Opioid peptide receptor studies. 3. Interaction of opioid peptides and other drugs with four subtypes of the κ2 receptor in guinea pig brain

Abstract Using guinea pig, rat, and human brain membranes depleted of μ and δ receptors by pretreatment with the site-directed acylating agents BIT (μ selective) and FIT (δ selective), previous studies from our laboratory resolved two subtypes of the κ 2 binding site, termed κ 2a and κ 2b . In more recent studies, we used 6β-[ 125 Iodo]-3,14-dihydroxy-17-cyclopropylmethyl-4,5α-epoxymorphinan ([ 125 I]IOXY) to characterize multiple κ 2 binding sites in rat brain. The results indicated that [ 125 I]IOXY, like [ 3 H]bremazocine, selectively labels κ 2 binding sites in rat brain membranes pretreated with BIT and FIT. In the rat brain, using 100 n M [ d -Ala 2 -MePhe 4 ,Gly-ol 5 ]enkephalin to block [ 125 I]IOXY binding to the κ 2b site, we resolved two subtypes of the κ 2a binding site. In the present study we examined the binding of [ 125 I]IOXY to the κ 2 receptors of guinea pig brain. As observed in rat brain, [ 125 I]IOXY, under appropriate assay conditions, selectively labels κ 2 binding sites. Quantitative binding studies readily demonstrated the presence of κ 2a and κ 2b binding sites. The κ 2a binding sites were selectively assayed using 5 μ M [Leu 5 ]enkephalin to block [ 125 I]IOXY binding to the κ 2b sites, and κ 2b sites were selectively assayed using 5 μM (−)-(1 S ,2 S )-U50,488 to block [ 125 I]IOXY binding to the κ 2a sites. Under these conditions, two subtypes of the κ 2a site were resolved with high ( κ 2a-1 ) and low ( κ 2a-2 ) affinity for nor-BNI ( K i values = 0.88 and 476 n M ) and CI977 ( K i values = 17.5 and 95,098 n M ). Similarly, two subtypes of the κ 2b site were observed with high ( κ 2b-1 ) and low ( κ 2b-2 ) affinity for [ d -Ala 2 -MePhe 4 ,Gly-ol 5 ]enkephalin (DAMGO) ( K i values = 97 and 12,321 n M ) and α-neoendorphin ( K i values = 33 and 5308 n M ). Two-site models were also resolved in the presence of 100 μ M 5′-guanylyimidodiphosphate (GppNHp). We carried out detailed ligand selectivity analysis of the multiple κ 2 binding sites. Most test agents were either nonselective or selective for the κ 2a-1 site. Nalbuphine was moderately selective for the κ 2a-2 site. Similarly, although most test agents were either nonselective or selective for the κ 2b-1 site, butorphanol, and the delta antagonists naltrindole, naltriben, and 7-benzylidene-7-dehydronaltrexone were moderately selective for the κ 2b-2 site. Of the endogenous opioid peptides tested. BAM22P had the highest affinity for the κ 2b-2 site (31 n M ) and peptide E had the highest affinity for the κ 2a-a binding site (53 n M ). These data provide additional evidence for heterogeneity of the κ-opioid receptor and new targets for drug design, synthesis, and therapeutics.