title: Characterization of [3H]naltrindole binding to delta opioid receptors in mouse brain and mouse vas deferens: evidence for delta opioid receptor heterogeneity.
abstract: Naltrindole (NTI) is a potent and selective nonpeptide delta opioid receptor antagonist. This study reports on the binding characteristics of [3H]NTI (specific activity = 30.5 Ci/mmole) for mouse brain and vas deferens (MVD) tissues. In brain, [3H]NTI had unusually high specific binding to delta receptors (80% at its Kd concentration) relative to other selective delta receptor radioligands. Saturation Kd values with 95% confidence intervals for mouse brain and MVD tissue preparations were 56.2 (41.8-75.7) and 104 (25.8-420) pM, respectively. These Kd values were significantly different (P = .028) and [3H]NTI binding to both tissues was best fit by a one-site model. Receptor densities were 83.9 (66.8-106) fmol/mg of protein for mouse brain and 14.8 (7.03-31.2) fmol/mg of protein for the MVD. Binding inhibition studies showed that NTI and the delta opioid receptor agonists [4'-Cl-Phe4]DPDPE and [D-Ala2, Glu4]deltorphin had high affinity for the sites labeled by [3H]NTI in both tissue preparations whereas mu [Tyr-Pro-psi-MePhe-D-Pro-NH2 (PL-17)] and kappa (U-69593) agonists had micromolar affinity. Both agonists recognized multiple sites in mouse brain under control (with 5 mM Mg++) and treatment (with 50 microM guanylyl-5'-imido-diphosphate and 100 mM NaCl) conditions but only single-site binding was observed for MVD (only control condition tested). [D-Ala2, Glu4]deltorphin showed about 6.5-fold selectivity for a portion (approximately 33%) of mouse brain sites (Ki = 130 pM) compared to sites labeled by [3H]NTI in MVD (Ki = 1200 pM) under control conditions. No significant difference was observed for [4'-Cl-Phe4]DPDPE binding affinity to both tissues (Ki = 450-680 pM) under control conditions. The affinity of opioid agonists, but not antagonists at [3H]NTI binding sites in mouse brain, was substantially reduced by the presence of guanylyl-5'-imidodiphosphate and sodium ions consistent with guanine nucleotide-binding protein regulation of the delta receptors. The portions of high- and low-affinity sites recognized by [4'-Cl-Phe4]DPDPE and [D-Ala2, Glu4]deltorphin in mouse brain labeled by [3H]NTI under treatment conditions were not significantly different (each subtype represented approximately 50% of the total population) suggesting delta receptor heterogeneity in this tissue. It is concluded that [3H]NTI binds to delta opioid receptor affinity states and subtypes with equal affinity and can be used for their characterization in conjunction with different treatment conditions and ligands.
authors.affiliation: Department of Pharmacology, University of Arizona Health Sciences, Center, Tucson.
authors.forename: L
authors.initials: L
authors.lastname: Fang
authors.forename: R J
authors.initials: RJ
authors.lastname: Knapp
authors.forename: R
authors.initials: R
authors.lastname: Horvath
authors.forename: T O
authors.initials: TO
authors.lastname: Matsunaga
authors.forename: R C
authors.initials: RC
authors.lastname: Haaseth
authors.forename: V J
authors.initials: VJ
authors.lastname: Hruby
authors.forename: F
authors.initials: F
authors.lastname: Porreca
authors.forename: H I
authors.initials: HI
authors.lastname: Yamamura
mesh_terms: D000818:Animals; D001921:Brain / Q000378:metabolism*; D020881:Enkephalin, D-Penicillamine (2,5)-; D004745:Enkephalins / Q000378:metabolism; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D066298:In Vitro Techniques; D008297:Male; D051379:Mice; D008813:Mice, Inbred ICR; D009271:Naltrexone / Q000031:analogs & derivatives* / Q000378:metabolism; D009292:Narcotic Antagonists / Q000378:metabolism*; D017465:Receptors, Opioid, delta / Q000032:analysis / Q000378:metabolism*; D014649:Vas Deferens / Q000378:metabolism*
chemical_list: D004745:Enkephalins; D009292:Narcotic Antagonists; D017465:Receptors, Opioid, delta; D006165:Guanylyl Imidodiphosphate; D009271:Naltrexone; D020881:Enkephalin, D-Penicillamine (2,5)-; C055382:naltrindole