bluuebunny/crossref_metadata_2025 huggingface dataset:
Parietal cells express high levels of Na-K-2Cl cotransporter on migrating into the gastric gland neck
Authors: McDaniel Nichole , Lytle Christian
Published: December 2017

Na-K-2Cl cotransport and Cl/[Formula: see text] exchange are prominent mechanisms for Cl−uptake in Cl−-secreting epithelial cells. We used immunofluorescence microscopy to delineate the distributions of Na-K-2Cl cotransporter-1 (NKCC1) and anion exchanger-2 (AE2) proteins in rat gastric mucosa (zymogenic zone). Parietal cells (PCs) above the neck of the gastric gland contained abundant AE2 but little or no NKCC1, whereas those in the neck and base contained high NKCC1 but diminished AE2. Lower levels of NKCC1 were detected in surface mucous cells and in cells comprising the blind ends of all glands. Pulse labeling of proliferating cells with bromodeoxyuridine indicated that new PCs originate in the isthmus with scant NKCC1; the subset of PCs that migrate downward expresses NKCC1 abruptly on entering the neck, within 7 days of cell division. Our results suggest that downwardly migrating PCs replace one mechanism for Cl−entry (Cl/[Formula: see text] exchange) with another (Na-K-2Cl cotransport).
Acute carbon tetrachloride feeding induces damage of large but not small cholangiocytes from BDL rat liver
Authors: LeSage Gene D. , Glaser Shannon S. , Marucci Luca , Benedetti Antonio , Phinizy Jo Lynne , Rodgers Rebecca , Caligiuri Alessandra , Papa Emanuela , Tretjak Ziga , Jezequel Anne-Marie , Holcomb Leigh A. , Alpini Gianfranco
Published: December 2017

Bile duct damage and/or loss is limited to a range of duct sizes in cholangiopathies. We tested the hypothesis that CCl4damages only large ducts. CCl4or mineral oil was given to bile duct-ligated (BDL) rats, and 1, 2, and 7 days later small and large cholangiocytes were purified and evaluated for apoptosis, proliferation, and secretion. In situ, we measured apoptosis by morphometric and TUNEL analysis and the number of small and large ducts by morphometry. Two days after CCl4administration, we found an increased number of small ducts and reduced number of large ducts. In vitro apoptosis was observed only in large cholangiocytes, and this was accompanied by loss of proliferation and secretion in large cholangiocytes and loss of choleretic effect of secretin. Small cholangiocytes de novo express the secretin receptor gene and secretin-induced cAMP response. Consistent with damage of large ducts, we detected cytochrome P-4502E1 (which CCl4converts to its radicals) only in large cholangiocytes. CCl4induces selective apoptosis of large ducts associated with loss of large cholangiocyte proliferation and secretion.
Ca-sensitive Na transport in sheep omasum
Authors: Schultheiss Gerhard , Martens Holger
Published: December 2017

Na transport across a preparation of sheep omasum was studied. All tissues exhibited a serosa-positive short-circuit current ( Isc), with a range of 1–4 μeq ⋅ h−1⋅ cm−2. A Michaelis-Menten-type kinetic was found between the Na concentration and the Isc(Michaelis-Menten constant for transport of Na = 6.7 mM; maximal transport capacity of Na = 4.16 μeq ⋅ h−1⋅ cm−2). Mucosal amiloride (1 mM), phenamil (1 or 10 μ), or serosal aldosterone (1 μM for 6 h) did not change Isc. Removal of divalent cations (Ca and Mg) enhanced Iscconsiderably from 2.61 ± 0.24 to a peak value of 11.18 ± 1.1 μeq ⋅ h−1⋅ cm−2. The peak Isc(overshoot) immediately declined to a plateau Iscof ∼6–7 μeq ⋅ h−1⋅ cm−2. Na flux measurements showed a close correlation between changes in Iscand Na transport. Transepithelial studies demonstrated that K, Cs, Rb, and Li are transported, indicating putative nonselective cation channels, which are inhibited by divalent cations (including Ca, Mg, Sr, Ba) and by (trivalent) La. Intracellular microelectrode recordings from the luminal side clearly showed changes of voltage divider ratio when mucosal divalent cations were removed. The obtained data support the assumption of a distinct electrogenic Na transport mechanism in sheep omasum.
Saturable stimulation of fatty acid transport through model cytoplasm by soluble binding protein
Authors: Weisiger Richard A.
Published: December 2017

To better define the role of soluble binding proteins in the cytoplasmic transport of amphipathic molecules, we measured the diffusional mobility of a fluorescent long-chain fatty acid, 12- N-methyl-(7-nitrobenz-2-oxa-1,3-diazol)aminostearate (NBD-stearate), through model cytoplasm as a function of soluble binding protein concentration. Diffusional mobilities were correlated with the partition of the fatty acid between membrane and protein binding sites. Cytoplasm was modeled as a dense suspension of liposomes, and albumin was used as a model binding protein. Albumin saturably increased NBD-stearate mobility through the membrane suspension approximately eightfold. Fatty acid mobility in the absence of albumin was identical to the mobility of the membrane vesicles (1.99 ± 0.33 × 10−8cm2/s), whereas the mobility at saturating concentrations was identical to the mobility of albumin (1.65 ± 0.12 × 10−7cm2/s). The protein concentration producing half-maximal stimulation of NBD-stearate diffusion (42.8 ± 0.3 μM) was unexpectedly greater than that required to solubilize half of the NBD-stearate (17.9 ± 3.0 μM). These results support a proposed mechanism for cytoplasmic transport of small amphipathic molecules in which aqueous diffusion of the protein-bound form of the molecule largely determines the transport rate. However, slow interchange of fatty acid between the binding protein and membranes also appears to influence the transport rate in this model system.
A novel nitric oxide scavenger decreases liver injury and improves survival after hemorrhagic shock
Authors: Menezes John , Hierholzer Christian , Watkins Simon C. , Lyons Valerie , Peitzman Andrew B. , Billiar Timothy R. , Tweardy David J. , Harbrecht Brian G.
Published: December 2017

We tested the ability of a nitric oxide (NO) scavenger to reduce tissue injury in a rodent model of hemorrhagic shock. Rats were hemorrhaged to a mean arterial blood pressure (MAP) of 40 mmHg and then resuscitated when either 30% of their shed blood had been returned ( group 1) or after 100 min of continuous shock ( group 2). Selected animals were treated with the NO scavenger NOX (30 mg ⋅ kg−1 ⋅ h−1) infused over 4 h. Hemorrhaged rats had a lower MAP after resuscitation compared with sham-shock control rats. NOX treatment significantly increased MAP after resuscitation from hemorrhage. Hemorrhagic shock also increased liver injury as reflected by elevated ornithine carbamoyltransferase (OCT) plasma levels, and NOX treatment significantly reduced OCT release. In addition, NOX was associated with significantly decreased hepatic neutrophil infiltration and improved 24-h survival ( n = 8 of 9) compared with saline-treated shock animals ( n = 3 of 9). These data suggest that excess NO mediates shock-induced tissue injury and that suppression of NO availability with NO scavengers may reduce the pathophysiological sequelae of severe hemorrhage.
Mechanism of action of cholera toxin on the opossum internal anal sphincter smooth muscle
Authors: Fan Ya-Ping , Chakder Sushanta , Rattan Satish
Published: December 2017

Cholera toxin (CTX), an activator of Gsprotein, is an important pharmacological tool in G protein research. The effect and the mechanism of action of CTX in the gastrointestinal smooth muscle, including the internal anal sphincter (IAS), are not known. The present investigation was carried out to examine the effects of CTX on the signal transduction associated with the adenylate cyclase (AC) pathway on the basal tone of the IAS smooth muscle. CTX caused a prompt and dose-dependent fall in the basal tone of the IAS that was not affected by the neurotoxins TTX and ω-conotoxin or the nitric oxide synthase inhibitor NG-nitro-l-arginine. The cyclooxygenase inhibitor indomethacin, cAMP-dependent protein kinase inhibitor Rp-8-bromoadenosine 3′,5′ cyclic monophosphorothioate inhibited CTX-induced IAS smooth muscle relaxation. Furthermore, CTX caused a concentration-dependent relaxation of the isolated smooth muscle cells (SMC) of the IAS, which was blocked by Gsα antibody (Gsα-Ab). The IAS smooth muscle relaxation was accompanied with an increase in the GTPase activity that was also specifically blocked by Gsα-Ab. We conclude that a major part of the inhibitory action of CTX in the IAS is via the direct response of the SMC that is linked with Gsprotein to the AC pathway. A part of the inhibitory action of CTX on the smooth muscle occurs via the activation of cyclooxygenase pathway. The relative contribution of such actions of CTX in the smooth muscle in the gastrointestinal motility disturbances following cholera infection remains to be determined.
Autoimmune gastritis results in disruption of gastric epithelial cell development
Authors: Judd Louise M. , Gleeson Paul A. , Toh Ban-Hock , van Driel Ian R.
Published: December 2017

We have investigated the underlying basis of the lesion in murine autoimmune gastritis, a model of the human disease pernicious anemia. The disease is mediated by T lymphocytes and characterized by selective depletion of parietal and zymogenic cells from the gastric unit (gland) together with gastric epithelial cell hyperplasia. The gastric units of gastritic stomachs contained 2.3-fold more cells than normal and accumulated rapidly dividing, short-lived gastric epithelial stem cells and mucous neck cells. Most of these immature cells failed to differentiate into end-stage cells but rather appeared to die by apoptosis. We also found no correlation between anti-parietal cell autoantibody titers and the degree of gastric pathology, providing further evidence that autoantibodies do not play a direct role in the pathogenesis of gastritis. Taken together, the normal developmental pathways of the gastric mucosa are disrupted in autoimmune gastritis, resulting in an amplification of immature cell types. The differentiation of these immature cells appears to be blocked, contributing to depletion of end-stage cells. This scenario provides an explanation for depletion of not only parietal cells but also zymogenic cells even though they are not directly targeted by the immune system.
Effects of LPS on transport of indocyanine green and alanine uptake in perfused rat liver
Authors: Lund Mette , Kang Leslie , Tygstrup Niels , Wolkoff Allan W. , Ott Peter
Published: December 2017

Lipopolysaccharide (LPS) initiates cholestasis. Whether this process is mediated by tumor necrosis factor-α (TNF-α) and whether the cholestatic response to LPS is associated with intrahepatic accumulation of possibly toxic substances are under debate. To study these questions the hepatic uptake and biliary excretion of indocyanine green (ICG) was examined in the isolated perfused rat liver 18 h after intravenous treatment of rats with either saline, 1 mg/kg body wt LPS, or LPS and intraperitoneal pentoxifylline (POF) ( n = 6 in each group). POF inhibits TNF-α release after LPS administration. LPS induced a typical acute-phase response with increased mRNA for acute-phase proteins, reduced albumin mRNA, and increased hepatic uptake of alanine. Intrinsic hepatic clearance of ICG in controls (1.01 ± 0.05 ml ⋅ min−1⋅ g liver−1) was similarly decreased by LPS alone (0.62 ± 0.04 ml ⋅ min−1⋅ g−1; P = 0.002 vs. control) or combined with POF (0.66 ± 0.06 ml ⋅ min−1⋅ g−1). A kinetic analysis indicated that LPS reduced both uptake and excretion processes in a balanced manner, so that intrahepatic ICG content was unaffected or even slightly reduced, as confirmed by measurement of ICG contents in the perfused livers. In livers from parallel-treated nonperfused rats, mRNA for the organic anion transporting protein-1 (Oatp1, which is likely to mediate ICG uptake) was unaffected by LPS, whereas the concentration of Oatp1 protein was reduced. Thus LPS induced an acute-phase response that included downregulation of ICG uptake by reduction of Oatp1 protein concentration, possibly at a posttranscriptional level. TNF-α appears not to be the mediator because POF did not modify these LPS effects.
Myogenic mechanism for peristalsis in the cat esophagus
Authors: Preiksaitis Harold G. , Diamant Nicholas E.
Published: December 2017

A myogenic control system (MCS) is a fundamental determinant of peristalsis in the stomach, small bowel, and colon. In the esophagus, attention has focused on neuronal control, the potential for a MCS receiving less attention. The myogenic properties of the cat esophagus were studied in vitro with and without nerves blocked by 1 μM TTX. Muscle contraction was recorded, while electrical activity was monitored by suction electrodes. Spontaneous, nonperistaltic, electrical, and mechanical activity was seen in the longitudinal muscle and persisted after TTX. Spontaneous circular muscle activity was minimal, and peristalsis was not observed without pharmacological activation. Direct electrical stimulation (ES) in the presence of bethanechol or tetraethylammonium chloride (TEA) produced slow-wave oscillations and spike potentials accompanying smooth muscle contraction that progressed along the esophagus. Increased concentrations of either drug in the presence of TTX produced slow waves and spike discharges, accompanied by peristalsis in 5 of 8 TEA- and 2 of 11 bethanechol-stimulated preparations without ES. Depolarization of the muscle by increasing K+ concentration also produced slow waves but no peristalsis. We conclude that the MCS in the esophagus requires specific activation and is manifest by slow-wave oscillations of the membrane potential, which appear to be necessary, but are not sufficient for myogenic peristalsis. In vivo, additional control mechanisms are likely supplied by nerves.
Expression of AE2 anion exchanger in mouse intestine
Authors: Alper Seth L. , Rossmann Heidi , Wilhelm Sabine , Stuart-Tilley Alan K. , Shmukler Boris E. , Seidler Ursula
Published: December 2017

We have characterized expression of anion exchanger 2 (AE2) mRNA and protein in the mouse intestine. AE2 mRNA abundance was higher in colon than in more proximal segments. AE2a mRNA was more abundant than AE2b mRNA throughout the intestine, and AE2c mRNA was expressed at very low levels. This AE2 mRNA pattern contrasted with that in mouse stomach, in which AE2c > AE2b > AE2a. AE2 polypeptide abundance as detected by immunoblot qualitatively paralleled that of mRNA, whereas AE2 immunostaining exhibited a more continuous decrease in intensity from colon to duodenum. AE2 polypeptide was more abundant in colonic surface cells than in crypts, whereas ileal crypts and villi exhibited similar AE2 abundance. AE2 was also observed in mural and vascular smooth muscle. Localization of AE2 epitopes was restricted to the basolateral membranes of epithelial cells throughout the intestine with three exceptions. Under mild fixation conditions, anti-AE2 amino acids (aa) 109–122 detected nonpolarized immunostaining of ileal enterocytes and of Paneth cell granule membranes. An epitope detected by anti-AE2 aa 1224–1237 was also localized to subapical regions of Brunner’s gland ducts of duodenum and upper jejunum. These localization studies will aid in the interpretation of anion exchanger function measured in epithelial sheets, isolated cells, and membrane vesicles.
Intestinal adaptation and enterocyte apoptosis following small bowel resection is p53 independent
Authors: Shin Cathy E. , Falcone Richard A. , Kemp Christopher J. , Erwin Christopher R. , Litvak David A. , Evers B. Mark , Warner Brad W.
Published: December 2017

Adaptation following small bowel resection (SBR) signals enterocyte proliferation and apoptosis. Because p53-induced p21waf1/cip1may be important for apoptosis in many cells, we hypothesized that these genes are required for increased enterocyte apoptosis during adaptation. Male C57BL/6 (wild-type) or p53-null mice underwent 50% proximal SBR or sham operation (bowel transection-reanastomosis). Adaptation (DNA-protein content, villus height-crypt depth, enterocyte proliferation), appearance of apoptotic bodies, and p53 and p21waf1/cip1protein expression were measured in the ileum after 5 days. Adaptation was equivalent after SBR in both wild-type and p53-null mice as monitored by significantly increased ileal DNA-protein content, villus height, and enterocyte proliferation. The number of crypt apoptotic bodies increased significantly after SBR evenly in both wild-type and p53-null mice. In the p53-null mice, SBR substantially induced the expression of p21waf1/cip1protein in villus enterocytes. The p53-independent induction of p21waf1/cip1may account for the similar intestinal response to SBR between wild-type and p53-null mice. Intestinal adaptation and increased enterocyte apoptosis following intestinal resection occur via a p53-independent mechanism.
Somatostatin peptides inhibit basolateral potassium channels in human colonic crypts
Authors: Sandle Geoffrey I. , Warhurst Geoffrey , Butterfield Ian , Higgs Norman B. , Lomax Richard B.
Published: December 2017

Somatostatin is a powerful inhibitor of intestinal Cl−secretion. We used patch-clamp recording techniques to investigate the effects of somatostatin on low-conductance (23-pS) K+channels in the basolateral membrane of human colonic crypts, which are an important component of the Cl−secretory process. Somatostatin (2 μM) elicited a >80% decrease in “spontaneous” K+channel activity in cell-attached patches in nonstimulated crypts (50% inhibition =∼8 min), which was voltage-independent and was prevented by pretreating crypts for 18 h with pertussis toxin (200 ng/ml), implicating a G protein-dependent mechanism. In crypts stimulated with 100–200 μM dibutyryl cAMP, 2 μM somatostatin and its synthetic analog octreotide (2 μM) both produced similar degrees of K+channel inhibition to that seen in nonstimulated crypts, which was also present under low-Cl−(5 mM) conditions. In addition, 2 μM somatostatin abolished the increase in K+channel activity stimulated by 2 μM thapsigargin but had no effect on the thapsigargin-stimulated rise in intracellular Ca2+. These results indicate that somatostatin peptides inhibit 23-pS basolateral K+channels in human colonic crypt cells via a G protein-dependent mechanism, which may result in loss of the channel's inherent Ca2+sensitivity.
Nerve-mediated motility of ileal segments isolated from NK1 receptor knockout mice
Authors: Saban R. , Nguyen N.-B. , Saban M. R. , Gerard N. P. , Pasricha P. J.
Published: December 2017

Tachykinins such as substance P (SP) and neurokinin A (NKA) acting on neurokinin (NK) receptors modulate the nonadrenergic noncholinergic (NANC) neurotransmission in the gastrointestinal tract of several species, but the information about the mouse small intestine is scanty. Both SP and NKA induced concentration-dependent contractions of ileal segments isolated from wild-type mice that were blocked by NK1 and NK2 antagonists, respectively. In contrast, segments isolated from NK1receptor (NK1-R) knockout mice responded only to elevated concentrations of SP. To reveal the inhibitory NANC (iNANC) responses, tissues were pretreated with atropine and guanethidine. Under these conditions, a tetrodotoxin-sensitive relaxation in response to electrical field stimulation (EFS) was observed. NK1-R knockout mice presented a trend toward an increase in iNANC responses, whereas the NK1-R antagonist significantly potentiated iNANC relaxation in tissues isolated from wild-type mice. N G-nitro-l-arginine methyl ester (100 μM) transformed the relaxant response to EFS into a tetrodotoxin-sensitive, frequency-dependent contraction characteristic of an excitatory NANC (eNANC) system. A NK1-R antagonist abolished the contractile responses of the mouse ileum to EFS, whereas a NK2 receptor antagonist had a trend toward reducing EFS-induced contraction. The eNANC component was absent in NK1-R knockout mice. Measurement of SP-like immunoreactivity indicated similar amounts of SP per gram of tissue isolated from wild-type and NK1-R knockout mice, indicating that the observed differences in response to EFS were not due to a differential peptide content. It is concluded that, in the mouse ileum, both NK1 and NK2 receptors modulated the responses to exogenous tachykinins, whereas NK1 is the primary tachykinin receptor involved in both iNANC and eNANC transmission.
Evidence for Gd3+inhibition of membrane ATP permeability and purinergic signaling
Authors: Roman Richard M. , Feranchak Andrew P. , Davison Amy K. , Schwiebert Erik M. , Fitz J. Gregory
Published: December 2017

Extracellular ATP functions as an important autocrine and paracrine signal that modulates a broad range of cell and organ functions through activation of purinergic receptors in the plasma membrane. Because little is known of the cellular mechanisms involved in ATP release, the purpose of these studies was to evaluate the potential role of the lanthanide Gd3+as an inhibitor of ATP permeability and to assess the physiological implications of impaired purinergic signaling in liver cells. In rat hepatocytes and HTC hepatoma cells, increases in cell volume stimulate ATP release, and the localized increase in extracellular ATP increases membrane Cl−permeability and stimulates cell volume recovery through activation of P2receptors. In cells in culture, spontaneous ATP release, as measured by a luciferin-luciferase-based assay, was always detectable under control conditions, and extracellular ATP concentrations increased 2- to 14-fold after increases in cell volume. Gd3+(200 μM) inhibited volume-sensitive ATP release by >90% ( P < 0.001), inhibited cell volume recovery from swelling ( P < 0.01), and uncoupled cell volume from increases in membrane Cl−permeability ( P < 0.01). Moreover, Gd3+had similar inhibitory effects on ATP release from other liver and epithelial cell models. Together, these findings support an important physiological role for constitutive release of ATP as a signal coordinating cell volume and membrane ion permeability and suggest that Gd3+might prove to be an effective inhibitor of ATP-permeable channels once they are identified.
Role of neuropeptide-sensitive L-type Ca2+channels in histamine release in gastric enterochromaffin-like cells
Authors: Zeng Ningxin , Athmann Christoph , Kang Tao , Walsh John H. , Sachs George
Published: December 2017

Peptides release histamine from enterochromaffin-like (ECL) cells because of elevation of intracellular Ca2+concentration ([Ca2+]i) by either receptor-operated or voltage-dependent Ca2+channels (VDCC). To determine whether VDCCs contribute to histamine release stimulated by gastrin or pituitary adenylate cyclase-activating polypeptide (PACAP), the presence of VDCCs and their possible modulation by peptides was investigated in a 48-h cultured rat gastric cell population containing 85% ECL cells. Video imaging of fura 2-loaded cells was used to measure [Ca2+]i, and histamine was assayed by RIA. Cells were depolarized by increasing extracellular K+concentrations or by 20 mM tetraethylammonium (TEA+). Cell depolarization increased transient and steady-state [Ca2+]iand resulted in histamine release, dependent on extracellular Ca2+. These K+- or TEA+-dependent effects on histamine release from ECL cells were coupled to activation of parietal cells in intact rabbit gastric glands, and L-type channel blockade by 2 μM nifedipine inhibited 50% of [Ca2+]ielevation and histamine release. N-type channel blockade by 1 μM ω-conotoxin GVIA inhibited 25% of [Ca2+]ielevation and 14% of histamine release. Inhibition was additive. The effects of 20 mM TEA+were fully inhibited by 2 μM nifedipine. Both classes of Ca2+channels were found in ECL cells, but not in parietal cells, by RT-PCR. Nifedipine reduced PACAP-induced (but not gastrin-stimulated) Ca2+entry and histamine release by 40%. Somatostatin, peptide YY (PYY), and galanin dose dependently inhibited L-type Ca2+channels via a pertussis toxin-sensitive pathway. L-type VDCCs play a role in PACAP but not gastrin stimulation of histamine release from ECL cells, and the channel opening is inhibited by somatostatin, PYY, and galanin by interaction with a Gior Goprotein.
NOX, a novel nitric oxide scavenger, reduces bacterial translocation in rats after endotoxin challenge
Authors: Dickinson Eva , Tuncer Recep , Nadler Evan , Boyle Patricia , Alber Sean , Watkins Simon , Ford Henri
Published: December 2017

Endotoxemia promotes gut barrier failure and bacterial translocation (BT) by upregulating inducible nitric oxide synthase (iNOS) in the gut. We hypothesized that administration of a dithiocarbamate derivative, NOX, which scavenges nitric oxide (NO), may reduce intestinal injury and BT after lipopolysaccharide (LPS) challenge. Sprague-Dawley rats were randomized to receive NOX or normal saline via subcutaneously placed osmotic pumps before or after LPS challenge. Mesenteric lymph nodes, liver, spleen, and blood were cultured 24 h later. Transmucosal passage of Escherichia coli C-25or fluorescent beads were measured in an Ussing chamber. Intestinal membranes were examined morphologically for apoptosis, iNOS expression, and nitrotyrosine immunoreactivity. NOX significantly reduced the incidence of bacteremia, BT, and transmucosal passage of bacteria and beads when administered before or up to 12 h after LPS challenge. LPS induced enterocyte apoptosis at the villus tips where bacterial entry was demonstrated by confocal microscopy. NOX significantly decreased the number of apoptotic nuclei and nitrotyrosine residues. NOX prevents LPS-induced gut barrier failure by scavenging NO and its toxic derivative, peroxynitrite.
Immunolocalization of anion exchanger AE2 and Na+- HCO 3 − cotransporter in rat parotid and submandibular glands
Authors: Roussa Eleni , Romero Michael F. , Schmitt Bernhard M. , Boron Walter F. , Alper Seth L. , Thévenod Frank
Published: December 2017

Salivary glands secrete K+and[Formula: see text] and reabsorb Na+and Cl−, but the identity of transporters involved in[Formula: see text] transport remains unclear. We investigated localization of Cl−/[Formula: see text] exchanger isoform AE2 and of Na+-[Formula: see text] cotransporter (NBC) in rat parotid gland (PAR) and submandibular gland (SMG) by immunoblot and immunocytochemical techniques. Immunoblotting of PAR and SMG plasma membranes with specific antibodies against mouse kidney AE2 and rat kidney NBC revealed protein bands at ∼160 and 180 kDa for AE2 and ∼130 kDa for NBC, as expected for the AE2 full-length protein and consistent with the apparent molecular mass of NBC in several tissues other than kidney. Immunostaining of fixed PAR and SMG tissue sections revealed specific basolateral staining of PAR acinar cells for AE2 and NBC, but in SMG acinar cells only basolateral AE2 labeling was observed. No AE2 expression was detected in any ducts. Striated, intralobular, and main duct cells of both glands showed NBC expression predominantly at basolateral membranes, with some cells being apically stained. In SMG duct cells, NBC staining exhibited a gradient of distribution from basolateral localization in more proximal parts of the ductal tree to apical localization toward distal parts of the ductal tree. Both immunoblotting signals and immunostaining were abolished in preabsorption experiments with the respective antigens. Thus the mechanisms of fluid and anion secretion in salivary acinar cells may be different between PAR and SMG, and, because NBC was detected in acinar and duct cells, it may play a more important role in transport of [Formula: see text] by rat salivary duct cells than previously believed.
A novel human organic anion transporting polypeptide localized to the basolateral hepatocyte membrane
Authors: König Jörg , Cui Yunhai , Nies Anne T. , Keppler Dietrich
Published: December 2017

We cloned and expressed a new organic anion transporting polypeptide (OATP), termed human OATP2, (OATP-C, LST-1; symbol SLC21A6), involved in the uptake of various lipophilic anions into human liver. The cDNA encoding OATP2 comprised 2073 base pairs, corresponding to a protein of 691 amino acids, which were 44% identical to the known human OATP. An antibody directed against the carboxy terminus localized OATP2 to the basolateral membrane of human hepatocytes. Northern blot analysis indicated a strong expression of OATP2 only in human liver. Transport mediated by recombinant OATP2 and its localization were studied in stably transfected Madin-Darby canine kidney strain II (MDCKII) and HEK293 cells. Confocal microscopy localized recombinant OATP2 protein to the lateral membrane of MDCKII cells. Substrates included 17β-glucuronosyl estradiol, monoglucuronosyl bilirubin, dehydroepiandrosterone sulfate, and cholyltaurine. 17β-Glucuronosyl estradiol was a preferred substrate, with a Michaelis-Menten constant value of 8.2 μM; its uptake was Na+ independent and was inhibited by sulfobromophthalein, with a inhibition constant value of 44 nM. Our results indicate that OATP2 is important for the uptake of organic anions, including bilirubin conjugates and sulfobromophthalein, in human liver.
Modulation by colonic fermentation of LES function in humans
Authors: Piche Thierry , Zerbib Frank , Varannes Stanislas Bruley Des , Cherbut Christine , Anini Younès , Roze Claude , le Quellec Alain , Galmiche Jean-Paul
Published: December 2017

Colonic fermentation of carbohydrate has been shown to influence gastric and intestinal motility. Our aim was to investigate the effects of colonic infusion of lactose and short-chain fatty acids (SCFAs) on lower esophageal sphincter (LES) function in humans. LES pressure (LESP), transient relaxations of LES (TLESRs), and esophageal pH were monitored over 6 h on 4 different days in 7 healthy volunteers. After 1 h of baseline recording, the effects of different colonic infusions (270 ml of isotonic or hypertonic saline, 30 g lactose, or 135 mmol SCFAs) were tested in fasting conditions and after a standard meal. Peptide YY (PYY) and oxyntomodulin (OLI) were also measured in plasma. Both lactose and SCFA infusions increased the number of TLESRs as well as the proportion of TLESRs associated with acid reflux episodes, but saline solutions did not. The postprandial fall of LESP was enhanced by previous SCFA infusion. Plasma PYY and OLI increased similarly after all colonic infusions. Colonic fermentation of lactose markedly affected LES function, and this effect was reproduced by SCFA infusion. Whether the mechanisms of this feedback phenomenon are of hormonal nature, neural nature, or both remains to be determined.
III. Visceral afferent pathways: a source of new therapeutic targets for abdominal pain
Authors: Buéno Lionel , Fioramonti Jean , Garcia-Villar Rafael
Published: December 2017

Visceral pain is the major cause of consulting in gastroenterology and the principal symptom of functional bowel disorders. This symptom is often associated with gut hypersensitivity to distension. The use of animal models has recently permitted the identification of some mediators supposed to play a pivotal role in the genesis of visceral hypersensitivity. Serotonin, through different receptor subtypes, as well as kinins and calcitonin gene-related peptide, are known to be involved, but other putative transmitters arise and are new potential targets for the development of efficacious treatments. This themes article addresses both physiological and preclinical issues of interest for the selection of active new drugs in regard to the clinical pharmacology of visceral pain.
Role of caspases and NF-κB signaling in hydrogen peroxide- and superoxide-induced hepatocyte apoptosis
Authors: Jones Brett E. , Lo Chau R. , Liu Hailing , Pradhan Zehra , Garcia Lydia , Srinivasan Anu , Valentino Karen L. , Czaja Mark J.
Published: December 2017

Reactive oxygen intermediates (ROI) have been implicated as mediators of hepatocyte death resulting from a variety of forms of liver injury. To delineate the mechanisms that underlie ROI-induced apoptosis, the roles of caspase activation and nuclear factor-κB (NF-κB) signaling were determined in the rat hepatocyte cell line RALA255-10G after treatment with H2O2or the superoxide generator menadione. By 8 h, H2O2and menadione caused 26% and 33% cell death, respectively. Death from both ROI occurred by apoptosis as indicated by morphology under fluorescence microscopy, the induction of caspase activation and DNA fragmentation, and the cleavage of poly(ADP-ribose) polymerase. Despite the presence of caspase activation in both forms of apoptosis, caspase inhibition blocked H2O2- but not menadione-induced apoptosis. In contrast, inhibition of NF-κB activation decreased cell death from both ROI. Different ROI, therefore, induce distinct apoptotic pathways in RALA hepatocytes that are both caspase dependent and independent. In contrast to the known protective effect of NF-κB activation in tumor necrosis factor-α-induced hepatocyte apoptosis, NF-κB promotes hepatocellular death from ROI in these cells.
Induction of transcriptional activity of AP-1 and NF-κB in the gastric mucosa during aging
Authors: Xiao Zhi-Qiang , Majumdar Adhip P. N.
Published: December 2017

Although aging enhances expression and tyrosine kinase activity of epidermal growth factor receptor (EGFR) in the gastric mucosa, there is no information about EGFR signaling cascades. We examined the age-related changes in mitogen-activated protein kinases (MAPKs) [extracellular signal-related kinases (ERKs), c-Jun NH2-terminal kinases (JNKs), and p38], an EGFR-induced signaling cascade, and activator protein-1 (AP-1) and nuclear factor-κB (NF-κB) transcriptional activity in the gastric mucosa of 4- to 6-, 12- to 14-, and 22- to 24-mo-old Fischer 344 rats. AP-1 and NF-κB transcriptional activity in the gastric mucosa rose steadily with advancing age. This can be further induced by transforming growth factor-α. The age-related activation of AP-1 and NF-κB in the gastric mucosa was associated with increased levels of c-Jun, c-Fos, and p52, but not p50 or p65. Total and phosphorylated IκBα levels in the gastric mucosa were unaffected by aging. Aging was also associated with marked activation of ERKs (p42/p44) and JNK1. In contrast, aging decreased p38 MAPK activity in the gastric mucosa. Our observation of increased activation of ERKs and JNK1 in the gastric mucosa of aged rats suggests a role for these MAPKs in regulating AP-1 and NF-κB transcriptional activity. These events may be responsible for the age-related rise in gastric mucosal proliferative activity.
Salivary epidermal growth factor and intestinal adaptation in male and female mice
Authors: Stern Lawrence E. , Falcone Richard A. , Kemp Christopher J. , Braun Margaret C. , Erwin Christopher R. , Warner Brad W.
Published: December 2017

Salivary epidermal growth factor (sEGF) levels are increased in male mice after small bowel resection (SBR) and may be important during intestinal adaptation. Since males have greater sEGF than females, the influence of sex on postresection adaptation was tested. Females had lower sEGF; however, sEGF substantially increased in both sexes after a massive (50%) SBR. Adaptive increases in DNA and protein content, villus height, and crypt depth, as well as crypt cell proliferation rates in the remnant ileum, were not different between males and females. Although significant postresection increases in sEGF were identified, EGF mRNA and protein did not change within the submandibular gland. Glandular kallikrein-13 and ileal EGF receptor expression were greater after SBR in female mice. Intestinal adaptation is equivalent in female and male mice after SBR. Despite lower sEGF, females demonstrated increased expression of a kallikrein responsible for sEGF precursor cleavage as well as amplified ileal EGF receptor expression. These results endorse an important differential response between sexes regarding sEGF mobilization and intestinal receptor availability during adaptation.
Heterogeneity of detergent-insoluble membranes from human intestine containing caveolin-1 and ganglioside GM1
Authors: Badizadegan Kamran , Dickinson Bonny L. , Wheeler Heidi E. , Blumberg Richard S. , Holmes Randall K. , Lencer Wayne I.
Published: December 2017

In intestinal epithelia, cholera and related toxins elicit a cAMP-dependent chloride secretory response fundamental to the pathogenesis of toxigenic diarrhea. We recently proposed that specificity of cholera toxin (CT) action in model intestinal epithelia may depend on the toxin's cell surface receptor ganglioside GM1. Binding GM1enabled the toxin to elicit a response, but forcing the toxin to enter the cell by binding the closely related ganglioside GD1arendered the toxin inactive. The specificity of ganglioside function correlated with the ability of GM1to partition CT into detergent-insoluble glycosphingolipid-rich membranes (DIGs). To test the biological plausibility of these hypotheses, we examined native human intestinal epithelia. We show that human small intestinal epithelia contain DIGs that distinguish between toxin bound to GM1and GD1a, thus providing a possible mechanism for enterotoxicity associated with CT. We find direct evidence for the presence of caveolin-1 in DIGs from human intestinal epithelia but find that these membranes are heterogeneous and that caveolin-1 is not a structural component of apical membrane DIGs that contain CT.
The endothelin system in normal human colon
Authors: Egidy Giorgia , Juillerat-Jeanneret Lucienne , Korth Petra , Bosman Fred T. , Pinet Florence
Published: December 2017

Endothelin (ET)-1 is a potent vasoconstrictor and mitogenic peptide that has a variety of biological effects in noncardiovascular tissues. The precise cellular distribution of the ET-1 system in the wall of the normal human colon was studied to identify the physiological role of ET in the gut. In situ hybridization revealed ET-converting enzyme-1 (ECE-1) mRNA in all vessels, the colon epithelium, and macrophages. Prepro-ET-1 (PPET-1) mRNA had a similar distribution except for a scattered signal in mucosal microvessels. ETAand ETBreceptor mRNAs were mainly in the lamina propria, pericryptal myofibroblasts, microvessels, and mononuclear cells, with ETAmRNA more abundant than ETBmRNA.125I-ET-1 binding showed ETBalong the crypts and in nerve fibers descending from the ganglionic plexus that contained PPET-1, ECE-1, and ETBtranscripts, whereas glia contained ETAreceptors. The finding of the entire ET system in the normal mucosa suggests its implication in some characteristic functions of the colon and its secretion as both a neuroactive and a vasoactive peptide.
Disruption of primary and secondary esophageal peristalsis by afferent stimulation
Authors: Bardan Eytan , Xie Pengyan , Aslam Muhammad , Kern Mark , Shaker Reza
Published: December 2017

Recent studies have shown that afferent signals originating from the pharynx inhibit progression of primary esophageal peristalsis. Our aim was to further elucidate the effect of esophageal and pharyngeal afferent stimulation on primary and secondary esophageal peristalsis. We studied the effect of esophageal air distension and pharyngeal water stimulation on progression of primary and secondary peristalsis in nine healthy volunteers aged 27 ± 2 yr (4 men, 5 women). At a threshold volume, rapid injection of water into the pharynx, directed posteriorly, resulted in complete halt of the progressing secondary and primary esophageal peristalses in both the proximal and distal esophagus. The threshold volume of injected water for inducing inhibition was similar for secondary (0.6 ± 0.2 ml) and primary (0.5 ± 0.1 ml) esophageal peristalsis. Progression of primary peristalsis induced by a dry swallow and secondary peristalsis induced by intraesophageal air distension were completely inhibited by intraesophageal injection of 15 ± 2 ml of air in 70% and 75% of the trials, respectively. We conclude that afferent signals induced by esophageal air distension and pharyngeal water stimulation inhibit propagation of both primary and secondary esophageal peristalsis, suggesting a shared neural control mechanism for these types of peristalsis.
EAAT1 is involved in transport ofl-glutamate during differentiation of the Caco-2 cell line
Authors: Mordrelle Agnès , Jullian Eric , Costa Cyrille , Cormet-Boyaka Estelle , Benamouzig Robert , Tomé Daniel , Huneau Jean-François
Published: December 2017

Little is known concerning the expression of amino acid transporters during intestinal epithelial cell differentiation. The transport mechanism ofl-glutamate and its regulation during the differentiation process were investigated using the human intestinal Caco-2 cell line. Kinetic studies demonstrated the presence of a single, high-affinity,d-aspartate-sensitive l-glutamate transport system in both confluent and fully differentiated Caco-2 cells. This transport was clearly Na+dependent, with a Hill coefficient of 2.9 ± 0.3, suggesting a 3 Na+-to-1 glutamate stoichiometry and corresponding to the well-characterized XA,G−system. The excitatory amino acid transporter (EAAT)1 transcript was consistently expressed in the Caco-2 cell line, whereas the epithelial and neuronal EAAT3 transporter was barely detected. In contrast with systems B0and y+, which have previously been reported to be downregulated when Caco-2 cells stop proliferating, l-glutamate transport capacity was found to increase steadily between day 8 and day 17. This increase was correlated with the level of EAAT1 mRNA, which might reflect an increase in EAAT1 gene transcription and/or stabilization of the EAAT1 transcript.
Vagal inhibition in the antral region of guinea pig stomach
Authors: Dickens Emma J. , Edwards F. R. , Hirst G. D. S.
Published: December 2017

The effects of vagal stimulation in the presence of a muscarinic antagonist were examined on three distinct rhythmically active cells located in guinea pig antrum. Vagal stimulation inhibited contractions of the circular muscle layer but did not change their rate of occurrence. With the use of intracellular recording techniques, these stimuli were found to initiate inhibitory junction potentials in the circular layer but produced smaller potential changes in driving and follower cells. Inhibition of the circular muscle layer involved two separate components. The dominant component was independent of changes in membrane potential and was abolished by nitro-l-arginine. After abolishing Ca2+ entry into smooth muscle cells with a Ca2+ antagonist, vagal stimulation continued to inhibit the residual contractions associated with each slow wave. When the cyclic changes in intracellular Ca2+ concentration associated with each slow wave were measured, they were found to be unchanged by vagal stimulation. The observations suggest that vagal inhibition of stomach movements does not alter pacemaker activity in the stomach; rather, it results from a change in the sensitivity of smooth muscle contractile proteins to Ca2+.
Error analysis of classic colonic transit time estimates
Authors: Bouchoucha Michel , Thomas S. Randall
Published: December 2017

Estimates of colonic transit times (CTT) through the three colonic segments, right colon, left colon, and rectosigmoid, are commonly based on radiopaque markers. For a given segment, CTT is usually calculated from just the number of markers visible in that segment on abdominal X-rays. This procedure is only strictly valid for the theoretical, but unrealistic, case of continuous marker ingestion (i.e., not for a single or once-daily ingestion). CTT was analyzed using the usual estimate of the mean CTT of one marker and also using a new, more realistic estimate based on the kinetic coefficients of a three-compartment colonic model. We directly compared our compartmental approach to classic CTT estimates by double-marker studies in six patients. We also retrospectively studied CTT in 148 healthy control subjects (83 males, 65 females) and 1,309 subjects with functional bowel disorders (irritable bowel syndrome or constipation). Compared with the compartmental estimates, the classic approach systematically underestimates CTT in both populations, i.e., in patients and in healthy control subjects. The relative error could easily reach 100% independent of the site of colonic transit delay. The normal values of total CTT are then 44.3 ± 29.3 instead of 30.1 ± 23.6 h for males and 68.2 ± 54.4 instead of 47.1 ± 28.2 h for females.
Cerulein upregulates ICAM-1 in pancreatic acinar cells, which mediates neutrophil adhesion to these cells
Authors: Zaninovic Vjekoslav , Gukovskaya Anna S. , Gukovsky Ilya , Mouria Michelle , Pandol Stephen J.
Published: December 2017

Neutrophil infiltration into the pancreas is a key event in pancreatitis. Here we show that intercellular adhesion molecule-1 (ICAM-1), which regulates neutrophil adhesion, is present on rat pancreatic acinar cells, is upregulated by a hormone (cerulein) and mediates direct binding of neutrophils to acinar cells. ICAM-1 was upregulated in pancreas of rats with experimental pancreatitis induced by supramaximal doses of cerulein. Furthermore, cerulein time and dose dependently stimulated expression of ICAM-1 mRNA and protein in isolated pancreatic acinar cells. Inhibitory analysis showed that activation of transcription factor nuclear factor-κB (NF-κB) was involved in ICAM-1 upregulation by cerulein, but NF-κB did not mediate basal expression of ICAM-1 mRNA in acinar cells. With an adhesion assay, we found that neutrophils bind to isolated pancreatic acinar cells and that cerulein upregulates the extent of adhesion. Neutralizing ICAM-1 antibody blocked neutrophil binding to both control and cerulein-stimulated acinar cells, suggesting ICAM-1 involvement in this adhesion. Thus the acinar cell is capable of targeting neutrophils to its surface, a process that may be important for inflammatory and cell death responses in pancreatitis and other pancreatic disorders.
Nitrergic and purinergic regulation of the rat pylorus
Authors: Ishiguchi Tadashi , Takahashi Toku , Itoh Hidekazu , Owyang Chung
Published: December 2017

The role of nitric oxide (NO) and ATP in the regulation of nonadrenergic, noncholinergic (NANC) inhibitory transmission in the pylorus remains unclear. In the presence of atropine and guanethidine, electric field stimulation induced NANC relaxations in a frequency-dependent manner (1–20 Hz) in the rat pylorus. NANC relaxations were significantly inhibited by NG-nitro-l-arginine methyl ester (l-NAME; 10−4M). P2Xpurinoceptor antagonist pyridoxal phosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS; 3 × 10−5M) and P2Ypurinoceptor antagonist reactive blue 2 (2 × 10−5M) had no effect on NANC relaxations. However, the combined administration of l-NAME and PPADS, but not reactive blue 2, evoked greater inhibitory effects on NANC relaxation than that evoked by l-NAME alone. α-Chymotrypsin and vasoactive intestinal polypeptide antagonist did not affect NANC relaxations. ATP (10−5–10−3M) and P2Xpurinoceptor agonist α,β-methyleneadenosine 5′-triphosphate (10−7−10−5M), but not P2Ypurinoceptor agonist 2-methylthioadenosine 5′-triphosphate (10−7−10−5M), induced muscle relaxations in a dose-dependent manner, and relaxations were significantly reduced by PPADS and unaffected by TTX. These studies suggest that NO and ATP act in concert to mediate NANC relaxation of the rat pylorus. ATP-induced relaxation appears to be mediated by P2Xpurinoceptors located on smooth muscle cells.