3H-morphine-6beta-glucuronide binding in brain membranes and an MOR-1-transfected cell line.

Morphine-6beta-glucuronide (M6G) is a potent morphine metabolite. In an effort to further explore its mechanisms of action, we synthesized 3H-M6G of high specific activity and examined its binding. Although its affinity toward traditional mu receptors is similar to morphine in binding assays in brain and in Chinese hamster ovary cells stably transfected with MOR-1, M6G is >100-fold more potent than morphine in analgesic assays. This apparent discrepancy cannot be explained by differing intrinsic activities of the two drugs because both agents are partial agonists with similar efficacies in adenylyl cyclase assays in the transfected cell lines. Behavioral studies have implied the possibility of a distinct M6G receptor. Detailed binding studies in brain tissue reveal evidence for heterogeneity. Nonlinear regression analysis of 3H-M6G saturation studies reveals two components. The lower-affinity component (K(D) = 1.93 +/- 0.6 nM) corresponds to labeling of traditional mu receptors. In addition, 3H-M6G labels another site of low abundance with very high affinity (K(D) = 68 +/- 7 pM). Competition studies indicate that both sites are relatively mu selective. However, several compounds clearly distinguish between the two sites. These binding studies support the concept of a unique M6G receptor responsible for its analgesic activity.