Chemotactic factor-induced superoxide radical generation by human neutrophils: requirement for proteinase (esterase) activity.
The requirement for proteinase (esterase) activity in the generation of O2- by human peripheral neutrophils was investigated. Neutrophils were activated by exposure to the chemotactic peptide FMLP and superoxide generation was assessed by ferricytochrome C reduction. Inhibition of O2- generation was observed by pretreating cells with the chloromethyl ketone derivatives of tosyl-phenylalanine (TPCK, ID50 2.2 X 10(-5)M) and tosyl-lysine (TLCK, ID50 1.9 X 10(-4)M). Dose-dependent inhibition was also noted with synthetic proteinase substrates, especially those of chymotrypsin-like specificity, phenylalanine and tryptophan methyl esters (ID50S approximately 1.5 X 10(-4)M), as compared to derivatives of basic amino acids, arginine and acetyl-lysine methyl esters, which caused negligible inhibition at 10(-3M. The inhibition of O2- generation demonstrated by TryME was time- and temperature-dependent and reversible with washing of the cells, whereas the inhibition seen with TPCK was irreversible. DFP at high concentrations, 10(-3)M or greater, caused moderate inhibition of O2- generation that was unaffected by exogenous serine and almost completely reversible by washing the cells. TPCK and TryME, at concentrations that caused marked inhibition of O2- generation, had no effect on the fmlp-induced calcium-45 uptake by the cells. These studies suggest that intact proteinase function is required for O2- generation and that this step follows the calcium influx in the activation sequence induced by FMLP.
D002118:Calcium / Q000097:blood; D002634:Chemotaxis, Leukocyte; D010450:Endopeptidases / Q000097:blood*; D006801:Humans; D066298:In Vitro Techniques; D007531:Isoflurophate / Q000494:pharmacology; D008715:Methionine / Q000031:analogs & derivatives*; D009239:N-Formylmethionine / Q000031:analogs & derivatives* / Q000494:pharmacology; D009504:Neutrophils / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*; D009842:Oligopeptides / Q000494:pharmacology*; D010100:Oxygen / Q000097:blood*; D011480:Protease Inhibitors / Q000494:pharmacology; D013481:Superoxides / Q000097:blood*; D014107:Tosyllysine Chloromethyl Ketone / Q000494:pharmacology; D014108:Tosylphenylalanyl Chloromethyl Ketone / Q000494:pharmacology
D009842:Oligopeptides; D011480:Protease Inhibitors; D013481:Superoxides; D007531:Isoflurophate; D014107:Tosyllysine Chloromethyl Ketone; D014108:Tosylphenylalanyl Chloromethyl Ketone; D009239:N-Formylmethionine; D008715:Methionine; D010450:Endopeptidases; D010100:Oxygen; D002118:Calcium