Uliginosin B, a natural phloroglucinol derivative with antidepressant-like activity, increases Na+,K+-ATPase activity in mice cerebral cortex

ABSTRACT Uliginosin B, a phloroglucinol isolated from Hypericum polyanthemum Klotzsch ex Reichardt, Hypericaceae, has antidepressant-like effect in the forced swimming test in rodents and inhibits monoamines neuronal reuptake without binding to their neuronal carriers. Studies showed the involvement of Na+,K+-ATPase brain activity in depressive disorders, as well as the dependence of neuronal monoamine transport from Na+ gradient generated by Na+,K+-ATPase. This study aimed at evaluating the effect of uliginosin B on Na+,K+-ATPase activity in mice cerebral cortex and hippocampus (1 and 3 h after the last administration) as well as the influence of veratrine, a Na+ channel opener, on the antidepressant-like effect of uliginosin B. Mice were treated (p.o.) with uliginosin B single (10 mg/kg) or repeated doses (10 mg/kg/day, 3 days). Acute administration reduced the immobility in the forced swimming test and tail suspension test and increased Na+,K+-ATPase activity in cerebral cortex 1 h after treating, whereas the repeated treatment induced the antidepressant-like effect and increased the Na+,K+-ATPase activity at both times evaluated. None treatment affected the hippocampus enzyme activity. Veratrine pretreatment prevented uliginosin B antidepressant-like effect in the forced swimming test, suggesting the involvement of Na+ balance regulation on this effect. Altogether, these data indicate that uliginosin B reduces the monoamine uptake by altering Na+ gradient.

Severe Hyperhomocysteinemia Decreases Respiratory Enzyme and Na(+)-K(+) ATPase Activities, and Leads to Mitochondrial Alterations in Rat Amygdala.

Severe hyperhomocysteinemia is caused by increased plasma levels of homocysteine (Hcy), a methionine derivative, and is associated with cerebral disorders. Creatine supplementation has emerged as an adjuvant to protect against neurodegenerative diseases, due to its potential antioxidant role. Here, we examined the effects of severe hyperhomocysteinemia on brain metabolism, and evaluated a possible neuroprotective role of creatine in hyperhomocysteinemia, by concomitant treatment with Hcy and creatine (50 mg/Kg body weight). Hyperhomocysteinemia was induced in young rats (6-day-old) by treatment with homocysteine (0.3-0.6 µmol/g body weight) for 23 days, and then the following parameters of rat amygdala were evaluated: (1) the activity of the respiratory chain complexes succinate dehydrogenase, complex II and cytochrome c oxidase; (2) mitochondrial mass and membrane potential; (3) the levels of necrosis and apoptosis; and (4) the activity and immunocontent of Na(+),K(+)-ATPase. Hcy treatment decreased the activities of succinate dehydrogenase and cytochrome c oxidase, but did not alter complex II activity. Hcy treatment also increased the number of cells with high mitochondrial mass, high mitochondrial membrane potential, and in late apoptosis. Importantly, creatine administration prevented some of the key effects of Hcy administration on the amygdala. We also observed a decrease in the activity and immunocontent of the α1 subunit of the Na(+),K(+)-ATPase in amygdala after Hcy- treatment. Our findings support the notion that Hcy modulates mitochondrial function and bioenergetics in the brain, as well as Na(+),K(+)-ATPase activity, and suggest that creatine might represent an effective adjuvant to protect against the effects of high Hcy plasma levels.

Ammonia impairs glutamatergic communication in astroglial cells: protective role of resveratrol.

Ammonia is a key toxin in the precipitation of hepatic encephalopathy (HE), a neuropsychiatric disorder associated with liver failure. In response to ammonia, various toxic events are triggered in astroglial cells, and alterations in brain glutamate communication are common. Resveratrol is a polyphenolic compound that has been extensively studied in pathological events because it presents several beneficial effects, including some in the central nervous system (CNS). We previously described that resveratrol is able to significantly modulate glial functioning and has a protective effect during ammonia challenge in vitro. In this study, we addressed the mechanisms by which resveratrol can protect C6 astroglial cells from glutamatergic alterations induced by ammonia. Resveratrol was able to prevent all the effects triggered by ammonia: (i) decrease in glutamate uptake activity and expression of the EAAC1 glutamate transporter, the main glutamate transporter present in C6 cells; (ii) increase of glutamate release, which was also dependent on the activation of the Na(+)-K(+)-Cl(-) co-transporter NKCC1; (iii) reduction in GS activity and intracellular GSH content; and (iv) impairment of Na(+)K(+)-ATPase activity. Interestingly, resveratrol, per se, also positively modulated the astroglial functions evaluated. Moreover, we demonstrated that heme oxygenase 1 (HO1), an enzyme that is part of the cellular defense system, mediated some of the effects of resveratrol. In conclusion, the mechanisms of the putative protective role of resveratrol against ammonia toxicity involve the modulation of pathways and molecules related to glutamate communication in astroglial cells.

Lipid, Oxidative and Inflammatory Profile and Alterations in the Enzymes Paraoxonase and Butyrylcholinesterase in Plasma of Patients with Homocystinuria Due CBS Deficiency: The Vitamin B12 and Folic Acid Importance.

Cystathionine-ß-synthase (CBS) deficiency is the main cause of homocystinuria. Homocysteine (Hcy), methionine, and other metabolites of Hcy accumulate in the body of affected patients. Despite the fact that thromboembolism represents the major cause of morbidity in CBS-deficient patients, the mechanisms of cardiovascular alterations found in homocystinuria remain unclear. In this work, we evaluated the lipid and inflammatory profile, oxidative protein damage, and the activities of the enzymes paraoxonase (PON1) and butyrylcholinesterase (BuChE) in plasma of CBS-deficient patients at diagnosis and during the treatment (protein-restricted diet supplemented with pyridoxine, folic acid, betaine, and vitamin B12). We also investigated the effect of folic acid and vitamin B12 on these parameters. We found a significant decrease in HDL cholesterol and apolipoprotein A1 (ApoA-1) levels, as well as in PON1 activity in both untreated and treated CBS-deficient patients when compared to controls. BuChE activity and IL-6 levels were significantly increased in not treated patients. Furthermore, significant positive correlations between PON1 activity and sulphydryl groups and between IL-6 levels and carbonyl content were verified. Moreover, vitamin B12 was positively correlated with PON1 and ApoA-1 levels, while folic acid was inversely correlated with total Hcy concentration, demonstrating the importance of this treatment. Our results also demonstrated that CBS-deficient patients presented important alterations in biochemical parameters, possibly caused by the metabolites of Hcy, as well as by oxidative stress, and that the adequate adherence to the treatment is essential to revert or prevent these alterations.

Hyperprolinemia induces DNA, protein and lipid damage in blood of rats: antioxidant protection.

The present study investigated the effects of hyperprolinemia on oxidative damage to biomolecules (protein, lipids and DNA) and the antioxidant status in blood of rats. The influence of the antioxidants on the effects elicited by proline was also examined. Wistar rats received two daily injections of proline and/or vitamin E plus C (6th-28th day of life) and were killed 12h after the last injection. Results showed that hyperprolinemia induced a significant oxidative damage to proteins, lipids and DNA demonstrated by increased carbonyl content, malondialdehyde levels and a greater damage index in comet assay, respectively. The concomitant antioxidants administration to proline treatment completely prevented oxidative damage to proteins, but partially prevented lipids and DNA damage. We also observed that the non-enzymatic antioxidant potential was decreased by proline treatment and partially prevented by antioxidant supplementation. The plasma levels of vitamins E and C significantly increased in rats treated exogenously with these vitamins but, interestingly, when proline was administered concomitantly with vitamin E plus C, the levels of these vitamins were similar to those found in plasma of control and proline rats. Our findings suggest that hyperprolinemia promotes oxidative damage to the three major classes of macromolecules in blood of rats. These effects were accomplished by decrease in non-enzymatic antioxidant potential and decrease in vitamins administered exogenously, which significantly decreased oxidative damage to biomolecules studied. These data suggest that antioxidants may be an effective adjuvant therapeutic to limit oxidative damage caused by proline.

Effect of physical exercise on changes in activities of creatine kinase, cytochrome c oxidase and ATP levels caused by ovariectomy.

The reduction in the secretion of ovarian hormones, principally estrogen, is a consequence of menopause. Estrogens act primarily as female sex hormones, but also exert effects on different physiological systems including the central nervous system. The treatment normally used to reduce the symptoms of menopause is the hormone therapy, which seems to be effective in treating symptoms, but it may be responsible for adverse effects. Based on this, there is an increasing demand for alternative therapies that minimize signs and symptoms of menopause. In the present study we investigated the effect of ovariectomy and/or physical exercise on the activities of energy metabolism enzymes, such as creatine kinase (cytosolic and mitochondrial fractions), pyruvate kinase, succinate dehydrogenase, complex II, cytochrome c oxidase, as well as on ATP levels in the hippocampus of adult rats. Adult female Wistar rats with 90 days of age were subjected to ovariectomy (an animal model widely used to mimic the postmenopausal changes). Thirty days after the procedure, the rats were submitted to the exercise protocol, which was performed three times a week for 30 days. Twelve hours after the last training session, the rats were decapitated for subsequent biochemical analyzes. Results showed that ovariectomy did not affect the activities of pyruvate kinase, succinate dehydrogenase and complex II, but decreased the activities of creatine kinase (cytosolic and mitochondrial fractions) and cytochrome c oxidase. ATP levels were also reduced. Exercise did not produce the expected results since it was only able to partially reverse the activity of creatine kinase cytosolic fraction. The results of this study suggest that estrogen deficiency, which occurs as a result of ovariectomy, affects generation systems and energy homeostasis, reducing ATP levels in hippocampus of adult female rats.

Creatine prevents the imbalance of redox homeostasis caused by homocysteine in skeletal muscle of rats.

Homocystinuria is a neurometabolic disease caused by severe deficiency of cystathionine beta-synthase activity, resulting in severe hyperhomocysteinemia. Affected patients present several symptoms including a variable degree of motor dysfunction, being that the pathomechanism is not fully understood. In the present study we investigated the effect of chronic hyperhomocysteinemia on some parameters of oxidative stress, namely 2'7'dichlorofluorescein (DCFH) oxidation, levels of thiobarbituric acid-reactive substances (TBARS), antioxidant enzyme activities (SOD, CAT and GPx), reduced glutathione (GSH), total sulfhydryl and carbonyl content, as well as nitrite levels in soleus skeletal muscle of young rats subjected to model of severe hyperhomocysteinemia. We also evaluated the effect of creatine on biochemical alterations elicited by hyperhomocysteinemia. Wistar rats received daily subcutaneous injection of homocysteine (0.3-0.6 µmol/g body weight), and/or creatine (50mg/kg body weight) from their 6th to the 28th days age. Controls and treated rats were decapitated at 12h after the last injection. Chronic homocysteine administration increased 2'7'dichlorofluorescein (DCFH) oxidation, an index of production of reactive species and TBARS levels, an index of lipoperoxidation. Antioxidant enzyme activities, such as SOD and CAT were also increased, but GPx activity was not altered. The content of GSH, sulfhydril and carbonyl were decreased, as well as levels of nitrite. Creatine concurrent administration prevented some homocysteine effects probably by its antioxidant properties. Our data suggest that the oxidative insult elicited by chronic hyperhomocystenemia may provide insights into the mechanisms by which homocysteine exerts its effects on skeletal muscle function. Creatine prevents some alterations caused by homocysteine.

Neonatal environmental intervention alters the vulnerability to the metabolic effects of chronic palatable diet exposure in adulthood.

Previous studies have demonstrated that early environmental interventions influence the consumption of palatable food and the abdominal fat deposition in female rats chronically exposed to a highly caloric diet in adulthood. In this study, we verified the metabolic effects of chronic exposure to a highly palatable diet, and determine the response to its withdrawal in adult neonatally handled and non-handled rats. Consumption of foods (standard lab chow and chocolate), body weight gain, abdominal fat deposition, plasma triglycerides, and leptin, as well as serum butyrylcholinesterase (BuChE), and cerebral acetylcholinesterase (AChE) activities were measured during chronic chocolate exposure and after deprivation of this palatable food in female rats exposed or not to neonatal handling (10 minutes/day, 10 first days of life). Handled rats increased rebound chocolate consumption in comparison to non-handled animals after 1 week of chocolate withdrawal; these animals also decreased body weight in the first 24 hours but this effect disappeared after 7 days of withdrawal. Chocolate increased abdominal fat in non-handled females, and this effect remained after 30 days of withdrawal; no differences in plasma leptin were seen after 7 days of withdrawal. Chocolate also increased serum BuChE activity in non-handled females, this effect was still evident after 7 days of withdrawal, but it disappeared after 30 days of withdrawal. Chocolate deprivation decreased cerebral AChE activity in both handled and non-handled animals. These findings suggest that neonatal handling modulates the preference for palatable food and induces a specific metabolic response that may be more adaptive in comparison to non-handled rats.

Mild hyperhomocysteinemia increases brain acetylcholinesterase and proinflammatory cytokine levels in different tissues.

Mild hyperhomocysteinemia is considered to be a risk factor for cerebral and cardiovascular disorders and can be modeled in experimental rats. Inflammation has been implicated in the toxic effects of homocysteine. Cholinergic signaling controls cytokine production and inflammation through the "cholinergic anti-inflammatory pathway," and brain acetylcholinesterase activity plays a role in this regulation. The aim of this present study is to investigate the effect of mild chronic hyperhomocysteinemia on proinflammatory cytokine levels in the brain, heart, and serum of rats. Activity, immunocontent, and gene expression of acetylcholinesterase in the brain and butyrylcholinesterase activity in serum were also evaluated. Mild hyperhomocysteinemia was induced in Wistar rats by homocysteine administration (0.03 µmol/g of body weight) twice a day, from the 30th to the 60th days of life. Controls received saline in the same volumes. Results demonstrated an increase in tumor necrosis factor-alpha (TNF-α), interleukin-1ß (IL-1ß), interleukin-6 (IL-6), and the chemokine monocyte chemotactic protein-1 (MCP-1) in the hippocampus, as well as an increase in IL-1ß and IL-6 levels in cerebral cortex. Acetylcholinesterase activity was increased in rats subjected to mild hyperhomocysteinemia in both cerebral structures tested; the immunocontent of this enzyme was also increased in the cerebral cortex and decreased in the hippocampus. Levels of acetylcholinesterase mRNA transcripts were not altered. Peripherally, homocysteine increased TNF-α, IL-6, and MCP-1 levels in the heart and IL-6 levels in serum. Taken altogether, these findings suggest that homocysteine promotes an inflammatory status that can contribute, at least in part, to neuronal and cardiovascular dysfunctions observed in mild hyperhomocysteinemia.

Experimental lung injury promotes alterations in energy metabolism and respiratory mechanics in the lungs of rats: prevention by exercise.

In the present study we investigated the effects of lung injury on energy metabolism (succinate dehydrogenase, complex II, cytochrome c oxidase, and ATP levels), respiratory mechanics (dynamic and static compliance, elastance and respiratory system resistance) in the lungs of rats, as well as on phospholipids in bronchoalveolar lavage fluid. The protective effect of physical exercise on the alterations caused by lung injury, including lung edema was also evaluated. Wistar rats were submitted to 2 months of physical exercise. After this period the lung injury was induced by intratracheal instillation of lipopolysaccharide. Adult Wistar rats were submitted to 2 months of physical exercise and after this period the lung injury was induced by intratracheal instillation of lipopolysaccharide in dose 100 µg/100 g body weight. The sham group received isotonic saline instillation. Twelve hours after the injury was performed the respiratory mechanical and after the rats were decapitated and samples were collected. The rats subjected to lung injury presented a decrease in activities of the enzymes of the electron transport chain and ATP levels in lung, as well as the formation of pulmonary edema. A decreased lung dynamic and static compliance, as well as an increase in respiratory system resistance, and a decrease in phospholipids content were observed. Physical exercise was able to totally prevent the decrease in succinate dehydrogenase and complex II activities and the formation of pulmonary edema. It also partially prevented the increase in respiratory system resistance, but did not prevent the decrease in dynamic and static compliance, as well as in phospholipids content. These findings suggest that the mitochondrial dysfunction may be one of the important contributors to lung damage and that physical exercise may be beneficial in this pathology, although it did not prevent all changes present in lung injury.

Contextual fear conditioning in maternal separated rats: the amygdala as a site for alterations.

The first 2 weeks of life are a critical period for neural development in rats. Repeated long-term separation from the dam is considered to be one of the most potent stressors to which rat pups can be exposed, and permanently modifies neurobiological and behavioral parameters. Prolonged periods of maternal separation (MS) usually increase stress reactivity during adulthood, and enhance anxiety-like behavior. The aim of this study was to verify the effects of maternal separation during the neonatal period on memory as well as on biochemical parameters (Na(+), K(+)-ATPase and antioxidant enzymes activities) in the amygdala of adult rats. Females and male Wistar rats were subjected to repeated maternal separation (incubator at 32 °C, 3 h/day) during postnatal days 1-10. At 60 days of age, the subjects were exposed to a Contextual fear conditioning task. One week after the behavioral task, animals were sacrificed and the amygdala was dissected for evaluation of Na(+), K(+)-ATPase and antioxidant enzymes activities. Student-t test showed significant MS effect, causing an increase of freezing time in the three exposures to the aversive context in both sexes. Considering biochemical parameters Student-t test showed significant MS effect causing an increase of Na(+), K(+)-ATPase activity in both sexes. On the other hand, no differences were found among the groups on the antioxidant enzymes activities [superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT)] in male rats, but in females, we found a significant MS effect, causing an increase of CAT activity and no differences were found among the groups on SOD and GPx activities. Our results suggest a role of early rearing environment in programming fear learning and memory in adulthood. An early stress experience such as maternal separation may increase activity in the amygdala (as pointed by the increased activity of Na(+), K(+)-ATPase), affecting behaviors related to fear in adulthood, and this effect could be task-specific.

Alterations on Na⁺,K⁺-ATPase and acetylcholinesterase activities induced by amyloid-ß peptide in rat brain and GM1 ganglioside neuroprotective action.

Alzheimer's disease (AD) is a neurodegenerative disorder whose pathogenesis involves production and aggregation of amyloid-ß peptide (Aß). Aß-induced toxicity is believed to involve alterations on as Na(+),K(+)-ATPase and acetylcholinesterase (AChE) activities, prior to neuronal death. Drugs able to prevent or to reverse these biochemical changes promote neuroprotection. GM1 is a ganglioside proposed to have neuroprotective roles in AD models, through mechanisms not yet fully understood. Therefore, this study aimed to investigate the effect of Aß1-42 infusion and GM1 treatment on recognition memory and on Na(+),K(+)-ATPase and AChE activities, as well as, on antioxidant defense in the brain cortex and the hippocampus. For these purposes, Wistar rats received i.c.v. infusion of fibrilar Aß1-42 (2 nmol) and/or GM1 (0.30 mg/kg). Behavioral and biochemical analyses were conducted 1 month after the infusion procedures. Our results showed that GM1 treatment prevented Aß-induced cognitive deficit, corroborating its neuroprotective function. Aß impaired Na(+),K(+)-ATPase and increase AChE activities in hippocampus and cortex, respectively. GM1, in turn, has partially prevented Aß-induced alteration on Na(+),K(+)-ATPase, though with no impact on AChE activity. Aß caused a decrease in antioxidant defense, specifically in hippocampus, an effect that was prevented by GM1 treatment. GM1, both in cortex and hippocampus, was able to increase antioxidant scavenge capacity. Our results suggest that Aß-triggered cognitive deficit involves region-specific alterations on Na(+),K(+)-ATPase and AChE activities, and that GM1 neuroprotection involves modulation of Na(+),K(+)-ATPase, maybe by its antioxidant properties. Although extrapolation from animal findings is difficult, it is conceivable that GM1 could play an important role in AD treatment.

Mild hyperhomocysteinemia reduces the activity and immunocontent, but does not alter the gene expression, of catalytic α subunits of cerebral Na+,K+-ATPase.

Na(+),K(+)-ATPase is a membrane protein which plays a key role in the maintenance of ion homeostasis that is necessary to neuronal excitability, secondary transport and neurotransmitter uptake. Mild hyperhomocysteinemia leads to several clinical manifestations and particularly cerebral diseases; however, little is known about the mechanisms of homocysteine on cerebral Na(+),K(+)-ATPase. In the present study, we investigated the effect of mild hyperhomocysteinemia on the activity, the immunocontent of catalytic subunits (α1, α2, and α3) and the gene expression of this enzyme. We used the experimental model of mild hyperhomocysteinemia that was induced by homocysteine administration (0.03 µmol/g of body weight) twice a day, from the 30th to the 60th postpartum day. Controls received saline in the same volumes. Results showed that mild hyperhomocysteinemia significantly decreased the activity and the immunocontent of the α 1 and α 2 subunits of the Na(+),K(+)-ATPase in cerebral cortex and hippocampus of adult rats. On the other hand, we did not observe any change in levels of Na(+),K(+)-ATPase mRNA transcripts in such cerebral structures of rats after chronic exposure to homocysteine. The present findings support that the homocysteine modulates the Na(+),K(+)-ATPase and this could be associated, at least in part, with the risk to the development of cerebral diseases in individuals with mild hyperhomocysteinemia.

Homocysteine induces energy imbalance in rat skeletal muscle: is creatine a protector?

Homocystinuria is a neurometabolic disease caused by a severe deficiency of cystathionine beta-synthase activity, resulting in severe hyperhomocysteinemia. Affected patients present several symptoms including a variable degree of motor dysfunction. In this study, we investigated the effect of chronic hyperhomocysteinemia on the cell viability of the mitochondrion, as well as on some parameters of energy metabolism, such as glucose oxidation and activities of pyruvate kinase, citrate synthase, isocitrate dehydrogenase, malate dehydrogenase, respiratory chain complexes and creatine kinase in gastrocnemius rat skeletal muscle. We also evaluated the effect of creatine on biochemical alterations elicited by hyperhomocysteinemia. Wistar rats received daily subcutaneous injections of homocysteine (0.3-0.6 µmol/g body weight) and/or creatine (50 mg/kg body weight) from the 6th to the 28th days of age. The animals were decapitated 12 h after the last injection. Homocysteine decreased the cell viability of the mitochondrion and the activities of pyruvate kinase and creatine kinase. Succinate dehydrogenase was increased other evaluated parameters were not changed by this amino acid. Creatine, when combined with homocysteine, prevented or caused a synergistic effect on some changes provoked by this amino acid. Creatine per se or creatine plus homocysteine altered glucose oxidation. These findings provide insights into the mechanisms by which homocysteine exerts its effects on skeletal muscle function, more studies are needed to elucidate them. Although creatine prevents some alterations caused by homocysteine, it should be used with caution, mainly in healthy individuals because it could change the homeostasis of normal physiological functions.

Cytoskeleton of cortical astrocytes as a target to proline through oxidative stress mechanisms.

Hyperprolinemia is an inherited disorder of proline (Pro) metabolism and patients affected by this disease may present neurological manifestations. However, the mechanisms of neural excitotoxicity elicited by hyperprolinemia are far from being understood. Considering the pivotal role of cytoskeletal remodeling in several neurodegenerative pathologies and the potential links between cytoskeleton, reactive oxygen species production and cell death, the aim of the present work was to study the effects of Pro on astrocyte and neuron cytoskeletal remodeling and the possible oxidative stress involvement. Pro induced a shift of actin cytoskeleton in stress fibers together with increased RhoA immunocontent and ERK1/2 phosphorylation/activation in cortical astrocytes. Unlike astrocytes, results evidenced little susceptibility of neuron cytoskeleton remodeling, since Pro-treated neurons presented unaltered neuritogenesis. We observed increased hydrogen peroxide production characterizing oxidative stress together with decreased superoxide dismutase (SOD) and catalase (CAT) activities in cortical astrocytes after Pro treatment, while glutathione peroxidase (GSHPx) activity remained unaltered. However, coincubation with Pro and Trolox/melatonin prevented decreased SOD and CAT activities in Pro-treated astrocytes. Accordingly, these antioxidants were able to prevent the remodeling of the actin cytoskeleton, RhoA increased levels and ERK1/2 phosphorylation in response to high Pro exposure. Taken together, these findings indicated that the cytoskeleton of cortical astrocytes, but not of neurons in culture, is a target to Pro and such effects could be mediated, at least in part, by redox imbalance, RhoA and ERK1/2 signaling pathways. The vulnerability of astrocyte cytoskeleton may have important implications for understanding the effects of Pro in the neurotoxicity linked to inborn errors of Pro metabolism.

Methylphenidate induces lipid and protein damage in prefrontal cortex, but not in cerebellum, striatum and hippocampus of juvenile rats.

The use of psychostimulant methylphenidate has increased in recent years for the treatment of attention-deficit hyperactivity disorder in children and adolescents. However, the behavioral and neurochemical changes promoted by its use are not yet fully understood, particularly when used for a prolonged period during stages of brain development. Thus, the aim of this study was to determine some parameters of oxidative stress in encephalic structures of juvenile rats subjected to chronic methylphenidate treatment. Wistar rats received intraperitoneal injections of methylphenidate (2.0 mg/kg) once a day, from the 15th to the 45th day of age or an equivalent volume of 0.9% saline solution (controls). Two hours after the last injection, animals were euthanized and the encephalic structures obtained for determination of oxidative stress parameters. Results showed that methylphenidate administration increased the activities of superoxide dismutase and catalase, but did not alter the levels of reactive species, thiobarbituric acid reactive substances levels and sulfhydryl group in cerebellum of rats. In striatum and hippocampus, the methylphenidate-treated rats presented a decrease in the levels of reactive species and thiobarbituric acid reactive substances, but did not present changes in the sulfhydryl groups levels. In prefrontal cortex, methylphenidate promoted an increase in reactive species formation, SOD/CAT ratio, and increased the lipid peroxidation and protein damage. These findings suggest that the encephalic structures respond differently to methylphenidate treatment, at least, when administered chronically to young rats. Notably, the prefrontal cortex of juvenile rats showed greater sensitivity to oxidative effects promoted by methylphenidate in relation to other encephalic structures analyzed.

Evidence that hyperprolinemia alters glutamatergic homeostasis in rat brain: neuroprotector effect of guanosine.

This study investigated the effects of acute and chronic hyperprolinemia on glutamate uptake, as well as some mechanisms underlying the proline effects on glutamatergic system in rat cerebral cortex. The protective role of guanosine on effects mediated by proline was also evaluated. Results showed that acute and chronic hyperprolinemia reduced glutamate uptake, Na(+), K(+)-ATPase activity, ATP levels and increased lipoperoxidation. GLAST and GLT-1 immunocontent were increased in acute, but not in chronic hyperprolinemic rats. Our data suggest that the effects of proline on glutamate uptake may be mediated by lipid peroxidation and disruption of Na(+), K(+)-ATPase activity, but not by decreasing in glutamate transporters. This probably induces excitotoxicity and subsequent energy deficit. Guanosine was effective to prevent most of the effects promoted by proline, reinforcing its modulator role in counteracting the glutamate toxicity. However, further studies are needed to assess the modulatory effects of guanosine on experimental hyperprolinemia.

Chronic mild hyperhomocysteinemia alters ectonucleotidase activities and gene expression of ecto-5'-nucleotidase/CD73 in rat lymphocytes.

Since mild hyperhomocysteinemia is a risk factor for cardiovascular and cerebral diseases and extracellular nucleotides/nucleosides, which are controlled by the enzymatic action of ectonucleotidases, can induce an immune response, in the present study, we investigated the effect of chronic mild hyperhomocysteinemia on ectonucleotidase activities and expression in lymphocytes from mesenteric lymph nodes and serum of adult rats. For the chronic chemically induced mild hyperhomocysteinemia, Hcy (0.03 µmol/g of body weight) or saline (control) were administered subcutaneously from the 30th to the 60th day of life. Results showed that homocysteine significantly decreased ATP, ADP, and AMP hydrolysis in lymphocytes of adult rats. E-NTPDases transcriptions were not affected, while the ecto-5'-nucleotidase transcription was significantly decreased in mesenteric lymph nodes of hyperhomocysteinemic rats. ATP, ADP, and AMP hydrolysis were not affected by homocysteine in rat serum. Our findings suggest that Hcy in levels similar to considered risk factor to development of vascular diseases modulates the ectonucleotidases, which could lead to a pro-inflammatory status.

Reduction of butyrylcholinesterase activity in plasma from patients with disorders of propionate metabolism is prevented by treatment with L-carnitine and protein restriction.

OBJECTIVE: We investigated the relationship between butyrylcholinesterase (BuChE) activity and lipid oxidative damage in patients with disorders of propionate metabolism, before and after treatment with protein restriction and L-carnitine. DESIGN AND METHODS: BuChE activity and malondialdehyde (MDA) were measured in plasma from eight untreated patients (at diagnosis) and from seven patients under treatment with protein restriction and L-carnitne supplementation (100mg/kg/day). RESULTS: We verified a significant reduction of butyrylcholinesterase activity, as well as an increased MDA formation in plasma from untreated patients. However, treated patients presented MDA and BuChE activity similar to controls. Furthermore, butyrylcholinesterase activity was negatively correlated with MDA concentrations in these patients. CONCLUSION: The results suggest that an increased free radicals formation may be involved in the decrease of butyrylcholinesterase activity, possibly contributing to the neurological damage of these disorders, and that treatment with L-carnitine and low-protein diet possibly is able to prevent this damage.