Short-term inverse-agonist treatment induces reciprocal changes in delta-opioid agonist and inverse-agonist binding capacity

This study assessed the effects of short-term treatment (30-min) with inver se agonists on receptor protein levels and on the ability of agonists, inve rse agonists, and neutral antagonists to bind to the human delta -opioid re ceptor (h delta OR). Incubation of human embryonic kidney 293s cells stably expressing h delta OR with the inverse agonist ICI174864 (1 muM) induced r eciprocal changes in agonist and inverse-agonist binding. The total number of binding sites recognized by the agonists [H-3]bremazocine and [H-3][D-Pe n(2),D-Pen(5)]-enkephalin was reduced by 33 and 57%, respectively, whereas binding capacity for the radio-labeled inverse-agonist [H-3]Tyr-TicY[CH2NH] Cha-Phe-OH increased by 44%. In contrast, total receptor protein and sites labeled by neutral antagonists [H-3]naltrindole and [H-3]Tyr-D-Tic-Phe-Phe- OH remained unchanged. Pertussis toxin (PTX) and 5-guanylylimidodiphosphate (GppNHp) mimicked the outcome of ICI174864 pretreatment in promoting the l oss of agonist binding sites. The lack of an additive effect on [H-3]bremaz ocine binding when these three agents were combined indicates that inverse agonists may, in part, share the mechanism by which GppNHp and PTX reduce a gonist binding capacity. Spontaneous recovery of maximal agonist binding ca pacity after inverse-agonist treatment was slow, suggesting a decrease in t he isomerization rate between agonist- and inverse agonist-preferring confo rmations. Overall, the data presented are consistent with the idea that h d elta ORs exist in multiple states capable of discriminating among ligands o f different levels of efficacy and show that, after short-term treatment wi th an inverse agonist, the receptor ability to adopt conformations preferen tially induced by agonist ligands is reduced.