Expression of naloxone-resistant β-endorphin binding sites on A20 cells: effects of concanavalin A and dexamethasone

Abstract

β-Endorphin affects mononuclear cell proliferation, cytokine production and calcium uptake in a naloxone-resistant manner. The presence of naloxone-insensitive binding sites for β-endorphin have been demonstrated on murine EL4-thymoma cells, transformed human mononuclear cells and normal murine splenocytes. Since murine splenic B cells have been shown to express naloxone-resistant receptors for β-endorphin in response to the mitogen, concanavalin A (Con A), the A20 B-cell lymphoma line was used to further study regulation of this site by Con A and dexamethasone. Analyses showed two sites: a high-affinity site, Kd1 = (8.7 ± 2.3) × 10−11 M and binding capacity (Bmax1) of (2.6 ± 2.0) × 103 receptors/cell; and a low-affinity site, Kd2 = (2.2 ± 0.8) × 10−8 M with Bmax2 of (1.5 ± 0.8) × 105 receptors/cell. Competition studies showed that N-acetyl-β-endorphin was approx. 5-fold and β-endorphin6−31 10-fold less potent than β-endorphin1−31. Neither β-endorphin1−27 nor naloxone, morphine or other opioid receptor agonists displaced [125I]β-endorphin. Con A (20 μg/ml) significantly increased the Bmax (3.5-fold; expressed per cell) and resulted in a loss of the higher-affinity site. However, the increased Bmax occurred in proportion to the Con-A-induced increase in protein/cell. Dexamethasone (Dex) also increased Bmax, primarily by increasing (2–3-fold) the number of lower affinity sites. In contrast to Con A, two binding sites persisted after treatment with Dex, which exerted a minimal effect on protein/cell. Therefore, binding/cell and binding/protein/cell were both significantly enhanced by Dex. The combined effects of Dex and Con A on binding failed to show additivity or synergy. When binding was analyzed per protein/cell, the effect of Con A appeared to dominate; the Dex-enhanced binding/protein/cell was no longer evident in the presence of Dex plus Con A. Thus, Dex and Con A may enhance binding by independent mechanisms.