bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence for a functional link between stress response and vascular control in hepatic portal circulation
Authors: Bauer M. , Pannen B. H. , Bauer I. , Herzog C. , Wanner G. A. , Hanselmann R. , Zhang J. X. , Clemens M. G. , Larsen R.
Published: December 2017

Heme oxygenase (HO)-derived carbon monoxide (CO) may contribute to vascular control through elevation of guanosine 3',5'-cyclic monophosphate. In the present study, we investigated the functional significance of expression of the isoenzyme HO-1 (heat-shock protein 32) in liver after hemorrhage/resuscitation (H/R) in rats anesthetized with pentobarbital sodium. An increase of mRNA levels for HO-1 was observed at 3 h after resuscitation, followed by induction of the protein at 6 h in pericentral hepatocytes and sinusoidal lining cells. Concomitantly, lower portal resistance was observed in H/R (0.33 +/- 0.060 mmHg.ml-1.min) compared with control rats (0.47 +/- 0.035 mmHg.ml-1.min). Blockade of the HO-CO pathway by tin protoporphyrin-IX (SnPP-IX) led to a transient increase in portal pressure with no effect on portal low in controls, whereas an increase in pressure and a decrease in flow contributed to the sustained increase in portal resistance after H/R. These results indicate that HO contributes to maintenance of hepatic perfusion in vivo under stressful conditions, suggesting a functional link between stress response and vascular control in portal circulation.
Ca2+ role in myogenic and neurogenic activities of canine ileum circular muscle
Authors: Cayabyab F. S. , deBruin H. , Jimenez M. , Daniel E. E.
Published: December 2017

The role of Ca2+ in myogenic and neural activation in canine ileum circular muscle (CM) was studied during simultaneous recordings of contractile and electrical activity in cross-sectioned slabs of muscularis externa or of isolated CM with deep muscular plexus (DMP) intact. Ca(2+)-free Krebs solution abolished inhibitory junction potentials (IJP) and contractions before changes in CM membrane potentials and while slow waves (SW) persisted at lower amplitude and frequency. This medium abolished SW more rapidly in isolated CM than in intact muscle strips and affected triggered SW (TSW) by 100-ms pulses recorded near myenteric plexus or near DMP differentially in the full-thickness preparation; TSW did not occur in isolated CM. Ni2+, a nonselective Ca2+ channel antagonist, left IJP unchanged and reduced contractions, frequencies, and amplitudes of spontaneous SW and TSW, but increased their durations. Nifedipine abolished contractions but SW, TSW, and IJP were unaffected. Cyclopiazonic acid (CPA) increased SW frequency, produced spikes on SW plateaus, and increased CM tone, but did not affect IJP or resting membrane potentials. In nifedipine-pretreated strips, CPA decreased SW frequencies and amplitudes, evoked less tone, depolarized membrane potentials, and left IJP unaltered. The neuronal N-type Ca2+ channel blocker omega-conotoxin GVIA abolished IJP, without affecting SW or TSW. We conclude that Ca2+ influx, not through L- or N-type Ca2+ channels, helps initiate ileal SW; L-type Ca2+ channels provide Ca2+ for contraction and N-type Ca2+ channels provide Ca2+ for IJP mediator release. Frequencies of SW may be modulated by uptake of Ca2+ into pacemaker stores.
CFTR expression and mucin secretion in cultured mouse gallbladder epithelial cells
Authors: Peters R. H. , French P. J. , van Doorninck J. H. , Lamblin G. , Ratcliff R. , Evans M. J. , Colledge W. H. , Bijman J. , Scholte B. J.
Published: December 2017

Dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) in humans is frequently associated with progressive liver disease, which appears to result from obstruction of biliary ducts with mucous material. CFTR in the liver is expressed in the biliary epithelium. With the use of a mouse model for cystic fibrosis (CF) we have studied the relationship between CFTR expression and glycoprotein secretion in primary culture of mouse gallbladder epithelial cells (MGBC) MGBC in culture maintain a well-differentiated phenotype as shown by microscopy. The cells produce CFTR mRNA to levels comparable to the intact tissue. With patch-clamp analysis we could frequently observe a linear protein kinase A-regulated Cl- channel that shows all the major characteristics of human CFTR, although its conductance is lower (5 pS compared with 8 pS). MGBC in culture produce and secrete high molecular weight glycoproteins (HMG) in a time-dependent and temperature-sensitive manner. Secretion of HMG was not stimulated significantly by either adenosine 3',5'-cyclic monophosphate (cAMP), Ca2+, or protein kinase C agonists in this system. High concentrations (3 mM) of extracellular ATP stimulated secretion threefold, but low concentrations (0.3 mM) had no effect. Approximately one-third of the HMG produced and secreted consisted of mucin. Cultured MGBC from CFTR-deficient mice produced and secreted mucin to a similar extent as normal cells. We conclude that cultured mouse gallbladder cells are a convenient model to study both CFTR function and mucin secretion. In this system, we found no evidence for a direct link between mucin secretion and CFTR activity, as has been suggested for other cell types.
Characterization of two distinct chloride channels in cultured dog pancreatic duct epithelial cells
Authors: Nguyen T. D. , Koh D. S. , Moody M. W. , Fox N. R. , Savard C. E. , Kuver R. , Hille B. , Lee S. P.
Published: December 2017

Cl- secretion by pancreatic duct epithelial cells (PDEC) regulates cellular HCO3- secretion, an important component of the exocrine pancreas. In cystic fibrosis, for example, impaired function of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel results in decreased pancreatic secretion and secondary pancreatic insufficiency. Studies of ion transport by PDEC have been hindered by the lack of a practical in vitro model. We have successfully cultured nontransformed dog PDEC on Vitrogen-coated permeable membranes overlying a feeder layer of myofibroblasts and report the characterization of Cl- channels in these cells. Cl- conductance, assessed through efflux of 125I from PDEC, was stimulated by agents acting via adenosine 3',5'-cyclic monophosphate (cAMP) or cytosolic Ca2+. The Cl- conductances activated by cAMP and Ca2+ were distinct, since they were differentially inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and, to a lesser extent, by 5-nitro-2-(3-phenylpropylamino)benzoic acid and diphenylamine-2 carboxylate. Patch-clamp studies confirmed the presence of Cl- channels activated by cAMP and Ca2+, with differential inhibition by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. The presence of CFTR Cl- channels in PDEC was confirmed by immunoblotting. These cultured PDEC are an optimal model for studies of pancreatic duct secretion.
Relaxant effect of xenin on rat ileum is mediated by apamin-sensitive neurotensin-type receptors
Authors: Clemens A. , Katsoulis S. , Nustede R. , Seebeck J. , Seyfarth K. , Morys-Wortmann C. , Feurle G. E. , Folsch U. R. , Schmidt W. E.
Published: December 2017

The action of xenin, a novel 25-residue peptide of the neurotensin (NT)/xenopsin family, was investigated in isolated rat ileal muscle strips and in dispersed longitudinal smooth muscle cells of rat small intestine in vitro. Xenin relaxes KCl-precontracted ileal strips dose dependently (1 nM-3 microM). The order of potency of the investigated peptides was as follows: xenopsin = NT = xenin > neuromedin N. Kinetensin was inactive. Tetrodotoxin, hexamethonium, tetraethylammonium, 4-aminopyridine, and NG-nitro-L-arginine did not influence the relaxant effects of xenin or NT, whereas the K+ channel blocker apamin nearly abolished their effects. Desensitization against one of the peptides or blockade of NT receptors by SR-48692 prevented the effect of xenin and NT. Structure-activity experiments revealed that the COOH-terminal part of the molecules of xenin and NT is essential for biological activity. Experiments with isolated dispersed smooth muscle cells and binding studies on intestinal smooth muscle cell membranes confirmed and extended the results obtained with muscle strips. In conclusion, xenin relaxes rat ileal smooth muscle via a muscular NT-type apamin-sensitive receptor.
Both free and complexed trypsin inhibitors stimulate pancreatic secretion and change duodenal enzyme levels
Authors: Pusztai A. , Grant G. , Bardocz S. , Baintner K. , Gelencser E. , Ewen S. W.
Published: December 2017

Secretion of pancreatic digestive enzymes was measured in pancreatic cannulated rats after duodenal stimulation with Kunitz or Bowman-Birk protease inhibitors or their complexes with trypsin and/or chymotrypsin. Free and complexed inhibitors were bound by the duodenal epithelium, stimulated the discharge of cholecystokinin, and significantly increased secretion rates of alpha-amylase, trypsinogen, and chymotrypsinogen. Inasmuch as secretion rates returned to basal levels with cholecystokinin-A receptor antagonists, the stimulation was likely to be mediated by cholecystokinin. Soya factors also influenced the duodenal concentration of pancreatic enzymes under simulated feeding conditions. Thus the level of alpha-amylase increased while the trypsin concentration decreased in rats gavaged with free or complexed inhibitors. The same was true for chymotrypsin when the Bowman-Birk inhibitor was used, but the Kunitz inhibitor and its trypsin complex actually raised the luminal concentration of chymotrypsin. Accordingly, because soya inhibitors remained effective in stimulating pancreatic secretion after elimination of their inhibitory activity by complex formation, it is questionable whether the signal for cholecystokinin secretion was solely due to lowering of duodenal protease levels.
Enhanced metabolism of arginine and glutamine in enterocytes of cortisol-treated pigs
Authors: Flynn N. E. , Wu G.
Published: December 2017

This study was designed to determine whether cortisol plays a role in arginine and glutamine metabolism in enterocytes and, more specifically, whether cortisol regulates metabolic changes in these cells during weaning. Twenty-eight 21-day-old suckling pigs were randomly assigned to one of four groups (7 animals in each) and received intramuscular injections of vehicle solution (sesame oil) (control group), hydrocortisone 21-acetate (HYD) (25 mg/kg body wt), RU-486 (10 mg/kg body wt) (a potent blocker of glucocorticoid receptors), or HYD plus RU-486. At 29 days of age, pigs were killed for preparation ofjejunal enterocytes. During the entire experimental period, pigs were nursed by sows. Activities of argininosuccinate synthase, argininosuccinate lyase (ASL), arginase, and pyrroline-5-carboxylate (P5C) synthase were measured. For metabolic studies, enterocytes were incubated for 30 min at 37 degrees C in 2 ml of Krebs-bicarbonate buffer (pH 7.4) containing 0, 0.5, or 2 mM [U-(14)C]arginine or [U-(14)C]glutamine. Compared with control, cortisol administration increased 1) the activities of ASL and arginase and the production of CO(2), ornithine, and proline from arginine, and 2) P5C synthase activity and the formation of glutamate, alanine, aspartate, ornithine, citrulline, proline, and CO(2) from glutamine in enterocytes. The stimulating effects of cortisol on the enzyme activities and the metabolism of arginine and glutamine were abolished by coadministration of RU-486. Our data suggest that cortisol plays an important role in regulating arginine and glutamine metabolism in enterocytes via a glucocorticoid receptor-mediated mechanism.
Expression of insulin-like growth factor I receptors and binding proteins by colonic smooth muscle cells
Authors: Zeeh J. M. , Ennes H. S. , Hoffmann P. , Procaccino F. , Eysselein V. E. , Snape W. J. , McRoberts J. A.
Published: December 2017

We recently demonstrated upregulation of insulin-like growth factor I (IGF-I) binding sites in the smooth muscle layer of inflamed rat colon. The increase in binding sites was due to increased expression of IGF binding proteins (IGFBPs), which modulate the effects of IGF. To further study the role of IGF in the colon, we investigated whether cultured colonic smooth muscle cells (SMC) express IGF-I receptors and IGFBPs. SMC were isolated by collagenase digestion from rat colonic smooth muscle and grown in primary culture. Equilibrium binding experiments using (125)I-labeled IGF-I showed the presence of an IGF-I receptor with a dissociation constant of 1.96 nM and a maximal binding constant of 53,000 receptors/cell. Competition binding studies with IGF-II and insulin, together with chemical cross-linking experiments, corroborated this conclusion. Western ligand blotting of conditioned medium and Northern analysis of total RNA demonstrated that the cells expressed and secreted IGFBP-4, -5, and -3 with molecular masses of 25, 31, and 45 kDa, respectively. These results, together with our in vivo studies in the rat, support a role for IGF in tissue fibrosis and stricture formation during chronic intestinal inflammation.
Effects of nitric oxide synthesis inhibition on mesenteric perfusion in young pigs
Authors: Alemany C. A. , Oh W. , Stonestreet B. S.
Published: December 2017

We tested the hypothesis that nitric oxide (NO) modulates postprandial hyperemia in young pigs. To test this hypothesis, we studied five groups of 3-wk-old pigs: group 1: milk fed, N(G)-monomethyl-L-arginine (L-NMMA) and L-arginine (L-Arg) treated (n = 10); group 2: milk fed, placebo treated (n = 8); group 3: water fed, L-NMMA and L-Arg treated (n = 6); group 4: water fed, placebo treated (n = 4); and group 5: fasted sham fed, L-NMMA and L-Arg treated (n = 6). After catheter placement and electromagnetic flow probe instrumentation of the mesenteric artery, systemic blood pressure and mesenteric artery blood flow were measured during preprandial baseline, postprandial, and postprandial intra-arterial L-NMMA- and L-Arg-infused study periods. The same measurements were made in the milk- and water-fed placebo-treated groups except that diluent replaced the L-NMMA and L-Arg infusions. In the milk- and water-fed placebo-treated groups, a significant (analysis of variance, P < 0.05), postprandial hyperemia was observed. The change in blood flow was greater (P < 0.05) in the milk-fed group than in the water-fed group. Inhibition of NO synthesis with L-NMMA diminished (P < 0.05) the hyperemic responses to both water and milk feeding and resulted in a decrease (P < 0.05) in mesenteric artery perfusion in the fasted sham-fed state. We conclude that, in young pigs, NO is a modulator of mesenteric vascular tone in both the postprandial and fasted states.
Biological determinants of intestinotrophic properties of GLP-2 in vivo
Authors: Tsai C. H. , Hill M. , Drucker D. J.
Published: December 2017

Glucagon-like peptide 2 (GLP-2) appears to regulate growth of the small bowel epithelium. The importance of dose, vehicle, and route of administration for GLP-2 bioactivity was examined in vivo. Mouse small bowel weight increased following subcutaneous injection of GLP-2, 0.25 to 5.0 microg twice daily, with an increment in jejunal crypt plus villus height detected with only 250 ng GLP-2 twice daily for 14 days (1.35-fold greater than control, P < 0.05). Small bowel weight was increased by 6 days of GLP-2 administration (1.4-fold greater than control, P < 0.05) and further increased after up to 14 days of GLP-2 treatment (P < 0.001, GLP-2 vs. control). GLP-2 increased small bowel weight in either 12% gelatin or phosphate-buffered saline (PBS) formulations (P < 0.01 vs. P < 0.05, gelatin vs. PBS, respectively), and GLP-2 was intestinotrophic following either intramuscular, intraperitoneal, or subcutaneous administration (P < 0.05 for im and ip, P < 0.001 for sc, GLP-2 vs. control). GLP-2 also induced bowel growth when administered daily or every other day. These results define important biological parameters for the activity of GLP-2 in vivo.
Molecular mechanisms for somatostatin inhibition of c-fos gene expression
Authors: Todisco A. , Takeuchi Y. , Yamada J. , Sadoshima J. I. , Yamada T.
Published: December 2017

We reported previously that somatostatin inhibits the expression of the immediate early gene c-fos. Accordingly, we characterized the molecular mechanisms by which somatostatin inhibits c-fos gene expression. Because growth factors activate c-fos through a region of its promoter known as the serum response element [SRE; base pairs (bp) -357 to -276] we transfected rat pituitary adenoma cells (GH3) with plasmids containing the SRE or the SRE core fragment (bp -320 to -298) upstream of the luciferase reporter gene. Epidermal growth factor (EGF) stimulated SRE-luciferase activity, and this effect was inhibited by somatostatin and by the analog MK-678. Identical results were obtained with the SRE core plasmid, demonstrating that the sequence between bp -320 and -298 of the c-fos promoter is a somatostatin response element. Because the extracellular signal-regulated protein kinases (ERKs) induce the SRE via phosphorylation of transcription factors such as Elk-1, we examined the effect of somatostatin on ERK phosphorylation and activation. EGF stimulated tyrosine phosphorylation of ERK2, and MK-678 attenuated this effect. In experiments using in-gel kinase assays, MK-678 also inhibited EGF-stimulated ERK activity via a pertussis toxin sensitive pathway, and this effect resulted in inhibition of Elk-1 transcriptional activity. Our data suggest that one mechanism of somatostatin action involves inhibition of ERK activity, Elk-1 phosphorylation and transcriptional activation, and ultimately c-fos gene transcription.
Gastrin regulates the human histidine decarboxylase promoter through an AP-1-dependent mechanism
Authors: Hocker M. , Zhang Z. , Merchant J. L. , Wang T. C.
Published: December 2017

The histidine decarboxylase (HDC) gene is regulated transcriptionally by gastrin and phorbol 12-myristate 13-acetate (PMA) through a protein kinase C (PKC)-related pathway. To determine the role of AP-1 (fos/jun) in the regulation of the HDC promoter, gastric cancer (AGS-B) cells stably expressing the cholecystokinin-B/ gastrin receptor and the 1.8-kb human (h) HDC-luciferase (luc) construct were cotransfected with constructs expressing c-fos and c-jun. Overexpression of c-fos and c-jun activated the HDC promoter in a dose-dependent fashion in 1.8-kb hHDC-luc/AGS-B cells as well as in transfected F9 embryonal carcinoma cells, which lack endogenous AP-1 activity. PMA was unable to activate the HDC promoter in F9 cells, which were not transfected with c-fos and c-jun. Gastrin stimulation increased c-fos and c-jun mRNA abundance and AP-1-dependent transcriptional activity, as assessed by a reporter construct in which the CAT reporter gene is under the control of a 12-O-tetradecanoylphorbol-13-acetate response element multimer. Gastrin-stimulated HDC promoter activity was blocked by transfection of c-fos antisense and dominant negative c-jun expression constructs. Finally, overexpression of c-fos and c-jun activated the hHDC promoter through a downstream cis-acting element (gastrin response element), which does not bind AP-1. In conclusion, activation of AP-1 is essential for gastrin-stimulated HDC transcription, but the mechanism appears to be indirect.
Programming of hepatic insulin-sensitive enzymes in offspring of rat dams fed a protein-restricted diet
Authors: Desai M. , Byrne C. D. , Zhang J. , Petry C. J. , Lucas A. , Hales C. N.
Published: December 2017

Hepatic enzymes associated with glucose hemostasis were studied in offspring of dams fed either a 20% protein (control) or an isocaloric 8% protein (low-protein) diet during pregnancy and lactation. Additionally, offspring were exposed to maternal 8% protein diet only during gestation (recuperated) or lactation (postnatal low-protein). Glucokinase activity decreased (approximately 50%), whereas phosphoenolpyruvate carboxykinase (PEPCK) activity increased (approximately 100%), in the low-protein and recuperated offspring compared with controls (P < 0.001) at 21 days of age. However, the postnatal low-protein offspring had enzyme activities comparable with those of controls. These changes were still evident in 11-mo-old offspring weaned onto a normal laboratory chow. Parallel changes were apparent in mRNA levels of glucokinase and PEPCK in the low-protein male offspring. Thus the effect of programming metabolism extends not only to protein biochemistry but possibly also to the regulation of gene expression. Furthermore, these changes could not be attributed to glucagon or insulin, because ratios of these hormones were comparable between the control and low-protein groups.
GH elevates serum IGF-I levels but does not alter mucosal atrophy in parenterally fed rats
Authors: Peterson C. A. , Carey H. V. , Hinton P. L. , Lo H. C. , Ney D. M.
Published: December 2017

Growth hormone (GH) action is primarily mediated by insulin-like growth factor I (IGF-I), although both growth factors show tissue-selective effects. We investigated the effects of GH, IGF-I, and GH plus IGF-I on jejunal growth and function in rats maintained with total parenteral nutrition (TPN) and given recombinant human GH (rhGH) (400 micrograms/day sc, twice daily) and/or rhIGF-I (800 micrograms/day in TPN solution) for 5 days. Administration of GH or IGF-I alone produced similar increases in serum IGF-I levels and body weight; GH plus IGF-I further increased these parameters. TPN reduced mucosal mass, protein and DNA content, villus height, crypt depth, and enterocyte migration rate. IGF-I or GH plus IGF-I produced equivalent increases in all intestinal growth parameters; GH alone had no effect. GH, IGF-I, or GH plus IGF-I reduced TPN-induced increases in sucrase-specific activity. IGF-I, but not GH, attenuated TPN-induced increases in tissue conductance and carbachol-stimulated ion secretion. In contrast to IGF-I, GH does not stimulate intestinal growth during TPN and has less effect on normalizing TPN-induced changes in epithelial function.
Kinetic and molecular identification of sodium-dependent glucose transporter in normal rat cholangiocytes
Authors: Lazaridis K. N. , Pham L. , Vroman B. , de Groen P. C. , LaRusso N. F.
Published: December 2017

While previous work has demonstrated that monosaccharides can be absorbed from bile, studies of sugar transport by the biliary, epithelia (i.e., cholangiocytes) are lacking. Using a novel model of polarized rat cholangiocytes in primary culture, designated normal rat cholangiocytes (NRC), we examined directly the uptake and transcellular transport of a nonmetabolizable monosaccharide, methyl alpha-D-glucopyranoside (AMG). When the apical or basolateral domain of cholangiocytes was exposed to radiolabeled AMG or sucrose (control), only apical absorption of AMG was evident. This apical uptake was time dependent, saturable, and significantly inhibited (> or = 90%) by removal of Na+ or in the presence of phlorizin (0.1 mM), a competitive inhibitor of the Na(+)-glucose cotransporter. The transcellular flux of AMG was also polar (i.e., apical to basolateral). Reverse transcriptase-polymerase chain reaction (RT-PCR) revealed the presence of the transcript for the specific Na(+)-glucose cotransporter SGLT1 in NRC and in freshly isolated cholangiocytes but not in purified hepatocytes; in contrast, the transcript for SGLT2 was absent in all liver samples. In situ RT-PCR on frozen sections of normal rat liver showed that SGLT1 was expressed exclusively in cholangiocytes. Immunoblot analysis using a specific polyclonal antibody for the facilitative glucose transporter GLUT1 demonstrated it to be present in vesicles derived from NRC enriched in basolateral plasma membrane domains. Our data are consistent with the concept that SGLT1 is present on the apical domain of biliary epithelia and, in conjunction with GLUT1 on the basolateral domain, accounts for glucose absorption from bile.
Modulation of hormone-induced calcium oscillations by intracellular pH in rat hepatocytes
Authors: Chatton J. Y. , Liu H. , Stucki J. W.
Published: December 2017

Single isolated rat hepatocytes were used to investigate the influence of intracellular pH (pHi) on hormone-induced cytosolic Ca2+ oscillations, using videofluorescence microscopy. Although pHi did not vary after alpha-adrenergic stimulation, manipulations of pHi induced pronounced alterations in the frequency of oscillations. Increasing the resting pHi with ammonium chloride (5-20 mM), trimethylammonium (2-10 mM), or triethylammonium (1.2-8 mM) reduced the frequency of oscillations. A change in pHi of > 0.25 was sufficient to reversibly inhibit oscillations. This effect could be overcome by increasing the agonist concentration or by adding 8-bromoadenosine 3',5'-cyclic monophosphate, an agent known to potentiate the alpha-adrenergic response. Cellular acidification, obtained by the ammonium prepulse method as well as by application of acetate or the ionophore nigericin, in the continuous presence of agonist was accompanied by a modest frequency increase of the oscillations, leading in some cases to an overstimulated state. This study indicates that pHi, within a range of values expected to occur in vivo (0.1-0.2 pH units), exerts a chronotropic effect on phenylephrine-induced Ca2+ oscillations. In contrast, oscillations induced by ADP or vasopressin were pHi invariant.
Intracisternal antisense oligonucleotides to TRH receptor abolish TRH-evoked gastric motor excitation
Authors: Sivarao D. V. , Krowicki Z. K. , Abrahams T. P. , Hornby P. J.
Published: December 2017

Thyrotropin-releasing hormone (TRH) from the nucleus raphe obscurus (nROb) innervates the dorsal vagal complex (DVC) and activates gastric motor function. Assessment of the importance of TRH has been hampered by the lack of TRH receptor antagonists. To overcome this, rats were given intracisternal antisense oligonucleotides against the first 18 bases of TRH receptor mRNA, mismatch oligonucleotides, or saline. Rats were anesthetized, and L-glutamate (15 nmol), TRH (1 and 10 pmol), and saline were microinjected into the DVC and nROb while gastric motor function was monitored. Intracisternal TRH mRNA antisense oligonucleotides abolished the gastric excitatory affects of microinjection of TRH, but not L-glutamate, into the DVC, and the response to TRH recovered after 2 wk of no antisense treatment. Chemical stimulation of the nROb increased intragastric pressure in saline- and mismatch- but not antisense-treated animals. These studies demonstrate that intracisternal TRH receptor antisense oligonucleotides produce a selective and reversible "knockdown" of responsiveness to exogenous TRH in the DVC, as well as to excitation of an endogenous TRH pathway controlling gastric function. It also provides a new tool for assessment of TRH pathways in hindbrain control of gastric function.
Bombesin-stimulated ceramide production and MAP kinase activation in rabbit rectosigmoid smooth muscle cells
Authors: Sbrissa D. , Yamada H. , Hajra A. , Bitar K. N.
Published: December 2017

We have investigated the hypotheses that 1) bombesin activation of protein kinase C (PKC) results in the hydrolysis of sphingolipids and the production of ceramide and that 2) ceramide produced on activation by bombesin mediates sustained contraction of smooth muscle cells by activation of PKC and mitogen-activated protein (MAP) kinase. Ceramide production was assessed using a technique that involved benzoylation of purified ceramide extracts, followed by reverse-phase high-performance liquid chromatography. Contraction of smooth muscle cells isolated from the rabbit rectosigmoid and stimulated with bombesin gave a significant increase in newly formed ceramide (38 +/- 3.5%). 12-O-tetradecanoylphorbol-13-acetate also induced production of ceramide, which was blocked by calphostin C. The short-chain permeable C2 ceramide induced a sustained contraction and activation of MAP kinase, which was blocked by calphostin C. The increase in MAP kinase activity was maximal at 30 s and declined at 2 min. The data suggest that stimulation of smooth muscle cells by bombesin results in a functional coupling between sn-1,2-diacylglycerol (DAG)/ PKC and a sphingomyelinase, whereby DAG activates the hydrolysis of sphingomyelin to produce ceramide. Ceramide in turn activates PKC, which then activates MAP kinase. This could be the basis for the sustained contraction observed with bombesin.
Effect of cholecystokinin-A receptor blockade on lipid-induced gastric relaxation in humans
Authors: Mesquita M. A. , Thompson D. G. , Troncon L. E. , D'Amato M. , Rovati L. C. , Barlow J.
Published: December 2017

The purpose of this study was to assess the role of endogenous cholecystokinin (CCK) in regulating fat-induced changes in human gastric relaxation. Proximal gastric pressure-volume relationships were determined in 12 healthy volunteers during a series of gastric distensions, both fasting and after intragastric instillation of 250 ml of 10% Intralipid. All subjects were studied twice, in a randomized, double-blind study, during intravenous infusion of either loxiglumide (CCK-A antagonist) or saline. For each distension, intragastric pressure and compliance were determined together with perception intensity. During saline infusion, Intralipid reduced intragastric pressure (prelipid, 11.7 +/- 0.8; postlipid, 9.7 +/- 0.6 mmHg; P = 0.002) and increased compliance (pressure-volume slope values: prelipid, 87.6 +/- 9.7; postlipid, 47.2 +/- 7; P < 0.01). Loxiglumide infusion during fasting exerted no effect on either intragastric pressure or compliance. After lipid, however, loxiglumide abolished the expected postlipid reduction in intragastric pressure (prelipid, 12.1 +/- 0.7; postlipid, 11.5 +/- 0.8 mmHg; P = 0.4) but did not consistently abolish the postlipid increase in compliance. Loxiglumide exerted no effect on the cumulative perception score or on the volume at perception threshold, although it prevented the fat-induced reduction in pressure at perception threshold [control: prelipid, 15.4 +/- 1.1; postlipid, 10.7 +/- 0.5 (P < 0.05); loxiglumide: prelipid, 13.8 +/- 1.5; postlipid, 12.2 +/- 0.9 (P > 0.05)]. Endogenous CCK or CCK-A receptors therefore play a role in the fat-induced reduction of intragastric pressure and might also modulate gastric perception after lipid.
Exposure to cigarette smoke delays the plasma clearance of chylomicrons and chylomicron remnants in rats
Authors: Pan X. M. , Staprans I. , Hardman D. A. , Rapp J. H.
Published: December 2017

We investigated the effects of cigarette smoke exposure on the clearance of chylomicrons (CM) and CM remnants in rats after administration of a fat-containing meal. There was a decrease in clearance of both postprandial CM and exogenous radiolabeled CM in smoke-exposed animals. For exogenous CM, clearance (t1/2) increased significantly for both triglyceride and cholesterol labels and correlated with the delay in liver uptake. This decrease in lipid clearance could not be explained by decreased lipoprotein lipase (LPL) activity because smoke exposure resulted in a significant increase in LPL activity. When the hydrolysis of CM by endothelial LPL was tested in a heart perfusion system, there was no difference in CM hydrolysis between the two groups. Hepatic lipase activity was also unchanged in smoke-exposed animals. However, there was a significant delay in the CM remnant uptake into livers isolated from smoke-exposed rats. Thus the delay in CM clearance in smoke-exposed animals cannot be attributed to reduced lipase activities but results from impaired hepatic uptake of CM remnants.
Polyamines are important for attachment of IEC-6 cells to extracellular matrix
Authors: Santos M. F. , Viar M. J. , McCormack S. A. , Johnson L. R.
Published: December 2017

The inhibition of ornithine decarboxylase, a rate-limiting enzyme of polyamine biosynthesis, with alpha-difluoromethylornithine in IEC-6 cells (small intestinal crypt cell line) reduces cell migration by 70%, inhibits protein cross-linking, and affects the cytoskeletal assembly. The current study examines the effects of intracellular polyamine depletion on attachment of IEC-6 cells to different matrices. Polyamine deficiency inhibited cell attachment to plastic, laminin, fibronectin, collagen IV, and Matrigel by different extents. Intracellular putrescine restored attachment to all matrices. The presence of a specific inhibitor of protein cross-linking also inhibited attachment to laminin in a dose-dependent manner. The inhibition of cell attachment to plastic and Matrigel was correlated with the inhibition of cell migration. Immunofluorescence studies showed that polyamines are essential for the correct expression of the integrin subunit alpha 2 but not for the expression of the alpha 1-subunit. This study demonstrates that polyamines are important for cell attachment and expression of the integrin alpha 2 beta 1, a putative receptor for collagen and laminin. The impairment of protein cross-linking and the inhibition of the expression of cell surface receptors that bind extracellular matrix (ECM) proteins may be part of the mechanism by which polyamine deficiency retards cell migration in the small intestine.
Rapid in vivo hydrolysis of fatty acid ethyl esters, toxic nonoxidative ethanol metabolites
Authors: Saghir M. , Werner J. , Laposata M.
Published: December 2017

Fatty acid ethyl esters (FAEE), esterification products of fatty acids and ethanol, are in use as fatty acid supplements, but they also have been implicated as toxic mediators of ethanol ingestion. We hypothesized that hydrolysis of orally ingested FAEE occurs in the gastrointestinal (GI) tract and in the blood to explain their apparent lack of toxicity. To study the in vivo inactivation of FAEE by hydrolysis to free fatty acids and ethanol, we assessed the hydrolysis of FAEE administered as an oil directly into the rat stomach and when injected within the core of low-density lipoprotein particles into the circulation of rats. Our studies demonstrate that FAEE are rapidly degraded to free fatty acids and ethanol in the GI tract at the level of the duodenum with limited hydrolysis in the stomach. In addition, FAEE are rapidly degraded in the circulation, with a half-life of only 58 s. Thus the degradation of FAEE in the GI tract and in the blood provides an explanation for the apparent lack of toxicity of orally ingested FAEE.
Expression of insulin receptors and of 60-kDa receptor substrate in rat mature and immature enterocytes
Authors: Buts J. P. , De Keyser N. , Marandi S. , Maernoudt A. S. , Sokal E. M. , Rahier J. , Hermans D.
Published: December 2017

The mechanism(s) by which rat immature enterocytes exhibit increased responsiveness to insulin before weaning is unknown. Therefore, we have analyzed the distribution, ontogeny, and molecular properties of insulin receptors (IR) and of related substrates in immature and mature enterocytes. IR were studied by radioligand binding assays, cross-linking labeling, immunohistochemistry, and in vitro phosphorylated substrates by immunoprecipitation. Regardless of age, 125I-insulin binding to IR was five times higher in crypt cells than in villus cells and two times higher in the ileum than in the jejunum. Binding capacity to villus cells from sucklings (day 14) exceeded three times that of older animals (day 30 and day 60). Scatchard analysis of equilibrium binding data confirmed an age-related decrease in low- and high-affinity receptor classes without change in affinity constants. In concordance, both alpha- and beta-IR subunits were more abundant in immature than in mature membranes. In vitro, insulin elicited the phosphorylation of three membrane proteins (96, 60 and 42 kDa), whose signals were virtually inhibited by preincubating membranes with antireceptor monoclonal antibodies. By immunoprecipitation, the 60-kDa signal was rapidly detected as a tyrosine-phosphorylated protein, expressed in mature and immature membranes, and identified as a receptor substrate phosphorylated in vitro by the IR tyrosine kinase. In conclusion, 1) increased responsiveness of rat immature enterocytes to insulin could be related to high membrane concentrations of IR and 2) normal rat enterocytes express a 60-kDa phosphotyrosine protein identified as a direct substrate of the IR tyrosine kinase.
P-selectin-deficient mice are protected from PAF-induced shock, intestinal injury, and lethality
Authors: Sun X. , Rozenfeld R. A. , Qu X. , Huang W. , Gonzalez-Crussi F. , Hsueh W.
Published: December 2017

In a previous study, we showed that anti-CD11b or anti-CD18 antibody markedly attenuated platelet-activating factor (PAF)-induced shock and intestinal necrosis in rats, whereas anti-P-selectin anti-body was ineffective. Here we used genetically altered mice to study the mechanism of PAF in mice. We found that P-selectin-deficient mice are completely protected from the adverse effects of PAF with no mortality or intestinal injury and only mild hemoconcentration and transient hypotension. In contrast, CD18- or intercellular adhesion molecule 1 (ICAM-1)-deficient mice were not protected from PAF-induced tissue injury and death. However, when ICAM-1-, but not CD18-, deficient mice were pretreated with fucoidin, the adverse effects of PAF were markedly reduced; survival was 100%, although hypotension still developed. Neutrophil-depleted mice were protected from PAF-induced intestinal injury but still developed hypotension and hemoconcentration. PAF increases peripheral blood neutrophil counts, probably by inducing granulopoiesis, since neutrophil-depleted mice still showed granulocytosis 60 min after PAF. Thus P-selectin plays an important role in PAF-induced injury in mice, and the selectins and the integrin-ICAM-1 system work in concert to mediate the inflammatory response to PAF in vivo.
Motilin and OHM-11526 activate a calcium current in human and canine jejunal circular smooth muscle
Authors: Farrugia G. , Macielag M. J. , Peeters T. L. , Sarr M. G. , Galdes A. , Szurszewski J. H.
Published: December 2017

Motilin is a potent agonist for gastrointestinal smooth muscle contraction and has been proposed to regulate the onset of phase III of the migrating motor complex in dogs and humans. The effects of motilin and OHM-11526, a motilin antagonist in rabbit smooth muscle strips, were examined in isolated canine and human jejunal circular smooth muscle cells using whole cell patch-clamp techniques with Ba2+ as the charge carrier. Effects of both drugs on inward current through L-type Ca2+ channels (ICaL) in both canine and human cells were first observed at 10(-3) M. At 10(-6) M, motilin increased ICaL in canine and human jejunal circular smooth muscle cells by 43 +/- 20 and 45 +/- 11%, respectively, and OHM-11526 increased ICaL by 54 +/- 8 and 54 +/- 14%, respectively. The increase in inward current was blocked by nifedipine and by guanosine 5'-O-(2-thiodiphosphate) but not by pertussis toxin. Washout of both drugs resulted in a further increase in ICaL. These data suggest that both motilin and OHM-11526 activate and ICaL in human and canine jejunal circular smooth muscle cells through a G protein-coupled mechanism.
Inhibition of mechanical activity by neurotensin in rat proximal colon: involvement of nitric oxide
Authors: Mule F. , Serio R.
Published: December 2017

The aim of the present study was to define the nature of inhibitory action of neurotensin in rat proximal colon. Mechanical activity was detected as changes of intraluminal pressure. Neurotensin (10(-10) to 10(-7) M), in the presence of atropine (10(-6) M), guanethidine (10(-6) M), and nifedipine (10(-8) M), induced a tetrodotoxin-insensitive inhibitory effect characterized by the complete disappearance of the spontaneous phasic contractions. The inhibitory effect of neurotensin (10(-7) M) was abolished by scorpion venom (Leiurus quinquestriatus hebraeus) (10(-6) g/ml) or high K+ (40 mM KCl), whereas it persisted in the presence of omega-conotoxin GVIA, (10(-7) M). N omega-nitro-L-arginine methyl ester (L-NAME) (10(-4) M to 3 x 10(-4) M) antagonized the inhibitory response to neurotensin but did not affect the response to norepinephrine. L-Arginine (5 x 10(-3) M) prevented the effect of L-NAME. The contractile action of neurotensin, observed in the absence of atropine and nifedipine, was potentiated by L-NAME (10(-4) M); L-arginine (5 x 10(-3) M) prevented the L-NAME effect. The present results suggest that in rat proximal colon the inhibitory action of neurotensin is largely due to production of nitric oxide (NO), likely released from neural prejunctional sites. Ca2+ influx through N-type channels is not a required step in the mechanism of neurotensin inhibitory action.
Identification of a bile acid response element in the cholesterol 7 alpha-hydroxylase gene CYP7A
Authors: Stroup D. , Crestani M. , Chiang J. Y.
Published: December 2017

The transcriptional activity of the cholesterol 7 alpha-hydroxylase gene CYP7A is repressed by bile acids. Taurine conjugates of chenodeoxycholate and deoxycholate, but not cholate and ursodeoxycholate, inhibited the CYP7A promoter/luciferase reporter activity in transient transfection assays in Hep G2 cells. A region from nucleotide (nt) -74 to -55 was found to mediate bile acid response. However, deletion of this bile acid response element (BARE-I) enhanced reporter activity but did not eliminate the bile acid response. This is due to the presence of another BARE-II located in a conserved region between nt -149 and -128. Deletion or mutations of these sequences reduced promoter activity and abolished bile acid repression. This BARE-II shares an identical AGTTCAAG core sequence with BARE-I. Electrophoretic mobility shift assays of BARE-I and BARE-II probes using Hep G2 nuclear extract and the partially purified binding activity of nt -65/-54 DNA-affinity column revealed that the same or a similar nuclear protein might bind to both BAREs. BARE-II is the major BARE involved in the transcriptional repression of the CYP7A gene by hydrophobic bile acids.
Potassium currents in atrial and ventricular myocytes from a rat model of cirrhosis
Authors: Ward C. A. , Ma Z. , Lee S. S. , Giles W. R.
Published: December 2017

The underlying mechanisms for the electrophysiological abnormalities that develop as a consequence of cirrhosis of the liver have been studied by recording three different K+ currents in mammalian heart tissue. Single myocytes from the atria and ventricles of sham-operated and bile ductligated (BDL) cirrhotic adult rats were current and voltage clamped using standard whole cell methods. In ventricular myocytes from cirrhotic animals, measurements of the current-voltage relationships, voltage dependence of inactivation, and reactivation kinetics of K+ currents showed that the only significant functional changes (within the physiological range of membrane potentials) were decreases in the density of expression of 1) I(t), a Ca(2+)-independent transient outward K+ current, and 2) Isus, a delayed rectifier K+ current. The decreases in I(t) and Isus contribute to the prolonged Q-T interval of the electrocardiogram that has been described in cirrhotic patients. Measurement of K+ currents in atrial myocytes demonstrated that there were no significant differences in any of the K+ current densities between sham and BDL animals, although reactivation kinetics of I(t) were slowed somewhat.
In vitro analysis of rat intestinal wall movements at rest and during propagated contraction: a new method
Authors: Benard Thierry , Bouchoucha Michel , Dupres Michel , Cugnenc Paul-Henri
Published: December 2017

Intestinal wall motions are not easily studied and are frequently deduced from manometric and electromyographic measurements. This study aimed to establish a method of wall movement analysis based on an automatic technique of image processing. Segments of rat jejunum were fixed in an organ bath under isometric conditions. A real-time edge-detection algorithm was used to find the contours of the intestine using video imaging. After the measurement, a mapping of intestinal wall movements was performed based on diameter variations. In the 260 experiments without stimulation, intestinal wall activity was always detected. Propagated activity was found in 40% of the experiments and periodic wall motion in 60%, with 0.5-Hz activity found more frequently (41%) than 0.24-Hz activity (19%). These cyclic activities, related to intestinal slow waves, had their amplitude decreased by acetylcholine and were modified by vapreotide. Analysis of a propagated wave after cholinergic stimulation showed that it is characterized by an increase of the diameter of the intestine followed by a decrease. Moreover, this methodology allows analysis of the initiation of a propagated wave.
Hydrogen peroxide-induced liver cell necrosis is dependent on AP-1 activation
Authors: Xu Yang , Bradham Cynthia , Brenner David A. , Czaja Mark J.
Published: December 2017

To determine whether intracellular signaling events involved in apoptosis may also mediate necrosis, the role of the transcription factor AP-1 was investigated in a hepatoma cell model of cellular necrosis induced by oxidant stress. Treatment of the human hepatoma cell line HuH-7 with H2O2caused dose-dependent necrosis as determined by light microscopy, fluorescent staining, and an absence of DNA fragmentation. H2O2treatment led to increases in c- fosand c- jun mRNA levels, Jun nuclear kinase activity, and AP-1 DNA binding. AP-1 transcriptional activity measured with an AP-1-driven luciferase reporter gene was also increased. To determine whether this AP-1 activation contributed to H2O2-induced cell necrosis, HuH-7 cells were stably transfected with an antisense c- jun expression vector. Cells expressing antisense c- jun had decreased levels of AP-1 activation and significantly increased survival after H2O2exposure. These data indicate that AP-1 activation occurs during oxidant-induced cell necrosis and contributes to cell death. Necrosis is therefore not always a passive process but may involve the activation of intracellular signaling pathways similar to those that mediate apoptosis.
Endothelin-1 selectively contracts portal vein through both ETAand ETBreceptors in isolated rabbit liver
Authors: Wang Hong-Gang , Shibamoto Toshishige , Miyahara Takashige
Published: December 2017

We determined the constrictive effects of endothelin (ET)-1 on the hepatic vascular resistance distribution and the receptor subtype responsible for the effect in isolated rabbit livers perfused via the portal vein with 5% albumin-Krebs solution. The sinusoidal pressure was estimated using the double vascular occlusion pressure. The basal portal venous resistance comprised 59% of the total portal-hepatic venous resistance. In response to a bolus injection of ET-1 (0.05–5 μg), which led to a final concentration of 0.1–10 nM in the recirculating perfusate, the portal venous resistance increased in a dose-dependent manner, whereas the hepatic venous resistance did not change significantly at any concentration. This hepatic vasoconstriction was associated with liver weight loss. The selective portal venous constriction induced by ET-1 was confirmed in livers perfused retrogradely from the hepatic vein to the portal vein. The ET-1-induced hepatic vasoconstriction was significantly attenuated by the selective ETAreceptor antagonist BQ-123 (1 μM). The ETBreceptor antagonist BQ-788 (1 μM) also attenuated the constriction at ET-1 concentrations less than 10 nM. The combination of BQ-123 and BQ-788 tended to inhibit the hepatic vasoconstriction more effectively than BQ-123 alone. These results suggest that ET-1 selectively constricts the portal vein via both ETAand ETBreceptors, with predominance of ETAreceptor in isolated albumin-Krebs-perfused rabbit livers.
V. Transcriptional regulation, development, and neoplasia of the intestinal epithelium
Authors: Traber Peter G.
Published: December 2017

Coordination of gene transcription is a critical regulatory step in orchestrating developmental, differentiation, and adaptation processes in the mammalian intestinal epithelium. An understanding of the regulatory network of nuclear proteins that direct transcriptional initiation of intestinal genes will provide insight into the mechanisms of normal development and differentiation as well as disease processes such as neoplasia.