Lippincott Williams & Wilkins, Inc., Philadelphia
Purine Uptake and Release in Rat C6 Glioma Cells
Nucleoside Transport and Purine Metabolism Under ATP-Depleting Conditions
Article first published online: 4 JAN 2002
Journal of Neurochemistry
Volume 75, Issue 4, pages 1528–1538, October 2000
How to Cite
Sinclair, C. J. D., LaRivière, C. G., Young, J. D., Cass, C. E., Baldwin, S. A. and Parkinson, F. E. (2000), Purine Uptake and Release in Rat C6 Glioma Cells. Journal of Neurochemistry, 75: 1528–1538. doi: 10.1046/j.1471-4159.2000.0751528.x
Abbreviations used: BCX-34, 2-amino-1,5-dihydro-7-(3-pyridinyl-methyl)-4H-pyrrolo[3,2-d]pyridin-4-one; DPR, dipyridamole; EHNA, erythro-9-(2-hydroxy-3-nonyl)adenine hydrochloride; ENT, equilibrative nucleoside transporter; IAA, iodoacetate; NaCN, sodium cyanide; NBMPR, nitrobenzylmercaptopurine riboside (nitrobenzylthioinosine); NMG, N-methylglucamine; PNP, purine nucleoside phosphorylase.
- Issue published online: 4 JAN 2002
- Article first published online: 4 JAN 2002
- Nucleoside transport;
- ATP depletion
Abstract: Adenosine, through activation of membrane-bound receptors, has been reported to have neuroprotective properties during strokes or seizures. The role of astrocytes in regulating brain interstitial adenosine levels has not been clearly defined. We have determined the nucleoside transporters present in rat C6 glioma cells. RT-PCR analysis, 3H-nucleoside uptake experiments, and [3H]nitrobenzylthioinosine ([3H]NBMPR) binding assays indicated that the primary functional nucleoside transporter in C6 cells was rENT2, an equilibrative nucleoside transporter (ENT) that is relatively insensitive to inhibition by NBMPR. [3H]Formycin B, a poorly metabolized nucleoside analogue, was used to investigate nucleoside release processes, and rENT2 transporters mediated [3H]formycin B release from these cells. Adenosine release was investigated by first loading cells with [3H]adenine to label adenine nucleotide pools. Tritium release was initiated by inhibiting glycolytic and oxidative ATP generation and thus depleting ATP levels. Our results indicate that during ATP-depleting conditions, AMP catabolism progressed via the reactions AMP IMP inosine hypoxanthine, which accounted for >90% of the evoked tritium release. It was surprising that adenosine was not released during ATP-depleting conditions unless AMP deaminase and adenosine deaminase were inhibited. Inosine release was enhanced by inhibition of purine nucleoside phosphorylase; ENT2 transporters mediated the release of adenosine or inosine. However, inhibition of AMP deaminase/adenosine deaminase or purine nucleoside phosphorylase during ATP depletion produced release of adenosine or inosine, respectively, via the rENT2 transporter. This indicates that C6 glioma cells possess primarily rENT2 nucleoside transporters that function in adenosine uptake but that intracellular metabolism prevents the release of adenosine from these cells even during ATP-depleting conditions.