Cloning and pharmacological characterization of the equine adenosine A _2A receptor: A potential therapeutic target for the treatment of equine endotoxemia

The aim of the current study was to clone the equine adenosine A _2A receptor gene and to establish a heterologous expression system to ascertain its pharmacologic profile via radioligand binding and functional assays. An eA_2A-R expression construct was generated by ligation of the eA_2A cDNA into the pcDNA3.1 expression vector, and stably transfected into human embryonic kidney cells (HEK). Binding assays identified those clones expressing the eA_2A-R, and equilibrium saturation isotherm experiments were utilized to determine dissociation constants (K _D), and receptor densities (B_max) of selected clones. Equilibrium competition binding revealed a rank order of agonist potency of ATL > CV-1808 > NECA > 2-CADO > CGS21680, and a rank order of antagonist potency as ZM241385 > 8-phenyltheophylline > p-sulfophenyltheophylline > caffeine. Furthermore, adenylate cyclase assays using selective A _2A-R agonists revealed that the eA_2A-R functionally coupled to G_αs as indicated by an increase in intracellular [³H]cAMP upon receptor activation. Finally, NF-κB reporter gene assays revealed a CGS21680 concentration-dependent inhibition of NF-κB activity. These results indicate that the heterologously expressed eA _2A-R has a pharmacological profile similar to that of other mammalian A_2A receptors and thus can be utilized for further characterization of the eA_2A-R to ascertain whether it can serve as a suitable pharmacological target for equine inflammatory disease.