The major objective of this proposal is to develop highly sensitive fluorescent methods for the detection of opioid receptors on neurons and cells of the immune system. Preliminary studies have shown that by using a fluorescein-labelled opioid followed by a phycoerythrin-based amplification procedure, specific binding of the fluorescent opioid to kappa receptors on the mouse thymoma R1.1 cell line and mouse thymocytes can be detected by fluorescent microscopy and by fluorescent-activated cell sorting (FACS). This application will extend these preliminary observations to the synthesis and characterization of new fluorescein-labelled opioids that are specific for mu, delta, and kappa receptors. Selective ligands for mu, delta, and kappa receptors that are both agonists and antagonists will be synthesized by incorporating a fluorescein group in the molecule of the ligand. Since many of the selective ligands are small polypeptides, the fluorescein will be coupled to an appropriate internal amino acid. Both agonists and antagonists will be prepared since it is possible that the binding of the agonists to immune cells will be diminished since the experiments will be performed in a saline solution. On the other hand, the binding capacity of the antagonists should not be diminished in such a medium. Fluorescein labelled compounds will be tested for their affinity and selectivity for the opioid receptors using guinea pig brain membranes. Ki values for the ligands will be determined for these three major types of opioid receptors. Neuroblastoma and lymphoma cell lines, expressing mu, delta, or kappa receptors will be tested with specific fluorescent-labelled compounds. Labelled cells will be viewed by both fluorescence microscopy and FACS. Once methods have been optimized with cell lines the fluorescent ligands will be used with cells isolated from the immune system, such as thymocytes and splenocytes. These studies will show whether mu, delta, and kappa receptors can be detected on subpopulations of lymphocytes. The method will also be applied to primary neuronal cell cultures to determine whether neurons that express a specific type of opioid receptor can be detected. Finally the method will be applied to brain slices to determine histochemically the localization of mu, delta, and kappa receptors.
| Ignatowski, T A; Bidlack, J M (1998) Detection of kappa opioid receptors on mouse thymocyte phenotypic subpopulations as assessed by flow cytometry. J Pharmacol Exp Ther 284:298-306 |
| Ignatowski, T A; Bidlack, J M (1998) Changes in kappa opioid receptor expression during maturation of mouse lymphocytes. Adv Exp Med Biol 437:117-24 |
| Bidlack, J M; Lawrence, D M; Ignatowski, T A (1996) Kappa Opioid receptors on immune cells as studied by fluorescent ligands. Adv Exp Med Biol 402:13-22 |
