A subset of kappa opioid ligands bind to the membrane glucocorticoid receptor in an amphibian brain

Previous studies demonstrated that a membrane receptor for glucocorticoids (mGR) exists in neuronal membranes from the roughskin newt (Taricha granulosa) and that this receptor appears to be a G protein-coupled receptor (GPCR). The present study investigated the question of whether this mGR recognizes nonsteroid ligands that bind to cognate receptors in the GPCR superfamily. To address this question, ligand-binding competition studies evaluated the potencies of various ligands to displace [H-3]corticosterone (CORT) binding to neuronal membranes. Initial screening studies tested 21 different competitors and found that [H-3]CORT binding was displaced only by dynorphin 1-13 amide (an endogenous kappa-selective opioid peptide), U50,488 (a synthetic kappa-specific agonist) and naloxone (a nonselective opioid antagonist). Follow-up studies revealed that the kappa agonists bremazocine (BRE) and ethylketocyclazocine (EKC) also displaced [H-3]CORT binding to neuronal membranes, but that U69,593 (a kappa specific agonist) and nor-BNI (a kappa specific antagonist) were ineffective. The K-i values measured for the opioid competitors were in the subnanomolar to low micromolar range and had the following rank-order: dynorphin > U50,488 > naloxone > BRE > EKC. Because these ligands displaced, at most, only 70% of [H-3]CORT specific binding, it appears that some [H-3]CORT binding sites are opioid insensitive. Kinetic analysis of [H-3]CORT off-rates in the presence of U50,488 and/or CORT revealed no differences in dissociation rate constants, suggesting that there is a direct, rather than allosteric, interaction with the [H-3]CORT binding site. In summary, these results are consistent with the hypothesis that the high-affinity membrane binding site for [H-3] CORT is located on a kappa opioid-like receptor.