Received for publication October 18, 2005.
Revised January 13, 2006.
Accepted for publication January 13, 2006.

Chronic opioid agonist-induced adenylyl cyclase superactivation is dependent on receptor localized within lipid rafts and is independent on receptor internalization

Abstract

Chronic opioid agonist treatment results in adenylyl cyclase superactivation. A recent "Rave" theory implicates a direct correlation between the ability of agonist to induce receptor internalization and the magnitude of adenylyl cyclase superactivation. We decided to test such a theory by examining the adenylyl cyclase superactivation after chronic activation of µ-opioid receptor (MOR) in an EcR293 cell model. We examined the magnitudes of adenylyl cyclase superactivation in the presence of naloxone after chronic treatment with morphine, etorphine and methadone, three agonists reported to have differential activities in promoting MOR internalization. It can be shown that the magnitudes of adenylyl cyclase superactivation after treating with these three agonists, that though different were dependent on MOR density. Blunting MOR internalization with the dominant negative mutant of dynamin, K44E, did not alter the magnitude of either morphine- or etorphine-induced adenylyl cyclase superactivation. In the presence of diprenorphine, the magnitude of adenylyl cyclase superactivation after etorphine treatment was identical to that observed with morphine. It could be demonstrated further that adenylyl cyclase superactivation is dependent upon the cell surface located MOR. Sucrose gradient fractionation demonstrated the colocalization of MOR and adenylyl cyclase V/VI with caveolin-1, a marker for lipid rafts. After chronic agonist treatment, the majority of MOR remained at the lipid rafts. Methyl--cyclodextrin (MCD) completely blunted the adenylyl cyclase superactivation and agonist-induced receptor internalization. These MCD actions were reversed by incubating the cells with cholesterol. Thus, the adenylyl cyclase superactivation is not dependent on agonist-induced receptor internalization. Rather, the location of MOR at lipid rafts is an absolute requirement for the observed adenylyl cyclase superactivation.

Key words: Neuropeptides, Opioid, Gi family, cAMP, Lipid rafts/microdomains, Sequestration/Internalization

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