Nitrogen monoxide and oxidative stress: composition and intensity of cellular oxidative bursts cocktail. A study through artificial electrochemical synapses on single human fibroblasts

Abstract

Positioning an ultramicroelectrode at micrometric distances from an isolated living cell ensures that any electroactive material released by the cell is collected and analyzed by the electrode surface. The film of extracellular fluid comprised between the cell and the electrode surfaces defines an artificial synaptic cleft of a few hundred femtoliters volume, in which the release of minute molecular amounts of chemicals produces a sudden and important concentration rise. This guarantees the detection of the released species with an extremely high signalto-noise ratio, as well as a determination of its instant released flux since the collection efficiency is quantitative. In other words, the assembly cell/liquid cleft/ultramicroelectrode behaves as an artificial neuronal synapse. In this review we wish to elaborate on how this artificial synaptic arrangement may be used to monitor an oxidative stress response stimulated in a human fibroblast, and demonstrate that such long-time conjectured oxidative cellular bursts involve a subtle cocktail of femtomoles of hydrogen peroxide, nitrogen monoxide, peroxynitrite and nitrite ions. The analysis performed establishes that this delicate cocktail results from the ultimate combinations of equimolar primary productions of superoxide ion and nitrogen monoxide by two distinct enzymatic systems which are presumably operating in distinct compartments of the cell: NADPH-oxidase type enzymes for the generation of superoxide, and NO-synthases for that of nitrogen monoxide.Article available at http://analusis.edpsciences.org or http://dx.doi.org/10. 1051/analusis:2000280506 Electrodes pick up electrical noise through their capacitance, viz., through their overall conducting surface area, while the analytical information (viz., the Faradaic current) may arise only from the surface area exposed to the cell release. Thus, using an electrode with an active surface matching that of the examined cell decreases the noise while it does not affect at all the quality and intensity of the analytical information. This increases the signal-to-noise ratio and ensures simultaneously a quantitative collection efficiency since the artificial synaptic cleft covers the cell emitting surface. Cells being of micrometric dimensions, it is understood that positioning an ultramicroelectrode at micrometric distances from an isolated living cell ( Fig. 1 a,b) ensures the most adequate signal-to-noise ratio and guarantees that the collection of electroactive chemicals released by a single living cell surface is total [1][2][3][4][5][6] Application of artificial synapses to oxidative stressThe biological problem and its biological and medical importanceAerobic cells are known to produce superoxide ion (O 2 -) and nitrogen monoxide (NO) in response to a stress created by a drastic change in the cell activity provoked by an aggression of its membrane integrity (during a viral or bacterial intrusion for example) or following a sudden and drastic change in its surrounding medium. The released NO is thought to ...

Monitoring in Real Time with a Microelectrode the Release of Reactive Oxygen and Nitrogen Species by a Single Macrophage Stimulated by its Membrane Mechanical Depolarization

“…The first one consisted of plotting the reconstructed voltammogram of the cell response in the zone of potentials where the two electrochemical waves overlap (E + 500 mV versus SSCE). [43] Application of this method involving 11 different measurement potentials (every 25 mV between + 250 and + 500 mV versus SSCE) on more than 300 macrophages led to the voltammogram in Figure 4 b, which can be very well described by the arithmetic addition of the independent voltammograms of ONOO À and H 2 O 2 at 2.4 mM and 1.2 mM, respectively, detected in in vitro experiments with the same microelectrodes (Figure 4 b). Addition of the two currents is justified by 1) the absence in vitro of any reaction between ONOO À and H 2 O 2 when they are mixed together in an equivalent molar ratio (1 mM each) and 2) the fact that the in vitro oxidation wave for the mixture corresponds to the addition of the oxidation currents from each species as determined from independent solutions of each species.…”

“…[19,25,43] and, on the other hand, the oxidation wave of nitrite starts at + 650 mV versus SSCE, that is, at the plateau potential of NOC oxidation. Consequently, the nitric oxide wave amplitude can be obtained with excellent precision by subtracting the amperometric signal detected at + 650 mV from the one detected at + 450 mV.…”

“…Besides this, we have previously determined that, in the configuration employed for the present cell-response detection, wherein the platinized carbon surface of the microelectrode, with 10 mm diameter, is placed at a distance of 5 mm from the cell membrane, the collection efficiency by the microelectrode is nearly quantitative. [31,43] Therefore, the current intensities calculated above provide a quantitative measurement of the fluxes of emission of each compound by the cell. These fluxes (F) were then calculated from the subtracted current traces in Figure 5 a, according to Faraday's law [Eq.…”

Some Applications of Electrochemistry in Biomedical Chemistry. Emphasis on the Correlation of Electrochemical and Bioactive Properties

“…29 However what is less understood is how these chemical messengers are released by the cell in its outer-cytoplasmic fluids, due mainly to the fact that those releases occur in the atto-or femtomole ranges which prevent the use of classical analytical methods. 29 Ultramicroelectrodes may prove extremely useful for monitoring such events. They allow fundamental biological events to be monitored in real time at the single cell level.…”

“…They allow fundamental biological events to be monitored in real time at the single cell level. 29,30 Because an ultramicroelectrode, normally a platinized carbon fiber, may only probe a volume that is comparable to its own size, it behaves like a fluxmicroscope observing concentration changes in its vicinity. The acquired information is thus essentially dynamic.…”