Morphometrical and immunocytochemical characterization of peri-insular and tele-insular acinar cells in the rat pancreas

Morphometrical and immunocytochemical techniques have been applied in order to characterize the pancreatic acinar cells located in peri-insular and tele-insular regions of the pancreas. The results obtained, have shown that the acinar cells of the peri-insular regions are twice as large as those of the tele-insular. On the other hand, the volume density of all organelles, except that of the zymogen granules, remains constant implying that the larger the cell, the larger are its organelles. For the zymogen granules however, their volume density was found to be higher in peri-insular acinar cells. The immunofluorescence technique applied for the demonstration of amylase and chymotrypsinogen has confirmed the presence of an inhomogeneity in the staining. Acinar cells in peri-insular regions show a brighter fluorescent staining. At the electron microscope level, both amylase and chymotrypsinogen were demonstrated in all organelles of acinar cells involved in protein secretion. Quantitative evaluations demonstrate no major differences in the intensity of labeling per micron2 between organelles of peri-insular and tele-insular cells. These results put together demonstrate that peri-insular acinar cells contain higher amounts of secretory proteins because their organelles are larger and their zymogen granules are more numerous. The partition of the exocrine pancreas into peri- and tele-insular regions, confirmed herein through morphometrical and cytochemical techniques, is discussed in relation to the possible influence of the endocrine secretion arising from the islets of Langerhans on the surrounding acinar cells.     

Effects of hypothyroidism on the ultrastructure of rat pancreatic acinar cells: a stereological analysis.

The morphological and stereological characteristics of the exocrine pancreas subcellular organelles from healthy and thyroidectomized rats have been studied. The acinar tissue from hypothyroid rats showed an interstitial edema and evidence of degenerative processes. Stereological parameters of zymogen granules were significantly reduced in thyroidectomized rats. The hypothyroidism induced degenerative changes in the pancreatic acinar cells as well as a decrease in the number and size of the zymogen granules. These modifications probably cause functional alterations.     

Analysis of ZAPs,Zymogen Granule Membrane Associated Proteins,in the Regulated Exocytosis of the Pancreas

To characterize molecules involved in the intracellular sorting and regulated exocytosis of digestive enzymes in the pancreas, proteins that are specifícially associated with the zymogen granule membranes were analyzed. Zymogen granules, the major secretory organelles in the pancreas, were highly purified. SDS-PAGE analysis found at least 7 protein components in the zymogen granule membranes including ZAP (zymogen granule membrane associated protein) 75, 54, 47, 36, 32, 29, 25 (numbers refer to their apparent kDas). ZAP75 is identical to the glycophosphatidylinositol (GPI)-anchored protein, GP2. Partial amino acid sequencing of ZAP47 and ZAP36 found similarities to a preprocarboxypeptidase B and annexins, respectively. The method we used was a useful tool for structural analysis of the members of ZAPs.     

Development of pancreatic acini in embryos of the grass snake Natrix natrix (Lepidosauria,Serpentes)

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.     

Localization and neurophysiological properties of motoneurones of the M. triceps surae of the rat after retrograde labelling with evans blue

Summary The effect of vinblastine on the intracellular transport of newly synthesized protein in the mouse exocrine pancreas in vivo was studied by electron microscopic autoradiography after administration of ³H-leucine. Vinblastine (1.1 mole/mouse; i.v. injection) was in general given 1 h before radioleucine and 2–4 h before fixation of the pancreas by perfusion with glutaraldehyde.Vinblastine causes the disappearance of microtubules, mainly present in controls in the apical portion of the acinar cell. After injection of vinblastine, zymogen granules form clusters located throughout the cell but often associated with Golgi areas. The latter are enlarged mainly due to the accumulation of small vesicles. In addition, Golgi areas are displaced, most often in an apical direction.Electron microscopic autoradiography demonstrated that vinblastine delays the appearance of labeled protein in zymogen granules; even 2 h after injection of radioleucine the majority of silver grains is located over the rough endoplasmic reticulum while very few grains are related to zymogen granules. This finding might be related to the structural changes of the Golgi areas observed. Although intracellular migration of protein is retarded, zymogen granules are formed. However, many of the labeled granules are found in peculiar locations, often distant from the acinar lumen.The present study suggests that vinblastine, possibly due to its effect on microtubules, influences both the formation and the translocation of zymogen granules.Supported by Swedish Medical Research Council, Grant No. 12X-537     

Intermediate cell tumour of the pancreas in rat

A malignant tumour of the rat pancreas with features of both acinar and endocrine cells is presented. This consisted of a continuous cytoplasmic mass with numerous dispersed nuclei and branches protruding from its borders invading the surrounding exocrine tissue. The most prominent characteristic of the tumour was the co-existence of zymogen and endocrine secretory granules and cytoplasmic organelles typical of both acinar and islet cells. Some hypotheses are put forward concerning the origin of the tumour and its vasculature.     

Secretion of gamma-glutamyltranspeptidase by the pancreas: evidence for a membrane shedding process during exocytosis

Gamma Glutamyltranspeptidase (GGT) is a membrane-bound enzyme involved in glutathione metabolism. It is present in rat exocrine pancreas at a level which is only exceeded by the kidney. It has been previously shown that most of the enzyme activity is located in the apical area of the acinar cell, more precisely at the level of zymogen granules and plasma membrane. The aim of the present study was to examine the secretory behavior of that enzyme. Under resting conditions, in vivo, high levels of GGT were found in pancreatic juice and its level was not related to protein concentration. Under secretin infusion, a relatively constant level of GGT was released, and again, there was no correlation between enzyme activity and protein concentration. Following a bolus injection of caerulein, an analog of cholecystokinin, marked and concomitant rises in protein and GGT levels were observed. Ultracentrifugation, as well as gel filtration on Sepharose 4B, demonstrated that the enzyme was not released in a soluble form. This observation is in agreement with in vitro determinations on isolated zymogen granules showing that GGT is totally associated with the ZG membrane and undetect-able in the content of these organelles. The present data show that 1 degree GGT is released from the rat pancreas acinar cells in a particulate form; 2 degree GGT release is elicited by hormonal stimulation coinciding with the exocytotic release of secretory proteins. Our observations lead us to propose that in rat pancreas, ZG membrane fragments are released along with secretory proteins during exocytosis.     

Discharge effect on pancreatic exocrine secretion produced by toxins purified from Tityus serrulatus scorpion venom

Three toxic polypeptides were purified from the venom of the Brazilian scorpion Tityus serrulatus by means of gel filtration in Sephadex G-50 and ion-exchange chromatography in carboxymethylcellulose. The peptides are basic molecules with molecular weights in the range of 7000 for which the amino acid compositions and sequences were determined. The effect of the purified peptides on pancreatic exocrine secretion in the guinea pig was studied. Biochemical measurements show that the cells are stimulated by these peptides to discharge their zymogen granules. Light and electron microscopic images confirm the biochemical measurements. At the light microscope level, acinar cells show dramatically fewer zymogen granules than in control pancreas with the appearance of large vacuoles and some loss of morphological integrity. Electron micrographs display apical regions devoid of zymogen granules and condensing vacuoles whereas acinar lumina contain crystalline secretory material. The secretory effect observed in vitro is comparable to that of carbamylcholine and that of the peptidergic secretagogue cholecystokinin-pancreozymin.     

Absence of the major zymogen granule membrane protein, GP2, does not affect pancreatic morphology or secretion

The majority of digestive enzymes in humans are produced in the pancreas where they are stored in zymogen granules before secretion into the intestine. GP2 is the major membrane protein present in zymogen granules of the exocrine pancreas. Numerous studies have shown that GP2 binds digestive enzymes such as amylase, thereby supporting a role in protein sorting to the zymogen granule. Other studies have suggested that GP2 is important in the formation of zymogen granules. A knock-out mouse was generated for GP2 to study the impact of the protein on pancreatic function. GP2-deficient mice displayed no gross signs of nutrient malab-sorption such as weight loss, growth retardation, or diarrhea. Zymogen granules in the GP2 knock-out mice appeared normal on electron microscopy and contained the normal complement of proteins excluding GP2. Primary cultures of pancreatic acini appropriately responded to secretagogue stimulation with the secretion of digestive enzymes. The course of experimentally induced pancreatitis was also examined in the knock-out mice because proteins known to associate with GP2 have been found to possess a protective role. When GP2 knock-out mice were subjected to two different models of pancreatitis, no major differences were detected. In conclusion, GP2 is not essential for pancreatic exocrine secretion or zymogen granule formation. It is unlikely that GP2 serves a major intracellular role within the pancreatic acinar cell and may be functionally active after it is secreted from the pancreas.     

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.     

Separation of cell organelles in density gradients based on their permeability characteristics

The buoyant density of intracellular organelles is dependent in part on the nature of the buffer composition of the density gradient and the permeability characteristics of the organelle membrane to the constituents of this buffer. Therefore, knowledge of the transport properties of different organelles allows the design of density gradients useful for their purification. We have used this approach to significantly decrease mitochondrial contamination of pancreatic zymogen granules in a one-step purification procedure on a 40% Percoll density gradient. These gradients, prepared with isoosmotic sucrose, yield a narrow band of zymogen granules and mitochondria. However, by substitution of sucrose with salts to which mitochondria but not zymogen granules are permeable, the densities of mitochondria are altered to give a significant separation. For example, the incorporation of 100 mM sodium succinate in the Percoll gradient can produce a 70% reduction in mitochondrial contamination. The increased ionic strength has an additional beneficial effect on zymogen granule yield by 5-10%. The recognition and utilization of transport pathways in organelle membranes is the principal feature of this technique and should prove to be widely applicable to other isolation procedures.     

Studies on intracellular transport of secretory proteins in the rat exocrine pancreas

Summary The previous finding that intracellular transport of secretory proteins in the rat exocrine pancreas is accelerated by in vivo stimulation with a pancreatic secretagogue has been further analyzed. Using a radioassay for discharge of newly synthesized proteins, the rate of release was compared in control and prestimulated lobules. In control preparations discharge occurred with an initial lag period of 30 minutes and a maximum after two hours of incubation. After in vivo infusion of 5 × 10^-8 g/hr. caerulein for 24 h in vitro discharge started after 10 minutes of in vitro incubation and attained a maximal rate after one hour. Using the same radioassay and several inhibitors of intracellular transport and granule discharge, it could be demonstrated that both processes were reduced to the same extent in controls and in lobules with accelerated transport. To obtain direct evidence for the degree of acceleration of the different transport steps between rough endoplasmic reticulum, Golgi complex and zymogen granules, the respective subcellular fractions of these organelles prepared and characterized ultrastructurally and biochemically. The rate of disappearance of newly formed proteins from rough microsomes and the appearance in smooth microsomes and zymogen granules were significantly increased after in vivo stimulation. The data substantiate an acceleration of the regular transport steps by the secretagogue. There was no indication that a high level of secretory activity leads to a rerouting of secretory proteins or to an omission of one of the regular steps in intracellular transport.Supported by a grant from Deutsche Forschungsgemeinschaft Bonn-Bad Godesberg (Ke 113/10) The expert technical assistance of Miss Hiltraud Hosser and Miss Helga Hollerbach is gratefully acknowledged     

Morphometry and elemental analysis of rat exocrine pancreas following administration of trypsin inhibitor

The morphological responses of the exocrine pancreas of the adult male rat to soybean trypsin inhibitor (STI) were studied by ultrastructural morphometry and electron probe X-ray microanalysis. STI administered orally in drinking water for 14 days resulted in a 72% increase in the wet weight of the pancreas. This enlargement was due, largely, to an increase in acinar cell mass. Volume increases in the acinar cell mass and extra-acinar cell compartment were 72 and 30%, respectively. The estimated total number of acinar cells in the mean exocrine pancreas was 500 million in the control and 630 million in the experimental group, representing an increase of 27%. Acinar cell volume was 1,790 microns 3 for the control and 2,457 microns 3 for the STI group. The pronounced morphometric changes of the organelles in the STI group were: the mean nucleolar volume increased by 56%; the volume of zymogen granular mass per cell increased by 93%; the volume of the Golgi complex and the condensing vacuoles per cell increased by 52 and 100%, respectively, whereas the membrane area of the Golgi complex and the condensing vacuoles increased by 98 and 47%, respectively. Spectral analysis of seven elements (Na, Mg, P, S, Cl, K and Ca) showed significant changes for nuclei, zymogen granules and mitochondria following STI: nuclei showed Na, P, K increased; zymogen granules showed Na, P, S, K increased, Cl decreased; mitochondrial particles showed Mg, P, Cl, Ca increased, and the mitochondrial matrix showed S decreased. The persistent uptake of STI probably resulted in a continual release of a trophic hormone acting on pancreatic tissue components, consequently causing hyperplasia and hypertrophy of the exocrine pancreas to accommodate a heightened demand for synthesis of exportable proteins.     

Development of the neural apparatus of the human pancreas in prenatal ontogeny

In human embryos, prefetuses and fetuses from 6 to 40 weeks of development 82 preparations of the pancreas have been studied; 39 series of slices of the human embryos have been impregnated after Bielschowsky-Boeke. For the first time the nerves grow into the pancreas anlage in embryos of 16 mm long (6 weeks). The main sources supplying the pancreas with the nerves in the period studied are nervus vagus, celiac, hepatic, splenic, right adrenal, superior mesenteric and intermesenteric plexuses. Three stages in formation of the innervational apparatus of the pancreas are distinguished during the intrauterine development. The first stage is characterized by beginning of the nerve growing into the pancreas anlage. For the second stage an increased development of the intraorganic innervational apparatus and appearance of the all main sources of the organ's innervation are specific. The third stage is characterized with an increased differentiation of receptor zones in various parts of the pancreas. The development of the pancreatic innervational apparatus by the time of birth is not completed.     

Pancreatic damage induced by injecting a large dose of arginine

Male Wistar rats were injected intraperitoneally with a large dose of arginine (500 mg/100 g body weight) and were sacrificed 24, 48 and 72 h later. Pancreatic tissue was examined by electron microscopy to study the resulting process of degeneration. Degeneration started with disorganization of the rough endoplasmic reticulum into whorls with a concomitant decrease in the numbers of zymogen granules. The main changes in acinar cells after 24 h were partial distension of the endoplasmic reticulum, whorls of agranular membranes encircling zymogen granules and perinuclear vacuoles. At this time large sequestered areas in the cytoplasm contained disarranged rough endoplasmic reticulum and degraded zymogen granules. The mitochondria showed only slight changes. After 48 h, dissociation and necrosis of acinar cells were noted. Subsequently, the necrotic cells were replaced by interstitial tissue composed of leucocytes and fibroblasts. It was concluded that a large dose of arginine is toxic to the rat pancreas when injected intraperitoneally. The early morphological changes of the acinar cells may be related to metabolic alterations associated with the endoplasmic reticulum. The disorganization of the endoplasmic reticulum and the reduced number of zymogen granules may indicate disturbance of protein synthesis. The focal sequestration and degradation of the cytoplasm seemed to represent changes of the acinar cells associated with removal of damaged organelles.     

Large-scale purification of calf pancreatic zymogen granule membranes.

A protocol for isolating milligram quantities of highly purified zymogen granule membranes from calf pancreas was developed. The method provides a fivefold enriched zymogen granule fraction that is virtually free from major isodense contaminants, such as mitochondria and erythrocytes. Isolated granules are osmotically stable in isosmotic KCl buffers with half-lives between 90 and 120 min. They display specific ion permeabilities that can be demonstrated using ionophore probes to override intrinsic control mechanisms. A Cl- conductance, a Cl-/anion exchanger, and a K+ conductance are found in the zymogen granule membrane, as previously reported for rat pancreatic, rat parotid zymogen granules, and rabbit pepsinogen granules. Lysis of calf pancreatic secretory granules in hypotonic buffers and subsequent isolation of pure zymogen granule membranes yield about 5-10 mg membrane protein from approximately 1000 ml pancreas homogenate. The purified zymogen granule membranes are a putative candidate for the rapid identification and purification of epithelial Cl- channels and regulatory proteins, since they contain fewer proteins than plasma membranes.     

On the isolation of zymogen granules from rat panereas by zonal ultracentrifugation and their nucleic acid content

The isolation of zymogen granules from rat pancreas by a one-step zonal centrifugation procedure is described. The high purity of the isolated granules was established by electron microscopy and the use of marker enzymes. The DNA and RNA contents of the zymogen granules as well as the contents of mitochondria and microsome-containing fractions were measured. The zymogen granules contained low levels of both DNA and RNA.     

A cyto-chemical study on the pancreas of the guinea pig. III. In vivo incorporation of leucine-1-C14 into the proteins of cell fractions

DL-leucine-1-C(14) was administered by intracardiac injection to guinea pigs and its in vivo incorporation into the proteins of various pancreatic cell fractions followed over a period of 2 hours. The pancreas was homogenized in 0.88 M sucrose and fractionated by differential centrifugation to give nuclear, zymogen, mitochondrial, microsomal, postmicrosomal, and final supernatant fractions. The proteins of these fractions, obtained by precipitation with trichloroacetic acid followed by washing, were counted. The proteins of the microsomal fraction showed the highest early specific activity and were followed by those of the zymogen and mitochondrial fractions. The microsomal fraction was broken up into two subfractions: one consisting of detached RNP particles, the other representing mainly the microsomal content and membranes. The incorporation of labelled leucine into the proteins of microsomal subtractions and in those of postmicrosomal fractions was studied comparatively in the pancreas of fasted and fed guinea pigs as well as in the liver and pancreas of fasted animals. A tentative cytological picture of protein synthesis and transport based on these findings is presented.     

Proteolysis in isolated autophagic vacuoles from the rat pancreas. Effects of chloroquine administration.

Administration of the antimalaria drug chloroquine increased the number of autophagic vacuoles (AVs) in the rat pancreas. Ultrastructural analysis showed that AVs contained segregated organelles such as mitochondria, zymogen granules, peroxisomes and small portions of cytoplasm. The maximum number of AVs was observed after 3 h of chloroquine treatment. The effect lasted for 12 h and almost disappeared after 16 h. The increase in AVs caused by chloroquine made it possible to isolate them in a discontinuous Metrizamide gradient with high purity. The proteolytic capacity of the AVs isolated after different chloroquine exposure times was measured after prelabeling pancreatic proteins with an injection of L-(1-14C)leucine 16 h before sacrifice. Protein degradation in isolated AVs increased during the first 6 h of chloroquine exposure and then returned to control values 16 h after the administration. In addition, the activities of two lysosomal enzymes, acid phosphatase and cathepsin B, increased in the AV-fractions following chloroquine treatment. It is concluded that the augmented proteolysis in the isolated AVs is due to a combination of increased substrate content and increased proteolytic lysosomal enzyme activities.     

Establishment and immunocharacterization of an immortalized pancreatic cell line derived from the H-2Kb-tsA58 transgenic mouse

Summary This study describes the establishment and characterization of an immortalized cell line derived from the pancreas of an adult H-2K^b-tsA58 transgenic mouse. These cells, designated IMPAN for IMmortalized PANcreatic cells, displayed a cobblestone appearance typical of confluent epithelial cells and a distinct polarity in the organization of their cytoplasmic organelles. Immunocytochemical studies revealed that all IMPAN cells stained positively for a wide range of markers characteristic of pancreatic acinar cells, namely the secretory products α-amylase, chymotrypsinogen, DNAse, the lectinlike secretory protein PAP (pancreatitis associated protein), and the zymogen granule membrane proteins GP-2 and gp300. They also stained positively for carbonic anhydrase II and cytokeratin 19, two proteins characteristic of pancreatic duct cells, as well as for rab3A, a small GTP-binding protein specifically localized in pancreatic islet cells. No reactivity was ever obtained with insulin antibodies. Taken together, these results show that the IMPAN cells exhibit a phenotype comparable to exocrine pancreatic acinar cells. However the expression of some proteins more specific to duct and islet cells make them similar to in vivo or in vitro growing acinar cells. The cell line should be a valuable model to study the mechanisms of growth, differentiation, and transformation of the exocrine pancreatic acinar cell.