Expression of naloxone-resistant beta-endorphin binding sites on A20 cells: effects of concanavalin A and dexamethasone.
beta-Endorphin affects mononuclear cell proliferation, cytokine production and calcium uptake in a naloxone-resistant manner. The presence of naloxone-insensitive binding sites for beta-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 beta-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) x 10(-11) M and binding capacity (Bmax1) of (2.6 +/- 2.0) x 10(3) receptors/cell; and a low-affinity site, Kd2 = (2.2 +/- 0.8) x 10(-8) M with Bmax2 of (1.5 +/- 0.8) x 10(5) receptors/cell. Competition studies showed that N-acetyl-beta-endorphin was approx. 5-fold and beta-endorphin6-31 10-fold less potent than beta-endorphin1-31. Neither beta-endorphin1-27 nor naloxone, morphine or other opioid receptor agonists displaced [125I]beta-endorphin. Con A (20 micrograms/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.
Shaker
M
M
Endocrine-Neuroscience and Neuroimmunomodulation Research Laboratory, Minneapolis Medical Research Foundation, MN.
Shahabi
N A
NA
Sharp
B M
BM
D000818:Animals; D001402:B-Lymphocytes / Q000187:drug effects* / Q000378:metabolism*; D001665:Binding Sites; D001667:Binding, Competitive; D002460:Cell Line; D003208:Concanavalin A / Q000494:pharmacology; D003907:Dexamethasone / Q000494:pharmacology; D004351:Drug Resistance; D006801:Humans; D007700:Kinetics; D051379:Mice; D009270:Naloxone / Q000494:pharmacology*; D010446:Peptide Fragments / Q000378:metabolism; D011869:Radioligand Assay; D013936:Thymidine / Q000378:metabolism; D001615:beta-Endorphin / Q000031:analogs & derivatives / Q000378:metabolism*
D010446:Peptide Fragments; D003208:Concanavalin A; D009270:Naloxone; D001615:beta-Endorphin; D003907:Dexamethasone; D013936:Thymidine