bluuebunny/crossref_metadata_2025 huggingface dataset:
Rat liver myofibroblasts and hepatic stellate cells differ in CD95-mediated apoptosis and response to TNF-α
Authors: Saile Bernhard , Matthes Nina , Neubauer Katrin , Eisenbach Christoph , El-Armouche Hammoudeh , Dudas Joszef , Ramadori Giuliano
Published: March 2015

Hepatic stellate cells (HSC), particularly activated HSC, are thought to be the principle matrix-producing cell of the diseased liver. However, other cell types of the fibroblast lineage, especially the rat liver myofibroblasts (rMF), also have fibrogenic potential. A major difference between the two cell types is the different life span under culture conditions. Although nearly no spontaneous apoptosis could be shown in rMF cultures, 18 ± 2% of the activated HSC ( day 7) were apoptotic. Compared with activated HSC, CD95R was expressed in 70% higher amounts in rMF. CD95L could only be detected in activated HSC. Stimulation of the CD95 system by agonistic antibodies (1 ng/ml) led to apoptosis of all rMF within 2 h, whereas activated HSC were more resistant (5.3 h/ 40% of total cells). Although transforming growth factor-β downregulated apoptosis in both activated HSC and rMF, tumor necrosis factor-α (TNF-α) upregulated apoptosis in rMF. Lack of spontaneous apoptosis and CD95L expression in rMF and the different reaction on TNF-α stimulation reveal that activated HSC and rMF belong to different cell populations.
TNF-α-induced cell death in ethanol-exposed cells depends on p38 MAPK signaling but is independent of Bid and caspase-8
Authors: Pastorino John G. , Shulga Nataly , Hoek Jan B.
Published: March 2015

Alcoholic liver disease is associated with an increase in the number of necrotic and apoptotic liver parenchymal cells. Part of this injury is mediated by TNF-α. Ethanol exposure sensitizes cells to the cytotoxic effects of TNF-α. This may be due, in part, to the increased propensity of the mitochondria in ethanol-exposed cells to induction of mitochondrial permeability transition (MPT) by various agents, including the proapoptotic protein Bax. This idea is supported by the observation that increased cell death induced by TNF-α in ethanol-exposed cells was dependent on development of the MPT. In the present study, we elucidate the pathways through which ethanol exposure enhances TNF-α induction of the MPT and the resulting cytotoxicity. Specifically, ethanol-exposed cells display caspase-8- and Bid-independent cell killing during TNF-α treatment. Moreover, the ethanol-enhanced pathway is dependent on p38 MAPK signaling, which brings about caspase-3 activation, mitochondrial depolarization, accumulation of cytochrome c in the cytosol, and the translocation of Bax to the mitochondria. Additionally, ethanol-exposed cells display a blunting of TNF-α-induced Akt activation and Bcl-2 antagonist of cell death phosphorylation that may account, in part, for the increased sensitivity of the mitochondria to Bax-mediated damage.
Intestinal epithelial CD23 mediates enhanced antigen transport in allergy: evidence for novel splice forms
Authors: Yu Linda C. H. , Montagnac Guillaume , Yang Ping-Chang , Conrad Daniel H. , Benmerah Alexandre , Perdue Mary H.
Published: March 2015

We previously demonstrated enhanced transepithelial antigen transport in the intestine of allergic rodents associated with elevated expression of the low-affinity IgE receptor CD23 on enterocytes. Here, we examined the role of CD23 in the transport phenomenon using CD23–/–mice and characterized the isoform of intestinal epithelial CD23. Jejunal segments of sensitized mice were challenged with antigen. Enhanced transepithelial antigen transport and transmucosal antigen flux were found in the intestine of sensitized CD23+/+but not CD23–/–mice. RT-PCR showed that enterocytes expressed only the isoform b of CD23. Sequencing revealed classic and alternative CD23 b transcripts lacking exon 5 ( bΔ 5) or 6, all of which were translated into functional IgE receptors. The protein encoded by bΔ 5 but not the classic b transcript was able to mediate the uptake of anti-CD23 or IgE, whereas both CD23 proteins were internalized after binding to IgE/antigen complexes. Our results suggest that the classic and alternative forms of CD23 b display distinct endocytic properties, suggesting that they are likely to play different roles in transepithelial transport of IgE and allergens.
Regulatory volume decrease in human esophageal epithelial cells
Authors: Orlando Geraldine S. , Tobey Nelia A. , Wang Paul , Abdulnour-Nakhoul Solange , Orlando Roy C.
Published: March 2015

In vivo human esophageal epithelial cells are regularly exposed to hyposmolal stress. This stress, however, only becomes destructive when the surface epithelial cell (barrier) layers are breached and there is contact of the hyposmolal solution with the basolateral cell membranes. The present investigation was designed to examine the effects of hyposmolal stress in the latter circumstance using as a model for human esophageal epithelial cells the noncancer-derived HET-1A cell line. Cell volume and the response to hyposmolal stress in suspensions of HET-1A cells were determined by cell passage through a Coulter Counter Multisizer II. HET-1A cells behaved as osmometers over the range of 280 to 118 mosmol/kgH2O with rapid increases in cell volume ≤15–20% above baseline. Following swelling, the cells exhibited regulatory volume decrease (RVD), restoring baseline volume within 30 min, despite continued hyposmolal stress. With the use of pharmacologic agents and ion substitutions, RVD appeared to result from rapid activation of parallel K+and Cl−conductance pathways and this was subsequently joined by activation of a KCl cotransporter. Exposure to hyposmolal stress in an acidic environment, pH 6.6, inhibited, but did not abolish, RVD. These data indicate that human esophageal epithelial cells can protect against hyposmolal stress by RVD and that the redundancy in mechanisms may, to some extent, serve as added protection in patients with reflux disease when hyposmolal stress may occur in an acidic environment.
Motilin effects on the proximal stomach in patients with functional dyspepsia and healthy volunteers
Authors: Kamerling Ingrid M. C. , Van Haarst Aernout D. , Burggraaf Jacobus , Schoemaker Rik C. , Biemond Izak , Heinzerling Hartmut , Jones Richard , Cohen Adam F. , Masclee Ad A. M.
Published: March 2015

This study investigates motilin effects on the proximal stomach in patients with functional dyspepsia (FD) and healthy volunteers. Eight healthy volunteers and 12 patients with FD were infused with synthetic motilin or placebo. Proximal gastric volume was measured with a barostat at constant pressure and during isobaric distensions. Abdominal symptoms were scored by visual analog scales. Plasma motilin concentrations were measured by radioimmunoassay. Motilin concentrations and baseline gastric volumes were similar for patients and healthy volunteers. Motilin, compared with placebo, reduced gastric volume by 112 ml [ F(29,195); confidence interval (CI) 95%] in patients and by 96 ml [ F(−7,200); CI 95%] in healthy volunteers. In patients, motilin decreased compliance by 76 ml/mmHg [ F(9,143); CI 95%] compared with placebo, which was similar in volunteers [66 ml/mmHg; F(11,120); CI 95%]. Patients were more nauseous during motilin compared with placebo ( P = 0.04), whereas healthy volunteers did not experience nausea. We conclude that in a fasted condition, FD patients have a similar proximal gastric motor response to motilin as healthy volunteers, but experience an exaggerated sensation of nausea.
An evolutionarily ancient Oatp: insights into conserved functional domains of these proteins
Authors: Cai Shi-Ying , Wang Wei , Soroka Carol J. , Ballatori Nazzareno , Boyer James L.
Published: March 2015

Cellular uptake of organic solutes is mediated in large part by a gene family of membrane transporters called OATPs (SLC21A). To study the structural determinants and evolutionary development of the SLC21A family, we have cloned and functionally characterized a highly expressed evolutionarily primitive Oatp from the liver of the small skate, Raja erinacea. A full-length cDNA (2.3 kb) was obtained that encodes a protein of 689 amino acids. The characteristics of this novel skate Oatp, including tissue expression, subcellular localization, substrate selectivity, Na+ dependence, and inhibitor selectivity were generally similar to liver-specific human OATP-C and rat Oatp4. However, sequence comparisons with other OATPs indicate that this skate Oatp shares only ∼40–50% amino acid identity with the liver-specific OATPs/Oatps and with human OATP-F. Further computer analysis revealed that the highest amino acid identities reside in the first external (78%) and internal loops (75%) and transmembrane domains 2 (76%), 3 (62%), 4 (70%), and 11 (64%). We propose that the conserved regions of the SLC21A transporter family may be critical structural determinants of substrate specificity and function.
Glutamine preserves protein synthesis and paracellular permeability in Caco-2 cells submitted to ”luminal fasting“
Authors: Le Bacquer Olivier , Laboisse Christian , Darmaun Dominique
Published: March 2015

This study used polarized cell line Caco-2 as a model of human enterocytes to determine: 1) whether deprivation of nutrients on the apical (luminal) side of the epithelium (fasting) alters protein synthesis in enterocytes; 2) if so, whether glutamine can attenuate the effects of fasting; and 3) whether the effects of glutamine depend on its route (i.e., apical vs. basolateral) of supply. Caco-2 cells were submitted to nutrient deprivation on the apical side to mimic the effects of fasting, whereas the basolateral side of the epithelium remained exposed to regular medium. Cells were then incubated with [2H3]leucine with or without glutamine, and the fractional synthesis rate (FSR) of total cell protein was determined from [2H3]leucine enrichments in protein-bound and intracellular free leucine measured by gas chromatography/mass spectrometry. A 24-h apical nutrient deprivation (luminal fasting) was associated with a decline in intracellular glutamine, glutamate, and glutathione concentrations (–38, –40, and –40%, respectively), protein FSR (–20%), and a rise in passage of dextran, an index of transepithelial permeability. In fasted cells, basolateral or luminal glutamine supplementation did not alter the glutathione pool, but it restored protein FSR and improved permeability. The effects of glutamine were abolished by 6-diazo-oxo-l-norleucine, an inhibitor of glutaminase, and was mimicked by glutamate. We conclude that in Caco-2 cells, protein synthesis depends on nutrient supply on the apical side, and glutamine regardless of the route of supply corrects some of the deleterious effects of fasting in a model of human enterocytes through its deamidation into glutamate.
Hepatic leukocyte recruitment in a model of acute colitis
Authors: Scott Jeffrey R. , Fox-Robichaud Alison E.
Published: March 2015

There is a close relationship between inflammatory bowel disease (IBD) and various hepatobiliary disorders. The objective of this study was to determine whether hepatic leukocyte recruitment occurs in experimental colitis. We used the murine model of colitis induced by 2,4-dinitrobenezenesulfonic acid (DNBS). Male C57Bl/6 mice received an intrarectal injection of 4 mg DNBS in 100 μl 50% ethanol. Controls received 100 μl 50% ethanol. The hepatic microcirculation was examined at 3 and 14 days post-DNBS by intravital video microscopy. Three days post-DNBS, when mice had developed acute colitis, there was associated hepatic leukocyte recruitment. Within the postsinusoidal venules there was a fourfold increase in the flux of rolling leukocytes that was P-selectin dependent but not α4-integrin dependent. There was also an increase in stationary leukocytes within the sinusoids, although this was not associated with an increase in serum alanine transaminase. By 14 days post-DNBS when macroscopic evidence of colonic inflammation was resolved, rolling within the postsinusoidal venules had returned to control levels. In this murine model of colitis, we describe a link between acute colonic inflammation and remote hepatic leukocyte recruitment that is P-selectin dependent. Active IBD may lead to remote hepatic inflammation.
Role of extrinsic innervation in modulating nitrergic transmission in the canine ileocolonic region
Authors: Leelakusolvong Somchai , Sarr Michael G. , Miller Steven M. , Phillips Sidney F. , Bharucha Adil E.
Published: March 2015

The human colon can dilate, often to life-threatening proportions. Our aim was to explore nitrergic mechanisms underlying colonic dilation in conscious dogs with enterically isolated ileocolonic loops either extrinsically innervated ( n = 4) or extrinsically denervated ( n= 4). We recorded phasic pressures in ileum and ileocolonic sphincter (ICS), colonic tone, compliance, and relaxation during ileal distension. By NADPH-diaphorase histochemistry, we assessed effects of extrinsic denervation and enteric isolation on nitrergic fibers. Extrinsic denervation increased phasic pressures in ileum, ICS, and colon and abolished ICS and colonic relaxation in response to ileal distension. The nitric oxide synthase (NOS) inhibitor N ω-nitro-l-arginine (l-NNA) increased phasic pressures at all sites and ICS tone but did not abolish colonic relaxation during ileal distension in innervated loops. l-NNA reduced compliance and induced colonic high-amplitude propagated contractions in denervated loops. The NOS substrate donor l-arginine reversed effects ofl-NNA. The number of NADPH-diaphorase fibers increased in both enterically isolated preparations. Nonnitrergic extrinsic nerve pathways mediate reflex colonic relaxation during ileal distension. Enteric isolation augments the number of NOS fibers, an effect not modified by extrinsic denervation.
Multichannel intraluminal impedance accurately detects fasting, recumbent reflux events and their clearing
Authors: Shay Steven S. , Bomeli Steven , Richter Joel
Published: March 2015

Multichannel intraluminal impedance (MII) is a new diagnostic test for gastroesophageal reflux disease (GERD). The objective of this report is to determine the accuracy of MII in detecting individual reflux events (REs) identified by pH probe and manometry, as well as their clearing in patients with severe GERD compared with normal volunteers. Ten severe GERD patients and 10 normal volunteers underwent simultaneous manometry [7 sites: gastric, lower esophageal sphincter, esophagus (4), pharynx], pH, and MII (6 sites in esophagus) for 15 min in the left and right recumbent posture while fasting. We found that patients had 30-fold more REs than normal volunteers (41 ± 11 vs. 1.3 ± 0.4), and 95% of all REs were detected by MII. An average 15-fold fall in impedance with liquid and fivefold rise with gas made REs and their composition easy to detect with MII. In the right recumbent posture, nearly all REs detected by MII were liquid (98%, 98/100). In contrast, all 283 REs detected by MII in the left recumbent posture were gas. Nearly all REs detected by MII were cleared (98%, 368/374). Mean acid clearing time was threefold longer (47 s) than clearing time by either manometry (15 s) or MII (13 s), primarily due to acid rereflux, i.e., additional acid REs during acid clearing. We conclude that MII is accurate in detecting REs identified by manometry and/or pH probe, their composition, and their clearing.
Fundamental role of ClC-3 in volume-sensitive Cl- channel function and cell volume regulation in AGS cells
Authors: Jin Nan Ge , Kim Jin Kyoung , Yang Dong Ki , Cho Soo Jin , Kim Jung Mogg , Koh Eun Ju , Jung Hyun Chae , So Insuk , Kim Ki Whan
Published: March 2015

Volume regulation is essential for cell function, but it is unknown which channels are involved in a regulatory volume decrease (RVD) in human gastric epithelial cells. Exposure to a hypotonic solution caused the increase in AGS cell volume, followed by the activation of a current. The reversal potential of the swelling-induced current suggested that Cl- was the primary charge carrier. The selectivity sequence for different anions was I- > Br- > Cl- > F- > gluconate. This current was inhibited by flufenamate, DIDS, tamoxifen, and 5-nitro-2-(3-phenylpropylamino)benzoate. Intracellular dialysis of three different anti-ClC-3 antibodies abolished or attenuated the Cl- current and disrupted RVD, whereas the current and RVD was unaltered by anti-ClC-2 antibody. Immunoblot studies demonstrated the presence of ClC-3 protein in Hela and AGS cells. RT-PCR analysis detected expression of ClC-3, MDR-1, and pICln mRNA in AGS cells. These results suggest a fundamental role of endogenous ClC-3 in the swelling-activated Cl- channels function and cell volume regulation in human gastric epithelial cells.
High-level activation by a duodenum-specific enhancer requires functional GATA binding sites
Authors: Dusing Mary R. , Florence Elizabeth A. , Wiginton Dan A.
Published: March 2015

The purine metabolic gene adenosine deaminase ( ADA) is expressed at high levels in a well-defined spatiotemporal pattern in the villous epithelium of proximal small intestine. A duodenum-specific enhancer module responsible for this expression pattern has been identified in the second intron of the human ADA gene. It has previously been shown that binding of the factor PDX-1 is essential for function of this enhancer. The studies presented here examine the proposed roles of GATA factors in the enhancer. Site-directed mutagenesis of the enhancer's GATA binding sites crippled enhancer function in 10 lines of transgenic mice, with 9 of the lines demonstrating <1% of normal activity. Detailed studies along the longitudinal axis of mouse small intestine indicate that GATA-4 and GATA-5 mRNA levels display a reciprocal pattern, with low levels of GATA-6 throughout. Interestingly, gel shift studies with duodenal nuclear extracts showed binding only by GATA-4.
Controversy: PPARγ as a target for treatment of colorectal cancer
Authors: Gupta Rajnish A. , Dubois Raymond N.
Published: March 2015

Colorectal cancer (CRC) represents a significant cause of morbidity and mortality worldwide. Recently, ligands for the nuclear hormone receptor peroxisome proliferator-activated receptor γ (PPARγ) have exhibited promise in the treatment of CRC. For example, activation of PPARγ reduces the proliferation of cultured CRC cells grown in vitro or in vivo using the nude mouse xenograft model of tumor growth. Furthermore, agonists of the receptor also reduce the development of preneoplastic lesions in a model of carcinogen-induced CRC in rats. However, ligands for the receptor paradoxically enhance intestinal adenoma formation in another murine model of intestinal polyposis, the APCMinmice. These disparate results may be due to the inherent limitations of the APCMinmouse as a model for humans with CRC. Finally, genetic studies identifying loss of function mutations of PPARγ in human CRC specimens strongly suggest a tumor suppressive role for the receptor during the development of CRC.
Helicobacter pyloriinfection activates NF-κB signaling pathway to induce iNOS and protect human gastric epithelial cells from apoptosis
Authors: Kim Jung Mogg , Kim Joo Sung , Jung Hyun Chae , Oh Yu-Kyoung , Chung Hee-Young , Lee Chul-Hoon , Song In Sung
Published: March 2015

Helicobacter pylori infection induces apoptosis and inducible nitric oxide synthase (iNOS) expression in gastric epithelial cells. In this study, we investigated the effects of NF-κB activation and iNOS expression on apoptosis in H. pylori-infected gastric epithelial cells. The suppression of NF-κB significantly increased caspase-3 activity and apoptosis in H. pylori-infected MKN-45 and Hs746T gastric epithelial cell lines as well as primary gastric epithelial cells. An NF-κB signaling pathway via NF-κB-inducing kinase and IκB kinase-β activation was found to be involved in the inhibition of apoptosis in H. pylori-infected gastric epithelial cells. In gastric epithelial cells transfected with retrovirus containing IκBα superrepressor, iNOS mRNA and protein levels were reduced, indicating that H. pylori infection induced the expression of iNOS by activating NF-κB. Moreover, a NO donor, S-nitroso- N-acetylpenicillamine (100 μM), decreased caspase-3 activity and apoptosis in NF-κB-suppressed cells infected with H. pylori. These results suggest that NF-κB activation may play a role in protecting gastric epithelial cells from H. pylori-induced apoptosis by upregulating endogenous iNOS.
Molecular evidence for a glycine-gated chloride channel in macrophages and leukocytes
Authors: Froh Matthias , Thurman Ronald G. , Wheeler Michael D.
Published: March 2015

Recent studies have demonstrated that glycine blunts the response of Kupffer cells to endotoxin. Based on pharmacological evidence, it was hypothesized that Kupffer cells and other macrophages contain a glycine-gated chloride channel similar to the glycine receptor expressed in neuronal tissues. Moreover, glycine stimulates influx of radiolabeled chloride in Kupffer cells in a dose-dependent manner. RT-PCR was used to identify mRNA of both α- and β-subunits of the glycine receptor in rat Kupffer cells, peritoneal neutrophils, and splenic and alveolar macrophages, similar to the sequence generated from rat spinal cord. Importantly, the sequence of the cloned Kupffer cell glycine receptor fragment for the β-subunit was >95% homologous with the receptor from the spinal cord. Membranes of these cells also contain a protein that is immunoreactive with antibodies against the glycine-gated chloride channel. These data demonstrate that Kupffer cells, as well as other macrophages and leukocytes, express mRNA and protein for a glycine-gated chloride channel with both molecular and pharmacological properties similar to the channel expressed in the central nervous system.
Apical EGF receptors regulate epithelial barrier to gastric acid: endogenous TGF-α is an essential facilitator
Authors: Chen Monica C. , Solomon Travis E. , Kui Robert , Soll Andrew H.
Published: March 2015

In previous studies, we found that apical and basolateral EGF receptors (EGFR) on primary canine gastric monolayers decreased paracellular permeability, evident by increased transepithelial electrical resistance (TER) and decreased flux of [3H]mannitol (MF). After studying monolayers in Ussing chambers, we now report that treatment with apical, but not basolateral, EGF enhanced tolerance to apical H+, evident by a slower decay in TER and an attenuated rise in MF. Enhanced tolerance to apical acid was evident within 10 min of treatment with apical EGF. Immunoneutralization of endogenous transforming growth factor (TGF)-α accelerated the drop in TER and the rise in MF in response to apical acidification; apical EGF reversed these effects. Study of monolayers cultured in Transwell inserts showed that immunoblockade of basolateral, but not apical, EGFR also impaired the resistance to apical acidification and enhanced MF. We conclude that apical EGFR regulates the barrier to apical acidification via effects on paracellular resistance. Although exogenous basolateral EGF has a less apparent effect on the barrier to acid, endogenous ligand active at basolateral EGFR plays an important role in maintaining the barrier to apical acid. Our data implicate a role for an apical EGFR ligand, which may be EGF or another member of the EGF family.
Copper deficiency increases iron absorption in the rat
Authors: Thomas Carla , Oates Phillip S.
Published: March 2015

Release of iron from enterocytes and hepatocytes is thought to require the copper-dependent ferroxidase activity of hephaestin (Hp) and ceruloplasmin (Cp), respectively. In swine, copper deficiency (CD) impairs iron absorption, but whether this occurs in rats is unclear. By feeding a diet deficient in copper, CD was produced, as evidenced by the loss of copper-dependent plasma ferroxidase I activity, and in enterocytes, CD reduced copper levels and copper-dependent oxidase activity. Hematocrit was reduced, and liver iron was doubled. CD reduced duodenal mucosal iron and ferritin, whereas CD increased iron absorption. Duodenal mucosal DMT1-IRE and ferroportin1 expression remained constant with CD. When absorption in CD rats was compared with that seen normally and in iron-deficient anemic animals, strong correlations were found among mucosal iron, ferritin, and iron absorption, suggesting that the level of iron absorption was appropriate given that the erythroid and stores stimulators of iron absorption are opposed in CD. Because CD reduced the activity of Cp, as evidenced by copper-dependent plasma ferroxidase I activity and hepatocyte iron accumulation, but iron absorption increased, it is unlikely that the ferroxidase activity of Hp is important and suggests another function for this protein in the export of iron from the enterocyte during iron absorption. Also, the copper-dependent ferroxidase activity of Cp does not appear important for iron efflux from macrophages, because Kupffer cells of the liver and nonheme iron levels of the spleen were normal during copper deficiency, suggesting another role for Cp in these cells.
II. Proteomics: core technologies and applications in physiology
Authors: Witzmann Frank A. , Li Junyu
Published: March 2015

Technologies for proteomics, e.g., studies examining the protein complement of the genome, have been in development for over 20 years. More recently, proteomics has become formalized by combining techniques for large-scale protein separation with very precise, high-fidelity approaches that analyze, identify, and characterize the separated proteins. These methods bring to reality the powerful scope of proteomics, enabling researchers to investigate cellular function at the protein level and thus representing one of proteomics' most fitting applications. In this review, we take a brief and concise look at some of the current, physiologically relevant technologies that comprise proteomics and report specific applications in which proteomics has provided valuable biological insight.
H+-peptide cotransport in the human bile duct epithelium cell line SK-ChA-1
Authors: Knütter Ilka , Rubio-Aliaga Isabel , Boll Michael , Hause Gerd , Daniel Hannelore , Neubert Klaus , Brandsch Matthias
Published: March 2015

This study describes for the first time the presence of H+-peptide cotransport in cells of the bile duct. Uptake of [glycine-1-14C]glycylsarcosine ([14C]Gly-Sar) in human extrahepatic cholangiocarcinoma SK-ChA-1 cells was stimulated sevenfold by an inwardly directed H+ gradient. Transport was mediated by a low-affinity system with a transport constant ( K t) value of 1.1 mM. Several dipeptides, cefadroxil, and δ-aminolevulinic acid, but not glycine and glutathione, were strong inhibitors of Gly-Sar uptake. SK-ChA-1 cells formed tight, polarized monolayers on permeable membranes. The transepithelial electrical resistance was 856 ± 29 Ω × cm2. The transepithelial flux of [14C]Gly-Sar in apical-to-basolateral direction exceeded the basolateral-to-apical flux 11-fold. Uptake was 20-fold higher from the apical side. RT-PCR analysis using primer pairs specific for the intestinal-type peptide transporter (PEPT1) or kidney-type (PEPT2) revealed that the transport system expressed in SK-ChA-1 and also in cells of the native rabbit bile duct is PEPT1. Immunohistochemistry localized PEPT1 to the apical membrane of cholangiocytes of mouse extrahepatic biliary duct. We conclude that the cells of the mammalian extrahepatic biliary tract epithelium express the intestinal-type H+-peptide cotransporter in their apical membrane. SK-ChA-1 cells represent a convenient model to study the physiological and clinical aspects of peptide transport in cholangiocytes.
Role of luminal ATP in regulating electrogenic Na+absorption in guinea pig distal colon
Authors: Yamamoto Takeshi , Suzuki Yuichi
Published: March 2015

Extracellular ATP regulates a variety of functions in epithelial tissues by activating the membrane P2-receptor. The purpose of this study was to investigate the autocrine/paracrine regulation by luminal ATP of electrogenic amiloride-sensitive Na+absorption in the distal colon from guinea pigs treated with aldosterone by measuring the amiloride-sensitive short-circuit current ( Isc) and22Na+flux in vitro with the Ussing chamber technique. ATP added to the luminal side inhibited the amiloride-sensitive Iscand22Na+absorption to a similar degree. The concentration dependence of the inhibitory effect of ATP on amiloride-sensitive Ischad an IC50value of 20–30 μM, with the maximum inhibition being ∼50%. The effects of different nucleotides and of a nucleoside were also studied, the order of potency being ATP = UTP > ADP > adenosine. The effects of ATP were slightly, but significantly, reduced in the presence of suramin in the luminal solution. The inhibitory effect of luminal ATP was more potent in the absence of both Mg2+and Ca2+from the luminal solution. Pretreatment of the tissue with ionomycin or thapsigargin in the absence of serosal Ca2+did not affect the percent inhibition of amiloride-sensitive Iscinduced by ATP. Mechanical perturbation with a hypotonic luminal solution caused a reduction in amiloride-sensitive Isc, this effect being prevented by the presence of hexokinase, an ATP-scavenging enzyme. These results suggest that ATP released into the luminal side by hypotonic stimulation could exert an inhibitory effect on the electrogenic Na+absorption. This effect was probably mediated by a P2Y2receptor on the apical membrane of colonic epithelial cells, and a change in the intracellular Ca2+concentration may not be necessary for this process.
Helicobacter pyloriinduces apoptosis of rat gastric parietal cells
Authors: Neu Bruno , Randlkofer Pamela , Neuhofer Mathilde , Voland Petra , Mayerhofer Artur , Gerhard Markus , Schepp Wolfgang , Prinz Christian
Published: March 2015

Gastric Helicobacter pylori infection may lead to multifocal atrophic corpus gastritis associated with loss of epithelial cells as well as glandular structures. The current work investigated H. pylori effects on cell death of isolated, nontransformed rat parietal cells (PC). Highly enriched rat PC (>97%) were isolated from gastric mucosa and cultured in serum-free medium over 24 h. The cells were cocultured over 8 h with cytotoxin-associated immunodominant protein (cagA)+/vacuolating toxin (vacA)+or with cagA−/vacA−H. pylori laboratory strains and also with H. pylori mutants deleted in several genes of the cag pathogenicity island. Staphylococcus aureus or Campylobacter jejuni were used as controls. Apoptosis was determined by terminal deoxynucleotidyl transferase dUTP nick-end labeling staining and electron microscopy. Interleukin (IL)-8 and cytokine-induced neutrophil chemoattractant (CINC)-1 secretion was measured by ELISA. Activation of nuclear factor-κB (NF-κB) was studied in nuclear extracts of PC by electrophoretic mobility shift assay. Apoptosis of PC was induced in a concentration- and time-dependent manner by cagA+/vacA+H. pylori strains but not by cagA−/vacA−negative strains or by the cagE knockout mutant. S. aureusand C. jejuni had no effect. PC showed no IL-8 or CINC-1 secretion on exposure to cagA+/vacA+H. pylori. cagA+/vacA+strains induced activation of NF-κB complexes in nuclear extracts of PC, which were composed of p65 and p50 subunits. No significant stimulation of NF-κB activation was detected by incubation of PC with the cagE knockout mutant. Preincubation of PC with antisense but not missense oligodeoxynucleotides against the p65 subunit significantly reduced DNA binding to the κB recognition sequence. The p65 oligonucleotides as well as the proteasome inhibitor N-CBZ-isoleucin-glutamin-(o-t-butyl-)-alanin-leucin and the nitric oxide synthase inhibitor NG-monomethyl-l-arginine completely prevented PC apoptosis induced by cagA+/vacA+strains. In summary, cagE presence appears to be essential for H. pylori-induced apoptosis of gastric parietal cells, and this effect is dependent on the activation of NF-κB and production of nitric oxide.
Lipopolysaccharides in liver injury: molecular mechanisms of Kupffer cell activation
Authors: Su Grace L.
Published: March 2015

Endogenous gut-derived bacterial lipopolysaccharides have been implicated as important cofactors in the pathogenesis of liver injury. However, the molecular mechanisms by which lipopolysaccharides exert their effect are not entirely clear. Recent studies have pointed to proinflammatory cytokines such as tumor necrosis factor-α as mediators of hepatocyte injury. Within the liver, Kupffer cells are major sources of proinflammatory cytokines that are produced in response to lipopolysaccharides. This review will focus on three important molecular components of the pathway by which lipopolysaccharides activate Kupffer cells: CD14, Toll-like receptor 4, and lipopolysaccharide binding protein. Within the liver, lipopolysaccharides bind to lipopolysaccharide binding protein, which then facilitates its transfer to membrane CD14 on the surface of Kupffer cells. Signaling of lipopolysaccharide through CD14 is mediated by the downstream receptor Toll-like receptor 4 and results in activation of Kupffer cells. The role played by these molecules in liver injury will be examined.
Effect of maternal chronic hypoxic exposure during gestation on apoptosis in fetal rat heart
Authors: Bae Soochan , Xiao Yuhui , Li Guohu , Casiano Carlos A. , Zhang Lubo
Published: March 2015

Chronic hypoxia during pregnancy is one of the most common insults to fetal development. We tested the hypothesis that maternal hypoxia induced apoptosis in the hearts of near-term fetal rats. Pregnant rats were divided into two groups, normoxic control and continuous hypoxic exposure (10.5% O2) from day 15 to 21 of gestation. Hearts were isolated from fetal rats of 21-day gestational age. Maternal hypoxia increased hypoxia-inducible factor-1α protein in fetal hearts. Chronic hypoxia significantly increased the percentage and size of binucleated myocytes and increased apoptotic cells from 1.4 ± 0.14% to 2.7 ± 0.3% in the fetal heart. In addition, the active cleaved form of caspase 3 was significantly increased in the hypoxic heart, which was associated with an increase in caspase 3 activity. There was a significant increase in Fas protein levels in the hypoxic heart. Chronic hypoxia did not change Bax protein levels but significantly decreased Bcl-2 proteins. In addition, chronic hypoxia significantly suppressed expression of heat shock protein 70. However, chronic hypoxia significantly increased expression of the anti-apoptotic protein 14–3-3 θ, among other 14–3-3 isoforms. Chronic hypoxia differentially regulated β-adrenoreceptor (β-AR) subtypes with an increase in β1-AR levels but no changes in β2-AR. The results demonstrate that maternal hypoxia increases apoptosis in fetal rat heart, which may be mediated by an increase in Fas and a decrease in Bcl-2 proteins. Chronic hypoxia-mediated increase in β1-AR and decrease in heat shock proteins may also play an important role in apoptosis in the fetal heart.
Central command blunts the baroreflex bradycardia to aortic nerve stimulation at the onset of voluntary static exercise in cats
Authors: Komine Hidehiko , Matsukawa Kanji , Tsuchimochi Hirotsugu , Murata Jun
Published: March 2015

To examine whether the central characteristics of the aortic baroreflex alter from moment to moment during static exercise, we identified the dynamic changes in the sizes of the bradycardia and depressor response evoked by stimulation of the aortic depressor nerve (ADN). Three conscious cats were trained to voluntarily extend the right forelimb and press a bar for 31 ± 1 s with a peak force of 337 ± 22 g while maintaining a sitting posture. The ADN stimulation-induced bradycardia was attenuated at the initial period of exercise (up to 8 s from the exercise onset) to 62 ± 5% of the preexercise bradycardia and remained blunted until the end of exercise. The most blunted bradycardia was observed immediately before or when the forelimb was extended before force development. The baroreflex-induced bradycardia was suppressed again at cessation of exercise when the forelimb was retracted and recovered within a few seconds. In contrast, static exercise did not affect the ADN stimulation-induced depressor response. The ADN stimulation-induced bradycardia was also blunted at the beginning of naturally occurring body movement such as spontaneous postural change or grooming behavior. Thus it is likely that the central characteristics of the aortic baroreflex dynamically change from moment to moment during voluntary static exercise and during natural body movement and that particularly a central inhibition of the cardiac component of the aortic baroreflex is induced by central command at the onset of static exercise, whereas the central property of the vasomotor component of the baroreflex is preserved.
Cardioprotection by multiple preconditioning cycles does not require mitochondrial KATP channels in pigs
Authors: Schwartz Lisa M. , Welch Timothy S. , Crago Mark S.
Published: March 2015

To test whether cardioprotection induced by ischemic preconditioning depends on the opening of mitochondrial ATP-sensitive K+(KATP) channels, the effect of channel blockade was studied in barbital-anesthetized open-chest pigs subjected to 30 min of complete occlusion of the left anterior descending coronary artery and 3 h of reflow. Preconditioning was elicited by two cycles of 5-min occlusion plus 10-min reperfusion before the 30-min occlusion period. 5-Hydroxydecanoate (5 mg/kg iv) was injected 15 min before preconditioning or pharmacological preconditioning induced by diazoxide (3.5 mg/kg, 1 ml/min iv). Infarct size (percentage of the area at risk) after 30 min of ischemia was 35.1 ± 9.9% ( n = 7). Preconditioning markedly limited myocardial infarct size (2.7 ± 1.6%, n = 7), and 5-hydroxydecanoate did not abolish protection (2.4 ± 0.9%, n = 8). Diazoxide infusion also significantly limited infarct size (14.6 ± 7.4%, n = 7), and 5-hydroxydecanoate blocked this effect (30.8 ± 8.0%, n = 7). Thus the opening of mitochondrial KATP channels is cardioprotective in pigs, but these data do not support the hypothesis that opening of mitochondrial KATP channels is required for the endogenous protection afforded by preconditioning.
ACE and non-ACE mediated effect of angiotensin I on intracellular calcium mobilization in rat glomerular arterioles
Authors: Marchetti Jeannine , Helou Claudia M. B. , Chollet Catherine , Rajerison Rabary , Alhenc-Gelas François
Published: March 2015

Because renin and angiotensin I (ANG I) level are high in the renal circulation, the conversion of ANG I is a critical step in the regulation of glomerular hemodynamics. We studied this conversion by investigating the effect of ANG I on intracellular Ca2+concentration ([Ca2+]i) in rat juxtamedullary glomerular afferent and efferent arterioles (AA and EA, respectively). Two types of EA were considered, thin EA and muscular EA, terminating as peritubular capillaries and vasa rectae, respectively. In all arterioles, ANG I elicited [Ca2+]ielevations. Maximal responses of 171 ± 28 (AA), 183 ± 7 (muscular EA), and 78 ± 11 nM (thin EA) ( n = 6), similar to those obtained with ANG II, were observed with 100 nM ANG I. The EC50values were 20 times higher for ANG I than for ANG II in AA (10.2 vs. 0.5) and muscular EA (6.8 vs. 0.4 nM) and 150 times higher in thin EA (15.2 vs. 0.1 nM). ANG I effect was blocked by losartan, indicating that AT1receptors were involved. The ANG-converting enyzme (ACE) inhibitor lisinopril inhibited the maximal response to ANG I in AA and muscular EA by 75 ± 9% ( n = 13) and 70 ± 7% ( n = 13), respectively, but had no effect in thin EA ( n = 14). The serine protease inhibitor aprotinin, the chymase inhibitor chymostatin, and the cysteine protease inhibitors E64 and leupeptin had no effect on ANG I action. These data show that ANG I effects are mainly mediated by ACE in AA and muscular EA but not in thin EA. The lisinopril-insensitive response may be related to conversion by unknown enzyme(s) and/or to activation of AT1receptors by ANG I.
Right ventricular volume measurement by conductance catheter
Authors: Danton Mark H. D. , Greil Gerald F. , Byrne John G. , Hsin Michael , Cohn Lawrence , Maier Stephan E.
Published: March 2015

Continuous ventricular volume measurement by the conductance method assumes a homogeneous electrical field dispersed throughout and contained within the ventricle. Because of dense trabeculation and complex geometry, right ventricular (RV) volume description by this method may be seriously compromised. This study sought to determine the accuracy and limitations of RV volume measurement by conductance, with magnetic resonance (MR) imaging (MRI) used as a reference, in the porcine RV. Anesthetized pigs ( n = 5, 45–55 kg) were placed in a 1.5-T magnet, and ECG-gated transverse MR images (5-mm slices) were acquired during the complete cardiac cycle. RV cavity volumes were subsequently determined by Simpson's technique. Animals were then instrumented with an RV conductance catheter and an ultrasonic pulmonary artery flow probe. Conductance catheter signals were recorded using single- and dual-field (SF and DF) excitation, and the saline-dilution technique was used to correct volumes for parallel conductance. The gain factor (α) was calculated as the ratio of conductance- to MRI-derived stroke volume (αSV). Variation of α during the cardiac cycle was computed by comparing RV conductance volumes with 1) MRI volumes at isochronal time points within the cardiac cycle [α( t)] and 2) the pulmonary flow integral during ejection. After calibration, the conductance-MRI volume relation was modeled linearly with good correlation [ r = 0.96 (SF) and r = 0.94 (DF)], close to the line of identity. Individual conductance-MRI plots displayed a slight curvilinear relation that was concave toward the MRI axis. Consistent with this finding, α( t) varied significantly during the cardiac cycle (0.49 and 0.39 by SF for end systole and end diastole, respectively, P = 0.011). DF excitation resulted in improved volume measurement [αSV = 0.41 (SF) and 0.96 (DF)], with less variation in α( t) (1.0 and 0.92 by DF for end systole and end diastole, respectively, P = 0.66). These results indicate that, with calibration, the conductance method can measure absolute RV volume under steady-state conditions. However, the curvilinearity and α( t) variation would indicate the potential for nonlinearity when RV volumes are varied over a wider range.
Effect of cardiac pacing on forearm vascular responses and nitric oxide function
Authors: Green Daniel , Cheetham Craig , Henderson Chelsea , Weerasooriya Rukshen , O'Driscoll Gerard
Published: March 2015

We examined the hypothesis that changes in heart rate at rest influence bioactivity of nitric oxide (NO) in humans by examining forearm blood flow responses during cardiac pacing in six subjects. Peak forearm and mean forearm blood flows across the cardiac cycle were continuously recorded at baseline and during pacing, with the use of high-resolution brachial artery ultrasound and Doppler flow velocity measurement. The brachial artery was cannulated to allow continuous infusion of saline or N G-monomethyl-l-arginine (l-NMMA). As heart rate increased, no changes in pulse pressure and mean or peak blood flow were evident. l-NMMA had no effect on brachial artery diameter, velocity, or flows compared with saline infusion. These results contrast with our recent findings that exercise involving the lower body, associated with increases in heart rate and pulse pressure, also increased forearm blood flow, the latter response being diminished by l-NMMA. These data suggest that changes in blood pressure, rather than pulse frequency, may be the stimulus for shear stress-mediated NO release in vivo.
Inducible nitric oxide synthase dimerization inhibitor prevents cardiovascular and renal morbidity in sheep with combined burn and smoke inhalation injury
Authors: Enkhbaatar Perenlei , Murakami Kazunori , Shimoda Katsumi , Mizutani Akio , Traber Lillian , Phillips Gary , Parkinson John , Salsbury John R. , Biondo Nettie , Schmalstieg Frank , Burke Ann , Cox Robert , Hawkins Hal , Herndon David , Traber Daniel
Published: March 2015

Inducible nitric oxide synthase (iNOS) is implicated in the pathogenesis of acute respiratory distress syndrome (ARDS). ARDS treatment is frequently complicated by significant extrapulmonary comorbidity. This study was designed to clarify the role of iNOS in mediating extrapulmonary comorbidity in sheep after combined burn and smoke inhalation injuries using a potent and highly selective iNOS dimerization inhibitor, BBS-2. Twenty-two female sheep were operatively prepared. After 5 days of recovery, tracheostomy was performed under ketamine-halothane anesthesia. Sheep were given a 40% total body surface third-degree burns and insufflated with cotton smoke (48 breaths, <40°C). Sheep were divided into four groups: noninjured and nontreated (sham group; n = 6), noninjured but treated with BBS-2 (sham/BBS-2 group; n = 4), injured but nontreated (control group, n = 6), and injured but treated with 100 μg · kg–1 · h–1 BBS-2 (BBS-2 group; n = 6). Evaluation was in a laboratory intensive care unit setting for 48 h. The sham group had stable cardiopulmonary and systemic hemodynamics. Control animals showed multiple signs of morbidity. Decreased left ventricular stroke work index and stroke volume index with elevated left atrial pressure indicated myocardial depression. Systemic vascular leak was evidenced by robust hemoconcentration, decreased plasma oncotic pressure, and increased transvascular fluid flux into the lymphatic system. Finally, severely impaired renal function (urinary output) was associated with adverse net fluid balance. Treatment with BBS-2 prevented all these morbidities without adversely effecting cardiovascular hemodynamics such as cardiac index and mean arterial pressure. The results identify a major role for iNOS in mediating extrapulmonary comorbidity in a clinically relevant and severe trauma model and support the use of highly selective iNOS inhibitors as novel treatments in critical care medicine.
Dynamics of microvascular oxygen pressure during rest-contraction transition in skeletal muscle of diabetic rats
Authors: Behnke Bradley J. , Kindig Casey A. , McDonough Paul , Poole David C. , Sexton William L.
Published: March 2015

Type I diabetes reduces dramatically the capacity of skeletal muscle to receive oxygen (Q˙o 2). In control (C; n = 6) and streptozotocin-induced diabetic (D: n = 6, plasma glucose = 25.3 ± 3.9 mmol/l and C: 8.3 ± 0.5 mmol/l) rats, phosphorescence quenching was used to test the hypothesis that, in D rats, the decline in microvascular Po 2 [PmO2 , which reflects the dynamic balance between O2 utilization (V˙o 2) andQ˙o 2] of the spinotrapezius muscle after the onset of electrical stimulation (1 Hz) would be faster compared with that of C rats. PmO2 data were fit with a one or two exponential process (contingent on the presence of an undershoot) with independent time delays using least-squares regression analysis. In D rats, PmO2 at rest was lower (C: 31.2 ± 3.2 mmHg; D: 24.3 ± 1.3 mmHg, P < 0.05) and at the onset of contractions decreased after a shorter delay (C: 13.5 ± 1.8 s; D: 7.6 ± 2.1 s, P < 0.05) and with a reduced mean response time (C: 31.4 ± 3.3 s; D: 23.9 ± 3.1 s, P < 0.05). PmO2 exhibited a marked undershoot of the end-stimulation response in D muscles (D: 3.3 ± 1.1 mmHg, P < 0.05), which was absent in C muscles. These results indicate an alteredV˙o 2-to-Q˙o 2matching across the rest-exercise transition in muscles of D rats.