bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of cholera toxin on glutamine metabolism and transport in rabbit ileum
Authors: Abely M. , Dallet P. , Boisset M. , Desjeux J. F.
Published: December 2017

The aim of the present study was to evaluate the effect of cholera toxin on energy balance from intestinal glutamine metabolism and oxidation, glutamine-dependent sodium absorption, and cholera toxin-dependent ion flux. Cholera toxin-stimulated sodium andl-glutamine ileal transport and metabolism were studied in Ussing chambers. Glutamine (10 mM) transport and metabolism were simultaneously studied using 14C flux and HPLC. In the same tissues, the flux of each amino acid was studied by HPLC, and glutamine metabolism and oxidation were studied by the determination of amino acid specific activity and 14CO2production. In control tissues, glutamine stimulated sodium absorption and was mainly oxidized. The transepithelial flux of intact glutamine represented 45% of glutamine flux across the luminal membrane. The other metabolites were glutamate and, to a lesser degree, citrulline, ornithine, and proline. Cholera toxin did not alter glutamine-stimulated sodium absorption, glutamine oxidation, transport, and metabolism. In conclusion, the present results indicate that cholera toxin does not alter glutamine intestinal function and metabolism. In addition, ∼95% of the energy provided by glutamine oxidation remains available to the enterocyte.
Pancreatic CCKBreceptors: their potential roles in somatostatin release and δ-cell proliferation
Authors: Morisset Jean , Wong Helen , Walsh John H. , Lainé J. , Bourassa Judith
Published: December 2017

In rodents, cholecystokinin (CCK) induces pancreatic enzyme secretion and pancreas growth through its CCKAreceptors. It is unknown whether occupation of the CCKBreceptors present in pig and human pancreas can cause the same effects. This study evaluates CCKBreceptor expression in rat, mouse, pig, and fetal human pancreata using Northern blot, Western blot, and immunofluorescence techniques. The reported 2.7-kb CCKBreceptor mRNA transcript in the rat brain and gastric fundus is absent in pancreas; the message was, however, detected by RT-PCR and by a CCKBreceptor antibody as an 80-kDa protein present uniquely in islet δ-cells. Proteins of 50 and 80 kDa appear in mouse pancreas, and proteins of 50 and 115 kDa appear in pig and human pancreas, respectively, all localized in islet δ-cells. Gastrin mRNAs are strongly present in fetal rat pancreas, and the hormone is localized in islets; both are repressed 10 days after birth. In conclusion, the CCKBreceptors are present in pancreas of four species with exclusive location in islet δ-cells. In such a location, they could be indirectly involved in the control of enzyme secretion.
Sustained muscle contraction induced by agonists, growth factors, and Ca2+mediated by distinct PKC isozymes
Authors: Murthy K. S. , Grider J. R. , Kuemmerle J. F. , Makhlouf G. M.
Published: December 2017

The role of protein kinase C (PKC) in sustained contraction was examined in intestinal circular and longitudinal muscle cells. Initial contraction induced by agonists (CCK-8 and neuromedin C) was abolished by 1) inhibitors of Ca2+mobilization (neomycin and dimethyleicosadienoic acid), 2) calmidazolium, and 3) myosin light chain (MLC) kinase (MLCK) inhibitor KT-5926. In contrast, sustained contraction was not affected by these inhibitors but was abolished by 1) the PKC inhibitors chelerythrine and calphostin C, 2) PKC-ε antibody, and 3) a pseudosubstrate PKC-ε inhibitor. GDPβS abolished both initial and sustained contraction, whereas a Gαq/11antibody inhibited only initial contraction, implying that sustained contraction was dependent on activation of a distinct G protein. Sustained contraction induced by epidermal growth factor was inhibited by calphostin C, PKC-α,β,γ antibody, and a pseudosubstrate PKC-α inhibitor. Ca2+(0.4 μM) induced an initial contraction in permeabilized muscle cells that was blocked by calmodulin and MLCK inhibitors and a sustained contraction that was blocked by calphostin C and a PKC-α,β,γ antibody. Thus initial contraction induced by Ca2+, agonists, and growth factors is mediated by MLCK, whereas sustained contraction is mediated by specific Ca2+-dependent and -independent PKC isozymes. G protein-coupled receptors are linked to PKC activation via distinct G proteins.
Increased nuclear translocation of catalytically active PKC-ζ during mouse colonocyte hyperproliferation
Authors: Umar Shahid , Sellin Joseph H. , Morris Andrew P.
Published: December 2017

Protein kinase (PK) C-ζ is implicated in the control of colonic epithelial cell proliferation in vitro. However, less is known about its physiological role in vivo. Using the transmissible murine colonic hyperplasia (TMCH) model, we determined its expression, subcellular localization, and kinase activity during native crypt hyperproliferation. Enhanced mitosis was associated with increased cellular 72-kDa holoenzyme (PKC-ζ, 3.2-fold), 48-kDa catalytic subunit (PKM-ζ, 3- to 9-fold), and 24-kDa membrane-bound fragment (Mf-ζ, >10-fold) expression. Both PKC-ζ and PKM-ζ exhibited intrinsic kinase activity, and substrate phosphorylation increased 4.5-fold. No change in cellular PKC-ι/PKM-ι expression occurred. The subcellular distribution of immunoreactive PKC-ζ changed significantly: neck cells lost their basal subcellular pole filamentous staining, whereas proliferating cell nuclear antigen-positive cells exhibited elevated cytoplasmic, lateral membrane, and nuclear staining. Subcellular fractionation revealed increased PKC-ζ and PKM-ζ expression and activity within nuclei, which preferentially accumulated PKM-ζ. These results suggest separate cellular and nuclear roles, respectively, for PKC-ζ in quiescent and mitotically active colonocytes. PKM-ζ may specifically act as a modulator of proliferation during TMCH.
Expression and canalicular localization of two isoforms of the ClC-3 chloride channel from rat hepatocytes
Authors: Shimada Kazuo , Li Xinhua , Xu Guiyan , Nowak David E. , Showalter Lori A. , Weinman Steven A.
Published: December 2017

The molecular identities of functional chloride channels in hepatocytes are largely unknown. We examined the ClC-3 chloride channel in rat hepatocytes and found that mRNA for two different isoforms is present. A short form is identical to the previously reported sequence for rat ClC-3, and a long form contains a 176-bp insertion immediately upstream of the translation initiation site. This predicts a 58-amino acid NH2 terminal insertion. Both long and short form mRNA was expressed in diverse tissues of the rat. Transient transfection of the long form in CHO-K1 cells resulted in currents with an I− > B− > Cl− selectivity sequence, outward rectification, and inactivation at positive voltages. Short form currents had identical ionic selectivity but displayed a more extreme outward rectification and showed no voltage-dependent inactivation. Immunofluorescence and immunoblots localized native ClC-3 preferentially but not exclusively to the canalicular membrane. We have therefore identified a new isoform of rat ClC-3 and shown that expression of both isoforms produces functional channels. In hepatocytes, ClC-3 is located in association with the canalicular membrane.
Cellular mechanism of sodium oleate-stimulated secretion of cholecystokinin and secretin
Authors: Chang Cecilia H. , Chey William Y. , Chang Ta-Min
Published: December 2017

Long-chain fatty acids are potent stimulants of secretin and CCK release. The cellular mechanisms of fatty acid-stimulated secretion of these two hormones are not clear. We studied the stimulatory effect and mechanism of sodium oleate (SO) on secretin- and CCK-producing cells. SO stimulated the release of secretin or CCK from isolated rat mucosal cell preparations enriched in either secretin- or CCK-producing cells, respectively. SO also time- and dose-dependently stimulated secretin and CCK release from STC-1 cells. In STC-1 cells, SO-stimulated secretin and CCK release was potentiated by IBMX and inhibited by a protein kinase A-selective inhibitor and a cAMP-specific antagonist. SO-stimulated releases of the two hormones were also inhibited by downregulation or inhibitors of protein kinase C, a calmodulin antagonist and an inhibitor of calmodulin-dependent protein kinase II. Chelating of extracellular Ca2+or addition of an L-type calcium channel blocker diminished SO-stimulated hormone releases. SO caused an increase in intracellular Ca2+concentration that was partially reversed by diltiazem but had no effect on production of cAMP, cGMP, or inositol-1,4,5-triphosphate. These results indicate that SO acts on secretin- and CCK-producing cells. Its stimulatory effect is potentiated by endogenous protein kinase A and mediated by activation of Ca2+influx through the L-type channels and of protein kinase C and Ca2+/calmodulin-dependent protein kinase II.
Myenteric neurons activate submucosal vasodilator neurons in guinea pig ileum
Authors: Vanner S.
Published: December 2017

This study examined whether myenteric neurons activate submucosal vasodilator pathways in in vitro combined submucosal-myenteric plexus preparations from guinea pig ileum. Exposed myenteric ganglia were electrically stimulated, and changes in the outside diameter of submucosal arterioles were monitored in adjoining tissue by videomicroscopy. Stimulation up to 18 mm from the recording site evoked large TTX-sensitive vasodilations in both orad and aborad directions. In double-chamber baths, which isolated the stimulating myenteric chamber from the recording submucosal chamber, hexamethonium or the muscarinic antagonist 4-diphenylacetoxy- N-(2-chloroethyl)-piperdine hydrochloride (4-DAMP) almost completely blocked dilations when superfused in the submucosal chamber. When hexamethonium was placed in the myenteric chamber ∼50% of responses were hexamethonium sensitive in both orad and aborad orientations. The addition of 4-DAMP or substitution of Ca2+-free, 12 mM Mg2+ solution did not cause further inhibition. These results demonstrate that polysynaptic pathways in the myenteric plexus projecting orad and aborad can activate submucosal vasodilator neurons. These pathways could coordinate intestinal blood flow and motility.
Characterization of mouse A33 antigen, a definitive marker for basolateral surfaces of intestinal epithelial cells
Authors: Johnstone Cameron N. , Tebbutt Niall C. , Abud Helen E. , White Sara J. , Stenvers Kaye L. , Hall Nathan E. , Cody Stephen H. , Whitehead Robert H. , Catimel Bruno , Nice Edouard C. , Burgess Antony W. , Heath Joan K.
Published: December 2017

The murine A33 antigen is emerging as a definitive marker of intestinal epithelial cells. Cloning and sequence determination of cDNAs encoding mA33 antigen predict a novel type 1 transmembrane protein of 298 amino acids, comprising an extracellular domain with two immunoglobulin-like domains, a single-span transmembrane domain, and a highly acidic cytoplasmic domain. On the basis of conservation of amino acid sequence and genomic structure, the mA33 antigen is a member of a growing subfamily within the immunoglobulin superfamily, which includes transmembrane proteins CTX/ChT1, CTM/CTH, and CAR. During embryonic development, mA33 antigen expression is first observed in the inner cell mass of blastocysts before implantation. Intestinal expression of mA33 antigen is initiated in the hindgut at E14.5 and increases steadily throughout late embryonic and postnatal life into adulthood. The protein is specifically expressed on the basolateral surfaces of intestinal epithelial cells of all lineages, independent of their position along the rostrocaudal and crypt-villus axes. Thus the mA33 antigen appears to be a novel marker for both proliferating and differentiating intestinal epithelial cells.
Fibrogenesis IV. Fibrosis and inflammatory bowel disease: cellular mediators and animal models
Authors: Pucilowska Jolanta B. , Williams Kristen L. , Lund P. Kay
Published: December 2017

The cellular mediators of intestinal fibrosis and the relationship between fibrosis and normal repair are not understood. Identification of the types of intestinal mesenchymal cells that produce collagen during normal healing and fibrosis is vital for elucidating the answers to these questions. Acute injury may cause normal mesenchymal cells to convert to a fibrogenic phenotype that is not maintained during normal healing but may lead to fibrosis when inappropriately sustained. Proliferation of normal or fibrogenic mesenchymal cells may lead to muscularis overgrowth associated with fibrosis. The presence of increased numbers of vimentin-positive cells within fibrotic, hypertrophied muscularis in Crohn's disease suggests that changes in mesenchymal cell phenotype and number may indeed be associated with fibrosis. Fibrosis is induced in rats by peptidoglycan polysaccharides or trinitrobenzene sulfonic acid-ethanol administration, but inducing fibrosis in mice has been technically challenging. The development of current mouse models of colitis, such as dextran sodium sulfate or trinitrobenzene sulfonic acid-ethanol administration, into models of fibrosis will allow us to use genetic manipulation to study molecular mediators of fibrosis.
Endothelin-mediated vasoconstriction in postischemic newborn intestine
Authors: Nankervis Craig A. , Schauer Gail M. , Miller Charles E.
Published: December 2017

We previously suggested that the profound, sustained vasoconstriction noted in 3-day-old swine intestine after a moderate episode of ischemia-reperfusion (I/R) reflects the unmasking of underlying constrictor tone consequent to a loss of endothelium-derived nitric oxide (NO). In this study, we sought to determine whether endothelin-1 (ET-1) was the unmasked constrictor and whether selective loss of endothelial ETBreceptors, which mediate NO-based vasodilation, participated in the hemodynamic consequences of I/R in newborn intestine. Studies were performed in innervated, autoperfused intestinal loops in 3- and 35-day-old swine. Selective blockade of ETAreceptors with BQ-610 had no effect on hemodynamics under control conditions; however, when administered before and during I/R, BQ-610 significantly attenuated the post-I/R vasoconstriction and reduction in arteriovenous O2difference in the younger group. In 3-day-old intestine, reduction of intestinal O2uptake to a level similar to that noted after I/R by lowering tissue temperature had no effect on the response to BQ-610 or ET-1, indicating that the change in response to BQ-610 noted after I/R was not simply consequent to the reduction in tissue O2demand. In studies in mesenteric artery rings suspended in myographs, we observed a leftward shift in the dose-response curve for ET-1 after selective blockade of ETBreceptors with BQ-788 in 3- but not 35-day-old swine. Rings exposed to I/R in vivo behaved in a manner similar to control rings treated with BQ-788 or endothelium-denuded non-I/R rings.
NK-1 receptor desensitization and neutral endopeptidase terminate SP-induced pancreatic plasma extravasation
Authors: Maa John , Grady Eileen F. , Kim Ed H. , Yoshimi Shandra K. , Hutter Matthew M. , Bunnett Nigel W. , Kirkwood Kimberly S.
Published: December 2017

Substance P (SP) induces plasma extravasation and neutrophil infiltration by activating the neurokinin-1 receptor (NK1-R). We characterized the mechanisms regulating this response in the rat pancreas. Anesthetized rats were continuously infused with SP, and plasma extravasation was quantified using Evans blue (EB) dye. Continuous infusion of SP (8 nmol · kg−1· h−1) resulted in a threshold increase in EB at 15 min, a peak effect at 30 min (150% increase), and a return to baseline by 60 min. The NK1-R antagonist CP-96,345 blocked SP-induced plasma extravasation. After 60 min, the NK1-R was desensitized to agonist challenge. Resensitization was first detected at 20 min and increased until full recovery was seen at 30 min. Inhibition of the cell-surface protease neutral endopeptidase (NEP) by phosphoramidon potentiated the effect of exogenous SP; therefore endogenous NEP attenuates SP-induced plasma extravasation. Thus the continuous infusion of SP stimulates plasma extravasation in the rat pancreas via activation of the NK1-R, and these effects are terminated by both desensitization of the NK1-R and the cell-surface protease NEP.
Identification of γ-aminobutyric acid receptor subunit types in human and rat liver
Authors: Erlitzki Ronit , Gong Yuewen , Zhang Manna , Minuk Gerald
Published: December 2017

GABA is a potent inhibitory neurotransmitter that binds to heterooligomeric receptors in the mammalian brain. In a previous study, we documented specific GABA binding to isolated rat hepatocytes that resulted in inhibition of hepatocyte proliferation. The purpose of the present study was to define the nature of hepatic GABAAreceptors and to document their expression during rapid liver growth (after partial hepatectomy). PCRs with gene-specific primers derived from published sequences were performed with Marathon-ready human and rat liver cDNA. Two GABAAreceptor subunit types (β3 and ε) were expressed in human liver and one subunit type (β3) in rat liver. PCR amplification of the human GABAAreceptorβ3-subunit produced a single product (molecular mass 53–59 kDa). In the case of the ε-subunit, two PCR products were identified. After partial hepatectomy, GABAAreceptorβ3-subunit expression inversely correlated with regenerative activity ( r = −0.527, P = 0.006). In conclusion, these results indicate that in the human liver GABAAreceptors consist of the β3- and ε-subunit types, whereas in the rat liver only the β3-subunit type is expressed. The results also support the hypothesis that GABAergic activity serves to maintain hepatocytes in a quiescent state.
Actions of reactive oxygen species on AH/type 2 myenteric neurons in guinea pig distal colon
Authors: Wada-Takahashi S. , Tamura K.
Published: December 2017

With conventional intracellular recording methods, we investigated the mechanism of actions of reactive oxygen species (ROS) derived from hypoxanthine and xanthine oxidase (HX/XO) reactions on AH/type 2 myenteric neurons in the guinea pig distal colon. Of the 54 neurons to which HX/XO was applied, 32 neurons showed a transient membrane hyperpolarization(s) followed by a long-lasting membrane depolarization. Two additional groups of 10 myenteric neurons exhibited only a membrane hyperpolarization(s) or a late-onset membrane depolarization, respectively, and the remaining two neurons did not show any response to HX/XO. Analysis of changes of the input resistance induced by HX/XO indicated that suppression and augmentation of the conductance of Ca2+-dependent K+ channels are the ionic mechanisms underlying the membrane hyperpolarization and depolarization, respectively. The effects of HX/XO on myenteric neurons were mimicked by application of caffeine or H2O2. The results suggest that OH·, but neither H2O2 nor O2 · −, is responsible for HX/XO-induced responses. The intracellular Ca2+ store may be the acting site of ROS in colonic AH/type 2 neurons.
Whole cell current and membrane potential regulation by a human smooth muscle mechanosensitive calcium channel
Authors: Holm Adrian N. , Rich Adam , Sarr Michael G. , Farrugia Gianrico
Published: December 2017

Mechanotransduction is required for a wide variety of biological functions. The aim of this study was to determine the effect of activation of a mechanosensitive Ca2+ channel, present in human jejunal circular smooth muscle cells, on whole cell currents and on membrane potential. Currents were recorded using patch-clamp techniques, and perfusion of the bath (10 ml/min, 30 s) was used to mechanoactivate the L-type Ca2+ channel. Perfusion resulted in activation of L-type Ca2+ channels and an increase in outward current from 664 ± 57 to 773 ± 72 pA at +60 mV. Membrane potential hyperpolarized from −42 ± 4 to −50 ± 5 mV. In the presence of nifedipine (10 μM), there was no increase in outward current or change in membrane potential with perfusion. In the presence of charybdotoxin or iberiotoxin, perfusion of the bath did not increase outward current or change membrane potential. A model is proposed in which mechanoactivation of an L-type Ca2+ channel current in human jejunal circular smooth muscle cells results in increased Ca2+ entry and cell contraction. Ca2+ entry activates large-conductance Ca2+-activated K+channels, resulting in membrane hyperpolarization and relaxation.
Swallowing reflex and brain stem neurons activated by superior laryngeal nerve stimulation in the mouse
Authors: Sang Q. , Goyal Raj K.
Published: December 2017

The purpose of the present study was to identify vagal subnuclei that participate in reflex swallowing in response to electrical stimulation of the left superior laryngeal nerve (SLN). SLN stimulation at 10 Hz evoked primary peristalsis, including oropharyngeal and esophageal peristalsis, and LES relaxation. It also induced c- fos expression in interneurons in the interstitial (SolI), intermediate (SolIM), central (SolCe), dorsomedial (SolDM) and commissural (SolC) solitary subnuclei. Neurons in parvicellular reticular nucleus (PCRt) and area postrema (AP) and motoneurons in the semicompact (NAsc), loose (NAl), and compact (NAc) formations of the nucleus ambiguus and both rostral (DMVr) and caudal (DMVc) parts of the dorsal motor nucleus of vagus were also activated. The activated neurons represent all neurons concerned with afferent SLN-mediated reflexes, including the swallowing-related neurons. SLN stimulation at 5 Hz elicited oropharyngeal and LES but not esophageal responses and evoked c- fos expression in neurons in SolI, SolIM, SolDM, PCRt, AP, NAsc, NAl, and DMVc but not in SolCe, NAc, or DMVr. These data are consistent with the role of SolI, SolIM, SolDM, NAsc, NAl, and DMVc circuit in oropharyngeal peristalsis and LES relaxation and SolCe, NAc, DMVc, and DMVr in esophageal peristalsis and LES responses.
Circadian coupling between pancreatic secretion and intestinal motility in humans
Authors: Keller Jutta , Gröger Gabriele , Cherian Leelamma , Günther Britt , Layer Peter
Published: December 2017

Human interdigestive intestinal motility follows a circadian rhythm with reduced nocturnal activity, but circadian pancreatic exocrine secretion is unknown. To determine whether circadian changes in interdigestive pancreatic secretion occur and are associated with motor events, pancreatic enzyme outputs, proximal jejunal motility, and plasma pancreatic polypeptide concentrations were measured during consecutive daytime and nighttime periods (12 h each) in seven healthy volunteers using orojejunal multilumen intubation. Studies were randomly started in the morning or evening. Nocturnally, motility decreased (motor quiescence: 67 ± 22 vs. 146 ± 37 min; motility index: 3.59 ± 0.33 vs. 2.78 ± 0.40 mmHg/min; both P < 0.05) but amylase output increased (273 ± 78 vs. 384 ± 100 U/min; P < 0.05) and protease output remained unchanged ( P > 0.05); consequently, enzyme/motility ratio increased. Amylase outputs were always lowest during phase I. Motor but not pancreatic circadian activities were associated with sleep. Pancreatic polypeptide plasma concentrations were unchanged. Consequently, intestinal motor and pancreatic exocrine functions may have different circadian rhythms, i.e., decreased motor and stable secretory activity during the night. However, the association between individual phases of interdigestive motor and secretory activity is preserved. The nocturnal increase in enzyme/motility ratio is probably not caused by increased cholinergic tone.
Endothelial E- and P-selectin expression in iNOS- deficient mice exposed to polymicrobial sepsis
Authors: Lush Cameron W. , Cepinskas Gediminas , Sibbald William J. , Kvietys Peter R.
Published: December 2017

In vitro, nitric oxide (NO) decreases leukocyte adhesion to endothelium by attenuating endothelial adhesion molecule expression. In vivo, lipopolysaccharide-induced leukocyte rolling and adhesion was greater in inducible NO synthase (iNOS)−/− mice than in wild-type mice. The objective of this study was to assess E- and P-selectin expression in the microvasculature of iNOS−/− and wild-type mice subjected to acute peritonitis by cecal ligation and perforation (CLP). E- and P-selectin expression were increased in various organs within the peritoneum of wild-type animals after CLP. This CLP-induced upregulation of E- and P-selectin was substantially reduced in iNOS−/− mice. Tissue myeloperoxidase (MPO) activity was increased to a greater extent in the gut of wild-type than in iNOS−/− mice subjected to CLP. In the lung, the reduced expression of E-selectin in iNOS−/− mice was not associated with a decrease in MPO. Our findings indicate that NO derived from iNOS plays an important role in sepsis-induced increase in selectin expression in the systemic and pulmonary circulation. However, in iNOS−/− mice, sepsis-induced leukocyte accumulation is affected in the gut but not in the lungs.
Transient vs. prolonged histone hyperacetylation: effects on colon cancer cell growth, differentiation, and apoptosis
Authors: Wu James T. , Archer Sonia Y. , Hinnebusch Brian , Meng Shufen , Hodin Richard A.
Published: December 2017

The role of histone hyperacetylation in regard to growth, differentiation, and apoptosis in colon cancer cells was assessed in an in vitro model system. HT-29 cells were grown in ±10% fetal bovine serum with either 5 mM sodium butyrate or 0.3 μM trichostatin A [single dose (T) or 3 doses 8 h apart (TR)] for 24 h. Serum-starved HT-29 cells were further treated with epidermal growth factor or insulin-like growth factor I for an additional 24 h. Apoptosis was quantified with propidium iodide and characterized by electron microscopy. Northern blot analyses were performed with cDNA probes specific for intestinal alkaline phosphatase, Na-K-2Cl cotransporter, the cell cycle inhibitor p21, and the actin control. Flow cytometric analysis revealed a time-dependent growth suppression along with early induction of p21 mRNA in the butyrate, T, and TR groups. Histone hyperacetylation, assessed by acid-urea-triton gel electrophoresis, was transient in the T group but persisted for up to 24 h in the butyrate and TR groups. Induction of apoptosis, growth factor unresponsiveness, and differentiation occurred in the butyrate- and TR-treated cells but not those treated with a single dose of trichostatin A. Thus transient hyperacetylation of histones is sufficient to induce p21 expression and produce cellular growth arrest, but prolonged histone hyperacetylation is required for induction of the programs of differentiation, apoptosis, and growth factor unresponsiveness.
Nutrient-induced spatial patterning of human duodenal motor function
Authors: Andrews Jane M. , Doran Selena M. , Hebbard Geoffrey S. , Malbert Charles H. , Horowitz Michael , Dent John
Published: December 2017

The spatiotemporal patterning of duodenal motor function has been evaluated comprehensively for the first time in humans, with a novel 21-lumen manometric assembly. In nine young, healthy volunteers (6 male, 3 female), duodenal motility was recorded during fasting and three 45-min intraduodenal (ID) nutrient infusion periods (Intralipid at 0.25, 0.5, and 1.5 kcal/min). Pressures were recorded along the length of the duodenum with an array of 18 sideholes at 1.5-cm intervals. Pressure patterns were compared for the final 20 min of each of the four periods. Compared with fasting, ID lipid was associated with regional variation in pressure wave (PW) sequences, with fewer proximally and more distally; this was not observed during fasting ( P < 0.001). During fasting and all rates of lipid infusion, most (87–90%) PW sequences were short (1.5–4.5 cm), with a small number (2–4%) of 10.5 cm or longer. At all times, antegrade PW sequences occurred more frequently than retrograde sequences over all distances examined (3, 4.5, and >6 cm), and the proportion of antegrade sequences increased with greater PW sequence length ( P = 0.0001). Increasing ID lipid rates appeared to produce dose-related suppression of PW sequences ( P< 0.001). The frequency and spatial patterning of human duodenal motor function show substantial variability in response to different nutrient delivery rates. These complex patterns are likely to be involved in duodenal modulation of flow and gastric emptying rate.
Endothelin ETAand ETBreceptors in postnatal intestine
Authors: Nankervis Craig A. , Dunaway David J. , Miller Charles E.
Published: December 2017

We aimed to characterize endothelin (ET) receptors in the swine intestinal vasculature and to determine ischemia-reperfusion (I/R) effects on these receptors. Saturation and competitive binding assays were performed on mesenteric artery protein membranes from 1- and 40-day-old animals, both control and those subjected to 1 h of partial ischemia followed by 6 h of reperfusion in vivo. Scatchard analysis of saturation binding with125I-labeled ET-1 in membranes from endothelium-denuded (E−) vessels revealed that the maximum number of binding sites was greater in younger animals. Competitive125I-ET-1 binding was significant for a one-site model with ET-1, ET-3, and sarafotoxin S6c (S6c) in membranes from endothelium-intact (E+) and E−vessels in both age groups. The maximum number of ET-1 binding sites was significantly greater in younger animals. In the presence of the ETAreceptor antagonist BQ-123, competitive125I-ET-1 binding was significant for a one-site model with ET-1 and S6c in membranes from E+vessels in both age groups. The maximum number of ET-1 binding sites was significantly greater in younger animals. After I/R, the maximum number of ET-1 binding sites was unchanged. In the presence of BQ-123, specific binding by ET-1 and S6c was eliminated in both age groups after I/R. These results suggest that both ET receptor populations are expressed to a greater degree in younger animals and I/R significantly affects the ETBreceptor.
Immunolocalization, ontogeny, and regulation of microsomal triglyceride transfer protein in human fetal intestine
Authors: Levy E. , Stan S. , Garofalo C. , Delvin E. E. , Seidman E. G. , Ménard D.
Published: December 2017

To examine the multiple stages of lipoprotein packaging during development, we studied localization, ontogeny, and regulation of microsomal transfer protein (MTP), a crucial protein for lipid transport. With the use of immunofluorescence, MTP was identified in villus and crypt epithelial cells in different regions of human fetal intestine, including colon. Staining was detected as early as the 13th wk of gestation in all gut segments and was almost entirely confined to the columnar epithelial cells of the jejunum and colon. Unlike immunofluorescence, which provides qualitative but not quantitative information on MTP signal, enzymatic assays revealed a decreasing gradient from proximal small intestine to distal, as confirmed by immunoblot. Activity of MTP in small intestinal explants cultured for different incubation periods (0, 4, 8, and 24 h) peaked at 4 h but remained insensitive to different concentrations of oleic acid. Also, a trend toward increasing MTP activity was observed at 20–22 wk of gestation. Finally, in strong contrast to jejunal efficiency, colonic explants displayed impaired lipid production, apolipoprotein biogenesis, and lipoprotein assembly, in association with poor expression of MTP. These findings provide the first evidence that human fetal gut is able to express MTP and emphasize the distinct regional distribution, regulation by oleic acid, and ontogeny of MTP.
Diphenyleneiodonium sulfate, an NADPH oxidase inhibitor, prevents early alcohol-induced liver injury in the rat
Authors: Kono Hiroshi , Rusyn Ivan , Uesugi Takehiko , Yamashina Shunhei , Connor Henry D. , Dikalova Anna , Mason Ronald P. , Thurman Ronald G.
Published: December 2017

The oxidant source in alcohol-induced liver disease remains unclear. NADPH oxidase (mainly in liver Kupffer cells and infiltrating neutrophils) could be a potential free radical source. We aimed to determine if NADPH oxidase inhibitor diphenyleneiodonium sulfate (DPI) affects nuclear factor-κB (NF-κB) activation, liver tumor necrosis factor-α (TNF-α) mRNA expression, and early alcohol-induced liver injury in rats. Male Wistar rats were fed high-fat liquid diets with or without ethanol (10–16 g · kg−1· day−1) continuously for up to 4 wk, using the Tsukamoto-French intragastric enteral feeding protocol. DPI or saline vehicle was administered by subcutaneous injection for 4 wk. Mean urine ethanol concentrations were similar between the ethanol- and ethanol plus DPI-treated groups. Enteral ethanol feeding caused severe fat accumulation, mild inflammation, and necrosis in the liver (pathology score, 4.3 ± 0.3). In contrast, DPI significantly blunted these changes (pathology score, 0.8 ± 0.4). Enteral ethanol administration for 4 wk also significantly increased free radical adduct formation, NF-κB activity, and TNF-α expression in the liver. DPI almost completely blunted these parameters. These results indicate that DPI prevents early alcohol-induced liver injury, most likely by inhibiting free radical formation via NADPH oxidase, thereby preventing NF-κB activation and TNF-α mRNA expression in the liver.
P2Y11, a purinergic receptor acting via cAMP, mediates secretion by pancreatic duct epithelial cells
Authors: Nguyen T. D. , Meichle S. , Kim U. S. , Wong T. , Moody M. W.
Published: December 2017

Pancreatic duct epithelial cells (PDEC) mediate the exocrine secretion of fluid and electrolytes. We previously reported that ATP and UTP interact with P2Y2 receptors on nontransformed canine PDEC to increase intracellular free Ca2+ concentration ([Ca2+]i) and stimulate Ca2+-activated Cl− and K+channels. We now report that ATP interacts with additional purinergic receptors to increase cAMP and activate Cl−channels. ATP, 2-methylthio-ATP, and ATP-γ-S stimulated a 4- to 10-fold cAMP increase with EC50 of 10–100 μM. Neither UTP nor adenosine stimulated a cAMP increase, excluding a role for P2Y2 or P1 receptors. Although UTP stimulated an125I− efflux that was fully inhibited by 1,2-bis(2-aminophenoxy)ethane- N,N,N′,N′-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA-AM), ATP stimulated a partially resistant efflux, suggesting activation of additional Cl−conductances through P2Y2-independent and Ca2+-independent pathways. In Ussing chambers, increased cAMP stimulated a much larger short-circuit current ( I sc) increase from basolaterally permeabilized PDEC monolayers than increased [Ca2+]i. Luminal ATP and UTP and serosal UTP stimulated a small Ca2+-type I sc increase, whereas serosal ATP stimulated a large cAMP-type I scresponse. Serosal ATP effect was inhibited by P2 receptor blockers and unaffected by BAPTA-AM, supporting ATP activation of Cl−conductances through P2 receptors and a Ca2+-independent pathway. RT-PCR confirmed the presence of P2Y11 receptor mRNA, the only P2Y receptor acting via cAMP.
Key role of PKC and Ca2+in EGF protection of microtubules and intestinal barrier against oxidants
Authors: Banan A. , Fields J. Z. , Zhang Y. , Keshavarzian A.
Published: December 2017

Using monolayers of human intestinal (Caco-2) cells, we showed that growth factors (GFs) protect microtubules and barrier integrity against oxidative injury. Studies in nongastrointestinal cell models suggest that protein kinase C (PKC) signaling is key in GF-induced effects and that cytosolic calcium concentration ([Ca2+]i) is essential in cell integrity. We hypothesized that GF protection involves activating PKC and maintaining normal [Ca2+]i. Monolayers were pretreated with epidermal growth factor (EGF) or PKC or Ca2+modulators before exposure to oxidants (H2O2or HOCl). Oxidants disrupted microtubules and barrier integrity, and EGF protected from this damage. EGF caused rapid distribution of PKC-α, PKC-βI, and PKC-ζ isoforms to cell membranes, enhancing PKC activity of membrane fractions while reducing PKC activity of cytosolic fractions. EGF enhanced45Ca2+efflux and prevented oxidant-induced (sustained) rises in [Ca2+]i. PKC inhibitors abolished and PKC activators mimicked EGF protection. Oxidant damage was mimicked by and potentiated by a Ca2+ionophore (A-23187), exacerbated by high-Ca2+media, and prevented by calcium removal or chelation or by Ca2+channel antagonists. PKC activators mimicked EGF on both45Ca2+efflux and [Ca2+]i. Membrane Ca2+-ATPase pump inhibitors prevented protection by EGF or PKC activators. In conclusion, EGF protection of microtubules and the intestinal epithelial barrier requires activation of PKC signal transduction and normalization of [Ca2+]i.
Regulation of apolipoprotein secretion by long-chain polyunsaturated fatty acids in newborn swine enterocytes
Authors: Wang Heng , Lu Song , Du Jianhui , Yao Ying , Berschneider Helen M. , Black Dennis D.
Published: December 2017

Long-chain polyunsaturated fatty acids (LC-PUFA) are important in the development of the immature nervous system, and adding these fatty acids to infant formula has been proposed. To determine the effect of n-3 LC-PUFA on apolipoprotein secretion and lipid synthesis in newborn swine enterocytes, differentiated IPEC-1 cells were incubated for 24 h with docosahexaenoic acid (DHA; 22:6) or eicosapentaenoic acid (EPA; 20:5) complexed with albumin at a fatty acid concentration of 0.8 mM or albumin alone (control) added to the apical medium. Oleic acid (OA; 18:1) was used a control for lipid-labeling studies. Both DHA and EPA reduced apolipoprotein (apo) B secretion by one-half, whereas EPA increased apo A-I secretion. The increased apo A-I secretion occurred primarily in the high-density lipoprotein fraction. These changes in apoprotein secretion were not accompanied by significant changes in synthesis. Modest decreases in apo B mRNA levels were observed for DHA and EPA, whereas there were no changes in apo A-I mRNA abundance. EPA reduced cellular triacylglycerol labeling by one-half, and DHA and EPA decreased cellular phospholipid labeling compared with OA. Labeled triacylglycerol secretion was decreased 75% by EPA, and DHA doubled labeled phospholipid secretion. If present in vivo, these effects should be considered before supplementing infant formula with these fatty acids.
Interleukin-6 expression and regulation in rat enteric glial cells
Authors: Rühl A. , Franzke S. , Collins S. M. , Stremmel W.
Published: December 2017

As yet, little is known about the function of the glia of the enteric nervous system (ENS), particularly in an immune-stimulated environment. This prompted us to study the potential of cultured enteroglial cells for cytokine synthesis and secretion. Jejunal myenteric plexus preparations from adult rats were enzymatically dissociated, and enteroglial cells were purified by complement-mediated cytolysis and grown in tissue culture. Cultured cells were stimulated with recombinant rat interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α, and IL-6 mRNA expression and secretion were assessed using RT-PCR and a bioassay, respectively. Stimulation with TNF-α did not affect IL-6 mRNA expression, whereas IL-1β stimulated IL-6 mRNA and protein synthesis in a time- and concentration-dependent fashion. In contrast, IL-6 significantly and dose-dependently suppressed IL-6 mRNA expression. In summary, we have presented evidence that enteric glial cells are a potential source of IL-6 in the myenteric plexus and that cytokine production by enteric glial cells can be regulated by cytokines. These findings strongly support the contention that enteric glial cells act as immunomodulatory cells in the enteric nervous system.
Cell-specific basolateral membrane sorting of the human liver Na+-dependent bile acid cotransporter
Authors: Sun An-Qiang , Swaby I'Kyori , Xu Shuhua , Suchy Frederick J.
Published: December 2017

The human Na+-taurocholate cotransporting polypeptide (Ntcp) is located exclusively on the basolateral membrane of hepatocyte, but the mechanisms underlying its membrane sorting domain have not been fully elucidated. In the present study, a green fluorescent protein-fused human NTCP (NTCP-GFP) was constructed using the polymerase chain reaction and was stably transfected into Madin-Darby canine kidney (MDCK) and Caco-2 cells. Taurocholate uptake studies and confocal microscopy demonstrated that the polarity of basolateral surface expression of NTCP-GFP was maintained in MDCK cells but was lost in Caco-2 cells. Nocodazole (33 μM), an agent that causes microtubular depolymerization, partially disrupted the basolateral localization of NTCP-GFP by increasing apical surface expression to 33.5% compared with untreated cells ( P < 0.05). Brefeldin A (BFA; 1–2 μM) disrupted the polarized basolateral localization of NTCP, but monensin (1.4 μM) had no affect on NTCP-GFP localization. In addition, low-temperature shift (20°C) did not affect the polarized basolateral surface sorting of NTCP-GFP and repolarization of this protein after BFA interruption. In summary, these data suggest that the polarized basolateral localization of human NTCP is cell specific and is mediated by a novel sorting pathway that is BFA sensitive and monensin and low-temperature shift insensitive. The process may also involve microtubule motors.
Mechanism of thiamine uptake by human colonocytes: studies with cultured colonic epithelial cell line NCM460
Authors: Said Hamid M. , Ortiz Alvaro , Subramanian Veedamali S. , Neufeld Ellis J. , Moyer Mary Pat , Dudeja Pradeep K.
Published: December 2017

Thiamine (vitamin B1) is essential for normal cellular functions and growth. Mammals cannot synthesize thiamine and thus must obtain the vitamin via intestinal absorption. The intestine is exposed to a dietary thiamine source and a bacterial source in which the vitamin is synthesized by the normal microflora of the large intestine. Very little is known about thiamine uptake in the large intestine. The aim of this study was, therefore, to address this issue. Our results with human-derived colonic epithelial NCM460 cells as a model system showed thiamine uptake to be 1) temperature- and energy dependent, 2) Na+independent, 3) increased with increasing buffer pH from 5 to 8 and after cell acidification but inhibited by amiloride, 4) saturable as a function of concentration, 5) inhibited by thiamine structural analogs but not by unrelated organic cations, and 6) inhibited by modulators of a Ca2+/calmodulin-mediated pathway. NCM460 cells and native human colonic mucosa expressed the recently cloned human thiamine transporter THTR-1 (product of the SLC19A2 gene) at both mRNA and protein levels. These results demonstrate for the first time that human NCM460 colonocytes possess a specific carrier-mediated system for thiamine uptake that appears to be under the regulation of an intracellular Ca2+/calmodulin-mediated pathway. It is suggested that bacterially synthesized thiamine in the large intestine may contribute to thiamine nutrition of the host, especially toward cellular nutrition of the local colonocytes.
Scavenging nitric oxide reduces hepatocellular injury after endotoxin challenge
Authors: Nadler Evan P. , Dickinson Eva C. , Beer-Stolz Donna , Alber Sean M. , Watkins Simon C. , Pratt David W. , Ford Henri R.
Published: December 2017

Sustained upregulation of inducible nitric oxide (NO) synthase in the liver after endotoxin [lipopolysaccharide (LPS)] challenge may result in hepatocellular injury. We hypothesized that administration of a NO scavenger, NOX, may attenuate LPS-induced hepatocellular injury. Sprague-Dawley rats received NOX or saline via subcutaneous osmotic pumps, followed 18 h later by LPS challenge. Hepatocellular injury was assessed using biochemical assays, light, and transmission electron microscopy (TEM). Interleukin (IL)-6 mRNA was measured by RT-PCR. Tumor necrosis factor (TNF)-α protein expression was determined by immunohistochemistry. NOX significantly reduced serum levels of ornithine carbamoyltransferase and aspartate aminotransferase. TNF-α and IL-6 expression were increased in the livers of saline-treated but not NOX-treated rats. Although there was no difference between groups by light microscopy, TEM revealed obliteration of the space of Disse in saline-treated but not in NOX-treated animals. Electron paramagnetic resonance showed the characteristic mononitrosyl complex in NOX-treated rats. We conclude that NOX reduces hepatocellular injury after endotoxemia. NOX may be useful in the management of hepatic dysfunction secondary to sepsis or other diseases associated with excessive NO production.
Regression of cholangiocyte proliferation after cessation of ANIT feeding is coupled with increased apoptosis
Authors: Lesage Gene , Glaser Shannon , Ueno Yoshiyuki , Alvaro Domenico , Baiocchi Leonardo , Kanno Noriatsu , Phinizy Jo Lynne , Francis Heather , Alpini Gianfranco
Published: December 2017

Cholangiocyte proliferation and loss through apoptosis occur in cholestatic liver diseases. Our aim was to determine the mechanisms of apoptosis in an animal model of ductal hyperplasia. Rats were fed α-naphthylisothiocyanate (ANIT) for 2 wk and subsequently fed normal chow for 1, 2, and 4 wk. Proliferation was assessed in sections by morphometry and in small and large cholangiocytes by proliferating cellular nuclear antigen immunoblots and measurement of cAMP levels. Apoptosis and reactive oxygen species (ROS) levels were also assessed. ANIT feeding increased small and large cholangiocyte proliferation and apoptosis. Cessation of ANIT feeding was associated with decreased proliferation and a further increase in apoptosis in small and large cholangiocytes. Cholangiocytes from ANIT-fed rats or exposed to ANIT in vitro showed increased apoptosis and ROS generation. ANIT-induced duct injury results in enhanced proliferation and apoptosis in small and large cholangiocytes. The mechanism of ANIT-induced apoptosis may be due to ROS generation induced directly by ANIT. Our model has implications for understanding the pathophysiology of cholangiopathies (characterized by the coexistence of cholangiocyte apoptosis and proliferation).
Vagal innervation modulates motor pattern but not initiation of canine gastric migrating motor complex
Authors: Tanaka Toshiyuki , Kendrick Michael L. , Zyromski Nicholas J. , Meile Tobias , Sarr Michael G.
Published: December 2017

To determine the role of vagal nerves in initiation and modulation of the gastric migrating motor complex (MMC), motor activity was recorded in four dogs before and after total abdominal vagotomy during fasting, after exogenous intravenous motilin and insulin, and after feeding. After vagotomy, a temporally coordinated cyclic gastric and small bowel MMC persisted with an unchanged period. During gastric phase III, vagotomy decreased number of contractions (42 ± 4 vs. 16 ± 2), number of groupings of contractions (14 ± 1 vs. 7 ± 1), and motility index (12 ± 1 vs. 10 ± 1) and increased the duration between groupings (1 ± 1 vs. 3 ± 1 min) ( P< 0.05 in each). Before and after vagotomy, motilin and insulin induced a premature MMC with minor changes in contractile pattern. A 200-g liver meal but not a 50-g liver meal inhibited the gastric MMC after vagotomy. A cyclic MMC persisted after vagotomy, but the contractile pattern during gastric phase III was altered. After a short recovery period, vagal innervation to the stomach modulates the pattern but not the presence of gastric interdigestive motility during phase III.