects of peak I and II on Na⁺,K⁺-ATPase were reversed by catecholamines. The extent of Na⁺,K⁺-ATPase inhibition by peak II was dependent on K⁺ concentration, thus suggesting an interference with the K⁺ site of the enzyme. Peak II was able to induce the release of neurotransmitter stored in the synaptic vesicles in a way similar to ouabain. Taking into account that peak II inhibits only Na⁺,K⁺-ATPase, increases diuresis and natriuresis, blocks high affinity³H-ouabain binding, and induces neurotransmitter release, it is suggested that it contains an ouabain-like substance.     

The influence of potassium ion (K+) on digoxin-induced inhibition of porcine cerebral cortex Na+/K+-ATPase

The in vitro influence of potassium ion modulations, in the concentration range 2 mM–500 mM, on digoxin-induced inhibition of porcine cerebral cortex Na⁺/K⁺-ATPase activity was studied. The response of enzymatic activity in the presence of various K⁺ concentrations to digoxin was biphasic, thereby, indicating the existence of two Na⁺/K⁺-ATPase isoforms, differing in the affinity towards the tested drug. Both isoforms showed higher sensitivity to digoxin in the presence of K⁺ ions below 20 mM in the medium assay. The IC₅₀ values for high/low isoforms 2.77 × 10^{? 6} M / 8.56 × 10^{? 5} M and 7.06 × 10^{? 7} M /1.87 × 10^{? 5} M were obtained in the presence of optimal (20 mM) and 2 mM K⁺, respectively. However, preincubation in the presence of elevated K⁺ concentration (50 – 500 mM) in the medium assay prior to Na⁺/K⁺-ATPase exposure to digoxin did not prevent the inhibition, i.e. IC₅₀ values for both isoforms was the same as in the presence of the optimal K⁺ concentration. On the contrary, addition of 200 mM K⁺ into the medium assay after 10 minutes exposure of Na⁺/K⁺-ATPase to digoxin, showed a time-dependent recovery effect on the inhibited enzymatic activity. Kinetic analysis showed that digoxin inhibited Na⁺/K⁺-ATPase by reducing maximum enzymatic velocity (V_max) and K_m, implying an uncompetitive mode of interaction.     

Effect of tissue specificity of brain soluble fractions on Na+, K+-ATPase activity

Previous evidence from this laboratory indicated that catecholamines and brain endogenous factors modulate Na⁺, K⁺-ATPase activity of the synaptosomal membranes. The filtration of a brain total soluble fraction through Sephadex G-50 permitted the separation of two fractions-peaks I and II-which stimulated and inhibited Na⁺, K⁺-ATPase, respectively (Rodríguez de Lores Arnaiz and Antonelli de Gomez de Lima, Neurochem. Res.11, 1986, 933). In order to study tissue specificity a rat kidney total soluble was fractionated in Sephadex G-50 and kidney peak I and II fractions were separated; as control, a total soluble fraction prepared from rat cerebral cortex was also processed. The UV absorbance profile of the kidney total soluble showed two zones and was similar to the profile of the brain total soluble. Synaptosomal membranes Na⁺, K⁺- and Mg²⁺-ATPases were stimulated 60–100% in the presence of kidney and cerebral cortex peak I; Na⁺, K⁺-ATPase was inhibited 35–65% by kidney peak II and 60–80% by brain peak II. Mg²⁺-ATPase activity was not modified by peak II fractions. ATPases activity of a kidney crude microsomal fraction was not modified by kidney peak I or brain peak II, and was slightly increased by kidney peak II or brain peak I. Kidney purified Na⁺, K⁺-ATPase was increased 16–20% by brain peak I and II fractions. These findings indicate that modulatory factors of ATPase activity are not exclusive to the brain. On the contrary, there might be tissue specificity with respect to the enzyme source.     

Digitalis receptor sugar binding site characteristics: A model based upon studies of Na+, K+-ATPase preparations with differing digitalis sensitivities

The structure-activity relationships of the genin moieties of digitalis glycosides are commonly elucidated by determining the inhibitory potency of a variety of genins toward the plasma membrane Na⁺, K⁺-ATPase; qualitatively these relationships appear to be fairly independent of the specific Na⁺, K⁺-ATPase preparation utilized for the analysis. To determine whether this is the case with regard to the sugar moieties of glycosides, the inhibitory effects of 12 monoglycosides of digitoxigenin toward four Na⁺, K⁺-ATPase preparations of different origin were measured. It was found that while recognition of the major structural determinants of sugar activity appeared to be independent of enzyme source, recognition of the minor structural determinants of activity showed some source dependence. It was also observed that the intrinsic sensitivity to sugar potentiation may be source dependent and unrelated to intrinsic sensitivity to inhibition by digitoxigenin. These observations are compatible with a model of the Na⁺, K⁺-ATPase sugar binding site(s) in which intrinsic sensitivity to sugar attachment as well as recognition characteristics (for sugar structural features) both determine the extent to which a sugar moiety may contribute to the activity of monoglycosides. Further, in these studies one of the Na⁺, K⁺-ATPase preparations employed was obtained from rat brain, a tissue known to contain a mixture of ouabain sensitive and insensitive isoforms. We have observed that the rigorous purification techniques employed appear to have selectively removed from or denatured the less ouabain sensitive al isoform found in this enzyme preparation.     

Pressure adaptation of teleost gill Na+/K+-adenosine triphosphatase: role of the lipid and protein moieties

Summary The effects of temperature and pressure on Na⁺/K⁺-adenosine triphosphatases (Na⁺/K⁺-ATPases) from gills of marine teleost fishes were examined over a range of temperatures (10–25°C) and pressures (1–680 atm). The relationship between gill membrane fluidity and Na⁺/K⁺-ATPase activity was studied using the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH). The increase in temperature required to offset the membrane ordering effects of high pressure was 0.015–0.025°C·atm^-1, the same coefficient that applied to Na⁺/K⁺-ATPase activities. Thus, temperature-pressure combinations yielding the same Na⁺/K⁺-ATPase activity also gave similar estimates of membrane fluidity. Substituion of endogenous lipids with lipids of different composition altered the pressure responses of Na⁺/K⁺-ATPase. Na⁺/K⁺-adenosine triphosphatase became more sensitive to pressure in the presence of chicken egg phosphatidylcholine, but phospholipids isolated from fish gills reduced the inhibition by pressure of Na⁺/K⁺-ATPase. Cholesterol increased enzyme pressure sensitivity. Membrane fluidity and pressure sensitivity of Na⁺/K⁺-ATPase were correlated, but the effects of pressure also dependent on the source of the enzyme. Our results suggest that pressure adaptation of Na⁺/K⁺-ATPase is the result of both changes in the primary structure of the protein and homeoviscous adaptation of the lipid environment.Abbreviations EDTA; DPH 1,6-diphenyl-1,3,5-hexatriene - PC phosphatidylcholine - PL phospholipid - SDH succinate dehydrogenase     

Differential Properties Between an Endogenous Brain Na+, K+-ATPase Inhibitor and Ouabain

By means of a Sephadex G-50 column and anionic exchange HPLC a cerebral cortex soluble fraction (II-E) which highly inhibits neuronal Na⁺-K⁺-ATPase activity has been previously obtained. Herein, II-E properties are compared with those of the cardenolide ouabain, the selective and specific Na⁺, K⁺-ATPase inhibitor. It was observed that alkali treatment destroyed II-E but not ouabain inhibitory activity. II-E presented a maximal absorbance at 265 nm both at pH 7 and pH 2 which diminished at pH 10. Ouabain showed a maximum at 220 nm which was not altered by alkalinization. II-E was not retained in a C-18 column, indicating its hydrophilic nature, whereas ouabain presented a 26-min retention time in reverse phase HPLC. Therefore, it is concluded that the inhibitory factor present in II-E is structurally different to ouabain.     

Nitration as a Mechanism of Na+, K+-ATPase Modification During Hypoxia in the Cerebral Cortex of the Guinea Pig Fetus

Previous studies have shown that hypoxia induces nitric oxide synthase-mediated generation of nitric oxide free radicals leading to peroxynitrite production. The present study tests the hypothesis that hypoxia results in NO-mediated modification of Na⁺, K⁺-ATPase in the fetal brain. Studies were conducted in guinea pig fetuses of 58-days gestation. The mothers were exposed to FiO₂ of 0.07% for 1 hour. Brain tissue hypoxia in the fetus was confirmed biochemically by decreased ATP and phosphocreatine levels. P₂ membrane fractions were prepared from normoxic and hypoxic fetuses and divided into untreated and treated groups. The membranes were treated with 0.5 mM peroxynitrite at pH 7.6. The Na⁺, K⁺-ATPase activity was determined at 37°C for five minutes in a medium containing 100 mM NaCl, 20 mM KCl, 6.0 mM MgCl₂, 50 mM Tris HCl buffer pH 7.4, 3.0 mM ATP with or without 10 mM ouabain. Ouabain sensitive activity was referred to as Na⁺, K⁺-ATPase activity. Following peroxynitrite exposure, the activity of Na⁺, K⁺-ATPase in guinea pig brain was reduced by 36% in normoxic membranes and further 29% in hypoxic membranes. Enzyme kinetics was determined at varying concentrations of ATP (0.5 mM-2.0 mM). The results indicate that peroxynitrite treatment alters the affinity of the active site of Na⁺, K⁺-ATPase for ATP and decreases the Vmax by 35% in hypoxic membranes. When compared to untreated normoxic membranes Vmax decreases by 35.6% in treated normoxic membranes and further to 52% in treated hypoxic membranes. The data show that peroxynitrite treatment induces modification of Na⁺, K⁺-ATPase. The results demonstrate that peroxynitrite decreased activity of Na⁺, K⁺-ATPase enzyme by altering the active sites as well as the microenvironment of the enzyme. We propose that nitric oxide synthase-mediated formation of peroxynitrite during hypoxia is a potential mechanism of hypoxia-induced decrease in Na⁺, K⁺-ATPase activity.