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

Inhalation of tumour necrosis factor-α (TNF-α) induced a bronchial hyperreactivity to contractile agonists. However, the mechanisms of TNF-α involved in the pathogenesis of bronchial hyperreactivity were not completely understood. Therefore, we investigated the effect of TNF-α on bradykinin (BK)-induced inositol phosphate (IP) accumulation and Ca²⁺ mobilization, and up-regulation of BK receptor density in canine cultured tracheal smooth muscle cells (TSMCs). Pretreatment of TSMCs with TNF-α potentiated BK-induced IP accumulation and Ca²⁺ mobilization. However, there was no effect on the IP response induced by endothelin-1 (ET-1), 5-hydroxytryptamine (5-HT), and carbachol. Pretreatment with PDGF B-chain homodimer (PDGF-BB) also enhanced BK-induced IP response. These enhancements induced by TNF-α and PDGF-BB might be due to an increase in BK B₂ receptor density (B_max), since [³H]BK binding to TSMCs was inhibited by the B₂ selective agonist and antagonist, BK and Hoe 140, but not by the B₁ selective reagents. The enhancing effects of TNF-α and PDGF-BB were attenuated by PD98059 (an inhibitor of activation of MAPK kinase, MEK) and cycloheximide (an inhibitor of protein synthesis), suggesting that TNF-α may share a common signalling pathway with PDGF-BB via protein(s) synthesis in TSMCs. Furthermore, overexpression of dominant negative mutants, H-Ras-15A and Raf-N4, significantly suppressed p42/p44 mitogen-activated protein kinase (MAPK) activation induced by TNF-α and PDGF-BB and attenuated the effect of TNF-α on BK-induced IP response, indicating that Ras and Raf may be required for activation of these kinases. These results suggest that the augmentation of BK-induced responses produced by TNF-α might be, at least in part, mediated through activation of Ras/Raf/MEK/MAPK pathway in TSMCs.