白三烯C4的特异性放射受体测定法及钙离子载体诱导人白细胞产生白三烯C4的测定

A specific radioreceptor assay for leukotriene C4 and the measurement of calcium ionophore-induced leukotriene C4 production from human leukocytes.

作者信息

Tamura N, Agrawal D K, Townley R G

机构信息

Department of Medicine, Creighton University School of Medicine, Omaha, NE 68178.

出版信息

J Pharmacol Methods. 1987 Dec;18(4):327-33. doi: 10.1016/0160-5402(87)90064-7.

DOI:10.1016/0160-5402(87)90064-7

PMID:3695541

Abstract

A highly specific radioreceptor assay (RRA) for leukotriene (LT) C4 using BC3H-1 cell membrane as a source of LTC4 receptors was developed and demonstrated its use in the measurement of calcium ionophore A23187-induced LTC4 production from purified eosinophils, neutrophils, and mononuclear cells. Unlabeled LTC4 competed for the specific [3H]LTC4 binding to the BC3H-1 cell membranes in a dose-dependent manner. Under the experimental conditions used in this study, the calculated IC50 value of unlabeled LTC4 was 27.2 +/- 1.2 nM (n = 5). The sensitivity of the method for LTC4 was 0.6 pmol. The cross-reactivities of LTD4, LTE4, and FPL 55712 were negligible. The recovery of the exogenously added LTC4 with eosinophils was greater than 90% in both the presence and the absence of calcium ionophore A23187. Calcium ionophore induced 73.0 +/- 17.0 ng of LTC4 production/10(6) eosinophils (n = 8), and this was about 20-40 times more than those from neutrophils and from mononuclear cells.

摘要

利用BC3H-1细胞膜作为白三烯（LT）C4受体来源，开发了一种用于LT C4的高特异性放射受体分析（RRA），并证明了其在测量钙离子载体A23187诱导的纯化嗜酸性粒细胞、中性粒细胞和单核细胞产生LT C4中的应用。未标记的LT C4以剂量依赖性方式竞争与BC3H-1细胞膜的特异性[3H]LT C4结合。在本研究使用的实验条件下，未标记的LT C4的计算IC50值为27.2±1.2 nM（n = 5）。该方法对LT C4的灵敏度为0.6 pmol。LTD4、LTE4和FPL 55712的交叉反应可忽略不计。在有和没有钙离子载体A23187的情况下，嗜酸性粒细胞中外源添加的LT C4的回收率均大于90%。钙离子载体诱导73.0±17.0 ng LT C4产生/10(6)嗜酸性粒细胞（n = 8），这比中性粒细胞和单核细胞产生的量多约20-40倍。