Dynamics of surfactant release in alveolar type II cells
Dynamics of surfactant release in alveolar type II cells
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DOI：
10.1073/pnas.95.4.1579
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发表时间：
1998-02-17
影响因子：
11.1
通讯作者：
Dietl, P
中科院分区：
文献类型：
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作者：
Haller, T;Ortmayr, J;Dietl, P
肺表面活性物质由肺泡II型（AT II）细胞通过板层体（LB）的胞吐分泌，维持低肺泡表面张力，因此对正常肺功能至关重要。在这里，我们描述了通过可视化和量化LB与质膜融合（PPI I）来实时监测这些细胞的胞吐活性。采用两种方法，一种是当染料胞外释放后LB中LysoTracker Green ddn -26 （LTG）的荧光消失，另一种是当该染料通过融合孔进入LB时，fm1 -43对磷脂进行染色，产生明亮的荧光，这两种过程都局限于LB，并与LB共定位，同时发生。fm1 -43具有独特的优势，可以独立地确定单个胞外事件的时刻和细胞位置以及释放的物质数量，并监测其胞外命运。此外，这两种染料都可以与fura-2联合使用。结果表明，LB胞外分泌的动力学具有相当大的多样性，在大多数受到ATP和异丙肾上腺素刺激的细胞中，LB的第一次融合与[Ca(2+)]的上升相吻合(i)。然而，在其他细胞中，胞吐的开始延迟了几分钟。在LB融合后，表面活性剂从LB释放到水溶液中是缓慢的。总之，AT II细胞中受刺激的胞吐发生的速度比大多数其他分泌细胞慢得多，但仍然是一个比释放到细胞上清液中的表面活性剂的传统测量所预测的更动态的过程。
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 (PPI I). 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 [Ca(2+)](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 hut is still a more dynamic process than predicted from conventional measurements of surfactant released into cell supernatants.