The intent of this proposal is to study the mechanism of action of the toxic effect of mercury on transport by chloride transporting epithelia. Mercury inhibits chloride transport in both the shark rectal gland and the mammalian thick ascending limb but its mechanism of action mechanism of action is unknown. In the proposed set of experiments we will study the effect of organic and inorganic mercury on the transport of chloride by the shark rectal gland and compare it with that in the thick ascending limb of the loop of Henle. The mechanism of the inhibition of mercury, mercurial (both inorganic and organic) or of any compound on chloride transport by the rectal gland or the thick ascending limb can be evaluated with reference to our present understanding of transport by these cells. Inhibitory effects are possible at five different sites: 1) The site of entry of chloride into the cell, the Na-K-2CI carrier; 2) Inhibition of Na-K-ATPase with the resulting reduction in the driving force for entry of sodium, chloride and potassium via the cotransporter; 3) Inhibition of cellular metabolism resulting in insufficient ATP to power the pump; 4) Inhibition of the exit of chloride at the apical membrane by a reduction in the apical membrane conductance for chloride; 5) inhibition of potassium recycling through the potassium conductive pathway. These possibilities can be distinguished using specific transport inhibitors in combination with mercury. An additive effect of mercurial to inhibit chloride transport in the presence of specific inhibitors of these transport steps will suggest a site of action of distinct from that of the specific inhibitor. Because we have found unexpected and striking differences in the response of these two epithelia to inorganic and organic mercurial, we believe that the comparative approach will be particularly fruitful. We will examine the mechanism and the cellular site of action of mercury by determining its effect in the isolated perfused rectal gland, isolated rectal gland tubules, rectal gland membrane vesicles, rectal gland plasma membranes, thick ascending limb cells, thick ascending limb vesicles, thick ascending limb plasma membranes. Specifically we will examine the binding of mercury by plasma membranes and cytosolic proteins; the effect of mercury on the chloride cotransporter, the efflux of potassium, the chloride conductance, and its effect on transport related enzymes.
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