Abstract

KDN21 is a bivalent ligand that contains δ and κ opioid antagonist pharmacophores linked through a 21-atom spacer. It has been reported that KDN21 bridges δ and κ receptors that are organized as heterodimers. We have shown previously that when using [³H]diprenorphine as radioligand, KDN21 displayed greatly enhanced affinity in this series for coexpressed δ and κ opioid receptors (CDK). The present study used in vitro expression systems to investigate interactions of members of the KDN series with δ-κ heterodimers through competition binding using selective ligands and the mitogen-activated protein kinase (MAPK) assay. In this regard, the use of the selective radioligands [³H]naltrindole and [³H]norbinaltorphimine (nor-BNI) in competition binding studies revealed that KDN21 has much higher affinity than other KDN members for CDK and bound to CDK more selectively relative to mixed δ and κ opioid receptors or singly expressed δ and κ opioid receptors. Other experiments revealed that the binding of naltrindole to δ opioid receptors could increase the binding of nor-BNI to κ opioid receptors and vice versa, suggesting reciprocal allosteric modulation of receptors in the heterodimer. Regarding the selectivity of KDN21 for phenotypic δ and κ opioid receptors, we investigated the effect of KDN21 on the activation of MAPKs [extracellular signal-regulated kinases 1 and 2 (ERK1/2)] by δ- or κ-selective agonists. KDN21 inhibited the activation of ERK1/2 by [d-Pen²,d-Pen⁵]-enkephalin (δ₁) and bremazocine (κ₂) but had no effect on the activation by deltorphin II (δ₂) and (+)-(5α,7α,8β)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro[4.5]dec-8-yl]benzeneacetamide (U69593, κ₁). 7-Benzylidenenaltrexone (δ₁) and bremazocine (κ₂) significantly reduced the binding of KDN21 to CDK, whereas naltriben (δ₂) and U69593 produced no such change. Taken together, these data support the idea that the organization of δ and κ receptors as heterodimers gives rise to δ₁ and κ₂ phenotypes.