VOLUME REGULATION IN NORMAL AND CATARACTOUS LENSES

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DESCRIPTION: (Investigator's Abstract) The lens exists in a physiologically challenging state with no blood supply, little circulation, limited oxidative metabolism and almost no protein turn-over. Thus to homoeostatically maintain its cells in a transparent state is indeed a remarkable feat. The long term goal is to contribute to the prevention of cataracts, but we must first understand how a normal lens remains transparent (i.e., avoids cataracts). One dramatic factor is a standing internal circulation of ions and fluid that enters the normal lens at both anterior and posterior surfaces, then exists at the equator. The PI hypothesizes these internal circulatory fluxes are essential to homeostasis and, furthermore, their diminution with age may lead to the senile cataract. The goal of this proposal is to understand the structural and molecular basis of these fluxes and how they are regulated. Background data suggest: The circulating current is generated by a difference in electromotive potential of surface and fiber cells; it is directed in the equator to poles pattern by gap junctions in a peripheral shell of fiber cells; and it is followed by a circulation of fluid. Other preliminary data suggest neurotransmitters can alter the circulation, so one of the major aims of this proposal is to determine which transmitters, acting through which pathways, affect which target proteins and what is the end effect on the fluxes. To achieve this goal, a chamber was designed and built to allow separate monitoring of anterior, posterior and equatorial currents in intact lenses, while superfusing agents of interest. Whole lens impedance techniques will determine the location of regulatory changes in membrane conductance and gap junctional coupling. Whole cell patch clamp of acutely isolated lens epithelial cells will determine effects of agents on Na/K pumps and ion channels. Moreover, this technique will be used to determine the Na/K pump's dependence on the ionic environment and voltage. Since recent studies suggest polar and equatorial epithelial cells express different -isoforms of the Na/K pump, these separate populations of isolated cells will be studied. The role of fiber cells will be evaluated by expressing clones of membrane transport and gap junction proteins, then measuring their functional properties in occytes and/or a cell line.