LOW TEMPERATURE SEM AND X RAY MICROANALYSIS OF ASL

这个项目有两个目标。首先，在正常组织中

This project has two objectives. First, in normal tissues we will characterize how epithelial processes (gland secretion, surface epithelial ion transport, goblet cell discharge) affect the depth of airway surface liquid. We will then determine how the regulation of ASL depth is altered in cystic fibrosis (CF). Second, we will determine how the ion and mucin concentrations of ASL and gland secretions are altered in CF. The depth of ASL will be measured by low temperature scanning electron microscopy (LTSEM) of frozen hydrated tissues fractured perpendicular to the plane of the surface epithelium. Ion contents will be measured by X-ray microanalysis of specimens in the LTSEM. Mucin concentrations will be estimated from the sulfur peak in the X-ray microanalysis spectrum. As an alternative to X-ray microanalysis we will estimate Na and Cl levels in ASL of surface epithelial cultures by adding tritiated water (or mannitol), /36 Cl and /22 Na to the basolateral side of cell cultures. Na and Cl concentrations are then estimated from the ration of equilibrium uptake for 22/Na or 36/C1 to the equilibrium uptakes of 3/H/2/0 (or /3 H- mannitol). To determine concentrations of Na, Cl and mucin in gland secretions, frozen hydrated sections will be fractured in a plane parallel to, and just below the surface epithelium. This will result in profiles of gland ducts of approximately 50 mum diameter and 1 per mm/2, over which the electron beam will be centered. Finally, we will estimate the forces of surface tension holding the periciliary sol in place. When solute is added to the basolateral medium, there should come a point at which the osmotic grandient exceeds the forces of surface tension. At this point, the sol should disappear and the cilia collapse onto the epithelium. The information on depth and composition of ASL obtained in this project will serve as guidelines for the other projects.