bluuebunny/crossref_metadata_2025 huggingface dataset:
Responses of ferret lower esophageal sphincter to 5-hydroxytryptamine: pathways and receptor subtypes
Authors: Blackshaw L. A. , Nisyrios V. , Dent J.
Published: December 2017

In urethananesthetized ferrets, basal lower esophageal sphincter pressure (LESP) was unaffected by the 5-hydroxytryptamine3 (5-HT3) receptor antagonist granisetron (0.5 mg/kg) or by greater splanchnic nerve section (GSX), but increased after bilateral vagotomy. Peripheral vagal nerve stimulation caused LES relaxation, often followed by a brief contraction and a prolonged inhibition of LESP. Close intra-arterial injection of 5-HT (5-100 micrograms) had a biphasic effect on LESP, with a brief drop followed by a prolonged increase. Granisetron (0.5 mg/kg i.v.) abolished the initial relaxation and revealed an earlier peak of excitation. This was not influenced by subsequent vagotomy and GSX. In a series of eight additional experiments (series 2), granisetron was given after vagotomy and GSX. In series 2, 5-HT-induced relaxation was unaffected by vagotomy but was significantly reduced after GSX and was further reduced after granisetron, indicating that 5-HT3 receptor mechanisms may lie on a sympathetic neural pathway. Vagotomy had no effect on the excitatory component. GSX had no effect on the amplitude of excitation, but reduced its latency. Granisetron had no further effect on excitation in series 2. In a separate series of 13 experiments (series 3), the excitatory component of the LES response to 5-HT was abolished by ketanserin (2.5 mg/kg i.v.) , after which only relaxation occurred. Both 5-HT2 and 5-HT3 antagonists in combination abolished all effects of 5-HT on LESP. Atropine (400 micrograms/kg i.v., n = 7) had no effect on 5-HT-induced LES responses.(ABSTRACT TRUNCATED AT 250 WORDS)
Central vagal activation by an analogue of TRH stimulates gastric nitric oxide release in rats
Authors: Saperas E. , Mourelle M. , Santos J. , Moncada S. , Malagelada J. R.
Published: December 2017

In the stomach nitric oxide (NO) appears to be involved in vagally induced cholinergic vasodilation and nonadrenergic, noncholinergic relaxation of the fundus. We investigated whether central vagal activation by intracisternal injection of a thyrotropin-releasing hormone (TRH) analogue stimulates gastric NO release in anesthetized rats. To quantitate gastric NO production, the luminal release of NO breakdown products, nitrite (NO2-) and nitrate (NO3-), were measured by the Griess method. Intracisternal injection of RX-77368 (30-300 ng) dose dependently stimulated gastric NO2- and NO3- release (P < 0.05) along with a significant acid secretory response (P < 0.05). The specific inhibitor of NO synthesis, NG-nitro-L-arginine methyl ester (10 mg/kg ip), completely blocked gastric luminal NO2- and NO3- release without affecting the acid secretory response to the highest dose of RX-77368. Either bilateral cervical vagotomy, hexamethonium (15 mg/kg ip), or atropine (1 mg/kg ip) abolished both gastric luminal release of NO-derived metabolites and the acid secretory responses to RX-77368. These results indicate that intracisternal injection of RX-77368 stimulates gastric release of NO through vagal nicotinic pathways and peripheral activation of muscarinic receptors. These findings provide evidence for central nervous system regulation of NO-mediated functions in the rat stomach through TRH-sensitive vagal pathways.
Effect of NSAIDs on pepsinogen secretion and calcium mobilization in isolated chief cells
Authors: Fiorucci S. , Santucci L. , Gresele P. , Luinetti O. , Morelli A.
Published: December 2017

Acid and pepsin are thought to play an important role in the process of gastrointestinal side effects induced by nonsteroidal anti-inflammatory drugs (NSAIDs). Although NSAIDs increase basal gastric acid secretion, the effect they exert on pepsinogen secretion is unknown. Because pepsin plays a key role in many acid-related diseases, we investigated whether NSAIDs directly stimulate pepsinogen secretion from isolated chief cells. Exposure of guinea pig gastric chief cells to indomethacin (10 microM) did not reduce cell viability as evaluated by lactate dehydrogenase and 51Cr release and trypan blue incorporation. Indomethacin (10 microM) caused two- to threefold increases in pepsinogen secretion and intracellular calcium concentrations ([Ca2+]i). Both effects were concentration dependent. Removal of extracellular Ca2+ or pretreatment of the cells with 0.5 mM lanthanum blocked both pepsinogen secretion and the [Ca2+]i increase in chief cells stimulated with 10 microM indomethacin. Exposure of isolated chief cells to indomethacin caused a 90% inhibition of prostaglandin (PG) E2 generation, but a 12-fold increase in leukotriene (LT) B4 release. Incubating chief cells with exogenously added LTB4, LTC4, LTD4, and LTE4 provoked a concentration-dependent stimulation of pepsinogen release (mean effective concentration of 0.05-0.1 nM). Maximally effective concentrations of all LTs (10 microM) increased [Ca2+]i two- to threefold. Pretreating the cells with a 5-lipoxygenase inhibitor abolished LTB4 generation induced by Ca2+ ionophore and indomethacin and reduced indomethacin-induced pepsinogen secretion 20%. In conclusion, indomethacin induced a concentration-dependent stimulation of pepsinogen secretion and [Ca2+]i in isolated chief cells. Indomethacin inhibits PGE2 generation, but increases LTB4 release. This "lipoxygenase shunt" may contribute to the effect indomethacin exerts on isolated chief cells.
Endogenous ET-1 contributes to liver injury induced by galactosamine and endotoxin in isolated perfused rat liver
Authors: Ohuchi T. , Tada K. , Akamatsu K.
Published: December 2017

Injury to hepatocytes most likely occurs via disturbances in the microcirculation. The role of vasoconstriction due to the effect of endogenous endothelin-1 (ET-1) in the development of galactosamine (GalN)- and lipopolysaccharide (LPS)-induced liver injury was investigated. Using the multiple indicator dilution technique, we measured the volume of the hepatic sinusoids and the apparent Disse space as indicators of overall hepatic microcirculation. Serum purine nucleoside phosphorylase activity as a marker of damage to nonparenchymal cells increased and the volume of the sinusoids and the Disse space decreased prior to hepatocyte damage in rats treated intraperitoneally with GalN and LPS. Moreover, the amount of ET-1 release was elevated. When livers from untreated rats were perfused with ET-1 in a recirculating system, hepatocyte damage was observed similar to experiments with GalN and LPS. A monoclonal anti-endothelin antibody, AwETN40, diminished the extent of liver injury caused by GalN and LPS in isolated perfused rat liver. The present study suggests that vasoconstriction is an early event in GalN- and LPS-induced liver injury and that the development of hepatocyte damage is mediated via microcirculatory disturbances due to endogenous ET-1.
A novel colon-specific steroid prodrug enhances sodium chloride absorption in rat colitis
Authors: Fedorak R. N. , Cui N. , Friend D. R. , Madsen K. L. , Empey L. R.
Published: December 2017

A recently synthesized novel colon-specific dexamethasone prodrug, dexamethasone-beta-D-glucuronide, delivers efficacious amounts of dexamethasone to the colon with limited adrenal suppressive effects. During experimentally induced colitis in rats, the dexamethasone prodrug is significantly more potent than free dexamethasone in improving colonic fluid and electrolyte absorptive injury. The present studies examined whether the improvement in colonic absorption seen with the prodrug occurred as a consequence of alterations in sodium and chloride epithelial transport. The efficacy of the dexamethasone prodrug and free dexamethasone were tested in an acetic acid-induced rat model of colitis. Healing of the induced colitis was assessed by measuring net colonic fluid absorption and surface area ulceration. Transmural unidirectional fluxes of 22Na and 36Cl across sheets of colonic mucosa were measured in Ussing chambers. Treatment of colitis with the prodrug delivered a sixfold higher concentration of dexamethasone to the colon than did treatment with the free drug. The prodrug accelerated healing of colitis by returning in vivo colonic fluid absorption to normal and virtually eliminated colonic macroscopic ulceration, whereas the free drug did not. In vitro transmural fluxes demonstrated that, in addition to repair of mucosal integrity, the prodrug enhanced electroneutral sodium chloride absorption over and above that seen in control animals or after treatment with the free drug. Both the prodrug and the free drug limited theophylline-mediated net chloride and sodium secretion, an effect that would be consistent with the antidiarrheal effect induced by these drugs in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)
Cellular hypercalcemia is an early event in deoxycholate injury of rabbit gastric mucosal cells
Authors: Dziki A. J. , Batzri S. , Harmon J. W. , Molloy M.
Published: December 2017

Ca2+ entry into the cell may be an early event in the pathophysiology of bile salt-induced gastric mucosal injury. The aim of this study was to characterize the rise in cytosolic free Ca2+ associated with bile salt injury and its association with cell injury and death. Rabbit gastric mucosal cells were preloaded with the Ca2+ indicator fura 2-acetoxymethyl ester (fura 2-AM) for 20 min at 37 degrees C and then exposed to graded concentrations of the bile salt deoxycholate (DC). Cytosolic free Ca2+ concentration ([Ca2+]i) was estimated by spectrofluorometry. The resting [Ca2+]i in gastric cells was 177 +/- 15 nM (n = 6). When cells were subjected to 0.5 mM DC, there was a time-dependent rise in [Ca2+]i. An increase in [Ca2+]i was observed within 2 min, at which time [Ca2+]i rose from 177 +/- 15 to 480 +/- 30 nM. The maximal increase in [Ca2+]i was observed after 20 min of exposure to 0.5 mM DC (639 +/- 49 nM), and [Ca2+]i remained unchanged for at least 2 h. The increase in [Ca2+]i depended on the concentration of DC. The minimum effective dose of DC was 0.2 mM, with which [Ca2+]i was increased by 1.6-fold (from 177 to 285 nM). At 0.5 mM DC also caused a rise in 45Ca2+ influx into the cells and reduced the viability of gastric cells from 96% to 58% at 2 h. The DC-induced rise in cytosolic free Ca2+ depended on the presence of extracellular Ca2+. In the absence of extracellular Ca2+ there was no rise in cytosolic Ca2+ and gastric cells were protected from cell death caused by DC. The DC-induced cell death was reduced from 26% to 10% and from 37% to 16% at 60 and 90 min, respectively, by removal of extracellular Ca2+. The association of DC with gastric cells was not altered by removing extracellular Ca2+. This suggests decreased DC-induced injury in the absence of extracellular Ca2+ is due to the protection from cellular hypercalcemia rather than some other mechanism related to reduced binding and/or association of DC to gastric cells. These experiments show that rising [Ca2+]i appears to be an early pathophysiological event in bile salt-induced cellular injury and that extracellular Ca2+ is critical to produce this effect.
Regulation of cholecystokinin secretion by intraluminal releasing factors
Authors: Liddle R. A.
Published: December 2017

Ingested nutrients stimulate secretion of gastrointestinal hormones that are necessary for the coordinated processes of digestion and absorption of food. One of the most important hormonal regulators of the digestive process is cholecystokinin (CCK). This hormone is concentrated in the proximal small intestine and is secreted into the blood on the ingestion of proteins and fats. The physiological actions of CCK include stimulation of pancreatic secretion and gallbladder contraction, regulation of gastric emptying, and induction of satiety. Therefore, in a highly coordinated manner CCK regulates the ingestion, digestion, and absorption of nutrients. The manner by which foods affect enteric hormone secretion is largely unknown. However, it has recently become apparent that two CCK-releasing factors are present in the lumen of the proximal small intestine. One of these factors, known as monitor peptide, has been chemically characterized. Monitor peptide is produced by pancreatic acinar cells and is secreted by way of the pancreatic duct into the duodenum. On reaching the small intestine, monitor peptide interacts with CCK cells to induce hormone secretion. A CCK-releasing factor of intestinal origin has been partially characterized and is responsible for stimulation of CCK secretion after 1) ingestion of protein or fats, 2) instillation of protease inhibitors into the duodenum, or 3) diversion of bile-pancreatic juice from the upper small intestine. Together, these releasing factors provide positive and negative feedback mechanisms for regulation of CCK secretion. This review discusses the physiological observations that have led to the chemical characterization of the CCK-releasing factors and the potential implications of this work to other hormones of the gastrointestinal tract.
Actions of Rab3 effector domain peptides in chief cells from guinea pig stomach
Authors: Singh G. , Raffaniello R. D. , Eng J. , Raufman J. P.
Published: December 2017

Rab3 proteins are low molecular weight guanine nucleotide-binding proteins that belong to the Ras superfamily and are believed to play a role in the final steps of exocytosis. To examine potential interactions of these proteins with signaling pathways that mediate pepsinogen secretion from gastric chief cells, we synthesized peptides corresponding to the effector domain of Rab3. In the absence of added calcium [calcium concentration ([Ca2+]) < 1 nM], a maximal concentration (15 microM) of the Rab3 effector domain peptide or Rab3AL peptide, containing alanine and leucine substitutions, stimulated the release of 62 and 66%, respectively, of total pepsinogen from streptolysin O-permeabilized chief cells. A Rab2AL peptide, corresponding to the Rab2 effector domain, and modified (scrambled and truncated) Rab3AL peptides did not alter secretion from permeabilized cells. An additive secretory response was observed when 5 microM Rab3AL peptide was combined with increasing calcium ([Ca2+] < 1 nM to 3 microM). In contrast, adding up to 3 mM adenosine 3',5'-cyclic monophosphate (cAMP) had no effect on Rab3AL peptide-induced secretion, and Rab3AL peptide did not alter endogenous cAMP production. The addition of a nonhydrolyzable GTP analogue [0.01 to 100 microM guanosine 5'-O-(3-thiotriphosphate)] potentiated the secretory response to Rab3AL peptide. This potentiated response indicates that other GTP-binding proteins are involved in calcium-independent secretion. Preincubation of cells with streptolysin O (10-30 min), to allow egress of cytosolic constituents, enhanced the response to Rab3AL peptide, suggesting that the target(s) for this peptide is (are) anchored to chief cell membranes.(ABSTRACT TRUNCATED AT 250 WORDS)
Impaired hepatocanalicular organic anion transport in endotoxemic rats
Authors: Roelofsen H. , Schoemaker B. , Bakker C. , Ottenhoff R. , Jansen P. L. , Elferink R. P.
Published: December 2017

To investigate the mechanism of sepsis-associated hyperbilirubinemia we have studied hepatocanalicular transport of organic anions in a rat model of endotoxemia. Rats were injected intravenously with lipopolysaccharides (LPS), and at different times after injection, canalicular transport of 2,4-dinitrophenyl-S-glutathione (GS-DNP), as a model organic anion, was measured in perfused livers and isolated hepatocytes. In isolated liver perfusion experiments the initial biliary GS-DNP secretion rate was found to be significantly decreased 18 h after injection with 2 mg/kg LPS. In isolated hepatocytes from these rats, GS-DNP efflux rate was also significantly decreased (193.0 +/- 67 and 448.3 +/- 53 nmol.min-1.g dry wt-1 in endotoxemic and normal hepatocytes, respectively). Inhibition of GS-DNP effluxin isolated endotoxemic hepatocytes was dose dependent and reached a maximum with 0.25 mg/kg LPS. Inhibition was maximal at 12 h after LPS injection. Transport activity gradually returned to normal in 4-5 days after endotoxemia was induced. Dexamethasone pretreatment partially reversed the inhibition of GS-DNP transport in isolated endotoxemic hepatocytes. The phorbol ester phorbol 12-myristate 13-acetate increased GS-DNP efflux by 73 +/- 16 and 24 +/- 8% in endotoxemic and control hepatocytes, respectively, but could not restore the transport activity of endotoxemic hepatocytes to control levels. These results show that canalicular organic anion transport is decreased in the endotoxemic liver; this may play a role in the frequently observed hyperbilirubinemia during sepsis.
A novel serine-specific kinase activity associated with exocrine secretory granules
Authors: Tang L. H. , Modlin I. M. , Caulfield T. A. , Goldenring J. R.
Published: December 2017

Zymogen secretion from exocrine cells involves an exocytotic process that is highly regulated by the modification of cytoplasmic components at different cellular levels. In the present studies, purified secretory granules were prepared from rabbit gastric chief cells, rat pancreatic acinar cells, and parotid glands to characterize a Mg(2+)-dependent protein kinase activity. In chief cell granules, endogenous pepsinogen, a fortuitous substrate, was phosphorylated at optimal Mg2+ and K+ concentrations of 40 and 50 mM, respectively. Adenosine 3',5'-cyclic monophosphate, Ca2+, and calmodulin had no significant effects on the kinase activity. In contrast, Mn2+ or Zn2+ inhibited the kinase activity. In addition to pepsinogen, the exogenous substrates casein, myelin basic protein, and lysine-rich histone were also phosphorylated by the granule-associated kinase. All substrates were exclusively phosphorylated on serine residues. ATP, but not GTP, served as the donor in the phosphorate transfer reaction. Casein kinase (CK) inhibitors CKI-7 and dibromoribofuransylbenzimidazole at concentrations (10 microM) that significantly inhibited CK activities in the tissue homogenate failed to inhibit the granule-associated kinase activity. The kinase activity was localized to the granule membrane and could be removed from the membrane with either 5 mM EDTA or alkaline carbonate extraction. Furthermore, protease digestion sensitivity revealed that the kinase was localized on the cytoplasmic face of the granules. Our results therefore indicate that the secretory granules of exocrine gastric chief cells, pancreatic acini, and parotid acini possess a unique serine-specific protein kinase activity. The cytoplasmic orientation of the kinase activity suggests a possible role in vesicle processing or the exocytotic process.
Metabolism and influence of gastrin-52 on gastric acid secretion in humans
Authors: Hansen C. P. , Stadil F. , Rehfeld J. F.
Published: December 2017

It has been shown recently that the two largest alpha-carboxyamidated progastrin products are gastrin-71 and gastrin-52. Human gastrin-52 has now been synthesized, and the effect on gastric acid secretion and elimination from plasma was examined and compared with gastrin-17 in 12 normal subjects. The peptides were infused separately in four consecutive doses; the maximum response of gastrin-17 and gastrin-52 was 25.2 +/- 2.8 and 22.2 +/- 2.8 mmol H+/50 min, respectively (P< 0.01). This difference in efficacy was presumably related to nonequilibrium of gastrin-52 between plasma and receptor. The elimination of gastrin-17 was monoexponential with a half-life of 4.7 +/- 0.3 min; clearance and apparent volume of distribution were 16.7 +/- 1.5 ml.kg-1.min-1 and 106.0 +/- 9.2 ml/kg, respectively. The elimination of gastrin-52 was biexponential, the half-lives were 4.9 +/- 0.7 and 49.9 +/- 4.2 min, and clearance and apparent volume of distribution were 1.9 +/- 0.2 ml.kg-1.min-1 and 106.3 +/- 10.1 ml/kg, respectively. Gel chromatography of plasma samples drawn during infusion of gastrin-52 revealed that most of the immunoreactivity eluted in the position of the intact peptide. Small peaks in the positions of gastrin-34 and the NH2-terminal pentapeptide fragment of gastrin-52 indicate that a minor part of gastrin-52 is degraded to smaller peptides in vivo. It is concluded that gastrin-52 is bioactive with an efficacy close to or similar to that of gastrin-17. A minor fraction of gastrin-52 undergoes postsecretory cleavage either in plasma or after capillary transit.
Activation of MAP kinase and translocation with HSP27 in bombesin-induced contraction of rectosigmoid smooth muscle
Authors: Yamada H. , Strahler J. , Welsh M. J. , Bitar K. N.
Published: December 2017

We have investigated whether mitogen-activated protein (MAP) kinase cascade is essential for sustained contraction of smooth muscle cells of the rabbit rectosigmoid. We have identified MAP kinase as one of the enzymes activated by bombesin, performed immunologic studies blocking the activation of MAP kinase, and conducted confocal localization of MAP kinase in relation to heat-shock protein (HSP27), postulated to be involved in the sustained contraction of smooth muscle. Immunoblotting revealed two forms of MAP kinase (42 and 44 kDa). Activation of MAP kinase by bombesin was rapid, reaching a maximum in 30 s and subsequently declining. [D-Phe6,Leu13,psi(CH2NH),Phe14]BN-(6-14), a potent bombesin antagonist, and protein kinase C (PKC) inhibitors 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, calphostin C, and chelerythrine inhibited the increase in MAP kinase induced by bombesin. Immunofluorescent dual labeling and confocal microscopy indicate that these two proteins are closely distributed in resting cells and that during bombesin-induced contraction MAP kinase translocates accompanied by HSP27. In conclusion, a series of events involving PKC activation, MAP kinase activation, and MAP kinase-HSP27 translocation could be the signaling pathway involved in bombesin-induced sustained contraction.
Haustral septations increase axial and radial distribution of luminal contents in glass models of the colon
Authors: Brown B. P. , Schrier J. E. , Berbaum K. S. , Shirazi S. S. , Schulze-Delrieu K.
Published: December 2017

We used two glass models of the colon to test the hypothesis that luminal septations increase efficiency of flow. Each was a straight glass tube 4 cm in diameter and filled with water. One had four septations narrowing the lumen to 2 cm; the other had no septations. For each run, liquid dye and one solid test particle were placed at one end of the model. A single pressure pulse was applied to the dye and particle. The distance each traveled was compared using analysis of variance. The nonseptated model had poor mixing of dye, and the particle moved only short distances. In the septated model, there was thorough mixing of dye and the particle moved rapidly along what appeared as a central core of high-velocity liquid flow from one septal opening to another. Compared with the nonseptated model, this produced statistically significant increases in the distances traveled by means of 32 (dye) and 90% (particle). Our studies suggest that septations in a tubular organ facilitate intraluminal mixing and flow of liquids and solids.
Posttranscriptional regulation of mRNA levels in rat liver associated with deoxycholic acid feeding
Authors: Kren B. T. , Rodrigues C. M. , Setchell K. D. , Steer C. J.
Published: December 2017

We investigated the effects of bile acid feeding on the mRNA levels and transcriptional activity of genes involved in various facets of hepatic cell function. Rats were maintained for 10 days on standard diet supplemented with combinations of 1 and 0.4% deoxycholic acid and ursodeoxycholic acid. Significant reductions in mRNA levels for liver fatty acid binding protein, albumin, the asialoglycoprotein receptor, connexins 32 and 26, and cytochromes P-450IIB1 and P-450IIE1 were associated with 1% deoxycholic acid feeding. Conversely, the 1% deoxycholic acid-fed animals exhibited increased mRNA levels for cholesterol 7 alpha-hydroxylase, 3-hydroxy-3-methylglutaryl-CoA reductase, multidrug resistance, procollagens, extracellular matrix, protooncogenes, tumor suppressors, and cyclins. The 0.4% deoxycholic acid-fed animals exhibited increased mRNA levels for c-jun, H-ras, p53, cyclins D1 and D3, fibronectin, and procollagens alpha 1(I) and alpha 1(III). Transcriptional rate changes could not account for the observed changes in steady-state mRNA levels. Ursodeoxycholic acid feeding had no significant effect on gene expression and almost completely inhibited the changes associated with 1% deoxycholic acid when coadministered. The results indicate that dietary ingestion of deoxycholic acid profoundly affects hepatic gene expression in the rat, and regulation occurs primarily at the posttranscriptional level.
Mechanisms behind changes in gastric acid and bicarbonate outputs during the human interdigestive motility cycle
Authors: Dalenback J. , Fandriks L. , Olbe L. , Sjovall H.
Published: December 2017

Human gastric interdigestive acid and bicarbonate outputs vary cyclically in association with the migrating motor complex (MMC). These phenomena were studied in 26 healthy volunteers by constant-flow gastric perfusion, with continuous recording of pH and Pco2 in mixed gastric effluent and concomitant open-tip manometry of gastroduodenal motility. Stable acid and bicarbonate outputs were registered during less than 50% of the MMC cycle. Acid secretion started to increase 71 +/- 3% into the cycle, with maximum output during antral phase III. Bicarbonate output increased biphasically 1) 40 +/- 5% into the cycle, coinciding with reflux of bile, and 2) at the end of duodenal phase III when the aspirate was devoid of bile. The bicarbonate peak associated with phase III was abolished by atropine (0.01 mg/kg iv, n = 8) and by pyloric occlusion (n = 9) but remained unchanged after omeprazole (n = 10). The acid peak was abolished by both atropine and omeprazole. It is concluded that the MMC-related changes in acid and alkaline outputs represent two different and independent phenomena. Acid secretion cyclicity is due to periodical variations in cholinergic stimulation of the parietal cells. In contrast, the phase III-associated increase in bicarbonate output is due to duodenogastric reflux.
CCK-evoked hyperemia in rat gastric mucosa involves neural mechanisms and nitric oxide
Authors: Heinemann A. , Jocic M. , Peskar B. M. , Holzer P.
Published: December 2017

This study was performed to identify the possible neural mechanisms and mediators that underlie the gastric mucosal hyperemia evoked by cholecystokinin octapeptide (CCK-8). Gastric mucosal blood flow in anesthetized rats was assessed by the clearance of hydrogen and gastric acid secretion determined in the luminally perfused stomach. The gastric mucosal hyperemic effect of a low dose of CCK-8 (0.04 nmol/min iv infusion for 7 min) was abolished by inhibition of nitric oxide synthesis with NG-nitro-L-arginine methyl ester (15 mg/kg iv) and significantly blunted by defunctionalization of afferent neurons with a neurotoxic dose of capsaicin (125 mg/kg sc). The hyperemic reaction to a high dose of CCK-8 (0.2 nmol/min) was not significantly affected by these pharmacological maneuvers. The vasodilator response to low-dose CCK-8 (0.04 nmol/min) was further analyzed and found to be inhibited by acute bilateral subdiaphragmatic vagotomy, atropine (1 mumol/kg ip), and the antagonistic calcitonin gene-related peptide (CGRP) fragment CGRP-(8-37) (6 nmol/ min ia). Cyclooxygenase inhibition with indomethacin (10 mg/kg ip) was ineffective. The CCK-8-induced increment of gastric acid secretion was not significantly altered by any of these procedures. These results indicate that the gastric vasodilator effect of submaximal doses of CCK-8 is brought about by a vagovagal reflex that involves acetylcholine, CGRP or a related peptide, and nitric oxide as vasodilator messengers.
Beta-adrenergic regulation of cholecystokinin secretion in STC-1 cells
Authors: Scott L. , Prpic V. , Capel W. D. , Basavappa S. , Mangel A. W. , Gettys T. W. , Liddle R. A.
Published: December 2017

Previously, it has been shown that an increase in adenosine 3',5'-cyclic monophosphate (cAMP) levels stimulates intestinal secretion of cholecystokinin (CCK); however, the mechanisms for increasing intracellular cAMP levels are not known. Using the CCK-secreting intestinal cell line, STC-1, we evaluated whether beta-adrenergic receptors (beta-ARs) might be present on STC-1 cells and whether they stimulated CCK release through increases in cAMP. Photoaffinity labeling of beta-ARs from solubilized STC-1 cell membranes revealed photoincorporation of the agonist [125I]iodocyanopindolol into an approximately 75-kDa band. Addition of the beta-AR agonist, isoproterenol, in the presence of 3-isobutyl-1-methylxanthine, produced a concentration-dependent increase in both cAMP levels and CCK release. Blockade of beta 1- and/or beta 2-ARs significantly inhibited isoproterenol-stimulated increases in cAMP production and CCK release. With the use of fura 2-loaded cells to measure changes in intracellular Ca2+ concentration ([Ca2+]i), isoproterenol stimulation was found to increase cytosolic Ca2+ levels. To evaluate whether this increase in [Ca2+]i was due to release of Ca2+ or influx of Ca2+, cells were treated with the L-type calcium channel blocker, diltiazem, which inhibited isoproterenol-stimulated CCK secretion. Furthermore, in patch-clamp studies with inside-out membrane patches, addition of the catalytic subunit of protein kinase A activated diltiazem-sensitive Ca2+ channels. It is concluded that beta-ARs are present on STC-1 cells and are coupled to the production of cAMP, which may increase CCK release through a calcium-dependent process.
Deregulation of hexose transporter expression in Caco-2 cells by ras and polyoma middle T oncogenes
Authors: Baron-Delage S. , Mahraoui L. , Cadoret A. , Veissiere D. , Taillemite J. L. , Chastre E. , Gespach C. , Zweibaum A. , Capeau J. , Brot-Laroche E. , Cherqui G.
Published: December 2017

We investigated whether the oncogenic activation of p21ras or pp60c-src, which is frequently observed in colorectal cancers, induced alterations of sugar uptake in human colonic cells. We therefore examined hexose transporter expression and/or activity in Caco-2 cells transfected either with an activated human (Val-12) Ha-ras gene or with the polyoma middle T (PyMT) oncogene, a constitutive activator of pp60c-src tyrosine kinase activity. Experiments were performed at day 20 of culture, when Caco-2 cells express enterocyte-specific GLUT-2, GLUT-5, and SGLT-1 transporters in addition to GLUT-1 and GLUT-3. Along with increased glucose consumption rates, both oncogene-transfected cells exhibited increased levels of GLUT-1 and GLUT-3 mRNAs and/or immunoreactive proteins compared with control vector Caco-2 cells. In contrast, oncogene-transfected cells lost GLUT-2, GLUT-5, and SGLT-1 expression as determined by Northern and/or Western blot analyses and/or specific transport assays. The oncogene-induced repressive effect on these enterocyte-specific hexose transporters extended to brush-border hydrolases and villin but not to tight junctional protein ZO-1. In conclusion, oncogenic p21ras and PyMT/pp60c-src induce severe deregulation of hexose transporter expression in Caco-2 cells, which is manifested by 1) increased GLUT-1 and GLUT-3 expression and 2) repression of GLUT-2, GLUT-5, and SGLT-1, which parallels repression of some markers of the enterocyte-like differentiated phenotype of Caco-2 cells.
cAMP stimulates fluorescent bile acid uptake into hepatocytes by membrane hyperpolarization
Authors: Grune S. , Meng X. J. , Weinman S. A.
Published: December 2017

Elevation of intracellular adenosine 3',5'-cyclic monophosphate (cAMP) hyperpolarizes hepatocytes and increases the uptake rate of bile acids. The purpose of this study was to determine to what extent these two phenomena are linked. Fluorescent bile acid analogues (FBA) were used to probe bile acid transport into whole cell patch-clamped hepatocytes. Na(+)-dependent uptake of cholyl-nitrobenz-2-oxa-1,3-diazol-4-yl-lysine (C-NBD-L), an FBA with a net charge of -1, was shown to be electrogenic, whereas uptake of cholylglycylamidofluorescein (CGamF), an FBA with a net charge of -2, was neutral. Incubation of hepatocytes with 8-bromo-cAMP (8-BrcAMP; 100 microM) increased the uptake rate of the electrogenically transported FBA by 25% (P = 0.002), but had no effect on the uptake rate of the electroneutrally transported FBA. Microelectrode impalements revealed that 8-BrcAMP or forskolin hyperpolarized hepatocytes by 6-8 mV. To determine if hyperpolarization is responsible for the cAMP-induced increase in uptake rate, cAMP was directly introduced into hepatocytes during whole cell patch clamp under voltage-clamp conditions. As long as voltage clamp was maintained at -30 mV there was no stimulation of C-NBD-L uptake. However, when voltage clamp was terminated by either pipette removal or current clamp, cAMP increased the uptake rate by 25-34% (P < 0.002). In both of these protocols, cAMP had no effect on uptake of the electroneutrally transported FBA, CGamF. Finally, in voltage-clamped hepatocytes in the absence of cAMP, a 10-mV hyperpolarization increased the uptake rate of C-NBD-L by 23%. We therefore conclude that short-term cAMP-induced stimulation of fluorescent bile acid uptake in hepatocytes is a direct consequence of membrane hyperpolarization.
Measurements of the jejunal unstirred layer in normal subjects and patients with celiac disease
Authors: Strocchi A. , Corazza G. , Furne J. , Fine C. , Di Sario A. , Gasbarrini G. , Levitt M. D.
Published: December 2017

Normal intestinal absorption of nutrients requires efficient luminal mixing to deliver solute to the brush border. Lacking such mixing, the buildup of thick unstirred layers over the mucosa markedly retards absorption of rapidly transported compounds. Using a technique based on the kinetics of maltose hydrolysis, we measured the unstirred layer thickness of the jejunum of normal subjects and patients with celiac disease, as well as that of the normal rat. The jejunum of humans and rats was perfused with varying maltose concentrations, and the apparent Michaelis constant (Km) and maximal velocity (Vmax) of maltose hydrolysis were determined from double-reciprocal plots. The true Km of intestinal maltase was determined on mucosal biopsies. Unstirred layer thickness was calculated from the in vivo Vmax and apparent Km and the in vitro Km of maltase. The average unstirred layer thickness of 11 celiac patients (170 micron) was seven times greater than that of 3 controls (25 micron). The unstirred layer of each celiac exceeded that of the controls. A variety of factors could account for the less efficient luminal stirring observed in celiacs. Although speculative, villous contractility could be an important stirring mechanism that would be absent in celiacs with villous atrophy. This speculation was supported by the finding of a relatively thick unstirred layer (mean: 106 micron) in rats, an animal that lacks villous contractility. Because any increase in unstirred layer slows transport of rapidly absorbed compounds, poor stirring appears to represent a previously unrecognized defect that could contribute to malabsorption in celiac disease and, perhaps, in other intestinal disorders.
Pyruvate reduces anoxic injury and free radical formation in perfused rat hepatocytes
Authors: Borle A. B. , Stanko R. T.
Published: December 2017

The effects of 5 mM pyruvate on anoxic injury, superoxide (O2-.) and hydrogen peroxide (H2O2) generation, and lactate dehydrogenase (LDH) release during reoxygenation after 2.5 h anoxia were studied in perfused rat hepatocytes. When pyruvate was present during anoxia and reoxygenation, there was little anoxic injury, and the generation of free radicals and LDH release during reoxygenation were reduced 50-60%. When Pyruvate was added during reoxygenation, there was no decrease in O2-. or LDH release, although H2O2 formation was depressed. Free radical formation and anoxic/reperfusion injury were significantly reduced when pyruvate was added during the anoxic period only. Pyruvate reduced the deleterious effects of 10 microM antimycin A by preventing the increase in O2-. formation and LDH release evoked by the inhibitor. These results indicate that pyruvate protected hepatocytes against anoxic injury and that its protective action occurred principally during anoxia and not during reoxygenation. Pyruvate appeared to act at a mitochondrial site, since it reduced the deleterious effects of antimycin A.
Soybean lectin stimulates pancreatic exocrine secretion via CCK-A receptors in rats
Authors: Jordinson M. , Deprez P. H. , Playford R. J. , Heal S. , Freeman T. C. , Alison M. , Calam J.
Published: December 2017

Rats fed raw soy flour (RSF) show pancreatic growth due to excessive cholecystokinin (CCK) release. Soybean trypsin inhibitors are implicated, but rats fed soybean lectin also showed pancreatic growth. Therefore, we studied the effect of soybean lectin on pancreatic protein secretion in anesthetized rats. Intraduodenal administration of 30 mg of RSF stimulated a 1-h integrated rise in pancreatic protein output of 2.2 +/- 1.1 mg/h (mean +/- SE) in rats with bile pancreatic (BP) juice returned to the duodenum. Selective removal of the lectin by affinity to N-acetyl-D-galactosamineagarose abolished the response (-0.1 +/- 0.2 mg/h). Adding back the 84 micrograms of lectin restored the output of 2.2 +/- 0.9 mg/h. With BP juice returned to the duodenum, 84 micrograms of lectin required the added presence of protein and protease inhibitors to have this effect. However, when BP juice was not returned, 84 micrograms of lectin given alone produced a pancreatic response of 3.2 +/- 1.3 mg/h. Plasma CCK concentrations rose significantly from 6.6 +/- 1.9 to 14.3 +/- 2.9 pmol/l, and the pancreatic response was abolished by CCK-A receptor blockade (0.0 +/- 0.1 mg/h). We conclude that soybean lectin plays a major role in the acute stimulation of pancreatic protein secretion by RSF. The lectin releases CCK and the effect is mediated by CCK-A receptors.
5-HT released by mucosal stimuli initiates peristalsis by activating 5-HT4/5-HT1p receptors on sensory CGRP neurons
Authors: Grider J. R. , Kuemmerle J. F. , Jin J. G.
Published: December 2017

The intestinal peristaltic reflex can be elicited by mucosal stimulation or circular muscle stretch. Muscle stretch activates extrinsic, whereas mucosal stimulation activates intrinsic calcitonin gene-related peptide (CGRP)-containing sensory neurons. The present study examined the role of 5-hydroxytryptamine (5-HT) in sensory transmission. A three-compartment preparation of rat colon was used that enables separate measurement of sensory transmitters and modulators. Mucosal stimuli (2-8 brush strokes) caused concurrent increase in 5-HT and CGRP release in proportion to the intensity of stimulation. Release of both 5-HT and CGRP occurred exclusively into the central compartment where the stimuli were applied. Exogenous 5-HT caused a concentration-dependent release of CGRP. Release of CGRP induced by exogenous 5-HT or mucosal stimulation was inhibited by selective 5-HT4 and 5-HT1p antagonists but was not affected by 5-HT1A, 5-HT2, and 5-HT3 antagonists. Ascending contraction and descending relaxation of circular muscle measured in the peripheral orad and caudad compartments, respectively, were also selectively inhibited by 5-HT4 and 5-HT1p antagonists added to the central but not peripheral compartments. In contrast, muscle stretch elicited CGRP but not 5-HT release; the ascending contraction and descending relaxation components of the peristaltic reflex induced by muscle stretch were not affected by 5-HT antagonists. We conclude that 5-HT released by mucosal stimulation initiates the peristaltic reflex by activating 5-HT4/5-HT1p receptors on sensory CGRP-containing neurons.
Role of 5-HT in cholera toxin-induced mucin secretion in the rat small intestine
Authors: Moore B. A. , Sharkey K. A. , Mantle M.
Published: December 2017

We examined the role of 5-hydroxytryptamine (5-HT) in cholera toxin (CT)-induced mucin secretion in the proximal and distal regions of the rat small intestine. Neither the 5-HT2 receptor antagonist ketanserin nor the cyclooxygenase inhibitor indomethacin was capable of inhibiting choleraic mucin secretion. However, in the presence of the mixed 5-HT3/4 receptor antagonist tropisetron at doses that block both receptor subtypes, the secretory response was reduced to baseline levels in the proximal and distal small intestine. The selective 5-HT3 receptor antagonist ondansetron had no significant effect. These findings suggest that choleraic mucin secretion is mediated primarily through the activation of a 5-HT4-like receptor. Mucin secretion in response to the exogenous application of 5-HT occurs via two pathways: one is mediated by a 5-HT4-like receptor and is capsaicin sensitive but tetrodotoxin (TTX) insensitive, and one lacks the capsaicin-sensitive 5-HT4-mediated response but is TTX sensitive. Both converge on a common pathway that is cholinergic. No significant differences were observed between proximal and distal intestinal segments.
Active microtubule-dependent secretion of a fluorescent bile salt derivative in skate hepatocyte clusters
Authors: Miller D. S. , Fricker G. , Schramm U. , Henson J. H. , Hager D. N. , Nundy S. , Ballatori N. , Boyer J. L.
Published: December 2017

Fluorescence microscopy and video image analysis were used to study the transport of a fluorescent bile acid derivative [N-[7-(4-nitrobenzo-2-oxa-1,3-diazol)]-7 beta-amino-3 alpha, 12 alpha-dihydroxy-5 beta-cholan-24-oyl-2-aminoethanesulfonate (NBD-TC)] in isolated clusters of hepatocytes from the little skate Raja erinacea. Analysis of images of hepatocyte clusters that were incubated in medium with 0.5-1 microM NBD-TC showed that the fluorescent derivative accumulated in the cells and that the clusters retained a patent canalicular lumen as well as the ability to actively transport the bile acid derivative from the cells into the lumen; i.e., the lumen-to-cell fluorescence ratio greatly exceeded unity. NBD-TC uptake by hepatocytes was inhibited by several organic anions, of which taurocholate was the most effective. Uptake was also blocked by metabolic inhibitors and by incubation in the cold. Neither Na replacement nor increased medium K, which depolarizes the membrane electrical potential [potential difference (PD)], affected NBD-TC accumulation by hepatocytes. Transport of NBD-TC into the canalicular lumen was inhibited by incubation in the cold and was substantially reduced by high-K medium; these blocks were removed by warming and transfer to normal-K medium, respectively. Treatment of hepatocytes with 20-40 microM nocodazole, a drug that reversibly depolymerizes microtubules, reduced cellular NBD-TC accumulation and blocked its secretion into the canalicular lumen; nocodazole effects were reversed by washing the hepatocyte clusters in drug-free medium. Thus uptake of NBD-TC by skate hepatocytes is active and carrier mediated but not dependent on the PD or Na gradient. NBD-TC secretion from cell to canalicular lumen also appears to be active and carrier mediated. Canalicular secretion appears to be driven at least in part by the PD and is highly dependent on an intact microtubular system in this marine species.
Significance of peptic digestion in rat pancreatic secretory response to dietary protein
Authors: Guan D. , Green G. M.
Published: December 2017

The importance of peptic digestion of dietary protein in pancreatic enzyme secretion and cholecystokinin (CCK) release was investigated in conscious rats. Native casein and native bovine serum albumin (BSA) were infused intragastrically and intra-intestinally, and the effect of peptic predigestion on the pancreatic secretory and plasma CCK responses to BSA were determined (all infused at 450 mg/h). When dietary proteins were infused intraduodenally, native casein was a much stronger stimulant of CCK release (5.8 +/- 0.6 vs. 1.6 +/- 0.2 pM) and pancreatic protein secretion [5,592 +/- 736 vs. 750 +/- 461 (0-180)mg.kg-1.min] than native BSA. Infusion by the intragastric route markedly increased pancreatic protein secretion for BSA but not for casein. HCl-pepsin treatment of BSA significantly increased its ability to increase pancreatic secretion and plasma CCK. Pancreatic protease binding to native BSA was inferior compared with casein. Peptic digestion of BSA increased its protease binding activity more than threefold. The results indicate that peptic digestion of dietary proteins enhances the proteins' ability to elicit the pancreatic feedback stimulatory response.
Immunologic distribution of an organic anion transport protein in rat liver and kidney
Authors: Bergwerk A. J. , Shi X. , Ford A. C. , Kanai N. , Jacquemin E. , Burk R. D. , Bai S. , Novikoff P. M. , Stieger B. , Meier P. J. , Schuster V. L. , Wolkoff A. W.
Published: December 2017

A Na(+)-independent organic anion transport protein was recently cloned from rat liver using a Xenopus laevis oocyte expression system [E. Jacquemin, B. Hagenbuch, B. Stieger, A.W. Wolkoff, and P.J. Meier, Proc. Natl. Acad. Sci. USA 91: 133-137, 1994]. Although expression of this protein is sufficient for cells to transport the organic anion bromosulfophthalein, little is known about its cell biology or biochemical characteristics. Northern blot analysis performed under high-stringency conditions revealed hybridization with RNA only from liver and kidney; transcripts appeared the same in these two organs. Within kidney, hybridization was greatest when RNA extracted from the outer medulla was used. Immunoblot analysis revealed that in liver, the transporter was enriched in 0.1 M Na2CO3-extracted membranes and sinusoidal plasma membrane preparations, consistent with its being an integral membrane protein. This 80-kDa protein migrated as a 65-kDa protein after treatment with N-glycanase. Immunomorphological examination of liver revealed basolateral plasma membrane localization. In 0.1 M Na2CO3-extracted membranes of kidney, the transporter migrated as an 83-kDa protein on nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). On reduction, it resolved into peptides of 33 and 37 kDa. SDS-PAGE migration of the liver protein was unaffected by reduction. Immunomorphological examination of kidney revealed apical plasma membrane localization in the S3 segment of the proximal tubule of the outer medulla. Differential processing and trafficking of this transporter in liver and kidney may have important functional and regulatory consequences.
Stoichiometry of neurally induced VIP release, NO formation, and relaxation in rabbit and rat gastric muscle
Authors: Jin J. G. , Murthy K. S. , Grider J. R. , Makhlouf G. M.
Published: December 2017

Vasoactive intestinal peptide (VIP) release, nitric oxide (NO) formation, and relaxation induced by nerve stimulation were examined in rabbit and rat gastric muscle. VIP stimulated NO formation in muscle strips, whereas NO stimulated VIP release. Nerve stimulation (0.025-16 Hz or 2-940 pulses) elicited frequency-dependent stimulation of VIP release, NO formation, and relaxation, all significant at two to three pulses. NG-nitro-L-arginine (L-NMA) abolished NO formation, abolished VIP release and relaxation at low frequencies, and partly inhibited them at higher frequencies. Oxyhemoglobin (oxy-Hb) inhibited VIP release and relaxation by 80% at low frequencies and 20-30% at higher frequencies. VIP-(10-28) abolished NO formation and relaxation at lower frequencies and partly inhibited them at higher frequencies; in contrast, VIP-(10-28) augmented VIP release in both species. The pattern of inhibition was similar in both species. Inhibition of maximal NO formation by VIP-(10-28) (82% in rabbit; 48% in rat) implied that a major component of NO is formed in muscle cells by the action of VIP. Thus 1) inhibition of relaxation by L-NNA reflects suppression of NO and VIP release from nerve terminals and NO formation in muscle cells, 2) inhibition by VIP-(10-28) partly reflects suppression of NO formation in muscle cells, and 3) inhibition by oxy-Hb reflects neutralization of extracellular NO and suppression of VIP release. The study demonstrates the dual origin of NO from nerves and muscle and its interplay with VIP in regulating relaxation.