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 o f CC16 kD protein specific for these cells; we studied their capacity to generate endothelin 1 (ET-1). Using monoclonal antibody and immunof luorescence techniques, ET-1 was localized in these cultured Clara cel ls. The basal release of immunoreactive endothelin (ir-ET), measured b y 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, respe ctively. Treatment of Clara cells with phosphoramidon (100 mu M), an i nhibitor of the endothelin-converting enzyme, caused a significant red uction 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 decreas ed 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 su pernatant. 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 treat ment of the cells with phosphoramidon (1 mM) but not with thiorphan (1 mM). Our results suggest that Clara cells release ET-1, a potent bron choconstrictive 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 indirectl y, in lung pathophysiology.