Lipopolysaccharide enhances bradykinin-induced signal transduction via activation of Ras/Raf/MEK/MAPK in canine tracheal smooth muscle cells.
Bacterial lipopolysaccharide (LPS) was found to induce inflammatory responses and to enhance bronchial hyperreactivity to several contractile agonists. However, the implication of LPS in the pathogenesis of bronchial hyperreactivity was not completely understood. Therefore, in this study, we investigated the effect of LPS on mitogen-activated protein kinase (MAPK) activation associated with potentiation of bradykinin (BK)-induced inositol phosphates (IPs) accumulation and Ca(2+) mobilization in canine cultured tracheal smooth muscle cells (TSMCs). LPS stimulated phosphorylation of p42/p44 MAPK in a time- and concentration-dependent manner using a Western blot analysis against a specific phosphorylated form of MAPK antibody. Maximal stimulation of the p42 and p44 MAPK isoforms occurred after 7 min-incubation and the maximal effect was achieved with 100 microg ml(-1) LPS. Pretreatment of TSMCs with LPS potentiated BK-induced IPs accumulation and Ca(2+) mobilization. However, there was no effect on the IPs response induced by endothelin-1, 5-hydroxytryptamine, and carbachol. In addition, pretreatment with PDGF-BB enhanced BK-induced IPs response. 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 inhibition of [(3)H]-BK binding using B(1) and B(2) receptor-selective reagents. The enhancing effects of LPS and PDGF-BB were attenuated by PD98059, an inhibitor of MAPK kinase (MEK), suggesting that the effect of LPS may share a common signalling pathway with PDGF-BB in TSMCs. Furthermore, overexpression of dominant negative mutants, H-Ras-15A and Raf-N4, significantly suppressed p42/p44 MAPK activation induced by LPS and PDGF-BB, 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 LPS might be, at least in part, mediated through activation of Ras/Raf/MEK/MAPK pathway in TSMCs.
Luo
S F
SF
Department of Internal Medicine, College of Medicine, Chang Gung University, 259 Wen-Hwa 1 Road, Kwei-San, Tao-Yuan, Taiwan.
Wang
C C
CC
Chiu
C T
CT
Chien
C S
CS
Hsiao
L D
LD
Lin
C H
CH
Yang
C M
CM
D000818:Animals; D001667:Binding, Competitive / Q000187:drug effects; D001920:Bradykinin / Q000378:metabolism / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D005419:Flavonoids / Q000494:pharmacology; D007093:Imidazoles / Q000494:pharmacology; D007295:Inositol Phosphates / Q000378:metabolism; D007527:Isoenzymes / Q000187:drug effects / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008297:Male; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009130:Muscle, Smooth / Q000166:cytology / Q000187:drug effects* / Q000502:physiology; D010766:Phosphorylation / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism; D011725:Pyridines / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects; D013997:Time Factors; D014132:Trachea / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology; D014316:Tritium; D018631:ras Proteins / Q000187:drug effects* / Q000378:metabolism
D004791:Enzyme Inhibitors; D005419:Flavonoids; D007093:Imidazoles; D007295:Inositol Phosphates; D007527:Isoenzymes; D008070:Lipopolysaccharides; D011725:Pyridines; D014316:Tritium; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D020928:Mitogen-Activated Protein Kinases; D020929:Mitogen-Activated Protein Kinase Kinases; D018631:ras Proteins; C093642:SB 203580; D001920:Bradykinin; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one; D002118:Calcium