Resumo

Introduction and Objectives: Adenosine (Ado) is an important modulator of neuronal survival and differentiation in the CNS. Our previous work showed that Ado transporters are present in cultures of chick retinal cells, but little is known about the mechanisms regulating Ado transport in these cultures. Our aim in this work was to characterize and compare the Ado uptake systems in retinal mixed and purified glial cultures and to investigate their mechanisms of modulation. Methods: The uptake was measured after incubation of cultures with (3H)-Ado for 15 minutes and cell lysis to determine intracellular radioactivity. Results: Kinetic analysis of Ado uptake indicated the existence of 2 components, one saturable and one linear, in mixed or glial cultures, but the saturable component was much smaller in glial cultures. In order to investigate which is the main Ado transporter system in the cultures, we tested the effect of NBMPR, an inhibitor of equilibrative nucleoside transporters, and found a strong reduction of uptake. Preincubation of (3H)Ado with 2 Units/ml adenosine deaminase for 45 minutes before addition to cultures promoted a strong inhibition of uptake in mixed or glial cultures (64.2% ± 3.6, n=3 and 71.3% ± 2.0, n=3, respectively). Adenosine kinase inhibition with iodotubercidin (100 nM for mixed and 300 nM for glial cultures) also dramatically reduced the uptake in mixed or glial cultures (47.7% ± 1.8, n=3 and 41.7 ± 1.9, n=2, respectively), indicating the importance of this enzyme activity as a driving force in Ado uptake by retinal cells. Conclusions: This study showed that the equilibrative nucleoside transporter (ENT1) is the main transporter present in mixed neuronal/glial or purified cultures of retinal glial cells, and that this transporter has low affinity for inosine. Our results also show that besides ENT1 being a potential target to control Ado levels, adenosine kinase could be another target to regulate Ado levels in normal or pathological conditions. Financial Support: CAPES, CNPq, FAPERJ and PRONEX-MCT.