Biomedical subjects

R D Dias

Publications and source records attributed to R D Dias.

At least 19 recordsLinked to original sources

PubMed2001

Characterisation of an ATP diphosphohydrolase (Apyrase, EC 3.6.1.5) activity in Trichomonas vaginalis.

In the present report the enzymatic properties of an ATP diphosphohydrolase (apyrase, EC 3.6.1.5) in Trichomonas vaginalis were determined. The enzyme hydrolyses purine and pyrimidine nucleoside 5'-di- and 5'-triphosphates in an optimum pH range of 6.0--8.0. It is Ca(2+)-dependent and is insensitive to classical ATPase inhibitors, such as ouabain (1 mM), N-ethylmaleimide (0.1 mM), orthovanadate (0.1 mM) and sodium azide (5 mM). A significant inhibition of ADP hydrolysis (37%) was observed in the presence of 20 mM sodium azide, an inhibitor of ATP diphosphohydrolase. Levamisole, a specific inhibitor of alkaline phosphatase, and P(1), P(5)-di (adenosine 5'-) pentaphosphate, a specific inhibitor of adenylate kinase, did not inhibit the enzyme activity. The enzyme has apparent K(m) (Michaelis Constant) values of 49.2+/-2.8 and 49.9+/-10.4 microM and V(max) (maximum velocity) values of 49.4+/-7.1 and 48.3+/-6.9 nmol of inorganic phosphate x min(-1) x mg of protein(-1) for ATP and ADP, respectively. The parallel behaviour of ATPase and ADPase activities and the competition plot suggest that ATP and ADP hydrolysis occur at the same active site. The presence of an ATP diphosphohydrolase activity in T. vaginalis may be important for the modulation of nucleotide concentration in the extracellular space, protecting the parasite from the cytolytic effects of the nucleotides, mainly ATP.

Adenosine Diphosphate↗

PubMed2000

Brain ischemia alters platelet ATP diphosphohydrolase and 5'-nucleotidase activities in naive and preconditioned rats.

The effects of transient forebrain ischemia, reperfusion and ischemic preconditioning on rat blood platelet ATP diphosphohydrolase and 5'-nucleotidase activities were evaluated. Adult Wistar rats were submitted to 2 or 10 min of single ischemic episodes, or to 10 min of ischemia 1 day after a 2-min ischemic episode (ischemic preconditioning) by the four-vessel occlusion method. Rats submitted to single ischemic insults were reperfused for 60 min and for 1, 2, 5, 10 and 30 days after ischemia; preconditioned rats were reperfused for 60 min 1 and 2 days after the long ischemic episode. Brain ischemia (2 or 10 min) inhibited ATP and ADP hydrolysis by platelet ATP diphosphohydrolase. On the other hand, AMP hydrolysis by 5'-nucleotidase was increased after 2, but not 10, min of ischemia. Ischemic preconditioning followed by 10 min of ischemia caused activation of both enzymes. Variable periods of reperfusion distinctly affected each experimental group. Enzyme activities returned to control levels in the 2-min group. However, the decrease in ATP diphosphohydrolase activity was maintained up to 30 days of reperfusion after 10-min ischemia. 5'-Nucleotidase activity was decreased 60 min and 1 day following 10-min ischemia; interestingly, enzymatic activity was increased after 2 and 5 days of reperfusion, and returned to control levels after 10 days. Ischemic preconditioning cancelled the effects of 10-min ischemia on the enzymatic activities. These results indicate that brain ischemia and ischemic preconditioning induce peripheral effects on ecto-enzymes from rat platelets involved in nucleotide metabolism. Thus, ATP, ADP and AMP degradation and probably the generation of adenosine in the circulation may be altered, leading to regulation of microthrombus formation since ADP aggregates platelets and adenosine is an inhibitor of platelet aggregation.

5'-Nucleotidase↗

PubMed1999

Pre-conditioning to global cerebral ischemia changes hippocampal acetylcholinesterase in the rat.

This study shows the effect of transient global cerebral ischemia (ISC) on hippocampal acetylcholinesterase (AChE) activity. Naive adult Wistar rats received either a brief (2 min) or a long (10 min) ischemic episode by the four-vessel occlusion method. Pre-conditioned rats received double ischemia: a 10 min episode inflicted 24 h after a 2 min event, a condition known to confer cytoprotection to CA1 pyramidal cells of hippocampus. 2 min of ischemia caused an increase in acetylcholinesterase activity both immediately and 30 min after the episode, however enzyme activity was significantly decreased after 24 h of reperfusion. 10 min of ischemia caused an increase in activity both 60 min and 24 h after ischemia. Conversely, pre-conditioned rats displayed lower activity both immediately and 60 min after ischemia. Our results suggest that: a) neuronal death, that follows 10 min of ischemia, is associated to a late increase in acetylcholinesterase activity; b) pre-conditioning is related to diminished acetylcholinesterase activity. This is in agreement with previous evidence that acetylcholinesterase inhibition and maintenance of acetylcholine levels are beneficial for cell surviving after cerebral ischemia.

Acetylcholine↗

PubMed1998

In vitro effect of central nervous system active drugs on the ATPase-ADPase activity and acetylcholinesterase activity from cerebral cortex of adult rats.

1. The effect of several central nervous system active drugs was studied in vitro on ATPase-ADPase activity and acetylcholinesterase (AChE) activity from the cerebral cortex of adult rats. 2. Lithium (1.0-10.0 mM) had no effect on either ATPase-ADPase or acetylcholinesterase activity. 3. Imipramine (0.5-5.0 mM), desipramine (0.5-5.0 mM), amitriptyline (0.1-1.0 mM) and diazepam (0.5-2.0 mM) inhibited ATP and ADP hydrolysis at all concentrations tested. 4. AChE activity was altered by imipramine (1.0-2.0 mM) and by diazepam (0.5-2.0 mM). 5. The possible participation of ATP diphosphohydrolase and AChE in the action of these drugs cannot be ruled out. The probable reduction of ATP, ADP and acetylcholine hydrolysis by the inhibitory effect of these drugs is discussed.

Acetylcholinesterase↗

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↗

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↗

PubMed1997

Free radical-induced inhibition of ATP diphosphohydrolase activity (EC 3.6.1.5) from rat blood platelets.

In the present report we demonstrate the in vitro effects of free radicals on an ecto-ATP diphosphohydrolase activity (apyrase, EC 3.6.1.5) from rat blood platelets. Rat blood platelets were exposed to an oxidant-generating system (H2O2/Fe2+/ascorbate) and the ATP diphosphohydrolase activity was inhibited. The enzyme inhibition was prevented by glutathione (GSH) and cysteine but not by trolox as a vitamin E analogue. The TBARS (thiobarbituric acid reactive substances) assay and the determination of sulphydryl groups indicate that the inhibition of the enzyme activity in resting platelets is not related to lipid peroxidation or to oxidation of sulphydryl residues. These results demonstrate the susceptibility of ATP diphosphohydrolase activity from platelets to free radicals and suggest that amino acid residues which are essential for the enzyme function are probably modified.

Animals↗

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

Effects of aluminum chloride on the kinetics of rat cortex synaptosomal ATP diphosphohydrolase (EC 3.6.1.5).

Aluminum chloride (AlCl3), a neurotoxic compound, inhibited ATP diphosphohydrolase activity of synaptosomes obtained from cerebral cortex of adult rats. The metal ion significantly inhibited ATPase and ADPase activities of the enzyme at all concentrations tested in vitro (0.01, 0.05, 0.5, 5, and 10 mM) in the presence of 1.5 mM calcium. When tested in the absence of Ca2+, and with increasing amounts of Al3+, enzyme activity remained below basal levels, suggesting that the trivalent cation Al3+ is not a substitute for the divalent cation Ca2+ in ATP-Ca2+ and ADP-Ca2+ complexes. The Al3+ inhibition was competitive with respect to Ca2+. The enzyme inhibition was reversed by the addition of deferoxamine (DFO). NaF significantly inhibited ATP diphosphohydrolase activity, and this inhibition was reversed by the addition of Ca2+ to the medium. Such inhibition was not potentiated by AlF4, which is an inhibitor of cation-transport ATPases.

Adenosine Triphosphatases↗

PubMed1995

Inhibition and kinetic alterations by excess free ATP and ADP of the ATP diphosphohydrolase activity (EC 3.6.1.5) from rat blood platelets.

ATP diphosphohydrolase (EC 3.6.1.5) catalyzes the hydrolysis of diphospho- and triphosphonucleosides and is activated by divalent cations. The enzyme described in rat blood platelets hydrolyzes Ca(2+)-ATP and Ca(2+)-ADP with a high affinity for these Ca(2+)-nucleotide complexes as substrates. In the present paper, we demonstrate that free ATP or free ADP induces inhibition and kinetic alterations of the enzyme from rat blood platelets. From these results, we draw conclusions about the binding of free nucleotides to the enzyme and their action as inhibitors with respect to calcium-nucleotide complex.

Adenosine Diphosphate↗

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↗

PubMed1994

Activity of synaptosomal ATP diphosphohydrolase from hippocampus of rats tolerant to forebrain ischemia.

Cerebral ischemia causes cell death of vulnerable neurons in mammalian brain. Wistar adult rats (male and female, weighing 180-280 g) were submitted to 2 min, 10 min, or to 2 and 10 min (separated by a 24-h interval) of transient forebrain ischemia by the four-vessel occlusion method. Animals subjected to the longer ischemic episodes had massive necrosis of pyramidal CA1 cells of the hippocampus, while animals receiving double ischemia (2 + 10 min) showed neuronal tolerance to the ischemic insult. ATP-diphosphohydrolase activity from hippocampal synaptosomes was assayed in these three groups (N = 6 animals/group) under two conditions: no reperfusion and 5-min of reperfusion. The control values for ATPase and ADPase activities were 144.7 +/- 18.8 and 60.6 +/- 5.24 nmol Pi min-1 mg protein-1, respectively. The 10-min group without reperfusion showed an enhancement of approximately 20% for ATPase and ADPase activities. In reperfused rats, only the 2-min group had a 20% increase in both enzymatic activities. We suggest that modulation of ATP-diphosphohydrolase activity might be involved in molecular events that follow both ischemia and reperfusion.

Adenosine Triphosphatases↗

PubMed1993

Characterization of an ATP diphosphohydrolase activity (APYRASE, EC 3.6.1.5) in rat blood platelets.

In the present report we describe an apyrase (ATP diphosphohydrolase, EC 3.6.1.5) in rat blood platelets. The enzyme hydrolyses almost identically quite different nucleoside di- and triphosphates. The calcium dependence and pH requirement were the same for the hydrolysis of ATP and ADP and the apparent Km values were similar for both Ca(2+)-ATP and Ca(2+)-ADP as substrates. Ca(2+)-ATP and Ca(2+)-ADP hydrolysis could not be attributed to the combined action of different enzymes because adenylate kinase, inorganic pyrophosphatase and nonspecific phosphatases were not detected under our assay conditions. The Ca(2+)-ATPase and Ca(2+)-ADPase activity was insensitive to ATPase, adenylate kinase and alkaline phosphatase classical inhibitors, thus excluding these enzymes as contaminants. The results demonstrate that rat blood platelets contain an ATP diphosphohydrolase involved in the hydrolysis of ATP and ADP which are vasoactive and platelet active adenine nucleotides.

Adenosine↗

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↗

PubMed1992

Undernutrition during suckling has no effect on the rat locomotor activity response to caffeine.

It is known that early malnutrition causes hyposensitivity to serotonergic, gabaergic, catecholaminergic and opioid stimulation. In the present study, we determined whether adult rats undernourished during suckling presented an altered response to caffeine administration in a locomotor activity test. Rats were undernourished during suckling by feeding their dams a 7% casein diet. During the same period, well-nourished dams were fed a 28% casein diet. Animals (90-100 days of age) were habituated to the apparatus. Thereafter, a dose-response curve for caffeine (2.5, 10.0, 20.0, 40.0 and 120.0 mumol/kg, ip) was determined. During handling sessions, undernourished rats presented lower activity scores than well-nourished animals (average values: 44.2 +/- 16.4 vs 57.9 +/- 15.4). Well-nourished and undernourished rats responded in a similar way to caffeine administration by increasing the locomotor activity in a dose-dependent manner. Although undernourished animals present an altered sensitivity to various neuropharmacological compounds, the present results indicate that their sensitivity to the locomotor-activating effect of caffeine is the same as that of rats well-nourished during suckling.

Animals↗

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↗