bluuebunny/crossref_metadata_2025 huggingface dataset:
Acidosis induces hyperpermeability in Caco-2BBe cultured intestinal epithelial monolayers
Authors: Menconi M. J. , Salzman A. L. , Unno N. , Ezzell R. M. , Casey D. M. , Brown D. A. , Tsuji Y. , Fink M. P.
Published: December 2017

We previously demonstrated that ileal mucosal acidosis in pigs reversibly increases intestinal permeability to hydrophilic macromolecules, even in the absence of tissue hypoxia [A.L. Salzman, H. Wang, P.S. Wollert, T.J. Vandermeer, C.C. Compton, A.G. Denenberg, and M.P. Fink. Am. J. Physiol. 266 (Gastrointest, Liver Physiol, 29): G633-G646, 1994]. In an effort to further explore the mechanism(s) underlying this phenomenon, we examined the effect of acidic pH on the permeability characteristics of cultured Caco-2BBe (human intestinal epithelial) cells grown as monolayers on permeable supports. Permeability was determined by measuring the mucosal-to-basolateral flux of fluorescein disulfonic acid (FS; mol wt 478 Dal, fluorescein isothiocyanate-labeled dextran (FD4; average mol wt 4 kDa), or [3H]mannitol. Incubation of monolayers under hypercarbic conditions or with acidific bicarbonate-free medium significantly increased permeability to FS, FD4, and mannitol in a manner dependent on both time and pH. Incubation in medium at pH 5.43 for 24 h increased the release of lactate dehydrogenase and decreased the intensity of staining with calcein-acetoxymethyl ester, findings that are indicative of plasma membrane injury; nevertheless, the percentage of nonviable cells did not increase. Ultrastructural analyses revealed evidence of increased paracellular trafficking of horseradish peroxidase after incubation of monolayers under acidic conditions. Fluorescence confocal microscopy and temperature studies demonstrated that incubation at pH 5.43 induced an increase in both the intracellular uptake of FD4 and the activation energy for FS permeation across Caco-2BBe monolayers, respectively, suggesting increased transcellular permeation. Exposure to acidic conditions also decreased cellular levels of ATP. We conclude that acidosis increases both paracellular and transcellular permeability to hydrophilic macromolecules and leads to depletion of ATP.
Characterization of proteases and protease inhibitors in the rat stomach
Authors: Nagy L. , Johnson B. R. , Hauschka P. , Szabo S.
Published: December 2017

Previously our laboratory reported increased activity of the thiol proteinase cathepsin B in gastric juice after ethanol-induced mucosal injury. In this study we measured proteinase activity (PA) and proteinase inhibitory activity (PIA) with the general substrates hemoglobin, azocasein, and bovine serum albumin (BSA) at optimal pH (2.0, 5.6, and 7.4) of aspartic, cysteine, and serine proteinases. Homogenates of glandular stomach mucosa and gastric juice from fasted rats were incubated in the presence or absence of specific inhibitors and sulfhydryl (SH) alkylators N-ethylmaleimide and iodoacetate. PIA was measured after acid and heat inactivation of endogenous proteinases and addition of 20 micrograms/ml pepsin, 20 or 100 micrograms/ml thiol proteinase papain, or 20 micrograms/ml trypsin for 5 min before digestion at 37 degrees C. The highest proteolytic activity was found at pH 2.0 (pepsin) in juice and mucosal homogenate, but proteases were also found at pH 5.6 and 7.4, where pepsin was inactive. Pepstatin inhibited most proteolytic activity at pH 2.0. The SH protease inhibitor leupeptin diminished PA mainly at pH 5.6. N-ethylmaleimide or iodoacetate substantially reduced the PA in acidic milieu, with maximum effect at pH 5.6. Endogenous PIA, expressed as inhibition of the effect of 1 microgram of pepsin, papain, and trypsin on BSA, was 13.1, 1.4, and 9.2% in gastric mucosa and 15.3, 22.5, and 6.2% in gastric juice at pH 2.0, 5.6, and 7.4, respectively. We have concluded that 1) endogenous proteinases and inhibitors in rat stomach can be measured using BSA and hemoglobin as substrates, 2) of the proteinases found in the stomach, 98% was pepsin at pH 2.0 and up to 27% or 17% was SH sensitive at pH 5.6 or 7.4, respectively, and 3) proteinases and their specific endogenous inhibitors may play a role in gastric mucosal injury and protection.
Epidermal growth factor activates phospholipase C-gamma 1 via G(i)1-2 proteins in isolated pancreatic acinar membranes
Authors: Piiper A. , Stryjek-Kaminska D. , Zeuzem S.
Published: December 2017

In the present study, isolated pancreatic acinar membranes were used to investigate the mechanism of epidermal growth factor (EGF)-induced activation of phospholipase C (PLC). The data show that EGF caused a rapid and strong increase in tyrosine phosphorylation of the EGF receptor, with a maximum 5-15 s after the beginning of the incubation followed by a decline. With use of [3H]phosphatidylinositol 4,5-bisphosphate as an exogenous substrate, PLC activity increased fourfold on exposure of the membranes to EGF (85 nM). In contrast, EGF-induced tyrosine phosphorylation of PLC-gamma 1 was rather small, indicating that tyrosine phosphorylation of PLC-gamma 1 is not proportional to changes in PLC activity. EGF-induced activation of PLC was strongly inhibited by pretreatment of the membranes with pertussis toxin, by an antibody raised against a COOH-terminal sequence shared by alpha-subunits of the inhibitory G proteins G(i)1 and G(i)2, and by an anti-PLC-gamma 1 antibody, whereas anti-G(i) alpha 3, anti-Gq/11 alpha, and anti-PLC-beta 1 antibodies had no effect. In contrast, pertussis toxin or the anti-G(i) alpha 1-2 antibody had no effect on EGF-induced tyrosine phosphorylation of PLC-gamma 1. EGF promoted association of G(i) proteins with both the EGF receptor and PLC-gamma 1 with similar kinetics as EGF-receptor autophosphorylation. All EGF-induced responses were abolished by the specific tyrosine kinase inhibitor pp60v-arc (137-157), suggesting that EGF-receptor tyrosine kinase activity is essential for G(i)1-2-mediated activation of PLC-gamma 1. However, there was no evidence of tyrosine phosphorylation of G(i) alpha 1-2. Taken together, these data show that EGF causes activation of PLC-gamma 1 by a mechanism requiring activation of G(i)1-2 and only a small increase in tyrosine phosphorylation of PLC-gamma 1.
Hepatobiliary transport mechanism for the cyclopentapeptide endothelin antagonist BQ-123
Authors: Shin H. C. , Kato Y. , Yamada T. , Niinuma K. , Hisaka A. , Sugiyama Y.
Published: December 2017

The hepatobiliary transport of an anionic cyclopentapeptide endothelin antagonist, BQ-123, was studied in rats. Biliary excretion of [3H]BQ-123 was extensive in vivo (approximately 75% of intravenous infusion rates). Liver-to-plasma and bile-to-liver concentration ratios at steady state were approximately 3 and 200, respectively, suggesting that hepatic uptake and biliary excretion are concentrative processes. The biliary excretion clearance exhibited a saturation at a hepatic concentration of > 100 nmol/g liver and was markedly reduced in Eisai hyperbilirubinemic rats, which have a hereditary defect of canalicular multispecific organic anion transporter. An ATP-dependent and saturable uptake of BQ-123 by isolated canalicular membrane vesicles was observed in vitro. Impaired transport of BQ-123 was also confirmed in canalicular membrane vesicles prepared from Eisai hyperbilirubinemic rats. These results demonstrate that the biliary excretion process is ATP-driven primary active transport. It is proposed that a canalicular multispecific organic anion transporter is mainly responsible for the biliary excretion of BQ-123.
Synthesis of citrulline and arginine from proline in enterocytes of postnatal pigs
Authors: Wu G.
Published: December 2017

The synthesis of ornithine, citrulline, and arginine from proline was quantified in enterocytes of newborn (0-day-old) pigs, 2- to 21-day-old suckling pigs, and 29- to 58-day-old pigs weaned at 21 days of age. Mitochondria were prepared from enterocytes for measurement of proline oxidase activity. For metabolic studies, cells were incubated at 37 degrees C for 30 min in Krebs bicarbonate buffer (pH 7.4) containing 5 mM D-glucose and 0.0-5.0 mM L-[U-14C]proline with or without 2.0 mM L-glutamine. Proline oxidase activity and rates of synthesis of citrulline and arginine from proline were high in enterocytes of newborn pigs and markedly decreased in 7-day-old pigs. With increasing piglet age from 7 to 21 days, enterocyte proline oxidase activity and rate of conversion of proline into citrulline progressively increased, but the values in 21-day-old pigs remained much lower than in newborn pigs. The synthesis of ornithine, citrulline, and arginine from 0.5-5 mM proline was concentration dependent and required the addition of glutamine. About 80-90% of utilized proline carbons were recovered in ornithine plus citrulline plus arginine, with CO2 being a minor product. These results demonstrate a hitherto unrecognized pathway for the synthesis of ornithine, citrulline, and arginine via proline oxidase in enterocytes and challenge the current concept that pyrroline-5-carboxylate (P5C) is synthesized in the small intestine only from glutamine/glutamate via P5C synthase on the basis of previous work with postnatal rats.
Nontraditional effects of protein binding and hematocrit on uptake of indocyanine green by perfused rat liver
Authors: Ott P. , Weisiger R. A.
Published: December 2017

We used a novel parameter-free approach to study the role of protein binding in the hepatic clearance of indocyanine green (ICG) from reconstituted pig blood by perfused rat liver. Either perfusate total plasma protein concentration or hematocrit was changed. By analyzing protein concentration ratios or plasma volume ratios relative to ratios of intrinsic hepatic clearance of ICG (K), it was possible to evaluate current models of hepatic uptake of protein-bound ligands without precise knowledge of some of the model parameters. A four-fold increase in the total plasma protein concentration produced only a 36% decrease in K. This was substantially less than predicted by the traditional model, where K is proportional to the free concentration of ligand. Because an unstirred water layer effect could not by itself account for the observations, the effects of binding disequilibrium in the sinusoids or uptake directly from the bound pool had to be considered. To discriminate, hematocrit was increased from 15% to 29%, causing a 20% decrease in the sinusoidal plasma volume. A significant reduction in K strongly suggested a sinusoidal binding disequilibrium effect. The dissociation rate constant predicted by this model was confirmed by in vitro measurement, further supporting this interpretation. The simple experimental design and its parameter-free evaluation provide a new tool for investigating the hepatic uptake of protein-bound ligands.
Tyrosine phosphorylation in contraction of opossum esophageal longitudinal muscle in response to SNP
Authors: Hirano I. , Kakkar R. , Saha J. K. , Szymanski P. T. , Goyal R. K.
Published: December 2017

Sodium nitroprusside (SNP) has been shown to elicit a guanosine 3',5'-cyclic monophosphate (cGMP)-mediated, indomethacin-sensitive contraction of the opossum esophageal longitudinal muscle. We examined the role of tyrosine phosphorylation in the signal transduction pathway of contractions induced by SNP and cGMP in longitudinal muscle strips in vitro. Force of isometric contractions was expressed as the percentage of responses to KCl (73 mM). SNP (100 microM)-induced contractions were 75 +/- 5% before and 3 +/- 2% after 50 microM genistein (P < 0.005) and 86 +/- 16% before and 0 +/- 0% after 50 microM tyrphostin B46. Contractions in response to 8-bromo-cGMP (8-BrcGMP; 1 mM) were 74 +/- 15% before and 3 +/- 2% after genistein (P < 0.01) and 63 +/- 15% before and 18 +/- 4% after tyrphostin B46 (P < 0.05). In contrast, KCl-induced contractions were 82 +/- 8% and 96 +/- 9% of the control value after genistein and tyrphostin B46 treatments, respectively (P > 0.05 for both). Carbachol contractions were partially suppressed by genistein (106 +/- 8% vs. 79 +/- 8%; P < 0.05) but unaffected by tyrphostin B46 (114 +/- 10% vs. 107 +/- 12%; P > 0.05). Western blot analysis revealed a 116-kDa phosphotyrosine protein in the control muscle strips. The level of this protein was increased to 206 +/- 15% of control after SNP treatment. Both genistein and tyrphostin B46 blocked this increase. These studies show that contractions of the esophageal longitudinal muscle induced by SNP and cGMP utilize a signal transduction pathway different from that used by the depolarizing agent KCl and the muscarinic agonist carbachol. Contractions induced by SNP and cGMP involve tyrosine phosphorylation of a protein, possibly identified as a 116-kDa protein, as a key step in the signaling pathway.
Characterization of ion transport mechanisms regulating intracellular pH in hepatic stellate cells
Authors: Di Sario A. , Baroni G. S. , Bendia E. , D'Ambrosio L. , Ridolfi F. , Marileo J. R. , Jezequel A. M. , Benedetti A.
Published: December 2017

The aim of this study was to evaluate intracellular pH (pHi) regulation in nonactivated and activated rat hepatic stellate cells (HSC). The fluorescent pHi indicator 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein was used to measure pHi in the presence and absence of HCO3-. In the absence of HCO3-, baseline pHi was significantly higher (P < 0.001) in activated than in nonactivated HSC (7.1 +/- 0.1 vs. 6.9 +/- 0.2) and decreased, in both groups, after amiloride administration and after Na+ removal. After an acid-loading maneuver, pHi recovery was significantly higher (P < 0.03) in activated than in nonactivated HSC (H+ flux = 11.0 +/- 3.8 vs. 7.7 +/- 2.9 mM/min at pHi 6.6) and was inhibited by amiloride and Na+ removal. In the presence of HCO3-, baseline pHi was higher in both groups and decreased after amiloride administration. Amiloride and Na+ removal inhibited pHi recovery after an intracellular acid load by 77 and 93%, respectively, in nonactivated and by 82 and 92%, respectively, in activated HSC, whereas 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid inhibited pHi recovery by only 27%. Acute Cl- removal increased pHi by 0.07 +/- 0.01 pH unit/min in the absence but not in the presence of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid in nonactivated and activated HSC in an Na(+)-independent manner. In activated HSC, 24 h of incubation with 25 ng/ml platelet-derived growth factor (PDGF)-BB (in 0.5% serum) did not modify baseline pHi (7.07 +/- 0.1 vs. 7.08 +/- 0.1 in HSC cultured in 0.5% serum only) but significantly (P < 0.02) increased, with respect to controls, pHi recovery after an acute acid load. Incubation with PDGF for 24 h induced a fivefold increase in HSC proliferation expressed as percentage of bromodeoxyuridine-positive cells (30.8 +/- 6.7 vs. 6.1 +/- 1.9% in controls). When amiloride (0.1 mM) was present, PDGF-induced HSC proliferation was significantly inhibited (8.1 +/- 0.4%, P < 0.001). Our results show that 1) the Na+/H+ exchanger is the main pHi regulator in rat HSC, 2) activation of HSC is associated with an increase in pHi and in the activity of the Na+/H+ exchanger, 3) PDGF increases the activity of this exchanger, and 4) amiloride is able to inhibit HSC proliferation induced by PDGF.
IL-11 inhibits Clostridium difficile toxin A enterotoxicity in rat ileum
Authors: Castagliuolo I. , Kelly C. P. , Qiu B. S. , Nikulasson S. T. , LaMont J. T. , Pothoulakis C.
Published: December 2017

Interleukin-11 (IL-11) is a stromal cell-derived cytokine with several biological activities against hematopoietic cells. Recent results indicated that IL-11 reduced mucosal damage in animal models of colitis. This study aimed to explore the action of recombinant human IL-11 (rhIL-11) on the intestinal effects of Clostridium difficile toxin A, an inflammatory enterotoxin, and cholera toxin, a noninflammatory enterotoxin in rat ileum. We administered rhIL-11 subcutaneously to rats before injection of toxin A into ileal loops and measured fluid secretion, epithelial permeability to mannitol, histopathological damage, and release of rat mast cell protease II (RMCP II) from intestinal mast cells and of tumor necrosis factor-alpha (TNF-alpha) and macrophage inflammatory protein-2 (MIP-2) from lamina propria macrophages. rhIL-11 (50-1,000 micrograms/kg) inhibited toxin A but not cholera toxin-mediated secretion and permeability in a dose-dependent fashion and reduced toxin A-induced epithelial cell damage. Rats treated with rhIL-11 also showed reduced RMCP II, TNF-alpha, and MIP-2 release in response to toxin A. Exposure of rat peripheral monocytes in vitro to rhIL-11 (1 microgram/ml) inhibited lipopolysaccharide and toxin A-mediated increases in TNF-alpha mRNA and protein levels. We conclude that rhIL-11 blocks the intestinal effects of C. difficile toxin A, possibly by inhibiting release of inflammatory mediators from mucosal mast cells and intestinal macrophages.
CCK-8 infusion increases plasma LMW alkaline phosphatase coincident with enterohepatic circulation of bile acids
Authors: Solter P. F. , Hoffmann W. E. , Chambers M. D. , Schaeffer D. J.
Published: December 2017

Bile acids may facilitate the release of liver alkaline phosphatase (ALP) from its hydrophobic membrane anchor. The purpose of this study was to determine whether such a facilitatory role could be observed during the enterohepatic circulation of bile acids in dogs. Increased hepatic ALP activity was induced in four dogs by daily injections of 4 mg.kg-1.day-1 of prednisone for 10 days. Intravenous infusions of cholecystokinin octapeptide (CCK-8) were given before treatment and on treatment days 3, 5, 7, and 10 to induce gallbladder emptying and the enterohepatic circulation of bile acids. Blood samples were taken hourly for 4 h before and for 4 h after CCK-8 infusion. These showed that plasma ALP activity increased significantly only after CCK-8 infusion. Gel exclusion chromatography, polyacrylamide gel electrophoresis, and octyl Sepharose phase separation showed that the increased ALP activity was a hydrophilic, low-molecular-weight (LMW) isoform, which is consistent with phospholipase release. Histochemical staining of endogenous ALP activity showed increased ALP activity over sinusoidal surfaces of prednisone-treated dogs. There was also an increased serum-to-tissue ratio of ALP activity in the prednisone-treated dogs, suggestive of increased release of ALP into blood. It was concluded that bile acids probably play a facilitatory role in the enzymatic release of ALP from the sinusoidal surface of hepatocytes, which may be accentuated by the presence of increased amounts of ALP on the sinusoidal surface in some disease states.
Nitric oxide as modulator of cholinergic neurotransmission in gastric muscle of rabbits
Authors: Baccari M. C. , Iacoviello C. , Calamai F.
Published: December 2017

The effects of the nitric oxide (NO) synthesis inhibitors, NG-nitro-L-arginine (L-NNA) and NG-nitro-L-arginine methyl ester (L-NAME), on the electrical field stimulation (EFS)-induced inhibitory responses were investigated. EFS caused, in strips contracted by means of substance P (SP), prostaglandin F2 alpha (PGF2 alpha), or carbachol (CCh), a fast relaxant response that, depending on stimulation frequency and strip tension, could be followed by a slower, sustained relaxation. The NO synthesis inhibitors blocked the EFS-induced fast relaxations and often reversed them into contractions; these effects were greatly counteracted in SP- or PGF2 alpha-treated strips by scopolamine or atropine. In CCh-precontracted strips, either L-NNA or L-NAME became progressively unable to block the EFS-induced fast relaxations as the CCh concentration was increased. The NO synthesis inhibitors greatly reduced the sustained relaxant responses elicited either by EFS or exogenous vasoactive intestinal polypeptide (VIP). The results indicate that the NO synthesis inhibitors abolish the neurally induced fast relaxation by interfering with the cholinergic excitatory pathway. The involvement of both VIP and NO in sustained relaxations is also suggested.
Cholinergic nerves mediate stress-induced intestinal transport abnormalities in Wistar-Kyoto rats
Authors: Saunders P. R. , Hanssen N. P. , Perdue M. H.
Published: December 2017

We have previously reported that acute stress alters intestinal transport physiology in Wistar-Kyoto rats, a stress-susceptible strain. In this study, we tested the hypothesis that the abnormalities in these rats are due to cholinergic mechanisms. Atropine- or saline-treated rats were exposed to acute restraint stress, and, subsequently, electrophysiological parameters of excised jejunal segments were assessed in Ussing chambers. Compared with the parent Wistar rat strain, Wistar-Kyoto rats demonstrated significantly greater stress-induced changes in ion secretion and permeability. The activity of cholinesterase in intestinal mucosal homogenates was significantly less in Wistar-Kyoto than in Wistar rats. Atropine pretreatment of rats before stress corrected the epithelial pathophysiology. Our results suggest that stress stimulated the release of acetylcholine, resulting in altered epithelial function in these genetically predisposed rats.
Gene transfer into hepatoma cell lines via the serpin enzyme complex receptor
Authors: Ziady A. G. , Perales J. C. , Ferkol T. , Gerken T. , Beegen H. , Perlmutter D. H. , Davis P. B.
Published: December 2017

The serpin enzyme complex receptor (SECR) expressed on hepatocytes binds to a conserved sequence in alpha 1-antitrypain (alpha 1-AT) and other serpins. A molecular conjugate consisting of a synthetic peptide (C1315) based on the SECR binding motif of human alpha 1-AT covalently coupled to poly-L-lysine was used to introduce reporter genes into hepatoma cell lines in culture. This conjugate condensed DNA into spheroidal particles 18-25 nm in diameter. When transfected with the SECR-directed complex containing pGL3, Hep G2 cells that express the receptor, but not Hep G2 cells that do not, expressed a peak luciferase activity of 538,731 +/- 144,346 integrated light units/mg protein 4 days after transfection. Free peptide inhibited uptake and expression in a dose-dependent manner. Complexes of DNA condensed with polylysine or LC-sulfo-N-succinimidyl-3-(2-pyridyldithio)propionate-substituted polylysine were ineffective. Transfection with a plasmid encoding human factor IX produced expression in Hep G2 (high) and HuH7 cells that express SECR but not Hep G2 (low) cells that lack the receptor. Fluorescein-labeled C1315 peptide labeled 9-31% of Hep G2 (high), 10-14% of HuH7, and 0.6-3.4% of Hep G2 (low) cells, and when the lac Z gene was transfected, only these cells expressed beta-galactosidase. SECR-mediated gene transfer gives efficient, specific uptake and high-level expression of three reporter genes, and the system merits further study for gene therapy.
Regulation of hepatic enzymes and insulin levels in offspring of rat dams fed a reduced-protein diet
Authors: Desai Mina , Byrne Christopher D. , Meeran Karim , Martenz Nick D. , Bloom Steven R. , Hales C. Nicholas
Published: December 2017

We have hypothesized that permanent changes caused by poor growth during early development due to maternal malnutrition may be exacerbated by overnutrition of offspring in later life. To test this hypothesis, rats were exposed to a maternal 20% protein diet or an isocaloric 8% protein diet during fetal and postnatal life. All offspring were weaned onto laboratory chow. At 6 wk, rats were fed laboratory chow or a highly palatable diet (high fat and high calorie with adequate protein) and studied at 12 wk after a 48-h fast. The highly palatable diet resulted in excess weight gain and higher plasma insulin levels in all animals. Plasma insulin concentrations were significantly increased in male offspring of dams fed a reduced-protein diet compared with male offspring of dams fed an adequate-protein diet, but no differences were observed between the female offspring. The key hepatic enzymes of glucose homeostasis programmed in offspring of protein-restricted rat dams retained the ability to respond to overnutrition during adult life. In these offspring, however, the enzymes were regulated around a “set point” that was different from that in the controls.
Demonstration of changes in fetal liver erythropoiesis using echo-planar magnetic resonance imaging
Authors: Duncan K. R. , Baker P. N. , Gowland P. A. , Issa B. , Moore R. , Worthington B. , Johnson I. R.
Published: December 2017

This study investigated the variation in magnetic resonance characteristics of the fetal liver during a time of changing erythropoietic function. Echo-planar imaging was carried out in 25 normal pregnant women at 20 and 26 wk gestation. The signal intensity from regions of the fetal liver, background image, and maternal back muscle and the highest signal intensity from the maternal spinal cord were measured and compared with the signal intensity of amniotic fluid. Data are expressed as ratios, in arbitrary units (median pixel values; interquartile range shown in parentheses), and analyzed with the use of Wilcoxon’s signed-rank test. At 20 wk, the signal intensity ratio of liver to amniotic fluid was 0.309 (0.231–0.365). At 26 wk, the ratio was 0.544 (0.429–0.616). The change was highly significant ( P< 0.0001). No change in the signal intensity ratios of amniotic fluid compared with other measured parameters was noted. These data are consistent with known changes in fetal liver erythropoiesis occurring between 20 and 26 wk gestation and have potential use in early noninvasive physiological assessment of the fetus.
Epidermal growth factor attenuatesClostridium difficiletoxin A- and B-induced damage of human colonic mucosa
Authors: Riegler Martin , Sedivy Roland , Sogukoglu Tacettin , Castagliuolo Ignazio , Pothoulakis Charalabos , Cosentini Enrico , Bischof Georg , Hamilton Gerhard , Teleky Bela , Feil Wolfgang , Lamont J. Thomas , Wenzl E.
Published: December 2017

Epidermal growth factor (EGF) exhibits a cytoprotective effect on gastrointestinal epithelia via a receptor-mediated mechanism. We investigated the effect of EGF on Clostridium difficile toxin A (TxA)- and toxin B (TxB)-induced damage of human colon. Ussing-chambered colonic mucosa was exposed serosally to EGF before and during luminal exposure to TxA and TxB. Resistance was calculated from potential difference and short-circuit current. Epithelial damage was assessed by light microscopy and alteration of F-actin by fluoresceinated phalloidin. Luminal exposure of colonic strips to TxA and TxB caused a time- and dose-dependent decrease in electrical resistance, necrosis and dehiscence of colonocytes, and disruption and condensation of enterocyte F-actin. These effects were inhibited by prior, but not simultaneous, serosal application of EGF (20 nM). Administration of the tyrosine kinase inhibitor genistein (10−6M) inhibited the protective effects of EGF. We conclude that EGF protects against TxA and TxB probably by stabilizing the cytoskeleton, the main target of these toxins.
Neuronal release of endogenous dopamine from corpus of guinea pig stomach
Authors: Shichijo Kazuko , Sakurai-Yamashita Yasuko , Sekine Ichiro , Taniyama Kohtaro
Published: December 2017

Neuronal release of endogenous dopamine was identified in mucosa-free preparations (muscle layer including intramural plexus) from guinea pig stomach corpus by measuring tissue dopamine content and dopamine release and by immunohistochemical methods using a dopamine antiserum. Dopamine content in mucosa-free preparations of guinea pig gastric corpus was one-tenth of norepinephrine content. Electrical transmural stimulation of mucosa-free preparations of gastric corpus increased the release of endogenous dopamine in a frequency-dependent (3–20 Hz) manner. The stimulated release of dopamine was prevented by either removal of external Ca2+ or treatment with tetrodotoxin. Dopamine-immunopositive nerve fibers surrounding choline acetyltransferase-immunopositive ganglion cells were seen in the myenteric plexus of whole mount preparations of gastric corpus even after bilateral transection of the splanchnic nerve proximal to the junction with the vagal nerve (section of nerves between the celiac ganglion and stomach). Domperidone and sulpiride potentiated the stimulated release of acetylcholine and reversed the dopamine-induced inhibition of acetylcholine release from mucosa-free preparations. These results indicate that dopamine is physiologically released from neurons and from possible dopaminergic nerve terminals and regulates cholinergic neuronal activity in the corpus of guinea pig stomach.
Phenylalanine utilization by the gut and liver measured with intravenous and intragastric tracers in pigs
Authors: Stoll Barbara , Burrin Douglas G. , Henry Joseph , Jahoor Farook , Reeds Peter J.
Published: December 2017

To investigate intestinal and hepatic metabolism of phenylalanine, four conscious pigs (7.5 kg), bearing arterial, venous, and hepatic portal catheters, were fasted for 12 h and infused with [ phenyl-2H5]phenylalanine via a peripheral vein and [ carboxyl-13C]phenylalanine via the stomach. During the first 6 h of the infusion, the pigs remained fasted and received only the intravenous tracer. During the second 6 h, they received an intragastric infusion of milk replacer and both tracers. In the fasted state, the portal-drained viscera extracted 10% ( P < 0.025) of the arterial [2H5]phenylalanine flow of the pigs. In the fed state, the splanchnic tissues metabolized 45% of the enteral tracer and intestinal metabolism accounted for 76% of the total splanchnic extraction. The tracer-to-tracee ratio of both tracers in apolipoprotein B-100 (apo B-100) phenylalanine was twofold ( P < 0.001) higher than that of hepatic free phenylalanine. The ratios of the two tracers in portal (13C/2H; 1.66) and apo B-100 (1.76) phenylalanine were similar but higher ( P < 0.05) than that of arterial phenylalanine (1.29). We conclude that intestinal metabolism dominates the splanchnic extraction of enteral phenylalanine and that in the fed state, the hepatic protein synthetic precursor pool derives from portal phenylalanine.
Role of glutathione and catalase in H2O2detoxification in LPS-activated hepatic endothelial and Kupffer cells
Authors: Spolarics Zoltán , Wu Jun-Xi
Published: December 2017

The present study investigated the effect of lipopolysaccharide (LPS; from Escherichia coli, 2 mg/kg body wt ip) on selected aspects of the antioxidant status in Kupffer and sinusoidal endothelial cells. Cells were isolated 18 h after the injection of saline or LPS. In fresh suspension cultures, cellular reduced glutathione (GSH) and H2O2were determined by monochlorobimane, and 2′,7′-dichlorofluorescein diacetate, respectively, using a fluorescence plate reader. LPS injection increased GSH content two- to threefold in Kupffer cells compared with cells from control rats. Cellular GSH content was higher in endothelial than Kupffer cells. However, LPS did not increase GSH content in endothelial cells. Addition of H2O2(40–200 μM) to Kupffer or endothelial cells caused a transient decrease in GSH, which was more pronounced in cells from control rats (∼45% drop) than in LPS-exposed cells (∼25% drop). Depleted GSH levels were accompanied by a proportional increase in cellular H2O2. After inhibition of catalase by 3-amino-1,2,4-triazole, the presence of 0.2 mM H2O2depleted GSH content by 75% and 40% in Kupffer cells from saline- or LPS-injected rats, respectively. The same treatments caused a similar 50% decrease in both activated and control endothelial cells. LPS decreased catalase activity by 45% in Kupffer cells, whereas it had no effect on catalase in endothelial cells. Glutathione reductase activity was not altered by LPS in either cell type. These data show that in activated Kupffer cells the elevated level of cellular glutathione plays an augmented role in the protection against reactive oxygen species, whereas the contribution of catalase to H2O2detoxification is attenuated. In LPS-stimulated endothelial and Kupffer cells, the efficient maintenance of GSH is consistent with upregulated production of reducing power through the hexose phosphate shunt observed previously.
CGRP upregulation in dorsal root ganglia and ileal mucosa duringClostridium difficiletoxin A-induced enteritis
Authors: Keates Andrew C. , Castagliuolo Ignazio , Qiu Bosheng , Nikulasson Sigfus , Sengupta Ashok , Pothoulakis Charalabos
Published: December 2017

We have previously reported that pretreatment of rats with capsaicin (an agent that ablates sensory neurons) or CP-96345 (a substance P receptor antagonist) dramatically inhibits fluid secretion and intestinal inflammation in ileal loops exposed to Clostridium difficile toxin A. The aim of this study was to determine whether calcitonin gene-related peptide (CGRP), a neuropeptide also found in sensory afferent neurons, participates in the enterotoxic effects of toxin A. Administration of toxin A was also found to increase CGRP content in dorsal root ganglia and ileal mucosa 60 min after toxin exposure. Furthermore, immunohistochemical studies demonstrated increased neuronal staining for CGRP 2 h after toxin A treatment. Pretreatment of rats with CGRP-(8—37), a specific CGRP antagonist, before instillation of toxin A into ileal loops significantly inhibited toxin-mediated fluid secretion (by 48%), mannitol permeability (by 83%), and histological damage. We conclude that CGRP, like substance P, contributes to the secretory and inflammatory effects of toxin A via increased production of this peptide from intestinal nerves, including primary sensory afferent neurons.
Impaired G protein function in gallbladder muscle from progesterone-treated guinea pigs
Authors: Chen Qian , Chitinavis Vikas , Xiao Zouliang , Yu Peirong , Oh Sangik , Biancani Piero , Behar Jose
Published: December 2017

This study was designed to elucidate the mechanism of action of progesterone on gallbladder smooth muscle in guinea pigs. Adult male guinea pigs were treated with either progesterone (2 mg ⋅ kg−1⋅ day−1) or saline for 7 days. Gallbladder muscle cells were isolated by enzymatic digestion with collagenase. Contractile responses to agonists were expressed as percent shortening from control cell length. [35S]guanosine 5′- O-(3-thiotriphosphate) ([35S]GTPγS)-binding properties of G proteins were assessed in crude membranes of gallbladder muscle with or without cholecystokinin octapeptide (CCK-8) stimulation. Gallbladder muscle cells from progesterone-treated guinea pigs exhibited an impaired contractile response to CCK-8, GTPγS, or aluminum fluoride but a normal response to potassium chloride ord- myo-inositol 1,4,5-trisphosphate compared with controls. Western blot analysis of gallbladder muscle revealed the presence of Gi 1–2, Gi 3, Gq/11, and Gsproteins. The maximal contraction induced by CCK-8 was blocked by pertussis toxin and Giα3-specific antibodies, but not by Giα1–2or Gq/11α antibodies. CCK-8 caused a significant increase in [35S]GTPγS binding to Giα3, but not to Gq/11α or Giα1–2. The stimulation of Giα3binding, however, was significantly reduced in gallbladder muscle membranes from progesterone-treated guinea pigs compared with that in control animals. In conclusion, progesterone might cause gallbladder hypomotility by downregulating Gi 3proteins.
Pressure and flow characteristics of terminal mesenteric arteries in postnatal intestine
Authors: Reber Kristina M. , Nowicki Philip T.
Published: December 2017

The responses of buffer-perfused terminal mesenteric arteries from 3- and 35-day-old swine to manipulation of intravascular pressure and flow rate were determined. Under in vivo conditions, these vessels demonstrated age-dependent differences in resting diameter (182 vs. 301 μm in 3- vs. 35-day-old swine). A proximal-to-distal pressure gradient was present in vessels from both age groups (Δ13 vs. Δ16 mmHg in 3- vs. 35-day-old swine), suggesting their functional role as resistance vessels. Vessels were mounted within an in vitro perfusion apparatus that allowed independent regulation of inflow and outflow pressure. Vessels from both age groups demonstrated the development of active tone in response to an incremental rise in pressure, applied in the absence of flow. However, myogenic vasoconstriction was only observed in younger arterioles. Similarly, both groups demonstrated dilation in response to a flow stimulus generated in the absence of a net change in intravascular pressure, although the magnitude of this response was significantly greater in younger vessels (+27 vs. +7% in 3- vs. 35-day-old swine). The dilatory response to flow was eliminated by N G-monomethyl-l-arginine (10−4 M) but restored by coadministration of l-arginine (10−3 M). Myogenic vasoconstriction was overridden by flow-mediated dilation in terminal mesenteric arteries from 3- but not 35-day-old swine after concomitant application of pressure and flow stimuli. We conclude that the hemodynamic characteristics of terminal mesenteric arteries are age dependent in postnatal swine.
Contractions move contents by changing the configuration of the isolated cat stomach
Authors: Schulze-Delrieu Konrad , Herman Robert J. , Shirazi Siroos S. , Brown Bruce P.
Published: December 2017

To understand how contractions move gastric contents, we measured, in isolated cat stomachs, the effects of contractions on gastric length, diameters, pressures, and emptying. Movements of the stomach and of gastric contents were monitored by video camera and ultrasound and were related to mechanical events. Based on pressures, we defined the following four phases of contractions: 1) Po, a steady pressure associated with tonic contraction of proximal stomach; 2) P′, a pressure wave during which the contraction indents the gastric body; 3) a pressure nadir while the contraction lifts the gastric sinus toward the incisura; and 4) a second pressure wave, P”, as the contraction advances through the antrum. In open preparations, liquid output and shortening of the greater curvature are large during Po, stop during P′, and resume with P”. Contractions generate higher pressures when gastric volume is held steady. Contractions increase wall thickness and decrease gastric diameters at sites they involve and have opposite effects at remote sites. Contractions move the incisura and hence redraw the borders between gastric segments and shift volumes back and forth within the gastric lumen. Contractions furthermore stir up, compress, and disperse particulate beans without moving them to the pylorus. We conclude that gastric contractions 1) reverse changes in gastric length that occur during gastric filling, 2) move gastric contents directly through local contact and indirectly by changing the configuration of the stomach, and 3) interact with structures such as the incisura in retaining and breaking up solid gastric contents.
Chronic hypoxia alters glucose utilization during GSH-dependent detoxication in rat small intestine
Authors: Legrand Terry S. , Aw Tak Yee
Published: December 2017

We showed that hypoxia alters glutathione (GSH)-dependent detoxication and induces mucosal metabolic instability. To determine the impact of these changes and the role of reductant supply in intestinal lipid peroxide disposition, pair-fed (16 g/day) Sprague-Dawley rats were exposed to air (20.9% O2; n = 6) or 10% O2( n = 6) for 10 days. Jejunal and ileal everted sacs were exposed to 75 μM peroxidized fish oil with or without 10 mM glucose or 1 mM GSH. Peroxide transport was determined as the abluminal recovery of thiobarbituric acid-reactive substances. Peroxide recovery in hypoxic intestine was twice that in normoxic intestine. Addition of GSH and glucose did not affect peroxide recovery, indicating reduced intracellular GSH-dependent metabolism and enhanced output by the hypoxic intestine. Glucose uptake by normoxic and hypoxic intestine is similar, whereas its utilization for detoxication is decreased in hypoxic cells. Determination of NADPH supply indicates that decreased glucose availability for NADPH production during hypoxia impairs GSH disulfide reduction, compromises hydroperoxide metabolism, and increases peroxide output from hypoxic intestine.
Nociceptive inhibition of migrating myoelectric complex by nitric oxide and monoaminergic pathways in the rat
Authors: Hellström Per M. , Thollander Mikael , Theodorsson Elvar
Published: December 2017

This study investigated the role of nitric oxide (NO) and adrenergic and dopaminergic mechanisms in reflex inhibition of the migrating myoelectric complex (MMC) after intraperitoneal administration of acid in rats. Acid instilled immediately after an activity front inhibited the migrating complex and prolonged the cycle length from 13.0 ± 0.7 to 98.5 ± 17.2 min ( P < 0.001). Administration of Nω-nitro-l-arginine, reserpine, or guanetidine before acid decreased the prolonged cycle length to 18.1 ± 2.8 ( P < 0.001), 19.0 ± 2.0 ( P < 0.001), and 27.5 ± 9.3 min ( P < 0.001), respectively. Similarly, haloperidol given before acid shortened the prolonged cycle length to 46.7 ± 5.2 min ( P < 0.05). There was no effect of phentolamine in combination with propranolol or hexamethonium given alone. After intraperitoneal instillation of acid there was an increase in the plasma levels of somatostatin and a decrease of calcitonin gene-related peptide, but there was no change of neuropeptide Y, vasoactive intestinal peptide, substance P, neurokinin A, or neurotensin. The results indicate that NO and adrenergic, dopaminergic, and somatostatinergic mechanisms cooperate in inhibiting the MMC after nociceptive stimulation of the peritoneum.
Hepatic pyruvate dehydrogenase activity in humans: effect of cirrhosis, transplantation, and dichloroacetate
Authors: Shangraw Robert E. , Rabkin John M. , Lopaschuk Gary D.
Published: December 2017

The liver is the major site for lactate clearance, and liver disease exacerbates lactic acidosis during orthotopic liver transplantation (OLT). This study assessed pyruvate dehydrogenase (PDH) activity in control, cirrhotic, and graft liver to test the hypotheses that 1) liver disease decreases hepatic PDH activity, 2) graft PDH activity is inhibited due to protracted ischemia, and 3) dichloroacetate (DCA) reverses functional PDH inhibition in cirrhotic and graft liver. After having given their informed consent, 43 patients received either DCA (80 mg/kg) or aqueous 5% glucose during OLT. Six patients without apparent liver dysfunction that were undergoing subtotal hepatic resection served as controls. Liver biopsy PDH activity was assayed by measuring [14C]citrate synthesis from [14C]oxaloacetate and PDH-derived acetyl-CoA. PDH in the active form (PDHa) in cirrhotic and control liver was 5.6 ± 1.3 (SE) and 57 ± 10 nmol ⋅ g wet wt−1 ⋅ min−1, respectively ( P < 0.001). Total PDH activity (PDHt) was 21.5 ± 3.6 and 264 ± 27 nmol ⋅ g wet wt−1 ⋅ min−1, respectively ( P < 0.001). DCA increased PDHa in cirrhotic liver to 22.3 ± 4.1 nmol ⋅ g wet wt−1 ⋅ min−1( P < 0.05 vs. no DCA) without altering PDHt. Graft liver PDHa was 166 ± 19 nmol ⋅ g wet wt−1 ⋅ min−1, which was not altered by DCA. We conclude that decreased hepatic PDH activity secondary to decreased content may underlie lactic acidosis during OLT, which can be partially compensated by DCA administration. There is no apparent inhibition of graft liver PDH activity after reperfusion.
Effect of ethanol on the structure and function of rabbit esophageal epithelium
Authors: Bor Serhat , Caymaz-Bor Canan , Tobey Nelia A. , Abdulnour-Nakhoul Solange , Marten Esteban , Orlando Roy C.
Published: December 2017

Epidemiological studies indicate a relationship between alcohol consumption and esophageal epithelial disease. We therefore sought the contribution of the direct effects of ethanol on esophageal epithelial structure and (transport and barrier) function. Epithelium from the rabbit was mounted in Ussing chambers and exposed luminally for 1 h to 1–40% ethanol. At concentrations of 1–5% potential difference (PD) increased, and at 10–40% PD decreased. The increase in PD with 1–5% ethanol was accompanied by an increase in short-circuit current ( Isc), and this increase in Isccould be blocked by ouabain pretreatment. The decrease in PD with 10–40% ethanol was associated with a decrease in electrical resistance ( R), and this decrease in R was paralleled by an increase in transepithelial [14C]mannitol flux. Reversibility of these changes was limited at ethanol concentrations ≥10%, and these were associated morphologically by patchy or diffuse tissue edema. Moreover, as with ethanol exposure in vitro, exposure in vivo produced dose-dependent changes in PD, Isc, R, and morphology. These observations indicate that exposure to ethanol in concentrations and under conditions reflecting alcohol consumption in humans can alter and impair esophageal epithelial transport and barrier functions. Such impairments are likely to contribute to the observed increase in risk of esophageal disease with regular consumption of alcoholic beverages.
TNF-α inhibits liver collagen-α1(I) gene expression through a tissue-specific regulatory region
Authors: Houglum Karl , Buck Martina , Kim Dong Joon , Chojkier Mario
Published: December 2017

Although tumor necrosis factor-α (TNF-α) inhibits collagen-α1(I) gene expression in cultured hepatic stellate cells, assessment of its effects on hepatic collagen expression is complicated by the confounding variables of tissue necrosis and inflammation. Therefore, we analyzed whether chronically elevated serum TNF-α affects constitutive hepatic collagen metabolism in vivo by inoculating nude mice with Chinese hamster ovary (CHO) cells secreting TNF-α (TNF-α mice) or with control CHO cells (control mice). Before the onset of weight loss, collagen synthesis and collagen gene expression were inhibited in the liver of TNF-α mice. In transgenic mice, after 8 h, TNF-α (500 ng at 0 and 5 h) inhibited the liver expression of the collagen-α1(I)-human growth hormone (hGH) transgene containing the first intron and −440 bp of the 5′ region. Similarly, in cultured hepatic stellate cells isolated from these transgenic animals, the −440 bp collagen-α1(I)-hGH transgene was responsive to TNF-α treatment independent of the activation of these cells. Transfection studies in stellate cells allowed further characterization of this TNF-α-responsive segment to −220 bp of the 5′ region. Because in the skin the inhibitory effect of TNF-α involves a regulatory region of the collagen-α1(I) gene beyond −440 bp, we herein identify a novel tissue-specific regulation of collagen-α1(I) gene by TNF-α.
Central CRF inhibits gastric emptying of a nutrient solid meal in rats: the role of CRF2receptors
Authors: Martinez V. , Barquist E. , Rivier J. , Taché Y.
Published: December 2017

Corticotropin-releasing factor (CRF)-related peptides exhibit different affinity for the receptor subtypes 1 and 2 cloned in the rat brain. We investigated, in conscious rats, the effects of intracisternal (IC) injection of CRF (rat/human) on the 5-h rate of gastric emptying of a solid nutrient meal (Purina chow and water ad libitum for 3 h) and the CRF receptor subtype involved. CRF, urotensin I (suckerfish), and sauvagine (frog) injected IC inhibited gastric emptying in a dose-dependent manner, with ED50 values of 0.31, 0.13, and 0.08 μg/rat, respectively. Rat CRF-(6—33) (0.1–10 μg ic) had no effect. The nonselective CRF1and CRF2 receptor antagonist, astressin, injected IC completely blocked the inhibitory effect of IC CRF, urotensin I, and sauvagine with antagonist-to-agonist ratios of 3:1, 10:1, and 16:1, respectively. The CRF1-selective receptor antagonist NBI-27914 injected IC at a ratio of 170:1 had no effect. These data show that central CRF and CRF-related peptides are potent inhibitors of gastric emptying of a solid meal with a rank order of potency characteristic of the CRF2receptor subtype affinity (sauvagine > urotensin I > CRF). In addition, the reversal by astressin but not by the CRF1-selective receptor antagonist further supports the view that the CRF2 receptor subtype is primarily involved in central CRF-induced delayed gastric emptying.