title: Electrochemical detection of nitric oxide production in human polymorphonuclear neutrophil leukocytes.
abstract: The detection of nitric oxide (NO) release by human polymorphonuclear neutrophil leukocytes (PMNs) presents several difficulties, mainly due to concomitant production of O2- and H2O2, which could interfere with the measurements. A Nafion and nickel porphyrin-coated microelectrode was used to measure NO production in PMNs in vitro. It allowed detection of 6.3 +/- 1.9 nM NO in a PMN-containing system and was unaffected by added chemicals. Addition of the chemotactic oligopeptide f-met-leu-phe (fMLP; 100 nM) induced a NO release which reached a value of 71 +/- 30 pmol NO/10(6) PMN x ml(-1) 5 min after stimulation in the presence of SOD (150 U/ml). If SOD was omitted, the corresponding value was 36 +/- 20 pmol NO/10(6) PMN x ml(-1). Presence or absence of catalase did not alter the amount of NO measured. Addition of the NO-synthase inhibitor N(G)-monomethyl-L-arginine (LNMMA; 1 mM) reduced the current by 82 +/- 20%. These results agree with the rate of NO production in human PMNs when measured spectrophotometrically using the NO-dependent oxidation of oxyhaemoglobin to methaemoglobin. The NO production in human PMN was dependent on fMLP concentrations, but independent of cell-concentrations of 0.5-3.5 x 10(6)/ml. This paper shows that a electrochemical method, e.g. Nafion and porphyrin-coated microelectrode, is suitable for studies of NO release from stimulated human PMNs.
authors.affiliation: Department of Hematology and the Center for Inflammation and Hematology Research, Karolinska Institute at Huddinge University Hospital, Sweden. gerd.larfars@telia.com
authors.forename: G
authors.initials: G
authors.lastname: Lärfars
authors.forename: F
authors.initials: F
authors.lastname: Lantoine
authors.forename: M A
authors.initials: MA
authors.lastname: Devynck
authors.forename: H
authors.initials: H
authors.lastname: Gyllenhammar
mesh_terms: D004563:Electrochemistry; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism*; D009569:Nitric Oxide / Q000096:biosynthesis*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors; D019323:omega-N-Methylarginine / Q000494:pharmacology
chemical_list: D004791:Enzyme Inhibitors; D019323:omega-N-Methylarginine; D009569:Nitric Oxide; D009240:N-Formylmethionine Leucyl-Phenylalanine; D019001:Nitric Oxide Synthase