bluuebunny/crossref_metadata_2025 huggingface dataset:
Fibrogenesis I. New insights into hepatic stellate cell activation: the simple becomes complex
Authors: Eng Francis J. , Friedman Scott L.
Published: December 2017

Hepatic stellate cell activation is a complex process. Paradoxes and controversies include the origin(s) of hepatic stellate cells, the regulation of membrane receptor signaling and transcription, and the fate of the cells once liver injury resolves. Major themes have emerged, including the dominance of autocrine signaling and the identification of counterregulatory stimuli that oppose key features of activated cells. Advances in analytical methods including proteomics and gene array, coupled with powerful bioinformatics, promise to revolutionize how we view cellular responses. Our understanding of stellate cell activation is likely to benefit from these advances, unearthing modes of regulating cellular behavior that are not even conceivable on the basis of current paradigms.
Molecular Physiology and Pathophysiology of Tight Junctions I. Tight junction structure and function: lessons from mutant animals and proteins
Authors: Mitic Laura L. , Van Itallie Christina M. , Anderson James M.
Published: December 2017

Tight junctions form the major paracellular barrier in epithelial tissues. Barrier-sealing properties are quite variable among cell types in terms of electrical resistance, solute and water flux, and charge selectivity. A molecular explanation for this variability appears closer following identification of the transmembrane proteins occludin and members of the claudin multigene family. For example, the human phenotype of mutations in claudin-16 suggests that it creates a channel that allows magnesium to diffuse through renal tight junctions. Similarly, a mouse knockout of claudin-11 reveals its role in formation of tight junctions in myelin and between Sertoli cells in testis. The study of other claudins is expected to elucidate their contributions to creating junction structure and physiology in all epithelial tissues.
Endothelial nitric oxide synthase modulates gastric ulcer healing in rats
Authors: Ma Li , Wallace John L.
Published: December 2017

Nitric oxide has been shown to be beneficial for gastric ulcer healing. We determined the relative effects of endothelial and inducible nitric oxide synthases on gastric ulcer healing in rats. Ulcers were induced by serosal application of acetic acid. Ulcer severity, angiogenesis, and nitric oxide synthase expression were assessed 3–10 days later. The effects of inhibitors of nitric oxide synthase were also examined. Inducible nitric oxide synthase mRNA was only detected in ulcerated tissue (maximal at day 3), whereas the endothelial isoform mRNA was detected in normal tissue and increased during ulcer healing. Inducible nitric oxide synthase was expressed in inflammatory cells in the ulcer bed, whereas endothelial nitric oxide synthase was found in the vascular endothelium and in some mucosal cells in both normal and ulcerated tissues. Angiogenesis changed in parallel with endothelial nitric oxide synthase expression. N 6-(iminoethyl)-l-lysine did not affect angiogenesis or ulcer healing, while N G-nitro-l-arginine methyl ester significantly reduced both. In conclusion, endothelial nitric oxide synthase, but not the inducible isoform, plays a significant role in gastric ulcer healing.
Low glutathione peroxidase activity inGpx1knockout mice protects jejunum crypts from γ-irradiation damage
Authors: Esworthy R. Steven , Mann Jeffrey R. , Sam Mindy , Chu Fong-Fong
Published: December 2017

Gpx1 knockout (KO) mice had a higher number of regenerating crypts in the jejunum than did Gpx2-KO or wild-type mice analyzed 4 days after ≥10 Gy γ-irradiation. Without γ-irradiation, glutathione peroxidase (GPX) activity in the jejunal and ileal epithelium of Gpx1-KO mice was <10 and ∼35%, respectively, of that of the wild-type mice. Four days after exposure to 11 Gy, GPX activity in wild-type and Gpx1-KO ileum was doubled and tripled, respectively. However, jejunal GPX activity was not changed. Thus the lack of GPX activity in the jejunum is associated with better regeneration of crypt epithelium after radiation. Gpx2 gene expression was solely responsible for the increase in GPX activity in the ileum, since radiation did not alter GPX activity in Gpx2-KO mice. The intestinal Gpx2mRNA levels of Gpx1-KO and wild-type mice increased up to 14- and 7-fold after radiation, respectively. Although the Gpx1-KO jejunum had higher levels of PGE2than the wild-type jejunum after exposure to 0 or 15 Gy, these differences were not statistically significant. Thus whether GPX inhibits PG biosynthesis in vivo remains to be established. We can conclude that the Gpx2 gene compensates for the lack of Gpx1gene expression in the ileal epithelium. This may have abolished the protective effect in Gpx1-KO mice against the radiation damage in the ileum.
Gastric acid secretion in aquaporin-4 knockout mice
Authors: Wang Kasper S. , Komar Alex R. , Ma Tonghui , Filiz Ferda , McLeroy Jeff , Hoda Kaveh , Verkman A. S. , Bastidas J. Augusto
Published: December 2017

The aquaporin-4 (AQP4) water channel has been proposed to play a role in gastric acid secretion. Immunocytochemistry using anti-AQP4 antibodies showed strong AQP4 protein expression at the basolateral membrane of gastric parietal cells in wild-type (+/+) mice. AQP4 involvement in gastric acid secretion was studied using transgenic null (−/−) mice deficient in AQP4 protein. −/− Mice had grossly normal growth and appearance and showed no differences in gastric morphology by light microscopy. Gastric acid secretion was measured in anesthetized mice in which the stomach was luminally perfused (0.3 ml/min) with 0.9% NaCl containing [14C]polyethylene glycol ([14C]PEG) as a volume marker. Collected effluent was assayed for titratable acid content and [14C]PEG radioactivity. After 45-min baseline perfusion, acid secretion was stimulated by pentagastrin (200 μg · kg−1· h−1iv) for 1 h or histamine (0.23 mg/kg iv) + intraluminal carbachol (20 mg/l). Baseline gastric acid secretion (means ± SE, n = 25) was 0.06 ± 0.03 and 0.03 ± 0.02 μeq/15 min in +/+ and −/− mice, respectively. Pentagastrin-stimulated acid secretion was 0.59 ± 0.14 and 0.70 ± 0.15 μeq/15 min in +/+ and −/− mice, respectively. Histamine plus carbachol-stimulated acid secretion was 7.0 ± 1.9 and 8.0 ± 1.8 μeq/15 min in +/+ and −/− mice, respectively. In addition, AQP4 deletion did not affect gastric fluid secretion, gastric pH, or fasting serum gastrin concentrations. These results provide direct evidence against a role of AQP4 in gastric acid secretion.
Role of cGMP as a mediator of nerve-induced motor functions of the opossum esophagus
Authors: Shahin W. , Murray J. A. , Clark E. , Conklin J. L.
Published: December 2017

Stimulation of esophageal nerves produces biphasic relaxation of the lower esophageal sphincter (LES) and an off response of circular esophageal muscle. Previously, we proposed that cGMP mediates nerve-induced hyperpolarization of circular LES muscle but not LES relaxation. These experiments explore whether cGMP mediates LES relaxation or the off response. Strips of muscle from the opossum esophagus and LES were connected to force-displacement transducers, placed in tissue baths containing oxygenated Krebs solution at 37°C, and stimulated by an electrical field. 1H-[1,2,4]oxadiazolo-[4,3- a]quinoxalin-1-one (ODQ), a selective inhibitor of guanylyl cyclase, antagonized the off response, shortened its latency, and blocked the first phase of LES relaxation. ODQ also antagonized LES relaxation by exogenous nitric oxide (NO) but not relaxations by vasoactive intestinal polypeptide (VIP). Part of the nerve-induced LES relaxation and the off response appear to be mediated by the second messenger cGMP. These studies indicate that VIP-induced LES relaxation is not mediated by cGMP and therefore do not support the hypothesis that VIP produces LES relaxation by causing the generation of NO.
Control of CCK gene transcription by PACAP in STC-1 cells
Authors: Deavall Damian G. , Raychowdhury Raktima , Dockray Graham J. , Dimaline Rod
Published: December 2017

The mechanisms by which neuroendocrine stimulants regulate CCK gene transcription are unclear. We examined promoter activation by pituitary adenylate cyclase-activating polypeptide (PACAP), a known CCK secretagogue, in the enteroendocrine cell line STC-1. The promoter region from −70 to −87 bp, relative to the transcriptional start site, contains a composite calcium/cyclic AMP response element (CRE)/activator protein 1 (AP1) site that may bind CRE binding protein (CREB) and AP1. PACAP (with IBMX) stimulated expression of an 87-bp construct 3.35 ± 0.36-fold but had no effect on a −70 construct. The effect was blocked by the protein kinase A inhibitor H-89 and by a dominant-negative CREB plasmid. Mutation of the CRE/AP1 site to a canonical CRE site did not affect the response to PACAP, but mutation to a canonical AP1 site prevented it. CREB phosphorylation was increased after PACAP treatment. Electrophoretic mobility shift assay and supershift analysis revealed that CREB and not AP1 bound to the CRE/AP1 site and that PACAP increased the proportion of phosphorylated CREB that was bound. We conclude that PACAP increases CCK gene expression via a cAMP-mediated pathway involving CREB phosphorylation by protein kinase A and activation of a composite CRE/AP1 site.
Rotavirus alters paracellular permeability and energy metabolism in Caco-2 cells
Authors: Dickman Kathleen G. , Hempson Scott J. , Anderson Joseph , Lippe Scott , Zhao Liming , Burakoff Robert , Shaw Robert D.
Published: December 2017

Rotaviruses infect epithelial cells of the small intestine, but the pathophysiology of the resulting severe diarrhea is incompletely understood. Histological damage to intestinal epithelium is not a consistent feature, and in vitro studies showed that intestinal cells did not undergo rapid death and lysis during viral replication. We show that rotavirus infection of Caco-2 cells caused disruption of tight junctions and loss of transepithelial resistance (TER) in the absence of cell death. TER declined from 300 to 22 Ω · cm2between 8 and 24 h after infection and was accompanied by increased transepithelial permeability to macromolecules of 478 and 4,000 Da. Distribution of tight junction proteins claudin-1, occludin, and ZO-1 was significantly altered during infection. Claudin-1 redistribution was notably apparent at the onset of the decline in TER. Infection was associated with increased production of lactate, decreased mitochondrial oxygen consumption, and reduced cellular ATP (60% of control at 24 h after infection), conditions known to reduce the integrity of epithelial tight junctions. In conclusion, these data show that rotavirus infection of Caco-2 intestinal cells altered tight junction structure and function, which may be a response to metabolic dysfunction.
Keratinocyte growth factor-2 (FGF-10) promotes healing of experimental small intestinal ulceration in rats
Authors: Han Dong Soo , Li Fengling , Holt Lisa , Connolly Kevin , Hubert Melissa , Miceli Reneé , Okoye Zebedee , Santiago Gemma , Windle Kathleen , Wong Eling , Sartor R. Balfour
Published: December 2017

Keratinocyte growth factor-2 (KGF-2, repifermin) is a homolog of KGF-1 with epithelial mitogenic activities. We investigated the therapeutic role of KGF-2 in intestinal ulceration and its mechanisms of protection. KGF-2 (0.3–5 mg/kg) was administered before or after induction of small intestinal ulceration by indomethacin (Indo) in prevention and treatment protocols. In acute studies, KGF-2 was injected for up to 7 days before or daily for 5 days after Indo. In a 15-day chronic study, KGF-2 was injected intravenously daily beginning before or 7 days after Indo. Injury was evaluated by blinded macroscopic and microscopic inflammatory scores, epithelial BrdU staining, tissue IL-1β, PGE2, and hydroxyproline concentrations, and collagen type I RNA expression. In vitro effects of KGF-2 were evaluated by epithelial cellular proliferation, restitution of wounded monolayers, PGE2secretion, and expression of COX-2 and collagen mRNA. Intravenous KGF-2 significantly decreased acute intestinal injury by all parameters and significantly decreased chronic ulceration. Pretreatment, daily infusion, and delayed treatment were effective. KGF-2 promoted in vitro epithelial restitution with only modest effects on epithelial cell proliferation, stimulated COX-2 expression in cultured epithelial cells, and upregulated in vitro and in vivo PGE2production. KGF-2 did not affect in vivo fibrosis, although it induced collagen expression in cultured intestinal myofibroblasts. These results suggest that KGF-2 inhibits intestinal inflammation by stimulating epithelial restitution and protective PGs.
Disruption of cyclooxygenase-1 gene results in an impaired response to radiation injury
Authors: Houchen Courtney W. , Stenson William F. , Cohn Steven M.
Published: December 2017

Prostaglandins may play an important role in regulating normal renewal of gastrointestinal epithelium, epithelial injury repair, and initiation or progression of intestinal neoplasia. Synthesis of prostaglandins is catalyzed by either of two cyclooxygenase isoforms, Cox-1 and Cox-2. Cox-1 is the predominant cyclooxygenase isoform found in the normal intestine. In contrast, Cox-2 is present at low levels in normal intestine but is elevated at sites of inflammation and in adenomas and carcinomas. To determine directly whether prostaglandins synthesized by Cox-1 or Cox-2 regulate crypt epithelial cell fate after genotoxic or cytotoxic injury, we examined apoptosis, prostaglandin synthesis, and crypt stem cell survival after γ-irradiation in Cox-1−/−and Cox-2−/−mice. Cox-1−/−mice had increased crypt epithelial cell apoptosis and decreased clonogenic stem cell survival compared with wild-type littermates. PGE2synthesis was also diminished in Cox-1−/−mice compared with wild-type controls in unstressed intestine and after radiation injury. In contrast, apoptosis, stem cell survival, and intestinal PGE2synthesis in Cox-2−/−mice after irradiation were the same as in wild-type littermates. Crypt stem cell survival after irradiation was inhibited by a highly specific neutralizing antibody to PGE2, suggesting that this prostaglandin mediates stem cell fate in vivo. These data suggest that prostaglandins synthesized by Cox-1 regulate multiple steps that determine the fate of crypt epithelial cell after genotoxic or cytotoxic injury.
Regulation of DRA and AE1 in rat colon by dietary Na depletion
Authors: Rajendran Vazhaikkurichi M. , Black Joel , Ardito Thomas A. , Sangan Pitchai , Alper Seth L. , Schweinfest Clifford , Kashgarian Michael , Binder Henry J.
Published: December 2017

Two distinct Cl/anion exchange activities (Cl/HCO3and Cl/OH) identified in apical membranes of rat distal colon are distributed in cell type-specific patterns. Cl/HCO3exchange is expressed only in surface cells, whereas Cl/OH exchange is localized in surface and crypt cells. Dietary Na depletion substantially inhibits Cl/HCO3but not Cl/OH exchange. We determined whether anion exchange isoforms (AE) and/or downregulated in adenoma (DRA) are expressed in and related to apical membrane anion exchanges by examining localization of AE isoform-specific and DRA mRNA expression in normal and Na-depleted rats. Amplification of AE cDNA fragments by RT-PCR with colonic mRNA as template indicates that AE1 and AE2 but not AE3 mRNAs are expressed. In situ hybridization study revealed that AE1 mRNA is expressed predominantly in surface but not crypt cells. In contrast, AE2 polypeptide is expressed in basolateral membranes and DRA protein is expressed in apical membranes of both surface and crypt cells. AE1 mRNA is only minimally present in proximal colon, and DRA mRNA abundance is similar in distal and proximal colon. Dietary Na depletion reduces AE1 mRNA abundance but did not alter DRA mRNA abundance. This indicates that AE1 encodes surface cell-specific aldosterone-regulated Cl/HCO3exchange, whereas DRA encodes aldosterone-insensitive Cl/OH exchange.
Opposing roles of K+and Cl−channels in maintenance of opossum lower esophageal sphincter tone
Authors: Zhang Yong , Miller David V. , Paterson William G.
Published: December 2017

The ionic basis underlying the maintenance of myogenic tone of lower esophageal sphincter circular muscle (LES) was investigated in opossum with the use of standard isometric tension and conventional intracellular microelectrode recordings in vitro. In tension recording studies, nifedipine (1 μM) reduced basal tone to 27.7 ± 3.8% of control. The K+channel blockers tetraethylammonium (TEA, 2 mM), charybdotoxin (100 nM), and 4-aminopyridine (4-AP, 2 mM) enhanced resting tone, whereas apamin and glibenclamide were without affect. Cl−channel blockers DIDS (500 μM) and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (500 μM), as well as niflumic acid (0.1–300 μM), decreased basal tone, but tamoxifen was without effect. Intracellular microelectrode recordings revealed ongoing, spontaneous, spike-like action potentials (APs). Nifedipine abolished APs and depolarized resting membrane potential (RMP). Both TEA and 4-AP significantly depolarized RMP and augmented APs, whereas niflumic acid dose-dependently hyperpolarized RMP and abolished APs. These data suggest that, in the opossum, basal tone is associated with continuous APs and that K+and Ca2+-activated Cl−channels have important opposing roles in the genesis of LES tone.
Intestinal absorption and postabsorptive metabolism of linoleic acid in rats with short-term bile duct ligation
Authors: Minich Deanna M. , Havinga Rick , Stellaard Frans , Vonk Roel J. , Kuipers Folkert , Verkade Henkjan J.
Published: December 2017

We investigated in bile duct-ligated (BDL) and sham-operated control rats whether the frequent presence of essential fatty acid deficiency in cholestatic liver disease could be related to linoleic acid malabsorption, altered linoleic acid metabolism, or both. In plasma of BDL rats, the triene-to-tetraene ratio, a biochemical marker for essential fatty acid deficiency, was increased compared with controls (0.024 ± 0.004 vs. 0.013 ± 0.001; P < 0.05). Net and percentage of dietary linoleic acid absorbed were decreased in BDL rats compared with control rats (1.50 ± 0.16 mmol/day and 81.3 ± 3.3% vs. 2.08 ± 0.07 mmol/day and 99.2 ± 0.1%, respectively; each P < 0.001). At 24 h after [13C]linoleic acid administration, BDL rats had a similar ratio of plasma [13C]arachidonic acid to plasma [13C]linoleic acid concentration compared with control rats. Δ6-Desaturase activity was not significantly different in hepatic microsomes from control or BDL rats. At 3 h after [13C]linoleic acid administration, plasma appearance of [13C]linoleic acid and cumulative expiration of13CO2were decreased in BDL rats, compared with controls (by 54% and 80%, respectively). The present data indicate that the impaired linoleic acid status in cholestatic liver disease is mainly due to decreased net absorption and not to quantitative alterations in postabsorptive metabolism.
IGF-I and procollagen α1(I) are coexpressed in a subset of mesenchymal cells in active Crohn's disease
Authors: Pucilowska Jolanta B. , McNaughton Kirk K. , Mohapatra Nirupama K. , Hoyt Eileen C. , Zimmermann Ellen M. , Sartor R. Balfour , Lund P. Kay
Published: December 2017

This study tested the hypothesis that insulin-like growth factor I (IGF-I) expression is increased at sites of fibrosis in diseased intestine of patients with Crohn's disease (CD). IGF-I mRNA was quantified by RNase protection assay in uninvolved and involved intestine of 13 CD patients (10 ileum, 3 colon) and 7 ulcerative colitis (UC) patients (colon). In situ hybridization histochemistry compared the localization of IGF-I and procollagen α1(I) mRNAs. Masson's trichrome staining and immunohistochemistry for IGF-I precursor, α-smooth muscle actin (A), vimentin (V), desmin (D), and c- kit were used to examine the mesenchymal cell subtypes that express IGF-I and collagen in uninvolved and involved ileum and colon of CD patients and “normal” ileum and colon from noninflammatory controls. IGF-I mRNA was elevated in involved ileum and colon of patients with CD but not in involved colon of patients with UC. IGF-I and procollagen α1(I) mRNA showed overlapping distribution within fibrotic submucosa and muscularis propria of involved CD ileum and colon. In involved CD intestine, increased IGF-I precursor expression localized to mesenchymal cells in regions of tissue disorganization and fibrosis in muscularis mucosa, submucosa, and muscularis propria. In these regions, there were increased numbers of V+ cells relative to normal or uninvolved intestine. Increased IGF-I expression was localized to cells with a phenotype typical of fibroblasts (V+/A−/D−), myofibroblasts (V+/A+/D+), and, to a lesser extent, cells with normal enteric smooth muscle phenotype (V−/A+/D+). We conclude that increased IGF-I expression in multiple mesenchymal cell subtypes and increased numbers of cells with fibroblast/myofibroblast phenotype are involved in fibrosis associated with CD.
Polyamine regulation of ornithine decarboxylase and its antizyme in intestinal epithelial cells
Authors: Yuan Qing , Ray Ramesh M. , Viar Mary Jane , Johnson Leonard R.
Published: December 2017

Ornithine decarboxylase (ODC) is feedback regulated by polyamines. ODC antizyme mediates this process by forming a complex with ODC and enhancing its degradation. It has been reported that polyamines induce ODC antizyme and inhibit ODC activity. Since exogenous polyamines can be converted to each other after they are taken up into cells, we used an inhibitor of S-adenosylmethionine decarboxylase, diethylglyoxal bis(guanylhydrazone) (DEGBG), to block the synthesis of spermidine and spermine from putrescine and investigated the specific roles of individual polyamines in the regulation of ODC in intestinal epithelial crypt (IEC-6) cells. We found that putrescine, spermidine, and spermine inhibited ODC activity stimulated by serum to 85, 46, and 0% of control, respectively, in the presence of DEGBG. ODC activity increased in DEGBG-treated cells, despite high intracellular putrescine levels. Although exogenous spermidine and spermine reduced ODC activity of DEGBG-treated cells close to control levels, spermine was more effective than spermidine. Exogenous putrescine was much less effective in inducing antizyme than spermidine or spermine. High putrescine levels in DEGBG-treated cells did not induce ODC antizyme when intracellular spermidine and spermine levels were low. The decay of ODC activity and reduction of ODC protein levels were not accompanied by induction of antizyme in the presence of DEGBG. Our results indicate that spermine is the most, and putrescine the least, effective polyamine in regulating ODC activity, and upregulation of antizyme is not required for the degradation of ODC protein.
Clusterin gene transcription is activated by caudal-related homeobox genes in intestinal epithelium
Authors: Suh Eunran , Wang Zhengqi , Swain Gary P. , Tenniswood Martin , Traber Peter G.
Published: December 2017

Caudal-related homeobox (Cdx) proteins play an important role in development and differentiation of the intestinal epithelium. Using cDNA differential display, we identified clusterin as a prominently induced gene in a Cdx2-regulated cellular model of intestinal differentiation. Transfection experiments and DNA-protein interaction assays showed that clusterin is an immediate downstream target gene for Cdx proteins. The distribution of clusterin protein in the intestine was assessed during development and in the adult epithelium using immunohistochemistry. In the adult mouse epithelium, clusterin protein was localized in both crypt and villus compartments but not in interstitial cells of the intestinal mucosa. Together, these data suggest that clusterin is a direct target gene for Cdx homeobox proteins, and the pattern of clusterin protein expression suggests that it is associated with the differentiated state in the intestinal epithelium.
Effects of fibrogenic mediators on the development of pancreatic fibrosis in a TGF-β1 transgenic mouse model
Authors: Vogelmann R. , Ruf D. , Wagner M. , Adler G. , Menke A.
Published: December 2017

The pancreas morphology of transgenic mice that overexpress transforming growth factor-β1 (TGF-β1) in the pancreas resembles partially morphological features of chronic pancreatitis, such as progressive accumulation of extracellular matrix (ECM). Using this transgenic mouse model, we characterized the composition of pancreatic fibrosis and involved fibrogenic mediators. On day 14 after birth, fibrotic tissue was mainly composed of collagen type I and III. At this time, mRNA levels of TGF-β1 were increased. On day 70, the ECM composition was expanded by increased deposition of fibronectin, whereas connective tissue growth factor, fibroblast growth factor (FGF)-1, and FGF-2 mRNA expression levels were elevated in addition to TGF-β1. In parallel, the number of pancreatic stellate cells (PSC) increased over time. In vitro, TGF-β1 stimulated collagen type I expression but not fibronectin expression in PSC, in contrast to FGF-2, which stimulated both. This confirms that TGF-β1 mediates pancreatic fibrosis through activation of PSC and deposition of collagen type I and III at early time points. Furthermore, this points to an indirect mechanism in which TGF-β regulates pancreatic ECM assembly by induction of additional growth factors.
Modulation of gastric distension-induced sensations by small intestinal receptors
Authors: Feinle Christine , Grundy David , Fried Michael
Published: December 2017

Duodenal lipid exacerbates gastrointestinal sensations during gastric distension. Using luminal application of the local anesthetic benzocaine, we investigated the role of intestinal receptors in the induction of these sensations. Nine healthy subjects were studied on five occasions, during which isotonic saline or 20% lipid (2 kcal/min), combined with (duodenal or jejunal) 0.75% benzocaine or vehicle at 2.5 ml/min, was infused intraduodenally before and during gastric distension. Intragastric pressures and volumes, gastrointestinal sensations, and plasma CCK levels were determined. Duodenal lipid combined with vehicle increased gastric volume (in ml: saline, −10 ± 18; lipid/vehicle, 237 ± 30) and plasma CCK [mean levels (pmol/l): saline, 2.0 ± 0.2; lipid/vehicle, 8.0 ± 1.6] and, during distensions, induced nausea (scores: saline, 3 ± 2: lipid/vehicle, 58 ± 19) and decreased pressures at which fullness and discomfort occurred. Duodenal but not jejunal benzocaine attenuated the effect of lipid on gastric volume, plasma CCK, and nausea during distension (135 ± 38 and 216 ± 40 ml, 4.6 ± 0.6 pmol/l and not assessed, and 37 ± 12 and 64 ± 21 for lipid + duodenal benzocaine and lipid + jejunal benzocaine, respectively) and on pressures for sensations. In conclusion, intestinal receptors modulate gastrointestinal sensations associated with duodenal lipid and gastric distension. There is also the potential for local neural mechanisms to regulate CCK release and thereby reduce afferent activation indirectly.
Role of promoter methylation in increased methionine adenosyltransferase 2A expression in human liver cancer
Authors: Yang Heping , Huang Zong-Zhi , Zeng Zhaohui , Chen Changjin , Selby Robert R. , Lu Shelly C.
Published: December 2017

Methionine adenosyltransferase (MAT), an essential enzyme that catalyzes the formation of S-adenosylmethionine (SAM), is encoded by two genes, MAT1A (liver-specific) and MAT2A (non-liver-specific). We showed a switch from MAT1A to MAT2A expression in human liver cancer, which facilitates cancer cell growth. The present work examined the role of methylation in MAT2A transcriptional regulation. We found that the human MAT2A promoter is hypomethylated in hepatocellular carcinoma, in which the gene is upregulated transcriptionally, but hypermethylated in normal liver, in which the gene is minimally expressed. Luciferase activities driven by in vitro methylated MAT2A promoter constructs were 75–95% lower than activities driven by unmethylated constructs. SAM treatment of Hep G2 cells reduced MAT2A endogenous expression by 75%, hypermethylated the MAT2A promoter, and reduced luciferase activities driven by MAT2A promoter constructs by 65–75% while not affecting MAT1A's promoter activity. Treatment of adult rat and human hepatocytes with trichostatin A, an inhibitor of histone deacetylase, upregulated MAT2Aexpression by more than fourfold. Collectively, these results suggest that MAT2A expression is regulated by promoter methylation and histone acetylation.
Molecular mechanisms for the antiapoptotic action of gastrin
Authors: Todisco Andrea , Ramamoorthy Saravanan , Witham Thomas , Pausawasdi Nonthalee , Srinivasan Shanti , Dickinson Chris J. , Askari Frederick K. , Krametter Dieter
Published: December 2017

Gastrin (G17) has a CCK-B receptor-mediated growth-promoting effect on the AR42J rat acinar cell line. We examined whether G17 inhibits apoptosis induced by serum withdrawal of AR42J cells and CHO-K1 cells stably expressing CCK-B receptors (CHO-K1/CCK-B cells). Cellular apoptosis was measured by flow cytometry and the terminal deoxynucleotidyltransferase-mediated dUTP-FITC nick end-labeling method. Serum withdrawal induced AR42J and CHO-K1/CCK-B cell apoptosis. Addition of 10 nM G17 reversed these effects. We examined the action of G17 (10 nM) on phosphorylation and activation of protein kinase B/Akt, a kinase known to promote cell survival. Akt phosphorylation and activation were measured by kinase assays and Western blots with an anti-phospho-Akt antibody. G17 stimulated Akt phosphorylation and activation. G17 induction of Akt phosphorylation was inhibited by the phosphoinositide 3-kinase (PI 3-kinase) inhibitors LY-294002 (10 μM) and wortmannin (200 nM) but not by the mitogen-activated protein kinase kinase 1 inhibitor PD-98059 (50 μM). To study the role of p38 kinase in G17 signaling to Akt, we examined the effect of G17 on p38 kinase activation and phosphorylation using kinase assays and Western blots with an anti-phospho-p38 kinase antibody. G17 induced p38 kinase activity at doses and with kinetics similar to those observed for Akt induction. The p38 kinase inhibitor SB-203580 inhibited G17 induction of Akt phosphorylation and activation at a concentration (10 μM) 10-fold higher than necessary to block p38 kinase (1 μM), suggesting the possible involvement of kinase activities other than p38 kinase. Transduction of AR42J cells with the adenoviral vector Adeno-dn Akt, which overexpresses an inhibitor of Akt, reversed the antiapoptotic action of G17. In conclusion, G17 promotes AR42J cell survival through the induction of Akt via PI 3-kinase and SB-203580-sensitive kinase activities.
Effects of central and peripheral urocortin on fed and fasted gastroduodenal motor activity in conscious rats
Authors: Kihara Naoki , Fujimura Masaki , Yamamoto Ikuo , Itoh Etsuro , Inui Akio , Fujimiya Mineko
Published: December 2017

Since few previous studies have examined the effects of urocortin on physiological fed and fasted gastrointestinal motility, we administered urocortin intracerebroventricularly (icv) or intravenously (iv) in freely moving conscious rats and examined the changes in antral and duodenal motility. Icv and iv injection of urocortin disrupted fasted motor patterns of gastroduodenal motility, which were replaced by fed-like motor patterns. When urocortin was given icv and iv in the fed state, the motor activity remained like the fed patterns but % motor index (%MI) was decreased in the antrum and increased in the duodenum. Increase in the %MI in the duodenum induced by urocortin was shown as a nonpropagated event, since the transit of nonnutrient contents in the duodenum was decreased by icv and iv injection of urocortin. Changes in the gastroduodenal motility induced by icv injection of urocortin were abolished in animals with truncal vagotomy but not altered in animals with mechanical sympathectomy, suggesting that the vagal pathway may mediate the central action of urocortin. Neither urocortin antiserum nor α-helical CRF-(9–41) affected fed and fasted gastroduodenal motility, suggesting that endogenous urocortin is not involved in regulation of basal gastroduodenal motility.
Roles of 5-HT receptors in the release and action of secretin on pancreatic secretion in rats
Authors: Li James P. , Chang Ta-Min , Chey William Y.
Published: December 2017

5-Hydroxytryptamine (serotonin, 5-HT) is a hormone and neurotransmitter regulating gastrointestinal functions. 5-HT receptors are widely distributed in gastrointestinal mucosa and the enteric nervous system. Duodenal acidification stimulates not only the release of both 5-HT and secretin but also pancreatic exocrine secretion. We investigated the effect of 5-HT receptor antagonists on the release of secretin and pancreatic secretion of water and bicarbonate induced by duodenal acidification in anesthetized rats. Both the 5-HT2receptor antagonist ketanserin and the 5-HT3receptor antagonist ondansetron at 1–100 μg/kg dose-dependently inhibited acid-induced increases in plasma secretin concentration and pancreatic exocrine secretion. Neither the 5-HT1receptor antagonists pindolol and 5-HTP-DP nor the 5-HT4receptor antagonist SDZ-205,557 affected acid-evoked release of secretin or pancreatic secretion. None of the 5-HT receptor antagonists affected basal pancreatic secretion or plasma secretin concentration. Ketanserin or ondansetron at 10 μg/kg or a combination of both suppressed the pancreatic secretion in response to intravenous secretin at 2.5 and 5 pmol · kg−1· h−1by 55–75%, but not at 10 pmol · kg−1· h−1. Atropine (50 μg/kg) significantly attenuated the inhibitory effect of ketanserin on pancreatic secretion but not on the release of secretin. These observations suggest that 5-HT2and 5-HT3receptors mediate duodenal acidification-induced release of secretin and pancreatic secretion of fluid and bicarbonate. Also, regulation of pancreatic exocrine secretion through 5-HT2receptors may involve a cholinergic pathway in the rat.
Mast cell-independent impairment of host defense and muscle contraction inT. spiralis-infected W/WVmice
Authors: Vallance Bruce A. , Blennerhassett Patricia A. , Huizinga Jan D. , Collins Stephen M.
Published: December 2017

In response to nematode infection, the host presumably attempts to create an unfavorable environment to prevent larval penetration of the host and to expedite parasite expulsion from the gut. In this study, we have used W/WVmice with or without mast cells after bone marrow reconstitution (BMR-W/WV) to examine the role of mast cells in the host response. W/WV, BMR-W/WV, and wild-type (+/+) mice were infected with Trichinella spiralis. Infected W/WVmice exhibited less tissue damage and experienced a delay in worm expulsion and a greater degree of larval penetration of the gut leading to encystment in skeletal muscle. Tissue injury was greater and worm expulsion was normalized in BMR-W/WVmice, but larval penetration remained unchanged. Spontaneous contractile activity of jejunal muscle was disrupted in W/WVmice, as was the contractile response to carbachol. These abnormalities were also present in BMR-W/WVmice. These results indicate that mast cells mediate tissue damage and contribute to the timely expulsion of nematodes from the gut during primary infection.
Ca2+signaling in porcine duodenal glands by muscarinic receptor activation
Authors: Teraoka Hiroki , Maruyama Yutaka , Takehana Kazushige , Iwanaga Toshihiko , Hiraga Takeo , Fujita Shoichi , Ohta Toshio
Published: December 2017

The duodenal glands have been thought to play an important role in defense against proximal duodenal ulcer; however, the secretory mechanisms of these glands remain to be determined. In isolated duodenal acinar cells of the pig, we investigated the effects of ACh on intracellular Ca2+concentration ([Ca2+]i) and on membrane currents with fura 2 fluorometry and the patch clamp technique. ACh caused a transient increase in [Ca2+]i, and the increase was markedly inhibited by atropine or 4-diphenylacetoxy- N-methylpiperidine methiodide but not by hexamethonium, pirenzepine, or methoctramine. The expression of mRNA for the M3subtype far exceeded that for either M1or M2as revealed by real-time quantitative PCR and in situ hybridization. The rise in [Ca2+]ievoked by ACh was largely inhibited by thapsigargin but slightly affected by extracellular Ca2+deprivation. Caffeine had no effect on [Ca2+]i. ACh elicited Ca2+-dependent K+currents, a finding similar to the response to inositol 1,4,5,-trisphosphate applied intracellularly. These results suggest the presence of M3receptors linked to Ca2+release in porcine duodenal glands.
The human Na+/H+exchangerNHE2gene: genomic organization and promoter characterization
Authors: Malakooti Jaleh , Dahdal Refka Y. , Dudeja Pradeep K. , Layden Thomas J. , Ramaswamy Krishnamurthy
Published: December 2017

The Na+/H+exchanger (NHE) 2 belongs to a family of plasma membrane transporters involved in intracellular pH and cell volume regulation. We recently reported cloning of human NHE2 ( hNHE2) from a colonic cDNA library. Northern blot analysis has identified NHE2 mRNA only in small intestine, prostate, kidney, colon, and skeletal muscle. In this study, we describe the structure and 5′-regulatory region of the hNHE2 gene. The hNHE2 gene spans >90 kb and is organized in 12 exons intervened by 11 introns. All introns contain the conserved GT and AG dinucleotides at the donor and acceptor sites, respectively. The hNHE2 gene was mapped to chromosome 2q11.2. Primer extension analysis revealed a single transcription initiation site in human colonic adenocarcinoma cell lines. Analysis of the DNA nucleotide sequences of a 1.4-kb fragment of the 5′-flanking region shows no canonical TATA or CAAT boxes. However, the promoter region contains several potential cis-regulatory elements such as Sp1, early growth response-1, activator protein-2, MyoD, p300, nuclear factor-κB, myeloid zinc finger protein-1, caudal-related homeobox (Cdx) gene A, and Cdx protein-2 binding sites. In transient transfection studies, a reporter construct containing the 1.4-kb promoter region exhibited low luciferase activity levels. However, after deletion upstream of −664, its activity increased approximately threefold. Thus our data suggest that an inhibitory element may exist in the NHE2 promoter 5′-upstream region.
I. Receptors on visceral afferents
Authors: Kirkup Anthony J. , Brunsden Alan M. , Grundy David
Published: December 2017

Visceral afferents are the information superhighway from the gut to the central nervous system. These sensory nerves express a wide range of membrane receptors that can modulate their sensitivity. In this themes article, we concentrate on those receptors that enhance the excitability of visceral afferent neurons. Some receptors are part of a modality-specific transduction pathway involved in sensory signaling. Others, which are activated by substances derived from multiple cellular sources during ischemia, injury, or inflammation, act in a synergistic fashion to cause acute or chronic sensitization of the afferent nerves to mechanical and chemical stimuli. Such hypersensitivity is the hallmark of conditions such as irritable bowel syndrome. Accordingly, these receptors represent a rational target for drug treatments aimed at attenuating both the inappropriate visceral sensation and the aberrant reflex activity that are the foundation for alterations in bowel function.
Assessment of antral grinding of a model solid meal with echo-planar imaging
Authors: Marciani Luca , Gowland Penny A. , Fillery-Travis Annette , Manoj Pretima , Wright Jeff , Smith Andrew , Young Paul , Moore Rachel , Spiller Robin C.
Published: December 2017

Mathematical modeling of how physical factors alter gastric emptying is limited by lack of precise measures of the forces exerted on gastric contents. We have produced agar gel beads (diameter 1.27 cm) with a range of fracture strengths (0.15–0.90 N) and assessed their breakdown by measuring their half-residence time (RT1/2) using magnetic resonance imaging. Beads were ingested either with a high (HV)- or low (LV)-viscosity liquid nutrient meal. With the LV meal, RT1/2was similar for bead strengths ranging from 0.15 to 0.65 N but increased from 22 ± 2 min (bead strength <0.65 N) to 65 ± 12 min for bead strengths >0.65 N. With the HV meal, emptying of the harder beads was accelerated. The sense of fullness after ingesting the LV meal correlated linearly (correlation coefficient = 0.99) with gastric volume and was independently increased by the harder beads, which were associated with an increased antral diameter. We conclude that the maximum force exerted by the gastric antrum is close to 0.65 N and that gastric sieving is impaired by HV meals.
Expression of multidrug resistance-associated protein 2 in small intestine from pregnant and postpartum rats
Authors: Mottino Aldo D. , Hoffman Tim , Jennes Lothar , Cao Jingsong , Vore Mary
Published: December 2017

We analyzed the expression of multidrug resistance-associated protein 2 (mrp2) in the small intestine of control female rats and in rats during late pregnancy (19–20 days of pregnancy) and lactation (2–4, 10–14, and 21 days after delivery). Western blot analysis was performed on brush-border membranes prepared from different regions of the small intestine. Expression of mrp2 was maximal in the proximal segments for all experimental groups, was preserved in pregnant rats, and increased by 100% in postpartum rats by late lactation with respect to control animals. Northern blot analysis of mrp2 mRNA revealed a positive correlation with protein levels. Transport of S-glutathione-dinitrophenol (DNP-SG) from the intestinal cell to the lumen was analyzed in the everted intestinal sac model. Secretion of DNP-SG was not altered in pregnant rats but increased in lactating animals by late lactation. Intestinal mrp2 mRNA, protein, and transport activity are increased in lactating rats, suggesting that this may represent an adaptive mechanism to minimize the toxicity of dietary xenobiotics in response to increased postpartum food consumption.
Role of nuclear factor-κB in gastric ulcer healing in rats
Authors: Takahashi Satoru , Fujita Takuya , Yamamoto Akira
Published: December 2017

We investigated the role of nuclear factor-κB (NF-κB) in gastric ulcer healing in rats. NF-κB was activated in ulcerated tissue but not in normal mucosa, and the level of the activation was decreased with ulcer healing. NF-κB activation was observed in fibroblasts, monocytes/macrophages, and neutrophils. Treatment of gastric fibroblasts, isolated from the ulcer base, with interleukin-1β activated NF-κB and the subsequently induced cyclooxygenase-2 and cytokine-induced neutrophil chemoattractant-1 (CINC-1) mRNA expression. Inhibition of activated NF-κB action resulted in suppression of both their mRNA expression and increases in PGE2 and CINC-1 levels induced by interleukin-1β. Persistent prevention of NF-κB activation caused an impairment of ulcer healing in rats. Gene expression of interleukin-1β, CINC-1, cyclooxygenase-2, and inducible nitric oxide synthase in ulcerated tissue had been inhibited before the delay in ulcer healing became manifest. The increased levels of cyclooxygenase-2 protein and PGE2 production were also reduced. These results demonstrate that NF-κB, activated in ulcerated tissue, might upregulate the expression of healing-promoting factors responsible for gastric ulcer healing in rats.
Mechanism of internal anal sphincter smooth muscle relaxation by phorbol 12,13-dibutyrate
Authors: Chakder Sushanta , Sarma D. N. K. , Rattan Satish
Published: December 2017

We investigated the mechanism of the inhibitory action of phorbol 12,13-dibutyrate (PDBu), one of the typical protein kinase C (PKC) activators, in in vitro smooth muscle strips and in isolated smooth muscle cells of the opossum internal anal sphincter (IAS). The inhibitory action of PDBu on IAS smooth muscle (observed in the presence of guanethidine + atropine) was partly attenuated by tetrodotoxin, suggesting that a part of the inhibitory action of PDBu is via the nonadrenergic, noncholinergic neurons. A major part of the action of PDBu in IAS smooth muscle was, however, via its direct action at the smooth muscle cells, accompanied by a decrease in free intracellular Ca2+concentration ([Ca2+]i) and inhibition of PKC translocation. PDBu-induced IAS smooth muscle relaxation was unaffected by agents that block Ca2+mobilization and Na+-K+-ATPase. The PDBu-induced fall in basal IAS smooth muscle tone and [Ca2+]iresembled that induced by the Ca2+channel blocker nifedipine and were reversed specifically by the Ca2+channel activator BAY K 8644. We speculate that a major component of the relaxant action of PDBu in IAS smooth muscle is caused by the inhibition of Ca2+influx and of PKC translocation to the membrane. The specific role of PKC downregulation and other factors in the phorbol ester-mediated fall in basal IAS smooth muscle tone remain to be determined.
Epidermal growth factor inhibits glycylsarcosine transport and hPepT1 expression in a human intestinal cell line
Authors: Nielsen Carsten Uhd , Amstrup Jan , Steffansen Bente , Frokjaer Sven , Brodin Birger
Published: December 2017

The human intestinal cell line Caco-2 was used as a model system to study the effects of epidermal growth factor (EGF) on peptide transport. EGF decreased apical-to-basolateral fluxes of [14C]glycylsarcosine ([14C]Gly-Sar) up to 50.2 ± 3.6% ( n = 6) of control values. Kinetic analysis of the fluxes showed that maximal flux ( Vmax) of transepithelial transport decreased from 3.00 ± 0.17 nmol · cm−2· min−1in control cells to 0.50 ± 0.07 nmol · cm−2· min−1in cells treated with 5 ng/ml EGF ( n = 6, P < 0.01). The apparent Michaelis-Menten constant ( Km) was 2.71 ± 0.31 mM ( n= 6) in control cells and 1.89 ± 0.28 mM ( n = 6, not significantly different from control) in EGF-treated cells. Similarly, apical uptake of [14C]Gly-Sar decreased in cells treated with EGF, with an ED50value of 0.36 ± 0.06 ng/ml ( n = 6) EGF and a maximal inhibition of 80 ± 0.02% ( n = 6). Vmaxdecreased from 2.61 ± 0.4 to 1.06 ± 0.1 nmol · cm−2· min−1( n = 3, P < 0.05), whereas Kmremained constant. Basolateral Gly-Sar uptake showed no changes in Vmaxor Kmafter EGF treatment ( n = 3). RT-PCR showed a decrease in hPepT1 mRNA (using glucose-6-phosphate dehydrogenase mRNA as control) in cells treated with EGF. Western blotting indicated a decrease in hPepT1 protein in cell lysates. We conclude that EGF treatment decreases Gly-Sar transport in Caco-2 cells by decreasing the number of peptide transporter molecules in the apical membrane.
Protease-activated receptor-1 stimulates Ca2+-dependent Cl−secretion in human intestinal epithelial cells
Authors: Buresi M. C. , Schleihauf E. , Vergnolle N. , Buret A. , Wallace J. L. , Hollenberg M. D. , MacNaughton W. K.
Published: December 2017

The thrombin receptor, protease-activated receptor-1 (PAR-1), has wide tissue distribution and is involved in many physiological functions. Because thrombin is in the intestinal lumen and mucosa during inflammation, we sought to determine PAR-1 expression and function in human intestinal epithelial cells. RT-PCR showed PAR-1 mRNA expression in SCBN cells, a nontransformed duodenal epithelial cell line. Confluent SCBN monolayers mounted in Ussing chambers responded to PAR-1 activation with a Cl−-dependent increase in short-circuit current. The secretory effect was blocked by BaCl2and the Ca2+-ATPase inhibitor thapsigargin, but not by the L-type Ca2+channel blocker verapamil or DIDS, the nonselective inhibitor of Ca2+-dependent Cl−transport. Responses to thrombin and PAR-1-activating peptides exhibited auto- and crossdesensitization. Fura 2-loaded SCBN cells had increased fluorescence after PAR-1 activation, indicating increased intracellular Ca2+. RT-PCR showed that SCBN cells expressed mRNA for the cystic fibrosis transmembrane conductance regulator (CFTR) and hypotonicity-activated Cl−channel-2 but not for the Ca2+-dependent Cl−channel-1. PAR-1 activation failed to increase intracellular cAMP, suggesting that the CFTR channel is not involved in the Cl−secretory response. Our data demonstrate that PAR-1 is expressed on human intestinal epithelial cells and regulates a novel Ca2+-dependent Cl−secretory pathway. This may be of clinical significance in inflammatory intestinal diseases with elevated thrombin levels.