bluuebunny/crossref_metadata_2025 huggingface dataset:
Activation of VEGF and Ras genes in gastric mucosa during angiogenic response to ethanol injury
Authors: Jones Michael K. , Itani Rabiha M. , Wang Hongtao , Tomikawa Morimasa , Sarfeh I. James , Szabo Sandor , Tarnawski Andrzej S.
Published: December 2017

Our previous studies demonstrated that ethanol injury triggers the angiogenic response in gastric mucosa bordering necrosis. The present study was aimed to determine whether vascular endothelial growth factor (VEGF) (a potent angiogenic peptide selectively acting on endothelial cells) and Ras (a mediator of cell proliferation and a putative regulator of VEGF expression) are involved in gastric angiogenesis after ethanol injury. We studied the angiogenic response and expression of VEGF and Ras in gastric mucosa after ethanol injury. Ethanol damage triggered 1) angiogenesis in the gastric mucosa bordering necrosis, 2) significant increases in VEGF mRNA and protein expression, and 3) significant increases in the expression of Ki- ras mRNA and Ras proteins. Neutralizing anti-VEGF antibody significantly reduced (by greater than threefold) the angiogenic response to ethanol-induced injury. Moreover, mevastatin, an inhibitor of Ras activation, completely blocked the induction of VEGF expression in cultured primary endothelial cells. Because, in other tissues, VEGF is one of the most potent angiogenic factors and VEGF expression is dependent on Ras, our data indicate that Ras and VEGF are involved in gastric mucosal angiogenesis after ethanol injury.
Intestinal plasma membrane calcium pump protein and its induction by 1,25(OH)2D3decrease with age
Authors: Armbrecht H. J. , Boltz M. A. , Kumar V. B.
Published: December 2017

The plasma membrane Ca pump of intestinal absorptive cells has been proposed as a component in the vitamin D-dependent active transport of Ca. Because intestinal Ca transport declines with age, the purpose of this study was to determine if changes in Ca pump expression parallel this decline. Intestinal levels of the plasma membrane Ca pump protein were measured by Western blotting in Fischer 344 rats that were 2, 12, and 24 mo of age. Ca pump protein levels declined by 90% in the duodenum and 65% in the ileum between 2 and 12 mo of age, the time during which active Ca transport declines markedly. The effect of age on the induction of the Ca pump by 1,25-dihydroxyvitamin D3[1,25(OH)2D3], the active metabolite of vitamin D, was determined. Rats were made deficient in 1,25(OH)2D3by feeding a high-strontium diet, and they were then dosed with 1,25(OH)2D3or vehicle at 48, 24, and 6 h. In 12-mo-old rats 1,25(OH)2D3induced duodenal Ca pump protein to only 39% and active Ca transport to 33% of that seen in 2-mo-old animals. These studies demonstrate that decreased expression of the plasma membrane Ca pump protein, along with calbindin protein, parallels the decline in intestinal Ca transport and its response to 1,25(OH)2D3with age.
Stimulation of the paraventricular nucleus modulates the activity of gut-sensitive neurons in the vagal complex
Authors: Zhang Xueguo , Fogel Ronald , Renehan William E.
Published: December 2017

There is good evidence that stimulation of the lateral hypothalamus excites neurons in the dorsal vagal complex (DVC), but the data regarding the role of the paraventricular nucleus (PVN) in vagal function are less clear. The purpose of this study was to clarify the effect of PVN stimulation on the activity of neurons in the DVC. We utilized extracellular and intracellular neuronal recordings with intracellular injections of a neuronal tracer to label individual, physiologically characterized neurons in the DVC of rats anesthetized with pentobarbital sodium. Most (80%) of the gut-sensitive dorsal motor nucleus of the vagus (DMNV) neurons characterized in this study exhibited a change in activity during electrical stimulation of the PVN. Stimulation of the PVN caused an increase in the spontaneous activity of 59% of the PVN-sensitive DMNV neurons, and the PVN was capable of modulating the response of a small subset of DMNV neurons to gastrointestinal stimuli. This study also demonstrated that the PVN was capable of influencing the activity of neurons in the nucleus of the solitary tract (NST). Electrical stimulation of the PVN decreased the basal activity of 66% of the NST cells that we characterized and altered the gastrointestinal response of a very small subset of NST neurons. It is likely that these interactions play a role in the modulation of a number of gut-related homeostatic processes. Increased or decreased activity in the descending pathway from the PVN to the DVC has the potential to alter ascending satiety signals, modulate vago-vagal reflexes and the cephalic phase of feeding, and affect the absorption of nutrients from the gastrointestinal tract.
IV. Paneth cell antimicrobial peptides and the biology of the mucosal barrier
Authors: Ouellette Andre J.
Published: December 2017

The hypothesis that epithelial cells release preformed antibiotic peptides as components of mucosal innate immunity has gained experimental support in recent years. In the mammalian small intestine, Paneth cells secrete granules that are rich in α-defensins and additional antimicrobial peptides into the lumen of the crypt. The α-defensins are homologues of peptides that function as mediators of nonoxidative microbial cell killing in phagocytic leukocytes, and they are potent microbicidal agents in in vitro assays. Because certain mouse α-defensins stimulate cultured epithelial cells to secrete chloride ion, those peptides appear to be capable of interacting directly with the apical membranes of neighboring cells and perhaps influencing crypt physiology. In instances of crypt disruption or induced Paneth cell deficiency, crypt intermediate cells appear to compensate by accumulating and secreting Paneth cell antimicrobial peptides. Challenges for the future will be to understand the mechanisms of this epithelial plasticity and to show that Paneth cells contribute directly to innate immunity in the crypt microenvironment.
Neuronal NOS provides nitrergic inhibitory neurotransmitter in mouse lower esophageal sphincter
Authors: Kim Chi Dae , Goyal Raj K. , Mashimo Hiroshi
Published: December 2017

To identify the enzymatic source of nitric oxide (NO) in the lower esophageal sphincter (LES), studies were performed in wild-type and genetically engineered endothelial nitric oxide synthase [eNOS(−)] and neuronal NOS [nNOS(−)] mice. Under nonadrenergic noncholinergic (NANC) conditions, LES ring preparations developed spontaneous tone in all animals. In the wild-type mice, electrical field stimulation produced frequency-dependent intrastimulus relaxation and a poststimulus rebound contraction. NOS inhibitor N ω-nitro-l-arginine methyl ester (100 μM) abolished intrastimulus relaxation and rebound contraction. In nNOS(−) mice, both the intrastimulus relaxation and rebound contraction were absent. However, in eNOS(−) mice there was no significant difference in either the relaxation or rebound contraction from the wild-type animal. Both nNOS(−) and eNOS(−) tissues showed concentration-dependent relaxation to NO donor diethylenetriamine-NO and there was no difference in the sensitivity to the NO donor in nNOS(−), eNOS(−), or wild-type animals. These results indicate that in mouse LES, nNOS rather than eNOS is the enzymatic source of the NO that mediates NANC relaxation and rebound contraction.
Corticotropin-releasing hormone mimics stress-induced colonic epithelial pathophysiology in the rat
Authors: Santos Javier , Saunders Paul R. , Hanssen Nico P. M. , Yang Ping-Chang , Yates Derrick , Groot Jack A. , Perdue Mary H.
Published: December 2017

We examined the effect of stress on colonic epithelial physiology, the role of corticotropin-releasing hormone (CRH), and the pathways involved. Rats were restrained or injected intraperitoneally with CRH or saline. Colonic segments were mounted in Ussing chambers, in which ion secretion and permeability (conductance and probe fluxes) were measured. To test the pathways involved in CRH-induced changes, rats were pretreated with hexamethonium, atropine, bretylium, doxantrazole, α-helical CRH-(9—41) (all intraperitoneally), or aminoglutethimide (subcutaneously). Restraint stress increased colonic ion secretion and permeability to ions, the bacterial peptide FMLP, and horseradish peroxidase (HRP). These changes were prevented by α-helical CRH-(9—41) and mimicked by CRH (50 μg/kg). CRH-induced changes in ion secretion were abolished by α-helical CRH-(9—41), hexamethonium, atropine, or doxantrazole. CRH-stimulated conductance was significantly inhibited by α-helical CRH-(9—41), hexamethonium, bretylium, or doxantrazole. CRH-induced enhancement of HRP flux was significantly reduced by all drugs but aminoglutethimide. Peripheral CRH reproduced stress-induced colonic epithelial pathophysiology via cholinergic and adrenergic nerves and mast cells. Modulation of stress responses may be relevant to the management of colonic disorders.
IL-5 contributes to worm expulsion and muscle hypercontractility in a primaryT. spiralisinfection
Authors: Vallance Bruce A. , Blennerhassett Patricia A. , Deng Yikang , Matthaei Klaus I. , Young Ian G. , Collins Stephen M.
Published: December 2017

Enteric nematode infections lead to increased interleukin (IL)-5 expression, eosinophilic inflammation, and intestinal smooth muscle hypercontractility. Although eosinophils release inflammatory mediators that cause smooth muscle contraction, the role of IL-5 and eosinophils in enteric smooth muscle hypercontractility is unclear. IL-5-deficient mice and their wild-type controls were infected with the nematode Trichinella spiralis. Intestinal parasites and eosinophils were counted, and jejunal longitudinal muscle contractility was assessed. During infection, IL-5 gene expression increased significantly in wild-type mice and was accompanied by significant intestinal eosinophilia in wild-type but not IL-5-deficient mice. Although both strains developed increased muscle contractility during infection, contraction was significantly less in the IL-5-deficient mice at days 16 and 21 postinfection. In addition, parasite expulsion was transiently delayed at day 16 in IL-5-deficient mice. Thus, in the nematode-infected mouse, IL-5 appears essential for intestinal eosinophilia and contributes to, but is not essential for, the development of muscle hypercontractility. IL-5 also appears to play a minor role in expelling a primary T. spiralis infection from the gut.
HSP27 in signal transduction and association with contractile proteins in smooth muscle cells
Authors: Ibitayo Adenike I. , Sladick Jeanette , Tuteja Sony , Louis-Jacques Otto , Yamada Hirotaka , Groblewski Guy , Welsh Michael , Bitar Khalil N.
Published: December 2017

Sustained smooth muscle contraction is mediated by protein kinase C (PKC) through a signal transduction cascade leading to contraction. Heat-shock protein 27 (HSP27) appears to be the link between these two major events, i.e., signal transduction and sustained smooth muscle contraction. We have investigated the involvement of HSP27 in signal transduction and HSP27 association with contractile proteins (e.g., actin, myosin, tropomyosin, and caldesmon) resulting in sustained smooth muscle contraction. We have carried out confocal microscopy to investigate the cellular reorganization and colocalization of proteins and immunoprecipitation of HSP27 with actin, myosin, tropomyosin, and caldesmon as detected by sequential immunoblotting. Our results indicate that 1) translocation of Raf-1 to the membrane when stimulated with ceramide is inhibited by vasoactive intestinal peptide (VIP), a relaxant neuropeptide; 2) PKC-α and mitogen-activated protein kinase translocate and colocalize on the membrane in response to ceramide, and PKC-α translocation is inhibited by VIP; 3) HSP27 colocalizes with actin when contraction occurs; and 4) HSP27 immunoprecipitates with actin and with the contractile proteins myosin, tropomyosin, and caldesmon. We propose a model in which HSP27 is involved in sustained smooth muscle contraction and modulates the interaction of actin, myosin, tropomyosin, and caldesmon.
Canine pancreatic juice stimulates the release of secretin and pancreatic secretion in the dog
Authors: Song Y. , Li P. , Lee K. Y. , Chang T.-M. , Chey W. Y.
Published: December 2017

A secretin-releasing factor (SRF) was found in canine pancreatic juice that increases plasma secretin and stimulates pancreatic secretion in rats, suggesting that a positive feedback mechanism may be involved in the regulation of pancreatic secretion. In the present study, we investigated to determine whether or not SRF releases endogenous secretin and stimulates exocrine pancreatic secretion in conscious dogs. Fresh pancreatic juice was collected from four dogs by intravenous administration of secretin at 0.5 μg ⋅ kg−1⋅ h−1and CCK at 0.2 μg ⋅ kg−1⋅ h. The juice was boiled for 10 min at 100°C. Experiments were carried out in phase I of spontaneous cycle of interdigestive pancreatic secretion. The testing solutions were infused intraduodenally in separate experiments: NaHCO3solution (0.1 M, 4.5 ml/min, 60 min), a corn oil (Lipomul, 2 ml/min, 10 min), boiled pancreatic juice (BPJ, 4.5 ml/min, 60 min), and mixture of BPJ and Lipomul. Pancreatic secretion of fluid and bicarbonate was significantly increased by either BPJ or a mixture of BPJ and Lipomul (34- and 31-fold or 41- and 38-fold, respectively). Plasma secretin level also significantly increased by 164.7 ± 13.4% and 223.1 ± 35.0%, respectively, from basal concentration of 1.7 ± 0.5 pM. In contrast, neither bicarbonate solution nor Lipomul influenced the plasma secretin level or pancreatic secretion. In addition, when Lipomul was incubated with BPJ, no fatty acid was produced. Thus the increased pancreatic secretion in the dog infused with a combination of BPJ and Lipomul was caused by SRF in BPJ, which released endogenous secretin. Moreover, the increases by BPJ of both plasma secretin level and bicarbonate secretion were completely blocked by intravenous administration of an antisecretin antibody in these dogs. The observations suggest that SRF in pancreatic juice exerts a positive feedback effect on exocrine pancreatic secretion that is mediated by the release of secretin in the interdigestive state in dogs.
Trypsin and activation of circulating trypsinogen contribute to pancreatitis-associated lung injury
Authors: Hartwig Werner , Werner Jens , Jimenez Ramon E. , Z'graggen Kaspar , Weimann Jörg , Lewandrowski Kent B. , Warshaw Andrew L. , Castillo Carlos Fernández-Del
Published: December 2017

Pancreatic proteases are secreted in acute pancreatitis, but their contribution to associated lung injury is unclear. Applying models of mild edematous (intravenous caerulein) and severe necrotizing (intraductal glycodeoxycholic acid) pancreatitis in rats, we showed that both trypsinogen and trypsin concentrations in peripheral blood, as well as lung injury, correlate with the severity of the disease. To isolate the potential contribution of proteases to lung injury, trypsin or trypsinogen was injected into healthy rats or trypsinogen secreted in caerulein pancreatitis was activated by intravenous enterokinase. Pulmonary injury induced by protease infusions was dose dependent and was ameliorated by neutrophil depletion. Trypsinogen activation worsened lung injury in mild pancreatitis. In vitro incubation of leukocytes with trypsinogen showed that stimulated leukocytes can convert trypsinogen to trypsin. In conclusion, this study demonstrates that the occurrence and severity of pancreatitis-associated lung injury (PALI) corresponds to the levels of circulating trypsinogen and its activation to trypsin. Neutrophils are involved in both protease activation and development of pulmonary injury.
Induction of mucin gene expression in human colonic cell lines by PMA is dependent on PKC-ε
Authors: Hong D.-H. , Petrovics G. , Anderson W. B. , Forstner J. , Forstner G.
Published: December 2017

Treatment of HT-29 cells with phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC), induces MUC2 expression. To investigate the role of PKC in regulating mucin genes in intestinal cells, we examined the regulation of MUC1, MUC2, MUC5AC, MUC5B, and MUC6 expression in two human mucin-producing colonic cell lines, T84 and HT29/A1. T84 and HT29/A1 cells (at 80–90% confluency) were exposed to 100 nM PMA for 0, 3, and 6 h. Twofold or greater increases in mRNA levels for MUC2 and MUC5AC were observed in both cell lines during this time period, whereas the levels of MUC1, MUC5B, and MUC6 mRNAs were only marginally affected. These results indicated that PKC differentially regulates mucin gene expression and that it may be responsible for altered mucin expression. Our previous results suggested that the Ca2+-independent PKC-ε isoform appeared to mediate PMA-regulated mucin exocytosis in these cell lines. To determine if PKC-ε was also involved in MUC2/MUC5AC gene induction, HT29/A1 cells were stably transfected with either a wild-type PKC-ε or a dominant-negative ATP-binding mutant of PKC-ε (PKC-ε K437R). Overexpression of the dominant-negative PKC-ε K437R blocked induction of both mucin genes, whereas PMA-induced mucin gene expression was not prevented by overexpression of wild-type PKC-ε. PMA-dependent MUC2 mucin secretion was also blocked in cells overexpressing the dominant-negative PKC-ε K437R. On the basis of these observations, PKC-ε appears to mediate the expression of two major gastrointestinal mucins in response to PMA as well as PMA-regulated mucin exocytosis.
Carbon monoxide overproduced by heme oxygenase-1 causes a reduction of vascular resistance in perfused rat liver
Authors: Wakabayashi Yoshiyuki , Takamiya Rina , Mizuki Akira , Kyokane Takanori , Goda Nobuhito , Yamaguchi Tokio , Takeoka Shinji , Tsuchida Eishun , Suematsu Makoto , Ishimura Yuzuru
Published: December 2017

This study aimed to examine whether livers overexpressing heme oxygenase (HO)-1 could alter the vascular resistance through the vasorelaxing action of carbon monoxide (CO). The relationship among HO-1 expression, CO generation, and the vascular resistance was assessed in perfused rat livers pretreated with hemin, an inducer of HO-1. At 18 h after the hemin treatment, livers displayed marked increases in HO-1 expression in hepatocytes and venous CO flux and a reduction of the basal resistance. The reduction of the resistance in hemin-treated livers was canceled by administration of oxyhemoglobin, a reagent trapping both CO and nitric oxide (NO), but not by methemoglobin, which captures NO but not CO. Liposome-encapsulated oxyhemoglobin, which cannot access the space of Disse, did not cause vasoconstriction. Furthermore, these livers became less sensitive to endothelin-1, a vasoconstrictive peptide, than the untreated controls through mechanisms involving CO. On the other hand, at 12 or 24 h after the treatment when the HO-1 induction was not accompanied by CO overproduction, neither a decrease in the basal resistance nor vascular hyporeactivity to endothelin-1 was observed. These results suggest that CO overproduced in the extrasinusoidal compartment is a determinant of the HO-1-mediated vasorelaxation in the liver.
Induction of G1 checkpoint in the gastric mucosa of aged rats
Authors: Xiao Zhi-Qiang , Yu Yingjie , Khan Ahmed , Jaszewski Richard , Ehrinpreis Murray N. , Majumdar Adhip P. N.
Published: December 2017

Although in Fischer 344 rats aging is found to be associated with increased gastric mucosal proliferative activity, little is known about specific changes in the regulatory mechanisms of this process. To determine whether changes in cell cycling events could partly contribute to the age-related rise in gastric mucosal proliferative activity, the present investigation examines changes in cyclin-dependent kinase (Cdk2) activity and the regulation of this process in the gastric mucosa of Fischer 344 rats aged 4 (young), 13 (middle aged), and 24 (old) mo. We observed that aging is associated with a progressive rise in activity and protein levels of Cdk2 in the gastric mucosa. This is also found to be accompanied by a concomitant increase in cyclin E but not cyclin D1 levels. On the other hand, the levels of p21Waf1/Cip1 (total as well as the fraction associated with Cdk2), a nuclear protein that is known to inhibit different cyclin-Cdk complexes, are found to decline in the gastric mucosa with advancing age. In contrast, with aging, there was a steady rise in p53 levels in the gastric mucosa. We have also observed that the levels of phosphorylated retinoblastoma protein, a form that participates in regulating progression through the S phase, are markedly elevated in the gastric mucosa of aged rats. In conclusion, our data suggest that, in the gastric mucosa, aging enhances transition of G1 to S phase as well as progression through the S phase of the cell cycle. However, the age-related decline in p21Waf1/Cip1 in the gastric mucosa appears to be independent of p53 status.
Endothelin-1 stimulation of endothelial nitric oxide synthase in the pathogenesis of hepatopulmonary syndrome
Authors: Zhang Ming , Luo Bao , Chen Shi-Juan , Abrams Gary A. , Fallon Michael B.
Published: December 2017

Biliary cirrhosis in the rat triggers intrapulmonary vasodilatation and gas exchange abnormalities that characterize the hepatopulmonary syndrome. This vasodilatation correlates with increased levels of pulmonary microcirculatory endothelial nitric oxide synthase (eNOS) and hepatic and plasma endothelin-1 (ET-1). Prehepatic portal hypertension induced by portal vein ligation (PVL) does not cause similar changes, suggesting that ET-1 in cirrhosis may modulate pulmonary eNOS and vascular tone. We assessed whether ET-1 altered eNOS expression and nitric oxide production in bovine pulmonary artery endothelial cells (BPAECs) and if a 2-wk low-level intravenous ET-1 infusion in PVL animals modulated pulmonary eNOS levels, microcirculatory tone, and gas exchange. ET-1 caused a 2.5-fold increase in eNOS protein in BPAECs, inhibitable with an endothelin B receptor antagonist, and an increase in eNOS mRNA and nitrite production. ET-1 infusion in PVL animals caused increased pulmonary eNOS levels, intrapulmonary vasodilatation, and gas exchange abnormalities without increasing pulmonary arterial pressure. ET-1 produced during hepatic injury may contribute to the hepatopulmonary syndrome by modulating eNOS and inducing pulmonary microcicrulatory vasodilatation.
Copper treatment alters the permeability of tight junctions in cultured human intestinal Caco-2 cells
Authors: Ferruzza Simonetta , Scarino Maria-Laura , Rotilio Giuseppe , Ciriolo Maria Rosa , Santaroni Paolo , Muda Andrea Onetti , Sambuy Yula
Published: December 2017

The effects of copper on tight-junction permeability were investigated in human intestinal Caco-2 cells, monitoring transepithelial electrical resistance and transepithelial passage of mannitol. Apical treatment of Caco-2 cells with 10–100 μM CuCl2(up to 3 h) produced a time- and concentration-dependent increase in tight-junction permeability, reversible after 24 h in complete medium in the absence of added copper. These effects were not observed in cells treated with copper complexed to l-histidine [Cu(His)2]. The copper-induced increase in tight-junction permeability was affected by the pH of the apical medium, as was the apical uptake of64CuCl2, both exhibiting a maximum at pH 6.0. Treatment with CuCl2produced a concentration-dependent reduction in the staining of F actin but not of the junctional proteins zonula occludens-1, occludin, and E-cadherin and produced ultrastructural alterations to microvilli and tight junctions that were not observed after treatment with up to 200 μM Cu(His)2for 3 h. Overall, these data point to an intracellular effect of copper on tight junctions, mediated by perturbations of the F actin cytoskeleton.
VIII. Absorption and secretion of ions in the gastrointestinal tract
Authors: Shull Gary E. , Miller Marian L. , Schultheis Patrick J.
Published: December 2017

Absorption and secretion of ions in gastrointestinal and other epithelial tissues require the concerted activities of ion pumps, channels, symporters, and exchangers, which operate in coupled systems to mediate transepithelial transport. Our understanding of the identities, membrane locations, and biochemical activities of epithelial ion transporters has advanced significantly in recent years, but major gaps and uncertainties remain in our understanding of their physiological functions. Increasingly, this problem is being addressed by the analysis of mutant mouse models developed by gene targeting. In this review, we discuss gene knockout studies of the secretory isoform of the Na+-K+-2Cl−cotransporter, isoforms 1, 2, and 3 of the Na+/H+exchanger, and the colonic H+-K+-ATPase. This approach is leading to a clearer understanding of the functions of these transporters in the living animal.
c-Myb modulates transcription of the α-smooth muscle actin gene in activated hepatic stellate cells
Authors: Buck Martina , Kim Dong Joon , Houglum Karl , Hassanein Tarek , Chojkier Mario
Published: December 2017

Expression of α-smooth muscle actin (α-SMA) defines the phenotype of activated (myofibroblastic) hepatic stellate cells. These cells, but not quiescent stellate cells, have a high level of α-SMA and c-Myb expression, as well as increased c-Myb-binding activities to the proximal α-SMA E box. Therefore, we analyzed the role of c-Myb in α-SMA transcription and stellate cell activation. Activated primary rat stellate cells displayed a high expression of the −724 and −271 α-SMA/luciferase (LUC) chimeric genes, which contain c-Myb binding sites (−223/−216 bp). α-SMA/LUC minigenes with mutation (−219/−217 bp), truncation (−224 bp), or deletion (−191 bp) of the c-Myb binding site were not efficiently transcribed. Transfection of wild-type c-Myb into quiescent stellate cells, which do not express endogenous c-Myb, induced a ∼10-fold stimulation of −724 α-SMA/LUC expression. Conversely, expression of either a dominant-negative c-Myb basic domain mutant (Cys43→ Asp) or a c-Myb antisense RNA blocked transcription from the −724 α-SMA/LUC or −271 α-SMA/LUC in activated cells. Moreover, transfection of c- myb antisense, but not sense, RNA inhibited both expression of the endogenous α-SMA gene and stellate cell activation, whereas transfection of c- myb stimulated α-SMA expression in quiescent stellate cells. These findings suggest that c-Myb modulates the activation of stellate cells and that integrity of the redox sensor Cys43in c-Myb is required for this effect.
Role of endothelin-1 in regulation of the postnatal intestinal circulation
Authors: Nankervis Craig A. , Nowicki Philip T.
Published: December 2017

Newborn intestine is uniquely prone to vasoconstriction in response to a wide variety of perturbations. To test the hypothesis that endothelin (ET)-1 is an important factor in this process, we determined the effects of exogenous ET-1 administration and blockade of endogenous ET-1 in vivo and in vitro in 3- and 35-day-old swine. Intramesenteric artery administration of exogenous ET-1 to vascularly isolated in vivo gut loops (10−9 M/kg bolus) caused vasoconstriction and reduced gut O2 uptake similarly in these age groups. Selective blockade of ETA or ETB receptors with BQ-610 or BQ-788, respectively, in vascularly isolated in vivo gut loops had no effect on gut vascular resistance or O2 uptake in either age group; within in vitro gut loops, BQ-610 significantly increased vasoconstriction when perfusion pressure was reduced below baseline, but only in 3-day-old animals; i.e., it impaired the autoregulatory response to perfusion pressure reduction. Exogenous ET-1 significantly decreased capillary perfusion within in vitro gut loops, as evidenced by a decrease in capillary filtration coefficient, but only in 3-day-old animals; furthermore, blockade of endogenous ET-1 activity with BQ-610 significantly enhanced capillary filtration coefficient in 3-day-old animals and increased O2extraction ratio. ET-1 did not depress intestinal metabolic rate, as evidenced by its effect on the O2 uptake-blood flow relationship; it did compromise tissue oxygenation because of its effects on intestinal O2 transport. ET-1 concentration in mesenteric venous effluent exceeded arterial concentration, but only in 3-day-old intestine, suggesting production of ET-1 by newborn intestine. We conclude that ET-1 exerts an age-dependent effect on intestinal hemodynamics in postnatal intestine, having a greater impact in 3- than in 35-day-old intestine.
Acid-base effects on electrolyte transport in CA II-deficient mouse colon
Authors: Goldfarb David S. , Sly William S. , Waheed Abdul , Charney Alan N.
Published: December 2017

To determine the role of carbonic anhydrase (CA) in colonic electrolyte transport, we studied Car-20mice, mutants deficient in cytosolic CA II. Ion fluxes were measured under short-circuit conditions in an Ussing chamber. CA was analyzed by assay and Western blots. In Car-20mouse colonic mucosa, total CA activity was reduced 80% and cytosolic CA I and membrane-bound CA IV activities were not increased. Western blots confirmed the absence of CA II in Car-20mice. Normal mouse distal colon exhibited net Na+and Cl−absorption, a serosa-positive PD, and was specifically sensitive to pH. Decrease in pH stimulated active Na+and Cl−absorption whether it was caused by increasing solution [Formula: see text], reducing [Formula: see text] concentration, or reducing pH in CO2/[Formula: see text]-free HEPES-Ringer solution. Membrane-permeant methazolamide, but not impermeant benzolamide, at 0.1 mM prevented the effects of pH. Car-20mice exhibited similar basal transport rates and responses to pH and CA inhibitors. We conclude that basal and pH-stimulated colonic electrolyte absorption in mice requires CA I. CA II and IV may have accessory roles.
Coupling of M2 muscarinic receptors to ERK MAP kinases and caldesmon phosphorylation in colonic smooth muscle
Authors: Cook Amy K. , Carty Michael , Singer Cherie A. , Yamboliev Ilia A. , Gerthoffer William T.
Published: December 2017

Coupling of M2 and M3 muscarinic receptors to activation of mitogen-activated protein (MAP) kinases and phosphorylation of caldesmon was studied in canine colonic smooth muscle strips in which M3 receptors were selectively inactivated by N, N-dimethyl-4-piperidinyl diphenylacetate (4-DAMP) mustard (40 nM). ACh elicited activation of extracellular signal-regulated kinase (ERK) 1, ERK2, and p38 MAP kinases in control muscles and increased phosphorylation of caldesmon (Ser789), a putative downstream target of MAP kinases. Alkylation of M3 receptors with 4-DAMP had only a modest inhibitory effect on ERK activation, p38 MAP kinase activation, and caldesmon phosphorylation. Subsequent treatment with 1 μM AF-DX 116 completely prevented activation of ERK and p38 MAP kinase and prevented caldesmon phosphorylation. Caldesmon phosphorylation was blocked by the MAP kinase/ERK kinase inhibitor PD-98509 but not by the p38 MAP kinase inhibitor SB-203580. These results indicate that colonic smooth muscle M2 receptors are coupled to ERK and p38 MAP kinases. Activation of ERK, but not p38 MAP kinases, results in phosphorylation of caldesmon in vivo, which is a novel function for M2receptor activation in smooth muscle.
II. Genetic approaches to pain therapy
Authors: Wood John N.
Published: December 2017

New analgesic drugs are necessary because a number of pain states are untreatable. Genetic approaches to the identification of analgesic drug targets include mapping genes involved in human pain perception (e.g., trkA involved in hereditary neuropathies), identifying regulators of sensory neuron function in simple multicellular organisms and then investigating the activity of their mammalian homologs (e.g., POU domain transcription factors that specify sensory cell fate), as well as difference, expression, and homology cloning of receptors, ion channels, and transcription factors present in sensory neurons. After target validation through the construction of null mutant mice, high-throughput cell-based screens can be used to identify potential drug candidates. As a result of these approaches, a number of receptors and ion channels present in sensory neurons such as voltage-gated sodium channels [sensory neuron specific (SNS) and Na channel novel] and ATP-gated (P2X3), capsaicin-gated [vanilloid receptor 1(VR1)], and proton-gated [acid-sensing ion channel (ASIC)] channels are now under investigation as potential new analgesic drug targets.
Effect of exogenous ATP on canine jejunal smooth muscle
Authors: Xue L. , Farrugia G. , Szurszewski J. H.
Published: December 2017

Intracellular recordings were made from the circular smooth muscle cells of the canine jejunum to study the effect of exogenous ATP and to compare the ATP response to the nonadrenergic, noncholinergic (NANC) inhibitory junction potential (IJP) evoked by electrical field stimulation (EFS). Under NANC conditions, exogenous ATP evoked a transient hyperpolarization (6.5 ± 0.6 mV) and EFS evoked a NANC IJP (17 ± 0.4 mV). ω-Conotoxin GVIA (100 nM) and a low-Ca2+, high-Mg2+ solution abolished the NANC IJP but had no effect on the ATP-evoked hyperpolarization. The ATP-evoked hyperpolarization and the NANC IJP were abolished by apamin (1 μM) and N G-nitro-l-arginine (100 μM). Oxyhemoglobin (5 μM) partially (38.8 ± 5.5%) reduced the amplitude of the NANC IJP but had no effect on the ATP-evoked hyperpolarization. Neither the NANC IJP nor the ATP-evoked hyperpolarization was affected by P2 receptor antagonists or agonists, including suramin, reactive blue 2, 1-( N, O-bis-[5-isoquinolinesulfonyl]- N-methyl-l-tyrosyl)-4-phenylpiperazine, pyridoxal phosphate-6-azophenyl-2′,4′-disulfonic acid, α,β-methylene ATP, 2-methylthioadenosine 5′-triphosphate tetrasodium salt, and adenosine 5′- O-2-thiodiphosphate. The data suggest that ATP evoked an apamin-sensitive hyperpolarization in circular smooth muscle cells of the canine jejunum via local production of NO in a postsynaptic target cell.
Dextran sulfate sodium-induced murine colitis activates NF-κB and increases galanin-1 receptor expression
Authors: Marrero Jorge A. , Matkowskyj Kristina A. , Yung Kenny , Hecht Gail , Benya Richard V.
Published: December 2017

Galanin is widely distributed in enteric nerve terminals and acts to modulate intestinal motility by altering smooth muscle contraction. This ligand causes Cl−secretion when colonic epithelial cells express the galanin-1 receptor (Gal1-R) subtype. Because Gal1-R expression by colonic epithelia is upregulated by the transcription factor nuclear factor-κB (NF-κB), increasingly appreciated as critical in the pathophysiology of inflammatory bowel disease, we wondered whether the diarrhea associated with this condition could be due to NF-κB-mediated increases in Gal1-R expression. To test this hypothesis, we provided oral dextran sulfate sodium (DSS) to C57BL/6J mice. Although Gal1-R are not normally expressed by epithelial cells lining the mouse colon, DSS treatment resulted in increased NF-κB activation and Gal1-R expression. Whereas galanin had no effect on murine colonic tissues studied ex vivo, it progressively increased short-circuit current and colonic fluid secretion in DSS-treated mice. Concomitant parenteral administration of the NF-κB inhibitor dexamethasone attenuated the activation of this transcription factor by DSS, inhibiting the increase in Gal1-R expression. Although Gal1-R-specific antagonists do not exist, intracolonic administration of commercially available galanin antibody diminished the DSS-induced increase in colonic fluid accumulation. Overall, these data demonstrate that a significant component of the excessive fluid secretion observed in DSS-treated mice is due to increased Gal1-R expression.
Kupffer cell-derived prostaglandin E2is involved in alcohol-induced fat accumulation in rat liver
Authors: Enomoto Nobuyuki , Ikejima Kenichi , Yamashina Shunhei , Enomoto Ayako , Nishiura Teruhiro , Nishimura Tetsuro , Brenner David A. , Schemmer Peter , Bradford Blair U. , Rivera Chantal A. , Zhong Zhi , Thurman Ronald G.
Published: December 2017

Destruction of Kupffer cells with gadolinium chloride (GdCl3) and intestinal sterilization with antibiotics diminished ethanol-induced steatosis in the enteral ethanol feeding model. However, mechanisms of ethanol-induced fatty liver remain unclear. Accordingly, the role of Kupffer cells in ethanol-induced fat accumulation was studied. Rats were given ethanol (5 g/kg body wt) intragastrically, and tissue triglycerides were measured enzymatically. Kupffer cells were isolated 0–24 h after ethanol, and PGE2production was measured by ELISA, whereas inducible cyclooxygenase (COX-2) mRNA was detected by RT-PCR. As expected, ethanol increased liver triglycerides about threefold. This increase was blunted by antibiotics, GdCl3, the dihydropyridine-type Ca2+channel blocker nimodipine, and the COX inhibitor indomethacin. Ethanol also increased PGE2production by Kupffer cells about threefold. This increase was also blunted significantly by antibiotics, nimodipine, and indomethacin. Furthermore, tissue triglycerides were increased about threefold by PGE2treatment in vivo as well as by a PGE2EP2/EP4receptor agonist, whereas an EP1/EP3agonist had no effect. Moreover, permeable cAMP analogs also increased triglyceride content in the liver significantly. We conclude that PGE2derived from Kupffer cells, which are activated by ethanol, interacts with prostanoid receptors on hepatocytes to increase cAMP, which causes triglyceride accumulation in the liver. This mechanism is one of many involved in fatty liver caused by ethanol.
Fibrogenesis II. Metalloproteinases and their inhibitors in liver fibrosis
Authors: Arthur Michael J. P.
Published: December 2017

Liver fibrosis is characterized by activation of hepatic stellate cells, which are then involved in synthesis of matrix proteins and in regulating matrix degradation. In the acute phases of liver injury and as liver fibrosis progresses, there is increased expression of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs). Among the changes described, striking features include increased expression of gelatinase A (MMP-2) and membrane type 1-MMP (MT1-MMP; MMP-14) as well as TIMP-1 and TIMP-2. These molecules and other family members are involved in regulating degradation of both normal and fibrotic liver matrix. This article outlines recent progress in this field and discusses the mechanisms by which MMPs and TIMPs may contribute to the progression and regression of liver fibrosis. Recently described properties of MMPs and TIMPs of relevance to the pathogenesis of liver fibrosis are outlined. The proposal that regression of liver fibrosis is mediated by decreased expression of TIMPs and involves degradation of fibrillar collagens by a combination of MT1-MMP and gelatinase A, in addition to interstitial collagenase, is explored.
Presynaptic nicotinic acetylcholine receptors in the myenteric plexus of guinea pig intestine
Authors: Schneider David A. , Galligan James J.
Published: December 2017

Presynaptic nicotinic acetylcholine receptors (nAChRs) were studied in myenteric plexus preparations from guinea pig ileum using intracellular electrophysiological methods. Microapplication of nicotine (1 mM) caused a biphasic depolarization in all AH neurons ( n = 30) and in 36 of 49 S neurons. Cytisine (1 mM) caused fast depolarizations in S neurons and no response in AH neurons. Mecamylamine (10 μM) blocked all responses caused by nicotine and cytisine. TTX (0.3 μM) blocked slow excitatory synaptic potentials in S and AH neurons but had no effect on fast depolarizations caused by nicotine. Nicotine-induced slow depolarizations were reduced by TTX in two of twelve AH neurons (79% inhibition) and four of nine S neurons (90 ± 12% inhibition). Slow nicotine-induced depolarizations in the remaining neurons were TTX resistant. TTX-resistant slow depolarizations were inhibited after neurokinin receptor 3 desensitization caused by senktide (0.1 μM); senktide desensitization inhibited the slow nicotine-induced depolarization by 81 ± 5% and 63 ± 15% in AH and S neurons, respectively. A low-calcium and high-magnesium solution blocked nicotine-induced slow depolarizations in AH neurons. In conclusion, presynaptic nAChRs mediate the release of substance P and/or neurokinin A to cause slow depolarizations of myenteric neurons.
Inhibition of poly(ADP-ribose) polymerase attenuates inflammation in a model of chronic colitis
Authors: Jijon Humberto B , Churchill Thomas , Malfair David , Wessler Andreas , Jewell Laurence D , Parsons Howard G , Madsen Karen L
Published: December 2017

Crohn's disease is a chronic disease characterized by oxidant-induced tissue injury and increased intestinal permeability. A consequence of oxidative damage is the accumulation of DNA strand breaks and activation of poly(ADP-ribose) polymerase (PARP), which subsequently catalyzes ADP-ribosylation of target proteins. In this study, we assessed the role of PARP in the colitis seen in interleukin (IL)-10 gene-deficient mice. IL-10 gene-deficient mice demonstrated significant alterations in colonic cellular energy status in conjunction with increased permeability, proinflammatory cytokine release, and nitrosative stress. After 14 days of treatment with the PARP inhibitor 3-aminobenzamide, IL-10 gene-deficient mice demonstrated normalized colonic permeability; reduced tumor necrosis factor-α and interferon-γ secretion, inducible nitric oxide synthase expression, and nitrotyrosine levels; and significantly attenuated inflammation. Time course studies demonstrated that 3-aminobenzamide rapidly altered cellular metabolic activity and decreased cellular lactate levels. This was associated with normalization of colonic permeability and followed by a downregulation of proinflammatory cytokine release. Our data demonstrate that inhibition of PARP activity results in a marked improvement of colonic inflammatory disease and a normalization of cellular metabolic function and intestinal permeability.
Adenovirus-mediated human pancreatic lipase gene transfer to rat bile: gene therapy of fat malabsorption
Authors: Kuhel David G. , Zheng Shuqin , Tso Patrick , Hui David Y.
Published: December 2017

This study explored the potential of using the gene therapy approach, based on adenovirus-mediated expression of pancreatic lipase in the hepatobiliary tract, to increase lipid digestion in the intestinal lumen and promote lipid absorption through the gastrointestinal tract. Recombinant adenovirus containing the human pancreatic lipase cDNA (Ad PL) was shown to transduce and mediate pancreatic lipase biosynthesis in rat IEC-6 epithelial cells in vitro. Retrograde infusion of recombinant adenovirus (3 × 108plaque-forming units) containing the bacterial LacZ gene (Ad LacZ) into the bile duct of rats resulted in positive X-gal reaction products in the periportal liver cells 7 days after Ad LacZ infusion. A high level of human pancreatic lipase was detected in bile after retrograde bile duct infusion of rats with Ad PL but not in the bile of animals infused with Ad LacZ. Triglyceride hydrolytic activity in the bile of Ad PL-infused rats was equivalent to that present in pancreatic juice. In contrast, serum obtained from these animals did not contain any detectable pancreatic lipase activity. These results suggest that ectopic expression of pancreatic enzymes in the hepatobiliary tract may be an alternative therapeutic strategy for treating fat malabsorption due to pancreatic insufficiency.
Human esophageal smooth muscle cells express muscarinic receptor subtypes M1 through M5
Authors: Wang Jian , Krysiak Pawel S. , Laurier Lisanne G. , Sims Stephen M. , Preiksaitis Harold G.
Published: December 2017

Receptor characterization in human esophageal smooth muscle is limited by tissue availability. We used human esophageal smooth muscle cells in culture to examine the expression and function of muscarinic receptors. Primary cultures were established using cells isolated by enzymatic digestion of longitudinal muscle (LM) and circular muscle (CM) obtained from patients undergoing esophagectomy for cancer. Cultured cells grew to confluence after 10–14 days in medium containing 10% fetal bovine serum and stained positively for anti-smooth muscle specific α-actin. mRNA encoding muscarinic receptor subtypes M1–M5 was identified by RT-PCR. The expression of corresponding protein for all five subtypes was confirmed by immunoblotting and immunocytochemistry. Functional responses were assessed by measuring free intracellular Ca2+ concentration ([Ca2+]i) using fura 2 fluorescence. Basal [Ca2+]i, which was 135 ± 22 nM, increased transiently to 543 ± 29 nM in response to 10 μM ACh in CM cells ( n = 8). This response was decreased <95% by 0.01 μM 4-diphenylacetoxy- N-methylpiperidine, a M1/M3-selective antagonist, whereas 0.1 μM methoctramine, a M2/M4-selective antagonist, and 0.1 μM pirenzepine, a M1-selective antagonist, had more modest effects. LM and CM cells showed similar results. We conclude that human smooth muscle cells in primary culture express five muscarinic receptor subtypes and respond to ACh with a rise in [Ca2+]i mediated primarily by the M3 receptor and involving release of Ca2+ from intracellular stores. This culture model provides a useful tool for further study of esophageal physiology.
Cytochalasin B modulation of Caco-2 tight junction barrier: role of myosin light chain kinase
Authors: Ma Thomas Y. , Hoa Neil T. , Tran Daniel D. , Bui Vuong , Pedram Ali , Mills Susan , Merryfield Margaret
Published: December 2017

The intracellular mechanisms that mediate cytochalasin-induced increase in intestinal epithelial tight junction (TJ) permeability are unclear. In this study, we examined the involvement of myosin light chain kinase (MLCK) in this process, using the filter-grown Caco-2 intestinal epithelial monolayers. Cytochalasin B (Cyto B) (5 μg/ml) produced an increase in Caco-2 MLCK activity, which correlated with the increase in Caco-2 TJ permeability. The inhibition of Cyto B-induced MLCK activation prevented the increase in Caco-2 TJ permeability. Additionally, myosin-Mg2+-ATPase inhibitor and metabolic inhibitors (which inhibit MLCK induced actin-myosin contraction) also prevented the Cyto B-induced increase in Caco-2 TJ permeability. Cyto B caused a late-phase (15–30 min) aggregation of actin fragments into large actin clumps, which was also inhibited by MLCK inhibitors. Cyto B produced a morphological disturbance of the ZO-1 TJ proteins, visually correlating with the functional increase in Caco-2 TJ permeability. The MLCK and myosin-Mg2+-ATPase inhibitors prevented both the functional increase in TJ permeability and disruption of ZO-1 proteins. These findings suggested that Cyto B-induced increase in Caco-2 TJ permeability is regulated by MLCK activation.
Portal hypertension induces sodium channel expression in colonocytes from the distal colon of the rat
Authors: Fraser Gerald M. , Blendis Laurence M. , Smirnoff Patricia , Sikular Emanuel , Niv Yaron , Schwartz Betty
Published: December 2017

Cellular mechanisms for Na+retention in portal hypertension are undefined, but epithelial Na+channels (ENaC) may be involved. Under high-salt diet, ENaC are absent from distal colon of rat but can be induced by mineralocorticoids such as aldosterone. Presence of rat ENaC was determined by amiloride inhibition of22Na+uptake in surface colonocytes 7 and 14 days after partial portal vein ligation (PVL) or sham surgery. At both times, uptake inhibition was significantly increased in PVL rats. Presence of mRNA transcripts, determined by RT-PCR, demonstrated that channel α- and γ-subunits were similarly expressed in both groups but that β-subunit mRNA was increased in PVL rats. This confirms that there was induction of rat ENaC and indicates that β-subunit has a regulatory role. Urinary Na+was decreased for 3 days after PVL but was not different at other times, and serum aldosterone levels were elevated at 7 days, at a time when urinary Na+output was similar to that of sham-operated rats. We conclude that PVL leads to induction of ENaC in rat distal colon. An increase in aldosterone levels may prevent natiuresis and is probably one of several control mechanisms involved in Na+retention in portal hypertension.