bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of prolactin and growth hormone receptor genes and their isoforms in the gastrointestinal tract
Authors: Nagano M. , Chastre E. , Choquet A. , Bara J. , Gespach C. , Kelly P. A.
Published: December 2017

Distribution of transcripts for prolactin and growth hormone receptors (PRLR and GHR) and their isoforms was characterized in the gastrointestinal (GI) tract from several species by reverse transcription-polymerase chain reaction combined with Southern analysis. Human, rabbit, and fetal and adult rat PRLR and GHR transcripts were detected in isolated gastric glands, gastric cell fractions, and intestinal mucosa lineages. Human PRLR and GHR transcripts were also observed throughout the cancerous progression of the colonic and gastric mucosa from adenomas to colonic liver metastasis and gastrointestinal cancer cell lines at various stages of growth and differentiation. Prolactin (PRL) produced no detectable effect on M1 gastric mucin secretion in HT-29 cells adapted to methotrexate (HT-29-MTX) or on acid secretion in isolated rabbit parietal cells. GHRd3, an isoform of human GHR transcript missing exon 3, was also broadly expressed and was the only form found in gastric and colorectal adenocarcinomas. Interestingly, several extra bands of polymerase chain reaction products of the human PRLR, which were smaller than the expected size, were observed not only in the GI tract but also in liver and T-47D breast cancer cells. These products from human intestinal and breast cancer cell lines were subsequently subcloned and sequenced, and we isolated six isoforms of the receptor transcripts. One of these clones encodes a putative human PRL binding protein. The expression of PRL and PRLR transcripts was also clearly observed in intraepithelial lymphocytes purified from the mouse intestine. The widespread expression of the PRL and GH receptor transcripts in gastric and intestinal mucosal lineages, particularly in epithelia, suggests regulatory roles of these hormones on digestive and immune functions, including metabolism, growth, or differentiation.
Enhanced endothelium-dependent vasodilation in patients with cirrhosis
Authors: Albillos A. , Rossi I. , Cacho G. , Martinez M. V. , Millan I. , Abreu L. , Barrios C. , Escartin P.
Published: December 2017

Experimental evidence indicates that an increased production of nitric oxide could play a role in the peripheral vasodilation of portal hypertension. To test this hypothesis in humans, we studied basal serum NO(2-) + NO3- levels and the response of forearm resistance vessels to increasing concentrations of methacholine chloride, sodium nitroprusside, and phenylephrine infused into the brachial artery of 12 cirrhotic patients and 10 controls. Forearm vascular resistance (FVR) was calculated from mean arterial pressure and forearm blood flow (FBF). Cirrhotics showed higher NO(2-) + NO3- levels (P < 0.05), higher FBF (P < 0.01), and lower FVR (P < 0.01) than controls. The reduction of FVR in response to every dose of methacholine was greater in cirrhotics than in controls; this was significant (P < 0.05) at the 3 and 10 micrograms/min doses. This response to methacholine was not modified by blockade of vascular prostacyclin. The response to nitroprusside was similar in both groups. The increase in FVR in response to every dose of phenylephrine was significantly (P < 0.01) lower in cirrhotics than in controls. In cirrhotics, a significant correlation (r = -0.81, P < 0.01) was found between the FVR response to the highest doses of methacholine and phenylephrine. In conclusion, cirrhotic patients show an enhanced endothelium-mediated vasodilation, which suggests an increased synthesis of nitric oxide. This defect may mediate the peripheral vasodilation and hyporeactivity to vasopressors of these patients.
Central cholinergic and alpha-adrenergic mediation of gastric slow wave dysrhythmias evoked during motion sickness
Authors: Hasler W. L. , Kim M. S. , Chey W. D. , Stevenson V. , Stein B. , Owyang C.
Published: December 2017

Motion sickness is associated with gastric slow wave disruption. Animal models of slow wave disturbances show dependence on neural and prostaglandin pathways. Roles of these pathways in circular vection-evoked gastric dysrhythmias and nausea were tested. Eight volunteers with histories of motion sickness underwent vection (60 degrees/s), during which nausea (0 = none to 3 = severe) and electrogastrographic parameters were assessed. Tachygastric activity was expressed as the signal percentage at frequencies of > 4.5 cycles/min. Circular vection induced a maximal nausea score of 2.8 +/- 0 at 513 +/- 66 s. Tachygastric activity increased from 18 +/- 2 to 37 +/- 4% (P < 0.05) and peaked before maximal nausea. Atropine reduced nausea scores to 0 +/- 0 (P < 0.01) with no increase in tachygastric activity (14 +/- 6%). In contrast, the peripheral muscarinic antagonist methscopolamine did not reduce tachygastric activity (46 +/- 4%), nausea (1.8 +/- 0.5), or time to maximal tachygastric activity (504 +/- 80 s) with vection. Phentolamine reduced nausea (1.5 +/- 0.3, P < 0.01) and tachygastric activity, and delayed their onset, whereas propranolol and naloxone had no effect. Pretreatment with oral indomethacin (50 mg) three times daily for 3 days had no effect on vection-evoked tachygastric activity or nausea. To conclude, circular vection evokes gastric dysrhythmias that correlate temporally with maximal nausea and are suppressed by atropine, but not methscopolamine, and are reduced by phentolamine. In contrast to other models of slow wave disruption, endogenous prostaglandins play no role. Thus central cholinergic pathways mediate vection-evoked dysrhythmias with additional modulation by alpha-adrenergic pathways.
Identification of CCK-A receptors on chief cells with use of a novel, highly selective ligand
Authors: Pradhan T. K. , Qian J. M. , Sutliff V. E. , Mantey S. A. , Jensen R. T.
Published: December 2017

Functional studies suggest that guinea pig chief cells have both cholecystokinin-A (CCK-A) and CCK-B receptors (CCK-A-R and CCK-B-R, respectively). However, all efforts to directly characterize the specific CCK-A-R using binding have been unsuccessful. Recent studies describe specific CCK-A-R agonists such as A-71378 ([desamino-Nle28,31-(N-methyl)Asp32]CCK heptapeptide]. In the present study, [D-Tyr-Gly]A-71378 was synthesized, which has > 300-fold selectivity for CCK-A-R and can be iodinated. [D-Tyr-Gly]A-71378 was equipotent to A-71378 in stimulating pepsinogen release from purified guinea pig chief cells. Binding of 125I-labeled [D-Tyr-Gly]A-71378 was saturable and specific. Potencies for inhibiting binding were as follows: [D-Tyr-Gly]A-71378 = A-71378 = 4x CCK octapeptide (CCK-8) > 1,000x des(SO4)-CCK-8, gastrin. In contrast, for 125I-gastrin binding they were CCK-8 > gastrin-17-I > des(SO4)-CCK-8 >> A-71378 or [D-Tyr-Gly]A-71378. Binding of [D-Tyr-Gly]A-71378 was best fitted by a two-site model. In contrast, 125I-gastrin binding was fitted with a single-site model. For inhibiting binding of 125I-[D-Tyr-Gly]A-71378, the CCK antagonists had relative affinities of L-364,718 >> L-365,260, and the reverse was true with 125I-gastrin. Correlation of binding with changes in biological activity suggested low-affinity CCK-A-R were mediating these changes. These results demonstrate directly for the first time that guinea pig chief cells possess CCK-A-R and CCK-B-R. The pharmacology of these CCK-A-R resembles those on other tissues. This novel, highly selective CCK-A ligand should be useful because it will identify CCK-A-R when they make up as little as 0.2% of the total CCK receptor number.
ATP-dependent bile acid transport across microvillous membrane of human term trophoblast
Authors: Marin J. J. , Bravo P. , el-Mir M. Y. , Serrano M. A.
Published: December 2017

The main fate for fetal bile acids is to be transferred to the mother by the trophoblast. In this study, ATP-dependent bile acid transport across the maternal- and the fetal-facing plasma membranes (mTPM and fTPM, respectively) of the human trophoblast was investigated. With the use of [14C]glycocholate (GC) and a rapid-filtration technique, GC transport by mTPM and fTPM was measured in the absence or the presence of 3 mM ATP plus an ATP-regenerating system. GC efflux from preloaded mTPM or fTPM vesicles was found to be insensitive to ATP. By contrast, GC uptake by mTPM, but not by fTPM, was significantly increased (approximately threefold) by ATP. This was temperature sensitive and occurred into an osmotically reactive space. Kinetic analysis revealed that GC uptake by mTPM was saturable and fit the Michaelis-Menten equation both in the absence and in the presence of ATP. ATP-dependent transport was not abolished by a protonophore (carbonyl cyanide p-trifluormethoxyphenyl hydrazone) together with 100 mM K+ (in = out) plus a K+ ionophore (valinomycin). It specifically required hydrolyzable ATP, although CTP had a slight stimulatory effect. Neither Na+ nor Cl- (100 mM, in = out) was mandatory. Moreover, 100 mM gradients of either Na+ (in << out) or Cl- (in >> out) had no effect on ATP-dependent GC uptake. This was inhibited by vanadate and bile acid analogues but not by several cholephilic organic anions and a variety of adenosine triphosphatase inhibitors. These results provide strong evidence for the existence of an ATP-dependent transport system for bile acids across the apical membrane of human trophoblast, which may play an important role in the control of the overall fetal-maternal bile acid traffic.
Mechanism of bile acid facilitation of biliary protoporphyrin excretion in rat liver
Authors: Berenson M. M. , el-Mir M. Y. , Zhang L. K.
Published: December 2017

The mechanism(s) by which bile acids increase biliary protoporphyrin excretion was characterized using perfused rat livers. We determined 1) relationships between biliary bile acids, phospholipid, and protoporphyrin, using rapid kinetic analyses; 2) protoporphyrin excretion in livers with defective canalicular multispecific organic anion transport; 3) effects of intracellular vesicular transport inhibition with colchicine and monensin; and 4) the role of luminal bile acids, using retrograde intrabiliary taurocholate injections. Biliary protoporphyrin excretion peaked with phospholipid excretion 14-18 min after loading. Protoporphyrin excretion induced by taurocholate was not related to effects on intracellular transport, including colchicine- and monensin-inhibitable vesicular systems. Eisai hyperbilirubinemic rat livers excreted protoporphyrin similarly to controls. Retrograde intrabiliary taurocholate injections increased protoporphyrin output. Collectively, these data suggest that 1) intracellular protoporphyrin transport is mediated by nonvesicular carriers targeted to the canalicular membrane, and 2) bile acid facilitates protoporphyrin translocation into bile in the same manner it effects phospholipid excretion.
PYY in brain stem nuclei induces vagal stimulation of gastric acid secretion in rats
Authors: Yang H. , Tache Y.
Published: December 2017

The influence of peptide YY (PYY) microinjected into brain stem nuclei on gastric acid secretion (GAS) was investigated in urethan-anesthetized rats with gastric cannula. PYY (30-200 ng) microinjected into the dorsal motor nucleus of the vagus (DMN) induces a dose-related and vagal-dependent stimulation of GAS (net increase from 13 +/- 4 to 59 +/- 12 mumol/90 min). PYY (200 ng) injected intravenously intracisternally into sites adjacent to the DMN had no effect. GAS induced by PYY into the DMN was potentiated by coinjection of thyrotropin-releasing hormone (TRH, 30 ng) or the serotonin receptor (5-HT2) agonist (+/-)-1-(4-methyl-1-piperazinyl)-pyrrolo(1,2-a)quinoxaline (357 ng) and by microinjection of kainic acid (1 ng) into the raphe pallidus. Prepro-TRH-(160-169) (200 ng into the DMN) did not influence the stimulatory effect of PYY. PYY (200 ng) microinjected into the raphe pallidus, raphe obscurus, and nucleus ambiguous also increased GAS, although the response was of shorter duration than that in the DMN. These results indicate that PYY acts in brain stem nuclei involved in the vagal regulation of GAS and that PYY action in the DMN is potentiated by TRH or 5-HT2 receptor agonist acting at this site.
Cellular and regional expression of transcripts of the plasma membrane calcium pump PMCA1 in rabbit intestine
Authors: Freeman T. C. , Howard A. , Bentsen B. S. , Legon S. , Walters J. R.
Published: December 2017

The plasma membrane Ca(2+)-pumping adenosinetriphosphatase (PMCA) is the energy-dependent step in the active vitamin D-dependent absorption of dietary Ca2+ by the enterocyte. Studies of the various PMCA genes and splicing variants in humans and rats have indicated that the isoform known as PMCA1b is the predominant form expressed in small intestine. Using an oligonucleotide probe, we have studied the regional and cellular distribution of PMCA1 transcripts in rabbit intestinal tissues by in situ hybridization. On small intestinal RNA blots, this hybridized to species similar in size to those detected by PMCA1-specific cDNA probes; an additional larger transcript was present in rabbit than in rat or human. In situ hybridization signals were principally in the enterocyte population of the mucosa and were maximal in differentiating enterocytes on the lower part of the villus, a pattern similar to that previously demonstrated for other nutrient transporters. Reflecting the capacity of the different small intestinal segments to transport Ca2+, much higher levels of transcript were detected by both methods proximally (in duodenum) than distally (in jejunum and ileum) and were also higher in cecum and ascending colon mucosa than in descending colon. We conclude that as enterocytes differentiate in regions that absorb Ca2+, they express high levels of mRNA for PMCA1. These results confirm the importance of transcriptional regulation of this gene for active Ca2+ absorption.
Vessel- and target cell-specific actions of endothelin-1 and endothelin-3 in rat liver
Authors: Zhang J. X. , Bauer M. , Clemens M. G.
Published: December 2017

We studied the sinusoidal and extrasinusoidal constrictor response of hepatic microcirculation to endothelin-1 (ET-1) and endothelin-3 (ET-3) and the possible role of Ito cells vs. Kupffer cells or endothelial cells in mediating this response, using isolated rat livers under high-power intravital microscopy. Rats were pretreated by injection of 2.6 x 10(8) fluorescent latex beads (1 micron) intravenously to label Kupffer cells. Three hours later livers were isolated and perfused before and during the infusion of 1 nM ET-1 or ET-3 with or without the endothelin type A (ETA) receptor antagonist BQ-123 or ETB antagonist IRL-1038. Alternatively, the perfused livers were infused with the ETB agonist sarafotoxin 6c (S6c, 1 or 5 nM). Sinusoid diameters were quantitated at the sites of Ito cells (identified by vitamin A fluorescence) or Kupffer cells (phagocytosed fluorescent latex beads) or where neither cell type was found (endothelial cells). ET-1 was found to induce significant sinusoid constriction at the sites of Ito cells (13.21 +/- 0.58 microns control vs. 10.47 +/- 0.48 microns during ET-1 infusion) but not at the sites of Kupffer cells or endothelial cells (13.26 +/- 0.79 vs. 12.92 +/- 0.61 microns and 12.20 +/- 0.71 vs. 11.98 +/- 0.40 microns, respectively), whereas neither ET-3 nor S6c had any effect on sinusoid narrowing, despite a 1.8-fold (ET-3) or 5.6-fold (5 nM S6c) greater increase in total portal resistance compared with ET-1.(ABSTRACT TRUNCATED AT 250 WORDS)
Rhythmic electrical and mechanical activity in stomach of toad and frog
Authors: Prosser C. L.
Published: December 2017

Rhythmic slow spikes and contractions at 2-5/min occur in frog/toad stomach when longitudinal muscle is present, not in the circular layer after removal of the longitudinal layer. Interstitial cells of Cajal occur in frog stomach in the longitudinal layer and in toad stomach mainly in the longitudinal layer, with some between layers. They were identified by staining with methylene blue, the fluorescent dye rhodamine 123, toluidine blue, and by electron microscopy. Interstitial cells resemble in ultrastructure those observed in mammalian intestine by small cell diameter, long processes forming a network, large nuclei, mitochondria, caveolae, endoplasmic reticulum, and absence of fibrillae. Rhythmic activity in stomachs stained with methylene blue is abolished by bright illumination. Rhythmic slow spikes and contractions are reduced and frequency is lowered when Ca2+ concentration in the medium is significantly decreased; in high Ca2+ these functions are increased. Threshold concentration of Ca2+ for maintaining slow spikes was between 10(-7) and 10(-6) M Ca2+. Ba2+ and 2.5 mM increases slow spike frequency and amplitude; Ba2+ increases contractions proportionately more than spikes. Slow spikes are reduced or blocked by verapamil, nifedipine, and Cd2+ and are enhanced by BAY K 8644. Quaternary ammonium compounds tetraethylammonium and tetrapentyl-ammonium bromide prolong slow spikes and enhance contractions. Replacement of NaCl by LiCl, methyl glutamine, or treatment with amiloride reduces slow spike amplitude and to a lesser extent frequency. This indicates efflux of Ca2+ by Na+/Ca2+ exchange. Contractions, but not spikes, are reduced by the calmodulin blocker trifluoroperazine. Contractions are enhanced, but slow spikes are not altered by ouabain. This suggests retention of Ca2+ when a tonic Na+/K+ pump is blocked. A model for rhythmicity includes influx of Ca2+ via L-channels, repolarization by IKCa2+, efflux of Ca2+ by Na+/Ca2+ exchange, and efflux of Na+ by a Na+/K+ pump. Frequency is determined by rate constants for both influx and efflux of Ca2+.
Human colonic smooth muscle electrical activity during and after recovery from postoperative ileus
Authors: Condon R. E. , Cowles V. E. , Ferraz A. A. , Carilli S. , Carlson M. E. , Ludwig K. , Tekin E. , Ulualp K. , Ezberci F. , Shoji Y. , et al.
Published: December 2017

Colon smooth muscle electrical control (ECA) and response activities (ERA) were recorded for up to 4 wk postoperatively for 48 patients after major abdominal operations. Bipolar electrodes were implanted into right and left colon circular muscle and exteriorized through the flanks, and signals were tape recorded for 2-24 h daily beginning on the 1st postoperative day. A computer program was used for data reduction and analysis. Recorded signals were digitized and filtered. The ECA frequency components were identified by fast Fourier transformation, and their relative tenancy in low, mid, and high frequency ranges was determined. Short and long ERA burst duration and frequency and number and velocity of propagating long ERA bursts were determined. ECA was omnipresent and exhibited a downshift of the dominant frequency from the mid to the low range as recovery from postoperative ileus progressed. Concurrently, first in the right and then in the left colon, the frequency of long ERA bursts increased, followed by the appearance of propagating long ERA. After the 6th postoperative day, no further significant changes in parameters of colon electrical activity occurred with time.
Rat hepatocytes and Kupffer cells interact to produce interleukin-8 (CINC) in the setting of ethanol
Authors: Maher J. J.
Published: December 2017

Interleukin-8 is a neutrophil chemoattractant that has been implicated in the pathogenesis of alcoholic hepatitis. The mechanism of ethanol-induced interleukin-8 production in liver is uncertain, although hepatocytes and Kupffer cells have both been proposed as sources of the chemokine. In this study we investigated whether short-term ethanol exposure stimulates production of rat interleukin-8 [cytokine-induced neutrophil chemoattractant (CINC)] by normal rat hepatocytes and Kupffer cells in primary culture. Initial experiments verified that hepatocytes and Kupffer cells produce CINC in response to cytokines or lipopolysaccharide. Ethanol, by contrast, failed to stimulate CINC secretion by either cell type even at concentrations as high as 100 mM. Although ethanol had no direct effect on liver cell CINC production, conditioned medium from ethanol-treated hepatocytes induced a threefold rise in CINC production by Kupffer cells. The increase was abrogated when hepatocytes were treated with ethanol and the metabolic inhibitor 4-methylpyrazole. The results suggest that the mechanism of ethanol-induced CINC production is indirect, involving ethanol oxidation and communication between hepatocytes and Kupffer cells.
NHE2 and NHE3 are human and rabbit intestinal brush-border proteins
Authors: Hoogerwerf W. A. , Tsao S. C. , Devuyst O. , Levine S. A. , Yun C. H. , Yip J. W. , Cohen M. E. , Wilson P. D. , Lazenby A. J. , Tse C. M. , Donowitz M.
Published: December 2017

Rabbit NHE2 and NHE3 are two epithelial isoform Na+/H+ exchangers (NHE), the messages for which are found predominantly and entirely, respectively, in renal, intestinal, and gastric mucosa. The current studies used Western analysis and immunohistochemistry to identify and characterize the apical vs. basolateral membrane distribution of NHE2 and NHE3 in intestinal epithelial cells. Based on Western analysis, NHE2 and NHE3 both are present in brush-border but not basolateral membranes of small intestine. Both NHE2 and NHE3 are 85-kDa proteins. Consistent with Western analysis, NHE2 and NHE3 are immunolocalired to the brush-border but not basolateral membranes of villus epithelial cells, but not goblet cells, in human jejunum and ileum and in surface epithelial cells in the ascending and descending colon and rectum. In addition, NHE2 and NHE3 are present in small amounts in the crypt cell brush border of human jejunum, ileum, ascending and descending colon, and rectum. In rabbit jejunum, ileum, and ascending colon, NHE2 and NHE3 are present in the brush border of epithelial and not goblet cells, again much more in the villus (small intestine)/ surface cells (colon) than the crypt. NHE2 but not NHE3 is present in the brush border of rabbit descending colon surface cells and in small amounts in crypt cells. NHE2 and NHE3 are both human and rabbit small intestinal and colonic epithelial cell brush-border Na+/H+ exchanger isoforms that colocalize in all intestinal segments except rabbit descending colon, which lacks NHE3.
Oleate uptake by isolated hepatocytes and the perfused rat liver is competitively inhibited by palmitate
Authors: Sorrentino D. , Stump D. D. , Van Ness K. , Simard A. , Schwab A. J. , Zhou S. L. , Goresky C. A. , Berk P. D.
Published: December 2017

Competition for uptake between long-chain free fatty acids has been difficult to document, because there has been no algorithm for computing unbound concentrations of two fatty acids simultaneously in solution with albumin. We modified an iterative procedure to permit this computation and studied initial [3H]oleate uptake by isolated hepatocytes and steady-state uptake by the single-pass perfused rat liver from 600 microM bovine serum albumin solutions containing various concentrations of oleate in the presence and absence of palmitate. In both systems, the Michaelis-Menten constant was significantly higher in the presence of palmitate than in its absence, whereas the maximal reaction velocity was unaltered, indicating competitive inhibition. In additional experiments employing the multiple transhepatic indicator-dilution technique, the influx rate constant and permeability-surface area product for oleate influx were significantly reduced by palmitate, confirming that the competition observed in the conventional perfused liver studies was at the influx step. Long-chain fatty acid uptake has now been shown to exhibit all the kinetic properties of facilitated transport and cannot be attributed solely to passive diffusion.
Prostaglandin endoperoxide synthase: why two isoforms?
Authors: Williams C. S. , DuBois R. N.
Published: December 2017

Prostaglandin endoperoxide synthase-1 [prostaglandin G/H synthase-1 (PGHS-1)] and PGHS-2 are key enzymes in the conversion of arachidonic acid to prostaglandins and other eicosanoids. We refer to these isoforms as cyclooxygenase-1 (COX-1) and COX-2 in this review. This brief review focuses on recent developments in the study of these enzymes. Alterations in the expression levels of COX-2 result in distinct phenotypic changes in intestinal epithelial cells. Overexpression of COX-2 in intestinal epithelial cells results in increased adhesion to extracellular matrix proteins and inhibition of apoptosis. Disruption of the COX-2 gene in mice results in renal dysplasia, cardiac fibrosis, and defects in the ovary. Interestingly, disruption of the COX-1 gene results in distinct phenotypic changes different from those observed for COX-2. COX-1 null mice survive well, have no gastric pathology, and show less indomethacin-induced gastric ulceration than wild-type mice. These two closely related enzymes must have distinct functions in the organisms, since lack of their expression causes distinct phenotypic changes for each respective isoform.
Glucose transporters in the regulation of intestinal, renal, and liver glucose fluxes
Authors: Thorens B.
Published: December 2017

Five functional mammalian facilitated hexose carriers (GLUTs) have been characterized by molecular cloning. By functional expression in heterologous systems, their specificity and affinity for different hexoses have been defined. There are three high-affinity transporters (GLUT-1, GLUT-3 and GLUT-4) and one low-affinity transporter (GLUT-2), and GLUT-5 is primarily a fructose carrier. Because their Michaelis constants (Km) are below the normal blood glucose concentration, the high-affinity transporters function at rates close to maximal velocity. Thus their level of cell surface expression greatly influences the rate of glucose uptake into the cells. In contrast, the rate of glucose uptake by GLUT-2 (Km = 17 mM) increases in parallel with the rise in blood glucose over the physiological concentration range. High-affinity transporters are found in almost every tissue, but their expression is higher in cells with high glycolytic activity. Glut-2, however, is found in tissues carrying large glucose fluxes, such as intestine, kidney, and liver. As an adaptive response to variations in metabolic conditions, the expression of these transporters is regulated by glucose and different hormones. Thus, because of their specific characteristics and regulated expression, the facilitated glucose transporters control fundamental aspects of glucose homeostasis. I review data pertaining to the structure and regulated expression of the glucose carriers present in intestine, kidney, and liver and discuss their role in the control of glucose flux into or out of these different tissues.
Identification of an EGF/TGF-alpha receptor in primary cultures of guinea pig gastric mucous epithelial cells
Authors: Rutten M. J. , Dempsey P. J. , Luttropp C. A. , Hawkey M. A. , Sheppard B. C. , Crass R. A. , Deveney C. W. , Coffey R. J.
Published: December 2017

Binding and localization of transforming growth factor-alpha (TGF-alpha) and epidermal growth factor (EGF) were assessed using in vitro primary cultures of guinea pig gastric mucous epithelial cells (GMEC). GMEC were isolated and cultured in six-well plates with Dulbecco's modified Eagle's medium + 10% serum and then changed to serum-free medium for 24 h for binding studies. The binding time course of 125I-labeled EGF and 125I-TGF-alpha in GMEC cultures at 4 degrees C was saturable, reaching a plateau within 4-6 h. Competition-binding curves revealed that the amount of unlabeled EGF and TGF-alpha to reduce 125I-EGF binding by 50% was 0.35 and 0.23 nM, respectively. The amount of unlabeled EGF and TGF-alpha to decrease 125I-TGF-alpha binding by 50% was 0.30 and 0.21 nM, respectively. A Scatchard analysis of the data disclosed that a single class of high-affinity binding sites (dissociation constant = 0.24 nM) was present. The maximal binding capacity was approximately 20 fmol/10(6) cells or approximately 12,000 receptors per cell. The binding of 125I-EGF and 125I-TFG-alpha to GMEC cultures was maximal between pH 7.0 and 8.5. No specific binding of EGF or TGF-alpha could be detected below pH 5.0. The half-maximal pH dissociation value for EGF and TGF-alpha was pH 5.89 and pH 6.83, respectively. We found no difference in the final amounts of membrane-bound or internalized 125I-EGF and 125I-TGF-alpha. However, there was a significant difference (P < 0.05) at 5-30 min in the rate of dissociated and internalized 125I-EGF- and 125I-TGF-alpha. Immunofluorescence microscopy of GMEC cultures for EGF/TGF-alpha receptors showed increased fluorescence at the leading edges and around the perimeter of cells. Detection of an EGF/TGF-alpha receptor was also confirmed by Western blotting. Our findings demonstrate that guinea pig GMEC possess a specific EGF/TGF-alpha receptor, which further supports a physiological role for EFG and TFG-alpha as mitogens in these cells.
Transcriptional regulation of hepatic sterol 27-hydroxylase by bile acids
Authors: Vlahcevic Z. R. , Jairath S. K. , Heuman D. M. , Stravitz R. T. , Hylemon P. B. , Avadhani N. G. , Pandak W. M.
Published: December 2017

The study objective was to determine whether and to what extent sterol 27-hydroxylase, the initial step in the "acidic" pathway of bile acid biosynthesis, is regulated by bile acids. Rats were fed diets supplemented with cholestyramine (CT, 5%), cholate (CA, 1%), chenodeoxycholate (CDCA, 1%), or deoxycholate (DCA, 0.25%). When compared with paired controls, sterol 27-hydroxylase and cholesterol 7 alpha-hydroxylase specific activities increased after CT administration by 188 +/- 20% (P < 0.05) and 415 +/- 36% (P < 0.01), respectively. Similarly, mRNA levels increased by 159 +/- 14% (P < 0.05) and 311 +/- 106% (P < 0.05), respectively. Feeding CA, CDCA, or DCA decreased sterol 27-hydroxylase specific activity to 57 +/- 6, 61 +/- 8, and 74 +/- 8% of controls, respectively (P < 0.05). By comparison, the specific activity of cholesterol 7 alpha-hydroxylase decreased to 46 +/- 7 , 32 +/- 10, and 26 +/- 8% (P = 0.001). mRNA levels and transcriptional activities for sterol 27-hydroxylase and cholesterol 7 alpha-hydroxylase transcriptional activity were changed to the same extent as the specific activities after CT or bile acid feeding. We conclude that sterol 27-hydroxylase and cholesterol 7 alpha-hydroxylase are subject to negative feedback regulation by hydrophobic bile acids at the level of transcription. However, the responses of sterol 27-hydroxylase to manipulation of the bile acid pool are less prominent than those of cholesterol 7 alpha-hydroxylase. During the diurnal cycle the specific activities of sterol 27-hydroxylase and cholesterol 7 alpha-hydroxylase changed in tandem, suggesting that both may be under control of glucocorticoids.
Dexamethasone-regulated expression of pancreatic lipase and two related proteins in AR42J cells
Authors: Kullman J. , Gisi C. , Lowe M. E.
Published: December 2017

The pancreas makes two pancreatic proteins [pancreatic lipase-related protein-1 (PLRP-1) and PLRP-2] with marked homology to pancreatic lipase (PL). To determine if a pancreatic acinar cell line, AR42J, also expresses PLRP-1 and PLRP-2, we examined the cells for the presence of PL, PLRP-1, and PLRP-2. RNA blot analysis with specific probes and immunoblot analysis with antipeptide antibodies demonstrated the presence of mRNA and protein for all three homologues in AR42J cells. Additionally, we showed that dexamethasone decreased PLRP-1 mRNA levels twofold and increased PLRP-2 mRNA 20-fold but had little effect on PL or colipase mRNA. Extracellular PLRP-2 protein levels increased threefold, and intracellular PLRP-2 protein levels increased about fourfold. The characteristics of the dexamethasone-induced increase in PLRP-2 mRNA, a rapid change requiring new protein synthesis independent of mRNA turnover, suggested that dexamethasone regulated transcription. We conclude that AR42J cells synthesize and secrete PL, PLRP-1, and PLRP-2 and that dexamethasone discoordinately regulates the expression of the three genes.
LPS-mediated NF-kappa beta activation in rat Kupffer cells can be induced independently of CD14
Authors: Bellezzo J. M. , Britton R. S. , Bacon B. R. , Fox E. S.
Published: December 2017

Lipopolysaccharide (LPS) activation of macrophages occurs after LPS complexed with serum LPS-binding protein (LBP) binds CD14. Activation of the nuclear transcription factor NF-kappa B is directly related to this event. Since the role of CD14 in LPS signaling has not been evaluated in Kupffer cells, the resident hepatic macrophage, the purpose of this study was to characterize LPS-mediated NF-kappa B activation under CD14-dependent (1% serum, as a source of LBP) and CD14-independent (serum-free) conditions. Classic CD14-dependent signaling was seen in peritoneal macrophages where serum potentiated NF-kappa B activation. However, in Kupffer cells, NF-kappa B was activated by LPS under CD14-independent conditions, and this response was not potentiated by serum. The activation of NF-kappa B in Kupffer cells, by 1 ng/ml LPS, reached a maximum within 60 min of stimulation. However, peritoneal macrophage NF-kappa B activation occurred only in serum and increased progressively through 240 min of stimulation. These results suggest a novel mechanism of LPS-mediated activation in Kupffer cells that may represent an adaptation to their role in clearance and detoxification of gut-derived endotoxin.
Insulin potentiates mitogenic effect of epidermal growth factor on cultured guinea pig gastric mucous cells
Authors: Ogihara S. , Yamada M. , Saito T. , Shono M. , Rokutan K.
Published: December 2017

Epidermal growth factor (EGF) stimulated proliferation of gastric mucous epithelial cells from guinea pigs in serum-free culture conditions. Western blot analysis with antiphosphotyrosine antibody showed that EGF initiated tyrosine phosphorylation of a 170-kDa protein, and this protein was identical to the EGF receptor. Insulin was not mitogenic, but it potentiated the mitogenic effect of EGF. Tyrosine phosphorylation of additional proteins was not induced by the combined actions of insulin and EGF. Stimulation by EGF and/or insulin did not cause a calcium response. However, when insulin was added to cells pretreated with EGF for > 6 h, it elicited a rapid intracellular calcium concentration rise that was reproducible in both cell suspension and single cell analyses. This calcium response coincided with the translocation of protein kinase C (PKC) from the cytosolic to the particulate fraction. Phorbol 12-myristate 13-acetate also caused the translocation and stimulated proliferation of the cells. These results suggest that the calcium-dependent activation of PKC may participate in the potentiation of the mitogenic effect of EGF by insulin.
Effect of endotoxin on bile acid transport in rat liver: a potential model for sepsis-associated cholestasis
Authors: Moseley R. H. , Wang W. , Takeda H. , Lown K. , Shick L. , Ananthanarayanan M. , Suchy F. J.
Published: December 2017

Intrahepatic cholestasis in the setting of extrahepatic bacterial infection has been attributed to the effects of endotoxin and cytokines such as tumor necrosis factor-alpha (TNF-alpha) on bile acid transport. To define the mechanism of sepsis-associated cholestasis, taurocholate transport was examined in basolateral (bLPM) and canalicular (cLPM) rat liver plasma membrane vesicles derived from control and endotoxin [lipopolysaccharide (LPS)]-treated animals and in plasma membrane vesicles prepared after TNF-alpha treatment. Na(+)-dependent [3H]taurocholate uptake and both membrane-potential-dependent and ATP-dependent [3H]taurocholate transport were reduced in bLPM and cLPM vesicles, respectively, after LPS treatment. In membrane vesicles from TNF-alpha-treated animals, Na(+)-dependent [3H]taurocholate uptake was also reduced. Northern blot hybridization, using cDNA probes for the putative sinusoidal bile acid transporter (Ntcp) and canalicular ecto-adenosinetriphosphatase, demonstrated decreased mRNA levels after LPS and TNF-alpha treatment. Immunoblot analysis of membrane extracts from LPS-treated animals revealed decreased levels of these putative bile acid transporters. Impaired bile acid transport at the sinusoidal and canalicular membrane domains by these and other mediators of the inflammatory response may account for sepsis-associated cholestasis.
Carbachol, but not forskolin, increases mucosal-to-serosal transport of intact protein in rat ileum in vitro
Authors: Bijlsma P. B. , Kiliaan A. J. , Scholten G. , Heyman M. , Groot J. A. , Taminiau J. A.
Published: December 2017

The effects of the secretagogues forskolin and carbachol on protein uptake in isolated ileum of rats were studied. The mucosal-to-serosal transport of horseradish peroxidase (HRP, mol mass 40 kDa) was measured in Ussing chambers, and afterwards tissues were processed for electron microscopy. In the absence of secretagogues, the flux of enzymatically active HRP was 5 pmol.cm-2.h-1 at a mucosal concentration of 10 microM. Electron micrographs showed vesicles filled with active HRP in enterocytes but no HRP activity in intercellular spaces. Forskolin decreased HRP activity in the cells. Carbachol increased the amount of HRP-filled vesicles in enterocytes and induced HRP filling in some intercellular spaces and tight junctions in the upper parts of the villi. The transepithelial flux of intact HRP increased more than 2.5-fold. This effect was suppressed by atropine. We conclude that cholinergic activation can increase the uptake of intact protein via endocytosis and the transepithelial passage by the induction of a diffusional paracellular pathway. We speculate that the increased transport of intact protein through the intestinal barrier may influence immunologic sensitization to food allergens.
Measurement of proximal and distal gastric motility with magnetic resonance imaging
Authors: Schwizer W. , Fraser R. , Borovicka J. , Asal K. , Crelier G. , Kunz P. , Boesiger P. , Fried M.
Published: December 2017

The precise motor mechanisms associated with gastric emptying of nutrient liquids are unclear, in part because of difficulties in measuring the motility from the proximal and distal stomach simultaneously. We have now examined proximal and distal gastric motility, using a novel magnetic resonance imaging (MRI) technique. In seven healthy volunteers (4 males, 3 females; 27-37 yr), gastric emptying and motility were determined on two occasions after ingestion of 500 ml 10% and 25% dextrose labeled with 1 mM gadolinium tetraazacyclododecane tetraacetic acid, using a 1.5-tesla Philips Gyroscan ACS II scanner. Gastric emptying was determined every 15 min with a series of transaxial scans. After each series of transaxial scans, 120 coronal scans, 1.2 s apart, were performed through the antrum and proximal stomach. For each coronal slice the diameters of the proximal stomach and the antrum were measured to determine the number of contractions per minute and depth (%basal diameter). Gastric emptying (half-emptying time) was faster after ingestion of 10% compared with 25% dextrose (49 +/- 15 vs. 118 +/- 37 min; P < 0.01). After both meals, the diameter of the proximal stomach remained relatively constant, whereas there were marked fluctuations in the diameter of the antrum. Mean (+/- SD) frequency (2.8 +/- 0.6 vs. 2.0 +/- 0.8/min; P < 0.001) and depth (40 +/- 17% vs. 34 +/- 16%; P < 0.04) of antral contractions were higher after 10% dextrose compared with 25% dextrose. Rapid MRI techniques allow simultaneous measurement of both gastric emptying and motor function of different gastric regions. The increase in the frequency and depth of distal gastric contractions during ingestion of 10% compared with 25% dextrose supports the concept that the antrum contributes to the regulation of gastric emptying of nutrient liquids.
Regulation of colonic propulsion by enteric excitatory and inhibitory neurotransmitters
Authors: Foxx-Orenstein A. E. , Grider J. R.
Published: December 2017

The contribution of excitatory and inhibitory motor neurotransmitters to colonic propulsion was examined in isolated segments of guinea pig colon. Synthetic fecal pellets were inserted at the proximal end of the segment, and the velocity of pellet propulsion across a fixed distance was measured in the presence and absence of selective neurotransmitter antagonists. The control velocity (0.97 +/- 0.02 mm/s) was inhibited in a concentration-dependent fashion by atropine and the neurokinin (NK)-2a antagonist MEN-10,376 [half-maximal inhibitory concentration (IC50), 1 microM; maximal inhibition, 98 +/- 1%]. The NK-1 antagonist GR-82,334 (10 microM) also inhibited velocity by 65 +/- 9%, consistent with involvement of acetylcholine, neurokinin A (NK-2 agonist), and substance P (NK-1 agonist) in the contractile components of the peristaltic reflex. Velocity was also inhibited in a concentration-dependent fashion by the nitric oxide synthase inhibitor NG-nitro-L-arginine (L-NNA; IC50, 1 microM; maximal inhibition, 96 +/- 2%) and by the vasoactive intestinal peptide (VIP) antagonist VIP-(10-28) (IC50, 30 nM; maximal inhibition, 64 +/- 6%), consistent with involvement of both nitric oxide and VIP in descending relaxation of circular muscle and contraction of longitudinal muscle. A combination of threshold concentrations of L-NNA and the NK-2a antagonist was synergistic (53 +/- 7% inhibition). The potentiation implied that the ascending and descending phases were functionally coupled in series. We conclude that blockade of neurotransmitters that mediate either phase of the peristaltic reflex inhibits colonic propulsive activity. Serial coupling of the phases leads to synergism between inhibitors, a condition of potential therapeutic importance.
Regulation of Fe absorption by cultured intestinal epithelia (Caco-2) cell monolayers with varied Fe status
Authors: Tapia V. , Arredondo M. , Nunez M. T.
Published: December 2017

Body Fe homeostasis is maintained through the regulation of Fe absorption by the intestinal epithelia. Working under the hypothesis that the intracellular concentration of Fe is instrumental in the control of its transepithelial flux, we investigated in vitro which steps in Fe absorption are regulated by cellular Fe content. For that study, Caco-2 cells containing different concentrations of intracellular 55Fe were grown in porous filters, and the apical-to-cell-to- basolateral flux of 59Fe was then determined. We found that 1) at low (up to 0.1 mM) intracellular Fe content the apical-to-basal Fe transport was primarily regulated by a decrease in apical Fe uptake (first stage of regulation), 2) at higher levels of intracellular Fe (0.1-1 mM) the transepithelial Fe flux was regulated by intracellular factors that sequester most of the Fe taken up at the apical surface (second stage of regulation), and 3) a fraction of the apical-to-basolateral Fe flux was not regulated by the intracellular concentration of Fe. Ferritin synthesis preceded the onset of the second stage of regulation, suggesting a causal relationship between intracellular Fe levels, ferritin levels, and regulation of Fe absorption.
Acute alcohol produces hypoxia directly in rat liver tissue in vivo: role of Kupffer cells
Authors: Arteel G. E. , Raleigh J. A. , Bradford B. U. , Thurman R. G.
Published: December 2017

Previous studies using liver slices and isolated perfused rat liver have suggested that ethanol causes hypoxia by increasing oxygen consumption. However, ethanol also increases blood flow to the liver, a phenomenon that may counteract the effects of hypermetabolism by increasing oxygen delivery. Thus whether ethanol causes hypoxia in vivo remains unclear. To clarify this important point, female Sprague-Dawley rats (100-125 g) simultaneously received pimonidazole (120 mg/kg ip), a 2-nitroimidazole hypoxia marker, and one large dose of ethanol (5 g/kg ig), which increase hepatic oxygen uptake dramatically and elevate ethanol metabolism (swift increase in alcohol metabolism) in 2-3 h. After 2 h, ethanol significantly increased the accumulation of bound pimonidazole in pericentral regions of the liver lobule. Treatment of animals with the Kupffer cell-specific toxicant, GdCl3 (10 mg/kg iv, 24 h before experiment), blocked ethanol-induced increases in pimonidazole binding. It is concluded that one large dose of ethanol causes pericentral hypoxia in rat liver tissue in vivo and that Kupffer cells are involved.
Interaction between prostanoids, NO, and VIP in modulation of duodenal alkaline secretion and motility
Authors: Sababi M. , Hallgren A. , Nylander O.
Published: December 2017

The relation between duodenal motility and duodenal mucosal alkaline secretion (DMAS) was examined in anesthetized rats. The duodenum was perfused with saline, and DMAS was determined by titration. Duodenal motility, assessed by intraluminal pressure measurements, was induced by indomethacin and/or N omega-nitro-L-arginine methyl ester (L-NAME) and inhibited by iloprost or vasoactive intestinal peptide (VIP). Six of 66 rats showed spontaneous duodenal contractions. Basal DMAS was higher in these rats than in those without contractions. Rats treated with indomethacin and L-NAME before abdominal operation exhibited duodenal motility postoperatively and had higher DMAS than in controls. Iloprost abolished both the duodenal motility increase and increase in DMAS induced by indomethacin. L-NAME-induced motility and increase in DMAS were antagonized by L-arginine. VIP increased DMAS without affecting motility. VIP abolished indomethacin-induced motility and augmented indomethacin-stimulated DMAS. VIP reduced L-NAME-induced motility and slightly increased L-NAME-stimulated DMAS. It is concluded that DMAS varies with duodenal motility. Prostaglandins and NO inhibit duodenal motility, thereby indirectly reducing DMAS. VIP may have dual effects on DMAS, an inhibitory action mediated via smooth muscle relaxation and a stimulatory action independent of motility.
Effects of neuropeptide Y and substance P on antigen-induced ion secretion in rat jejunum
Authors: McKay D. M. , Berin M. C. , Fondacaro J. D. , Perdue M. H.
Published: December 2017

We previously described a model of intestinal hypersensitivity in which isolated gut segments from sensitized rats demonstrated a rapid epithelial secretory response to luminal antigen that was mediated by mucosal mast cells and capsaicin-sensitive nerves. In this study, we examined the ability of the inhibitory neuropeptide, neuropeptide Y (NPY), to diminish the antigen-induced secretory response. Rats were sensitized to egg albumin (EA), and 12-14 days later, jejunal tissue was excised and mounted in Ussing chambers. NPY inhibited the short-circuit current (Isc) increase and Cl- secretion evoked by addition of EA to the luminal side of the tissue; neural blockade with tetrodotoxin (TTX) had a similar inhibitory effect. In contrast, NPY was much less effective, and TTX was completely ineffective, on the response to serosal antigen. Additional experiments examined the cell target for NPY action. NPY and TTX almost abolished the Isc response to electrical transmural stimulation of enteric nerves, suggesting a possible neural site of action. In addition, NPY significantly reduced baseline Isc; this inhibition involved both TTX-dependent and TTX-independent components. Because nerves were previously shown to facilitate antigen uptake and substance P was implicated in the response to only luminal antigen, we postulated that NPY was inhibiting nerves that facilitate antigen transport from the lumen to effector cells in the lamina propria. We therefore examined the effect of exogenous substance P added after NPY inhibition. Substance P restored the luminal antigen-induced secretory response to pretreatment values. We conclude that the neuropeptides play a significant role in immunophysiology by acting at neural and epithelial sites in the intestinal mucosa.