Advantages and limits of the electrochemical method using Nafion and Ni-porphyrin-coated microelectrode to monitor NO release from cultured vascular cells

Abstract

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.

Physiologically Relevant Measurements of Nitric Oxide in Cardiovascular Research Using Electrochemical Microsensors

“…For further information about specific types of NO sensors and how they are constructed, readers are referred to previous reviews [18, 22, 27, 30, 33,40,41,42]. The authors encourage those who have the expertise to develop their own NO sensors to improve on the performance and produce less expensive and more specific NO sensors than those commercially available.…”

“…The decay of the NO signal did not appear to be caused by local destruction of NO, and it was concluded that in cultured endothelial cells, histamine and thrombin initiate a brief pulse of NO. However, NOS in these cells remains capable of generating sustained NO release, as was observed on administration of thapsigargin [42]. It appears that the NO release characteristics of endothelial cells change when they are grown in culture, and this point will have to be considered when interpreting such data.…”

“…Evidence for the involvement of calcium calmodulin-dependent protein kinase II in NO formation has been obtained by showing slight inhibition of NO formation following the addition of a blocker of the enzyme [67]. Induction of inducible NOS (iNOS) gave a 5-fold increase in the capacity of cultured internal mammary artery smooth muscle cells to generate NO in response to L -arginine [42]. Endothelium stimulants such as acetylcholine and calcimycin can generate superoxide, thus reducing the net output of NO [64, 65,68,69,70].…”

Analysis Of Agonist‐Evoked Nitric Oxide Release From Human Endothelial Cells: Role Of Superoxide Anion

“…An unsolved question concerns the mechanisms controlling the duration of the ·NO signal. At these low ·NO concentrations, the exchange of ·NO with the atmosphere is negligible and its spontaneous oxidation is also very slow, as indicated by the rate constant determined at room temperature (in the 10 6 (L/mol) 2 per s range) 20 and by the experimental comparison with the stability of a similar concentration of authentic ·NO 12 . However, dilution of the near membrane ·NO concentration by diffusion from the cell layer is an obligatory event.…”

“…This was confirmed by agonist‐induced ONOO – formation, as well as by the modulation of amplitude and time‐course of ·NO release associated with changes in ·O 2 – metabolism. This study was made possible by the use of an electrochemical approach that does not significantly disturb ·NO metabolism and its consequences, in contrast with studies using indicators that bind ·NO with high affinity and compete with its physiological targets 12 . Among the regulatory functions of ·NO are its negative feedback on NOS activity, 13 the modulation of superoxide‐dependent oxidation and hydroxylation reactions 14 and the inhibition of catalase activity 15 .…”