Research

Abstract: The aim of this study was to use both a theoretical and experimental approach to determine the influence of the sarco-endoplasmic Ca2+-ATPase (SERCA) activity and mitochondria Ca2+ uptake on Ca2+ homeostasis in airway myocytes. Experimental studies were performed on myocytes freshly isolated from rat trachea. [Ca2+]i was measured by microspectrofluorimetry using indo-1. Stimulation by caffeine for 30 s induced a concentration-graded response characterized by a transient peak followed by a progressive decay to a plateau phase. The decay phase was accelerated for 1-s stimulation, indicating ryanodine receptor closure. In Na2+-Ca2+-free medium containing 0.5 mM La3+, the [Ca2+]i response pattern was not modified, indicating no involvement of transplasmalemmal Ca2+ fluxes. The mathematical model describing the mechanism of Ca2+ handling upon RyR stimulation predicts that after Ca2+ release from the sarcoplasmic reticulum, the Ca2+ is first sequestrated by cytosolic proteins and mitochondria, and pumped back into the sarcoplasmic reticulum after a time delay. Experimentally, we showed that the [Ca2+]i decay after Ca2+ increase was not altered by the SERCA inhibitor cyclopiazonic acid, but was slightly but significantly modified by the mitochondria uncoupler carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone. The experimental and theoretical results indicate that, although Ca2+ pumping back by SERCA is active, it is not primarily involved in [Ca2+]i decrease that is due, in part, to mitochondrial Ca2+ uptake.

Elsevier	Roux E, Noble PJ, Noble D, Marhl M Modelling of calcium handling in airway myocytes Progress in Biophysics & Molecular Biology 90 (2006) 64-87	*Abstract:* Airway myocytes are the primary effectors of airway reactivity which modulates airway resistance and hence ventilation. Stimulation of airway myocytes results in an increase in the cytosolic Ca2+ concentration ([Ca2+](i)) and the subsequent activation of the contractile apparatus. Many contractile agonists, including acetylcholine, induce [Ca2+](i) increase via Ca2+ release from the sarcoplasmic reticulum through InsP(3) receptors. Several models have been developed to explain the characteristics of InsP(3)-induced [Ca2+](i) responses, in particular Ca2+ oscillations. The article reviews the modelling of the major structures implicated in intracellular Ca2+ handling, i.e., InsP(3) receptors, SERCAs, mitochondria and Ca2+-binding cytosolic proteins. We developed theoretical models specifically dedicated to the airway myocyte which include the major mechanisms responsible for intracellular Ca2+ handling identified in these cells. These biocomputations pointed out the importance of the relative proportion of InsP(3) receptor isoforms and the respective role of the different mechanisms responsible for cytosolic Ca2+ clearance in the pattern of [Ca2+](i) variations. We have developed a theoretical model of membrane conductances that predicts the variations in membrane potential and extracellular Ca2+ influx. Stimulation of this model by simulated increase in [Ca2+](i) predicts membrane depolarisation, but not great enough to trigger a significant opening of voltage-dependant Ca2+ channels. This may explain why airway contraction induced by cholinergic stimulation does not greatly depend on extracellular calcium. The development of such models of airway myocytes is important for the understanding of the cellular mechanisms of airway reactivity and their possible modulation by pharmacological agents.

The Biophysical Society	*E. Roux and M. Marhl* Role of sarcoplasmic reticulum and mitochondria in Ca2+ removal in airway myocytes Biophysical Journal 86 (2004) 2583-2595	Abstract: The aim of this study was to use both a theoretical and experimental approach to determine the influence of the sarco-endoplasmic Ca2+-ATPase (SERCA) activity and mitochondria Ca2+ uptake on Ca2+ homeostasis in airway myocytes. Experimental studies were performed on myocytes freshly isolated from rat trachea. [Ca2+]i was measured by microspectrofluorimetry using indo-1. Stimulation by caffeine for 30 s induced a concentration-graded response characterized by a transient peak followed by a progressive decay to a plateau phase. The decay phase was accelerated for 1-s stimulation, indicating ryanodine receptor closure. In Na2+-Ca2+-free medium containing 0.5 mM La3+, the [Ca2+]i response pattern was not modified, indicating no involvement of transplasmalemmal Ca2+ fluxes. The mathematical model describing the mechanism of Ca2+ handling upon RyR stimulation predicts that after Ca2+ release from the sarcoplasmic reticulum, the Ca2+ is first sequestrated by cytosolic proteins and mitochondria, and pumped back into the sarcoplasmic reticulum after a time delay. Experimentally, we showed that the [Ca2+]i decay after Ca2+ increase was not altered by the SERCA inhibitor cyclopiazonic acid, but was slightly but significantly modified by the mitochondria uncoupler carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone. The experimental and theoretical results indicate that, although Ca2+ pumping back by SERCA is active, it is not primarily involved in [Ca2+]i decrease that is due, in part, to mitochondrial Ca2+ uptake.

Elsevier	*T. Haberichter, E. Roux, M. Marhl, J.-P. Mazat* The influence of different InsP3 receptor isoforms on Ca2+ signaling in tracheal smooth muscle cells Bioelectrochemistry 57 (2002) 129-138	Abstract: In airway myocytes, like in many cells, Ca2+ signaling is controlled by inositol 1,4,5-trisphosphate (InsP(3)) via InsP(3) receptors (InsP(3)R) located in the sarco-endoplasmic reticulum. Three types of InsP(3)R exist, labeled Types 1, 2, and 3, which differ in their gating kinetics.-We analyze a possible impact of the different gating kinetics of Type 1 and Type 3 InsP(3)R on the time course of cytosolic Ca2+ concentration in tracheal smooth muscle cells upon agonist stimulation. Previous experimental data in rat tracheal myocytes showed that upon gradually increased stimulation with acetylcholine (ACh), a contractile agonist that acts via InsP(3) production, signal spikes, several spikes with declining maxima, and sustained oscillations appear. Our model reproduces the time courses of cytosolic Ca2+ measured in tracheal myocytes. Moreover, by postulating slight variations in the model parameters which determine the total number of receptors expressed and the ratio between Type 1 and Type 3 InsP(3)R, it offers an explanation to the experimental observation of qualitatively different responses of cells within a presumably homogeneous tissue.