Andreas C.C. Wagner

p38 Map Kinase Is Expressed in the Pancreas and Is Immediately Activated following Cerulein Hyperstimulation

Background: The pathophysiology of pancreatitis and the pancreatic stress response are not well understood. In the pancreas, mitogen-activated protein kinase (MAPK) and stress-activated protein kinase (SAPK) are reportedly regulated by secretagogue stimulation and hyperstimulation. However, no data exist on the expression and regulation of pancreatic p38 Map kinase. Aims: Pancreatic expression of p38 Map kinase and MAPK, SAPK and p38 regulation during pancreatic stress were investigated. Methods: For hyperstimulation and secretory stress, cerulein was given intravenously, while hyperthermia preconditioning stress was induced by whole body hyperthermia (42°C). Results: In addition to MAPK and SAPK, p38 Map kinase was found to be expressed in the rat pancreas. Cerulein regulated all kinases time- and dose-dependently. MAPK and p38 Map kinase showed basal activity and were further activated at low cerulein doses. SAPK had no basal activity and its activation required maximal secretory to supramaximal amounts of cerulein. Cerulein hyperstimulation, inducing pancreatitis, activated p38 more rapidly than SAPK and more strongly than MAPK. In contrast to cerulein hyperstimulation stress, hyperthermia stress only activated p38 Map kinase. Conclusions: p38 Map kinase is expressed in the pancreas and is most rapidly activated following cerulein hyperstimulation. Both SAPK and p38 Map kinase are possibly important regulators during the onset of cerulein pancreatitis.

Caerulein pancreatitis increases mRNA but reduces protein levels of rat pancreatic heat shock proteins

We have recently reported that preconditioning through hyperthermia induces expression of pancreatic heat shock proteins (HSPs) and protects against caerulein pancreatitis. Here, we investigate caerulein-mediated effects on pancreatic HSPs without prior hyperthermia. Caerulein time and dose dependently increased pancreatic mRNA levels of the constitutive isoform of HSP70 (HSC70). However, pancreatic HSC70 protein levels were decreased, as were HSP60 protein levels. Also, in contrast to hyperthermia preconditioning, caerulein did not induce measurable levels of mRNA or protein of the inducible isoform of HSP70. Thus the pancreas reacts to different kinds of stress (hyperthermia vs. hyperstimulation) with differential induction of HSP mRNAs. Clearly, hyperthermia leads to induction of HSP protein expression, whereas caerulein treatment does not. Therefore, our current study further supports the idea that hyperthermia-induced protection against caerulein pancreatitis may be mediated through increased protein levels of pancreatic HSPs. It is further tempting to hypothesize that failure to appropriately increase HSP protein levels in response to high doses of caerulein might be a factor in the development of pancreatitis.We have recently reported that preconditioning through hyperthermia induces expression of pancreatic heat shock proteins (HSPs) and protects against caerulein pancreatitis. Here, we investigate caerulein-mediated effects on pancreatic HSPs without prior hyperthermia. Caerulein time and dose dependently increased pancreatic mRNA levels of the constitutive isoform of HSP70 (HSC70). However, pancreatic HSC70 protein levels were decreased, as were HSP60 protein levels. Also, in contrast to hyperthermia preconditioning, caerulein did not induce measurable levels of mRNA or protein of the inducible isoform of HSP70. Thus the pancreas reacts to different kinds of stress (hyperthermia vs. hyperstimulation) with differential induction of HSP mRNAs. Clearly, hyperthermia leads to induction of HSP protein expression, whereas caerulein treatment does not. Therefore, our current study further supports the idea that hyperthermia-induced protection against caerulein pancreatitis may be mediated through increased protein levels of pancreatic HSPs. It is further tempting to hypothesize that failure to appropriately increase HSP protein levels in response to high doses of caerulein might be a factor in the development of pancreatitis.

Hyperthermia Preconditioning Induces Renal Heat Shock Protein Expression, Improves Cold Ischemia Tolerance, Kidney Graft Function and Survival in Rats

Background: Evidence indicates that hyperthermia preconditioning (HP) can be protective in kidney transplantation, possibly through increased heat shock protein (HSP) expression. A detailed study about individual HSPs and functional preservation is lacking, however. Therefore, we studied the effects of HP on kidney graft survival, function and HSP expression. Methods: Male Lewis rats were or were not subjected to whole-body hyperthermia 24 h prior to kidney procurement. Kidneys were stored in UW solution at 4°C for 32, 40 or 45 h. Recipient kidneys were both removed and single isografts transplanted orthotopically. Results: HP strongly induced HSP72 and HSP32 expression. Following 32-hour cold ischemia, most animals survived even without prior HP. However, HP strongly reduced functional impairment induced by cold ischemia. Following 40-hour cold ischemia, kidneys from donors without HP did not recover function and all animals died within 3 days. In contrast, HP-exposed kidneys tolerated 40-hour storage significantly better, with 44% of rats surviving until sacrifice on day 7. In these animals, renal function was still better compared to animals with 32-hour-stored kidneys without HP. Histological alterations were also diminished following HP. Conclusion: Our data show that HP induces renal HSP72 and, for the first time, HSP32. HP increases survival following transplantation and acts by improving several parameters of kidney function including proteinuria, volume output and creatinine clearance.

Treatment of hepatic hydrothorax and reduction of chest tube output with octreotide.

Hepatic hydrothorax is a dreaded complication in patients with liver cirrhosis. Placement of chest tubes can alleviate respiratory distress, but patients often succumb due to excessive fluid and protein loss via the open drain. Our case illustrates that high-dose octreotide can strongly reduce hepatic hydrothorax drainage volume. This allows removal of the chest tube, which would otherwise not have been possible.

Pancreatic Zymogen Granule Membrane Proteins: Molecular Details Begin to Emerge

Regulated secretion and exocytosis require the controlled docking and fusion of secretory storage organelles with the plasma membrane. Proteins resident in secretory organelle membranes are, therefore, likely to have critical functions as a part of these processes. The exocrine pancreas with its secretory storage organelle, the zymogen granule, represents a classical model of regulated secretion and exocytosis. Until recently, however, little has been known of zymogen granule membrane protein structure and function. With the recent cloning of two zymogen granule membrane proteins, GP-2 and GP-3, and with the identification of the presence of low molecular weight guanosine triphosphate binding proteins, it is now possible to approach the role of the zymogen granule membrane proteins at the molecular level.

Hyperthermia, inducing pancreatic heat-shock proteins, fails to prevent cerulein-induced stress kinase activation.

The dually phosphorylated c-jun kinase and p38 mitogen-activated protein (MAP) kinase, also termed stress kinases, are members of the MAP kinase family. They are activated early during cerulein pancreatitis induction and have been proposed as regulators during pancreatitis development by us and others. We recently showed that hyperthermia preconditioning induces expression of pancreatic heat-shock proteins (HSP) and protects against cerulein pancreatitis. Because it was further reported that HSP70 can prevent activation of stress kinases in lymphoid tumor cells, we investigated whether hyperthermia preconditioning might reduce hyperstimulation-mediated activation of pancreatic stress kinases. Pancreatic HSP expression was induced by whole-body hyperthermia preconditioning. Without prior HSP induction, cerulein led to a rapid and dose-dependent increase in serum lipase and amylase levels, pancreatic wet weight through edema formation, and activation of pancreatic MAP kinases. Hyperthermia preconditioning, although strongly inducing HSP70 and almost completely preventing edema formation, as well as the increase of serum amylase and lipase, did not reduce cerulein-mediated stress kinase activation. This indicates that in the pancreas, cerulein can strongly activate MAP kinases even when pancreatitis development is greatly inhibited, and that pancreatic HSPs do not inhibit activation of pancreatic stress kinases in vivo.

Effects of calyculin A on amylase release in streptolysin-O permeabilized acinar cells

The effects of the phosphatase inhibitors calyculin A and okadaic acid on amylase release from streptolysin-O permeabilized rat pancreatic acini were investigated. Both agents induced similar biphasic effects with moderate potentiation of calcium-stimulated amylase release at medium and strong inhibition at higher concentrations. Calyculin A was thirty times more potent than okadaic acid and at 100 nM totally inhibited calcium-induced amylase release while 3 microM okadaic acid reduced amylase release by 78%. 100nM calyculin A also completely inhibited GTP gamma S-potentiated amylase release and partially inhibited phorbol ester potentiated secretion. The data indicate that inhibition of a serine/threonine phosphatase, probably a type 1 phosphatase, leads to inhibition of calcium-induced amylase release in permeabilized pancreatic acini.

Expression and regulation of calpain in rat pancreatic acinar cells.

Introduction Calpains, cytosolic Ca 2+ -dependent cysteine proteases, are expressed in a variety of mammalian cells and have been found to participate in stimulus–secretion coupling in platelets and alveolar cells. Aims In pancreatic acinar cells, expression of calpains and their role in the secretory process have not yet been elucidated. Both subjects, therefore, were examined in the current study. Methodology &mgr;-calpain and m-calpain were detected immunochemically. Calpain activation was measured by fluorescence spectrophotometry and single-cell fluorometry using Suc-Leu-Leu-Val-Tyr-AMC as substrate. Amylase secretion and cell damage, characterized by lactate dehydrogenase release, were measured by colorimetric assays. Results Immunochemistry revealed cytoplasmic localization of both calpain isoforms. Immediately after increasing the cytosolic Ca 2+ concentration with ionomycin, a marked dose-dependent protease activation and cellular damage were observed. Inhibition of ionomycin-mediated enzyme activation through preincubation of cells with Ca 2+ -free medium, BAPTA-AM, or Z-Leu-Leu-Tyr-CHN 2 significantly reduced cell injury. Cholecystokinin (100 pM) also induced proteolytic activity, preceding cholecystokinin-stimulated amylase secretion. Protease activity and amylase release were significantly inhibited by Z-Leu-Leu-Tyr-CHN 2 pretreatment. Conclusion Calpains are expressed in pancreatic acinar cells and may participate in stimulus–secretion coupling. In addition, our study indicates that pathologic calpain activation may contribute to Ca 2+ -mediated acinar cell damage.

Endothelin increases (Ca2+)i in rat pancreatic acinar cells by intracellular release but fails to increase amylase secretion

In individual fura-2 loaded cells of rat pancreatic acini endothelin-1 (ET-1) (10-50 nM) induced sustained oscillations in [Ca2+]i. At higher concentrations a larger, but transient increase in [Ca2+]i was observed, which was largely unaffected by removal of extracellular Ca2+. ET-1 induced the release of Ca2+i from the same store as cholecystokinin (CCK), but with less potency. At concentrations of endothelin which transiently increased Ca2+, ET-1 increased the accumulation of inositol phosphates. Specific binding sites for 125I-endothelin were demonstrated on rat pancreatic acini. A single class of binding sites was identified with an apparent Kd 108 +/- 12 pM and Bmax of 171 +/- 17 fmol/mg for ET-1. The relative potency order for displacing [125I]ET was ET-1 greater than ET-2 greater than ET-3. In contrast to CCK and the non-phorbol ester tumour promoter Thapsigargin (TG) which induce both transient and sustained components of [Ca2+]i elevation, ET-1 failed to increase amylase release over the range 100 pM-1 microM.

Map kinase phosphatases (MKPS) are early responsive genes during induction of cerulein hyperstimulation pancreatitis

Mitogen-activated protein kinase (MAPK) family members such as c-jun N-terminal kinase (JNK) may act as signal transducers early during pancreatitis development and evidence indicates that MAPK phosphatases (MKP) downregulate MAPK. We therefore investigated expression and regulation of pancreatic MKP in vivo. Pancreatic MKP mRNA levels were near or below the detection threshold in unstimulated animals. Cerulein hyperstimulation strongly induced MKP-1, MKP-3, and MKP-5 expression, peaking 30 to 60 min after treatment. Thus, MKPs clearly are early responsive genes during pancreatitis induction. Interestingly, inhibition of MKP-1 expression by Ro-31-8220 maximally induced activation of JNK but not of p38 and ERK in acutely isolated acini. These effects indicate that JNK may indeed be a preferred MKP-1 substrate in vivo.