Lysophospholipids in pig pancreatic zymogen granules in relation to exocytosis.

A hypothesis to explain the stimulatory role of cyclic AMP (adenosine 3':5'-monophosphate) in pancreatic enzyme secretion. has been tested. In this hypothesis cyclic AMP would activate a phospholipase activity, which would lead to a locally increased lysophospholipid formation, resulting in a fusion between the zymogen granule membrane and the apical plasma membrane. Cyclic AMP added to isolated pig pancreatic zymogen granules leads to an increased lysis of these granules, but the slowness of this effect makes its physiological significance dubious. In pancreatic homogenates or zymogen granules no stimulating effect of cyclic AMP on lipase of phospholipase activity could be demonstrated. Isolated zymogen granules have a high lysophospholipid content (27% of total phospholipids), consisting of the 1-acyl and 2-acyl forms of lysophosphatidylcholine and lysophosphatidylethanolamine. Experiments with radioactive phosphatidylcholine indicate that the lysophospholipids are due to the action of endogenous (phospho)lipases during the isolation procedure. It is concluded that these experiments do not lend support to the above hypothesis for the mechanism of action of cyclic AMP in pancreatic enzyme secretion     

Protein kinase C and an endogenous substrate associated with adenohypophyseal secretory granules.

Secretory granules isolated from anterior pituitary glands were examined for Ca2+/phospholipid-dependent protein kinase (protein kinase C) activity as well as the occurrence of granule-associated substrate proteins. Sheep adenohypophyses were fractionated by differential and sucrose-density-gradient centrifugation to yield a granule fraction enriched for luteinizing-hormone (lutropin)-containing secretory granules. Marker-enzyme analysis showed no detectable cytosolic contamination, although there were small amounts of plasma membranes (2-4%) and lysosomes (4-6%) associated with the preparation. As determined by histone-H1 phosphorylation after DEAE-cellulose DE-52 chromatography, protein kinase C activity with a marked dependence on Ca2+ and lipid (4-fold increase in their presence) was evident in the secretory-granule fraction. Phosphorylation in vitro of the secretory-granule fraction by endogenous and exogenous protein kinase C revealed a protein of Mr 36,000, which by two-dimensional SDS/polyacrylamide-gel electrophoresis showed multiple sites of phosphorylation. The Mr-36,000 protein was not found in cytosolic or plasma-membrane fractions and was not phosphorylated by the catalytic subunit of cyclic AMP-dependent protein kinase. Several secretory-granule proteins served as substrates for the catalytic subunit, the most prominent of which were of Mr 63,000, 23,000 and 21,000. From these data, we suggest that phosphorylation of secretory-granule-associated proteins by protein kinase C and by cyclic AMP-dependent protein kinase may be important in secretion regulation in the anterior pituitary gland.     

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.     

Protein concentration in the pancreatic zymogen granule

We have estimated the concentration of protein in the zymogen granule, a major storage depot for secretory protein in the pancreatic acinar cell, in four different ways. Each of these approaches yielded roughly similar values. The protein concentration in the granule is approx. 135-270 mg protein/ml granule volume in rat and rabbit, as compared to an average value for the protein concentration of tissue of 135 mg/g tissue for rabbits and 183 mg/g tissue for rats. This is equivalent to an average molarity for the contained proteins of between 4-9 mM based on an estimated average molecular weight for the mixture of 30 000-40 000. An upper limit for the concentration of protein in these granules can reasonably be set at about double the overall concentration of protein in the non-granule portion of the cell.     

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.     

Dual modulation of chloride conductance by nucleotides in pancreatic and parotid zymogen granules.

The regulation of Cl- conductance by cytoplasmic nucleotides was investigated in pancreatic and parotid zymogen granules. Cl- conductance was assayed by measuring the rate of cation-ionophore-induced osmotic lysis of granules suspended in iso-osmotic salt solutions. Both inhibition and stimulation were observed, depending on the type and concentration of nucleotide. Under optimal conditions, the average inhibition measured in different preparations was 1.6-fold, whereas the average stimulation was 4.4-fold. ATP was inhibitory at 1-10 microM but stimulated Cl- conductance above 50 microM. Stimulation by ATP was more pronounced in granules with low endogenous Cl- conductance. The potency of nucleotides in terms of inhibition was ATP greater than adenosine 5'-[gamma-thio]triphosphate (ATP[S]) greater than UTP much greater than or equal to CTP much greater than or equal to GTP much greater than or equal to guanosine 5'-[gamma-thio]triphosphate (GTP[S]) much greater than or equal to ITP. The potency with respect to stimulation had the following order: adenosine 5'-[beta gamma-methylene]triphosphate (App[CH2]p) greater than ATP greater than guanosine 5'-[beta-thio]diphosphate (GDP[S]). Adenosine 5'-[beta gamma-imido]triphosphate (App[NH]p) was also stimulatory, and was more potent than ATP in the parotid granules, but less potent in the pancreatic granules. Aluminium fluoride stimulated Cl- conductance maximally at 15-30 microM-Al3+ and 10-15 mM-F. F was less effective at higher concentrations. Protein phosphorylation by kinases was apparently not involved, since the nucleotide effects (1) could be mimicked by non-hydrolysable analogues of ATP and GTP, (2) showed reversibility, and (3) were not abolished by the protein kinase inhibitors 1-(5-isoquinolinesulphonyl)-2-methylpiperazine (H-7) or staurosporine. The data suggest the presence of at least two binding sites for nucleotides, whereby occupancy of one induces inhibition and occupancy of the other induces stimulation.     

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.     

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.     

Proteomic analysis of pancreatic zymogen granules: identification of new granule proteins

The composition of zymogen granules from rat pancreas was determined by LC-MS/MS. Enriched intragranular content, peripheral membrane, and integral membrane protein fractions were analyzed after one-dimensional SDS-PAGE and tryptic digestion of gel slices. A total of 371 proteins was identified with high confidence, including 84 previously identified granule proteins. The 287 remaining proteins included 37 GTP-binding proteins and effectors, 8 tetraspan membrane proteins, and 22 channels and transporters. Seven proteins, pantophysin, cyclic nucleotide phosphodiesterase, carboxypeptidase D, ecto-nucleotide phosphodiesterase 3, aminopeptidase N, ral, and the potassium channel TWIK-2, were confirmed by immunofluorescence microscopy or by immunoblotting to be new zymogen granule membrane proteins.     

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.     

Protein kinase activity associated with polyoma virus middle T antigen in vitro.

A protein kinase activity can be detected in immunoprecipitates of extracts from polyoma virus (Py)-infected cells using antiserum raised against Py-transformed cells (anti-T serum). The activity is not detected in uninfected cells or when using control serum. Using rat anti-T serum both Py middle T and the heavy chain of rat IgG are phosphorylated, whereas using hamster anti-T serum only Py middle T is phosphorylated. Experiments using a number of different mutants of Py indicate that the kinase activity detected is under viral control and is associated with Py middle T. Consistent with this the kinase, like middle T, can be detected in purified preparations of plasma membranes. The kinase can also be detected in a large number of Py-transformed cells, but not in untransformed cells or in cells transformed by other viruses. Some of the Pytransformed cells which contain kinase activity lack full sized Py large T but all contain middle T. Kinase activity is not detected in a cell line (18.37) which contains integrated viral DNA of a nontransforming hr-t deletion mutant and which contains Py large T but not middle T or small t. These results show that Py middle T or a protein which specifically binds to it has protein kinase activity in vitro. Although these results raise the possibility that protein kinases play an essential role in Py-induced transformation, however, thus far we have no data which show unequivocally that the results are physiologically significant.     

Proteomic analysis of zymogen granules

Zymogen granules (ZGs) are specialized storage organelles in the exocrine pancreas that allow the sorting, packaging and regulated apical secretion of digestive enzymes. ZG constituents play important roles in pancreatic injury and disease. The molecular mechanisms underlying these processes are still poorly defined. Thus, there is currently great interest in the identification and characterization of ZG components. Recent proteomic studies have greatly enhanced our knowledge regarding potential new ‘players’ in ZG biogenesis and regulated secretion. In this article, we present the latest advancements in and insights into the analysis of the ZG proteome by the combination of organelle isolation, protein separation, mass spectrometry and validation of protein identification. Recent developments in the analysis of ZG proteins from pancreatic juice and related proteins from saliva are also discussed.     

In vitro stability of pancreatic zymogen granules: roles of pH and calcium

Purified preparations of pancreatic zymogen granules have the peculiar property of lysing instantaneously at neutral pH, a property clearly irreconcilable with the cytoplasmic pH of the acinar cell. Two important factors known for regulating the stability of secretory granules are calcium and pH. Fluorescence microscopy of acinar cells in the presence of weak bases showed that zymogen granules have an acidic pH. In vivo, abolition of the delta pH by NH4Cl did not induce any lysis of the granules. In vitro, with purified granules, an acidic intragranular pH was measured. This delta pH was produced by a Donnan potential. The importance for granule stability of keeping the intragranular pH acidic has been confirmed in vitro by addition of K+ and nigericin to the suspension medium. These conditions produced alkalinization of the granule matrix and caused instantaneous solubilization of the granules. Concentrations of 15 mM total, and 10 mM free calcium were measured in purified granules. The importance of intragranular Ca2+ was evaluated by means of the ionophore A23187 which induced calcium efflux and granule lysis. The lysis induced by the calcium ionophore was in direct relation with the calcium efflux, since addition of Ca2+ to the medium, at concentrations corresponding to that measured in the granule, relieved the effect. The role of calcium-binding sites on the cytoplasmic surface of the granules was investigated with Ca2+, EGTA, and La3+. Calcium did not have any damaging effects; EGTA induced a slight lysis, while lanthanum yielded a strong and spontaneous lysis at micromolar concentrations. In addition to calcium-binding sites, La3+ would bind to specific sites on the granule that would be directly coupled to maintenance of its stability. These findings suggest that the intragranular acidic pH and calcium are both important for the in vitro stability of the zymogen granule and that purified granules have lost, in the course of purification, some cytoplasmic factors that in vivo, control the permeability of the membrane to protons, and chloride more particularly. Calcium-binding sites and other specific sites probed with La3+, presumably on proteins at the surface of the granule, are also believed to have key roles in preserving the integrity of the membrane and the resulting stability of the granule.     

Proteomic analysis of zymogen granules

Zymogen granules (ZGs) are specialized storage organelles in the exocrine pancreas that allow the sorting, packaging and regulated apical secretion of digestive enzymes. ZG constituents play important roles in pancreatic injury and disease. The molecular mechanisms underlying these processes are still poorly defined. Thus, there is currently great interest in the identification and characterization of ZG components. Recent proteomic studies have greatly enhanced our knowledge regarding potential new 'players' in ZG biogenesis and regulated secretion. In this article, we present the latest advancements in and insights into the analysis of the ZG proteome by the combination of organelle isolation, protein separation, mass spectrometry and validation of protein identification. Recent developments in the analysis of ZG proteins from pancreatic juice and related proteins from saliva are also discussed.     

Cytoplasmic dynein and dynactin as likely candidates for microtubule-dependent apical targeting of pancreatic zymogen granules.

The critical role of microtubules in vectorial delivery of post-Golgi carrier vesicles to the apical cell surface has been established for various polarized epithelial cell types. In the present study we used secretory granules of the rat and chicken pancreas, termed zymogen granules, as model system for apically bound post-Golgi carrier vesicles that underlie the regulated exocytotic pathway. We found that targeting of zymogen granules to the apical cell surface requires an intact microtubule system which contains its colchicine-resistant organizing center and, thus, the microtubular minus ends close to the apical membrane domain. Purified zymogen granules and their membranes were found to be associated with cytoplasmic dynein intermediate and heavy chain and to contain the major components of the dynein activator complex, dynactin, i.e. p150Glued, p62, p50, Arp1, and beta-actin. Kinesin heavy chain and the kinesin receptor, 160 kD kinectin, were not detected as components of zymogen granules. Immunofluorescence staining showed a zymogen granule-like distribution for dynein and dynactin (p150Glued, p62, p50, Arpl) in the apical cytoplasm, whereas kinesin and kinectin were largely concentrated in the basal half of the cells in a pattern similar to the distribution of calreticulin, a component of the endoplasmic reticulum. Secretory granules of non-polarized chromaffin cells of the bovine adrenal medulla, that are assumed to underlie microtubular plus end targeting from the Golgi apparatus to the cell periphery, were not found to be associated with dynein or dynactin. To our knowledge, this is the first demonstration of major components of the dynein-dynactin complex associated with the membrane of a biochemically and functionally well-defined organelle which is considered to underlie a vectorial minus end-driven microtubular transport critically involved in precise delivery of digestive enzymes to the apically located acinar lumen.     

Ultrastructural immunocytochemical localization of l-glutamate decarboxylase and GABA in rat pancreatic zymogen granules

Summary The ultrastructural immunohistochemical localization of gamma aminobutyric acid (GABA) and its regulating enzymes, l-glutamate decarboxylase (GAD) and gamma aminobutyrate--ketoglutarate transaminase, was determined utilizing an immunogold post-embedding protocol in pancreatic exocrine tissue. Within the acinar cell, GABA and its biosynthetic enzyme, GAD, were localized in zymogen granules. Quantitative analysis of the GABA immunoreactivity in the acinar cell revealed 1.7±0.5 gold particles/m² over the cytoplasm, 36.6±14.1 gold particles/ m² over the zymogen granules, and 2.9±2.1 gold particles /m² over the mitochondria. Quantitative analysis of the distribution of colloidal gold particles, representing glutamate decarboxylase immunoreactivity in the acinar cells, revealed 38.4±2.5 gold particles/m² over the zymogen granules, 4.7±1.1 gold particles/m² over the mitochondria and 6.3±0.5 gold particles/m² over the remainder of the cytoplasm. Substitution of normal sheep serum for the sheep anti-glutamate decarboxylase serum revealed a significant (p< 0.001) decrease of the colloidal gold particle distribution over the zymogen granules and cytoplasmic compartments of the acini. Gamma aminobutyrate --ketoglutarate transaminase, the catabolic enzyme for GABA, was not detected in the mitochondria, zymogen granules, and cytoplasm of the acinar cell, suggesting that GABA is not catabolized within the acinar cell. Preabsorption and substitution controls resulted in an absence of labeling. These results suggest that GABA may act extracellularly and/or have a role within the zymogen granule in the exocrine pancreas.