Activation of the mitogen-activated protein kinase/cytosolic phospholipase A2 pathway in a rat mast cell line. Indications of different pathways for release of arachidonic acid and secretory granules.
The role of mitogen-activated protein (MAP) kinase in the release of arachidonic acid was examined in a mutated mast cell (RBL-2H3(m1)) line that expressed both native Fc epsilon R1 and the G protein-coupled muscarinic m1 receptor. Stimulation of these cells with Ag, carbachol, Ca(2+)-ionophore, or thapsigargin resulted in the phosphorylation of Raf1, MEK1, p42mapk MAP kinase, and the recently cloned cytosolic phospholipase A2 (PLA2) and increased activities of both MAP kinase and PLA2, as well as release of arachidonic acid. Because this cascade of reactions was inhibited by guanosine 5'-(2-thiodiphosphate), it appeared to be dependent on a GTP-binding protein(s). These reactions, however, were not dependent on protein kinase C; the cascade was totally resistant to the actions of a selective protein kinase C inhibitor, Ro31-7549, whereas release of the secretory granule marker, hexosaminidase, was blocked by this agent. Differences between the stimulatory pathways for release of arachidonic acid and hexosaminidase were evident also from the effects of the kinase inhibitor, quercetin. The above cascade of reactions, including release of arachidonic acid, was inhibited by 50% with approximately 5 microM quercetin, whereas secretion was inhibited only at higher concentrations of inhibitor. Moreover, inhibition of the activation of MAP kinase and release of arachidonic acid were closely correlated. This and previous findings suggested that release of arachidonic acid was attributable to the regulation of cytosolic PLA2 by MAP kinase (for activation of PLA2) and Ca2+ (for association of PLA2 with the membrane), whereas release of hexosaminidase was regulated primarily by Ca2+ and protein kinase C.
Hirasawa
N
N
Laboratory of Molecular Immunology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Santini
F
F
Beaven
M A
MA
D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D003594:Cytoplasmic Granules / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D019204:GTP-Binding Proteins / Q000502:physiology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000502:physiology; D015151:Immunoblotting; D007211:Indoles / Q000494:pharmacology; D008301:Maleimides / Q000494:pharmacology; D008407:Mast Cells / Q000378:metabolism*; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000378:metabolism*; D011794:Quercetin / Q000494:pharmacology; D051381:Rats; D015398:Signal Transduction / Q000502:physiology*
D007211:Indoles; D008301:Maleimides; D047428:Protein Kinase Inhibitors; D016718:Arachidonic Acid; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D011794:Quercetin; D011494:Protein Kinases; D010741:Phospholipases A; D054467:Phospholipases A2; D019204:GTP-Binding Proteins; D002118:Calcium; C064755:Ro 31-7549