title: Modulation of neutrophil-reduced pyridine nucleotide content following stimulation with phorbol myristate acetate and chemotactic factors.
abstract: Modulation of NAD(P)H in human neutrophils (PMN) following stimulation with phorbol myristate acetate (PMA) or chemotactic factors was determined by flow cytometry. Stimulation of PMN with 1 microgram/ml of PMA results in a time-dependent decrease in fluorescence, attributable to the oxidation of NAD(P)H. The decrease in fluorescence did not occur with PMN from a patient with chronic granulomatous disease (CGD) and was observed in only half of PMN from the mother of the patient. Loss of fluorescence in normal PMN was maximal following 7-15 min of stimulation with PMA. Simultaneous measurement of PMA-stimulated NAD(P)H oxidation and H2O2 production showed that NAD(P)H oxidation occurred as an all-or-none response while H2O2 production showed a graded response. These data suggest that with PMA stimulation, a threshold exists beyond which constitutive NAD(P)+ reduction is suppressed and complete oxidation of NAD(P)H occurs, while H2O2 production is proportional to the concentration of PMA. PMA-stimulated oxidation of NAD(P)H was reversible, and fluorescence returned to the initial level or higher after 40-60 min. Oxidation of NAD(P)H also occurred when cytochalasin B-treated PMN were stimulated with 25 nM C5a or 100 nM formyl-methionyl-leucyl-phenylalanine (f-MLP), but occurred more rapidly, peaking at 1 to 3 min. Fluorescence also returned by 5-6 min. This response to C5a and f-MLP was graded and proportional to the concentration of chemotactic factor used. Comparative studies showed that the cytochalasin-B treatment was essential for measurement of NAD(P)H oxidation, in response to C5a and F-MLP.
mesh_terms: D003182:Complement C5 / Q000494:pharmacology; D015936:Complement C5a; D003571:Cytochalasin B / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D006105:Granulomatous Disease, Chronic / Q000097:blood; D006801:Humans; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009249:NADP / Q000378:metabolism*; D009504:Neutrophils / Q000187:drug effects / Q000502:physiology*; D010084:Oxidation-Reduction; D013481:Superoxides / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D003182:Complement C5; D013481:Superoxides; D003571:Cytochalasin B; D009249:NADP; D009240:N-Formylmethionine Leucyl-Phenylalanine; D015936:Complement C5a; D013755:Tetradecanoylphorbol Acetate