AGA Abstracts
are involved in lubiprostone activation of hClC-2. To rule out involvement of PKA phosphorylation of hClC-2 in lubiprostone activation of Cl- currents, a mutant hClC-2 Cl- channel lacking the two unique PKA consensus phosphorylation sites (RRAT and RGET) essential for PKA activation of ClC-2, was used [Cuppoletti, et al JBC 279:21849, 2004]. In wildtype hClC-2 expressing HEK293 cells, control Cl- currents at -140 mV (n=6) of -27.2 ± 3.5 pA/pF, significantly increased (P<0.0005) to -100.9 ± 6.5 pA/pF with 20nM lubiprostone and subsequently were reduced with 500µM CdCl2 to control levels, -29.9 ± 5.6 pA/pF (P<0.0005). Cl- currents in mutant hClC-2 expressing HEK293 cells was also significantly (P<0.0005) increased with 20 nM lubiprostone and inhibited by CdCl2 n=6): control, -27.1 ± 6.9 pA/pF; with lubiprostone, -81.9 ± 5.1 (P<0.0005); after CdCl2, -29.4 ± 3.7 pA/pF (P<0.0005). Conclusions. Activation of Cl- currents by lubiprostone is independent of [Ca2+]i and cAMP signaling and PKA mediated phosphorylation of hClC-2. More direct action of lubiprostone on hClC-2 Cl- channels may be involved. Supported by Sucampo Pharmaceuticals, Inc.
T1879 PAR2-Induced Epithelial Chloride Secretion Involves Phospholipase C (PLC) and Both Protein Kinase C (PKC)β1 and PKCδ Signaling Jacques Q. van der Merwe, Wallace K. MacNaughton Background: Proteinase-activated receptor (PAR)2 is activated by trypsin-like serine proteinases and selective synthetic peptides. It has been implicated in intestinal inflammation, but its role in the regulation of intestinal mucosal function remains unclear. We have previously shown that PAR2-induced chloride secretion involves both calcium- and cAMPmediated signaling, and Src-dependent EGFr transactivation leading to subsequent ERK1/ 2 activation in a cell culture model (SCBNs). In the present study, we have further examined the stimulus-secretion coupling mechanisms of PAR2 activation on epithelial chloride transport in SCBNs, focusing on specific PLC and PKC isoforms. Methods: 1. SCBN monolayers were grown on Snapwell supports and mounted in modified Ussing chambers. Short circuit current (Isc) was monitored as a measure of net electrogenic ion transport. The roles of specific PLC and PKC isoforms in PAR2-induced chloride secretion were determined by the use of selective PLC (U73122, ET-18 and D609) and PKC inhibitors (GFX, Gö6976, rottlerin, PKCζ pseudosubstrate inhibitor). 2. Immunoblot analysis was conducted for both PLCβ and PLCγ following PAR2 activation by the activating peptide, SLIGRL-NH2 (50 µM), for 5 minutes. Immunoblots for total amounts of each protein were conducted as controls. 3. Confocal immunofluorescence microscopy and cellular fractionation with subsequent immunoblot analysis was conducted for specific PKC isoforms (PKCα,βI/II,δ,ε,ζ) following PAR2 activation by SLIGRL-NH2 (50 µM), for 40, 60 and 90 seconds. Results: 1. Pretreatment with U71322, D609 or ET-18 resulted in a significant decrease in PAR2-induced ion secretion, suggesting a role for both phosphatidylcholine-specific PLC and phosphatidylinositol-specific PLC in the Isc response. Furthermore, PAR2 stimulation by SLIGRL-NH2 resulted in significant increases in PLCβ and PLCγ phosphorylation. Pretreatment of the cells with GFX, rottlerin and Gö6976, but not the PKCζ pseudosubstrate inhibitor, resulted in a significant reduction in the chloride secretory response to PAR2 activation by SLIGRL-NH2, thereby suggesting a role for PKCδ and/or PKCβ1 in PAR2-induced chloride secretion. Furthermore, activation of PAR2 by SLIGRL-NH2 resulted in a rapid (<40 s) redistribution of PKCβI, PKCδ and PKCε, but not PKCα or PKCζ, from the cytosol to the cell membrane. Conclusions: PAR2 activation induces epithelial chloride secretion that is mediated by both PC-PLC and PI-PLC, and involves increases in PLCβ/γ phosphorylation. Furthermore PKCδ and PKCβ1, but not PKCζ or PKCα are important in PAR2-induced epithelial chloride secretion.
T1877 Clc-2-Targeted SIRNA Eliminates Lubiprostone Activation of Cl- Currents in T84 Cells John Cuppoletti, Danuta H. Malinowska, Kirti Tewari, Jayati Chakrabarti, Ryuji Ueno Introduction. Lubiprostone stimulates electrogenic Cl- transport in human intestinal T84 cells and Cl- currents in HEK293 cells stably transfected with recombinant human ClC-2 (hClC-2) [Cuppoletti et al AJP 287:C1173, 2004]. The purpose of this study was to determine whether ClC-2 is the Cl- ion channel in T84 cells responsible for lubiprostone's effects by employing siRNA (small interfering RNA) to knockdown hClC-2. SiRNA targeting hClC-2 was expressed in T84 cells and HEK293 cells stably transfected with hClC-2 and the effect of lubiprostone on Cl- currents was determined. Loss of lubiprostone-activated Cl- currents was expected if ClC-2 is the target of lubiprostone. Methods. Short hairpin sequences (shRNAs) producing siRNAs targeting hClC-2 (bp1302-1322), control vector (V) and a nontarget (NT) sequence in lentiviral vector pLKO.1 were used. HEK293 cells stably expressing hClC-2 were transfected with the shRNAs using lipofectamine. T84 cells were transduced with lentiviral particles containing the shRNAs using ViroMag R/L. Puromycin was used for selection. Cl- currents were measured by whole cell patch clamp. Results. In hClC-2expressing HEK293 cells, control Cl- currents at -140 mV (n=6) of -27.2 ± 3.5 pA/pF, increased significantly to -100.9 ± 6.5 pA/pF (P<0.0005) with 20nM lubiprostone and then were reduced by 500 µM CdCl2 (-29.9 ± 5.6 pA/pF, P<0.0005). When expressing siRNA targeting hClC-2 in this HEK cell line, lubiprostone-activated Cl- currents were absent (n= 6): control, -25.5 ± 3.1 pA/pF; with lubiprostone, -26.4 ± 3.3 pA/pF; with CdCl2, -19.5.0 ± 4.8 pA/pF. In contrast, in hClC-2 HEK293 cells expressing vector alone (V) or non-target (NT) siRNA, 20 nM lubiprostone continued to stimulate Cd2+-inhibitable Cl- currents: in V (n=6) and NT (n=7) cells respectively: control, -31.3 ± 3.4; -24.34 ± 4.2 pA/pF, with lubiprostone, -83.9 ± 4.1 (P<0.0005); -66.3 ± 9.1 (P<0.0025) pA/pF; with CdCl2, -34.8 ± 4.2; 24.8 ± 4.3 pA/pF. In T84 cells (n=6), Cl- currents were significantly activated (P<0.0025) with 20 nM lubiprostone (control, -34.2 ± 3.4 pA/pF; with lubiprostone, 78.2 ± 9.0 pA/ pF), and inhibited by CdCl2 (P<0.001) to -25.8 ± 3.5 pA/pF. Upon expressing siRNA targeting hClC-2 in T84 cells, lubiprostone-activated Cl- currents (n=7) were absent: control, -48.9 ± 5.7 pA/pF, with lubiprostone, -50.2 ± 7.6 pA/pF, with CdCl2, -25.8 ± 6.0 pA/pF. Conclusions. Activation of Cl- currents by lubiprostone in human T84 cells and in HEK293 expressing recombinant hClC-2 is due to the opening of hClC-2 Cl- channels. Supported by Sucampo Pharmaceuticals, Inc.
T1880 Mechanisms of Metformin-Induced Inhibition of Intestinal D-Glucose Absorption Yassine Sakar, Bouchra Meddah, My A Faouzi, Y. Cherrah, André Bado, Robert A. Ducroc Dimethylbiguanide Metformin (Met) is an orally administered drug that lowers blood glucose concentration and improves insulin sensitivity in patients with non insulino-dependent diabetes. Met belongs to the “top ten” of the most prescribed generic drugs with ~35 millions doses in 2006. The effect of Met on intestinal glucose transport is however poorly documented and the mechanism of action of Met on enterocyte remains elusive. This study was designed to study the effect of Met action on enterocyte. Methods : Male Wistar rats were fasted 16h. Sodium-glucose transporter-1 activity was followed as glucose-induced Isc in jejunal mucosa isolated in Ussing chambers. Met was introduced 3 min before glucose challenge in mucosal bath. Met was also injected in jejunal loops of anesthetized rats 3 min before glucose challenge. After 5 min, mucosa was scrapped and brush border membranes (BBM) further prepared to study SGLT-1 by western blotting. Phosphorylation status of AMPK in enterocyte was studied in total protein extracts. Finally, acute effect of Met was studied by oral glucose tolerance test (OGTT) in awake fasted rats. Met (300 mg/kg) was given orally before glucose (2 g/kg) and blood glucose was monitored during 2 hrs with an AccuCheck reader (Roche Diagnostics France). Results : Met markedly inhibited Na+-induced Isc (~77%) in rat jejunal mucosa after addition from mucosal side. Inhibition was rapid (3 min) and dose dependent. Quantitative analysis of sugar transporters in BBM demonstrated that Met blocked the glucose-induced abundance of SGLT1 in apical membrane. At the same time, Met strongly increased AMPK phosphorylation as previously shown (Walker J et al. Biochem J. 385: 485, 2005). In Vivo, Met markedly reduced glycemia in OGTT (-19.1%). This increase in glucose tolerance was observed after acute administration of Met. Discussion : These data demonstrate that Met rapidly increases AMPK phosphorylation, decreases abundance of SGLT-1 in BBM and markedly inhibits SGLT1-mediated absorption of glucose. As previously shown, shortterm inhibition of SGLT-1 action is also accompanied by an increase in glucose tolerance. Conclusion : Met, a drug commonly used for the treatment of type 2 diabetes, has a shortterm effect on intestinal glucose absorption by reducing SGLT1 abundance in BBM, possibly through the control of AMPK in enterocyte. These data give new insight into the mechanisms of drug-induced regulation of glucose absorption in small intestine.
T1878 Hydroxylases Regulate Intestinal Epithelial Secretory Function Karen Lawler, Cormac T. Taylor, Stephen J. Keely BACKGROUND: Inflammation of the gastrointestinal tract is commonly associated with tissue hypoxia. In this low O2 environment, the activity of key oxygen sensing enzymes known as prolyl hydroxylases (PHDs) are decreased, resulting in activation of transcription factors such as hypoxic inducible factor (HIF-1α) and Nuclear Factor-κB. While recent studies have shown that PHD inhibition is protective in murine colitis, little is known about the role of PHDs in regulating epithelial transport function. AIM: Using human colonic T84 cells grown as monolayers on permeable supports, we aimed to characterise the effects of hydroxylase inhibition on epithelial secretory function. METHODS: Hydroxylase activity was inhibited using the pan specific inhibitor, dimethyloxalylglycine, (DMOG). Protein expression was analyzed by western blotting and chloride secretory responses were measured as changes in short circuit current (Isc) across voltage clamped monolayers of T84 cells. RESULTS: Treatment of T84 cells with DMOG (1 mM) increased expression of HIF-1α, demonstrating its efficacy as an hydroxylase inhibitor. Pretreatment of T84 cells with DMOG (1 mM; 24 hrs) attenuated subsequent secretory responses to the Ca2+- and cAMP-dependent agonists, carbachol (CCh; 100 µM) and forskolin (FSK; 10 µM), respectively. After DMOG treatment responses to CCh and FSK were 25.5 ± 2.2% (p < 0.001) and 43.8 ± 4.1% (p < 0.05) of those in control cells, respectively (n = 8). DMOG did not alter transepithelial resistance implying it was not toxic to the cells. The effects of DMOG were both concentrationand time-dependent with maximal antisecretory effects occurring at 100 µM and being apparent within 3 hrs and maximal by 12 hrs. In selectively permeabilized monolayers, DMOG did not alter apical Cl- or basolateral K+ conductances but significantly inhibited (by 69.5 ± 4.9 % n = 4; p < 0.001) Na+/K+ ATPase pump activity. Examination of transport protein expression revealed that DMOG decreased the expression of both the catalytic subunit of the Na+/K+ ATPase pump and the NKCC1 cotransporter. CONCLUSIONS: These studies demonstrate a novel role for hydroxylases in regulating intestinal epithelial secretory function. Our data suggest that by virtue of their ability to modulate transport protein expression, hydroxylases are likely to be important regulators of intestinal fluid and electrolyte transport in hypoxic conditions.
AGA Abstracts
T1881 Pyridoxine Uptake By Colonocytes: A Specific and Regulated Carrier-Mediated Process Zainab M. Said, Veedamali S. Subramanian, Nosratola D. Vaziri, Hamid M. Said Background: The water-soluble vitamin B6 (pyridoxine) is important for normal cellular functions, growth and development via its involvement in variety of metabolic reactions. Humans and other mammals cannot synthesize pyridoxine, and thus, must obtain it from exogenous sources. Two exogenous sources of pyridoxine are available to the intestine: The dietary source, which is absorbed in the small intestine and the bacterial source where the vitamin is synthesized in considerable quantities by the normal microflora of the large intestine. Bacterially synthesized pyridoxine is released into the large intestinal lumen and thus is available for absorption. Nothing, however, is known about the mechanism or regulation of pyridoxine absorption in the large intestine. These issues were addressed in the present investigations. Method: We used the young adult mouse colonic epithelial YAMC cells, and colonic apical membrane vesicles (AMV) isolated from human organ donors as
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