Identification of vimentin in rat peritoneal mast cells and its phosphorylation in association with histamine release.
Stimulation of rat peritoneal mast cells with histamine releasers, such as compound 48/80 and substance P, caused a similar pattern of protein phosphorylations: the molecular weights of the two major phosphorylated proteins were 45 kDa and 59 kDa. When rat mast cells permeabilized with beta-escin were exposed to Ca2+ at concentrations higher than 0.6 microM, phosphorylated proteins of identical molecular weight were also detected. By a radioimmunoprecipitation assay using anti-vimentin mouse monoclonal antibody, the 59 kDa protein was identified as vimentin, one of the intermediate cytoskeletal proteins. Moreover, it became apparent that the phosphoamino acid in phosphorylated vimentin was a serine residue. Sequential changes in vimentin phosphorylation were similar to that of histamine release elicited by histamine releasers: phosphorylation took place within 5 s of stimulation and reached a maximum within 10 s. When permeabilized mast cells were treated with calphostin C, a specific protein kinase C inhibitor, phosphorylation was markedly inhibited. Fluorescence images of mast cells stained with FITC-labelled anti-vimentin antibody showed filamentous structures surrounding the granules in the cytoplasm. However, after exposure to compound 48/80, the filamentous structures promptly disappeared and a dim fluorescence was observed homogeneously in the cell indicating that a rapid depolymerization of vimentin had taken place. From the present study, it became clear that when rat peritoneal mast cells were stimulated, vimentin was rapidly phosphorylated by protein kinase C and this phosphorylation process seems to be related to histamine release.
Izushi
K
K
Department of Pharmacology, Faculty of Pharmaceutical Sciences, Okayama University, Japan.
Fujiwara
Y
Y
Tasaka
K
K
D000818:Animals; D002147:Calmodulin / Q000037:antagonists & inhibitors; D002463:Cell Membrane Permeability; D000242:Cyclic AMP / Q000494:pharmacology; D006636:Histamine Release; D008297:Male; D008407:Mast Cells / Q000737:chemistry / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008970:Molecular Weight; D009281:Naphthalenes; D010766:Phosphorylation; D011083:Polycyclic Compounds / Q000494:pharmacology; D011233:Precipitin Tests; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D051381:Rats; D011919:Rats, Inbred Strains; D013373:Substance P / Q000494:pharmacology; D014746:Vimentin / Q000032:analysis / Q000378:metabolism*; D003189:p-Methoxy-N-methylphenethylamine / Q000494:pharmacology
D002147:Calmodulin; D009281:Naphthalenes; D011083:Polycyclic Compounds; D014746:Vimentin; D013373:Substance P; D003189:p-Methoxy-N-methylphenethylamine; D000242:Cyclic AMP; D011493:Protein Kinase C; C058819:calphostin C