Chronic morphine treatment increases stimulatory beta-2 adrenoceptor signaling in A431 cells stably expressing the mu opioid receptor.
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/mu 13; Bmax = 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 microM; 2 d) resulted in homologous desensitization of MOR function as well as heterologous sensitization of adenylate cyclase (AC). Up-regulation of AC in A431/mu 13 cells is characterized by an increased capacity rather than an increased sensitivity of beta-2 AR-stimulated AC. Moreover, opioid withdrawal falls 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/ mu13 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.
Ammer
H
H
Institute of Pharmacology, Toxicology and Pharmacy, University of Munich, Germany.
Schulz
R
R
D000262:Adenylyl Cyclases / Q000378:metabolism; D000701:Analgesics, Opioid / Q000494:pharmacology*; D000818:Animals; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000096:biosynthesis; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D004745:Enkephalins / Q000378:metabolism; D006801:Humans; D007545:Isoproterenol / Q000494:pharmacology; D009020:Morphine / Q000494:pharmacology*; D051381:Rats; D018343:Receptors, Adrenergic, beta-2 / Q000187:drug effects*; D017450:Receptors, Opioid, mu / Q000235:genetics / Q000502:physiology*; D013375:Substance Withdrawal Syndrome / Q000378:metabolism; D014162:Transfection; D015854:Up-Regulation
D000701:Analgesics, Opioid; D004745:Enkephalins; D018343:Receptors, Adrenergic, beta-2; D017450:Receptors, Opioid, mu; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D009020:Morphine; D000242:Cyclic AMP; D000262:Adenylyl Cyclases; D007545:Isoproterenol