Molecular and Cellular PharmacologyStimulation of nucleoside efflux and inhibition of adenosine kinase by A1 adenosine receptor activation
Section snippets
Materials
[3H]Adenine was purchased from NEN Life Sciences. PMA, adenine, adenosine, UTP, histamine, and IAA were purchased from the Sigma Chemical Co. CGS 21680 (2-[p-(2-carboxyethyl) phenylethylamino]-5′-N-ethylcarboxamidoadenosine), DPCPX, NECA, CHA, and NBMPR were purchased from Research Biochemicals International. Chelerythrine chloride was purchased from Calbiochem. Dulbecco’s modified Eagle’s medium and fetal bovine serum were obtained from Gibco BRL.
Cell culture
DDT1 MF-2 smooth muscle cells were obtained
Results
DDT1 MF-2 smooth muscle cells, loaded with [3H]adenine, were subjected to glycolytic inhibition with IAA (5 mM) (Fig. 1). IAA increased tritium efflux by 63% (P < 0.01) over release into buffer alone. The es transport inhibitor NBMPR (1 μM) was used to determine whether the increase in tritium release was due to increased efflux of [3H]purine nucleosides. NBMPR significantly blocked the IAA-induced increase (P < 0.05). As both adenosine and its metabolite, inosine, are permeants of es
Discussion
The main findings of this study were that adenosine A1 receptor activation increased the efflux of [3H]purines from DDT1 MF-2 cells during glycolytic inhibition. Stimulation of PLC by histamine H1 and P2Y nucleotide receptor activation as well as direct PKC activation with PMA mimicked this increase in efflux. Adenosine kinase assays showed that CHA or PMA treatment inhibited adenosine metabolism, an effect that could elevate intracellular adenosine levels and enhance adenosine efflux.
Depleting
Acknowledgements
This work was supported by the Medical Research Council (MRC) of Canada. F. E. P. is an MRC/RPP Investigator. C. J. D. S. is the recipient of a studentship award from the Manitoba Health Research Council and the Natural Sciences and Engineering Council of Canada. P. N. S. is the recipient of a studentship award from the MRC.
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