ION FLUOROPHOTOMETRY OF CILIARY BODY EPITHELIA

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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 human production. This humor is generated by the syncytially arranged dual layered epithelium that covers the ciliary body. 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 noninvasive optical techniques for intracellular ion tracking (based on cell entrappable, fluorescent dyes exhibiting ion sensitive spectra) to generate a thorough characterization of 1) calcium mobilization phenomena and its regulation in each layer of the CBE and in the whole tissue; 2) the ionic phenomena controlling intracellular pH and bicarbonate flux; 3) the functional properties and biochemical basis of the communication between the two CBE layers; and the pharmacological regulation of these activities. Specialized equipment and dissection techniques allowing intracellular measurements of H+ and Ca2+ in either single (or small groups of) cells of each type or, in both cell types simultaneously, will be used. Electrophysiological and immunohistological investigation will be used to complement the fluorescence-based methods. The emerging knowledge will be applied to generate plausible models for the aqueous human 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.