Selective opioid agonist and antagonist competition for [3H]-naloxone binding in amphibian spinal cord

Abstract

Opioids elicit antinociception in mammals through three distinct types of receptors designated as mu, kappa and delta. However, it is not clear what type of opioid receptor mediates antinociception in non-mammalian vertebrates. Radioligand binding techniques were employed to characterize the site(s) of opioid action in the amphibian, Rana pipiens. Naloxone is a general opioid antagonist that has not been characterized in Rana pipiens. Using the non-selective opioid antagonist, [3H]-naloxone, opioid binding sites were characterized in amphibian spinal cord. Competitive binding assays were done using selective opioid agonists and highly-selective opioid antagonists. Naloxone bound to a single-site with an affinity of 11.3 nM and 18.7 nM for kinetic and saturation studies, respectively. A B(max) value of 2725 fmol/mg protein in spinal cord was observed. The competition constants (K(i)) of unlabeled mu, kappa and delta ranged from 2.58 nM to 84 microM. The highly-selective opioid antagonists yielded similar K(i) values ranging from 5.37 to 31.1 nM. These studies are the first to examine opioid binding in amphibian spinal cord. In conjunction with previous behavioral data, these results suggest that non-mammalian vertebrates express a unique opioid receptor which mediates the action of selective mu, kappa and delta opioid agonists.

Fig. 1

Association kinetics of [³H]-naloxone (10 nM) binding in Rana pipiens spinal cord (A). Dissociation kinetics in Rana pipiens spinal cord (B).

Fig. 2

Saturation analysis of [³H]-naloxone in spinal cord tissue homogenates. The membrane preparation was incubated with various concentrations of [³H]-naloxone. Measured binding is the difference between total and nonspecific binding. Values represent the mean of three independent determinations, each performed in triplicate. K_D and B_max values were determined by the rectangular hyperbole using GraphPad Prism. Inset shows Scatchard analysis of the saturation data.

Fig. 3

Inhibition of 10 nM [³H]-naloxone binding with various unlabeled opioid receptor ligands in Rana pipiens spinal cord tissue homogenates. (A) depicts competition with μ agonists, (B) shows competition with κ ligands and (C) with δ agonists. Aliquots of tissue homogenates were incubated with radioligand in the presence of various concentrations (0.01 nM–100 μM) of cold competitor. Data was normalized to aid comparisons defining the smallest value in the data set as 0% and the largest value as 100% of specific binding. K_i values for these competitors are shown in Table 2. Data points are the means of one representative experiment in triplicate determinations, which was repeated three times.

Fig. 4

Correlation plot of Rana pipiens spinal cord K_i values versus brain K_i values against [³H]-naloxone. Numerical results of regression analysis are shown in the figure.

Fig. 5

Competition of 10 nM [³H]-naloxone binding with increasing concentrations (0.01 nM–10 μM) of selective antagonists in Rana pipiens spinal cord. β-funaltrexamine is a μ-selective antagonist, naltrindole is δ-selective and nor-binaltorphimine is a κ-selective antagonist. Data was normalized to aid comparisons defining the smallest value in the data set as 0% and the largest value as 100% of specific binding. Data points are the means of one representative experiment in triplicate determinations, which was repeated three times.