Ursodeoxycholic acid inhibits ENaC and Na/K pump activity to restore airway surface liquid height in cystic fibrosis bronchial epithelial cells.

Cystic fibrosis (CF) is a disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) that in the airways result in reduced Cl- secretion and increased Na+ absorption, airway surface liquid (ASL) dehydration, decreased mucociliary clearance, infection and inflammation leading to lung injury. Cystic fibrosis patients often present with bile acids in the lower airways, however the effects of bile acids on ASL and ion transport in CF airways are not known. Secondary bile acids, such as ursodeoxycholic acid (UDCA), have been shown to modulate immune responses and epithelial ion transport. Here we investigated the effects of UDCA in normal and CF airway epithelial cell models. NuLi-1 (normal genotype) and CuFi-1 (CF genotype, Δ508/Δ508) primary immortalized airway epithelial cells were grown under an air-liquid interface. Electrogenic transepithelial ion transport was measured by short-circuit current (Isc) across cell monolayers mounted in Ussing chambers. We observed that UDCA (500 µM, 60 min, bilateral) decreased the basal Isc and ENaC currents in both NuLi-1 and CuFi-1 cells. UDCA inhibited the amiloride-sensitive ENaC current by 44% in NulI-1 monolayers and by 30% in CuFi-1 cells. Interestingly, UDCA also inhibited currents through the basolateral Na/K pump in both Nuli-1 and CuFi-1 monolayers without alterting the expression of ENaC or Na+/K+-ATPase proteins. The airway surface liquid height is regulated by transpeithelial Na+ absorption (ENaC) and Cl- secretion (CFTR) in normal airway but mainly by ENaC activity in CF epithelia when Cl- secretion is compromised by CFTR mutations. UDCA increased ASL height by 50% in Nuli-1 and by 40% in CUFI-1 monolayers. In conclusion, we demonstrate a previously unknown effect of UDCA to inhibit ENaC activity and increase ASL height in normal and CF human airway epithelial cells suggesting a therapeutic potential for UDCA in CF lung disease.

The bile acids, deoxycholic acid and ursodeoxycholic acid, regulate colonic epithelial wound healing.

The intestinal epithelium constitutes an innate barrier which, upon injury, undergoes self-repair processes known as restitution. Although bile acids are known as important regulators of epithelial function in health and disease, their effects on wound healing processes are not yet clear. Here we set out to investigate the effects of the colonic bile acids, deoxycholic acid (DCA) and ursodeoxycholic acid (UDCA), on epithelial restitution. Wound healing in T84 cell monolayers grown on transparent, permeable supports was assessed over 48 h with or without bile acids. Cell migration was measured in Boyden chambers. mRNA and protein expression were measured by RT-PCR and Western blotting. DCA (50-150 µM) significantly inhibited wound closure in cultured epithelial monolayers and attenuated cell migration in Boyden chamber assays. DCA also induced nuclear accumulation of the farnesoid X receptor (FXR), whereas an FXR agonist, GW4064 (10 µM), inhibited wound closure. Both DCA and GW4064 attenuated the expression of CFTR Cl- channels, whereas inhibition of CFTR activity with either CFTR-inh-172 (10 µM) or GlyH-101 (25 µM) also prevented wound healing. Promoter/reporter assays revealed that FXR-induced downregulation of CFTR is mediated at the transcriptional level. In contrast, UDCA (50-150 µM) enhanced wound healing in vitro and prevented the effects of DCA. Finally, DCA inhibited and UDCA promoted mucosal healing in an in vivo mouse model. In conclusion, these studies suggest bile acids are important regulators of epithelial wound healing and are therefore good targets for development of new drugs to modulate intestinal barrier function in disease treatment. NEW & NOTEWORTHY The secondary bile acid, deoxycholic acid, inhibits colonic epithelial wound healing, an effect which appears to be mediated by activation of the nuclear bile acid receptor, FXR, with subsequent downregulation of CFTR expression and activity. In contrast, ursodeoxycholic acid promotes wound healing, suggesting it may provide an alternative approach to prevent the losses of barrier function that are associated with mucosal inflammation in IBD patients.

Bile acids stimulate chloride secretion through CFTR and calcium-activated Cl- channels in Calu-3 airway epithelial cells.

Bile acids resulting from the aspiration of gastroesophageal refluxate are often present in the lower airways of people with cystic fibrosis and other respiratory distress diseases. Surprisingly, there is little or no information on the modulation of airway epithelial ion transport by bile acids. The secretory effect of a variety of conjugated and unconjugated secondary bile acids was investigated in Calu-3 airway epithelial cells grown under an air-liquid interface and mounted in Ussing chambers. Electrogenic transepithelial ion transport was measured as short-circuit current (Isc). The taurine-conjugated secondary bile acid, taurodeoxycholic acid (TDCA), was found to be the most potent modulator of basal ion transport. Acute treatment (5 min) of Calu-3 cells with TDCA (25 µM) on the basolateral side caused a stimulation of Isc, and removal of extracellular Cl(-) abolished this response. TDCA produced an increase in the cystic fibrosis transmembrane conductance regulator (CFTR)-dependent current that was abolished by pretreatment with the CFTR inhibitor CFTRinh172. TDCA treatment also increased Cl(-) secretion through calcium-activated chloride (CaCC) channels and increased the Na(+)/K(+) pump current. Acute treatment with TDCA resulted in a rapid cellular influx of Ca(2+) and increased cAMP levels in Calu-3 cells. Bile acid receptor-selective activation with INT-777 revealed TGR5 localized at the basolateral membrane as the receptor involved in TDCA-induced Cl(-) secretion. In summary, we demonstrate for the first time that low concentrations of bile acids can modulate Cl(-) secretion in airway epithelial cells, and this effect is dependent on both the duration and sidedness of exposure to the bile acid.

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 Ca(2+) 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 Ca(2+) 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.

Ursodeoxycholic acid attenuates colonic epithelial secretory function.

Dihydroxy bile acids, such as chenodeoxycholic acid (CDCA), are well known to promote colonic fluid and electrolyte secretion, thereby causing diarrhoea associated with bile acid malabsorption. However, CDCA is rapidly metabolised by colonic bacteria to ursodeoxycholic acid (UDCA), the effects of which on epithelial transport are poorly characterised. Here, we investigated the role of UDCA in the regulation of colonic epithelial secretion. Cl(-) secretion was measured across voltage-clamped monolayers of T84 cells and muscle-stripped sections of mouse or human colon. Cell surface biotinylation was used to assess abundance/surface expression of transport proteins. Acute (15 min) treatment of T84 cells with bilateral UDCA attenuated Cl(-) secretory responses to the Ca(2+) and cAMP-dependent secretagogues carbachol (CCh) and forskolin (FSK) to 14.0 ± 3.8 and 40.2 ± 7.4% of controls, respectively (n = 18, P < 0.001). Investigation of the molecular targets involved revealed that UDCA acts by inhibiting Na(+)/K(+)-ATPase activity and basolateral K(+) channel currents, without altering their cell surface expression. In contrast, intraperitoneal administration of UDCA (25 mg kg(-1)) to mice enhanced agonist-induced colonic secretory responses, an effect we hypothesised to be due to bacterial metabolism of UDCA to lithocholic acid (LCA). Accordingly, LCA (50-200 µm) enhanced agonist-induced secretory responses in vitro and a metabolically stable UDCA analogue, 6α-methyl-UDCA, exerted anti-secretory actions in vitro and in vivo. In conclusion, UDCA exerts direct anti-secretory actions on colonic epithelial cells and metabolically stable derivatives of the bile acid may offer a new approach for treating intestinal diseases associated with diarrhoea.

Epidermal growth factor chronically upregulates Ca(2+)-dependent Cl(-) conductance and TMEM16A expression in intestinal epithelial cells.

Dysregulated epithelial fluid and electrolyte transport is a common feature of many intestinal disorders. However, molecular mechanisms that regulate epithelial transport processes are still poorly understood, thereby limiting development of new therapeutics. Previously, we showed that epidermal growth factor (EGF) chronically enhances intestinal epithelial secretory function. Here, we investigated a potential role for altered expression or activity of apical Cl(−) channels in mediating the effects of EGF. Cl(−) secretion across monolayers of T(84) colonic epithelia was measured as changes in short-circuit current. Protein expression/phosphorylation was measured by RT-PCR and Western blotting. Under conditions that specifically isolate apical Ca(2+)-activated Cl(−) channel (CaCC) currents, EGF pretreatment (100 ng ml(−1) for 15 min) potentiated carbachol (CCh)-induced responses to 173 ± 25% of those in control cells, when measured 24 h later (n = 26; P < 0.01). EGF-induced increases in CaCC currents were abolished by the transmembrane protein 16A (TMEM16A) inhibitor, T16A(inh)-A01 (10 µm). Furthermore, TMEM16A mRNA and protein expression was increased by EGF to 256 ± 38% (n = 7; P < 0.01) and 297 ± 46% (n = 9, P < 0.001) of control levels, respectively. In contrast, EGF did not alter CFTR expression or activity. EGF-induced increases in Cl(−) secretion, CaCC currents and TMEM16A expression were attenuated by a PKCδ inhibitor, rottlerin (20 µm), and a phosphatidylinositol 3-kinase (PI3K) inhibitor, LY290042 (25 µm). Finally, LY290042 inhibited EGF-induced phosphorylation of PKCδ. We conclude that EGF chronically upregulates Ca(2+)-dependent Cl(−) conductances and TMEM16A expression in intestinal epithelia by a mechanism involving sequential activation of PI3K and PKCδ. Therapeutic targeting of EGF receptor-dependent signalling pathways may provide new approaches for treatment of epithelial 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 T(84) 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 microM) inhibited responses to Ca(2+) 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 microM) 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.

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.