Abstract
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.
Highlights
7294 JOURNAL OF BIOLOGICAL CHEMISTRY characterized by a linear, negative-sense, single-stranded RNA genome [1]
Our results indicate that nitric oxide (NO) or reactive oxygen nitrogen species formed by the interaction of NO with reactive oxygen species are partially responsible for the down-regulation of epithelial Na؉ channels (ENaC) activity in Respiratory syncytial virus (RSV)-infected cells
H441 Cells Are Infected with RSV rRA2—Significant numbers of green fluorescent protein (GFP) positive (i.e. H441 cells incubated with rRA2-GFP and expressing green fluorescence; GFP(ϩ)) cells were seen after infection of H441 cells with normal but not UV-inactivated rRA2 (Fig. 1A)
Summary
7294 JOURNAL OF BIOLOGICAL CHEMISTRY characterized by a linear, negative-sense, single-stranded RNA genome [1]. Because of previous reports showing that RSV infection of epithelial cells up-regulates iNOS [10] and the well demonstrated inhibition of ENaC by nitric oxide (NO) and reactive oxygen nitrogen intermediates via cGMP-dependent and independent mechanisms [11, 12], we tested the hypothesis that increased levels of NO from iNOS were at least partially responsible for the observed decrease of ENaC function To accomplish this goal we measured ENaC activity, NF-B activity, iNOS expression, and levels of nitrite in the medium in RSV-infected cells pretreated with either 1400W (an iNOS inhibitor) or vehicle.
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
