| Abstrakt: |
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 μm), benzamidine (1 mm), pepstatin-A (30 μm), aprotinin (1 μg/ml), bacitracin (20 μg/ml) or leupeptin (50 μm), but was inhibited by phosphoramidon, the neutral metalloprotease inhibitor (IC50=16.8 μm), 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 μm), significantly increased ir-ET release, whereas TNF-α (0.1–10 ng/ml), RANTES (0.1–100 nm), IL-1 (0.01–10 ng/ml), bradykinin (1 nm–10 μm), CGRP (0.01 nm–1 μm), 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 μm), LTD4(1 nm–10 μm) or major basic protein (10 nm–1 μm) were without stimulatory effect. The results indicate that the enzyme responsible for the basal release of ET from cultured GPTECs is a Ca2+-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. |
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