REGULATION OF SPONTANEOUS ACTIVITY OF THE DELTA-OPIOID RECEPTOR - STUDIES OF INVERSE AGONISM IN INTACT-CELLS

Adenylyl cyclase activity was measured following labelling of the cell ular ATP pool with [H-3] adenine in intact Rat-1 fibroblasts that had been stably transfected to express the murine S-opioid receptor (clone D2). Basal [H-3]cyclic AMP accumulation was low and was increased sub stantially by the addition of the diterpene forskolin. The synthetic e nkephalin D-Ala(2),D-Leu(5) enkephalin (DADLE) produced strong inhibit ion of forskolin-amplified [H-3]cyclic AMP production, whereas the S-o pioid ligand ICI174864 augmented forskolin-amplified adenylyl cyclase activity. Naloxone was unable to mimic the effects of ICI174864, and c oincubation of the cells with these two ligands attenuated the effect of ICI174864. The EC50 (9.4 +/- 0.6 x 10(-8) M) for ICI174864 augmenta tion of forskolin-stimulated adenylyl cyclase was equal to its estimat ed Ki. Pertussis toxin pretreatment of clone D2 cells prevented both t his effect of ICI174864 and the inhibition produced by DADLE. Use of a Cytosensor microphysiometer demonstrated that treatment of clone D2 c ells with DADLE increased and that with ICI174864 decreased the basal rate of cellular proton extrusion, By using these two distinct experim ental strategies, ICI174864 was shown to function in a manner anticipa ted for an inverse agonist, demonstrating that such effects can be obs erved in intact cells and are not restricted to assays performed on me mbrane preparations.