Vol. 280, Issue 1, 512-520, 1997

Chronic Morphine Treatment Increases Stimulatory Beta-2 Adrenoceptor Signaling in A431 Cells Stably Expressing the Mu Opioid Receptor

Hermann Ammer and Rüdiger Schulz

Institute of Pharmacology, Toxicology and Pharmacy, University of Munich, München, Germany

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 rat mu 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 of beta-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 in beta-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 the beta-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.