Title: Adenosine A3 receptor activation inhibits pro-nociceptive N-type Ca2+ currents and cell excitability in dorsal root ganglion neurons.

Recently, studies have focused on the antihyperalgesic activity of the A(3) adenosine receptor (A(3)AR) in several chronic pain models, but the cellular and molecular basis of this effect is still unknown. Here, we investigated the expression and functional effects of A(3)AR on the excitability of small-to medium-sized, capsaicin-sensitive, dorsal root ganglion (DRG) neurons isolated from 3- to 4-week-old rats. Real-time quantitative polymerase chain reaction experiments and immunofluorescence analysis revealed A(3)AR expression in DRG neurons. Patch-clamp experiments demonstrated that 2 distinct A(3)AR agonists, Cl-IB-MECA and the highly selective MRS5980, inhibited Ca2+-activated K+ (K-Ca) currents evoked by a voltage-ramp protocol. This effect was dependent on a reduction in Ca2+ influx via N-type voltage-dependent Ca2+ channels, as Cl-IB-MECA-induced inhibition was sensitive to the N-type blocker PD173212 but not to the L-type blocker, lacidipine. The endogenous agonist adenosine also reduced N-type Ca2+ currents, and its effect was inhibited by 56% in the presence of A(3)AR antagonist MRS1523, demonstrating that the majority of adenosine's effect is mediated by this receptor subtype. Current-clamp recordings demonstrated that neuronal firing of rat DRG neurons was also significantly reduced by A(3)AR activation in a MRS1523-sensitive but PD173212-insensitive manner. Intracellular Ca2+ measurements confirmed the inhibitory role of A(3)AR on DRG neuronal firing. We conclude that pain-relieving effects observed on A(3)AR activation could be mediated through N-type Ca2+ channel block and action potential inhibition as independent mechanisms in isolated rat DRG neurons. These findings support A(3)AR-based therapy as a viable approach to alleviate pain in different pathologies.