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 beta-endorphin on immune cells are resistant to inhibition by naloxone. To further characterize the beta-[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 beta-[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 beta-endorphin and N-acetyl (Ac)-beta-endorphin but not by naloxone, morphine, or selective opiate receptor agonists. Competition studies showed that beta-endorphin-(6-31) and beta-endorphin-(28-31) were approximately 5- and 100-fold less potent, respectively, whereas beta-endorphin-(1-16) or -(1-27) was ineffective. Covalent cross-linking of beta-[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 beta-endorphin and N-Ac-beta-endorphin. Increasing concentrations of mono (Na+, K+)- and divalent (Ca2+, Mg2+, Mn2+) cations reduced the binding of beta-[125I]endorphin to U937 membrane; beta-[125I]endorphin binding to rat brain membrane showed similar cation sensitivity. GTP gamma-sulfate (GTP gamma S; 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 gamma S reduced binding by an additional 50%. Moreover, GTP gamma S (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, beta-endorphin binds to sites on human U937 cells similar to those observed on normal murine splenocytes. 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.