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The onset of the respiratory burst in human neutrophils real time studies of hydrogen peroxide formation reveal a rapid agonist induced transduction process



The onset of the respiratory burst in human neutrophils real time studies of hydrogen peroxide formation reveal a rapid agonist induced transduction process



Journal of Biological Chemistry 262(25): 12048-12053



A real-time study of the initiation of the respiratory burst in human neutrophils was made. The cells were stimulated with fMet-Leu-Phe (fMLP) C5a, platelet-activating factor, leukotriene B4, phorbol myristate acetate (PMA), or ionomycin, and H2O2 production was determined by chemiluminescence. Identical average onset times (2.4 s) and closely comparable values for the apparent first-order rate constant (kapp) for the induction of NADPH-oxidase activity (0.21-0.29 s-1) were obtained following stimulation with fMLP, C5a, platelet-activating factor, or leukotriene B4, suggesting that different agonists act through a common transduction sequence. Much longer onset times and lower kapp values were obtained upon stimulation with PMA or ionomycin. Pretreatment with PMA consistently shortened the onset time of the neutrophil's responses to agonists by about 1 s. When H2O2 production was initiated with PMA, a subsequent stimulation with the agonist fMLP elicited an immediate response (onset time < 0.2 s) which preceded further changes in the fura-2-detected [Ca2+]i. The results are consistent with a mechanism in which agonist signals appear to be transduced by two sequences acting in concert.sbd.a rate-limiting one liberating Ca2+ and diacylglycerol and turning on the Ca2+/phospholipid-dependent enzyme protein kinase C, and an essentially instantaneous one which does not appear to require further change in cytosolic Ca2+.

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