The aims of the project are to understand the mechanisms, cellular and molecular by means of which the corneal epithelium, ciliary epithelium and retina pigment epithelium perform their functions of ion and non-electrolyte transport and water in normal and arranged states of ocular function. The consequences of the study will lead to further comprehension of basic mechanisms and the effect of specific drugs and chemicals on these functions that can lead to production of eventual ocular therapeutic agents. The regulatory process controlled by specific receptor proteins in the cells membranes and the channels affecting traffic across these membranes will provide a detailed account that will permit the formulation of eventual modes of control in the disease states affecting the corneal epithelium the production of aqueous humor and the control of the microenvironment of the retina. The methods to be used are of two natures: (1) electrophysiological and water flow methodology consisting in the use of transepithelial current and flux measurements, use of specific intercellular electrodes for C1, Na and Ca, together with the use of the new method of voltage patching. These measurements in different experimental conditions will provide the function of the apical and basolateral membrane of the epithelial cells with respect to ion movements and the effects of drugs and specific agents controlling channels and pumps. The water movements of isolated epithelia will be determined with a nanoliter injector method permitting electrical measurements simultaneously. The combination of these methods will provide a cellular picture for the epithelial functions and their modifications in altered states. (2) The molecular approach will consist of the use mainly of fluorescently labelled probes of receptors and channels. These biochemical or physicochemical approaches will use whole cells obtained from the epithelia or cell membrane fragments. Proteins will be purified, such as specific receptors and their properties and incorporation into cell membranes followed by fluorescence spectroscopy or microscopy. The characterization of these membrane proteins will require the preparation of specific antibodies, use of cultures and the utilization of monoclonally produced antibodies.
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