Methylphenidate treatment increases Na<Superscript>+</Superscript>, K<Superscript>+</Superscript>-ATPase activity in the cerebrum of young and adult rats

Methylphenidate is a central nervous system stimulant used for the treatment of attention-deficit hyperactivity disorder. Na⁺, K⁺-ATPase is a membrane-bound enzyme necessary to maintain neuronal excitability. Considering that methylphenidate effects on central nervous system metabolism are poorly known and that Na⁺, K⁺-ATPase is essential to normal brain function, the purpose of this study was to evaluate the effect of this drug on Na⁺, K⁺-ATPase activity in the cerebrum of young and adult rats. For acute administration, a single injection of methylphenidate (1.0, 2.0, or 10.0 mg/Kg) or saline was given to rats on postnatal day 25 or postnatal day 60, in the young and adult groups, respectively. For chronic administration, methylphenidate (1.0, 2.0, or 10.0 mg/Kg) or saline injections were given to young rats starting at postnatal day 25 once daily for 28 days. In adult rats, the same regimen was performed starting at postnatal day 60. Our results showed that acute methylphenidate administration increased Na⁺, K⁺-ATPase activity in hippocampus, prefrontal cortex, and striatum of young and adult rats. In young rats, chronic administration of methylphenidate also enhanced Na⁺, K⁺-ATPase activity in hippocampus and prefrontal cortex, but not in striatum. When tested in adult rats, Na⁺, K⁺-ATPase activity was increased in all cerebral structures studied. The present findings suggest that increased Na⁺, K⁺-ATPase activity may be associated with neuronal excitability caused by methylphenidate.     

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.     

Effects of lithium and valproate on hippocampus citrate synthase activity in an animal model of mania

Some studies suggest that mitochondrial dysfunction may be related to the pathophysiology of bipolar disorder. In this work, we evaluated the activity of citrate synthase in rats, and the effects of the treatment with mood stabilizers (lithium and valproate) on the enzyme activity. In the first experiment (reversal treatment), amphetamine or saline were administered to rats for 14 days, and between day 8 and 14, rats were treated with either lithium, valproate or saline. In the second experiment (prevention treatment), rats were pretreated with lithium, valproate or saline, and between day 8 and 14, rats were administered amphetamine or saline. In reversal and prevention models, amphetamine administration significantly inhibited citrate synthase activity in rat hippocampus. In amphetamine-pretreated animals, valproate administration reversed citrate synthase activity inhibition induced by amphetamine. In the prevention model, pretreatment with lithium prevented amphetamine-induced citrate synthase inhibition. Our results showed that amphetamine inhibited citrate synthase activity and that valproate reversed and lithium prevented the enzyme inhibition.     

Severe Hyperhomocysteinemia Decreases Respiratory Enzyme and Na<Superscript>+</Superscript>-K<Superscript>+</Superscript> ATPase Activities,and Leads to Mitochondrial Alterations in Rat Amygdala

Severe hyperhomocysteinemia is caused by increased plasma levels of homocysteine (Hcy), a methionine derivative, and is associated with cerebral disorders. Creatine supplementation has emerged as an adjuvant to protect against neurodegenerative diseases, due to its potential antioxidant role. Here, we examined the effects of severe hyperhomocysteinemia on brain metabolism, and evaluated a possible neuroprotective role of creatine in hyperhomocysteinemia, by concomitant treatment with Hcy and creatine (50 mg/Kg body weight). Hyperhomocysteinemia was induced in young rats (6-day-old) by treatment with homocysteine (0.3–0.6 µmol/g body weight) for 23 days, and then the following parameters of rat amygdala were evaluated: (1) the activity of the respiratory chain complexes succinate dehydrogenase, complex II and cytochrome c oxidase; (2) mitochondrial mass and membrane potential; (3) the levels of necrosis and apoptosis; and (4) the activity and immunocontent of Na⁺,K⁺-ATPase. Hcy treatment decreased the activities of succinate dehydrogenase and cytochrome c oxidase, but did not alter complex II activity. Hcy treatment also increased the number of cells with high mitochondrial mass, high mitochondrial membrane potential, and in late apoptosis. Importantly, creatine administration prevented some of the key effects of Hcy administration on the amygdala. We also observed a decrease in the activity and immunocontent of the α1 subunit of the Na⁺,K⁺-ATPase in amygdala after Hcy- treatment. Our findings support the notion that Hcy modulates mitochondrial function and bioenergetics in the brain, as well as Na⁺,K⁺-ATPase activity, and suggest that creatine might represent an effective adjuvant to protect against the effects of high Hcy plasma levels.     

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     

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.     

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.     

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.     

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.     

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.     

Role of opioidergic and monoaminergic neurotransmission in the GnRH release mechanism of EBP-primed OVX rats

We examined the effect of intracerebroventricular (i.c.v.) administration of μ-opioid agonist, morphine, and its antagonist naloxone followed by morphine on the activities of monoamine-metabolizing enzymes, namely tyrosine hydroxylase (TH) and monoamine oxidase (MAO) along with adenosinetriphosphatase (Na⁺, K⁺ -ATPase), the enzyme responsible for the maintenance of ionic gradients across the membrane, in seven discrete regions of brain from estrogen- and progesterone-primed ovariectomized rats. TH activity decreased after morphine treatment in some areas such as the median eminence-arcuate region (ME-ARC), the amygdala, and the thalamus, showing statistically significant change. MAO activity increased in all the areas studied, but more appreciable change was observed in medial preoptic area (mPOA), the ME-ARC region, and the cortex. Pronounced increase in Na⁺, K⁺ -ATPase enzyme activity was observed after the drug treatment. Naloxone given prior to morphine injection resulted in recovery of the enzyme activities in most of the areas studied. Our study may provide insights into the precise opioidergic modulation of gonadotropin releasing hormone (GnRH) release mechanisms through the involvement of monoaminergic system, elucidating the basis of various neuronal dysfunctions and their management in opioid addicts.     

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.     

Effects of lithium and valproate on amphetamine-induced oxidative stress generation in an animal model of mania

OBJECTIVE: Previous studies have suggested that oxidative stress may play a role in the pathophysiology of bipolar disorder (BD). Moreover, recent studies indicate that lithium and valproate exert neuroprotective effects against oxidative stress. We studied the effects of the mood stabilizers lithium and valproate on amphetamine-induced oxidative stress in an animal model of mania. METHODS: In the first model (reversal treatment), adult male Wistar rats received d-amphetamine or saline for 14 days, and between the 8th and 14th days, they were treated with lithium, valproate or saline. In the second model (prevention treatment), rats were pretreated with lithium, valproate or saline, and between the 8th and 14th days, they received d-amphetamine or saline. We assessed locomotor activity with the open-field task. We measured thiobarbituric acid reactive substances (TBARS) and protein carbonyl formation, as parameters of oxidative stress, and superoxide dismutase (SOD) and catalase (CAT), the major antioxidant enzymes, in the prefrontal cortex and hippocampus. RESULTS: Lithium and valproate reversed (reversal treatment model) and prevented (prevention treatment model) amphetamine-induced hyperactivity and reversed and prevented amphetamine-induced TBARS formation in both experiments. However, the co-administration of lithium or valproate with amphetamine increased lipid peroxidation, depending on the brain region and treatment regimen. No changes in protein carbonyl formation were observed. SOD activity varied with different treatment regimens, and CAT activity increased when the index of lipid peroxidation was more robust. CONCLUSION: Our findings suggest that lithium and valproate exert protective effects against amphetamine-induced oxidative stress in vivo, further supporting the hypothesis that oxidative stress may be associated with the pathophysiology of BD.     

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     

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.     

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.     

Na, K-ATPase in the rat olfactory bulb: evidence for a higher enzymatic activity in the glomerular layer

Protein content and ATPase activities have been determined in the superficial and deep layers of the rat olfactory bulb. Protein levels, Mg²⁺-ATPase and Na⁺, K⁺-ATPase activities were significantly higher in the whole homogenate of the superficial layers. These differences were amplified when activity was expressed on a wet weight basis in a crude microsomal preparation isolated by differential centrifugation. Specific activities, however, showed similar values in the microsomal fractions from superficial and deep layers. The results are discussed in terms of differences in the density of neuronal processes and efficiency of K⁺ reuptake mechanisms.     

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.     

A G301R Na<Superscript>+</Superscript>/K<Superscript>+</Superscript>-ATPase mutation causes familial hemiplegic migraine type 2 with cerebellar signs

Familial hemiplegic migraine (FHM) is an autosomal dominant subtype of migraine with hemiparesis during the aura. In over 50% of cases the causative gene is CACNA1A (FHM1), which in some cases produces a phenotype with cerebellar signs, including ataxia and nystagmus. Recently, mutations in ATP1A2 on chromosome 1q23 encoding a Na⁺/K⁺-ATPase subunit were identified in four families (FHM2). We now describe an FHM2 pedigree with a fifth ATP1A2 mutation coding for a G301R substitution. The phenotype was particularly severe and included hemiplegic migraine, seizure, prolonged coma, elevated temperature, sensory deficit, and transient or permanent cerebellar signs, such as ataxia, nystagmus, and dysarthria. A mild crossed cerebellar diaschisis during an attack further supported the clinical evidence of a cerebellar deficit. This is the first report suggesting cerebellar involvement in FHM2. A possible role for CACNA1A in producing the phenotype in this family was excluded by linkage studies to the FHM1 locus. The study of this family suggests that the absence of cerebellar signs may not be a reliable indicator to clinically differentiate FHM2 from FHM1.