The lens is a functional syncytium in which, for transparency, the vast majority of the cells are differentiated into fibers. The differentiation process emphasizes tissue transparency but deemphasizes the cell's ability for ion transport. Electrolyte balance for the entire lens is accomplished primarily by Na,K-ATPase which is present in varying amounts throughout the different cells. The proposed experiments examine how sodium transport capability and membrane permeability mechanisms change as the lens epithelium differentiates to become fibers. They will pinpoint how cells in some lens epithelium differentiates to become fibers. They will pinpoint how cells in some lens regions retain sufficient transport ability to perform electrolyte transport duties for other regions. Three hypotheses will be evaluated: Hypothesis 1. For sodium-potassium balance, the lens needs to have functional pump sites (Na-K-ATPase molecules) in some fiber cells as well as pump sites distributed throughout the epithelial monolayer. To test this hypothesis, 3H-ouabain binding and immunoblotting will be used to measure the number and determine the types of pump sites in different lens cells. Functional transport of ions by fiber cell Na,K-ATPase will be determined by tracer flux studies. Hypothesis 2. Lens electrolyte balance is very dependent upon transport mechanisms at the lens equator. Furthermore, transport mechanisms in different regions of the lens may be more or less susceptible to perturbations such as oxidation. To test this hypothesis, regional ion changes will be quantified following immersion of the lens in solutions containing ouabain, a Na,K-ATPase inhibitor. A divided chamber will be used to separately expose the anterior, posterior and equatorial surfaces of the lens to ouabain, hydrogen peroxide and calcium. Hypothesis 3. Membrane transport properties change as lens epithelial cells differentiate to become fibers. To test this hypothesis, Na,K- ATPase activity and isoform expression will be measured in differentiating cells in an FGF-treated rat lens epithelial explant model. Using tracer fluxes and microelectrode techniques, sodium- potassium pump activity and membrane permeability parameters will be quantified at different states of differentiation. These studies may help define the specific events leading to failure of lens electrolyte balance during cataract formation so that medical therapies can be developed to prevent or retard the opacification process.
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