AJP - Gastrointestinal and Liver Physiology

Ma, Jie; Harnett, Karen M.; Behar, Jose; Biancani, Piero; Cao, Weibiao

doi: 10.1152/ajpgi.00409.2009pmid: 19959817

Abstract Transient receptor potential channel, vanilloid subfamily member 1 (TRPV1) receptors were identified in human esophageal squamous epithelial cell line HET-1A by RT-PCR and by Western blot. In fura-2 AM-loaded cells, the TRPV1 agonist capsaicin caused a fourfold cytosolic calcium increase, supporting a role of TRPV1 as a capsaicin-activated cation channel. Capsaicin increased production of platelet activating factor (PAF), an important inflammatory mediator that acts as a chemoattractant and activator of immune cells. The increase was reduced by the p38 MAP kinase (p38) inhibitor SB203580, by the cytosolic phospholipase A2 (cPLA 2 ) inhibitor AACOCF3, and by the lyso-PAF acetyltransferase inhibitor sanguinarin, indicating that capsaicin-induced PAF production may be mediated by activation of cPLA 2 , p38, and lyso-PAF acetyltransferase. To establish a sequential signaling pathway, we examined the phosphorylation of p38 and cPLA 2 by Western blot. Capsaicin induced phosphorylation of p38 and cPLA 2 . Capsaicin-induced p38 phosphorylation was not affected by AACOCF3. Conversely, capsaicin-induced cPLA 2 phosphorylation was blocked by SB203580, indicating that capsaicin-induced PAF production depends on sequential activation of p38 and cPLA 2 . To investigate how p38 phosphorylation may result from TRPV1-mediated calcium influx, we examined a possible role of calmodulin kinase (CaM-K). p38 phosphorylation was stimulated by the calcium ionophore A23187 and by capsaicin, and the response to both agonists was reduced by a CaM inhibitor and by CaM-KII inhibitors, indicating that calcium induced activation of CaM and CaM-KII results in P38 phosphorylation. Acetyl-CoA transferase activity increased in response to capsaicin and was inhibited by SB203580, indicating that p38 phosphorylation in turn causes activation of acetyl-CoA transferase to produce PAF. Thus epithelial cells produce PAF in response to TRPV1-mediated calcium elevation. capsaicin esophagus Copyright © 2010 the American Physiological Society

Milona, Alexandra; Owen, Bryn M.; van Mil, Saskia; Dormann, Dirk; Mataki, Chikage; Boudjelal, Mohamed; Cairns, William; Schoonjans, Kristina; Milligan, Stuart; Parker, Malcolm; White, Roger; Williamson, Catherine

doi: 10.1152/ajpgi.00336.2009pmid: 19815629

Abstract Rodents undergo gestational hepatomegaly to meet the increased metabolic demands on the maternal liver during pregnancy. This is an important physiological process, but the mechanisms and signals driving pregnancy-induced liver growth are not known. Here, we show that liver growth during pregnancy precedes maternal body weight gain, is proportional to fetal number, and is a result of hepatocyte hypertrophy associated with cell-cycle progression, polyploidy, and altered expression of cell-cycle regulators p53, Cyclin-D1, and p27. Because circulating reproductive hormones and bile acids are raised in normal pregnant women and can cause liver growth in rodents, these compounds are candidates for the signal driving gestational liver enlargement in rodents. Administration of pregnancy levels of reproductive hormones was not sufficient to cause liver growth, but mouse pregnancy was associated with increased serum bile acid levels. It is known that the bile acid sensor Fxr is required for normal recovery from partial hepatectomy, and we demonstrate that Fxr − / − mice undergo gestational liver growth by adaptive hepatocyte hyperplasia. This is the first identification of any component that is required to maintain the normal mechanisms of gestational hepatomegaly and also implicates Fxr in a physiologically normal process that involves control of the hepatocyte cell cycle. Understanding pregnancy-induced hepatocyte hypertrophy in mice could suggest mechanisms for safely increasing functional liver capacity in women during increased metabolic demand. cholestasis bile acids liver regeneration cell cycle Footnotes Copyright © 2010 the American Physiological Society

Urquhart, Bradley L.; Gregor, Jamie C.; Chande, Nilesh; Knauer, Michael J.; Tirona, Rommel G.; Kim, Richard B.

doi: 10.1152/ajpgi.00224.2009pmid: 19762432

Abstract Folic acid is a vitamin essential for thymidylate and purine synthesis. The human proton-coupled folate transporter (hPCFT) has recently been identified as a pH-dependent folic acid transporter, and mutations in this transporter have been linked to hereditary folic acid malabsorption. In this study, we assessed hPCFT-mediated transport activity in vitro, intersubject variability of intestinal expression in relation to blood folates, and the relationship of proton-pump inhibitor (PPI) therapy on hPCFT expression in vivo. We created a Madin-Darby canine kidney strain II (MDCKII) cell line stably expressing hPCFT to evaluate its drug substrates and inhibitors. Intestinal pinch biopsies (duodenum, ileum, colon) were collected from patients undergoing routine endoscopy procedures, and expressed levels of hPCFT were determined by RT-PCR. When assessed using MDCKII-hPCFT cells, folic acid and methotrexate were found to be high-affinity hPCFT substrates. Sulfasalazine and pyrimethamine were noted to inhibit hPCFT activity with Ki values of 42.3 and 161.7 μmol/l, respectively. hPCFT was localized to the brush-border membrane of enterocytes with highest expression in the duodenum and reduced levels in the ileum and colon. When we assessed hPCFT expression in a subset of patients who were receiving PPI therapy, a near 50% reduction in duodenal hPCFT mRNA expression was noted. These results suggest that hPCFT transporter activity can be modulated by many drugs in clinical use, and expression of this transporter in the gastrointestinal tract is higher in the duodenum than more distal sites (duodenum > ileum > colon). Importantly, we note that PPI drug use appears to be associated with reduced hPCFT expression in vivo. folic acid absorption vitamin Copyright © 2010 the American Physiological Society

Mizushima, Takashi; Sasaki, Makoto; Ando, Tomoaki; Wada, Tsuneya; Tanaka, Mamoru; Okamoto, Yasuyuki; Ebi, Masahide; Hirata, Yosikazu; Murakami, Kenji; Mizoshita, Tsutomu; Shimura, Takaya; Kubota, Eiji; Ogasawara, Naotaka; Tanida, Satoshi; Kataoka, Hiromi; Kamiya, Takeshi; Alexander, J. S.; Joh, Takashi

Musch, Mark W.; Arvans, Donna L.; Wang, Yunwei; Nakagawa, Yasushi; Solomaha, Elena; Chang, Eugene B.

doi: 10.1152/ajpgi.00379.2009pmid: 19926819

Abstract The apical membrane Na + -H + exchanger (NHE)3 is regulated by cAMP-dependent phosphorylation, which inhibits its activity through membrane endocytosis. The clathrin complex adaptor protein synaptotagmin 1 (Syt 1) appears to be essential to this process, but little is known about its expression in intestinal epithelial cells or interaction with NHE3. The intestinal epithelial expression and apical location of Syt 1 were determined by Syt 1 mRNA profiling and immunolocalization. Tandem mass spectrometry was used for protein identification. Bis (sulfosuccinimidyl) suberate (BS 3 ) cross linking suggested that NHE3 and Syt 1 were in a membrane complex following cAMP stimulation of Caco2BBE (Brush Border Expressions) cells. To investigate the regulation of NHE3 appearance in a Syt 1-containing membrane compartment, doxycycline-inducible hemaglutinin (HA)-tagged NHE3 was expressed in Caco2BBE cells. HA-NHE3 correctly targeted to the apical membrane, where, upon cAMP stimulation, it was internalized with a Syt 1-containing compartment. Site-directed mutagenesis of NHE3 showed that serine 605 (S605) was pivotal to NHE3 and Syt 1 association and internalization. Direct Syt 1 interaction with NHE3 was suggested by fluorescence resonance energy transfer (FRET) analysis. The physiological role of S552 was less clear. By FRET, this serine residue appeared to be involved in cAMP-induced Syt 1 binding of NHE3. However, when HA-tagged NHE3 S552A was expressed in Caco2 cells, the mutated construct was not inserted into the apical membrane. We conclude that intestinal epithelial Syt 1 plays an important role in cAMP-stimulated endocytosis of apical NHE3 through cAMP-dependent phosphorylation of S605 that is required for NHE3 and Syt 1 association. protein trafficking fluorescence resonance energy transfer Na + transport diarrheal disease clathrin and adaptor protein 2 complex Na + -H + exchanger Copyright © 2010 The American Physiological Society

Chow, Jimmy Y. C.; Ban, Makiko; Wu, Helen L.; Nguyen, Flang; Huang, Mei; Chung, Heekyung; Dong, Hui; Carethers, John M.

doi: 10.1152/ajpgi.00344.2009pmid: 19940030

Abstract TGF-β utilizes receptor-activated SMAD signaling to mediate growth suppression; however, non-SMAD signaling that modulates the TGF-β response in epithelial cells become apparent when the SMAD signaling is abrogated, a common occurrence in pancreatic cancers. Here, we examined whether TGF-β utilized NF-κB to downregulate PTEN , a gene that is rarely mutated in pancreatic cancers. SMAD4 -null BxPc3 and CAPAN-1 pancreatic cancer cells were treated with TGF-β (10 ng/ml) and lysed, and cellular proteins were analyzed by Western blots using p-IκB, p65, and PTEN antibodies. PTEN promoter and NF-κB activities were assessed by PTEN -luc and p-NF-luc constructs, respectively. Dominant negative p-IκB-α-M (NF-κB superrepressor) was used to block activation of NF-κB. Cell motility was assessed by Boyden chamber migration assay. TGF-β induced IκB-α phosphorylation followed by NF-κB p65 subunit nuclear translocation and increased NF-κB activity. IκB-α-M blocked TGF-β-induced NF-κB activity, reversed downregulated PTEN promoter activity and PTEN expression, and prevented augmentation of cell motility induced by TGF-β. SMAD4 restoration, but not knockdown of SMAD2 and/or 3, reversed TGF-β-induced NF-κB activity. Thus TGF-β suppresses PTEN in pancreatic cancer cells through NF-κB activation and enhances cell motility and invasiveness in a SMAD4-independent manner that can be counteracted when TGF-β-SMAD signaling is restored. The TGF-β/NF-κB/PTEN cascade may be a critical pathway for pancreatic cancer cells to proliferate and metastasize. pancreatic cancer TGF-β NF-κB PTEN cell motility Footnotes Copyright © 2010 the American Physiological Society

Hoogerwerf, Willemijntje A.; Shahinian, Vahakn B.; Cornélissen, Germaine; Halberg, Franz; Bostwick, Jonathon; Timm, John; Bartell, Paul A.; Cassone, Vincent M.

doi: 10.1152/ajpgi.00402.2009pmid: 19926812

Abstract Human bowel movements usually occur during the day and seldom during the night, suggesting a role for a biological clock in the regulation of colonic motility. Research has unveiled molecular and physiological mechanisms for biological clock function in the brain; less is known about peripheral rhythmicity. This study aimed to determine whether clock genes such as period 1 ( per1 ) and period2 ( per2 ) modulate rhythmic changes in colonic motility. Organ bath studies, intracolonic pressure measurements, and stool studies were used to examine measures of colonic motility in wild-type and per1per2 double-knockout mice. To further examine the mechanism underlying rhythmic changes in circular muscle contractility, additional studies were completed in neuronal nitric oxide synthase (nNOS) knockout mice. Intracolonic pressure changes and stool output in vivo, and colonic circular muscle contractility ex vivo, are rhythmic with greatest activity at the start of night in nocturnal wild-type mice. In contrast, rhythmicity in these measures was absent in per1per2 double-knockout mice. Rhythmicity was also abolished in colonic circular muscle contractility of wild-type mice in the presence of N ω -nitro- l -arginine methyl ester and in nNOS knockout mice. These findings suggest that rhythms in colonic motility are regulated by both clock genes and a nNOS-mediated inhibitory process and suggest a connection between these two mechanisms. biological rhythms clock genes circadian nNOS Copyright © 2010 the American Physiological Society

Singla, Amika; Dwivedi, Alka; Saksena, Seema; Gill, Ravinder K.; Alrefai, Waddah A.; Ramaswamy, Krishnamurthy; Dudeja, Pradeep K.

doi: 10.1152/ajpgi.00345.2009pmid: 19910524

Abstract Lysophosphatidic acid (LPA), a potent bioactive phospholipid, is a natural component of food products like soy and egg yolk. LPA modulates a number of epithelial functions and has been shown to inhibit cholera toxin-induced diarrhea. Antidiarrheal effects of LPA are known to be mediated by inhibiting chloride secretion. However, the effects of LPA on chloride absorption in the mammalian intestine are not known. The present studies examined the effects of LPA on apical Cl − /OH − exchangers known to be involved in chloride absorption in intestinal epithelial cells. Caco-2 cells were treated with LPA, and Cl − /OH − exchange activity was measured as DIDS-sensitive 36 Cl − uptake. Cell surface biotinylation studies were performed to evaluate the effect of LPA on cell surface levels of apical Cl − /OH − exchangers, downregulated in adenoma (DRA) (SLC26A3), and putative anion transporter-1 (SLC26A6). Treatment of Caco-2 cells with LPA (100 μM) significantly stimulated Cl − /OH − exchange activity. Specific agonist for LPA2 receptor mimicked the effects of LPA. LPA-mediated stimulation of Cl − /OH − exchange activity was dependent on activation of phosphatidylinositol 3-kinase/Akt signaling pathway. Consistent with the functional activity, LPA treatment resulted in increased levels of DRA on the apical membrane. Our results demonstrate that LPA stimulates apical Cl − /OH − exchange activity and surface levels of DRA in intestinal epithelial cells. This increase in Cl − /OH − exchange may contribute to the antidiarrheal effects of LPA. downregulated in adenoma chloride absorption human intestine LPA receptor 2 phosphatidylinositol 3-kinase/Akt Copyright © 2010 the American Physiological Society

Dickson, Eamonn J.; Heredia, Dante J.; McCann, Conor J.; Hennig, Grant W.; Smith, Terence K.

doi: 10.1152/ajpgi.00399.2009pmid: 19959818

Abstract Colonic migrating motor complexes (CMMCs) propel fecal contents and are altered in diseased states, including slow-transit constipation. However, the mechanisms underlying the CMMCs are controversial because it has been proposed that disinhibition (turning off of inhibitory neurotransmission) or excitatory nerve activity generate the CMMC. Therefore, our aims were to reexamine the mechanisms underlying the CMMC in the colon of wild-type and neuronal nitric oxide synthase (nNOS) −/− mice. CMMCs were recorded from the isolated murine large bowel using intracellular recordings of electrical activity from circular muscle (CM) combined with tension recording. Spontaneous CMMCs occurred in both wild-type (frequency: 0.3 cycles/min) and nNOS −/− mice (frequency: 0.4 cycles/min). CMMCs consisted of a hyperpolarization, followed by fast oscillations (slow waves) with action potentials superimposed on a slow depolarization (wild-type: 14.0 ± 0.6 mV; nNOS −/− : 11.2 ± 1.5 mV). Both atropine (1 μM) and MEN 10,376 neurokinin 2 (NK2) antagonist; 0.5 μM added successively reduced the slow depolarization and the number of action potentials but did not abolish the fast oscillations. The further addition of RP 67580 (NK1 antagonist; 0.5 μM) blocked the fast oscillations and the CMMC. Importantly, none of the antagonists affected the resting membrane potential, suggesting that ongoing tonic inhibition of the CM was maintained. Fecal pellet propulsion, which was blocked by the NK2 or the NK1 antagonist, was slower down the longer, more constricted nNOS −/− mouse colon (wild-type: 47.9 ± 2.4 mm; nNOS −/− : 57.8 ± 1.4 mm). These observations suggest that excitatory neurotransmission enhances pacemaker activity during the CMMC. Therefore, the CMMC is likely generated by a synergistic interaction between neural and interstitial cells of Cajal networks. cholinergic transmission NK2 receptors NK1 receptors tachykinins neuronal nitric oxide synthase circular muscle longitudinal muscle smooth muscle interstitial cells of Cajal slow waves Copyright © 2010 the American Physiological Society

van der Hoven, Ben; van Pelt, Hans; Swart, Eleonore L.; Bonthuis, Fred; Tilanus, Huug W.; Bakker, Jan; Gommers, Diederik

doi: 10.1152/ajpgi.90688.2008pmid: 19940028

Abstract Plasma clearance of d -sorbitol, a nontoxic polyol, occurs predominantly in the liver and has been used to measure functional liver blood flow after bolus and steady- state intravenous administration. However, it is not known which of these two administration methods is superior. Therefore, plasma d -sorbitol clearance was studied in an animal model both after a bolus dose and under steady-state (SS) conditions and compared directly with liver blood flow, under normal conditions, and after the induction of endotoxin (LPS) sepsis. Adult male Wistar rats (526 ± 38 g body wt; n = 27) were anesthetized and mechanically ventilated. Hemodynamics, hepatic arterial flow, and portal venous flow were measured. Two groups were studied, namely healthy animals that served as controls and a sepsis group that received 5 mg/kg LPS intravenously ( Escherichia coli O127:B8). Each animal received either a SS infusion (0.1 mg/100 g body wt per min) or a bolus (3 mg/100 g body wt) of a 5% d -sorbitol solution intravenously in a randomized order. After the initial measurements and a 60-min pause time in between ( T 1/2,sorbitol = 9 min), a crossover was done. The hepatic clearance of d -sorbitol in the control group showed a good correlation between bolus and SS (Spearman's r = 0.7681, P = 0.0004), and both techniques correlated well with total liver blood flow (TLBF) ( r = 0.7239, P = 0.0023 and r = 0.7226, P = 0.0023, respectively). Also in the sepsis group there was a good correlation between bolus and SS sorbitol clearance ( r = 0.6655, P = 0.0182). In the sepsis group, only the SS clearance correlated with TLBF ( r = 0.6434, P = 0.024). In conclusion, in normal and under septic conditions, hepatic clearance of d -sorbitol either by bolus or a SS infusion is comparable. In healthy animals, this also correlated well with TLBF but not in septic conditions. However, this is expected because of the changes in the liver microcirculation, shunting, and decreased hepatocyte function in sepsis. microcirculation rats sepsis Copyright © 2010 the American Physiological Society