Acute respiratory distress syndrome is a life threatening condition triggered by a variety ofpulmonary and extrapulmonary causes, that is characterized by pulmonary edema andsubsequently impaired gas exchange. Due to lung protective ventilation strategies, itstreatment is often associated with systemic accumulation of CO2, a condition termedpermissive hypercapnia. Recent studies report a negative effect of CO2 on alveolar fluidclearance, a process mediated by its two key elements the Na+,K+-ATPase and epithelialNa+-channels (ENaCs). A reduced activity of the Na+,K+-ATPase during hypercapnia hasalready been demonstrated, but regulation of ENaC has never been directly linked to CO2.Many molecular signaling events that are activated during hypercapnia are known toregulate ENaC function, so the present study aimed to generate and subsequently applytechniques to investigate a possible contribution of ENaC to the reduction of alveolarepithelial fluid transport upon hypercapnia.ENaC function was studied in H441 cells by Ussing chamber experiments which revealedno significant regulation during short term hypercapnia, but a clear reduction of ENaCfunction during sustained hypercapnia.To identify the signaling mechanism on the molecular level, epitope-tagged human ENaCconstructs for the α-, β- and γ-subunit were cloned and initially expressed in A549 cells.Exposition to hypercapnia up to 4 hours did not significantly reduce cell surface expressionof the ENaC-subunits, but after 24 hours, a significant decrease of β-ENaC was observed.Since the molecular sizes of α- and γ-ENaC expressed in A549 cells were differing frompreviously published studies, transfection of ENaC was continued in other cells. H441 cellsare commonly used for ENaC studies, so their transfection was established, yielding anefficiency of about 60 %. The molecular sizes of transfected ENaC subunits matched thepattern that was expected, but expression levels were evanescent and too low for furtherexperiments. Since ENaC detection in these two cell lines remained problematic, a novelmethodology was applied. Since the primary site of ENaC expression in the lung areepithelial cells, rat primary alveolar epithelial cells type II were used as recipients forENaC plasmids. Non-viral transfection of ATII cells has been inefficient in the past, butduring the present study a protocol was generated to efficiently deliver nucleic acids toexactly this cell type. ENaC expression was largely increased in ATII cells, compared tothe cell lines used, indicating that established system might be extremely useful for furtherstudies involving ENaC turnover.Thus, a new and highly relevant, non-viral transfection technique for primary alveolarepithelial type II cells was established, providing ground-breaking opportunities for futurepulmonary research.
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