Phenylmethylsulfonyl fluoride inhibits chemotactic peptide-induced actin polymerization and oxidative burst activity in human neutrophils by an effect unrelated to its anti-proteinase activity.
Stimulation of polymorphonuclear leukocytes with the chemotactic peptide N-formylmethionylleucylphenylalanine (fMet-Leu-Phe) causes conversion of monomeric actin to polymeric actin. We studied the role of proteinase inhibitors phenylmethylsulfonyl fluoride PMSF) and diisopropyl fluorophosphate in fMet-Leu-Phe-induced actin polymerization in polymorphonuclear leukocytes. Pre-incubation of cells with PMSF (2 mM) for 1 min caused inhibition of fMet-Leu-Phe-induced actin polymerization, as studied by 7-nitrobenz-2-oxa-1,3-diazole (NBD) -phallacidin labeling and flow cytometry. PMSF also inhibited fMet-Leu-Phe-induced hydrogen peroxide release, superoxide anion generation and chemiluminescence. In contrast, diisopropyl fluorophosphate (5 mM) was unable to inhibit fMet-Leu-Phe-induced actin polymerization and superoxide generation, but was effective in inhibiting hydrogen peroxide production and chemiluminescence. PMSF did not cause any change in membrane potential by itself and failed to inhibit the membrane potential changes induced by fMet-Leu-Phe, indicating that PMSF does not affect the binding of fMet-Leu-Phe to the receptors. The high concentration of PMSF required coupled with the fact that diisopropyl fluorophosphate was unable to inhibit fMet-Leu-Phe-induced actin polymerization suggested that this activity of PMSF might be unrelated to proteinase inhibitory activity. Polymyxin B, a membrane-active antibiotic, had an effect similar to PMSF on fMet-Leu-Phe-induced actin polymerization. This suggests that PMSF may also be acting via its membrane effect rather than its anti-proteinase effect.
Rao
K M
KM
Department of Pathology, West Virginia University, Morgantown.
Castranova
V
V
D000199:Actins / Q000378:metabolism*; D006801:Humans; D006861:Hydrogen Peroxide / Q000097:blood; D066298:In Vitro Techniques; D007700:Kinetics; D046911:Macromolecular Substances; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism*; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology*; D013450:Sulfones / Q000494:pharmacology*; D013481:Superoxides / Q000097:blood*
D000199:Actins; D046911:Macromolecular Substances; D013450:Sulfones; D013481:Superoxides; D010664:Phenylmethylsulfonyl Fluoride; D009240:N-Formylmethionine Leucyl-Phenylalanine; D006861:Hydrogen Peroxide