Probe-based confocal laser endomicroscopy for the diagnosis of pancreatic ductal structures.

BACKGROUND AND AIM: The image-based diagnosis of pancreatic diseases can be difficult and requires pathological evaluation. Probe-based confocal laser endomicroscopy (pCLE) enables real-time observation of the microscopic tissue pattern of lesion and may be a useful assistance for the diagnosis. This study aimed to evaluate the feasibility and utility of pCLE for the diagnosis of pancreatic diseases. METHODS: Thirty patients who underwent endoscopic retrograde cholangiopancreatography with pCLE for the evaluation of indeterminate pancreatic diseases from June 2015 to October 2018 were included in this study. The pCLE findings were interpreted according to the Miami Classification. RESULTS: Among a total of 30 patients, 12, 10, 4, and 4 patients received the definitive diagnoses of pancreatic ductal adenocarcinoma (PDAC), main duct intrapapillary mucinous neoplasm, autoimmune pancreatitis, and chronic pancreatitis, respectively. The diagnostic accuracy of pCLE for PDAC and pancreatitis (96.7% and 93.3%, respectively) was higher than that of cytology (76.7% and 63.3%, respectively) (P = 0.0227 and 0.0048, respectively). The sensitivity of pCLE for PDAC was significantly higher (91.7%) than that of cytology (41.7%) (P = 0.0094). Moreover, the specificity of pCLE for pancreatitis was significantly higher than that of cytology (90.9% vs 50%; P = 0.0029). However, the diagnostic accuracies of pCLE and cytology for main duct intrapapillary mucinous neoplasm did not differ significantly (96.7% and 86.7%, respectively). CONCLUSIONS: Probe-based confocal laser endomicroscopy may be effective for the diagnosis of pancreatic diseases as adjunct modality. It requires technical learning and further evaluation of its usefulness.

Decellularized Pancreas Matrix Scaffolds for Tissue Engineering Using Ductal or Arterial Catheterization.

INTRODUCTION: Diabetes is known as a worldwide disease with a great burden on society. Since therapeutic options cover a limited number of target points, new therapeutic strategies in the field of regenerative medicine are considered. Bioscaffolds along with islet cells would provide bioengineered tissue as a substitute for ß-cells. The perfusion-decellularization technique is considered to create such scaffolds since they mimic the compositional, architectural, and biomechanical nature of a native organ. In this study, we investigated 2 decellularization methods preserving tissue microarchitecture. METHODS: Procured pancreas from Sprague-Dawley rats was exposed to different percentages of detergent for 2, 4, and 6 h after cannulation via the common bile duct or aorta. RESULTS: High concentrations of sodium dodecyl sulfate (SDS), i.e., > 0.05%, resulted in tissue disruption or incomplete cell removal depending on the duration of exposure. In both methods, 6-h exposure to 0.05% SDS created a bioscaffold with intact extracellular matrices and proper biomechanical characteristics. Tissue-specific stainings revealed that elastic, reticular, and collagen fiber concentrations were well preserved. Quantitative findings showed that glycosaminoglycan content was slightly different, but hydroxyproline was in the range of native pancreas tissue. Dye infusion through ductal and vascular cannulation proved that the vascular network was intact, and scanning electron microscopy indicated a homogeneous porous structure. CONCLUSIONS: Using the detergent-based method, an effective and time-efficient procedure, a whole pancreas extracellular matrix bioscaffold can be developed that can be used as a 3D structure for pancreas tissue engineering-based studies and regenerative medicine applications.

Immunohistochemical Analysis and Electron Microscopy of Glial Cells in the Pancreas of Fetuses and Children.

Schwann cells forming peri-insular glial sheath of the pancreatic islets in some mammals can be involved in the pathogenesis of diabetes mellitus. Human pancreatic cells contain small elongated or oval cells of unknown origin with S100-immunopositive processes. We found that cells with processes located in pancreatic islets of human fetuses and children are similar to Schwann cells by their morphological and ultrastructural characteristics, immunopositive reaction for S100, and integration with nerve endings. In the pancreas of fetuses and children, Schwann cells are often seen in forming pancreatic islets and around the pancreatic ducts. The data suggests that Schwann cells can participate in the morphogenesis of human pancreatic islets.

Subcellular fractionation enhances proteome coverage of pancreatic duct cells.

OBJECTIVES: Subcellular fractionation of whole cell lysates offers a means of simplifying protein mixtures, potentially permitting greater depth of proteomic analysis. Here we compare proteins identified from pancreatic duct cells (PaDC) following organelle enrichment to those identified from PaDC whole cell lysates to determine if the additional procedures of subcellular fractionation increase proteome coverage. METHODS: We used differential centrifugation to enrich for nuclear, mitochondrial, membrane, and cytosolic proteins. We then compared - via mass spectrometry-based analysis - the number of proteins identified from these four fractions with four biological replicates of PaDC whole cell lysates. RESULTS: We identified similar numbers of proteins among all samples investigated. In total, 1658 non-redundant proteins were identified in the replicate samples, while 2196 were identified in the subcellular fractionation samples, corresponding to a 30% increase. Additionally, we noted that each organelle fraction was in fact enriched with proteins specific to the targeted organelle. CONCLUSIONS: Subcellular fractionation of PaDC resulted in greater proteome coverage compared to PaDC whole cell lysate analysis. Although more labor intensive and time consuming, subcellular fractionation provides greater proteome coverage, and enriches for compartmentalized sub-populations of proteins. Application of this subcellular fractionation strategy allows for a greater depth of proteomic analysis and thus a better understanding of the cellular mechanisms of pancreatic disease.

Pancreatic duct glands are distinct ductal compartments that react to chronic injury and mediate Shh-induced metaplasia.

BACKGROUND & AIMS: Pancreatic intraepithelial neoplasia (PanIN) are pancreatic cancer precursor lesions of unclear origin and significance. PanIN aberrantly express sonic hedgehog (Shh), an initiator of pancreatic cancer, and gastrointestinal mucins. A majority of PanIN are thought to arise from ducts. We identified a novel ductal compartment that is gathered in gland-like outpouches (pancreatic duct glands [PDG]) of major ducts and characterized its role in injury and metaplasia. METHODS: The ductal system was analyzed in normal pancreata and chronic pancreatitis in humans and mice. Anatomy was assessed by serial hematoxylin and eosin sections and scanning electron microscopy of corrosion casts. Expression of mucins and developmental genes and proliferation were assessed by immunohistochemistry or real-time quantitative polymerase chain reaction. Effects of Shh on ductal cells were investigated by exposure to Shh in vitro and transgenic misexpression in vivo. RESULTS: Three-dimensional analysis revealed blind-ending outpouches of ducts in murine and human pancreata. These PDG are morphologically and molecularly distinct from normal ducts; even in normal pancreata they display PanIN and metaplastic features, such as expression of Shh and gastric mucins. They express other developmental genes, such as Pdx-1 and Hes-1. In injury, Shh is up-regulated along with gastric mucins. Expansion of the PDG compartment results in a mucinous metaplasia. Shh promotes this transformation in vitro and in vivo. CONCLUSIONS: PDG are distinct gland-like mucinous compartments with a distinct molecular signature. In response to injury, PDG undergo an Shh-mediated mucinous gastrointestinal metaplasia with PanIN-like features. PDG may provide a link between Shh, mucinous metaplasia, and neoplasia.

Pancreatic exocrine insufficiency in LXRbeta-/- mice is associated with a reduction in aquaporin-1 expression.

Liver X receptors (LXRs) alpha and beta are nuclear oxysterol receptors with a key role in cholesterol, triglyceride, and glucose metabolism. In LXRbeta(-/-) mice on a normal diet, there is a reduction in size of perigonadal fat pad and, on high-fat diet there is resistance to obesity. In the present study, we investigated the reason for the resistance of LXRbeta(-/-) mice to weight gain. In LXRbeta(-/-) mice we found pancreatic exocrine insufficiency with reduced serum levels of amylase and lipase, reduced proteolytic activity in feces, chronic inflammatory infiltration, and, in the ductal epithelium, an increased apoptosis without compensatory proliferation. Electron microscopy revealed ductal dilatation with intraductal laminar structures characteristic of cystic fibrosis. To investigate the relationship between LXRbeta and pancreatic secretion, we studied the expression of LXRbeta and the water channel, aquaporin-1 (AQP1), in the ductal epithelium of the pancreas. In WT mice, ductal epithelial cells expressed LXRbeta in the nuclei and AQP1 on the plasma membrane. In LXRbeta(-/-) mice neither LXRbeta nor AQP1 was detectable. Moreover, in WT mice the LXR agonist (T2320) increased AQP1 gene expression. These data demonstrate that in LXRbeta(-/-) mice dietary resistance to weight gain is caused by pancreatic insufficiency and that LXRbeta regulates pancreatic exocrine secretion through the control of AQP1 expression. Pancreatic exocrine insufficiency is the main cause of malabsorption syndrome responsible for weight loss in adults and growth failure in children. Several genes are known to be involved in the pathogenesis and susceptibility to pancreatic insufficiency. LXRbeta should be included in that list.

Apical Restriction of the Planar Cell Polarity Component VANGL in Pancreatic Ducts Is Required to Maintain Epithelial Integrity.

Cell polarity is essential for the architecture and function of numerous epithelial tissues. Here, we show that apical restriction of planar cell polarity (PCP) components is necessary for the maintenance of epithelial integrity. Using the mammalian pancreas as a model, we find that components of the core PCP pathway, such as the transmembrane protein Van Gogh-like (VANGL), become apically restricted over a period of several days. Expansion of VANGL localization to the basolateral membranes of progenitors leads to their death and disruption of the epithelial integrity. VANGL basolateral expansion does not affect apico-basal polarity but acts in the cells where Vangl is mislocalized by reducing Dishevelled and its downstream target ROCK. This reduction in ROCK activity culminates in progenitor cell egression, death, and eventually pancreatic hypoplasia. Thus, precise spatiotemporal modulation of VANGL-dependent PCP signaling is crucial for proper pancreatic morphogenesis.

Autocrine stimulation of human pancreatic duct-like development by soluble isoforms of epimorphin in vitro.

Epimorphin was recently described as a mesenchymal factor modulating morphogenesis of murine mammary ducts, skin, liver, and lung in vitro. In this study epimorphin was analyzed in a human, pancreatic adenocarcinoma cell line (A818-6) which develops single layer epithelial hollow spheres resembling normal pancreatic ductal structures in vitro. Soluble 34- and 31-kD isoforms of epimorphin were found in the culture supernatant of A818-6 cells. In lysates of A818-6 cells we detected the 34-and 31-kD isoforms and the dimers, and in lysates of fibroblasts the 150-kD tetramers of epimorphin additionally. A neutralizing monoclonal antibody against epimorphin (MC-1) efficiently blocked the development of hollow sphere structures from A818-6 cells. Coculture of A818-6 cells with fibroblasts stimulated the development of hollow sphere structures in general and increased differentiation in 5-6-d-old hollow spheres. A818-6 hollow sphere development in the presence of fibroblasts was also blocked by MC-1. In this novel system for human duct-like differentiation of pancreatic epithelial cells, we provide evidence for an autocrine and paracrine function of epimorphin as a major mediator for morphogenesis.

Biphenotypic Differentiation of Pancreatic Cancer in 3-Dimensional Culture.

OBJECTIVE: Pancreatic ductal adenocarcinoma (PDAC) is the third most common cause of cancer death in the United States. Improved characterized models of PDAC are needed for drug screening. METHODS: We grew 4 established pancreatic cancer cell lines in hanging drop cultures to produce spheroids. We also grew organoids from explanted xenografted PDAC and surgically resected primary PDAC. We performed transmission and scanning electron microscopy and compared findings with those of the normal pancreatic duct. We also performed single-cell cloning to determine the potential options for differentiation. RESULTS: Spheroids contained tight junctions and desmosomes but lacked zymogen granules, as expected. The former features were present in normal pancreatic duct but absent from PDAC cell lines grown in standard 2-dimensional culture. Spheroids functionally excluded macromolecules in whole mounts. Cells on the surface of PDAC spheroids were carpeted by microvilli except for rare cells with prominent stereocilia. Carpets of microvilli were also seen in low passage organoids produced from xenografts and surgically resected human PDAC, in addition to normal human pancreatic duct. We performed single-cell cloning and resulting spheroids produced both cell phenotypes at the same approximate ratios as those from bulk cultures. CONCLUSIONS: Pancreatic cancer spheroids/organoids are capable of biphenotypic differentiation.

Variaciones de la configuración ductal pancreática: factor predisponente de pancreatitis recurrente / Variations of pancreatic ductal configuration: predisposing factor of recurrent pancreatitis

El páncreas es un órgano de origen endodérmico, que se desarrolla de dos esbozos intestinales separados, uno dorsal y otro ventral,a partir de la cuarta o quinta semana de gestación. El páncreas se origina en el intestino anterior en la parte correspondiente a lafutura segunda porción duodenal. Allí se originan dos brotes: uno posterior o dorsal que aparece a principios de la cuarta semanay crece rápidamente en el mesenterio dorsal y el otro brote es anterior o ventral. Este último da origen a dos brotes, uno origina unaparte del páncreas y otro a la vía biliar e hígado. Existen diferentes variantes anatómicas, derivadas de este desarrollo embrionario;su conocimiento es de vital importancia en pacientes con persistencia de dolor abdominal y episodios de pancreatitis recurrente.El objetivo de este artículo es una revisión de las variantes anatómicas del conducto pancreático que pueden manifestarse como pancreatitis idiopática recurrente. (AU)

The pancreas is an organ of endodermal origin, which develops from two separate intestinal sketches, one dorsal and one ventral,from the fourth or fifth week of gestation. The pancreas originates in the anterior intestine in the part corresponding to the futuresecond duodenal portion. There two shoots originate: one posterior or dorsal that appears at the beginning of the fourth week andgrows rapidly in the dorsal mesentery and the other outbreak is anterior or ventral. The latter gives rise to two outbreaks, oneoriginates a part of the pancreas and another to the bile duct and liver. There are different anatomical variants, derived from thisembryonic development; their knowledge is of vital importance in patients with persistent abdominal pain and episodes of recurrentpancreatitis. The objective of this article is a review of the anatomical variants of the pancreatic duct that can manifest as recurrent idiopathic pancreatitis. (AU)

Development of an in vitro pancreatic tissue model to study regulation of islet neogenesis associated protein expression.

Restoration of a functional beta-cell mass in a patient with diabetes may hold the key for curing the disease. In recent years, there has been increasing interest in the development of new strategies to induce beta-cell regeneration and new islet formation in situ and a role for Reg proteins has been suggested. One such protein, islet neogenesis associated protein (INGAP), is a member of the Reg3 family of proteins and has been shown to induce islet neogenesis. Elucidation of the mechanisms and factors involved in the regulation of expression of INGAP and related proteins is, therefore, of great importance. Here, we report the establishment of the first in vitro tissue model of INGAP expression that consists of epithelial cystic structures derived from hamster pancreatic acinar tissue cultured in collagen matrix. The objective of this study was to characterize INGAP expression in this model and to investigate the role of pro-inflammatory cytokines and growth factors. Using quantitative reverse transcriptase PCR, we show that INGAP expression correlates with cyst formation and size suggesting the involvement of intra-luminal pressure associated with cyst growth. We also demonstrate for the first time that INGAP gene expression was significantly induced by treatment with interleukin (IL)-6 and further enhanced by a combination of IL-6 with dexamethazone and nicotinamide. Additionally, our data suggest that the effect of IL-6 on INGAP expression is mediated via the JAK/STAT3 signaling pathway. In summary, the in vitro model of INGAP expression described here represents an important step in the development of strategies for the use of INGAP and related proteins as islet neogenic agents in the pharmacotherapy of both type-1 and type-2 diabetes.

Optical coherence tomography compared with histology of the main pancreatic duct structure in normal and pathological conditions: an 'ex vivo study'.

BACKGROUND AND STUDY AIMS: Optical coherence tomography permits high-resolution imaging of tissue microstructures by a probe inserted into the main pancreatic duct through a standard ERCP catheter. The aim of this study was to compare optical coherence tomography images of the main pancreatic duct with histology and identify the optical coherence tomography pattern of the normal and pathological structure of the main pancreatic duct. PATIENTS AND METHODS: Multiple sections of neoplastic and non-neoplastic segments of 10 consecutive surgical pancreatic specimens obtained from patients with pancreatic head adenocarcinoma were investigated by optical coherence tomography scanning within 1h of resection. One hundred optical coherence tomography findings were then compared with the corresponding histopathological diagnoses. RESULTS: Main pancreatic duct wall architecture appeared at optical coherence tomography investigation as a three-layer structure with a different back-scattered signal from each layer. Optical coherence tomography imaging was concordant with histology in 81.8% and 18.75% of sections with normal tissue and chronic inflammatory changes. The K statistic between the two procedures was equal to 0.059 for non-neoplastic main pancreatic duct wall appearance. In all neoplastic sections optical coherence tomography showed a subverted layer architecture with heterogeneous back-scattering of the signal and was concordant with histology. CONCLUSIONS: Optical coherence tomography provided images of main pancreatic duct wall structure that were concordant with histology in 100% of cases in presence of neoplastic ductal changes and did not have false-positive or negative results. Optical coherence tomography images were also concordant with histology in about 80% of cases with normal main pancreatic duct structure; however, the differential diagnosis between normal tissue and chronic pancreatitis or dysplastic changes appeared very difficult.

Electron microscopic autoradiography of the pancreas in the hamster treated with tritiated N-nitroso-2,6-dimethylmorpholine.

Syrian golden hamsters were given a single dose of [3H]-N-nitroso-2,6-dimethylmorpholine and killed 8 hr later. The pancreas was processed for electron microscopic autoradiography to detect binding of radioactivity to cellular constituents. The pancreatic acinar cells and duct epithelia were found to be labeled, while islet cells, centroacinar cells, and all nonepithelial elements were not. Acinar cells active in secreting zymogen had a high concentration of grains over the zymogen granules and the rough endoplasmic reticulum. Their nonsecreting counterparts contained abundant bound material in the nuclei and rough endoplasmic reticulum. Labeling was lower in the duct epithelia than in acinar cells, with the majority of grains associated with the heterochromatin. Our findings suggest that the acinar cells are the principle site of metabolic activation in this organ.

Membrane localization of H+ and HCO3- transporters in the rat pancreatic duct.

The pancreatic duct secretes alkaline fluid that is rich in HCO3- and poor in Cl-. The molecular mechanisms that mediate ductal secretion and are responsible for the axial gradients of Cl- and HCO3- along the ductal tree are not well understood because H+ and HCO3- transport by duct cells have not been characterized or localized. To address these questions, we microdissected the intralobular, main, and common segments of the rat pancreatic duct. H+ and HCO3- transporters were characterized and localized by following intracellular pH while perfusing the bath and the lumen of the ducts. In intralobular ducts, Na(+)-dependent and amiloride-sensitive recovery from acid load in the absence of HCO3- was used to localize a Na+/H+ exchanger to the basolateral membrane (BLM). Modification of Cl- gradients across the luminal (LM) and BLM in the presence of HCO3- showed the presence of Cl-/HCO3- exchangers on both membranes of intralobular duct cells. Measurement of the effect of Cl- on one side of the membrane on the rate and extent of pHi changes caused by removal and addition of Cl- to the opposite side suggested that both exchangers are present in the same cell. In the presence of HCO3-, intralobular duct cells used three separate mechanisms to extrude H+: (a) BLM-located Na+/H+ exchange, (b) Na(+)-independent vacuolar-type H+ pump, and (c) BLM-located, Na(+)-dependent, amiloride-insensitive, and 4',4'-diisothiocyanatostilbene-2,2'-disulfonic acid sensitive mechanism, possibly a Na(+)-dependent HCO3- transporter. The main and common segments of the duct displayed similar mechanisms and localization of H+ and HCO3- transporters to the extent studied in the present work. In addition to the transporters found in intralobular ducts, the main and common ducts showed Na+/H+ exchange activity in the LM. Three tests were used to exclude a significant luminal to basolateral Na+ leak as the cause for an apparent luminal Na+/H+ exchange in an HCO3- secreting cells: (a) addition of amiloride and removal of Na+ from the LM had a profound effect on Na+/H+ exchange activity on the BLM and vice versa; (b) inhibition of all transporters in the BLM by bathing the duct in the inert hydrocarbon Fluorinert FC-75 did not prevent cytosolic acidification caused by removal of luminal Na+; and (c) luminal Na+ did not activate the basolateral Na(+)-dependent HCO3- transporter. An Na(+)-independent, bafilomycin-sensitive H+ pumping activity was marginal in the absence of HCO3-.(ABSTRACT TRUNCATED AT 400 WORDS)

Expression of a wild-type CFTR maintains the integrity of the biosynthetic/secretory pathway in human cystic fibrosis pancreatic duct cells.

The structural integrity of the Golgi complex is essential to its functions in the maturation, sorting, and transport of plasma membrane proteins. Previously, we demonstrated that in pancreatic duct CFPAC-1 cells, which express DeltaF508 CFTR (cystic fibrosis transmembrane conductance regulator), the intracellular trafficking of carbonic anhydrase IV (CA IV), a membrane protein involved in HCO(3)(-) secretion, was impaired. To determine whether these abnormalities were related to changes in the Golgi complex, we examined the ultrastructure and distribution of Golgi compartments with regard to the microtubule cytoskeleton in CFPAC-1 cells transfected or not with the wild-type CFTR. Ultrastructural and immunocytochemical analysis showed that in polarized CFPAC-1 cells, Golgi stacks were disconnected from one another and scattered throughout the cytoplasm. The colocalization of CA IV with markers of Golgi compartments indicated the ability of stacks to transfer this enzyme. This Golgi dispersal was associated with abnormal microtubule distribution and multiplicity of the microtubule-organizing centers (MTOCs). In reverted cells, the normalization of Golgi structure, microtubule distribution, and MTOC number was observed. These observations suggest that the entire biosynthetic/secretory pathway is disrupted in CFPAC-1 cells, which might explain the abnormal intracellular transport of CA IV. Taken together, these results point to the fact that the expression of DeltaF508 CFTR affects the integrity of the secretory pathway.

Cytochemical and immunocytochemical characterization of a fibrillar network (GP2) in pancreatic juice: possible role as a sieve in the pancreatic ductal system.

The secretory product of the exocrine pancreas contains sedimentable and non-sedimentable materials. Electron microscopy of the pellet obtained after ultracentrifugation reveals two major components: microvesicles (pancreasomes) and a fibrillar network of small mesh size. Negative staining of an unfixed pellet demonstrated that these structures are not fixation artifacts. Cytochemical analysis showed that pancreasomes are reactive to osmication and uranyl acetate staining, whereas the fibrillar network was unreactive thereby indicating that the latter does not contain lipids; however, lead citrate staining reveals the network. Alcian blue, known to bind sulfate groups of mucosubstances, reacted strongly with the fibrillar network. The pellet was also characterized by immunocytochemistry with specific antibodies to amylase and glycoprotein 2 (GP2). Both antibodies were located only on the fibrillar network. Washing of the pellet with 100 mM KCl-250 mM NaBr had little effect on GP2 content, but reduced considerably alpha-amylase associated with the reticular matrix. It appeared that GP2 was the major component of the scaffolding that gives rise to the fibrillar network and that other proteins such as alpha-amylase could reversibly bind to it. When double-labeling immunocytochemistry was carried out on the unwashed pellet, labeling of the first antigen reduced the labeling of the second. Removal of amylase by washing the pellet increased the GP2 signal. These results indicate that amylase is bound on the GP2 network. Although the function of the GP2 network is still not clearly defined several possibilities could be envisaged at the level of the pancreatic duct system: 1) The network could drain off any aggregates or precipitates forming in small ducts. 2) The small mesh of the network would present a physical barrier to infecting bacteria that could enter into the duct system from the intestine, especially in conditions of low flow rates. 3) The network may exert a mechanical pressure on the membranes bordering the acinar lumen and small ducts thereby preventing their collapse in basal conditions.

Pancreatic acinar cells in culture: expression of acinar and ductal antigens in a growth-related manner.

In the current study two monoclonal antibodies (mAb) were used to investigate the expression of adult acinar and duct cell-specific antigens and their relationship with cell growth in primary acinar cell cultures. We have previously found that adult mouse pancreatic acinar cells divide in primary culture. Furthermore, during growth the cells lose their differentiated morphology and exhibit decreased expression of secretory proteins, followed by some degree of morphological redifferentiation after reaching confluency. A mAb specific in the adult pancreas for acinar cells (mAb Acinar-1) and another specific in the adult pancreas for duct cells (mAb Duct-1) were generated using such cultures as the immunogen. The starting material for the cultures consisted of predominantly Acinar-1 positive cells which incorporated [3H]thymidine, as determined by autoradiography and immunofluorescence labeling. However, expression of the acinar antigen persisted for only the first 3 to 7 days in culture. By contrast, expression of the duct antigen was rare until after 5 days in culture and was highest at day 9, the peak of cell growth. Dual label immunofluorescence showed that during the growth phase fewer cells expressed the acinar antigen, most expressed the duct antigen, and occasional cells expressed both antigens. After reaching confluency, the growth rate declined from days 15 to 21, and the cells progressively regained the acinar antigen with a concomitant loss of the duct antigen. mAb labeling was morphometrically quantitated and showed that more than 97% of the labeled area was Acinar-1 positive at 3 days, which decreased to approximately 16% at day 9, and then returned to over 97% by day 21 of culture. Ultrastructural immunolabeling showed that Acinar-1 positive cells at 21 days had well organized rough endoplasmic reticulum and small apical vesicles, while Duct-1 positive cells were undifferentiated in appearance (day 9) or had numerous mitochondria (day 21). Thus, changes in cell-specific antigens were paralleled by cell type associated morphological characteristics and indicate that adult acinar cells can retrodifferentiate to a more duct-like cell while retaining the potential to express an acinar-specific antigen.

Quantitative immunoelectron microscopic localization of (Na+,K+)ATPase on rat exocrine pancreatic cells.

Distribution of (Na+,K+)ATPase in rat exocrine pancreatic cells was investigated quantitatively by immunoelectron microscopy using the post-embedding protein A-gold technique. We found that in acinar and duct cells (Na+,K+)ATPase exists on both the luminal and the basolateral surfaces, with higher particle density on the luminal surface (4.4 times in the acinar cells and 5.6 times in the duct cells). According to Bolender (J Cell Biol 61:269, 1974), the luminal surface represents only 5% of the total cell surface of an average pancreatic acinar cell. It is roughly estimated, therefore, that approximately 80% of the plasma membrane (Na+,K+)ATPase in the acinar cells exists on the basolateral surface. When the acinar and duct cells were compared, more than twice as many particles were found on acinar cells than on duct cells. The enzyme existed on all the cell surfaces, preferentially on the microvilli or on the cell membrane folds, and no clustering was detected. We suggest that the (Na+,K+)ATPase on the basolateral surface is mainly responsible for the extrusion of a large number of sodium ions that are incorporated into the cytoplasm accompanying the secondary active transport of various organic substances and inorganic ions, whereas that on the luminal surface is responsible for active extrusion of sodium ions that are partially responsible for the fluid secretion of the pancreatic cells.

Distribution of gamma-glutamyl transpeptidase in human pancreas: immunohistochemical study with a monoclonal antibody.

We studied in detail the distribution pattern of gamma-glutamyl transpeptidase (gamma-GT) in human pancreas, using the immunoperoxidase technique and a monoclonal antibody to human kidney gamma-GT. Positive reaction was confined exclusively to the luminal surface of the centroacinar cells and the epithelia of the intercalated, intralobular, and interlobular ducts. The immunoreaction was stronger in the intercalated and intralobular ducts than in the interlobular ducts. The acinar cells did not show any reaction. The islets of Langerhans were heavily surrounded by the ducts, and normal islet cells showed no reaction. The course of the ducts, from the acinar lumina to the interlobular ducts, was delineated by using reaction sites positive for gamma-GT as markers. The courses of the ducts, which comprise the pathway for pancreatic juice, were found to vary widely in their connections with each other, especially between the intralobular and interlobular ducts. At least three separate routes could be identified.

Carbonic anhydrase II associated with plasma membrane in a human pancreatic duct cell line (CAPAN-1).

The subcellular distribution of carbonic anhydrase II, either throughout the cytosol or in the cytoplasm close to the apical plasma membrane or vesicular compartments, suggests that this enzyme may have different roles in the regulation of pH in intra- or extracellular compartments. To throw more light on the role of pancreatic carbonic anhydrase II, we examined its expression and subcellular distribution in Capan-1 cells. Immunocytochemical analysis by light, confocal, and electron microscopy, as well as immunoblotting of cell homogenates or purified plasma membranes, was performed. A carbonic anhydrase II of 29 kD associated by weak bonds to the inner leaflet of apical plasma membranes of polarized cells was detected. This enzyme was co-localized with markers of Golgi compartments. Moreover, the defect of its targeting to apical plasma membranes in cells treated with brefeldin A was indicative of its transport by the Golgi apparatus. We show here that a carbonic anhydrase II is associated with the inner leaflet of apical plasma membranes and with the cytosolic side of the endomembranes of human cancerous pancreatic duct cells (Capan-1). These observations point to a role for this enzyme in the regulation of intra- and extracellular pH.