Hans Kosterlitz - US grants

OBJECTIVES: 1. To continue the investigation into the physiological roles of the endogenous opioid peptides, methionine-enkephalin, leucine-enkephalin and beta-endorphin. 2. To continue with the characterization of the binding of opiates and opioid peptides to mu-, delta- and kappa-receptor sites. To search for agonists and antagonists which interact specifically with each type of these receptors. 3. To use specific agonists or antagonists for the study of the differential distribution of the various opiate receptors in the central and peripheral nervous system of various species. 4. To continue to study the mechanisms mediating the release of the enkephalins and to examine the differences in the release of methionine-and leucine-enkephalin. To investigate the modulation of release by drugs. 5. To study the biosynthesis of the two enkephalins and, in particular, the kinetics of the interactions between the enkephalins and their putative precursors. 6. To continue to assess the agonist and antagonist characteristics of new synthetic narcotic analgesic drugs and to pay special attention to their differential interactions with the mu, delta, and kappa-receptors. 7. To continue the investigation of the role of the interaction between the endogenous opioid peptides and exogenously applied narcotic analgesics in the development of tolerance and dependence.

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.

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.

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.

Our principal object is the continuation of 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. One of the important problems is the coupling mechanism between binding of an opioid ligand and the response of the effector system. In this respect, the investigation of the differences found in the binding of selective ligands at mu-, delta-, and kappa-sites and their modulation by monovalent and divalent cations and by guanyl nucleotides will be continued. The selective agonists are the mu-ligand (3H)-(D-Ala2,MePhe4,Gly-ol5)enkephalin, the delta-ligand (3H)-(D-Pen2,D-Pen5)enkephalin and the kappa- ligands (3H)-dynorphin A (1-9) and (3H)-U-69,593. It is of particular importance to extend this investigation to use the selective mu-antagonist D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP; V.J. Hruby), the selective delta-antagonist naltrindole and the selective kappa-antagonist norbinaltorphimine, both developed in the laboratory of Portoghese. The other important problem is the mechanism involved in the release of fragments of pro-enkephalin and Pro-dynorphin. The basis for the investigation of the release is published in J. Neurochemistry (in press). The principle of the method is the use of two successive HPLC systems which separate the endogenous opioid peptides for subsequent assay in the mouse vas deferens. It is now possible to determine the evoked release of (Met)enkephalin, (Leu)enkephalin, (Met)enkephalyl-Arg-Gly-Leu, and (Met)enkephalyl- Arg, Phe. BAM 8 amide, (Met)enkephalyl-Arg-Arg-Val-NH;, is also released but there is no detectable release of BAM 18 although it is present in the non-stimulated tissue. The amount of released opioid varies between 18 to 30% of the tissue content.