Search

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-1β (IL-1β), interleukin-6 (IL-6), and the chemokine monocyte chemotactic protein-1 (MCP-1) in the hippocampus, as well as an increase in IL-1β 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.

Parkinson's disease (PD) is characterized by a progressive neurodegeneration in the substantia nigra and a striatal dopamine decrease. Striatal extracellular adenosine and ATP modulate the dopaminergic neurotransmission whereas guanosine has a protective role in the brain. Therefore, the regulation of their levels by enzymatic activity may be relevant to the clinical feature of PD. Here it was evaluated the extracellular nucleotide hydrolysis from striatal slices 4 weeks after a unilateral infusion with 6-OHDA into the medial forebrain bundle. This infusion increased ADP, AMP, and GTP hydrolysis by 15, 25, and 41%, respectively, and decreased GDP hydrolysis by 60%. There was no change in NTPDases1, 2, 3, 5, 6, and 5'-nucleotidase transcription. Dopamine depletion changes nucleotide hydrolysis and, therefore, alters the regulation of striatal nucleotide levels. These changes observed in 6-OHDA-lesioned animals may contribute to the symptoms observed in the model and provide evidence to indicate that extracellular purine hydrolysis is a key factor in understanding PD, giving hints for new therapies.

Adenosine, a well-known neuromodulator, can act as an endogenous anticonvulsant via the activation of adenosine A1 receptors. This adenine nucleoside can be produced in the synaptic cleft by the ectonucleotidase cascade, which includes the nucleoside triphosphate diphosphohydrolase (NTPDase) family and ecto-5'-nucleotidase. It has been previously reported that ectonucleotidase activities are increased in female adult rats submitted to the pilocarpine model of epilepsy. Several studies have suggested that the immature brain is less vulnerable to morphologic and physiologic alterations after status epilepticus (SE). Here, we evaluate the ectonucleotidase activities of synaptosomes from the hippocampus and cerebral cortex of male and female rats at different ages (7-9, 14-16 and 27-30-day old) submitted to the pilocarpine model of epilepsy. Our results show that ATP and ADP hydrolysis in the hippocampus and cerebral cortex were not altered by the pilocarpine treatment in female and male rats at 7-9, 14-16 and 27-30 days. There were no changes in AMP hydrolysis in female and male rats submitted to the model at different ages, but a significant increase in AMP hydrolysis (71%) was observed in synaptosomes from the cerebral cortex of male rats at 27-30 days. Pilocarpine-treated male rats (60-70-day old) presented an enhancement in ectonucleotidase activities in the synaptosomes of the cerebral cortex (33, 40 and 64% for ATP, ADP and AMP hydrolysis, respectively) and hippocampus (55, 98 and 101% for ATP, ADP and AMP hydrolysis, respectively). These findings highlight differences between the purinergic system of young and adult rats submitted to the pilocarpine model of epilepsy.

In the present study, the glucose concentration in the haemolymph and glycogen levels were determined in the various body parts of the Helix aspersa snail after feeding lettuce ad libitum and after various periods of starvation. To characterize the effect of starvation on nucleotidase activity, enzyme assays were performed on membranes of the nervous ganglia and digestive gland. Results demonstrated the maintenance of the haemolymph glucose concentration for up to 30 days of starvation, probably due to the consumption of glycogen from the mantle. In the nervous ganglia, depletion of glycogen occurs progressively during the different periods of starvation. No significant changes were observed on ATP and ADP hydrolysis in the membranes of nervous ganglia and no alterations in Ca2+ -ATPase and Mg2+ -ATPase occurred in the membranes of the digestive gland of H. aspersa during the different periods of starvation. Although there were no changes in the enzyme activities during starvation, they could be modulated by effectors in situ with concomitant changes in products/reactants during starvation.

Hypermethioninemic patients may exhibit different neurological dysfunctions, and the mechanisms underlying these pathologies remain obscure. Glutamate and ATP are important excitatory neurotransmitters co-released at synaptic clefts, and whose activities are intrinsically related. Adenosine—the final product of ATP breakdown—is also an important neuromodulator. Here, we investigated the effects of long-term (7-day) exposure to 1.5 or 3 mM methionine (Met) on glutamate uptake in brain tissues (telencephalon, optic tectum, and cerebellum) and on ATP, ADP, and AMP catabolism by ecto-nucleotidases found in brain membrane samples, using a zebrafish model. Also, we evaluated the expression of ecto-nucleotidase (ntdp1, ntdp2mg, ntdp2mq, ntdp2mv, ntdp3, and nt5e) and adenosine receptor (adora1, adora2aa, adora2ab, adora2b) genes in the brain of zebrafish exposed to Met. In animals exposed to 3.0 mM Met, glutamate uptake in the telencephalon decreased significantly. Also, ATP and ADP (but not AMP) catabolism decreased significantly at both Met concentrations tested. The messenger RNA (mRNA) levels of ntpd genes and of the adenosine receptors adora1 and adora2aa increased significantly after Met exposure. In contrast, adora2ab mRNA levels decreased after Met exposure. Our data suggest that glutamate and ATP accumulate at synaptic clefts after Met exposure, with potential detrimental effects to the nervous system. This phenomenon might explain, at least in part, the increased susceptibility of hypermethioninemic patients to neurological symptoms.

Hyperprolinemia is an inherited disorder of proline metabolism and hyperprolinemic patients can present neurological manifestations, such as seizures, cognitive dysfunctions, and schizoaffective disorders. However, the mechanisms related to these symptoms are still unclear. In the present study, we evaluated the in vivo and in vitro effects of proline on acetylcholinesterase (AChE) activity and gene expression in the zebrafish brain. For the in vivo studies, animals were exposed at two proline concentrations (1.5 and 3.0mM) during 1h or 7 days (short- or long-term treatments, respectively). For the in vitro assays, different proline concentrations (ranging from 3.0 to 1000 μM) were tested. Long-term proline exposures significantly increased AChE activity for both treated groups when compared to the control (34% and 39%). Moreover, the proline-induced increase on AChE activity was completely reverted by acute administration of antipsychotic drugs (haloperidol and sulpiride), as well as the changes induced in ache expression. When assessed in vitro, proline did not promote significant changes in AChE activity. Altogether, these data indicate that the enzyme responsible for the control of acetylcholine levels might be altered after proline exposure in the adult zebrafish. These findings contribute for better understanding of the pathophysiology of hyperprolinemia and might reinforce the use of the zebrafish as a complementary vertebrate model for studying inborn errors of amino acid metabolism.

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 (α 1, α 2, and α 3) 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.

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.

Hypermethioninemic patients exhibit a variable degree of neurological dysfunction. However, the mechanisms involved in these alterations have not been completely clarified. Cholinergic system has been implicated in many physiological processes, including cognitive performances, as learning, and memory. Parameters of cholinergic signaling have already been characterized in zebrafish brain. Since zebrafish is a small freshwater teleost which is a vertebrate model for modeling behavioral and functional parameters related to human pathogenesis and for clinical treatment screenings, in the present study we investigated the effects of short- and long-term methionine exposure on cognitive impairment, AChE activity and gene expression in zebrafish. For the studies, animals were exposed at two methionine concentrations (1.5 and 3.0 mM) during 1 h or 7 days (short- or long-term treatments, respectively). We observed a significant increase in AChE activity of zebrafish brain membranes after long-term methionine exposure at 3.0 mM. However, AChE gene expression decreased significantly in both concentrations tested after 7 days of treatment, suggesting that post-translational events are involved in the enhancement of AChE activity. Methionine treatment induces memory deficit in zebrafish after long-term exposure to this amino acid, which could be related, at least in part, with cognitive impairment observed in hypermethioninemia. Therefore, the results here presented raise a new perspective to use the zebrafish as a complementary vertebrate model for studying inborn errors of metabolism, which may help to better understand the pathophysiology of this disease.

Hyperprolinemia is an inherited disorder of proline metabolism and patients affected by this disease may present neurological manifestations, including seizures and cognitive dysfunctions. Moreover, an association between adulthood schizoaffective disorders and moderate hyperprolinemia has been reported. However, the mechanisms underlying these behavioral phenotypes still remain unclear. In the present study, we investigated the effect of proline treatments on behavioral parameters in zebrafish, such as locomotor activity, anxiety, and social interaction. Adult zebrafish (Danio rerio) were exposed to proline (1.5 and 3.0 mM) during 1h or 7 days (short- or long-term treatments, respectively). Short-term proline exposure did not promote significant changes on the behavioral parameters observed. Long-term exposure at 1.5 mM proline significantly increased the number of line crossing (47%), the total distance (29%), and the mean speed (33%) when compared to control group. A significant increase in the time spent in the upper portion of the test tank was also observed after this treatment (91%), which may be interpreted as an indicator of anxiolytic behavior. Proline at 1.5 mM also induced social interaction impairment (78%), when compared to the untreated group after long-term treatment. Moreover, these proline-induced behavioral changes in zebrafish were completely reversed by acute administration of an atypical antipsychotic drug (sulpiride), but not by a typical (haloperidol). These findings demonstrate that proline is able to induce schizophrenia-like symptoms in zebrafish, which reinforce the use of this species as a complementary vertebrate model for studying behavioral phenotypes associated with neurological dysfunctions characteristic of metabolic diseases.

Studies have shown that seizures in young animals lead to later cognitive deficits. There is evidence that long-term potentiation (LTP) and long-term depression (LTD) might contribute to the neural basis for learning and memory mechanism and might be modulated by ATP and/or its dephosphorylated product adenosine produced by a cascade of cell-surface transmembrane enzymes, such as E-NTPDases (ecto-nucleoside triphosphate diphosphohydrolases) and ecto-5'-nucleotidase. Thus, we have investigated if hippocampal ecto-nucleotidase activities are altered at different time periods after one episode of seizure induced by kainic acid (KA) in 7 days old rats. We also have evaluated if 90 day-old rats previously submitted to seizure induced by KA at 7 days of age presented cognitive impairment in Y-maze behavior task. Our results have shown memory impairment of adult rats (Postnatal day 90) previously submitted to one single seizure episode in neonatal period (Postnatal day 7), which is accompanied by an increased ATP hydrolysis in hippocampal synaptosomes. The metabolism of ATP evaluated by HPLC confirmed that ATP hydrolysis was faster in adult rats treated with KA in neonatal period than in controls. Surprisingly, the mRNA and protein levels as seen by PCR and Western blot, respectively, were not altered by the KA administration in early age. Since we have found an augmented hydrolysis of ATP and this nucleotide seems to be important to LTP induction, we could assume that impairment of memory and learning observed in adult rats which have experienced a convulsive episode in postnatal period may be a consequence of the increased ATP hydrolysis. These findings correlate the purinergic signaling to the cognitive deficits induced by neonatal seizures and contribute to a better understanding about the mechanisms of seizure-induced memory dysfunction.

Aims To investigate the effect of Nω-Nitro- l -arginine methyl ester ( l -NAME) treatment, known to induce a sustained elevation of blood pressure, on ectonucleotidase activities in kidney membranes of rats. Main methods l -NAME (30 mg/kg/day) was administered to Wistar rats for 14 days in the drinking water. Enzyme activities were determined colorimetrically and their gene expression patterns were analyzed by semi-quantitative RT-PCR. The metabolism of ATP and the accumulation of adenosine were evaluated by HPLC in kidney membranes from control and hypertensive rats. PKC phosphorylation state was investigated by Western blot. Key findings We observed an increase in systolic blood pressure from 115 ± 12 mmHg (control group) to 152 ± 18 mmHg ( l -NAME-treated group). Furthermore, the hydrolysis of ATP, ADP, AMP, and p-Nph-5′TMP was also increased (17%, 35%, 27%, 20%, respectively) as was the gene expression of NTPDase2, NTPDase3 and NPP3 in kidneys of hypertensive animals. Phospho-PKC was increased in hypertensive rats. Significance The general increase in ATP hydrolysis and in ecto-5′-nucleotidase activity suggests a rise in renal adenosine levels and in renal autoregulatory responses in order to protect the kidney against the threat presented by hypertension.

Submitted by Biblioteca Suporte PUCRS (biblioteca.suporte@pucrs.br) on 2022-09-06T12:06:03Z No. of bitstreams: 2 Influence_of_antidepressant_drugs_on_Ectonucleotide_pyrophosphatasephosphodiesterases_ENPPs_from_salivary_glands_of.pdf: 265416 bytes, checksum: d28afb46b27608d3fa637cacf0c53d24 (MD5) Influence_of_antidepressant_drugs_on_Ectonucleotide_pyrophosphatasephosphodiesterases_ENPPs_from_salivary_glands_of.pdf: 265416 bytes, checksum: d28afb46b27608d3fa637cacf0c53d24 (MD5) Made available in DSpace on 2022-09-06T12:06:03Z (GMT). No. of bitstreams: 2 Influence_of_antidepressant_drugs_on_Ectonucleotide_pyrophosphatasephosphodiesterases_ENPPs_from_salivary_glands_of.pdf: 265416 bytes, checksum: d28afb46b27608d3fa637cacf0c53d24 (MD5) Influence_of_antidepressant_drugs_on_Ectonucleotide_pyrophosphatasephosphodiesterases_ENPPs_from_salivary_glands_of.pdf: 265416 bytes, checksum: d28afb46b27608d3fa637cacf0c53d24 (MD5) Previous issue date: 2009