bluuebunny/crossref_metadata_2025 huggingface dataset:
Soluble ICAM-1 reduces leukocyte adhesion to vascular endothelium in ischemia-reperfusion injury in mice
Authors: Kusterer Klaus , Bojunga Jörg , Enghofer Michael , Heidenthal Edmund , Usadel Klaus H. , Kolb Hubert , Martin Stephan
Published: December 2017

Ischemia-reperfusion injury is a pathogenic factor in the course of many clinical disorders, such as myocardial infarction, stroke, organ transplantation, burns, and circulatory shock. The extent of ischemia-reperfusion injury is dependent on the number of infiltrating leukocytes. With in vivo microscopy, we evaluated the effect of the recombinant form of soluble murine intercellular adhesion molecule-1 (ICAM-1) on ischemia-reperfusion injury in an animal model. A mesenteric vein was occluded with a clamp for 45 min. During a reperfusion period of 30 min, the number of leukocytes rolling along the endothelium and the number of adherent leukocytes were measured with and without pretreatment with recombinant ICAM-1. The number of leukocytes rolling along the endothelial surface increased more than twofold during postischemic perfusion ( P < 0.05). Recombinant ICAM-1 had no effect on leukocyte rolling. In the control group, firm adherence of leukocytes was increased 10-fold. Recombinant ICAM-1 dose dependently reduced firm adhesion to the endothelium in response to prior ischemia. After 30 min, reperfusion pretreatment with recombinant ICAM-1 inhibited leukocyte adherence from 512 ± 123 to 166 ± 34 leukocytes/mm2( P < 0.01). We demonstrate here for the first time that soluble recombinant ICAM-1 is able to reduce leukocyte adherence to mesenteric venules in postischemic reperfusion injury dose dependently. Because soluble ICAM-1 is naturally circulating in human serum, the therapeutic use of soluble recombinant forms of ICAM-1 may represent a physiological way to protect against ischemiareperfusion injury.
Strain induces Caco-2 intestinal epithelial proliferation and differentiation via PKC and tyrosine kinase signals
Authors: Han Okhee , Li Guang Di , Sumpio Bauer E. , Basson Marc D.
Published: December 2017

Although the intestinal epithelium undergoes complex deformations during normal function, nutrient absorption, fasting, lactation, and disease, the effects of deformation on intestinal mucosal biology are poorly understood. We previously demonstrated that 24 h of cyclic deformation at an average 10% deformation every 6 s stimulates proliferation and modulates brush-border enzyme activity in human intestinal Caco-2 cell monolayers. In the present study we sought potential mechanisms for these effects. Protein kinase C (PKC) activity increased within 1 min after initiation of cyclic deformation, and the PKC-α and -ζ isoforms translocated from the soluble to the particulate fraction. Cyclic deformation also rapidly increased tyrosine kinase activity. Tyrosine phosphorylation of several proteins was increased in the soluble fraction but decreased in the particulate fraction by cyclic deformation for 30 min. Inhibition of PKC and tyrosine kinase signals by calphostin C, G-06967, and erbstatin attenuated or blocked cyclic deformation-mediated modulation of Caco-2 DNA synthesis and differentiation. These results suggest that cyclic deformation may modulate intestinal epithelial proliferation and brush-border enzyme activity by regulating PKC and tyrosine kinase signals.
Indomethacin increases susceptibility to injury in human gastric cells independent of PG synthesis inhibition
Authors: Kokoska Evan R. , Smith Gregory S. , Deshpande Yashwant , Wolff Andrew B. , Miller Thomas A.
Published: December 2017

Indomethacin and other nonsteroidal anti-inflammatory drugs are commonly used to indirectly deduce the possible role of PGs in a process being studied. The objective of this study was to determine if indomethacin, at concentrations comparable to plasma and tissue levels obtained in humans taking therapeutic doses, predisposes human gastric cells to injury through inhibition of PGs or acts through an alternate mechanism. The role of intracellular Ca2+in this damaging process was also assessed. Indomethacin pretreatment, although by itself nondamaging, was associated with elevated intracellular Ca2+concentrations and an increased cellular permeability, an effect that was dependent on extracellular Ca2+. Furthermore, indomethacin pretreatment significantly predisposed AGS cells to injury induced by two dissimilar agents (deoxycholate and A-23187), both of which are associated with intracellular Ca2+accumulation. The addition of exogenous PGs did not reverse the predisposition to injury induced by indomethacin. The observed effects of indomethacin were dependent on concentration and not on ability to inhibit PG synthesis. Similar effects were not observed with equipotent concentrations of ibuprofen or aspirin. Finally, the exacerbation of deoxycholate-induced injury induced by indomethacin was not observed when extracellular Ca2+was removed. Indomethacin, by disturbing intracellular Ca2+homeostasis, predisposes human gastric cells to injury through mechanisms independent of PG synthesis. The current study suggests that data resulting from studies employing only indomethacin as a PG synthesis inhibitor should be interpreted with caution.
Binding of [3H]palmitate to BSA
Authors: Elmadhoun B. M. , Wang G. Q. , Templeton J. F. , Burczynski F. J.
Published: December 2017

Determination of the BSA-palmitate high-affinity binding constant ( Ka) traditionally relied on the heptane-water partitioning technique. We used this technique to calculate Kafor the BSA-[3H]palmitate complex, to determine if Kawas independent of protein concentration, and to determine if the unbound [3H]palmitate concentration is constant at different BSA concentrations using constant BSA-to-palmitate molar ratios (range 1:1 to 1:4). After extensive extraction of non-[3H]palmitate radiolabeled substances, the heptane-to-buffer partition ratio, in the absence of BSA, was 702 ± 19 (mean ± SD, n = 6). This value was much lower than the predicted value of 1,376 and was highly dependent on which phase (organic or aqueous) initially contained the [3H]palmitic acid. The data were consistent with the notion of self-association of [3H]palmitate in the aqueous phase. Kafor the BSA-[3H]palmitate complex was determined to be similar (2.2 ± 0.1) × 108M−1(mean ± SD, P > 0.05) at all BSA concentrations studied. At each BSA-to-palmitate molar ratio, the equilibrium unbound ligand concentration was constant only at low BSA concentrations (<10 μM) and at low BSA-to-palmitate molar ratios (i.e., 1:1 and 1:2). At higher BSA concentrations and molar ratios, the unbound ligand concentration increased with an increase in protein concentration. Hepatocyte uptake using the manufacturer-supplied radiolabeled product was significantly higher than with the purified product, suggesting that a non-[3H]palmitate radiolabel is also a substrate for the uptake process.
Endothelin-1 inhibits secretin-stimulated ductal secretion by interacting with ETAreceptors on large cholangiocytes
Authors: Caligiuri Alessandra , Glaser Shannon , Rodgers Rebecca E. , Phinizy Jo Lynne , Robertson Willie , Papa Emanuela , Pinzani Massimo , Alpini Gianfranco
Published: December 2017

We studied the expression of endothelin-1 (ET-1) receptors (ETAand ETB) and the effects of ET-1 on cholangiocyte secretion. The effects of ET-1 on cholangiocyte secretion were assessed in normal and bile duct-ligated (BDL) rats by measuring 1) basal and secretin-induced choleresis in vivo, 2) secretin receptor gene expression and cAMP levels in small and large cholangiocytes, and 3) luminal expansion in response to secretin in intrahepatic bile duct units (IBDU). ETAand ETBreceptors were expressed by small and large cholangiocytes. ET-1 had no effect on basal bile flow or bicarbonate secretion in normal or BDL rats but decreased secretin-induced bicarbonate-rich choleresis in BDL rats. ET-1 decreased secretin receptor gene expression and secretin-stimulated cAMP synthesis in large cholangiocytes and secretin-induced luminal expansion in IBDU from normal or BDL rats. The inhibitory effects of ET-1 on secretin-induced cAMP synthesis and luminal duct expansion were blocked by specific inhibitors of the ETA(BQ-610) receptor. ET-1 inhibits secretin-induced ductal secretion by decreasing secretin receptor and cAMP synthesis, two important determinants of ductal secretion.
Localization and secretion of tissue kallikrein in peptidoglycan-induced enterocolitis in Lewis rats
Authors: Stadnicki Antoni , Chao Julie , Stadnicka Iwona , Van Tol Eric , Lin Kuei-Fu , Li Fengling , Sartor R. Balfour , Colman Robert W.
Published: December 2017

The plasma kallikrein-kinin system is a mediator of intestinal inflammation induced by peptidoglycan-polysaccharide from group A streptococci (PG-APS) in rats. In this study we investigated the participation of intestinal tissue kallikrein (ITK). Lewis rats were injected intramurally with PG-APS. ITK was visualized by immunohistochemical staining. Cecal ITK concentration was measured by radioimmunoassay, and gene expression was evaluated by RNase protection assay. Kallikrein-binding protein (KBP) was evaluated in plasma by ELISA. Tissue kallikrein was identified in cecal goblet cells in both control and PG-APS-injected rats and in macrophages forming granulomas in inflamed tissues. Cecal ITK was significantly lower in acute and chronic phases of inflammation and in supernatant from in vitro cultures of inflamed cecum. ITK mRNA levels were not significantly different. Plasma KBP levels were significantly reduced in inflamed rats. The presence of tissue kallikrein in macrophages suggests participation in experimental colitis. The decrease of ITK in the inflamed intestine associated with unchanged mRNA levels suggests ITK release during intestinal inflammation.
Intracellular pH regulation in bombesin-stimulated secretion in isolated bile duct units from rat liver
Authors: Cho Won Kyoo , Mennone Albert , Boyer James L.
Published: December 2017

Bombesin, a neuropeptide, stimulates fluid and [Formula: see text] secretion from cholangiocytes, but the underlying mechanisms are poorly understood. In this study, we aimed to examine the effects of bombesin on ion transport processes involved in the regulation of intracellular pH (pHi) and[Formula: see text] secretion in polarized cholangiocytes. Isolated bile duct units from normal rat liver were used to measure pHiby 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein 495 nm-to-440 nm dual ratio methods. Bombesin increased Cl−-[Formula: see text] exchange activity but did not affect basal pHior the activities of Na+/H+exchange or Na+-[Formula: see text] symport. Depolarization of cholangiocytes increased basal pHiand the activity of Cl−/[Formula: see text]exchange, suggesting that an electrogenic Na+-[Formula: see text]symport might function as a counterregulatory pHimechanism. Na+-independent acid-extruding mechanisms were not observed. We conclude that bombesin stimulates biliary secretion from cholangiocytes by activating luminal Cl−/[Formula: see text]exchange, which may be coupled to basolateral electrogenic Na+-[Formula: see text] symport.
Different acid secretagogues activate different Na+/H+exchanger isoforms in rabbit parietal cells
Authors: Bachmann O. , Sonnentag T. , Siegel W.-K. , Lamprecht G. , Weichert A. , Gregor M. , Seidler U.
Published: December 2017

Rabbit parietal cells express three Na+/H+exchanger isoforms (NHE1, NHE2, and NHE4). We investigated the effects of carbachol, histamine, and forskolin on Na+/H+exchange activity and acid formation in cultured rabbit parietal cells and tested the effect of NHE isoform-specific inhibition on agonist-induced Na+/H+exchange. Carbachol (10−4M) was the weakest acid secretagogue but caused the strongest Na+/H+exchange activation, which was completely blocked by 1 μM HOE-642 (selective for NHE1); histamine (10−4M) and forskolin (10−5M) were stronger stimulants of [14C]aminopyrine accumulation but weaker stimulants of Na+/H+exchange activity. HOE-642 (1 μM) reduced forskolin-stimulated Na+/H+exchange activity by 35%, and 25 μM HOE-642 (inhibits NHE1 and -2) inhibited an additional 13%, but 500 μM dimethyl amiloride (inhibits NHE1, -2, and -4) caused complete inhibition. The presence of 5% CO2-[Formula: see text]markedly reduced agonist-stimulated H+efflux rates, suggesting that the anion exchanger is also activated. Hyperosmolarity also activated Na+/H+exchange. Our data suggest that, in rabbit parietal cells, Ca2+-dependent stimulation causes a selective activation of NHE1, whereas cAMP-dependent stimulation activates NHE1, NHE2, and more strongly NHE4. Because intracellular pH (pHi) did not change in the presence of CO2-[Formula: see text]and concomitant activation of Na+/H+and anion exchange is one of the volume regulatory mechanisms, we speculate that the physiological significance of secretagogue-induced Na+/H+exchange activation may not be related to pHibut to volume regulation during acid secretion.
Evidence for NO ⋅ redox form of nitric oxide as nitrergic inhibitory neurotransmitter in gut
Authors: Goyal Raj K. , He Xue D.
Published: December 2017

A nitric oxide (NO)-like product of thel-arginine NO synthase pathway has been shown to be a major inhibitory neurotransmitter that is involved in the slow component of the inhibitory junction potential (IJP) elicited by stimulation of nonadrenergic, noncholinergic nerves. However, the exact nature of the nitrergic transmitter, the role of cGMP, and the involvement of a potassium or a chloride conductance in the slow IJP remain unresolved. We examined the effects of soluble guanylate cyclase inhibitors LY-83583 and 1 H-[1,2,4]oxadiazolo[4,3- a]quinoxalin-1-one (ODQ), potassium-channel blockers and putative chloride-channel blockers diphenylamine-2-carboxylate (DPC) and niflumic acid (NFA) on the hyperpolarization elicited by an NO ⋅ donor, diethylenetriamine/NO adduct (DNO), NO in solution, and an NO+ donor, sodium nitroprusside (SNP), in the guinea pig ileal circular muscle. Effects of these blockers on purinergic (fast) and nitrergic (slow) IJP were also examined. DNO-induced hyperpolarization and nitrergic slow IJP were suppressed by LY-83583 or ODQ and DPC or NFA but not by the potassium-channel blocker apamin. In contrast, hyperpolarization caused by SNP or solubilized NO gas and purinergic fast IJP were antagonized by apamin but not by inhibitors of guanylate cyclase or chloride channels. These results demonstrate biological differences in the actions of different redox states of NO and suggest that NO ⋅ is the nitrergic inhibitory neurotransmitter.
Scintigraphic measurement of regional gastrointestinal transit in the dog
Authors: Iwanaga Yuji , Wen Jipu , Thollander Mikael S. , Kost Louis J. , Thomforde George M. , Allen Richard G. , Phillips Sidney F.
Published: December 2017

Scintigraphic techniques can measure sequentially gastric emptying, small bowel transit, and colonic transit in humans, and comparable methods for experimental studies in animals would be useful. We developed such a method in dogs and examined the effects of prokinetic drugs on regional transit. Two isotopes were given to fasting dogs. Polystyrene pellets labeled with99mTc were mixed in a can of dog food and111In- labeled pellets were given in a gelatin capsule coated with a pH-sensitive polymer, designed to dissolve in the distal bowel. Gamma camera images were obtained for up to 24 h. Prokinetic drugs were given by intravenous injection. Duplicate baseline studies showed good agreement in seven dogs. In a second group ( n = 4), intra- and interanimal variabilities were established. Two novel prokinetic drugs (AU-116 and AU-130) accelerated small bowel and colonic transit. A simple noninvasive method for measuring whole gut transit in dogs was developed and validated. Two new prokinetics accelerated small bowel and colonic transit.
Role of insulin and nutritional factors in intestinal glycoprotein fucosylation during postnatal development
Authors: Biol Marie-Claire , Lenoir Didier , Greco Sandrine , Galvain Dany , Hugueny Irene , Louisot Pierre
Published: December 2017

This study deals with the role of insulin in the regulation of the intestinal glycoprotein fucosylation process during postnatal development in the rat. Circulating insulin level was found to increase at weaning time in parallel with α-1,2-fucosyltransferase activity and with the appearance of α-1,2-fucoproteins in brush-border membranes. Insulin treatment of young suckling rats induced a precocious increase in fucosyltransferase activity and in the biosynthesis of its substrate (GDP-fucose), but the sensitivity to insulin disappeared after weaning. The insulin level was lower in 22-day-old rats that received prolonged nursing (on a high-fat diet) compared with age-matched normally weaned rats (on a high-carbohydrate diet), whereas the appearance of α-1,2-fucoproteins and the increase in activity of α-1,2-fucosyltransferase were delayed, as was the decrease in the degradation of GDP-fucose. In 22-day-old animals that received prolonged nursing and insulin treatment, the α-1,2-fucosyltransferase activity reached a level close to that observed in age-matched weaned rats, and several α-1,2-fucoproteins appeared in brush-border membranes with a molecular mass similar to that found in weaned rats. These results suggest that changes in insulin levels at weaning time (as caused, in the present case, by dietary modifications) may be responsible for the regulation of the glycoprotein fucosylation process, essentially by increasing fucosyltransferase activity.
Differential expression of EGFR during early reparative phase of the gastric mucosa between young and aged rats
Authors: Liu Lei , Turner Jerrold R. , Yu Yingjie , Khan Ahmed J. , Jaszewski Richard , Fligiel Suzanne E. G. , Majumdar Adhip P. N.
Published: December 2017

Aging is associated with decreased reparative ability of the gastric mucosa. Our recent data suggest a role for epidermal growth factor receptor (EGFR) in the mucosal reparative processes. Thus we examined changes in EGFR tyrosine kinase activity as well as expression and subcellular localization of EGFR and its ligand transforming growth factor-α (TGF-α) in the gastric mucosa of young (4-mo-old) and aged (24-mo-old) Fischer 344 male rats during the early reparative phase after acute injury induced by 2 M NaCl. Within 240 min of injury, significant epithelial restitution was observed in the gastric mucosa of young but not of aged rats. Expansion of the neck region and initiation of foveolar cell migration could be seen within 45 min of injury in young rats but not until 90 min in aged rats. In young rats mucosal EGFR tyrosine kinase activity increased at 45 min after injury and subsequently fell to basal levels. Mucosal EGFR mRNA increased throughout the reparative phase as did content of the EGFR ligand TGF-α. In contrast, although the basal tyrosine kinase activity and levels of EGFR mRNA and TGF-α were elevated in the gastric mucosa of aged rats, injury did not cause increases in these parameters. Immunofluorescent localization suggests that internalization and/or degradation of EGFR may be higher in aged than in young rats. We suggest that diminished induction of EGFR tyrosine kinase activity and increased EGFR internalization after injury may in part be responsible for the age-related decrease in the reparative capacity of the gastric mucosa.
Role of transcellular pathway in ileal Ca2+ absorption: stimulation by low-Ca2+ diet
Authors: Auchère D. , Tardivel S. , Gounelle J.-C. , Drüeke T. , Lacour B.
Published: December 2017

The present study was performed to determine the respective involvement of the cellular and paracellular routes in ileal Ca2+ transport. Two groups of rats were either fed a normal Ca2+ diet (1.0%) or a Ca2+-deficient diet (0.02%) for 14 days. Ileal Ca2+absorption was determined using both an in situ method of continuous luminal perfusion and an in vitro method (Ussing chamber model). The low-Ca2+ diet stimulated net Ca2+ flux in the ileum twofold, associated with a twofold increase of the mucosal-to-serosal Ca2+ flux in both models. This effect was observed in the absence of concomitant changes in Na+ or water flux in the in situ model or mannitol flux in the in vitro model, excluding the participation of the paracellular pathway in Ca2+ transport. Thus only cellular Ca2+ flux was stimulated. These data suggest that the ileum plays a major role in the adaptation to low dietary Ca2+. Whereas under physiological conditions with usual Ca2+ intakes the transcellular pathway of Ca2+ transport is negligible, it becomes of major importance in the case of Ca2+ deficiency, at least under the present conditions of severe Ca2+ deprivation.
II. Regulation of CFTR by small molecules including HCO 3 −
Authors: Illek Beate , Fischer Horst , Machen Terry E.
Published: December 2017

Cystic fibrosis (CF) affects a number of epithelial tissues, including those in the gastrointestinal tract. The goal of this review is to summarize data related to regulation of the protein product of the CF gene, CF transmembrane conductance regulator (CFTR), by a variety of small molecules. There has been a surge of interest in discovering small molecules that could be exogenously added to cells and tissues to regulate CFTR and could potentially be used alone or in combination with genetic approaches for therapy in CF. We will discuss the apparent mechanisms of action of genistein, milrinone, 8-cyclopentyl-1,3-dipropylxanthine, IBMX, and NS-004; several of which appear to interact directly with one or both nucleotide binding domains of CFTR. We also discuss how [Formula: see text]interacts with CFTR as both a permeating anion and a potential regulator of Cl−permeation through the CFTR ion channel. It is likely that there are complicated interactions between Cl−and[Formula: see text] in the secretion of both ions through the CFTR and the anion exchanger in intestinal cells, and these may yield a role of CFTR in regulation of intestinal[Formula: see text] secretion as well as of intra- and extracellular pH.
Hepatic production and intestinal uptake of IGF-I: response to infection
Authors: Lang Charles H. , Frost Robert A. , Ejiofor Joseph , Lacy D. Brooks , McGuinness Owen P.
Published: December 2017

The role of the liver and gut in contributing to the infection-induced fall in circulating insulin-like growth factor I (IGF-I) was examined in chronically catheterized conscious dogs. Two weeks before study, catheters and Doppler flow probes were implanted to assess hepatic and gut balance of IGF-I. To control nutrient intake, dogs were placed on total parenteral nutrition (TPN) as their sole caloric source. After dogs received TPN for 5 days, net hepatic and intestine IGF-I balances were assessed. A hypermetabolic infected state was then induced by the intraperitoneal implantation of a fibrin clot containing Escherichia coli. TPN was continued, and organ IGF-I balance was assessed 24 and 48 h after induction of infection. Arterial IGF-I levels were significantly decreased following infection (111 ± 18, 62 ± 10, and 63 ± 8 ng/ml before and 24 and 48 h after, respectively). Net hepatic IGF-I output decreased markedly (221 ± 73, to 73 ± 41 and 41 ± 17 ng ⋅ kg−1 ⋅ min−1before and 24 and 48 h after, respectively). The infection-induced decrease in hepatic IGF-I output could not be explained by concomitant alterations in plasma cortisol or insulin levels. The gut demonstrated a net uptake of IGF-I before infection (178 ± 29 ng ⋅ kg−1 ⋅ min−1). However, after infection, intestinal IGF-I uptake was completely suppressed (−10 ± 15 and −8 ± 36 ng ⋅ kg−1 ⋅ min−1). In summary, infection decreases net hepatic IGF-I release 65–80% and completely suppresses net IGF-I uptake by the intestine. As a consequence of these reciprocal changes in IGF-I balance across the liver and intestine, splanchnic production of IGF-I was unchanged by infection. These data suggest that changes in the clearance and/or production of IGF-I by extrasplanchnic tissues contribute to the infection-induced decrease in circulating IGF-I levels.
Intrahepatic STAT-3 activation and acute phase gene expression predict outcome after CLP sepsis in the rat
Authors: Andrejko Kenneth M. , Chen Jodi , Deutschman Clifford S.
Published: December 2017

Interleukin-6 (IL-6) regulates hepatic acute phase responses by activating the transcription factor signal transducer and activator of transcription (STAT)-3. IL-6 also may modulate septic pathophysiology. We hypothesize that 1) STAT-3 activation and transcription of α2-macroglobulin (A2M) correlate with recovery from sepsis and 2) STAT-3 activation and A2M transcription reflect intrahepatic and not serum IL-6. Nonlethal sepsis was induced in rats by single puncture cecal ligation and puncture (CLP) and lethal sepsis via double-puncture CLP. STAT-3 activation and A2M transcription were detected at 3–72 h and intrahepatic IL-6 at 24–72 h following single-puncture CLP. All were detected only at 3–16 h following double-puncture CLP and at lower levels than following single-puncture CLP. Loss of serum and intrahepatic IL-6 activity after double-puncture CLP correlated with mortality. Neither intrahepatic nor serum IL-6 levels correlated with intrahepatic IL-6 activity. STAT-3 activation following single-puncture CLP inversely correlated with altered transcription of gluconeogenic, ketogenic, and ureagenic genes. IL-6 may have both beneficial and detrimental effects in sepsis. Fulminant sepsis may decrease the ability of hepatocytes to respond to IL-6.
Distribution and regulation of apical Cl/anion exchanges in surface and crypt cells of rat distal colon
Authors: Rajendran Vazhaikkurichi M. , Binder Henry J.
Published: December 2017

Na depletion inhibits electroneutral Na-Cl absorption in intact tissues and Na/H exchange in apical membrane vesicles (AMV) of rat distal colon. Two anion (Cl/HCO3and Cl/OH) exchanges have been identified in AMV from surface cells of rat distal colon. To determine whether Cl/HCO3and/or Cl/OH exchange is responsible for vectorial Cl movement, this study examined the spatial distribution and the effect of Na depletion on anion-dependent36Cl uptake by AMV in rat distal colon. These studies demonstrate that HCO3concentration gradient-driven36Cl uptake (i.e., Cl/HCO3exchange) is 1) primarily present in AMV from surface cells and 2) markedly reduced by Na depletion. In contrast, OH concentration gradient-driven36Cl uptake (i.e., Cl/OH exchange) present in both surface and crypt cells is not affected by Na depletion. In Na-depleted animals HCO3also stimulates36Cl via Cl/OH exchange with low affinity. These results suggest that Cl/HCO3exchange is responsible for vectorial Cl absorption, whereas Cl/OH exchange is involved in cell volume and/or cell pH homeostasis.
Regulation of cytosolic free calcium concentration by extracellular nucleotides in human hepatocytes
Authors: Schöfl Christof , Ponczek Martin , Mader Thilo , Waring Mark , Benecke Heike , Von Zur Mühlen Alexander , Mix Heiko , Cornberg Markus , Böker Klaus H. W. , Manns Michael P. , Wagner Siegfried
Published: December 2017

The effects of extracellular ATP and other nucleotides on the cytosolic free Ca2+concentration ([Ca2+]i) have been studied in single primary human hepatocytes and in human Hep G2 and HuH-7 hepatoma cells. ATP, adenosine 5′- O-(3-thiotriphosphate) (ATPγS), and UTP caused a concentration-dependent biphasic increase in [Ca2+]iwith an initial peak followed by a small sustained plateau in most cells. In some cells, however, repetitive Ca2+transients were observed. The rank order of potency was ATP ≥ UTP > ATPγS, and complete cross-desensitization of the Ca2+responses occurred between ATP and UTP. The initial transient peak in [Ca2+]iwas resistant to extracellular Ca2+depletion, which demonstrates mobilization of internal Ca2+by inositol 1,4,5-trisphosphate whose formation was enhanced by ATP and UTP. In contrast, the sustained plateau phase required influx of external Ca2+. Ca2+influx occurs most likely through a capacitative Ca2+entry mechanism, which was shown to exist in these cells by experiments performed with thapsigargin. On the molecular level, specific mRNA coding for the human P2Y1, P2Y2, P2Y4, and P2Y6receptors could be detected by RT-PCR in Hep G2 and HuH-7 cells. However, ADP and UDP, which are agonists for P2Y1and P2Y6receptors, respectively, caused no changes in [Ca2+]i, demonstrating that these receptors are not expressed at a functional level. Likewise, α,β-methylene-ATP, β,γ-methylene-ATP, AMP, and adenosine were inactive in elevating [Ca2+]i, suggesting that the ATP-induced increase in [Ca2+]iwas not caused by activation of P2X or P1 receptors. Thus, on the basis of the pharmacological profile of the nucleotide-induced Ca2+-responses, extracellular ATP and UTP increase [Ca2+]iby activating P2Y2and possibly P2Y4receptors coupled to the Ca2+-phosphatidylinositol signaling cascade in human hepatocytes. This suggests that extracellular nucleotides from various sources may contribute to the regulation of human liver cell functions.
Downregulated in adenoma gene encodes a chloride transporter defective in congenital chloride diarrhea
Authors: Moseley Richard H. , Höglund Pia , Wu Gary D. , Silberg Debra G. , Haila Siru , De La Chapelle Albert , Holmberg Christer , Kere Juha
Published: December 2017

Congenital chloride diarrhea (CLD) is a recessively inherited disorder characterized by massive loss of chloride in stool. We previously identified mutations in the downregulated in adenoma ( DRA) gene in patients with CLD and demonstrated that DRA encodes an intestine-specific sulfate transporter. To determine whether DRA is an intestinal chloride transporter and how mutations affect transport, Xenopus oocytes were injected with wild-type and mutagenized DRA cRNA and uptake of Cl−and[Formula: see text] was assayed. Both Cl−and[Formula: see text] were transported by wild-type DRA and an outwardly directed pH gradient stimulated Cl−uptake, consistent with Cl−/OH−exchange. Among three mutants, C307W transported both anions as effectively as wild-type, whereas transport activity was lost in V317del and the double mutant identified in 32 of 32 Finnish CLD patients. We conclude that DRA is a chloride transporter defective in CLD and that V317del is a functional mutation and C307W a silent polymorphism.
Secretin induces the apical insertion of aquaporin-1 water channels in rat cholangiocytes
Authors: Marinelli Raúl A. , Tietz Pamela S. , Pham Linh D. , Rueckert Lisa , Agre Peter , LaRusso Nicholas F.
Published: December 2017

Aquaporin-1 (AQP1) water channels are present in the apical and basolateral plasma membrane domains of bile duct epithelial cells, or cholangiocytes, and mediate the transport of water in these cells. We previously reported that secretin, a hormone known to stimulate ductal bile secretion, increases cholangiocyte osmotic water permeability and stimulates the redistribution of AQP1 from an intracellular vesicular pool to the cholangiocyte plasma membrane. Nevertheless, the target plasma membrane domain (i.e., basolateral or apical) for secretin-regulated trafficking of AQP1 in cholangiocytes is unknown, as is the functional significance of this process for the secretion of ductal bile. In this study, we used primarily an in vivo model (i.e., rats with cholangiocyte hyperplasia induced by bile duct ligation) to address these issues. AQP1 was quantitated by immunoblotting in apical and basolateral plasma membranes prepared from cholangiocytes isolated from rats 20 min after intravenous infusion of secretin. Secretin increased bile flow (78%, P < 0.01) as well as the amount of AQP1 in the apical cholangiocyte plasma membrane (127%, P < 0.05). In contrast, the amount of AQP1 in the basolateral cholangiocyte membrane and the specific activity of an apical cholangiocyte marker enzyme (i.e., γ-glutamyltranspeptidase) were unaffected by secretin. Similar observations were made when freshly isolated cholangiocytes were directly exposed to secretin. Immunohistochemistry for AQP1 in liver sections from secretin-treated rats showed intensified staining at the apical region of cholangiocytes. Pretreatment of rats with colchicine (but not with its inactive analog β-lumicolchicine) inhibited both the increases of AQP1 in the cholangiocyte plasma membrane (94%, P < 0.05) and the bile flow induced by secretin (54%, P < 0.05). Our results in vivo indicate that secretin induces the microtubule-dependent insertion of AQP1 exclusively into the secretory pole (i.e., apical membrane domain) of rat cholangiocytes, a process that likely accounts for the ability of secretin to stimulate ductal bile secretion.
A novel plant-derived inhibitor of cAMP-mediated fluid and chloride secretion
Authors: Gabriel S. E. , Davenport S. E. , Steagall R. J. , Vimal V. , Carlson T. , Rozhon E. J.
Published: December 2017

We have identified an agent (SP-303) that shows efficacy against in vivo cholera toxin-induced fluid secretion and in vitro cAMP-mediated Cl−secretion. Administration of cholera toxin to adult mice results in an increase in fluid accumulation (FA) in the small intestine (FA ratio = 0.63 vs. 1.86 in control vs. cholera toxin-treated animals, respectively). This elevation in FA induced by cholera toxin was significantly reduced (FA ratio = 0.70) in animals treated with a 100 mg/kg dose of SP-303 at the same time as the cholera treatment. Moreover, when SP-303 was administered 3 h after cholera toxin, a dose-dependent inhibition of FA levels was observed with a half-maximal inhibitory dose of 10 mg/kg. In Ussing chamber studies of Caco-2 or T84 monolayer preparations, SP-303 had a significant effect on both basal current and forskolin-stimulated Cl−current. SP-303 also induced an increase in resistance that paralleled the observed decrease in current. These data suggest that SP-303 has an inhibitory effect on cAMP-mediated Cl−and fluid secretion. Thus SP-303 may prove to be a useful broad-spectrum antidiarrheal agent.
III. Congenital chloride diarrhea
Authors: Kere Juha , Lohi Hannes , Höglund Pia
Published: December 2017

Congenital chloride diarrhea (CLD) is a recessively inherited disorder of intestinal electrolyte absorption that involves, specifically, Cl−/[Formula: see text]exchange. CLD is caused by mutations in a chromosome 7 gene, first known as DRA (for downregulated in adenoma). The disease occurs in all parts of the world but is more common in some populations with genetic founder effects. More than 20 mutations in the gene are known to date. The CLD (or DRA) gene encodes a transmembrane protein belonging to the sulfate transporter family with three known members in humans, all associated with a distinct genetic disease. Members of the gene family can transport other anions as well that may turn out to be physiologically more important than sulfate transport. The gene family is well conserved in many prokaryotic and eukaryotic species and is expected to be much larger than presently known.
Role of nitric oxide in fasting gastric fundus tone and in 5-HT1 receptor-mediated relaxation of gastric fundus
Authors: Coulie Bernard , Tack Jan , Sifrim Daniel , Andrioli Antonius , Janssens Jozef
Published: December 2017

Fasting gastric fundus tone is maintained by continuous cholinergic input. 5-Hydroxytryptamine-1 (5-HT1) receptor activation decreases gastric fundus tone in humans. Whether this fundus-relaxing effect is mediated via inhibition of cholinergic input or via activation of a nitrergic pathway is unknown. The aim of the present study was to determine the effect of nitrergic inhibition on feline gastric fundus tone and on 5-HT1receptor-mediated relaxation of the fundus. Administration of N ω-nitro-l-arginine methyl ester (l-NAME) alone caused a significant decrease of the mean baseline volume ( P < 0.005), which was restored completely by addition ofl-arginine. Sumatriptan caused a dose-dependent relaxation of the gastric fundus ( P < 0.0005). This relaxation was inhibited by l-NAME ( P < 0.02) and was restored by prior administration of l-arginine. Buspirone did not cause any change in mean baseline volume, whereas the sumatriptan-induced relaxation was not affected by prior administration of NAN-190. Our data indicate that fasting fundus tone relies not only on continuous cholinergic input but also on continuous nitrergic input, implying that fasting fundus tone is maintained by the balance of a cholinergic and nitrergic drive. Furthermore, fundus relaxation via 5-HT1 receptor activation is mediated through activation of a nitrergic pathway.
Normalization of atropine-induced postprandial dysrhythmias with gastric pacing
Authors: Qian Liwei , Lin Xuemei , Chen J. D. Z.
Published: December 2017

Gastric pacing has received increasing attention recently. However, few studies have systematically assessed the effect of pacing on gastric dysrhythmias. The aims of this study were to investigate the effect of gastric pacing on gastric dysrhythmia and to explore whether the effect of gastric pacing was mediated via cholinergic nerves. Eight hound dogs implanted with three pairs of serosal electrodes were studied. Three study sessions were performed on each dog. The experiment was conducted sequentially as follows: a 30-min myoelectrical recording immediately after a meal, intravenous injection of atropine or saline, and three sequential 20-min myoelectrical recordings with or without gastric pacing during the second 20-min recording. The percentage of regular slow waves (3.5–7.0 cycles/min) was calculated using spectral analysis. The percentage of the regular slow waves was progressively reduced from 96.7 ± 1.7% at baseline to 29.6 ± 9.0 ( P < 0.001), 23.1 ± 7.1 ( P < 0.001), and 27.3 ± 4.3% ( P < 0.001), respectively, during the first, second, and third 20 min after atropine injection. Normalization of the gastric slow wave was achieved with gastric pacing 2.3 ± 1.0 min after the initiation of pacing. The percentage of regular slow waves was significantly increased both during pacing (93.6 ± 2.4 vs. 23.1 ± 7.1%, P < 0.002) and after pacing (70.9 ± 6.8 vs. 27.3 ± 4.3%, P < 0.003) in comparison with the session without pacing. We conclude that 1) atropine induces gastric myoelectric dysrhythmia in the fed state, 2) gastric pacing is able to normalize gastric postprandial dysrhythmia induced by atropine, and 3) the effect of gastric pacing is not mediated by vagal cholinergic mechanism.
Kinetic analysis of59Fe movement across the intestinal wall in duodenal rat segments ex vivo
Authors: Schümann Klaus , Elsenhans Bernd , Forth Wolfgang
Published: December 2017

Duodenal segments from iron-deficient and iron-adequate rats were luminally perfused ex vivo with solutions containing 1, 10, 50, 100, 200 and 500 μmol59Fe/l. When duodenal tissue load and mucosal-to-serosal transport had reached a steady state, perfusion was continued without luminal59Fe supply. Mobilization of59Fe from the duodenal tissue into the serosally released absorbate followed first-order rate kinetics, which permitted calculation of the asymptotic maximum, the rate constant, and the initial mobilization rate for tissue-to-absorbate transfer. There was no evidence for adaptation of59Fe tissue binding in iron-deficient segments.59Fe tissue-to-absorbate transfer increased in proportion to the mobilizable fraction of recently absorbed iron in the tissue, which is indicative of simple diffusion or carrier-mediated transport below saturation. Regulation of the mucosal uptake step appears to determine the mobilizable59Fe fraction and thus the adaptation of the overall iron absorption process to the demand. Iron retention in the duodenal tissue and iron transfer from here into the body appear not to be either regulated or rate limited.
Differential regulation of NHE isoforms by sodium depletion in proximal and distal segments of rat colon
Authors: Ikuma Mutsuhiro , Kashgarian Michael , Binder Henry J. , Rajendran Vazhaikkurichi M.
Published: December 2017

Dietary sodium depletion has multiple diverse effects on ion transport in the rat colon, including both the induction and inhibition of electroneutral NaCl absorption in proximal and distal colon of rat, respectively. To establish the mechanism of the differential regulation of Na+absorption by sodium depletion, this study utilized 1) HOE-694, a dose-dependent inhibitor of Na+/H+exchanger (NHE) isoforms, in studies of proton gradient-driven22Na uptake (i.e., Na+/H+exchange) by apical membrane vesicles (AMV); 2) Northern blot analyses of NHE isoform-specific mRNA abundance; and 3) Western blot analyses of NHE isoform-specific protein expression. HOE-694 inhibition studies establish that 25 μM HOE-694-sensitive (NHE2) and 25 μM HOE-694-insensitive (NHE3) Na+/H+exchange activities are present in AMV of both proximal and distal colon of normal rats. In proximal colon, dietary sodium depletion enhanced both NHE2 and NHE3 isoform-specific Na+/H+exchange activities, protein expression, and mRNA abundance. In contrast, in distal colon both NHE2 and NHE3 isoform-specific Na+/H+exchange activities, protein expression, and mRNA abundance were inhibited by sodium depletion. NHE1 isoform-specific mRNA abundance in proximal or distal colon was not altered by sodium depletion. Differential effects by sodium depletion on Na+/H+exchange in rat colon are tissue specific and isoform specific; sodium depletion both induces and inhibits apical Na+/H+exchange at a pretranslational level.
Evidence of T cell receptor β-chain patterns in inflammatory and noninflammatory bowel disease states
Authors: Saubermann Lawrence J. , Probert Christopher S. J. , Christ Andreas D. , Chott Andreas , Turner Jerrold R. , Stevens A. Christopher , Balk Steven P. , Blumberg Richard S.
Published: December 2017

T cell activation, as defined by expression of relevant cell surface molecules, such as the interleukin-2 receptor (CD25), is increased in many chronic relapsing diseases, including inflammatory bowel disease (IBD). These T cells are generally activated through contact of their clonotypic T cell receptor (TCR) with a peptide antigen presented by a major histocompatibility complex molecule. One of the putative antigenic contact sites for the TCR is the third complementarity determining region (CDR3) of the TCR β-chain variable region (TCRBV). Therefore, analysis of the TCRBV CDR3 provides insight into the diversity of antigens encountered by a given T cell population. This study evaluated the TCRBV CDR3 usage of the activated intestinal lymphocytes from human subjects with IBD, diverticulitis (inflammatory control), and a normal tissue control. Public patterns, as demonstrated by shared TCRBV CDR3 amino acid sequences of activated intestinal T cell subpopulations, were observed. In particular, a public pattern of TCRBV22, a conserved valine in the fifth position, and use of TCRBJ2S1 or TCRBJ2S5 was present in three of four Crohn’s disease subjects while not present in the ulcerative colitis subjects. However, the private patterns of TCRBV CDR3 region amino acid sequences were far more striking and easily demonstrated in all individuals studied, including a normal noninflammatory control. Thus we conclude that selective antigenic pressures are prevalent among an individual’s activated intestinal lymphocytes.
Rat liver endothelial cell glutamine transporter and glutaminase expression contrast with parenchymal cells
Authors: Lohmann Rüdiger , Souba Wiley W. , Bode Barrie P.
Published: December 2017

Despite the central role of the liver in glutamine homeostasis in health and disease, little is known about the mechanism by which this amino acid is transported into sinusoidal endothelial cells, the second most abundant hepatic cell type. To address this issue, the transport ofl-glutamine was functionally characterized in hepatic endothelial cells isolated from male rats. On the basis of functional analyses, including kinetics, cation substitution, and amino acid inhibition, it was determined that a Na+-dependent carrier distinct from system N in parenchymal cells, with properties of system ASC or B0, mediated the majority of glutamine transport in hepatic endothelial cells. These results were supported by Northern blot analyses that showed expression of the ATB0transporter gene in endothelial but not parenchymal cells. Concurrently, it was determined that, whereas both cell types express glutamine synthetase, hepatic endothelial cells express the kidney-type glutaminase isozyme in contrast to the liver-type isozyme in parenchymal cells. This represents the first report of ATB0and kidney-type glutaminase isozyme expression in the liver, observations that have implications for roles of specific cell types in hepatic glutamine homeostasis in health and disease.
Enteropathogenic E. coli attenuates secretagogue-induced net intestinal ion transport but not Cl− secretion
Authors: Hecht Gail , Koutsouris Athanasia
Published: December 2017

Enteric bacterial pathogens often increase intestinal Cl− secretion. Enteropathogenic Escherichia coli(EPEC) does not stimulate active ion secretion. In fact, EPEC infection decreases net ion transport in response to classic secretagogues. This has been presumed to reflect diminished Cl− secretion. The aim of this study was to investigate the influence of EPEC infection on specific intestinal epithelial ion transport processes. T84 cell monolayers infected with EPEC were used for these studies. EPEC infection significantly decreased short-circuit current ( I sc) in response to carbachol and forskolin, yet125I efflux studies revealed no difference in Cl− channel activity. There was also no alteration in basolateral K+ channel or Na+-K+-2Cl−cotransport activity. Furthermore, net36Cl−flux was not decreased by EPEC. No alterations in either K+ or Na+ transport could be demonstrated. Instead, removal of basolateral bicarbonate from uninfected monolayers yielded an I sc response approximating that observed with EPEC infection, whereas bicarbonate removal from EPEC-infected monolayers further diminished I sc. These studies suggest that the reduction in stimulated I sc is not secondary to diminished Cl−secretion. Alternatively, bicarbonate-dependent transport processes appear to be perturbed.
Primary active transport of organic anions on bile canalicular membrane in humans
Authors: Niinuma Kayoko , Kato Yukio , Suzuki Hiroshi , Tyson Charles A. , Weizer Valorie , Dabbs Jack E. , Froehlich Ritchie , Green Carol E. , Sugiyama Yuichi
Published: December 2017

Biliary excretion of several anionic compounds was examined by assessing their ATP-dependent uptake in bile canalicular membrane vesicles (CMV) prepared from six human liver samples. 2,4-Dinitrophenyl- S-glutathione (DNP-SG), leukotriene C4(LTC4), sulfobromophthalein glutathione (BSP-SG), E3040 glucuronide (E-glu), β-estradiol 17-(β-d-glucuronide) (E2–17G), grepafloxacin glucuronide (GPFXG), pravastatin, BQ-123, and methotrexate, which are known to be substrates for the rat canalicular multispecific organic anion transporter, and taurocholic acid (TCA), a substrate for the bile acid transporter, were used as substrates. ATP-dependent and saturable uptake of TCA, DNP-SG, LTC4, E-glu, E2–17G, and GPFXG was observed in all human CMV preparations examined, suggesting that these compounds are excreted in the bile via a primary active transport system in humans. Primary active transport of the other substrates was also seen in some of CMV preparations but was negligible in the others. The ATP-dependent uptake of all the compounds exhibited a large inter-CMV variation, and there was a significant correlation between the uptake of glutathione conjugates (DNP-SG, LTC4, and BSP-SG) and glucuronides (E-glu, E2–17G, and GPFXG). However, there was no significant correlation between TCA and the other organic anions, implying that the transporters for TCA and for organic anions are different also in humans. When the average value for the ATP-dependent uptake by each preparation of human CMVs was compared with that of rat CMVs, the uptake of glutathione conjugates and nonconjugated anions (pravastatin, BQ-123, and methotrexate) in humans was ∼3- to 76-fold lower than that in rats, whereas the uptake of glucuronides was similar in the two species. Thus there is a species difference in the primary active transport of organic anions across the bile canalicular membrane that is less marked for glucuronides.