bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression and processing of human preprogastrin in murine medullary thyroid carcinoma cells
Authors: Daugherty D. F. , Dickinson C. J. , Takeuchi T. , Bachwich D. , Yamada T.
Published: December 2017

Gastrin, the primary hormonal mediator of postprandial gastric acid secretion, is produced from its precursor progastrin by a series of posttranslational processing reactions including dibasic residue cleavage and carboxyl-terminal alpha-amidation. Progastrin contains three dibasic cleavage signals, Arg57Arg58, Lys74Lys75, and Arg94Arg95, that appear to be cleaved differently in different tissues. Differential processing is a potential means by which the production of biologically active peptides may be regulated in a tissue-specific manner. To study these reactions further, we used the pZipNeo SV(X) retroviral vector to express human gastrin cDNA in a heterologous cell line (MTC 6-23) known to be capable of processing other peptide precursors. The psi 2 packaging cell line transfected with the gastrin cDNA-retroviral construct (pSVXgas) produced progastrin, but no substantial amounts of processed amidated gastrin were detected. amounts of processed amidated gastrin were detected. In contrast, MTC 6-23 cells infected with the viral stock obtained from the supernatant of pSVXgas-transfected psi 2 cells produced carboxyl-terminally amidated gastrin in all of its standard molecular forms, including sulfated and nonsulfated forms of tetratriacontagastrin (G-34), heptadecagastrin (G-17), and tetradecagastrin (G-14). These studies indicate that heterologous endocrine cell lines infected with a retroviral-peptide cDNA construct can serve as useful models for peptide hormone posttranslational processing.
Distinct receptors for epidermal growth factor-urogastrone in cultured gastric smooth muscle cells
Authors: Yang S. G. , Hollenberg M. D.
Published: December 2017

We describe a propagable cell strain from guinea pig gastric circular muscle (GCM), which we have characterized in terms of its smooth muscle phenotype and its binding and biological response (thymidine incorporation) to epidermal growth factor-urogastrone (EGF-URO) and transforming growth factor-alpha (TGF-alpha). The binding of 125I-labeled EGF-URO to the GCM cells exhibited high affinity and an appropriate peptide specificity. A curvilinear Scatchard plot of the binding data indicated two classes of high-affinity binding sites (dissociation constants of 0.69 and 4.3 nM) and a maximal binding capacity of 24,000 sites/cell. Binding competition data demonstrated that the binding affinity of TGF-alpha was greater than that of EGF-URO by a factor of 2. These relative binding affinities agreed with the two- to threefold greater potency of TGF-alpha, compared with EGF-URO, for the stimulation of GCM thymidine incorporation. The relative order of binding affinity and biological potency (TGF-alpha greater than EGF-URO) was distinct from the relative order of binding affinities (EGF-URO greater than TGF-alpha) observed using guinea pig liver and human placental membrane preparations. We conclude that the cultured smooth muscle-derived GCM cells possess a receptor subtype that is in accord with contractile bioassay data obtained previously with intact gastric circular muscle strips. This receptor (TGF-alpha greater than EGF-URO) appears distinct from the one previously characterized in nonmuscle tissues.
Inhibition of muscle cell relaxation by somatostatin: tissue-specific, cAMP-dependent, pertussis toxin-sensitive
Authors: McHenry L. , Murthy K. S. , Grider J. R. , Makhlouf G. M.
Published: December 2017

The direct action of somatostatin on smooth muscle was examined in muscle cells isolated from the stomach and intestine of human and guinea pig. Somatostatin inhibited relaxation in gastric but not intestinal muscle cells of the two species, and its mechanism of action was explored in more detail in gastric muscle cells of the guinea pig. Somatostatin inhibited relaxation induced by vasoactive intestinal peptide (VIP, 83 +/- 7%, P less than 0.001) and isoproterenol (85 +/- 5%, P less than 0.001), as well as the concomitant increase in adenosine 3',5'-cyclic monophosphate (cAMP) production [81 +/- 25% inhibition with VIP (P less than 0.02) and 68 +/- 12% inhibition with isoproterenol (P less than 0.01)]. Inhibition of relaxation and cAMP production was abolished by pretreatment of the cells with pertussis toxin. Relaxation induced by the permeant derivative of cAMP, N6,2'-O-dibutyryladenosine 3',5'-cyclic monophosphate, by sodium nitroprusside, which acts by increasing levels of guanosine 3',5'-cyclic monophosphate, or by ATP, which acts by opening of K+ channels, was not affected by somatostatin. The fact that inhibition by somatostatin and its reversal by pertussis toxin was confined to agonists that stimulate an increase in the levels of cAMP implied that somatostatin acts by inhibiting the generation and not the action of cAMP. It is concluded that somatostatin receptors on gastric muscle cells mediate inhibition via a GTP-binding, pertussis-sensitive regulatory protein, Gi, coupled to adenylate cyclase.
Binding, internalization, and processing of bombesin by rat pancreatic acini
Authors: Zhu W. Y. , Goke B. , Williams J. A.
Published: December 2017

The binding and subsequent fate of 125I-labeled bombesin was studied in rat pancreatic acini. At both 37 degrees C and 4 degrees C binding occurred to a single saturable site, with Kd equal to 1.7 nM. The amount of steady-state tracer binding was reduced at 4 degrees C (5.7%/mg protein) compared with 37 degrees C (8.7%/mg), with a similar relative change in calculated binding capacity. With the use of an acid wash procedure to remove surface bound ligand, 55% of cell-associated 125I-bombesin was internalized in the steady state at 37 degrees C but only 5% at 4 degrees C. Preincubation at 4 degrees C followed by an increase to 37 degrees C led to rapid internalization of bound bombesin, which was blocked by the metabolic inhibitor antimycin. 125I-bombesin was found to be degraded by two acinar systems. One was not related to receptor binding but could be inhibited with bacitracin. The other occurred after internalization and was partially blocked with chloroquine. Thus, after binding, bombesin is internalized, and the degradation products are released from the cell. Exposure to bombesin is also accompanied by a subsequent decrease in cell surface binding (54% after 1 h exposure to 100 nM bombesin), suggesting that the bombesin receptor may also be internalized in a ligand-dependent manner.
A novel transcellular transport pathway for non-bile salt cholephilic organic anions
Authors: Aoyama N. , Tokumo H. , Ohya T. , Chandler K. , Holzbach R. T.
Published: December 2017

Non-bile salt cholephilic organic anions comprise a single class of nonhomologous ligands having a range of hydrophobicity. Hydrophobicity enhances the hepatic extraction of cholephiles as well as their partitioning into secreted biliary lipid particles. When hydrophobicity is correlated with patterns of biliary excretion for studying transcellular transport, however, the more hydrophobic probes are unsuitable. Specifically, with the isolated perfused rat liver technique, the excretory times for sulfobromophthalein and rose bengal were significantly longer compared with that for the much more hydrophilic analogue phenol red (PR), which showed only a single, nearly symmetrical excretory peak at 10 min. Colchicine affected the apparently well-defined PR pathway only at a saturation dose (10,000 times the tracer dose). In contrast, the effect of a different perturbant, monensin, was striking at a tracer dose of PR, but was less evident at a saturation dose. The combined administration of colchicine and monensin had no additive inhibitory effect on PR excretion at tracer doses. At a saturation dose of PR, where monensin is less inhibiting, however, a significant additive inhibitory effect was observed.(ABSTRACT TRUNCATED AT 250 WORDS)
O2-. release by activated Kupffer cells upon hypoxia-reoxygenation
Authors: Rymsa B. , Wang J. F. , de Groot H.
Published: December 2017

Primary cultures of rat liver Kupffer cells generated large amounts of superoxide anion radical (O2-.) when subjected to reoxygenation after a hypoxic period of at least 2 h. O2-. formation reached its maximum rate of approximately 25 nmol/10(6) cells within 1 h after reoxygenation. Two to four hours after reoxygenation, the number of injured cells began to increase and after 10 h approximately 60% of the cells were dead. During the period of O2-. release no significant difference in cell viability was observed between reoxygenated and hypoxically incubated cells, indicating a distinct time lag between O2-. release and onset of cell damage. Addition of diphenyliodonium, a specific inhibitor of the neutrophilic NADPH oxidase, to the Kupffer cells just before reoxygenation diminished both O2-. formation and cell injury up to 70%. Reoxygenation injury was completely prevented when superoxide dismutase and catalase were added immediately before reoxygenation. The results indicate that Kupffer cells subjected to hypoxia-reoxygenation generate a burst of reactive oxygen species and that this kind of "activation," probably by activating the NADPH oxidase, contributes to the self-destruction of the cells.
Dipeptidyl peptidase IV expression in rat jejunal crypt-villus axis is controlled at mRNA level
Authors: Darmoul D. , Rouyer-Fessard C. , Blais A. , Voisin T. , Sapin C. , Baricault L. , Cibert C. , Geraud G. , Couvineau A. , Laburthe M. , et al.
Published: December 2017

The expression of dipeptidyl peptidase IV (DPP IV), an intestinal brush-border hydrolase, has been studied in the rat jejunum crypt-villus axis. DPP IV enzyme activity is lower in crypt cells than in villus cells. Indirect immunofluorescence studies, using a polyclonal antibody raised against purified DPP IV, show a gradient of immunoreactivity from the crypts to the villi that was quantified using confocal laser scanning microscopy. Accordingly, Western blot analysis demonstrates that the steady-state amount of DPP IV is much lower in crypt cells than in villus cells. Northern blot analysis was performed using our cDNA probe for human DPP IV that presents more than 94% homology with rat DPP IV cDNA. Results clearly show that there is approximately seven times less DPP IV mRNA in crypt cells than in villus cells. We conclude that the differentiation-dependent expression of DPP IV in rat jejunum is primarily controlled at the mRNA level.
Hepatic uptake of protein-bound ligands: extended sinusoidal perfusion model
Authors: Weisiger R. A. , Pond S. , Bass L.
Published: December 2017

Traditional transport models have failed to account for uptake of many protein-bound ligands by liver and other tissues when the concentration of plasma albumin or other binding protein is varied. In this paper, we extend the standard sinusoidal perfusion model to include the effects of slow dissociation of the ligand from albumin and of diffusion across extracellular barriers such as the unstirred layer. We then use experimental data for uptake of oleate from albumin solutions (1:10 molar ratio) by perfused female rat livers to test this model. Unlike the standard model, the extended model closely conformed to observed uptake rates over a wide concentration range (0.015-0.45 mM albumin). The extension, which is conceptually simple, is based on widely accepted physiological principles. It requires only the introduction of two dimensionless ratios into the standard model: the ratio of the mean unbound ligand concentration actually present within the capillary or sinusoid to its equilibrium value, and the ratio of the permeability of the membrane plus associated extracellular diffusion barriers to the permeability of the membrane alone. The resulting model simplifies to the standard sinusoidal perfusion model when both ratios approximate unity. We first develop the model for steady influx alone because our data suggest that little efflux of oleate occurred over the time course of the current study. In an appendix, we extend the model to include the effects of efflux and metabolism. The new model offers an alternative for explaining uptake kinetics of protein-bound ligands that cannot be explained by less complete traditional models.
Bacterial deconjugation and enterohepatic circulation of norursocholic acid conjugates in rats
Authors: Lillienau J. , Borgstrom B.
Published: December 2017

Experiments were performed to define the metabolism of norusocholic acid (nUC) conjugates and to quantify to what extent the bile acid pool can be enriched in these bile acids. In vitro incubations of norusocholylglycine (nUCG) and -taurine (nUCT) with small intestinal or cecal content showed deconjugation with only cecal content. Cholylglycine (CG) was deconjugated by small intestinal and cecal content. Infusion of nUCG and CG showed that only a small proportion of nUCG was deconjugated after 24 h of enterohepatic circulation, whereas all CG was deconjugated. When nUCT was administered orally, deconjugation was shown to take place mainly in the cecum. Chronic feeding of nUCT enriched the bile acid pool with only 20% nUCT. We conclude that nUC conjugates are deconjugated primarily by bacteria in the cecum and colon, in contrast to CG, which, in addition to cecum and colon, is deconjugated in the distal small intestine. nUCT and its metabolites do not enrich in the circulating bile acid pool mainly for the following reasons: 1) nUC conjugates have a low affinity for the ileal transport system; 2) nUC, even if formed by deconjugation, is not passively absorbed at a sufficient rate; 3) the small amount of norursodeoxycholic acid formed from nUC is glucuronidated in the liver and glucuronide conjugates do not undergo enterohepatic circulation; and 4) nUC conjugates do not suppress bile acid biosynthesis.
Antioxidant defenses of cultured gastric cells against oxygen metabolites: role of GSH redox cycle and endogenous catalase
Authors: Hiraishi H. , Terano A. , Ota S. , Mutoh H. , Sugimoto T. , Razandi M. , Ivey K. J.
Published: December 2017

Gastric mucous epithelial cells may represent a first line of defense against reactive oxygen species that are generated within the gastric lumen. However, little is known about their defenses against oxidant species. This study examined the importance of the glutathione (GSH) redox cycle and of endogenous catalase as antioxidant defenses in cultured gastric mucous cells. Cultured rat gastric mucous cells were exposed to H2O2 generated by glucose oxidase acting on glucose or to nascent H2O2 for 5 h. Cytotoxicity was quantified by measuring 51Cr release from prelabeled cells. The effects of inhibition of the GSH redox cycle and of endogenous catalase were examined. Glucose oxidase caused a dose-dependent increase of 51Cr release. Similarly, nascent H2O2 damaged the cells dose dependently. Pretreatment with 1,3-bis(chloroethyl)-1-nitrourea (inhibitor of GSH reductase) dose dependently increased glucose oxidase-induced 51Cr release. Preincubation with buthionine sulfoximine (inhibitor of gamma-glutamyl-cysteine synthetase), which lowered intracellular GSH content, enhanced glucose oxidase-induced damage in a dose-dependent manner. Pretreatment with diethyl maleate, which covalently binds GSH as catalyzed by GSH transferase, also enhanced the sensitivity to lysis by glucose oxidase. However, inhibition of endogenous catalase activity by 3-amino-1,2,4-triazole did not significantly alter glucose oxidase- or nascent H2O2-induced 51Cr release. These results suggest that the GSH redox cycle rather than endogenous catalase plays a critical role in intracellular antioxidant defense in cultured gastric mucous cells.
Identification of longitudinal muscle activity in opossum lower esophageal sphincter
Authors: Harrington S. S. , Dodds W. J. , Mittal R. K.
Published: December 2017

The aim of this study was to characterize lower esophageal sphincter (LES) longitudinal muscle contraction during changes in LES pressure in opossums. LES pressure was used as a marker of LES circular muscle contraction. Longitudinal muscle contraction was measured by strain gauge transducers. For precise placement of the strain gauge on the LES, the high-pressure zone was identified using a manometric-laser illumination technique. Measurements were made during 1) spontaneous LES pressure fluctuations, 2) LES pressure changes induced by pharmacological agents, and 3) LES relaxation induced by vagal nerve stimulation. These studies show that 1) spontaneous LES pressure fluctuations correlated with spontaneous changes in LES longitudinal muscle contraction; 2) pharmacologically induced contraction and relaxation of LES pressure correlated with contraction and relaxation of LES longitudinal muscle, respectively; 3) a close temporal relationship existed between changes in LES longitudinal muscle contraction and LES pressure that occurred spontaneously or were induced pharmacologically; and 4) vagal stimulation caused LES longitudinal muscle contraction and LES pressure relaxation. We propose that LES longitudinal muscle may play a role in the modulation of intraluminal LES pressure.
Release of cholecystokinin and secretin by sodium oleate in dogs: molecular form and bioactivity
Authors: Sun G. , Chang T. M. , Xue W. J. , Wey J. F. , Lee K. Y. , Chey W. Y.
Published: December 2017

The release of cholecystokinin (CCK) and secretin into both circulation and duodenal lumen, after intraduodenal perfusion with sodium oleate or oral ingestion of fat, was studied in anesthetized and conscious dogs, respectively. Intraduodenal infusion with sodium oleate (4 mmol.kg.-1.h-1, pH 9.5) in anesthetized dogs with diversion of bile and pancreatic juice stimulated the release of both CCK and secretin not only into the circulation but also into the duodenal lumen. The concentration of CCK and secretin in the luminal perfusate increased from 0.2 +/- 0.1 to 2.1 +/- 0.4 nM and 0.34 +/- 0.16 to 2.59 +/- 0.63 nM, respectively. Intraduodenal infusion of NaHCO3 solution at pH 9.5 did not result in release of either hormone. Luminal release of both hormones was also observed by intraduodenal infusion of sodium oleate in the dogs without diversion of bile and pancreatic juice, albeit at lower concentrations than those released in the dogs with diversion. Analysis of the molecular form of luminal secretin by gel filtration, ion-exchange chromatography, and high-performance liquid chromatography showed only a single form of secretin with molecular size, hydrophobicity, and charge similar to those of natural porcine secretin. In contrast, multiple forms of CCK were released into both circulation and duodenal lumen with CCK-58 as the predominant form. In conscious dogs, CCK-58 was also found to be the predominant form of CCK released into the circulation after oral ingestion of fat.(ABSTRACT TRUNCATED AT 250 WORDS)
Both crypt and villus intestinal goblet cells secrete mucin in response to cholinergic stimulation
Authors: Phillips T. E.
Published: December 2017

Computer-assisted morphometric analysis was used to quantify the effects of cholinergic stimulation on intestinal goblet cells. Within 5 min of stimulation (250 micrograms/kg carbachol sc), many crypt goblet cells were depleted of mucin secretory granules and their apical membranes had the deep cavitation that accompanies recent compound exocytotic activity. The percentage of crypt epithelial volume occupied by mucin secretory granules was decreased by 58.4% at 5 min and 45.9% at 60 min. Although villus goblet cells never showed signs of recent compound exocytosis, morphometric analysis revealed a 22.4% decrease in the percentage of villus epithelial volume occupied by mucin secretory granules within 5 min of stimulation and a 32.4% decrease by 60 min. The decrease in villus mucin stores was due to both a reduction in the volume of mucin in an average villus goblet cell and a drop in the number of recognizable goblet cells per square micrometer of villus epithelium. Mucin stores in both the crypt and villus regions were largely replenished by 4 h poststimulation.
Taurine transport in skate hepatocytes. II. Volume activation, energy, and sulfhydryl dependence
Authors: Ballatori N. , Boyer J. L.
Published: December 2017

Isolated skate (Raja erinacea) hepatocytes swollen in hypotonic media exhibited a regulatory volume decrease (RVD) that was associated with only a small increase in K+ or 86Rb+ efflux but a substantial increase in the release of taurine, an amino acid found in high concentrations in skate hepatocytes. Taurine efflux was stimulated in media made hypotonic by addition of H2O or removal of NaCl, as well as in cells swollen in isotonic media containing rapidly penetrating solutes (202 mM ethylene glycol or 202 mM additional urea substituted for 101 mM NaCl), suggesting that cell swelling rather than hyposmolarity is the stimulus for the activation of taurine release. In contrast, release of glutathione, L-[14C]alanine and other alpha-amino acids (e.g., threonine, serine, glutamate, glutamine, glycine, or valine) was unaffected by dilution with 40% H2O. Taurine efflux was not altered by replacement of extracellular Na+ with choline+ or K+ and was only slightly diminished by replacing Cl- with NO3-. Addition of 50 mM taurine or hypotaurine to the incubation media also had no effect on volume-stimulated [14C]taurine efflux, suggesting that the taurine concentration gradient across the plasma membrane is not the driving force. Volume-stimulated taurine transport was temperature sensitive, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid inhibitable (0.5 mM), and nearly completely blocked by metabolic inhibitors (2,4-dinitrophenol, KCN, sodium azide, oligomycin, carbonyl cyanide m-chlorophenylhydrazone, and antimycin A), suggesting an active energy-dependent process. Sulfhydryl-reactive reagents (N-ethylmaleimide, diamide, iodoacetate, tert-butyl hydroperoxide, and mercury) also blocked volume-stimulated taurine efflux, whereas efflux was unaffected by Ca2+ ionophore, phorbol ester, dibutyryl-adenosine 3',5'-cyclic monophosphate, vasopressin, or pretreatment with ouabain or furosemide. N-ethylmaleimide, diamide, 2,4-dinitrophenol, and iodoacetate plus KCN also inhibited the RVD. These findings suggest that, in contrast to hepatocytes from most vertebrate species, RVD in skate hepatocytes is associated with the release of only a small fraction of intracellular K+ but a substantial fraction of intracellular taurine and perhaps other organic osmolytes. This volume-activated taurine transport mechanism is energy and sulfhydryl group dependent and is not related to the taurine concentration gradient across the skate hepatocyte plasma membrane.
Distribution of mast cells in intestinal muscle of nematode-sensitized rats
Authors: Marzio L. , Blennerhassett P. , Vermillion D. , Chiverton S. , Collins S.
Published: December 2017

We examined the distribution and functional integrity of mast cells in intestinal longitudinal muscle in rats sensitized by two previous infections with Trichinella spiralis. A segment of jejunum was excluded from the gut before infection, and the remainder of the gut was anastomosed. Few mast cells were seen in muscle of noninfected control rats except in the region of the jejunal anastomosis. In rats sensitized by T. spiralis infection, mast cells were increased in number in the jejunum and the number of mast cells followed an aboral gradient down the entire length of the gut in continuity. In addition, mast cells were present in muscle of the excluded segment of sensitized rats. All mast cells were stained red with safranin. Functional integrity was assessed by the ability of mast cells to induce contraction after degranulation by antigen. In muscle from sensitized rats, contraction was induced in each region after exposure to T. spiralis antigen but not Nippostrongylus brasiliensis antigen. Contraction was inhibited by the mast cell stabilizer doxantrazole and the 5-hydroxytryptamine (5-HT) antagonist cyproheptadine. When antigen-induced contraction was expressed as a percentage of the maximum response of the tissue to exogenous 5-HT, the magnitude of contraction decreased along an aboral gradient down the intestine and correlated well (r2 = 0.878) with mast cell numbers. These results suggest that the increase in connective tissue mast cells in gut muscle after T. spiralis infection involves both local and systemic mechanisms.
Gastric microvascular actions of platelet-activating factor: role of leukocytes
Authors: Wood J. G. , Yan Z. Y. , Cheung L. Y.
Published: December 2017

This study examined the effects of platelet-activating factor (PAF) on the gastric microcirculation. We measured changes in vascular resistance and filtration during intra-arterial infusion of graded doses of PAF and its metabolite, lyso-PAF, to an ex vivo gastric segment of alpha-chloralose-anesthetized dogs. PAF produced dose-related sustained increases in vascular resistance (2-150 nM; n = 6). Filtration and venous hematocrit were also both significantly increased by PAF. In contrast, there were no statistically significant changes in these measurements with lyso-PAF (n = 6). Filtration and venous hematocrit were not significantly changed by norepinephrine at doses that increased perfusion pressure to the same degree as PAF (n = 4). PAF also increased filtration in the absence of changes in perfusion pressure (during maximal vasodilation induced with papaverine). Finally, removal of leukocytes from gastric arterial blood significantly attenuated these responses to PAF. Our results suggest that PAF-induced mucosal ischemia is primarily due to vasoconstriction and may involve edema formation due to increased filtration as well. In addition, these responses to PAF appear to be largely dependent on circulating leukocytes.
Effect of intravascular ethanol on modulation of gastric mucosal integrity: possible role of endothelin-1
Authors: Masuda E. , Kawano S. , Nagano K. , Tsuji S. , Ishigami Y. , Tsujii M. , Hayashi N. , Fusamoto H. , Kamada T.
Published: December 2017

The influences of intravascular ethanol on gastric mucosal integrity and its relation to gastric circulation were investigated in rats. Ulcer formation of the gastric mucosa correlated with the blood ethanol concentration in the presence of 150 mM HCl in the rat stomach. Furthermore, the gastric mucosal hemoglobin concentration (IHb) and hemoglobin oxygen saturation (ISO2), estimated using organ reflectance spectrophotometry, decreased in a concentration-dependent manner with blood ethanol. In isolated, vascularly perfused rabbit stomach, various concentrations (10-400 mM) of ethanol infused into the celiac artery increased the perfusion pressure and released endothelin-1 (ET-1) from the gastric vasculature in a concentration-dependent manner. Moreover, a significant correlation existed between changes in the perfusion pressure and ET-1 concentration in effluents from the gastric vasculature. Furthermore, anti-ET-1 antibody reduced 100 mM ethanol-induced vasoconstriction in a concentration-dependent manner. The results indicate that intravascular ethanol increases the susceptibility of gastric mucosa to injury induced by intraluminal HCl by causing gastric vasoconstriction mediated by ET-1. Thus intravascular ethanol may play an important role in the mechanism of ethanol-induced gastric mucosal injury.
Role of calcium in monitor peptide-stimulated cholecystokinin release from perifused intestinal cells
Authors: Bouras E. P. , Misukonis M. A. , Liddle R. A.
Published: December 2017

Monitor peptide stimulates cholecystokinin (CCK) release from the intestine, but the cellular mechanisms responsible for this effect are uncertain. In the present study, the roles of membrane potential difference and calcium influx in monitor peptide-mediated CCK release were examined in a perifusion system containing isolated mucosal cells from the rat duodenum. This method represents an in vitro system in which CCK-releasing cells can be challenged with secretagogues or other maneuvers to study the dynamics of hormone secretion. High concentrations of KCl (50 mM), which reduce electrical potential difference across the cell membrane, caused the release of CCK. This effect was inhibited by the calcium channel blocker MnCl2. Monitor peptide stimulated CCK release in a dose-dependent manner at concentrations from 3 x 10(-12) to 3 x 10(-8) M. The requirement for extracellular calcium in secretagogue-stimulated release of CCK was investigated using ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), a calcium chelator, and MnCl2. A calcium-free environment supplemented with 2 mM EGTA completely inhibited CCK secretion in response to stimulatory doses of monitor peptide. CCK secretion was restored when calcium was reintroduced into the system. Similarly, MnCl2 completely blocked monitor peptide-stimulated CCK release. These data indicate that membrane depolarization and monitor peptide stimulate the release of CCK through calcium-dependent mechanisms, suggesting that increases in intracellular calcium within CCK cells are likely to be important in CCK release.
Macromolecular transport by rat gastric mucosa
Authors: Curtis G. H. , Gall D. G.
Published: December 2017

We previously demonstrated that the stomach is capable of mounting a type I hypersensitivity reaction to luminal antigen challenge. These findings imply that antigenically intact macromolecules cross the gastric mucosa. To test this hypothesis, rat gastric mucosa was mounted in Ussing chambers, and bovine serum albumin (BSA, 0.5 mg/ml) and 125I-labeled BSA (10 microCi) were added to mucosal fluids. After equilibration, serosal fluids were sampled for two 30-min periods, and fluxes of immunologically intact BSA (determined by an enzyme-linked immunosorbent assay) and total BSA (125I-BSA) were calculated under basal conditions and in the presence of NaF and colchicine, and at 4 degrees C. Additional experiments examined macromolecular permeability in sensitized-challenged tissues. Immunologically intact BSA (21.3 +/- 4.5 ng.30 min-1.cm-2) crossed the gastric mucosa as approximately one-fourth of the total BSA flux (78.2 +/- 7.5 ng.30 min-1.cm-2). The uptake of immunologically intact BSA was significantly reduced by NaF, an inhibitor of ATP production and endocytosis; colchicine, which inhibits polymerization of cytoskeletal microtubules; and at 4 degrees C, a general metabolic inhibitor. The transmural passage of antigen was not significantly altered by immunoglobulin E-mediated anaphylaxis. These findings indicate that intact protein antigens cross the gastric mucosa by an active, energy-dependent mechanism that uses the microtubular network.
In vitro hyporeactivity to methoxamine in portal hypertensive rats: reversal by nitric oxide blockade
Authors: Sieber C. C. , Groszmann R. J.
Published: December 2017

The endothelial cell plays an important role in the local control of vascular smooth muscle tone. Portal hypertension is accompanied by systemic vasodilatation and a decreased response to vasoconstrictors, changes especially evident in the superior mesenteric arterial bed. To evaluate a possible effect of the locally released endothelium-derived relaxing factor nitric oxide (NO), we tested the effect of NO blockade in in vitro perfused superior mesenteric arterial beds of normal (sham) and portal hypertensive (PVL) rats, induced by partial portal vein ligation. A significant (n = 7/group; P = 0.02) hyporeactivity to the vasoconstrictive properties of the alpha-adrenoceptor agonist methoxamine (3 x 10(-6) to 3 x 10(-4) M) was prevented by blocking NO formation in PVL compared with sham rats, using the stereospecific biosynthesis antagonist N omega-nitro-L-arginine (10(-4) M, n = 7/group; NS for all methoxamine concentrations tested). This effect was reversed by the NO precursor L-arginine (10(-3) M, n = 5/group). In conclusion, these in vitro results in mesenteric vessels demonstrate that 1) portal hypertension is accompanied by a hyporeactivity to the vasopressor methoxamine and 2) locally released NO in this preparation is responsible for the decreased vasoconstrictive response.
Duodenal intramural nerves in control of pyloric motility and gastric emptying
Authors: Treacy P. J. , Jamieson G. G. , Dent J. , Devitt P. G. , Heddle R.
Published: December 2017

We have investigated the role of ascending duodenal intramural nerves in the control of isolated pyloric pressure waves (IPPW) and liquid gastric emptying. In six pigs, the proximal duodenum was transected to interrupt intramural nerves. A further six pigs had a sham operation. Four weeks after operation, motility of the antrum, pylorus, and duodenum was recorded by side holes and a sleeve sensor. Gastric emptying of a 1,000-ml test meal was significantly more rapid in duodenum-transected animals (P less than 0.0001) during intraduodenal infusion of each of isosmolar dextrose (424 ml emptied), fatty acid (335 ml), and amino acids (396 ml) than in duodenum-intact animals (dextrose: 128 ml; fatty acid: 57 ml; amino acids: 192 ml). Associated with this, in duodenum-transected animals infusion of each of isosmolar dextrose, fatty acid, amino acids, and hyperosmolar saline failed to produce the stimulation of IPPW seen in duodenum-intact animals. In both duodenum-intact and -transected animals, supramaximal stimulation by intraduodenal infusion of 25% dextrose slowed gastric emptying and stimulated IPPW. Ascending duodenal intramural nerves are a major physiological pathway for nutrient- and osmolar-stimulated feedback control of pyloric motility and gastric emptying.
Peptidergic nerves mediate post-nerve stimulation hyperemia in rat gut
Authors: Hottenstein O. D. , Remak G. , Jacobson E. D.
Published: December 2017

Cessation of perivascular nerve stimulation (NS) elicits a transient increase in intestinal blood flow above the prestimulatory value. This enhancement of blood flow constitutes the phenomenon of post-nerve stimulation hyperemia (PSH). We investigated the involvement of peptidergic sensory nerves in intestinal PSH. In anesthetized rats the velocity of blood flowing through the anterior mesenteric artery (VBF) was measured with a pulsed Doppler velocimeter. PSH was induced by 4 min of postganglionic electrical NS (5 Hz). PSH was abolished by distal periarterial application of tetrodotoxin and intra-arterial lidocaine, which suggests a peripheral sensory nervous mechanism for PSH. The increase in conductance at peak PSH was blocked by pretreatment with the selective, primary afferent neurotoxin capsaicin administered as 1) subcutaneous injection in neonatal life, 2) topical application to periarterial nerves, or 3) injection into the jejunal lumen. In rats pretreated with reserpine, NS evoked a hyperemic response, which was blocked by capsaicin. Treatment with adenosine deaminase inhibited PSH considerably less than capsaicin, suggesting a lesser role for adenosine in PSH. Our findings support the hypothesis that postganglionic NS activates both adrenergic and peptidergic nerves and that the latter release vasodilator peptides in the gut during PSH.
Gallbladder mucosal function: studies in absorption and secretion in humans and in dog gallbladder epithelium
Authors: Igimi H. , Yamamoto F. , Lee S. P.
Published: December 2017

The gallbladder is conventionally regarded as an absorptive organ such that dilute hepatic bile is both stored and concentrated. We studied 35 patients who had recovered from a percutaneous transhepatic gallbladder drainage performed for acute cholecystitis. After an overnight fast, gallbladder bile was dark brown in color and had a wide scatter in the lipid composition. Two hours after a meal, the gallbladder bile was opalescent white in color and had the composition of an extracellular fluid. This phenomenon was uniformly observed in all 35 patients and was also consistently reproducible when five patients were repeatedly studied. We used normal dog gallbladder epithelial cell monolayers grown in culture and examined sodium flux. Control gallbladder cells absorbed sodium. When secretin (0.5-2.5 x 10(-7) M) was added, there was a prompt reversal of sodium flux, resulting in net secretion. We conclude that secretion is a physiological function of the gallbladder mucosa. After feeding, the neural and humoral factors divert stored and newly secreted bile into the duodenum and induce active de novo secretion thus producing a gallbladder bile that is opalescent white with no lipids. Our results also have important implications on the origin of the pathological "white bile," the pathogenesis and treatment of gallbladder sludge, as well as the kinetic analysis of compounds undergoing enterohepatic recirculation.
Submucosal reflexes: distension-evoked ion transport in the guinea pig distal colon
Authors: Frieling T. , Wood J. D. , Cooke H. J.
Published: December 2017

Muscle-stripped segments of distal colon from guinea pigs were mounted in modified flux chambers to determine the effect of distension on mucosal secretion. Ion secretion was monitored as changes in short-circuit current (Isc). Distending forces were pressure gradients established by controlled reduction in liquid volume of the submucosal compartment of the chamber. Volume removal for 10 s or 5 min evoked a monophasic or biphasic increase in Isc, which returned to baseline within 5-20 min. The amplitude of the response correlated with the volume removed and was reduced by bumetanide and Cl-free solutions but not by tetraethylammonium or amiloride. Tetrodotoxin and atropine also suppressed the response. Neither the nicotinic receptor antagonist mecamylamine, the 5-hydroxytryptamine3 (5-HT3) receptor antagonist ICS 205-930, or the prostaglandin synthesis inhibitor piroxicam altered the response. Addition of prostaglandin D2 to the submucosal bath significantly enhanced the response. The results suggest that distension of the colon evokes anion secretion by activation of reflex circuits with cholinergic neurons and muscarinic synapses. Prostaglandins and 5-hydroxytryptamine acting at 5-HT3 receptors appear not to be signal substances in the reflex pathway, which evokes the secretory response to distension.
Characterization of colonic circular smooth muscle cells in culture
Authors: Ennes H. S. , McRoberts J. A. , Hyman P. E. , Snape W. J.
Published: December 2017

The receptor-binding properties of isolated rabbit colonic circular smooth muscle cells in primary culture have been investigated. In intact smooth muscle, acetylcholine, acting through M2 muscarinic receptors, and vasoactive intestinal polypeptide (VIP), acting through VIP receptors, are two of the principal neurotransmitters mediating contraction and relaxation, respectively. The muscarinic receptor was present in very high levels (600,000 receptors/cell) on freshly isolated colonic smooth muscle cells as shown by binding of the muscarinic receptor antagonist N-methylscopolamine (NMS). However, NMS binding sites decreased rapidly when the cells were placed in primary culture. After 21 h in culture, specific binding of [3H]NMS decreased to 20%, and after 48 h to less than 10% that of preculture values. This loss was not associated with a change in receptor affinity, since Kd was unchanged for the receptors still present. In contrast, high-affinity VIP receptors were expressed on cultured smooth muscle cells but could not be detected on freshly isolated cells. Cultured cells responded to VIP with an increase in intracellular adenosine 3',5'-cyclic monophosphate (cAMP), indicating that the VIP receptors were functionally coupled to adenylate cyclase. Cultured cells also responded to calcitonin gene-related peptide (CGRP) and forskolin with increased production of intracellular cAMP. In contrast, neither VIP nor CGRP elicited an increase in intracellular cAMP when added to freshly isolated cells. Furthermore, freshly isolated cells had a greatly diminished response to forskolin, suggesting that the isolation procedure not only destroyed cell surface receptors for VIP and CGRP, but also damaged the cells sufficiently to decrease cellular adenylate cyclase activity.(ABSTRACT TRUNCATED AT 250 WORDS)
Development of Ca2+ homeostasis in epithelial cells from embryonic and neonatal intestine
Authors: Black B. L. , Rogers J. O.
Published: December 2017

The fluorescent probe fura-2 was used to assay Ca2+ levels in epithelial cell suspensions from embryonic and neonatal chick duodenum. Cell preparations maintained high viability, completely hydrolyzed fura-2/AM to fura-2, retained 92% of cellular fura-2 within the cytoplasmic compartment, and gave low autofluorescence values during assay. Fura-2 leakage from loaded cells occurred at all ages, but could be corrected for in subsequent calculations of cellular Ca2+. Cytoplasmic Ca2+ concentration was 76-80 nM in cells from 14- to 16-day embryonic intestine, rose significantly to 92-98 nM at 17-20 days, and reached 209 nM at 1-day post-hatch when assayed in buffers containing 1.3 mM Ca2+. The developmental rise in cytoplasmic Ca2+ was accompanied by an enhanced ability of cells to maintain a constant Ca2+ concentration at increased levels of extracellular Ca2+ and by a highly correlated rise in alkaline phosphatase (ALP) activity. Epithelial Ca2+ subsequently decreased to the "adult" value of 133-142 nM and was constant along the crypt-villus axis of neonatal intestine. These results verify that fura-2 can be used to compare baseline cytoplasmic Ca2+ values of epithelial cells from developing intestine, reveal that significant changes in Ca2+ homeostasis occur during ontogeny, and suggest that epithelial Ca2+ may modulate ALP activity during the differentiation of embryonic enterocytes.
Interleukin-1 beta acts at hypothalamic sites to inhibit gastric acid secretion in rats
Authors: Saperas E. , Yang H. , Tache Y.
Published: December 2017

It has been established that interleukin-1 beta (IL-1 beta) injected into the cerebrospinal fluid inhibits gastric acid secretion in rats. Brain sites of action of IL-1 beta were investigated in conscious rats implanted unilaterally with chronic hypothalamic cannula. Gastric acid secretion was monitored 2 h after pylorus ligation. Human recombinant IL-1 beta (10 ng) microinjected into the medial preoptic area, anterior hypothalamus, and paraventricular nucleus inhibited gastric acid secretion by 76-83%. IL-1 beta microinjected into the ventromedial hypothalamus and other hypothalamic sites outside of responsive sites had no effect. IL-1 beta inhibitory action in the medial preoptic area was dose related (0.1-10 ng), prevented by indomethacin (5 mg/kg ip), and mimicked by prostaglandin E2. These results show that IL-1 beta acts in the medial preoptic area/anterior hypothalamus and paraventricular nucleus to inhibit acid secretion in pylorus-ligated rats and that IL-1 beta action is likely to involve prostaglandin E2.
Mechanism of action of cholecystokinin octapeptide on cat lower esophageal sphincter
Authors: Salapatek A. M. , Hynna-Liepert T. , Diamant N. E.
Published: December 2017

In five cats we examined 1) the role of a cholinergic mechanism in cholecystokinin octapeptide (CCK-OP) excitation of the lower esophageal sphincter (LES) and 2) the interaction between CCK-OP-induced excitation and inhibition of the LES. Under ketamine anesthesia, LES pressure was monitored with a sleeve catheter. With CCK-OP, LES excitation was seen in four of five cats, and inhibition was seen in three of five cats. (4-Hydroxy-2-butynyl)trimethylammonium chloride (McNeil-A343) produced similar responses in the same cats but was less potent than CCK-OP. Atropine and/or hexamethonium reduced the response to CCK-OP. Pirenzepine had no effect on any CCK-OP response but reduced relaxation and enhanced excitation produced by McNeil-A343. Phentolamine increased CCK-OP-induced relaxation. In conclusion, CCK-OP can produce LES contraction through a preganglionic cholinergic mechanism involving a nicotinic synapse; however, induction of relaxation occurs predominantly at a postganglionic site involving adrenergic modulation. There is animal-to-animal variability in the balance of excitatory and inhibitory mechanisms to the LES, which determines the effect of a drug capable of activating both mechanisms.
Ontogeny of intestinal lactase: posttranslational regulation by thyroxine
Authors: Liu T. , Reisenauer A. M. , Castillo R. O.
Published: December 2017

To assess the molecular mechanisms underlying the regulation of lactase ontogeny by thyroxine (T4), we performed an in vivo study of lactase catalytic activity, synthesis, subunit structure, degradation, and enterocyte migration rates in propylthiouracil-induced hypothyroid rat pups, hypothyroid pups injected with T4, and normally weaned rats. Although lactase catalytic activity remained elevated in the hypothyroid rats and declined normally in the other two groups, lactase synthesis was constant among the groups. Lactase subunit structure was identical in normally weaned and T4-injected animals, but the 100-kDa moiety, characteristic of weaned rats, was absent in the hypothyroid pups. The turnover of lactase enzyme was more rapid in euthyroid and T4-injected rats than in hypothyroid animals (t1/2 = 17, 20, and 30 h, respectively). In addition, enterocyte migration was accelerated in the T4-injected rats and reduced in the hypothyroid group compared with controls. However, transit rate was not directly related to lactase activity. Our results suggest that T4 regulates lactase ontogeny by posttranslational mechanisms that include altered processing and increased degradation of the lactase enzyme.
Effect of increased tissue oxygen uptake on autoregulation in postnatal intestine
Authors: Nowicki P. T. , Miller C. E.
Published: December 2017

To determine whether the rate of tissue oxygen utilization affects pressure-flow autoregulation in an age-dependent fashion in postnatal swine intestine, in vitro gut loops from 3- and 35-day-old swine were subjected to a 35% step reduction in arterial pressure under control conditions, during intra-arterial infusion of 2,4-dinitrophenol (DNP), and 30 min after luminal instillation of predigested artificial swine milk. Autoregulation was quantitated by determining the effect of pressure reduction on vascular resistance, and also by calculating Gf, a flow-controlling gain factor that relates pressure and flow. DNP infusion increased oxygen uptake 77 and 58% in gut from 3- and 35-day-old swine, respectively, whereas feeding increased oxygen uptake approximately 50% in both groups. Under control conditions, arterial pressure reduction had no effect on vascular resistance in either group. During DNP infusion and 30 min after feeding, however, intestine from 35- but not from 3-day-old swine demonstrated significant vasodilation in response to pressure reduction. Gf averaged -0.06 +/- 0.11 vs. 0.21 +/- 0.08 (P < 0.05) before vs. DNP infusion, and 0.06 +/- 0.03 vs. 0.22 +/- 0.06 (P < 0.05) before vs. 30 min after feeding in intestine from 35-day-old swine; these increases in Gf indicate that the intensity of the pressure-flow response increased during experimental treatments. In contrast, Gf averaged -0.11 +/- 0.07 vs. -0.23 +/- 0.08 before vs. DNP infusion, and -0.23 +/- 0.06 vs. -0.23 +/- 0.09 before vs. 30 min after feeding in intestine from 3-day-old swine.(ABSTRACT TRUNCATED AT 250 WORDS)