Tosylamido-phenylethyl-chloromethylketone (TPCK) inhibits superoxide (O₂)production by stimulated polymorphonuclear leucocytes (PMN) but does not affect receptor mediated signal transduction

Abstract

Previous studies with inhibitors such as TPCK have suggested,that a serine protease is involved in neutrophil activation (Kitagawa,JCI,1980 but the step involved in inhibition has not been defined.Recently ithas been shown that interaction of the chemotactic peptide fmlp with its cell receptor generates two signals:Inositol 1,4,5-trisphosphate(IP3) which releases calcium from intracellular stores and diacylglycerol which activates protein kinase C and thereby protein phosphorylation. Both pathways are necessary for PMN activation. In this study we demonstrate that TPCK 10 μM did not affect ingestion of endotoxin coated oil red O particles by PMN, but it inhibited O₂⁻production of PMN stimulated with fmlp or Phorbol-myristate-acetate(PMA) by 97 and 70% respectively.The release of secondary (marker:Vit.B12 binding protein) and primary(markerβ-glucuronidase) granules was inhibited by 37-58% and 34-53% respectively,depending on the stimulus (PMA,fmlp,ionomycin). Under identical conditions, cytosolic free calcium levels (assessed with quin2),IP3 generation(in 3H-myoinositol labelled PMN) and protein phosphorylation (incorporation of 32Pi) in PMN stimulated with fmlp were unaffected.These findings demonstrate,that although PMN exposed to TPCK 10μMphagocytose normally, O₂⁻production is inhibited markedly and degranulation to a smaller extent. In contrast to this, intracellular signals are generated normally, which indicates that TPCK selectively inhibits PMN function at a step distal to calcium mobilisation and protein kinase C activation.

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