bluuebunny/crossref_metadata_2025 huggingface dataset:
Uptake of taurocholic acid and cholic acid in isolated hepatocytes from rainbow trout
Authors: Rabergh C. M. , Ziegler K. , Isomaa B. , Lipsky M. M. , Eriksson J. E.
Published: December 2017

The uptake of the bile acids cholate (CHA) and taurocholate (TCHA) was studied in isolated hepatocytes from rainbow trout (Oncorhynchus mykiss). Both CHA and TCHA were taken up in a concentration- and temperature-dependent manner with optimum temperature at 15 degrees C and a strikingly efficient uptake even at low temperatures (0-5 degrees C). The total uptake was a combination of a saturable [Michaelis-Menten constant (Km) for CHA, 20 microM; Km for TCHA, 19 microM] and a nonsaturable component. The maximal uptake rate of the saturable component was 416 and 805 pmol.mg protein-1.min-1 for CHA and TCHA, respectively. The uptake of both bile acids was shown to be energy dependent, since it was inhibited by the metabolic inhibitors antimycin A, oligomycin and carbonyl cyanide m-chlorophenylhydrazone. The uptake was clearly Na+ independent, since isosmotic replacement of extracellular Na+ by Li+, choline, or K+ did not inhibit the uptake. Furthermore, it seemed to be independent of the presence of extracellular Cl-, since it was not inhibited by replacement of Cl- with sodium gluconate. On the whole, our results show that the hepatocellular uptake of bile acids in rainbow trout is mediated by a Na(+)-independent carrier system, with characteristics resembling the corresponding transport component in mammalian hepatocytes, but with high efficiency even at low temperatures.
Isotonic volume reduction associated with cAMP stimulation of 36Cl efflux from jejunal crypt epithelial cells
Authors: MacLeod R. J. , Lembessis P. , Hamilton J. R.
Published: December 2017

To determine the effect of 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) on the isotonic volume of jejunal crypt epithelial cells, we isolated these cells by sequential perfusion using a hyperosmolar Ca(2+)-free solution and measured cell volume electronically. 8-BrcAMP caused rapid shrinkage to a reduced but stable cell volume; this isotonic volume reduction was prevented by either a Cl(-)-channel blocker, anthracene-9-carboxylate (A-9C), or Ba2+, a K(+)-channel blocker. 8-BrcAMP substantially increased the rate of A-9C-sensitive 36Cl efflux from crypt cells; this increased rate of efflux was not influenced by Ba2+ but was abolished by alterations in membrane potential. Following 8-BrcAMP-stimulated isotonic volume reduction, addition of either Ba2+ or A-9C caused the crypt cells to reswell. In contrast, 8-BrcAMP added to enterocytes isolated from the villus compartment did not result in A-9C-sensitive volume reduction or in an increased rate of 36Cl efflux. Our data demonstrate that epithelial cells isolated from jejunal crypt compartments respond directly to cAMP with a rapid volume reduction that is paralleled by an increase in 36Cl efflux through a conductive pathway. Unlike other Cl- secretory epithelial cells, the intestinal crypt cell does not appear to regulate its volume in an isotonic medium after cAMP-induced shrinkage.
Regulation of intracellular pH in crypt cells from rabbit distal colon
Authors: Abrahamse S. L. , Vis A. , Bindels R. J. , van Os C. H.
Published: December 2017

H+ secretory mechanisms and intrinsic intracellular buffering capacity were studied in crypt cells from rabbit distal colon. To this end crypts of Lieberkuhn were isolated by microdissection, and intracellular pH (pHi) was measured using digital imaging fluorescence microscopy and the pH-sensitive fluorescent dye 2',7'-bis(2-carboxyethyl)- 5(6)-carboxyfluorescein. In the absence of HCO(3-)-CO2 and presence of Na+, resting pHi was 7.51 +/- 0.04 (n = 237/23, cells/crypts). However, 6 min after superfusion with a solution containing zero Na+, 1 x 10(5) M Sch-28080 and 5 x 10(-8) M bafilomycin A1, pHi in cells at the bottom of the crypts was significantly reduced, whereas pHi in cells at the top of the crypts remained unchanged. The intrinsic buffering capacity of cells from the middle to the top portion of crypts was significantly higher in the pHi range 7.2-7.6 than of cells at the bottom of the crypt. H+ secretion after an NH(4+)-NH3 pulse amounted to 245 +/- 53 microM/s (n = 73/7) at pHi 7.1 and was largely Na+ dependent and ethylisopropylamiloride sensitive. The Na(+)-independent recovery of pHi after an acid load was insensitive to Sch-28080 and bafilomycin A1. In conclusion, pHi in colonic crypt cells is regulated through Na+/H+ exchange activity in the absence of HCO3-. In addition, intracellular buffering capacity varied with the position along the crypt axis, whereas Na+/H+ exchange activity and pHi did not.
Role of 100-kDa cytosolic PLA2 in ACh-induced contraction of cat esophageal circular muscle
Authors: Sohn U. D. , Kim D. K. , Bonventre J. V. , Behar J. , Biancani P.
Published: December 2017

We have shown that acetylcholine (ACh)-induced contraction of esophageal circular muscle cells is mediated by activation of protein kinase C (PKC). We now examine the role of phospholipase A2 (PLA2). ACh increases [3H]arachidonic acid release in esophageal but not in lower esophageal sphincter (LES) muscle. In addition, ACh-induced contraction of esophageal but not of LES cells was reduced by the PLA2 antagonist dimethyleicosadienoic acid and by antiserum to a 100-kDa cytosolic PLA2 (cPLA2). These data suggest that the 100-kDa cPLA2 plays a role in ACh-induced contraction of esophageal but not of LES muscle. In esophageal cells, arachidonic acid produced by PLA2 caused little contraction by itself but potentiated contraction induced by the PKC agonist diacylglycerol (DAG). The free fatty acids linoleic acid and linolenic acid also potentiated DAG-induced contraction. Indomethacin and nordihydroguaiaretic acid had no effect on arachidonic acid-induced potentiation of DAG. The potentiation of DAG-induced contraction by arachidonic acid was inhibited by the PKC inhibitor H-7, but it was not affected by the calmodulin inhibitor CGS-9343B. We conclude that a 100-kDa cPLA2 participates in ACh-induced esophageal contraction by producing arachidonic acid and potentiating DAG-induced activation of a PKC-dependent pathway.
1,25(OH)2 vitamin D3 activates PKC-alpha in Caco-2 cells: a mechanism to limit secosteroid-induced rise in [Ca2+]i
Authors: Bissonnette M. , Tien X. Y. , Niedziela S. M. , Hartmann S. C. , Frawley B. P. , Roy H. K. , Sitrin M. D. , Perlman R. L. , Brasitus T. A.
Published: December 2017

Our laboratory recently reported that 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] rapidly increases the breakdown of membrane phosphoinositides, raises intracellular calcium concentration ([Ca2+]i), and translocates protein kinase C (PKC) from the cytosolic to the particulate fraction of Caco-2 cells. In the present experiments, we found that Caco-2 cells contained predominantly the alpha- and zeta-isoforms of PKC, with minimally detectable amounts of PKC-beta and -epsilon by Western blotting. 1,25(OH)2D3 and the PKC activator 12-O-tetradecanoylphorbol 13-acetate (TPA) each caused time-dependent translocations of PKC-alpha, but not PKC-zeta. TPA treatment of these cells for 24 h induced a significant concentration-dependent downregulation of PKC-alpha, but not PKC-zeta. Since PKC inhibits phospholipase C-induced mobilization of Ca2+ in other cells, we examined the effects of staurosporine and H-7, PKC inhibitors, and TPA on 1,25(OH)2D3-stimulated increase in [Ca2+]i. As previously demonstrated by our laboratory, 1,25(OH)2D3 caused a biphasic increase in [Ca2+]i, with an initial elevation (transient phase) followed by a sustained increase (plateau phase). We previously demonstrated that the transient phase is mediated, at least in part, by an increase in inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] stimulated by the secosteroid. Acute pretreatment with staurosporine or H-7 caused a significant stimulation, whereas acute TPA pretreatment caused a significant inhibition of the 1,25(OH)2D3-induced increase in the transient phase of [Ca2+]i. Preincubation of Caco-2 cells with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxy-methyl ester (BAPTA-AM) abolished both the rise in [Ca2+]i and the increase in particulate-associated PKC-alpha stimulated by 1,25(OH)2D3. Moreover, downregulation of PKC-alpha by chronic TPA treatment significantly augmented the transient phase of the 1,25(OH)2D3-stimulated rise in [Ca2+]i but had no effect on the 1,25(OH)2D3-induced change in Ins(1,4,5)P3 concentration. Furthermore, in these PKC-alpha downregulated cells staurosporine no longer increased the secosteroid-stimulated transient rise in [Ca2+]i. These results indicate that 1,25(OH)2D3, which increases [Ca2+]i and diacylglycerol, activates PKC-alpha, but not PKC-zeta. The alpha-isoform, in turn, limits the secosteroid-stimulated rise in [Ca2+]i, at a step distal to Ins(1,4,5)P3 accumulation in Caco-2 cells.
Inhibition of carbon tetrachloride-induced liver injury by liposomes containing vitamin E
Authors: Yao T. , Degli Esposti S. , Huang L. , Arnon R. , Spangenberger A. , Zern M. A.
Published: December 2017

We tested a variety of antioxidants as possible therapeutic agents in an acute CCl4 mouse model of hepatotoxicity. Liver damage, gauged by the amount of serum aminotransferase released into the blood, morphological changes, lethal dose response, and presence of thiobarbituric acid-reactive substances (TBARS), were significantly inhibited in a dose-dependent manner by liposomes containing vitamin E (LVE) or by Rocavit E, a water-soluble emulsion of alpha-tocopherol. Serum aminotransferase levels in LVE- or Rocavit E-treated animals were always > 10-fold lower than levels in corresponding CCl4 controls. Other liposome-associated antioxidants, butylated hydroxytoluene, vitamin E succinate, catalase, desferoxamine, superoxide dismutase, and ascorbic acid 6-palmitate, were also able to elicit a decrease in damage; however, they were substantially less effective. Intravenous therapy with LVE decreased mortality by nearly 90% when a lethal dose of CCl4 was given. When the biodistribution of the liposomes was examined, it was determined that the vast majority were localized in the Kupffer cell population. This approach of delivering nontoxic therapeutic agents selectively to the liver offers a variety of clinical applications in humans.
Evidence for age-associated reduction in acetylcholine release and smooth muscle response in the rat colon
Authors: Roberts D. , Gelperin D. , Wiley J. W.
Published: December 2017

The effect of aging was examined on cholinergically mediated contractions and acetylcholine (ACh) release in isolated colonic segments from Fischer (F344 x BN) F1 rats, 4-8 mo (postpubertal) and 22-28 mo (senescent) of age. This species demonstrates age-dependent slowing of colonic transit. Muscle tension response to electrical stimulation of cholinergic neural pathways and application of ACh was significantly decreased in preparations from senescent compared with postpubertal animals. We focused on the hypothesis that aging was associated with reduced ACh release that resulted from decreased calcium influx through membrane calcium channels. Aging did not affect either the synthesis of [3H]ACh from [3H]choline or the percentage of 3H released in the form of [3H]ACh. However, elevated KCl-evoked release of [3H]ACh was significantly reduced in tissue from senescent compared with postpubertal animals. Treatment with the calcium ionophore ionomycin increased [3H]ACh release in tissue from senescent animals to near postpubertal levels. However, increasing extracellular calcium concentration ([Ca2+]o) from 1.2 to 5 mM did not increase the amount of transmitter release in tissue from senescent animals to the levels observed with 1.2 mM [Ca2+]o in postpubertal tissue. The neuronal calcium channel antagonist omega-conotoxin GVIA inhibited acetylcholine release in a concentration-dependent manner with half-maximal inhibitory values of 1.8 and 8.2 nM for senescent and postpubertal preparations, respectively. In summary, age-dependent reduction in ACh release was observed in the rat colon myenteric plexus that may, in part, be associated with decreased calcium influx via membrane calcium channels.
Ileal tone in humans: effects of locoregional distensions and eating
Authors: Coffin B. , Lemann M. , Flourie B. , Picon L. , Rambaud J. C. , Jian R.
Published: December 2017

Using an electronic barostat in the human terminal ileum, we studied ileal tone in response to various stimuli in 10 healthy volunteers. Phasic activity was also measured by manometry. Graded orad (ileal) and caudad (cecal) balloon distensions did not produce any significant changes in ileal tone at all levels tested, even when a sensation of discomfort was felt. A linear relationship between pressure and volume was observed during in situ isobaric ileal distensions. Meal induced a biphasic tonic response with a brief (20 min) and immediate decrease in intrabag volume (64 +/- 7% of preprandial value, P < 0.05), followed by a prolonged increase in intrabag volume (158 +/- 10% of preprandial value, P < 0.05). A significant correlation was found between volume variations and phasic activities during the first phase (r = 0.88, P < 0.05), whereas no correlation was found during the second phase (r = 0.55, NS). The administration of glucagon induced a significant increase in intrabag volume from 45 +/- 5 to 70 +/- 8 ml (P < 0.05). We conclude that 1) regional distensions do not influence ileal tone and phasic motility, 2) the ileum does not show accommodation to distensions, and 3) meal produces an immediate increase in phasic and tonic activity followed by a prolonged relaxation.
Expression of IGF-II and IGF-binding proteins by colon cancer cells in relation to growth response to IGFs
Authors: Singh P. , Dai B. , Yallampalli C. , Xu Z.
Published: December 2017

We previously reported that even though virtually all human colon cancers were positive for IGF-I receptors, only 50% responded to growth effects of insulin-like growth factor (IGF)-I (1-100 nM). The present studies were undertaken to determine whether expression and secretion of IGFs (IGF-I, IGF-II) and IGF-binding proteins (BPs; 1-6) were perhaps different in IGF-responsive (COLO 205, COLO 320, Caco-2) and IGF-nonresponsive (HCT 116, HT-29, DLD-1) cells. Several bands (2.0-6.0 kb) of IGF-II mRNA transcripts were detected in all the cell lines; none expressed IGF-I. Significant concentrations of IGF-II (0.2-0.9 ng/10(6) cells) were measured in the conditioned media (CM) of the cells. All cell lines expressed BP2 and/or BP4 mRNA and secreted BP4 (24 kDa) and/or BP2 (32.5 kDa); BP1 was not detected in any cell line. Interestingly, BP3 mRNA was measured only in the responsive cell lines. The relative concentration of total BPs tended to be higher in the CM of nonresponsive cells. Interestingly, a large concentration of 44- to 48-kDa BP (BP3?) was associated with the membranes of only the responsive cell lines. Our present studies thus demonstrate that human colon cancers do not secrete IGF-I and BP1. Of all the IGF-related factors examined, the quantity and the type of BPs expressed by the human colon cancer cell lines (especially BP2, BP4, and BP3) may significantly dictate the growth response of the cells to exogenous IGF-I.
Interplay of somatostatin, opioid, and GABA neurons in the regulation of the peristaltic reflex
Authors: Grider J. R.
Published: December 2017

The interplay of somatostatin, gamma-aminobutyric acid (GABA), and opioid neurons in the regulation of the descending relaxation phase of peristalsis was examined in isolated rat colonic segments. Release of somatostatin, GABA, vasoactive intestinal peptide (VIP), and L-[3H]citrulline [coproduct and index of nitric oxide (NO) production] increased, and release of Met-enkephalin decreased, during descending relaxation. Somatostatin antiserum (1:50) inhibited GABA and L-[3H]citrulline and reversed Met-enkephalin from decrease below to increase above basal level; exogenous somatostatin had the opposite effect. Bicuculline (GABAA antagonist) inhibited L-[3H]citrulline, had no effect on somatostatin, and reversed Met-enkephalin from decrease below to increase above basal level; exogenous GABA had the opposite effect. Naloxone increased GABA and L-[3H]citrulline but had no effect on somatostatin; exogenous Met-enkephalin had the opposite effect. In all instances the changes in L-[3H]citrulline paralleled those previously obtained with VIP. The results are consistent with the operation of a circuit in which somatostatin neurons inhibit the activity of opioid neurons, causing a decrease in Met-enkephalin. The decrease in Met-enkephalin initiated by somatostatin is accentuated by a reciprocal inhibitory pathway linking GABA and opioid neurons. The decrease in Met-enkephalin eliminates the inhibitory influence of opioid neurons on VIP/NO neurons and leads to increase in VIP, NO, and descending relaxation.
Expression of intestinal fatty acid binding protein in intestinal epithelial cell lines, hBRIE 380 cells
Authors: Hallden G. , Holehouse E. L. , Dong X. , Aponte G. W.
Published: December 2017

Intestinal fatty acid binding protein (I-FABP) is a cytosolic protein present only in differentiated intestinal epithelial cells. Here we report on an intestinal cell culture system expressing I-FABP during cell differentiation and the modulation of expression by extracellular factors. An I-FABP-expressing cell line (hBRIE 380i) was generated from Berkeley rat intestinal epithelial cells (hBRIE 380). Time- and substratum-dependent changes in I-FABP mRNA expression were paralleled by changes in protein levels. Induction of I-FABP levels observed on collagen type I gels in the presence of limiting serum was prevented by insulin. When cells were grown on collagen gels containing fibronectin and laminin, a stimulation of ultrastructural characteristics of cell differentiation was observed with no further induction of I-FABP expression. The data show that I-FABP expression is limited to a differentiated population of hBRIE 380i cells and that the expression can be regulated by factors present in the extracellular matrix as well as involved in regulation of replication or metabolic state of the cell.
Mechanisms of PTH-induced rise in cytosolic calcium in adult rat hepatocytes
Authors: Klin M. , Smogorzewski M. , Khilnani H. , Michnowska M. , Massry S. G.
Published: December 2017

Available data indicate that the liver is a target organ for parathyroid hormone (PTH) and that this effect is most likely mediated by PTH-induced calcium entry into hepatocytes. The present study examined the effects of both PTH-(1-84) and its amino-terminal fragment [PTH-(1-34)] on cytosolic calcium concentration ([Ca2+]i) of hepatocytes and explored the cellular pathways that mediate this potential action of PTH. Both moieties of PTH produced a dose-dependent rise in [Ca2+]i, but the effect of PTH-(1-84) was greater (P < 0.01) than an equimolar amount of PTH-(1-34). This effect required calcium in the medium and was totally [PTH-(1-34)] or partially [PTH-(1-84)] blocked by PTH antagonist ([Nle8,18,Tyr34]bPTH-(7-34)-NH2] and by verapamil or nifedipine. Sodium or chloride channel blockers did not modify this effect. 12-O-tetradecanoylphorbol 13-acetate (TPA), an activator of protein kinase C, dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP), and G protein activator also produced a dose-dependent rise in [Ca2+]i. Staurosporine abolished the effect of TPA, and both staurosporine and calphostin C partially inhibited the effect of PTH. Staurosporine and verapamil together produced greater inhibition of PTH action than each alone. Rp-cAMP, a competitive inhibitor of cAMP binding to the R subunit of protein kinase A, and N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), a protein kinase A inhibitor, blocked the effect of both DBcAMP and PTH, but the effect of these agents was greater (P < 0.01) on DBcAMP action. G protein inhibitor and pertussis toxin partially blocked the action of PTH. The data indicate that 1) PTH increases [Ca2+]i of hepatocytes; 2) this action of the hormone is receptor mediated; 3) the predominant pathway for this PTH action is the stimulation of a G protein-adenylate cyclase-cAMP system, which then leads to stimulation of a calcium transport system inhibitable by verapamil or nifedipine or activation of L-type calcium channels; 4) activation of protein kinase C is also involved; and 5) the PTH-induced rise in [Ca2+]i is due, in major parts, to movement of extracellular calcium into the cell.
Evidence for an apical, nonregulated protein secretion in pig exocrine pancreas
Authors: Viau G. , Laine J. , Levenez F. , Gueugneau A. M. , Corring T. , Morisset J. , Lebel D.
Published: December 2017

Secretory proteins are segregated into two pathways out of the trans-Golgi network of regulated secretory cells. To identify proteins specifically secreted by pathways other than the one leading to zymogen granule exocytosis in the exocrine pancreas, conscious permanently cannulated pigs were perfused with atropine to inhibit the regulated fusion of granules. Atropine almost totally inhibited the protein secretion after 1 h of perfusion. The secretion of GP-2, a glycosyl phosphatidylinositol-anchored protein of the zymogen granule membrane, was partially inhibited but was never totally abolished by atropine perfusion. The pattern of proteins secreted under atropine was almost totally different. Soluble GP-2 was the major secretory product. Its specific activity increased 60 times over its normal level in all other conditions. This secretion clearly originated from nonregulated pathways. Results suggest that during the atropine block the apical plasmalemma could be the source of the released GP-2 and that the sustained nature of this release is compatible with a replenishment of the plasmalemma with GP-2 by the continuous exocytosis of vesicles from the nonregulated pathways.
Intestinal myoelectric alterations in rats chronically infected with the tapeworm Hymenolepis diminuta
Authors: Dwinell M. B. , Bass P. , Oaks J. A.
Published: December 2017

This study determined that intestinal myoelectric activity was profoundly altered during a strictly luminal, chronic, tapeworm infection. Chronically implanted bipolar electrodes were attached to five sites on the serosal surface of the rat small intestine. One was placed on the duodenum, three on the jejunum, and the fifth on the ileum. Electromyographic recording in nonfasted unanesthetized animals was begun at day 5 postsurgery. All electromyographic recordings were analyzed for slow wave (SW) frequency, phase III frequency, duration of phase III, and percentage of SW with spike potentials. Three initial control recordings prior to infection confirmed the presence of normal interdigestive motility characterized by the three phases (I, II, III) of the migrating myoelectric complex (MMC). Two nonpropulsive myoelectric alterations were observed in infected animals: the repetitive bursts of action potentials (RBAP) and periods of sustained spike potentials (SSP). Myoelectric activity from infected animals indicated decreased cycling of the interdigestive MMC. RBAP and SSP were more prevalent in the distal small intestine corresponding to tapeworm location. The percent of spike potential activity indicated that there was a reversal in the spike potential gradient on the small intestine. The number of spike potentials was maximal in caudal and minimal in oral intestine. We propose that overall localized increases in myoelectric spike potential activity represent increased contractility and decreased propulsion triggered by the presence of the tapeworm. These motility changes were surprising, since the tapeworm Hymenolepis diminuta does not penetrate the intestinal mucosa. This interaction between parasite and host may prevent expulsion of the tapeworm from the small intestine.
Characterization of a Y1-preferring NPY/PYY receptor in HT-29 cells
Authors: Mannon P. J. , Mervin S. J. , Sheriff-Carter K. D.
Published: December 2017

Equilibrium binding studies showed that butyrate-treated HT-29 cells express a high-affinity 125I-labeled peptide YY (125I-PYY) binding site with a dissociation constant of 0.32 +/- 0.12 nM (mean +/- SE, n = 4). This site was Y1 preferring because neuropeptide Y (NPY) and the Y1-selective agonist [Leu31,Pro34]NPY were equipotent to PYY at displacing 125I-PYY; PYY-(13-36) and pancreatic polypeptide were > 1,000- and 10,000-fold less potent at displacing the radioligand. PYY and [Leu31,Pro34]NPY inhibited forskolin-stimulated adenosine 3',5'-cyclic monophosphate production 63% and 48%, respectively, with a half-maximal inhibitory concentration between 0.1 and 1.0 nM. PYY and [Leu31,Pro34]NPY had no effect on release of intracellular calcium alone or on the increase in intracellular calcium concentration caused by carbachol or neurotensin. Northern blot analysis of poly(A)+ RNA from HT-29 cells demonstrated a single transcript of 2.5 kb that hybridized to a human Y1-receptor cDNA probe. Sequence analysis of a reverse transcription-polymerase chain reaction product amplified with primers based on human Y1-receptor cDNA confirmed that these cells contained mRNA encoding the human Y1 receptor. These studies show that butyrate-treated HT-29 cells constitutively express the Y1-preferring NPY/PYY receptor and Y1 mRNA and provide a new model for studies of PYY-regulated epithelial cell function and tissue-specific expression of the human Y1-receptor gene.
Hepatic ischemia/reperfusion injury: importance of oxidant/tumor necrosis factor interactions
Authors: Scales W. E. , Campbell D. A. , Green M. E. , Remick D. G.
Published: December 2017

Previous studies have demonstrated a role for both tumor necrosis factor (TNF) and reactive oxygen intermediates (ROI) in hepatic ischemia/reperfusion (I/R) injury. Biologically active TNF was present in liver homogenates in ischemic and nonischemic lobes after 2 h of ischemia but without reperfusion. Using an in situ liver perfusion model, we measured ROI, TNF, and hepatic enzymes in the effluent after 2 h of ischemia. Increased reduction of ferricytochrome C was observed in the hepatic effluent, indicative of the formation of ROI. Treatment of animals with TNF neutralizing antisera significantly reduced both ROI and aspartate aminotransferase (AST). Animals treated with superoxide dismutase (SOD), or SOD + catalase (CAT) had greater TNF in the hepatic effluent compared with I/R alone; however, SOD or SOD + CAT did not cause additional release of AST.SOD + CAT plus anti-TNF serum resulted in significant protection compared with SOD + CAT plus control serum. Reperfusion of ischemic liver with 4 mM H2O2 increased both TNF and AST. Optimal protection of hepatocellular injury from reperfusion injury is achieved with a combination of antioxidants and inhibition of TNF.
Effects of continued NO inhibition on portal hypertensive syndrome after portal vein stenosis in rat
Authors: Garcia-Pagan J. C. , Fernandez M. , Bernadich C. , Pizcueta P. , Pique J. M. , Bosch J. , Rodes J.
Published: December 2017

The present study investigated whether chronic nitric oxide (NO) inhibition prevents the hyperdynamic circulatory syndrome that appears in rats after partial portal vein ligation (PPVL). N omega-nitro-L-arginine methyl ester (L-NAME; 30 micrograms.kg-1.min-1, n = 17), a NO biosynthesis inhibitor, or vehicle (n = 17) was infused continuously from PPVL through subcutaneously osmotic pumps. Studies were performed, in ketamine-anesthetized Sprague-Dawley rats, in one-half of the animals at 4 days and in the remaining one-half at 8 days from PPVL. At 4 days, PPVL rats treated with L-NAME had higher mean arterial pressure (MAP), systemic vascular resistance (SVR), and splanchnic arteriolar resistance (SAR) and lower cardiac output and portal venous inflow (PVI) than PPVL rats treated with vehicle (P < 0.05). Similarly, at 8 days PPVL rats treated with L-NAME had higher MAP and SVR and lower cardiac output (P < 0.05) than PPVL rats treated with vehicle. In contrast, PVI and SAR were similar. At 4 days plasma volume and mesenteric-systemic shunting were lower, although nonsignificantly, in PPVL rats treated with L-NAME. This trend completely disappeared at 8 days. L-NAME did not change portal pressure at either 4 or 8 days. After 4 days of continuous treatment with L-NAME, nonportal hypertensive control rats had a significantly higher MAP, lower cardiac index and PVI, and higher SVR and SAR than nonportal hypertensive rats treated with vehicle. Contrary to PPVL rats, these effects were maintained after 8 days of treatment. The present study shows that NO contributes to the systemic disturbances of portal hypertension. However, NO inhibition delayed but did not prevent the splanchnic vasodilation that appears after PPVL, suggesting that other factors could also be involved.
PGE generates intracellular cAMP and accelerates mucin secretion by cultured dog gallbladder epithelial cells
Authors: Kuver R. , Savard C. , Oda D. , Lee S. P.
Published: December 2017

Mucin is the main secretory product of gallbladder epithelial cells. Increased gallbladder mucus secretion has been implicated in gallstone formation in humans. The mechanisms underlying control of mucin synthesis and secretion by the gallbladder are not known. This study aimed to elucidate the efficacy of a panel of secretagogues to stimulate mucin secretion and to determine the intracellular second messengers involved. Studies were carried out on normal well-differentiated epithelial cells from dog gallbladder grown in monolayer culture. Intracellular adenosine 3',5'-cyclic monophosphate (cAMP) as measured by radioimmunoassay increased in response to prostaglandin (PG) E2, PGE1, vasoactive intestinal peptide, epinephrine, and isoproterenol. The greatest effect, a 37-fold increase in cAMP level, was noted with PGE2 at 1.0 microM concentration. In contrast, three breakdown products of phosphatidylinositol (inositol triphosphate, inositol bisphosphate and inositol monophosphate) were not detected with any of the secretagogues tested. Assay of mucin secretion using tritiated N-acetyl-D-glucosamine, a mucin precursor, showed that the same secretagogues noted to increase intracellular cAMP led to an increase in mucin secretion. No correlation was noted, however, between the magnitude of the intracellular cAMP rise and the amount of mucin secreted. A membrane-permeable form of cAMP, dibutyryl cAMP, mimicked PGE2-induced mucin secretion. The results unequivocally show that secretagogue-stimulated mucin secretion in these normal gallbladder epithelial cells can proceed via a cAMP signal transduction pathway.
Prolactin increases mRNA encoding Na(+)-TC cotransport polypeptide and hepatic Na(+)-TC cotransport
Authors: Liu Y. , Ganguly T. , Hyde J. F. , Vore M.
Published: December 2017

We have shown that prolactin (Prl) increases the transhepatic transport of taurocholate (TC) in postpartum rats and following treatment of ovariectomized (Ovx) rats with ovine Prl (oPrl). The present studies were designed to determine if treatment of Ovx rats with oPrl (100, 300, or 600 micrograms/day, 7 days iv) 1) increases Na(+)-TC cotransport in basolateral plasma membrane vesicles (bLPM), 2) induces a corresponding increase in the steady-state levels of Na(+)-TC cotransport polypeptide (Ntcp mRNA), and 3) if the oPrl-mediated increase in Na(+)-TC cotransport activity is blocked by cycloheximide, an inhibitor of protein synthesis. oPrl (300 micrograms/day) induced a twofold increase in the maximal velocity for Na(+)-TC cotransport in both hepatocytes and bLPM vesicles with little change in the Michaelis constant. Infusion of oPrl at a dose of 100, 300, or 600 micrograms/day increased steady-state Ntcp mRNA four-, ten-, and twofold, respectively. Finally, cycloheximide blocked the oPrl-mediated increase in Na(+)-TC cotransport but did not affect basal activity. These data support the hypothesis that an increase in Ntcp mRNA followed by increased synthesis and incorporation of Ntcp in the plasma membrane is responsible for the oPrl-mediated increase in Na(+)-TC cotransport in the basolateral plasma membrane domain of the hepatocyte.
Phenylalanine-stimulated secretion of cholecystokinin is calcium dependent
Authors: Mangel A. W. , Prpic V. , Wong H. , Basavappa S. , Hurst L. J. , Scott L. , Garman R. L. , Hayes J. S. , Sharara A. I. , Snow N. D. , et al.
Published: December 2017

The secretion of cholecystokinin was examined in STC-1 cells, an intestinal cholecystokinin (CCK)-secreting cell line. Exposure to the amino acid L-phenylalanine increased release of CCK by 135%, 180%, and 251% of control levels after 15-min treatments with 5, 20, and 50 mM phenylalanine, respectively. L-Phenylalanine-induced secretion of CCK was inhibited by the calcium channel blocker diltiazem (10 microM). L-Phenylalanine (20 mM) also significantly increased cytosolic calcium levels in fura 2-acetoxymethyl ester (fura 2-AM)-loaded cells, and this increase was diltiazem sensitive. D-Phenylalanine, over the dose range of 5-50 mM, produced nonsignificant increases in CCK release. Treatment of STC-1 cells with 300 ng/ml of pertussis toxin for either 4 or 24 h did not significantly affect either basal release of CCK or L-phenylalanine-stimulated secretion. Patch-clamp recordings from cell-attached membrane patches showed a stimulation in calcium channel activity after L-phenylalanine. These results indicate that, in STC-1 cells, L-phenylalanine stimulates release of cholecystokinin via a calcium-dependent process.
Characterization of PGE2 receptors in isolated rabbit colonic crypt cells
Authors: Homaidan F. R. , Zhao L. , Burakoff R.
Published: December 2017

The physiological effects of prostaglandins (PG) are mediated through their interactions with specific receptors on effector cells. In this study the properties of PGE2 receptors in the rabbit distal colon were examined. We report the presence of specific, saturable, and high-affinity binding sites of PGE2 of the EP2 subtype in isolated colonic crypts. Scatchard analysis revealed the presence of two binding sites with dissociation constants of 0.3 and 10.8 nM and corresponding maximum number of receptors of 15 and 134 fmol/10(6) cells. From competition experiments in the presence of guanosine 5'-O-(3-thiotriphosphate), PGE2 binding was decreased, suggesting that the receptor is coupled to a G protein. No PGE2 binding sites were detected in surface cells. Levels of adenosine 3',5'-cyclic monophosphate (cAMP) were measured in isolated epithelial cells after being exposed to different concentrations of PGE2. cAMP levels were significantly increased only in the crypt cells when exposed to PGE2. These data provide the first demonstration for the existence of PGE2 receptors on colonic crypt cells, which when activated lead to increased levels of cAMP.
The phrenic ampulla: distal esophagus or potential hiatal hernia?
Authors: Lin S. , Brasseur J. G. , Pouderoux P. , Kahrilas P. J.
Published: December 2017

The mechanics of phrenic ampullary emptying were analyzed to determine whether this structure functions in a manner similar to the tubular esophagus or a hiatal hernia. Simultaneous videofluoroscopy and intraluminal manometry of the gastroesophageal junction were done during barium swallows in 18 normal volunteers. Esophageal emptying was studied without any external influences, during abdominal compression with a cuff inflated to 100 mmHg, during a Muller maneuver, and after medication with atropine. The key finding of the study was that ampullary emptying was distinct from esophageal bolus transport in several ways: the propagation velocity of the clearing wave was slower, the maximal contact pressures achieved after luminal closure were lower and unaffected by atropine or outflow obstruction, and ampulary emptying was driven by a hydrostatic pressure difference between the ampulla and stomach rather than by a peristaltic contraction. Increased bolus volume slightly enlarged the ampulla. Taken together, these findings suggest that ampullary emptying occurs, in part, as a result of the restoration of esophageal length (presumably by tension from the phrenoesophageal membrane) rather than as a result of an aborally propagated contraction. As such, a normal phrenic ampulla is analogous to a small reducing hiatal hernia. We speculate that overt hernia formation occurs as a result of progressive degeneration of the phrenoesophageal membrane.
Chyme transport patterns in human duodenum, determined by multiple intraluminal impedancometry
Authors: Nguyen H. N. , Silny J. , Wuller S. , Marschall H. U. , Rau G. , Matern S.
Published: December 2017

To get information about the mechanisms involved in chyme transport during the fasting and postprandial states, the novel procedure of multiple intraluminal impedancometry was evaluated in 14 healthy subjects (6 during fasting, 8 after a test meal). All main features of the migrating motor complex (MMC) cycle were determined. During phase II of the MMC cycle and the postprandial period, different transport patterns of chyme, termed bolus transport events (BTEs), were determined. These were 1) simple long-distance propulsive transport (spreading distance > 16 cm), 2) short-distance propulsive transport, and 3) retrograde transport. A significantly lower number of BTEs was recorded during fasting than postprandially. Short-distance propulsive BTEs predominated during fasting (72%), and long-distance propulsive BTEs predominated after the test meal (76%). Retrograde BTEs were recorded during fasting (4%) and postprandially (8%). In the latter state, complex long-distance propulsive BTEs were also observed (5%), consisting of multiple components. A major proportion of gastric contents was found to be continuously transported to jejunum. In conclusion, impedancometry enables us to determine patterns and parameters of chyme transport during fasting and postprandial states.
K+ channel opening mediates hyperpolarizations by nitric oxide donors and IJPs in opossum esophagus
Authors: Cayabyab F. S. , Daniel E. E.
Published: December 2017

The ionic mechanisms by which nitric oxide (NO) or a related compound mediates the inhibitory junction potentials (IJPs) of the opossum esophageal circular smooth muscle were studied using microelectrodes and double sucrose gap. The NO donors, 3-morpholino-sydnonimine hydrochloride and sodium nitroprusside, induced 15- to 20-mV hyperpolarizations that reversed near the potassium equilibrium potential as did the IJPs. They inhibited the IJPs and decreased electrotonic potentials (increased conductance) even during restoration of the resting membrane potential by application of depolarizing current. Quinine was more efficacious than apamin in inhibiting the IJPs or NO donor hyperpolarizations, whereas the other K+ channel blockers tested (tetraethylammonium, charybdotoxin, 4-aminopyridine, Cs+, and glibenclamide) were without effect. Glibenclamide abolished the hyperpolarizing effects of the K+ channel opener BRL-34915. Low Cl- Krebs (isethionate substitutions) caused hyperpolarizations, increased electrotonic potentials, and reduced IJPs. The neural blockers, tetrodotoxin, omega-conotoxin GVIA, and N omega-nitro-L-arginine methyl ester, inhibited IJPs but not the responses to NO donors, indicating a postjunctional effect. Methylene blue and cystamine, soluble guanylate cyclase inhibitors, suppressed IJPs and responses to NO donors. We conclude that NO mediates esophageal IJPs, which depend on guanosine 3',5'-cyclic monophosphate elevation and activation of quinine- and apamin-sensitive K+ channels.
Interferon-gamma primes neutrophil-mediated gastric surface cell cytotoxicity
Authors: Lieser M. J. , Kozol R. A. , Tennenberg S. D.
Published: December 2017

The T lymphocyte product interferon-gamma (IFN-gamma) upregulates or primes polymorphonuclear leukocyte (PMN) oxidative responses to the receptor-initiated stimulant N-formyl-methionyl-leucyl-phenylalanine (FMLP) but not to the transduction-mediated stimulant phorbol myristate acetate (PMA). We sought a functional correlation between IFN-gamma-induced oxidative priming of PMNs and PMN-mediated cytotoxicity, using an in vitro assay of 51Cr release from rabbit gastric surface cells. Compared with control PMNs, IFN-gamma-primed PMNs exhibited a significant increase in cytotoxicity when stimulated with FMLP, but not when stimulated with PMA. IFN-gamma-induced, FMLP-stimulated, PMN-mediated cytotoxicity was reduced by adding superoxide dismutase to scavenge superoxide anion or by adding catalase or glutathione peroxidase to scavenge hydrogen peroxide. Cytotoxicity was also reduced by inhibiting myeloperoxidase activity with azide or scavenging HOCl with alanine or methionine. Cytotoxicity was blocked by a monoclonal antibody against the CD11/CD18 integrin of PMNs. The results indicate that the immunoregulatory lymphokine IFN-gamma primes the FMLP-stimulated cytotoxic activity of PMNs via the increased generation of reactive oxygen metabolites and indicate that cytotoxicity may require effector-target cell adherence. Therefore, T lymphocyte-derived IFN-gamma may have a role in the pathogenesis of PMN-mediated injury to gastric and gastrointestinal tract mucosa.
Galactosylated proteins are recognized by the liver according to the surface density of galactose moieties
Authors: Nishikawa M. , Miyazaki C. , Yamashita F. , Takakura Y. , Hashida M.
Published: December 2017

The recognition of 111In-labeled galactosylated superoxide dismutase (Gal-SOD) and galactosylated bovine serum albumin (Gal-BSA) by the liver was investigated in mice after intravenous injection. 111In-labeled galactosylated proteins were recovered in the liver by amounts that were highly dependent on the degree of galactose modification and the administered dose. The distribution patterns were analyzed based on a physiological pharmacokinetic model including an uptake process with Michaelis-Menten kinetics in the liver and hepatic plasma flow. The Michaelis constant of hepatic uptake of 111In-Gal-SOD was observed to inversely correlate with the number of galactose residues, without a significant change in maximum rate of uptake or extrahepatic clearance. This relation could be applied to 111In-Gal-BSA and other galactosylated proteins by using the surface density of galactose residues as a degree of modification, suggesting galactose density controls ligand recognition by the asialoglycoprotein receptor. The analysis also indicated that increasing galactose density higher than 1.0 x 10(-3) molecules/A2 did not affect the distribution of galactosylated proteins due to limitation by the hepatic plasma flow rate. In conclusion, efficient delivery of proteins modified with galactose to the liver will be achieved by controlling both the galactose density on the protein surface and the administered dose.
Secretin at physiological doses inhibits gastric motility via a vagal afferent pathway
Authors: Lu Y. , Owyang C.
Published: December 2017

Secretin is an important modulator of gastric motility. In this study, we investigated the site(s) and mechanism(s) of action of secretin to inhibit gastric motility, using an in vivo rat model. Intragastric pressure response to graded doses of secretin was recorded in anesthetized rats by a balloon attached to a catheter passed through an incision in the duodenum into the body of the stomach. The intragastric pressure was set at 10 cmH2O with balloon distension. Intravenous infusion of secretin (1.4, 2.8, 5.6, 11.2, and 22.4 pmol.kg-1.h-1) decreased intragastric pressure in a dose-dependent manner. The threshold dose was 2.8 pmol.kg-1.h-1, and the effective dose at 50% (ED50) was 5.6 pmol.kg-1.h-1, which produced physiological levels of plasma secretin. Pretreatment with hexamethonium (10 mg/kg) markedly reduced gastric motor response to secretin (5.6 pmol.kg-1.h-1). Bilateral truncal vagotomy also significantly diminished gastric motor responses to secretin. In contrast, secretin (5.6 pmol.kg-1.h-1) had no effect on gastric contraction evoked by electrical vagal stimulation (1.25-5 Hz) or carbachol (10(-6) to 3 x 10(-5) M). These observations indicate that physiological concentrations of secretin act via stimulation of presynaptic cholinergic neurons in a vagally mediated pathway. In subsequent studies, we demonstrated that perivagal treatment 4 days before with the sensory neurotoxin, capsaicin, abolished gastric motor response to secretin but did not affect contraction evoked by electrical vagal stimulation. Similarly, we also showed that gastroduodenal application of capsaicin for 30 min also markedly reduced gastric response to secretin. These observations indicate that physiological doses of secretin act on vagal afferent pathways originating from the gastroduodenal mucosa to induce gastric relaxation.
Activation of MAP kinase kinase (MEK) and Ras by cholecystokinin in rat pancreatic acini
Authors: Duan R. D. , Zheng C. F. , Guan K. L. , Williams J. A.
Published: December 2017

Cholecystokinin (CCK) has recently been shown to activate mitogen-activated protein (MAP) kinase in rat pancreatic acini [Duan and Williams, Am. J. Physiol. 267 (Gastrointest. Liver Physiol. 30): G401-G408, 1994]. To evaluate the mechanism of MAP kinase activation, we studied the effects of CCK on MAP kinase kinase (MEK) in rat pancreatic acini. Two forms of MEK were identified by immunoblotting, using antibodies specific to MEK1 and MEK2. MEK activity in acinar extracts and after immunoprecipitation with anti-MEK was detected using a recombinant fusion protein, glutathione S-transferase-MAP kinase, as a substrate. MEK activity rapidly increased after stimulation of acini by CCK, with significant stimulation at 1 min and a maximal effect at 5 min, followed by a slow decline to slightly above control levels after 30 min. The threshold concentration of CCK was approximately 10 pM, and the maximal effect was induced by 1 nM CCK, which increased MEK activity by 120%. In addition to CCK, bombesin and carbachol, but not secretin or vasoactive intestinal peptide, enhanced MEK activity. Phorbol ester mimicked the effect of CCK, whereas ionomycin and thapsigargin failed to activate MEK. We further studied the activation of Ras, an important component leading to activation of MEK by growth factors. Ras in acini was immunoprecipitated and identified by Western blotting. CCK and 12-O-tetradecanoylphorbol-13-acetate stimulated the incorporation of GTP into Ras, a requirement for its activation, reaching a maximum at 10 min of approximately 120% over control. In conclusion, the activation of MAP kinase by CCK can be explained by activation of MEK and may involve the activation of Ras by a protein kinase C-dependent mechanism.
Dual regulation of cGMP formation by calcium in pancreatic acinar cells
Authors: Gukovskaya A. S. , Pandol S. J.
Published: December 2017

Regulation of guanosine 3',5'-cyclic monophosphate (cGMP) formation by calcium and calcium-binding proteins was studied at the levels of nitric oxide synthase (NOS) and guanylyl cyclase (GC) in dispersed pancreatic acini isolated from guinea pig. In intact cells, in the cytosol, and on diethylaminoethyl fractions from cytosolic proteins, GC activity was negatively regulated by Ca2+. An increase in Ca2+ concentration ([Ca2+]) from 25 to 950 nM suppressed cGMP formation by 85%. On the other hand, NOS was stimulated by agents increasing cytosolic [Ca2+] and inhibited by intracellular Ca2+ chelators. Thus Ca2+ regulates cGMP production in opposite directions by activating NOS and inhibiting GC. Calmodulin antagonists W-7, trifluoperazine, and R-24571 inhibited NOS, suggesting that the enzyme is regulated by calmodulin as in other cell types. Calmodulin antagonists appeared to inhibit GC. In particular, 200 microM W-7 completely abolished the cGMP rise evoked by the nitric oxide donor, nitroprusside. The effect was not reversed by addition of excess calmodulin. The findings suggest that the negative regulation of GC by Ca2+ is due to factors other than calmodulin but affected by calmodulin antagonists.