198 Bitter Stimuli Induce ERK Activation in Mouse and Human Cell Lines

Abstract

Bacterial toxins such as cholera toxin induce diarrhea by both direct epithelial cell generation of cyclic nucleotides as well as stimulation of the enteric nervous system (ENS) to release secretagogues. We have previously demonstrated that extracellular calcium-sensing receptor (CaSR) agonists can abrogate toxin-stimulated fluid secretion in ENS-absent colonic epithelial crypts by increasing phosphodiesterase-dependent cyclic-nucleotide degradation. Based on our observation that CaSR is expressed in tetrodotoxin (TTX)-sensitive neurons comprising the ENS, we hypothesized that CaSR agonists may also function through neuronal pathways. To examine this possibility, rat colon segments containing intact ENS were isolated, mounted on Ussing chambers and perfused. Basal and cyclic nucleotide-stimulated anion secretions were monitored by measuring changes in short circuit current (Isc). CaSR was activated by R-568 either before or after cyclic nucleotide stimulation to mimic diarrhea prevention or treatment, respectively, and its effects were compared in the presence and absence of TTX. Consistent with active regulation of anion secretion by the ENS, a significant proportion of Isc in the proximal and distal colon was inhibited by serosal TTX, both at basal and under cyclic AMP-stimulated conditions. Activation of CaSR with R-568 significantly reduced basal and forskolin-induced Isc. A similar inhibitor effect of R-568 on Isc was achieved following the forskolin stimulation, suggesting that this agonist might be useful both for diarrhea prevention and treatment. Interestingly, when colon tissues were pretreated with TTX to inhibit the ENS activity, the inhibitory effects of R-568 were either significantly attenuated or abolished, indicating that R-568 exerts its antisecretory effect on the intestine primarily by inhibiting the ENS. The antisecretory effect of R-568 appears to be largely mediated by the submucosal Meissner's plexus because removal of the muscular layers and the myenteric Auerbach's plexus from the colon did not significantly reduce the inhibitory effect of R568. Our results suggest a new model in which the neuronal component of intestinal fluid transport can be modulated by the inhibitory effects of CaSR on the ENS. The ability of CaSR agonist to reduce secretagogue-stimulated anion secretion suggests that modulation of CaSR activity may provide a new therapy for secretory diarrheas and other ENS-mediated conditions.