The molecular mechanism of chronic delta-opioid-mediated adenylyl cyclase superactivation in Chinese hamster ovary cells stably expressing the delta-opiod receptor: A cellular model for tolerance and withdrawal

We are studying the pharmacology of the human delta-opioid receptor stably expressed in Chinese hamster ovary cells (hDOR/CHO). The delta-opioid receptor mediates analgesia, without the negative side effects noted by mu- and kappa-opioid agonists. However, tolerance to delta-opioid agonists still occurs. One mechanism of drug tolerance in CHO cells involves a compensatory response by adenylyl cyclase VI. This response, termed adenylyl cyclase (AC) superactivation, arises from the observation that acute hDOR activation leads to inhibition of AC, whereas chronic activation of the receptor (greater than 4 hours) leads to a compensatory increase in AC activity, effectively negating the acute inhibition normally seen in the presence of the delta-opioid agonist. The increased AC activity also causes an overshoot of cAMP formation upon the removal of the agonistic inhibitory influence. The loss of receptor-mediated AC inhibition after chronic agonist treatment is thought to contribute to in vivo drug tolerance, and the resulting cAMP overshoot may contribute to opioid withdrawal. In studying this phenomenon, we have demonstrated a requirement for G-protein betagamma subunits (Gbetagamma) by expressing scavengers of Gbetagamma, such as alpha-transducin and phosducin. Additionally, we have shown AC VI phosphorylation by chronic agonist treatment, which may cause AC superactivation, and that this phosphorylation is sensitive to calmodulin antagonists and inhibitors of the atypical protein kinase C enzymes. We have also recently postulated the involvement of Raf-1 kinase. Inhibitors of Raf-1, as well as pathways that lead to activation of this enzyme, significantly attenuate the cAMP overshoot, suggesting that hDOR-activated Raf-1 can phosphorylate and superactivate AC VI in CHO cells