Electrochemical detection of nitric oxide production in human polymorphonuclear neutrophil leukocytes

The detection of nitric oxide (NO) release by human polymorphonuclear neutr ophil leukocytes (PMNs) presents several difficulties, mainly due to concom itant production of O-2(-) and H2O2, which could interfere with the measure ments. A Nafion and nickel porphyrin-coated microelectrode was used to meas ure 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. Additi on of the chemotactic oligopeptide f-met-leu-phe (fMLP; 100 nM) induced a N O release which reached a value of 71 +/- 30 pmol NO/10(6) PMN x ml(-1) 5 m in 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 o f the NO-synthase inhibitor NG-monomethyl-L-arginine (LNMMA; 1 mM) reduced the current by 82 +/- 20%. These results agree with the rate of NO producti on in human PMNs when measured spectrophotometrically using the NO-dependen t oxidation of oxyhaemoglobin to methaemoglobin. The NO production ih human PMN was dependent on fMLP concentrations, but independent of cell-concentr ations of 0.5-3.5 x 10(6)/ml. This pager shows that a electrochemical metho d, e.g. Nafion and porphyrin-coated microelectrode, is suitable for studies of NO release from stimulated human PMNs.