Anales RANF

S16-05 THE SELECTIVE STIMULATION OF ADENOSINE A 3 RECEPTOR INHIBITS N-TYPE CA 2+ CURRENTS IN RAT DORSAL ROOT GANGLION NEURONS F. Cherchi 1 , E. Coppi 1 , I. Fusco 1 , M. Scortchini 2 , K.A. Jacobson 2 , F. Pedata 1 and A.M. Pugliese 1 . 1 University of Florence, Florence, Italy; 2 National Institutes of Health, Bethesda, Maryland, United States. Introduction: Interest has been focused in recent years on the analgesic effects exerted by adenosine and its receptors, A 1 , A 2A , A 2B and A 3 (AR, adenosine receptor) subtypes, in different in vivo models of chronic pain. We recently demonstrated that selective A 3 AR agonists block pro-nociceptive N-type Ca 2+ calcium channels in dorsal root ganglion (DRG) neurons isolated from rats (Coppi et al. 2019, Pain, 160(5):1103-18). In the present study, we investigate the effect of the first reported irreversibly binding A 3 AR agonist, ICBM, on N-type Ca 2+ currents in rat DRG neurons. Material and methods : Whole-cell patch-clamp recordings from primary DRG neurons isolated from Sprague Dawley rats were performed as described (Coppi et al., 2019). Results: The A 3 AR agonist ICBM, which contains a reactive isothiocyanate group, significantly inhibited N-type Ca 2+ currents. The effect was concentration dependent (0.01 - 3 μM), with an EC 50 of 22.9 nM, and blocked by the selective A 3 AR antagonist MRS1523 (100 nM). In order to demonstrate whether ICBM acts as an irreversible ligand on A 3 AR, in a subsequent experimental series we pre-incubated (10 minutes) DRG neurons with 100 nM ICBM or Cl-IB-MECA, the most used A 3 AR agonist, and then we washed out the A 3 AR agonist for 15 min, before applying 30 nM Cl-IB-MECA again to test its ability to inhibit Ca 2+ currents. ICBM pre-incubated cells maintained a prolonged suppression of Ca 2+ currents, and no further effect of Cl-IB-MECA on these currents was detected. In contrast, after Cl-IB-MECA pre-incubation, the ability to inhibit Ca 2+ currents upon a subsequent Cl-IB-MECA application was restored. Conclusions: Present data demonstrate that ICBM, an A 3 AR agonist designed for covalent binding to the receptor, concentration-dependently inhibits N-type Ca 2+ currents in rat DRG neurons. This effect is irreversible and might represent an innovative, favourable strategy to achieve efficacious pain control upon selective A 3 AR activation.