Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, PCA108

Poster Communications

Hypoxic inhibition of alveolar epithelial Na-transport is aggravated by hypocapnia

F. Huber1, M. Sahinoz1, A. Luetge1, E. Ermis-Kaya1, E. Swenson2, H. Mairbäurl1

1. Medical Clinic VII, Sports Medicine, University Hospital Heidelberg, Heidelberg, Germany. 2. Pulm.Sec., VA Med.Center, Univ.Washington, Seattle, Washington, United States.


Hypoxia stimulates ventilation resulting in exposure of alveolar epithelium to both hypoxia and hypocapnia. Hypoxia inhibits Na-reabsorption across the alveolar epithelium by decreasing the activity of epithelial Na channels (ENaC) and Na/K-ATPase, due to decreased mRNA and protein expression as well as their abundance in the plasma membrane. Effects of hypocapnia in combination with hypoxia on alveolar ion transport are not well understood. To test this, primary alveolar epithelial cells were isolated from male Sprague Dawley rats (~150g), were cultured on transwell filters, and kept in normoxia and hypoxia (1.5% O2) in combination with normocapnia (5% CO2) and hypocapnia (3% CO2) for 24 hours in presence of various modulators (see below). Acute exposures were done by equilibrating cells cultured in normoxia with the respective gas mixture for 30 min in Ussing chambers prior to measuring transepithelial electrical currents (Ieq). Normocapnic hypoxia for 24h decreased ENaC (-80%) and Na/K-ATPase (-42%) activity measured as amiloride- and amphotericin B sensitive electrical currents, respectively. ENaC activity was even further decreased by hypocapnic hypoxia (-60%), whereas hypocapnia did not affect Na/K-ATPase. Effects were small after acute exposure but inhibition by hypocapnic hypoxia was already detectable. The decreased ENaC activity was not prevented by inhibition of carbonic anhydrase. In 24h experiments, addition of HEPES to bicarbonate-containing medium to keep extracellular pH independent of hypocapnia-induced changes in pH did not prevent aggravated inhibition of ENaC. However, culturing cells in bicarbonate-free, HEPES-buffered media for 24h decreased ENaC activity in normocapnic hypoxia but prevented the additional decrease in hypocapnic hypoxia. Culturing cells in bicarbonate-buffered media together with inhibitors of protein kinase C (chelerythrine and GO6976), which is involved in inhibition of alveolar epithelial Na-transport in hypercapnia (Vadasz & Snajder, Front Immunol; doi: 10.3389/fimmu.2017.00757), and of ERK-1/2 (U0126), did not prevent the inhibition of ENaC by normocapnic hypoxia nor its aggravation by hypocapnia. Hypoxia (24h) decreased α-, β-, and γ- ENaC mRNA expression by ~70%, and decreased αENaC protein abundance in the apical membrane and in the intracellular compartment (measured after membrane biotinylation) by approximately 50%. However, hypocapnia did not affect mRNA and protein expression. These results indicate that hypocapnia aggravates hypoxic inhibition of alveolar epithelial ENaC-mediated Na-reabsorption. Aggravated inhibition was independent of alkalosis, PKC, ERK, and of mRNA and protein expression of ENaC, but seems to be caused by increased intracellular chloride concentrations upon decreasing cellular bicarbonate in hypocapnia.

Where applicable, experiments conform with Society ethical requirements