Protons Released During Pancreatic Acinar Cell Secretion Acidify the Lumen and Contribute to Pancreatitis in Mice

doi:10.1053/j.gastro.2010.07.051

Gastroenterology

Volume 139, Issue 5, November 2010, Pages 1711-1720.e5

https://doi.org/10.1053/j.gastro.2010.07.051 Get rights and content

Background & Aims

Secretory granules are acidic; cell secretion will therefore lead to extracellular acidification. We propose that during secretion, protons co-released with proteins from secretory granules of pancreatic acinar cells acidify the restricted extracellular space of the pancreatic lumen to regulate normal physiological and pathophysiological functions in this organ

Methods

Extracellular changes in pH were quantified in real time using 2-photon microscopy analysis of pancreatic tissue fragments from mouse models of acute pancreatitis (mice given physiological concentrations [10 -20 pM] of cholecystokinin or high concentrations of [100 nM] cerulein). The effects of extracellular changes in pH on cell behavior and structures were measured.

Results

With physiological stimulation, secretory granule fusion (exocytosis) caused acidification of the pancreatic lumen. Acidifications specifically affected intracellular calcium responses and accelerated the rate of recovery from agonist-evoked calcium signals. Protons therefore appear to function as negative-feedback, extracellular messengers during coupling of cell stimuli with secretion. At high concentrations of cerulein, large increases in secretory activity were associated with extreme, prolonged acidification of the luminal space. These pathological changes in pH led to disruption of intercellular junctional coupling, measured by movement of occludin and E-cadherin.

Conclusions

By measuring changes in extracellular pH in pancreas of mice, we observed that luminal acidification resulted from exocytosis of zymogen granules from acinar cells. This process is part of normal organ function but ccould contribute to the tissue damage in cases of acute pancreatitis.

Section snippets

Cell Preparation

Mice were humanely killed according to local animal ethics procedures. Isolated mouse pancreatic tissue was prepared by a collagenase digestion method in normal NaCl-rich extracellular solution,²¹ modified to reduce the time in collagenase and limit mechanical trituration. The resulting preparation was composed mainly of pancreatic lobules and fragments (50–100 cells), which were plated onto poly-l-lysine–coated glass coverslips.

Live-Cell Two-Photon Imaging

We used a custom-made, video-rate, 2-photon microscope²¹ with a

Pancreatic Zymogen Granules Are Acidic

A previous report showed zymogen granules are acidic at their genesis, neutralizing as they mature.¹³ To investigate this we labeled live mouse pancreatic tissue fragments with 30 μmol/L Acridine Orange (Sigma, Castle Hill, NSW, Australia) for 20 minutes and imaged acinar cell lobules (Figure 1A). Acridine Orange distributes to acid compartments, dimerizes, and shifts its fluorescence emission from green to red. Our data show red fluorescence consistent with labeling of zymogen granules.

Discussion

The major finding of this work is that protons, co-released during exocytosis, cause significant extracellular acidification. We prove that it is the loss of protons from secretory granules that is the source of luminal acidification. Under physiological stimulation a decrease of up to 1 pH unit is observed that significantly accelerates the recovery phase of intracellular Ca²⁺ spikes. Importantly, we show that with supramaximal cerulein stimulation, a model for acute pancreatitis, the acid

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Pancreatic exocrine secretory processes are challenging to investigate on primary epithelial cells. Pancreatic organoid cultures may help to overcome shortcomings of the current models, however the ion secretory processes in pancreatic organoids—and therefore their physiological relevance or their utility in disease modeling—are not known. To answer these questions, we provide side-by-side comparison of gene expression, morphology, and function of epithelial cells in primary isolated pancreatic ducts and organoids. We used mouse pancreatic ductal fragments for experiments or were grown in Matrigel to obtain organoid cultures. Using PCR analysis we showed that gene expression of ion channels and transporters remarkably overlap in primary ductal cells and organoids. Morphological analysis with scanning electron microscopy revealed that pancreatic organoids form polarized monolayers with brush border on the apical membrane. Whereas the expression and localization of key proteins involved in ductal secretion (cystic fibrosis transmembrane conductance regulator, Na⁺/H⁺ exchanger 1 and electrogenic Na⁺/HCO₃⁻ cotransporter 1) are equivalent to the primary ductal fragments. Measurements of intracellular pH and Cl⁻ levels revealed no significant difference in the activities of the apical Cl⁻/HCO₃⁻ exchange, or in the basolateral Na⁺ dependent HCO₃⁻ uptake. In summary we found that ion transport activities in the mouse pancreatic organoids are remarkably similar to those observed in freshly isolated primary ductal fragments. These results suggest that organoids can be suitable and robust model to study pancreatic ductal epithelial ion transport in health and diseases and facilitate drug development for secretory pancreatic disorders like cystic fibrosis, or chronic pancreatitis.
Early Intra-Acinar Events in Pathogenesis of Pancreatitis
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Premature activation of digestive enzymes in the pancreas has been linked to development of pancreatitis for more than a century. Recent development of novel models to study the role of pathologic enzyme activation has led to advances in our understanding of the mechanisms of pancreatic injury. Colocalization of zymogen and lysosomal fraction occurs early after pancreatitis-causing stimulus. Cathepsin B activates trypsinogen in these colocalized organelles. Active trypsin increases permeability of these organelles resulting in leakage of cathepsin B into the cytosol leading to acinar cell death. Although trypsin-mediated cell death leads to pancreatic injury in early stages of pancreatitis, multiple parallel mechanisms, including activation of inflammatory cascades, endoplasmic reticulum stress, autophagy, and mitochondrial dysfunction in the acinar cells are now recognized to be important in driving the profound systemic inflammatory response and extensive pancreatic injury seen in acute pancreatitis. Chymotrypsin, another acinar protease, has recently been shown be play critical role in clearance of pathologically activated trypsin protecting against pancreatic injury. Mutations in trypsin and other genes thought to be associated with pathologic enzyme activation (such as serine protease inhibitor 1) have been found in familial forms of pancreatitis. Sustained intra-acinar activation of nuclear factor κB pathway seems to be key pathogenic mechanism in chronic pancreatitis. Better understanding of these mechanisms will hopefully allow us to improve treatment strategies in acute and chronic pancreatitis.
Structure-Function Relationships in the Pancreatic Acinar Cell
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The pancreatic acinar cell synthesizes, stores, and secretes the enzymes and enzyme precursors required for the digestion of dietary proteins, carbohydrates, and lipids. To meet the daily needs for digestion, the acinar cell has the highest average rates of protein synthesis in the body. Enzymes are stored in zymogen granules that are localized to the apical region of the cell. Signals generated by food in the intestine reach the acinar cell by neural and hormonal routes stimulate the secretion of enzymes from zymogen granules by exocytosis into the pancreatic duct. The acinar cell has been a model system for examining mechanisms of protein synthesis and export. Thus, nascent digestive enzyme proteins are transported from the endoplasmic reticulum to the Golgi complex, segregated from lysosomal enzymes, and then directed to zymogen granules by vesicular transport in a time-dependent and vectorial manner.
The exocrine pancreas has two major physiologic functions: it supplies the enzymes and enzyme precursors (zymogens) that are needed for digesting dietary lipids, carbohydrates, and proteins and secretes a bicarbonate-rich fluid that neutralizes acidic gastric secretions and thus provides the correct pH for intestinal digestion by pancreatic enzymes. Two major cell types form the exocrine pancreas; acinar cells and duct cells. The chapter focuses on the acinar cell which synthesizes, stores, and secretes digestive enzymes while duct cells secrete chloride and bicarbonate. Reasons to focus on the acinar cell include its critical physiologic function and its history as the model for the scientific studies that described the steps responsible for regulating protein synthesis and export and cell signaling. Thus, after electron microscopy was developed, cell biologists first visualized acinar cell organelles, they often then determined their cellular function by studying this cell. Although this review is largely based on the data from rodent acinar cells, limited data from human acinar cells suggest they will exhibit the same fundamental responses.
Pancreatitis and pancreatic cystosis in Cystic Fibrosis
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In non-CF mouse models, acinar exocytosis of zymogens is associated with acidification of the lumen secondary to impaired neutralization resulting from HCO3– deficiency. Impaired control of luminal pH is believed to contribute to tissue damage and pancreatitis [11]. Furthermore, separate animal and human models have shown that CFTR Cl− channel and anion exchangers are inhibited by trypsin resulting in decreased luminal pH that could promote premature zymogen activation resulting in acute and/or chronic pancreatitis [12].
HCO<inf>3</inf><sup>-</sup> transport through anoctamin/transmembrane protein ANO1/TMEM16A in pancreatic acinar cells regulates luminal pH
2016, Journal of Biological Chemistry
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To test this possibility, we incubated the cells in Lysosensor Yellow/Blue DND-160. The dye enters acidic compartments within the cell (Fig. 3C) that, in acinar cells, are dominated by the zymogen granules, although it would include lysosomes (5). After calibration, the Lysosensor dye showed that the intragranular pH was not different in control cells compared with cells treated with T16Ainh-A01 (Fig. 3, C and D).
The identification of ANO1/TMEM16A as the likely calcium-dependent chloride channel of exocrine glands has led to a more detailed understanding of its biophysical properties. This includes a calcium-dependent change in channel selectivity and evidence that HCO₃⁻ permeability can be significant. Here we use freshly isolated pancreatic acini that preserve the luminal structure to measure intraluminal pH and test the idea that ANO1/TMEM16A contributes to luminal pH balance. Our data show that, under physiologically relevant stimulation with 10 pm cholesystokinin, the luminal acid load that results from the exocytic fusion of zymogen granules is significantly blunted by HCO₃⁻ buffer in comparison with HEPES, and that this is blocked by the specific TMEM16A inhibitor T16inh-A01. Furthermore, in a model of acute pancreatitis, we observed substantive luminal acidification and provide evidence that ANO1/TMEM16A acts to attenuate this pH shift. We conclude that ANO1/TMEM16A is a significant pathway in pancreatic acinar cells for HCO₃⁻ secretion into the lumen.
Spontaneous Pancreatitis Caused by Tissue-Specific Gene Ablation of Hhex in Mice
2015, Cellular and Molecular Gastroenterology and Hepatology
Citation Excerpt :
In addition to peptides, an extensive list of other signaling molecules has been described; among these are Ca2+ and adenosine-5′-triphosphate (ATP), capable of mediating signals on ductal cells via luminal calcium-sensing G-protein coupled and iono-/metabotropic purinergic receptors, respectively.55,56 Moreover, Behrendorff et al57 reported that exaggerated intraluminal acidification caused by proton release from secretory granules of acinar cells in response to supraphysiologic activation directly contributes to pancreatitis via perturbation of tight junctions. Although the function of intraluminal acinar acidification is not entirely clear at this time, it may serve as a negative feedback mechanism to prevent acinar hypersecretion by inhibiting acinar cell endocytosis;58 thus, this report highlights a direct link between paracrine mediators and disease pathogenesis.
Perturbations in pancreatic ductal bicarbonate secretion cause chronic pancreatitis. The physiologic mechanism of ductal secretion is known, but its transcriptional control is not. We determine the role of the transcription factor hematopoietically expressed homeobox protein (Hhex) in ductal secretion and pancreatitis.
We derived mice with pancreas-specific, Cre-mediated Hhex gene ablation to determine the requirement of Hhex in the pancreatic duct in early life and in adult stages. Histologic and immunostaining analyses were used to detect the presence of pathology. Pancreatic primary ductal cells were isolated to discover differentially expressed transcripts upon acute Hhex ablation on a cell autonomous level.
Hhex protein was detected throughout the embryonic and adult ductal trees. Ablation of Hhex in pancreatic progenitors resulted in postnatal ductal ectasia associated with acinar-to-ductal metaplasia, a progressive phenotype that ultimately resulted in chronic pancreatitis. Hhex ablation in adult mice, however, did not cause any detectable pathology. Ductal ectasia in young mice did not result from perturbation of expression of Hnf6, Hnf1β, or the primary cilia genes. RNA-seq analysis of Hhex-ablated pancreatic primary ductal cells showed mRNA levels of the G-protein coupled receptor natriuretic peptide receptor 3 (Npr3), implicated in paracrine signaling, up-regulated by 4.70-fold.
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: Conflicts of interest The authors disclose no conflicts.

: Funding Supported by grants from the Australian Research Council (DP0771481) and a Research Infrastructure Block Grant from The University of Queensland (P.T.).

View full text

Basic—Liver, Pancreas, and Biliary TractProtons Released During Pancreatic Acinar Cell Secretion Acidify the Lumen and Contribute to Pancreatitis in Mice

Background & Aims

Methods

Results

Conclusions

Section snippets

Cell Preparation

Live-Cell Two-Photon Imaging

Pancreatic Zymogen Granules Are Acidic

Discussion

Gastroenterology

Gastroenterology

Cell

J Biol Chem

Archiv Biochem Biophys

J Biol Chem

Neuron

Brain Res

J Biol Chem

Gastroenterology

Influence of exocrine pancreatic insufficiency on the intraluminal pH of the proximal small intestine

Dig Dis Sci

Clinical physiology of acid-base and electrolyte disorders

The pH of the secretory pathway, measurement, determinants and regulation

Physiology (Bethesda)

Ion channels in secretory granules of the pancreas and their role in exocytosis and release of secretory proteins

Am J Physiol

Reconstitution in vitro of the pH-dependent aggregation of pancreatic zymogens en route to the secretory granule: implication of GP-2

Biochem J

Intraorganellar calcium and pH control proinsulin cleavage in the pancreatic β cell via two distinct site-specific endopeptidases

Nature

The condensing vacuole of exocrine cells is more acidic than the mature secretory vesicle

Nature

Basic—Liver, Pancreas, and Biliary Tract
Protons Released During Pancreatic Acinar Cell Secretion Acidify the Lumen and Contribute to Pancreatitis in Mice