Published ahead of print on January 26, 2006, doi:10.1165/rcmb.2005-0418OC Am. J. Respir. Cell Mol. Biol., Volume 34, Number 6, June 2006, 677-687 A more recent version of this article appeared on June 1, 2006
Submitted on November 8, 2005 Effect of Cholesterol Depletion on Exocytosis of Alveolar Type II CellsNarendranath Reddy Chintagari1,1 Department of Physiological Sciences, Oklahoma State University, Stillwater, OK, USA * To whom correspondence should be addressed. 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 excluded the non-raft 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-CD 44 antibodies, the CD 44 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.
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