Abstract
The elevated level of thrombin has been detected in the airway fluids of asthmatic patients and shown to stimulate cell proliferation in tracheal smooth muscle cells (TSMCs). However, the implication of thrombin in the cell proliferation was not completely understood. In this study, thrombin stimulated [3H]thymidine incorporation and p42/p44 mitogen-activated protein kinase (MAPK) phosphorylation in a time- and concentration-dependent manner in TSMCs. Pretreatment of TSMCs with pertussis toxin (PTX) significantly inhibited [3H]thymidine incorporation and phosphorylation of MAPK induced by thrombin. These responses were attenuated by tyrosine kinase inhibitors genistein and herbimycin A, phosphatidyl inositide (PI)-phospholipase C (PLC) inhibitor U73122, protein kinase C inhibitor GF109203X, removal of Ca2+ by addition of BAPTA/AM plus EGTA, PI 3-kinase inhibitors wortmannin and LY294002, and inhibitor of MEK1/2 PD98059. Furthermore, overexpression of dominant negative mutants, H-Ras-15A and Raf-N4, significantly suppressed p42/p44 MAPK activation induced by thrombin and PDGF-BB, indicating that Ras and Raf may be required for activation of these kinases. These results conclude that the mitogenic effect of thrombin was mediated through the activation of Ras/Raf/MEK/MAPK pathway. Thrombin-mediated MAPK activation was modulated by PI-PLC, Ca2+, PKC, tyrosine kinase, and PI 3-kinase associated with cell proliferation in canine cultured TSMCs.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Calcium / metabolism
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Cell Division / drug effects
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Cells, Cultured
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DNA / biosynthesis
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Dogs
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Enzyme Activation
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Enzyme Inhibitors / pharmacology
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Estrenes / pharmacology
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Female
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MAP Kinase Kinase 1
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MAP Kinase Kinase 2
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MAP Kinase Signaling System / physiology*
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Male
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Mitogen-Activated Protein Kinase 1 / metabolism*
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Mitogen-Activated Protein Kinase 3
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Mitogen-Activated Protein Kinase Kinases / antagonists & inhibitors
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Mitogen-Activated Protein Kinases / metabolism*
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Muscle, Smooth, Vascular / cytology
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Pertussis Toxin
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Phosphatidylinositol Diacylglycerol-Lyase
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Phosphoinositide Phospholipase C
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Phosphoinositide-3 Kinase Inhibitors
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Phosphorylation
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Protein Kinase C / antagonists & inhibitors
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Protein Serine-Threonine Kinases / antagonists & inhibitors
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Protein-Tyrosine Kinases / antagonists & inhibitors
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Proto-Oncogene Proteins c-raf / genetics
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Proto-Oncogene Proteins c-raf / metabolism*
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Proto-Oncogene Proteins p21(ras) / genetics
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Proto-Oncogene Proteins p21(ras) / metabolism*
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Pyrrolidinones / pharmacology
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Thrombin / metabolism*
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Thrombin / pharmacology
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Trachea / cytology
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Type C Phospholipases / antagonists & inhibitors
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Virulence Factors, Bordetella / pharmacology
Substances
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Enzyme Inhibitors
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Estrenes
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Phosphoinositide-3 Kinase Inhibitors
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Pyrrolidinones
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Virulence Factors, Bordetella
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1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione
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DNA
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Pertussis Toxin
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Protein-Tyrosine Kinases
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Protein Serine-Threonine Kinases
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Proto-Oncogene Proteins c-raf
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Protein Kinase C
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Mitogen-Activated Protein Kinase 1
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Mitogen-Activated Protein Kinase 3
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Mitogen-Activated Protein Kinases
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MAP Kinase Kinase 1
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MAP Kinase Kinase 2
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Mitogen-Activated Protein Kinase Kinases
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Type C Phospholipases
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Phosphoinositide Phospholipase C
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Thrombin
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Proto-Oncogene Proteins p21(ras)
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Phosphatidylinositol Diacylglycerol-Lyase
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Calcium