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.     

ATPase and phosphatase activities from human red cell membranes III. Stimulation of K+-activated phosphatase by phospholipase C

Summary Treatment of red cell membranes with pure phospholipase C inactivates (Na⁺+K⁺)-ATPase activity and Na⁺-dependent phosphorylation but increases K⁺-dependent phosphatase activity. When phospholipase A₂ replaces phospholipase C, all activities are lost. Activation of K⁺-dependent phosphatase by treatment with phospholipase C is caused by an increase in the maximum rate of hydrolysis ofp-nitrophenylphosphate and in the maximum activating effect of K⁺, the apparent affinities for substrate and cofactors being little affected. After phospholipase C treatment K⁺-dependent phosphatase is no longer sensitive to ouabain but becomes more sensitive to N-ethylmaleimide. In treated membranes Na⁺ partially replaces K⁺ as an activator of the phosphatase. Although ATP still inhibits phosphatase activity, neither ATP nor ATP+Na⁺ are able to modify the apparent affinity for K⁺ of K⁺-dependent phosphatase in these membranes.     

Na+,K+-ATPase in developing fetal guinea pig brain and the effect of maternal hypoxia

Na⁺,K⁺-ATPase activity was determined in fetal guinea pig brain at 35, 40, 45, 50, 55, and 60 days of gestation. The activity remained at a constant level during the early periods (35–45 days) of gestation and increased significantly during 45–60 days. Following maternal hypoxia, the activity of Na⁺,K⁺-ATPase in the term (60 days) fetal brain was reduced by 50% whereas the preterm (50 days) brain activity was unaffected. Under identical hypoxic conditions, the enzymatic activity of adult brain was significantly reduced by 20%. Na⁺,K⁺-ATPase obtained from fetal brain (50 days of gestation) has both a low and a high affinity for ATP (K _m values =0.50 and 0.053 mM and correspondingV _max values =10.77 and 2.82 umoles Pi/mg protein/hr), whereas the enzyme in the adult brain has only a low affinity (K _m=1.67 mM andV _max=20.32 umoles Pi/mg protein/hr). The high and low affinity sites for ATP in the fetal brain suggests a mechanism essential for the maintenance of cellular ionic gradients at low concentrations of ATP and which would provide the fetal brain with a greater tolerance to hypoxia. The high sensitivity of Na⁺,K⁺-ATPase activity to hypoxia in guinea pig brain at term suggests that the cell membrane functions of the fetal brain may be more susceptible to hypoxia at term than it is earlier in gestation.     

Gel electrophoretic identity of the (Na+ + Mg2+)- and (Na+ + Ca2+)-stimulated phosphorylations of rat brain ATPase

The classical E₂-P intermediate of (Na⁺ + K⁺)-ATPase dephosphorylates readily in the presence of K⁺ and is not affected by the addition of ADP. To determine the significane in the reaction cycle of (Na⁺ + K⁺)-ATPase of kinetically atypical phosphorylations of rat brain (Na⁺ + K⁺)-ATPase we compared these phosphorylated components with the classical E₂-P intermediate of this enzyme by gel electrophoresis. When rat brain (Na⁺ + K⁺)-ATPase was phosphorylated in the presence of high concentrations of Na⁺ a proportion of the phosphorylated material formed was sensitive to ADP but resistant to K⁺. Similarly, if phosphorylation was carried out in the presence of Na⁺ and Ca²⁺ up to 300 pmol/mg protein of a K⁺-resistant, ADP-sensitive material were formed. If phosphorylation was from [γ-³²P]CTP up to 800 pmol ³²P/mg protein of an ADP-resistant, K⁺-sensitive phosphorylated matterial were formed. On gel electrophoresis these phosphorylated materials co-migrated with authentic Na⁺-stimulated, K⁺-sensitive, E₂-P-phosphorylated intermediate of (Na⁺ + K⁺)-ATPase, supporting suggestions that they represent phosphorylated intermediates in the reaction sequence of this enzyme.     

Studies of (Na+ + K+)-sensitive ATPase activity in pig lymphocytes. Effects of concanavalin A

(Na⁺ + K⁺)-ATPase activity is demonstrated in plasma membranes from pig mesenteric lymph nodes. After dodecyl sulfate treatment plasma membranes have an 18-fold higher (Na⁺ + K⁺)-ATPase activity, while their ouabain-insensitive Mg²⁺-ATPase is markedly lowered. A solubilized (Na⁺ + K⁺)-ATPase fraction, obtained by Lubrol WX treatment of the membranes, has very high specific activity (21μmol P_i/h per mg protein). Concanavalin A has no effect on these partially purified (Na⁺ + K⁺)-ATPase, while it inhibits (40%) this activity in less purified fractions which still contain Mg²⁺-ATPase activity.     

Na+,K+-ATPase Na+ Affinity in Rat Skeletal Muscle Fiber Types

Previous studies in expression systems have found different ion activation of the Na⁺/K⁺-ATPase isozymes, which suggest that different muscles have different ion affinities. The rate of ATP hydrolysis was used to quantify Na⁺,K⁺-ATPase activity, and the Na⁺ affinity of Na⁺,K⁺-ATPase was studied in total membranes from rat muscle and purified membranes from muscle with different fiber types. The Na⁺ affinity was higher (K _m lower) in oxidative muscle compared with glycolytic muscle and in purified membranes from oxidative muscle compared with glycolytic muscle. Na⁺,K⁺-ATPase isoform analysis implied that heterodimers containing the β₁ isoform have a higher Na⁺ affinity than heterodimers containing the β₂ isoform. Immunoprecipitation experiments demonstrated that dimers with α₁ are responsible for approximately 36% of the total Na,K-ATPase activity. Selective inhibition of the α₂ isoform with ouabain suggested that heterodimers containing the α₁ isoform have a higher Na⁺ affinity than heterodimers containing the α₂ isoform. The estimated K _m values for Na⁺ are 4.0, 5.5, 7.5 and 13 mM for α₁β₁, α₂β₁, α₁β₂ and α₂β₂, respectively. The affinity differences and isoform distributions imply that the degree of activation of Na⁺,K⁺-ATPase at physiological Na⁺ concentrations differs between muscles (oxidative and glycolytic) and between subcellular membrane domains with different isoform compositions. These differences may have consequences for ion balance across the muscle membrane.     

Control of brain slice respiration by (Na+ + K+)-activated adenosine triphosphatase and the effects of enzyme inhibitors

The involvement of membrane (Na⁺ + K⁺)-ATPase (Mg²⁺-dependent, (Na⁺ + K⁺)-activated ATP phosphohydrolase, E.C. 3.6.1.3) in the oxygen consumption of rat brain cortical slices was studied in order to determine whether (Na⁺ + K⁺)-ATPase activity in intact cells can be estimated from oxygen consumption. The stimulation of brain slice respiration with K⁺ required the simultaneous presence of Na⁺. Ouabain, a specific inhibitor of (Na⁺ + K⁺)-ATPase, significantly inhibited the (Na⁺ + K⁺)-stimulation of respiration. These observations suggest that the (Na⁺ + K⁺)-stimulation of brain slice respiration is related to ADP production as a result of (Na⁺ + K⁺)-ATPase activity. However, ouabain also inhibited non-K⁺-stimulated respiration. Additionally, ouabain markedly reduced the stimulation of respiration by 2,4-dinitrophenol in a high (Na⁺ + K⁺)-medium. Thus, ouabain depresses brain slice respiration by reducing the availability of ADP through (Na⁺ + K⁺)-ATPase inhibition and acts additionally by increasing the intracellular Na⁺ concentration. These studies indicate that the use of ouabain results in an over-estimation of the respiration related to (Na⁺ + K⁺)-ATPase activity. This fraction of the respiration can be estimated more precisely from the difference between slice respiration in high Na⁺ and K⁺ media and that in choline, K⁺ media. Studies were performed with two (Na⁺ + K⁺)-ATPase inhibitors to determine whether administration of these agents to intact rats would produce changes in brain respiration and (Na⁺ + K⁺)-ATPase activity. The intraperitoneal injection of digitoxin in rats caused an inhibition of brain (Na⁺ + K⁺)-ATPase and related respiration, but chlorpromazine failed to alter either (Na⁺ + K⁺)-ATPase activity or related respiration.     

Gel electrophoretic and density gradient analysis of the (K + Ca)-ATPase and the (Na + K)-ATPase activities of cardiac membrane vesicles

The two major ATPase activities of intact and leaky cardiac membrane vesicles (microsomes) were characterized with respect to ionic activation requirements. The predominant ATPase activity of intact vesicles was (K⁺ + Ca²⁺)-ATPase, an enzymic activity localized to sarcoplasmic reticulum, whereas the predominant ATPase activity of leaky, sodium dodecyl sulfate-pretreated vesicles was (Na⁺ + K⁺)-ATPase, an enzymic activity localized to sarcolemma. The (K⁺ + Ca²⁺)-ATPase activity was stimulated 4- to 5-fold by 100 mM K⁺ in the presence of 50 μM Ca²⁺. Phosphorylation of the (K⁺ + Ca²⁺)-ATPase of intact vesicles with [γ-³²P]ATP was Ca²⁺ dependent, and monovalent cations including K⁺ increased the level of [³²P]phosphoprotein by up to 50% when phosphorylation was measured at 5°C. After the intact vesicles were treated with SDS (0.30 mg/ml), (K⁺ + Ca²⁺)-ATPase was inactivated, as was Ca²⁺-dependent ³²P incorporation. The monovalent cation-stimulated ATPase activity of the particulate residue (SDS-extracted membrane vesicles) displayed the usual characteristics of ouabain-sensitive (Na⁺ + K⁺)-ATPase and the activity was increased 9- to 14-fold over the small amount of patent (Na⁺ + K⁺)-ATPase activity of intact membrane vesicles. ³²P incorporation by the (Na⁺ + K⁺)-ATPase of SDS-extracted vesicles was Na⁺ dependent, and Na⁺-stimulated incorporation was increased 7- to 9-fold over that of intact vesicles.Slab gel polyacrylamide electrophoresis of both intact and SDS-extracted crude vesicle preparations revealed at least 40 distinct Coomassie Blue-positive protein bands and provided evidence for a possible heterogeneous membrane origin of the vesicles. Periodic acid-Schiff staining of the gels revealed at least two major glycoproteins. Simultaneous electrophoresis of the ³²P-intermediates of the (K⁺ + Ca²⁺)-ATPase and the (Na⁺ + K⁺)-ATPase in the same gels did not resolve the two enzymes clearly. With sucrose gradient centrifugation of intact membrane vesicles, it was possible to physically resolve the two ATPase activities. Latent (Na⁺ + K⁺)-ATPase activity (unmasked by exposing the various fractions to SDS) was found in the higher regions of the gradient, whereas (K⁺ + Ca²⁺)-ATPase activity was primarily in the denser regions. A reasonable interpretation of the data is that cardiac microsomes consist of membrane vesicles derived both from sarcolemma and sarcoplasmic reticulum. (Na⁺ + K⁺)-ATPase is localized to intact vesicles of sarcolemma but is mainly latent, whereas (K⁺ + Ca²⁺)-ATPase is mostly patent and is localized to vesicles of sarcoplasmic reticulum.     

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.     

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.     

Effects of two-vessel forebrain ischemia and of administration of indomethacin and quinacrine on Na<Superscript>+</Superscript>, K<Superscript>+</Superscript>-ATPase activity in various rat brain areas

The effect of a two-vessel forebrain ischemia (induced by occlusion of carotid arteries and hypotension), subsequent reperfusion, and administration of indomethacin and quinacrine on the Na⁺,K⁺-ATPase activity and diene conjugate content was studied in various rat forebrain fields. The most pronounced metabolic alterations were observed during ischemia and reperfusion. Under these effects, there was a statistically significant reduction of the Na⁺,K⁺-ATPase activity in the brain cortex and striatum and an increase of the diene conjugate content in the rat brain cortex in comparison with sham-operated animals. Injection of indomethacin, a cyclooxygenase inhibitor, to rats subjected to ischemia and reperfusion, resulted to a statistically significant increase of the Na⁺,K⁺-ATPase activity in the brain cortex, hippocampus, and striatum (p < 0.02) as compared with control animals. The diene conjugate content in the rat brain cortex during brain ischemia and reperfusion was statistically significantly lower in the rats injected with indomethacin. The effect of quinacrine (a blocker of phospholipase A₂) was similar to that of indomethacin in the rat cortex, whereas in the rat striatum and hippocampus, the quinacrine effect during ischemia and reperfusion was less marked than that of indomethacin. The obtained data indicate the ability of inhibitors of the arachidonic pathway of free radical formation to normalize the Na⁺, K⁺-ATPase activity during brain ischemia. There also revealed local peculiarities of metabolic disturbances in different regions of the rat forebrain during ischemia and reperfusion.Translated from Zhurnal Evolyutsionnoi Biokhimii i Fiziologii, Vol. 41, No. 1, 2005, pp. 33–38.Original Russian Text Copyright © 2005 by Molchanova, Moskvin, Zakharova, Yurlova, Nosova, Avrova.     

Human Brain Capillary Endothelium: Modulation of K+ Efflux and K+, Ca2+ Uptake by Endothelin

This report describes K⁺ efflux, K⁺ and Ca²⁺ uptake responses to endothelins (ET-1 and ET-3) in cultured endothelium derived from capillaries of human brain (HBEC). ET-1 dose dependently increased K⁺ efflux, K⁺ and Ca²⁺ uptake in these cells. ET-1 stimulated K⁺ efflux occurred prior to that of K⁺ uptake. ET-3 was ineffective. The main contributor to the ET-1 induced K⁺ uptake was ouabain but not bumetanide-sensitive (Na⁺-K⁺-ATPase and Na⁺-K⁺-Cl^– cotransport activity, respectively). All tested paradigms of ET-1 effects in HBEC were inhibited by selective antagonist of ET_A but not ET_B receptors and inhibitors of phospholipase C and receptor-operated Ca²⁺ channels. Activation of protein kinase C (PKC) decreased whereas inhibition of PKC increased the ET-1 stimulated K⁺ efflux, K⁺ and Ca²⁺ uptake in HBEC. The results indicate that ET-1 affects the HBEC ionic transport systems through activation of ET_A receptors linked to PLC and modulated by intracellular Ca²⁺ mobilization and PKC.     

Preparation of renal cortex basal-lateral and brush border membranes. Localization of adenylate cyclase and guanylate cyclase activities

Luminal brush border and contraluminal basal-lateral segments of the plasma membrane from the same kidney cortex were prepared. The brush border membrane preparation was enriched in trehalase and γ-glutamyltranspeptidase, whereas the basal-lateral membrane preparation was enriched in (Na⁺ + K⁺)-ATPase. However, the specific activity of (Na⁺ + K⁺)-ATPase in brush border membranes also increased relative to that in the crude plasma membrane fraction, suggesting that (Na⁺ + K⁺)-ATPase may be an intrinsic constituent of the renal brush border membrane in addition to being prevalent in the basal-lateral membrane. Adenylate cyclase had the same distribution pattern as (Na⁺ + K⁺)-ATPase, i.e. higher specific activity in basal-lateral membranes and present in brush border membranes. Adenylate cyclase in both membrane preparations was stimulated by parathyroid hormone, calcitonin, epinephrine, prostaglandins and 5′-guanylylimidodiphosphate. When the agonists were used in combination enhancements were additive. In contrast to the distribution of adenylate cyclase, guanylate cyclase was found in the cytosol and in basal-lateral membranes with a maximal specific activity (NaN₃ plus Triton X-100) 10-fold that in brush border membranes. ATP enhanced guanylate cyclase activity only in basal-lateral membranes. It is proposed that guanylate cyclase, in addition to (Na⁺ + K⁺)-ATPase, be used as an enzyme “marker” for the renal basal-lateral membrane.     

Tissue specificity of dopamine effects on brain ATPases

Dopamine inhibits Mg²⁺,Na⁺,K⁺- and Na⁺,K⁺-ATPase activities but does not modify Mg²⁺-ATPase activity of nerve ending membranes isolated from rat cerebral cortex. In the presence of the soluble fraction of brain, dopamine activates total, Na⁺,K⁺-, and Mg²⁺-ATPases. Dopamine stimulation of nerve ending membrane ATPases is achieved when soluble fractions of brain, kidney, or liver are used. On the other hand, dopamine effects are not observed on kidney or heart ATPase preparations. These results indicate tissue specificity of dopamine effects with respect to the enzyme source; there is no tissue specificity for the requirement of the soluble fraction to achieve stimulation of ATPases by dopamine.     

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.     

Activation energy and phospholipid requirements of membrane-bound adenosine triphosphatases

In order to evaluate the role of lipids in the function of membrane ATPase reactions, the apparent activation energies of membrane-bound (Na⁺ + K⁺)-ATPase and membrane-bound Mg²⁺-ATPase have been measured under conditions frequently supposed to alter the membrane lipids in vitro.In the case of (Na⁺ + K⁺)-ATPase, the untreated enzyme was shown to have two different activation energies as shown by an Arrhenius plot comprising two straight lines which intersect at the “critical temperature.” Treatment of the preparation with detergents or with phospholipase C causes some alteration in the spécifie activity of the enzyme but did not significantly alter the activation energies or the critical temperature. After treatment with phospholipase A, however, the Arrhenius plot appeared linear over the entire temperature range studied. Subsequent treatment of phospholipase A-treated preparations with phosphatidylserine restored the control response.Conversely, untreated preparations of Mg²⁺-ATPase give an Arrhenius plot which is neither linear nor composed of two intersecting straight lines. This plot, which we regard as curvilinear, does not permit a unique value of the activation energy to be determined. The shape of this plot is unaltered by detergent or by treatment with phospholipase C. In contrast to (Na⁺ + K⁺)-ATPase, it is also unaffected by treat-with phospholipase A or phospholipase A followed by phosphatidylserine.We conclude that although (Na⁺ + K⁺)-ATPase and Mg²⁺-ATPase are frequently closely associated in many membranes, their functions involve the presence of different membrane lipids.     

Interactions of K+ ATP channel blockers with Na+/K+-ATPase

Two K⁺ _ATP channel blockers, 5-hydroxydecanoate (5-HD) and glyburide, are often used to study cross-talk between Na⁺/K⁺-ATPase and these channels. The aim of this work was to characterize the effects of these blockers on purified Na⁺/K⁺-ATPase as an aid to appropriate use of these drugs in studies on this cross-talk. In contrast to known dual effects (activating and inhibitory) of other fatty acids on Na⁺/K⁺-ATPase, 5-HD only inhibited the enzyme at concentrations exceeding those that block mitochondrial K⁺ _ATP channels. 5-HD did not affect the ouabain sensitivity of Na⁺/K⁺-ATPase. Glyburide had both activating and inhibitory effects on Na⁺/K⁺-ATPase at concentrations used to block plasma membrane K⁺ _ATP channels. The findings justify the use of 5-HD as specific mitochondrial channel blocker in studies on the relation of this channel to Na⁺/K⁺-ATPase, but question the use of glyburide as a specific blocker of plasma membrane K⁺ _ATP channels, when the relation of this channel to Na⁺/K⁺-ATPase is being studied.     

(Na++K+)-adenosinetriphosphatase in the brain of Shiverer (Shi/Shi) mice

The myelin-deficient Shiverer (Shi/Shi) mutant mouse may be a useful model in assessing the dependence of brain (Na⁺+K⁺)-ATPase concentration and composition on myelin membrane formation. Brain microsomal membranes from age-matched control (+/+) and Shiverer (Shi/Shi) mice were fractionated by differential centrifugation and sucrose gradient sedimentation. No reduction in (Na⁺+K⁺)-ATPase specific activity was measured in whole homogenates, high-and low-speed fractions or gradient fractions from brains of Shi/Shi mice as compared to those of +/+ mice. In addition, sodium dodecylsulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting with antisera specific for mouse brain (Na⁺+K⁺)-ATPase revealed no significant difference in catalytic subunit composition between fractions of +/+ and Shi/Shi brains. The similar results obtained for both +/+ and myelin-deficient Shi/Shi mice suggest that myelin contributes little to total brain (Na⁺+K⁺)-ATPase.     

Kinetics of Na+-, K+-ATPase Inhibition by an Endogenous Modulator (II-A)

We have previously reported the isolation by gel filtration and anionic exchange HPLC of two brain Na⁺, K⁺-ATPase inhibitors, II-A and II-E, and kinetics of enzyme interaction with the latter. In the present study we evaluated the kinetics of synaptosomal membrane Na⁺, K⁺-ATPase with II-A and found that inhibitory activity was independent of ATP (2–8 mM), Na⁺ (3.1–100 mM), or K⁺ (2.5–40 mM) concentration. Hanes-Woolf plots showed that II-A decreases V_max in all cases; K_M value decreased for ATP but remained unaltered for Na⁺ and K⁺, indicating respectively uncompetitive and noncompetitive interaction. However, II-A became a stimulator at 0.3 mM K⁺ concentration. It is postulated that brain endogenous factor II-A may behave as a sodium pump modulator at the synaptic region, an action which depends on K⁺ concentration.     

Ro le of phospholipids in the NA + ,K + -stimulated adenosine triphosphatase system of brain microsomes

Removal of phospholipids from brain microsomes using a purified, protease-free phospholipase C preparation led to proportional losses of net Na⁺,K⁺-stimulated adenosine triphosphatase, K⁺-stimulated p-nitrophenylphosphatase, and Na⁺-stimulated ADP-ATP exchange activities. These enzymatic activities were restored to 60–100% of control values by the addition of a variety of purified phospholipids, but not by detergents or EGTA. These findings support the concept of a general phospholipid requirement for this enzyme system. This work further suggests that phospholipids are important both for formation and decomposition of the phosphorylated intermediate (s) which probably participate in the net reaction.