In situ electrochemical monitoring of reactive oxygen and nitrogen species released by single MG63 osteosarcoma cell submitted to a mechanical stress.

The oxidative stress responses of single MG63 osteosarcoma cells submitted to a brief mechanical stress have been investigated by amperometry at platinized carbon fiber electrodes for monitoring and characterizing the nature and the amounts of the various reactive oxygen (ROS) and reactive nitrogen species (RNS) released. It was thus shown that, on average, a single MG63 cell released prominent amounts of reactive nitrogen species (17 fmol NO(*), 6 fmol ONOO(-), and 5 fmol NO(2)(-)) together with a comparatively small quantity of H(2)O(2) (2 fmol). These species resulted from the primary production of 13 fmol for O(2)(*-) and 28 fmol for NO(*) per single cell as reconstructed from the stoichiometries of the ROS and RNS releases. The high NO(*)/H(2)O(2) and NO(*)/O(2)(*-) ratios thus found are perfectly consistent with previous claims that the malignant bone formation ability of the osteosarcoma cells is related to a specific high production of NO* associated to a small one of O(2)(*-).

Prediction of local pH variations during amperometric monitoring of vesicular exocytotic events at chromaffin cells.

Electrochemical monitoring of the exocytosis process is generally performed through amperometric oxidation of the electroactive messengers released by single living cells. Herein, we consider the vesicular release of catecholamines by chromaffin cells. Each exocytotic event is thus detected as a current spike whose morphology (intensity, duration, area, etc.) features the efficiency of the secretion process. However, the electrochemical oxidation of catechols produces quinone derivatives and protons. As a consequence, unless specific mechanisms may be adopted by a cell to regulate the pH near its membrane, the local pH between the cell membrane and the electrode necessarily drops within the electrode-cell cleft. Though this consequence of amperometric detection is generally ignored, it has been investigated in this work through simulation of the local pH drop created during the amperometric recording of a sequence of exocytotic events. This was performed based on frequencies and magnitudes of release detected at chromaffin cells. The corresponding acidification was shown to severely depend on the microelectrode radius. For usual 10 µm diameter carbon fiber electrodes, pH values below six were predicted to be reached within the electrode-cell cleft after monitoring a few current spikes.

Quantitative investigations of amperometric spike feet suggest different controlling factors of the fusion pore in exocytosis at chromaffin cells.

Around 30% of exocytosis events recorded by amperometry at carbon fiber microelectrodes exhibit a pre-spike feature (PSF) termed a "foot". This wave is associated with the release of the neurotransmitters via a transitory fusion pore, whilst the large, main exocytotic spike is due to complete release. The amperometric data reported herein were obtained using bovine chromaffin cells stimulated with either potassium or barium ions, two commonly-employed elicitors of exocytosis. Identical trends are observed with both activators: (i) they induce the same ratio (close to 30%) of events with a foot in the population of amperometric spikes, and (ii) spikes with a foot can be divided into two primary categories, depending on the temporal variation of the current wave (viz. as a ramp, or a ramp followed by a plateau). Correlations between the characteristics of the whole current spike, and of its observed foot, have been sought; such analyses demonstrate that the maximum current of both foot and spike signals are highly correlated, but, in contrast, the integrated charges of both are poorly correlated. Moreover, the temporal duration of the PSF is fully uncorrelated with any parameter pertaining to the main current spike. On the basis of these reproducible observations, it is hypothesized that the characteristics (dimensions and topology, at least) of each secretory vesicle determine the probability of formation of the fusion pore and its maximum size, whilst molecular factors of the cell membrane control its duration, and, consequently, the amount delivered prior to the massive exocytosis of catecholamines observed as a spike in amperometry.

Invariance of exocytotic events detected by amperometry as a function of the carbon fiber microelectrode diameter.

Etched carbon fiber microelectrodes of different radii have been used for amperometric measurements of single exocytotic events occurring at adrenal chromaffin cells. Frequency, kinetic, and quantitative information on exocytosis provided by amperometric spikes were analyzed as a function of the surface area of the microelectrodes. Interestingly, the percentage of spikes with foot (as well as their own characteristics), a category revealing the existence of sufficient long-lasting fusion pores, was found to be constant whatever the microelectrode diameter was, whereas the probability of overlapping spikes decreased with the electrode size. This confirmed that the prespike foot could not feature accidental superimposition of separated events occurring at different places. Moreover, the features of amperometric spikes investigated here (charge, intensity and kinetics) were found constant for all microelectrode diameters. This demonstrated that the electrochemical measurement does not introduce significant bias onto the kinetics and thermodynamics of release during individual exocytotic events. All in all, this work evidences that information on exocytosis amperometrically recorded with the usual 7 microm diameter carbon fiber electrodes is biologically relevant, although the frequent overlap between spikes requires a censorship of the data during the analytical treatment.

The nature and efficiency of neurotransmitter exocytosis also depend on physicochemical parameters.

Exocytosis is an important biological process used by cells to deliver messengers or effectors to target cells with high spatial, quantitative, and kinetic precision. This process occurs by interaction and fusion of vesicles containing the (bio)chemical information with the cell membrane to release their contents into the surrounding medium. Because of its importance for life, this mechanism underlies many biological controlling factors, including different families of proteins and enzymes. Tremendous efforts have been made over the last decade toward their determination. However, in parallel, many studies have also shown that the physical and chemical characteristics of the exocytosis actors (vesicle, membrane, and extracellular medium) could directly affect the quantitative or kinetic features of secretion. The major pieces evidence for this influence, which have been reported in the literature, are reviewed herein. It demonstrates undoubtedly that pure biological aspects cannot be segregated from the physicochemical context of living mechanisms.

Relationship between amperometric pre-spike feet and secretion granule composition in chromaffin cells: an overview.

Amperometry is a simple and powerful technique to study exocytosis at the single cell level. By positioning and polarizing (at an appropriate potential at which the molecules released by the cell can be oxidized) a carbon fiber microelectrode at the top of the cell, each exocytotic event is detected as an amperometric spike. More particularly, a portion of these spikes has previously been shown to present a foot, i.e. a small pedestal of current that precedes the spike itself. Among the important number of works dealing with the monitoring of exocytosis by amperometry under different conditions, only a few studies focus on amperometric spikes with a foot. In this work, by coupling our previous and recent experiments on chromaffin cells (that release catecholamines after stimulation) with literature data, we bring more light on what an amperometric foot and particularly its features, represents.

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.

Assessment of the electrochemical behavior of two-dimensional networks of single-walled carbon nanotubes.

Scanning electrochemical microscopy (SECM) has been employed in the feedback mode to assess the electrochemical behavior of two-dimensional networks of single-walled carbon nanotubes (SWNTs). It is shown that, even though the network comprises both metallic and semiconducting SWNTs, at high density (well above the percolation threshold for metallic SWNTs) and with approximately millimolar concentrations of redox species the network behaves as a thin metallic film, irrespective of the formal potential of the redox couple. This result is particularly striking since the fractional surface coverage of SWNTs is only approximately 1% and SECM delivers high mass transport rates to the network. Finite element simulations demonstrate that under these conditions diffusional overlap between neighboring SWNTs is significant so that planar diffusion prevails in the gap between the SECM tip and the underlying SWNT substrate. The SECM feedback response diminishes at higher concentrations of the redox species. However, wet gate measurements show that at the solution potentials of interest the conductivity is sufficiently high that lateral conductivity is not expected to be limiting. This suggests that reaction kinetics may be a limiting factor, especially since the low surface coverage of the SWNT network results in large fluxes to the SWNTs, which are characterized by a low density of electronic states. For electroanalytical purposes, significantly, two-dimensional SWNT networks can be considered as metallic films for typical millimolar concentrations employed in amperometry and voltammetry. Moreover, SWNT networks can be inexpensively and easily formed over large scales, opening up the possibility of further electroanalytical applications.

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.

Modelling release of nitric oxide in a slice of rat's brain: describing stimulated functional hyperemia with diffusion-reaction equations.

The physicochemical process of nitric oxide (NO degrees ) release from an active neuron is modelled based on the results obtained experimentally in independent series of experiments reported elsewhere in which the NO degrees release elicited by patch-clamping a single neuron (stellate neuron from cerebellum area) is monitored by an ultramicroelectrode introduced into a slice of living rat's brain. This process is believed to be central to brain behaviour by coupling neuronal activity with the blood supply to active areas of the living brain through precise control of NO degrees -mediated dilatation of blood capillary vessels. This work, based on the conformal mapping approach initially proposed in a previous work, aims to model the overall physicochemical and diffusional processes giving rise to the release of NO degrees by a neuron and during its collection at an electrode sensor. Fitting simulated currents to experimental ones published previously yields indeed the gross kinetic information which represents the overall neuron activation and defines the instant value of the concentration of NO degrees at the neuron surface. This allows reconstructing the NO degrees fluxes around the neuron body as they would have been in the absence of the electrode sensor. This permits one to appreciate how far NO degrees is released by the neuron at concentrations which greatly exceed their basal values. The success of this procedure is exemplified using a set of three experimental data reported elsewhere.

Nitric oxide release during evoked neuronal activity in cerebellum slices: detection with platinized carbon-fiber microelectrodes.

Nitric oxide is an important biological messenger that particularly induces the relaxation of smooth muscle cells surrounding vessels, and, hence, controls the flow of blood. This mechanism is essential for brain function, and its fine control, termed functional hyperemia, is supposed to be realized by certain neurons that may release bursts of NO*. The aim of the present study is to examine the advantages of platinized carbon-fiber microelectrodes (5-7 microm tip diameter) for the direct and in situ electrochemical detection of NO* released by neurons into ex vivo cerebellum slices. After establishing the different analytical properties of the platinized carbon-fiber microelectrodes in vitro on NO* solutions at 50 nM to 1 mM concentration, they were characterized using DEA-NONOate solutions that chemically decompose into NO*, and therefore mimic the measurement of transient variations of NO* concentration in biological samples. This validated the present approach, so that direct, in situ ex vivo measurements of nitric oxide released by neurons in a rat cerebellar slice are presented and discussed.

Dynamics of full fusion during vesicular exocytotic events: release of adrenaline by chromaffin cells.

Vesicular exocytosis is important in the communication between cells in complex organisms. It controls the release of specific chemical or biochemical messengers stored in the emitting cell, which elicit a response upon detection by the target cells. Secretion of a messenger molecule (a neurotransmitter) was measured electrochemically, which allowed the quantification of cellular events and the validation of current physicochemical models. This model led us to formulate predictions about the occurrence and kinetics of vesicular exocytotic events based on the physicochemical meaning of its key parameters. These predictions were tested successfully through a series of experiments on chromaffin cells, involving changes of osmotic conditions, presence of trivalent ions and cholesterol-induced structuring of the cell plasmic membrane.