IntroductionIn order for the mechanisms by which the airways stay clear and free from infection to function normally, there must be strict control of the pH of the liquid bathing the luminal surface of the airway (pH_ASL). The mechanisms that regulate pH_ASL are not well understood, but our recent studies suggest that both glandular and surface epithelial regions are involved, and specifically that glandular secretions are relatively alkaline, whilst the surface epithelium secretes acid equivalents through the activity of bafilomycin A1-sensitive vacuolar type H⁺-ATPase. (Inglis et al 2003). The aim of this study was to test the hypothesis that H⁺-ATPase transporters are preferentially located in the plasma membrane of surface epithelial cells.Immunohistochemistry was carried out on sections of bronchus, which possess both glandular and surface epithelia, and these were fixed/embedded to see the presence and cellular distribution of subunits of the V1 sector of the vacuolar-type H⁺-ATPase in the glands and epithelium. MethodCotswold pigs (15kg) were obtained from a local supplier, sedated with inhaled halothane, and killed with an intravenous overdose of pentobarbital sodium in accordance with UK and institutional regulations. Apical and middle lobes of the right and left lungs were excised and placed in KRB solution. Distal bronchi were carefully dissected free and fixed in a formalin solution. Porcine kidney was also dissected and placed in the fixative. As a secondary control rat kidneys were removed from animals that had been sacrificed for work in other areas of research following the recommended guidelines. The bronchi and kidney were embedded in paraffin wax prior to sectioning and immunohistochemistry using microwave antibody retrieval. Primary antibody was applied at a concentration of 1:1400.Positive and negative controls were carried out on rat kidney and porcine kidney, and also negative controls on porcine bronchi.ResultsResults in these tissues demonstrate the presence of a vacuolar-type H⁺-ATPase in the apical surface of the epithelial cells in porcine bronchi. There also seems to be a presence in the submucosal glands, although this does appear to be much more diffuse throughout the cell. Positive and negative controls confirmed the efficacy of this technique.