AJP - Gastrointestinal and Liver Physiology

Sundaram, Uma; Wisel, Sheik; Coon, Steven

doi: 10.1152/ajpgi.00503.2005pmid: 17290012

Neutral Na-amino acid cotransport by system ATB 0 e.g., Na-alanine cotransport (NAcT) is an important means of assimilation of amino acids in the intestine. NAcT is inhibited during chronic intestinal inflammation by an alteration in the affinity for the amino acid. How glucocorticoids, a standard of treatment for diseases characterized by chronic intestinal inflammation, may affect NAcT during chronic enteritis is not known. Thus we first demonstrated that methylprednisolone (MP) stimulated NAcT in the normal intestine. The mechanism of stimulation was secondary to an increase in cotransporter numbers without an alteration in the affinity for the amino acid. Treatment with MP reversed the reduction in NAcT in villus cells from the chronically inflamed intestine. MP also alleviated the decrease in Na-K-ATPase activity in villus cells during chronic enteritis. However, MP treatment reversed the NAcT inhibition in villus cell brush border membrane vesicles from the inflamed intestine, which suggested an effect of MP at the level of the cotransporter itself. Kinetic studies demonstrated that the reversal of NAcT inhibition by MP was secondary to restoration in the affinity for the amino acid without a change in the V max . Unaltered steady-state mRNA and immunoreactive protein levels of NAcT also indicated that the number of cotransporters was unchanged after MP treatment in the chronically inflamed intestine. These results indicated that MP reversed NAcT inhibition in the chronically inflamed intestine by restoring the affinity of the transporter for the amino acid while it stimulated NAcT in the normal intestine by increasing the cotransporter numbers. Therefore, MP differentially regulates NAcT in the normal and chronically inflamed intestine. glucocorticoids; inflammation; amino acid transport; sodium transport; intestinal absorption Address for reprint requests and other correspondence: U. Sundaram, Section of Digestive Diseases, Dept. of Medicine, West Virginia Univ. School of Medicine, One Medical Center Drive, Morgantown, WV 26506 (e-mail: [email protected] )

Neish, Andrew S.

doi: 10.1152/ajpgi.00274.2006pmid: 17082224

Flagellin is bacterial protein that serves as a danger signal across a wide variety of eukaryotes and is a potent inducer of inflammatory effector responses in the mammalian gut. Recent findings utilizing purified flagellin and flagellate/aflagellate bacteria in in vitro and in vivo systems have revealed the important roles played by flagellin in the initial encounter between mucosa and flagellate bacteria, specifically in the modulation of apoptotic responses. Toll-like receptor 5; inflammation; apoptosis; bacterial pathogenesis; colitis; epithelia Address for reprint requests and other correspondence: A. S. Neish, Dept. of Pathology, Emory Univ. School of Medicine, 105-F Whitehead Memorial Research Bldg., 615 Michaels St., Atlanta, GA 30322 (e-mail: [email protected] )

Reid, Kaye M.; Tsung, Allan; Kaizu, Takahashi; Jeyabalan, Geetha; Ikeda, Atsushi; Shao, Lifang; Wu, Guoyao; Murase, Noriko; Geller, David A.

doi: 10.1152/ajpgi.00227.2006pmid: 17023552

Liver ischemia-reperfusion (I/R) injury is associated with profound arginine depletion due to arginase release from injured hepatocytes. The purpose of this study was to determine whether arginase inhibition with N ω -hydroxy-nor- L -arginine (nor-NOHA) would increase circulating arginine levels and decrease hepatic damage during liver I/R injury. The effects of nor-NOHA were initially tested in normal animals to determine in vivo toxicity. In the second series of experiments, orthotopic syngeneic liver transplantation (OLT) was performed after 18 h of cold ischemia time in Lewis rats. Animals were given nor-NOHA (100 mg/kg) or saline before and after graft reperfusion. In normal animals treated with nor-NOHA, there were no histopathological changes to organs, liver enzymes, serum creatinine, or body weight. In the OLT model, animals treated with saline exhibited markedly elevated serum transaminases and circulating arginase protein levels. Nor-NOHA administration blunted the increase in serum arginase activity by 80% and preserved serum arginine levels at 3 h after OLT. Nor-NOHA treatment reduced post-OLT serum liver enzyme release by 50%. Liver histology (degree of necrosis) in nor-NOHA-treated animals was markedly improved compared with the saline-treated group. Furthermore, use of the arginase inhibitor nor-NOHA did not influence polyamine synthesis owing to the decrease in ornithine levels. Arginase blockade represents a potentially novel strategy to combat hepatic I/R injury associated with liver transplantation. liver transplantation; arginine; nitric oxide; preservation injury Address for reprint requests and other correspondence: D. A. Geller, Starzl Transplantation Institute, 3459 Fifth Ave., Univ. of Pittsburgh, Pittsburgh, PA 15213 (e-mail: [email protected] )

Guo, Lili; Sans, Maria Dolors; Gurda, Grzegorz T.; Lee, Sae-Hong; Ernst, Stephen A.; Williams, John A.

doi: 10.1152/ajpgi.00433.2006pmid: 17095753

Endogenous CCK release induced by a synthetic trypsin inhibitor, camostat, stimulates pancreatic growth; however, the mechanisms mediating this growth are not well established. Early response genes often couple short-term signals with long-term responses. To study their participation in the pancreatic growth response, mice were fasted for 18 h and refed chow containing 0.1% camostat for 1–24 h. Expression of 18 early response genes were evaluated by quantitative PCR; mRNA for 17 of the 18 increased at 1, 2, 4, or 8 h. Protein expression for c-jun, c-fos, ATF-3, Egr-1, and JunB peaked at 2 h. Nuclear localization was confirmed by immunohistochemistry of c-fos, c-jun, and Egr-1. Refeeding regular chow induced only a small increase of c-jun and none in c-fos expression. JNKs and ERKs were activated 1 h after camostat feeding as was the phosphorylation of c-jun and ATF-2. AP-1 DNA binding evaluated by EMSA showed a significant increase 1–2 h after camostat feeding with participation of c-jun, c-fos, ATF-2, ATF-3, and JunB shown by supershift. The CCK antagonist IQM-95,333 blocked camostat feeding-induced c-jun and c-fos expression by 67 and 84%, respectively, and AP-1 DNA binding was also inhibited. In CCK-deficient mice, the maximal response of c-jun induction and AP-1 DNA binding were reduced by 64 and 70%, respectively. These results indicate that camostat feeding induces a spectrum of early response gene expression and AP-1 DNA binding and that these effects are mainly CCK dependent. CCK; camostat; c-jun; c-fos; AP-1 Address for reprint requests and other correspondence: J. A. Williams, Dept. of Molecular and Integrative Physiology, Univ. of Michigan Medical School, 7744 Medical Sciences II, 1301 E. Catherine St., Ann Arbor, MI 48109-0622 (e-mail: [email protected] )

Spencer, Nick J.; Bayguinov, Peter; Hennig, Grant W.; Park, Kyu Joo; Lee, Hyun-Tai; Sanders, Kenton M.; Smith, Terence K.

doi: 10.1152/ajpgi.00352.2006pmid: 17023548

In mammals that develop rectal aganglionosis, the aganglionic segment still exhibits spontaneous phasic contractions that contribute to dysmotility and pseudoobstruction in this region. However, almost nothing is known about the mechanisms that generate these myogenic contractions or the effects of aganglionosis on the generation of Ca 2+ waves that underlie contractions of the longitudinal muscle (LM) and circular muscle (CM). In a mouse model of Hirschsprung's disease endothelin type B receptor-deficient ( Ednrb s–l / Ednrb s–l ) mice, the Ca 2+ indicator fluo-4 was used to simultaneously monitor the temporal activation and spread of intercellular Ca 2+ waves in the LM and CM during spontaneous colonic motor activities. During the intervals between colonic migrating motor complexes (CMMCs) in control mice, Ca 2+ waves discharged asynchronously between the LM and CM. However, in these same mice, during CMMCs, a burst of discreet Ca 2+ waves fired simultaneously in both muscle layers, where the propagation velocity of Ca 2+ waves significantly increased, as did the rate of initiation and number of collisions between Ca 2+ waves. Hexamethonium (300 µM) or atropine (1 µM) prevented synchronized firing of Ca 2+ waves. In the aganglionic distal colon of Ednrb s–l / Ednrb s–l mice, not only were CMMCs absent, but Ca 2+ waves between the two muscle layers fired asynchronously, despite increased propagation velocity. The generation of CMMCs in control mice involves synchronized firing of enteric motor nerves to both the LM and CM, explaining the synchronized firing of discreet Ca 2+ waves between the two muscle layers. Aganglionosis results in a sporadic and sustained asynchrony in Ca 2+ wave firing between the LM and CM and an absence of CMMCs. colon; myenteric; peristalsis; colonic migrating motor complexes Address for reprint requests and other correspondence: N. Spencer, Dept. of Physiology and Cell Biology, Univ. of Nevada School of Medicine, Reno, NV 89557 (e-mail: [email protected] )

Boivin, Michel A.; Ye, Dongmei; Kennedy, John C.; Al-Sadi, Rana; Shepela, Chris; Ma, Thomas Y.

doi: 10.1152/ajpgi.00252.2006pmid: 17068119

A defective intestinal epithelial tight junction (TJ) barrier has been proposed as an important pathogenic factor contributing to the intestinal inflammation of Crohn's disease. Glucocorticoids are first-line therapeutic agents for the treatment of moderate to severe Crohn's disease. Glucocorticoid treatment has been shown to induce retightening of the intestinal TJ barrier defect in Crohn's disease patients. However, the mechanisms that mediate the glucocorticoid therapeutic action on intestinal TJ barrier function remain unknown. The aim of this study was to elucidate the mechanism of glucocorticoid modulation of the intestinal epithelial TJ barrier using an in vitro model system. Filter-grown Caco-2 intestinal epithelial cells were used as an in vitro model to examine the effects of glucocorticoids on basal intestinal epithelial TJ barrier function and on TNF-α-induced disruption of the TJ barrier. Glucocorticoids (prednisolone and dexamethasone) did not have a significant effect on baseline Caco-2 TJ barrier function but prevented the TNF-α-induced increase in Caco-2 TJ permeability. The glucocorticoid protective effect against the TNF-α-induced increase in Caco-2 TJ permeability required activation of the glucocorticoid receptor (GR) complex. The activation of the GR complex resulted in GR complex binding to the glucocorticoid response element (GRE) site on DNA and activation of a GR-responsive promoter. Glucocorticoids inhibited the TNF-α-induced increase in myosin light chain kinase (MLCK) protein expression, a key process mediating the TNF-α increase in intestinal TJ permeability. The glucocorticoid inhibition of the TNF-α-induced increase in MLCK protein expression was due to the binding of the GR complex to a GRE binding site on the MLCK promoter region suppressing the TNF-α-induced activation. Glucocorticoids inhibit the TNF-α-induced increase in Caco-2 TJ permeability. The prednisolone protective action was mediated by binding of activated GR complex to the GRE site on the MLCK promoter, suppressing the TNF-α-induced increase in MLCK gene activity, protein expression, and subsequent opening of the intestinal TJ barrier. prednisolone; inflammation; tumor necrosis factor-α; glucocorticoid receptor; glucocorticoid response element; Crohn's disease Address for reprint requests and other correspondence: T. Y. Ma, MSC 10 5550, Univ. of New Mexico, Albuquerque, NM 87131 (e-mail: [email protected] )

Yan, Yutao; Dalmasso, Guillaume; Sitaraman, Shanthi; Merlin, Didier

doi: 10.1152/ajpgi.00385.2006pmid: 17023546

Growing evidence that epithelial CD98 plays an important role in intestinal inflammation focused our interest to investigate the transcriptional regulation of CD98. Our mouse-based in vivo and in vitro experiments revealed that epithelial colonic CD98 mRNA expression was transcriptionally increased in intestinal inflammation. We then isolated and characterized a 5'-flanking fragment containing the promoter region required for CD98 gene transcription. Primer extension and rapid amplification of 5'-cDNA ends were used to map a transcriptional initiation site 129 bp upstream from the translational start codon (ATG). Direct sequencing of the 5'-flanking region revealed the presence of four GC-rich stimulating protein (Sp)1 binding domains, one NF-κB binding domain, and no TATA box. Binding of Sp1 Sp1(–874), SP1(–386), Sp1(–187), and Sp1(–177) and NF-κB NF-κB(–213) to the promoter was confirmed by EMSA and supershift assays. Furthermore, chromatin immunoprecipitation experiments showed the in vivo DNA-Sp1 and DNA-NF-κB interactions under basal and IFN-γ-stimulated conditions. Reporter genes driven by serially truncated and site-mutated CD98 promoters were used to examine basal and IFN-γ-responsive transcription in transiently transfected Caco2-BBE cells. Our results revealed that Sp1(–187), Sp1(–177), and the NF-κB binding site were essential for basal and IFN-γ-stimulated CD98 promoter activities, whereas Sp1(–874) and Sp1(–386) were not. The results from additional site-mutated CD98 promoters suggested that Sp1(–187), Sp1(–177), and the NF-κB site may cooperate in mediating basal and IFN-γ-stimulated CD98 promoter activities. Finally, we demonstrated that a reduction of Sp1 or NF-κB expression reduced CD98 protein expression in unstimulated and IFN-γ-stimulated Caco2-BBE cells. Collectively, these findings indicate that the Sp1 and NF-κB transcription factors are likely to play a significant role in IFN-γ-mediated transcriptional regulation of CD98 in the intestinal epithelium, providing new insights into the regulation of CD98 expression in intestinal inflammation. Caco2-BBE; mice; promoter; stimulating protein 1; nuclear factor-κB Address for reprint requests and other correspondence: D. Merlin, Div. of Digestive Diseases, Dept. of Medicine, Emory Univ., 615 Michael St., Atlanta, GA 30322 (e-mail: [email protected] )

Moeser, Adam J.; Nighot, Prashant K.; Engelke, Kory J.; Ueno, Ryuji; Blikslager, Anthony T.

doi: 10.1152/ajpgi.00183.2006pmid: 17053162

Previous studies utilizing an ex vivo porcine model of intestinal ischemic injury demonstrated that prostaglandin (PG)E 2 stimulates repair of mucosal barrier function via a mechanism involving Cl – secretion and reductions in paracellular permeability. Further experiments revealed that the signaling mechanism for PGE 2 -induced mucosal recovery was mediated via type-2 Cl – channels (ClC-2). Therefore, the objective of the present study was to directly investigate the role of ClC-2 in mucosal repair by evaluating mucosal recovery in ischemia-injured intestinal mucosa treated with the selective ClC-2 agonist lubiprostone. Ischemia-injured porcine ileal mucosa was mounted in Ussing chambers, and short-circuit current ( I sc ) and transepithelial electrical resistance (TER) were measured in response to lubiprostone. Application of 0.01–1 µM lubiprostone to ischemia-injured mucosa induced concentration-dependent increases in TER, with 1 µM lubiprostone stimulating a twofold increase in TER (ΔTER = 26 Ω·cm 2 ; P < 0.01). However, lubiprostone (1 µM) stimulated higher elevations in TER despite lower I sc responses compared with the nonselective secretory agonist PGE 2 (1 µM). Furthermore, lubiprostone significantly ( P < 0.05) reduced mucosal-to-serosal fluxes of 3 H-labeled mannitol to levels comparable to those of normal control tissues and restored occludin localization to tight junctions. Activation of ClC-2 with the selective agonist lubiprostone stimulated elevations in TER and reductions in mannitol flux in ischemia-injured intestine associated with structural changes in tight junctions. Prostones such as lubiprostone may provide a selective and novel pharmacological mechanism of accelerating recovery of acutely injured intestine compared with the nonselective action of prostaglandins such as PGE 2 . ischemia; type 2 chloride channels; repair Address for reprint requests and other correspondence: A. T. Blikslager, Coll. of Veterinary Medicine, North Carolina State Univ., 4700 Hillsborough St., Raleigh, NC 27606 (e-mail: [email protected] )

Przemeck, S. M. C.; Duckworth, C. A.; Pritchard, D. M.

doi: 10.1152/ajpgi.00391.2006pmid: 17068116

Unlike the small intestine and colon where γ-radiation-induced apoptosis has previously been well characterized, the response of murine gastric epithelium to γ-radiation has not been investigated in detail. Apoptosis was therefore assessed on a cell positional basis in gastric antral and corpus glands from adult male mice following γ-radiation. Maximum numbers of apoptotic cells were observed in both antrum and corpus at 48 h and at radiation doses greater than 12 Gy. However, the number of apoptotic cells observed in the gastric epithelium was much lower than observed in the small intestine or colon after similar doses of radiation. Hematoxylin and eosin, caspase 3 immunohistochemistry, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling detected similar numbers and cell positional distributions of apoptotic cells, hence hematoxylin and eosin was used for subsequent studies. The highest numbers of apoptotic cells were observed at cell positions 5–6 in the antrum and cell positions 15–18 in the corpus. These distributions coincided with the distributions of PCNA-labeled proliferating cells, but not with the distributions of H + -K + -ATPase-labeled parietal cells or TFF2-labeled mucous neck cells. Decreased numbers of apoptotic gastric epithelial cells were observed in p53 -null, bak -null, and bax -null mice compared with wild-type counterparts 6 and 48 h after 12 Gy γ-radiation. Significantly increased numbers of apoptotic gastric epithelial cells were observed in bcl-2 -null mice compared with wild-type littermates 6 h after 12 Gy γ-radiation. Radiation therefore induces apoptosis in the proliferative zone of mouse gastric epithelium. This response is regulated by the expression of p53, bak, bax, and bcl-2. apoptosis; stomach; p53; bcl-2 family; γ-radiation Address for reprint requests and other correspondence: D. M. Pritchard, 5 th Floor UCD Bldg., Univ. of Liverpool, Daulby St., Liverpool L69 3GA, UK (e-mail: [email protected] )

Reed, David E.; Vanner, Stephen

doi: 10.1152/ajpgi.00364.2006pmid: 17008553

This study examined whether mucosal stimulation activates long secretomotor neural reflexes and, if so, how they are organized. The submucosa of in vitro full thickness guinea pig ileal preparations was exposed in the distal portion and intracellular recordings were obtained from electrophysiologically identified secretomotor neurons. Axons in the intact mucosa of the oral segment were stimulated by a large bipolar stimulating electrode. In control preparations, a single stimulus pulse evoked a fast excitatory postsynaptic potential (EPSP) in 86% of neurons located 0.7–1.0 cm anal to the stimulus site. A stimulus train evoked multiple fast EPSPs, but slow EPSPs were not observed. To examine whether mucosal stimulation specifically activated mucosal sensory nerve terminals, the mucosa/submucosa was severed from the underlying layers and repositioned. In these preparations, fast EPSPs could not be elicited in 89% of cells. Superfusion with phorbol dibutyrate enhanced excitability of sensory neurons and pressure-pulse application of serotonin to the mucosa increased the fast EPSPs evoked by mucosal stimulation, providing further evidence that sensory neurons were involved. To determine whether these reflexes projected through the myenteric plexus, this plexus was surgically lesioned between the stimulus site and the impaled neuron. No fast EPSPs were recorded in these preparations following mucosal stimulation whereas lesioning the submucosal plexus had no effect. These results demonstrate that mucosal stimulation triggers a long myenteric pathway that activates submucosal secretomotor neurons. This pathway projects in parallel with motor and vasodilator reflexes, and this common pathway may enable coordination of intestinal secretion, blood flow, and motility. secretomotor reflexes; submucosa; secretion Address for reprint requests and other correspondence: S. Vanner, Hotel Dieu Hospital, 166 Brock St., Kingston, Ontario, Canada K7L 5G2 (e-mail: [email protected] )