The overall objective of the proposed research is to explain the molecular mechanism of adenylate cyclase-activating enterotoxins (e.g., cholera toxin). If time permits, additional experiments will also be performed with purified Salmonella enterotoxin, Escherichia coli LT, and Aeromonas hydrophila enterotoxin. Preliminary studies indicate that synthesis of prostaglandins and protein is markedly enhanced in enterotoxin-stimulated cells and that levels of prostaglandins and H3-leucine incorporation are closely regulated by cyclic AMP concentration. The involvement of prostaglandins in the mode of action of enterotoxins provides a mechanism whereby the effect of enterotoxins on the adenylate cyclase system is substantially amplified. Available data has led to the formulation of a working hypothesis to explain these metabolic interactions in enterotoxin-affected cells involving the adenylate cyclase system and the synthesis of proteins and prostaglandins. In order to test the hypothesis that prostaglandins and protein synthesis are important in the mode of action of adenylate cyclase-activating enterotoxins, antimetabolites will be used in conjunction with tissue culture cells exposed to cholera toxin. Specifically, enterotoxin-treated and control cells will be subjected to protein synthesis blockade using actinomycin D and cycloheximide, while levels of cyclic AMP, H3-leucine incorporation, and prostaglandins will be measured. Similarly, prostaglandin synthesis will be blocked by such drugs as aspirin and indomethacin, and levels of cyclic AMP, H3-leucine incorporation, and prostaglandins will be determined. The sequential effect on cellular levels of cyclic AMP, H3-leucine incorporation, and prostaglandins in enterotoxin-treated cells will be measured in both Chinese hamster ovary (CHO) cells and epithelial cells from rabbit intestinal loops challenged with enterotoxin-producing bacteria. Cholera toxin B subunit will be used to block the binding of cholera toxin to control against membrane perturbation as a cause of prostaglandin synthesis. Finally, selected combinations of drugs, exhibiting antagonistic effects in enterotoxin-treated cells, will be tested for their capacity to protect rabbits against challenge with cholera toxin and enterotoxin-producing bacteria. In summary, this proposal strives to clarify and extend information relating to adenylate cyclase-activating enterotoxins as well as search for possible points for metabolic intervention.
