bluuebunny/crossref_metadata_2025 huggingface dataset:
Separate pathways for cellular uptake of ferric and ferrous iron
Authors: Conrad Marcel E. , Umbreit Jay N. , Moore Elizabeth G. , Hainsworth Lucille N. , Porubcin Michael , Simovich Marcia J. , Nakada Marian T. , Dolan Kevin , Garrick Michael D.
Published: December 2017

Separate pathways for transport of nontransferrin ferric and ferrous iron into tissue cultured cells were demonstrated. Neither the ferric nor ferrous pathway was shared with either zinc or copper. Manganese shared the ferrous pathway but had no effect on cellular uptake of ferric iron. We postulate that ferric iron was transported into cells via β3-integrin and mobilferrin (IMP), whereas ferrous iron uptake was facilitated by divalent metal transporter-1 (DMT-1; Nramp-2). These conclusions were documented by competitive inhibition studies, utilization of a β3-integrin antibody that blocked uptake of ferric but not ferrous iron, development of an anti-DMT-1 antibody that blocked ferrous iron and manganese uptake but not ferric iron, transfection of DMT-1 DNA into tissue culture cells that showed enhanced uptake of ferrous iron and manganese but neither ferric iron nor zinc, hepatic metal concentrations in mk mice showing decreased iron and manganese but not zinc or copper, and data showing that the addition of reducing agents to tissue culture media altered iron binding to proteins of the IMP and DMT-1 pathways. Although these experiments show ferric and ferrous iron can enter cells via different pathways, they do not indicate which pathway is dominant in humans.
Role of gut-enriched Krüppel-like factor in colonic cell growth and differentiation
Authors: Shie Jue-Lon , Chen Zhi Y. , O'Brien Michael J. , Pestell Richard G. , Lee Mu-En , Tseng Chi-Chuan
Published: December 2017

Cancer cells differ from normal cells in many aspects, including hyperproliferation and loss of differentiation. Recent research has focused on the role of transcription factors in regulating abnormal cell growth. Gut-enriched Krüppel-like factor (GKLF) is a newly identified eukaryotic zinc finger protein expressed extensively in the gastrointestinal tract. In the current study, we demonstrated that GKLF mRNA levels were significantly decreased in the dysplastic epithelium of the colon, including adenomatous polyp and cancer. GKLF immunostains in the normal colon were higher at the surface epithelium and gradually decreased toward the crypt, but this gradient was not present in the adenomatous and cancerous mucosa. Constitutive overexpression of GKLF DNA in a human colonic adenocarcinoma cell line (HT-29) decreased [3H]thymidine incorporation, whereas suppression of GKLF gene increased DNA synthesis, indicating that downregulation of the GKLF gene might contribute to cellular hyperproliferation. Cyclin D1 (CD1) protein level and CD1-associated kinase activity were decreased in HT-29 cell overexpressed GKLF cDNA, and CD1 promoter activity was profoundly suppressed by GKLF. When HT-29 cells were cultured in the presence of sodium butyrate, GKLF mRNA levels increased as cells acquired more differentiated phenotypes. These results suggest that GKLF plays an important role in regulating cell growth and differentiation in the colonic epithelium and that downregulation of GKLF expression may cause colonic cells to become hyperproliferative. Furthermore, GKLF appears to be a transcriptional repressor of the CD1 gene.
Inwardly rectifying K+ channels in esophageal smooth muscle
Authors: Ji Junzhi , Salapatek Anne Marie F. , Diamant Nicholas E.
Published: December 2017

The whole cell patch-clamp technique was used to investigate whether there were inwardly rectifying K+(Kir) channels in the longitudinal muscle of cat esophagus. Inward currents were observable on membrane hyperpolarization negative to the K+ equilibrium potential ( E k) in freshly isolated esophageal longitudinal muscle cells. The current-voltage relationship exhibited strong inward rectification with a reversal potential ( E rev) of −76.5 mV. Elevation of external K+ increased the inward current amplitude and positively shifted its E rev after the E k, suggesting that potassium ions carry this current. External Ba2+ and Cs+ inhibited this inward current, with hyperpolarization remarkably increasing the inhibition. The IC50 for Ba2+ and Cs+ at −60 mV was 2.9 and 1.6 mM, respectively. Furthermore, external Ba2+ of 10 μM moderately depolarized the resting membrane potential of the longitudinal muscle cells by 6.3 mV while inhibiting the inward rectification. We conclude that Kir channels are present in the longitudinal muscle of cat esophagus, where they contribute to its resting membrane potential.
I. Stress and hepatic inflammation
Authors: Swain Mark G.
Published: December 2017

Stress is an ever-present part of modern life. The “stress response” constitutes an organism's mechanism for coping with a given stress and is mediated via the release of glucocorticoids and catecholamines. Patients often complain of stress-related worsening of their liver disease; however, the interrelationship between stress and hepatic inflammation is incompletely understood and has received little scientific attention. Considering the broad impact glucocorticoids and catecholamines have on immune cell function, it is very likely that stress has a significant impact on the hepatic inflammatory response. This themes article discusses studies of the stress response and its peripheral effectors (glucocorticoids and catecholamines) in liver disease and their impact on hepatic inflammation and outlines potential areas for future scientific investigation.
NO donor sodium nitroprusside inhibits excitation-contraction coupling in guinea pig taenia coli
Authors: Kwon Seong-Chun , Ozaki Hiroshi , Karaki Hideaki
Published: December 2017

In guinea pig taenia coli, the nitric oxide (NO) donor sodium nitroprusside (SNP, 1 μM) reduced the carbachol-stimulated increases in muscle force in parallel with a decrease in intracellular Ca2+concentration ([Ca2+]i). A decrease in the myosin light chain phosphorylation was also observed that was closely correlated with the decrease in [Ca2+]i. With the patch-clamp technique, 10 μM SNP decreased the peak Ba2+current, and this effect was blocked by an inhibitor of soluble guanylate cyclase. Carbachol (10 μM) induced an inward current, and this effect was markedly inhibited by SNP. SNP markedly increased the depolarization-activated outward K+currents, and this current was completely blocked by 0.3 μM iberiotoxin. SNP (1 μM) significantly increased cGMP content without changing cAMP content. Decreased Ca2+sensitivity by SNP of contractile elements was not prominent in the permeabilized taenia, which was consistent with the [Ca2+]i-force relationship in the intact tissue. These results suggest that SNP inhibits myosin light chain phosphorylation and smooth muscle contraction stimulated by carbachol, mainly by decreasing [Ca2+]i, which resulted from the combination of the inhibition of voltage-dependent Ca2+channels, the inhibition of nonselective cation currents, and the activation of Ca2+-activated K+currents.
Interferon-γ downregulates ion transport in murine small intestine cultured in vitro
Authors: Yoo Dongjin , Lo Winson , Goodman Stephen , Ali Wasif , Semrad Carol , Field Michael
Published: December 2017

Effects of IFN-γ on mammalian small intestinal ion transport were studied in vitro using incubated sheets of murine small intestine in Ussing chambers. In oxygenated standard culture medium containing hydrocortisone and antibiotics, they maintained their short-circuit current ( Isc) responses to glucose and theophylline for 48 h. Histological examination revealed a 50% diminution of villus height over 36 h but no change in crypts. Height was better maintained during a 36-h incubation of small intestine from SCID mice, suggesting a role for B or T lymphocytes in villus atrophy. Exposure of small intestine to 100 U/ml IFN-γ for 36 h decreased basal Iscby 40% and Iscresponses to glucose and theophylline by ∼70%; at 1,000 U/ml for 36 h, IFN-γ inhibited these Iscresponses by 90%. An inhibitor of inducible NO synthase did not reverse these effects, suggesting that they are not mediated by NO. Tissue resistance, mucosal K+content, and epithelial morphology were not affected. Ouabain-sensitive ATPase activity in homogenates was inhibited 60% by IFN-γ (100 U/ml for 36 h). IFN-γ inhibition of Iscresponses to glucose and theophylline also occurred in SCID mouse small intestine. Thus murine small intestinal sheets can be maintained viable in vitro for at least 48 h, although villus blunting develops (but less so in SCID mouse small intestine). Also, prolonged exposure to IFN-γ downregulates Na+-coupled glucose absorption, active Cl−secretion, and Na+-K+-ATPase activity, effects unlikely to be mediated by enhanced NO.
Expression of small heat shock protein αB-crystallin is induced after hepatic stellate cell activation
Authors: Lang Alon , Schrum Laura W. , Schoonhoven Robert , Tuvia Shmuel , Solís-Herruzo Jose A. , Tsukamoto Hidekazu , Brenner David A. , Rippe Richard A.
Published: December 2017

Using the differential PCR display method to select cDNA fragments that are differentially expressed after hepatic stellate cell (HSC) activation, we have isolated from activated HSCs a cDNA that corresponds to rat αB-crystallin. Northern blots confirmed expression of αB-crystallin in culture-activated HSCs but not in quiescent HSCs. Western blot analysis and immunocytochemical staining confirmed expression of αB-crystallin protein in activated but not quiescent HSCs. αB-crystallin is induced as early as 6 h after plating HSCs on plastic and continues to be expressed for 14 days in culture. Expression of αB-crystallin was also induced in vivo in activated HSCs from experimental cholestatic liver fibrosis. Confocal microscopy demonstrated a cytoplasmic distribution of αB-crystallin in a cytoskeletal pattern. Heat shock treatment resulted in an immediate perinuclear redistribution that in time returned to a normal cytoskeletal distribution. The expression pattern of αB-crystallin was similar to that of HSP25, another small heat shock protein, but differed from the classic heat shock protein HSP70. Therefore, αB-crystallin represents an early marker for HSC activation.
Microbes and Microbial Toxins: Paradigms for Microbial- Mucosal Interactions I. Pathophysiological aspects of enteric infections with the lumen-dwelling protozoan pathogenGiardia lamblia
Authors: Eckmann Lars , Gillin Frances D.
Published: December 2017

Giardia lamblia is one of the most important causes of waterborne diarrheal disease worldwide, and giardiasis is the most common protozoan infection of the human small intestine. Symptomatic infection is characterized by diarrhea, abdominal pain, and malabsorption, leading to malnutrition and weight loss, particularly in children. The pathogen resides strictly in the lumen of the small intestine, and infection is typically not accompanied by significant mucosal inflammation. Clinical and experimental studies indicate that B cell-dependent host defenses, particularly IgA, are important for controlling and clearing Giardia infection, although B cell-independent mechanisms also contribute to this outcome. In contrast to antigiardial host defenses, much less is known about the pathophysiological mechanisms underlying the clinical symptoms of giardiasis, partly because of the current lack of suitable model systems. In addition to being an important human enteric pathogen, Giardia is an interesting model organism for gaining basic insights into genetic innovations that led to evolution of eukaryotic cells, since it belongs to the earliest diverging eukaryotic lineage known. The completion of the giardial genome project will increase understanding of the basic biology of the protozoan and will help us to better understand host pathogen-interactions as a basis for developing new vaccination and therapeutic strategies.
Integrin and FAK-mediated MAPK activation is required for cyclic strain mitogenic effects in Caco-2 cells
Authors: Li Wei , Duzgun Aydin , Sumpio Bauer E. , Basson Marc D.
Published: December 2017

Rhythmic strain stimulates Caco-2 proliferation. We asked whether mitogen-activated protein kinase (MAPK) activation mediates strain mitogenicity and characterized upstream signals regulating MAPK. Caco-2 cells were subjected to strain on collagen I-precoated membranes or antibodies to integrin subunits. Twenty-four hours of cyclic strain increased cell numbers compared with static conditions. MAPK-extracellular signal-regulated kinase (ERK) kinase inhibition (20 μM PD-98059) blocked strain mitogenicity. p38 Inhibition (10 μM SB-202190) did not. Strain rapidly and time-dependently activated focal adhesion kinase (FAK), paxillin, ERK1 and 2, and p38 on collagen. c-Jun NH2-terminal kinase (JNK)1 and 2 exhibited delayed activation. Similar activation occurred when Caco-2 cells were subjected to strain on a substrate of functional antibody to the α2-, α3-, α6-, or β1-integrin subunits but not on a substrate of functional antibody to the α5-subunit. FAK inhibition by FAK397 transfection blocked ERK2 and JNK1 activation by in vitro kinase assays, but pharmacological protein kinase C inhibition did not block ERK1 or 2 activation by strain. Strain-induced ERK signals mediate strain's mitogenic effects and may require integrins and FAK activation.
Processing of vitamin A and E in the human gastrointestinal tract
Authors: Borel Patrick , Pasquier Berengere , Armand Martine , Tyssandier Viviane , Grolier Pascal , Alexandre-Gouabau Marie-Cecile , Andre Marc , Senft Michele , Peyrot Jacques , Jaussan Veronique , Lairon Denis , Azais-Braesco Veronique
Published: December 2017

We aimed to provide basic data on the processing of vitamin A and E in the human gastrointestinal tract and to assess whether the size of emulsion fat globules affects the bioavailability of these vitamins. Eight healthy men received intragastrically two lipid formulas differing in their fat-globule median diameter (0.7 vs. 10.1 μm). Formulas provided 28 mg vitamin A as retinyl palmitate and 440 mg vitamin E as all- rac α-tocopherol. Vitamins were measured in gastric and duodenal aspirates, as well as in chylomicrons, during the postprandial period. The gastric emptying rate of lipids and vitamin A and E was similar. The free retinol/total vitamin A ratio was not significantly modified in the stomach, whereas it was dramatically increased in the duodenum. The proportion of ingested lipid and vitamins was very similar in the duodenal content. The chylomicron response of lipids and vitamins was not significantly different between the two emulsions. Our main conclusions are as follows: 1) there is no significant metabolism of vitamin A and E in the human stomach, 2) the enzyme(s) present in the duodenal lumen is significantly involved in the hydrolysis of retinyl esters, and 3) the size of emulsion fat globules has no major effect on the overall absorption of vitamin A and E.
Sequential activation of heterotrimeric and monomeric G proteins mediates PLD activity in smooth muscle
Authors: Murthy K. S. , Zhou H. , Grider J. R. , Makhlouf G. M.
Published: December 2017

The identity of G proteins mediating CCK-stimulated phospholipase D (PLD) activity was determined in intestinal smooth muscle cells. CCK-8 activated Gq/11, G13, and G12, and the monomeric G proteins Ras-homology protein (RhoA) and ADP ribosylation factor (ARF). Activation of RhoA, but not ARF, was mediated by G13and inhibited by Gα13antibody. CCK-stimulated PLD activity was partly mediated by RhoA and could be inhibited to the same extent (47 ± 2% to 53 ± 6%) by 1) a dominant negative RhoA mutant, 2) RhoA antibody or Gα13antibody, and 3) Clostridium botulinum C3 exoenzyme. PLD activity was also inhibited by ARF antibody, and the effect was additive to that of RhoA antibody or C3 exoenzyme. PLD activity was inhibited by calphostin C, bisindolylmaleimide I, and a selective protein kinase C (PKC)-α inhibitor; the inhibition was additive to that of ARF and RhoA antibodies and C3 exoenzyme. In contrast, activated G12was not coupled to RhoA or ARF, and Gα12antibody augmented PLD activity. Thus agonist-stimulated PLD activity is mediated additively by G13-dependent RhoA and by ARF and PKC-α and is modulated by an inhibitory G12-dependent pathway.
Insulin-like growth factor I and insulin-like growth factor binding protein 5 in Crohn's disease
Authors: Zimmermann E. M. , Li L. , Hou Y. T. , Mohapatra N. K. , Pucilowska J. B.
Published: December 2017

Insulin-like growth factor (IGF)-I and its binding protein IGF binding protein 5 (IGFBP-5) were highly expressed in inflamed and fibrotic intestine in experimental Crohn's disease. IGF-I induced proliferation and increased collagen synthesis by smooth muscle cells and fibroblasts/myofibroblasts in vitro. Here we studied IGF-I and IGFBP-5 in Crohn's disease tissue. Tissue was collected from patients undergoing intestinal resection for Crohn's disease. IGF-I and IGFBP-5 mRNAs were quantitated by RNase protection assay and Northern blot analysis, respectively. In situ hybridization was performed to localize mRNA expression, and Western immunoblot was performed to quantitate protein expression. IGF-I and IGFBP-5 mRNAs were increased in inflamed/fibrotic intestine compared with normal-appearing intestine. IGF-I mRNA was expressed in multiple cell types in the lamina propria and fibroblast-like cells of the submucosa and muscularis externa. IGFBP-5 mRNA was highly expressed in smooth muscle of the muscularis mucosae and muscularis externa as well as fibroblast-like cells throughout the bowel wall. Tissue IGFBP-5 protein correlated with collagen type I ( r = 0.82). These findings are consistent with a mechanism whereby IGF-I acts on smooth muscle and fibroblasts/myofibroblasts to increase collagen synthesis and cellular proliferation; its effects may be modulated by locally expressed IGFBP-5.
Adenovirus-mediated gene transfer of dominant-negative Smad4 blocks TGF-β signaling in pancreatic acinar cells
Authors: Zhang Lizhi , Graziano Kathleen , Pham Trinh , Logsdon Craig D. , Simeone Diane M.
Published: December 2017

Transforming growth factor-β (TGF-β) is a potent inhibitor of pancreatic acinar cell growth. Smad4 is a central mediator in the TGF-β signaling pathway. To study the effect of Smad4 on pancreatic growth, cell cycle protein expression, and the expression of a TGF-β-responsive promoter in vitro, we constructed an adenovirus containing dominant-negative COOH terminal truncated Smad4 (AddnSmad4) downstream of the rat elastase promoter. Acinar cells expressed dominant-negative Smad4 within 8 h after infection, and expression persisted for 72 h. Mouse pancreatic acini were infected with either AddnSmad4 or control adenovirus expressing green fluorescent protein, and TGF-β was added 8 h after infection. Acinar cells were then incubated for 1, 2, or 3 days, and [3H]thymidine incorporation was determined. AddnSmad4 significantly reduced TGF-β inhibition of [3H]thymidine incorporation, with maximal effects on day 3. AddnSmad4 also completely blocked TGF-β-mediated growth inhibition in the presence of basic fibroblast growth factor. We next examined the effects of AddnSmad4 on TGF-β-induced expression of the cell cycle regulatory proteins p21Cip1and p27Kip1. TGF-β induced upregulation of p21Cip1, which was completely blocked by AddnSmad4. AddnSmad4 also inhibited TGF-β-induced expression of the TGF-β-responsive luciferase reporter 3TP-Lux. These results show that Smad4 is essential in TGF-β-mediated signaling in pancreatic acinar cells.
Role of taurine in preventing acetaminophen-induced hepatic injury in the rat
Authors: Waters Eoin , Wang Jiang Huai , Redmond H. Paul , Wu Qiong Di , Kay Elaine , Bouchier-Hayes David
Published: December 2017

Acetaminophen overdose causes acute liver injury in both humans and animals. This study was designed to investigate the potential role of the conditionally essential amino acid taurine in preventing acetaminophen-induced hepatotoxicity. Male Sprague-Dawley rats were administered acetaminophen (800 mg/kg) intraperitoneally. Taurine (200 mg/kg) was given 12 h before, at the time of, and 1 or 2 h after acetaminophen injection. Acetaminophen treatment increased the plasma levels of aspartate transaminase, alanine aminotransferase, and alkaline phosphatase and caused hepatic DNA fragmentation and hepatocyte necrosis. Taurine administered before, simultaneously with, or 1 h after acetaminophen resulted in significant improvement in hepatic injury as represented by decrease of hepatocellular enzyme release and attenuation of hepatocyte apoptosis and necrosis, and this correlated with taurine-mediated attenuation of hepatic lipid peroxidation. These results indicate that taurine possesses prophylactic and therapeutic effects in acetaminophen-induced hepatic injury.
ICAM-1 is involved in the mechanism of alcohol-induced liver injury: studies with knockout mice
Authors: Kono Hiroshi , Uesugi Takehiko , Froh Matthias , Rusyn Ivan , Bradford Blair U. , Thurman Ronald G.
Published: December 2017

To test the hypothesis that leukocyte infiltration mediated by intercellular adhesion molecule (ICAM)-1 is involved in early alcohol-induced liver injury, male wild-type or ICAM-1 knockout mice were fed a high-fat liquid diet with either ethanol or isocaloric maltose-dextrin for 4 wk. There were no differences in mean urine alcohol concentrations between the groups fed ethanol. Alcohol administration significantly increased liver size and serum alanine aminotransferase levels in wild-type mice over high-fat controls, effects that were blunted significantly in ICAM-1 knockout mice. Dietary ethanol caused severe steatosis, mild inflammation, and focal necrosis in livers from wild-type mice. Furthermore, livers from wild-type mice fed ethanol showed significant increases in the number of infiltrating leukocytes, which were predominantly lymphocytes. These pathological changes were blunted significantly in ICAM-1 knockout mice. Tumor necrosis factor (TNF)-α mRNA expression was increased in wild-type mice fed ethanol but not in ICAM-1 knockout mice. These data demonstrate that ICAM-1 and infiltrating leukocytes play important roles in early alcohol-induced liver injury, most likely by mechanisms involving TNF-α.
Increased in vitro activation of EGFR by membrane-bound TGF-α from gastric and colonic mucosa of aged rats
Authors: Xiao Zhi-Qiang , Majumdar Adhip P. N.
Published: December 2017

Although aging is associated with increased epidermal growth factor receptor (EGFR) tyrosine kinase activity in Fischer 344 rat gastric and colonic mucosa, the regulatory mechanisms for the age-related rise in EGFR tyrosine kinase are poorly understood. Transmembrane transforming growth factor-α (TGF-α) may modulate EGFR function through an autocrine/juxtacrine mechanism. The present study aimed to determine the contribution of membrane-bound precursors of TGF-α in enhancing EGFR activation in the gastric and colonic mucosa during aging. The extent of EGFR tyrosine phosphorylation, a measure of EGFR activation, was substantially higher (300–350%) in the gastric and colonic mucosa of 23- (aged) vs. 4-mo-old (young) Fischer 344 rats. This was accompanied by an increase (200–1,000%) in the relative concentration of 18- to 20-kDa membrane-bound precursor forms of TGF-α. The amount of TGF-α bound to EGFR was also higher (150–250%) in the gastric and colonic mucosa of aged vs. young rats. In vitro studies revealed that exposure of HCT 116 cells (a colon cancer cell line) to TGF-α from gastric and colonic mucosal membranes of aged rats caused a 200–250% higher activation of EGFR and extracellular signal-related kinases (p42/44) compared with young rats. Our data suggest that the membrane-bound precursor form(s) of TGF-α may partly be responsible for enhancing EGFR activation in the gastric and colonic mucosa of aged rats, probably though an autocrine/juxtacrine mechanism(s).
Biochemical, histological, and inhibitor studies of membrane carbonic anhydrase in frog gastric acid secretion
Authors: Swenson Erik R. , Tewson Timothy W. , Wistrand Per J. , Ridderstrale Yvonne , Tu Chingkuang
Published: December 2017

Gastric acid secretion is dependent on carbonic anhydrase (CA). To define the role of membrane-bound CA, we used biochemical, histochemical, and pharmacological approaches in the frog ( Rana pipiens). CA activity and inhibition by membrane-permeant and -impermeant agents were studied in stomach homogenates and microsomal fractions. H+secretion in the histamine-stimulated isolated mucosa was measured before and after mucosal addition of a permeant CA inhibitor (methazolamide) and before and after mucosal or serosal addition of two impermeant CA inhibitors of differing molecular mass: a 3,500-kDa polymer linked to aminobenzolamide and p-fluorobenzyl-aminobenzolamide (molecular mass, 454 kDa). Total CA activity of frog gastric mucosa is 2,280 U/g, of which 10% is due to membrane-bound CA. Membrane-bound CA retains detectable activity below pH 4. Histochemically, there is membrane-associated CA in surface epithelial, oxynticopeptic, and capillary endothelial cells. Methazolamide reduced H+secretion by 100%, whereas the two impermeant inhibitors equally blocked secretion by 40% when applied to the mucosal side and by 55% when applied to the serosal side. The presence of membrane-bound CA in frog oxynticopeptic cells and its relative resistance to acid inactivation and inhibition by impermeant inhibitors demonstrate that it subserves acid secretion at both the apical and basolateral sides.
Bile salt export pump is highly conserved during vertebrate evolution and its expression is inhibited by PFIC type II mutations
Authors: Cai Shi-Ying , Wang Lin , Ballatori Nazzareno , Boyer James L.
Published: December 2017

Bile secretion is a fundamental function of the liver of all vertebrates and is generated by ATP-dependent transport proteins at the canalicular membrane of hepatocytes, particularly by the bile salt export pump BSEP. To determine the evolutionary origin and structure-function relationship of this transport mechanism, a liver cDNA library from the marine skate Raja erinacea, a 200 million-year-old vertebrate, was screened for BSEP orthologues. A full-length clone was isolated that encodes for 1,348 amino acids and shares 68.5% identity to human BSEP. Northern blot analysis revealed a 5-kb transcript only in skate liver. Expression of skate Bsep in Sf9 cells demonstrated a sixfold stimulation of ATP-dependent taurocholate transport compared with controls, with a Michaelis-Menten constant of 15 μM, which is comparable to rat Bsep. Sequences at the site of published mutations in human BSEP are also conserved in skate Bsep. When two of these mutations were introduced into the skate Bsep cDNA, this resulted in defective expression of the mutant proteins in Sf9 cells. These studies demonstrate that Bsep is a liver-specific ATP-dependent export pump that is highly conserved throughout evolution and provide insights into critical determinants for the function of this transporter in higher vertebrates.
Characterization of a mouse colonic system B0+amino acid transporter related to amino acid absorption in colon
Authors: Ugawa Shinya , Sunouchi Yoko , Ueda Takashi , Takahashi Eri , Saishin Yoshitsugu , Shimada Shoichi
Published: December 2017

Previous experiments have shown that an amino acid transport system B0+transporter in cultured colonic epithelial cells mediates amino acid absorption. Here we describe the cloning and functional characterization of a system B0+transporter selectively expressed in the colon. Using the combination of an expressed sequence tag database search and RT-PCR approaches, we cloned a mouse colonic amino acid transporter, designated mCATB0+. Northern blot analysis revealed that mCATB0+was selectively expressed in the large intestine. In situ hybridization showed the mCATB0+mRNA to be localized in absorptive epithelial cells. When expressed in Xenopus oocytes, mCATB0+exhibited a Na+-dependent stereoselective uptake and a broad specificity for neutral and cationic amino acids, which is characteristic of amino acid transport system B0+. In vivo [3H]glycine uptake assay demonstrated that a system B0+-like transporter protein was expressed on the apical surface of the colonic absorptive cells. Our data suggest that a mouse colonic amino acid transporter mCATB0+may absorb amino acids from the intestinal contents in the colon.
Differential expression and regulation of Na+/H+exchanger isoforms in rabbit parietal and mucous cells
Authors: Rossmann Heidi , Sonnentag Thorsten , Heinzmann Alexander , Seidler Barbara , Bachmann Oliver , Vieillard-Baron Dorothee , Gregor Michael , Seidler Ursula
Published: December 2017

Several Na+/H+exchanger (NHE) isoforms are expressed in the stomach, and NHE1 and NHE2 knockout mice display gastric mucosal atrophy. This study investigated the cellular distribution of the NHE isoforms NHE1, NHE2, NHE3, and NHE4 in rabbit gastric epithelial cells and their regulation by intracellular pH (pHi), hyperosmolarity, and an increase in cAMP. Semiquantitative RT-PCR and Northern blot experiments showed high NHE1 and NHE2 mRNA levels in mucous cells and high NHE4 mRNA levels in parietal and chief cells. Fluorescence optical measurements in cultured rabbit parietal and mucous cells using the pH-sensitive dye 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein and NHE isoform-specific inhibitors demonstrated that in both cell types, intracellular acidification activates NHE1 and NHE2, whereas hyperosmolarity activates NHE1 and NHE4. The relative contribution of the different isoforms to pHi- and hyperosmolarity-activated Na+/H+exchange in the different cell types paralleled their relative expression levels. cAMP elevation also stimulated NHE4, whereas an increase in osmolarity above a certain threshold further increased NHE1 and not NHE4 activity. We conclude that in rabbit gastric epithelium, NHE1 and NHE4 regulate cell volume and NHE1 and NHE2 regulate pHi. The high NHE1 and NHE2 expression levels in mucous cells may reflect their special need for pHiregulation during high gastric acidity. NHE4 is likely involved in volume regulation during acid secretion.
Aquaporin-2, a regulated water channel, is expressed in apical membranes of rat distal colon epithelium
Authors: Gallardo Pedro , Cid L. Pablo , Vio Carlos P. , Sepúlveda Francisco V.
Published: December 2017

Aquaporin-2 (AQP-2) is the vasopressin-regulated water channel expressed in the apical membrane of principal cells in the collecting duct and is involved in the urinary concentrating mechanism. In the rat distal colon, vasopressin stimulates water absorption through an unknown mechanism. With the hypothesis that AQP-2 could contribute to this vasopressin effect, we studied its presence in rat colonic epithelium. We used RT-PCR, in situ hybridization, immunoblotting, and immunocytochemistry to probe for AQP-2 expression. An AQP-2 amplicon was obtained through RT-PCR of colon epithelium RNA, and in situ hybridization revealed AQP-2 mRNA in colonic crypts and, to a lesser extent, in surface absorptive epithelial cells. AQP-2 protein was localized to the apical membrane of surface absorptive epithelial cells, where it colocalized with H+-K+-ATPase but not with Na+-K+-ATPase. AQP-2 was absent from the small intestine, stomach, and liver. Water deprivation increased the hybridization signal and the protein level (assessed by Western blot analysis) for AQP-2 in distal colon. This was accompanied by increased p-chloromercuriphenylsulfonic acid-sensitive water absorption. These results indicate that AQP-2 is present in the rat distal colon, where it might be involved in a water-sparing mechanism. In addition, these results support the idea that AQP-2, and probably other aquaporins, are involved in water absorption in the colon.
Norepinephrine-induced hepatocellular dysfunction in early sepsis is mediated by activation of α2-adrenoceptors
Authors: Yang Shaolong , Zhou Mian , Chaudry Irshad H. , Wang Ping
Published: December 2017

Gut-derived norepinephrine (NE) has been shown to play a critical role in producing hepatocellular dysfunction in early sepsis, but it is not known whether α2-adrenoceptor activation mediates this dysfunction. We infused normal male adult rats with NE, NE plus the specific α2-adrenergic antagonist rauwolscine (RW), or vehicle (normal saline) for 2 h. Hepatocellular function was determined by in vivo indocyanine green (ICG) clearance. An isolated perfused liver preparation was also used to assess hepatocellular function by in vitro ICG clearance; NE alone or with RW was added to the perfusate. Rats were subjected to sepsis by cecal ligation and puncture (CLP). At 1 h after CLP, RW was infused for 15 min. At 5 h after CLP, we measured hepatocellular function and serum tumor necrosis factor-α (TNF-α) levels. Intraportal NE infusion in normal rats produced hepatocellular dysfunction, which was prevented by RW and NE infusion. This is confirmed by findings with the isolated perfused liver preparation. RW administration in early sepsis maintained hepatocellular function and downregulated TNF-α production at 5 h after CLP. These results suggest that NE-induced hepatocellular dysfunction in early sepsis is mediated by α2-adrenoceptor activation, which appears to upregulate TNF-α production. Modulation of hepatic responsiveness to NE by α2-adrenergic antagonists should provide a novel approach for maintaining cell and organ functions during sepsis.
nNOS in canine lower esophageal sphincter: colocalized with Cav-1 and Ca2+-handling proteins?
Authors: Daniel E. E. , Jury J. , Wang Y. F.
Published: December 2017

Immunochemical studies with light microscopy, confocal microscopy, and electron microscopy were used to examine proteins associated with caveolin (Cav) in canine lower esophageal sphincter. The main Cav was Cav-1. It appeared to be colocalized at the cell periphery, in punctate sites, with immunoreactivity to antibodies against different COOH- and NH2-terminal epitopes of neuronal nitric oxide (NO) synthase (nNOS). One COOH-terminal-directed antibody, made in guinea pig, was used to colocalize other immunoreactivities. Those that apparently colocalized with nNOS were L-Ca2+channels, the PM Ca2+pump, and, in part, calreticulin and calsequestrin. The large-conductance Ca2+-activated K+(BKCa) channels were located in discrete peripheral sites, some with Cav. Immunoreactivites not fully colocalized with nNOS were to the sarcoplasmic reticulum Ca2+pump, connexins 43, 40, and 45, and vinculin. In patch-clamp studies, NO-driven outward currents, mainly through BKCachannels, were inhibited by antibodies to Cav-1 and not by calmodulin and were restored by an NO donor. Several Ca2+-handling molecules are localized at the PM with and/or near Cav. This may allow intracellular calcium concentration levels to be controlled differently than those in the cytosol near caveolae.
cAMP inhibits IP3-dependent Ca2+release by preferential activation of cGMP-primed PKG
Authors: Murthy Karnam S.
Published: December 2017

The singular effects and interplay of cAMP- and cGMP-dependent protein kinase (PKA and PKG) on Ca2+mobilization were examined in dispersed smooth muscle cells. In permeabilized muscle cells, exogenous cAMP and cGMP inhibited inositol 1,4,5-trisphosphate (IP3)-induced Ca2+release and muscle contraction via PKA and PKG, respectively. A combination of cAMP and cGMP caused synergistic inhibition that was exclusively mediated by PKG and attenuated by PKA. In intact muscle cells, low concentrations (10 nM) of isoproterenol and sodium nitroprusside (SNP) inhibited agonist-induced, IP3-dependent Ca2+release and muscle contraction via PKA and PKG, respectively. A combination of isoproterenol and SNP increased PKA and PKG activities: the increase in PKA activity reflected inhibition of phosphodiesterase 3 activity by cGMP, whereas the increase in PKG activity reflected activation of cGMP-primed PKG by cAMP. Inhibition of Ca2+release and muscle contraction by the combination of isoproterenol and SNP was preferentially mediated by PKG. In light of studies showing that PKG phosphorylates the IP3receptor in intact and permeabilized muscle cells, whereas PKA phosphorylates the receptor in permeabilized cells only, the results imply that inhibition of IP3-induced Ca2+release is mediated exclusively by PKG. The effect of PKA on agonist-induced Ca2+release probably reflects inhibition of IP3formation.
Colonic H+-K+-ATPase in K+conservation and electrogenic Na+absorption during Na+restriction
Authors: Spicer Zachary , Clarke Lane L. , Gawenis Lara R. , Shull Gary E.
Published: December 2017

Upregulation of the colonic H+-K+- ATPase (cHKA) during hyperaldosteronism suggests that it functions in both K+conservation and electrogenic Na+absorption in the colon when Na+-conserving mechanisms are activated. To test this hypothesis, wild-type ( cHKA+/+) and cHKA-deficient ( cHKA−/−) mice were fed Na+-replete and Na+-restricted diets and their responses were analyzed. In both genotypes, Na+restriction led to reduced plasma Na+and increased serum aldosterone, and mRNAs for the epithelial Na+channel (ENaC) β- and γ-subunits, channel-inducing factor, and cHKA were increased in distal colon. Relative to wild-type controls, cHKA−/−mice on a Na+-replete diet had elevated fecal K+excretion. Dietary Na+restriction led to increased K+excretion in knockout but not in wild-type mice. The amiloride-sensitive, ENaC-mediated short-circuit current in distal colon was significantly reduced in knockout mice maintained on either the Na+-replete or Na+-restricted diet. These results demonstrate that cHKA plays an important role in K+conservation during dietary Na+restriction and suggest that cHKA-mediated K+recycling across the apical membrane is required for maximum electrogenic Na+absorption.
Role of nitric oxide in capillary perfusion and oxygen delivery regulation during systemic hypoxia
Authors: Bertuglia Silvia , Giusti Andrea
Published: December 2017

The role of nitric oxide (NO) and reactive oxygen species (ROS) in regulating capillary perfusion was studied in the hamster cheek pouch model during normoxia and after 20 min of exposure to 10% O2-90% N2. We measured Po2 by using phosphorescence quenching microscopy and ROS production in systemic blood. Identical experiments were performed after treatment with the NO synthase inhibitor NG-monomethyl-l-arginine (l-NMMA) and after the reinfusion of the NO donor 2,2′-(hydroxynitrosohydrazono)bis-etanamine (DETA/NO) after treatment with l-NMMA. Hypoxia caused a significant decrease in the systemic Po2. During normoxia, arteriolar intravascular Po2 decreased progressively from 47.0 ± 3.5 mmHg in the larger arterioles to 28.0 ± 2.5 mmHg in the terminal arterioles; conversely, intravascular Po2 was 7–14 mmHg and approximately uniform in all arterioles. Tissue Po2 was 85% of baseline. Hypoxia significantly dilated arterioles, reduced blood flow, and increased capillary perfusion (15%) and ROS (72%) relative to baseline. Administration of l-NMMA during hypoxia further reduced capillary perfusion to 47% of baseline and increased ROS to 34% of baseline, both changes being significant. Tissue Po2 was reduced by 33% versus the hypoxic group. Administration of DETA/NO after l-NMMA caused vasodilation, normalized ROS, and increased capillary perfusion and tissue Po2. These results indicate that during normoxia, oxygen is supplied to the tissue mostly by the arterioles, whereas in hypoxia, oxygen is supplied to tissue by capillaries by a NO concentration-dependent mechanism that controls capillary perfusion and tissue Po2, involving capillary endothelial cell responses to the decrease in lipid peroxide formation controlled by NO availability during low Po2 conditions.
Specific desensitization (tachyphylaxis) of the guinea pig ileum to angiotensin II
Authors: Paiva T. B. , Mendes G. B. , Paiva A. C.
Published: December 2017

Tachyphylaxis to [Ile5]angiotensin II (angiotensin) in the isolated guinea pig ileum was found to be more severe when the Ca2+ concentration or the temperature of the medium were lowered, or when glucose was absent. Incubation with indomethacin or prostaglandin E2 did not affect the onset of tachyphylaxis or recovery from the tachyphylactic state. The angiotensin dose-response curves of tachyphylactic organs were shifted to the right, and the maximum responses were depressed in proportion to the conditioning doses of the hormone. The recovery from tachyphylaxis followed zero-order kinetics and was not affected by Ca2+ concentration or pH. The temperature dependence of the rate of recovery yielded a value of 14.6 kcal/mol for the activation energy in the physiological temperature range. It is concluded that tachyphylaxis results from the tight binding of angiotensin to superficial calcium-binding sites in the smooth muscle cell membrane. Recovery from tachyphylaxis appears to involve displacement of angiotensin by calcium in a process that is dependent on active transport.
Some pressure and fluid dynamic characteristics of the canine epidural space
Authors: Bengis R. G. , Guyton A. C.
Published: December 2017

Pressure measured in teh epidural space of 50 dogs was consistently negative (subatmospheric). Acutely implanted epidural catheters gave an average reading of -2.72 mmHg (n = 44, SE = 0.18), whereas chronically implanted catheters gave more negative readings, averaging -5.8 mmHg (n = 6, SE = 0.49). Since the chronically implanted catheters permit healing, resolution of hemorrhage, and resealing of the epidural space to take place, resulting in equilibration of microcirulatory forces, these readings are believed to represent more closely the true epidural pressure. Also these results approximate closely the calculated and measured interstitial fluid pressures obtained by different techniques in other loose areolar connective tissue systems. Compliance studies, using a double-lumen catheter for simultaneous recording and infusing in the epidural space, showed increasing compliance as the pressure rose into the positive pressure range, with an eventual plateau of the volume-pressure curve at the level of the concomitantly measured cerebrospinal fluid pressure (avg = +5.73 mmHg, n = 10, SE = 0.48). Intravenous volume loading using Tyrode solution caused a large rise in epidural pressure, and volume depletion with diuretics caused a significant drop in epidural pressure.
Determinants of hypoxic and posthypoxic myocardial contracture
Authors: Greene H. L. , Weisfeldt M. L.
Published: December 2017

The relationship between contracture (a rise in resting tension at constant length) and contractile activity was studied in isometric rat trabeculae carneae and cat right ventricular papillary muscles. Muscles at Lmax were paced at 12/min in solutions equilibrated at 29 degrees C with 95% O2-5% CO2. In rat myocardium, exposure to 60 min hypoxia (95% N2-5% CO2) at alkalotic pH produced contracture. In cat myocardium contracture was produced by the combination of exposure to digitalis and the recovery from hypoxia. Delayed relaxation was not responsible for the rise in resting tension in either preparation. In muscles not paced during hypoxia, contracture appeared despite total absence of contractile activity. Exposure to low calcium or acidosis reversed or prevented the rise in resting tension. Thus, contracture can be: 1) dynamic, reversible, and modified by factors which affect intracellular calcium availability; 2) independent of contractile activity and apparent activity of the relaxing system; and 3) distinct from delayed or incomplete relaxation. The extent of contracture during the recovery from hypoxia is not simply the function of the level of contractile activity during hypoxia but appears related to calcium availability at a critical time.
Capillary supply of skeletal muscle fibers in untrained and endurance-trained men
Authors: Brodal P. , Ingjer F. , Hermansen L.
Published: December 2017

Muscle fiber diameters and numbers of capillaries per fiber, per square millimeter, and around each fiber were determined in needle biopsies from the lateral part of the quadriceps muscle of 23 young men. Twelve subjects were untrained (UT) and eleven were endurance-trained (ET) athletes. Average values for maximal oxygen uptake were 51.3 (UT) and 72.0 ml/kg-min (ET). Mean fiber diameters were not significantly different in the two groups (48.8 and 49.1 micron). The capillaries per fiber ratios were 1.77+/-0.10 and 2.49+/-0.08 (mean+/-SE) in the UT and ET groups, respectively. The numbers of capillaries around each fiber were 4.43+/-0.19 (UT) and 5.87+/-0.18 (ET). The numbers of capillaries per mm2 were 585+/-40 (UT) and 821+/-28 (ET). Fiber diameters were 28% smaller in ultrathin than in fresh-frozen sections from the same biopsies. After correction for this difference, the numbers of capillaries per mm2 were 305 and 425 in the UT and ET, respectively. The capillaries per fiber ratio increased with increasing fiber diameter, but not sufficiently to maintain the number of capillaries per mm2. Fibers containing many mitochondria are surrounded by more capillaries than fibers with few mitochondria.
Effect of hematocrit variations on coronary hemodynamics and oxygen utilization
Authors: Jan K. M. , Chien S.
Published: December 2017

Twenty-five closed-chest pentobarbitalized dogs were used for studying coronary flow dynamics and myocardial oxygen utilization following variations of hematocrit (Hct) by isovolumetric exchange of blood with plasma or packed red cells. Coronary blood flow (133Xe washout) and cardiac output varied inversely with Hct. Coronary systemic, and pulmonary flow resistances varied in the same direction with Hct. Blood viscosity played a significant role in determining the flow resistances in these three regions. Analysis of vascular hindrance (vascular resistance/blood viscosity) suggested that coronary vasodilation occurred following Hct changes beyond the range of 20-60%. In systemic and pulmonary circulations, however, there was vasoconstriction following hemodilution. The range of optimum Hct for maximum O2 transport (blood flow X arterial O2 content) was much wider in coronary (20-60% Hct) than in systemic circulation (40-60% Hct). The O2 consumptions in total body and in myocardium were essentially constant over a wide range of Hct (20-60%). The maintenance of total body O2 consumption over the Hct range of 20-40% was attributable to an increase in A-V O2 extraction. The O2 extraction ratio in the coronary circulation was constant over the entire range of Hct studied, suggesting that the myocardial O2 consumption was primarily determined by the coronary O2 transport.
Renin release and pressor response to renal arterial hypotension: effect of dietary sodium
Authors: Fray J. C. , Johnson M. D. , Barger A. C.
Published: December 2017

Changes in plasma renin activity (PRA) and mean arterial pressure (MAP) produced by renal arterial hypotension were studied in conscious, adrenalectomized dogs maintained on low-, normal-, or high-Na diet during constant steroid replacement therapy. In animals maintained on a low-Na diet, reduction of renal perfusion pressure to 50 mmHg for 45 min increased MAP 40 +/- 3 (SE) mmHg, while PRA rose rapidly by 36.5 +/- 6 ng ml-1 h-1. Similar renal hypotension in dogs maintained on a normal-Na diet increased MAP only 21 +/- 3 mmHg while PRA rose 5.5 +/- 0.9 ng ml-1 h-1; dogs on high-Na intake had a 6 +/- 1 mmHg pressure rise without a significant change in PRA. The rise in MAP correlated well with the log deltaPRA. Calculated open-loop gain was -1.2, -0.7, and -0.1 in dogs on low-, normal-, and high-Na diets, respectively. Nonpeptide angiotensin I converting enzyme inhibitor (CEI) reversed the elevated MAP observed during reduction of renal perfusion pressure in dogs on low- and normal-Na diets, but had little effect in dogs on high-Na intake. These observations suggest that the renin-angiotensin system becomes quantitatively more important in the regulation of blood pressure as Na intake is reduced.