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1 INSERM 426, Faculte de Medecine Xavier Bichat, Universite Paris 7, Paris, France; Department of Physiology, Universite de Versailles Saint Quentin, Versailles, France
2 Departement de Pharmacologie et Toxicologie, Universite de Lausanne, Lausanne, Switzerland
3 Department of Anesthesiology, Tokyo Medical and Dental University, Tokyo, Japan
4 Cardiovascular Research Institute, University of California, San Francisco, San Fancisco, California, USA
5 INSERM 426, Faculte de Medecine Xavier Bichat, Universite Paris 7, Paris, France
* To whom correspondence should be addressed. E-mail: carole.planes{at}apr.ap-hop-paris.fr.
The amiloride-sensitive epithelial sodium channel (ENaC) constitutes a rate-limiting step for sodium (Na+) and water absorption across lung alveolar epithelium. Recent reports suggested that ENaC is regulated by membrane-bound extracellular serine proteases, such as channel-activating proteases (CAPs). The objectives of this study were to examine the role of serine proteases in the regulation of transepithelial alveolar Na+ and water transport in vitro and in vivo and the expression of CAPs in rodent distal lung. In vitro experiments showed that inhibition of endogenous serine proteases by apical aprotinin (i) decreased ENaC-mediated currents in primary cultures of rat and mouse alveolar epithelial cells without affecting the abundance nor the electrophoretic migration pattern of biotinylated 
- and 
-ENaC expressed at the cell surface, and (ii) suppressed the increase in amiloride-sensitive short-circuit current induced by the 2-agonist terbutaline. RT-PCR experiments indicated that CAP1, CAP2 and CAP3 mRNAs were expressed in mouse alveolar epithelial cells, whereas CAP1 was also expressed in alveolar macrophages recovered by bronchoalveolar lavage. CAP1 protein was detected by Western-blotting in rat and mouse alveolar epithelial cells, alveolar macrophages and bronchoalveolar lavage fluid. Finally, in vivo experiments revealed that intra-alveolar treatment with aprotinin abolished the increase in Na+-driven alveolar fluid clearance induced by terbutaline in an in situ mouse lung model, while trypsin potentiated it. These results show that endogenous membrane-bound and/or secreted serine proteases such as CAPs regulate alveolar Na+ and fluid transport in vitro and in vivo in rodent lung.
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