ABSTRACT
Title
ATP and adenosine release under physiological and ischemic conditions in the rat striatum in vivo
Authors
F. Corti
Doctorate School in Neuroscience
Dept. of Preclinical and Clinical Pharmacology, Section of Neuropharmacology - University of Florence, Italy.
Abstract
In the central nervous system (CNS), ATP and adenosine act on their own receptors behaving as transmitters and neuromodulators.The role of ATP as a neurotransmitter has been largely demonstrated both in the peripheral and the CNS [1]. Adenosine is considered one of the most important modulators of neurotransmission [2,3]. Despite a vast literature on the modalities of release of adenosine and ATP from CNS cells [1,3], it is still unknown which part of adenosine derives extracellularly from released ATP. Enzymes ecto-nucleotidases are present extracellularly, membrane bound and may account for ATP conversion to adenosine [4].
The aim of this study was to investigate the contribution of extracellular ATP to the adenosine formation under basal (i.e. physiological) and ischemic conditions in vivo by using a new inhibitor of ecto-ATPases, [TiW11CoO40]8- (PV4), which is the most potent inhibitor of different ecto-ATPase enzymes, recently synthesized by Muller and coworkers [5]. Moreover it was investigated the presence of the concentrative nucleoside transporter 2, CNT2, on subcellular compartments by immunoelectron microscopy.
Extracellular concentrations of adenosine and ATP were measured in rat striatal microdialysates by High Pressure Liquid Chromatography (HPLC) and by luciferine/luciferase assay respectively.
Under physiological conditions (i.e. when neurons spontaneously fire), in the presence of PV4, adenosine extracellular concentrations (mean value: ~140 nM, n=5) were not decreased respect to control values, indicating that adenosine is released “per se” from cells. ATP extracellular concentrations, in the presence of PV4, were increased by 12 fold (mean values from ~30 to ~360 nM, n=4-6). Therefore, although more than 90% ATP is hydrolyzed, it remains in the intermediate products ADP and AMP without further conversion to adenosine.
Under focal ischemia, induced by medial cerebral artery occlusion (MCAo), in the presence of PV4 extracellular ATP concentration increased from ~50 nM to ~450 nM, while adenosine decreased from ~690 nM (n=5) to ~275 nM (n=10). A time-related analysis of adenosine extracellular levels indicates that the contribution of extracellular ATP to extracellular adenosine was maximal in the first 20 min after ischemia.
In this study, the presence of the CNT2 nucleoside transporter was demonstrated, by immunoelectron microscopy, on plasma and vesicle membranes isolated from the rat striatum. These results suggest that adenosine is transported in vesicles and is released in an excitation-secretion manner under in vivo physiological conditions.
Early after ischemia, extracellular ATP hydrolyzed by ecto-nucleotidases might significantly contribute to an increase in extracellular adenosine. Degradation of ATP by ecto-nucleotidases after ischemia might represent an important mechanism in regulation of ATP and adenosine extracellular levels in the first hours after ischemia. In view of the important roles of extracellular adenosine and ATP in ischemia, such information is important in devising putative purinergic strategies aimed at protection from ischemic damage.
1) Burnstock G (2007) Physiol Rev. 87: 659.
2) Sebastiao AM and Ribeiro JA (2000) Trends Pharmacol. Sci. 21: 341.
3) Latini S and Pedata F (2001) J Neurochem. 79: 463.
4) Zimmermann H (2000). Naunyn Schmiedebergs Arch. Pharmacol. 362: 299.
5) Muller CE et al. (2006) Bioorg. Med Chem Lett. 16: 5943.
The aim of this study was to investigate the contribution of extracellular ATP to the adenosine formation under basal (i.e. physiological) and ischemic conditions in vivo by using a new inhibitor of ecto-ATPases, [TiW11CoO40]8- (PV4), which is the most potent inhibitor of different ecto-ATPase enzymes, recently synthesized by Muller and coworkers [5]. Moreover it was investigated the presence of the concentrative nucleoside transporter 2, CNT2, on subcellular compartments by immunoelectron microscopy.
Extracellular concentrations of adenosine and ATP were measured in rat striatal microdialysates by High Pressure Liquid Chromatography (HPLC) and by luciferine/luciferase assay respectively.
Under physiological conditions (i.e. when neurons spontaneously fire), in the presence of PV4, adenosine extracellular concentrations (mean value: ~140 nM, n=5) were not decreased respect to control values, indicating that adenosine is released “per se” from cells. ATP extracellular concentrations, in the presence of PV4, were increased by 12 fold (mean values from ~30 to ~360 nM, n=4-6). Therefore, although more than 90% ATP is hydrolyzed, it remains in the intermediate products ADP and AMP without further conversion to adenosine.
Under focal ischemia, induced by medial cerebral artery occlusion (MCAo), in the presence of PV4 extracellular ATP concentration increased from ~50 nM to ~450 nM, while adenosine decreased from ~690 nM (n=5) to ~275 nM (n=10). A time-related analysis of adenosine extracellular levels indicates that the contribution of extracellular ATP to extracellular adenosine was maximal in the first 20 min after ischemia.
In this study, the presence of the CNT2 nucleoside transporter was demonstrated, by immunoelectron microscopy, on plasma and vesicle membranes isolated from the rat striatum. These results suggest that adenosine is transported in vesicles and is released in an excitation-secretion manner under in vivo physiological conditions.
Early after ischemia, extracellular ATP hydrolyzed by ecto-nucleotidases might significantly contribute to an increase in extracellular adenosine. Degradation of ATP by ecto-nucleotidases after ischemia might represent an important mechanism in regulation of ATP and adenosine extracellular levels in the first hours after ischemia. In view of the important roles of extracellular adenosine and ATP in ischemia, such information is important in devising putative purinergic strategies aimed at protection from ischemic damage.
1) Burnstock G (2007) Physiol Rev. 87: 659.
2) Sebastiao AM and Ribeiro JA (2000) Trends Pharmacol. Sci. 21: 341.
3) Latini S and Pedata F (2001) J Neurochem. 79: 463.
4) Zimmermann H (2000). Naunyn Schmiedebergs Arch. Pharmacol. 362: 299.
5) Muller CE et al. (2006) Bioorg. Med Chem Lett. 16: 5943.