The applicant's overall research objective is to define essential components in the mechanism of action of cholera toxin (CT) on water and electrolyte secretion in the small intestine. Various mediator responses to CT in the rabbit and porcine models were characterized in the last project period, which enhanced our understanding of the mechanism of cholera. The proposed research is targeted toward CT-induced effects on cell proteins, as they impact intestinal physiology. Antimetabolites (e.g., actinomycin D and cycloheximide) and nonsteroidal anti-inflammatory drugs (e.g., indomethacin) block both CT- induced secretion and eicosanoid synthesis. One CT-induced acute-phase reactant, known as phospholipase A2- activating protein (PLAP), has been identified by the investigator in cultured cells and mouse intestinal tissue. His hypothesis is that cell proteins, such as PLAP, participate in the pathogenesis of cholera. The pathognomonic role of PLAP in stimulating eicosanoid synthesis by activating PLA2 will be assessed with PLAP antisense DNA and PLAP antibodies. In addition, other cell proteins affected by CT, such as those phosphorylated by CT-induced protein kinase activity, will be characterized. Using immunocytochemistry, the investigator will determine the intestinal cell types that synthesize these proteins and other mediators following luminal challenge with CT, Vibrio cholerae, and Salmonella typhimurium. Mutant CT proteins will be used to determine whether the active site of the CT-A subunit that catalyzes the ADP- ribosylation of GS alpha is involved in the induction of PLAP synthesis. The effects of CT on eicosanoid synthesis will be evaluated in a cyc- (GS alpha-deficient) mutant S49 cell line (compared to wild-type control cells), to separate CT effects on the cAMP system from those on arachidonic acid (AA) metabolism. Rapid induction of PLAP synthesis suggests the involvement of transcriptional activators that could enable CT to signal the rapid de novo synthesis of PLAP. Consequently, the mechanism by which CT rapidly signals the synthesis of PLAP mRNA, which, in turn, stimulates AA release from membrane phospholipids and leads to increased eicosanoid synthesis, will be defined. The DNA sequence 5' to the plap gene will be examined for conserved transcription factor- binding motifs, and a DNA mobility shift assay with transcription factor-binding [32P]- deoxyoligonucleotides will be used to test nuclear extracts from CT-treated cells for transcription factor activity. Studies on the role of Ca++ in the mechanism of action of CT have also been proposed, because CT-induced secretion is diminished by calcium channel blockers and CT elicits the synthesis of LTC4, a Ca++ channel activator. Knowledge about the molecular events in cholera should improve future strategies to control the hypersecretion of water and electrolytes in cholera and other diarrheal diseases.
