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

1 Bacterial lipopolysaccharide (LPS) was found to induce inflammatory respo nses and to enhance bronchial hyperreactivity to several contractile agonis ts. However, the implication of LPS in the pathogenesis of bronchial hyperr eactivity was not completely understood. Therefore, in this study, we inves tigated the effect of LPS on mitogen-activated protein kinase (MAPK) activa tion associated with potentiation of bradykinin (BK)-induced inositol phosp hates (IPs) accumulation and Ca2+ mobilization in canine cultured tracheal smooth muscle cells (TSMCs). 2 LPS stimulated phosphorylation of p42/p44 MAPK in a time- and concentrati on-dependent manner using a Western blot analysis against a specific phosph orylated form of MAPK antibody. Maximal stimulation of the p42 and p44 MAPK isoforms occurred after 7 min-incubation and the maximal effect was achiev ed with 100 mu g ml(-1) LPS. 3 Pretreatment of TSMCs with LPS potentiated BK-induced IPs accumulation an d Ca2+ mobilization. However, there was no effect on the IPs response induc ed by endothelin-1, 5-hydroxytryptamine, and carbachol. In addition, pretre atment with PDGF-BB enhanced BK-induced IPs response. 4 These enhancements by LPS and PDGF-BB might be due to an increase in BK B -2 receptor density (B-max) in TSMCs, characterized by competitive inhibiti on of [H-3]-BK binding using B-1 and B-2 receptor-selective reagents. 5 The enhancing effects of LPS and PDGF-BB were attenuated by PD98059, an i nhibitor of MAPK kinase (MEK), suggesting that the effect of LPS may share a common signalling pathway with PDGF-BB in TSMCs. 6 Furthermore, overexpression of dominant negative mutants, H-Ras-15A and R af-N4, significantly suppressed p42/p44 MAPK activation induced by LPS and PDGF-BB, indicating that Ras and Raf may be required for activation of thes e kinases. 7 These results suggest that the augmentation of PR-induced responses produ ced by LPS might be, at least in part, mediated through activation of Ras/R af/MEK/MAPK pathway in TSMCs.