bluuebunny/crossref_metadata_2025 huggingface dataset:
IV. Nonalcoholic fatty liver disease abnormalities in macrophage function and cytokines
Authors: Diehl Anna Mae
Published: March 2015

Macrophage products, such as cytokines, prostanoids, nitric oxide, and reactive oxygen intermediates, influence the function and viability of macrophages and neighboring cells. Given that the liver has one of the largest resident macrophage populations in the body, it is not surprising that hepatic macrophages [i.e., Kupffer cells (KC)] are involved in the pathogenesis of many kinds of liver disease. This review summarizes the abnormalities that have been demonstrated in bone marrow, peritoneal and hepatic macrophage of leptin-resistant (fa/fa) rats and leptin-deficient (ob/ob) mice, two animal models for nonalcoholic fatty liver disease (NAFLD). Evidence supports the concept that altered KC function influences the viability of other cells, such as lymphocytes and hepatocytes, in fatty livers, thereby contributing to the pathogenesis of NAFLD in animals with reduced leptin activity. Further work is needed to determine whether KC dysfunction is a component of more generalized mechanisms that lead to NAFLD.
Anion-dependent Mg2+influx and a role for a vacuolar H+-ATPase in sheep ruminal epithelial cells
Authors: Schweigel Monika , Martens Holger
Published: March 2015

The K+-insensitive component of Mg2+influx in primary culture of ruminal epithelial cells (REC) was examined by means of fluorescence techniques. The effects of extracellular anions, ruminal fermentation products, and transport inhibitors on the intracellular free Mg2+concentration ([Mg2+]i), Mg2+uptake, and intracellular pH were determined. Under control conditions (HEPES-buffered high-NaCl medium), the [Mg2+]iof REC increased from 0.56 ± 0.14 to 0.76 ± 0.06 mM, corresponding to a Mg2+uptake rate of 15 μM/min. Exposure to butyrate did not affect Mg2+uptake, but it was stimulated (by 84 ± 19%) in the presence of [Formula: see text]. In contrast, Mg2+uptake was strongly diminished if REC were suspended in [Formula: see text]-buffered high-KCl medium (22.3 ± 4 μM/min) rather than in HEPES-buffered KCl medium (37.5 ± 6 μM/min). After switching from high- to low-Cl–solution, [Mg2+]iwas reduced from 0.64 ± 0.09 to 0.32 ± 0.16 mM and the [Formula: see text]-stimulated Mg2+uptake was completely inhibited. Bumetanide and furosemide blocked the rate of Mg2+uptake by 64 and 40%, respectively. Specific blockers of vacuolar H+-ATPase reduced the [Mg2+]i(36%) and Mg2+influx (38%) into REC. We interpret this data to mean that the K+-insensitive Mg2+influx into REC is mediated by a cotransport of Mg2+and Cl–and is energized by an H+-ATPase. The stimulation of Mg2+transport by ruminal fermentation products may result from a modulation of the H+-ATPase activity.
Differential changes in human pharyngoesophageal motor excitability induced by swallowing, pharyngeal stimulation, and anesthesia
Authors: Fraser Christopher , Rothwell John , Power Maxine , Hobson Anthony , Thompson David , Hamdy Shaheen
Published: March 2015

We investigated the effects of water swallowing, pharyngeal stimulation, and oropharyngeal anesthesia on corticobulbar and craniobulbar projections to human swallowing musculature. Changes in pathway excitability were measured via electromyography from swallowed intraluminal pharyngeal and esophageal electrodes to motor cerebral and trigeminal nerve magnetic stimulation. After both water swallowing and pharyngeal stimulation, pharyngoesophageal corticobulbar excitability increased (swallowing: pharynx = 59 ± 12%, P < 0.001; esophagus = 45 ± 20%, P < 0.05; pharyngeal stimulation: pharynx = 76 ± 19%, P < 0.001; esophagus = 45 ± 23%, P = 0.05), being early with swallowing but late with stimulation. By comparison, craniobulbar excitability increased early after swallowing but remained unaffected by pharyngeal stimulation. After anesthesia, both corticobulbar (pharynx =–24 ± 10%, P < 0.05; esophagus = –28 ± 7%, P < 0.01) and craniobulbar excitability showed a late decrease. Thus swallowing induces transient early facilitation of corticobulbar and craniobulbar projections, whereas electrical stimulation promotes delayed facilitation mainly in cortex. With removal of input, both corticobulbar and craniobulbar projections show delayed inhibition, implying a reduction in motoneuron and/or cortical activity.
Interdigestive transpyloric fluid transport assessed by intraluminal impedance recording
Authors: Savoye G. , Savoye-Collet C. , Oors J. , Smout A. J. P. M.
Published: March 2015

Our aim was to explore the use of intraluminal impedance recording for assessment of interdigestive transpyloric fluid movements. Twenty healthy volunteers were studied with a catheter allowing the recording of five antropyloroduodenal impedance signals simultaneously with six pressure signals. Patterns induced by air were verified by standard ultrasound. Transpyloric Doppler ultrasound was used to validate impedance patterns associated with transpyloric fluid transports. Impedance changes caused by air (short-lived increases) occupied 14 ± 12% of the time in the antrum and 0.8 ± 0.5% in the duodenum ( P < 0.005). All fluid transport events lasting >4 s were recorded by both Doppler and impedance techniques. Transpyloric fluid transport was observed in all three phases of the antral migrating motor complex. The total number of transport events was significantly higher ( P < 0.05) in phase II (18 ± 7) than in phases I (2.6 ± 2) and III(6.1 ± 3). Retrograde transport was observed mainly in antral phase I (54% of fluid movements, compared with 2.5% in phase II and 18.5% in phase III, P < 0.05). During phase II, 80 ± 13% of the impedance changes were associated with manometric events and 72 ± 9% of the antral contractions were associated with transpyloric fluid transport. Prolonged assessment of interdigestive transpyloric fluid transport events using intraluminal measurement of impedance is possible. Manometrically detectable contractions are the most frequent, but not the only, driving forces of these events.
Asynchronous expression and colocalization of Bsep and Mrp2 during development of rat liver
Authors: Zinchuk Vadim S. , Okada Teruhiko , Akimaru Kunihiro , Seguchi Harumichi
Published: March 2015

In the liver, function of the bile salt export pump (Bsep), a major canalicular exporter of bile salts, is complemented by activity of the multidrug resistance protein 2 (Mrp2), a canalicular organic anions transporter. Mrp2 was found capable of transporting various anticancer drugs out of cells, eventually undermining their therapeutic potential and contributing to multidrug resistance. We employed a RT-PCR, immunoblotting, and immunofluorescence to examine their gene, protein expression, and distribution of antigenic sites in the rat liver during development from 16-day-old fetus to adult animal. Bsep mRNA was almost undetectable before birth. It was first clearly expressed in the liver of newborn rats. On the contrary, Mrp2 mRNA was seen before birth, although at low levels. In concert with mRNA expression, Bsep protein was undetectable before birth, while Mrp2 protein was already expressed. Both proteins were clearly detectable in the postnatal period. Confocal immunofluorescent microscopy showed asynchronous appearance of Bsep and Mrp2 proteins during development but their colocalization in the bile canaliculi once each one is expressed. During the gestational period, a weak immunofluorescence for Mrp2 was observed only in livers of 16-day-old embryos. No fluorescence for Bsep was seen. Both proteins were clearly visualizable after birth, although the pattern of immunostaining varied. These findings provide molecular evidence that expression of both Bsep and Mrp2 during development is transcriptionally regulated. They also point out the differences in relevance to the liver function of the systems responsible for canalicular transport of bile salts versus organic anions.
LPA protects intestinal epithelial cells from apoptosis by inhibiting the mitochondrial pathway
Authors: Deng Wenlin , Wang De-An , Gosmanova Elvira , Johnson Leonard R. , Tigyi Gabor
Published: March 2015

We previously showed ( Gastroenterology 123: 206–216, 2002) that lysophosphatidic acid (LPA) protects and rescues rat intestinal epithelial cells (IEC-6) from apoptosis. Here, we provide evidence for the LPA-elicited inhibition of the mitochondrial apoptotic pathway leading to attenuation of caspase-3 activation. Pretreatment of IEC-6 cells with LPA inhibited campothecin-induced caspase-9 and caspase-3 activation and DNA fragmentation. A caspase-9 inhibitor peptide mimicked the LPA-elicited antiapoptotic activity. LPA elicited ERK1/ERK2 and PKB/Akt phosphorylation. The LPA-elicited antiapoptotic activity and inhibition of caspase-9 activity were abrogated by pertussis toxin, PD 98059, wortmannin, and LY 294002. LPA reduced cytochrome c release from mitochondria and prevented activation of caspase-9. LPA prevented translocation of Bax from cytosol to mitochondria and increased the expression of the antiapoptotic Bcl-2 mRNA and protein. LPA had no effect on Bcl-xl, Bad, and Bak mRNA or protein expression. These data indicate that LPA protects IEC-6 cells from camptothecin-induced apoptosis through Gi-coupled inhibition of caspase-3 activation mediated by the attenuation of caspase-9 activation due to diminished cytochrome c release, involving upregulation of Bcl-2 protein expression and prevention of Bax translocation.
Different modes of NF-κB/Rel activation in pancreatic lobules
Authors: Algül Hana , Tando Yusuke , Beil Michael , Weber Christoph K. , Von Weyhern Claus , Schneider Günter , Adler Guido , Schmid Roland M.
Published: March 2015

The eukaryotic transcription factor nuclear factor-κB (NF-κB)/Rel is activated by a large variety of stimuli. It has been demonstrated that NF-κB/Rel is induced during the course of cerulein pancreatitis. Here, we show that NF-κB/Rel is differentially activated in pancreatic lobules. Cerulein induces NF-κB/Rel via activation of IκB kinase (IKK), which causes degradation of IκBα but not IκBβ. Tumor necrosis factor-α-mediated IKK activation leads to IκBα and IκBβ degradation. In contrast, oxidative stress induced by H2O2activates NF-κB/Rel independent of IKK activation and IκBα degradation; instead IκBα is phosphorylated on tyrosine. H2O2but not cerulein-mediated NF-κB/Rel activation can be blocked by stabilizing microtubules with Taxol. Inhibition of tubulin polymerization with nocodazole causes NF-κB/Rel activation in pancreatic lobules. These results propose three different pathways of NF-κB/Rel activation in pancreatic acinar cells. Furthermore, these data demonstrate that microtubules play a key role in IKK-independent NF-κB/Rel activation following oxidative stress.
Peristalsis regulation by tachykinin NK1 receptors in the rabbit isolated distal colon
Authors: Onori Luciano , Aggio Annalisa , Taddei Gennaro , Loreto Maria F. , Ciccocioppo Rachele , Vicini Riccardo , Tonini Marcello
Published: March 2015

In the gastrointestinal tract, tachykinin NK1 receptors are widely distributed in a number of neuronal and nonneuronal cells involved in the control of gut motor activity. In particular, in the rabbit isolated distal colon, which is a suitable model system to investigate the contribution of tachykinins as noncholinergic excitatory transmitters, the influence of NK1 receptors in the regulation of peristalsis is not known. The selective NK1-receptor antagonists SR-140333 (0.3 and 1 nM) and MEN-10930 (0.3–10 nM) significantly enhanced the velocity of rabbit colonic propulsion to submaximal stimulation. The prokinetic effect of SR-140333 was prevented by Nω-nitro-l-arginine (l-NNA), a nitric oxide synthase inhibitor, indicating that NK1 receptors located on nitrergic innervation exert a functional inhibitory restraint on the circular muscle and probably on descending excitatory and inhibitory pathways during propulsion. Conversely, the selective NK1-receptor agonist septide (3–10 nM) significantly inhibited colonic propulsion. In the presence of l-NNA, the inhibitory effect of septide was reverted into a prokinetic effect, which is probably mediated by the activation of postjunctional excitatory NK1 receptors.
Peroxynitrite inhibits enterocyte proliferation and modulates Src kinase activity in vitro
Authors: Potoka Douglas A. , Upperman Jeffrey S. , Zhang Xiao-Ru , Kaplan Joshua R. , Corey Seth J. , Grishin Anatoly , Zamora Ruben , Ford Henri R.
Published: March 2015

Overproduction of nitric oxide (NO) or its toxic metabolite, peroxynitrite (ONOO-), after endotoxemia promotes gut barrier failure, in part, by inducing enterocyte apoptosis. We hypothesized that ONOO-may also inhibit enterocyte proliferation by disrupting the Src tyrosine kinase signaling pathway, thereby blunting repair of the damaged mucosa. We examined the effect of ONOO-on enterocyte proliferation and Src kinase activity. Sprague-Dawley rats were challenged with LPS or saline, whereas intestinal epithelial cell line cells were treated with ONOO-or decomposed ONOO-in vitro. Enterocyte proliferation in vivo and in vitro was measured by 5-bromo-2′-deoxyuridine (BrdU) or [3H]thymidine incorporation. Src kinase activity in cell lysates was determined at various times. LPS challenge in vivo and ONOO-treatment in vitro inhibited enterocyte proliferation. ONOO-treatment blunted the activity of Src and its downstream target, focal adhesion kinase, in a time-dependent manner. ONOO-blocked mitogen (FBS, EGF)-induced enterocyte proliferation and Src phosphorylation while increasing Src nitration. Thus ONOO-may promote gut barrier failure not only by inducing enterocyte apoptosis but also by disrupting signaling pathways involved in enterocyte proliferation.
Raf-1 activation suppresses neuroendocrine marker and hormone levels in human gastrointestinal carcinoid cells
Authors: Sippel Rebecca S. , Carpenter Jennifer E. , Kunnimalaiyaan Muthusamy , Lagerholm Sara , Chen Herbert
Published: March 2015

Gastrointestinal carcinoid cells secrete multiple neuroendocrine markers and hormones including 5-HT and chromogranin A. The intracellular signaling pathways that regulate production of bioactive molecules are not completely understood. Our aim was to determine whether activation of the raf-1/MEK/MAPK signal transduction pathway in carcinoid cells could modulate production of neuroendocrine markers and hormones. Human pancreatic carcinoid cells (BON) were stably transduced with an estrogen-inducible raf-1 construct creating BON-raf cells. Activation of raf-1 in BON-raf cells led to a marked induction of phosphorylated MEK and ERK1/2 within 48 h. Importantly, raf-1 activation resulted in morphological changes accompanied by a marked decrease in neuroendocrine secretory granules by electronmicroscopy. Moreover, induction of raf-1 in BON-raf cells led to significant reductions in 5-HT, chromogranin A, and synaptophysin levels. Furthermore, treatment of BON-raf cells with MEK inhibitors PD-98059 and U-0126 blocked raf-1-mediated morphological changes and hormone suppression but not ERK1/2 phosphorylation. These results show that raf-1 induction suppresses neuroendocrine marker and hormone production in human gastrointestinal carcinoid cells via a pathway dependent on MEK activation.
New method to study oxidative damage and antioxidants in the human small bowel: effects of iron application
Authors: Troost Freddy J. , Saris Wim H. M. , Haenen Guido R. M. M. , Bast Aalt , Brummer Robert-Jan M.
Published: March 2015

Iron may induce oxidative damage to the intestinal mucosa by its catalyzing role in the formation of highly reactive hydroxyl radicals. This study aimed to determine iron-induced oxidative damage provoked by a single clinical dosage of ferrous sulfate and to elucidate the antioxidant defense mechanisms in the human small intestine in vivo. A double-lumen perfusion tube was positioned orogastrically into a 40-cm segment of the proximal small intestine in six healthy volunteers (25 ± 5 yr). The segment was perfused with saline and subsequently with saline containing 80 mg iron as ferrous sulfate at a rate of 10 ml/min. Intestinal fluid samples were collected at 15-min intervals. Thiobarbituric acid reactive substances concentrations as an indicator of lipid peroxidation increased significantly from 0.07 μM (range, 0–0.33 μM) during saline perfusion to 3.35 μM (range, 1.19–7.27 μM) during iron perfusion ( P < 0.05). Nonprotein antioxidant capacity increased significantly from 474 μM (range, 162–748 μM) to 1,314 μM (range, 674–1,542 μM) ( P < 0.05). These data show that a single dosage of ferrous sulfate induces oxidative damage and the subsequent release of an antioxidant in the small intestine in vivo in healthy volunteers.
Effects of bile acids on dog pancreatic duct epithelial cell secretion and monolayer resistance
Authors: Okolo Charles , Wong Thomas , Moody Mark W. , Nguyen Toan D.
Published: March 2015

Pancreatic duct epithelial cells (PDEC) mediate the secretion of fluid and electrolytes and are exposed to refluxed bile. In nontransformed cultured dog PDEC, which express many ion transport pathways of PDEC, 1 mM taurodeoxycholic acid (TDCA) stimulated an125I−efflux inhibited by DIDS and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and a86Rb+efflux inhibited by charybdotoxin. Inhibition by 1,2-bis(2-aminophenoxy)ethane- N,N,N′,N′-tetraacetic acid (BAPTA)-AM suggests mediation via increased intracellular Ca2+concentration, whereas the absence of lactate dehydrogenase release excludes cellular toxicity. At 1 mM, TDCA stimulated a larger125I−efflux than glycodeoxycholate; two dihydroxy bile acids, taurochenodeoxycholate and TDCA, were similarly effective, whereas a trihydroxy bile acid, taurocholate, was ineffective. In Ussing chambers, 1 mM serosal or 2 mM luminal TDCA stimulated an Iscincrease from confluent PDEC monolayers. TDCA also stimulated 1) a short-circuit current ( Isc) increase from basolaterally permeabilized PDEC subject to a serosal-to-luminal Cl−gradient that was inhibited by BAPTA-AM, DIDS, and NPPB and 2) an Iscincrease from apically permeabilized PDEC subject to a luminal-to-serosal K+gradient inhibited by BAPTA-AM and charybdotoxin. Along with the efflux studies, these findings suggest that TDCA interacts directly with PDEC to stimulate Ca2+-activated apical Cl−channels and basolateral K+channels. Monolayer transepithelial resistance was only minimally affected by 1 mM serosal and 2 mM luminal TDCA but decreased after exposure to higher TDCA concentrations (2 mM serosal and 4 mM luminal). A secretory role for bile acids should be considered in pancreatic diseases associated with bile reflux.
Therapeutic effect of anti-OX40L and anti-TNF-α MAbs in a murine model of chronic colitis
Authors: Totsuka T. , Kanai T. , Uraushihara K. , Iiyama R. , Yamazaki M. , Akiba H. , Yagita H. , Okumura K. , Watanabe M.
Published: March 2015

Interaction of OX40 (CD134) on T cells with its ligand (OX40L) on antigen-presenting cells has been implicated in pathogenic T cell activation. This study was performed to explore the involvement of OX40/OX40L in the development of T cell-mediated chronic colitis. We evaluated both the preventive and therapeutic effects of neutralizing anti-OX40L MAb on the development of chronic colitis in SCID mice induced by adoptive transfer of CD4+CD45RBhighT cells as an animal model of Crohn's disease. We also assessed the combination of anti-OX40L and anti-TNF-α MAbs to improve the therapeutic effect. Administration of anti-OX40L MAb markedly ameliorated the clinical and histopathological disease in preventive and therapeutic protocols. In vivo treatment with anti-OX40L MAb decreased CD4+T cell infiltration in the colon and suppressed IFN-γ, IL-2, and TNF-α production by lamina propria CD4+T cells. The combination with anti-TNF-α MAb further improved the therapeutic effect by abolishing IFN-γ, IL-2, and TNF-α production by lamina propria CD4+T cells. Our present results suggested a pivotal role of OX40/OX40L in the pathogenesis of T cell-mediated chronic colitis. The OX40L blockade, especially in combination with the TNF-α blockade, may be a promising strategy for therapeutic intervention of Crohn's disease.
Modulatory effects of estrogen in two murine models of experimental colitis
Authors: Verdú Elena F. , Deng Yikang , Bercik Premysl , Collins Stephen M.
Published: March 2015

The association between oral contraceptives or pregnancy and inflammatory bowel disease is unclear. We investigated whether 17β-estradiol modulates intestinal inflammation in two models of colitis. Female mice were treated with 17β-estradiol alone or with tamoxifen, tamoxifen alone, 17α-estradiol, or placebo. Dinitrobenzene sulfonic acid (DNB)- or dextran sodium sulfate (DSS)-induced colitis were assessed macroscopically, histologically, and by myeloperoxidase (MPO) activity. Malondialdehyde and mRNA levels of intercellular adhesion molecule-1 (ICAM-1), interferon-γ (IFN-γ), and interleukin-13 (IL-13) were determined. In DNB colitis, 17β-estradiol alone, but not 17β-estradiol plus tamoxifen, or 17α-estradiol reduced macroscopic and histological scores, MPO activity and malondialdehyde levels. 17β-Estradiol also decreased the expression of ICAM-1, IFN-γ, and IL-13 mRNA levels compared with placebo. In contrast, 17β-Estradiol increased the macroscopic and histological scores compared with placebo in mice with DSS colitis. These results demonstrate anti-inflammatory and proinflammatory effects of 17β-estradiol in two different models of experimental colitis. The net modulatory effect most likely reflects a combination of estrogen receptor-mediated effects and antioxidant activity and may explain, in part, conflicting results from clinical trials.
Interstitial cells of Cajal and electrical activity in ganglionic and aganglionic colons of mice
Authors: Ward Sean M. , Gershon Michael D. , Keef Kathleen , Bayguinov Yulia R. , Nelson Cheryl , Sanders Kenton M.
Published: March 2015

An antibody directed against Kit protein was used to investigate the distribution of interstitial cells of Cajal (ICC) within the murine colon. The ICC density was greatest in the proximal colon and decreased along its length. The distribution of the different classes of ICC in the aganglionic colons of lethal spotted ( ls/ls) mice was found to be similar in age-matched wild-type controls. There were marked differences in the electrical activities of the colons from ls/ls mutants compared with wild-type controls. In ls/ls aganglionic colons, the circular muscle was electrically quiescent compared with the spontaneous spiking electrical activity of wild-type tissues. In ls/ls aganglionic colons, postjunctional neural responses were greatly affected. Inhibitory junction potentials were absent or excitatory junction potentials inhibited by atropine were observed. In conclusion, the distribution of ICC in the ganglionic and aganglionic regions of the colons from ls/ls mutants appeared similar to that of wild-type controls. The electrical activity and neural responses of the circular layer are significantly different in aganglionic segments of ls/ls mutants.
NO-flurbiprofen maintains duodenal blood flow, enhances mucus secretion contributing to lower mucosal injury
Authors: Holm L. , Phillipson M. , Perry M. A. ,   (With the Technical Assistance of A. Jägare)
Published: March 2015

This study investigates possible mechanisms behind the reduced gastrointestinal ulcerogenicity of nitric oxide (NO)-flurbiprofen compared with flurbiprofen. The duodenal mucosa of Inactin-anaesthetised rats was exteriorized for intravital microscopy. Blood flow was measured with laser-Doppler flowmetry (LDF), mucus thickness with micropipettes, ICAM-1 and P-selectin expression with dual-labeled antibody technique, and mucosal integrity by51Cr-EDTA permeability. Exposure of the duodenum to flurbiprofen (1.0 mg/ml) for 90 min significantly reduced LDF to 70 ± 4%, whereas NO-flurbiprofen (1.3 mg/ml) had no significant effect. Mucus accumulation after 60-min exposure was 75 ± 23 μm (control), −1 ± 17 μm (flurbiprofen), and 104 ± 35 μm (NO-flurbiprofen). Mucosal permeability to51Cr-EDTA was unchanged in the control and NO-flurbiprofen groups but increased significantly from 1.0 ± 0.2 to 3.7 ± 0.7 μl · min−1· g−1after 90-min exposure to flurbiprofen. Expression of ICAM-1 was significantly increased after oral flurbiprofen but not by NO-flurbiprofen. Positive effects of NO-flurbiprofen compared with flurbiprofen on mucus formation, blood flow, and adhesion molecule expression likely contribute to the reduced mucosal injury observed with NO-flurbiprofen.
Serotonin availability is increased in mucosa of guinea pigs with TNBS-induced colitis
Authors: Linden David R. , Chen Jing-Xian , Gershon Michael D. , Sharkey Keith A. , Mawe Gary M.
Published: March 2015

5-HT released from enterochromaffin cells acts on enteric nerves to initiate motor reflexes. 5-HT's actions are terminated by a serotonin reuptake transporter (SERT). In this study, we tested the hypothesis that inflammation leads to altered mucosal 5-HT signaling. Colitis was induced by 2,4,6-trinitrobenzene sulfonic acid (TNBS), and experiments were conducted on day 6. 5-HT content, number of 5-HT-immunoreactive cells, and the proportion of epithelial cells that were 5-HT-immunoreactive increased twofold in colitis. The amount of 5-HT released under basal and stimulated conditions was significantly increased in colitis. SERT inhibition increased the 5-HT concentration in media bathing-stimulated control tissue to a level comparable to that of the stimulated colitis tissue. mRNA encoding SERT and SERT immunoreactivity were reduced during inflammation. Slower propulsion and reduced sensitivity to 5-HT-receptor antagonism were observed in colitis. These data suggest that colitis alters 5-HT signaling by increasing 5-HT availability while decreasing 5-HT reuptake. Altered 5-HT availability may contribute to the dysmotility of inflammatory bowel disease, possibly due to desensitization of 5-HT receptors.
IL-4 inhibits vasoactive intestinal peptide production by macrophages
Authors: Metwali Ahmed , Blum Arthur M. , Elliott David E. , Weinstock Joel V.
Published: March 2015

In schistosomiasis, eggs induce granulomas that have a vasoactive intestinal peptide (VIP) immunoregulatory circuit. This study explored the regulation of VIP production at sites of inflammation. Splenocytes from uninfected C57BL/6 mice expressed VIP mRNA and protein, which stopped following egg deposition. Eggs induce a Th2 response, suggesting that Th2 cytokines like interleukin (IL)-4 can regulate VIP. To address this issue, splenocytes from uninfected mice were incubated for 4 h with or without recombinant IL-4. IL-4 inhibited VIP mRNA expression. F4/80+macrophages were the source of constitutively expressed VIP, subject to IL-4 regulation. In IL-4 knockout mice, splenic VIP production did not downmodulate during schistosome infection, suggesting that IL-4 is a critical cytokine regulating VIP production in wild-type mouse spleen. IL-4-producing granulomas in schistosomiasis made VIP. Experiments showed that granuloma VIP derived from F4/80−(nonmacrophage) cell populations, explaining this paradox. Granuloma F4/80+cells from IL-4 knockout mice expressed VIP. Thus macrophages can make VIP, which is subject to IL-4 regulation. However, in the Th2 granulomas, other cell types produce VIP, which compensates for loss of macrophages as a source of this molecule.
Melatonin-induced calcium signaling in clusters of human and rat duodenal enterocytes
Authors: Sjöblom Markus , Säfsten Bengt , Flemström Gunnar
Published: March 2015

The amount of melatonin present in enterochromaffin cells in the alimentary tract is much higher than that in the central nervous system, and melatonin acting at MT2receptors mediates neural stimulation of mucosal HCO[Formula: see text]secretion in duodenum in vivo. We have examined effects of melatonin and receptor ligands on intracellular free calcium concentration ([Ca2+]i) signaling in human and rat duodenal enterocytes. Clusters of interconnecting enterocytes (10–50 cells) were isolated by mild digestion (collagenase/dispase) of human duodenal biopsies or rat duodenal mucosa loaded with fura-2 AM and attached to the bottom of a temperature-controlled perfusion chamber. Clusters provided viable preparations and respond to stimuli as a syncytium. Melatonin and melatonin receptor agonists 2-iodo- N-butanoyl-5-methoxytryptamine and 2-iodomelatonin (1.0–100 nM) increased enterocyte [Ca2+]i, EC50of melatonin being 17.0 ± 2.6 nM. The MT2receptor antagonists luzindole and N-pentanoyl-2-benzyltryptamine abolished the [Ca2+]iresponses. The muscarinic antagonist atropine (1.0 μM) was without effect on basal [Ca2+]iand did not affect the response to melatonin. In the main type of response, [Ca2+]ispiked rapidly and returned to basal values within 4–6 min. In another type, the initial rise in [Ca2+]iwas followed by rhythmic oscillations of high amplitude. Melatonin-induced enterocyte [Ca2+]isignaling as well as mucosal cell-to-cell communication may be involved in stimulation of duodenal mucosal HCO[Formula: see text] secretion.
Substance P-evoked Cl−secretion in guinea pig distal colonic epithelia: interaction with PGE2
Authors: Hosoda Yutaka , Karaki Shin-Ichiro , Shimoda Yukiko , Kuwahara Atsukazu
Published: March 2015

Interaction between substance P (SP) and PGE2on Cl−secretion in the guinea pig distal colonic epithelia was investigated. A short-circuit current ( Isc) was measured as an index of ion transport. Mucosa preparations deprived of muscle and submucosa of distal colon were mounted in the Ussing flux chamber and treated with TTX and piroxicam to remove the influences of neuronal activity and endogenous PG synthesis, respectively. Although SP (10−7M) itself evoked little increase in Isc, exogenous PGE2concentration dependently enhanced the response of SP. The effect of PGE2on the SP-evoked response was mimicked by forskolin and 8-bromoadenosine cAMP. Depletion of Ca2+from the bathing solution reduced the PGE2-dependent response of SP. Effects of PGE2, SP, and SP in the presence of PGE2on intracellular Ca2+concentration ([Ca2+]i) in isolated crypt cells were measured by the confocal microscope fluorescence imaging system. SP, but not PGE2, temporally evoked an increase in [Ca2+]ibut declined to the baseline within 3 min. A return of the SP-evoked increase in [Ca2+]iwas slower in the presence of PGE2than SP alone. These results suggest that PGE2synergistically enhances SP-evoked Cl−secretion via an interaction between the intracellular cAMP and [Ca2+]iin the epithelial cells. In conclusion, SP and PGE2could cooperatively induce massive Cl−secretion in guinea pig distal colon at epithelial levels.
I. Global gene expression profiling using DNA microarrays
Authors: Arcellana-Panlilio Mayi , Robbins Stephen M.
Published: March 2015

Having the complete human genomic sequence poses a new challenge: to use genomic structural information to display and analyze biological processes on a genome-wide scale to assign gene function. DNA microarrays are a miniaturized, ordered arrangement of nucleic acid fragments from individual genes located at defined positions on a solid support, enabling the analysis of thousands of genes in parallel by specific hybridization. This review describes technical aspects, discusses relevant applications, and suggests factors affecting the use of this technology and how it fits in the grand scheme of meeting the needs of the postgenomic era.
PG-mediated closure of paracellular pathway and not restitution is the primary determinant of barrier recovery in acutely injured porcine ileum
Authors: Gookin Jody L. , Galanko Joseph A. , Blikslager Anthony T. , Argenzio Robert A.
Published: March 2015

Small bowel epithelium is at the frontline of intestinal barrier function. Restitution is considered to be the major determinant of epithelial repair, because function recovers in parallel with restitution after acute injury. As such, studies of intact mucosa have largely been replaced by migration assays of cultured epithelia. These latter studies fail to account for the simultaneous roles played by villous contraction and paracellular permeability in recovery of barrier function. NSAIDs result in increased intestinal permeability and disease exacerbation in patients with inflammatory bowel disease (IBD). Thus we examined the reparative attributes of endogenous PGs after injury of ileal mucosa by deoxycholate (6 mM) in Ussing chambers. Recovery of transepithelial electrical resistance (TER) from 20-40 Ω·cm2 was abolished by indomethacin (Indo), whereas restitution of 40-100% of the villous surface was unaffected despite concurrent arrest of villous contraction. In the presence of PG, resident crypt and migrating epithelial cells were tightly apposed. In tissues treated with Indo, crypt epithelial cells had dilated intercellular spaces that were accentuated in the migrating epithelium. TER was fully rescued from the effects of Indo by osmotic-driven collapse of the paracellular space, and PG-mediated recovery was significantly impaired by blockade of Cl- secretion. These studies are the first to clearly distinguish the relative contribution of paracellular resistance vs. restitution to acute recovery of epithelial barrier function. Restitution was ineffective in the absence of PG-mediated paracellular space closure. Failure of PG-mediated repair mechanisms may underlie barrier failure resulting from NSAID use in patients with underlying enteropathy.
CaM kinase II regulation of CRHSP-28 phosphorylation in cultured mucosal T84 cells
Authors: Kaspar Kala M. , Thomas Diana D. H. , Taft William B. , Takeshita Eriko , Weng Ning , Groblewski Guy E.
Published: March 2015

Ca2+-regulated heat-stable protein of 28 kDa (CRHSP-28; a member of the tumor protein D52 family) is highly expressed in exocrine glands and was shown to regulate digestive enzyme secretion from pancreatic acinar cells. We found CRHSP-28 highly expressed in cultured mucosal secretory T84 cells, consistent with an important regulatory role in apical membrane trafficking. Stimulation of cells with carbachol (CCh) induced rapid, concentration-dependent phosphorylation of CRHSP-28 on at least two serine residues. Isoelectric focusing and immunoblotting were used to characterize cellular mechanisms governing CRHSP-28 phosphorylation. Phosphorylation depends on elevated cellular Ca2+, being maximally induced by ionomycin and thapsigargin and fully inhibited by BAPTAAM. In vitro phosphorylation of recombinant CRHSP-28 was 10-fold greater by casein kinase II (CKII) than Ca2+/calmodulin-dependent protein kinase II (CaMKII). However, phosphopeptide mapping studies demonstrated that CaMKII induced an identical phosphopeptide profile to endogenous CRHSP-28 immunoprecipitated from T84 cells. Although calmodulin antagonists had no effect on CCh-stimulated phosphorylation, disruption of actin filaments by cytochalasin D inhibited phosphorylation by 50%. Confocal microscopy indicated that CRHSP-28 is expressed in perinuclear regions of cells and accumulates immediately below the apical membrane of polarized monolayers following CCh stimulation. CaMKII was also localized to the subapical cytoplasm and was clearly displaced following actin filament disruption. These data suggest that CRHSP-28 phosphorylation is regulated by a CaMKII-like enzyme and likely involves a translocation of the protein within the apical cytoplasm of epithelial cells.
Bile acids activate EGF receptor via a TGF-α-dependent mechanism in human cholangiocyte cell lines
Authors: Werneburg Nathan W. , Yoon Jung-Hwan , Higuchi Hajime , Gores Gregory J.
Published: March 2015

Bile acids transactivate the EGF receptor (EGFR) in cholangiocytes. However, the mechanisms by which bile acids transactivate the EGFR remain unknown. Our aims were to examine the effects of bile acids on EGFR activation in human cholangiocyte cell lines KMBC and H-69. Bile acids stimulated cell growth and induced EGFR phosphorylation in a ligand-dependent manner. Although cells constitutively expressed several EGFR ligands, only transforming growth factor-α (TGF-α) antisera effectively blocked bile acid-induced EGFR phosphorylation. Consistent with the concept that matrix metalloproteinase (MMP) activity is requisite for TGF-α membrane release and ligand function, bile acid transactivation of EGFR and cell growth was blocked by an MMP inhibitor. In conclusion, bile acids activate EGFR via a TGF-α-dependent mechanism, and this EGFR activation promotes cellular growth.
Regulation of parietal cell migration by gastrin in the mouse
Authors: Kirton C. M. , Wang T. , Dockray G. J.
Published: March 2015

Recent studies suggest that gastrin regulates parietal cell maturation. We asked whether it also regulates parietal cell life span and migration along the gland. Dividing cells were labeled with 5′-bromo-2′-deoxyuridine (BrdU), and parietal cells were identified by staining with Dolichos biflorus lectin. Cells positive for D. biflorus lectin and BrdU were reliably identified 10–30 days after BrdU injection in mice in which the gastrin gene had been deleted by homologous recombination (Gas-KO) and wild-type (C57BL/6) mice. The time course of labeling was similar in the two groups. The distribution of BrdU-labeled parietal cells in wild-type mice was consistent with migration to the base of the gland, but in Gas-KO mice, a higher proportion of BrdU-labeled cells was found more superficially 20 and 30 days after BrdU injection. Conversely, in transgenic mice overexpressing gastrin, BrdU-labeled parietal cells accounted for a higher proportion of the labeled pool in the base of the gland 10 days after BrdU injection. Gastrin, therefore, stimulates movement of parietal cells along the gland axis but does not influence their life span.
Unimpaired osmotic water permeability and fluid secretion in bile duct epithelia of AQP1 null mice
Authors: Mennone Albert , Verkman Alan S. , Boyer James L.
Published: March 2015

The mechanisms by which fluid moves across the luminal membrane of cholangiocyte epithelia are uncertain. Previous studies suggested that aquaporin-1 (AQP1) is an important determinant of water movement in rat cholangiocytes and that cyclic AMP mediates the movement of these water channels from cytoplasm to apical membrane, thereby increasing the osmotic water permeability. To test this possibility we measured agonist-stimulated fluid secretion and osmotically driven water transport in isolated bile duct units (IBDUs) from AQP1 wild-type (+/+) and null (−/−) mice. AQP1 expression was confirmed in a mouse cholangiocyte cell line and +/+ liver. Forskolin-induced fluid secretion, measured from the kinetics of IBDU luminal expansion, was 0.05 fl/min and was not impaired in −/− mice. Osmotic water permeability (Pf), measured from the initial rate of IBDU swelling in response to a 70-mosM osmotic gradient, was 11.1 × 10−4cm/s in +/+ mice and 11.5 × 10−4cm/s in −/− mice. Pfvalues increased by ∼50% in both +/+ and −/− mice following preincubation with forskolin. These findings provide direct evidence that AQP1 is not rate limiting for water movement in mouse cholangiocytes and does not appear to be regulated by cyclic AMP in this species.
Acid- and bile-induced PGE2release and hyperproliferation in Barrett's esophagus are COX-2 and PKC-ε dependent
Authors: Kaur Baljeet S. , Triadafilopoulos George
Published: March 2015

Barrett's esophagus (BE) results from acid and bile reflux and predisposes to cancer. To further understand the mechanisms of acid- and bile-induced hyperproliferation in BE, we investigated the release of PGE2in response to acid or bile salt exposure. Biopsies of esophagus, BE, and duodenum were exposed to a bile salt mixture as a 1-h pulse and compared with exposure to pH 7.4 for up to 24 h, and PGE2release, cyclooxygenase-2 (COX-2), and protein kinase C (PKC) expression were compared. Similar experiments were also performed with acidified media (pH 3.5) alone, in the presence or absence of bisindolylmaleimide (BIM), a selective PKC inhibitor, and NS-398, a COX-2 inhibitor. One-hour pulses of bile salts or acid significantly enhanced proliferation, COX-2 expression, and PGE2release in BE. In contrast, the combination pulse of acid and bile salts had no such effect. Treatment with either BIM or NS-398 led to a dramatic decrease in PGE2release in BE explants and a suppression of proliferation. The acid- or bile salt-mediated hyperproliferation is related to PGE2release. Acid- and bile salt-induced induction of COX-2 and PKC may explain, at least in part, the tumor-promoting effects of acid and bile in BE.
Functional role of specific amino acid residues in human thiamine transporter SLC19A2: mutational analysis
Authors: Balamurugan Krishnaswamy , Said Hamid M.
Published: March 2015

SLC19A2 is a membrane thiamine transporter expressed in a variety of human tissues, including the gastrointestinal tract. Little is currently known about the structure/function relationship of SLC19A2. We examined the effect of introducing mutations in SLC19A2 identical to those found in thiamine-responsive megaloblastic anemia syndrome (TRMA), on functional activity and membrane expression of the transporter. We also examined the effect of mutating the only conserved anionic residue (E138) in the transmembrane (TM) domains of the SLC19A2 and that of the putative glycosylation sites (N63, N314). Northern blot analysis showed SLC19A2 mRNA was expressed at the same level in HeLa cells transfected with wild-type or mutated SLC19A2. Introducing the clinically relevant mutations (D93H, S143F, G172D) or mutation at the conserved anionic residue (E138A) of SLC19A2 led to a significant ( P < 0.01) inhibition of thiamine uptake. Mutations of the two potential N-linked glycosylation sites (N63Q, N314Q) of SLC19A2 did not affect functional activity; they did, however, lead to a noticeable reduction in apparent molecular weight of protein. Western blot analysis showed all proteins (except D93H) were expressed in the membrane (not the cytoplasmic) fraction of HeLa cells. These results provide direct confirmation that clinically relevant mutations in SLC19A2 observed in TRMA cause malfunctioning of the transporter and/or a defect in its translation/stability. Results also show conserved TM anionic residue of the SLC19A2 protein is critical for its function. Furthermore, native SLC19A2 is glycosylated, but this is not important for its function.
III. JNK/AP-1 regulation of hepatocyte death
Authors: Czaja Mark J.
Published: March 2015

Activation of the JNK/activator protein-1 (AP-1)-signaling pathway is a common mediator of hepatocyte death from a variety of stimuli. Although the mechanism by which JNK or AP-1 promotes death is unknown, it results when activation of this signaling pathway is unusually prolonged. Although JNK/AP-1 mediates TNF-induced cell death at or above the level of the mitochondria, the ability of JNK/AP-1 to promote death from necrosis as well as apoptosis suggests that JNK/AP-1 may induce death by several mechanisms. Recognition of JNK/AP-1 signaling as a critical promoter of hepatocyte death raises the possibility that the therapeutic manipulation of this pathway may be effective in the treatment of human liver disease.
Free nitric oxide diffusion in the bronchial microcirculation
Authors: Condorelli Peter , George Steven C.
Published: March 2015

Theoretical mass transfer rates and concentration distributions were determined for transient diffusion of free nitric oxide (NO) generated in vivo from vascular endothelial cells. Our analytical framework is typical of the bronchial circulation in the human pulmonary system but is applicable to the microvascular circulation in general. We characterized mass transfer rates in terms of the fractional mass flux across a boundary relative to the total endothelial NO production rate. NO concentration in the tissue surrounding blood vessels was expressed in terms of fractional soluble guanylate cyclase (sGC) activity. Our results suggest that endothelium-derived free NO is capable of vascular smooth muscle dilation despite its rapid consumption by hemoglobin in blood. An optimal blood vessel radius of 20 μm was estimated for NO signaling. We hypothesize intermittent generation of endothelial NO as a possible mechanism for sGC activation in vascular smooth muscle. This mechanism enhances the efficacy of NO-modulated vascular smooth muscle dilation while minimizing NO losses to blood and surrounding tissue.
Measurement of aortic input impedance in mice: effects of age on aortic stiffness
Authors: Reddy Anilkumar K. , Li Yi-Heng , Pham Thuy T. , Ochoa Lyssa N. , Treviño Maria T. , Hartley Craig J. , Michael Lloyd H. , Entman Mark L. , Taffet George E.
Published: March 2015

Mice are used with increasing frequency as models of human cardiovascular diseases, but significant gaps exist in our knowledge of vascular function in the aging mouse. We determined aortic input impedance spectra, pulse wave velocity, and augmentation index in adult (8-mo-old) and old (29-mo-old) mice to determine whether arterial stiffening occurred with age in mice as it does in humans. Pressure and blood velocity signals measured simultaneously from the same location in the ascending aorta were used to determine input impedance spectra (0–10 harmonics). The first minimum of the impedance modulus occurred at the second harmonic in adult mice but shifted to the fourth harmonic in old mice. Characteristic impedance (average of 2nd–10th harmonic) was 57% higher in old mice: 471 ± 62 vs. 299 ± 10 (SE) dyn · s · cm–3 ( P < 0.05). Pulse pressure and augmentation index, determined from the aortic pressure signals, were also higher in old mice: 42 ± 2.2 vs. 29 ± 4.9 mmHg ( P < 0.05) and 37 ± 5 vs. 14 ± 2% ( P < 0.005). Aortic pulse wave velocity measured from the timing of upstrokes of the Doppler velocity signals was 45% higher in old mice: 416 ± 22 vs. 286 ± 14 cm/s ( n = 3, P < 0.01). These results reproduce age-related findings reported in humans and confirm that mice may be used as models of age-related vascular stiffening.
Activation of focal adhesion kinase by protein kinase Cϵ in neonatal rat ventricular myocytes
Authors: Heidkamp Maria C. , Bayer Allison L. , Scully Brian T. , Eble Diane M. , Samarel Allen M.
Published: March 2015

Focal adhesion kinase (FAK) is a nonreceptor protein tyrosine kinase critical for both cardiomyocyte survival and sarcomeric assembly during endothelin (ET)-induced cardiomyocyte hypertrophy. ET-induced FAK activation requires upstream activation of one or more isoenzymes of protein kinase C (PKC). Therefore, with the use of replication-defective adenoviruses (Adv) to overexpress constitutively active (ca) and dominant negative (dn) mutants of PKCs, we examined which PKC isoenzymes are necessary for FAK activation and which downstream signaling components are involved. FAK activation was assessed by Western blot analysis with an antibody specific for FAK autophosphorylated at Y397 (Y397pFAK). ET (10 nmol/l; 2–30 min) resulted in the time-dependent activation of FAK which was inhibited by chelerythrine (5 μmol/l; 1 h pretreatment). Adv-caPKCϵ, but not Adv-caPKCδ, activated FAK compared with a control Adv encoding β-galactosidase. Conversely, Adv-dnPKCϵ inhibited ET-induced FAK activation. Y-27632 (10 μmol/l; 1 h pretreatment), an inhibitor of Rho-associated coiled-coil-containing protein kinases (ROCK), prevented ET- and caPKCϵ-induced FAK activation as well as cofilin phosphorylation. Pretreatment with cytochalasin D (1 μmol/l, 1 h pretreatment) also inhibited ET-induced Y397pFAK and cofilin phosphorylation and caPKCϵ-induced Y397pFAK. Neither inhibitor, however, interfered with ET-induced ERK1/2 activation. Finally, PP2 (50 μmol/l; 1 h pretreatment), a highly selective Src inhibitor, did not alter basal or ET-induced Y397pFAK. PP2 did, however, reduce basal and ET-induced phosphorylation of other sites on FAK, namely, Y576, Y577, Y861, and Y925. We conclude that the ET-induced signal transduction pathway resulting in downstream Y397pFAK is partially dependent on PKCϵ, ROCK, cofilin, and assembled actin filaments, but not ERK1/2 or Src.