q Birkha¨user Verlag, Basel, 1997 Inflamm. res. 46, Supplement 2 (1997) S171–S172 1023-3830/97/02S171-02 $ 1.50+0.20/0
Inflammation Research
VIII. C. Gordon Van Arman Scholarship Competition Neutral endopeptidase degrades endothelins in guinea pig tracheal epithelial cells Q. Yang, T. Kawaguchi, B. Battistini and P. Sirois Department of Pharmacology, Faculty of Medicine, University of Sherbrooke, 3001 North 12th Avenue, Sherbrooke, PQ, J1H 5N4, Canada, Fax +1 819 564 5400, e-mail:
[email protected]
Introduction Endothelins (ETs: ET-1, -2, -3) are 21 amino acid peptides that are produced from distinct precursors (big ET-1, -2, -3) through proteases called endothelin-converting enzymes (ECEs: -1a, -1b, -2) [1]. Numerous cells produce and release ETs amongst which human bronchial epithelial cells [2], guinea pig Clara cells [3], dog and pig tracheal epithelial cells [4]. Once produced and released, ETs act on specific receptors (ETA , ETB ) to elicit their pleiotropic effects, including potent vasoconstriction, growth promotion and/or bronchoconstriction [5]. ET-1 gene expression and levels are increased in bronchial epithelial cells and bronchoalveolar lavage fluid of asthmatics [6]. These data suggest that ETs are involved in the pathogenesis of asthma. As peptides, ETs are susceptible to degradation. Their metabolism in the lung is not clearly established. In human isolated bronchi, endothelins-induced contractions are potentiated by removal of the epithelial layer [7] where neutral endopeptidases (NEP; EC 3.4.24.11) are predominantly located. Airway epithelial cells also cleave and inactivate other bronchocontractile peptides such as bradykinin and neurokinins. Incubation of ET-1 with human recombinant N.E.P. also degrades the peptide within minutes [8]. We report that ET-1 is the only one, amongst all three ETs, to be released by guinea pig tracheal epithelial cells (GPTEpC) in culture, that the basal release is inhibited by phosphoramidon, but conversely potentiated by the presence of distinct NEP inhibitors. Materials and methods Male Hartley guinea pigs (300–350 g) were killed by cervical dislocation. The trachea was dissected into sterile Krebs Henseleit buffer at 20 8C. GPTEpC were obtained through a 1 h incubation at 37 8C with a solution of 0.15% protease type XXIV in Krebs-Henseleit buffer. GPTEpC were then mechanically removed from the mucosal
Correspondence to: P. Sirois
surface by gentle scraping with a policeman. The cells were centrifuged (2800 RPM) and washed twice with the DMEM/F12 containing 10% of fetal bovine serum, penicillin (100 u/ml), streptomycin (0.08%) and fungizone (1%). The cells were then resuspended in the same culture medium and seeded at a concentration of 5 × 105 cells/well in 24 wells (2 cm2 /well) culture plates at 37 8C in an atmosphere containing 5% CO2 . The medium was replaced after 48 h with DMEM/F12 without serum. The cells reached confluence after 4–5 days and characterized immunohistochemically for cytokeratin. The cells were incubated with either phosphoramidon (1 mM; a metalloendopeptidase and ECEs inhibitor; N-(a-rhamnopyranosyloxyhydroxyphosphinyl}-LLeu-L-Trp), thiorphan (1 mM; a NEP inhibitor) or SQ28603 (100 mM; a more specific NEP inhibitor; N-[2-(mercaptomethyl)-1-oxo-3-phenylpropyl]-
