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Epidermal (EGF) and hepatocyte (HGF) growth factors stimulate chemotaxis in an intestinal epithelial cell line
A902 AGAABSTRACTS • G3697 MOLECULAR CHARACTERIZATION OF THE HUMAN FECAL FLORA USING rRNA-TARGETED HYBRIDIZATION PROBES. P. Pochart, G. Gramet, I. Goderel, J.Dor~. INRA, Jouy-en-Josas ; INSERM U290, St. Lazare Hospital, and CNAM, Paris, France. Culture-based anaerobic techniques of microbial identification and enumeration are central to studies of the human fecal flora. Nevertheless, they are time-consuming, require fresh samples and are not suited to large epidemiological investigations. Moreover, comparison of culture- and microscopic-counts indicate that a significant fraction of the human fecal flora is not yet culturable. Molecular techniques should allow more accurate characterizations of extensive numbers of frozen fecal samples. The ai___m_mof this study was to assess rRNA-based quantitative dot-blot hybridization for the characterization of Bacteroides, Bifidobacterium, and Escherichia. coli populations of the human fecal flora in comparison with cultural counts. Methods: Total anaerobes, Bacteroides, Bifidobacterium and E. coli populations were enumerated from fresh fecal samples obtained from 10 healthy volunteers. Total RNA was extracted from the same fecal samples after storage at -20°C; hybridized with specific 32p-labeled oligonucleotide probes, and quantified by radio-imaging. Results are expressed as percent of total viable anaerobes for culture-counts, and as percent of total bacterial rRNA for dot-blot quantification. They are given as mean ± SE. Results: CoUntsof total viable anaerobes were 7.9 109 cellsdg feces (wet weight). Total Bacteroides represented 59.9± 16.7 % of total viable anaerobes (3.4 109/g) and 35.5 ± 4.5 % of bacterial rRNA, Bifidobacterium represented 10.6 ± 3.3 % of total viable anaerobes (7.4 10S/g) and 4.1 ± 0.7 % of bacterial rRNA, and Escherichia coli 5.4 ± 3.5 % of total viable anaerobes (1.4 10S/g) and 2.7 ± 0.7 % of bacterial rRNA. Conclusion: Relative rRNA proportions for the different bacterial groups were in good aggreement with accepted levels based on cultural counts. The two-fold higher proportion of all bacterial groups analyzed by culture compared to quantitative dot-blot was likely due to a systematic underestimation of counts of total viable anaerobes. The present work demonstrates that the rRNA approach bears great potential for the analysis of the human fecal flora and circumvents the limitations of culture-based techniques. • G3698
EFFECT OF ELEMENTAL DIETS ± ADDITIONAL GLUTAMINE ON SMALL INTESTINAL MORPHOLOGY AND RESISTANCE TO INDOMETHACIN-INDUCED INJURY. T Podas l, M Khatri l, CE MacDonald 1, W Johnson2, RA Goodlad 3, RJ PlayfordL University Division Of Gastroenterology, Leicester General Hospital, Gwendolen Rd. Leicester l, Scientific Hospital Supplies, Liverpool 2, Imperial Cancer Research Fund, London3, UK. INTRODUCTION: The effects of elemental diets +/- glutamine supplementation on gut growth and resistance to injury are poorly understood. We therefore examined the effects of a standard elemental diet (E028), and an isocaloric glutamine-supplemented variant (containing 4% glutamine) on small bowel morphometry and resistance to NSAID injury. METHODS: Three groups of mice (N=20 per group) received standard chow diet (C), standard elemental diet (E) or the glutamine supplemented variant (E+G) for 2 weeks. 24 h prior to killing half the animals also received indomethacin 85mgs/kg sc. To assess changes in proliferation, all animals received vincristine 2h prior to killing. Morphometry was assessed using microdissected villi and crypts. RESULTS: Small intestine: Animals not given indomethacin showed no differences in small intestinal length or weight between diets. However, villus height in E or E+G groups had 20% longer villi than group C and had a 10% reduction in 2h-metaphase accumulation throughout the SI (all p < 0.01). Indomethacin caused a 10% reduction in villus height in group C, and a 20% reduction in group E+G so that no significant difference in height was seen between C and E+G after indomethacin. However, indomethacin only caused a 10% reduction in villus height of group E, so that the villus height in animals in E group treated with indomethacin was still greater than those seen in C fed group not given indomethacin (p < 0.01). Colon: E and E+G groups had about a 25% reduction in colonic length and wt compared to C (all p< 0.01). There was no significant difference in metaphase accumulation between dietary groups and indomethacin did not affect any of these parameters. CONCLUSION: A standard elemental diet preserved small intestinal, but not colonic morphology and reduced the damaging effect of indomethacin. Addition of glutamine was counterproductive. Elemental diets may provide a useful tool to study the mechanisms underlying NSAID-induced gut injury. • G3699 EPIDERMAL (EGF) AND HEPATOCYTE (HGF) GROWTH FACTORS STIMULATE CHEMOTAXIS IN AN INTESTINAL EPITHELIAL CELL LINE. D.B. Polk and W. Tong. Department of Pediatrics, Division of Gastroenterology & Nutrition, Vanderbilt University School of Medicine, Nashville, TN. Directed migration of epithelial cells plays a significant role in growth and development. Chemotactic movement of cells towards a growth factor
GASTROENTEROLOGYVol. 114, No. 4 concentration gradient may provide one means for directed migration of epithelial cells along the crypt/villus axis. The purpose of this study was to determine growth factor-mediated chemotaxis of an intestinal epithelial cell line (MSIE). Cells cultured under non-transforming conditions were studied for migration across an 8 pM pore polycarbonate membrane in a modified Boyden chamber, by direct counting & optical density, toward various concentrations (1-I00 ng/ml) of EGF, transforming growth factor (TGF) 131, HGF, keratinocyte growth factor (KGF), fibroblast growth factor (FGF) and insulin like growth factor (IGF)-I for various periods of time (1-24 h). The migration across membranes coated with mouse laminin, fibronectin, collagen IV and rat tail collagen 1 were compared. Chemotaxis required matrix on both sides of the membrane suggesting that cellular adhesion was necessary prior to migration. Of the matrices studied, chemotaxis was 2-fold greater on fibronectin and least on rat collagen. EGF and HGF enhanced chemotaxis by up to 30-fold within 2h, while a modest increase in chemotaxis occurred with TGF131 after 18h. No chemotaxis was seen for KGF, FGF nor IGF-1. Effects on cell proliferation or on activation of Erkl/Erk2 mitogen activated protein (MAP) kinases were shown for all growth factors studied demonstrating receptor-responsive signaling for each. Co-culture with inhibitors of EGF receptor tyrosine kinase activity (AG1478, PD15305) blocked EGF-, but not HGF- nor TGF131-stimulated chemotaxis. The tyrosine kinase inhibitor genistein inhibited chemotaxis toward all 3 growth factors. The PLC inhibitors (U73122 & L108) inhibited EGF- and HGF-, but not TGF131stimulated chemotaxis while neither protein kinase C nor phosphatidylinositol 3-kinase inhibitors had an effects on chemotaxis. The MAP kinase inhibitor (PD98059) completely blocked activation of Erkl/Erk2 by EGF & HGF but had no inhibitory effect on their chemotactic activities. These findings demonstrate that EGF & HGF uniquely stimulate intestinal cell chemotaxis by a mechanism requiring tyrosine kinase and PLC, but not PKC, PI 3-kinase nor MAP kinase activities, with implications for regulation of epithelial cell migration in growth, development and differentiation. • G3700 EPIDERMAL GROWTH FACTOR (EGF) STIMULATES PROTEIN KINASE C~ IN AN INTESTINAL EPITHELIAL CELL LINE. D.B. Polk, M.R. Frey and J.D. Black. Department of Pediatrics, Division of Gastroenterology & Nutrition, Vanderbilt University School of Medicine, Nashville, TN 37232 and Department of Experimental Therapeutics, Roswell Park Cancer Institute, Buffalo, NY. We have recently shown that EGF-stimulated intestinal epithelial cell migration in a wound-healing model requires both phosphatidylinositol phospholipase C (PLC) and PKC activities, and that PKC activation by phorbol ester stimulates migration in the presence of PLC inhibition. The purpose of these studies was to determine EGF regulation of PKC isozymes in the same intestinal cell line. The expression and activation of PKC isozymes was determined in EGF-treated (100 ng/ml) MSIE cells, under nontransforming conditions, in the presence of various inhibitors or agonists by cell fractionation and/or Western blot analysis. Cells were cultured at 37°C for 24 h in media supplemented with 0.5% serum prior to study. Cell lysates were prepared by scraping into a digitonin buffer and centrifugation at 275,000 x g for 12 min with the supernatant designated cytosolic fraction. The pellet was resuspended into buffer containing 1% Triton X-100 and centrifuged at 16,000 x g for 30 min with the supernatant designated Tritonsoluble membrane and the pellet resuspended in 10 M urea was designated as cytoskeletal/nuclear fraction. The cells express the PKC isozymes ct, 1311,c, & t by Western blotting. Phorbol ester (10 ng/ml phorbol 12-myristate 13-acetate) treatment of cells stimulated membrane translocation (activation) of PKCs a, 1311& e, but not ~ or t, while EGF however, only stimulated activation of PKCe. A slower migrating PKC~band appeared in the membrane fraction on Western blot analysis with either EGF or phorbol ester treatment. This band likely represents activationinduced autophosphorylation of PKCe. Pretreatment of ceils for 45 min with inhibitors of EGF receptor tyrosine kinase activity (AG1478 or genistein) or PLC activity (1 pM U73122), but not phosphatidylinositol 3-kinase activity (10 nM wortmannin), blocked activation and the appearance of the slower migrating form of PKC~ by EGF. These data suggest that PKCe is a downstream target of EGF-stimulated PLC activity and may therefore play a regulatory role in EGF-stimulated cell migration. • G3701 NEURONAL NITRIC OXIDE SYNTItASE EXPRESSION DECLINES WITH AGE IN MURNE SMALL INTESTINE. A.R. Powell and R.R, Reddix, Dept. of Pharmacology and Experimental Therapeutics, LSU Medical Center, New Orleans, LA 70112. In the gastrointestinal tract, constitutive nitric oxide production plays an important role in the regulation of motility, blood flow and ion transport. An age-related decline in constitutive endothelial (eNOS) and neuronal nitric oxide synthase (nNOS) expression and activity in both central and peripheral neural pathways have been reported in various species. AIM: To determine whether enteric expression of neuronal nitric oxide synthase is altered with age in murine small intestine. METHODS: Whole thickness segments of small intestine and whole brain were isolated from young (3 month) and old (24 month) male C57BL/6J mice.
EFFECT OF ELEMENTAL DIETS ± ADDITIONAL GLUTAMINE ON SMALL INTESTINAL MORPHOLOGY AND RESISTANCE TO INDOMETHACIN-INDUCED INJURY. T Podas l, M Khatri l, CE MacDonald 1, W Johnson2, RA Goodlad 3, RJ PlayfordL University Division Of Gastroenterology, Leicester General Hospital, Gwendolen Rd. Leicester l, Scientific Hospital Supplies, Liverpool 2, Imperial Cancer Research Fund, London3, UK. INTRODUCTION: The effects of elemental diets +/- glutamine supplementation on gut growth and resistance to injury are poorly understood. We therefore examined the effects of a standard elemental diet (E028), and an isocaloric glutamine-supplemented variant (containing 4% glutamine) on small bowel morphometry and resistance to NSAID injury. METHODS: Three groups of mice (N=20 per group) received standard chow diet (C), standard elemental diet (E) or the glutamine supplemented variant (E+G) for 2 weeks. 24 h prior to killing half the animals also received indomethacin 85mgs/kg sc. To assess changes in proliferation, all animals received vincristine 2h prior to killing. Morphometry was assessed using microdissected villi and crypts. RESULTS: Small intestine: Animals not given indomethacin showed no differences in small intestinal length or weight between diets. However, villus height in E or E+G groups had 20% longer villi than group C and had a 10% reduction in 2h-metaphase accumulation throughout the SI (all p < 0.01). Indomethacin caused a 10% reduction in villus height in group C, and a 20% reduction in group E+G so that no significant difference in height was seen between C and E+G after indomethacin. However, indomethacin only caused a 10% reduction in villus height of group E, so that the villus height in animals in E group treated with indomethacin was still greater than those seen in C fed group not given indomethacin (p < 0.01). Colon: E and E+G groups had about a 25% reduction in colonic length and wt compared to C (all p< 0.01). There was no significant difference in metaphase accumulation between dietary groups and indomethacin did not affect any of these parameters. CONCLUSION: A standard elemental diet preserved small intestinal, but not colonic morphology and reduced the damaging effect of indomethacin. Addition of glutamine was counterproductive. Elemental diets may provide a useful tool to study the mechanisms underlying NSAID-induced gut injury. • G3699 EPIDERMAL (EGF) AND HEPATOCYTE (HGF) GROWTH FACTORS STIMULATE CHEMOTAXIS IN AN INTESTINAL EPITHELIAL CELL LINE. D.B. Polk and W. Tong. Department of Pediatrics, Division of Gastroenterology & Nutrition, Vanderbilt University School of Medicine, Nashville, TN. Directed migration of epithelial cells plays a significant role in growth and development. Chemotactic movement of cells towards a growth factor
GASTROENTEROLOGYVol. 114, No. 4 concentration gradient may provide one means for directed migration of epithelial cells along the crypt/villus axis. The purpose of this study was to determine growth factor-mediated chemotaxis of an intestinal epithelial cell line (MSIE). Cells cultured under non-transforming conditions were studied for migration across an 8 pM pore polycarbonate membrane in a modified Boyden chamber, by direct counting & optical density, toward various concentrations (1-I00 ng/ml) of EGF, transforming growth factor (TGF) 131, HGF, keratinocyte growth factor (KGF), fibroblast growth factor (FGF) and insulin like growth factor (IGF)-I for various periods of time (1-24 h). The migration across membranes coated with mouse laminin, fibronectin, collagen IV and rat tail collagen 1 were compared. Chemotaxis required matrix on both sides of the membrane suggesting that cellular adhesion was necessary prior to migration. Of the matrices studied, chemotaxis was 2-fold greater on fibronectin and least on rat collagen. EGF and HGF enhanced chemotaxis by up to 30-fold within 2h, while a modest increase in chemotaxis occurred with TGF131 after 18h. No chemotaxis was seen for KGF, FGF nor IGF-1. Effects on cell proliferation or on activation of Erkl/Erk2 mitogen activated protein (MAP) kinases were shown for all growth factors studied demonstrating receptor-responsive signaling for each. Co-culture with inhibitors of EGF receptor tyrosine kinase activity (AG1478, PD15305) blocked EGF-, but not HGF- nor TGF131-stimulated chemotaxis. The tyrosine kinase inhibitor genistein inhibited chemotaxis toward all 3 growth factors. The PLC inhibitors (U73122 & L108) inhibited EGF- and HGF-, but not TGF131stimulated chemotaxis while neither protein kinase C nor phosphatidylinositol 3-kinase inhibitors had an effects on chemotaxis. The MAP kinase inhibitor (PD98059) completely blocked activation of Erkl/Erk2 by EGF & HGF but had no inhibitory effect on their chemotactic activities. These findings demonstrate that EGF & HGF uniquely stimulate intestinal cell chemotaxis by a mechanism requiring tyrosine kinase and PLC, but not PKC, PI 3-kinase nor MAP kinase activities, with implications for regulation of epithelial cell migration in growth, development and differentiation. • G3700 EPIDERMAL GROWTH FACTOR (EGF) STIMULATES PROTEIN KINASE C~ IN AN INTESTINAL EPITHELIAL CELL LINE. D.B. Polk, M.R. Frey and J.D. Black. Department of Pediatrics, Division of Gastroenterology & Nutrition, Vanderbilt University School of Medicine, Nashville, TN 37232 and Department of Experimental Therapeutics, Roswell Park Cancer Institute, Buffalo, NY. We have recently shown that EGF-stimulated intestinal epithelial cell migration in a wound-healing model requires both phosphatidylinositol phospholipase C (PLC) and PKC activities, and that PKC activation by phorbol ester stimulates migration in the presence of PLC inhibition. The purpose of these studies was to determine EGF regulation of PKC isozymes in the same intestinal cell line. The expression and activation of PKC isozymes was determined in EGF-treated (100 ng/ml) MSIE cells, under nontransforming conditions, in the presence of various inhibitors or agonists by cell fractionation and/or Western blot analysis. Cells were cultured at 37°C for 24 h in media supplemented with 0.5% serum prior to study. Cell lysates were prepared by scraping into a digitonin buffer and centrifugation at 275,000 x g for 12 min with the supernatant designated cytosolic fraction. The pellet was resuspended into buffer containing 1% Triton X-100 and centrifuged at 16,000 x g for 30 min with the supernatant designated Tritonsoluble membrane and the pellet resuspended in 10 M urea was designated as cytoskeletal/nuclear fraction. The cells express the PKC isozymes ct, 1311,c, & t by Western blotting. Phorbol ester (10 ng/ml phorbol 12-myristate 13-acetate) treatment of cells stimulated membrane translocation (activation) of PKCs a, 1311& e, but not ~ or t, while EGF however, only stimulated activation of PKCe. A slower migrating PKC~band appeared in the membrane fraction on Western blot analysis with either EGF or phorbol ester treatment. This band likely represents activationinduced autophosphorylation of PKCe. Pretreatment of ceils for 45 min with inhibitors of EGF receptor tyrosine kinase activity (AG1478 or genistein) or PLC activity (1 pM U73122), but not phosphatidylinositol 3-kinase activity (10 nM wortmannin), blocked activation and the appearance of the slower migrating form of PKC~ by EGF. These data suggest that PKCe is a downstream target of EGF-stimulated PLC activity and may therefore play a regulatory role in EGF-stimulated cell migration. • G3701 NEURONAL NITRIC OXIDE SYNTItASE EXPRESSION DECLINES WITH AGE IN MURNE SMALL INTESTINE. A.R. Powell and R.R, Reddix, Dept. of Pharmacology and Experimental Therapeutics, LSU Medical Center, New Orleans, LA 70112. In the gastrointestinal tract, constitutive nitric oxide production plays an important role in the regulation of motility, blood flow and ion transport. An age-related decline in constitutive endothelial (eNOS) and neuronal nitric oxide synthase (nNOS) expression and activity in both central and peripheral neural pathways have been reported in various species. AIM: To determine whether enteric expression of neuronal nitric oxide synthase is altered with age in murine small intestine. METHODS: Whole thickness segments of small intestine and whole brain were isolated from young (3 month) and old (24 month) male C57BL/6J mice.