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

Keating N, Mroz MS, Scharl MM, Marsh C, Ferguson G, Hofmann AF, Keely SJ - J. Cell. Mol. Med. (2009)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Medicine, Royal College of Surgeons in Ireland, Dublin, Ireland.

ABSTRACT
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.

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Inhibition of protein synthesis but not ERK or p38 MAPK blocks the antisecretory effect of DCA in colonic epithelial cells. (A) Cells were treated bilaterally with DCA (50 μM) for the times indicated after which cell lysates were analysed by Western blotting with anti‐phospho‐ERK or anti‐phospho‐p38 antibodies. Each blot is representative of three similar experiments. (B–D) Cells were pretreated with either (B) PD98059 (20 μM), (C) SB203580 (10 μM) or (D) cycloheximide (100 ng/ml) for 30 min prior to DCA (50 μM). After a further 24 hrs incubation, cells were mounted in Ussing chambers and Isc responses to CCh (100 μM) were measured. The insets to Fig. 7B and D show that PD98059 and SB203580 inhibited DCA‐stimulated ERK and p38 phosphorylation, respectively (these blots are representative of three similar experiments). Responses to CCh in DCA‐treated cells were expressed as percentage of vehicle or inhibitor‐treated controls, as appropriate (n= 5; *P < 0.05; **P < 0.01; ***P < 0.001 compared to cells stimulated with CCh alone).
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f7: Inhibition of protein synthesis but not ERK or p38 MAPK blocks the antisecretory effect of DCA in colonic epithelial cells. (A) Cells were treated bilaterally with DCA (50 μM) for the times indicated after which cell lysates were analysed by Western blotting with anti‐phospho‐ERK or anti‐phospho‐p38 antibodies. Each blot is representative of three similar experiments. (B–D) Cells were pretreated with either (B) PD98059 (20 μM), (C) SB203580 (10 μM) or (D) cycloheximide (100 ng/ml) for 30 min prior to DCA (50 μM). After a further 24 hrs incubation, cells were mounted in Ussing chambers and Isc responses to CCh (100 μM) were measured. The insets to Fig. 7B and D show that PD98059 and SB203580 inhibited DCA‐stimulated ERK and p38 phosphorylation, respectively (these blots are representative of three similar experiments). Responses to CCh in DCA‐treated cells were expressed as percentage of vehicle or inhibitor‐treated controls, as appropriate (n= 5; *P < 0.05; **P < 0.01; ***P < 0.001 compared to cells stimulated with CCh alone).

Mentions: Our previous work has identified the epidermal growth factor receptor (EGFr) and mitogen‐activated protein kinases (MAPKs) as important regulators of epithelial secretory responses [29, 30]. Furthermore, others have shown that bile acids induce EGFr and extracellular signal‐regulated kinase (ERK) activation in colonic epithelia [5, 31, 32]. Thus, we examined a potential role for the EGFr and MAPKs in mediating antisecretory effects of DCA. As shown in Fig. 6A and B, DCA (50 μM) rapidly stimulated EGFr phosphorylation and pretreatment with the EGFr inhibitor, tyrphostin AG1478 (100 nM), reversed the antisecretory effects of the bile acid (Fig. 6C). DCA also activated both ERK and p38 MAPKs (Fig. 7A). However, although PD98059 (20 μM) and SB203580 (10 μM) effectively prevented DCA‐induced ERK and p38 phosphorylation, respectively, neither inhibitor prevented the antisecretory actions of DCA (Fig. 7B and C). However, inhibition of protein translation with cycloheximide (CHX; 100 ng/ml), was found to attenuate the antisecretory actions of DCA (Fig. 7D).


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

Keating N, Mroz MS, Scharl MM, Marsh C, Ferguson G, Hofmann AF, Keely SJ - J. Cell. Mol. Med. (2009)

Inhibition of protein synthesis but not ERK or p38 MAPK blocks the antisecretory effect of DCA in colonic epithelial cells. (A) Cells were treated bilaterally with DCA (50 μM) for the times indicated after which cell lysates were analysed by Western blotting with anti‐phospho‐ERK or anti‐phospho‐p38 antibodies. Each blot is representative of three similar experiments. (B–D) Cells were pretreated with either (B) PD98059 (20 μM), (C) SB203580 (10 μM) or (D) cycloheximide (100 ng/ml) for 30 min prior to DCA (50 μM). After a further 24 hrs incubation, cells were mounted in Ussing chambers and Isc responses to CCh (100 μM) were measured. The insets to Fig. 7B and D show that PD98059 and SB203580 inhibited DCA‐stimulated ERK and p38 phosphorylation, respectively (these blots are representative of three similar experiments). Responses to CCh in DCA‐treated cells were expressed as percentage of vehicle or inhibitor‐treated controls, as appropriate (n= 5; *P < 0.05; **P < 0.01; ***P < 0.001 compared to cells stimulated with CCh alone).
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f7: Inhibition of protein synthesis but not ERK or p38 MAPK blocks the antisecretory effect of DCA in colonic epithelial cells. (A) Cells were treated bilaterally with DCA (50 μM) for the times indicated after which cell lysates were analysed by Western blotting with anti‐phospho‐ERK or anti‐phospho‐p38 antibodies. Each blot is representative of three similar experiments. (B–D) Cells were pretreated with either (B) PD98059 (20 μM), (C) SB203580 (10 μM) or (D) cycloheximide (100 ng/ml) for 30 min prior to DCA (50 μM). After a further 24 hrs incubation, cells were mounted in Ussing chambers and Isc responses to CCh (100 μM) were measured. The insets to Fig. 7B and D show that PD98059 and SB203580 inhibited DCA‐stimulated ERK and p38 phosphorylation, respectively (these blots are representative of three similar experiments). Responses to CCh in DCA‐treated cells were expressed as percentage of vehicle or inhibitor‐treated controls, as appropriate (n= 5; *P < 0.05; **P < 0.01; ***P < 0.001 compared to cells stimulated with CCh alone).
Mentions: Our previous work has identified the epidermal growth factor receptor (EGFr) and mitogen‐activated protein kinases (MAPKs) as important regulators of epithelial secretory responses [29, 30]. Furthermore, others have shown that bile acids induce EGFr and extracellular signal‐regulated kinase (ERK) activation in colonic epithelia [5, 31, 32]. Thus, we examined a potential role for the EGFr and MAPKs in mediating antisecretory effects of DCA. As shown in Fig. 6A and B, DCA (50 μM) rapidly stimulated EGFr phosphorylation and pretreatment with the EGFr inhibitor, tyrphostin AG1478 (100 nM), reversed the antisecretory effects of the bile acid (Fig. 6C). DCA also activated both ERK and p38 MAPKs (Fig. 7A). However, although PD98059 (20 μM) and SB203580 (10 μM) effectively prevented DCA‐induced ERK and p38 phosphorylation, respectively, neither inhibitor prevented the antisecretory actions of DCA (Fig. 7B and C). However, inhibition of protein translation with cycloheximide (CHX; 100 ng/ml), was found to attenuate the antisecretory actions of DCA (Fig. 7D).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Medicine, Royal College of Surgeons in Ireland, Dublin, Ireland.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus