Limits...
Bile acid effects are mediated by ATP release and purinergic signalling in exocrine pancreatic cells.

Kowal JM, Haanes KA, Christensen NM, Novak I - Cell Commun. Signal (2015)

Bottom Line: Taurine and glycine conjugated forms of CDCA had smaller effects on ATP release in Capan-1 cells.CDCA evokes significant ATP release that can stimulate purinergic receptors, which in turn increase [Ca(2+)]i.We propose that purinergic signalling could be taken into consideration in other cells/organs, and thereby potentially explain some of the multifaceted effects of BAs.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Section for Cell Biology and Physiology, August Krogh Building, University of Copenhagen, Universitetsparken 13, DK-2100, Copenhagen, Denmark. justyna.kowal@bio.ku.dk.

ABSTRACT

Background: In many cells, bile acids (BAs) have a multitude of effects, some of which may be mediated by specific receptors such the TGR5 or FXR receptors. In pancreas systemic BAs, as well as intra-ductal BAs from bile reflux, can affect pancreatic secretion. Extracellular ATP and purinergic signalling are other important regulators of similar secretory mechanisms in pancreas. The aim of our study was to elucidate whether there is interplay between ATP and BA signalling.

Results: Here we show that CDCA (chenodeoxycholic acid) caused fast and concentration-dependent ATP release from acini (AR42J) and duct cells (Capan-1). Taurine and glycine conjugated forms of CDCA had smaller effects on ATP release in Capan-1 cells. In duct monolayers, CDCA stimulated ATP release mainly from the luminal membrane; the releasing mechanisms involved both vesicular and non-vesicular secretion pathways. Duct cells were not depleted of intracellular ATP with CDCA, but acinar cells lost some ATP, as detected by several methods including ATP sensor AT1.03(YEMK). In duct cells, CDCA caused reversible increase in the intracellular Ca(2+) concentration [Ca(2 +)]i, which could be significantly inhibited by antagonists of purinergic receptors. The TGR5 receptor, expressed on the luminal side of pancreatic ducts, was not involved in ATP release and Ca(2+) signals, but could stimulate Na(+)/Ca(2+) exchange in some conditions.

Conclusions: CDCA evokes significant ATP release that can stimulate purinergic receptors, which in turn increase [Ca(2+)]i. The TGR5 receptor is not involved in these processes but can play a protective role at high intracellular Ca(2+) conditions. We propose that purinergic signalling could be taken into consideration in other cells/organs, and thereby potentially explain some of the multifaceted effects of BAs.

No MeSH data available.


Related in: MedlinePlus

The acute and chronic effects of CDCA on intracellular ATP concentration in AR42J and Capan-1 cells. a Original trace for luminometric measurements of ATP based on Capan-1 measurement. Baseline values were recorded every 20 s for 2 min. Cells were then stimulated with 0.3 mM (Capan-1) or 0.5 mM (AR42J) with CDCA or with vehicle. Stimulated ATP release was recorded every 1 sec for 1 min directly after addition of CDCA, or after 12 min or 24 h of incubation. Finally, cells were permeabilized with digitonin (50 μM), added automatically using pump to release remaining ATP. Panel b and c show the values of released and remaining ATP in Capan-1 and AR42J cells, respectively. These values were calculated per cell after 1, 12 min and 24 h (n = 8, 7, 10 and n = 5, 5, 10) of incubation with CDCA. Data are shown as mean values ± SEM; ***= P < 0.001, N.S. - not significant
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4459444&req=5

Fig5: The acute and chronic effects of CDCA on intracellular ATP concentration in AR42J and Capan-1 cells. a Original trace for luminometric measurements of ATP based on Capan-1 measurement. Baseline values were recorded every 20 s for 2 min. Cells were then stimulated with 0.3 mM (Capan-1) or 0.5 mM (AR42J) with CDCA or with vehicle. Stimulated ATP release was recorded every 1 sec for 1 min directly after addition of CDCA, or after 12 min or 24 h of incubation. Finally, cells were permeabilized with digitonin (50 μM), added automatically using pump to release remaining ATP. Panel b and c show the values of released and remaining ATP in Capan-1 and AR42J cells, respectively. These values were calculated per cell after 1, 12 min and 24 h (n = 8, 7, 10 and n = 5, 5, 10) of incubation with CDCA. Data are shown as mean values ± SEM; ***= P < 0.001, N.S. - not significant

Mentions: The above ATPi measurement methods are dynamic, but difficult to calibrate in acinar cells. Therefore, we also used luciferin/luciferase assay to determine ATPi. Cell membranes were permeabilized with digitonin after CDCA treatment and intracellular ATPi was quantified (Fig. 5a). ATPi concentrations were measured at different time points (1 and 12 min), correlating with the peak and plateau for MgGreen (Fig. 3). Additionally, the long-term effect was also determined by incubating the cells with CDCA for 24 h. After stimulation with 0.3 mM CDCA, Capan-1 released 68 ± 26 nM ATP to extracellular medium, which corresponds to a calculated decrease of 0.08 ± 0.03 mM ATPi in a cell (n = 8). Fig. 5b shows that after incubation of duct cells for 1 min and 12 min, the total content of remaining ATPi was not significantly changed and remained at 2.47 ± 0.32 mM calculated per cell; (n = 8) and 2.22 ± 0.5 mM (n = 7) compared to their respect controls of 2.72 ± 0.38 mM and 2.46 ± 0.45 mM (n = 7). Furthermore, the long-term exposure of Capan-1 to 0.3 mM CDCA did not cause significant changes in ATPi concentrations, i.e., 2.08 ± 0.24 mM compared to control 1.85 ± 0.18 mM (n = 10). For AR42J cells, stimulation with 0.5 mM CDCA (Fig. 5c) caused ATP release of 358 ± 52 nM to extracellular medium, which corresponds to a calculated decrease of 0.68 ± 0.1 mM in the cell (n = 5). Similar to the Capan-1 cells, we did not observe significant changes in remaining ATPi concentrations after 1 min (1.36 ± 0.1 mM; n = 5). However, after 12 min there was a tendency, though not significant, of lower intracellular ATP levels (0.68 ± 0.06 mM; n = 5) with CDCA compared to their respective controls (1.12 ± 0.01 mM and 0.97 ± 0.13 mM; n = 5). Furthermore, after 24 h incubation of AR42J with 0.5 mM CDCA, there was a significant decrease of ATPi to 0.66 ± 0.06 mM compared to the control 1.3 ± 0.08 mM (n = 10).Fig. 5


Bile acid effects are mediated by ATP release and purinergic signalling in exocrine pancreatic cells.

Kowal JM, Haanes KA, Christensen NM, Novak I - Cell Commun. Signal (2015)

The acute and chronic effects of CDCA on intracellular ATP concentration in AR42J and Capan-1 cells. a Original trace for luminometric measurements of ATP based on Capan-1 measurement. Baseline values were recorded every 20 s for 2 min. Cells were then stimulated with 0.3 mM (Capan-1) or 0.5 mM (AR42J) with CDCA or with vehicle. Stimulated ATP release was recorded every 1 sec for 1 min directly after addition of CDCA, or after 12 min or 24 h of incubation. Finally, cells were permeabilized with digitonin (50 μM), added automatically using pump to release remaining ATP. Panel b and c show the values of released and remaining ATP in Capan-1 and AR42J cells, respectively. These values were calculated per cell after 1, 12 min and 24 h (n = 8, 7, 10 and n = 5, 5, 10) of incubation with CDCA. Data are shown as mean values ± SEM; ***= P < 0.001, N.S. - not significant
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4459444&req=5

Fig5: The acute and chronic effects of CDCA on intracellular ATP concentration in AR42J and Capan-1 cells. a Original trace for luminometric measurements of ATP based on Capan-1 measurement. Baseline values were recorded every 20 s for 2 min. Cells were then stimulated with 0.3 mM (Capan-1) or 0.5 mM (AR42J) with CDCA or with vehicle. Stimulated ATP release was recorded every 1 sec for 1 min directly after addition of CDCA, or after 12 min or 24 h of incubation. Finally, cells were permeabilized with digitonin (50 μM), added automatically using pump to release remaining ATP. Panel b and c show the values of released and remaining ATP in Capan-1 and AR42J cells, respectively. These values were calculated per cell after 1, 12 min and 24 h (n = 8, 7, 10 and n = 5, 5, 10) of incubation with CDCA. Data are shown as mean values ± SEM; ***= P < 0.001, N.S. - not significant
Mentions: The above ATPi measurement methods are dynamic, but difficult to calibrate in acinar cells. Therefore, we also used luciferin/luciferase assay to determine ATPi. Cell membranes were permeabilized with digitonin after CDCA treatment and intracellular ATPi was quantified (Fig. 5a). ATPi concentrations were measured at different time points (1 and 12 min), correlating with the peak and plateau for MgGreen (Fig. 3). Additionally, the long-term effect was also determined by incubating the cells with CDCA for 24 h. After stimulation with 0.3 mM CDCA, Capan-1 released 68 ± 26 nM ATP to extracellular medium, which corresponds to a calculated decrease of 0.08 ± 0.03 mM ATPi in a cell (n = 8). Fig. 5b shows that after incubation of duct cells for 1 min and 12 min, the total content of remaining ATPi was not significantly changed and remained at 2.47 ± 0.32 mM calculated per cell; (n = 8) and 2.22 ± 0.5 mM (n = 7) compared to their respect controls of 2.72 ± 0.38 mM and 2.46 ± 0.45 mM (n = 7). Furthermore, the long-term exposure of Capan-1 to 0.3 mM CDCA did not cause significant changes in ATPi concentrations, i.e., 2.08 ± 0.24 mM compared to control 1.85 ± 0.18 mM (n = 10). For AR42J cells, stimulation with 0.5 mM CDCA (Fig. 5c) caused ATP release of 358 ± 52 nM to extracellular medium, which corresponds to a calculated decrease of 0.68 ± 0.1 mM in the cell (n = 5). Similar to the Capan-1 cells, we did not observe significant changes in remaining ATPi concentrations after 1 min (1.36 ± 0.1 mM; n = 5). However, after 12 min there was a tendency, though not significant, of lower intracellular ATP levels (0.68 ± 0.06 mM; n = 5) with CDCA compared to their respective controls (1.12 ± 0.01 mM and 0.97 ± 0.13 mM; n = 5). Furthermore, after 24 h incubation of AR42J with 0.5 mM CDCA, there was a significant decrease of ATPi to 0.66 ± 0.06 mM compared to the control 1.3 ± 0.08 mM (n = 10).Fig. 5

Bottom Line: Taurine and glycine conjugated forms of CDCA had smaller effects on ATP release in Capan-1 cells.CDCA evokes significant ATP release that can stimulate purinergic receptors, which in turn increase [Ca(2+)]i.We propose that purinergic signalling could be taken into consideration in other cells/organs, and thereby potentially explain some of the multifaceted effects of BAs.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Section for Cell Biology and Physiology, August Krogh Building, University of Copenhagen, Universitetsparken 13, DK-2100, Copenhagen, Denmark. justyna.kowal@bio.ku.dk.

ABSTRACT

Background: In many cells, bile acids (BAs) have a multitude of effects, some of which may be mediated by specific receptors such the TGR5 or FXR receptors. In pancreas systemic BAs, as well as intra-ductal BAs from bile reflux, can affect pancreatic secretion. Extracellular ATP and purinergic signalling are other important regulators of similar secretory mechanisms in pancreas. The aim of our study was to elucidate whether there is interplay between ATP and BA signalling.

Results: Here we show that CDCA (chenodeoxycholic acid) caused fast and concentration-dependent ATP release from acini (AR42J) and duct cells (Capan-1). Taurine and glycine conjugated forms of CDCA had smaller effects on ATP release in Capan-1 cells. In duct monolayers, CDCA stimulated ATP release mainly from the luminal membrane; the releasing mechanisms involved both vesicular and non-vesicular secretion pathways. Duct cells were not depleted of intracellular ATP with CDCA, but acinar cells lost some ATP, as detected by several methods including ATP sensor AT1.03(YEMK). In duct cells, CDCA caused reversible increase in the intracellular Ca(2+) concentration [Ca(2 +)]i, which could be significantly inhibited by antagonists of purinergic receptors. The TGR5 receptor, expressed on the luminal side of pancreatic ducts, was not involved in ATP release and Ca(2+) signals, but could stimulate Na(+)/Ca(2+) exchange in some conditions.

Conclusions: CDCA evokes significant ATP release that can stimulate purinergic receptors, which in turn increase [Ca(2+)]i. The TGR5 receptor is not involved in these processes but can play a protective role at high intracellular Ca(2+) conditions. We propose that purinergic signalling could be taken into consideration in other cells/organs, and thereby potentially explain some of the multifaceted effects of BAs.

No MeSH data available.


Related in: MedlinePlus