Abstract
Pulmonary surfactant, secreted via exocytosis of lamellar bodies (LB) by alveolar type II (AT II) cells, maintains low alveolar surface tension and is therefore essential for normal lung function. Here we describe real-time monitoring of exocytotic activity in these cells by visualizing and quantifying LB fusion with the plasma membrane (PM). Two approaches were used. First, fluorescence of LysoTracker Green DND-26 (LTG) in LB disappeared when the dye was released after exocytosis. Second, phospholipid staining by FM 1-43 resulted in bright fluorescence when this dye entered the LB through the fusion pore. Both processes were restricted to and colocalized with LB and occurred simultaneously. In AT II cells, FM 1-43 offered the unique advantage to independently define the moment and cellular location of single exocytotic events as well as the amount of material released, and to monitor its extracellular fate. Furthermore, both dyes could be used in combination with fura-2. The results indicate considerable diversity in the dynamics of LB exocytosis. In the majority of cells stimulated with ATP and isoproterenol, the first fusion of LB coincided with the rise of [Ca2+]i, but subsequent response of other LB in the same cell considerably outlasted this signal. In other cells, however, the onset of exocytosis was delayed by several minutes. After LB fusion, release of surfactant from LB into an aqueous solution was slow. In summary, stimulated exocytosis in AT II cells occurs at a much slower rate than in most other secretory cells but is still a more dynamic process than predicted from conventional measurements of surfactant released into cell supernatants.