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)

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.

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.

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.

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.

The three-dimensional microanatomy of the pancreatic duct system in the Japanese monkey, Macaca fuscata, with special reference to fine proximal passages of a high species-specificity.

We have previously shown the duct system in the rat pancreas to consist of two parts: a fine proximal (intercalated) duct and thicker distal (intralobular and interlobular) duct, with the latter part displaying morphological signs indicative of a bicarbonate-rich fluid secretion. In this study the pancreatic duct system in the Japanese monkey Macaca fuscata was observed by scanning electron microscopy after the hydrolytic exposure of cell surfaces as well as by transmission electron microscopy of ultrathin sections. Cellular expression of the water channel aquaporin 1 (AQP1) was also examined immunohistochemically. In contrast to the segmented duct system in the rat, all the duct cells in the monkey pancreas consistently displayed rich mitochondria in the cytoplasm, elaborate interdigitations of cell processes, and an intense immunoreactivity for AQP1 on the apical and basolateral cell membrane to favor active ion transport and osmotic water movement across the epithelium. Both the existence of secretory canaliculi and basal trabeculae in the duct epithelium and randomized localization of primary cilia on the luminal cell surfaces were demonstrated for the first time in monkeys, and the physiological implications of these phenomena are discussed.

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.

The ontogeny of the endocrine pancreas in the fetal/newborn baboon.

Erratic regulation of glucose metabolism including hyperglycemia is a common condition in premature infants and is associated with increased morbidity and mortality. The objective of this study was to examine histological and ultrastructural differences in the endocrine pancreas in fetal (throughout gestation) and neonatal baboons. Twelve fetal baboons were delivered at 125 days (d) gestational age (GA), 140d GA, or 175d GA. Eight animals were delivered at term (185d GA); half were fed for 5 days. Seventy-three nondiabetic adult baboons were used for comparison. Pancreatic tissue was studied using light microscopy, confocal imaging, and electron microscopy. The fetal and neonatal endocrine pancreas islet architecture became more organized as GA advanced. The percent areas of α-ß-δ-cell type were similar within each fetal and newborn GA (NS) but were higher than the adults (P<0.05) regardless of GA. The ratio of ß cells within the islet (whole and core) increased with gestation (P<0.01). Neonatal baboons, which survived for 5 days (feeding), had a 2.5-fold increase in pancreas weight compared with their counterparts killed at birth (P=0.01). Endocrine cells were also found in exocrine ductal and acinar cells in 125, 140 and 175d GA fetuses. Subpopulation of tissue that coexpressed trypsin and glucagon/insulin shows the presence of cells with mixed endo-exocrine lineage in fetuses. In summary, the fetal endocrine pancreas has no prevalence of a α-ß-δ-cell type with larger endocrine cell percent areas than adults. Cells with mixed endocrine/exocrine phenotype occur during fetal development. Developmental differences may play a role in glucose homeostasis during the neonatal period and may have long-term implications.

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.

Primary cilia deletion in pancreatic epithelial cells results in cyst formation and pancreatitis.

BACKGROUND & AIMS: Defects in cilia formation or function have been implicated in several human genetic diseases, including polycystic kidney disease (PKD), Bardet-Biedl syndrome, and primary ciliary dyskinesia. Pancreatic lesions are found in approximately 10% of PKD patients, suggesting a connection between cilia defects and pancreatic pathologies. Here, we investigate the role of cilia in pancreas formation and function by analyzing mice that lack cilia in pancreatic cells. METHODS: Using Cre/lox technology, we conditionally inactivated Kif3a, the gene encoding for a subunit of the kinesin-2 complex that is essential for cilia formation, in pancreatic epithelia. Kif3a mice were studied by immunohistochemical and biochemical methods to assess the morphology and differentiation status of pancreatic cells. RESULTS: Tissue-specific loss of Kif3a in pancreatic cells resulted in severe pancreatic abnormalities including acinar-to-ductal metaplasia, fibrosis, and lipomatosis. Ductal metaplasia appears to be due to expansion of ductal cells rather than transdifferentiation of acinar cells. Cyst formation, aberrant ductal morphology, and extensive fibrosis associated with severe adhesion to adjacent organs were commonly observed in aged Kif3a mutant mice. Deletion of Kif3a using different pancreas-specific Cre strains suggests that these pancreatic phenotypes might be caused by the absence of cilia in ductal cells. Activation of transforming growth factor beta and Mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK) pathways may play a role in these phenotypes. CONCLUSIONS: These results demonstrate that the absence of cilia in pancreatic cells produces pancreatic lesions that resemble those found in patients with chronic pancreatitis or cystic fibrosis.

Anatomical features of the minor duodenal papilla in pancreas divisum.

BACKGROUND: Clinical expression of pancreas divisum is often explained as a consequence of relative or true stenosis of the minor papilla with dorsal duct obstruction. This anatomo-functional study of the minor papilla in pancreas divisum has included its topographical, functional and structural features. MATERIALS AND METHODS: The study was carried out on 37 human autopsy specimens of duodenopancreas, which underwent pancreatography, manometrically controlled perfusion and light microscopy. RESULTS: One pancreas divisum was detected in the study group. In this case, the distances between the minor and the major papilla was 24.0 mm, and between the minor papilla and the superior duodenal flexure 27.4 mm. The minor papilla was patent when perfused under pressure of 10 mmHg, and its light microscopy revealed regular global histological organization with only light fibrosis and no cellular atypia. CONCLUSIONS: The structure and position of the minor papilla in pancreas divisum did not significantly differ from the ones in fused pancreases.

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.

Stimulation of cAMP signalling allows isolation of clonal pancreatic precursor cells from adult mouse pancreas.

AIMS/HYPOTHESIS: Duct cells of the pancreas are thought to include latent progenitors of islet endocrine cells that can be induced to differentiate by appropriate morphogens. Here we developed a method for isolating pancreatic ductal epithelial cells from adult mice that overcomes the shortcomings of previous methods. MATERIALS AND METHODS: Pancreatic ductal cells were grown in serum-free DMEM/F12 medium in the presence of cholera toxin or 8-bromo-cyclic adenosine monophosphate, which is known to be an intracellular cAMP generator. Single cell cloning was performed by limiting dilution in serum-free medium. RESULTS: The isolated clonal cells expressed high levels of cytokeratin and Ipf1 (formerly known as Pdx-1). Adenovirus-mediated expression of ngn3 (also known as Neurog3) and Ptf1a in these cells induced expression of insulin and somatostatin, and of carboxypeptidase A, respectively. Furthermore, albumin production was induced by dexamethasone or by long-term culture in serum-containing medium. CONCLUSIONS/INTERPRETATION: Stimulation of the cAMP-dependent signalling allowed us to isolate clonal pancreatic ductal cells from adult mice. These cells are able to partially differentiate into endocrine cells, exocrine cells and hepatocyte-like cells and are therefore considered to have the characteristics of endodermal progenitor cells.

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.

Morphological changes of pancreatic ducts in Otsuka Long-evans Tokushima Fatty rats.

OBJECTIVE: To determine if pancreatic ductal changes characteristic of chronic pancreatitis occur in male Otsuka Long-Evans Tokushima Fatty (OLETF) rats and to elucidate the 3-dimensional structure of the pancreatic ducts in those rats. METHODS: Male OLETF rats aged 10, 20, and 40 weeks were used. The pancreas was injected with a 120% barium sulfate suspension into the pancreatic ducts, after which radiograph images of the injected pancreas were taken using radiography of the soft parts. Conventional fixed pancreatic tissues were observed by the use of light and scanning electron microscopy, whereas corrosion casts of the pancreatic ducts were observed by the use of scanning electron microscopy. RESULTS: In 20- and 40-week-old male OLETF rats, deletion of acini and formation of tubular complexes and tortuous or helical, deformed, dilated pancreatic ducts were found, whereas the inner surfaces of pancreatic ducts had many craterlike depressions and long cilia. CONCLUSIONS: Male OLETF rats demonstrated the same morphological changes in pancreatic ductal lumina and histological changes in pancreatic tissues as the rats were subjected to pancreatic duct ligation. These results suggested that increased pancreatic duct pressure occurs in male OLETF rats and induces the chronic pancreatitis-like lesions, including the above-mentioned findings.

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.

ATP and ATPase secretion by exocrine pancreas in rat, guinea pig, and human.

ATP is an extracellular regulator in numerous physiological and pathologic processes. Recently, 7 different subtypes of purinoceptors were identified on either the basolateral or the luminal membrane of pancreatic duct cells. However, the in vivo regulatory role of ATP in pancreatic function has not been established. We investigated the possible regulatory role of endogenous ATP in pancreatic function by measuring ATP concentrations and ATPase activity in pancreatic juice obtained from anesthetized rats and guinea pigs and from human patients undergoing endoscopy. Juice was collected from the main pancreatic duct in rats and guinea pigs under basal conditions or during stimulation with CCK, bombesin, or secretin. In guinea pigs, CCK, bombesin, and secretin did not affect ATP output, although they did stimulate fluid secretion. ATPase activity in the juice was evaluated by measuring the rate of hydrolysis of added ATP. Consistent with the low ATP concentrations in rat pancreatic juice, we found high levels of ATPase activity in this species. This was confirmed by HPLC, which also showed the metabolites of ATP hydrolysis. Ecto-ATPase activity was demonstrated by enzyme histochemistry in both the pancreatic acini and ducts in rats, but it was not detectable in guinea pigs and humans. These differences in ATP levels and ATPase expression may indicate significant species differences in the purinergic regulation of pancreatic secretion.

Immunohistochemical localization of type IV collagen alpha chains in the basement membrane of the pancreatic duct in human normal pancreas and pancreatic diseases.

The type IV collagen (Col-IV) consists of 3 alpha chains. Six different alpha chains [alpha1(IV), alpha2(IV), alpha3(IV), alpha4(IV), alpha5(IV), and alpha6(IV)] have been identified, and their combination is considered to be organ specific. We investigated the immunohistochemical localization of alpha (IV) chains in the basement membrane (BM) of the pancreatic duct in human normal pancreas (NP) and pancreatic diseases. Fifty specimens [10 NP, 10 chronic pancreatitis (CP), 10 intraductal papillary mucinous tumor (IPMT), and 20 pancreatic adenocarcinoma (PAC)] were examined. Alpha 1(IV), alpha2(IV), alpha5(IV), and alpha6(IV) were linearly immunostained in NP, CP, and IPMT. In PAC, alpha(IV) and alpha2(IV) were immunostained, but alpha5(IV) and alpha6(IV) were not stained in 30% and 40% of the cases, respectively. In conclusion, immunohistochemically, the Col-IV of human normal pancreatic duct consisted of alpha1(IV), alpha2(IV), alpha5(IV), and alpha6(IV). alpha5(IV) and alpha6(IV) were frequently absent in PAC, and their absence might be related to the invasion of cancer cells.