bluuebunny/crossref_metadata_2025 huggingface dataset:
HCO3(-)-dependent conformational change in gastric parietal cell AE2, a glycoprotein naturally lacking sialic acid
Authors: Zolotarev A. S. , Townsend R. R. , Stuart-Tilley A. , Alper S. L.
Published: December 2017

Although the AE1 chloride/bicarbonate exchanger of the red blood cell is among the most thoroughly investigated of membrane transport proteins, less is known about the related AE2 polypeptide of parietal cells. We have studied enzymatic deglycosylation of native AE2 polypeptide in gastric mucosal membranes from pig and rabbit. Deglycosylation of AE2 was maximal at low ionic strength. Deglycosylation of AE2 in membranes was preferentially inhibited by bicarbonate compared with other anions. This inhibition was maximal at alkaline pH and was not evident after detergent solubilization of AE2. Deglycosylation of AE2 increased its susceptibility to proteolytic degradation, but the presence of bicarbonate protected against this degradation. Bicarbonate failed to inhibit deglycosylation of the membrane glycoproteins AE1 and gastric H(+)-K(+)-adenosinetriphosphatase beta-subunit or deglycosylation of the soluble glycoproteins fetuin and ribonuclease B. These data suggest that bicarbonate induces a conformational change in AE2 that can protect the polypeptide from deglycosylation and proteolysis. Pig AE2 was purified in sodium dodecyl sulfate, and its monosaccharide composition was determined after blotting onto polyvinylidene fluoride membrane. AE2 was found to be devoid of sialic acid, with a composition suggestive of the presence of lactosamine-type chains.
Rab3D localizes to zymogen granules in rat pancreatic acini and other exocrine glands
Authors: Ohnishi H. , Ernst S. A. , Wys N. , McNiven M. , Williams J. A.
Published: December 2017

Rab3 proteins are members of the family of Ras-like monomeric GTP-binding proteins that have been implicated in secretion in neuronal cells. Although an isoform of Rab3 has been assumed to exist in pancreatic acini, its identity has not yet been established. We now report that Rab3D is present in rat pancreatic acini and is localized to the zymogen granule membrane. Reverse transcription-polymerase chain reaction (PCR) was used with primers based on mouse Rab3D to amplify Rab3D from rat pancreas. The PCR product without primer sites consisted of 580 base pairs and was 94% identical to the mouse Rab3D cDNA sequence previously cloned from adipocytes. Western blotting with a polyclonal antiserum raised against Rab3D-specific carboxyterminal amino acids identified Rab3D in rat pancreatic acini and revealed its concentration on zymogen granule membranes. Immunocytochemistry of pancreatic lobules showed that Rab3D localized to the apical region in a pattern similar to amylase. Confocal fluorescence microscopy of lobules double immunolabeled with antibodies to Rab3D and the granule membrane marker protein glycoprotein-2 (GP-2) revealed a similar localization of these proteins to zymogen granules. Immunocytochemistry also revealed the presence of Rab3D in chief and enterochromaffin-like cells in the stomach, acinar cells in lacrimal and parotid gland, and Paneth cells in the intestine. These results show that Rab3D is expressed in rat pancreatic acini and other exocrine secretory cells. Its location implies it may be involved in regulated exocytosis.
Tumor necrosis factor-alpha induces Cl- and K+ secretion in human distal colon driven by prostaglandin E2
Authors: Schmitz H. , Fromm M. , Bode H. , Scholz P. , Riecken E. O. , Schulzke J. D.
Published: December 2017

Increased levels of tumor necrosis factor-alpha (TNF-alpha) have been found in, for example, inflammatory bowel disease (IBD) and human immunodeficiency virus (HIV) infection. To investigate a possible contribution of TNF-alpha to the pathogenesis of diarrhea in these diseases, ion transport of human distal colon was studied in the Ussing chamber in vitro. Serosal addition of TNF-alpha increased short-circuit current (Isc) of partially stripped tissues in a dose-dependent manner. Maximum Isc increase of 1.8 +/- 0.2 mumol.h-1.cm-2 was reached after 60 +/- 9 min at 200 ng/ml TNF-alpha. Bidirectional tracer flux measurements revealed that TNF-alpha induced an increase in 36 Cl serosal-to-mucosal flux, a decrease in 36Cl- mucosal-to-serosal flux, and a slight increase in K+ secretion indicated by an increased secretory 86Rb net flux. In the highly differentiated colonic epithelial cell line HT-29/B6, TNF-alpha had no effect on Isc, suggesting a mediation step located in the subepithelium. This supposition was supported by measurements on totally stripped human tissues, since removal of subepithelial layers by total stripping reduced the TNF-alpha effect by 40%. Experiments with tetrodotoxin (10(-6)M) indicated that the TNF-alpha effect was not mediated by the enteric nervous system. The specific 5-lipoxygenase blocker ICI-230487 (5 x 10(-8)M) also had no effect on TNF-alpha action. In contrast, inhibition of cyclooxygenase by indomethacin (10(-6)M inhibited the effect of TNF-alpha. Radioimmunoassay of prostaglandin E2 (PGE2) in the serosal bathing solution revealed an increase in PGE2 production/release after addition of TNF-alpha, which paralleled the Isc response. We conclude that TNF-alpha changed Cl- and K+ transport toward secretion in human colon. This effect was mediated by PGE2 produced by subepithelial cells. Thus TNF-alpha could be a mediator of diarrhea during intestinal inflammation, e.g., in IBD and HIV infection.
Effect of morphine and naloxone on motor response of the human esophagus to swallowing and distension
Authors: Penagini R. , Picone A. , Bianchi P. A.
Published: December 2017

The effect of morphine on esophageal motility has been little explored. In eight healthy volunteers, we studied the effect of intravenous morphine (100 micrograms/kg) followed 60 min later by intravenous naloxone (80 micrograms/kg) and of intravenous naloxone alone (80 micrograms/kg) on the esophageal motor response to swallowing and 30-s intraluminal distensions(4, 6, 8, and 10 ml) during two separate experiments. Morphine increased (P < 0.01) the velocity but did not alter the amplitude or duration of primary peristalsis, and it decreased the duration and magnitude of swallow-induced lower esophageal sphincter (LES) relaxation (P < 0.01). It also markedly increased contractile activity below the balloon at high distending volumes (P < 0.05) and decreased the magnitude of distension-induced LES relaxation (P < 0.05) but did not affect contractile activity above the balloon. All effects were reversed by naloxone. The latter alone did not influence the esophageal response to swallowing or distension. The conclusions are that 1) morphine exerts effects on the response of the human esophagus to swallowing and intraluminal distension that are consistent with an action at the level of the inhibitory neural pathways, 2) these effects occur through opioid receptors, and 3) endogenous opioids do not seem to control esophageal motility, at least through mu-receptors.
Expression of genes involved in iron metabolism in mouse intestine
Authors: McKie A. T. , Raja K. B. , Peters T. J. , Farzaneh F. , Simpson R. J.
Published: December 2017

Intestinal nonheme iron levels and mRNA levels of genes implicated in iron metabolism were measured in mice with altered iron metabolism [chronic (4 wk) and acute (4 days) dietary iron deficiency; iron overload and hypoxia] to investigate their role in the process and regulation of intestinal iron absorption. Mucosal nonheme iron levels were decreased by both chronic and acute iron deficiency and increased by iron overload but were not affected by hypoxia. There was evidence of a gradient of mucosal nonheme iron along the small intestine (duodenum, jejunum > ileum). There were also regional differences in H-ferritin (duodenum > ileum) and transferrin receptor (ileum > duodenum) mRNA levels. Iron overload produced a decrease in transferrin receptor (TfR) mRNA in the duodenum, with ferritin mRNA levels unaffected in both the duodenum and ileum. Chronic iron deficiency induced a twofold increase in TfR mRNA levels in both the duodenum and ileum, whereas H- and L-ferritin mRNA levels did not change significantly. The ratio of H- to L-ferritin mRNA decreased significantly during exposure to hypoxia; however, individual ferritin and TfR mRNA levels were not significantly altered. Calreticulin (mobilferrin), cysteine-rich intestinal protein, and H(+)-adenosinetriphosphatase mRNA levels were virtually unchanged in all models. A comparison with previously published data on changes in iron absorption leads us to conclude that 1) iron absorption can be altered independently of effects on transcripts of genes for iron-related proteins, and 2) it is not essential for iron absorption to be coordinated with regulation of mucosal iron metabolism.
Products of enteropathogenic E. coli inhibit lymphokine production by gastrointestinal lymphocytes
Authors: Klapproth J. M. , Donnenberg M. S. , Abraham J. M. , James S. P.
Published: December 2017

Previously we have shown that lysates of enteropathogenic Escherichia coli (EPEC) inhibit lymphokine production by mitogen-activated peripheral blood mononuclear cells (PBMCs). The aim of the present study was to determine whether products of EPEC alter lymphokine expression by gastrointestinal mucosal lymphocytes. Lysates from EPEC clones inhibited mitogen-stimulated interleukin-2 (IL-2), IL-4, IL-5, and interferon-gamma (IFN-gamma) but not IL-8 mRNA expression by lamina propria mononuclear cells isolated from surgically resected colon specimens. Inhibitory lysates did not significantly change CD25 expression on either CD4, CD8, or CD45R0 lymphocytes by flow cytometry. Bacterial supernatants of EPEC inhibited IL-2 and IL-5 protein secretion by mitogen-stimulated PBMCs. EPEC lysates inhibited IL-2 mRNA expression induced by lysates of nonpathogenic E. coli. In conclusion, EPEC contains a novel gene(s) that encodes factors that selectively inhibit IL-2, IL-4, IL-5, and IFN-gamma expression by mucosal mononuclear cells without affecting CD25 or IL-8 expression. Thus enteric bacteria can produce factors that may regulate the function of the gastrointestinal mucosal immune system.
Tachykinin-induced increase in gastric mucosal resistance: role of primary afferent neurons, CGRP, and NO
Authors: Stroff T. , Plate S. , Ebrahim J. S. , Ehrlich K. H. , Respondek M. , Peskar B. M.
Published: December 2017

The tachykinins [Ala5,beta-Ala8]neurokinin A-(4-10) -[Ala5,beta-Ala8]NKA-(4-10) inverted question mark and NKA-(4-10) dose dependently protected against ethanol-induced gastric mucosal damage in rats (half-maximal inhibitory dose, 46 and 48 nmol/kg, respectively). These effects were abolished by primary afferent nerve denervation, calcitonin gene-related peptide (CGRP) immunoneutralization, the CGRP receptor antagonist human (h) hCGRP-(8-37), and inhibition of nitric oxide (NO) biosynthesis by NG-nitro-L-arginine methyl ester. Tachykinin-induced protection occurred despite marked depression of gastric mucosal blood flow and was not associated with increased acid secretion. NK2-receptor blockade antagonized the protective effects of [Ala5,beta-Ala8]NKA-(4-10) and NKA-(4-10), whereas NK1-receptor blockade was ineffective. Blockade of NK2 but not NK1 receptors prevented by 65% the protection evoked by topical capsaicin without affecting capsaicin-induced hyperemia. We conclude that the increase in gastric mucosal resistance evoked by tachykinins is NK2 receptor-mediated and involves primary afferent neurons, CGRP, and NO. Gastric mucosal hyperemia and increased acid secretion do not participate in the effect. Tachykinins activating NK2 receptors contribute to the increase in gastric mucosal resistance but not the increment in mucosal blood flow after primary afferent nerve stimulation by capsaicin.
CCK, carbachol, and bombesin activate distinct PLC-beta isoenzymes via Gq/11 in rat pancreatic acinar membranes
Authors: Piiper A. , Stryjek-Kaminska D. , Klengel R. , Zeuzem S.
Published: December 2017

Four different isoforms of phospholipase C-beta (PLC-beta 1-4) have been discovered, raising the important question of whether a distinct receptor activates a single PLC-beta isoenzyme or a subset of PLC-beta isoenzymes. The present study was designed to investigate activation of PLC-beta isoenzymes by three different PLC-activating agonists that bind to different receptor entities, i.e., cholecystokinin octapeptide (CCK-8), bombesin, and carbachol in rat pancreatic acinar membranes. PLC activity was measured using exogenous [3H]phosphatidylinositol 4,5-bisphosphate as substrate. Western blot analysis of pancreatic acinar membranes revealed the presence of PLC-beta 1, -beta 3, -gamma 1, and -delta 1, but not of PLC-beta 2, -beta 4, -gamma 2, and -delta 2. Preincubation of the membranes with anti-PLC-beta 1 or -beta 3 antibody reduced agonist-induced activation of PLC. The order of sensitivity toward inhibition by anti-PLC-beta 1 antibody was CCK-8 > bombesin > carbachol. An opposite order of sensitivity was found for inhibition of PLC activity by anti-PLC-beta 3 antibody (carbachol > bombesin > CCK-8). Anti-PLC-beta 2, -beta 4, -gamma 1, -gamma 2, -delta 1, and -delta 2 antibodies had no effect. Preincubation of the membranes with an antibody raised against the COOH terminus of the alpha-subunit of Gq/11 proteins inhibited PLC activity in response to all three different receptor agonists to a similar extent, whereas anti-Gi alpha 1-2 and anti-Gi alpha 3 antibodies had no effect. In conclusion, the data of the present study indicate that CCK-8 and carbachol activate PLC-beta 1 and PLC-beta 3, respectively, whereas bombesin activates both PLC-beta 1 and PLC-beta 3. Activation of PLC-beta by these receptor agonists is mediated by Gq/11.
Intestinal anaphylaxis induces Fos immunoreactivity in myenteric plexus of rat small intestine
Authors: Miampamba M. , Tan D. T. , Oliver M. R. , Sharkey K. A. , Scott R. B.
Published: December 2017

Fos immunohistochemistry was used to identify myenteric neurons activated as a consequence of intestinal anaphylaxis in Hooded-Lister rats sensitized to egg albumin (EA 10 micrograms ip). After incubation in test solutions, or after in vivo challenge, jejunal tissues were processed for immunohistochemistry with an anti-Fos antibody (1:500, TF161). The neuronal identity of the Fos-labeled nuclei was confirmed by double labeling with neuron-specific enclose (1:1,000). In in vitro studies, exposure of control tissue to 50 mM K(+)-Krebs-EA (2 x 10(-5) M) solutions significantly increased Fos immunoreactivity in the myenteric plexus, whereas a basal level of Fos was seen in control tissue incubated in Krebs solution, sham-sensitized tissue exposed to bovine serum albumin (BSA, 2 x 10(-5) M), or EA and sensitized tissue exposed to BSA. Pretreatment of sensitized tissue with doxantrazole (10(-4) M) markedly reduced Fos immunoreactivity observed after EA exposure. In in vivo studies, there was negligible Fos immunoreactivity in the myenteric plexus of control, sham-sensitized, or sensitized rats challenged with saline. A low level of Fos was seen in neurons of sham-sensitized rats challenged with BSA or EA and in sensitized rats challenged with BSA. Significantly greater levels of Fos were observed in the myenteric plexus of sensitized animals challenged with EA, even after pretreatment with capsaicin (125 mg/kg). These results suggest a role for myenteric neurons in intestinal anaphylaxis. In sensitized rats, activation of myenteric neurons is dependent on antigen-induced mast cell activation and occurs independently of capsaicin-sensitive afferent nerves.
Regulation of motilin release: studies with ex vivo perfused canine jejunum
Authors: Poitras P. , Trudel L. , Miller P. , Gu C. M.
Published: December 2017

The regulatory process of motilin release was studied in segments of canine jejunum isolated and perfused ex vivo. The secretion of motilin in the effluent venous system of the isolated intestine was measured by radioimmunoassay in response to various pharmacological agents injected intra-arterially. Muscarinie agonist and antagonist, respectively, increased and decreased the release of motilin. The stimulatory effect of carbachol was still documented after tetrodotoxin (10(-5) M) was injected in the system to block neural influence on M cells. Bombesin and morphine also increased the release of motilin. The effect of bombesin was still documented in the presence of atropine or tetrodotoxin, but the stimulatory morphine effect was blocked by atropine. Both phenylephrine and octreotide decreased the release of motilin stimulated by carbachol in a jejunal segment pretreated and denervated with tetrodotoxin. Therefore, a revised model for the regulation of motilin release from M cells of intestinal mucosa can now be proposed. Cholinergic and bombesin receptors are present on M cells to encode a stimulatory signal, whereas adrenergic and somatostatin receptors are responsible for inhibitory transmission. The stimulatory effect of morphine is mediated via a muscarinic transmitter.
Purinergic Ca2+ signaling in myenteric neurons via P2 purinoceptors
Authors: Christofi F. L. , Guan Z. , Wood J. D. , Baidan L. V. , Stokes B. T.
Published: December 2017

Fura 2 microfluorimetry was used to test the hypothesis that ATP acts at P1 and P2 purinoceptors to elevate cytosolic free Ca2+ concentrations [Ca2+]i) in calbindin-immunoreactive cultured myenteric neurons from adult guinea pig small intestine. Local "micro-puff" application of ATP or ATP(gamma)S caused an increase in [Ca2+]i in 99% of 200 multipolar neurons. The potency profile of agonists for the rise in [Ca2+]i was ATP(gamma)S = ATP >> ADP >> AMP, adenosine, 5'-(N-ethylcarboxamido)adenosine, and 2-chloro-N(6)-cyclopentyladenosine. Tetrodotoxin-sensitive synaptic transmission could contribute as much as 25% to the ATP response. The P1 antagonist 8-cyclopentyl-1,3-dipropylxanthine blocked 50% of the peakATP Ca2+ response. P2 antagonists blocked the ATP response: pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid > reactive blue 2 > suramin. Suramin enhanced the ATP response in 27.5% of neurons. Some neurons (<15%) displayed distinct multiphasic Ca2+ signatures. About 54% of ATP-responsive neurons expressed calbindin. The data support the following hypotheses: 1) two distinct P2 purinoceptors are linked to the rise in [Ca2+]i in myenteric neurons; 2) purinergic Ca2+ signaling is not restricted to one neuronal phenotype; and 3) intraneuronal Ca2+ is not involved in adenosinergic hyperpolarization in AH/type 2 neurons.
Intestinal absorption of sodium dodecyl sulfate in the rodent: evidence for paracellular absorption
Authors: Amelsberg A. , Schteingart C. D. , Stein J. , Simmonds W. J. , Sawada G. A. , Ho N. F. , Hofmann A. F.
Published: December 2017

Experiments were performed to define the mechanism of intestinal absorption of dodecyl sulfate (DS), an amphipathic organic anion whose chemical structure resembles that of dodecanoate, a C12 fatty acid anion. With jejunal segments perfused in single-pass fashion in the anesthetized rat, steady-state absorption of DS was concentration dependent, with the apparent permeability constant (P(app)) ranging from 4 to 22 x 10(-5) cm/s. When DS concentration was held constant and net water absorption was induced by decreasing perfusate osmolality, DS absorption increased in direct proportion to water absorption, suggesting absorption by solvent drag via the paracellular route. However, DS absorption continued even when water secretion was induced by a hypertonic perfusate. Consequently, for all experiments, DS absorption could be empirically described as the sum of two terms: 1) absorption in the absence of water absorption (P(app) = 5.6 x 10(-5) cm/s) and 2) absorption induced by water movement [(delta P(app)/delta water absorption) = 0.2 x 10(-5) cm x s(-1) x microl segment(-1) x min(-1)]. In a polarized epithelial monolayer of renal epithelial cells (Madin-Darby canine kidney cells), DS was absorbed predominantly by a paracellular pathway, as the absorption rate increased threefold when paracellular junction pore size was increased by the addition of cytochalasin D. The calculated apparent radius was 2.9 A, indicating that the cross section of the molecule, not its length, determined the rate of absorption. It is concluded that absorption of DS in the intact animal occurs slowly and mostly via the paracellular route, because the fixed negative charge on the molecule retards rapid passive entry into the enterocyte, as occurs with protonated fatty acids. That absorption of DS persisted despite net water secretion suggests a low level of transcellular absorption across the jejunal enterocyte also occurs.
Characterization of synthesis and storage of TGF-alpha in rat parotid acinar and intercalated duct cells
Authors: Login G. R. , Yang J. , Bryan K. P. , Digenis E. C. , McBride J. , Elovic A. , Quissell D. O. , Dvorak A. M. , Wong D. T.
Published: December 2017

Although the expression and biological role of transforming growth factor-alpha (TGF-alpha) have been explored in a variety of normal cells in mammalian species, little is known about the storage of TGF-alpha in secretory cells of exocrine organs. Parotid glands from four rats were homogenized for RNA isolation followed by reverse transcription-polymerase chain reaction to determine the presence of TGF-alpha message. In situ hybridization using a hamster-specific TGF-alpha riboprobe was done on paraffin sections. Parotid gland and isolated acinar cells were processed for transmission electron microscopy (TEM) and postembedding immunogold labeled for TGF-alpha. Gold particles were counted on approximately 200 granules in 10 acinar cells and in 10 intercalated duct cells. Labeling density was calculated as the number of gold particles per square micrometer +/- SD. Statistical significance was calculated using one-way analysis of variance. Using multiple technologies, we have established that rat parotid acinar and intercalated duct cells synthesize TGF-alpha and store the precursor form of this cytokine in their secretory granules.
Glycolysis is a source of pyruvate for transamination of glutamine amino nitrogen in jejunal epithelial cells
Authors: Cremin J. D. , Fleming S. E.
Published: December 2017

Previous research has shown that glucose increases transamination of glutamine amino nitrogen with pyruvate. It is unclear whether glucose or glutamine provides the pyruvate used for transamination. In the current study, it was hypothesized that glucose provides pyruvate for transamination of glutamine amino nitrogen. This hypothesis was tested by tracing the metabolism of [2-(13)C]glucose in these cells incubated in the presence of [2-(13)C] glucose or [2-(13)C] glucose and glutamine using (13)C nuclear magnetic resonance. Glutamine supplementation increased alanine production but did not affect lactate production. The 1-(13)C, 2-(13)C, 3-(13)C, 1,2-(13)C, and 2,3-(13)C isotopomers of alanine and lactate were produced when glutamine was supplemented. Glutamine supplementation increased production of 2-(13)C, 1,2-(13)C, and 2,3-(13)C isotopomers of alanine but did not affect the production of isotopomers of lactate. The ratio of production of [2-(13)C]alanine to [3-(13)C]alanine was 37:1 when glutamine was present. The predominance of production of [2-(13)C]alanine vs. all other isotopomers demonstrates that a large proportion of the pyruvate used for transamination of glutamine amino nitrogen was derived from glycolysis.
Diminished retinoic acid signaling in hepatic stellate cells in cholestatic liver fibrosis
Authors: Ohata M. , Lin M. , Satre M. , Tsukamoto H.
Published: December 2017

Hepatic stellate cells (HSC) participate in liver fibrogenesis via myofibroblastic activation, the extent of which appears to correlate with the loss of cellular vitamin A. The present study has tested a hypothesis that HSC activation is associated with diminished retinoic acid (RA) signaling. Pure HSC were isolated from rats with cholestatic liver fibrosis induced by bile duct ligation (BDL) and sham-operated animals (Sham). Northern blot analysis of HSC RNA from BDL confirmed fibrogenic activation of the cells with enhanced mRNA levels for procollagen-alpha1(I) and transforming growth factor-beta1 (TGF-beta1). Competitive polymerase chain reaction analysis showed selective reductions in the mRNA levels of RA receptor (RAR)-beta and retinoid X receptor (RXR)-alpha to 20 and 17% of the Sham HSC. The mRNA level for cellular retinol binding protein I, a gene with RA responsive element (RARE), was also suppressed by 78% in BDL. The concentrations of all-trans-RA and 9-cis-RA were decreased in HSC from BDL. Nuclear extracts of these cells showed diminished binding activity to the RARE, whereas activity of AP-1, a transcription factor known to be antagonized by RAR and RXR, was enhanced. These results demonstrate diminished RA signaling in HSC from cholestatic liver fibrosis, which appeared to have resulted from RA deficiency and suppressed expression of RAR-beta and RXR-alpha. Furthermore, the reciprocal enhancement of AP-1 activity and coordinately increased expression of an AP-1 responsive gene, TGF-beta1, suggest a permissive role of the diminished RA signaling in promoting AP-1 activity and TGF-beta1 expression.
Effect of chronic inflammation on electrolyte transport in rabbit ileal villus and crypt cells
Authors: Sundaram U. , West A. B.
Published: December 2017

The effect of chronic inflammation on electrolyte transport in rabbit ileal villus and crypt cells was determined with the use of a rabbit model of chronic ileitis. In both cells, Na+/H+ exchange was monitored by following recovery from an acid load, and Cl-/HCO3- exchange was monitored by following recovery from an alkaline load. In villus cells, recovery from an acid load was not affected; however, recovery from an alkaline load was slowed. These data suggest that chronic inflammation inhibits Cl-/HCO3- exchange in villus cells. In contrast, in crypt cells, recovery from an alkaline load was unaffected, whereas recovery from an acid load was accelerated. These data suggest that chronic inflammation stimulates Na+/H+ exchange in crypt cells. Inhibition of Cl-/HCO3- exchange in villus cells would be expected to inhibit coupled NaCl absorption, which occurs by the coupling of brush-border membrane (BBM) Na+/H+ and Cl-/HCO3- exchange. Stimulation of Na+/H+ exchange in crypt cells, known to be present only on the basolateral membrane, alkalinizes the cell. This alkalinization may stimulate BBM Cl-/HCO3- exchange, resulting in HCO3- secretion. Thus these unique alterations in transporter activity suggest that different endogenous immune-inflammatory mediators may have differing effects on specific transporters in villus and crypt cells in the chronically inflamed ileum.
Insights into human colonic physiology obtained from the study of flatus composition
Authors: Suarez F. , Furne J. , Springfield J. , Levitt M.
Published: December 2017

To better understand the physiology of colonic gas production, each flatus passage of 16 subjects over a 4-h period was analyzed by gas chromatography for N2, O2, H2, CO2, CH4, and for odoriferous sulfur-containing gases. Appreciable intraindividual and enormous interindividual variability was observed, indicating that each gas passage reflected the interaction of highly variable liberation and/or removal mechanisms. The predominant flatus gas was CO2, H2, and N2 in seven, six, and three subjects, respectively. Gases produced intraluminally (H2, CO2, and CH4) comprised approximately 74% of flatus, and rapid CO2 and H2 productions were responsible for high passage rates. A positive correlation between flatus H2 and CO2 suggested that CO2, like H2, mainly was a bacterial product. Whereas methanogens and H2S-producing bacteria usually are mutually exclusive in feces, CH4 and H2S did not negatively correlate, indicating coexistence of both organisms in the colon. We conclude that analysis of flatus composition provides a novel means of assessing colonic physiology, particularly ongoing bacterial metabolism throughout the unperturbed colon.
Characterization of sphingosine 1-phosphate-induced actions and its signaling pathways in rat hepatocytes
Authors: Im D. S. , Fujioka T. , Katada T. , Kondo Y. , Ui M. , Okajima F.
Published: December 2017

Sphingosine 1-phosphate (S-1-P) and lysophosphatidic acid (LPA) stimulated glycogen phosphorylase, a rate-limiting enzyme responsible for glycogenolysis, in association with Ca2+ mobilization and phospholipase C (PLC) activation in rat hepatocytes. S-1-P, but not LPA, also inhibited adenosine 3',5'-cyclic monophosphate accumulation reflecting adenylyl cyclase inhibition. S-1-P-induced PLC activation, Ca2+ mobilization, and phosphorylase activation were markedly enhanced by primary culture of the cells for 24 h, whereas the inhibitory adenosine 3',5'-cyclic monophosphate response was unchanged by increasing culture time. Activation of the PLC-Ca2+ system during primary culture was specific to the lysosphingolipid; PLC and Ca2+ responses to LPA and NaF were unchanged or slightly attenuated by increasing culture time. Pertussis toxin treatment almost completely suppressed the S-1-P-induced inhibition of adenylyl cyclase but hardly influenced the lipid-induced activation of PLC and its cascade reactions. We conclude that S-1-P, through an LPA receptor-independent mechanism, stimulates two signaling pathways, i.e., activation of the PLC-Ca2+ system and inhibition of adenylyl cyclase, through distinct S-1-P receptor-transducer systems, resulting in the modulation of glycogenolysis in rat hepatocytes.
Hindbrain effects of PACAP on gastric motor function in the rat
Authors: Krowicki Z. K. , Arimura A. , Nathan N. A. , Hornby P. J.
Published: December 2017

Pituitary adenylate cyclase-activating polypeptide (PACAP)-like immunoreactive cell bodies and fibers are visualized in hindbrain nuclei that are involved in the regulation of autonomic function, yet little is known about the gastric and cardiovascular effects of this peptide in the dorsal vagal complex, nucleus raphe obscurus, and nucleus ambiguus. Therefore, multiple-barreled micropipettes were used to inject PACAP-38 (1-100 pmol) into each of these nuclei in alpha-chloralose anesthetized rats, while intragastric pressure, pyloric and greater curvature smooth muscle contractile activity, blood pressure, and heart rate were recorded. For comparison, the effect of L-glutamate (15 nmol) microinjected into the same sites on gastric motor activity was also assessed. L-Glutamate microinjected into each nucleus before PACAP-38 significantly increased intragastric pressure, both in terms of the peak increase and the total area of the response. Microinjections of PACAP-38 (10 and 100 pmol) into each of the nuclei significantly increased peak intragastric pressure, but the total area of the response was only significantly increased by the highest dose (100 pmol) in the case of the dorsal vagal complex and nucleus raphe obscurus. No consistent changes in heart rate and mean arterial blood pressure were noted after microinjection of PACAP-38 into each of the three nuclei. Bilateral vagotomy abolished the increase in intragastric pressure in response to microinjection of PACAP-38 into the dorsal vagal complex and nucleus raphe obscurus. We conclude that PACAP-38 in the dorsal vagal complex and nucleus raphe obscurus is involved in vagally mediated gastric motor excitation.
Demonstration and characterization of motilin-binding sites in the rabbit cerebellum
Authors: Depoortere I. , Peeters T. L.
Published: December 2017

This is the first report on central motilin receptors. Autoradiography on cerebellar slices revealed specific motilin-binding sites in the molecular layer of the cortex. Scatchard analysis of cold saturation studies showed the existence of a high-(pKd,hi = 9.07 +/- 0.09, where pKd is the negative logarithm of the dissociation constant) and a low-affinity binding site (pKd,lo = 6.56 +/- 0.09). Similar affinities were found with rabbit motilin and with the porcine (po) antagonist [Phe3, Leu13]po-motilin. Feline and canine motilin had a markedly lower affinity for the low-affinity site (pKd,lo = 5.29 and 4.58, respectively); chicken motilin had a lower affinity for both sites (pKd,hi = 8.36, pKd,lo = 3.97). Erythromycin A and its derivative N-trimethyl erythromycin A cnol ether also bound to cerebellar motilin receptors (pKd,hi = 7.29 and 8.91, respectively). Structure-activity studies with motilin fragments and the potency ranking of agonists suggest that a novel subtype receptor of motilin may exist in the brain. Guanosine 5'-O-(3-thiotriphosphate) (0.1 mM) reduced the number and the affinity for the high-affinity binding sites, which is evidence for G protein-coupled receptors. Our findings open new perspectives for the study of the physiological role of motilin.
Molecular aspects of hepatobiliary transport
Authors: Muller M. , Jansen P. L.
Published: December 2017

Generation of bile flow is a regulated, ATP-dependent process and depends on the coordinated action of a number of transporter proteins in the sinusoidal and canalicular domains of the hepatocyte. Dysfunction of any of these proteins leads to retention of substrates, with conjugated hyperbilirubinemia or cholestasis as a result. In recent years many of the transport proteins involved in bile formation have been identified, cloned, and functionally characterized. The hepatocyte sinusoidal membrane contains transport proteins for the hepatic uptake of organic anions and cations and for the uptake of bile acids. The multispecific organic anion transporting polypeptide (OATP) mediates the hepatic uptake of organic anions and a variety of organic amphiphilic compounds, including organic cations. The organic cation transporter OCT1 more specifically transports small organic cations. NTCP is the Na(+)-bile acid cotransporting protein that mediates the hepatic uptake of bile acids. The canalicular transport proteins are able to transport endogenous and exogenous metabolites into the bile against steep concentration gradients. Most of these transporters are members of the large ATP-binding cassette (ABC) superfamily, and their transport function directly depends on the hydrolysis of Mg2+/ATP. At least five ABC transporter proteins have been characterized so far: 1) the human multidrug resistance protein MDR1 mediates the excretion of hydrophobic, mostly cationic, metabolites; 2) MDR3 is involved in phosphatidylcholine secretion; 3) the canalicular bile acid transporter cBAT mediates secretion of monovalent bile salts and provides the molecular basis of bile acid-dependent bile flow; 4) SPGP, product of the P-glycoprotein sister gene, is exclusively expressed in the liver but its function is currently unknown; and 5) the human multidrug resistance protein MRP2 mediates the excretion of multivalent anionic conjugates.
Characterization of PACAP receptors and signaling pathways in rabbit gastric muscle cells
Authors: Murthy K. S. , Jin J. G. , Grider J. R. , Makhlouf G. M.
Published: December 2017

Pituitary adenylate cyclase-activating peptide (PACAP) receptors and their signaling pathways were characterized in dispersed rabbit gastric muscle cells. 125I-PACAP-27 and 125I-vasoactive intestinal peptide (VIP) binding to muscle cells were inhibited equally by PACAP and VIP (mean inhibitory concentration 0.8 to 1.3 nM) and desensitized to the same extent (70-80%) by exposure to either peptide. PACAP, like VIP, increased cytosolic free Ca2+ and the formation of L-[3H]citrulline, NO-3/NO-2, guanosine 3',5'-cyclic monophosphate (cGMP), and adenosine 3'5'-cyclic monophosphate (cAMP) and induced relaxation (mean effective concentration 1.8 +/- 0.1 nM) that was partly inhibited by NG-nitro-L-arginine (L-NNA), VIP-(10-28), and PACAP 6-38. L-[3H]citrulline and cGMP formation were blocked by nifedipine, L-NNA, and pertussis toxin (PTx), implying activation of a G protein-coupled, Ca(2+)-calmodulin-dependent nitric oxide (NO) synthase. PACAP-induced relaxation was inhibited to the same extent (46-49%) by nifedipine, L-NNA, PTx, and the protein kinase G inhibitor KT-5823; the inhibition reflected the component of relaxation mediated by the NO-cGMP pathway. The residual relaxation was abolished by the protein kinase A inhibitor H-89. The pattern of inhibition of all responses was identical to that observed with VIP. Desensitization with VIP or PACAP abolished cAMP formation but had no effect on L-[3H]citrulline and cGMP formation induced by either peptide. Receptor protection with VIP or PACAP preserved fully all responses (L-[3H]citrulline, cGMP, and cAMP formation and relaxation) to either peptide. The complete cross-competition, cross-desensitization, cross-antagonism, and cross-protection of receptors by either VIP or PACAP are consistent with interaction of both peptides with the same receptors; the receptors consist of two classes, each coupled to a distinct signaling pathway.
Carrier-mediated transport of conjugated bile acids across the basolateral membrane of biliary epithelial cells
Authors: Benedetti A. , Di Sario A. , Marucci L. , Svegliati-Baroni G. , Schteingart C. D. , Ton-Nu H. T. , Hofmann A. F.
Published: December 2017

When secreted into bile, unconjugated dihydroxy bile acids are absorbed passively by cholangiocytes according to the cholehepatic circulation hypothesis. A fraction of these are likely to be conjugated during transcellular transport. Experiments were performed using fluorescent conjugated bile acids to test whether carrier-mediated transport of conjugated bile acids is present in the basolateral domains of polarized cholangiocytes of intrahepatic bile ductules isolated from rat liver. The time course of the cellular localization of cholyl-NBDAB-Gly and chenodeoxycholyl-NBDAB-Gly, which are anionic fluorescent derivatives of the corresponding glycine-conjugated bile acids, was characterized using an image-analysis system. With 0.3-3 microM solutions, fluorescence was present at 1 and 3 min in the basolateral area of cholangiocytes. Staining in the apical region occurred later, with a peak after 15 min of incubation. The basolateral uptake of the two fluorescent bile acids was temperature dependent and Na+ independent, and was not influenced by the addition of amiloride, by lowering of the medium pH to 6.0, or by preincubation with valinomycin. Uptake was partially inhibited by the absence of Cl- or HCO3- in the perfusate, by preincubation with 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), and by the presence of different organic anions or unconjugated and conjugated bile acids in the medium. When cells were preloaded with an ethyl ester of chenodeoxycholyl-NBDAB-Gly, which is hydrolyzed by intracellular esterases, the decrease of cell fluorescence was partly inhibited by H2DIDS, whereas it was stimulated by the presence of 20 microM cholyltaurine in the medium. It is concluded that transport of conjugated bile acid anions across the basolateral membrane of the polarized rat cholangiocyte is carrier mediated. The conjugated bile acid transporter is likely to be an anion exchanger and is likely to be involved in bile secretion whenever conjugated bile acids or other organic anions are transported from the base of the biliary ductular epithelial cells into the plasma of the periductular capillary plexus.
Dual effects of PACAP on guinea pig gallbladder muscle via PACAP-preferring and VIP/PACAP-preferring receptors
Authors: Parkman H. P. , Pagano A. P. , Ryan J. P.
Published: December 2017

The aims of this study were to determine the effect and mechanism of action of pituitary adenylate cyclase-activating peptide (PACAP) on gallbladder muscle. Guinea pig gallbladder muscle strips were studied isometrically. In noncontracted muscle strips, PACAP-27 and PACAP-38 caused dose-dependent contractions, whereas vasoactive intestinal peptide (VIP) caused dose-dependent relaxation. PACAP-27 contractions were resistant to tetrodotoxin, atropine, and the substance P receptor antagonist [D-Arg1,D-Trp7,9,Leu11]substance P (Spantide) but were inhibited by the selective PACAP receptor antagonist PACAP-(6-38) and slightly increased with the VIP receptor antagonist [4-chloro-D-Phe6,Leu17]VIP. In cholecystokinin-precontracted muscle strips, both VIP and PACAP caused relaxations. This relaxant effect of PACAP-27 was inhibited by PACAP-(6-38) and [4-chloro-D-Phe6,Leu17]VIP, but not by tetrodotoxin. These studies suggest that PACAP has dual excitatory and inhibitory effects on guinea pig gallbladder muscle. The contractile effect of PACAP is a direct action on muscle through PACAP-preferring receptors. The relaxant effect of PACAP is seen in precontracted muscle strips and mediated through VIP/ PACAP-preferring receptors.
Transcriptional regulation of human inducible nitric oxide synthase gene in an intestinal epithelial cell line
Authors: Linn S. C. , Morelli P. J. , Edry I. , Cottongim S. E. , Szabo C. , Salzman A. L.
Published: December 2017

The inducible form of nitric oxide synthase (iNOS) is expressed during inflammation of the intestine and may contribute to tissue injury. We have examined iNOS transcriptional regulation in DLD-1 cells, a human intestinal epithelial line that produces large amounts of nitric oxide and iNOS mRNA in response to a combination of the proinflammatory cytokines interleukin-1 beta (IL-1 beta) and interferon-gamma (IFN-gamma). Levels of iNOS mRNA are extremely low in unstimulated DLD-1 cells but increase dramatically after cytokine treatment. Nuclear run-on analyses demonstrated that transcriptional activation, which accounts for a portion of this increase, is dependent on both IL-1 beta and IFN-gamma and requires de novo protein synthesis. Transfection of DLD-1 cells with reporter constructs containing deletions of the iNOS promoter showed that sequences located between 8.7 and 10.7 kb upstream of the transcription initiation site are necessary for cytokine responsiveness. This region contains potential binding sites for several cytokine-induced transcription factors and was shown to function in either orientation when placed upstream of a basal iNOS promoter segment terminating at-1.1 kb. The extremely distal location of the cytokine-responsive region contrasts with the reported positions of elements involved in the regulation of iNOS transcription in other cell types. Our data also suggest that posttranscriptional events could play a significant role in regulating iNOS gene expression in human intestinal epithelia.
Hepatocytes in the bile duct-ligated rat express Bcl-2
Authors: Kurosawa H. , Que F. G. , Roberts L. R. , Fesmier P. J. , Gores G. J.
Published: December 2017

Hepatocytes do not express Bcl-2, a repressor of apoptosis. In contrast, cholangiocytes, which are in direct contact with bile, do express Bcl-2. Because cholestasis results in the retention of bile within hepatocytes, we reasoned cholestasis may induce hepatocellular expression of Bcl-2. Thus our aim was to determine whether hepatocytes express Bcl-2 or alter expression of other Bcl-2 family members in cholestasis using the bile duct-ligated (BDL) rat as a model of cholestasis. De novo Bcl-2 expression was observed in hepatocytes of BDL rats assessed by reverse transcriptase-polymerase chain reaction and immunoblot analysis. Immunohistochemistry demonstrated that Bcl-2 expression in hepatocytes was greater in periportal hepatocytes than pericentral hepatocytes. Expression of Bcl-x (an antiapoptotic Bcl-2 family protein) was not altered by bile duct ligation, whereas expression of Bax (a proapoptotic Bcl-2 family protein) increased slightly as determined by Northern and Western blot analyses. Bcl-2-positive hepatocytes isolated from BDL rats were resistant to induction of apoptosis by 50 microM glycochenodeoxycholate. Our results demonstrate, for the first time, expression of Bcl-2 by hepatocytes during cholestasis. We suggest that hepatocellular expression of Bcl-2 during cholestasis is an adaptive phenomenon to resist apoptosis by toxic bile salts.
Pancreatic spasmolytic polypeptide protects the gastric mucosa but does not inhibit acid secretion or motility
Authors: McKenzie C. , Marchbank T. , Playford R. J. , Otto W. , Thim L. , Parsons M. E.
Published: December 2017

The objectives of these studies were to examine whether the trefoil peptide porcine pancreatic spasmolytic polypeptide (PSP) had gastric mucosal protectant properties similar to its human equivalent human spasmolytic polypeptide (hSP) and to confirm the antisecretory and antimotility action of the peptide. PSP and recombinant hSP reduced gastric mucosal damage caused by a combination of subcutaneous indomethacin and restraint stress in the conscious rat. At a dose of 500 micrograms/kg bolus plus 500 micrograms.kg-1.h-1 sc, PSP significantly reduced the total area of damage by 58%. PSP at a dose of 150 micrograms/kg iv had no inhibitory effect on pentagastrin-stimulated gastric acid secretion in the perfused stomachs of anesthetized rats. This lack of antisecretory activity was confirmed in vitro using an isolated stomach preparation from the immature rat. PSP and hSP at concentrations up to 800 nM did not inhibit electrically or chemically evoked contractions of the guinea pig ileum and duodenum in vitro. Thus antisecretory and antimotility actions do not underlie the mucosal protectant properties of PSP. PSP did, however, stimulate cell migration, and this may, at least in part, account for its protectant properties.
Bile acids stimulate proliferative and secretory events in large but not small cholangiocytes
Authors: Alpini G. , Glaser S. , Robertson W. , Phinizy J. L. , Rodgers R. E. , Caligiuri A. , LeSage G.
Published: December 2017

Accumulation of bile acids (BA) and cholangiocyte proliferation occur in cholestasis, but BA effects on the proliferative and secretory capacity of cholangiocytes are undefined. Cholangiocyte proliferation coupled with increased expression of H3 histone and secretin receptor (SR) genes and secretin-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) levels is limited to large cholangiocytes. We isolated pooled small and large cholangiocytes and studied the effect of taurocholic (TC) and taurolithocholic (TLC) acids on proliferation, by measurement of H3 histone gene expression, and secretion, by measurement of SR gene expression, cAMP levels, and Cl-/HCO3- exchanger activity. In pooled cholangiocytes, TC and TLC increased H3 histone (12-fold) and SR (3-fold) gene expression and both spontaneous (1.4-fold) and secretin-induced (4-fold) cAMP response. TC and TLC increased H3 histone (10-fold) and SR (2-fold) gene expression and secretin-induced cAMP response and Cl-/HCO3- exchanger activity (3-fold) only in large cholangiocytes. In large cholangiocytes, BA may have a signaling function in the modulation of ductal secretion.