RNA interference for alpha-ENaC inhibits rat lung fluid absorption in vivo.
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.
Li
Tianbo
T
Dept. of Physiology and Pharmacology, Northeastern Ohio Universities College of Medicine, Rootstown, OH 44272-0095, USA.
Folkesson
Hans G
HG
D000042:Absorption / Q000187:drug effects; D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D001826:Body Fluids / Q000378:metabolism*; D002199:Capillary Permeability; D004305:Dose-Response Relationship, Drug; D004730:Endothelium, Vascular / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism; D053503:Epithelial Sodium Channels; D004848:Epithelium / Q000378:metabolism; D020128:Inhibitory Concentration 50; D008168:Lung / Q000201:enzymology / Q000378:metabolism*; D008297:Male; D010539:Permeability; D010957:Plasmids / Q000008:administration & dosage / Q000494:pharmacology; D012333:RNA, Messenger / Q000037:antagonists & inhibitors / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D015222:Sodium Channels / Q000187:drug effects / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D013726:Terbutaline / Q000494:pharmacology; D014018:Tissue Distribution
D000318:Adrenergic beta-Agonists; D053503:Epithelial Sodium Channels; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D015222:Sodium Channels; D000584:Amiloride; D013726:Terbutaline