Differential agonist regulation of the human ≃-opioid receptor

Abstract

Opiates are potent analgesics used clinically in the treatment of pain. A significant drawback to the chronic use and clinical effectiveness of opiates is the development of tolerance. To investigate the cellular mechanisms of tolerance, the cloned human K-opioid receptor was stably expressed in human embryonic kidney (HEK 293) cells, and the effects of opioid agonist treatment were examined. The receptor-expressing cells showed specific high-affinity membrane binding for a κ-selective opioid, 3H- labeled (+)-(5α,7α,8β)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro[4,5]dec- 8-yl]benzeneacetamide ([3H] U69,593), and a nonselective opioid antagonist, [3H]diprenorphine. Pretreatment with pertussis toxin or guanosine 5'-O-(3- thiotriphosphate) reduced [3H]69,593 binding, indicating that the human κ receptor coupled to G proteins of the G(i) or G(o) families in HEK 293 cells. The receptor-mediated inhibition of adenylyl cyclase was abolished by pertussis toxin pretreatment and was blocked by a K-selective antagonist, norbinaltorphimine. A 3-h pretreatment with a κ-selective agonist, (±)- trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamide (U50,488), caused receptor down-regulation, whereas no receptor down- regulation was found after levorphanol pretreatment. U50,488 or dynorphin A1-17 pretreatments (3 h) desensitized the ability of U50,488 or dynorphin A1-17 to inhibit cyclic AMP accumulation, as evidenced by a decrease in functional potency. Also, U50,488 pretreatment desensitized the ability of levorphanol to inhibit forskolin-stimulated cyclic AMP accumulation. In contrast, pretreatment of cells with either levorphanol or a potent nonselective opioid, etorphine, resulted in no apparent receptor desensitization. Taken together, these results demonstrate that the human κ receptor is differentially regulated by selective and nonselective opioid agonists, with selective agonists able to desensitize the receptor.