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The alveolar surface of the lung is made up of alveolar type1 (AT1) and type 2 (AT2) cells.
AT1 and AT2 cells both express functional ENaC.
Lung ENaC plays a crucial role in maintaining alveolar fluid balance; a-ENaC knock-out mice die due to an inability to clear lung fluid (Hummler 1996).
Primary cell culture: Lung tissue purification was based on differential adherence of cells to dishes coated with rat IgG. Non-adherent ATII cells were collected, centrifuged, and seeded in enriched medium (50:50 Dulbecco’s modified Eagle’s medium/F12 w/ 10% fetal bovine serum, 2mM l-glutamine, 1mM Dexamethasone, 84mM gentamicin, and 20units/ml penicillin-streptomycin). AT2 cells were grown on a specialized culture support and transdifferentiated in AT1-like cells.
Standard western blot techniques: Protein lysate was collected and electrophoresed on 7.5% or 15% acrylamide gels under denaturing conditions and then transferred to a nitrocellulose membrane. Blots were incubated with a 1:1000 fold dilution of anti-NOX antibodies (gp91phox, p67phox, and p47phox) obtained from Millipore, and anti-rac1 antibody from Santa Cruz followed by incubation with IgG-alkaline phosphatase-labeled secondary antibody purchased from KPL.
Transepithelial current measurements: The potential difference (PD) and transepithelial resistance (RTE) across confluent A6 cell monolayers were measured using an epithelial voltammeter equipped with stick electrodes (World Precision Instruments). The equivalent short-circuit current (Isc) was calculated according to Ohm's law where (Isc = PD/RTE).
Figure 4. Expression profiles of NOX enzymes in alveolar type 1 and type 2 cells. Expression levels of gp91phox and rac1 correspond to observed increases in superoxide production as alveolar type 2 cells transform into an alveolar type 1 phenotype. p47 Phox and p67 Phox seemingly decrease in the level of expression as cells transdifferentiate from AT2 to AT1 phenotype. Rac1 expression levels increase in AT-1 like cells compared to cultured AT2 cells.
Figure 5. Specific Rac1 inhibitor, NSC23766, significantly decreases transepithelial Na current (Isc) in a bi-phasic manner. Withing 30min-1hour 100mM Rac1 inhibition significantly decreases Isc in A6 cells. Longer term treatment with Rac1 inhibitor (10-24 hours) may be altering redox sensitive protein expression in sodium transporting epithelia to downregulate ENaC function.
Alveolar Type 2 cells transdifferentiate into Alveolar Type 1 cells. Significantly greater amounts of superoxide are produced by Type 1 cells.
ENaC function is inhibited by nitric oxide in Type 2 cells, but is not inhibited by nitric oxide in superoxide-producing Type 1 cells. Differences in the redox state could account for these differences in response to nitric oxide.
Expression levels of gp91phox and Rac1 correspond to observed increases in superoxide production as alveolar type 2 cells transform into an alveolar type 1 phenotype. p47 Phox and p67 Phox seemingly decrease in the level of expression as cells transdifferentiate from AT2 to AT1 phenotype. It is possible that p47 Phox and p67Phox are increasingly phosphorylated, and hence activated, by increased expression levels of rac1 GTPase in alveolar type 1 cells.
Seemingly, Rac1 mediated production of superoxide anions regulate ENaC in sodium transporting epithelia. Inhibition of Rac1 significantly decreases amiloride-sensitive current in A6 cells.
This research was supported by NIH grants 4R37DK-37963 awarded to D.C.E., K99/R00HL092226 to M.N.H. and Howard Hughes Medical Institute Grant No. 52005873 S.U.R.E Program.
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