bluuebunny/crossref_metadata_2025 huggingface dataset:
Apoptosis in activated rat pancreatic stellate cells
Authors: Klonowski-Stumpe Hanne , Fischer Richard , Reinehr Roland , Lüthen Reinhard , Häussinger Dieter
Published: March 2015

Proliferation and matrix synthesis by activated pancreatic stellate cells (PSC) participate in the development of chronic pancreatitis. Apoptosis of PSC may terminate this process but has not yet been studied in this particular cell type and was the aim of the present study. PSC were isolated from rat pancreas and characterized for expression of glial fibrillary acidic protein, α-smooth muscle actin, CD95, and tumor necrosis factor-α-related apoptosis-inducing ligand (TRAIL) receptors. Apoptosis was determined by TdT-UTP nick end-labeling reaction, annexin V binding, and caspase-8 activation. Both CD95L and TRAIL induced apoptosis in PSC. The apoptotic response was minor in PSC cultured for 7 days but increased markedly thereafter. Sensitization of PSC with culture duration was accompanied by increased expression of CD95 and TRAIL receptor 2 and no alterations of Flip expression or protein kinase B phosphorylation but was paralleled by the appearance of a COOH-terminal cleavage product of receptor-interacting protein. PSC apoptosis was also induced by PK-11195, a ligand of the peripheral benzodiazepine receptor. PSC apoptosis may be important in terminating the wound-healing response after pancreas injury and exhibits features distinct from apoptosis induction in hepatic stellate cells.
Differential transcriptional expression of Ca2+BP superfamilies in murine gastrointestinal smooth muscles
Authors: Ohya Susumu , Horowitz Burton
Published: March 2015

Calmodulin (Cal) plays important roles for contractile activity in smooth muscles. Recently, two distinct Ca2+-binding protein superfamilies with sequence similarities to Cal have been identified in neuronal cells: neuronal Ca2+-binding proteins (NCBPs) and Cal-like Ca2+-binding proteins (CaBPs). Some NCBPs and CaBPs play significant roles for Ca2+-dependent cellular signaling in the nervous system. In gastrointestinal smooth muscles (GISMs), Cal functions as the regulator of contractile behavior and electrical rhythmicity. However, the molecular identification of NCBPs and CaBPs has not been elucidated in GISMs. Here, we have identified NCBPs and CaBPs expressed in GISMs and determined the expression levels of their transcripts by quantitative RT-PCR. Of 12 NCBPs, the transcripts for neuronal Ca2+sensor 1, neural visinin-like proteins 1, 2, and 3, and K+channel-interacting proteins 1 and 3 were detected in proximal colon, gastric fundus, gastric antrum, and jejunum. On the other hand, of seven CaBPs including alternatively spliced variants, only CaBP1L transcripts were detected in GISMs.
Local, exendin-(9—39)-insensitive, site of action of GLP-1 in canine ileum
Authors: Daniel E. E. , Anvari M. , Fox-Threlkeld J. E. T. , McDonald T. J.
Published: March 2015

Glucagon-like peptide-1 (GLP-1) modulates glucose levels following a meal, including by inhibition of gastric emptying and intestinal transport. Intra-arterial injection of GLP-1 into the gastric corpus, antrum, or pylorus of anesthetized dogs had no effect on the contractile activity of the resting or neurally activated stomach. GLP-1 injected intra-arterially inhibited intestinal segments when activated by enteric nerve stimulation but not by acetylcholine. Isolated ileum segments were perfused intra-arterially, instrumented with strain gauges to record circular muscle activity and with subserosal electrodes to stimulate enteric nerves. GLP-1 caused concentration-dependent inhibition of nerve-stimulated phasic but not tonic activity. This was absent during TTX-induced activity and partly prevented by N G-nitro-l-arginine. Exendin-(9—39), the GLP-1 antagonist, had no intrinsic activity and did not affect the actions of GLP-1. Capsaicin mimicked the effects of GLP-1 and may have reduced the effect of subsequent GLP-1. GLP-1 may mediate paracrine action on afferent nerves in the canine ileal mucosa using an unusual receptor.
CRHSP-24 phosphorylation is regulated by multiple signaling pathways in pancreatic acinar cells
Authors: Schäfer Claus , Steffen Hanna , Krzykowski Karen J. , Göke Burkhard , Groblewski Guy E.
Published: March 2015

Ca2+-regulated heat-stable protein of 24 kDa (CRHSP-24) is a serine phosphoprotein originally identified as a physiological substrate for the Ca2+-calmodulin regulated protein phosphatase calcineurin (PP2B). CRHSP-24 is a paralog of the brain-specific mRNA-binding protein PIPPin and was recently shown to interact with the STYX/dead phosphatase protein in developing spermatids (Wishart MJ and Dixon JE. Proc Natl Acad Sci USA 99: 2112–2117, 2002). Investigation of the effects of phorbol ester (12- o-tetradecanoylphorbol-13-acetate; TPA) and cAMP analogs in32P-labeled pancreatic acini revealed that these agents acutely dephosphorylated CRHSP-24 by a Ca2+-independent mechanism. Indeed, cAMP- and TPA-mediated dephosphorylation of CRHSP-24 was fully inhibited by the PP1/PP2A inhibitor calyculin A, indicating that the protein is regulated by an additional phosphatase other than PP2B. Supporting this, CRHSP-24 dephosphorylation in response to the Ca2+-mobilizing hormone cholecystokinin was differentially inhibited by calyculin A and the PP2B-selective inhibitor cyclosporin A. Stimulation of acini with secretin, a secretagogue that signals through the cAMP pathway in acini, induced CRHSP-24 dephosphorylation in a concentration-dependent manner. Isoelectric focusing and immunoblotting indicated that elevated cellular Ca2+dephosphorylated CRHSP-24 on at least three serine sites, whereas cAMP and TPA partially dephosphorylated the protein on at least two sites. The cAMP-mediated dephosphorylation of CRHSP-24 was inhibited by low concentrations of okadaic acid (10 nM) and fostriecin (1 μM), suggesting that CRHSP-24 is regulated by PP2A or PP4. Collectively, these data indicate that CRHSP-24 is regulated by diverse and physiologically relevant signaling pathways in acinar cells, including Ca2+, cAMP, and diacylglycerol.
PI3K signaling is required for prostaglandin-induced mucosal recovery in ischemia-injured porcine ileum
Authors: Little Dianne , Dean Rebecca A. , Young Karen M. , McKane Shaun A. , Martin Linda D. , Jones Samuel L. , Blikslager Anthony T.
Published: March 2015

We have previously shown that PGE2and PGI2induce recovery of transepithelial resistance (TER) in ischemia-injured porcine ileal mucosa, associated with initial increases in Cl−secretion. We believe that the latter generates an osmotic gradient that stimulates resealing of tight junctions. Because of evidence implicating phosphatidylinositol 3-kinase (PI3K) in regulating tight junction assembly, we postulated that this signaling pathway is involved in PG-induced mucosal recovery. Porcine ileum was subjected to 45 min of ischemia, after which TER was monitored for a 180-min recovery period. Endogenous PG production was inhibited with indomethacin (5 μM). PGE2(1 μM) and PGI2(1 μM) stimulated recovery of TER, which was inhibited by serosal application of the osmotic agent urea (300 mosmol/kgH2O). The PI3K inhibitor wortmannin (10 nM) blocked recovery of TER in response to PGs or mucosal urea. Immunofluorescence imaging of recovering epithelium revealed that PGs restored occludin and zonula occludens-1 distribution to interepithelial junctions, and this pattern was disrupted by pretreatment with wortmannin. These experiments suggest that PGs stimulate recovery of paracellular resistance via a mechanism involving transepithelial osmotic gradients and PI3K-dependent restoration of tight junction protein distribution.
Colocalization of the apical Cl−/HCO 3 − exchanger PAT1 and gastric H-K-ATPase in stomach parietal cells
Authors: Petrovic Snezana , Wang Zhaohui , Ma Liyun , Seidler Ursula , Forte John G. , Shull Gary E. , Soleimani Manoocher
Published: March 2015

The apical Cl−/HCO[Formula: see text] exchanger called the putative anion transporter (PAT1; SLC26A6) is expressed on apical membranes of villus cells in the duodenum, but its location in the stomach remains unknown. Here we examined the cell distribution and membrane location of PAT1 in mouse stomach. Immunofluorescence labeling studies with anti-PAT1 antibodies and Dolichos biflorusagglutinin indicated the exclusive expression of PAT1 in gastric parietal cells. Double immunocytochemical staining revealed colocalization of PAT1 with the gastric H-K-ATPase, consistent with expression in tubulovesicles and/or the secretory canaliculus. Radiolabeled36Cl flux studies demonstrated the functional presence of Cl−/HCO[Formula: see text] exchange in purified tubulovesicles of parietal cells. The expression of PAT1 was significantly decreased in parietal cells of gastric H-K-ATPase-null mice, which exhibit a sharp reduction in tubulovesicle membranes. These data indicate that the Cl−/HCO[Formula: see text]exchanger PAT1 is localized on tubulovesicular membranes, and they are consistent with the hypothesis that it functions in the maintenance of intravesicular ion concentrations in the resting state and dehydration of vesicles derived from the secretory membranes following the transition from the stimulated to the resting state.
N ω-nitro-l-arginine inhibits inducible HSP-70 via Ca2+, PKC, and PKA in human intestinal epithelial T84 cells
Authors: Kiang Juliann G. , Kiang Sharon C. , Juang Yuang-Taung , Tsokos George C.
Published: March 2015

The nitric oxide (NO) synthase inhibitor N ω-nitro-l-arginine (l-NNA) inhibits heat stress (HS)-induced NO production and the inducible 70-kDa heat shock protein (HSP-70i) in many rodent organs. We used human intestinal epithelial T84 cells to characterize the inhibitory effect of l-NNA on HS-induced HSP-70i expression. Intracellular Ca2+ concentration ([Ca2+]i) was measured using fura-2, and protein kinase C (PKC), and PKA activities were determined. HS increased HSP-70i mRNA and protein in T84 cells exposed to 45°C for 10 min and allowed to recover for 6 h. l-NNA treatment for 1 h before HS inhibited the induction of HSP-70i mRNA and protein, with an IC50 of 0.0471 ± 0.0007 μM. Because the HS-induced increase in HSP-70i mRNA and protein is Ca2+ dependent, we measured [Ca2+]i after treating cells withl-NNA. l-NNA at 100 μM significantly decreased resting [Ca2+]i. Likewise, treatment with 1 μM GF-109203X or H-89 (inhibitors of PKC and PKA, respectively) for 30 min also significantly decreased [Ca2+]i and inhibited HS-induced increase in HSP-70i. GF-109203X- or H-89-treated cells failed to respond tol-NNA by further decreasing [Ca2+]i and HSP-70i. l-NNA effectively blocked heat shock factor-1 (HSF1) translocation from the cytosol to the nucleus, a process requiring PKC phosphorylation. These results suggest that l-NNA inhibits HSP-70i by reducing [Ca2+]i and decreasing PKC and PKA activity, thereby blocking HSF1 translocation from the cytosol to the nucleus.
Critical role for NHE1 in intracellular pH regulation in pancreatic acinar cells
Authors: Brown David A. , Melvin James E. , Yule David I.
Published: March 2015

The primary function of pancreatic acinar cells is to secrete digestive enzymes together with a NaCl-rich primary fluid which is later greatly supplemented and modified by the pancreatic duct. A Na+/H+exchanger(s) [NHE(s)] is proposed to be integral in the process of fluid secretion both in terms of the transcellular flux of Na+and intracellular pH (pHi) regulation. Multiple NHE isoforms have been identified in pancreatic tissue, but little is known about their individual functions in acinar cells. The Na+/H+exchange inhibitor 5-( N-ethyl- N-isopropyl) amiloride completely blocked pHirecovery after an NH4Cl-induced acid challenge, confirming a general role for NHE in pHiregulation. The targeted disruption of the Nhe1 gene also completely abolished pHirecovery from an acid load in pancreatic acini in both [Formula: see text]-containing and [Formula: see text]-free solutions. In contrast, the disruption of either Nhe2 or Nhe3 had no effect on pHirecovery. In addition, NHE1 activity was upregulated in response to muscarinic stimulation in wild-type mice but not in NHE1-deficient mice. Fluctuations in pHicould potentially have major effects on Ca2+signaling following secretagogue stimulation; however, the targeted disruption of Nhe1 was found to have no significant effect on intracellular Ca2+homeostasis. These data demonstrate that NHE1 is the major regulator of pHiin both resting and muscarinic agonist-stimulated pancreatic acinar cells.
Physiological effects of enteral and parenteral feeding on pancreaticobiliary secretion in humans
Authors: O'Keefe Stephen J. D. , Lee Ronzo B. , Anderson Frank P. , Gennings Chris , Abou-Assi Souheil , Clore John , Heuman Douglas , Chey William
Published: March 2015

In the nutritional management of digestive disorders, it is important to know the relative secretory and metabolic responses to enteral and parenteral feeding. Twenty-seven healthy volunteers were studied while receiving either oral drinks or duodenal infusions of a complex formula diet, duodenal or intravenous infusions of elemental (protein as free amino acids, low fat) formulae, or saline. Pancreaticobiliary secretory responses were measured by nasoduodenal polyethylene glycol perfusion and aspiration, while monitoring blood hormone and nutrient levels. Diets were matched for protein (1.5 g · kg−1· d−1) and energy (40 kcal · kg−1· d−1). Compared with placebo, all oroenteral diets stimulated amylase, lipase, trypsin, and bile acid secretion and increased plasma concentrations of gastrin and cholecystokinin, whereas intravenous feeding did not. The complex formula produced a similar response whether given as drinks or duodenal infusions. Changing the duodenal formula to elemental reduced enzyme secretion by 50%, independently of CCK. Higher increases in plasma insulin, glucose, and amino acids were noted with intravenous feeding. Delivering food directly to the intestine by a feeding tube does not reduce pancreaticobiliary secretion. Enteral “elemental” formulae diminish, but only intravenous feeding avoids pancreatic stimulation. Intravenous administration impairs metabolic clearance.
Effect of solid meal on gastric emptying of, and glycemic and cardiovascular responses to, liquid glucose in older subjects
Authors: Berry Melanie K. , Russo Antonietta , Wishart Judith M. , Tonkin Anne , Horowitz Michael , Jones Karen L.
Published: March 2015

Gastric emptying is a determinant of the postprandial glycemic and cardiovascular responses to oral carbohydrate. We evaluated the effects of a solid meal on gastric emptying and the glycemic and cardiovascular responses to oral glucose in healthy older subjects. Ten subjects aged 72.1 ± 1.9 yr were studied. Each subject had measurements of gastric emptying, blood glucose, serum insulin, blood pressure, and heart rate after ingestion of a 50-g glucose drink (300 ml) with (mixed meal) or without (liquid only) a solid meal (300 g ground beef). Gastric emptying of liquid was initially slightly more rapid ( P < 0.05) after the mixed meal compared with liquid only at 5 min (92.0 ± 1.5 vs. 96.0 ± 1.3%) and much slower ( P < 0.05) after 120 min. The time to peak blood glucose was less (39.0 ± 4.0 vs. 67.5 ± 10.3 min; P < 0.01) and blood glucose subsequently lower ( P < 0.01) after the mixed meal. The increase in serum insulin was greater ( P < 0.001) after the mixed meal. Blood pressure fell ( P < 0.05) in the first 30 min, with no difference between the two meals. Increase in heart rate after both meals ( P < 0.005), was greater ( P< 0.05) after the mixed meal. The presence of a noncarbohydrate solid meal had discrepant effects on early and subsequent emptying of a nutrient liquid, which affects postprandial glycemia and increased heart rate.
Regulation and function of COX-2 gene expression in isolated gastric parietal cells
Authors: Pausawasdi Nonthalee , Ramamoorthy Saravanan , Crofford Leslie J. , Askari Frederick K. , Todisco Andrea
Published: March 2015

We examined expression, function, and regulation of the cyclooxygenase (COX)-2 gene in gastric parietal cells. COX-2-specific mRNA was isolated from purified (>95%) canine gastric parietal cells in primary culture and measured by Northern blots using a human COX-2 cDNA probe. Carbachol was the most potent inducer of COX-2 gene expression. Gastrin and histamine exhibited minor stimulatory effects. Carbachol-stimulated expression was inhibited by intracellular Ca2+chelator 1,2-bis(2-aminophenoxy)ethane- N,N,N′,N′-tetraacetic acid-AM (90%), protein kinase C (PKC) inhibitor GF-109203X (48%), and p38 kinase inhibitor SB-203580 (48%). Nuclear factor (NF)-κB inhibitor 1-pyrrolidinecarbodithioic acid inhibited carbachol-stimulated expression by 80%. Similar results were observed in the presence of adenoviral vector Ad.dom.neg.IκB, which expresses a repressor of NF-κB. Addition of SB-203580 with Ad.dom.neg.IκB almost completely blocked carbachol stimulation of COX-2 gene expression. We examined the effect of carbachol on PGE2release by enzyme-linked immunoassay. Carbachol induced PGE2 release. Ad.dom.neg.IκB, alone or with SB-203580, produced, respectively, partial (70%) and almost complete (>80%) inhibition of carbachol-stimulated PGE2 production. Selective COX-2 inhibitor NS-398 blocked carbachol-stimulated PGE2 release without affecting basal PGE2production. In contrast, indomethacin inhibited both basal and carbachol-stimulated PGE2 release. Carbachol induces COX-2 gene expression in the parietal cells through signaling pathways that involve intracellular Ca2+, PKC, p38 kinase, and activation of NF-κB. The functional significance of these effects seems to be stimulation of PGE2 release.
Effects of ginger on motion sickness and gastric slow-wave dysrhythmias induced by circular vection
Authors: Lien Han-Chung , Sun Wei Ming , Chen Yen-Hsueh , Kim Hyerang , Hasler William , Owyang Chung
Published: March 2015

Ginger has long been used as an alternative medication to prevent motion sickness. The mechanism of its action, however, is unknown. We hypothesize that ginger ameliorates the nausea associated with motion sickness by preventing the development of gastric dysrhythmias and the elevation of plasma vasopressin. Thirteen volunteers with a history of motion sickness underwent circular vection, during which nausea (scored 0–3, i.e., none to severe), electrogastrographic recordings, and plasma vasopressin levels were assessed with or without ginger pretreatment in a crossover-design, double-blind, randomized placebo-controlled study. Circular vection induced a maximal nausea score of 2.5 ± 0.2 and increased tachygastric activity and plasma vasopressin. Pretreatment with ginger (1,000 and 2,000 mg) reduced the nausea, tachygastria, and plasma vasopressin. Ginger also prolonged the latency before nausea onset and shortened the recovery time after vection cessation. Intravenous vasopressin infusion at 0.1 and 0.2 U/min induced nausea and increased bradygastric activity; ginger pretreatment (2,000 mg) affected neither. Ginger effectively reduces nausea, tachygastric activity, and vasopressin release induced by circular vection. In this manner, ginger may act as a novel agent in the prevention and treatment of motion sickness.
Demonstration of a functional apical sodium hydrogen exchanger in isolated rat gastric glands
Authors: Kirchhoff Philipp , Wagner Carsten A. , Gaetzschmann Florian , Radebold Klaus , Geibel John P.
Published: March 2015

Previous studies have shown that gastric glands express at least sodium-hydrogen exchanger (NHE) isoforms 1-4. Our aim was to study NHE-3 localization in rat parietal cells and to investigate the functional activity of an apical membrane NHE-3 isoform in parietal cells of rats. Western blot analysis and immunohistochemistry showed expression of NHE-3 in rat stomach colocalizing the protein in parietal cells together with the β-subunit of the H+-K+-ATPase. Functional studies in luminally perfused gastric glands demonstrated the presence of an apical NHE isoform sensitive to low concentrations of 5-ethylisopropyl amiloride (EIPA). Intracellular pH measurements in parietal cells conducted in omeprazole-pretreated superfused gastric glands showed an Na+-dependent proton extrusion pathway that was inhibited both by low concentrations of EIPA and by the NHE-3 specific inhibitor S3226. This pathway for proton extrusion had a higher activity in resting glands and was inhibited on stimulation of histamine-induced H+-K+-ATPase proton extrusion. We conclude that the NHE-3 isoform located on the apical membrane of parietal cells offers an additional pathway for proton secretion under resting conditions. Furthermore, the gastric NHE-3 appears to work under resting conditions and inactivates during periods of H+-K+-ATPase activity.
Impaired Ras membrane association and activation in PPARα knockout mice after partial hepatectomy
Authors: Wheeler Michael D. , Smutney Olivia M. , Check Jennifer F. , Rusyn Ivan , Schulte-Hermann R. , Thurman Ronald G.
Published: March 2015

Liver regeneration after partial hepatectomy (PH) involves several signaling mechanisms including activation of the small GTPases Ras and RhoA in response to mitogens leading to DNA synthesis and cell proliferation. Peroxisome proliferator-activated receptor-α (PPARα) regulates the expression of several key enzymes in isoprenoid synthesis, which are key events for membrane association of Ras and RhoA. Thus the role of PPARα in cell proliferation after PH was tested. After PH, an increase in PPARα DNA binding was observed in wild-type mice, correlating with an increase in the PPARα-regulated enzyme acyl-CoA oxidase. In addition, the PPARα-regulated genes farnesyl pyrophosphate synthase and 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase were significantly increased in wild-type mice. However, these increases were not observed in PPARα knockout (PPARα −/−) mice. The peak in DNA synthesis observed 42 h after PH was reduced by ∼60% in PPARα −/− mice, despite increases in TNF-α and IL-1. Also, under these conditions, membrane association of Ras was high in wild-type mice after PH but was impaired in PPARα −/− mice. Accordingly, Ras was significantly elevated in the cytosol in PPARα −/− mice. This observation correlated with lower levels of active GTP-bound Ras after PH in PPARα −/− mice compared with wild-type mice. Similar observations were made for RhoA. Moreover, deletion of PPARα blunted the activation of cyclin-dependent kinase (cdk)2/cyclin E and cdk4/cyclin D complexes. Collectively, these results support the hypothesis that PPARα is necessary for cell cycle progression in regenerating mouse liver via mechanisms involving prenylation of small GTPases Ras and RhoA.
Phorbol ester-mediated neurotensin secretion is dependent on the PKC-α and -δ isoforms
Authors: Li Jing , Hellmich Mark R. , Greeley George H. , Townsend Courtney M. , Evers B. Mark
Published: March 2015

Neurotensin (NT) plays an important role in gastrointestinal secretion, motility, and growth. The mechanisms regulating NT secretion are not entirely known. Our purpose was to define the role of the PKC signaling pathway in secretion of NT from BON cells, a human pancreatic carcinoid cell line that produces and secretes NT peptide. We demonstrated expression of all 11 PKC isoforms at varying levels in untreated BON cells. Expression of PKC-α, -β2, -δ, and -μ isoforms was most pronounced. Immunofluorescent staining showed PKC-α and -μ expression throughout the cytoplasm and in the membrane. Also, significant fluorescence of PKC-δ was noted in the nucleus and cytoplasm. Treatment with PMA induced translocation of PKC-α, -δ, and -μ from cytosol to membrane. Activation of PKC-α, -δ, and -μ was further confirmed by kinase assays. Addition of PKC-α inhibitor Gö-6976 at a nanomolar concentration, other PKC inhibitors Gö-6983 and GF-109203X, or PKC-δ-specific inhibitor rottlerin significantly inhibited PMA-mediated NT release. Overexpression of either PKC-α or -δ increased PMA-mediated NT secretion compared with control cells. We demonstrated that PMA-mediated NT secretion in BON cells is associated with translocation and activation of PKC-α, -δ, and -μ. Furthermore, inhibition of PKC-α and -δ blocked PMA-stimulated NT secretion, suggesting a critical role for these isoforms in NT release.
ETAand ETBreceptor function in pancreatitis-associated microcirculatory failure, inflammation, and parenchymal injury
Authors: Plusczyk T. , Witzel B. , Menger M. D. , Schilling M.
Published: March 2015

The role of endothelin (ET)Aand ETBreceptor function in experimental pancreatitis is still not fully understood. Using a rat model of sodium taurocholate-induced pancreatitis and intravital microscopy, we therefore studied whether selective inhibition of ETAreceptor function or combined ETAand ETBreceptor blockade affects the development of pancreatitis-associated microcirculatory failure, inflammation, and parenchymal injury. Pretreatment with 10 mg/kg body wt of a combined ETA/Breceptor antagonist, which is thought to mediate a simultaneous inhibition of both receptors, did not attenuate the pancreatitis-induced microcirculatory failure, inflammatory response, and parenchymal tissue injury. In contrast, pretreatment with a low concentration of the combined ETA/Breceptor antagonist (4 mg/kg body wt), which predominantly inhibits the ETAreceptor, revealed an improvement of some microcirculatory disorders and a significant attenuation of leukocyte recruitment and tissue injury. Furthermore, pretreatment with a selective ETAreceptor antagonist (1 μg/kg body wt) almost abolished pancreatitis-associated capillary constriction, restored functional capillary density, and, consequently, improved overall nutritive perfusion. Importantly, the maintenance of an appropriate microcirculation by selective ETAreceptor inhibition was accompanied by a significant attenuation of the inflammation-associated leukocytic response and by a marked reduction of parenchymal injury. Thus our study indicates that pancreatitis-associated development of microcirculatory failure, inflammation, and parenchymal injury is caused by ETs coupling onto the ETAreceptor, which therefore may represent a promising target for novel strategies in the treatment of pancreatitis.
Coordinated regulation of gastric chloride secretion with both acid and alkali secretion
Authors: Coskun Tamer , Baumgartner Heidi K. , Chu Shaoyou , Montrose Marshall H.
Published: March 2015

Gastric secretion of hydrochloric acid requires protons and chloride, yet the mechanisms and regulation of gastric chloride secretion remain unclear. We developed an in vivo technique to simultaneously measure acid/base and chloride secretion into the gastric lumen of anesthetized rats. The cannulated stomach lumen was perfused with weakly pH-buffered chloride-free solution containing a chloride-sensitive fluorophore [5 μM N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE)]. Gastric acid and chloride secretion was detected in gastric effluents by 1) flow-through pH electrode and 2) MQAE fluorescence. Gastric effluent was also collected at 1-min intervals for independent determination of chloride amount by chloridometer. In all conditions, both optical and chemical determinations of chloride report similar amounts of secreted chloride. During luminal perfusion with pH 5 solution, net acid and chloride secretion into the lumen was observed. Pentagastrin stimulated both secretions. In contrast, proton pump inhibition (omeprazole) caused alkalinization of the gastric effluent, but chloride secretion was not diminished. During luminal pH 3 perfusion, net alkali secretion was observed, and chloride secretion at luminal pH 3 was greater than pH 5. When tissue is pretreated with omeprazole at luminal pH 3, the addition of prostaglandin E2 synchronously stimulates both alkali and chloride secretion. Results suggest that both acid and alkali secretions are separately coupled with chloride secretion.
Inhibition of gastric cancer cell proliferation by resveratrol: role of nitric oxide
Authors: Holian Oksana , Wahid Shahid , Atten Mary Jo , Attar Bashar M.
Published: March 2015

Resveratrol is a dietary phytochemical that has been shown to inhibit proliferation of a number of cell lines, and it behaves as a chemopreventive agent in assays that measure the three stages of carcinogenesis. We tested for its chemopreventive potential against gastric cancer by determining its interaction with signaling mechanisms that contribute to the proliferation of transformed cells. Low levels of exogenous reactive oxygen (H2O2) stimulated [3H]thymidine uptake in human gastric adenocarcinoma SNU-1 cells, whereas resveratrol suppressed both synthesis of DNA and generation of endogenous O[Formula: see text] but stimulated nitric oxide (NO) synthase (NOS) activity. To address the role of NO in the antioxidant action of resveratrol, we measured the effect of sodium nitroprusside (SNP), an NO donor, on O[Formula: see text] generation and on [3H]thymidine incorporation. SNP inhibited DNA synthesis and suppressed ionomycin-stimulated O[Formula: see text] generation in a concentration-dependent manner. Our results revealed that the antioxidant action of resveratrol toward gastric adenocarcinoma SNU-1 cells may reside in its ability to stimulate NOS to produce low levels of NO, which, in turn, exert antioxidant action. Resveratrol-induced inhibition of SNU-1 proliferation may be partly dependent on NO formation, and we hypothesize that resveratrol exerts its antiproliferative action by interfering with the action of endogenously produced reactive oxygen. These data are supportive of the action of NO against reactive oxygen and suggest that a resveratrol-rich diet may be chemopreventive against gastric cancer.
Uncoupling protein-1: involvement in a novel pathway for β-adrenergic, cAMP-mediated intestinal relaxation
Authors: Shabalina Irina , Wiklund Claes , Bengtsson Tore , Jacobsson Anders , Cannon Barbara , Nedergaard Jan
Published: March 2015

The pathway for adrenergic relaxation of smooth muscle is not fully understood. As mitochondrial uncoupling protein-1 (UCP1) expression has been reported in cells within the longitudinal smooth muscle layer of organs exhibiting peristalsis, we examined whether the absence of UCP1 affects adrenergic responsiveness. Intestinal (ileal) segments were obtained from UCP1-ablated mice and from wild-type mice (C57Bl/6, 129/SvPas, and outbred NMRI). In UCP1-containing mice, isoprenaline totally inhibited contractions induced by electrical field stimulation, but in intestine from UCP1-ablated mice, a significant residual contraction remained even at a high isoprenaline concentration; the segments were threefold less sensitive to isoprenaline. Also, when contraction was induced by carbachol, there was a residual isoprenaline-insensitive contraction. Similar results were obtained with the β3-selective agonist CL-316,243 and with the adenylyl cyclase stimulator forskolin. Thus the UCP1 reported to be expressed in the longitudinal muscle layer of the mouse intestine is apparently functional, and UCP1, presumably through uncoupling, may be involved in a novel pathway leading from increased cAMP levels to relaxation in organs exhibiting peristalsis.
Epidermal growth factor reduces the development of necrotizing enterocolitis in a neonatal rat model
Authors: Dvorak Bohuslav , Halpern Melissa D. , Holubec Hana , Williams Catherine S. , McWilliam Debra L. , Dominguez Jessica A. , Stepankova Renata , Payne Claire M. , McCuskey Robert S.
Published: March 2015

Necrotizing enterocolitis (NEC) is the most common gastrointestinal disease of prematurely born infants. Maternal milk plays an important protective role against NEC development and is the major source of epidermal growth factor (EGF) for neonates. The aim of this study was to examine the effect of orally administered EGF on the incidence of NEC in a neonatal rat model. Newborn rats were artificially fed either with growth factor-free rat milk substitute (RMS) or RMS supplemented with 500 ng/ml of EGF (RMS+EGF). Experimental NEC was induced by exposure to asphyxia and cold stress. Development of NEC was evaluated by gross and histological scoring of damage in the ileum. Ileal EGF receptor (EGF-R), EGF, and transforming growth factor-α mRNA expression was assessed by RT competitive-PCR, and the EGF-R was localized by immunohistochemistry. EGF supplementation of formula reduced the incidence and severity of NEC in rats (13/16 RMS vs. 4/13 RMS+EGF). Ileal EGF-R mRNA expression was markedly increased in the RMS group compared with RMS+EGF. Enhanced EGF-R expression in the RMS group was localized predominantly in the epithelial cells of injured ileum. These data suggest a new potential therapeutic approach for the prevention and treatment of NEC.
PAF-like lipids- and PAF-induced gallbladder muscle contraction is mediated by different pathways in guinea pigs
Authors: Guarino Michele P. L. , Xiao Zuo-Liang , Biancani Piero , Behar Jose
Published: March 2015

H2O2stimulates gallbladder muscle contraction and scavengers of free radicals through the generation of PGE2. Oxidative stress causes lipid peroxidation and generation of platelet-activating factor (PAF) or PAF-like lipids. The present studies therefore were aimed at determining whether either one induced by H2O2mediates the increased generation of PGE2. Dissociated muscle cells of guinea pig gallbladder were obtained by enzymatic digestion. Both PAF-like lipids and PAF-induced muscle contraction was blocked by the PAF receptor antagonist CV-3988. This antagonist also blocked the increased PGE2production caused by PAF-like lipids or PAF. Actions of PAF-like lipids were completely inhibited by indomethacin, but those of PAF were only partially reduced by indomethacin or by nordihydroguaiaretic acid and completely blocked by their combination. PAF-like lipids-induced contraction was inhibited by AACOCF3(cystolic phospholipase A2inhibitor), whereas the actions of PAF were blocked by MJ33(secretory phospholipase A2inhibitor). Receptor protection studies showed that pretreatment with PAF-like lipids before N-ethylmaleimide protected the contraction induced by a second dose of PAF-like lipids or PGE2but not by PAF. In contrast, pretreatment with PAF protected the actions of PAF and PGE2but not that of PAF-like lipids. Both PAF-like lipids and PAF-induced contractions were inhibited by anti-Gαq/11antibody and by inhibitors of MAPK and PKC. In conclusion, PAF-like lipids seem to activate a pathway different from that of PAF probably by stimulating a different PAF receptor subtype.
cAMP-mediated regulation of murine intestinal/pancreatic Na+/HCO 3 − cotransporter subtype pNBC1
Authors: Bachmann O. , Rossmann H. , Berger U. V. , Colledge W. H. , Ratcliff R. , Evans M. J. , Gregor M. , Seidler U.
Published: March 2015

Basolateral Na+-HCO[Formula: see text] cotransport is essential for intestinal anion secretion, and indirect evidence suggests that it may be stimulated by a rise of intracellular cAMP. We therefore investigated the expression, activity, and regulation by cAMP of the Na+-HCO[Formula: see text] cotransporter isoforms NBC1 and NBCn1 in isolated murine colonic crypts. Na+-HCO[Formula: see text] transport rates were measured fluorometrically in BCECF-loaded crypts, and mRNA expression levels and localization were determined by semiquantitative PCR and in situ hybridization. Acid-activated Na+-HCO[Formula: see text]cotransport rates were 5.07 ± 0.7 mM/min and increased by 62% after forskolin stimulation. NBC1 mRNA was more abundant in colonic crypts than in surface cells, and crypts expressed far more NBC1 than NBCn1. To investigate whether the cAMP-induced Na+-HCO[Formula: see text] cotransport activation was secondary to secretion-associated changes in HCO[Formula: see text] or cell volume, we measured potential forskolin-induced changes in intracellular pH and assessed Na+-HCO[Formula: see text]transport activity in CFTR −/− crypts (in which no forskolin-induced cell shrinkage occurs). We found 30% reduced Na+-HCO[Formula: see text] transport rates in CFTR −/− compared with CFTR +/+ crypts but similar Na+-HCO[Formula: see text] cotransport activation by forskolin. These studies establish the existence of an intracellular HCO[Formula: see text] concentration- and cell volume-independent activation of colonic NBC by an increase in intracellular cAMP.
Human hepatic stellate cells express CCR5 and RANTES to induce proliferation and migration
Authors: Schwabe Robert F. , Bataller Ramón , Brenner David A.
Published: March 2015

Activated hepatic stellate cells (HSCs) are the main producers of extracellular matrix in the fibrotic liver and are involved in the regulation of hepatic inflammation. The aim of this study was to characterize the role of regulated on activation, normal T-cell expressed, and presumably secreted (RANTES) in activated HSCs. RANTES mRNA and protein secretion were strongly induced after stimulating HSCs with TNF-α, IL-1β, or CD40L. RANTES production was NF-κB dependent, because inhibitor-κB (IκB) superrepressor and dominant-negative IκB kinase-2 almost completely blocked RANTES expression. NF-κB activation was sufficient to drive RANTES expression as demonstrated by the strong induction of RANTES in HSCs expressing NF-κB-inducing kinase. The JNK/activator protein-1 pathway also contributed to RANTES expression as demonstrated by the blocking effects of the JNK inhibitor SP600125. HSCs responded to stimulation with recombinant human (rh)RANTES with an increase in intracellular calcium concentration and a rapid increase in free radical formation. Furthermore, rhRANTES induced ERK phosphorylation, ERK-dependent [3H]thymidine incorporation, and HSC proliferation. Additionally, rhRANTES induced focal adhesion kinase phosphorylation and a substantial increase in HSC migration. HSCs functionally expressed chemokine receptor-5 (CCR5), as shown by flow-cytometric analysis and RT-PCR, and the inhibitory effects of a blocking CCR5 antibody on rhRANTES-induced ERK activation, proliferation, and migration. Diphenylene iodonium and N-acetylcysteine inhibited rhRANTES-induced ERK activation and HSC proliferation, indicating that NADPH oxidase-dependent production of reactive oxygen species was required. In conclusion, RANTES and CCR5 represent potential mediators of 1) HSC migration and proliferation and 2) a cross-talk between HSCs and leukocytes during fibrogenesis.
Activation of group II mGlu receptors inhibits voltage-gated Ca2+ currents in myenteric neurons
Authors: Chen Wei-Ping , Kirchgessner Annette L.
Published: March 2015

The enteric nervous system (ENS) contains functional ionotropic and group I metabotropic glutamate (mGlu) receptors. In this study, we determined whether enteric neurons express group II mGlu receptors and the effects of mGlu receptor activation on voltage-gated Ca2+ currents in these cells. (2R,4R)-4-aminopyrrolidine-2,4-dicarboxylate (2R,4R-APDC), a group II mGlu receptor agonist, reversibly suppressed the Ba2+current in myenteric neurons isolated from the guinea pig ileum. Significant inhibition was also produced by l-glutamate and the group II mGlu receptor agonists, (2S,2′R,3′R)-2-(2′,3′-dicarboxycyclopropyl)glycine (DCG-IV) and (2S,1′S,2′S)-2-(2-carboxycyclopropyl)glycine (l-CCG-I), with a rank order potency of 2R,4R-APDC > DCG-IV >l-glutamate > l-CCG-I, and was reduced by the group II mGlu receptor antagonist LY-341495. Pretreatment of neurons with pertussis toxin (PTX) reduced the action of mGlu receptor agonists, suggesting participation of Gi/Goproteins. Finally, ω-conotoxin GVIA blocked current suppression by DCG-IV, suggesting modulation of N-type calcium channels. mGlu2/3 receptor immunoreactivity was displayed by neurons in culture and in the submucosal and myenteric plexus of the ileum. A subset of these cells displayed a glutamatergic phenotype as shown by the expression of vesicular glutamate transporter 2. These results provide the first evidence for functional group II mGlu receptors in the ENS and show that these receptors are PTX sensitive and negatively coupled to N-type calcium channels. Inhibition of N-type calcium channels produced by activation of group II mGlu receptors may modulate enteric neurotransmission.
Site-specific gene expression of nNOS variants in distinct functional regions of rat gastrointestinal tract
Authors: Saur Dieter , Neuhuber Winfried L. , Gengenbach Bernd , Huber Andrea , Schusdziarra Volker , Allescher Hans-Dieter
Published: March 2015

5′ mRNA variants of neuronal nitric oxide synthase (nNOS) are generated either by alternative promoter usage resulting in different mRNAs that encode for the same protein (nNOSα) or alternative splicing encoding NH2-terminally truncated proteins (nNOSβ/γ) that lack the PDZ/GLGF domain for protein-protein interaction of nNOSα. We studied the expression of 5′ nNOS mRNA forms and nNOS-interacting proteins (postsynaptic density protein-95; PSD-95) in the rat gastrointestinal tract and analyzed the more distinct localization of nNOS protein variants in the duodenum by immunohistochemistry with COOH- and NH2-terminal nNOS antibodies. 5′ nNOS mRNA variants showed a site-specific expression along the gastrointestinal tract with presence of all forms (nNOSα-a, -b, -c; nNOSβ) in the muscle layer of esophagus, stomach, duodenum, longitudinal muscle layer of jejunum/ileum, proximal colon, and rectum. In contrast, a lack of nNOSα-a and nNOSβ mRNA was observed in pylorus, circular muscle layer of jejunum/ileum, and cecum. Expression of nNOSα and nNOSβ cDNAs revealed proteins of ∼155 kDa and 135/125 kDa, respectively. Immunohistochemistry showed a differential distribution of COOH- and NH2-terminal nNOS immunoreactivity in distinct layers of rat duodenum, suggesting a cell-specific expression and distinct compartmentalization of nNOS proteins. Observed distribution of 5′ nNOS mRNA variants and proteins argue for a complex control of nNOS expression by usage of separate promoters, cell- and site-specific splicing mechanisms, and translational initiation. These mechanisms could be involved in gastrointestinal motor diseases and may explain the phenotype of nNOSα knockout mice with gastric stasis and pyloric stenosis, due to a total loss of nNOS in the pyloric sphincter region.
Enhanced capacitative calcium entry and TRPC channel gene expression in human LES smooth muscle
Authors: Wang Jian , Laurier Lisanne G. , Sims Stephen M. , Preiksaitis Harold G.
Published: March 2015

Transient receptor potential channel ( TRPC) genes encode Ca2+-permeable channels mediating capacitative Ca2+entry (CCE), which maintains intracellular Ca2+stores. We compared TRPC gene expression and CCE in human esophageal body (EB) and lower esophageal sphincter (LES), because these smooth muscles have distinct contractile functions that are likely associated with different Ca2+regulatory mechanisms. Circular layer smooth muscle cells were grown in primary culture. Transcriptional expression of TRPC genes was compared by semiquantitative RT-PCR. CCE was measured by fura 2 Ca2+fluorescence after blockade of sarcoplasmic reticulum Ca2+-ATPase with thapsigargin. mRNA for TRPC1, TRPC3, TRPC4, TRPC5, and TRPC6was identified in EB and LES. TRPC3 and TRPC4were more abundant in LES than EB. Basal concentration of free intracellular Ca2+([Ca2+]i) was similar in cells from LES (138 ± 8 nmol/l) and EB (110 ± 6 nmol/l) and increased with ACh (10 μmol/l; 650 ± 28 and 590 ± 21 nmol/l, respectively). With zero Ca2+in bath, thapsigargin (2 μmol/l) increased [Ca2+]imore in LES (550 ± 22 nmol/l) than EB (250 ± 15 nmol/l, P < 0.001). Subsequent external application of 1 mmol/l Ca2+increased [Ca2+]imore in LES (585 ± 35 nmol/l) than EB (295 ± 21 nmol/l, P < 0.001), indicating enhanced CCE in LES. This demonstrates CCE and TRPC transcriptional expression in human esophageal smooth muscle. In LES cells, enhanced CCE and expression of TRPC3 and TRPC4 may contribute to the physiological characteristics that distinguish LES from EB.
Cortical processing of human gut sensation: an evoked potential study
Authors: Hobday David I. , Hobson Anthony R. , Sarkar Sanchoy , Furlong Paul L. , Thompson David G. , Aziz Qasim
Published: March 2015

The rectum has a unique physiological role as a sensory organ and differs in its afferent innervation from other gut organs that do not normally mediate conscious sensation. We compared the central processing of human esophageal, duodenal, and rectal sensation using cortical evoked potentials (CEP) in 10 healthy volunteers (age range 21–34 yr). Esophageal and duodenal CEP had similar morphology in all subjects, whereas rectal CEP had two different but reproducible morphologies. The rectal CEP latency to the first component P1 (69 ms) was shorter than both duodenal (123 ms; P = 0.008) and esophageal CEP latencies (106 ms; P = 0.004). The duodenal CEP amplitude of the P1-N1 component (5.0 μV) was smaller than that of the corresponding esophageal component (5.7 μV; P = 0.04) but similar to that of the corresponding rectal component (6.5 μV; P = 0.25). This suggests that rectal sensation is either mediated by faster-conducting afferent pathways or that there is a difference in the orientation or volume of cortical neurons representing the different gut organs. In conclusion, the physiological and anatomic differences between gut organs are reflected in differences in the characteristics of their afferent pathways and cortical processing.
Identification of an apical Cl-/HCO3-exchanger in gastric surface mucous and duodenal villus cells
Authors: Xu Jie , Barone Sharon , Petrovic Snezana , Wang Zhaohui , Seidler Ursula , Riederer Brigitte , Ramaswamy Krishnamurthy , Dudeja Pradeep K. , Shull Gary E. , Soleimani Manoocher
Published: March 2015

The molecular identity of the apical HCO3--secreting transporter in gastric mucous cells remains unknown despite its essential role in preventing injury and ulcer by gastric acid. Here we report the identification of a Cl-/HCO3-exchanger that is located on apical membranes of gastric surface epithelial cells. RT-PCR studies of mouse gastrointestinal tract mRNAs demonstrated that this transporter, known as anion exchanger isoform 4 (AE4), is expressed in both stomach and duodenum. Northern blot analysis of RNA from purified stomach epithelial cells indicated that AE4 is expressed at higher levels in mucous cells than in parietal cells. Immunoblotting experiments identified AE4 as a ∼110- to 120-kDa protein in membranes from stomach epithelium and apical membranes from duodenum. Immunocytochemical staining demonstrated that AE4 is expressed in apical membranes of surface cells in both mouse and rabbit stomach and duodenum. Functional studies in oocytes indicated that AE4 functions as a Cl-/HCO3-exchanger. These data show that AE4 is an apical Cl-/HCO3-exchanger in gastric mucous cells and duodenal villus cells. On the basis of its function and location, we propose that AE4 may play an important role in mucosal protection.
Reactive oxygen species (H2O2): effects on the gallbladder muscle of guinea pigs
Authors: Xiao Zuo-Liang , Pozo Andrada Maria J. , Biancani Piero , Behar Jose
Published: March 2015

Reactive oxygen species (ROS) have been implicated in the pathogenesis of muscle dysfunction in acute inflammatory processes. The aim of these studies was to determine the effects of ROS on gallbladder muscle function in vitro. Single muscle cells were obtained by enzymatic digestion. H2O2(70 μM) caused maximal contraction of up to 14% and blocked the response to CCK-8, ACh, and KCl. It did not affect the contractions induced by guanosine 5′- O-(3-thiotriphosphate), diacylglycerol, and inositol 1,4,5-trisphosphate that circumvent membrane receptors. The contraction induced by H2O2was inhibited by AACOCF3[cytosolic phospholipase A2(cPLA2) inhibitor], indomethacin (cyclooxygenase inhibitor), chelerythrine [protein kinase C (PKC) inhibitor], or PD-98059 [mitogen-activated protein kinase (MAPK) inhibitor]. H2O2also reduced the CCK receptor binding capacity from 0.36 ± 0.05 pmol/mg protein (controls) to 0.17 ± 0.03 pmol/mg protein. The level of lipid peroxidation as well as the PGE2content was significantly increased after H2O2pretreatment. Unlike superoxide dismutase, the free radical scavenger catalase prevented the H2O2induced contraction, and its inhibition of the CCK-8 induced contraction. It is concluded that ROS cause damage to the plasma membrane of the gallbladder muscle and contraction through the generation of PGE2induced by cPLA2-cyclooxygenase and probably mediated by the PKC-MAPK pathway.