Opioid Peptide Receptor Studies. 13. Characterization of Opioid Antagonists With the [35S]GTP-γ-S Binding Assay

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Abstract
The major purpose of this study was to develop and validate assay conditions suitable for determining the apparent functional K i, values of opioid antagonists at (μ, δ, and κ1 receptors using the [35S]GTP-γ-S binding assay. The apparent K i values of selected opioid antagonists were determined by measuring their inhibition of DAMGO (μ) -, SNC80 (δ)-, and U69,593 (κ1)-stimulated [35S]GTP-γ-S binding using guinea pig caudate membranes. The κ1-selective antagonist, nor-BNI, had high affinity for the κ1 receptor (K i = 0.038 nM) and was 440- and 268-fold selective for the κ1 receptor relative to the μ and δ receptors, respectively. The δ-selective antagonist, naltrindole, had high affinity for the δ receptor (K i = 0.062 nM) and was 52- and 143-fold selective for the δ receptor relative to the μ and κ1 receptors, respectively. The peptidic μ-selective antagonist CTAP had moderate affinity for the μ receptor (K i = 65.7 nM) and was about 100-fold selective for the μ receptor relative to the δ and κ1 receptors. The phenylpiperdine antagonist RTI-5989-25 was the most potent μ antagonist tested (K i = 0.013 nM) and was about 13- and 25-fold μ. selective relative to the δ and κ1 receptors, respectively. Oxymorphindole, which has δ agonist activity in vivo, tested as an antagonist in this system, having K i values of 1583, 23 . 1 , and 1514 nM at the μ, δ, and κ1 receptors, respectively. These results indicate that the [35S]GTP-γ-S binding assay can be used to functionally characterize opioid antagonists in vitro.

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