The objective of this proposal is to establish the existence of volume regulatory responses in corneal epithelium. The tissue will be subjected to conditions fostering cell volume change. Alterations in electrogenic or electroneutral transport processes induced by these conditions will be followed by the changes in transepithelial electrical events and ion fluxes in both intact epithelia and epithelia permeabilized apically to monovalent ions by Amphotericin B. Studies of plausible cytosolic mediators of the volume regulatory responses, limited at this stage to Ca2+ and pH, will be initiated using a number of intracellular effectors. Additionally, the application of spectrofluorimetric techniques using cytosol entrapped dyes for measurement of cellular pH and Ca2+ will be explored. The gross morphology and cellular ultrastructure associated with each of the cell swelling or cell shrinking conditions applied will be monitored in histological sections and by transmission electron microscopy following tissue fixation. The feasibility of quantitation of cell volume changes by morphometric measurement will then be assessed. The successful completion of this pilot project will a) yield information leading to the determination of the nature and loci of transport alterations and their significance for volume regulation; b) help develop useful methodologies for the study of the phenomenon in question; and c) provide a base for more comprehensive studies of volume regulatory responses in the epithelium, in particular, its significance in pathological states associated with cellular edema.
NIH ExPorter
NIH ExPorter