REGULATION OF RESPIRATORY TRACT FLUID LAYERS

显微镜检查显示，气道内的液体由两部分组成

Microscopy has shown the fluid lining the airways to consist of two layers. A mucous blanket of variable depth lies on top of the periciliary sol, a clear layer which bathes the cilia. The sources and regulation of these layers are incompletely understood. However, in cystic fibrosis, it is believed that abnormal ion transport by the surface epithelium alters the relationship or hydration of these layers in such a way as to impair mucociliary clearance. In this proposal we will test two hypotheses. Firstly, we suggest that ion transport by the surface epithelium is of little importance in regulating the depth of the periciliary sol, which is set precisely at the length of the cilia by forces of capillarity generated by the large combined circumferential length (600 m per cm2 of epithelial surface) of the cilia. Secondly, we propose that defective Cl and fluid secretion by the glands is mainly responsible for the initial accumulation of mucus in cystic fibrosis. These defects result in dehydrated mucus which will not detach fully from the gland openings and/or will not be effectively transported by the cilia. In addition malfunction of CFTR in glands may alter the sulfation or other properties of gland mucus, again impairing mucociliary clearance. To test these hypotheses, we will investigate how osmotic and active fluid flows across the surface epithelium alter the depth of the mucus layers, and how these changes affect mucus transport. The fluid layers will be visualized in transverse sections of rapidly frozen tissues in the Scanning electron microscope. We will measure mediator-induced fluid secretion across CF and non-CF cell cultures using a capacitance probe technique, and from intact glands by micropipette sampling from duct openings. We will determine if the sulfation and rheology of mucins from CF gland and surface cultures is abnormal. Finally, we will determine the localization and total levels of CFTR in our cultures, and correlate hormonally-induced alterations in these parameters with changes in function. Most of these proposed experiments will be performed on our recently developed primary cultures of surface and gland epithelia, which show high levels of ultrastructural and functional differentiation.