Selective A3AR Agonists and Channel Blockers

2-Chloro-N⁶-(3-iodobenzyl)-adenosine-5′-N-methyluronamide (Cl-IB-MECA; Merck Life Science S.r.l.) was used as a selective A₃AR agonist. N⁶-(3-Isothiocyanatobenzyl)-5′-N-methylcarboxamidoadenosine (ICBM) was synthesized as reported [40 (link)] and was used as a selective A₃AR agonist. Stock solutions of ICBM in DMSO were prepared and stored as small aliquots at − 20 °C and warmed to RT immediately before use, to avoid decomposition associated with freeze–thaw cycles [41 ]. The K_i values of this compound were described in rat cloned A₁AR, A_2AAR and A₃AR stably transfected in CHO cells (K_i values are 145, 272, and 10.0 nM, respectively).
Tetrodotoxin (TTX; Tocris, Bio-Techne S.r.l., Bristol, UK) was used to block Na⁺ channels. 5-(4-butoxy-3-chlorophenyl)-N-[[2-(4-morpholinyl)-3-pyridinyl]methyl]-3-pyridinecarboxamide (A887826: Merck Life Science S.r.l.) was used to block TTX-insensitive Na⁺ channels (Nav1.5; Nav1.8; Nav1.9). 3-Propyl-6-ethyl-5-[(ethylthio)carbonyl]-2-phenyl-4-propyl-3-pyridinecarboxylate (MRS1523; Sigma-Aldrich, St. Louis, MO, USA) was used as a selective A₃AR antagonist. 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX; Merck Life Science S.r.l.) was used as a selective A₁AR antagonist. DPCPX was added to all electrophysiological solutions to prevent A₁AR activation [32 (link)].