期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 284, 期 11, 页码 7294-7306出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M806816200
关键词
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资金
- National Institutes of Health [HL-31197, HL-51173]
- NIEHS, Public Health Service [U01ES015676, 1U54NS063739]
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-kappa B p65 subunit and NF-kappa B activation were also up-regulated. iNOS up-regulation in GFP(+) cells was prevented by knocking down I kappa B kinase gamma before infection. Furthermore, pretreatment of H441 cells with the specific iNOS inhibitor 1400W (1 mu M) resulted in a doubling of the amiloride-sensitive Na+ current in GFP(+) cells. Additionally, preincubation of H441 cells with A77-1726 (20 mu M), 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.
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