Stable expression and functional characterization of the cloned rat mu-opioid receptor in human epidermoid carcinoma (A431) cells.
Regulation of intracellular cAMP levels serves as a cellular model for chronic drug action. Since the adenylate cyclase effector system is under dual control of both stimulatory as well as inhibitory receptor systems, a permanent cell line was created in order to allow evaluation of acute and chronic opioid effects on stimulatory receptor function. For this purpose, the cloned rat mu-opioid receptor was stably expressed in human epidermoid carcinoma (A431) cells, which carries high levels of endogenous beta 2-adrenoceptors. Four out of 16 cell clones were found to express considerable amounts of [3H]diprenorphine binding sites and were further characterized. Scatchard analysis of saturation binding data revealed maximal binding capacities (Bmax) between 242.2 +/- 11 and 1,271.8 +/- 221 fmol/mg of membrane protein, whereas drug affinity was found similar among all cell clones tested (Kd = 1.4 +/- 0.2 nM). The expressed mu-receptors also mediated agonist inhibition of adenylate cyclase, indicating that these receptors are functionally coupled to intracellular signalling pathways. Long-term exposure of the cells to morphine (10 microM; 2 days) produced cellular correlates of chronic opioid action as displayed by both a decrease in the maximal degree of adenylate cyclase inhibition (tolerance) as well as an increase in overall effector activity (dependence). Thus, based on these parameters, mu-opioid receptor expressing A431 cells provide a promising tool to investigate cellular mechanisms of chronic drug action.
Ammer
H
H
Institute of Pharmacology, Toxicology and Pharmacy, School of Veterinary Medicine, University of Munich, Germany.
Schulz
R
R
D000262:Adenylyl Cyclases / Q000502:physiology; D000818:Animals; D001665:Binding Sites; D002294:Carcinoma, Squamous Cell / Q000737:chemistry / Q000235:genetics* / Q000473:pathology*; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000502:physiology; D004247:DNA / Q000032:analysis / Q000235:genetics; D004273:DNA, Neoplasm / Q000032:analysis / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D051381:Rats; D011943:Receptors, Adrenergic, beta / Q000032:analysis / Q000235:genetics / Q000502:physiology; D017450:Receptors, Opioid, mu / Q000032:analysis / Q000235:genetics* / Q000502:physiology*; D014162:Transfection; D014407:Tumor Cells, Cultured
D004273:DNA, Neoplasm; D011943:Receptors, Adrenergic, beta; D017450:Receptors, Opioid, mu; D004247:DNA; D000242:Cyclic AMP; D000262:Adenylyl Cyclases