ATP release, generation and hydrolysis in exocrine pancreatic duct cells
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Extracellular adenosine triphosphate (ATP) regulates pancreatic duct function via P2Y and P2X receptors. It is well known that ATP is released from upstream pancreatic acinar cells. The ATP homeostasis in pancreatic ducts, which secrete bicarbonate-rich fluid, has not yet been examined. First, our aim was to reveal whether pancreatic duct cells release ATP locally and whether they enzymatically modify extracellular nucleotides/sides. Second, we wished to explore which physiological and pathophysiological factors may be important in these processes. Using a human pancreatic duct cell line, Capan-1, and online luminescence measurement, we detected fast ATP release in response to pH changes, bile acid, mechanical stress and hypo-osmotic stress. ATP release following hypo-osmotic stress was sensitive to drugs affecting exocytosis, pannexin-1, connexins, maxi-anion channels and transient receptor potential cation channel subfamily V member 4 (TRPV4) channels, and corresponding transcripts were expressed in duct cells. Direct stimulation of intracellular Ca2+ and cAMP signalling and ethanol application had negligible effects on ATP release. The released ATP was sequentially dephosphorylated through ecto-nucleoside triphosphate diphosphohydrolase (NTPDase2) and ecto-5′-nucleotidase/CD73 reactions, with respective generation of adenosine diphosphate (ADP) and adenosine and their maintenance in the extracellular medium at basal levels. In addition, Capan-1 cells express counteracting adenylate kinase (AK1) and nucleoside diphosphate kinase (NDPK) enzymes (NME1, 2), which contribute to metabolism and regeneration of extracellular ATP and other nucleotides (ADP, uridine diphosphate (UDP) and uridine triphosphate (UTP)). In conclusion, we illustrate a complex regulation of extracellular purine homeostasis in a pancreatic duct cell model involving: ATP release by several mechanisms and subsequent nucleotide breakdown and ATP regeneration via counteracting nucleotide-inactivating and nucleotide-phosphorylating ecto-enzymes. We suggest that extracellular ATP homeostasis in pancreatic ducts may be important in pancreas physiology and potentially in pancreas pathophysiology.
KeywordsPannexin CD39 Ecto-nucleotidase Adenylate kinase UTP VNUT Connexin Pancreatitis
We appreciate assistance of M. Zuccarini in autoradiographic assays and opportunity to use some of the facilities of S. Jalkanen’s laboratory. We are grateful to Prof. J. Hanrahan for providing us with the CFTR plasmid, Prof. Jean Sevigny for providing anti-NTPDase2 antibody and Prof. P. A. Pedersen and D. Sørensen for use of their facilities. The technical assistance of P. Roshof is greatly appreciated. Imaging experiments were done the Center for Advanced Bioimaging (CAB), University of Copenhagen, Denmark.
JMK performed all experiments and analysis on ATP release and enzyme activity. Enzyme assays were performed in collaboration with GY. IN performed imaging studies. The project was planned by IN, JMK and GY. The manuscript was written jointly by JMK and IN. All authors were involved in critically revising and approved the final version of the manuscript.
Conflict of interest
The authors declare that they have no competing interests.
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