Mouse Pancreatic Duct Research Articles

222-LB: Canonical Wnt Signaling Regulates Beta-Cell Neogenesis from Pancreatic Duct Cells

Diabetes results from loss or insufficient numbers of insulin producing beta cells. Thus, restoration of beta cell mass can offer a lasting cure for diabetes. Physiological conditions such as obesity and pregnancy are known to induce compensatory increase in beta cell mass; suggesting the presence of an endogenous mechanism for beta cell regeneration. Significant progress has been made in understanding the regeneration of beta-cells through proliferation of existing beta-cells. In addition to proliferation of existing beta-cells, there is mounting evidence, from both animal models and human tissue, indicating that adult pancreatic duct cells can regenerate endocrine cells, including insulin producing cells. However, the molecular mechanisms governing the regeneration of pancreatic endocrine cells, especially beta cell from pancreatic duct cells remain to be defined. We have found that adult pancreatic duct cells are active in both Notch and Wnt/beta-catenin signaling. Transcriptional profiling revealed Hes1 as the predominant Notch target gene in adult mouse pancreatic duct cells. Using lineage-tracing mouse models, we found that duct-specific deletion of Hes1 in vivo resulted in the conversion of pancreatic duct cells into predominantly somatostatin+ cells. Conversely, pancreatic duct-specific deletion of the canonical Wnt signaling effector, beta-catenin, in vivo resulted in downregulation of Hes1 expression, and the conversion of pancreatic duct cells into predominantly insulin producing cells. Taken together our results demonstrate that Notch and Wnt signaling maintain the adult pancreatic duct state, with duct-derived beta-cell neogenesis requiring the loss of Wnt/beta-catenin signaling. Disclosure S. Afelik: None. Z. Yu: None.

A Method for Identifying Mouse Pancreatic Ducts.

Proper identification of pancreatic ducts is a major challenge for researchers performing partial duct ligation (PDL), because pancreatic ducts, which are covered with acinar cells, are translucent and thin. Although damage to pancreatic ducts may activate quiescent ductal stem cells, which may allow further investigation into ductal stem cells for therapeutic use, there is a lack of effective techniques to visualize pancreatic ducts. In this study, we report a new method for identifying pancreatic ducts. First, we aimed to visualize pancreatic ducts using black, waterproof fountain pen ink. We injected the ink into pancreatic ducts through the bile duct. The flow of ink was observed in pancreatic ducts, revealing their precise architecture. Next, to visualize pancreatic ducts in live animals, we injected fluorescein-labeled bile acid, cholyl-lysyl-fluorescein into the mouse tail vein. The fluorescent probe clearly marked not only the bile duct but also pancreatic ducts when observed with a fluorescent microscope. To confirm whether the pancreatic duct labeling was successful, we performed PDL on Neurogenin3 (Ngn3)-GFP transgenic mice. As a result, acinar tissue is lost. PDL tail pancreas becomes translucent almost completely devoid of acinar cells. Furthermore, strong activation of Ngn3 expression was observed in the ligated part of the adult mouse pancreas at 7 days after PDL.

303. Deletion of Pdx1 in Mouse Pancreatic Ducts by CRISPR-Cas9 Mediated Gene Editing

Introduction: The homeodomain transcription factor Pdx1 is crucial for pancreas formation. Pdx1-expressing cells are first observed at embryonic day 8.5 (E8.5), prior even to the earliest indication of morphogenesis, in endodermal cells designated to give rise to the pancreas. In adult mouse, Pdx1 protein is transiently expressed when pancreas is injured, such as Langerhans islets damage in alloxan induced diabetic mice, implying Pdx1 may be necessary for the neogenetic formation of β-cells from mature ducts. In order to study the role of pdx1 in the pancreatic duct for pancreatic regeneration, most investigators use the Cre-lox system to generate duct-specific Pdx1 deletions in mice. This method is able to delete Pdx1specifically from tissue or cells with or without a tamoxifen-inducible (cre-ERT2) system; however, several caveats and limitations exist with tamoxifen-induced depletion. In addition, the cre-ERT2 system may be leaky, resulting in constitutive rather than inducible activation in some cells. To overcome these flaws from the Cre-ERT2/lox system, we decided to use a novel CRISPR-Cas9 gene editing technique to delete pdx1 in pancreatic ducts. Methods: We previously used a mouse short Sox9 promoter (468 bp) to generate a recombinant adeno-associated virus (AAV) carrying a reporter GFP (AAV6-Sox9-GFP), and this recombinant virus construct was infused into the pancreatic ducts, through the common bile/pancreatic duct, to specifically tag the duct cells. In order to address pancreatic duct specific deletion of Pdx1 after alloxan ablation of beta-cells, we first to applied the CRISPR-Cas9 gene editing technique on the HEK293-CMV-GFP cell line in vitro (Figure 2Figure 2) and ROSA26 LSL tomato reporter mice ex vivo (Figure 3Figure 3), and then employed AAV6 mediated CRISPR-SaCas9 under the control of the mouse sox9 short promoter, coupled with paired guide RNAs (gRNAs) flanking the Pdx1 exon2 (Figure 4Figure 4) through pancreatic duct infusion into the pancreas. RESULTS AND CONCLUSIONS: These CRISPR-SaCas9 and gRNAs AAV viruses were co-infused into pancreases of C57BL/6J mice 2 days after alloxan treatment. One week after AAV6-SaCas9+gRNAs infusion, it appeared that only the Pdx1 in pancreatic ductal cells were deleted. Comparatively, PBS or AAV6-CMV-GFP viral infused C57BL/6J mice and ROSA26 LSL tomato reporter mice expressed fluorescence in the entire pancreas as a result of the deleted loxP sites that removed the stop cassette, the Pdx1 is not deleted. Thus, we first report here a highly efficient and specific ablation of Pdx1 in adult mouse pancreatic ducts using the CRISPR-Cas9 technique. Figure 1Figure 1. Construction of AAV CRISPR-SaCas9 and gRNA vectors. Figure 2Figure 2. Deletion of ZsGeen in HEK293-CMV-GFP cells using SacCas9 and gRNA in vitro. Figure 3Figure 3. Valiation of SaCas9 and gRNA in vivo deletion of loxP sites in ROSA26-LSL tomato mice pancreas. Figure 4Figure 4. Deletion of Pdx1 exon2 in alloxan treated C57 pancreatic ducts.View Large Image | Download PowerPoint SlideView Large Image | Download PowerPoint SlideView Large Image | Download PowerPoint SlideView Large Image | Download PowerPoint Slide

758 3-D Imaging of Mouse Pancreatic Duct Lesion and Neurovascular Remodeling

Pancreas features extensive ductal and neurovascular networks in association with acini and islets to perform exocrine and endocrine functions. However, due to the dispersed nature of the network architecture, the standard microtome-based histology cannot provide a globe view of the pancreatic tissue networks in health and disease. To overcome this challenge, we prepared transparent pancreatic specimens by optical clearing (use of immersion solution of high refractive index to promote tissue photon penetration; Tang et al., Diabetes, Obesity and Metabolism, 2014, doi: 10.1111/dom.12342) and performed 3-D microscopy with tile scanning to reveal the spatial features of the pancreatic ductal and neurovascular networks with high definition. Mouse pancreatic intraepithelial neoplasia (PanIN) was induced by activation of oncogenic Kras in acinar cells and/or caerulein injections (chronic pancreatitis). Taking advantage of the transparent tissue, we simultaneously revealed the 3-D PanIN microstructure and the associated neurovascular environment in an integrated fashion. Perilesional neural tissue outgrowth (sympathetic neurite outgrowth and gliosis) and an increase in pericyte marker density were identified via in-depth image projection, indicating the reactivity of the pancreatic neurovascular tissues in response to lesion formation. Overall, the global characterization of duct lesions highlights the advantage of using 3-D microscopy to explore the unknown details of the pancreatic microenvironment in PanIN development.

Specific transduction and labeling of pancreatic ducts by targeted recombinant viral infusion into mouse pancreatic ducts

Specific labeling of pancreatic ducts has proven to be quite difficult. Such labeling has been highly sought after because of the power it would confer to studies of pancreatic ductal carcinogenesis, as well as studies of the source of new insulin-producing β-cells. Cre-loxp recombination could, in theory, lineage-tag pancreatic ducts, but results have been conflicting, mainly due to low labeling efficiencies. Here, we achieved a high pancreatic duct labeling efficiency using a recombinant adeno-associated virus (rAAV) with a duct-specific sox9 promoter infused into the mouse common biliary/pancreatic duct. We saw rapid, diffuse duct-specific labeling, with 50 and 89% labeling in the pancreatic tail and head region, respectively. This highly specific labeling of ducts should greatly enhance our ability to study the role of pancreatic ducts in numerous aspects of pancreatic growth, development and function.

Irbit Mediates Synergy Between Ca2+ and cAMP Signaling Pathways During Epithelial Transport in Mice

The cyclic adenosine monophosphate (cAMP) and Ca(2+) signaling pathways synergize to regulate many physiological functions. However, little is known about the mechanisms by which these pathways interact. We investigated the synergy between these signaling pathways in mouse pancreatic and salivary gland ducts. We created mice with disruptions in genes encoding the solute carrier family 26, member 6 (Slc26a6(-/-) mice) and inositol 1,4,5-triphosphate (InsP3) receptor-binding protein released with InsP3 (Irbit(-/-)) mice. We investigated fluid secretion by sealed pancreatic ducts and the function of Slc26a6 and the cystic fibrosis transmembrane conductance regulator (CFTR) in HeLa cells and in ducts isolated from mouse pancreatic and salivary glands. Slc26a6 activity was assayed by measuring intracellular pH, and CFTR activity was assayed by measuring Cl(-) current. Protein interactions were determined by immunoprecipitation analyses. Irbit mediated the synergistic activation of CFTR and Slc26a6 by Ca(2+) and cAMP. In resting cells, Irbit was sequestered by InsP3receptors (IP3Rs) in the endoplasmic reticulum. Stimulation of Gs-coupled receptors led to phosphorylation of IP3Rs, which increased their affinity for InsP3 and reduced their affinity for Irbit. Subsequent weak stimulation of Gq-coupled receptors, which led to production oflow levels of IP3, caused dissociation of Irbit from IP3Rsand allowed translocation of Irbit to CFTR and Slc26a6 in the plasma membrane. These processes stimulated epithelial secretion of electrolytes and fluid. These pathways were not observed in pancreatic and salivary glands from Irbit(-/-) or Slc26a6(-/-) mice, or in salivary gland ducts expressing mutant forms of IP3Rs that could not undergo protein kinase A-mediated phosphorylation. Irbit promotes synergy between the Ca(2+) and cAMP signaling pathways in cultured cells and in pancreatic and salivary ducts from mice. Defects in this pathway could be involved in cystic fibrosis, pancreatitis, or Sjögren syndrome.

Deletion of Slc26a6 alters the stoichiometry of apical Cl−/HCO3− exchange in mouse pancreatic duct

To define the stoichiometry and molecular identity of the Cl(-)/HCO(3)(-) exchanger in the apical membrane of pancreatic duct cells, changes in luminal pH and volume were measured simultaneously in interlobular pancreatic ducts isolated from wild-type and Slc26a6-null mice. Transepithelial fluxes of HCO(3)(-) and Cl(-) were measured in the presence of anion gradients favoring rapid exchange of intracellular HCO(3)(-) with luminal Cl(-) in cAMP-stimulated ducts. The flux ratio of Cl(-) absorption/HCO(3)(-) secretion was ∼0.7 in wild-type ducts and ∼1.4 in Slc26a6(-/-) ducts where a different Cl(-)/HCO(3)(-) exchanger, most likely SLC26A3, was found to be active. Interactions between Cl(-)/HCO(3)(-) exchange and cystic fibrosis transmembrane conductance regulator (CFTR) in cAMP-stimulated ducts were examined by measuring the recovery of intracellular pH after alkali-loading by acetate prepulse. Hyperpolarization induced by luminal application of CFTRinh-172 enhanced HCO(3)(-) efflux across the apical membrane via SLC26A6 in wild-type ducts but significantly reduced HCO(3)(-) efflux in Slc26a6(-/-) ducts. In microperfused wild-type ducts, removal of luminal Cl(-), or luminal application of dihydro-4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid to inhibit SLC26A6, caused membrane hyperpolarization, which was abolished in Slc26a6(-/-) ducts. In conclusion, we have demonstrated that deletion of Slc26a6 alters the apparent stoichiometry of apical Cl(-)/HCO(3)(-) exchange in native pancreatic duct. Our results are consistent with SLC26A6 mediating 1:2 Cl(-)/HCO(3)(-) exchange, and the exchanger upregulated in its absence, most probably SLC26A3, mediating 2:1 exchange.

Isolation of mouse pancreatic alpha, beta, duct and acinar populations with cell surface markers

Tools permitting the isolation of live pancreatic cell subsets for culture and/or molecular analysis are limited. To address this, we developed a collection of monoclonal antibodies with selective surface labeling of endocrine and exocrine pancreatic cell types. Cell type labeling specificity and cell surface reactivity were validated on mouse pancreatic sections and by gene expression analysis of cells isolated using FACS. Five antibodies which marked populations of particular interest were used to isolate and study viable populations of purified pancreatic ducts, acinar cells, and subsets of acinar cells from whole pancreatic tissue or of alpha or beta cells from isolated mouse islets. Gene expression analysis showed the presence of known endocrine markers in alpha and beta cell populations and revealed that TTR and DPPIV are primarily expressed in alpha cells whereas DGKB and GPM6A have a beta cell specific expression profile.

Experimental acute biliary pancreatitis induced by retrograde infusion of bile acids into the mouse pancreatic duct

Mechanistic studies of acute pancreatitis require animal models because clinical material is generally not available during the early phases of the disease. Here we describe a protocol to induce biliary pancreatitis by retrogradely infusing bile acids into the pancreatic duct of anesthetized mice. The resulting model replicates events believed to be responsible for the onset of clinical biliary (i.e., gallstone) pancreatitis and creates highly reproducible pancreatitis with a severity that depends on the concentration of infused bile acid. Pancreatitis reaches its maximal level of severity within 24 h of induction, and it resolves over the subsequent week. This protocol enables the investigator to use genetically modified strains of mice, and it requires only relatively simple and easily learned techniques of small animal surgery. With practice and gentle technique, the surgery (from induction of anesthesia to completion of the infusion) can be completed within 25 min per animal.

Effects of Slc26a6 deletion and CFTR inhibition on HCO3- secretion by mouse pancreatic duct

Pancreatic duct epithelium secretes HCO(3)(-)-rich fluid, which is dependent on cystic fibrosis transmembrane conductance regulator (CFTR). HCO(3)(-) transport across the apical membrane is thought to be mediated by both SLC26A6 Cl(-)-HCO(3)(-) exchange and CFTR HCO(3)(-) conductance. In this study we examined the relative contribution and interaction of SLC26A6 and CFTR in apical HCO(3)(-) transport. Interlobular pancreatic ducts were isolated from slc26a6 null mice. Intracellular pH (pH(i)) was measured by BCECF microfluorometry. Duct cells were stimulated with forskolin and alkalinized by acetate pre-pulse in the presence of HCO(3)(-)-CO(2). Apical HCO(3)(-) secretion was estimated from the recovery rate of pH(i) from alkaline load. When the lumen was perfused with high-Cl(-) solution, the rate of apical HCO(3)(-) secretion was increased by luminal application of CFTRinh-172 in ducts from wild-type mice but it was decreased in ducts from slc26a6 -/- mice. This suggests that slc26a6 and CFTR compensate/compete with each other for apical HCO(3)(-) secretion with high Cl(-) in the lumen. With high HCO(3)(-) in the lumen, luminal CFTRinh-172 reduced the rate of apical HCO(3)(-) secretion in both wild-type and slc26a6 -/- ducts. This suggests that HCO(3)(-) conductance of CFTR mediates a significant portion of apical HCO(3)(-) secretion with high HCO(3)(-) in the lumen.

Molecular characterization of Slc26a3 and Slc26a6 anion transporters in guinea pig pancreatic duct

HCO3 in pancreatic juice arises from the pancreatic duct cells. Secretin stimulates HCO3 secretion via a mechanism that involves activation of the adenylate cyclase pathway, activation of both basolateral K channels and the apical CFTR Cl channel, and stimulation of an apical Slc26mediated Cl/HCO3 exchanger (1). The Slc26 anion exchangers, Slc26a3 and Slc26a6, have both been localized to the apical membrane of human pancreatic duct and mouse pancreatic duct although their functional roles in pancreatic HCO3 secretion remain uncertain. The high rates of pancreatic bicarbonate secretion shared by guinea pig (gp) and human, in contrast to the low rates in mouse, prompted us to study the functional characteristics of gp Slc26 anion transporters. Furthermore, recent data from isolated guinea pig pancreatic duct experiments demonstrated that during stimulated HCO3 secretion an apical Slc26a6-like Cl/HCO3 exchanger is functionally coupled with CFTR (2). This study prompted us to clone cDNAs encoding Slc26a3 and Slc26a6 polypeptides from the gp pancreas and to compare their functional characteristics in Xenopus oocytes with those of their mouse and human orthologs (3-5), and with those of luminal Cl/HCO3 exchange measured in isolated gp pancreatic interlobular ducts (2). Whereas gp Slc26a6 mediated Cl/Cl exchange and bidirectional exchange of Cl for oxalate and HCO3, gp Slc26a6 mediated only sulfate influx but not sulfate efflux in preliminary experiments. Gp Slc26a3 mediated robust Cl/Cl exchange and minimal oxalate efflux in exchange for Cl, but did not mediate detectable influx of oxalate or sulfate. Measurement of intracellular pH by BCECF ratio fluorimetry in HEK cells transfected with Slc26a6 or in Slc26a3-expressing Xenopus oocytes during removal and restoration of Clo in the presence of HCO3 indicated that both gp Slc26a6 and Slc26a3 function as Cl/HCO3 exchangers. These substrate specificity experiments reveal that whereas Slc26a3 functions predominantly as a Cl/HCO3 exchanger, Slc26a6 can exchange a wider range of substrates, functioning as a Cl/HCO3 exchanger or a Cl/anion exchanger. This finding allows for the possibility that Slc26a6 may have alternative roles in transepithelial solute transport in the pancreatic duct. The apparent affinity of gp Slc26a6 for extracellular Cl (K1/2) was 2 mM during Cli/Clo exchange and 30 mM during C-oxalatei/Clo exchange. These experiments reveal that binding and/ or translocation rates of extracellular Cl with respect to gp slc26a6 is/are dependent on the internal transanion substrate and show species-specificity (5). The high affinity for extracellular Clo suggests that even when duct luminal Cl is low, such as might occur with passage of pancreatic juice along the duct lumen, Slc26a6-mediated Cl/HCO3 exchange would still be active. Whereas gp Slc26a6-mediated isotopic flux was moderately sensitive to DNDS (500 μM), niflumic acid (100 μM) and DIDS (500 μM), gp Slc26a3 was minimally sensitive to inhibition by DIDS and DNDS but strongly sensitive to niflumic acid. These pharmacological data, combined with EXPANDED ABSTRACT

A mouse model of acute biliary pancreatitis induced by retrograde pancreatic duct infusion of Na-taurocholate

Objective:Most mechanistic studies of pancreatitis in mice employ the secretagogue-induced model. The currently reported studies were designed to develop an alternative, and possibly more clinically relevant, mouse model of pancreatitis.Design:Na-taurocholate...

Nestin-lineage cells contribute to the microvasculature but not endocrine cells of the islet.

To clarify the lineage relationship between cells that express the neural stem cell marker nestin and endocrine cells of the pancreas, we analyzed offspring of a cross between mice carrying a nestin promoter/enhancer-driven cre-recombinase (Nestin-cre) and C57BL/6J-Gtrosa26(tm1Sor) mice that carry a loxP-disrupted beta-galactosidase gene (Rosa26). In nestin-cre(+/tg);R26R(loxP/+) embryos, cre-recombinase was detected in association with nestin-positive cells in the pancreatic mesenchyme with some of the nestin-positive cells lining vascular channels. In postnatal mice, pancreatic beta-galactosidase expression was restricted to vascular endothelial cells of the islet and a subset of cells in the muscularis of arteries in a distribution identical to endogenous nestin expression. Ex vivo explants of mouse pancreatic ducts grew dense cultures that costained for nestin and beta-galactosidase, demonstrating recombination in vitro. The cultures could be differentiated into complex stereotypic structures that contain nestin- and insulin-expressing cells. Nestin-cre(+/tg);R26R(loxP/+)-derived duct cultures showed that insulin-positive cells were negative for beta-galactosidase. These results indicate that both in vivo and in vitro pancreatic endocrine cells arise independently of nestin-positive precursors. The apparent vascular nature of the nestin-positive cell population and the close association with endocrine cells suggest that nestin-positive cells play an important role in the growth and maintenance of the islet.

Autonomic nerve changes in the mouse pancreas after pancreatic duct ligation.

The mouse pancreas exhibits distinct atrophy of the exocrine tissue following pancreatic duct ligation. To investigate changes of innervation in the whole pancreas after pancreatic duct ligation. The mouse pancreatic duct was ligated at 6 weeks of age. Pancreatic tissues were removed 7 days and 14 days after the ligation, fixed by perfusion and immersion with Zamboni solution, and embedded in gelatin. The whole organ was serially sectioned at a thickness of 100 microm, histochemically stained for cholinesterase, and observed by light microscopy. The number and volume of intrapancreatic ganglia, number of ganglion cells, and volume of each ganglion cell in the whole pancreas were quantitated. Some sections were analyzed using transmission electron microscopy after histochemically staining for cholinesterase. In the normal pancreas, ganglia were often situated on the outer surface of the islets of Langerhans. Thick nerve bundles ran along the arteries and emanated thin nerve fibers that surrounded the arterioles. In the atrophied pancreas following pancreatic duct ligation, ganglia remained on the islets of Langerhans as in normal mice, while the nerve fibers appeared dense, bending and curling in a more complex manner. The thin nerves also crossed each other in a complex network. Using morphometry in the pancreas following pancreatic duct ligation, the total ganglion cell number was found to decrease from normal levels. The mean ganglion cell volume in the ligated pancreas was significantly smaller than that in normal mice. As observed by transmission electron microscopy, some ganglion cells in the ligated pancreas were negative for cholinesterase activity but were surrounded by positive staining around the surface. These results suggest that the function of pancreatic ganglion cells changes with organ atrophy after pancreatic duct ligation.

Up-regulation of the expression of activins in the pancreatic duct by reduction of the beta-cell mass.

Activins expressed in progenitor cells of the pancreas regulate differentiation of endocrine cells during development. Neogenesis of beta-cells takes place in adult animals under some conditions, and beta-cells are thought to arise from precursors locating in the pancreatic duct. In the present study, we investigated whether or not activins are expressed in the duct where beta-cell neogenesis is initiated. mRNA for the beta(A)- and beta(B)-subunits was expressed in isolated mouse pancreatic ducts. Immunohistochemically, the beta(A)-subunit was detected in the pancreatic duct and colocalized with cytokeratin, a marker of ductal cells. The beta(A)-subunit was also expressed in nestin-positive cells in the duct. Likewise, the beta(B)-subunit was detected in the pancreatic duct. In addition, mRNA for the type II and type IIB activin receptors was expressed in the duct. Expression of mRNA for two activin subunits was markedly increased after streptozotocin injection. Similarly, the mRNA expression was up-regulated after partial pancreatectomy. These results indicate that activins are expressed in the pancreatic duct and are up-regulated shortly after the reduction of the beta-cell mass. Induction of activins in the duct may be a critical step in the initiation of beta-cell neogenesis.

Regulatory Interaction between the Cystic Fibrosis Transmembrane Conductance Regulator and HCO[formula omitted] Salvage Mechanisms in Model Systems and the Mouse Pancreatic Duct

The pancreatic duct expresses cystic fibrosis transmembrane conductance regulator (CFTR) and HCO3- secretory and salvage mechanisms in the luminal membrane. Although CFTR plays a prominent role in HCO3- secretion, the role of CFTR in HCO3- salvage is not known. In the present work, we used molecular, biochemical, and functional approaches to study the regulatory interaction between CFTR and the HCO3- salvage mechanism Na+/H+ exchanger isoform 3 (NHE3) in heterologous expression systems and in the native pancreatic duct. We found that CFTR regulates NHE3 activity by both acute and chronic mechanisms. In the pancreatic duct, CFTR increases expression of NHE3 in the luminal membrane. Thus, luminal expression of NHE3 was reduced by 53% in ducts of homozygote DeltaF508 mice. Accordingly, luminal Na+-dependent and HOE694- sensitive recovery from an acid load was reduced by 60% in ducts of DeltaF508 mice. CFTR and NHE3 were co-immunoprecipitated from PS120 cells expressing both proteins and the pancreatic duct of wild type mice but not from PS120 cells lacking CFTR or the pancreas of DeltaF508 mice. The interaction between CFTR and NHE3 required the COOH-terminal PDZ binding motif of CFTR, and mutant CFTR proteins lacking the C terminus were not co-immunoprecipitated with NHE3. Furthermore, when expressed in PS120 cells, wild type CFTR, but not CFTR mutants lacking the C-terminal PDZ binding motif, augmented cAMP-dependent inhibition of NHE3 activity by 31%. These findings reveal that CFTR controls overall HCO3- homeostasis by regulating both pancreatic ductal HCO3- secretory and salvage mechanisms.

The stoichiometry of the electrogenic sodium bicarbonate cotransporter pNBC1 in mouse pancreatic duct cells is 2 HCO(3)(-):1 Na(+).

The electrogenic sodium bicarbonate cotransporter pNBC1 is believed to play a major role in the secretion of bicarbonate by pancreatic duct cells, by transporting bicarbonate into the cell across the basolateral membrane. Thermodynamics predict that this function can be achieved only if the reversal potential of the cotransporter is negative to the cell's membrane potential, or equivalently that the HCO3-:Na+ stoichiometry is not larger then 2:However, there are no data available on either the reversal potential or the HCO3-:Na+ stoichiometry of pNBC1 in pancreatic cells. We studied pNBC1 function in mouse pancreatic duct cells. RT-PCR analysis of total RNA revealed that these cells contain the message for pNBC1, but not for kNBC1, NBC2 or NBC3. To measure cotransporter activity, mouse pancreatic duct cells were grown to confluence on a porous substrate, mounted in an Ussing chamber, and the apical plasma membrane permeabilized with amphotericin B. Ion flux through pNBC1 was achieved by applying Na+ concentration gradients across the basolateral plasma membrane. The current through the cotransporter was isolated as the difference current due to the reversible inhibitor dinitrostilbene disulfonate (DNDS). Current-voltage relationships for the cotransporter, measured at three different Na+ concentration gradients, were linear over a range of about 100 mV. The reversal potential data, obtained from these current-voltage relationships, all corresponded to a 2 HCO3-:1 Na+ stoichiometry. The data indicate that pNBC1 is functionally expressed in mouse pancreatic duct cells. The cotransporter operates with a 2 HCO3-:1 Na+ stoichiometry in these cells, and mediates the transport of bicarbonate into the cell across the basolateral membrane.

Cystic Fibrosis Transmembrane Conductance Regulator Regulates Luminal Cl−/HCO3−Exchange in Mouse Submandibular and Pancreatic Ducts

We have demonstrated previously the regulation of Cl-/HCO3- exchange activity by the cystic fibrosis transmembrane conductance regulator (CFTR) in model systems of cells stably or transiently transfected with CFTR (Lee, M. G., Wigley, W. C., Zeng, W., Noel, L. E., Marino, C. R., Thomas, P. J., and Muallem, S. (1999) J. Biol. Chem. 274, 3414-3421). In the present work we examine the significance of this regulation in cells naturally expressing CFTR. These include the human colonic T84 cell line and the mouse submandibular gland and pancreatic ducts, tissues that express high levels of CFTR in the luminal membrane. As in heterologous expression systems, stimulation of T84 cells with forskolin increased the Cl-/HCO3- exchange activity independently of CFTR Cl- channel activity. Freshly isolated submandibular gland ducts from wild type mice showed variable Cl-/HCO3- exchange activity. Measurement of [Cl-]i revealed that this was largely the result of variable steady-state [Cl-]i. Membrane depolarization with 5 mM Ba2+ or 100 mM K+ increased and stabilized [Cl-]i. Under depolarized conditions wild type and DeltaF/DeltaF mice had comparable basal Cl-/HCO3- exchange activity. Notably, stimulation with forskolin increased Cl-/HCO3- exchange activity in submandibular gland ducts from wild type but not DeltaF/DeltaF mice. Microperfusion of the main pancreatic duct showed Cl-/HCO3- exchange activity in both the basolateral and luminal membranes. Stimulation of ducts from wild type animals with forskolin had no effect on basolateral but markedly stimulated luminal Cl-/HCO3- exchange activity. By contrast, forskolin had no effect on either basolateral or luminal Cl-/HCO3- exchange activity of ducts from DeltaF/DeltaF animals. We conclude that CFTR regulates luminal Cl-/HCO3- exchange activity in CFTR-expressing cells, and we discuss the possible physiological significance of these findings regarding cystic fibrosis.

Increased expression of sulfated gp300 and acinar tissue pathology in pancreas of CFTR(-/-) mice

The CFTR (-/-) mouse model of cystic fibrosis (CF) has revealed that the mouse pancreatic duct has a Ca(2+)-regulated chloride conductance that allows ductal electrolyte transport to remain unaffected by loss of the cystic fibrosis transmembrane conductance regulator (CFTR). Therefore, this model provides a unique opportunity to investigate effects of CF on the acinar tissue. It has been reported that exocrine secretions contain higher levels of sulfate in CF. We discovered in CFTR(-/-) acini that gp300, the major sulfated glycoprotein of the mouse acinar cell, has increased steady-state and biosynthetic levels. However, there are no apparent changes in sulfate or carbohydrate composition of gp300, indicating that posttranslational processing of this sulfated glycoprotein is not altered in CF. In addition to the increase in gp300, the morphology of CF acinar tissue is dramatically altered: acinar lumina of CFTR(-/-) mice are greatly dilated and filled with aggregated protein. gp300, which in the normal tissue is mainly localized to the zymogen granule membrane, was found to line the distended luminal membranes in the CF tissue. These results demonstrate that the acinar tissue is affected in the CF mouse and that expression of the major sulfated glycoprotein is also increased. It is suggested that increased expression of gp300 in CFTR(-/-) mice may cause poorly soluble exocrine protein secretion, contributing to the development of CF in the pancreas.

Carbonic anhydrase II gene expression in mouse pancreatic duct cells.

Our goal is to create a transgenic mouse model for human pancreatic duct cell adenocarcinoma using the promoter/enhancer region of the carbonic anhydrase (CA) II gene to drive the expression of SV-40 T-antigen in pancreatic duct cells. This requires that the CA II gene be expressed in mouse pancreatic duct cells and not in other pancreatic cells, as has already been shown to be the case in the human and guinea pig pancreas. We have shown with an enzyme histochemical assay that mouse pancreatic duct cells contain CA activity in both intact pancreas and cultured interlobular duct epithelium. In addition, CA activity was detected with a biochemical assay in homogenates of cultured duct epithelium. The specific activity of duct cells was 2.75-fold greater than in whole pancreas, suggesting that a substantial amount of total pancreatic CA activity is contributed by duct cells. At least some of the CA in cultured duct cells was inferred to be CA II by Northern blot analysis of RNA extracted from the cells. The concentration of CA II mRNA in the cultured duct cells was substantially greater than in whole pancreas and would appear to account for the majority, if not all, of the CA II in the mouse pancreas.