Methionine Exposure Alters Glutamate Uptake and Adenine Nucleotide Hydrolysis in the Zebrafish Brain.

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.

Long-term proline exposure alters nucleotide catabolism and ectonucleotidase gene expression in zebrafish brain.

Hyperprolinemia is an inherited disorder of proline metabolism and hyperprolinemic patients can present neurological manifestations, such as seizures cognitive dysfunctions, and psychotic disorders. However, the underlying mechanisms of these symptoms are still unclear. Since adenine nucleotides play crucial roles in neurotransmission and neuromodulation, we evaluated the in vivo and in vitro effects of proline on ectonucleotidase activities and gene expression in zebrafish brain. For the in vivo studies, animals were exposed at two proline concentrations (1.5 and 3.0 mM) during 1 h 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. Short-term proline exposure did not promote significant changes on the ectonucleotidase activities and gene expression. Long-term proline exposure significantly increased ATP catabolism in both concentrations tested (14 % and 22 %, respectively), whereas ADP and AMP hydrolysis were increased only at 3.0 mM proline (21 % and 17 %, respectively) when compared to control. Moreover, the relative gene expression of enpd3 increased in both treated groups after long-term proline, whereas enptd1 increased only at 3.0 mM proline. Proline in vitro did not promote significant changes on ectonucleotidase activities. Altogether, these data indicate that the enzymes responsible for the control of extracellular nucleotides levels might be altered after proline exposure in zebrafish, contributing to better understand the pathophysiology of this disease. Moreover, such findings might facilitate the use of the zebrafish as a complementary vertebrate model for studying inborn errors of amino acid metabolism.

Long-term methionine exposure induces memory impairment on inhibitory avoidance task and alters acetylcholinesterase activity and expression in zebrafish (Danio rerio).

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.

Behavioral changes induced by long-term proline exposure are reversed by antipsychotics in zebrafish.

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.

Adenosine A(2A) receptor agonist (CGS-21680) prevents endotoxin-induced effects on nucleotidase activities in mouse lymphocytes.

Adenosine 5'-triphosphate (ATP) released during inflammation presents proinflammatory properties. Adenosine, produced by catabolism of ATP, is an anti-inflammatory compound. Considering the role of ATP and adenosine in inflammation and the importance of ectonucleotidases in the maintenance of their extracellular levels, we investigated the effect of a selective agonist of the adenosine A(2A) receptor (CGS-21680) on ectonucleotidase activities and gene expression patterns in lymphocytes from mice submitted to an endotoxemia model. Animals were injected intraperitoneally with 12mg/kg Lipopolyssacharide (LPS) and/or 0.5mg/kg CGS-21680 or saline. Nucleotidase activities were determined in lymphocytes from mesenteric lymph nodes and analysis of ectonucleotidase expression was carried out by a semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) assay. Exposure to endotoxemia promoted an increase in nucleotide hydrolysis. When CGS-21680 was administered concomitantly with LPS, this increase was prevented for ATP, adenosine 5'-monophosphate (AMP), and p-Nitrophenyl thymidine 5'-monophosphate (p-Nph-5'-TMP) hydrolysis. However, when CGS-21680 was administered 24h after LPS injection, the increase was not reversed. The expression pattern of ectonucleotidases was not altered between LPS and LPS plus CGS-21680 groups, indicating that the transcriptional control was not involved on the effect exerted for CGS-21680. These results showed an enhancement of extracellular nucleotide catabolism in lymphocytes after induction of endotoxemia, which was prevented, but not reversed by CGS-21680 administration. These findings suggest that the control of nucleotide and nucleoside levels exerted by CGS-21680 could contribute to the modulation of the inflammatory process promoted by adenosine A(2A) agonists.

The hydrolysis of striatal adenine- and guanine-based purines in a 6-hydroxydopamine rat model of Parkinson's disease.

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.

L-NAME-treatment alters ectonucleotidase activities in kidney membranes of rats.

AIMS: To investigate the effect of N(omega)-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.

Exercise effects on activities of Na(+),K(+)-ATPase, acetylcholinesterase and adenine nucleotides hydrolysis in ovariectomized rats.

Hormone deficiency following ovariectomy causes activation of Na(+),K(+)-ATPase and acetylcholinesterase (AChE) that has been related to cognitive deficits in experimental animals. Considering that physical exercise presents neuroprotector effects, we decide to investigate whether exercise training would affect enzyme activation in hippocampus and cerebral cortex, as well as adenosine nucleotide hydrolysis in synaptosomes from cerebral cortex of ovariectomized rats. Female adult Wistar rats were assigned to one of the following groups: sham (submitted to surgery without removal of the ovaries), exercise, ovariectomized (Ovx) and Ovx plus exercise. Thirty days after surgery, animals were submitted to one month of exercise training, three times per week. After, rats were euthanized, blood serum was collected and hippocampus and cerebral cortex were dissected. Data demonstrated that exercise reversed the activation of Na(+),K(+)-ATPase and AChE activities both in hippocampus and cerebral cortex of ovariectomized rats. Ovariectomy decreased AMP hydrolysis in cerebral cortex and did not alter adenine nucleotides hydrolysis in blood serum. Exercise per se decreased ADP and AMP hydrolysis in cerebral cortex. On the other hand, AMP hydrolysis in blood serum was increased by exercise in ovariectomized adult rats. Present data support that physical exercise might have beneficial effects and constitute a therapeutic alternative to hormone replacement therapy for estrogen deprivation.

Influence of antidepressant drugs on Ecto-nucleotide pyrophosphatase/phosphodiesterases (E-NPPs) from salivary glands of rats.

Xerostomia is commonly caused by antidepressant drugs and ATP can influence the saliva production. Adenosine is the product of extracellular hydrolysis of adenine nucleotides in submandibular gland cells, which occurs by the action of ectonucleotidases. In this study, we have evaluated the effect of three different antidepressants in ecto-nucleotide pyrophosphatase/phosphodiesterase (E-NPP1-3) activities in cultured cells from salivary glands. Rats received imipramine (10mg/ml), fluoxetine (20mg/ml) or moclobemide (30mg/ml) by oral gavage. The drugs were administered once a day for 14 days. Our results have shown that the hydrolysis of p-nitrophenyl-5'-thymidine monophosphate increased in all treatments. These effects were not consequence of transcriptional control of E-NPP1-3 genes. The results reported here can highlight the importance of ectonucleotidases in the most common side effect caused by antidepressant therapy.

Endotoxemia alters nucleotide hydrolysis in platelets of rats.

Platelets play a critical role in homeostasis and blood clotting at sites of vascular injury, and also in various ways in innate immunity and inflammation. Platelets are one of the first cells to accumulate at an injured site, and local release of their secretome at some point initiate an inflammatory cascade that attracts leukocytes, activates target cells, stimulates vessel growth and repair. The level of exogenous ATP in the body may be increased in various inflammatory and shock conditions, primarily as a consequence of nucleotide release from platelets, endothelium and blood vessel cells. An increase of ATP release has been described during inflammation and this compound presents proinflammatory properties. ADP is a nucleotide known to induce changes in platelets shape and aggregation, to promote the exposure of fibrinogen-binding sites and to inhibit the stimulation of adenylate cyclase. Adenosine, the final product of the nucleotide hydrolysis, is a vasodilator and an inhibitor of platelet aggregation. There is a group of ecto-enzymes responsible for extracellular nucleotide hydrolysis named ectonucleotidases, which includes the NTPDase (nucleoside triphosphate diphosphohydrolase) family, the NPP (nucleoside pyrophosphatase/phosphodiesterase) family and an ecto-5'-nucleotidase. Therefore, we have aimed to investigate the effect of lipopolysaccharide endotoxin from Escherichia coli on ectonucleotidases in platelets from adult rats in order to better understand the role of extracellular adenine nucleotides and nucleosides in the maintenance of blood homeostasis in inflammatory processes. LPS administered in vitro was not able to alter the ATP, ADP, AMP and rho-Nph-5'-TMP hydrolysis of platelets from untreated rats in all concentrations tested (25-100 microg/ml). There was a significant decrease in ATP, ADP, AMP and rho-Nph-5'-TMP hydrolysis in rat platelets after 48 hours of LPS exposure (2 mg/Kg, i.p.). ATP and ADP hydrolysis has been reduced about 28% whereas it has been observed a significant 30% and 26% decrease on AMP and rho-Nph-5'-TMP hydrolysis. Platelet aggregation and platelet number have shown a significant decrease in LPS-treated rats (40% and 55%, respectively) when compared to control group. These results suggest that changes observed in platelet count and, consequently, in nucleotidase activities from circulatory system could alter extracellular nucleotide and nucleoside levels, which might modulate the inflammatory process.

Lipopolysaccharide alters nucleotidase activities from lymphocytes and serum of rats.

ATP exerts a proinflammatory role and induces cytokine release by acting at P2X(7) receptors. The product of ATP hydrolysis is the nucleoside adenosine, an important immunomodulator. The main source of extracellular adenosine is the hydrolysis of extracellular ATP by a group of ecto-enzymes: ENTPDase family, NPP family and ecto-5'-nucleotidase. Considering the role of ATP and adenosine in inflammatory processes, we investigated the effect of lipopolysaccharide on ectonucleotidases activities and expression in lymphocytes from mesenteric lymph nodes and serum of rats, in order to better understand the involvement of extracellular nucleotide hydrolysis in an endotoxemia model. We observed significant changes on nucleotidase activities from lymphocytes and serum of rats after in vitro and in vivo exposure to LPS. In vitro results have shown an increase on nucleotide hydrolysis in lymphocytes and a decrease on the enzyme activity of NPP in blood serum. In vivo, we observed an increase on nucleotide hydrolysis in lymphocytes and a decrease in the hydrolysis of all nucleotides tested in blood serum. After 24 and 48 h of LPS treatment, there was a reduction in NTPDase1, 2, 3 and ecto-5'-nucleotidase transcripts. These results suggest that there is a time-dependent enhancement of extracellular nucleotides metabolism in lymphocytes and blood serum after the induction of an endotoxemic model. The changes observed suggest that these enzymes can act in the regulation of extracellular nucleosides and nucleotides in a model able to trigger inflammatory process.

Ontogenetic profile of ectonucleotidase activities from brain synaptosomes of pilocarpine-treated rats.

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.

Nucleotidase activities in membrane preparations of nervous ganglia and digestive gland of the snail Helix aspersa.

Nucleotide-metabolizing enzymes play an important role in the regulation of nucleotide levels. In the present report, we demonstrated an enzyme activity with different kinetic properties in membrane preparations of the nervous ganglia and digestive gland from Helix aspersa. ATPase and ADPase activities were dependent on Ca2+ and Mg2+ with pH optima approximately 7.2 and between 6.0 and 8.0 in digestive gland and nervous ganglia, respectively. The enzyme activities present in membrane preparations of these tissues preferentially hydrolyzed triphosphate nucleotides. In nervous ganglia, the enzyme was insensitive to the classical ATPases inhibitors. In contrast, in digestive gland, N-ethylmaleimide (NEM) produced 45% inhibition of Ca(2+)-ATP hydrolysis. Sodium azide, at 100 microM and 20 mM, inhibited Mg(2+)-ATP hydrolysis by 36% and 55% in digestive gland, respectively. The presence of nucleotide-metabolizing enzymes in these tissues may be important for the modulation of nucleotide and nucleoside levels, controlling their actions on specific purinoceptors in these species.

Effects of starvation on haemolymphatic glucose levels, glycogen contents and nucleotidase activities in different tissues of Helix aspersa (Müller, 1774) (Mollusca, Gastropoda).

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.

Different sensitivity of Ca(2+)-ATPase and cholinesterase to pure and commercial pesticides in nervous ganglia of Phyllocaulis soleiformis (Mollusca).

We measured the effects in vitro of pure and commercial pesticides on Ca(2+)-activated ATPase and cholinesterase (ChE) activities in the nervous system of the slug Phyllocaulis soleiformis. The pesticides used in this study included carbamate and organophosphates, which acts as reversible and irreversible anticholinesterases, respectively. Both enzymes were insensitive to pure carbofuran (1 mM), glyphosate (1 mM) and malathion (120 microM). However, the carbamate carbofuran, in the commercial formulation Furandan 350S, inhibited ATPase and ChE activities. The organophosphate glyphosate used in the commercial preparation of Gliz 480CS inhibited ATPase activity and increased cholinesterase activity. These effects are likely due to the action of adjuvant substances of the chemical formulation. The commercial formulation (Malatol 500CE) did not alter enzymes activities. Our results suggest that cholinesterase present in the slug nervous tissue has a different behavior to those identified in vertebrate nervous tissue, since it was insensitive to pure compounds, known as anticholinesterases in vertebrates. Considering the insensitivity of the Ca(2+)-activated ATPase, we suggested that the purinergic neurotransmission and other roles of ATP might not be affected by the pure pesticides tested.