Abstract

Chronic opioid regulation of stimulatory beta-2 adrenoceptor (beta-2 AR) signaling was investigated in human mammary epidermoid carcinoma A431 cells stably expressing the cloned ratmu opioid receptor. In the cell clone used (A431/μ13;B _max = 302.9 ± 46 fmol/mg membrane protein), the addition of morphine acutely attenuated basal as well as (−)-isoproterenol-stimulated cAMP accumulation. Prolonged exposure of the cells to morphine (10 μM; 2 d) resulted in homologous desensitization of MOR function as well as heterologous sensitization of adenylate cyclase (AC). Up-regulation of AC in A431/μ13 cells is characterized by an increased capacity rather than an increased sensitivity of beta-2 AR-stimulated AC. Moreover, opioid withdrawal fails to precipitate a cAMP overshoot in this cell system. Sensitization of stimulatory AC signaling by chronic morphine develops in a time- and dose-dependent manner and is blocked by both naloxone and pertussis toxin. Investigation into the mechanism leading to up-regulation of AC revealed a 40% increase in the number ofbeta-2 ARs as assessed by [¹²⁵I]-cyanopindolol binding experiments. No additional quantitative changes were found for stimulatory G proteins and the effector enzyme itself. Sensitization of AC appears to be mediated solely by the increase inbeta-2 AR numbers, because (±)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol hydrochloride, which acts as an “inverse agonist” at thebeta-2 AR, completely reversed elevated basal AC activities, and because the ratio between functional active beta-2 ARs and stimulatory G proteins remained unchanged. In conclusion, chronic exposure of clonal A431/μ13 cells to morphine increases the capacity of stimulatory AC signaling by up-regulating beta-2 AR number. These results demonstrate participation of stimulatory receptor systems in the cellular mechanisms underlying opioid dependence.