Previously, our laboratory has identified a human colonic epithelial cell line, T84, as a suitable model for investigation of Cl- secretory mechanisms. The cell line, composed of only secretory cells and free of influence from paracrine, neurocrine or endocrine controls, is ideal for studies of regulatory controls of Cl- secretion at the cellular level. Our studies in the past four years identified transport pathways involved in various Cl- secretory processes and unmasked a synergistic phenomenon between cyclic nucleotide and Ca++ -related secretions. Recent studies suggest a more complicated interaction of secondary messengers in Ca++ - related secretory mechanisms and implicate involvement of phospholipid turnovers. A candidate messenger (phospholipid metabolite) which co- activates Cl- secretion with free cytosolic Ca++ is arachidonic acid. A candidate messenger which inhibits Cl- secretion is diacylglycerol. This renewal application proposes to sort out the contribution of secondary messengers, particularly phospholipid metabolites using cell monolayers and plasma membrane preparations. Three approaches will be utilized: 1) Correlative studies of the time courses and dose-responses on each secondary messenger and a biological response to identify potential candidates. Activators or inhibitors of phospholipases, diglyceride lipase, lipoxygenase, cyclooxygenase or G-proteins will also be used in the studies under varying [Ca++]i or [Ca++] (for plasma membrane). Initial studies will focus on the mechanism of action of carbachol, histamine, 4- Br-A23187, ionomycin, taurodeoxy-cholate and adenosine. 2) Reconstitution studies will be carried out after a better method to deliver fat-soluble substances to the cell or plasma membrane is developed. The biological actions of each candidate messenger will be directly tested both alone and in combination with others to mimic the responses of each secretagogue listed in correlative studies. Again, the use of specific enzymes and their inhibitors will complement the studies proposed under varying [Ca++]i or [Ca++]. 3) Comparative studies will be carried out in other epithelial cell lines that secrete Cl-, e.g., CaCO and HT-29 clones to test whether the same mechanism also applies to them. It is expected that some cell lines with a defective secondary messenger or a defective transport pathway will provide useful complementary insights into the role of that messenger on the transport pathway.
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