Angiotensin II signaling through the AT1a and AT1b receptors does not have a role in the development of cerulein-induced chronic pancreatitis in the mouse.

The intraorgan renin-angiotensin system (RAS) plays an important role in the pathophysiology of a variety of diseases and has been implicated in fibrogenesis. The role of RAS in the development of chronic pancreatitis is not well established. The blockade of RAS in rat models with angiotensin-converting enzyme inhibitors (ACEi) or angiotensin receptor 1 (AT1) blockers (ARBs) mostly have reduced pancreatic inflammation and fibrosis with a few exceptions. At the same time, the use of ACEi and ARBs in humans is associated with a modest risk of acute pancreatitis. The aim of this study was to elucidate the effect of the AT1 signaling pathway in the development of pancreatitis using AT1a- and AT1b-deficient mice as well as the ARB losartan. Chronic pancreatitis was induced by repetitive cerulein administration in C57BL/6J wild-type (WT) and AT1a- and AT1b-deficient mice (AT1a-/- and AT1b-/-), and pancreatic injury was assessed at day 10. Pancreatic weight of cerulein treated groups was significantly reduced. There was severe parenchymal atrophy and fibrosis assessed by histological examination. Fibrosis was accompanied by activation of pancreatic stellate cells (PSC) evaluated by Western blot analysis for alpha-smooth muscle actin. No differences were seen between cerulein-treated WT, AT1a-/- , AT1b-/- mice, or losartan treated-WT mice with regards to morphological or molecular alterations induced by cerulein. Our results demonstrate that AT1a and AT1b receptor pathways do not seem to be essential for the development of pancreatitis in the mouse model of pancreatitis induced by repetitive cerulein injury.

Induced thrombospondin expression in the mouse pancreas during pancreatic injury.

Chronic pancreatitis is a disease characterized by pancreatic fibrogenesis in response to sustained or repetitive injury. Pancreatic stellate cells (PSC) are interstitial cells that produce excessive extracellular matrix components during the process of fibrogenesis and therefore play a central role in the pathogenesis of chronic pancreatitis. Because the matricellular proteins thrombospondin-1 (TSP-1) and TSP-2 have a role in regulating fibrogenesis in other tissues, the expression of these major TSP isoforms in the whole pancreas was measured in a mouse model of repetitive pancreatic injury. Specifically, mice were treated with cerulein, 50 microg/kg/h x 6h with treatments repeated once or twice every 48 h. Expression was also evaluated in cultured PSC. PSC were isolated by outgrowth from normal mouse pancreas and expression of TSP-1 and TSP-2 was evaluated after serum-activation. The mRNA transcripts for TSP-1 and TSP-2 were increased, 16-fold and 87-fold respectively, in the pancreas in response to repetitive injury. In cultured PSC, these transcripts were also increased in response to serum and increases in mRNA were reflected by the secretion of TSP-1 and TSP-2 proteins by PSC into culture media. In summary, PSC may be an important source of both TSP-1 and TSP-2 in the pancreas in response to injury. These modulators of fibrogenesis could play a role in the development of pancreatic fibrosis that characterizes chronic pancreatitis.

Differential expression of the trypsin inhibitor SPINK3 mRNA and the mouse ortholog of secretory granule protein ZG-16p mRNA in the mouse pancreas after repetitive injury.

A mouse model using repetitive acinar cell injury caused by supraphysiologic doses of cerulein to induce the characteristic fibrosis and loss of acinar cell mass found in human chronic pancreatitis was employed to identify early changes in gene expression. A gene array was used to detect changes in 18,000 expressed sequence tags; changes in specific transcripts were confirmed by RNase protection assays. These methods identified SPINK3, the mouse homologue of human and rat protease inhibitor genes, as being highly expressed in the pancreas and induced after pancreatic injury. Because SPINK3 may be an important serine protease inhibitor, its up-regulation may reflect an important endogenous cytoprotective mechanism in preventing further injury. The up-regulation of SPINK3 was specific; the mouse homologue of the zymogen-processing protein ZG-16p was also highly expressed in the pancreas but sharply down-regulated early in the course of injury. These findings suggest that the pancreatic acinar cell may respond to injury with a program of self-preservation and loss of normal function.

The hepatocellular bile acid transporter Ntcp facilitates uptake of the lethal mushroom toxin alpha-amanitin.

Hepatotoxicity caused by the mushroom poison alpha-amanitin is an unusual but serious cause of death and liver transplantation. Understanding the mechanisms of alpha-amanitin uptake may lead to rational therapeutic approaches. Because older data suggested that a sodium-dependent bile acid transporter is responsible for alpha-amanitin uptake, we tested the hypothesis that Na(+)-taurocholate cotransporter polypeptide (Ntcp) facilitates hepatocellular alpha-amanitin uptake. Human hepatoblastoma cells (HepG2), cells that have lost native Ntcp expression, were stably transfected with the rat Ntcp gene. Taurocholate uptake by the transfected cells exhibited a physiologically normal K(m) and V(max). A toxicologically relevant functional assay for alpha-amanitin uptake was developed by measuring its ability to block cytokine-induced synthesis of interleukin-1 receptor antagonist (IL-1Ra) mRNA. Treatment with interleukin-1beta (10 ng/ml) and interleukin-6 (100 ng/ml) increased IL-1Ra mRNA abundance 8.6-fold and 15.6-fold in HepG2 cells and Ntcp-transfected cells, respectively. Pretreatment of transfected cells with 1 micro M alpha-amanitin for 6-10 h almost completely blocked induction of IL-1Ra mRNA (1.9-fold induction) whereas pretreatment of non-transfected cells did not block induction of IL-1Ra mRNA (21.6-fold induction, P<0.02 compared with stimulated transfected cells without alpha-amanitin). These findings demonstrate that Ntcp may be an important mediator of alpha-amanitin uptake by the liver.