DYNAMICS OF SURFACTANT RELEASE IN ALVEOLAR TYPE-II CELLS

Pulmonary surfactant, secreted via exocytosis of lamellar bodies (LB) by alveolar type II (AT II) cells, maintains low alveolar surface tens ion and is therefore essential for normal lung function, Here we descr ibe real-time monitoring of exocytotic activity in these cells by visu alizing and quantifying LB fusion with the plasma membrane (PPI I). Tw o approaches were used, First, fluorescence of LysoTracker Green DND-2 6 (LTG) in LB disappeared when the dye was released after exocytosis, Second, phospholipid staining by FM 1-43 resulted in bright fluorescen ce when this dye entered the LB through the fusion pore, Both processe s were restricted to and colocalized with LB and occurred simultaneous ly, In AT II cells, FM 1-43 offered the unique advantage to independen tly define the moment and cellular location of single exocytotic event s as well as the amount of material released, and to monitor its extra cellular fate, Furthermore, both dyes could be used in combination wit h fura-2, The results indicate considerable diversity in the dynamics of LB exocytosis, In the majority of cells stimulated with ATP and iso proterenol, the first fusion of LB coincided with the rise of [Ca2+](i ), but subsequent response of other LB in the same cell considerably o utlasted this signal, In other cells, however, the onset of exocytosis was delayed by several minutes, After LB fusion, release of surfactan t from LB into an aqueous solution was slow, In summary, stimulated ex ocytosis in AT II cells occurs at a much slower rate than in most othe r secretory cells hut is still a more dynamic process than predicted f rom conventional measurements of surfactant released into cell superna tants.