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(ml)) line that expressed both native FcεR1 and the G protein-coupled muscarinic ml receptor. Stimulation of these cells with Ag, carbachol, Ca²⁺-ionophore, or thapsigargin resulted in the phosphorylation of Raf1, MEK1, p42(mapk) 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 ~5 μM 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.