This application seeks support through the Center for Membrane Toxicology Studies (CMTS) at the Mount Desert Island Biological Laboratory for studies on the effects of three heavy metals (cadmium, cobalt and nickel) on signal transduction pathways of hormones regulating chloride secretion in the shark rectal gland. The shark rectal gland is a homogenous, single cell type, highly specialized epithelium that is a model system for hormone regulated chloride secretion. Our hypothesis is that the toxic effects of heavy metals results from interactions at different specific sites of stimulatory as opposed to inhibitory hormonal signal transduction pathways. We have determined that cadmium reversibly blocks receptor-mediated inhibition of chloride secretion and that a major component of this effect occurs by a novel and unexpected mechanism- and augmentation of the response to stimulatory hormones. We will determine the specific site(s) of this metal-protein interaction by distinguishing between effects of cadmium on (a) an extracellular receptor for Cd mediating activation of inositol triphosphates and release of intracellular calcium; (b) direct effects on specific isozyme of cyclic nucleotide phosphodiesterases; and (c) a post-receptor/kinase mechanism- translocation of DFTR-chloride channels from an intracellular site to the apical plasma membrane. In contrast to Cd, cobalt and nickel inhibit VIP and forskolin stimulated chloride secretion in the perfused rectal gland. In collaboration with others in the center we will determine the protein interactive site(s) of these metals and distinguish between toxic effects on the calcium messenger system and direct actions on apical DFTR channels. Studies will be carried out in the in vitro perfused rectal gland, in primary culture monolayers of rectal gland cells, and in Xenopus oocytes expressing the DFTR chloride channel.
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