Horst F. Kern

Acute interstitial pancreatitis in the rat induced by excessive doses of a pancreatic secretagogue.

Conscious rats were infused via a jugular vein catheter with 5×10−6 g/kg/h caerulein for periods up to 24 h. On macroscopic inspection a progressive interstitial oedema is seen to develop in the pancreas, from one hour of infusion on and is most marked at twelve hours. This oedema is largely reabsorbed after 24 h treatment, but the pancreas is considerably indurated by this time. Serum amylase levels increase consistently to reach a tenfold elevation above controls after three, six or twelve hours infusion. Premature fusion of condensing vacuoles and secretory granules leads to formation of large vacuoles in the cytoplasm of exocrine pancreatic cells. These vacuoles fuse with the lateral and basal plasma membrane and realease their content into the extracellular space. Regular discharge of zymogen granules at the cell apex into the duct system does not occur. Vacuole formation is associated with cytoplasmic destruction of the pancreatic cells. The rate of protein synthesis decreases consistently as a result of these structural alterations and this change corresponds largely to a reduction of cellular respiration. Release of amylase from isolated pancreatic lobules of caerulein infused animals shows a progressive increase of unstimulated discharge, while in vitro stimulation with 5×10−6 M carbamylcholine gives secretion patterns of wash-out kinetics. Stimulated discharge of labeled secretory proteins indicates a progressive reduction in the in vitro sensitivity of the pancreatic cells to secretagogues. After 24 h infusion of 5×10−6 g/kg/h caerulein the pancreatic lobules are totally insensitive to the in vitro effect of carbamylcholine or caerulein.

Course and spontaneous regression of acute pancreatitis in the rat

Rat exocrine pancreatic function was studied structurally and biochemically after the in vivo production of acute interstitial pancreatitis by supramaximal stimulation with caerulein. Two major phases in the reaction of the gland were observed: During the first two days after cessation of the supramaximal stimulation a progressive infiltration of the interstitium and the pancreatic tissue with polymorphonuclear leucocytes, lymphocytes and macrophages occurred which led to further destruction of the gland and to decreased functional response. From two days after the cessation of the treatment, hypertrophy of centro-acinar cells and an increased rate of mitotic activity indicated regeneration of the pancreas. This was combined with an accelerated in vitro discharge of newly synthesized proteins over a period of four days. Between days three and six after the initial treatment mitotic activity was also observed in fully differentiated exocrine cells. Total structural and functional recovery of the pancreas was achieved nine to twelve days after the cessation of the supramaximal stimulation.

Time course and cellular source of pancreatic regeneration following acute pancreatitis in the rat

The regenerative capacity of the different cell types in the rat exocrine pancreas has been studied in a model of hormone-induced acute pancreatitis in which pancreatic edema, inflammation, and acinar cell destruction were induced within 12 h of infusion of supramaximal concentrations of cerulein (5 μg/kg/h). A sequential biochemical and structural analysis of the pancreas in daily intervals was combined with the autoradiographic quantitation of labeling indices of five cell populations following 3H-thymidine injection at days 1–7 after induction of pancreatitis. Desquamation of acinar cell apical cytoplasm and release of cytoplasmic segments into the acinar lumen on the first day following induction of pancreatitis led to formation of duct-like tubular complexes. Enzyme content in the pancreas decreased progressively following the formation of the edema to levels 15–20% of controls and remained reduced during the initial 5 days. Thymidine incorporation into total DNA showed a biphasic pattern with a distinct peak at day 1 and a second broader peak between days 4 and 7. Autoradiographic quantitation of labeling indices demonstrated the exclusive incorporation into intercalated duct cells and interstitial cells during the initial 24 h, while the second peak was predominantly due to labeling of acinar cells. Larger interlobular ducts and islets did not show changes in labeling index. In vivo labeling with 3H-thymidine during the first day and analysis of labeling indices 14 days later showed the persistence of label in intercalated duct cells and interstitial cells and argued against the stem cell hypothesis and against transformation of duct cells into acinar cells. It was concluded that regeneration following acute pancreatitis is mainly due to mitotic activity of remaining acinar cells. The initial and transient increase in replication of intercalated duct cells and interstitial cells could be due to the inflammatory reaction.

Pancreatic Dysfunction in Cystic Fibrosis Occurs as a Result of Impairments in Luminal ph, Apical Trafficking of Zymogen Granule Membranes, and Solubilization of Secretory Enzymes

Recent progress in understanding the luminal biochemistry of regulated pancreatic exocrine secretion, including acid-base interactions between acinar and duct cells and pH-dependent processes that regulate membrane trafficking (endocytosis) at the apical plasma membrane, have led to the development of in vitro models of cystic fibrosis in the rat exocrine pancreas. Based on investigations in these model systems, a unifying hypothesis is presented that proposes that pancreatic dysfunction in cystic fibrosis occurs as a result of progressive acidification of the acinar and duct lumen, which leads to secondary defects in (i) apical trafficking of zymogen granule membranes and (ii) solubilization of secretory (pro)enzymes. By directly acidifying the pH of the acinar lumen in cholescystokinin-stimulated acini, the early cytological findings observed in cystic fibrosis, including (i) massive dilatation of the acinar lumen, (ii) decreased appearance of zymogen granules, (iii) loss of the apical pole of the acinar cell, and (iv) persistent aggregation of secretory (pro)enzymes released into the luminal space, have been reproduced in primary cultures of pancreatic tissue.

Fibroblast structure and function during regeneration from hormone-induced acute pancreatitis in the rat.

The regeneration of the rat exocrine pancreas from a hormone- induced pancreatitis was investigated. In a previous study it was shown that the [3H]thymidine labeling index of interstitial cells increases 20- to 30-fold on day 1.5 after the induction of pancreatitis. Here we show by electron microscopic autoradiography that 80% of the labeled interstitial cells are fibroblasts. Their replication, fine structure, and collagen biosynthesis was further investigated: By day 2.5 numerous mitotic figures were found, indicating an enhanced proliferative activity of fibroblasts at the early stage of pancreatic regeneration. The ultrastructural analysis revealed that many fibroblasts contain abundant cytoplasm with a well-developed rough endoplasmic reticulum, prominent Golgi complexes, and secretory granules filled with fibrillar material. In contrast, the pancreatic fibroblasts of saline-infused control animals were shown to be spindle-shaped and to contain only very little cytoplasmic organelles. The collagen biosynthesis was quantified by in vivo labeling with [3H]proline and quantification of [3H]hydroxyproline in pancreatic protein hydrolysates. The collagen biosynthesis of experimental pancreata was measured to be 15 times that of controls on days 1.5 and 2.5 after the induction of pancreatitis and to remain fourfold elevated on days 3.5 through 10.5. In pulse-chase experiments using [3H]proline as the labeled precursor for collagen, the newly synthesized collagen was shown to be degraded with a half-life of 35 h. We conclude that replication of pancreatic fibroblasts and collagen biosynthesis as well as collagen degradation play important roles in the early phase of pancreatic regeneration.

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

The possible role of microtubules and microfilaments in the secretory process of the rat exocrine pancreas was analysed in vitro using isolated pancreatic lobules. Colchicine and vinblastine as microtubule inhibitors, hexylene glycol as a microtubule stabilizer, and cytochalasin B as a disruptive agent for microfilaments were used in increasing concentrations to test their effects on protein synthesis, intracellular transport, zymogen discharge, and cellular respiration. Colchicine only at 10−2 M concentrations inhibits protein synthesis, while vinblastine inhibits at 10−6 and 10−5 M by 20% and at 10−4 M by 55%. A similar inhibition is observed with 1.5% concentrations of hexylene glycol while cytochalasine B at 1,5 and 10 μg/ml is without effect on protein synthesis. Colchicine and vinblastine have their major effects on intracellular transport both in secretion studies and cell fractionation experiments. Colchicine in concentrations between 10−3 to 10−5 M inhibits discharge of newly synthesized proteins by 50%, while vinblastine shows a dose-response relationship of 40% inhibition at 10−6 M to 90% at 10−4 M. Discharge of amylase is uniformly reduced by 30% by both colchicine and vinblastine in the whole dose range. The pronounced effect of colchicine and vinblastine is evident in cell fractionation studies: both drugs inhibit the disappearance of protein radioactivity from microsomes and its appearance in zymogen granules; similarly the peak radioactivity in smooth microsomes (Golgi) appears delayed. No differential effect on the secretory process was observed with 1.5% concentrations of hexylene glycol or cytochalasin B at 1.5 and 10 μg/ml concentrations. A fines tructural analysis of microtubules and microfilaments in the exocrine pancreatic cell reveals their distribution in all parts of the cytoplasm and in relation to all cell organelles. Both systems (microtubules, microfilaments) seem to be connected, at least in certain areas of the cytoplasm and at the plasma membrane. The reduction of transport efficiency by microtubule inhibitors results in a deposition of secretory material in the cisternal space of the rough endoplasmatic reticulum, which leads to the formation of paracrystals. Colchicine at 10−3 M concentrations leads to an enlargement of condensing vacuoles in the Golgi complex.

Characterization of clones of a human pancreatic adenocarcinoma cell line representing different stages of differentiation.

With a limiting dilution technique, clones have been established from the human pancreatic adenocarcinoma cell line, HPAF. Phenotypic analysis by a panel of murine monoclonal antibodies demonstrated distinct profiles of antigenic expression between the clones. However, identical isozyme patterns of different clones indicated their common origin from the parental HPAF cells. Two clones, CD11 and CD18, appeared to be arrested at different stages of secretory epithelial cell differentiation. CD11 cells demonstrated many characteristics of a well-differentiated state, including the formation of ductal structures with polarized, long columnar-shaped cells, the presence of secretory granules in the cytoplasm, high DU-PAN-2 antigen expression in nude mouse xenografts, and a longer doubling time (42 h) in tissue culture. In contrast, CD18 cells exhibited characteristics of a poorly differentiated state, including solid nests of isoprismatic cells without luminal spaces and cellular polarization, absence of secretory granules and DU-PAN-2 antigen expression in xenografts, and a shorter doubling time (26 h) in tissue culture. Since no culture systems of normal pancreatic ductal cells are currently available, these two pancreatic adenocarcinoma clones may provide a unique system to study genes and antigens related to pancreatic ductal cell differentiation.

In vitro condensation-sorting of enzyme proteins isolated from rat pancreatic acinar cells.

To study the process of granule formation in pancreatic acinar cells in more detail we have established an in vitro system in which the whole complement of enzyme proteins released from isolated zymogen granules is mixed with a tracer amount of the same biosynthetically labeled proteins and is incubated at conditions prevailing in either pre-Golgi (pH 7.5) or trans-Golgi (pH 5.9) compartments. Condensation of the proteins into dense cores is assayed and quantitated after centrifugation of the mixture at 13000g and separation of the proteins in both the supernatant and the pellet by 2D-gel electrophoresis. At pH 7.5 about 1% of the total protein-bound radioactivity can be sedimented into the pellet and this increases 5-fold at pH 5.9 with similar sedimentation efficiency for individual enzyme proteins. The usual assumption that all aggregated proteins can be sedimented and thus only the pellet is representative for pH-dependent condensation has to be modified by the fine structural analysis of both the supernatant and pellet fraction at pH 7.5 and 5.9. Small particulate complexes form already in the supernatant at pH 7.5 which are not sedimented to a large extent into the pellet. At pH 5.9 aggregates of a homogeneous size of about 0.6 to 0.8 microm formed in the supernatant while the pellet is composed of sheets and vesicles of membranes studded with dense core particles of about 20 to 30nm size. The pH-dependent protein condensation is a stepwise process starting with the formation of small dense core particles already at pH 8.0/7.5 which then progressively aggregate to form larger cores at pH 6.0/5.0. These aggregates can only be sedimented employing higher centrifugal forces. In the condensation process of pancreatic enzyme proteins calcium ions exert an effect only at pH 7.5, leading to somewhat larger dense particles, while potassium ions are inhibitory both in protein condensation and in the binding of particles to membranes. The process of pH-dependent protein condensation is reversible and can be performed repetitively. The sedimentation of condensed proteins can be increased by the addition of isolated zymogen granule membranes. Thus the in vitro system allows the analysis of two related processes in granule formation: the condensation of secretory proteins into granule cores and their binding to the granule membrane.

Repetitive Cerulein-Induced Pancreatitis and Pancreatic Fibrosis in the Rat

The effect of repetitive inductions of pancreatitis by supramaximal doses of cerulein on pancreatic morphology and collagen content was studied in the rat. Pancreatitis was induced nine times at intervals of about 20 days; 3 days after the last injection of cerulein, pancreatitis was still observed, as indicated by pancreatic weight loss, increase of protein-bound hydroxyproline content, acinar-cell destruction, cellular infiltration, and deposition of collagen fibers. However, 6 weeks later, no differences in the parameters mentioned above were observed between control and cerulein-treated animals. Thus, repetitive induction of pancreatitis in the rat, according to the experimental protocol we used, did not result in pancreatic fibrosis.

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.