Andréa Gisiane Kurek

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 (α‐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.

Proline reduces brain cytochrome c oxidase : prevention by antioxidants

In the present study, we initially investigated the in vivo (acute and chronic) and in vitro effects of proline on cytochrome c oxidase (complex IV) activity in rat cerebral cortex to test the hypothesis that proline might alter energy metabolism and that this alteration could be provoked by oxidative stress. The action of α‐tocopherol and ascorbic acid on the effects produced by proline was also evaluated. For acute administration, 29‐ and 60‐day‐old rats received one subcutaneous injection of proline (18.2 μmol/g body weight) or an equivalent volume of 0.9% saline solution (control) and were sacrificed 1 h later. For chronic treatment, proline was injected subcutaneously twice a day at 10 h intervals from the 6th to the 28th day of age. Rats were sacrificed 12 h (29th) or 31 days (60th) after the last injection. Results showed that acute administration of proline significantly diminished the activity of cytochrome c oxidase in the cerebral cortex of 29‐ and 60‐day‐old rats. On the other hand, chronic hyperprolinemia reduced this complex activity only on day 29, but not on the 60th day of life. In another set of experiments, 22‐day‐old rats or 53‐day‐old rats were pretreated for 1 week with daily intraperitoneal administration of α‐tocopherol (40 mg/kg) and ascorbic acid (100 mg/kg) or saline. Twelve hours after the last antioxidant injection, rats received a single injection of proline or saline and were killed 1 h later. In parallel to chronic treatment, rats received a daily intraperitoneal injection of α‐tocopherol and ascorbic acid from the 6th to the 28th day of life and were killed 12 h after the last injection. Results showed that the pretreatment with α‐tocopherol and ascorbic acid before acute proline administration or concomitant to chronic proline administration significantly prevented these effects. We also observed that proline (3.0 μM–1.0 mM) when added to the incubation medium (in vitro studies) did not alter cytochrome c oxidase activity. Data suggest that the inhibitory effect of proline on cytochrome c oxidase activity is possibly associated with oxidative stress and that this parameter may be involved in the brain dysfunction observed in hyperprolinemia.

Effects of chronic proline administration on lipid contents of rat brain.

In the present work we investigated the effects of chronic proline administration on ganglioside, cholesterol and phospholipid total contents, as well as on ganglioside profile in cerebral cortex, hippocampus, hypothalamus and cerebellum of rats. We also evaluated the ganglioside content and profile in detergent‐soluble and resistant microdomains isolated from synaptic membranes obtained from cerebral cortex. Proline solution (hyperprolinemic) or saline (control) were subcutaneously administered to rats from 6th to 28th post‐natal day, according to body weight. Twelve hours after the last injection, the animals were sacrificed by decapitation without anaesthesia. Brain structures were homogenized with chloroform:methanol for lipid extraction. Synaptic membranes were obtained by differential centrifugation and detergent‐soluble and resistant microdomains were isolated by cold Triton X‐100 treatment. Results showed that rats subjected to chronic proline treatment presented a significant increase of ganglioside content in cortex and hippocampus, while this membrane lipid content was not altered in hypothalamus and cerebellum. Besides, phospholipid and cholesterol contents were not modified in all structures studied. On the other hand, ganglioside content decreased in detergent‐soluble and resistant microdomains isolated from synaptic membrane obtained from hyperprolinemic cortex. Although ganglioside profiles were apparently not modified, the individual absolute quantities were altered in cortex and hippocampus total lipid extract and membrane microdomains. Our findings suggest that chronic proline treatment affects in a distinct manner different cerebral regions concerning the lipid composition of the cell membranes, reflecting on its distribution in the cortex membrane microdomains. Among these phenomena consequences, distinct modulations in synaptic transmission may be suggested which might contribute to the impairment in cognition and/or other neurological dysfunctions found in hyperprolinemia type II patients.