bluuebunny/crossref_metadata_2025 huggingface dataset:
Interferon-γ decreases barrier function in T84 cells by reducing ZO-1 levels and disrupting apical actin
Authors: Youakim Adel , Ahdieh Minoo
Published: December 2017

The effects of interferon-γ (IFN-γ) on tight junctions in T84 human intestinal epithelial cells were investigated. Treatment of T84 cells with IFN-γ caused a dose- and time-dependent increase in monolayer permeability as assessed by transepithelial electrical resistance measurements. Examination of specific proteins associated with tight junctions by immunoblotting and confocal microscopy revealed changes in the expression levels and localization of some of these proteins after exposure of the cells to IFN-γ. Specifically, IFN-γ treatment resulted in an almost total loss of zonula occludens (ZO)-1, whereas the levels of ZO-2 and occludin showed relatively modest decreases compared with untreated cells. Loss of ZO-1 was associated with the altered localization of ZO-2 and occludin. In IFN-γ-treated cells, ZO-2 and occludin were diffusely distributed, whereas, in control cells, they, along with ZO-1, were predominantly localized to the tight junctions. Analysis of ZO-1 protein and RNA by pulse chase and RT-PCR, respectively, showed an increase in protein turnover, a decrease in protein synthesis, and a reduction in RNA levels following IFN-γ treatment. In contrast to ZO-1, ZO-2 and occludin did not show any major changes in these parameters. In addition, the organization of actin in the apical and tight junction regions, but not in the mid- or basal regions, of the cells was also perturbed by IFN-γ treatment of cells. Time-course analysis of IFN-γ-induced alterations in ZO-1 expression and apical actin perturbation indicated that these two effects were intimately linked and could not be dissociated. These results suggest that IFN-γ affects barrier function in T84 cells by decreasing the levels of ZO-1 and perturbing apical actin organization, which leads to a disorganization of the tight junction and an increase in paracellular permeability.
Uptake of bromosulfophthalein via SO2-4/OH−exchange increases the K+conductance of rat hepatocytes
Authors: Wehner Frank , Tinel Hanna
Published: December 2017

In confluent primary cultures of rat hepatocytes, micromolar concentrations of bromosulfophthalein (BSP) lead to a sizeable hyperpolarization of membrane voltage. The effect is a saturable function of BSP concentration yielding an apparent value of 226 μmol/l and a Vmaxof −10.3 mV. The BSP-induced membrane hyperpolarization is inhibited by the K+channel blocker Ba2+, and in cable-analysis and ion-substitution experiments it becomes evident that the effect is due to a significant increase in cell membrane K+conductance. Voltage changes were attenuated by the simultaneous administration of [Formula: see text], succinate, and cholate ( cis-inhibition) and increased after preincubation with [Formula: see text] and succinate ( trans-stimulation), suggesting that the effect occurs via BSP uptake through the known[Formula: see text]/OH−exchanger. Microfluorometric measurements reveal that BSP-induced activation of K+conductance is not mediated by changes in cell pH, cell Ca2+, or cell volume. It is concluded that K+channel activation by BSP (as well as by DIDS and indocyanine green) may reflect a physiological mechanism linking the sinusoidal uptake of certain anions to their electrogenic canalicular secretion.
Rat intestinal α1-antitrypsin secretion is regulated by triacylglycerol feeding
Authors: Xie Qing-Mei , Shao Jian-Su , Alpers David H.
Published: December 2017

α1-Antitrypsin (AAT) is secreted by the enterocyte, but its regulation of expression, intramucosal distribution, and functional status are unclear. After corn oil gavage (plus Pluronic L-81 to block chylomicron release), rat intestine was examined for mRNA encoding AAT, immunoreactivity by light and electron microscopy, and protein content by Western blot. Species-specific antisera used were raised against both AAT and surfactant-like particle (SLP), a membrane secreted by the enterocyte in response to fat feeding. Purified luminal SLP was fractionated by Bio-Gel P-200 chromatography to assess its interaction with AAT. Triacylglycerol feeding maximally increased mucosal mRNA-encoding AAT and AAT intracellular protein content by 3 and 5 h, respectively. Immunocytochemistry revealed predominance of AAT in basolateral spaces around enterocytes and Pluronic-blocked extracellular accumulation of AAT, patterns nearly identical to those of secreted SLP. About 10% of AAT was reversibly associated with SLP. Luminal AAT was smaller (51 kDa) than mature AAT (55 kDa) and did not form a complex with pancreatic elastase. When the common bile duct was tied, excluding pancreatic proteases from the lumen, mature AAT that was cleaved by pancreatic elastase was secreted. The luminal secretion of AAT and its reversible association with SLP suggest an intracellular association and a possible role for AAT during lipid digestion and absorption.
Metabolism of short-chain fatty acids by rat colonic mucosa in vivo
Authors: Fitch Mark D. , Fleming Sharon E.
Published: December 2017

To determine the influence of substrate concentration and substrate interactions on short-chain fatty acid metabolism in vivo, a surgical procedure was established. Rats were surgically operated to cannulate a 5-cm segment of proximal colon, isolate the vasculature, and cannulate the right colic vein draining this segment. Thus metabolism was restricted to the defined colonic segment. The appearance of total14C and14CO2in the mesenteric blood stabilized after 30 min of perfusion. Increasing luminal concentrations of butyrate from 2 to 40 mmol/l resulted in linear increases in total14C, but14CO2production from [14C]butyrate increased as a function of concentration only up to 10 mmol/l and was stable at higher butyrate concentrations. In addition to CO2, 3-hydroxybutyrate and lactate were major metabolites of acetate and butyrate in vivo. The presence of a mixture of alternative substrates in the lumen had no influence on the metabolism of butyrate to CO2but significantly reduced the metabolism of acetate to CO2. When compared with young (4 mo old) animals, transport of butyrate was significantly lower for aged (48 mo old) animals, as evidenced by the rate of appearance in blood of total14C ( P = 0.04) and14C in butyrate ( P = 0.03), but metabolism was similar, since differences were not significant for14C in the major metabolites 3-hydroxybutyrate ( P = 0.06) and CO2( P = 0.17). These results show that important aspects of short-chain fatty acid transport and metabolism are not predicted from data using isolated colonocytes but require study using an in vivo model.
Microcirculatory perfusion deficits are not essential for remote parenchymal injury within the liver
Authors: Brock Robert W. , Carson Michael W. , Harris Kenneth A. , Potter Richard F.
Published: December 2017

A normotensive model of hindlimb ischemia-reperfusion in Wistar rats was used to test the hypothesis that microvascular perfusion deficits contribute to the initiation of remote hepatic injury during a systemic inflammatory response. Animals were randomly assigned to one of three groups: 4 h of ischemia with 6 h of reperfusion (I/R-6; n = 4), 4 h of ischemia with 3 h of reperfusion (I/R-3; n = 5), or no ischemia (naive; n = 5). With intravital fluorescence microscopy, propidium iodide (PI; 0.05 mg/100 g body wt) was injected for the in vivo labeling of lethally injured hepatocytes (number/10−1mm3). PI-positive hepatocytes increased progressively over the 6-h period (naive 32.9 ± 7.8 vs. I/R-3 92.8 ± 11.5 vs. I/R-6 232 ± 39.2), with no difference between periportal and pericentral regions of the lobule. Additionally, a significant decrease in continuously perfused sinusoids (naive 70.0 ± 1.5 vs. I/R-3 65.0 ± 1.0 vs. I/R-6 48.8 ± 0.9%) was measured. Regional sinusoidal perfusion differences were only observed after 3 h of limb reperfusion. Indirect measures of hepatocellular injury using alanine transaminase levels support the progressive nature of hepatic parenchymal injury (0 h 57.8 ± 6.5 vs. 3 h 115.3 ± 20.7 vs. 6 h 125.6 ± 19.5 U/l). Evidence from this study suggests that remote hepatic parenchymal injury occurs early and progresses after the induction of a systemic inflammatory response and that microvascular perfusion deficits are not essential for the initiation of such injury.
Effect of milrinone on small mesenteric artery vasoconstriction: role of K+channels
Authors: Taylor Mark S. , Benoit Joseph N.
Published: December 2017

We examined whether milrinone-mediated attenuation of small mesenteric artery vasoconstriction results predominantly from the activation of vascular smooth muscle K+channels. Resistance arteries (∼150 μm) were dissected from rat mesentery and were mounted on a wire myograph. Isometric force development in response to increasing concentrations of norepinephrine (NE) was monitored before and after treatment with the type 3 phosphodiesterase inhibitor milrinone. Milrinone significantly reduced NE-induced vasoconstriction, attenuating both NE sensitivity and maximal tension generation. Inhibition of ATP-sensitive K+channels or voltage-gated K+channels did not prevent the milrinone-induced attenuation of NE responses. Blockade of inwardly rectifying K+channels or Ca2+-sensitive K+channels prevented the milrinone-mediated reduction in NE sensitivity, but this effect was apparently due to direct enhancement of vasoconstrictor responsiveness rather than interference with the mechanism of milrinone action. In addition, milrinone elicited substantial relaxation in vessels preconstricted with 100 mM KCl. This effect was mimicked by the adenylyl cyclase activator forskolin and was reversed by the Rp diastereomer of cAMP, which is a cAMP-dependent protein kinase (PKA) inhibitor. Our results indicate that cAMP/PKA-mediated impairment of vasoconstriction may occur without the contribution of K+channel regulation.
Cytoplasmic transport of fatty acids in rat enterocytes: role of binding to fatty acid-binding protein
Authors: Luxon Bruce A. , Milliano Michael T.
Published: December 2017

The intracellular movement of fatty acids is thought to be facilitated through codiffusion with fatty acid-binding protein (FABP). This facilitation may occur by decreasing binding to immobile membranes, leading to faster cytoplasmic diffusion. The aims of this study were to measure the intracellular transport of 12- N-methyl-(7-nitrobenzo-2-oxa-1,3-diazol)aminostearate (NBD-stearate) in villus rat enterocytes and to determine 1) the mechanism of its cytoplasmic transport and 2) if its transport rate correlated with the known variation of FABP binding capacity along the length of the small intestine. Two-dimensional laser photobleaching was used to measure the movement of a fluorescent fatty acid NBD-stearate in enterocytes isolated from different segments of rat intestine. The fraction of NBD-stearate found in the cytostol of enterocytes was determined by differential centrifugation. Cytoplasmic transport of NBD-stearate occurred solely by diffusion and not by convection. Diffusion was homogeneous (nondirectional), consistent with isotropic diffusion. The diffusion rate varied with location along the intestine, correlating with the local FABP concentration and measured cytosolic binding. We conclude that cytoplasmic proteins like FABP promote the intracellular transport of fatty acids by enhancing their diffusive flux. We suggest that facilitation is not specific for a particular cell type but occurs in a variety of cells that transport fatty acids and may contain different types of FABP.
Molecular cloning, tissue distribution, and functional expression of the human Na+/H+exchanger NHE2
Authors: Malakooti Jaleh , Dahdal Refka Y. , Schmidt Larry , Layden Thomas J. , Dudeja Pradeep K. , Ramaswamy Krishnamurthy
Published: December 2017

In the present report, we describe the cloning of a human colonic cDNA that describes the full-length Na+/H+exchanger (NHE) 2 coding region. The human NHE2 (hNHE2) cDNA encodes for a polypeptide of 812 amino acids with a 90% overall identity to both rabbit and rat NHE2 isoforms. In comparison with SLC9A2, recently reported as the human NHE2, the hNHE2 polypeptide is 115 amino acids longer in the NH2-terminal end and shows only an 84% DNA nucleotide sequence identity. Northern blot analysis revealed that hNHE2 message has an uneven tissue distribution, with high levels in the skeletal muscle, colon, and kidney and lower levels in the testis, prostate, ovary, and small intestine. Protein expression studies with hNHE2 clone showed that a 75-kDa protein was expressed. Stable expression of transfected full-length hNHE2 cDNA in Na+/H+exchange-deficient LAP1 cells exhibited Na+-dependent pH recovery after an acid prepulse that was inhibited by 0.1 mM amiloride. These data indicate that this cDNA is the true human NHE2 cDNA and that the encoded protein is capable of catalyzing Na+/H+exchange activity.
Intestinal overexpression of EGF in transgenic mice enhances adaptation after small bowel resection
Authors: Erwin Christopher R. , Helmrath Michael A. , Shin Cathy E. , Falcone Richard A. , Stern Lawrence E. , Warner Brad W.
Published: December 2017

The effect of direct intestinal overexpression of epidermal growth factor (EGF) on postresection adaptation has been investigated by the production of transgenic mouse lines. A murine pro-EGF cDNA construct was produced, and expression of the EGF construct was targeted to the small intestine with the use of the rat intestinal fatty acid-binding protein promoter. An approximately twofold increase in intestinal EGF mRNA and protein was detected in heterozygous mice. No changes in serum EGF levels were noted. Except for a slightly shortened small intestine, no other abnormal phenotype was observed. Intestinal adaptation (increases in body weight, DNA, protein content, villus height, and crypt depth) was markedly enhanced after a 50% proximal small bowel resection in transgenic mice compared with nontransgenic littermates. This transgenic mouse model permits the study of intestinal adaptation and other effects of EGF in the small intestine in a more physiological and directed manner than has been previously possible. These results endorse a direct autocrine/paracrine mechanism for EGF on enterocytes as a means to enhance adaptation.
Intracisternal PYY increases gastric mucosal resistance: role of cholinergic, CGRP, and NO pathways
Authors: Yang Hong , Kawakubo Keishi , Taché Yvette
Published: December 2017

The influence of intracisternal injection of peptide YY (PYY) on gastric lesions induced by ethanol was studied in urethan-anesthetized rats. Gastric lesions covered 15–22% of the corpus as monitored 1 h after intragastric administration of 45% ethanol (5 ml/kg) in intracisternal vehicle control groups. PYY, at doses of 23, 47, or 117 pmol 30 min before ethanol, decreased gastric lesions by 27%, 63%, and 59%, respectively. Thyrotropin-releasing hormone (TRH) receptor antisense oligodeoxynucleotide pretreatment (intracisternally, 48 and 24 h before intracisternal PYY) did not influence the gastroprotective effect of intracisternal PYY (47 pmol) but abolished that of intracisternal TRH analog RX-77368 (4 pmol). RX-77368 (2.6 pmol) and PYY (6 pmol) were ineffective when injected intracisternally alone but reduced ethanol lesions by 44% when injected simultaneously. Atropine (subcutaneously), the calcitonin gene-related peptide (CGRP) receptor antagonist CGRP-(8—37) (intravenously), or the nitric oxide (NO) synthase inhibitor N G-nitro-l-arginine methyl ester (l-NAME, intravenously) completely abolished the gastroprotective effect of intracisternal PYY (47 pmol), whereas indomethacin (intraperitoneally) had no effect. The l-NAME action was reversed by l-arginine but not by d-arginine (intravenously). These results suggest that intracisternal PYY acts independently of medullary TRH to decrease ethanol-induced gastric lesions. The PYY action involves vagal cholinergic-mediated CGRP/NO protective mechanisms.
Lower esophageal sphincter monitoring with sphinctometer: in vitro and in vivo studies
Authors: Pehlivanov Nonko , Liu Jianmin , Arora Tania , Yamamoto Yoshihiro , Mittal Ravinder K.
Published: December 2017

The sphinctometer is a solid-state device system for prolonged ambulatory recordings of the lower esophageal sphincter (LES) pressure. The aims of this study were to determine 1) the pressure sensitivity and the latency of sphinctometer responses in vitro, 2) if the sphinctometer can record transient LES relaxations (TLESR) in vivo, and 3) if the currently accepted criteria for TLESR are applicable to sphinctometer recordings. Different segments of the sphinctometer were positioned in a chamber at known pressures to assess the sphinctometer readings as well as the latency of the response. Ten healthy subjects were investigated with the use of a solid-state transducer sphinctometer assembly and a pH probe for 4 h. The LES pressure was analyzed during baseline periods, swallow-induced LES relaxations (SILESR), and LES relaxations associated with acid reflux episodes (pH <4) (presumed TLESRs). Our results showed that sphinctometer readings were linearly related to the chamber pressure and the length of the segment exposed to the pressure; however, the latter was considerably underestimated. We also found that sphinctometer segments of equal length but at different levels showed different pressure readings, the mean response time of sphinctometer was 0.25 s, and sphinctometer output was susceptible to temperature changes of the environment. In humans, only 25 of 45 episodes (64%) and 17 of 22 episodes (77%) were detected by the sphinctometer as being SILESRs and TLESRs, respectively. The pattern of the LES pressure during acid reflux events resembled classical TLESR. We concluded that the sphinctometer is a useful device for determining qualitative changes in LES pressure. However, its major limitations are pressure underestimation, different sensitivity of various segments, pressure drifts, and underscoring of LES relaxations.
Oral IGF-I enhances nutrient and electrolyte absorption in neonatal piglet intestine
Authors: Alexander Andrew N. , Carey Hannah V.
Published: December 2017

The effect of orally administered insulin-like growth factor-I (IGF-I) on small intestinal structure and function was studied in 5-day-old colostrum-deprived piglets. Human recombinant IGF-I (3.5 mg ⋅ kg−1 ⋅ day−1) or control vehicle was given orogastrically for 4 days. Body weights, jejunal and ileal mucosa wet and dry weights, and serum IGF-I levels were similar in the two groups. Small intestinal villus height and crypt depth and jejunal enterocyte microvillar dimensions were also similar between groups. Oral IGF-I produced higher rates of jejunal ion transport because of increased basal Na+ absorption. Short-circuit current responses to mucosal addition ofd-glucose andl-alanine and net transepithelial absorption of 3- O-methylglucose were increased by IGF-I. Carrier-mediated uptake ofd-glucose per milligram in everted jejunal sleeves was greater in IGF-I-treated piglets because of a significantly greater maximal rate of uptake. We conclude that rates of net Na+ and Na+-dependent nutrient absorption are enhanced in piglets treated with oral IGF-I, and this effect is independent of changes in mucosal mass or surface area.
Requirement of the MAP kinase cascade for cell cycle progression and differentiation of human intestinal cells
Authors: Aliaga José Cristobal , Deschênes Claude , Beaulieu Jean-François , Calvo Ezéquiel L. , Rivard Nathalie
Published: December 2017

The intracellular signaling pathways responsible for cell cycle arrest and establishment of differentiated cells along the gut axis remain largely unknown. In the present study, we analyzed the regulation of p42/p44 mitogen-activated protein kinase (MAPK) in the process of proliferation and differentiation of human intestinal cells. In vitro studies were done in Caco-2/15 cells, a human colon cancer cell line that spontaneously differentiates into an enterocyte phenotype. In vivo studies were performed on cryostat sections of human fetal intestinal epithelium by indirect immunofluorescence. We found that inhibition of the p42/p44 MAPK signaling by the PD-98059 compound or by ectopic expression of the MAPK phosphatase-1 strongly attenuated E2F-dependent transcriptional activity in Caco-2/15 cells. p42/p44 MAPK activities dramatically decreased as soon as Caco-2/15 cells reached confluence. However, significant levels of activated p42 MAPK were detected in differentiated Caco-2/15 cells. Addition of PD-98059 during differentiation interfered with sustained activation of p42 MAPK and sucrase-isomaltase expression. Although p42/p44 MAPKs were expressed in both the villus tip and crypt cells, their phosphorylated and active forms were detected in the undifferentiated crypt cells. Our results indicate that elevated p42/p44 MAPK activities stimulate cell proliferation of intestinal cells, whereas low sustained levels of MAPK activities correlated with G1arrest and increased expression of sucrase-isomaltase.
Caerulein-induced NF-κB/Rel activation requires both Ca2+and protein kinase C as messengers
Authors: Tando Yusuke , Algül Hana , Wagner Martin , Weidenbach Hans , Adler Guido , Schmid Roland M.
Published: December 2017

The eukaryotic transcription factor NF-κB/Rel is activated by a large variety of stimuli. We have recently shown that NF-κB/Rel is induced during the course of caerulein pancreatitis. Here, we show that activation of NF-κB/Rel by caerulein, a CCK analog, requires increasing intracellular Ca2+levels and protein kinase C activation. Caerulein induces a dose-dependent increase of nuclear NF-κB/Rel binding activity in pancreatic lobules, which is paralleled by degradation of IκBα. IκBβ was only slightly affected by caerulein treatment. Consistent with an involvement of Ca2+, the endoplasmic reticulum-resident Ca2+-ATPase inhibitor thapsigargin activated NF-κB/Rel in pancreatic lobules. The intracellular Ca2+chelator TMB-8 prevented IκBα degradation and subsequent nuclear translocation of NF-κB/Rel induced by caerulein. BAPTA-AM was less effective. Cyclosporin A, a Ca2+/calmodulin-dependent protein phosphatase (PP2B) inhibitor, decreased caerulein-induced NF-κB/Rel activation and IκBα degradation. The inhibitory effect of bisindolylmaleimide suggests that protein kinase C activity is also required for caerulein-induced NF-κB/Rel activation. These data suggest that Ca2+- as well as protein kinase C-dependent mechanisms are required for caerulein-induced NF-κB/Rel activation.
PGE2stimulates O2uptake in hepatic parenchymal cells: involvement of the cAMP-dependent protein kinase
Authors: Qu Wei , Graves Lee M. , Thurman Ronald G.
Published: December 2017

The aim of this study was to determine which PGE2receptors and signal transduction pathways are responsible for the stimulation of oxygen uptake in liver. Hepatic parenchymal cells isolated from female Sprague-Dawley rats were incubated either with PGE2, 17-phenyl-omega-trinor PGE2(an EP1-specific agonist), or 11-deoxy PGE1(an EP2/EP4-specific agonist), and oxygen consumption was measured. Both PGE2and 11-deoxy PGE1stimulated oxygen consumption. However, an EP1agonist was without effect. Although PGE2elevated intracellular calcium, this occurred at concentrations ∼500-fold lower than that required to stimulate oxygen uptake. PGE2-stimulated increases in cAMP formation correlated well with the increase in oxygen consumption. Dibutyryl cAMP also increased oxygen consumption. Furthermore, N-[2-( p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide, a cell-permeable inhibitor of protein kinase A (PKA), reduced the stimulation of oxygen uptake by PGE2. Incubation of isolated parenchymal cell mitochondria with the purified catalytic subunit of PKA and ATP increased both state 3 rates of oxygen uptake and the respiratory control ratio by ∼50%. Activation of these events was prevented by incubation with the PKA inhibitory peptide, PKI. These findings are consistent with the hypothesis that PGE2stimulates oxygen consumption via an EP2and/or EP4subclass of receptors through the actions of cAMP on a cAMP-dependent protein kinase.
Influence of H. pylori infection on meal-stimulated gastric acid secretion and gastroesophageal acid reflux
Authors: Feldman Mark , Cryer Byron , Sammer Doug , Lee Edward , Spechler Stuart J.
Published: December 2017

Gastric acid secretion, gastrin release, gastric emptying, and gastroesophageal acid reflux were measured in asymptomatic individuals before and after elimination of Helicobacter pylorigastritis. After basal gastric acid secretion and serum gastrin concentrations were measured, meal-stimulated gastric acid secretion and gastrin release were assessed during in vivo intragastric titration to pH 3. Experiments were repeated 4 wk after treatment with lansoprazole, amoxicillin, and clarithromycin. Esophageal pH was also monitored for 24 h before and after therapy. Basal gastric acidity increased ∼20 mmol/l in subjects whose infection was eradicated ( P < 0.05) but not in those with persistent infection. Basal and meal-stimulated gastric acid secretion did not change after H. pylorieradication, despite a 41% reduction in meal-stimulated gastrin release ( P < 0.05). Gastroesophageal acid reflux increased two- to threefold after successful treatment ( P < 0.05) but did not change in subjects with persistent infection. Thus elimination of H. pylori gastritis increases gastric acidity, probably by reducing nonparietal alkaline secretion, and this may facilitate gastroesophageal acid reflux.
Hepatic fibronectin matrix turnover in rats: involvement of the asialoglycoprotein receptor
Authors: Rotundo Robert F. , Vincent Peter A. , McKeown-Longo Paula J. , Blumenstock Frank A. , Saba Thomas M.
Published: December 2017

Fibronectin (Fn) is a major adhesive protein found in the hepatic extracellular matrix (ECM). In adult rats, the in vivo turnover of plasma Fn (pFn) incorporated into the liver ECM is relatively rapid, i.e., <24 h, but the regulation of its turnover has not been defined. We previously reported that cellular Fn (cFn) and enzymatically desialylated plasma Fn (aFn), both of which have a high density of exposed terminal galactose residues, rapidly interact with hepatic asialoglycoprotein receptors (ASGP-R) in association with their plasma clearance after intravenous infusion. With the use of adult male rats (250–350 g) and measurement of the deoxycholate (DOC)-insoluble125I-labeled Fn in the liver, we determined whether the ASGP-R system can also influence the hepatic matrix retention of various forms of Fn. There was a rapid deposition of125I-pFn,125I-aFn, and125I-cFn into the liver ECM after their intravenous injection. Although125I-pFn was slowly lost from the liver matrix over 24 h, more than 90% of the incorporated125I-aFn and125I-cFn was cleared within 4 h ( P < 0.01). Intravenous infusion of excess nonlabeled asialofetuin to competitively inhibit the hepatic ASGP-R delayed the rapid turnover of both aFn and cFn already incorporated within the ECM of the liver. ECM retention of both125I-aFn and125I-cFn was also less than125I-pFn ( P < 0.01) as determined in vitro using liver slices preloaded in vivo with either tracer form of Fn. The hepatic ASGP-R appears to participate in the turnover of aFn and cFn within the liver ECM, whereas a non-ASGP-R-associated endocytic pathway apparently influences the removal of normal pFn incorporated within the hepatic ECM, unless it becomes locally desialylated.
Iron primes hepatic macrophages for NF-κB activation in alcoholic liver injury
Authors: Tsukamoto Hidekazu , Lin Min , Ohata Mitsuru , Giulivi Cecilia , French Samuel W. , Brittenham Gary
Published: December 2017

NF-κB activation induced by lipopolysaccharide (LPS) in cultured hepatic macrophages (HM) may be abrogated by pretreatment of cells with a lipophilic iron chelator, 1,2-dimethyl-3-hydroxypyrid-4-one (L1, deferiprone), suggesting a role for iron in this molecular event [M. Lin, M., R. A. Rippe, O. Niemelä, G. Brittenham, and H. Tsukamoto, Am. J. Physiol. 272 ( Gastrointest. Liver Physiol. 35): G1355–G1364, 1997]. To ascertain the relevance in vivo of this hypothesis, HM from an experimental model of alcoholic liver injury were examined for the relationship between nuclear factor (NF)-κB activation and iron storage. HM showed a significant increase in nonheme iron concentration (+70%), accompanied by enhanced generation of electron paramagnetic resonance-detected radicals (+200%), NF-κB activation (+100%), and tumor necrosis factor-α (+150%) and macrophage inflammatory protein-1 (+280%) mRNA induction. Treatment of the cells ex vivo with L1 normalized all these parameters. HM content of ferritin protein, ferritin L chain mRNA, and hemeoxygenase-1 mRNA and splenic content of nonheme iron were increased, suggesting enhanced heme turnover as a cause of the increased iron storage and NF-κB activation. To test this possibility, increased iron content in HM was reproduced in vitro by phagocytosis of heat-treated red blood cells. Treatment caused a 40% increase in nonheme iron concentration and accentuated LPS-induced NF-κB activation twofold. Both effects could be abolished by pretreatment of cells with zinc protoporphyrin, a hemeoxygenase inhibitor. To extend this observation, animals were splenectomized before 9-wk alcohol feeding. Splenectomy resulted in further increments in HM nonheme iron storage (+60%) and NF-κB activation (+90%) and mononuclear cell infiltration (+450%), particularly around the iron-loaded HM in alcohol-fed animals. These results support the pivotal role of heme-derived iron in priming HM for NF-κB activation and expression of proinflammatory genes in alcoholic liver injury.
Contribution of NK2 tachykinin receptors to propulsion in the rabbit distal colon
Authors: Onori L. , Aggio A. , Taddei G. , Tonini M.
Published: December 2017

The role of the tachykinin neurokinin (NK)2 receptors on rabbit distal colon propulsion was investigated by using two selective NK2-receptor antagonists, MEN-10627 and SR-48968. Experiments on colonic circular muscle strips showed that contractile responses to [β-Ala8]NKA-(4—10) (1 nM–1 μM), a selective NK2-receptor agonist, were competitively antagonized by MEN-10627 (1–100 nM), whereas SR-48968 (0.1–10 nM) caused an insurmountable antagonism, thus confirming the difference in the mode of action of the two compounds. Colonic propulsion was elicited by distending a mobile rubber balloon with 0.3 ml (submaximal stimulus) or 1.0 ml (maximal stimulus) of water. The velocity of anal displacement of the balloon (mm/s) was considered the main propulsion parameter. At low concentrations (1.0–100 nM and 0.1–10 nM, respectively), MEN-10627 and SR-48968 facilitated the velocity of propulsion, whereas at high concentrations (100 nM and 1 μM, respectively) they decelerated propulsion. The excitatory and inhibitory effects of both antagonists were observed only with submaximal stimulus. We focused on the hypothesis that the facilitatory effect on propulsion may result from blockade of neuronal NK2 receptors and the inhibitory effect from suppression of the excitatory transmission mediated by NK2 receptors on smooth muscle cells. In the presence of N G-nitro-l-arginine (300 μM), a nitric oxide synthase inhibitor, MEN-10627, at a concentration (10 nM) that was found to accelerate propulsion in control experiments inhibited the velocity of propulsion. In the presence of threshold (1–10 nM) or full (1 μM) concentration of atropine, which inhibited to a great extent the velocity of propulsion, the inhibitory effect of MEN-10627 (1 μM) was markedly increased. In conclusion, in the rabbit distal colon NK2 receptors may decelerate propulsion by activating a nitric oxide-dependent neuronal mechanism and may accelerate it by a postjunctional synergistic interaction with cholinergic muscarinic receptors.
Changes in 5-HT-mediated pathways in radiation-induced attenuation and recovery of ion transport in rat colon
Authors: François A. , Ksas B. , Gourmelon P. , Griffiths N. M.
Published: December 2017

Whole body exposure to high doses of ionizing radiation is associated with small intestinal and colonic dysfunction, the etiology of which remains unknown. In this study, we investigated the role of both neural and nonneural 5-hydroxytryptamine (5-HT)-mediated pathways in radiation-induced attenuation and recovery of colonic secretory function. Rats were exposed to whole body 10-Gy gamma irradiation, and distal colonic tissues were studied in Ussing chambers 1, 3, and 7 days after exposure. Tissue responses to exogenously added 5-HT (nonneural pathway) and electrical field stimulation (EFS; neural pathway) were performed, and 5-HT receptor subtypes implicated in both responses were determined using three different 5-HT receptor antagonists: methysergide (5-HT2/1C), granisetron (5-HT3), and SDZ-205,557 (5-HT4). Maximal responses to exogenously added 5-HT were decreased at 1 and 3 days and returned to control values at 7 days. Responses to exogenous 5-HT were insensitive to both 5-HT2/1C and 5-HT3 antagonists and to TTX but were totally inhibited by SDZ-205,557 in both control and irradiated tissues. Responses to EFS were decreased 1 and 3 days after exposure and returned to control values at 7 days. In control tissues and 1 and 3 days after exposure, EFS responses were insensitive to both 5-HT2/1C and 5-HT4 antagonists but reduced by granisetron in control (51%) and at 1 (64%) and 3 days (58%) after exposure. Granisetron was more effective at 7 days (73% inhibition), which was concomitant with the appearance of a 5-HT4antagonist-sensitive pathway (40% inhibition). In conclusion, neural and nonneural 5-HT-mediated pathways involve 5-HT3 and 5-HT4 receptors, respectively, in control as well as in irradiated tissues 1 and 3 days after exposure. Conversely, the recovery of colonic transport is associated with additional 5-HT3-mediated pathways, probably in combination with 5-HT4 receptors.
Properties of synaptic inputs from myenteric neurons innervating submucosal S neurons in guinea pig ileum
Authors: Moore B. A. , Vanner S.
Published: December 2017

This study examined synaptic inputs from myenteric neurons innervating submucosal neurons. Intracellular recordings were obtained from submucosal S neurons in guinea pig ileal preparations in vitro, and synaptic inputs were recorded in response to electrical stimulation of exposed myenteric plexus. Most S neurons received synaptic inputs [>80% fast (f) excitatory postsynaptic potentials (EPSP), >30% slow (s) EPSPs] from the myenteric plexus. Synaptic potentials were recorded significant distances aboral (fEPSPs, 25 mm; sEPSPs, 10 mm) but not oral to the stimulating site. When preparations were studied in a double-chamber bath that chemically isolated the stimulating “myenteric chamber” from the recording side “submucosal chamber,” all fEPSPs were blocked by hexamethonium in the submucosal chamber, but not by a combination of nicotinic, purinergic, and 5-hydroxytryptamine-3 receptor antagonists in the myenteric chamber. In 15% of cells, a stimulus train elicited prolonged bursts of fEPSPs (>30 s duration) that were blocked by hexamethonium. These findings suggest that most submucosal S neurons receive synaptic inputs from predominantly anally projecting myenteric neurons. These inputs are poised to coordinate intestinal motility and secretion.
Evidence for a peripheral mechanism of esophagocrural diaphragm inhibitory reflex in cats
Authors: Liu Jianmin , Yamamoto Yoshihiro , Schirmer Bruce D. , Ross Robert A. , Mittal Ravinder K.
Published: December 2017

The esophagogastric junction (EGJ) is guarded by two sphincters, a smooth muscle lower esophageal sphincter (LES) and a skeletal muscle crural diaphragm. These two sphincters relax simultaneously under certain physiological conditions, i.e., swallowing, belching, vomiting, transient LES relaxation, and esophageal distension. Esophageal distension-induced crural diaphragm relaxation is mediated through vagal afferents that are thought to exert inhibitory influence on the central mechanism (brain stem) of crural diaphragm contraction. We conducted studies in 10 cats to determine whether a mechanism of crural diaphragm relaxation was located at the level of the neuromuscular junction and/or muscle. Stimulation of the crural diaphragm neuromuscular junction through 1) the electrodes implanted in the muscle and 2) the bilateral phrenic nerve resulted in an increase in EGJ pressure. Nicotinic receptor blockade (pancuronium, 0.2 mg/kg) abolished the EGJ pressure increase caused by electrical stimulation of the neuromuscular junction. Esophageal distension and bolus-induced secondary esophageal peristalsis caused relaxation of the EGJ during the stimulation of the neuromuscular junction. Bilateral phrenicotomy and vagotomy had no influence on this relaxation. These data suggest the existence of a peripheral mechanism of crural diaphragm inhibition. This peripheral inhibitory mechanism may reside at the level of either the neuromuscular junction or the skeletal muscle.
Kir3.1/3.2 encodes an I KACh-like current in gastrointestinal myocytes
Authors: Bradley Karri K. , Hatton William J. , Mason Helen S. , Walker Rebecca L. , Flynn Elaine R. M. , Kenyon James L. , Horowitz Burton
Published: December 2017

Expression of the Kir3 channel subfamily in gastrointestinal (GI) myocytes was investigated. Members of this K+ channel subfamily encode G protein-gated inwardly rectifying K+ channels ( I KACh) in other tissues, including the heart and brain. In the GI tract, I KACh could act as a negative feedback mechanism to temper the muscarinic response mediated primarily through activation of nonselective cation currents and inhibition of delayed-rectifier conductance. Kir3 channel subfamily isoforms expressed in GI myocytes were determined by performing RT-PCR on RNA isolated from canine colon, ileum, duodenum, and jejunum circular myocytes. Qualitative PCR demonstrated the presence of Kir3.1 and Kir3.2 transcripts in all smooth muscle cell preparations examined. Transcripts for Kir3.3 and Kir3.4 were not detected in the same preparations. Semiquantitative PCR showed similar transcriptional levels of Kir3.1 and Kir3.2 relative to β-actin expression in the various GI preparations. Full-length cDNAs for Kir3.1 and Kir3.2 were cloned from murine colonic smooth muscle RNA and coexpressed in Xenopus oocytes with human muscarinic type 2 receptor. Superfusion of oocytes with ACh (10 μM) reversibly activated a Ba2+-sensitive and inwardly rectifying K+current. Immunohistochemistry using Kir3.1- and Kir3.2-specific antibodies demonstrated channel expression in circular and longitudinal smooth muscle cells. We conclude that an I KAChcurrent is expressed in GI myocytes encoded by Kir3.1/3.2 heterotetramers.
Characterization of inducible nature of MRP3 in rat liver
Authors: Ogawa Kotaro , Suzuki Hiroshi , Hirohashi Tomoko , Ishikawa Toshihisa , Meier Peter J. , Hirose Kenji , Akizawa Toshifumi , Yoshioka Masanori , Sugiyama Yuichi
Published: December 2017

We found previously that expression of multidrug resistance-associated protein (MRP) 3 is induced in a mutant rat strain (Eisai hyperbilirubinemic rats) whose canalicular multispecific organic anion transporter (cMOAT/MRP2) function is hereditarily defective and in normal Sprague-Dawley (SD) rats after ligation of the common bile duct. In the present study, the inducible nature of MRP3 was examined, using Northern and Western blot analyses, in comparison with that of other secondary active [Na+-taurocholic acid cotransporting polypeptide (Ntcp), organic anion transporting polypeptide 1 (oatp1), and organic cation transporter (OCT1)] and primary active [P-glycoprotein (P-gp), cMOAT/MRP2, and MRP6] transporters. α-Naphthylisothiocyanate treatment and common bile duct ligation induced expression of P-gp and MRP3, whereas expression of Ntcp, oatp1, and OCT1 was reduced by the same treatment. Although expression of MRP3 was also induced by administration of phenobarbital, that of cMOAT/MRP2, MRP1, and MRP6 was not affected by any of these treatments. Moreover, the mRNA level of MRP3, but not that of P-gp, was increased in SD rats after administration of bilirubin and in Gunn rats whose hepatic bilirubin concentration is elevated because of a defect in the expression of UDP-glucuronosyl transferase. However, the MRP3 protein level was not affected by bilirubin administration. Although the increased MRP3 mRNA level was associated with the increased concentration of bilirubin and/or its glucuronides in mutant rats and in SD rats that had undergone common bile duct ligation or α-naphthylisothiocyanate treatment, we must assume that factor(s) other than these physiological substances are also involved in the increased protein level of MRP3.
Cell-specific localization of insulin-like growth factor binding protein mRNAs in rat liver
Authors: Zimmermann Ellen M. , Li Lina , Hoyt Eileen C. , Pucilowska Jolanta B. , Lichtman Steven , Lund P. Kay
Published: December 2017

The liver is a major source of circulating insulin-like growth factor I (IGF-I), and it also synthesizes several classes of IGF binding proteins (IGFBPs). Synthesis of IGF-I and IGFBPs is regulated by hormones, growth factors, and cytokines. They are nutritionally regulated and expressed in developmentally specific patterns. To gain insight into cellular regulatory mechanisms that determine hepatic synthesis of IGF-I and IGFBPs and to identify potential target cells for IGF-I within the liver, we studied the cellular sites of synthesis of IGF-I, IGF receptor, growth hormone (GH) receptor, and IGFBPs in freshly isolated rat hepatocytes, endothelial cells, and Kupffer cells. We also localized cellular sites of IGFBP synthesis by in situ hybridization histochemistry. Western ligand and immunoblot analyses were used to determine IGFBP secretion by isolated cells. Two IGF-I mRNA subtypes with different 5′ ends (class 1 and class 2) were detected in all isolated liver cell preparations. Type 1 IGF receptor mRNA was detected in endothelial cells, indicating that these cells are a local target for IGF actions in liver. GH receptor was expressed in all cell preparations, consistent with GH regulation of IGF-I and IGFBP synthesis in multiple liver cell types. The IGFBPs expressed striking cell-specific expression. IGFBP-1 was synthesized only in hepatocytes, and IGFBP-3 was expressed in Kupffer and endothelial cells. IGFBP-4 was expressed at high levels in hepatocytes and at low levels in Kupffer and endothelial cells. Cell-specific expression of distinct IGFBPs in the liver provides the potential for cell-specific regulation of hepatic and endocrine actions of IGF-I.
Diphenhydramine transport by pH-dependent tertiary amine transport system in Caco-2 cells
Authors: Mizuuchi Hiroshi , Katsura Toshiya , Ashida Kayoko , Hashimoto Yukiya , Inui Ken-Ichi
Published: December 2017

Substrate specificity and pH dependence of the transport system for diphenhydramine were investigated in Caco-2 cell monolayers. Diphenhydramine uptake was not affected by any typical substrate for the renal organic cation transport system except procainamide. Along with procainamide, tertiary amine compounds with N-dimethyl or N-diethyl moieties in their structures inhibited the diphenhydramine uptake. Moreover, accumulation of diphenhydramine was stimulated by preloading the Caco-2 cells with these tertiary amines ( trans-stimulation effect), indicating the existence of the specific transport system for tertiary amines with N-dimethyl or N-diethyl moieties. Efflux of diphenhydramine from monolayers was enhanced by medium acidification. In addition, intracellular acidification resulted in marked stimulation of diphenhydramine accumulation. ATP depletion of the cells caused an enhancement of diphenhydramine accumulation, suggesting the involvement of an active secretory pathway. However, P-glycoprotein did not mediate the diphenhydramine transport. These findings indicate that a novel pH-dependent tertiary amine transport system that recognizes N-dimethyl or N-diethyl moieties is involved in diphenhydramine transport in Caco-2 cells.
Effect of metformin on the vascular and glucose metabolic actions of insulin in hypertensive rats
Authors: Santuré Marta , Pitre Maryse , Gaudreault Nathalie , Marette André , Nadeau André , Bachelard Hélène
Published: December 2017

We investigated the long-term effect of metformin treatment on blood pressure, insulin sensitivity, and vascular responses to insulin in conscious spontaneously hypertensive rats (SHR). The rats were instrumented with intravascular catheters and pulsed Doppler flow probes to measure blood pressure, heart rate, and blood flow. Insulin sensitivity was assessed by the euglycemic hyperinsulinemic clamp technique. Two groups of SHR received metformin (100 or 300 mg ⋅ kg− 1 ⋅ day− 1) for 3 wk while another group of SHR and a group of Wistar Kyoto (WKY) rats were left untreated. We found that vasodilation of skeletal muscle and renal vasculatures by insulin is impaired in SHR. Moreover, a reduced insulin sensitivity was detected in vivo and in vitro in isolated soleus and extensor digitorum longus muscles from SHR compared with WKY rats. Three weeks of treatment with metformin improves the whole-body insulin-mediated glucose disposal in SHR but has no blood pressure-lowering effect and no influence on vascular responses to insulin (4 mU ⋅ kg− 1 ⋅ min− 1). An improvement in insulin-mediated glucose transport activity was detected in isolated muscles from metformin-treated SHR, but in the absence of insulin no changes in basal glucose transport activity were observed. It is suggested that part of the beneficial effect of metformin on insulin resistance results from a potentiation of the hormone-stimulating effect on glucose transport in peripheral tissues (mainly skeletal muscle). The results argue against a significant antihypertensive or vascular effect of metformin in SHR.
Enteric locus of action of prokinetics: ABT-229, motilin, and erythromycin
Authors: Sarna Sushil K. , Gonzalez Asensio , Ryan Robert P.
Published: December 2017

We investigated the in vivo and in vitro locus of actions of prokinetics: motilin, erythromycin, and ABT-229. The test substances were infused close intra-arterially in short segments of the jejunum in the intact conscious state. Each prokinetic acted on a presynaptic neuron and utilized at least one nicotinic synapse to stimulate circular muscle contractions. The final neurotransmitter at the neuroeffector junction was ACh. Motilin and erythromycin, but not ABT-229, also released nitric oxide. Each prokinetic utilized somewhat different subtypes of muscarinic, serotonergic, tachykininergic, and histaminergic receptors, except for the M3 receptor, which was common to all of them. In contrast, none of the prokinetics stimulated contractions in mucosa-free or mucosa-attached muscle strips, or rings, even though methacholine or electrical field stimulation induced phasic contractions in all of them. The prokinetics also did not release ACh in longitudinal muscle-myenteric plexus preparations. Each prokinetic, however, decreased the length of enzymatically dispersed single cells. In conclusion, each prokinetic may act on a different subset of presynaptic neurons that converge on the postsynaptic cholinergic and nonadrenergic noncholinergic motoneurons. The presynaptic neurons may be impaired in the muscle bath environment.