Guinea pig Clara cells secrete endothelin 1 through a phosphoramidon-sensitive pathway.
We have developed a method to harvest and culture Clara cells isolated from guinea pig lungs. Their identity was confirmed by the presence of CC16kD protein specific for these cells; we studied their capacity to generate endothelin 1 (ET-1). Using monoclonal antibody and immunofluorescence techniques, ET-1 was localized in these cultured Clara cells. The basal release of immunoreactive endothelin (ir-ET), measured by radioimmunoassay, from cultured Clara cells incubated for 2, 6, and 10 h was 74.8 +/- 11.1, 230.0 +/- 32.0 and 331.0 +/- 22.9 pg/ml, respectively. Treatment of Clara cells with phosphoramidon (100 microM), an inhibitor of the endothelin-converting enzyme, caused a significant reduction of the ir-ET release by 40% after a 6-h incubation period (P<0.01). Following treatment with 1 mM phosphoramidon, ir-ET was decreased by 73% and 76% after 6- and 10-h incubation periods, respectively (P<0.01). In contrast, treatment with thiorphan (1 mM), an inhibitor of neutral endopeptidase, increased the levels of ir-ET in the cell supernatant. High-performance liquid chromatography of supernatants from cultured Clara cells revealed one peak corresponding to the retention time of synthetic ET-1. This peak was greatly reduced following treatment of the cells with phosphoramidon (1 mM) but not with thiorphan (1 mM). Our results suggest that Clara cells release ET-1, a potent bronchoconstrictive agent. Furthermore, the synthesis of ET-1 is dependent on a phosphoramidon-sensitive endothelin-converting enzyme. Secretion of this peptide by Clara cells may play a role, directly or indirectly, in lung pathophysiology.
Laporte
J
J
Department of Pharmacology, Medical School, University of Sherbrooke, Sherbrooke, Québec, Canada.
D'Orléans-Juste
P
P
Sirois
P
P
D000818:Animals; D000911:Antibodies, Monoclonal; D016282:Aspartic Acid Endopeptidases / Q000037:antagonists & inhibitors*; D002478:Cells, Cultured; D000072396:Endothelin-Converting Enzymes; D016232:Endothelins / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005455:Fluorescent Antibody Technique; D006020:Glycopeptides / Q000494:pharmacology*; D006168:Guinea Pigs; D008168:Lung / Q000166:cytology / Q000378:metabolism*; D008666:Metalloendopeptidases; D013047:Specific Pathogen-Free Organisms; D015244:Thiorphan / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D016232:Endothelins; D004791:Enzyme Inhibitors; D006020:Glycopeptides; D015244:Thiorphan; D016282:Aspartic Acid Endopeptidases; D008666:Metalloendopeptidases; D000072396:Endothelin-Converting Enzymes; C008890:phosphoramidon