Pancreatic Bicarbonate Secretion Involves Two Proton Pumps

Ivana Novak,Jing Wang,Katrine L Henriksen,Kristian A Haanes,Simon Krabbe,Roland Nitschke,Susanne E Hede

doi:10.1074/jbc.m110.136382

Ivana Novak, Jing Wang + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m110.136382

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Abstract

Pancreas secretes fluid rich in digestive enzymes and bicarbonate. The alkaline secretion is important in buffering of acid chyme entering duodenum and for activation of enzymes. This secretion is formed in pancreatic ducts, and studies to date show that plasma membranes of duct epithelium express H(+)/HCO(3)(-) transporters, which depend on gradients created by the Na(+)/K(+)-ATPase. However, the model cannot fully account for high-bicarbonate concentrations, and other active transporters, i.e. pumps, have not been explored. Here we show that pancreatic ducts express functional gastric and non-gastric H(+)-K(+)-ATPases. We measured intracellular pH and secretion in small ducts isolated from rat pancreas and showed their sensitivity to H(+)-K(+) pump inhibitors and ion substitutions. Gastric and non-gastric H(+)-K(+) pumps were demonstrated on RNA and protein levels, and pumps were localized to the plasma membranes of pancreatic ducts. Quantitative analysis of H(+)/HCO(3)(-) and fluid transport shows that the H(+)-K(+) pumps can contribute to pancreatic secretion in several species. Our results call for revision of the bicarbonate transport physiology in pancreas, and most likely other epithelia. Furthermore, because pancreatic ducts play a central role in several pancreatic diseases, it is of high relevance to understand the role of H(+)-K(+) pumps in pathophysiology.

Highlights

HCO3Ϫ from serosa to lumen and/or by Hϩ transport in the opposite direction
We used the smallest pancreatic ducts (Fig. 1), which are rich in carbonic anhydrase, CFTR and aquaporins [25,26,27,28], and considered the most likely site of HCO3Ϫ transport
We have shown that rat pancreatic ducts express gastric and non-gastric Hϩ-Kϩ pumps

Summary

EXPERIMENTAL PROCEDURES

Solutions and Chemicals—Control bathing solutions containing bicarbonate had the following composition (in mmol/ liter): Naϩ 145, ClϪ 125, HCO3Ϫ 25, Kϩ 4, Ca2ϩ 1.5, Mg2ϩ 1, phosphate 2, glucose 5; they were equilibrated with 5% CO2 in O2; pH was 7.4. Ducts were placed in an experimental chamber on an inverted microscope and used for pHi measurements or for secretion studies. PHi Measurements—Detailed methods for pHi measurement in pancreatic ducts are described elsewhere [21]. Pancreatic ducts were loaded with BCECF/AM (2 ␮M), for 20 –30 min; thereafter they were kept at 37 °C in perfused experimental chamber. Both the luminal and the basolateral sides were accessible to the bathing solution. RT-PCR and Western Blot Analysis—RNA from whole pancreatic tissue, pancreatic ducts, colon, and stomach was isolated using TRIzol reagent (Invitrogen), and method was optimized similar to published protocols [22].

RESULTS

Primer sets

DISCUSSION