Respiratory Physiology & Neurobiology 170 (2010) 113–119 Contents lists available at ScienceDirect Respiratory Physiology & Neurobiology journal homepage: www.elsevier.com/locate/resphysiol Epithelial Na + channels derived from human lung are activated by shear force Martin Fronius a,∗ , Roman Bogdan a , Mike Althaus a,b , Rory E. Morty b , Wolfgang G. Clauss a a Institute of Animal Physiology, University of Giessen Lung Center, Justus-Liebig-University, Wartweg 95, 35392 Giessen, Germany b Department of Internal Medicine, University of Giessen Lung Center, Justus-Liebig-University, Giessen, Germany article info Article history: Accepted 11 November 2009 Keywords: ENaC Epithelial Na + channel Shear force Mechanosensitive Pulmonary epithelium abstract During breathing the pulmonary epithelial cells are permanently exposed to physical forces and shear force (SF) in particular. In our present study we questioned whether the lung epithelial Na + channel (hENaC) responds to shear force. For this purpose ENaC was cloned from human lung tissue, expressed in Xenopus oocytes and functionally characterized by electrophysiological techniques. Shear force in physi- ological relevant ranges was applied via a fluid stream. By the application of SF we obtained an increased inward current indicating an activation of hENaC. The SF-induced effect was reversible and interestingly, the response to SF was augmented by trypsin due to proteolytic cleavage. The direct activation of hENaC by SF was confirmed in outside-out single channel experiments. In five out of nine recordings an increased NP O was observed. From our observations we conclude that lung-derived hENaCs are directly activated by SF and this may represent an important feature for the regulation of pulmonary Na + reabsorption and pulmonary fluid homeostasis. © 2009 Elsevier B.V. All rights reserved. 1. Introduction The lung of air breathing vertebrates is a highly dynamic organ composed of cells that are continually exposed to mechanical forces. In mammals, the pulmonary epithelial cells, which form a barrier between the external environment and the interior of the organism, are exposed to different mechanical stimuli including distention caused by the expansion of the chest due to tidal breath- ing (Wirtz and Dobbs, 2000) and shear forces at the surface of the cells generated by airflow through the airways and the movement of fluid that covers the epithelial layer (Tarran et al., 2006). Mechanical forces are important for lung function under physio- logical conditions. For example, fetal lung development depends on transmural pressures generated by liquid secretion into the alve- olar airspace (Olver et al., 2004) and tidal breathing movements of the chest (Kitterman, 1996). Furthermore, mechanical forces influence the differentiation of alveolar epithelial cells (Dobbs and Gutierrez, 2001), the release of surfactant components (Edwards et al., 1999) and induce the release of paracrine mediators such as ATP (Grygorczyk and Hanrahan, 1997; Homolya et al., 2000). The release of ATP introduces the possibility of multiple cellular responses by the activation of purinergic receptors (Bucheimer and Linden, 2004; Burnstock, 2007). Apart from forming the blood–air barrier, the pulmonary epithelium controls the viscosity and the volume of the fluid ∗ Corresponding author. Tel.: +49 641 99 35255; fax: +49 641 99 35059. E-mail address: martin.fronius@bio.uni-giessen.de (M. Fronius). that lines the epithelial surface (Matthay et al., 2002; Boucher, 2003). This is accomplished primarily through the activity of apical epithelial Na + channels (ENaCs) and the basolateral Na + /K + -ATPase (Matthay et al., 2002). The concerted activity of these proteins generates transepithelial osmotic gradients, which represent the physical driving force for water movement across the epithelium. The importance of these processes is evident in patients with cystic fibrosis (Thelin and Boucher, 2007) or pulmonary edema (Matthay et al., 2002), where perturbations to alveolar ion transport are observed. Although the function of pulmonary epithelia may be influenced by mechanical forces, little is known about the direct impact of mechanical forces on ion transport processes in general, and lung epithelial ion channels in particular. We have recently reported that exposure of native lung epithelia to increased hydrostatic pressure (5 cm H 2 O) altered the behavior of epithelial cells; includ- ing alterations to ion transport processes and the activation of a Na + conductance in particular (Bogdan et al., 2008). Taking this into consideration, together with the fact that mouse, Xeno- pus and rat ENaCs are activated by shear force (Satlin et al., 2001; Althaus et al., 2007), the focus of this study was to question, whether or not the human lung ortholog might also be mechanosensitive. For this approach we used , , and ENaC cloned from human lung tissue. ENaC was heterologously expressed in Xenopus oocytes and activity was measured by the two-electrode-voltage-clamp (TEVC) and the patch-clamp tech- nique. Channels were mechanically challenged by the activation of a fluid stream, producing shear forces at the surface of the cell membrane. 1569-9048/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.resp.2009.11.004