bluuebunny/crossref_metadata_2025 huggingface dataset:
Reduced tLESR elicitation in response to gastric distension in fundoplication patients
Authors: Scheffer R. C. H. , Tatum R. P. , Shi G. , Akkermans L. M. A. , Joehl R. J. , Kahrilas P. J.
Published: March 2015

Transient lower esophageal sphincter relaxations (tLESRs) are vagally mediated in response to gastric cardiac distension. Nine volunteers, eight gastroesophageal reflux disease (GERD) patients, and eight fundoplication patients were studied. Manometry with an assembly that included a barostat bag was done for 1 h with and 1 h without barostat distension to 8 mmHg. Recordings were scored for tLESRs and barostat bag volume. Fundoplication patients had fewer tLESRs (0.4 ± 0.3/h) than either normal subjects (2.4 ± 0.5/h) or GERD patients (2.0 ± 0.3/h). The tLESRs rate increased significantly in normal subjects (5.8 ± 0.9/h) and GERD patients (5.4 ± 0.8/h) during distension but not in the fundoplication group. All groups exhibited similar gastric accommodation (change in volume/change in pressure) in response to distension. Fundoplication patients exhibit a lower tLESR rate at rest and a marked attenuation of the response to gastric distension compared with either controls or GERD patients. Gastric accommodation was not impaired with fundoplication. This suggests that the receptive field for triggering tLESRs is contained within a wider field for elicitation of gastric receptive relaxation and that only the first is affected by fundoplication.
Development of the myogenic response in postnatal intestine: role of PKC
Authors: Su Baogen Y. , Reber Kristina M. , Nankervis Craig A. , Nowicki Philip T.
Published: March 2015

Previous attempts to determine developmental changes in the vascular myogenic response have been confounded by the presence of competing vasoactive stimuli or the use of isolated vessels with markedly different baseline diameters. To circumvent these issues, small mesenteric arteries (diameter ∼150 μm) from 1- and 10-day-old piglets were studied in vitro under no-flow conditions. In situ studies demonstrated that the intravascular pressure and diameter of these vessels were similar in both age groups, allowing an effective comparison of the myogenic response not obscured by differences in basal diameter. The pressure-diameter relationship was age specific. Thus, although small mesenteric arteries from both age groups demonstrated myogenic constriction in response to stepwise increases in pressure (0 to 100 mmHg, in 20-mmHg increments), the intensity of contraction was significantly greater in vessels from 1-day-old piglets particularly within the pressure range normally experienced by these vessels in situ. Attenuation or activation of PKC with calphostin C or indolactam, respectively, substantially altered the pressure-diameter relationship in 1-, but not 10-day-old arteries; thus calphostin C essentially eliminated the contractile response to pressure elevation in younger subjects, whereas indolactam significantly increased the intensity of the myogenic response and shifted its activation point to a lower pressure range. Immunoblots carried out on protein recovered from these arteries revealed the presence of α, β, ε, ι, and λ; notably, expression of the α- and ε-isoforms substantially decreased between postnatal days 1 and 10.
V. Aging and gastrointestinal smooth muscle: from signal transduction to contractile proteins
Authors: Bitar Khalil N.
Published: March 2015

The object of this theme is to offer new perspectives on the effect of aging on signal-transduction pathways associated with agonist-induced contraction of smooth muscle cells from the colon. Smooth muscle cells from old rats (32 mo old) exhibit limited cell length distribution and diminished contractility. The observed reduced contractile response may be due to the effect of aging on signal-transduction pathways, especially an inhibition of the tyrosine kinase-Src kinase pathway, a reduced activation of the PKC pathway, and a reduced association of contractile proteins [heat shock protein 27 (HSP27)-tropomyosin, HSP27-actin, actin-myosin]. Levels of HSP27 phosphorylation are also reduced compared with adult rats.
Vitamin D-inducible calcium transport and gene expression in three Caco-2 cell lines
Authors: Fleet James C. , Eksir Faria , Hance Kenneth W. , Wood Richard J.
Published: March 2015

The parental cell line (P) of Caco-2 cells and two clones, BBe and TC7, were studied at 11 days postconfluence to test the facilitated diffusion model of vitamin D-mediated intestinal calcium absorption (CaTx). Nuclear vitamin D receptor (nVDR) and calbindin D9k(CaBP) were measured by Western blot; 1,25-hydroxyvitamin D324-hydroxylase (CYP24), CaBP, plasma membrane Ca-ATPase (PMCA), and Ca transport channel (CaT1) mRNA levels were examined by RT-PCR; and net apical-to-basolateral CaTx was examined after treating cells with vehicle or 10 nM calcitriol for 8 (mRNA levels) or 48 h (protein, CaBP mRNA, CaTx). nVDR level was lowest in BBe (38% P value) and directly related to CYP24 induction (TC7 = P, which were 1.56 times greater than BBe). nVDR was inversely related to the vitamin D-induced levels of CaT1 mRNA, CaBP mRNA, PMCA mRNA, and net CaTx, with the highest induction seen in BBe. Basal CaBP mRNA (86 times greater than P) and protein levels were highest in TC7 cells and were not associated with higher net CaTx, suggesting CaBP may not be rate limiting for CaTx in these cells.
Syntaxin 3 is required for cAMP-induced acid secretion: streptolysin O-permeabilized gastric gland model
Authors: Ammar David A. , Zhou Rihong , Forte John G. , Yao Xuebiao
Published: March 2015

Gastric gland stimulation triggers H+,K+-ATPase translocation from cytoplasmic tubulovesicles to apical plasma membrane in parietal cells, resulting in HCl secretion. We studied the mechanisms involved in tubulovesicle translocation with a permeabilized gland system. Streptolysin O (SLO)-treated glands were permeabilized such that exogenous fluorescently labeled actin incorporated into cytoskeleton in a pattern mimicking endogenous F-actin. As shown by accumulation of the weak base aminopyrine (AP), SLO-permeabilized glands are stimulated to secrete acid by addition of cAMP and ATP and inhibited by proton pump inhibitors. Direct visualization with the fluorescent pH probe Lysosensor showed acid accumulation in glandular lumen and parietal cell canaliculi. ME-3407, an antiulcer drug with inhibitory action implicated to involve ezrin, inhibited AP uptake in and effectively released ezrin from intact and SLO-permeabilized glands. In contrast, wortmannin, an effective secretion inhibitor in intact glands, had minimal effects on ezrin or AP accumulation in SLO-permeabilized glands. The finding that SNARE protein syntaxin 3 is associated with H+,K+-ATPase-containing tubulovesicles suggested that it is involved in membrane fusion. Addition of recombinant syntaxin 3, but not syntaxin 5 or heat-denatured syntaxin 3, dose-dependently inhibited acid secretion. Our studies are consistent with a membrane recycling hypothesis that activation of protein kinase cascades leads to SNARE-mediated fusion of H+,K+-ATPase-containing tubulovesicles to apical plasma membrane.
PKC-ζ is required in EGF protection of microtubules and intestinal barrier integrity against oxidant injury
Authors: Banan A. , Fields J. Z. , Talmage D. A. , Zhang L. , Keshavarzian A.
Published: March 2015

Using monolayers of human intestinal (Caco-2) cells, we showed that epidermal growth factor (EGF) protects intestinal barrier integrity against oxidant injury by protecting the microtubules and that protein kinase C (PKC) is required. Because atypical PKC-ζ isoform is abundant in wild-type (WT) Caco-2 cells, we hypothesized that PKC-ζ mediates, at least in part, EGF protection. Intestinal cells (Caco-2 or HT-29) were transfected to stably over- or underexpress PKC-ζ. These clones were preincubated with low or high doses of EGF or a PKC activator [1-oleoyl-2-acetyl- sn-glycerol (OAG)] before oxidant (0.5 mM H2O2). Relative to WT cells exposed to oxidant, only monolayers of transfected cells overexpressing PKC-ζ (2.9-fold) were protected against oxidant injury as indicated by increases in polymerized tubulin and decreases in monomeric tubulin, enhancement of architectural stability of the microtubule cytoskeleton, and increases in monolayer barrier integrity toward control levels (62% less leakiness). Overexpression-induced protection was OAG independent and even EGF independent, but EGF significantly potentiated PKC-ζ protection. Most overexpressed PKC-ζ (92%) resided in membrane and cytoskeletal fractions, indicating constitutive activation of PKC-ζ. Stably inhibiting PKC-ζ expression (95%) with antisense transfection substantially attenuated EGF protection as demonstrated by reduced tubulin assembly and increased microtubule disassembly, disruption of the microtubule cytoskeleton, and loss of monolayer barrier integrity. We conclude that 1) activation of PKC-ζ is necessary for EGF-induced protection, 2) PKC-ζ appears to be an endogenous stabilizer of the microtubule cytoskeleton and of intestinal barrier function against oxidative injury, and 3) we have identified a novel biological function (protection) among the atypical isoforms of PKC.
Impairment of TGF-β signaling in T cells increases susceptibility to experimental autoimmune hepatitis in mice
Authors: Schramm Christoph , Protschka Martina , Köhler Heinz H. , Podlech Jürgen , Reddehase Matthias J. , Schirmacher Peter , Galle Peter R. , Lohse Ansgar W. , Blessing Manfred
Published: March 2015

In autoimmune hepatitis, strong TGF-β1 expression is found in the inflamed liver. TGF-β overexpression may be part of a regulatory immune response attempting to suppress autoreactive T cells. To test this hypothesis, we determined whether impairment of TGF-β signaling in T cells leads to increased susceptibility to experimental autoimmune hepatitis (EAH). Transgenic mice of strain FVB/N were generated expressing a dominant-negative TGF-β type II receptor in T cells under the control of the human CD2 promoter/locus control region. On induction of EAH, transgenic mice showed markedly increased portal and periportal leukocytic infiltrations with hepatocellular necroses compared with wild-type mice (median histological score = 1.8 ± 0.26 vs. 0.75 ± 0.09 in wild-type mice; P < 0.01). Increased IFN-γ production (118 vs. 45 ng/ml) and less IL-4 production (341 vs. 1,256 pg/ml) by mononuclear cells isolated from transgenic livers was seen. Impairment of TGF-β signaling in T cells therefore leads to increased susceptibility to EAH in mice. This suggests an important role for TGF-β in immune homeostasis in the liver and may teleologically explain TGF-β upregulation in response to T cell-mediated liver injury.
Stimulation of chloride secretion by baicalein in isolated rat distal colon
Authors: Ko W. H. , Law V. W. Y. , Yip W. C. Y. , Yue G. G. L. , Lau C. W. , Chen Z. Y. , Huang Y.
Published: March 2015

The effect of baicalein on mucosal ion transport in the rat distal colon was investigated in Ussing chambers. Mucosal addition of baicalein (1–100 μM) elicited a concentration-dependent short-circuit current ( Isc) response. The increase in Iscwas mainly due to Cl−secretion. The presence of mucosal indomethacin (10 μM) significantly reduced both the basal and subsequent baicalein-evoked Iscresponses. The baicalein-induced Iscwere inhibited by mucosal application of diphenylamine-2-carboxylic acid (100 μM) and glibenclamide (500 μM) and basolateral application of chromanol 293B (30 μM), a blocker of KvLQT1 channels and Ba2+ions (5 mM). Treatment of the colonic mucosa with baicalein elicited a threefold increase in cAMP production. Pretreating the colonic mucosa with carbachol (100 μM, serosal) but not thapsigargin (1 μM, both sides) abolished the baicalein-induced Isc. Addition of baicalein subsequent to forskolin induced a further increase in Isc. These results indicate that the baicalein evoked Cl−secretion across rat colonic mucosa, possibly via a cAMP-dependent pathway. However, the action of baicalein cannot be solely explained by its cAMP-elevating effect. Baicalein may stimulate Cl−secretion via a cAMP-independent pathway or have a direct effect on cystic fibrosis transmembrane conductance regulator.
IL-10 receptor and coreceptor expression in quiescent and activated hepatic stellate cells
Authors: Mathurin Philippe , Xiong Shigang , Kharbanda Kusum K. , Veal Nary , Miyahara Takeo , Motomura Kenta , Rippe Richard A. , Bachem Max G. , Tsukamoto Hidekazu
Published: March 2015

Interleukin (IL)-10 expression is induced in activated hepatic stellate cells (HSC) in vitro and in vivo. We analyzed expression of IL-10 receptor (IL-10R) and coreceptor cytokine receptor family (CRF2–4) in HSC. We aimed to clone and sequence partial cDNA for rat IL-10R and CRF2–4, determine their expression in activated rat HSC in vivo and in vitro, and examine the biological responsiveness of HSC to exogenous IL-10. PCR cloning and sequencing of partial rat IL-10R and CRF2–4 cDNAs revealed 86% homology with corresponding mouse sequences. In hepatic macrophages, Northern blot with cloned IL-10R cDNA detected an expected 3.5-kb transcript, and IL-10R and CRF2–4 mRNAs showed steady constitutive expression after in vitro lipopolysaccharide treatment or cholestatic liver injury. IL-10R mRNA expression, as confirmed by immunohistochemistry, was induced 20.1- and 8.6-fold in HSC from cholestatic livers and 7-day culture-activated HSC, respectively but CRF2–4 mRNA levels were unchanged. Under serum-free conditions, IL-10 had minimal effects on collagen production but reduced DNA synthesis, matrix metalloprotease-2 mRNA levels, and activity in HSC. With serum, IL-10 inhibited both collagen production and DNA synthesis but had no effect on procollagen-α1(I) mRNA levels. This shows concomitant induction of IL-10R but not CRF2–4 to that of IL-10 by activated HSC in vitro and in vivo and associated acquisition of the responsiveness to IL-10, entailing complex effects on HSC.
Regulation of hepatic connexins in cholestasis: possible involvement of Kupffer cells and inflammatory mediators
Authors: González Hernán E. , Eugenín Eliseo A. , Garcés Gladys , Solís Nancy , Pizarro Margarita , Accatino Luigi , Sáez Juan C.
Published: March 2015

Hepatocyte gap junction proteins, connexins (Cxs) 26 and 32, are downregulated during obstructive cholestasis (OC) and lipopolysaccharide hepatocellular cholestasis (LPS-HC). We investigated rat hepatic Cxs during ethynylestradiol hepatocellular cholestasis (EE-HC) and choledochocaval fistula (CCF) and compared them with OC and LPS-HC. Levels (immunoblotting) and cellular distribution (immunofluorescence) of Cx26, -32, and -43, as well as macrophage infiltration, were studied in livers of rats under each condition. Cx26 and -32 were reduced in LPS-HC, OC, and CCF. However, in EE-HC, Cx26 did not change and Cx32 was increased. Prominent inflammation occurred in LPS-HC, OC, and CCF, which was associated with increased levels of Cx43 in LPS-HC and OC but not CCF. No inflammation nor changes in Cx43 levels occurred during EE-HC. In cultured hepatocytes, dye coupling was reduced by tumor necrosis factor-α and interleukins-1β and -6, whereas reduction induced by LPS required coculture with Kupffer cells. Thus hepatocyte gap junctions are downregulated in forms of cholestasis associated with inflammation, and reduced intercellular communication might be induced in part by proinflammatory mediators.
Deglutitive inhibition affects both esophageal peristaltic amplitude and shortening
Authors: Shi Guoxiang , Pandolfino John E. , Zhang Qing , Hirano Ikuo , Joehl Raymond J. , Kahrilas Peter J.
Published: March 2015

Deglutitive inhibition attenuates ongoing esophageal contractions if swallows are separated by short time intervals. This study aimed to determine whether esophageal shortening, mediated by longitudinal muscle, was similarly affected. Eight healthy subjects with two distal esophageal segments demarcated by mucosal clips and manometric recording sites positioned within those segments underwent concurrent manometry and fluoroscopy. Peristaltic amplitude and change in distal segment lengths were quantified during single swallows, paired swallows separated by progressively prolonged intervals, and a series of rapid repetitive swallows. During grouped swallows, deglutitive inhibition with complete attenuation of both the manometric contraction and segment shortening was evident with short-interval swallows and rapid-sequence swallows. No inhibition of either was evident with long-interval pairs. With intermediate interswallow intervals, the occurrence and degree of deglutitive inhibition between peristaltic amplitude and segment shortening were closely correlated. Deglutitive inhibition affects both the longitudinal and circular muscle layers of the esophageal wall, and the occurrence of inhibition evident in one layer is strongly correlated with the other.
Neonatal maternal separation predisposes adult rats to colonic barrier dysfunction in response to mild stress
Authors: Söderholm Johan D. , Yates Derrick A. , Gareau Mélanie G. , Yang Ping-Chang , MacQueen Glenda , Perdue Mary H.
Published: March 2015

Intestinal dysfunction is related to stress and early life events, but the mechanisms are largely unknown. Our aim was to determine whether early trauma predisposes adult rats to intestinal mucosal dysfunction in response to stress. Neonatal Sprague-Dawley rats were individually separated from their mothers for 3 h/day at 4–21 days of age. Between days 80 and 90, separated and control rats were subjected to mild acute stress (30-min water avoidance) or sham stress. Mucosal barrier function and ion transport were assessed in colonic tissues mounted in Ussing chambers. Mild stress increased short-circuit current, conductance, and transepithelial transport of macromolecules in separated rats, while having minimal effects in controls. Pretreatment of the separated rats with a corticotropin-releasing hormone (CRH) antagonist, the peptide α-helical CRH(9–41) injected intraperitoneally 20 min before stress, abolished the stress-induced mucosal changes. Our results indicate that neonatal trauma can induce phenotypic changes in adulthood, including enhanced vulnerability of the gut mucosa to stress via mechanisms involving peripherally located CRH receptors.
Lactoferrin reduces colitis in rats via modulation of the immune system and correction of cytokine imbalance
Authors: Togawa Jun-Ichi , Nagase Hajime , Tanaka Katsuaki , Inamori Masahiko , Umezawa Tadashi , Nakajima Atsushi , Naito Makoto , Sato Shinobu , Saito Toshifumi , Sekihara Hisahiko
Published: March 2015

Natural immunomodulator lactoferrin is known to exert an anti-inflammatory effect. However, there have been no studies that examine the mode of action of lactoferrin in reducing intestinal damage. We investigated the effect of lactoferrin on a trinitrobenzenesulfonic acid (TNBS)-induced colitis model in rats. Bovine lactoferrin was given once daily through gavage, starting 3 days before (preventive mode) or just after TNBS administration (treatment mode) until death. The distal colon was removed to be examined. Colitis was attenuated by lactoferrin via both modes in a dose-dependent manner, as reflected by improvement in macroscopic and histological scores and myeloperoxidase activity. Lactoferrin caused significant induction of the anti-inflammatory cytokines interleukin (IL)-4 and IL-10, significant reductions in the proinflammatory cytokines tumor necrosis factor-α and IL-1β, and downregulation of the nuclear factor-κB pathway. We concluded that lactoferrin exerts a protective effect against colitis in rats via modulation of the immune system and correction of cytokine imbalance. Lactoferrin has potential as a new therapeutic agent for inflammatory bowel disease.
Double-stranded RNA activates a p38 MAPK-dependent cell survival program in biliary epithelia
Authors: Tadlock Laura , Yamagiwa Yoko , Marienfeld Carla , Patel Tushar
Published: March 2015

Double-stranded RNA (dsRNA) is produced during replicative viral infection or genotoxic stress. Thus knowledge of the cellular response to dsRNA is necessary to understand the effects of DNA damage or viral infection in biliary epithelia. We assessed the effect of dsRNA on biliary epithelial cell proliferation and apoptosis and the role of the stress-activated p38 MAPK signaling pathway in these responses. dsRNA did not induce apoptosis or proliferation in Mz-ChA-1 human malignant cholangiocytes, but decreased cytotoxicity induced by camptothecin or tumor necrosis factor-related apoptosis inducing ligand and decreased activity of caspases 3, 8, and 9. Furthermore, dsRNA increased p38 MAPK and JNK kinase active site phosphorylation but had no effect on either MAPK kinase (MEK)1/2 or protein kinase R phosphorylation. Inhibition of p38 MAPK with SB-203580 increased basal caspase activity. Thus dsRNA stimulates a p38 MAPK-dependent cell-survival pathway in biliary epithelial cells that may modulate the response of the biliary epithelia to dsRNA produced during genotoxic injury or virus infection.
Upregulation of CFTR expression but not SLC26A3 and SLC9A3 in ulcerative colitis
Authors: Lohi H. , Mäkelä S. , Pulkkinen K. , Höglund P. , Karjalainen-Lindsberg M.-L. , Puolakkainen P. , Kere J.
Published: March 2015

In inflamed colonic mucosa, the equilibrium between absorptive and secretory functions for electrolyte and salt transport is disturbed. We compared the expression of three major mediators of the intestinal salt transport between healthy and inflamed colonic mucosa to understand the pathophysiology of diarrhea in inflammatory bowel disease. Expression levels of the cystic fibrosis transmembrane regulator (CFTR) (Cl− channel), SLC26A3 (Cl−/HCO[Formula: see text] exchanger) and SLC9A3 (Na+/H+ exchanger) mRNAs were measured by real-time quantitative RT-PCR in peroperative colonic samples from controls ( n = 4) and patients with ulcerative colitis ( n = 10). Several samples were obtained from each individual. Tissue samples were divided into three subgroups according to their histological degree of inflammation. Expression of CFTR and SLC26A3 proteins were determined by immunohistochemistry and Western blotting from the same samples, respectively. Increased expression of CFTR mRNA was observed in all three groups of affected tissue samples, most pronounced in mildly inflamed colonic mucosa (5-fold increase in expression; P < 0.001). The expression of the CFTR protein was detected from health and inflamed colon tissue. Although the expression of the SLC26A3 mRNA was significantly decreased in severe ulcerative colitis ( P< 0.05), the SLC26A3 protein levels remained unchanged in all groups. The expression of SLC9A3 mRNA was significantly changed between the mild and severe groups. Intestinal inflammation modulates the expression of three major mediators of intestinal salt transport and may contribute to diarrhea in ulcerative colitis both by increasing transepithelial Cl− secretion and by inhibiting the epithelial NaCl absorption.
N-glycosylation controls functional activity of Oatp1, an organic anion transporter
Authors: Lee Thomas K. , Koh Albert S. , Cui Zhifeng , Pierce Robert H. , Ballatori Nazzareno
Published: March 2015

Rat Oatp1 (Slc21a1) is an organic anion-transporting polypeptide believed to be an anion exchanger. To characterize its mechanism of transport, Oatp1 was expressed in Saccharomyces cerevisiae under control of the GAL1 promoter. Protein was present at high levels in isolated S. cerevisiae secretory vesicles but had minimal posttranslational modifications and failed to exhibit taurocholate transport activity. Apparent molecular mass ( M) of Oatp1 in yeast was similar to that of unmodified protein, ∼62 kDa, whereas in liver plasma membranes Oatp1 has an M of ∼85 kDa. To assess whether underglycosylation of Oatp1 in yeast suppressed functional activity, Oatp1 was expressed in Xenopus laevis oocytes with and without tunicamycin, a glycosylation inhibitor. With tunicamycin, M of Oatp1 decreased from ∼72 to ∼62 kDa and transport activity was nearly abolished. Mutations to four predicted N-glycosylation sites on Oatp1 (Asn to Asp at positions 62, 124, 135, and 492) revealed a cumulative effect on function of Oatp1, leading to total loss of taurocholate transport activity when all glycosylation sites were removed. M of the quadruple mutant was ∼ 62 kDa, confirming that these asparagine residues are sites of glycosylation in Oatp1. Relatively little of the quadruple mutant was able to reach the plasma membrane, and most remained in unidentified intracellular compartments. In contrast, two of the triple mutants tested (N62/124/135D and N124/135/492D) were present in the plasma membrane fraction yet exhibited minimal transport activity. These results demonstrate that both membrane targeting and functional activity of Oatp1 are controlled by the extent of N-glycosylation.
Intestinal expression of genes involved in iron absorption in humans
Authors: Rolfs Andreas , Bonkovsky Herbert L. , Kohlroser James G. , McNeal Kristina , Sharma Ashish , Berger Urs V. , Hediger Matthias A.
Published: March 2015

Hereditary hemochromatosis (HHC) is one of the most frequent genetic disorders in humans. In healthy individuals, absorption of iron in the intestine is tightly regulated by cells with the highest iron demand, in particular erythroid precursors. Cloning of intestinal iron transporter proteins provided new insight into mechanisms and regulation of intestinal iron absorption. The aim of this study was to assess whether, in humans, the two transporters are regulated in an iron-dependent manner and whether this regulation is disturbed in HHC. Using quantitative PCR, we measured mRNA expression of divalent cation transporter 1 (DCT1), iron-regulated gene 1 (IREG1), and hephaestin in duodenal biopsy samples of individuals with normal iron levels, iron-deficiency anemia, or iron overload. In controls, we found inverse relationships between the DCT1 splice form containing an iron-responsive element (IRE) and blood hemoglobin, serum transferrin saturation, or ferritin. Subjects with iron-deficiency anemia showed a significant increase in expression of the spliced form, DCT1(IRE) mRNA. Similarly, in subjects homozygous for the C282Y HFE mutation, DCT1(IRE) expression levels remained high despite high serum iron saturation. Furthermore, a significantly increased IREG1 expression was observed. Hephaestin did not exhibit a similar iron-dependent regulation. Our data show that expression levels of human DCT1 mRNA, and to a lesser extent IREG1 mRNA, are regulated in an iron-dependent manner, whereas mRNA of hephaestin is not affected. The lack of appropriate downregulation of apical and basolateral iron transporters in duodenum likely leads to excessive iron absorption in persons with HHC.
Fish oil feeding alters liver gene expressions to defend against PPARα activation and ROS production
Authors: Takahashi Mayumi , Tsuboyama-Kasaoka Nobuyo , Nakatani Teruyo , Ishii Masami , Tsutsumi Shuichi , Aburatani Hiroyuki , Ezaki Osamu
Published: March 2015

Fish oil rich in n-3 polyunsaturated fatty acids has been shown to reduce the risk of cardiovascular diseases partly by reduction of blood triglyceride concentration. This favorable effect mainly results from the combined effects of inhibition of lipogenesis by decrease of SREBP-1 and stimulation of fatty acid oxidation by activation of peroxisome proliferator-activated receptor-α (PPARα) in liver. However, because fish oil is easily peroxidized to form hydroperoxides and increases oxidative stress, some defense mechanism(s) against oxidative stress might occur. To understand these complex effects of fish oil diet, the gene expression profile of mice liver was analyzed using high-density oligonucleotide arrays. High-fat diet (60% of total energy intake) as either safflower oil or fish oil (tuna) was given to mice. After 6 mo of feeding, expression levels of a total of 6,521 genes were analyzed. In fish oil diet compared with safflower oil diet, immune reaction-related genes, antioxidant genes (several glutathione transferases, uncoupling protein 2, and Mn-superoxide dismutase), and lipid catabolism-related genes upregulated, whereas cholesterol and fatty acid synthesis-related genes and 17-alpha hydroxylase/C17–20 lyase and sulfotransferases related to production of endogenous PPARα ligands and reactive oxygen species (ROS) downregulated markedly. Because upregulation of these antioxidant genes and downregulation of sulfotransferases were also observed in mice administered fenofibrate, altered gene expression related to antioxidant system observed in fish oil feeding was mediated directly and indirectly by PPARα activation. However, downregulation of 17-alpha hydroxylase/C17–20 lyase was not due to PPARα activation. These data indicate that fish oil feeding downregulated the endogenous PPARα-activation system and increased antioxidant gene expressions to protect against ROS excess.
Electrical stimulation reveals complex neuronal input and activation patterns in single myenteric guinea pig ganglia
Authors: Bisschops R. , Vanden Berghe P. , Bellon E. , Janssens J. , Tack J.
Published: March 2015

The myenteric plexus plays a key role in the control of gastrointestinal motility. We used confocal calcium imaging to study responses to electrical train stimulation (ETS) of interganglionic fiber tracts in entire myenteric ganglia of the guinea pig small intestine. ETS induced calcium transients in a subset of neurons: 52.2% responded to oral ETS, 65.4% to aboral ETS, and 71.7% to simultaneous oral and aboral ETS. A total of 41.3% of the neurons displayed convergence of oral and aboral ETS-induced responses. Responses could be reversibly blocked with TTX (10− 6 M), demonstrating involvement of neuronal conduction, and by removal of extracellular calcium. ω-Conotoxin (5 × 10−7 M) blocked the majority of responses and reduced the amplitude of residual responses by 45%, indicating the involvement of N-type calcium channels. Staining for calbindin and calretinin did not reveal different response patterns in these immunohistochemically identified neurons. We conclude that, at least for ETS close to a ganglion, confocal calcium imaging reveals complex oral and aboral input to individual myenteric neurons rather than a polarization in spread of activity.
Cloning of human agmatinase. An alternate path for polyamine synthesis induced in liver by hepatitis B virus
Authors: Mistry Sanjay K. , Burwell Tim J. , Chambers Rebecca M. , Rudolph-Owen Laura , Spaltmann Frank , Cook W. Jim , Morris Sidney M.
Published: March 2015

Agmatinase, which hydrolyzes agmatine to putrescine and urea, not only represents a potentially important mechanism for regulating the biological effects of agmatine in mammalian cells but also represents an alternative to ornithine decarboxylase for polyamine biosynthesis. We have isolated a full-length cDNA encoding human agmatinase whose function was confirmed by complementation in yeast. The single-copy human agmatinase gene located on chromosome 1 encodes a 352-residue protein with a putative mitochondrial targeting sequence at the NH3-terminus. Human agmatinase has about 30% identity to bacterial agmatinases and <20% identity to mammalian arginases. Residues required for binding of Mn2+at the active site in bacterial agmatinase and other members of the arginase superfamily are fully conserved in human agmatinase. Agmatinase mRNA is most abundant in human liver and kidney but also is expressed in several other tissues, including skeletal muscle and brain. Its expression in human liver is induced during hepatitis B virus infection, suggesting that agmatinase may play a role in the pathophysiology of this disease.
Modulation of Cl−/OH−exchange activity in Caco-2 cells by nitric oxide
Authors: Saksena Seema , Gill Ravinder K. , Syed Irfan A. , Tyagi Sangeeta , Alrefai Waddah A. , Ramaswamy K. , Dudeja Pradeep K.
Published: March 2015

The present studies were undertaken to determine the direct effects of nitric oxide (NO) released from an exogenous donor, S-nitroso- N-acetyl pencillamine (SNAP) on Cl−/OH−exchange activity in human Caco-2 cells. Our results demonstrate that NO inhibits Cl−/OH−exchange activity in Caco-2 cells via cGMP-dependent protein kinases G (PKG) and C (PKC) signal-transduction pathways. Our data in support of this conclusion can be outlined as follows: 1) incubation of Caco-2 cells with SNAP (500 μM) for 30 min resulted in ∼50% inhibition of DIDS-sensitive36Cl uptake; 2) soluble guanylate cyclase inhibitors Ly-83583 and (1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one significantly blocked the inhibition of Cl−/OH−exchange activity by SNAP; 3) addition of 8-bromo-cGMP (8-BrcGMP) mimicked the effects of SNAP; 4) specific PKG inhibitor KT-5823 significantly inhibited the decrease in Cl−/OH−exchange activity in response to either SNAP or 8-BrcGMP; 5) Cl−/OH−exchange activity in Caco-2 cells in response to SNAP was not altered in the presence of protein kinase A (PKA) inhibitor (Rp-cAMPS), demonstrating that the PKA pathway was not involved; 6) the effect of NO on Cl−/OH−exchange activity was mediated by PKC, because each of the two PKC inhibitors chelerythrine chloride and calphostin C blocked the SNAP-mediated inhibition of Cl−/OH−exchange activity; 7) SO[Formula: see text]/OH−exchange in Caco-2 cells was unaffected by SNAP. Our results suggest that NO-induced inhibition of Cl−/OH−exchange may play an important role in the pathophysiology of diarrhea associated with inflammatory bowel diseases.
Taurocholate prevents the loss of intrahepatic bile ducts due to vagotomy in bile duct-ligated rats
Authors: Marzioni Marco , LeSage Gene D. , Glaser Shannon , Patel Tushar , Marienfeld Carla , Ueno Yoshiyuki , Francis Heather , Alvaro Domenico , Tadlock Laura , Benedetti Antonio , Marucci Luca , Baiocchi Leonardo , Phinizy Jo Lynne , Alpini Gianfranco
Published: March 2015

The aim of this study was to determine whether taurocholate prevents vagotomy-induced cholangiocyte apoptosis. After bile duct ligation (BDL) + vagotomy, rats were fed taurocholate for 1 wk in the absence or presence of wortmannin. Caspase involvement was evaluated by measurement of caspase 8, 9, and 3 activities. Proliferation was determined by morphometry and PCNA immunoblots. Changes in phosphatidylinositol 3-kinase (PI3-kinase) activity were estimated by the expression of the phosphorylated Akt protein. Apically located Na+-dependent bile acid transporter (ABAT) expression and activity were evaluated by immunoblots and [3H]taurocholate uptake, respectively. Cholangiocyte apoptosis increased, whereas proliferation decreased in BDL + vagotomy rats. Taurocholate feeding prevented vagotomy effects on cholangiocyte functions, which were abolished by wortmannin. ABAT expression and activity as well as phosphorylated Akt protein expression were reduced by vagotomy but restored by taurocholate. The activities of caspase 8, 9, and 3 increased in BDL + vagotomy rats but were restored by taurocholate. The protective effect of taurocholate was associated with maintenance of ABAT activity, downregulation of caspase 8, 9, and 3, and activation of PI3-kinase. Bile acids are important in modulating cholangiocyte proliferation in denervated livers.
Gastric H-K-ATPase and acid-resistant surface proteins
Authors: Thangarajah Hariharan , Wong Aline , Chow Dar C. , Crothers James M. , Forte John G.
Published: March 2015

Despite the fact that mucus and bicarbonate are important macroscopic components of the gastric mucosal barrier, severe acidic and peptic conditions surely exist at the apical membrane of gastric glandular cells, and these membranes must have highly specialized adaptations to oppose external insults. Parietal cells abundantly express the heterodimeric, acid-pumping H-K-ATPase in their apical membranes. Its β-subunit (HKβ), a glycoprotein with >70% of its mass and all its oligosaccharides on the extracellular side, may play a protective role. Here, we show that the extracellular domain of HKβ is highly resistant to trypsin in the native state (much more than that of the structurally related Na-K-ATPase β-subunit) and requires denaturation to expose tryptic sites. Native HKβ also resists other proteases, such as chymotrypsin and V8 protease, which hydrolyze at hydrophobic and anionic amino acids, respectively. Removal of terminal α-anomeric-linked galactose does not appreciably alter tryptic sensitivity of HKβ. However, full deglycosylation makes HKβ much more susceptible to all proteases tested, including pepsin at pH <2.0. We propose that 1) intrinsic folding of HKβ, 2) bonding forces between subunits, and 3) oligosaccharides on HKβ provide a luminal protein domain that resists gastric lytic conditions. Protein folding that protects susceptible charged amino acids and is maintained by disulfide bonding and hydrophilic oligosaccharides would provide a stable structure in the face of large pH changes. The H-K-ATPase is an obvious model, but other gastric luminally exposed proteins are likely to possess analogous protective specializations.
Selective phosphorylation of the IP3R-I in vivo by cGMP-dependent protein kinase in smooth muscle
Authors: Murthy Karnam S. , Zhou Huiping
Published: March 2015

This study examined the expression of inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) types and PKG isoforms in isolated gastric smooth muscle cells and determined the ability of PKG and PKA to phosphorylate IP3Rs and inhibit IP3-dependent Ca2+release, which mediates the initial phase of agonist-induced contraction. PKG-Iα and PKG-Iβ were expressed in gastric smooth muscle cells, together with IP3-R-associated cG-kinase substrate, a protein that couples PKG-Iβ to IP3R-I. IP3R-I and IP3R-III were also expressed, but only IP3R-I was phosphorylated by PKA and PKG in vitro and exclusively by PKG in vivo. Sequential phosphorylation by PKA and by PKG-Iα in vitro showed that PKA phosphorylated the same site as PKG (presumably S1755) and an additional PKA-specific site (S1589). In intact muscle cells, agents that activated PKG or both PKG and PKA induced IP3R-I phosphorylation that was reversed by the PKG inhibitor (8R,9S,11s)-(−)-9-methoxy-carbamyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H,1H,-2,7b,11a-trizadizo-benzo9(a,g)cycloocta(c,d,e)-trinden-1-one. Agents that activated PKA induced IP3R-I phosphorylation in permeabilized but not intact muscle cells, implying that PKA does not gain access to IP3R-I in intact muscle cells. The pattern of IP3R-I phosphorylation in vivo and in vitro was more consistent with phosphorylation by PKG-Iα. Phosphorylation of IP3R-I in microsomes by PKG, PKA, or a combination of PKG and PKA inhibited IP3-induced Ca2+release to the same extent, implying that inhibition was mediated by phosphorylation of the PKG-specific site. We conclude that IP3R-I is selectively phosphorylated by PKG-I in intact smooth muscle resulting in inhibition of IP3-dependent Ca2+release.
Multiple transcription factors in 5′-flanking region of human polymeric Ig receptor control its basal expression
Authors: Solorzano-Vargas R. Sergio , Wang Jiafang , Jiang Lingling , Tsai Hugh V. , Ontiveros Luis O. , Vazir Mukta A. , Aguilera Renato J. , Martín Martín G.
Published: March 2015

The polymeric Ig receptor ( pIgR) is a critical component of the mucosal immune system and is expressed in largest amounts in the small intestine. In this study, we describe the initial characterization of the core promoter region of this gene. Expression of chimeric promoter-reporter constructs was supported in Caco-2 and HT-29 cells, and DNase I footprint analysis revealed a large protein complex within the core promoter region. Site-directed mutagenesis experiments determined that elements within this region serve to either augment or repress basal activity of the human pIgR promoter. Band shift assays of overlapping oligonucleotides within the core promoter identified eight distinct complexes; the abundance of most complexes was enhanced in post-confluent cells. In summary, we report the characterization of the human pIgR promoter and the essential role that eight different nuclear complexes have in controlling basal expression of this gene in Caco-2 cells.
Hepatic glutathione and nitric oxide are critical for hepatic insulin-sensitizing substance action
Authors: Guarino Maria P. , Afonso Ricardo A. , Raimundo Nuno , Raposo João F. , Macedo M. Paula
Published: March 2015

We tested the hypothesis that hepatic nitric oxide (NO) and glutathione (GSH) are involved in the synthesis of a putative hormone referred to as hepatic insulin-sensitizing substance HISS. Insulin action was assessed in Wistar rats using the rapid insulin sensitivity test (RIST). Blockade of hepatic NO synthesis with N G-nitro-l-arginine methyl ester (l-NAME, 1.0 mg/kg intraportal) decreased insulin sensitivity by 45.1 ± 2.1% compared with control (from 287.3 ± 18.1 to 155.3 ± 10.1 mg glucose/kg, P < 0.05). Insulin sensitivity was restored to 321.7 ± 44.7 mg glucose/kg after administration of an NO donor, intraportal SIN-1 (5 mg/kg), which promotes GSH nitrosation, but not after intraportal sodium nitroprusside (20 nmol · kg−1 · min−1), which does not nitrosate GSH. We depleted hepatic GSH using the GSH synthesis inhibitor l-buthionine-[ S,R]-sulfoximine (BSO, 2 mmol/kg body wt ip for 20 days), which reduced insulin sensitivity by 39.1%. Insulin sensitivity after l-NAME was not significantly different between BSO- and sham-treated animals. SIN-1 did not reverse the insulin resistance induced by l-NAME in the BSO-treated group. These results support our hypothesis that NO and GSH are essential for insulin action.
Proguanylin secretion and the role of negative-feedback inhibition in a villous epithelial cell line
Authors: Rudolph Jeffrey A. , Hawkins Jennifer A. , Cohen Mitchell B.
Published: March 2015

The mechanisms of proguanylin synthesis and secretion in the intestine are incompletely understood. We designed an in vitro model to study proguanylin secretion in a model of intestinal villous epithelial cells. The C2/bbe1 cell line, a differentiated subclone of Caco-2 cells, was used to examine the direction of proguanylin secretion and the potential for feedback regulation via activators of the guanylyl cyclase C signal transduction pathway. When cells were grown on Transwell inserts, proguanylin was secreted into the apical and basolateral media, consistent with other models of intestinal guanylin secretion. Proguanylin synthesis and secretion were not decreased on activation of guanylyl cyclase C-mediated chloride secretion, implying a regulatory system other than negative-feedback inhibition. These data describe the use of C2/bbe1 cells as a model for proguanylin secretion in villous epithelial cells and demonstrate their potential use for the study of the regulatory mechanisms governing proguanylin synthesis and secretion.
5-HT2Areceptors: location and functional analysis in intestines of wild-type and 5-HT2Aknockout mice
Authors: Fiorica-Howells Elena , Hen Rene , Gingrich Jay , Li Zhishan , Gershon Michael D.
Published: March 2015

The distribution and function of the 5-hydroxytryptamine (5-HT2A) receptor were investigated in the intestines of wild-type (5-HT2A+/+) and knockout (5-HT2A−/−) mice. In 5-HT2A+/+ mice, rats, and guinea pigs, 5-HT2Areceptor immunoreactivity was found on circular and longitudinal smooth muscle cells, neurons, enterocytes, and Paneth cells. Muscular 5-HT2Areceptors were concentrated in caveolae; neuronal 5-HT2Areceptors were found intracellularly and on the plasma membranes of nerve cell bodies and axons. Neuronal 5-HT2Aimmunoreactivity was detected as early as E14 in ganglia, intravillus nerves, and the deep muscle plexus. The 5-HT2A−/− colon did not express 5-HT2Areceptors and did not contract in response to exogenous 5-HT. 5-HT2A−/− enterocytes were smaller, Paneth cells fewer, and muscle layers thinner (and showed degeneration) compared with those of 5-HT2A+/+ littermates. The 5-HT2Areceptor may thus be required for the maintenance and/or development of enteric neuroeffectors and other enteric functions, although gastrointestinal and colonic transit times in 5-HT2A−/− and +/+ mice did not differ significantly.
Glucose alleviates ammonia-induced inhibition of short-chain fatty acid metabolism in rat colonic epithelial cells
Authors: Cremin John D. , Fitch Mark D. , Fleming Sharon E.
Published: March 2015

Ammonia decreased metabolism by rat colonic epithelial cells of butyrate and acetate to CO2and ketones but increased oxidation of glucose and glutamine. Ammonia decreased cellular concentrations of oxaloacetate for all substrates evaluated. The extent to which butyrate carbon was oxidized to CO2after entering the tricarboxylic acid (TCA) cycle was not significantly influenced by ammonia, suggesting there was no major shift toward efflux of carbon from the TCA cycle. Ammonia reduced entry of butyrate carbon into the TCA cycle, and the proportion of CoA esterified with acetate and butyrate correlated positively with the production of CO2and ketone bodies. Also, ammonia reduced oxidation of propionate but had no effect on oxidation of 3-hydroxybutyrate. Inclusion of glucose, lactate, or glutamine with butyrate and acetate counteracted the ability of ammonia to decrease their oxidation. In rat colonocytes, it appears that ammonia suppresses short-chain fatty acid (SCFA) oxidation by inhibiting a step before or during their activation. This inhibition is alleviated by glucose and other energy-generating compounds. These results suggest that ammonia may only affect SCFA metabolism in vivo when glucose availability is compromised.
Sexual dimorphic expression of ADH in rat liver: importance of the hypothalamic-pituitary-liver axis
Authors: Simon Francis R. , Fortune John , Iwahashi Mieko , Sutherland Eileen
Published: March 2015

Hepatic alcohol dehydrogenase (ADH) activity is higher in female than in male rats. Although sex steroids, thyroid, and growth hormone (GH) have been shown to regulate hepatic ADH, the mechanism(s) for sexual dimorphic expression is unclear. We tested the possibility that the GH secretory pattern determined differential expression of ADH. Gonadectomized and hypophysectomized male and female rats were examined. Hepatic ADH activity was 2.1-fold greater in females. Because protein and mRNA content were also 1.7- and 2.4-fold greater, results indicated that activity differences were due to pretranslational mechanisms. Estradiol increased ADH selectively in males, and testosterone selectively decreased activity and mRNA levels in females. Effect of sex steroids on ADH was lost after hypophysectomy; infusion of GH in males increased ADH to basal female levels, supporting a role of the pituitary-liver axis. However, GH andl-thyroxine (T4) replacements alone in hypophysectomized rats did not restore dimorphic differences for either ADH activity or mRNA levels. On the other hand, T4in combination with intermittent administration of GH reduced ADH activity and mRNA to basal male values, whereas T4plus GH infusion replicated female levels. These results indicate that the intermittent male pattern of GH secretion combined with T4is the principal determinant of low ADH activity in male liver.