bluuebunny/crossref_metadata_2025 huggingface dataset:
A novel motility effect of tachykinins in normal and inflamed colon
Authors: Tsukamoto M. , Sarna S. K. , Condon R. E.
Published: December 2017

The role of tachykinins in stimulating phasic and giant migrating contractions (GMCs) in the normal and inflamed colon in conscious dogs was investigated by close-intra-arterial infusions of test substances. At low doses (0.1 nmol), substance P and neurokinin (NK1) receptor agonist ([Sar9,Met(O2)11]substance P] stimulated phasic contractions only. At higher doses (2.0 nmol), they stimulated phasic contractions and GMCs. The phasic contractions were blocked partially but significantly by prior close-intra-arterial infusions of tetrodotoxin and atropine but not by hexamethonium. NK1 receptor antagonist partially but significantly inhibited the phasic contractile response to substance P, whereas NK2 and NK3 receptor antagonists had no significant effect. The contractile response to NK2 receptor agonist was less than one-half of the response to substance P; NK3 receptor agonist did not stimulate any contractile activity. The stimulation of GMCs by higher doses of substance P was not blocked by prior infusions of atropine, tetrodotoxin, or NK1, NK2, and NK3 receptor antagonists, nor was the contractile response to substance P blocked by H1 and H2 receptor antagonists. Inflammation depressed the phasic contractile response but enhanced the stimulation of GMCs by substance P. The ability of substance P to stimulate GMCs is novel and suggests its potential role in increasing the frequency of these contractions during colonic inflammation.
Effect of cholestasis on regulation of cAMP synthesis by glucagon and bile acids in isolated hepatocytes
Authors: Matsuzaki Y. , Bouscarel B. , Le M. , Ceryak S. , Gettys T. W. , Shoda J. , Fromm H.
Published: December 2017

Previously, we have reported that bile acids can directly inhibit hormone-induced adenosine 3',5'-cyclic monophosphate (cAMP) formation through a protein kinase C (PKC)-dependent mechanism [Bouscarel, B., T.W. Gettys, H. Fromm, and H. Dubner. Am. J. Physiol. 268 (Gastrointest. Liver Physiol. 31): G300-G310, 1995]. Therefore, the regulation of cAMP synthesis by glucagon and bile acids was investigated in hepatocytes isolated after 2-day ligation of the common bile duct in Golden Syrian hamsters. The bile acid concentration was increased 30-fold in the serum, whereas it was not significantly different in the bile of duct-ligated vs. sham-operated hamsters. The glycine/taurine and cholate/chenodeoxycholate ratios were significantly increased fourfold and sevenfold, respectively, only in the serum of bile duct-ligated hamsters. Ligation of the bile duct decreased the efficacy of glucagon-stimulated cAMP synthesis by 40-50% without changing its potency. This attenuation of cAMP synthesis, which was also observed with forskolin, remained in the absence of any detectable amount of bile acids in the hepatocytes. The decrease in glucagon-stimulated cAMP production was also not attributable to changes in either the affinity or the number of receptors for this hormone. The potency and efficacy of the bile acids to inhibit glucagon-induced cAMP formation was also reduced in bile duct-ligated hamsters. The inhibitory regulation of cAMP synthesis through angiotensin II was similarly diminished after bile duct ligation. Although the total expression of PKC-alpha was not affected, an increased translocation by 60% from the cytosol to the membrane fraction was observed in hepatocytes isolated after bile duct ligation. Therefore, during cholestasis and prolonged exposure of the liver to bile acids, both the stimulatory and inhibitory regulatory, mechanisms of cAMP synthesis are compromised in an irreversible manner because the effects persist even after isolation of the hepatocytes. This decreased regulation of cAMP synthesis is possibly mediated through PKC-alpha activation.
Intestinal physiology and pathology in gene-targeted mouse models of cystic fibrosis
Authors: Grubb B. R. , Gabriel S. E.
Published: December 2017

Cystic fibrosis (CF) is a fatal genetic disorder that affects approximately 1 in 2,500 live Caucasian births. The disease can be described as a generalized exocrine disease affecting a variety of epithelial tissues, with early manifestation as meconium ileus in a significant number of neonates. Cloning of the gene causing CF was accomplished in 1989, and the protein product, cystic fibrosis transmembrane conductance regulator (CFTR), has been conclusively shown to be an adenosine 3',5'-cyclic monophosphate (cAMP)-regulated Cl- channel. Subsequently, several mouse models of CF were generated by gene-targeting approaches in an attempt to further understand this disease. The initial excitement generated by the emergence of these mouse models was somewhat tempered by the finding that none of the models developed airway disease, which is currently responsible for most of the morbidity and mortality in the human CF population. However, the various CF mouse models, of which there are now 10, are remarkably similar to their human counterparts with respect to intestinal pathophysiology. Most importantly, the intestinal tract of the CF mouse models demonstrates the absence of cAMP-mediated Cl- transport, which is a hallmark of CF disease. Furthermore, the murine CF intestinal tract also shows an inability to secrete HCO3-, defective cAMP regulation of electroneutral NaCl absorption, and elevated electrogenic Na+ transport in the distal colon, as well as other ion transport perturbations. Besides the fundamental mechanisms of ion transport studied in the murine CF intestinal tract, these models have also been important in understanding other tissues with regard to CF. Mice heterozygous for the CFTR knockout gene have a reduced ability to secret Cl- and fluid and therefore provide further support for the CF "heterozygote advantage" hypothesis. Some CF mouse models maintain a limited ability to secrete Cl-, which may be due to accessory genes that are hypothesized to ameliorate disease severity in the intestines of these mice. This review describes the CF models generated and compares the murine defects in ion transport with observed abnormalities in the human CF intestine.
Increased expression of EGFR in gastric mucosa of aged rats
Authors: Tureaud J. , Sarkar F. H. , Fligiel S. E. , Kulkarni S. , Jaszewski R. , Reddy K. , Yu Y. , Majumdar A. P.
Published: December 2017

Although in Fischer-344 rats aging is found to be associated with increased gastric mucosal proliferative activity, little is known about the intracellular events that regulate this process. The present investigation examines the age-related changes in gastric mucosal tyrosine kinase activity and expression of epidermal growth factor receptor(EGFR) and its structural and functional analog p185c-erbB-2, the protein product of c-erbB-2/c-neu protooncogene. We observed a significantly higher intrinsic tyrosine kinase activity and tyrosine phosphorylation of EGFR and p185c-erbB-2 in the gastric mucosa of 24-mo-old (aged) rats than in that of their 4- or 12-mo-old counterparts. This was associated with increased levels of EGFR protein and steady-state mRNA levels of EGFR and p185c-erbB-2. In addition, we also observed threefold higher steady-state mRNA levels of transforming growth factor-alpha (TGF-alpha; one of the primary ligands of EGFR) in the gastric mucosa of aged rats than in that of 4-mo-old (young) animals. This was accompanied by a fivefold increase in the relative concentration of the 18-kDa precursor form of TGF-alpha in gastric mucosal membranes but not in the cytosol. In conclusion, our data demonstrate that aging is associated with increased tyrosine kinase activity of EGFR and p185c-erbB-2 in the gastric mucosa. Moreover, the observation that aging results in increased accumulation of TGF-alpha in gastric mucosal membranes raises the possibility that the membrane-bound TGF-alpha could partly be responsible for the constitutively active EGFR-induced signaling pathway in the gastric mucosa of aged rats and, in turn, for stimulation of mucosal proliferative activity.
Ca2+channel blockade by verapamil inhibits GMCs and diarrhea during small intestinal inflammation
Authors: Lee Ching-Wen , Sarna Sushil K. , Singaram Chandar , Casper Margaret A.
Published: December 2017

The aim of this study was to investigate whether the blockade of L-type Ca2+channels with verapamil suppresses giant migrating contractions (GMCs) and therefore diarrhea during small intestinal inflammation. Small intestinal inflammation was induced by infection with the nematode Trichinella spiralis. T. spiralisinfection alone significantly increased the frequency of GMCs and decreased the frequency of phase III activity in the small intestine for 9 days. The increased frequency of GMCs was associated with diarrhea. Immunohistochemical staining with specific antibodies indicated that the number of neutrophils and mast cells increased significantly in the jejunal lamina propria during T. spiralis infection. Only the neutrophils increased significantly in the muscularis externa of the jejunum. Myeloperoxidase (MPO) activity increased significantly in the jejunal and ileal lamina propria. Daily verapamil administration during T. spiralis infection significantly reduced the frequency of GMCs and diarrhea but had no further significant effect on the already reduced frequency of phase III activity. Verapamil administration, however, did not reduce MPO activity or immunocyte infiltration in the jejunum or ileum. We conclude that blockade of L-type Ca2+channels selectively reduces the frequency of GMCs and therefore diarrhea during small intestinal inflammation. The decreased frequency of GMCs is not secondary to a reduction in the inflammatory response.
Hepatocyte nuclear factor-1α regulates transcription of the guanylin gene
Authors: Hochman J. A. , Sciaky D. , Whitaker T. L. , Hawkins J. A. , Cohen M. B.
Published: December 2017

To study the molecular mechanisms controlling guanylin expression, we have cloned the mouse guanylin gene, including 2.7 kb of upstream sequence. We show that the first 133 base pairs (bp) of the upstream guanylin promoter are sufficient to drive near maximal (6-fold over basal) luciferase reporter gene expression in Caco-2 intestinal cells; at least 300 bp of upstream promoter are required for reporter gene expression in HT-29 intestinal cell lines. Using electromobility shift assays, we demonstrate that nuclear proteins bind to the hepatocyte nuclear factor-1 (HNF-1) consensus sequence in the guanylin promoter. The HNF-1 consensus sequence, located in the immediate 5′ flanking region, is required for transcriptional activation of the guanylin gene in both intestinal cell lines. Mutagenesis of the HNF-1 consensus sequence abolishes transcriptional activation of guanylin promoter-luciferase reporter gene constructs. Cotransfection of these constructs with HNF-1α augments transcriptional initiation of the reporter gene. In contrast, HNF-1β has no significant effect on transcription of the reporter gene. These experiments demonstrate that HNF-1α is an important regulatory element in the transcriptional activation of guanylin.
Caerulein pancreatitis increases mRNA but reduces protein levels of rat pancreatic heat shock proteins
Authors: Strowski M. Z. , Sparmann G. , Weber H. , Fiedler F. , Printz H. , Jonas L. , Göke B. , Wagner A. C. C.
Published: December 2017

We have recently reported that preconditioning through hyperthermia induces expression of pancreatic heat shock proteins (HSPs) and protects against caerulein pancreatitis. Here, we investigate caerulein-mediated effects on pancreatic HSPs without prior hyperthermia. Caerulein time and dose dependently increased pancreatic mRNA levels of the constitutive isoform of HSP70 (HSC70). However, pancreatic HSC70 protein levels were decreased, as were HSP60 protein levels. Also, in contrast to hyperthermia preconditioning, caerulein did not induce measurable levels of mRNA or protein of the inducible isoform of HSP70. Thus the pancreas reacts to different kinds of stress (hyperthermia vs. hyperstimulation) with differential induction of HSP mRNAs. Clearly, hyperthermia leads to induction of HSP protein expression, whereas caerulein treatment does not. Therefore, our current study further supports the idea that hyperthermia-induced protection against caerulein pancreatitis may be mediated through increased protein levels of pancreatic HSPs. It is further tempting to hypothesize that failure to appropriately increase HSP protein levels in response to high doses of caerulein might be a factor in the development of pancreatitis.
Use of measurements of ethanol absorption from stomach and intestine to assess human ethanol metabolism
Authors: Levitt Michael D. , Li Ricardo , Demaster Eugene G. , Elson Michael , Furne Julie , Levitt David G.
Published: December 2017

Controversy exists concerning the site (stomach vs. liver) and magnitude of first-pass metabolism of ethanol. We quantitated gastric and total ethanol absorption rates in five male subjects and utilized these measurements to evaluate first-pass metabolism. Gastric emptying of ethanol (0.15 g/kg) was determined via a gamma camera and gastric absorption from the ratio of gastric ethanol to [14C]polyethylene glycol. Gastric absorption accounted for 30% and 10% of ethanol administered with food and water, respectively. With food, estimated gastric mucosal ethanol concentrations fell from 19 to 5 mM over 2 h. Calculations using these concentrations and kinetic data for gastric alcohol dehydrogenase showed <2% of the dose underwent gastric metabolism. Application of observed ethanol absorption rates to a model of human hepatic ethanol metabolism indicated that only 30% and 4% of the dose underwent first-pass metabolism when administered with food and water, respectively. We conclude that virtually all first-pass ethanol metabolism occurs in the liver and first-pass metabolism accounts for only a small fraction of total clearance.
Neurokinin A increases duodenal mucosal permeability, bicarbonate secretion, and fluid output in the rat
Authors: Hällgren Anneli , Flemström Gunnar , Hellström Per M. , Lördal Mikael , Hellgren Sandra , Nylander Olof
Published: December 2017

The aim of this study was to examine the integrative response to neurokinin A (NKA) on duodenal mucosal permeability, bicarbonate secretion, fluid flux, and motility in an in situ perfusion model in anesthetized rats. Intravenous infusion of NKA (100, 200, and 400 pmol ⋅ kg−1 ⋅ min−1) induced duodenal motility. Furthermore, duodenal mucosal bicarbonate secretion, fluid output, and mucosal permeability increased in response to NKA. Pretreatment with the nicotinic antagonist hexamethonium did not change the response in any of the parameters investigated, whereas the NK2-receptor antagonist MEN 10,627 effectively inhibited all responses to NKA. Indomethacin induced duodenal motility and stimulated bicarbonate secretion. In indomethacin-treated rats, NKA further increased motility but decreased indomethacin-stimulated bicarbonate secretion by 70%. The NKA-induced increase in mucosal permeability was unaltered by indomethacin. It is concluded that NKA not only induces motility but also increases mucosal permeability and fluid output. Furthermore, the neuropeptide may have both stimulative and inhibitory effects on bicarbonate secretion. All responses to NKA are dependent on NK-2 receptor activation but are not mediated through nicotinic receptors.
Regulation of biliary secretion through apical purinergic receptors in cultured rat cholangiocytes
Authors: Schlenker Thorsten , Romac Joelle M.-J. , Sharara Ala I. , Roman Richard M. , Kim Stephen J. , Larusso Nicholas , Liddle Rodger A. , Fitz J. Gregory
Published: December 2017

To evaluate whether ATP in bile serves as a signaling factor regulating ductular secretion, voltage-clamp studies were performed using a novel normal rat cholangiocyte (NRC) model. In the presence of amiloride (100 μM) to block Na+channels, exposure of the apical membrane to ATP significantly increased the short-circuit current ( Isc) from 18.2 ± 5.9 to 52.8 ± 12.7 μA ( n = 18). The response to ATP is mediated by basolateral-to-apical Cl−transport because it is inhibited by 1) the Cl−channel blockers 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (1 mM), diphenylanthranilic acid (1.5 mM), or 5-nitro-2-(3-phenylpropylamino)benzoic acid (50 or 100 μM) in the apical chamber, 2) the K+channel blocker Ba2+(5 mM), or 3) the Na+-K+-2Cl−cotransport inhibitor bumetanide (200 μM) in the basolateral chamber. Other nucleotides stimulated an increase in Iscwith a rank order potency of UTP = ATP = adenosine 5′-O-(3)-thiotriphosphate, consistent with P2upurinergic receptors. ADP, AMP, 2-methylthioadenosine 5′-triphosphate, and adenosine had no effect. A cDNA encoding a rat P2ureceptor (rP2uR) was isolated from a liver cDNA library, and functional expression of the corresponding mRNA in Xenopus laevis oocytes resulted in the appearance of ATP-stimulated currents with a similar pharmacological profile. Northern analysis identified hybridizing mRNA transcripts in NRC as well as other cell types in rat liver. These findings indicate that exposure of polarized cholangiocytes to ATP results in luminal Cl−secretion through activation of P2ureceptors in the apical membrane. Release of ATP into bile may serve as an autocrine or paracrine signal regulating cholangiocyte secretory function.
Nitric oxide production duringVibrio choleraeinfection
Authors: Janoff Edward N. , Hayakawa Hiroshi , Taylor David N. , Fasching Claudine E. , Kenner Julie R. , Jaimes Edgar , Raij Leopoldo
Published: December 2017

Vibrio cholerae induces massive intestinal fluid secretion that continues for the life of the stimulated epithelial cells. Enhanced regional blood flow and peristalsis are required to adapt to this obligatory intestinal secretory challenge. Nitric oxide (NO) is a multifunctional molecule that modulates blood flow and peristalsis and possesses both cytotoxic and antibacterial activity. We demonstrate that, compared with those in asymptomatic control subjects, levels of stable NO metabolites ([Formula: see text]/[Formula: see text]) are significantly increased in sera from acutely ill Peruvian patients with natural cholera infection as well as from symptomatic volunteers from the United States infected experimentally with V. cholerae. In a rabbit ileal loop model in vivo, cholera toxin (CT) elicited fluid secretion and dose-dependent increases in levels of[Formula: see text]/[Formula: see text]in the fluid ( P < 0.01). In contrast, lipopolysaccharide (LPS) elicited no such effects when applied to the intact mucosa. NO synthase (NOS) catalytic activity also increased in toxin-exposed tissues ( P< 0.05), predominantly in epithelial cells. The CT-induced NOS activity was Ca2+dependent and was not suppressed by dexamethasone. In conclusion, symptomatic V. cholerae infection induces NO production in humans. In the related animal model, CT, but not LPS, stimulated significant production of NO in association with increases in local Ca2+-dependent NOS activity in the tissues.
Inflammation modulates in vitro colonic myoelectric and contractile activity and interstitial cells of Cajal
Authors: Lu G. , Qian X. , Berezin I. , Telford G. L. , Huizinga J. D. , Sarna S. K.
Published: December 2017

Inflammation suppresses phasic contractile activity in vivo. We investigated whether inflammation also suppresses in vitro phasic contractile activity and, if so, whether this could in part be due to the alteration of specific slow wave characteristics and morphology of the interstitial cells of Cajal (ICC). Circular muscle strips were obtained from normal and inflamed distal canine colon. Inflammation was induced by mucosal exposure to ethanol and acetic acid. The amplitudes of spontaneous, methacholine-induced, substance P-induced, and electrical field stimulation-induced contractions were smaller in inflamed muscle strips than in normal muscle strips. Inflammation reduced the resting membrane potential and the amplitude and duration of slow waves in circular muscle cells. Inflammation did not affect the amplitude of inhibitory junction potentials but did decrease their duration. Ultrastructural studies showed expansion of the extracellular space between circular muscle cells, reduction in the density of ICC and associated neural structures, damage to ICC processes, vacuolization of their cytoplasm, and blebbings of the plasma membrane. We conclude that inflammation-induced alterations of slow wave characteristics contribute to the suppression of phasic contractions. These alterations may, in part, be due to the damage to ICC. Inflammation impairs both the myogenic and neural regulation of phasic contractions.
Functional role of extracellular signal-regulated protein kinases in gastric acid secretion
Authors: Takeuchi Yoshiaki , Yamada Junko , Yamada Tadataka , Todisco Andrea
Published: December 2017

Epidermal growth factor (EGF) has acute inhibitory and chronic stimulatory effects on gastric acid secretion. Because a cascade of intracellular events culminating in the activation of a family of serine-threonine protein kinases called extracellular signal-regulated protein kinases (ERKs) is known to mediate the actions of EGF, we undertook studies to explore the functional role of the ERKs in gastric acid secretion. ERK2 was immunoprecipitated from cell lysates of highly purified (>95%) gastric canine parietal cells, and its activity was quantified using in-gel kinase assays. Of the primary gastric secretagogues, carbachol was the most potent inducer of ERK2 activity. Gastrin and EGF had weaker stimulatory effects, whereas no induction was noted in response to histamine. The effect of carbachol appeared to be independent of Ca2+signaling. PD-98059, a selective inhibitor of the upstream ERK activator mitogen-activated protein kinase/ERK kinase, dose-dependently inhibited both carbachol- and EGF-stimulated ERK2 activity, with a maximal effect observed between 50 and 100 μM. ERKs activation is required for induction of the early gene c- fos via phosphorylation of the transcription factor Elk-1 which binds to the c- fos serum response element (SRE). Carbachol stimulated a two- to threefold induction of luciferase activity in cultured parietal cells transfected with either a SRE-luciferase reporter plasmid or with a chimeric GAL4-ElkC expression vector and the 5×GAL-luciferase reporter plasmid. To examine the significance of ERK activation in gastric acid secretion, we tested the effect of PD-98059 on carbachol-stimulated uptake of14C-labeled aminopyrine (AP). Acute inhibition of the ERKs by PD-98059 led to a small increase in AP uptake and a complete reversal of the acute inhibitory effect of EGF on AP uptake induced by either carbachol or histamine. In contrast, exposure of the cells to PD-98059 for 16 h led to a reversal of the chronic stimulatory effect of EGF on AP uptake induced by carbachol. Our data led us to conclude that carbachol induces a cascade of events in parietal cells that results in ERK activation. Although the acute effect of the ERKs on gastric acid secretion appears to be inhibitory, the activation of transcription factors and of early gene expression could be responsible for its chronic stimulatory effects.
Protection against ethanol injury by prostaglandin in a human intestinal cell line: role of microtubules
Authors: Banan A. , Smith G. S. , Rieckenberg C. L. , Kokoska E. R. , Miller T. A.
Published: December 2017

Prostaglandins have been shown to protect the gastrointestinal (GI) epithelium from injury induced by various luminal insults independent of their known acid-inhibitory effects, a process termed “cytoprotection.” The mechanism of this protective action remains unknown. The present investigation determined the role of microtubules (a major cytoskeletal component) in GI injury induced by ethanol (EtOH) and its prevention by 16,16-dimethylprostaglandin E2(dmPGE2) using cells from a human colonic cell line known as Caco-2 cells. These cells were preincubated in Eagle’s minimum essential medium with and without dmPGE2(2.6 μM) for 15 min and subsequently incubated in media containing 1, 2.5, 5, 7.5, and 10% EtOH. The effects on cell viability and tubulin (the major protein backbone of microtubules) were then determined. EtOH concentrations ≥2.5% extensively disrupted the microtubules as demonstrated by fragmentation, kinking, and perturbation of the microtubule organizer center. EtOH treatment also led to a significant decrease in the S2 (polymerized) fraction and an increase in the S1 (monomeric) pool of tubulin. Concomitant with these effects were marked decreases in cellular viability. DmPGE2pretreatment abolished the disruption of microtubules, significantly increased the S2 fraction of tubulin, and increased cellular viability in cultures exposed to EtOH. Furthermore, pretreatment with colchicine, an inhibitor of microtubule assembly, prevented the cytoprotective action of dmPGE2. Taxol, a microtubule stabilizing agent, mimicked the effects of dmPGE2by also enhancing microtubule integrity and increasing cellular viability in cells exposed to EtOH. Our data indicate that organization and stabilization of microtubules may play an essential role in the mechanism of prostaglandin-induced protection.
Synergistic regulation of NOS II expression by IL-1β and TNF-α in cultured rat colonic smooth muscle cells
Authors: Kuemmerle John F.
Published: December 2017

Interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and lipopolysaccharide (LPS) were examined for their ability to regulate the activity and protein levels of inducible nitric oxide synthase (NOS II) in cultured rat colonic smooth muscle cells. Treatment with these agents resulted in a time-dependent increase in NOS II activity. After 48 h, NOS II activity, measured asl-[3H]citrulline production, was increased 24.3 ± 6.9 pmol ⋅ min−1⋅ mg protein−1by 1 nM IL-1β and 3.2 ± 1.1 pmol ⋅ min−1⋅ mg protein−1by 1 nM TNF-α, and increased synergistically by a combination of the two (51.8 ± 7.3 pmol ⋅ min−1⋅ mg protein−1). Measurement of NOS II activity as nitrite production yielded similar results: IL-1β, 27.2 ± 1.2; TNF-α, 1.6 ± 0.1; and IL-1β + TNF-α, 46.8 ± 3.2 pmol ⋅ min−1⋅ mg protein−1above basal. LPS (10 μg/ml) had a small but significant effect at 48 h that was only additive with that of IL-1β. The increase in NOS II activity induced by IL-1β and TNF-α was inhibited 73–86% by transforming growth factor-β1 (TGF-β1). The NOS isoform induced by IL-1β and TNF-α was identified as NOS II by Western immunoblot analysis and confirmed by its 66–97% inhibition by 100 μM S-methylisothiourea, a selective NOS II inhibitor, and its Ca2+-independent activity. We conclude that the cytokines IL-1β and TNF-α act independently and synergistically to stimulate NOS II expression and enzymatic activity in rat colonic smooth muscle through a mechanism sensitive to inhibition by TGF-β1.
I. Antigen presentation in the intestine: new rules and regulations
Authors: Mayer Lloyd
Published: December 2017

The recognition that immune responses in the intestine differ from those seen systemically has led to a search for novel pathways involved in mucosal immunoregulation. One cell type that has surfaced as a prime candidate for such a regulatory role is the intestinal epithelial cell. A number of laboratories have documented that intestinal epithelial cells sample luminal antigens and process and present these to primed T cells. However, several unique features have emerged, making their potential role as antigen-presenting cells a critical part of mucosal homeostasis.
Effects of meal volume and composition on gastric myoelectrical activity
Authors: Levanon Daniela , Zhang Ming , Orr William C. , Chen J. D. Z.
Published: December 2017

The absence of a standard meal in electrogastrography may limit its clinical significance. Different meals may fail to produce the expected postprandial motility pattern. The aim of this study was to investigate the effect of meal volume and composition on postprandial myoelectrical activity. Fourteen healthy subjects were given four meals that differed from a “reference meal” in one single parameter (volume, calorie, or fiber content). Gastric myoelectrical activity was measured using surface electrogastrography. Spectral and statistical analyses were performed to investigate the effect of food properties on electrogastrogram (EGG) parameters. It was found that the reference meal produced a postprandial increase in the dominant frequency ( P < 0.007), dominant power ( P < 0.04), and percentage of normal 2–4 cycle/min gastric slow waves ( P > 0.05). Similar changes were observed with the low-volume and high-fiber meals but not with the reduced-calorie meal. Fasting EGG parameters in all four sessions showed no significant difference. It was concluded that low-calorie meals do not result in expected postprandial physiological responses and thus are not appropriate for EGG tests. A volume reduction of down to one-half the volume of a regular meal does not affect postprandial changes of the EGG; thus a condensed test meal may be recommended for symptomatic patients.
Direct measurement of nitric oxide release in gastric mucosa during ischemia-reperfusion in rats
Authors: Wada Kouichirou , Kamisaki Yoshinori , Ohkura Tsuyoshi , Kanda Gaku , Nakamoto Kentaro , Kishimoto Yosuke , Ashida Kumiyo , Itoh Tadao
Published: December 2017

Nitric oxide (NO) generation in the rat gastric mucosa during ischemia-reperfusion was measured using an NO-sensitive electrode. Under pentobarbital sodium anesthesia, an electrode was inserted into the submucosa from the serous membrane side in the fundus. After steady-state baseline recording, the celiac artery was clamped for 30 min, and then ischemia-reperfusion was achieved by removing the clamp. The clamping of the celiac artery caused a decrease in blood flow and an increase in NO level in the gastric tissue. Just after the removal of the clamp, the NO level rapidly fell and returned to the baseline level. Administration of N G-nitro-l-arginine methyl ester (an NO synthase inhibitor, 30 mg/kg ip) before ischemia significantly attenuated both the increase in NO level during ischemia and the formation of acute gastric mucosal lesions observed after 60 min reperfusion. Administration of superoxide dismutase (a superoxide radical scavenger, 10,000 U/kg iv) at the end of ischemia inhibited both the rapid decrease in NO level during the reperfusion and the gastric mucosal erosions. Because NO and superoxide radical produce a highly reactive peroxynitrite, it can be argued that NO has an important pathological role in acute gastric mucosal injury induced by ischemia-reperfusion. Our conclusion was strongly supported by immunohistochemical staining of nitrotyrosine residues, an indication of peroxynitrite formation.
Role of mucosal mast cells in early vascular permeability changes of intestinal DTH reaction in the rat
Authors: Kraneveld Aletta D. , Muis Thea , Koster Andries S. , Nijkamp Frans P.
Published: December 2017

Previously, it was shown that depletion and stabilization of the mucosal mast cell around the time of challenge were very effective in reducing delayed-type hypersensitivity (DTH) reactions in the small intestine of the rat. The role of mucosal mast cells in the early component of intestinal DTH reaction was further investigated in this study. In vivo small intestinal vascular leakage and serum levels of rat mast cell protease II (RMCP II) were determined within 1 h after intragastric challenge of rats that had been sensitized with dinitrobenzene 5 days before. A separate group of rats was used to study vasopermeability in isolated vascularly perfused small intestine after in vitro challenge. To investigate the effects of mast cell stabilization on the early events of the DTH reaction, doxantrazole was used. The influence of sensory nerves was studied by means of neonatal capsaicin-induced depletion of sensory neuropeptides. Within 1 h after challenge, a significant increase in vascular permeability was found in vivo as well as in vitro. This was associated with a DTH-specific increase in RMCP II in the serum, indicating mucosal mast cell activation. In addition, doxantrazole treatment and caspaicin pretreatment resulted in a significant inhibition of the DTH-induced vascular leakage and an increase in serum RMCP II. These findings are consistent with an important role for mucosal mast cells in early vascular leakage changes of intestinal DTH reactions. In addition, sensory nervous control of mucosal mast cell activation early after challenge is demonstrated.
Localization and significance of pp55, a gastric mucosal membrane protein with tyrosine kinase activity
Authors: Majumdar Adhip P. N. , Goldenring James R.
Published: December 2017

In Fischer 344 rats, induction of gastric mucosal proliferative activity, whether the result of aging or injury or occurring after administration of epidermal growth factor, gastrin, or bombesin, is associated with a rise in tyrosine kinase activity and tyrosine phosphorylation of several mucosal proteins, including a protein with a molecular mass of 53–55 kDa. We hypothesized that this phosphotyrosine membrane protein (referred to as pp55) may play a role in regulating gastric mucosal cell proliferation and differentiation. Purification and subsequent immunoprecipitation studies now show that pp55 is a tyrosine kinase. In addition, the enzyme activity in the gastric mucosa is found to be fourfold higher in aged rats than in young rats. Incubation of gastric mucosal membranes with transforming growth factor-α (2 × 10−8M) stimulates tyrosine kinase activity of pp55. Immuolocalization studies reveal that pp55 immunoreactivity is predominantly present in mucous cells that are located just above the proliferative zone and spasmolytic peptide-immunoreactive mucous neck cells. Tyrosine kinase activity as well as expression of pp55 are also greatly increased in the gastric mucosa after hypertonic saline-induced injury, a condition that results in stimulation of surface mucosal cell proliferation and differentiation. Our current data suggest that pp55 is a tyrosine kinase, likely localized to pre-surface cells. The presence of pp55 in pre-surface mucous cells and the expression and tyrosine kinase activity of this protein, which can be stimulated during mucosal cell proliferation and differentiation, strongly suggest a role for pp55 in differentiation of gastric surface mucous cells.
Nitric oxide suppresses a Ca2+-stimulated Cl− current in smooth muscle cells of opossum esophagus
Authors: Zhang Yong , Vogalis Fivos , Goyal Raj K.
Published: December 2017

Nitric oxide (NO) hyperpolarizes visceral smooth muscles. Using the patch-clamp technique, we investigated the possibility that NO-mediated hyperpolarization in the circular muscle of opossum esophagus results from the suppression of a Ca2+-stimulated Cl− current. Smooth muscle cells were dissociated from the circular layer and bathed in high-K+Ca2+-EGTA-buffered solution. Macroscopic ramp currents were recorded from cell-attached patches. Contaminating K+-channel currents were blocked with tetrapentylammonium chloride (200 μM) added to all solutions. Raising bath Ca2+concentration above 150 nM in the presence of A-23187 (10 μM) activated a leak current ( I L-Ca) with an EC50 of 1.2 μM at −100 mV. The reversal potential ( E rev) of I L-Ca (−8.5 ± 1.8 mV, n = 8) was significantly different ( P < 0.05) from E rev of the background current (+4.2 ± 1.2 mV, n = 8). Equimolar substitution of 135 mM Cl− in the pipette solution with gluconate significantly shifted E rev of I L-Ca to +16.6 ± 3.4 mV ( n = 4) ( P < 0.05 compared with background), whereas replacement of total Na+with Tris+ suppressed I L-Ca but did not affect E rev(−15 ± 3 mV, n = 3; P > 0.05). I L-Ca was inhibited by DIDS (500 μM). Diethylenetriamine-NO adduct (200 μM), a NO• donor, and 8-bromo-cGMP (200 μM) suppressed I L-Ca by 59 ± 15% ( n = 5) and 62 ± 21% ( n = 4) at −100 mV, respectively. We conclude that in opossum esophageal smooth muscle NO-mediated hyperpolarization may be produced by suppression of a Ca2+-stimulated Cl−-permeable conductance via formation of cGMP.
Effect of CCK on proximal gastric motor function in humans
Authors: Straathof Jan Willem A. , Mearadji Banafsche , Lamers Cornelis B. H. W. , Masclee Ad A. M.
Published: December 2017

We have studied the effect of CCK on proximal gastric motor function in humans. Seven healthy volunteers participated in three experiments performed in random order during continuous intravenous infusion of 1) saline (control), 2) 0.5 IDU ⋅ kg−1 ⋅ h−1CCK, and 3) 1.0 IDU ⋅ kg−1 ⋅ h−1CCK. Proximal gastric mechanics were measured by an electronic barostat, and abdominal symptoms were scored by visual analog scales. Infusion of 0.5 and 1.0 IDU ⋅ kg−1 ⋅ h−1CCK resulted in plasma CCK levels (RIA) in the postprandial range. CCK induced gastric relaxation; at 2 mmHg above intra-abdominal pressure the intragastric volume during 1.0 IDU ⋅ kg−1 ⋅ h−1CCK was significantly increased over saline (363 ± 44 vs. 195 ± 34 ml; P < 0.01) but not during 0.5 IDU ⋅ kg−1 ⋅ h−1CCK (195 ± 14 ml; not significant). During both isovolumetric and isobaric distensions, 1.0 IDU ⋅ kg−1 ⋅ h−1CCK significantly ( P < 0.05) increased proximal gastric compliance compared with saline. However, 0.5 IDU ⋅ kg−1 ⋅ h−1CCK had no significant effect on gastric compliance. During volume distensions, but not during fixed pressure distensions, 1.0 IDU ⋅ kg−1 ⋅ h−1CCK significantly ( P < 0.05) reduced visceral perception. These results suggest that in humans CCK may have a physiological role in regulating proximal gastric mechanics.
The labile iron pool in hepatocytes: prooxidant-induced increase in free iron precedes oxidative cell injury
Authors: Stäubli Anna , Boelsterli Urs A.
Published: December 2017

The labile iron pool (LIP) represents the nonferritin-bound, redox-active iron that has been implicated in oxidative stress and cell injury. Here we examined whether alterations in LIP can be detected in cultured murine hepatocytes and whether increases in LIP are related to the oxidative damage inflicted by the redox cycling drug nitrofurantoin (NFT). Early changes in LIP were monitored with the metal-sensitive fluorescent probe calcein (CA), the fluorescence of which is quenched on binding to iron. Short-term exposure (<1 h) to NFT reduced the CA fluorescence signal by 30%, indicating that the amount of LIP-associated iron had increased. Prolonged exposure (≥2 h) to NFT caused oxidative cell injury. The addition of the cell-permeable ferrous iron chelator 2,2′-bipyridyl not only prevented the quenching of CA fluorescence but also partially protected from NFT toxicity. It is concluded that reductive stress-induced increase in LIP is an essential event that precedes oxidative cell damage in intact hepatocytes.
Pepsinogen secretion: coupling of exocytosis visualized by video microscopy and [Ca2+]iin single cells
Authors: Tao Chie , Yamamoto Masao , Mieno Hiroshi , Inoue Masaki , Masujima Tsutomu , Kajiyama Goro
Published: December 2017

Conventional in vitro studies of pepsinogen secretion have measured secretion into the bulk medium and have demonstrated the critical role of Ca2+ in the process. The present study was undertaken to obtain further details of the process of secretion and its relation to Ca2+changes over very short time periods. The relation between Ca2+ mobilization and exocytosis in an isolated individual peptic cell of the bullfrog was investigated by a method to measure both intracellular Ca2+([Ca2+]i), using a fluorescent Ca2+indicator, fura 2, and exocytosis from single cells using a video microscope analyzing system. Bombesin (3.2 × 10−7 M) and bethanechol (3.2 × 10−4 M) caused a rapid increase in [Ca2+]i(initial peak) and a corresponding high frequency of initial exocytosis. After the initial peak, [Ca2+]iwas maintained at a somewhat elevated level over the baseline (sustained phase), with a corresponding low frequency of exocytosis. Both the sustained phase of elevated [Ca2+]iand the related exocytosis were eliminated by the depletion of extracellular Ca2+. Low concentrations of bombesin (3.2 × 10−10 M) and bethanechol (3.2 × 10−7 M) caused sustained low-amplitude Ca2+oscillations with correspondingly low frequencies but also caused sustained exocytosis. These data show that 1) cellular response differs between high and low concentrations of stimulus, 2) there is a close relation between [Ca2+]iand exocytosis, 3) exocytosis follows elevation of [Ca2+]iby 14–45 s ( n = 6), and 4) there is a significant positive correlation between the peak [Ca2+]iand the number of exocytoses.
Secretory effects of ATP on nontransformed dog pancreatic duct epithelial cells
Authors: Nguyen Toan D. , Moody Mark W. , Savard Christopher E. , Lee Sum P.
Published: December 2017

Extracellular triphosphate nucleotides, such as ATP, may regulate various cellular functions through specific cell surface receptors. We examine in this report the different secretory effects of ATP and analogs on nontransformed dog pancreatic duct epithelial cells (PDEC). We observed that 1) ATP, UTP, adenosine 5′- O-(3-thiotriphosphate), and, to a lesser extent, β,γ-methylene-ATP, but not adenosine, stimulated125I−efflux from PDEC, suggesting a primary role for P2Y2receptors, 2) ATP-stimulated125I−efflux was inhibited by 5-nitro-2-(3-phenylpropylamino)benzoic acid, diphenylamine-2-carboxylate, and DIDS, suggesting mediation through Ca2+-activated Cl−channels, 3) ATP stimulated an86Rb+efflux sensitive to BaCl2and charybdotoxin, thus likely occurring through Ca2+-activated K+channels, 4) serosal or luminal addition of UTP activated apical Cl−conductance and basolateral K+conductance when nystatin-permeabilized PDEC were studied in an Ussing chamber, suggesting the expression of P2Y2receptors on both sides of the cell, 5) ATP stimulated mucin secretion, and 6) ATP increases intracellular Ca2+concentration ([Ca2+]i). In conclusion, ATP and UTP interact with P2Y2receptors on nontransformed PDEC to increase [Ca2+]i, stimulate mucin secretion, and activate ion conductances; these findings have implications for pancreatic exocrine function in both health and disease, such as cystic fibrosis.
Changes in methionine adenosyltransferase during liver regeneration in the rat
Authors: Huang Zong-Zhi , Mao Zebin , Cai Jiaxin , Lu Shelly C.
Published: December 2017

Liver-specific and non-liver-specific methionine adenosyltransferase (MAT) are products of two genes (MAT1A and MAT2A, respectively) that catalyze the formation of S-adenosylmethionine (SAM), the principal methyl donor. We previously showed that MAT2A expression was associated with more rapid cell growth. Here we examined changes in hepatic MAT gene expression and related consequences after two-thirds partial hepatectomy (PH) in rats. The mRNA levels of both MAT forms increased from 3 to 6 h, but the MAT1A level then fell below baseline from 12 to 24 h, whereas the MAT2A level remained elevated up to 4 days after PH. The increase in the MAT2A mRNA level was due to increased gene transcription and mRNA stabilization. The change in the MAT1A mRNA level was posttranscriptional and did not require de novo protein synthesis. Changes in MAT activity were consistent with an increased amount of MAT isozymes. SAM levels, the ratio of SAM to S-adenosylhomocysteine (SAH), and DNA methylation fell from 6 to 24 h, whereas SAH levels increased slightly at 12 and 24 h after PH. Both increased SAM utilization and MAT2A gene expression likely contributed to the fall in SAM.
Secretin inhibits gastric acid secretion via a vagal afferent pathway in rats
Authors: Li P. , Chang T. M. , Chey W. Y.
Published: December 2017

Secretin is an enterogastrone that inhibits gastric acid secretion and motility. Recently, it was reported that secretin inhibited gastric emptying via a capsaicin (Cap)-sensitive vagal afferent pathway. However, a possible role of the sensory afferent pathway in secretin-inhibited acid secretion has not been clarified. We investigated whether or not the acid secretion suppressed by secretin is modulated by a vagal and/or splanchnic afferent pathway in rats. Subdiaphragmatic perivagal (PV) or periceliac ganglionic (PCG) application of Cap (10 mg/ml) or vehicle was performed in both conscious and anesthetized rats 2 wk before experiments. Bilateral vagotomy was performed in some conscious rats 5 days before studies. Pentagastrin was administered intravenously at 0.6 μg ⋅ kg−1 ⋅ h−1. Secretin (20 pmol ⋅ kg−1 ⋅ h−1iv) or 0.03 N HCl (4.32 ml/h id) was infused in conscious rats with gastric cannulas or anesthetized rats with ligation of the pylorus, respectively. A rabbit antisecretin serum was injected in some anesthetized rats before duodenal acidification. Secretin significantly inhibited pentagastrin-stimulated acid secretion by 63% ( P < 0.01), which was abolished by both vagotomy and PV treatment of Cap in conscious rats. In anesthetized rats, duodenal infusion of 0.03 N HCl suppressed pentagastrin-induced acid secretion by 59.4% ( P < 0.01), which was reversed not only by antisecretin serum but also by PV application of Cap. However, PCG treatment with Cap did not influence the inhibition by secretin or duodenal acidification in either awake or anesthetized rats. These results indicate that the inhibition by secretin of pentagastrin-stimulated acid secretion is mediated by a Cap-sensitive vagal afferent pathway but not via a splanchnic afferent pathway in rats.
Short-chain fatty acids inhibit intestinal trefoil factor gene expression in colon cancer cells
Authors: Tran Chau P. , Familari Mary , Parker Lorraine M. , Whitehead Robert H. , Giraud Andrew S.
Published: December 2017

Intestinal trefoil factor (ITF) gene expression was detected in five colon cancer cell lines. ITF was synthesized by mucous cells of LIM 1215 and LIM 1863 lines, from which it is secreted constitutively. The ITF mRNA transcript was estimated to be 0.6 kb. In LIM 1215 cells, the expression of ITF was potently and dose-dependently inhibited by short-chain fatty acids (butyrate > propionate > acetate) within 8 h of application. The inhibitory effect of butyrate was ablated by actinomycin D and preceded its effects on differentiation of LIM 1215 cells as indicated by induction of alkaline phosphatase activity and counting of periodic acid-Schiff-positive cells. The human ITF promoter contained an 11-residue consensus sequence with high homology to the butyrate response element of the cyclin D1 gene. Mobility shift assays show specific binding of this response element to nuclear protein extracts of LIM 1215 cells. We conclude that butyrate inhibits ITF expression in colon cancer cells and that this effect may be mediated transcriptionally and independently of its effects on differentiation.
III. Role of the RET signal transduction pathway in development of the mammalian enteric nervous system
Authors: Pachnis V. , Durbec P. , Taraviras S. , Grigoriou M. , Natarajan D.
Published: December 2017

The enteric nervous system (ENS) in vertebrates is derived from the neural crest and constitutes the most complex part of the peripheral nervous system. Natural and induced mutagenesis in mammals has shown that the tyrosine kinase receptor RET and its functional ligand glial cell line-derived neurotrophic factor (GDNF) play key roles in the development of the ENS in humans and mice. We have developed and briefly describe here a number of assays that analyze the specific function of the RET receptor and its ligand. Our data suggest that the RET signal transduction pathway has multiple roles in the development of the mammalian ENS.