Pancreatic duct epithelium secretes HCO3--rich fluid, which is dependent on cystic fibrosis transmembrane conductance regulator (CFTR). HCO3- transport across the apical membrane is thought to be mediated by both SLC26A6 Cl--HCO3- exchange and CFTR HCO3- conductance. In this study we examined the relative contribution and interaction of SLC26A6 and CFTR in apical HCO3- transport. Interlobular pancreatic ducts were isolated from slc26a6 null mice. Intracellular pH (pHi) was measured by BCECF microfluorometry. Duct cells were stimulated with forskolin and alkalinized by acetate pre-pulse in the presence ofHCO3--CO2. Apical HCO3- secretion was estimated from the recovery rate of pHi from alkaline load. When the lumen was perfused with high-Cl- solution, the rate of apical HCO3- secretion was increased by luminal application of CFTRinh-172 in ducts from wild-type mice but it was decreased in ducts from slc26a6 -/- mice. This suggests that slc26a6 and CFTR compensate/compete with each other for apical HCO3- secretion with high Cl- in the lumen. With high HCO3- in the lumen, luminal CFTRinh-172 reduced the rate of apical HCO3- secretion in both wild-type and slc26a6 -/- ducts. This suggests that HCO3- conductance of CFTR mediates a significant portion of apical HCO3- secretion with high HCO3- in the lumen

Ishiguro, Hiroshi

Jin, Chun Xiang

Kondo, Takaharu

Song, Ying

Yamamoto, Akiko

Tokushima University Institutional Repository

SLC26A6 MEDIATES APICAL Cl--HCO3-EXCHANGE IN PANCREATIC DUCTThe pancreatic duct system of humans producesapproximately 2.5 liters per day of isotonic fluid con-taining140 mM HCO3-, which is dependent on cys-tic fibrosis transmembrane conductance regulator(CFTR). The HCO3--rich ductal secretion acts asboth a vehicle for acini-derived digestive enzymesand a buffer for duodenal acidity. We have beenstudying the mechanisms of HCO3- secretion by pan-creatic duct cell by using interlobular duct segmentsisolated from guinea-pig pancreas (1). The pancre-atic juice of guinea-pig contains140 mM HCO3-.Our data suggest that HCO3- secretion across theapical membrane is mediated by both Cl- -HCO3- ex-change and HCO3- conductance of CFTR and thatthe relative contribution of these 2 apical mecha-nisms varies depending on the anion compositionof the luminal fluid (Fig. 1). A computer model sug-gested that75% of apical HCO3- secretion is medi-ated by Cl- -HCO3- exchange in proximal pancreaticducts close to acini where the lumen is filled withacini-derived Cl- -rich fluid (2).MINI-REVIEWEffects of Slc26a6 deletion and CFTR inhibition on HCO3-secretion by mouse pancreatic ductYing Song1,2, Hiroshi Ishiguro2, Akiko Yamamoto2, Chun Xiang Jin1, andTakaharu Kondo21Second Clinical College of Norman Bethune Medical Division , Jilin University, Changchun, China ;and 2Department of Human Nutrition, Nagoya University Graduate School of Medicine, Nagoya, JapanAbstract : Pancreatic duct epithelium secretes HCO3- -rich fluid, which is dependent oncystic fibrosis transmembrane conductance regulator (CFTR). HCO3- transport across theapical membrane is thought to be mediated by both SLC26A6 Cl--HCO3- exchange andCFTR HCO3- conductance. In this study we examined the relative contribution and interac-tion of SLC26A6 and CFTR in apical HCO3- transport. Interlobular pancreatic ducts wereisolated from slc26a6 null mice. Intracellular pH (pHi) was measured by BCECF micro-fluorometry. Duct cells were stimulated with forskolin and alkalinized by acetate pre-pulsein the presence of HCO3--CO2. Apical HCO3- secretion was estimated from the recoveryrate of pHi from alkaline load. When the lumen was perfused with high-Cl- solution, therate of apical HCO3- secretion was increased by luminal application of CFTRinh-172 inducts from wild-type mice but it was decreased in ducts from slc26a6 -/- mice. This sug-gests that slc26a6 and CFTR compensate/compete with each other for apical HCO3- se-cretion with high Cl- in the lumen. With high HCO3- in the lumen, luminal CFTRinh-172reduced the rate of apical HCO3- secretion in both wild-type and slc26a6 -/- ducts. This sug-gests that HCO3- conductance of CFTR mediates a significant portion of apical HCO3- secre-tion with high HCO3- in the lumen. J. Med. Invest. 56 Suppl. : 332-335, December, 2009Keywords : pancreatic duct cell, HCO3- transport, CFTR, slc26A6Received for publication October 14, 2009 ; accepted October21, 2009.Address correspondence and reprint requests to Hiroshi Ishiguro,Research Center of Health, Physical Fitness, and Sports, NagoyaUniversity, (Human Nutrition, Nagoya University Graduate Schoolof Medicine), Furo-cho E5-2 (130), Chikusa-ku, Nagoya 464-8601, Japan and Fax : +81-52-789-3957.The Journal of Medical Investigation Vol. 56 Supplement 2009332It is now generally accepted that apical Cl- -HCO3-exchange of epithelial tissues including pancreaticduct is mediated by members of SLC26 family of an-ion transporters (3). SLC26A3 and SLC26A6 havebeen identified in pancreatic duct cells (4), butSLC26A6 is likely the major Cl- -HCO3- exchanger inthe apical membrane. This is because apical Cl- -HCO3- exchange is DIDS-sensitive (5) and SLC26A6and CFTR are co-localized in the apical membraneof rat pancreatic intralobular ducts (5).FUNCTIONAL CHARACTERISTICS OFSLC26A6 EXPRESSED IN THE HETEROLO-GOUS SYSTEMHeterologous expression studies have demon-strated that SLC26A6 operates in multiple transportmodes : Cl- -formate exchange, Cl- -oxalate exchange,sulfate-oxalate exchange, Cl- -OH- exchange, and Cl- -HCO3- exchange (6). Recently mouse slc26a6 wasreported to be electrogenic and mediate 1Cl- -2HCO3-exchange (7) and this stoichiometry/electrogenicitymay help achieve higher HCO3- concentrations inthe pancreatic juice. However, human SLC26A6 andmouse slc26a6 share only 78% amino acid identityand exhibit significant functional differences (8).PHENOTYPE OF SLC26A6 NULL MICESLC26A6 is expressed in the small intestine,pancreatic duct, kidney proximal tubule, and gas-tric parietal cells. Slc26a6 null mice appearedhealthy and exhibited normal growth, blood pres-sure, glomerular filtration rate, and serum electro-lyte profile. In the duodenum, prostaglandin E2-stimulated HCO3- secretion was reduced, whereasforskolin-stimulated component of HCO3- secretionwas not affected (9). In kidney proximal tubule,oxalate-stimulated NaCl absorption and apical Cl- -HCO3- exchange were reduced. Calcium oxalateurolithiasis was frequently found, which was prob-ably due to defective oxalate secretion in the smallintestine (10).FLUID AND HCO3- SECRETION BY PAN-CREATIC DUCT IN SLC26A6 NULL MICETwo laboratories investigated the effects of slc26a6deletion on HCO3- transport in pancreatic duct cellby using interlobular pancreatic ducts isolated fromslc26a6 null mice. In our study, there were no effectson basal and forskolin-stimulated fluid secretion insealed ducts and the effects on apical Cl- -HCO3- ex-change in luminally-microperfused ducts were op-posite in 2 alternative directions (11). HCO3- ef-flux in exchange for luminal Cl- was reduced, whileHCO3- influx from the lumen in exchange for intra-cellular Cl- was enhanced. The data suggested thatslc26a6 mediates Cl- -dependent HCO3- secretion andthe unidirectionality is consistent with 1Cl- -2HCO3-stoichiometry (11). Another laboratory found thatFig. 1 Hypothetical roles of SLC26A6 Cl- -HCO3- exchange andCFTR HCO3- conductance in HCO3- secretion by pancreatic ductIn proximal pancreatic ducts close to acini where the lumen isfilled with acini -derived Cl- -rich fluid, SLC26A6 Cl- -HCO3- ex-change and CFTR HCO3- conductance compensate/compete witheach other for apical HCO3- secretion. The former provides amajor route. As a result of HCO3- secretion across ductal epithe-lium, luminal [HCO3-] gradually rises with distance along the duct(and luminal [Cl-] decreases). According to the changes in lumi-nal anion composition, the relative contribution of Cl- -HCO3- ex-change and HCO3- conductance was reversed. As luminal [HCO3-]increases to 140 mM, Cl- -HCO3- exchanger of any stoichiometry(SLC26A3 and SLC26A6) approaches to their equilibrium andcannot support HCO3- secretion. Here HCO3- conductance ofCFTR provides a major route for apical HCO3- secretion.The Journal of Medical Investigation Vol. 56 Supplement December 2009 333the basal levels of Cl- -HCO3- exchange and HCO3-secretion were enhanced in sealed ducts fromslc26a6-/- mice and secretin failed to stimulateHCO3- secretion (12). The data suggest that slc26a6mediates most of cAMP-stimulated HCO3- secretion.Despite these discrepancies, volume and pH of thepancreatic juice collected in vivo under secretinstimulation was not affected by deletion of slc26a6.Slc26a6 Cl- -HCO3- exchange may operate only invery proximal part of the duct system and/or thecontribution of slc26a6 to overall ductal HCO3- se-cretion may be small in mouse pancreas.INTERACTION OF SLC26A6 AND CFTR INHETEROLOGOUS SYSTEMHeterologous expression studies have demon-strated that the activation of SLC26A6 Cl- -HCO3- ex-change by cAMP largely depends on the presenceof functional CFTR and that SLC26A6 reciprocallyincreases CFTR current (13). This mutual enhance-ment is achieved by physical interactions of 2 mole-cules through the STAS domain of SLC26A6 andthe R domain of CFTR.INTERACTION OF SLC26A6 AND CFTRIN PANCREATIC DUCTThe activation of apical Cl- -HCO3- exchange bycAMP depends on the presence of functional CFTRin pancreatic duct cell similarly to the findings inheterologous expression studies, whereas SLC26A6seems to have a tonic inhibitory effect on CFTR ac-tivity in basal unstimulated condition. Activation ofapical Cl- -HCO3- exchange by cAMP was abolishedin main pancreatic duct from ΔF cystic fibrosis mice(14). When human pancreatic duct cell-line CFPAC-1 cells, which lack functional CFTR, were trans-fected with wild-type CFTR, mRNA expression ofSLC26A6 was enhanced and apical Cl- -HCO3- ex-change activity was increased (4).Recently we investigated the functional interac-tion of SLC26A6 Cl- -HCO3- exchange and CFTR(once they are expressed and activated) in cAMP-stimulated HCO3- secretion across the apical mem-brane using interlobular ducts isolated from guineapig pancreas (15). Inhibition of CFTR by luminalapplication of CFTRinh-172 enhanced both apicalCl- -HCO3- exchange activity (probably mediated bySLC26A6) and Cl- -dependent HCO3- secretion. Thedata suggest that Cl- -HCO3- exchange by SLC26A6and HCO3- conductance of CFTR compensate orcompete with each other for HCO3- secretion withCl- -rich fluid in the lumen. Membrane hyperpolari-zation upon removal of luminal Cl- support the 1Cl- -2HCO3- stoichiometry proposed for SLC26A6.SLC26A6 and CFTR are possibly coupled function-ally via changes in membrane potential.EFFECTS OF SLC26A6 DELETION ANDCFTR INHIBITION ON HCO3- SECRETIONBY THE MOUSE PANCREATIC DUCTTo examine the relative contribution and inter-action of SLC26A6 and CFTR in apical HCO3- trans-port under different anion composition in the lumen,we examined apical HCO3- secretion in interlobularpancreatic ducts (diameter :100 μm) isolated fromslc26a6 null mice. The lumen was perfused eitherwith high-Cl- (125 mM Cl--25 mM HCO3-) or high-HCO3- (25 mM Cl--125 mM HCO3-) solution. Intra-cellular pH (pHi) was measured by microfluorome-try in duct cells loaded with BCECF. Duct cells werestimulated with forskolin and alkalinized by acetatepre-pulse in the presence of HCO3--CO2. ApicalHCO3- secretion was estimated from the recoveryrate of pHi from alkaline load in the presence ofdihydro-DIDS in the bath to inhibit basolateralHCO3- flux. When the lumen was perfused with thehigh-Cl- solution, luminal application of CFTRinh-172 enhanced HCO3- secretion in ducts from wild-type mice. This finding is consistent with our pre-vious data on guinea-pig pancreatic ducts (15).When ducts from slc26a6 -/- mice were luminally-perfused with the high-Cl- solution, the rate of api-cal HCO3- secretion was significantly faster com-pared with wild-type, and it was significantly inhib-ited by luminal CFTRinh-172. The data suggest thatslc26a6 Cl- -HCO3- exchange and CFTR HCO3- con-ductance compensate/compete with each other forapical HCO3- secretion in proximal pancreatic ductsclose to acini where the lumen is filled with acini-derived Cl- -rich fluid (Fig. 1). We repeated the sameprotocol in ducts luminally-perfused with the high-HCO3- solution. In this condition, luminal applica-tion of CFTRinh-172 inhibited apical HCO3- secre-tion both in wild-type and slc26a6 -/- ducts. Thissuggests that HCO3- conductance of CFTR mediatesa significant portion of apical HCO3- secretion indistal pancreatic ducts where the luminal HCO3-concentration is already high due to ductal HCO3-Y. Song, et al. Slc26a6 and CFTR in pancreatic duct334secretion (Fig. 1).ACKNOWLEDGEMENTSThis work was supported by grants from the Japa-nese Society for the Promotion of Science and theMinistry of Health, Labor, and Welfare, Japan.Ying Song is supported by Uehara MemorialFoundation.REFERENCES1. Ishiguro H, Steward MC, Sohma Y, Kubota T,Kitagawa M, Kondo T, Case RM, Hayakawa T,Naruse S : Membrane potential and bicarbon-ate secretion in isolated interlobular ducts fromguinea-pig pancreas. J Gen Physiol 120 : 617-628, 20022. Sohma Y, Gray MA, Imai Y, Argent BE : HCO3-transport in a mathematical model of the pan-creatic ductal epithelium. J Membr Biol 176 :77-100, 20003. Steward MC, Ishiguro H, Case RM : Mecha-nisms of bicarbonate secretion in the pancre-atic duct. Annu Rev Physiol 67 : 377-409, 20054. Greeley T, Shumaker H, Wang Z, SchweinfestCW, Soleimani M : Downregulated in adenomaand putative anion transporter are regulated byCFTR in cultured pancreatic duct cells. Am JPhysiol Gastrointest Liver Physiol 281 : G1301-G1308, 20015. Shcheynikov N, Wang Y, Park M, Ko SB,Dorwart M, Naruse S, Thomas PJ, MuallemS : Coupling modes and stoichiometry of Cl-/HCO3- exchange by slc26a3 and slc26a6. J GenPhysiol 127 : 511-524, 20066. Mount DB, Romero MF : The SLC26 genefamily of multifunctional anion exchangers.Pflugers Arch 447 : 710-721, 20047. Xie Q, Welch R, Mercado A, Romero MF,Mount DB : Molecular characterization of themurine Slc26a6 anion exchanger : functionalcomparison with Slc26a1. Am J Physiol RenalPhysiol 283 : F826-F838, 20028. Chernova MN, Jiang L, Friedman DJ, DarmanRB, Lohi H, Kere J, Vandorpe DH, Alper SL :Functional comparison of mouse slc26a6 anionexchanger with human SLC26A6 polypeptidevariants : differences in anion selectivity, regu-lation, and electrogenicity. J Biol Chem 280 :8564-8580, 20059. Tuo B, Riederer B, Wang Z, Colledge WH,Soleimani M, Seidler U : Involvement of the an-ion exchanger SLC26A6 in prostaglan din E2-but not forskolin-stimulated duodenal HCO3- se-cretion. Gastroenterology 130 : 349-358, 200610. Jiang Z, Asplin JR, Evan AP, Rajendran VM,Velazquez H, Nottoli TP, Binder HJ, AronsonPS : Calcium oxalate urolithiasis in mice lack-ing anion transporter. Nat Genet 38 : 474-478,200611. Ishiguro H, Namkung W, Yamamoto A, WangZ, Worrell RT, Xu J, Lee MG, Soleimani M :Effect of Slc26a6 deletion on apical Cl-/HCO3-exchanger activity and cAMP-stimulated bi-carbonate secretion in pancreatic duct. Am JPhysiol 292 : G447-G455, 200712. Wang Y, Soyombo AA, Shcheynikov N, ZengW, Dorwart M, Marino CR, Thomas PJ,Muallem S : Slc26a6 regulates CFTR activity invivo to determine pancreatic duct HCO3- secre-tion : relevance to cystic fibrosis. EMBO J 25 :5049-5057, 200613. Ko SB, Zeng W, Dorwart MR, Luo X, KimKH, Millen L, Goto H, Naruse S, Soyombo A,Thomas PJ, Muallem S : Gating of CFTR by theSTAS domain of SLC26 transporters. Nat CellBiol 6 : 343-350, 200414. Lee MG, Choi JY, Luo X, Strickland E, ThomasPJ, Muallem S : Cystic fibrosis transmembraneconductance regulator regulates luminal Cl-/HCO3- exchange in mouse submandibular andpancreatic ducts. J Biol Chem 274 : 14670-14677, 199915. Stewart AK, Yamamoto A, Nakakuki M, KondoT, Alper SL, Ishiguro H : Functional coupling ofapical exchange with CFTR in stimulated secre-tion by guinea pig interlobular pancreatic duct.Am J Physiol 296 : G1307-G1317, 2009The Journal of Medical Investigation Vol. 56 Supplement December 2009 335

Effects of Slc26a6 deletion and CFTR inhibition on HCO3- secretion by mouse pancreatic duct

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Effects of Slc26a6 deletion and CFTR inhibition on HCO3- secretion by mouse pancreatic duct

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