Respiratory syncytial virus inhibits lung epithelial Na+ channels by up-regulating inducible nitric-oxide synthase.
Respiratory syncytial virus (RSV) infection has been shown to reduce Na+-driven alveolar fluid clearance in BALB/c mice in vivo. To investigate the cellular mechanisms by which RSV inhibits amiloride-sensitive epithelial Na+ channels (ENaC), the main pathways through which Na+ ions enter lung epithelial cells, we infected human Clara-like lung (H441) cells with RSV that expresses green fluorescent protein (rRA2). 3-6 days later patch clamp recordings showed that infected cells (i.e. cells expressing green fluorescence; GFP+) had significantly lower whole-cell amiloride-sensitive currents and single channel activity (NPo) as compared with non-infected (GFP-), non-inoculated, or cells infected with UV-inactivated RSV. Both alpha and beta ENaC mRNA levels were significantly reduced in GFP+ cells as measured by real-time reverse transcription-PCR. Infection with RSV increased expression of the inducible nitric-oxide synthase (iNOS) and nitrite concentration in the culture medium; nuclear translocation of NF-kappaB p65 subunit and NF-kappaB activation were also up-regulated. iNOS up-regulation in GFP+ cells was prevented by knocking down IkappaB kinase gamma before infection. Furthermore, pretreatment of H441 cells with the specific iNOS inhibitor 1400W (1 microM) resulted in a doubling of the amiloride-sensitive Na+ current in GFP+ cells. Additionally, preincubation of H441 cells with A77-1726 (20 microM), a de novo UTP synthesis inhibitor, and 1400W completely reversed the RSV inhibition of amiloride-sensitive currents in GFP+ cells. Thus, both UTP- and iNOS-generated reactive species contribute to ENaC down-regulation in RSV-infected airway epithelial cells.
Song
Weifeng
W
Department of Anesthesiology, Center for Free Radical Biology, University of Alabama at Birmingham, Birmingham, Alabama 35233, USA.
Liu
Gang
G
Bosworth
Charles A
CA
Walker
John R
JR
Megaw
George A
GA
Lazrak
Ahmed
A
Abraham
Edward
E
Sullender
Wayne M
WM
Matalon
Sadis
S
D000814:Aniline Compounds / Q000494:pharmacology; D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology; D002460:Cell Line; D003437:Crotonates; D015536:Down-Regulation / Q000187:drug effects / Q000235:genetics; D004847:Epithelial Cells / Q000378:metabolism* / Q000821:virology; D053503:Epithelial Sodium Channels / Q000096:biosynthesis* / Q000235:genetics; D055785:Gene Knockdown Techniques; D006801:Humans; D006885:Hydroxybutyrates / Q000494:pharmacology; D051550:I-kappa B Kinase / Q000235:genetics / Q000378:metabolism; D008168:Lung; D051379:Mice; D008807:Mice, Inbred BALB C; D052247:Nitric Oxide Synthase Type II / Q000235:genetics / Q000378:metabolism*; D009570:Nitriles; D009573:Nitrites / Q000378:metabolism; D020545:Respiratory Mucosa / Q000378:metabolism* / Q000821:virology; D018357:Respiratory Syncytial Virus Infections / Q000235:genetics / Q000378:metabolism*; D012136:Respiratory Syncytial Viruses / Q000235:genetics / Q000378:metabolism*; D012964:Sodium; D014052:Toluidines; D051996:Transcription Factor RelA / Q000235:genetics / Q000378:metabolism; D015854:Up-Regulation / Q000187:drug effects / Q000235:genetics; D014544:Uridine Triphosphate / Q000235:genetics / Q000378:metabolism*
D000814:Aniline Compounds; D000894:Anti-Inflammatory Agents, Non-Steroidal; D003437:Crotonates; D053503:Epithelial Sodium Channels; D006885:Hydroxybutyrates; D009570:Nitriles; D009573:Nitrites; C498143:RELA protein, human; D014052:Toluidines; D051996:Transcription Factor RelA; C527525:teriflunomide; D012964:Sodium; C496319:NOS2 protein, human; D052247:Nitric Oxide Synthase Type II; D051550:I-kappa B Kinase; D014544:Uridine Triphosphate