bluuebunny/crossref_metadata_2025 huggingface dataset:
LPS receptor CD14 participates in release of TNF-α in RAW 264.7 and peritoneal cells but not in Kupffer cells
Authors: Lichtman Steven N. , Wang Jian , Lemasters John J.
Published: December 2017

Lipopolysaccharide (LPS) is a bacterial polymer that stimulates macrophages to release tumor necrosis factor-α (TNF-α). In macrophages (RAW 264.7 and peritoneal cells), LPS binds to the CD14 surface receptor as the first step toward signaling. Liver macrophages, Kupffer cells, are the most numerous fixed-tissue macrophage in the body. The presence of CD14 on Kupffer cells and its role in LPS stimulation of TNF-α were examined. TNF-α release by Kupffer cells after LPS stimulation was the same in the presence and absence of serum. RAW 264.7 and peritoneal cells, which utilize the CD14 receptor, released significantly less TNF-α after LPS stimulation in the absence of serum because of the absence of LPS-binding protein. Phosphatidylinositol-phospholipase C treatment, which cleaves the CD14 receptor, decreased LPS-stimulated TNF-α release by RAW 264.7 cells but not by Kupffer cells. Deacylated LPS (dLPS) competes with LPS at the CD14 receptor when incubated in a ratio of 100:1 (dLPS/LPS). Such competition blocked LPS-stimulated TNF-α release from RAW 264.7 cells but not from Kupffer cells. Western and fluorescence-activated cell sorter analysis directly demonstrated the presence of CD14 on RAW 264.7 cells and murine peritoneal cells but showed only minimal amounts of CD14 in murine Kupffer cells. LPS stimulation did not increase the amount of CD14 detectable on mouse Kupffer cells. CD14 expression is very low in Kupffer cells, and LPS-stimulated TNF-α release is independent of CD14 in these cells.
Expression of endothelial nitric oxide synthase in human and rabbit gastrointestinal smooth muscle cells
Authors: Teng B.-Q. , Murthy K. S. , Kuemmerle J. F. , Grider J. R. , Sase K. , Michel T. , Makhlouf G. M.
Published: December 2017

The aim of this study was to identify the nitric oxide synthase (NOS) isoform expressed in freshly dispersed rabbit gastric smooth muscle cells and in cultured rabbit gastric, human intestinal, and guinea pig taenia coli smooth muscle cells. RT-PCR products of the predicted size (354 bp) were obtained with endothelial NOS (eNOS)-specific primers, but not neuronal NOS (nNOS)- or inducible NOS (iNOS)-specific primers, in all smooth muscle preparations except guinea pig taenia coli. Control RT-PCR studies showed absence of the endothelial markers, platelet endothelial cell adhesion molecule-1 (PECAM-1) and vascular endothelial growth factor receptor (VEGFR), and the interstitial cell marker, c- kit, from cultures of smooth muscle cells. Cloning and sequence analysis showed that the predicted amino acid sequence (117 residues) in rabbit and human smooth muscle cells differed by only one residue from that of human eNOS. Northern blot analysis, using the PCR-generated and cloned eNOS cDNA from rabbits and humans as probes, demonstrated the expression of eNOS mRNA (4.4 kb) in both species. eNOS, but not nNOS or iNOS, transcripts were localized by in situ RT-PCR in single, freshly dispersed rabbit gastric smooth muscle cells; expression was evident in the majority of cells in each preparation. We conclude that eNOS is selectively expressed in rabbit gastric and human intestinal smooth muscle cells. The results confirm functional evidence for the existence of a constitutive NOS in smooth muscle cells of the gut in different species, except for guinea pig taenia coli.
Immunolocalization of gastrin-dependent histidine decarboxylase activity in rat gastric mucosa during feeding
Authors: Ohning Gordon V. , Song Min , Wong Helen C. , Wu S. Vincent , Walsh John H.
Published: December 2017

The localization of histidine decarboxylase (HDC) activity in the enterochromaffin-like (ECL) cells of the oxyntic mucosa was studied during fasting and refeeding using monoclonal (CURE no. 44178) and polyclonal (CURE no. 94211) antibodies directed against the COOH terminus of HDC (HDC-CT). Changes in HDC immunostaining were correlated with mucosal HDC enzyme activity. Immunoneutralization of circulating gastrin and atropine treatment during refeeding were used to determine the relative importance of gastrin and cholinergic mechanisms in the regulation of HDC activity and immunostaining. Fasting caused a rapid reduction in the number of ECL cells immunostaining for HDC that was correlated with an almost complete loss of mucosal HDC enzyme activity. Refeeding restored both HDC immunostaining and enzyme activity within 2–4 h, and this response was inhibited by gastrin immunoneutralization but not by atropine treatment. Immunostaining was uniformly decreased and restored in the lower half of the oxyntic mucosa, which corresponds to the predominant area of ECL cells in the gastric gland. Histamine immunostaining and mucosal histamine content were not significantly changed during fasting and refeeding or by gastrin antibody and/or atropine treatment during refeeding. These findings indicate that HDC activity correlates with HDC-CT immunostaining and that both HDC activity and HDC-CT immunostaining are regulated by gastrin during refeeding.
Inhibition of Na+/H+exchange stimulates CCK secretion in STC-1 cells
Authors: Prpic Veronica , Fitz J. Gregory , Wang Yu , Raymond John R. , Garnovskaya Maria N. , Liddle Rodger A.
Published: December 2017

It has been demonstrated that K+channel regulation of membrane potential is critical for control of CCK secretion. Because certain K+channels are pH sensitive, it was postulated that pH affects K+channel activity in the CCK-secreting cell line STC-1 and may participate in regulating CCK secretion. The present study examines the role of electroneutral Na+/H+exchange on extracellular acidification and hormone secretion. Treatment of STC-1 cells with the amiloride analog ethylisopropyl amiloride (EIPA) to inhibit Na+/H+exchange inhibited Na+-dependent H+efflux and increased basal CCK secretion. Substituting choline for NaCl in the extracellular medium elevated basal intracellular Ca2+concentration and stimulated CCK release. Stimulatory effects on hormone secretion were blocked by the L-type Ca2+channel blocker diltiazem, indicating that secretion was dependent on the influx of extracellular Ca2+. To determine whether the effects of EIPA and Na+depletion were due to membrane depolarization, we tested graded KCl concentrations. The ability of EIPA to increase CCK secretion was inhibited by depolarization induced by 10–50 mM KCl in the bath. Maneuvers to lower intracellular pH (pHi), including reducing extracellular pH (pHo) to 7.0 or treatment with sodium butyrate, significantly increased CCK secretion. To examine whether pH directly affects membrane K+permeability, we measured outward currents carried by K+, using whole cell patch techniques. K+current was significantly inhibited by lowering pHoto 7.0. These effects appear to be mediated through changes in pHi, because intracellular dialysis with acidic solutions nearly eliminated current activity. These results suggest that Na+/H+exchange and membrane potential may be functionally linked, where inhibition of Na+/H+exchange lowers pHiand depolarizes the membrane, perhaps through inhibition of pH-sensitive K+channels. In turn, K+channel closure and membrane depolarization open voltage-dependent Ca2+channels, leading to an increase in cytosolic Ca2+and CCK release. The effects of pHion K+channels may serve as a potent stimulus for hormone secretion, linking cell metabolism and secretory functions.
Effects of chronic ethanol consumption on cytokine regulation of liver regeneration
Authors: Yang Shi Qi , Lin Hui Zhi , Yin Ming , Albrecht Jeffrey H. , Diehl Anna Mae
Published: December 2017

Ethanol ingestion may interrupt the proregenerative signal transduction that is initiated by injury-related cytokines such as tumor necrosis factor (TNF)-α and TNF-α- inducible cytokines including interleukin (IL)-6. To test this theory, liver regeneration, TNF-α and IL-6 expression, and cytokine-regulated prereplicative events were compared in ethanol-fed rats and isocalorically fed controls after 70% partial hepatectomy (PH). Ethanol feeding inhibits hepatocyte replication and recovery of liver mass after PH but generally promotes induction of both cytokines in the liver and extrahepatic tissues (i.e., white adipose tissue). Cytokine-regulated events that occur early in the prereplicative period are influenced differentially. TNF-α-dependent increases in hepatic nuclear factor-κB (NF-κB) p50 and p65 expression and DNA binding activity are prevented, whereas IL-6-dependent inductions of hepatic Stat-3 phosphorylation and DNA binding activity occur normally. In contrast, events (e.g., induction of cyclin D1, cdk-1, cyclin D3, and p53 mRNA) that occur at the end of the prereplicative period are uniformly inhibited. These findings indicate that chronic ethanol ingestion arrests the regenerative process during the prereplicative period and demonstrate that increased TNF-α, IL-6 and Stat-3 are not sufficient to assure hepatocyte proliferation after PH.
Reduced folate derivatives are endogenous substrates for cMOAT in rats
Authors: Kusuhara Hiroyuki , Han Yong-Hae , Shimoda Minoru , Kokue Eiichi , Suzuki Hiroshi , Sugiyama Yuichi
Published: December 2017

We examined the role of the canalicular multispecific organic anion transporter (cMOAT) in the biliary excretion of reduced folate derivatives in vivo and in vitro using normal [Sprague-Dawley rats (SDR)] and mutant [Eisai hyperbilirubinemic rats (EHBR)] rats whose cMOAT is hereditarily deficient. In vivo, the biliary excretion of endogenous tetrahydrofolate (H4PteGlu), 5-methyltetrahydrofolate (5-CH3-H4PteGlu), and 5,10-methylenetetrahydrofolate (5,10-CH2-H4PteGlu) in EHBR was reduced to 8.2%, 1.9%, and 5.5% of those in SDR, respectively, whereas that of 10-formyltetrahydrofolate (10-HCO-H4PteGlu) was detected only in SDR and not in EHBR. Bile drainage caused reduction of endogenous plasma folate concentrations in SDR but not in EHBR. In vitro, significant ATP-dependent uptake of3H-labeled 5-CH3-H4PteGlu into canalicular membrane vesicles was observed only in SDR. This ATP-dependent uptake was saturable with a Michaelis constant ( Km) value of 126 μM, which was comparable with its inhibitor constant ( Ki) value of 121 μM for the ATP-dependent uptake of a typical cMOAT substrate, 2,4-dinitrophenyl- S-glutathione (DNP-SG). Vice versa, DNP-SG inhibited the uptake of 5-CH3-H4PteGlu with a Kiof 35 μM, which was similar to its Kmvalue. In addition, H4PteGlu and 5,10-CH2-H4PteGlu also inhibited the ATP-dependent uptake of DNP-SG. These results indicate that 5-CH3-H4PteGlu and other derivatives are transported via cMOAT. Therefore, reduced folate derivatives are the first endogenous substrates for cMOAT that do not contain glutathione, glucuronide, or sulfate moieties.
Physiological changes in blood glucose affect appetite and pyloric motility during intraduodenal lipid infusion
Authors: Andrews J. M. , Rayner C. K. , Doran S. , Hebbard G. S. , Horowitz M.
Published: December 2017

We evaluated the effects of varying blood glucose concentration within the normal postprandial range and its interaction with small intestinal nutrients on antropyloric motility and appetite. Eight healthy males (19–40 yr) underwent paired studies, with a blood glucose level of 5 or 8 mmol/l. Manometry and visual analog scales were used to assess motility and appetite, during fasting and intraduodenal lipid infusion (1.5 kcal/min). In the fasting state, antral waves were suppressed at 8 mmol/l compared with 5 mmol/l ( P = 0.018). However, pyloric motility was no different between the two blood glucose concentrations. Hunger was no different at 5 mmol/l compared with 8 mmol/l, but fullness was greater at 8 mmol/l ( P = 0.01). During intraduodenal lipid infusion, antral waves were suppressed ( P < 0.035) and isolated pyloric pressure waves (IPPWs) were stimulated ( P < 0.02) compared with during the fasting state, with no difference between blood glucose concentrations, although the temporal patterning of IPPWs varied between blood glucose concentrations. The amplitude of IPPWs was greater at 5 mmol/l compared with 8 mmol/l ( P < 0.001), and hunger decreased at 8 mmol/l compared with 5 mmol/l ( P = 0.02). We conclude that “physiological” hyperglycemia modifies gastric motor and sensory function and that synergy exists between blood glucose concentration and small intestinal nutrients in modulating gastric motility and appetite.
Stress stimulates transepithelial macromolecular uptake in rat jejunum
Authors: Kiliaan Amanda J. , Saunders Paul R. , Bijlsma Pieter B. , Berin M. Cecilia , Taminiau Jan A. , Groot Jack A. , Perdue Mary H.
Published: December 2017

Evidence suggests that stress may be a contributing factor in intestinal inflammatory disease; however, the involved mechanisms have not been elucidated. We previously reported that acute stress alters epithelial physiology of rat intestine. In this study, we documented stress-induced macromolecular transport across intestinal epithelium. After exposure of Wistar-Kyoto rats to acute restraint stress, transport of a model protein, horseradish peroxidase (HRP), was assessed in isolated segments of jejunum. The flux of intact HRP was significantly enhanced across intestine from stressed rats compared with controls. Electron microscopy revealed HRP-containing endosomes within enterocytes, goblet cells, and Paneth cells of stressed rats. The number and area of HRP endosomes within enterocytes were found to be significantly increased by stress. HRP was also visualized in paracellular spaces between adjacent epithelial cells only in intestine from stressed rats. Atropine treatment of rats prevented the stress-induced abnormalities of protein transport. Our results suggest that stress, via a mechanism that involves release of acetylcholine, causes epithelial dysfunction that includes enhanced uptake of macromolecular protein antigens. We speculate that immune reactions to such foreign proteins may initiate or exacerbate inflammation.
Vagal mechanisms underlying gastric protection induced by chemical activation of raphe pallidus in rats
Authors: Kaneko Hiroshi , Kaunitz Jonathan , Taché Yvette
Published: December 2017

Peripheral mechanisms involved in kainic acid injected into the raphe pallidus (Rpa)-induced gastric protection were investigated in urethan-anesthetized rats. Gastric mucosal blood flow (GMBF), acid secretion, and gastric injury induced by intragastric ethanol (60%) were measured in response to kainic acid (25 pg) injected into the Rpa. Kainic acid reduced ethanol-induced gastric lesions by 57%. The protective effect was blocked by vagotomy, capsaicin deafferentation, and intravenous injection of the calcitonin gene-related peptide (CGRP) antagonist CGRP-(8—37) and NG-nitro-l-arginine methyl ester (l-NAME).l- but notd-arginine reversed thel-NAME action. Kainic acid injected into the Rpa, unlike outside sites, increased basal GMBF but not acid secretion. Indomethacin unmasked an acid secretory response to kainic acid. These results show that kainic acid injected into the Rpa at a dose that did not stimulate acid secretion, due to the inhibitory effect of prostaglandins, protects against ethanol-induced gastric injury through vagal-dependent activation of CGRP contained in capsaicin-sensitive afferents and nitric oxide-mediated gastric vasodilatory mechanisms.
Capillary diffusion capacity and tissue distribution of pancreatic procolipase in rat
Authors: Rippe Catarina , Rippe Bengt , Erlanson-Albertsson Charlotte
Published: December 2017

The permeability-surface area product of procolipase and its apparent distribution volume in rat tissues were assessed using a tissue uptake technique. Procolipase was investigated together with51Cr-EDTA, used as an inert extracellular marker, and131I-albumin, used as a plasma volume marker. The tissue uptake of procolipase seemed to occur by passive transport in most of the organs studied, such as in muscle, liver, lung, adipose tissue, adrenal glands, colon, and skin. However, throughout the gastrointestinal tract, except in the colon, there was a high uptake of procolipase, greatly exceeding that of51Cr-EDTA. This was especially evident in the stomach, in which the procolipase uptake was nonsaturable within the experimental period. Also, in the central nervous system (CNS), there was evidence of specific, possibly carrier-mediated, transport. These results suggest that procolipase may have specific, conceivably receptor-mediated, transport pathways across the microvascular endothelium in the stomach, pancreas, duodenum, ileum, and the CNS.
I. Glucose Galactose Malabsorption
Authors: Wright Ernest M.
Published: December 2017

Glucose Galactose Malabsorption is a genetic disorder caused by a defect in glucose and galactose transport across the intestinal brush border. Normally, lactose in milk is broken down into glucose and galactose by lactase, an ectoenzyme on the brush border, and the hexoses are transported into the cell by the Na+-glucose cotransporter SGLT1. The mutations causing the defect in sugar transport have been identified in patients from 33 kindreds, and functional studies have established how these mutations cause the disease.
Thyroid hormone regulation of rat hepatocyte proliferation and polyploidization
Authors: Torres Santiago , Díaz Blanca P. , Cabrera Juan J. , Díaz-Chico Juan C. , Díaz-Chico Bonifacio N. , López-Guerra Antonio
Published: December 2017

The liver of adult mammals contains various classes of polyploid hepatocytes produced by a process that is partially regulated by hormones. However, it is not well understood how the hormones affect the rate of hepatocyte proliferation under physiological conditions. Here we have studied the specific roles of 3,5,3′-triiodothyronine (T3), growth hormone (GH), and sex steroids on the percentage of diploid nuclei in S phase and on the population of liver tetraploid (4C) cell nuclei in several rat model systems. Gonadal steroids had no effect on the S phase but account for gender differences in the 4C nuclei. Hypophysectomy in adult male rats produced a moderate decrease in 4C nuclei that was reversed by treatment with 25 μg T3⋅ kg−1⋅ day−1, whereas treatment with 200 μg human recombinant GH (hGH) ⋅ kg−1⋅ day−1was ineffective. Rats made hypothyroid by methimazole treatment of dams and pups until death showed a low S phase and only 5% of 4C nuclei at 70 days of age. T3significantly increased the S phase 24 h after administration and restored the adult normal level of 4C nuclei after 10 days of treatment. hGH did not affect the 4C nuclei or the S phase in the hypothyroid rats. These results suggest that the processes of hepatocyte proliferation and polyploidization of the rat liver are under endocrine control, with thyroid hormones playing the essential regulatory role.
β-Alanine and α-fluoro-β-alanine concentrative transport in rat hepatocytes is mediated by GABA transporter GAT-2
Authors: Liu Mengping , Russell Rosalind L. , Beigelman Leonid , Handschumacher Robert E. , Pizzorno Giuseppe
Published: December 2017

Studies on the compartmentalization of uridine catabolic metabolism in liver have indicated accumulation of β-alanine as well as α-fluoro-β-alanine (FβAL) for 5-fluorouracil in the hepatocytes. Using preparations of rat hepatocytes we were able to identify a Na+-dependent transport with high affinity for β-alanine and GABA with Michaelis constant ( Km) of 35.3 and 22.5 μM, respectively. A second Na+-dependent kinetic component with Km>1 mM was also identified. The sigmoidal profile of β-alanine uptake with respect to Na+shows the involvement of multiple ions of sodium in the transport process. A Hill coefficient of 2.6 ± 0.4 indicates that at least two sodium ions are cotransported with β-alanine. The flux of β-alanine was also shown to be chlorine dependent. The substitution of this anion with gluconate, even in the presence of Na+, reduced the intracellular concentrative accumulation of β-alanine to passive diffusion level, indicating that both Na+and Cl−are essential for the activity of this transporter. The transport of β-alanine was inhibited by GABA, hypotaurine, β-aminoisobutyric acid, and FβAL in a competitive manner. However, concentrations up to 1 mM ofl- andd-alanine, taurine, and α-aminoisobutyric acid did not affect β-alanine uptake. Considering the similarities in substrate specificity with the rat GAT-2 transporter, extracts of hepatocytes were probed with the anti-GABA transporter antibody R-22. A 80-kDa band corresponding to GAT-2 was present in the hepatocyte and in the GAT-2 transfected Madin-Darby canine kidney cell extract, confirming the extraneural localization of this transporter. In view of these results, the neurotoxic effects related to the administration of uridine and 5-fluorouracil could be explained with the formation of β-alanine and FβAL and their effect on the cellular reuptake of GABA.
Concurrent elevation of fundic somatostatin prevents gastrin stimulation by GRP
Authors: Zavros Yana , Fleming William R. , Shulkes Arthur
Published: December 2017

Gastrin-releasing peptide (GRP) can stimulate both gastrin and somatostatin (SOM) secretion, but, as gastrin increases SOM and SOM in turn inhibits gastrin, the overall endpoint in terms of gastrin output is variable. To examine the mechanisms involved, we compared the effects of GRP on gastrin secretion in normal sheep and sheep chronically immunized against SOM. In the normal animal, GRP had no effect on either plasma gastrin or SOM. However, in sheep immunized against SOM, GRP stimulated gastrin secretion, suggesting that the concurrent stimulation of SOM prevents the increase in gastrin secretion. To determine the local source of SOM, GRP was then infused into nonimmunized sheep with cannulas draining blood from the fundus and antrum. GRP stimulated fundic SOM output but inhibited antral SOM and gastrin secretion, demonstrating that the fundus was the source of the SOM. Because cholinergic interactions have a major influence on the effects of GRP, a cholinergic stimulus was administered, and we found that the responses were different: SOM output was inhibited in both the antrum and fundus, and antral gastrin secretion was increased. The present study demonstrates two further instances of the differential regulation of SOM from the antrum and fundus. GRP fails to stimulate gastrin because of an increase in fundic SOM, whereas gastrin levels increase following a cholinergic stimulus because of inhibition of both antral and fundic SOM secretion.
Dietary and systemic phenylalanine utilization for mucosal and hepatic constitutive protein synthesis in pigs
Authors: Stoll Barbara , Burrin Douglas G. , Henry Joseph F. , Jahoor Farook , Reeds Peter J.
Published: December 2017

The objective of this study was to quantify the utilization of dietary and systemic phenylalanine for mucosal and hepatic constitutive protein synthesis in piglets. Seven female piglets (7.6 kg) bearing arterial, portal, peripheral venous, and gastric catheters were fed a high-protein diet and infused intragastrically with U-13C-labeled protein and intravenously with [2H( phenyl)5]phenylalanine ([2H5]phenylalanine) for 6 h. The isotopic enrichment of the two phenylalanine tracers was measured in arterial and portal blood, in mucosal and hepatic-free and protein-bound phenylalanine, and in very low-density apolipoprotein B-100, albumin, and fibrinogen. The relative isotopic enrichments of the tracers in mucosal-free (ratio of2H5- to U-13C-labeled = 0.20 ± 0.05) and protein-bound (0.32 ± 0.08) phenylalanine differed significantly ( P < 0.01). Although this suggests preferential use of arterial phenylalanine for mucosal protein synthesis, on a molar basis, 59 ± 6% of the mucosal protein was derived from dietary phenylalanine. There were significant differences ( P < 0.025) between the relative labeling of the two tracers in arterial (ratio of2H5- to U-13C-labeled = 1.25 ± 0.48) and portal (ratio of2H5- to U-13C-labeled = 0.72 ± 0.18) phenylalanine. The mean ratio of the two tracers in all proteins of hepatic origin that were analyzed (0.69 ± 0.18) was similar to that of portal phenylalanine. We conclude that in the fed state portal phenylalanine is preferentially used for constitutive as well as secreted hepatic protein synthesis.
Ethanol dissociates hormone-stimulated cAMP production from inhibition of TNF-α production in rat Kupffer cells
Authors: Aldred Andrew , Nagy Laura E.
Published: December 2017

Ethanol impairs hormone-stimulated cAMP production in a number of cell types, yet the effects of ethanol on downstream responses mediated by cAMP-dependent protein kinase (PKA) are not understood. Here we have investigated the effects of ethanol feeding on cAMP-mediated inhibition of tumor necrosis factor-α (TNF-α) synthesis in rat Kupffer cells. Male Wistar rats were fed liquid diets containing 36% of calories as ethanol for 4 wk or were pair fed a control diet. Stimulation of cAMP production by the adenosine A2receptor agonist 5′-( N-ethylcarboxamido)-adenosine (NECA), prostaglandin E2, or forskolin was decreased to 25% of control values in Kupffer cells isolated from ethanol-fed rats. This decrease was associated with a reduction in the quantity of immunoreactive Gsα protein in ethanol-fed rats, with no changes observed in Giα or Gβ. TNF-α production was higher in ethanol-fed rats in response to stimulation with lipopolysaccharide or latex beads. Despite the profound reduction in the ability of hormone to increase cAMP production, NECA and prostaglandin E2inhibited TNF-α production to an equivalent degree in Kupffer cells from ethanol- and pair-fed rats. Total activity and immuoreactive protein quantity of PKA did not differ between groups. Activation of PKA in response to a 15-min treatment with 1 μM NECA was reduced by 50% in ethanol-fed rats compared with control. Despite this reduction in activation, translocation of the catalytic subunit of PKA to the nucleus and phosphorylation of cAMP response element binding protein in response to activation were observed in Kupffer cells from both ethanol- and pair-fed rats. These data demonstrate that there is a dissociation between ethanol-induced desensitization of hormone-stimulated cAMP production in rat Kupffer cells and the downstream inhibition of TNF-α production mediated by cAMP.
Cardiac impairment and nitric oxide synthase activity in the chronic portal vein-stenosed rat
Authors: Battarbee Harold D. , Zavecz James H. , Grisham Matthew B. , Maloney Ronald E. , Chandler L. Judson , Mercer John W. , Cady Francois M.
Published: December 2017

Decreased cardiac contractility and β-adrenergic responses have been observed in the chronic portal vein-stenosed (PVS) rat. Because nitric oxide (NO) may be increased in PVS and has been recognized as a negative inotropic agent, we investigated the induction of NO synthase (NOS2) and/or changes in constitutive NOS (NOS3) as factors in the cardiac dysfunction of the PVS rat. Ten to twelve days after portal vein stenosis or sham operation, cardiac function was evaluated in paced left ventricular papillary muscles (LVPM) and right ventricular strips (RV). To determine if NO modulation of contractile function was altered in PVS, we examined the increase in developed tension produced by the effect of Nω-nitro-l-arginine (l-NNA) on the myocardial force-frequency relationship. Cardiac tissue NOS2 and NOS3 activities were assayed, Western blot analyses of NOS2 and NOS3 expression were performed, and circulating nitrate-nitrite (NOX) levels (an indicator of in vivo NOS activity) were assayed. Basal LVPM and RV contractile indexes were significantly reduced in PVS (30–50%), without a change in the relaxation rate. No between-group differences in the cardiac NOS2 or NOS3 enzymatic activities of PVS and sham-operated (SO) rats were observed. Western blots revealed no cardiac NOS2 expression in either SO or PVS rats. In contrast, NOS3 was expressed in both SO and PVS rats, but there was no quantitative difference in expression between the two groups. Changes in the cardiac force-frequency relationship (staircase effect) after l-NNA were consistent with NOS3 modulation of contractile function in both SO and PVS rats, but there was no between-group difference in the modulation. Circulating NOXconcentrations did not differ between SO and PVS rats. In conclusion, protein expression data, enzymatic assays, end-product assays, and functional data indicate that between-group differences in NOS2 and NOS3 activity are not responsible for the cardiac impairment that has been observed in the chronic PVS rat.
Electrogenic Na+transport in rat late distal colon by natural and synthetic glucocorticosteroids
Authors: Grotjohann Ingo , Schulzke Jörg-Dieter , Fromm Michael
Published: December 2017

The potency of in vitro-added corticosteroids to stimulate electrogenic Na+absorption ( JNa, the Na+absorptive short-circuit current blockable by 10−4M amiloride) was determined in rat late distal colon. JNawas determined 8 h after steroid addition from the drop in short-circuit current caused by 10−4M amiloride. The concentration dependency of JNawas obtained for seven corticosteroids and compared with that established for aldosterone. Apparent mineralocorticoid potencies as determined from apparent Michaelis-Menten constant ( Km) values were as follows: aldosterone 1.2 nM ≫ RU-28362 20 nM = deoxycorticosterone 20 nM > deoxycortisol 36 nM ≥ dexamethasone 37 nM ≫ corticosterone 170 nM > cortisol 210 nM. These steroids exhibited Vmaxvalues of 9–13 μmol ⋅ h−1⋅ cm−2and similar concentration dependencies. Hill coefficients were between 1.6 and 2.1, suggesting cooperative effects between activated receptors. We conclude that corticosteroids exhibit graded mineralocorticoid potency instead of a sharp partition into exclusive groups of mineralocorticoid and nonmineralocorticoid hormones. The low apparent Kmvalue of RU-28362 for mineralocorticoid action and the need for high concentrations of the mineralocorticoid antagonist mespirenone to block this response indicated that JNain a native mammalian epithelium can be mediated by the glucocorticoid receptor. Glucocorticoid receptor-specific amounts of RU-28362 in combination with mineralocorticoid receptor-specific amounts of aldosterone or of the mineralocorticoid antagonist spironolactone showed cooperative action, suggesting a heterodimeric activation of JNaby the glucocorticoid receptor and mineralocorticoid receptor.
Effect of central corticotropin-releasing factor on carbon tetrachloride-induced acute liver injury in rats
Authors: Yokohama Shiro , Yoneda Masashi , Nakamura Kimihide , Makino Isao
Published: December 2017

Central neuropeptides play important roles in many instances of physiological and pathophysiological regulation mediated through the autonomic nervous system. In regard to the hepatobiliary system, several neuropeptides act in the brain to regulate bile secretion, hepatic blood flow, and hepatic proliferation. Stressors and sympathetic nerve activation are reported to exacerbate experimental liver injury. Some stressors are known to stimulate corticotropin-releasing factor (CRF) synthesis in the central nervous system and induce activation of sympathetic nerves in animal models. The effect of intracisternal CRF on carbon tetrachloride (CCl4)-induced acute liver injury was examined in rats. Intracisternal injection of CRF dose dependently enhanced elevation of the serum alanine aminotransferase (ALT) level induced by CCl4. Elevations of serum aspartate aminotransferase, alkaline phosphatase, and total bilirubin levels by CCl4were also enhanced by intracisternal CRF injection. Intracisternal injection of CRF also aggravated CCl4-induced hepatic histological changes. Intracisternal CRF injection alone did not modify the serum ALT level. Intravenous administration of CRF did not influence CCl4-induced acute liver injury. The aggravating effect of central CRF on CCl4-induced acute liver injury was abolished by denervation of hepatic plexus with phenol and by denervation of noradrenergic fibers with 6-hydroxydopamine treatment but not by hepatic branch vagotomy or atropine treatment. These results suggest that CRF acts in the brain to exacerbate acute liver injury through the sympathetic-noradrenergic pathways.
Goblet cell-specific expression mediated by theMUC2mucin gene promoter in the intestine of transgenic mice
Authors: Gum James R. , Hicks James W. , Gillespie Anne-Marie , Carlson Elaine J. , Kömüves Lazlo , Karnik Satyajit , Hong Joe C. , Epstein Charles J. , Kim Young S.
Published: December 2017

The regulation of MUC2, a major goblet cell mucin gene, was examined by constructing transgenic mice containing bases −2864 to +17 of the human MUC25′-flanking region fused into the 5′-untranslated region of a human growth hormone (hGH) reporter gene. Four of eight transgenic lines expressed reporter. hGH message expression was highest in the distal small intestine, with only one line expressing comparable levels in the colon. This contrasts with endogenous MUC2 expression, which is expressed at its highest levels in the colon. Immunohistochemical analysis indicated that goblet cell-specific expression of reporter begins deep in the crypts, as does endogenous MUC2 gene expression. These results indicate that the MUC2 5′-flanking sequence contains elements sufficient for the appropriate expression of MUC2 in small intestinal goblet cells. Conversely, elements located outside this region appear necessary for efficient colonic expression, implying that the two tissues utilize different regulatory elements. Thus many, but not all, of the elements necessary for MUC2 gene regulation reside between bases −2864 and +17 of the 5′-flanking region.
Bcl-2 inhibits ischemia-reperfusion-induced apoptosis in the intestinal epithelium of transgenic mice
Authors: Coopersmith Craig M. , O’Donnell David , Gordon Jeffrey I.
Published: December 2017

Little is known about the effects of ischemia-reperfusion on the inductive, commitment, or execution phases of apoptosis. We have created a genetically defined model to study the response of small intestinal epithelial cells to ischemia-reperfusion injury as a function of their proliferative status and differentiation. Occlusion of the superior mesenteric artery for 20 min in adult FVB/N or C57BL/6 mice results in the appearance of TUNEL-positive apoptotic cells in the jejunal epithelium within 4 h, with a maximum response occurring at 24 h. Stimulation of apoptosis is greater in postmitotic, differentiated epithelial cells located in the upper portions of villi compared with undifferentiated, proliferating cells in the crypts of Lieberkühn (7-fold vs. 2-fold relative to sham-operated controls). Comparisons of p53+/+and p53−/−mice established that the apoptosis is p53 independent. To further characterize this response, we generated FVB/N transgenic mice that express human Bcl-2 in epithelial cells distributed from the base of crypts to the tips of their associated villi. The fivefold elevation in steady-state Bcl-2 concentration is not accompanied by detectable changes in the levels or cellular distributions of the related anti-apoptotic regulator Bcl-xLor of the proapoptotic regulators Bax and Bak and does not produce detectable effects on basal proliferation, differentiation, or death programs. The apoptotic response to ischemia-reperfusion is reduced twofold in the crypts and villi of transgenic mice compared with their normal littermates. These results suggest that both undifferentiated and differentiated cells undergo a commitment phase that is sensitive to Bcl-2. Forced expression of Bcl-2 also suppressed the p53-dependent death that occurs in proliferating crypt epithelial cells following γ-irradiation. Thus suppressibility by Bcl-2 operationally defines a common feature of the apoptosis induced in the crypt epithelium by these two stimuli.
1,25-Dihydroxyvitamin D3but not TPA activates PLD in Caco-2 cells via pp60c-srcand RhoA
Authors: Khare Sharad , Bissonnette Marc , Wali Ramesh , Skarosi Susan , Boss Gerry R. , von Lintig Friederike C. , Scaglione-Sewell Beth , Sitrin Michael D. , Brasitus Thomas A.
Published: December 2017

In the accompanying paper [Khare et al., Am. J. Physiol. 276 ( Gastrointest. Liver Physiol. 39): G993–G1004, 1999], activation of protein kinase C-α (PKC-α) was shown to be involved in the stimulation of phospholipase D (PLD) by 1,25-dihydroxyvitamin D3[1,25(OH)2D3] and 12- O-tetradecanoylphorbol 13-acetate (TPA) in Caco-2 cells. Monomeric or heterotrimeric G proteins, as well as pp60c- srchave been implicated in PLD activation. We therefore determined whether these signal transduction elements were involved in PLD stimulation by 1,25(OH)2D3or TPA. Treatment with C3 transferase, which inhibits members of the Rho family of monomeric G proteins, markedly diminished the ability of 1,25(OH)2D3, but not TPA, to stimulate PLD. Brefeldin A, an inhibitor of ADP-ribosylation factor proteins, did not, however, significantly reduce the stimulation of PLD by either of these agents. Moreover, 1,25(OH)2D3, but not TPA, activated pp60c- srcand treatment with PP1, a specific inhibitor of the pp60c- srcfamily, blocked the ability of 1,25(OH)2D3to activate PLD. Pretreatment of cells with pertussis toxin (PTx) markedly reduced the stimulation of PLD by either agonist. PTx, moreover, inhibited the stimulation of pp60c- srcand PKC-α by 1,25(OH)2D3. PTx did not, however, block the membrane translocation of RhoA induced by 1,25(OH)2D3or inhibit the stimulation of PKC-α by TPA. These findings, taken together with those of the accompanying paper, indicate that although 1,25(OH)2D3and TPA each activate PLD in Caco-2 cells in part via PKC-α, their stimulation of PLD differs in a number of important aspects, including the requirement for pp60c- srcand RhoA in the activation of PLD by 1,25(OH)2D3, but not TPA. Moreover, the requirement for different signal transduction elements by 1,25(OH)2D3and TPA to induce the stimulation of PLD may potentially underlie differences in the physiological effects of these agents in Caco-2 cells.
TGF-β1 in liver fibrosis: an inducible transgenic mouse model to study liver fibrogenesis
Authors: Kanzler Stephan , Lohse Ansgar W. , Keil Andrea , Henninger Jürgen , Dienes Hans P. , Schirmacher Peter , Rose-John Stefan , Meyer Zum Büschenfelde Karl H. , Blessing Manfred
Published: December 2017

Transforming growth factor-β1 (TGF-β1) is a powerful stimulus for collagen formation in vitro. To determine the in vivo effects of TGF-β1 on liver fibrogenesis, we generated transgenic mice overexpressing a fusion gene [C-reactive protein (CRP)/TGF-β1] consisting of the cDNA coding for an activated form of TGF-β1 under the control of the regulatory elements of the inducible human CRP gene promoter. Two transgenic lines were generated with liver-specific overexpression of mature TGF-β1. After induction of the acute phase response (15 h) with lipopolysaccharide (100 μg ip), plasma TGF-β1 levels reached >600 ng/ml in transgenic animals, which is >100 times above normal plasma levels. Basal plasma levels of uninduced transgenic animals were about two to five times above normal. As a consequence of hepatic TGF-β1 expression, we could demonstrate marked transient upregulation of procollagen I and procollagen III mRNA in the liver 15 h after the peak of TGF-β1 expression. Liver histology after repeated induction of transgene expression showed an activation of hepatic stellate cells in both transgenic lines. The fibrotic process was characterized by perisinusoidal deposition of collagen in a linear pattern. This transgenic mouse model gives in vivo evidence for the important role of TGF-β1 in stellate cell activation and liver fibrogenesis. Due to the ability to control the level of TGF-β1 expression, this model allows the study of the regulation and kinetics of collagen synthesis and fibrolysis as well as the degree of reversibility of liver fibrosis. The CRP/TGF-β1 transgenic mouse model may finally serve as a model for the testing of antifibrogenic agents.
Cholinergic and GABAergic regulation of nitric oxide synthesis in the guinea pig ileum
Authors: Hebeiss Katalina , Kilbinger Heinz
Published: December 2017

Nitric oxide (NO) synthesis was examined in intact longitudinal muscle-myenteric plexus preparations of the guinea pig ileum by determining the formation of [3H]citrulline during incubation with [3H]arginine. Spontaneous [3H]citrulline production after 30 min was 80–90 dpm/mg, which constituted ∼1% of the tissue radioactivity. Electrical stimulation (10 Hz) led to a threefold increase in [3H]citrulline formation. Removal of calcium from the medium or addition of N G-nitro-l-arginine strongly inhibited both spontaneous and electrically induced production of [3H]citrulline. TTX reduced the electrically induced but not spontaneous [3H]citrulline formation. The electrically induced formation of [3H]citrulline was diminished by (+)-tubocurarine and mecamylamine and enhanced by scopolamine, which suggests that endogenous ACh inhibits, via muscarinic receptors, and stimulates, via nicotinic receptors, the NO synthesis in the myenteric plexus. The GABAA receptor agonist muscimol and GABA also reduced the electrically evoked formation of [3H]citrulline, whereas baclofen was without effect. Bicuculline antagonized the inhibitory effect of GABA. It is concluded that nitrergic myenteric neurons are equipped with GABAAreceptors, which mediate inhibition of NO synthesis.
Effects of growth factors and trefoil peptides on migration and replication in primary oxyntic cultures
Authors: Kato Kimitoshi , Chen Monica C. , Nguyen Minh , Lehmann Frank S. , Podolsky Daniel K. , Soll Andrew H.
Published: December 2017

Restitution, the lateral migration of cells over an intact basement membrane, maintains mucosal integrity. We studied the regulation of migration and proliferation of enzyme-dispersed canine oxyntic mucosa cells in primary culture. Confluent monolayers were wounded and cultured in serum-free medium, and cells migrating into the wound were counted. [3H]thymidine incorporation into DNA was studied using subconfluent cultures. Considerable migration occurred in untreated monolayers; however, epidermal growth factor (EGF), transforming growth factor (TGF)-α, basic fibroblast growth factor (bFGF), insulin-like growth factor I (IGF-I), two trefoil peptides, and interleukin (IL)-1β further enhanced migration. The specific EGF receptor (EGFR) monoclonal antibody, MAb-528, inhibited both basal and TGF-α- or IL-1β-stimulated migration, but not the response to trefoil peptide, bFGF, or IGF-I. Exogenous TGF-β inhibited cell proliferation but did not alter migration. Immunoneutralization with anti-TGF-β blocked the response to exogenous TGF-β and produced a small enhancement of basal thymidine incorporation but did not attenuate basal or TGF-α-stimulated migration. In conclusion, endogenous EGFR ligands regulate proliferation and migration. TGF-β inhibits mitogenesis; it did not upregulate migration in these cultures. Although bFGF, IGF-I, and IL-1β enhance gastric epithelial migration, only IL-1β acted in a TGF-α-dependent fashion.
Impairment of neutrophil emigration in CD18-null mice
Authors: Walzog Barbara , Scharffetter-Kochanek Karin , Gaehtgens Peter
Published: December 2017

This study was undertaken to investigate the requirement of β2-integrins (CD11/CD18) for extravasation of neutrophils in mice. After intraperitoneal thioglycollate injection, an in vivo model of inflammation, leukocyte extravasation into the peritoneal cavity was studied in CD18-deficient and wild-type control mice. Before the induction of peritonitis, total and differential leukocyte counts in the circulation were analyzed and found to be 10-fold elevated in CD18-deficient animals compared with wild-type animals. This was largely due to neutrophilia, with a 30-fold increase in neutrophil counts. In CD18-deficient animals, extravasated white blood cells in the peritoneal cavity were diminished by 46%. The neutrophil number in the peritoneal fluid was severely reduced to 13% compared with control animals. In contrast, the number of emigrated monocytes was enhanced and lymphocyte emigration was not altered in the absence of CD18. The emigration efficiency of the neutrophils, calculated as ratio of the cell number in the lavage fluid and the circulating blood, was reduced to 0.4% in CD18-deficient mice compared with wild-type animals. Thus efficient neutrophil emigration into the peritoneal cavity strongly required CD11/CD18.
Characteristics of the muscularis mucosae in the acid-secreting region of the rabbit stomach
Authors: Percy W. H. , Warren J. M. , Brunz J. T.
Published: December 2017

It has been suggested that muscularis mucosae excitation may augment gastric acid secretion, implying that this muscle should contract to secretagogues or stimulation of its motor innervation. The aim of this study was to characterize in vitro the responses of the muscularis mucosae in the rabbit gastric corpus to substances that modulate acid release and to intrinsic nerve stimulation. Muscularis mucosae from both fundic and antral ends of the corpus had identical mechanical properties, contracted to ACh, ADP, ATP, and histamine but relaxed to vasoactive intestinal polypeptide. Fundic but not antral muscularis mucosae contracted to bombesin and PGE2and PGF2α, whereas adenosine, AMP, CCK, gastrin, secretin, and somatostatin were without effect on any preparation. In both regions electrical field stimulation evoked TTX-sensitive responses consisting of an atropine-resistant contraction followed by an N G-nitro-l-arginine methyl ester- and indomethacin-resistant relaxation. It is concluded from the regional variability in the pharmacological properties of the gastric muscularis mucosae that if its motor activity is linked to acid secretion this would be achieved by a neurally mediated relaxation rather than a paracrine- and/or endocrine-induced alteration in tone.
Shear stress-induced nitric oxide antagonizes adenosine effects on intestinal metabolism
Authors: Han Chao , Ming Zhi , Lautt W. Wayne
Published: December 2017

The influence of nitric oxide (NO) on adenosine-induced metabolic effects was studied in the intestine. Blood flow supplied an in situ- isolated segment of small intestine in anesthetized cats via the superior mesenteric artery (SMA) and was controlled by a vascular circuit. The SMA and portal samples were taken for analysis of oxygen and lactate. Adenosine (0.4 mg ⋅ kg−1 ⋅ min−1, intra-SMA) reduced oxygen consumption by 25.1 ± 2.9 from 73.1 ± 10.8 μmol ⋅ min−1 ⋅ 100 g−1 and increased lactate production by 13.3 ± 3.0 from 12.8 ± 4.6 μmol ⋅ min−1 ⋅ 100 g tissue−1 during constant-flow (CF, decreased shear stress) but not during constant-pressure (CP, increased shear stress) perfusion. Blockade of NO synthase using N ω-nitro-l-arginine methyl ester did not affect the metabolic effects of adenosine during CF but eliminated the differences seen between CP and CF perfusion. A NO donor, 3-morpholinosydnonimine, attenuated the metabolic effects of adenosine during CF perfusion. The results suggested that shear-induced NO antagonized metabolic effects of adenosine but that the inhibition of vascular effects by NO was not shear dependent since it occurred in both CP and CF perfusion.