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T1875 Increased Glial Glutamate Transporter Eaat2 Expression Reduces Visceral Pain Response in Mice
been identified along the gastrointestinal tract, including NHE4 protein in cultured human cells with colonic origin. We recently demonstrated NHE4 activity in the rat colon and its role in regulating pHi in response to an acid load. In this study we present functional NHE4 activity in human colon in response to an acid load. Methods: Colonic samples were obtained from patients undergoing elective colon resections using healthy margins of resected specimens. Colonic crypts were isolated from the basement membrane using ethylenediaminetetraacetic acid. Intracellular pH changes in the crypts were monitored using the pHsensitive dye BCECF and a high speed real time video imaging system. To induce an acid load, individual crypts were exposed to an NH4Cl pulse followed by Na+ removal. To isolate NHE4 activity, one series of experiments were performed in the presence of 7µM EIPA, an amiloride analogue known to inhibit NHE1, 2 and 3 at 7µM concentration but allow NHE4 to remain active. Results: An intracellular acid load was induced with NH4Cl and removal of sodium which resulted in a rapid decrease in pHi. When Na+ was added to the perfusate we observed a steep recovery to baseline pHi. When 7µM EIPA was added to all solutions to eliminate NHE1, 2, and 3 activity, a Na+-dependent recovery was observed that was indicative of NHE4. The rate of NHE4-dependent pHi recovery was significantly less when compared to control crypts. Conclusions: We have experimentally demonstrated a functional NHE4 activity in normal human colonic tissue. This novel exchanger is capable of modulating intracellular pH in response to an acid load, and over a wide pH spectrum demonstrating that this protein may play an important role in maintain cellular pH homeostasis.
T1872 Alterations in Transporters and Regulators At the Base of Mouse Colonic Crypts After Genetic Disruption of the NHE2 Isoform Na+/H+ Exchanger Yanfang Guan, Michael P. Humphreys, Marshall H. Montrose BACKGROUND: NHE2 is the main apical epithelial Na+/H+ exchanger (NHE) at the colonic crypt base, but NHE2-knockout (KO) mice demonstrate adaptive, compensatory upregulation of one or more transporters that replace NHE2 function, yet are distinct from NHE3 (the only other known NHE isoform expressed in the colonic apical membrane). AIM: Evaluate the altered gene profile of crypt base colonocytes in NHE2-KO, to identify candidate upregulated transporters and regulatory pathways mediating the adaptive response. METHODS: Crypt base epithelium from cell position 1-6 was obtained by laser capture microdissection (Arcturus Veritas). RNA was pooled from multiple crypts from the same animal and amplified. mRNA expression profiles were measured with mouse full genome microarrays (Affymetrix). Outcomes from male NHE2-KO and NHE2 heterozygotes (Het) mice (n=3 each) were compared using Ingenuity Systems and GeneSpring software. RESULTS: 377 differentially expressed genes (P<0.05) were identified as having a greater than 1.4 change between genotypes. In the NHE gene family, only NHE5 was differentially expressed, being increased in NHE2-KO. Among other solute carrier (SLC) transporter families, only Slc4a7 (Na:HCO3cotransporter) was increased; Slc43a2 (L-amino acid transporter) and Slc10a1 (Na:bile acid cotransporter) were decreased. Increased regulators included beta-arrestin2 (shown to affect NHE5 expression at cell surface). Ingenuity Systems software revealed the top clustering of genes in networks of tissue and cellular development, cell signaling and cell cycle plus cell growth/proliferation. Greater than 100 differentially expressed genes were involved in cell development or differentiation, including transcriptional regulators known to affect epithelial differentiation or development (e.g. wnt2a, myc, cebp and cdx2). Twenty-six genes were involved in cell signaling and cell death related to tissue development (e.g. caspase 8 and phospholipase C beta). CONCLUSIONS: NHE5 is the strongest candidate for an NHE isoform that adaptively replaces NHE2 in the NHE2-KO crypt base. Loss of NHE2 causes surprisingly broad changes in gene expression of epithelial cells at the crypt base, suggesting that this transporter is linked more tightly to the metabolic function of crypt epithelium than previously suspected. Supported by NIH RO1 DK45457 and Digestive Health Center grant.
T1875 Increased Glial Glutamate Transporter Eaat2 Expression Reduces Visceral Pain Response in Mice Robert L. Stephens, C. G. Lin, Y. Lin, J. T. Travers Irritable bowel syndrome (IBS) is the leading digestive disease diagnosis among gastroenterologists and affects 15-20% of the US population. A significant component of IBS is recurrent abdominal pain. Central sensitization mediated by glutamate NMDA receptor activation is implicated in visceral pain pathophysiology. The glial glutamate transporter EAAT2 is the main mediator of glutamate clearance to terminate glutamate-mediated responses. Transgenic mice over-expressing human EAAT2 (EAAT2 mice), which exhibited a 1.4-2.5 fold enhanced glutamate uptake, showed 30% less writhing response to ip acetic acid than nontransgenic littermates [writhes/20 min (n): nontransgenic mice- 23 + 2 (37); EAAT2 mice- 16 + 2 (34); p<0.05]. Moreover, EAAT2 mice showed a 34-63% reduction in visceromotor response (VMR) to colo-rectal distension when assessing the response to 15- 60 mmHg pressures. Corroborating the involvement of enhanced glutamate uptake, wild-type animals treated for 1 week with ceftriaxone (200mg/kg daily), a stimulator of EAAT2 expression, showed a 5285% reduction in VMR to graded increases in colo-rectal distension (Figure 1). The data suggest that enhanced glutamate uptake provides protective effects against mechanical distension-induced pain. Future studies will explore 1) the physiological site and mechanism by which enhanced glutamate transport activity mediates the antinociceptive effect and 2) enhanced glutamate transport effects on inflammogen-augmented visceral pain response. Supported by DK071839.
T1873 Activation of Intestinal Na+/H+ Exchanger 2 (NHE2) By Platelet Activating Factor Amika Singla, Ali Esmaili, Shivani Katyal, Seema Saksena, Ravinder K. Gill, Waddah A. Alrefai, Krishnamurthy Ramaswamy, Pradeep K. Dudeja Platelet activating factor (PAF), a biologically active proinflammatory phospholipid mediator, is produced by many cells and tissues and exhibits various cellular physiological functions e.g. platelet and neutrophil aggregation, leukocyte adhesion and vasodilatation. PAF has also been implicated as a critical mediator in the pathology of various gastrointestinal disorders such as Crohn's disease, necrotizing enterocolitis and ulcerative colitis. Previous studies have shown that PAF directly activates Cl- secretion in intestinal epithelial cells. However, nothing is known about the effects of PAF on Na+/H+ exchange activity in human intestine. Therefore, the present studies examined the effect of PAF on Na+/H+ exchange activity utilizing small intestinal Caco-2 and colonic T84 cell monolayers as In Vitro models for the human intestinal epithelia. Na+/H+ exchange activity was measured as EIPA sensitive 22 Na+ uptake after acid loading the cells. Our results showed that PAF treatment resulted in a dose and time dependent increase (~ 3 fold at 100 µM for 30 min, p < 0.001) in Na+/ H+ exchange activity in Caco-2 cells. Specific agonist for PAF receptor, Carbamyl PAF (100 µM) mimicked the effects of PAF in Caco-2 cells. PAF induced stimulation of Na+/H+ exchange activity was abrogated by PAF receptor antagonist WEB 2086 (1 µM) in Caco-2 cells. PAF treatment of Caco-2 cells from the apical (but not baoslateral side) stimulated Na+/H+ exchange activity suggesting the apical localization of PAF receptor in intestinal cells. PAF treatment stimulated apical Na+/H+ exchange activity in T84 cells (express only NHE2), similar to the effects in Caco-2 cells indicating that effects of PAF are not cell line specific. Kinetic analysis of PAF mediated effect on Na+/H+ exchange activity showed an increase in Vmax (~ 2 fold) with no change in Km in T84 cells. An analysis of NHE isoforms affected by PAF in Caco2 cells utilizing EIPA and HOE694 (NHE2 inhibitor), and PS120 cells expressing NHE3 and PAF receptor indicated that PAF mainly stimulates NHE2 activity but not NHE3. Conclusion: Our results suggest that PAF stimulates intestinal Na+/H+ exchanger 2 (NHE2) through PAF receptor mediated process. Our studies suggest that PAF may play an important role in regulating human intestinal Na+ absorption. (Supported by Dept of Vet Affairs and NIDDK)
Effect of 1 week pre-treatment with ceftriaxone (200mg/kg daily) on viscero-motor (VMR) response to colo-rectal distension (CRD) in mice. T1876 Lubiprostone Activation of Cl- Currents Does Not Involve CA2+, cAMP, or PKA John Cuppoletti, Danuta H. Malinowska, Kirti Tewari, Jayati Chakrabarti, Katrin Mende, 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) with an EC50 of approximately 20 nM [Cuppoletti et al AJP 287:C1173, 2004]. The purpose of this study was to determine whether lubiprostone (over the concentration range that activates electrogenic Cl- transport) causes changes in [Ca2+]i, cAMP or PKA mediated phosphorylation. Methods. T84 cells, HEK293 cells and HEK293 cells stably expressing hClC-2 or mutant hClC-2 were used. T84 cell [Ca2+]i, was measured using the calcium indicator dye indo-1/AM and intracellular [cAMP] was determined using a commercial ELISA kit. A commercial FLIPER assay was used to measure [Ca2+]i, and [cAMP] in HEK293 cells. Cl- currents were measured by whole cell patch clamp. Results. Lubiprostone at 1, 10 or 100 nM did not increase [Ca2+]i, in T84 cells and only slightly increased [cAMP] from 0.56 ± 0.02 to 4.0 ± 0.3 pmole/105 cells (n=4, P<0.001) at 100 nM (5 times the EC50 for lubiprostone activation of Cl- currents). 1 µM PGE1 significantly (P<0.0005) increased [Ca2+]i from 80.8 ± 2.4 nM (control) to 1175.0 ± 18.7 nM (n=3) and greatly increased [cAMP] to 37.6 ± 0.6 pmole/105 cells (n=4)(P<0.0005). In contrast, 100 nM lubiprostone had no effect on [Ca2+]i or cAMP in HEK293 cells. Nevertheless hClC-2 in HEK293 cells is activated by lubiprostone, thereby demonstrating that neither [Ca2+]i or cAMP signaling
T1874 Functional Role of the Sodium Hydrogen Exchanger Nhe4 in Human Colon Elizabeth Arena, Walter Longo, John P. Geibel Background: The colon plays a critical role in maintaining water/Na+ homeostasis. Na+/H+ exchangers (NHE) are integral plasma membrane proteins essential for electrolyte absorption, cell volume regulation, and maintaining intracellular pH (pHi). Several NHE isoforms have
A-581
AGA Abstracts
AGA Abstracts
, p<0.05 and 0.47±0.08 vs. 0.31±0.03, in O2, n.s., respectively), as well as in NHE3 -/than -/- enterocytes (0.30±0.03 vs. 0.15±0.01 in CO2/HCO3-, p<0.05 and 0.49±0.05 vs. 0.32±0.01 in O2 , p<0.05, respectively. Conclusions: 1. NHE3, but not Slc26a6, is involved in pHi regulation after villous enterocyte acidification after glucose uptake, but its presence is not essential for either SGLT1-mediated glucose or salt and fluid uptake. 2. In contrast, both NHE3 and Slc26a6 are equally involved in pHi regulation after PEPT1-mediated uptake of H+/dipeptide over the api-cal BBM. 3. NHE3 is essential for both PEPT1-mediated dipeptide as well as salt and fluid absorption, but Slc26a6 is not. 4. Intracellular pH is not the coupling mechanism of NHE3 and PEPT1. The requirement of NHE3 for PEPT1-mediated transport may be explained by an unique H+-recycling function of NHE3 in conjunction with PEPT1.
T1872 Alterations in Transporters and Regulators At the Base of Mouse Colonic Crypts After Genetic Disruption of the NHE2 Isoform Na+/H+ Exchanger Yanfang Guan, Michael P. Humphreys, Marshall H. Montrose BACKGROUND: NHE2 is the main apical epithelial Na+/H+ exchanger (NHE) at the colonic crypt base, but NHE2-knockout (KO) mice demonstrate adaptive, compensatory upregulation of one or more transporters that replace NHE2 function, yet are distinct from NHE3 (the only other known NHE isoform expressed in the colonic apical membrane). AIM: Evaluate the altered gene profile of crypt base colonocytes in NHE2-KO, to identify candidate upregulated transporters and regulatory pathways mediating the adaptive response. METHODS: Crypt base epithelium from cell position 1-6 was obtained by laser capture microdissection (Arcturus Veritas). RNA was pooled from multiple crypts from the same animal and amplified. mRNA expression profiles were measured with mouse full genome microarrays (Affymetrix). Outcomes from male NHE2-KO and NHE2 heterozygotes (Het) mice (n=3 each) were compared using Ingenuity Systems and GeneSpring software. RESULTS: 377 differentially expressed genes (P<0.05) were identified as having a greater than 1.4 change between genotypes. In the NHE gene family, only NHE5 was differentially expressed, being increased in NHE2-KO. Among other solute carrier (SLC) transporter families, only Slc4a7 (Na:HCO3cotransporter) was increased; Slc43a2 (L-amino acid transporter) and Slc10a1 (Na:bile acid cotransporter) were decreased. Increased regulators included beta-arrestin2 (shown to affect NHE5 expression at cell surface). Ingenuity Systems software revealed the top clustering of genes in networks of tissue and cellular development, cell signaling and cell cycle plus cell growth/proliferation. Greater than 100 differentially expressed genes were involved in cell development or differentiation, including transcriptional regulators known to affect epithelial differentiation or development (e.g. wnt2a, myc, cebp and cdx2). Twenty-six genes were involved in cell signaling and cell death related to tissue development (e.g. caspase 8 and phospholipase C beta). CONCLUSIONS: NHE5 is the strongest candidate for an NHE isoform that adaptively replaces NHE2 in the NHE2-KO crypt base. Loss of NHE2 causes surprisingly broad changes in gene expression of epithelial cells at the crypt base, suggesting that this transporter is linked more tightly to the metabolic function of crypt epithelium than previously suspected. Supported by NIH RO1 DK45457 and Digestive Health Center grant.
T1875 Increased Glial Glutamate Transporter Eaat2 Expression Reduces Visceral Pain Response in Mice Robert L. Stephens, C. G. Lin, Y. Lin, J. T. Travers Irritable bowel syndrome (IBS) is the leading digestive disease diagnosis among gastroenterologists and affects 15-20% of the US population. A significant component of IBS is recurrent abdominal pain. Central sensitization mediated by glutamate NMDA receptor activation is implicated in visceral pain pathophysiology. The glial glutamate transporter EAAT2 is the main mediator of glutamate clearance to terminate glutamate-mediated responses. Transgenic mice over-expressing human EAAT2 (EAAT2 mice), which exhibited a 1.4-2.5 fold enhanced glutamate uptake, showed 30% less writhing response to ip acetic acid than nontransgenic littermates [writhes/20 min (n): nontransgenic mice- 23 + 2 (37); EAAT2 mice- 16 + 2 (34); p<0.05]. Moreover, EAAT2 mice showed a 34-63% reduction in visceromotor response (VMR) to colo-rectal distension when assessing the response to 15- 60 mmHg pressures. Corroborating the involvement of enhanced glutamate uptake, wild-type animals treated for 1 week with ceftriaxone (200mg/kg daily), a stimulator of EAAT2 expression, showed a 5285% reduction in VMR to graded increases in colo-rectal distension (Figure 1). The data suggest that enhanced glutamate uptake provides protective effects against mechanical distension-induced pain. Future studies will explore 1) the physiological site and mechanism by which enhanced glutamate transport activity mediates the antinociceptive effect and 2) enhanced glutamate transport effects on inflammogen-augmented visceral pain response. Supported by DK071839.
T1873 Activation of Intestinal Na+/H+ Exchanger 2 (NHE2) By Platelet Activating Factor Amika Singla, Ali Esmaili, Shivani Katyal, Seema Saksena, Ravinder K. Gill, Waddah A. Alrefai, Krishnamurthy Ramaswamy, Pradeep K. Dudeja Platelet activating factor (PAF), a biologically active proinflammatory phospholipid mediator, is produced by many cells and tissues and exhibits various cellular physiological functions e.g. platelet and neutrophil aggregation, leukocyte adhesion and vasodilatation. PAF has also been implicated as a critical mediator in the pathology of various gastrointestinal disorders such as Crohn's disease, necrotizing enterocolitis and ulcerative colitis. Previous studies have shown that PAF directly activates Cl- secretion in intestinal epithelial cells. However, nothing is known about the effects of PAF on Na+/H+ exchange activity in human intestine. Therefore, the present studies examined the effect of PAF on Na+/H+ exchange activity utilizing small intestinal Caco-2 and colonic T84 cell monolayers as In Vitro models for the human intestinal epithelia. Na+/H+ exchange activity was measured as EIPA sensitive 22 Na+ uptake after acid loading the cells. Our results showed that PAF treatment resulted in a dose and time dependent increase (~ 3 fold at 100 µM for 30 min, p < 0.001) in Na+/ H+ exchange activity in Caco-2 cells. Specific agonist for PAF receptor, Carbamyl PAF (100 µM) mimicked the effects of PAF in Caco-2 cells. PAF induced stimulation of Na+/H+ exchange activity was abrogated by PAF receptor antagonist WEB 2086 (1 µM) in Caco-2 cells. PAF treatment of Caco-2 cells from the apical (but not baoslateral side) stimulated Na+/H+ exchange activity suggesting the apical localization of PAF receptor in intestinal cells. PAF treatment stimulated apical Na+/H+ exchange activity in T84 cells (express only NHE2), similar to the effects in Caco-2 cells indicating that effects of PAF are not cell line specific. Kinetic analysis of PAF mediated effect on Na+/H+ exchange activity showed an increase in Vmax (~ 2 fold) with no change in Km in T84 cells. An analysis of NHE isoforms affected by PAF in Caco2 cells utilizing EIPA and HOE694 (NHE2 inhibitor), and PS120 cells expressing NHE3 and PAF receptor indicated that PAF mainly stimulates NHE2 activity but not NHE3. Conclusion: Our results suggest that PAF stimulates intestinal Na+/H+ exchanger 2 (NHE2) through PAF receptor mediated process. Our studies suggest that PAF may play an important role in regulating human intestinal Na+ absorption. (Supported by Dept of Vet Affairs and NIDDK)
Effect of 1 week pre-treatment with ceftriaxone (200mg/kg daily) on viscero-motor (VMR) response to colo-rectal distension (CRD) in mice. T1876 Lubiprostone Activation of Cl- Currents Does Not Involve CA2+, cAMP, or PKA John Cuppoletti, Danuta H. Malinowska, Kirti Tewari, Jayati Chakrabarti, Katrin Mende, 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) with an EC50 of approximately 20 nM [Cuppoletti et al AJP 287:C1173, 2004]. The purpose of this study was to determine whether lubiprostone (over the concentration range that activates electrogenic Cl- transport) causes changes in [Ca2+]i, cAMP or PKA mediated phosphorylation. Methods. T84 cells, HEK293 cells and HEK293 cells stably expressing hClC-2 or mutant hClC-2 were used. T84 cell [Ca2+]i, was measured using the calcium indicator dye indo-1/AM and intracellular [cAMP] was determined using a commercial ELISA kit. A commercial FLIPER assay was used to measure [Ca2+]i, and [cAMP] in HEK293 cells. Cl- currents were measured by whole cell patch clamp. Results. Lubiprostone at 1, 10 or 100 nM did not increase [Ca2+]i, in T84 cells and only slightly increased [cAMP] from 0.56 ± 0.02 to 4.0 ± 0.3 pmole/105 cells (n=4, P<0.001) at 100 nM (5 times the EC50 for lubiprostone activation of Cl- currents). 1 µM PGE1 significantly (P<0.0005) increased [Ca2+]i from 80.8 ± 2.4 nM (control) to 1175.0 ± 18.7 nM (n=3) and greatly increased [cAMP] to 37.6 ± 0.6 pmole/105 cells (n=4)(P<0.0005). In contrast, 100 nM lubiprostone had no effect on [Ca2+]i or cAMP in HEK293 cells. Nevertheless hClC-2 in HEK293 cells is activated by lubiprostone, thereby demonstrating that neither [Ca2+]i or cAMP signaling
T1874 Functional Role of the Sodium Hydrogen Exchanger Nhe4 in Human Colon Elizabeth Arena, Walter Longo, John P. Geibel Background: The colon plays a critical role in maintaining water/Na+ homeostasis. Na+/H+ exchangers (NHE) are integral plasma membrane proteins essential for electrolyte absorption, cell volume regulation, and maintaining intracellular pH (pHi). Several NHE isoforms have
A-581
AGA Abstracts
AGA Abstracts
, p<0.05 and 0.47±0.08 vs. 0.31±0.03, in O2, n.s., respectively), as well as in NHE3 -/than -/- enterocytes (0.30±0.03 vs. 0.15±0.01 in CO2/HCO3-, p<0.05 and 0.49±0.05 vs. 0.32±0.01 in O2 , p<0.05, respectively. Conclusions: 1. NHE3, but not Slc26a6, is involved in pHi regulation after villous enterocyte acidification after glucose uptake, but its presence is not essential for either SGLT1-mediated glucose or salt and fluid uptake. 2. In contrast, both NHE3 and Slc26a6 are equally involved in pHi regulation after PEPT1-mediated uptake of H+/dipeptide over the api-cal BBM. 3. NHE3 is essential for both PEPT1-mediated dipeptide as well as salt and fluid absorption, but Slc26a6 is not. 4. Intracellular pH is not the coupling mechanism of NHE3 and PEPT1. The requirement of NHE3 for PEPT1-mediated transport may be explained by an unique H+-recycling function of NHE3 in conjunction with PEPT1.