Molecular mechanisms contributing to necrotizing enterocolitis.

OBJECTIVE: To examine the cellular mechanisms involved in the pathogenesis of necrotizing enterocolitis (NEC). SUMMARY BACKGROUND DATA: Necrotizing enterocolitis is a major cause of death and complications in neonates; the cellular mechanisms responsible for NEC are unknown. The inducible form of cyclooxygenase (i.e., COX-2) is activated by the transcription factor nuclear factor (NF)-kappaB and is thought to play a role in inflammation. METHODS: Segments of perforated and adjacent uninvolved small intestine from neonates with NEC were analyzed for COX-2 expression by immunohistochemistry. NEC was induced in weanling (18 days old) rats by occlusion of superior mesenteric vessels for 1 hour and intraluminal injection of platelet activating factor (50 micro/kg). Small intestine was harvested for protein extraction. Western immunoblot was performed to determine expression of COX-2. Gel shift assays were performed to assess NF-kappaB binding activity. RESULTS: Immunohistochemical analysis showed increased COX-2 protein expression in the perforated intestinal sections of all 36 neonates but not in adjacent normal intestine. Increased expression of COX-2 protein and NF-kappaB binding activity was noted in the small intestine of weanling rats at 0 and 3 hours after induction of NEC. CONCLUSIONS: Increased COX-2 expression was identified in all neonatal intestinal segments resected for perforated NEC. In addition, a coordinate induction of COX-2 expression and NF-kappaB binding was noted in a rodent model of NEC. These findings suggest that the COX-2/NF-kappaB pathway may play a role in the pathogenesis of NEC. Therapeutic agents that target this pathway may prove useful in the treatment or possible prevention of NEC.

Acute pancreatitis results in induction of heat shock proteins 70 and 27 and heat shock factor-1.

Heat shock proteins (HSPs) 70 and 27 are stress-responsive proteins that are important for cell survival after injury; the expression of these HSPs is regulated primarily by the transcription factor heat shock factor-1 (HSF-1). The purpose of this study was to determine the effect of acute pancreatitis on pancreatic HSPs (70, 27, 60, and 90) expression and to assess potential mechanisms for HSP induction using a murine model of cerulein-induced pancreatitis. We found an increase of both HSP70 and HSP27 levels with expression noted throughout the pancreas after induction of pancreatitis. HSP60 and HSP90 levels were constitutively expressed in the pancreas and did not significantly change with acute pancreatitis. HSF-1 DNA binding activity increased in accordance with increased HSP expression. We conclude that acute pancreatitis results in a marked increase in the expression of HSP70 and HSP27. Furthermore, the induction of HSP70 and HSP27 expression was associated with a concomitant increase in HSF-1 binding activity. The increased expression of both HSP70 and HSP27 noted with pancreatic inflammation may confer a protective effect for the remaining acini after acute pancreatitis.

Gut peptide receptor expression in human pancreatic cancers.

OBJECTIVE: To determine the prevalence of gastrointestinal (GI) peptide receptor expression in pancreatic cancers, and to further assess signaling mechanisms regulating neurotensin (NT)-mediated pancreatic cancer growth. SUMMARY BACKGROUND DATA: Pancreatic cancer remains one of the leading causes of GI cancer death; novel strategies for the early detection and treatment of these cancers is required. Previously, the authors have shown that NT, an important GI hormone, stimulates the proliferation of an NT receptor (NTR)-positive pancreatic cancer. METHODS: A total of 26 human pancreatic adenocarcinomas, obtained after resection, and 5 pancreatic cancer xenografts were analyzed for expression of NTR, vasoactive intestinal peptide receptor (VIPR), substance P receptor (SPR), and gastrin-releasing peptide receptor (GRPR). In addition, NTR expression, [Ca2+]i mobilization, and growth in response to NT was determined in L3.6, a metastatic pancreatic cancer cell line. RESULTS: Neurotensin receptor was expressed in 88% of the surgical specimens examined and all five of the pancreatic cancer xenografts. In contrast, VIPR, SPR, and GRPR expression was detected in 31%, 27%, and 8% of pancreatic cancers examined, respectively. Expression of NTR, functionally coupled to the Ca2+ signaling pathway, was identified in L3.6 cells; treatment with NT (10 micromol/L) stimulated proliferation of these cells. CONCLUSIONS: The authors demonstrated NTR expression in most of the pancreatic adenocarcinomas examined. In contrast, VIPR, SPR, and GRPR expression was detected in fewer of the pancreatic cancers. The expression of NTR and other peptide receptors suggests the potential role of endocrine manipulation in the treatment of these cancers. Further, the presence of GI receptors may provide for targeted chemotherapy or radiation therapy or in vivo scintigraphy for early detection.

Mitochondrial DNA damage and altered membrane potential (delta psi) in pancreatic acinar cells induced by reactive oxygen species.

BACKGROUND: Reactive oxygen species (ROS) have been implicated in the induction of acute pancreatitis. Mitochondria possess a distinct genome (mtDNA) that is more susceptible to ROS-induced damage than nuclear DNA (nDNA). The purpose of our study was to determine the effect of ROS on mitochondrial function and membrane potential (delta psi mt), to identify signal transduction mechanisms activated by ROS, and to quantify damage to mtDNA in an in vitro pancreatitis model. METHODS: Pancreatic acinar cells, AR4-2J, were treated with saline solution (control) or hydrogen peroxide (H2O2), a representative ROS. Mitochondrial function was assessed with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay; to determine delta psi mt, rhodamine-123 uptake was measured. Intracellular calcium levels and c-Jun N-terminal kinase activity was determined; gel mobility shift assays were performed to assess induction of the transcription factor NF-kappa B. To quantitate DNA damage, a novel polymerase chain reaction-based procedure was performed. RESULTS: Mitochondrial function and delta psi mt were significantly decreased with oxidative damage. H2O2 treatment resulted in increased intracellular calcium levels, activation of c-Jun N-terminal kinase, and induction of NF-kappa B DNA binding. Treatment of AR4-2J cells with H2O2 resulted in selective mtDNA damage; nDNA was not affected. CONCLUSIONS: Our data demonstrate that pancreatic mtDNA is more susceptible to oxidative damage than nDNA; this damage is associated with decreases in mitochondrial function and delta psi mt and activation of downstream signal transduction pathways. Mitochondrial damage mediated by ROS may play a central role in pancreatic cell injury associated with acute pancreatitis.

Role of low molecular weight iron in functional preconditioning of the isolated rat heart.

Post-ischemic contractile dysfunction in the heart may be due to oxygen-derived free radicals catalyzed by low molecular weight iron (lmw Fe), which is thought to accumulate during ischemia and reperfusion. We tested the hypothesis that functional preconditioning with transient ischemia in the rat heart may be due to decreasing the myocardial lmw Fe pool, and consequently free radicals during ischemia or reperfusion. Hearts were preconditioned with two 5-min episodes of ischemia followed by 5 min of reperfusion. The lmw Fe pool of pre-ischemic hearts was 172 +/- 13pmol/mg protein. After 40 min of prolonged ischemia, the lmw Fe contents were 176 +/- 25 and 127 +/- 13 pmol/mg for non-conditioned and preconditioned hearts, respectively (P=N.S.). After 10 min of reperfusion, the lmw Fe contents were 246 +/- 26 and 228 +/- 23 pmol/mg protein, respectively (P=N.S.). We next tested the ability of deferoxamine, an iron chelator, to mimic functional preconditioning. The percentage recoveries of heart rate x developed pressure after 40 min of ischemia and 30 min of reperfusion were 38 +/- 6 and 25 +/- 5 for non-conditioned and deferoxamine-treated hearts, respectively (P=N.S.). We further tested the hypothesis by determining if iron-overloading by dietary enhancement and weekly iron injections would exacerbate post-ischemic contractile dysfunction and attenuate functional preconditioning with ischemia. The total iron contents of the high iron and normal groups were 10.3 +/- 0.6 and 4.4 +/- 0.2 nmol/mg protein (P<0.001). Percentage recoveries of heart rate x developed pressure were 36 +/- 6 and 33 +/- 5 for non-conditioned hearts in the high iron and normal iron groups, respectively (P=N.S.). Percentage recoveries of heart rate x developed pressure were 58 +/- 5 and 68 +/- 6 for ischemically preconditioned hearts in the high and normal iron groups, respectively (P= N.S.). The results suggest that functional preconditioning in the rat heart is not due to attenuation lmw Fe accumulation.