Selective fluorescence of zymogen granules of pancreatic acinar cells stained with hematoxylin and eosin

Following staining with hematoxylin and eosin Y, paraffin sections of mouse pancreas were examined by transmitted light, epifluorescence and confocal laser scanning microscopy. Light microscopy revealed that the nuclei of pancreatic acinar cells were located basally, while the apices of the cells appeared eosinophilic, although the secretory granules were difficult to visualize. Under violet-blue light excitation, the zymogen granules at the apices of the acinar cells showed strong yellowish fluorescence; the other part of the cytoplasm was only faintly fluorescent and the nuclei and the supporting tissues were nonfluorescent. Confocal laser scanning microscopy resulted in clear pictures of the zymogen granules and their distribution within the cell. The fluorescent emission of zymogen granules was certainly the result of eosin Y staining, because hematoxylin is not a fluorochrome and the zymogen granules are not autofluorescent. Staining with eosin Y alone, however, did not result in clear fluorescent images of zymogen granules or any other cellular structures. Our observation shows that the fluorescence emission of eosin Y allows easy and precise recognition of zymogen granules of pancreatic cells.     

Selective fluorescence of zymogen granules of pancreatic acinar cells stained with hematoxylin and eosin

Following staining with hematoxylin and eosin Y, paraffin sections of mouse pancreas were examined by transmitted light, epifluorescence and confocal laser scanning microscopy. Light microscopy revealed that the nuclei of pancreatic acinar cells were located basally, while the apices of the cells appeared eosinophilic, although the secretory granules were difficult to visualize. Under violet-blue light excitation, the zymogen granules at the apices of the acinar cells showed strong yellowish fluorescence; the other part of the cytoplasm was only faintly fluorescent and the nuclei and the supporting tissues were nonfluorescent. Confocal laser scanning microscopy resulted in clear pictures of the zymogen granules and their distribution within the cell. The fluorescent emission of zymogen granules was certainly the result of eosin Y staining, because hematoxylin is not a fluorochrome and the zymogen granules are not autofluorescent. Staining with eosin Y alone, however, did not result in clear fluorescent images of zymogen granules or any other cellular structures. Our observation shows that the fluorescence emission of eosin Y allows easy and precise recognition of zymogen granules of pancreatic cells.     

Growth of rat pancreatic acinar cells quantitated with a monoclonal antibody against the proliferating cell nuclear antigen

The monoclonal antibody PC10 raised against the proliferating cell nuclear antigen (PCNA) was used to study acinar cell replication in the pancreas of rats under different functional conditions. In Western blots, the antibody recognized a single band of 37 kDa in pancreatic homogenates indicating its specificity in this particular species and organ. Three conditions of growth were chosen for immunohistochemical analysis: pancreatic preand postnatal development, pancreatic regeneration after injury, and cholecystokinin-stimulated acinar cell proliferation. The time course of acinar cell replication under each condition was the same as that obtained after tritiated thymidine incorporation with subsequent autoradiography, indicating that the percentage of PCNA-positive cells reflects the pool of cycling cells in the models investigated. However, the absolute number of PCNA-positive cells was two to ten times higher than comparable labeling indices from ³H-thymidine autoradiography. This finding might reflect the half life of PCNA, which exceeds the duration of the S-phase. Thus, PCNA-positive cells not only represent S-phase cells, but also cells that have recently completed the cell cycle.     

In vitro transdifferentiation of adult pancreatic acinar cells into insulin-expressing cells

Despite a recent breakthrough in human islet transplantation for treating diabetes mellitus, the limited availability of insulin-producing tissue is still a major obstacle. Here, we studied whether adult pancreatic acinar cells have the potential to transdifferentiate into islet or beta cells. Pancreatic acini were isolated from 7- to 8-weeks-old male Sprague-Dawley rats and cultured in suspension. Within 1 week, most of the acinar cells lost amylase expression and converted to cells with a duct cell phenotype. Insulin-positive cells were also observed, mainly at the periphery of the acini-derived spheroids. Insulin gene and protein expression was increased. Presence of a few insulin-positive cells coexpressing cytokeratins suggests that a spontaneous acinar to ductal cell transdifferentiation process was further going on towards beta cells. This study provides the first evidence that adult pancreatic acinar cells could be differentiated into insulin-expressing cells in vitro.     

Functional aquaporin diversity in plants

Due to the fact that most plants are immobile, a rapid response of physiological processes to changing environmental conditions is essential for their survival. Thus, in comparison to many other organisms, plants might need a more sophisticated tuning of water balance. Among others, this is reflected by the comparable large amount of aquaporin genes in plant genomes. So far, aquaporins were shown to be involved in many physiological processes like root water uptake, reproduction or photosynthesis. Their classification as simple water pores has changed according to their molecular function into channels permeable for water, small solutes and/or gases. An adjustment of the corresponding physiological process could be achieved by regulation mechanisms. Concerning aquaporins these range from posttranslational modification, molecular trafficking to heteromerization of aquaporin isoforms. The aim of this review is to underline the function of the four plant aquaporin family subclasses with regard to the substrate specificity, regulation and physiological relevance.     

Scanning electron microscopy of dissociated pancreatic acinar cell surfaces

Summary The pancreatic acinar cell surfaces have been studied by SEM with a dissection technique and correlated with results obtained by TEM. The SEM results demonstrate characteristic arrangement of microplicae which in some areas are densely packed.In many areas, the microplicae are distributed in such a manner that they create zones with typical geometrical shapes and show a relatively smooth surface.These smooth areas may coincide, as indicated by correlated TEM results, with the limits of intimate contact between adjacent acinar cells which, in turn, represent part of the junctional complex. Another aspect revealed by these SEM preparations concerns the presence of groups of densely packed microplicae, arranged in regular rows and distributed along some grooves and/or infoldings of the cellular surface. On the basis of SEM and TEM information, it is likely that these structures correspond to intercellular (and possibly, in some cases, intracellular) canaliculi which topographically form a kind of extensive microlabyrinthine arrangement running along all the cell sides.One final point revealed by fractured samples concerns the finding of spherical zymogen droplets within the vesicles of the Golgi complex. Because in many scanning images these vesicles appear connected by small openings, it is suggested that they may represent a system of intercommunicating chambers (vacuoles) through which the zymogen droplets can be continuously accumulated and discharged into the acinar lumen.     

Aquaporin-9 is expressed in a mucus-secreting goblet cell subset in the small intestine

We analyzed the expression of aquaporins (AQPs) in the small intestine to elucidate their functions, and found that AQP9, which had not previously been detected there, is present in duodenum, jejunum, and ileum. AQP9 is expressed in colon as well, but not in stomach. Also, its expression in these intestinal sections is limited to the basolateral membranes of a goblet cell subset. Our finding that AQP9 is present specifically in goblet cells as mucus-secreting cells suggests its involvement in the synthesis and/or secretion of a certain kind of mucus which may protect the intestinal surface and smooth the flow of intestinal contents.     

Localization and trafficking of aquaporin 2 in the kidney

Aquaporins (AQPs) are membrane proteins serving in the transfer of water and small solutes across cellular membranes. AQPs play a variety of roles in the body such as urine formation, prevention from dehydration in covering epithelia, water handling in the blood-brain barrier, secretion, conditioning of the sensory system, cell motility and metastasis, formation of cell junctions, and fat metabolism. The kidney plays a central role in water homeostasis in the body. At least seven isoforms, namely AQP1, AQP2, AQP3, AQP4, AQP6, AQP7, and AQP11, are expressed. Among them, AQP2, the anti-diuretic hormone (ADH)-regulated water channel, plays a critical role in water reabsorption. AQP2 is expressed in principal cells of connecting tubules and collecting ducts, where it is stored in Rab11-positive storage vesicles in the basal state. Upon ADH stimulation, AQP2 is translocated to the apical plasma membrane, where it serves in the influx of water. The translocation process is regulated through the phosphorylation of AQP2 by protein kinase A. As soon as the stimulation is terminated, AQP2 is retrieved to early endosomes, and then transferred back to the Rab 11-positive storage compartment. Some AQP2 is secreted via multivesicular bodies into the urine as exosomes. Actin plays an important role in the intracellular trafficking of AQP2. Recent findings have shed light on the molecular basis that controls the trafficking of AQP2.     

A morphometric study of 24-hour variations in subcellular structures of the rat pancreatic acinar cell

Summary Subcellular structures of pancreatic acinar cells were examined at six evenly spaced time points in the 24-h period (light cycle: 06.00 h–18.00 h) in four Wistar male rats at each time point. At each sampling point, the area and circumference of acinar cell bodies and the area, number and circumference of their cytoplasmic organelles were measured using a semiautomatic computer system for morphometry and a point-counting method.The area, number and circumference-area ratio of the cytoplasmic organelles were subject to strong circadian variations, and the cellular area and circumference exhibited weak circadian variations. Variation pattern of the cytoplasmic organelles suggested an intracellular route for secretory proteins during a 24-h span. From the results it was possible to divide the 24-h period into three stages. 1. The resting or protein synthetic stage (00.00 h to 08.00h): the area of the rough surfaced endoplasmic reticulum (rER) was strongly increased, and that of zymogen granules was clearly decreased. 2. The granule accumulation stage (08.00h to 16.00h): the area of the rER was markedly decreased; that of zymogen granules was increased. 3. The secretion stage (16.00 h to 00.00): as a result of the release of zymogen granules from the acinar cell, the area of zymogen granules decreased, and that of the rER increased. The relationship between the area of the rER and zymogen granules varied in a reciprocal manner. Other cytoplasmic organelles, namely the Golgi complex, condensing vacuoles, mitochondria and lysosomes also varied prominently during the 24-h span, corresponding to variations in the rER and zymogen granules.     

Acinar cells and the development of pancreatic fibrosis

Pancreatic fibrosis is caused by excessive deposition of extracellular matrixes of collagen and fibronectin in the pancreatic tissue as a result of repeated injury often seen in patients with chronic pancreatic diseases. The most common causative conditions include inborn errors of metabolism, chemical toxicity and autoimmune disorders. Its pathophysiology is highly complex, including acinar cell injury, acinar stress response, duct dysfunction, pancreatic stellate cell activation, and persistent inflammatory response. However, the specific mechanism remains to be fully clarified. Although the current therapeutic strategies targeting pancreatic stellate cells show good efficacy in cell culture and animal models, they are not satisfactory in the clinic. Without effective intervention, pancreatic fibrosis can promote the transformation from pancreatitis to pancreatic cancer, one of the most lethal malignancies. In the normal pancreas, the acinar component accounts for 82% of the exocrine tissue. Abnormal acinar cells may activate pancreatic stellate cells directly as cellular source of fibrosis or indirectly via releasing various substances and initiate pancreatic fibrosis. A comprehensive understanding of the role of acinar cells in pancreatic fibrosis is critical for designing effective intervention strategies. In this review, we focus on the role of and mechanisms underlying pancreatic acinar injury in pancreatic fibrosis and their potential clinical significance.     

Acetylcholine-evoked uncoupling restricts the passage of Lucifer Yellow between pancreatic acinar cells

Summary Direct cell to cell movement of the fluorescent dye Lucifer Yellow CH (457 daltons) in exocrine acinar tissue is demonstrated by direct observation of living mouse pancreatic segments. Electrical uncoupling of pancreatic acinar cells by local application of a high concentration of acetylcholine significantly restricts cell to cell passage of the fluorescent dye. This result shows that a secretagogue can control direct movement of organic molecules between cells through junctional channels.     

Non-uniform distribution of mitochondria in pancreatic acinar cells

The distribution of mitochondria in pancreatic acinar cells was investigated using confocal fluorescence microscopy and transmission electron microscopy (EM). Acinar cells were studied either after enzymatic isolation or in small segments of undisassociated pancreatic tissue. Loading of isolated acinar cells with Mito Tracker Green or Red, a fluorescence mitochondrial probe, showed that mitochondria are predominantly situated in the perigranular, subplasmalemmal and perinuclear regions. Subsequent applications of EM fixatives induced a leak of the fluorescent indicator to the cytosol but did not change the distribution of mitochondria. EM was then performed on isolated acinar cells and on acinar cells of pancreatic tissue segments. The intracellular distribution of mitochondria was quantified by calculating the percentage of the cross-sectional area that was occupied by mitochondria. In isolated acinar cells the highest density of mitochondria was seen in the perigranular region, where mitochondria occupied 25.69±1.58% of the area, then the subplasmalemmal region with 12.61±0.77% and the perinuclear region with 9.07±0.97% (n=26). Similar results were obtained from acinar cells of pancreatic tissue segments: the perigranular 22.9±1.95%, subplasmalemmal 12.45±0.78% and perinuclear regions 9.07±0.97% (n=26). The outer mitochondrial membranes were frequently positioned close to membranes of the ER, which followed the outer contour of mitochondria. Mitochondria were never found in direct contact with the nuclear envelope: there were usually layers of ER between the mitochondrial and nuclear membranes. Subplasmalemmal mitochondria were found in a very close proximity to the plasma membrane with no ER layers between the mitochondrial and the corresponding plasma membranes. We conclude that in pancreatic acinar cells mitochondria are preferentially distributed to perigranular, subplasmalemmal and perinuclear regions and this distribution is not affected by isolation or fixation procedures.P.R. Johnson and N.J. Dolman contributed equally to the study. This work was supported by a Medical Research Council programme grant. P.R.J. is a Medical Research Council PhD student and N.J.D. is a Wellcome Trust PhD student.     

Distribution of aquaporin 4 on sarcolemma of fast-twitch skeletal myofibres

The aquaporin 4 (AQP4) water channel is present on the sarcolemma of fast-twitch-type skeletal myofibres. We have examined the distribution of AQP4 in relation to sarcolemmal domain structure and found that AQP4 protein is not evenly distributed on the sarcolemma. Immunofluorescence staining of isolated single myofibres indicated a punctate staining pattern overlapping with the dystrophin glycoprotein complex, but with the transverse tubule openings being left clear. Myotendinous and neuromuscular junctions also lacked AQP4, despite their high content of the dystrophin glycoprotein complex. The destruction of caveoli with methyl-β-cyclodextrin did not change the distribution of AQP4 at the sarcolemma. Moreover, AQP4 did not float with the caveolar marker caveolin-3 in sucrose gradients after Triton X-100 extraction at 4°C. These data indicated that AQP4 was not associated with caveoli. Surprisingly, m. flexor digitorum brevis fibres, although of the fast-twitch type, often lacked AQP4. Furthermore, those fibres harbouring AQP4 at the sarcolemma showed a regionalized distribution, suggesting that large areas were devoid of the protein. Blockage of the synthesized proteins in the endoplasmic reticulum with brefeldin A showed that, in spite of its regionalized sarcolemmal distribution, AQP4 was synthesized along the entire length of the fibres. These results suggest functional differences in the water permeability of the sarcolemma not only between the fast-twitch muscles, but also within single muscle fibres.     

Insulin protects pancreatic acinar cells from cytosolic calcium overload and inhibition of plasma membrane calcium pump

Acute pancreatitis is a serious and sometimes fatal inflammatory disease of the pancreas without any reliable treatment or imminent cure. In recent years, impaired metabolism and cytosolic Ca(2+) ([Ca(2+)](i)) overload in pancreatic acinar cells have been implicated as the cardinal pathological events common to most forms of pancreatitis, regardless of the precise causative factor. Therefore, restoration of metabolism and protection against cytosolic Ca(2+) overload likely represent key therapeutic untapped strategies for the treatment of this disease. The plasma membrane Ca(2+)-ATPase (PMCA) provides a final common path for cells to "defend" [Ca(2+)](i) during cellular injury. In this paper, we use fluorescence imaging to show for the first time that insulin treatment, which is protective in animal models and clinical studies of human pancreatitis, directly protects pancreatic acinar cells from oxidant-induced cytosolic Ca(2+) overload and inhibition of the PMCA. This protection was independent of oxidative stress or mitochondrial membrane potential but appeared to involve the activation of Akt and an acute metabolic switch from mitochondrial to predominantly glycolytic metabolism. This switch to glycolysis appeared to be sufficient to maintain cellular ATP and thus PMCA activity, thereby preventing Ca(2+) overload, even in the face of impaired mitochondrial function.     

Comparative simulations of aquaporin family: AQP1, AQPZ, AQP0 and GlpF

Molecular dynamics simulations were performed for four members of the aquaporin family (AQP1, AQPZ, AQP0, and GlpF) in the explicit membrane environment. The single-channel water permeability, pf, was evaluated to be GlpF approximately AQPZ > AQP1 > AQP0, while their relative pore sizes were GlpF > AQP1 > AQPZ > AQP0. This relation between pf and pore size indicates that water permeability was determined not only by the channel radius, but also another competing factor. Analysis of water dynamics revealed that this factor was the single-file nature of water transport.     

Membrane trafficking of AQP5 and cAMP dependent phosphorylation in bronchial epithelium

Phosphorylation pathway has been identified as an important step in membrane trafficking for AQP5. We generated stably transfected BEAS-2B human bronchial epithelial cells with various over-expression constructs on permeable support. In stable cells with wild-type AQP5 and S156A (AQP5 mutant targeting PKA consensus sequence), AQP5 expression was predominantly polarized to the apical membrane, whereas stable cells with N185D (AQP5 mutant targeting second NPA motif), mainly localized to the cytoplasm. Treatment with H89 and/or chlorophenylthio-cAMP (cpt-cAMP) did not affect membrane expression of AQP5 in any of three stable cells. In cells with wild-type AQP5 and N185D, AQP5s were phosphorylated by PKA, while phosphorylation of AQP5 was not detected in cells with S156A. These results indicate that, in AQP5, serine156 may be phosphorylated by PKA, but membrane expression of AQP5 may not be regulated by PKA phosphorylation. We conclude that AQP5 membrane targeting can include more than one mechanism besides cAMP dependent phosphorylation.     

Expression of aquaporin 1 and aquaporin 4 water channels in rat choroid plexus

The role of aquaporins in cerebrospinal fluid (CSF) secretion was investigated in this study. Western analysis and immunocytochemistry were used to examine the expression of aquaporin 1 (AQP1) and aquaporin 4 (AQP4) in the rat choroid plexus epithelium. Western analyses were performed on a membrane fraction that was enriched in Na⁺/K⁺-ATPase and AE2, marker proteins for the apical and basolateral membranes of the choroid plexus epithelium, respectively. The AQP1 antibody detected peptides with molecular masses of 27 and 32 kDa in fourth and lateral ventricle choroid plexus. A single peptide of 29 kDa was identified by the AQP4 antibody in fourth and lateral ventricle choroid plexus. Immunocytochemistry demonstrated that AQP1 is expressed in the apical membrane of both lateral and fourth ventricle choroid plexus epithelial cells. The immunofluorescence signal with the AQP4 antibody was diffusely distributed throughout the cytoplasm, and there was no evidence for AQP4 expression in either the apical or basolateral membrane of the epithelial cells. The data suggest that AQP1 contributes to water transport across the apical membrane of the choroid plexus epithelium during CSF secretion. The route by which water crosses the basolateral membrane, however, remains to be determined.     

In-vivo stimulation of rat pancreatic acinar cells by infusion of secretin

Summary Infusion of synthetic secretin in conscious unrestricted rats for periods up to 24 h was used to study the structural and functional adaptation of pancreatic acinar cells to this secretagogue. Initial dose-response studies established 16 clinical units (CU) per kg and h (corresponding to 4.64 ug x kg^-1 x h^-1) as optimal dose for persistent stimulation of enzyme discharge. Infusion of this dose led to a slow but progressive depletion of enzyme stores with minimal content by 12 h stimulation. As a result of persistent stimulation total protein synthesis in the acinar cells increased after a lag period of 3 h and reached maximal values 90% above controls by 6 and 12 h secretin infusion. No structural equivalent for pronounced fluid and bicarbonate secretion was observed for either acinar or duct cells over the entire dose range (1 to 64 CU x kg^-1 x h^-1) and infusion period (1–24 h), except an increased number of coated vesicles in duct cells.Discharge of enzymes from acinar cells was paralleled by a high frequency of exocytotic images at the luminal plasma membrane and was accompanied by the occurrence of membrane fragments in the luminal space, especially after 3 and 6 h secretin infusion. An increased number of lysosomal bodies at these time points especially in the vicinity of the Golgi complex was interpreted in relation to membrane recycling following massive exocytosis. This pattern of structural and functional adaptation of acinar cells following secretin infusion corresponds to previously described changes following caerulein and carbamylcholine stimulation.Supported by a grant from Deutsche Forschungsgemeinschaft (Ke 113/15-1)