Caffeine regulates GABA transport via AR blockade and cAMP signaling.

Caffeine is the most consumed psychostimulant drug in the world, acting as a non-selective antagonist of adenosine receptors AR and AR, which are widely expressed in retinal layers. We have previously shown that caffeine, when administered acutely, acts on AR to potentiate the NMDA receptor-induced GABA release. Now we asked if long-term caffeine exposure also modifies GABA uptake in the avian retina and which mechanisms are involved in this process. Chicken embryos aged E11 were injected with a single dose of caffeine (30 mg/kg) in the air chamber. Retinas were dissected on E15 for ex vivo neurochemical assays. Our results showed that [H]-GABA uptake was dependent on Na and blocked at 4 °C or by NO-711 and caffeine. This decrease was observed after 60 min of [H]-GABA uptake assay at E15, which is accompanied by an increase in [H]-GABA release. Caffeine increased the protein levels of AR without altering ADORA1 mRNA and was devoid of effects on AR density or ADORA2A mRNA levels. The decrease of GABA uptake promoted by caffeine was reverted by AR activation with N6-cyclohexyl adenosine (CHA) but not by AR activation with CGS 21680. Caffeine exposure increased cAMP levels and GAT-1 protein levels, which was evenly expressed between E11-E15. As expected, we observed an increase of GABA containing amacrine cells and processes in the IPL, also, cAMP pathway blockage by H-89 decreased caffeine mediated [H]-GABA uptake. Our data support the idea that chronic injection of caffeine alters GABA transport via AR during retinal development and that the cAMP/PKA pathway plays an important role in the regulation of GAT-1 function.