Transport mechanisms in corneal endothelium

[unreadable] DESCRIPTION (provided by applicant): Introduction. The mechanism of fluid transport across epithelia is not yet known with certainty. It is most frequently explained in terms of local osmosis. However, we have generated recent evidence suggesting that corneal endothelial fluid transport is instead due to electro-osmosis through the paracellular junctions. Hence, our specific aims are: 1) To investigate whether endothelial fluid transport behaves as a local osmotic or electro-osmotic process. Distinctive criteria for electro-osmosis include 1-1) presence of current-induced fluid movements; 1-2) much faster response of flow to imposed electrical currents, 1-3) dependence of the fluid movement/electrical current coupling ratio on junctional electrical charges, 1-4) dependence of fluid movement on ambient osmolarity, 1-5) dependence of fluid movement on intercellular junctional integrity, 1-6) presence of paracellular (rather than transcellular) fluid movements associated with fluid transport. Techniques include determinations of fluid transport by specular microscopy and volume clamp at high time resolution (~1 s), theoretical estimates of transendothelial osmotic flows driven by solute buildups, determinations of fluxes of paracellular markers, and determinations of the coupling ratio after junctional modification by agents including cadmium chloride, protamine, and polylysine. 2) To confirm and characterize novel routes for trans-endothelial membrane movements of ions which would contribute to local osmotic or electro-osmotic fluid transport. We propose to use fluorescent dyes to monitor intracellular electrolytes and intracellular potential of cultured cells, and radiolabeled Na+ to determine the sidedness of Na+ entry via phenamil-inhibitable pathways. Using immunocytochemistry and molecular biology, we will seek to pinpoint which cell membranes express electrogenic transport components consistent with electro-osmotic fluid movement. Through the use of genetically modified mice and cultured cells with transiently modified expression, we propose to determine to which extent fluid transport and osmotic permeability across cultured bovine corneal endothelial cells depend on functional expression of aquaporin 1, sodium bicarbonate cotransporter(s), and epithelial sodium channels. 3) To develop a model for fluid and electrolyte transport across the endothelial layer, and a theory for electro-osmotic coupling across the endothelial junctions. Clarification of local osmotic flows may require a time-dependent extension of the model. We propose to utilize Brinkman's ideas to describe electro-osmosis in narrow channels filled with a charged gel matrix, rather that in the conduits covered by the classical Helmholtz-Smoluchowski treatment. The understanding to be gained may provide insights on how to modulate fluid transport so as to offset endothelial dysfunction leading to the loss of normal vision.