Inhibition of NADPH oxidase by aminoacyl chloromethane protease inhibitors in phorbol-ester-stimulated human neutrophils: a reinvestigation. Are proteases really involved in the activation process?
Superoxide anion production by polymorphonuclear leukocytes stimulated with phorbol 12-myristate 13-acetate is known to be inhibited by a number of inhibitors and substrates of serine proteases, in particular by tosylphenylalanylchloromethane (TosPheCH2Cl) and to a lesser extent by tosyllysylchloromethane (TosLysCH2Cl). We have reinvestigated the characteristics of this inhibition, in view of the fact that other serine protease inhibitors with similar specificities, phenylmethanesulfonyl fluoride and leupeptin, were without effect. We found that the inhibition of phorbol-ester-induced superoxide production after cell preincubation with the chloromethanes followed saturation kinetics, with Kinact and kinact values of 100 microM and 31 min-1 for TosPheCH2Cl and 2 mM and 18 min-1 for TosLysCh2Cl. We also showed that the two compounds, which can inhibit protein kinase C in vitro, inhibited neither its activity in vivo, nor its translocation induced by phorbol myristate acetate. Furthermore the intracellular non-protein sulfhydryl group content was not affected by the treatment with the chloromethanes. Finally, addition of the inhibitors to stimulated cells also led to a time-dependent, concentration-dependent inhibition of superoxide production. Altogether, our results suggest that the chloromethane target is neither a protease nor protein kinase C and is not involved in NADPH oxidase activation, but rather in maintenance of its activity. The possible identity of this protein is discussed.
Conseiller
E C
EC
Institut National de la Santé et de la Recherche Médicale U 25, Hôpital Necker, Paris, France.
Lederer
F
F
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D009247:NADH, NADPH Oxidoreductases / Q000037:antagonists & inhibitors*; D019255:NADPH Oxidases; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology*; D010447:Peptide Hydrolases / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D013481:Superoxides / Q000096:biosynthesis; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014107:Tosyllysine Chloromethyl Ketone / Q000494:pharmacology; D014108:Tosylphenylalanyl Chloromethyl Ketone / Q000494:pharmacology*
D000590:Amino Acid Chloromethyl Ketones; D011480:Protease Inhibitors; D013481:Superoxides; D014107:Tosyllysine Chloromethyl Ketone; D014108:Tosylphenylalanyl Chloromethyl Ketone; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; D011493:Protein Kinase C; D010447:Peptide Hydrolases; D013755:Tetradecanoylphorbol Acetate