Matthieu Raoux

ATP signalling is crucial for the response of human keratinocytes to mechanical stimulation by hypo‐osmotic shock

Abstract:u2002 Touch is detected through receptors located in the skin and the activation of channels in sensory nerve fibres. Epidermal keratinocytes themselves, however, may sense mechanical stimulus and contribute to skin sensation. Here, we showed that the mechanical stimulation of human keratinocytes by hypo‐osmotic shock releases adenosine triphosphate (ATP) and increases intracellular calcium. We demonstrated that the release of ATP was found to be calcium independent because emptying the intracellular calcium stores did not cause ATP release; ATP release was still observed in the absence of external calcium and it persisted on chelating cytosolic calcium. On the other hand, the released ATP activated purinergic receptors and mobilized intracellular calcium stores. The resulting depletion of stored calcium led to the activation of capacitative calcium entry. Increase in cytosolic calcium concentration was blocked by the purinergic receptor blocker suramin, phospholipase C inhibitor and apyrase, which hydrolyses ATP. Collectively, our data demonstrate that human keratinocytes are mechanically activated by hypo‐osmotic shock, leading first to the release of ATP, which in turn stimulates purinergic receptors, resulting in the mobilization of intracellular calcium and capacitative calcium entry. These results emphasize the crucial role of ATP signalling in the transduction of mechanical stimuli in human keratinocytes.

Chemicals inducing acute irritant contact dermatitis mobilize intracellular calcium in human keratinocytes.

Intracellular Ca(2+) increase is a common feature of multiple cellular pathways associated with receptor and channel activation, mediator secretion and gene regulation. We investigated the possibility of using this Ca(2+) signal as a biomarker for a reaction to chemical irritants of normal human keratinocytes (NHK) in submerged primary cell culture. We tested 14 referenced chemical compounds classified as strong (seven), weak (four) or non- (three) irritants in acute irritant contact dermatitis. We found that the strong irritant compounds tested at 20-40 mM induced an intracellular Ca(2+) increase measurable by spectrofluorimetry in an automated test. Weak and non-irritant compounds however did not increase intracellular Ca(2+) concentration. We further investigated the mechanisms by which the amine heptylamine, classified as a R34 corrosive compound, increases intracellular Ca(2+). Heptylamine (20mM) induced an ATP release that persisted in the absence of intra- and extra-cellular Ca(2+). In addition, we found that this ATP activates NHK purinergic receptors that subsequently cause the increase in intracellular Ca(2+) from sarcoplasmic reticular stores. We conclude that measuring the intracellular Ca(2+) concentration in NHK is a suitable and easy way of determining any potential reaction to soluble chemical compounds.

Mechanosensitive Cation Currents and their Molecular Counterparts in Mammalian Sensory Neurons

Although all animals employ mechanical sensations to apprehend their external and internal environments, the molecular transduction mechanisms involved in the detection of mechanical stimuli remain obscure. Mechanoreceptive somatosensory neurons are responsible for the transduction of mechanical stimuli into action potentials that propagate to the central nervous system. The ability of these sensory neurons to detect mechanical information relies on the presence of mechanosensitive channels that rapidly transform external mechanical forces into electrical signals. In the few past years, genetic approaches coupled to functional studies have provided insights into the basic mechanisms by which the senses of touch and pain are transduced in mammals. This review summarizes the methodological approaches and properties of mechanically gated ion channels in mammalian somatosensory neurons.

Bioelectronic organ-based sensor for microfluidic real-time analysis of the demand in insulin

On-line and real-time analysis of micro-organ activity permits to use the endogenous analytical power of cellular signal transduction algorithms as biosensors. We have developed here such a sensor using only a few pancreatic endocrine islets and the avoidance of transgenes or chemical probes reduces bias and procures general usage. Nutrient and hormone-induced changes in islet ion fluxes through channels provide the first integrative read-out of micro-organ activity. Using extracellular electrodes we captured this read-out non-invasively as slow potentials which reflect glucose concentration-dependent (3-15u202fmM) micro-organ activation and coupling. Custom-made PDMS-based microfluidics with platinum black micro-electrode arrays required only some tens of islets and functioned at flow rates of 1-10u202fµl/min which are compatible with microdialysis. We developed hardware solutions for on-line real-time analysis on a reconfigurable Field-Programmable Gate Array (FPGA) that offered resource-efficient architecture and storage of intermediary processing stages. Moreover, real-time adaptive and reconfigurable algorithms accounted for signal disparities and noise distribution. Based on islet slow potentials, this integrated set-up allowed within less than 40u202fμs the discrimination and precise automatic ranking of small increases (2u202fmM steps) of glucose concentrations in real time and within the physiological glucose range. This approach shall permit further development in continuous monitoring of the demand for insulin in type 1 diabetes as well as monitoring of organs-on-chip or maturation of stem-cell derived islets.

Simultaneous monitoring of single cell and of micro-organ activity by PEDOT:PSS covered multi-electrode arrays

Continuous and long-term monitoring of cellular and micro-organ activity is required for new insights into physiology and novel technologies such as Organs-on-Chip. Moreover, recent advances in stem cell technology and especially in the field of diabetes call for non-invasive approaches in quality testing of the large quantities of surrogate pancreatic islets to be generated. Electrical activity of such a micro-organ results in single cell action potentials (APs) of high frequency and in low frequency changes in local field potentials (slow potentials or SPs), reflecting coupled cell activity and overall organ physiology. Each of them is indicative of different physiological stages in islet activation. Action potentials in islets are of small amplitude and very difficult to detect. The use of PEDOT:PSS to coat metal electrodes is expected to reduce noise and results in a frequency-dependent change in impedance, as shown here. Whereas detection of high-frequency APs improves, low frequency SPs are less well detected which is, however, an acceptable trade off in view of the strong amplitude of SPs. Using a dedicated software, recorded APs and SPs can be automatically diagnosed and analyzed. Concomitant capture of the two signals will considerably increase the diagnostic power of monitoring islets and islet surrogates in fundamental research as well as drug screening or the use of islets as biosensors.