Shahabi N A, Linner K M, Sharp B M
Endocrine-Neuroscience Research Laboratory, Minneapolis Medical Research Foundation, Minnesota.
Endocrinology. 1990 Mar;126(3):1442-8. doi: 10.1210/endo-126-3-1442.
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.
先前已在转化的外周血单核细胞和EL4-胸腺瘤细胞系上观察到β-内啡肽的纳洛酮抗性结合位点。这些位点可能与β-内啡肽的纳洛酮不敏感免疫调节作用有关。本研究旨在：1）确定正常脾细胞上是否存在这些位点；2）对其进行表征。用Ficoll-泛影葡胺纯化的小鼠脾细胞与[125I]β-内啡肽进行放射受体分析。无论是从无病毒抗体的小鼠获得的新鲜完整细胞还是膜制剂，均未显示出结合的证据。然而，在含5%胎牛血清中培养24-96小时的脾细胞，在完整细胞或膜上显示出结合位点（培养3小时后不存在结合位点）。完整的培养脾细胞表现出可饱和的结合等温线，Scatchard分析显示为单一结合位点（解离常数Kd = 4.1×10(-9) M）。竞争研究表明，N-乙酰-β-内啡肽（N-Ac-β-内啡肽）-(1-31)与β-内啡肽-(1-31)的效力相当。β-内啡肽-(6-31)和β-内啡肽-(28-31)的效力分别约低10倍和1000倍，而β-内啡肽-(1-27)和纳洛酮则完全无效。[125I]β-内啡肽与脾细胞的共价交联及凝胶电泳分离显示，在66K和57K处出现条带，随着β-内啡肽和N-Ac-β-内啡肽量的增加，这些条带被等量取代。β-内啡肽-(18-31)或(28-31)的效力较低，而纳洛酮或其他对受体亚型有选择性的阿片样物质配体则无效。因此，β-内啡肽的高亲和力、纳洛酮不敏感结合位点可在正常小鼠脾细胞上诱导产生，其竞争特性与脑阿片受体明显不同。由于N-乙酰-β-内啡肽不能结合脑阿片受体，推测它是β-内啡肽的失活产物，但它可能在这个纳洛酮不敏感的结合位点发挥作用。
