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TMEM16A may regulate pH in the pancreatic acinar lumen

Y. Han and P. Thorn, School of Biomedical Sciences, University of Queensland, St Lucia, QLD 4072, Australia.

Acidosis in the pancreatic fluid is an important component and likely a cause of pancreatitis and cystic fibrosis-related pancreatic problems, such as pancreatic insufficiency and pancreatitis. In vitro studies showed that the combination of protons released from acidic zymogen granules fused with the apical membrane of acinar cells during exocytosis and bicarbonate secreted from ductal cells controls pH in the pancreatic fluid (Behrendorff et al., 2010; Steward et al., 2005). Calcium activated chloride current (CaCC) in the apical membrane of the pancreatic acinar cell has been reported long ago (Park et al., 2001). Recent study confirmed that TMEM16A accounts for the CaCC in the pancreatic acinar cell (Ousingsawat et al., 2009). The permeability of TMEM16A to bicarbonate could be substantial at high free cytosolic calcium concentration (0.4 - 3μM) (Jung et al., 2013); cytosolic calcium in the apical area easily reaches such concentration during physiological stimulation (Ito et al., 1997). This project is aimed to prove that TMEM16A in the apical membrane of the acinar cell accounts for bicarbonate secretion into the pancreatic juice and therefore may lead a new way to restore pH in the acidified pancreatic fluid during pancreatic diseases.

Methods. Pancreatic acini were isolated from anaethetised c57bl/6j black mice following a previously described protocol (Behrendorff et al., 2010). Video-rate 2-photon microscope with a 60× oil immersion objective was used to image exocytotic events and the acinar lumen. To image acinar lumenal pH changes we used HPTS (800 μmol/L) excited at 950 nm and fluorescence detected at 420 - 520 nm.

Results. Acinar lumenal pH drop and recovery in areas near exocytotic events induced by physiological stimulation were observed in 2mM HEPES buffered solution (pH = 7.4) as previously described (Behrendorff et al., 2010). In 2mM HCO3 buffered solution (pH = 7.4), a similar pH drop but faster pH recovery was observed in the acinar lumen. Inhibition of TMEM16A by a selective inhibitor, T16A(inh)-A01, slowed down the pH recovery significantly in 2mM HCO3 buffered solution.

Conclusion. Bicarbonate secretion via TMEM16A helped pH recovery from an acid load in the acinar lumen caused by bulk proton release from exocytosis events.

Behrendorff N, Floetenmeyer M, Schwiening C & Thorn P. (2010) Gastroenterology, 139: 1711-20.

Steward MC, Ishiguro H & Case RM. (2005) Annual Review of Physiology, 67: 377-409.

Park MK, Lomax RB, Tepikin AV & Petersen OH. (2001) Proceedings of the National Academy of Sciences of the United States of America, 98: 10948-53.

Ousingsawat J, Martins JR, Schreiber R, Rock JR, Harfe BD & Kunzelmann K. (2009) The Journal of Biological Chemistry, 284: 28698-703.

Jung J, Nam JH, Park HW, Oh U, Yoon JH & Lee MG. (2013) Proceedings of the National Academy of Sciences of the United States of America 110: 360-5.

Ito K, Miyashita Y & Kasai H. (1997) The EMBO Journal, 16: 242-51.