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.     

Properties of pancreatic zymogen granules studied by quasi-elastic light scattering

Physico-chemical properties of isolated zymogen granules of the mouse pancreas were studied by means of quasi-elastic light scattering. The average diameter of the granules in 0.3 M sucrose was found to be 1.1 ± 0.1 μm from the correlation time of intensity fluctuation of the scattered light. The average diameter altered depending on the osmolality of the medium in a manner that the alteration was smaller than that expected from the van't Hoff relation. Aggregation of the granules induced by the increase of Ca²⁺ concentration or the decrease of pH in the medium was also detected. The aggregation started at a critical level of 1 mM CaCl₂ or at pH 5.4.     

Oxidative injury to isolated rat pancreatic acinar cells vs. isolated zymogen granules

This study compares the susceptibility of pancreatic acinar cells and zymogen granules against oxidative injury and analyzes the mechanisms involved. Zymogen granules and acinar cells, isolated from rat pancreas, were exposed to a reaction mixture containing xanthine oxidase, hypoxanthine, and chelated iron. Cell function and viability were assessed by various techniques. Trypsin activation was quantified by an Elisa for trypsinogen activating peptide. Integrity of granules was determined by release of amylase. The reaction mixture rapidly generated radicals as assessed by deoxyribose and luminol assays. This oxidative stress caused lysis of granules in a matter of minutes but significant cell death only after some hours. Nevertheless, radicals initiated intracellular vacuolization, morphological damage to zymogen granules and mitochondria, increase in trypsinogen activating peptide, and decrease in ATP already after 5–30 min. Supramaximal caerulein concentrations also caused rapid trypsin activation. Addition of cells but not of granules reduced deoxyribose oxidation, suggesting that intact cells act as scavengers. Caerulein pretreatment only slightly increased the susceptibility of cells but markedly that of granules. In conclusion, isolated zymogen granules are markedly more susceptible to oxidative injury than intact acinar cells, in particular, in early stages of caerulein pancreatitis. The results show that oxidative stress causes a rapid trypsin activation that may contribute to cell damage by triggering autodigestion. Zymogen granules and mitochondria appear to be important targets of oxidative damage inside acinar cells. The series of intracellular events initiated by oxidative stress was similar to changes seen in early stages of pancreatitis.     

Rab27b localizes to zymogen granules and regulates pancreatic acinar exocytosis

To understand the function of pancreatic zymogen granules, we performed a proteomics analysis to identify ZG membrane components. Here we report the identification of Rab27b through this proteomics study and validate its role in granule function. MALDI-MS peptide mass fingerprint was matched to rat Rab27b with 43% sequence coverage, and the identification was also confirmed by tandem mass spectrometry. The localization of Rab27b on ZGs was confirmed by Western blotting and immunocytochemistry. To examine the function of Rab27b in acinar secretion, we overexpressed wild type and mutant Rab27b protein in pancreatic acini using recombinant adenoviruses. Wild type Rab27b had no effect on amylase secretion, while Rab27b Q78L enhanced, and Rab27b N133I inhibited, CCK-induced amylase release by 92+/-13% and 53+/-8%, respectively. This enhancement and inhibition occurred at all points on the CCK dose-response curve and over a 30min time course. These results demonstrate that Rab27b is present on ZGs and plays an important role in regulating acinar exocytosis.     

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.     

Ethanol augments elevated-[Ca2+]C induced trypsin activation in pancreatic acinar zymogen granules

It has been long recognized that significant percentage of patients with acute pancreatitis often presents with a history of excessive alcohol consumption; however, the patho-physiological effect of ethanol on acute pancreatitis remains poorly understood. Abnormally elevated cytosolic Ca²⁺ ([Ca²⁺]_C) has been found to be a shared phenomenon in acute pancreatitis that could induce trypsin premature activation. Here, we present the effects of ethanol to sensitize zymogen granules (ZGs) of pancreas acinar cells to elevated [Ca²⁺]_C leading to zymogen premature activation that could result in acute pancreatitis. The pH fluctuations ([pH]_G), Ca²⁺ concentration ([Ca²⁺]_G), and premature trypsin activation inside the ZGs were monitored directly with specific fluorescence indicators. Our results showed that ethanol could act directly on ZGs and cause ZGs more receptive to elevated [Ca²⁺]_C that could induce premature activation of zymogen (trypsin). This alcohol-induced effect is concentration dependent and strongly influenced by the surrounding [Ca²⁺]_C. The K⁺ channels on ZGs membranes are required in the sensitization process. Our observations provide a mechanistic understanding of the role of ethanol in the initiation phase of alcoholic pancreatitis.     

Complexin 2 Modulates Vesicle-associated Membrane Protein (VAMP) 2-regulated Zymogen Granule Exocytosis in Pancreatic Acini

Complexins are soluble proteins that regulate the activity of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes necessary for vesicle fusion. Neuronal specific complexin 1 has inhibitory and stimulatory effects on exocytosis by clamping trans-SNARE complexes in a prefusion state and promoting conformational changes to facilitate membrane fusion following cell stimulation. Complexins are unable to bind to monomeric SNARE proteins but bind with high affinity to ternary SNARE complexes and with lower affinity to target SNARE complexes. Far less is understood about complexin function outside the nervous system. Pancreatic acini express the complexin 2 isoform by RT-PCR and immunoblotting. Immunofluorescence microscopy revealed complexin 2 localized along the apical plasma membrane consistent with a role in secretion. Accordingly, complexin 2 was found to interact with vesicle-associated membrane protein (VAMP) 2, syntaxins 3 and 4, but not with VAMP 8 or syntaxin 2. Introduction of recombinant complexin 2 into permeabilized acini inhibited Ca²⁺-stimulated secretion in a concentration-dependent manner with a maximal inhibition of nearly 50%. Mutations of the central α-helical domain reduced complexin 2 SNARE binding and concurrently abolished its inhibitory activity. Surprisingly, mutation of arginine 59 to histidine within the central α-helical domain did not alter SNARE binding and moreover, augmented Ca²⁺-stimulated secretion by 130% of control. Consistent with biochemical studies, complexin 2 colocalized with VAMP 2 along the apical plasma membrane following cholecystokinin-8 stimulation. These data demonstrate a functional role for complexin 2 outside the nervous system and indicate that it participates in the Ca²⁺-sensitive regulatory pathway for zymogen granule exocytosis.     

Identification of a novel aquaporin, AQP12, expressed in pancreatic acinar cells

Members of the aquaporin (AQP) water channel family are widely distributed in various tissues and contribute to the water permeability of epithelial and endothelial cells. Currently 11 members of the AQP family (AQP0-10) have been reported in mammals. Here we report the identification of AQP12, which we found by performing a BLAST program search. Northern blot analysis revealed that AQP12 was specifically expressed in the pancreas. Further analysis by in situ hybridization and RT-PCR studies showed that AQP12 was selectively localized in the acinar cells of the pancreas. To analyze the cellular localization and function of AQP12, we expressed AQP12 in Xenopus oocytes and cultured mammalian cells. Immunocytochemistry revealed that AQP12 was not targeted to the plasma membrane. The selective localization of AQP12 in pancreatic acinar cells and possibly in the intracellular organelles suggests a role of AQP12 in digestive enzyme secretion such as maturation and exocytosis of secretory granules.     

Histochemical staining of zymogen granules of pancreatic acinar cells using a permanganate-HID-technique

Summary Using a permanganate-HID technique specific staining of cystine-containing zymogen granules of exocrine pancreas is possible. This method is based upon breaking and oxidation of disulphide bonds into sulphonic acids which subsequently are demonstrable with the HID-reaction.N,N-dimethyl-p-phenylendiamine-dihydrochloride was obtained from Fluka AG/ Basel; N,N-dimethyl-m-phenylendiamine-dihydrochloride from Schuchardt/Munich.     

The perinuclear space of pancreatic acinar cells and the synthetic pathway of zymogen in Scorpaena scrofa L.: ultrastructural aspects

Electron microscopic examination of exocrine pancreatic tissues from the fish Scorpaena scrofa L., probably captured while replenishing the acinar cells, shows two main functional cell morphologies of the same cell type. One cell functional aspect contains numerous well-contrasted small vesicles, the zymogenic vesicles. The other functional morphology is mainly represented by a few cells containing large apical zymogen vesicles with many empty RER cisterns. In our observations, the zymogenic vesicles are always studded with ribosomes. The main cytological finding is to report that zymogenic vesicles can be extruded from the perinuclear space and it confirms the suspected, synthetic activity of this cell compartment. The pool of zymogenic vesicles, maintaining their coat of ribosomes, then fuses and transfers their content into the cis Golgi complex network. Finally, the zymogen vesicles are produced following the classical secretory pathway from the trans Golgi saccular network into the supranuclear, apical region of the acinar cells where the largest vesicles concentrate their content until secretion.     

Morphometric and fine-structural studies of rat pancreatic acinar cells during early postnatal life

Summary Pancreatic acinar cells of rats obtained at 1,2, 3, 5, 7 and 14 days of age were examined using fine structural and morphometric techniques. From 5 days of age onwards, the acinar cells were analysed twice per day, at 20.00 h and 08.00 h.The present study demonstrates changes in the average volume of the cell, nucleus and cytoplasm, and volume densities of various cytoplasmic organelles during the first two weeks after birth. During early postnatal life, the volume density of rER increases, whereas that of zymogen granules decreases. From 5 days of age onwards, the volume densities of these two organelles differ significantly at 20.00 h and 08.00 h. During the first 2–3 days after birth, inclusion body-like structures appear in the cytoplasm of acinar cells; they contain aggregated zymogen granules and, sometimes, amorphous structures or cytoplasmic organelles. These structures also occur in interstitial cells and cells located in the intercalated region between acinar and ductal epithelial cells. Serum level of -amylase activity is high at birth, compared with other stages during the first three weeks. Degenerating acinar cells and cell debris can be seen in the acinar and ductal lumina during these stages, a feature suggesting holocrine secretion. Cellular polarity appears to be incomplete during the first two or three days after birth.     

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.     

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.     

A morphometric study of developing pancreatic acinar cells of rats during prenatal life

Summary The pancreatic acinar cells of rat embryos obtained at 15, 17, 19 and 21 days of gestation have been examined using fine-structural and morphometric techniques.Morphometric analysis demonstrates significant variations in the average volume of the cell, nucleus and cytoplasm, and the volume, surface and numerical densities of various cytoplasmic organelles during fetal life. In particular, the volume and surface densities of rER exhibit maximal values at 19 days of gestation, suggesting that secretory proteins are produced most actively at this time. Further-more, membrane continuity between the nuclear envelope and rER is frequently discernible in acinar cells, indicating that at this stage the rER is mainly derived from the nuclear envelope. Zymogen granules first appear at 17 days of gesstation. By 21 days, they occupy the greater part of the cytoplasm of the acinar cells, no polarity being seen in their distribution pattern. No direct evidence for the secretion of zymogen granules has been observed during fetal life.It therefore appears that membrane transport involved with intracellular movement of newly synthesized proteins from rER via the Golgi complex to zymogen granules occurs in one direction and lacks regulation.     

Regulation of zymogen granule exocytosis by Ca2+, cAMP, and PKC in pancreatic acinar cells

The effect of cAMP and PKC on zymogen granule exocytosis was investigated by simultaneously measuring cytosolic Ca2+ concentration ([Ca2+]c) and individual zymogen granule exocytosis in isolated mouse pancreatic acini. When acinar cells were stimulated with acetylcholine (ACh, 10 microM), exocytic events were detected through granule-attached apical membranes with [Ca2+]c rise. Application of secretin, forskolin (an adenylate cyclase activator), or PMA (a PKC activator) alone did not elicit any [Ca2+]c rise or zymogen granule exocytosis, but co-stimulation with ACh led to exocytosis in that the total number of secreted granules increased markedly without a significant difference in [Ca2+]c rises. When we evoked exocytosis by [Ca2+]c ramps, pretreatment with forskolin or PMA elicited exocytosis at lower [Ca2+]c levels. These results indicate that PKC or cAMP alone could not directly elicit zymogen granule exocytosis, but that they increase the total releasable pool by rendering zymogen granules more sensitive to Ca2+.     

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.     

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.     

Ca-regulated secretory granule exocytosis in pancreatic and parotid acinar cells

Protein secretion from acinar cells of the pancreas and parotid glands is controlled by G-protein coupled receptor activation and generation of the cellular messengers Ca²⁺, diacylglycerol and cAMP. Secretory granule (SG) exocytosis shares some common characteristics with nerve, neuroendocrine and endocrine cells which are regulated mainly by elevated cell Ca²⁺. However, in addition to diverse signaling pathways, acinar cells have large ∼1 μm diameter SGs (∼30 fold larger diameter than synaptic vesicles), respond to stimulation at slower rates (seconds versus milliseconds), demonstrate significant constitutive secretion, and in isolated acini, undergo sequential compound SG–SG exocytosis at the apical membrane. Exocytosis proceeds as an initial rapid phase that peaks and declines over 3 min followed by a prolonged phase that decays to near basal levels over 20–30 min. Studies indicate the early phase is triggered by Ca²⁺ and involves the SG proteins VAMP2 (vesicle associated membrane protein2), Ca²⁺-sensing protein synatotagmin 1 (syt1) and the accessory protein complexin 2. The molecular details for regulation of VAMP8-mediated SG exocytosis and the prolonged phase of secretion are still emerging. Here we review the known regulatory molecules that impact the sequential exocytic process of SG tethering, docking, priming and fusion in acinar cells.