Characterization of endothelin release from guinea-pig tracheal epithelium: influence of proinflammatory mediators...

The characteristics of endothelin (ET) release from guinea-pig tracheal epithelium were investigated, including examination of the effects of several pro-inflammatory mediators. In confluent cultured guinea-pig tracheal epithelial cells (GPTECs) there was a time-dependent basal release of immunoreactive ET (ir-ET) from 4-48 h. Basal ir-ET release from GPTECs was unaffected by the peptidase inhibitors, thiorphan (10 microM), benzamidine (1 mM), pepstatin-A (30 microM), aprotinin (1 microgram/ml), bacitracin (20 micrograms/ml) or leupetin (50 microM), but was inhibited by phosphoramidon, the neutral metalloprotease inhibitor (IC50 = 16.8 microM), or the calcium chelator, EGTA (10 mM). There was little ir-ET release 1 day after placing GPTECs in culture, although appreciable release (> 10-fold higher) was detected on days 5 and 7. No significant release of ir-ET was demonstrated from intact guinea-pig trachea. Human thrombin (0.1-10 U/ml), LPS (0.3-10 ng/ml) and the phorbol ester, phorbol 12-myristate-13-acetate (0.1 nM-1 microM), significantly increased ir-ET release, whereas TNF-alpha (0.1-10 ng/ml), RANTES (0.1-100 nM), IL-1 (0.01-10 ng/ml), bradykinin (1 nM-10 microM), CGRP (0.01 nM-1 microM), PDGF (0.1-3 ng/ml), Sar9, Met(O2)11-Sub P, Nle10-NKA 4-10 and senktide (selective NK-1, NK-2 and NK-3 receptor agonists, respectively; 1 nM-10 microM), LTD4 (1 nM-10 microM) or major basic protein (10 nM-1 microM) were without stimulatory effect. The results indicate that the enzyme responsible for the basal release of ET from cultured GPTECs is a Ca(2+)-dependent, phosphoramidon-sensitive, neutral metalloprotease. Furthermore, normally there is minimal ET release from guinea-pig airway epithelium but this can be increased markedly by culturing the cells to confluence, and by select pro-inflammatory mediators.