Premium
Inhibition of rat lung fluid absorption by αENaC silencing in vivo
Author(s) -
Li Tianbo,
Folkesson Hans G
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.4.a745-d
Subject(s) - in vivo , epithelial sodium channel , gene silencing , chemistry , absorption (acoustics) , lung , microbiology and biotechnology , medicine , biology , physics , biochemistry , sodium , genetics , gene , organic chemistry , acoustics
The relative importance for ENaC during baseline and βAR‐stimulated conditions has not been fully tested. We used siRNA specifically generated to αENaC and measured AFC during baseline and terbutaline‐stimulated conditions in normal rat lungs. siRNA‐generating pDNA against αENaC (pSi‐4) or an irrelevant pDNA (pSi‐0) was instilled intratracheally (100 μg/kg body wt) into the lungs of adult rats. After 24 h, AFC was measured by mass balance with and without terbutaline. In pSi‐0‐instilled rats, terbutaline stimulated AFC by 165%, while terbutaline‐stimulation of AFC was attenuated in pSi‐4‐instilled rats. Baseline AFC was reduced by ~30% in pSi‐4‐instilled rats compared to pSi‐0. We measured lung αENaC, βENaC, and α 1 ‐Na,K‐ATPase mRNA and protein expression from pSi‐4‐ and pSi‐0‐instilled rats. Both αENaC mRNA and protein were undetectable in pSi‐4‐instilled lungs, while normal in pSi‐0‐instilled lungs. We then isolated alveolar epithelial type II (ATII) cells 24 h after in vivo pSi‐4 and pSi‐0 pretreatment. In ATII cells, αENaC mRNA was undetectable, thus demonstrating that alveolar epithelial ENaC expression was attenuated. A dose‐response curve for siRNA‐silencing of αENaC was carried out. The dose‐response curve demonstrated that, in order to reach 50% inhibition (IC 50 ) for αENaC mRNA expression, 32 μg/kg body wt pSi‐4 was needed and to reach IC 50 for αENaC protein expression, 59 μg/kg body wt pSi‐4 was needed. We tested for organ‐, ENaC subunit‐, and protein specificity and found no changes in kidney αENaC and βENaC mRNA and protein, lung βENaC, or lung α 1 ‐Na,K‐ATPase expression. Thus, we provide conclusive evidence that βAR stimulation of AFC depends exclusively on ENaC, while baseline AFC seems less ENaC‐dependent. (Supported by a research grant from the March of Dimes Foundation for Birth Defects (Grant No. 6‐FY03‐64)