Deprez-Roy I, Coge F, Bertry L, Galizzi J P, Feletou M, Vanhoutte P M, Canet E
Institut de Recherches Servier, 11 rue des Moulineaux, 92150 Suresnes, France.
Acta Pharmacol Sin. 2000 Jun;21(6):499-506.
This study was designed to characterize the endothelin pathway in an immortalized human adenocarcinoma-derived alveolar epithelial cell line (A549) and human umbilical vein endothelial cell line (HUVEC).
The release of ET-1 and big-ET-1 was measured in the incubation medium of both cell lines. The expression of mRNAs coding for the endothelin isoforms (hppET-1, -2, -3), the endothelin converting enzymes (hECE-1a, b, c, and d) and the hETA and hETB receptors was investigated using RT-PCR. The expression of ECE-1 mRNA in various human tissues and in A549 cells was investigated by Northern blot analysis and the subcellular localization of ECE-1 in A549 cells was investigated by immunoblotting using a polyclonal antibody.
Under control conditions, HUVEC release both ET-1 and big-ET-1 (ratio 5 to 1) while in A549 cells the big-ET-1 levels were below the threshold of detection. The release of these two peptides was minimally affected by various inhibitors of peptidases. However, in both cell lines phosphoramidon produced a concentration-dependent inhibition of ET-1 release and an enhanced accumulation of big-ET-1. Both HUVEC and A549 cells express the mRNAs for ppET-1, ET-A, and ET-B receptor subtypes and ECE-1 (isoforms ECE-1b, c and/or d). In addition, in HUVEC the mRNAs for ppET-2 and for the isoform ECE-1a were also detected. In A549 cells, ECE-1 had a preferential subcellular localization in the membrane fraction but was not detected in the cytosol.
Both A549 and HUVEC produce and release endothelin-1 through a specific enzymatic pathway, whether or not ECE-1 is the only enzyme involved remains to be determined. A549 might be used as a screening assay for drug discovery such as for inhibitors of endothelin-1 release.
本研究旨在对永生化人腺癌来源的肺泡上皮细胞系（A549）和人脐静脉内皮细胞系（HUVEC）中的内皮素途径进行特征描述。
在两种细胞系的培养液中检测ET-1和大内皮素-1的释放。采用逆转录聚合酶链反应（RT-PCR）研究编码内皮素同工型（hppET-1、-2、-3）、内皮素转化酶（hECE-1a、b、c和d）以及hETA和hETB受体的mRNA的表达。通过Northern印迹分析研究ECE-1 mRNA在各种人体组织和A549细胞中的表达，并使用多克隆抗体通过免疫印迹法研究ECE-1在A549细胞中的亚细胞定位。
在对照条件下，HUVEC释放ET-1和大内皮素-1（比例为5比1），而在A549细胞中，大内皮素-1水平低于检测阈值。这两种肽的释放受各种肽酶抑制剂的影响极小。然而，在两种细胞系中，磷酰胺素均产生浓度依赖性的ET-1释放抑制和大内皮素-1的积累增强。HUVEC和A549细胞均表达ppET-1、ET-A和ET-B受体亚型以及ECE-1（ECE-1b、c和/或d同工型）的mRNA。此外，在HUVEC中还检测到ppET-2和ECE-1a同工型的mRNA。在A549细胞中，ECE-1在膜部分有优先的亚细胞定位，但在胞质溶胶中未检测到。
A549和HUVEC均通过特定的酶促途径产生和释放内皮素-1，ECE-1是否是唯一涉及的酶仍有待确定。A549可作为药物发现的筛选试验，例如用于内皮素-1释放抑制剂的筛选。
