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 u nder dual control of both stimulatory as well as inhibitory receptor s ystems, 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 expres sed in human epidermoid carcinoma (A431) cells, which carries high lev els of endogenous beta(2)-adrenoceptors. Four out of 16 cell clones we re found to express considerable amounts of [H-3]diprenorphine binding sites and were further characterized. Scatchard analysis of saturatio n binding data revealed maximal binding capacities (B-max) between 242 .2 +/- 11 and 1,271.8 +/- 221 fmol/mg of membrane protein, whereas dru g affinity was found similar among all cell clones tested (K-d = 1.4 /- 0.2 nM). The expressed mu-receptors also mediated agonist inhibitio n of adenylate cyclase, indicating that these receptors are functional ly coupled to intracellular signalling pathways. Long-term exposure of the cells to morphine (10 mu M; 2 days) produced cellular correlates of chronic opioid action as displayed by both a decrease in the maxima degree of adenylate cyclase inhibition (tolerance) as well as an incr ease in overall effector activity (dependence). Thus, based on these p arameters, mu-opioid receptor expressing A431 cells provide a promisin g tool to investigate cellular mechanisms of chronic drug action.