Endothelin-1 pathway in human alveolar epithelial cell line A549 and human umbilical vein endothelial cells.
OBJECTIVE
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).


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Deprez-Roy
I
I
Institut de Recherches Servier, 11 rue des Moulineaux, 92150 Suresnes, France.
Coge
F
F
Bertry
L
L
Galizzi
J P
JP
Feletou
M
M
Vanhoutte
P M
PM
Canet
E
E
D000230:Adenocarcinoma / Q000473:pathology; D016282:Aspartic Acid Endopeptidases / Q000096:biosynthesis* / Q000235:genetics; D002478:Cells, Cultured; D019332:Endothelin-1 / Q000378:metabolism*; D000072396:Endothelin-Converting Enzymes; D016232:Endothelins / Q000096:biosynthesis* / Q000235:genetics; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D006020:Glycopeptides / Q000494:pharmacology; D006801:Humans; D008175:Lung Neoplasms / Q000473:pathology; D008666:Metalloendopeptidases; D011498:Protein Precursors / Q000096:biosynthesis* / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis; D014407:Tumor Cells, Cultured; D014471:Umbilical Veins / Q000166:cytology / Q000378:metabolism*
D019332:Endothelin-1; D016232:Endothelins; D006020:Glycopeptides; D011498:Protein Precursors; D012333:RNA, Messenger; D016282:Aspartic Acid Endopeptidases; D008666:Metalloendopeptidases; C000606923:ECE1 protein, human; D000072396:Endothelin-Converting Enzymes; C008890:phosphoramidon