Our 5-year plan continues to focus on, but is not limited to, the causes and consequences of disturbed ion homeostasis, with emphasis on calcium. Lens electrolytes, especially calcium are essential to normal lens function and have already been implicated in numerous pathological pathways. Our approach is to study in detail potentially important physiological processes which may depend upon calcium. Our investigation will concentrate on several established models of cataract,, including diabetic cataract as produced in alloxan treated rabbit or galactosemic rats, ultraviolet irradiation and x-irradiation cataracts in rabbits and several lipid-related cataracts: U18666A and lysophosphatidylcholine. To help ascertain whether intracellular calcium is always involved in the opacification proces, intracellular calcium measurements, both in clear and opaque regions of a given lens will be obtained. Measurements will also include free and bound levels of total calcium and intracellular pH, recently found to be associated with calcium-related opacification and aggregation of crystallins. In addition, the possibility of therapeutic intervention will be explored by studying a number of putative calcium- entry blockers, concentrating first on cultured lenses and epithelial cells exposed to calcium-enriched media and then various experimental cataracts. The other major area of study will focus on how ion homeostasis is disrupted and how the lens either adapts or attempts to restore equilibrium or achieve a new steady state. Thus, in cultured rabbit, rat and human lenses (postmortem), as well as in epithelial cells in tissue culture,k cell membranes will be investigated for normal physiologic function. Membrane integrity will be assessed by voltage and conductance measurements as well as flux analysis of the major ions and metabolites such as glucose, all of which may be sensitive to excess calcium. Three primary causes of membrane deterioration will be explored, viz. modification of membrane sulfhydryl groups and endogenous oxidants such as selenium or hydrogen peroxide;l alteration of membranes due to phospholipid degradation induced by calcium-activated phospholipase A2; and damage to membranes by osmotic stress already known to be a factor in diabetic models of cataract. Experiments are planned to investigate whether these types of insults might explain calcium accumulation characteristic of human cataracts.
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