Investigation of neutrophil signal transduction using a specific inhibitor of phosphatidylinositol 3-kinase.
Neutrophils contain a multicomponent NADPH oxidase system that is involved in the production of microbicidal oxidants. Stimulation of human neutrophils with the peptide FMLP activates this respiratory burst enzyme to produce superoxide and also has been shown to result in activation of phosphatidylinositol (Ptdlns) 3-kinase. Treatment of human neutrophils with 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), a potent and specific inhibitor of Ptdlns 3-kinase, resulted in complete inhibition of Ptdlns 3-kinase activity as well as in inhibition of superoxide production in FMLP-treated neutrophils in suspension; FMLP-stimulated oxidant production in adherent cells was also abolished. Treatment of human neutrophils with PMA resulted in production of superoxide without activation of Ptdlns 3-kinase; LY294002 did not block superoxide production in neutrophils exposed to PMA. In addition, LY294002 did not inhibit cellfree NADPH oxidase activation, CD11b-dependent adhesion, actin polymerization in response to FMLP, or FMLP-induced calcium flux. These results suggest that the signal transduction pathway of the FMLP-receptor involves activation of Ptdlns 3-kinase, which is required for subsequent superoxide production induced by the chemotactic peptide. Furthermore, Ptdlns 3-kinase may be located directly upstream of protein kinase C or other protein kinases, which in turn activate the NADPH oxidase system.
Vlahos
C J
CJ
Lilly Research Labortories, Eli Lilly and Company, Indianapolis, IN 46285.
Matter
W F
WF
Brown
R F
RF
Traynor-Kaplan
A E
AE
Heyworth
P G
PG
Prossnitz
E R
ER
Ye
R D
RD
Marder
P
P
Schelm
J A
JA
Rothfuss
K J
KJ
D000199:Actins / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002448:Cell Adhesion / Q000187:drug effects; D002474:Cell-Free System; D002867:Chromones / Q000494:pharmacology / Q000633:toxicity; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D066298:In Vitro Techniques; D008027:Light; D009025:Morpholines / Q000494:pharmacology / Q000633:toxicity; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism; D019255:NADPH Oxidases; D009504:Neutrophils / Q000187:drug effects / Q000502:physiology*; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000037:antagonists & inhibitors*; D012542:Scattering, Radiation; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D013481:Superoxides / Q000378:metabolism
D000199:Actins; D002867:Chromones; D009025:Morpholines; D013481:Superoxides; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D009240:N-Formylmethionine Leucyl-Phenylalanine; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); D002118:Calcium