Serotonin (5-HT) is an important neurotransmitter in the central nervous system and postsynaptic serotonin receptors and the serotonin uptake system have been implicated in the etiology and/or therapeutic treatment of human mental depression. This project will use two aryl azides as photoaffinity probes to characterize, in molecular terms, the 5-HT-1A receptor and the serotonin uptake carrier. This information should greatly aid in the understanding of mental depression. The two probes, which were developed in this laboratory, are 1-(2-(4-azidophenyl)ethyl)-4-(3-trifluoromethylphenyl)piperazine (p-azido-TFMPP) and serotonin azidobenzamidine (SABA). p-Azido-TFMPP has high affinity for the 5-HT-1A site and binding experiments with the tritiated compound will be performed with rat brain membranes to assess the specificity of the labeling. Once this is demonstrated, [3H]p-azido-TFMPP will be used to covalently label the 5-HT-1A receptor in photolysis experiments. The labeled proteins will be separated by SDS-polyacrylamide gel electrophoresis and serotonin receptor agonists and antagonists will be used to identify the polypeptide associated with the 5-HT-1A receptor. Pharmacological procedures will be used to demonstrate the synaptic location of the polypeptide. An active 5-HT-1A receptor will be solubilized with detergents and purified by affinity chromatography using the p-amino analogue of p-azido-TFMPP as the column ligand. Experiments will be performed with p-azido-TFMPP to assess the relationship between the 5-HT-1A receptor and the serotonin stimulated adenylate cyclase in rat hippocampus. Photolysis experiments with unlabeled p-azido-TFMPP will demonstrate whether the serotonin stimulated adenylate cyclase can be irreversibly inhibited concomitant with inhibition of [3H]p-azido-TFMPP binding to 5-HT-1A receptors. The specificity of the photoaffinity labeling of the 5-HT uptake site in rat brain syanptosomes and human platelets by [3H]SABA will be established [3H]SABA labeled polypeptides will be separated and identified by SDS-polyacrylamide gel electrophoresis of the photolyzed tissue samples. The ability of tricyclic antidepressants to protect against labeling of the carrier polypeptide will be evaluated to determine whether or not these drugs inhibit 5-HT uptake by binding to the same polypeptide that contains the substrate binding site. Comparison of the proteins specifically labeled by [3H]SABA and [3H]azidoimipramine will provide further information concerning the relationship of the carrier macromolecule and the tricyclic binding site.
Showing the most recent 10 out of 80 publications
