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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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The antisecretory effect of DCA is not mediated by muscarinic receptors or by alterations in agonist‐induced generation of the second messengers. (A) Cells were pretreated with atropine (1 μM) for 30 min prior to DCA (100 μM). After a further 24 hrs incubation, cells were washed and Isc responses to FSK (10 μM) were measured (n= 5). (B) T84 cells grown on glass coverslips were pretreated for 24 hrs with DCA (50 μM) after which CCh‐induced mobilization of intracellular Ca2+ was measured by Fura‐2 fluorescence. Data are expressed as mean fluorescence ratio at 340 and 380 nM (n= 3 coverslips for each condition). The inset shows the net change in mean fluorescence ratio (ΔF340/380) for these experiments. (C) Monolayers of T84 cells were treated with DCA (50 μM) for 24 hrs and then stimulated with FSK (10 μM) for 5 min, lysed, and intracellular cAMP was measured (n= 4). **P < 0.01.
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f5: The antisecretory effect of DCA is not mediated by muscarinic receptors or by alterations in agonist‐induced generation of the second messengers. (A) Cells were pretreated with atropine (1 μM) for 30 min prior to DCA (100 μM). After a further 24 hrs incubation, cells were washed and Isc responses to FSK (10 μM) were measured (n= 5). (B) T84 cells grown on glass coverslips were pretreated for 24 hrs with DCA (50 μM) after which CCh‐induced mobilization of intracellular Ca2+ was measured by Fura‐2 fluorescence. Data are expressed as mean fluorescence ratio at 340 and 380 nM (n= 3 coverslips for each condition). The inset shows the net change in mean fluorescence ratio (ΔF340/380) for these experiments. (C) Monolayers of T84 cells were treated with DCA (50 μM) for 24 hrs and then stimulated with FSK (10 μM) for 5 min, lysed, and intracellular cAMP was measured (n= 4). **P < 0.01.

Mentions: Previous studies suggest bile acids can exert biological effects through activation of muscarinic M3 receptors [28]. However, we found that although it abolished responses to CCh (data not shown), the muscarinic receptor antagonist, atropine (1 μM), did not prevent the antisecretory effect of DCA (Fig. 5A). We also examined the possibility that DCA exerts antisecretory effects by altering secretagogue‐induced mobilization of the second messengers. However, in cells pretreated with DCA (50 μM, 24 hrs), neither CCh‐stimulated mobilization of intracellular Ca2+ nor FSK‐induced accumulation of cAMP was altered (Fig. 5B and C). These data suggest that DCA exerts its antisecretory effects downstream from agonist‐induced mobilization of prosecretory second messengers.


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)

The antisecretory effect of DCA is not mediated by muscarinic receptors or by alterations in agonist‐induced generation of the second messengers. (A) Cells were pretreated with atropine (1 μM) for 30 min prior to DCA (100 μM). After a further 24 hrs incubation, cells were washed and Isc responses to FSK (10 μM) were measured (n= 5). (B) T84 cells grown on glass coverslips were pretreated for 24 hrs with DCA (50 μM) after which CCh‐induced mobilization of intracellular Ca2+ was measured by Fura‐2 fluorescence. Data are expressed as mean fluorescence ratio at 340 and 380 nM (n= 3 coverslips for each condition). The inset shows the net change in mean fluorescence ratio (ΔF340/380) for these experiments. (C) Monolayers of T84 cells were treated with DCA (50 μM) for 24 hrs and then stimulated with FSK (10 μM) for 5 min, lysed, and intracellular cAMP was measured (n= 4). **P < 0.01.
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f5: The antisecretory effect of DCA is not mediated by muscarinic receptors or by alterations in agonist‐induced generation of the second messengers. (A) Cells were pretreated with atropine (1 μM) for 30 min prior to DCA (100 μM). After a further 24 hrs incubation, cells were washed and Isc responses to FSK (10 μM) were measured (n= 5). (B) T84 cells grown on glass coverslips were pretreated for 24 hrs with DCA (50 μM) after which CCh‐induced mobilization of intracellular Ca2+ was measured by Fura‐2 fluorescence. Data are expressed as mean fluorescence ratio at 340 and 380 nM (n= 3 coverslips for each condition). The inset shows the net change in mean fluorescence ratio (ΔF340/380) for these experiments. (C) Monolayers of T84 cells were treated with DCA (50 μM) for 24 hrs and then stimulated with FSK (10 μM) for 5 min, lysed, and intracellular cAMP was measured (n= 4). **P < 0.01.
Mentions: Previous studies suggest bile acids can exert biological effects through activation of muscarinic M3 receptors [28]. However, we found that although it abolished responses to CCh (data not shown), the muscarinic receptor antagonist, atropine (1 μM), did not prevent the antisecretory effect of DCA (Fig. 5A). We also examined the possibility that DCA exerts antisecretory effects by altering secretagogue‐induced mobilization of the second messengers. However, in cells pretreated with DCA (50 μM, 24 hrs), neither CCh‐stimulated mobilization of intracellular Ca2+ nor FSK‐induced accumulation of cAMP was altered (Fig. 5B and C). These data suggest that DCA exerts its antisecretory effects downstream from agonist‐induced mobilization of prosecretory second messengers.

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