260 Probiotics Reverse TNFα-Mediated Down-Regulation of SLC5A8 Expression and Function

Abstract

Background and Aim: Dysregulated epithelial secretory function can lead to the clinical manifestation of diarrhea. Intestinal fluid secretion is driven by active Clion secretion and is dependent on the availability of O2 for generation of cellular energy. Hydroxylases are the primary intracellular sensors of O2 availability, and we have previously shown that hydroxylase inhibition attenuates colonic epithelial secretory capacity. Here, we sought to investigate the molecular mechanisms involved and to test the efficacy of hydroxylase inhibitors in preventing diarrhea In Vivo.Methods: The pan-hydroxylase inhibitor dimethyloxallyl glycine (DMOG) was used to inhibit hydroxylase activity. Clsecretion was measured as changes in short circuit current across monolayers of T84 cells or muscle-stripped segments of mouse colon. Western blotting and biotinylation techniques were used to assess protein expression. DMOG effects were also tested In Vivo in a mouse allergic (ovalbumin) model of diarrhea. Results: As previously reported treatment of T84 cells with DMOG (1 mM) reduced Clsecretory responses to the Ca2+ and cAMP-dependent agonists, carbachol (CCh) and forskolin (FSK), to 20.2 ± 2.6% and 38.8 ± 4.8% of controls, respectively (n=16; p<0.001). To determine molecular mechanisms involved we analysed the activity of transport proteins comprising the Clsecretory pathway. Hydroxylase inhibition did not alter apical Clor basolateral K+ conductances but significantly reduced the activity of the Na+/K+ATPase, the energy-dependent step of Clsecretion, to 42.7% ± 5.5% of controls (n=5; p≤0.01). However, DMOG did not alter total cellular or surface expression of the ATPase. Cellular ATP, required to drive pump activity, was reduced to 67% ± 8.5% of controls (n=6; p≤0.05) by DMOG. Intraperitoneal injection of mice with DMOG (320 mg/kg; 24 hrs) attenuated colonic secretory responses to CCh and FSK to 54.8 ± 6.1% (n=11; p≤0.001) and 72.4 ± 11.5% (n=11; p≤0.05) of those of controls, respectively. Furthermore, in an In Vivo mouse model of allergic diarrhea, DMOG pretreatment reduced the occurrence of diarrhea by 80% compared to controls (n=10). Conclusion: Our studies show that hydroxylase inhibition in colonic epithelial cells inhibits secretory function. This antisecretory effect is mediated by attenuation of Na+/K+-ATPase pump activity, either directly, or secondary to the depletion of cellular ATP. Our data suggest that by virtue of their ability to regulate epithelial transport In Vitro and In Vivo, hydroxylases present a promising target for the development of novel anti-diarrheal agents that act by directly modulating transport protein function.