Regulation of extracellular adenosine levels, role of nucleoside transporters, purine metabolism and the blood-brain-barrier

Abstract

Adenosine is an important neuromodulator in brain and, through activation of membrane bound receptors, it has been reported to have neuroprotective properties during strokes or seizures. The levels of adenosine have been reported to increase up to 100-fold during cerebral ischemia; however, the pathways involved in regulating intra- and extracellular concentrations of adenosine are poorly characterized. This thesis work has investigated how nucleoside transporters, purinergic enzymes and the blood-brain barrier (BBB) are involved in the regulation of extracellular adenosine levels. The first study demonstrated that lumenal administration of the nucleoside transport inhibitor dipyridamole inhibited the movement of [14C]adenosine from the interstitial space into the lumen of a dynamic 'in vitro' model of the BBB without permeating the BBB. This demonstrates that nucleoside transport inhibitors may be able to regulate CNS adenosine levels even without entering the brain parenchyma. The next study demonstrated that activation of the adenosine A 1 receptor during ATP depleting conditions in DDT1 MF-2 cells increased the cellular release of [3H]adenosine via a PKC-dependent pathway. These results demonstrate the autoregulatory effects of adenosine receptor activation on adenosine production. In a third study, expression of the rENT1 nucleoside transporter in rat C6 glioma cells facilitated the cellular permeability of the adenosine kinase inhibitor iodotubercidin. This demonstrates that the site selective effects of adenosine kinase inhibitors may be determined by nucleoside transporter distribution 'in vivo'. The rat C6 glioma cells were also used to demonstrate release of [ 3H]hypoxanthine but not [3H]adenosine during ATP depleting conditions. This result indicates that astrocytes may be more important as a site of salvage, rather than a source, of adenosine during conditions such as hypoxia/ischemia. To investigate this further, primary rat cortical astrocytes and neurons were subjected to hypoxia, iodoacetate or sodium cyanide to deplete the cells of ATP. The results indicate that adenosine is produced by an intracellular pathway in neurons and by an extracellular pathway in astrocytes. The overall conclusion of this thesis work is that the regulation of extracellular adenosine level is complex and varies among cell types.