Thiago Calcagnotto

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.

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 μM), 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 μmol/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.

Impairment of energy metabolism in hippocampus of rats subjected to chemically-induced hyperhomocysteinemia

Homocystinuria is an inherited metabolic disease biochemically characterized by tissue accumulation of homocysteine (Hcy). Mental retardation, ischemia and other neurological features, whose mechanisms are still obscure are common symptoms in homocystinuric patients. In this work, we investigated the effect of Hcy administration in Wistar rats on some parameters of energy metabolism in the hippocampus, a cerebral structure directly involved with cognition. The parameters utilized were 14CO2 production, glucose uptake, lactate release and the activities of succinate dehydrogenase and cytochrome c oxidase (COX). Chronic hyperhomocysteinemia was induced by subcutaneous administration of Hcy twice a day from the 6th to the 28th day of life in doses previously determined in our laboratory. Control rats received saline in the same volumes. Rats were killed 12 h after the last injection. Results showed that Hcy administration significantly diminished 14CO2 production and glucose uptake, as well as succinate dehydrogenase and COX activities. It is suggested that impairment of brain energy metabolism may be related to the neurological symptoms present in homocystinuric patients.

Brain Na+,K+-ATPase Inhibition Induced by Arginine Administration Is Prevented by Vitamins E and C

Hyperargininemia is a metabolic disorder caused by deficiency of arginase activity resulting in tissue accumulation of arginine and neurological dysfunction. We have previously demonstrated that arginine induces oxidative stress and decreases Na+,K+-ATPase in rat midbrain. In the present study we investigated the action of vitamins E and C on the inhibition of Na+,K+-ATPase provoked by arginine in the midbrain of 60-day-old rats. Animals were pretreated for 1 week with daily IP administration of saline (control) or vitamins E (40 mg/kg) and C (100 mg/kg). Twelve h after the last injection, animals received one injection of arginine (0.8 μmol/g of body weight) or saline. Chemiluminescence was significantly increased, whereas total antioxidant capacity and Na+,K+-ATPase activity were significantly decreased. Furthermore, treatment with vitamins E and C prevented these effects. If these effects also occur in the human condition, it is possible that antioxidant administration might slow the progression of neurodegeneration in this disorder.

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.