Stephen J. Keely

The Hydroxylase Inhibitor Dimethyloxalylglycine Is Protective in a Murine Model of Colitis

BACKGROUND & AIMS Prolyl and asparaginyl hydroxylases are key oxygen-sensing enzymes that confer hypoxic sensitivity to transcriptional regulatory pathways including the hypoxia inducible factor 1 (HIF-1) and nuclear factor-kappaB (NF-kappaB). Knockout of either HIF-1 or (IKKbeta-dependent) NF-kappaB pathways in intestinal epithelial cells promotes inflammatory disease in murine models of colitis. Both HIF-1 and NF-kappaB pathways are repressed by the action of hydroxylases through the hydroxylation of key regulatory molecules. METHODS In this study we have investigated the effects of the hydroxylase inhibitor dimethyloxalylglycine (DMOG) on Caco-2 intestinal epithelial cells in vitro and in a dextran sodium sulfate-induced model of murine colitis. RESULTS DMOG induces both HIF-1 and NF-kappaB activity in cultured intestinal epithelial cells, and is profoundly protective in dextran-sodium sulfate colitis in a manner that is at least in part reflected by the development of an anti-apoptotic phenotype in intestinal epithelial cells, which we propose reduces epithelial barrier dysfunction. CONCLUSIONS These data show that hydroxylase inhibitors such as DMOG represent a new strategy for the treatment of inflammatory bowel disease.

The autonomic nervous system and inflammatory bowel disease

Crohns disease and ulcerative colitis, collectively known as inflammatory bowel disease (IBD), are chronic, recurring, inflammatory conditions of the intestine. The precise mechanisms underlying the pathogenesis of IBD are not yet clear but they are believed to involve a number of precipitating factors, most notably genetic susceptibility and environmental influences. The autonomic nervous system (ANS) has long been known as a critical regulator of intestinal function and much evidence now exists to suggest that it also plays an important role in the development of IBD. Dramatic changes in the ANS in IBD are apparent from the cellular to the molecular level ultimately leading to altered communication between the ANS and effector cells of the intestine. This review aims to synthesize the current understanding of the pathogenesis of IBD with a particular emphasis on the role that the ANS plays in the progression of these diseases.

The bile acid receptor, TGR5, regulates basal and cholinergic-induced secretory responses in rat colon.

Bile acids (BA) are becoming increasingly appreciated as enteric hormones that regulate many aspects of intestinal physiology. The BA receptor, TGR5, has been recently shown to be expressed on enteric nerves and enterochromaffin cells (ECs), where its activation regulates small intestinal and colonic motility. Here, we show that TGR5 is also expressed on colonic epithelial cells and that its activation decreases basal secretory tone and inhibits cholinergic‐induced secretory responses. Our data demonstrate a new role for TGR5 in regulating colonic fluid and electrolyte transport and suggest that the receptor represents a good therapeutic target for intestinal transport disorders.

Farnesoid X receptor agonists attenuate colonic epithelial secretory function and prevent experimental diarrhoea in vivo

Objective Bile acids are important regulators of intestinal physiology, and the nuclear bile acid receptor, farnesoid X receptor (FXR), is emerging as a promising therapeutic target for several intestinal disorders. Here, we investigated a role for FXR in regulating intestinal fluid and electrolyte transport and the potential for FXR agonists in treating diarrhoeal diseases. Design Electrogenic ion transport was measured as changes in short-circuit current across voltage-clamped T84 cell monolayers or mouse tissues in Ussing chambers. NHE3 activity was measured as BCECF fluorescence in Caco-2 cells. Protein expression was measured by immunoblotting and cell surface biotinylation. Antidiarrhoeal efficacy of GW4064 was assessed using two in vivo mouse models: the ovalbumin-induced diarrhoea model and cholera toxin (CTX)-induced intestinal fluid accumulation. Results GW4064 (5 μmol/L; 24 h), a specific FXR agonist, induced nuclear translocation of the receptor in T84 cells and attenuated Cl− secretory responses to both Ca2+ and cAMP-dependent agonists. GW4064 also prevented agonist-induced inhibition of NHE3 in Caco-2 cells. In mice, intraperitoneal administration of GW4064 (50 mg/mL) also inhibited Ca2+ and cAMP-dependent secretory responses across ex vivo colonic tissues and prevented ovalbumin-induced diarrhoea and CTX-induced intestinal fluid accumulation in vivo. At the molecular level, FXR activation attenuated apical Cl− currents by inhibiting expression of cystic fibrosis transmembrane conductance regulator channels and inhibited basolateral Na+/K+-ATPase activity without altering expression of the protein. Conclusions These data reveal a novel antisecretory role for the FXR in colonic epithelial cells and suggest that FXR agonists have excellent potential for development as a new class of antidiarrheal drugs.

Epidermal growth factor chronically upregulates Ca2+-dependent Cl− conductance and TMEM16A expression in intestinal epithelial cells

Key points  •  Cl− secretion, the predominant driving force for fluid secretion in the intestine, can be dysregulated in conditions of disease, such as cystic fibrosis. •  We have previously shown that acute exposure to epidermal growth factor (EGF) chronically upregulates the capacity of colonic epithelial cells to secrete Cl−. •  Here, we show that the effects of EGF are mediated by upregulation of the Ca2+‐dependent Cl− channel, transmembrane protein 16A (TMEM16A), in the apical membrane of colonic epithelial cells. •  EGF‐induced TMEM16A expression is mediated by sequential activation of phosphatidylinositol 3‐kinase and PKCδ. •  These findings are among the first to elucidate molecular mechanisms that regulate TMEM16A expression in epithelial cells and suggest the channel represents a good target for development of new therapeutics for intestinal transport disorders.

Physiological concentrations of bile acids down‐regulate agonist induced secretion in colonic epithelial cells

In patients with bile acid malabsorption, high concentrations of bile acids enter the colon and stimulate Cl− and fluid secretion, thereby causing diarrhoea. However, deoxycholic acid (DCA), the predominant colonic bile acid, is normally present at lower concentrations where its role in regulating transport is unclear. Thus, the current study set out to investigate the effects of physiologically relevant DCA concentrations on colonic epithelial secretory function. Cl− secretion was measured as changes in short‐circuit current across voltage‐clamped T84 cell monolayers. At high concentrations (0.5–1 mM), DCA acutely stimulated Cl− secretion but this effect was associated with cell injury, as evidenced by decreased transepithelial resistance (TER) and increased lactate dehydrogenase (LDH) release. In contrast, chronic (24 hrs) exposure to lower DCA concentrations (10–200 μM) inhibited responses to Ca2+ and cAMP‐dependent secretagogues without altering TER, LDH release, or secretagogue‐induced increases in intracellular second messengers. Other bile acids – taurodeoxycholic acid, chenodeoxycholic acid and cholic acid – had similar antisecretory effects. DCA (50 μM) rapidly stimulated phosphorylation of the epidermal growth factor receptor (EGFr) and both ERK and p38 MAPKs (mitogen‐activated protein kinases). The EGFr inhibitor, AG1478, and the protein synthesis inhibitor, cycloheximide, reversed the antisecretory effects of DCA, while the MAPK inhibitors, PD98059 and SB203580, did not. In summary, our studies suggest that, in contrast to its acute prosecretory effects at pathophysiological concentrations, lower, physiologically relevant, levels of DCA chronically down‐regulate colonic epithelial secretory function. On the basis of these data, we propose a novel role for bile acids as physiological regulators of colonic secretory capacity.

Ursodeoxycholic acid attenuates colonic epithelial secretory function.

•  Although diarrhoeal diseases represent a significant health and economic burden to society, therapeutic options remain limited. •  While several bile acids are known to stimulate epithelial Cl− secretion, the major driving force for fluid secretion in the intestine, the effects of ursodeoxycholic acid (UDCA) on epithelial transport function are not well described. •  We report that in contrast to other bile acids, UDCA exerts anti‐secretory actions on colonic epithelial cells in vitro. •  In contrast, in vivo administration of UDCA enhances epithelial secretory function, an affect we ascribe to being due to its bacterial metabolism to lithocholic acid. In keeping with this hypothesis, in vivo administration of a metabolically stable analogue of UDCA, 6α‐methyl‐UDCA, was anti‐secretory. •  Our findings reveal novel anti‐secretory actions of UDCA and suggest that metabolically stable analogues of bile acid may be useful for development as a new class of anti‐diarrhoeal drug.

Induction of Na+/K+/2Cl- cotransporter expression mediates chronic potentiation of intestinal epithelial Cl- secretion by EGF.

Alterations in EGF receptor (EGFR) signaling occur in intestinal disorders associated with dysregulated epithelial transport. In the present study, we investigated a role for the EGFR in the chronic regulation of intestinal epithelial secretory function. Epithelial Cl(-) secretion was measured as changes in short-circuit current (Isc) across voltage-clamped monolayers of T84 cells in Ussing chambers. Acute treatment of T84 cells with EGF (100 ng/ml, 15 min) chronically enhanced Isc responses to a broad range of secretagogues. This effect was apparent within 3 h, maximal by 6 h, and sustained for 24 h after treatment with EGF. The Na+/K+/2Cl(-) cotransporter (NKCC1) inhibitor bumetanide (100 microM) abolished the effect of EGF, indicating increased responses are due to potentiated Cl(-) secretion. Neither basal nor agonist-stimulated levels of intracellular Ca2+ or PKA activity were altered by EGF, implying that the effects of the growth factor are not due to chronic alterations in levels of second messengers. EGF increased the expression of NKCC1 with a time course similar to that of its effects on Cl(-) secretion. This effect of EGF was maximal after 6 h, at which time NKCC1 expression in EGF-treated cells was 199.9 +/- 21.9% of that in control cells (n = 21, P < 0.005). EGF-induced NKCC1 expression was abolished by actinomycin D, and RT-PCR analysis demonstrated EGF increased expression of NKCC1 mRNA. These data increase our understanding of mechanisms regulating intestinal fluid and electrolyte transport and reveal a novel role for the EGFR in the chronic regulation of epithelial secretory capacity through upregulation of NKCC1 expression.

Oxygen in the regulation of intestinal epithelial transport

The transport of fluid, nutrients and electrolytes to and from the intestinal lumen is a primary function of epithelial cells. Normally, the intestine absorbs approximately 9 l of fluid and 1 kg of nutrients daily, driven by epithelial transport processes that consume large amounts of cellular energy and O2. The epithelium exists at the interface of the richly vascularised mucosa, and the anoxic luminal environment and this steep O2 gradient play a key role in determining the expression pattern of proteins involved in fluid, nutrient and electrolyte transport. However, the dynamic nature of the splanchnic circulation necessitates that the epithelium can evoke co‐ordinated responses to fluctuations in O2 availability, which occur either as a part of the normal digestive process or as a consequence of several pathophysiological conditions. While it is known that hypoxia‐responsive signals, such as reactive oxygen species, AMP‐activated kinase, hypoxia‐inducible factors, and prolyl hydroxylases are all important in regulating epithelial responses to altered O2 supply, our understanding of the molecular mechanisms involved is still limited. Here, we aim to review the current literature regarding the role that O2 plays in regulating intestinal transport processes and to highlight areas of research that still need to be addressed.

Missing link identified: GpBAR1 is a neuronal bile acid receptor.

Abstract  In addition to their classical functions in aiding the digestion and absorption of lipids, bile acids are increasingly gaining appreciation for their roles in regulating intestinal physiology. Bile acids are now widely considered as hormones that exert a wide range of physiological and pathophysiological effects both within and outside the gastrointestinal (GI) tract. The discovery of the bile acid receptor, GpBAR1, represented a major step forward in our understanding of how cells can sense and respond to bile acids. GpBAR1 is a cell surface G protein‐coupled receptor expressed on adipose tissue and skeletal muscle where it has been found to be an important regulator of cellular metabolism. In a paper published in the current issue of Neurogastroenterology and Motility, Poole et al. investigated the expression and function of GpBAR1 in mouse intestine. They found the receptor to be expressed throughout the GI tract but predominantly on nerves within the myenteric and submucosal plexuses. Employing in vitro and in vivo techniques they demonstrated that activation of GpBAR1 by bile acids inhibits small and large intestinal motor function and delays intestinal transit. The effects of GpBAR1 activation are mediated through activation of cholinergic and nitrergic interneurons. The data reported by Poole et al. provides novel and exciting insights into how bile acids exert their actions in the intestine. This Editorial Viewpoint aims to further consider the potential physiological and pathophysiological implications of their findings.