bluuebunny/crossref_metadata_2025 huggingface dataset:
Orexins in rat dorsal motor nucleus of the vagus potently stimulate gastric motor function
Authors: Krowicki Zbigniew K. , Burmeister Melissa A. , Berthoud Hans-Rudolf , Scullion Roisin T. , Fuchs Kristine , Hornby Pamela J.
Published: March 2015

Orexins regulate food intake, arousal, and the sleep-wake cycle. They are synthesized by neurons in the lateral hypothalamus and project to autonomic areas in the hindbrain. Orexin A applied to the dorsal surface of the medulla stimulates gastric acid secretion via a vagally mediated pathway. We tested the hypothesis that orexins in the dorsal motor nucleus (DMN) of the vagus regulate gastric motor function. Multibarelled micropipette assemblies were used to administer vehicle, l-glutamate, orexins A (1 and 10 pmol) and B (10 pmol), and a dye marker into this site in anesthetized rats. When the pipette was positioned in the DMN rostral to the obex (where excitation of neurons by l-glutamate evoked an increase in contractility), orexins A and B increased intragastric pressure and antral motility. In contrast, 10 pmol orexin A microinjected into the DMN caudal to the obex (wherel-glutamate evokes gastric relaxation through a vagal inhibitory pathway) did not significantly alter gastric motor function. In separate immunocytochemical studies, orexin receptor 1 was highly expressed in neurons in the DMN. Specifically, it was present in retrogradely labeled preganglionic neurons in the DMN that innervate the stomach. These data are consistent with the idea that orexin A stimulates vagal excitatory motor neurons. These are the first data to suggest that orexins in the DMN have potent and long-lasting effects to increase gastric contractility.
Role of thromboxane in producing portal hypertension following trauma-hemorrhage
Authors: Yokoyama Yukihiro , Toth Balazs , Kitchens William C. , Schwacha Martin G. , Bland Kirby I. , Chaudry Irshad H.
Published: March 2015

Thromboxane A2(TXA2) and endothelin-1 (ET-1) have been proposed as the important vasoconstrictors that increase portal venous resistance in paracrine or autocrine fashion. We hypothesized that the hepatic damage following trauma-hemorrhage (T-H) is induced by the impaired hepatic circulation due to the increased production of vasoconstrictors such as ET-1 and TXA2by the liver. To test this, male Sprague-Dawley rats ( n = 6/group) were subjected to trauma (i.e., midline laparotomy) and hemorrhage (35-40 mmHg for 90 min followed by fluid resuscitation) or sham operation. At 2 or 5 h after the end of resuscitation, the liver was isolated and perfused and portal inflow pressure, bile flow, and release of ET-1 and thromboxane B2(TXB2; a stable metabolite of TXA2) into the perfusate were measured. The level of portal pressure was higher at 5 h following T-H compared with 2 h after T-H and sham. The portal pressure was inversely correlated to the amount of bile production. Furthermore, the bile flow was significantly correlated to the hepatic damage as evidenced by release of lactate dehydrogenase into the perfusate. The level of ET-1 at 5 h following T-H in the perfusate after 30 min of recirculation did not show any difference from sham. However, the levels of TXB2in the T-H group were significantly higher than those in sham at that interval. These results indicate that the increased release of TXA2but not ET-1 following T-H might be responsible for producing the increased portal resistance, decreased bile production, and hepatic damage.
Molecular cloning and functional analysis of the human Na+/H+exchanger NHE3 promoter
Authors: Malakooti Jaleh , Memark V. C. , Dudeja Pradeep K. , Ramaswamy Krishnamurthy
Published: March 2015

Na+/H+exchanger (NHE) isoforms NHE2 and NHE3, colocalized to the brush border membrane of the epithelial cells, exhibit differences in their pattern of tissue expression and regulation by various molecular signals. To investigate the mechanisms involved in regulation of NHE3 gene expression, the human NHE3 promoter region was cloned and characterized. Primer extension experiments located the transcription start site to a position 116 nucleotides upstream from the translation start codon. The 5′-flanking region lacked a CCAAT box but contained a TATA-like sequence. Nucleotide sequencing of the 5′-flanking region revealed the presence of a number of cis elements including Sp1, AP-2, MZF-1, CdxA, Cdx-2, steroid and nonsteroid hormone receptor half sites, and a phorbol 12-myristate 13-acetate-response element. Transient transfection experiments using C2/bbe cell line defined a maximal promoter activity in −95/+5 region. The regulatory response elements clustered within this region include a potential transcription factor IID (TF IID), a CACCC, two Sp1, and two AP-2 motifs. Deletion of a fragment containing the AP-2 and Sp1 motifs resulted in a drastic decrease in promoter activity. In gel mobility shift assays, an oligonucleotide spanning from −78 to −56 bp bound a recombinant AP-2, and the corresponding binding activity in nuclear extracts was supershifted with anti-AP2α antibody. Our studies suggest that the NHE3 expression is regulated by a combination of ciselements and their cognate transcription factors that include the AP-2 and Sp1 family members.
Intraepithelial lymphocyte-derived interferon-γ evokes enterocyte apoptosis with parenteral nutrition in mice
Authors: Yang Hua , Fan Yongyi , Teitelbaum Daniel H.
Published: March 2015

Total parenteral nutrition (TPN) results in an increase in intraepithelial lymphocyte (IEL)-derived interferon-γ (IFN-γ) expression as well as an increase in epithelial cell (EC) apoptosis. This study examined the role that IEL-derived IFN-γ has in the increase in EC apoptosis. Mice received either TPN or oral feedings for 7 days. Small bowel EC apoptosis significantly rose in mice receiving TPN. The administration of TPN also significantly increased IEL-derived IFN-γ and Fas ligand (FasL) expression. EC apoptosis in IFN-γ knockout (IFNKO) mice that received TPN was significantly lower than in wild-type TPN mice. Sensitivity of EC to Fas-mediated apoptosis in IFNKO mice was significantly lower than in wild-type TPN mice. Apoptosis in Fas-deficient and FasL-deficient mice that received TPN was significantly lower than in wild-type mice that received TPN. The TPN-induced increase in IFN-γ expression appears to result in an increase in Fas-L expression and EC sensitivity to Fas, with a resultant increase in EC apoptosis. This may well be one of the mediators of increased EC apoptosis observed with TPN administration.
Stretch-activated neuronal pathways to longitudinal and circular muscle in guinea pig distal colon
Authors: Spencer Nick J. , Hennig Grant W. , Smith Terence K.
Published: March 2015

The role of the longitudinal muscle (LM) layer during the peristaltic reflex in the small and large intestine is unclear. In this study, we have made double and quadruple simultaneous intracellular recordings from LM and circular muscle (CM) cells of guinea pig distal colon to correlate the electrical activities in the two different muscle layers during circumferential stretch. Simultaneous recordings from LM and CM cells (<200 μm apart) at the oral region of the colon showed that excitatory junction potentials (EJPs) discharged synchronously in both muscle layers for periods of up to 6 h. Similarly, at the anal region of the colon, inhibitory junction potentials (IJPs) discharged synchronously in the two muscle layers. Quadruple recordings from LM and CM orally at the same time as from the LM and CM anally revealed that IJPs occurred synchronously in the LM and CM anally at the same time as EJPs in LM and CM located 20 mm orally. Oral EJPs and anal IJPs were linearly related in amplitude between the two muscle layers. Spatiotemporal maps generated from simultaneous video imaging of the movements of the colon, combined with intracellular recordings, revealed that some LM contractions orally could be correlated in time with IJPs in CM cells anally. N ω-nitro-l-arginine (l-NA; 100 μM) abolished the IJP in LM, whereas a prominent l-NA-resistant “fast” IJP was always observed in CM. In summary, in stretched preparations, synchronized EJPs in both LM and CM orally are generated by synchronized firing of many ascending interneurons, which simultaneously activate excitatory motor neurons to both muscle layers. Similarly, synchronized IJPs in both LM and CM anally are generated by synchronized firing of many descending interneurons, which simultaneously activate inhibitory motor neurons to both muscle layers. This synchronized motor activity ensures that both muscles around the entire circumference are excited orally at the same time as inhibited anally, thus producing net aboral propulsion.
Expression of 5-HT3 receptors by extrinsic duodenal afferents contribute to intestinal inhibition of gastric emptying
Authors: Raybould Helen E. , Glatzle Jorg , Robin Carla , Meyer James H. , Phan Thomas , Wong Helen , Sternini Catia
Published: March 2015

Intestinal perfusion with carbohydrates inhibits gastric emptying via vagal and spinal capsaicin-sensitive afferent pathways. The aim of the present study was to determine the role of 1) 5-hydroxytryptamine (5-HT)3receptors (5-HT3R) in mediating glucose-induced inhibition of gastric emptying and 2) 5-HT3R expression in vagal and spinal afferents in innervating the duodenum. In awake rats fitted with gastric and duodenal cannulas, perfusion of the duodenum with glucose (50 and 100 mg) inhibited gastric emptying. Intestinal perfusion of mannitol inhibited gastric emptying only at the highest concentration (990 mosm/kgH2O). Pretreatment with the 5-HT3R antagonist tropisetron abolished both glucose- and mannitol-induced inhibition of gastric emptying. Retrograde labeling of visceral afferents by injection of dextran-conjugated Texas Red into the duodenal wall was used to identify extrinsic primary afferents. Immunoreactivity for 5-HT3R, visualized with an antibody directed to the COOH terminus of the rat 5-HT3R, was found in >80% of duodenal vagal and spinal afferents. These results show that duodenal extrinsic afferents express 5-HT3R and that the receptor mediates specific glucose-induced inhibition of gastric emptying. These findings support the hypothesis that enterochromaffin cells in the intestinal mucosa release 5-HT in response to glucose, which activates 5-HT3R on afferent nerve terminals to evoke reflex changes in gastric motility. The primary glucose sensors of the intestine may be mucosal enterochromaffin cells.
Inhibitory effects of botulinum toxin on pyloric and antral smooth muscle
Authors: James Arlene N. , Ryan James P. , Parkman Henry P.
Published: March 2015

Botulinum toxin injection into the pylorus is reported to improve gastric emptying in gastroparesis. Classically, botulinum toxin inhibits ACh release from cholinergic nerves in skeletal muscle. The aim of this study was to determine the effects of botulinum toxin on pyloric smooth muscle. Guinea pig pyloric muscle strips were studied in vitro. Botulinum toxin type A was added; electric field stimulation (EFS) was performed every 30 min for 6 h. ACh (100 μM)-induced contractile responses were determined before and after 6 h. Botulinum toxin caused a concentration-dependent decrease of pyloric contractions to EFS. At a low concentration (2 U/ml), botulinum toxin decreased pyloric contractions to EFS by 43 ± 9% without affecting ACh-induced contractions. At higher concentrations (10 U/ml), botulinum toxin decreased pyloric contraction to EFS by 75 ± 7% and decreased ACh-induced contraction by 79 ± 9%. In conclusion, botulinum toxin inhibits pyloric smooth muscle contractility. At a low concentration, botulinum toxin decreases EFS-induced contractile responses without affecting ACh-induced contractions suggesting inhibition of ACh release from cholinergic nerves. At higher concentrations, botulinum toxin directly inhibits smooth muscle contractility as evidenced by the decreased contractile response to ACh.
Intestinal NaCl transport in NHE2 and NHE3 knockout mice
Authors: Gawenis Lara R. , Stien Xavier , Shull Gary E. , Schultheis Patrick J. , Woo Alison L. , Walker Nancy M. , Clarke Lane L.
Published: March 2015

Sodium/proton exchangers [Na+/H+(NHEs)] play an important role in salt and water absorption from the intestinal tract. To investigate the contribution of the apical membrane NHEs, NHE2 and NHE3, to electroneutral NaCl absorption, we measured radioisotopic Na+and Cl−flux across isolated jejuna from wild-type [NHE(+)], NHE2 knockout [NHE2(−)], and NHE3 knockout [NHE3(−)] mice. Under basal conditions, NHE(+) and NHE2(−) jejuna had similar rates of net Na+(∼6 μeq/cm2· h) and Cl−(∼3 μeq/cm2· h) absorption. In contrast, NHE3(−) jejuna had reduced net Na+absorption (∼2 μeq/cm2· h) but absorbed Cl−at rates similar to NHE(+) and NHE2(−) jejuna. Treatment with 100 μM 5-( N-ethyl- N-isopropyl) amiloride (EIPA) completely inhibited net Na+and Cl−absorption in all genotypes. Studies of the Na+absorptive flux ( J [Formula: see text]) indicated that J [Formula: see text] in NHE(+) jejunum was not sensitive to 1 μM EIPA, whereas J [Formula: see text] in NHE3(−) jejunum was equally sensitive to 1 and 100 μM EIPA. Treatment with forskolin/IBMX to increase intracellular cAMP (cAMPi) abolished net NaCl absorption and stimulated electrogenic Cl−secretion in all three genotypes. Quantitative RT-PCR of epithelia from NHE2(−) and NHE3(−) jejuna did not reveal differences in mRNA expression of NHE3 and NHE2, respectively, when compared with jejunal epithelia from NHE(+) siblings. We conclude that 1) NHE3 is the dominant NHE involved in small intestinal Na+absorption; 2) an amiloride-sensitive Na+transporter partially compensates for Na+absorption in NHE3(−) jejunum; 3) cAMPistimulation abolishes net Na+absorption in NHE(+), NHE2(−), and NHE3(−) jejunum; and 4) electroneutral Cl−absorption is not directly dependent on either NHE2 or NHE3.
Low venular shear rates promote leukocyte-dependent recruitment of adherent platelets
Authors: Russell Janice , Cooper Dianne , Tailor Anitaben , Stokes Karen Y. , Granger D. Neil
Published: March 2015

The influence of reductions in venular shear rate on platelet-endothelial (P/E) cell adhesion has not been previously addressed. The objectives of this study were to define the effects of reductions in venular shear rate on P/E cell adhesion and to determine the interdependence of P/E cell adhesion and leukocyte-endothelial (L/E) cell adhesion at low shear rates. Intravital videomicroscopy was used to quantify P/E and L/E cell adhesion in rat mesenteric venules exposed to shear rates ranging between 118 ± 9 and 835 ± 44 s−1. Shear rate was altered in postcapillary venules by rapid, graded blood withdrawal, without retransfusion of shed blood. Reducing shear rate from >600 s−1to <200 s−1resulted in an eightfold increase in L/E cell adhesion, whereas P/E cell adhesion increased 18-fold. A blocking antibody directed against P-selectin blunted both the P/E and L/E cell adhesion elicited by low shear rates. Immunoneutralization of CD11/CD18 on leukocytes or rendering animals neutropenic also blocked the shear rate-dependent recruitment of both platelets and leukocytes. These findings indicate that 1) low shear rates promote P/E and L/E cell adhesion in mesenteric venules, and 2) adherent neutrophils (mediated by CD11/CD18) create a platform onto which platelets can bind to the venular wall at low shear rates.
Amelioration of dextran sulfate colitis by butyrate: role of heat shock protein 70 and NF-κB
Authors: Venkatraman Aparna , Ramakrishna B. S. , Shaji R. V. , Kumar N. S. Nanda , Pulimood Anna , Patra Susama
Published: March 2015

Butyrate enemas have been demonstrated to ameliorate inflammation in ulcerative colitis. The mechanism of this protective effect of butyrate is not known, and this study examines the effect of butyrate on epithelial function, inducible heat shock protein 70 (HSP70) expression, and NF-κB activation in experimental colitis. Colitis was induced in rats by oral dextran sulfate sodium (DSS) and by butyrate or saline enemas. Mucosal barrier function was assessed by electrical resistance and [14C]mannitol permeability. HSP70 production was determined by [35S]methionine labeling, Western blot analysis, and immunohistochemistry. Activation of heat shock factors (HSFs) and NF-κB was evaluated by EMSA. Butyrate showed a significant protection against the decrease in cell viability, increase in mucosal permeability, and polymorphonuclear neutrophil infiltration seen in DSS colitis. Butyrate inhibited HSP70 expression in DSS colitis and also inhibited the activation of HSF and NF-κB. Thus butyrate enema was found to be cytoprotective in DSS colitis, an effect partly mediated by suppressing activation of HSP70 and NF-κB.
Expression of monosaccharide transporters in intestine of diabetic humans
Authors: Dyer J. , Wood I. S. , Palejwala A. , Ellis A. , Shirazi-Beechey S. P.
Published: March 2015

Noninsulin-dependent diabetes mellitus (NIDDM) is an increasingly common disease, which brings a number of life-threatening complications. In rats with experimentally induced diabetes, there is an increase in the capacity of the intestine to absorb monosaccharides. We have examined the activity and the expression of monosaccharide transporters in the intestine of patients suffering from NIDDM. Na+-dependent d-glucose transport was 3.3-fold higher in brush-border membrane (BBM) vesicles isolated from duodenal biopsies of NIDDM patients compared with healthy controls. Western analysis indicated that SGLT1 and GLUT5 protein levels were also 4.3- and 4.1-fold higher in diabetic patients. This was associated with threefold increases in SGLT1 and GLUT5 mRNA measured by Northern blotting. GLUT2 mRNA levels were also increased threefold in the intestine of diabetic patients. Analysis of other BBM proteins indicated that the activity and abundance of sucrase and lactase were increased by 1.5- to 2-fold and the level of the structural proteins villin and β-actin was enhanced 2-fold in diabetic patients compared with controls. The increase in the capacity of the intestine to absorb monosaccharides in human NIDDM is due to a combination of intestinal structural change with a specific increase in the expression of the monosaccharide transporters SGLT1, GLUT5, and GLUT2.
IGF-I elicits growth of human intestinal smooth muscle cells by activation of PI3K, PDK-1, and p70S6 kinase
Authors: Kuemmerle John F.
Published: March 2015

Endogenous IGF-I regulates growth of human intestinal smooth muscle cells by jointly activating phosphatidylinositol 3-kinase (PI3K) and ERK1/2. The 70-kDa ribosomal S6 kinase (p70S6 kinase) is a key regulator of cell growth activated by several independently regulated kinases. The present study characterized the role of p70S6 kinase in IGF-I-induced growth of human intestinal smooth muscle cells and identified the mechanisms of p70S6 kinase activation. IGF-I-induced growth elicited via either the PI3K or ERK1/2 pathway required activation of p70S6 kinase. IGF-I elicited concentration-dependent activation of PI3K, 3-phosphoinositide-dependent kinase-1 (PDK-1), and p70S6 kinase that was sequential and followed similar time courses. IGF-I caused time-dependent and concentration-dependent phosphorylation of p70S6 kinase on Thr421/Ser424, Thr389, and Thr229that paralleled p70S6 kinase activation. p70S6 kinase(Thr421/Ser424) phosphorylation was PI3K dependent and PDK-1 independent, whereas p70S6 kinase(Thr389) and p70S6 kinase(Thr229) phosphorylation and p70S6 kinase activation were PI3K dependent and PDK-1 dependent. IGF-I elicited sequential Akt(Ser308), Akt(Ser473), and mammalian target of rapamycin(Ser2448) phosphorylation; however, transfection of muscle cells with kinase-inactive Akt1(K179M) showed that these events were not required for IGF-I to activate p70S6 kinase and stimulate proliferation of human intestinal muscle cells.
Glucocorticoid regulation and glycosylation of mouse intestinal type IIb Na-Picotransporter during ontogeny
Authors: Arima Kayo , Hines Eric R. , Kiela Pawel R. , Drees Jason B. , Collins James F. , Ghishan Fayez K.
Published: March 2015

We sought to characterize expression of an apically expressed intestinal Na-Picotransporter (Na-Pi-IIb) during mouse ontogeny and to assess the effects of methylprednisolone (MP) treatment. In control mice, Na-Piuptake by intestinal brush-border membrane vesicles was highest at 14 days of age, lower at 21 days, and further reduced at 8 wk and 8–9 mo of age. Na-Pi-IIb mRNA and immunoreactive protein levels in 14-day-old animals were markedly higher than in older groups. MP treatment significantly decreased Na-Piuptake and Na-Pi-IIb mRNA and protein expression in 14-day-old mice. Additionally, the size of the protein was smaller in 14-day-old mice. Deglycosylation of protein from 14-day-old and 8-wk-old animals with peptide N-glycosidase reduced the molecular weight to the predicted size. We conclude that intestinal Na-Piuptake and Na-Pi-IIb expression are highest at 14 days and decrease with age. Furthermore, MP treatment reduced intestinal Na-Piuptake approximately threefold in 14-day-old mice and this reduction correlates with reduced Na-Pi-IIb mRNA and protein expression. We also demonstrate that Na-Pi-IIb is an N-linked glycoprotein and that glycosylation is age dependent.
II. Integration of afferent signaling from the viscera by the nodose ganglia
Authors: Browning Kirsteen N. , Mendelowitz David
Published: March 2015

To understand vago-vagal reflexes, one must have an appreciation of the events surrounding the encoding, integration, and central transfer of peripheral sensations by vagal afferent neurons. A large body of work has shown that vagal afferent neurons have nonuniform properties and that distinct subpopulations of neurons exist within the nodose ganglia. These sensory neurons display a considerable degree of plasticity; electrophysiological, pharmacological, and neurochemical properties have all been shown to alter after peripheral tissue injury. The validity of claims of selective recordings from populations of neurons activated by peripheral stimuli may be diminished, however, by the recent demonstration that stimulation of a subpopulation of nodose neurons can enhance the activity of unstimulated neuronal neighbors. To better understand the neurophysiological processes occurring after vagal afferent stimulation, it is essential that the electrophysiological, pharmacological, and neurochemical properties of nodose neurons are correlated with their sensory function or, at the very least, with their specific innervation target.
Role of TGF-β1 in the development of pancreatic fibrosis in Otsuka Long-Evans Tokushima Fatty rats
Authors: Yoshikawa Hiroyuki , Kihara Yasuyuki , Taguchi Masashi , Yamaguchi Taizo , Nakamura Hayato , Otsuki Makoto
Published: March 2015

Recently established Otsuka Long-Evans Tokushima Fatty (OLETF) rats, a model of naturally occurring obesity diabetes, exhibit progressive accumulation of connective tissue in the pancreas. The present study was designed to determine the pathogenic role of transforming growth factor-β1 (TGF-β1) in the development of pancreatic fibrosis in OLETF rats by investigating the serial changes in the expression of TGF-β1 and extracellular matrix (ECM) in the pancreas. Progressive proliferation of connective tissue arose from the interstitial region surrounding islets at 20 wk of age and extended to the exocrine pancreas adjacent to the islets. TGF-β1 mRNA levels in the pancreas increased at 20 wk of age and reached a peak value at 30 wk of age. Fibronectin (FN) and procollagen types I and III mRNAs peaked at 20 wk of age and remained at higher levels than those in the nondiabetic counterparts Long-Evans Tokushima Otsuka rats until 50 wk of age. Immunoreactivities for TGF-β1 and FN were found in islets of OLETF rats at 20 wk of age and were seen in acinar and interstitial cells at 50 wk of age. Moreover, α-smooth muscle actin was located at interstitial region surrounding the islets. Proliferation of the connective tissue in the pancreas of OLETF rats closely correlated with expression of TGF-β1 and ECM. Our results suggest that the development of pancreatic fibrosis in OLETF rats extends from endocrine to exocrine pancreas and that TGF-β1 is involved in pancreatic fibrosis of OLETF rats.
In vivo absorption of water and electrolytes in mouse intestine. Application to villin −/− mice
Authors: Athman Rafika , Tsocas Annick , Presset Olivier , Robine Sylvie , Rozé Claude , Ferrary Evelyne
Published: March 2015

This study was done to establish and validate a single-pass perfusion method for measuring the absorption of water and electrolytes by the mouse small intestine. The method was then used to study intestinal absorption in mice whose villin gene had been invalidated ( v−/−). The single-pass perfusion of the jejunum measures the absorption of water, Cl−, Na+, K+, HCO[Formula: see text], and glucose in anesthetized wild-type and v−/− mice in vivo. We measured absorption under basal and stimulated conditions (carbachol, vasoactive intestinal polypeptide, intralumen PGE2). Basal absorption and stimulated secretions were similar to those previously obtained in rats. There was no difference between wild-type and v−/− mice in animals with mixed genetic background or in pure C57BL6 mice. We conclude that this in vivo perfusion method is suitable for studying the absorption/secretion of electrolytes in the mouse intestine and that a lack of villin does not significantly alter basal and secretagogue-stimulated electrolyte movements across the epithelium of the mouse jejunum in vivo.
Identification of an apical Cl−/HCO3−exchanger in the small intestine
Authors: Wang Zhaohui , Petrovic Snezana , Mann Elizabeth , Soleimani Manoocher
Published: March 2015

HCO[Formula: see text]secretion is the most important defense mechanism against acid injury in the duodenum. However, the identity of the transporter(s) mediating apical HCO[Formula: see text] secretion in the duodenum remains unknown. A family of anion exchangers, which include downregulated in adenoma (DRA or SLC26A3), pendrin (PDS or SLC26A4), and the putative anion transporter (PAT1 or SLC26A6) has recently been identified. DRA and pendrin mediate Cl−/base exchange; however, the functional identity and distribution of PAT1 (SLC26A6) is not known. In these studies, we investigated the functional identity, tissue distribution, and membrane localization of PAT1. Expression studies in Xenopus oocytes demonstrated that PAT1 functions in Cl−/HCO[Formula: see text] exchange mode. Tissue distribution studies indicated that the expression of PAT1 is highly abundant in the small intestine but is low in the colon, a pattern opposite that of DRA. PAT1 was also abundantly detected in stomach and heart. Immunoblot analysis studies identified PAT1 as a ∼90 kDa protein in the duodenum. Immunohistochemical studies localized PAT1 to the brush border membranes of the villus cells of the duodenum. We propose that PAT1 is an apical Cl−/HCO[Formula: see text]exchanger in the small intestine.
Substrate specificities of rat oatp1 and ntcp: implications for hepatic organic anion uptake
Authors: Hata Soichiro , Wang Pijun , Eftychiou Nicole , Ananthanarayanan Meenakshisundaram , Batta Ashok , Salen Gerald , Pang K. Sandy , Wolkoff Allan W.
Published: March 2015

Transport of a series of3H-radiolabeled C23, C24, and C27bile acid derivatives was compared and contrasted in HeLa cell lines stably transfected with rat Na+/taurocholate cotransporting polypeptide (ntcp) or organic anion transporting polypeptide 1 (oatp1) in which expression was under regulation of a zinc-inducible promoter. Similar uptake patterns were observed for both ntcp and oatp1, except that unconjugated hyodeoxycholate was a substrate of oatp1 but not ntcp. Conjugated bile acids were transported better than nonconjugated bile acids, and the configuration of the hydroxyl groups (α or β) had little influence on uptake. Although cholic and 23 norcholic acids were transported by ntcp and oatp1, other unconjugated bile acids (chenodeoxycholic, ursodeoxycholic) were not. In contrast to ntcp, oatp1-mediated uptake of the trihydroxy bile acids taurocholate and glycocholate was four- to eightfold below that of the corresponding dihydroxy conjugates. Ntcp mediated high affinity, sodium-dependent transport of [35S]sulfobromophthalein with a Kmsimilar to that of oatp1-mediated transport of [35S]sulfobromophthalein ( Km= 3.7 vs. 3.3 μM, respectively). In addition, for both transporters, uptake of sulfobromophthalein and taurocholic acid showed mutual competitive inhibition. These results indicate that the substrate specificity of ntcp is considerably broader than previously suspected and caution the extrapolation of transport data obtained in vitro to physiological function in vivo.
IV. Genetic and animal models of GI motility disorders caused by loss of interstitial cells of Cajal
Authors: Sanders Kenton M. , Ördög Tamás , Ward Sean M.
Published: March 2015

Several human motility disorders have been shown to be associated with loss or defects in interstitial cells of Cajal (ICC) networks. Because tissue samples for these studies were taken from patients with well-advanced motility problems, it is difficult to determine whether the loss of ICC is a cause or a consequence of the disease process. To establish the cause-and-effect relationship of ICC loss in motility disorders, it may be feasible to use animal models in which ICC are lost as motility dysfunction develops. Several models with defects in ICC networks have been developed, and these include animals with defects in the Kit signaling pathway (e.g., white-spotting mutants that have defects in Kit receptors; steel mutants that have mutations in stem cell factor, the ligand for Kit; and animals that are chronically treated with reagents that block Kit or downstream signaling proteins). ICC do not die when Kit signaling is blocked, rather, they redifferentiate into a smooth musclelike phenotype. Diabetic animals (NOD/LtJ mice), animals with chronic bowel obstruction, and inflammatory bowel models also have defects in ICC networks that have been associated with motility disorders. By studying these models with molecular and genomic techniques it may be possible to determine the signals that cause loss of ICC and find ways of restoring ICC to dysfunctional tissues. This article discusses recent progress in the utilization of animal models to study the consequences of losing ICC on the development of motility disorders.
Cathepsin B inhibition prevents trypsinogen activation and reduces pancreatitis severity
Authors: van Acker Gijs J. D. , Saluja Ashok K. , Bhagat Lakshmi , Singh Vijay P. , Song Albert M. , Steer Michael L.
Published: March 2015

Intrapancreatic activation of trypsinogen is believed to play a critical role in the initiation of acute pancreatitis, but mechanisms responsible for intrapancreatic trypsinogen activation during pancreatitis have not been clearly defined. In previous in vitro studies, we have shown that intra-acinar cell activation of trypsinogen and acinar cell injury in response to supramaximal secretagogue stimulation could be prevented by the cell permeant cathepsin B inhibitor E64d (Saluja A, Donovan EA, Yamanaka K, Yamaguchi Y, Hofbauer B, and Steer ML. Gastroenterology 113: 304–310, 1997). The present studies evaluated the role of intrapancreatic trypsinogen activation, this time under in vivo conditions, in two models of pancreatitis by using another highly soluble cell permeant cathepsin B inhibitor,l-3-trans-(propylcarbamoyl)oxirane-2-carbonyl-l-isoleucyl-l-proline methyl ester (CA-074me). Intravenous administration of CA-074me (10 mg/kg) before induction of either secretagogue-elicited pancreatitis in mice or duct infusion-elicited pancreatitis in rats markedly reduced the extent of intrapancreatic trypsinogen activation and substantially reduced the severity of both pancreatitis models. These observations support the hypothesis that, during the early stages of pancreatitis, trypsinogen activation in the pancreas is mediated by the lysosomal enzyme cathepsin B. Our findings also suggest that pharmacological interventions that inhibit cathepsin B may prove useful in preventing acute pancreatitis or reducing its severity.
Increased renal expression of bilirubin glucuronide transporters in a rat model of obstructive jaundice
Authors: Tanaka Yuji , Kobayashi Yoshinao , Gabazza Esteban C. , Higuchi Kunihiro , Kamisako Toshinori , Kuroda Makoto , Takeuchi Keisuke , Iwasa Motoh , Kaito Masahiko , Adachi Yukihiko
Published: March 2015

Regulation of bilirubin glucuronide transporters during hyperbilirubinemia in hepatic and extrahepatic tissues is not completely clear. In the present study, we evaluated the regulation of the bilirubin glucuronide transporters, multidrug resistance-associated proteins (MRP)2 and 3, in rats with obstructive jaundice. Bile duct ligation (BDL) or sham operation was performed in Wistar rats. Liver and kidneys were removed 1, 3, and 5 days after BDL ( n = 4, in each group). Serum and urine were collected to measure bilirubin levels just before animal killing. MRP2 And MRP3 mRNA expressions were determined by real-time RT-PCR. Protein expression of MRP2 and MRP3 was determined by Western blotting. Renal MRP2 function was evaluated by para-aminohippurate (PAH) clearance. The effect of conjugated bilirubin, unconjugated bilirubin, human bile, and sulfate-conjugated bile acid on MRP2 gene expression was also evaluated in renal and hepatocyte cell lines. Serum bilirubin and urinary bilirubin excretion increased significantly after BDL. In the liver, the mRNA expression of MRP2 decreased 59, 86, and 82%, and its protein expression decreased 25, 74, and 93% compared with sham-operated animals after 24, 72, and 120 h of BDL, respectively. In contrast, the liver expression of MRP3 mRNA increased 138, 2,137, and 3,295%, and its protein expression increased 560, 634, and 612% compared with sham-operated animals after 24, 72, and 120 h of BDL, respectively. On the other hand, in the kidneys, the mRNA expression of MRP2 increased 162, 73, and 21%, and its protein expression increased 387, 558, and 472% compared with sham-operated animals after 24, 72, and 120 h of BDL, respectively. PAH clearance was significantly increased after BDL. The mRNA expression of MRP2 increased in renal proximal tubular epithelial cells after treatment with conjugated bilirubin, sulfate-conjugated bile acid or human bile. Upregulation of MRP2 in the kidneys and MRP3 in the liver may be a compensatory mechanism to improve bilirubin clearance during obstructive jaundice.
Upregulation of activin A gene by butyrate in human colon cancer cell lines
Authors: Sonoyama Kei , Pholnukulkit Pimara , Toyoda Masahiko , Rutatip Suriya , Kasai Takanori
Published: March 2015

Activin A has been reported to play a role in the progression of colorectal cancer. Because dietary fiber protects against colorectal cancer, we hypothesized that butyrate, a fermentation product of dietary fiber, may affect the expression of activin A in colon cancer cells. Semiquantitative RT-PCR demonstrated that the activin A gene was upregulated by sodium butyrate in the human colon cancer cell lines HT-29 and Caco-2 in a concentration- and time-dependent manner. However, the activin A gene did not respond to sodium butyrate in the human normal colonic cell line FHC, rat normal intestinal epithelial cell (IEC) line IEC-6, and the explant of rat colon. Flow cytometry and agarose gel electrophoresis of genomic DNA revealed that cell cycle arrest and apoptosis were induced by sodium butyrate but not exogenous activin A in HT-29 cells, indicating that activin A could not act as an autocrine factor in colon cancer cells. By assuming that activin A promotes colorectal cancer spread as a paracrine factor, our findings suggest that butyrate could act as a tumor promoter in some circumstances.
Alcohols enhance caerulein-induced zymogen activation in pancreatic acinar cells
Authors: Lu Zhao , Karne Suresh , Kolodecik Thomas , Gorelick Fred S.
Published: March 2015

Activation of zymogens within the pancreatic acinar cell is an early feature of acute pancreatitis. Supraphysiological concentrations of cholecystokinin (CCK) cause zymogen activation and pancreatitis. The effects of the CCK analog, caerulein, and alcohol on trypsin and chymotrypsin activation in isolated pancreatic acini were examined. Caerulein increased markers of zymogen activation in a time- and concentration-dependent manner. Notably, trypsin activity reached a peak value within 30 min, then diminished with time, whereas chymotrypsin activity increased with time. Ethanol (35 mM) sensitized the acinar cells to the effects of caerulein (10−10to 10−7M) on zymogen activation but had no effect alone. The effects of ethanol were concentration dependent. Alcohols with a chain length of ≥2 also sensitized the acinar cell to caerulein; the most potent was butanol. Branched alcohols (2-propanol and 2-butanol) were less potent than aliphatic alcohols (1-propanol and 1-butanol). The structure of an alcohol is related to its ability to sensitize acinar cells to the effects of caerulein on zymogen activation.
PAR-2 modulates pepsinogen secretion from gastric-isolated chief cells
Authors: Fiorucci Stefano , Distrutti Eleonora , Federici Barbara , Palazzetti Barbara , Baldoni Monia , Morelli Antonio , Cirino Giuseppe
Published: March 2015

In the present study, we investigated whether activation of protease-activated receptor type 2 (PAR-2) with SLIGRL (SL)NH2, a short mimetic agonistic peptide, directly stimulates pepsinogen secretion from gastric-isolated, pepsinogen-secreting (chief) cells. Immunostaining of gastric-dispersed chief cells with a specific anti-PAR-2 antibody demonstrated expression of PAR-2 receptors on membrane and cytoplasm. SL-NH2and trypsin potently stimulated pepsinogen secretion (EC50= 0.3 nM) and caused Ca2+mobilization (EC50= 0.6 nM). In contrast to SL-NH2, the scramble peptide LSIGRL-NH2failed to stimulate pepsinogen release. Exposure to SL-NH2also resulted in ERK1/2 phosphorylation and activation. Exposure of chief cells to phosphotyrosine kinase inhibitors and 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one, a selective MEK inhibitor, significantly reduced secretion induced by SL-NH2. Pepsinogen secretion induced by SL-NH2was desensitized by pretreating the cells with the mimetic peptide and trypsin, and exposure to SL-NH2abrogates pepsinogen secretion induced by carbachol and CCK-8, but not secretion induced by secretin and vasointestinal peptide. Exposure to Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2(substance P) but not to calcitonin gene-related peptide increased pepsinogen release. The neurokinin-1 receptor antagonist, N-acetyl-l-tryptophan 3,5-bis(trifluoromethyl)benzyl ester, inhibited substance P-stimulated pepsinogen secretion, whereas it did not affect secretion induced by SL-NH2. Collectively, these data indicate that PAR-2 is expressed on gastric chief cells and that its activation causes a Ca2+-ERK-dependent stimulation of pepsinogen secretion.
Endothelial COX-1 and -2 differentially affect reactivity of MVB in portal hypertensive rats
Authors: Potenza M. A. , Botrugno O. A. , De Salvia M. A. , Lerro G. , Nacci C. , Marasciulo F. L. , Andriantsitohaina R. , Mitolo-Chieppa D.
Published: March 2015

Expression of constitutive and inducible cyclooxygenase (COX-1 and COX-2, respectively) and the role of prostanoids were investigated in the aorta and mesenteric vascular bed (MVB) from the portal vein-ligated rat (PVL) as a model of portal hypertension. Functional experiments were carried out in MVB from PVL and sham-operated rats in the absence or presence of the nonselective COX inhibitor indomethacin or the selective inhibitors of COX-1 (SC-560) or COX-2 (NS-398). Western blots of COX-1 and COX-2 proteins were evaluated in aorta and MVB, and PGI2production by enzyme immunoassay of 6-keto-PGF1αwas evaluated in the aorta. In the presence of functional endothelium, decreased contraction to norepinephrine (NE) and increased vasodilatation to ACh were observed in MVB from PVL. Exposure of MVB to indomethacin, SC-560, or NS-398 reversed the hyporeactivity to NE and the increased endothelial vasodilatation to ACh in PVL, with NS-398 being more potent than the other two inhibitors. Upregulation of COX-1 and COX-2 expressions was detected in aorta and MVB from PVL portal hypertensive rats, and increased production of 6-keto-PGF1αwas observed in aorta from portal hypertensive rats. These results suggest that generation of endothelial vasodilator prostanoids, from COX-1 and COX-2 isoforms, accounts for the increased mesenteric blood flow in portal hypertension.
Expression and function of KCNH2 (HERG) in the human jejunum
Authors: Farrelly A. M. , Ro S. , Callaghan B. P. , Khoyi M. A. , Fleming N. , Horowitz B. , Sanders K. M. , Keef K. D.
Published: March 2015

Previous studies suggest that ether-a-go-go related gene (ERG) KCNH2 potassium channels contribute to the control of motility patterns in the gastrointestinal tract of animal models. The present study examines whether these results can be translated into a role in human gastrointestinal muscles. Messages for two different variants of the KCNH2 gene were detected: KCNH2 V1 human ERG (HERG) (28) and KCNH2 V2 (HERGUSO) (13). The amount of V2 message was greater than V1 in both human jejunum and brain. The base-pair sequence that gives rise to domains S3– S5 of the channel was identical to that previously published for human KCNH2 V1 and V2. KCNH2 protein was detected immunohistochemically in circular and longitudinal smooth muscle and enteric neurons but not in interstitial cells of Cajal. In the presence of TTX (10−6 M), atropine (10−6M). and l-nitroarginine (10−4 M) human jejunal circular muscle strips contracted phasically (9 cycles/min) and generated slow waves with superimposed spikes. Low concentrations of the KCNH2 blockers E-4031 (10−8 M) and MK-499 (3 × 10−8 M) increased phasic contractile amplitude and the number of spikes per slow wave. The highest concentration of E-4031 (10−6 M) produced a 10–20 mV depolarization, eliminated slow waves, and replaced phasic contractions with a small tonic contracture. E-4031 (10−6 M) did not affect [14C]ACh release from enteric neurons. We conclude that KCNH2 channels play a fundamental role in the control of motility patterns in human jejunum through their ability to modulate the electrical behavior of smooth muscle cells.
Regulation of triacylglycerol and phospholipid trafficking by fatty acids in newborn swine enterocytes
Authors: Yao Ying , Eshun John K. , Lu Song , Berschneider Helen M. , Black Dennis D.
Published: March 2015

We (Wang H, Berschneider HM, Du J, and Black DD. Am J Physiol Gastrointest Liver Physiol 272: G935–G942, 1997; Wang H, Lu S, Du J, Yao Y, Berschneider HM, and Black DD. Am J Physiol Gastrointest Liver Physiol 280: G1137–G1144, 2001) previously showed that different fatty acids influence synthesis and secretion of triacylglycerol (TG) and phospholipid (PL) in a newborn swine enterocyte cell line (IPEC-1). The most striking effects were produced by stearic acid (SA; 18:0), which modestly affected TG and PL synthesis but reduced TG and PL secretion, and by eicosapentaenoic acid (EPA; 20:5), which reduced TG and PL synthesis and TG secretion relative to oleic acid (OA; 18:1). To define the mechanism of these effects, differentiated IPEC-1 cells were incubated for 24 h with OA, SA, or EPA and [3H]glycerol. Endoplasmic reticulum (ER) and Golgi (G) content of labeled lipids and apolipoprotein (apo) B and apoAI protein were measured. Relative to OA, SA did not impair ER TG synthesis, but reduced movement of labeled TG from ER to G. EPA impaired both ER TG synthesis and movement of labeled TG from ER to G. PL followed the same pattern, except ER synthesis of PL was relatively unaffected by EPA. Carbonate treatment demonstrated decreased partitioning of labeled lipid from ER membrane to lumen in EPA-treated cells. Organelle apoB and apoAI content demonstrated opposite patterns after SA and EPA incubation. We conclude that SA and EPA adversely influence immature enterocyte ER to G lipid trafficking, compared with OA. Furthermore, EPA inhibits ER lipid synthesis and transfer of membrane lipid to luminal particles. Regulation of apoAI ER to G trafficking is independent of that of apoB.
Characterizing voltage-dependent Ca2+ channels coupled to VIP release and NO synthesis in enteric synaptosomes
Authors: Kurjak M. , Sennefelder A. , Aigner M. , Schusdziarra V. , Allescher H. D.
Published: March 2015

In enteric synaptosomes of the rat, the role of voltage-dependent Ca2+channels in K+-induced VIP release and nitric oxide (NO) synthesis was investigated. Basal VIP release was 39 ± 4 pg/mg, and cofactor-substituted NO synthase activity was 7.0 ± 0.8 fmol · mg−1 · min−1. K+ depolarization (65 mM) stimulated VIP release Ca2+ dependently (basal, 100%; K+, 172.2 ± 16.2%; P < 0.05, n = 5). K+-stimulated VIP release was reduced by blockers of the P-type (ω-agatoxin-IVA, 3 × 10−8 M) and N-type (ω-conotoxin-GVIA, 10−6 M) Ca2+ channels by ∼50 and 25%, respectively, but not by blockers of the L-type (isradipine, 10−8 M), Q-type (ω-conotoxin-MVIIC, 10−6 M), or T-type (Ni2+, 10−6 M) Ca2+ channels. In contrast, NO synthesis was suppressed by ω-agatoxin-IVA, ω-conotoxin-GVIA, and isradipine by ∼79, 70, and 70%, respectively, whereas Ni2+ and ω-conotoxin-MVIIC had no effect. These findings are suggestive of a coupling of depolarization-induced VIP release primarily to the P- and N-type Ca2+ channels, whereas NO synthesis is presumably dependent on Ca2+ influx not only via the P- and N- but also via the L-type Ca2+ channel. In contrast, none of the Ca2+ channel blockers affected VIP release evoked by exogenous NO, suggesting that NO induces VIP secretion by a different mechanism, presumably involving intracellular Ca2+ stores.
Physiology of iron transport and the hemochromatosis gene
Authors: Pietrangelo Antonello
Published: March 2015

Iron is essential for fundamental cell functions but is also a catalyst for chemical reactions involving free radical formation, potentially leading to oxidative stress and cell damage. Cellular iron levels are therefore carefully regulated to maintain an adequate substrate while also minimizing the pool of potentially toxic “free iron.” The main control of body iron homeostasis in higher organisms is placed in the duodenum, where dietary iron is absorbed, whereas no controlled means of eliminating unwanted iron have evolved in mammals. Hereditary hemochromatosis, the prototype of deregulated iron homeostasis in humans, is due to inappropriately increased iron absorption and is commonly associated to a mutated HFE gene. The HFE protein is homologous to major histocompatibility complex class I proteins but is not an iron carrier, whereas biochemical and cell biological studies have shown that the transferrin receptor, the main protein devoted to cellular uptake of transferrin iron, interacts with HFE. This review focuses on recent advances in iron research and presents a model of HFE function in iron metabolism.