DIFFERENTIAL AGONIST REGULATION OF THE HUMAN KAPPA-OPIOID RECEPTOR

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 cellul ar mechanisms of tolerance, the cloned human kappa-opioid receptor was stably expressed in human embryonic kidney (HEK 293) cells, and the e ffects of opioid agonist treatment were examined. The receptor-express ing cells showed specific high-affinity membrane binding for a kappa-s elective opioid, H-3-labeled (+)-(5 alpha,7 alpha,8 olidinyl)-1-oxaspi ro[4,5]dec-8-yl]benzeneacetamide ([H-3]U69,593), and a nonselective op ioid antagonist, [H-3]diprenorphine. Pretreatment with pertussis toxin or guanosine 5'-O-(3-thiotriphosphate) reduced [H-3]69,593 binding, i ndicating that the human kappa receptor coupled to G proteins of the G (i) or G(o) families in HEK 293 cells. The receptor-mediated inhibitio n of adenylyl cyclase was abolished by pertussis toxin pretreatment an d was blocked by a kappa-selective antagonist, norbinal-torphimine. A 3-h pretreatment with a kappa-selective agonist, l-N-[2-(1-pyrrolidiny l)cyclohexyl]benzeneacetamide (U50,488), caused receptor down-regulati on, whereas no receptor down-regulation was found after levorphanol pr etreatment. U50,488 or dynorphin A(1-17) pretreatments (3 h) desensiti zed the ability of U50,488 or dynorphin A(1-17) to inhibit cyclic AMP accumulation, as evidenced by a decrease in functional potency. Also, U50,488 pretreatment desensitized the ability of levorphanol to inhibi t forskolin-stimulated cyclic AMP accumulation. In contrast, pretreatm ent of cells with either levorphanol or a potent nonselective opioid, etorphine, resulted in no apparent receptor desensitization. Taken tog ether, these results demonstrate that the human kappa receptor is diff erentially regulated by selective and nonselective opioid agonists, wi th selective agonists able to desensitize the receptor.