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Published ahead of print on January 26, 2006, doi:10.1165/rcmb.2005-0418OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 34, pp. 677-687, 2006
© 2006 American Thoracic Society
DOI: 10.1165/rcmb.2005-0418OC

Effect of Cholesterol Depletion on Exocytosis of Alveolar Type II Cells

Narendranath Reddy Chintagari, Nili Jin, Pengcheng Wang, Telugu Akula Narasaraju, Jiwang Chen and Lin Liu

Department of Physiological Sciences, Oklahoma State University, Stillwater, Oklahoma

Correspondence and requests for reprints should be addressed to Lin Liu, Ph.D., Department of Physiological Sciences, Oklahoma State University, 264 McElroy Hall, Stillwater, OK 74078. E-mail: liulin{at}okstate.edu

Alveolar epithelial type II cells secrete lung surfactant via exocytosis. Soluble N-ethylmaleimide–sensitive factor attachment protein receptors (SNARE) are implicated in this process. Lipid rafts, the cholesterol- and sphingolipid-rich microdomains, may offer a platform for protein organization on the cell membrane. We tested the hypothesis that lipid rafts organize exocytotic proteins in type II cells and are essential for the fusion of lamellar bodies, the secretory granules of type II cells, with the plasma membrane. The lipid rafts, isolated from type II cells using 1% Triton X-100 and a sucrose gradient centrifugation, contained the lipid raft markers, flotillin-1 and -2, whereas they excluded the nonraft marker, Na+-K+ ATPase. SNAP-23, syntaxin 2, and VAMP-2 were enriched in lipid rafts. When type II cells were depleted of cholesterol, the association of SNAREs with the lipid rafts was disrupted and the formation of fusion pore was inhibited. Furthermore, the cholesterol-depleted plasma membrane had less ability to fuse with lamellar bodies, a process mediated by annexin A2. The secretagogue-stimulated secretion of lung surfactant from type II cells was also reduced by methyl-beta-cyclodextrin. When the raft-associated cell surface protein, CD44, was cross-linked using anti-CD44 antibodies, the CD44 clusters were observed. Syntaxin 2, SNAP-23, and annexin A2 co-localized with the CD44 clusters, which were cholesterol dependent. Our results suggested that lipid rafts may form a functional platform for surfactant secretion in alveolar type II cells, and raft integrity was essential for the fusion between lamellar bodies with the plasma membrane.

Key Words: alveolar type II cells • lipid rafts • membrane fusion • SNARE proteins • surfactant secretion




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