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 N 6 -cyclohexyladenosine (CHA) on DOR down-regulation was assessed using radioligand binding assay.]enkephalin (DPDPE)-induced inhibition of intracellular cAMP accumulation with a t 1/2 =2.56 (2.09-3.31) h. Pretreatment with 1 μmol/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 μmol/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 downregulation of DOR.

Protein phosphatase 2A activation attenuates inflammation in murine models of acute lung injury

“…1). Desensitization of adenosine A 1 receptors through both G-protein receptor uncoupling and receptor internalization acts as a homeostatic negative feedback loop and has been previously reported to occur following adenosine A 1 receptor stimulation with pharmacologic agonists (3,4). Here we observed a similar phenomenon whereby PP2A activity in cells that received more than 1 h of 10 M N 6 -CPA treatment returned to basal levels suggesting receptor desensitization (Fig.…”

Central activation of the A1 adenosine receptor in fed mice recapitulates only some of the attributes of daily torpor

“…Administering CHA to a mouse with a genotype that is unable to undergo daily torpor, such as a dbh -/- mouse (Swoap, Gutilla et al 2006) or a mouse with an ablated ARC (Gluck, Stephens et al 2006), might definitively distinguish between the two states. Further, we recognize the potential for off-target effects of CHA, such as such as phosphorylation, and subsequent de-sensitization, of delta-opioid receptors (Cheng, Tao et al 2010), which may be relevant as delta opioid receptors may be involved in hypothermia/torpor (Borlongan, Hayashi et al 2009). Future experiments should use specific knock-out mice or combine CHA with a specific A1-antagonist to rule out effects mediated by targets other than the A1-receptor.…”

The influences of reproductive status and acute stress on the levels of phosphorylated delta opioid receptor immunoreactivity in rat hippocampus

“…The CA2 region is also notable because it highly expresses adenosine A1 receptors (A 1 Rs), which have recently been shown to bind caffeine and act to induce long-lasting potentiation of synaptic transmission (Simons et al, 2011; Ochiishi et al, 1999). Moreover, activation of A 1 Rs causes phosphorylation and desensitization of DORs, suggesting a functional interaction between these two receptors (Cheng et al, 2010). …”