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

Abstract

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 N6-cyclohexyladenosine (CHA) on DOR down-regulation was assessed using radioligand binding assay.Results:CHA 1 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 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 EC 50 but no change in E max. Pretreatment with 1 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 (Kd) 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. © 2010 CPS and SIMM All rights reserved.