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Extracellular ATP protects pancreatic duct epithelial cells from alcohol-induced damage through P2Y1 receptor-cAMP signal pathway

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Abstract

Extracellular adenosine-5′-triphosphate (ATP) regulates cell death and survival of neighboring cells. The detailed effects are diverse depending on cell types and extracellular ATP concentration. We addressed the effect of ATP on ethanol-induced cytotoxicity in epithelial cells, the cell type that experiences the highest concentrations of alcohol. Using pancreatic duct epithelial cells (PDEC), we found that a micromolar range of ATP reverses all intracellular toxicity mechanisms triggered by exceptionally high doses of ethanol and, thus, improves cell viability dramatically. Out of the many purinergic receptors expressed in PDEC, the P2Y1 receptor was identified to mediate the protective effect, based on pharmacological and siRNA assays. Activation of P2Y1 receptors increased intracellular cyclic adenosine monophosphate (cAMP). The protective effect of ATP was mimicked by forskolin and 8-Br-cAMP but inhibited by a protein kinase A (PKA) inhibitor, H-89. Finally, ATP reverted leakiness of PDEC monolayers induced by ethanol and helped to maintain epithelial integrity. We suggest that purinergic receptors reduce extreme alcohol-induced cell damage via the cAMP signal pathway in PDEC and some other types of cells.

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Abbreviations

ADH:

Alcohol dehydrogenase

ALDH:

Acetaldehyde dehydrogenase

FAEE:

Fatty acid ethyl ester

FRET:

Fluorescence resonance energy transfer

GAPDH:

Glyceraldehyde-3-phosphate dehydrogenase

MMP:

Mitochondrial membrane potential

PDEC:

Pancreatic duct epithelial cells

PKA:

Protein kinase A

PKC:

Protein kinase C

P2 receptor:

Purinergic type 2 receptor

PMA:

Phorbol 12-myristate 13-acetate

ROS:

Reactive oxygen species

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Acknowledgments

We thank Mark Moody and Lea M. Miller for technical assistance; Drs. Eamonn J. Dickson, and Toan N. Nguyen for helpful discussion and reading of manuscript: and Drs. Martin Kruse, Oscar Vivas, Haijie Yu, Po-Ni Lai, and Sei-Hum Jang for comments and helpful discussion. This work was supported by National Institutes of Health grant (R01-DK080840 to D.S.K.).

Author contributions

J.B.S., S.R.J., B.H., and D.S.K. developed the concept and designed research; J.B.S. and S.R.J. performed the experiments; J.B.S., S.R.J., and D.S.K. analyzed data; J.B.S., S.R.J., B.H., and D.S.K. interpreted results of experiments; J.B.S. and S.R.J. prepared figures; J.B.S. drafted manuscript; and J.B.S., S.R.J., B.H., and D.S.K. edited manuscript.

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  1. Jong Bae Seo

    Present address: Department of Medicine, Division of Endocrinology and Metabolism, University of California San Diego, La Jolla, CA, 92093, USA

Authors and Affiliations

  1. Department of Physiology and Biophysics, University of Washington, Health Sciences Bldg. Rm. G-424, Seattle, WA, 98195-7290, USA

    Jong Bae Seo, Seung-Ryoung Jung, Bertil Hille & Duk-Su Koh

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  1. Jong Bae Seo
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Correspondence to Duk-Su Koh.

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Seo, J.B., Jung, SR., Hille, B. et al. Extracellular ATP protects pancreatic duct epithelial cells from alcohol-induced damage through P2Y1 receptor-cAMP signal pathway. Cell Biol Toxicol 32, 229–247 (2016). https://doi.org/10.1007/s10565-016-9331-3

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