BETA-ENDORPHIN BINDING TO NALOXONE-INSENSITIVE SITES ON A HUMAN MONONUCLEAR CELL-LINE (U937) - EFFECTS OF CATIONS AND GUANOSINE TRIPHOSPHATE

Some of the functional effects of β-endorphin on immune cells are resistant to inhibition by naloxone. To further characterize the β-[125I]endorphin-binding site mediating these effects and its response to cations and GTP, the human monocyte-like cell line U937 was used. Incubation of intact cells and -β-[125I]endorphin for 60 min at 4 C demonstrated a saturable, high affinity binding site [Kd = 1.2 ± 0.5 x 10-8 M (mean ± SE; n = 4] competed by equimolar β-endorphin and N-acetyl (Ac)-β-endorphin but not by naloxone, morphine, or selective opiate receptor agonists. Competition studies showed that β-endorphin-(6-31) and β-endorphin-(28-31) were approximately 5-and 100fold less potent, respectively, whereas β-endorphin-(l-16) or -(1-27) was ineffective. Covalent cross-linking of β-[125I]endorphin to intact cells and resolution by gel electrophoresis showed dominant bands at 59K and 44K and a minor band at 66K. The bands at 44K and 66K were completely displaced by increasing equivalent concentrations of β-endorphin and N-Ac-β-endor-phin. Increasing concentrations of mono (Na+K+)-and divalent (Ca2+Mg2+Mn2+) cations reduced the binding of β-[125I]endorphin to U937 membrane; β-[125I] endorphin binding to rat brain membrane showed similar cation sensitivity. GTP7-sulfate (GTPγS; 10-4 M) alone reduced binding to U937 membrane by 25%. In the presence of Na+ (100 or 150 mM) or Mg2+ (10 mM), GTP γS reduced binding by an additional 50%. Moreover, GTP γS (10-8-10-4 M) in the presence of Na+ (100 mM) reduced binding in a dose-dependent manner, whereas GMP was ineffective. In conclusion, β-endorphin binds to sites on human U937 cells similar to those observed on normal murine spleno-cytes. Although naloxone insensitive, these sites exhibit properties, such as size, salt sensitivity, and coupling to a GTP-binding protein, that are similar to those observed for agonist binding to brain opiate receptors. © 1990 by The Endocrine Society.