bluuebunny/crossref_metadata_2025 huggingface dataset:
Cloning and expression of the large-conductance Ca(2+)-activated K+ channel from colonic smooth muscle
Authors: Vogalis F. , Vincent T. , Qureshi I. , Schmalz F. , Ward M. W. , Sanders K. M. , Horowitz B.
Published: December 2017

We have cloned cDNAs encoding the alpha- and beta-subunits of a large-conductance Ca(2+)-activated K+ channel (BK channel) from canine colonic smooth muscle (cslo-alpha and cslo-beta). Nucleotide sequence homology of cslo-alpha with mslo and dslo suggests that it is the canine homologue of these genes. The carboxy-terminal end of the protein is the most diverse between species, and we have also found alternative exons in cslo-alpha in this region. We have identified a unique splice site in the carboxy-terminal region of cslo-alpha, which we term site 5. Northern analysis demonstrates expression of both alpha- and beta-subunits in all canine vascular and visceral smooth muscles tested. Expression of alpha-1 alone and alpha + beta-subunit cRNA in Xenopus oocytes results in a Ca(2+)- and voltage-dependent conductance. The activity of alpha/beta-channels, measured as either changes in the voltage of half-maximal activation (V0.5) in open probability (NP0) or in the normalized conductance (G/Cmax), was more sensitive to [Ca2+]free than channels composed of the alpha-subunit alone. Neither alpha- nor alpha/beta-channels expressed in membrane patches of Xenopus oocytes were found to be regulated by protein kinase G.
Phospholipase A2 stimulation of rat intestinal epithelial cell (IEC-6) migration
Authors: Minami T. , Zushi S. , Shinomura Y. , Matsuzawa Y.
Published: December 2017

The effect of phospholipase A2 (PLA2) on intestinal epithelial cell migration was investigated using an in vitro wounding model of confluent monolayers of IEC-6. PLA2 (0.001-2 U/ml) enhanced IEC-6 cell migration in a dose dependent manner. Addition of 4-bromophenacyl bromide (BPB) (a PLA2 inhibitor) to PLA2 completely blocked the migration-promoting effect. However, addition of piroxicam (a cyclooxygenase inhibitor) or nordihydroguaiaretic acid (a lipoxygenase inhibitor) had no influence on the effect. Lysophosphatidylcholine (lysoPC) (0.01-5,000 ng/ml), one of the products of phosphatidylcholine by PLA2, dose-dependently enhanced IEC-6 cell migration as well. A combination of PsLA2 (1 U/ml) and lysoPC (1,000 ng/ml) had no additive effect or migration. Moreover, the migration-promoting effect of PLA2 that was blocked by BPB was recovered by lysoPC. After pretreatment of IEC-6 cells with replication-inhibiting doses of mitomycin C, the enhanced migration induced by PLA2 or lysoPC was still observed. These observations suggest that PLA2 may, independently of proliferation, enhance intestinal epithelial cell migration mainly via lysoPC.
Intracerebral adrenoceptor agonists influence rat duodenal mucosal bicarbonate secretion
Authors: Larson G. M. , Jedstedt G. , Nylander O. , Flemstrom G.
Published: December 2017

We have studied the effects of intracerebral administration of selective alpha-adrenergic agonists on duodenal bicarbonate secretion. Duodenum free of Brunner's glands was cannulated in situ in anesthetized rats, and bicarbonate secretion into the luminal reperfusate was continuously titrated by pH stat. Infusion of the alpha 1-selective adrenoceptor agonist, phenylephrine (1,000–2,500 micrograms.kg-1.h-1), into a lateral brain ventricle increased (P< 0.01) duodenal bicarbonate secretion. Pretreatment with prazosin, an alpha 1-antagonist, significantly (P< 0.01) reduced the stimulatory effect when infused into the lateral ventricle (30 micrograms.kg-1.h-1), but not when administered intravenously (1,000 micrograms.kg-1.h-1). Hexamethonium (10 mg.kg-1.h-1 iv) abolished stimulation, whereas cervical vagotomy, epidural blockade, and naloxone were each without effect. Vasopressin, vasopressin antagonists, ts, and oxytocin did not affect basal secretion. Intracerebro-ventricular administration of the alpha 2-adrenoceptor agonist, clonidine (1,000 micrograms.kg-1.h-1), in contrast to alpha 1-receptor activation, decreased (P< 0.01) the secretion. Thus central nervous adrenoceptors influence duodenal mucosal bicarbonate te secretion, and alpha 1-adrenoceptor stimulation may provide protection against luminal acid. This potent stimulation was not mediated by the vagal nerves, spinal cord pathways, or the release of beta-endorphin but involves nicotinic, possibly enteric nervous transmission.
Acute stress causes mucin release from rat colon: role of corticotropin releasing factor and mast cells
Authors: Castagliuolo I. , Lamont J. T. , Qiu B. , Fleming S. M. , Bhaskar K. R. , Nikulasson S. T. , Kornetsky C. , Pothoulakis C.
Published: December 2017

We determined the effects of immobilization stress on rat colonic mucus release and mast cell degranulation and examined whether corticotropin releasing factor (CRF) was involved in these responses. After 30-min immobilization, rats were killed, colonic mucosal explants were cultured, and levels of rat mast cell protease II (RMCP II) and prostaglandin E2 (PGE2) were measured. Mucin release from explants was assayed by incorporation of [3H]glucosamine into colonic mucin and by histological evaluation of goblet cell depletion. Stress caused significant increases of colonic RMCP II, PGE2, and mucin release and fecal pellet output and caused an approximately 10-fold increase in colonic mucosal levels of cyclooxygenase-2 (COX-2) mRNA. These stress-associated changes were reproduced by intravenous or intracerebral injection of CRF in conscious, nonstressed rats. Pretreatment of rats with the CRF antagonist alpha-helical-CRF9-41, hexamethonium, atropine, or bretylium, or the mast cell stabilizer lodoxamide inhibited stress-induced release of RMCP II, PGE2, and mucin, whereas indomethacin prevented mucin release but not mast cell degranulation. Hexamethonium and CP-96,345, a substance P antagonist, inhibited fecal pellet output caused by stress. We conclude that CRF released during immobilization stress increases colonic transit via a neuronal pathway and stimulates colonic mucin secretion via activation of neurons and mast cells.
Endocytosis and Ca2+ are required for endotoxin-stimulated TNF-alpha release by rat Kupffer cells
Authors: Lichtman S. N. , Wang J. , Zhang C. , Lemasters J. J.
Published: December 2017

Endotoxin [lipopolysaccharide (LPS)] is a cell wall polymer derived from Gram-negative bacteria that stimulates macrophages to produce a variety of inflammatory mediators. In these studies, we examined LPS-stimulated formation of tumor necrosis factor-alpha (TNF-alpha) by cultured rat Kupffer cells. Cytochalasin B and methylpalmitate, blockers of endocytosis, decreased LPS-stimulated TNF-alpha release by > 92%. Bafilomycin A, monensin, and chloroquine, which prevent endosomal acidification, also blocked LPS-stimulated release of TNF-alpha by > 90%. Cytochalasin B and bafilomycin A decreased TNF-alpha mRNA levels by > 90% after LPS stimulation. Consistent with the requirement for LPS uptake and processing was the observation that Kupffer cells required 30 min of contact with LPS for maximal TNF-alpha release. LPS-stimulated TNF-alpha release was unaltered by incubation in Ca(2+)-free ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid medium, and A-23187, a Ca2+ ionophore, failed to stimulate TNF-alpha release in the absence of LPS. However, nisoldipine, a Ca2+ channel blocker, suppressed LPS-stimulated TNF-alpha release in cells cultured both in Ca(2+)-containing and Ca(2+)-free media. Although thapsigargin did not block TNF-alpha release, this depleter of intracellular Ca2+ stores blocked LPS-stimulated TNF-alpha synthesis in Ca(2+)-free medium and decreased TNF-alpha mRNA levels by 80%. Furthermore, LPS induced a late rise in intracellular free Ca2+ demonstrated by video microscopy of fura 2-loaded Kupffer cells. De novo protein and RNA synthesis were required, since cycloheximide and actinomycin D also inhibited LPS-stimulated TNF-alpha release. We compared free TNF-alpha secreted into culture supernatants with cell-associated TNF-alpha and found that cytochalasin B, bafilomycin A, chloroquine, monensin, and nisoldipine did not increase bound, cell-associated TNF-alpha. We conclude that endocytosis and endocytic processing may be necessary for LPS-stimulated TNF-alpha release from Kupffer cells. Ca2+ release, regulated by dihydropyridine-sensitive Ca2+ channels, also appears to be necessary for LPS-induced signaling and may arise from intracellular stores associated with the endosome/lysosome compartment.
Effect of vitamin E supplementation on hepatic fibrogenesis in chronic dietary iron overload
Authors: Brown K. E. , Poulos J. E. , Li L. , Soweid A. M. , Ramm G. A. , O'Neill R. , Britton R. S. , Bacon B. R.
Published: December 2017

It has been suggested that lipid peroxidation plays an important role in hepatic fibrogenesis resulting from chronic iron overload. Vitamin E is an important lipid-soluble antioxidant that has been shown to be decreased in patients with hereditary hemochromatosis and in experimental iron overload. The aim of this study was to determine the effects of vitamin E supplementation on hepatic lipid peroxidation and fibrogenesis in an animal model of chronic iron overload. Rats were fed the following diets for 4, 8, or 14 mo: standard laboratory diet (control), diet with supplemental vitamin E (200 IU/kg, control + E), diet with carbonyl iron (Fe), and diet with carbonyl iron supplemented with vitamin E (200 IU/kg. Fe + E). Iron loading resulted in significant decreases in hepatic and plasma vitamin E levels at all time points, which were overcome by vitamin E supplementation. Thiobarbituric acid-reactive substances (an index of lipid peroxidation) were increased three- to fivefold in the iron-loaded livers; supplementation with vitamin E reduced these levels by at least 50% at all time points. Hepatic hydroxyproline levels were increased twofold by iron loading. Vitamin E did not affect hydroxyproline content at 4 or 8 mo but caused an 18% reduction at 14 mo in iron-loaded livers. At 8 and 14 mo, vitamin E decreased the number of alpha-smooth muscle actin-positive stellate cells in iron-loaded livers. These results demonstrate a dissociation between lipid peroxidation and collagen production and suggest that the profibrogenic action of iron in this model is mediated through effects which cannot be completely suppressed by vitamin E.
Cryptdin gene expression in developing mouse small intestine
Authors: Darmoul D. , Brown D. , Selsted M. E. , Ouellette A. J.
Published: December 2017

In rodents, the four intestinal epithelial cell lineages differentiate and become morphologically distinct during the first 2-3 postnatal wk. In studies reported here, reverse transcriptase-polymerase chain reaction (RT-PCR)-based assays detected Paneth cell defensin mRNAs in intestinal RNA from 1-day-old (P1) mice before crypt formation and maturation of the epithelium. Analysis of these defensin-coding RT-PCR products from P1 mice showed that 69% of clones sequenced coded for cryptdin-6, suggesting that it is the most abundant enteric defensin mRNA in the newborn. Paneth cell mRNAs, including cryptdins-4 and -5, lysozyme, matrilysin, and defensin-related sequences, also were detected in RNA from P1 mouse intestine. Unlike adult mice, where only Paneth cells are immunopositive for cryptdin, cryptdin-containing cells were distributed throughout the newborn intestinal epithelium and not in association with rudimentary crypts. Cryptdin immunoreactivity in the P1 mouse intestine was specific for intracellular granule contents, and immunofluorescent detection of cryptdins on mucosal surfaces suggested that the peptides are released into the intestinal lumen in P1 mice Defensin secretion may contribute to innate immunity of the neonatal intestine before the presence of distinguishable Paneth cells.
Molecular and functional heterogeneity of cholangiocytes from rat liver after bile duct ligation
Authors: Alpini G. , Ulrich C. , Roberts S. , Phillips J. O. , Ueno Y. , Podila P. V. , Colegio O. , LeSage G. D. , Miller L. J. , LaRusso N. F.
Published: December 2017

Cholangiocytes, the epithelial cells that line intrahepatic bile ducts, participate in bile secretion via basal and agonist-stimulated transport of solutes and water. On the basis of subtle structural differences between cholangiocytes lining small vs. large bile ducts, as well as known phenotypic variations among transporting epithelia in other organs, we demonstrated that cholangiocytes are functionally heterogeneous along the intrahepatic biliary tree of normal rats. In studies reported here, we confirm and extend the concept of functional heterogeneity of cholangiocytes by employing the bile duct-ligated (BDL) rat model of cholestasis associated with selective cholangiocyte proliferation. Using novel isolation and separatory techniques, we prepared subpopulations of pure small, medium, and large cholangiocytes from BDL rats and compared them with regard to gene expression and basal or agonist-responsive transport activities. Although transcripts for gamma-glutamyl transpeptidase and cytokeratin 19, two cholangiocyte-specific proteins, and glyceraldehyde-3-phosphate dehydrogenase, a housekeeping gene, were in all three subpopulations, genes for several proteins involved in solute transport [Cl-/HCO3- exchanger, cystic fibrosis transmembrane conductance regulator (CFTR), and secretin receptor] were expressed only in medium and large cholangiocytes. Consistent with these findings, secretin increased intracellular levels of adenosine 3',5'-cyclic monophosphate (cAMP) and 36Cl- efflux rates in medium and large cholangiocytes but not in small cholangiocytes. Also, forskolin/8-(4-chlorophenylthio)-cAMP stimulated 36Cl- efflux rates only in medium and large cholangiocytes, consistent with selective functional expression of CFTR in these subpopulations. These results support the molecular and functional heterogeneity of cholangiocytes within the intrahepatic biliary ductal system and are consistent with the notion that hormone-regulated transport of solutes after BDL occurs principally in medium and large cholangiocytes in a fashion similar to that observed in normal rat liver.
cAMP modulation of a Ca2+-dependent K+ conductance in rat submandibular acinar cells
Authors: Ishikawa T.
Published: December 2017

Rat submandibular acinar cells exhibit a tetraethylammonium (TEA)-insensitive, Ca2+-activated K+ conductance, which may play an important role in Ca2+-dependent fluid secretion by this gland (T. Ishikawa, M. Murakami, and Y. Seo. Pfluegers Arch. 428: 516-525, 1994; T. Ishikawa and M. Murakami. Pfluegers Arch. 429: 748-750, 1995). We have now investigated whether this conductance would be modified during the potentiation of Ca2+-activated fluid secretion by an adenosine 3',5'-cyclic monophosphate (cAMP)-mediated mechanism in the rat submandibular gland. In isolated, vascularly perfused rat submandibular gland, we found that the adenylate cyclase activator forskolin (10 and 30 microM) enhanced a transient initial phase of a biphasic fluid secretion induced by the Ca2+ ionophore A-23187 (3 microM). We also unexpectedly found that forskolin reduced a smaller sustained phase of the secretion. The dual effects of forskolin were mimicked by the application of the cocktail of 3-isobutyl-1-methylxanthine (IBMX) (0.1 mM) and dibutyryl cAMP (DBcAMP) (0.1 and 0.5 mM). Basolateral K+ efflux studies showed that 1) forskolin significantly enhanced a large transient net K+ efflux induced by A-23187 in a dose-dependent manner; 2) the forskolin-induced enhancement was also mimicked by a cocktail of IBMX and DBcAMP; and 3) the A-23187-induced K+ efflux enhanced by these agents was not inhibited by TEA (5 or 10 mM), a concentration known to completely block Ca2+ voltage-dependent large-conductance K+ channels (maxi-K+ channels). With use of microspectrofluorometry with fura 2, we then showed that an increase in cytosolic Ca2+ concentration induced by the nonfluorescent, brominated derivative 4-bromo-A-23187 (3 microM) was not altered by forskolin (30 microM) in freshly isolated rat submandibular acinar cells. With use of whole cell patch-clamp techniques, we demonstrated that a TEA-insensitive K+ conductance induced by A-23187 in cells dialyzed with pipette solutions containing cAMP (0.1 mM) was significantly larger than that in cells treated with control pipette solutions. Taken together, these results indicate that potentiation of the Ca2+-activated fluid secretion by a cAMP-mediated mechanism is accompanied by potentiation of a TEA-insensitive, Ca2+-activated K+ conductance in this gland.
Fermentable carbohydrate exerts a urea-lowering effect in normal and nephrectomized rats
Authors: Younes H. , Remesy C. , Behr S. , Demigne C.
Published: December 2017

The influence of nondigestible carbohydrate on intestinal fermentations and on the route of nitrogen excretion has been investigated in normal rats and in unilaterally nephrectomized rats. Rats were adapted to 10% casein diets, either fiber free or containing different levels of two fermentable carbohydrates, inulin or crude potato starch. Ingestion of fermentable carbohydrate led to a considerable enlargement of the cecum because of hypertrophy of the cecal wall and an increase in cecal contents. Cecal digesta contained elevated concentrations of short-chain fatty acids, resulting in acidic pH. Diets containing fermentable carbohydrate enhanced fecal nitrogen excretion, which was more than doubled at the highest level of inulin or potato starch. In parallel, urinary nitrogen excretion was significantly decreased by fermentable carbohydrate. Although these changes were similar in all animals, there were quantitative differences in the response of nephrectomized animals to fermentable carbohydrate. In nephrectomized rats, plasma urea concentrations were more than 2.5 times higher than in normal rats (5.8 mM compared with 2.2 mM). Plasma urea concentrations were reduced by approximately 50% when normal rats were fed diets containing 7.5-15% inulin or 10-20% resistant starch. In nephrectomized animals fed the highest level of fermentable carbohydrate, plasma urea concentrations were also significantly decreased, but only by 30%. In nephrectomized rats, the concentration of nitrogen cycling in the cecum was greater (urea nitrogen transfer into the cecum was 50-60% greater and ammonia flux from the cecal lumen to the blood was two times higher than in normal rats), but fecal nitrogen excretion was equivalent in normal and nephrectomized animals. When expressed as a percentage of total nitrogen excretion, fecal nitrogen excretion was <20% in animals fed fiber-free diets, compared with 45-50% in normal animals and 40% in nephrectomized animals fed fermentable carbohydrate.
Maintenance of morphology and function of canine proximal colon smooth muscle in organ culture
Authors: Horner M. J. , Ward S. M. , Gerthoffer W. T. , Sanders K. M. , Horowitz B.
Published: December 2017

We have determined that serum source plays a critical role in optimizing conditions for an organ culture model of canine proximal colon. Previous studies using equine serum in the medium have shown that some properties of canine colonic smooth muscle can be maintained in organ culture. However, many characteristics of the tissue were altered by the culture conditions. The aims of the present study were to determine whether serum isolated from canine blood would improve the preservation of physiological properties of canine proximal colon in organ culture. Strips of canine colonic smooth muscle were cultured in 10% canine serum medium, and electrical, mechanical, morphological, and molecular analyses were performed after 0, 3, and 6 days in culture. Unlike organ culture in equine serum, in which Na+-K+-adenosinetriphosphatase (Na+-K+-ATPase) expression declined, culture in canine serum maintained Na+-K+-ATPase expression, and resting membrane potential of smooth muscle cells along the submucosal surface of the circular muscle in cultured tissue remained unchanged during the culture period. Increased sensitivity in the contractile response to acetylcholine, previously observed with tissues cultured in equine serum, was not observed. However, the mechanical performance of the muscle (maximal contractile activity) declined over time in culture. Ultrastructural organization of cellular organelles and myofilaments remained intact in the majority of cells; however, some cells possessed regions devoid of contractile filaments. The results of these studies suggest that organ cultured strips of smooth muscle may provide a useful tool for evaluating electrical and mechanical events in conjunction with molecular analysis of functional components.
Characterization of a beta 3-adrenoceptor stimulating gastrin and somatostatin secretions in rat antrum
Authors: Levasseur S. , Bado A. , Laigneau J. P. , Moizo L. , Reyl-Desmars F. , Lewin M. J.
Published: December 2017

The beta 3-adrenoceptor (beta 3-AR) agonist SR-58611A inverted question markethyl-[(7s)-7-[[(2R)-2-(3-chlorophenyl)-2-hydroxyethyl]amino]5, 6,7,8-tetrahydronaphth-2-yl]oxyacetate hydrochloride inverted question mark stimulated somatostatin and gastrin releases in isolated rat gastric antral epithelial cells. Stimulation was a concentration-dependent process with 50% effective concentrations of 2.7 +/- 1.1 and 3.8 +/- 1.9 nM compared with 209 +/- 71 and 230 +/- 51 nM for isoproterenol, respectively. It was inhibited by selective beta-AR antagonists with the following rank order of potency: SR-59230A 3-(2-ethylphenoxy)1-[(1S)-1,2,3,4-tetrahydronaphth- 1-ylamino]-(2S)-2-propranol oxalate; beta 3-AR antagonist > ICI-118551[erythro-(+/-)-1-(7-methylindan-4-yloxy)-3- isopropylaminobutan-2-ol-hydrochloride; beta 2-AR antagonist > CGP-20712A[(+/-)-[2-(3-carbarmoyl-4-hydroxyphenoxy)-et hyl- amino]-3-[4 (1-methyl-4-trifluoromethyl-2-imidazolyl)-phenoxy]- 2-propranol; beta 1-AR antagonist]. Furthermore, specific binding of 125I-cyanopindolol to the isolated cells was demonstrated and was displaced by the beta-AR antagonists according to the same rank order of potency and with apparent dissociation constants consistent with the 50% inhibitory concentrations for SR-58611A-stimulated somatostatin and gastrin releases. In addition, the presence of beta 3-AR mRNA was detected by reverse transcriptase polymerase chain reaction. These findings provide the first evidence for a gastric beta 3-AR mediating catecholamine stimulation of gastrin and somatostatin releases from antral cells.
Patterns of gastric myoelectrical activity in human subjects of different ages
Authors: Chen J. D. , Co E. , Liang J. , Pan J. , Sutphen J. , Torres-Pinedo R. B. , Orr W. C.
Published: December 2017

The aim of this study was to investigate the developmental change of gastric myoelectrical activity in humans. Five groups of healthy subjects were studied, including 10 preterm newborns, 8 full-term newborns, 8 full-term infants (ages 2-6 mo), 9 children (ages 4-11 yr), and 9 adults. Gastric myoelectrical activity was recorded using surface electrogastrography for 30 min before and 30 min after a test meal in each subject. Spectral analysis methods were applied to compute the parameters of the electrogastrogram (EGG). The results showed that the percentage of 2- to-4-cycles/min (cpm) slow waves was 26.6 +/- 3.9% in the preterm newborns, 30.0 +/- 4.0% in full-term newborns, 70 +/- 6.1% in 2- to 6-mo-old infants (P < 0.001 compared with newborns), 84.6 +/- 3.2% in 4- to 11-yr-old children (P < 0.03 compared with infants), and 88.9 +/- 2.2% in the adults (P > 0.05 compared with children). In conclusion, gastric slow waves are absent at birth, and there is a maturing process after birth. Age-matched controls are necessary for the interpretation of EGG data from neonates and infants, whereas EGG data in children are the same as in adults.
Hypothalamic nitric oxide synthase is depressed in cholestatic rats
Authors: Swain M. G. , Le T. , Tigley A. W. , Beck P.
Published: December 2017

We examined hypothalamic nitric oxide synthase (NOS) levels and release as well as steady-state mRNA levels in rats with cholestasis due to bile duct resection (BDR) and in sham-resected control rats. BDR rats had a significant reduction in hypothalamic NOS-containing neurons in the hypothalamic paraventricular nucleus as determined by NADPH-diaphorase staining, compared with sham-resected controls. In addition, NOS activity, measured indirectly by determining nitrite release from hypothalamic explants, was significantly lower in BDR rats compared with sham-resected animals. Hypothalamic steady-state NOS mRNA levels [brain constitutive NOS (bNOS)] were determined by semiquantitative reverse transcription-polymerase chain reaction and were found to be increased 1.5-fold in BDR rats compared with sham rats. In summary, BDR rats have diminished hypothalamic NOS levels and activity coupled with enhanced steady-state bNOS mRNA levels, suggesting that depressed hypothalamic NOS protein levels are due to posttranscriptional defects.
Effects of transmural field stimulation in isolated smooth muscle of human rectum and internal anal sphincter
Authors: Glavind E. B. , Forman A. , Madsen G. , Tottrup A.
Published: December 2017

Smooth muscle preparations from the circular muscle layer of the most distal rectum and the proximal and distal human internal anal sphincter (IAS) mounted in organ baths to record isometric tension developed spontaneous tension. Transmural electrical field stimulation (TMS) induced frequency- and impulse duration-dependent relaxations sensitive to tetrodotoxin in the stimulation range of 0.5-40 Hz and 0.04-0.6 ms. Poststimulus contractions were most frequent and prominent in rectal preparations. Maximal relaxations were comparable in the three locations and were achieved at 10 Hz and 0.4 ms. The frequency inducing half-maximal response was lower in rectal strips compared with IAS. Phentolamine (10-(6) M) enhanced relaxations and diminished off-contractions at 40 Hz in distal IAS. N omega-nitro-L-arginine (L-NNA) concentration dependently inhibited both relaxations and off-contractions (10 Hz, 0.4 ms). The pD2 values (-log EC50) of L-NNA were lower in rectal muscle compared with those in IAS. L-Arginine (10-(4) M) inhibited the blocking effect of L-NNA. In one-half of the preparations, L-NNA reversed the relaxations to duration contractions (15-40 Hz), which were inhibited by atropine in rectal preparations and by phentolamine in IAS. In conclusion, excitatory innervation of the IAS is alpha-adrenergic and cholinergic in the rectum. A product of the L-arginine-nitric oxide pathway mediates the TMS-induced inhibition of the muscle and is also involved in poststimulus contractions.
Correlation and comparison of magnetic and electric detection of small intestinal electrical activity
Authors: Bradshaw L. A. , Allos S. H. , Wikswo J. P. , Richards W. O.
Published: December 2017

The small intestinal basic electrical rhythm (BER) was detected simultaneously with serosal electrodes and a transabdominal superconducting quantum interference device (SQUID) magnetometer in anesthetized rabbits. We induced mesenteric ischemia to correlate serosal electrode recording of changes in BER with the SQUID magnetometer. The BER frequency was obtained by spectral analysis of the data using Fourier and autoregressive techniques. There was a high degree of correlation (r = 0.96) between the BER frequency determined using the serosal electrodes and the BER frequency ascertained from SQUID data. Additionally, the effects of an electrical insulator on the external electric and magnetic fields were studied in the rabbit model. The presence of an insulator profoundly attenuates external electric potentials recorded by cutaneous electrodes but does not significantly affect external magnetic fields or serosal potentials. We conclude that SQUID magnetometers could noninvasively record small intestinal BER that was highly correlated with the activity recorded by invasive serosal electrodes. The advantages of magnetic field measurements have encouraged us to investigate clinical applications.
L-glutamine stimulates intestinal cell proliferation and activates mitogen-activated protein kinases
Authors: Rhoads J. M. , Argenzio R. A. , Chen W. , Rippe R. A. , Westwick J. K. , Cox A. D. , Berschneider H. M. , Brenner D. A.
Published: December 2017

We studied the mechanisms by which L-glutamine (Gln), a major fuel for enterocytes, signals proliferation in intestinal epithelial cell lines. Gln was additive to epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) in stimulating DNA synthesis, as assessed by [3H]thymidine incorporation. Extracellular signal-regulated kinases (ERKs) p42mapk and p44mapk and Jun nuclear kinases (JNKs) phosphorylate and activate nuclear transcription factors. Proteins of the c-Jun, ATF-2, and c-Fos families aggregate to form DNA-binding homodimers or heterodimers called activating protein 1 (AP-1). In vitro assays and functional assays of phosphorylation demonstrated that Gln activates both ERKs and JNKs, resulting in a fourfold increase in AP-1-dependent gene transcription. Gln was required for EGF signaling through ERKs. Maximal stimulation of proliferation required approximately 2.5 mM Gln. c-Jun mRNA levels responded to Gln in "Gln-starved" porcine IPEC-J2 cells and in rat IEC-6 cells. Although Gln metabolism is required for the proliferative response, several Gln by-products did not stimulate [3H]thymidine incorporation, with the exception of arginine. Gln may be a unique nutrient for enterocytes, capable of dual signaling and augmenting the effects of growth factors that govern cellular proliferation and repair.
Role of iron in NF-kappa B activation and cytokine gene expression by rat hepatic macrophages
Authors: Lin M. , Rippe R. A. , Niemela O. , Brittenham G. , Tsukamoto H.
Published: December 2017

A redox-sensitive nuclear factor, NF-kappa B, induces transcription of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) in macrophages. The present study has investigated the role of iron in NF-kappa B activation and TNF-alpha and IL-6 expression by rat hepatic macrophages (HM). As an in vivo model, cholestatic liver injury was induced in rats by ligation of the common bile duct (BDL). During the first 2 wk after BDL, there was an increase in the hepatic level of thiobarbituric acid-reactive substances (TBARS) that was accompanied by the appearance of protein-malondialdehyde adducts in the periportal region. This increase was reduced after 3 wk. TNF-alpha and IL-6 mRNA levels in HM from the BDL rats were increased at 1 and 2 wk and attenuated at 3 wk. Gel mobility shift assay of HM nuclear extracts demonstrated the similar temporal pattern of enhanced NF-kappa B binding activity. Treatment of the BDL animals with 1,2-dimethyl-3-hydroxypyrid-4-one (L-1), a lipophilic iron chelator, suppressed the increases in hepatic TBARS by 64%, plasma alanine aminotransferase by 45%, and HM TNF-alpha and IL-6 mRNA by > 84%. Concomitantly, the HM NF-kappa B binding activity was reduced close to the level observed in sham-operated rats. Treatment of cultured HM with L-1 also blocked lipopolysaccharide-stimulated NF-kappa B activation and TNF-alpha and IL-6 expression at mRNA and protein levels. These results demonstrate that the iron chelator effectively blocks NF-kappa B activation and coordinate TNF-alpha and IL-6 gene upregulation by HM in cholestatic liver injury or under in vitro lipopolysaccharide stimulation. These findings support a pivotal role for iron in activation of NF-kappa B and cytokine gene expression by HM in vitro and in vivo.
Characterization of intracellular copper pools in rat hepatocytes using the chelator diamsar
Authors: Bingham M. J. , Sargeson A. M. , McArdle H. J.
Published: December 2017

When hepatocytes are incubated with the chelator diamsar, two pools can be identified, which we have termed extractable and nonextractable. On entering the hepatocyte, 67Cu first associates with the extractable pool and, after approximately 2 h, moves to the nonextractable pool. Both pools demonstrate saturation and are filled as a function of Cu concentration and incubation time. Using the Michaelis-Menten equation, we have estimated the size of the pools after incubation with 67Cu for 30 min and 4 h. During this period the extractable pool decreases in size from 200 +/- 27 to 116 +/- 5 pmol/microgram DNA, whereas the nonextractable pool increases from 28 +/- 9 to 77 +/- 11 pmol/microgram DNA. Movement of Cu from the nonextractable pool to the extractable pool is slow and incomplete. Using [3H]diamsar, we demonstrate that uptake of the chelator is not rate limiting and probably does not occur by pinocytosis. Incubation with diamsar does not affect the activity of superoxide dismutase or cytochrome-c oxidase, although it does prevent the incorporation of 67Cu into ceruloplasmin. Incubation with zinc, which induces metallothionein, results in an increase in 67Cu associated with the nonextractable pool, suggesting that 67Cu-metallothionein constitutes at least part of the nonextractable pool.
Effect of oxidative stress on cellular functions and cytosolic free calcium of rat pancreatic acinar cells
Authors: Klonowski-Stumpe H. , Schreiber R. , Grolik M. , Schulz H. U. , Haussinger D. , Niederau C.
Published: December 2017

The present study evaluates the effect of free radicals generated by xanthine oxidase-catalyzed oxidation of hypoxanthine on cellular function of isolated rat pancreatic acinar cells. The results show that a rapid and sustained increase in intracellular Ca2+ concentration ([Ca2+]i) preceded all other morphological and functional alterations investigated. Radical-induced [Ca2+]i increase was largely inhibited by 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester, which prevents Ca2+ release from intracellular stores, but not by Ca2(+)-depleted medium. Radicals released Ca2+ from thapsigargin-insensitive, ryanodine-sensitive intracellular stores, whereas the secretagogue caerulein at physiological concentrations mainly released Ca2+ from thapsigargin-sensitive stores. In contrast to effects of the secretagogue, radical-induced Ca2+ changes did not cause luminal protein secretion but cell death. In single-cell measurements, both secretagogue and radicals induced oscillations of [Ca2+]i. Radical-induced oscillations had a lower frequency but similar amplitude when compared with caerulein-induced oscillations. 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid and ryanodine, which prevented the radical-induced Ca2+ increase without altering the generation of radicals, markedly reduced the radical-induced cell damage. These results suggest that the Ca2+ increase mediates the radical-induced cell injury. The studies also indicate that not only the extent and duration but also the origin of [Ca2+]i release as well as the frequency of Ca2+ oscillations may determine whether a pancreatic acinar cell will secrete or die.
Temporal expression of trefoil peptides in the TGF-alpha knockout mouse after gastric ulceration
Authors: Cook G. A. , Yeomans N. D. , Giraud A. S.
Published: December 2017

Trefoil peptides are gut peptides that have been implicated in the repair of the gastric mucosa after injury. Previous studies suggest that epidermal growth factor (EGF) receptor ligands may induce the expression of trefoil peptides. Because transforming growth factor-alpha (TGF-alpha) is a major EGF receptor ligand in the gut, we tested the hypothesis that mice with a TGF-alpha null mutation (knockout) would have reduced trefoil peptide expression compared with wild-type controls after gastric ulceration. The rate of macroscopic ulcer healing was the same in knockout and wild-type mice. Spasmolytic polypeptide (SP) and intestinal trefoil factor (ITF) expression were quantified in tissue and gastric lavage. SP and ITF levels in tissue fell within 48 h of ulceration (P < 0.05), but secretion into gastric juice was unchanged. ITF peptide expression was increased (as was SP expression) in wild-type but not knockout mice 42 and 72 days after injury (P < 0.05). The induction of SP and ITF expression in the latter stages of injury repair has a TGF-alpha-dependent component and suggests a role for these peptides in gastric differentiation and cell positioning.
Glucocorticoids upregulate taurocholate transport by ileal brush-border membrane
Authors: Nowicki M. J. , Shneider B. L. , Paul J. M. , Heubi J. E.
Published: December 2017

The regulation of the enterohepatic circulation of bile acids has not been fully elucidated. Substrate availability has been shown to have a regulatory role on the ileal uptake of taurocholate (TC) by a positive feedback mechanism. Other mechanisms are likely to be involved in regulating ileal bile acid uptake. The present study was designed to test the hypothesis that the ileal bile acid transporter (iBAT) is glucocorticoid sensitive and that changes in expression are mediated by changes in iBAT synthesis. Adult Sprague-Dawley rats (300–400 g) received intraperitoneal injections with either corticosterone (5 mg/ 100 g body weight) or an equivalent vehicle (control) daily for 3 days. On day 4, ileal brush-border membrane vesicles (BBMV) and hepatic basolateral membrane vesicles (BLMV) were prepared, and TC transport was performed using the rapid filtration technique. Initial velocity was measured at selected time points, and kinetics were calculated over a range of TC concentrations. Ileal RNA was isolated, and Northern analysis of steady-state iBAT mRNA levels was determined. Western blot analysis was performed to quantitate the level of the 48-kDa iBAT protein. The initial velocity of Na(+)-dependent TC uptake at 30 s by ileal BBMV was higher in treated animals (264.3 +/- 64.6 pmol/mg protein) compared with control animals (148.3 +/- 41.1 pmol/mg protein; P = 0.07). The maximal velocity of uptake (Vmax) was significantly higher in treated vs. control animals (1,091 +/- 62.7 vs. 689.1 +/- 55.0 pmol.min-1.mg protein-1, respectively; P = 0.002), whereas there was no significant difference in the Michaelis constant (Km) between the control and treated animals (43.3 +/- 7.2 vs. 35.3 +/- 8.7 microM, respectively; P = not significant). Steady-state iBAT mRNA levels were increased twofold in the treated vs. control groups. Western blot analysis showed that the abundance of the 48-kDa iBAT protein was eightfold higher in the treated animals compared with control. Kinetic analysis of hepatic Na(+)-dependent TC uptake revealed nearly identical Vmax and Km between the study and control animals. Therefore, we conclude that TC transport by ileal BBMV is upregulated by administration of glucocorticoids. The increase in BBMV transport Vmax corresponds to an increase in both iBAT transcript and protein.
Models for the regulation of purine metabolism in rat hepatocytes: evaluation of tracer kinetic experiments
Authors: Schwendel A. , Grune T. , Holzhutter H. G. , Siems W. G.
Published: December 2017

Metabolic pathways are characterized by numerous regulatory mechanisms. Their study calls for the determination of the metabolite concentrations as well as the flux rates. Corresponding experiments using purified enzymes and an artificial environment frequently yield results that differ from findings for in vivo systems. To be more realistic, the tracer kinetic experiments presented here involved intact isolated hepatocytes. It is necessary to establish mathematical models to deduce the flux rates. With the presumption of metabolic steady-state conditions, the flux rates are determined by a possibly stiff system of linear differential equations. For the first time, the flux rate determination in the purine metabolism of rat hepatocytes was accomplished by applying a combination of a nonlinear least-square fit and a numerical integration. Because of the complexity of this pathway, it was necessary to use three different tracers requiring three partial models. By ensuring their compatibility and using a fit of high statistical quality, the experimental situation could be described adequately. Our flux rate analysis confirmed earlier experimental findings and also allows much more insight into the regulatory mechanisms of the metabolism studied.
Synaptic inputs to morphologically identified myenteric neurons in guinea pig rectum from pelvic nerves
Authors: Tamura K.
Published: December 2017

Neurobiotin-filled microelectrodes were used to investigate electrical and synaptic behavior and morphological characteristics of rectal myenteric neurons that received synaptic inputs from the pelvic nerves. Stimulation of the pelvic nerve at low frequencies (< 3.3 Hz) evoked nicotinic fast excitatory postsynaptic potentials (fast EPSPs) in 45.3% of rectal neurons. Pelvic fast EPSPs were found in S/type 1, AH/type 2, type 3, or single-spike neurons that had a single long process preferentially projecting in the orad direction. Stimulation of the pelvic nerve at higher frequencies (5–20 Hz) elicited slow membrane excitation in 13.9% of the neurons. They were either AH/type 2 neurons with Dogiel II morphology or S/type 1 neurons with a single long process. Hexamethonium (100 microM) blocked pelvic fast EPSPs more quickly than those evoked by fiber tract stimulation but did not affect slow excitatory response. The results suggested the presence of more than one nicotinic-cholinergic synapse in the pelvic nerve pathway and the possible release of a noncholinergic excitatory substance from the afferent nerve terminals. It is possible that a subpopulation of rectal neurons, which receive a fast EPSP and have a single long process that projects in the orad direction, might be interneurons that mediate the defecation reflex.
Expression cloning of NaDC-2, an intestinal Na(+)- or Li(+)-dependent dicarboxylate transporter
Authors: Bai L. , Pajor A. M.
Published: December 2017

A cDNA coding for a Na(+)-dicarboxylate cotransporter from Xenopus laevis intestine, NaDC-2, was isolated by functional expression cloning in Xenopus oocytes. NaDC-2 encodes a 622-residue polypeptide with a predicted mass of 68.6 kDa. The sequence and secondary structure of NaDC-2 are related to the mammalian renal Na(+)-dicarboxylate and Na(+)-sulfate cotransporters. NaDC-2 mRNA is expressed only in the intestine. Oocytes injected with NaDC-2 cRNA exhibit increased transport of succinate, citrate, and glutarate. Transport of succinate by NaDC-2 is stimulated by Na+ or Li+, with Michaelis-Menten constant values for succinate of 0.3 mM (in Na+) and 0.7 mM (in Li+). Na+ and Li+ activation curves show sigmoid kinetics, with Hill coefficients of 1.4 (nNa) and 1.7 (nLi), indicating that multiple cations are involved in the transport of succinate. The transport of succinate by NaDC-2 is insensitive to pH, whereas the transport of citrate is inhibited at high pH. The differences in functional properties between NaDC-2 and the structurally related Na(+)-dicarboxylate cotransporters NaDC-1 and hNaDC-1 will form the basis of detailed structure-function studies.
Intracellular iron regulates iron absorption and IRP activity in intestinal epithelial (Caco-2) cells
Authors: Arredondo M. , Orellana A. , Garate M. A. , Nunez M. T.
Published: December 2017

In vertebrates, body Fe homeostasis is maintained through the regulation of its intestinal absorption. In addition, because Fe is both essential and toxic, intracellular Fe levels are tightly regulated. Consequently, intestinal epithelial cells are in the unique position of being responsible simultaneously for the regulation of body Fe absorption and the regulation of their intracellular Fe levels to remain viable. We tested the hypothesis that the regulation of transepithelial Fe transport and the regulation of intracellular Fe levels are sensitive to a common effector. To this end, we used a recently developed protocol to obtain cultured intestinal epithelial cells with defined intracellular Fe concentrations. In these cells we tested Fe absorption and Fe regulatory protein (IRP) activities. We found that transepithelial Fe transport was inversely related to 20-200 microM intracellular Fe and that Caco-2 cells expressed Fe regulatory protein-1 and Fe regulatory protein-2 activities. Fe regulatory protein-1 activity, Fe regulatory protein-2 mass, transferrin receptor density, and ferritin levels were regulated by intracellular Fe in the same range (20-200 microM) that affected transepithelial Fe transport. These results suggest that a common Fe-responsive factor regulates both intracellular Fe levels and Fe absorption by Caco-2 cells.
L-type calcium channels regulate gastrin release from human antral G cells
Authors: Ray J. M. , Squires P. E. , Meloche R. M. , Nelson D. W. , Snutch T. P. , Buchan A. M.
Published: December 2017

In mRNA samples isolated from a gastrin (G) cell-enriched human antral cell preparation, reverse transcription-polymerase chain reaction identified products encoding part of the alpha 1-subunit of class C and D L-type voltage-dependent Ca2+ channels (VDCCs). Analysis of the polymerase chain reaction products demonstrated a 100% homology with the known human gene sequences. An antibody to the class D alpha 1-subunit immunostained 30-40% of the cultured cells; of these 90% were gastrin immunoreactive. Gastrin release stimulated by terbutaline (beta 2-agonist) and forskolin was abolished by blockade of L-type VDCCs; the effect of 3.6 mM extracellular Ca2+ was only partially reversed. In G cells the rise in intracellular Ca2+ observed in response to increasing extracellular Ca2+ from 0.5 to 3.6 mM was reduced by nitrendipine. These results indicated that human antral cells expressed class C and D L-type VDCCs. Activation of G cells with beta-adrenergic agonists required an influx of extracellular Ca2+ through these channels to stimulate gastrin release. However, activation of L-type channels was not the only mechanism underlying Ca(2+)-stimulated gastrin release.
Trefoil peptide expression and secretion is regulated by neuropeptides and acetylcholine
Authors: Ogata H. , Podolsky D. K.
Published: December 2017

Trefoil peptides are a family of small proteins expressed by goblet cells that are secreted onto the apical gastrointestinal mucosal surface, where they are present in high concentrations. These peptides appear to both protect the epithelium and promote healing after injury. However, the factors regulating the expression and secretion of these proteins contributing to mucosal defense have not been characterized. To determine the mechanisms controlling production of trefoil peptides, the human colon cancer-derived model cell line HT-29 was exposed to a variety of potential secretagogues. Expression and secretion of human intestinal trefoil factor (hITF) as well as the intestinal apomucin MUC2 were assessed by Northern and Western blot analysis. Carbachol, an analog of acetylcholine, and the neuroendocrine peptides somatostatin and vasoactive intestinal polypeptide (VIP) stimulated increased expression of hITF mRNA within 5 min. These same factors stimulated parallel secretion of the hITF peptide, with maximal stimulation observed at concentrations ranging from 10(-6) M (carbachol and somatostatin) to 10(-7) M (VIP). Expression and secretion of hITF in response to carbachol, VIP, and somatostatin was independent of production of apomucin. hITF was not regulated by other neuroendocrine transmitters including histamine and substance P. Similarly, hITF expression and secretion was not modulated by peptide growth factors (epidermal growth factor, transforming growth factor-beta, and keratinocyte growth factor), cytokines [interleukin (IL)-1 beta, IL-2, IL-7, and IL-11], or arachidonic acid metabolites (prostaglandin E1/E2 and leukotriene B4). In conclusion, trefoil peptides appear to be integrated into mechanisms of mucosal defense and repair through the enteric neuroendocrine system and independent of the classical mucosal immune cytokine network.