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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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Chronic exposure to low concentrations of DCA inhibits Cl− secretion across T84 cells. (A) Monolayers of T84 cells were exposed to bilateral DCA (50 μM) for 24 hrs and subsequent Isc responses to CCh (100 μM) were measured. (B) T84 cell monolayers were exposed to increasing concentrations of DCA for 24 hrs after which Isc responses to CCh (100 μM) were measured (n= 5). (C) Cells were exposed to DCA (50 μM) for varying periods after which Isc responses to CCh (100 μM; n= 4) or FSK (10 μM; n= 4) were measured. (D) Monolayers of T84 cells were exposed to increasing concentrations of DCA for 24 hrs after which TER was measured (n= 5). (E) Aliquots of supernatants from cells exposed to DCA at varying concentrations for 24 hrs were analysed for LDH. Data are expressed as sample absorbance at 490 nM (n= 3). *P < 0.05; **P < 0.01; ***P < 0.001.
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f2: Chronic exposure to low concentrations of DCA inhibits Cl− secretion across T84 cells. (A) Monolayers of T84 cells were exposed to bilateral DCA (50 μM) for 24 hrs and subsequent Isc responses to CCh (100 μM) were measured. (B) T84 cell monolayers were exposed to increasing concentrations of DCA for 24 hrs after which Isc responses to CCh (100 μM) were measured (n= 5). (C) Cells were exposed to DCA (50 μM) for varying periods after which Isc responses to CCh (100 μM; n= 4) or FSK (10 μM; n= 4) were measured. (D) Monolayers of T84 cells were exposed to increasing concentrations of DCA for 24 hrs after which TER was measured (n= 5). (E) Aliquots of supernatants from cells exposed to DCA at varying concentrations for 24 hrs were analysed for LDH. Data are expressed as sample absorbance at 490 nM (n= 3). *P < 0.05; **P < 0.01; ***P < 0.001.

Mentions: We first analysed acute effects of DCA on Cl− secretion across voltage‐clamped T84 cells. Bilateral addition of 1 mM DCA stimulated a transient increase in Isc that rapidly returned towards baseline over the course of 20 min (Fig. 1). The threshold concentration for this prosecretory effect of DCA was greater than 200 μM with an EC50 of 488.8 ± 45.6 μM (Fig. 1B). At concentrations greater than 500 μM, DCA reduced TER (EC50= 982.5 ± 194.2 μM; Fig. 1C), whereas at concentrations greater than 1 mM, DCA induced cell lysis and lactate dehydrogenase (LDH) release (EC50= 1.99 ± 9.5 mM; Fig. 1D). Figure 1E shows that within 1 hr after addition, DCA (500 μM) almost abolished TER across T84 monolayers. However, if the bile acids was washed from the cells after 40 min, there was a gradual restoration of TER. Twenty‐four hours after DCA exposure, the TER across monolayers from which DCA was removed had returned to 54.9 ± 7.3% of that in control cells although continued exposure to the bile acid obliterated TER (Figs. 1E and 2E). Thus, upon removal of DCA the barrier function of colonic epithelial cells can, at least partially, be restored.


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)

Chronic exposure to low concentrations of DCA inhibits Cl− secretion across T84 cells. (A) Monolayers of T84 cells were exposed to bilateral DCA (50 μM) for 24 hrs and subsequent Isc responses to CCh (100 μM) were measured. (B) T84 cell monolayers were exposed to increasing concentrations of DCA for 24 hrs after which Isc responses to CCh (100 μM) were measured (n= 5). (C) Cells were exposed to DCA (50 μM) for varying periods after which Isc responses to CCh (100 μM; n= 4) or FSK (10 μM; n= 4) were measured. (D) Monolayers of T84 cells were exposed to increasing concentrations of DCA for 24 hrs after which TER was measured (n= 5). (E) Aliquots of supernatants from cells exposed to DCA at varying concentrations for 24 hrs were analysed for LDH. Data are expressed as sample absorbance at 490 nM (n= 3). *P < 0.05; **P < 0.01; ***P < 0.001.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4940785&req=5

f2: Chronic exposure to low concentrations of DCA inhibits Cl− secretion across T84 cells. (A) Monolayers of T84 cells were exposed to bilateral DCA (50 μM) for 24 hrs and subsequent Isc responses to CCh (100 μM) were measured. (B) T84 cell monolayers were exposed to increasing concentrations of DCA for 24 hrs after which Isc responses to CCh (100 μM) were measured (n= 5). (C) Cells were exposed to DCA (50 μM) for varying periods after which Isc responses to CCh (100 μM; n= 4) or FSK (10 μM; n= 4) were measured. (D) Monolayers of T84 cells were exposed to increasing concentrations of DCA for 24 hrs after which TER was measured (n= 5). (E) Aliquots of supernatants from cells exposed to DCA at varying concentrations for 24 hrs were analysed for LDH. Data are expressed as sample absorbance at 490 nM (n= 3). *P < 0.05; **P < 0.01; ***P < 0.001.
Mentions: We first analysed acute effects of DCA on Cl− secretion across voltage‐clamped T84 cells. Bilateral addition of 1 mM DCA stimulated a transient increase in Isc that rapidly returned towards baseline over the course of 20 min (Fig. 1). The threshold concentration for this prosecretory effect of DCA was greater than 200 μM with an EC50 of 488.8 ± 45.6 μM (Fig. 1B). At concentrations greater than 500 μM, DCA reduced TER (EC50= 982.5 ± 194.2 μM; Fig. 1C), whereas at concentrations greater than 1 mM, DCA induced cell lysis and lactate dehydrogenase (LDH) release (EC50= 1.99 ± 9.5 mM; Fig. 1D). Figure 1E shows that within 1 hr after addition, DCA (500 μM) almost abolished TER across T84 monolayers. However, if the bile acids was washed from the cells after 40 min, there was a gradual restoration of TER. Twenty‐four hours after DCA exposure, the TER across monolayers from which DCA was removed had returned to 54.9 ± 7.3% of that in control cells although continued exposure to the bile acid obliterated TER (Figs. 1E and 2E). Thus, upon removal of DCA the barrier function of colonic epithelial cells can, at least partially, be restored.

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