Silvana Soriano Frassetto

ATP diphosphohydrolase activity (apyrase, EC 3.6.1.5) in human blood platelets

Human platelets contain an ATP diphosphohydrolase activity (apyrase, EC 3.6.1.5) that is Ca(2+) dependent, hydrolyses ATP and ADP and also GTP, ITP, CTP, GDP, IDP, CDP. The enzyme does not hydrolyse AMP, p-nitrophenylphosphate, inorganic phosphate or glucose-6-phosphate. Contaminant activities were ruled out because the enzyme was not inhibited by 2 μg/d ouabain, 1.0 μM levamisole, 10 μM ApSA or 1.0 mM azide. The enzyme was sensitive to 100 μM orthovanadate, 100μMApSA and 10 mM azide, reagents that have been described as inhibitors of some other apyrases. A strong inhibition by 1.0 mM NEM was observed, indicating that sulphydryl groups are involved in the enzyme activity. The parallel behaviour of ATPase and ADPase activities and the competition plot presented suggest that ATP and ADP hydrolysis occurs at the same active site. ATP diphosphohydrolase from human platelets may be involved in the modulation of nucleotide concentration in the circulation and thus in vascular tonus.

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 Ca2+-ATP and Ca2+-ADP as substrates. Ca2+-ATP and Ca2+-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 Ca2+-ATPase and Ca2+-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.

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.

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 (H2O2/Fe2+/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.

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 (H202 / 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 diphosphohydrotase activity from platelets to free radicals and suggest that amino acid residues which are essential for the enzyme function are probably modified.

Ischemic preconditioning reduces peripheral oxidative damage associated with brain ischemia in rats

Brain ischemia followed by reperfusion causes neuronal death related to oxidative damage. Furthermore, it has been reported that subjects suffering from ischemic cerebrovascular disorders exhibit changes in circulating platelet aggregation, a characteristic that might be important for their clinical outcome. In the present investigation we studied tert-butyl hydroperoxide-initiated plasma chemiluminescence and thiol content as measures of peripheral oxidative damage in naive and preconditioned rats submitted to forebrain ischemia produced by the 4-vessel occlusion method. Rats were submitted to 2 or 10 min of global transient forebrain ischemia followed by 60 min or 1, 2, 5, 10 or 30 days of reperfusion. Preconditioned rats were submitted to a 10-min ischemic episode 1 day after a 2-min ischemic event (2 + 10 min), followed by 60 min or 1 or 2 days of reperfusion. It has been demonstrated that such preconditioning protects against neuronal death in rats and gerbils submitted to a lethal (10 min) ischemic episode. The results show that both 2 and 10 min of ischemia cause an increase of plasma chemiluminescence when compared to control and sham rats. In the 2-min ischemic group, the effect was not present after reperfusion. In the 10-min ischemic group, the increase was present up to 1 day after recirculation and values returned to control levels after 2 days. However, rats preconditioned to ischemia (2 + 10 min) and reperfusion showed no differences in plasma chemiluminescence when compared to controls. We also analyzed plasma thiol content since it has been described that sulfhydryl (SH) groups significantly contribute to the antioxidant capacity of plasma. There was a significant decrease of plasma thiol content after 2, 10 and 2 + 10 min of ischemia followed by reperfusion when compared to controls. We conclude that ischemia may cause, along with brain oxidative damage and cell death, a peripheral oxidative damage that is reduced by the preconditioning phenomenon.