Astrocytes influence N-methyl-D-aspartate-evoked release of adenosine from neurons

Zamzow CR, Parkinson FE. Dept of Pharmacology, University of Manitoba, Winnipeg, MB, CANADA.

During a stroke, extracellular glutamate levels increase significantly and activate N-methyl-D-aspartate (NMDA) receptors causing neuronal death. The purine adenosine ( ADO) protects neurons in stroke models and NMDA receptor activation increases ADO in the CNS (Craig et al 1993, Lu et al 2003). Previous research from our laboratory has shown that cultured cortical neurons release ADO per se whereas astrocytes produce ADO through an extracellular pathway (Parkinson et al, 2004). It is not known what effect astrocytes have on NMDA-evoked ADO release from neurons. The aim of this study was to compare NMDA-evoked purine release from neurons cultured alone or in the presence of astrocytes.

P rimary cultures of Sprague Dawley E16 rat forebrain neurons or cultures containing both neurons and astrocytes were pre-loaded with 3Hadenine to radiolabel adenine nucleotides. Cells were treated with NMDA (100 µM) for 10 or 30 minutes at 37 ° C after which samples were collected and analyzed for ADO, inosine (INO), hypoxanthine (HX) and adenine nucleotides (AN). Data were analyzed with one-way ANOVA and Tukey’s post-hoc tests. Data expressed as mean ± SEM (n=5-6 per experiment).

ADO release from neurons was 0.294 ± 0.048 pmol/mg protein and was increased, by 29.3 ± 11.3%, with NMDA. The nucleoside transport inhibitor dipyridamole prevented NMDA-evoked ADO release whereas the ecto-5’-nucleotidase inhibitor alpha, beta-methylene ADP had no significant effect, indicating an intracellular source of ADO. In neuron cultures treated with NMDA, ADO, INO and HX accounted for 1 3.1 ± 2.3%, 48.7 ± 4.9%, and 7.2 ± 1.0%, respectively, of released purines. In co-cultures treated with NMDA, ADO, INO and HX accounted for 10.1 ± 1%, 24.4 ± 12.7%, and 52.9 ± 5.8%, respectively, of released purines.

Treatment with NMDA-evokes ADO release from neurons; however, most of the purine release is in the form of INO. In the presence of astrocytes, the predominant purine released is HX. This is likely a result of purine nucleoside phosphorylase activity, which converts INO to HX. These data indicate that purine nucleoside phosphorylase is present in astrocytes and has a major effect to reduce evoked-ADO release from mixed cultures.

Craig C.G. et al. (1993) J Neurochem 60, 1073-1080.
Lu Y. et al. (2003) Eur J Neurosci 17:1213-1222.
Parkinson F. E. et al. (2004) J Neurochem 88, 1305-1312.