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Title: Opiate receptor mechanisms : an in vitro analysis of multiple binding sites and interaction with adrenoceptors
Author: Robson, Linda E.
ISNI:       0000 0004 2667 8987
Awarding Body: University of Aberdeen
Current Institution: University of Aberdeen
Date of Award: 1982
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A series of novel experiments investigating the protection of opioid binding sites from irreversible inactivation was designed to establish, by direct means, whether there are mu, delta- and kappa-binding sites in homogenates of guinea-pig brain. Phenoxybenzamine was shown to cause a long-lasting inactivation of the binding of (3H)-dihydromorphine, ( 3H) -(D-Ala2, D-Leu5) enkephalin and (3H)-ethylketazocine. Unlabelled (D-Ala2, D-Leu5) enkephalin, a ligand with high affinity for the putative delta-binding site, protects the binding of (3H)-(D-Ala2, D-Leu 5) enkephalin, from this effect of phenoxy benzamine, more readily than the binding of (3H)-dihydro-morphine, which has high affinity for the putative mu-binding site. Conversely, unlabelled dihydromorphine protects (3H)-dihydromorphine binding sites more readily than those of (3H)-(D-Ala2, D-Leu5) enkephalin, whereas (D-Ala2, L-Leu-NH25)enkephalin, which has high affinities for both mu- and delta-binding sites, protects the binding of both tritiated ligands equally well. It is concluded that mu- and delta-binding sites exist as separate entities in guinea-pig brain. In a further series of protection experiments it was shown that ethylketazocine readily protects the mu- and delta-binding sites, and binds in addition, to a site, the kappa-binding site, which can be protected by only very high concentrations of dihydro-morphine and which is not protected by (D-Ala 2, D-Leu5) enkephalin at concentrations up to 4,900 nM. The results of the protection assays are in good agreement with those of competitive displacement assays in guinea-pig brain. The characteristics and significance of the mu-, delta- and kappa-binding sites are discussed. In view of the high degree of cross-reactivity of ketazocine-like compounds for mu- and delta-binding sites, selective labelling of kappa-binding sites can only be achieved, so far, by the measurement of the binding of labelled ketazocine-like compounds in the presence of unlabelled mu- and delta-ligands. By using this technique and tritiated selective and ligands, the characteristics of the subtypes of the binding sites were investigated in homogenates of the longitudinal muscle-myenteric plexus of the guinea-pig. By analysis of saturation curves, the proportion of the binding sites were found to be 24.3% mu-sites, 28.4% delta-sites and 4-7.3% kappa-sites. These findings are discussed in relation to the pharmacological profiles of prototype ligands in the electrically stimulated ileum. By selective labelling of the mu-, delta- and kappa-binding sites, with single concentrations of prototype ligands, it was shown that each has a different distribution in guinea-pig brain. The patterns of distribution differ in some respects from those reported in the rat and cow. These findings are discussed and the limitations of the technique are evaluated. The aforementioned investigations of binding to the recognition sites of the opiate receptor are presented and discussed in Section A of this thesis. Proposals for future research are outlined. The investigation of the effects of opioids and other drugs in in vitro bioassay preparations allows, in addition to investigation of interactions at the recognition sites of receptors, consideration of post-recognition events. In Section B of this thesis an interaction between presynaptic opiate receptors and alpha-adrenoceptors in the electrically stimulated ileum from guinea-pigs implanted with morphine pellets is described. Briefly, when morphine was absent from the bath fluid the inhibitory effects of the presynaptic alpha-adrenoceptor agonists, clonidine and oxymetazoline, were much reduced, and the dose-response curves were flat. This state of "withdrawal" was readily reversed by morphine and levorphanol, but not by its inactive (+)-isomer, dextorphan. Ketazocine and ethylketazocine and two opioid peptides, (D-Ala2, D-Leu 5)enkephalin (delta-selective peptide) and (D-Ala 2, MePhe4, Met(0-ol5)-enkephalin (mu-selective peptide) also restored the effects of clonidine. The inhibitory effects of adrenaline and adenosine 3', 5'-diphosphate were reduced at low, but not at high concentrations. The possible site and mechanism of this interaction between presynaptic opiate receptors and alpha-adrenoceptors are discussed, and the possible relationship of such interactions to the phenomena of tolerance and dependence are considered. Proposals for future research are outlined.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Pharmacology & pharmacy & pharmaceutical chemistry