Thomas Ducret

Involvement of TRPV1 and TRPV4 channels in migration of rat pulmonary arterial smooth muscle cells.

Pulmonary hypertension, the main disease of the pulmonary circulation, is characterized by an increase in pulmonary vascular resistance, involving proliferation and migration of pulmonary arterial smooth muscle cells (PASMC). However, cellular and molecular mechanisms underlying these phenomena remain to be identified. In the present study, we thus investigated in rat intrapulmonary arteries (1) the expression and the functional activity of TRPV1 and TRPV4, (2) the PASMC migration triggered by these TRPV channels, and (3) the associated reorganization of the cytoskeleton. Reverse transcriptase–polymerase chain reaction (RT-PCR) analysis demonstrated expression of TRPV1 and TRPV4 mRNA in rat intrapulmonary arteries. These results were confirmed at the protein level by western blot. Using microspectrofluorimetry (indo-1), we show that capsaicin and 4α-phorbol-12,13-didecanoate (4α-PDD), selective agonists of TRPV1 and TRPV4, respectively, increased the intracellular calcium concentration of PASMC. Furthermore, stimulation of TRPV1 and TRPV4 induced PASMC migratory responses, as assessed by two different methods (a modified Boyden chamber assay and a wound-healing migration assay). This response cannot seem to be attributed to a proliferative effect as assessed by BrdU and Wst-1 colorimetric methods. Capsaicin- and 4α-PDD-induced calcium and migratory responses were inhibited by the selective TRPV1 and TRPV4 blockers, capsazepine and HC067047, respectively. Finally, as assessed by immunostaining, these TRPV-induced migratory responses were associated with reorganization of the F-actin cytoskeleton and the tubulin and intermediate filament networks. In conclusion, these data point out, for the first time, the implication of TRPV1 and TRPV4 in rat PASMC migration, suggesting the implication of these TRPV channels in the physiopathology of pulmonary hypertension.

Expression and Physiological Roles of TRP Channels in Smooth Muscle Cells

Smooth muscles are widely distributed in mammal body through various systems such as circulatory, respiratory, gastro-intestinal and urogenital systems. The smooth muscle cell (SMC) is not only a contractile cell but is able to perform other important functions such as migration, proliferation, production of cytokines, chemokines, extracellular matrix proteins, growth factors and cell surface adhesion molecules. Thus, SMC appears today as a fascinating cell with remarkable plasticity that contributes to its roles in physiology and disease. Most of the SMC functions are dependent on a key event: the increase in intracellular calcium concentration ([Ca(2+)](i)). Calcium entry from the extracellular space is a major step in the elevation of [Ca(2+)](i) in SMC and involves a variety of plasmalemmal calcium channels, among them is the superfamily of transient receptor potential (TRP) proteins. TRPC (canonical), TRPM (melastatin), TRPV (vanilloid) and TRPP (polycystin), are widely expressed in both visceral (airways, gastrointestinal tract, uterus) and vascular (systemic and pulmonary circulation) smooth muscles. Mainly, TRPC, TRPV and TRPM are implicated in a variety of physiological and pathophysiological processes such as: SMC contraction, relaxation, growth, migration and proliferation; control of blood pressure, arterial myogenic tone, pulmonary hypertension, intestinal motility, gastric acidity, uterine activity during parturition and labor. Thus it is becoming evident that TRP are major element of SMC calcium homeostasis and, thus, appear as novel drug targets for a better management of diseases originating from SMC dysfunction.

Dehydroepiandrosterone reverses chronic hypoxia/reoxygenation-induced right ventricular dysfunction in rats

Dehydroepiandrosterone (DHEA) prevents chronic hypoxia-induced pulmonary hypertension and associated right ventricle dysfunction in rats. In this animal model, reoxygenation following hypoxia reverses pulmonary hypertension but not right ventricle dysfunction. We thus studied the effect of DHEA on the right ventricle after reoxygenation, i.e. after a normoxic recovery phase secondary to chronic hypoxia in rats. Right ventricle function was assessed in vivo by Doppler echocardiography and in vitro by the isolated perfused heart technique in three groups of animals: control, recovery (21 days of hypoxia followed by 21 days of normoxia) and recovery DHEA (30 mg·kg−1 every 2 days during the recovery phase). Right ventricle tissue was assessed by optical and electron microscopy. DHEA abolished right ventricle diastolic dysfunction, as the echographic E wave remained close to that of controls (mean±sd 76.5±2.4 and 79.7±1.7 cm·s−1, respectively), whereas it was diminished to 40.3±3.7 in the recovery group. DHEA also abolished right ventricle systolic dysfunction, as shown by the inhibition of the increase in the slope of the pressure–volume curve in isolated heart. The DHEA effect was related to cardiac myocytes proliferation. In conclusion, DHEA prevents right ventricle dysfunction in this animal model by preventing cardiomyocyte alteration.

Voltage-dependent ionic conductances in the human malignant astrocytoma cell line U87-MG.

Although the human malignant astrocytoma cell line U87-MG has been used in numerous studies, few findings are available on the properties of its membrane ion conductances. Characterization of the ion channels expressed in these cells will make it possible to study membrane ion conductance changes when a receptor is activated by its ligand. This will help to elucidate the functional properties of these receptors and their signal-transduction pathways in pathophysiological events. This work studied the voltage-dependent ionic conductances of U87-MG cells using the Whole-Cell Recording patch-clamp technique. Six types of voltage-dependent ionic currents were identified: (i) a TEA-, 4-AP- and CTX-sensitive Ca2+-dependent K+ current, (ii) a transient K+ current inhibited by 4-AP, (iii) an inwardly rectifying K+ current blocked by Ba2+ and 4-AP, (iv) a DIDS- and SITS-sensitive Cl− current, (v) a TTX-sensitive Na+ conductance and (vi) a L-type Ca2+ conductance activated by BayK-8644 and inhibited by Ni and the L-type Ca2+ channel inhibitor, nifedipine. In addition, electrical depolarizations elicited inward currents due to voltage-independent, Ca2+-dependent K+ influx against the electrochemical gradient, probably via an ouabain-sensitive Na+-K+ pump.

T-type voltage gated calcium channels are involved in endothelium-dependent relaxation of mice pulmonary artery

ABSTRACT In pulmonary arterial endothelial cells, Ca2+ channels and intracellular Ca2+ concentration ([Ca2+]i) control the release of vasorelaxant factors such as nitric oxide and are involved in the regulation of pulmonary arterial blood pressure. The present study was undertaken to investigate the implication of T‐type voltage‐gated Ca2+ channels (T‐VGCCs, Cav3.1 channel) in the endothelium‐dependent relaxation of intrapulmonary arteries. Relaxation was quantified by means of a myograph in wild type and Cav3.1−/− mice. Endothelial [Ca2+]i and NO production were measured, on whole vessels, with the fluo‐4 and DAF‐fm probes. Acetylcholine (ACh) induced a nitric oxide‐ and endothelium‐dependent relaxation that was significantly reduced in pulmonary arteries from Cav3.1−/− compared to wild type mice as well as in the presence of T‐VGCC inhibitors (NNC 55–0396 or mibefradil). ACh also increased endothelial [Ca2+]i and NO production that were both reduced in Cav3.1−/− compared to wild type mice or in the presence of T‐VGCC inhibitors. Immunofluorescence labeling revealed the presence of Cav3.1 channels in endothelial cells that co‐localized with endothelial nitric oxide synthase in arteries from wild type mice. TRPV4‐, beta2 adrenergic‐ and nitric oxide donors (SNP)‐mediated relaxation were not altered in Cav3.1−/− compared to wild type mice. Finally, in chronically hypoxic mice, a model of pulmonary hypertension, ACh relaxation was reduced but still depended on Cav3.1 channels activity. The present study thus demonstrates that T‐VGCCs, mainly Cav3.1 channel, contribute to intrapulmonary vascular reactivity in mice by controlling endothelial [Ca2+]i and ACh‐mediated relaxation.

CD95-Mediated Calcium Signaling

Intracellular calcium signals regulate cell function and cell survival by controlling many processes. CD95 engagement results in distinct intracellular calcium signals that control the cell fate, apoptosis, or survival, depending on the ligand (membrane or soluble). Intracellular calcium determination is an exquisite readout to explore the molecular mechanisms elicited by CD95 engagement. The most widely applied methods for studying calcium signaling pathways use fluorescent indicators and imaging methods with fluorescence microscopy. This technical approach, however, requires many precautions that we discuss in this chapter.

0158 : Benefits of a nutritherapy with glycerophospholipids-DHA versus triglycerides- DHA in preventing pulmonary hypertension and cardiac insufficiency in chronic hypoxemic rats

Background and objectives Chronic hypoxemia induces pulmonary hypertension (PH) leading to cardiac insufficiency, and fish oil (EPA+DHA) seems beneficial to prevent PH. We aimed at investigating the effects of DHA from glycerophospholipids (GPL-DHA) or from algal oil (triglycerides) in preventing experimental PH and heart insufficiency. Procedure The study was conducted in Wistar males rats (n=10 per group) over 7 weeks: a control group (C) in normoxic conditions, two groups maintained in normoxic conditions for 4 weeks and under hypoxic conditions (hypobaric chamber, 0.5 Bar) for the last 3 weeks receiving no DHA (H) or 60-120xa0mg/kg/ day of DHA as GPL (GPL-D60, GPL-D120) or triglycerides (TG-D120). The thickness of the right ventricle (RV) and the pulmonary artery acceleration time (PAAT) were measured weekly by echocardiography. At the end of the study, pulmonary artery pressure (PAP, invasive catheterization) and the fatty acid profile of lungs and heart were determined. Statistics were done by Mann-Whitney test (p Results Hypoxemia led to i) a high increase of the thickness of the RV and RV hypertrophy, ii) a decrease in PAAT (increase in vascular resistance), ii) a change in DHA level in heart (increase) and in lungs (decrease). The DHA supplementation i) limitated the development of RV dysfunction (–19%/–24% thickness), ii) attenuated the decrease in PAAT (+41% or +76% for GPLDHA60 or 120, +46% for TG-DHA120) and the vascular resistance (–12% PAP), iii) increased DHA level slightly in heart (C: 9.3±2.0a; H: 12.4±3.0b; GPL-D60: 14.2±4.4bc; GPL-D120: 15.6±2.8c; TG-DHA120: 14.3±1.5bc) and highly in lungs (C: 2.1±0.2a; H: 1.9±0.2b; GPL-D60: 2.5±0.4c; GPL-D120: 3.5±0.9d; TG-DHA120: 3.7±0.8d). Conclusions GPL-DHA and TG-DHA partially prevented PH and cardiac remodeling. The effects seem not related only to the total level of DHA in tissues suggesting plausible changes in phospholipid classes. The author hereby declares no conflict of interest