Functional coupling of a μ opioid receptor to G proteins and adenylyl cyclase: modulation by chronic morphine treatment

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Abstract
A cloned mu opioid receptor was used to study its coupling to signal transduction pathways and its involvement in morphine-induced opioid dependence in stably transfected Chinese hamster ovary (CHO) cells. Membrane binding assays with a mu-selective agonist [(3)H]DAMGO showed that one cell line expresses a high level of mu opioid receptors with a B(max) of approximately 630 fmol/mg membrane protein and a K(d) of 0.47 nM for DAMGO. Stimulation of the transfected cells with DAMGO led to an increase in the low K(m) GTPase activity, indicative of activation of guanine nucleotide regulatory proteins (G proteins), and this effect was blocked by the opioid antagonist naloxone. In addition, binding of the mu opioid receptor to DAMGO was affected by GTP and nonhydrolizable GTP analogs, Gp(NH)pp and GTP-gamma-S. These results suggest a functional coupling between the mu opioid receptor and G proteins. Furthermore, DAMGO treatment of the cells produced a dose-dependent inhibition of the intracellular cyclic adenosine monophosphate (cAMP) level, with an EC(50) value of approximately 30 nM. Chronic treatment of the cells with morphine not only elevated the basal and forskolin-stimulated cAMP levels after morphine withdrawal, but also increased the extent of the DAMGO-induced reduction of intracellular cAMP levels. The whole cell binding assay with [(3)H]DAMGO, on the other hand, did not detect receptor down-regulation after chronic morphine treatment. These results suggest that chronic morphine treatment may trigger a compensatory mechanism in cellular signaling pathways to offset the inhibitory input from the mu receptor without down-regulation of the surface receptor number, and that withdrawal of chronic inhibition leads to elevated activities of adenylyl cyclase to provide a basis for system sensitization.
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