bluuebunny/crossref_metadata_2025 huggingface dataset:
Upregulation of iNOS by COX-2 in muscularis resident macrophage of rat intestine stimulated with LPS
Authors: Hori Masatoshi , Kita Muneto , Torihashi Shigeko , Miyamoto Shigeki , Won Kyung-Jong , Sato Koichi , Ozaki Hiroshi , Karaki Hideaki
Published: December 2017

We investigated the effect of lipopolysaccharide (LPS) on the induction of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in muscularis resident macrophages of rat intestine in situ. When the tissue was incubated with LPS for 4 h, mRNA levels of iNOS and COX-2 were increased. The majority of iNOS and COX-2 proteins appeared to be localized to the dense network of muscularis resident macrophages immunoreactive to ED2. LPS treatment also increased the production of nitric oxide (NO), PGE2, and PGI2. The increased expression of iNOS mRNA by LPS was suppressed by indomethacin but not by NG-monomethyl-l-arginine (l-NMMA). The increased expression of COX-2 mRNA by LPS was affected neither by indomethacin nor by l-NMMA. Muscle contractility stimulated by 3 μM carbachol was significantly inhibited in the LPS-treated muscle, which was restored by treatment of the tissue with l-NMMA, aminoguanidine, indomethacin, or NS-398. Together, these findings show that LPS increases iNOS expression and stimulates NO production in muscularis resident macrophages to inhibit smooth muscle contraction. LPS-induced iNOS gene expression may be mediated by autocrine regulation of PGs through the induction of COX-2 gene expression.
Compensatory hepatic regeneration after mild, but not fulminant, intraperitoneal sepsis in rats
Authors: Weiss Yoram G. , Bellin Lisa , Kim Patrick K. , Andrejko Kenneth M. , Haaxma Charlotte A. , Raj Nichelle , Furth E. Elizabeth , Deutschman Clifford S.
Published: December 2017

Sepsis is the leading cause of death in surgical intensive care units. Although both mild sepsis secondary to cecal ligation and single puncture (CLP) and fulminant, double puncture CLP (2CLP) may provoke hepatocyte death, we hypothesize that regeneration compensates for cell death after CLP but not 2CLP. In male Sprague-Dawley rats, hepatic necrosis, as determined by serum α-glutathione S-transferase (α-GST) levels, was significantly but equally elevated over time after both CLP and 2CLP. Apoptosis, evaluated using both terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and morphological examination, was minimal after both CLP and 2CLP. Regeneration, assayed by staining tissue for incorporation of exogenously administered bromodeoxyuridine, was present after CLP but not after 2CLP. To further substantiate impaired regeneration, steady-state levels of mRNAs encoding JunB, LRF-1, and cyclin D1 were determined. After 2CLP, the absence of JunB, LRF-1, and cyclin D1 mRNAs confirmed failed activation of the mitogen-activated protein kinase-linked proliferative pathway and progression through the cell cycle. Therefore, failed hepatocyte regeneration may be a manifestation of hepatic dysfunction in fulminant sepsis.
NF-κB activation and susceptibility to apoptosis after polyamine depletion in intestinal epithelial cells
Authors: Li Li , Rao Jaladanki N. , Bass Barbara L. , Wang Jian-Ying
Published: December 2017

The maintenance of intestinal mucosal integrity depends on a balance between cell renewal and cell death, including apoptosis. The natural polyamines, putrescine, spermidine, and spermine, are essential for mucosal growth, and decreasing polyamine levels cause G1phase growth arrest in intestinal epithelial (IEC-6) cells. The present study was done to determine changes in susceptibility of IEC-6 cells to apoptosis after depletion of cellular polyamines and to further elucidate the role of nuclear factor-κB (NF-κB) in this process. Although depletion of polyamines by α-difluoromethylornithine (DFMO) did not directly induce apoptosis, the susceptibility of polyamine-deficient cells to staurosporine (STS)-induced apoptosis increased significantly as measured by changes in morphological features and internucleosomal DNA fragmentation. In contrast, polyamine depletion by DFMO promoted resistance to apoptotic cell death induced by the combination of tumor necrosis factor-α (TNF-α) and cycloheximide. Depletion of cellular polyamines also increased the basal level of NF-κB proteins, induced NF-κB nuclear translocation, and activated the sequence-specific DNA binding activity. Inhibition of NF-κB binding activity by sulfasalazine or MG-132 not only prevented the increased susceptibility to STS-induced apoptosis but also blocked the resistance to cell death induced by TNF-α in combination with cycloheximide in polyamine-deficient cells. These results indicate that 1) polyamine depletion sensitizes intestinal epithelial cells to STS-induced apoptosis but promotes the resistance to TNF-α-induced cell death, 2) polyamine depletion induces NF-κB activation, and 3) disruption of NF-κB function is associated with altered susceptibility to apoptosis induced by STS or TNF-α. These findings suggest that increased NF-κB activity after polyamine depletion has a proapoptotic or antiapoptotic effect on intestinal epithelial cells determined by the nature of the death stimulus.
Mechanisms in regulating the release of serotonin from the perfused rat stomach
Authors: Yu P.-L. , Fujimura M. , Hayashi N. , Nakamura T. , Fujimiya M.
Published: December 2017

The mechanisms regulating the release of serotonin into the portal circulation as well as into the gastric lumen were studied in the isolated vascularly and luminally perfused rat stomach. Immunohistochemical study of the rat stomach showed that serotonin-containing enterochromaffin (EC) cells were densely packed in the antral mucosa, sparsely scattered in the corpus, and not found in the fundus. Such morphological findings suggest that serotonin detected in this study may have originated from antral EC cells. Luminal acidification stimulated the vascular release of serotonin but did not affect the luminal release of serotonin. The basal release of serotonin into the vasculature was 10 times higher than that into the gastric lumen at intragastric pH 2. The vascular release of serotonin is regulated by stimulation from cholinergic nicotinic mechanisms, whereas inhibitory neurotransmitters such as vasoactive intestinal peptide and NO are probably not involved. Somatostatin and peptide YY originating from endocrine cells may exert direct inhibitory effects, possibly via somatostatin and peptide YY receptors on the EC cells, and a cholinergic muscarinic mechanism may exert indirect effects on the vascular release of serotonin via the muscarinic receptor on the endocrine cells.
Characterization of a human plasma membrane heme transporter in intestinal and hepatocyte cell lines
Authors: Worthington Mark T. , Cohn Steven M. , Miller Suzanne K. , Luo Roger Qi , Berg Carl L.
Published: December 2017

Heme is the most bioavailable form of dietary iron and a component of many cellular proteins. Controversy exists as to whether heme uptake occurs via specific transport mechanisms or passive diffusion. The aims of this study were to quantify cellular heme uptake with a fluorescent heme analog and to determine whether heme uptake is mediated by a heme transporter in intestinal and hepatic cell lines. A zinc-substituted porphyrin, zinc mesoporphyrin (ZnMP), was validated as a heme homolog in uptake studies of intestinal (Caco-2, I-407) and hepatic (HepG2) cell lines. Uptake experiments to determine time dependence, heme inhibition, concentration dependence, temperature dependence, and response to the heme synthesis inhibitor succinylacetone were performed. Fluorescence microscope images were used to quantify uptake and determine the cellular localization of ZnMP; ZnMP uptake was seen in intestinal and hepatic cell lines, with cytoplasmic uptake and nuclear sparing. Uptake was dose- and temperature dependent, inhibited by heme competition, and saturated over time. Preincubation with succinylacetone augmented uptake, with an increased initial uptake rate. These findings establish a new method for quantifying heme uptake in individual cells and provide strong evidence that this uptake is a regulated, carrier-mediated process.
Development of an animal model of chronic alcohol-induced pancreatitis in the rat
Authors: Kono Hiroshi , Nakagami Mikio , Rusyn Ivan , Connor Henry D. , Stefanovic Branko , Brenner David A. , Mason Ronald P. , Arteel Gavin E. , Thurman Ronald G.
Published: December 2017

This study was designed to develop an animal model of alcoholic pancreatitis and to test the hypothesis that the dose of ethanol and the type of dietary fat affect free radical formation and pancreatic pathology. Female Wistar rats were fed liquid diets rich in corn oil (unsaturated fat), with or without a standard or high dose of ethanol, and medium-chain triglycerides (saturated fat) with a high dose of ethanol for 8 wk enterally. The dose of ethanol was increased as tolerance developed, which allowed approximately twice as much alcohol to be delivered in the high-dose group. Serum pancreatic enzymes and histology were normal after 4 wk of diets rich in unsaturated fat, with or without the standard dose of ethanol. In contrast, enzyme levels were elevated significantly by the high ethanol dose. Increases were blunted significantly by dietary saturated fat. Fibrosis and collagen α1(I) expression in the pancreas were not detectable after 4 wk of enteral ethanol feeding; however, they were enhanced significantly by the high dose after 8 wk. Furthermore, radical adducts detected by electron spin resonance were minimal with the standard dose; however, the high dose increased carbon-centered radical adducts as well as 4-hydroxynonenal, an index of lipid peroxidation, significantly. Radical adducts were also blunted by ∼70% by dietary saturated fat. The animal model presented here is the first to demonstrate chronic alcohol-induced pancreatitis in a reproducible manner. The key factors responsible for pathology are the amount of ethanol administered and the type of dietary fat.
Localized pancreatic NF-κB activation and inflammatory response in taurocholate-induced pancreatitis
Authors: Vaquero Eva , Gukovsky Ilya , Zaninovic Vjekoslav , Gukovskaya Anna S. , Pandol Stephen J.
Published: December 2017

Transcription factor nuclear factor-κB (NF-κB) is activated in cerulein pancreatitis and mediates cytokine expression. The role of transcription factor activation in other models of pancreatitis has not been established. Here we report upregulation of NF-κB and inflammatory molecules, and their correlation with local pancreatic injury, in a model of severe pancreatitis. Rats received intraductal infusion of taurocholate or saline, and the pancreatic head and tail were analyzed separately. NF-κB and activator protein-1 (AP-1) activation were assessed by gel shift assay, and mRNA expression of interleukin-6, tumor necrosis factor-α, KC, monocyte chemoattractant protein-1, and inducible nitric oxide synthase was assessed by semiquantitative RT-PCR. Morphological damage and trypsin activation were much greater in the pancreatic head than tail, in parallel with a stronger activation of NF-κB and cytokine mRNA. Saline infusion mildly affected these parameters. AP-1 was strongly activated in both pancreatic segments after either taurocholate or saline infusion. NF-κB inhibition with N-acetylcysteine ameliorated the local inflammatory response. Correlation between localized NF-κB activation, cytokine upregulation, and tissue damage suggests a key role for NF-κB in the development of the inflammatory response of acute pancreatitis.
Effect of meal viscosity and nutrients on satiety, intragastric dilution, and emptying assessed by MRI
Authors: Marciani Luca , Gowland Penny A. , Spiller Robin C. , Manoj Pretima , Moore Rachel J. , Young Paul , Fillery-Travis Annette J.
Published: December 2017

The relationship between the intragastric distribution, dilution, and emptying of meals and satiety was studied using noninvasive magnetic resonance imaging techniques in 12 healthy subjects with four polysaccharide test meals of varying viscosity and nutrient content as follows: 1) low-viscosity nonnutrient, 2) low-viscosity nutrient, 3) high-viscosity nonnutrient, and 4) high-viscosity nutrient. Increasing the nutrient content of the high-viscosity meal delayed gastric emptying from 46 ± 9 to 76 ± 6 min ( P < 0.004), whereas increasing viscosity had a smaller effect. The volume of secretions within the stomach 60 min after ingestion was higher for the high-viscosity nutrient meal ( P < 0.04). A simple model to calculate the total volume of secretion added to the test meal is presented. Color-coded dilution map images showed the heterogeneous process of progressive gastric dilution of high-viscosity meals, whereas low-viscosity meals were uniformly diluted. Fullness was found to be linearly related to total gastric volumes for the nutrient meals ( R2= 0.98) and logarithmically related for the nonnutrient meals ( R2= 0.96). Fullness was higher for high- compared with low-viscosity meals ( P < 0.02), and with the nutrient meals this was associated with greater antral volumes ( P < 0.05).
IL-12 gene transfer alters gut physiology and host immunity in nematode-infected mice
Authors: Khan Waliul I. , Blennerhassett Patricia A. , Deng Yikang , Gauldie Jack , Vallance Bruce A. , Collins Stephen M.
Published: December 2017

Immune responses elicited by nematode parasite infections are characterized by T helper 2 (Th2) cell induction. The immunologic basis for changes in intestinal physiology accompanying nematode infection is poorly understood. This study examined whether worm expulsion and associated goblet cell hyperplasia and muscle contractility share a similar immune basis by shifting the response from Th2 to Th1 using interleukin-12 (IL-12) overexpression. We used a single administration of recombinant adenovirus vector expressing IL-12 (Ad5IL-12) in Trichinella spiralis-infected mice. Ad5IL-12 administered 1 day after infection prolonged worm survival and inhibited infection-induced muscle hypercontractility and goblet cell hyperplasia. This was correlated with upregulated interferon-γ (IFN-γ) expression and downregulated IL-13 expression in the muscularis externa layer. We also observed increased IFN-γ production and decreased IL-4 and IL-13 production from in vitro stimulated spleen and mesenteric lymph node cells of infected Ad5IL-12-treated mice. These results indicate that transfer and overexpression of the IL-12 gene during Th2-based nematode infection shifts the immune response toward Th1 and delays worm expulsion. Moreover, the immune response shift abrogated the physiological responses to infection, attenuating both muscle hypercontractility and goblet cell hyperplasia. These findings strongly indicate that worm expulsion, muscle hypercontractility, and goblet cell hyperplasia share a common immunologic basis and may be causally linked.
VIII. Pathological consequences of rotavirus infection and its enterotoxin
Authors: Morris Andrew P. , Estes Mary K.
Published: December 2017

Rotaviral infection in neonatal animals and young children leads to acute self-limiting diarrhea, but infected adults are mainly asymptomatic. Recently, significant in-roads have been made into our understanding of this disease: both viral infection and virally manufactured nonstructural protein (NSP)4 evoke intracellular Ca2+([Ca2+]i) mobilization in native and transformed gastrointestinal epithelial cells. In neonatal mouse pup mucosa models, [Ca2+]ielevation leads to age-dependent halide ion movement across the plasma membrane, transepithelial Cl−secretion, and, unlike many microbial enterotoxins, initial cyclic nucleotide independence to secretory diarrhea. Similarities between rotavirus infection and NSP4 function suggest that NSP4 is responsible for these enterotoxigenic effects. NSP4-mediated [Ca2+]imobilization may further facilitate diarrhea by signaling through other Ca2+-sensitive cellular processes (cation channels, ion and solute transporters) to potentiate fluid secretion while curtailing fluid absorption. Apart from these direct actions in the mucosa at the onset of diarrhea, innate host-mediated defense mechanisms, triggered by either or both viral replication and NSP4-induced [Ca2+]imobilization, sustain the diarrheal response. This secondary component appears to involve the enteric nervous system and may be cyclic nucleotide dependent. Both phases of diarrhea occur in the absence of significant inflammation. Thus age-dependent rotaviral disease represents an excellent experimental paradigm for understanding a noninflammatory diarrhea.
Phospholipase C-γ inhibition prevents EGF protection of intestinal cytoskeleton and barrier against oxidants
Authors: Banan A. , Fields J. Z. , Zhang Y. , Keshavarzian A.
Published: December 2017

Loss of intestinal barrier integrity is associated with oxidative inflammatory GI disorders including inflammatory bowel disease. Using monolayers of human intestinal epithelial (Caco-2) cells, we recently reported that epidermal growth factor (EGF) protects barrier integrity against oxidants by stabilizing the microtubule cytoskeleton, but the mechanism downstream of the EGF receptor (EGFR) is not established. We hypothesized that phospholipase C (PLC)-γ is required. Caco-2 monolayers were exposed to oxidant (H2O2) with or without pretreatment with EGF or specific inhibitors of EGFR tyrosine kinase (AG-1478, tyrphostin 25) or of PLC (L-108, U-73122). Other Caco-2 cells were stably transfected with a dominant negative fragment for PLC-γ (PLCz) to inhibit PLC-γ activation. Doses of EGF that enhanced PLC activity also protected monolayers against oxidant-induced tubulin disassembly, disruption of the microtubule cytoskeleton, and barrier leakiness as assessed by radioimmunoassay, quantitative Western blots, high-resolution laser confocal microscopy, and fluorometry, respectively. Pretreatment with either type of inhibitor abolished EGF protection. Transfected cells also lost EGF protection and showed reduced PLC-γ phosphorylation and activity. We conclude that EGF protection requires PLC-γ signaling and that PLC-γ may be a useful therapeutic target.
Transport of protein in the abdominal wall during intraperitoneal therapy. I. Theoretical approach
Authors: Flessner Michael F.
Published: December 2017

Intraperitoneal therapies such as peritoneal dialysis or regional chemotherapy use large volumes of solution within the peritoneal cavity. These volumes increase intraperitoneal hydrostatic pressure (Pip), which causes flow of the solution into tissues that surround the cavity. The goal of this paper is to integrate new experimental findings in a rigorous mathematical model to predict protein transport from the cavity into tissue. The model describes non-steady-state diffusion and convection of protein through a deformable porous medium with simultaneous exchange with the microcirculation and local tissue binding. Model parameters are dependent on local tissue pressure, which varies with Pip. Solute interactions with the tissue in terms of local distribution volume (solute void space), local binding, and retardation relative to solvent flow are demonstrated to be major determinants of tissue concentration profiles and protein penetration from the peritoneal cavity. The model predicts the rate of fluid loss from the cavity to the abdominal wall in dialysis patients to be 94 ml/h, within the observed range of 60–100 ml/h. The model is fitted to published transport data of IgG, and the retardation coefficient f is estimated to be 0.3, which markedly reduces the rate of protein penetration and is far lower than previously published estimates. With the value of f = 0.3, model calculations predict that Pipof 4.4 mmHg and dialysis duration of 24 h result in several millimeters of protein penetration into the tissue.
Cl−-dependent secretory mechanisms in isolated rat bile duct epithelial units
Authors: Singh Satish K. , Mennone Albert , Gigliozzi Alessandro , Fraioli Flavia , Boyer James L.
Published: December 2017

Cholangiocytes absorb and secrete fluid, modifying primary canalicular bile. In several Cl−-secreting epithelia, Na+-K+-2Cl−cotransport is a basolateral Cl−uptake pathway facilitating apical Cl−secretion. To determine if cholangiocytes possess similar mechanisms independent of CO2/HCO[Formula: see text], we assessed Cl−-dependent secretion in rat liver isolated polarized bile duct units (IBDUs) by using videomicroscopy. Without CO2/HCO[Formula: see text], forskolin (FSK) stimulated secretion entirely dependent on Na+and Cl−and inhibited by Na+-K+-2Cl−inhibitor bumetanide. Carbonic anhydrase inhibitor ethoxyzolamide had no effect on FSK-stimulated secretion, indicating negligible endogenous CO2/HCO[Formula: see text] transport. In contrast, FSK-stimulated secretion was inhibited ∼85% by K+channel inhibitor Ba2+and blocked completely by bumetanide plus Ba2+. IBDU Na+-K+-2Cl−cotransport activity was assessed by recording intracellular pH during NH4Cl exposure. Bumetanide inhibited initial acidification rates due to NH[Formula: see text] entry in the presence and absence of CO2/HCO[Formula: see text]. In contrast, when stimulated by FSK, a 35% increase in Na+-K+-2Cl−cotransport activity occurred without CO2/HCO[Formula: see text]. These data suggest a cellular model of HCO[Formula: see text]-independent secretion in which Na+-K+-2Cl−cotransport maintains high intracellular Cl−concentration. Intracellular cAMP concentration increases activate basolateral K+conductance, raises apical Cl−permeability, and causes transcellular Cl−movement into the lumen. Polarized IBDU cholangiocytes are capable of vectorial Cl−-dependent fluid secretion independent of HCO[Formula: see text]. Bumetanide-sensitive Na+-K+-2Cl−cotransport, Cl−/HCO[Formula: see text] exchange, and Ba2+-sensitive K+channels are important components of stimulated fluid secretion in intrahepatic bile duct epithelium.
I. Functional development and plasticity of interstitial cells of Cajal networks
Authors: Ward Sean M. , Sanders Kenton M.
Published: December 2017

Interstitial cells of Cajal (ICC) are the pacemaker cells in gastrointestinal (GI) muscles. They also mediate or transduce inputs from enteric motor nerves to the smooth muscle syncytium. What is known about functional roles of ICC comes from developmental studies based on the discovery that ICC express c- kit. Functional development of ICC networks depends on signaling via the Kit receptor pathway. Immunohistochemical studies using Kit antibodies have expanded our knowledge about the ICC phenotype, the structure of ICC networks, the interactions of ICC with other cells within the tunica muscularis, and the loss of ICC in some motility disorders. Manipulating Kit signaling with reagents to block the receptor or downstream signaling pathways or by using mutant mice in which Kit or its ligand, stem cell factor, are defective has allowed novel studies of the development of these cells within the tunica muscularisand also allowed the study of specific functions of different classes of ICC in several regions of the GI tract. This article examines the role of ICC in GI motility, focusing on the functional development and maintenance of ICC networks in the GI tract and the phenotypic changes that can occur when the Kit signaling pathway is disrupted.
Nitric oxide in gastrointestinal epithelial cell carcinogenesis: linking inflammation to oncogenesis
Authors: Jaiswal Meeta , LaRusso Nicholas F. , Gores Gregory J.
Published: December 2017

Chronic inflammation of gastrointestinal tissues is a well-recognized risk factor for the development of epithelial cell-derived malignancies. Although the inflammatory mediators linking chronic inflammation to carcinogenesis are numerous, current information suggests that nitric oxide (NO) contributes to carcinogenesis during chronic inflammation. Inducible nitric oxide synthase (iNOS), expressed by both macrophages and epithelial cells during inflammation, generates the bioreactive molecule NO. In addition to causing DNA lesions, NO can directly interact with proteins by nitrosylation and nitosation reactions. The consequences of protein damage by NO appear to be procarcinogenic. For example, NO inhibits DNA repair enzymes such as human 8-oxodeoxyguanosine DNA glycosylase 1 and blocks apoptosis via nitrosylation of caspases. These cellular events permit DNA damage to accumulate, which is required for the numerous mutations necessary for development of invasive cancer. NO also promotes cancer progression by functioning as an angiogenesis factor. Strategies to inhibit NO generation during chronic inflammation or to scavenge reactive nitrogen species may prove useful in decreasing the risk of cancer development in chronic inflammatory gastrointestinal diseases.
Persistent epithelial dysfunction and bacterial translocation after resolution of intestinal inflammation
Authors: Asfaha Samuel , MacNaughton Wallace K. , Appleyard Caroline B. , Chadee Kris , Wallace John L.
Published: December 2017

Epithelial secretion may play an important role in reducing bacterial colonization and translocation in intestine. If so, secretory dysfunction could result in increased susceptibility to infection and inflammation. We investigated whether long-term colonic secretory dysfunction occurs after a bout of colitis and if this is accompanied by an increase in bacterial colonization and translocation. Rats were studied 6 wk after induction of colitis with trinitrobenzene sulfonic acid when inflammation had completely resolved, and epithelial permeability was normal. Intestinal loops were stimulated with either Clostridium difficile toxin A or a phosphodiesterase inhibitor. In vitro, colonic tissue from previously sensitized rats was exposed to antigen (ovalbumin). Secretory responses to all three stimuli were suppressed in rats that had previously had colitis. These rats exhibited increased (16-fold) numbers of colonic aerobic bacteria and increased (>3-fold) bacterial translocation, similar to results in rats studied after resolution of enteritis. Postcolitis bacterial translocation was prevented by daily treatment with an inhibitor of inducible nitric oxide synthase. This study demonstrates that intestinal inflammation results in prolonged impairment of colonic epithelial secretion, which may contribute to increases in bacterial load and bacterial translocation. Epithelial dysfunction of this type could underlie an increased propensity for further bouts of inflammation, a hallmark of diseases such as inflammatory bowel disease.
Helicobacter-induced inflammatory bowel disease in IL-10- and T cell-deficient mice
Authors: Burich Andrew , Hershberg Robert , Waggie Kim , Zeng Weiping , Brabb Thea , Westrich Gina , Viney Joanne L. , Maggio-Price Lillian
Published: December 2017

Inflammatory bowel disease (IBD) is thought to result from a dysregulated mucosal immune response to luminal microbial antigens, with T lymphocytes mediating the colonic pathology. Infection with Helicobacter spp has been reported to cause IBD in immunodeficient mice, some of which lack T lymphocytes. To further understand the role of T cells and microbial antigens in triggering IBD, we infected interleukin (IL)-10−/−, recombinase-activating gene (Rag)1−/−, T-cell receptor (TCR)-α−/−, TCR-β−/−, and wild-type mice with Helicobacter hepaticus or Helicobacter bilis and compared the histopathological IBD phenotype. IL-10−/−mice developed severe diffuse IBD with either H. bilis or H. hepaticus, whereas Rag1−/−, TCR-α−/−, TCR-β−/−, and wild-type mice showed different susceptibilities to Helicobacter spp infection. Proinflammatory cytokine mRNA expression was increased in the colons of Helicobacter-infected IL-10−/−and TCR-α−/−mice with IBD. These results confirm and extend the role of Helicobacter as a useful tool for investigating microbial-induced IBD and show the importance, but not strict dependence, of T cells in the development of bacterial-induced IBD.
Gap junction expression and cell proliferation in differentiating cultures of Cx43 KO mouse hepatocytes
Authors: Kojima Takashi , Fort Alfredo , Tao Mingyuan , Yamamoto Masao , Spray David C.
Published: December 2017

Primary cultures of adult mouse hepatocytes are shown here to reexpress differentiated hepatocyte features following treatment with 2% DMSO and 10−7M glucagon. To examine the roles of gap junctional communication during hepatocyte growth and differentiation, we have compared treated and untreated hepatocytes from connexin (Cx)32-deficient [Cx32 knockout (KO)] and wild-type mice. In untreated cultures, DNA replication of Cx32 KO hepatocytes was markedly higher than of wild types. Although Cx26 mRNA levels remained high at all time points in wild-type and Cx32 KO hepatocytes, Cx32 mRNA and protein in wild-type hepatocytes underwent a marked decline, which recovered in 10-day treated cultures. Increased levels of Cx26 protein and junctional conductance were observed in Cx32 KO hepatocytes at 96 h in culture, a time when cell growth rate was high. Treatment with DMSO/glucagon highly reinduced Cx26 expression in Cx32 KO hepatocytes, and such treatment reinduced expression of both Cx32 and Cx26 expression in wild types. Dye transfer was not observed following Lucifer yellow injection into DMSO/glucagon-treated Cx32 KO hepatocytes, whereas the spread was extensive in wild types. Nevertheless, high junctional conductance values were observed in treated cells from both genotypes. These studies provide a method by which the differentiated phenotype can be obtained in cultured mouse hepatocytes and provide in vitro evidence that expression of gap junctions formed of Cx32 are involved in the regulation of growth of mouse hepatocytes.
Polarized expression and function of P2Y ATP receptors in rat bile duct epithelia
Authors: Dranoff Jonathan A. , Masyuk Anatoly I. , Kruglov Emma A. , LaRusso Nicholas F. , Nathanson Michael H.
Published: December 2017

Extracellular nucleotides may be important regulators of bile ductular secretion, because cholangiocytes express P2Y ATP receptors and nucleotides are found in bile. However, the expression, distribution, and function of specific P2Y receptor subtypes in cholangiocytes are unknown. Thus our aim was to determine the subtypes, distribution, and role in secretion of P2Y receptors expressed by cholangiocytes. The molecular subtypes of P2Y receptors were determined by RT-PCR. Functional studies measuring cytosolic Ca2+(Ca[Formula: see text]) signals and bile ductular pH were performed in isolated, microperfused intrahepatic bile duct units (IBDUs). PCR products corresponding to P2Y1, P2Y2, P2Y4, P2Y6, and P2X4receptor subtypes were identified. Luminal perfusion of ATP into IBDUs induced increases in Ca[Formula: see text] that were inhibited by apyrase and suramin. Luminal ATP, ADP, 2-methylthioadenosine 5′-triphosphate, UTP, and UDP each increased Ca[Formula: see text]. Basolateral addition of adenosine 5′- O-(3-thiotriphosphate) (ATP-γ-S), but not ATP, to the perifusing bath increased Ca[Formula: see text]. IBDU perfusion with ATP-γ-S induced net bile ductular alkalization. Cholangiocytes express multiple P2Y receptor subtypes that are expressed at the apical plasma membrane domain. P2Y receptors are also expressed on the basolateral domain, but their activation is attenuated by nucleotide hydrolysis. Activation of ductular P2Y receptors induces net ductular alkalization, suggesting that nucleotide signaling may be an important regulator of bile secretion by the liver.
I. Molecular mechanism for polyunsaturated fatty acid regulation of gene transcription
Authors: Clarke Steven D.
Published: December 2017

This review addresses the hypothesis that polyunsaturated fatty acids (PUFA), particularly those of the n-3 family, play pivotal roles as “fuel partitioners” in that they direct fatty acids away from triglyceride storage and toward oxidation and they enhance glucose flux to glycogen. In doing this, PUFA may reduce the risk of enhanced cellular apoptosis associated with excessive cellular lipid accumulation. PUFA exert their beneficial effects by upregulating the expression of genes encoding proteins involved in fatty acid oxidation while simultaneously downregulating genes encoding proteins of lipid synthesis. PUFA govern oxidative gene expression by activating the transcription factor peroxisome proliferator-activated receptor-α. PUFA suppress lipogenic gene expression by reducing the nuclear abundance and DNA binding affinity of transcription factors responsible for imparting insulin and carbohydrate control to lipogenic and glycolytic genes. In particular, PUFA suppress the nuclear abundance and expression of sterol regulatory element binding protein-1 and reduce the DNA binding activities of nuclear factor Y, stimulatory protein 1, and possibly hepatic nuclear factor-4. Collectively, the studies discussed suggest that the fuel “repartitioning” and gene expression actions of PUFA should be considered among the criteria used in defining the dietary needs of n-6 and n-3 fatty acids and in establishing the dietary ratio of n-6 to n-3 fatty acids needed for optimum health benefit.
Intestinal serotonin acts as paracrine substance to mediate pancreatic secretion stimulated by luminal factors
Authors: Li Y. , Wu X. Y. , Zhu J. X. , Owyang C.
Published: December 2017

We recently demonstrated that luminal factors such as osmolality, disaccharides, and mechanical stimulation evoke pancreatic secretion by activating 5-hydroxytryptamine subtype 3 (serotonin-3, 5-HT3) receptors on mucosal vagal afferent fibers in the intestine. We hypothesized that 5-HT released by luminal stimuli acts as a paracrine substance, activating the mucosal vagal afferent fibers to stimulate pancreatic secretion. In the in vivo rat model, luminal perfusion of maltose or hypertonic NaCl increased 5-HT level threefold in intestinal effluent perfusates. Similar levels were observed after intraluminal 10−5M 5-HT perfusion. These treatments did not affect 5-HT blood levels. In a separate study, intraduodenal, but not intraileal, 5-HT application induced a dose-dependent increase in pancreatic protein secretion, which was not blocked by the CCK-A antagonist CR-1409. Acute vagotomy, methscopolamine, or perivagal or intestinal mucosal application of capsaicin abolished 5-HT-induced pancreatic secretion. In conscious rats, luminal 10−5M 5-HT administration produced a 90% increase in pancreatic protein output, which was markedly inhibited by the 5-HT3antagonist ondansetron. In conclusion, luminal stimuli induce 5-HT release, which in turn activates 5-HT3receptors on mucosal vagal afferent terminals. In this manner, 5-HT acts as a paracrine substance to stimulate pancreatic secretion via a vagal cholinergic pathway.
Effects of alosetron on spontaneous migrating motor complexes in murine small and large bowel in vitro
Authors: Bush Toby G. , Spencer Nick J. , Watters Niamh , Sanders Kenton M. , Smith Terence K.
Published: December 2017

Alosetron (Lotronex) is a serotonin subtype 3 (5-HT3) receptor antagonist that alleviates symptoms of irritable bowel syndrome (IBS) in female patients. Alosetron may act centrally, involve the alteration of ascending pain sensation, or modulate peristaltic, secretory, or sensory function. To investigate further the mechanisms underlying its action and gender selectivity we recorded the effect of increasing concentrations of alosetron or ondansetron on spontaneous migrating motor complexes (MMCs) from isolated terminal ileum or colon from C57BL/6 mice. Both antagonists inhibited MMC frequency before affects on duration or amplitude. The threshold of inhibition for alosetron was 100-fold less in small intestine from females (20 nM) than from males. The opposite effect of gender was observed with ondansetron in the colon. All MMCs were abolished by either drug at 10 μM. Our results demonstrate that alosetron selectively inhibits MMC frequency in isolated preparations of murine bowel. Because contractile events in the ileum correlate with symptoms of IBS in humans, the gender selectivity of alosetron may be caused by a direct action within the small intestine.
Central neural mechanisms mediating human visceral hypersensitivity
Authors: Sarkar Sanchoy , Hobson Anthony R. , Furlong Paul L. , Woolf Clifford J. , Thompson David G. , Aziz Qasim
Published: December 2017

Although visceral hypersensitivity is thought to be important in generating symptoms in functional gastrointestinal disorders, the neural mechanisms involved are poorly understood. We recently showed that central sensitization (hyperexcitability of spinal cord sensory neurones) may play an important role. In this study, we demonstrate that after a 30-min infusion of 0.15 M HCl acid into the healthy human distal esophagus, we see a reduction in the pain threshold to electrical stimulation of the non-acid-exposed proximal esophagus (9.6 ± 2.4 mA) and a concurrent reduction in the latency of the N1 and P2 components of the esophageal evoked potentials (EEP) from this region (10.4 ± 2.3 and 15.8 ± 5.3 ms, respectively). This reduced EEP latency indicates a central increase in afferent pathway velocity and therefore suggests that hyperexcitability within the central visceral pain pathway contributes to the hypersensitivity within the proximal, non-acid-exposed esophagus (secondary hyperalgesia/allodynia). These findings provide the first electrophysiological evidence that central sensitization contributes to human visceral hypersensitivity.
Space-time pressure structure of pharyngo-esophageal segment during swallowing
Authors: Williams Rohan B. , Pal Anupam , Brasseur James G. , Cook Ian J.
Published: December 2017

We applied high-resolution manometry with spatiotemporal data interpolation and simultaneous videofluoroscopy to normal pharyngeal swallows to correlate specific features in the space-time intraluminal pressure structure with physiological events and normal deglutitive transsphincteric bolus flow to define normal biomechanical properties of the pharyngo-esophageal (PE) segment. Pressures were recorded by microperfused catheter, and the two-dimensional space-time data sets were plotted as isocontours. On these were superimposed bolus trajectories, anatomic segment movements, and hyo-laryngeal trajectories from concurrent videofluoroscopy. Correlation of the highly reproducible space-time-pressure structure with radiographic images confirmed that primary deglutitive PE segment functions (pressure profile, laryngeal elevation, axial sphincter motion, timing of relaxation, contraction) are accurately discernible from single isocontour pressure visualization. Pressure during bolus flow was highly dependent on axial location within PE segment and time instant. The intrabolus pressure domain, corresponding to the space-time region between bolus head and tail trajectories, demonstrated significant bolus volume dependence. High-resolution manometry accurately, comprehensively, and highly reproducibly depicts the PE segment space-time-pressure structure and specific physiological events related to upper esophageal sphincter opening and transsphincteric flow during normal swallowing. Intrabolus pressure variations are highly dependent on position within the PE segment and time.
Akt protects mouse hepatocytes from TNF-α- and Fas-mediated apoptosis through NK-κB activation
Authors: Hatano Etsuro , Brenner David A.
Published: December 2017

To determine the role of phosphatidylinositol 3-kinase (PI3K)/Akt and nuclear factor-κB (NF-κB) in protecting hepatocytes from tumor necrosis factor-α (TNF-α)- and Fas-mediated apoptosis, we pretreated primary cultures of mouse hepatocytes with pharmacological and adenovirus-mediated inhibitors of the PI3K/Akt and NF-κB pathways followed by treatment with TNF-α or Jo2, an anti-Fas antibody. Jo2 and, to a lesser extent, TNF-α phosphorylate Akt. The PI3K inhibitor LY-294002 blocks TNF-α- and Fas-mediated Akt phosphorylation. LY-294002 pretreatment reduces NF-κB binding activity and transcriptional activity and NF-κB-responsive gene expression by TNF-α or Jo2. LY-294002 promotes apoptosis after TNF-α or Jo2. The expression of dominant-negative Akt blocks NF-κB activation and sensitizes hepatocytes to TNF-α- and Fas-mediated apoptosis. The expression of constitutively active Akt rescues LY-294002-pretreated cells from TNF-α- and Fas-mediated apoptosis. Active Akt induces NF-κB transcriptional activity but not NF-κB binding activity or IκB degradation. Furthermore, LY-294002 pretreatment blocks TNF-α- and Jo2-induced Bcl-xL levels in hepatocytes, with no effect on the phosphorylation levels of Bad. Bcl-xL overexpression protects hepatocytes from Fas- but not TNF-α-induced apoptosis after sensitization by actinomycin D or the IκB superrepressor. Together, the PI3K/Akt pathway has a protective role in Fas-mediated apoptosis, which requires NF-κB activation, partially through the subsequent induction of Bcl-xL.
Loss of the NHE2 Na+/H+exchanger has no apparent effect on diarrheal state of NHE3-deficient mice
Authors: Ledoussal Clara , Woo Alison L. , Miller Marian L. , Shull Gary E.
Published: December 2017

The expression of NHE2 and NHE3 on intestinal-brush border membranes suggests that both Na+/H+exchangers serve absorptive functions. Studies with knockout mice showed that the loss of NHE3, but not NHE2, causes diarrhea, demonstrating that NHE3 is the major absorptive exchanger and indicating that any remaining absorptive capacity contributed by NHE2 is not sufficient to compensate fully for the loss of NHE3. To test the hypothesis that NHE2 provides partial compensation for the diarrheal state of NHE3-deficient mice, we crossed doubly heterozygous mice carrying null mutations in the Nhe2and Nhe3 genes and analyzed the phenotypes of their offspring. The additional loss of NHE2 in NHE3-deficient mice caused no apparent reduction in viability, no further impairment of systemic acid-base status or increase in aldosterone levels, and no apparent worsening of the diarrheal state. These in vivo phenotypic correlates of the absorptive defect suggest that the NaCl, HCO[Formula: see text], and fluid absorption that is dependent on apical Na+/H+exchange is due overwhelmingly to the activity of NHE3, with little contribution from NHE2.
TNF-α and interleukin 1 activate gastrin gene expression via MAPK- and PKC-dependent mechanisms
Authors: Suzuki T. , Grand E. , Bowman C. , Merchant J. L. , Todisco A. , Wang L. , Del Valle J.
Published: December 2017

Helicobacter pyloriand proinflammatory cytokines have a direct stimulatory effect on gastrin release from isolated G cells, but little is known about the mechanism by which these factors regulate gastrin gene expression. We explored whether tumor necrosis factor (TNF)-α and interleukin (IL)-1 directly regulate gastrin gene expression and, if so, by what mechanism. TNF-α and IL-1 significantly increased gastrin mRNA in canine G cells to 181 ± 18% and 187 ± 28% of control, respectively, after 24 h of treatment. TNF-α and IL-1 stimulated gastrin promoter activity to a maximal level of 285 ± 12% and 415 ± 26% of control. PD-98059 (a mitogen-activated protein kinase kinase inhibitor), SB-202190 (a p38 kinase inhibitor), and GF-109203 (a protein kinase C inhibitor) inhibited the stimulatory action of both cytokines on the gastrin promoter. In conclusion, both cytokines can directly regulate gastrin gene expression via a mitogen-activated protein kinase- and protein kinase C-dependent mechanism. These data suggest that TNF-α and IL-1 may play a direct role in Helicobacter pylori-induced hypergastrinemia.
Regional differences in the response of feline esophageal smooth muscle to stretch and cholinergic stimulation
Authors: Muinuddin Ahmad , Xue Shuwen , Diamant Nicholas E.
Published: December 2017

There are no objective differences in neural elements that explain regional differences in neural influences along the smooth muscle (SM) esophageal body (EB). Regional differences in muscle properties are present in the lower esophageal sphincter (LES). This study examines whether regional differences in SM properties exist along the EB and are reflected in length-tension relationships and responses to cholinergic excitation. Circular SM strips from feline EB at 1 cm (EB1) and 3 cm (EB3) above LES and from clasp and sling muscle bundles of LES were assessed in normal and calcium-free solutions with and without bethanechol stimulation. Neural inhibition was assessed by electrical field stimulation (EFS). EB3 developed significantly higher tension in response to stretch and to bethanechol than did EB1. The relaxation response to EFS in bethanechol-precontracted strips was less in EB3 than in EB1. In LES, clasp developed higher resting tension than sling but less active tension in response to bethanechol. EFS-induced relaxations of sling and clasp tissues precontracted by bethanechol were not different. In calcium-free solution, length-tension differences between EB3 and EB1 persisted, but those of LES clasp and sling were abolished. Therefore, regional myogenic differences exist in feline EB circular SM as well as in LES and may contribute to the nature of esophageal contraction.
Regulation of human jejunal transmucosal resistance and MLC phosphorylation by Na+-glucose cotransport
Authors: Berglund Jessica J. , Riegler Martin , Zolotarevsky Yevgeny , Wenzl Etienne , Turner Jerrold R.
Published: December 2017

Na+- nutrient cotransport-dependent regulation of paracellular permeability has been demonstrated in rodent intestine and human intestinal epithelial cell lines. In cell lines this regulation is associated with phosphorylation of myosin II regulatory light chain (MLC). However, the subcellular localization of phosphorylated MLC during this regulation has not been studied and regulation of paracellular permeability and MLC phosphorylation has not been studied in isolated human intestine. To evaluate these events in human jejunum, isolated mucosa was mounted in Ussing chambers, characterized electrophysiologically, and then immunostained using anti-phosphorylated MLC and anti-total MLC antisera. MLC phosphorylation was assessed by calculating the ratio of anti-phosphorylated MLC signal to anti-total MLC signal within defined regions. Transmucosal resistance of mucosae without active Na+-glucose cotransport was 37 ± 3% greater than that of mucosae with active Na+-glucose cotransport within 15 min. Quantitative double-label immunofluorescence showed that the phosphorylated MLC-to-total MLC ratio increased by 45 ± 4% within the perijunctional actomyosin ring when Na+-glucose cotransport was active. Thus regulation of transmucosal resistance by Na+-glucose cotransport is accompanied by increased MLC phosphorylation within the perijunctional actomyosin ring. These data support the proposed critical role of the perijunctional cytoskeleton in physiological regulation of human small intestinal paracellular permeability.
Chylomicron components mediate intestinal lipid-induced inhibition of gastric motor function
Authors: Glatzle Jörg , Kalogeris Theodore J. , Zittel Tilman T. , Guerrini Stephania , Tso Patrick , Raybould Helen E.
Published: December 2017

Lipid, particularly long-chain triglyceride, initiates feedback regulation of gastrointestinal function. To determine whether the site of action of lipid is pre- or postabsorptive, we investigated the ability of mesenteric lipid-fed lymph to inhibit gastric motor function. Lymph was collected from awake lymph-fistula rats during intestinal infusion with either a glucose-saline maintenance solution or lipid. Intra-arterial injection of lymph collected during intestinal lipid infusion significantly inhibited gastric motility in anesthetized recipient rats compared with injection of equivalent amounts of triglyceride or lymph collected during intestinal infusion of maintenance solution. Lymph collected from rats during lipid infusion with Pluronic L-81 [an inhibitor of chylomicron formation and apolipoprotein (apo) A-IV secretion] compared with lymph injection from donor animals treated with Pluronic L-63 (a noninhibitory control for Pluronic L-81) was significantly less potent. Injection of purified recombinant apo A-IV significantly inhibited gastric motility. Products of lipid digestion and absorption, other than fatty acids or triglyceride, released by the intestine during lipid digestion likely serve as signals to initiate intestinal feedback regulation of gastrointestinal function. Most likely, apo A-IV is one of the signals involved.