Evidence that oxidative stress is involved in the inhibitory effect of proline on Na(+),K(+)-ATPase activity in synaptic plasma membrane of rat hippocampus

In the present study, we investigated the effect of Vitamins E and C on the inhibition of Na(+),K(+)-ATPase activity provoked by proline (Pro) administration in rat hippocampus. Five-day-old rats were pretreated for 1 week with daily i.p. administration of saline (control) or Vitamin E (40 mg/kg) and Vitamin C (100 mg/kg). Twelve hours after the last injection, animals received one single injection of Pro (12.8 micromol/g of body weight) or saline and were killed 1h later. Results showed that Na(+),K(+)-ATPase activity was decreased in the Pro-treated rats and that the pretreatment with Vitamins E and C prevented this effect. In another set of experiments, we investigated the in vitro effect of 1.0 mM Pro on Na(+),K(+)-ATPase activity from synaptic membranes of hippocampus of rats. Pro significantly inhibited (30%) Na(+),K(+)-ATPase activity. We also evaluated the effect of preincubating glutathione, trolox and N(pi)-nitro-L-arginine methyl ester (L-NAME) alone or combined with Pro on Na(+),K(+)-ATPase activity. Tested drugs did not alter Na(+),K(+)-ATPase activity, but glutathione prevented the inhibitory effect of Pro on this enzyme activity. These results suggest that the in vivo and in vitro inhibitory effect of Pro on Na(+),K(+)-ATPase activity is probably mediated by free radicals that may be involved in the neurological dysfunction found in hyperprolinemic patients.     

Folic acid pretreatment prevents the reduction of Na(+),K(+)-ATPase and butyrylcholinesterase activities in rats subjected to acute hyperhomocysteinemia.

The main objective of the present study was to evaluate the effect of folic acid pretreatment on parietal cortex Na(+),K(+)-ATPase and serum butyrylcholinesterase activities in rats subjected to acute hyperhomocysteinemia. Animals were pretreated daily with an intraperitoneal injection of folic acid (5 mg/kg) or saline from the 22th to the 28th day of age. Twelve hours after the last injection of folic acid or saline, the rats received a single subcutaneous injection of homocysteine (0.6 micromol/g of weight body) or saline and were killed 1h later. Serum was collected and the brain was quickly removed and parietal cortex dissected. Results showed that acute homocysteine administration significantly decreased the activities of Na(+),K(+)-ATPase and butyrylcholinesterase on parietal cortex and serum, respectively. Furthermore, folic acid pretreatment totally prevented these inhibitory effects. We also evaluated the effect of acute homocysteine administration on some parameters of oxidative stress, namely thiobarbituric acid-reactive substances and total thiol content in parietal cortex of rats. No alteration of these parameters were observed in parietal cortex of homocysteinemic animals, indicating that these oxidative stress parameters were probably not responsible for the reduction of Na(+),K(+)-ATPase and butyrylcholinesterase activities. The presented results confirm previous findings that acute hyperhomocysteinemia produces an inhibition of Na(+),K(+)-ATPase and butyrylcholinesterase activities and that pretreatment with folic acid prevents such effects. Assuming that homocysteine might also reduce the activities of these enzymes in human beings, our results support a new potential therapeutic strategy based on folic acid supplementation to prevent the neurological damage found in hyperhomocysteinemia.     

Reduction of energy metabolism in rat hippocampus by arginine administration

Hyperargininemia is an inherited metabolic disease biochemically characterized by tissue accumulation of arginine. Mental retardation and other neurological features are common symptoms in hyperargininemic patients. Considering that the underlying mechanisms of brain damage in this disease are poorly established, in this work we investigated the effect of arginine administration to adult Wistar rats on some parameters of energy metabolism (CO(2) production, glucose uptake, lactate release and the activities of succinate dehydrogenase, complexes II and IV of the respiratory chain) in rat hippocampus. The action of L-NAME, an inhibitor of oxide nitric oxide synthase, on the effects produced by arginine was also tested. Sixty-day-old rats were treated with a single intraperitoneal injection of saline (group I, control), arginine (0.8 g/kg) (group II) or arginine (0.8 g/kg) plus L-NAME (2 mg/kg) (group III) and were killed 1 h later. Results showed that arginine administration significantly increased lactate release and diminished CO(2) production, glucose uptake, succinate dehydrogenase and complex II activities. In contrast, complex IV (cytochrome c oxidase) activity was not changed by this amino acid. Furthermore, simultaneous injection of L-NAME prevented some of these effects, except CO(2) production and lactate release. The present data indicate that in vivo arginine administration impairs some parameters of energy metabolism in hippocampus of rats probably through NO formation.     

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.     

Effects of an acute treatment with L-thyroxine on memory, habituation, danger avoidance, and on Na+, K+ -ATPase activity in rat brain

Thyroid hormones (THs) have a relevant action on brain development and maintenance. By using an acute treatment to induce a hyperthyroid animal model, we aimed at investigating the effect of an altered THs levels on learning and memory and on the activity of Na(+), K(+)-ATPase in the rat brain. Our results have shown that the acute treatment with L-T4 did not alter the retrieval of the inhibitory avoidance task, but had a significant effect on the elevated plus maze and on open-field performance in rats. We suggest that animals subjected to L-T4 administration improved the habituation to a novel environment as well as a better evaluation of a dangerous environment, respectively. Na(+), K(+)-ATPase activity is increased in parietal cortex (30%), but it is not altered in hippocampus in L-T4 treated group. These both brain structures are involved in memory processing and it was previously demonstrated that there is a double dissociation between them for spatial location information, perceptual and episodic memory. We propose the hypothesis that this increase of Na(+), K(+)-ATPase activity in parietal cortex may be correlated to our results in behavior tests, which suggest a role of THs as well as of the Na(+), K(+)-ATPase in the cognitive process.     

Inhibition of Na, K-ATPase activity in rat striatum by the metabolites accumulated in Lesch–Nyhan disease

In the present study, we investigated the in vitro effect of hypoxanthine, xanthine and uric acid, metabolites accumulating in tissue of patients with Lesch-Nyhan disease, on Na(+), K(+)-ATPase activity in striatum of neonate rats. Results showed that all compounds significantly inhibited Na(+), K(+)-ATPase activity. We also studied the kinetics of the inhibition of Na(+), K(+)-ATPase activity caused by hypoxanthine. The apparent K(m) and V(max) of Na(+), K(+)-ATPase activity for ATP as the substrate and hypoxanthine as the inhibitor were 0.97 mM and 0.69 nmol inorganic phosphate (Pi) released per min per mg of protein, respectively. K(i)-value was 1.9 microM, and the inhibition was of the non-competitive type. We also observed that the inhibitory effects of hypoxanthine, xanthine and uric acid probably occur through the same mechanism, suggesting a common binding site for these oxypurines on Na(+), K(+)-ATPase. Therefore, it is conceivable that inhibition of brain Na(+), K(+)-ATPase activity may be involved at least in part in the neuronal dysfunction characteristic of patients with Lesch-Nyhan disease.     

On the mechanism of the inhibition of Na, K-ATPase activity caused by homocysteine

In the present work, we investigated the kinetics of the inhibition of Na(+), K(+)-ATPase activity caused by homocysteine (Hcy) in rat hippocampus. We also studied the interaction between Hcy and phenylalanine (Phe) and the kinetics of alanine (Ala) reversal of the inhibition of Na(+), K(+)-ATPase caused by Hcy. The apparent K(m) and V(max) of Na(+), K(+)-ATPase for ATP as substrate were 0.55mM and 2.0nmol Pi released per min per mg of protein, respectively. K(i) value was approximately 0.1mM, and the inhibition was of the non-competitive type. The results also showed a competition between Hcy and Phe. Ala per se did not alter this enzyme, but prevented the inhibitory effect caused by Hcy, suggesting a common binding site for these substances. It is proposed that the inhibition of Na(+), K(+)-ATPase by Hcy may be one of the mechanisms related to the neuronal dysfunction observed in human homocystinuria.     

Inhibition of Na, K-ATPase activity in rat striatum by guanidinoacetate

The aim of this work was to investigate the effect of guanidinoacetate (GAA), the principal metabolite accumulating in guanidinoacetate methyltransferase (GAMT)-deficiency, on Na(+), K(+)-ATPase, Mg(2+)-ATPase and acetylcholinesterase (AChE) activities in striatum of young rats. We also studied the kinetics of the inhibition of Na(+), K(+)-ATPase activity caused by guanidinoacetate. Guanidinoacetate did not alter acetylcholinesterase and Mg(2+)-ATPase activities, but significantly inhibited Na(+), K(+)-ATPase activity. The apparent K(m) and V(max) of Na(+), K(+)-ATPase for ATP as substrate were 0.20mM and 0.82nmol inorganic phosphate (Pi) released per min per mg of protein, respectively. K(i) value was 7.18mM, and the inhibition was of the uncompetitive type. The results also showed a competition between guanidinoacetate and argininic acid (AA), suggesting a common binding site for the guanidino compounds (GC) on the enzyme. It is proposed that Na(+), K(+)-ATPase inhibition by guanidinoacetate may be one of the mechanisms involved in the neuronal dysfunction observed in GAMT-deficiency and in other diseases which accumulate guanidinoacetate.     

Effects of thyroid hormones on memory and on Na(+), K(+)-ATPase activity in rat brain

Thyroid hormones (THs), including triiodothyronine (T3) and tetraiodothyronine (T4), are recognized as key metabolic hormones of the body. THs are essential for normal maturation and function of the mammalian central nervous system (CNS) and its deficiency, during a critical period of development, profoundly affects cognitive function. Sodium-potassium adenosine 5'-triphosphatase (Na(+), K(+)-ATPase) is a crucial enzyme responsible for the active transport of sodium and potassium ions in the CNS necessary to maintain the ionic gradient for neuronal excitability. Studies suggest that Na(+), K(+)-ATPase might play a role on memory formation. Moreover, THs were proposed to stimulate Na(+), K(+)-ATPase activity in the heart of some species. In this work we investigated the effect of a chronic administration of L-thyroxine (L-T4) or propylthiouracil (PTU), an antithyroid drug, on some behavioral paradigms: inhibitory avoidance task, open field task, plus maze and Y-maze, and on the activity of Na(+), K(+)-ATPase in the rat parietal cortex and hippocampus. By using treatments which have shown to induce alterations in THs levels similar to those found in hyperthyroid and hypothyroid patients, we aimed to understand the effect of an altered hyperthyroid and hypothyroid state on learning and memory and on the activity of Na(+), K(+)-ATPase. Our results showed that a hyper and hypothyroid state can alter animal behavior and they also might indicate an effect of THs on learning and memory.     

Mechanisms involved in morphine-induced activation of synaptosomal Na+,K+-ATPase

Morphine through mu-opioid receptors and G(i/o) proteins modulates several cellular effector systems; however, the mechanisms involved in the regulation of Na(+),K(+)-ATPase are not well known. We evaluated the effect of two mu-opioid receptor agonists on ouabain-sensitive Na(+),K(+)-ATPase activity in mice forebrain synaptosomes, and examined the modulation of this effect by antagonists of opioid receptors and a blocker of G(i/o) proteins. Incubation of synaptosomes with morphine (10(-9) to 10(-4) M) or buprenorphine (10(-10) to 10(-5) M) concentration-dependently stimulated Na(+),K(+)-ATPase activity, morphine being less potent but more efficacious than buprenorphine. Morphine did not displace [3H]ouabain from its binding site (Na(+),K(+)-ATPase) to forebrain membranes, whereas ouabain did so in a concentration-dependent manner. Naloxone, an opioid antagonist (10(-6) M), added to the synaptosomal medium, antagonized the enhancement of Na(+),K(+)-ATPase activity induced by morphine, producing a parallel shift to the right of the morphine concentration-response curve. Treatment with beta-funaltrexamine, a mu antagonist (2.5 and 10 microg/mouse, i.c.v.) and naloxonazine, a mu1 antagonist (35 mg/kg, s.c.), 24 h before the synaptosomes were obtained, produced a dose-dependent reduction in the E(max) of the morphine-induced increase in Na(+),K(+)-ATPase activity in vitro, but did not significantly modify its EC(50). Pertussis toxin (G(i/o) protein blocker) treatment at a dose of 0.5 microg/mouse, administered i.c.v. 5 days before the synaptosomes were obtained, completely abolished the enhancement of Na(+),K(+)-ATPase activity induced by morphine. A lower dose (0.25 microg/mouse) decreased the E(max) of morphine by 50% but did not significantly affect its EC(50). These results suggest that morphine indirectly enhances Na(+),K(+)-ATPase activity in the brain by activating mu-opioid receptors and G(i/o) proteins.     

Phylogenetic preservation of alpha3 Na+,K+-ATPase distribution in vertebrate peripheral nervous systems

The alpha(3) isoform of Na(+),K(+)-ATPase is uniquely expressed in afferent and efferent neurons innervating muscle spindles in the peripheral nervous system (PNS) of adult rats, but the distribution pattern of this isoform in other species has not been investigated. We compared expression of alpha(3) Na(+),K(+)-ATPase in lumbar dorsal root ganglia (DRG), spinal roots, and skeletal muscle samples of amphibian (frog), reptilian (turtle), avian (pigeon and chicken), and mammalian (mouse and human) species. In all species studied, the alpha(3) Na(+),K(+)-ATPase isoform was nonuniformly expressed in peripheral ganglia and nerves. In spinal ganglia, only 5-20% of neurons expressed this isoform, and, in avian and mammalian species, these alpha(3) Na(+),K(+)-ATPase-expressing neurons belonged to a subpopulation of large DRG neurons. In ventral root fibers of pigeons, mice, and humans, the alpha(3) Na(+),K(+)-ATPase was abundantly expressed predominantly in small myelinated axons. In skeletal muscle samples from turtles, pigeons, mice, and humans, alpha(3) Na(+),K(+)-ATPase was detected in intramuscular myelinated axons and in profiles of nerve terminals associated with the equatorial and polar regions of muscle spindle intrafusal fibers. These results show that the expression profiles for alpha(3) Na(+),K(+)-ATPase in the peripheral nervous system of a wide variety of vertebrate species are similar to the profile of rats and suggest that stretch receptor-associated expression of alpha(3) Na(+),K(+)-ATPase is preserved through vertebrate evolution.     

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.     

Suppression of sodium pump activity and an increase in the intracellular Ca2+ concentration by dexamethasone in acidotic mouse brain

The effects of dexamethasone on adenosine 5'-triphosphatase (ATPase) activity and the intracellular Ca(2+) concentration ([Ca(2+)](i)) were investigated in acidotic mouse brain. Dexamethasone (3 mg/kg, i.p.) or vehicle was administered 3 h before decapitation ischemia, and the brain concentration of adenosine 5'-triphosphate (ATP) was determined 0.5-2 min after ischemia. The effects of dexamethasone (0.3-3 mg/kg, i.p.) on Na(+),K(+)-activated ATPase (Na(+),K(+)-ATPase) and Ca(2+)-ATPase activities were evaluated at pH 7.4 and 6.8. Changes in [Ca(2+)](i) in an acidic medium were determined in hippocampal slices by microfluorometry using rhod-2 acetoxymethyl ester as a Ca(2+) marker, and the effects of dexamethasone (240 microg/l) was evaluated. Decapitation ischemia for 0.5 and 1 min reduced the brain ATP contents to 32% and 16% of the basal level, respectively. Dexamethasone slightly suppressed the extent of the decrease in the ATP level. Although dexamethasone did not affect Na(+),K(+)-ATPase activity at pH 7.4, the activity was suppressed by dexamethasone (3 mg/kg) to 68% at pH 6.8. The activity of Ca(2+)-ATPase was not affected by dexamethasone at either pH 7.4 or pH 6.8. When the pH of the medium of the brain slices was changed from 7.4 to 6.8, almost no increase in [Ca(2+)](i) was observed in the control group. The dexamethasone treatment increased [Ca(2+)](i) in the CA1 field and dentate gyrus immediately after induction of the acidic medium, the effect being significant after 150 s. Because anaerobic glucose metabolism in the early stage of ischemia enhances intracellular lactic acidosis, the findings may suggest a mechanism for the aggravation of ischemic neuronal damage by glucocorticoids.     

Suppression of sodium pump activity and an increase in the intracellular Ca concentration by dexamethasone in acidotic mouse brain

The effects of dexamethasone on adenosine 5'-triphosphatase (ATPase) activity and the intracellular Ca(2+) concentration ([Ca(2+)](i)) were investigated in acidotic mouse brain. Dexamethasone (3 mg/kg, i.p.) or vehicle was administered 3 h before decapitation ischemia, and the brain concentration of adenosine 5'-triphosphate (ATP) was determined 0.5-2 min after ischemia. The effects of dexamethasone (0.3-3 mg/kg, i.p.) on Na(+),K(+)-activated ATPase (Na(+),K(+)-ATPase) and Ca(2+)-ATPase activities were evaluated at pH 7.4 and 6.8. Changes in [Ca(2+)](i) in an acidic medium were determined in hippocampal slices by microfluorometry using rhod-2 acetoxymethyl ester as a Ca(2+) marker, and the effects of dexamethasone (240 microg/l) was evaluated. Decapitation ischemia for 0.5 and 1 min reduced the brain ATP contents to 32% and 16% of the basal level, respectively. Dexamethasone slightly suppressed the extent of the decrease in the ATP level. Although dexamethasone did not affect Na(+),K(+)-ATPase activity at pH 7.4, the activity was suppressed by dexamethasone (3 mg/kg) to 68% at pH 6.8. The activity of Ca(2+)-ATPase was not affected by dexamethasone at either pH 7.4 or pH 6.8. When the pH of the medium of the brain slices was changed from 7.4 to 6.8, almost no increase in [Ca(2+)](i) was observed in the control group. The dexamethasone treatment increased [Ca(2+)](i) in the CA1 field and dentate gyrus immediately after induction of the acidic medium, the effect being significant after 150 s. Because anaerobic glucose metabolism in the early stage of ischemia enhances intracellular lactic acidosis, the findings may suggest a mechanism for the aggravation of ischemic neuronal damage by glucocorticoids.     

Creatine reduces oxidative stress markers but does not protect against seizure susceptibility after severe traumatic brain injury

Achievements made over the last years have highlighted the important role of creatine in health and disease. However, its effects on hyperexcitable circuit and oxidative damage induced by traumatic brain injury (TBI) are not well understood. In the present study we revealed that severe TBI elicited by fluid percussion brain injury induced oxidative damage characterized by protein carbonylation, thiobarbituric acid reactive species (TBARS) increase and Na(+),K(+)-ATPase activity inhibition 4 and 8 days after neuronal injury. Statistical analysis showed that after TBI creatine supplementation (300 mg/kg, p.o.) decreased the levels of protein carbonyl and TBARS but did not protect against TBI-induced Na(+),K(+)-ATPase activity inhibition. Electroencephalography (EEG) analysis revealed that the injection of a subconvulsant dose of PTZ (35 mg/kg, i.p.), 4 but not 8 days after neuronal injury, decreased latency for the first clonic seizures and increased the time of spent generalized tonic-clonic seizures compared with the sham group. In addition, creatine supplementation had no effect on convulsive parameters induced by a subconvulsant dose of PTZ. Current experiments provide evidence that lipid and protein oxidation represents a separate pathway in the early post-traumatic seizures susceptibility. Furthermore, the lack of consistent anticonvulsant effect exerted by creatine in this early phase suggests that its apparent antioxidant effect does not protect against excitatory input generation induced by TBI.     

MK-801 alters Na+, K+-ATPase activity and oxidative status in zebrafish brain: reversal by antipsychotic drugs

Schizophrenia is a debilitating mental disorder with a global prevalence of 1% and its etiology remains poorly understood. In the current study we investigated the influence of antipsychotic drugs on the effects of MK-801 administration, which is a drug that mimics biochemical changes observed in schizophrenia, on Na(+), K(+)-ATPase activity and some parameters of oxidative stress in zebrafish brain. Our results showed that MK-801 treatment significantly decreased Na(+), K(+)-ATPase activity, and all antipsychotics tested prevented such effects. Acute MK-801 treatment did not alter reactive oxygen/nitrogen species by 2'7'-dichlorofluorscein (H2DCF) oxidation assay, but increased the levels of thiobarbituric acid reactive substances (TBARS), when compared with controls. Some antipsychotics such as sulpiride, olanzapine, and haloperidol prevented the increase of TBARS caused by MK-801. These findings indicate oxidative damage might be a mechanism involved in the decrease of Na(+), K(+)-ATPase activity induced by MK-801. The parameters evaluated in this study had not yet been tested in this animal model using the MK-801, suggesting that zebrafish is an animal model that can contribute for providing information on potential treatments and disease characteristics.     

Creatine increases hippocampal Na(+),K(+)-ATPase activity via NMDA-calcineurin pathway

Achievements made over the past few years have demonstrated the important role of the creatine and phosphocreatine system in the buffering and transport of high-energy phosphates into the brain; however, the non-energetic processes elicited by this guanidine compound in the hippocampus are still poorly understood. In the present study we disclosed that the incubation of rat hippocampal slices with creatine (10mM) for 30 min increased Na(+),K(+)-ATPase activity. In addition, intrahippocampal injection of creatine (5 nmol/site) also increased the above-mentioned activity. The incubation of hippocampal slices with N-methyl-d-aspartate (NMDA; MK-801, 10 μM) and NMDA Receptor 2B (NR2B; ifenprodil, 3 μM) antagonists but not with the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPA)/kainate antagonist (DNQX, 10 μM) and nitric oxide synthase inhibitor (NOS; l-NAME, 100 μM), blunted the effect of creatine on Na(+),K(+)-ATPase activity. Furthermore, the calcineurin inhibitor (cyclosporine A, 200 nM) as well as the Protein Kinase C (PMA, 100 nM) and Protein Kinase A (8-Br-cAMP, 30 μM) activators attenuated the creatine-induced increase of Na(+),K(+)-ATPase activity. In addition, the incubation of hippocampal slices with creatine (10mM) for 30 min increased calcineurin activity. The results presented here suggest that creatine increases Na(+),K(+)-ATPase activity via NMDA-calcineurin pathway, proposing an putative underlying non-energetic role of this guanidine compound. However, more studies are needed to assess the contribution of this putative alternative role in neurological diseases that present decreased Na(+),K(+)-ATPase activity.     

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.     

The role of nitric oxide on the convulsive behavior and oxidative stress induced by methylmalonate: an electroencephalographic and neurochemical study

Methylmalonic acidemias consist of a group of inherited metabolic disorders caused by deficiency of methylmalonyl-CoA mutase activity and biochemically characterized by methylmalonate (MMA) accumulation, impairment mitochondrial oxidative metabolism and reactive species production. Preliminary studies with nitric oxide synthase (NOS) inhibitors have suggested that nitric oxide (NO) plays a role in the convulsant effect of MMA. However, definitive biochemical and electrophysiological evidence of the involvement of NO in the convulsions induced by MMA are lacking. In this study, we investigated whether the inhibition of NOS by 7-nitroindazole (7-NI, 3-60mg/kg, i.p.) altered the convulsions, protein oxidative damage, NO(x) (NO(2) plus NO(3)) production and Na(+),K(+)-ATPase activity inhibition induced by MMA. 7-NI decreased striatal NO(x) content, but increased seizures and protein carbonylation induced by MMA (6mumol/striatum). The intrastriatal injection of l-arginine (50nmol/0.5mul), but not of d-arginine (50nmol/0.5mul), increased striatal NO(x) content and protected against MMA-induced electroencephalographic seizures, striatal protein carbonylation and Na(+),K(+)-ATPase inhibition. Furthermore, l-arginine (50nmol/0.5mul) and MMA had no additive effect on NO(x) increase. These results are experimental evidence that endogenous NO plays a protective role in the convulsions and acute neurochemical alterations induced by this organic acid.     

Novel regimen through combination of memantine and tea polyphenol for neuroprotection against brain excitotoxicity

NMDA receptors are abundant, ubiquitously distributed throughout the brain, fundamental to excitatory neurotransmission, and critical for normal CNS function. However, excessive glutamate overstimulates NMDA receptors, leading to increased intracellular calcium and excitotoxicity. Mitochondrial dysfunction associated with loss of Ca(2+)homeostasis and enhanced cellular oxidative stress has long been recognized to play a major role in cell damage associated with excitotoxicity. In this experiment, we attempted to explore whether treatment with memantine (an NMDA receptor antagonist) and tea polyphenol (an antioxidant and anti-inflammatory agent), either alone or in combination, is effective in neuroprotection in a mouse excitotoxic injury model. Memantine (10 mg/kg/day), tea polyphenol (60 mg/kg/day), or a combination (memantine 5 mg/kg/day plus tea polyphenol 30 mg/kg/day) was administered by oral gavage for 2 consecutive days before causing excitotoxic injury. Mice received a 0.3-microL NMDA [335 mM (pH 7.2)] injection into the left striatum. Locomotor activity was assessed 24 hr before and after excitotoxic injury. Brain synaptosomes were harvested 24 hr after excitotoxic injury for assessment of Na(+), K(+)-ATPase and Mg(2+)-ATPase activity, reactive oxygen species production, mitochondrial membrane potential (Delta Psi m), mitochondrial reductase activity (MTT test), and Ca(2+)concentration. The results showed that treatment with memantine could significantly rescue mitochondrial function by attenuating the decreased mitochondrial membrane potential (Delta Psi m) and mitochondrial reductase activity in mouse excitotoxic injury. Treatment with tea polyphenol could significantly decrease the increased production of synaptosomal reactive oxygen species (ROS) and thus reduced the deteriorative ROS-sensitive Na(+), K(+)-ATPase and Mg(2+)-ATPase activity. However, neither memantine nor tea polyphenol alone could significantly improve the impaired locomotor activity unless treatment was combined. Combined treatment with memantine and tea polyphenol could significantly protect mice against excitotoxic injury by reducing the increased synaptosomal ROS production, attenuating the decreased Na(+), K(+)-ATPase and Mg(2+)-ATPase activity, the mitochondrial membrane potential (Delta Psi m), the mitochondrial reductase activity, and the increased synaptosomal Ca(2+)concentration. In addition, the impairment in locomotor activity was also significantly improved. Therefore, the combined treatment of memantine and tea polyphenol is more effective in neuroprotection than either memantine or tea polyphenol alone in mouse excitotoxic injury. These findings provide useful information about the potential application of memantine and tea polyphenols in preventing clinical excitotoxic injury such as brain trauma, brain ischemia, epilepsy, and Alzheimer's disease.