bluuebunny/crossref_metadata_2025 huggingface dataset:
Chronic ethanol feeding impairs endothelin-1-stimulated glucose uptake via decreased Gα11expression in rat adipocytes
Authors: Rachdaoui Nadia , Sebastian Becky M. , Nagy Laura E.
Published: March 2015

Chronic ethanol feeding decreases insulin-stimulated glucose uptake in rat adipocytes. Here, we show that chronic ethanol also decreases endothelin-stimulated glucose uptake. Endothelin-1 increased uptake of 2-deoxyglucose 2.4-fold in adipocytes isolated from pair-fed rats. However, in adipocytes isolated from rats that had consumed a diet containing 35% ethanol for 4 wk, endothelin-1 did not increase glucose uptake. Although endothelin-1 increased GLUT4 quantity at the plasma membrane in adipocytes from pair-fed rats, there was no increase in GLUT4 after chronic ethanol feeding. Loss of endothelin-1-stimulated glucose uptake after ethanol feeding was associated with a specific decrease in the quantity of Gα11in plasma membranes, with no change in Gαqquantity. Activation of proline-rich tyrosine kinase 2 (PYK2), a downstream target of Gαq/11that is required for endothelin-1-stimulated GLUT4 translocation in 3T3-L1 adipocytes, was also suppressed after chronic ethanol feeding. In contrast, activation of p38 MAPK by endothelin-1 was not affected by chronic ethanol exposure. These data demonstrate that chronic ethanol feeding suppresses endothelin-1-stimulated glucose uptake and suggest that decreased expression of Gα11coupled to impaired endothelin-1-dependent activation of PYK2 contributes to this response.
Sepsis-induced muscle growth hormone resistance occurs independently of STAT5 phosphorylation
Authors: Hong-Brown Ly Q. , Brown C. Randell , Cooney Robert N. , Frost Robert A. , Lang Charles H.
Published: March 2015

Growth hormone (GH) stimulates insulin-like growth factor I (IGF-I) synthesis in both liver and muscle. During sepsis, proinflammatory cytokines inhibit GH action in liver, but it is unknown whether sepsis also produces GH resistance in muscle. Sepsis was induced by cecal ligation and puncture, and 18 h later the effect of GH on signal transducer and activator of transcription (STAT) phosphorylation and IGF-I mRNA content was assessed in rat gastrocnemius and liver. The relative abundance of phosphorylated (p)STAT5a, pSTAT5b, pSTAT3, and pSTAT1 was increased in liver from control rats after GH. Sepsis alone also increased hepatic pSTAT5a, pSTAT3, and pSTAT1. Sepsis dramatically impaired the ability of GH to stimulate the phosphorylation of STAT5a and -5b, as well as to increase IGF-I mRNA in liver. In muscle from control rats, GH increased pSTAT5a and -5b, whereas content of pSTAT3 and pSTAT1 was not affected. Sepsis increased basal content of pSTAT3 but not pSTAT5a, pSTAT5b, or pSTAT1 in muscle. The GH-induced increase of pSTAT5a and -5b in muscle from septic rats was not inhibited, suggesting that muscle was not GH resistant. In contrast to these changes in pSTAT5, the ability of GH to increase IGF-I mRNA was completely absent in muscle from septic rats. Because the suppressor of cytokine signaling (SOCS) proteins may function as negative regulators of GH signaling, we examined the content of these proteins. Sepsis produced small (30–50%), albeit statistically significant, increases in SOCS-1, -2, and -3 protein in muscle. In contrast to muscle, the SOCS proteins in the liver did not change under the various experimental conditions, suggesting that these proteins are not responsible for the impaired phosphorylation of STAT5 by GH. In conclusion, sepsis produces GH resistance in both muscle and liver, with the locus of this impairment in muscle differing from that in liver and being independent of a defect in STAT5 phosphorylation.
A mathematical model of metabolic insulin signaling pathways
Authors: Sedaghat Ahmad R. , Sherman Arthur , Quon Michael J.
Published: March 2015

We develop a mathematical model that explicitly represents many of the known signaling components mediating translocation of the insulin-responsive glucose transporter GLUT4 to gain insight into the complexities of metabolic insulin signaling pathways. A novel mechanistic model of postreceptor events including phosphorylation of insulin receptor substrate-1, activation of phosphatidylinositol 3-kinase, and subsequent activation of downstream kinases Akt and protein kinase C-ζ is coupled with previously validated subsystem models of insulin receptor binding, receptor recycling, and GLUT4 translocation. A system of differential equations is defined by the structure of the model. Rate constants and model parameters are constrained by published experimental data. Model simulations of insulin dose-response experiments agree with published experimental data and also generate expected qualitative behaviors such as sequential signal amplification and increased sensitivity of downstream components. We examined the consequences of incorporating feedback pathways as well as representing pathological conditions, such as increased levels of protein tyrosine phosphatases, to illustrate the utility of our model for exploring molecular mechanisms. We conclude that mathematical modeling of signal transduction pathways is a useful approach for gaining insight into the complexities of metabolic insulin signaling.
Inducible nitric oxide synthase mediates early epithelial repair of porcine ileum
Authors: Gookin Jody L. , Rhoads J. Marc , Argenzio Robert A.
Published: March 2015

Reports conflict regarding the effect of nitric oxide (NO) on intestinal epithelium. In chronic injury, NO appears detrimental by combining with reactive oxygen to form potent-free radicals. In contrast, inhibition of NO synthesis after acute injury exacerbates damage and inflammation. Recent studies have disclosed constitutive expression of inducible NO synthase (iNOS) by normal intestinal epithelia, yet little attention has been given to the role of iNOS in acute epithelial repair. We studied the local effects of iNOS on early epithelial repair of porcine ileal mucosa injured by deoxycholate within Ussing chambers. iNOS was constitutively expressed by the villous epithelium, and after deoxycholate injury, iNOS was expressed by injured and detaching enterocytes. Selective inhibition of iNOS abolished increases in NO synthesis and villous reepithelialization after injury. Exogenous l-arginine rescued baseline reepithelialization from NOS inhibitors but was only capable of stimulating additional repair in the presence of serum. These results demonstrate that iNOS-derived NO is a key mediator of early villous reepithelialization following acute mucosal injury.
Polyphenols fromCamellia sinenesisattenuate experimental cholestasis-induced liver fibrosis in rats
Authors: Zhong Zhi , Froh Matthias , Lehnert Mark , Schoonhoven Robert , Yang Liu , Lind Henrik , Lemasters John J. , Thurman Ronald G.
Published: March 2015

Accumulation of hydrophobic bile acids during cholestasis leads to generation of oxygen free radicals in the liver. Accordingly, this study investigated whether polyphenols from green tea Camellia sinenesis, which are potent free radical scavengers, decrease hepatic injury caused by experimental cholestasis. Rats were fed a standard chow or a diet containing 0.1% polyphenolic extracts from C. sinenesis starting 3 days before bile duct ligation. After bile duct ligation, serum alanine transaminase increased to 760 U/l after 1 day in rats fed a control diet. Focal necrosis and bile duct proliferation were also observed after 1–2 days, and fibrosis developed 2–3 wk after bile duct ligation. Additionally, procollagen-α1(I) mRNA increased 30-fold 3 wk after bile duct ligation, accompanied by increased expression of α-smooth muscle actin and transforming growth factor-β and the accumulation of 4-hydroxynenonal, an end product of lipid peroxidation. Polyphenol feeding blocked or blunted all of these bile duct ligation-dependent changes by 45–73%. Together, the results indicate that cholestasis due to bile duct ligation causes liver injury by mechanisms involving oxidative stress. Polyphenols from C. sinenesis scavenge oxygen radicals and prevent activation of stellate cells, thereby minimizing liver fibrosis.
Resistance to Fas-induced apoptosis in hepatocytes: role of GSH depletion by cell isolation and culture
Authors: Musallam Lina , Éthier Chantal , Haddad Pierre Selim , Denizeau Francine , Bilodeau Marc
Published: March 2015

The involvement of reduction/oxidation (redox) state in cell sensitivity to apoptosis has been suggested by several studies in which induction of apoptosis was shown to require oxidative stress or GSH extrusion. On the other hand, biochemical studies of caspases revealed that their activation necessitates a reduced cysteine in their active site. This is ensured by maintaining intact intracellular glutathione status during apoptotic induction as reported by in vivo studies. Therefore, we investigated the relationship between intracellular glutathione levels and the sensitivity of mouse hepatocytes in culture to Fas-induced apoptosis as well as potential mechanisms responsible for this sensitivity. We found that total and reduced glutathione levels are decreased by one-half after cell isolation procedure and further decline by 25% during cell culture for 2 h in normal Williams' E medium. Cell culture in medium supplemented with cysteine and methionine maintains glutathione at a level similar to that measured just after cell isolation. Results show that the capacity of Fas to activate caspase-8 and to induce apoptosis requires important intracellular glutathione levels and high GSH/total glutathione ratio. In conclusion, the present study shows that intracellular glutathione plays an important role in maintaining the apoptotic machinery functional and is thus capable of transmitting the apoptotic signal.
Physiological and morphological effects of alendronate on rabbit esophageal epithelium
Authors: Dobrucali A. , Tobey N. A. , Awayda M. S. , Argote C. , Abdulnour-Nakhoul S. , Shao W. , Orlando R. C.
Published: March 2015

Alendronate, an aminobisphosphonate, produces as a side effect a topical (pill induced) esophagitis. To gain insight into this phenomenon, we assessed the effects of luminal alendronate on both esophageal epithelial structure and function. Sections of rabbit esophageal epithelium were exposed to luminal alendronate at neutral or acidic pH while mounted in Ussing chambers to monitor transmural electrical potential difference (PD), short-circuit current ( Isc), and resistance ( R). Morphological changes were sought by light microscopy in hematoxylin and eosin-stained sections. Impedance analysis was used for localization of alendronate-induced effects on ion transport. Luminal, but not serosal, alendronate (pH 6.9–7.2), increased PD and Iscin a dose- and time-dependent manner, with little change in R and mild edema of surface cell layers. The changes in Isc(and PD) were reversible with drug washout and could be prevented either by inhibition of Na,K-ATPase activity with serosal ouabain or by inhibition of apical Na channels with luminal acidification to pH 2.0 with HCl. An effect on apical Na channel activity was also supported by impedance analysis. Luminal alendronate at acidic pH was more damaging than either alendronate at neutral pH or acidic pH alone. These data suggest that alendronate stimulates net ion (Na) transport in esophageal epithelium by increasing apical membrane sodium channel activity and that this occurs with limited morphological change and no alteration in barrier function. Also alendronate is far more damaging at acidic than at neutral pH, suggesting its association with esophagitis requires gastric acid for expression. This expression may occur either by potentiation between the damaging effects of (refluxed) gastric acid and drug or by acid-induced conversion of the drug to a more toxic form.
Ethyl pyruvate modulates inflammatory gene expression in mice subjected to hemorrhagic shock
Authors: Yang Runkuan , Gallo David J. , Baust Jeffrey J. , Uchiyama Takashi , Watkins Simon K. , Delude Russell L. , Fink Mitchell P.
Published: March 2015

Administration of pyruvate, an effective scavenger of reactive oxygen species, has been shown to be salutary in numerous models of redox-mediated tissue or organ injury. Pyruvate, however, is unstable in solution and, hence, is not attractive for development as a therapeutic agent. Herein, ethyl pyruvate, which is thought to be more stable than the parent compound, was formulated in a calcium-containing balanced salt solution [Ringer ethyl pyruvate solution (REPS)] and evaluated in a murine model of hemorrhagic shock and resuscitation (HS/R). Resuscitation with REPS instead of Ringer lactate solution (RLS) significantly improved survival at 24 h and abrogated bacterial translocation to mesenteric lymph nodes and the development of increased ileal mucosal permeability to FITC-labeled dextran (4,000 Da) at 4 h. Mice treated with REPS instead of RLS also had lower circulating levels of alanine aminotransferase at 4 h. Treatment with REPS instead of RLS decreased activation of nuclear factor-κB in liver and colonic mucosa after HS/R and also decreased the expression of inducible nitric oxide synthase, tumor necrosis factor, cyclooxygenase-2, and interleukin-6 mRNA in liver, ileal mucosa, and/or colonic mucosa. These data support the view that resuscitation with REPS modulates the inflammatory response and decreases hepatocellular and gut mucosal injury in mice subjected to HS/R.
Role for complement in mediating intestinal nitric oxide synthase-2 and superoxide dismutase expression
Authors: Montalto Michael C. , Hart Melanie L. , Jordan James E. , Wada Koichiro , Stahl Gregory L.
Published: March 2015

Inducible nitric oxide synthase (iNOS) and superoxide dismutase (SOD) play an important role in the pathology of ischemia-reperfusion. This study sought to determine if the proinflammatory effects of complement modulate iNOS and SOD in the rat after gastrointestinal ischemia and reperfusion (GI/R). An inhibitory or noninhibitory anti-complement component 5 (C5) monoclonal antibody (18A or 16C, respectively) was administered before GI/R. RT-PCR revealed a significant increase in intestinal iNOS mRNA compared with sham after GI/R that was attenuated significantly by 18A. Immunohistochemistry demonstrated increased iNOS protein expression within the intestinal crypts after GI/R. Cu/Zn SOD (mRNA and protein) was unaffected by GI/R, whereas Cu/Zn SOD activity was reduced significantly. Mn SOD protein expression was decreased significantly by GI/R. Anti-C5 preserved Cu/Zn SOD activity and Mn SOD protein expression. Staining for nitrotyrosine showed that anti-C5 treatment reduced protein nitration in the reperfused intestine. Immunohistochemistry demonstrated prominent phosphorylated (p) inhibitory factor-κB (IκB)-α staining of intestinal tissue after GI/R, whereas anti-C5 reduced p-IκB-α expression. These data indicate that complement may mediate tissue damage during GI/R by increasing intestinal iNOS and decreasing the activity and protein levels of Cu/Zn SOD and Mn SOD, respectively.
Intrabolus pressure gradient identifies pathological constriction in the upper esophageal sphincter during flow
Authors: Pal Anupam , Williams Rohan B. , Cook Ian J. , Brasseur James G.
Published: March 2015

Propulsion of a bolus through the upper esophageal sphincter (UES) is driven by a pressure drop in the direction of flow against frictional resisting force. Basic mechanics suggest that the axial rate of drop in intrabolus pressure (IBP), i.e., the intrabolus pressure gradient (IBPG), should be locally sensitive to abnormal constriction. We sought to quantify space-time patterns of IBP and IBPG that correlate with pathological disruption to transsphincteric bolus transport. High-resolution high-fidelity perfused manometry was applied concurrent with videofluoroscopy in 6 healthy controls and 10 patients with restricted UES opening and 4 bolus volumes. Pressures were interpolated spatially and displayed as space-time isocontours with bolus head and tail trajectories superimposed to identify the IBP domain. IBP and IBPG were averaged over an approximately steady period of transsphincteric flow. The axial location and magnitude of maximum IBPG were quantified for each swallow relative to the location of the abnormal restriction. We found that average hypopharyngeal IBP and locally maximal IBPG were significantly higher in the patient group ( P < 0.001), whereas the maximum IBPG was insensitive to bolus volume, and the locations of maximum IBPG in the patient group were well correlated with axial locations of maximal UES constriction ( r = 0.84, P < 0.01). Space-time structure of IBP and IBPG correlated qualitatively with swallow dysfunction. Because IBPG reflects pressure force driving the bolus against frictional force in the UES, IBPG reflects local changes in frictional resistance from pathological constriction during bolus flow. Consequently, the location and magnitude of IBPG reflect the existence and location of abnormal constriction, and IBP and IBPG structure reflect decompensation of the pharyngeal swallow.
Fasting in α1-antitrypsin deficient liver: constitutive activation of autophagy
Authors: Teckman Jeffrey H. , An Jae-Koo , Loethen Scott , Perlmutter David H.
Published: March 2015

α1-Antitrypsin (α1-AT) deficiency causes severe liver injury in a subgroup of patients. Liver injury is thought to be caused by retention of a polymerized mutant α1-ATZ molecule in the endoplasmic reticulum (ER) of hepatocytes and is associated with an intense autophagic response. However, there is limited information about what physiologic stressors might influence liver injury. In this study, we examined the effect of fasting in the PiZ mouse model of α1-AT deficiency, because fasting is a well-characterized physiological stressor and a known stimulus for autophagy. Results show that there is a marked increase in fat accumulation and in α1-AT-containing globules in the liver of the PiZ mouse induced by fasting. Although fasting induced a marked autophagic response in wild-type mice, the autophagic response was already activated in PiZ mice and did not further increase with fasting. PiZ mice also had a significantly decreased tolerance for prolonged fasting compared with wild-type mice (PiZ mice 0% survival of 72-h fast; wild-type 100% survivial). These results demonstrate an altered response to stress in the α1-AT-deficient liver, including inability to further increase an activated autophagic response, a developmental state-specific increase in α1-AT-containing globules, and increased mortality.
Inflammatory reaction without endogenous antioxidant response in Caco-2 cells exposed to iron/ascorbate-mediated lipid peroxidation
Authors: Bernotti Sandra , Seidman Ernest , Sinnett Daniel , Brunet Sylvain , Dionne Serge , Delvin Edgard , Levy Emile
Published: March 2015

To characterize the role of intestinal epithelial cells in mucosal host defense, we have examined endogenous antioxidant reactivity and inflammatory response in Caco-2 cell line. When differentiated Caco-2 cells were incubated with iron/ascorbate for 1–24 h, they exhibited increased malondialdehyde levels and decreased polyunsaturated fatty acid proportion in favor of saturated fatty acids. These modifications were accompanied with alterations in membrane fluidity and permeability. The oxidative stress did not induce changes in the antioxidant enzyme activity of superoxide dismutase, catalase, glutathione peroxidase, and glutathione transferase, or in cellular glutathione content. However, iron/ascorbate-mediated lipid peroxidation promoted inhibitor-κB degradation and NF-κB activation, as well as gave rise to IL-8, cyclooxygenase-2, and ICAM-1. These results support the importance of oxidant/antioxidant balance in the epithelial cell inflammatory response.
Mechanisms by which cAMP increases bile acid secretion in rat liver and canalicular membrane vesicles
Authors: Misra Suniti , Varticovski Lyuba , Arias Irwin M.
Published: March 2015

Bile acid secretion induced by cAMP and taurocholate is associated with recruitment of several ATP binding cassette (ABC) transporters to the canalicular membrane. Taurocholate-mediated bile acid secretion and recruitment of ABC transporters are phosphatidylinositol 3-kinase (PI3K) dependent and require an intact microtubular apparatus. We examined mechanisms involved in cAMP-mediated bile acid secretion. Bile acid secretion induced by perfusion of rat liver with dibutyryl cAMP was blocked by colchicine and wortmannin, a PI3K inhibitor. Canalicular membrane vesicles isolated from cAMP-treated rats manifested increased ATP-dependent transport of taurocholate and PI3K activity that were reduced by prior in vivo administration of colchicine or wortmannin. Addition of a PI3K lipid product, phosphoinositide 3,4-bisphosphate, but not its isomer, phosphoinositide 4,5-bisphosphate, restored ATP-dependent taurocholate in these vesicles. Addition of a decapeptide that activates PI3K to canalicular membrane vesicles increased ATP-dependent transport above baseline activity. In contrast to effects induced by taurocholate, cAMP-stimulated intracellular trafficking of the canalicular ABC transporters was unaffected by wortmannin, and recruitment of multidrug resistance protein 2, but not bile salt excretory protein (bsep), was partially decreased by colchicine. These studies indicate that trafficking of bsep and other canalicular ABC transporters to the canalicular membrane in response to cAMP is independent of PI3K activity. In addition, PI3K lipid products are required for activation of bsep in the canalicular membrane. These observations prompt revision of current concepts regarding the role of cAMP and PI3K in intracellular trafficking, regulation of canalicular bsep, and bile acid secretion.
Role of Ca2+-activated Cl−channels and MLCK in slow IJP in opossum esophageal smooth muscle
Authors: Zhang Yong , Paterson William G.
Published: March 2015

The possible contribution of Ca2+-activated Cl−channel [ICl(Ca)] and myosin light-chain kinase (MLCK) to nonadrenergic, noncholinergic slow inhibitory junction potentials (sIJP) was studied using conventional intracellular microelectrode recordings in circular smooth muscle of opossum esophageal body and guinea pig ileum perfused with Krebs solution containing atropine (3 μM), guanethidine (3 μM), and substance P (1 μM). In opossum esophageal circular smooth muscle, resting membrane potential (MP) was −51.9 ± 0.7 mV ( n = 89) with MP fluctuations of 1–3 mV. A single square-wave nerve stimulation of 0.5 ms duration and 80 V induced a sIJP with amplitude of 6.3 ± 0.2 mV, half-amplitude duration of 635 ± 19 ms, and rebound depolarization amplitude of 2.4 ± 0.1 mV ( n = 89). 9-Anthroic acid (A-9-C), niflumic acid (NFA), wortmannin, and 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine (ML-9) abolished MP fluctuations, sIJP, and rebound depolarization in a concentration-dependent manner. A-9-C and NFA but not wortmannin and ML-9 hyperpolarized MP. In guinea pig ileal circular smooth muscle, nerve stimulation elicited an IJP composed of both fast (fIJP) and slow (sIJP) components, followed by rebound depolarization. NFA (200 μM) abolished sIJP and rebound depolarization but left the fIJP intact. These data suggest that in the tissues studied, activation of ICl(Ca), which requires MLCK, contributes to resting MP, and that closing of ICl(Ca)is responsible for sIJP.
Permeability transition in rat liver mitochondria is modulated by the ATP-Mg/Picarrier
Authors: Hagen Thilo , Lagace Christopher J. , Modica-Napolitano Josephine S. , Aprille June R.
Published: March 2015

Mitochondrial permeability transition, due to opening of the permeability transition pore (PTP), is triggered by Ca2+in conjunction with an inducing agent such as phosphate. However, incubation of rat liver mitochondria in the presence of low micromolar concentrations of Ca2+and millimolar concentrations of phosphate is known to also cause net efflux of matrix adenine nucleotides via the ATP-Mg/Picarrier. This raises the possibility that adenine nucleotide depletion through this mechanism contributes to mitochondrial permeability transition. Results of this study show that phosphate-induced opening of the mitochondrial PTP is, at least in part, secondary to depletion of the intramitochondrial adenine nucleotide content via the ATP-Mg/Picarrier. Delaying net adenine nucleotide efflux from mitochondria also delays the onset of phosphate-induced PTP opening. Moreover, mitochondria that are depleted of matrix adenine nucleotides via the ATP-Mg/Picarrier show highly increased susceptibility to swelling induced by high Ca2+concentration, atractyloside, and the prooxidant tert-butylhydroperoxide. Thus the ATPMg/Picarrier, by regulating the matrix adenine nucleotide content, can modulate the sensitivity of rat liver mitochondria to undergo permeability transition. This has important implications for hepatocytes under cellular conditions in which the intramitochondrial adenine nucleotide pool size is depleted, such as in hypoxia or ischemia, or during reperfusion when the mitochondria are exposed to increased oxidative stress.
Stimulation of the gastrin-cholecystokininBreceptor promotes branching morphogenesis in gastric AGS cells
Authors: Pagliocca A. , Wroblewski L. E. , Ashcroft F. J. , Noble P. J. , Dockray G. J. , Varro A.
Published: March 2015

Epithelial organization is maintained by cell proliferation, migration, and differentiation. In the case of the gastric epithelium, at least some of these events are regulated by the hormone gastrin. In addition, gastric epithelial cells are organized into characteristic tubular structures (the gastric glands), but the cellular mechanisms regulating the organization of tubular structures (sometimes called branching morphogenesis) are uncertain. In the present study, we examined the role of the gastrin-cholecystokininB receptor in promoting branching morphogenesis of gastric epithelial cells. When gastric cancer AGS-GR cells were cultured on plastic, gastrin and PMA stimulated cell adhesion, formation of lamellipodia, and extension of long processes in part by activation of protein kinase C (PKC) and phosphatidylinositol (PI)-3 kinase. Branching morphogenesis was not observed in these circumstances. However, when cells were cultured on artificial basement membrane, the same stimuli increased the formation of organized multicellular arrays, exhibiting branching morphogenesis. These effects were reversed by inhibitors of PKC but not of PI-3 kinase. We conclude that, in the presence of basement membrane, activation of PKC by gastrin stimulates branching morphogenesis.
PAR-2 agonists induce contraction of murine small intestine through neurokinin receptors
Authors: Zhao Aiping , Shea-Donohue Terez
Published: March 2015

Protease-activated receptor-2 (PAR-2) is a G protein-coupled receptor and is expressed throughout the gut. It is well known that PAR-2 participates in the regulation of gastrointestinal motility; however, the results are inconsistent. The present study investigated the effect and mechanism of PAR-2 activation on murine small intestinal smooth muscle function in vitro. Both trypsin and PAR-2-activating peptide SLIGRL induced a small relaxation followed by a concentration-dependent contraction. The sensitivity to trypsin was greater than that to SLIGRL (EC50 = 0.03 vs. 40 μM), but maximal responses were similar (12.3 ± 1.6 vs. 13.7 ± 1.3 N/cm2). Trypsin-evoked contraction (1 μM) exhibited a rapid desensitization, whereas the desensitization of response to SLIGRL was less even at high concentration (50 μM). Atropine had no effect on PAR-2 agonist-induced contractions. In contrast, TTX and capsaicin significantly attenuated those contractions, implicating a neurogenic mechanism that may involve capsaicin-sensitive sensory nerves. Furthermore, contractions induced by trypsin and SLIGRL were reduced by neurokinin receptor NK1 antagonist SR-140333 or NK2 antagonist SR-48968 alone or were further reduced by combined application of SR-140333 and SR-48968, indicating the involvement of neurokinin receptors. In addition, desensitizing neurokinin receptors with substance P and/or neurokinin A decreased the PAR-2 agonist-evoked contraction. We concluded that PAR-2 agonists induced a contraction of murine intestinal smooth muscle that was mediated by nerves. The excitatory effect is also dependent on sensory neural pathways and requires both NK1 and NK2 receptors.
Impaired gastrocolonic response and peristaltic reflex in slow-transit constipation: role of 5-HT3 pathways
Authors: Björnsson Einar S. , Chey William D. , Hooper Forrest , Woods Michelle L. , Owyang Chung , Hasler William L.
Published: March 2015

Colonic motility is modulated by the 5-hydroxytryptamine (5-HT)3-dependent gastrocolonic response and 5-HT3-independent peristaltic reflex. We compared descending colon tone responses to antral distension, duodenal lipid perfusion, and colonic distension after double-blind placebo or granisetron in 13 healthy volunteers and nine slow-transit constipated patients. Antral distension (100–300 ml) and duodenal lipids (3 kcal/min) evoked increases in colon tone in volunteers, which were blunted in constipated patients ( P < 0.05). Granisetron (10 μg/kg) reduced responses to antral distension and lipids in volunteers and to lipids in constipated patients ( P < 0.05). The ascending contraction of the peristaltic reflex was blunted in constipated patients ( P < 0.05), whereas descending responses were similar. Granisetron did not modify the peristaltic reflex. Colonic responses to bethanechol were similar in patients and volunteers. In conclusion, antral distension- and duodenal lipid-activated gastrocolonic responses and ascending contractions of the peristaltic reflex are impaired with slow-transit constipation with loss of both 5-HT3-dependent and -independent function. Thus abnormalities of neural reflex modulation of colonic motor function may play pathophysiological roles in slow-transit constipation.
Independence of apical Cl-/HCO3-exchange and anion conductance in duodenal HCO3-secretion
Authors: Spiegel S. , Phillipper M. , Rossmann H. , Riederer B. , Gregor M. , Seidler U.
Published: March 2015

Reduced gastrointestinal [Formula: see text] secretion contributes to malabsorption and obstructive syndromes in cystic fibrosis. The apical [Formula: see text] transport pathways in these organs have not been defined. We therefore assessed the involvement of apical Cl-/[Formula: see text] exchangers and anion conductances in basal and cAMP-stimulated duodenal [Formula: see text] secretion. Muscle-stripped rat and rabbit proximal duodena were mounted in Ussing chambers, and electrical parameters, [Formula: see text] secretion rates, and36Cl-,22Na+, and3H+mannitol fluxes were assessed. mRNA expression levels were measured by a quantitative PCR technique. Removal of Cl-from or addition of 1 mM DIDS to the luminal perfusate markedly decreased basal [Formula: see text] secretion but did not influence the [Formula: see text] secretory response to 8-bromo-cAMP, which was inhibited by luminal 5-nitro-2-(3-phenylpropylamino)-benzoate. Bidirectional22Na+and36Cl-flux measurements demonstrated an inhibition rather than a stimulation of apical anion exchange during cAMP-stimulated [Formula: see text] secretion. The ratio of Cl-to [Formula: see text] in the anion secretory response was compatible with both Cl-and [Formula: see text] being secreted via the CFTR anion channel. CFTR expression was very high in the duodenal mucosa of both species. We conclude that in rat and rabbit duodena, an apical Cl-/[Formula: see text] exchanger mediates a significant part of basal [Formula: see text] secretion but is not involved in the [Formula: see text] secretory response to cAMP analogs. The inhibitor profile, the strong predominance of Cl-over [Formula: see text] in the anion secretory response, and the high duodenal CFTR expression levels suggest that a major portion of cAMP-stimulated duodenal [Formula: see text] secretion is directly mediated by CFTR.
Fat malabsorption in essential fatty acid-deficient mice is not due to impaired bile formation
Authors: Werner Anniek , Minich Deanna M. , Havinga Rick , Bloks Vincent , Van Goor Harry , Kuipers Folkert , Verkade Henkjan J.
Published: March 2015

Essential fatty acid (EFA) deficiency induces fat malabsorption, but the pathophysiological mechanism is unknown. Bile salts (BS) and EFA-rich biliary phospholipids affect dietary fat solubilization and chylomicron formation, respectively. We investigated whether altered biliary BS and/or phospholipid secretion mediate EFA deficiency-induced fat malabsorption in mice. Free virus breed (FVB) mice received EFA-containing (EFA+) or EFA-deficient (EFA−) chow for 8 wk. Subsequently, fat absorption, bile flow, and bile composition were determined. Identical dietary experiments were performed in multidrug resistance gene-2-deficient [ Mdr2(−/−)] mice, secreting phospholipid-free bile. After 8 wk, EFA−-fed wild-type [ Mdr2(+/+)] and Mdr2(−/−)mice were markedly EFA deficient [plasma triene (20:3n-9)-to-tetraene (20:4n-6) ratio >0.2]. Fat absorption decreased (70.1 ± 4.2 vs. 99.1 ± 0.3%, P < 0.001), but bile flow and biliary BS secretion increased in EFA−mice compared with EFA+controls (4.87 ± 0.36 vs. 2.87 ± 0.29 μl · min−1· 100 g body wt−1, P < 0.001, and 252 ± 30 vs. 145 ± 20 nmol · min−1· 100 g body wt−1, P < 0.001, respectively). BS composition was similar in EFA+- and EFA−-fed mice. Similar to EFA−Mdr2(+/+)mice, EFA−Mdr2(−/−)mice developed fat malabsorption associated with twofold increase in bile flow and BS secretion. Fat malabsorption in EFA−mice is not due to impaired biliary BS or phospholipid secretion. We hypothesize that EFA deficiency affects intracellular processing of dietary fat by enterocytes.
Coupling of M2muscarinic receptors to Src activation in cultured canine colonic smooth muscle cells
Authors: Singer Cherie A. , Vang Sa , Gerthoffer William T.
Published: March 2015

The purpose of this study was to determine whether Src tyrosine kinases are one of the signaling intermediaries linking M2receptor stimulation to extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) in cultures of canine colonic smooth muscle cells (CSMC). RT-PCR studies demonstrate expression of multiple Src tyrosine kinases, including Src, Fyn, and Yes, in CSMC. Muscarinic stimulation of CSMC with 10 μM ACh results in a twofold increase in Src activity within 10 min but does not increase the activity of Fyn. Treatment with the M2antagonist AF-DX 116 (10 μM) blocks ACh-stimulated Src activation in primary CSMC cultures that express both M2and M3receptors and in first-passage CSMC cultures that express predominantly M2receptors. Alkylation of M3receptors with 100 nM N,N-dimethyl-4-piperidinyl diphenylacetate mustard has no effect on Src activity. Treatment with the pyrazolopyrimidine Src inhibitor PP1 (10 μM) or AF-DX 116 (10 μM) blocks ACh-stimulated ERK phosphorylation. Together these results indicate that M2receptors are coupled to Src tyrosine kinase and subsequent activation of ERK in cultured CSMC.
Mechanism of active repolarization of inhibitory junction potential in murine colon
Authors: Baker Salah A. , Mutafova-Yambolieva Violeta , Monaghan Kevin , Horowitz Burton , Sanders Kenton M. , Koh Sang Don
Published: March 2015

Enteric inhibitory responses in gastrointestinal (GI) smooth muscles involve membrane hyperpolarization that transiently reduce the excitability of GI muscles. We examined the possibility that an active repolarization mechanism participates in the restoration of resting membrane potential after fast inhibitory junction potentials (IJPs) in the murine colon. Previously, we showed these cells express a voltage-dependent nonselective cation conductance (NSCC) that might participate in active repolarization of IJPs. Colonic smooth muscle cells were impaled with micro-electrodes and voltage responses to nerve-evoked IJPs, and locally applied ATP were recorded. Ba2+ (500 μM), a blocker of the NSCC, slowed the rate of repolarization of IJPs. We also tested the effects of Ba2+, Ni2+, and mibefradil, all blockers of the NSCC, on responses to locally applied ATP. Spritzes of ATP caused transient hyperpolarization, and the durations of these responses were significantly increased by the blockers of the NSCC. We considered whether NSCC blockers might affect ATP metabolism and found that Ni2+ decreased ATP breakdown in colonic muscles. Mibefradil had no effect on ATP metabolism. Because both Ni2+ and mibefradil had similar effects on prolonging responses to ATP, it appears that restoration of resting membrane potential after ATP spritzes is not primarily due to ATP metabolism. Neurally released enteric inhibitory transmitter and locally applied ATP resulted in transient hyperpolarizations of murine colonic muscles. Recovery of membrane potential after these responses appears to involve an active repolarization mechanism due to activation of the voltage-dependent NSCC expressed by these cells.
Gastrin stimulates the growth of gastric pit cell precursors by inducing its own receptors
Authors: Nakajima Toshio , Konda Yoshitaka , Izumi Yoshio , Kanai Masashi , Hayashi Naoki , Chiba Tsutomu , Takeuchi Toshiyuki
Published: March 2015

Gastrin/CCK-B receptors (CCKB-Rs) are present on parietal and enterochromaffin-like cells in the gastric mucosa but not on pit cells in the proliferative zone. Because serum gastrin levels are well correlated with the growth of the gastric pit, we examined whether pit precursor cells express CCKB-Rs using hypergastrinemic transgenic mice and a mouse pit precursor cell line, GSM06. In situ hybridization indicated that CCKB-R mRNA was limited to the lower one-third of the mucosa in control mice, whereas it was faintly distributed along the mid- to low glandular region in the hypergastrinemic transgenic mouse mucosa. CCKB-R-positive midglandular cells appear to have a pit cell lineage; therefore, GSM06 cells were used for an [125I]gastrin binding study. [125I]gastrin bound to the membrane fraction of the GSM06 cells when precultured with gastrin. Gastrin dose dependently induced CCKB-R expression in GSM06 cells and stimulated their growth. Thus these findings suggest that gastrin directly stimulates the growth of the pit cell lineage by inducing its own receptor in pit cell precursors.
Localization and function of metabotropic glutamate receptor 8 in the enteric nervous system
Authors: Tong Qingchun , Kirchgessner Annette L.
Published: March 2015

The enteric nervous system (ENS) contains glutamatergic neurons, transporters, and functional ionotropic and groups I and II metabotropic glutamate receptors (mGluRs). The aim of this study was to determine whether the ENS contains functional group III mGluRs. RT-PCR demonstrated the expression of mGluR7 and mGluR8 mRNA in rat myenteric ganglia. Western blot analysis confirmed the presence of mGluR8 protein. Immunocytochemistry, in conjunction with confocal microscopy, demonstrated mGluR8 immunoreactivity in the ENS of several species, including humans. mGluR8 immunoreactivity was localized to the membrane of nerve cell bodies that received glutamatergic input. Significant receptor internalization of mGluR8 was observed on activation, and localization to membrane was observed on blocking with the mGluR III antagonist (RS)-cyclopropyl-4-phosphonophenylglycine (CPPG). mGluR8-positive myenteric neurons contained glutamate or nitric oxide synthase (NOS), a marker of inhibitory motorneurons. Enteric group III mGluRs are functional because mGluR8 agonists inhibited forskolin-induced accumulation of cAMP in isolated myenteric ganglia, and CPPG reduced this effect. In addition, an accelerating effect on guinea pig colonic motility was observed after the application of mGluR8 agonists. Increase in motility was specific, because CPPG inhibited it. Moreover, in the presence of hexamethonium or Nω-nitro-l-arginine methyl ester, an inhibitor of NOS, responses caused by mGluR8 agonists were abolished. mGluR8 agonists also increased longitudinal muscle contractions. These findings suggest that mGluR8 agonists increase motility by inhibiting nitrergic relaxation and possibly by facilitating cholinergic contractions.
Musings on the Wanderer: What's New in Our Understanding of Vago-Vagal Reflexes? V. Remodeling of vagus and enteric neural circuitry after vagal injury
Authors: Li Ying , Owyang Chung
Published: March 2015

The vago-vagal reflexes mediate a wide range of digestive functions such as motility, secretion, and feeding behavior. Previous articles in this series have discussed the organization and functions of this important neural pathway. The focus of this review will be on some of the events responsible for the adaptive changes of the vagus and the enteric neutral circuitry that occur after vagal injury. The extraordinary plasticity of the neural systems to regain functions when challenged with neural injury will be discussed. In general, neuropeptides and transmitter-related enzymes in the vagal sensory neurons are downregulated after vagal injury to protect against further injury. Conversely, molecules previously absent or present at low levels begin to appear or are upregulated and are available to participate in the survival-regeneration process. Neurotrophins and other related proteins made at the site of the lesion and then retrogradely transported to the soma may play an important role in the regulation of neuropeptide phenotype expression and axonal growth. Vagal injury also triggers adaptive changes within the enteric nervous system to minimize the loss of gastrointestinal functions resulting from the interruption of the vago-vagal pathways. These may include rearrangement of the enteric neural circuitry, changes in the electrophysiological properties of sensory receptors in the intramural neural networks, an increase in receptor numbers, and changes in the affinity states of receptors on enteric neurons.
Ion channel diversity in the feline smooth muscle esophagus
Authors: Salapatek Anne Marie F. , Ji Junzhi , Diamant Nicholas E.
Published: March 2015

We have characterized ion-channel identity and density differences along the feline smooth muscle esophagus using patch-clamp recording. Current clamp recording revealed that the resting membrane potential (RMP) of esophageal smooth muscle cells (SMC) from the circular layer at 4 cm above the lower esophageal sphincter (EBC4; LES) were more depolarized than at 2 cm above LES. Higher distal Na+permeability (but not Cl−permeability) contributes to this RMP difference. K+channels but not large-conductance Ca2+-activated K+(BKCa) channels contribute to RMP at both levels, because nonspecific K+-channel blockers depolarize all SMC. Depolarization of SMC under voltage clamp revealed that the density of voltage-dependent K+channels (KV) was greatest at EBC4 due to increased BKCa.Delayed rectifier K+channels (KDR), compatible with subtype KV1.2, were present at both levels. Differences in KCa-to-KDRchannel ratios were also manifest by predictable shifts in voltage-dependent inactivation at EBC4 when BKCachannels were blocked. We provide the first evidence for regional electrophysiological differences along the esophageal body resulting from SMC ion channel diversity, which could allow for differential muscular responses to innervation and varied muscular contribution to peristaltic contractions along the esophagus.
Expression of rat hepatic multidrug resistance-associated proteins and organic anion transporters in pregnancy
Authors: Cao Jingsong , Stieger Bruno , Meier Peter J. , Vore Mary
Published: March 2015

The expression of hepatic multidrug resistance-associated protein (Mrp)1, 2, 3, and 6 and organic anion transporting polypeptides (Oatp)1 and 2 were examined in control and 20- to 21-day pregnant rats. Western analysis showed that expression of Oatp2 was decreased 50% in pregnancy, whereas expression of Oatp1 did not change. Expression of Mrp2 protein determined by Western analysis of total liver homogenate decreased to 50% of control levels in pregnant rats, consistent with studies using plasma membranes. Confocal immunohistochemistry showed that Mrp2 expression was confined to the canalicular membrane in both control and pregnant rats and was not detectable in intracellular compartments. In isolated perfused liver, the biliary excretion of 2,4-dintrophenyl-glutathione was significantly decreased in pregnancy, consistent with decreased expression of Mrp2. The expression of the basolateral transporter Mrp1 was not altered in pregnancy, whereas expression of Mrp6 mRNA was decreased by 60%. Expression of Mrp3 was also decreased by 50% in pregnant rat liver, indicating differential regulation of Mrp isoforms in pregnancy. These data also demonstrate that decreased Mrp2 expression is not necessarily accompanied by increased Mrp3 expression.
Cholecystokinin secretagogue-induced gastroprotection: role of nitric oxide and blood flow
Authors: West Sonlee D. , Helmer Kenneth S. , Chang Lily K. , Cui Yan , Greeley George H. , Mercer David W.
Published: March 2015

This study was done to examine the role of CCK in gastric mucosal defense and to assess the gastroprotective roles of nitric oxide and blood flow. In rats, the CCK secretagogues oleate and soybean trypsin inhibitor augmented gastric mucosal blood flow and prevented gastric injury from luminal irritants. Type A CCK receptor blockade negated CCK secretagogue-induced gastroprotection and exacerbated gastric injury from bile and ethanol but did not block adaptive cytoprotection. CCK secretagogue-induced gastroprotection and hyperemia were negated by nonselective nitric oxide synthase (NOS) inhibition ( NG-nitro-l-arginine methyl ester) but not by selective inducible NOS inhibition (aminoguanidine). Gastric mucosal calcium-dependent NOS activity, but not calcium-independent NOS activity, was increased following CCK and CCK secretagogues. The release of endogenous CCK plays a role in the intrinsic gastric mucosal defense system against injury from luminal irritants. The protective mechanism appears to involve increased production of nitric oxide from primarily the constitutive isoforms of NOS and a resultant increase in blood flow.
Identification of the biomechanical factors associated with the perception of distension in the human esophagus
Authors: Barlow J. D. , Gregersen H. , Thompson D. G.
Published: March 2015

Current techniques used to investigate the mechanisms responsible for the sensory responses to distension of the human esophagus provide limited information because the degree of circumferential stretch required to determine tension can only be inferred. We used impedance planimetry to measure the cross-sectional area during esophageal distension to ascertain the degree of stretch and tension that initiated motor and sensory responses. Hyoscine- N-butyl bromide (HBB), a cholinergic muscarinic receptor blocker, was also used to alter esophageal tension during distension. Motor activity was initiated at a lower degree of stretch and tension than that which initiated sensory awareness; both increased directly with increasing distension. HBB reduced both esophageal motility and tension during distension without altering the relationship between sensation intensity and cross-sectional area. Esophageal stretch, rather than tension, thus appears to be the major factor influencing sensory responses to esophageal distension.