Transforming growth factor-beta-induced upregulation of transforming growth factor-beta receptor expression in pancreatic regeneration. (1/463)

The transforming growth factor-beta (TGFbeta) signaling pathway is one important player in the regulation of extracellular matrix turnover and cell proliferation in epithelial regeneration. We used cerulein-induced pancreatitis in rats as a model to investigate the regulation of TGFbeta receptor type I and type II expression on protein and messenger RNA level during regeneration. In the regenerating pancreas, mRNA levels of TGFbeta receptor I and II were significantly increased with a maximum after 2 days. On protein level, expression of TGFbeta receptor II was significantly increased after three to 3-5 days. This elevated expression could be inhibited by neutralizing the endogenous biological activity of TGFbeta1 with a specific antibody. In cultured pancreatic epithelial cells, TGFbeta1 reduced cell proliferation as measured by [3H]thymidine incorporation. Furthermore the transcript levels of TGFbeta1 as well as mRNA and protein concentrations of type I and type II receptor increased during TGFbeta stimulation in vitro. These results indicate that epithelial pancreatic cells contribute to the enhanced TGFbeta1 synthesis during pancreatic regeneration by an autocrine mechanism. TGFbeta1, furthermore, upregulates the expression of its own receptors during the regenerative process, thereby contributing to the increase of the TGFbeta-induced cellular responses.  (+info)

Secretagogue-induced digestive enzyme activation and cell injury in rat pancreatic acini. (2/463)

The mechanisms responsible for intrapancreatic digestive enzyme activation as well as the relationship between that activation and cell injury during pancreatitis are not understood. We have employed an in vitro system in which freshly prepared pancreatic acini are exposed to a supramaximally stimulating concentration of the CCK analog caerulein to explore these issues. We find that in vitro trypsinogen activation depends on the continued presence of Ca2+ in the suspending medium and that it is half-maximal in the presence of 0.3 mM Ca2+. Caerulein-induced trypsinogen activation can be halted by removal of Ca2+ from the suspending medium or by chelation of intracellular Ca2+. Increasing intracellular Ca2+ with either ionomycin or thapsigargin does not induce trypsinogen activation. We have monitored cell injury by measuring the leakage of lactate dehydrogenase (LDH) from acini and by quantitating intercalation of propidium iodide (PI) into DNA. Leakage of LDH and intercalation of PI in response to supramaximal stimulation with caerulein can be detected only after caerulein-induced trypsinogen activation has already occurred, and these indications of cell injury can be prevented by addition of a cell-permeant protease inhibitor. Our findings indicate that caerulein-induced intra-acinar cell activation of trypsinogen depends on a rise in intracellular Ca2+, which reflects entry of Ca2+ from the suspending medium. Intra-acinar cell activation of trypsinogen is an early as well as a critical event in pancreatitis. The subsequent cell injury in this model is mediated by activated proteases.  (+info)

Myofibroblast proliferation, fibrosis, and defective pancreatic repair induced by cyclosporin in rats. (3/463)

BACKGROUND: Full recovery is always achieved after caerulein induced pancreatitis. Cyclosporin stimulates transforming growth factor beta (TGF-beta) and may interfere with pancreatic regeneration. AIM: To investigate the effects of cyclosporin after caerulein induced pancreatitis or after caerulein injury. METHODS: Protocol A: rats received cyclosporin daily (20 mg/kg) and caerulein pancreatitis was induced on days 2 and 8. Protocol B: six courses of caerulein pancreatitis were induced at weekly intervals. Cyclosporin was administered on induction and the day before. Rats recovered for two weeks before being killed. Control groups received saline, cyclosporin, or caerulein alone. RESULTS: Protocol A: plasma TGF-beta1 and tissue collagenase rose after pancreatitis but decreased towards baseline values on day 15, matching a low collagen content. Morphology disclosed minimal inflammatory infiltration and some interstitial cells immunoreactive for smooth muscle alpha-actin (SMA). TGF-beta1 increased, and remained high in cyclosporin treated groups (cyclosporin alone and cyclosporin plus caerulein). Rats treated with cyclosporin and caerulein showed severe pancreatic weight reduction, abundant inflammatory infiltrates, increased SMA immunoreactive interstitial cells, high collagen content, and delayed collagenase response. No SMA immunoreactive cells were detected in normal rats. Cyclosporin alone also increased SMA immunoreactive cells, despite the absence of inflammatory infiltration and fairly conserved pancreatic structure. Protocol B: the combined pulse treatment induced appreciable collagen deposition and resulted in a smaller pancreas than controls. Morphological examination showed atrophy, fibrosis, fibroblast proliferation, and mononuclear infiltrates. CONCLUSION: Cyclosporin greatly distorts pancreatic repair, transforming caerulein induced pancreatitis into a fibrotic chronic-like disease. The mechanism involves TGF-beta, myofibroblasts, and defective collagenase activation.  (+info)

Chemotactic peptide uptake in acute pancreatitis: correlation with tissue accumulation of leukocytes. (4/463)

Chemotactic peptides bind specifically to receptors on leukocyte membranes. This property makes them prospective vehicles to evaluate inflammation and infection. We used two well-established models of acute pancreatitis to quantitate the binding of the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine-lysine (fMLFK) to leukocytes and its correlation to degree of organ inflammation. Uptake of the (99m)Tc-labeled nicotinyl hydrazine-derivatized chemotactic peptide analog fMLFK-HYNIC was measured in blood, pancreas, lung, and muscle specimens in rats with edematous or necrotizing pancreatitis and was compared with neutrophil sequestration assessed by myeloperoxidase activity and histology. Chemotactic peptide uptake in the pancreas was increased in mild and severe pancreatitis compared with controls, with higher levels in severe than in mild disease, and correlated with tissue myeloperoxidase activity (r = 0.7395, P < 0.001). Increased pulmonary uptake only in severe pancreatitis reflected pancreatitis-induced neutrophil sequestration in the lungs. Muscle uptake was unchanged compared with controls. Edema formation did not affect chemotactic peptide uptake. The data suggest that uptake of chemotactic peptides can contribute to quantitative assessment of neutrophils in localized inflammatory processes and is independent of associated edema formation or microcirculatory compromise.  (+info)

Caerulein-induced NF-kappaB/Rel activation requires both Ca2+ and protein kinase C as messengers. (5/463)

The eukaryotic transcription factor NF-kappaB/Rel is activated by a large variety of stimuli. We have recently shown that NF-kappaB/Rel is induced during the course of caerulein pancreatitis. Here, we show that activation of NF-kappaB/Rel by caerulein, a CCK analog, requires increasing intracellular Ca2+ levels and protein kinase C activation. Caerulein induces a dose-dependent increase of nuclear NF-kappaB/Rel binding activity in pancreatic lobules, which is paralleled by degradation of IkappaBalpha. IkappaBbeta was only slightly affected by caerulein treatment. Consistent with an involvement of Ca2+, the endoplasmic reticulum-resident Ca2+-ATPase inhibitor thapsigargin activated NF-kappaB/Rel in pancreatic lobules. The intracellular Ca2+ chelator TMB-8 prevented IkappaBalpha degradation and subsequent nuclear translocation of NF-kappaB/Rel induced by caerulein. BAPTA-AM was less effective. Cyclosporin A, a Ca2+/calmodulin-dependent protein phosphatase (PP2B) inhibitor, decreased caerulein-induced NF-kappaB/Rel activation and IkappaBalpha degradation. The inhibitory effect of bisindolylmaleimide suggests that protein kinase C activity is also required for caerulein-induced NF-kappaB/Rel activation. These data suggest that Ca2+- as well as protein kinase C-dependent mechanisms are required for caerulein-induced NF-kappaB/Rel activation.  (+info)

Trypsin and activation of circulating trypsinogen contribute to pancreatitis-associated lung injury. (6/463)

Pancreatic proteases are secreted in acute pancreatitis, but their contribution to associated lung injury is unclear. Applying models of mild edematous (intravenous caerulein) and severe necrotizing (intraductal glycodeoxycholic acid) pancreatitis in rats, we showed that both trypsinogen and trypsin concentrations in peripheral blood, as well as lung injury, correlate with the severity of the disease. To isolate the potential contribution of proteases to lung injury, trypsin or trypsinogen was injected into healthy rats or trypsinogen secreted in caerulein pancreatitis was activated by intravenous enterokinase. Pulmonary injury induced by protease infusions was dose dependent and was ameliorated by neutrophil depletion. Trypsinogen activation worsened lung injury in mild pancreatitis. In vitro incubation of leukocytes with trypsinogen showed that stimulated leukocytes can convert trypsinogen to trypsin. In conclusion, this study demonstrates that the occurrence and severity of pancreatitis-associated lung injury (PALI) corresponds to the levels of circulating trypsinogen and its activation to trypsin. Neutrophils are involved in both protease activation and development of pulmonary injury.  (+info)

Acinar cell apoptosis and the origin of tubular complexes in caerulein-induced pancreatitis. (7/463)

The interrelationship between acinar cell apoptosis and tubular complex formation was examined in caerulein-induced pancreatitis using histology, immunohistochemistry, electron microscopy and DNA gel electrophoresis. Rats were given 8 hourly subcutaneous injections of caerulein, 24 micrograms/kg, for up to 2 days. Morphologically and biochemically typical apoptosis affected 4.6 and 8.9% of acinar cells at 1 and 2 days, respectively, resulting in removal of most acinar cells by 2 days. Consequently, pancreatic ducts, the lining cells expressing bcl-2 and therefore resistant to apoptosis, became much more closely approximated to form the basis of tubular complexes; small numbers of immunohistochemically discrete acinar cells in their lining were either pre-apoptotic resistant to it or newly formed. Proliferation of duct-like lining cells was associated with apoptosis, an increase in islet cells and acinar cell regeneration. There was evidence of duct to acinar cell differentiation but the main increase in acinar cell numbers appeared to derive from proliferation of newly formed acinar cells.  (+info)

HSP27 expression regulates CCK-induced changes of the actin cytoskeleton in CHO-CCK-A cells. (8/463)

We investigated how heat shock protein 27 (HSP27) and its phosphorylation are involved in the action of cholecystokinin (CCK) on the actin cytoskeleton by genetic manipulation of Chinese hamster ovary (CHO) cells stably transfected with the CCK-A receptor. In these cells, as in rat acini, CCK activated p38 mitogen-activated protein (MAP) kinase and increased the phosphorylation of HSP27. This effect could be blocked with the p38 MAP kinase inhibitor SB-203580. Examination by confocal microscopy of cells stained with rhodamine phalloidin showed that CCK dose-dependently induced changes of the actin cytoskeleton, including cell shape changes, which were coincident with actin cytoskeleton fragmentation and formation of actin filament patches in the cells. To further evaluate the role of HSP27, CHO-CCK-A cells were transfected with expression vectors for either wild-type (wt) or mutant (3A, 3G, and 3D) human HSP27. Overexpression of wt-HSP27 and 3D-HSP27 inhibited the effects on the actin cytoskeleton seen after high-dose CCK stimulation. In contrast, overexpression of nonphosphorylatable mutants, 3A- and 3G-HSP27, or inhibition of phosphorylation of HSP27 by preincubation of wt-HSP27 transfected cells with SB-203580 did not protect the actin cytoskeleton. These results suggest that phosphorylation of HSP27 is required to stabilize the actin cytoskeleton and to protect the cells from the effects of high concentrations of CCK.  (+info)