Abstract

In this study, we examined whether the human kappaopioid receptor stably expressed in Chinese hamster ovary cells underwent desensitization and down-regulation after prolonged exposure to the agonist (−)U50,488H. Pretreatment with (−)U50,488H led to a reduction in the magnitude of increase in [³⁵S]GTPγS binding by the subsequent application of (−)U50,488H. The extent of desensitization was related to duration of exposure and (−)U50,488H concentration. Pretreatment with (−)U50,488H also reduced the potency of (−)U50,488H in inhibiting forskolin-stimulated adenylate cyclase. In membranes of (−)U50,488H-pretreated cells, the affinity of (−)U50,488H was lower than that in the untreated control, and GTPγS had no effect on (−)U50,488H affinity, consistent with the notion of uncoupling of the receptor-G protein complex by (−)U50,488H treatment. Down-regulation of the kappa opioid receptor also occurred on exposure to (−)U50,488H. Higher (−)U50,488H concentrations and/or longer incubation periods were required for down-regulation than for desensitization. The degree of down-regulation depended on the agonist concentration and incubation time. (−)U50,488H-induced desensitization and down-regulation were blocked by naloxone. (+)U50,488H, an inactive stereoisomer, did not cause desensitization or down-regulation. These results indicate that both processes were receptor-mediated. After incubation with (−)U50,488H and removal of (−)U50,488H, both (−)U50,488H-induced [³⁵S]GTPγS binding and receptor number returned to the control level, which indicates that both processes were reversible. Thus, desensitization and down-regulation of the kappaopioid receptor occur after agonist exposure and represent two different adaptation mechanisms.