Search

The sequence of Aquaporin 12 (AQP12) cDNA was amplified from spiny dogfish (Squalus acanthias) cDNAs using degenerate PCR, followed by 5′ and 3′ RACE PCR. The AQP12 nucleotide sequence had an open reading frame of 300 amino acids, which included one or more N-glycosylation sites. Degenerate and tissue PCRs revealed that AQP12 is expressed at the highest levels in the liver, followed by the pyloric stomach and the esophagus/cardiac stomach, with a small amount potentially present in the eye. A polyclonal antibody was made using a peptide from the derived amino acid sequence. Western blotting with the antibody showed faint banding around the size expected (33 kDa) by the 300 amino acid protein. A few more intense bands were seen at around 40 kDa and larger sizes. Immunohistochemistry in cardiac stomach tissue sections showed staining in a few sporadic paneth-like secretory cells along the surface of the epithelium. High-magnification imaging showed that the AQP12 staining was located in the membrane of secretory granules in the apical pole of the cells. This localization is reminiscent of the AQP12 localization in pancreatic acinar cells, where it is found in the membrane of zymogen granules containing digestive enzymes. [ABSTRACT FROM AUTHOR]

The sequence of Aquaporin 12 (AQP12) cDNA was amplified from spiny dogfish (Squalus acanthias) cDNAs using degenerate PCR, followed by 5′ and 3′ RACE PCR. The AQP12 nucleotide sequence had an open reading frame of 300 amino acids, which included one or more N-glycosylation sites. Degenerate and tissue PCRs revealed that AQP12 is expressed at the highest levels in the liver, followed by the pyloric stomach and the esophagus/cardiac stomach, with a small amount potentially present in the eye. A polyclonal antibody was made using a peptide from the derived amino acid sequence. Western blotting with the antibody showed faint banding around the size expected (33 kDa) by the 300 amino acid protein. A few more intense bands were seen at around 40 kDa and larger sizes. Immunohistochemistry in cardiac stomach tissue sections showed staining in a few sporadic paneth-like secretory cells along the surface of the epithelium. High-magnification imaging showed that the AQP12 staining was located in the membrane of secretory granules in the apical pole of the cells. This localization is reminiscent of the AQP12 localization in pancreatic acinar cells, where it is found in the membrane of zymogen granules containing digestive enzymes.

Diabetes mellitus (DM) is a metabolic disorder with rising incidences worldwide. Gastric symptoms of DM have been reported, including nausea, vomiting, bloating, and epigastric pain. Moreover, acute to chronic gastritis and atrophic gastritis occur in DM can affect the chief cells of the gastric gland. Chief cells are vital because of their ability to digest and separate vitamin B12 from protein. Lack of vitamin B12 leads to impaired DNA synthesis and abnormal metabolism in red blood cells, and eventually leading to pernicious anemia. Furthermore, decreased vibratory and positional senses, numbness, ataxia with subacute combined degeneration, and dementia are present in pernicious anemic patients. Twenty-four male adult Sprague-Dawley rats were used in this study. The rats were divided into control (n = 12) and diabetic (n = 12) groups. The rats were further separated into two categories: short-term (4 weeks) and long-term (24 weeks) groups. DM model was induced by manually injecting intraperitoneally with streptozotocin in citrate buffer at a dose of 60 mg/kg body weight. The same amount of buffer was injected into the control group. After sacrifice, three regions of the stomach (the cardia, body, and pylorus) were dissected. Histopathology was performed by staining with toluidine blue. Image analysis was used to quantify the zymogen granule accumulation in chief cells. The data were compared between the control and DM rats in each period using Student's t-test. In addition, transmission electron microscopy (TEM) was also used to examine the ultrastructures. There was a significant decrease in the percentage of zymogen granules in DM rats. Under TEM, the destructions of mitochondria, rough endoplasmic reticulum, and Golgi apparatus in the DM rat were observed in the chief cells. In rats with uncontrolled diabetes, there is damage to the chief cells all over the area of the stomach, affecting digestion and malabsorption of vitamin B12. Therefore, this result helps clinicians recognize that diabetic patients with gastric symptoms may have hidden pernicious anemia. [ABSTRACT FROM AUTHOR]

This study was conducted on the pancreas of 06 turkey birds. The pancreas of turkey is located between the descending and ascending parts of the duodenum with total length of 9.82 ± 0.28 cm, width of 2.17 ± 0.10 cm and thickness of 1.12 ± 0.08 cm. Histologically, the parenchyma of pancreas consisted of both exocrine and endocrine portion. The exocrine part composed of serous tubulo-acinar glands. Acinar cells were pyramidal to tall columnar in shape with round, basally situated nucleus with a prominent nucleolus and apically located acidophilic zymogen granules. The average longer diameter was 50.73 ± 5.59 µ whereas the smaller diameter was 35.08 ± 3.80 µ. The average height of acinar cell was 15.63 ± 1.59 µ with average nuclear diameter of 6.87 ± 0.35 µ. The ducts located within the lobules were small and lined by simple cuboidal to low columnar epithelium. The endocrine part consisted of islets of Langerhans. The average longer diameter was 49.26 ± 1.41 µ whereas the smaller diameter was 43.61 ± 1.23 µ. [ABSTRACT FROM AUTHOR]

We herein describe a rare case of low‐grade endobronchial tumor that exhibited two distinct features of typical carcinoid and acinic cell carcinoma (ACC) by immunohistochemical and ultrastructure study. ACC was suspected on transbronchial biopsy. The resected specimen showed that the tumor surface comprised an acinic cell component (40% of the tumor), and the central area comprised typical carcinoid (60% of the tumor). The acinic cell component was positive for chromogranin A, synaptophysin and alpha‐1‐antichymotrypsin. Additionally, this component showed focal apical membranous staining for DOG1 and weak positivity for BCL10 and SOX10. Conversely, the carcinoid component was negative for all proteins except for chromogranin A and synaptophysin. Electron microscopy indicated zymogen‐type granules (600–800 nm in diameter) in the acinic cell component, whereas neuroendocrine‐type granules (200–300 nm in diameter) were observed in the carcinoid component. Nuclear NR4A3 immunostaining, which is highly specific for ACC of the salivary gland, was negative in this case. We conclude that the pulmonary carcinoid tumor with true zymogen‐type granules could be seen but showed superficial similarities to ACC based on negative nuclear staining for NR4A3. Pulmonary carcinoids encompass a wide morphological spectrum and may exhibit prominent acinic cell differentiation. [ABSTRACT FROM AUTHOR]

Pancreas gets affected by fibrosis associated with aging. This study analyzed the age-related fibrotic changes in the exocrine as well as endocrine system of the pancreas. After obtaining necessary ethical clearances 30 post-natal and adult pancreases were collected and processed to obtain resin-embedded sections for transmission electron microscopy and paraffin-embedded sections for H and E staining and light microscopy. The sections were analyzed qualitatively and quantitatively. It was observed with ageing, the ductal epithelial cytoplasm contained many lipoid bodies. The basal lamina and the connective tissue around ducts increased. Periductal fibrosis appeared during fourth decade in Indian population whereas it appears sixth decade in Europeans. There was a direct correlation between area of the ducts and increasing age. Stellate cells and centroacinar cells increased with aging. The cytoplasmic processes of the centroacinar cells covered the acini and ductal epithelial cells, indicating their important function. The centroacinar cells have a regulatory role in secretory process during normal and pathological conditions. Increased fibrosis was noted in and around the islets of Langerhans. Epithelial hyperplasia, papillary projections, and periductal fibrosis around small and medium sized duct started very early in Indian populations indicating the vulnerability to pancreatic diseases in the Indian population in early ages.

Pancreas gets affected by fibrosis associated with aging. This study analyzed the age-related fibrotic changes in the exocrine as well as endocrine system of the pancreas. After obtaining necessary ethical clearances 30 post-natal and adult pancreases were collected and processed to obtain resin-embedded sections for transmission electron microscopy and paraffin-embedded sections for H&E staining and light microscopy. The sections were analyzed qualitatively and quantitatively. It was observed with ageing, the ductal epithelial cytoplasm contained many lipoid bodies. The basal lamina and the connective tissue around ducts increased. Periductal fibrosis appeared during fourth decade in Indian population whereas it appears sixth decade in Europeans. There was a direct correlation between area of the ducts and increasing age. Stellate cells and centroacinar cells increased with aging. The cytoplasmic processes of the centroacinar cells covered the acini and ductal epithelial cells, indicating their important function. The centroacinar cells have a regulatory role in secretory process during normal and pathological conditions. Increased fibrosis was noted in and around the islets of Langerhans. Epithelial hyperplasia, papillary projections, and periductal fibrosis around small and medium sized duct started very early in Indian populations indicating the vulnerability to pancreatic diseases in the Indian population in early ages. [ABSTRACT FROM AUTHOR]

The comparative structure and expression of salivary components and vesicular transport proteins in the canine major salivary glands were investigated. Histochemical analysis revealed that the morphology of the five major salivary glands—parotid, submandibular, polystomatic sublingual, monostomatic sublingual, and zygomatic glands—was greatly diverse. Immunoblot analysis revealed that expression levels of α-amylase and antimicrobial proteins, such as lysozyme, lactoperoxidase, and lactoferrin, differed among the different glands. Similarly, Rab proteins (Rab3d, Rab11a, Rab11b, Rab27a, and Rab27b) and soluble N -ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) proteins VAMP4, VAMP8, syntaxin-2, syntaxin-3, syntaxin-4, and syntaxin-6 were expressed at various levels in individual glands. mmunohistochemistry of Rab3d, Rab11b, Rab27b, VAMP4, VAMP8, syntaxin-4, and syntaxin-6 revealed their predominant expression in serous acinar cells, demilunes, and ductal cells. The VAMP4/syntaxin-6 SNARE complex, which is thought to be involved in the maturation of secretory granules in the Golgi field, was found more predominantly in the monostomatic sublingual gland than in the parotid gland. These results suggest that protein expression profiles in canine salivary glands differ among individual glands and reflect the properties of their specialized functions. [ABSTRACT FROM AUTHOR]

Small G protein Rab27B is expressed in various secretory cell types and plays a role in mediating secretion. In pancreatic acinar cells, Rab27B was found to be expressed on the zymogen granule membrane and by overexpression to regulate the secretion of zymogen granules. However, the effect of Rab27B deletion on the physiology of pancreatic acinar cells is unknown. In the current study, we utilized the Rab27B KO mouse model to better understand the role of Rab27B in the secretion of pancreatic acinar cells. Our data show that Rab27B deficiency had no obvious effects on the expression of major digestive enzymes and other closely related proteins, e.g. similar small G proteins, such as Rab3D and Rab27A, and putative downstream effectors. The overall morphology of acinar cells was not changed in the knockout pancreas. However, the size of zymogen granules was decreased in KO acinar cells, suggesting a role of Rab27B in regulating the maturation of secretory granules. The secretion of digestive enzymes was moderately decreased in KO acini, compared with the WT control. These data indicate that Rab27B is involved at a different steps of zymogen granule maturation and secretion, which is distinct from that of Rab3D. [ABSTRACT FROM AUTHOR]

Zymogen granules (ZG) are specialized organelles in the exocrine pancreas which allow digestive enzyme storage and regulated secretion. The molecular mechanisms of their biogenesis and the sorting of zymogens are still incompletely understood. Here, we investigated the role of proteoglycans in granule formation and secretion of zymogens in pancreatic AR42J cells, an acinar model system. Cupromeronic Blue cytochemistry and biochemical studies revealed an association of proteoglycans primarily with the granule membrane. Removal of proteoglycans by carbonate treatment led to a loss of membrane curvature indicating a supportive role in the maintenance of membrane shape and stability. Chemical inhibition of proteoglycan synthesis impaired the formation of normal electron-dense granules in AR42J cells and resulted in the formation of unusually small granule structures. These structures still contained the zymogen carboxypeptidase, a cargo molecule of secretory granules, but migrated to lighter fractions after density gradient centrifugation. Furthermore, the basal secretion of amylase was increased in AR42J cells after inhibitor treatment. In addition, irregular-shaped granules appeared in pancreatic lobules. We conclude that the assembly of a proteoglycan scaffold at the ZG membrane is supporting efficient packaging of zymogens and the proper formation of stimulus-competent storage granules in acinar cells of the pancreas. [ABSTRACT FROM AUTHOR]

Abstract: 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 ∼1μm 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 3min followed by a prolonged phase that decays to near basal levels over 20–30min. 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. [Copyright &y& Elsevier]

Abstract: Zymogen granules (ZG) are specialized organelles in the exocrine pancreas which allow digestive enzyme storage and regulated secretion. To investigate ZG biogenesis, cargo sorting and packaging, suitable cellular model systems are required. Here, we demonstrate that granule formation in pancreatic AR42J cells, an acinar model system, can be modulated by altering the growth conditions in cell culture. We find that cultivation of AR42J cells in Panserin™ 401, a serum-free medium, enhances the induction of granule formation in the presence or absence of dexamethasone when compared to standard conditions including serum. Biochemical and morphological studies revealed an increase in ZG markers on the mRNA and protein level, as well as in granule size compared to standard conditions. Our data indicate that this effect is related to pronounced differentiation of AR42J cells. To address if enhanced expression of ZG proteins promotes granule formation, we expressed several zymogens and ZG membrane proteins in unstimulated AR42J cells and in constitutively secreting COS-7 cells. Neither single expression nor co-expression was sufficient to initiate granule formation in AR42J cells or the formation of granule-like structures in COS-7 cells as described for neuroendocrine cargo proteins. The importance of our findings for granule formation in exocrine cells is discussed. [Copyright &y& Elsevier]

Rab3D is a low molecular weight GTP-binding protein that associates with secretory granules in exocrine cells. AR42J cells are derived from rat pancreatic exocrine tumor cells and develop an acinar cell-like phenotype when treated with dexamethasone (Dex). In the present study, we examined the role of Rab3D in Dex-treated AR42J cells. Rab3D expression and localization were analyzed by subcellular fractionation and immunoblotting. The role of Rab3D was examined by overexpressing myc-labeled wild-type-Rab3D and a constitutively active form of Rab3D (Rab3D-Q81L) in AR42J cells. We found that Rab3D is predominantly membrane-associated in AR42J cells and co-localizes with zymogen granules (ZG). Following CCK-8-induced exocytosis, amylase-positive ZGs appeared to move towards the periphery of the cell and co-localization between Rab3D and amylase was less complete when compared to basal conditions. Overexpression of WT, but not mutant Rab3D, resulted in an increase in cellular amylase levels. Overexpression of mutant and WT Rab3D did not affect granule morphology, CCK-8-induced secretion, long-term (48 hr) basal amylase release or granule density. We conclude that Rab3D is not involved in agonist-induced exocytosis in AR42J cells. Instead, Rab3D may regulate amylase content in these cells. [ABSTRACT FROM AUTHOR]

We developed a novel method for enhancing light-microscopic visualization of pancreatic zymogen granules in a selective manner on hematoxylin and eosin-stained sections. By using an absorption filter that transmits light with wavelength from 510 to 550 nm, corresponding to the narrow absorption spectrum of eosin, only eosinophilic tissue and cellular components were remarkably highlighted as distinct shadows against lighter background consisting of basophilic components. Using a pair of mirror sections of the pancreas, immunocytochemistry with anti-amylase antibody confirmed that the shadows observed through the filter represented zymogen granules. Immersion in formalin for 36 h at room temperature was the optimal fixation condition. Here we designate the procedure as the 'eosin-shadow method' and propose that this technique is convenient and useful to help investigators identify zymogen granules more easily in routine pathological examination and histological studies. [ABSTRACT FROM AUTHOR]

The heat shock protein 70 family members Hsc70 and Hsp70 are known to play a protective role against the onset of experimental pancreatitis, yet their molecular function in acini is unclear. Cysteine string protein (CSP-α) is a zymogen granule (ZG) membrane protein characterized by an NH2-terminal "J domain" and a central palmitoylated string of cysteine residues. The J domain functions as a cochaperone by modulating the activity of Hsc70/Hsp70 family members. A role for CSP-α in regulating digestive enzyme exocytosis from pancreas was investigated by introducing CSP-α truncations into isolated following their permeabilization with Perfringolysin O. Incubation of acini with CSP-α1-82, containing the J domain, significantly augmented Ca2+-stimulated amylase secretion. Effects of CSP-α1-82 were concentration dependent, with a maximum 80% increase occurring at 200 μg/ml of protein. Although CSP-α1-82 had no effects on basal secretion measured in the presence of ≤ 10 nM free Ca2+, it did significantly augment GTP-γS-induced secretion under basal Ca2+ conditions by -25%. Mutation of the J domain to abolish its cochaperone activity failed to augment Ca2+-stimulated secretion, implicating the CSP-α/Hsc70 cochaperone system as a regulatory component of the secretory pathway. CSP-α physically associates with vesicle-associated membrane protein 8 (VAMP 8) on ZGs, and the CSP-α- VAMP 8 interaction was dependent on amino acids 83-112 of CSP-α. Immunofluorescence analysis of acinar lobules or purified ZGs confirmed the CSP-α colocalization with VAMP 8. These data establish a role for CSP-α in regulating digestive enzyme secretion and suggest that CSP-α and Hsc70 modulate specific soluble N-ethylmaleimide- sensitive attachment receptor interactions necessary for exocytosis. [ABSTRACT FROM AUTHOR]

Abstract: Trypsin premature activation has been thought to be a key event in the initiation phase of acute pancreatitis. Here we test a hypothesis that a sustained increase of cytosolic Ca2+ concentration ([Ca2+]C) can trigger K+ influx into pancreas acinar zymogen granules (ZGs) via a Ca2+-activated K+ channel (KCa), and this influx of K+ then mobilizes bound-Ca2+ by K+/Ca2+ ion-exchange to increase free Ca2+ concentration in the ZGs ([Ca2+]G) and release bound-H+ by K+/H+ ion-exchange to decrease the pH in ZGs (pHG). Both the increase of [Ca2+]G and the decrease of pHG will facilitate trypsinogen autoactivation and stabilize active trypsin inside ZGs that could lead to acute pancreatitis. The experimental results are consistent with our hypothesis, suggesting that K+ induced ion-exchanges play a critical role in the initiation of trypsin premature activation in ZGs. [Copyright &y& Elsevier]

Fumarase, a mitochondrial matrix protein, is previously indicated to be present in substantial amounts in the cytosol as well. However, recent studies show that newly synthesized human fumarase is efficiently imported into mitochondria with no detectable amount in the cytosol. To clarify its subcellular localization, the subcellular distribution of fumarase in mammalian cells/tissues was examined by a number of different methods. Cell fractionation using either a mitochondria fraction kit or extraction with low concentrations of digitonin, detected no fumarase in a 100,000 g supernatant fraction. Immunoflourescence labeling with an affinity-purified antibody to fumarase and an antibody to the mitochondrial Hsp60 protein showed identical labeling pattern with labeling seen mainly in mitochondria. Detailed studies were performed using high-resolution immunogold electron microscopy to determine the subcellular localization of fumarase in rat tissues, embedded in LR White resin. In thin sections from kidney, liver, heart, adrenal gland and anterior pituitary, strong and specific labeling due to fumarase antibody was only detected in mitochondria. However, in the pancreatic acinar cells, in addition to mitochondria, highly significant labeling was also observed in the zymogen granules and endoplasmic reticulum. The observed labeling in all cases was completely abolished upon omission of the primary antibody indicating that it was specific. In a western blot of purified zymogen granules, a fumarase-antibody cross-reactive protein of the same molecular mass as seen in the mitochondria was present. These results provide evidence that fumarase in mammalian cells/tissues is mainly localized in mitochondria and significant amounts of this protein are not present in the cytosol. However, these studies also reveal that in certain tissues, in addition to mitochondria, this protein is also present at specific extramitochondrial sites. Although the cellular function of fumarase at these extramitochondrial locations is not known, the appearance/localization of fumarase outside mitochondria may help explain how mutations in this mitochondrial protein can give rise to a number of different types of cancers. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

The author describes a case of pancreatic heterotopia in the gastric antrum detected after histopathological and ultrastructural studies. Bioptic fragments were processed in part for optic microscopy and in part for electron transmission microscopy. Regressive changes were found in the cytoplasm of the exocrine secreting epithelia that led to necrosis of the cells. The changes are consequential and more markedly involve the base area of the secretory epithelia. The changes observed are to be attributed to the absence of excretory activity by the epithelia due to regressive damage of the organelles carrying out this activity. [ABSTRACT FROM AUTHOR]

Cytochrome c oxidase (COX) complex is an integral part of the electron transport chain. Three subunits of this complex (COX I, COX II and COX III) are encoded by mitochondrial (mit-) DNA. High-resolution immunogold electron microscopy has been used to study the subcellular localization of COX I and COX II in rat tissue sections, embedded in LR Gold resin, using monoclonal antibodies for these proteins. Immunofluorescence labeling of BS-C-1 monkey kidney cells with these antibodies showed characteristic mitochondrial labeling. In immunogold labeling studies, the COX I and COX II antibodies showed strong and specific mitochondrial labeling in the liver, kidney, heart and pancreas. However, in rat pancreatic acinar tissue, in addition to mitochondrial labeling, strong and specific labeling was also observed in the zymogen granules (ZGs). In the anterior pituitary, strong labeling with these antibodies was seen in the growth hormone secretory granules. In contrast to these compartments, the COX I or COX II antibodies showed only minimal labeling (five- to tenfold lower) of the cytoplasm, endoplasmic reticulum and the nucleus. Strong labeling with the COX I or COX II antibodies was also observed in highly purified ZGs from bovine pancreas. The observed labeling, in all cases, was completely abolished upon omission of the primary antibodies. These results provide evidence that, similar to a number of other recently studied mit-proteins, COX I and COX II are also present outside the mitochondria. The presence of mit-DNA encoded COX I and COX II in extramitochondrial compartments, provides strong evidence that proteins can exit, or are exported, from the mitochondria. Although the mechanisms responsible for protein exit/export remain to be elucidated, these results raise fundamental questions concerning the roles of mitochondria and mitochondrial proteins in diverse cellular processes in different compartments. [ABSTRACT FROM AUTHOR]

Abstract: 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, 10μM), 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+. [Copyright &y& Elsevier]

The exocrine pancreas releases secretory products essential for nutrient assimilation. In addition to digestive enzymes, the release of lipoprotein-like particles containing the membrane trafficking protein caveolin-1 from isolated pancreatic explants has been reported. The present study examined: (1) if gastrointestinal hormones induce the apical secretion of phospholipid in vivo and (2) a potential association of caveolin-1 and the lipid-soluble vitamin K analog menaquinone-4 (MK-4) with these structures. Analysis of isolated acinar cells, purified zymogen granules, and pancreatic juice collected in vivo indicated the presence a caveolin-1 immunoreactive protein that was acutely released in response hormone stimulation. Chloroform-extracted fractions of pancreatic juice also contained high concentrations of MK-4 that was secreted in parallel to protein and phospholipid. The presence of caveolin-1 and MK-4 in the phospholipid fraction of pancreatic juice places these molecules in the secretory pathway of exocrine cells and suggests a physiological role in digestive enzyme synthesis and/or processing. [Copyright &y& Elsevier]

A long-standing impediment for applications of optical techniques in cellular biology is the inability to characterize subcellular structures whose dimensions are much less than about 1 μm. In this paper, we describe a method based on light scattering spectroscopy that can find the size distribution of subcellular organelles as small as 100 nm with an accuracy of 20 nm. We report experiments using aqueous suspensions of subcellular organelles enriched in mitochondria, zymogen granules, and microsomes. From the observed light scattering spectra, we extract size distributions that are in excellent agreement with the results of electron microscopy. Further studies are underway to extract the shapes of organelles in addition to their sizes. [ABSTRACT FROM PUBLISHER]

Hsp10 (10-kDa heat shock protein, also known as chaperonin 10 or Cpn10) is a co-chaperone for Hsp60 in the protein folding process. This protein has also been shown to be identical to the early pregnancy factor, which is an immunosuppressive growth factor found in maternal serum. In this study we have used immunogold electron microscopy to study the subcellular localization of Hsp10 in rat tissues sections embedded in LR Gold resin employing polyclonal antibodies raised against different regions of human Hsp10. In all rat tissues examined including liver, heart, pancreas, kidney, anterior pituitary, salivary gland, thyroid, and adrenal gland, antibodies to Hsp10 showed strong labeling of mitochondria. However, in a number of tissues, in addition to the mitochondrial labeling, strong and highly specific labeling with the Hsp10 antibodies was also observed in several extramitochondrial compartments. These sites included zymogen granules in pancreatic acinar cells, growth hormone granules in anterior pituitary, and secretory granules in PP pancreatic islet cells. Additionally, the mature red blood cells which lack mitochondria, also showed strong reactivity with the Hsp10 antibodies. The observed labeling with the Hsp10 antibodies, both within mitochondria as well as in other compartments/cells, was abolished upon omission of the primary antibodies or upon preadsorption of the primary antibodies with the purified recombinant human Hsp10. These results provide evidence that similar to a number of other recently described mitochondrial proteins (viz., Hsp60, tumor necrosis factor receptor-associated protein-1, P32 (gC1q-R) protein, and cytochrome c), Hsp10 is also found at a variety of specific extramitochondrial sites in normal rat tissue. These results raise important questions as to how these mitochondrial proteins are translocated to other compartments and their possible function(s) at these sites. The presence of these proteins at extramitochondrial sites in normal tissues has important implications concerning the role of mitochondria in apoptosis and genetic diseases. [ABSTRACT FROM AUTHOR]

We describe the preparation of a biological tissue for imaging in a transmission soft X-ray microscope. Sections of exocrine pancreas embedded in glycol methacrylate polymer, an embedding medium widely used in visible light and electron microscopy, were examined. Contrast was based primarily on the nitrogen content of the tissue, and dual-wavelength imaging at the nitrogen K-shell absorption edge was used to map the distribution and provide quantitative densitometry of both the protein and embedding matrix components of the sample. The measurements were calibrated by obtaining the absorption spectrum of protein near the nitrogen edge. The contrast was consistent and reproducible, making possible the first large-scale X-ray microscopic study on sections of plastic-embedded soft tissue. At radiation doses of up to 108 Gray, much more than required for routine imaging, no distortion and little mass loss were observed. This sample preparation method should permit routine imaging of tissues in X-ray microscopes, previously a difficult task, as well as multimodal imaging (using visible light, X-ray, electron, and scanned probe microscopies) on the same sample. [ABSTRACT FROM AUTHOR]

BackgroundThe amount of enzymes stored in individual zymogen granules and the glycosylation of their membrane have been analysed in rats with acute pancreatitis induced by caerulein after hydrocortisone treatment. The consequences of prolonging hydrocortisone administration after pancreatitis and the use of the cholecystokinin (CCK) receptor antagonist, L-364,718, have also been evaluated. Materials and methodsAnalysis was performed using flow cytometry. ResultsCaerulein-induced pancreatitis in rats previously treated for 7 days with hydrocortisone (10 mg kg-1 per day) revealed alterations in enzyme storage in the pancreas. Significant increases in amylase and trypsinogen contents in zymogen granules were observed, an effect associated with a reduction in l-fucose glycoconjugates. Pancreatitis persists 7 days later if hydrocortisone treatment is prolonged. At this stage, a reduced granule fucosylation was still observed, and a significant decrease in the amount of trypsinogen stored in the granules was found. However, hydrocortisone administration led to an increase in intragranular amylase quantities up to normal values, even when L-364,718 was simultaneously administered, but it reverted to plasma as a consequence of pancreatitis. The amount of N-acetyl d-glucosamine in the zymogen granule membrane was not altered by caerulein acute pancreatitis induced under continuous hydrocortisone treatment, but it was decreased by the administration of L-364,718 over 7 days after pancreatitis induction. ConclusionsThe administration of hydrocortisone after the development of pancreatitis prevented recurrence of the disease. L-364,718 proved to be detrimental, not only failing to reduce the symptoms of pancreatitis but also altering the glycoproteins of zymogen granule membrane. [ABSTRACT FROM AUTHOR]

Little is known about the changes in pancreatic enzyme storage in acute pancreatitis. We have performed flow cytometric studies of zymogen granules from rats with acute pancreatitis induced by hyperstimulation with caerulein. A comparison was made with rats treated with hydrocortisone (10 mg/kg/day) over 7 days before inducing pancreatitis in order to find out whether the amount of enzymes stored in the pancreas plays a key role in the development of pancreatitis. The potentially therapeutic effect of L-364,718 (0.1 mg/kg/day, for 7 days), a CCK receptor antagonist, was assayed in the rats with caerulein-induced pancreatitis which had previously received the hydrocortisone treatment. A significant increase in the intragranular enzyme content was observed 5 h after hyperstimulation with caerulein. The highest values were reached in the rats previously treated with hydrocortisone. The greatest pancreatic enzyme load was parallel to the highest values in plasma amylase, edema and haematocrit observed. Acute pancreatitis was reversed seven days later. At this stage smaller granules appeared in the pancreas whose enzyme content was similar to that of controls when no treatment was applied after pancreatitis. In contrast, L-364,718 administration prevented the favourable evolution of pancreatitis since the antagonism exerted on CCK receptors induced a blockade of secretion of the large amounts of enzymes stored in the pancreas. Moreover, the enzyme content in zymogen granules was below normal values since the stimulatory CCK action on enzyme synthesis can be inhibited by L-364,718. Our results suggest that the efficiency of CCK antagonists, as potential therapy, would also depend on the load of enzymes in the pancreas when acute pancreatitis is produced. [ABSTRACT FROM AUTHOR]

In the present study on the exocrine pancreas, stereological techniques were applied at the level of electron microscopy to confirm morphological differences between juxta-insular and tele-insular acinar cells of normal rats and to evaluate the effect of B cell secretion on these differences in streptozotocin-diabetic rats. As no similar data have been previously published, we conducted a systematic sampling analysis with special reference to the acinar cell volume, the granule content and the nuclear size. In the normal rats, the juxta-insular acinar cell had an average volume of 918μm and contained 420 zymogen granules which occupied 10.2% of the cell. In tele-insular acini, the average cell volume was estimated at 755 fan3 and the cell contained 231 zymogen granules which amounted to 6.8% of the cell. The juxta-insular acinar cell was 18% larger in size and contained 45% more zymogen granules, but no difference was observed in the size of zymogen granules. In addition, the numerical density of cell nuclei was slightly larger in the tele-insular acini in spite of the similarity in the volume density and diameter of the nuclei in the cells of the two areas. In the streptozotocin-induced diabetic rats, no differences were observed in the cell volume and the zymogen granule content between the juxta- and tele-insular acini. These data suggest that the morphological inhomogeneity in the pancreatic acini is caused by the influence of B cell secretion on the activity of the juxta-insular acini. [ABSTRACT FROM PUBLISHER]

In the present study fine structural changes of acinar zymogen granules were investigated in human acute pancreatitis. Pancreatic tissue was obtained at surgery from 6 patients, prepared for ultrastructural analysis, and stained immunocytochemically for trypsinogen. Stereological parameters of zymogen granules were evaluated. The density of the immunocytochemical labelling for trypsinogen was estimated over zymogen granules, the rough endoplasmic reticulum, Golgi apparatus and the acinar lumina. In acute pancreatitis the number of zymogen granules was diminished and their size reduced. The density of the labelling for trypsinogen was unchanged over zymogen granules but showed a significant reduction over the rough endoplasmic reticulum, Golgi apparatus, and the acinar lumina. In general the integrity of zymogen granules was well preserved. Focally degenerative changes of zymogen granules and large autophagosomes were observed. From the immunogold labelling a disturbance of enzyme synthesis and secretion was suggested. Evidence is given that a disruption of the zymogen granule membranes and a fusion with lysosomal bodies might contribute to the pathogenesis of human acute pancreatitis. [ABSTRACT FROM AUTHOR]

An organ explant culture system has been developed for long term maintenance of adult pancreatic tissue from the Syrian golden hamster. Gastric and duodenal lobe explants of up to 0.5 cm size were placed in tissue culture dishes (60 mm) on Gelfoam sponge rafts to which was added 5 ml of CMRL medium 1066 supplemented with heat inactivated newborn bovine serum, l-glutamine hydrocortisone, insulin, and antibiotics. Dishes were placed in a controlled atmosphere chamber, which was gassed with 45% O 50% N, and 5% CO and incubated at 36.5°C. Viability of the tissues was determined by light and electron microscopy as well as by []thymidine incorporation. Explants were viable for up to 70 d. Zymogen granule-containing cells characteristic of acinar cells and mucuscontaining cells characteristic of ductal cells were present throughout this period. However, endocrine cells were only present for the 1st wk in culture. [ABSTRACT FROM AUTHOR]

The influence of n-3 and n-6 PUFA on the fatty acid composition and the enzyme content of zymogen granules of the normal exocrine pancreas was tested on rats. The animals were fed on different diets comprising 5% fish oil (FO), safflower oil (SFO), and evening primrose oil (EPO) used singly or in combination as dietary fats. The results were compared with those from animals fed 5% hydrogenated beef tallow (HBT). The fatty acid composition and digestive enzyme content were analyzed after a 6-wk feeding period. Differences in the pancreatic fatty acid profiles were related to the fatty acid composition of the ingested fats. Equivalent levels of n-3 fatty acids and 20:3n-6 were obtained with either EPO or FO fed singly or in combination. Similar results were observed with SFO/FO. Higher C20:3n-6/C20:4n-6 ratios were obtained with the oil mixtures. An increase in amylase levels, but a decrease in serine protease (Band 21 kdalton) levels, was associated with EPO. An elevation in procarboxypeptidase levels paralleled an increase in 18:0 levels, whereas the proportion of lipase (Band 49 kdalton) varied inversely with the proportion of C20:3n-6. The SFO/FO mixture elevated the proportions of protease II and proelastase. These results suggest that specific fatty acids influence the proportion of specific digestive enzymes in the zymogen granules. [ABSTRACT FROM AUTHOR]

The number or the size of the zymogen granules was calculated in a determined volume slice of pigeon pancreas and in a total cellular fraction of zymogen granules isolated from a part of this organ having the same volume. The obtained values were not significantly different. These results are discussed in relation to the mechanism of digestive enzyme secrtion. They confirm that amylase can be transported molecule by molecule across the intact apical membrane of the pancreatic exocrine cells. [ABSTRACT FROM AUTHOR]

Particle microeleetrophoresis has been used to measure the surface charge of secretory granules isolated from the exocrine and endocrine pancreas of guineapigs. Zymogen granules were found to have a surface charge density of 2378 esu.cm and islet granules 4177 esu.cm. Calcium ions decreased the surface charge density of both types of granule; binding of calcium at the zymogen granule surface was found to fit a Langmuir adsorption isotherm. The results are discussed with respect to the role of calcium in exocytosis. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 α-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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Stimulation of the exocrine pancreas by the secretagogue urecholine causes degranulation of the acinar cells. Under in vivo conditions, this degranulation is not uniform throughout the tissue. Indeed some of the acini are almost completely depleted of their granules while others display the appearance of resting acini. A noticeable feature is that all the cells of the same acinus display a comparable degree of degranulation. Moreover, groups of neighbouring acini seem to respond simultaneously suggesting that the secretory stimulus is propagated from one acinus to the other. In vitro stimulation of dispersed acini also showed that some of the acini are more responsive than others indicating that this phenomenon can not be attributed to accessibility of the secretagogue to its receptor. These observations lead us to the concept that the response of the pancreatic acinar cell is controlled at the level of the acinus. [ABSTRACT FROM AUTHOR]

In the first descriptions of pancreatic enzyme secretion about 100 years ago, it was noticed that zymogen granules became smaller and disappeared from the apical region of acinar cells after feeding. We have repeated these experiments and characterized changes in granule size by quantitative electron microscopy 90 min after feeding previously fasted rats. In fasted animals, granules occupied the apical portion of the cell, had an average number of 45±3 granules per cell section (±SE), and measured 0.85±0.15 μm in diameter (±SD). After feeding, the number and size of granules decreased. Individual samples showed either a decrease in size alone or a decrease in both size and number, but in no case did they show a reduction in granule number alone. The mean diameter of granules decreased to 0.65±0.15 μm (±SD) or about a 55% reduction in average granule volume as compared to controls (0.32 vs. 0.14 μm). The size distributions were unimodal and normal in both fasted and fed rats; however, in fed animals, the distribution was shifted to lower values (diameter range 0.40-1.40 μm for fasted rats vs. 0.10-1.30 μm for fed rats). The number of granules decreased to an average of 29±2 granules per cell section (±SE) after feeding, and, on the average, samples with the most granules had larger ones than samples with the fewest granules. The present results support the original observations on live rabbit pancreas that individual granules decrease in size in response to feeding. We suggest that these size changes reflect the loss of proteins across the granule membrane as proposed by the equilibrium hypothesis for digestive enzyme secretion. [ABSTRACT FROM AUTHOR]

Electron microscopy of unstained, fresh air-dried spreads of the mouse pancreas has disclosed various substructures of acinar cells. The nucleus with a nucleolus, rough endoplasmic reticulum with ribosomes, mitochondria with dense granules, lysosomes, intracisternal granules, and zymogen granules with a core surrounded by a halo, and a boundary membrane were observable under a conventional electron microscope. Five zymogen granules of an acinar cell were examined by energy dispersive X-ray microanalysis. Peaks for phosphorus, sulfur, chlorine and potassium were detected in all of the zymogen granules examined, which confirmed a previous report (Takaya, 1975b). By increasing the integrated pulse counting time up to 300 seconds, calcium and magnesium were detected in two of the granules in addition to the elements mentioned above. No peaks for either copper or zinc were observed when nylon grids were used. The electrolyte content of zymogen granules and of acinar initial fluid is discussed in relation to the secretory mechanism of the exocrine pancreas. [ABSTRACT FROM AUTHOR]

In isolated pancreatic acinar cells application of the proton-potassium ionophore nigericin or the proton-sodium ionophore monensin led to a reduction of acidity inside the zymogen granules which could be visualized in an imaging system by a rapid reduction in the intragranular quinacrine fluorescence. Cytosolic Ca spikes in response to acetylcholine stimulation or intra-cellular inositol trisphosphate application were assessed by recording Ca -sensitive ionic currents in the patch clamp whole-cell recording configuration. Both nigericin and monensin evoked marked reductions in frequency and amplitude of spikes and in many experiments abolished spiking altogether. The Ca -sensitive membrane currents could still be activated after nigericin or monensin treatment since subsequent application of the Ca ionophore ionomycin evoked a large current response. The decrease in intragranular acidity would appear to inhibit intracellular Ca release perhaps due to a reduction in the free intragranular Ca concentration. [ABSTRACT FROM AUTHOR]

This study report about the differentiation of pancreatic acinar tissue in grass snake, Natrix natrix, embryos using light microscopy, transmission electron microscopy, and immuno-gold labeling. Differentiation of acinar cells in the embryonic pancreas of the grass snake is similar to that of other amniotes. Pancreatic acini occurred for the first time at Stage VIII, which is the midpoint of embryonic development. Two pattern of acinar cell differentiation were observed. The first involved formation of zymogen granules followed by cell migration from ducts. In the second, one zymogen granule was formed at the end of acinar cell differentiation. During embryonic development in the pancreatic acini of N. natrix, five types of zymogen granules were established, which correlated with the degree of their maturation and condensation. Within differentiating acini of the studied species, three types of cells were present: acinar, centroacinar, and endocrine cells. The origin of acinar cells as well as centroacinar cells in the pancreas of the studied species was the pancreatic ducts, which is similar as in other vertebrates. In the differentiating pancreatic acini of N. natrix, intermediate cells were not present. It may be related to the lack of transdifferentiation activity of acinar cells in the studied species. Amylase activity of exocrine pancreas was detected only at the end of embryonic development, which may be related to animal feeding after hatching from external sources that are rich in carbohydrates and presence of digestive enzymes in the egg yolk. Mitotic division of acinar cells was the main mechanism of expansion of acinar tissue during pancreas differentiation in the grass snake embryos., (© 2019 Wiley Periodicals, Inc.)

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 Ca(2+), diacylglycerol and cAMP. Secretory granule (SG) exocytosis shares some common characteristics with nerve, neuroendocrine and endocrine cells which are regulated mainly by elevated cell Ca(2+). 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 Ca(2+) and involves the SG proteins VAMP2 (vesicle associated membrane protein2), Ca(2+)-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., (Copyright © 2014 Elsevier Ltd. All rights reserved.)