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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

Effect of the TGR5 ligands on [Ca2+]i transients in duct epithelia. Capan-1 cells were incubated with nominal 0 mM Ca2+ buffer (a, b) and thapsigargin (1 μM) to deplete intracellular Ca2+ stores. Thereafter, cells were gently perfused with physiological buffer to refill intracellular Ca2+ stores and after the fluorescence was relatively stable, solutions were changed to GPBAR-A (30 μM), CDCA (0.3 mM), or control. c, d The contribution of the sodium-calcium exchanger (NCX) was tested. Cells were perfused with 5 mM Na+ buffer which increased [Ca2+]i and this response was potentiated in the presence of GPBAR-A (30 μM). b, d Summary of data given as mean values ± SEM of 7–15 cells per each independent experiment (n). *= P < 0.05, ***= P < 0.001, N.S = not significant
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Fig9: Effect of the TGR5 ligands on [Ca2+]i transients in duct epithelia. Capan-1 cells were incubated with nominal 0 mM Ca2+ buffer (a, b) and thapsigargin (1 μM) to deplete intracellular Ca2+ stores. Thereafter, cells were gently perfused with physiological buffer to refill intracellular Ca2+ stores and after the fluorescence was relatively stable, solutions were changed to GPBAR-A (30 μM), CDCA (0.3 mM), or control. c, d The contribution of the sodium-calcium exchanger (NCX) was tested. Cells were perfused with 5 mM Na+ buffer which increased [Ca2+]i and this response was potentiated in the presence of GPBAR-A (30 μM). b, d Summary of data given as mean values ± SEM of 7–15 cells per each independent experiment (n). *= P < 0.05, ***= P < 0.001, N.S = not significant

Mentions: Above we observed that CDCA appeared to protect Capan-1 cells from thapsigargin effects (Fig. 7d, f). This could occur if, for example, CDCA stimulated SERCA (or protected the pumps from thapsigargin) or if CDCA stimulated alternative Ca2+ efflux pathways. Our hypothesis was that the TGR5 receptor could be involved in these effects and we tested this in the following experiments. First, intracellular Ca2+ stores were emptied by thapsigargin in low Ca2+ medium (Fig. 9a). Thereafter, extracellular Ca2+ was reintroduced, and since SERCA pumps were inhibited, [Ca2+]i increased to very high levels in control cells. Notably, perfusion of the cells with CDCA caused a fast and marked reduction in [Ca2+]i from 1253 ± 117 to 244 ± 28 nM (Fig. 9 a, b, n = 3). A similar response was observed with perfusion of GPBAR-A (Fig. 9a-b), which lowered the [Ca2+]i from 1249 ± 69 nM to 647 ± 71 nM (n = 5).Fig. 9


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)

Effect of the TGR5 ligands on [Ca2+]i transients in duct epithelia. Capan-1 cells were incubated with nominal 0 mM Ca2+ buffer (a, b) and thapsigargin (1 μM) to deplete intracellular Ca2+ stores. Thereafter, cells were gently perfused with physiological buffer to refill intracellular Ca2+ stores and after the fluorescence was relatively stable, solutions were changed to GPBAR-A (30 μM), CDCA (0.3 mM), or control. c, d The contribution of the sodium-calcium exchanger (NCX) was tested. Cells were perfused with 5 mM Na+ buffer which increased [Ca2+]i and this response was potentiated in the presence of GPBAR-A (30 μM). b, d Summary of data given as mean values ± SEM of 7–15 cells per each independent experiment (n). *= P < 0.05, ***= 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

Fig9: Effect of the TGR5 ligands on [Ca2+]i transients in duct epithelia. Capan-1 cells were incubated with nominal 0 mM Ca2+ buffer (a, b) and thapsigargin (1 μM) to deplete intracellular Ca2+ stores. Thereafter, cells were gently perfused with physiological buffer to refill intracellular Ca2+ stores and after the fluorescence was relatively stable, solutions were changed to GPBAR-A (30 μM), CDCA (0.3 mM), or control. c, d The contribution of the sodium-calcium exchanger (NCX) was tested. Cells were perfused with 5 mM Na+ buffer which increased [Ca2+]i and this response was potentiated in the presence of GPBAR-A (30 μM). b, d Summary of data given as mean values ± SEM of 7–15 cells per each independent experiment (n). *= P < 0.05, ***= P < 0.001, N.S = not significant
Mentions: Above we observed that CDCA appeared to protect Capan-1 cells from thapsigargin effects (Fig. 7d, f). This could occur if, for example, CDCA stimulated SERCA (or protected the pumps from thapsigargin) or if CDCA stimulated alternative Ca2+ efflux pathways. Our hypothesis was that the TGR5 receptor could be involved in these effects and we tested this in the following experiments. First, intracellular Ca2+ stores were emptied by thapsigargin in low Ca2+ medium (Fig. 9a). Thereafter, extracellular Ca2+ was reintroduced, and since SERCA pumps were inhibited, [Ca2+]i increased to very high levels in control cells. Notably, perfusion of the cells with CDCA caused a fast and marked reduction in [Ca2+]i from 1253 ± 117 to 244 ± 28 nM (Fig. 9 a, b, n = 3). A similar response was observed with perfusion of GPBAR-A (Fig. 9a-b), which lowered the [Ca2+]i from 1249 ± 69 nM to 647 ± 71 nM (n = 5).Fig. 9

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