Acute renal failure potentiates methylmalonate-induced oxidative stress in brain and kidney of rats.

Tissue methylmalonic acid (MMA) accumulation is the biochemical hallmark of methylmalonic acidemia. The disease is clinically characterized by progressive neurological deterioration and kidney failure, whose pathophysiology is still unclear. In the present work we investigated the effects of acute MMA administration on various parameters of oxidative stress in cerebral cortex and kidney of young rats, as well as the influence of acute renal failure on MMA-elicited effects on these parameters. Acute renal failure was induced by gentamicin, an aminoglycoside antibiotic whose utilization over prolonged periods causes nephrotoxicity. The administration of gentamicin alone increased carbonyl content and inhibited superoxide dismutase (SOD) activity in cerebral cortex, as well as increased thiobarbituric acid-reactive substances (TBA-RS) and sulfhydryl levels and diminished glutathione peroxidase activity in kidney. On the other hand, MMA administration increased TBA-RS levels in cerebral cortex and decreased SOD activity in kidney. Furthermore, the simultaneous administration of MMA and gentamicin to the rats provoked an augment in TBA-RS levels and superoxide generation in cerebral cortex and in TBA-RS, carbonyl and sulfhydryl levels in kidney, while diminished SOD activity in both studied tissues. Finally, nitrate/nitrite content, reduced glutathione levels, 2',7'-dihydrodichlorofluorescein oxidation and catalase activity were not affected by this animal treatment in either tissue. In conclusion, our present data are in line with the hypothesis that MMA acts as a toxin in brain and kidney of rats and suggest that renal injury potentiates the toxicity of MMA on oxidative stress parameters in brain and peripheral tissues.

Nitric oxide synthase inhibition by L-NAME prevents the decrease of Na+,K+-ATPase activity in midbrain of rats subjected to arginine administration.

In the present study we investigated the effect of acute administration of L-arginine on Na(+),K(+)-ATPase and Mg(2+)-ATPase activities and on some parameters of oxidative stress (chemiluminescence and total radical-trapping antioxidant parameter-TRAP) in midbrain of adult rats. We also tested the effect of L-NAME on the effects produced by arginine. Sixty-day-old rats were treated with an acute intraperitoneal injection of saline (group I, control), arginine (0.8 g/kg) (group II), L-NAME (2 mg/kg) (group III) or arginine (0.8 g/kg) plus L-NAME (2 mg/kg) (group IV). Na(+),K(+)-ATPase activity was significantly reduced in the arginine-treated rats, but was not affected by other treatments. In contrast, Mg(2+)-ATPase activity was not altered by any treatment. Furthermore, chemiluminescence was significantly increased and TRAP was significantly decreased in arginine-treated rats, whereas the simultaneous injection of L-NAME prevented these effects. These results demonstrate that in vivo arginine administration reduces Na(+),K(+)-ATPase activity possibly through free radical generation induced by NO formation.

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.

Effect of phenylalanine and p-chlorophenylalanine on Na+, K+-ATPase activity in the synaptic plasma membrane from the cerebral cortex of rats.

Na+, K+-ATPase activity was measured in synaptic plasma membrane from cerebral cortex of Wistar rats subjected to experimental phenylketonuria, i.e., chemical hyperphenylalaninemia induced by subcutaneous administration of 5.2 micromol phenylalanine / g body weight (twice a day) plus 0.9 micromol p-chlorophenylalanine / g body weight (once a day). The treatment was performed from the 6th to the 14th postpartum day and rats were killed 12 h after the last injection. Synaptic plasma membrane from cerebral cortex was prepared by a discontinuous density sucrose gradient for Na+, K+-ATPase activity determination. The results showed that the enzyme activity was decreased by 30% in animals subjected to experimental phenylketonuria when compared to control. The in vitro effects of the drugs on Na+, K+-ATPase activity were also investigated. Phenylalanine and p-chlorophenylalanine inhibited the enzyme activity and this inhibition was reversed by alanine. In addition, competition between phenylalanine and p-chlorophenylalanine for binding to the enzyme was observed, suggesting a common binding site for these substances. Our results suggest that reduction of Na+, K+-ATPase activity may be one of the mechanisms related to the brain dysfunction observed in human PKU.

Preconditioning prevents the inhibition of Na+,K+-ATPase activity after brain ischemia.

Application of single transient forebrain ischemia (ISC) in adult Wistar rats, lasting 2 or 10 min, caused inhibition of Na+,K+-ATPase activity in cytoplasmic membrane fractions of hippocampus and cerebral cortex immediately after the event. In the 2-min ISC group followed by 60 min of reperfusion, the enzyme inhibition was maintained in the cortex, while there was an increase in hippocampal enzyme activity; both effects were over 1 day after the event. However, in the 10-min ISC group enzyme inhibition had been maintained for 7 days in both cerebral structures. Interestingly, ischemic preconditioning (2-min plus 10-min ISC, with a 24-hour interval in between) prevented the inhibitory effect of ischemia/reperfusion on Na+,K+-ATPase activity observed either after a single insult of 2 min or 10 min ischemia. We suggest that the maintenance of Na+,K+-ATPase activity afforded by preconditioning be related to cellular neuroprotection.

Methylmalonate administration decreases Na+,K+-ATPase activity in cerebral cortex of rats.

Buffered methylmalonate (MMA) was injected s.c. into rats twice a day at 8 h intervals from 5 to 25 days of age (chronic treatment), or into 10-day-old rats three times a day at 1 h intervals (acute treatment). Control rats received saline in the same volumes. Na+,K+-ATPase and Mg2+-ATPase activities were determined in the synaptic plasma membranes from cerebral cortex of rats. Na+,K+-ATPase activity was reduced by 30-40% in MMA-treated rats, whereas Mg2+-ATPase activity was not. In contrast, MMA at final concentrations ranging from 0.1 to 2.0 mM had no in vitro effect on these enzyme activities. However, when brain homogenates were incubated with 2 mM MMA before membrane preparation, Na+,K+-ATPase activity was decreased by 44%. Furthermore, this reduction was totally prevented by the simultaneous addition of glutathione and MMA, suggesting that oxidation of thiol groups or other oxidative damage to the enzyme could be responsible for this effect.

In vitro inhibition of Na+,K(+)-ATPase activity from rat cerebral cortex by guanidino compounds accumulating in hyperargininemia.

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.

Proline administration decreases Na+,K+-ATPase activity in the synaptic plasma membrane from cerebral cortex of rats.

Buffered proline was injected subcutaneously into rats twice a day at 8 h intervals from the 6th to the 28th day of age. Control rats received saline in the same volumes. The animals were weighed and killed by decapitation 12 h after the last injection. Cerebral cortex was used for the determination of Na+,K+-ATPase and Mg2+-ATPase activities. Body, whole brain and cortical weights were similar in the two groups. Na+,K+-ATPase activity was significantly reduced (by 20%) in membranes from the proline-treated group compared to the controls, whereas Mg2+-ATPase activity was not affected by proline. In another set of experiments, synaptic plasma membranes were prepared from cerebral cortex of 29-day-old rats and incubated with proline at final concentrations ranging from 0.1 to 2.0 mM. Na+,K+-ATPase activity, but not Mg2+-ATPase activity, was inhibited by 20-30%. Since proline concentrations in plasma of chronically treated rats and of type 11 hyperprolinemic children are of the same order of magnitude as those tested in vitro, the results suggest that reduction of Na+,K+-ATPase activity may contribute to the neurological dysfunction found in some patients affected by type II hyperprolinemia.

Inhibition of Na+,K+-ATPase from rat brain cortex by propionic acid.

Buffered propionic acid was injected s.c. into rats twice a day at 8 h intervals from the 6 to 21 days of age. Control rats received saline in the same volumes. The animals were weighed and killed by decapitation at 23 days. Whole brain and cerebral cortex were weighed and synaptic plasma membranes were prepared from cortex for the determination of Na+,K+-ATPase and Mg2+-ATPase activities. Body, whole brain and cortical weights were similar in the two groups, suggesting that propionic acid does not cause malnutrition in rats. Na+,K+-ATPase activity was significantly reduced by 30% in membranes from the propionate-treated group, whereas Mg2+-ATPase activity was not. In another set of experiments, synaptic plasma membranes were prepared from cerebral cortex of 23-day-old rats and incubated with propionic acid at final concentrations ranging from 0.1 to 2.0 mM. Na+,K+-ATPase activity, but not Mg2+-ATPase activity, was inhibited by 22-32%. Since propionic acid concentrations in plasma of chronically treated rats and of propionic acidemic children are of the same order of magnitude as those tested in vitro, the results suggest that the inhibition of Na+,K+-ATPase activity may be related to the neurological dysfunction of patients affected by propionic acidaemia.