Effects of mammalian brain extracts and chlormadinone acetate on neuronal Na, K-ATPase and electrogenic Na, K-pump activity in vitro

Acid-acetone extracts of brain (from beef and guinea pig) and chlormadinone acetate (CMA) were compared with ouabain for their ability to inhibit the electrogenic Na⁺,K⁺-pump and the Na⁺,K⁺-ATPase of neuronal tissues. The membrane potential of neurones in the paravertebral sympathetic ganglion of the bullfrog was recorded in K⁺-free Ringer's solution by means of the sucrose gap technique. The potassium activated hyperpolarization (K_H⁺), induced by the re-introduction of potassium, was used as an index of electrogenic Na⁺,K⁺-pumping. The K_H⁺ was blocked by 1 μM ouabain. Na⁺,K⁺-ATPase activity was measured in microsomal membrane preparations of frog and beef brain using a continuous spectrophotometric assay. Although ouabain consistently inhibited beef brain Na⁺,K⁺-ATPase (IC₅₀ = 2.2 μM), acid-acetone extracts prepared from guinea pig and beef brain produced only partial inhibition. Neither of the extracts significantly reduced the K_H⁺ of the frog ganglion. CMA inhibited Na⁺,K⁺-ATPase prepared from bullfrog brain and spinal cord with slightly greater potency (IC₅₀ = 4.5 μM) than did ouabain (IC₅₀ = 10 μM). In contrast, electrogenic Na⁺,K⁺-pumping (i.e. the K_H⁺) in the frog ganglion was not affected by this steroid. It is concluded that although both the extracts and CMA inhibited Na⁺,K⁺-ATPase, neither can be considered ouabain-like due to their failure to affect the electrogenic Na⁺,K⁺-pump in situ.     

Glial contribution to seizure: K activation of (Na, K)-ATPase in bulk isolated glial cells and synaptosomes of epileptogenic cortex

Glial and neuronal (Na⁺, K⁺)-ATPase have dissimilar apparent affinities for K⁺ ions. Glial (Na⁺, K⁺)-ATPase is maximally activated by 20 mM K⁺ while neuronal (Na⁺, K⁺)-ATPase is maximally stimulated by 3–5 mM K⁺. Because this glial property may play an important role in the clearance of [K⁺]₀ during seizures, we investigated the K⁺ activation of (Na⁺, K⁺)-ATPase within bulk isolated glial cells and synaptosomes isolated from epileptogenic brains. The primary focus (F), the homolateral brain area around the focus (PF) and the mirror (M) or secondary focus induced by freezing lesions were studied.Results show that both glial and synaptosomal enzyme activities in the epileptogenic state change in comparison with controls, i.e. sham-operated cats. In F and M., glial enzyme decreased reaction velocities between 3 and 18 mM K⁺. In PF, maximum velocities increased in glial (Na⁺, K⁺)-ATPase. These results indicate that in actively firing epileptogenic tissue (F, M), glial (Na⁺, K⁺)-ATPase decreased rate reactions while the catalytic activity was increased in cortical tissues surrounding the focus. These phenomena appeared early, i.e. 1–3 days after production of the freezing lesion, and was associated with a sharp decrease in absolute levels of enzyme activity.Synaptosomal (Na⁺, K⁺)-ATPase from controls always exhibited a saturation curve at 3–6 mM K⁺. Synaptosomal enzyme activities in the primary (F) lesion increased slightly 24 h after lesion production, then progressively decreased 3 days after lesion production. No significant changes were seen in M and PF.     

Alterations of cerebromicrovascular Na, K-ATPase activity due to fatty acids and acute hypertension

Acute hypertension, induced in rats by intravenous injection of angiotensin II, previously has been shown to increase cerebrovascular permeability to macromolecules. The purpose of this study was to examine the effect of acute hypertension on Na⁺, K⁺-ATPase, the enzyme responsible for controlling ionic permeability of the cerebromicrovascular endothelium. The K⁺-dependent p-nitrophenylphosphatase activity of the cerebromicrovascular Na⁺, K⁺-ATPase was determined using microvessels prepared from hypertensive and normotensive rats. When compared to controls, a 70% decrease (P < 0.02) in the maximum rate (V_max) of the Na⁺, K⁺-ATPase from hypertensive rats was evident with no change in the Michaelis constant (K_M). In contrast, γ-glutamyltranspeptidase, a marker enzyme for cerebral endothelic cells, was not significantly affected. Sodium arachidonate (1–100 μM) inhibited the phosphatase activity of the Na⁺, K⁺-ATPase in microvessels isolated from both normotensive and hypertensive rats in a dose-dependent manner. Furthermore, poly-unsaturated fatty acids (sodium linoleate and arachidonate) evoked the greatest inhibition of the enzyme, while sodium oleate and sodium palmitate inhibited the Na⁺, K⁺-ATPase to lesser extents. This regulation of enzyme activity by fatty acids was comparable in control and hypertensive groups. In summary, the data indicate that the cerebromicrovascular Na⁺, K⁺-ATPase was altered as a consequence of acute hypertension and that poly-unsaturated fatty acids can modulate this enzyme in microvessels derived from hypertensive or control rats     

Erythrocyte membrane sodium — potassium adenosine triphosphatase activity in affective disorders

Summary Erythrocyte membrane Na⁺,K⁺-ATPase activity was studied in drug naive patients with bipolar (BP) mania (n=62) and unipolar (UP) depression (n=60) and normal controls (n=66). Compared to controls there was a significantly decreased Na⁺,K⁺-ATPase activity in UP depressives but no change in BP manics. However, lithium treatment caused a significant increase in Na⁺,K⁺-ATPase activity although there was no correlation between plasma lithium levels and enzyme activity. Plasma cortisol correlated inversely with Na⁺,K⁺-ATPase in UP depressives. Interestingly, the lithium responders [<50% Beck Rafaelson's Mania Rating Scale (BRMS) score] showed a significant increase in Na⁺,K⁺-ATPase activity compared to lithium nonresponders (>50% BRMS score). These observations indicate that monitoring of Na⁺,K⁺-ATPase activity during lithium therapy is useful to predict a therapeutic response.     

Na<Superscript>+</Superscript>,K<Superscript>+</Superscript>-ATPase activity in an animal model of mania

We evaluated Na⁺,K⁺-ATPase activity in hippocampus of rats submitted to an animal model of mania which included the use of lithium and valproate. In the acute treatment, amphetamine or saline was administered to rats for 14 days, between day 8 and 14, rats were treated with lithium, valproate or saline. In the maintenance treatment, rats were treated with lithium, valproate or saline, between day 8 and 14, amphetamine or saline were administered. Locomotor activity was assessed by open field test and Na⁺,K⁺-ATPase activity was measured. Our results showed that mood stabilizers reversed and prevented amphetamine-induced behavioral effects. Moreover, amphetamine (acute treatment) increased Na⁺,K⁺-ATPase activity, and administration of lithium or valproate reversed this effect. In the maintenance treatment, amphetamine increased Na⁺,K⁺-ATPase activity in saline-pretreated rats. Amphetamine administration in lithium- or valproate-pretreated animals did not alter Na⁺,K⁺-ATPase activity. The findings suggest that amphetamine-induced hyperactivity may be associated with an increase in Na⁺,K⁺-ATPase.     

Immunohistochemical localization of Na, K-ATPase in the choroid plexus

To determine if canine and rat choroid plexus Na⁺, K⁺-ATPase can be localized by immunoperoxidase staining after fixation and embedding, we prepared rabbit antiserum to purified canine kidney medulla Na⁺, K⁺-ATPase. When sodium dodecylsulfate polyacrylamide electrophoretic gels of purified canine kidney Na⁺, K⁺-ATPase and canine kidney microsomes were treated with antiserum followed by [¹²⁵I]protein A and autoradiography, the canine microsomes and purified Na⁺, K⁺-ATPase showed a prominent radioactive band coincident with the α-, β- and γ-subunits of the purified canine kidney enzyme.When the rabbit immunoglobulin that was purified from the Na⁺, K⁺-ATPase antiserum through DEAE-cellulose ion exchange chromatography was used for immunoperoxidase staining of the choroid plexus fixed with Bouin's fixative, intense immunoreactive staining was present on the epithelial cells of both choroid plexuses but was not found in the tissue around the vessel. The staining was especially confined to apical surfaces of the epithelial cells. The same procedure was performed in the canine kidney, and immunostaining was obtained in the tubules where Baskin and Stahl described the enzyme localization. No staining was seen with pre-immune serum of the normal rabbit. We concluded that both the canine and rat choroid plexus are rich in Na⁺, K⁺-ATPase, which plays an important role in cerebrospinal fluid (CSF) secretion.     

Effect of allopurinol on NMDA receptor modification following recurrent asphyxia in newborn piglets

The present study tests the hypothesis that repeated episodes of asphyxia will lead to alterations in the characteristics of the N-methyl-d-aspartate (NMDA) receptor in the brain cell membrane of newborn piglets and that pre-treatment with allopurinol, a xanthine oxidase inhibitor, will prevent these modifications. Eighteen newborn piglets were studied. Six untreated and six allopurinol treated animals were subjected to eight asphyxial episodes and compared to six normoxic, normocapneic controls. Brain cell membrane Na⁺,K⁺-ATPase activity was determined to assess membrane function. Na⁺,K⁺-ATPase activity was decreased from control following asphyxia in both the untreated and treated animals (47.7±3.2 vs. 43.0±2.2 and 41.0±5.3 μmol Pi/mg protein/h, p<0.05, respectively). ³H-MK-801 binding studies were performed to measure NMDA receptor binding characteristics. The receptor density (B_max) in the untreated asphyxia group was decreased compared to control animals (0.80±0.11 vs. 1.13±0.33, p<0.05); furthermore, the dissociation constant (K_d) was also decreased (3.8±0.7 vs. 9.2±2.2, p<0.05), indicating an increase in receptor affinity. In contrast, B_max in the allopurinol treated asphyxia group was similar to control (1.06±0.37); and K_d was higher (lower affinity) than in the untreated group (6.5±1.4, p<0.05). The data indicate that recurrent asphyxial episodes lead to alterations in NMDA receptor characteristics; and that despite cell membrane dysfunction as seen by a decrease in Na⁺,K⁺-ATPase activity, allopurinol prevents modification of NMDA receptor–ion channel binding characteristics induced by repeated episodes of asphyxia.     

Microsphere embolism-induced changes in noradrenaline uptake of the cerebral cortex in rats

The present study was undertaken to elucidate pathophysiological changes in noradrenaline (NA) transporter and Na⁺/K⁺-ATPase, key regulators of cation gradient across the plasma membrane, in nerve terminals of the cerebral cortex after microsphere-induced cerebral embolism in rats. The V_max value of NA uptake, when analyzed by the Eadie–Hofstee plot, tended to decrease on the 1st day and decreased on the 3rd and 7th days after the embolism without any change in the K_m value. The NA content in cerebrocortical synaptosomes did not alter on the 1st day, but decreased on the 3rd and 7th days after the embolism. Ouabain (1 mM) inhibited NA uptake on the 1st day, but did not alter the uptake on the 3rd and 7th days after the embolism. The activity of Na⁺/K⁺-ATPase of cerebrocortical synaptosomes increased on the 1st day and gradually decreased up to the 7th day after the embolim. These results suggest that NA uptake in nerve terminals of the cerebral cortex decreased after microsphere embolism, which may be due to a reduction in function of NA transporters. The changes in Na⁺/K⁺-ATPase following microsphere embolism may represent a compensatory action to maintain ion homeostasis in nerve terminals at an early stage of ischemic injury.     

Transport mechanism of glutamate by hypotonic-treated glial plasmalemmal vesicles from rat hippocampus

Recently, we isolated a novel subcellular fraction of glial plasmalemmal vesicles (GPV), which showed a higher activity of Na⁺-dependent glutamate transport than synaptosomes (Nakamura et al., 1993). In order to study kinetically the glutamate transport mechanism, we measured the reaction under various ionic conditions both inside and outside the vesicles. The vesicles treated hypotonically and preloaded with KCl could take up glutamate in the presence of external Na⁺. The level of glutamate uptake was dependent on external concentrations of NaCl ([NaCl]_o) and competitively inhibited by [KCl]_o. However, it was dependent on [KCl]_i, and competitively inhibited by [NaCl]_i. The activation and inhibition constants of K⁺ were about 30 mM inside and 20 mM outside, respectively, whereas those of Na⁺ were 140 mM outside and 4 mM inside, respectively. These results suggest that the transport carrier molecules work asymmetrically to the membranes. Nigericin and monensin, acidic ionophores for K⁺ and Na⁺, respectively, inhibited the glutamate uptake. On the other hand, valinomycin, a neutral ionophore for K⁺, elevated the uptake level, suggesting that the inside-negative membrane potential induced by K⁺ diffusion enhances the uptake activity. We conclude that glutamate transport by glial cells requires both external Na⁺ and internal K⁺ and is regulated by the membrane potential.     

Inhibition of purified (Na,K)-ATPase activity from porcine cerebral cortex by NO generating drugs

We tested the effects of several nitric oxide (NO) generating compounds on the activity of sodium-potassium adenosine 5′-triphosphatase [(Na⁺,K⁺)-ATPase] purified from porcine cerebral cortex. Sodium nitroprusside (SNP),S-nitroso-N-acetylpenicillamine (SNAP), 3-morpholinosydnonimine (SIN-1) and (dl)-(E)-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexeneamide (NOR 3) inhibited the (Na⁺,K⁺)-ATPase activity dose dependently. Superoxide dismutase, a NO scavenger, and sulfhydryl (SH) compounds, reduced-form glutathione (rGSH) and dithiothreitol (DTT), prevented the inhibitory action of SNAP, SIN-1 and NOR 3 but not of SNP, when applied simultaneously with NO generating compounds, and this enzyme inhibition could be reactivated by the incubation with these SH compounds but not with SOD. The inhibitory action by SNP was magnified by simultaneous application of DTT. These results suggest that NO generating compounds, SNAP, SIN-1 and NOR 3 but not SNP, may release NO or NO-derived products and may inhibit (Na⁺,K⁺)-ATPase activity by interacting with a SH group at the active site of the enzyme.     

Ultracytochemical localisation of Na+, K+-ATPase in cerebral endothelium in acute hypertension

Summary This ultracytochemical study was undertaken to determine whether increased arteriolar permeability in acute hypertension is accompanied by altered localisation of the ouabain-sensitive, K⁺-dependent p-nitrophenylphosphatase (K⁺-NPPase), a component of the Na⁺, K⁺-ATPase system. Rats were injected with horseradish peroxidase (HRP) intravenously and acute hypertension was induced by a 2-min infusion of angiotensin amide. Rats were killed at 3 and 15 min, following which brains were sliced and reacted for demonstration of K⁺-NPPase and HRP reaction product. Vessels of normotensive and hypertensive rats that were nonpermeable to HRP showed discontinuous distribution of K⁺-NPPase on the outer plasma membranes of endothelial and adventitial cells of arterioles and endothelial cells and pericytes of capillaries. Arterioles of the hypertensive rats which were permcable to HRP showed marked reduction of K⁺-NPPase localisation in their walls at 3 min while at 15 min when the blood pressure had returned to resting levels the enzyme localisation was similar to controls. This study demonstrates transient alteration of the NA⁺, K⁺-ATPase system during increased endothelial permeability in acute hypertension. The implication of this finding and our previous observation of reduced Ca²⁺-ATPase localisation in endothelial plasma membranes in acute hypertension has been discussed.Supported by the Heart and Stroke Foundation of Ontario     

Brain (Na, K)-ATPase and nonadrenergic function: recovery of enzyme activity after norepinephrine depletion

We examined the time course of K⁺-p-nitrophenylphosphatase and ouabain binding associated with cerebral cortex (Na⁺,K⁺) -AT-Pase after depletion of norepinephrine. Norepinephrine depletion by the norepinephrine-selective neurotoxin DSP4 initially reduced the indices of (Na⁺,K⁺)-ATPase, with a significant correlation between ouabain binding and tissue norepinephrine levels 16 h after DSP4. Tissue norepinephrine content and DMI binding rapidly declined after DSP4 and remained essentially unchanged for at least 8 weeks. By contrast, the indices of (Na⁺,K⁺)-ATPase remained low for about two weeks but then gradually increased, returning to baseline levels by 8 weeks after DSP4. These data indicate that, while usually regulated in part by exposure to norepinephrine, brain (Na⁺,K⁺)-ATPase undergoes adaptation to prolonged noradrenergic depletion.     

Brain Na, K-ATPase activity possibly regulated by a specific serotonin receptor

Na⁺, K⁺-ATPase activity in 6 regions of adult brain was measured after incubation with varying concentrations of serotonin. A concentration-dependent increase in enzyme activity was observed in 4 regions, with cerebral cortex and cerebellum showing the largest response. These results together with previous ones suggest that serotoninmmodulates brain Na⁺, K⁺-ATPase activity through a specific receptor located in target neurons or glial cells.     

Cholesterol oxidation reduces Ca + Mg-ATPase activity, interdigitation, and increases fluidity of brain synaptic plasma membranes

These experiments examined effects of cholesterol oxidation on Ca²⁺+Mg²⁺-ATPase activity, Na⁺+K⁺-ATPase activity, and membrane structure of brain synaptic plasma membranes (SPM). Cholesterol oxidase [E.C.1.1.3.6 fromBrevibacterium sp.] was used to oxidize cholesterol. Two cholesterol pools were identified in synaptosomal membranes based on their accessibility to cholesterol oxidase. A rapidly oxidized cholesterol pool was observed with a¹t₁₂ of 1.19±0.09 min and a second pool with a²t₁₂ of 38.30±4.16 min. Activity of Ca²⁺+Mg²⁺-ATPase was inhibited by low levels of cholesterol oxidation. Ten percent cholesterol oxidation, for example, resulted in approximately 35% percent inhibition of Ca²⁺+Mg²⁺-ATPase activity. After 13% cholesterol oxidation, further inhibition of Ca²⁺+Mg²⁺-ATPase activity was not observed. Activity of Na⁺+K⁺-ATPase was not affected by different levels of cholesterol oxidation (5%–40%). SPM interdigitation was significantly reduced and fluidity was significantly increased by cholesterol oxidation. The relatiobship observed between SPM interdigitation and Ca²⁺+Mg²⁺-ATPase activity was consistent with studies using model membranes [7]. Brain SPM function and structure were altered by relatively low levels of cholesterol oxidation and is a new approach to understanding cholesterol dynamics and neuronal function. The sensitivity of brain SPM to cholesterol oxidation may be important with respect to the proposed association between oxygen free radicals and certain neurodegenerative diseases.     

Effect of repeated restraint stress and clomipramine on Na/K-ATPase activity and behavior in rats

Activation of the limbic-hypothalamic-pituitary-adrenal axis (LHPA) and the release of glucocorticoids are fundamental for the adaptive response and immediate survival of an organism in reaction to acute stimuli. However, high levels of glucocorticoids in the brain may produce neuronal injury and a decrease of Na⁺/K⁺-ATPase activity, with effects on neurotransmitter signaling, neural activity, as well as the whole animal behavior. Clomipramine is a tricyclic antidepressant that inhibits the reuptake of serotonin and norepinephrine by indirect actions on the dopaminergic system and LHPA axis. Its chronic use increases the body's ability to cope with stress; however, high doses can potentiate its side effects on memory, learning, and sensory motor function. The purpose of the present study was to compare the effect of repeated restraint stress and clomipramine treatment on Na⁺/K⁺-ATPase activity and on the behavior of male rats. Changes in the behavioral response were evaluated by measuring the memory, learning, anxiety, and exploratory responses. Our results showed that exposure to repeated restraint stress reduced levels of Na⁺/K⁺-ATPase in brain structures and changed short and long-term memory, learning, and exploratory response when compared to the control group. Exposure to clomipramine treatment increased anxiety levels and reduced Na⁺/K⁺-ATPase activity in the cerebral cortex as well as short term memory, learning, and exploratory response. In conclusion, the present results provide additional evidence concerning how repeated restraint stress and clomipramine chronically administered at higher dose levels affect the neural activity and behavior of male rats.     

Acute enhancement of spinal cord synaptosomal (Na + K)-ATPase activity in cats following intravenous methylprednisolone

The acute intravenous administration of high doses of methylprednisolone sodium succinate (15–90 mg/kg) to cats enhanced (Na⁺ + K⁺)-ATPase activity as much as 2-fold in synaptosomes prepared from lumbar spinal cord removed following drug administration. The effect was apparent within 5 min and was statistically significant at 1 h following a single injection of methylprednisolone 90 mg/kg. (Na⁺ + K⁺)-ATPase activity had returned to control levels by 24 h post-injection. The implication of these findings to the treatment of spinal cord trauma and the reported neurophysiological effects of glucocorticoids are discussed.     

Endogenous modulators of brain Na, K-atpase at early postnatal stages of rat development

The presence of endogenous modulators (peaks I and II) of synaptosomal Na⁺, K⁺-ATPase activity from adult rat cerebral cortex was previously suggested. In this study, the presence of such modulators at different postnatal stages of rat development was examined and their effect was tested on Na⁺, K⁺-ATPase activity. Synaptosomal membrane Na⁺, K⁺-ATPase activity was enhanced 20–30% by peak I and inhibited 70–75% by peak II obtained from 4-, 10-, 20- and 35–40-day-old rats. A fraction purified from peak II by anionic exchange HPLC (termed II-E) highly inhibits enzyme activity and behaves as a ouabain-like factor. Inhibitory activity of a 4-day-old II-E fraction proved higher than the corresponding fraction obtained from adult rats. Since expression of cerebral Na⁺, K⁺-ATPase has been shown to increase 10-fold during development whereas peak II concentration was observed to remain constant, and given the higher potency of purified neonatal II-E fraction, the effect of the latter may be greater at early postnatal stages of development than during adult life. It is suggested that the II-E fraction, which contains an ouabain-like factor, may play a role in neuronal development.     

Cyclic AMP-induced depolarization measured by bis-oxonol fluorescence in bovine adrenal medullary chromaffin cells

Summary Effects of cyclic AMP on membrane potentials were examined by measuring the changes of bis-oxonol fluorescence in bovine adrenal medullary chromaffin cells. 8-Bromo cyclic AMP (8Br-cAMP) or forskolin caused a gradual and long lasting increase of the fluorescence intensity. The effects of 8br-cAMP was blocked by cyclic AMP-dependent protein kinase inhibitor, adenosine-3, 5-cyclic monophosphothioate, Rp-diastereomer (Rp-cAMPS) and there was no further increase in the fluorescence by 8br-cAMP in the cells depolarized with 56 mM KCl or gramicidin D. Ouabain or the removal of extracellular K⁺ ([K⁺]₀free) which block Na⁺, K⁺-ATPase also increased the fluorescence. The effect of 8br-cAMP on the fluorescence was counteracted by ouabain or [K⁺]₀ free and was blocked in the absence of extracellular Na⁺ but not by tetrodotoxin or the removal of Ca²⁺ from the medium. These results may suggest that cyclic AMP causes the membrane depolarization by accumulating Na⁺ through the inhibition of Na⁺, K⁺-ATPase in adrenal chromaffin cells.     

Depression of neuronal excitability and epileptic activities by group II metabotropic glutamate receptors in the medial entorhinal cortex

Whereas the ionotropic glutamate receptors are the major mediator in glutamatergic transmission, the metabotropic glutamate receptors (mGluRs) usually play a modulatory role. Whereas the entorhinal cortex (EC) is an essential structure involved in the generation and propagation of epilepsy, the roles and mechanisms of mGluRs in epilepsy in the EC have not been determined. Here, we studied the effects of activation of group II metabotropic glutamate receptors (mGluRs II) on epileptiform activity induced by picrotoxin or deprivation of extracellular Mg²⁺ and neuronal excitability in the medial EC. We found that activation of mGluRs II by application of the selective agonist, LY354740, exerted robust inhibition on epileptiform activity. LY354740 hyperpolarized entorhinal neurons via activation of a K⁺ conductance and inhibition of a Na⁺‐permeable channel. LY354740‐induced hyperpolarization was G protein‐dependent, but independent of adenylyl cyclase and protein kinase A. However, the function of Gβγ was involved in mGluRs II‐mediated depression of both neuronal excitability and epileptiform activity. Our results provide a novel cellular mechanism to explain the antiepileptic effects of mGluRs II in the treatment of epilepsy. © 2015 Wiley Periodicals, Inc.