bluuebunny/crossref_metadata_2025 huggingface dataset:
Sources and implications of basal nitric oxide in spontaneous contractions of guinea pig taenia caeci
Authors: Caballero-Alomar Catalina , Santos Carmen , Lopez Diego , Mitjavila M. Teresa , Puig-Parellada Pere
Published: March 2015

We examined in vitro the source and role of basal nitric oxide (NO) in proximal segments of guinea pig taenia caeci in nonadrenergic, noncholinergic (NANC) conditions. Using electron paramagnetic resonance (EPR), we measured the effect of the NO synthase inhibitor NG-nitro-l-arginine methyl ester (l-NAME, 10–4 M), the neuronal blocker tetrodotoxin (TTX, 10–6 M), or both on spontaneous contractions and on the production of basal NO. Both l-NAME and TTX, when tested alone, increased the amplitude and frequency of contractions. NO production was abolished by l-NAME and was inhibited by 38% by TTX. When tested together, l-NAME in the presence of TTX or TTX in the presence of l-NAME had no further effect on the amplitude or frequency of spontaneous contractions, and the NO production was inhibited. These findings suggest that basal NO consists of TTX-sensitive and TTX-resistant components. The TTX-sensitive NO has an inhibitory effect on spontaneous contractions; the role of TTX-resistant NO is unknown.
Role of 5-HT1B/D receptors in canine gastric accommodation: effect of sumatriptan and 5-HT1B/D receptor antagonists
Authors: De Ponti Fabrizio , Crema Francesca , Moro Elisabetta , Nardelli Giuseppe , Frigo Gianmario , Crema Antonio
Published: March 2015

The 5-HT1B/D receptor agonist sumatriptan has been proposed to treat dyspeptic symptoms, because it facilitates gastric accommodation. It is unknown whether stimulation of 5-HT1B/D receptors is involved. Thus, in four conscious dogs, we compared the effects of sumatriptan alone or combined with N-[4-methoxy-3-(4-methyl-1-piperazinyl) phenyl]-2′-methyl-4′-(5-methyl-1,2,4-oxadiazol-3-yl)-[1,1-biphenyl]-4-carboxamide hydrocloride (GR-127935), N-[3-[3 (dimethylamino)-ethoxy]-4-methoxyphenyl]-2′-[methyl-4′-(5-methyl-1,2,4-oxadiazol-3-yl)]-[1,1-biphenyl]-4-carboxamide hydrocloride (SB-216641 hydrochloride), or 3-[4-(4-chloro-phenyl)piperazin-1-yl]-1,1-diphenyl-2-propanol hydrochloride (BRL-15572 hydrochloride) (respectively, nonselective 5-HT1B/D, selective 5-HT1B, and selective 5-HT1D receptor antagonists) on gastric accommodation to isobaric distensions performed with a barostat. An exponential and a linear model were used to fit the pressure-volume relationship. An exponential equation fitted the data better than a linear equation. Sumatriptan (800 nmol/kg iv) induced an immediate gastric relaxation (Δvolume: 112 ± 44 ml, P < 0.05). After sumatriptan, the pressure-volume curve was shifted toward significantly higher volumes. This effect was fully reversed by GR-127935 or SB-216641 but not by BRL-15572. In conclusion, 5-HT1B receptors seem to play an important role in modulating gastric accommodation to a distending stimulus. An exponential model for pressure-volume curves fits well with the concept of gastric adaptive relaxation.
Human urocortin II, a new CRF-related peptide, displays selective CRF2-mediated action on gastric transit in rats
Authors: Million Mulugeta , Maillot Céline , Saunders Paul , Rivier Jean , Vale Wylie , Taché Yvette
Published: March 2015

Human urocortin (hUcn) II is a new member of the corticotropin-releasing factor (CRF) family that selectively binds to the CRF2receptor. We investigated the CRF receptors involved in mediating the effects of hUcn II and human/rat CRF (h/rCRF) on gut transit. Gastric emptying, 4 h after a solid meal, and distal colonic transit (bead expulsion time) were monitored simultaneously in conscious rats. CRF antagonists were given subcutaneously 30 min before intravenous injection of peptides or partial restraint (for 90 min). hUcn II (3 or 10 μg/kg iv) inhibited gastric emptying (by 45% and 55%, respectively) and did not influence distal colonic transit. The CRF2 peptide antagonist astressin2-B blocked hUcn II action. h/rCRF, rat Ucn, and restraint delayed gastric emptying while accelerating distal colonic transit. The gastric response to intravenous h/rCRF and restraint was blocked by the CRF2 antagonist but not by the CRF1 antagonist CP-154,526, whereas the colonic response was blocked only by CP-154,526. None of the CRF antagonists influenced postprandial gut transit. These data show that intravenous h/rCRF and restraint stress-induced delayed gastric emptying involve CRF2 whereas stimulation of distal colonic transit involves CRF1. The distinct profile of hUcn II, only on gastric transit, is linked to its CRF2 selectivity.
Enhanced calcium signaling and acid secretion in parietal cells isolated from gastrin-deficient mice
Authors: Hinkle Karen L. , Bane Gina C. , Jazayeri Ali , Samuelson Linda C.
Published: March 2015

Gastrin-deficient mice have impaired basal and agonist-stimulated gastric acid secretion. To analyze whether an intrinsic parietal cell defect contributed to the reduced acid secretion, we analyzed parietal cell calcium responses and acid secretory function in vitro. Parietal cells were purified by light-scatter cell sorting and calcium responses to gastrin, histamine, and carbachol were measured in gastrin-deficient and wild-type mice cell preparations. Surprisingly, basal and histamine-induced calcium concentrations were higher in the mutant cell preparations. [14C]aminopyrine uptake analysis in acutely isolated gastric glands revealed that basal acid accumulation was enhanced in gastrin-deficient cell preparations as well as on treatment with carbachol or histamine. These results suggested that an intrinsic parietal cell defect was not responsible for the reduced acid secretion in gastrin-deficient mice. Flow cytometric analysis of dispersed, H+-K+-ATPase-immunostained gastric mucosal preparations revealed a marked increase in parietal cell number in gastrin-deficient mice, which may have accounted for the enhanced in vitro acid secretion detected in this study. Parietal cells were found to be significantly smaller in the mutant cell preparations, suggesting that gastrin stimulation modulates parietal cell morphology.
Bile salts potentiate adenylyl cyclase activity and cAMP-regulated secretion in human gallbladder epithelium
Authors: Chignard Nicolas , Mergey Martine , Veissière Danielle , Poupon Raoul , Capeau Jacqueline , Parc Rolland , Paul Annick , Housset Chantal
Published: March 2015

Fluid and ion secretion in the gallbladder is mainly triggered by the intracellular second messenger cAMP. We examined the action of bile salts on the cAMP-dependent pathway in the gallbladder epithelium. Primary cultures of human gallbladder epithelial cells were exposed to agonists of the cAMP pathway and/or to bile salts. Taurochenodeoxycholate and tauroursodeoxycholate increased forskolin-induced cAMP accumulation to a similar extent, without affecting cAMP basal levels. This potentiating effect was abrogated after PKC inhibition, whereas both taurochenodeoxycholate and tauroursodeoxycholate induced PKC-α and -δ translocation to cell membranes. Consistent with a PKC-mediated stimulation of cAMP production, the expression of six adenylyl cyclase isoforms, including PKC-regulated isoforms 5 and 7, was identified in human gallbladder epithelial cells. cAMP-dependent chloride secretion induced by isoproterenol, a β-adrenergic agonist, was significantly increased by taurochenodeoxycholate and by tauroursodeoxycholate. In conclusion, endogenous and therapeutic bile salts via PKC regulation of adenylyl cyclase activity potentiate cAMP production in the human gallbladder epithelium. Through this action, bile salts may increase fluid secretion in the gallbladder after feeding.
Regulation of NHE3 by nitric oxide in Caco-2 cells
Authors: Gill Ravinder K. , Saksena Seema , Syed Irfan Ali , Tyagi Sangeeta , Alrefai Waddah A. , Malakooti Jaleh , Ramaswamy Krishnamurthy , Dudeja Pradeep K.
Published: March 2015

The effect of nitric oxide (NO) on Na+/H+exchange (NHE) activity was investigated utilizing Caco-2 cells as an experimental model. Incubation of Caco-2 cells with 10−3M S-nitroso- N-acetylpenicillamine (SNAP), a conventional donor of NO, for 20 min resulted in a ∼45% dose-dependent decrease in NHE activity, as determined by assay of ethylisopropylamiloride-sensitive22Na uptake. A similar decrease in NHE activity was observed utilizing another NO-specific donor, sodium nitroprusside. SNAP-mediated inhibition of NHE activity was not secondary to a loss of cell viability. NHE3 activity was significantly reduced by SNAP ( P < 0.05), whereas NHE2 activity was essentially unaltered. The effects of SNAP were mediated by the cGMP-dependent signal transduction pathway as follows: 1) LY-83583 and 1 H-(1,2,4)oxadiazolo(4,3- a)quinoxalin-1-one (ODQ), specific inhibitors of soluble guanylate cyclase, blocked the inhibitory effect of SNAP on NHE; 2) 8-bromo-cGMP mimicked the effects of SNAP on NHE activity; 3) the SNAP-induced decrease in NHE activity was counteracted by a specific protein kinase G inhibitor, KT-5823 (1 μM); 4) chelerythrine chloride (2 μM) or calphostin C (200 nM), specific protein kinase C inhibitors, did not affect inhibition of NHE activity by SNAP; 5) there was no cross activation by the protein kinase A-dependent pathway, as the inhibitory effects of SNAP were not blocked by Rp-cAMPS (25 μM), a specific protein kinase A inhibitor. These data provide novel evidence that NO inhibits NHE3 activity via activation of soluble guanylate cyclase, resulting in an increase in intracellular cGMP levels and activation of protein kinase G.
Stimulation of gastrin-CCKB receptor promotes migration of gastric AGS cells via multiple paracrine pathways
Authors: Noble Peter J. M. , Wilde Geraint , White Michael R. H. , Pennington Steven R. , Dockray Graham J. , Varro Andrea
Published: March 2015

Responses to G protein-coupled receptor stimulation may be mediated by paracrine factors. We have developed a coculture system to study paracrine regulation of migration of gastric epithelial (AGS) cells after stimulation of gastrin-CCKBreceptors. In cells expressing this receptor, G-17 stimulated migration by activation of protein kinase C. However, G-17 also stimulated the migration of cells expressing green fluorescent protein, but not the receptor, when they were cocultured with receptor-expressing cells consistent with activation of paracrine signals. The use of various pharmacological inhibitors indicated that gastrin stimulated migration via activation of the EGF receptor (EGR-R), the erbB-2 receptor tyrosine kinase, and the MAP kinase pathway. However, gastrin also released fibroblast growth factor (FGF)-1, and migration was inhibited by the FGF receptor tyrosine kinase inhibitor SU-5402. Flow cytometry indicated that in both cell types, gastrin increased MAP kinase via activation of EGF-R but not FGF-R1 or erbB-2. We conclude that gastrin-CCKB receptors stimulate epithelial cell migration partly via paracrine mechanisms; transactivation of EGF-R is only one component of the paracrine pathway.
A phosphorylated phloretin derivative. Synthesis and effect on intestinal Na+-dependent phosphate absorption
Authors: Peerce Brian E. , Clarke Rebecca
Published: March 2015

2′-Phosphophloretin (2′-PP), a phosphorylated derivative of the plant chalcone, was synthesized. The effect of 2′-PP, on Na+-dependent phosphate uptake into intestinal brush-border membrane vesicles (BBMV) isolated from rabbit and rat duodenum and jejunum was examined. 2′-PP decreased Na+-dependent phosphate uptake into rabbit BBMV with an IC50 of 55 nM and into rat BBMV with an IC50 of 58 nM. 2′-PP did not affect Na+-dependent glucose, Na+-dependent sulfate, or Na+-dependent alanine uptake by rabbit intestinal BBMVs. 2′-PP inhibition of rabbit intestinal BBMV Na+-dependent phosphate uptake was sensitive to external phosphate concentration, suggesting that 2′-PP inhibition of Na+-dependent phosphate uptake was competitive with respect to phosphate. Binding of [3H]2′-PP to rabbit intestinal BBMV was examined. Binding of [3H]2′-PP was Na+-dependent with a K 0.5 for Na+(Na+concentration for 50% 2′-PP binding) of 30 mM. The apparent K s for Na+-dependent [3H]2′-PP binding to rabbit BBMVs was 58 nM in agreement with the IC50 for 2′-PP inhibition of Na+-dependent phosphate uptake. These results indicate that 2′-PP bound to rabbit or rat intestinal BBMV Na+-phosphate cotransporter and inhibited Na+-dependent phosphate uptake. In rats treated with 2′-PP by daily gavage, the effect of 2′-PP on serum phosphate, serum glucose, and serum calcium was examined. In a concentration-dependent manner, 2′-PP reduced serum phosphate by 45% 1 wk after starting treatment. 2′-PP did not alter serum calcium or serum glucose. The apparent IC50 for 2′-PP in vivo was 3 μM.
rHuKGF ameliorates symptoms in DSS and CD4+CD45RBHiT cell transfer mouse models of inflammatory bowel disease
Authors: Byrne Fergus R. , Farrell Catherine L. , Aranda Richard , Rex Karen L. , Scully Sheila , Brown Heather L. , Flores Silvia A. , Gu Li H. , Danilenko Dimitry M. , Lacey David L. , Ziegler Thomas R. , Senaldi Giorgio
Published: March 2015

There is an acute need for effective therapy for inflammatory bowel disease (IBD), particularly at the level of repair of the damaged epithelium. We evaluated the efficacy of recombinant human keratinocyte growth factor (rHuKGF) in both the dextran sodium sulfate (DSS) and the CD4+CD45RBHiT cell transfer models of IBD. Disease was induced either by the ad libitum administration to normal mice of 4% DSS in the drinking water or by the injection of 4 × 105CD4+CD45RBHiT cells into immunodeficient scid/scid mice. rHuKGF was administered by subcutaneous injection at doses of 1.0 or 3.0 mg/kg in both preventative and therapeutic regimens during both studies. rHuKGF significantly improved survival and body weight loss in the DSS model in both preventative and therapeutic dosing regimens. It also improved diarrhea, hematochezia, and hematological parameters, as well as large intestine histopathology. In the T cell transfer model, rHuKGF improved body weight loss, diarrhea, and levels of serum amyloid A, as well as large intestine histopathology. In both models of IBD, the colonic levels of intestinal trefoil factor (ITF) were elevated by the disease state and further elevated by treatment with rHuKGF. These data suggest that rHuKGF may prove useful in the clinical management of IBD and its effects are likely mediated by its ability to locally increase the levels of ITF.
Role of thromboxane A2in early BDL-induced portal hypertension
Authors: Yokoyama Yukihiro , Xu Hongzhi , Kresge Nicole , Keller Steve , Sarmadi Amir H. , Baveja Rajiv , Clemens Mark G. , Zhang Jian X.
Published: March 2015

Although the mechanisms of cirrhosis-induced portal hypertension have been studied extensively, the role of thromboxane A2(TXA2) in the development of portal hypertension has never been explicitly explored. In the present study, we sought to determine the role of TXA2in bile duct ligation (BDL)-induced portal hypertension in Sprague-Dawley rats. After 1 wk of BDL or sham operation, the liver was isolated and perfused with Krebs-Henseleit bicarbonate buffer at a constant flow rate. After 30 min of nonrecirculating perfusion, the buffer was recirculated in a total volume of 100 ml. The perfusate was sampled for the enzyme immunoassay of thromboxane B2(TXB2), the stable metabolite of TXA2. Although recirculation of the buffer caused no significant change in sham-operated rats, it resulted in a marked increase in portal pressure in BDL rats. The increase in portal pressure was found concomitantly with a significant increase of TXB2in the perfusate (sham vs. BDL after 30 min of recirculating perfusion: 1,420 ± 803 vs. 10,210 ± 2,950 pg/ml; P < 0.05). Perfusion with a buffer containing indomethacin or gadolinium chloride for inhibition of cyclooxygenase (COX) or Kupffer cells, respectively, substantially blocked the recirculation-induced increases in both portal pressure and TXB2release in BDL group. Hepatic detection of COX gene expression by RT-PCR revealed that COX-2 but not COX-1 was upregulated following BDL, and this upregulation was confirmed at the protein level by Western blot analysis. In conclusion, these results clearly demonstrate that increased hepatic TXA2release into the portal circulation contributes to the increased portal resistance in BDL-induced liver injury, suggesting a role of TXA2in liver fibrosis-induced portal hypertension. Furthermore, the Kupffer cell is likely the source of increased TXA2, which is associated with upregulation of the COX-2 enzyme.
Increased activity and expression of iNOS in human duodenal enterocytes from patients with celiac disease
Authors: Murray Iain A. , Daniels Ian , Coupland Kathryn , Smith Julie A. , Long Richard G.
Published: March 2015

The activity of nitric oxide synthase (NOS) was assayed in enterocytes isolated from human duodenal biopsies to determine its role in celiac disease. Patients were categorized into groups with irritable bowel syndrome, iron-deficiency anemia, B12/folate deficiency, and treated and untreated celiac disease. Enterocytes isolated from all groups showed 1400W-inhibitable Ca2+-independent NOS activity with a pH level and temperature optimum of 9.4 and 37°C, respectively. Western blotting showed that enterocytes expressed the inducible NOS protein and proteins with nitrated tyrosine residues, the latter being indicative of nitric oxide-driven peroxynitrite and/or free-radical damage. Endothelial NOS was seen only in the lamina propria. Patients with celiac disease had higher NOS activity than other patient groups. Treatment of the condition led to a fall in activity. Enzyme-linked immunosorbent assay demonstrated cGMP production by the enterocyte fraction, but cGMP levels did not correlate with NOS activity. These results suggest that inducible NOS is constitutively expressed in human duodenal enterocytes, is increased in patients with untreated celiac disease, and is partially corrected when such patients are treated. We found no evidence to support a role for nitric oxide in the formation of cGMP within the small intestine. Furthermore, we were unable to demonstrate a role for peroxynitrite/free radical damage in the pathophysiology of celiac disease.
Apoptosis is a major mechanism of deoxycholate-induced gastric mucosal cell death
Authors: Redlak Maria J. , Dennis Miranda S. , Miller Thomas A.
Published: March 2015

This study was undertaken to determine whether necrosis or apoptosis was the predominant mechanism responsible for gastric mucosal cellular death using the cell line known as AGS cells. Cells were exposed to various concentrations of deoxycholate (DC; 50–500 μM) for periods ranging from 30 min to 24 h. Lactic dehydrogenase (LDH) activity was used as a marker for necrotic cell death, whereas apoptosis was characterized by 4′,6-diamidino-2 phenylindole staining, DNA gel electrophoresis, terminal deoxynucleotidyl transferase dUTP nick-end labeling assay and DNA-histone-associated complex formation. When cells were bathed in Hank's balanced salt solution, DC-induced necrosis was the predominant mechanism of cell death. In contrast, when cells were bathed in Ham's F-12 solution (a more physiologically relevant medium), no evidence of cytotoxicity (by LDH assay) was discernible when cells were exposed to DC (50–300 μM) for periods as long as 8 h; instead, clear evidence of apoptosis was noted that was time and dose dependent. When cells were exposed for 24 h to these DC concentrations, cytotoxicity was also present, indicating necrosis as well. Furthermore, acidification of the ambient environment also evoked a necrotic response when exposed to DC. We demonstrated that apoptosis induced by DC shows early activation of caspase-3 that is dependent on both receptor and mitochondrial pathways. Our results indicate that physiological concentrations of DC (50–300 μM) primarily induce cellular death through an apoptotic process. Only after prolonged exposure to DC or acidification of the bathing solution does necrosis also occur.
Ghrelin/motilin-related peptide is a potent prokinetic to reverse gastric postoperative ileus in rat
Authors: Trudel L. , Tomasetto C. , Rio M. C. , Bouin M. , Plourde V. , Eberling P. , Poitras P.
Published: March 2015

A novel peptide called ghrelin or motilin-related-peptide (MTLRP) was found in the stomach of various mammals. We studied its effect on the motor function of the rat gastrointestinal tract. In normal, conscious unoperated animals, ghrelin/MTLRP (5 or 20 μg/kg iv) significantly accelerated the gastric emptying of a methylcellulose liquid solution (gastric residue after 15 min: 57 ± 7, 42 ± 11, 17 ± 4, and 9 ± 3% of the ingested meal with doses of 0, 1, 5, and 20 μg/kg iv, respectively) Transit of the methylcellulose liquid solution was also accelerated by ghrelin/MTLRP in the small intestine but not in the colon. Des-[Gln14]ghrelin, also found in the mammalian stomach, was as potent as ghrelin in emptying the stomach (gastric residue after 15 min: 12 ± 3% at a dose of 20 μg/kg iv). In rats in which postoperative gastrointestinal ileus had been experimentally induced, ghrelin/MTLRP (20 μg/kg iv) reversed the delayed gastric evacuation (gastric residue after 15 min: 28 ± 7% of the ingested meal vs. 82 ± 9% with saline). In comparison, the gastric ileus was not modified by high doses of motilin (77 ± 7%) or erythromycin (82 ± 6%) and was only partially improved by calcitonin gene-related peptide (CGRP) 8–37 antagonist (59 ± 7%). Ghrelin/MTLRP, therefore, accelerates the gastric emptying and small intestinal transit of a liquid meal and is a strong prokinetic agent capable of reversing the postoperative gastric ileus in rat.
Thyroid hormone regulates the activity and expression of the peptide transporter PEPT1 in Caco-2 cells
Authors: Ashida Kayoko , Katsura Toshiya , Motohashi Hideyuki , Saito Hideyuki , Inui Ken-Ichi
Published: March 2015

An oligopeptide transporter (PEPT1) in the small intestine plays an important role in the absorption of small peptides and peptide-like drugs. We examined the effect of thyroid hormone 3,5,3′-l-triiodothyronine (T3) on the activity and expression of PEPT1 in human intestinal Caco-2 cells. Treatment of Caco-2 cells with T3 inhibited [14C]glycylsarcosine uptake in a time- and dose-dependent manner. [14C]glycylsarcosine uptake was reduced by pretreatment of the cells with 100 nM T3 for 4 days (67% of control value), whereas methyl-α-d-[U-14C]glucopyranoside and [3H]threonine uptake were not decreased. Kinetic analysis showed that T3 treatment significantly decreased the maximum uptake (Vmax) value for [14C]glycylsarcosine uptake but had no effect on the K m value. Moreover, T3 treatment caused a significant decrease in the amount of PEPT1 mRNA (25% of the control). Western blotting indicated that the amount of PEPT1 protein in the apical membrane was decreased (70% of the control). These findings indicate that T3 treatment inhibits the uptake of [14C]glycylsarcosine by decreasing the transcription and/or stability of PEPT1 mRNA.
Electrical rhythmicity and spread of action potentials in longitudinal muscle of guinea pig distal colon
Authors: Spencer Nick J. , Hennig Grant W. , Smith Terence K.
Published: March 2015

Using simultaneous intracellular recordings, we have characterized 1) electrical activity in the longitudinal muscle (LM) of isolated segments of guinea pig distal colon free to contract spontaneously and 2) extent of propagation of spontaneous action potentials around the circumference of the colon. In all animals, rhythmical spontaneous depolarizations (SDs) were recorded that are usually associated with the generation of action potentials. Recordings from pairs of LM cells, separated by 100 μm in the circumferential axis, revealed that each action potential was phase locked at the two electrodes (mean propagation velocity: 3 mm/s). However, at an increased electrode separation distance of 1 mm circumferentially, action potentials and SDs became increasingly uncoordinated at the two recording sites. No SDs or action potentials ever propagated from one circumferential edge to the other (i.e., 13 mm apart). When LM strips were separated from the myenteric plexus and circular muscle, rhythmically firing SDs and action potentials were still recorded. Atropine (1 μM) or tetrodotoxin (1 μM) either reduced the frequency of SDs or temporily abolished activity, whereas nifedipine (1 μM) always abolished SDs and action potentials. Kit-positive interstitial cells of Cajal were present at the level of the myenteric plexus and circular and longitudinal muscle. In summary, SDs and action potentials in LM propagate over discrete localized zones, usually <1 mm around the circumference of the colon. Furthermore, in contrast to the classic slow wave, rhythmic depolarizations in LM appear to be generated by an intrinsic property of the smooth muscle itself and are critically dependent on opening of L-type Ca2+ channels.
Somatostatin sst2 receptors inhibit peristalsis in the rat and mouse jejunum
Authors: Abdu Faiza , Hicks Gareth A. , Hennig Grant , Allen Jeremy P. , Grundy David
Published: March 2015

Somatostatin [somatotropin release-inhibitory factor (SRIF)] has widespread actions throughout the gastrointestinal tract, but the receptor mechanisms involved are not fully characterized. We have examined the effect of selective SRIF-receptor ligands on intestinal peristalsis by studying migrating motor complexes (MMCs) in isolated segments of jejunum from rats, mice, and sst2-receptor knockout mice. MMCs were recorded in 4- to 5-cm segments of jejunum mounted horizontally in vitro. MMCs occurred in rat and mouse jejunum with intervals of 104.4 ± 10 and 131.2 ± 8 s, respectively. SRIF, octreotide, and BIM-23027 increased the interval between MMCs, an effect fully or partially antagonized by the sst2-receptor antagonist Cyanamid154806. A non-sst2 receptor-mediated component was evident in mouse as confirmed by the observation of an inhibitory action of SRIF in sst2 knockout tissue. Blocking nitric oxide generation abolished the response to SRIF in rat but not mouse jejunum. sst2 Receptors mediate inhibition of peristalsis in both rat and mouse jejunum, but a non-sst2component also exists in the mouse. Nitrergic mechanisms are differentially involved in rat and mouse jejunum.
Effects of a κ-opioid agonist, asimadoline, on satiation and GI motor and sensory functions in humans
Authors: Delgado-Aros Silvia , Chial Heather J. , Camilleri Michael , Szarka Lawrence A. , Weber Frank T. , Jacob Jutta , Ferber Irene , McKinzie Sanna , Burton Duane D. , Zinsmeister Alan R.
Published: March 2015

To compare the effects of the κ-opioid agonist asimadoline and placebo on visceral sensation and gastrointestinal (GI) motor functions in humans, 91 healthy participants were randomized in a double-blind fashion to 0.15, 0.5, or 1.5 mg of asimadoline or placebo orally twice a day for 9 days. We assessed satiation (nutrient drink test), colonic compliance, tone, perception of colonic distension (barostat), and whole gut transit (scintigraphy). Treatment effect was assessed by analysis of covariance. Asimadoline increased nutrient drink intake ( P = 0.03). Asimadoline decreased colonic tone during fasting ( P = 0.03) without affecting postprandial colonic contraction, compliance, or transit. Gas scores in response to colonic distension were decreased with 0.5 mg of asimadoline at low levels (8 mmHg above operating pressure) of distension ( P = 0.04) but not at higher levels of distension. Asimadoline at 1.5 mg increased gas scores at 16 mmHg of distension ( P = 0.03) and pain scores at distensions of 8 and 16 mmHg ( P = 0.003 and 0.03, respectively) but not at higher levels of distension. Further studies of this compound in diseases with altered satiation or visceral sensation are warranted.
Contribution of capsaicin-sensitive sensory neurons to stress-induced increases in gastric tissue levels of prostaglandins in rats
Authors: Harada Naoaki , Okajima Kenji , Uchiba Mitsuhiro , Katsuragi Takeshi
Published: March 2015

We examined whether capsaicin-sensitive sensory neurons might be involved in the increase in the gastric tissue level of prostaglandins, thereby contributing to the reduction of water immersion restraint stress (WIR)-induced gastric mucosal injury in rats. Gastric tissue levels of calcitonin gene-related peptide (CGRP), 6-keto-PGF1α, and PGE2were transiently increased 30 min after WIR. These increases were significantly inhibited by subcutaneous injection of capsazepine (CPZ), a vanilloid receptor antagonist, and by functional denervation of capsaicin-sensitive sensory neurons induced by the administration of high-dose capsaicin. The administration of capsaicin (orally) and CGRP (intravenously) significantly enhanced the WIR-induced increases in the gastric tissue level of prostaglandins 30 min after WIR, whereas CGRP-(8-37), a CGRP receptor antagonist, significantly inhibited them. Pretreatment with Nω-nitro-l-arginine methyl ester (l-NAME), a nonselective inhibitor of nitric oxide (NO) synthase (NOS), and that with indomethacin inhibited the WIR-induced increases in gastric tissue levels of prostaglandins, whereas either pretreatment with aminoguanidine (AG), a selective inhibitor of the inducible form of NOS, or that with NS-398, a selective inhibitor of cyclooxygenase (COX)-2, did not affect them. CPZ, the functional denervation of capsaicin-sensitive sensory neurons, and CGRP-(8-37) significantly increased gastric MPO activity and exacerbated the WIR-induced gastric mucosal injury in rats subjected to 4-h WIR. The administration of capsaicin and CGRP significantly increased the gastric tissue levels of prostaglandins and inhibited both the WIR-induced increases in gastric MPO activity and gastric mucosal injury 8 h after WIR. These effects induced by capsaicin and CGRP were inhibited by pretreatment with l-NAME and indomethacin but not by pretreatment with AG and NS-398. These observations strongly suggest that capsaicin-sensitive sensory neurons might release CGRP, thereby increasing the gastric tissue levels of PGI2and PGE2by activating COX-1 through activation of the constitutive form of NOS in rats subjected to WIR. Such activation of capsaicin-sensitive sensory neurons might contribute to the reduction of WIR-induced gastric mucosal injury mainly by inhibiting neutrophil activation.
Inhibition of cyclooxygenase-2 ameliorates the severity of pancreatitis and associated lung injury
Authors: Song Albert M. , Bhagat Lakshmi , Singh Vijay P. , Van Acker Gijs G. D. , Steer Michael L. , Saluja Ashok K.
Published: March 2015

Cyclooxygenase-2 (COX-2), a widely distributed enzyme, plays an important role in inflammation. We have studied the role of COX-2 in acute pancreatitis and pancreatitis-associated lung injury using both the pharmacological inhibition of COX-2 and genetic deletion of COX-2. Pancreatitis was induced in mice by 12 hourly injections of cerulein. The severity of pancreatitis was assessed by measuring serum amylase, pancreatic trypsin activity, intrapancreatic sequestration of neutrophils, and acinar cell necrosis. The severity of lung injury was evaluated by measuring lactate dehydrogenase levels in the bronchoalveolar lavage fluid and by quantitating neutrophil sequestration in the lung. In both the pharmacologically inhibited and genetically altered mice, the severity of pancreatitis and pancreatitis-associated lung injury was reduced compared with the noninhibited strains of COX-2-sufficient mice. This reduction in injury indicates that COX-2 plays an important proinflammatory role in pancreatitis and its associated lung injury. Our findings support the concept that COX-2 inhibitors may play a beneficial role in the prevention of acute pancreatitis or in the reduction of its severity.
Hemodynamic and proinflammatory actions of endothelin-1 in guinea pig small intestine submucosal microcirculation
Authors: King-VanVlack C. E. , Mewburn J. D. , Chapler C. K. , MacDonald P. H.
Published: March 2015

The hemodynamic and proinflammatory effects of endothelin-1 (ET-1) in proximal (1st/2nd order) and terminal (3rd/4th order) arterioles and venules were examined in small intestine submucosa of anesthetized guinea pigs. Vessel diameter (D), red blood cell velocity, and blood flow (Q) were determined in eight proximal and eight terminal microvessels before and at 20 min of ET-1 suffusion (10−10, 10−9, and 10−8 M) and then with endothelin-A (ETA)-receptor blockade with BQ-123 (10−5 M). This protocol was repeated with platelet-activating factor (PAF) inhibition (WEB-2086, 1.0 mg/kg iv; n = 16). The ET-1-mediated microvascular responses were also examined with endothelin-B (ETB)-receptor blockade using BQ-788 (10−5 M; n = 11) alone or with ETA+B-receptor blockade with BQ-123 + BQ-788 ( n = 10). Microvascular permeability was assessed by FITC-albumin (25 mg/kg iv) extravasation in seven series: 1) buffered modified Krebs solution suffusion ( n = 6), 2) histamine suffusion (HIS; 10−3 M, n = 5), 3) ET-1 suffusion (10−8M, n = 5), 4) BQ-123 (10−5 M) plus ET-1 suffusion ( n = 5), 5) PAF inhibition before ET-1 suffusion ( n = 5), 6) histamine-1 (H1)-receptor blockade (diphenhydramine, 20 mg/kg iv) before ET-1 suffusion ( n = 5), and 7) ETB-receptor blockade before (BQ-788 10−5 M; n = 3) or with ET-1 suffusion ( n = 3). D and Q decreased at 10−8 M ET-1 and returned to control values with BQ-123 and BQ-123+BQ788 but not with BQ-788 in proximal microvessels. D did not change in terminal microvessels with ET-1 (10−8 M) but decreased with BQ-788 and increased with BQ-123. PAF inhibition did not affect the D and Q responses of proximal microvessels to ET-1 but prevented the fall in Q in terminal microvessels with ET-1. ET-1 increased vascular permeability to ∼1/3 of that with HIS; this response was prevented with BQ-123 and WEB-2086 but not with H1-receptor blockade. This is the first evidence that submucosal terminal microvessel flow is reduced with ET-1 independent of vessel diameter changes and that this response is associated with increased microvascular permeability mediated via ETA-receptor stimulation and PAF activation.
Fas is not essential for lamina propria T lymphocyte homeostasis
Authors: Boone David L. , Dassopoulos Themistocles , Chai Sophia , Chien Marcia , Lodolce James , Ma Averil
Published: March 2015

IL-2 receptor α-deficient (IL2Rα-/-) mice spontaneously accumulate vast numbers of intestinal lamina propria (LP) T cells and develop bowel inflammation. The accumulation of T cells in IL2Rα-/-mice is thought to result, in part, from defective Fas-induced cell death. To understand the role of cell proliferation and death in regulating LP T cells in IL2Rα-/-mice, we have directly examined the proliferation and Fas sensitivity of wild-type, lpr/lpr, and IL2Rα-/-LP T cells. In wild-type mice, 5′-bromodeoxyuridine (BrdU) labeling and Fas susceptibility are greatest in CD44HiLP T cells. Fas-deficient lpr/lpr mice have normal total numbers of LP T cells, despite an increased proportion of BrdU+T cells. By contrast, IL2Rα-/-mice possess increased total numbers of LP T cells, despite normal proportions of BrdU+LP T cells. Finally, wild-type and IL2Rα-/-LP T cells are equivalently Fas sensitive. These results demonstrate that LP T cells proliferate and are Fas-sensitive cells. IL2Rα-/-mice accumulate a large number of these Fas-sensitive LP T cells and clearly differ from Fas-deficient lpr/lpr mice in this regard. Thus our studies reveal that Fas is dispensable for LP T cell homeostasis and suggest that the intestinal inflammation observed in IL2Rα-/-mice is independent of defective Fas-induced cell death.
Effect of GLP-2 on mucosal morphology and SGLT1 expression in tissue-engineered neointestine
Authors: Ramsanahie Anthony , Duxbury Mark S. , Grikscheit Tracy C. , Perez Alexander , Rhoads David B. , Gardner-Thorpe James , Ogilvie Jennifer , Ashley Stanley W. , Vacanti Joseph P. , Whang Edward E.
Published: March 2015

Using tissue-engineering techniques, we have developed a neointestine that regenerates the structural and dynamic features of native small intestine. In this study, we tested neointestinal responsiveness to glucagon-like peptide 2 (GLP-2). Neointestinal cysts were engineered by seeding biodegradable polymers with neonatal rat intestinal organoid units. The cysts were matured and anastomosed to the native jejunum of syngeneic adult recipients. Animals were treated with GLP-2 [Gly2] (twice daily, 1 μg/g body wt) or vehicle alone (control) for 10 days. Rats were then killed, and tissues were harvested for analysis. Na+-glucose cotransporter (SGLT1) mRNA expression was assessed with Northern blotting and in situ hybridization. SGLT1 protein was localized by using immunofluorescence. GLP-2 administration resulted in 1.8- and 1.7-fold increases ( P < 0.05) in neointestinal villus height and crypt depth, respectively. GLP-2 administration also resulted in a 2.4-fold increase ( P < 0.01) in neomucosal SGLT1 mRNA expression. SGLT1 mRNA expression was localized to enterocytes throughout the villi, and SGLT1 protein was localized to the brush border of enterocytes along the entire length of villi from the neointestine of GLP-2-treated animals. The response of tissue-engineered neointestine to exogenous GLP-2 includes mucosal growth and enhanced SGLT1 expression. Therefore, tissue-engineering principles may help in dissecting the regulatory mechanisms mediating complex processes in the intestinal epithelium.
Serotonin stimulates endotoxin translocation via 5-HT3 receptors in the rat ileum
Authors: Yamada Tadanori , Inui Akio , Hayashi Naoki , Fujimura Masaki , Fujimiya Mineko
Published: March 2015

Because few previous studies have investigated the mechanisms of endotoxin translocation induced by intestinal obstruction, we aimed to clarify whether or not serotonin [5-hydroxytryptamine (5-HT)], which is released from enterochromaffin (EC) cells, is responsible for alterations of the mucosal permeability to endotoxin and to identify the 5-HT receptor subtypes that mediate this action. FITC-labeled LPS (FITC-LPS) was injected into the ileum of rats, and the FITC-LPS level in the superior mesenteric vein was subsequently measured by using a fluorescence spectrophotometer. To measure the 5-HT release induced by high intraluminal pressure, ex vivo preparation of vascularly and luminally perfused rat ileum was used. Results demonstrated that elevated intraluminal pressure stimulates the translocation of FITC-LPS and the release of 5-HT from the EC cells into the intestinal lumen but not into the portal circulation. This FITC-LPS translocation, which was stimulated by exogenously applied 5-HT in the lumen and the jugular vein, was inhibited by 5-HT3 receptor antagonist administration both intaluminally and intravenously. The stimulatory effect of elevated intraluminal pressure on the translocation of FITC-LPS was inhibited by the intraluminal and intravenous administration of 5-HT3receptor antagonist. These results suggest that 5-HT released from EC cells may be involved in the translocation of FITC-LPS induced by elevated intraluminal pressure and that this effect is mediated by 5-HT3 receptors that may be located in the intestinal epithelium.
A novel ultrasound technique to study the biomechanics of the human esophagus in vivo
Authors: Takeda Torahiko , Kassab Ghassan , Liu Jianmin , Puckett James L. , Mittal Rishi R. , Mittal Ravinder K.
Published: March 2015

The objectives of this study were to validate a novel ultrasound technique and to use it to study the circumferential stress-strain properties of the human esophagus in vivo. A manometric catheter equipped with a high-compliance bag and a high-frequency intraluminal ultrasonography probe was used to record esophageal pressure and images. Validation studies were performed in vitro followed by in vivo studies in healthy human subjects. Esophageal distensions were performed with either an isovolumic (5–20 ml of water) or with an isobaric (10–60 mmHg) technique. Sustained distension was also performed for 3 min in each subject. The circumferential wall stress and strain were calculated. In vitro studies indicate that the ultrasound technique can make measurements of the esophageal wall with an accuracy of 0.01 mm. The in vivo studies provide the necessary data to compute the Kirchhoff's stress, Green's strain, and Young's elastic modulus during esophageal distensions. The stress-strain relationship revealed a linear shape, the slope of which corresponds to the Young's modulus. During sustained distensions, we found dynamic changes of stress and strain during the period of distension. We describe and validate a novel ultrasound technique that allows measurement of biomechanical properties of the esophagus in vivo in humans.
Regulation of postischemic liver injury following different durations of ischemia
Authors: Hines Ian N. , Hoffman Jason M. , Scheerens Heleen , Day Brian J. , Harada Hirohisa , Pavlick Kevin P. , Bharwani Sulaiman , Wolf Robert , Gao Bifeng , Flores Sonia , McCord Joe M. , Grisham Matthew B.
Published: March 2015

The objective of this study was to define the relationship among Kupffer cells, O[Formula: see text]production, and TNF-α expression in the pathophysiology of postischemic liver injury following short and long periods of ischemia. Using different forms of superoxide dismutase with varying circulating half-lives, a monoclonal antibody directed against mouse TNF-α, and NADPH oxidase-deficient mice, we found that 45 or 90 min of partial (70%) liver ischemia and 6 h of reperfusion (I/R) produced time-dependent increases in liver injury and TNF-α expression in the absence of neutrophil infiltration. Furthermore, we observed that hepatocellular injury induced by short periods of ischemia were not dependent on formation of TNF-α but were dependent on Kupffer cells and NADPH oxidase-independent production of O[Formula: see text]. However, liver injury induced by extended periods of ischemia appeared to require the presence of Kupffer cells, NADPH oxidase-derived O[Formula: see text], and TNF-α expression. We conclude that the sources for O[Formula: see text] formation and the relative importance of TNF-α in the pathophysiology of I/R-induced hepatocellular injury differ depending on the duration of ischemia.
TNBS ileitis evokes hyperexcitability and changes in ionic membrane properties of nociceptive DRG neurons
Authors: Moore Beverley A. , Stewart Timothy M. R. , Hill Ceredwyn , Vanner Stephen J.
Published: March 2015

This study examines whether intestinal inflammation leads to changes in the properties of ion channels in dorsal root ganglia (DRG) neurons. Ileitis was induced by injection of trinitrobenzene sulfonic acid (TNBS), and DRG neurons innervating the ileum were labeled using fast blue. Intracellular recording techniques were used to measure electrophysiological properties of acutely dissociated neurons 12–24 h after dissection. Nociceptive neurons were identified by sensitivity to capsaicin, tetrodotoxin resistance, and size (<30 μm). The action potential threshold in neurons from TNBS-treated animals was reduced by >70% compared with controls ( P < 0.001), but the resting membrane potential was unchanged. Cell diameter, input resistance (67%), and action potential upstroke velocity (22%) increased in the TNBS group ( P < 0.05). The number of action potentials discharged increased in the TNBS group ( P < 0.001), whereas application of 4-aminopyridine to control cells mimicked this effect. This study demonstrates that ileitis induces hyperexcitability in nociceptive DRG neurons and changes in the properties of Na+ and K+channels at the soma, which persist after removal from the inflamed environment.
Copper repletion enhances apical iron uptake and transepithelial iron transport by Caco-2 cells
Authors: Han Okhee , Wessling-Resnick Marianne
Published: March 2015

The influence of copper status on Caco-2 cell apical iron uptake and transepithelial transport was examined. Cells grown for 7–8 days in media supplemented with 1 μM CuCl2had 10-fold higher cellular levels of copper compared with control. Copper supplementation did not affect the integrity of differentiated Caco-2 cell monolayers grown on microporous membranes. Copper-repleted cells displayed increased uptake of iron as well as increased transport of iron across the cell monolayer. Northern blot analysis revealed that expression of the apical iron transporter divalent metal transporter-1 (DMT1), the basolateral transporter ferroportin-1 (Fpn1), and the putative ferroxidase hephaestin (Heph) was upregulated by copper supplementation, whereas the recently identified ferrireductase duodenal cytochrome b (Dcytb) was not. These results suggest that DMT1, Fpn1, and Heph are involved in the iron uptake process modulated by copper status. Although a clear role for Dcytb was not identified, an apical surface ferrireductase was modulated by copper status, suggesting that its function also contributes to the enhanced iron uptake by copper-repleted cells. A model is proposed wherein copper promotes iron depletion of intestinal Caco-2 cells, creating a deficiency state that induces upregulation of iron transport factors.
Regulation of copper absorption by copper availability in the Caco-2 cell intestinal model
Authors: Zerounian Nora R. , Redekosky Carmen , Malpe Rashmi , Linder Maria C.
Published: March 2015

Relatively little is known about the individual steps in intestinal copper absorption and whether or how they may be regulated. Polarized Caco-2 cell monolayers with tight junctions offer an already tested model in which to study intestinal metal transport. This model was used to examine potential effects of cellular copper availability on copper absorption. Uptake and transport were determined on application of64Cu(II) to the brush border. In the range of 0.2–2 μM, uptake was dose dependent and was ∼20% of dose/90 min. Overall transport of64Cu across the basolateral surface was ∼0.3%. When cellular copper levels were depleted 40% by 18-h pretreatment with the specific copper chelator triethylenetetraamine, uptake and overall transport were markedly increased, going to 80 and 65% of dose, respectively. Cellular retention of64Cu fell fourfold, from 6 to 1.5%. Depletion of copper with the chelator was rapid and preceded initial changes in uptake and overall transport by 4 h. A lesser depletion of cellular copper (13%) failed to enhance copper uptake but doubled the rate of overall transport, as measured with64Cu and by atomic absorption. As previously reported, preexposure of the cells to excess copper (10 μM, 18 h) also enhanced copper uptake (∼3-fold). In contrast, ascorbate (10–1,000 μM) failed to significantly alter uptake and transport of 1 μM64Cu. Our findings are consistent with the concepts that, in the low physiological range, copper availability alters the absorption capacity of the intestine to support whole body homeostasis and that basolateral transport is more sensitively regulated than uptake.
In situ correlation of cytokine secretion and apoptosis inHelicobacter pylori-associated gastritis
Authors: Lehmann Frank Serge , Terracciano Luigi , Carena Ilaria , Baeriswyl Christian , Drewe Jürgen , Tornillo Luigi , De Libero Gennaro , Beglinger Christoph
Published: March 2015

Tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) are important for the pathogenesis of Helicobacter pylori-associated gastritis and peptic ulcer disease. Gastric biopsies from H. pylori-positive and -negative patients were used to examine the in situ correlation of TNF-α and IFN-γ with epithelial cell apoptosis, bacterial load, and histological parameters of gastritis. From the same patients, we isolated H. pylori-specific T cell lines and clones and examined their ex vivo release of proinflammatory cytokines. We found a highly significant correlation of TNF-α and IFN-γ production with activity and grade of gastritis ( P < 0.01), H. pylori density ( P = 0.01), epithelial cell apoptosis ( P < 0.001), and Fas/Fas-ligand expression ( P < 0.001). T cell lines and clones were all TCR-αβ+and showed T helper 1 functional phenotype. With the use of serial histological sections, this study showed for the first time the in situ correlation of TNF-α and IFN-γ with epithelial cell apoptosis, bacterial load, and histological severity of disease and emphasizes the role of these cytokines in the pathophysiology of H. pylori-associated disease.