The al. (13, 14) indicates that STB induces a rapid short-circuit current across porcine intestinal mucosa. Evidence for chloride transport was not observed, yet electrogenic anion secretion, possibly bicarbonate, was observed in the absence of elevated cyclic nucleotides (14,15). Beyond these few studies, nothing is known of the mechanism of STB action.Evidence that suggests a receptor-mediated mode of STB action includes short-circuit current responses when toxin is delivered to the mucosal, but not serosal, side of intestinal tissue mounted in Ussing chambers (13); a rapid STB response in experiments utilizing either mounted tissue or whole animal models (15 min and 2 hr, respectively) (13,14,16); and the lack of apparent damage to the intestinal mucosa following STB treatment (17,18). Known mediators of membrane signal transduction include cAMP, cGMP, the phosphatidylinositol 4,5-bisphosphate-derived metabolites inositol 1,4,5-trisphosphate (InsP3) and diacyl glycerol, and Ca2W (for reviews, see refs. 19-21

The intestinal secretory action of Escherichia coli heat-stable enterotoxin B (STb) is poorly defined. Previous work indicates that STb causes loss of intestinal fluid and electrolytes by a mechanism independent of elevated levels of cyclic nucleotides, the hallmark of other E. coli cytotonic enterotoxins. In the work described in this report, we observed that treatment of ligated rat intestinal loops with purified STb of E. coli resulted in a dose-dependent rise in intestinal secretion concomitant with dose-related increases in levels of serotonin (5-hydroxytryptamine [5-HT]) and prostaglandin E 2 (PGE 2). Treatment of rats with the 5-HT 2 receptor antagonist ketanserin prior to STb challenge resulted in significant (P < 0.05) reduction in intestinal secretion. Blockage of 5-HT 2 receptors with ketanserin also reduced (P < 0.05) the level of PGE 2 observed following STb treatment, indicating that at least a portion of the PGE 2 was formed in response to 5-HT 2 receptor stimulation. In a similar fashion, indomethacin, an inhibitor of cyclooxygenase activity, significantly reduced the level of secretion (P < 0.05) observed following STb treatment yet had no effect on 5-HT levels. Treatment of rats with both ketanserin and indomethacin further reduced STb-mediated secretion to a level not attained by either drug alone. Taken together, our data suggest that secretion due to STb involves both 5-HT and PGE 2 as intestinal secretagogues. Furthermore, PGE 2 formation appears to arise through both 5-HT-dependent and 5-HT-independent pathways.

No abstract

Heat-stable enterotoxins (STs) of Escherichia coli are peptides which alter normal gut physiology by stimulating the loss of water and electrolytes. The action of heat-stable toxin B (STb) is associated with an increase in levels of lumenal 5-hydroxytryptamine and prostaglandin E 2 , known mediators of intestinal secretion. In addition, the toxin is responsible for elevation of cytosolic calcium ion levels in cultured cells. STb is a 48-amino-acid basic peptide containing four cysteine residues and two disulfide bonds. Previous work indicates that disulfide bonds are required for intestinal secretory activity, and yet the relative contribution of the two bonds to toxin stability and action is presently unclear. Site-directed mutagenesis was used to alter the cysteine residues of STb to assess the role of the individual disulfide bonds in toxin activity. Our results indicate that loss of a single disulfide bond was sufficient to abolish the intestinal secretory and G protein-coupled calcium ion influx activities associated with STb toxicity. Loss of toxin action was not a function of increased sensitivity of STb mutants to proteolysis, since mutant toxins displayed proteolytic decay rates equivalent to that of wild-type STb. Circular dichroism spectroscopy of mutant STb toxins indicated that single-disulfide-bond elimination did not apparently affect the toxin secondary structure of one mutant, STbC33S,C71S. In contrast, the ␣-helical content of the other disulfide bond mutant, STbC44S,C59G, was significantly altered, as was that of reduced and alkylated authentic STb. Since both Cys-Cys mutant STbs were completely nontoxic, the absence of biological activity cannot be explained by dramatic secondary structural changes alone; keys to the conformational requirements for STb toxicity undoubtedly reside in the three-dimensional structure of this peptide.