Hanne Klonowski-Stumpe

Menadione Induces Both Necrosis and Apoptosis in Rat Pancreatic Acinar AR4-2J Cells

This study evaluated the action of menadione on cell proliferation and integrity of the rat pancreatic acinar cell line, AR4-2J. Menadione at 1-20 microM dose- and time-dependently inhibited cell proliferation of AR4-2J cells. In contrast, a high concentration of menadione (100 microM) caused rapid cell death (> 90% of cells took up trypan blue within 4-h). While the high concentration of menadione (100 microM) induced DNA smear in electrophoresis indicative of necrosis, lower concentrations (10-20 microM) induced a DNA ladder indicative of apoptosis. Similar results were obtained using a DNA fragmentation ELISA. Glutathione (1 mM), the calcium chelator EGTA (500 microM), and the cysteine protease inhibitor NCO-700 (5 mM) partly inhibited the effect of 1-10 microM menadione on cell proliferation and DNA fragmentation. Menadione at 1-20 microM induced wild-type P53, whereas the 100 microM menadione had a minor effect on wild-type P53. It is concluded that menadione induced necrosis at high concentrations and apoptosis at low concentrations in AR4-2J cells. Apoptosis induced by lower concentrations of menadione may be mediated by wild-type P53, intracellular calcium, and mechanisms which decrease the intracellular concentration of reduced glutathione.

Differential and synergistic effects of platelet-derived growth factor-BB and transforming growth factor-beta1 on activated pancreatic stellate cells.

Objective: The cytokines platelet-derived growth factor (PDGF) and transforming growth factor (TGF)-β1 are major factors influencing the transformation from the quiescent to the activated phenotype of pancreatic stellate cells (PSC), a process involved in the pathogenesis of chronic pancreatitis. Albeit much effort has been made to study the effects of PDGF and TGF-β1 on PSCs, their interaction is still unclear, because these cytokines show both differential and synergistic effects as outlined by this study. Methods: Culture-activated PSCs of rats were treated with PDGF-BB and TGF-β1. Subsequent changes of cell proliferation and migration were determined by cell counting, (+)-bromo-2′-deoxyuridine enzyme-linked immunosarbant assay (ELISA), and migration assay. Gene expression, synthesis of proteins, and activation of kinases were further studied by reverse transcription-polymerase chain reaction, real-time polymerase chain reaction, ELISA, and Western blot. Results: PDGF-BB increased PSC proliferation and migration, accompanied by elevated expression of matrix metalloproteinases (MMP)-13 and MMP-3. The mRNA amount of procollagen α2(I), α-smooth muscle actin (α-SMA), tissue inhibitor of metalloproteinase (TIMP)-1, and TGF-β1 was also increased by PDGF-BB. In contrast, PDGF-BB reduced collagen type I in culture medium and synthesis of α-SMA. Treatment of PSC with TGF-β1 decreased proliferation, had no significant effect on migration and MMP expression, but increased expression and synthesis of procollagen α2(I) and α-SMA. Both cytokines induced phosphorylation of extracellular signal regulated kinase (ERK)-1/2 and p38MAPK, but only PDGF-BB activated the protein kinase B signaling pathway. Conclusion: PDGF-BB augments effects of TGF-β1 on the mRNA level presumably because of up-regulation of TGF-β1 synthesis and common signaling pathways of the 2 cytokines. However, at the protein level, PDGF-BB impairs typical TGF-β1 effects such as increased synthesis of collagen (type I) and α-SMA. Moreover, PDGF-BB facilitates degradation of extracellular matrix proteins by enhancement of MMP synthesis, but MMP activity was probably limited because of elevated tissue inhibitor of metalloproteinase 1 expression.

LONG-TERM EFFECTS OF CCK-AGONIST AND -ANTAGONIST ON FOOD INTAKE AND BODY WEIGHT IN ZUCKER LEAN AND OBESE RATS

The present study evaluates long-term effects of the CCK-agonist caerulein and the CCK-A antagonist loxiglumide in obese and lean Zucker rats. Caerulein and loxiglumide altered food intake neither in obese nor in lean rats. By as yet unknown mechanisms, however, weight increase was accelerated by loxiglumide and reduced by caerulein in obese and lean rats. Caerulein increased pancreatic weight only in lean but not in obese rats. Thus, obese rats show a resistance of pancreatic CCK-A receptors. The failure of CCK-agonist and -antagonist to alter food intake suggests that this CCK-resistance is not responsible for obesity in the genetically altered rats.

Disruption of Paracellular Sealing is an Early Event in Acute Caerulein-Pancreatitis

Caerulein-induced pancreatitis is a widely used experimental model for studies on acute pancreatitis, however, the molecular mechanisms underlying pancreatitis in response to caerulein hyperstimulation are incompletely understood. We therefore studied early effects of caerulein on tight junctional integrity. Mice were injected with the cholecystokinin analogue caerulein (50&mgr;g/kg BW/h) to induce pancreatitis. In pancreatic tissue occludin, claudin 1, zonula occludens protein 1 (ZO-1) were stained immunohistochemically and F-actin was visualized with phalloidin-TRITC. Stained sections and isolated acini were studied by confocal laser scanning microscopy. Under control conditions occludin, claudin1, ZO-1, and F-actin showed a linear staining pattern delineating the apical membranes of intralobular duct cells and of acinar cells. While in vitro caerulein hyperstimulation induced within 10 minutes disassembly of both occludin and ZO-1, in vivo caerulein hyperstimulation induced disassembly of occludin and claudin1 but not of ZO-1 from the tight junctions. Subsequent progressive disruption of ZO-1 was detected in a time dependent manner. Disruption of the transmembrane tight junction proteins occludin and claudin1 is an early event of caerulein hyperstimulation and may allow evasion of noxious luminal content into the interstitium, which may augment edema formation in acute pancreatitis.

Effects of angiotensin II on rat pancreatic stellate cells.

The aim of the study was to identify pancreatic stellate cells (PSCs) as a potential target of angiotensin II (ATII) action because recently a local renin-angiotensin system (RAS) has been described in the pancreas. PSCs were isolated from male Wistar rats and investigated for ATII receptor expression and ATII-induced calcium transients, contractions, proliferation, and &agr;-smooth muscle actin expression. Quiescent and activated PSCs expressed the ATII receptor subtype AT1 but not AT2. Addition of ATII led to a rapid elevation of intracellular calcium ([Ca2+]i). The sensitivity toward ATII with respect to calcium transients did not change during the transdifferentiation process. In activated PSCs, ATII dose dependently induced PSC cell contraction. Furthermore, ATII induced an activation of the c-Jun-N-terminal kinase (JNK) and extracellular regulated kinase (Erk), which was inhibited after intracellular calcium chelation by BAPTA-AM. The p38 mitogen-activated protein kinase (p38MAPK) was also activated by ATII. BAPTA-AM itself induced p38MAPK activation, which was not further enhanced by ATII. ATII stimulated PSC proliferation, while PSC transdifferentiation, as indicated by &agr;-smooth muscle actin expression and collagen type I secretion, was not enhanced. The data suggest that PSCs are targets of ATII action with potential pathophysiological relevance.

Cytotoxicity of peroxynitrite in rat pancreatic acinar AR4-2J cells.

Peroxynitrite (0.5–50 μM) induced dose-dependent cytotoxic effects in rat pancreatic acinar AR4–2J cells. Glutathione (2 mM) and ebselen (10 μM) partially reduced the cytotoxicity caused by 1–10 μM concentrations of peroxynitrite. Higher concentrations (10–50 μM) of peroxynitrite induced DNA smear suggestive of necrosis, while lower concentrations (2–5 μM) induced DNA fragmentations suggestive of apoptosis. The effects of peroxynitrite on [Ca2+]ishowed a similar dose dependency. Peroxynitrite concentrations >10 μM rapidly increased [Ca2+]i in a dose-dependent manner, while concentrations <5 μM did not affect [Ca2+]i. In contrast, the presentation of wild-type P53 was accelerated at lower concentrations of peroxynitrite (≤10 μM) but not at higher concentrations (50 μM). The present study suggests that peroxynitrite at lower concentrations (2–5 μM) induces wild-type P53 and apoptosis, which is potentially a protective response toward the DNA damage caused by peroxynitrite. On the other hand, higher concentrations of peroxynitrite (10–50 μM) rapidly increase [Ca2+]i and eventually induce necrosis.

Production and effects of endothelin-1 in rat pancreatic stellate cells

Introduction Proliferation and matrix synthesis of activated pancreatic stellate cells (PSCs) participate in the development of chronic pancreatitis. Besides other substances, endothelin-1 (ET-1) may influence the activation process of PSCs. Until now, ET-1 has not been studied in this particular cell type. Aims To characterize PSCs in rat pancreas with respect to expression of ETA-receptors, production of ET-1, and physiological effects induced by ET-1 during PSC activation. Methodology Immunocytochemical and ELISA techniques and cDNA microarray analysis were used. Physiologic effects were characterized by single cell measurements of free cytosolic Ca2+-concentration and of PSC contractility on collagen lattices. Results Activation of PSCs in vitro, as assessed by &agr;-smooth muscle actin expression, was accompanied by the de novo expression of ETA-receptors and synthesis of ET-1 mRNA and protein. Cytosolic Ca2+-concentration was increased upon ET-1 stimulation in activated but not in quiescent PSCs. Contractility of activated PSCs was significantly reduced by the selective ETA-receptor antagonist BQ123 but not by the ETB-receptor antagonist IRL-1038. Conclusions The results suggest that ET-1 may act as a paracrine and autocrine factor for activated PSCs and may mediate contractions of activated, but not quiescent, PSCs.

Inhibition of Cathepsin B Does Not Affect the Intracellular Activation of Trypsinogen by Cerulein Hyperstimulation in Isolated Rat Pancreatic Acinar Cells

Activation of trypsinogen is thought to trigger the autodigestive process in acute pancreatitis. The lysosomal enzyme cathepsin B was suggested to cause the activation of trypsinogen because it is known that cathepsin B is able to activate trypsinogen in special circumstances and that lysosomal and digestive enzymes are colocalized within intracellular vacuoles in the early stage of pancreatitis. As yet this hypothesis has been difficult to prove because activated trypsin is difficult to quantify in pancreatitis by conventional enzymatic measurements. We therefore employed an ELISA for trypsin activating peptide (TAP), which is a small peptide cleaved during the activation of trypsinogen and can be determined reliably. Supraphysiological concentrations of cerulein (1 nM-1 microM) resulted in a marked increase in TAP in freshly isolated pancreatic acinar cells, indicating activation of trypsinogen. This activation as determined by the TAP increase was significantly reduced by the serine protease inhibitor Fut-175 but not by the cathepsin B inhibitors E-64 and NCO-700. The concentrations of NCO-700 and E-64 abolished the cathepsin B activity of pancreatic acinar cells but did not significantly reduce the trypsin activity (after enterokinase preincubation); correspondingly the concentrations of Fut-175 used abolished the trypsin activity but did not reduce the cathepsin B activity. The results indicate that an autoactivation of trypsin rather than an activation of trypsinogen by cathepsin B triggers trypsin activation by supramaximal cerulein concentrations.

Glutathione might exert an important function in caerulein-stimulated amylase release in isolated rat pancreatic acini.

Aims The effect of different modes of thiol depletion on pancreatic exocrine secretory function and potential mechanisms of interference with the secretory process in pancreatic acinar cells were investigated. Methodology After incubation with three thiol-modulating agents (L-buthionine sulfoximine, ethacrynic acid, and diamide) for 30 minutes, caerulein-stimulated amylase release and cholecystokinin (CCK) receptor binding characteristics were assessed in isolated rat pancreatic acini. The level of thiol groups (glutathione and protein thiols) and cytosolic-free calcium were measured in pancreatic acinar cells. Results All three thiol-modulating agents decreased caerulein (10 −10M)-stimulated amylase release and the level of pancreatic acinar glutathione in a dose-dependent fashion without a marked increase in cell damage. Diamide also diminished the level of protein thiols. Ethacrynic acid and diamide, but not L-buthionine sulfoximine, inhibited the caerulein (10 −9M)-induced Ca 2+ mobilization in pancreatic acinar cells. None of the three thiol-modulating agents altered the CCK receptor binding characteristics. Conclusion The present findings strongly suggest an important role of glutathione in the secretory process in pancreatic acinar cells and in the secretory blockade observed in acute pancreatitis. A decrease in caerulein-induced Ca 2+ mobilization might participate in the inhibition of amylase release by some oxidative agents, but it is not the prominent cause in general.