Christian Amatore

Electrochemical Monitoring of Single Cell Secretion: Vesicular Exocytosis and Oxidative Stress

Communication between cellular organisms occurs, among other mechanisms, through the release of specific biochemical or chemical messengers by an emitting cell, generally coupled to a specific detection of these messengers by a receiving cell. According to the target or the scope of the information exchanged, these messengers are released into biological fluids (for instance, into the blood flow), a restricted volume (i.e., a * To whom correspondence should be addressed. Tel: 33-1-4432-3388. Fax: 33-1-4432-3863. E-mail: Christian.Amatore@ens.fr. Chem. Rev. 2008, 108, 2585–2621 2585

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

Macrophages are key cells of the immune system. During phagocytosis, the macrophage engulfs a foreign bacterium, virus, or particle into a vacuole, the phagosome, wherein oxidants are produced to neutralize and decompose the threatening element. These oxidants derive from in situ production of superoxide and nitric oxide by specific enzymes. However, the chemical nature and sequence of release of these compounds is far from being completely determined. The aim of the present work was to study the fundamental mechanism of oxidant release by macrophages at the level of a single cell, in real time and quantitatively. The tip of a microelectrode was positioned at a micrometric distance from a macrophage in a culture to measure oxidative‐burst release by the cell when it was submitted to physical stimulation. The ensuing release of electroactive reactive oxygen and nitrogen species was detected by amperometry and the exact nature of the compounds was characterized through comparison with in vitro electrochemical oxidation of H2O2, ONOO−, NO., and NO2− solutions. These results enabled the calculation of time variations of emission flux for each species and the reconstruction of the original flux of production of primary species, O2.− and NO., by the macrophage.

Glutamatergic Control of Microvascular Tone by Distinct GABA Neurons in the Cerebellum

The tight coupling between increased neuronal activity and local cerebral blood flow, known as functional hyperemia, is essential for normal brain function. However, its cellular and molecular mechanisms remain poorly understood. In the cerebellum, functional hyperemia depends almost exclusively on nitric oxide (NO). Here, we investigated the role of different neuronal populations in the control of microvascular tone by in situ amperometric detection of NO and infrared videomicroscopy of microvessel movements in rat cerebellar slices. Bath application of an NO donor induced both NO flux and vasodilation. Surprisingly, endogenous release of NO elicited by glutamate was accompanied by vasoconstriction that was abolished by inhibition of Ca2+-phopholipase A2 and impaired by cyclooxygenase and thromboxane synthase inhibition and endothelin A receptor blockade, indicating a role for prostanoids and endothelin 1 in this response. Interestingly, direct stimulation of single endothelin 1-immunopositive Purkinje cells elicited constriction of neighboring microvessels. In contrast to glutamate, NMDA induced both NO flux and vasodilation that were abolished by treatment with a NO synthase inhibitor or with tetrodotoxin. These findings indicate that NO derived from neuronal origin is necessary for vasodilation induced by NMDA and, furthermore, that NO-producing interneurons mediate this vasomotor response. Correspondingly, electrophysiological stimulation of single nitrergic stellate cells by patch clamp was sufficient to release NO and dilate both intraparenchymal and upstream pial microvessels. These findings demonstrate that cerebellar stellate and Purkinje cells dilate and constrict, respectively, neighboring microvessels and highlight distinct roles for different neurons in neurovascular coupling.

Electrochemical Study of Methyl 2-[p-Nitrophenyl(hydroxy)methyl]acrylate An Anticancer Drug and Its Reactivity Toward GSH and Oxygen

Electrochemical experiments with methyl 2-[p-nitrophenyl(hydroxy)methyl]acrylate (1) were performed in protic (EtOH + phosphate buffer 1:9, 0.1 mol L -1 , pH 6.9; EtOH + phosphate buffer + NaOH 1:9, 0.1 mol L -1 or 0.2 mol L -1 , pH 9.4 and EtOH + NaCO 3 + NaOH 2:8, 0.18 mol L -1 , pH 9.6) and aprotic [dimethylformamide (DMF) + tetrabutylammonium perchlorate (TBAP), 0.1 mol L -1 ] media. The primary reduction behavior in aprotic medium was typical of nitroaromatics along with an additional wave related to the reduction of the acrylate function. Kinetic analysis carried out in aprotic and aqueous basic media pointed out to the high stability of the electrogenerated nitro radical anion, especially in DMF + TBAP. Reduced (GSH) and oxidized (GSSG) gluthatione in phosphate buffer influenced the reduction behavior of 1, due mainly to protonation effects. Direct reduction of 1, in the presence of GSH, led to a transient nitroso-GS adduct. In the presence of GSSG, hydrogen-bonding-associated GSSG-hydroxylamine was the main product. Electrochemical studies of 1, in the presence of oxygen, showed no chemical reactivity between O 2 and 1. These electrochemical results help in the understanding of the anticancer activity of 1 that can be considered a bioreductive agent with a glutathione depleting function.

Electrochemical Study of Pharmacological Activity at Single Cells: Beta-lapachone Effect on Oxidative Stress of Macrophages

Beta-lapachone [1], a natural ortho-naphthoquinone, has been widely used for its large pharmacological activities, particularly against cancers. In the present study, amperometry at platinized carbon microelectrode was used to investigate the activity of [1], at different concentrations after various incubation times, on the oxidative bursts release of single macrophages. The results show that the presence of 0.1 to 100 μM of beta-lapachone, within one hour of incubation, leads to a decrease of reactive oxygen and nitrogen species release, comparatively to control. Conversely, when the incubation time increases (4 hours and more), the quantity of the species released in presence of 1 μM of [1] increases. Moreover, macrophage membranes became withered after 4 hours of incubation in presence of 10 μM of [1] and leading to cell death. These studies demonstrated the advantage of electrochemical methods for analyzing in real-time and quantitatively the effect of beta-lapachone on oxidative burst.

Modification of Cephalosporins Bearing Substituents at C(3’)Position via Electrogenerated Radical-Cation of Sulfide

Chemoselective electrooxidations of 3-(3, 5-Di tert-butyl-4-methoxyphenylsulfenylmethyl)-△3-cephem la was successfully achieved to affored 3-methoxymethyl-△3-cephem 2 On the other hand, electrooxidations of 3-(3,5-dimethyl-4-methoxyphenylsulfenylmethyl)-△3-cephem 1b was afforded 3dimethoxymethyl-△3-cephem 3

CHAPTER 6:Real Time Monitoring of Peroxynitrite by Stimulation of Macrophages with Ultramicroelectrodes

In the two last decades, electrochemical techniques have been shown to be an efficient tool to investigate oxidative stress and the production of reactive oxygen and nitrogen species. Among them, the “artificial synapse” configuration involving platinized carbon fiber UMEs is of particular interest because it allows one to quantify in real time the very harmful and unstable peroxynitrite anion within the oxidative burst at the single cell level. In this chapter, the main studies dealing with this method of electrochemical detection of peroxynitrite will be summarized and commented upon. Additionally, because some drawbacks may remain, recent works devoted to the use of microsystems or nanotools will also be considered.

A new approach to the determination of the stellate neuron activity function in rat’s brain

In this work, we present the results of a mathematical modelling of NO· release by neurons and its transport in the brain by diffusion. The model is applied to analyze the experimental data on NO· release from a neuron monitored during its patch-clamp stimulation by an ultramicroelectrode introduced into a slice of living rat’s brain. The neuron activity function was obtained by numerical deconvolution of the experimental data using the response function of the electrode to an instantaneous spike of neuronal activity. The Gaussian decomposition of NO· release activity function allows qualitative and quantitative conclusions to be drawn about neuron activity. Since the integral activity function is readily obtained by deconvolution, the decomposition can be performed using other more relevant descriptions of NO· bursts emerging from active neurons.

Chemical Reactivity of Molecular Systems in Media Organized at the Molecular Level

Direct experimental investigation of chemical transformations undergone by molecular substances over the considerable time-scales involved in the evolution of archaeological samples is by definition inaccessible.

Ultramicroelectrodes: Their Use in Semi-Artificial Synapses

Among their numerous other important properties, ultramicroelectrodes allow fundamental biological events to be monitored in real time at the single cell level. This is explained and discussed here based on the presentation of the first kinetic characterization of oxidative stress response from living cells.