733 The Anion Transporter SLC26A6 (Putative Anion Transporter-1) Regulates Murine Small Intestinal...
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were analyzed by real-time PCR and Western blotting, respectively. DRA promoter activity was measured by luciferase reporter activity assays after transiently transfecting Caco-2 cells with DRA promoter construct (-1183/+144). Treatment of Caco-2 cells with 10 μM concentration of ATRA significantly induced DRA mRNA levels within 8 h of incubation, which increased to ~3 fold by 24 h. A concomitant increase (140 ± 5 % of control P ,0.001) in DRA protein expression was also observed. In addition, ATRA treatment significantly increased DRA promoter activity (258.8 ± 3.4 % of control, P ,0.001) indicating that ATRA effects are transcriptionally mediated. ATRA is known to mediate its effects via specific retinoic acid receptors (RAR's) α, β and γ. A remarkable increase (~11 fold) in mRNA levels of RAR-β receptor subtype were found in response to ATRA treatment with no change in levels of RAR'- α and γ. Treatment of cells with RAR- β agonist (CH-55, 1μM) mimicked ATRA effects on DRA promoter. RAR- β antagonist (LE-135, 1μM) blocked the stimulatory effects of ATRA on DRA promoter activity. Since, DRA expression is known to be modulated by hepatic nuclear factors (HNF's), the potential mediation of ATRA effects via HNFs was examined. A significant increase in HNF-1 β mRNA levels (but not HNF-1 α or HNF4) in response to ATRA treatment was observed. The ability of ATRA to induce DRA expression was blocked by silencing HNF-1β using siRNA. In summary, ATRA increased DRA expression via RAR-β through HNF-1β activation. We speculate that ATRA may act as a potential therapeutic tool under inflammatory conditions where DRA expression is altered (Supported by NIDDK and Dept. of Veteran Affairs). 730 Cyclic GMP Kinase II (CKII) Inhibits NHE3 by Affecting Trafficking via Phosphorylating NHE3 At Three Sites: Identification of a Multifunctional Phosphorylation Site Tian-e Chen, Rakhilya Murtazina, Boyoung Cha, Rafiquel Sarker, Jianbo Yang, Robert N. Cole, Peter S. Aronson, Hugo de Jonge, Mark Donowitz Background: The epithelial brush border (BB) Na/H exchanger NHE3 is inhibited by cGMP. While it is known that this is mediated via the BB protein cGKII, how cGKII regulates NHE3 is unknown. Previous studies of cGKII have identified a number of its phosphorylated substrates, all of which have also been phosphorylated by PKA. This study tested the hypothesis that cGMP inhibits NHE3 activity by phosphorylating NHE3 at specific sites which are unique to cGKII to affect NHE3 membrane trafficking. Methods: Studies were carried out in PS120/NHERF2, polarized epithelial OK/NHERF2 and Caco-2/Bbe cells, all cell lines stably overexpressing HA-NHE3 and transiently expressing recombinant cGKII (adenoviral vector), and also in mouse ileum. NHE3 activity was measured with BCECF/ fluorometry. Surface NHE3 was determined by cell surface biotinylation. Identification of NHE3 phosphorylation sites was by IP/LC-MS/MS with TiO2 enrichment and IB with specific anti-phospho NHE3552 and S605 antibodies. NHE3 mutations were generated by site directed mutagenesis. Results: cGMP inhibited NHE3 in all cell types studied, requiring NHERF2 and cGKII, and occurring in a time dependent manner (present 15mins after cGMP addition, maxium in 45 min and present at 60 min.) The cGMP inhibition was associated with a 46% reduction of cell surface NHE3. Quantitative mass spec identified cGMP increased NHE3 phosphorylation at three serines (S552, S605, S663). Inhibiting PKA with H89 did not prevent cGKII phosphorylation of S552, S605 determined by IB. In contrast to WT NHE3 expressed in PS120/NHERF2/cGKII cells, both NHE3 S552A and S605A double mutant and S663A single mutant lost cGMP inhibition of NHE3; however, S663D was inhibited by cGKII. Basal NHE3 activity was increased in both S663A (by 56%) and S663D (by 80%) cell lines. S663 was previously shown to be necessary for dexamethasone (dex) stimulation of NHE3 by SGK1. Dex stimulated wild type NHE3 activity and increased surface expression but failed to stimulate NHE3 activity or increase surface expression when NHE3 was mutated to S663A or S663D. Conclusions: 1) cGMP rapidly inhibits NHE3 activity and decreases surface expression by a cGKII and NHERF2 dependent process in epithelial cells. 2) The inhibition is associated with phosphorylation of NHE3 at S552, S605 and S663 all of which must be phosphorylated for cGMP/cGKII to inhibit NHE3. 3) While both cGKII and PKA phosphorylate NHE3 at S552, and S605, cGKII but not PKA phosphorylates S663 4) Dex stimulates NHE3 by phosphorylation of a single site, S663 . The requirement of three phosphorylation sites in NHE3 for cGKII inhibition, the phosphorylation of one of which is necessary for Dex stimulation of NHE3, is a unique form of regulation by phosphorylation, which almost certainly relates to differences in formation of NHE3 signal- ing complexes. 731 Notch Signaling Regulates Expression of Bestrophin-4, a Novel Absorptive Cell Specific HCO3 - /Cl - Channel, in Human Intestinal Epithelial Cells Go Ito, Ryuichi Okamoto, Tatsuro Murano, Hiromichi Shimizu, Satoru Fujii, Kiichiro Tsuchiya, Tetsuya Nakamura, Mamoru Watanabe Background & Aim: Intestinal epithelial cells (IECs) regulate absorption and secretion of anions, such as HCO3- or Cl-, thereby contributing to the proper fluid exchange between the bloodstream and gut lumen. Disruption of such function is the underlying cause of severe diarrhea in various diseases. Bestrophin genes represent a newly identified group of calcium-activated Cl- channels (CaCCs). Recent study has shown that Bestrophin-2 is expressed specifically in goblet cells of the human intestine, and regulates secretion of HCO3-/Cl- at its basolateral surface. Studies have suggested that Bestrophin-4 (BEST4) is also expressed specifically in the human colon. However, its precise expression within the human intestinal tissue, or the mechanism regulating its expression has never been described. In this study, we aimed to identify BEST4 expressing-cells in the human intestinal tissue, and also examine the molecular mechanism regulating BEST4 expression in IECs. Methods: Expression of BEST4 in human intestinal tissues was identified by immunohistochemical analysis (IHC). Role of Notch signaling in expression of BEST4 was examined by using cell- lines in which forced expression of NICD, an activated form of the Notch receptor, can be induced by Doxycycline. Such a cell-line was established from a human colonic epithelial cell-line LS174T, and analyzed by quantitative RT-PCR or immunocytochemistry. Results: IHC of human small intestinal and colonic tissues revealed that BEST4 is predominantly expressed at the basolateral membrane of pan-cytokeratin or E-cadherin positive IECs. Such S-131 AGA Abstracts BEST4-positive IECs were completely negative for Ki-67 expression, and resided exclusively at the villus of the small intestine, or at the surface epithelium of the colon, suggesting that these cells are all post-mitotic. Co-staining analysis revealed that BEST4-positive IECs are completely negative for secretory lineage markers such as MUC2, Chromogranin-A, Lysozyme or HPGDS. In sharp contrast, BEST4-positive IECs completely co-localized with absorptive cell specific markers such as Villin or CD10, which clearly suggested that BEST4 is an absorptive cell-specific CaCC. Consistently, forced activation of Notch signaling, a key molecular pathway for absorptive cell differentiation, significantly increased BEST4 protein expression at the basolateral membrane of LS174T cells. Such a Notch-dependent up- regulation of BEST4 expression in LS174T cells appeared to be regulated at the mRNA level, as confirmed by quantitative RT-PCR analysis. Conclusion: Notch signaling promotes expression of a novel absorptive cell specific HCO3-/Cl- channel, BEST4, in human IECs. Such a lineage-specific expression of BEST4 suggests its possible involvement in absorptive cell specific regulation of water and electrolyte absorption, via HCO3-/Cl- exchange. 732 Large Conductance K + (BK) Channel-Mediated K + Secretion Provides the Driving Force for Water Secretion in Rat Distal Colon Deeban Ganesan, Kevin J. Engels, Geoffrey I. Sandle, Vazhaikkurichi M. Rajendran Background: In secretory diarrhea, electrogenic Cl - secretion is regarded as the main driving force for intestinal water secretion, but high levels of K + secretion may underlie high stool volumes in patients with colonic pseudo-obstruction (Gastroenterology 129:1268-73, 2005; Nephrol Dial Transplant 23:3350-2, 2008). Recent studies suggest that cAMP-induced colonic K + secretion via large conductance apical K + (BK) channels may account for excessive fecal K + losses in secretory diarrhea, but it is unclear whether BK channel-mediated K + secretion also provides a driving force for water secretion. Aim: To determine whether BK channel-mediated K + secretion provides a driving force for colonic water secretion. Methods: Net water transport was evaluated in the distal colon of anaesthetized control and aldosterone (‘aldo') SD rats by in vivo perfusion of Ringer solution (0.5 ml/min). ‘Aldo' rats were fed a Na + -free diet for 6-7 days. 3 H-PEG 4000 was used as a non-absorbable volume marker. Positive and negative values represent net water absorption and secretion, respectively. Results: Under basal condition, net water absorption was present in both control and ‘aldo' animals. In controls, amiloride (10 μM, to inhibit Na + channels) did not change water absorption, but amiloride (1 mM, to inhibit Na-H exchange) abolished water absorption (89.6 ± 6.4 μl/h.cm versus -2.6 ± 1.8 μl/h.cm; p , 0.001). In ‘aldo' animals, in which there was both Na + channel-mediated Na + absorption and BK channel-mediated K + secretion, 10 μM amiloride reversed net water absorption to net secretion (92.6 ± 6.7 μl/h.cm versus -38.9 ± 4.8 μl/h.cm; p , 0.001) and 1 mM amiloride had no further effect. In presence of 10 μM amiloride, net water secretion in ‘aldo' animals was completely abolished by 3 mM Ba 2+ (a nonspecific K + channel blocker) and iberiotoxin (a specific BK channel blocker), but inhibited by only 16% by clotrimazole (an IK channel blocker). Furthermore, in the absence of amiloride, iberiotoxin increased net water absorption in ‘aldo' animals (92.6 ± 6.7 μl/h. cm versus 118.8 ± 7.1 μl/h.cm; p , 0.05), consistent with inhibition of a secretory water flux driven by BK channel-mediated K + secretion. In controls (1 mM amiloride in perfusate), BMS (a BK channel opener) significantly increased net water secretion (-2.6 ± 1.8 μl/h.cm versus -8.8 ± 3.4 μl/h.cm; p , 0.05). Conclusions: In rat distal colon, enhancement of apical BK channel expression secondary to chronic hyperaldosteronism, and acute activation of apical BK channels in controls, stimulate K + secretion sufficiently to increase water secretion. On the other hand, BK channel inhibition has the opposite effect on net water transport. Taken together, these results suggest that BK channel-mediated K + secretion may provide a driving force for water secretion in some types of secretory diarrhea. 733 The Anion Transporter SLC26A6 (Putative Anion Transporter-1) Regulates Murine Small Intestinal Na+HCO3- Absorption Weiliang Xia, Qin Yu, Brigitte Riederer, Anurag K. Singh, Regina Engelhardt, Penghong Song, Dean Tian, Manoocher Soleimani, Ursula Seidler Background: Slc26a6 (PAT-1), a multi anion transporter, is strongly expressed in the apical membrane of the upper small intestine, where it is colocalized with another member of the same gene family, Slc26a3 (DRA). Experiments in heterologous expression systems suggest that Pat-1 may exchange HCO3- and Cl- in an electrogenic fashion, and experiments in native duodenum suggest that after intracellular acidification by H+-dipeptide transport, PAT-1 may import HCO3-. Aim: This study investigates whether PAT-1 is essential for small intestinal fluid absorption under the unique ionic conditions of the upper GI tract, where high luminal CO2 as well as high luminal HCO3- concentrations can be found after the pancreatic juice has mixed with the acidic milieu of the proximal duodenum. Methods and Results: Small intestinal fluid absorption was therefore assessed by single pass perfusion of prewarmed, isotonic solutions of different ionic compositions but constant pH of 7.4 through jejunal segments of anesthetized acid/base status controlled mice, and the volume and pH of the inflowing and outflowing solution was measured. WT jejunum absorbed fluid and acidified the lumen. DRA-deficient jejunum absorbed less fluid than WT, and acidified the lumen more strongly, consistent with its action as a Cl-/HCO3- exchanger. PAT-1-deficient jejunum also absorbed less fluid, but resulted in less luminal acidification. Switching the luminal solution to a 5% CO2/HCO3- buffer, pH 7.4, resulted in a persistent decrease in strongly augmented fluid absorption in a PAT-1- and Na+/H+ exchanger 3 (NHE3)- but not DRA-dependent manner. In the absence of luminal Cl-, luminal CO2/HCO3- also augumented fluid absorption in WT and DRA-deficient, but not in PAT-1- and NHE3- deficient mice, indicating that PAT-1 likely serves to import HCO3- under these circum- stances. Conclusion: The results suggest that PAT-1 may play an important role in jejunal Na+HCO3- and fluid absorption. AGA Abstracts
Transcript of 733 The Anion Transporter SLC26A6 (Putative Anion Transporter-1) Regulates Murine Small Intestinal...
were analyzed by real-time PCR and Western blotting, respectively. DRA promoter activitywas measured by luciferase reporter activity assays after transiently transfecting Caco-2cells with DRA promoter construct (-1183/+144). Treatment of Caco-2 cells with 10 μMconcentration of ATRA significantly induced DRA mRNA levels within 8 h of incubation,which increased to ~3 fold by 24 h. A concomitant increase (140 ± 5 % of control P ,0.001)in DRA protein expression was also observed. In addition, ATRA treatment significantlyincreased DRA promoter activity (258.8 ± 3.4 % of control, P ,0.001) indicating that ATRAeffects are transcriptionally mediated. ATRA is known to mediate its effects via specificretinoic acid receptors (RAR's) α, β and γ. A remarkable increase (~11 fold) in mRNA levelsof RAR-β receptor subtype were found in response to ATRA treatment with no change inlevels of RAR'-α and γ. Treatment of cells with RAR-β agonist (CH-55, 1μM) mimickedATRA effects on DRA promoter. RAR-β antagonist (LE-135, 1μM) blocked the stimulatoryeffects of ATRA on DRA promoter activity. Since, DRA expression is known to be modulatedby hepatic nuclear factors (HNF's), the potential mediation of ATRA effects via HNFs wasexamined. A significant increase in HNF-1β mRNA levels (but not HNF-1α or HNF4) inresponse to ATRA treatment was observed. The ability of ATRA to induce DRA expressionwas blocked by silencing HNF-1β using siRNA. In summary, ATRA increased DRA expressionvia RAR-β through HNF-1β activation. We speculate that ATRA may act as a potentialtherapeutic tool under inflammatory conditions where DRA expression is altered (Supportedby NIDDK and Dept. of Veteran Affairs).
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Cyclic GMP Kinase II (CKII) Inhibits NHE3 by Affecting Trafficking viaPhosphorylating NHE3 At Three Sites: Identification of a MultifunctionalPhosphorylation SiteTian-e Chen, Rakhilya Murtazina, Boyoung Cha, Rafiquel Sarker, Jianbo Yang, Robert N.Cole, Peter S. Aronson, Hugo de Jonge, Mark Donowitz
Background: The epithelial brush border (BB) Na/H exchanger NHE3 is inhibited by cGMP.While it is known that this is mediated via the BB protein cGKII, how cGKII regulates NHE3is unknown. Previous studies of cGKII have identified a number of its phosphorylatedsubstrates, all of which have also been phosphorylated by PKA. This study tested thehypothesis that cGMP inhibits NHE3 activity by phosphorylating NHE3 at specific siteswhich are unique to cGKII to affect NHE3 membrane trafficking. Methods: Studies werecarried out in PS120/NHERF2, polarized epithelial OK/NHERF2 and Caco-2/Bbe cells, allcell lines stably overexpressing HA-NHE3 and transiently expressing recombinant cGKII(adenoviral vector), and also in mouse ileum. NHE3 activity was measured with BCECF/fluorometry. Surface NHE3 was determined by cell surface biotinylation. Identification ofNHE3 phosphorylation sites was by IP/LC-MS/MS with TiO2 enrichment and IB with specificanti-phospho NHE3552 and S605 antibodies. NHE3 mutations were generated by sitedirected mutagenesis. Results: cGMP inhibited NHE3 in all cell types studied, requiringNHERF2 and cGKII, and occurring in a time dependent manner (present 15mins after cGMPaddition, maxium in 45 min and present at 60 min.) The cGMP inhibition was associatedwith a 46% reduction of cell surface NHE3. Quantitative mass spec identified cGMP increasedNHE3 phosphorylation at three serines (S552, S605, S663). Inhibiting PKA with H89 didnot prevent cGKII phosphorylation of S552, S605 determined by IB. In contrast to WTNHE3 expressed in PS120/NHERF2/cGKII cells, both NHE3 S552A and S605A doublemutant and S663A single mutant lost cGMP inhibition of NHE3; however, S663D wasinhibited by cGKII. Basal NHE3 activity was increased in both S663A (by 56%) and S663D(by 80%) cell lines. S663 was previously shown to be necessary for dexamethasone (dex)stimulation of NHE3 by SGK1. Dex stimulated wild type NHE3 activity and increased surfaceexpression but failed to stimulate NHE3 activity or increase surface expression when NHE3was mutated to S663A or S663D. Conclusions: 1) cGMP rapidly inhibits NHE3 activity anddecreases surface expression by a cGKII and NHERF2 dependent process in epithelial cells.2) The inhibition is associated with phosphorylation of NHE3 at S552, S605 and S663 allof which must be phosphorylated for cGMP/cGKII to inhibit NHE3. 3) While both cGKIIand PKA phosphorylate NHE3 at S552, and S605, cGKII but not PKA phosphorylates S6634) Dex stimulates NHE3 by phosphorylation of a single site, S663 . The requirement ofthree phosphorylation sites in NHE3 for cGKII inhibition, the phosphorylation of oneof which is necessary for Dex stimulation of NHE3, is a unique form of regulation byphosphorylation, which almost certainly relates to differences in formation of NHE3 signal-ing complexes.
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Notch Signaling Regulates Expression of Bestrophin-4, a Novel AbsorptiveCell Specific HCO3-/Cl- Channel, in Human Intestinal Epithelial CellsGo Ito, Ryuichi Okamoto, Tatsuro Murano, Hiromichi Shimizu, Satoru Fujii, KiichiroTsuchiya, Tetsuya Nakamura, Mamoru Watanabe
Background & Aim: Intestinal epithelial cells (IECs) regulate absorption and secretion ofanions, such as HCO3- or Cl-, thereby contributing to the proper fluid exchange betweenthe bloodstream and gut lumen. Disruption of such function is the underlying cause ofsevere diarrhea in various diseases. Bestrophin genes represent a newly identified group ofcalcium-activated Cl- channels (CaCCs). Recent study has shown that Bestrophin-2 isexpressed specifically in goblet cells of the human intestine, and regulates secretion ofHCO3-/Cl- at its basolateral surface. Studies have suggested that Bestrophin-4 (BEST4) isalso expressed specifically in the human colon. However, its precise expression within thehuman intestinal tissue, or the mechanism regulating its expression has never been described.In this study, we aimed to identify BEST4 expressing-cells in the human intestinal tissue,and also examine the molecular mechanism regulating BEST4 expression in IECs. Methods:Expression of BEST4 in human intestinal tissues was identified by immunohistochemicalanalysis (IHC). Role of Notch signaling in expression of BEST4 was examined by using cell-lines in which forced expression of NICD, an activated form of the Notch receptor, can beinduced by Doxycycline. Such a cell-line was established from a human colonic epithelialcell-line LS174T, and analyzed by quantitative RT-PCR or immunocytochemistry. Results:IHC of human small intestinal and colonic tissues revealed that BEST4 is predominantlyexpressed at the basolateral membrane of pan-cytokeratin or E-cadherin positive IECs. Such
S-131 AGA Abstracts
BEST4-positive IECs were completely negative for Ki-67 expression, and resided exclusivelyat the villus of the small intestine, or at the surface epithelium of the colon, suggesting thatthese cells are all post-mitotic. Co-staining analysis revealed that BEST4-positive IECs arecompletely negative for secretory lineage markers such as MUC2, Chromogranin-A, Lysozymeor HPGDS. In sharp contrast, BEST4-positive IECs completely co-localized with absorptivecell specific markers such as Villin or CD10, which clearly suggested that BEST4 is anabsorptive cell-specific CaCC. Consistently, forced activation of Notch signaling, a keymolecular pathway for absorptive cell differentiation, significantly increased BEST4 proteinexpression at the basolateral membrane of LS174T cells. Such a Notch-dependent up-regulation of BEST4 expression in LS174T cells appeared to be regulated at the mRNAlevel, as confirmed by quantitative RT-PCR analysis. Conclusion: Notch signaling promotesexpression of a novel absorptive cell specific HCO3-/Cl- channel, BEST4, in human IECs.Such a lineage-specific expression of BEST4 suggests its possible involvement in absorptivecell specific regulation of water and electrolyte absorption, via HCO3-/Cl- exchange.
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Large Conductance K+ (BK) Channel-Mediated K+ Secretion Provides theDriving Force for Water Secretion in Rat Distal ColonDeeban Ganesan, Kevin J. Engels, Geoffrey I. Sandle, Vazhaikkurichi M. Rajendran
Background: In secretory diarrhea, electrogenic Cl- secretion is regarded as the main drivingforce for intestinal water secretion, but high levels of K+ secretion may underlie high stoolvolumes in patients with colonic pseudo-obstruction (Gastroenterology 129:1268-73, 2005;Nephrol Dial Transplant 23:3350-2, 2008). Recent studies suggest that cAMP-inducedcolonic K+ secretion via large conductance apical K+ (BK) channels may account for excessivefecal K+ losses in secretory diarrhea, but it is unclear whether BK channel-mediated K+
secretion also provides a driving force for water secretion. Aim: To determine whether BKchannel-mediated K+ secretion provides a driving force for colonic water secretion. Methods:Net water transport was evaluated in the distal colon of anaesthetized control and aldosterone(‘aldo') SD rats by in vivo perfusion of Ringer solution (0.5 ml/min). ‘Aldo' rats were fed aNa+-free diet for 6-7 days. 3H-PEG 4000 was used as a non-absorbable volume marker.Positive and negative values represent net water absorption and secretion, respectively.Results: Under basal condition, net water absorption was present in both control and ‘aldo'animals. In controls, amiloride (10 μM, to inhibit Na+ channels) did not change waterabsorption, but amiloride (1 mM, to inhibit Na-H exchange) abolished water absorption(89.6 ± 6.4 μl/h.cm versus -2.6 ± 1.8 μl/h.cm; p , 0.001). In ‘aldo' animals, in which therewas both Na+ channel-mediated Na+ absorption and BK channel-mediated K+ secretion, 10μM amiloride reversed net water absorption to net secretion (92.6 ± 6.7 μl/h.cm versus-38.9 ± 4.8 μl/h.cm; p , 0.001) and 1 mM amiloride had no further effect. In presence of10 μM amiloride, net water secretion in ‘aldo' animals was completely abolished by 3 mMBa2+ (a nonspecific K+ channel blocker) and iberiotoxin (a specific BK channel blocker),but inhibited by only 16% by clotrimazole (an IK channel blocker). Furthermore, in theabsence of amiloride, iberiotoxin increased net water absorption in ‘aldo' animals (92.6 ±6.7 μl/h. cm versus 118.8 ± 7.1 μl/h.cm; p , 0.05), consistent with inhibition of a secretorywater flux driven by BK channel-mediated K+ secretion. In controls (1 mM amiloride inperfusate), BMS (a BK channel opener) significantly increased net water secretion (-2.6 ± 1.8μl/h.cm versus -8.8 ± 3.4 μl/h.cm; p , 0.05). Conclusions: In rat distal colon, enhancement ofapical BK channel expression secondary to chronic hyperaldosteronism, and acute activationof apical BK channels in controls, stimulate K+ secretion sufficiently to increase watersecretion. On the other hand, BK channel inhibition has the opposite effect on net watertransport. Taken together, these results suggest that BK channel-mediated K+ secretion mayprovide a driving force for water secretion in some types of secretory diarrhea.
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The Anion Transporter SLC26A6 (Putative Anion Transporter-1) RegulatesMurine Small Intestinal Na+HCO3- AbsorptionWeiliang Xia, Qin Yu, Brigitte Riederer, Anurag K. Singh, Regina Engelhardt, PenghongSong, Dean Tian, Manoocher Soleimani, Ursula Seidler
Background: Slc26a6 (PAT-1), a multi anion transporter, is strongly expressed in the apicalmembrane of the upper small intestine, where it is colocalized with another member of thesame gene family, Slc26a3 (DRA). Experiments in heterologous expression systems suggestthat Pat-1 may exchange HCO3- and Cl- in an electrogenic fashion, and experiments innative duodenum suggest that after intracellular acidification by H+-dipeptide transport,PAT-1 may import HCO3-. Aim: This study investigates whether PAT-1 is essential for smallintestinal fluid absorption under the unique ionic conditions of the upper GI tract, wherehigh luminal CO2 as well as high luminal HCO3- concentrations can be found after thepancreatic juice has mixed with the acidic milieu of the proximal duodenum. Methods andResults: Small intestinal fluid absorption was therefore assessed by single pass perfusion ofprewarmed, isotonic solutions of different ionic compositions but constant pH of 7.4 throughjejunal segments of anesthetized acid/base status controlled mice, and the volume and pHof the inflowing and outflowing solution was measured. WT jejunum absorbed fluid andacidified the lumen. DRA-deficient jejunum absorbed less fluid than WT, and acidified thelumen more strongly, consistent with its action as a Cl-/HCO3- exchanger. PAT-1-deficientjejunum also absorbed less fluid, but resulted in less luminal acidification. Switching theluminal solution to a 5% CO2/HCO3- buffer, pH 7.4, resulted in a persistent decrease instrongly augmented fluid absorption in a PAT-1- and Na+/H+ exchanger 3 (NHE3)- butnot DRA-dependent manner. In the absence of luminal Cl-, luminal CO2/HCO3- alsoaugumented fluid absorption in WT and DRA-deficient, but not in PAT-1- and NHE3-deficient mice, indicating that PAT-1 likely serves to import HCO3- under these circum-stances. Conclusion: The results suggest that PAT-1 may play an important role in jejunalNa+HCO3- and fluid absorption.
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