ACTIVATION OF THE MITOGEN-ACTIVATED PROTEIN-KINASE CYTOSOLIC PHOSPHOLIPASE A(2) 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 a rachidonic acid was examined in a mutated mast cell (RBL-2H3(ml)) line that expressed both native Fc epsilon R1 and the G protein-coupled mu scarinic mi receptor. Stimulation of these cells with Ag, carbachol, C a2+-ionophore, or thapsigargin resulted in the phosphorylation of Raf1 , MEK1, p42(mapk) MAp kinase, and the recently cloned cytosolic phosph olipase A(2) (PLA(2)) and increased activities of both MAP kinase and PLA(2), as well as release of arachidonic acid. Because this cascade o f reactions was inhibited by guanosine 5'-(2-thiodiphosphate), it appe ared to be dependent on a GTP-binding protein(s). These reactions, how ever, were not dependent on protein kinase C; the cascade was totally resistant to the actions of a selective protein kinase C inhibitor, Ro 31-7549, whereas release of the secretory granule marker, hexosaminida se, was blocked by this agent. Differences between the stimulatory pat hways for release of arachidonic acid and hexosaminidase were evident also from the effects of the kinase inhibitor, quercetin. The above ca scade of reactions, including release of arachidonic acid, was inhibit ed by 50% with similar to 5 ELM quercetin, whereas secretion was inhib ited only at higher concentrations of inhibitor. Moreover, inhibition of the activation of MAP kinase and release of arachidonic acid were c losely correlated. This and previous findings suggested that release o f arachidonic acid was attributable to the regulation of cytosolic PLA (2) by MAP kinase (for activation of PLA(2)) and Ca2+ for association of PLA(2) with the membrane), whereas release of hexosaminidase was re gulated primarily by Ca2+ and protein kinase C.