Alexandra I. Zugno

Inhibition of Na+,K+-ATPase Activity in Hippocampus of Rats Subjected to Acute Administration of Homocysteine Is Prevented by Vitamins E and C Treatment

In the present study we evaluated the effect of acute homocysteine (Hcy) administration on Na+,K+-ATPase activity, as well as on some parameters of oxidative stress such as total radical-trapping antioxidant potential (TRAP) and on activities of antioxidant enzymes catalase (CAT), superoxide dismutase and glutathione peroxidase in rat hippocampus. Results showed that Hcy significantly decreased TRAP, Na+,K+-ATPase and CAT activities, without affecting the activities of superoxide dismutase and glutathione peroxidase. We also verified the effect of chronic pretreatment with vitamins E and C on the reduction of TRAP, Na+,K+-ATPase and CAT activities caused by Hcy. Vitamins E and C per se did not alter these parameters, but prevented the reduction of TRAP, Na+,K+-ATPase and CAT activities caused by Hcy. Our results indicate that oxidative stress is probably involved in the pathogenesis of homocystinuria and that reduction of Na+,K+-ATPase activity may be related to the neuronal dysfunction found in homocystinuric patients.

Training in inhibitory avoidance causes a reduction of Na+,K+-ATPase activity in rat hippocampus.

Compelling evidence has indicated the involvement of Na(+),K(+)-ATPase in the mechanisms of synaptic plasticity. In the present study, we investigated the effect of inhibitory avoidance training on Na(+),K(+)-ATPase activity, at different times after training, in the rat hippocampus. Male adult Wistar rats were trained in a step-down inhibitory avoidance task and compared to those submitted to isolated footshock (0.4 mA) or placed directly onto the platform. Na(+),K(+)-ATPase activity decreased, by 60%, in hippocampus of rats sacrificed immediately after the isolated footshock, as well as immediately (0 min) and 6 h after training; this effect was not present 24 h after training. We also verified that enzyme activity was not altered in rats killed after just being on the platform. These findings suggest that Na(+),K(+)-ATPase activity may be involved in the memory consolidation of step-down inhibitory avoidance in the hippocampus.

Inhibition of rat brain Na+, K+-ATPase activity induced by homocysteine is probably mediated by oxidative stress.

The objective of the present study was to investigate the effects of preincubation of hippocampus homogenates in the presence of homocysteine or methionine on Na+, K+-ATPase and Mg2+-ATPase activities in synaptic membranes of rats. Homocysteine significantly inhibited Na+, K+-ATPase activity, whereas methionine had no effect. Mg2+-ATPase activity was not altered by the metabolites. We also evaluated the effect of incubating glutathione, cysteine, dithiothreitol, trolox, superoxide dismutase and GM1 ganglioside alone or incubation with homocysteine on Na+, K+-ATPase activity. Tested compounds did not alter Na+, K+-ATPase and Mg2+-ATPase activities, but except for trolox, prevented the inhibitory effect of homocysteine on Na+, K+-ATPase activity. These results suggest that inhibition of this enzyme activity by homocysteine is possibly mediated by free radicals and may contribute to the neurological dysfunction found in homocystinuric patients.

Inhibition of Na+, K+-ATPase activity by the metabolites accumulating in homocystinuria.

Homocystinuria is an inborn error of sulfur amino acid metabolism characterized predominantly by vascular and nervous system dysfunction. In this study we determined the in vitro effects of homocysteine and methionine, metabolites which accumulate in homocystinuria, on Na+, K+-ATPase, and Mg2+-ATPase activities in synaptic membranes from the hippocampus of rats. The results showed that both metabolites significantly inhibit Na+, K+-ATPase but not Mg2+-ATPase activity at concentrations usually observed in plasma of homocystinuric patients. Furthermore, incubation of hippocampal homogenates with homocysteine also elicited an inhibition of the enzyme activity which was however prevented by the simultaneous addition of cysteine to the medium. In addition, cysteine or methionine per se did not modify the two enzymatic activities. These findings indicate that oxidation of critical groups in the enzyme may possibly be involved in homocysteine inhibitory effect. Moreover, kinetic studies performed to investigate the interaction between homocysteine and methionine on Na+, K+-ATPase inhibition suggested a common site for the two amino acids in the enzyme. Considering the critical role exerted by Na+, K+-ATPase in brain, it is proposed that the inhibition provoked by homocysteine and methionine on the enzyme activity may be possibly related to the brain dysfunction characteristic of homocystinuria.

Intrastriatal administration of guanidinoacetate inhibits Na+, K+-ATPase and creatine kinase activities in rat striatum

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 this disease is predominantly characterized by severe neurological findings, the underlying mechanisms of brain injury are not yet established. In the present study, we investigated the effect of intrastriatal administration of GAA on Na+, K+-ATPase activity, total (tCK), cytosolic (Cy-CK), and mitochondrial (Mi-CK) creatine kinase (CK) activities in rat striatum. We verified that Na+, K+-ATPase, tCK, and Mi-CK activities were significantly inhibited by GAA, in contrast to Cy-CK which was not affected by this guanidino compound. Since these enzyme activities can be affected by reactive species, we also investigated the effect of intrastriatal administration of GAA on thiobarbituric acid reactive substances (TBARS), an index of lipid peroxidation in rats. We found that this metabolite significantly increased this oxidative stress parameter. Considering the importance of Na+, K+-ATPase and CK activities for brain metabolism homeostasis, our results suggest that the inhibition of these enzymes by increased intracerebral levels of GAA may contribute to the neuropathology observed in patients with GAMT-deficiency.

Guanidinoacetate Decreases Antioxidant Defenses and Total Protein Sulfhydryl Content in Striatum of Rats

Guanidinoacetate methyltransferase (GAMT) deficiency is an inherited neurometabolic disorder biochemically characterized by tissue accumulation of guanidinoacetate (GAA) and depletion of creatine. Affected patients present epilepsy and mental retardation whose pathogeny is unclear. In the present study we investigated the inxa0vitro and inxa0vivo (intrastriatal administration) effects of GAA on some oxidative stress parameters in rat striatum. Sixty-day-old rats were used for intrastriatal infusion of GAA. For the inxa0vitro studies, 60-day-old Wistar rats were killed by decapitation and the striatum was pre-incubated for 1xa0h at 37°C in the presence of GAA at final concentrations ranging from 10 to 100xa0μM. Parameters of oxidative stress such as total radical-trapping antioxidant potential (TRAP), antioxidant enzymes (SOD, GPx, and CAT), protein carbonyl and thiol contents were measured. DNA damage was also evaluated. Results showed that GAA administration (inxa0vivo studies) or the addition of 100xa0μM GAA to assays (inxa0vitro studies) significantly decreased TRAP, SOD activity, and total thiol levels in rat striatum. In contrast, this guanidino compound did not alter protein carbonyl content and the activities of CAT and GPx. DNA damage was not found after intrastriatal administration of GAA. The data indicate that the metabolite accumulating in GAMT deficiency decreases antioxidant capacity and total thiol content in the striatum. It is therefore presumed that this pathomechanism may contribute at least in part to the pathophysiology of the brain injury observed in patients affected by GAMT deficiency.

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, Nϖ‐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.

In vivo and in vitro effect of imipramine and fluoxetine on Na+,K+-ATPase activity in synaptic plasma membranes from the cerebral cortex of rats

The effects of in vivo chronic treatment and in vitro addition of imipramine, a tricyclic antidepressant, or fluoxetine, a selective serotonin reuptake inhibitor, on the cortical membrane-bound Na+,K+-ATPase activity were studied. Adult Wistar rats received daily intraperitoneal injections of 10 mg/kg of imipramine or fluoxetine for 14 days. Twelve hours after the last injection rats were decapitated and synaptic plasma membranes (SPM) from cerebral cortex were prepared to determine Na+,K+-ATPase activity. There was a significant decrease (10%) in enzyme activity after imipramine but fluoxetine treatment caused a significant increase (27%) in Na+,K+-ATPase activity compared to control (P<0.05, ANOVA; N = 7 for each group). When assayed in vitro, the addition of both drugs to SPM of naive rats caused a dose-dependent decrease in enzyme activity, with the maximal inhibition (60-80%) occurring at 0.5 mM. We suggest that a) imipramine might decrease Na+,K+-ATPase activity by altering membrane fluidity, as previously proposed, and b) stimulation of this enzyme might contribute to the therapeutic efficacy of fluoxetine, since brain Na+,K+-ATPase activity is decreased in bipolar patients.

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, Mg2+‐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 Mg2+‐ATPase activities, but significantly inhibited Na+, K+‐ATPase activity. The apparent Km and Vmax of Na+, K+‐ATPase for ATP as substrate were 0.20 mM and 0.82 nmol inorganic phosphate (Pi) released per min per mg of protein, respectively. Ki value was 7.18 mM, 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.

Association Between Na+,K+-ATPase Activity and the Vulnerability/Resilience to Mood Disorders induced by Early Life Experience

There is increasing evidence that early life events can influence neurodevelopment and later susceptibility to disease. Chronic variable stress (CVS) has been used as a model of depression. The objective of this study was to evaluate the interaction between early experience and vulnerability to chronic variable stress in adulthood, analyzing emotional, metabolic and neurochemical aspects related to depression. Pups were (1) handled (10xa0min/day) or (2) left undisturbed from day 1 to 10 after birth. When the animals reached adulthood, the groups were subdivided and the rats were submitted or not to CVS, which consisted of daily exposure to different stressors for 40xa0days, followed by a period of behavioral tasks, biochemical (plasma corticosterone and insulin sensitivity) and neurochemical (Na+,K+-ATPase activity in hippocampus, amygdala and parietal cortex) measurements. Neonatally-handled rats demonstrated shorter immobility times in the forced swimming test, independently of the stress condition. There was no difference concerning basal corticosterone or insulin sensitivity between the groups. Na+,K+-ATPase activity was decreased in hippocampus and increased in the amygdala of neonatally-handled rats. CVS decreased the enzyme activity in the three structures, mainly in the non-handled group. These findings suggest that early handling increases the ability to cope with chronic variable stress in adulthood, with animals showing less susceptibility to neurochemical features associated with depression, confirming the relevance of the precocious environment to vulnerability to psychiatric conditions in adulthood.