Characterization of μ, κ, and δ opioid binding in amphibian whole brain tissue homogenates

Opioid agonists produce analgesia in mammals through the activation of μ, κ, or δ opioid receptors. Previous behavioral and binding studies from our laboratory using an amphibian model suggested that μ, κ, or δ opioid agonists may activate a single type of opioid receptor in the grass frog, Rana pipiens. In the present study, kinetic, saturation, and competitive binding profiles for three opioid radioligands, [³H]DAMGO ([D-Ala², N-Me-Phe⁴, Gly⁵-ol]-enkephalin) (μ-selective), [³H]U65953 [(5α, 7α,8β)-(+)-N-methyl-N-[7-(1-pyrrolidinyl) -1-oxaspiro[4.5]dec-8-yl]-benzeneacetamide] (κ-selective), and [³H]DPDPE ([D-Pen²,D-Pen⁵]-enkephalin) (δ-selective) were determined using frog whole brain homogenates. Kinetic analyses and experimentally derived values from saturation experiments gave affinity constants (K_D) in the low nanomolar range. The density of opioid binding sites (B_max) was 224.4, 118.6, and 268.9 fmol/mg for μ, κ and δ opioid radioligands, respectively. The affinity values did not significantly differ among the three opioid radioligands, but the κ radioligand bound to significantly fewer sites than did the μ or δ radioligands. K_i values for unlabeled μ, κ, and δ competitors, including highly selective opioid antagonists, were consistent with each radioligand selectivity profile, The present data suggest that μ, κ, and δ opioid radioligands bind to distinct opioid receptors in amphibians that are surprisingly similar to those found in mammalian brain.