Biomedical subjects

A M Battastini

Publications and source records attributed to A M Battastini.

At least 19 recordsLinked to original sources

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↗

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↗

PubMed1993

Postnatal development of ATPase-ADPase activities in synaptosomal fraction from cerebral cortex of rats.

ATPase-ADPase activities in synaptosomes from cerebral cortex was measured in rats of various ages (0-, 7-, 10-, 14- and 21- and 60-90-days). The activities (nmol Pi/min/mg) increased steadily from birth, reaching maximum values at 21 days of age. The increase was primarily due to increases in Vmax; the Km values are the same from birth until adult age. The developmental profile was similar for ATPase-ADPase activities and acetylcholinesterase from the same fraction. Several specific ATPase inhibitors and Ap5A (P1P5-di(adenosine-5)-pentaphosphate) did not interfere with the hydrolysis of ATP and ADP at all ages studied, suggesting that classical ATPases and adenylate kinase were not involved in the degradation of both nucleotides by synaptosomal fraction in the assay conditions. Other phosphatases were also ruled out. It is conceivable that ATPase-ADPase activities play an important role in neurotransmitter metabolism.

Adenosine Triphosphatases↗

PubMed1991

Characterization of an ATP diphosphohydrolase (EC 3.6.1.5) in synaptosomes from cerebral cortex of adult rats.

Data from the literature have demonstrated that synaptosomal preparations from various sources can hydrolyze externally added ATP. Various authors characterized this activity as an ecto-ATPase. In the present report, we demonstrate that synaptosomal preparations obtained from the cerebral cortex of rats show ATPase activity that could not be dissociated from ADPase activity, suggesting that an ATP-diphosphohydrolase is involved in ATP and ADP hydrolysis. Furthermore, the ATP and ADP hydrolysis could not be attributed to associations of enzymes that could mimic an ATP-diphosphohydrolase because none of the following activities were detected in our assay conditions inorganic pyrophosphatase, adenylate kinase, or nonspecific phosphatases. A possible association between an ATPase and an ADPase was excluded on the basis of both the kinetics and much additional data on inhibitors, ion dependence, pH, etc. The present results demonstrate that in synaptosomal preparations from cerebral cortex an ATP-diphosphohydrolase is involved, at least in part, in ATP and ADP hydrolysis.

Adenosine Diphosphate↗

PubMed1991

Effects of undernutrition during suckling on ATP and ADP hydrolysis by synaptosomes from the cerebral cortex of adult rats.

1. Early undernutrition can cause permanent functional changes in the central nervous system. Alterations in enzymes involved in neurotransmitter metabolism have been reported to result from early undernutrition. 2. In a previous study, we demonstrated that undernutrition during suckling decreases ATP and ADP hydrolysis by synaptosomes from cerebral cortex by about 20% of the value found in 20-day-old well-nourished rats (J. B. T. Rocha, C. F. Mello, J. J. F. Sarkis and R. D. Dias, British Journal of Nutrition, 63:273-283, 1990). In the present study, we investigated whether this deficit persists in synaptosomes from cerebral cortex of nutritionally rehabilitated adult rats. 3. Rats were undernourished from birth to 25 days of life by feeding their dams a 7% casein (w/w) diet, while well-nourished offspring were fed by mothers maintained on a 28% casein diet. 4. In contrast to the results previously obtained in young rats, the synaptosomes obtained from the cerebral cortex of early undernourished adult rats hydrolyzed ATP and ADP more efficiently than did those obtained from well-nourished rats. Specific activity (nmol min-1 mg protein-1, mean +/- SD) was 114.9 +/- 9.5 for undernourished rats (N = 8) vs 94.1 +/- 9.5 for well-nourished rats (N = 8) for ATP, and 50.4 +/- 6.1 (N = 8) vs 38.8 +/- 4.5 (N = 8) for ADP. These results suggest that the deficits found in young rats disappear in rehabilitated adult rats.

Adenosine Diphosphate↗

PubMed1990

Ontogeny of ATP and ADP hydrolysis by cerebral cortex synaptosomes from rats.

In the present study, we examined the ontogeny of ATP and ADP hydrolysis by cerebral cortex synaptosomes from rats of various ages (0-, 7-, 14-, 21- and 60 to 90-day-old rats) in order to learn whether hydrolytic activity increases during the period of intense brain growth, as has been reported for other enzymes involved in neurotransmitter metabolism. The results demonstrate that ATP and ADP hydrolyzing activities increase in parallel from birth until the second postnatal week (about 4-fold), followed by a slight and statistically insignificant increase until the animal reaches adulthood. The maximum increase in nucleotide hydrolysis coincided with maximum brain growth, which may indicate a role for the enzyme in neurotransmission. Furthermore, the parallel development of both activities (ATPase and ADPase) strongly suggest that a single enzyme, an ATP diphosphohydrolase, is involved in ATP and ADP hydrolysis by the synaptosomal fraction.

Adenosine Diphosphate↗