headspace solid-phase microextraction in conjunction with gas chromatography and flame ionization detection. (2002) Analytica Chimica Acta, 458 (1). 111-117.
Electrochemical monitoring with a porphyrinic microsensor of in situ nitric oxide production from cultured cells offers numerous advantages but requires cautious analysis, repeated calibration and accurate localisation of the electrode. It also had some limitations. We describe here some characteristics of this method, the stability of the electrode response during experiments and its application to nitric oxide production by constitutive and inducible nitric oxide synthases. Real-time measurements of NO concentration allow the study of the kinetics of NO production. This is illustrated by the time-course of NO release from cultured human endothelial cells. How NO production by inducible nitric oxide synthase in cultured smooth muscle cells obtained from human internal mammary artery can be evaluated by Nafion-and Ni-porphyrin-coated electrode is also described. The results thus obtained are compared to the cumulated NO 2 -production evaluated by the Griess method.
1. Dichlorofluorescein oxidation and electrochemical monitoring of in situ nitric oxide (NO) release from cultured human endothelial cells reveals that agonists such as thrombin and histamine simultaneously stimulate transient superoxide production. 2. The duration of *NO release was increased only in the simultaneous presence of extracellular L-arginine and exogenous superoxide dismutase. In contrast, the inhibition of membrane reduced nicotinamide adenine dinucleotide (phosphate) oxidases, the major source of *O2- in endothelial cells, did not prolong *NO release, although extracellular L-arginine was also present. Comparison of these two experimental conditions suggested that H2O2 was involved in the extension of the *NO signal. 3. The present study demonstrates that, in the absence of external L-arginine, *O2- production does not constitute the major pathway controlling the duration of agonist-induced *NO signal. These results suggest that L-arginine and H2O2 act jointly to maintain nitric oxide synthase in an activated form.
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