Glycoconjugate pattern of membranes in the acinar cell of the rat pancreas

Lectin-binding studies were performed at the ultrastructural level to characterize glycoconjugate patterns on membrane systems in pancreatic acinar cells of the rat. Five lectins reacting with different sugar moieties were applied to ultrathin frozen sections: concanavalin A (ConA): glucose, mannose; wheat-germ agglutinin (WGA): N-acetylglucosamine, sialic acid; Ricinus communis agglutinin I (RCA I): galactose; Ulex europaeus agglutinin I (UEA I): L-fucose; soybean agglutinin (SBA): N-acetylgalactosamine). Binding sites of lectins were visualized either by direct conjugation to colloidal gold or by the use of a three-step procedure involving additional immune reactions. The rough endoplasmic reticulum and the nuclear envelope of acinar cells was selectively labelled for ConA. The membranes of the Golgi apparatus bound all lectins applied with an increasing intensity proceeding from the cis- to the trans-Golgi area for SBA, UEA I and WGA. In contrast RCA I selectively labelled the trans-Golgi cisternae. The membranes of condensing vacuoles and zymogen granules were labelled for all lectins used although the density of the label differed between the lectins. In contrast the content of zymogen granules failed to bind SBA and WGA. Lysosomal bodies (membranes and content) revealed binding sites for all lectins used. The plasma membranes were heavily labelled by all lectins except for SBA which showed only a weak binding to the lateral and the apical plasma membrane. These results are in accordance to current biochemical knowledge of the successive steps in the glycosylation of membrane proteins. It could be demonstrated, that the cryo-section technique is suitable for the fine structural localisation of surface glycoconjugates of plasma membranes and internal membranes in pancreatic acinar cells using plant lectins.     

Diurnal pattern of rat pancreatic acinar cell replication

Fully differentiated pancreatic acinar cells can enter the cell cycle under appropriate conditions in the rat. The aim of this study was to analyse the diurnal pattern of acinar cell proliferation as a function of food intake and the release of cholecystokinin (CCK), because the peptide hormone CCK is a major physiological regulator of rat pancreatic acinar cell replication. Pancreatic acinar cell replication was quantitated using an antibody against the S-phase marker proliferating cell nuclear antigen (PCNA). In addition, acinar cells in S-phase were detected after injecting bromodeoxyuridine (BrdU) and subsequent immunohistochemical staining of BrdU-positive nuclei. Rat pancreata were analysed during the day under standard diet conditions, as well as after various schedules of fasting and refeeding and after the application of the CCK receptor antagonist L-364,718. Between 6 a.m. and 6 p.m., the PCNA labeling index was 4.4±0.9%, while between 9 p.m. and 3 a.m. the PCNA labeling index was elevated and reached peak values of 11.4% (mean value: 7.8±2.5%) around midnight. BrdU-positive cells also doubled around midnight, compared to the 9:00 a.m. value. In fasted rats, acinar cell proliferation was completely suppressed and this suppression could be overcome by injection of the CCK analog cerulein. In addition, the CCK antagonist L-364,718 led to the same results as fasting. Here we show for the first time that there is a diurnal pattern of pancreatic acinar cell proliferation in rats, which is dependent on food intake and is mediated by CCK.     

Calcium transport in dispersed acinar cells from rat pancreas

The present studies were performed to attempt to elucidate the basis for the discrepancy between results of Kondo and Schulz (1976, Biochim. Biophys Acta 419, 76–92), who found that cholecystokinin and cholinergic agents increase uptake of ⁴⁵Ca by dispersed acinar cells from rat pancreas, and results of others (Matthews, E.K., Petersen, O.H. and Williams, J.A. (1973) J. Physiol. 234, 689–701; Chandler, D.E. and Williams, J.A. (1974) J. Physiol. 243, 831–846; Case, R.M. and Clausen, T. (1973) J. Physiol. 235, 75–102; Gardner, J.D., Conlon, T.P., Klaeveman, H.L., Adams, T.D. and Ondetti, M.A. (1975) J. Clin. Invest. 56, 366–375; Christophe, J.P., Frandsen, E.K., Conlon, T.P., Krishna, G. and Gardner, J.D. (1976) J. Biol. Chem. 251, 4640–4645; Shelby, H.T., Gross, L.P., Lichty, P. and Gardner, J.D. (1976) J. Clin. Invest. 58, 1482–1493 and Deschodt-Lanckman, M., Robberecht, P., de Neef, P., Lammens, M. and Christophe, J. (1976) J. Clin. Invest. 58, 891–898). They have reported that cholecystokinin and cholinergic agents do not alter or cause a slight decrease in uptake of ⁴⁵Ca by pancreatic acinar cells. Our present results indicate that increased uptake of ⁴⁵Ca by acinar cells incubated with cholecystokinin occurs only in cells washed with iced, 160 mM choline chloride and reflects increased cellular uptake of radioactivity from the wash solution but not from the incubation medium. We detected no effect of cholecystokinin on uptake of ⁴⁵Ca by cells washed with 160 mM choline chloride containing 5 mM ethylenediaminetetraacetate or by cells washed with Krebs-Ringer bicarbonate. Furthermore, cells washed with 160 mM choline chloride accumulated a substantial amount of ⁴⁵Ca from the wash solution and this accumulation was increased in cells that had been preincubated with cholecystokinin. Cells washed with Krebs-Ringer bicarbonate did not take up ⁴⁵Ca from the wash solution.     

Heterogeneity of the zymogen granule membranes in rat pancreas

Zymogen granules (ZG) of rat pancreas have been isolated by the procedure of Paquet et al. The granules lysed when exposed to alkaline pH (pH 8.2), and their membranes could be subfractionated by centrifugation on a sucrose gradient. Four discrete types of membranes corresponding to densities of 1.105, 1.085, 1.075, and 1.020 were obtained, designated types A, B, C, and D, respectively and characterized both by morphological and biochemical criteria. Electrophoretic profiles showed that they contain the same protein bands but in different proportions. Type A membranes are comprised of four major bands corresponding to molecular weights of 80, 69, 54, and 20 kDa, being in higher concentration than the others. Types B and C contain three major bands at 80, 54 and 20 kDa whereas type D is comprised of only two major bands at 69 and 54 kDa, the latter polypeptide corresponding to ATP-diphosphohydrolase activity which is present in all four membrane types. Freeze-fracture of rapidly frozen membranes, followed by transmission electron microscopy (TEM) showed that type A are large superimposed sheets of membranes with amorphous material between sheets. The surface area of these sheets corresponds grossly to the surface of an intact ZG with a few intramembrane particles (IMP) distributed at random or in small aggregates on large smooth fracture planes. Types B and C exhibit a totally different aspect, forming closed vesicles about the size of a small ZG with few IMP distributed at random or in small aggregates on smooth fracture planes. Type D membranes are very small vesicles with no detectable IMP on relatively smooth fracture planes.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Phorbol ester or diacylglycerol modulates somatostatin binding to its receptors on rat pancreatic acinar cell membranes

We and others have suggested previously that the binding of somatostatin to its receptors in the pancreas is regulated by not only somatostatin analogs but also cholecystokinin analogs in proportion to their known biological potencies. To clarify the precise mechanism by which unrelated peptides modulate somatostatin binding, the effect of a phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), or a synthetic diacylglycerol analog, 1-oleyl-2-acetylglycerol (OAG), on [125I-Tyr1]somatostatin binding to pancreatic acinar cell membranes was examined. Pretreatment of pancreatic acini for 120 min at 37 degrees C with 100 ng/ml TPA maximally reduced subsequent labeled somatostatin binding to acinar membranes. The inhibitory effect of TPA on the somatostatin binding was dependent on the dose used or the time and temperature of pretreatment. These effects of TPA were almost mimicked by the treatment of acini with OAG. Scatchard analysis of [125I-Tyr1]somatostatin binding demonstrated that the decrease in the labeled somatostatin binding induced by TPA or OAG pretreatment was due to the decrease in the maximum binding capacity without a significant change in the binding affinity. A specifically labeled single band of Mr = 90,000 obtained with a photoaffinity cross-linking study indicates that the somatostatin-binding sites are the same somatostatin receptor as previously described. Moreover, the intensity of the Mr = 90,000 band was dramatically decreased when acini were treated with increasing concentrations of TPA, a finding consistent with TPA-induced decrease in binding capacity. Such an inhibitory effect of TPA was abolished when pretreatment of acini with TPA was performed in the presence of Ca2+-chelating compounds such as EDTA and EGTA or phospholipid-interacting drugs such as chlorpromazine and tetracaine. Interestingly, the combined treatment of TPA and Ca2+ ionophore A23187 caused synergistic inhibition of the subsequent labeled somatostatin binding to acinar membranes, although Ca2+ ionophore itself almost failed to affect the somatostatin binding. These results suggest, therefore, that TPA or OAG can modulate somatostatin binding to its receptors on rat pancreatic acinar cell membranes, presumably through activation of Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C); and the activated protein kinase C and intracellular Ca2+ mobilization presumably act to modulate the pancreatic acinar somatostatin receptors synergistically.     

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.     

Detection and characterization of microvesicles in the acinar lumen and in juice of unstimulated rat pancreas

Small vesicles were visualized in the lumen of rat pancreas acini by freeze-substitution and conventional electron microscopy. Microvesicles were subsequently isolated from pancreatic juice. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that these vesicles contain only one major protein. The major protein was identified by an immunoblot technique as GP-2, an 80 kD glycoprotein also found in the zymogen granule membrane. The immunocytochemical localization of rabbit anti-GP-2 and anti-amylase by the protein A-gold technique confirmed that GP-2 was associated with clusters of microvesicles, whereas amylase was virtually excluded. Freeze-fracture of the microvesicles revealed that their membrane was devoid of intramembrane particles. Biochemical analysis indicated also that the membrane did not contain any detectable cholesterol. These results demonstrate that GP-2 is released from the acinar cell in the gland lumen within microvesicles by a hitherto undescribed mode of secretion.     

Ultrastructural morphometric study on developing acinar cells of the rat pancreas and parotid gland

The fraction of cellular volume occupied by the cytoplasm has been assessed by point-counting volumetry in 0.5 micrometer araldite sections. Measurement of the transection radius of the nuclei of acinar cells allowed the assessment of the mean nuclear volume according to the method of Bach. With these data, the cytoplasm volume was calculated in cubic micrometers. Ultrastructural morphometric data were obtained by means of superimposing on the electron photomicrographies (x 21,000 or x 28,500) a test system of 84 evenly spaced segments according to Weibel et al. The analysis of the values obtained for the pancreas and parotid gland allowed for the supposition that the process of maturation in the rat, either for the pancreas or the parotid gland, occurs simultaneously with the mitotic process, this last being held as the predominating one in the first 3--4 postnatal weeks. The cytodifferentiation process becomes more marked in the last phases studied and on the 40th day it already shows parameters held as similar to those of the adult animal.     

The development of the acinar heterotopic pattern of phosphoenolpyruvate carboxykinase activity in the newborn rat

Summary The postnatal appearance of phosphoenolpyruvate carboxykinase activity (PEPCK) and acinar heterotopy was investigated in newborn rats aged 2 h, 12 h, 24 h and 3 days, as well as in juvenile rats aged 25 days. The livers showed an almost homogeneous distribution of activity along the sinusoidal length at the beginning of extrauterine life where energy needs are greatest. Compared to rats aged 2 h, the PEPCK activity was higher in the livers from rats aged 12 h. The increase in activity was most pronounced in the intermediary zone. After 24 h of extrauterine life the activity decreased again creating a homogeneous acinar activity pattern. By day 3 activity had increased in the periportal zone, while decreasing in the perivenous zone, resulting in a periportal to perivenous gradient. By day 25 total activity had reached highest values both in males and females, due to a relatively high perivenous activity. The more prominent acinar gradient corresponded approximately to the one seen in adult animals.     

Structural characterization of a rat acinar cell tumor

A transplantable acinar cell tumor of the rat pancreas has been examined by light and electron microscopy. The tumor cells, though highly cytodifferentiated and characterized by the presence of abundant rough-surfaced endoplasmic reticulum, elements of the Golgi complex, and zymogen granules, undergo mitosis in a manner similar to that seen in the developing pancreas. Cells in the parenchyma of the tumor grow as disarrayed cords and sheets, are randomly oriented with respect to each other, and do not form acinar structures. However, when in contact with the adventitial surface of blood vessels, the tumor cells palisade and form a polarized layer of cells with their zymogen granule-rich poles oriented away from the vessel lumen. Only in this area of the tumor is a basal lamina present that underlies the basal plasmalemma of the reoriented epithelial cells. Freeze-fracture electron microscopy of tumor cells in the parenchyma shows extensive disruption of tight junctions whose sealing strands are randomly distributed over the entire plasmalemma. Gap junctions are infrequent and when present are often enclosed by tight-junctional strands. Intramembrane particles are randomly distributed over the cell surface. Both the absence of basal lamina and derangement of the junctional complexes may account in part for the altered morphogenesis of this tumor.     

The development of the acinar heterotopic pattern of phosphoenolpyruvate carboxykinase activity in the newborn rat

The postnatal appearance of phosphoenolpyruvate carboxykinase activity (PEPCK) and acinar heterotopy was investigated in newborn rats aged 2 h, 12 h, 24 h and 3 days, as well as in juvenile rats aged 25 days. The livers showed an almost homogeneous distribution of activity along the sinusoidal length at the beginning of extrauterine life where energy needs are greatest. Compared to rats aged 2 h, the PEPCK activity was higher in the livers from rats aged 12 h. The increase in activity was most pronounced in the intermediary zone. After 24 h of extrauterine life the activity decreased again creating a homogeneous acinar activity pattern. By day 3 activity had increased in the periportal zone, while decreasing in the perivenous zone, resulting in a periportal to perivenous gradient. By day 25 total activity had reached highest values both in males and females, due to a relatively high perivenous activity. The more prominent acinar gradient corresponded approximately to the one seen in adult animals.     

p53-dependent acinar cell apoptosis triggers epithelial proliferation in duct-ligated murine pancreas

The mechanisms linking acinar cell apoptosis and ductal epithelial proliferation remain unknown. To determine the relationship between these events, pancreatic duct ligation (PDL) was performed on p53(+/+) and p53(-/-) mice. In mice bearing a wild-type p53 allele, PDL resulted in upregulation of p53 protein in both acinar cells and proliferating duct-like epithelium. In contrast, upregulation of Bcl-2 occurred only in duct-like epithelium. Both p21(WAF1/CIP1) and Bax were also upregulated in duct-ligated lobes. After PDL in p53(+/+) mice, acinar cells underwent widespread apoptosis, while duct-like epithelium underwent proliferative expansion. In the absence of p53, upregulation of p53 target genes and acinar cell apoptosis did not occur. The absence of acinar cell apoptosis in p53(-/-) mice also eliminated the proliferative response to duct ligation. These data demonstrate that PDL-induced acinar cell apoptosis is a p53-dependent event and suggest a direct link between acinar cell apoptosis and proliferation of duct-like epithelium in duct-ligated pancreas.     

Freeze-fracture and deep-etching studies on zymogen-granule membranes of the rat pancreas

Summary Whole pancreatic zymogen granules or their membrane fraction were examined by freeze-fracture or deep-etching under different experimental conditions. The granules were fixed for different time periods, or not fixed, and were cryoprotected with glycerol or DMSO; 3% glutaraldehyde followed by 30% glycerol were finally chosen for giving the best resolution and the highest density of intramembrane particles (IMP). IMP are present on the PF and EF leaflets. Their number decreases with the duration of the fixation. Several granules exhibit IMP-free blebs.Incubation of the granules with protamine sulfate causes an aggregation of IMP and of the rough-textured background on the EF leaflet. A second fracture plane can be formed and has been shown by deep-etching to be intercalated between PF and EF. Deep-etching has also shown that particles attached to the perimeter of the granules and of the blebs are, in fact, large nodules on the PS face which partially extend onto the blebs and do not aggregate with the IMP after protamine treatment. Fusion is also indicated between membrane vesicles. Freeze-fracture of the purified membrane fraction seems to indicate the formation of an IMP cap during the lysis of the granule. Moreover, large nodules remain present on the PS face on these membrane fractions but the majority disappear after washing at pH 11.2 with Na₂CO₃ and EDTA.Supported by a research grant from the Medical Research Council of Canada (F.L.) (J.S.H.)     

Effects of pancreatic acinar cell surface antibodies and complement on isolated rat acinar cells in vitro

Surface directed pancreatic acinar cell antibodies raised by immunization of rabbits with suspensions of viable isolated rat acinar cells were utilized to study immune cytolytic processes as a model of in vitro pancreatic injury. The antibodies produced were bound to rat pancreatic acinar cell surface determinants and significantly damaged freshly separated acinar cells by immune cytolytic mechanisms. Addition of complement accelerated the cytolytic effects on the target cells in a dose-dependent manner. The decline of acinar cells was dependent only on the presence of the immune cytolytic potential and not on the number of already damaged cells. Morphologic changes in the cells induced by the agents applied were revealed by both transmission and scanning electron microscopy. The presented experimental model seems a valuable tool for further investigations at the cellular level into the contribution of primarily occurring acinar cell injury in triggering the subsequent pathophysiological mechanisms initiating autodigestion of the pancreatic gland in the pathogenesis of acute pancreatitis. Dedicated to Professor P. Heinrich on the occasion of his 60th birthday     

Effects of pancreatic acinar cell surface antibodies and complement on isolated rat acinar cells in vitro

Surface directed pancreatic acinar cell antibodies raised by immunization of rabbits with suspensions of viable isolated rat acinar cells were utilized to study immune cytolytic processes as a model of in vitro pancreatic injury. The antibodies produced were bound to rat pancreatic acinar cell surface determinants and significantly damaged freshly separated acinar cells by immune cytolytic mechanisms. Addition of complement accelerated the cytolytic effects on the target cells in a dose-dependent manner. The decline of acinar cells was dependent only on the presence of the immune cytolytic potential and not on the number of already damaged cells. Morphologic changes in the cells induced by the agents applied were revealed by both transmission and scanning electron microscopy. The presented experimental model seems a valuable tool for further investigations at the cellular level into the contribution of primarily occurring acinar cell injury in triggering the subsequent pathophysiological mechanisms initiating autodigestion of the pancreatic gland in the pathogenesis of acute pancreatitis.     

Particle displacement in epithelial cell membranes of rat prostate and pancreas induced by routine low temperature fixation

When carried out at 4 °C in contrast to 37 °C, glutaraldehyde fixation for routine freeze-fracture specimen preparation results in displacement of membrane particles of epithelial cells of rat ventral prostate. Particle-free areas are observed in endoplasmic reticulum (ER), nuclear envelope, and Golgi membranes; particle aggregation is observed in lateral and basal portions of the plasma membrane. Multilamellar arrangement of lipids can be observed in some of the large particle-free areas. Thin section observation indicates that in the ER, particle-free areas coexist with segregation of cytoplasmic ground substance from the membrane surfaces. Particle-free patches are also observed in the ER membranes of pancreatic tissues fixed at 4 °C.