Characterization of endothelin release from guinea-pig tracheal epithelium: influence of proinflammatory mediators including major basic protein.
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.
Department of Pulmonary Pharmacology, SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406, USA. douglas_w_hay@sbphrd.com
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DW
Hay
M R
MR
Van Scott
R M
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Muccitelli
D000818:Animals; D018929:Cell Culture Techniques; D016207:Cytokines / Q000494:pharmacology; D016232:Endothelins / Q000276:immunology / Q000378:metabolism*; D004727:Endothelium / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism; D006168:Guinea Pigs; D006801:Humans; D007249:Inflammation / Q000276:immunology; D008297:Male; D008666:Metalloendopeptidases / Q000378:metabolism*; D014132:Trachea / Q000378:metabolism*; D014470:Umbilical Cord
D016207:Cytokines; D016232:Endothelins; D008666:Metalloendopeptidases