Two Isoenzymes of Na+, K+-ATPase Have Different Kinetics of K+ Dephosphorylation in Normal Cat and Human Brain Cortex

Analysis of purified Na+,K+-ATPase from cat and human cortex by sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals two large catalytic subunits called alpha (-) (lower molecular weight) and alpha (+) (higher molecular weight). Differences in K+ dephosphorylation of these two molecular forms have been investigated by measuring the phosphorylation level of each protein after their separation on sodium dodecyl sulfate gels. In the presence of Na+, Mg2+, and ATP, both subunits are phosphorylated. Increasing concentrations (from 0 to 3 mM) of K+ induce progressive dephosphorylation of both alpha-subunits, although the phosphoprotein content of alpha (-) is decreased significantly less than that of alpha (+). Ka values of alpha (-) for K+ are 40% and 50% greater in cat and human cortex, respectively, than values of alpha (+). alpha (-) and alpha (+) are thought to be localized in specific cell types of the brain: alpha (-) is the exclusive form of nonneuronal cells (astrocytes), whereas alpha (+) is the only form of axolemma. Our results support the hypothesis that glial and neuronal Na+,K+-ATPases are different molecular entities differing at least by their K+ sensitivity. Results are discussed in relation to the role of glial cells in the regulation of extracellular K+ in brain.     

Ouabain Binding, ATP Hydrolysis, and Na+,K+-Pump Activity During Chemical Modification of Brain and Muscle Na+,K+-ATPase

The effects of 16 group-specific, amino acid-modifying agents were tested on ouabain binding, catalytical activity of membrane-bound (rat brain microsomal), sodium dodecyl sulfate-treated Na+,K(+)-ATPase, and Na+,K(+)-pump activity in intact muscle cells. With few exceptions, the potency of various tryptophan, tyrosine, histidine, amino, and carboxy group-oriented drugs to suppress ouabain binding and Na+,K(+)-ATPase activity correlated with inhibition of the Na+,K(+)-pump electrogenic effect. ATP hydrolysis was more sensitive to inhibition elicited by chemical modification than ouabain binding (membrane-bound or isolated enzyme) and than Na+,K(+)-pump activity. The efficiency of various drugs belonging to the same "specificity" group differed markedly. Tyrosine-oriented tetranitromethane was the only reagent that interfered directly with the cardiac receptor binding site as its inhibition of ouabain binding was completely protected by ouabagenin preincubation. The inhibition elicited by all other reagents was not, or only partially, protected by ouabagenin. It is surprising that agents like diethyl pyrocarbonate (histidine groups) or butanedione (arginine groups), whose action should be oriented to amino acids not involved in the putative ouabain binding site (represented by the -Glu-Tyr-Thr-Trp-Leu-Glu- sequence), are equally effective as agents acting on amino acids present directly in the ouabain binding site. These results support the proposal of long-distance regulation of Na+,K(+)-ATPase active sites.     

Inhibition of Protein Kinase C Restores Na+, K+-ATPase Activity in Sciatic Nerve of Diabetic Mice

We have tested if inhibition of protein kinase C is able to prevent and/or to restore the decrease of Na+,K(+)-ATPase activity in the sciatic nerve of alloxan-induced diabetic mice. Mice were made diabetic by subcutaneous injection of 200 mg of alloxan/kg of body weight. The activity of Na+,K(+)-ATPase decreased rapidly (43% after 3 days) and slightly thereafter (58% at 11 days). We show that intraperitoneal injection of 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7), an inhibitor of protein kinase C, prevents completely the loss of Na+,K(+)-ATPase activity produced by alloxan. Also, H7 injected into diabetic mice, 4-9 days after the injection of alloxan, restores the activity of the enzyme. The amount of activity recovered depends on the dose of H7 administered; complete recovery was reached with injection of 15 mg of H7/kg of body weight. The effect of H7 is transient, with a half-life of approximately 1 h.     

Quantification of Rat Cerebral Cortex Na+,K+-ATPase: Effect of Age and Potassium Depletion

Na+,K(+)-ATPase concentration in rat cerebral cortex was studied by vanadate-facilitated [3H]ouabain binding to intact samples and by K(+)-dependent 3-O-methylfluorescein phosphatase activity determinations in crude homogenates. Methodological errors of both methods were evaluated. [3H]Ouabain binding to cerebral cortex obtained from 12-week-old rats measured incubating samples in buffer containing [3H]ouabain, and ouabain at a final concentration of 1 x 10(-6) mol/L gave a value of 11,351 +/- 177 (n = 5) pmol/g wet weight (mean +/- SEM) without any significant variation between the lobes. Evaluation of affinity for ouabain was in agreement with a heterogeneous population of [3H]ouabain binding sites. K(+)-dependent 3-O-methylfluorescein phosphatase activity in crude cerebral homogenates of age-matched rats was 7.24 +/- 0.14 (n = 5) mumol/min/g wet weight, corresponding to a Na+,K(+)-ATPase concentration of 12,209 +/- 236 pmol/g wet weight. It was concluded that the present methods were suitable for quantitative studies of cerebral cortex Na+,K(+)-ATPase. The concentration of rat cerebral cortex Na+,K(+)-ATPase showed approximately 10-fold increase within the first 4 weeks of life to reach a plateau of approximately 11,000-12,000 pmol/g wet weight, indicating a larger synthesis of Na+,K+ pumps than tissue mass in rat cerebral cortex during the first 4 weeks of development. K+ depletion induced by K(+)-deficient fodder for 2 weeks resulted in a slight tendency toward a reduction in K+ content (6%, p > 0.5) and Na+,K(+)-ATPase concentration (3%, p > 0.4) in cerebral cortex, whereas soleus muscle K+ content and Na+,K(+)-ATPase concentration were decreased by 30 (p < 0.02) and 32% (p < 0.001), respectively. Hence, during K+ depletion, cerebral cortex can maintain almost normal K+ homeostasis, whereas K+ as well as Na+,K+ pumps are lost from skeletal muscles.     

Immunocytochemical Localization of (Na+,K+)-ATPase in the Goldfish Optic Nerve

Antiserum to the catalytic subunit of goldfish brain (Na+, K+)-ATPase has been employed at the electron microscopic level by means of the peroxidase-antiperoxidase immunohistochemical method. In optic nerve, antigenic sites are restricted to the nodes of Ranvier. No reaction product is detected in underlying internodal neurolemma. Outgrowing neurites for cultured retinal explants devoid of glial ensheathment exhibit a continuous distribution of the enzyme subunit. Antibodies against eel electroplax (Na+, K+)-ATPase cross-react with the goldfish brain enzyme and show a similar immunocytochemical distribution pattern.     

Highly Ouabain-Sensitive α3 Isoform of Na+,K+-ATPase in Human Brain

The Na+,K(+)-ATPase alpha 3 isoform has recently been demonstrated immunochemically in human brain. Conclusive biochemical evidence, however, is still lacking. In this study, a unique 50-kDa polypeptide, which is known to be specific to the rat alpha 3 isoform, has been found in human brainstem Na+,K(+)-ATPase following formic acid treatment of the purified alpha isoform proteins. Human alpha 3 Na+,K(+)-ATPase is also highly sensitive to ouabain inhibition, with a 50% ouabain inhibition value of 1.0 x 10(-7) M. These results provide clear and direct evidence for the existence of the alpha 3 isoform in human brain.     

Activity-Dependent Regulation of Na+,K+-ATPase α Isoform mRNA Expression In Vivo

To investigate the functional role of the different Na+, K(+)-ATPase alpha (catalytic) subunit isoforms in neuronal cells, we used quantitative in situ hybridization with riboprobes specific for alpha 1, alpha 2, and alpha 3 isoforms to measure the level of alpha isoform-specific expression in the neuroendocrine cells of the supraoptic (SON) and paraventricular (PVN) nuclei of rat hypothalamus. A prolonged increase in electrical activity of these cells, achieved by 5 days of salt treatment, increased the amount of alpha 1 isoform mRNA in the SON and PVN by 50%. Levels of alpha 1 mRNA in other brain regions and levels of alpha 2 and alpha 3 mRNAs were not affected by salt treatment. We conclude that the alpha 1 isoform Na+, K(+)-ATPase may be specifically adapted to pump out Na+, which enters the cells through voltage-gated channels during neuronal depolarization.     

Functional Significance of the Effects of Neurotransmitters on the Na+,K+-ATPase System

The aim of the present experiments was to study the effects of the neurotransmitters acetylcholine, noradrenaline, 5-hydroxytryptamine, and dopamine on the Na+,K+-ATPase of rat brain synaptosomal fractions. It is shown that dopamine at low concentrations specifically inhibits the Na+,K+-ATPase of synaptic membranes from the brain regions rich in dopaminergic endings, but has no effect on the synaptosomal Na+,K+-ATPase from the other parts of brain. Acetylcholine and noradrenaline have similar specific effects on Na+,K+-ATPase from cholinergic and adrenergic synaptosomes. The Na+,K+-ATPase of synaptic membranes from the different brain regions, characterised by different distributions of cholinergic, adrenergic, and 5-hydroxytryptaminergic endings, show different reactions with neurotransmitters. These data indicate a functional significance of the effects of the neurotransmitters on the synaptosomal Na+,K+-ATPase.     

Nerve Growth Factor Induces Na+, K+-ATPase in a Nerve Cell Line

The effects of nerve growth factor (NGF) on induction of Na+,K+-ATPase were examined in a rat pheochromocytoma cell line, PC12h. Na+,K+-ATPase activity in a crude particulate fraction from the cells increased from 0.37 +/- 0.02 (n = 19) to 0.55 +/- 0.02 (n = 20) (means +/- SEM, mumol Pi/min/mg of protein) when cultured with NGF for 5-11 days. The increase caused by NGF was prevented by addition of specific anti-NGF antibodies. Epidermal growth factor and insulin had only a small effect on induction of Na+,K+-ATPase. A concentration of basic fibroblast growth factor three times higher than that of NGF showed a similar potency to NGF. The molecular form of the enzyme was judged as only the alpha form in both the untreated and the NGF-treated cells by a simple pattern of low-affinity interaction with cardiotonic steroids: inhibition of enzyme activity by strophanthidin (Ki approximately 1 mM) and inhibition of Rb+ uptake by ouabain (Ki approximately 100 microM). As a consequence, during differentiation of PC12h cells to neuron-like cells, NGF increases the alpha form of Na+,K+-ATPase, but does not induce the alpha(+) form of the enzyme, which has a high sensitivity for cardiotonic steroid and is a characteristic form in neurons.     

Distribution of Na+, K+-ATPase α-Subunit Isoforms in Rat Pituitary

The distributions of alpha-subunit isoforms of the Na+,K(+)-ATPase in rat pituitary were determined by immunoblotting and immunohistochemistry. Immunoreactivity for all three forms is present in the neural lobe, whereas the anterior lobe contains only alpha 1 and alpha 2. Most areas of the intermediate lobe exhibit faint immunoreactivity for only alpha 1, but thin strands of cells which stain strongly for all three isoforms are also present in this lobe. The previously reported ouabain inhibitable Na+,K(+)-ATPase activity in the neural lobe is consistent with the presence of both alpha 2 and alpha 3 subunits.     

Activation of (Na+, K+)-ATPase by Nanomolar Concentrations of GM1 Ganglioside

GM1 ganglioside binding to the crude mitochondrial fraction of rat brain and its effect on (Na+, K+)-ATPase were studied, the following results being obtained: (a) the binding process followed a biphasic kinetics with a break at 50 nM-GM1; GM1 at concentrations below the break was stably associated, while over the break it was loosely associated; (b) stably bound GM1 activated (Na+, K+)-ATPase up to a maximum of 43%; (c) the activation was dependent upon the amount of bound GM1 and was highest at the critical concentration of 20 pmol bound GM1 X mg protein-1; (d) loosely bound GM1 suppressed the activating effect on (Na+, K+)-ATPase elicited by firmly bound GM1; (e) GM1-activated (Na+, K+)-ATPase had the same pH optimum and apparent Km (for ATP) as normal (Na+, K+)-ATPase but a greater apparent Vmax; (f) under identical binding conditions (2 h, 37 degrees C, with 40 nM substance) all tested gangliosides (GM1, GD1a, GD1b, GT1b) activated (Na+, K+)-ATPase (from 26-43%); NeuNAc, sodium dodecylsulphate, sulphatide and cerebroside had only a very slight effect. It is suggested that the ganglioside activation of (Na+-K+)-ATPase is a specific phenomenon not related to the amphiphilic and ionic properties of gangliosides, but due to modifications of the membrane lipid environment surrounding the enzyme.     

Regulation of Na+, K+ -ATPase Isoforms in Rat Neostriatum by Dopamine and Protein Kinase C

Our previous studies showed that dopamine inhibits Na+,K+-ATPase activity in acutely dissociated neurons from striatum. In the present study, we have found that in this preparation, dopamine inhibited significantly (by approximately 25%) the activity of the alpha3 and/or alpha2 isoforms, but not the alpha1 isoform, of Na+,K+-ATPase. Dopamine, via D1 receptors, activates cyclic AMP-dependent protein kinase (PKA) in striatal neurons. Dopamine is also known to activate the calcium- and phospholipid-dependent protein kinase (PKC) in a number of different cell types. The PKC activator phorbol 12,13-dibutyrate reduced the activity of Na+,K+-ATPase alpha3 and/or alpha2 isoforms (by approximately 30%) as well as the alpha1 isoform (by approximately 15%). However, dopamine-mediated inhibition of Na+,K+-ATPase activity was unaffected by calphostin C, a PKC inhibitor. Dopamine did not affect the phosphorylation of Na+,K+-ATPase isoforms at the PKA-dependent phosphorylation site. Phorbol ester treatment did not alter the phosphorylation of alpha2 or alpha3 isoforms of Na+,K+-ATPase in neostriatal neurons but did increase the phosphorylation of the alpha1 isoform. Thus, in rat neostriatal neurons, treatment with either dopamine or PKC activators results in inhibition of the activity of specific (alpha3 and/or alpha2) isoforms of Na+,K+-ATPase, but this is not apparently mediated through direct phosphorylation of the enzyme. In addition, PKC is unlikely to mediate inhibition of rat Na+,K+-ATPase activity by dopamine in neostriatal neurons.     

An Activation of Synaptosomal Na+, K+-ATPase by a Novel Dibenzoxazepine Derivative (BY-1949) in the Rat Brain: Its Functional Role in the Neurotransmitter Uptake Systems

In search of factors mitigating the final outcome of ischemic and epileptic brain damage, we tested a novel dibenzoxazepine derivative (BY-1949), as the compound has been shown to be effective under these two conditions. First, using rat brain, we assessed whether or not BY-1949 affects the Na+,K(+)-ATPase activity. Although in vitro applications of either BY-1949 or its three major metabolites did not cause any apparent effects, both acute and chronic oral administrations of the compound (10 mg/kg) invariably increased the Na+,K(+)-ATPase activity in the synaptosomal plasma membranes by increasing Vmax values. Second, it was shown by this study that the drug treatment caused marked increases in the uptake of both glutamic acid and gamma-aminobutyric acid into the synaptosomes. These results suggest that the activity against ischemic/epileptic brain damage by BY-1949 is explicable, at least partly, in terms of improvement of ionic derangements across the neural membranes via Na+,K(+)-ATPase activation.     

Effects of Systemic Kainic Acid Administration on Regional Na+, K+-ATPase Activity in Rat Brain

Changes in the activity of Na+,K+-ATPase and in the water, Na+, and K+ levels in the parietal cortex, hippocampus, and thalamus were investigated in rats 1, 3, 6, and 24 h following systemic kainic acid injection. An increase in Na+,K+-ATPase activity was observed in all three regions 3 h after the treatment, with a subsequent decrease in enzyme activity. The elevation in Na+,K+-ATPase activity was accompanied by an increase in the Na+ content and a decrease in the K+ content. These changes are presumed to occur because of repeated discharges and excessive prolonged depolarization in response to kainic acid. The decreases in Na+,K+-ATPase activity 6 and 24 h following kainic acid treatment coincide with neuropathological damage and edema formation, mainly in the hippocampus and thalamus.     

Na+,K+-ATPase and Mg2+-ATPase Activities in Different Regions of Rat Brain During Alloxan Diabetes

The effect of alloxan diabetes on the activities of Na+,K+-ATPase and Mg2+-ATPase was studied in three regions of rat brain at various time intervals after the onset of diabetes. It was observed that Na+,K+-ATPase activity increased at early time intervals after diabetes, followed by a recovery to near control levels in all three regions of the brain. There was an overall increase in Mg2+-ATPase activity in all the regions. A reversal of the effect was observed with insulin administration to the diabetic rats.     

Kinetics of the Mechanism of Action of Monoamine Oxidase in the Regulation of Na+, K+-ATPase Activity in Rat Brain

Kinetic studies on the action of monoamine oxidase (MAO) in the regulation of Na+,K+-ATPase were performed using 3-methoxy-4-hydroxybenzaldehyde (MHB), which is an analogue of 3-methoxy-4-hydroxy-phenylacetylaldehyde (product of MAO-catalysed reaction with dopamine as substrate). It was observed that at 2.6 microM MHB, the activation of Na+,K+-ATPase may be the result of the removal of the inhibitory Ca2+, thereby increasing the Vmax. Double-reciprocal plots of Pi versus MHB showed that Ca2+ counteracted the effect of the aldehyde not by changing the Km, but be decreasing the Vmax of the Na+,K+-ATPase stimulation. The removal of 3',5'-cyclic AMP-dependent protein kinase from the microsomes by sodium dodecyl sulphate treatment abolished the activation and/or inhibition of the Na+,K+-ATPase by aldehyde; it can therefore be inferred that 3',5'-cyclic AMP-dependent protein kinase is involved in the regulation of Na+,K+-ATPase.     

Phenytoin Dephosphorylates the Catalytic Subunit of the (Na+,K+)-ATPase in C57/BL Mice

The effects of phenytoin, a potent antiepileptic drug, on the active transport of cations within membranes remain controversial. To assess the direct effects of phenytoin on the Na+,K+ pump, we studied the drug's influence on the phosphorylation of partially purified (Na+,K+)-ATPase from mouse brain. (Na+,K+)-ATPase subunits were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Phenytoin, in vitro, decreased net phosphorylation of the (Na+,K+)-ATPase catalytic subunit in a dose-dependent manner (approximately 50% at 10(-4) M). When the conversion of E1-P to E2-P, e.g., the two major phosphorylated conformational states of (Na+,K+)-ATPase, was blocked by oligomycin or N-ethylmaleimide, phenytoin had no effect. The results suggest that phenytoin acts on the phosphatasic component of the reaction cycle, decreasing the phosphorylation level of the enzyme.     

Subacute Noradrenergic Agonist Infusions In Vivo Increase Na+, K+-ATPase and Ouabain Binding in Rat Cerebral Cortex

In order to investigate the specificity of noradrenergic effects on Na+, K+-ATPase, we infused noradrenergic agonists into the cerebral ventricles of rats, with or without depletion of forebrain norepinephrine. Infusion of norepinephrine, isoproterenol, or phenylephrine increased ouabain binding in intact rats, whereas clonidine infusion decreased binding. Depletion of forebrain norepinephrine by destruction of the dorsal noradrenergic bundle reduced ouabain binding. Norepinephrine infusion reversed the effect of dorsal bundle lesion; isoproterenol and phenylephrine increased ouabain binding in lesioned rats, but did not restore the effect of the lesions. Clonidine had no effect in lesioned rats. Effects on Na+, K+-ATPase activity were similar, but smaller. These results suggest that stimulation of both alpha 1- and beta-noradrenergic receptors may be necessary for optimal Na+, K+-ATPase, and that clonidine reduces Na+, K+-ATPase indirectly through decreased norepinephrine release.     

Neuronal Na+, K+-ATPase in the peripheral nerve fibers

ATPase activity was localized by means of Wachstein-Meisel's method in rat sciatic nerve fibers. Using controls with ouabain, the presence of alpha + (neuronal) Na+, K+-ATPase was examined. The enzyme occurs in the ATPase reaction of the myelin-forming membranes, axoplasm and Schwann cell cytoplasm. Its presence in the Schwann cell plasma membrane is only admittable. The ATPase activity of the compact myelin and axolemma was exclusively of alpha + type of Na+, K+-ATPase.     

Structural peculiarities of Na+,K+-ATPase isozymes from the calf brain

Functionally active preparations of Na+,K(+)-ATPase isozymes from calf brain that contain catalytic subunits of three types (alpha 1, alpha 2, and alpha 3) were obtained using two approaches: a selective removal of contaminating proteins by the Jorgensen method and a selective solubilization of the enzyme with subsequent reconstitution of the membrane structure by the Esmann method. The ouabain inhibition constants were determined for the isozymes. The real isozyme composition of the Na+ pump from the grey matter containing glial cells and the brain stem containing neurons was determined. The plasma membranes of glial cells were shown to contain mainly Na+,K(+)-ATPase of the alpha 1 beta 1 type and minor amounts of isozymes of the alpha 2 beta 2 (beta 1) and the alpha 3 beta 1 (beta 2) type. The axolemma contains alpha 2 beta 1- and alpha 3 beta 1 isozymes. A carbohydrate analysis indicated that alpha 1 beta 1 enzyme preparations from the brain grey matter substantially differ from the renal enzymes of the same composition in the glycosylation of the beta 1 isoform. An enhanced sensitivity of the alpha 3 catalytic subunit of Na+,K(+)-ATPase from neurons to endogenous proteolysis was found. A point of specific proteolysis in the amino acid sequence PNDNR492 decreases Y493 was localized (residue numbering is that of the human alpha 3 subunit). This sequence corresponds to one of the regions of the greatest variability in alpha 1, alpha 2, alpha 3, and alpha 4-subunits, but at the same time, it is characteristic of the alpha 3 isoforms of various species. The presence of the beta 3 isoform of tubulin (cytoskeletal protein) was found for the first time in the high-molecular-mass Na+,K(+)-ATPase alpha 3 beta 1 isozyme complex isolated from the axolemma of brain stem neurons, and its binding to the alpha 3 catalytic subunit was shown.