Interaction of dynorphin with kappa opioid receptors in bovine adrenal medulla

Abstract

Dynorphin (Dyn) and various prototypic κ opioid ligands were tested for their ability to bind to opioid receptors in a membrane preparation of bovine adrenal medulla and to modulate the release of catecholamines (CA) from isolated adrenal chromaffin cells. Saturation binding studies with [ 3H]-ethylketocyclazocine ([ 3H]-EKC) were performed at 37°C for 30 min in the presence of [D-Ala 2, Me-Phe 4, Gly-ol 5]-enkephalin (DAGO) and [D-Ser 2, Thr 6]-Leu-enkephalin (DSLET), two specific ligands for crossreacting μ and δ opioid receptors, respectively. Scatchard plot analysis of the data revealed the presence of two receptor sites: a high affinity binding site (κ) with a K D of 0.66 nM and a Bmax of 12 pmoles/g protein and a low affinity binding site (κ 2) with a K D of 11.1 nM and a Bmax of 56 pmoles/g protein. The presence of κ opioid receptors in the membrane preparation was also supported by competition studies. U-50,488H and Dyn-(1–13), two selective κ opioid ligands, were potent inhibitors of [ 3H]-EKC binding with Ki (high affinity binding sites) of 2.5 and 2.3 nM, respectively. Among the various ligands tested for each class of opioid receptors (μ, δ, κ), U-50,488H and Dyn-(1–13) were the most potent inhibitors of the acetylcholine-evoked CA secretions from isolated adrenal chromaffin cells with IC 50 of 0.31 and 1.14 μM, respectively. The inhibitory effect of U-50,488H was significantly antagonized by diprenorphine and MR-2266, two opioid antagonists with a high affinity for the κ opioid receptor. Among various fragments of Dyn, Dyn-(1–8) and larger N-terminal peptides retained a significant part of the potency of Dyn-(1–13) (> 6%) while Dyn-(1–5), [Arg 6, Phe 7]-Met-enkephalin and Dyn-(6–13) were relatively ineffective (relative potency < 1%). These results suggest a putative modulatory role for Dyn in the adrenal medulla, possibly mediated through the stimulation of κ opioid receptors.