bluuebunny/crossref_metadata_2025 huggingface dataset:
Histamine stimulates ion transport by dog pancreatic duct epithelial cells through H1receptors
Authors: Nguyen Toan D. , Okolo Charles N. , Moody Mark W.
Published: December 2017

Histamine affects pancreatic secretion, but its direct action on ion transport by pancreatic duct epithelial cells (PDEC) has not been defined. We now characterize the secretory effects of histamine on cultured, well-differentiated, and nontransformed dog PDEC. Histamine stimulated, in a concentration-dependent manner (1–100 μM), a cellular125I−efflux that was inhibited by 500 μM 5-nitro-2-(3-phenylpropylamino)benzoic acid, 2.5 mM diphenylamine-2-carboxylate, and 500 μM DIDS and thus mediated through Ca2+-activated Cl− channels. Histamine-stimulated125I−efflux was 1) inhibited by 100 μM diphenhydramine, an H1receptor antagonist, 2) resistant to 1 mM cimetidine, an H2 receptor antagonist, 3) not reproduced by 1 mM dimaprit, an H2 agonist, and 4) inhibited by 50 μM 1,2-bis(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid-AM, a Ca2+ chelator, suggesting that it was mediated through H1 receptors acting via increased cytosolic Ca2+. Histamine also stimulated a86Rb+efflux that was sensitive to 100 nM charybdotoxin and thus mediated through Ca2+-activated K+ channels. When PDEC monolayers were studied in Ussing chambers, a short-circuit current of 21.7 ± 3.1 μA/cm2 was stimulated by 100 μM histamine. This effect was inhibited by diphenhydramine but not cimetidine, was not reproduced with dimaprit, and was observed only after serosal addition of histamine, suggesting that it was mediated by basolateral H1 receptors on PDEC. In conclusion, histamine, acting through basolateral H1 receptors, activates both Ca2+-activated Cl− and K+ channels; in this manner, it may regulate PDEC secretion in normal or inflamed pancreas.
Estimation of specific hepatic arterial water space
Authors: Sahin Selma , Rowland Malcolm
Published: December 2017

The aim of this study was to estimate the specific arterial water space and associated blood flow using statistical moments of the frequency versus time outflow profile, with a model with specific spaces for hepatic arterial (HA) and portal venous (PV) flows in parallel with a common space. Studies were performed in the in situ dual-perfused rat liver ( n = 6–10), using Krebs-bicarbonate buffer with constant PV flow (12 ml/min) and various HA flow rates (3–6 ml/min). An impulse input-output technique was employed, varying the route of input, using [14C]urea as the reference indicator. Regardless of flow conditions, the frequency outflow profile after HA input was flatter and broader and the mean transit time longer than after PV input. Excellent recovery of marker was obtained in all cases. Applying the above model, the specific arterial space was estimated to be 9.7 ± 2.3 of total water space and receives ∼17% of the HA flow, with the remainder mixing with portal blood in the common space. The estimated total water content of liver (0.67–0.72 ml/g liver) agrees well with that determined by desiccation (0.72 ± 0.01 ml/g liver).
Autonomic nervous control of venous pressure and secretion in submandibular gland of anesthetized dogs
Authors: Lung Mary A.
Published: December 2017

In dogs anesthetized with pentobarbital sodium, hilar venous pressure (Phv) and secretion were measured from the submandibular gland receiving spontaneous blood flow or vascular perfusion at the normal resting flow rate. Parasympathetic nerve stimulation and ACh-induced secretion increased Phv and its pulse pressure; Phv also showed an obvious arterial (or perfusion pressure)-like waveform. Vasoactive intestinal polypeptide (VIP) exerted similar effects on Phv but produced negligible secretion. Sympathetic nerve stimulation, phenylephrine, and clonidine did not induce secretion and had no significant action on Phv, whereas isoproterenol provoked secretion and changed Phvas with parasympathetic stimulation. Background or superimposed sympathetic nerve stimulation reduced the parasympathetic nerve-induced responses; the sympathetic inhibition was abolished by phentolamine and yohimbine but not by prazosin and propranolol. The results suggest a direct relationship between Phvand secretion during parasympathetic salivation: the elevation in Phv was primarily independent of the concurrent blood flow response, mediated via muscarinic and peptidergic mechanisms, and related to an opening of arteriovenous anastomoses. Sympathetic inhibition of parasympathetic salivation may be related to prevention of an increased Phv exerted primarily via the α2-adrenergic mechanism.
Helicobacter pylori-infected human antral primary cell cultures: effect on gastrin cell function
Authors: Richter-Dahlfors Agneta , Heczko Ursula , Meloche R. Mark , Finlay B. Brett , Buchan Alison M. J.
Published: December 2017

Although Helicobacter pylori infection increases gastrin secretion, it is unknown whether this is a direct effect or requires activation of the immune system. We developed an H. pylori-infected human primary antral epithelial cell culture model to address this question. This culture protocol favors growth of H. pylori, and infected cultures could be maintained for up to 48 h. These cultures were enriched for gastrin (10–40%), somatostatin (2–5%), and gastric mucin (60–80%) cells but did not contain immunocytes. Bacterial attachment occurred in a random manner within 2 h of infection, although bacterial density was lower than in sections from infected patients. After 24 or 48 h, the bacterial microcolonies were similar in size to those seen in vivo, and at 24 h ultrastructural studies demonstrated well-developed pedestal formation underlying the bacteria. Coculture with H. pylori increased basal but not stimulated gastrin secretion at all time points >2 h. In conclusion, a newly developed cell culture model has been used to characterize the interactions between H. pylori and normal human antral epithelial cells.
Regulation of PC4/TIS7 expression in adapting remnant intestine after resection
Authors: Rubin Deborah C. , Swietlicki Elzbieta A. , Wang Joseph L. , Levin Marc S.
Published: December 2017

The adaptive response of the small intestine to loss of functional surface area includes enhanced crypt cell proliferation and enterocyte differentiation. To better define the underlying molecular and cellular mechanisms, we have cloned rat genes that are specifically regulated in the adaptive gut after 70% small intestinal resection. One of these is the immediate early gene PC4/TIS7. Compared with sham-resected control ileum, PC4/TIS7 mRNA levels in the adaptive remnant ileum were markedly increased at 16 and 48 h but not 1 wk after resection. Greater augmentation of PC4/TIS7 mRNA levels occurred in the ileum compared with the duodenum and proximal jejunum. After resection, the changes in intestinal PC4/TIS7 mRNA levels also exceeded changes in extraintestinal levels. The demonstration by in situ hybridization that villus-associated, but not crypt, cells express PC4/TIS7 mRNA is consistent with a role in regulating cytodifferentiation. The pattern of expression in the Caco-2 cell line is also consistent with such a role. Although the precise function of PC4/TIS7 in adaptation remains unclear, the early and intestine-specific changes in mRNA levels after 70% resection suggest that it might augment the adaptive response by stimulating the production of differentiated enterocytes.
Effect of feeding diets of varying fatty acid composition on apolipoprotein expression in newborn swine
Authors: Wang Heng , Hunter Felicia , Black Dennis D.
Published: December 2017

The purpose of this study was to determine the effect of chronic (1 wk) feeding of dietary triacylglycerol (TG) of varying fatty acid composition on small intestinal and hepatic apolipoprotein expression, as well as serum lipid and apolipoprotein concentrations, in newborn swine. Two-day-old female swine were fed one of three diets by gavage with the following lipid composition: medium-chain TG (MCT; MCT oil), intermediate-chain saturated TG (ICST; coconut oil), and long-chain polyunsaturated TG (LCPUT; safflower oil) at 753 kJ ⋅ kg−1⋅ day−1with 51% of energy from fat. After 1 wk, serum lipids and apolipoprotein concentrations were measured, and jejunal apolipoprotein B (apo B) and apo A-I mass and apo B, apo A-I, apo A-IV, and apo C-III synthesis were measured. Liver was processed for determination of apo B and apo A-I mass and apo B, apo A-I, apo C-III, and β-actin mRNA abundance by slot blot hybridization. Compared with the MCT and LCPUT groups, the ICST group had higher total serum cholesterol, TG, high-density lipoprotein (HDL)-cholesterol, and apo A-I concentrations. There were no differences among the three groups for intestinal apolipoprotein mass or synthesis. In liver, apo A-I mass was highest in the ICST group. Liver apo A-I and apo C-III mRNA abundance was highest in the ICST group. Among all three groups, hepatic apo A-I mass correlated significantly with plasma HDL-cholesterol concentrations, and serum TG concentrations correlated with hepatic apo C-III mRNA abundance. In conclusion, we found that in the newborn piglet, chronic feeding of ICST increases serum total cholesterol, TG, HDL-cholesterol, and apo A-I concentrations and hepatic expression of apo A-I and apo C-III mRNA, compared with feeding of MCT or LCPUT. We speculate that increased hepatic apo A-I expression may contribute to the higher serum HDL and apo A-I concentrations in the ICST animals. Increased hepatic expression of apo C-III with ICST feeding may contribute to the higher serum TG concentrations by apo C-III-mediated inhibition of the catabolism of triacylglycerol-rich lipoproteins.
Src kinase and PI 3-kinase as a transduction pathway in ceramide-induced contraction of colonic smooth muscle
Authors: Ibitayo Adenike I. , Tsunoda Yasuhiro , Nozu Fumihiko , Owyang Chung , Bitar Khalil N.
Published: December 2017

Ceramide mediates sustained contraction of smooth muscle cells. C2ceramide induced a rapid increase in Src kinase activity within 15 s, peaked at 1 min, and was sustained up to 8 min. Contraction and Src kinase activity were inhibited in cells incubated in Ca2+-free medium containing 2 mM EGTA and in cells preincubated with herbimycin A, a Src kinase inhibitor. Immunoblotting using a phosphospecific anti-Src (416Y) antibody showed a ceramide-induced increase in pp60srctyrosine phosphorylation. Immunoprecipitation using an anti-phosphotyrosine antibody followed by Western immunoblotting using a monoclonal IgG anti-phosphoinositide 3-kinase NH2terminal-SH2 domain antibody showed a ceramide-induced increase in phosphoinositide 3-kinase (PI 3-kinase) tyrosine phosphorylation at a protein mass corresponding to 85 kDa, the regulatory subunit of PI 3-kinase, which contains the Src kinase binding site. PI 3-kinase phosphorylation was inhibited by herbimycin A and by the PI 3-kinase inhibitors wortmannin and LY-294002. Preincubation of cells with herbimycin A or PI 3-kinase inhibitors also resulted in an inhibition of mitogen-activated protein (MAP) kinase p42 and p44 activities as seen on Western blots. In summary, we found that 1) the maintenance of sustained contraction is dependent on extracellular Ca2+; 2) ceramide activates a nonreceptor tyrosine kinase pathway through activation of pp60srcand PI 3-kinase; and 3) the converging signals are probably through activation of MAP kinase.
Effect of gastrin on antroduodenal motility: role of intraluminal acidity
Authors: Verkijk M. , Gielkens H. A. J. , Lamers C. B. H. W. , Masclee A. A. M.
Published: December 2017

The effect of gastrin on the migrating motility complex (MMC) was studied in seven healthy subjects. It was hypothesized that a potential effect of gastrin on the MMC may result from intraluminal acidification through increased gastric acid secretion. Therefore, antroduodenal manometry and intraluminal acidity were recorded simultaneously. The effect of gastric acid inhibition, with and without administration of gastrin, on antroduodenal motility and intraluminal acidity was also evaluated and compared with saline infusion (control). Continuous infusion of gastrin-17 (20 pmol ⋅ kg−1⋅ h−1) increased intragastric and intraduodenal acidity and suppressed phase II and phase III motor activity in both antrum and duodenum. Concomitant gastric acid inhibition with intravenous famotidine, as demonstrated by intragastric neutralization of pH, completely antagonized the effect of gastrin on the MMC. In fact, famotidine infusion, both with and without administration of gastrin, significantly shortened MMC cycle length. It is concluded that the effect of gastrin on interdigestive antroduodenal motility results from increased intraluminal acidity.
Rat gastroduodenal motility in vivo: involvement of NO and ATP in spontaneous motor activity
Authors: Glasgow Ian , Mattar Kamal , Krantis Anthony
Published: December 2017

Our studies of fasted anesthetized rats have shown that all spontaneous relaxations of the antrum are nitric oxide (NO) dependent. Duodenal motility is patterned into propagating “grouped” motor activity interposed with “intergroup” periods of nonpropagating motor activity; in the duodenum, only intergroup relaxations are NO dependent. We examined the involvement of NO and ATP in spontaneous motor activities of the gastroduodenum in vivo: contractions and relaxations were recorded and analyzed simultaneously from the antrum (S1) and proximal duodenum (D1) of anesthetized Sprague-Dawley rats ( n = 10/group), using extraluminal foil strain gauges. Treatment with the NO synthase inhibitor N G-nitro-l-arginine methyl ester (l-NAME; 10 mg/kg iv) attenuated ( P < 0.05) antral and intergroup relaxations, whereas grouped relaxations were enhanced ( P < 0.05). These effects were reversed with l-arginine (300 mg/kg iv). l-NAME also increased ( P < 0.05) the amplitude of duodenal contractions. ATP (8 mg ⋅ kg−1 ⋅ min−1iv) stimulated relaxations at S1and D1 that were blocked by the P2-purinoceptor antagonist suramin (60 mg/kg iv). This treatment did not affect spontaneous antral relaxations; however, duodenal grouped relaxations were attenuated. Desensitization to the P2x-purinoceptor agonist α,β-methylene ATP (300 μg/kg iv) gave results similar to suramin. In contrast, the P2y-purinoceptor agonist 2-methylthio-ATP (2-MeS-ATP; 360 μg/kg iv) evoked duodenal relaxations that were attenuated byl-NAME, and desensitization to 2-MeS-ATP attenuated intergroup relaxations. Spontaneous relaxations of the rat antrum and duodenal intergroup relaxations are NO dependent. Both gut regions relax in response to systemically administered ATP; this response is sensitive to suramin. Grouped duodenal relaxations display functional sensitivity to suramin and P2x- purinoceptor desensitization, indicative of the involvement of ATP and P2x purinoceptors. P2y purinoceptors must also be present; however, these occur on elements releasing NO. Although NO does not mediate grouped relaxations or duodenal contractions, the sensitivity of these responses tol-NAME indicates that the pathway(s) controlling these responses is modulated by NO.
Dopamine-dependent inhibition of jejunal Na+-K+-ATPase during high-salt diet in young but not in adult rats
Authors: Vieira-Coelho M. Augusta , Teixeira Vera A. Lucas , Finkel Yigael , Soares-Da-Silva Patricio , Bertorello Alejandro M.
Published: December 2017

During high-salt diet endogenous dopamine (DA) reduces jejunal sodium transport in young but not in adult rats. This study was designed to evaluate whether this effect is mediated, at the cellular level, by inhibition of Na+-K+-ATPase activity. Enzyme activity was determined in isolated jejunal cells by the rate of [γ-32P]ATP hydrolysis. Cells were obtained from weanling and adult rats fed either with high- or normal-salt diet. In 20-day-old but not in 40-day-old rats Na+-K+-ATPase activity was significantly reduced during high-salt diet. This inhibition was abolished by a blocker of DA synthesis. The decreased activity was associated with a decreased α1-subunit at the plasma membrane. During high-salt diet there was an increase in DA content in jejunal cells from 20-day-old rats, associated with a parallel decrease in 5-hydroxytryptamine, compared with normal-salt diet. In 40-day-old rats, however, the catecholamine level remained unchanged during high-salt diet. Incubation of isolated jejunal cells with DA resulted in a dose-dependent inhibition of Na+-K+-ATPase activity in 20- but not in 40-day-old rats. We conclude that during high-salt diet, jejunal Na+-K+-ATPase in 20-day-old rats is inhibited, and this effect is likely to be mediated by locally formed DA.
Rho A regulates sustained smooth muscle contraction through cytoskeletal reorganization of HSP27
Authors: Wang Pinglang , Bitar Khalil N.
Published: December 2017

The ras-related protein Rho p21 regulates various actin-dependent functions, including smooth muscle contraction. However, the precise mechanism of action of Rho p21 is still not clear. We report here that Rho A is a key regulator of agonist-induced contractile effects in rabbit colonic smooth muscle. Endothelin-1 and C2ceramide were used. Both seem to activate phosphoinositide 3-kinase (PI 3-kinase) through G protein and pp60src, respectively. Immunoprecipitation and immunoblotting revealed one form of 21-kDa Rho A that translocated from the cytosol to the membrane in response to stimulation by either endothelin (10−7M) or ceramide (10−7M) (∼30% increase at 30 s that was sustained at 4 min). The translocation of Rho A to the membrane was confirmed by immunostaining. The translocation of Rho A was inhibited by Clostridium botulinum C3 exoenzyme, which ADP ribosylated Rho A, but was not inhibited by the pp60srcinhibitor herbimycin A or by the protein kinase C (PKC) inhibitor calphostin C, suggesting that Rho A may be upstream of pp60srcand PKC or may belong to a different pathway than these proteins. Both ceramide- and endothelin-induced PI 3-kinase activation was inhibited by C3 exoenzyme pretreatment. However, the C3 exoenzyme inhibited endothelin- but not ceramide-induced mitogen-activated protein kinase phosphorylation, indicating that Rho regulates ceramide- and endothelin-induced contraction through different pathways. Furthermore, the dominant negative form of Rho (N19Rho) inhibited the actin binding protein, 27-kDa heat shock protein (HSP27), reorganization in response to ceramide and endothelin observed under confocal microscopy.
Regulation of intracellular pH and blood flow in rat duodenal epithelium in vivo
Authors: Akiba Yasutada , Kaunitz Jonathan D.
Published: December 2017

Duodenal mucosal defense was assessed by measuring blood flow and epithelial intracellular pH (pHi) of rat proximal duodenum in vivo. Fluorescence microscopy was used to measure epithelial pHiusing the trapped, pHi-indicating dye 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-AM. Blood flow was measured with laser-Doppler flowmetry. The mucosa was briefly superfused with NH4Cl, pH 2.2 buffer, the potent Na+/H+exchange inhibitor 5-( N, N-dimethyl)-amiloride (DMA), or the anion exchange and Na+-[Formula: see text]cotransport inhibitor DIDS. Cryostat sections localized dye fluorescence to the villus tip. Steady-state pHiwas 7.02 ± 0.01, which remained stable for 60 min. Interventions that load the cells with protons without affecting superfusate pH (NH4Cl prepulse, nigericin with low superfusate K+concentration, DMA, and DIDS) all decreased pHi, supporting our contention that the dye was faithfully measuring pHi. An acid pulse decreased pHi, followed by a DIDS-inhibitable overshoot over baseline. Intracellular acidification increased duodenal blood flow independent of superfusate pH, which was inhibited by DMA, but not by DIDS. We conclude that we have established a novel in vivo microscopy system enabling simultaneous measurements of pHiand blood flow of duodenal epithelium. Na+/H+exchange and Na+-[Formula: see text]cotransport regulate baseline duodenal epithelial pHi. Intracellular acidification enhances duodenal blood flow by a unique, amiloride-inhibitable, superfusate pH-independent mechanism.
Slow waves in circular muscle of porcine ileum: structural and electrophysiological studies
Authors: Jiménez M. , Borderies J. R. , Vergara P. , Wang Y.-F. , Daniel E. E.
Published: December 2017

The structural and functional bases of pacemaking (slow waves) in porcine ileal circular muscle were studied. The myenteric plexus contained two, structurally distinct types of interstitial cells of Cajal (ICC) interconnected by gap junctions and connected by close contacts to muscle layers. At the deep muscular plexus, ICC were present, not regularly close to nerve axons or in gap junction contact with one another or outer circular muscle, which had many gap junctions. Slow waves (5.2 ± 2 mV amplitude and 4.6 ± 0.7 s duration) occurred at 9.9 ± 1.1 counts/min. Tissue length and time constants were 2.00 ± 0.3 mm and 111 ± 37 ms, respectively. Large electrical field-induced hyperpolarizations or depolarizations reduced amplitudes but not frequencies or durations of slow waves; hyperpolarizations progressively reduced inhibitory junction potentials as if the K+channel opening mediated them. In conclusion, the myenteric plexus ICC of pig ileum, which appears to pace the muscle layers, appears insensitive to voltages applied to the syncytium of circular muscle cells. Limited coupling between ICC and circular muscle or voltage-insensitive pacemaking activity may explain these findings.
Polyamine depletion is associated with an increase in JunD/AP-1 activity in small intestinal crypt cells
Authors: Patel Anami R. , Wang Jian-Ying
Published: December 2017

Activator protein 1 (AP-1) is a group of dimeric transcription factors composed of protooncogene (Jun and Fos) subunits that bind to a common DNA site, the AP-1 binding site. The proteins of c-Jun, JunB, and Fos are essential for initiation of the cell cycle. Conversely, the activation of the junD gene slows cell growth in some cell types. The current study tests the hypothesis that polyamines influence cell growth by altering the balance of positive and negative Jun/AP-1 activities in intestinal epithelial cells. Studies were conducted in the IEC-6 cell line derived from rat small intestinal crypt cells. Administration of α-difluoromethylornithine (DFMO), a specific inhibitor for polyamine synthesis, for 4 and 6 days completely depleted cellular polyamine levels, while AP-1 binding activity was significantly increased. Spermidine, when given together with DFMO, restored AP-1 binding activity toward normal. The increased AP-1 complexes in polyamine-deficient cells were dramatically supershifted by the anti-JunD antibody but not by antibodies against c-Jun, JunB, or Fos proteins. There were significant increases in JunD mRNA and protein in DFMO-treated cells, although expression of the c- fos, c- jun, and junB genes decreased. The increase in JunD/AP-1 activity in DFMO-treated cells was associated with a significant decrease in cell division. Exposure of control quiescent cells to 5% dialyzed serum increased c-Jun/AP-1 but not JunD/AP-1 activities. DFMO prevented the stimulation of c-Jun/AP-1 activity induced by 5% dialyzed serum. These results indicate that 1) polyamine depletion is associated with an increase in AP-1 binding activity and 2) the increase in AP-1 activity in the DFMO-treated cells was primarily contributed by an increase in the JunD/AP-1. These findings suggest that polyamines regulate cell growth at least partially by modulating the balance of positive and negative Jun/AP-1 activities in the intestinal mucosa.
Activation of neuronal adenosine A1 receptors suppresses secretory reflexes in the guinea pig colon
Authors: Cooke Helen J. , Wang Y.-Z. , Liu C. Y. , Zhang H. , Christofi F. L.
Published: December 2017

The role of adenosine A1 receptors (A1R) in reflex-evoked short-circuit current ( I sc) indicative of chloride secretion was studied in the guinea pig colon. The A1R antagonist 8-cyclopentyltheophylline (CPT) enhanced reflex-evoked I sc. Adenosine deaminase and the nucleoside transport inhibitor S-(4-nitrobenzyl)-6-thioinosine enhanced and reduced reflex-induced I sc, respectively. The A1R agonist 2-chloro- N 6-cyclopentyladenosine (CCPA) inhibited reflex-evoked I sc at nanomolar concentrations, and its action was antagonized by CPT. In the presence of either N-acetyl-5-hydroxytryptophyl-5-hydroxytryptophan amide to block the 5-hydroxytryptamine (5-HT)-mediated pathway or piroxicam to block the prostaglandin-mediated pathway, CCPA reduced the residual reflex-evoked I sc. CCPA reduced the response to a 5-HT pulse without affecting the tetrodotoxin-insensitive I sc responses to carbachol or forskolin. Immunoreactivity for A1R was detected in the membrane (10% of neurons) and cytoplasm (90% of neurons) of neural protein gene product 9.5-immunoreactive (or S-100-negative) submucosal neurons, in glia, and in the muscularis mucosa. A1R immunoreactivity in a majority of neurons remained elevated in the cytoplasm despite preincubation with adenosine deaminase or CPT. A1R immunoreactivity colocalized in synaptophysin-immunoreactive presynaptic varicose nerve terminals. The results indicate that endogenous adenosine binding to high-affinity A1R on submucosal neurons acts as a physiological brake to suppress reflex-evoked I scindicative of chloride secretion.
Expression of multiple Na+/H+exchanger isoforms in rat parotid acinar and ductal cells
Authors: Park Keerang , Olschowka John A. , Richardson Linda A. , Bookstein Crescence , Chang Eugene B. , Melvin James E.
Published: December 2017

Several members of the Na+/H+exchanger gene family (NHE1, NHE2, NHE3, and NHE4) with unique functional properties have been cloned from rat epithelial tissues. The present study examined the molecular and pharmacological properties of Na+/H+exchange in rat parotid salivary gland cells. In acinar cells superfused with a physiological salt solution (145 mM Na+), Na+/H+exchanger activity was inhibited by low concentrations of the amiloride derivative ethylisopropyl amiloride (EIPA; IC50= 0.014 ± 0.005 μM), suggesting the expression of amiloride-sensitive isoforms NHE1 and/or NHE2. Semiquantitative RT-PCR confirmed that NHE1 transcripts are most abundant in this cell type. In contrast, the intermediate sensitivity of ductal cells to EIPA indicated that inhibitor-sensitive and -resistant Na+/H+exchanger isoforms are coexpressed. Ductal cells were about one order of magnitude more resistant to EIPA (IC50= 0.754 ± 0.104 μM) than cell lines expressing NHE1 or NHE2 (IC50= 0.076 ± 0.013 or 0.055 ± 0.015 μM, respectively). Conversely, ductal cells were nearly one order of magnitude more sensitive to EIPA than a cell line expressing the NHE3 isoform (IC50= 6.25 ± 1.89 μM). Semiquantitative RT-PCR demonstrated that both NHE1 and NHE3 transcripts are expressed in ducts. NHE1 was immunolocalized to the basolateral membranes of acinar and ductal cells, whereas NHE3 was exclusively seen in the apical membrane of ductal cells. Immunoblotting, immunolocalization, and semiquantitative RT-PCR experiments failed to detect NHE2 expression in either cell type. Taken together, our results demonstrate that NHE1 is the dominant functional Na+/H+exchanger in the plasma membrane of rat parotid acinar cells, whereas NHE1 and NHE3 act in concert to regulate the intracellular pH of ductal cells.
Participation of reactive oxygen metabolites inClostridium difficiletoxin A-induced enteritis in rats
Authors: Qiu Bosheng , Pothoulakis Charalabos , Castagliuolo Ignazio , Nikulasson Sigfus , LaMont J. Thomas
Published: December 2017

Reactive oxygen metabolites (ROMs) contribute to the pathophysiology of intestinal inflammation. Our aim was to ascertain the involvement of ROMs in experimental ileitis in rats produced by toxin A of Clostridium difficile. Intraluminal toxin A caused a significant increase in hydroxyl radical and hydrogen peroxide production by ileal microsomes starting 1 h following toxin exposure and peaking at 2–3 h, and this was inhibited by pretreatment with DMSO, a ROM scavenger, or superoxide dismutase (SOD), which inactivates ROMs. In contrast, mucosal xanthine oxidase increased only slightly after toxin A exposure, and allopurinol, an inhibitor of xanthine oxidase, had no effect on toxin A-associated intestinal responses. Induction of neutropenia resulted in reduction of toxin-mediated free radical formation, fluid secretion, and permeability. The enterotoxic effects of C. difficile toxin A were associated with increased ROM release in ileal tissues, and the ROM inhibitors DMSO and SOD inhibited these effects. This suggests that ROMs released during toxin A enteritis are released primarily from neutrophils invading the inflamed bowel segment.
Adenovirus-mediated gene transfer of RasN17 inhibits specific CCK actions on pancreatic acinar cells
Authors: Nicke Barbara , Tseng Min-Jen , Fenrich Marycarol , Logsdon Craig D.
Published: December 2017

CCK stimulates pleiotrophic responses in pancreatic acinar cells; however, the intracellular signaling pathways involved are not well understood. To evaluate the role of the ras gene product in CCK actions, a strategy involving in vitro adenoviral-mediated gene delivery of a dominant-negative mutant Ras (RasN17) was utilized. Isolated acini were infected with various titers of either a control adenovirus or an adenoviral construct expressing RasN17 for 24 h before being treated with CCK. Titer-dependent expression of RasN17 in the acini was confirmed by Western blotting. Infection with control adenovirus [106–109plaque-forming units/mg acinar protein (multiplicity of infection of ∼1–1,000)] had no effect on CCK stimulation of acinar cell amylase release, extracellular-regulated kinase (ERK) or c-Jun kinase (JNK) kinases, or DNA synthesis. In contrast, infection with adenovirus bearing ras N17 increased basal amylase release, inhibited CCK-mediated JNK activation, had no effect on CCK activation of ERK, and inhibited DNA synthesis. These data demonstrate important roles for Ras in specific actions of CCK on pancreatic acinar function.
Experimental enteropathy in athymic and euthymic rats: synergistic role of lipopolysaccharide and indomethacin
Authors: Koga Hideki , Aoyagi Kunihiko , Matsumoto Takayuki , Iida Mitsuo , Fujishima Masatoshi
Published: December 2017

The aim of this study was to investigate the immunologic and microbiological bases of indomethacin enteropathy. Athymic nude and euthymic specific pathogen-free (SPF) rats were reared under conventional or SPF conditions. In each group, indomethacin was given intrarectally for 2 days. Indomethacin enteropathy was evaluated using a previously described ulcer index and tissue myeloperoxidase activity. Both euthymic and athymic nude rats developed intestinal ulcers to the same degree under conventional conditions but no or minimal ulcer under SPF conditions. Pretreatment of conventional rats with intragastric kanamycin sulfate, an aminoglycoside antibiotic, attenuated indomethacin enteropathy in a dose-dependent fashion. Interestingly, when lipopolysaccharide was injected intraperitoneally in kanamycin-pretreated rats, it fully restored enteropathy in these rats in a dose-dependent manner. We confirmed that kanamycin decreased the number of gram-negative bacteria and endotoxin concentration of the small intestine in a dose-dependent fashion. These results indicate that indomethacin enteropathy is bacteria dependent and does not require a T cell function. Synergy between indomethacin and bacterial lipopolysaccharide may play a major role in this enteropathy.
Prolonged colonic epithelial hyporesponsiveness after colitis: role of inducible nitric oxide synthase
Authors: Asfaha Samuel , Bell Cameron J. , Wallace John L. , MacNaughton Wallace K.
Published: December 2017

Colonic epithelial secretion is an important host defense mechanism. We examined whether a bout of colitis would produce long-lasting changes in epithelial function that persisted after resolution of mucosal inflammation. Colitis was induced in rats with intracolonic trinitrobenzenesulfonic acid. Six weeks later, colonic damage and inducible nitric oxide synthase (iNOS) mRNA expression and activity were measured. Segments of distal colon were mounted in Ussing chambers for measurement of permeability and responsiveness to secretory stimuli. Basal electrolyte transport parameters and permeability were not different from untreated controls. Despite normal macroscopic and histological appearance, secretory responses to electrical field stimulation (EFS), isobutylmethylxanthine (IBMX), and carbachol were significantly depressed (by 60–70%) relative to controls. iNOS mRNA expression and enzyme activity were significantly elevated. Dexamethasone reversed epithelial hyporesponsiveness and significantly reduced iNOS mRNA expression. A selective iNOS inhibitor normalized the secretory responses to EFS and IBMX but not to carbachol. These data suggest that ongoing synthesis of nitric oxide by iNOS contributes to chronic suppression of epithelial secretory function after episodes of colitis.
Regulation of IGF binding protein-1 in Hep G2 cells by cytokines and reactive oxygen species
Authors: Lang Charles H. , Nystrom Gerald J. , Frost Robert A.
Published: December 2017

The liver is a major site of synthesis for insulin-like growth factor binding protein (IGFBP)-1. Because IGFBP-1 inhibits many anabolic actions of IGF-I, increases in IGFBP-1 may be partly responsible for the decrease in lean body mass observed in catabolic/inflammatory conditions. This study aimed to determine in Hep G2 cells 1) the sensitivity of IGFBP-1 synthesis to treatment with interleukin (IL)-1, tumor necrosis factor-α (TNF-α), and IL-6, 2) the ability of reactive oxygen species (ROS) to enhance IGFBP-1 production, and 3) the role of ROS in mediating cytokine-induced increases in IGFBP-1. Hep G2 cells responded to IL-1β, TNF-α, and IL-6 with maximal 8- to 10-fold increases in IGFBP-1 production. Although the maximal responsiveness of cells treated with TNF-α and IL-6 was 20–30% less than that with IL-1β, cells demonstrated a similar sensitivity to all cytokines (half-maximal responsive dose of ∼10 ng/ml). A low concentration (3 ng/ml) of all three cytokines had an additive effect on IGFBP-1 production. Cytokines also increased IGFBP-1 mRNA. The half-life of IGFBP-1 mRNA was ∼4 h and not altered by IL-1β. Incubation with ROS, including H2O2and nitric oxide (NO) donors, resulted in a relatively smaller increase in IGFBP-1. However, preincubating Hep G2 cells with various free radical scavengers and NO synthase and eicosanoid inhibitors failed to prevent or attenuate cytokine-induced increases in IGFBP-1. Finally, preincubating cells with pyrrolidinedithiocarbamate (PDTC) but not SN50 (inhibitors of nuclear factor-κB activation and nuclear translocation, respectively) attenuated increases in IGFBP-1 induced by IL-1. These results indicate that 1) proinflammatory cytokines directly enhance IGFBP-1 synthesis by stimulating transcription without altering mRNA stability, 2) addition of exogenous ROS also stimulates IGFBP-1 production but to a smaller extent than cytokines, and 3) the cytokine-induced increase in IGFBP-1 production is not mediated by endogenous production of ROS or eicosanoids but appears to at least partially involve a PDTC-sensitive pathway.
Peripheral urocortin delays gastric emptying: role of CRF receptor 2
Authors: Nozu Tsukasa , Martinez Vicente , Rivier Jean , Taché Yvette
Published: December 2017

Urocortin, a new mammalian member of the corticotropin-releasing factor (CRF) family has been proposed to be the endogenous ligand for CRF receptor 2 (CRF-R2). We studied the influence of intravenous urocortin on gastric emptying and the role of CRF-R2 in peptide action and postoperative gastric ileus in conscious rats. The intravenous doses of rat CRF and rat urocortin producing 50% inhibition of gastric emptying were 2.5 and 1.1 μg/kg, respectively. At these intravenous doses, CRF and urocortin have their actions fully reversed by the CRF-R1/CRF-R2 antagonist astressin at antagonist/agonist ratios of 5:1 and 67:1, respectively. Astressin (12 μg/kg iv) completely prevented abdominal surgery-induced 54% inhibition of gastric emptying 3 h after surgery while having no effect on basal gastric emptying. The selective nonpeptide CRF-R1 antagonists antalarmin (20 mg/kg ip) and NBI-27914 (400 μg/kg iv) did not influence intravenous CRF-, urocortin- or surgery-induced gastric stasis. These results as well as earlier ones showing that α-helical CRF9—41(a CRF-R2 more selective antagonist) partly prevented postoperative ileus indicate that peripheral CRF-R2 may be primarily involved in intravenous urocortin-, CRF-, and abdominal surgery-induced gastric stasis.
Microvascular perfusion deficits are not a prerequisite for mucosal injury in septic rats
Authors: Nevière R. R. , Pitt-Hyde M. L. , Piper R. D. , Sibbald W. J. , Potter R. F.
Published: December 2017

Our major objective was to investigate whether injury to the mucosa of the small intestine occurred in a normotensive model of sepsis and whether such injury was associated with microvascular perfusion deficits. Using fluorescence intravital microscopy, we show direct evidence of cell injury within the mucosa (pneumonia 12.4 ± 2.6 cells/field, sham 2.2 ± 0.7 cells/field), whereas use of51Cr-labeled EDTA showed evidence of increased mucosal permeability (pneumonia 1.90 ± 0.67 ml · min−1 · 100 g−1; sham 0.24 ± 0.04 ml · min−1 · 100 g−1), 48 h following induction of pneumonia. Despite such injury the capillary density in the ileal mucosa and submucosa of pneumonic rats (1,027 ± 77 and 1,717 ± 86 mm2) was not significantly different compared with sham (998 ± 63 and 1,812 ± 101 mm2). However, a modest albeit significant decrease in capillary perfusion was measured in the muscularis layer of pneumonia (11.0 ± 1.3 mm) compared with sham (13.9 ± 0.63 mm) and appeared to be associated with leukocyte entrapment. Pretreatment using low doses of endotoxin to induce endotoxin tolerance not only increased muscularis capillary density but reduced the number of leukocytes trapped within the microvasculature, decreased myeloperoxidase activity within the ileum in pneumonic rats, and prevented mucosal injury. In conclusion, we have shown that pneumonia results in remote injury to the mucosa of the ileum and that such injury was not associated with concurrent mucosal perfusion deficits.
Excretion of progastrin products in human urine
Authors: Hansen C. Palnaes , Goetze J. P. , Stadil F. , Rehfeld J. F.
Published: December 2017

The renal handling of carboxyamidated gastrins, NH2-terminal progastrin fragments, and glycine-extended gastrins was examined in healthy volunteers. The respective urinary clearances after a meal amounted to 0.09 ± 0.02%, 0.17 ± 0.04% ( P< 0.05), and 0.04 ± 0.01% ( P< 0.01) of the glomerular filtration rate. During intravenous infusion of carboxyamidated gastrin-17, progastrin fragment-(1—35), and glycine-extended gastrin-17, the respective urinary clearances amounted to 0.08 ± 0.02, 0.46 ± 0.08, and 0.02 ± 0.01%, respectively, of the glomerular filtration rate. The metabolic clearance rate of the three peptides was 24.4 ± 1.3, 6.0 ± 0.4, and 8.6 ± 0.7 ml ⋅ kg−1⋅ min−1. A maximum rate for tubular transport or degradation of the peptides could not be determined, nor was a renal plasma threshold recorded. Plasma concentrations and urinary excretion rates correlated for gastrin-17 and progastrin fragment-(1—35) ( r = 0.94 and 0.97, P < 0.001), whereas the excretion of glycine-extended gastrin diminished with increasing plasma concentrations. We conclude that renal excretion of progastrin products is negligible compared with renal metabolism and that renal handling of the peptides depends on their molecular structure. Hence, the kidneys exhibited a higher excretion of NH2-terminal progastrin fragments than of carboxyamidated and especially glycine-extended gastrins.
I. Mucosal dendritic cells: their specialized role in initiating T cell responses
Authors: Iwasaki Akiko , Kelsall Brian L.
Published: December 2017

Dendritic cells (DCs) are the most competent antigen-presenting cells known for the induction of primary T cell responses. Functional studies of tissue-resident DCs have been impaired by the rarity of these cells in any given organ. Recent development of isolation procedures allowing extraction of highly purified fresh DC populations has made it possible to study mucosal DCs in distinct mucosa-associated lymphoid tissues. Here, we discuss several recent studies by us and others that describe the tissue-specific phenotype and function of mucosal DCs and speculate on the mechanism by which the resident DCs regulate tissue-specific T cell responses.
Dissociation between growth arrest and differentiation in Caco-2 subclone expressing high levels of sucrase
Authors: Tian Jean Q. , Quaroni Andrea
Published: December 2017

Growth arrest and cell differentiation are generally considered temporally and functionally linked phenomena in small intestinal crypt cells and colon tumor cell lines (Caco-2, HT-29). We have derived a Caco-2 subclone (NGI3) that deviates from such a paradigm. In striking contrast with the parental cells, proliferative and subconfluent NGI3 cells were found to express sucrase-isomaltase (SI) mRNA and to synthesize relatively high levels of SI, dipeptidyl peptidase IV, and aminopeptidase N (APN). In postconfluent cells, little difference was seen in SI mRNA levels between Caco-2 and NGI3 cells, but the latter still expressed much higher levels of SI that could be attributed to higher rates of translation. APN expression was also greatly enhanced in NGI3 cells. To determine whether high levels of brush-border enzymes correlated with expression of cell-cycle regulatory proteins, we investigated their relative cellular levels in growing and growth-arrested cells. The results showed that the cyclin-dependent kinase inhibitors (p21 and p27) and D-type cyclins (D1 and D3) were all induced in postconfluent cells, but NGI3 cells expressed much higher levels of p21. This study demonstrated that cell growth and expression of differentiated traits are not mutually exclusive in intestinal epithelial cells and provided evidence indicating that posttranscriptional events play an important role in regulation of SI expression.
Effect of liver disease and transplantation on urea synthesis in humans: relationship to acid-base status
Authors: Shangraw Robert E. , Jahoor Farook
Published: December 2017

It has been suggested that hepatic urea synthesis, which consumes[Formula: see text], plays an important role in acid-base homeostasis. This study measured urea synthesis rate (Raurea) directly to assess its role in determining the acid-base status in patients with end-stage cirrhosis and after orthotopic liver transplantation (OLT). Cirrhotic patients were studied before surgery ( n = 7) and on the second postoperative day ( n = 11), using a 5-h primed-constant infusion of [15N2]urea. Six healthy volunteers served as controls. Raurea was 5.05 ± 0.40 (SE) and 3.11 ± 0.51 μmol ⋅ kg−1⋅ min−1, respectively, in controls and patients with cirrhosis ( P < 0.05). Arterial base excess was 0.6 ± 0.3 meq/l in controls and −1.1 ± 1.3 meq/l in cirrhotic patients (not different). After OLT, Raurea was 15.05 ± 1.73 μmol ⋅ kg−1⋅ min−1, which accompanied an arterial base excess of 7.0 ± 0.3 meq/l ( P < 0.001). We conclude that impaired Raurea in cirrhotic patients does not produce metabolic alkalosis. Concurrent postoperative metabolic alkalosis and increased Raurea indicate that the alkalosis is not caused by impaired Raurea. It is consistent with, but does not prove, the concept that the graft liver responds to metabolic alkalosis by augmenting Raurea, thus increasing[Formula: see text] consumption and moderating the severity of metabolic alkalosis produced elsewhere.
High plasma cholesterol in drug-induced cholestasis is associated with enhanced hepatic cholesterol synthesis
Authors: Chisholm Jeffrey W. , Nation Patrick , Dolphin Peter J. , Agellon Luis B.
Published: December 2017

In α-naphthylisothiocyanate-treated mice, plasma phospholipid (PL) levels were elevated 10- and 13-fold at 48 and 168 h, respectively, whereas free cholesterol (FC) levels increased between 48 h (17-fold) and 168 h (39-fold). Nearly all of these lipids were localized to lipoprotein X-like particles in the low-density lipoprotein density range. The PL fatty acyl composition was indicative of biliary origin. Liver cholesterol and PL content were near normal at all time points. Hepatic hydroxymethylglutaryl CoA reductase activity was increased sixfold at 48 h, and cholesterol 7α-hydroxylase activity was decreased by ∼70% between 24 and 72 h. These findings suggest a metabolic basis for the appearance of abnormal plasma lipoproteins during cholestasis. Initially, PL and bile acids appear in plasma where they serve to promote the efflux of cholesterol from hepatic cell membranes. Hepatic cholesterol synthesis is then likely stimulated in the response to the depletion of hepatic cell membranes of cholesterol. We speculate that the enhanced synthesis of cholesterol and impaired conversion to bile acids, particularly during the early phase of drug response, contribute to the accumulation of FC in the plasma.
Calcium waves in colonic myocytes produced by mechanical and receptor-mediated stimulation
Authors: Young S. H. , Ennes H. S. , McRoberts J. A. , Chaban V. V. , Dea S. K. , Mayer E. A.
Published: December 2017

The mechanisms underlying intracellular Ca2+waves induced by either mechanical or receptor-mediated stimulation of myocytes isolated from the longitudinal muscle layer of the rabbit distal colon were compared using fura 2 and fluorescence videomicroscopy. Light focal mechanical deformation of the plasma membrane or focal application of substance P resulted in localized intracellular Ca2+concentration ([Ca2+]i) transients that propagated throughout the cell. In both cases, the Ca2+response consisted of a transient peak response followed by a delayed-phase response. Substance P-mediated [Ca2+]iresponses involved generation of inositol 1,4,5-trisphosphate and release of Ca2+from thapsigargin-sensitive stores, whereas mechanically induced responses were partially (29%) dependent on La3+-sensitive influx of extracellular Ca2+and partially on release of intracellular Ca2+from thapsigargin-insensitive stores gated by ryanodine receptors. The delayed-phase response in both cases was dependent on extracellular Ca2+. However, although the response to substance P was sensitive to La3+, that after mechanical stimulation was not. In the later case, the underlying mechanism may involve capacitative Ca2+entry channels that are activated after mechanical stimulation but not by substance P.
Effects of acute graded exercise on human colonic motility
Authors: Rao Satish S. C. , Beaty Jennifer , Chamberlain Mindi , Lambert Patrick G. , Gisolfi Carl
Published: December 2017

Whether physical exercise stimulates colonic motility is unclear. Our aim was to determine the immediate effects of graded exercise on colonic motility. Colonic motility was recorded at six sites in 11 untrained subjects, by colonoscopically placing a solid-state probe. Subjects were free to ambulate. The next day, subjects exercised on a bicycle at 25, 50, and 75% of peak oxygen uptake for 15 min, with each followed by a 15-min rest. Motor patterns, motility indexes, and regional variations before, during exercise, during rest, and during postexercise periods were compared. During exercise, there was an intensity-dependent decrease ( P < 0.001) in the number and area under the curve of pressure waves. The incidence of propagated or simultaneous pressure waves and cyclical events also decreased ( P < 0.05). After exercise, the pressure activity reverted to baseline, but the number and amplitude of propagated waves increased ( P < 0.01), whereas the simultaneous waves and cyclical events remained lower. Acute graded exercise decreases colonic phasic activity. This may offer less resistance to colonic flow, whereas the postexercise increase in propagated activity may enhance colonic propulsion.