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Expression of naloxone-resistant beta-endorphin binding sites on A20 cells: effects of concanavalin A and
M Shaker1, N A Shahabi, B M Sharp
1Endocrine-Neuroscience and Neuroimmunomodulation Research Laboratory, Minneapolis Medical Research Foundation, MN.
Abstract:
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.
Insights
Beta-endorphin binding sites on B cells are regulated by concanavalin A (Con A) and dexamethasone (Dex). Con A increases binding capacity, while Dex enhances binding affinity and capacity, suggesting independent regulatory mechanisms.
Area of Science:
- Immunology
- Neuroendocrinology
- Cell Biology
Background:
- Beta-endorphin influences immune cells via naloxone-resistant pathways.
- Naloxone-insensitive beta-endorphin binding sites are present on various immune cells, including murine splenocytes.
Purpose of the Study:
- To investigate the regulation of naloxone-resistant beta-endorphin binding sites on A20 B-cell lymphoma cells.
- To determine the effects of concanavalin A (Con A) and dexamethasone (Dex) on these binding sites.
Main Methods:
- Characterization of beta-endorphin binding sites using radioligand binding assays.
- Competition studies with various beta-endorphin analogues and opioid receptor agonists.
- Treatment of A20 cells with Con A and/or Dex to assess changes in binding parameters.
Main Results:
- Two binding sites were identified: a high-affinity site (Kd1) and a low-affinity site (Kd2).
- Con A increased the Bmax (binding capacity) per cell, predominantly affecting the low-affinity site and reducing the high-affinity site.
- Dex increased Bmax, primarily by upregulating the low-affinity sites, and enhanced binding per protein per cell.
Conclusions:
- Con A and Dex differentially regulate beta-endorphin binding sites on B cells.
- Con A's effect on binding per protein per cell dominated over Dex's effect when used in combination.
- These findings suggest independent mechanisms by which Con A and Dex modulate beta-endorphin binding.