bluuebunny/crossref_metadata_2025 huggingface dataset:
Bile duct epithelium: frontiers in transport physiology
Authors: Boyer J. L.
Published: December 2017

The bile duct epithelium is known to modify hepatic bile by both secretory and absorptive processes. However, identification and characterization of the transport systems that carry out these physiological functions at the cellular and molecular level have been slow compared with progress in understanding hepatic bile production. Recently, techniques have been developed that enable bile duct cells to be isolated in substantial number and purity and as intact polarized units that can be studied in vitro. These newer preparations have enabled classic physiological approaches to be performed directly with the bile duct epithelial tissues. Progress in this important area of biliary physiology is now rapid and forms the basis for this review.
Calyculin A, a phosphoprotein phosphatase inhibitor, stimulates acid secretion in isolated gastric glands
Authors: Urushidani T. , Nagao T.
Published: December 2017

The effects of pkadaic acid (OKA) and calyculin A (CLA), inhibitors of protein phosphatases type 1 (PrPase1) and type 2A (PrPase2A), an acid secretion were examined in rabbit isolated gastric gland, CLA, but not OKA, strongly stimulated acid secretion by itself without affecting glandular adenosine 3',5'-cyclic monophosphate (cAMP) contents. CLA-induced secretion was suggested to be mainly due to the increase in the phosphorylation of protein kinase A substrates via the inhibition of PrPase1 in the parietal cell, since 1) CLA-induced secretion was not inhibited by cimetidine or atropine, 2) a protein kinase A inhibitor inhibited the secretion, whereas a protein kinase C inhibitor did not, 3) CLA augmented dibutyryl cAMP-induced secretion in some cases, and 4) OKA, which is 100 times more selective to PrPase2A than to PrPase1, was not a secretagogue. Unexpectedly, CLA did not augment the secretion by histamine, possibly because the inhibitor augmented the phosphorylation-mediating negative feedback pathway as well. Both CLA and OKA markedly increased phosphorylation of ezrin, a putative protein kinase A substrate, in the course of secretory activation.
Adaptation to fat markedly increases pancreatic secretory response to intraduodenal fat in rats
Authors: Spannagel A. W. , Nakano I. , Tawil T. , Chey W. Y. , Liddle R. A. , Green G. M.
Published: December 2017

Exposure to higher levels of fat in the diet increases the secretion of fat-digesting enzymes in pancreatic juice. This study examines the functional consequences of this phenomenon and demonstrates that adapting rats to high fat (triglyceride) loads increases the release of cholecystokinin (CCK) and the pancreatic secretory response to intraduodenal fat. Lipolytic activity in the small intestine was also higher in adapted rats. Exchanging pancreatic juice from unadapted rats with pancreatic juice from adapted rats decreased the response to fat in adapted rats and increased the response to fat in unadapted rats. Infusing oleic acid into unadapted rats stimulated CCK secretion and pancreatic exocrine secretion to levels observed with triglycerides in adapted rats. Pancreatic exocrine secretion in response to intraduodenal fat in rats adapted to a high-fat (20%) diet were significantly higher than the responses seen in rats fed a low-fat (5%) diet. Adaptation to fat increases the pancreatic secretory and plasma CCK responses to fat, apparently by increasing the efficiency of triglyceride digestion and thereby increasing CCK release.
Depolarization-stimulated cholecystokinin secretion is mediated by L-type calcium channels in STC-1 cells
Authors: Mangel A. W. , Scott L. , Liddle R. A.
Published: December 2017

To examine the role of calcium channels in depolarization-activated cholecystokinin (CCK) release, studies were performed in an intestinal CCK-secreting cell line, STC-1. Blockade of potassium channels with barium chloride (5 mM) increased the release of CCK by 374.6 +/- 46.6% of control levels. Barium-induced secretion was inhibited by the L-type calcium-channel blocker, nicardipine. Nicardipine (10(-9)-10(-5) M) produced a dose-dependent inhibition in barium-stimulated secretion with a half-maximal inhibition (IC50) value of 0.1 microM. A second L-type calcium-channel blocker, diltiazem (10(-9)-10(-4) M), also inhibited barium-induced CCK secretion with an IC50 value of 5.1 microM. By contrast, the T-type calcium-channel blocker, nickel chloride (10(-7)-10(-8) M), failed to significantly inhibit barium-induced CCK secretion. To further evaluate a role for L-type calcium channels in the secretion of CCK, the effects of the L-type calcium channel opener, BAY K 8644, were examined. BAY K 8644 (10(-8)-10(-4) M) produced a dose-dependent stimulation in CCK release with a mean effective concentration value of 0.2 microM. Recordings of single-channel currents from inside-out membrane patches showed activation of calcium channels by BAY K 8644 (1 microM), with a primary channel conductance of 26.0 +/- 1.2 pS. It is concluded that inhibition of potassium channel activity depolarizes the plasma membrane, thereby activating L-type, but not T-type, calcium channels. The corresponding influx of calcium serves to trigger secretion of CCK.
Defective iron uptake by the duodenum of Belgrade rats fed diets of different iron contents
Authors: Oates P. S. , Morgan E. H.
Published: December 2017

Homozygous Belgrade rats have an inherited hypochromic, microcytic anemia that is due to impaired iron transport into immature erythrocytes. There is also evidence for abnormal iron transport in other tissues such as the intestine. This study was aimed at investigating the intestinal defect in rats that had been fed diets for 12 days that are normal, low, or high in iron. The duodenal uptake, transfer, and absorption of Fe(III)-nitrilotriacetate and Fe(II)-ascorbate were studied using in vivo tied-off gut sacs in genetically normal rats and in heterozygous or homozygous Belgrade rats. In normal and heterozygous Belgrade rats, the handling of Fe(III) and Fe(II) was similar; uptake, transfer, and absorption of Fe(III) and Fe(II) changed inversely with the iron content of the diet. In contrast, in homozygous Belgrade rats the uptake of both Fe(III) and Fe(II) was markedly reduced and absorption of Fe(III) did not change when animals were fed an iron-deficient diet. Since absorption of Fe(II) was similar to Fe(III), there is no evidence that the defect in iron absorption is due to failure of a mechanism for reduction of Fe(III). The lowered uptake of Fe(III) and Fe(II) in homozygous Belgrade rats probably involves a defective iron carrier associated with the microvillous membrane of the duodenum.
Pancreatic secretion stimulated by CCK is not mediated by capsaicin-sensitive vagal afferent pathway in awake rats
Authors: Guan D. , Phillips W. T. , Green G. M.
Published: December 2017

A capsaicin-sensitive vagal afferent pathway was reported to mediate the effect of endogenous cholecystokinin (CCK) on pancreatic secretion in anesthetized rats. Because neural blockade affects pancreatic secretion much less in awake than in anesthetized rats, the effect of capsaicin ablation of vagal afferent pathways on pancreatic secretion stimulated by endogenous CCK was examined in awake rats. During surgery, abdominal vagal trunks were exposed, and 0.1 ml of capsaicin (10 mg/ml) was applied to the vagal trunks. Ablation of the vagal afferent pathway was assessed by the ability of intraperitoneal cholecystokinin octapeptide (CCK-8) to suppress food intake and inhibit gastric emptying. Endogenous CCK release was stimulated by diversion of bile pancreatic juice from the intestine and by intraduodenal infusion of casein. Pancreatic protein and fluid secretion were significantly increased by both treatments, and the responses were unaffected by capsaicin. Intraperitoneal CCK-8 markedly inhibited food intake and gastric emptying, and both effects were significantly attenuated in capsaicin-treated rats, indicating that capsaicin treatment successfully ablated vagal afferent fibers. It is concluded that CCK-stimulated pancreatic secretion in rats is not mediated by a vagal afferent pathway.
Hyperpermeability and ATP depletion induced by chronic hypoxia or glycolytic inhibition in Caco-2BBe monolayers
Authors: Unno N. , Menconi M. J. , Salzman A. L. , Smith M. , Hagen S. , Ge Y. , Ezzell R. M. , Fink M. P.
Published: December 2017

Previous studies have documented that the barrier function of epithelial or endothelial monolayers is deranged when cellular ATP levels are rapidly decreased to very low levels by inhibitors of mitochondrial and glycolytic function. We hypothesized that lesser degrees of ATP depletion also might affect epithelial permeability, particularly if the perturbation were sustained for a prolonged interval. Using Caco-2BBe cells grown on permeable supports mounted in bicameral chambers, we assessed permeability by measuring the apical-to-basolateral clearance (flux divided by apical compartment concentration) of fluorescein disulfonic acid. ATP was depleted by incubating cells in glucose-free (Glu-) medium containing 10 mM 2-deoxyglucose (2-DOG) for 12, 24, or 48 h or under an anoxic atmosphere for 24, 48, or 72 h. Although both models of energy depletion were characterized by significant derangements in barrier function, metabolic inhibition with 2-DOG/ Glu- resulted in greater increases in permeability and more profound decrements in cellular ATP content. Morphological studies using electron and confocal fluorescence microscopy showed structural changes in individual cells and derangements in the normal distribution of perijunctional actin after monolayers were incubated with 2-DOG/Glu- but not after incubation under an anoxic atmosphere. Addition of 10 mM lactic acid (final pH 6.7) provided significant protection against both hyperpermeability and ATP depletion induced by 2-DOG/Glu-. We conclude that moderate degrees of ATP depletion are sufficient to increase the permeability of Caco-2BBe monolayers and that lactic acidosis helps to preserve ATP content, barrier function, and morphological integrity in hypoxic intestinal epithelial cells.
Initiation of esophageal secondary peristalsis by slow fluid infusion in the opossum: effect of hydrochloric acid
Authors: Kusano M. , Hogan W. J. , Lang I. M. , Bonnevier J. L. , Massey B. T. , Shaker R.
Published: December 2017

We investigated the mechanisms of slow fluid infusion-induced secondary peristalsis and the effects of hydrochloric acid on this response. In 13 chronically esophagostomized opossum, acidic and neutral barium sulfate were infused into the distal esophagus at a rate of 1.1 ml/min, while recording the esophageal dimension by videofluoroscopy and esophageal intraluminal pressure concurrently. The effects of atropine, tetrodotoxin, capsaicin, and bilateral cervical vagotomy on the response to slow fluid infusion were examined. Acidic barium initiated secondary peristalsis more frequently and at shorter latency with less increase of preperistaltic intraesophageal pressure than neutral barium (P < 0.05). Atropine abolished secondary peristalsis initiated by neutral barium. For acidic barium, atropine decreased the incidence of secondary peristalsis, increased the latency for initiation of secondary peristalsis, and initiated secondary peristalsis more distally (P < 0.05). Tetrodotoxin or vagotomy and capsaicin abolished activation of secondary peristalsis. We concluded that secondary peristalsis can be stimulated in response to slow distension by minute amounts of fluid. This peristalsis is atropine and capsaicin sensitive and vagally mediated. The presence of acid significantly lowers the threshold for stimulation of secondary peristalsis induced by slow fluid distension. This effect seems to be atropine resistant.
Differential effects of GTP gamma S on acid and pepsinogen secretion by permeable gastric glands
Authors: Miller M. D. , Hersey S. J.
Published: December 2017

Gastric glands isolated from rabbit stomach were permeabilized with Staphylococcus aureus alpha-toxin. Acid secretion by parietal cells, as measured by the accumulation of weak base, was inhibited by incubation with alpha-toxin but could be restored by addition of exogenous ATP (1 mM). The permeable glands were found to retain acid secretory responses to receptor-linked secretagogues, histamine and carbachol, as well as to intracellular mediators, forskolin and 8-bromoadenosine 3',5'-cyclic monophosphate, indicating the presence of intact, functional intracellular coupling mechanisms. Both basal and stimulated acid secretion by the permeable glands were blocked by the Mg2+ chelator, trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA; 5 mM), whereas CDTA had no effect on nonpermeabilized glands. These results are interpreted to show that alpha-toxin permeabilizes parietal cells to moderate sized molecules without causing a loss of critical intracellular components. The acid secretory responses to histamine and carbachol persisted in media containing low ( < 50 nM) levels of free Ca2+ buffered by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (0.5 mM), indicating that changes in bulk Ca2+ are not required for these responses. Inclusion of the nonhydrolyzable analogue of GTP, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S; 100 microM), resulted in inhibition of spontaneous acid secretion, blocked responses to all agents tested, and inhibited stimulated acid secretion. GTP gamma S had no effect on nonpermeabilized glands. No effects on acid secretion by either permeable or nonpermeable glands were observed with GTP, guanosine diphosphate, or guanosine 5'-O-(2-thiodiphosphate). GTP gamma S had no effect on H+ gradient formation by gastric membrane vesicles, showing that it does not inhibit the gastric H(+)-K(+)-adenosinetriphosphatase directly. These results are interpreted to show that GTP gamma S interacts at a postreceptor site to inhibit or reverse a critical step in stimulus-secretion coupling in parietal cells. In contrast to the effect on parietal cells, GTP gamma S was found to stimulate pepsinogen secretion by alpha-toxin-permeabilized chief cells. The differential effects of GTP gamma S on acid and pepsinogen secretions suggest unique roles for GTP binding proteins in these two secretory processes. The use of alpha-toxin-permeabilized gastric glands should prove useful in defining the stimulus-secretion coupling mechanisms involved in both acid and pepsinogen secretions.
Edema and intrapancreatic trypsinogen activation precede glutathione depletion during caerulein pancreatitis
Authors: Grady T. , Saluja A. , Kaiser A. , Steer M.
Published: December 2017

Acute pancreatitis is characterized by hyperamylasemia, pancreatic edema, and the presence of activated digestive enzymes within the pancreas. The secretagogue-induced model of acute pancreatitis is also characterized by pancreatic acinar cell vacuolation, subcellular redistribution of lysosomal hydrolases, and a fall in pancreatic glutathione levels. We have performed time-dependence studies to determine the sequence with which these phenomena appear and to establish their cause-and-effect relationship. Evidence of lysosomal enzyme redistribution and trypsinogen activation within the pancreas could be detected within 10-15 min of the onset of supramaximal secretagogue stimulation, while hyperamylasemia (30 min), pancreatic edema (60 min), and acinar cell vacuolation (60 min) occurred at later times. Pancreatic glutathione levels were either unchanged (15 and 30 min) or elevated (60 min) during the early times of supramaximal stimulation and were only noted to be decreased at a later time. These results support the conclusion that intrapancreatic digestive enzyme activation, possibly occurring by a mechanism involving lysosomal hydrolase redistribution, is an early and likely a critical event in the evolution of secretagogue-induced pancreatitis but that glutathione depletion is neither early nor critical to the evolution of this model of pancreatitis.
Membrane cholesterol alters gallbladder muscle contractility in prairie dogs
Authors: Yu P. , Chen Q. , Biancani P. , Behar J.
Published: December 2017

The cause and effect relationship between membrane cholesterol and gallbladder muscle contractility was examined by altering membrane cholesterol to phospholipid mole ratio using cholesterol-rich or cholesterol-free liposomes. Gallbladder single muscle cells, from prairie dogs that were fed either a regular or high-cholesterol (1.2%) diet, were isolated enzymatically with collagenase. Plasma membranes of gallbladder muscle were purified in sucrose gradient. Cholesterol was measured using the cholesterol oxidase method. Phospholipids were measured with the method of G.R. Bartlett (J. Biol. Chem. 234: 466-468, 1959). The results of this experiment are 1) after high-cholesterol feeding, cholesterol contents and cholesterol/ phospholipid mole ratio in plasma membranes of gallbladder muscle increased 90%, and muscle cell contraction in response to cholecystokinin octapeptide decreased 58%; 2) similar changes were observed when normal gallbladder muscle cells were incubated with cholesterol-rich liposomes for 2 h; and 3) the changes induced either in vivo or in vitro were reversed when muscle cells were subsequently incubated with cholesterol-free liposomes for 2-6 h. We conclude that gallbladder muscle may incorporate excess cholesterol into its plasma membrane when exposed to a cholesterol-rich environment, that excess membrane cholesterol impairs muscle contractility, and that these changes appear to be reversible.
Action potential generation in the small intestine of W mutant mice that lack interstitial cells of Cajal
Authors: Malysz J. , Thuneberg L. , Mikkelsen H. B. , Huizinga J. D.
Published: December 2017

The small intestine of W/Wv mice lacks both the network of interstitial cells of Cajal (ICC), associated with Auerbach's plexus, and pacemaker activity, i.e., it does not generate slow-wave-type action potentials. The W/Wv muscle preparations showed a wide variety of electrical activities, ranging from total quiescence to generation of action potentials at regular or irregular frequency with or without periods of quiescence. The action potentials consisted of a slow component with superimposed spikes, preceded by a slowly developing depolarization and followed by a transient hyperpolarization. The action potentials were completely abolished by L-type Ca2+ channel blockers. W/Wv mice responded to K+ channel blockade (0.5 mM Ba2+ or 10 mM tetraethylammonium chloride) with effects on amplitude, frequency, rate of rise, and duration of the action potentials. In quiescent tissues from W/Wv mice, K+ channel blockade evoked the typical spikelike action potentials. Electron microscopy identified few methylene blue-positive cells in the W/Wv small intestine associated with Auerbach's plexus as individual ICC. Numbers of resident macrophage-like cells (MLC) and fibroblast-like cells (FLC) were significantly changed. Neither FLC nor MLC were part of a network nor did they form specialized junctions with neighboring cells as ICC do. Hence no cell type had replaced ICC at their normal morphological position associated with Auerbach's plexus. ICC were present in W/Wv mice at the deep muscular plexus in normal organization and numbers, indicating that they are not dependent on the Kit protein and do not take part in generation of pacemaker activity.
The effect of GIP and glucagon-like peptides on intestinal basolateral membrane hexose transport
Authors: Cheeseman C. I. , Tsang R.
Published: December 2017

The effect of gastric inhibitory polypeptide (GIP) and the related glucagon-like peptides-1 and -2 (GLP-1 and GLP-2) on jejunal basolateral membrane glucose transport was investigated to determine if the upregulation produced by luminal hexoses could be explained by the release of one or more of these peptides. Luminal perfusion of the rat jejunum for 4 h, under pentobarbital sodium anesthesia, with 100 mM D-glucose produced a significant increase in plasma GIP levels. Vascular infusion of saline containing 100-800 pM GIP also increased the maximal transport rate for carrier-mediated glucose uptake in jejunal basolateral membrane vesicles. The effect of vascular 400 pM GIP was maximal after 1 h and maintained out to 4 h. The effect of luminal glucose could be blocked by preinjection with anti-GIP antibodies, whereas an antineurotensin antibody had no effect. Vascular infusion with 800 pM GLP-1-(7-36) amide had no effect, but GLP-2 (400 and 800 pM) increased the D-glucose maximal transport rate. An anti-GLP antibody was able to block the response to luminal glucose.
Inhibition of nitric oxide synthase gene expression in vivo and in vitro by repeated doses of endotoxin
Authors: Chang C. C. , McCormick C. C. , Lin A. W. , Dietert R. R. , Sung Y. J.
Published: December 2017

We have examined the effects of repeated endotoxin administration in vivo and in vitro on the induction of nitric oxide synthase (NOS). In vivo, hepatic NOS activity and mRNA were increased markedly by the administration of Escherichia coli lipopolysaccharide (LPS). The change in hepatic NOS activity coincided with a marked accumulation of hepatic citrulline. Both enzyme activity and citrulline concentration returned to normal by 12 h after LPS administration. At this time, a subsequent administration of endotoxin caused no change in either NOS mRNA, NOS activity, or citrulline concentration, and thus an endotoxin-refractory state for nitric oxide (NO) synthesis was established. Normal sensitivity was reestablished by 24 h after the initial dose. In vitro studies using both a macrophage cell line (HD11) and primary macrophages indicated that LPS pretreatment caused cells in culture to become completely refractory to subsequent stimulation by LPS. Finally, we tested the hypothesis that NO may be involved in the development of the refractory state. Various inhibitors blocked the initial synthesis of NO by > 90% but failed to influence the development of the refractory state. Our study demonstrates both in vivo and in vitro that NO synthesis is completely blocked after repeated exposure to endotoxin by a mechanism that appears to be pretranslational. This model of early endotoxin tolerance may provide insight into the molecular mechanisms that regulate expression of the NOS gene.
Relationship between polyamines, actin distribution, and gastric healing in rats
Authors: Banan A. , Wang J. Y. , McCormack S. A. , Johnson L. R.
Published: December 2017

ntragastric administration of 3.4 M NaCl damages the gastric mucosa and increases the activity of ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine synthesis. Polyamines are essential for the repair of gastric erosions. Little is known about the restitution of damaged mucosa except that cell migration is essential. Actin is the principal cytoskeletal protein and is essential for migration. This investigation determines the relationship between polyamines, actin, and gastric healing. Rats were fasted for 22 h and given 1.0 ml of 3.4 M NaCl intragastrically and killed 1, 2, 4, 8, and 10 h later. The mucosa was assayed for ODC activity and stained for G- and F-actin. F-actin was concentrated below the damaged mucosa at 1.5, 2, and 4 h. There was no increase in F-actin distribution at any time point, when NaCl-treated animals were given alpha-difluoromethylornithine (DFMO), a specific inhibitor of ODC. In addition, DFMO significantly prevented the healing of the mucosal lesions. Spermidine treatment after DFMO + NaCl significantly prevented the effects of DFMO. Cytochalasin D, a potent actin-disrupting drug, significantly delayed normal gastric mucosal healing. The endogenous polyamines increased significantly in NaCl animals. Data indicate that increases in polyamine synthesis after damage influence the distribution of F-actin in vivo, which may play a part in the healing of mucosal erosions.
Molecular cloning of canalicular multispecific organic anion transporter defective in EHBR
Authors: Ito K. , Suzuki H. , Hirohashi T. , Kume K. , Shimizu T. , Sugiyama Y.
Published: December 2017

Several organic anions are excreted into the bile via a canalicular multispecific organic anion transporter (cMOAT), which is hereditarily defective in mutant rats, such as the Eisai hyperbilirubinemic rat (EHBR) and TR- rat. In the present study, we cloned cMOAT from the Sprague-Dawley rat liver cDNA library based on the homology with human multidrug resistance-associated protein (hMRP). cMOAT was encoded by 4,623-base pair (bp) cDNA with a homology of 53.0 and 46.3% with hMRP at the cDNA and deduced amino acid level, respectively. The deduced amino acid sequence was the same as that cloned in Wistar rats (C. C. Paulusma, P. J. Bosma, G. J. Zaman, C. T. Bakker, M. Otter, G. L. Sceffer, P. Borst, and R. P. Oude Elferink. Science Wash. DC 271: 1126, 1996) except for four amino acid substitutions. By screening the library, three kinds of cDNA species for cMOAT with the same open reading frame and different 3'-untranslated region lengths (0.2, 1.5, and 3.5 kbp) were isolated. The Northern blot analysis of poly(A)+ RNA from the liver revealed that the expression of plural bands (approximately 5, 6, and 8 kb) was defective in EHBR, and this may be due to the presence of these cDNA species. Expression of cMOAT was observed almost exclusively in the liver and to a lesser extent in the duodenum, kidney, and jejunum. Reverse transcription-polymerase chain reaction (RT-PCR) and subsequent sequence analysis of EHBR liver, kidney, duodenum, and jejunum revealed that 1-bp replacement from G to A at nucleotide 2564 resulted in the introduction of the premature stop codon in all tissues examined. This mutation was different from that observed in TR (C. C. Paulusma, P. J. Bosma, G. J. Zaman, C. T. Bakker, M. Otter, G. L. Sceffer, P. Borst, and R. P. Oude Elferink. Science Wash. DC 271: 1126, 1996). Because EHBR and TR- are allelic mutants and both strains exhibit an autosomal recessive inheritance in the biliary excretion of organic anions it was concluded that the impaired expression of this particular protein is related to the pathogenesis of hyperbilirubinemia in the mutant animals.
Stress-induced enhancement of colitis in rats: CRF and arginine vasopressin are not involved
Authors: Gue M. , Bonbonne C. , Fioramonti J. , More J. , Del Rio-Lacheze C. , Comera C. , Bueno L.
Published: December 2017

Because exacerbation of colitis seems to be associated with stress, we proposed evaluating the influence of stress and the involvement of corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) on experimental colitis in rats. Partial restraint stress was applied during 4 consecutive days, before or after intracolonic 2,4,6-trinitrobenzenesulfonic acid (TNB) instillation (15 mg) in rats. Finally, two groups of rats were centrally injected with alpha-helical CRF-(9-41) (5 micrograms) or AVP antagonist (5 micrograms) before each session of stress. Stress was applied before or right after TNB enhanced colitis, with an increase in macroscopic and histological scores and myeloperoxidase activity, alpha-Helical CRF-(9-41) or AVP antagonist had no effect on TNB-induced colitis but enhanced the effects of stress on colitis. These results show that stress may exacerbate experimental colitis in rats and that CRF and AVP are not responsible for this effect.
Substance P as a mediator of colonic secretory reflexes
Authors: Cooke H. J. , Sidhu M. , Fox P. , Wang Y. Z. , Zimmermann E. M.
Published: December 2017

The role of substance P in neural reflex pathways activated by stroking was investigated in muscle-stripped segments of distal colon from guinea pigs. Stroking the mucosal surface with a brush at 1 stroke/s evoked an increase in short-circuit current (Isc) indicative of chloride secretion. The response to mucosal stroking was maximally reduced by 69-75% by the antagonist GR-82334. The agonist [Sar9,Met(O2)11] substance P caused a bumetanide-sensitive increase in Isc when added to the mucosal or serosal bath. Ablation of extrinsic afferents with acute or chronic administration of capsaicin did not alter the mucosal stroking response. Reverse transcription-polymerase chain reaction and in situ hybridization revealed the presence of neurokinin1 (NK1) receptor messenger RNA in isolated colonocytes or crypt glands. Ligand binding of 125I-Bolton-Hunter-labeled substance P was inhibited by GR-82334. The 50% inhibitory concentration was 0.84 nM. The results demonstrate a role for substance P released from capsaicin-insensitive submucosal neurons and in mucosal stroking reflexes. The presence of NK1 receptors on isolated colonocytes suggests that appropriate elements are present for axon reflex activation of intestinal epithelial cells.
A role for tissue transglutaminase in hepatic injury and fibrogenesis, and its regulation by NF-kappaB
Authors: Mirza A. , Liu S. L. , Frizell E. , Zhu J. , Maddukuri S. , Martinez J. , Davies P. , Schwarting R. , Norton P. , Zern M. A.
Published: December 2017

This study was undertaken to delineate a possible role for tissue transglutaminase (tTG), an enzyme that catalyzes protein cross-linking, in hepatic fibrogenesis. Rats were treated with CCl4 solution and then killed at different stages of liver injury and fibrogenesis. Liver tTG mRNA levels were markedly increased as early as 6 h after the first injection, peaked at 4 days and 1 wk, and remained increased for 8 wk. The enzymatic activity of tTG was increased in livers of rats treated with CCl4, in a fashion that paralleled the Northern blot results. Cell isolation experiments indicated that all hepatic cell types synthesize tTG mRNA. Increased binding to the nuclear factor-kappaB (NF-kappaB) motif of the tTG promoter was found in the nuclear extracts prepared from CCl4-treated samples. These data demonstrate an increase in tTG gene expression during hepatic injury and fibrosis, suggesting a possible role for this enzyme in stabilizing the fibrotic bands during hepatic fibrogenesis. Moreover, increased NF-kappaB binding to the tTG promoter may represent one of the mechanisms by which cell injury induces tTG transcription and thus potentiates the process of fibrogenesis.
CCK activates p90rsk in rat pancreatic acini through protein kinase C
Authors: Bragado M. J. , Dabrowski A. , Groblewski G. E. , Williams J. A.
Published: December 2017

The presence of the 90-kDa ribosomal S6 protein kinase (p90(rsk)) in isolated rat pancreatic acini was demonstrated by Western blotting and immunoprecipitation with anti-p90(rsk). Cholecystokinin (CCK) activated p90(rsk) activity in a time- and dose-dependent manner and increased its phosphorylation. The threshold concentration of CCK was 10 pM and the maximal effect was seen at 1 nM. An increase in p90(rsk) was observed 1 min after 1 nM CCK stimulation, reaching a maximum at 10 min, when p90(rsk) activity was increased 5.4-fold. Carbachol and bombesin, but not vasoactive intestinal peptide, also activated p90(rsk). CCK-induced activation of p90(rsk) appears to be mediated by protein kinase C (PKC), since 12-O-tetradecanoylphorbol-13-acetate increased p90(rsk) activity 5.3-fold. GF-109293X, a potent inhibitor of PKC, strongly inhibited CCK-evoked p90(rsk) activity. Treatment of acini with ionomycin or 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid had no effect, indicating that mobilization of intracellular Ca2+ by CCK is not important in p90(rsk) activation. Although there were some quantitative differences in the extent of inhibition, the specific inhibitors [rapamycin, wortmannin, mitogen-activated protein kinase (MAPK) kinase inhibitor PD98059, and GF-109293X] had parallel effects on p90(rsk) and p42(mapk) activities, consistent with a model in which p90(rsk) can be regulated in acini by MAPK.
Cellular handling of unoccupied and agonist-stimulated cholecystokinin receptor determined by immunolocalization
Authors: Fischer de Toledo C. , Roettger B. F. , Morys-Wortmann C. , Schmidt W. E. , Miller L. J.
Published: December 2017

Cellular handling of receptor molecules is an important mechanism for the regulation of appropriately sensitive hormone-stimulated signaling. Until now, our understanding of the cellular handling of the cholecystokinin (CCK) receptor has been largely limited to following a tagged ligand through the cell. In the present work, we report the application of unique CCK receptor antisera directed toward intracellular domains, which permitted the immunolocalization of this molecule independently of its occupation with ligand. The CCK receptor antisera were also useful in Western blotting and immunoprecipitation of this receptor. Unstimulated CCK receptors remained on the surface of both recombinant stable rat CCK-A receptor-bearing Chinese hamster ovary cell line (CHO-CCKR) cells and native rat pancreatic acinar cells and did not constitutively internalize. Agonist stimulation of the CHO-CCKR cells resulted in the prompt internalization of a subset of surface receptors, representing those that were occupied with ligand. Unoccupied receptors remained on the surface, uninfluenced by the stimulated signaling pathways. Consistent with this, CCK receptor phosphorylation induced by 12-O-tetradecanoylphorbol-13-acetate treatment did not stimulate receptor internalization. After internalization, we observed substantial receptor recycling to the plasma membrane. These insights provide the first evidence that CCK receptor internalization occurs as a direct result of an induced conformational change and presumed bimolecular interaction, rather than as an effect of a signaling event.
Presynaptic alpha2-adrenoceptors inhibit excitatory synaptic transmission in rat brain stem
Authors: Bertolino M. , Vicini S. , Gillis R. , Travagli A.
Published: December 2017

The synaptic connection between the commissural portion of the nucleus tractus solitarius (ComNTS) and the dorsal motor nucleus of the vagus (DMV) was studied in rat brain stem slices, using the patch-clamp technique. The excitatory postsynaptic currents (EPSC) evoked by stimulation of the ComNTS were blocked by kynurenic acid (1 mM) and, in Mg2+-free solution, were sensitive to both the N-methyl-D-aspartic acid (NMDA) receptor blocker 3-[(RS)-2-carboxypiperazine-4-yl] -propyl-1-phosphonic acid (20 microM) and the non-NMDA receptor blocker 2,3-dihydro-6-nitro-7-sulfamoyl-benzo(f)quinoxaline (5 microM). Norepinephrine (NE, 1-100 microM) inhibited the EPSC, and the inhibition was attenuated by the alpha2-adrenoceptor antagonists idazoxan (1 microM) and yohimbine (10 microM) but not by the beta-adrenoceptor antagonist nadolol (50 microM). The NE-releasing agent tyramine (100 microM) reduced the EPSC, and the inhibition was attenuated by 1 microM idazoxan. NE (30 microM) did not affect the membrane input resistance but reduced the paired-pulse depression, demonstrating that NE acts on presynaptic alpha2-adrenoceptors. The results indicate the existence of a glutamatergic pathway from the ComNTS to the DMV neurons modulated by presynaptic NE receptors. This pathway might be a component of the vagovagal reflex regulating gastrointestinal function.
ME-3407, a new antiulcer agent, inhibits acid secretion by interfering with redistribution of H(+)-K(+)-ATPase
Authors: Urushidani T. , Muto Y. , Nagao T. , Yao X. , Forte J. G.
Published: December 2017

ME-3407 is a newly developed antiulcer drug that markedly promoted the healing of acetic acid-induced chronic ulcers in rats presumably due to potent inhibition of acid secretion. ME-3407 and its metabolites, the sulfoxide of which was preserved, produced dosedependent inhibition of aminopyrine accumulation by rabbit gastric glands stimulated by any agonist, suggesting that the site of their action was downstream from the production of second messengers. Although one of the metabolites, EF-4025, showed some inhibitory effects on functional activities of H(+)-K(+)-ATPase, ME-3407 itself was not a proton pump inhibitor. ME-3407, but not omeprazole, inhibited the stimulation-associated redistribution of H(+)-K(+)-ATPase from microsomes into the apical membranes in addition to delocalizing ezrin, a putative F-actin-membrane linker, from apical plasma membrane. ME-3407 and EF-4025 inhibited myosin light chain kinase (MLCK) and protein kinase A activities. Because another MLCK inhibitor, wortmannin, showed the same properties as ME-3407, i.e., inhibition of aminopyrine accumulation, inhibition of stimulation-associated redistribution of H(+)-K(+)-ATPase, and abnormal distribution of ezrin, we hypothesize that MLCK is one of the potential targets for the drug. We conclude that ME-3407 is a promising drug for treating peptic ulcers, as well as a useful tool for studying mechanisms of parietal cell activation, especially related to the recruitment and recycling of the proton pump.
Spontaneous activity of guinea pig ileum longitudinal muscle regulated by Ca(2+)-activated K+ channel
Authors: Hong S. J. , Roan Y. F. , Chang C. C.
Published: December 2017

Isolated guinea pig ileum displayed spontaneous contraction and fluctuation of membrane potential originating from the longitudinal muscle. Transmural stimulation of the enteric nerve plexus evoked contractions that were followed by lowered muscle tone and decreased spontaneous activity. The Na+ channel blocker tetrodotoxin, N-type Ca2+ channel blockers omega-conotoxins GVIA and MVIIA, the muscarinic receptor antagonist atropine, and inhibitory mediators alpha, beta-methylene-ATP and sodium nitroprusside all inhibited stimulation-evoked contraction while restoring spontaneous motility. L-type Ca2+ channel blockers nifedipine and calciseptine completely suppressed evoked and spontaneous contractions. Blockade of the large-conductance Ca(2+)-activated K+ (Kca) channel with charybdotoxin (but not of small-conductance Kca channel by apamin or of ATP-regulated K+ channel by glybenclamide) induced spikelike depolarization, inhibited nerve stimulation-evoked membrane hyperpolarization, and increased spontaneous activity. The results suggest that the large-conductance Kca channel is constitutively activated for modulations of spontaneous activity, and that muscle excitation, through elevation of Ca2+ levels, stimulates the large-conductance Kca channel to suppress spontaneous activity.
Regional differences in cholinergic activity of muscle fibers from the human gastroesophageal junction
Authors: Preiksaitis H. G. , Diamant N. E.
Published: December 2017

Muscles of the gastroesophageal junction that contribute to the lower esophageal sphincter (LES) include clasplike semicircular fibers on the right and slinglike oblique gastric fibers on the left. This study examined whether in vitro differences between the sling and clasp muscles could account for the in vivo asymmetry of LES pressure and its cholinergic contribution. Isometric tension was recorded from muscle strips of the sling, clasp, and circular layers of the esophagus and gastric fundus isolated from surgical specimens. The sling developed less spontaneous tension (8.9 +/- 4.3 mN/mm2) than the clasp (25.0 +/- 7.4 mN/mm2, P < 0.01) but showed a fivefold greater increase in response to carbachol. Eserine (1 microM) increased tension in the sling muscle (64.5 +/- 29.7%), but not in the clasp, whereas 1 microM atropine or 1 microM tetrodotoxin had no significant effect in either muscle. In both muscles, tension was reduced by 10 microM sodium nitroprusside. Sling or clasp muscle differed from circular muscle of the esophagus or gastric fundus in spontaneous tension, carbachol response, or responses to electrical stimulation. Thus the clasp muscle develops greater spontaneous tension, whereas the sling is more sensitive to cholinergic stimulation, providing a potential explanation for the in vivo asymmetry of the LES pressure and its response to cholinergic blockade.
Isolation and culture of bovine pancreatic duct epithelial cells
Authors: Cotton C. U. , al-Nakkash L.
Published: December 2017

We describe a method to isolate and culture epithelial cells from the main duct of the bovine pancreas. In primary cultures, secretin caused a dose-dependent increase in intracellular adenosine 3',5'-cyclic monophosphate (cAMP) and stimulated electrogenic transepithelial ion transport. Elevation of intracellular cAMP increased the rate coefficient for 36Cl- efflux from 0.14 +/- 0.03 to 0.47 +/- 0.12 min-1, and plasma membrane conductance, measured by the whole cell patchclamp technique, was increased from 0.7 +/- 0.1 to 6.9 +/- 0.8 nS. The cAMP-activated anion currents had properties similar to those mediated by the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Cells grown on permeable supports formed confluent monolayers with high transepithelial electrical resistance (1.004 +/- 96 omega. cm2) and generated a lumen negative transepithelial voltage difference (-2.5 +/- 0.6 mV). The short-circuit current (Isc) was increased by forskolin or secretin and was inhibited 87 +/- 4% by addition of ouabain (100 microM) to the basolateral bathing solution. Replacement of bathing solution Cl- by cyclamate reduced the forskolin-induced steady-state increase in Isc from 5.3 +/- 0.5 to 0.2 +/- 0.2 microA/cm2, suggesting that the stimulated current is due to anion secretion. The results of these studies demonstrate that large numbers of pancreatic ductal cells can be isolated and grown in primary cell culture. The monolayers express differentiated functions and will be useful for studies of acute and chronic regulation of ion transport in pancreatic duct epithelial cells.
Evidence for the existence of a carrier-mediated folate uptake mechanism in human colonic luminal membranes
Authors: Dudeja P. K. , Torania S. A. , Said H. M.
Published: December 2017

Colonic bacteria synthesize significant amounts of folate. The current studies were undertaken to examine the presence of a possible folate transporter and its characterization in apical membranes of the human colon. Apical membrane vesicles were purified from mucosal scrapings of proximal organ donor colons using a differential centrifugation and divalent cation precipitation technique. [3H]folate (PteGlu) uptake was measured by a rapid filtration technique. Our results demonstrate that [3H]PteGlu uptake into these vesicles 1) was significantly increased with decreasing pH of the incubation buffer, 2) was markedly inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid but not by acetazolamide, amiloride, bumetanide, furosemide, and diphenyl 2-carboxylic acid, 3) was sensitive to temperature and osmolarity of the incubation medium, 4) was inhibited by structural analogs methotrexate and 5-methyltetrahydrofolate, 5) exhibited trans-stimulation phenomenon, 6) was potential insensitive, and 7) exhibited saturation kinetics with an apparent Michaelis constant for PteGlu of 8.2 +/- 2.4 microM and a maximal enzyme reaction rate of 19.8 +/- 2.9 pmol.mg protein-1.10 s-1. Studies on distal colonic membranes also demonstrated the presence of a folate transporter with similar kinetic characteristics. These results demonstrate the existence of a pH-dependent, DIDS-sensitive, electroneutral carrier-mediated mechanism for folate absorption in the human colon.
Insulin induces Ca2+ influx into isolated rat hepatocyte couplets
Authors: Benzeroual K. , van de Werve G. , Meloche S. , Mathe L. , Romanelli A. , Haddad P.
Published: December 2017

Isolated rat hepatocyte couplets were used to study the direct effect of insulin on intracellular Ca2+ homeostasis. Insulin induced a dose-dependent increase in hepatocellular Ca2+ that was gradual, generally monophasic, and reversible. Chelation of extracellular Ca2+ abolished the insulin-induced Ca2+ response, and this suppression was not related to an effect on insulin binding, as indicated by displacement studies. We thus tested the effect of several Ca2+ channel inhibitors on insulin-induced Ca2+ influx. Verapamil at 20 or 200 microM was without effect, whereas 500 microM nickel and 50 microM gadolinium strongly inhibited insulin-induced Ca2+ entry. Finally, we tested whether insulin-induced Ca2+ movements were implicated in the stimulation of mitogen-activated protein kinase (MAPK) activity, which we measured with the use of an immune-complex assay. Verapamil was without effect on the insulin-dependent stimulation of p44mapk activity, whereas addition of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, nickel, or gadolinium strongly inhibited the effect of the peptide hormone. Our results indicate that insulin triggers Ca2+ influx into hepatocytes, possibly through the opening of channels on the plasma membrane, and that this effect is important for insulin activation of MAPK.
Molecular and functional characterization of intestinal Na(+)-dependent neutral amino acid transporter B0
Authors: Kekuda R. , Torres-Zamorano V. , Fei Y. J. , Prasad P. D. , Li H. W. , Mader L. D. , Leibach F. H. , Ganapathy V.
Published: December 2017

We have cloned the Na(+)-dependent neutral amino acid transporter B0 (ATB0) from rabbit jejunum and from the human intestinal cell line Caco-2. Rabbit intestinal ATB0 (riATB0) cDNA codes for a protein of 541 amino acids with 10 potential transmembrane domains. When expressed in HeLa cells, riATB0 mediates the transport of several neutral amino acids, including glutamine, in a Na(+)-dependent manner. Anionic amino acids, cationic amino acids, and N-methylated amino acids are excluded by riATB0. When expressed in Xenopus laevis oocytes, riATB0 increases the transport of neutral amino acids severalfold. The induced transport activity is specific for neutral amino acids, with no noticeable interaction with anionic, cationic, and N-methylated amino acids. However, riATB0 does interact with anionic amino acids at acidic pH. In oocytes expressing riATB0, the neutral amino acid threonine evokes inward currents at a holding potential of -50 mV. The amino acid-evoked current is sensitive to membrane potential. The inward current increases as the membrane potential is hyperpolarized, but the current reverses at about -30 to -40 mV. Threonine evokes outward currents if the membrane potential is depolarized beyond this value. We have also cloned the ATB0 from the human intestinal cell line Caco-2. The Caco-2 ATB0 cDNA also codes for a protein of 541 amino acids that is essentially identical to the ATB0 expressed in the human choriocarcinoma cell line JAR. Reverse transcription-polymerase chain reaction (RT-PCR) and restriction analysis of the RT-PCR products indicate that the human intestine and the human kidney proximal tubular cell line HKPT express an ATB0 identical to the ATB0 expressed in Caco-2 cells.
Kupffer cells contain a glycine-gated chloride channel
Authors: Ikejima K. , Qu W. , Stachlewitz R. F. , Thurman R. G.
Published: December 2017

Here the effect of glycine on intracellular Ca2+ concentration ([Ca2+]i) in cultured Kupffer cells stimulated with lipopolysaccharide (LPS) was investigated to assess the possibility that they contain a glycine-gated chloride channel. LPS (10 micrograms/ml) increased [Ca2+]i rapidly, with peak values reaching 307 +/- 29 nM. Glycine (1 mM) prevented this increase nearly completely. Low concentrations of strychnine (1 microM), a glycine receptor antagonist, reversed the inhibitory effect of glycine completely; however, high concentrations of strychnine (1 mM) mimicked glycine. The effects of glycine and high-dose strychnine were prevented when cells were incubated in chloride-free buffer. Furthermore, potassium (25 mM) and LPS depolarized the Kupffer cell plasma membrane, whereas glycine caused hyperpolarization and prevented depolarization due to potassium and LPS. Moreover, tumor necrosis factor-alpha (TNF-alpha) production in cultured Kupffer cells due to LPS was decreased significantly by glycine. Therefore, it is concluded that Kupffer cells contain a glycine-gated chloride channel similar to that described previously in the central nervous system. Prevention of increases in [Ca2+]i due to LPS by activation of chloride influx reduced synthesis and release of toxic mediators by Kupffer cells.