Endothelin-1 pathway in human alveolar epithelial cell line A549 and humanumbilical vein endothelial cells

AIM: This study was designed to characterize the endothelin pathway in an i mmortalized human adenocarcinoma-derived alveolar epithelial cell line (A54 9) and human umbilical vein endothelial cell line (HUVEC). METHODS: The rel ease of ET-1 and big-ET-1 was measured in the incubation medium of both cel l 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 th e hET(A) and hET(B) 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. RESU LTS: 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 var ious inhibitors of peptidases. However, in both cell lines phosphoramidon p roduced a concentration-dependent inhibition of ET-1 release and an enhance d 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. CONCLUSION: Both A549 and HUVEC produce and release endothelin-1 through a specific enzymatic pathway, whether or not ECE-1 is the only enzyme involve d remains to be determined. A549 might be used as a screening assay for dru g discovery such as for inhibitors of endothelin-l release.