dynorphin-(1-17) and bremazocine

The binding of radiolabelled ligands with high affinity for kappa-opioid binding sites has been studied in homogenates of lumbo-sacral spinal cord from the rat. The unselective ligands [3H]bremazocine and [3H]diprenorphine labelled a large number of sites which could not be fully resolved in terms of mu-, delta- and kappa-types by displacement assays. In particular binding at the kappa-site appeared anomalous in that sites which could be identified as high affinity kappa-type represented only 40% of total kappa-binding, defined using the unselective [3H]ligands. This was confirmed by the low levels of binding seen with the kappa-agonists [3H]dynorphin A(1-9) and [3H]U-69593. In guinea-pig cord, under conditions in which binding to mu- and delta-sites was suppressed, [3H]dynorphin A(1-9) and [3H]U-69593 labelled only 60% of the kappa population, defined by the [3H]unselective ligands. The reasons for the observed discrepancies are discussed.

Topics: Animals; Benzeneacetamides; Benzomorphans; Captopril; Diprenorphine; Dynorphins; Guinea Pigs; In Vitro Techniques; Leucine; Ligands; Male; Peptide Fragments; Pyrrolidines; Rats; Rats, Inbred Strains; Receptors, Opioid; Receptors, Opioid, kappa; Spinal Cord

Following incubation of [3H]dynorphin A (1-8) and [3H]dynorphin A (1-9) with suspensions of guinea pig brain membranes, analysis of the supernatants by HPLC has shown that both peptides are degraded at 25 degrees C and at 0 degrees C. Bestatin and captopril reduce degradation at 0 degrees C but for a similar degree of protection at 25 degrees C arginine-containing dipeptides are also required. The effects of these peptidase inhibitors on the degradation profiles indicate that [3H]dynorphin A (1-8) has three main sites of cleavage: the Tyr1-Gly2, Arg6-Arg7, and Leu5-Arg6 bonds. With [3H]dynorphin A (1-9) as substrate the Arg7-Ile8 and Ile8-Arg9 bonds are also liable to cleavage. In binding assays, in contrast to the effects of peptidase inhibitors on the degradation of unbound [3H]dynorphin A (1-8) and [3H]dynorphin A (1-9), bestatin and captopril have little effect on the binding characteristics of the tritiated dynorphin A fragments at the kappa-site at 0 degrees C. However, at 25 degrees C binding is low in the absence of peptidase inhibitors. When binding at mu- and delta-sites is prevented, the maximal binding capacities of [3H]dynorphin A (1-8), [3H]dynorphin A (1-9), and [3H](-)-bremazocine at the kappa-site are similar; [3H]dynorphin A (1-9) has 5-10 times higher affinity for the kappa-site than [3H]dynorphin A (1-8). Comparison of the effects of peptidase inhibitors on unbound dynorphin A fragments with their effects in binding assays suggests that the bound peptides are protected from the action of peptidases.

Topics: Animals; Benzomorphans; Binding Sites; Brain; Dynorphins; Guinea Pigs; Kinetics; Membranes; Peptide Fragments; Protease Inhibitors; Receptors, Opioid; Receptors, Opioid, kappa; Substrate Specificity; Temperature