In vivo and in vitro effects of homocysteine on Na,K-ATPase activity in parietal, prefrontal and cingulate cortex of young rats

In the present study we determined the effect of chronic administration of homocysteine on Na+,K+-ATPase activity in synaptic membranes from parietal, prefrontal and cingulate cortex of young rats. We also studied the in vitro effect of homocysteine on this enzyme activity and on some oxidative stress parameters, namely thiobarbituric acid-reactive substances (TBA-RS) and total radical-trapping antioxidant potential (TRAP) in the same cerebral structures. For the in vivo studies, we induced elevated levels of homocysteine in blood (500 microM), comparable to those of human homocystinuria, and in brain (60 nmol/g wet tissue) of young rats by injecting subcutaneously homocysteine (0.3-0.6 micromol/g of body weight) twice a day at 8 h intervals from the 6th to the 28th postpartum day. Controls received saline in the same volumes. Rats were killed 12 h after the last injection. Chronic administration of homocysteine significantly decreased (50%) Na+,K+-ATPase activity in parietal, increased (36%) in prefrontal and did not alter in cingulate cortex of young rats. In vitro homocysteine decreased Na+,K+-ATPase activity and TRAP and increased TBA-RS in all cerebral structures studied. It is proposed that the alteration of Na+,K+-ATPase and induction of oxidative stress by homocysteine in cerebral cortex may be one of the mechanisms related to the neuronal dysfunction observed in human homocystinuria.     

The effect of vanadate on Na, K-ATPase activity of mouse cerebral cortex during bicuculline-induced seizures

The effect of bicuculline-induced seizures on Na+,K+-ATPase activity of mouse cerebral cortex homogenates, using two different procedures of sample preparation (freezing in situ or decapitation of animals without freezing) is described. Regardless of tissue treatment Na+,K+-ATPase activities during bicuculline-induced seizures did not differ significantly from the appropriate controls when vanadate-free ATP was used as substrate. The response of Na+,K+-ATPase to K+ activation was also similar; the increase in potassium concentration from 2 to 20 mM caused a 33.0 and 32.3% increase of enzyme activity in cortical homogenates from control and convulsing mice, respectively. Vanadate added to the assay medium inhibited Na+,K+-ATPase activity in a dose-dependent manner; with both types of tissue treatment there was, however, a tendency towards lesser inhibition of the enzyme from convulsing mice and at 1 X 10(-7) M vanadate this difference, though slight, was statistically significant: -22.59 vs -27.55% (freezing) and -28.73 vs -38.42% (decapitation) for seizures vs controls, respectively. The reduced sensitivity of Na+,K+-ATPase towards vanadate inhibition in cortical homogenates prepared from mice with convulsions suggests that vanadate might play a role in the modulation of enzyme activity during seizures in vivo.     

Influence of lactate accumulation on Na,K-ATPase activity of ischemic and post-ischemic brain

In this study the cerebral Na+, K+-ATPase activity as well as selected parameters of oxidative metabolism and electrophysiological function were assessed in normoglycemic and hyperglycemic rats which were exposed to ischemia produced by electrocautery of the vertebral arteries and reversible occlusion of the carotid arteries. In hyperglycemic animals 0.5 h of ischemia was associated with massive accumulation of lactate (34 mumol X g-1) and enhanced Na+, K+-ATPase activity (116% control), whereas normoglycemic animals showed more moderate lactate accumulation (17 mumol X g-1) and normal Na+, K+-ATPase activity (102% control). In normoglycemic animals release of the carotid clamps and recirculation for 0.5-1.5 h was associated with a normalization of the lactate levels and a decrease in Na+, K+-ATPase activity (68-72% control). Restituted hyperglycemic animals showed metabolic changes which seemed related to the blood pressure, with hypotensive hyperglycemic animals showing continuing massive lactacidosis (30-35 mumol X g-1) and enhanced Na+, K+-ATPase activity (108-110% control), whereas normotensive hyperglycemic animals showed progressive decreases in lactate level (14-20 mumol X g-1) and normal or mildly suppressed Na+, K+-ATPase activity (88-97% control). These patterns of change suggest that the reperfusion of the post-ischemic hyperglycemic-hyperlactacidotic brain was inadequate or non-homogeneous.     

Na+, K+ ATPase activity is markedly reduced by cis-4-decenoic acid in synaptic plasma membranes from cerebral cortex of rats

We have previously demonstrated that octanoic (OA) and decanoic acids (DA) inhibit Na+, K+ ATPase activity in synaptic plasma membranes from rat brain. The objective of the present study was to investigate the in vitro effects of the other metabolites that accumulate in tissues of medium-chain acyl-CoA dehydrogenase (MCAD)-deficient patients, namely cis-4-decenoic acid (cDA), octanoylcarnitine (OC), hexanoylcarnitine (HC), hexanoylglycine (HG), phenylpropionylglycine (PPG) and suberoylglycine (SG), on Na+, K+ ATPase activity in synaptic plasma membrane from cerebral cortex of 30-day-old rats. cDA, the pathognomonic compound found in this disorder, provoked the strongest inhibition on this enzyme activity at concentrations as low as 0.25 mM, whereas OC inhibited this activity at 1.0 mM and higher concentrations in a dose-dependent manner. In contrast, HC, HG, PPG and SG did not affect Na+, K+ ATPase activity. Furthermore, pre-treatment of cortical homogenates with the antioxidant enzymes catalase plus superoxide dismutase totally prevented cDA-induced Na+, K+ ATPase inhibition. We also provided evidence that cDA, as well as OA and DA, caused lipid peroxidation, which may explain, at least in part, the inhibitory properties of these compounds towards Na+, K+ ATPase. Considering that Na+, K+ ATPase is a critical enzyme for normal brain development and functioning, it is presumed that these findings, especially those regarding to the marked inhibitory effect of cDA, may be involved in the pathophysiology of the neurological dysfunction of MCAD-deficient patients.     

Neurochemical correlates of selective neuronal loss following cerebral ischemia: role of decreased Na, K-ATPase activity

In order to investigate the role of Na+,K(+)-ATPase in the development of neuronal necrosis following cerebral ischemia, ischemia was induced in gerbils by occluding the common carotid artery unilaterally for 10 min. A time-course analysis revealed that significant reductions of the Na+,K(+)-ATPase activity in the cerebral cortex and hippocampus were manifested at 15 min, 30 min, and 1 h, and returned to the control level one day following recirculation. No apparent alterations of the Mg(2+)-ATPase activity, on the other hand, were obtained throughout the experimental period. Furthermore, Scatchard analyses of [3H]ouabain binding to the cerebral cortex membranes disclosed that the Bmax values invariably decreased without any change of Kd values following ischemia. It has also been shown that treatment of the animals with an agent known to mitigate ischemic neuronal necrosis, i.e. BY-1949, significantly reversed such derangements. These results suggest that the recovery of decreased Na+,K(+)-ATPase activity shortly after ischemia exerts a protective effect against ischemic brain damage.     

Expression of citrulline-nitric oxide cycle in lipopolysaccharide and cytokine-stimulated rat astroglioma C6 cells

Hyperargininemia is a metabolic disorder biochemically characterized by tissue accumulation of arginine (Arg) and other guanidino compounds (GC). Convulsions, lethargy and psychomotor delay are predominant clinical features of this disease. Considering that some GC are epileptogenic and cause a decrease in membrane fluidity and that Na+,K(+)-ATPase, a membrane-bound enzyme, is essential for cellular excitability and is decreased in experimental and human epilepsy, in the present study we determined the in vitro effects of Arg, N-acetylarginine (NAA), argininic acid (AA) and homoarginine (HA) on the activity of Na+,K(+)-ATPase in the synaptic plasma membrane from cerebral cortex of young rats in the hope to identify a possible mechanism for the brain damage in hyperargininemia. The results showed that all GC tested, except Arg, significantly inhibited Na+,K(+)-ATPase activity at concentrations similar to those observed in plasma and CSF of patients with hyperargininemia. In addition, competition between NAA, AA and HA for the binding to the enzyme was observed, suggesting a common binding site for the GC. It is therefore possible that the inhibitory effect of GC on Na+,K(+)-ATPase may be related to the brain dysfunction observed in hyperargininemia.     

Evaluation of the mechanism underlying the inhibitory effect of guanidinoacetate on brain Na, K-ATPase activity

Guanidinoacetate methyltransferase deficiency (GAMT-deficiency) is an inherited neurometabolic disorder clinically characterized by epilepsy and mental retardation and biochemically by accumulation of guanidinoacetate (GAA) and depletion of creatine. Although the neurological symptoms are predominant, the pathogenesis of the brain dysfunction in this disorder is not yet established. In the present study we investigated the in vitro effect of GAA on Na+, K+-ATPase and Mg2+-ATPase activities in synaptic plasma membrane from hippocampus of young rats. Results showed that GAA significantly inhibited Na+, K+-ATPase activity without affecting Mg2+-ATPase activity. We also evaluated the effect of glutathione (GSH), trolox, Nomega-nitro-L-arginine methyl ester (L-NAME) and taurine (Tau) on the inhibition elicited by GAA on Na+, K+-ATPase activity. GSH, trolox, L-NAME and Tau per se did not alter Na+, K+-ATPase activity. However, L-NAME and taurine prevented the inhibitory effect of GAA on this enzyme activity. Our findings suggest that the inhibition of Na+, K+-ATPase activity caused by GAA is possibly mediated by nitric oxide (NO) formation and/or synaptic membrane alteration. The present data may contribute to the understanding of the neurological dysfunction characteristic of GAMT-deficient patients.     

Effects of ethanol on Na-dependent amino acid uptake: dependence on rat age and Na, K-ATPase activity

Acute effects of ethanol on Na(+)-dependent transport of gamma-aminobutyric acid (GABA) and glutamic acid (GLU) were investigated in crude synaptosomal preparations from rat cerebral cortex. In experiments with 30-40-day-old (peripubertal) rats, the overall dose responses of the GABA and GLU transport systems to ethanol were biphasic. Stimulation was observed at ethanol concentrations (40-160 mM) relevant to intoxication. Inhibition was observed at higher concentrations of ethanol. The stimulatory phase of the dose response was not observed in 60-100-day-old (adult) rats. In preparations from peripubertal rats, other alcohols also had biphasic dose response curves with stimulation at low alcohol concentrations. The relative efficacy of the different alcohols appeared to correlate with the relative membrane-buffer partition coefficient. In synaptosomal membrane vesicles, where artificial ion concentration gradients rather than Na+,K(+)-ATPase activity provide the driving force for uptake, ethanol did not stimulate GABA uptake. In direct measures of Na+,K(+)-ATPase activity, both Rb+ uptake and ATP hydrolysis were enhanced by 80 mM ethanol. We conclude that stimulation of Na(+)-dependent uptake of amino acids by ethanol was secondary to enhanced Na+,K(+)-ATPase activity and may be associated with a specific developmental stage in the rat.     

Neonatal hypothyroidism alters the kinetic properties of Na+, K+-ATPase in synaptic plasma membranes from rat brain

Neonatal hypothyroidism was induced in rat pups by injecting 131I within two days of birth and the effects on kinetic properties of Na+, K+-ATPase from synaptic plasma membranes were examined. Neonatal hypothyroidism resulted in a generalized decrease in V(max) with ATP, Na+, K+ and Mg2+ together with an increase in the K(m) for ATP, appearance of a low affinity component for Na+ and allosteric characteristic for the Mg2+-dependent activity at high Mg2+ concentrations. Binding pattern for Na+ and Mg2+ changed. Our results suggest that impairment of Na+, K+-ATPase activity together with altered kinetic properties could be one of the underlying biochemical mechanism leading to central nervous system (CNS) dysfunctions as a consequence of thyroid hormone deprivation during critical stages of brain development.     

Methionine alters Na,K-ATPase activity, lipid peroxidation and nonenzymatic antioxidant defenses in rat hippocampus

In the present study we investigated the effect of methionine exposure of hippocampus homogenates on Na+,K+-ATPase activity from synaptic plasma membrane of rats. Results showed that methionine significantly decreased this enzyme activity. We also evaluated the effect of incubating glutathione (GSH) and trolox (alpha-tocopherol) alone or combined with methionine on Na+,K+-ATPase activity. The tested antioxidants per se did not alter the enzymatic activity, but prevented the inhibitory action of methionine on Na+,K+-ATPase activity, indicating that Met inhibitory effect was probably mediated by free radical formation. Besides, we tested the in vitro effect of methionine on some parameters of oxidative stress, namely chemiluminescence, thiobarbituric acid reactive substances (TBARS), total radical-trapping antioxidant potential (TRAP), as well as on the antioxidant enzyme activities catalase, glutathione peroxidase and superoxide dismutase in rat hippocampus. We observed that methionine significantly increased chemiluminescence and TBARS, decreased TRAP, but did not change the activity of the antioxidant enzymes. These findings suggest that reduction of Na+,K+-ATPase activity and induction of oxidative stress may be involved in the brain damage observed in human hypermethioninemia.     

GM1 ganglioside prevents seizures, Na+,K+-ATPase activity inhibition and oxidative stress induced by glutaric acid and pentylenetetrazole

Monosialoganglioside (GM1) is a glycosphingolipid that protects against some neurological conditions, such as seizures and ischemia. Glutaric acidemia type I (GA-I) is an inherited disease characterized by striatal degeneration, seizures, and accumulation of glutaric acid (GA). In this study, we show that GA inhibits Na+,K+-ATPase activity and increases oxidative damage markers (total protein carbonylation and thiobarbituric acid-reactive substances-TBARS) production in striatal homogenates from rats in vitro and ex vivo. It is also shown that GM1 (50 mg/kg, i.p., twice) protects against GA-induced (4 micromol/striatum) seizures, protein carbonylation, TBARS increase, and inhibition of Na+,K+-ATPase activity ex vivo. Convulsive episodes induced by GA strongly correlated with Na+,K+-ATPase activity inhibition in the injected striatum but not with oxidative stress marker measures. Muscimol (46 pmol/striatum), but not MK-801 (3 nmol/striatum) and DNQX (8 nmol/striatum) prevented GA-induced convulsions, increase of TBARS and protein carbonylation and inhibition of Na+,K+-ATPase activity. The protection of GM1 and muscimol against GA-induced seizures strongly correlated with Na+,K+-ATPase activity maintenance ex vivo. In addition, GM1 (50-200 microM) protected against Na+,K+-ATPase inhibition induced by GA (6 mM) but not against oxidative damage in vitro. GM1 also decreased pentylenetetrazole (PTZ)-induced (1.8 micromol/striatum) seizures, Na+,K+-ATPase inhibition, and increase of TBARS and protein carbonyl in the striatum. These data suggest that Na+,K+-ATPase and GABA(A) receptor-mediated mechanisms may play important roles in GA-induced seizures and in their prevention by GM1.     

Cytochemical study on enzyme activity associated with cerebrospinal fluid secretion in the choroid plexus and ventricular ependyma

Normal adult albino and Sprague-Dawley rats, under intraperitoneal Nembutal anesthesia, were used to demonstrate enzymatic activity in the choroid plexus and ventricular ependyma. The brain tissues were perfused or immersed with cold 2% glutaraldehyde and 8% sucrose in 0.1 M cacodylate buffer (pH 7.2-7.4) for 30 min and washed overnight in the same buffer solution., The choroid plexus (lateral and fourth ventricles) and ventricular ependyma (lateral ventricle) were trimmed from the fixed and washed brain tissues, which were frozen and sectioned. For histo- and cyto-chemical study, the sections were immersed in the following incubation media; for Na+, K+-ATPase (ouabain-sensitive, K+-dependent, p-nitrophenylphosphatase: p-NPPase) according to the one-step method of Mayahara et al. (1978): for Mg2+- ATPase, Wachstein-Meisel's incubation medium (1957); for adenylate cyclase (AC), following Araki and Saito's lead citrate method (1979). The cytochemical findings gave the following results. In the choroid plexus, the ouabain-sensitive electron-dense reaction products of NA+, K+-ATPase (p-NPPase) were strongly positive in the microvilli and along the inner surface of microvilli, without showing any Mg2+-ATPase and AC activities, and all three enzymatic activities were positive along the basal plasmalemmas and negative along the lateral and apical (not including microvilli) plasmalemmas. In the ventricular ependyma, Na+,K+-ATPase (P-NPPase) activity was not found, and the reaction product of AC was observed on the apical plasmalemmas and those of Mg2+-ATPase along the basal plasmalemmas. These cytochemical findings are helpful in understanding the regulation of cerebrospinal fluid production through Na+, K+-ATPase (p-NPPase) and cyclic AMP (AC).     

Glial cells influence membrane-associated enzyme activity at the blood-brain barrier

Glial cells have been shown to influence several cerebral endothelial cell properties in vitro. A situation similar to the endothelial-astrocyte relationship existing at the blood-brain barrier (BBB) can be produced by growing cultured cerebral endothelium on one side of a filter and glial cells on the other in an enclosed double chamber. In this setting membrane-associated reaction product on the cerebral endothelial cell for both Na+,K+-ATPase and non-specific alkaline phosphatase was markedly increased when the endothelial cells were co-cultured with glial cells. In addition, the distribution of reaction product on the cerebral endothelial cell membrane was similar to that reported in vivo. These observations support a glial influence on enzyme activity at the BBB.     

Polarity of the blood-brain barrier: Distribution of enzymes between the luminal and antiluminal membranes of brain capillary endothelial cells

The subcellular distribution in brain capillaries of alkaline phosphatase and Na+, K+-ATPase was investigated by two methods. Cytochemical studies using whole brain perfusion and electron microscopic examination indicated that alkaline phosphatase activity was located in both the luminal and antiluminal cytoplasmic membranes of the brain capillary endothelial cells. By contrast, the K+-dependent phosphatase activity associated with Na+, K+-ATPase was located in only the antiluminal membrane. Biochemical studies using membranes prepared by homogenization of isolated brain capillaries and density gradient centrifugation resulted in identification of two plasma membrane fractions. The light fraction contained alkaline phosphatase but very little Na+, K+-ATPase while the heavier fraction contained both enzyme activities. In addition, gamma-glutamyl transpeptidase showed a distribution similar to alkaline phosphatase while 5'-nucleotidase activity was distributed with the Na+, K+-ATPase activity. We conclude that the luminal and antiluminal membranes of brain capillaries are biochemically and functionally different. This polarity should permit active solute transport across brain capillary endothelial cells which are the cells responsible for the blood-brain barrier.     

Rapid decrease of high affinity ouabain binding sites in hippocampal CA1 region following short-term global cerebral ischemia in rat

High affinity [3H]ouabain binding was examined in the hippocampal CA1 region and frontal cortex of rats subjected to 5 min complete cerebral ischemia in a clinical death model, and to subsequent resuscitation. A decrease of Bmax directly after ischemia and its further gradual decrease during 120 min of reperfusion were noted in the ischemia-vulnerable CA1 region, whereas no change of Bmax was observed in frontal cortex. The apparent Kd constant showed insignificant fluctuations in either of the two brain regions. Since ouabain binds with high affinity to the neuronal (alpha +)-form of Na+/K+-ATPase, the results indicate a rapid enzyme loss in CA1 neurons. The high affinity ouabain binding test proved to be a sensitive detector of premorphological changes in nerve cell membranes in ischemia.     

Intrastriatal hypoxanthine administration affects Na,K-ATPase, acetylcholinesterase and catalase activities in striatum, hippocampus and cerebral cortex of rats

The aim of this study was to investigate the effects of a single intrastriatal injection of hypoxanthine, the major metabolite accumulating in Lesch-Nyhan disease, on Na(+),K(+)-ATPase, acetylcholinesterase and catalase activities in striatum, cerebral cortex and hippocampus of rats at different post-infusion periods. Adult Wistar rats were divided in two groups: (1) vehicle-injected group (control) and (2) hypoxanthine-injected group. For Na(+),K(+)-ATPase activity determination, the animals were sacrificed 3h, 24h and 7 days after drug infusion. For the evaluation of acetylcholinesterase and catalase activities, the animals were sacrificed 30min, 3h, 24h and 7 days after hypoxanthine infusion. Results show regional and time dependent effects of hypoxanthine on Na(+),K(+)-ATPase, acetylcholinesterase and catalase activities. The in vitro effect of hypoxanthine on the same enzymes in striatum was also investigated. Results showed that hypoxanthine inhibited Na(+),K(+)-ATPase, but not the activities of acetylcholinesterase and catalase in rat striatum. We suggest that these modification on cerebral biochemical parameters (Na(+),K(+)-ATPase, acetylcholinesterase and catalase activities) induced by intrastriatal administration of hypoxanthine in all cerebral structures studied, striatum, hippocampus and cerebral cortex, could be involved in the pathophysiology of Lesch-Nyhan disease.     

Intrastriatal methylmalonic acid administration induces convulsions and TBARS production,and alters Na+,K+-ATPase activity in the rat striatum and cerebral cortex

PURPOSE: Methylmalonic acid (MMA) inhibits succinate dehydrogenase (SDH) and beta-hydroxybutyrate dehydrogenase activity in vitro. Acute intrastriatal administration of MMA induces convulsions through glutamatergic mechanisms probably involving primary adenosine triphosphate (ATP) depletion and free radical generation. In this study we investigated whether the intrastriatal administration of MMA causes lipoperoxidation and alteration in Na+, K+-ATPase activity ex vivo and characterized the electrographic changes elicited by the intrastriatal administration of this organic acid. METHODS: MMA-induced lipoperoxidation, alterations in Na+, K+-ATPase activity and electrographic changes were measured by measuring total thiobarbituric acid-reacting substances and inorganic phosphate release by spectrophotometry, and by depth electrode recording, respectively. RESULTS: We demonstrated that intrastriatal MMA (6 mmol) injection causes convulsive behavior and electrographically recorded convulsions that last approximately 2 h. Concomitant with the increase of thiobarbituric acid-reacting substances (TBARS) content, we observed a significant inhibition of Na+,K+-ATPase activity in the striatum, and activation of Na+,K+-ATPase activity in the ipsilateral cerebral cortex. Intrastriatal MMA injection increased the content of TBARS in the striatum measured 30 min (32.4 +/- 12.0%, compared with the noninjected contralateral striatum) and 3 h (39.7 +/- 5.1%, compared with the noninjected contralateral striatum) after MMA injection. TBARS content of the ipsilateral cerebral cortex increased after MMA injection at 30 min (42.1 +/- 6.0%) and 3 h (40.4 +/- 20.2%), and Na+,K+-ATPase activity in the ipsilateral cerebral cortex increased during ictal activity (113.8 +/- 18%) and returned to basal levels as electrographic convulsions vanished in the cortex. Interestingly, intrastriatal MMA administration induced a persistent decrease in Na+,K+-ATPase activity only in the injected striatum (44.9 +/- 8.1% at 30 min and 68.7 +/- 9.4 at 3 h). CONCLUSIONS: These data suggest that MMA induces lipoperoxidation associated with Na+,K+-ATPase inhibition or activation, depending on the cerebral structure analyzed. It is suggested that Na+,K+-ATPase inhibition may play a primary role in generating MMA-induced convulsions.     

Increased ouabain binding after repeated noradrenergic stimulation

Acute noradrenergic stimulation has previously been shown to stimulate brain (Na+, K+)-adenosine triphosphatase activity. Effects of repeated stimulation with piperoxane were examined in the present study. Daily piperoxane increased ouabain binding, measured 24 h after the last dose, after 4 days or 3 weeks treatment; K+-p-nitrophenylphosphatase was increased after 3 weeks. Prazosin, which, like piperoxane, activates presynaptic noradrenergic neurons but, unlike piperoxane, blocks postsynaptic receptors, did not increase K+-p-nitrophenylphosphatase, and decreased ouabain binding after 3 weeks.     

Effects of inhibitors of Na,K-ATPase on the membrane potentials and neurotransmitter efflux in rat brain slices

The potassium potential EK, of rat brain slices was estimated by determining the uptake of 86Rb+. The ERb was the same for slices prepared from five rostral brain regions, the average value being 66.4 mV. The ERb values in the presence of 20 microM ouabain were only slightly lower than the resting values; increasing concentrations of ouabain above 20 microM resulted in a graded depolarization in all five brain regions. High concentrations (1 mM) of two other inhibitors of Na+,K+-ATPase, dihydro-ouabain and strophanthidin, produced no more depolarization than did 20 microM ouabain. Competitive binding studies indicated that the differential effects were due to the relative binding to brain slices. Erythrosin B, an inhibitor of Na+,K+-ATPase, had no measurable effect on ERb. Intermediate concentrations of the Na+/H+ ionophore monensin slightly hyperpolarized striatal slices, whereas the same monensin concentrations plus 20 microM ouabain, 1 mM strophanthidin or 70 microM erythrosin B resulted in marked depolarization. Measurement of the membrane potential via uptake of methyltriphenylphosphonium cation indicated that ERb was indeed a valid estimation of the membrane potential. EK was measured directly by monitoring 42K+ uptake in striatal slices and was found to be essentially identical to ERb. Uptake of 22Na+ was consistent with the values for ERb or EK. Several conditions that resulted in little or no measurable depolarization of striatal slices did induce efflux of exogenously loaded GABA and dopamine; these conditions included 20 microM ouabain, 1 mM dihydro-ouabain or strophanthidin, and 70 microM erythrosin B. Neurotransmitter efflux in the absence of general cell depolarization was not accompanied by altered rates of respiration or decreased ATP levels.     

Na+,K+-ATPase activity impairment after experimental traumatic brain injury: relationship to spatial learning deficits and oxidative stress

Traumatic brain injury (TBI) is a devastating disease that commonly causes persistent mental disturbances and cognitive deficits. Although studies indicate that oxidative stress and functional deficits occurring after TBI are interrelated events, the knowledge of the mechanisms underlying the development of such cognitive deficits has been limited. Thus, in the present study, we investigated the effect of fluid percussion brain injury (FPI) on a spatial learning task and levels of oxidative stress markers, namely, protein carbonylation and thiobarbituric acid-reactive substances (TBARS) and Na(+),K(+)-ATPase activity 1 or 3 months after FPI in rats. Statistical analysis revealed that FPI increased the scape latency and mean number of error in Barnes maze test 1 and 3 months after FPI. We also found that protein carbonylation and TBARS content increased in the parietal cortex 1 and 3 months after FPI. In addition, 3 months after FPI, protein carbonylation levels increased both in ipsilateral and contralateral cortices of FPI animals. Indeed, statistical analysis revealed a decrease in Na(+),K(+)-ATPase activity in the cerebral cortex of 1 month FPI animals. Furthermore, the decrease in enzyme activity found 3 months was larger, when compared with 1 month after FPI. These results suggest that cognitive impairment following TBI may result, at least in part, from increase of two oxidative stress markers, protein carbonylation and TBARS that occurs concomitantly to a decrease in Na(+),K(+)-ATPase activity.