Abstract
Adenosine, through activation of its A1 receptors, has neuroprotective effects during hypoxia and ischemia. Recently, using transgenic mice with neuronal expression of human equilibrative nucleoside transporter 1 (hENT1), we reported that nucleoside transporter-mediated release of adenosine from neurons was not a key mechanism facilitating the actions of adenosine at A1 receptors during hypoxia/ischemia. The present study was performed to test the importance of CD73 (ecto-5′-nucleotidase) for basal and hypoxic/ischemic adenosine production. Hippocampal slice electrophysiology was performed with CD73+/+ and CD73−/− mice. Adenosine and ATP had similar inhibitory effects in both genotypes, with IC50 values of approximately 25 µM. In contrast, ATP was a less potent inhibitor (IC50 = 100 µM) in slices from mice expressing hENT1 in neurons. The inhibitory effects of ATP in CD73+/+ and CD73−/− slices were blocked by the adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) and were enhanced by the nucleoside transport inhibitor S-(4-nitrobenzyl)-6-thioinosine (NBTI), consistent with effects that are mediated by adenosine after metabolism of ATP. AMP showed a similar inhibitory effect to ATP and adenosine, indicating that the response to ATP was not mediated by P2 receptors. In comparing CD73−/− and CD73+/+ slices, hypoxia and oxygen-glucose deprivation produced similar depression of synaptic transmission in both genotypes. An inhibitor of tissue non-specific alkaline phosphatase (TNAP) was found to attenuate the inhibitory effects of AMP and ATP, increase basal synaptic activity and reduce responses to oxygen-glucose deprivation selectively in slices from CD73−/− mice. These results do not support an important role for CD73 in the formation of adenosine in the CA1 area of the hippocampus during basal, hypoxic or ischemic conditions, but instead point to TNAP as a potential source of extracellular adenosine when CD73 is absent.
Highlights
ATP and adenosine inhibit synaptic transmission in electrically stimulated hippocampal slices [1]
Primer set 2, which targets the region of the gene that was mutated to produce the knock out phenotype, amplified a 1 kb sequence from genomic DNA isolated from CD732/2 mice
The main findings were that (1) CD73 metabolizes AMP to adenosine in cortical membranes and hippocampal slices; (2) CD73 is not required for the inhibitory effects of ATP in hippocampal slices; and (3) CD73 is not required for the inhibitory effects of adenosine observed in hypoxia or OGD
Summary
ATP and adenosine inhibit synaptic transmission in electrically stimulated hippocampal slices [1]. Ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases; ecto-apyrases; CD39), ecto-nucleotide pyrophosphatase/phosphodiesterases (E-NPPs) and alkaline phosphatases metabolize ATP and ADP to AMP, whereas alkaline phosphatases and CD73 (ecto-59-nucleotidase; EC 3.1.3.5) can metabolize AMP to adenosine [4]. Inhibitors of these enzymes have modest efficacy to decrease the effects of ATP or AMP and can have inhibitory effects of their own [3,5,6,7]. It has been difficult to demonstrate conclusively that the inhibitory effects of exogenous adenine nucleotides result from their metabolism extracellularly to adenosine, in part, because their slow metabolism of variable efficacy is in contrast to their rapid inhibition of synaptic activity [2,7]
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