Stéphane Arbault

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.

The Effects of Vesicular Volume on Secretion through the Fusion Pore in Exocytotic Release from PC12 Cells

Many spikes in amperometric records of exocytosis events initially exhibit a prespike feature, or foot, which represents a steady-state flux of neurotransmitter through a stable fusion pore spanning both the vesicle and plasma membranes and connecting the vesicle lumen to the extracellular fluid. Here, we present the first evidence indicating that vesicular volume before secretion is strongly correlated with the characteristics of amperometric foot events. l-3,4-Dihydroxyphenylalanine and reserpine have been used to increase and decrease, respectively, the volume of single pheochromocytoma cell vesicles. Amperometry and transmission electron microscopy have been used to determine that as vesicle size is decreased the frequency with which foot events are observed increases, the amount and duration of neurotransmitter released in the foot portion of the event decreases, and vesicles release a greater percentage of their total contents in the foot portion of the event. This previously unidentified correlation provides new insight into how vesicle volume can modulate the activity of the exocytotic fusion pore.

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.

Characterization of the Electrochemical Oxidation of Peroxynitrite: Relevance to Oxidative Stress Bursts Measured at the Single Cell Level

The electrochemical signature of peroxynitrite oxidation is reported for the first time, and its mechanism discussed in the light of data obtained by steady-state and transient voltammetry at microelectrodes. Peroxynitrite is an important biological species generated by aerobic cells presumably via the near diffusion-limited coupling of nitric oxide and superoxide ion. Its production by living cells has been previously suspected during cellular oxidative bursts as well as in several human pathologies (arthritis, inflammation, apoptosis, ageing, carcinogenesis, Alzheimer disease, AIDS, etc.). However, this could only be inferred on the basis of characteristic patient metabolites or through indirect detection, or by observation of follow-up species resulting supposedly from its chemical reactions in vivo. In this work, thanks to the independent knowledge of the electrochemical characteristics of ONO2- oxidation, the kinetics and intensity of this species released by single human fibroblasts could be established directly and quantitatively based on the application of the artificial synapse method. It was then observed and established that fibroblasts submitted to mechanical stresses produce oxidative bursts, which involve the release within less than a tenth of a second of a complex cocktail composed of several femtomoles of peroxynitrite, hydrogen peroxide, nitric oxide, and nitrite ions.

Real-Time Amperometric Analysis of Reactive Oxygen and Nitrogen Species Released by Single Immunostimulated Macrophages

Macrophages are key cells of the immune system. Immunologically activated macrophages are known to release a cocktail of reactive oxygen and nitrogen species. In this work, RAW 264.7 macrophages were activated by interferon‐γ and lipopolysaccharide, and the reactive mixture released by single cells was analyzed, in real time, by amperometry at platinized carbon microelectrodes. In comparison with untreated macrophages, significant increases in amperometric responses were observed for activated macrophages. Nitric oxide (NO.), nitrite (NO2−), and peroxynitrite (ONOO−) were the main reactive species detected. The amounts of these reactive species were quantified, and their average fluxes released by a single, activated macrophage were evaluated. The detection of ONOO− is of particular interest, as its role and implications in various physiological conditions have been widely debated. Herein, direct evidence for the formation of ONOO− in stimulated macrophages is presented. Finally, the presence of 1400W, a selective inducible nitric oxide synthase (iNOS) inhibitor, led to an almost complete attenuation of the amperometric response of activated RAW 264.7 cells. The majority of the reactive species released by a macrophage are thus likely to be derived from NO. and superoxide (O2.−) co‐produced by iNOS.

Electrochemical detection in a microfluidic device of oxidative stress generated by macrophage cells

The release of reactive oxygen species (ROS) or reactive nitrogen species (RNS), i.e., the initial phase of oxidative stress, by macrophage cells has been studied by electrochemistry within a microfluidic device. Macrophages were first cultured into a detection chamber containing the three electrodes system and were subsequently stimulated by the microinjection of a calcium ionophore (A23187). Their production of ROS and RNS was then measured by amperometry at the surface of a platinized microelectrode. The fabricated microfluidic device provides an accurate measurement of oxidative release kinetics with an excellent reproducibility. We believe that such a method is simple and versatile for a number of advanced applications based on the detection of biological processes of secretion by a few or even a single living cell.

Regulation of Exocytosis in Chromaffin Cells by Trans-Insertion of Lysophosphatidylcholine and Arachidonic Acid into the Outer Leaflet of the Cell Membrane

Vesicular exocytosis is an important complex process in the communication between cells in organisms. It controls the release of chemical and biochemical messengers stored in an emitting cell. In this report, exocytosis is studied amperometrically (at carbon fiber ultramicroelectrodes) at adrenal chromaffin cells, which release catecholamines after appropriate stimulation, while testing the effects due to trans‐insertion of two exogenous compounds (lysophosphatidylcholine (LPC) and arachidonic acid (AA)) on the kinetics of exocytotic events. Amperometric analyses showed that, under the present conditions (short incubation times and micromolar LPC or AA solutions), LPC favors catecholamine release (rate, event frequency, charge released) while AA disfavors the exocytotic processes. The observed kinetic features are rationalized quantitatively by considering a stalk model, for the fusion pore formation, and the physical constraints applied to the cell membrane by the presence of small fractions of LPC and AA diluted in its external leaflet (trans‐insertion). We also observed that the detected amount of neurotransmitters in the presence of LPC was larger than under control conditions, while the opposite trend is observed with AA.

Simultaneous Detection of Reactive Oxygen and Nitrogen Species Released by a Single Macrophage by Triple Potential-Step Chronoamperometry

Macrophages produce reactive oxygen and nitrogen species (ROS/RNS) in response to immunological challenges. We have previously reported the real-time detection and quantification of released ROS/RNS by immunostimulated macrophages using constant potential amperometry, at four different potentials, with platinized carbon microelectrodes. As a methodological extension to that work, we sought to develop an electroanalytical method that would allow for the simultaneous monitoring of several ROS/RNS. Triple potential-step chronoamperometry at platinized carbon microelectrodes was found to provide satisfactory sensitivity and signal/noise ratio for this purpose. The title method was applied to the detection of endogenously produced ROS/RNS by single IFN-gamma/LPS/PMA stimulated RAW 264.7 macrophages. Significantly higher fluxes of H(2)O(2), ONOO(-), and NO* responses were detected over stimulated macrophages as compared to unactivated macrophages, consistent with the endogenous production of primary NO* and O(2)(*-) by both the inducible isoform of nitric oxide synthase (iNOS) and reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase enzymatic systems in stimulated cells. Crucially, significant temporal variations in the release of each of the aforementioned species was evidenced using this method, which would not have been achievable with the use of either constant potential amperometry or classical biochemical methods such as the Griess assay.

Coupling Amperometry and Total Internal Reflection Fluorescence Microscopy at ITO Surfaces for Monitoring Exocytosis of Single Vesicles

Water-soluble hormones and neurotransmitters are packaged in secretory vesicles and secreted into the extracellular medium by exocytosis, a process involving the fusion of the vesicle membrane with the cell membrane. Transport of the secretory vesicles to the cell s periphery, the maturation stages they undergo there to acquire fusion competence, and the factors controlling the fusion process itself (including the dynamics of the fusion pore) are important biological questions that are not fully understood. To elucidate secretory mechanisms at the single-vesicle level, currently only a few analytical methods exist, which can be grouped into electrical or optical recordings. The great advantage of electrical recordings (patch–clamp membrane capacitance and electrochemical amperometry) is their excellent time resolution (ca. tens of microseconds), which allows studies of the dynamics of the fusion pore itself. However, a major disadvantage is the fact that signals appear only after fusion has commenced; that is, the dynamics of the secretory vesicle itself or any labeled regulatory protein prior to the fusion event cannot be detected. In contrast, optical recordings allow secretory vesicles or regulatory proteins to be visualized and tracked prior to their fusion, yet generally they lack the time resolution required to follow the dynamics of the fusion pore (typical time resolution is ca. 100 ms). In addition, depending on the technique, secretion may be probed from different areas of a cell (top or bottom), which makes comparison of the results obtained by different approaches difficult. Because of their complementary nature, it would be a great advance if electrical and optical measurements could be made simultaneously from the same side of a cell at the singlevesicle level. This will enable a comprehensive and precise analysis of the whole exocytotic event, from predocking through fusion steps up to the dynamics of vesicular release. Herein, we report a device based on transparent indium tin oxide (ITO) electrodes, which allows simultaneous total internal reflection fluorescence microscopy (TIRFM) and amperometric measurements (Figure 1). As a proof of concept, the ability of our device in the coupled optical and electrochemical detections of exocytotic events is demonstrated using enterochromaffin BON cells. Amperometry is based on detection at a microelectrode surface positioned near the emitting cell of electroactive vesicular contents that are released into the extracellular medium. With very high temporal resolution and sensitivity, the flux of the vesicular content (released through an initial fusion pore that is only a few nanometers wide) corresponding to an exocytotic event appears as a current spike, which features (frequency, time length, area, magnitude) the dynamics of release from single vesicles. Generally, amperometry involves placing a large collecting electrode near the investigated cell. The whole cell active surface area is covered so the spatial localization of a particular exocytotic event cannot be achieved. Nevertheless, a few studies involving smaller microelectrodes or microelectrode arrays allowed amperometric signals from different releasing sites to be identified, but with a random positioning for the small microelectrode and a spatial resolution necessarily limited by the array dimensions, respectively. Coupling of amperometric and optical recordings would allow precise localization of exocytosis events in space and time. The most widely used optical approach to study exocytosis, TIRFM, is based on the total internal reflection of a laser beam at the glass/water interface, which creates an evanescent field in the aqueous medium whose characteristic decay length (ca. 100 nm) provides a high signal-to-noise ratio and an axial resolution of about 10 nm. When a vesicle fuses with the plasma membrane, its labeled contents are released toward the glass/water interface where the excitation [*] A. Meunier, Dr. R. Fulcrand, Dr. S. Arbault, Dr. M. Guille, Dr. F. Lema tre, Prof. C. Amatore D partement de Chimie, Ecole Normale Sup rieure UMR 8640 (CNRS-ENS-UPMC Univ Paris 06) 24 rue Lhomond, 75005 Paris (France) Fax: (+33)1-4432-3863 E-mail: Christian.Amatore@ens.fr