bluuebunny/crossref_metadata_2025 huggingface dataset:
Functional vagal input to gastric myenteric plexus as assessed by vagal stimulation-induced Fos expression
Authors: Zheng Huiyuan , Berthoud Hans-Rudolf
Published: December 2017

Immunohistochemical detection of c-Fos expression was used to identify gastric myenteric plexus neurons that receive excitatory input from vagal efferent neurons activated by electrical stimulation of the cervical vagi in anesthetized rats. Vagal stimulation-induced Fos expression increased with higher pulse frequency, so that with 16 Hz (rectangular pulses of 1 mA/0.5 ms for 30 min) ∼30% and with 48 Hz 90% of all neurons near the lesser curvature were Fos positive. In sham-stimulated rats there was no Fos expression. The percentage of Fos-activated neurons was only slightly smaller (85% with 48 Hz) near the greater curvature. Prior atropine administration (1 mg/kg ip) had little effect on vagal stimulation-induced Fos expression, and in unilaterally stimulated rats there was no Fos expression on the contralateral (noninnervated) side of the stomach, ruling out mediation by gastric motility or secretory responses. However, polysynaptic recruitment of third- and higher-order neurons cannot be ruled out completely. These results support the idea that, at least in the stomach, functional excitatory innervation of myenteric plexus neurons by the efferent vagus is profuse and widespread, refuting the notion of only a few vagal “command neurons.”
Basolateral outward rectifier chloride channel in isolated crypts of mouse colon
Authors: Mignen Olivier , Egee Stéphane , Liberge Martine , Harvey Brian J.
Published: December 2017

Single channel patch-clamp techniques were used to demonstrate the presence of outwardly rectifying chloride channels in the basolateral membrane of crypt cells from mouse distal colon. These channels were rarely observed in the cell-attached mode and, in the inside-out configuration, only became active after a delay and depolarizing voltage steps. Single channel conductance was 23.4 pS between −100 and −40 mV and increased to 90.2 pS between 40 and 100 mV. The channel permeability sequence for anions was: I−> SCN−> Br−> Cl−> NO3−> F−≫ SO42−≈ gluconate. In inside-out patches, the channel open probability was voltage dependent but insensitive to intracellular Ca2+concentration. In cell-attached mode, forskolin, histamine, carbachol, A-23187, and activators of protein kinase C all failed to activate the channel, and activity could not be evoked in inside-out patches by exposure to the purified catalytic subunit of cAMP-dependent protein kinase A. The channel was inhibited by 5-nitro-2-(3-phenylpropylamino)benzoate, 9-anthracenecarboxylic acid, and DIDS. Stimulation of G proteins with guanosine 5′- O-(3-thiotriphosphate) decreased the channel open probability and conductance, whereas subsequent addition of guanosine 5′- O-(2-thiodiphosphate) reactivated the channel.
Enteral nutrient intake level determines intestinal protein synthesis and accretion rates in neonatal pigs
Authors: Stoll Barbara , Chang Xiaoyan , Fan Ming Z. , Reeds Peter J. , Burrin Douglas G.
Published: December 2017

Our objective was to determine the minimum enteral intake level necessary to increase the protein accretion rate (PAR) in the neonatal small intestine. Seven-day-old piglets received an equal total daily intake of an elemental diet, with different proportions given enterally (0, 10%, 20%, 40%, 60%, 80%, and 100%). After 7 days, piglets were infused intravenously with [2H3]leucine for 6 h, and the fractional protein synthesis rate (FSR) was measured in the proximal (PJ) and distal jejunum (DJ) and the proximal (PI) and distal ileum (DI). The jejunal FSR increased from 45%/day to 130%/day between 0 and 60% enteral intake, whereas the FSR in the ileum was less sensitive to enteral intake level. At 0% enteral intake, PAR was significantly negative in the PJ, DJ, and PI (range −70 to −43 mg/day) and positive in the DI (49 mg/day), whereas intestinal protein balance occurred at 20% enteral intake. At 100% enteral intake, the PAR was greatest in the DI, even though the rates of protein turnover were 50% lower than in the PJ. We conclude that there is net intestinal protein loss at 0% enteral intake, protein balance at 20% enteral intake, and maximal intestinal protein accretion at 60% enteral intake.
Gene transfer of recombinant endothelial nitric oxide synthase to liver in vivo and in vitro
Authors: Shah Vijay , Chen Alex F. , Cao Sheng , Hendrickson Helen , Weiler Deb , Smith Leslie , Yao Janet , Katusic Zvonimir S.
Published: December 2017

Endothelial nitric oxide synthase (eNOS)-derived nitric oxide (NO) contributes to hepatic vascular homeostasis. The aim of this study was to examine whether delivery of an adenoviral vector encoding eNOS gene to liver affects vasomotor function in vivo and the mechanism of NO production in vitro. Rats were administered adenoviruses encoding β-galactosidase (AdCMVLacZ) or eNOS (AdCMVeNOS) via tail vein injection and studied 1 wk later. In animals transduced with AdCMVLacZ, β-galactosidase activity was increased in the liver, most prominently in hepatocytes. In AdCMVeNOS-transduced animals, eNOS protein levels and catalytic activity were significantly increased. Overexpression of eNOS diminished baseline perfusion pressure and constriction in response to the α1-agonist methoxamine in the perfused liver. Transduction of cultured hepatocytes with AdCMVeNOS resulted in the targeting of recombinant eNOS to a perinuclear distribution and binding with the NOS-activating protein heat shock protein 90. These events were associated with increased ionomycin-stimulated NO release. In summary, this is the first study to demonstrate successful delivery of the recombinant eNOS gene to liver in vivo and in vitro with ensuing NO production.
A duodenum-specific enhancer regulates expression along three axes in the small intestine
Authors: Dusing Mary R. , Brickner Anthony G. , Lowe Sharon Y. , Cohen Mitchell B. , Wiginton Dan A.
Published: December 2017

Adenosine deaminase (ADA) is expressed at high levels in the epithelium of proximal small intestine. Transgenic mice were used to characterize the regulatory region governing this activation. A duodenum-specific enhancer is located in intron 2 of the human ADA gene at the central site among a cluster of seven DNase I-hypersensitive sites present in duodenal DNA. Flanking DNA, including the remaining hypersensitive sites, is required for consistent high-level enhancer function. The enhancer activates expression in a pattern identical to endogenous ADA along both the anterior-posterior axis of the small intestine and the crypt-villus differentiation axis of the intestinal epithelium. Timing of activation by the central enhancer mimics endogenous mouse ADA activation, occurring at 2–3 wk of age. However, two upstream DNA segments, one proximal and one distal, collaborate to change enhancer activation to a perinatal time point. Studies with duodenal nuclear extracts identified five distinct DNase I footprints within the enhancer. Protected regions encompass six putative binding sites for the transcription factor PDX-1, as well as proposed CDX, hepatocyte nuclear factor-4, and GATA-type sites.
Somatostatin receptor subtype-5 mediates inhibition of peptide YY secretion from rat intestinal cultures
Authors: Chisholm Connie , Greenberg Gordon R.
Published: December 2017

Somatostatin-14 (S-14) and somatostatin-28 (S-28) bind to five distinct membrane receptors (SSTRs), but S-28 has higher affinity for SSTR-5. Whether S-28 acting through SSTR-5 regulates inhibition of peptide YY (PYY) secretion was tested in fetal rat intestinal cell cultures. S-28 and S-14 caused dose-dependent inhibition of PYY secretion stimulated by gastrin-releasing peptide, but S-28 was more potent than S-14 (EC500.04 vs. 13.2 nM). PYY was inhibited by two analogs with affinity for SSTR-5, BIM-23268 and BIM-23052, more potently than S-14 and as effectively as S-28. The SSTR-5 analog L-362855 suppressed PYY equivalent only to S-14, but the structurally related peptide L-372588 (Phe to Tyr at position 2) was equipotent to S-28, whereas L-372587 (Phe to Tyr at position 7) caused no inhibition. An SSTR-2 analog decreased PYY secretion similar to S-14, and an SSTR-3 analog was ineffective. PYY secretion stimulated by phorbol 12-myristate 13-acetate and by forskolin was also more potently suppressed by S-28 and the octapeptide SSTR-5 analogs. The results indicate that S-28 mediates inhibition of gastrin-releasing peptide-stimulated PYY secretion through activation of SSTR-5 and includes suppression of cAMP- and protein kinase C-dependent pathways. Substitution of a single hydroxyl group confers differences in SSTR-5 agonist properties, suggesting region specificity for the intrinsic activity of this receptor subtype.
Molecular Physiology and Pathophysiology of Tight Junctions V. Assault of the tight junction by enteric pathogens
Authors: Sears Cynthia L.
Published: December 2017

Studies of the impact of enteric pathogens and their virulence factors on the proteins comprising the tight junction and zonula adherens offer a novel approach to dissection of tight junctional complex regulation. Most studies to date provide only tantalizing clues that select pathogens may indeed assault the tight junctional complex. Information on critical human pathogens such as Campylobacter jejuni and Shigella and Salmonella subspecies is lacking. Mechanistic studies are currently sparse, but available results on pathogenic Escherichia coli and specific virulence factors such as the Rho-modifying and protease bacterial toxins indicate four major mechanisms by which these pathogens may act: 1) direct cleavage of tight junctional structural proteins; 2) modification of the actin cytoskeleton; 3) activation of cellular signal transduction; and 4) triggering transmigration of polymorphonuclear cells across the epithelial cell barrier. New therapeutics may evolve from detailed studies of these pathogens and the cellular processes and proteins they disrupt.
Autocrine expression of activated transforming growth factor-β1 induces apoptosis in normal rat liver
Authors: Schrum Laura W. , Bird Mark A. , Salcher Olga , Burchardt Elmar-Reinhold , Grisham Joe W. , Brenner David A. , Rippe Richard A. , Behrns Kevin E.
Published: December 2017

The aim of this study was to determine the differential effects of latent and activated transforming growth factor (TGF)-β1in growth control of normal and proliferating hepatocytes in vivo. Rats were injected with adenoviruses expressing control transgenes (Ctrl), latent TGF-β1 [TGF-β(L)], or activated TGF-β1 [TGF-β(A)]. Additional animals underwent two-thirds partial hepatectomy (PH) 24 h after injection. Increased hepatocyte apoptosis was observed in TGF-β(A)-injected but not TGF-β(L)-injected animals 24 h postinjection (10.5%) compared with Ctrl animals (0.37%). The percent of apoptotic cells increased to 32.1% in TGF-β(A)-injected animals 48 h after injection. Furthermore, TGF-β(A)-injected rats did not survive 24 h after PH. Four hours after PH, 0.25 and 14.1% apoptotic hepatocytes were seen in Ctrl- and TGF-β(A)-injected rats, respectively. TGF-β(A)-induced apoptosis in primary rat hepatocytes was blocked with a pancaspase inhibitor. Thus autocrine expression of TGF-β(A) but not TGF-β(L) induces hepatocyte apoptosis in the normal rat liver. Rats overexpressing TGF-β(A) do not survive two-thirds PH due to hepatic apoptosis. Thus activation of TGF-β1 may be a critical step in the growth control of normal and proliferating rat hepatocytes.
Role of heme oxygenase-carbon monoxide pathway in pathogenesis of cirrhotic cardiomyopathy in the rat
Authors: Liu Hongqun , Song Daisheng , Lee Samuel S.
Published: December 2017

The enzyme heme oxygenase (HO), which exists in inducible (HO-1) and constitutive (HO-2) isoforms, degrades heme to biliverdin and CO. CO depresses cardiac contraction via cGMP. We aimed to clarify a possible role for the HO-CO pathway in the pathogenesis of cirrhotic cardiomyopathy in bile duct-ligated rats. Four weeks after bile duct ligation or sham operation, rat ventricles were examined for HO-1 and HO-2 mRNA by RT-PCR and for protein expression by Western blotting. Total HO enzyme activity and cGMP levels were also measured. The effects of a HO inhibitor, zinc protoporphyrin IX (ZnPP), on ventricular cGMP levels and isolated papillary muscle contractility were studied. We found that HO-1 mRNA transcription and protein expression were significantly augmented in cirrhotic hearts compared with sham-operated controls, whereas there was no difference in HO-2 mRNA or protein levels. Total HO activity and cGMP levels were significantly increased in cirrhotic ventricles vs. controls. In cirrhotic ventricles, treatment with ZnPP significantly decreased cGMP production and improved the blunted papillary muscle contractility, whereas it had no effect on control muscles. CO perfusion inhibited papillary muscle contractility, an effect completely blocked by methylene blue and partially blocked by ZnPP. These results indicate that activation of the HO-CO-cGMP pathway is involved in the pathogenesis of cirrhotic cardiomyopathy.
Effects of nutrients and serotonin 5-HT3 antagonism on symptoms evoked by distal gastric distension in humans
Authors: Ladabaum Uri , Brown Morton B. , Pan Wenqin , Owyang Chung , Hasler William L.
Published: December 2017

Distal gastric distension may contribute to meal-related dyspeptic symptoms. This study's aims were to determine the effects of distinct nutrient classes on symptoms induced by distal gastric distension and their dependence on 5-hydroxytryptamine3 (5-HT3) receptors. Nine healthy subjects rated pain, nausea, and bloating induced by isobaric distal gastric distensions (6–24 mmHg) during duodenal lipid, carbohydrate, protein, or saline perfusion after treatment with placebo or the 5-HT3 receptor antagonist granisetron (10 μg/kg iv). Distensions produced greater pain, nausea, and bloating with lipid at 1.5 kcal/min compared with saline ( P≤ 0.02), primarily because of greater distal gastric volumes at each distending pressure. In contrast, carbohydrate and protein had no significant effect. At 3 kcal/min, lipid increased symptoms through a volume-independent as well as a volume-dependent effect. Granisetron did not affect symptom perception or gastric pressure-volume relationships. In conclusion, isobaric distal gastric distension produces more intense symptoms during duodenal lipid compared with saline perfusion. Symptom perception during distal gastric distension is unaffected by 5-HT3 receptor antagonism.
Neuronal nitric oxide synthase: expression in rat parietal cells
Authors: Premaratne Shyamal , Xue Chun , McCarty John M. , Zaki Muhammad , McCuen Robert W. , Johns Roger A. , Schepp Wolfgang , Neu Bruno , Lippman Robert , Melone P. D. , Schubert Mitchell L.
Published: December 2017

Nitric oxide synthases (NOS) are enzymes that catalyze the generation of nitric oxide (NO) from l-arginine and require nicotinamide adenine dinucleotide phosphate (NADPH) as a cofactor. At least three isoforms of NOS have been identified: neuronal NOS (nNOS or NOS I), inducible NOS (iNOS or NOS II), and endothelial NOS (eNOS or NOS II). Recent studies implicate NO in the regulation of gastric acid secretion. The aim of the present study was to localize the cellular distribution and characterize the isoform of NOS present in oxyntic mucosa. Oxyntic mucosal segments from rat stomach were stained by the NADPH-diaphorase reaction and with isoform-specific NOS antibodies. The expression of NOS in isolated, highly enriched (>98%) rat parietal cells was examined by immunohistochemistry, Western blot analysis, and RT-PCR. In oxyntic mucosa, histochemical staining revealed NADPH-diaphorase and nNOS immunoreactivity in cells in the midportion of the glands, which were identified as parietal cells in hematoxylin and eosin-stained step sections. In isolated parietal cells, decisive evidence for nNOS expression was obtained by specific immunohistochemistry, Western blotting, and RT-PCR. Cloning and sequence analysis of the PCR product confirmed it to be nNOS (100% identity). Expression of nNOS in parietal cells suggests that endogenous NO, acting as an intracellular signaling molecule, may participate in the regulation of gastric acid secretion.
ClC-2 chloride channels contribute to HTC cell volume homeostasis
Authors: Roman Richard M. , Smith Roderic L. , Feranchak Andrew P. , Clayton Gerald H. , Doctor R. Brian , Fitz J. Gregory
Published: December 2017

Membrane Cl−channels play an important role in cell volume homeostasis and regulation of volume-sensitive cell transport and metabolism. Heterologous expression of ClC-2 channel cDNA leads to the appearance of swelling-activated Cl−currents, consistent with a role in cell volume regulation. Since channel properties in heterologous models are potentially modified by cellular background, we evaluated whether endogenous ClC-2 proteins are functionally important in cell volume regulation. As shown by whole cell patch clamp techniques in rat HTC hepatoma cells, cell volume increases stimulated inwardly rectifying Cl−currents when non-ClC-2 currents were blocked by DIDS (100 μM). A cDNA closely homologous with rat brain ClC-2 was isolated from HTC cells; identical sequence was demonstrated for ClC-2 cDNAs in primary rat hepatocytes and cholangiocytes. ClC-2 mRNA and membrane protein expression was demonstrated by in situ hybridization, immunocytochemistry, and Western blot. Intracellular delivery of antibodies to an essential regulatory domain of ClC-2 decreased ClC-2-dependent currents expressed in HEK-293 cells. In HTC cells, the same antibodies prevented activation of endogenous Cl−currents by cell volume increases or exposure to the purinergic receptor agonist ATP and delayed HTC cell volume recovery from swelling. These studies provide further evidence that mammalian ClC-2 channel proteins are functional and suggest that in HTC cells they contribute to physiological changes in membrane Cl−permeability and cell volume homeostasis.
Endogenous corticosteroids modulateClostridium difficiletoxin A-induced enteritis in rats
Authors: Castagliuolo Ignazio , Karalis Katia , Valenick Leyla , Pasha Asiya , Nikulasson Sigfus , Wlk Michael , Pothoulakis Charalabos
Published: December 2017

We examined the role of glucocorticoids in acute inflammatory diarrhea mediated by Clostridium difficile toxin A. Toxin A (5 μg) or buffer was injected in rat ileal loops, and intestinal responses were measured after 30 min to 4 h. Ileal toxin A administration increased plasma glucocorticoids after 1 h, at which time the toxin-stimulated secretion was not significant. Administration of the glucocorticoid analog dexamethasone inhibited toxin A-induced intestinal secretion and inflammation and downregulated toxin A-mediated increase of macrophage inflammatory protein-2. Adrenalectomy followed by replacement with glucocorticoids at various doses suggested that intestinal responses to toxin A were related to circulating levels of glucocorticoids. Administration of the glucocorticoid receptor antagonist RU-486 enhanced toxin A-mediated intestinal secretion and inflammation. We conclude that C. difficile toxin A causes increased secretion of endogenous glucocorticoids, which diminish the intestinal secretory and inflammatory effects of toxin A.
Ambulatory 24-h colonic manometry in healthy humans
Authors: Rao Satish S. C. , Sadeghi Pooyan , Beaty Jennifer , Kavlock Renae , Ackerson Kris
Published: December 2017

Our aim was to investigate motor activity of the healthy, relatively unprepared colon in the ambulatory state. Twenty-five age- and gender-matched adults had a six-sensor solid-state probe inserted into the proximal transverse colon without sedation. Subjects ambulated freely and ate standard meals. In 528 h of recording, we found a lower ( P < 0.05) area under the curve during the night. Waking induced a threefold increase in motility, whereas meals induced a twofold increase. Women showed less activity ( P < 0.05) in the transverse/descending colon than men. The transverse/descending colon showed more ( P < 0.05) activity than the rectosigmoid colon. Seven patterns were recognized; predominantly, they were simultaneous, propagated, or periodic bursts of 3-cycles/min (cpm) waves. A specialized propagating pressure wave with a high amplitude (>105 mmHg) and a prolonged duration (>14 s) occurred in all subjects (mean 10/day), mostly after waking, after meals, or with defecation. A 3-cpm motor activity was seen in the rectosigmoid region predominantly at night. The colon exhibits a wide spectrum of pressure activity around the clock, with gender and regional differences and circadian rhythm. This comprehensive study provides qualitative and quantitative normative data for colonic manometry.
SCFA increase intestinal Na absorption by induction of NHE3 in rat colon and human intestinal C2/bbe cells
Authors: Musch Mark W. , Bookstein Cres , Xie Yue , Sellin Joseph H. , Chang Eugene B.
Published: December 2017

Short-chain fatty acids (SCFA), produced by colonic bacterial flora fermentation of dietary carbohydrates, promote colonic Na absorption through mechanisms not well understood. We hypothesized that SCFA promote increased expression of apical membrane Na/H exchange (NHE), serving as luminal physiological cues for regulating colonic Na absorptive capacity. Studies were performed in human colonic C2/bbe (C2) monolayers and in vivo. In C2 cells exposed to butyrate, acetate, proprionate, or the poorly metabolized SCFA isobutyrate, apical membrane NHE3 activity and protein expression increased in a time- and concentration-dependent manner, whereas no changes were observed for NHE2. In contrast, no significant changes in brush-border hydrolase or villin expression were noted. Analogous to the in vitro findings, rats fed the soluble fiber pectin exhibited a time-dependent increase in colonic NHE3, but not NHE2, protein, mRNA, and brush-border activity. These changes were region-specific, as no changes were observed in the ileum. We conclude that luminal SCFA are important physiological cues for regulating colonic Na absorptive function, allowing the colon to adapt to chronic changes in dietary carbohydrate and Na loads.
Kupffer cell-derived cyclooxygenase-2 regulates hepatocyte Bcl-2 expression in choledocho-venous fistula rats
Authors: Souto Enrico O. , Miyoshi Hideyuki , Dubois Raymond N. , Gores Gregory J.
Published: December 2017

We have previously demonstrated that after bile duct ligation hepatocytes express Bcl-2, although the mechanisms regulating Bcl-2 expression were not identified. Our aim was to determine if biliary constituents induce hepatocellular expression of Bcl-2 by a cyclooxygenase-2 (COX-2)-dependent mechanism. We used the choledocho-venous fistula (CVF) rat model for these studies and inhibited COX-2 by feeding the animals nimesulide, a selective inhibitor of COX-2 activity. Serum bile acids were 70-fold greater in CVF animals compared with controls, although liver histology and serum alanine aminotransferase values remained normal for the duration of the study. Neither Bcl-2 nor COX-2 was detected in sham-operated animals. However, Bcl-2 was expressed in hepatocytes but not in other liver cells in the CVF animals. In contrast, COX-2 protein was identified in Kupffer cells but not in hepatocytes of CVF animals. Hepatic Bcl-2 protein expression was fourfold lower in the livers from nimesulide-treated CVF rats. In conclusion, high circulating concentrations of biliary constituents are associated with stimulation of de novo hepatocyte expression of Bcl-2 and Kupffer cell expression of COX-2. These data suggest Kupffer cell-derived prostanoids may regulate Bcl-2 expression in the hepatocyte.
Modulation of gastric motor activity by a centrally acting stimulus, circular vection, in humans
Authors: Faas Henryk , Feinle Christine , Enck Paul , Grundy David , Boesiger Peter
Published: December 2017

The aims of this study were to investigate gastric motor correlates of vection, a centrally acting stimulus, and relate these responses to the induction of motion sickness symptoms. Antral contractile activity and gastric volume retained after a liquid nutrient meal (600 ml) were assessed by magnetic resonance imaging in healthy subjects during two different protocols. Vection was induced by an optokinetic drum, and subjects repeatedly rated the intensity of vection and nausea on 0–10 analog scales. Vection delayed gastric emptying {99% (89–102%) [median (interquartile ranges)] of volume retained at 28 min; control situation: 79% (69–81%), P < 0.05}. Antral contractile activity followed a distinct time course of rapid decrease [−64% (−72 to −59%) change from baseline activity] immediately after onset of drum rotation followed by gradual recovery upon withdrawal of the stimulus. No relationship was found between the severity of nausea and inhibition of gastric emptying or antral contractile activity. The inhibition of antral contractile activity appears to be a good measure of the peripheral response to vection but is probably independent of subjective symptom induction.
Endotoxin-induced reduction in biliary indocyanine green excretion rate in a chronically catheterized rat model
Authors: Beno David W. A. , Uhing Michael R. , Goto Masakatsu , Chen Yong , Jiyamapa-Serna Vanida A. , Kimura Robert E.
Published: December 2017

Using a nonstressed chronically catheterized rat model in which the common bile duct was cannulated, we studied endotoxin-induced alterations in hepatic function by measuring changes in the maximal steady-state biliary excretion rate of the anionic dye indocyanine green (ICG). Biliary excretion of ICG was calculated from direct measurements of biliary ICG concentrations and the bile flow rate during a continuous vascular infusion of ICG. Despite significant elevations in mean peak serum tumor necrosis factor-α (TNF-α) concentrations (90.9 ± 16.2 ng/ml), there was no effect on mean rates of bile flow or biliary ICG clearance after administration of 100 μg/kg endotoxin at 6 or 24 h. Significant differences from mean baseline rates of bile flow and biliary ICG excretion did occur after administration of 1,000 μg/kg endotoxin (mean peak TNF-α 129.6 ± 24.4 ng/ml). Furthermore, when rats were treated with up to 16 μg/kg of recombinant TNF-α, there was no change in mean rates of bile flow or ICG biliary clearance compared with baseline values. These data suggest that the complex regulation of biliary excretion is not mediated solely by TNF-α.
Expression of apoptosis on rat liver by hepatic vagus hyperactivity after ventromedial hypothalamic lesioning
Authors: Kiba Takayoshi , Saito Satoru , Numata Kazushi , Kon Yasuhiro , Mizutani Tetsuya , Sekihara Hisahiko
Published: December 2017

We examined whether the Fas (APO-1/CD95)/Fas ligand system mediates apoptosis in rats with ventromedial hypothalamus (VMH) lesions. Northern and Western blotting indicated that VMH lesions lead to a significant increase in Fas mRNA and protein expression from day 1 to day 7 and in Fas ligand mRNA and protein expression from day 2 to day 7. Immunohistochemistry indicated that the region of strongest Fas expression shifted from acinar zone 1 to zones 2 and 3 by day 7 after VMH lesioning and that at days 2–7Fas-ligand-positive hepatocyte cell membranes and cytoplasm were randomly distributed in acinar zones 1–3. We also analyzed activation of caspase 3-like proteases in hepatocytes, Kupffer cells, and sinusoidal endothelial cells. Spectrofluorometric assay demonstrated that caspase 3-like activity significantly increased only in hepatocytes after VMH lesioning. Moreover, electron microscopy and TUNEL assay showed that VMH lesions induced apoptosis. All of these effects were completely inhibited by hepatic vagotomy and administration of atropine. Vagal firing after VMH lesioning may stimulate Fas/Fas ligand system-mediated apoptosis through the cholinergic system in the rat liver.
Activated Kupffer cells cause a hypermetabolic state after gentle in situ manipulation of liver in rats
Authors: Schemmer Peter , Enomoto Nobuyuki , Bradford Blair U. , Bunzendahl Hartwig , Raleigh James A. , Lemasters John J. , Thurman Ronald G.
Published: December 2017

Harvesting trauma to the graft dramatically decreases survival after liver transplantation. Since activated Kupffer cells play a role in primary nonfunction, the purpose of this study was to test the hypothesis that organ manipulation activates Kupffer cells. To mimic what occurs with donor hepatectomy, livers from Sprague-Dawley rats underwent dissection with or without gentle organ manipulation in a standardized manner in situ. Perfused livers exhibited normal values for O2uptake (105 ± 5 μmol · g−1· h−1) measured polarigraphically; however, 2 h after organ manipulation, values increased significantly to 160 ± 8 μmol · g−1· h−1and binding of pimonidazole, a hypoxia marker, increased about threefold ( P < 0.05). Moreover, Kupffer cells from manipulated livers produced three- to fourfold more tumor necrosis factor-α and PGE2, whereas intracellular calcium concentration increased twofold after lipopolysaccharide compared with unmanipulated controls ( P < 0.05). Gadolinium chloride and glycine prevented both activation of Kupffer cells and effects of organ manipulation. Furthermore, indomethacin given 1 h before manipulation prevented the hypermetabolic state, hypoxia, depletion of glycogen, and release of PGE2from Kupffer cells. These data indicate that gentle organ manipulation during surgery activates Kupffer cells, leading to metabolic changes dependent on PGE2from Kupffer cells, which most likely impairs liver function. Thus modulation of Kupffer cell function before organ harvest could be beneficial in human liver transplantation and surgery.
A silencer inhibitor confers specific expression of intestinal trefoil factor in gobletlike cell lines
Authors: Iwakiri Dai , Podolsky Daniel K.
Published: December 2017

Intestinal trefoil factor (ITF) is selectively expressed in intestinal goblet cells. Previous studies identified cis-regulatory elements in the proximal promoter of ITF, but these were insufficient to recapitulate the exquisite tissue- and cell-specific expression of native ITF in vivo. Preliminary studies suggested that goblet cell-specific expression of murine ITF requires elements far upstream that include a silencer element that effectively prevents ITF expression in non-goblet cells. Transient transfection studies using native or mutant ITF 5′-flanking sequences identified a region that restores expression in goblet cells. This element, designated goblet cell silencer inhibitor (GCSI) element, enables human and murine goblet cell-like cell lines to override the silencing effect of more proximal elements. The GCSI has no intrinsic enhancer activity and regulates expression only when the silencer element is present. Ligation of GCSI and silencer elements to sucrase-isomaltase conferred goblet cell-specific expression. Goblet cells but not non-goblet cells possess a nuclear protein that binds to the GCSI regulatory element (GCSI binding protein; GCSI-BP). Both transient transfection and gel mobility shift assay studies localize the GCSI and GCSI-BP to −2216 to −2204. We conclude that goblet cell-specific transcription of ITF in vivo depends on a regulatory element designated GCSI.
Role of EGF receptor tyrosine kinase activity in antiapoptotic effect of EGF on mouse hepatocytes
Authors: Musallam Lina , Éthier Chantal , Haddad Pierre S. , Bilodeau Marc
Published: December 2017

The apoptotic Fas pathway is potentially involved in the pathogenesis of liver diseases. Growth factors, such as epidermal growth factor (EGF), can protect cells against apoptosis induced by a variety of stimuli, including Fas receptor stimulation. However, the underlying mechanisms of this protection have yet to be determined. We investigated the involvement of EGF receptor (EGFR) tyrosine kinase (TK) activity in the antiapoptotic effect of EGF on primary mouse hepatocyte cultures. Cells undergoing apoptosis after treatment with anti-Fas antibody were protected by EGF treatment. This protection was significantly but partially decreased when cells were treated with two specific inhibitors of the TK activity of EGFR. Evaluation of the efficacy of these compounds indicated that they were able to abolish EGFR autophosphorylation and postreceptor events such as activation of mitogen-activated protein kinases and the phosphatidylinositol 3′-kinase pathways as well as increases in Bcl-xLmRNA and protein levels. This leads us to postulate that EGF exerts its antiapoptotic action partially through the TK activity of EGFR. In addition, our results suggest that Bcl-xLprotein upregulation caused by EGF is linked to the TK activity of its receptor.
Adaptation of stress-induced mucosal pathophysiology in rat colon involves opioid pathways
Authors: Yates Derrick A. , Santos Javier , Söderholm Johan D. , Perdue Mary H.
Published: December 2017

Acute stress increases ion secretion and permeability of rat colonic epithelium. However, it is not known if stress-induced mucosal changes are subject to adaptation. Wistar-Kyoto rats were exposed to either continuous water-avoidance stress (CS) for 60 min or intermittent stress (IS) for three 20-min periods. Distal colonic segments were mounted in Ussing Chambers, and ion-transport [short-circuit current ( I sc)] and permeability [conductance and flux of horseradish peroxidase (HRP)] parameters were measured. CS significantly increased I sc, conductance, and HRP flux compared with control values. In contrast, in IS rats these variables were similar to those in nonstressed controls. To study the pathways involved in IS-induced adaptation, rats were pretreated intraperitoneally with the opioid antagonists naloxone or methylnaloxone. Opioid antagonists had no effect on values in control or CS rats. However, in the IS group, naloxone and methylnaloxone reversed the adaptive responses, and all variables increased to CS values. We conclude that stress-induced colonic mucosal pathophysiology is subject to rapid adaptation, which involves opioid pathways.
Insulin and IGF-I inhibit calcium-dependent chloride secretion by T84 human colonic epithelial cells
Authors: Chang Nelson , Uribe Jorge M. , Keely Stephen J. , Calandrella Sean , Barrett Kim E.
Published: December 2017

d- Myo-inositol (3,4,5,6) tetrakisphosphate [Ins(3,4,5,6)P4] or phosphatidylinositol 3-kinase (PI 3-kinase) activity acts to inhibit calcium-dependent chloride secretion in T84 colonic epithelial cells. To further distinguish between the contributions of these two signaling pathways to the inhibition of secretion, we studied effects of insulin, because the insulin receptor links to PI 3-kinase but not to pathways postulated to generate Ins(3,4,5,6)P4. Chloride secretion across T84 cell monolayers was studied in Ussing chambers. Activation of PI 3-kinase was assessed by Western blotting. Basolateral, but not apical, addition of insulin inhibited carbachol- and thapsigargin-induced chloride secretion in a time- and concentration-dependent fashion. Insulin-like growth factor-I (IGF-I) had similar effects. Insulin had no effect on Ins(3,4,5,6)P4levels, and the inhibitory effects of insulin and IGF-I on chloride secretion were fully reversed by the PI 3-kinase inhibitors wortmannin and LY-294002. Western blot analysis showed that both insulin and IGF-I recruited the 85-kDa regulatory and 110-kDa catalytic subunits of PI 3-kinase to anti-phosphotyrosine immunoprecipitates. In conclusion, insulin and IGF-I act to inhibit calcium-dependent chloride secretion through a PI 3-kinase-dependent pathway. Because insulin is released in a pulsatile fashion postprandially and IGF-I levels are elevated in pathological settings, our findings may have physiological and/or pathophysiological significance.
III. μ-Opioid receptors in the enteric nervous system
Authors: Sternini Catia
Published: December 2017

G protein-coupled receptors are cell surface signal-transducing proteins, which elicit a variety of biological functions by the activation of different intracellular effector systems. Many of these receptors, including the μ-opioid receptor (μOR), have been localized in the gastrointestinal tract. μOR is the target of opioids and alkaloids, potent analgesic drugs with high potential for abuse. μOR is expressed by enteric neurons, and it undergoes ligand-selective endocytosis. It is of clinical importance because it mediates tolerance and other major side effects of opiate analgesics, including impairment of gastrointestinal propulsion. An important observation of μOR is its differential trafficking and desensitization properties in response to individual agonists, which might have long-term physiological consequences and be involved in the development of opiate side effects. Receptor activation by agonists is the basis for signaling, and alterations of the mechanisms controlling cellular responses of G protein-coupled receptors to agonists might be the basis of several diseases, including gastrointestinal diseases. Therefore, understanding these basic cellular mechanisms is important for developing appropriate therapeutic agents.
Relationship between ultrasonically detected phasic antral contractions and antral pressure
Authors: Hveem K. , Sun W. M. , Hebbard G. , Horowitz M. , Doran S. , Dent J.
Published: December 2017

The relationships between gastric wall motion and intraluminal pressure are believed to be major determinants of flows within and from the stomach. Gastric antral wall motion and intraluminal pressures were monitored in five healthy subjects by concurrent antropyloroduodenal manometry and transabdominal ultrasound for 60 min after subjects drank 500 ml of clear soup. We found that 99% of antral contractions detected by ultrasound were propagated aborally, and 68% of contractions became lumen occlusive at the site of the ultrasound marker. Of the 203 contractions detected by ultrasound, 53% were associated with pressure events in the manometric reference channel; 86% of contractions had corresponding pressure events detectable somewhere in the antrum. Contractions that occluded the lumen were more likely to be associated with a pressure event in the manometric reference channel ( P < 0.01) and to be of greater amplitude ( P < 0.01) than non-lumen-occlusive contractions. We conclude that heterogeneous pressure event patterns in the antrum occur despite a stereotyped pattern of contraction propagation seen on ultrasound. Lumen occlusion is more likely to be associated with higher peak antral pressure events.
Deletion of neutral endopeptidase exacerbates intestinal inflammation induced byClostridium difficiletoxin A
Authors: Kirkwood Kimberly S. , Bunnett Nigel W. , Maa John , Castagliolo Ignazio , Liu Bao , Gerard Norma , Zacks Jeff , Pothoulakis Charalabos , Grady Eileen F.
Published: December 2017

Toxin A (TxA) of Clostridium difficile induces acute inflammation of the intestine initiated by release of substance P (SP) and activation of the neurokinin-1 receptor. However, the mechanisms that terminate this response are unknown. We determined whether the SP-degrading enzyme neutral endopeptidase (NEP, EC 3.4.24.11 ) terminates TxA-induced enteritis. We used both genetic deletion and pharmacological inhibition of NEP to test this hypothesis. In wild-type mice, instillation of TxA (0.5–5 μg) into ileal loops for 3 h dose dependently increased ileal fluid secretion, stimulated granulocyte transmigration determined by myeloperoxidase activity, and caused histological damage characterized by depletion of enterocytes, edema, and neutrophil accumulation. Deletion of NEP reduced the threshold secretory and inflammatory dose of TxA and exacerbated the inflammatory responses by more than twofold. This exacerbated inflammation was prevented by pretreatment with recombinant NEP. Conversely, pretreatment of wild-type mice with the NEP inhibitor phosphoramidon exacerbated enteritis. Thus NEP terminates enteritis induced by C. difficile TxA, underlying the importance of SP degradation in limiting neurogenic inflammation.
Intestinal peptide transport: ex vivo uptake studies and localization of peptide carrier PEPT1
Authors: Groneberg David A. , Döring Frank , Eynott Paul R. , Fischer Axel , Daniel Hannelore
Published: December 2017

The nature of protein breakdown products and peptidomimetic drugs such as β-lactams is crucial for their transmembrane transport across apical enterocyte membranes, which is accomplished by the pH-dependent high-capacity oligopeptide transporter PEPT1. To visualize oligopeptide transporter-mediated uptake of oligopeptides, an ex vivo assay using the fluorophore-conjugated dipeptide derivatived-Ala-Lys- N ε-7-amino-4-methylcoumarin-3-acetic acid (d-Ala-Lys-AMCA) was established in the murine small intestine and compared with immunohistochemistry for PEPT1 in murine and human small intestine. d-Ala-Lys-AMCA was accumulated by enterocytes throughout all segments of the murine small intestine, with decreasing intensity from the top to the base of the villi. Goblet cells did not show specific uptake. Inhibition studies revealed competitive inhibition by the β-lactam cefadroxil, the angiotensin-converting enzyme inhibitor captopril, and the dipeptide glycyl-glutamine. Controls were performed using either the inhibitor diethylpyrocarbonate or an incubation temperature of 4°C to exclude unspecific uptake. Immunohistochemistry for PEPT1 localized immunoreactivity to the enterocytes, with the highest intensity at the apical membrane. This is the first study that visualizes dipeptide transport across the mammalian intestine and indicates that uptake assays using d-Ala-Lys-AMCA might be useful for characterizing PEPT1-specific substrates or inhibitors.
NSAIDs induce both necrosis and apoptosis in guinea pig gastric mucosal cells in primary culture
Authors: Tomisato Wataru , Tsutsumi Shinji , Rokutan Kazuhito , Tsuchiya Tomofusa , Mizushima Tohru
Published: December 2017

A major clinical problem encountered with the use of nonsteroidal anti-inflammatory drugs (NSAIDs) such as indomethacin is gastropathy. In this study, we examined, using guinea pig gastric mucosal cells in primary culture, how NSAIDs damage gastric mucosal cells. The short-term treatment of cells with high concentrations of indomethacin decreased cell viability in the absence of apoptotic DNA fragmentation, chromatin condensation, or caspase activation. Cells lost membrane integrity with this short-term indomethacin treatment, suggesting that indomethacin induced necrosis under these conditions. In contrast, the long-term treatment of cells with low concentrations of indomethacin decreased cell viability and was accompanied by apoptotic DNA fragmentation, chromatin condensation, and caspase activation. Pretreatment of cells with inhibitors of caspases or protein synthesis suppressed cell death caused by long-term indomethacin treatment, suggesting that apoptosis was induced when the inhibitors were not present. These results imply that NSAIDs cause gastric mucosal damage through both necrosis and apoptosis of gastric mucosal cells.
Epidermal growth factor induces Egr-1 promoter activity in hepatocytes in vitro and in vivo
Authors: Tsai Jo C. , Liu Lixin , Zhang Jie , Spokes Katherine C. , Topper James N. , Aird William C.
Published: December 2017

Early growth response-1 (Egr-1) is a transcription factor that couples short-term changes in the extracellular milieu to long-term changes in gene expression. Under in vitro conditions, the Egr-1 gene has been shown to respond to many extracellular signals. In most cases, these findings have not been extended to the in vivo setting. The goal of the present study was to explore the role of epidermal growth factor (EGF) in mediating Egr-1 expression in hepatocytes under both in vitro and in vivo conditions. In HepG2 cells, Egr-1 protein and mRNA were upregulated in the presence of EGF. In stable transfections of HepG2 cells, a 1,200-bp Egr-1 promoter contained information for EGF response via a protein kinase C-independent, mitogen-activated protein kinase-dependent signaling pathway. A promoter region containing the two most proximal serum response elements was sufficient to transduce the EGF signal. In transgenic mice that carry the Egr-1 promoter coupled to the LacZ reporter gene, systemic delivery of EGF by intraperitoneal injection resulted in an induction of the endogenous Egr-1 gene and the Egr-1- lacZ transgene in hepatocytes. Together, these results suggest that the 1,200-bp promoter contains information for EGF response in hepatocytes both in vitro and in intact animals.
Increased lymphatic lipid transport in genetically diabetic obese rats
Authors: Hayashi Hiroshi , Sato Yuko , Kanai Setsuko , Ichikawa Mineko , Funakoshi Akihiro , Miyasaka Kyoko
Published: December 2017

Otsuka Long-Evans Tokushima fatty (OLETF) rats are a model for noninsulin-dependent diabetes mellitus (NIDDM), which is first manifested at 18 wk of age. We assessed age-related changes in lymphatic lipid transport in the intestine of OLETF rats and compared them with those of control Long-Evans Tokushima Otsuka (LETO) rats. Olive oil was infused into the rats with a mesenteric lymph fistula, which was created under ethrane anesthesia. A significant increase in lymphatic triglyceride (TG) transport in OLETF rats was observed at 18–19 wk compared with under 17 wk, but no age-related change was observed in LETO rats. Food restriction, exercise training, or troglitazone treatment in OLETF rats prevented the age-related increase in lipid transport. Biliary phosphatidylcholine concentration was higher in OLETF rats than in LETO rats, but no difference was seen in bile acid concentrations or the activity of microsomal TG transfer protein between the two strains. This study shows that increased lipid transport in the intestine may occur in NIDDM.
Adrenergic receptors regulating patterns of blood flow through the gills of trout
Authors: Payan P. , Girard J. P.
Published: December 2017

A technique of perfusing the isolated head of trout with a constant-fluid flow was used to study adrenergic factors controlling the vasomotricity of the gill. The use of adrenergic blockers (phentolamine 10(-4) M and propranolol 10(-4) M) enabled the vascular effect of epinephrine to be split into two components: an alpha-vasoconstrictor effect superimposed upon the dominant beta-vasodilator effect. Effect alpha appears more rapidly than effect beta and causes an initial vasoconstriction. Under constant-flow perfusion the effect of epinephrine on the efferent arterial and venous flows is a diminution of the venous flow and an increase of the arterial. This is the result of closure of the anastomoses between the efferent artery and the central compartment of the filament. It was shown that only alpha-vasoconstrictor adrenoreceptors occur in this region.
Comparison of arteries with longitudinal and circular venous muscle from the rat
Authors: Cohen M. L. , Wiley K. S.
Published: December 2017

Rat portal, mesenteric, renal, and femoral veins possess functionally responsive circular and longitudinal smooth muscle layers in vitro. In contrast to the dominant rhythmically active longitudinal muscle of portal veins, rat longitudinal mesenteric veins lacked rhythmic activity and developed maximal force equivalent to that of mesenteric circular muscle. Renal and femoral veins exhibited predominantly circular smooth muscle responses. Rat veins must be subjected to between 1 g (renal and femoral) and 4 g (circular portal and mesenteric) of passive forse for optimal responsiveness. Contractile response to vasoactive agent including carbamylcholine, serotonin, and norepinephrine was quantitatively different among veins. Femoral veins developed greater maximal force in response to norepinephrine than to KCL, and the responses to norepinephrine were not altered by cocaine. In contrast, cocaine markedly potentiated responses to norepinephrine in portal, mesenteric, and renal veins and, to a lesser extent, in the mesenteric artery. These data demonstrate heterogeneity in rat venous tissue and suggest that neuronal innervation may markedly influence responses to norepinephrine in some, but not all, rat blood vessels.