RESTITUTION OF PANCREATIC ACINAR CELLS FOLLOWING ETHIONINE

The regeneration of the pancreatic acinar cell was studied at four time periods after ethionine had destroyed most of the acinar cells. Within 2 days of the last ethionine injection, small basophilic cells (pre-acinar cells) with whorls of ergastoplasm or nebenkern were present. These cells also contained a decreased amount of Golgi substance, small zymogen granules, and a fine granularity of the nuclear matrix. They showed persistence of the characteristic ergastoplasm lesion produced by ethionine. Eight days after the last ethionine injection, the nebenkern was replaced by approximately normal appearing ergastoplasm and the nucleoli and Golgi bodies were enlarged. Zymogen granules were less dense but more abundant. Mitochondria were considerably enlarged. Most cells showed no ethionine lesions or only small foci of damage. Eighteen days after the cessation of ethionine, a good approximation of the normal acinar cell was present. The whorls of ergastoplasm appeared at a time (day 12) when basophilia was pronounced. Other studies showed that nucleic acid and protein precursors began to show an increased concentration in acinar cells at this time. The appearance of nebenkern during a phase of cellular recovery and its absence during a phase of replication when mitotic indices were high suggest that its presence is more indicative of ergastoplasmic synthesis than of cell multiplication as such. Possibly the increased density of zymogen granules was a reflection of this increased protein synthesis. The increase in size of Golgi apparatus occurred prior to the replenishment of zymogen granules and thus satisfied a precursor relationship for a possible role in the formation of these secretory structures. Evidence suggests that some injured acinar cells recover from the ethionine and protein-free regimen and give rise to most of the new acinar cells formed. It is possible that, under the severe conditions which prevailed, the centroacinar ductule cells may also have given rise to some acinar cells.     

Some properties of a Ca2+- and (or) Mg2+-requiring nucleoside di- and tri-phosphatase(s) associated with the membranes of rat pancreatic zymogen granules.

Membranes of rat pancreatic zymogen granules have been purified and found to be essentially free of contamination by mitochondria, microsomes, and plasma membranes. They possessed an acid phosphatase activity which derived probably from lysed lysosomes contaminating the purified zymogen granules from which the membranes were prepared. These membranes were found to contain a strong Ca2+- and (or) Mg2+-requiring activity toward all nucleoside di- and tri-phosphates. Various data support the tentative conclusion that a single protein catalyzes the hydrolysis of the nucleoside di- and tri-phosphates. This protein appears to be intrinsic with its active site localized on the internal face of the membranes.     

Glycoprotein synthesis in the adult rat pancreas. IV. Subcellular distribution of membrane glycoproteins

Zymogen granule membranes from the rat exocrine pancreas displays distinctive, simple protein and glycoprotein compositions when compared to other intracellular membranes. The carbohydrate content of zymogen granule membrane protein was 5-10-fold greater than that of membrane fractions isolated from smooth and rough microsomes, mitochondria and a preparation containing plasma membranes, and 50-100-fold greater than the zymogen granule content and the postmicrosomal supernate. The granule membrane glycoprotein contained primarily sialic acid, fucose, mannose, galactose and N-acetylglucosamine. The levels of galactose, fucose and sialic acid increased in membranes in the following order: rough microsomes less than smooth microsomes less than zymogen granules. Membrane polypeptides were analyzed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The profile of zymogen granule membrane polypeptides was characterized by GP-2, a species with an apparent molecular weight of 74 000. Radioactivity profiles of membranes labeled with [3H]glucosamine or [3H]leucine, as well as periodic acid-Schiff stain profiles, indicated that GP-2 accounted for approx. 40% of the firmly bound granule membrane protein. Low levels of a species similar to GP-2 were detected in membranes of smooth microsomes and the preparation enriched in plasma membranes but not in other subcellular fractions. These results suggest that GP-2 is a biochemical marker for zymogen granules. Membrane glycoproteins of intact zymogen granules were resistant to neuraminidase treatment, while those in isolated granule membranes were readily degraded by neuraminidase. GP-2 of intact granules was not labeled by exposure to galactose oxidase followed by reduction with NaB3H4. In contrast, GP-2 in purified granule membranes was readily labeled by this procedure. Therefore GP-2 appears to be located on the zymogen granule interior.     

Glycoprotein synthesis in the adult rat pancreas: IV. Subcellular distribution of membrane glycoproteins

Zymogen granule membranes from the rat exocrine pancreas displays distinctive, simple protein and glycoprotein compositions when compared to other intracellular membranes. The carbohydrate content of zymogen granule membrane protein was 5–10-fold greater than that of membrane fractions isolated from smooth and rough microsomes, mitochondria and a preparation containing plasma membranes, and 50–100-fold greater than the zymogen granule content and the postmicrosomal supernate. The granule membrane glycoprotein contained primarily sialic acid, fucose, mannose, galactose and $\text{N-}\text{acetylglucosamine}$. The levels of galactose, fucose and sialic acid increased in membranes in the following order: rough microsomes < smooth microsomes < zymogen granules.Membrane polypeptides were analyzed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The profile of zymogen granule membrane polypeptide was characterized by GP-2, a species with an apparent molecular weight of 74 000. Radioactivity profiles of membranes labeled with [³H]glucosamine or [³H]leucine, as well as periodic acid-Schiff stain profiles, indicated that GP-2 accounted for approx. 40% of the firmly bound granule membrane protein. Low levels of a species similar to GP-2 were detected in membranes of smooth microsomes and the preparation enriched in plasma membranes but not in other subcellular fractions. These results suggest that GP-2 is a biochemical marker for zymogen granules.Membrane glycoproteins of intact zymogen granules were resistant to neuraminidase treatment, while those in isolated granule membranes were readily degraded by neuraminidase. GP-2 of intact granules was not labeled by exposure to galactose oxidase followed by reduction with NaB³H₄. In contrast, GP-2 in purified granule membranes was readily labeled by this procedure. Therefore GP-2 appears to be located on the zymogen granule interior.     

An assessment of the role of protein kinase and zymogen granule phosphorylation during secretion by the rat exocrine pancreas.

1. The levels of protein kinase activity and zymogen granule phosphorylation were studied in the adult rat during stimulus-coupled secretion in vitro. 2. The specific activity of protein kinase associated with intact zymogen granules was 11 pmol [32P]phosphate transferred to histone per min per mg protein. Most of this activity was recovered in purified granule membranes. 2. The addition of 10(-6) M cyclic AMP to a mixture of zymogen granules and the postmicrosomal supernatant resulted in a 5-fold increase in protein kinase activity associated with zymogen granules. The adsorbed activity was eluted from granules by 0.15 M NaCl. Cyclic GMP did not promote protein kinase binding to isolated granules. 4. Incubation of tissues with carbachol (10(-5) M), pancreozymin (0.1 unit/ml), caerulein (10(-8) M) or dibutyryl cyclic AMP (2.10(-4) M) between 2.5 and 60 min did not increase the levels of protein kinase activity in isolated zymogen granules above control values. 5. Protein phosphorylation of zymogen granule membranes and granule content was not detectable in tissues incubated with carbachol, pancreozymin-C-octapeptide, or caerulein. 6. These results suggest that neither the phosphorylation of zymogen granule membrane protein nor the adsorption of protein kinase activity to zymogen granules is an obligatory step in secretion.     

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.     

Topographical and planar distribution of Helix pomatia lectin-binding glycoconjugates in secretory granules and plasma membrane of pancreatic exocrine acinar cells of the rat: demonstration of membrane heterogeneity

The lectin-gold technique was used to detect Helix pomatia lectin (HPL) binding sites directly on thin sections of rat pancreas embedded in Lowicryl K4M and on freeze-fractured preparations of rat pancreas submitted to fracture label. On thin sections of acinar cells, whereas the content of zymogen granules was negative or weakly labeled, the limiting membrane displayed a high degree of labeling. In the Golgi complex, labeling by HPL was localized on the trans saccules and the limiting membrane of the condensing vacuoles. The latter appeared to be more intensely labeled than the membrane of the zymogen granules. Intense labeling by HPL was also observed along the microvilli and the plasma membrane. In contrast to the weak labeling of the zymogen-granule content, labeling of the acinar lumen was intense. Fracture-label preparations revealed preferential partition of HPL-binding sites to the exoplasmic half of the zymogen-granule and plasma membranes. The population of zymogen granules was, however, heterogeneous with respect to labeling intensity; the exoplasmic fracture-face of the plasma membrane was intensely and uniformly labeled, while the protoplasmic membrane halves were only weakly labeled. These observations were further confirmed and extended by the thin-section fracture-label approach. In addition, favorable profiles of thin sections of freeze-fractured zymogen granules showed that the labeling was not associated with the external surface of the limiting membrane, but rather localized over the exoplasmic fracture-face. We conclude that 1) zymogen granules contain little HPL-binding glycoconjugates, 2) HPL-binding sites are preferentially associated with the exoplasmic half of the zymogen-granule and plasma membranes, and 3) the limiting membrane of the immature condensing vacuoles carries a greater number of HPL-binding sites than that of the mature zymogen granules. These last, in turn, constitute a heterogenous population with respect to labeling density. These results support the current view that glycoconjugates are directed toward the lumen in secretory granules but become external to the cell surface after fusion of the secretory-granule membrane with the plasma membrane. Also, the results reflect membrane modifications during the maturation process of secretory granules in the exocrine pancreas in which glycoproteins are removed from the limiting membrane of the granule to become soluble and secreted with the content.     

RAPID RELEASE OF THE ZYMOGEN GRANULE PROTEIN BY OSMIUM TETROXIDE AND ITS RETENTION DURING FIXATION BY GLUTARALDEHYDE

Fixation by osmium tetroxide and glutaraldehyde of zymogen granules isolated from rat parotid and pancreas was investigated. Protein determinations showed that osmium tetroxide caused rapid release of most of the soluble protein of the granule during fixation in buffered isotonic sucrose. Such granules when examined in the electron microscope after shadow casting appeared quite flat, indicating that most of the contents had indeed been removed. Numerous damaged membranes of the granules were also observed. In contrast, zymogen granules fixed by glutaraldehyde and shadow cast essentially retained the spherical shape and the protein contents. The application of the shadow-casting technique in quantitative studies on the protein content of zymogen granules is discussed.     

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

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

THE STAINING OF THIN SECTIONS OF MOUSE PANCREAS PREPARED BY THE FERNÁNDEZ-MORÁN HELIUM II FREEZE-SUBSTITUTION METHOD

Small pieces of mouse pancreas were rapidly frozen in helium II, substituted in methanol at -75°C., and embedded in methacrylate by ultraviolet polymerization in the cold. The unstained cells show a structure similar to that after OsO₄ fixation, except that the RNP particles have little or no contrast and the mitochondria and Golgi zones appear as grey areas without internal structure. After staining the sections by floating them on solutions of lead acetate or osmium tetroxide, there is an increase in contrast of RNP particles, ergastoplasmic membranes, and zymogen granules. Mitochondrial and Golgi membranes, zymogen granule membranes, and a membrane along the outside of the ergastoplasmic cisterna appear in negative contrast. The structure of the ergastoplasm, the existence of RNP particles, and the production of negative contrast are discussed. A modification of Gomori's method for acid phosphatase produces a lead deposit around the periphery of the zymogen granules. Possibly this deposit does not represent the true site of the enzyme, but the results show the feasibility of histochemistry at the level of resolution of the electron microscope.     

Modulation of phospholipase A2 activity in zymogen granule membranes by GTP[S]; evidence for GTP-binding protein regulation

In membranes associated with purified pancreatic zymogen granules, GTP[S] elicited a concentration-dependent activation of phospholipase A2 (PLA2), which was converted to inhibition in the presence of added Ca2+. The GTP-binding protein inhibitor GDP[S] blocked both the stimulatory and inhibitory actions of GTP[S]. We conclude that in zymogen granule membranes GTP-binding proteins exert a dual regulation of PLA2 activity.     

Calcium binding properties of purified zymogen granule membrane of pig pancreas. Evidence for calcium binding proteins

Ca2+ binding properties of purified zymogen granule membranes of pig pancreas have been measured: Binding increased linearly with Ca2+ concentration in the medium up to the micromolar range; in the millimolar range a sharp rise in binding capacity was observed. Binding increased with pH both at low and high concentrations of Ca2+. It was insensitive to Na+ and K+ ions at concentrations up to 100 mM. Mg2+ was inhibitory in the millimolar range whereas La2+ and Tb3+ were inhibitory in the micromolar range. The Ca2+ binding components of zymogen granule membranes were identified by two methods: (1) by measuring 45Ca2+ binding after counter-ion electrophoresis and (2) by Stain's-all (forms a complex with Ca2+ binding proteins absorbing maximally at 600 nm), after SDS-polyacrylamide gel electrophoresis. The first method, counter-ion electrophoresis, indicated that most of the 45Ca2+ was associated with an acidic band which could be subsequently subfractionated by SDS-polyacrylamide gel electrophoresis in five bands: 66, 57, 30, 27 and 22.5 kDa. The second method, Stain's-all, revealed six positive polypeptides after SDS-polyacrylamide gel electrophoresis of native zymogen granule membranes' two were unreactive after neuraminidase treatment (130 and 92 kDa, respectively), whereas four other bands were still reactive (66, 57, 43, 30 kDa, respectively.) Ca2+ binding was also measured on intact zymogen granules: the binding capacity was higher than for zymogen granule membranes. Among the Ca2+ binding proteins of the zymogen granule membrane only one is apparently located on the granule external surface: the 30 kDa polypeptide. If Ca2+ directly facilitates fusion of zymogen granules with plasma membrane by a Ca2+-protein interaction, then this protein is a presumptive candidate to play such a key role.     

Quantitative ultrastructural studies on rat gastric zymogen cells under different physiological and experimental conditions

Summary Quantitative electron microscopical measurements performed on gastric zymogen cells were aimed at determinations of size and volume density of the zymogen granules, and size of cell and nuclear profiles. 17 groups of rats each comprising 6–8 animals were investigated: five of these groups were used to study the influence of fasting and feeding, three groups were killed at different intervals after a pilocarpine injection, and four other groups were investigated after an atropine injection. The remaining five groups of rats were operated on: vagotomy was performed on one group, vagotomy + pyloroplasty on another, pyloroplasty on the third group, and antrectomy on the last two groups. The operated rats were sacrificed 4 or 10 weeks after the operations.Pilocarpine was more effective than feeding in reducing the size and the amount of zymogen granules. After atropine the size and amount of zymogen granules tended to increase. Ten weeks after pyloroplasty, vagotomy + pyloroplasty, or antrectomy the mean size of the zymogen cells was reduced.Loss of trophic vagal impulses, duodenal regurgitation, and abnormal serum gastrin levels are factors which might be responsible for the zymogen cell hypotrophy in operated rats.Supported by grants from the Swedish Medical Research council (project no 12X-2298), M. Bergvalls Stiftelse, and Julins Fond. Excellent technical assistance was provided by Mrs. Sigrid Kilter and Miss Ulla Hedlund     

Caerulein-induced acute pancreatitis in rats: changes in glycoprotein-composition of subcellular membrane systems in acinar cells

Summary Caerulein-induced acute pancreatitis is characterized by the occurrence of two membrane-bound vacuolar systems in acinar cells. Beside digestive enzymes containing secretory vacuoles, lysosomal autophagic structures can be identified at the ultrastructural level. In the present study glycoconjugate patterns of the surrounding membranes were characterized by ultrastructural lectin-binding experiments using five colloidal-gold labeled lectins with distinct sugar specificities. Furthermore, the profile of membrane glycoproteins of isolated vacuolar fractions was studied by SDS-PAGE and lectin-blotting. In pancreatitis, membranes of secretory vacuoles showed a significant lower degree of lectin-binding compared to normal zymogen granules. In contrast, newly appearing autophagic vacuoles in pancreatitis revealed a strong membrane labelling for most lectins used. The pattern of membrane glycoproteins of secretory and autophagic vacuoles as determined by SDS-PAGE and lectin-blotting differed from those of normal zymogen granules resembling the protein profile of smooth microsomes. Since this pattern requires a previous passage through Golgi stacks, it is assumed that the two types of vacuoles derive from Golgi elements. For the pathogenesis of caerulein pancreatitis these vacuolar post-Golgi structures seem to play an important role.     

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.     

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     

Characterization of phospholipase A2 and acyltransferase activities in purified zymogen granule membranes

Phospholipase A2 and acyltransferase activities were identified in membranes associated with purified pancreatic zymogen granules. In homogenate and granule membranes, phospholipase activity was linearly related to protein concentration and was Ca2(+)-dependent with an alkaline pH optimum. The Ca2+ sensitivity was observed over the range of concentrations through which intracellular ionic Ca2+ is elevated by physiological stimuli in intact cells. Intact zymogen granules and granule membranes also demonstrated reacylating activity in the presence and absence of an exogenous acceptor. Reacylating activity was related to the concentration of lyosphospholipid added and was optimally activated at alkaline pH. A more rapid rate of reacylation was observed when [14C]arachidonoyl CoA was employed as the donor molecule rather than [3H]arachidonate (plus coenzyme A); this suggests the absence of acyl-CoA synthetase in the purified granule membranes. We conclude that granule membrane phospholipase A2 and acyltransferases may be involved in arachidonic acid turnover in exocrine pancreas and perhaps in membrane fusion events associated with exocytosis.     

Redistribution of a rab3-like GTP-binding protein from secretory granules to the Golgi complex in pancreatic acinar cells during regulated exocytosis

Regulated secretion from pancreatic acinar cells occurs by exocytosis of zymogen granules (ZG) at the apical plasmalemma. ZGs originate from the TGN and undergo prolonged maturation and condensation. After exocytosis, the zymogen granule membrane (ZGM) is retrieved from the plasma membrane and ultimately reaches the TGN. In this study, we analyzed the fate of a low M(r) GTP-binding protein during induced exocytosis and membrane retrieval using immunoblots as well as light and electron microscopic immunocytochemistry. This 27-kD protein, identified by a monoclonal antibody that recognizes rab3A and B, may be a novel rab3 isoform. In resting acinar cells, the rab3-like protein was detected primarily on the cytoplasmic face of ZGs, with little labeling of the Golgi complex and no significant labeling of the apical plasmalemma or any other intracellular membranes. Stimulation of pancreatic lobules in vitro by carbamylcholine for 15 min, resulted in massive exocytosis that led to a near doubling of the area of the apical plasma membrane. However, no relocation of the rab3-like protein to the apical plasmalemma was seen. After 3 h of induced exocytosis, during which time approximately 90% of the ZGs is released, the rab3- like protein appeared to translocate to small vesicles and newly forming secretory granules in the TGN. No significant increase of the rab3-like protein was found in the cytosolic fraction at any time during stimulation. Since the protein is not detected on the apical plasmalemma after stimulation, we conclude that recycling may involve a membrane dissociation-association cycle that accompanies regulated exocytosis.     

FINE STRUCTURE OF DIFFERENTIATING MOUSE PANCREATIC EXOCRINE CELLS IN TRANSFILTER CULTURE

Fine structural observations have been made in the 11-day embryonic mouse of exocrine cells in pancreatic epithelium developing in tissue culture transfilter from salivary gland mesenchyme of the 13-day embryonic mouse. After 2 days in culture, the exocrine cells show increased cytoplasmic density, abundant ribosomes in aggregate or "rosette" form, and expanded profiles of rough-surfaced endoplasmic reticulum. After 3 and 4 days in culture, the cells exhibit continued expansion of the profiles of endoplasmic reticulum, increased amounts of Golgi membranes, and large areas of light density (prozymogen granules). After 5 days in culture, dense zymogen granules are present in the most highly differentiated cells. In addition, at the filter-epithelial surface, at 2 days, small fibers can be discerned which, after 4 days in culture, show obvious periodicity and are thought to be collagen. The significance of these changes, in relation to the mesenchymal effect, to the onset of specific synthesis and to the stabilization of differentiation is discussed.