Biomedical subjects

A M Battastini

Publications and source records attributed to A M Battastini.

At least 19 recordsLinked to original sources

PubMed2001

Effect of nitric oxide donors on extracellular ATP, ADP, and AMP catabolism in rat hippocampal synaptosomes.

Extracellular adenine nucleotides acting as signaling molecules are inactivated by hydrolysis catalyzed by ectonucleotidases. Adenosine triphosphate (ATP) diphosphohydrolase (apyrase, EC 3.6.1.5) and 5'-nucleotidase (EC 3.1.3.5) are involved in an enzymatic chain for the hydrolysis of ATP to adenosine in the synaptic cleft. In this study, we investigated the in vitro effect of nitric oxide (NO) donors on extracellular ATP, adenosine diphosphate (ADP), and adenosine monophosphate (AMP) catabolism in hippocampal synaptosomes of rats. We evaluated the effect of the incubation time on ATP, ADP, and AMP hydrolysis in the absence and in the presence of 1 mM sodium nitroprusside (SNP). The inhibitory effect of SNP increased with the incubation time and the maximal inhibition was observed after 180 min for both enzyme activities. The inhibition observed attained a maximum at 1 mM SNP for ATP, ADP, and AMP hydrolysis, with the enzyme activities being markedly reduced at this concentration of SNP. However, other NO donors tested, such as S-nitroso-N-acetyl-penicillamine and isosorbide dinitrate, did not affect the enzyme activities. The effect of the NO donor, SNP, on extracellular ATP and ADP catabolism was increased by the addition of the thiol glutathione but this effect was not observed on extracellular AMP catabolism. The results suggest that the increased production of NO could have a modulatory role on the ectonucleotidase activities.

Adenine Nucleotides↗

PubMed2001

Changes in cortical and hippocampal ectonucleotidase activities in mice lacking cellular prion protein.

Animals lacking cellular prion protein (PrP(c)) expression are more susceptible to seizures. Adenosine is an endogenous anticonvulsant agent and it levels in the synaptic cleft are regulated by ectonucleotidases. We evaluated ectonucleotidase activities in synaptosomes from hippocampus and cerebral cortex of adult PrP(c) null mice and wild-type mice (genetic background 129/Sv X C57BL/6J). There was an increase (47%) in adenosine triphosphate (ATP) hydrolysis in hippocampal synaptosomes of PrP(c) knockout mice as compared with the wild-type animals. In cortical synaptosomes, ATP hydrolysis was similar in both PrP(c) mice and controls. However, there was a significant decrease in adenosine diphosphate (ADP) hydrolysis in both hippocampal (-39%) and cortical (-25%) synaptosomes in PrP(c) null animals compared to wild-type mice. Changes in brain ectonucleotidases activities related to modifications in the PrP(c) expression may contribute, at least in part, to the higher sensitivity to seizures of PrP(c) null mice.

Adenosine Diphosphate↗

PubMed2001

Phenylalanine and phenylpyruvate inhibit ATP diphosphohydrolase from rat brain cortex.

The main objective of the present study was to characterize the inhibition by phenylalanine and phenylpyruvate of ATP diphosphohydrolase activity in synaptosomes from the brain cortex of rats. This enzyme participates together with a 5'-nucleotidase in adenosine formation from the neurotransmitter, ATP, in the synaptic cleft. The inhibition of ATP diphosphohydrolase was competitive for nucleotide hydrolysis but 5'-nucleotidase was not affected by these metabolites. Furthermore, the two substances inhibited enzyme activity by acting at the same binding site. If the enzyme inhibition observed in vitro also occurs in the brain of PKU patients, it may promote an increase in ATP levels in the synaptic cleft. In this case, the neurotoxicity of ATP could possibly be one of the mechanisms leading to the characteristic brain damage of phenylketonuria.

5'-Nucleotidase↗

PubMed2001

Chronic treatment with clozapine, but not haloperidol, increases striatal ecto-5'-nucleotidase activity in rats.

In the search for differential mechanisms underlying clozapine's superior antipsychotic efficacy, the purinergic system has been considered, since an antagonist of the adenosine receptor A(2A) was shown to block clozapine acute effects on c-fos expression in rat striatum. Further investigating the interaction of clozapine with the purinergic system, we studied the effects of chronic treatment (28 days, intraperitoneal) with clozapine (25 mg/kg) and haloperidol (1.5 mg/kg) on the activity of ectonucleotidases in the striatum and hippocampus of rats. Clozapine selectively increased striatal 5'-nucleotidase activity (22%) compared to control and haloperidol groups. In vitro, neither drug affected enzyme activities. These results reinforce the differential effects of clozapine compared to haloperidol on the purinergic system.

5'-Nucleotidase↗

PubMed2001

Ectonucleotidase activities in Sertoli cells from immature rats.

Sertoli cells have been shown to be targets for extracellular purines such as ATP and adenosine. These purines evoke responses in Sertoli cells through two subtypes of purinoreceptors, P2Y2 and P A1. The signals to purinoreceptors are usually terminated by the action of ectonucleotidases. To demonstrate these enzymatic activities, we cultured rat Sertoli cells for four days and then used them for different assays. ATP, ADP and AMP hydrolysis was estimated by measuring the Pi released using a colorimetric method. Adenosine deaminase activity (EC 3.5.4.4) was determined by HPLC. The cells were not disrupted after 40 min of incubation and the enzymatic activities were considered to be ectocellularly localized. ATP and ADP hydrolysis was markedly increased by the addition of divalent cations to the reaction medium. A competition plot demonstrated that only one enzymatic site is responsible for the hydrolysis of ATP and ADP. This result indicates that the enzyme that acts on the degradation of tri- and diphosphate nucleosides on the surface of Sertoli cells is a true ATP diphosphohydrolase (EC 3.6.1.5) (specific activities of 113 +/- 6 and 21 +/- 2 nmol Pi mg(-1) min(-1) for ATP and ADP, respectively). The ecto-5'-nucleotidase (EC 3.1.3.5) and ectoadenosine deaminase activities (specific activities of 32 +/- 2 nmol Pi mg(-1) min(-1) for AMP and 1.52 +/- 0.13 nmol adenosine mg(-1) min(-1), respectively) were shown to be able to terminate the effects of purines and may be relevant for the physiological control of extracellular levels of nucleotides and nucleosides inside the seminiferous tubules.

5'-Nucleotidase↗

PubMed2000

Learning-specific decrease in synaptosomal ATP diphosphohydrolase activity from hippocampus and entorhinal cortex of adult rats.

Considering the involvement of extracellular ATP in the memory formation, we analyzed the effect of inhibitory avoidance training on ectonucleotidase activities in synaptosomes from hippocampus, entorhinal cortex and parietal cortex. ATP diphosphohydrolase activity presented a decrease (33%) in hippocampal synaptosomes of rats sacrificed 180 min after training. Our results also showed a decrease in synaptosomal ATP diphosphohydrolase (30% and 42% for ATP and ADP, respectively) in entorhinal cortex immediately after training. These findings suggest an integrated action of ATP diphosphohydrolase from hippocampus and entorhinal cortex in the formation of inhibitory avoidance memory.

Animals↗

PubMed2000

Changes in synaptosomal ectonucleotidase activities in two rat models of temporal lobe epilepsy.

Adenosine has been proposed as an endogenous anticonvulsant which can play an important role in seizure initiation, propagation and arrest. Besides the release of adenosine per se, the ectonucleotidase pathway is an important metabolic source of extracellular adenosine. Here we evaluated ATP diphosphohydrolase and 5'-nucleotidase activities in synaptosomes from hippocampus and cerebral cortex at different periods after induction of status epilepticus (SE) by intraperitoneal administration of pilocarpine or kainate. Ectonucleotidase activities from synaptosomes of hippocampus and cerebral cortex of rats were significantly increased at 48-52 h, 7-9 days and 45-50 days after induction of SE by pilocarpine. In relation to kainate model, both hippocampal enzymes were enhanced at 7-9 days and 45-50 days, but only 5'-nucleotidase remained elevated at 100-110 days after the treatment. In cerebral cortex, an increase in ATP diphosphohydrolase was observed at 48-52 h, 7-9 days and 45-50 days after induction of SE by kainate. However, 5'-nucleotidase activity only presented significant changes at 45-50 and 100-110 days. Our results suggest that SE can induce late and prolonged changes in ectonucleotidases activities. The regulation of the ectonucleotidase pathway may play a modulatory role during the evolution of behavioral and pathophysiological changes related to temporal lobe epilepsy.

5'-Nucleotidase↗

PubMed2000

Effect of protein-modifying reagents on ecto-apyrase from rat brain.

We have tested several chemical modifiers to investigate which amino acid residues, present in the primary structure of the ecto-apyrase, could be involved in catalysis. Synaptosomes from cerebral cortex of rats were prepared and the ATP diphosphohydrolase activity was assayed in absence or the presence of the modifiers. Percentages of residual activity for ATPase and ADPase obtained when the following reagents were tested, are respectively: phenylglyoxal (an arginine group modifier) 17 and 30%; Woodward's reagent (a carboxylic group modifier) 33 and 23%; Koshland's reagent (a tryptophan group modifier) 10 and 12%; maleic anhidride (an amino group modifier) 11 and 25% and carbodiimide reagent (a carboxylic group modifier) 56 and 72%. Otherwise, PMSF, a seryl protein modifier and DTNB, a SH-group modifier did not affect either ATPase or ADPase activity. Inhibitions observed after treatment with phenylglyoxal and Woodward's reagent were significantly prevented when the synaptosomal fraction was preincubated with ATP and ADP, indicating that the arginine and the side chain of glutamate or aspartate (carboxyl groups) participate in the structure of the active site. This interpretation was confirmed by using GTP and GDP, two other apyrase substrates. Phenylglyoxal and Woodward's reagent also inhibited the GTPase and GDPase activities and this inhibition was prevented by preincubation with these substrates.

2-Hydroxy-5-nitrobenzyl Bromide↗

PubMed2000

Identification of brain ecto-apyrase as a phosphoprotein.

Ecto-apyrase is a transmembrane glycoprotein that hydrolyzes extracellular nucleoside tri- or diphosphates. Apyrase activity is affected by several physiological and pathological conditions indicating the existence of regulatory mechanisms. Considering that apyrase presents consensus phosphorylation sites, we studied the phosphorylation of this enzyme. We found an overlay of the immunoblotting and phosphorylated bands in three different preparations from rat brain: (a) hippocampal slices, (b) synaptic plasma membrane fragments and (c) cultured astrocytes. In addition, two-dimensional electrophoresis separations with human astrocytoma cells were done to identify unequivocally the coincidence between the immunodetected and phosphorylated protein. These observations indicate that apyrase can be detected as a phosphoprotein, with obvious implications in the regulation of this enzyme.

Animals↗

PubMed2000

Altered ATP hydrolysis induced by pentylenetetrazol kindling in rat brain synaptosomes.

The ectonucleotidase pathway is an important metabolic source of extracellular adenosine. Adenosine has potent anticonvulsant effects on various models of epilepsy. One of these models is pentylenetetrazol (PTZ) kindling, in which repeated administration of subconvulsive doses of this drug induces progressive intensification of seizure activity. In this study, we examine the effect of a single convulsive injection (60 mg/kg, i.p.) or 10 successive (35 mg/kg, i.p.) injections of PTZ on synaptosomal ectonucleotidases. Our results have shown that no changes in ectonucleotidase activities were seen at 0, 1, and 24 h or at 5 days after a single convulsive PTZ injection. However, after PTZ-kindling, rats which were more resistant to seizure development presented an increase in ATP hydrolysis in synaptosomes from hippocampus and cerebral cortex (44% and 28%, respectively). These results suggest that changes in nucleotide hydrolysis may represent an important mechanism in the modulation of chronic epileptic activity in this model.

Adenosine Triphosphate↗

PubMed1999

Effects of suramin on hippocampal apyrase activity and inhibitory avoidance learning of rats.

The action of suramin on apyrase activity in hippocampal synaptosomes and its effects on retention of inhibitory avoidance learning were evaluated. Suramin, a P2-purinoceptor antagonist, significantly inhibited in a noncompetitive manner the ATP and ADP hydrolysis promoted by apyrase in hippocampal synaptosomes of adult rats. The Ki values obtained were 72.8 and 109 microM for ATP and ADP hydrolysis, respectively. Intrahippocampal infusion of suramin (0.01, 0.1, 1, and 10 microg) immediately posttraining, in a dose-dependent effect, significantly reduced the response latency during the retention test applied 24 h after the rats received step-down inhibitory avoidance training. The amnesic effects promoted by suramin probably occur by its antagonist action on hippocampal P2-purinoceptors and NMDA receptors. In view of the fact that ATP-metabolizing enzymes and P2-purinoceptors have similar binding domains, these results suggest that suramin can either alter ATP degradation and/or block purinergic neurotransmission.

Adenosine Triphosphate↗

PubMed1998

Studies on the anchorage of ATP diphosphohydrolase in synaptic plasma membranes from rat brain.

ATP diphosphohydrolases are described as ecto-enzymes in several tissues. In the present study, synaptic plasma membrane (SPM) was exposed to a series of agents used to distinguish between peripheral (hydrophilic), G-PI-anchored and transmembrane-polypeptide-anchored membrane proteins. These procedures included: (a) nondetergent extraction, (b) Triton X-114 phase partitioning, (c) phosphatidylinositol-specific phospholipase C (PI-PLC) extraction and (d) protease incubation. In cases (a), (c) and (d) the SPM was incubated with different agents and the ATPase-ADPase activities and the protein concentration was determined in the original sample, in the pellet and in the supernatant obtained after 100,000 g centrifugation. In procedure (b), the SPM was solubilized in 1% triton X-114 and submitted to phase separation onto a sucrose cushion. The aqueous and detergent rich phases obtained by this treatment were assayed for ATPase-ADPase activities and protein determination. The results obtained suggest an intrinsic behaviour for ATP diphosphohydrolase since none of the nondetergent treatments was efficient in removing the enzyme from SPM. Moreover, ATPase and ADPase activities were recovered predominantly (> 50%) in the detergent-rich phase obtained by Triton X-114 partitioning. The enzyme was not released by PI-PLC or proteases. These results indicate that the enzyme is not a GPI-anchored protein, but is probably deeply anchored on the plasma membrane in agreement with the amino acid sequence of the enzyme recently published.

Animals↗

PubMed1998

Inhibitory avoidance learning inhibits ectonucleotidases activities in hippocampal synaptosomes of adult rats.

Several lines of evidence indicate that ATP may play an important role in Long-Term Potentiation. In this investigation we evaluated the effect of a memory task (step-down inhibitory avoidance) on the synaptosomal ecto-enzymes (ATP diphosphohydrolase and 5'-nucleotidase) involved in the degradation of ATP to adenosine. After the training session, a decrease in the ATPase (40%) and ADPase (29%) activities of ATP diphosphohydrolase as well as was a decrease in 5'-nucleotidase activity (31%) was observed in hippocampal synaptosomes of rats trained and killed immediately after training. In synaptosomes of rats killed 30 minutes after training, a decrease in ATPase activity (28%) was observed. In the test session, no significant changes were observed in the enzyme activities studied. These results provide new information about the activity of ecto-enzymes involved in nucleotide degradation and their possible participation in mechanisms of acquisition and modulation of memory processing.

5'-Nucleotidase↗

PubMed1998

Catabolism of Ap4A and Ap5A by rat brain synaptosomes.

Adenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) and adenosine 5',5"'-P1,P5-pentaphosphate (Ap5A) are stored in and released from rat brain synaptic terminals. In the present study we investigated the hydrolysis of dinucleotides (Ap4A and Ap5A) in synaptosomes from the cerebral cortex of adult rats. Ap4A and Ap5A, but not Ap3A, were hydrolyzed at pH 7.5 in the presence of 20 mM Tris/HCl, 2.0 mM MgCl2, 10 mM glucose and 225 mM sucrose at 37 degrees C. The disappearance of the substrates measured by FPLC on a mono-Q HR column was both time and protein dependent. Since synaptosome integrity was at least 90% at the end of the assay, hydrolysis probably occurred by the action of an ecto-enzyme. Extracellular actions of adenine dinucleotides at central nervous system terminate due to the existence of ecto-nucleotidases which specifically cleave these dinucleotides. These enzymes in association with an ATP diphosphohydrolase and a 5'-nucleotidase are able to promote the complete hydrolysis of dinucleotides to adenosine in the synaptic cleft.

Animals↗

PubMed1997

Effects of 9-amino-1,2,3,4-tetrahydroacridine (THA) on ATP diphosphohydrolase (EC 3.6.1.5) and 5'-nucleotidase (EC 3.1.3.5) from rat brain synaptosomes.

1. 9-Amino-1,2,3,4-tetrahydroacridine (THA), an acetylcholinesterase inhibitor, significantly inhibited in vitro the ATP diphosphohydrolase activity of synaptosomes from the cerebral cortex and hippocampus of adult rats. 2. THA did not inhibit in vitro the 5'-nucleotidase activity of synaptosomes from cerebral cortex and hippocampus of rats. 3. THA exerted an uncompetitive inhibition on ATP diphosphohydrolase activity. This mechanism of inhibition was the same in the 2 different synaptosomal fractions (cerebral cortex and hippocampus) studied. 4. THA, proposed as a drug for the treatment of Alzheimer's disease, can alter in vitro ATP degradation in synaptosomes from the central nervous system.

5'-Nucleotidase↗

PubMed1996

Sensitivity of ATPase-ADPase activities from synaptic plasma membranes of rat forebrain to lipid peroxidation in vitro and the protective effect of vitamin E.

The in vitro effects of membrane lipid peroxidation on ATPase-ADPase activities in synaptic plasma membranes from rat forebrain were investigated. Treatment of synaptic plasma membranes with an oxidant generating system (H(2)0(2)/Fe(2+)/ascorbate) resulted in lipid peroxidation and inhibition of the enzyme activity. Besides, trolox as a water soluble vitamin E analogue totally prevented lipid peroxidation and the inhibition of enzyme activity. These results demonstrate the susceptibility of ATPase-ADPase activities of synaptic plasma membranes to free radicals and suggest that the protective effect against lipid peroxidation by trolox prevents the inhibition of enzyme activity. Thus, inhibition of ATPase-ADPase activities of synaptic plasma membranes in cerebral oxidative stress probably is related to lipid peroxidation in the brain.

Adenosine Triphosphatases↗

PubMed1995

Solubilization and characterization of an ATP diphosphohydrolase (EC 3.6.1.5) from rat brain synaptic plasma membranes.

The effect of different detergents on the ATPase and ADPase activities from synaptic plasma membrane were investigated. Triton X-100, deoxycholate, CHAPS, Nonidet, N-octylglucoside and C12E8, which is commonly used to solubilize plasma membrane proteins, easily inactivated the ATPase and ADPase activities, while digitonin was not harmful to the enzyme. Treatment of the synaptic plasma membrane from rat brain with 0.5% digitonin solubilizes 80% of the proteins and 50% and 60% of ATPase and ADPase, respectively, with the following characteristics: stimulation by Ca2+ in the millimolar range, insensitivity to ATPase inhibitors (ouabain, olygomicyn, orthovanadate), inhibition with sodium azide and NEM and broad substrate specificity for the hydrolysis of nucleoside di- and triphosphate. To further characterize the enzyme solubilized, polyclonal antibodies specific for ATP diphosphohydrolase from potato tuber were tested. Western blot showed that two electrophoretic bands with a molecular mass close to 60-70 kDa had cross-immunoreactivity with antibodies against potato apyrase. The results presented here demonstrate for the first time the solubilization of ATPase and ADPase activities with characteristics of a true ATP diphosphohydrolase from synaptic plasma membrane from rat brain and with cross-immunoreactivity with antibodies against potato apyrase.

Animals↗

PubMed1994

Inhibitory effect of cadmium acetate on synaptosomal ATP diphosphohydrolase (EC 3.6.1.5; apyrase) from adult rat cerebral cortex.

ATP diphosphohydrolase (EC 3.6.1.5; apyrase) is an enzyme that can promote ATP and ADP hydrolysis to AMP plus inorganic phosphate and depends on divalent cations such as Ca2+ or Mg2+. In previous papers we described this enzyme in the synaptosomal fraction from the central and peripheral nervous system. The present report examines whether cadmium acetate could affect the in vitro activity of the enzyme in the synaptosomal fraction from the cerebral cortex of adult male Wistar rats. Cadmium (Cd2+), a heavy metal with neurotoxic effects, inhibited the enzyme in a concentration-dependent manner. All concentrations tested (0.05-1.0 mM) significantly inhibited the hydrolysis of both substrates (ATP and ADP), with the exception of 0.05 mM on ATP hydrolysis. The kinetic data indicate a noncompetitive inhibition between the cations Cd2+ and Ca2+.

Acetates↗