INVERSE AGONISTIC EFFECT OF ICI-174,864 ON THE CLONED DELTA-OPIOID RECEPTOR - ROLE OF G-PROTEIN AND ADENYLYL-CYCLASE ACTIVATION

Previous studies have established that the delta-selective antagonist ICI-174,864 exhibits negative intrinsic activity at the delta-opioid r eceptors in NG108-15 membranes. To determine whether 101-174,864 can f unction as a true inverse agonist in intact cells, its ability to stim ulate cAMP accumulation was examined in a human embryonic kidney 293 c ell line (293/DOR) expressing the cloned murine delta-opioid receptor. Forskolin-stimulated cAMP accumulation in the 293/DOR cells was dose- dependently suppressed by the delta-selective agonist [D-Pen(2), D-Pen (5)]enkephalin, and such inhibition was abolished by pertussis toxin o r the opiate antagonist naloxone. In contrast, ICI-174,864 significant ly potentiated the forskolin response. The ICI-174,864-induced enhance ment of the forskolin response exhibited dose-dependency and was antag onized by [D-Pen(2),D-Pen(5)]enkephalin and blocked by pertussis toxin . Neither ICI-174,864 nor pertussis toxin elevated the basal level of cAMP accumulation in the absence of forskolin. Other opiate antagonist s, such as naloxone and naltrindole, were ineffective in enhancing the forskolin-stimulated cAMP accumulation. Elevation of cAMP levels in r esponse to the activation of G(s) (through either ligand-bound recepto r or point mutation on alpha(s)) was also potentiated by ICI-174,864. Our results indicate that ICI-174,864 behaves as an inverse agonist in human embryonic kidney 293 cells stably expressing the delta-opioid r eceptor. The inverse agonistic effect of ICI-174,864 seemed to require G(i) proteins and was clearly manifested when adenylyl cyclase was ac tivated.