Characterization of a (+)-azidophenazocine-sensitive sigma receptor on splenic lymphocytes.

Abstract

A study was undertaken to structurally define and functionally assess sigma receptors on splenocytes using the highly selective sigma ligand (+)-azidophenazocine. Radioreceptor assays under reduced lighting show (+)-azidophenazocine can effectively block the binding of sigma ligands [3H]haloperidol (IC50 = 30 nM, Ki = 19.0 nM) and [3H](+)-pentazocine (IC50 = 40 nM, Ki = 350 nM), but not the dopamine (D2 and [3H]spiperone (IC50 > 5 microM) to splenic lymphocytes. [3H](+)-1-Propyl-3-(3-hydroxyphenyl)piperidine ([3H](+)-PPP) sites (Kd = 40.8 nM, Bmax = 2.32 pmol/mg) were also present on these lymphocytes. Additional studies using [3H](+)-azidophenazocine indicated the presence of saturable sites (Kd = 29.7 nM, Bmax = 760 fmol/mg) on splenic lymphocytes. There are no significant differences in affinity between sites found on T-enriched (Kd = 59 +/- 47 nM) and B-enriched lymphocytes (Kd = 23 +/- 5 nM). Photoaffinity labeling studies of splenocyte membranes with [3H](+)-azidophenazocine revealed a protein migrating at an apparent m.w. of 57 kDa under reducing and nonreducing conditions on SDS-PAGE. The labeling was specific because pretreatment with unlabeled haloperidol, (+)-PPP, 1,3 di(2-tolyl)guanidine, (+)-pentazocine, and (+)-azidophenazocine before cross-linking competed away > 75% of the radioactivity associated with the protein, whereas (-)-pentazocine and naloxone were significantly less effective. This data together with the observation that both (+)-azidophenazocine or haloperidol inhibit Con A-induced production of IFN by splenocytes, indicates that lymphocytes possess a biologically relevant sigma receptor.