Cell Calcium 48 (2010) 251–259 Contents lists available at ScienceDirect Cell Calcium journal homepage: www.elsevier.com/locate/ceca Stretch-activated channels in pulmonary arterial smooth muscle cells from normoxic and chronically hypoxic rats Thomas Ducret a,b,∗ , Jalila El Arrouchi a,b , Arnaud Courtois a,b , Jean-Franc ¸ ois Quignard a,b , Roger Marthan a,b , Jean-Pierre Savineau a,b a Université Bordeaux 2, Laboratoire de Physiologie Cellulaire Respiratoire, F-33076 Bordeaux, France b INSERM, U885, F-33076 Bordeaux, France article info Article history: Received 19 February 2010 Received in revised form 10 September 2010 Accepted 30 September 2010 Available online 29 October 2010 Keywords: Mechanosensitive cation channels Hypotonic stimulation-induced calcium influx Membrane stretch Myogenic tone Vascular smooth muscle abstract Stretch-activated channels (SACs) act as membrane mechanotransducers since they convert physical forces into biological signals and hence into a cell response. Pulmonary arterial smooth muscle cells (PASMCs) are continuously exposed to mechanical stimulations e.g., compression and stretch, that are enhanced under conditions of pulmonary arterial hypertension (PAH). Using the patch-clamp tech- nique (cell-attached configuration) in PASMCs, we showed that applying graded negative pressures (from 0 to -60 mmHg) to the back end of the patch pipette increases occurrence and activity of SACs. The current–voltage relationship (from -80 to +40 mV) was almost linear with a reversal potential of 1 mV and a slope conductance of 34 pS. SACs were inhibited in the presence of GsMTx-4, a specific SACs blocker. Using microspectrofluorimetry (indo-1), we found that hypotonic-induced cell swelling increases intracellular Ca 2+ concentration ([Ca 2+ ] i ). This [Ca 2+ ] i increase was markedly inhibited in the absence of external Ca 2+ or in the presence of the following blockers of SACs: gadolinium, streptomycin, and GsMTx-4. Interestingly, in chronically hypoxic rats, an animal model of PAH, SACs were more active and hypotonic-induced calcium response in PASMCs was significantly higher (nearly a two-fold increase). Moreover, unlike in normoxic rats, intrapulmonary artery rings from hypoxic rats mounted in a Mulvany myograph, exhibited a myogenic tone sensitive to SAC blockers. In conclusion, this work demonstrates that SACs in rat PASMCs can be activated by membrane stretch as well as hypotonic stimulation and are responsible for [Ca 2+ ] i increase. The link between SACs activation-induced calcium response and myo- genic tone in chronically hypoxic rats suggests that SACs are an important element for the increased pulmonary vascular tone in PAH and that they may represent a molecular target for PAH treatment. © 2010 Elsevier Ltd. All rights reserved. 1. Introduction Stretch-activated channels (SACs) at the site of the cell mem- brane are referred to as mechanotransducers since they convert physical forces into biological signals and, hence into a cell response. Since their original characterization in embryonic chick skeletal muscle [1], SACs have been identified in a variety of tis- sues and species including mammalian smooth muscle cells (SMCs) (for review see [2,3]). A common characteristic of SACs is that their open probability increases with the applied pressure i.e., their gat- ing depends on membrane stretch [4,5]. Moreover it is known that SACs may provide an effective pathway for Ca 2+ influx from the extracellular medium to the cytosol [6]. However, the identity of ∗ Corresponding author at: Laboratoire de Physiologie Cellulaire Respiratoire, INSERM U885, Université Bordeaux 2, 146 rue Léo Saignat, F-33076 Bordeaux Cédex, France. Tel.: +33 5 57 57 16 94; fax: +33 5 57 57 16 95. E-mail address: thomas.ducret@u-bordeaux2.fr (T. Ducret). these mechanosensitive channels remains elusive. Best candidate channels for SACs are proteins of the transient receptor potential (TRP) superfamily, which were initially recognized in Drosophila [7] and then also in mammalian cells [8]. In vascular smooth muscle cells, electrophysiological experi- ments have shown that SACs have similar general characteristics (for review see [2]). The evoked currents have a similar reversal potential (around 0 mV) which is close to the theoretical equilib- rium potential of monovalent cations under these experimental conditions, showing that SACs are non-selective cationic channels. According to the cation used as the charge carrier, unitary con- ductance ranges from 26 to 36 pS. Because of their position in a complex biomechanical environment, vascular SMCs are contin- uously exposed to mechanical stimulations such as compression, shear stress, and stretch. In the arterial wall, SMCs are exposed to blood flow and intraluminal pressure. Altered vascular tone is responsible for elevation of intraluminal pressure, and conversely, increased intraluminal pressure induces an intrinsic vasomo- tor mechanism termed “myogenic tone” [9]. Indeed, change in 0143-4160/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.ceca.2010.09.011