Identification of Opioid Receptors in the Immune System Using a Novel Combination of Selective Opioid Ligands and Indirect Phycoerythrin Immunofluorescence

Abstract

Although there is substantial evidence for direct, in vitro opioid effects on the immune system, radioligand binding studies have not definitively described brain opioid receptors on these cells (1). It is possible that the density of receptors required for immunomodulation is too low to detect with radioligands, or that only a small percentage of cells may express opioid receptors in heterogeneous leukocyte populations. In addition, the level of receptor expression in leukocytes may depend on the developmental stage or the activation state of the cells. The use of fluorescent opioid ligands would allow visualization of opioid receptors by microscopy, analysis of cell subpopulations for opioid receptor expression, or separation of cells expressing opioid receptors by flow cytometry. Several groups have synthesized fluorescent opioid probes, using fluorophores such as 1-dimethylaminonaphthalene-5-sulfonic acid (dansyl) (2, 3), rhodamine (4, 5), fluorescein (5), pyrene (6), and nitrobenzodiazole (7). Although these compounds showed high affinity and biological activity at opioid receptors, the peak emission wavelengths of some may overlap with autofluorescence (3). In addition, irradiation at wavelengths near the excitation peak for dansyl compounds can greatly reduce opioid receptor affinity by irreversibly altering the structures of opioid receptors and ligands (8). The longer excitation and emission wavelengths of the rhodamine-and fluorescein-labeled opioid probes (4, 5) avoid the problems of irradiation-induced receptor alteration and tissue autofluo-rescence, but specific histological labeling of opioid receptors with these fluorescent ligands has not been demonstrated.