bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of the epithelial Ca2+channels in small intestine as studied by quantitative mRNA detection
Authors: van Abel Monique , Hoenderop Joost G. J. , van der Kemp Annemiete W. C. M. , van Leeuwen Johannes P. T. M. , Bindels René J. M.
Published: March 2015

The epithelial Ca2+channels TRPV5 and TRPV6 are localized to the brush border membrane of intestinal cells and constitute the postulated rate-limiting entry step of active Ca2+absorption. The aim of the present study was to investigate the hormonal regulation of these channels. To this end, the effect of 17β-estradiol (17β-E2), 1,25-dihydroxyvitamin D3[1,25(OH)2D3], and dietary Ca2+on the expression of the duodenal Ca2+transport proteins was investigated in vivo and analyzed using realtime quantitative PCR. Supplementation with 17β-E2increased duodenal gene expression of TRPV5 and TRPV6 but also calbindin-D9Kand plasma membrane Ca2+-ATPase (PMCA1b) in ovariectomized rats. 25-Hydroxyvitamin D3-1α-hydroxylase (1α-OHase) knockout mice are characterized by hyperparathyroidism, rickets, hypocalcemia, and undetectable levels of 1,25(OH)2D3and were used to study the 1,25(OH)2D3-dependency of the stimulatory effects of 17β-E2. Treatment with 17β-E2upregulated mRNA levels of duodenal TRPV6 in these 1α-OHase knockout mice, which was accompanied by increased serum Ca2+concentrations from 1.69 ± 0.10 to 2.03 ± 0.12 mM ( P < 0.05). In addition, high dietary Ca2+intake normalized serum Ca2+in these mice and upregulated expression of genes encoding the duodenal Ca2+transport proteins except for PMCA1b. Supplementation with 1,25(OH)2D3resulted in increased expression of TRPV6, calbindin-D9K, and PMCA1b and normalization of serum Ca2+. Expression levels of duodenal TRPV5 mRNA are below detection limits in these 1α-OHase knockout mice, but supplementation with 1,25(OH)2D3upregulated the expression to significant levels. In conclusion, TRPV5 and TRPV6 are regulated by 17β-E2and 1,25(OH)2D3, whereas dietary Ca2+is positively involved in the regulation of TRPV6 only.
Alterations in capsaicin-evoked electrolyte transport during the evolution of guinea pig TNBS ileitis
Authors: Miceli Paula , Morris Gerald P. , MacNaughton Wallace K. , Vanner Stephen
Published: March 2015

The efferent secretomotor activity of capsaicin-sensitive nerves was monitored during the evolution of 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced ileitis in the guinea pig by recording changes in short-circuit current (Δ I sc) in response to capsaicin, substance P (SP), and carbachol. Submucosal-mucosal preparations mounted in standard Ussing chambers were studied at time 0, at 8 h, and 1, 3, 5, 7, 14, and 30 days following the intraluminal instillation of TNBS or saline. Maximal Δ I scresponses to capsaicin were dramatically attenuated (54%) by 24 h. By day 7, SP- and TTX-insensitive carbachol-stimulated Δ I sc were also significantly reduced. Similar attenuation in capsaicin and carbachol responses was observed in jejunal tissue 20 cm proximal to the inflamed site at day 7. These studies demonstrate that efferent secretomotor function of capsaicin-sensitive nerves is maintained early in TNBS ileitis but significantly reduced by 24 h. By day 7, defects in enterocyte secretory function at inflamed and noninflamed sites also occurred, an effect that may be mediated by circulating cytokines.
Involvement of Rho/Rho kinase pathway in regulation of apoptosis in rat hepatic stellate cells
Authors: Ikeda Hitoshi , Nagashima Kayo , Yanase Mikio , Tomiya Tomoaki , Arai Masahiro , Inoue Yukiko , Tejima Kazuaki , Nishikawa Takako , Omata Masao , Kimura Satoshi , Fujiwara Kenji
Published: March 2015

Hepatic stellate cells (HSCs) play a central role in the development of hepatic fibrosis. Recent evidence has revealed that HSCs also play a role in its resolution, where HSC apoptosis was determined. Moreover, induction of HSC apoptosis caused a reduction of experimental hepatic fibrosis in rats. Thus knowing the mechanism of HSC apoptosis might be important to clarify the pathophysiology and establish the therapeutic strategy for hepatic fibrosis. In HSCs, Rho and Rho kinase are known to regulate contraction, migration, and proliferation with modulation of cell morphology. Controversy exists as to the participation of Rho and Rho kinase on cell survival, and little is known regarding this matter in HSCs. In this study, we directed our focus on the role of the Rho pathway in the regulation of HSC survival. C3, an inhibitor of Rho, increased histone-associated DNA fragmentation and caspase 3 activity with enhanced condensation of nuclear chromatin in rat cultured HSCs. Moreover, Y-27632, an inhibitor of Rho kinase, had the same effects, suggesting that inhibition of the Rho/Rho kinase pathway causes HSC apoptosis. On the other hand, lysophosphatidic acid, which stimulates the Rho/Rho kinase pathway, decreased histone-associated DNA fragmentation in HSCs. Inhibition of the Rho/Rho kinase pathway did not affect p53, Bcl-2, or Bax levels in HSCs. Thus we concluded that the Rho/Rho kinase pathway may play a role in the regulation of HSC survival.
Caspase 8-mediated cleavage of plectin precedes F-actin breakdown in acinar cells during pancreatitis
Authors: Beil Michael , Leser Jürgen , Lutz Manfred P. , Gukovskaya Anna , Seufferlein Thomas , Lynch Grit , Pandol Stephen J. , Adler Guido
Published: March 2015

Pancreatic acinar cells depend on the integrity of the cytoskeleton for regulated secretion. Stimulation of isolated rat pancreatic acini with the secretagogue CCK serves as a model for human acute edematous pancreatitis. It induces the breakdown of the actin filament system (F-actin) with the consecutive inhibition of secretion and premature activation of digestive enzymes. However, the mechanisms that regulate F-actin breakdown are largely unknown. Plectin is a versatile cytolinker protein regulating F-actin dynamics in fibroblasts. It was recently demonstrated that plectin is a substrate of caspase 8. In pancreatic acinar cells, plectin strongly colocalizes with apical and basolateral F-actin. Supramaximal secretory stimulation of acini with CCK leads to a rapid redistribution and activation of caspase 8, followed by degradation of plectin that in turn precedes the F-actin breakdown. Inhibition of caspase 8 before CCK hyperstimulation prevents plectin cleavage, stabilizes F-actin morphology, and reverses the inhibition of secretion. Thus we propose that the caspase 8-mediated degradation of plectin represents a critical biochemical event during CCK-induced secretory blockade and cell injury.
NSAIDs counteractH. pyloriVacA toxin-induced cell vacuolation in MKN 28 gastric mucosal cells
Authors: Ricci Vittorio , Manzo Barbara A. , Tuccillo Concetta , Boquet Patrice , Ventura Ulderico , Romano Marco , Zarrilli Raffaele
Published: March 2015

The relationship between nonsteroidal anti-inflammatory drugs (NSAIDs) and Helicobacter pylori-induced gastric mucosal injury is still under debate. VacA toxin is an important H. pylori virulence factor that causes cytoplasmic vacuolation in cultured cells. Whether and how NSAIDs affect VacA-induced cytotoxicity is unclear. This study was designed to evaluate the effect of NSAIDs on H. pylori VacA toxin-induced cell vacuolation in human gastric mucosal cells in culture (MKN 28 cell line). Our data show that 1) NSAIDs (indomethacin, aspirin, and NS-398) inhibit VacA-induced cell vacuolation independently of inhibition of cell proliferation and prostaglandin synthesis; 2) NSAIDs impair vacuole development/maintenance without affecting cell binding and internalization of VacA; and 3) NSAIDs, as well as the chloride channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid, also inhibit cell vacuolation induced by ammonia. We thus hypothesize that NSAIDs might protect MKN 28 cells against VacA-induced cytotoxicity by inhibiting VacA channel activity required for vacuole genesis.
Exposure to schistosome eggs protects mice from TNBS-induced colitis
Authors: Elliott David E. , Li Jie , Blum Arthur , Metwali Ahmed , Qadir Khurram , Urban Joseph F. , Weinstock Joel V.
Published: March 2015

Crohn's disease results from dysregulated T helper (Th)1-type mucosal inflammation. Crohn's disease is rare in tropical countries but prevalent in developed countries with temperate climates, in which its incidence rose after 1940. In contrast, exposure to helminthic parasites is common in tropical countries but is rare in developed countries. Helminthic parasites induce immunomodulatory T cell responses in the host. We hypothesize that immunomodulatory responses due to helminths may attenuate excessive Th1-type inflammation. To test that hypothesis, mice were exposed to eggs of the helminth Schistosoma mansoni and then challenged rectally with trinitrobenzesulfonic acid (TNBS) to induce colitis. Schistosome egg exposure attenuated TNBS colitis and protected mice from lethal inflammation. Schistosome egg exposure diminished IFN-γ and enhanced IL-4 production from αCD3-stimulated spleen and mesenteric lymph node cells of TNBS-treated mice. Schistosome egg exposure decreased colonic IFN-γ but increased IL-10 mRNA expression in TNBS-treated mice. Intact signal transducer and activator of transcription 6 was required for attenuation of colitis. Exposure to helminths can decrease murine colonic inflammation.
Hypoxia differentially regulates nutrient transport in rat jejunum regardless of luminal nutrient present
Authors: Kles K. A. , Tappenden K. A.
Published: March 2015

Aggressive enteral nutrition and poor intestinal perfusion are hypothesized to play an important pathogenic role in nonocclusive small bowel necrosis. This study tests the hypothesis that glucose and glutamine transport are differentially regulated during hypoxia regardless of the luminal nutrient present. Sprague-Dawley rats (247 ± 3 g; n = 16) were randomized to receive 1 h of intestinal hypoxia or serve as normoxic controls. During this hour, jejunal loops were randomized to receive in situ perfusions of mannitol, glucose, or glutamine. When compared with normoxic groups, glucose but not glutamine transport was impaired ( P < 0.001) during hypoxia. Messenger RNA abundance of the sodium glucose cotransporter sodium-dependent glucose cotransporter-1 (SGLT-1) and neutral basic amino acid transporter Bo did not differ with hypoxia or nutrient perfused. Jejunal brush-border SGLT-1 abundance was decreased ( P= 0.039) with hypoxia; however, total cellular SGLT-1 protein abundance did not differ among treatment groups. These data indicate that SGLT-1 activity is regulated during hypoxia at the posttranslational level. Additional information regarding the mechanisms regulating nutrient transport in the hypoperfused intestine is critical for optimizing the composition of enteral nutrient formulas.
Human ENS regulates the intestinal epithelial barrier permeability and a tight junction-associated protein ZO-1 via VIPergic pathways
Authors: Neunlist Michel , Toumi Férial , Oreschkova Tsvetelina , Denis Marc , Leborgne Joel , Laboisse Christian L. , Galmiche Jean-Paul , Jarry Anne
Published: March 2015

Although the enteric nervous system (ENS) has been shown to regulate various mucosal functions, its role in the physiological control of the human intestinal epithelial barrier is unknown. The aim of this study was to investigate whether the ENS is able to modulate epithelial barrier permeability and a key tight junction-associated protein, zonula occludens-1 (ZO-1). Therefore, we developed a co-culture model, consisting of human submucosa containing the submucosal neuronal network and human polarized colonic epithelial monolayers (HT29-Cl.16E or Caco-2). Submucosal neurons were activated by electrical field stimulation (EFS). Permeability was assessed by measuring the flux of paracellular permeability markers (FITC-dextran or FITC-inulin) across epithelial monolayers. Expression of ZO-1 was determined by immunofluorescence, quantitative immunoblot analysis, and real time RT-PCR. Using the coculture model, we showed that EFS of submucosal neurons resulted in a reduction in FITC-dextran or FITC-inulin fluxes, which was blocked by TTX. In HT29-Cl.16E, the effect of submucosal neuron activation was blocked by a VIP receptor antagonist (VIPra) and reproduced by VIP. Furthermore, ZO-1 expression (mRNA, protein) assessed in HT29-Cl.16E, was significantly increased after submucosal neuron activation by EFS. These effects on ZO-1 expression were blocked by TTX and VIPra and reproduced by VIP. In conclusion, our results strongly suggest a modulatory role of VIPergic submucosal neuronal pathways on intestinal epithelial barrier permeability and ZO-1 expression.
Heparin-binding EGF-like growth factor gene transcription regulated by Cdx2 in the intestinal epithelium
Authors: Uesaka Toshihiro , Lu Huimei , Katoh Osamu , Watanabe Hiromitsu
Published: March 2015

Development and differentiation of the intestinal epithelium appear to be regulated by various growth factors. Using cDNA microarrays, we identified heparin-binding EGF-like growth factor (HB-EGF) as one of the genes induced by intestinal-specific transcription factor Cdx2 in an intestinal undifferentiated rat cell line, intestinal epithetial cell (IEC)-6. Both Cdx2 and HB-EGF stimulated cell proliferation and migration, and their effects were inhibited partially by an EGF receptor-specific tyrosine kinase inhibitor, PD-153035. HB-EGF may function as one of the mediators of Cdx2 and may be associated with the proliferation and migration in the intestinal epithelium. The Cdx2 protein can bind to the Cdx2-binding element of the HB-EGF gene. Reporter gene analyses showed that the HB-EGF gene promoter is Cdx2 responsive and that the activity of the promoter in the IEC-6 cells depends on the number of consensus Cdx2-binding site-like sequences. These data indicate that HB-EGF gene expression can be regulated by Cdx2 and serves to mediate the control of Cdx2 of the proliferation and migration of IEC-6 cells.
Chloride transport in rabbit esophageal epithelial cells
Authors: Abdulnour-Nakhoul Solange , Nakhoul Nazih L. , Caymaz-Bor Canan , Orlando Roy C.
Published: March 2015

We investigated Cl− transport pathways in the apical and basolateral membranes of rabbit esophageal epithelial cells (EEC) using conventional and ion-selective microelectrodes. Intact sections of esophageal epithelium were mounted serosal or luminal side up in a modified Ussing chamber, where transepithelial potential difference and transepithelial resistance could be determined. Microelectrodes were used to measure intracellular Cl− activity (a[Formula: see text]), basolateral or apical membrane potentials ( V mBL or V mC), and the voltage divider ratio. When a basal cell was impaled, V mBL was −73 ± 4.3 mV and a[Formula: see text] was 16.4 ± 2.1 mM, which were similar in presence or absence of bicarbonate. Removal of serosal Cl−caused a transient depolarization of V mBL and a decrease in a[Formula: see text] of 6.5 ± 0.9 mM. The depolarization and the rate of decrease of a[Formula: see text] were inhibited by ∼60% in the presence of the Cl−-channel blocker flufenamate. Serosal bumetanide significantly decreased the rate of change of a[Formula: see text] on removal and readdition of serosal Cl−. When a luminal cell was impaled, V mC was −65 ± 3.6 mV and a[Formula: see text] was 16.3 ± 2.2 mM. Removal of luminal Cl− depolarized V mC and decreased a[Formula: see text] by only 2.5 ± 0.9 mM. Subsequent removal of Cl− from the serosal bath decreased a[Formula: see text]in the luminal cell by an additional 6.4 ± 1.0 mM. A plot of V mBL measurements vs. log a[Formula: see text]/log a[Formula: see text] (a[Formula: see text] is the activity of Cl− in a luminal or serosal bath) yielded a straight line [slope ( S) = 67.8 mV/decade of change in a[Formula: see text]/a[Formula: see text]]. In contrast, V mC correlated very poorly with log a[Formula: see text]/a[Formula: see text] ( S = 18.9 mV/decade of change in a[Formula: see text]/a[Formula: see text]). These results indicate that 1) in rabbit EEC, a[Formula: see text] is higher than equilibrium across apical and basolateral membranes, and this process is independent of bicarbonate; 2) the basolateral cell membrane possesses a conductive Cl− pathway sensitive to flufenamate; and 3) the apical membrane has limited permeability to Cl−, which is consistent with the limited capacity for transepithelial Cl− transport. Transport of Cl− at the basolateral membrane is likely the dominant pathway for regulation of intracellular Cl−.
VI. Pathogenesis and therapeutic approaches to human gastric dysrhythmias
Authors: Owyang Chung , Hasler William L.
Published: March 2015

This review describes recent advances in our knowledge about the pathogenesis and therapeutic approaches to human gastric dysrhythmias. A number of clinical conditions has been found to be associated with gastric slow-wave rhythm disturbances that may relate to the induction of nausea and vomiting. Human and animal studies indicate that multiple neurohumoral factors are involved in the generation of gastric dysrhythmias. Antral distension and increased intestinal delivery of lipids may cause slow-wave disruption and development of nausea. This may be mediated by cholinergic and serotonergic pathways. Similarly, progesterone and estrogen may also disrupt gastric slow-wave rhythm in susceptible individuals. Prostaglandin overproduction in gastric smooth muscle appears to mediate slow-wave disruption in diabetes and with tobacco smoking. On the other hand, central cholinergic pathways play an important role in the genesis of gastric dysrhythmias associated with motion sickness. This may be mediated by vasopressin released from the pituitary. Although it is difficult to ascribe with certainty a causative role of slow-wave rhythm disturbances in the genesis of nausea and vomiting, the search has begun for novel antiemetic therapies based on their abilities to ablate or prevent gastric dysrhythmia formation. This includes the use of prostaglandin synthesis inhibitors, central muscarinic receptor antagonists, and dopamine receptor antagonists. Finally direct gastric electrical stimulation using a surgically implanted neurostimulator has shown promise in reducing emesis in patients with gastroparesis and gastric dysrhythmias.
Primary sensory neurons: a common final pathway for inflammation in experimental pancreatitis in rats
Authors: Nathan Jaimie D. , Peng Ruth Y. , Wang Yu , McVey Douglas C. , Vigna Steven R. , Liddle Rodger A.
Published: March 2015

We hypothesized that neurogenic inflammation is a common final pathway for parenchymal inflammation in pancreatitis and evaluated the role of primary sensory neurons in secretagogue-induced and obstructive pancreatitis. Neonatal rats received either the primary sensory neuron-denervating agent capsaicin (50 mg/kg sc) or vehicle. At 8 wk of age, pancreatitis was produced by six hourly injections of caerulein (50 μg/kg ip) or by common pancreaticobiliary duct ligation (CPBDL). The severity of pancreatitis was assessed by serum amylase, pancreatic myeloperoxidase (MPO) activity, histological grading, pancreatic plasma extravasation, and wet-to-dry weight ratio. Caerulein significantly increased MPO activity and wet-to-dry weight ratio, produced histological evidence of edematous pancreatitis, induced plasma extravasation, and caused hyperamylasemia. CPBDL increased MPO activity and produced histological evidence of pancreatitis. Neonatal capsaicin administration significantly reduced tissue MPO levels, histological severity scores, and wet-to-dry weight ratio and abolished plasma extravasation. These results demonstrate that primary sensory neurons play a significant role in the inflammatory cascade in experimental pancreatitis and appear to constitute a common final pathway for pancreatic parenchymal inflammation.
Δ9-Tetrahydrocannabinol selectively acts on CB1 receptors in specific regions of dorsal vagal complex to inhibit emesis in ferrets
Authors: Van Sickle Marja D. , Oland Lorraine D. , Mackie Ken , Davison Joseph S. , Sharkey Keith A.
Published: March 2015

The aim of this study was to investigate the efficacy, receptor specificity, and site of action of Δ9-tetrahydrocannabinol (THC) as an antiemetic in the ferret. THC (0.05-1 mg/kg ip) dose-dependently inhibited the emetic actions of cisplatin. The ED50 for retching was ∼0.1 mg/kg and for vomiting was 0.05 mg/kg. A specific cannabinoid (CB)1 receptor antagonist SR-141716A (5 mg/kg ip) reversed the effect of THC, whereas the CB2 receptor antagonist SR-144528 (5 mg/kg ip) was ineffective. THC applied to the surface of the brain stem was sufficient to inhibit emesis induced by intragastric hypertonic saline. The site of action of THC in the brain stem was further assessed using Fos immunohistochemistry. Fos expression induced by cisplatin in the dorsal motor nucleus of the vagus (DMNX) and the medial subnucleus of the nucleus of the solitary tract (NTS), but not other subnuclei of the NTS, was significantly reduced by THC rostral to obex. At the level of the obex, THC reduced Fos expression in the area postrema and the dorsal subnucleus of the NTS. The highest density of CB1 receptor immunoreactivity was found in the DMNX and the medial subnucleus of the NTS. Lower densities were observed in the area postrema and dorsal subnucleus of the NTS. Caudal to obex, there was moderate density of staining in the commissural subnucleus of the NTS. These results show that THC selectively acts at CB1 receptors to reduce neuronal activation in response to emetic stimuli in specific regions of the dorsal vagal complex.
Cholecystokinin activates both A- and C-type vagal afferent neurons
Authors: Simasko Steven M. , Ritter Robert C.
Published: March 2015

Patch-clamp electrophysiological methods were used on dissociated rat nodose neurons maintained in culture to determine whether responses to cholecystokinin (CCK) were associated with capsaicin-resistant (A type) or capsaicin-sensitive (C type) neurons. Nodose neurons were classified as A or C type on the basis of the characteristics of the Na+current, a hyperpolarization-activated current, and sensitivity to a low concentration of capsaicin to ascertain the presence of vanilloid receptor 1 that has been associated with C-type neurons in sensory ganglia. It was expected that only capsaicin-sensitive C-type neurons would respond to CCK, because most vagally mediated actions of CCK are blocked by capsaicin treatment. However, we found that subpopulations of both A- and C-type neurons responded to CCK (24 and 38%, respectively). Thus some vagally mediated actions of CCK may be mediated by capsaicin insensitive A-type neurons.
Intestinal digestive resistance of immunodominant gliadin peptides
Authors: Hausch Felix , Shan Lu , Santiago Nilda A. , Gray Gary M. , Khosla Chaitan
Published: March 2015

Two recently identified immunodominant epitopes from α-gliadin account for most of the stimulatory activity of dietary gluten on intestinal and peripheral T lymphocytes in patients with celiac sprue. The proteolytic kinetics of peptides containing these epitopes were analyzed in vitro using soluble proteases from bovine and porcine pancreas and brush-border membrane vesicles from adult rat intestine. We showed that these proline-glutamine-rich epitopes are exceptionally resistant to enzymatic processing. Moreover, as estimated from the residual peptide structure and confirmed by exogeneous peptidase supplementation, dipeptidyl peptidase IV and dipeptidyl carboxypeptidase I were identified as the rate-limiting enzymes in the digestive breakdown of these peptides. A similar conclusion also emerged from analogous studies with brush-border membrane from a human intestinal biopsy. Supplementation of rat brush-border membrane with trace quantities of a bacterial prolyl endopeptidase led to the rapid destruction of the immunodominant epitopes in these peptides. These results suggest a possible enzyme therapy strategy for celiac sprue, for which the only current therapeutic option is strict exclusion of gluten-containing food.
MAPK mediates PKC-dependent contraction of cat esophageal and lower esophageal sphincter circular smooth muscle
Authors: Cao Weibiao , Sohn Uy Dong , Bitar Khalil N. , Behar Jose , Biancani Piero , Harnett Karen M.
Published: March 2015

Esophageal (ESO) circular muscle contraction and lower esophageal sphincter (LES) tone are PKC dependent. Because MAPKs may be involved in PKC-dependent contraction, we examined ERK1/ERK2 and p38 MAPKs in ESO and LES. In permeabilized LES muscle cells, ERK1/2 antibodies reduced 1,2-dioctanoylglycerol (DG)- and threshold ACh-induced contraction, which are PKC dependent, but not maximal ACh, which is calmodulin dependent. LES tone was reduced by the ERK1/2 kinase inhibitor PD-98059 and by the p38 MAPK inhibitor SB-203580. In permeable ESO cells, ACh contraction was reduced by ERK1/ERK2 and p38 MAPK antibodies and by PD-98059 and SB-203580. ACh increased MAPK activity and phosphorylation of MAPK and of p38 MAPK. The 27-kDa heat shock protein (HSP27) antibodies reduced ACh contraction. HSP27 and p38 MAPK antibodies together caused no greater inhibition than either one alone. p38 MAPK and HSP27 coprecipitated after ACh stimulation, suggesting that HSP27 is linked to p38 MAPK. These data suggest that PKC-dependent contraction in ESO and LES is mediated by the following two distinct MAPK pathways: ERK1/2 and HSP27-linked p38 MAPK.
Gastrin biosynthesis in canine G cells
Authors: Stepan Vinzenz , Sugano Kentaro , Yamada Tadataka , Park Jung , Dickinson Chris J.
Published: March 2015

Gastrin requires extensive posttranslational processing for full biological activity. It is presumed that progastrin is cleaved at pairs of basic amino acids by a prohormone convertase to form a glycine-extended intermediate (G-Gly) that serves as a substrate for peptidyl-glycine α-amidating monooxygenase (PAM), resulting in COOH-terminally amidated gastrin. To confirm the nature of progastrin processing in a primary cell line, we performed [35S]methionine-labeled pulse-chase biosynthetic experiments in canine antral G cells. Radiolabeled progastrin reached a peak earlier than observed for G-Gly or amidated gastrin. G-Gly radioactivity accumulated in G cells and preceded the appearance of radioactivity in amidated gastrin. The conversion of G-Gly to amidated gastrin was enhanced by the PAM cofactor ascorbic acid. To determine whether one member of the prohormone convertase family (PC2) was responsible for progastrin cleavage, G cells were incubated with PC2 antisense oligonucleotide probes. Cells treated with antisense probes had reduced PC2 expression, an accumulation of radiolabeled progastrin, and a delay in the formation of amidated gastrin. Progastrin in antral G cells is cleaved via PC2 to form G-Gly that is converted to amidated gastrin via the actions of PAM.
Ethanol modulates gut ischemia/reperfusion-induced liver injury in rats
Authors: Yamagishi Yoshiyuki , Horie Yoshinori , Kato Shinzo , Kajihara Mikio , Tamai Hironao , Granger D. Neil , Ishii Hiromasa
Published: March 2015

Whereas both ethanol and gut ischemia/reperfusion (I/R) are known to alter hepatic microvascular function, little is known about the influence of ethanol consumption on the hepatic microvascular responses to I/R. The objective of this study was to determine whether acute ethanol administration exacerbates the hepatic microvascular dysfunction induced by gut I/R. Rats were exposed to gut ischemia for 30 min followed by reperfusion. Intravital videomicroscopy was used to monitor leukocyte recruitment and the number of nonperfused sinusoids (NPS). Plasma alanine aminotransferase (ALT), tumor necrosis factor-α (TNF-α), and endotoxin concentrations were monitored. In separate experiments, ethanol was administered 15 min or 24 h before gut ischemia. In control rats, gut I/R increased the number of stationary leukocytes and NPS. It also elevated the plasma ALT, TNF-α, and endotoxin with a corresponding increase in intestinal mucosal permeability. Low-dose ethanol consumption 15 min before gut ischemia blunted the gut I/R-induced leukostasis and elevations in plasma TNF-α and ALT. However, high-dose ethanol consumption aggravated the gut I/R-induced increases in leukostasis and increases in plasma endotoxin and ALT. When ethanol was administered 24 h before, high-dose ethanol aggravated the gut I/R-induced hepatocellular injury, but low-dose ethanol did not have any effects on it. These results suggest that low-dose ethanol consumption shortly before gut ischemia attenuates the hepatic inflammatory responses, microvascular dysfunction, and hepatocellular injury elicited by gut I/R, whereas high-dose ethanol consumption appears to significantly aggravate these gut I/R-induced responses.
Cyclooxygenase blockade and exogenous glutamine enhance sodium absorption in infected bovine ileum
Authors: Cole Jeffrey , Blikslager Anthony , Hunt Elaine , Gookin Jody , Argenzio Robert
Published: March 2015

We have previously shown that prostanoids inhibit electroneutral sodium absorption in Cryptosporidium parvum-infected porcine ileum, whereas glutamine stimulates electroneutral sodium absorption. We postulated that glutamine would stimulate sodium absorption via a cyclooxygenase (COX)-dependent pathway. We tested this hypothesis in C. parvum-infected calves, which are the natural hosts of cryptosporidiosis. Tissues from healthy and infected calves were studied in Ussing chambers and analyzed via immunohistochemistry and Western blots. Treatment of infected tissue with selective COX inhibitors revealed that COX-1 and -2 must be blocked to restore electroneutral sodium absorption, although the transporter involved did not appear to be the expected Na+/H+exchanger 3 isoform. Glutamine addition also stimulated sodium absorption in calf tissue, but although this transport was electroneutral in healthy tissue, sodium absorption was electrogenic in infected tissue and was additive to sodium transport uncovered by COX inhibition. Blockade of both COX isoforms is necessary to release the prostaglandin-mediated inhibition of electroneutral sodium uptake in C. parvum-infected calf ileal tissue, whereas glutamine increases sodium uptake by an electrogenic mechanism in this same tissue.
Biomechanics of failed deglutitive upper esophageal sphincter relaxation in neurogenic dysphagia
Authors: Williams Rohan B. H. , Wallace Karen L. , Ali Galib N. , Cook Ian J.
Published: March 2015

Our aims were to examine the etiology and biomechanical properties of the nonrelaxing upper esophageal sphincter (UES) and the relationship between UES opening and failed relaxation. We examined the relationships among swallowed bolus volume, intrabolus pressure, sagittal UES diameter, the pharyngeal swallow response, and geniohyoid shortening in 18 patients with failed UES relaxation, 23 healthy aged controls, and 15 with Zenker's diverticulum. Etiology of failed UES relaxation was 56% medullary disease, 33% Parkinson's or extrapyramidal disease; and 11% idiopathic. Extent of UES opening ranged from absent to normal and correlated with preservation of the pharyngeal swallow response ( P = 0.012) and geniohyoid shortening ( P = 0.046). Intrabolus pressure was significantly greater compared with aged controls ( P < 0.001) or Zenker's diverticulum ( P < 0.001). The bolus volume-dependent increase in intrabolus pressure evident in controls was not observed in failed UES relaxation. The nonrelaxing UES therefore displays a constant loss of sphincter compliance throughout the full, and potentially normal, range of expansion during opening. Adequacy of UES opening is influenced by the degree of preservation of the pharyngeal swallow response and hyolaryngeal traction. In contrast, the stenotic UES displays a static loss of compliance, only apparent once the limit of sphincter expansion is reached.
Cholinergic inhibition of electrogenic sodium absorption in the guinea pig distal colon
Authors: Hayashi Hisayoshi , Suzuki Tomoko , Yamamoto Takeshi , Suzuki Yuichi
Published: March 2015

Submucosal cholinergic and noncholinergic neurons in intestines have been shown to be involved in regulating epithelial transport functions, particularly stimulating Cl−secretion. This study investigates the role of submucosal cholinergic neurons in regulating electrogenic Na+absorption in distal colon. Amiloride-sensitive short-circuit current ( Isc) and22Na+flux were measured in mucosal and mucosal-submucosal preparations mounted in Ussing chambers. In the mucosal preparation, carbachol (CCh) added to the serosal side inhibited amiloride-sensitive Iscand amiloride-sensitive22Na+absorption. The inhibitory effect of CCh was observed at ∼0.1 μM, and maximum inhibition of ∼70% was attained at ∼30 μM (IC50= ∼1 μM). CCh-induced inhibition of amiloride-sensitive Iscwas almost totally abolished by 10 μM atropine. Treatment of the tissue with ionomycin markedly reduced amiloride-sensitive Isc, but a subsequent addition of CCh further decreased it. Also, CCh still had an inhibitory effect, although significantly attenuated, after the tissue had been incubated with a low-Ca2+solution containing ionomycin and BAPTA-AM. Applying electrical field stimulation to submucosal neurons in the mucosal-submucosal preparation resulted in inhibition of amiloride-sensitive Isc, ∼33% of this inhibition being atropine sensitive. Physostigmine inhibited amiloride-sensitive Isc, this effect being abolished by atropine. In conclusion, submucosal cholinergic and noncholinergic neurons were involved in inhibiting electrogenic Na+absorption in colon. This inhibition by cholinergic neurons was mediated by muscarinic receptor activation.
Animal model for angiotensin II effects in the internal anal sphincter smooth muscle: mechanism of action
Authors: Fan Ya-Ping , Puri Rajinder N. , Rattan Satish
Published: March 2015

Effect of ANG II was investigated in in vitro smooth muscle strips and in isolated smooth muscle cells (SMC). Among different species, rat internal and sphincter (IAS) smooth muscle showed significant and reproducible contraction that remained unmodified by different neurohumoral inhibitors. The AT1antagonist losartan but not AT2antagonist PD-123319 antagonized ANG II-induced contraction of the IAS smooth muscle and SMC. ANG II-induced contraction of rat IAS smooth muscle and SMC was attenuated by tyrosine kinase inhibitors genistein and tyrphostin, protein kinase C (PKC) inhibitor H-7, Ca2+channel blocker nicardipine, Rho kinase inhibitor Y-27632 or p44/42mitogen-activating protein kinase (MAPK44/42) inhibitor PD-98059. Combinations of nicardipine and H-7, Y-27632, and PD-98059 caused further attenuation of the ANG II effects. Western blot analyses revealed the presence of both AT1and AT2receptors. We conclude that ANG II causes contraction of rat IAS smooth muscle by the activation of AT1receptors at the SMC and involves multiple intracellular pathways, influx of Ca2+, and activation of PKC, Rho kinase, and MAPK44/42.
Induction of short heterodimer partner 1 precedes downregulation of Ntcp in bile duct-ligated mice
Authors: Zollner Gernot , Fickert Peter , Silbert Dagmar , Fuchsbichler Andrea , Stumptner Conny , Zatloukal Kurt , Denk Helmut , Trauner Michael
Published: March 2015

Cholestasis is associated with retention of bile acids and reduced expression of the Na+/taurocholate cotransporter (Ntcp), the major hepatocellular bile acid uptake system. This study aimed to determine whether downregulation of Ntcp in obstructive cholestasis 1) is a consequence of bile acid retention and 2) is mediated by induction of the transcriptional repressor short heterodimer partner 1 (SHP-1). To study the time course for the changes in serum bile acid levels as well as SHP-1 and Ntcp steady-state mRNA levels, mice were subjected to common bile duct ligation (CBDL) for 3, 6, 12, 24, 72, and 168 h and compared with sham-operated controls. Serum bile acid levels were determined by radioimmunoassay. SHP-1 and Ntcp steady-state mRNA expression were assessed by Northern blotting. In addition, Ntcp protein expression was studied by Western blotting and immunofluorescence microscopy. Increased SHP-1 mRNA expression paralleled elevations of serum bile acid levels and was followed by downregulation of Ntcp mRNA and protein expression in CBDL mice. Maximal SHP-1 mRNA expression reached a plateau phase after 6-h CBDL (12-fold; P < 0.001) and preceded the nadir of Ntcp mRNA levels (12%, P < 0.001) by 6 h. In conclusion, bile acid-induced expression of SHP-1 may, at least in part, mediate downregulation of Ntcp in CBDL mice. These findings support the concept that downregulation of Ntcp in cholestasis limits intracytoplasmatic accumulation of potentially toxic bile acids.
Expression and localization of organic cation/carnitine transporter OCTN2 in Caco-2 cells
Authors: Elimrani Ihsan , Lahjouji Karim , Seidman Ernest , Roy Marie-Josée , Mitchell Grant A. , Qureshi Ijaz
Published: March 2015

l-Carnitine is derived both from dietary sources and biosynthesis. Dietary carnitine is absorbed in the small intestine and then distributed to other organs. Previous studies using Caco-2 cells demonstrated that the transport ofl-carnitine in the intestine involves a carrier-mediated system. The purpose of this study was to determine whether the uptake of l-carnitine in Caco-2 cells is mediated by the recently identified organic cation/carnitine transporter (OCTN2). Kinetics ofl-[3H]carnitine uptake were investigated with or without specific inhibitors. l-Carnitine uptake in mature cells was sodium dependent and linear with time. Kmand Vmaxvalues for saturable uptake were 14.07 ± 1.70 μM and 26.3 ± 0.80 pmol · mg protein−1· 6 min−1, respectively. l-carnitine uptake was inhibited ( P < 0.05–0.01) by valproate and other organic cations. Anti-OCTN2 antibodies recognized a protein in the brush-border membrane (BBM) of Caco-2 cells with an apparent molecular mass of 60 kDa. The OCTN2 expression was confirmed by double immunostaining. Our results demonstrate that l-carnitine uptake in differentiated Caco-2 cells is primarily mediated by OCTN2, located on the BBM.
Role of ICAM-1 in chronic hepatic allograft rejection in the rat
Authors: Wong John , Kubes Paul , Zhang Yikun , Li Yang , Urbanski Stefan J. , Bennett C. Frank , Lee Samuel S.
Published: March 2015

The pathogenesis of hepatic allograft rejection remains unclear. We aimed to clarify the early role of intercellular adhesion molecule-1 (ICAM-1)-mediated cell recruitment in chronic hepatic rejection. Liver transplantation was performed from Lewis to Lewis rats (isograft controls) and from Lewis to Brown Norway rats (allograft rejection group). The allografted rats were treated with either ICAM-1 antisense oligonucleotides (10 mg · kg−1· day−1× 6 days ip) or a control preparation (either ICAM-1 missense oligonucleotide or normal saline). Hepatic leukocyte recruitment in vivo was studied on day 6 by using intravital microscopy. Liver histology, biochemistry, and survival rates were also examined. Leukocyte adhesion in terminal hepatic venules was significantly increased in the rejection group compared with isograft controls. Antisense ICAM-1 in the allografted group effectively reduced leukocyte adhesion. Histology and liver chemistry were less deranged in the antisense-treated groups compared with control-treated allografted rats. In the allograft groups, survival was significantly prolonged in the antisense-treated rats (42.3 ± 1.2 days) compared with the controls (25.2 ± 2.7 days). These results showed that early leukocyte recruitment in the hepatic microvasculature of rejecting allografts is ICAM-1 dependent and suggest that impacting on early cell recruitment can significantly ameliorate chronic rejection.
Neonatal maternal separation alters stress-induced responses to viscerosomatic nociceptive stimuli in rat
Authors: Coutinho S. V. , Plotsky P. M. , Sablad M. , Miller J. C. , Zhou H. , Bayati A. I , McRoberts J. A. , Mayer E. A.
Published: March 2015

This study investigated the combined effect of neonatal maternal separation and acute psychological stress on pain responses in adult rats. Long-Evans dams and their male pups were reared under two conditions: 1) 180 min daily maternal separation (MS180) on postnatal days 2–14 or 2) no handling or separation (NH). At 2 mo of age, visceromotor responses to graded intensities of phasic colorectal distension (10–80 mmHg) at baseline as well as following acute 60 min water avoidance stress (WA) were significantly higher in MS180 rats. Both groups showed similar stress-induced visceral hyperalgesia in the presence of naloxone (20 mg/kg ip). MS180 rats had smaller stress-induced cutaneous analgesia in the tail-flick test compared with NH rats, with a residual naloxone-resistant component. MS180 rats showed an enhanced fecal pellet output following WA or exposure to a novel environment. These data suggest that early life events predispose adult Long-Evans rats to develop visceral hyperalgesia, reduced somatic analgesia, and increased colonic motility in response to an acute psychological stressor, mimicking the cardinal features of irritable bowel syndrome.
Acid infusion enhances duodenal mechanosensitivity in healthy subjects
Authors: Simrén Magnus , Vos Rita , Janssens Jozef , Tack Jan
Published: March 2015

Duodenal acid has been suggested to be of importance for dyspeptic symptoms. We investigated the effects of acid on duodenal mechanosensitivity and antroduodenal motility in 10 healthy subjects before and during duodenal infusion of acid (0.1 N HCl) or water by using a combined barostat-manometry assembly. During acid infusion, increased sensitivity to balloon distension was seen, with reduced perception ( P = 0.04) and discomfort thresholds ( P = 0.06) and higher intensity of discomfort ( P = 0.02) compared with water. Higher balloon volumes were seen during acid infusion, indicating decreased tone ( P = 0.05). Large volume waves were more prevalent during acid than water infusion ( P = 0.009). The acid infusion suppressed antral contractions ( P = 0.04) and increased the number of contractions in the proximal duodenum ( P = 0.02) compared with before the infusion. In conclusion, duodenal acid enhances mechanical sensitivity in the duodenum, affects gastroduodenal motor function, and might be of importance for dyspeptic symptoms.
Total parenteral nutrition adversely affects gut barrier function in neonatal piglets
Authors: Kansagra Ketan , Stoll Barbara , Rognerud Cheryl , Niinikoski Harri , Ou Ching-Nan , Harvey Roger , Burrin Douglas
Published: March 2015

Sepsis is the most common morbidity in preterm infants, who often receive total parenteral nutrition (TPN). We hypothesized that gut barrier function is compromised in TPN-fed compared with enterally fed newborn piglets (ENT pigs). Colostrum-deprived newborn pigs were implanted with jugular venous and bladder catheters under general anesthesia. Pigs were either administered TPN ( n = 15) or fed formula (ENT pigs, n = 15). After 6 days, pigs were gavaged a solution of mannitol, lactulose, and polyethylene glycol 4000 (PEG 4000) and urine was collected for 24 h. At 7 days, small bowel samples were assayed for myeloperoxidase activity, morphometry, and tight junction protein abundance. Intestinal contents and peripheral organ sites were cultured for bacteria. Urinary recovery (%dose) of mannitol (53 vs. 68) was lower, whereas that of lactulose (2.93 vs. 0.18) and PEG 4000 (12.78 vs. 0.96) were higher in TPN vs. ENT pigs, respectively ( P < 0.05). Incidence of translocation was similar in TPN and ENT pigs. Myeloperoxidase activity was increased in TPN vs. ENT pigs in the jejunum ( P < 0.001) and was weakly correlated with lactulose ( R2= 0.32) and PEG 4000 ( R2= 0.38) recovery. Goblet cell counts did not change, but intraepithelial lymphocyte numbers decreased with TPN. Only claudin-1 protein abundance was increased in the TPN group. We conclude that TPN is associated with impairment of neonatal gut barrier function as measured by permeability but not translocation.
MCP-1 causes leukocyte recruitment and subsequently endotoxemic ileus in rat
Authors: Türler Andreas , Schwarz Nicolas T. , Türler Esther , Kalff Jörg C. , Bauer Anthony J.
Published: March 2015

Endotoxemia causes an inflammatory response within the intestinal muscularis and gastrointestinal dysmotility. We hypothesize that the resident macrophage-derived chemokine monocyte chemoattractant protein-1 (MCP-1) plays a significant role in the recruitment of leukocytes into the lipopolysaccharide (LPS)-stimulated rat intestinal muscularis. MCP-1 mRNA expression was investigated by RT-PCR. Leukocyte extravasation and MCP-1 protein localization were determined by immunohistochemistry. Contractile activity was assessed by using a standard organ bath in rats that were treated with saline, recombinant MCP-1, LPS, LPS + nonspecific antibody, or LPS + MCP-1 antibody. Endotoxemia caused a significant 280-fold increase in MCP-1 mRNA expression in the muscularis, peaking at 3 h. MCP-1 protein was immunohistochemically located to muscularis macrophages. LPS application caused significant leukocyte recruitment into the muscularis and a 51% decrease in muscle contractility. MCP-1 antibody treatment significantly averted leukocyte recruitment and significantly prevented muscle dysfunction. These parameters were not significantly altered by the nonspecific antibody. Results show that resident muscularis macrophage-derived MCP-1 plays a major role in the recruitment of monocytes during endotoxemia, which then subsequently secrete kinetically active substances that cause ileus.
Selective effects of serotonergic psychoactive agents on gastrointestinal functions in health
Authors: Chial Heather J. , Camilleri Michael , Burton Duane , Thomforde George , Olden Kevin W. , Stephens Debra
Published: March 2015

This study evaluated the effects of serotonergic psychoactive agents on gastrointestinal functions in healthy human subjects. Participants received one of four regimens in a randomized, double-blind manner: buspirone, a 5-HT1Areceptor agonist (10 mg twice daily); paroxetine, a selective serotonin reuptake inhibitor (20 mg daily); venlafaxine-XR, a selective serotonin and norepinephrine reuptake inhibitor (75 mg daily); or placebo for 11 days. Physiological testing performed on days 8–11included scintigraphic assessment of gastrointestinal and colonic transit, the nutrient drink test, and assessment of the postprandial change in gastric volume. Fifty-one healthy adults (40 females, 11 males) participated in this study. No effects on gastric emptying or colonic transit were identified with any agent. Small bowel transit of a solid meal was accelerated by paroxetine. Buspirone decreased postprandial aggregate symptom and nausea scores. Venlafaxine-XR increased the postprandial change in gastric volume. Buspirone, paroxetine, and venlafaxine-XR affect upper gastrointestinal functions in healthy humans. These data support the need for clinical and physiological studies of these agents in functional gastrointestinal disorders.
Chelerythrine stimulates GSH transport by rat Mrp2 (Abcc2) expressed in canine kidney cells
Authors: Lou Huan , Ookhtens Murad , Stolz Andrew , Kaplowitz Neil
Published: March 2015

Rat multidrug resistant protein 2 (Mrp2; Abcc2), an ATP-driven pump located on the canalicular domain of hepatocytes, exports glutathione S-conjugates (GS-X) and GSH among its wide variety of substrates. Previous studies have shown that chelerythrine (CHEL), a quaternary benzophenanthridine cation, reacts with GSH to form a reversible adduct under physiological conditions. Here we report that CHEL can strongly stimulate GSH efflux by Mrp2, when it is constitutively expressed in polarized canine kidney cells, thereby leading to the depletion of cellular GSH. Transepithelial transport experiments indicate that Mrp2 transports GSH and CHEL with a 1:1 stoichiometry, which can be readily inhibited by GS-bimane, a GS-X substrate for Mrp2. Moreover, CHEL can block Mrp2-mediated leukotriene C4uptake by membrane vesicles with an IC50≈ 100 μM in the presence of GSH, but not S-methyl GSH or ophthalmic acid. Thus the thiol group of GSH is required for inhibition of Mrp2 in the presence of CHEL. Our results suggest that CHEL stimulates GSH efflux by forming a reversible GS-CHEL adduct, which is transported by Mrp2 and dissociates extracellularly.