Ca2+ induced surfactant secretion in alveolar type II cultures isolated from the H-2Kb-tsA58 transgenic mouse

There is a need for the development of transgenic mice to elucidate molecular mechanisms in surfactant secretion. However at present very little is known about the regulation of surfactant exocytosis in murine alveolar type II (AT II) cells.

We brought AT II cells isolated from the Immorto mouse into culture at 33 degrees C, in the presence of interferon, to generate immortal mouse AT II cells (iMAT II). Surfactant secretion was measured using real-time fluorescence imaging.

iMAT II cells stained with lysotracker green (LTG), a dye specific for lamellar body related vesicles in rat AT II cells. Expression of densely layered structures, characteristic of LBs, was confirmed by transmission electron microscopy. Flash photolysis of caged Ca2+, which specifically elevates intracellular Ca2+ concentration ([Ca2+]i), resulted in LB fusion to the plasma membrane, as analysed using the lipid staining dye FM 1-43. Purinergic stimulation with ATP (10 microM), also resulted in a rise in [Ca2+]i (measured by fura-2), which was followed by LB fusion.

iMATII cells maintain the expression of LBs over several passages. Surfactant secretion in these cells is regulated by [Ca2+]i, and exhibits similar characteristics to that of rat AT II cells. These cells will be beneficial in studying the impact of genetic modifications on regulated surfactant secretion.