Tumour necrosis factor-alpha enhances bradykinin-induced signal transduction via activation of Ras/Raf/MEK/MAPK in canine tracheal smooth muscle cells

Inhalation of tumour necrosis factor-alpha (TNF-alpha) induced a bronchial hyperreactivity to contractile agonists. However, the mechanisms of TNF-alp ha involved in the pathogenesis of bronchial hyperreactivity were not compl etely understood. Therefore, we investigated the effect of TNF-alpha on bra dykinin (BK)-induced inositol phosphate (IP) accumulation and Ca2+ mobiliza tion, and up-regulation of BK receptor density in canine cultured tracheal smooth muscle cells (TSMCs). Pretreatment of TSMCs with TNF-alpha potentiat ed BK-induced IP accumulation and Ca2+ mobilization. However, there was no effect on the IP response induced by endothelin-1 (ET-1), 5-hydroxytryptami ne (5-HT), and carbachol. Pretreatment with PDGF B-chain homodimer (PDGF-BB ) also enhanced BK-induced TP response. These enhancements induced by TNF-a lpha and PDGF-BB might be due to an increase in BK B-2 receptor density (B- max), since [H-3]BK binding to TSMCs was inhibited by the B-2 selective ago nist and antagonist, BK and Hoe 140, but not by the B-1 selective reagents. The enhancing effects of TNF-alpha and PDGF-BB were attenuated by PD98059 (an inhibitor of activation of MAPK kinase, MEK) and cycloheximide (an inhi bitor of protein synthesis), suggesting that TNF-alpha may share a common s ignalling pathway with PDGF-BB via protein(s) synthesis in TSMCs. Furthermo re, overexpression of dominant negative mutants, H-Ras-15A and Raf-N4, sign ificantly suppressed p42/p44 mitogen-activated protein kinase (MAPK) activa tion induced by TNF-alpha and PDGF-BB and attenuated the effect of TNF-alph a on BK-induced IP response, indicating that Ras and Raf may be required fo r activation of these kinases. These results suggest that the augmentation of BK-induced responses produced by TNF-alpha might be, at least in part, m ediated through activation of Ras/Raf/MEK/MAPK pathway in TSMCs. (C) 2001 E lsevier Science Inc. All rights reserved.