AJP: Gastrointestinal and Liver Physiology recent issues

Mechanism of leukotriene D4 inhibition of Na-alanine cotransport in intestinal epithelial cells

Talukder, J. R., Kekuda, R., Saha, P., Sundaram, U. — 2008-07-14

In a rabbit model of chronic intestinal inflammation, we previously demonstrated inhibition of neutral Na-amino acid cotransport. The mechanism of the inhibition was secondary to a decrease in the affinity for amino acid rather than the number of cotransporters. Since leukotriene (LT)D₄ is known to be elevated in enterocytes during chronic intestinal inflammation, we used rat intestinal epithelial cell (IEC-18) monolayers to determine the mechanism of regulation of Na-alanine cotransport (alanine, serine, cysteine transporter 1: ASCT1) by LTD₄. Na-alanine cotransport was inhibited by LTD₄ in IEC-18 cells. The mechanism of inhibition of ASCT1 (solute carrier, SLC1A4) by LTD₄ is secondary to a decrease in the affinity of the cotransporter for alanine without a significant change in cotransporter numbers and is not secondary to an alteration in the Na⁺ extruding capacity of the cells. Real-time quantitative PCR and Western blot analysis results indicate that ASCT1 message and protein levels are also unchanged in LTD₄-treated IEC-18 cells. These results indicate that LTD₄ inhibits Na-dependent neutral amino acid cotransport in IEC. The mechanism of inhibition is secondary to a decrease in the affinity for alanine, which is identical to that seen in villus cells from the chronically inflamed rabbit small intestine, where LTD₄ levels are significantly increased.

Identification and characterization of rabbit small intestinal villus cell brush border membrane Na-glutamine cotransporter

Talukder, J. R., Kekuda, R., Saha, P., Arthur, S., Sundaram, U. — 2008-07-14

Glutamine, the primary metabolic fuel for the mammalian small intestinal enterocytes, is primarily assimilated by Na-amino acid cotransporters. Although Na-solute cotransport has been shown to exist in the brush border membrane (BBM) of the absorptive villus cells, the identity of Na-glutamine cotransport in rabbit small intestinal villus cells was unknown. Na-dependent glutamine uptake is present in villus BBM vesicles. An intravesicular proton gradient did not stimulate this Na-dependent glutamine uptake, whereas Li⁺ did not significantly suppress this uptake. These observations in concert with amino acid substitution studies suggested that Na-glutamine cotransporter in the villus cell BBM was the newly identified cotransporter B⁰AT1 (SLC6A19). Quantitative real-time PCR identified the message for this cotransporter in villus cells. Thus a full-length cDNA of B⁰AT1 was cloned and expressed in MDA-MB-231 cells. This expressed cotransporter exhibited characteristics similar to those observed in villus cells from the rabbit small intestine. Antibody was generated for B⁰AT1 that demonstrated the presence of this cotransporter protein in the villus cell BBM. Kinetic studies defined the kinetic parameters of this cotransporter. Thus this study describes the identification, cloning, and characterization of the Na-amino acid cotransporter responsible for the assimilation of a critical amino acid by the absorptive villus cells in the mammalian small intestine.

Cholangiocyte expression of {alpha}2{beta}1-integrin confers susceptibility to rotavirus-induced experimental biliary atresia

2008-07-14

Inoculation of BALB/c mice with rhesus rotavirus (RRV) in the newborn period results in biliary epithelial cell (cholangiocyte) infection and the murine model of biliary atresia. Rotavirus infection of a cell requires attachment, which is governed in part by cell-surface expression of integrins such as ₂β₁. We hypothesized that cholangiocytes were susceptible to RRV infection because they express ₂β₁. RRV attachment and replication was measured in cell lines derived from cholangiocytes and hepatocytes. Flow cytometry was performed on these cell lines to determine whether ₂β₁ was present. Cholangiocytes were blocked with natural ligands, a monoclonal antibody, or small interfering RNA against the ₂-subunit and were infected with RRV. The extrahepatic biliary tract of newborn mice was screened for the expression of the ₂β₁-integrin. Newborn mice were pretreated with a monoclonal antibody against the ₂-subunit and were inoculated with RRV. RRV attached and replicated significantly better in cholangiocytes than in hepatocytes. Cholangiocytes, but not hepatocytes, expressed ₂β₁ in vitro and in vivo. Blocking assays led to a significant reduction in attachment and yield of virus in RRV-infected cholangiocytes. Pretreatment of newborn pups with an anti-₂ monoclonal antibody reduced the ability of RRV to cause biliary atresia in mice. Cell-surface expression of the ₂β₁-integrin plays a role in the mechanism that confers cholangiocyte susceptibility to RRV infection.

Hepatic uptake and metabolism of galactose can be quantified in vivo by 2-[18F]fluoro-2-deoxygalactose positron emission tomography

2008-07-14

Metabolism of galactose is a specialized liver function. The purpose of this PET study was to use the galactose analog 2-[¹⁸F]fluoro-2-deoxygalactose (FDGal) to investigate hepatic uptake and metabolism of galactose in vivo. FDGal kinetics was studied in 10 anesthetized pigs at blood concentrations of nonradioactive galactose yielding approximately first-order kinetics (tracer only; n = 4), intermediate kinetics (0.5–0.6 mmol galactose/l blood; n = 2), and near-saturation kinetics (>3 mmol galactose/l blood; n = 4). All animals underwent liver C¹⁵O PET (blood volume) and FDGal PET (galactose kinetics) with arterial and portal venous blood sampling. Flow rates in the hepatic artery and the portal vein were measured by ultrasound transit-time flowmeters. The hepatic uptake and net metabolic clearance of FDGal were quantified by nonlinear and linear regression analyses. The initial extraction fraction of FDGal from blood-to-hepatocyte was unity in all pigs. Hepatic net metabolic clearance of FDGal, K^FDGal, was 332–481 ml blood·min^–1·l^–1 tissue in experiments with approximately first-order kinetics and 15.2–21.8 ml blood·min^–1·l^–1 tissue in experiments with near-saturation kinetics. Maximal hepatic removal rates of galactose were on average 600 µmol·min^–1·l^–1 tissue (range 412–702), which was in agreement with other studies. There was no significant difference between K^FDGal calculated with use of the dual tracer input (K_dual^FDGal) or the single arterial input (K_arterial^FDGal). In conclusion, hepatic galactose kinetics can be quantified with the galactose analog FDGal. At near-saturated kinetics, the maximal hepatic removal rate of galactose can be calculated from the net metabolic clearance of FDGal and the blood concentration of galactose.

Targeted disruption of the Lasp-1 gene is linked to increases in histamine-stimulated gastric HCl secretion

Chew, C. S., Chen, X., Bollag, R. J., Isales, C., Ding, K. H., Zhang, H. — 2008-07-14

Lasp-1 (LIM and SH3 domain protein 1) is a multidomain actin-binding protein that is differentially expressed within epithelial tissues and brain. In the gastric mucosa, Lasp-1 is highly expressed in the HCl-secreting parietal cell, where it is prominently localized within the F-actin-rich subcellular regions. Histamine-induced elevation of parietal cell [cAMP]_i increases Lasp-1 phosphorylation, which is correlated with activation of HCl secretion. To determine whether Lasp-1 is involved in the regulation of HCl secretion in vivo, we generated a murine model with a targeted disruption of the Lasp-1 gene. Lasp-1-null mice had slightly lower body weights but developed normally and had no overt phenotypic abnormalities. Basal HCl secretion was unaffected by loss of Lasp-1, but histamine stimulation induced a more robust acid secretory response in Lasp-1-null mice compared with wild-type littermates. A similar effect of histamine was observed in isolated gastric glands on the basis of measurements of accumulation of the weak base [¹⁴C]aminopyrine. In addition, inhibition of the acid secretory response to histamine by H2 receptor blockade with ranitidine proceeded more slowly in glands from Lasp-1-null mice. These findings support the conclusion that Lasp-1 is involved in the regulation of parietal HCl secretion. We speculate that cAMP-dependent phosphorylation of Lasp-1 alters interactions with F-actin and/or endocytic proteins that interact with Lasp-1, thereby regulating the trafficking/activation of the H⁺, K⁺-ATPase (proton pump).

Stem cell factor and c-kit are involved in hepatic recovery after acetaminophen-induced liver injury in mice

Hu, B., Colletti, L. M. — 2008-07-14

Stem cell factor (SCF) and its receptor c-kit are important in hematopoiesis and cellular proliferation. c-kit has also been identified as a cell surface marker for progenitor cells. We have previously shown that there is a large reservoir of hepatic SCF, and this molecule plays a significant role in liver regeneration after 70% hepatectomy. In the current study, we further examined the expression of SCF and c-kit in acetaminophen (APAP)-induced liver injury in C57BL/6J mice or SCF-deficient sl-sld mice and their appropriate wild-type controls. Following APAP-induced liver injury, c-kit mRNA expression increased, with peak levels detected 48 h postinjury. Hepatic SCF mRNA levels after APAP injury were also increased, with peak levels seen 16 h post-APAP. The mortality rate in SCF-deficient mice treated with APAP was significantly higher than that of wild-type mice; furthermore, administration of exogenous SCF significantly reduced the mortality of APAP-treated wild-type mice. Bromodeoxyuridine incorporation experiments showed that SCF significantly increased hepatocyte proliferation at 48 and 72 h in APAP-treated mice. SCF inhibited APAP-induced hepatocyte apoptosis and increased Bcl-2 and Bcl-xL expression, suggesting that this decrease in hepatocyte apoptosis is mediated through Bcl-2 and Bcl-xL. In summary, SCF and c-kit expression was increased after APAP-induced liver injury. Administration of exogenous SCF reduces mortality in APAP-treated mice, increases hepatocyte proliferation, and prevents hepatocyte apoptosis induced by APAP, suggesting that these molecules are important in the liver's recovery from these injuries.

Cytosol-nucleus traffic and colocalization with FXR of conjugated bile acids in rat hepatocytes

2008-07-14

Bile acids (BAs) are natural ligands of nuclear receptors, in particular farnesoid X receptor (FXR). Whether, in addition to protein-mediated cytosolic-nuclear BA translocation, other mechanisms are involved in the access of BAs to nuclear FXR was investigated. When rat hepatocytes were incubated with radiolabeled taurocholic acid, taurodeoxycholic acid, taurochenodeoxycholic acid, and tauroursodeoxycholic acid, their nuclear accumulation was proportional to their intracellular levels. With the use of flow cytometry analysis, the accumulation by nuclei isolated from rat liver cells was found to differ for several fluorescent compounds of similar molecular weight and different charge, including fluorescein-tagged BAs [cholylglycyl amidofluorescein (CGamF), ursodeoxycholylglycyl amidofluorescein, or chenodeoxycholylglycyl amidofluorescein]. When we varied nuclear volume by incubation with different sucrose concentrations, a similar relationship between nuclear volume and content of FITC and 4-kDa FITC-dextran was found. In contrast, this relationship was markedly lower for CGamF. Confocal microscopy studies revealed that fluorescein-tagged BAs, but also FITC or 10-kDa FITC-dextran were found in the nuclear envelope and concentrated in regions where DNA was less densely packed. In contrast to the cytosolic subcellular localization of peroxisome proliferator-activated receptor-, FXR and nucleolin (a marker of transcriptional active chromatin) were also localized by immunoreactivity in these intranuclear regions. In conclusion, although intranuclear levels of small organic molecules including conjugated BAs depend on their concentrations in the extranuclear space, the existence of certain molecular selectivity (not strictly dependent on molecular weight or charge) suggests that, in addition to simple diffusional exchange, other mechanisms may be also involved in determining their overall nuclear content in regions where these compounds coincide and may interact with nuclear receptors such as FXR.

Colonic gene expression profile in NHE3-deficient mice: evidence for spontaneous distal colitis

2008-07-14

Na⁺/H⁺ exchanger 3 (NHE3) provides a major route for intestinal Na⁺ absorption. NHE3 has been considered a target of proinflammatory cytokines and enteropathogenic bacteria, and impaired NHE3 expression and/or activity may be responsible for inflammation-associated diarrhea. However, the possibility of loss of NHE3 function reciprocally affecting gut immune homeostasis has not been investigated. In this report, we describe that NHE3-deficient mice spontaneously develop colitis restricted to distal colonic mucosa. NHE3^–/– mice housed in a conventional facility exhibited phenotypic features such as mild diarrhea, occasional rectal prolapse, and reduced body weight. Genomewide microarray analysis identified not only a large group of transport genes that potentially represent an adaptive response, but also a considerable number of genes consistent with an inflammatory response. Histological examination demonstrated changes in the distal colon consistent with active inflammation, including crypt hyperplasia with an increased number of 5-bromo-2'-deoxyuridine-positive cells, diffuse neutrophilic infiltrate with concomitant 15-fold increase in matrix metalloproteinase 8 expression, an increased number of pSer²⁷⁶-RelA-positive cells, and a significant decrease in periodic acid-Schiff-positive goblet cells. Real-time PCR demonstrated elevated expression of inducible nitric oxide synthase (38-fold), TNF- (6-fold), macrophage inflammatory protein-2 (48-fold), and IL-18 (3-fold) in the distal colon of NHE3^–/– mice. NHE3^–/– mice showed enhanced bacterial adhesion and translocation in the distal colon. Colitis was ameliorated by oral administration of broad-spectrum antibiotics. In conclusion, NHE3 deficiency leads to an exacerbated innate immune response, an observation suggesting a potentially novel role of NHE3 as a modifier gene, which when downregulated during infectious or chronic colitis may modulate the extent and severity of colonic inflammation.

Cannabinoid CB2 receptors in the enteric nervous system modulate gastrointestinal contractility in lipopolysaccharide-treated rats

2008-07-14

Enhanced intestinal transit due to lipopolysaccharide (LPS) is reversed by cannabinoid (CB)₂ receptor agonists in vivo, but the site and mechanism of action are unknown. We have tested the hypothesis that CB₂ receptors are expressed in the enteric nervous system and are activated in pathophysiological conditions. Tissues from either saline- or LPS-treated (2 h; 65 µg/kg ip) rats were processed for RT-PCR, Western blotting, and immunohistochemistry or were mounted in organ baths where electrical field stimulation was applied in the presence or absence of CB receptor agonists. Whereas the CB₂ receptor agonist JWH133 did not affect the electrically evoked twitch response of the ileum under basal conditions, in the LPS-treated tissues JWH133 was able to reduce the enhanced contractile response in a concentration-dependent manner. Rat ileum expressed CB₂ receptor mRNA and protein under physiological conditions, and this expression was not affected by LPS treatment. In the myenteric plexus, CB₂ receptors were expressed on the majority of neurons, although not on those expressing nitric oxide synthase. LPS did not alter the distribution of CB₂ receptor expression in the myenteric plexus. In vivo LPS treatment significantly increased Fos expression in both enteric glia and neurons. This enhanced expression was significantly attenuated by JWH133, whose action was reversed by the CB₂ receptor antagonist AM630. Taking these facts together, we conclude that activation of CB₂ receptors in the enteric nervous system of the gastrointestinal tract dampens endotoxin-induced enhanced intestinal contractility.

Modulation of mouse intestinal epithelial cell turnover in the absence of angiotensin converting enzyme

Haxhija, E. Q., Yang, H., Spencer, A. U., Koga, H., Sun, X., Teitelbaum, D. H. — 2008-07-14

Angiotensin converting enzyme (ACE) has been shown to be involved in regulation of apoptosis in nonintestinal tissues. This study examined the role of ACE in the modulation of intestinal adaptation utilizing ACE knockout mice (ACE^–/–). A 60% small bowel resection (SBR) was used, since this model results in a significant increase in intestinal epithelial cell (EC) apoptosis as well as proliferation. Baseline villus height, crypt depth, and intestinal EC proliferation were higher, and EC apoptosis rates were lower in ACE^–/– compared with ACE^+/+ mice. After SBR, EC apoptosis rates remained significantly lower in ACE^–/– compared with ACE^+/+ mice. Furthermore, villus height and crypt depth after SBR continued to be higher in ACE^–/– mice. The finding of a lower bax-to-bcl-2 protein ratio in ACE^–/– mice may account for reduced EC apoptotic rates after SBR in ACE^–/– compared with ACE^+/+ mice. The baseline higher rate of EC proliferation in ACE^–/– compared with ACE^+/+ mice may be due to an increase in the expression of several EC growth factor receptors. In conclusion, ACE appears to have an important role in the modulation of intestinal EC apoptosis and proliferation and suggests that the presence of ACE in the intestinal epithelium has a critical role in guiding epithelial cell adaptive response.

Sonic hedgehog is associated with H+-K+-ATPase-containing membranes in gastric parietal cells and secreted with histamine stimulation

Zavros, Y., Orr, M. A., Xiao, C., Malinowska, D. H. — 2008-07-14

Sonic hedgehog (Shh) is found within gastric parietal cells and processed from a 45-kDa to a 19-kDa bioactive protein by an acid- and protease-dependent mechanism. To investigate whether Shh is associated with the parietal cell membrane compartment that becomes exposed to both acid and proteolytic enzymes during acid secretion, the cellular location of Shh within resting and stimulated gastric parietal cells was examined. Immunofluorescence microscopy of rabbit stomach sections showed that Shh colocalized predominantly with parietal and pit, not chief/zymogen or neck, cell markers. In resting and histamine-stimulated rabbit gastric glands Shh was expressed only in parietal cells close to H⁺-K⁺-ATPase-containing tubulovesicular and secretory membranes with some colocalizing with -actin at the basolateral membrane. Gastric gland microsomal membranes were prepared by differential and sucrose gradient centrifugation and immunoisolation with an anti-H⁺-K⁺-ATPase- subunit antibody. The 45- and 19-kDa Shh proteins were detected by immunoblot in immunopurified H⁺-K⁺-ATPase-containing membranes from resting and stimulated gastric glands, respectively. Incubating glands with a high KCl concentration removed Shh from the membranes. Histamine stimulated 19-kDa Shh secretion from gastric glands into the medium. In human gastric cancer 23132/87 cells cultured on permeable membranes, histamine increased 19-kDa Shh secretion into both apical and basolateral media. These findings show that Shh is a peripheral protein associated with resting and stimulated H⁺-K⁺-ATPase-expressing membranes. In addition, Shh appears to be expressed at or close to the basolateral membrane of parietal cells.

Degradation of submandibular gland AQP5 by parasympathetic denervation of chorda tympani and its recovery by cevimeline, an M3 muscarinic receptor agonist

2008-07-14

By chorda tympani denervation (CTD, parasympathectomy), the aquaporin 5 (AQP5), but not AQP1, protein level in the rat submandibular gland (SMG) was significantly decreased, dropping to 37% of that of the contralateral gland at 4 wk. The protein levels of AQP5 and AQP1 were not significantly affected by denervation of the cervical sympathetic trunk (sympathectomy). Administration of cevimeline hydrochloride, an M3 muscarinic receptor agonist (10 mg/kg for 7 days po), but not pilocarpine (0.3 mg/kg for 7 days po), recovered the AQP5 protein level reduced by CTD and increased the AQP1 protein level above the control one. The mRNA level of AQP5 was scarcely affected by CTD and cevimeline hydrochloride administration. Administration of chloroquine (50 mg/kg for 7 days po), a denaturant of lysosomes, increased the AQP5 protein level reduced by CTD. An extract obtained from the submandibular lysosomal fraction degraded the AQP5 protein in the total membrane fraction in vitro. These results suggest the possible regulation of the AQP5 protein level in the SMG by the parasympathetic nerves/M3 muscarinic receptor agonist and imply the involvement of lysosomal enzymes, but not a transcriptional mechanism, in this regulation.

Progesterone stimulates the proliferation of female and male cholangiocytes via autocrine/paracrine mechanisms

2008-07-14

During cholestatic liver diseases, cholangiocytes express neuroendocrine phenotypes and respond to a number of hormones and neuropeptides by paracrine and autocrine mechanisms. We examined whether the neuroendocrine hormone progesterone is produced by and targeted to cholangiocytes, thereby regulating biliary proliferation during cholestasis. Nuclear (PR-A and PR-B) and membrane (PRGMC1, PRGMC2, and mPR) progesterone receptor expression was evaluated in liver sections and cholangiocytes from normal and bile duct ligation (BDL) rats, and NRC cells (normal rat cholangiocyte line). In vivo, normal rats were chronically treated with progesterone for 1 wk, or immediately after BDL, rats were treated with a neutralizing progesterone antibody for 1 wk. Cholangiocyte growth was measured by evaluating the number of bile ducts in liver sections. The expression of the progesterone synthesis pathway was evaluated in liver sections, cholangiocytes and NRC. Progesterone secretion was evaluated in supernatants from normal and BDL cholangiocytes and NRC. In vitro, NRC were stimulated with progesterone and cholangiocyte supernatants in the presence or absence of antiprogesterone antibody. Aminoglutethimide was used to block progesterone synthesis. Cholangiocytes and NRC express the PR-B nuclear receptor and PRGMC1, PRGMC2, and mPR. In vivo, progesterone increased the number of bile ducts of normal rats, whereas antiprogesterone antibody inhibited cholangiocyte growth stimulated by BDL. Normal and BDL cholangiocytes expressed the biosynthetic pathway for and secrete progesterone. In vitro, 1) progesterone increased NRC proliferation; 2) cholangiocyte supernatants increased NRC proliferation, which was partially inhibited by preincubation with antiprogesterone; and 3) inhibition of progesterone steroidogenesis prevented NRC proliferation. In conclusion, progesterone may be an important autocrine/paracrine regulator of cholangiocyte proliferation.

The response of fenestrations, actin, and caveolin-1 to vascular endothelial growth factor in SK Hep1 cells

2008-07-14

To study the regulation of fenestrations by vascular endothelial growth factor in liver sinusoidal endothelial cells, SK Hep1 cells were transfected with green fluorescence protein (GFP)-actin and GFP-caveolin-1. SK Hep1 cells had pores; some of which appeared to be fenestrations (diameter 55 ± 28 nm, porosity 2.0 ± 1.4%), rudimentary sieve plates, bristle-coated micropinocytotic vesicles and expressed caveolin-1, von Willebrand factor, vascular endothelial growth factor receptor-2, endothelial nitric oxide synthase and clathrin, but not CD31. There was avid uptake of formaldehyde serum albumin, consistent with endocytosis. Vascular endothelial growth factor caused an increase in porosity to 4.8 ± 2.6% (P < 0.01) and pore diameter to 104 ± 59 nm (P < 0.001). GFP-actin was expressed throughout the cells, whereas GFP-caveolin-1 had a punctate appearance; both responded to vascular endothelial growth factor by contraction toward the nucleus over hours in parallel with the formation of fenestrations. SK Hep1 cells resemble liver sinusoidal endothelial cells, and the vascular endothelial growth factor-induced formation of fenestration-like pores is preceded by contraction of actin cytoskeleton and attached caveolin-1 toward the nucleus.

Carbon monoxide activates NF-{kappa}B via ROS generation and Akt pathways to protect against cell death of hepatocytes

Kim, H. S., Loughran, P. A., Rao, J., Billiar, T. R., Zuckerbraun, B. S. — 2008-07-14

Heme oxygenase overexpression or exogenous carbon monoxide (CO) protects against hepatocyte apoptosis and fulminant hepatitis. The prevention of hepatocyte apoptosis by CO has been shown to require activation of NF-B. The purpose of these investigations was to determine the mechanism of CO-induced hepatocyte NF-B activation and protection against apoptosis. Primary rat or mouse hepatocytes and Hep3B cells were utilized. CO exposure was performed at 250 parts per million. Main outcome measures included cell viability, reactive oxygen species (ROS) generation, and changes in the levels of the intracellular antioxidants glutathione and ascorbate. Western blotting was performed for phospho-Akt, total Akt, and IB. NF-B activation was determined by electrophoretic mobility shift assay and luciferase reporter assays. We found that CO treatment of hepatocytes prevents spontaneous apoptosis and leads to an increase in ROS production in association with Akt phosphorylation and IB degradation. CO did not increase ROS production in respiration-deficient (⁰) Hep3B cells. Both Akt phosphorylation and IB degradation can be inhibited by the addition of antioxidants. Furthermore, CO-induced NF-B activation is reversed by phosphatidylinositol 3-kinase (PI3-K) inhibitor (LY294002) or antioxidants. Additionally, prevention of spontaneous hepatocyte apoptosis by CO is reversed by PI3-K inhibition and antioxidants. In conclusion, these data implicate a survival pathway of CO-induced ROS, Akt phosphorylation, and NF-B activation in cultured hepatocytes. This pathway may prove to be important in maintenance of hepatic function in both physiological and pathophysiological conditions.

Human gastrin-releasing peptide receptor gene regulation requires transcription factor binding at two distinct CRE sites

Chinnappan, D., Qu, X., Xiao, D., Ratnasari, A., Weber, H. C. — 2008-07-14

Ectopic expression of the gastrin-releasing peptide (GRP) receptor (GRP-R) occurs frequently in human malignancies of the gastrointestinal tract. Owing to paracrine and autocrine interaction with its specific high-affinity ligand GRP, tumor cell proliferation, migration, and invasion might ensue. Here we provide the first insights regarding molecular mechanisms of GRP-R regulation in gastrointestinal cancer cells. We identified by EMSA and chromatin immunoprecipitation assays two cAMP response element (CRE) binding sites that recruited transcription factor CRE binding protein (CREB) to the human GRP-R promoter. Transfection studies with a wild-type human GRP-R promoter reporter and corresponding CRE mutants showed that both CRE sites are critical for basal transcriptional activation in gastrointestinal cancer cells. Forced expression of cAMP-dependent effectors CREB and PKA resulted in robust upregulation of human GRP-R transcriptional activity, and this overexpression strictly required intact wild-type CRE sites. Direct cAMP stimulation with forskolin resulted in enhanced human GRP-R promoter activity only in HuTu-80 cells, but not in Caco-2 cells, coinciding with forskolin-induced CREB phosphorylation occurring only in HuTu-80 but not Caco-2 cells. In summary, CREB is a critical regulator of human GRP-R expression in gastrointestinal cancer and might be activated through different upstream intracellular pathways.

Upregulation of GRAIL is associated with remission of ulcerative colitis

2008-07-14

Abrogating tolerance against unidentified antigens is a critical step in the pathogenesis of ulcerative colitis (UC). T cell anergy, one of the main mechanisms of tolerance, has been shown to be induced by E3 ubiquitin ligases, such as gene related to anergy in lymphocytes (GRAIL), Itch, and c-Cbl in mice. However, it is not well known whether these E3 ligases play roles in human diseases. The pathophysiological role of the E3 ligases in patients with UC was investigated. At first, the expression of GRAIL, Itch, and c-Cbl in human anergic T cells was analyzed by quantitative RT-PCR and Western immunoblotting. Next, the mRNA expression of the E3 ligases was analyzed in peripheral CD4⁺ T cells of 20 patients with UC and 10 healthy volunteers (HV). mRNA expression was analyzed in patients with active UC before and after treatment with prednisolone and leukocytapheresis. Anergic human CD4⁺ T cells expressed significantly higher levels of GRAIL, Itch, and c-Cbl than nonanergic cells. GRAIL expression was significantly higher in patients with UC in remission than in patients with active disease and in HV (P < 0.01). The level of GRAIL expression was also significantly increased in patients with active disease whose clinical activity index scores improved after treatment (P < 0.05). There were no significant differences in Itch and c-Cbl expression among patients with active UC, patients with UC in remission, and HV. These data suggest that GRAIL plays an important role in maintaining remission in patients with UC.

Regulation of transforming growth factor {beta}-induced responses by protein kinase A in pancreatic acinar cells

Yang, H., Lee, C. J., Zhang, L., Sans, M. D., Simeone, D. M. — 2008-07-14

TGF-β is an important regulator of growth and differentiation in the pancreas and has been implicated in pancreatic tumorigenesis. We have recently demonstrated that TGF-β can activate protein kinase A (PKA) in mink lung epithelial cells (Zhang L, Duan C, Binkley C, Li G, Uhler M, Logsdon C, Simeone D. Mol Cell Biol 24: 2169–2180, 2004). In this study, we sought to determine whether TGF-β activates PKA in pancreatic acinar cells, the mechanism by which PKA is activated, and PKA's role in TGF-β-mediated growth regulatory responses. TGF-β rapidly activated PKA in pancreatic acini while having no effect on intracellular cAMP levels. Coimmunoprecipitation experiments demonstrated a physical interaction between a Smad3/Smad4 complex and the regulatory subunits of PKA. TGF-β also induced activation of the PKA-dependent transcription factor CREB. Both the specific PKA inhibitor H89 and PKI peptide significantly blocked TGF-β's ability to activate PKA and CREB. TGF-β-mediated growth inhibition and TGF-β-induced p21 and SnoN expression in pancreatic acinar cells were blocked by H89 and PKI peptide. This study demonstrates that this novel cross talk between TGF-β and PKA signaling pathways may play an important role in regulating TGF-β signaling in the pancreas.

Ost{alpha}-Ost{beta} is required for bile acid and conjugated steroid disposition in the intestine, kidney, and liver

Ballatori, N., Fang, F., Christian, W. V., Li, N., Hammond, C. L. — 2008-07-14

Mice deficient in the organic solute transporter (Ost)- subunit of the heteromeric organic solute and steroid transporter, Ost-Ostβ, were generated and were found to be viable and fertile but exhibited small intestinal hypertrophy and growth retardation. Bile acid pool size and serum levels were decreased by more than 60% in Ost–/– mice, whereas fecal bile acid excretion was unchanged, suggesting a defect in intestinal bile acid absorption. In support of this hypothesis, when [³H]taurocholic acid or [³H]estrone 3-sulfate were administered into the ileal lumen, absorption was lower in Ost–/– mice. Interestingly, serum cholesterol and triglyceride levels were also ~15% lower in Ost–/– mice, an effect that may be related to the impaired intestinal bile acid absorption. After intraperitoneal administration of [³H]estrone 3-sulfate or [³H]dehydroepiandrosterone sulfate, Ost–/– mice had higher levels of radioactivity in their liver and urinary bladder and less in the duodenum, indicating altered hepatic, renal, and intestinal disposition. Loss of Ost was associated with compensatory changes in the expression of several genes involved in bile acid homeostasis, including an increase in the multidrug resistance-associated protein 3, (Mrp3)/Abcc3, an alternate basolateral bile acid export pump, and a decrease in cholesterol 7-hydroxylase, Cyp7a1, the rate-limiting enzyme in bile acid synthesis. The latter finding may be explained by increased ileal expression of fibroblast growth factor 15 (Fgf15), a negative regulator of hepatic Cyp7a1 transcription. Overall, these findings provide direct support for the hypothesis that Ost-Ostβ is a major basolateral transporter of bile acids and conjugated steroids in the intestine, kidney, and liver.

Gene therapy of Cav1.2 channel with VIP and VIP receptor agonists and antagonists: a novel approach to designing promotility and antimotility agents

Shi, X.-Z., Sarna, S. K. — 2008-07-14

Recent findings show that the enteric neurotransmitter VIP enhances gene transcription of the _1C subunit of Ca_v1.2 (L-type) Ca²⁺ channels in the primary cultures of human colonic circular smooth muscle cells and circular smooth muscle strips. In this study, we investigated whether systemic infusion of VIP in intact animals enhances the gene transcription and protein expression of these channels to accelerate colonic transit. We also investigated whether similar systemic infusions of VPAC_1/2 receptor antagonist retards colonic transit by repressing the constitutive gene expression of the _1C subunit. We found that the systemic infusion of VIP for 7 days by a surgically implanted osmotic pump enhances the gene and protein expression of the _1C subunit and circular muscle contractility in the proximal and the middle rat colons, but not in the distal colon. A similar systemic infusion of VPAC_1/2 receptor antagonist represses the expression of the _1C subunit and circular smooth muscle contractility in the proximal and the middle colons. The VIP infusion accelerates colonic transit and pellet defecation by rats, whereas the infusion of VPAC_1/2 receptor antagonist retards colonic transit and pellet defecation. VPAC₁ receptors, but not VPAC₂ receptors, mediate the above gene transcription-induced promotility effects of VIP. We conclude that VIP and VPAC₁ receptor agonists may serve as potential promotility agents in constipation-like conditions, whereas VPAC receptor antagonists may serve as potential antimotility agents in diarrhea-like conditions produced by enhanced motility function.

The role of hepatic arterial flow on portal venous and hepatic venous wedged pressure in the isolated perfused CCl4-cirrhotic liver

Zipprich, A., Loureiro-Silva, M. R., D'Silva, I., Groszmann, R. J. — 2008-07-14

In cirrhosis, hepatic venous pressure gradient is used to measure portal venous and sinusoidal pressures, as well as drug-induced decreases of elevated pressures. The aim of this study was to investigate the influence of hepatic arterial flow (HAF) changes on portal venous perfusion (PVPP) and wedged hepatic venous pressure (WHVP). Normal and CCl₄-cirrhotic rats were subjected to a bivascular liver perfusion with continuous measurements of PVPP, WHVP, and hepatic arterial perfusion pressure. Flow-pressure curves were performed with the use of different flows either through the portal vein (PVF: 20–32 ml/min) or HAF (5–15 ml/min). Increases in HAF lead to significant absolute and relative increases in PVPP (P = 0.002) and WHVP (P < 0.001). Absolute changes in HAF correlated to absolute changes in PVPP (cirrhosis: r = 0.64, P < 0.001; control: r = 0.67, P < 0.001) and WHVP (cirrhosis: r = 0.71, P < 0.001; control: r = 0.82, P < 0.001). Changes in PVPP correlated to changes in WHVP due to changes in PVF only in cirrhosis (r = 0.75, P < 0.001), whereas changes in HAF correlated in both cirrhosis (r = 0.92, P < 0.001) and control (r = 0.77, P < 0.001). In conclusion, increases and decreases in HAF lead to respective changes in PVPP and WHVP. This suggests a direct influence of HAF on PVPP and WHVP most likely due to changes in sinusoidal perfusion.

Regulation of direct transintestinal cholesterol excretion in mice

2008-07-14

Biliary secretion is generally considered to be an obligate step in the pathway of excess cholesterol excretion from the body. We have recently shown that an alternative route exists. Direct transintestinal cholesterol efflux (TICE) contributes significantly to cholesterol removal in mice. Our aim was to investigate whether the activity of this novel pathway can be influenced by dietary factors. In addition, we studied the role of cholesterol acceptors at the luminal side of the enterocyte. Mice were fed a Western-type diet (0.25% wt/wt cholesterol; 16% wt/wt fat), a high-fat diet (no cholesterol; 24% wt/wt fat), or high-cholesterol diet (2% wt/wt), and TICE was measured by isolated intestinal perfusion. Bile salt-phospholipid mixtures served as cholesterol acceptor. Western-type and high-fat diet increased TICE by 50 and 100%, respectively. In contrast, the high-cholesterol diet did not influence TICE. Intestinal scavenger receptor class B type 1 (Sr-B1) mRNA and protein levels correlated with the rate of TICE. Unexpectedly, although confirming a role for Sr-B1, TICE was significantly increased in Sr-B1-deficient mice. Apart from the long-term effect of diets on TICE, acute effects by luminal cholesterol acceptors were also investigated. The phospholipid content of perfusate was the most important regulator of TICE; bile salt concentration or hydrophobicity of bile salts had little effect. In conclusion, TICE can be manipulated by dietary intervention. Specific dietary modifications might provide means to stimulate TICE and, thereby, to enhance total cholesterol turnover.

Gallbladder nitric oxide

McKirdy, H. C. — 2008-07-14

Functional characterization, localization, and molecular identification of rabbit intestinal N-amino acid transporter

2008-06-09

We have characterized the Na-glutamine cotransporter in the rabbit intestinal crypt cell brush border membrane vesicles (BBMV). Substrate specificity experiments showed that crypt cell glutamine uptake is mediated by system N. Real-time PCR experiments showed that SN2 (SLC38A5) mRNA is more abundant in crypt cells compared with SN1 (SLC38A3), indicating that SN2 is the major glutamine transporter present in the apical membrane of the crypt cells. SN2 cDNA was obtained by screening a rabbit intestinal cDNA library with human SN1 used as probe. Rabbit SN2 cDNA encompassed a 473-amino-acid-long open reading frame. SN2 protein displayed 87% identity and 91% similarity to human SN2. Functional characterization studies of rabbit SN2 were performed by using vaccinia virus-mediated transient expression system. Substrate specificity of the cloned transporter was identical to that of SN2 described in the literature and matched well with substrate specificity experiments performed using crypt cell BBMV. Cloned rabbit SN2, analogous to its human counterpart, is Li⁺ tolerant. Hill coefficient for Li⁺ activation of rabbit SN2-mediated uptake was 1. Taken together, functional data from the crypt cell BBMV and the cloned SN2 cDNA indicate that the crypt cell glutamine transport is most likely mediated by SN2.

Effect of the nitric oxide donor V-PYRRO/NO on portal pressure and sinusoidal dynamics in normal and cirrhotic mice

Edwards, C., Feng, H.-Q., Reynolds, C., Mao, L., Rockey, D. C. — 2008-06-09

Reduced sinusoidal endothelial nitric oxide (NO) production contributes to increased intrahepatic resistance and portal hypertension after liver injury. We hypothesized that V-PYRRO/NO, an NO donor prodrug metabolized "specifically" in the liver, would reduce portal venous pressure (PVP) without affecting the systemic vasculature. Liver injury was induced in male BALB/c mice by weekly CCl₄ gavage. PVP and mean arterial pressure were recorded during intravenous administration of V-PYRRO/NO. In vivo microscopy was used to monitor sinusoidal diameter and flow during drug administration. Mean PVP was increased in CCl₄-treated mice compared with sham-treated mice. In dose-response experiments, the minimum dose of PYRRO/NO required to acutely lower PVP by 20%, the amount believed to yield a clinically meaningful outcome, was 200 nmol/kg. This dose decreased portal pressure in cirrhotic (23.4 ± 2.0%, P < 0.001 vs. vehicle) and sham-treated (19.5 ± 2.3%, P < 0.001 vs. vehicle) animals by a similar magnitude. This concentration also led to dilation of hepatic sinusoids and an increase in sinusoidal volumetric flow, consistent with a reduction of intrahepatic resistance. The effect of V-PYRRO/NO on mean arterial pressure was significant at all concentrations tested, including the lowest, 30 nmol/kg (P < 0.001 vs. vehicle for all doses). We conclude that V-PYRRO/NO had widespread vascular effects and, as such, is unlikely to be suitable for treatment of portal hypertension. As the potential of this or other similar compounds for treatment of portal hypertension is evaluated, effects on the systemic vasculature will also need to be considered.

CFTR inhibition augments NHE3 activity during luminal high CO2 exposure in rat duodenal mucosa

Mizumori, M., Choi, Y., Guth, P. H., Engel, E., Kaunitz, J. D., Akiba, Y. — 2008-06-09

We hypothesized that the function of duodenocyte apical membrane acid-base transporters are essential for H⁺ absorption from the lumen. We thus examined the effect of inhibition of Na⁺/H⁺ exchanger-3 (NHE3), cystic fibrosis transmembrane regulator (CFTR), or apical anion exchangers on transmucosal CO₂ diffusion and HCO₃^– secretion in rat duodenum. Duodena were perfused with a pH 6.4 high CO₂ solution or pH 2.2 low CO₂ solution with the NHE3 inhibitor, S3226, the anion transport inhibitor, DIDS, or pretreatment with the potent CFTR inhibitor, CFTR_inh-172, with simultaneous measurements of luminal and portal venous (PV) pH and carbon dioxide concentration ([CO₂]). Luminal high CO₂ solution increased CO₂ absorption and HCO₃^– secretion, accompanied by PV acidification and PV Pco₂ increase. During CO₂ challenge, CFTR_inh-172 induced HCO₃^– absorption, while inhibiting PV acidification. S3226 reversed CFTR_inh-associated HCO₃^– absorption. Luminal pH 2.2 challenge increased H⁺ and CO₂ absorption and acidified the PV, inhibited by CFTR_inh-172 and DIDS, but not by S3226. CFTR inhibition and DIDS reversed HCO₃^– secretion to absorption and inhibited PV acidification during CO₂ challenge, suggesting that HCO₃^– secretion helps facilitate CO₂/H⁺ absorption. Furthermore, CFTR inhibition prevented CO₂-induced cellular acidification reversed by S3226. Reversal of increased HCO₃^– loss by NHE3 inhibition and reduced intracellular acidification during CFTR inhibition is consistent with activation or unmasking of NHE3 activity by CFTR inhibition, increasing cell surface H⁺ available to neutralize luminal HCO₃^– with consequent CO₂ absorption. NHE3, by secreting H⁺ into the luminal microclimate, facilitates net transmucosal HCO₃^– absorption with a mechanism similar to proximal tubular HCO₃^– absorption.

Cyclic AMP accelerates calcium waves in pancreatic acinar cells

2008-06-09

Cytosolic Ca²⁺ (Ca_i²⁺) flux within the pancreatic acinar cell is important both physiologically and pathologically. We examined the role of cAMP in shaping the apical-to-basal Ca²⁺ wave generated by the Ca²⁺-activating agonist carbachol. We hypothesized that cAMP modulates intra-acinar Ca²⁺ channel opening by affecting either cAMP-dependent protein kinase (PKA) or exchange protein directly activated by cAMP (Epac). Isolated pancreatic acinar cells from rats were stimulated with carbachol (1 µM) with or without vasoactive intestinal polypeptide (VIP) or 8-bromo-cAMP (8-Br-cAMP), and then Ca_i²⁺ was monitored by confocal laser-scanning microscopy. The apical-to-basal carbachol (1 µM)-stimulated Ca²⁺ wave was 8.63 ± 0.68 µm/s; it increased to 19.66 ± 2.22 µm/s (*P < 0.0005) with VIP (100 nM), and similar increases were observed with 8-Br-cAMP (100 µM). The Ca²⁺ rise time after carbachol stimulation was reduced in both regions but to a greater degree in the basal. Lag time and maximal Ca²⁺ elevation were not significantly affected by cAMP. The effect of cAMP on Ca²⁺ waves also did not appear to depend on extracellular Ca²⁺. However, the ryanodine receptor (RyR) inhibitor dantrolene (100 µM) reduced the cAMP-enhancement of wave speed. It was also reduced by the PKA inhibitor PKI (1 µM). 8-(4-chloro-phenylthio)-2'-O-Me-cAMP, a specific agonist of Epac, caused a similar increase as 8-Br-cAMP or VIP. These data suggest that cAMP accelerates the speed of the Ca²⁺ wave in pancreatic acinar cells. A likely target of this modulation is the RyR, and these effects are mediated independently by PKA and Epac pathways.

Cellular cross talk in the small intestinal mucosa: postnatal lymphocytic immigration elicits a specific epithelial transcriptional response

2008-06-09

During the early postnatal period lymphocytes migrate into the mouse small intestine. Migrating infiltrative lymphocytes have the potential to affect the epithelial cells via secreted cytokines. Such cross talk can result in the elicitation of an epithelial transcriptional response. Knowledge about such physiological cross talk between the immune system and the epithelium in the postnatal small intestinal mucosa is lacking. We have investigated the transcriptome changes occurring in the postnatal mouse small intestine using DNA microarray technology, immunocytochemistry, and quantitative real-time RT-PCR analysis. The DNA microarray data were analyzed bioinformatically by using a combination of projections to latent structures analysis and functional annotation analysis. The results show that infiltrating lymphocytes appear in the mouse small intestine in the late postweaning period and give rise to distinct changes in the epithelial transcriptome. Of particular interest is the expression of three genes encoding a mucin (Muc4), a mucinlike protein (16000D21Rik), and ATP citrate lyase (Acly). All three genes were shown to be expressed by the epithelium and to be upregulated in response to lymphocytic migration into the small intestinal mucosa.

The novel protein kinase C isoforms -{delta} and -{varepsilon} modulate caerulein-induced zymogen activation in pancreatic acinar cells

2008-06-09

Isoforms of protein kinase C (PKC) have been shown to modulate some cellular responses such as pathological secretion and generation of inflammatory mediators during acute pancreatitis (AP). We propose that PKC also participates in premature zymogen activation within the pancreatic acinar cell, a key event in the initiation of AP. This hypothesis was examined in in vivo and cellular models of caerulein-induced AP using PKC activators and inhibitors. Phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA, 200 nM), a known activator of PKC, enhanced zymogen activation at both 0.1 nM and 100 nM caerulein, concentrations which mimic physiological and supraphysiological effects of the hormone cholecystokinin, respectively, in preparations of pancreatic acinar cells. Isoform-specific PKC inhibitors for PKC- and PKC- reduced supraphysiological caerulein-induced zymogen activation. Using a cell-free reconstitution system, we showed that inhibition of PKC- and -, reduced zymogen activation in both zymogen granule-enriched and microsomal fractions. In dispersed acinar cells, 100 nM caerulein stimulation caused PKC- and - isoform translocation to microsomal membranes using cell fractionation and immunoblot analysis. PKC translocation was confirmed with in vivo studies and immunofluorescence microscopy in pancreatic tissues from rats treated with or without 100 nM caerulein. PKC- redistributed from an apical to a supranuclear region following caerulein administration. The signal for PKC- overlapped with granule membrane protein, GRAMP-92, an endosomal/lysosomal marker, in a supranuclear region where zymogen activation takes place. These results indicate that PKC- and - isoforms translocate to specific acinar cell compartments and modulate zymogen activation.

Role of Sp1 and HNF1 transcription factors in SGLT1 regulation during chronic intestinal inflammation

Kekuda, R., Saha, P., Sundaram, U. — 2008-06-09

In a rabbit model of chronic intestinal inflammation, we previously demonstrated that the activity of Na-glucose cotransporter (SGLT1), SLC5A1, is inhibited. This inhibition is secondary to a decrease in the number of cotransporters, indicating that the regulation of SGLT1 during chronic inflammation is at the level of transcription. However, the regulation of SGLT1 expression and the transcription factors involved in the regulation are not yet known. In this report, we describe the cloning and characterization of rabbit SGLT1 promoter and the identification of transcription factors affected in villus cells during chronic intestinal inflammation. The promoter sequence for SGLT1 gene was identified by using the publicly available rabbit genomic sequence. Even though rabbit SGLT1 promoter did not have considerable overall homology with other mammalian SGLT1 promoters, two specificity protein 1 (Sp1) and a hepatocyte nuclear factor 1 (HNF1) binding sites were highly conserved among the species. Rabbit SGLT1 cDNA was encoded by 15 exons. Minimal promoter region determination showed that 196 nucleotides upstream of the transcription start site were sufficient for optimal promoter activity. This region encompassed two transcription factor binding sites, Sp1 and HNF1. For maximal SGLT1 promoter activity, these two transcription factor binding sites were essential, and their effect was synergistic, indicating that two separate regulatory pathways might be involved in their regulation. Using mobility shift assays, we further demonstrated that the binding of both Sp1 and HNF1 transcription factors to SGLT1 promoter regions were affected during chronic intestinal inflammation. Thus this report demonstrates that Sp1 and HNF1 transcription factors act in concert to regulate SGLT1 transcription in the chronically inflamed intestine.

ATP induces guinea pig gallbladder smooth muscle excitability via the P2Y4 receptor and COX-1 activity

Bartoo, A. C., Nelson, M. T., Mawe, G. M. — 2008-06-09

The purpose of this study was to elucidate the mechanisms by which ATP increases guinea pig gallbladder smooth muscle (GBSM) excitability. We evaluated changes in membrane potential and action potential (AP) frequency in GBSM by use of intracellular recording. Application of ATP (100 µM) caused membrane depolarization and a significant increase in AP frequency that were not sensitive to block by tetrodotoxin (0.5 µM). The nonselective P2 antagonist, suramin (100 µM), blocked the excitatory response, resulting in decreased AP frequency in the presence of ATP. The excitatory response to ATP was not altered by pyridoxal-phosphate-6-azophenyl-2,4-disulfonic acid (30 µM), a nonselective P2X antagonist. UTP also caused membrane depolarization and increased AP frequency, with a similar dose-response relationship as ATP. RT-PCR demonstrated that the P2Y₄, but not P2Y₂, receptor subtype is expressed in guinea pig gallbladder muscularis. ATP induced excitation was blocked by indomethacin (10 µM) and the cyclooxygenase (COX)-1 inhibitor SC-560 (300 nM), but not the COX-2 inhibitor nimesulide (500 nM). These data suggest that ATP stimulates P2Y₄ receptors within the gallbladder muscularis and, in turn, stimulate prostanoid production via COX-1 leading to increased excitability of GBSM.

Constitutive nitric oxide differentially regulates Na-H and Na-glucose cotransport in intestinal epithelial cells

Coon, S., Kekuda, R., Saha, P., Talukder, J. R., Sundaram, U. — 2008-06-09

Previous in vivo studies suggest that constitutive nitric oxide (cNO) can regulate Na- glucose cotransport (SGLT1) and Na-H exchange (NHE3) in rabbit intestinal villus cells. Whether these two primary Na absorbing pathways are directly regulated by cNO and the mechanisms of this regulation in the enterocyte is not known. Thus nontransformed rat small intestinal epithelial cells (IEC-18) were treated with N^G-nitro-l-arginine methyl ester (l-NAME), which directly decreased cNO in these cells. l-NAME treatment decreased SGLT1 in IEC-18 cells. Kinetic studies demonstrated that the mechanism of inhibition was secondary to a decrease in the affinity of the cotransporter for glucose without a change in the number of cotransporters. In contrast, l-NAME treatment increased NHE3 in IEC-18 cells. Kinetic studies demonstrated that the mechanism of stimulation was by increasing the number of the exchangers without a change in the affinity for Na. Quantitative RT-PCR (RTQ-PCR) and Western blot analysis of SGLT1 demonstrated no change in mRNA and protein, respectively. RTQ-PCR and Western blot analysis of NHE3 indicated that NHE3 was increased by l-NAME treatment by an increase in mRNA and protein, respectively. These results indicate that decreased cNO levels directly mediate the inhibition of SGLT1 and stimulation of NHE3 in intestinal epithelial cells. Thus cNO directly but uniquely regulates the two primary Na-absorptive pathways in the mammalian small intestine.

5-HT4 receptor activation facilitates recovery from synaptic rundown and increases transmitter release from single varicosities of myenteric neurons

Ren, J., Zhou, X., Galligan, J. J. — 2008-06-09

5-HT₄ receptor agonists facilitate synaptic transmission in the enteric nervous system, and these drugs are used to treat constipation. In the present study, we investigated the effects of the 5-HT₄ receptor agonist, renzapride, on rundown and recovery of fast excitatory postsynaptic potentials (fEPSPs) during and after trains of stimulation and on transmitter release from individual myenteric neuronal varicosities. Intracellular electrophysiological methods were used to record fEPSPs from neurons in longitudinal muscle myenteric plexus preparations of guinea pig ileum in vitro. During trains of supramaximal electrical stimulation (10 Hz, 2 s), fEPSP amplitude declined (time constant = 0.6 ± 0.1 s) from 17 ± 2 mV to 0.7 ± 0.3 mV. Renzapride (0.1 µM) did not change the time constant for fEPSP rundown, but it decreased the time constant for recovery of fEPSP amplitude after the stimulus train from 7 ± 2 s to 1.6 ± 0.2 s (P < 0.05). 5-HT (0.1 µM) also increased fEPSPs and facilitated recovery from rundown. The adenylate cyclase activator, forskolin (1 µM), mimicked the actions of renzapride and 5-HT, whereas H-89, a protein kinase A (PKA) inhibitor, blocked the effects of renzapride. We used nicotinic acetylcholine receptor containing outside-out patches obtained from myenteric neurons maintained in primary culture to detect acetylcholine release from single varicosities. Renzapride (0.1 µM) increased release probability twofold. We conclude that 5-HT₄ receptors activate the adenylyl cyclase-PKA pathway to increase acetylcholine release from single varicosities and to accelerate recovery from synaptic rundown. These responses may contribute to the prokinetic actions of 5-HT₄ receptor agonists.

Differences in intragastric pH in diabetic vs. idiopathic gastroparesis: relation to degree of gastric retention

2008-06-09

Evidence suggests that distinct mechanisms underlie diabetic and idiopathic gastroparesis. Differences in gastric acid in gastroparesis of different etiologies and varying degrees of gastric stasis are uninvestigated. We tested the hypotheses that 1) gastric pH profiles show differential alteration in diabetic vs. idiopathic gastroparesis and 2) abnormal pH profiles relate to the severity of gastric stasis. Sixty-four healthy control subjects and 44 gastroparesis patients (20 diabetic, 24 idiopathic) swallowed wireless transmitting capsules and then consumed ^99mTc-sulfur colloid-labeled meals for gastric scintigraphy. Gastric pH from the capsule was recorded every 5 s. Basal pH was higher in diabetic (3.64 ± 0.41) vs. control subjects (1.90 ± 0.18) and idiopathic subjects (2.41 ± 0.42; P < 0.05). Meals evoked initial pH increases that were greater in diabetic (4.98 ± 0.32) than idiopathic patients (3.89 ± 0.39; P = 0.03) but not control subjects (4.48 ± 0.14). pH nadirs prior to gastric capsule evacuation were higher in diabetic patients (1.50 ± 0.23) than control subjects (0.58 ± 0.11; P = 0.003). Four-hour gastric retention was similar in diabetic (18.3 ± 0.5%) and idiopathic (19.4 ± 0.5%) patients but higher than control subjects (2.2 ± 0.5%; P < 0.001). Compared with control subjects, those with moderate-severe stasis (>20% retention at 4 h) had higher basal (3.91 ± 0.55) and nadir pH (2.23 ± 0.42) values (P < 0.05). In subgroup analyses, both diabetic and idiopathic patients with moderate-severe gastroparesis exhibited increased pH parameters vs. those with mild gastroparesis. In conclusion, diabetic patients with gastroparesis exhibit reduced gastric acid, an effect more pronounced in those with severely delayed gastric emptying. Idiopathic gastroparetic subjects exhibit nearly normal acid profiles, although those with severely delayed emptying show reduced acid vs. those with mild delays. Thus both etiology and degree of gastric stasis determine gastric acidity in gastroparesis.

Salmonella enterica serovar Typhimurium modulates P-glycoprotein in the intestinal epithelium

Siccardi, D., Mumy, K. L., Wall, D. M., Bien, J. D., McCormick, B. A. — 2008-06-09

Studies over the last decade have shown that Salmonella enterica serovar Typhimurium (S. typhimurium) is able to preferentially locate to sites of tumor growth and modulate (shrink) the growth of many cancers. Given this unique association between S. typhimurium and cancer cells, the objective of this study was to investigate the capacity of this microorganism to modulate the plasma membrane multidrug resistance (MDR) protein P-glycoprotein (P-gp), an ATP-binding cassette transporter responsible for effluxing many cancer drugs. Using an in vitro model of S. typhimurium infection of polarized human cancer intestinal cell lines, we have found that this enteric pathogen functionally downregulates the efflux capabilities of P-gp. Specifically, we show that S. typhimurium infection of human intestinal cancer cells results in the enhanced intracellular accumulation of a number of P-gp substrates that corresponds to the posttranscriptional downregulation of P-gp expression. Furthermore, cells expressing small interfering RNAs against MDR1, the gene encoding P-gp, were significantly more susceptible to the cytotoxic effects of bacterial infection. This result is consistent with our observation that S. typhimurium was significantly less able to invade cells overexpressing MDR1. Taken together, these results reveal a novel role for P-gp in the maintenance of homeostasis in the gastrointestinal tract in regard to bacterial infection. Thus the regulation of P-gp by S. typhimurium has important implications not only for the development of new cancer therapeutics aimed at reversing drug resistance but also in the understanding of how microbes have evolved diverse strategies to interact with their host.

PI3K/Akt activation is critical for early hepatic regeneration after partial hepatectomy

2008-06-09

Hepatic resection is associated with rapid proliferation and regeneration of the remnant liver. Phosphatidylinositol 3-kinase (PI3K), composed of a p85 regulatory and a p110 catalytic subunit, participates in multiple cellular processes, including cell growth and survival; however, the role of PI3K in liver regeneration has not been clearly delineated. In this study, we used the potent PI3K inhibitor wortmannin and small interfering RNA (siRNA) targeting the p85 and p110 subunits to determine whether total or selective PI3K inhibition would abrogate the proliferative response of the liver after partial hepatectomy in mice. Hepatic resection is associated with an induction in PI3K activity; total PI3K blockade with wortmannin and selective inhibition of p85 or p110 with siRNA resulted in a significant decrease in hepatocyte proliferation, especially at the earliest time points. Fewer macrophages and Kupffer cells were present in the regenerating liver of mice treated with wortmannin or siRNA to p85 or p110, as reflected by a paucity of F4/80-positive cells. Additionally, PI3K inhibition led to an aberrant architecture in the regenerating hepatocytes characterized by vacuolization, lipid deposition, and glycogen accumulation; these changes were not noted in the sham livers. Our data demonstrate that PI3K/Akt pathway activation plays a critical role in the early regenerative response of the liver after resection; inhibition of this pathway markedly abrogates the normal hepatic regenerative response, most likely by inhibiting macrophage infiltration and cytokine elaboration and thus hepatocyte priming for replication.

Pathophysiological basis of liver disease in cystic fibrosis employing a {Delta}F508 mouse model

2008-06-09

The molecular pathogenesis of cystic fibrosis (CF) liver disease is unknown. This study investigates its earliest pathophysiological manifestations employing a mouse model carrying F508, the commonest human CF mutation. We hypothesized that, if increased bile salt spillage into the colon occurs as in the human disease, then this should lead to a hydrophobic bile salt profile and to "hyperbilirubinbilia" because of induced enterohepatic cycling of unconjugated bilirubin. Hyperbilirubinbilia may then lead to an increased bile salt-to-phospholipid ratio in bile and, following hydrolysis, precipitation of divalent metal salts of unconjugated bilirubin. We document in CF mice elevated fecal bile acid excretion and biliary secretion of more hydrophobic bile salts compared with control wild-type mice. Biliary secretion rates of bilirubin monoglucuronosides, bile salts, phospholipids, and cholesterol are increased significantly with an augmented bile salt-to-phospholipid ratio. Quantitative histopathology of CF livers displays mild early cholangiopathy in 53% of mice and multifocal divalent metal salt deposition in cholangiocytes. We conclude that increased fecal bile acid loss leads to more hydrophobic bile salts in hepatic bile and to hyperbilirubinbilia, a major contributor in augmenting the bile salt-to-phospholipid ratio and endogenous β-glucuronidase hydrolysis of bilirubin glucuronosides. The confluence of these perturbations damages intrahepatic bile ducts and facilitates entrance of unconjugated bilirubin into cholangiocytes. This study of the earliest stages of CF liver disease provides a framework for investigating the molecular pathophysiology of more advanced disease in murine models and in humans with CF.

Specific overexpression of IL-7 in the intestinal mucosa: the role in intestinal intraepithelial lymphocyte development

Yang, H., Madison, B., Gumucio, D. L., Teitelbaum, D. H. — 2008-06-09

IL-7 plays a crucial role in controlling T cell development and homeostasis. Since IL-7 may be derived from extraintestinal sources, and exogenous IL-7 broadly affects lymphoid populations, the actions of epithelial cell (EC)-derived IL-7 are not fully understood. The effect of intestinal specific expression of IL-7 on intestinal mucosal lymphocytes was investigated by using an IL-7 transgenic mouse model. We generated an intestinal EC-specific overexpressing IL-7 transgenic mouse model (IL-7^vill) and compared their phenotype and function to wild-type C57BL/6J mice. EC-derived IL-7 overexpression was found to be exclusively in the small and large intestine. Numbers and subtypes of mucosal lymphocytes, including intraepithelial lymphocytes (IEL) and lamina propria lymphocytes (LPL), significantly changed in IL-7^vill mice. From a functional standpoint, IEL proliferation also significantly increased in IL-7^vill mice. IEL cytokine expression significantly changed in both T cell receptor (TCR)-β⁺ and TCR-⁺ IEL subpopulations, including a significant increase in IFN- and TNF- as well as an increase in keratinocyte growth factor expression. EC expression of CD103 (integrin _Eβ₇), the ligand of E-cadherin, markedly upregulated and may account for a mechanism of the massive expansion of IEL in transgenic mice. Systemic lymphoid populations did not change in transgenic mice. IL-7 overexpression by intestinal EC significantly affected IEL phenotype and function. These results offer insight into the role of IL-7 in IEL development and suggest a critical role of EC-derived expression of IL-7 in the phenotype and function of IEL.

Use of a hanging-weight system for liver ischemic preconditioning in mice

2008-06-09

Ischemic preconditioning (IP) represents a powerful experimental strategy to identify novel molecular targets to attenuate hepatic injury during ischemia. As a result, murine studies of hepatic IP have become an important field of research. However, murine IP is technically challenging, and experimental details can alter the results. Therefore, we systematically tested a novel model of hepatic IP by using a hanging-weight system for portal triad occlusion. This system has the benefit of applying intermittent hepatic ischemia and reperfusion without manipulation of a surgical clamp or suture, thus minimizing surgical trauma. Systematic evaluation of this model revealed a close correlation of hepatic ischemia time with liver damage as measured by alanine (ALT) and aspartate (AST) aminotransferase serum levels. Using different numbers of IP cycles and times intervals, we found optimal liver protection with four cycles of 3 min ischemia/3 min reperfusion as measured by ALT, AST, lactate dehydrogenase, and interleukin-6. Similarly, ischemia-associated increases in hepatic infarct size, neutrophil infiltration, and histological injury were maximally attenuated with the above regimen. To demonstrate transcriptional consequences of liver IP, we isolated RNA from preconditioned liver and confirmed transcriptional modulation of known target genes (equilibrative nucleoside transporters, acute-phase complement genes). Taken together, these studies confirm highly reproducible liver injury and protection by IP when using the hanging-weight system for hepatic ischemia and intermittent reperfusion. Further studies of murine IP may consider this technique.

Subdiaphragmatic vagal afferent nerves modulate visceral pain

Chen, S. L., Wu, X. Y., Cao, Z. J., Fan, J., Wang, M., Owyang, C., Li, Y. — 2008-06-09

Activation of the vagal afferents by noxious gastrointestinal stimuli suggests that vagal afferents may play a complex role in visceral pain processes. The contribution of the vagus nerve to visceral pain remains unresolved. Previous studies reported that patients following chronic vagotomy have lower pain thresholds. The patient with irritable bowel syndrome has been shown alteration of vagal function. We hypothesize that vagal afferent nerves modulate visceral pain. Visceromotor responses (VMR) to graded colorectal distension (CRD) were recorded from the abdominal muscles in conscious rats. Chronic subdiaphragmatic vagus nerve sections induced 470, 106, 51, and 54% increases in VMR to CRD at 20, 40, 60 and 80 mmHg, respectively. Similarly, at light level of anesthesia, topical application of lidocaine to the subdiaphragmatic vagus nerve in rats increased VMR to CRD. Vagal afferent neuronal responses to low or high-intensity electrical vagal stimulation (EVS) of vagal afferent A or C fibers were distinguished by calculating their conduction velocity. Low-intensity EVS of A fibers (40 µA, 20 Hz, 0.5 ms for 30 s) reduced VMR to CRD at 40, 60, and 80 mmHg by 41, 52, and 58%, respectively. In contrast, high-intensity EVS of C fibers (400 µA, 1 Hz, 0.5 ms for 30 s) had no effect on VMR to CRD. In conclusion, we demonstrated that vagal afferent nerves modulate visceral pain. Low-intensity EVS that activates vagal afferent A fibers reduced visceral pain. Thus EVS may potentially have a role in the treatment of chronic visceral pain.

Arginine-induced pancreatitis: involvement of the autonomic nervous system?

Lechin, F., van der Dijs, B. — 2008-06-09

Reply to Lechin and van der Dijs

Dawra, R., Saluja, A. — 2008-06-09