Activation of p38 mitogen-activated protein kinase by formyl-methionyl-leucyl-phenylalanine in rat neutrophils

The signaling pathways leading to p38 mitogen-activated protein kinase (MAPK) activation in formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated rat neutrophils were examined. Immunoblot analysis with antibodies against a phosphorylated form of p38 MAPK showed that fMLP-stimulated p38 MAPK activation was dependent on a pertussis toxin-sensitive G protein. Two phosphatidylinositol 3-kinase inhibitors, wortmannin and 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), did not affect the p38 MAPK activation. Phosphorylation of p38 MAPK was concentration dependently attenuated by a tyrosine kinase inhibitor, genistein, and by a Ca2+-dependent protein kinase C inhibitor, 13-cyanoethyl-12-methyl-6,7,12,13-tetrahydroindolo[2,3-a]pyrrolo[3,4-c]carbazole-7-one (Go6976). However, the protein kinase C inhibitors with a broader spectrum, 2-[1-(3-dimethylaminopropyl)-5 -wmethoxy-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide (Go6983) and 2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide (GF109203X), had no inhibitory effect. fMLP-stimulated p38 MAPK phosphorylation was also reduced in cells pretreated with a phospholipase C inhibitor, 1-[6-((17 beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione (U73122), or preloaded with an intracellular Ca2+ chelator, 1,2-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA). We conclude that phosphorylation of p38 MAPK by fMLP stimulation in rat neutrophils is dependent on G(i/o) protein, nonreceptor tyrosine kinase, phospholipase C/Ca2+, and probably Ca2+-dependent protein kinase C pathways. (C) 2000 Elsevier Science B.V. All rights reserved.