bluuebunny/crossref_metadata_2025 huggingface dataset:
2-Deoxyglucose transport and metabolism in Caco-2 cells
Authors: Bissonnette P. , Gagne H. , Blais A. , Berteloot A.
Published: December 2017

We investigated the kinetics of 2-deoxy-D-glucose (DG) uptake and metabolism in Caco-2 cells, because this human cell line may represent a valid enterocyte model to assess the dynamics between sugar transport and metabolism and hence to obtain insights into the factors involved during the intracellular phase of glucose absorption. When studied in 14-day-old monolayers, DG uptake is characterized by a lag phase with a time course matching the decrease in intracellular glucose concentrations, and no intracellular glucose 6-phosphate (G-6-P) can be detected at any time during incubation. After 1 h of preincubation of Caco-2 cells in substrate-free transport medium, however, steady-state DG uptake matches 2-deoxy-D-glucose 6-phosphate (DG-6-P) accumulation with undetectable levels of free DG. This complex behavior in DG uptake is linked to high hexokinase activity in Caco-2 cells, and the enzyme has a Michaelis-Menten constant K(m) for glucose that is typical of hexokinase type II (0.120 +/- 0.003 mM). Caco-2 cells also contain low-level glucose-6-phosphatase (G-6-Pase) activity, which may account for the leveling off in DG uptake, and the kinetics of DG transport may be attributed to the existence of a predominant pathway with a K(m) of 1.7 +/- 0.2 mM. Finally, analysis of the growth-related expression of DG transport and hexokinase activity clearly shows that DG uptake is lowest in postconfluent cells when hexokinase is at its highest levels. We thus conclude that 1) transport is the rate-limiting step during DG accumulation, 2) G-6-P is a potent inhibitor of hexokinase activity compared with DG-6-P, so that enzyme inhibition may have physiological relevance in diverting glucose from metabolism during its active reabsorption in the small intestine, and 3) low levels of G-6-Pase activity seem to exclude this enzyme, and hence the endoplasmic reticulum, as important factors during the intracellular phase of glucose transport.
Polarized GP2 secretion in MDCK cells via GPI targeting and apical membrane-restricted proteolysis
Authors: Fritz B. A. , Lowe A. W.
Published: December 2017

The major zymogen granule membrane protein in the exocrine pancreas is glycoprotein 2 (GP2), a glycosyl phosphatidylinositol (GPI)-linked membrane protein. Despite its GPI anchor, GP2 is secreted into the pancreatic duct. We examined the mechanism underlying the secretion of GP2 in isolated pancreatic acini and transfected Madin-Darby canine kidney (MDCK) cells (MDCK-GP2). MDCK-GP2 cells release GP2 almost exclusively (> 95%) from the apical membrane. Using GP2 as a model, we defined a novel mechanism of polarized protein secretion in which a secretory protein is targeted via a GPI anchor to the apical plasma membrane, whereupon the mature form is released by proteolysis. Furthermore, we described two features of MDCK cells that enhance the polarized release of GP2: an apical plasma membrane-restricted distribution of the protease responsible for GP2 membrane cleavage, and a transcytotic pathway to reroute basolateral plasma membrane GP2 to the apical cell surface.
Impairment of the gastric hyperemic response to luminal acid in cirrhotic rats
Authors: Nishizaki Y. , Guth P. H. , Sternini C. , Kaunitz J. D.
Published: December 2017

Liver cirrhosis impairs gastric mucosal resistance to luminal acid in humans and in animal models. Because we have previously shown that pentagastrin enhances defensive as well as aggressive factors implicated in mucosal injury, we examined the hypothesis that the pentagastrin-mediated enhancement of mucosal defense mechanisms may be impaired in cirrhotic rats. Increased acid backdiffusion and susceptibility to gross mucosal injury, associated with an elimination of the hyperemic response to gastric barrier disruption, was observed in cirrhotic rats. In in vivo microscopic studies in anesthetized rats, cirrhosis had no effect on pentagastrin-associated enhancement of mucus gel thickness or baseline gastric mucosal blood flow, although baseline mucus gel thickness was decreased. Cirrhosis did, however, abolish the luminal acid-related hyperemic response to pentagastrin, which was associated with impaired intracellular pH homeostasis during acid superfusion. Cirrhosis did not alter submucosal calcitonin gene-related peptide immunoreactive nerves. We conclude that acid backdiffusion and pentagastrin-associated hyperemic responses are important mucosal defensive factors that are specifically impaired by cirrhosis.
Cytokines regulate apolipoprotein B secretion by Caco-2 cells: differential effects of IL-6 and TGF-beta 1
Authors: Murthy S. , Mathur S. N. , Varilek G. , Bishop W. , Field F. J.
Published: December 2017

A variety of cytokines are found in the intestinal mucosa of individuals with inflammatory diseases. The potential role of cytokines in mediating lipoprotein assembly and secretion in the human intestinal cell line, Caco-2, was investigated. Interleukin-1 beta, interleukin-6 (IL-6), and tumor necrosis factor-alpha all decreased the basolateral secretion of apolipoprotein B (apo B), with IL-6 being the most potent. IL-6 was also found to inhibit triacylglycerol secretion. In contrast, transforming growth factor-beta 1 (TGF-beta 1) increased the secretion of apo B and triacylglycerol. In pulse-chase experiments, IL-6 decreased the rate of synthesis and secretion of apo B-100 and apo B-48 without altering the rate of apo B degradation, whereas TGF-beta 1 increased the rate of synthesis and secretion of apo B-100 and apo B-48. Degradation of apo B was also not affected by TGF-beta 1. The abundance of apo B mRNA in cells incubated with IL-6 was decreased, whereas cells incubated with TGF-beta 1 had higher levels of apo B mRNA. In conditions of small intestinal inflammation, cytokines could contribute to the observed malabsorption of fat and other nutrients by the small intestine.
Role of nonmuscle myosin II in polyamine-dependent intestinal epithelial cell migration
Authors: Wang J. Y. , McCormack S. A. , Johnson L. R.
Published: December 2017

The current study determines whether nonmuscle myosin II is involved in the process requiring polyamines for the stimulation of cell migration in an in vitro model that mimics the early stages of epithelial restitution. Treatment with alpha-difluoromethylornithine (DFMO), a specific inhibitor of ornithine decarboxylase (ODC), for 4 days totally inhibited ODC activity and depleted intracellular polyamines in the IEC-6 cells. Nonmuscle myosin II concentrations in DFMO-treated cells were decreased by 75%, and stress fibers were sparse or absent. The most striking feature of DFMO-treated cells was the appearance of many small punctate foci of myosin II in the cell interior. Migration of DFMO-treated cells was reduced by 80%. In the presence of DFMO, exogenous putrescine not only returned nonmuscle myosin II levels and distribution toward normal but also restored cell migration to control levels. The administration of wortmannin, an inhibitor of myosin light chain kinase, significantly inhibited cell migration over the denuded area in control cells and in those treated with DFMO + polyamines. These results indicate that 1) polyamine depletion by DFMO is associated with decreased concentration and reorganization of nonmuscle myosin II in IEC-6 cells and 2) exogenous spermidine reverses the inhibitory effects of DFMO.
Hyperosmolarity inhibits the Na+/H+ exchanger isoforms NHE2 and NHE3: an effect opposite to that on NHE1
Authors: Nath S. K. , Hang C. Y. , Levine S. A. , Yun C. H. , Montrose M. H. , Donowitz M. , Tse, C. M.
Published: December 2017

The effect of hyperosmolarity on cloned Na+/H+ exchanger (NHE) isoforms NHE2 and NHE3 was studied in stably transfected PS120 fibroblasts. Na+/H+ exchanger activity was determined spectrofluorometrically in acidified cells that were exposed to isosmolar (300 mosmol/kg) or hyperosmolar (450 mosmol/kg) media, in which the only difference is the presence or absence of 150 mM mannitol. Hyperosmolar solution reversibly inhibited NHE2 and NHE3 with a delay of approximately 15 s. Hyperosmolarity significantly reduced their maximal reaction velocity compared with isosmolar medium but did not alter their Michaelis-Menten constant for intracellular H+. The Michaelis-Menten constant of the exchangers for extracellular Na+ in hyperosmolar medium was not different from that in isosmolar medium. Pretreatment of PS120/NHE3 cells with the protein kinase C inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, the tyrosine kinase inhibitor genistein, and the serine/threonine protein phosphatase inhibitor okadaic acid did not affect the hyperosmolar inhibition of NHE3. Hyperosmolar inhibition of Na+/H+ exchanger activity was also observed in PS120 cells transfected with truncated NHE3 cDNAs (E3/585, E3/543, E3509, and E3/475) and NHE2 cDNA (E2/499). We conclude that 1) hyperosmolarity inhibits NHE2 and NHE3, in contrast to the stimulatory effect on the housekeeping isoform NHE1, 2) this inhibition is reversible, and 3) the COOH termini of NHE2 and NHE3 are not necessary for hyperosmolar inhibition of NHE2 and NHE3.
Effects of dietary polyunsaturated fat on ethanol-induced Ito cell activation
Authors: Tsukamoto H. , Cheng S. , Blaner W. S.
Published: December 2017

Ito cells, vitamin A-storing perisinusoidal cells, are believed to undergo myofibroblastic transformation in liver fibrogenesis. Our previous studies have shown that a diet high in polyunsaturated fat was key for induction of experimental alcoholic liver fibrosis. To investigate the cellular basis for this fibrogenic effect of a high-fat diet, we analyzed the content of vitamin A and cellular retinol binding protein (CRBP), the steady-state mRNA levels of procollagen-alpha1(I), transforming growth factor-beta1 (TGF-beta1), interleukin-6 (IL-6), and smooth muscle alpha-actin (alpha-SM) in freshly isolated Ito cells from rats given isocaloric amounts of ethanol and a low- or high-fat diet. After 10 wk, the Ito cell content of retinyl palmitate was severely reduced in both the high- and low-fat diet-ethanol-fed animals to 13-17% of those measured in respective pair-fed controls. On the other hand, the content of CRBP was reduced in the high-fat-ethanol rats but not in the low-fat-ethanol group. The cells from the high-fat-ethanol but not low-fat ethanol rats showed an 18-fold increase in procollagen-alpha1(I) mRNA at 17 wk, which was accompanied by 2.8- and 2.3-fold enhancement of TGF-beta1 and alpha-SM transcripts. IL-6 mRNA was not detected in the cells from any groups. These results demonstrate 1) myofibroblastic activation of Ito cells is evident in rats given a high-fat diet and ethanol but not in the low-fat-ethanol animals; 2) vitamin A depletion of Ito cells is the early and general effect of chronic ethanol intake but does not necessarily predict subsequent myofibroblastic activation; 3) reduced CRBP level is more closely associated with the subsequent cellular activation seen under the high-fat-ethanol regimen; and 4) IL-6 is not expressed in vivo by Ito cells from either normal livers or livers with alcoholic liver fibrosis.
Differential expression of cysteine-rich intestinal protein in liver and intestine in CCl4-induced inflammation
Authors: Khoo C. , Hallquist N. A. , Samuelson D. A. , Cousins R. J.
Published: December 2017

Cysteine-rich intestinal protein (CRIP) is a double zinc finger (LIM domain) protein that is developmentally regulated but has an unknown function. CRIP is highly expressed in the intestine, but expression is low in liver. To determine if CRIP expression is regulated under altered physiological status, we used CCl4-induced injury as a model to produce hepatic injury and systemic effects associated with inflammation. Since CRIP is a zinc finger protein and zinc decreases the hepatic response to CCl4, the effect of supplemental dietary zinc (300 mg/kg diet) was also examined. Our results show that this supplemental level of dietary zinc did not affect the index of hepatic injury (plasma alanine aminotransferase), indicating zinc did not have a protective effect. Liver CRIP mRNA increased with CCl4 and CRIP protein was shown by immunohistochemistry to be localized in hepatocytes near the vascular supply. In the intestine, CCl4 caused a transient decrease in CRIP mRNA, but supplemental dietary zinc treatment prevented this decrease. These current results show that CRIP expression changes in response to cellular damage due to acute hepatic injury and are consistent with a functional role for CRIP in proliferation, differentiation, or turnover.
Rat histidine decarboxylase promoter is regulated by gastrin through a protein kinase C pathway
Authors: Hocker M. , Zhang Z. , Fenstermacher D. A. , Tagerud S. , Chulak M. , Joseph D. , Wang T. C.
Published: December 2017

The enzyme L-histidine decarboxylase (HDC; EC 4.1.1.22), which converts L-histidine to histamine, plays a key role in the regulation of acid secretion. In the rat and human stomach, the peptide hormone gastrin appears to be one of the main regulators of HDC expression. In rats, marked elevation of gastric HDC mRNA abundance was observed within 12 h after induction of hypergastrinemia by a single injection of the proton-pump blocker omeprazole. In situ hybridization revealed that HDC expression occurred in the basal third of gastric glands where enterochromaffin-like cells are localized. To study the regulation of HDC gene transcription, 1,291 nucleotides of the 5'-flanking region of the rat HDC gene and the noncoding portion of exon 1 were cloned and sequenced. Gastrin and cholecystokinin (CCK) octapeptide equipotently stimulated the transcriptional activity of the rat HDC promoter three- to fourfold, and deletion analysis revealed the presence of a gastrin response element within 201 nucleotides upstream of the translational start site. Time-course studies revealed maximal activation of the HDC promoter after 12-36 h. Direct stimulation of protein kinase C (PKC) with the phorbol ester phorbol 12-myristate 13-acetate (PMA) substantially elevated rat HDC promoter activity, whereas induction of Ca2+ -dependent signaling pathways with thapsigargin was without effect. Downregulation or blockade of PKC abolished the effects of gastrin and PMA on the HDC promoter. These data indicate that stimulation of the CCK-B/gastrin receptor activates the rat HDC promoter in a time- and dose-dependent fashion and that this effect is primarily mediated via a PKC-dependent signaling pathway. Use of HDC as a model gene will allow further investigation of the intracellular pathways that are involved in gastrin-dependent gene regulation.
Alterations in mRNA stability during rat liver regeneration
Authors: Kren B. T. , Trembley J. H. , Steer C. J.
Published: December 2017

We examined the in vivo transcriptional and posttranscriptional regulation of various genes involved in hepatocyte growth and replication that exhibited changes in steady-state mRNA levels after 70% partial hepatectomy (PH). Of the 19 genes examined by nuclear run-on assay, 17 demonstrated no change in transcriptional activity through the first 96 h of regeneration. However, results from in vivo half-life determinations indicated that changes in mRNA stability played a critical role in regulating transcript levels during liver regeneration. For many of the genes, alterations in transcript abundance correlated with similar changes in mRNA half-lives. Inhibition of protein synthesis by cycloheximide was generally associated with increased levels of mRNA expression, but no detectable changes in transcriptional rates in both control and regenerating rat liver. Finally, genomic methylation status was investigated by Southern analysis for several genes that displayed changes in mRNA stability. Interestingly, increases in mRNA half-lives for the genes p53, c-myc, H-ras, and ornithine decarboxylase were associated with decreased genomic methylation. In conclusion, regulation of gene expression beyond the immediate early phase of te cell cycle during rat liver regeneration after PH occurs predominantly at the posttranscriptional level. mRNA stability appears to be a significant factor in this control, and may itself be modulated by the methylation status of the corresponding genomic DNA.
Kinetic separation and characterization of three sugar transport modes in Caco-2 cells
Authors: Bissonnette P. , Gagne H. , Coady M. J. , Benabdallah K. , Lapointe J. Y. , Berteloot A.
Published: December 2017

The question of sugar transport heterogeneity in the human intestinal Caco-2 cell line was addressed using alpha-methyl-D-glucose (AMG) and 2-deoxy-D-glucose (DG) as substrate analogues for D-glucose, the transport inhibitors phlorizin (PZ) and phloretin (PT), and NaCl or choline chloride uptake media. The data are compatible with the existence of three distinct pathways that can be isolated kinetically according to specific characteristics: 1) an “:AMG-strict” system, strictly Na+ dependent and specific for AMG [Michaelis-Menten constant value (K(m)) = 2.0 +/- 0.3 mM] but sensitive to both PZ and PT, with PZ being more potent than PT, 2) a “DG-strict” system, strictly Na+ independent and specific for both DG (K(m) = 5.2 +/- 0.5 mM) and PT; and 3) a “DG/AMG-mixed” system, strictly Na+ dependent, with loose specificities for the glucose analogues DG (K(m) = 0.81 +/- 0.07 mM) and AMG (K(m) = 8.1 +/- 0.8 mM), and the inhibitors PZ and PT, but with PT being more potent than PZ. Since SGLT-1 obtained by polymerase chain reaction from either Caco-2 cells or normal human jejunum demonstrated identical transport properties when expressed in Xenopus laevis oocytes, we conclude that the “AMG-strict” system represents the expression of human SGLT-1 activity in this cell line. Moreover, Western blot analysis revealed that SGLT-1 is located exclusively in the apical membrane. In contrast, neither the nature nor the membrane location of both the DG-strict and DG/AMG-mixed pathways could be resolved unambiguously. Still it has been demonstrated that expression of the latter system is constitutive to all Caco-2 cells and that its Na+ dependence is not the consequence of H(+)-dependent transport activity. Aside from the presence of the DG/AMG-mixed system, a salient feature of Caco-2 cells is that the GLUT-3 protein is located exclusively in the brush-border membrane. Due to these limitations, it is concluded that the Caco-2 cell line cannot be considered as equivalent to either fetal colonic cells or normal enterocytes.
Inhibition of binding to fatty acid binding protein reduces the intracellular transport of fatty acids
Authors: Luxon B. A.
Published: December 2017

Male livers, containing lesser amounts of fatty acid binding protein (FABP), utilize fatty acids more slowly than female livers. Conventional wisdom dictates that FABP stimulates fatty acid use by increasing cytoplasmic transport rates. Previously, we showed that the cytoplasmic diffusion of a fatty acid analogue [12-N-methyl-7-nitrobenzo-2-oxa-1,3-diazol-amino stearate (NBD-stearate)] is faster in female hepatocytes, paralleling the larger amounts of FABP. Sex differences in other cytoplasmic factors could also lead to faster diffusion, independent of FABP levels. The aim of this study was to determine the effect of inhibition of fatty acid binding to FABP on the directly measured intracellular transport rate of NBD-stearate. The binding of NBD-stearate to FABP was reduced by incubating hepatocytes isolated from male and female rats with alpha-bromo-palmitate (0-1,500 microM), a modified long-chain fatty acid that binds to FABP. The inhibition by alpha-bromo-palmitate on NBD-stearate binding to FABP was measured with the use of centrifugation to separate cytosol from cytoplasmic membranes. Laser photobleaching (fluorescence recovery after photobleaching) was used to measure the cytoplasmic diffusion of NBD-stearate in hepatocytes. Alpha-Bromo-palmitate incubation reduced NBD-stearate binding to FABP in a dose-dependent manner. The measured diffusion rate was also reduced in proportion to the degree of binding inhibition. We conclude that cytoplasmic transport of NBD-stearate is modulated by binding to soluble proteins like FABP. FABP enhances diffusive transport by reducing binding to immobile cytosolic membranes.
Peptide YY receptor in submucosal and myenteric plexus synaptosomes of canine small intestine
Authors: Mao Y. K. , Wang Y. F. , Ward G. , Cipris S. , Daniel E. E. , McDonald T. J.
Published: December 2017

PYY receptors were characterized and their loci determined in canine small intestine. The density of 125I-labeled peptide tyrosine tyrosine (PYY) binding was highest in myenteric (MY) and submucosal (SUB) plexus fractions enriched in synaptosomes. Two binding sites [high affinity (H) and low affinity (L)] were found in the submucosal synaptosome-enriched membrane: dissociation constant (Kd)H = 7.6 pM, maximal binding capacity (Bmax)H = 28 fmol/mg; KdL = 0.18 nM, BmaxL = 120 fmol/mg protein. The binding of 125I-PYY reached a maximum within 30 min; dissociation was incomplete in the presence of unlabeled PYY. The rate of dissociation was enhanced after exposure of synaptosomes to guanosine 5'-O-(3-thiotriphosphate). Binding of 125I-PYY was completely inhibited by neuropeptide Y (NPY)-(13-36) (in SUB and MY) and by [Leu31,Pro34]NPY (in MY) but only partially by [Leu31,Pro34]NPY in SUB, suggesting the presence of Y2 receptor in SUB and the presence of Y1 and Y2 receptors in MY. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the PYY receptor complex revealed a radioactive band at 70 kDa. The PYY receptors in the canine small intestinal myenteric and submucosal plexus correspond in location to that of PYY in synaptosomes and are coupled with G proteins. Different subtypes are present in different loci.
Neural integration of jejunal motility and ion transport in nematode-infected ferrets
Authors: Greenwood B. , Palmer J. M.
Published: December 2017

Jejunal motility measured manometrically and coupled fluctuations in electrogenic ion transport measured as transmural potential differences (PD) were simultaneously studied in ferrets infected with enteric stages of the parasitic nematode, Trichinella spiralis. Vagotomy in uninfected ferrets abolished jejunal motility clusters and associated PD oscillations. Conversely, in infected ferrets on days 8-12 postinfection (PI), vagotomy did not abolish jejunal motility and PD. Calculated motility indexes (MI) indicated that postvagotomy MI decreased to 12% of prevagotomy MI in uninfected ferrets, whereas, in T.spiralis-infected ferrets, postvagotomy MI declined only to 48% of prevagotomy MI. Atropine abolished all vagotomy-resistant residual jejunal motility clusters and PD oscillations in T. spiralis-infected ferrets. Decreased intestinal content of substance P (27% of control) and vasoactive intestinal peptide (41% of control) and increased myeloperoxidase activity (262% of control) were detected in T. spiralis-infected ferrets. Our results suggest that integrated neural control of muscular and epithelial effectors in the small bowel is altered by nematode-induced inflammation.
Hepatocellular protein kinase C activation by bile acids: implications for regulation of cholesterol 7 alpha-hydroxylase
Authors: Stravitz R. T. , Rao Y. P. , Vlahcevic Z. R. , Gurley E. C. , Jarvis W. D. , Hylemon P. B.
Published: December 2017

We have recently shown that taurocholate (TCA) represses the transcriptional activity of cholesterol 7 alpha-hydroxylase, the rate-limiting enzyme of the bile acid biosynthetic pathway, through a protein kinase C (PKC)-dependent mechanism in primary cultures of rat hepatocytes. The present studies sought to determine the mechanisms by which bile acids activate hepatic PKC activity and the consequences of this activation on isoform distribution and cholesterol 7 alpha-hydroxylase mRNA levels. TCA (12.5-100 microM for 15 min) increased membrane-associated "classic" isoenzyme cPKC-alpha and "novel" isoenzymes nPKC-delta, and nPKC by two- to sixfold. Membrane-associated PKC progressively increased, and cytosolic PKC decreased, for 1 h after the addition of TCA (50 microM); after 24 h whole cell cPKC-alpha, nPKC-delta, and nPKC were downregulated by 35-55% compared with untreated controls. In a reconstituted assay system, TCA or taurodeoxycholate (10-100 microM) increased calcium-dependent and -independent PKC activity by three- and fourfold, respectively. Taurine-conjugated bile acids stimulated PKC activity in proportion to their hydrophobicity index (r = 0.99). Finally, cholesterol 7 alpha-hydroxylase mRNA was repressed > 75% by phorbol 12-myristate 13-acetate (100 nM for 3 h), a nonselective activator of PKC isoforms. In contrast, selective cPKC-alpha activation with thymeleatoxin (100 nM for 3 h) had no significant effect on cholesterol 7 alpha-hydroxylase mRNA levels. We conclude that bile acids activate hepatocellular PKC, resulting in sequential redistribution and down-regulation of calcium-dependent and -independent isoforms. The calcium-independent PKC isoforms may mediate the repression of cholesterol 7 alpha-hydroxylase mRNA by TCA.
Intrahepatic blood flow distribution in the perfused rat liver: effect of hepatic artery perfusion
Authors: Burczynski F. J. , Luxon B. A. , Weisiger R. A.
Published: December 2017

Variations in blood flow to different sinusoids within the liver can prevent uniform uptake of solutes from plasma and contribute to cellular ischemia in low-flow states. However, the degree of variability and the role of hepatic artery perfusion in maintaining uniform flow are poorly defined. We used an indicator dilution technique to compare the distribution of sinusoidal transit times in isolated rat livers perfused through the portal vein alone with livers perfused using both portal vein and hepatic artery. Physiological flow rates were used in each case (1.2 +/- 0.3 ml.min-1.g liver-1), but the second group received 32% of flow through the hepatic artery. Intralobular flow heterogeneity was further assessed by gamma counting of small (approximately 100 mg) pieces of the liver after bolus injection of approximately 5 mCi of a highly extracted compound ([125I])triiodothyronine) into the portal vein. Hepatic artery perfusion had no significant effect on mean sinusoidal transit time or intrahepatic distribution volume for 51Cr-labeled red blood cells or 125I-albumin. Analysis of the outflow profiles indicated that hepatic artery perfusion did not affect transit time dispersion. However, heterogeneity of flow to individual portions of the liver, measured as the coefficient of variation, increased from 19 to 30%. These results indicate relatively uniform perfusion of the sinusoids in the portally perfused rat liver and that additional perfusion of the hepatic artery does not further improve hemodynamics. These results have significance for the design and interpretation of transport studies with the use of the perfused rat liver model.
Ondansetron reduces nausea induced by gastroduodenal stimulation without changing gastric motility
Authors: Feinle C. , Read N. W.
Published: December 2017

The involvement of 5-hydroxytryptamine3 (5-HT3) receptors in gastric motor and sensory responses to distension and duodenal lipid was investigated. Subjects were studied on four occasions during which isotonic saline or 20% Intralipid (2 kcal/ml) was infused intraduodenally (1 ml/min) while the proximal stomach was distended with air (100 ml/min). Subjects received either 8 mg ondansetron (5-HT3 antagonist) or placebo orally in random order. Intragastric pressure was recorded continuously, and subjects reported gastric sensations. Gastric motor and sensory responses to distension during duodenal saline were similar with placebo and ondansetron. Intraduodenal lipid decreased gastric tonic and phasic pressure activity, and this was not influenced by ondansetron. Lipid also induced meal-like fullness followed by nausea during distensions. Ondansetron reduced nausea and did not affect meal-like fullness but increased volumes and pressures at which sensations were reported. Intestinal 5-HT3 receptors are involved in induction of nausea but not of meal-like fullness by intraduodenal lipid and gastric distension. 5-HT3 receptor antagonism reduces gastric sensitivity to distension during intraduodenal lipid infusion.
Histamine H2 receptor activates adenylate cyclase and PLC via separate GTP-dependent pathways
Authors: Wang L. , Gantz I. , DelValle J.
Published: December 2017

Previously, we demonstrated that a single histamine H2 receptor can couple to both the adenosine 3',5'-cyclic monophosphate and inositol 1,4,5-trisphosphate/intracellular Ca2+ signaling pathways in a stimulatory manner. We undertook the present studies to fur her characterize the postreceptor events involved in H2 receptor dual signaling. Histamine H2 receptor-mediated signal transduction was examined in isolated cell membranes prepared from purified canine parietal cells and HEPA cells (rat hepatoma cell line) stably transfected to express the canine H2 histamine receptor cDNA. Histamine dose-dependently stimulated both adenylate cyclase [AC; mean effective concentration (EC50) = 2 x 10(-7) M] and phospholipase C (PLC; EC50 = 3.1 +/- 0.5 x 10(-7) M) activity in an H2-specific and GTP-dependent manner. Cholera toxin pretreatment abolished the stimulatory effect of histamine on PLC activity in isolated membranes without altering binding of the H2 receptor antagonist tiotidine. Anti-Gs alpha dose-dependently inhibited histamine-stimulated AC activity while leaving the effect of this secretagogue on PLC activity unaltered. Although anti-Gq alpha inhibited vasopressin-stimulated PLC activity in HEPA cells and carbachol-stimulated PLC in parietal cells, this antibody did not alter the action of histamine on PLC in the same membrane preparations. Antibody against the NH2 and COOH terminals of the common beta-subunit of heterotrimeric G proteins did not inhibit histamine-stimulated PLC activity. Our studies demonstrate for the the first time that activation of the H2 receptor leads to stimulation of both AC and PLC via separate GTP-dependent mechanisms.
Excessive NO production dose not account for the inhibition of hepatic gluconeogenesis in endotoxemia
Authors: Ou J. , Molina L. , Kim Y. M. , Billiar T. R.
Published: December 2017

The pattern of inhibition of gluconeogenesis in hepatocytes was compared between endotoxemia in vivo and nitric oxide (NO) exposure in vitro. Fasted rats were injected with lipopolysaccharide (LPS; 12 mg/kg) or with vehicle alone. After 2-24 h, hepatocytes were isolated, placed in suspension, and incubated for 1 h with various gluconeogenic substrates that enter at different sites of the gluconeogenic pathway. Hepatocytes from LPS-treated rats exhibited up to a 50% decrease in gluconeogenesis for substrates that enter proximal to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) beginning at 6 h, followed by a nadir at 12 h after LPS. Although hepatocytes exposed to exogenous NO (S-nitroso-N-acetylpenicillamine) also exhibited a depressed gluconeogenesis, the pattern was not the same with inhibition in gluconeogenesis for substrates that enter the pathway both before and after GAPDH. Furthermore, when rats injected with LPS were subjected to a constant portal infusion (Alzet pump) of the NO synthase (NOS) inhibitors, NG-monomethyl-L-arginine or aminoguanidine, no changes in the LPS-induced gluconeogenesis suppression were seen. In addition, no difference in LPS-induced inhibition of gluconeogenesis was detected when hepatocytes from inducible NO synthase (NOS-2) knockout mice were compared with cells obtained from wild-type mice. Minimal decreases in GAPDH activity were measured in hepatocytes from the LPS-treated rats, whereas the activity of phosphoenol pyruvate carboxykinase (PEPCK) declined up to 40%, independent of NO synthesis. These data indicate that NO does not account for the inhibition of gluconeogenesis in endotoxemia, and they provide support for NO-independent reduction in PEPCK activity as a more plausible explanation.
Cell cycle protein suppression and p21 induction in differentiating Caco-2 cells
Authors: Evers B. M. , Ko T. C. , Li J. , Thompson E. A.
Published: December 2017

Despite intensive efforts, the exact cellular mechanisms leading to gut differentiation and development remain largely undefined. The cyclins, the cyclin-dependent kinases (Cdks), and the Cdk inhibitors (e.g., p21 and p27) are proteins that are important for cell cycle progression, subsequent growth inhibition, and differentiation of various cell types. The purpose of our study was to better define the role of these cell cycle proteins in gut differentiation using the Caco-2 human cell line, which spontaneously differentiates to a small bowel phenotype, as demonstrated by induction of sucrase-isomaltase (SI) gene expression. We found that protein levels of the cyclins (both D- and E-type) and the Cdks (both Cdk2 and Cdk4) progressively decreased in postconfluent Caco-2 cells. Moreover, cyclin E-associated histone H1 kinase activity decreased in an analogous fashion as the cyclins and Cdks. In contrast, induction of the Cdk inhibitor p21 occurred by 3 days postconfluency, which was before the increase in SI mRNA levels. These changes in the cell cycle proteins, which include a progressive decrease of the cyclins and Cdks and a concomitant induction of p21, suggest an important role for these proteins in Caco-2 cell differentiation. Identifying the cell cycle mechanisms responsible for intestinal cell differentiation will be important to our understanding of both normal gut development as well as gut neoplasia, which involves aberrant regulation of cell cycle arrest.
Impairment of Kit-dependent development of interstitial cells alters contractile responses of murine intestinal tract
Authors: Sato D. , Lai Z. F. , Tokutomi N. , Tokutomi Y. , Maeda H. , Nishikawa S. , Nishikawa S. , Ogawa M. , Nishi K.
Published: December 2017

We examined developmental changes in responses of the isolated segment of the ileum of BALB/c mice treated with a monoclonal antibody (ACK2) to the receptor tyrosine kinase (Kit) for 4 days postnatally to pharmacological agents in vitro. Rhythmic contraction and relaxation of the isolated ileum started to appear on day 4 postpartum, and the sensitivity to acetylcholine (ACh) decreased gradually after birth. Treatment with ACK2 induced augmentation of contractile responses and receptor sensitivity of the longitudinal muscle of the ileum to ACh, bradykinin, and prostaglandin F2 alpha. ACh induced larger depolarization in smooth muscle cells of the ileum in the ACK2-treated mice than in the control. Circular muscle responses to these substances, as measured by changes in intraluminal pressure, were not altered by ACK2 treatment. Results suggest that interstitial cells play an important role not only in the development of the pacemaking system of the small intestine but also in the functional development of the contractile properties of the intestinal smooth muscle.
Hypoxia/reoxygenation selectively impairs alpha 1b-adrenoceptor function in small mesenteric arteries
Authors: Gao H. , Korthuis R. J. , Benoit J. N.
Published: December 2017

The present study examined whether hypoxia/reoxygenation (H/R) attenuates norepinephrine (NE) effectiveness in small arteries by interfering with function of alpha 1a- and/or alpha 1b- adrenoceptor subtypes. Small mesenteric arteries (approximately 150 microns) were obtained from rats mounted on a small vessel myograph in oxygenated physiological salt solution (PSS), and the relationship between NE concentrations and contractile tension was assessed. Hypoxia was induced by bubbling the vessels with 95% N2-5% CO2 for 15 min. Vessels were then reoxygenated for 30 min, and NE responses were reevaluated. Superoxide dismutase (SOD) and catalase (CAT) were added to the PSS in one group of vessels to investigate the role of reactive oxygen metabolites. In other groups, alpha 1b-receptors were blocked with chloroethylclonidine and alpha 1a-receptors were blocked with 5-methylurapidil or WB-4101 to produce exclusive alpha 1a- or alpha 1b-responses to NE. H/R decreased the NE negative logarithm of the mean effective concentration (pD2: i.e., -log[EC50], where EC50 is mean effective concentration) from 6.26 +/- 0.24 to 5.84 +/- 0.12 (P < 0.05). SOD and CAT prevented the H/R-induced contractile dysfunction. alpha 1a-Receptor responses to NE were not altered by H/R. In contrast, alpha 1b-receptor responses were significantly attenuated after H/R. The results indicate that alterations in NE responsiveness after H/R are due to dysfunction of the alpha 1b signal transduction pathway.
Involvement of ICE family proteases in apoptosis induced by reoxygenation of hypoxic hepatocytes
Authors: Shimizu S. , Eguchi Y. , Kamiike W. , Akao Y. , Kosaka H. , Hasegawa J. , Matsuda H. , Tsujimoto Y.
Published: December 2017

Cell death due to reoxygenation after hypoxia was characterized in primary cultured hepatocytes. Fluorescence and electron microscopic analyses of reoxygenated hepatocytes revealed morphological characteristics of apoptosis, including chromatin condensation, nuclear fragmentation, and formation of apoptotic bodies. Few necrotic hepatocytes, defined by loss of plasma membrane integrity, mitochondrial swelling, and formation of large vacuoles, were observed. Activation of interleukin-1 beta-converting enzyme (ICE)-like and CPP32/Yama-like proteases, which are known to drive apoptosis, was observed during reoxygenation, and addition of their respective inhibitors inhibited the induction of apoptosis, indicating the involvement of ICE family proteases in apoptosis by reoxygenation. Production of oxygen radicals was enhanced by reoxygenation of hypoxic cells, and reoxygenation-induced apoptosis was inhibited by oxygen radical scavengers, suggesting a role for reactive oxygen species as a triggering factor in cell death. Electrophoretic analysis revealed the presence of 50-kb DNA fragments but not oligonucleosomal DNA fragments in reoxygenation-induced apoptotic hepatocytes.
Local regulation of ileal tone in healthy humans
Authors: Coffin B. , Lemann M. , Flourie B. , Jouet P. , Rambaud J. C. , Jian R.
Published: December 2017

We previously showed that a meal induced, in the human terminal ileum, a delayed tonic relaxation, which could be related to the ileal delivery of meal residues and/or endogenous secretions released by a meal. In this study, we assessed the effects of some components of the ileal contents on its motor activity. In six healthy subjects, we studied ileal tonic and phasic motility in response to the infusion into the terminal ileum of different isotonic solutions: saline, glycochenodeoxycholic acid (GDCA), triglycerides, and short-chain fatty acids (SCFA). Tonic activity was not modified by saline, whereas it was significantly decreased by GDCA and triglycerides (maximal increase in intrabag volume 139 +/- 7% and 152 +/- 16%, respectively, P < 0.01), and significantly increased by SCFA (maximal decrease in intrabag volume 72 +/- 4%, P < 0.01). No significant change of phasic activity was evidenced with either solution. We conclude that 1) bile acids and triglycerides not absorbed in the more proximal gut could be involved in the ileal relaxation occurring after eating and 2) local stimulation of chemoreceptors is of importance in the regulation of ileal motility.
Gastric mucus of the guinea pig: proton carrier and diffusion barrier
Authors: Schreiber S. , Scheid P.
Published: December 2017

Proton transport with the gastric mucus was investigated in the guinea pig in vitro by use of three experimental series. In series I, pH profiles were obtained in the mucus and mucosa of a gastric explant with fine-tipped double-barreled microelectrodes. With a luminal pH of 1.8, pH increased across this layer to approximately 6 at the epithelial surface. Thickness of the gastric mucous gel layer increased continuously by 170 +/- 100 microns/h in the unstimulated and by 450 +/- 120 microns/h in the histamine-stimulated preparation (means +/- SD). In series II, fresh guinea pig gastric mucus was obtained from the gastric mucosa and titrated at 10 degrees C from pH 6.5 to 0.7, followed by an incubation period of 30 min at 37 degrees C. During this incubation period, a spontaneous acidic shift was observed, corresponding to a proton release from the mucus of 130 +/- 19 mM. This proton release could be blocked by the pepsin inhibitor pepstatin and was not observed when titrating down to only pH 3. Buffer values calculated as the mean slope of the titration curves in the pH range of 7 to 3 averaged 40 mM/pH unit. In series III, when titration was repeated with purified porcine mucin, no proton release was observed during incubation at pH 1.0, unless pepsinogen (375 U/ml) had been added before titration. Proton release during incubation at pH 1.0 and 37 degrees C in the presence of pepsinogen averaged 50 mM. The data suggest that protons secreted by the gastric mucosa are buffered by the continuously secreted mucus and transported, bound to the proteins of the mucus, toward the gastric lumen. During this transport, pepsinogen is converted within the mucus to pepsin. Pepsin modifies the buffering properties of the mucus, whereby protons are released from the protein binding. Thus the mucus forms a vehicle for proton transport toward the gastric lumen while, at the same time, constituting a diffusion barrier to prevent proton backdiffusion toward the gastric epithelium.
Protein tyrosine kinase activity regulates nitric oxide synthase induction in rat hepatocytes
Authors: Miller D. R. , Collier J. M. , Billings R. E.
Published: December 2017

Regulation of induced nitric oxide synthase (NOS) in isolated rat hepatocytes is poorly understood. The specific protein tyrosine kinase inhibitor genistein was used to determine if NOS induction is dependent on protein tyrosine kinase activation. Genistein inhibited tumor necrosis factor-alpha (TNF-alpha)-stimulated induction of NOS activity and NOS protein in a dose-dependent manner. Genistein also impaired TNF-alpha-induced NOS mRNA accumulation, suggesting protein tyrosine kinase regulation of NOS induction occurred at the level of transcription-translation. Like TNF-alpha, genistein inhibited induction of NOS protein by a second proinflammatory cytokine, interleukin-1beta, suggesting similar activation mechanisms by proinflammatory cytokines. NOS induction by other stimuli, including phorbol 12-myristate 13-acetate and the superoxide-generating system xanthine/xanthine oxidase, was also inhibited by genistein. Finally, cytokine-stimulated protein tyrosine kinase activity in hepatocytes was demonstrated by increased tyrosine phosphorylation of five high molecular mass protein bands. Genistein inhibited this cytokine-induced phosphotyrosine increase. The commonality of genistein inhibition suggests that protein tyrosine kinase activity is critical for NOS induction by a variety of stimuli.
Lack of evidence for vesicle trafficking of fluorescent bile salts in rat hepatocyte couplets
Authors: El-Seaidy A. Z. , Mills C. O. , Elias E. , Crawford J. M.
Published: December 2017

The role of intracellular vesicles in the movement of bile salts through hepatocytes from blood to bile has not been resolved. To determine whether bile salts are sequestered during transit, rat hepatocyte couplets were incubated with the fluorescent bile salts cholyl-lysyl-fluorescein (CLF) and chenodeoxycholyl-lysyl-fluorescein (CDCLF). Cellular and canalicular fluorescence were measured by confocal scanning fluorescence microscopy; inhomogeneity in intracellular fluorescence was used to evaluate potential sequestering of bile salts. Mean cellular and canalicular fluorescence increased in parallel over 10 min, slightly exceeding (P < 0.05) the degree of increase in intracellular inhomogeneity. The microtubule inhibitor colchicine had no effect on cellular or canalicular fluorescence patterns. In contrast, the nonfluorescent bile salt taurocholate enhanced the recovery of microtubules from cold-induced depolymerization, measured by confocal immunofluorescence of beta-tubulin. Thus no evidence was obtained for intracellular sequestering of bile salts or microtubule-dependent trafficking before canalicular secretion; cellular uptake and distribution occurred in parallel with canalicular secretion. The previously documented dependence of bile salt secretion on intact microtubule function therefore appears to be an indirect rather than a direct consequence of microtubule-dependent events. In particular, enhanced microtubule assembly may play a role in bile salt-induced delivery of bile salt transporters to the canalicular membrane.
Central inhibition of nitric oxide synthase modulates upper gastrointestinal motor activity
Authors: Ohta D. , Lee C. W. , Sarna S. K. , Condon R. E. , Lang I. M.
Published: December 2017

We investigated the role of central nitric oxide (NO) synthase inhibition in the modulation of fasting gastrointestinal motor activity and gastric emptying rate of solid nutrient meals in conscious dogs. N(omega)-nitro-L-arginine methyl ester and N(omega)-nitro-L-arginine were infused intracerebroventricularly. Circular muscle contractions were recorded with surgically implanted strain gauge transducers. Gastric emptying was measured by a radiolabeled meal. The inhibition of NO synthase in the brain suppressed phase III activity in the stomach and the duodenum but not in the jejunum or the ileum. However, the central inhibition of NO synthase had no significant effect on the rate of gastric emptying of solid nutrient meals or the duration of postprandial disruption of migrating motor complex cycling. The central actions of NO synthase inhibition were blocked by bilateral truncal vagotomy but not by intravenous administration of propranolol or phentolamine. We conclude that the inhibition of NO synthase in the brain generates a stimulus that selectively inhibits gastric and duodenal phase III activities. This stimulus, however, may not affect the postprandial gastroduodenal motor activity or the rate of gastric emptying of solid nutrient meals.
Potentiation to vasodilators by nitric oxide synthase blockade in superior mesenteric but not hepatic artery
Authors: Macedo M. P. , Lautt W. W.
Published: December 2017

Our objective was to determine the vasodilator effect of adenosine and isoproterenol on the hepatic artery (HA) and superior mesenteric artery (SMA) before and after blockade of nitric oxide (NO) production to evaluate the possibility of organ specificity. Vascular circuits supplied blood flow to the liver or intestine in cats under pentobarbital sodium anesthesia. The NO synthase (NOS) antagonist N(G)-nitro-L-arginine methyl ester (L-NAME; 2.5 mg/kg iv) increased arterial pressure from 106.4 +/- 7.6 to 141.4 +/- 8.1 mmHg and raised basal vascular tone in the SMA but not in the HA. The NOS substrate L-arginine (75 mg/kg) reversed these effects. The decrease in perfusion pressure in response to adenosine was 51.7 +/- 2.9, 135.2 +/- 6.1, and 16.7 +/- 2.4 mmHg, respectively, for control and after L-NAME and L-arginine. Isoproterenol was also potentiated in the SMA. Adenosine and isoproterenol were not potentiated in the HA by L-NAME. Potentiation did not occur when HA or SMA basal tone was elevated by norepinephrine. In conclusion, L-NAME increased basal tone for the SMA and potentiated the dilation induced by adenosine and isoproterenol in the SMA but not in the HA. This study provides evidence that there is a highly organ-specific compensatory mechanism in which the absence of NO promotes potentiation of other vasodilators.