The upstream regulation of p38 mitogen-activated protein kinase phosphorylation by arachidonic acid in rat neutrophils

The signal transduction pathways activated by arachidonic acid that lead to p38 mitogen-activated protein kinase (MAPK) activation in neutrophils rema ins unclear. In this study, selective inhibitors of several signalling path ways were utilized to investigate the mechanisms of activation of p38 MAPK by arachidonic acid in rat neutrophils. Stimulation of p38 MAPK phosphorylation by arachidonic acid and its trifluo romethyl ketone analogue AACOCF(3) was transient, peaking at 1 min, and was concentration-dependent. Arachidonic acid-stimulated p38 MAPK phosphorylat ion was attenuated in cells pretreated with the G(i/o) inhibitor (pertussis toxin), but not with the dual cyclooxygenase/lipoxygenase inhibitor (BW755 C) or the leukotriene biosynthesis inhibitor (MK886). Tyrosine kinase inhib itor (genistein), but not the extracellular signal-regulated kinase kinase inhibitors (PD98059 and U0126), attenuated the phosphorylation of p38 MAPK by arachidonic acid. Phosphoinositide 3-kinase inhibitors (wortmannin and L Y294002) did not affect the arachidonic acid-induced response. After pretre atment of the cells with protein kinase C inhibitors (Go6976, Go6983 and GF 109203X), only Go6976 significantly attenuated the phosphorylation of p38 M APK by arachidonic acid. In addition, phosphorylation of p38 MAPK by arachi donic acid was greatly attenuated by the phospholipase C inhibitor (U73122) and the Ca2+ chelator BAPTA ((1,2-bis-o-aminophenoxy)-ethane-N,N,N',N'-tet raacetic acid), but not altered by the nitric oxide synthase inhibitor, N-n itro-L-arginine methyl ester. Arachidonic acid did not cause an increase in cellular cyclic GMP level. This study revealed the involvement of pertussis toxin-sensitive G protein, non-receptor tyrosine kinase, phospholipase C/Ca2+, and probably Ca2+-depe ndent protein kinase C in arachidonic acid-stimulated p38 MAPK activation.