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

Abstract

The signal transduction pathways activated by arachidonic acid that lead to p38 mitogen-activated protein kinase (MAPK) activation in neutrophils remains unclear. In this study, selective inhibitors of several signalling pathways 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 trifluoromethyl ketone analogue AACOCF3 was transient, peaking at 1 min, and was concentration-dependent. Arachidonic acid-stimulated p38 MAPK phosphorylation was attenuated in cells pretreated with the Gi/o inhibitor (pertussis toxin), but not with the dual cyclooxygenase/lipoxygenase inhibitor (BW755C) or the leukotriene biosynthesis inhibitor (MK886). Tyrosine kinase inhibitor (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 LY294002) did not affect the arachidonic acid-induced response. After pretreatment of the cells with protein kinase C inhibitors (Gö6976, Gö6983 and GF109203X), only Gö6976 significantly attenuated the phosphorylation of p38 MAPK by arachidonic acid. In addition, phosphorylation of p38 MAPK by arachidonic 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'-tetraacetic acid), but not altered by the nitric oxide synthase inhibitor, N-nitro-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+-dependent protein kinase C in arachidonic acid-stimulated p38 MAPK activation.