Murine splenocytes express a naloxone-insensitive binding site for beta-endorphin.
Naloxone-resistant binding sites for beta-endorphin have previously been observed on transformed peripheral blood mononuclear cells and on the EL4-thymoma cell line. These sites may be related to the naloxone-insensitive immunomodulatory effects of beta-endorphin. The present study was performed 1) to determine whether these sites are present on normal splenocytes and 2) to characterize them. Ficoll-hypaque-purified murine splenocytes were used in a RRA with [125I]beta-endorphin. Neither fresh intact cells obtained from viral antibody-free mice nor membrane preparations showed evidence of binding. However, splenocytes cultured in 5% fetal bovine serum for 24-96 h showed sites on intact cells or membranes (after 3 h in culture no sites were present). Intact cultured splenocytes demonstrated a saturable binding isotherm, and Scatchard analysis showed a single site (Kd = 4.1 X 10(-9) M). Competition studies showed that N-acetyl-beta-endorphin (N-Ac-beta-endorphin)-(1-31) was equipotent to beta-endorphin-(1-31). beta-Endorphin-(6-31) and beta-endorphin-(28-31) were approximately 10- and 1000-fold less potent, respectively, whereas beta-endorphin-(1-27) and naloxone were completely ineffective. Covalent cross-linking of [125I]beta-endorphin to splenocytes and resolution by gel electrophoresis showed bands at 66K and 57K which were displaced equipotently by increasing amounts of beta-endorphin and N-Ac-beta-endorphin. beta-Endorphin-(18-31) or (28-31) were less potent, and naloxone or other opioid ligands selective for receptor subtypes were ineffective. Thus, high affinity, naloxone-insensitive binding sites for beta-endorphin, which show competition characteristics distinctively different from brain opiate receptors, are inducible on normal mouse splenocytes. N-Ac-beta-endorphin, presumed to be an inactivation product of beta-endorphin because it fails to bind brain opiate receptors, may be functional at this naloxone-insensitive binding site.
Shahabi
N A
NA
Endocrine-Neuroscience Research Laboratory, Minneapolis Medical Research Foundation, Minnesota.
Linner
K M
KM
Sharp
B M
BM
D000818:Animals; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism; D004351:Drug Resistance; D051379:Mice; D009270:Naloxone / Q000494:pharmacology*; D010446:Peptide Fragments / Q000378:metabolism; D013154:Spleen / Q000166:cytology / Q000378:metabolism*; D013156:Splenectomy; D001615:beta-Endorphin / Q000031:analogs & derivatives / Q000378:metabolism*
D010446:Peptide Fragments; D009270:Naloxone; D001615:beta-Endorphin; C035101:N-acetyl-beta-endorphin