bluuebunny/crossref_metadata_2025 huggingface dataset:
Hyperpermeability of intestinal epithelial monolayers is induced by NO: effect of low extracellular pH
Authors: Unno N. , Menconi M. J. , Smith M. , Aguirre D. E. , Fink M. P.
Published: December 2017

Nitric oxide (NO.) increases the permeability of Caco-2BBe enterocytic monolayers. Many of the toxic effects of NO. are thought to be mediated by the peroxynitrite anion (ONOO-), which, under mildly acidic conditions, can rearrange to yield an intermediate with reactivity similar to toxic OH.. Accordingly, we assessed the permeability of Caco-2BBe cells grown on permeable supports for 24 h in media titrated to normal or acidic extracellular pH (pHo) with or without the NO. donors 3-morpholinosydnonimine (SIN-1) or sodium nitroprusside (SNP). Incubation with 2 mM SIN-1 at pHo 6.8 or 0.6 mM SNP at pHo 6.5 increased permeability (apical-to-basolateral flux of fluorescein sulfonic acid), whereas at pHo 7.4 permeability was unaffected by these concentrations of NO. donors. Accumulation of NO2/NO3 in medium (index of NO. release) was not increased by incubation of cells with SIN-1 or SNP under mildly acidic conditions. Under acidic but not control conditions, incubation with SIN-1 caused disruption of perijunctional actin filaments as assessed by fluorescence microscopy. At pHo 6.8 and 6.5 (but not 7.4), SIN-1 significantly decreased intracellular levels of both ATP and glutathione. Incubation with 5 mM deferoxamine or 500 uM ascorbic acid (ONOO- scavengers) abrogated SIN-1-induced hyperpermeability. We conclude that mild acidosis augments NO.-induced intestinal epithelial permeability, possibly by promoting oxidant-mediated cytoskeletal damage and/or ATP depletion.
Sodium-dependent nucleoside transport in the human intestinal brush-border membrane
Authors: Patil S. D. , Unadkat J. D.
Published: December 2017

The objective of the study was to determine the identity and kinetic characteristics of nucleoside transporters present in the brush-border membrane of the human jejunum. With use of brush-border membrane vesicles, uptake of [3H]uridine was stimulated two- to threefold by an inwardly directed Na+ gradient and was inhibited by both 100 microM thymidine and 100 microM guanosine nucleosides, which serve as model substrates for purine (N1, cif) and pyrimidine (N2, cit) transporters, respectively. [3H]thymidine and [3H]guanosine transport exhibited an overshoot phenomenon only in the presence of a Na+ gradient. Na(+)-thymidine uptake was inhibited by 100 microM cytidine or thymidine but not by guanosine, inosine, formycin B, or hypoxanthine. [3H]guanosine uptake was inhibited by 100 microM inosine, guanosine, or formycin B but not by thymidine or cytidine. Both adenosine and uridine inhibited uptake of [3H]thymidine and [3H]guanosine to a similar extent, indicating that both N1, cif and N2, cit Na(+)-nucleoside transporters are expressed in human jejunum. Enhanced uptake of Na(+)-thymidine by an inside-negative potential difference generated by K+ and valinomycin provides evidence that nucleoside transport is rheogenic, involving net transfer of a positive charge. The Hill coefficient was unity for all three substrates, indicating a Na(+)-nucleoside coupling stoichiometry of 1:1. At saturating Na+ concentration (150 mM) the kinetic parameters (n = 3-4) Michaelis-Menten constant and maximum velocity for uridine, thymidine, and guanosine uptake were 4.15 +/- 1.79, 2.74 +/- 0.58, 12.02 +/- 1.34 microM and 25.93 +/- 7.38, 16.10 +/- 3.64, 63.92 +/- 10.23 pmol.mg-1.10 s-1, respectively. These results suggest that, in contrast to the human kidney that expresses the N4 nucleoside transporter, the human jejunum expresses both N1 and N2 Na(+)-nucleoside transporters.
IL-1 beta induces synthesis of phospholipase A2-activating protein in rabbit distal colon
Authors: Homaidan F. R. , Zhao L. , Burakoff R.
Published: December 2017

In inflammatory bowel disease, the colonic mucosa is infiltrated by inflammatory cells that secrete a variety of inflammatory mediators such as interleukin-1 beta (IL-1 beta). IL-1 beta caused a delayed increase in Cl- secretion and in prostaglandin E2 (PGE2) release in rabbit distal colon. Both of these effects were abolished with cycloheximide, implying a role for protein synthesis in mediating IL-1 beta's effect. With the use of Western blot assays, the protein was identified as the phospholipase A2 (PLA2)-activating protein (PLAP). IL-1 beta caused a concentration-dependent and a time-dependent increase in PLAP levels as well as in PLA2 activity, with the maximal increase observed at an IL-1 beta concentration between 10 and 30 ng/ml reached in 2-10 min. The PLAP mRNA levels were also regulated by IL-1 beta with a similar time course. PLAP is constitutively present in the epithelial cells and in the subepithelial layer of the distal colon. These findings suggest a direct effect of IL-1 beta on intestinal epithelial cells to cause an increase in PLAP levels and phospholipase A2 activity and subsequent increase in PGE2 levels.
Salutary effects of ATP-MgCl2 on altered hepatocyte signal transduction after hemorrhagic shock
Authors: Mahmoud M. S. , Wang P. , Chaudry I. H.
Published: December 2017

Although previous studies indicate that hepatocyte P2 purinoceptors, macrophage adenosine 3',5'-cyclic monophosphate (cAMP), and beta-adrenergic receptors decrease after hemorrhage and that administration of ATP-MgCl2 after hemorrhage normalizes these parameters, it is not known whether other aspects of hepatocyte signal transduction processes, such as transmembrane coupling, are also affected by hemorrhage and, if so, whether ATP-MgCl2 has any beneficial effects on signal transduction. To study this, rats underwent a 5-cm midline laparotomy (i.e., trauma induced) and were bled to and maintained at a mean arterial pressure of 40 mmHg until 40% of a maximum bleed-out volume was returned in the form of Ringer lactate (RL). They were then resuscitated with three times the volume of shed blood with RL over 45 min followed by two times the volume with RL+ATP-MgCl2 (50 mumol/kg body wt) or an equivalent volume of saline over 95 min. Hepatocytes were isolated at 4 and 27 h after resuscitation, and basal as well as stimulated levels of cAMP and inositol 1,4,5-trisphosphate (IP3) were determined. The results indicate that basal levels of cAMP decreased whereas IP3 increased after hemorrhage and resuscitation. Receptor-dependent stimuli (i.e., glucagon and vasopressin) failed to elicit cAMP or IP3 accumulation after hemorrhage. In contrast, receptor-independent stimulation was not impaired. ATP-MgCl2 treatment, however, prevented the decreased basal levels of cAMP and IP3 and the ability of hepatocytes to respond to receptor-dependent stimulation. Thus ATP-MgCl2 treatment of animals after trauma-hemorrhage and resuscitation attenuates the impaired second messengers cAMP and IP3 and their membrane transduction processes.
Reciprocal inhibitory paracrine pathways link histamine and somatostatin secretion in the fundus of the stomach
Authors: Vuyyuru L. , Harrington L. , Arimura A. , Schubert M. L.
Published: December 2017

The present study was designed to examine the functional linkage between histamine and somatostatin secretion in the fundus of the stomach. In segments of rat fundic mucosa, superfusion with thioperamide (H3 antagonist) increased somatostatin and decreased histamine secretion; superfusion with (R)(-)-alpha-methylhistamine (H3 agonist) had the opposite effect, decreasing somatostatin and increasing histamine secretion. The pattern implied that endogenous histamine, acting via H3 receptors, exerts an inhibitory paracrine influence on somatostatin secretion. Superfusion with somatostatin antibody (1:250) increased histamine secretion, implying that endogenous somatostatin, in turn, exerts an inhibitory paracrine influence on histamine secretion. Somatostatin antibody also abolished the decrease in histamine secretion induced by thioperamide and the increase in histamine secretion induced by (R)(-)-alpha-methylhistamine, implying that changes in histamine secretion induced by activation of H3 receptors reflect changes in somatostatin secretion. Superfusion with the muscarinic agonist methacholine alone and in the presence of either H3 agonist or H3 antagonist confirmed the existence of reciprocal inhibitory pathways linking somatostatin and histamine. We conclude that fundic histamine and somatostatin secretion are linked via reciprocal inhibitory paracrine pathways that serve to amplify the regulatory influence of somatostatin.
Regulation of inducible nitric oxide synthase and nitric oxide during hepatic injury and fibrogenesis
Authors: Rockey D. C. , Chung J. J.
Published: December 2017

Nitric oxide (NO) production via inducible NO synthase (iNOS) is prominent in the liver after stimulation with cytokines and/or lipopolysaccharide. The aim of this study was to investigate the production of NO via iNOS in specific liver cell populations during toxin-mediated and obstructive hepatic injury and fibrogenesis. After a single dose of carbon tetrachloride, iNOS mRNA and nitrite (a metabolic product of NO) were detected only in Kupffer cells. They were not detectable in any cell type after recurrent administration of carbon tetrachloride, including in animals with far advanced cirrhosis (i.e., portal hypertension and/or ascites). After bile duct ligation, a mechanistically different form of liver injury and fibrogenesis, iNOS mRNA and nitrite were identified in all nonparenchymal cells but not in hepatocytes. Twenty-four hours after bile duct ligation, iNOS mRNA and NO production were greatest in Kupffer cells, but after prolonged bile duct ligation, iNOS was found predominantly in sinusoidal endothelial cells. These data indicate that iNOS expression varies temporally and spatially in the liver after injury and also varies with the type of insult.
Ursodeoxycholate inhibits induction of NOS in human intestinal epithelial cells and in vivo
Authors: Invernizzi P. , Salzman A. L. , Szabo C. , Ueta I. , O'Connor M. , Setchell K. D.
Published: December 2017

Ursodeoxycholate (UDCA) has anti-inflammatory and chemoprotective effects in animal models of inflammatory bowel disease and colon cancer. Because overproduction of nitric oxide (NO) by the inducible isoform of NO synthase (iNOS) is implicated in the pathogenesis of these conditions, we investigated the ability of UDCA to inhibit NO production in transformed human intestinal epithelial (DLD-1) cells. Nitrite/nitrate production was measured by the Griess reaction, enzymatic activity of iNOS was assessed by conversion of L-arginine to L-citrulline, and protein and mRNA were measured by Western and Northern blotting. Dose-dependent inhibition of interleukin-1 beta- and interferon-gamma-stimulated nitrite/nitrate production was observed when cells were preincubated for 6 h with UDCA (0–800 microM), and a substantial inhibition (81 +/- 3.2%) was seen at 500 microM. In cytokine-stimulated cells, UDCA reduced iNOS mRNA, protein, and enzyme activity without exerting cytotoxicity. UDCA had a minimal direct inhibitory effect on iNOS enzyme activity. UDCA pretreatment also reduced the expression of iNOS in the colonic epithelium of rats treated with bacterial lipopolysaccharide. Thus UDCA inhibits the induction of epithelial iNOS in vitro and in vivo, and this effect may contribute to the anti-inflammatory and chemoprotective actions of UDCA.
Determinants of triacylglycerol transport from the endoplasmic reticulum to the Golgi in intestine
Authors: Kumar N. S. , Mansbach C. M.
Published: December 2017

The ability of the intestinal cell to export triacylglycerol (TG) is a physiologically regulatable function. The intracellular site where this occurs is unknown, although available evidence suggests that the step between the endoplasmic reticulum (ER) and the Golgi is the most likely. We studied this process in rat enterocytes that were isolated from the proximal intestine. A novel system was developed in which [3H]TG was transported from ER to the Golgi. This process was time, ATP, temperature, and cytosol dependent. The cytosolic factor(s) was heat and trypsin sensitive. TG transport was directly proportional to the amount of added nonradiolabeled acceptor Golgi. The rate of TG transported to the Golgi was the fastest in cells isolated from rats that had been intraduodenally infused in vivo with glyceryltrioleate (TO) plus phosphatidylcholine and slowest in cells isolated from bile-fistulated rats infused with TO in vivo compared with cells from in vivo TO-infused, bile duct intact rats, mimicking the relative transport rates seen in vivo. TG transport in vitro could not be quenched by adding TG emulsions, chylomicrons, liposomes, or guanosine 5'-O-(3-thiotriphosphate). Cytosol from the liver and kidney supported TG transport, but the Golgi from liver or kidney did not accept TG from intestinal ER. We conclude that an intestinally specific, active transport mechanism transports TG from the ER to the Golgi and that this might be a regulatory step in TG export from the intestinal cell.
Calcitonin gene-related peptide in viscerosensitive response to colorectal distension in rats
Authors: Plourde V. , St-Pierre S. , Quirion R.
Published: December 2017

The role of calcitonin gene-related peptide (CGRP) on colorectal distension-induced visceral pain was investigated in conscious rats. Intracolonic administration of acetic acid (0.6%) resulted in a significantly increased number of abdominal contractions in response to colorectal balloon distension from 5.8 +/- 1.2 in controls to 16.6 +/- 1.0 in acetic acid-treated animals (P < 0.05), evidencing sensitization of visceral afferent pathways and subsequently visceral hyperalgesia. This sensitization phenomenon was not observed in animals previously treated with systemic capsaicin. Likewise, in animals not treated with capsaicin, use of an intravenous antagonist for CGRP [human CGRP-(8-37)], completely reversed the sensitizing effects of acetic acid. Furthermore, intravenous administration of CGRP dose dependently increased the number of abdominal contractions in response to colorectal distension from 3.0 +/- 1.1 (CGRP 250 ng) to 17.0 +/- 1.2 (CGRP 500 ng, P < 0.05), as previously observed in acetic acid-treated animals. Finally, intrathecal administration of hCGRP-(8–37) (mid-lumbar) also resulted in a total dose-dependent reversal of CGRP (500 ng) or acetic acid-induced visceral hypersensitivity. These results demonstrate that CGRP plays a major role in this model of visceral afferent nerve sensitization from gastrointestinal origin.
Ontogeny and location of HMG-CoA reductase, ACAT, and MGAT in human small intestine
Authors: Loirdighi N. , Menard D. , Delvin E. , Levy E.
Published: December 2017

Because a few enzymes are in most tissues, enabling them to produce lipids necessary for growth and differentiation, the development of their activity in the intestine, an important organ of fat transport and metabolism, is of great interest. In this investigation, the ontogeny and location of 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase (the key regulatory enzyme in the cholesterol pathway), acyl-CoA:cholesterol acyltransferase (ACAT; responsible for cholesterol esterification), and monoacylglycerol acyltransferase (MGAT; the more representative enzyme of the neutral lipid pathway) were examined in the human fetal small intestine. The developing gut exhibited high levels (pmol.mg-1.min-1) of HMG-CoA reductase (7.65 +/- 0.35), ACAT (16.98 +/- 1.12), and MGAT (689.74 +/- 37.54). Significant positive correlations were recorded between fetal age (8–22 wk) and the enzyme activities of HMG-CoA reductase in the proximal (P < 0.005) and middle (P < 0.01) segments, ACAT in the distal segment (P < 0.03), and MGAT in the proximal segment (P < 0.03) of the gut. Age-specific changes were found in the location of the three enzymes in the contiguous intestinal segments that were investigated. We concluded that the fetal small intestine has substantial HMG-CoA reductase, ACAT, and MGAT activity, which displays specific patterns during development.
Enterocytes are the primary source of the chemokine ENA-78 in normal colon and ulcerative colitis
Authors: Keates S. , Keates A. C. , Mizoguchi E. , Bhan A. , Kelly C. P.
Published: December 2017

Epithelial cell-derived neutrophil-activating protein-78 (ENA-78) is a neutrophil-directed C-X-C chemokine. We report that Caco-2 and T84 human intestinal epithelial cells produce ENA-78 after stimulation by interleukin (IL)-1 beta or tumor necrosis factor-alpha. Caco-2 cells show increased IL-8 production at 4-12 h and increased ENA-78 production at 8-24 h after cytokine stimulation. Immunohistochemical studies in normal human colon and in ulcerative colitis demonstrate ENA-78 immunoreactivity principally associated with crypt epithelial cells. Furthermore, human colonic tissues from patients with ulcerative colitis show elevated levels of ENA-78 mRNA (24-fold increase, P < 0.01) and protein (4-fold increase, P < 0.05) compared with normal controls. Thus ENA-78 is produced in normal colon and in ulcerative colitis and is predominantly of enterocyte origin. The kinetics of ENA-78 induction in human colon epithelial cell lines are delayed and prolonged compared with IL-8. We propose that ENA-78 and IL-8 serve complementary and sequential roles in neutrophil recruitment in ulcerative colitis. ENA-78 as an enterocyte-derived, neutrophil-activating chemokine may be especially important in neutrophil recruitment from the lamina propria into the epithelial layer.
Signal transduction pathways via guanylin and uroguanylin in stomach and intestine
Authors: London R. M. , Krause W. J. , Fan X. , Eber S. L. , Forte L. R.
Published: December 2017

Guanylin and uroguanylin are peptides that activate receptor guanylate cyclases (GCs) and elicit increased intestinal secretion. Bacteria that cause traveler's diarrhea produce heat-stable toxins (STs) that mimic this action. Investigation of the distribution and identity of receptor GCs in the gastrointestinal tract of rats revealed that receptors were localized to epithelial cells in stomach and intestine. Clusters of cells in gastric mucosa and enterocytes lining the intestine exhibited specific binding of 125I-labeled ST. Ligated loops of stomach and intestine treated with intraluminal ST had significant increases in guanosine 3',5'-cyclic monophosphate (cGMP), with duodenum exhibiting the greatest response. Expression of guanylate cyclase C (GCC) mRNA and a truncated, GCC-like mRNA was found in both stomach and intestine. Both mRNAs were isolated as cDNAs encoding the GC catalytic domain. The 0.9-kilobase (kb) cDNA is 99.8% identical to GCC, whereas the truncated, 0.75-kb GCC-like cDNA has a 159-nucleotide deletion and is 96.6% identical to GCC at the protein level. Uroguanylin and guanylin mRNAs were detected in stomach and intestine. Uroguanylin mRNA was most abundant in small intestine, whereas guanylin mRNA was highest in large intestine. Thus the stomach and intestine are targets for regulation of transport by guanylin and uroguanylin via cGMP.
Nuclear Fos immunoreactivity in guinea pig myenteric neurons following activation of motor activity
Authors: Ritter R. C. , Costa M. , Brookes S. H.
Published: December 2017

To identify enteric neurons activated during intestinal motor activity, we examined myenteric plexus of guinea pig small intestinal segments for expression of the immediate early gene product, Fos. Fos immunoreactivity was detected immunohistochemically following in vitro manipulations, which included distension, electrical stimulation, exposure to forskolin, and peristalsis. All of these manipulations induced neuronal Fos expression, which was prevented by tetrodotoxin, indicating that expression depended on nerve activity. Distension-induced Fos expression was blocked by omega-conotoxin and significantly reduced by hexamethonium, indicating that neurons expressing Fos immunoreactivity were activated synaptically. Blocking smooth muscle contraction with nicardipine reduced expression of neuronal Fos, suggesting that muscle tone influences neuronal activity. Calbindin-immunoreactive putative sensory neurons did not express Fos during distension, peristalsis, or exposure to forskolin and expressed Fos only weakly after strong electrical stimulation. Conversely, calretinin-immunoreactive ascending excitatory interneurons and longitudinal muscle motoneurons exhibited Fos immunoreactivity after all experimental manipulations. These results indicate that Fos expression can, with some caution, be used to identify classes of enteric neurons activated by different stimuli under various experimental conditions.
Effect of acute hyperglycemia on colorectal motor and sensory function in humans
Authors: Maleki Dordaneh , Camilleri Michael , Zinsmeister Alan R. , Rizza Robert A.
Published: December 2017

Increased use of laxatives and constipation are more common among people with diabetes mellitus than matched nondiabetic people in the same community. The mechanism of constipation in diabetes is unclear. Acute hyperglycemia was previously reported to reduce the gastrocolonic response. Our aim was to determine the effects of acute hyperglycemia on the colon compliance and motor response to feeding and on the sensory function of the colon and rectum in healthy human subjects. Eleven healthy individuals were studied under conditions of hyperglycemia (mean blood glucose 280 ± 13 mg/dl) and euglycemia. We evaluated three parameters: 1) colonic motility and compliance by a multilumen manometry and barostatic balloon assembly in the descending colon (motility was studied during fasting and for 2 h postprandially); 2) perception of isobaric distensions of polyethylene balloons in the rectum and colon; and 3) rectal compliance. Initial tonic response to meal ingestion (0–5 min) was slightly lower during hyperglycemia ( P = 0.3). However, colonic tone, motility, compliance, and sensation, as well as rectal compliance and sensation, were not significantly different under the conditions of euglycemia and acute hyperglycemia. In healthy individuals, acute hyperglycemia does not significantly change colonic or rectal motor functions or the perception of mechanosensory stimuli in the colon or rectum compared with euglycemia. These results do not support the hypothesis that hyperglycemia abolishes the colonic response to feeding.
Effects of a panel of dietary lectins on cholecystokinin release in rats
Authors: Jordinson Mark , Playford Raymond J. , Calam John
Published: December 2017

We previously showed that soybean lectin (SBL) releases cholecystokinin (CCK) and have now asked whether other dietary lectins have this effect and if extracellular calcium is involved. Lectins and vehicle were first infused into the duodenum of anesthetized rats. The CCK response to vehicle was 3.1 ± 0.6 pmol/l ( P < 0.05 vs. basal). SBL and peanut lectin (PNL) (84 μg/ml) significantly increased plasma CCK concentrations from 2.0 ± 0.4 pmol/l to a maximum of 8.4 ± 0.5 pmol/l ( P < 0.01 vs. vehicle, mean ± SE) and from 1.9 ± 0.5 to 7.0 ± 0.6 pmol/l ( P < 0.05 vs. vehicle, mean ± SE), respectively. Wheat germ lectin (WGL) (840 μg) also increased plasma CCK levels from 1.5 ± 0.3 pmol/l to a maximum of 9.7 ± 1.3 pmol/l ( P < 0.05 vs. vehicle, mean ± SE). Corresponding increases in pancreatic protein output occurred. Broad bean lectin (BBL) had no effect on either parameter. Dose-dependent responses were seen with SBL, PNL, and WGL (1, 10, and 100 μg/ml) in perifused rat intestinal cells. These responses were abolished in calcium-free medium and in the presence of the competing sugars of the lectins. Therefore, SBL, PNL, and WGL, which bind to motifs including N-acetyl-d-galactosamine, galactose, and N-acetylglucosamine, respectively, released CCK, but BBL, which binds to mannose and glucose, did not. Ingestion of lectins may have major CCK-mediated effects on gastrointestinal function and growth.
Protein tyrosine phosphorylation in pancreatic acini: differential effects of VIP and CCK
Authors: Lutz Manfred P. , Piiper Albrecht , Gaisano Herbert Y. , Stryjek-Kaminska Danuta , Zeuzem Stefan , Adler Guido
Published: December 2017

Cholecystokinin (CCK) and vasoactive intestinal peptide (VIP) stimulate enzyme secretion from pancreatic acini by binding to heptahelical receptors without intrinsic tyrosine kinase activity. Signal transduction by the CCK receptor involves activation of phospholipase C by Gqproteins and activation of tyrosine kinases, whereas occupation of VIP receptors stimulates adenylyl cyclase through binding to Gsproteins. Here, we use electrophoretic separation of cellular proteins and antiphosphotyrosine immunoblotting to demonstrate a VIP-stimulated rapid and dose-dependent increase in tyrosine phosphorylation of proteins migrating at 130, 115, and 93 kDa in freshly isolated rat pancreatic acini. Phosphorylation of these proteins was increased after direct stimulation of adenylyl cyclase or the adenosine 3′,5′-cyclic monophosphate (cAMP)-dependent protein kinase with forskolin or dibutyryl cAMP and was inhibited by the tyrosine kinase inhibitors genistein or tyrphostin 23. Compared with VIP, CCK stimulated tyrosine phosphorylation of additional proteins migrating at 60, 66, and 72/78 kDa. Using two-dimensional electrophoretic separation or immunoprecipitation, the 72/78-kDa phosphoprotein was identified as paxillin. We propose that paxillin might be involved in CCK- but not in VIP-induced exocytosis.
Translocation of verotoxin-1 across T84 monolayers: mechanism of bacterial toxin penetration of epithelium
Authors: Philpott Dana J. , Ackerley Cameron A. , Kiliaan Amanda J. , Karmali Mohamed A. , Perdue Mary H. , Sherman Philip M.
Published: December 2017

Verotoxin-producing Escherichia coli (VTEC) are pathogenic bacteria associated with diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome. Verotoxins (VTs) elaborated by these organisms produce cytopathic effects on a restricted number of cell types, including endothelial cells lining the microvasculature of the bowel and the kidney. Because human intestinal epithelial cells lack the globotriaosylceramide receptor for VT binding, it is unclear how the toxin moves across the intestinal mucosa to the systemic circulation. The aims of this study were to determine the effects of VT-1 on intestinal epithelial cell function and to characterize VT-1 translocation across monolayers of T84 cells, an intestinal epithelial cell line. VT-1 at concentrations up to 1 μg/ml had no effect on the barrier function of T84 monolayers as assessed by measuring transmonolayer electrical resistance (102 ± 8% of control monolayers). In contrast, both VT-positive and VT-negative VTEC bacterial strains lowered T84 transmonolayer resistance (45 ± 7 and 38 ± 6% of controls, respectively). Comparable amounts of toxin moved across monolayers of T84 cells, exhibiting high-resistance values, as monolayers with VTEC-induced decreases in barrier function, suggesting a transcellular mode of transport. Translocation of VT-1 across T84 monolayers paralleled the movement of a comparably sized protein, horseradish peroxidase. Immunoelectron microscopy confirmed transcellular transport of VT-1, since the toxin was observed within endosomes and associated with specific intracellular targets, including the Golgi network and endoplasmic reticulum. These data present a mode of VT-1 uptake by toxin-insensitive cells and suggest a general mechanism by which bacterial toxins lacking specific intestinal receptors can penetrate the intestinal epithelial barrier.
Activity-dependent changes in intracellular calcium in myenteric neurons
Authors: Hanani M. , Lasser-Ross N.
Published: December 2017

The spatial distribution and changes in intracellular calcium concentration ([Ca2+]i) in myenteric neurons were measured using fura 2 in the longitudinal muscle-myenteric plexus preparation from the guinea pig duodenum. These measurements were made simultaneously with intracellular voltage recordings. The generation of action potentials in the cell bodies of both S- and AH-type neurons increased [Ca2+]iin the processes and cell bodies. There was no measurable delay between the [Ca2+]ichanges in the somata and the processes, indicating that these changes were caused by the spread of electrical signals and not by diffusion. The rate of Ca2+ removal was faster in the processes than in the somata, apparently due to the large surface-to-volume ratio in the former. In AH neurons, the [Ca2+]itransient was shorter than the duration of the after-spike hyperpolarization. It is concluded that the two main types of myenteric neurons possess voltage-gated Ca2+channels in both somata and processes.
Distinct effects of tetragastrin, histamine, and CCh on rat gastric mucin synthesis and contribution of NO
Authors: Ichikawa Takafumi , Ishihara Kazuhiko , Kusakabe Tatsumi , Kurihara Makoto , Kawakami Tadashi , Takenaka Toshifumi , Saigenji Katsunori , Hotta Kyoko
Published: December 2017

Although gastrin, histamine, and carbachol (CCh) accelerate gastric mucin metabolism, information about their target cells of mucin production is lacking. To clarify this, we examined the effects of these stimulants, including the possible participation of nitric oxide (NO), on mucin biosynthesis in distinct sites and layers of rat gastric mucosa. Pieces of tissue obtained from the corpus and antrum were incubated in a medium containing radioactive precursors and each stimulant, with or without NO synthase (NOS) inhibitor. Distribution of NOS was compared with that of the specific mucins by immunostaining using specific antiserum and monoclonal antibodies. In the full-thickness corpus mucosa, tetragastrin enhanced [3H]glucosamine incorporation into mucin but had no effect on [14C]threonine incorporation. Both histamine and CCh dose dependently increased3H- and14C-labeled corpus mucin. Only CCh stimulated antral mucin biosynthesis. CCh stimulation was noted in the corpus mucosa after removal of surface mucous cells, but stimulation by tetragastrin or histamine disappeared as a result of this pretreatment. Only tetragastrin-induced activation was completely blocked by the NOS inhibitor. NOS immunoreactivity was limited to surface mucous cells. Mucus-producing cells present in the different sites and layers of the gastric mucosa have distinct mechanisms for regulation of mucin biosynthesis. Gastrin-stimulated mucin biosynthesis mediated by NO is limited to surface mucous cells of rat gastric oxyntic mucosa.
Inhibitory effect of central dopamine on basal pancreatic secretion in conscious rats
Authors: Masuda Masao , Kanai Setsuko , Miyasaka Kyoko
Published: December 2017

We examined the role and the peripheral mechanism of action of central dopamine on basal pancreatic exocrine secretion in conscious rats. Rats were fitted with bile and pancreatic catheters to collect bile and pancreatic juice separately and also with a left lateral brain ventricle and external jugular vein catheters. After 90-min basal collection, the D1- and D2-receptor antagonists (Sch-23390 and eticlopride, respectively) and dopamine were administered into the lateral brain ventricle. Sch-23390 (30, 100, and 300 nmol/rat), but not eticlopride (300 nmol/rat), stimulated pancreatic fluid and protein secretion. Dopamine (30, 100, and 300 nmol/rat) inhibited pancreatic secretion dose dependently. Pretreatment with Sch-23390 prevented the inhibitory effect of dopamine. Intravenously injected Sch-23390 or dopamine had no effect on pancreatic secretion. The inhibitory effect of dopamine was blocked by bretylium, an inhibitor of norepinephrine release, and phentolamine, an α-blocker, but not by vagotomy. The β-antagonist propranolol alone significantly inhibited basal pancreatic secretion, and dopamine did not modify the inhibitory effect of propranolol. The proton pump inhibitor omeprazole partially but not completely reduced the inhibition by dopamine. These results suggest that central dopamine inhibits pancreatic exocrine secretion via D1-like receptors and that the inhibitory effect is mediated via sympathetic nerves, especially α-adrenoceptors.
Substrate specificity of the rat liver Na+-bile salt cotransporter inXenopus laevisoocytes and in CHO cells
Authors: Schroeder Alice , Eckhardt Uta , Stieger Bruno , Tynes Ronald , Schteingart Claudio D. , Hofmann Alan F. , Meier Peter J. , Hagenbuch Bruno
Published: December 2017

It has been proposed that the hepatocellular Na+-dependent bile salt uptake system exhibits a broad substrate specificity in intact hepatocytes. In contrast, recent expression studies in mammalian cell lines have suggested that the cloned rat liver Na+-taurocholate cotransporting polypeptide (Ntcp) may transport only taurocholate. To characterize its substrate specificity Ntcp was stably transfected into Chinese hamster ovary (CHO) cells. These cells exhibited saturable Na+-dependent uptake of [3H]taurocholate [Michaelis constant ( Km) of ∼34 μM] that was strongly inhibited by all major bile salts, estrone 3-sulfate, bumetanide, and cyclosporin A. Ntcp cRNA-injected Xenopus laevis oocytes and the transfected CHO cells exhibited saturable Na+-dependent uptake of [3H]taurochenodeoxycholate ( Kmof ∼5 μM), [3H]tauroursodeoxycholate ( Kmof ∼14 μM), and [14C]glycocholate ( Kmof ∼27 μM). After induction of gene expression by sodium butyrate, Na+-dependent transport of [3H]estrone 3-sulfate ( Kmof ∼27 μM) could also be detected in the transfected CHO cells. However, there was no detectable Na+-dependent uptake of [3H]bumetanide or [3H]cyclosporin A. These results show that the cloned Ntcp can mediate Na+-dependent uptake of all physiological bile salts as well as of the steroid conjugate estrone 3-sulfate. Hence, Ntcp is a multispecific transporter with preference for bile salts and other anionic steroidal compounds.
Role of mast cell-derived mediators in acid-induced shortening of the esophagus
Authors: Paterson W. G.
Published: December 2017

It has recently been demonstrated that acid-induced esophageal mucosal injury leads to esophageal shortening, raising the possibility that reflux esophagitis per se may contribute to the development of hiatal hernia. The aim of the present study was to determine whether mast cell-derived mediators are involved in this acid-induced esophageal shortening. Changes in esophageal length were continuously monitored in anesthetized opossums while the esophageal lumen was perfused with 100 mmol/l HCl or normal saline. Changes in esophageal length were compared between animals perfused with acid, with or without pretreatment with the mast cell stabilizers doxantrazole or disodium cromoglycate (DSCG), and animals perfused with normal saline, with or without pretreatment with DSCG. In separate in vitro studies the effect of the mast cell stabilizers on electrical field stimulation-induced esophageal longitudinal muscle contraction was determined. Gradual esophageal lengthening occurred during saline perfusion, irrespective of whether animals were pretreated with DSCG. In contrast, acid perfusion induced esophageal shortening, which was abolished by pretreatment with either doxantrazole or DSCG in doses sufficient to attenuate the acid-induced mucosal histamine release. In vitro, the mast cell stabilizers had no effect on electrical field stimulation-induced esophageal shortening. This study suggests that esophageal shortening associated with acute acid-induced esophageal mucosal injury in the opossum is dependent on mast cell-derived mediators.
Spinal and pudendal nerve modulation of human corticoanal motor pathways
Authors: Hamdy Shaheen , Enck Paul , Aziz Qasim , Rothwell John C. , Uengoergil Samet , Hobson Anthony , Thompson David G.
Published: December 2017

We investigated the effects of lumbosacral and pudendal nerve stimulation on the corticofugal pathways to the human external anal sphincter. In 11 healthy subjects, anal sphincter electromyographic responses, evoked to transcranial magnetic stimulation of the motor cortex, were recorded 5–500 ms after lumbosacral root or pudendal nerve stimulation. Lumbosacral and pudendal nerve stimulation alone evoked responses with amplitudes of 293 ± 73 and 401 ± 153 μV and latencies of 3.2 ± 0.2 and 2.2 ± 0.2 ms, respectively. Cortical stimulation also evoked responses with amplitudes of 351 ± 104 μV and latencies of 20.9 ± 1.1 ms. When lumbosacral or pudendal nerve stimulation preceded cortical stimulation, the cortically evoked responses were facilitated ( P < 0.01), with the effect appearing greatest at 5–20 ms after both lumbosacral and pudendal excitation and at 50–100 ms after lumbosacral excitation alone. Our results demonstrate that cortical pathways to the external anal sphincter are facilitated by prior lumbosacral and pudendal nerve stimulation, indicating that sensorimotor interactions are important in the central neural control of sphincter function.
III. Ontogeny and function of γδ T cells in the intestine
Authors: Kagnoff Martin F.
Published: December 2017

γδ T cells are located in the paracellular space between epithelial cells. In the human colon and small intestine, 5–40% of intraepithelial lymphocytes (IEL) are γδ T cells, and in mice an even greater proportion of IEL are γδ T cells. The γδ T cell receptor repertoire in the human intestine undergoes marked changes in V region gene usage and junctional diversity during development from fetus to newborn to adult, suggesting that γδ T cells may mediate qualitatively or quantitatively different functions at various stages of development. γδ IEL have been shown to produce cytokines and growth factors and to influence epithelial cell proliferation and differentiation, as well as the mucosal development of immunoglobulin A B cells. γδ IEL also manifest cytolytic activity. However, the ligands recognized by intestinal γδ T cells and the role they play in intestinal immune responses, in immune defense to enteric pathogens, and in the pathogenesis of intestinal disease are thus far largely unknown.
Alteration of gene expression by intestinal epithelial cells precedes colitis in interleukin-2-deficient mice
Authors: Meijssen Maarten A. C. , Brandwein Steven L. , Reinecker Hans-Christian , Bhan Atul K. , Podolsky Daniel K.
Published: December 2017

Intestinal epithelial cells may be actively involved in the immunoregulatory pathways leading to intestinal inflammation. The aim of this study was to assess expression by intestinal epithelial cells of cytokines with potential involvement in the development of intestinal inflammation in interleukin (IL)-2-deficient [(−/−)] mice. Wild-type mice, mice heterozygous for the disrupted IL-2 gene, and IL-2(−/−) mice were studied at 6, 16, and 24 wk of age. The mRNA levels of transforming growth factor-β1 (TGF-β1), tumor necrosis factor-α (TNF-α), IL-1β, IL-6, IL-15, KC, JE, and CD14 in colonic and small intestinal epithelial cells were assessed by Northern blot analysis. CD14 was also measured by Western blotting and reverse transcriptase polymerase chain reaction (RT-PCR). TGF-β1 mRNA was constitutively expressed in both colonic and small intestinal epithelial cells with increased expression in the colonic epithelium of colitic mice. CD14 was detected only in colonic epithelial cells, and mRNA levels increased severalfold in IL-2(−/−) mice with colitis. Northern analysis demonstrated increased levels of TGF-β1 and CD14 mRNA in colonic epithelial cells of IL-2(−/−) mice before the development of signs of colitis. CD14 mRNA and protein expression in the epithelial cells of colitic mice were confirmed by RT-PCR and Western blot analysis of isolated cells. In addition, IL-2(−/−) mice also expressed increased levels of IL-15 mRNA in small intestinal and colonic epithelial cells compared with heterozygous control mice. TNF-α, IL-1β, IL-6, KC, and JE mRNAs were only detectable in colonic epithelial cells of mice after the onset of colitis. Enhanced expression of TGF-β1, IL-15, and CD14 by colonic epithelial cells may play a role in the subsequent development of colitis in IL-2(−/−) mice.
Vagal involvement in dietary regulation of nutrient transport
Authors: Bates Sarah L. , Sharkey Keith A. , Meddings Jon B.
Published: December 2017

In omnivores, gradual alterations in dietary nutrient composition are observed. To efficiently absorb dietary nutrients these animals alter intestinal nutrient transporter expression to match the pattern of nutrient intake. This often involves reprogramming the crypt cell to express greater numbers of the relevant transport system. The aim of this study was to determine whether vagal afferents are involved in this adaptive process. Guinea pigs were habituated to a low-carbohydrate diet and then switched to a high-carbohydrate diet. The resultant increase in glucose transporter expression was assessed by determining rates of glucose transport in jejunal brush-border membrane vesicles. Ablation of vagal afferents was accomplished by application of capsaicin to exposed cervical vagi and confirmed using Fast blue tracer studies. We found that animals in which vagal afferents were ablated with capsaicin were unable to alter rates of glucose transport in response to an increase in dietary carbohydrate. This suggests that vagal afferents are involved in this adaptive process. These findings support a role for the vagus nerve in regulating intestinal transport function, which may be important to consider in clinical disease that involves the vagus nerve.
Intestinal fibroblasts regulate intestinal epithelial cell proliferation via hepatocyte growth factor
Authors: Göke Michael , Kanai Michiyuki , Podolsky Daniel K.
Published: December 2017

Although the presence of subepithelial intestinal fibroblasts has been well recognized, the effects of fibroblasts on intestinal epithelial cell (IEC) growth are incompletely understood. In vitro studies were undertaken to evaluate the effects of fibroblasts on the proliferation of model IEC lines. IECs (Caco-2, T84, and IEC-6) were grown alone or in the presence of human intestinal (CCD-18), lung (CCD-37), or skin explant-derived fibroblasts. Cocultures were carried out directly on irradiated fibroblasts or by Transwell coculture technique with fibroblasts and epithelial cells separated by a porous filter. Cell proliferation was assessed by [3H]thymidine incorporation and cell counts. Hepatocyte growth factor (HGF) and c- met transcript expression in IECs and fibroblasts was examined by RT-PCR and Northern blotting; protein expression was evaluated by immunoblotting. Intestinal as well as lung and skin fibroblasts substantially stimulated proliferation of Caco-2, T84, and IEC-6 cells in both direct and Transwell cocultures. In addition, fibroblast-conditioned medium stimulated IEC proliferation, suggesting a paracrine mechanism. Anti-human HGF-neutralizing antibodies blocked the growth-promoting effects in both fibroblasts and fibroblast-conditioned medium. Recombinant human HGF dose dependently promoted IEC proliferation. HGF mRNA and protein expression was restricted to fibroblasts. High levels of c- met expression were found in Caco-2 and T84 cells; in contrast, expression in fibroblasts was weak. In summary, fibroblasts stimulate IEC proliferation through a paracrine mechanism mediated predominantly by HGF.
Discrete roles of hepatocytes and nonparenchymal cells in uridine catabolism as a component of its homeostasis
Authors: Liu M. P. , Beigelman L. , Levy E. , Handschumacher R. E. , Pizzorno G.
Published: December 2017

Previous studies indicated that uridine is essentially cleared in a single pass through a rat liver and replaced in a highly regulated manner by uridine formed presumably by de novo synthesis. We report a cellular basis for the catabolic component of this apparent paradox by dissociation of the liver with collagenase into two cell fractions, hepatocytes and a nonparenchymal cell population. Suspensions of the nonparenchymal cells rapidly cleave uridine to uracil, whereas in hepatocytes this activity was <5% of that in nonparenchymal cells. Conversely, hepatocytes cause extensive degradation of uracil to β-alanine. These differences correlate with the uridine phosphorylase and dihydrouracil dehydrogenase activity in cell-free extracts of each cell type. We have documented the existence of a Na+-dependent, nitrobenzylthioinosine-insensitive transport system for uridine in the parenchymal cells (Michaelis constant 46 ± 5 μM) that achieves a three- to fourfold concentration gradient in hepatocytes. A similar system is present in the nonparenchymal cell population. In addition, a highly specific and active Na+-dependent transport system for β-alanine, the primary catabolic metabolite of uracil, has been demonstrated in hepatocytes.
Effects of intraduodenal glucose infusion on gastric myoelectrical activity and antropyloroduodenal motility
Authors: Verhagen M. A. M. T. , Samsom M. , Smout A. J. P. M.
Published: December 2017

Intraduodenal nutrient infusions cause an inhibition of antral motility and an increase in pyloric motility. The involvement of gastric myoelectrical activity in this intestinogastric feedback was studied. Electrogastrography and antropyloroduodenal manometry were performed in 10 healthy volunteers. The effects of 20-min infusions of 25% glucose (4 kcal/min) and saline were compared. Intraduodenal glucose infusions caused a decrease in the power of the dominant frequency in the electrogastrogram ( P = 0.028), but the frequency itself remained unchanged. The total number of dysrhythmias increased ( P = 0.035). An inhibition of antral motor activity ( P = 0.001), an increase in the number of isolated pyloric pressure waves ( P = 0.027), and an increase in basal pyloric tone ( P = 0.001) were simultaneously recorded. The change in power during glucose infusion correlated positively with the change in the antral motility index ( rs= 0.50, P = 0.001). It is concluded that inhibition of gastric myoelectrical activity is one of the mechanisms underlying an inhibition of motor activity in the gastric antrum.
Changes in G protein expression account for impaired modulation of hepatic cAMP formation after BDL
Authors: Bouscarel Bernard , Matsuzaki Yasushi , Le Man , Gettys Thomas W. , Fromm Hans
Published: December 2017

The regulation of cAMP synthesis by hormones and bile acids is altered in isolated hamster hepatocytes 2 days after bile duct ligation (BDL) [Y. Matsuzaki, B. Bouscarel, M. Le, S. Ceryak, T. W. Gettys, J. Shoda, and H. Fromm. Am. J. Physiol. 273 ( Gastrointest. Liver Physiol. 36): G164–G174, 1997]. Therefore, studies were undertaken to elucidate the mechanism(s) responsible for this impaired modulation of cAMP formation. Hepatocytes were isolated 48 h after either a sham operation or BDL. Both preparations were equally devoid of cholangiocyte contamination. Although the basal cAMP level was not affected after BDL, the ability of glucagon to maximally stimulate cAMP synthesis was decreased by ∼40%. This decreased glucagon effect after BDL was not due to alteration of the total glucagon receptor expression. However, this effect was associated with a parallel 50% decreased expression of the small stimulatory G protein α-subunit (GsαS). The expression of either the large subunit (GsαL) or the common β-subunit remained unchanged. The expression of Giα2and Giα3was also decreased by 25 and 46%, respectively, and was associated with the failure of ANG II to inhibit stimulated cAMP formation. Therefore, alterations of the expression of GsαSand Giα are, at least in part, responsible for the attenuated hormonal regulation of cAMP synthesis. Because cAMP has been reported to stimulate both bile acid uptake and secretion, impairment of cAMP synthesis and bile acid uptake may represent an initial hepatocellular defense mechanism during cholestasis.