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H+ exchanger isoform NHE-2
A394 AGA ABSTRACTS
GASTROENTEROLOGY Vol. 114, No. 4
• G1607 INDUCIBLE NITRIC OXIDE SYNTHASE (iNOS) DEPRESSES NEURALLY EVOKED CHLORIDE SECRETION IN TNBS-INDUCED COLITIS IN THE MOUSE. W.K. MacNaughton. S.S. Lowe, K. Cushing. Gastrointestinal Research Group, University of Calgary, Calgary, AB, Canada. Inflammatory bowel disease (IBD) is associated with expression of iNOS, and it has been suggested that iNOS-derived NO may participate in inflammationinduced mucosal dysfunction. The aim of this study was to determine the role of iNOS-derived NO in altered electrolyte transport in a mouse model of colitis. Methods. Colitis was induced in C57BL mice by intrarectal administration of 6 mg trinitrobenzenesulfonic acid (TNBS) in 0.1 ml 30% ethanol. Controls received 0.1 ml of saline. Mice were studied 7 days later. First, segments of colon were removed and either processed for determination of myeloperoxidase activity (MPO) or homogenized in Trizol reagent for extraction of RNA. Expression of iNOS mRNA was confirmed by RT-PCR, with GAPDH mRNA as internal control. NOS activity was determined by measuring the conversion of 14C-L-arginine to 14C-L-citmlline in a radioenzymatic assay. To study electrolyte transport responses, segments of unstripped colon from colitic and control mice were mounted in Ussing chambers under voltage clamp conditions. Short circuit current (lsc) was measured as an indicator of net active electrolyte transport. In some experiments tissues were pretreated with either the nNOS inhibitor 7-nitroindazole (7-NI) or the iNOS inhibitor L-Nt-(1-iminoethyl)-lysine (L-NIL) 10 min prior to electrical field stimulation (EFS). Results. At 7 days post-treatment, MPO activity was 9-fold higher in TNBS-treated mice than in controls (saline, 0.46+0.16 U/mg/min; TNBS, 4.27+0.79 U/mg/min; p < 0.01). There was a basal level of iNOS mRNA expression in controls. Expression was significantly enhanced in colons from TNBS-treated mice. iNOS activity was elevated > 14-fold (p<0.01) in inflamed colons (1.29 -+0.20 nmol/g/min) vs. controls (0.09 -+0.02 nmol/g/min). Conversely, eNOS activity was significantly decreased in inflamed colon (0.79 -+0.10 nmol/g/min) vs. controls (2.4-+ 0.25 nmol/g/min). In transport studies, EFS evoked secretion was significantly reduced in inflamed colon compared to controls (Alsc, pA/cm2: saline, 68-+ 21; TNBS, 13-+ 4; p < 0.01). This was reversed by acute, in vitro administration of L-NIL (40 -+ 15 pA/cm2), but not by 7-NI (12_+2 pA/cm2). L-NIL (3 pM) blocked TNBS-induced iNOS activity by 54%, but did not affect cNOS activity. The Isc response tO the muscarinic agonist carbachol was also significantly reduced in inflamed colons (1 _+1 pA/cm2) vs. controls (41 _+7 pA/cm2). This effect was unaltered by L-NIL pretreatment. Conclusions. NO derived from iNOS contributes to suppression of neurally evoked transport in inflamed mouse colon. Because inhibition of iNOS did not affect carbachol responses, the effect of iNOSderived NO is likely at the level of the enteric nervous system. Supported by the Crohn's and Colitis Foundation of Canada. • G1608 EFFECT OF AN HEPATIC SINUSOIDAL ENDOTHELIAL CELL FRACTION ON KUPFFER CELL ANTIGEN PRESENTATION. K~ Maemura, ZL Sun, N. Sakamoto, M. Ozaki, GB Bulkley, AS Klein. Department of Surgery, Johns Hopkins Medical Institutions, Baltimore, MD Background: Kupffer cells (KC), by virtue of their capability to phagocytose and kill microorganisms, and present foreign peptides to immunocompetent lymphocytes, play a major role in host defense against circulating pathogens. We have previously reported that hepatic endothelial cells (HEC) potentiate KC phagocytic killing. However, the mechanism of this interaction is not fully understood. Study design: An ovalbumin (OVA)-sensitized T-ceU line was generated from Lewis rats by footpad injection of OVA. Rat KC and HEC enriched fractions were isolated by a two-step Percoll gradient technique. The in vitro response of these OVA-sensitized T-cells to OVA presentation was measured using a [3H]-thymidine incorporation assay. Antigen presentation was evaluated in: a) irradiated, spleen-derived antigen presenting cells (APC); b) irradiated rat KC c) normal rat KC; d) normal rat HEC; and e) normal rat KC in coculture with normal HEC. Results: Irradiated KC presented OVA to OVA-sensitized T lymphocytes even more effectively than conventional (splenic) APC. On the other hand, non-irradiated KC suppressed OVA-sensitized T cell proliferation. Moreover, whereas HEC alone increased the OVA-sensitized T cell response, the combination of HEC and (unirradiated) KC suppressed this proliferation significantly.
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HE(: KC HEC~KC *T Une ÷T line ÷T line
Conclusion: 1) KC present antigen quite efficiently. 2) Normal (unirradiatcd) KC do not. This may help explain antigen-specific tolerance. 3) HEC increase antigen-specific T cell proliferation. 4) HEC may regulate this KC lymphocyte response. • G1609 MOLECULAR CHARACTERIZATION AND EXPRESSION OF THE HUMAN Na÷/H+ EXCHANGER ISOFORM NHE-2. J. Malakooti, R. Y. Dahdal, L. Schmidt, T. J. Layden, P.K. Dudeja and K. Ramaswamy. Dept. of Medicine, University of Illinois at Chicago and Westside VAMC, Chicago, IL.
We have recently reported cloning of the human Na+/H+ exchanger NHE-2 (hNHE-2) from a colonic cDNA library. The hNHE-2 cDNA encoded a polypeptide of 812 amino acids with an overall 90% homology to both the rabbit and rat NHE-2. The hNHE-2 mRNA was distributed along the gastrointestinal tract in the order colon > stomach > small intestine. Our current studies focus on the molecular characterization and the functional activity of the hNHE-2 isoform. Studies of chromosomal localization were carried out by fluorescence in situ hybridization (FISH) using a 20 kb genomic NHE-2 DNA probe. The hNHE-2 gene was found to be localized to a position immediately adjacent to the centromere on the long arm of chromosome 2, which corresponds to the band 2ql 1.2. The genomic structure of NHE-2 spanning > 35 kb (excluding intron-1 sequence) was determined by a combination of a conventional library screening and genomic polymerase chain reaction. The human NHE-2 gene contains 12 exons intervened by 11 introns. The size of exons is 71-534 bp with similar intrordexon boundaries as hNHE-1, while introns range from 0.55-12.5 kb (excluding intron-1) and are larger than those of hNHE-1. Protein expression studies using an in vitro transcription-translation coupled system in wheat germ extract showed that a protein of -80 kDa was expressed using two different constructs harboring hNHE-2 cDNA. The size of the protein obtained for hNHE-2 was similar to that for hNHE1 expressed in the same system. For functional studies, hNHE-2 cDNA was stably transfected into Na+/H+ exchanger deficient mouse fibroblast LAP-I cells, and functional activity of the stable transfectants was measured as Na+-dependent recovery of intracellular pH from an acid load using the pH sensitive dye BCECF. The NHE-2/LAP-1 transfectants exhibited Na+-dependent pH recovery after an acid prepulse in contrast to LAP-I cells transfected with an empty vector. The pH recovery of hNHE-2 transfected cells was inhibited by 0.1 mM amiloride. Our results demonstrate the successful expression of a Na+/H+ exchanger activity in these cells. Studies of hNHE-2 transfeeted cells will yield important information on the physiological function and regulation of this apical Na+/H+ exchanger isoform. Supported by NIDDK and the Dept. of Veterans Affairs • G1610 EFFECT OF CHRONIC INFLAMMATION ON ILEAL SHORT CHAIN FATTY ACID: BICARBONATE EXCHANGE. T. Manokas. J.J. Fromkes, and U. Sundaram. Division of Digestive Diseases, Depts. of Internal Medicine and Physiology, The Ohio State Univ. School of Medicine, Columbus, OH. Background: The crypt-villus distribution and the effect of chronic inflammation on short chain fatty acid:bicarbonate (SCFA:HCO3) exchange in the intestine is unknown. We have previously demonstrated unique alterations in electrolyte and nutrient transport processes in a rabbit model of chronic ileal inflammation. Aims: Determine the distribution of SCFA:HCO 3 exchange along the cryptvillus axis of the normal rabbit ileum and the effect of chronic inflammation on ileal SCFA:HCO3exchange. Methods: Villus and crypt cells were isolated from the normal and the chronically inflamed rabbit ileum by a Ca++ chelation technique. Brush border membrane vesicles (BBMV) were prepared by Ca++ precipitation and differential centrifugation. Chronic inflammation of the rabbit ileum was produced by coccidial infection. Uptake of ~4C-butyrate was performed by rapid filtration. Results: In the normal ileum bicarbonate and pH gradient dependent 14C-butyrate uptake (e.g. SCFA:HCO 3 exchange) was present in the BBMV of villus cells (770 +-147 pmol/mg protein • 3 seconds with gradient vs 118 + 33 without gradient, n=10, p < 0~01). However, it was not present in crypt cells (156 _+48 pmol/mg protein • 3 seconds with gradient vs 91 +_27 without gradient, n=3, p=ns). An anion exchange inhibitor, DIDS (1 raM), significantly inhibited villus cell BBMV SCFA:HCO 3 exchange (856 ± 129 pmol/mg protein • 3 seconds vs 50 + 19 with DIDS, n=4, p < 0.01). Extravesicular CI did not significantly affect 14C-butyrate uptake in villus cell BBMV which suggested that this is an anion exchanger that is distinct from CI:HCO3 exchange which is known to be present on the BBM of these cells. In the chronically inflamed ileum SCFA:HCO3 exchange was reduced (694 -+ 137 pmol/mg protein • 3 seconds in normal and 127 + 51 in inflamed, n=6, p < 0.01) while remaining sensitive to DIDS (103 + 34 pmol/mg protein • 3 seconds vs 2.7 _+0.3 with DIDS, n=4, p < 0.05). Kinetic studies demonstrated that the maximal rate of uptake of butyrate (Vmax), but not the affinity for butyrate was reduced in the chronically inflamed rabbit ileum (Vmax: 14.8 nmol/mg protein • 3 seconds in normal and 3.5 in inflamed). Conclusions: These data demonstrate that a distinct SCFA:HCO 3 exchange is present on the BBMV of villus, but not crypt cells in the normal rabbit ileum. SCFA:HCO3 exchange is inhibited in the chronically inflamed rabbit ileum.
GASTROENTEROLOGY Vol. 114, No. 4
• G1607 INDUCIBLE NITRIC OXIDE SYNTHASE (iNOS) DEPRESSES NEURALLY EVOKED CHLORIDE SECRETION IN TNBS-INDUCED COLITIS IN THE MOUSE. W.K. MacNaughton. S.S. Lowe, K. Cushing. Gastrointestinal Research Group, University of Calgary, Calgary, AB, Canada. Inflammatory bowel disease (IBD) is associated with expression of iNOS, and it has been suggested that iNOS-derived NO may participate in inflammationinduced mucosal dysfunction. The aim of this study was to determine the role of iNOS-derived NO in altered electrolyte transport in a mouse model of colitis. Methods. Colitis was induced in C57BL mice by intrarectal administration of 6 mg trinitrobenzenesulfonic acid (TNBS) in 0.1 ml 30% ethanol. Controls received 0.1 ml of saline. Mice were studied 7 days later. First, segments of colon were removed and either processed for determination of myeloperoxidase activity (MPO) or homogenized in Trizol reagent for extraction of RNA. Expression of iNOS mRNA was confirmed by RT-PCR, with GAPDH mRNA as internal control. NOS activity was determined by measuring the conversion of 14C-L-arginine to 14C-L-citmlline in a radioenzymatic assay. To study electrolyte transport responses, segments of unstripped colon from colitic and control mice were mounted in Ussing chambers under voltage clamp conditions. Short circuit current (lsc) was measured as an indicator of net active electrolyte transport. In some experiments tissues were pretreated with either the nNOS inhibitor 7-nitroindazole (7-NI) or the iNOS inhibitor L-Nt-(1-iminoethyl)-lysine (L-NIL) 10 min prior to electrical field stimulation (EFS). Results. At 7 days post-treatment, MPO activity was 9-fold higher in TNBS-treated mice than in controls (saline, 0.46+0.16 U/mg/min; TNBS, 4.27+0.79 U/mg/min; p < 0.01). There was a basal level of iNOS mRNA expression in controls. Expression was significantly enhanced in colons from TNBS-treated mice. iNOS activity was elevated > 14-fold (p<0.01) in inflamed colons (1.29 -+0.20 nmol/g/min) vs. controls (0.09 -+0.02 nmol/g/min). Conversely, eNOS activity was significantly decreased in inflamed colon (0.79 -+0.10 nmol/g/min) vs. controls (2.4-+ 0.25 nmol/g/min). In transport studies, EFS evoked secretion was significantly reduced in inflamed colon compared to controls (Alsc, pA/cm2: saline, 68-+ 21; TNBS, 13-+ 4; p < 0.01). This was reversed by acute, in vitro administration of L-NIL (40 -+ 15 pA/cm2), but not by 7-NI (12_+2 pA/cm2). L-NIL (3 pM) blocked TNBS-induced iNOS activity by 54%, but did not affect cNOS activity. The Isc response tO the muscarinic agonist carbachol was also significantly reduced in inflamed colons (1 _+1 pA/cm2) vs. controls (41 _+7 pA/cm2). This effect was unaltered by L-NIL pretreatment. Conclusions. NO derived from iNOS contributes to suppression of neurally evoked transport in inflamed mouse colon. Because inhibition of iNOS did not affect carbachol responses, the effect of iNOSderived NO is likely at the level of the enteric nervous system. Supported by the Crohn's and Colitis Foundation of Canada. • G1608 EFFECT OF AN HEPATIC SINUSOIDAL ENDOTHELIAL CELL FRACTION ON KUPFFER CELL ANTIGEN PRESENTATION. K~ Maemura, ZL Sun, N. Sakamoto, M. Ozaki, GB Bulkley, AS Klein. Department of Surgery, Johns Hopkins Medical Institutions, Baltimore, MD Background: Kupffer cells (KC), by virtue of their capability to phagocytose and kill microorganisms, and present foreign peptides to immunocompetent lymphocytes, play a major role in host defense against circulating pathogens. We have previously reported that hepatic endothelial cells (HEC) potentiate KC phagocytic killing. However, the mechanism of this interaction is not fully understood. Study design: An ovalbumin (OVA)-sensitized T-ceU line was generated from Lewis rats by footpad injection of OVA. Rat KC and HEC enriched fractions were isolated by a two-step Percoll gradient technique. The in vitro response of these OVA-sensitized T-cells to OVA presentation was measured using a [3H]-thymidine incorporation assay. Antigen presentation was evaluated in: a) irradiated, spleen-derived antigen presenting cells (APC); b) irradiated rat KC c) normal rat KC; d) normal rat HEC; and e) normal rat KC in coculture with normal HEC. Results: Irradiated KC presented OVA to OVA-sensitized T lymphocytes even more effectively than conventional (splenic) APC. On the other hand, non-irradiated KC suppressed OVA-sensitized T cell proliferation. Moreover, whereas HEC alone increased the OVA-sensitized T cell response, the combination of HEC and (unirradiated) KC suppressed this proliferation significantly.
2~00--
$
~
Ulo-
s-
+T~
~'T~
'I'T l i n e
*nonotl T
I
HE(: KC HEC~KC *T Une ÷T line ÷T line
Conclusion: 1) KC present antigen quite efficiently. 2) Normal (unirradiatcd) KC do not. This may help explain antigen-specific tolerance. 3) HEC increase antigen-specific T cell proliferation. 4) HEC may regulate this KC lymphocyte response. • G1609 MOLECULAR CHARACTERIZATION AND EXPRESSION OF THE HUMAN Na÷/H+ EXCHANGER ISOFORM NHE-2. J. Malakooti, R. Y. Dahdal, L. Schmidt, T. J. Layden, P.K. Dudeja and K. Ramaswamy. Dept. of Medicine, University of Illinois at Chicago and Westside VAMC, Chicago, IL.
We have recently reported cloning of the human Na+/H+ exchanger NHE-2 (hNHE-2) from a colonic cDNA library. The hNHE-2 cDNA encoded a polypeptide of 812 amino acids with an overall 90% homology to both the rabbit and rat NHE-2. The hNHE-2 mRNA was distributed along the gastrointestinal tract in the order colon > stomach > small intestine. Our current studies focus on the molecular characterization and the functional activity of the hNHE-2 isoform. Studies of chromosomal localization were carried out by fluorescence in situ hybridization (FISH) using a 20 kb genomic NHE-2 DNA probe. The hNHE-2 gene was found to be localized to a position immediately adjacent to the centromere on the long arm of chromosome 2, which corresponds to the band 2ql 1.2. The genomic structure of NHE-2 spanning > 35 kb (excluding intron-1 sequence) was determined by a combination of a conventional library screening and genomic polymerase chain reaction. The human NHE-2 gene contains 12 exons intervened by 11 introns. The size of exons is 71-534 bp with similar intrordexon boundaries as hNHE-1, while introns range from 0.55-12.5 kb (excluding intron-1) and are larger than those of hNHE-1. Protein expression studies using an in vitro transcription-translation coupled system in wheat germ extract showed that a protein of -80 kDa was expressed using two different constructs harboring hNHE-2 cDNA. The size of the protein obtained for hNHE-2 was similar to that for hNHE1 expressed in the same system. For functional studies, hNHE-2 cDNA was stably transfected into Na+/H+ exchanger deficient mouse fibroblast LAP-I cells, and functional activity of the stable transfectants was measured as Na+-dependent recovery of intracellular pH from an acid load using the pH sensitive dye BCECF. The NHE-2/LAP-1 transfectants exhibited Na+-dependent pH recovery after an acid prepulse in contrast to LAP-I cells transfected with an empty vector. The pH recovery of hNHE-2 transfected cells was inhibited by 0.1 mM amiloride. Our results demonstrate the successful expression of a Na+/H+ exchanger activity in these cells. Studies of hNHE-2 transfeeted cells will yield important information on the physiological function and regulation of this apical Na+/H+ exchanger isoform. Supported by NIDDK and the Dept. of Veterans Affairs • G1610 EFFECT OF CHRONIC INFLAMMATION ON ILEAL SHORT CHAIN FATTY ACID: BICARBONATE EXCHANGE. T. Manokas. J.J. Fromkes, and U. Sundaram. Division of Digestive Diseases, Depts. of Internal Medicine and Physiology, The Ohio State Univ. School of Medicine, Columbus, OH. Background: The crypt-villus distribution and the effect of chronic inflammation on short chain fatty acid:bicarbonate (SCFA:HCO3) exchange in the intestine is unknown. We have previously demonstrated unique alterations in electrolyte and nutrient transport processes in a rabbit model of chronic ileal inflammation. Aims: Determine the distribution of SCFA:HCO 3 exchange along the cryptvillus axis of the normal rabbit ileum and the effect of chronic inflammation on ileal SCFA:HCO3exchange. Methods: Villus and crypt cells were isolated from the normal and the chronically inflamed rabbit ileum by a Ca++ chelation technique. Brush border membrane vesicles (BBMV) were prepared by Ca++ precipitation and differential centrifugation. Chronic inflammation of the rabbit ileum was produced by coccidial infection. Uptake of ~4C-butyrate was performed by rapid filtration. Results: In the normal ileum bicarbonate and pH gradient dependent 14C-butyrate uptake (e.g. SCFA:HCO 3 exchange) was present in the BBMV of villus cells (770 +-147 pmol/mg protein • 3 seconds with gradient vs 118 + 33 without gradient, n=10, p < 0~01). However, it was not present in crypt cells (156 _+48 pmol/mg protein • 3 seconds with gradient vs 91 +_27 without gradient, n=3, p=ns). An anion exchange inhibitor, DIDS (1 raM), significantly inhibited villus cell BBMV SCFA:HCO 3 exchange (856 ± 129 pmol/mg protein • 3 seconds vs 50 + 19 with DIDS, n=4, p < 0.01). Extravesicular CI did not significantly affect 14C-butyrate uptake in villus cell BBMV which suggested that this is an anion exchanger that is distinct from CI:HCO3 exchange which is known to be present on the BBM of these cells. In the chronically inflamed ileum SCFA:HCO3 exchange was reduced (694 -+ 137 pmol/mg protein • 3 seconds in normal and 127 + 51 in inflamed, n=6, p < 0.01) while remaining sensitive to DIDS (103 + 34 pmol/mg protein • 3 seconds vs 2.7 _+0.3 with DIDS, n=4, p < 0.05). Kinetic studies demonstrated that the maximal rate of uptake of butyrate (Vmax), but not the affinity for butyrate was reduced in the chronically inflamed rabbit ileum (Vmax: 14.8 nmol/mg protein • 3 seconds in normal and 3.5 in inflamed). Conclusions: These data demonstrate that a distinct SCFA:HCO 3 exchange is present on the BBMV of villus, but not crypt cells in the normal rabbit ileum. SCFA:HCO3 exchange is inhibited in the chronically inflamed rabbit ileum.