In fetal sheep, the liquid that fills the lung lumen has a pH of ≈6.3. Recent studies in our laboratory showed that inhibition of H+-ATPase slowed the rate of acidification of lung liquid. We therefore hypothesized that the fetal lung epithelium contains H+- ATPases that help to regulate acidification of liquid within the lung lumen. To test this hypothesis, we used lung tissue from 5 fetal lambs (126 ± 6 d gestation; 2.63 ± 0.68kg) to identify the cellular distribution of H+-ATPase. Paraffin-embedded tissue sections were incubated with a rabbit anti-bovine H+-ATPase polyclonal antibody to the B1 subunit isoform (1:1000 and 1:2000 in phosphate buffered saline). Antigen detection was done by the avidinbiotin-horseradish peroxidase method. Immunostaining controls included omission of the primary antibody, omission of the secondary antibody (biotinylated IgG), and immunostaining of kidney sections obtained from the same fetal lambs. We used the fetal kidney tissue as a positive immunostaining control because the H+-ATPase polyclonal antibody specifically stains intercalated cells of renal collecting tubules. The immunostaining pattern of the H+-ATPase antibody in fetal lamb lung was limited to epithelial cells of the small airways and distal airspaces. Many of the airway epithelial cells had immunoperoxidase staining that was localized to the luminal membrane. Similar polarization of immunoperoxidase staining was seen among the cuboidal epithelial cells in the corners of the developing distal airspaces. There also was some evidence of patchy immunostaining of the luminal surface of the squamous epithelial cells that lined the developing distal airspaces. These results indicate that H+-ATPases are present on the respiratory epithelium of fetal sheep, where they provide an important mechanism for acidification of liquid within the lung lumen during development.

[Supported by NHLBI Grants 28165, 40802, 02706].