Colonization of the small intestine by Vibrio cholerae causes the potentially lethal disease cholera due to massive salt and water secretion. Although the dehydrating diarrhea of cholera is attributed to secretion stimulated by cholera toxin, two other toxins of V. cholerae that alter short circuit current or resistance in Ussing chambers have been identified. V. cholerae ACE (accessory cholera enterotoxin) was initially described in 1993 as a toxin which increased short circuit current and potential difference in rabbit ileum mounted in Ussing chambers and caused fluid secretion in ligated rabbit ileal loops. We have investigated the mechanism of action of ACE utilizing monolayers of polarized intestinal epithelial cells (T84 cells) mounted in modified Ussing chambers. In these studies we identified novel physiology stimulated by ACE including that ACE is dependent on calcium as a second messenger; that although it is a calcium-dependent agonist it is unique in that it has a prolonged current response and acts synergistically with other calcium-dependent agonists; and finally, that it stimulates secretion that is equally dependent on chloride and bicarbonate ions (a newly emerging mechanism of secretion). We hypothesize that ACE interacts with the polarized intestinal epithelial cell, through a receptor present on the apical surface and that through this interaction it initiates signal transduction with calcium as a second messenger. We also hypothesize that ACE potentiates the Cl- secretory activity of carbachol by blocking the normal inhibitory pathway stimulated by carbachol.
The Specific Aims are 1) Identify the ACE receptor; 2) Examine the signal transduction pathways stimulated by ACE; and 3) Examine the interaction of ACE with calcium-mediated inhibitory pathways. We will use molecular genetic, biochemical and cell physiology methods to examine the mechanism of action of ACE. The long term objectives of this proposal are to enhance our understanding of the role of ACE in cholera pathogenesis and to identify novel mechanisms of action of bacterial toxins. The utility of ACE in investigating the epithelial transport pathways in the intestine, and perhaps other tissue types lies in ACE's novel physiology. The characterization of the ion channels activated by ACE may provide valuable information, which can be used to develop pharmacological modulators of chloride and bicarbonate secretion useful in the treatment of diarrheal diseases (excessive secretion) or cystic fibrosis (defective secretion).
