Effects of 5′-guanylylimidodiphosphate on the ligand binding of membrane-bound and solubilized opioid receptors of frog ( Rana esculenta) brain

Abstract

The effect of the nonhydrolysable analogue of GTP, 5′-guanylylimidodiphosphate (GppNHp) was investigated on the binding of mu and kappa opioid agonists and antagonists in frog brain membrane preparations. The greatest inhibition with GppNHp was observed with labelled dihydromorphine, ([ 3H]DHM), a mu opioid receptor agonist. The binding of [ 3H]U-69593, a specific kappa opioid receptor agonist was inhibited to a smaller extent by GppNHp. Conversely, [ 3H]naloxone (opioid antagonist) binding displayed a dose-dependent increase with GppNHp, which effect was larger at 0°C than at 24°C. The binding of [ 3H]ethylketocyclazocine (EKC), a ligand with agonist-antagonist character was not affected by GppNHp either in the absence or in the presence of sodium ions at 0°C. Slight inhibition of [ 3H]EKC binding in the presence of sodium ions was observed with GppNHp at 24°C. In displacement experiments GppNHp decreased the ability of mu and kappa agonists to compete for [ 3H]naloxone binding sites. This effect was maintained after solubilization with 1% digitonin. However, since the binding of [ 3H]naloxone was also reduced, there was no agonist specificity of the GppNHp induced inhibition of binding to solubilized receptors. These results suggest that frog brain opioid receptors are coupled to a GTP-regulatory protein similar to mammalian opioid receptors.