Abstract

A new class of highly selective δ-opioid receptor antagonists has been recently developed, termed the TIP(P) peptides. Two prototypical compounds in this class are TIPP (H-Tyr-Tic-Phe-Phe-OH) and a derivative, TIPP-ψ (H-Tyr-Tic[CH₂NH]-Phe-Phe-OH). Surprisingly, both TIPP and TIPP-ψ demonstrated inhibition of adenylyl cyclase activity in GH₃ cells transfected with δ-opioid receptors (GH₃DORT), an effect normally observed by agonists. The agonist activity was δ-selective, because no inhibition occurred in wild-type GH₃ or GH₃MOR (μ-opioid receptor) cells. Both TIPP and TIPP-ψ exhibited concentration-dependent inhibition of adenylyl cyclase activity; however, TIPP-ψ was found to be less potent (IC₅₀ = 3.97 versus 0.162 nM) and less efficacious (I_max = 50% versus 70%) than TIPP. Pretreatment of cells with pertussis toxin attenuated the inhibition of maximally effective concentrations of TIPP and TIPP-ψ, indicating the involvement of G_iα/G_oα G-proteins. Other δ-antagonists, naltriben, naloxone, and ICI 174864, attenuated the inhibition of adenylyl cyclase activity mediated by TIPP. Coadministration of TIPP with the selective δ-agonist [d-Pen^2,5]enkephalin resulted in an additive interaction. Both TIPP and TIPP-ψ exhibited significant inhibition of adenylyl cyclase activity in different GH₃DORT clones expressing a 28-fold range of δ-opioid receptor densities, and in cell lines expressing endogenous (i.e., N1E115 and NG108-15) and transfected (i.e., Chinese hamster ovary-DOR and human embryonic kidney-DOR) δ-opioid receptors, with densities ranging from 0.12 to 6.67 pmol/mg. These results suggest that compounds previously thought to be purely δ-opioid receptor antagonists also demonstrate agonist activity in several in vitro models.