Adenosine A1 receptor agonist N6-cyclohexyladenosine induced phosphorylation of delta oploid receptor and desensitization of its signaling opioid

Aim: To define the effect of adenosine A(1) receptor (A(1)R) on delta opioid receptor (DOR)-mediated signal transduction. Methods: CHO cells stably expressing HA-tagged A(1)R and DOR-CFP fusion protein were used. The localization of receptors was observed using confocal microscope. DOR-mediated inhibition of adenylyl cyclase was measured using cyclic AMP assay. Western blots were employed to detect the phosphorylation of Akt and the DOR. The effect of A(1)R agonist N-6-cyclohexyladenosine (CHA) on DOR down-regulation was assessed using radioligand binding assay. Results: CHA 1 mu mol/L time-dependently attenuated DOR agonist [D-Pen(2,5)] enkephalin (DPDPE)-induced inhibition of intracellular cAMP accumulation with a t(1/2)=2.56 (2.09-3.31) h. Pretreatment with 1 mu mol/L CHA for 24 h caused a right shift of the dose-response curve of DPDPE-mediated inhibition of cAMP accumulation, with a significant increase in EC50 but no change in E-max. Pretreatment with 1 mu mol/L CHA for 1 h also induced a significant attenuation of DPDPE-stimulated phosphorylation of Akt. Moreover, CHA time-dependently phosphorylated DOR (Ser363), and this effect was inhibited by A(1)R antagonist 1,3-Dipropyl-8-cyclopentylxanthine (DPCPX) but not by DOR antagonist naloxone. However, CHA failed to produce the down-regulation of DOR, as neither receptor affinity (K-d) nor receptor density (B-max) of DOR showed significant change after chronic CHA exposure. Conclusion: Activation of A(1)R by its agonist caused heterologous desensitization of DOR-mediated inhibition of intracellular cAMP accumulation and phosphorylation of Akt. Activation of A(1)R by its agonist also induced heterologous phosphorylation but not down-regulation of DOR.