AJP: Gastrointestinal and Liver Physiology current issue

Epithelial cells in fetal intestine produce chemerin to recruit macrophages

2009-06-26

Macrophages are first seen in the fetal intestine at 11–12 wk and rapidly increase in number during the 12- to 22-wk period of gestation. The development of macrophage populations in the fetal intestine precedes the appearance of lymphocytes and neutrophils and does not require the presence of dietary or microbial antigens. In this study, we investigated the role of chemerin, a recently discovered, relatively selective chemoattractant for macrophages, in the recruitment of macrophage precursors to the fetal intestine. Chemerin mRNA/protein expression was measured in jejunoileal tissue from 10- to 24-wk human fetuses, neonates operated for intestinal obstruction, and adults undergoing bariatric surgery. The expression of chemerin in intestinal epithelial cells (IECs) was confirmed by using cultured primary IECs and IEC-like cell lines in vitro. The regulatory mechanisms involved in chemerin expression were investigated by in silico and immunolocalization techniques. IECs in the fetal, but not mature, intestine express chemerin. Chemerin expression peaked in the fetal intestine at 20–24 wk and then decreased to original low levels by full term. During the 10- to 24-wk period, chemerin accounted for most of the macrophage chemotactic activity of cultured fetal IECs. The maturational changes in chemerin expression correlated with the expression of retinoic acid receptor-β in the intestine. Chemerin is an important mediator of epithelial-macrophage cross talk in the fetal/premature, but not in the mature, intestine. Understanding the regulation of the gut macrophage pool is an important step in development of novel strategies to boost mucosal immunity in premature infants and other patient populations at risk of microbial translocation.

Follicle-stimulating hormone increases cholangiocyte proliferation by an autocrine mechanism via cAMP-dependent phosphorylation of ERK1/2 and Elk-1

2009-06-26

Sex hormones regulate cholangiocyte hyperplasia in bile duct-ligated (BDL) rats. We studied whether follicle-stimulating hormone (FSH) regulates cholangiocyte proliferation. FSH receptor (FSHR) and FSH expression was evaluated in liver sections, purified cholangiocytes, and cholangiocyte cultures (NRICC). In vivo, normal female and male rats were treated with FSH or immediately after BDL with antide (a gonadotropin-releasing hormone antagonist blocking FSH secretion) or a neutralizing FSH antibody for 1 wk. We evaluated 1) cholangiocyte proliferation in sections and cholangiocytes and 2) changes in secretin-stimulated cAMP (functional index of cholangiocyte growth) levels, and ERK1/2 and Elk-1 phosphorylation. NRICC were stimulated with FSH before evaluation of proliferation, cAMP/IP₃ levels, and ERK1/2 and Elk-1 phosphorylation. To determine whether FSH regulates cholangiocyte proliferation by an autocrine mechanism, we evaluated the effects of 1) cholangiocyte supernatant (containing FSH) on NRICC proliferation and 2) FSH silencing in NRICC before measuring proliferation and ERK1/2 and Elk-1 phosphorylation. Cholangiocytes and NRICC express FSHR and FSH and secrete FSH. In vivo administration of FSH to normal rats increased, whereas administration of antide and anti-FSH antibody to BDL rats decreased 1) ductal mass and 2) secretin-stimulated cAMP levels, proliferation, and ERK1/2 and Elk-1 phosphorylation in cholangiocytes compared with controls. In NRICC, FSH increased cholangiocyte proliferation, cAMP levels, and ERK1/2 and Elk-1 phosphorylation. The supernatant of cholangiocytes increased NRICC proliferation, inhibited by preincubation with anti-FSH antibody. Silencing of FSH gene decreases cholangiocyte proliferation and ERK1/2 and Elk-1 phosphorylation. Modulation of cholangiocyte FSH expression may be important for the management of cholangiopathies.

Endothelin antagonism in portal hypertensive mice: implications for endothelin receptor-specific signaling in liver disease

Feng, H.-Q., Weymouth, N. D., Rockey, D. C. — 2009-06-26

Endothelin-1 (ET-1), a potent vasoactive peptide, plays an important role in the pathogenesis of liver disease and portal hypertension. Two major endothelin receptors (ET-A and ET-B) mediate biological effects, largely on the basis of their known downstream signaling pathways. We hypothesized that the different receptors are likely to mediate divergent effects in portal hypertensive mice. Liver fibrosis and cirrhosis and portal hypertension were induced in 8-wk-old male BALB/c mice by gavage with carbon tetrachloride (CCl₄). Portal pressure was recorded acutely during intravenous infusion of endothelin receptor antagonists in normal or portal hypertensive mice. In vivo microscopy was used to monitor sinusoidal dynamics. Additionally, the effect of chronic exposure to endothelin antagonists was assessed in mice during induction of fibrosis and cirrhosis with CCl₄ for 8 wk. Intravenous infusion of ET-A receptor antagonists into normal and cirrhotic mice reduced portal pressure whereas ET-B receptor antagonism increased portal pressure. A mixed endothelin receptor antagonist also significantly reduced portal pressure. Additionally, the ET-A receptor antagonist caused sinusoidal dilation, whereas the ET-B receptor antagonist caused sinusoidal constriction. Chronic administration of each the endothelin receptor antagonists during the induction of fibrosis and portal hypertension led to reduced fibrosis, a significant reduction in portal pressure, and altered sinusoidal dynamics relative to controls. Acute effects of endothelin receptor antagonists are likely directly on the hepatic and sinusoidal vasculature, whereas chronic endothelin receptor antagonism appears to be more complicated, likely affecting fibrogenesis and the hepatic microcirculation. The data imply a relationship between hepatic fibrogenesis or fibrosis and vasomotor responses.

TRPA1 in mast cell activation-induced long-lasting mechanical hypersensitivity of vagal afferent C-fibers in guinea pig esophagus

Yu, S., Gao, G., Peterson, B. Z., Ouyang, A. — 2009-06-26

Sensitization of esophageal sensory afferents by inflammatory mediators plays an important role in esophageal nociception. We have shown esophageal mast cell activation induces long-lasting mechanical hypersensitivity in vagal nodose C-fibers. However, the roles of mast cell mediators and downstream ion channels in this process are unclear. Mast cell tryptase via protease-activated receptor 2 (PAR2)-mediated pathways sensitizes sensory nerves and induces hyperalgesia. Transient receptor potential A1 (TRPA1) plays an important role in mechanosensory transduction and nociception. Here we tested the hypothesis that mast cell activation via a PAR2-dependent mechanism sensitizes TRPA1 to induce mechanical hypersensitivity in esophageal vagal C-fibers. The expression profiles of PAR2 and TRPA1 in vagal nodose ganglia were determined by immunostaining, Western blot, and RT-PCR. Extracellular recordings from esophageal nodose neurons were performed in ex vivo guinea pig esophageal-vagal preparations. Action potentials evoked by esophageal distention and chemical perfusion were compared. Both PAR2 and TRPA1 expressions were identified in vagal nodose neurons by immunostaining, Western blot, and RT-PCR. Ninety-one percent of TRPA1-positive neurons were of small and medium diameters, and 80% coexpressed PAR2. Esophageal mast cell activation significantly enhanced the response of nodose C-fibers to esophageal distension (mechanical hypersensitivity). This was mimicked by PAR2-activating peptide, which sustained for 90 min after wash, but not by PAR2 reverse peptide. TRPA1 inhibitor HC-030031 pretreatment significantly inhibited mechanical hypersensitivity induced by either mast cell activation or PAR2 agonist. Collectively, our data provide new evidence that sensitizing TRPA1 via a PAR2-dependent mechanism plays an important role in mast cell activation-induced mechanical hypersensitivity of vagal nodose C-fibers in guinea pig esophagus.

Adrenomedullin reduces intestinal epithelial permeability in vivo and in vitro

2009-06-26

Leakage of the gut mucosal barrier in the critically ill patient may allow translocation of bacteria and their virulence factors, thereby perpetuating sepsis and inflammation. Present evidence suggests that adrenomedullin (AM) improves endothelial barrier function and stabilizes circulatory function in systemic inflammation. We tested the hypothesis that exogenously applied AM stabilizes gut epithelial barrier function. Infusion of Staphylococcus aureus -toxin induced septic shock in rats. AM infusion in a therapeutic setting reduced translocation of labeled dextran from the gut into the systemic circulation in this model. AM also reduced -toxin and hydrogen peroxide (H₂O₂)-related barrier disruption in Caco-2 cells in vitro and reduced H₂O₂-related rat colon barrier malfunction in Ussing chamber experiments. AM was shown to protect endothelial barrier function via cAMP elevation, but AM failed to induce cAMP accumulation in Caco-2 cells. cAMP is degraded via phosphodiesterases (PDE), and Caco-2 cells showed high activity of cAMP-degrading PDE3 and 4. However, AM failed to induce cAMP accumulation in Caco-2 cells even in the presence of sufficient PDE3/4 inhibition, whereas adenylyl cyclase activator forskolin induced strong cAMP elevation. Furthermore, PDE3/4 inhibition neither amplified AM-induced epithelial barrier stabilization nor affected AM cAMP-related rat colon short-circuit current, furthermore indicating that AM may act independently of cAMP in Caco-2 cells. Finally, experiments using chemical inhibitors indicated that PKC, phosphatidylinositide 3-kinase, p38, and ERK did not contribute to AM-related stabilization of barrier function in Caco-2 cells. In summary, during severe inflammation, elevated AM levels may substantially contribute to the stabilization of gut barrier function.

Uptake and fate of ganglioside GD3 in human intestinal Caco-2 cells

Schnabl, K. L., Larcelet, M., Thomson, A. B. R., Clandinin, M. T. — 2009-06-26

Ganglioside GD3 is a glycosphingolipid found in colostrum, developing tissues, and tumors and is known to regulate cell growth, differentiation, apoptosis, and inflammation. Feeding a GD3-enriched diet to rats increases GD3 in intestinal lipid rafts and blood. The mechanism, efficiency, and fate of ganglioside absorption by human enterocytes have not been investigated. A model to study GD3 uptake by human intestinal cells was developed to test the hypothesis that enterocyte GD3 uptake is time and concentration dependent, with uptake efficiency and fate influenced by route of delivery. Caco-2 cells were exposed to GD3 on the apical or basolateral membrane (BLM) side for 6, 24, and 48 h. GD3 uptake, retention, transfer, and metabolism was determined. GD3 uptake across the apical and BLM was time and concentration dependent and reached a plateau. GD3 uptake across the BLM was more efficient than apical delivery. Apical GD3 was metabolized with some cell retention and transfer, whereas basolateral GD3 was mostly metabolized. This study demonstrates efficient GD3 uptake by enterocytes and suggests that the route of delivery influences ganglioside uptake and fate.

Serine proteases decrease intestinal epithelial ion permeability by activation of protein kinase C{zeta}

2009-06-26

Epithelial permeability to ions and larger molecules in the gut is essential for fluid balance, and its dysregulation contributes to intestinal pathology. We investigated the effect of digestive serine proteases on epithelial paracellular permeability. Trypsin, chymotrypsin, and elastase elicited sustained increases in transepithelial resistance (R_TE) in polarized monolayers of three intestinal epithelial cell lines. This effect was reflected by decreases in paracellular conductances of Na⁺ and Cl^– and a concomitant decrease in permeability to 3,000 molecular weight dextran. The enzyme activities of the proteases were required, yet activators of known protease-activated receptors (PARs) did not reproduce the effect of these proteases on R_TE. PKC isoform-specific inhibitor significantly reduced the trypsin-induced increase in R_TE whereas PKC activity was increased in cells treated with trypsin and chymotrypsin compared with control cells; this activity was reduced to control levels in the presence of PKC-specific inhibitor. Ca²⁺ chelators and pharmacological inhibitors of cell signaling support the role for PKC in the protease-induced effect. Finally, we showed that treatment with the serine proteases increased occludin immunostaining and zonula occludin-1 coimmunoprecipitation with occludin in the detergent-insoluble fraction of cell lysates, and these increases were ablated by pretreatment with PKC-specific inhibitor. This finding indicates increased insertion of occludin into the cell junctional complex. These data demonstrate a role for serine proteases in the facilitation of epithelial barrier function through a mechanism that is independent of PARs and is mediated by activation of PKC.

Terminating motor events for TLESR are influenced by the presence and distribution of refluxate

2009-06-26

Transient lower esophageal sphincter relaxation (TLESR) is frequently associated with reflux events and terminates with a primary or secondary peristaltic wave. However, it is unclear whether the presence and properties of the refluxate affect TLESR-termination events. The aims of this study were to determine the pattern of terminating esophageal motor activity after TLESR in healthy subjects and factors affecting the type of terminating motor event. Fifteen healthy subjects (7 men, age 18–56) were studied. High-resolution manometry and impedance/pH monitoring were performed simultaneously in supine position for 2 h after subjects took a 1,000-kcal meal (Awake Study). This procedure was repeated during the night under polysomnographic recording for 6–8 h after consuming a 1,000-kcal meal (Sleep Study). We categorized three types of TLESR-terminating motor events, primary peristalsis (PP), full secondary contraction (FSC), which propagated the entire esophagus, and partial secondary contractions (PSC), which started distal to the upper esophageal sphincter. Overall, 289 TLESR events were found. The percentages of TLESR events terminated by PP, FSC, and PSC were 22%, 14%, and 64%, respectively. TLESR events terminated by PP were less likely to be accompanied by reflux events. TLESR events terminated by FSC were significantly more likely to have evidence for proximal esophageal reflux and esophago-pharyngeal reflux. Findings were similar in awake and sleep states. We concluded that, in healthy recumbent subjects, the most common TLESR-termination event is a secondary contraction, rather than PP. Presence and distribution of the refluxate is a major influence on the type of terminating contraction.

Platelet-activating factor induces the processing of nuclear factor-{kappa}B p105 into p50, which mediates acute bowel injury in mice

Liu, S. X. L., Tian, R., Baskind, H., Hsueh, W., De Plaen, I. G. — 2009-06-26

Platelet-activating factor (PAF), an endogenous proinflammatory phospholipid, when injected intravascularly to rats and mice, causes shock, acute bowel injury, and a rapid activation of NF-B p50-p50 with upregulation of the chemokine CXCL2 in the intestine. In this study, we investigate the mechanism of NF-B activation and the role of the NF-B p50 subunit in PAF-induced shock and acute bowel injury. NF-B p50-deficient mice and wild-type mice were anesthetized and tracheotomized, and their carotid artery was cannulated for blood pressure monitoring, blood sampling, and PAF administration. For determination of bowel injury, shock, and survival, PAF (2.2 µg/kg, intra-arterially, i.a.) was injected. Two hours later, animals were euthanized, and their small intestines were removed for histological examination. For biochemical studies, PAF (1.5 µg/kg i.a.) was administered and the small intestine removed after 15–60 min. We found that PAF induced an increase in p105 processing within 30 min, but there were no changes in the levels of the NF-B inhibitory proteins IB and β. NF-B p50-deficient mice were protected against PAF-induced mortality, shock, intestinal hypoperfusion, and injury compared with wild-type animals. We also found that p50-deficient mice had decreased gene expression of CXCL2 and TNF and a decrease in CXCL2 protein production compared with wild-type mice. Our study suggests that PAF increases the processing of NF-B p105 into p50, with upregulation of proinflammatory cytokines, which leads to PAF-induced systemic inflammatory response and acute bowel injury.

Activation of PPAR{gamma} by rosiglitazone attenuates intestinal Cl- secretion

Bajwa, P. J., Lee, J. W., Straus, D. S., Lytle, C. — 2009-06-26

The thiazolidinedione (TZD) drugs rosiglitazone (Ro) and pioglitazone (Po) are PPAR agonists in widespread clinical use as insulin-sensitizing agents in Type 2 diabetes. On the basis of recent evidence implicating PPAR as a positive modulator of intestinal epithelial differentiation, we hypothesized that TZD drugs might attenuate intestinal secretory function. To evaluate this possibility, we examined the effects of Ro and Po on electrogenic Cl^– secretion [short-circuit current (I_sc)] in mouse intestinal segments and in cultured human intestinal epithelial cells (HT29-Cl.19A). As hypothesized, oral administration of Ro (20 mg·kg^–1·day^–1) to mice for 8 days markedly reduced intestinal I_sc responses to cAMP (forskolin)- and Ca²⁺ (carbachol)-dependent stimuli. In these Ro-treated mice, cholera toxin-induced intestinal fluid accumulation was reduced 65%. With continued Ro treatment, the I_sc response to carbachol recovered significantly, whereas that to forskolin remained attenuated. Treatment of HT29 cells for 5 days with 10 µM Ro or Po in vitro brought about a similar hyposecretory state. In HT29 cells, the loss of cAMP-dependent Cl^– secretion was attributable to a reduced expression of CFTR Cl^– channel, KCNQ1 K⁺ channel, and Na-K-2Cl cotransporter-1 proteins. The transient loss of Ca²⁺-dependent Cl^– secretion involved an impairment of basolateral Ca²⁺-stimulated K⁺ channel activity without a detectable loss of K_Ca3.1 channel protein. Our results establish TZD drugs as important modulators of intestinal Cl^– secretory function.

Bile acids inhibit duodenal secretin expression via orphan nuclear receptor small heterodimer partner (SHP)

Lam, I. P. Y., Lee, L. T. O., Choi, H.-S., Alpini, G., Chow, B. K. C. — 2009-06-26

Small heterodimer partner (SHP) is an orphan nuclear receptor in which gene expression can be upregulated by bile acids. It regulates its target genes by repressing the transcriptional activities of other nuclear receptors including NeuroD, which has been shown to regulate secretin gene expression. Here, we evaluated the regulation on duodenal secretin gene expression by SHP and selected bile acids, cholic acid (CA) and chenodeoxycholic acid (CDCA). In vitro treatment of CDCA or fexaramine elevated the SHP transcript level and occupancy on secretin promoter. The increase in the SHP level, induced by bile acid treatment or overexpression, reduced secretin gene expression, whereas this gene inhibitory effect was reversed by silencing of endogenous SHP. In in vivo studies, double-immunofluorescence staining demonstrated the coexpression of secretin and SHP in mouse duodenum. Feeding mice with 1% CA-enriched rodent chow resulted in upregulation of SHP and a concomitant decrease in secretin transcript and protein levels in duodenum compared with the control group fed with normal chow. A diet enriched with 5% cholestyramine led to a decrease in SHP level and a corresponding increase in secretin expression. Overall, this study showed that bile acids via SHP inhibit duodenal secretin gene expression. Because secretin is a key hormone that stimulates bile flow in cholangiocytes, this pathway thus provides a novel means to modulate secretin-stimulated choleresis in response to intraduodenal bile acids.

Portal pressure responses and angiotensin peptide production in rat liver are determined by relative activity of ACE and ACE2

2009-06-26

Angiotensin converting enzyme (ACE) 2 activity and angiotensin-(1-7) [Ang-(1-7)] levels are increased in experimental cirrhosis; however, the pathways of hepatic Ang-(1-7) production have not been studied. This study investigated the role of ACE2, ACE, and neutral endopeptidase (NEP) in the hepatic formation of Ang-(1-7) from angiotensin I (Ang I) and Ang II and their effects on portal resistance. Ang I or Ang II were administered to rat bile duct ligated (BDL) and control livers alone and in combination with the ACE inhibitor lisinopril, the ACE and NEP inhibitor omapatrilat, or the ACE2 inhibitor MLN4760 (n = 5 per group). BDL markedly upregulated ACE, ACE2, and NEP. Ang-(1-7) was produced from Ang II in healthy and in BDL livers and was increased following ACE inhibition and decreased by ACE2 inhibition. In contrast, Ang-(1-7) production from Ang I was minimal and not affected by ACE or NEP inhibition. Surprisingly, ACE2 inhibition in BDLs dramatically increased Ang-(1-7) production from Ang I, an effect abolished by ACE2/NEP inhibition. Ang II and Ang I induced greater portal pressure increases in BDL livers than controls. The effects of Ang I were closely correlated with Ang II production and were strongly attenuated by both ACE and ACE/NEP inhibition. These findings show that the major substrate for hepatic production of Ang-(1-7) is Ang II and this is catalyzed by ACE2. Ang I is largely converted to Ang II by ACE, and net conversion of Ang I to Ang-(1-7) is small. NEP has the ability to generate large amounts of Ang-(1-7) in the BDL liver from Ang I only when ACE2 activity is greatly decreased or inhibited.

Molecular and functional characterization of Kv7 K+ channel in murine gastrointestinal smooth muscles

Jepps, T. A., Greenwood, I. A., Moffatt, J. D., Sanders, K. M., Ohya, S. — 2009-06-26

Members of the K_v7 voltage-gated K⁺ channel family are important determinants of cardiac and neuronal membrane excitability. Recently, we and others have shown that K_v7 channels are also crucial regulators of smooth muscle activity. The aim of the present study was to assess the K_v7 expression in different parts of the murine gastrointestinal (GI) tract and to assess their functional roles by use of pharmacological agents. Of KCNQ/K_v7 members, both KCNQ4/K_v7.4 and KCNQ5/K_v7.5 genes and proteins were the most abundantly expressed K_v7 channels in smooth muscles throughout the GI tract. Immunohistochemical staining also revealed that K_v7.4 and K_v7.5 but not K_v7.1 were expressed in the circular muscle layer of the colon. In segments of distal colon circular muscle exhibiting spontaneous phasic contractions, the nonselective K_v7 blockers XE991 and linopirdine increased the integral of tension. Increases in the integral of tension were also observed under conditions of neuronal blockade. Similar effects, although less marked, were observed in the proximal colon. As expected, the K_v7.1-selective blocker chromanol 293B had no effect in either type of segment. These data show that K_v7.x especially K_v7.4 and K_v7.5 are expressed in different regions of the murine gastrointestinal tract and blockers of K_v7 channels augment inherent contractile activity. Drugs that selectively block K_v7.4/7.5 might be promising therapeutics for the treatment of motility disorders such as constipation associated with irritable bowel syndrome.

Morphological adaptation with preserved proliferation/transporter content in the colon of patients with short bowel syndrome

2009-06-26

In short bowel syndrome (SBS), although a remaining colon improves patient outcome, there is no direct evidence of a mucosal colonic adaptation in humans. This prospective study evaluates morphology, proliferation status, and transporter expression level in the epithelium of the remaining colon of adult patients compared with controls. The targeted transporters were Na⁺/H⁺ exchangers (NHE2 and 3) and oligopeptide transporter (PepT1). Twelve adult patients with a jejuno-colonic anastomosis were studied at least 2 yr after the last surgery and compared with 11 healthy controls. The depth of crypts and number of epithelial cells per crypt were quantified. The proliferating and apoptotic cell contents were evaluated by revealing Ki67, PCNA, and caspase-3. NHE2, NHE3, PepT1 mRNAs, and PepT1 protein were quantified by quantitative RT-PCR and Western blot, respectively. In patients with SBS compared with controls, 1) hyperphagia and severe malabsorption were documented, 2) crypt depth and number of cells per crypt were 35% and 22% higher, respectively (P < 0.005), whereas the proliferation and apoptotic levels per crypt were unchanged, and 3) NHE2 mRNA was unmodified; NHE3 mRNA was downregulated near the anastomosis and unmodified distally, and PepT1 mRNA and protein were unmodified. We concluded that, in hyperphagic patients with SBS with severe malabsorption, adaptive colonic changes include an increased absorptive surface with an unchanged proliferative/apoptotic ratio and well-preserved absorptive NHE2, NHE3, and PepT1 transporters. This is the first study showing a controlled nonpharmacological hyperplasia in the colon of patients with SBS.

Hepatocyte nuclear factor 4{alpha} contributes to an intestinal epithelial phenotype in vitro and plays a partial role in mouse intestinal epithelium differentiation

Babeu, J.-P., Darsigny, M., Lussier, C. R., Boudreau, F. — 2009-06-26

Hepatocyte nuclear factor 4 (HNF4) is a regulator of hepatocyte and pancreatic transcription. Hnf4 deletion in the mouse is embryonically lethal with severe defects in visceral endoderm formation. It has been concluded in the past that the role of Hnf4 in the developing colon was much less important than in the liver. However, the precise role of Hnf4 in the homeostasis of the small intestinal epithelium remains unclear. Our aim was to evaluate the potential of Hnf4 to support an intestinal epithelial phenotype. First, Hnf4 potential to dictate this phenotype was assessed in nonintestinal cell lines in vitro. Forced expression of Hnf4 in fibroblasts showed an induction of features normally restricted to epithelial cells. Combinatory expression of Hnf4 with specific transcriptional regulators of the intestine resulted in the induction of intestinal epithelial genes in this context. Second, the importance of Hnf4 in maintaining the homeostasis of the intestinal epithelium was investigated in mice. Mice conditionally deficient for intestinal Hnf4 developed normally throughout adulthood with an epithelium displaying normal morphological and functional structures with minor alterations. Subtle but statistical differences were observed at the proliferation and the cytodifferentiation levels. Hnf4 mutant mice displayed an increase in the number of goblet and enteroendocrine cells compared with controls. Given the fundamental role of this transcription factor in other tissues, these findings dispute the crucial role for this regulator in the maintenance of intestinal epithelial cell function at a period of time that follows cytodifferentiation but may suggest a functional role in instructing cells to become specific to the intestinal epithelium.

HCl-activated neural and epithelial vanilloid receptors (TRPV1) in cat esophageal mucosa

2009-06-26

To test whether transient receptor potential channel vanilloid subfamily member-1 (TRPV1) mediates acid-induced inflammation in the esophagus, a tubular segment of esophageal mucosa was tied at both ends, forming a sac. The sac was filled with 0.01 N HCl (or Krebs buffer for control) and kept in oxygenated Krebs buffer at 37°C. The medium around the sac (supernatant) was collected after 3 h. Supernatant of the HCl-filled sac abolished contraction of esophageal circular muscle strips in response to electric field stimulation. Contraction was similarly abolished by supernatant of mucosal sac filled with the TRPV1 agonist capsaicin (10^–6 M). These effects were reversed by the selective TRPV1 antagonist 5'-iodoresiniferatoxin (IRTX) and by the platelet-activating factor (PAF) receptor antagonist CV9388. Substance P and CGRP levels in mucosa and in supernatant increased in response to HCl, and these increases were abolished by IRTX and by tetrodotoxin (TTX) but not affected by CV9388, indicating that substance P and CGRP are neurally released and PAF independent. In contrast, the increase in PAF was blocked by IRTX but not by TTX. Presence of TRPV1 receptor was confirmed by RT-PCR and by Western blot analysis in whole mucosa and in esophageal epithelial cells enzymatically isolated and sorted by flow cytometry or immunoprecipitated with cytokeratin antibodies. In epithelial cells PAF increased in response to HCl, and the increase was abolished by IRTX. We conclude that HCl-induced activation of TRPV1 receptors in esophageal mucosa causes release of substance P and CGRP from neurons and release of PAF from epithelial cells.

Hepatic bile acid metabolism in the neonatal hamster: expansion of the bile acid pool parallels increased Cyp7a1 expression levels

Burke, K. T., Horn, P. S., Tso, P., Heubi, J. E., Woollett, L. A. — 2009-06-26

Intraluminal concentrations of bile acids are low in newborn infants and increase rapidly after birth, at least partly owing to increased bile acid synthesis rates. The expansion of the bile acid pool is critical since bile acids are required to stimulate bile flow and absorb lipids, a major component of newborn diets. The purpose of the present studies was to determine the mechanism responsible for the increase in bile acid synthesis rates and the subsequent enlargement of bile acid pool sizes (BAPS) during the neonatal period, and how changes in circulating hormone levels might affect BAPS. In the hamster, pool size was low just after birth and increased modestly until 10.5 days postpartum (dpp). BAPS increased more significantly (~3-fold) between 10.5 and 15.5 dpp. An increase in mRNA and protein levels of cholesterol 7-hydroxylase (Cyp7a1), the rate-limiting step in classical bile acid synthesis, immediately preceded an increase in BAPS. In contrast, levels of oxysterol 7-hydroxylase (Cyp7b1), a key enzyme in bile acid synthesis by the alternative pathway, were relatively elevated by 1.5 dpp. farnesyl X receptor (FXR) and short heterodimeric partner (SHP) mRNA levels remained relatively constant at a time when Cyp7a1 levels increased. Finally, although simultaneous increases in circulating cortisol and Cyp7a1 levels occurred, precocious expression of Cyp7a1 could not be induced in neonatal hamsters with dexamethasone. Thus the significant increase in Cyp7a1 levels in neonatal hamsters is due to mechanisms independent of the FXR and SHP pathway and cortisol.

Effect of esophageal acid exposure on the cortical swallowing network in healthy human subjects

Kern, M., Chai, K., Lawal, A., Shaker, R. — 2009-06-26

Recent studies have demonstrated common cortical activity regions associated with esophageal acidification and swallowing. The effect of sensory signals imparted on these regions by esophageal acidification on swallow-related brain activity has physiological and clinical ramifications. Our aim in this study was to determine the effect of prior, unperceived esophageal acid exposure on cortical activity associated with swallowing. Functional magnetic resonance imaging (fMRI) techniques monitored brain activity associated with volitional swallowing before and after subliminal esophageal acid stimulation. Studies were carried out in two phases. In phase I (15 healthy, right-handed subjects, age 21–49 yr, 7 female) using whole brain imaging, we documented the potentiating effects of esophageal acidification alone on swallow-related cortical activity. In phase II (10 healthy, right-handed subjects, age 20–54 yr, 5 female) using high-resolution fMRI, we measured swallow-induced regional brain activity within the cortical swallowing network before and after esophageal acidification. Unlike the phase I studies, we also tested the effect of saline perfusion alone on the cortical swallowing network in the phase II studies. Because of constraints imposed by high-resolution MRI for region-of-interest (ROI) analysis, we studied only the left hemisphere in this phase. None of the subjects developed heartburn during acid perfusion. In phase I, the number of swallow-induced activated voxels increased by 43% following esophageal acid stimulation (preacid, 44 ± 3 voxels; postacid, 63 ± 6 voxels; means ± SE, P < 0.05) In phase II, contrary to saline perfusion, ROI analysis showed significantly increased regional swallow-related fMRI activity volumes as well as percent maximum signal change after esophageal acid perfusion in cingulate, prefrontal, insula, and sensory/motor regions (P < 0.05). The precuneus showed no significant change. We concluded that subliminal esophageal acid stimulation has a potentiating effect on the cortical swallowing network in healthy individuals.

Regulation of colonic apical potassium (BK) channels by cAMP and somatostatin

Perry, M. D., Sandle, G. I. — 2009-06-26

High-conductance apical K⁺ (BK) channels are present in surface colonocytes of mammalian (including human) colon. Their location makes them well fitted to contribute to the excessive intestinal K⁺ losses often associated with infective diarrhea. Since many channel proteins are regulated by phosphorylation, we evaluated the roles of protein kinase A (PKA) and phosphatases in the modulation of apical BK channel activity in surface colonocytes from rat distal colon using patch-clamp techniques, having first increased channel abundance by chronic dietary K⁺ enrichment. We found that PKA activation using 50 µmol/l forskolin and 5 mmol/l 3-isobutyl-1-methylxanthine stimulated BK channels in cell-attached patches and the catalytic subunit of PKA (200 U/ml) had a similar effect in excised inside-out patches. The antidiarrheal peptide somatostatin (SOM; 2 µmol/l) had a G protein-dependent inhibitory effect on BK channels in cell-attached patches, which was unaffected by pretreatment with 10 µmol/l okadaic acid (an inhibitor of protein phosphatase type 1 and type 2A) but completely prevented by pretreatment with 100 µmol/l Na⁺ orthovanadate and 10 µmol/l BpV (inhibitors of phosphoprotein tyrosine phosphatase). SOM also inhibited apical BK channels in surface colonocytes in human distal colon. We conclude that cAMP-dependent PKA activates apical BK channels and may enhance colonic K⁺ losses in some cases of secretory diarrhea. SOM inhibits apical BK channels through a phosphoprotein tyrosine phosphatase-dependent mechanism, which could form the basis of new antidiarrheal strategies.

Fibroblast growth factor receptor-3 regulates Paneth cell lineage allocation and accrual of epithelial stem cells during murine intestinal development

2009-06-26

Fibroblast growth factor receptor 3 (FGFR-3) is expressed in the lower crypt epithelium, where stem cells of the intestine reside. The role of FGFR-3 signaling in regulating features of intestinal morphogenesis was examined in FGFR-3-null (FGFR-3^–/–) mice. FGFR-3^–/– mice had only about half the number of intestinal crypts and a marked decrease in the number of functional clonogenic stem cells, as assessed by an in vivo microcolony-forming assay, compared with wild-type littermates. A marked deficit in allocation of progenitor cells to Paneth cell differentiation was noted, although all the principal epithelial lineages were represented in FGFR-3^–/– mice. The total cellular content and nuclear localization of β-catenin protein were reduced in FGFR-3^–/– mice, as was expression of cyclin D1 and matrix metalloproteinase-7, major downstream targets of β-catenin/T cell factor-4 (Tcf-4) signaling. Activation of FGFR-3 in Caco-2 cells, an intestinal epithelial cell line, abrogated the fall in β-catenin/Tcf-4 signaling activity that is normally observed in these cells as cultures become progressively more confluent. These findings are consistent with the hypothesis that, during intestinal development, FGFR-3 signaling regulates crypt epithelial stem cell expansion and crypt morphogenesis, as well as Paneth cell lineage specification, through β-catenin/Tcf-4-dependent and -independent pathways.

Electrical stimulation of the mucosa evokes slow EPSPs mediated by NK1 tachykinin receptors and by P2Y1 purinoceptors in different myenteric neurons

Gwynne, R. M., Bornstein, J. C. — 2009-06-26

Slow excitatory postsynaptic potentials (EPSPs) in enteric neurons arise from diverse sources, but which neurotransmitters mediate specific types of slow EPSPs is unclear. We investigated transmitters and receptors mediating slow EPSPs in myenteric neurons evoked by electrical stimulation of the mucosa in guinea pig small intestine. Segments of ileum or jejunum were dissected to allow access to the myenteric plexus adjacent to intact mucosa, in vitro. AH and S neurons were impaled with conventional intracellular electrodes. Trains of stimuli delivered to the mucosa evoked slow EPSPs in AH neurons that were blocked or depressed by the neurokinin-1 (NK1) tachykinin antagonist SR140333 (100 nM) in 10 of 11 neurons; the NK3 tachykinin receptor antagonist SR142801 (100 nM) had no effect on slow EPSPs in seven of nine AH neurons. Single pulses to the mucosa evoked fast EPSPs and slow depolarizations in S neurons. The depolarizations were divided into intermediate (durations 300–900 ms) or slow (durations 1.3–9 s) EPSPs. The slow EPSPs were blocked by pyridoxal phosphate-6-axophenyl-2–4-disulfonic acid (30 µM, N = 3) or the specific P2Y₁ antagonist MRS 2179 (10 µM, N = 6) and were predominantly in anally projecting S neurons that were immunoreactive for nitric oxide synthase (NOS). In contrast, intermediate EPSPs were predominantly evoked in NOS-negative neurons; these were abolished by MRS 2179 (N = 8). Thus activation of pathways running from the mucosa excites three different types of slow EPSP in myenteric neurons, which are mediated by either a tachykinin (NK1, AH neurons) or a purine nucleotide (P2Y₁, S neurons).

Intestinal ischemia-reperfusion injury: reversible and irreversible damage imaged in vivo

Guan, Y., Worrell, R. T., Pritts, T. A., Montrose, M. H. — 2009-06-26

The early events in an intestinal ischemic episode have been difficult to evaluate. Using in vivo microscopy we have analyzed in real-time the effects of short (15 min) and long (40–50 min) ischemia with subsequent reperfusion (IR), evaluating structure, integrity, and functioning of the mouse jejunal mucosa while monitoring blood flow by confocal microscopy. IR was imposed by inflation/deflation of a vascular occluder, and blood flow was monitored and confirmed with scanning confocal imaging. After short ischemia, villus tip cells revealed a rapid increase (23%) in the intracellular NAD(P)H concentration (confocal autofluorescence microscopy), and the pH-sensitive probe BCECF showed a biphasic response of the intracellular pH (pH_i), quickly alkalinizing from the resting value of 6.8 ± 0.1 to 7.1 ± 0.1 but then strongly acidifying to 6.3 ± 0.1. Upon reperfusion, values returned toward control. In contrast, results were heterogeneous after long IR. During long ischemia, one-third of the epithelial cells remained viable with reversible changes upon reperfusion, but remaining cells lost membrane integrity (Lucifer Yellow uptake, LY) and had membrane blebs during ischemia. These effects became more pronounced as the reperfusion interval progressed when cells exhibited more severely affected NAD(P)H and pH_i values, larger blebs, and more LY uptake and eventually were shed from the villus. Results from stereo microscopy suggest that these irreversible effects of IR may have occurred as a result of incomplete restorations of local blood flow, especially at the antimesenteric side of the intestine. We conclude that the adverse effects of short ischemia on the jejunum epithelium are fully reversible during the reperfusion interval. However, after long ischemia, reperfusion cannot restore normal structure and functioning of a majority of cells, which deteriorate further. Our results provide a basis for defining the cellular events that cause tissue to transit from reversible to irreversible damage during IR.

Pancreatic beta cells and islets take up thiamin by a regulated carrier-mediated process: studies using mice and human pancreatic preparations

2009-06-26

Thiamin is essential for the normal function of the endocrine pancreas, but very little is known about uptake mechanism(s) and regulation by beta cells. We addressed these issues using mouse-derived pancreatic beta-TC-6 cells, and freshly isolated primary mouse and human pancreatic islets. Results showed that thiamin uptake by beta-TC-6 cells involves a pH (but not Na⁺)-dependent carrier-mediated process that is saturable at both the nanomolar (apparent K_m = 37.17 ± 9.9 nM) and micromolar (apparent K_m = 3.26 ± 0.86 µM) ranges, cis-inhibited by thiamin structural analogs, and trans-stimulated by unlabeled thiamin. Involvement of carrier-mediated process was also confirmed in primary mouse and human pancreatic islets. Both THTR-1 and THTR-2 were found to be expressed in these mouse and human pancreatic preparations. Maintaining beta-TC-6 cells in the presence of a high level of thiamin led to a significant (P < 0.01) decrease in thiamin uptake, which was associated with a significant downregulation in level of expression of THTR-1 and THTR-2 at the protein and mRNA levels and a decrease in transcriptional (promoter) activity. Modulators of intracellular Ca²⁺/calmodulin- and protein-tyrosine kinase-mediated pathways also altered thiamin uptake. Finally, confocal imaging of live beta-TC-6 cells showed that clinical mutants of THTR-1 have mixed expression phenotypes and all led to impairment in thiamin uptake. These studies demonstrate for the first time that thiamin uptake by the endocrine pancreas is carrier mediated and is adaptively regulated by the prevailing vitamin level via transcriptional mechanisms. Furthermore, clinical mutants of THTR-1 impair thiamin uptake via different mechanisms.

Role of lysophosphatidylcholine in brush-border intestinal alkaline phosphatase release and restoration

2009-06-26

Intestinal alkaline phosphatase (IAP) is a brush-border membrane ectoenzyme (BBM-IAP) that is released into the lumen (L-IAP) after a high-fat diet. We examined the effects of oil feeding and the addition of mixed-lipid micelles on the formation of L-IAP in oil-fed rat intestine, Caco-2 cell monolayers, and mouse intestinal loops. We localized IAP in the duodenum of rats fed corn oil using fluorescence microscopy with enzyme-labeled fluorescence-97 as substrate. Four hours after oil feeding, L-IAP increased ~10-fold accompanied by the loss of BBM-IAP, consistent with BBM-IAP release. Rat IAP isozyme mRNAs progressively increased 4–6 h after oil feeding, followed by the increase of IAP activity in the subapical location at 6 h, consistent with the restoration of IAP protein. Postprandial lipid-micelle components, sodium taurocholate with or without oleic acid, mono-oleylglycerol, cholesterol, or lysophosphatidylcholine (lysoPC) were applied singly or as mixed-lipid micelles to the apical surface of polarized Caco-2 cell monolayers. LysoPC increased L-IAP >10-fold over basal release. LysoPC released IAP into the apical medium more than other intestinal brush-border enzymes, 5'-nucleotidase, sucrase, aminopeptidase N, and lactase, without comparable lactate dehydrogenase release or cell injury. LysoPC increased human IAP mRNA levels by 1.5-fold in Caco-2 cells. Luminally applied lysoPC also increased release of IAP preferentially in mouse intestinal loops. These data show that lysoPC accelerates the formation of L-IAP from BBM-IAP, followed by enhanced IAP synthesis, suggesting the role that lysoPC might play in the turnover of brush-border proteins.

Cortagine, a CRF1 agonist, induces stresslike alterations of colonic function and visceral hypersensitivity in rodents primarily through peripheral pathways

2009-06-26

Corticotropin-releasing factor (CRF) 1 receptor (CRF₁) activation in the brain is a core pathway orchestrating the stress response. Anatomical data also support the existence of CRF signaling components within the colon. We investigated the colonic response to intraperitoneal (ip) injection of cortagine, a newly developed selective CRF₁ peptide agonist. Colonic motor function and visceral motor response (VMR) were monitored by using a modified miniaturized pressure transducer catheter in adult conscious male Sprague-Dawley rats and C57Bl/6 mice. Colonic permeability was monitored by the Evans blue method and myenteric neurons activation by Fos immunohistochemistry. Compared with vehicle, cortagine (10 µg/kg ip) significantly decreased the distal colonic transit time by 45% without affecting gastric transit, increased distal and transverse colonic contractility by 35.6 and 66.2%, respectively, and induced a 7.1-fold increase in defecation and watery diarrhea in 50% of rats during the first hour postinjection whereas intracerebroventricular (icv) cortagine (3 µg/rat) had lesser effects. Intraperitoneal (ip) cortagine also increased colonic permeability, activated proximal and distal colonic myenteric neurons, and induced visceral hypersensitivity to a second set of phasic colorectal distention (CRD). The CRF antagonist astressin (10 µg/kg ip) abolished ip cortagine-induced hyperalgesia whereas injected icv it had no effect. In mice, cortagine (30 µg/kg ip) stimulated defecation by 7.8-fold, induced 60% incidence of diarrhea, and increased VMR to CRD. Stresslike colonic alterations induced by ip cortagine in rats and mice through restricted activation of peripheral CRF₁ receptors support a role for peripheral CRF₁ signaling as the local arm of the colonic response to stress.

Discrete responses of myenteric neurons to structural and functional damage by neurotoxins in vitro

Lourenssen, S., Miller, K. G., Blennerhassett, M. G. — 2009-06-26

Damage to the enteric nervous system is implicated in human disease and animal models of inflammatory bowel disease, diabetes, and Parkinson's disease, but the mechanism of death and the response of surviving neurons are poorly understood. We explored this in a coculture model of myenteric neurons, glia, and smooth muscle during exposure to the established or potential neurotoxins botulinum A, hydrogen peroxide, and acrylamide. Neuronal survival, axonal degeneration and regeneration, and neurotransmitter release were assessed during acute exposure (0–24 h) to neurotoxin and subsequent recovery (96–144 h). Unique and selective responses to each neurotoxin were found with acrylamide (0.5–2.0 mM) causing a 30% decrease in axon number without neuronal loss, whereas hydrogen peroxide (1–200 µM) caused a parallel loss in both axon and neuron number. Immunoblotting identified the loss of synaptic vesicle proteins that paralleled axon damage and was associated with marked suppression of depolarization-induced release of acetylcholine (ACh). The caspase inhibitor zVAD, but not DEVD, significantly prevented neuronal death, implying a largely caspase-3/7-independent mechanism of apoptotic death that was supported by staining for annexin V and cleaved caspase-3. In contrast, botulinum A (2 µg/ml) caused a 40% decrease in ACh release without effect on neuronal survival or axon structure. By 96 h after exposure to acrylamide or hydrogen peroxide, axon number was restored to or even surpassed the level of time-matched controls, regardless of partial neuronal loss, but ACh release remained markedly suppressed. Neural responses to toxic factors are initially unique but then converge upon robust axonal regeneration, whereas neurotransmitter release is both vulnerable to damage and slow to recover.