FLUOROPHOTOMETRY OF ION TRANSPORT IN CILIARY BODY

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The steady state intraocular pressure is the result of a balance between fluid production and outflow. Elevated intraocular pressure (IOP), a condition which could have devastating effects for vision due to tissue degeneration, occurs when the outflow facility is reduced. However, clinical intervention to relieve elevated IOP relies mostly in pharmacological manipulation of aqueous humor production. This humor is generated by the dual layered epithelium that covers the ciliary body. Both, paracellular filtration between the cells (driven by the blood to aqueous hydrostatic pressure difference) and active movement of fluid by the cells could contribute to the flow. The cellular active transport, which has been shown to be the primary process, is poorly understood. In great part, this limited knowledge is due to the difficulties encountered in the application of classical electrical and physiological approaches to the study of ion and fluid transport in a tissue endowed with an intricate anatomy and a complex cell arrangement. This proposal aims to apply novel techniques for intracellular ion tracking (based on cell entrapable, fluorescent dyes exhibiting ion sensitive spectra) to generate a thorough characterization of 1) the individual translocation systems present in both epithelial cell layers; 2) the nature of the communication between the layers; and 3) the pharmacological regulation of these activities. Specialized equipment and dissection techniques allowing intracellular measurements of H+, Na+, K+, Cl- and Ca2+ in single (or small groups of) cells of each type in intact ciliary body strips will be used. The strips will be mounted in perfusion chambers allowing control of the extracellular ionic environment in each side of the tissue and the changes in intracellular concentrations induced by extracellular changes or by pharmacological effectors will be used to identify, and characterize each individual transporter. The emerging knowledge will be applied to generate plausible models for the aqueous humor secretory process. These models and the information gathered on the pharmacological modulation of individual transport functions by hypotensive agents, will improve our capacity to manipulate aqueous humor secretion in glaucoma patients or individuals exhibiting an elevated IOP.