Time-course of oxygen free radical production in acinar cells during acute pancreatitis induced by pancreatic duct obstruction

The time-course of oxygen free radicals (OFR) generation within acinar cells was studied at different stages of acute pancreatitis (AP) induced in rats by duct obstruction (PDO) for 48 h by flow cytometry, using dihydrorhodamine-123 (DHR) as fluorescent dye. Parallel measurements of the most common markers of oxidative stress such as glutathione (GSH) depletion and malondialdehyde (MDA) levels in pancreas were also performed. OFR production significantly increased within acinar cells at early stages of AP, concomitant with a marked depletion in pancreatic GSH. Lipid peroxidation was significantly enhanced 6 h after PDO, suggesting that the antioxidant defence system of the cell is overwhelmed by OFR production. Both MDA and OFR production in acinar cells decreased to normal values at late AP stages, thus allowing the recovery of pancreatic GSH levels 48 h after PDO. Among the two types of acinar cells differentiated by flow cytometry, R1 and R2, it was the R2 population that showed higher values of DHR dye. However, no differences between the two cell types were found regarding the amount of OFR generation. Our results demonstrate that individual acinar cells significantly contribute to produce large amounts of OFR at early stages of AP. The two existing populations of acinar cells displayed similar behaviour regarding oxidative stress over the course of the disease.     

Dye-coupling between cells of the salivary glands in the cockroach Periplaneta americana

Cockroaches have acinar salivary glands. The acini consist of peripheral cells specialized for electrolyte and water transport and central cells contributing proteinaceous components to the saliva. Salivary duct cells probably modify the primary saliva. The acinar cells in Nauphoeta cinerea had been shown to be electrically coupled and dye-coupled. Since intercellular communication via gap junctions between acinar cells is difficult to reconcile with previous findings that dopamine and serotonin selectively stimulate the secretion of either protein-free or protein-rich saliva in Periplaneta americana, we investigated whether dye-coupling occurs between both acinar cell types and between duct cells. We iontophoretically loaded Lucifer yellow into impaled cells and tested for dye-coupling by confocal laser scanning microscopy. We found that: (1) peripheral and central cells within an acinar lobulus of the gland in P. americana are dye-coupled; and (2) salivary duct cells are dye-coupled.     

Quantitative immunoelectron microscopic localization of (Na+,K+)ATPase on rat exocrine pancreatic cells

Distribution of (Na+,K+)ATPase in rat exocrine pancreatic cells was investigated quantitatively by immunoelectron microscopy using the post-embedding protein A-gold technique. We found that in acinar and duct cells (Na+,K+)ATPase exists on both the luminal and the basolateral surfaces, with higher particle density on the luminal surface (4.4 times in the acinar cells and 5.6 times in the duct cells). According to Bolender (J Cell Biol 61:269, 1974), the luminal surface represents only 5% of the total cell surface of an average pancreatic acinar cell. It is roughly estimated, therefore, that approximately 80% of the plasma membrane (Na+,K+)ATPase in the acinar cells exists on the basolateral surface. When the acinar and duct cells were compared, more than twice as many particles were found on acinar cells than on duct cells. The enzyme existed on all the cell surfaces, preferentially on the microvilli or on the cell membrane folds, and no clustering was detected. We suggest that the (Na+,K+)ATPase on the basolateral surface is mainly responsible for the extrusion of a large number of sodium ions that are incorporated into the cytoplasm accompanying the secondary active transport of various organic substances and inorganic ions, whereas that on the luminal surface is responsible for active extrusion of sodium ions that are partially responsible for the fluid secretion of the pancreatic cells.     

Histochemical localization and analysis of blood group-related antigens in human pancreas using immunostaining with monoclonal antibodies and exoglycosidase digestion

We examined the distribution of blood group-related antigens using an indirect immunoperoxidase method with monoclonal antibodies (MAb) directed to A, B, H, Lewis a (Lea), Lewis b (Leb), Lewis x (Lex), and Lewis y (Ley) antigens and Type 1 precursor chain in human pancreas. Effects of prior digestion with exoglycosidases on MAb stainings were simultaneously investigated. A, B, H, Leb, and Ley antigens were detected in acinar cells and interlobular duct cells but not in centroacinar cells, intercalated duct cells, and islet of Langerhans cells. The expression of these antigens in acinar cells was not dependent on Lewis type and secretor status of the tissue donors, whereas that in interlobular duct cells was strictly dependent on secretor status. The distribution pattern of these antigens in acinar cells was not homogeneous, i.e., cells producing H antigens expressed both Leb and Ley antigens but not A or B antigens, whereas those producing A or B antigens did not secrete Leb and Ley as well as H antigens. Digestion with alpha-N-acetylgalactosaminidase or alpha-galactosidase resulted in the appearance of Leb and Ley antigens as well as H antigen in acinar cells producing A and/or B antigens. Type 1 precursor chain was not detected in pancreatic tissues from secretors but appeared in acinar cells producing H antigen after alpha-L-fucosidase digestion, which also disclosed Lex but not Lea antigen in acinar cells expressing both Leb and Ley. In some non-secretors, MAb against Type 1 precursor chain reacted with acinar cells without enzyme digestion. Although Lea antigen was not detected in acinar cells, it was found in centroacinar cells, intercalated duct cells, and interlobular duct cells from all individuals examined except two Le(a-b-) secretors. After sialidase digestion, Lex antigen appeared in centroacinar and intercalated duct cells from some individuals. Sialidase digestion also elicited reactivity with MAb against Type 1 precursor chain in islet of Langerhans cells from some individuals. These results demonstrate the complexity in the pattern of expression and regulation of blood group-related antigens in different cell types of human pancreas. Such complexity may largely be ascribed to differences in individual genotypes and in gene expression patterns of different cell types.     

Early observations of pancreatic ultrastructure during hemorrhagic necrosis in the rat with closed duodenal pouch]

Many problems are still unanswered in the pathogenesis of acute clinical and experimental pancreatic necrosis. A new technique which can be performed in the rat seems a suitable model for reflux pancreatic necrosis without artificial pressure changes in the ductal system. A closed duodenal loop is obtained with ligation proximal and distal to Vater's ampulla and a gastroenteroanastomosis is associated to avoid intestinal obstruction. All the rats die with hemorrhagic pancreatic necrosis in 36 hours. After 12 hours from the operation ductal and acinar lumina are enlarged. In the centroacinar and intercalated duct cells some lysosomes and mitochondria with clear matrix and reduced cristae are detected. Intercellular junctions in ducts and acini have normal morphology. In the basal cytoplasm of acinar cells some prominent autophagic vacuoles are detectable. After 24 hours in the acinar cells autophagic vacuoles are greatly increased and basal cytoplasmic degeneration often occurs, with plasmalemma and basal lamina interruptions. Intercellular junctions are apparently unaffected until cell necrosis sets in. In blood capillaries endothelial cells are swollen, fibrin thrombosis, hemorrhage and leucocyte infiltration are often detectable. As lysosomal activity occurs also in different kinds of experimental pancreatic necrosis, it could be a common pathogenetic factor, responsible for hydrolytic enzyme activation and for vascular damage in the early stages of hemorrhagic pancreatic necrosis.     

The roles of apoptosis and mitosis in atrophy of the rat sublingual gland

The roles of apoptosis and mitosis of acinar and duct cells in the atrophy of the sublingual gland of rat induced by double duct ligation was investigated using immunohistochemistry for proliferating cell nuclear antigen (PCNA), terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick end labeling (TUNEL), and transmission electron microscopy (TEM). Many PCNA-positive duct cells were observed 3 days after duct ligation, and the numbers decreased thereafter. At 3 and 5 days, several TUNEL-positive acinar cells were observed and typical apoptotic acinar cells were identified by TEM. Necrotic acinar cells were also observed ultrastructurally. After 7 days, there were few acini but many ducts, as well as many structures representing transition from acinus to duct. These observations demonstrate that acinar cell loss by apoptosis and duct cell proliferation by mitosis occur in atrophic sublingual glands as well as in other atrophic salivary glands. In addition, it appears that the transition from acinar to duct cell and the necrosis of acinar cells play important roles in the atrophy of the sublingual gland.     

Chlamydia-like organisms in digestive and duct cells of mussels from the Basque coast.

Chlamydia-like organisms have been detected in digestive cells and duct cells of the digestive gland of mussels, Mytilus galloprovincialis, collected from the Basque coast. The organisms appeared as basophilic inclusion bodies within digestive cells and consisted of elongate initial reticulate bodies and previously undescribed condensed forms, interpreted as intermediate bodies. Of the 414 mussels examined by light microscopy, 5.31% showed this type of infection. A second type of chlamydia-like organism was found in nonciliated duct cells. The microorganisms were found mostly free in duct cells and large elongate reticulate bodies, intermediate condensing forms, and fully condensed elementary bodies were clearly distinguished. No serious histopathological or ultrastructural changes were observed in host cells but evidences of a possible localized metabolic damage within infected digestive cells is presented.     

Ionic components of secretory cell surfaces in relation to secretory function

Synopsis Rat pancreas was examined by ultrastructural cytochemical methods for localizing cations and anions, as well as polyanions and, more specifically, sulphated mucosubstance. Exceptionally abundant antimonate-precipitable cation was demonstrated between pancreatic acinar cells and at the base of the centro-acinar and other duct epithelial cells. Precipitates of nuclear heterochromatin appeared lighter whereas those of mitochondria and cytoplasm were coarser and more conspicuous in acinar that duct cells. Stimulation with synthetic secretin at a low level diminished antimonate reactivity of nuclei as well as the precipitation at the basement membrane of centro-acinar cells. At a higher dose, secretin selectively eliminated precipitation between and below centro-acinar and other duct cells while inducing increased antimonate-reactive cation in centro-acinar cells and the acinar lumens. Pancreozymin stimulation elimated antimonate-precipitable cations between acinar cells and, to a much lesser extent, those between duct cells and increased cytoplasmic precipitates on granular reticulum of acinar cells.Silver-precipitable anions were localized on the luminal surface of the apical plasma lemma and the outer surface of the latero-basal plasmalemma of centro-acinar cells but not on acinar cell surfaces. Silver precipitates also occurred on junctional complexes of acinar and duct epithelial cells and at tight junctions of acinar cells and on the inner face of the lateral plasmalemma of acinar cells.Dialysed iron staining demonstrated the most number of sites of acid mucosubstance on the luminal surface of the plasmalemma of acinar cells. Lateral and basal plasmalemmas of centro-acinar and more distal duct cells stained lightly with dialysed iron but those of acinar cells did not. Dialysed iron visualized acid mucosubstance in the lamina lucida of the basement membrane of duct but not of acinar cells. Dialysed iron staining of the plasma membranes succumbed to prior sialidase treatment whereas that of basement membrane resisted digestion. High iron diamine staining demonstrated sulphated mucosubstance in the lamina lucida of the duct basement membrane exclusively. The cytochemical results implicate centro-acinar cells as primarily responsible for contributing fluid and electrolytes to pancreatic secretion.     

Carbonic anhydrase isozyme VI in rat lacrimal gland

Using monoclonal antibody specific to rat carbonic anhydrase isozyme VI (CA VI), the isozyme was localized in the lacrimal gland. A minority of acini (less than 10% of the total) contained a few immunoreactive acinar cells. Enzyme histochemistry indicated that the CA VI-positive cells were the only cells possessing CA in the lacrimal acini. In the acinar cells, the reaction product for CA VI was distributed in the secretory granules and cytosol between secretory granules. Except for mitochondrial enzyme (CA V) activity, the intracellular distribution of enzyme activity was similar to that of CA VI immunoreactivity, suggesting that rat lacrimal acinar cells contain only CA VI and CA V. CA VI in the secretory granules was discharged into the acinar lumen and is considered to carry out its function on the surface of the conjunctiva and cornea. The cytosolic CA VI may function in situ and be involved in electrolyte and water secretion by the acinar cells. Polyclonal antibody to rat erythrocyte CA (CA I and CA II) stained only the interlobular ducts. In contrast, all the ductal elements exhibited CA enzyme activity. This discrepancy between immunohistochemistry and enzyme histochemistry suggests the presence of CA isozyme(s) other than CA I, CA II and CA VI in the lacrimal duct.     

Redistribution of carbonic anhydrase VI expression from ducts to acini during development of ovine parotid and submandibular glands

 Carbonic anhydrase VI (CA VI) is a secreted enzyme produced predominantly by serous acinar cells of submandibular and parotid glands. We have investigated the developmental pattern of CA VI production by these glands in the sheep, from fetal life to adulthood, using immunohistochemistry. Also, a specific radioimmunoassay for CA VI was used to measure changes in enzyme expression in the parotid gland postnatally. CA VI is detectable by immunohistochemistry in parotid excretory ducts from 106 days gestation (term is 145 days), in striated ducts from 138 days and in acinar cells from 1 day postnatal. The duct cell content of CA VI declined as the acinar cell population increased, a feature also of CA VI immunoreactivity in the submandibular gland. Production of CA VI by submandibular duct cells was detectable initially at 125 days gestation, and acinar production was not seen before 29 days post-natal. Apart from the differing ontogeny of CA VI production in ducts and acini of parotid and submandibular glands, there was a parallel pattern of CA VI expression during the development of these major salivary glands.With the development of the acinar tissues in the postnatal lamb, there was a dramatic increase (about 600-fold) in the level of expression of CA VI in the parotid gland between days 7 and 59 as measured by radioimmunoassay. Accepted: 19 December 1996     

In-vivo stimulation of rat pancreatic acinar cells by infusion of secretin

Infusion of synthetic secretin in conscious unrestricted rats for periods up to 24 h was used to study the structural and functional adaptation of pancreatic acinar cells to this secretagogue. Initial dose-response studies established 16 clinical units (CU) per kg and h (corresponding to 4.64 micrograms X kg-1 X h-1) as optimal dose for persistent stimulation of enzyme discharge. Infusion of this dose led to a slow but progressive depletion of enzyme stores with minimal content by 12 h stimulation. As a result of persistent stimulation total protein synthesis in the acinar cells increased after a lag period of 3 h and reached maximal values 90% above controls by 6 and 12 h secretin infusion. No structural equivalent for pronounced fluid and bicarbonate secretion was observed for either acinar or duct cells over the entire dose range (1 to 64 CU X kg-1 X h-1) and infusion period (1-24 h), except an increased number of coated vesicles in duct cells. Discharge of enzymes from acinar cells was paralleled by a high frequency of exocytotic images at the luminal plasma membrane and was accompanied by the occurrence of membrane fragments in the luminal space, especially after 3 and 6 h secretin infusion. An increased number of lysosomal bodies at these time points especially in the vicinity of the Golgi complex was interpreted in relation to membrane recycling following massive exocytosis. This pattern of structural and functional adaptation of acinar cells following secretin infusion corresponds to previously described changes following caerulein and carbamylcholine stimulation.     

SULFATE METABOLISM IN PANCREATIC ACINAR CELLS

The metabolism of inorganic sulfate in pancreatic acinar cells was studied by electron microscope radioautography in mice injected with sulfate-³⁵S. Labeled sulfate was concentrated in the Golgi complex at 10 min. Within 30 min, much of the radioactive material had been transferred to condensing vacuoles. These were subsequently transformed into zymogen granules. By 4 hr after injection, some of the zymogen granules with radioactive contents were undergoing secretion, and labeled material was present in the pancreatic duct system. The Golgi complex in pancreatic acinar cells is known to be responsible for concentrating and packaging digestive enzymes delivered to it from the endoplasmic reticulum. Our work demonstrates that the Golgi complex in these cells is also engaged in the manufacture of sulfated materials, probably sulfated mucopolysaccharides, which are packaged along with the enzymes in zymogen granules and released with them into the pancreatic secretion.     

Comparative studies on the localization of esteroproteases and kallikrein-like activity in primate organs

Summary Various organs of three species of monkey were screened histochemically for esteroproteases usingN-acethyl-l-methionine--naphthylester ( N-O-met) as the substrate and also for enzymes with kallikrein-like activity usingd-Val-Leu-Arg-4-methoxy-2-naphthylamide as the substrate. Characteristic differences were found in the localization of the reaction products obtained with both substrates. In the main salivary glands, esteroproteases ( N-O-met reactivity) were found in mucous cells (submandibular gland), intercalated duct cells (parotid gland), acinar cells (sublingual gland), striated and interlobular duct cells (all glands). They were also localized in superficial lining epithelial cells of the digestive system, in liver cells, and acinar cells of the pancreas.Enzymes with kallikrein-like activity were found only in the striated and interlobular duct cells of salivary glands, in acinar cells of the pancreas, and in proximal tubular cells of the kidney. Free cells (including mast cells) normally distributed in the connective tissue of various organs showed reactivity towards N-O-met. Some of these cells were also reactive against Val-Leu-Arg-4-MNA.     

Dose-response for inhibition by amylin of cholecystokinin-stimulated secretion of amylase and lipase in rats

BACKGROUND AND AIMS: The neuroendocrine hormone amylin, cosecreted with insulin from pancreatic beta-cells in response to nutrient ingestion, has several physiologic actions to limit the rate of nutrient uptake, including the slowing of gastric emptying. METHODS: To investigate whether amylin might modulate digestive enzyme secretion from the exocrine pancreas, anesthetized Sprague Dawley rats were cannulated via the pancreatic duct and the secretory response (flow, amylase and lipase) to cholecystokinin (1 microg s.c.) was measured in the absence and in the presence of 0.1, 0.3 and 1 microg s.c. doses of amylin. RESULTS: Amylin alone did not affect pancreatic secretion, but it dose-dependently inhibited cholecystokinin-stimulated amylase secretion by up to 58% and lipase secretion by up to 67%. The ED50's for these responses were 0.21 microg+/-0.18 log and 0.11 microg+/-0.05 log, respectively, doses that result in excursions of plasma amylin concentration that are within the reported physiological range. Amylin did not evoke cell signalling in the Ar42j model of pancreatic acinar cells, and responses to amylin were not observed in either Ar42j cells or isolated pancreatic acini in a microphysiometer indicating that the effect of amylin was indirect. CONCLUSIONS: Inhibition of stimulated pancreatic enzyme secretion is likely to be a physiological, extrapancreatic, action of amylin. Amylinergic mechanisms modulating both gastric emptying and pancreatic enzyme secretion may thus match, respectively, the appearance of substrate and enzymes in the gut lumen.     

Apoptosis and mitosis of parenchymal cells in the duct-ligated rat submandibular gland

Apoptosis and proliferation of parenchymal cells during atrophy of rat submandibular gland induced by double duct ligation were investigated using immunohistochemistry for proliferating cell nuclear antigen (PCNA), terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick end labelling (TUNEL) and transmission electron microscopy (TEM). At 2 and 3 days after ligation, increased PCNA positive cells and mitoses were seen in ducts; thereafter PCNA positive cells decreased in number. At 3 and 4 days, the acinar cell population rapidly decreased, with many remaining TUNEL positive acinar cells. During this period, TEM showed typical apoptotic acinar cells that were phagocytosed by adjacent acinar cells or intraepithelial macrophages. After 7 days, most acinar cells had disappeared, leaving prominent residual ducts; a few acinar cells remained, especially at the lobule periphery. Submandibular gland duct ligation thus induced marked depletion of acinar cell by apoptosis and a concurrent short-lived cycle of duct cell proliferation.     

Rab8 is involved in zymogen granule formation in pancreatic acinar AR42J cells

Zymogen granules (ZGs) are specialized storage organelles in the exocrine pancreas, which allow digestive enzyme storage and regulated apical secretion. To understand the function of these important organelles, we are conducting studies to identify and characterize ZG membrane proteins. Small guanosine triphosphatases (GTPases) of the Rab family are key protein components involved in vesicular/granular trafficking and membrane fusion in eukaryotic cells. In this study, we show by morphological studies that Rab8 (Rab8A) localizes to ZGs in acinar cells of the pancreas. We find that Rab8 is present on isolated ZGs from rat pancreas and in the ZG membrane fraction obtained after granule subfractionation. To address a putative role of Rab8 in granule biogenesis, we conducted RNA interference experiments to 'knock down' the expression of Rab8 in pancreatic AR42J cells. Silencing of Rab8 (but not of Rab3) resulted in a decrease in the number of ZGs and in an accumulation of granule marker proteins within the Golgi complex. By contrast, the trafficking of lysosomal and plasma membrane proteins was not affected. These data provide first evidence for a role of Rab8 early on in ZG formation at the Golgi complex and thus, apical trafficking of digestive enzymes in acinar cells of the pancreas.