The chymotrypsin inhibitor carbobenzyloxy-leucine-tyrosine-chloromethylketone interferes with the neutrophil respiratory burst mediated by a signaling pathway independent of PtdInsP2 breakdown and cytosolic free calcium.
The effects of carbobenzyloxy-leucine-tyrosine-chloromethylketone (zLYCK), an inhibitor of chymotrypsin, were investigated on the activation pathways of the human neutrophil respiratory burst. At 10 microM zLYCK, a parallel inhibition was observed of superoxide production stimulated with the chemo-attractant FMLP and of chymotrypsin-like activity of human neutrophils. By contrast, superoxide production induced by PMA was minimally affected by zLYCK. The known transduction pathways triggered by FMLP were analyzed. zLYCK did not affect either the FMLP-induced cytosolic free calcium transient, inositol 1,4,5 trisphosphate formation, nor the PMA-induced phosphorylation of the 47-kDa substrate of protein kinase C. zLYCK did not affect the activity of protein kinase C extracted from neutrophils. In Ca(2+)-depleted cells, in which phosphatidylinositol 4,5-biphosphate breakdown does not occur, zLYCK inhibited the FMLP-induced respiratory burst in cells primed by low doses of PMA. The activity of the NADPH oxidase tested with active membranes from stimulated neutrophils or in a cell-free system was not inhibited by zLYCK. We conclude that: 1) zLYCK inhibits superoxide production through the inhibition of a chymotrypsin-like protease of the neutrophil, 2) zLYCK inhibits FMLP-induced activation of NADPH oxidase through a pathway independent of PtdInsP2 breakdown and cytosolic free calcium, and 3) zLYCK may prove a useful probe for the characterization of its target protease in neutrophil activation.
Gervaix
A
A
Children's Hospital, Department of Pediatrics, Switzerland.
Kessels
G C
GC
Suter
S
S
Lew
P D
PD
Verhoeven
A J
AJ
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology*; D002118:Calcium / Q000502:physiology*; D002918:Chymotrypsin / Q000037:antagonists & inhibitors* / Q000378:metabolism; D003593:Cytoplasm / Q000502:physiology; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D006801:Humans; D066298:In Vitro Techniques; D015544:Inositol 1,4,5-Trisphosphate / Q000096:biosynthesis; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism; D019255:NADPH Oxidases; D009504:Neutrophils / Q000187:drug effects*; D010587:Phagocytosis / Q000187:drug effects; D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols / Q000502:physiology*; D013481:Superoxides / Q000378:metabolism; D013997:Time Factors; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism
D000590:Amino Acid Chloromethyl Ketones; D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols; D013481:Superoxides; D014443:Tyrosine; C070517:carbobenzyloxyleucyl-tyrosine chloromethyl ketone; C022787:benzoyltyrosine 4-nitroanilide; D015544:Inositol 1,4,5-Trisphosphate; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; D002918:Chymotrypsin; D002118:Calcium