DIFFERENTIATION OF TRANSPORT MECHANISMS IN LENS CELLS

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.