Bárbara Tagliari

Pretreatment with vitamins E and C prevent the impairment of memory caused by homocysteine administration in rats

Homocystinuria is a metabolic disorder caused by deficiency of cystathione β-synthase activity leading to tissue accumulation of homocysteine (Hcy); affected patients present neurological dysfunction. Considering that Hcy induces free radical formation and that memory is impaired by oxidative stress, in the present study we investigated the effect of an acute administration of Hcy on retrieval of step-down inhibitory avoidance in adult rats. The action of vitamins E and C on the effects produced by Hcy was also tested. Adult Wistar rats were pretreated for 1 week with daily i.p. administration of saline (control group) and vitamins E and C (vitamin E 40 mg/kg and vitamin C 100 mg/kg). Hcy (11 mmol/kg) or an equivalent volume of 0.9% saline were administered 1 h before training, 1 h before testing, or immediately after training sessions. Memory was significantly impaired in Hcy-treated group, whereas the rats chronically treated with vitamins E and C had this effect prevented. Present data strongly indicate that Hcy administration impairs memory, an effect probably mediated by oxidative stress since treatment with vitamins E and C prevented amnesia. Assuming the possibility that this might occur in the human condition, reported results may be relevant to explain, at least in part, neurologic dysfunction associated with homocystinuria.

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.

Oxidative stress mediated by NMDA, AMPA/KA channels in acute hippocampal slices: neuroprotective effect of resveratrol.

Glutamate is the major excitatory neurotransmitter in the brain and over-stimulation of the glutamate receptors, NMDA, AMPA and kainate (KA), may cause neuronal death in epilepsy, seizures and neurodegenerative diseases. Mitochondria have critical cellular functions that influence neuronal excitability, such as regulation of Ca(2+) homeostasis and ATP production to maintain Na(+)K(+)-ATPase in the central nervous system (CNS). However, mitochondria are also the primary site of reactive oxygen species (ROS) production, and oxidative stress can induce cellular damage. Resveratrol, a polyphenol found in grapes and wines, presents antioxidant and neuroprotective effects on brain pathologies. This study sought to determine the neuroprotective effect of resveratrol against glutamate toxicity in acute hippocampal slices, using specific inhibitors of glutamate channels, and to investigate the targets of glutamate excitotoxicity, such as mitochondrial membrane potential (ΔΨ(m)), Na(+)K(+)-ATPase and glutamine synthetase (GS) activity. Resveratrol decreases intracellular ROS production, most likely by mechanisms involving NMDA, AMPA/KA, intracellular Ca(2+) and the heme oxygenase 1 (HO1) pathway, and prevents mitochondrial dysfunction and impairments in Na(+)K(+)-ATPase and GS activity after glutamate activation. Taken together, these results show that resveratrol may exhibit an important neuroprotective mechanism against neuropsychiatric disorders, focusing on mitochondrial bioenergetics and oxidative stress, as well as inhibitory effects on ionic channels.

Brain energy metabolism is compromised by the metabolites accumulating in homocystinuria

Homocystinuria is an inborn error of metabolism caused by severe deficiency of cystathionine beta-synthase activity. It is biochemically characterized by tissue accumulation of homocysteine (Hcy) and methionine (Met). Homocystinuric patients present a variable degree of neurological dysfunction whose pathophysiology is poorly understood. In the present study, we investigated the in vitro effect of Hcy and Met on some parameters of energy metabolism in hippocampus of rats. CO(2) production from [U-14C] acetate, glucose uptake and lactate release were assessed by incubating hippocampus prisms from 28-day-old rats in Krebs-Ringer bicarbonate buffer, pH 7.4, in the absence (controls) or presence of Hcy (10-500 microM) or Met (0.2-2.0mM). Hcy and Met decreased CO(2) production in a dose-dependent manner and increased lactate release. In contrast, glucose uptake was not altered by the metabolites. The effect of Hcy and Met on cytochrome c oxidase activity was also studied. It was observed that Met did not alter this enzyme activity, in contrast with Hcy, which significantly inhibited cytochrome c oxidase activity. It is suggested that impairment of brain energy metabolism caused by the metabolites accumulating in homocystinuria may be related to the neurological symptoms present in homocystinuric patients.

Reduction of butyrylcholinesterase activity in rat serum subjected to hyperhomocysteinemia.

In the present study we investigate the effect of homocysteine (Hcy) administration, the main metabolite accumulating in homocystinuria, on butyrylcholinesterase (BuChE) activity in serum of rats. For the acute treatment, 29-day-old Wistar rats received one subcutaneous injection of Hcy (0.6 μmol/g) or saline (control) and were killed 1 h later. For the chronic treatment, Hcy was administered subcutaneously to rats from the 6th to the 28th day of life. Control rats received saline. The rats were killed 12 h after the last injection. In another set of experiments, rats were pretreated for one week with vitamins E and C or saline and 12 h after the last injection received one single injection of Hcy or saline, being killed 1 h later. Serum was used to determine BuChE activity. Our results showed that acute and chronic administration of Hcy significantly decreased BuChE activity. Furthermore, vitamins E and C per se did not alter BuChE activity, but prevented the reduction of this enzyme activity caused by acute administration of Hcy. The data suggest that the inhibitory effect of Hcy on BuChE activity is probably mediated by free radicals, since vitamins E and C administration prevented such effect.

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.

Homocysteine alters glutamate uptake and Na+,K+-ATPase activity and oxidative status in rats hippocampus: protection by vitamin C

In the present study we investigate the effect of homocysteine on glutamate uptake, Na+,K+-ATPase, enzymatic antioxidant defenses, as well as reactive species levels in hippocampus of rats. The influence of vitamin C, a classic antioxidant, on the effects elicited by homocysteine was also tested. Results showed that chronic hyperhomocysteinemia decreased glutamate uptake and the activities of Na+,K+-ATPase, catalase and superoxide dismutase in hippocampus of rats. Reactive species levels were increased by chronic homocysteine administration. Concomitant administration of vitamin C significantly prevented these alterations caused by homocysteine. According to our results, it seems possible to suggest that the reduction in glutamate uptake and Na+,K+-ATPase activity may be mediated by oxidative stress, since vitamin C prevented these effects. We suggest that the administration of antioxidants should be considered as an adjuvant therapy to specific diet in homocystinuria.

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.