Abstract
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.
Highlights
ATP is the energy molecule of cells
To assay nucleoside release in response to ischemia-like conditions, cells were washed to remove extracellular [3H] adenine treated with buffer, glucose deprivation (GD), or oxygen-glucose deprivation (OGD) in the absence or presence of AOPCP (50 μM), to inhibit eN, or DPR (30 μM), to inhibit ENT1 and ENT2
For OGD treatment, cells were treated with 2DG containing buffer and were placed in a humidified chamber containing 95 % N2 and 5 % CO2 for 1 h at 37 °C
Summary
ATP is the energy molecule of cells. ATP levels fall during conditions characterized by reduced oxygen and glucose availability and/or increased cellular energy demand. Adenosine (ADO) is a metabolite of ATP and increased metabolism of intracellular ATP can directly increase intracellular ADO levels, which can be released from cells through equilibrative nucleoside transport proteins 1 and/or 2 (ENT1, ENT2) [1–3]. This ADO activates specific receptors of the G protein-coupled receptor family, principally A1 and A2A receptors [4]. In addition to its role in energy requiring intracellular processes, ATP is a signaling molecule and is released from diverse cell types, including astrocytes and neurons, in response to stimuli such as increases in cytosolic calcium levels [3, 5]. The final enzyme in this cascade is ecto-5′-nucleotidase (eN); the protein corresponding to this enzyme is known as CD73 whereas the gene is known as 5NTE [7]
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