The goal of this study is to define how endogenous or pharmacological signals from outside the ciliary epithelium alter the actual machinery of sodium transport, influencing the rate of this key solute transport mechanism involved in aqueous humor formation. We plan to examine sodium transport by three experimental approaches; intracellular microelectrode studies with intact ciliary processes, Na, K-ATPase experiments with plasma membranes prepared from ciliary processes, and tracer flux studies with segments of iris-ciliary body. While most of the experiments will use rabbit tissue, a limited number of comparative studies will be performed with human tissue. Running through the proposal is the theme that a signal which tells the epithelium to modulate aqueous production is relayed inside the cell by a second messenger such as cAMP or calcium. We plan Na,K-ATPase studies to examine whether cAMP, calcium, protein kinases, calmodulin and adrenergic agents are capable of directly altering the activity of the sodium pump enzyme. Some Ca-ATPase studies will be performed because active calcium extrusion could modulate calcium-mediated effects. Changes in sodium transport can also result when membrane passive permeability is altered. We plan microelectrode measurements of cell potential in the intact ciliary epithelium and propose ion-substitution experiments to explore membrane permeability characteristics. Membrane permeability will be assessed in the epithelium following manipulation of cAMP and calcium, challenge with phorbol esters, calmodulin antagonists and adrenergic agents. Studies are also planned to test whether epithelial responses are modified by calcium channel antagonists and conditions where HCO3 and intracellular pH are altered. The electrophysiological and Na,K-ATPase studies are designed to examine the changes that take place in the cell membrane machinery used for sodium transport. As an index of the actual rate of sodium transport, 86 Rb will be used as a tracer to measure the rate of ouabain-inhibitable potassium uptake by iris-ciliary body segments. 86 Rb efflux studies will be performed to assess passive potassium permeability.
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