- Email: [email protected]
T1866 Lactose Malabsorption During Methotrexate-Induced Gastrointestinal Mucositis in a Rat Model
respectively, each p<0.01). Conclusions: In our rat model, the intestinal capacity to absorb lactose during MTX-induced mucositis is severly reduced due to defective lactose hydrolysis. In contrast, the intestinal capacity to absorb glucose is still intact during mucositis when supplied in trace amounts. Whether glucose can function as an appropriate source of dietary energy during mucositis is currenlty being investigated in our lab. T1867 Proteome of the Small Intestinal Brush Border of the NHERF2 Knock out (Ko) Mouse: Changes in Transporters and Signaling Molecules Mark Donowitz, Siddharth Singh, Prashant Singh, Molee Chakraborty, Marjan Gucek, Nicholas C. Zachos, Olga Kovbasnjuk, James R. Goldenring, Ursula Seidler, Hugo de Jonge, Boris M. Hogema, Xuhang Li, Rakhilya Murtazina The NHERF Family of multi-PDZ domains is made up of 4 closely related scaffolding proteins which are found primarily in the apical domain of polarized epithelial cells. Murine KO models of NHERFs 1and 2 have shown involvement in multiple aspects of small intestinal absorptive and secretory function, including basal and acutely regulated NHE3 activity. The hypothesis tested here was that functions of NHERF2 could be predicted by determining changes in the proteome of the small intestinal BB in the NHERF2 KO mouse. Methods: Villus epithelial cells were isolated from small intestine of wild type and NHERF2 null male FVB mice by vibration. BB were prepared by double Mg precipitation and matched for protein enrichment and changes in membrane marker enzymes. BB were trypsinized, labeled with iTRAQ reagents (8 dye) and examined by shotgun 2D LC- MS/MS analysis with altered protein expression identified using criteria for increase, NHERF2 null/wild type >1.20 and decrease, <0.80. In some cases, confirmation was by IB and IF, with emphasis on changes in transport proteins and signaling molecules. Results: Up Regulated in NHERF 2 -/-: Transport proteins: Changes also in NHERF1 Null. DRA (SLC26a3), sulfate transporter (SLC26a3), Na myoinositol co-transporter (SLCa11), TRP4, monocarboxylate transporter1 (SLC16a1), L-type amino acid transporter2 (SLC7a8), choline transporters 4 (SLC44a4). Only in NHERF2. Na nucleotide co-transporter 1(CNT1), Aquaporins 1,7, VAT1, Ca activated non-selective cation channel 1. Signaling Molecules: Changes also in NHERF1 .Cea Cam1, EH domain protein I, Lano Adapter protein, galectin-4. Only in NHERF2. NHERF1, NHERF3, PAPYF5, galectin 3, coronin 3, transmembrane protein 39, tetratricopeptide 21B . Down Regulated in NHERF 2 -/-: Transport proteins: Changes also in NHERF1 Null. Transmembrane channel like protein 5, choline transporter (SLC44a1), hephaestin precursor, cystein rich protein 1. Signaling Molecules: Changes also in NHERF1 null. intestinal alk phos precur, Enigma like protein (PDZ domain and LIM domain containing protein 5), Xaa-Pro aminopeptidase 1, B-catenin, E-cadherin, p120. Confirmed by IB: Increased, NHERF1, NHERF3, coronin 3, Cea Cam 1. Decreased, B-catenin, E-cadherin, p120. Confirmed by IF. B-catenin, E-cadherin, p120. Conclusions: The proteome of the NHERF2 null mouse small intestinal BB demonstrates up and downregulation of multiple transport proteins, cytoskeletal proteins and signaling molecules suggesting involvement of NHERF2 in multiple apical regulatory processes and interactions with luminal contents.
T1865 Differential Involvement of SLC26A3 (DRA), CFTR, and Other HCO3Transporters in Murine Ileal, Proximal and Distal Colonic HCO3- Secretion Fang Xiao, Anurag K. Singh, Mingmin Chen, Junhua Li, Brigitte Riederer, Janina Bonhagen, Marina Juri, Oliver Bachmann, Manoocher Soleimani, Ursula Seidler Backround: Intestinal HCO3- secretion is necessary for mucin and paneth cell secretion, mucosal protection and repair, and the HCO3- mediated-luminal alkalinisation in the distal small intestine is used for the pH-dependent release of drugs. We therefore had previously studied chronic inflammation-associated changes in intestinal HCO3- secretion in a TNF-α overex-pressing mouse model, and had observed dramatic, but completely different changes in the HCO3- secretory rate and response to stimulation in the inflamed ileum and colon, along with a decrease in Slc26a3 (DRA) mRNA and increase in CFTR mRNA expression. Aim and methods: In order to understand these complex inflammation-associated disturbances, we studied basal and FSK-stimulated HCO3- secretion in isolated distal ileal, proximal and distal colonic mucosa of mice deficient for the Cl-/HCO3- exchanger Slc26a3 and the anion channel CFTR, before and after luminal Cl- substitution. The obtained results were compared with the results of identical experiments performed in the in-flamed distal ileal, proximal and distal colonic mucosa of TNFΔare +/- and noninflamed WT littermates. Results: Ileal and distal colonic mucosa displayed high basal HCO3- secretory rates, which were dramatically decreased in the absence of Slc26a3 or removal of luminal Cl-. FSK induced an identical increase in HCO3- secretion both in Slc26a3-deficient and WT mucosa, which was completely CFTR-dependent in the ileum but not in the distal colon. In the proximal colon, basal HCO3- secretion was markedly lower than in the ileum or distal co-lon, neither reduced in Slc26a3 nor in CFTR-deficient mucosa, but briskly stimulated by FSK in a Slc26a3- as well as CFTR inde-pendent fashion. Chronic inflammation abolished the Cl-dependent, Slc26a3-mediated HCO3- secretion and interfered with FSK-stimulated, CFTRdependent HCO3- secretion, but stimulated the colonic CFTR-independent HCO3- secretory response. Conclusions: In the murine distal ileum and distal colon, the high basal HCO3secretory rates are largely Slc26a3-mediated, whereas the low basal rates are Cl--independent and not Slc26a3-mediated in the proximal colon. FSK elicits a CFTR-dependent HCO3secretory re-sponse in the ileum and a CFTR-independent one in the colon. Chronic inflamma-tion dramatically decreases Slc26a3-mediated as well as CFTR-dependent HCO3secretion, but but enhances the colonic CFTR-independent HCO3- secretory response to FSK. These data help understand inflammation-associated disturbances in intestinal barrier function and explain why pHi-dependent drug release is often defective in the inflamed gut.
T1868 The Flavanone Naringenin Inhibits Epithelial Chloride Secretion in Rat and Human Colon Danielle Collins, Aisling Hogan, Sascha Kopic, Mohammad Reza Boroumand, Alan W. Baird, John P. Geibel, Des Winter Naringenin (4',5,7-trihydroxyflavanone) is a naturally derived flavanone found predominantly in citrus fruits. Interest into the therapeutic potential of bioflavanoids is growing as epidemiological evidence demonstrates beneficial effects of these compounds in colorectal cancer, mutagenesis and inflammation. The aim of this study was to investigate effects of naringenin on colonic ion transport. Human mucosal epithelium from colectomy specimens was mounted in Ussing chambers. Cl- secretion was quantified as changes in short circuit current (ΔIsc) in microamperes per cm2. Rat colonic crypts were isolated by calcium chelation, and intracellular pH (pHi) and intracellular chloride (Cl-i) were measured by BCECF and MQAE fluorescence respectively. Naringenin (100μM) induced a cytosolic acidification of rat colonic crypts (ΔpHi/min = 0.05±0.004). This acidification did not occur following blockade of Na+/K+/2Cl- cotransporter (NKCC) with 100 μM bumetanide or in the absence of K+ in the perfusing solution. In addition, inhibition of protein kinase A with 10μM H-89 prevented the naringenin induced acidification. In MQAE loaded crypts, naringenin inhibited forskolin stimulated chloride efflux (control: 13.7±1.3 AFU/min vs. 2.5±1.1 AFU/min, P=0.008). In both rat and human colon in ussing chambers, naringenin caused a concentration dependent decrease in chloride secretion with an IC50 of 0.4mM and 0.9mM respectively, n=6) The flavanone naringenin has antisecretory effects in both rat and human colon and may be a potential treatment for diarrhoeal disorders.
T1866 Lactose Malabsorption During Methotrexate-Induced Gastrointestinal Mucositis in a Rat Model Margot Fijlstra, Edmond H. Rings, Julius F. Baller, Theo H. van Dijk, Theo S. Boer, Frans Stellaard, Hilda Van der Molen, Henkjan J. Verkade, Wim Tissing Background: Gastrointestinal mucositis is a severe side effect of chemotherapy, leading to an increased morbidity and mortality. Mucositis probably causes nutrient malabsorption because of villus atrophy and epithelial damage. Ultimately, we want to define the optimal nutrient composition for patients with chemotherapy-induced mucositis. Therefore, we have chosen to assess the intestinal capacity to absorb nutrients during methotrexate (MTX)induced mucositits in a rat model. In the present study, we concentrated on the absorption of carbohydrates because of its major role in dietary energy supply in Western diets. Aim: To assess the intestinal capacity to absorb lactose and glucose during MTX-induced mucositis in a rat model. Methods: At day 0, rats were i.v. injected with MTX (60 mg/kg, n=14) or with NaCl (controls, n=7). At day 4, carbohydrate absorption was assessed by stable isotope methodology. To differentiate between absorption of the disaccharide lactose and the monosaccharide glucose, 1-13C-lactose and U-13C-glucose were intragastrically administered (200 mg/kg and 100 mg/kg respectively). Blood samples were frequently collected for 3 hours after administration to determine plasma appearance of glucose derived from 1-13C-lactose and of U-13C-glucose. Subsequently, rats were terminated and jejunal samples were collected for assessing histology, lactase-phlorizin hydrolase (LPH) activity and mRNA expression. Results: MTX induced mucositis in rats, varying from rather mild to severe. Rats with severe mucositis (n=7) showed severe villus atrophy (villus length <55% compared with controls) and epithelial damage. In these rats, total plasma appearance of glucose derived from 113 C-lactose was severely reduced, compared with controls (-91%, p<0.01). In MTX treated rats, total plasma appearance of glucose derived from 1-13C-lactose correlated strongly with the severity of mucositis as measured by villus length (r=0.86, p<0.01). Interestingly, severe mucositis did not reduce total plasma appearance of U-13C-glucose, compared with controls. In line with lactose absorption data, jejunal activity and mRNA expression of LPH were markedly reduced in rats with severe mucositis, compared with controls (-97% and -99%
T1869 Bile Acid Uptake Capacity in Ileal Mucosa Correlates With the Expression of Apical but Not Intracellular Bile Acid Transporter Antal Bajor, Anita Fae, Anders Kilander, Lena Ohman, David Pazooki, Henrik Sjovall, Kjell-Arne Ung, Olof Jonsson Bakground:The apical sodium dependent bile acid transporter (ASBT) is responsible for bile acid (BA) uptake in the distal ileum. High intracellular BA-s are potentially toxic and therefore have to be shuttled through the enterocyte by an intracytoplasmatic transporter, the ileal bile acid binding protein (IBABP). Aims: The aim of the current study was to test if increased expression of ASBT is associated with increased expression of IBABP and also to correlate these two transporters to the mucosal BA uptake capacity In Vitro. Methods: Multiple biopsies were taken during open surgery from patients with normal bowel function operated for continent urinary diversion with an ileal reservoir. The expression of ASBT and IBABP was assayed with western blot analyses and the runs were also normalized for the expression of villin. Maximal BA uptake capacity was assessed in ileal biopsies using a
S-595
AGA Abstracts
AGA Abstracts
transport experiments. L-NG-Nitroarginine methyl ester (L-NAME, 1 mM) was used to inhibit cNO in cells. IEC18 cells were transfected with SGLT-1 siRNA along with a siRNA control. Rat specific NHE3 antibodies were used for Western blot studies. Results: L-NAME treatment of IEC-18 increased NHE3 activity as did SGLT-1 siRNA transfection compared to control cells (control 992.6±116 pmol/mg protein; +L-NAME 2432±402; +SGLT-1siRNA 1958±213; +L-NAME+SGLT-1siRNA 5279±650; n=5, p<0.05). Kinetic studies showed that NHE3 was stimulated by SGLT-1 siRNA transfection as well as L-NAME treatment by an increase in the maximal rate of uptake (Vmax) without a change in the affinity of NHE3 for Na (Km). Kinetic studies also showed that in SGLT siRNA transfected cells L-NAME treatment stimulated NHE3 by increasing Vmax. Western blot studies demonstrated increased NHE3 protein expression on the IEC-18 cell BBM in L-NAME treated cells and SGLT-1 siRNA transfected cells. It also showed a larger increase in NHE3 protein expression in SGLT silenced cells treated with L-NAME. Conclusions: Decreasing cNO stimulates BBM NHE3 secondary to an increase in the number of exchangers rather than a change in the affinity for Na. Silencing SGLT-1 also stimulates BBM NHE3 secondary to an increase in the number of exchangers rather than a change in the affinity. When both cNO is inhibited and SGLT-1 is silenced BBM NHE3 is stimulated additively. Thus, this data indicates that cNO is likely involved in the regulation of BBM NHE3 by SGLT-1 in intestinal epithelial cells.
T1865 Differential Involvement of SLC26A3 (DRA), CFTR, and Other HCO3Transporters in Murine Ileal, Proximal and Distal Colonic HCO3- Secretion Fang Xiao, Anurag K. Singh, Mingmin Chen, Junhua Li, Brigitte Riederer, Janina Bonhagen, Marina Juri, Oliver Bachmann, Manoocher Soleimani, Ursula Seidler Backround: Intestinal HCO3- secretion is necessary for mucin and paneth cell secretion, mucosal protection and repair, and the HCO3- mediated-luminal alkalinisation in the distal small intestine is used for the pH-dependent release of drugs. We therefore had previously studied chronic inflammation-associated changes in intestinal HCO3- secretion in a TNF-α overex-pressing mouse model, and had observed dramatic, but completely different changes in the HCO3- secretory rate and response to stimulation in the inflamed ileum and colon, along with a decrease in Slc26a3 (DRA) mRNA and increase in CFTR mRNA expression. Aim and methods: In order to understand these complex inflammation-associated disturbances, we studied basal and FSK-stimulated HCO3- secretion in isolated distal ileal, proximal and distal colonic mucosa of mice deficient for the Cl-/HCO3- exchanger Slc26a3 and the anion channel CFTR, before and after luminal Cl- substitution. The obtained results were compared with the results of identical experiments performed in the in-flamed distal ileal, proximal and distal colonic mucosa of TNFΔare +/- and noninflamed WT littermates. Results: Ileal and distal colonic mucosa displayed high basal HCO3- secretory rates, which were dramatically decreased in the absence of Slc26a3 or removal of luminal Cl-. FSK induced an identical increase in HCO3- secretion both in Slc26a3-deficient and WT mucosa, which was completely CFTR-dependent in the ileum but not in the distal colon. In the proximal colon, basal HCO3- secretion was markedly lower than in the ileum or distal co-lon, neither reduced in Slc26a3 nor in CFTR-deficient mucosa, but briskly stimulated by FSK in a Slc26a3- as well as CFTR inde-pendent fashion. Chronic inflammation abolished the Cl-dependent, Slc26a3-mediated HCO3- secretion and interfered with FSK-stimulated, CFTRdependent HCO3- secretion, but stimulated the colonic CFTR-independent HCO3- secretory response. Conclusions: In the murine distal ileum and distal colon, the high basal HCO3secretory rates are largely Slc26a3-mediated, whereas the low basal rates are Cl--independent and not Slc26a3-mediated in the proximal colon. FSK elicits a CFTR-dependent HCO3secretory re-sponse in the ileum and a CFTR-independent one in the colon. Chronic inflamma-tion dramatically decreases Slc26a3-mediated as well as CFTR-dependent HCO3secretion, but but enhances the colonic CFTR-independent HCO3- secretory response to FSK. These data help understand inflammation-associated disturbances in intestinal barrier function and explain why pHi-dependent drug release is often defective in the inflamed gut.
T1868 The Flavanone Naringenin Inhibits Epithelial Chloride Secretion in Rat and Human Colon Danielle Collins, Aisling Hogan, Sascha Kopic, Mohammad Reza Boroumand, Alan W. Baird, John P. Geibel, Des Winter Naringenin (4',5,7-trihydroxyflavanone) is a naturally derived flavanone found predominantly in citrus fruits. Interest into the therapeutic potential of bioflavanoids is growing as epidemiological evidence demonstrates beneficial effects of these compounds in colorectal cancer, mutagenesis and inflammation. The aim of this study was to investigate effects of naringenin on colonic ion transport. Human mucosal epithelium from colectomy specimens was mounted in Ussing chambers. Cl- secretion was quantified as changes in short circuit current (ΔIsc) in microamperes per cm2. Rat colonic crypts were isolated by calcium chelation, and intracellular pH (pHi) and intracellular chloride (Cl-i) were measured by BCECF and MQAE fluorescence respectively. Naringenin (100μM) induced a cytosolic acidification of rat colonic crypts (ΔpHi/min = 0.05±0.004). This acidification did not occur following blockade of Na+/K+/2Cl- cotransporter (NKCC) with 100 μM bumetanide or in the absence of K+ in the perfusing solution. In addition, inhibition of protein kinase A with 10μM H-89 prevented the naringenin induced acidification. In MQAE loaded crypts, naringenin inhibited forskolin stimulated chloride efflux (control: 13.7±1.3 AFU/min vs. 2.5±1.1 AFU/min, P=0.008). In both rat and human colon in ussing chambers, naringenin caused a concentration dependent decrease in chloride secretion with an IC50 of 0.4mM and 0.9mM respectively, n=6) The flavanone naringenin has antisecretory effects in both rat and human colon and may be a potential treatment for diarrhoeal disorders.
T1866 Lactose Malabsorption During Methotrexate-Induced Gastrointestinal Mucositis in a Rat Model Margot Fijlstra, Edmond H. Rings, Julius F. Baller, Theo H. van Dijk, Theo S. Boer, Frans Stellaard, Hilda Van der Molen, Henkjan J. Verkade, Wim Tissing Background: Gastrointestinal mucositis is a severe side effect of chemotherapy, leading to an increased morbidity and mortality. Mucositis probably causes nutrient malabsorption because of villus atrophy and epithelial damage. Ultimately, we want to define the optimal nutrient composition for patients with chemotherapy-induced mucositis. Therefore, we have chosen to assess the intestinal capacity to absorb nutrients during methotrexate (MTX)induced mucositits in a rat model. In the present study, we concentrated on the absorption of carbohydrates because of its major role in dietary energy supply in Western diets. Aim: To assess the intestinal capacity to absorb lactose and glucose during MTX-induced mucositis in a rat model. Methods: At day 0, rats were i.v. injected with MTX (60 mg/kg, n=14) or with NaCl (controls, n=7). At day 4, carbohydrate absorption was assessed by stable isotope methodology. To differentiate between absorption of the disaccharide lactose and the monosaccharide glucose, 1-13C-lactose and U-13C-glucose were intragastrically administered (200 mg/kg and 100 mg/kg respectively). Blood samples were frequently collected for 3 hours after administration to determine plasma appearance of glucose derived from 1-13C-lactose and of U-13C-glucose. Subsequently, rats were terminated and jejunal samples were collected for assessing histology, lactase-phlorizin hydrolase (LPH) activity and mRNA expression. Results: MTX induced mucositis in rats, varying from rather mild to severe. Rats with severe mucositis (n=7) showed severe villus atrophy (villus length <55% compared with controls) and epithelial damage. In these rats, total plasma appearance of glucose derived from 113 C-lactose was severely reduced, compared with controls (-91%, p<0.01). In MTX treated rats, total plasma appearance of glucose derived from 1-13C-lactose correlated strongly with the severity of mucositis as measured by villus length (r=0.86, p<0.01). Interestingly, severe mucositis did not reduce total plasma appearance of U-13C-glucose, compared with controls. In line with lactose absorption data, jejunal activity and mRNA expression of LPH were markedly reduced in rats with severe mucositis, compared with controls (-97% and -99%
T1869 Bile Acid Uptake Capacity in Ileal Mucosa Correlates With the Expression of Apical but Not Intracellular Bile Acid Transporter Antal Bajor, Anita Fae, Anders Kilander, Lena Ohman, David Pazooki, Henrik Sjovall, Kjell-Arne Ung, Olof Jonsson Bakground:The apical sodium dependent bile acid transporter (ASBT) is responsible for bile acid (BA) uptake in the distal ileum. High intracellular BA-s are potentially toxic and therefore have to be shuttled through the enterocyte by an intracytoplasmatic transporter, the ileal bile acid binding protein (IBABP). Aims: The aim of the current study was to test if increased expression of ASBT is associated with increased expression of IBABP and also to correlate these two transporters to the mucosal BA uptake capacity In Vitro. Methods: Multiple biopsies were taken during open surgery from patients with normal bowel function operated for continent urinary diversion with an ileal reservoir. The expression of ASBT and IBABP was assayed with western blot analyses and the runs were also normalized for the expression of villin. Maximal BA uptake capacity was assessed in ileal biopsies using a
S-595
AGA Abstracts
AGA Abstracts
transport experiments. L-NG-Nitroarginine methyl ester (L-NAME, 1 mM) was used to inhibit cNO in cells. IEC18 cells were transfected with SGLT-1 siRNA along with a siRNA control. Rat specific NHE3 antibodies were used for Western blot studies. Results: L-NAME treatment of IEC-18 increased NHE3 activity as did SGLT-1 siRNA transfection compared to control cells (control 992.6±116 pmol/mg protein; +L-NAME 2432±402; +SGLT-1siRNA 1958±213; +L-NAME+SGLT-1siRNA 5279±650; n=5, p<0.05). Kinetic studies showed that NHE3 was stimulated by SGLT-1 siRNA transfection as well as L-NAME treatment by an increase in the maximal rate of uptake (Vmax) without a change in the affinity of NHE3 for Na (Km). Kinetic studies also showed that in SGLT siRNA transfected cells L-NAME treatment stimulated NHE3 by increasing Vmax. Western blot studies demonstrated increased NHE3 protein expression on the IEC-18 cell BBM in L-NAME treated cells and SGLT-1 siRNA transfected cells. It also showed a larger increase in NHE3 protein expression in SGLT silenced cells treated with L-NAME. Conclusions: Decreasing cNO stimulates BBM NHE3 secondary to an increase in the number of exchangers rather than a change in the affinity for Na. Silencing SGLT-1 also stimulates BBM NHE3 secondary to an increase in the number of exchangers rather than a change in the affinity. When both cNO is inhibited and SGLT-1 is silenced BBM NHE3 is stimulated additively. Thus, this data indicates that cNO is likely involved in the regulation of BBM NHE3 by SGLT-1 in intestinal epithelial cells.