Dieterich K D, Grigoriadis D E, De Souza E B
Neurocrine Biosciences, Inc., San Diego, California 92121.
Endocrinology. 1994 Oct;135(4):1551-8. doi: 10.1210/endo.135.4.7925116.
Numerous peptides, growth factors, and receptors have been identified in small cell lung carcinoma (SCLC) cells. The present study was designed to examine the radioligand binding, second messenger, and messenger RNA (mRNA) characteristics of CRF receptors in a variety of SCLC lines and to compare their characteristics to CRF receptors in the mouse pituitary tumor AtT-20 cells. The human SCLC cell lines NCI-H69, H82, H146, H209, H345, H446, and H510A and control AtT-20 cells all demonstrated specific [125I]Tyr(o)-ovine CRF ([125I]oCRF) binding, which was linear with increasing protein concentrations, saturable, reversible, and of high affinity. NCI-H82 cells showed the highest level of specific [125I]oCRF binding (approximately 60% of the total binding). Scatchard analysis revealed a single homogeneous class of binding sites in NCI-H82 and AtT-20 cells, with Kd values of 263 +/- 48 and 285 +/- 75 pM, respectively, and binding capacities of 74 +/- 7 and 70 +/- 13 fmol/mg protein, respectively. [125I]oCRF-binding sites on NCI-H82 and AtT-20 cells had comparable pharmacological characteristics with the following rank order of inhibitory potencies: rat/human CRF approximately ovine CRF approximately bovine CRF > alpha-helical oCRF-(9-41) > bovine CRF-(1-41)OH >> vasoactive intestinal peptide, secretin, GH-releasing hormone. [125I]oCRF binding in the cell lines was inhibited by guanine nucleotides, suggesting a coupling of receptors to guanine nucleotide-binding proteins. The functional nature of the CRF receptor was demonstrated in second messenger studies in which rat/human CRF stimulated cAMP production in NCI-H82 and AtT-20 cells with comparable EC50 values of about 3 nM; the percent stimulation over basal activity was significantly higher in NCI-H82 cells (approximately 30-fold increase) than in AtT-20 cells (approximately 12-fold increase). Northern blot analysis of total RNA revealed the presence of a 2.6-kilobase mRNA band in NCI-H82 cells corresponding to the recently cloned human CRF receptor. In summary, the data demonstrate the presence of CRF receptors in SCLC cell lines with kinetic, pharmacological, second messenger, and mRNA characteristics comparable to those in pituitary and brain and suggest a possible role for CRF as a regulatory peptide in human SCLC.
在小细胞肺癌（SCLC）细胞中已鉴定出许多肽、生长因子和受体。本研究旨在检测多种SCLC细胞系中促肾上腺皮质激素释放因子（CRF）受体的放射性配体结合、第二信使和信使核糖核酸（mRNA）特征，并将其特征与小鼠垂体瘤AtT - 20细胞中的CRF受体进行比较。人SCLC细胞系NCI - H69、H82、H146、H209、H345、H446和H510A以及对照AtT - 20细胞均表现出特异性的[125I]酪氨酸（o）-羊CRF（[125I]oCRF）结合，其与蛋白质浓度增加呈线性关系，具有饱和性、可逆性且亲和力高。NCI - H82细胞显示出最高水平的特异性[125I]oCRF结合（约占总结合的60%）。Scatchard分析显示NCI - H82和AtT - 20细胞中存在单一均匀的结合位点类别，Kd值分别为26
3±48和285±75 pM，结合容量分别为74±7和70±13 fmol/mg蛋白质。NCI - H82和AtT - 20细胞上的[125I]oCRF结合位点具有可比的药理学特征，抑制效力的等级顺序如下：大鼠/人CRF≈羊CRF≈牛CRF＞α - 螺旋oCRF - (9 - 41)＞牛CRF - (1 - 41)OH＞＞血管活性肠肽、促胰液素、生长激素释放激素。细胞系中的[125I]oCRF结合受到鸟嘌呤核苷酸的抑制，表明受体与鸟嘌呤核苷酸结合蛋白偶联。在第二信使研究中证实了CRF受体的功能性质，其中大鼠/人CRF刺激NCI - H82和AtT - 20细胞中的环磷酸腺苷（cAMP）产生，可比的半数有效浓度（EC50）值约为3 nM；NCI - H82细胞中相对于基础活性的刺激百分比显著高于AtT - 20细胞（约增加30倍）（约增加12倍）。对总RNA的Northern印迹分析显示NCI - H82细胞中存在一条2.6千碱基的mRNA条带，对应于最近克隆的人CRF受体。总之，数据表明SCLC细胞系中存在CRF受体，其动力学、药理学、第二信使和mRNA特征与垂体和脑中的相当，并提示CRF作为一种调节肽在人SCLC中可能发挥作用。
