RNA interference for alpha-ENaC inhibits rat lung fluid absorption in vivo

• Published in: American journal of physiology. Lung cellular and molecular physiology Vol. 290; no. 4; pp. L649 - L660
• Main Authors: Li, Tianbo, Folkesson, Hans G
• Format: Journal Article
• Language: English
• Published: United States 01.04.2006
• Subjects: Absorption - drug effects; Adrenergic beta-Agonists - pharmacology; Amiloride - pharmacology; Animals; Body Fluids - metabolism; Capillary Permeability; Dose-Response Relationship, Drug; Endothelium, Vascular - metabolism; Epithelial Cells - metabolism; Epithelial Sodium Channels; Epithelium - metabolism; Inhibitory Concentration 50; Lung - enzymology; Lung - metabolism; Male; Permeability; Plasmids - administration & dosage; Plasmids - pharmacology; Rats; Rats, Sprague-Dawley; RNA, Messenger - antagonists & inhibitors; RNA, Messenger - metabolism; RNA, Small Interfering - genetics; RNA, Small Interfering - pharmacology; Sodium Channels - drug effects; Sodium Channels - genetics; Sodium Channels - metabolism; Sodium Channels - physiology; Terbutaline - pharmacology; Tissue Distribution
• ISSN: 1040-0605; 1522-1504
• DOI: 10.1152/ajplung.00205.2005

We used siRNA against the alpha-ENaC (epithelial Na channel) subunit to investigate ENaC involvement in lung fluid absorption in rats by the impermeable tracer technique during baseline and after beta-adrenoceptor stimulation by terbutaline. Terbutaline stimulation of lung fluid absorption increased fluid absorption by 165% in pSi-0-pretreated rat lungs (irrelevant siRNA-generating plasmid). Terbutaline failed to increase lung fluid absorption in rats given the specific alpha-ENaC siRNA-generating plasmid (pSi-4). pSi-4 pretreatment reduced baseline lung fluid absorption by approximately 30%. alpha-ENaC was undetectable in pSi-4-pretreated lungs, regardless of condition but was normal in pSi-0-pretreated lungs. We carried out a dose-response analysis where rats were given 0-200 microg/kg body wt pSi-4, and alpha-ENaC mRNA and protein expressions were analyzed. To reach IC(50) for alpha-ENaC mRNA expression, 32 microg/kg body wt pSi-4 was needed, and to reach IC(50) for alpha-ENaC protein expression, 59 microg/kg body wt pSi-4 was needed. We tested for lung tissue specificity and found no changes in beta-ENaC expression, at either mRNA or protein level, as well as no changes in alpha(1)-Na-K-ATPase protein expression. We isolated alveolar epithelial type II cells 24 h after in vivo pSi-4 pretreatment. In these cells, alpha-ENaC mRNA was undetectable, demonstrating that alveolar epithelial ENaC expression was attenuated after intratracheal alpha-ENaC siRNA-generating plasmid DNA instillation. We tested for organ specificity and found no changes in kidney alpha- and beta-ENaC mRNA and protein expression. Thus we provide conclusive evidence that beta-adrenoceptor stimulation of lung fluid absorption is critically ENaC dependent, whereas baseline lung fluid absorption seemed less ENaC dependent.