The opioid binding profile and in vitro activity of the endogenous opioid peptide dynorphin A(1-8) have been studied. At opioid receptors in guinea-pig brain dynorphin A(1-8) was nonselective, although with some preference for the delta receptor (Ki 4.6 nM) over µ (Ki 18 nM) and kappa (Ki 40 nM) receptors. However, a high degree of metabolism was observed, with less than 10% of added dynorphin A(1-8) remaining at the end of the binding assay. In the presence of peptidase inhibitors to prevent breakdown of the N- and C-termini and the Gly3-Phe4 bond the major metabolite was [Leu5]enkephalin (representing 49% recovered material). This was reduced by inclusion of an inhibitor of endopeptidase EC 3.4.24.15. In the presence of all the peptidase inhibitors the affinity for kappa receptors (Ki 0.5 nM) relative to µ and delta receptors increased, but no selectivity of binding was observed. This lack of selectivity was confirmed using membranes from C6 glioma cells expressing rat opioid receptors. The agonist effect of dynorphin A(1-8) in the mouse vas deferens (EC50 116 nM) and guinea-pig ileum (EC50 38 nM) was mediated through the kappa receptor as evidenced by the rightward shifts afforded by the kappa -selective antagonist norbinaltorphimine. In the presence of peptidase inhibition potency was improved 2-fold in the mouse vas deferens and 20-fold in the guinea-pig ileum, but this agonist activity was mediated through delta receptors in the vas deferens and µ receptors in the ileum, as a result of the formation and stabilization of [Leu5]enkephalin. The results confirm the absence of receptor selectivity of dynorphin A(1-8) in binding assays but show that its agonist effects, at least in vitro, are mediated exclusively through the kappa opioid receptor.Key words: dynorphin A(1-8), opioid receptors, peptide metabolism, mouse vas deferens, guinea-pig ileum.
ASSESSMENT IN THE GUINEA-PIG ILEUM AND MOUSE VAS DEFERENS OF BENZOMORPHANS WHICH HAVE STRONG ANTINOCICEPTIVE ACTIVITY BUT DO NOT SUBSTITUTE FOR MORPHINE IN THE DEPENDENT MONKEY
