A thin fluid layer in alveoli is normal and results from a balance of fluid entry and fluid uptake by transepithelial salt and water reabsorption. Conventional wisdom suggests the reabsorption is via epithelial Na channels (ENaC), but if all Na reabsorption were via ENaC, then amiloride, an ENaC inhibitor, should block alveolar fluid clearance (AFC). However, amiloride blocks only half of AFC. The reason for failure to block is clear from single-channel measurements from alveolar epithelial cells: ENaC channels are observed, but another channel is present at the same frequency that is nonselective for Na over K, has a larger conductance, and has shorter open and closed times. These two channel types are known as highly selective channels (HSC) and nonselective cation channels (NSC). HSC channels are made up of three ENaC subunits since knocking down any of the subunits reduces HSC number. NSC channels contain α-ENaC since knocking down α-ENaC reduces the number of NSC (knocking down β- or γ-ENaC has no effect on NSC, but the molecular composition of NSC channels remains unclear). We show that NSC channels consist of at least one α-ENaC and one or more acid-sensing ion channel 1a (ASIC1a) proteins. Knocking down either α-ENaC or ASIC1a reduces both NSC and HSC number, and no NSC channels are observable in single-channel patches on lung slices from ASIC1a knockout mice. AFC is reduced in knockout mice, and wet wt-to-dry wt ratio is increased, but the percentage increase in wet wt-to-dry wt ratio is larger than expected based on the reduction in AFC.

Downs CA, Kreiner L, Zhao XM, Trac P, Johnson NM, Hansen JM, Brown LA, Helms MN. Oxidized glutathione (GSSG) inhibits epithelial sodium channel activity in primary alveolar epithelial cells.

MOLECULAR CHAPERONES of the endoplasmic reticulum (ER) facilitate folding, oligomerization, maturation, and posttranslational modification of nascent polypeptide chains. ER protein 29 (ERp29) is a chaperone protein with high sequence homology to the protein disulfide isomerase family of enzymes, which catalyze posttranslational modification of disulfide exchange. However, ERp29 lacks the thioredoxin motif and is, therefore, regarded as a redox-insensitive member of this family of compounds (8). Although ERp29 is ubiquitously expressed, it is abundantly enriched in the rough ER of actively secreting epithelial cells and complex neurons, where it plays a major role in protein folding (7, 9). Epithelial Na ϩ channel (ENaC) subunits (␣, ␤, and ␥) are synthesized in the ER before further folding, glycosylation, assembly, and transport to the plasma membrane as an oligomer (reviewed in Ref. 2). Regulation of synthesis and maturation of ENaC plays a critically important role in main

At birth, the lung undergoes a profound phenotypic switch from secretion to absorption, which allows for adaptation to breathing independently. Promoting and sustaining this phenotype is critically important in normal alveolar growth and gas exchange throughout life. Several in vitro studies have characterized the role of key regulatory proteins, signaling molecules, and steroid hormones that can influence the rate of lung fluid clearance. However, in vivo examinations must be performed to evaluate whether these regulatory factors play important physiological roles in regulating perinatal lung liquid absorption. As such, the utilization of real time X-ray imaging to determine perinatal lung fluid clearance, or pulmonary edema, represents a technological advancement in the field. Herein, we explain and illustrate an approach to assess the rate of alveolar lung fluid clearance and alveolar flooding in C57BL/6 mice at post natal day 10 using X-ray imaging and analysis. Successful implementation of this protocol requires prior approval from institutional animal care and use committees (IACUC), an in vivo small animal X-ray imaging system, and compatible molecular imaging software.