Confocal imaging of time-dependent internalization and localization of NBD-PC in intact rat lungs

Morphological localization of a fluorescent analogue of phosphatidylcholine (PC), C12-NBD-PC ([1-palmitoyl-2-[12-(7-nitro-2-1,3-benzoxadiazol-4-yl) amino] dodecanoyl]PC) was evaluated in adult rat lung. Fluorescence was observed at various times after intratracheal liposome instillation. Fluorometric analysis of C12-NBD-PC in lung homogenate showed an initial rapid uptake followed by a more gradual uptake phase. Microscopic observation of fixed lung slices showed C12-NBD-PC uptake in alveolar type II cells (identified by surfactant protein A and Maclura pomifera agglutinin colocalization), nonciliated distal airway epithelial cells, but not in subepithelial tissue. Macrophages showed an initial uptake that did not increase with time. Fluorescence measured in type II cells was diffuse at 5 min, became punctate, and intensified gradually by 2 h. Isolation of type II cells and lamellar bodies from instilled lung confirmed localization of fluorophore in them. This study shows time-dependent uptake of C12-NBD-PC by type II and bronchiolar cells and preferential internalization to lung lamellar bodies as previously described for surfactant PC.