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

Aim： To define the effect of adenosine A1 receptor （AIR） on delta opioid receptor （DOR）-mediated signal transduction. Methods： CHO cells stably expressing HA-tagged AIR 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 A1R agonist N6-cyclohexyladenosine （CHA） on DOR down-regulation was assessed using radioligand binding assay. Results： CHA I pmol/L time-dependently attenuated DOR agonist [D-Pen2.5]enkephalin （DPDPE）-induced inhibition of intracellular cAMP accumulation with a t1/2=2.56 （2.09-3.31） h. Pretreatment with I pmol/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 ECso but no change in Emax. Pretreatment with I pmol/L CHA for I 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 A1R 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 （Kd） nor receptor density （Bmax） of DOR showed significant change after chronic CHA exposure. Conclusion： Activation of A1R by its agonist caused heterologous desensitization of DOR-mediated inhibition of intracellular cAMP accumulation and phosphorylation of Akt. Activation of A1R by its agonist also induced heterologous phosphorylation but not downregulation of DOR.