Effect of the synthetic inhibitor tosylamino-phenylethyl-chloromethylketone on chemotactic peptide receptor activation and superoxide production in human neutrophils.
It was previously shown that inhibitors such as tosylamido-phenylethyl-chloromethylketone (TPCK) inhibit superoxide production by human neutrophils. These studies suggested that a chymotrypsin-like protease inhibited by TPCK was involved in the activation of the neutrophils oxidative system. In this study, we attempted to define the step in cellular activation and/or cell function inhibited by TPCK. TPCK 10(-5) M did not inhibit the following early events thought to be involved in the activation of oxidase. 1) f-met-leu-phe-induced activation of phospholipase C assessed by the production of inositol-tris-phosphate (IP3), 2) f-met-leu-phe-induced membrane potential changes, 3) f-met-leu-phe-induced increase in free cytosolic calcium, and 4) phorbol-myristate acetate-induced protein phosphorylation in 32P labeled neutrophils. We also showed that TPCK 10(-5) M inhibited bactericidal activity of neutrophils on Staphylococcus aureus, whereas it did not inhibit the ingestion rate of endotoxin-coated Oil red O particles. We conclude that 1) TPCK at the concentration of 10(-5) M inhibits superoxide production but not ingestion of Oil red O particles and 2) TPCK inhibits superoxide production at a step distal from calcium mobilization and protein phosphorylation. Radiolabeled TPCK may therefore be a useful tool to study, whether a protease is involved in the activation of the oxidative system distal to second messenger generation.
D000328:Adult; D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology*; D002118:Calcium / Q000097:blood; D002462:Cell Membrane / Q000187:drug effects / Q000502:physiology; D003600:Cytosol / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008564:Membrane Potentials / Q000187:drug effects; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000378:metabolism; D009504:Neutrophils / Q000187:drug effects / Q000276:immunology / Q000502:physiology*; D010587:Phagocytosis / Q000187:drug effects; D010716:Phosphatidylinositols / Q000097:blood; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic / Q000187:drug effects / Q000378:metabolism*; D013211:Staphylococcus aureus; D013481:Superoxides / Q000097:blood*; D014108:Tosylphenylalanyl Chloromethyl Ketone / Q000494:pharmacology*
D000590:Amino Acid Chloromethyl Ketones; D010716:Phosphatidylinositols; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic; D013481:Superoxides; D014108:Tosylphenylalanyl Chloromethyl Ketone; D009240:N-Formylmethionine Leucyl-Phenylalanine; D002118:Calcium