Adenosine A(1) receptor agonist N(6)-cyclohexyl-adenosine induced phosphorylation of delta opioid receptor and desensitization of its signaling.
OBJECTIVE
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 micromol/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 micromol/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 micromol/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.


CONCLUSIONS
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.
Cheng
Yun
Y
Shanghai Institute of Materia Medica, Chinese Academy of Sciences, China.
Tao
Yi-min
YM
Sun
Jian-feng
JF
Wang
Yu-hua
YH
Xu
Xue-jun
XJ
Chen
Jie
J
Chi
Zhi-qiang
ZQ
Liu
Jing-gen
JG
D000241:Adenosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D058907:Adenosine A1 Receptor Agonists; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D000242:Cyclic AMP / Q000378:metabolism; D006207:Half-Life; D006801:Humans; D010766:Phosphorylation / Q000187:drug effects; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D011869:Radioligand Assay; D017465:Receptors, Opioid, delta / Q000187:drug effects* / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D013997:Time Factors
D058907:Adenosine A1 Receptor Agonists; D017465:Receptors, Opioid, delta; C027513:N(6)-cyclohexyladenosine; D000242:Cyclic AMP; D051057:Proto-Oncogene Proteins c-akt; D000241:Adenosine