Caffeine regulates GABA transport via A₁R blockade and cAMP signaling

Caffeine is the most consumed psychostimulant drug in the world, acting as a non-selective antagonist of adenosine receptors A₁R and A_2AR, which are widely expressed in retinal layers. We have previously shown that caffeine, when administered acutely, acts on A₁R 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 [³H]-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 [³H]-GABA uptake assay at E15, which is accompanied by an increase in [³H]-GABA release. Caffeine increased the protein levels of A₁R without altering ADORA1 mRNA and was devoid of effects on A_2AR density or ADORA2A mRNA levels. The decrease of GABA uptake promoted by caffeine was reverted by A₁R activation with N6-cyclohexyl adenosine (CHA) but not by A_2AR 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 [³H]-GABA uptake. Our data support the idea that chronic injection of caffeine alters GABA transport via A₁R during retinal development and that the cAMP/PKA pathway plays an important role in the regulation of GAT-1 function.