Fiona E. Parkinson

ATP is a gliotransmitter released from astrocytes. Extracellularly, ATP is metabolized by a series of enzymes, including ecto-5′-nucleotidase (eN; also known as CD73) which is encoded by the gene 5NTE and functions to form adenosine (ADO) from adenosine monophosphate (AMP). Under ischemic conditions, ADO levels in brain increase up to 100-fold. We used astrocytes cultured from 5NTE ^+/+ or 5NTE ^−/− mice to evaluate the role of eN expressed by astrocytes in the production of ADO and inosine (INO) in response to glucose deprivation (GD) or oxygen-glucose deprivation (OGD). We also used co-cultures of these astrocytes with wild-type neurons to evaluate the role of eN expressed by astrocytes in the production of ADO and INO in response to GD, OGD, or N-methyl-d-aspartate (NMDA) treatment. As expected, astrocytes from 5NTE ^+/+ mice produced adenosine from AMP; the eN inhibitor α,β-methylene ADP (AOPCP) decreased ADO formation. In contrast, little ADO was formed by astrocytes from 5NTE ^−/− mice and AOPCP had no significant effect. GD and OGD treatment of 5NTE ^+/+ astrocytes and 5NTE ^+/+ astrocyte-neuron co-cultures produced extracellular ADO levels that were inhibited by AOPCP. In contrast, these conditions did not evoke ADO production in cultures containing 5NTE ^−/− astrocytes. NMDA treatment produced similar increases in ADO in both 5NTE ^+/+ and 5NTE ^−/− astrocyte-neuron co-cultures; dipyridamole (DPR) but not AOPCP inhibited ADO production. These results indicate that eN is prominent in the formation of ADO from astrocytes but in astrocyte-neuron co-cultures, other enzymes or pathways contribute to rising ADO levels in ischemia-like conditions.

Adenosine is a neuromodulator with its level increasing up to 100-fold during ischemic events, and attenuates the excitotoxic neuronal injury. Adenosine is produced both intracellularly and extracellularly, and nucleoside transport proteins transfer adenosine across plasma membranes. Adenosine levels and receptor-mediated effects of adenosine are regulated by intracellular ATP consumption, cellular release of ATP, metabolism of extracellular ATP (and other adenine nucleotides), adenosine influx, adenosine efflux and adenosine metabolism. Recent studies have used genetically modified mice to investigate the relative contributions of intra- and extracellular pathways for adenosine formation. The importance of cortical or hippocampal neurons as a source or a sink of adenosine under basal and hypoxic/ischemic conditions was addressed through the use of transgenic mice expressing human equilibrative nucleoside transporter 1 (hENT1) under the control of a promoter for neuron-specific enolase. From these studies, we conclude that ATP consumption within neurons is the primary source of adenosine in neuronal cultures, but not in hippocampal slices or in vivo mice exposed to ischemic conditions.