bluuebunny/crossref_metadata_2025 huggingface dataset:
In vitro evidence that rabbit distal colonic muscularis mucosae has aClostridium difficiletoxin A receptor
Authors: Percy W. H. , Burakoff R. , Rose K. , Desai H. P. , Pothoulakis C. , Eglow R.
Published: December 2017

In the rabbit ileum Clostridium difficile toxin A causes inflammation and mucosal damage via a specific glycoprotein receptor that contains α-d-galactose. In rabbit colon toxin A also causes inflammation, and this is associated with increased myoelectric activity and eicosanoid production. The present in vitro study was undertaken to determine if a toxin A receptor on one or more layers of colonic smooth muscle could mediate the motor effects of this agent. Toxin A (20–100 μg/ml) was without effect on longitudinal and circular muscle but had two different effects on the muscularis mucosae. Initial exposure to the toxin caused increased numbers of spontaneous contractions and a small, atropine-, tetrodotoxin-, and indomethacin-resistant increase in resting tone. More importantly, however, 30-min exposure to toxin A resulted in attenuated muscularis mucosae responses to acetylcholine and K+. Both the small excitatory and the larger inhibitory effects of toxin A were abolished by pretreatment with the lectin BS-1, which binds to toxin A receptors, but not by the nonreceptor-binding lectin DBA. These data strongly suggest that toxin A causes significant motor effects on the distal colonic muscularis mucosae via a receptor-mediated mechanism. These mechanical data were supported by the presence of histologically demonstrable toxin A and BS-1 binding sites on the muscularis mucosae but not on either the longitudinal or circular muscle layers, both of which were unresponsive to the toxin. By depressing muscularis mucosae function and, ultimately, mucosal movement as a result of toxin A production, C. difficile may promote its own proliferation, thus further contributing to the development of antibiotic-associated colitis.
Persistence ofHelicobacter pyloriVacA toxin and vacuolating potential in cultured gastric epithelial cells
Authors: Sommi Patrizia , Ricci Vittorio , Fiocca Roberto , Necchi Vittorio , Romano Marco , Telford John L. , Solcia Enrico , Ventura Ulderico
Published: December 2017

The vacuolating toxin A (VacA) is one of the most important virulence factors in Helicobacter pylori-induced damage to human gastric epithelium. Using human gastric epithelial cells in culture and broth culture filtrate from a VacA-producing H. pylori strain, we studied 1) the delivery of VacA to cells, 2) the localization and fate of internalized toxin, and 3) the persistence of toxin inside the cell. The investigative techniques used were neutral red dye uptake, ultrastructural immunocytochemistry, quantitative immunofluorescence, and immunoblotting. We found that VacA 1) is delivered to cells in both free and membrane-bound form (i.e., as vesicles formed by the bacterial outer membrane), 2) localizes inside the endosomal-lysosomal compartment, in both free and membrane-bound form, 3) persists within the cell for at least 72 h, without loss of vacuolating power, which, however, becomes evident only when NH4Cl is added, and 4) generally does not degrade into fragments smaller than ∼90 kDa. Our findings suggest that, while accumulating inside the endosomal-lysosomal compartment, a large amount of VacA avoids the main lysosomal degradative processes and retains its apparent molecular integrity.
Distension-related responses in circular and longitudinal muscle of the human esophagus: an ultrasonographic study
Authors: Yamamoto Yoshihiro , Liu Jianmin , Smith Terrence K. , Mittal Ravinder K.
Published: December 2017

Both circular muscles (CM) and longitudinal muscles (LM) of the esophagus participate in peristalsis. Various measurement techniques have yielded conflicting information as to the temporal correlation between contraction in the two muscle layers. High-frequency intraluminal ultrasound (HFIUS) is a novel technique to detect contraction of LM and CM of the esophagus. We investigated the temporal correlation between the CM and LM contraction during ascending excitatory and descending inhibitory reflexes using HFIUS. A manometric catheter equipped with two balloons and a 12.5-MHz ultrasound transducer catheter was used to study 10 normal healthy subjects. The changes in muscle thickness and pressure, proximal and distal to esophageal distension, were recorded at 5 and 10 cm above the lower esophageal sphincter (LES). The esophageal distension induced an increase in pressure and an increase in muscle thickness of both CM and LM layers proximal to the distension site. The onset of increase in muscle thickness and peak muscle thickness in two layers occurred at the same time. There was a close temporal correlation between the changes in pressure and changes in muscle thickness. Atropine inhibited the distension-related pressure and muscle thickness increase in both layers. Distal to the esophageal distension, there was no change in pressure but a decrease in the thickness of the two muscle layers. The decrease in muscle thickness of the two layers occurred at the same time. The responses of the two muscle layers to distension were similar at 5- and 10-cm sites above the LES. HFIUS is a relatively noninvasive technique to study the LM layer response during peristalsis in vivo. Our data indicate that the two muscle layers may contract and relax together during distension-related peristaltic reflexes in the esophagus.
Effects of nitroglycerin on liquid gastric emptying and antropyloroduodenal motility
Authors: Sun W.-M. , Doran S. , Jones K. L. , Ooi E. , Boeckxstaens G. , Hebbard G. S. , Lingenfelser T. , Morley J. E. , Dent J. , Horowitz M.
Published: December 2017

The effects of the nitric oxide donor nitroglycerin on gastric emptying and antropyloroduodenal motility were evaluated in nine healthy male subjects (ages 19–36 yr). Antropyloroduodenal pressures were recorded with a manometric assembly that had nine side holes spanning the antrum and proximal duodenum and a pyloric sleeve sensor; gastric emptying was quantified scintigraphically. In each subject, the emptying of 300 ml of 25% glucose labeled with99mTc was assessed on two separate days during intravenous infusion of either nitroglycerin (5 μg/min in 5% dextrose) or 5% dextrose (control). Studies were performed with the subject in the supine position; blood pressure and heart rate were monitored. Nitroglycerin had no significant effect on blood pressure or heart rate. Nitroglycerin slowed gastric emptying ( P < 0.02), and this was associated with greater retention of the drink in the proximal stomach ( P < 0.05). In both nitroglycerin and control studies, ingestion of the drink was associated with an increase in the number of isolated pyloric pressure waves ( P < 0.05) and antral pressure wave sequences ( P < 0.05). Nitroglycerin reduced the number of isolated pyloric pressure waves ( P < 0.05), basal pyloric pressure ( P < 0.05), and the number of antral pressure wave sequences ( P < 0.05), but not the total number of antral pressure waves. The rate of gastric emptying and the number of isolated pyloric pressure waves were inversely related during control ( P = 0.03) and nitroglycerin ( P < 0.05) infusions. We conclude that in normal subjects, 1) gastric emptying of 300 ml of 25% glucose is inversely related to the frequency of phasic pyloric pressure waves, and 2) nitroglycerin in a dose of 5 μg/min inhibits pyloric motility, alters the organization but not the number of antral pressure waves, and slows gastric emptying and intragastric distribution of 25% glucose.
A neurokinin-1 receptor antagonist reduced hypersalivation and gastric contractility related to emesis in dogs
Authors: Furukawa Naohiro , Fukuda Hiroyuki , Hatano Mizue , Koga Tomoshige , Shiroshita Yasuteru
Published: December 2017

The roles of tachykinin neurokinin-1 (NK1) receptors in the induction of fictive retching, hypersalivation, and gastric responses associated with emesis induced by abdominal vagal stimulation were studied in paralyzed, decerebrated dogs. Vagal stimulation induced gradual increases in salivary secretion and activity of the parasympathetic postganglionic fibers to the submandibular gland, relaxation of the gastric corpus and antrum, and fictive retching. However, hypersalivation and increased nerve activity were suppressed and antral contractility was enhanced during fictive retching. An NK1 receptor antagonist, GR-205171, abolished the enhancement of antral contractility and fictive retching but had no effect on corpus and antral relaxation. Hypersalivation and increased nerve activity were inhibited by GR-205171 but were not completely abolished. Reflex salivation by lingual nerve stimulation was unaffected. These results suggest that GR-205171 acts on the afferent pathway in the bulb and diminishes hypersalivation and antral contraction related to emesis as well as fictive retching but does not affect gastric relaxation or hypersalivation induced by the vagovagal, vagosalivary, and linguosalivary reflexes.
Glucagon-like peptide-1 inhibits gastropancreatic function by inhibiting central parasympathetic outflow
Authors: Wettergren André , Wøjdemann Morten , Holst Jens Juul
Published: December 2017

Glucagon-like peptide (GLP)-1 inhibits acid secretion and gastric emptying in humans, but the effect on acid secretion is lost after vagotomy. To elucidate the mechanism involved, we studied its effect on vagally stimulated gastropancreatic secretion and motility in urethan-anesthetized pigs with cut splanchnic nerves, in which insulin-induced hypoglycemia elicited a marked stimulation of gastropancreatic secretion and antral motility. In addition, we studied vagally stimulated motility and pancreatic secretion in isolated perfused preparations of the porcine antrum and pancreas. GLP-1 infusion (2 pmol ⋅ kg−1⋅ min−1) strongly and significantly inhibited hypoglycemia-induced antral motility, gastric acid secretion, pancreatic bicarbonate and protein secretion, and pancreatic polypeptide (PP) secretion. GLP-1 (at 10−10–10−8mol/l) did not inhibit vagally induced antral motility, pancreatic exocrine secretion, or gastrin and PP secretion in isolated perfused antrum and pancreas. We conclude that the inhibitory effect of peripheral GLP-1 on upper gastrointestinal secretion and motility is exerted via interaction with centers in the brain or afferent neural pathways relaying to the vagal motor nuclei.
Role of endotoxin in the hypermetabolic state after acute ethanol exposure
Authors: Rivera Chantal A. , Bradford Blair U. , Seabra Vitor , Thurman Ronald G.
Published: December 2017

This study investigated the role of endotoxin in the hypermetabolic state or swift increase in alcohol metabolism (SIAM) due to acute ethanol exposure. Female Sprague-Dawley rats (100–120 g) were given ethanol (5 g/kg) by gavage. Endotoxin measured in plasma from portal blood was not detectable in saline-treated controls; however, 90 min after ethanol, endotoxin was increased to 85 ± 14 pg/ml, and endotoxin clearance was diminished by ∼50%. Oxygen uptake in perfused livers was increased 48% by ethanol, and production of PGE2by isolated Kupffer cells was increased similarly. These effects were blunted by elimination of gram-negative bacteria and endotoxin with antibiotics before ethanol administration. To reproduce ethanol-induced endotoxemia, endotoxin was infused via the mesenteric vein at a rate of 2 ng ⋅ kg−1⋅ h−1. Endotoxin mimicked the effect of ethanol on oxygen uptake. The specific Kupffer cell toxicant GdCl3completely prevented increases in oxygen uptake due to endotoxin. These findings demonstrate that endotoxin plays a pivotal role in SIAM, most likely by stimulating eicosanoid release from Kupffer cells.
Nitric oxide inhibits potassium transport in the rat distal colon
Authors: Aizman Roman , Brismar Hjalmar , Celsi Gianni
Published: December 2017

The effect of the nitric oxide (NO) pathway on K+ (measured using 86Rb) transport in adult rat distal colon was investigated in muscle-stripped segments of colons mounted in Ussing chambers. When added to the mucosal solution, the endogenous precursor of NO, l-arginine (30 mM), inhibited both mucosal-to-serosal and serosal-to-mucosal 86Rb fluxes and caused a prolonged decrease of short-circuit current ( I sc). This effect was significantly reduced by the NO synthase inhibitor N G-nitro-l-arginine methyl ester (l-NAME) but not by d-NAME. Mucosal application of S-nitroso- N-acetyl-penicillamine (SNAP) inhibited mucosal-to-serosal 86Rb flux without affecting serosal-to-mucosal transport. Serosal addition of two different exogenous NO donors, sodium nitroprusside (0.1 mM) and SNAP (0.2 mM), decreased serosal-to-mucosal 86Rb flux, whereas I sc increased. The SNAP-induced decrease in86Rb flux was abolished by 1H-(1,2,4)oxodiazolo(4,3-a)quinoxalin-1-one (0.2 mM), a selective inhibitor of NO-stimulated soluble guanylyl cyclase, and by methylene blue (0.01 mM). Addition of 8-bromo-cGMP (2 × 10−4 M) in the presence of an inhibitor of cGMP-specific phosphodiesterase mimicked the effects of NO-donating compounds. This study provides evidence that NO inhibits K+ transport in the rat distal colon via a cGMP-dependent pathway. The effect on net K+transport may depend on the side of NO action.
Regulation of intracellular pH gradients by identified Na/H exchanger isoforms and a short-chain fatty acid
Authors: Gonda Tamas , Maouyo Djikolngar , Rees Sharon E. , Montrose Marshall H.
Published: December 2017

Colonic luminal short-chain fatty acids (SCFA) stimulate electroneutral sodium absorption via activation of apical Na/H exchange. HT29-C1 cells were used previously to demonstrate that transepithelial SCFA gradients selectively activate polarized Na/H exchangers. Fluorometry and confocal microscopy (with BCECF and carboxy SNARF-1, respectively) are used to measure intracellular pH (pHi) in HT29-C1 cells, to find out which Na/H exchanger isoforms are expressed and if results are due to pHigradients. Inhibition of Na/H exchange by HOE-694 identified 1) two inhibitory sites [50% inhibitory dose (ID50) = 1.6 and 0.05 μM] in suspended cells and 2) one inhibitory site each in the apical and basolateral membranes of filter-attached cells (apical ID50= 1.4 μM, basolateral ID50= 0.3 μM). RT-PCR detected mRNA of Na/H exchanger isoforms NHE1 and NHE2 but not of NHE3. Confocal microscopy of filter-attached cells reported HOE-694-sensitive pHirecovery in response to luminal or serosal 130 mM propionate. Confocal analysis along the apical-to-basal axis revealed that 1) luminal or serosal propionate establishes transcellular pHigradients and 2) the predominant site of pHiacidification and pHirecovery is the apical portion of cells. Luminal propionate produced a significantly greater acidification of the apical vs. basal portion of the cell (compared with serosal propionate), but no other dependence on the orientation of the SCFA gradient was observed. Results provide direct evidence for a subcellular response that assures robust activation of apical NHE2 and dampening of basolateral NHE1 during pHiregulation.
Calcium signaling in rat pancreatic acinar cells: a role for Gαq, Gα11, and Gα14
Authors: Yule David I. , Baker Christopher W. , Williams John A.
Published: December 2017

Stimulus-secretion coupling in the pancreatic acinar cell is initiated by the secretagogues CCK and ACh and results in the secretion by exocytosis of the contents of zymogen granules. A key event in this pathway is the G protein-activated production of second messengers and the subsequent elevation of cytosolic-free Ca2+. The aim of this study was therefore to define the heterotrimeric G protein α-subunits present and participating in this pathway in rat pancreatic acinar cells. RT-PCR products were amplified from pancreatic acinar cell mRNA with primers specific for Gαq, Gα11, and Gα14but were not amplified with primers specific for Gα15. The sequences of these PCR products confirmed them to be portions of the rat homologues of Gαq, Gα11, and Gα14. The pancreatic-derived cell line AR42J similarly expressed Gαq, Gα11, and Gα14; however, the Chinese hamster ovary (CHO) cell line only expressed Gα11and Gαq. These data indicate that caution should be exercised when comparing signal transduction pathways between different cell types. The expression of these proteins in acinar cells was confirmed by immunoblotting samples of acinar membrane protein using specific antisera to the individual G protein α-subunits. The role of these proteins in Ca2+signaling events was investigated by microinjecting a neutralizing antibody directed against a homologous sequence in Gαq, Gα11, and Gα14into acinar cells and CHO cells. Ca2+signaling was inhibited in acinar cells and receptor-bearing CHO cells in response to both physiological and supermaximal concentrations of agonists. The inhibition was >75% in both cell types. These data indicate a role for Gαqand/or Gα11in intracellular Ca2+concentration signaling in CHO cells, and in addition to Gαqand Gα11, Gα14may also fulfill this role in rat pancreatic acinar cells.
Basal pressure patterns and reflexive motor responses in the human ileocolonic junction
Authors: Dinning P. G. , Bampton P. A. , Kennedy M. L. , Kajimoto T. , Lubowski D. Z. , de Carle D. J. , Cook I. J.
Published: December 2017

This study aimed to determine whether a sustained high-pressure zone exists at the human ileocolonic junction (ICJ) and whether the motor responses of ICJ are consistent with sphincteric function. In 10 subjects with temporary ileostomies, a high-pressure zone was identified using a manometric pull-through with a mean pressure of 9.7 ± 3.2 mmHg and length of 4.8 ± 1.2 cm. Prolonged recordings using a sleeve sensor confirmed sustained tone in the ICJ and superimposed phasic pressure waves (4–8 counts/min) occupying 35% of fasted state. A meal increased ICJ tone ( P = 0.0001) and the proportion of time occupied by phasic activity to 50% ( P = 0.013). Terminal ileal propagating pressure wave sequences inhibited ICJ phasic activity, and sequences not extending to the cecum reduced ICJ tone (9.0 ± 7.2 to 5.6 ± 6.3 mmHg; P = 0.04). Cecal distension increased ICJ tone (8.9 ± 4.4 mmHg to 11.7 ± 4.9 mmHg; P = 0.005). The ICJ response to ileal distension was variable and depended on resting tone at the time of distension. We conclude that the human ICJ has sustained tone with superimposed phasic activity. Tone is augmented by cecal distension or a meal and is inhibited by ileal propagating pressure waves. Response to ileal distension is variable but suggests control by descending excitatory and inhibitory pathways.
Gastrin induces c-fosgene transcription via multiple signaling pathways
Authors: Stepan Vinzenz M. , Tatewaki Makoto , Matsushima Masashi , Dickinson Chris J. , del Valle John , Todisco Andrea
Published: December 2017

We previously observed that the trophic actions of gastrin (G17) on the AR42J rat acinar cell line are mediated by mitogen-activated protein kinase (MAPK)-induced c- fos gene transcription via protein kinase C (PKC)-dependent and -independent pathways. In this study, we further investigated the signaling pathways that target c- fos in response to G17. G17 led to a sixfold induction in luciferase activity in cells transfected with plasmids containing the −356+109 sequence of the murine c- fos promoter, which includes the Sis-inducible element (SIE), serum response element (SRE), and the Ca2+/cAMP response element (CRE) regulatory elements. Addition of either the selective PKC inhibitor GF-109203X or the MAPK/extracellular signal-regulated kinase inhibitor PD-98059 resulted in an 80% reduction in luciferase activity. G17 induced the transcriptional activity of both Elk-1 and Sap-1a, transcription factors that bind to the E26 transformation specific (Ets) DNA sequence of the SRE, and this effect was inhibited by both GF-109203X and PD-98059. Point mutations in the Ets sequence led to a 4-fold induction of c- fos transcription stimulated by G17 and to a 1.3-fold induction in response to epidermal growth factor (EGF). In contrast, mutations in the CA rich G (CArG) sequence of the SRE prevented transcriptional activation by both G17 and EGF. G17 induction of the Ets mutant construct was unaffected by either GF-109203X or PD-98059. Because activation of the SRE involves the small GTP-binding protein Rho A, we examined the role of Rho A in G17 induction of c- fos transcription. Inactivation of Rho A by either the specific inhibitor C3 or by expression of a dominant negative Rho A gene inhibited G17 induction of both the wild-type and the Ets mutant constructs by 60%. C3 also inhibited G17-stimulated AR42J cell proliferation. Thus G17 targets the c- fos promoter CArG sequence via Rho A-dependent pathways, and Rho A appears to play an important role in the regulation of the trophic action of G17.
Mechanisms of basolateral Na+ transport in rabbit esophageal epithelial cells
Authors: Abdulnour-Nakhoul Solange , Bor Serhat , Imeryuz Nese , Orlando Roy C.
Published: December 2017

We examined the mechanisms of cellular Na+ transport, both Cl− dependent and Cl− independent, in the mammalian esophageal epithelium. Rabbit esophageal epithelium was dissected from its muscular layers and mounted in a modified Ussing chamber for impalement with ion-selective microelectrodes. In bicarbonate Ringer, transepithelial potential difference was −14.9 ± 0.9 mV, the transepithelial resistance ( R TE) was 1,879 ± 142 Ω ⋅ cm2, the basolateral membrane potential difference ( V mBL) was −53 ± 1.5 mV, and the intracellular activity of Na+([Formula: see text]) was 24.6 ± 2.1 mM. Removal of Na+ and Cl− from the serosal and luminal baths decreased [Formula: see text] to 6.6 ± 0.6 mM. Readdition of Na+ to the serosal bath in the absence of Cl− increased[Formula: see text] by 21.8 ± 3.0 mM, whereas V mBL and R TE remained unchanged. When serosal Na+ was readded in the presence of amiloride the increase in[Formula: see text] and the rate of Na+ entry were decreased by ∼50%. 5-( N-ethyl- N-isopropyl)amiloride mimicked the effect of amiloride, whereas phenamil did not. Subsequent readdition of Cl− to the serosal bath further increased [Formula: see text] by 4.4 ± 1.9 mM. When the cells were acid loaded by pretreatment with[Formula: see text] in nominally[Formula: see text]-free Ringer, intracellular pH measurements showed a pHi recovery that is dependent on the presence of Na+ in the serosal bath and that can be blocked by amiloride. These data indicate that esophageal epithelial cells possess a Na+-dependent, amiloride-sensitive electroneutral mechanism for Na+entry consistent with the presence of a basolateral Na+/H+ exchanger. The ability of Cl− to further enhance Na+ entry supports the existence of at least one additional Cl−-dependent component of basolateral Na+entry.
Evidence for an anion exchange mechanism for uptake of conjugated bile acid from the rat jejunum
Authors: Amelsberg Andree , Jochims Christina , Richter Claus Peter , Nitsche Rolf , Fölsch Ulrich R.
Published: December 2017

Absorption of conjugated bile acids from the small intestine is very efficient. The mechanisms of jejunal absorption are not very well understood. The aim of this study was to clarify the mechanism of absorption of conjugated bile acid at the apical membrane of jejunal epithelial cells. Brush-border membrane vesicles from intestinal epithelial cells of the rat were prepared. Absorption of two taurine-conjugated bile acids that are representative of endogenous bile acids in many variate vertebrate species were studied. In ileal, but not jejunal brush-border membrane vesicles, transport of conjugated bile acids was cis-stimulated by sodium. Transport of conjugated bile acids was trans-stimulated by bicarbonate in the jejunum. Absorption of conjugated dihydroxy-bile acids was almost twice as fast as of trihydroxy-bile acids. Coincubation with other conjugated bile acids, bromosulfophthalein, and DIDS, as well as by incubation in the cold inhibited the transport rate effectively. Absorption of conjugated bile acids in the jejunum from the rat is driven by anion exchange and is most likely an antiport transport.
Physiological changes in blood glucose do not affect gastric compliance and perception in normal subjects
Authors: Verhagen M. A. M. T. , Rayner C. K. , Andrews J. M. , Hebbard G. S. , Doran S. M. , Samsom M. , Horowitz M.
Published: December 2017

Marked hyperglycemia (blood glucose ∼14 mmol/l) slows gastric emptying and affects the perception of sensations arising from the gut. Elevation of blood glucose within the physiological range also slows gastric emptying. This study aimed to determine whether physiological changes in blood glucose affect proximal gastric compliance and/or the perception of gastric distension in the fasting state. Paired studies were conducted in 10 fasting healthy volunteers. On a single day, isovolumetric and isobaric distensions of the proximal stomach were performed using an electronic barostat while the blood glucose concentration was maintained at 4 and 9 mmol/l in random order. Sensations were quantified using visual analog scales. The blood glucose concentration had no effect on the pressure-volume relationship during either isovolumetric or isobaric distensions or the perception of gastric distension. At both blood glucose concentrations, the perceptions of fullness, nausea, bloating, and abdominal discomfort, but not hunger or desire to eat, were related to intrabag volume ( P ≤ 0.002) and pressure ( P ≤ 0.01). We conclude that, in the fasted state, elevations of blood glucose within the physiological range do not affect proximal gastric compliance or the perception of gastric distension.
Differential activation of phosphoinositide 3-kinase by endothelin and ceramide in colonic smooth muscle cells
Authors: Su Xuehui , Wang Pinglang , Ibitayo Adenike , Bitar Khalil N.
Published: December 2017

We have investigated the hypothesis that different contractile agonists activate distinct catalytic subunits of phosphoinositide (PI) 3-kinase in smooth muscle cells. Endothelin (10−7M) induced a sustained increase in PI 3-kinase activity at both 30 s and 4 min of stimulation (151.5 ± 8.5% at 30 s and 175.8 ± 8.7% at 4 min, P < 0.005). Preincubation of smooth muscle cells with the tyrosine kinase inhibitor genistein (3 μM) resulted in a significant inhibition of both C2ceramide-induced and endothelin-induced PI 3-kinase activation and contraction. Preincubation with herbimycin A, an Src kinase inhibitor (3 μM), inhibited only C2ceramide-induced PI 3-kinase activation and contraction. Western blotting using Src kinase antibody showed that C2ceramide, not endothelin, stimulated the phosphorylation of Src kinase. Western blotting and immunoprecipitation with PI 3-kinase antibodies to the regulatory subunit p85 and the catalytic subunits p110α and p110γ indicated that both endothelin and C2ceramide interacted with the regulatory subunit p85; endothelin interacted with the catalytic subunits p110α and p110γ, whereas C2ceramide interacted only with the catalytic subunit p110α. In summary, C2ceramide activated PI 3-kinase p110α subunit by a tyrosine kinase-mediated pathway, whereas endothelin-induced contraction, unlike C2ceramide, was not mediated by the activation of Src kinase but was mediated by G protein activation of both p110α and p110γ subunits (type IA and IB) of PI 3-kinase.
Cloning and functional expression of an SGLT-1-like protein from theXenopus laevisintestine
Authors: Nagata Katsumi , Hori Naohiro , Sato Kenzo , Ohta Kunimasa , Tanaka Hideaki , Hiji Yasutake
Published: December 2017

A cDNA encoding an Na+-glucose cotransporter type 1 (SGLT-1)-like protein was cloned from the Xenopus laevis intestine by the 5′- and 3′-rapid amplification of cDNA ends method. The deduced amino acid sequence was 673 residues long, with a predicted mass of 74.1 kDa and 52–53% identity to mammalian SGLT-1s. This gene was expressed in the small intestine and kidney, reflecting a tissue distribution similar to that of SGLT-1. The function of the protein was studied using the two-microelectrode voltage-clamp technique after injection of cRNA into Xenopus laevis oocytes. Perfusion with myo-inositol elicited about twofold larger inward currents than perfusion withd-glucose. The order of the substrate specificity was myo-inositol > d-glucose >d-galactose ≥ α-methyl-d-glucoside. The current induced by myo-inositol increased with membrane hyperpolarization and depended on external myo-inositol and Na+: the apparent Michaelis-Menten constant was 0.25 ± 0.07 (SD) mM with myo-inositol, whereas the apparent concentration for half-maximal activation was 12.5 ± 1.0 mM and the Hill coefficient was 1.6 ± 0.1 with Na+. In conclusion, the cloned protein shares features with both SGLT-1 and the Na+- myo-inositol cotransporter.
III. The mucosal antigen barrier: cross talk with mucosal cytokines
Authors: Perdue Mary H.
Published: December 2017

We have known for many years that mucosal responses to antigens are regulated by immune cells and their molecular signals. More recently, it has become clear that epithelial cells also synthesize and secrete chemokines and cytokines. A sophisticated system of bidirectional cytokine signals is responsible for immune activation in the case of enteropathogens vs. immune suppression to food and commensal microbial antigens. A key factor in determining antigen handling is the route taken by antigens across the epithelial barrier. Cytokines and other mucosal messenger molecules play a critical role in the regulation of transepithelial antigen transport.
Kex2 family endoprotease furin is expressed specifically in pit-region parietal cells of the rat gastric mucosa
Authors: Kamimura Hitoshi , Konda Yoshitaka , Yokota Hiromi , Takenoshita Sei-Ichi , Nagamachi Yukio , Kuwano Hiroyuki , Takeuchi Toshiyuki
Published: December 2017

The proprotein-processing endoprotease furin is localized in the gastric epithelial cells of the pit region in the rat gastric gland. The gastric pit is composed of several cell types, including gastric surface mucosal (GSM) cells and parietal cells. Furin converts many growth- or differentiation-related proproteins to their active forms. We examined identification of furin-positive cells by immunostaining of rat gastric mucosa and regulators of the furin expression by measuring the furin promoter activity by luciferase assay. Furin-positive cells were stained for H+-K+-ATPase, indicating that they are parietal cells. Furin-positive parietal cells were not stained for transforming growth factor-α (TGF-α) but were surrounded by TGF-α-positive GSM cells. In contrast, parietal cells below the proliferative zone were positive for TGF-α but not for furin. Furin-positive parietal cells expressed a high level of epidermal growth factor receptor (EGFR). TGF-α stimulated the furin promoter activity highly in a mouse GSM cell line GSM06. Thus we suggest that the parietal cells of the pit region have furin-mediated functions that can be stimulated by EGFR signaling.
Blood flow-dependent prostaglandin F2αregulates intestinal glucose uptake from the blood
Authors: Han Chao , Ming Zhi , Lautt W. Wayne
Published: December 2017

Intestinal glucose uptake (GUi) from blood increased when blood flow (BF) was increased. The increase in BF could elevate shear stress. Therefore, we hypothesize that shear stress-induced release of autacoids mediates the increase in GUi. A surgically separated segment of small intestine was perfused in situ with the use of an arterial circuit in anesthetized cats. Arterial and portal blood samples were taken simultaneously for assessment of GUi. Adenosine was used to elevate intestinal BF. The GUiincreased by 45.0 ± 18.3 from 25.3 ± 3.8 μmol ⋅ min−1⋅ 100 g tissue−1when the BF increased about four times. It was not a direct effect of adenosine because GUiwas not altered if the flow was held constant. This increase was blocked by a cyclooxygenase inhibitor, indomethacin, but not by nitric oxide synthase blocker NG-nitro-l-arginine methyl ester. Furthermore, prostaglandin F2α(PGF2α) but not PGE2or PGI2reversed the blockade of the increase in GUiafter indomethacin during elevated blood flow, whereas they had no influence on basal uptake. The results suggest that shear stress-induced release of PGF2αmediated the increase in GUiwhen blood flow was elevated.
Adherent surface mucus gel restricts diffusion of macromolecules in rat duodenum in vivo
Authors: Flemström Gunnar , Hällgren Anneli , Nylander Olof , Engstrand Lars , Wilander Erik , Allen Adrian
Published: December 2017

The aim of this study was to investigate the permeability of the adherent mucus gel layer in rat duodenum in vivo to macromolecules applied in the lumen. Rats were anesthetized with thiobarbiturate, and the duodenum was perfused with isotonic NaCl solution containing large-molecular-size secretagogues. Effects on mucosal [Formula: see text]secretion and blood-to-lumen51chromium-labeled EDTA clearance were used as indexes that compounds had migrated across the mucus layer. Exposure to a low concentration of papain (10 U/100 ml) for 30 min removed the mucus layer without damage to the epithelium and induced or markedly enhanced [Formula: see text]secretory responses to cholera toxin (molecular mass of 85 kDa) or glucagon (3.5 kDa). Water extracts from a VacA cytotoxin (89 kDa) producing Helicobacter pylori strain, but not from a toxin-negative isogenic mutant, caused a small increase in[Formula: see text] secretion but only after the mucus layer had been removed by papain. The duodenal surface mucus gel thus significantly restricts migration of macromolecules to the duodenal surface. Release of bacterial toxins at the cell-mucus interface may enhance or be a prerequisite for their effects on the gastrointestinal mucosa.
Azoxymethane-induced fulminant hepatic failure in C57BL/6J mice: characterization of a new animal model
Authors: Matkowskyj Kristina A. , Marrero Jorge A. , Carroll Robert E. , Danilkovich Alexey V. , Green Richard M. , Benya Richard V.
Published: December 2017

Without transplantation, ∼50–90% of all patients with fulminant hepatic failure (FHF) die. This poor outcome is due in part to the absence of an appropriate animal model, which would allow for a greater understanding of the pathophysiology of this syndrome. Given the reports of liver injury in humans and livestock fed cycad palm nuts on the island of Guam, we hypothesized that the active ingredient azoxymethane (AOM) could cause FHF. We therefore evaluated AOM in C57BL/6J mice. Histologically, we observed microvesicular steatosis 2 h, sinusoidal dilatation 4 h, and centrilobular necrosis 20 h after AOM administration, and transmission electron microscopy showed that this agent causes mitochondrial injury. FHF was associated with all four stages of encephalopathy, as well as by a prodromal period of decreased eating and drinking lasting ∼15 h before the development of stage I encephalopathy (i.e., loss of scatter reflex). Late encephalopathy was associated with increased arterial ammonia, decreased serum glucose, and evidence of brain edema (astrocyte swelling). We show that AOM-induced FHF is highly reproducible, without evidence of lot-to-lot variability, and is dose dependent. These findings therefore suggest that AOM is an excellent agent for the study of FHF, as well as indicate that Guamanian FHF may be due to AOM found in unwashed cycad palm nuts.
Expression of voltage-dependent K+channel genes in mesenteric artery smooth muscle cells
Authors: Xu Chuanli , Lu Yanjie , Tang Guanghua , Wang Rui
Published: December 2017

Molecular basis of native voltage-dependent K+(Kv) channels in smooth muscle cells (SMCs) from rat mesenteric arteries was investigated. The whole cell patch-clamp study revealed that a 4-aminopyridine-sensitive delayed rectifier K+current ( IK) was the predominant K+conductance in these cells. A systematic screening of the expression of 18 Kv channel genes using RT-PCR technique showed that six IK-encoding genes (Kv1.2, Kv1.3, Kv1.5, Kv2.1, Kv2.2, and Kv3.2) were expressed in mesenteric artery. Although no transient outward Kv currents ( IA) were recorded in the studied SMCs, transcripts of multiple IA-encoding genes, including Kv1.4, Kv3.3, Kv3.4, Kv4.1, Kv4.2, and Kv4.3 as well as IA-facilitating Kv β-subunits (Kvβ1, Kvβ2, and Kvβ3), were detected in mesenteric arteries. Western blot analysis demonstrated that four IK-related Kv channel proteins (Kv1.2, Kv1.3, Kv1.5, and Kv2.1) were detected in mesenteric artery tissues. The presence of Kv1.2, Kv1.3, Kv1.5, and Kv2.1 channel proteins in isolated SMCs was further confirmed by immunocytochemistry study. Our results suggest that the native IKin rat mesenteric artery SMCs might be generated by heteromultimerization of Kv genes.
Development of a test to measure gastric accommodation in humans
Authors: Kuiken Sjoerd D. , Samsom Melvin , Camilleri Michael , Mullan Brian P. , Burton Duane D. , Kost Louis J. , Hardyman Timothy J. , Brinkmann Benjamin H. , O'connor Michael K.
Published: December 2017

Postprandial symptoms of bloating, distension, early satiety, and nausea are associated with impaired postprandial gastric accommodation, which is detectable by means of an intragastric, barostatically controlled balloon in the proximal stomach and by ultrasound in the distal stomach. Our aim was to develop a noninvasive method to measure the entire gastric accommodation reflex. In 10 healthy volunteers, we used single photon emission computed tomography (SPECT) to measure fasting and postprandial gastric volumes. This method involved intravenous injection of99mTc pertechnetate and gastric reconstruction of tomographic images with Analyze software. SPECT-Analyze imaging detects the postprandial gastric accommodation reflex in vivo. Mean fasting gastric volume was 182 ± 11 (SE) ml and mean postprandial volume was 690 ± 32 ml ( P < 0.001). Both proximal and distal segments of stomach showed a two- to almost fourfold difference in volumes postprandially. Intraobserver coefficients of variation in estimated fasting and postprandial volumes were 9 and 8%; interobserver variations were 13 and 12%, respectively. SPECT-Analyze noninvasively measures postprandial gastric (total, proximal, and distal) accommodation in humans. This method appears promising to compare the accommodation response in health and disease and to perform mechanistic studies of the accommodation response.
Increased lactulose/rhamnose ratio during fluid load is caused by increased urinary lactulose excretion
Authors: Hallemeesch Marcella M. , Lamers Wouter H. , Soeters Peter B. , Deutz Nicolaas E. P.
Published: December 2017

Noninvasive assessment of intestinal permeability in vivo is based on the measurement of urinary excretion of orally administered sugar probes. It is expressed as a ratio, usually lactulose/rhamnose or 3-O-methyl-d-glucose (3-OMG)/rhamnose. In both endotoxemic and control rats that were receiving fluid, we observed an increase in the recovery of lactulose and 3-OMG but not rhamnose in both groups, suggesting an enhancement of intestinal permeability. In the measurement of intestinal permeability, all pre- and postmucosal factors are considered equal for all sugars. We hypothesized that postmucosal factors and not changes in intestinal permeability caused the increased urinary lactulose and 3-OMG recoveries observed during fluid loading. Therefore, the effects of fluid loading on urinary excretion of the sugar probes were studied in healthy rats receiving the sugars intravenously. After intravenous injection, fluid loading increased urinary lactulose recovery threefold but not that of 3-OMG and rhamnose. In conclusion, fluid loading increases the lactulose/rhamnose ratio independent of changes in intestinal permeability. The 3-OMG/rhamnose ratio is not influenced by fluid loading.
Effects of cholecystokinin on appetite and pyloric motility during physiological hyperglycemia
Authors: Rayner C. K. , Park H. S. , Doran S. M. , Chapman I. M. , Horowitz M.
Published: December 2017

Recent studies suggest that the interaction between small intestinal nutrient stimulation and the blood glucose concentration is important in the regulation of gastric motility and appetite. The purpose of this study was to determine whether the effects of cholecystokinin octapeptide (CCK-8) on antropyloric motility and appetite are influenced by changes in the blood glucose concentration within the normal postprandial range. Seven healthy volunteers were studied on 4 separate days. A catheter incorporating a sleeve sensor was positioned across the pylorus, and the blood glucose was stabilized at either 4 mmol/l (2 days) or 8 mmol/l (2 days). After the desired blood glucose had been maintained for 90 min, an intravenous infusion of either CCK-8 (2 ng ⋅ kg− 1 ⋅ min− 1) or saline (control) was given for 60 min. Thirty minutes after the infusion began, the catheter was removed and subjects drank 400 ml of water with guar gum before being offered a buffet meal. The amount of food consumed (kcal) was quantified. The order of the studies was randomized and single-blinded. There were fewer antral waves at a blood glucose of 8 than at 4 mmol/l during the 90-min period before the infusions ( P < 0.05) and during the first 30 min of CCK-8 or saline infusion ( P = 0.07). CCK-8 suppressed antral waves ( P < 0.05), stimulated isolated pyloric pressure waves (IPPWs) ( P < 0.01), and increased basal pyloric pressure ( P < 0.005) compared with control. During administration of CCK-8, basal pyloric pressure ( P < 0.01), but not the number of IPPWs, was greater at a blood glucose of 8 mmol/l than at 4 mmol/l. CCK-8 suppressed the energy intake at the buffet meal ( P < 0.01), with no significant difference between the two blood glucose concentrations. We conclude that the acute effect of exogenous CCK-8 on basal pyloric pressure, but not appetite, is modulated by physiological changes in the blood glucose concentration.
X. Trefoil peptide and EGF receptor/ligand transgenic mice
Authors: Giraud Andrew S.
Published: December 2017

The use of genetically engineered mice with both gain-of-function and loss-of-function mutations has been particularly informative about the normal and pathophysiological actions of a number of regulatory peptides of the gastrointestinal tract. This review highlights some of the major findings pertinent to the epidermal growth factor (EGF) receptor and its ligands, particularly the major gut ligand transforming growth factor-α, as well as the trefoil peptides. Both of these peptide families have important local actions in maintaining tissue homeostasis and repair after injury, and when mechanisms governing their regulation are disrupted they may contribute to disease progression. Future applications of transgenic technology to these areas are likely to be productive in furthering our understanding of the biology of these peptides in health and disease.
Escherichia coliShiga toxins induce apoptosis in epithelial cells that is regulated by the Bcl-2 family
Authors: Jones Nicola L. , Islur Avinash , Haq Rizwan , Mascarenhas Mariola , Karmali Mohamed A. , Perdue Mary H. , Zanke Brent W. , Sherman Philip M.
Published: December 2017

Human intestinal cells lack globotriaosylceramide (Gb3), the receptor for Shiga toxin-1 (Stx1) and Shiga toxin-2 (Stx2). Therefore, the role of these toxins in mediating intestinal disease during infection with Shiga toxin-producing Escherichia coli is unclear. The aims of this study were to determine whether Stx1 and Stx2 induce apoptosis in epithelial cells expressing (HEp-2, Caco-2) or lacking (T84) Gb3and to characterize the role of the Bcl-2 family. Stx1 (12.5 ng/ml) induced apoptosis in both HEp-2 (21.9 ± 7.9% vs. 0.8 ± 0.3%, P = 0.01) and Caco-2 (10.1 ± 1.2% vs. 3.1 ± 0.4%, P = 0.006) cells but not in Gb3-deficient T84 cells. Toxin-mediated apoptosis of HEp-2 cells was associated with enhanced expression of the proapoptotic protein Bax. Inhibition of caspase activation prevented toxin-stimulated apoptosis. In addition, overexpression of Bcl-2 by transient transfection blocked Stx1-stimulated cell death. These findings indicate that Shiga toxins produced by E. coli signal Gb3-expressing epithelial cells to undergo apoptosis in association with enhanced Bax expression, thereby resulting in activation of the caspase cascade.
Iron-ascorbate alters the efficiency of Caco-2 cells to assemble and secrete lipoproteins
Authors: Courtois F. , Suc I. , Garofalo C. , Ledoux M. , Seidman E. , Levy E.
Published: December 2017

Although oxidative stress has been implicated in development of gut pathologies, its role in intestinal fat transport has not been investigated. We assessed the effect of Fe2+-ascorbate-mediated lipid peroxidation on lipid synthesis, apolipoprotein biogenesis, and lipoprotein assembly and secretion. Incubation of postconfluent Caco-2 cells with iron(II)-ascorbate (0.2 mM/2 mM) in the apical compartment significantly promoted malondialdehyde formation without affecting sucrase activity, transepithelial resistance, DNA and protein content, and cell viability. However, addition of the oxygen radical-generating system reduced 1) [14C]oleic acid incorporation into cellular triglycerides (15%, P < 0.0002) and phospholipids (16%, P < 0.0005); 2) de novo synthesis of cellular apolipoprotein A-I (apo A-I) (18%, P < 0.05), apo A-IV (38%, P < 0.05), and apo B-48 (45%, P< 0.003) after [35S]methionine addition; and 3) production of chylomicrons (50%), VLDL (40%), LDL (37%), and HDL (30%) (all P < 0.0001). In contrast, increased total cellular cholesterol formation (96%, P < 0.0001), assayed by [14C]acetate incorporation, was noted, attributable to marked elevation (70%, P < 0.04) in activity of dl-3-hydroxy-3-methyl-glutaryl-CoA reductase, the rate-limiting enzyme in cholesterol synthesis. The ratio of Acyl-CoA to cholesterol acyltransferase, the esterifying cholesterol enzyme, remained unchanged. Fe2+-ascorbate-mediated lipid peroxidation modifies intracellular fat absorption and may decrease enterocyte efficiency in assembling and transporting lipids during gut inflammation.
Treatment of gastric ulcers and diarrhea with the Amazonian herbal medicine sangre de grado
Authors: Miller Mark J. S. , MacNaughton Wallace K. , Zhang Xiao-Jing , Thompson Jane H. , Charbonnet Randi M. , Bobrowski Paul , Lao Juan , Trentacosti Ann Marie , Sandoval Manuel
Published: December 2017

Sangre de grado is an Amazonian herbal medicine used to facilitate the healing of gastric ulcers and to treat gastritis, diarrhea, skin lesions, and insect stings. This study was designed to evaluate the gastrointestinal applications. Gastric ulcers were induced in rats by brief serosal exposure of the fundus to acetic acid (80%). Sangre de grado was administered in drinking water at 1:1,000 and 1:10,000 dilutions from the postoperative period to day 7. Guinea pig ileum secretory responses to capsaicin, electrical field stimulation, and the neurokinin-1 (NK-1) agonist [Sar9,Met(O2)11]substance P were examined in Ussing chambers. Sangre de grado facilitated the healing of experimental gastric ulcer, reducing myeloperoxidase activity, ulcer size, and bacterial content of the ulcer. The expression of proinflammatory genes tumor necrosis factor-α, inducible nitric oxide synthase (iNOS), interleukin (IL)-1β, IL-6, and cyclooxygenase-2 was upregulated by ulcer induction but reduced by sangre de grado treatment, particularly iNOS and IL-6. In Ussing chambers, sangre de grado impaired the secretory response to capsaicin but not to electrical field stimulation or the NK-1 agonist. We conclude that sangre de grado is a potent, cost-effective treatment for gastrointestinal ulcers and distress via antimicrobial, anti-inflammatory, and sensory afferent-dependent actions.