title: Activation of the respiratory burst and tyrosine phosphorylation of proteins in human neutrophils: no direct relationship and involvement of protein kinase C-dependent and -independent signaling pathways.
abstract: Human neutrophils maximally stimulated with the optimal concentration (100 ng/ml) of phorbol myristate acetate (PMA), a direct activator of protein kinase C (PKC), for 5 min at 37 degrees C did not respond with superoxide (O2-) release to the later addition of PMA itself or the Ca2+ ionophore ionomycin. However, these cells did respond with enhanced release of O2- to the later addition of N-formyl-methionyl-leucyl-phenylalanine (FMLP) or concanavalin A (Con A). In these PMA-pretreated cells, an increase in cytoplasmic free Ca2+ ([Ca2+]i) induced by ionomycin was unaffected, whereas that induced by FMLP was inhibited by 50-60% and that induced by Con A was completely abolished. A 42-kDa protein was predominantly and consistently tyrosine-phosphorylated by FMLP, PMA and ionomycin with the different kinetics according to the stimuli. The dose-response curves showed that tyrosine phosphorylation and O2- release were stimulated in parallel by PMA, whereas tyrosine phosphorylation and an increase in [Ca2+]i, but not O2- release, were stimulated in parallel by FMLP or ionomycin. The potency of inducing tyrosine phosphorylation was ionomycin > FMLP = PMA, whereas the potency of triggering of O2- release was PMA > ionomycin = FMLP. UCN-01, a PKC inhibitor, inhibited O2- release and tyrosine phosphorylation induced by PMA, but not by FMLP or ionomycin. In contrast, pertussis toxin inhibited O2- release and tyrosine phosphorylation induced by FMLP, but not by PMA. Tyrosine kinase inhibitors (erbstatin and genistein) inhibited O2- release induced by FMLP, but not by PMA. However, both tyrosine kinase inhibitors did not impair FMLP- or PMA-induced tyrosine phosphorylation of a 42-kDa protein. Increased tyrosine phosphorylation of a 42-kDa protein was also detected in immature myeloid cells (HL-60 cells) stimulated by PMA, but not by ionomycin. These findings suggest that FMLP and Con A trigger the respiratory burst in human neutrophils by activating the definite pathway which include other signals than activation of PKC and an increase in [Ca2+]i; tyrosine phosphorylation of a 42-kDa protein is induced by the PKC-dependent and independent mechanisms according to the stimuli, and the PKC-independent and ionomycin-sensitive mechanism is inoperative in HL-60 cells; and tyrosine phosphorylation of a 42-kDa protein is unlikely to be causally related to activation of the respiratory burst.
authors.affiliation: Division of Hemopoiesis, Jichi Medical School, Tochigi, Japan.
authors.forename: E K
authors.initials: EK
authors.lastname: Azuma
authors.forename: S
authors.initials: S
authors.lastname: Kitagawa
authors.forename: A
authors.initials: A
authors.lastname: Yuo
authors.forename: H
authors.initials: H
authors.lastname: Mizoguchi
authors.forename: K
authors.initials: K
authors.lastname: Umezawa
authors.forename: F
authors.initials: F
authors.lastname: Takaku
authors.forename: M
authors.initials: M
authors.lastname: Saito
mesh_terms: D003208:Concanavalin A / Q000494:pharmacology; D019833:Genistein; D006801:Humans; D006873:Hydroquinones / Q000494:pharmacology; D015759:Ionomycin / Q000494:pharmacology; D007529:Isoflavones / Q000494:pharmacology; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism*; D037342:Pertussis Toxin; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D016897:Respiratory Burst; D015398:Signal Transduction; D013481:Superoxides / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014443:Tyrosine / Q000378:metabolism*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
chemical_list: D006873:Hydroquinones; D007529:Isoflavones; D010566:Virulence Factors, Bordetella; D003208:Concanavalin A; D013481:Superoxides; D014443:Tyrosine; D015759:Ionomycin; D009240:N-Formylmethionine Leucyl-Phenylalanine; D019833:Genistein; D037342:Pertussis Toxin; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; C048212:erbstatin