TY  - THES
T1  - Impact of hypercapnia on alveolar Na+-transport : Establishing a system for ENaC-protein detection
A1  - Buchbinder,Benno Andreas
Y1  - 2013/12/06
N2  - Acute respiratory distress syndrome is a life threatening condition triggered by a variety of
pulmonary and extrapulmonary causes, that is characterized by pulmonary edema and
subsequently impaired gas exchange. Due to lung protective ventilation strategies, its
treatment is often associated with systemic accumulation of CO2, a condition termed
permissive hypercapnia. Recent studies report a negative effect of CO2 on alveolar fluid
clearance, a process mediated by its two key elements the Na+,K+-ATPase and epithelial
Na+-channels (ENaCs). A reduced activity of the Na+,K+-ATPase during hypercapnia has
already been demonstrated, but regulation of ENaC has never been directly linked to CO2.
Many molecular signaling events that are activated during hypercapnia are known to
regulate ENaC function, so the present study aimed to generate and subsequently apply
techniques to investigate a possible contribution of ENaC to the reduction of alveolar
epithelial fluid transport upon hypercapnia.
ENaC function was studied in H441 cells by Ussing chamber experiments which revealed
no significant regulation during short term hypercapnia, but a clear reduction of ENaC
function during sustained hypercapnia.
To identify the signaling mechanism on the molecular level, epitope-tagged human ENaC
constructs 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 expression
of 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 from
previously published studies, transfection of ENaC was continued in other cells. H441 cells
are commonly used for ENaC studies, so their transfection was established, yielding an
efficiency of about 60 %. The molecular sizes of transfected ENaC subunits matched the
pattern that was expected, but expression levels were evanescent and too low for further
experiments. Since ENaC detection in these two cell lines remained problematic, a novel
methodology was applied. Since the primary site of ENaC expression in the lung are
epithelial cells, rat primary alveolar epithelial cells type II were used as recipients for
ENaC plasmids. Non-viral transfection of ATII cells has been inefficient in the past, but
during the present study a protocol was generated to efficiently deliver nucleic acids to
exactly this cell type. ENaC expression was largely increased in ATII cells, compared to
the cell lines used, indicating that established system might be extremely useful for further
studies involving ENaC turnover.
Thus, a new and highly relevant, non-viral transfection technique for primary alveolar
epithelial type II cells was established, providing ground-breaking opportunities for future
pulmonary research.
KW  - Hyperkapnie
KW  - ENaC
KW  - Nucleofection
KW  - Primärzellen
CY  - Gießen
PB  - Universitätsbibliothek
AD  - Otto-Behaghel-Str. 8, 35394 Gießen
UR  - http://geb.uni-giessen.de/geb/volltexte/2013/10417
ER  -