Tubular complexes in cerulein- and oleic acid-induced pancreatitis in rats: glycoconjugate pattern, immunocytochemical, and ultrastructural findings.

Ductlike tubular complexes in cerulein-induced pancreatitis and oleic acid-induced pancreatic insufficiency were studied to analyze further their origin and development. Immunocytochemistry for pancreatic enzymes, lectin-binding studies, and ultrastructural investigations were combined with autoradiographic quantitation of labeling indices of ductlike cells in tubular complexes. In one group of rats, pancreatitis was induced by infusion of cerulein (10 micrograms kg-1 h-1). In a second group, pancreatic insufficiency was induced by intraductal injection of oleic acid (50 microliters). The investigations were carried out at distinct intervals following induction of pancreatic injury. In both groups of animals, after 3 days, a significant widening of acinar lumina was paralleled by a decreasing height of acinar cells, which showed pronounced retrogressive changes. At this time, acinar cells bound all of the lectins used and retained their immunoreactivity for amylase, trypsinogen, chymotrypsinogen, and lipase. At further intervals, acinar structures formed typical ductlike complexes, with a progressive loss of immunoreactivity for pancreatic enzymes and a reduced lectin-binding for L-fucose and N-acetylgalactosamine. Autoradiographic quantitation demonstrated no significant labeling of acinar cells undergoing tubular dedifferentiation. In both models, tubular complexes were removed by macrophages. It is concluded that lining cells in tubular complexes represent degenerating acinar cells that have no regenerative potency and have lost their secretory and membrane characteristics.

Recurrent acute pancreatitis and intraluminal duodenal diverticulum.

An intraluminal duodenal diverticulum (IDD) and a partial pancreas divisum were diagnosed in a 22-year-old man who exhibited recurrent attacks of acute pancreatitis. Resection of the diverticulum resulted in a complete disappearance of symptoms. The possible etiological relationship between IDD and recurrent acute pancreatitis is discussed.

Rat pancreatitis-associated protein is expressed in relation to severity of experimental pancreatitis.

To study the relation between expression of the pancreatitis-associated protein (PAP) and severity of pancreatitis in rats, different degrees of experimental pancreatitis were induced by a 1-, 3-, or 5-h cerulein infusion (5 micrograms kg-1 h-1). This treatment decreased pancreatic volume secretion to below 10%. Immediately after infusion, the secretion rate increased to approximately 50% of control. Within 1 day, volume and bicarbonate secretion rates were not different from controls. At this point, protein secretion amounted to 30% of control, but only in animals receiving the 3- or 5-h dose. The values increased to 40-60% within 3 days. In all groups, the isoenzyme pattern was not influenced by the cerulein treatment. One day after induction of pancreatitis, the PAP was found in pancreatic juice in concentrations related to the dose of cerulein given. By immunohistochemical techniques, the protein was localized over acinar cells, but was not detectable in interstitial tissue, islets, or in the healthy exocrine pancreas. Pathomorphologic alterations in the pancreas were quantified by a scoring system. One and 2 days after the treatment, a more severe pancreatitis and more elevated levels of PAP were found in animals treated with the higher dose of cerulein. It is concluded that PAP is expressed in the pancreas in relation to the severity of cerulein-induced pancreatitis.

Histochemical and ultrastructural characteristics of tubular complexes in human acute pancreatitis.

The morphologic characteristics of ductlike tubular complexes were studied in human acute pancreatitis. Pancreatic specimens were obtained from 10 patients who were operated on for acute pancreatitis. Immunocytochemistry for pancreatic enzymes, keratin, actin, and carcinoembryonic antigen were combined with lectin-binding studies and ultrastructural investigations. Irrespective of clinical onset and duration of pancreatitis, tubular complexes situated in the vicinity of fat necrosis were observed in all patients. Intermediate forms of ductlike structures were characterized by widening of acinar lumina, decreased height of acinar cells, and large autophagic vacuoles. These structures bound all of the lectins employed and retained their immunoreactivity to secretory proteins. Typical tubular complexes were composed of low cuboidal or flattened cells surrounding a large acinar lumen. They revealed a loss for pancreatic enzymes, a reduced lectin-binding for L-fucose and N-acetylgalactosamine, and an increase for cytoskeletal proteins (keratin, actin). It is concluded that tubular complexes in human acute pancreatitis represent degenerating acinar cells which lost their secretory and membrane characteristics.

Mechanism of acute pancreatitis. Cellular and subcellular events.

A membrane-bound system through which secretory and lysosomal proteins travel in a vectorial fashion is essential for the preserved integrity of pancreatic acinar cells. This system is composed of an ordered array of compartments, such as the rough endoplasmic reticulum, the Golgi complex, lysosomes, and secretory granules. As a principle, in acute pancreatitis the final steps of this transport seem to be disturbed. Caerulein-induced pancreatitis is a valuable experimental model for studying altered intracellular transport, and compartmentation of lysosomal and digestive enzymes. The formation of enlarged secretory vacuoles containing lysosomal and digestive enzymes is paralleled by the activation of lysosomes and degradation of cellular organelles in autophagosomes. On the level of secretory and autophagic vacuoles, activation of serine proteases occurs, which in addition to increasing lysosomal enzyme activities can represent the initial stage for acinar cell destruction and the development of pancreatitis.

[Age-related changes in glycosaminoglycans in the cell nuclei of the rat liver--biochemical and histochemical pilot studies]. / Alternsabhängige Veränderungen der Glycosaminoglycane in Zellkernen der Rattenleber--biochemische und histochemische Pilotstudien.

Cell nuclei of the livers of 12 male Wistar rats aged 24 months, and 16 male Wistar rats aged 4 months were isolated. 0.4 microCi [3H]-glucosamine/kg body weight were administered intraperitoneally. The livers of four animals were used for each biochemical analysis. The tissue was degraded by proteolysis, the glycosaminoglycans (GAGs) were isolated and the GAG types were determined by enzyme digestion, nitrite degradation and radiometry. The amount of GAGs was photometrically determined in single samples of pooled material of each of the two age groups. In paraffin sections (from other male Wistar rats of a different age), the influence of enzymes on specific GAG staining was observed. Most of the radioactivity was found in heparan sulfate (HS) and a lower content in chondroitin sulfates (CSs). In HS the incorporation increased with increasing age. The amount of HS showed no age-related differences. The results indicate an age-related activation of the HS metabolism. The GAG pattern and the age-related changes of the GAG types in total tissue (earlier results) are different from those in the nuclei. By histochemical methods, we observed a small but distinct effect of heparitinase in the cell nuclei.

Localization of lysosomal and digestive enzymes in cytoplasmic vacuoles in caerulein-pancreatitis.

Intracellular localization and enzymatic activities of lysosomal enzymes (cathepsin B, N-acetyl-beta-glucosaminidase, and beta-glucuronidase) were studied in control rats and after induction of caerulein pancreatitis. In control rats high enzymatic activities were found in the postnuclear 1000 g fraction (purified zymogen granules). The corresponding subcellular fraction in pancreatitis animals additionally contained larger secretory vacuoles and autophagosomes and revealed a marked increase in lysosomal enzyme activities. Immunolabelling studies at the ultrastructural level for trypsinogen and cathepsin B demonstrated a colocalization of lysosomal and digestive enzymes in zymogen granules in healthy controls. After induction of pancreatitis immunolabelling still demonstrated a colocalisation of cathepsin B and trypsinogen in secretory granules and newly formed Golgi-derived secretory vacuoles. Concomitantly appearing autophagosomes were, however, only labelled for cathepsin B. It is concluded that segregation of lysosomal and digestive enzymes is incomplete in normal acinar cells resulting in a colocalization in zymogen granules. In pancreatitis colocalization in secretory granules is maintained, whereas only lysosomal enzymes were sufficiently transferred into autophagic vacuoles. No indication for impaired mechanisms of molecular sorting of lysosomal and digestive enzymes in caerulein-induced pancreatitis was found.

Immunocytochemical and morphometric analysis of acinar zymogen granules in human acute pancreatitis.

In the present study fine structural changes of acinar zymogen granules were investigated in human acute pancreatitis. Pancreatic tissue was obtained at surgery from 6 patients, prepared for ultrastructural analysis, and stained immunocytochemically for trypsinogen. Stereological parameters of zymogen granules were evaluated. The density of the immunocytochemical labelling for trypsinogen was estimated over zymogen granules, the rough endoplasmic reticulum, Golgi apparatus and the acinar lumina. In acute pancreatitis the number of zymogen granules was diminished and their size reduced. The density of the labelling for trypsinogen was unchanged over zymogen granules but showed a significant reduction over the rough endoplasmic reticulum, Golgi apparatus, and the acinar lumina. In general the integrity of zymogen granules was well preserved. Focally degenerative changes of zymogen granules and large autophagosomes were observed. From the immunogold labelling a disturbance of enzyme synthesis and secretion was suggested. Evidence is given that a disruption of the zymogen granule membranes and a fusion with lysosomal bodies might contribute to the pathogenesis of human acute pancreatitis.

[Age dependence of the early reaction in glycosaminoglycan metabolism of the rat liver on damage following a single dose of thioacetamide]. / Die Altersabhängigkeit der frühen Reaktion im Glycosaminoglycan-Stoffwechsel der Rattenleber auf eine einzeitige Thioacetamidschädigung.

Wistar rats aged 7 or 22 months resp. were treated with 50 mg/kg of thioacetamide (TAA) i.p. The TAA-treated animals as well as control groups of the same ages and containing the same number of animals were injected i.p. with 3H-glucosamine (0.2 microCi/g body weight) 2 hours before sacrifice. Previously to sacrifice, the livers had been perfused in narcosis with NaCl (0.9%). Following degradation of the tissue by pronase, specimen of the substances precipitable by ethanol were digested with chondroitinases, streptomyces-hyaluronidase, keratanase, and nitrite. The quantities of substances were estimated photometrically using Alcian blue; radiometry was performed following precipitation with cetylpyridiniumchloride. In aging animals the amount of dermatan-sulphate (DS) and of the total acidic glycosaminoglycans (a.GAG) is increased as compared to the younger animals, although there ist no age related difference in the incorporation of glucosamine. The incorporation per microgram a.GAG (spec. act.) is decreased. Following TAA the amount of DS is increased in both age groups, whereas the total amount of GAG is increased only in the younger animals. The incorporation per mg dry weight is also increased in all GAG types of both age groups which is also the case with the spec. act. (with the exception of HS in the younger animals). The increase of the incorporation per mg dry weight in DS is significantly lower in the older TAA-animals as compared to the younger ones. HS shows the same tendency, but there is no significance. The age related increase of hydroxyproline parallels that of DS.(ABSTRACT TRUNCATED AT 250 WORDS)

Hormone-induced pancreatitis.

Intravenous infusion of the synthetic cholecystokinin analogue cerulein at a dose of 0.25 micrograms/kg/h causes maximal stimulation of pancreatic exocrine secretion. The infusion of supramaximal doses of cerulein (5 and 10 micrograms/kg/h) induces a significant increase in pancreatic enzymes in blood, and interstitial edema and inflammatory cell infiltration. This model of hormone-induced pancreatitis works in rats, mice, dogs and hamsters. Besides intravenous infusion, repeated intraperitoneal injections can also be used for induction of pancreatitis. In the early phase of cerulein-induced pancreatitis, large autophagic vacuoles result from fusion of zymogen granules within the acinar cell. This is accompanied by an increase in lysosomal enzyme activity and activation of trypsinogen which finally leads to cellular necrosis. All animals survive the induction of pancreatitis. The pancreas completely regenerates within 6 days after induction of pancreatitis. This model of experimental pancreatitis favors the analysis of intracellular events in the early phase of pancreatitis.

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

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.

Lung injury in acute experimental pancreatitis in rats. I. Morphological studies.

The pathogenesis of pancreatitis-related pulmonary injury was studied at the light- and electronmicroscopic level. Experimental pancreatitis was induced in rats by infusion of supramaximal doses of cerulein for 12 h. Investigations were carried out 3, 6, and 12 h after the start of infusion and 12, 48, and 72 h after the end of pancreatitis induction. Initial manifestations of pancreatitis-associated lung injury revealed a pronounced clustering of polymorphonuclear leukocytes in pulmonary microvessels, followed by severe damage of alveolar endothelial cells. Consecutively, the increase in vascular permeability of the lung resulted in interstitial edema formation. Structural changes were maximal after 12 h and reversed completely after 84 h. In conclusion, the structural appearance of pulmonary injury in cerulein-induced pancreatitis was similar to that reported in early stages of the adult respiratory distress syndrome (ARDS). It is suggested that polymorphonuclear granulocytes play a crucial role in the pathogenesis of pancreatitis-related lung injury.

Evidence of intracellular activation of serine proteases in acute cerulein-induced pancreatitis in rats.

It is believed that activation of zymogen proteases occurs in the early development of acute pancreatitis. This hypothesis was proved on subcellular fractions of rat pancreas after induction of pancreatitis by infusion of high doses of cerulein for 2 h. Secretory enzyme activities were measured spectrophotometrically in subcellular fractions obtained by differential ultracentrifugation. Additionally, trypsin and chymotrypsin activities were detected by enzyme blots after isoelectric focusing. Finally immunoblotting (Western-blot analysis) for amylase, lipase, trypsin/ogen, and chymotrypsin/ogen was carried out on fractions separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). In cerulein pancreatitis, subcellular fractions of secretory granules and vacuoles showed significant amounts of free trypsin and chymotrypsin activities compared with controls. The presence of free activities of serine proteases was paralleled by the appearance of numerous low molecular weight peptides detected by 2-dimensional electrophoresis and SDS-PAGE, which in part represented proteolytically cleaved secretory proteins. It is concluded that the intracellular activation of serine proteases that occurs in cerulein pancreatitis could contribute to further acinar cell destruction.

Cerulein-induced acute pancreatitis in rats: evidence for reduced calcium affinity of secretory granule membranes.

Membranes of secretory granules in pancreatic acinar cells seem to be interrelated in the regulation of intragranule Ca2+ concentrations. Since low intragranule Ca2+ levels are involved in zymogen stabilization versus autoactivation of proteases, a disturbance of the Ca2(+)-regulating system in secretory granules could be invoked to account for uncontrolled proenzyme activation. This is proposed as the initial mechanism in the pathogenesis of acute pancreatitis. Using pancreatic subcellular fractions obtained from control rats and after induction of acute cerulein pancreatitis we found a markedly reduced Ca2+ affinity of membranes from the secretory granule fraction in pancreatitis. The strong Ca2+ binding of control zymogen granule membranes primarily seemed to be a function of non-proteinacous membrane components, e.g. phosphatidylinositols. It is suggested, that part of the inner surface of membranes from secretory granules acts as a calcium-buffering system that works in synergy with other protective mechanisms to stabilize the zymogen granule population. In cerulein pancreatitis there seemed to be an imbalance of this system.

Influence of experimental hyperthyroidism on blood and myocardial serotonin in rats.

Recent reports suggest a role for serotonin in the pathogenesis of primary hypertension and left ventricular (LV) hypertrophy. In this study, we have induced LV hypertrophy by oral feeding of thyroxine at increasing dosages (150-450 micrograms/kg b.wt.) over a 5-week period. The effects of hyperthyroidism on cardiovascular parameters, blood and myocardial serotonin concentrations were assessed. Water-fed rats and formerly hyperthyroid recovered animals served as controls. Thyroxine caused a significant LV hypertrophy: hyperthyroid rats 2.19 +/- 0.16*; controls 1.65 +/- 0.13 g/kg b.wt. (mean +/- SD; *P less than 0.05). An almost complete regression of LV hypertrophy occurred in the recovery group (1.66 +/- 0.20 g/kg b.wt.) 3 weeks after cessation of thyroid hormone application. Thyroxine-treated animals showed a significant increase of serotonin blood levels (thyroxine rats: 2108 +/- 781*, recovery: 1132 +/- 726, controls: 705 +/- 480 ng/ml; *P less than 0.05). The concentrations of serotonin in left ventricular myocardium were increased after thyroid hormone application, whereas the highest levels were found in the recovery group (thyroxine rats: 139.1 +/- 30.4, recovery: 167.2 +/- 43.1, controls: 68.9 +/- 27.9 mg/ml homogenate). Serotonin-containing cells in the left ventricular myocardium were stained immunohistochemically. They were localized perivascularly and were assumed to represent tissue mast cells. In experimental hyperthyroidism the serotonin levels in blood and heart are increased possibly indicating an interaction of both hormones in thyroxine-induced cardiomyopathy.

Pancreatic lesions and hormonal profile of pancreatic tumors in multiple endocrine neoplasia type I. An immunocytochemical study of nine patients.

Pancreatic specimens of nine patients suffering from multiple endocrine neoplasia type I (MEN I) were investigated with regard to tumor frequency and growth pattern, islet hyperplasia and endocrine cell neoformation, immunocytochemical hormone profile of the tumors, and correlation to clinical symptoms. The majority of the 201 tumors were microadenomas (diameter less than 0.5 cm), which frequently displayed a trabecular growth pattern. Microadenomatosis was considered the most distinct feature of the MEN I pancreas. Additional larger tumors (diameter greater than 1.0 cm) were found in five patients. Whereas islet hyperplasia appears not to belong to the spectrum of the pancreatic lesions in MEN I, nesidioblastosis was occasionally observed. Immunocytochemical screening revealed that among hormone-positive tumors (approximately 80% of the tumors), pancreatic polypeptide tumors (PPomas), glucagonomas, and insulinomas were the most frequent. The high incidence of PPomas in these pancreases probably accounts for the elevated serum PP levels found in many MEN I patients. Somatostatinomas, gastrinomas, vasoactive intestinal polypeptide tumors (VIPomas), and neurotensinomas were rare. Clinically overt hyperinsulinism, observed in two patients and associated with a large insulinoma, was cured by tumor resection. Eight of nine patients presented a Zollinger-Ellison's syndrome (ZES), but only in two patients were gastrin-producing tumors found. The source of gastrin in MEN I patients with a ZES, in whom no gastrinoma could be detected, remains unclear.

[Ultrastructural diagnosis of neuroendocrine tumors using fine needle biopsy]. / Ultrastrukturelle Diagnostik neuroendokriner Tumoren an Feinnadelpunktaten.

Ultrasonic-guided fine-needle-biopsies of primary pancreatic tumours or liver metastasis were performed in 13 patients with neuroendocrine tumours of the gastrointestinal tract (5 carcinoids, 3 gastrinoma, 1 PPoma, 1 calcitoninoma, 1 insulinoma, and 2 non-functional tumours). Specimens obtained were examined on the light- and electronmicroscopic level. In all cases ultrastructural examination sufficiently revealed the correct diagnosis, due to the presence of cytoplasmic neuroendocrine granules within the tumour cells. Additionally performed immunocytochemical investigations at the ultrastructural level enabled the discrimination of gastrinomas, insulinomas, and PPomas. In contrast, light-microscopic examination was less sensitive for tumour classification. It is concluded that ultrastructural investigation of fine needle biopsies represents a valuable method to sufficiently discriminate neuroendocrine neoplasms.

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.

Lung injury in acute experimental pancreatitis in rats. II. Functional studies.

In this study we report the functional changes in isolated perfused lungs from rats with cerulein-induced experimental pancreatitis. Rat lungs isolated immediately after the cerulein infusion demonstrated decreased pressor responses to angiotensin II (A II) and acute hypoxia (FIO2: 0.0). The lung wet- to dry-weight ratio was increased, as was the lung-leak index, consistent with high-permeability edema formation in the lung. Neither saline-solution infusion for 12 h nor perfusion with cerulein of rat lungs isolated from untreated animals caused lung injury or functional alterations. The changes in pulmonary vascular reactivity were normalized 48-72 h after induction of pancreatitis. In conclusion, we describe an animal model of pancreatitis and reversible, ARDS-like lung injury.