bluuebunny/crossref_metadata_2025 huggingface dataset:
Mucosal mast cells are involved in CCK disruption of MMC in the rat intestine
Authors: Juanola Carme , Giralt Magda , Jiménez Marcel , Mourelle Marisabel , Vergara Patri
Published: December 2017

Our aim was to determine if mucosal mast cells could be activated by endogenous CCK and, as a consequence, mediate CCK actions in the small intestine. Rats were prepared for electromyography to record electrical activity in the small intestine. In another group of animals, the duodenum was perfused to measure rat mast cell protease II (RMCP II) as indicative of mast cell degranulation. Endogenous CCK release was induced by administration of soybean trypsin inhibitor (SBTI) in conscious rats or by intraduodenal perfusion of ovalbumin hydrolysate (OVH) in anesthetized rats. CCK concentration was measured by bioassay on pancreatic acini. SBTI in control rats disrupted migrating motor complexes (MMC) for >40 min. In rats treated with the mast cell stabilizer ketotifen, SBTI did not induce any change in the MMC pattern. RMCP II concentration in the duodenal perfusate significantly increased after OVH. Perfusate from ketotifen-treated animals did not show any significant increase in RMCP II values during OVH perfusion, although CCK plasma concentration was not different from the control group. Furthermore, infusion of the CCK-B receptor antagonist L-365,260 significantly blocked the increase of RMCP II concentration after OVH. Our results indicate that mucosal mast cells are degranulated by endogenous CCK release through stimulation of CCK-B receptors. Therefore mucosal mast cells participate in CCK intestinal actions.
Modulation of secretin release by neuropeptides in secretin-producing cells
Authors: Chang Cecilia H. , Chey William Y. , Erway Brian , Coy David H. , Chang Ta-Min
Published: December 2017

Nerve fibers containing bombesin (BB)/gastrin-releasing polypeptide (GRP), pituitary adenylate cyclase-activating polypeptide (PACAP), vasoactive intestinal polypeptide (VIP), or galanin are known to innervate the mucosa of the upper small intestine. Both BB/GRP and PACAP have been shown to elicit secretin secretion in vivo. We studied whether the above-mentioned neuropeptides can act directly on secretin-producing cells, including the murine neuroendocrine cell line STC-1 and a secretin cell-enriched preparation isolated from rat upper small intestinal mucosa. Secretin release from both cell types was stimulated by various agents known to elicit secretin release and by the neuropeptides BB, GRP, and PACAP, suggesting a comparable response between the two cell preparations. The effects of neuropeptides were further studied in STC-1 cells. BB, GRP, and PACAP stimulated secretin release time and concentration dependently. VIP also stimulated secretin release concentration dependently. Stimulation by BB/GRP or PACAP was accompanied by elevation of inositol-1,4,5-trisphosphate (IP3) or cAMP, respectively. The stimulatory effect of PACAP on secretin release was synergistically enhanced by BB without any synergistic increase in IP3or cAMP production, suggesting cross talk between different signal transduction pathways downstream of the production of these two second messengers. The L-type Ca2+channel blocker diltiazem (10 μM) and the Ca2+chelator EGTA (1 mM) significantly inhibited BB-stimulated secretin release by 64% and 59%, respectively, and inhibited PACAP-stimulated release by 75% and 55%, respectively. The protein kinase A-specific inhibitor Rp-cAMPS (100 μM) also inhibited both BB- and PACAP-stimulated secretin release by 30% and 62%, respectively. Galanin inhibited BB- and PACAP-stimulated secretin release and production of second messengers in a concentration-dependent and pertussis toxin-sensitive manner. These results suggested that the neuropeptides BB/GRP, PACAP, VIP, and galanin can modulate secretin release in secretin-producing cells and that STC-1 cells can serve as a useful model for studying the cellular mechanism of secretin secretion elicited by luminal secretagogues and neuropeptides.
Duodenal Ca2+absorption is not stimulated by calcitriol during early postnatal development of pigs
Authors: Schroeder Bernd , Dahl Maria Regina , Breves Gerhard
Published: December 2017

The role of calcitriol in stimulating intestinal active Ca2+absorption during postnatal life was studied in newborn, suckling, and weaned control (Con) piglets and piglets suffering from inherited calcitriol deficiency (Def piglets). In addition, a group of Def piglets was treated with vitamin D3(Def-D3piglets), which normalized plasma calcitriol levels. Regardless of age, duodenal calbindin-D9kconcentrations ranged between 1,839 and 2,846 μg/g mucosa in Con piglets, between 821 and 1,219 μg/g mucosa in Def piglets, and between 2,960 and 3,692 μg/g mucosa in Def-D3animals. In weaned animals, active Ca2+absorption as calculated from in vitro45Ca2+flux rate measurements in Ussing chambers could be related to calbindin-D9klevels. Thus active Ca2+absorption was completely absent in Def animals but was reconstituted in Def-D3animals. In contrast, in newborn Def piglets active Ca2+absorption functioned normally despite the low plasma calcitriol and mucosal calbindin-D9klevels and could not be affected by treatment with vitamin D3. Similar results were obtained from suckling Def piglets. The microtubule-disrupting agent colchicine caused significant inhibition of transepithelial net Ca2+absorption in duodenal epithelia from newborn piglets without exerting an effect in suckling and weaned animals. Colchicine had no effect on Ca2+uptake across the brush border membrane of mucosal enterocytes or on glucose-dependent electrogenic net ion flux rates in duodenal preparations from newborn Con piglets. In conclusion, our findings reveal intestinal active Ca2+absorption during early postnatal life of pigs that involves calcitriol-independent mechanisms and that may include intact microtubule actions.
Determination of lingual myoarchitecture in whole tissue by NMR imaging of anisotropic water diffusion
Authors: Gilbert Richard J. , Reese Timothy G. , Daftary Shrenik J. , Smith R. Neal , Weisskoff Robert M. , Wedeen Van J.
Published: December 2017

The muscular anatomy of the tongue consists of a complex three-dimensional array of fibers, which together produce the variations of shape and position necessary for deglutition. To define the myoarchitecture of the intact mammalian tongue, we have utilized NMR techniques to assess the location and orientation of muscle fiber bundles through measurement of the direction-specific diffusional properties of water molecules. Whole sheep tongues were excised and imaged with a slice-selective stimulated-echo diffusion sequence in the midline sagittal plane, and three-dimensional diffusion tensors were determined for each voxel. The derived diffusion tensors were depicted graphically as octahedra whose long axes indicate local muscle fiber orientation. Two distinct groups of midline fibers were identified: 1) in-plane sagittal fibers originating in the posteroinferior region of the tongue, radiating with a fanlike projection anteriorly and superiorly and merging with vertically oriented fibers, and 2) cross-plane (transverse) fibers, oriented at right angles to the vertically aligned fibers, predominantly in the anterior and superior regions of the tongue. Regional comparison of diffusion anisotropy revealed uniform and parallel alignment (high anisotropy) in the posteroinferior region of the tongue, corresponding to the base of the genioglossus, and less uniform, orthogonally aligned fibers (low anisotropy) in the anterosuperior region of the tongue, corresponding to the core intrinsic muscles. These data indicate that lingual myoarchitecture, determined through direction-dependent mobility of water molecules, can be depicted as discrete regions of muscle fibers, whose orientation and extent of diffusion anisotropy predict local contractility.
iNOS expression in human intestinal microvascular endothelial cells inhibits leukocyte adhesion
Authors: Binion David G. , Fu Sidong , Ramanujam Kalathur S. , Chai Yuh Cherng , Dweik Raed A. , Drazba Judith A. , Wade Justin G. , Ziats Nicholas P. , Erzurum Serpil C. , Wilson Keith T.
Published: December 2017

Increased nitric oxide (NO) production by inducible nitric oxide synthase (iNOS) has been associated with intestinal inflammation, including human inflammatory bowel disease. However, NO can downregulate endothelial activation and leukocyte adhesion, critical steps in the inflammatory response. Using primary cultures of human intestinal microvascular endothelial cells (HIMEC), we determined the role of NO in the regulation of HIMEC activation and interaction with leukocytes. Both nonselective ( NG-monomethyl-l-arginine) and specific ( N-iminoethyl-l-lysine) competitive inhibitors of iNOS significantly increased binding of leukocytes by HIMEC activated with cytokines and lipopolysaccharide. Increased adhesion was reversible with the NOS substratel-arginine and was not observed in human umbilical vein endothelial cells (HUVEC). Activation of HIMEC significantly upregulated HIMEC iNOS expression and NO production. NOS inhibitors did not augment cell adhesion molecule levels in activated HIMEC but did result in sustained increases in intracellular reactive oxygen species. In addition, antioxidant compounds reversed the effect of NOS inhibitors on HIMEC-leukocyte interaction. Taken together, these data suggest that after HIMEC activation, iNOS-derived NO is an endogenous antioxidant, downregulating leukocyte binding and potentially downregulating intestinal inflammation.
Glutathione depletion is associated with decreased Bcl-2 expression and increased apoptosis in cholangiocytes
Authors: Celli Adriane , Que Florencia G. , Gores Gregory J. , LaRusso Nicholas F.
Published: December 2017

Cholangiocytes are the target of a group of liver diseases termed the cholangiopathies that include conditions characterized by periductal inflammation and cholangiocyte apoptosis. Because inflammation is associated with oxidative stress, we developed the hypothesis that cholangiocytes exposed to oxidative stress will be depleted of endogenous cytoprotective molecules, leading to cholangiocyte apoptosis. To begin to test this hypothesis, we explored the relationships among glutathione (GSH) depletion, expression of Bcl-2 (a protooncogene that inhibits apoptosis), and apoptosis in a nonmalignant human cholangiocyte cell line. Monolayers of human bile duct epithelial cells, derived from normal liver and immortalized by SV40 transformation, were depleted of GSH using buthionine sulfoximine (BSO). Bcl-2 expression was assessed by quantitative immunoblot analysis, and apoptosis quantified by fluorescence microscopy using the DNA binding dye 4′,6′-diamidino-2-phenylindole. Bcl-2 message was assessed by RNase protection assay, and Bcl-2 protein synthesis and half-life by pulse-chase analysis. Exposure of human cholangiocytes in culture to BSO reduced GSH levels by 93 ± 3% ( P < 0.01). In addition, treatment of cholangiocytes with BSO reduced Bcl-2 levels by 87 ± 2% ( P < 0.01) and was associated with a time-dependent increase in the number of cells undergoing apoptosis; ∼11 ± 1% of cultured cells demonstrated morphological changes of apoptosis by 72 h compared with 1.5 ± 0.1% in untreated cholangiocytes ( P < 0.01). Maintenance of GSH levels by addition of glutathione ethyl ester in the presence of BSO blocked the BSO-associated increase in apoptosis in BSO-treated cholangiocytes and also prevented the decrease in Bcl-2 protein. BSO treatment of cholangiocytes did not change steady-state levels of bcl-2 mRNA or Bcl-2 protein synthesis. However, Bcl-2 protein half-life decreased 57% in BSO-treated vs. untreated cells. Our results using a human cholangiocyte cell line demonstrate that reduction in the cellular levels of an antioxidant such as GSH results in increased degradation of Bcl-2 protein and an increase in apoptosis. These data provide a mechanistic link between the consequences of oxidative stress and cholangiocyte apoptosis, an observation that may be important in the pathogenesis of the inflammatory cholangiopathies.
Effects of sustained flow reduction on postnatal intestinal circulation
Authors: Nowicki Philip T.
Published: December 2017

Studies were conducted to determine the effect of mechanically induced sustained flow reduction on intestinal hemodynamics and oxygenation in 3- and 35-day-old swine. In vitro gut loops were perfused under controlled-pressure conditions from an oxygenated blood reservoir at age-appropriate perfusion pressures; pressure was rapidly reduced to a level that lowered flow rate to ∼50% of its baseline value, and pressure was then kept at that level for 2 h. In 3-day-old intestine, vascular resistance ( R i) increased by 20% immediately after pressure and flow reduction but then stabilized for 3–4 min; thereafter, flow began to decrease despite maintenance of perfusion pressure, so that R i increased an additional 15% by 30 min after flow reduction. Flow continued to diminish over the next 90 min, though at much slower rate. Intestine from 35-day-old swine demonstrated an immediate increase in R i after pressure and flow reduction, but thereafter R i increased very little. The protocol was repeated within in vitro gut loops perfused under controlled-flow conditions, and within autoperfused, innervated gut loops developed in vivo and similar observations were made in both preparations. In 3-day-old intestine, pretreatment with thel-arginine analog N ω-monomethyl-l-arginine (10−4 M) had no effect on the immediate rise in resistance occurring in the first 1 min but substantially attenuated the subsequent slow, progressive rise noted thereafter. Pretreatment with the angiotensin 1A receptor antagonist losartan (2 × 10−6 M) had no effect on hemodynamic changes during the first 60 min after mechanical perfusion pressure reduction but attenuated the very slight increase in resistance noted during the final 60 min of the protocol. The postnatal intestinal circulation demonstrates progressive vasoconstriction when its flow rate is mechanically reduced in a sustained manner, and this effect is age specific, occurring in 3- but not 35-day-old swine. These changes in gut vascular resistance may be consequent to loss of nitric oxide production and/or local production of angiotensin.
Involvement of pituitary adenylate cyclase-activating peptide in opossum internal anal sphincter relaxation
Authors: Chakder Sushanta , Rattan Satish
Published: December 2017

Despite its widespread distribution and significance in the gut, the role of pituitary adenylate cyclase-activating peptide (PACAP) in internal anal sphincter (IAS) relaxation has not been examined. This study examined the role of PACAP in nonadrenergic noncholinergic (NANC) nerve-mediated relaxation of IAS smooth muscle. Circular smooth muscle strips from the opossum IAS were prepared for measurement of isometric tension. The influence of PACAP and vasoactive intestinal peptide (VIP) antagonists and tachyphylaxis on the neurally mediated IAS relaxation was examined either separately or in combination. The release of these neuropeptides in response to NANC nerve stimulation before and after the nitric oxide (NO) synthase inhibitor Nω-nitro-l-arginine and NO was also investigated. Both PACAP and VIP antagonists caused significant attenuation of IAS relaxation by NANC nerve stimulation. The combination of the antagonists, however, did not have an additive effect on IAS relaxation. VIP tachyphylaxis caused significant suppression of IAS relaxation by NANC nerve stimulation. PACAP and VIP were found to be released by NANC nerve stimulation and exogenous NO. The data suggest the involvement of PACAP in IAS relaxation primarily by the activation of PACAP1/VIP receptor and lack of its independent role in the relaxation. Furthermore, NO may regulate the presynaptic release of PACAP and VIP.
5-HT activates nitric oxide-generating neurons to stimulate chloride secretion in guinea pig distal colon
Authors: Kuwahara Atsukazu , Kuramoto Hirofumi , Kadowaki Makoto
Published: December 2017

The participation of nitric oxide (NO) in serotonin (5-hydroxytryptamine; 5-HT)-evoked chloride secretion in guinea pig distal colon was examined. Submucosal/mucosal segments were mounted in Ussing flux chambers, and an increase in short-circuit current ( I sc) was used as an index of secretion. Addition of 5-HT to the serosal side produced a concentration-dependent (10−7–10−5M) increase in I sc caused by chloride secretion. N G-nitro-l-arginine (l-NNA) significantly reduced the 5-HT-evoked early (P-1) and late (P-2) responses to 61.1 and 70.6% of control, respectively. Neurally evoked response was also inhibited by l-NNA. The NO donor sodium nitroprusside (SNP, 10−4 M) increased basal I sc mainly because of chloride secretion. The SNP-evoked response was significantly reduced by tetrodotoxin but was unchanged by atropine or indomethacin. These results suggest that the 5-HT-evoked increase in I sc is associated with an NO-generating mechanism. Atropine significantly reduced the 5-HT (10−5 M)-evoked P-1 and P-2 responses to 71.8 and 19.7% of control, respectively. Simultaneous application of atropine andl-NNA further decreased the 5-HT-evoked responses more than either drug alone; application ofl-NNA and atropine decreased the 5-HT-evoked P-1 and P-2 responses to 68.5 and 39.2% of atropine-treated tissues, respectively. These results suggest that noncholinergic components of P-1 and P-2 responses are 71.8 and 19.7% of control, respectively, and that NO components of P-1 and P-2 responses are 32 and 61%, respectively, of the noncholinergic component of the 5-HT-evoked responses. The results provide evidence that NO may participate as a noncholinergic mediator of 5-HT-evoked chloride secretion in guinea pig distal colon.
Nitric oxide mediates hepatocyte injury
Authors: Wang Jiang Huai , Redmond H. Paul , Di Wu Qiong , Bouchier-Hayes David
Published: December 2017

The degree of acute hepatic failure after severe trauma and sepsis is related to the extent of hepatocyte (HC) damage and cell death resulting from either necrosis or apoptosis. We have previously demonstrated that tumor necrosis factor-α (TNF-α) and lipopolysaccharide (LPS) can directly lead to HC necrosis, but not apoptosis. To date, the reactive oxygen intermediates (ROI) and nitric oxide (NO) have been shown to play a potential role in the induction of cell apoptosis. However, it is unknown whether ROI and NO are involved in HC cell death. Therefore, in this study we tested the hypothesis that NO and ROI exert different effects on HC cell death. TNF-α and LPS alone failed to induce HC apoptosis but when combined with antioxidants resulted in HC apoptosis and DNA fragmentation, which is correlated with an increase in NO production. This effect was attenuated by the NO synthase inhibitor NG-monomethyl-l-arginine (l-NMMA). Moreover, the NO donor sodium nitroprusside resulted in HC apoptosis and cell damage as represented by hepatocellular enzyme release. Antioxidants inhibited TNF-α- and LPS-mediated ROI generation and peroxynitrite formation in HC. TNF-α- and LPS-induced HC damage could be further reduced by the combination of antioxidants andl-NMMA. These results indicate that NO is involved in HC injury, primarily through the induction of HC apoptosis.
Cholinergic ion secretion in human colon requires coactivation by cAMP
Authors: Mall M. , Bleich M. , Schürlein M. , Kühr J. , Seydewitz H. H. , Brandis M. , Greger R. , Kunzelmann K.
Published: December 2017

Cl−secretion in the colon can be activated by an increase of either intracellular Ca2+ or cAMP. In this study we examined a possible interdependence of the two second-messenger pathways in human colonic epithelium. When measured in a modified Ussing chamber, carbachol (CCH; 100 μmol/l, basolateral), via an increase in cytosolic Ca2+concentration ([Ca2+]i), activated a transient lumen-negative equivalent short-circuit current ( I sc) [change (Δ) in I sc = −79.4 ± 7.5 μA/cm2]. Previous studies indicated that intracellular Ca2+ directly acts on basolateral K+ channels, thus enhancing driving force for luminal Cl− exit. Increased intracellular cAMP (by basolateral addition of 100 μmol/l IBMX and 1 μmol/l forskolin) activated a sustained lumen-negative current (Δ I sc = −42.4 ± 7.2 μA/cm2) that was inhibited by basolateral trans-6-cyano-4-( N-ethylsulfonyl- N-methylamino)-3-hydroxy-2,2-dimethyl&2-chromane (10 μmol/l), a blocker of KvLQT1 channels. In the presence of elevated cAMP, the CCH-activated currents were augmented (Δ I sc = 167.7 ± 32.7 μA/cm2), suggesting cooperativity of the Ca2+- and cAMP-mediated responses. Inhibition of endogenous cAMP production by indomethacin (10 μmol/l) significantly reduced CCH-activated currents and even reversed the polarity in 70% of the experiments. The transient lumen-positive I sc was probably due to activation of apical K+channels because it was blocked by luminal Ba2+ (5 mmol/l) and tetraethylammonium (10 mmol/l). In the presence of indomethacin (10 μmol/l, basolateral), an increase of cAMP activated a sustained negative I sc. Under these conditions, CCH induced a large further increase in lumen-negative I sc(Δ I sc = −100.0 ± 21.0 μA/cm2). We conclude that CCH acting via [Ca2+]ican induce Cl− secretion only in the presence of cAMP, i.e., when luminal Cl− channels are already activated. The activation of a luminal and basolateral K+ conductance by CCH may be essential for transepithelial KCl secretion in human colon.
Early NF-κB activation is associated with hormone-induced pancreatitis
Authors: Gukovsky Ilya , Gukovskaya Anna S. , Blinman Thane A. , Zaninovic Vjekoslav , Pandol Stephen J.
Published: December 2017

Inflammation and cell death are critical to pathogenesis of acute pancreatitis. Here we show that transcription factor nuclear factor-κB (NF-κB), which regulates these processes, is activated and plays a role in rat cerulein pancreatitis. NF-κB was strongly activated in the pancreas within 30 min of cerulein infusion; a second phase of NF-κB activation was prominent at 3–6 h. This biphasic kinetics could result from observed transient degradation of the inhibitory protein IκBα and slower but sustained degradation of IκBβ. The hormone also caused NF-κB translocation and IκB degradation in vitro in dispersed pancreatic acini. Both p65/p50 and p50/p50, but not c-Rel, NF-κB complexes were manifest in pancreatitis and in isolated acini. Coinfusion of CCK JMV-180, which abolishes pancreatitis, prevented cerulein-induced NF-κB activation. The second but not early phase of NF-κB activation was inhibited by a neutralizing tumor necrosis factor-α antibody. Antioxidant N-acetylcysteine (NAC) blocked NF-κB activation and significantly improved parameters of pancreatitis. In particular, NAC inhibited intrapancreatic trypsin activation and mRNA expression of cytokines interleukin-6 and KC, which were dramatically induced by cerulein. The results suggest that NF-κB activation is an important early event that may contribute to inflammatory and cell death responses in acute pancreatitis.
Short-chain fatty acids modify colonic motility through nerves and polypeptide YY release in the rat
Authors: Cherbut Christine , Ferrier Laurent , Rozé Claude , Anini Younès , Blottière Hervé , Lecannu Gérard , Galmiche Jean-Paul
Published: December 2017

Short-chain fatty acids (SCFAs) are recognized as the major anions of the large intestinal content in humans, but their effect on colonic motility is controversial. This study explores the colonic motor effect of SCFAs and their mechanisms in the rat. Colonic motility (electromyography) and transit time (plastic markers) were measured in conscious rats while SCFAs were infused into the colon, either alone or after administration of neural antagonists or immunoneutralization of circulating polypeptide YY (PYY). SCFA-induced PYY release was measured by RIA and then simulated by infusing exogenous PYY. Intracolonic infusion of 0.4 mmol/h SCFAs had no effect, whereas 2 mmol/h SCFAs reduced colonic motility (36 ± 3 vs. 57 ± 4 spike bursts/h with saline, P< 0.05) by decreasing the ratio of nonpropulsive to propulsive activity. This resulted in an increased transit rate ( P < 0.01). Neither α-adrenoceptor blockade nor nitric oxide synthase inhibition prevented SCFA-induced motility reduction. Intraluminal procaine infusion suppressed the SCFA effect, indicating that a local neural mechanism was involved. SCFA colonic infusion stimulated PYY release in blood. Immunoneutralization of circulating PYY abolished the effect of SCFAs on colonic motility, whereas exogenous PYY infusion partly reproduced this effect. SCFAs modify colonic motor patterns in the rat and increase transit rate; local nerve fibers and PYY are involved in this effect.
Effect of sorbin on electrolyte transport in rat and human intestine
Authors: Eto Bruno , Boisset Michel , Griesmar Bertrand , Desjeux Jehan-François
Published: December 2017

Stimulating water absorption in the colon represents an important target to reduce stool output in secretory diarrhea. Recently, a 153-amino-acid peptide was isolated from porcine upper small intestine and purified, taking into account the increase of water absorption in guinea pig gallbladder. Accordingly, this peptide was named sorbin. The aim of the present study was to determine if the COOH-terminal heptapeptide of sorbin (C7-sorbin) participates in the regulation of electrolyte transport in the colon. Different regions (from duodenum to colon) of stripped intestinal mucosa from rats or humans were mounted in Ussing chambers to measure the changes in short-circuit current (Δ Isc) and net22Na and36Cl fluxes ([Formula: see text] and[Formula: see text]) after serosal exposure of 10−7to 10−3M C7-sorbin. In fasted rat intestine, C7-sorbin (10−4M) induced an immediate reduction in Iscin the distal ileum and proximal and distal colon but not in the duodenum and jejunum. In the colon, Iscreduction and[Formula: see text] and[Formula: see text] stimulation were dose dependent (EC50= 2 × 10−5M). At 10−3M, maximal effect was observed (Δ Isc= −1.14 ± 0.05, Δ[Formula: see text] = +4.97 ± 1.38, and Δ[Formula: see text] = +9.25 ± 1.44 μeq ⋅ h−1⋅ cm−2). C7-sorbin (10−3M) inhibited the increase in Iscinduced by a series of 10 secretory agents such as secretin, vasoactive intestinal peptide, PGE2, and serotonin. In HT-29-Cl19A cells, C7-sorbin induced an increase in Isc, with a maximal effect at 10−3M (Δ Isc= 0.29 ± 0.10 μeq ⋅ h−1⋅ cm−2). In human intestine, a dose-dependent decrease in Iscwas observed in right and sigmoid colons in basal and stimulated conditions (EC50≅ 10−5M; at 10−4M, Δ Isc= −2.66 ± 0.17 μeq ⋅ h−1⋅ cm−2) but not in the jejunum. The results indicate that C7-sorbin stimulated NaCl neutral absorption and inhibited electrogenic Cl−in rat and human intestinal epithelia. In addition, the antisecretory effect was essentially observed in the distal part of both rat and human intestine and the magnitude of the proabsorptive effect was directly related to the magnitude of the previously induced secretion.
Peripheral and central actions of orphanin FQ (nociceptin) on murine colon
Authors: Osinski M. A. , Bass P. , Gaumnitz E. A.
Published: December 2017

Orphanin FQ (OFQ), also known as nociceptin, is a recently isolated endogenous peptide with a structure similar to the endogenous opioid peptides. The present study examines the actions of centrally administered OFQ on in vivo murine gastrointestinal and colonic transit as well as the actions of OFQ on the isolated colon. Intracerebroventricular injections of OFQ dose dependently inhibited colonic propulsive activity. OFQ inhibition of colonic propulsion was unaffected by coadministration of the competitive opioid receptor antagonist naltrexone. A subadditive response was observed when approximately equipotent doses of either morphine sulfate or the δ-agonist DPDPE were coadministered with OFQ. No subadditivity was observed with coadministration of the μ-agonist DAMGO, suggesting a functional interaction between OFQ and δ-opioid central pathways regulating colonic transit. High, but not low, doses of OFQ also inhibited the transit of a nonabsorbable charcoal marker through the stomach and/or small intestine. OFQ potently contracted isolated colon preparations; contractile activity was abolished by TTX or chlorpromazine. Our results suggest that OFQ may be an important peptide ligand acting on a novel inhibitory neural pathway that modulates gastrointestinal transit.
Region-specific antiproliferative effect of VIP and PACAP-(1—38) on rabbit enteric smooth muscle
Authors: Van Assche Gert , Depoortere Inge , De Vos Rita , Geboes Karel , Janssens Jozef J. , Collins Stephen M. , Peeters Theo L.
Published: December 2017

The ability of neuropeptides to modulate enteric smooth muscle proliferation was examined in primary explant cultures of rabbit gastric antrum and colon smooth muscle. Cell proliferation was determined by [3H]thymidine incorporation measurements and cell counting. Subcultured rabbit antrum and colon myocytes ( passages 2–6) preserved a smooth muscle phenotype, as verified by immunohistochemistry for α-smooth muscle actin and electron microscopy. Both vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating peptide-(1—38) [PACAP-(1—38)] concentration dependently (10−10to 10−6M) inhibited the serum-induced [3H]thymidine incorporation [in colon, 48.2 ± 5.8 and 55.6 ± 9.3% of control with 10−6M VIP and 10−7M PACAP-(1—38)] and inhibited increase in cell numbers in cultures derived from the colon but not in those from the antrum. Effects of VIP and PACAP-(1—38) were mimicked by forskolin (10−7to 10−6M) but not by 8-bromo-cGMP, whereas theophylline enhanced the effects of VIP. Inhibition of nitric oxide synthase with NG-nitro-l-arginine methyl ester (10−3.5M) did not alter the effects of VIP. Substance P, motilin, calcitonin gene-related peptide, and somatostatin had no effect. A single class of125I-labeled VIP binding sites was found in antrum and colon myocyte cultures with an equal affinity for VIP and PACAP-(1—38) [dissociation constant ( Kd) in antrum = 3.4 ± 0.8 nM for VIP and 2.0 ± 1.0 nM for PACAP-(1—38); Kdin colon = 2.0 ± 1.0 nM for VIP and 2.8 ± 1.6 nM for PACAP-(1—38)]. Density of binding sites in the antrum was higher than in the colon. In disease states such as inflammatory bowel disease, inhibition of myocyte proliferation by VIP and PACAP may serve to control smooth muscle hyperplasia in the colon but not in the antrum.
Cholestatic effects of the K+channel blockers Ba2+and TEA occur through different pathways in the rat liver
Authors: Hill Ceredwyn Elizabeth , Jacques Jody Elisabeth
Published: December 2017

The role of K+channels in bile acid-independent bile flow (BAIF) was studied in the isolated and bile duct-cannulated perfused rat liver by changing the driving force on K+and by using a variety of K+channel blockers. Bile flow rate, effluent perfusate K+content, and portal pressure were measured. Increase in perfusate K+from 5.9 to 80 mM caused inhibition of bile flow that could be fitted to a Boltzmann distribution, indicating partial dependence of bile formation on the K+equilibrium potential and hence K+channel activity. To investigate this further, the effects of compounds established as K+channel blockers in liver or other tissues were surveyed. Ba2+(1–5 mM) inhibited mean bile flow by 20%. Tetraethylammonium (TEA) inhibition of basal bile flow was biphasic with saturable (IC50∼0.7 mM) and linear components. In contrast, infusion of the K+channel blockers 4-aminopyridine (5 mM), cesium (2.5 mM), quinidine (0.1 mM), iberiotoxin (90 nM), or paxilline (100 nM) did not affect bile flow. As expected for a K+channel blocker, Ba2+caused a net K+uptake. Conversely, TEA did not affect basal K+fluxes, although TEA-induced cholestasis was accompanied by a 26% decrease in biliary glutathione excretion. These results suggest that the partial cholestasis induced by the K+channel blockers Ba2+and TEA occurs by significantly different mechanisms. Whereas the Ba2+response implicates K+channel activity as a significant driving force in BAIF, TEA-sensitive K+channels are not present or are not involved in bile formation.
Human [Gly2]GLP-2 reduces the severity of colonic injury in a murine model of experimental colitis
Authors: Drucker Daniel J. , Yusta Bernardo , Boushey Robin P. , DeForest Lorraine , Brubaker Patricia L.
Published: December 2017

The pathology of Crohn’s disease and ulcerative colitis is characterized by chronic inflammation and destruction of the gastrointestinal epithelium. Although suppression of inflammatory mediators remains the principle component of current disease therapeutics, strategies for enhancing repair and regeneration of the compromised intestinal epithelium have not been widely explored. The demonstration that a peptide hormone secreted by the intestinal epithelium, glucagon-like peptide-2 (GLP-2), is a potent endogenous stimulator of intestinal epithelial proliferation in the small bowel prompted studies of the therapeutic efficacy of GLP-2 in CD1 and BALB/c mice with dextran sulfate (DS)-induced colitis. We report here that a human GLP-2 analog (h[Gly2]GLP-2) significantly reverses weight loss, reduces interleukin-1 expression, and increases colon length, crypt depth, and both mucosal area and integrity in the colon of mice with acute DS colitis. The effects of h[Gly2]GLP-2 in the colon are mediated in part via enhanced stimulation of mucosal epithelial cell proliferation. These observations suggest that exploitation of the normal mechanisms used to regulate intestinal proliferation may be a useful adjunct for healing mucosal epithelium in the presence of active intestinal inflammation.
Paracrine upregulation of VEGF receptor mRNA in endothelial cells by hypoxia-exposed Hep G2 cells
Authors: Suzuki Hidekazu , Seto Koichi , Shinoda Yuichi , Mori Mikiji , Ishimura Yuzuru , Suematsu Makoto , Ishii Hiromasa
Published: December 2017

Although vascular endothelial growth factor (VEGF) plays a role in the growth of hypervascular tumors, mechanisms for paracrine regulation of its receptor expression on vascular endothelial cells remain unknown. This study aimed to investigate whether VEGF released from hypoxia-exposed Hep G2 cells alters expression of the two distinct receptors, kinase insert domain-containing receptor (KDR) and fms-like tyrosine kinase 1 ( flt-1), in human umbilical venous endothelial cells (HUVEC). Hep G2 cells were cultured in 20% or 1% O2 for 16 h to examine induction of VEGF mRNA and its protein expression. Conditioned medium from Hep G2 cells (CM) was applied to HUVEC under normoxic conditions, and expression of mRNA for the VEGF receptors was determined by RT-PCR. In response to the hypoxic challenge, Hep G2 cells upregulated VEGF mRNA and the release of VEGF. Hypoxia-CM preferentially stimulated the mRNA expression of flt-1 but not that of KDR in HUVEC. When the VEGF release from hypoxia-exposed Hep G2 cells was blocked by its antisense oligodeoxynucleotide, the endothelial flt-1 mRNA upregulation elicited by the hypoxia-CM was still maintained. These results suggest that hypoxia-exposed Hep G2 cells not only produce VEGF but also evolve paracrine induction of flt-1 through VEGF-independent mechanisms.
Epithelial injury induces Egr-1 and Fos expression by a pathway involving protein kinase C and ERK
Authors: Dieckgraefe Brian K. , Weems Danielle M.
Published: December 2017

The signaling pathways activated in response to gastrointestinal injury remain poorly understood. Previous work has implicated the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase as a mediator of wound-signal transduction and a possible regulator of epithelial restitution. Monolayer injury resulted in rapid activation of p42 and p44 ERK. Injury-induced ERK activation was blocked by protein kinase C inhibition or by disruption of the cell cytoskeleton. Significant increases in Fos and early growth response (Egr)-1 mRNA levels were stimulated by injury, peaking by 20 min. ERK activation and the induction of Egr-1 mRNA were inhibited in a dose-dependent fashion with PD-98059. Fos mRNA expression was partially blocked by PD-98059. Western blot analysis demonstrated strong expression and nuclear localization of Fos and Egr after wounding. Electrophoretic mobility shift assays demonstrated that nuclear extracts contained a protein that specifically bound double-stranded oligonucleotides containing the Egr consensus binding element. Gel supershift assays demonstrated that the protein-DNA complexes were recognized by anti-Egr antibody. Inhibition of injury-induced ERK activation by PD-98059 or direct interference with Egr by expression of a dominant negative mutant led to significantly reduced in vitro monolayer restitution.
Prechylomicron transport vesicle: isolation and partial characterization
Authors: Kumar N. Suresh , Mansbach C. M.
Published: December 2017

The intestine is able to regulate its output rate of chylomicrons, the major intestinal triacylglycerol (TG) transport vehicle. We have proposed that a vesicle, transporting the developing chylomicron from the endoplasmic reticulum (ER) to the Golgi, is the rate-limiting step in the process of TG transit through the enterocyte [ Am. J. Physiol. 273 ( Gastrointest. Liver Physiol. 36): G18–G30, 1997]. We wished to isolate and characterize this vesicle. The apical portion of rat intestinal cells were avulsed, and the mucosa was stirred in buffer. The supernatant was centrifuged in two different sucrose gradients, and the top 2.5 ml of the last gradient were collected and concentrated. Electron microscopy showed a 200-nm vesicle. The vesicle contained immunoidentifiable apolipoprotein (apo) B48and apo A-IV but very little apo A-I, although apo A-I was present in the ER and Golgi. [3H]TG-loaded vesicles delivered [3H]TG to the Golgi but not the ER. Marker enzyme assays also indicate that the isolated fraction is different from the ER and Golgi fractions. We conclude that we have isolated a vesicle that is post-ER but pre-Golgi that vectorially transports TG to the Golgi.
Protein kinase C mediates experimental colitis in the rat
Authors: Brown James F. , Chang Qing , Soper Brian D. , Tepperman Barry L.
Published: December 2017

Protein kinase C (PKC) plays an important role in the cell signal transduction of many physiological processes. In contrast to these physiological responses, increases in PKC activity have also been associated with inflammatory disease states, including ulcerative colitis. The objective of this study was to examine the role of PKC as a causative mediator in initiation of experimentally induced colitis in the rat. Colitis was induced in rats by intrarectal (0.6 ml) instillation of 2,4,6-trinitrobenzenesulfonic acid (TNBS; 75 mg/kg in 50% ethanol) or the PKC activator phorbol 12-myristate 13-acetate (PMA; 1.5–3.0 mg/kg in 20% ethanol). Gross and histological mucosal damage, mucosal neutrophil infiltration, mucosal PKC activity, and PKC protein content for PKC isoforms α, β, δ, and ε were assessed 2 h to 14 days after an inflammatory challenge. Both PKC activity and mucosal injury increased significantly within 4 h of TNBS treatment. PKC activity was maximal at 7 days and declined at 14 days, whereas mucosal damage became maximal at 1 day and declined after 7 days. In contrast, neutrophil infiltration as assessed by myeloperoxidase activity only increased 12 h after TNBS treatment, became maximal 1 day after TNBS administration, and declined thereafter. PKCβ, -δ, and -ε were increased in response to TNBS, whereas PKCα protein content was decreased. The PKC antagonists staurosporine and GF-109203X (25 ng/kg iv) reduced TNBS-induced changes in mucosal PKC activity and the degree of mucosal damage. In contrast, neutropenia induced by antineutrophil serum treatment did not significantly affect the degree of injury or mucosal PKC activity. Furthermore, activation of mucosal PKC activity with PMA also induced mucosal damage, which was also inhibited by pretreatment with a PKC antagonist. In conclusion, these results suggest that increases in PKC activity play a causative role in TNBS-induced colitis. The PKC-mediated response to TNBS does not appear to involve neutrophil infiltration.
Susceptibility of Lewis and Fischer rats to stress-induced worsening of TNB-colitis: protective role of brain CRF
Authors: Million Mulugeta , Taché Yvette , Anton Peter
Published: December 2017

We assessed the role of central corticotropin-releasing factor (CRF) in stress-induced worsening of colitis in inbred rat strains with hypo (Lewis/N) and hyper (Fischer344/N) CRF responses to stress. Intracolonic administration of 2,4,6-trinitrobenzenesulfonic acid (TNB) induced colitis of similar severity in both strains as assessed on day 7 by macroscopic scoring, histological evaluation, tissue myeloperoxidase (MPO) activity, and decrease in food intake and body weight. Colitis was inhibited by daily intracerebroventricular injections of CRF in both strains. Chronic stress (3 h/day, water avoidance or wrap restraint on alternate days for 6 days) aggravated colitis more in Lewis than Fischer rats (71 and 22% further increase in MPO activity, respectively). The CRF antagonist astressin injected intracerebroventricularly enhanced the colitis response to stress and caused mortality in both strains. Fischer rats had higher plasma corticosterone levels 20 min after stress alone on day 1 and after TNB plus stress on days 1 and 3 compared with Lewis. These data show that central CRF restrains the proinflammatory action of stress in experimental colitis.
A novel Sp1-relatedciselement involved in intestinal alkaline phosphatase gene transcription
Authors: Kim Jeong H. , Meng Shufen , Shei Amy , Hodin Richard A.
Published: December 2017

We have used sodium butyrate-treated HT-29 cells as an in vitro model system to study the molecular mechanisms underlying intestinal alkaline phosphatase (IAP) gene activation. Transient transfection assays using human IAP-CAT reporter genes along with DNase I footprinting were used to localize a critical cis element (IF-III) corresponding to the sequence 5′-GACTGGGCGGGGTCAAGATGGA-3′. Deletion of the IF-III element resulted in a dramatic reduction in reporter gene activity, and IF-III was shown to function in the context of a heterologous (SV40) promoter in a cell type-specific manner, further supporting its functional role in IAP transactivation. Electrophoretic mobility shift assays revealed that IF-III binds Sp1 and Sp3, but these factors comprise only a portion of the total nuclear binding and appear to mediate only a small portion of its transcriptional activity. IF-III does not correspond to any previously characterized regulatory region from other intestine-specific genes. We have thus identified a novel, Sp1-related cis-regulatory element in the human IAP gene that appears to play a role in its transcriptional activation during differentiation in vitro.
COOH-terminally extended secretins are potent stimulants of pancreatic secretion
Authors: Solomon Travis E. , Walsh John H. , Bussjaeger Louis , Zong Yumei , Hamilton James W. , Ho F. J. , Lee Terry D. , Reeve Joseph R.
Published: December 2017

Posttranslational processing of preprosecretin generates several COOH-terminally extended forms of secretin and α-carboxyl amidated secretin. We used synthetic canine secretin analogs with COOH-terminal -amide, -Gly, or -Gly-Lys-Arg to examine the effects of COOH-terminal extensions of secretin on bioactivity and detection in RIA. Synthetic products were purified by reverse-phase and ion-exchange HPLC and characterized by reverse-phase isocratic HPLC and amino acid, sequence, and mass spectral analyses. Secretin and secretin-Gly were noted to coelute during reverse-phase HPLC. In RIA using eight different antisera raised against amidated secretin, COOH-terminally extended secretins had little or no cross-reactivity. Bioactivity was assessed by measuring pancreatic responses in anesthetized rats. Amidated canine and porcine secretins were equipotent. Secretin-Gly and secretin-Gly-Lys-Arg had potencies of 81 ± 9% ( P > 0.05) and 176 ± 13% ( P < 0.01), respectively, compared with amidated secretin, and the response to secretin-Gly-Lys-Arg lasted significantly longer. These data demonstrate that 1) amidated secretin and secretin-Gly are not separable under some chromatographic conditions, 2) current RIA may not detect bioactive COOH-terminally extended forms of secretin in tissue extracts or blood, and 3) the secretin receptor mediating stimulation of pancreatic secretion recognizes both amidated and COOH-terminally extended secretins.
Autocrine and paracrine actions of intestinal fibroblast-derived insulin-like growth factors
Authors: Simmons James G. , Pucilowska Jolanta B. , Lund P. Kay
Published: December 2017

Paracrine and autocrine actions of the insulin-like growth factors (IGFs) are inferred by local expression within the bowel. CCD-18Co cells, IEC-6 cells, and immunoneutralization were used to analyze whether IGFs have direct autocrine or paracrine effects on proliferation of cultured intestinal fibroblasts and epithelial cells. Growth factor expression was analyzed by ribonuclease protection assay and RT-PCR. Extracellular matrix (ECM) was analyzed for effects on cell proliferation. CCD-18Co cells express IGF-II mRNAs and low levels of IGF-I mRNA. Conditioned medium from CCD-18Co cells (CCD-CM) stimulated proliferation of IEC-6 and CCD-18Co cells. Neutralization of IGF immunoreactivity in CCD-CM reduced but did not abolish this effect. RT-PCR and immunoneutralization demonstrated that other growth factors contribute to mitogenic activity of CCD-CM. Preincubation of CCD-CM with ECM prepared from IEC-6 or CCD-18Co cells reduced its mitogenic activity. ECM from CCD-18Co cells enhanced growth factor-dependent proliferation of IEC-6 cells. IEC-6 cell ECM inhibited IGF-I action on CCD-18Co cells. We conclude that IGF-II is a potent autocrine mitogen for intestinal fibroblasts. IGF-II interacts with other fibroblast-derived growth factors and ECM to stimulate proliferation of intestinal epithelial cells in a paracrine manner.
Characterization and regulation of Ca2+-dependent K+ channels in human esophageal smooth muscle
Authors: Hurley Bernard R. , Preiksaitis Harold G. , Sims Stephen M.
Published: December 2017

We examined the properties of K+ channels in smooth muscle cells dissociated from human esophagus using patch-clamp recording in the cell-attached configuration. The predominant channel observed had a conductance of 224 ± 4 pS, and current reversal was dependent on K+ concentration. Channel activity was voltage dependent and increased with elevation of intracellular free Ca2+ concentration ([Ca2+]i), consistent with this being the large-conductance Ca2+-dependent K+(KCa) channel. ACh as well as caffeine caused transient increases in KCa channel activity, and the effects of ACh persisted in Ca2+-free solution, indicating that Ca2+ release from stores contributed to channel activation. Simultaneous patch clamp and fluorescence revealed that KCachannel activity was well correlated with elevation of [Ca2+]i. The functional role of KCachannels in esophagus was studied by measuring ACh-induced contraction of strips of muscle. Tetraethylammonium and iberiotoxin, blockers of KCa channels, increased ACh-induced contraction, consistent with a role for K+ channels in limiting excitation and contraction. These studies are the first to characterize KCa channels and their regulation in human esophageal smooth muscle.