Research Project
3206809
Our principal objective is the investigation of the physiological role of the endogenous opioid peptides by correlating the results obtained from the binding at the three major mu-, delta- and kappa-sites with those obtained in pharmacological experiments. Most recently, it has been shown in our laboratories that the hamster vas deferens has delta-receptors but no mu- or kappa-receptors. It has powerful peptidases and inhibitors are required in bioassays. Thus, there are now tissues selective for delta- and kappa-receptors (rabbit vas deferens) and possibly mu-receptors (rat vas deferens). Selective peptidase-resistant opioid ligands are [D-Ala-2, MePhe-4, Gly-ol-5]enkephalin for the mu-site and [D-Pen-2, D-Pen-5]enkephalin for the delta-site. As far as the kappa-site is concerned, the endogenous dynorphins A and B, but not dynorphin A (1-8), are sufficiently selective. The non-endogenous dynorphin A (1-9) is selective but as all dynorphins, with the exception of dynorphin A, is peptidase-sensitive. Another approach to this problem is the use of the guinea-pig cerebellum which has mainly kappa-sites (our laboratories) or the rabbit cerebellum which has mainly mu-sites. The coupling mechanisms between the mu-binding sites and the effector system in the guinea-pig ileum is still not well understood. We are now investigating the differential inhibitory effects of Na+ and other monovalent and divalent ions and GPT in the binding of mu-, delta- and kappa-sites after different degrees of homogenisation. This will lead to an analysis of the reasons for the lack of effects of Na+ and GPT on the binding of antagonists and on binding of agonists at the kappa-site. Recent work in this laboratory has developed HPLC procedures with columns of different properties for the separation of all fragments of the opioid precursors in one sample of tissue. The eluate will be assayed with the mouse vas deferens. The content of the endogenous opioid peptides will be determined in brain regions and in the myenteric plexus. The effect of electrical stimuation will be measured by the amount of opioid peptides released into the bath fluid. The results will be correlated with those obtained by radioimmuno-assay to be established in our laboratories.
