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Colonic Anion Secretory Defects and Metabolic Acidosis in Mice Lacking the NBC1 Na + / HCO 3 - Cotransporter

2006; Elsevier BV; Volume: 282; Issue: 12 Linguagem: Inglês

10.1074/jbc.m607041200

ISSN

1083-351X

Autores

Lara R. Gawenis, Emily Bradford, Vikram Prasad, John N. Lorenz, Janet E. Simpson, Lane L. Clarke, Alison L. Woo, Christina Grisham, Lynn Sanford, Thomas Doetschman, Marian L. Miller, Gary E. Shull,

Tópico(s)

Ion channel regulation and function

Resumo

The NBC1 Na+/HCO3- cotransporter is expressed in many tissues, including kidney and intestinal epithelia. NBC1 mutations cause proximal renal tubular acidosis in humans, consistent with its role in HCO3- absorption in the kidney. In intestinal and colonic epithelia, NBC1 localizes to basolateral membranes and is thought to function in anion secretion. To test the hypothesis that NBC1 plays a role in transepithelial HCO3- secretion in the intestinal tract, null mutant (NBC1-/-) mice were prepared by targeted disruption of its gene (Slc4a4). NBC1-/- mice exhibited severe metabolic acidosis, growth retardation, reduced plasma Na+, hyperal-dosteronism, splenomegaly, abnormal dentition, intestinal obstructions, and death before weaning. Intracellular pH (pHi) was not altered in cAMP-stimulated epithelial cells of NBC1-/- cecum, but pHi regulation during sodium removal and readdition was impaired. Bioelectric measurements of NBC1-/- colons revealed increased amiloride-sensitive Na+ absorption. In Ringer solution containing both Cl- andHCO3-, the magnitude of cAMP-stimulated anion secretion was normal in NBC1-/- distal colon but increased in proximal colon, with the increase largely supported by enhanced activity of the basolateral NKCC1 Na+-K+-2Cl- cotransporter. Anion substitution studies in which carbonic anhydrase was inhibited and transepithelial anion conductance was limited to HCO3- revealed a sharp decrease in both cAMP-stimulated HCO3- secretion and SITS-sensitive current in NBC1-/- proximal colon. These results are consistent with the known function of NBC1 in HCO3- absorption in the kidney and demonstrate that NBC1 activity is a component of the basolateral mechanisms for HCO3- uptake during cAMP-stimulated anion secretion in the proximal colon. The NBC1 Na+/HCO3- cotransporter is expressed in many tissues, including kidney and intestinal epithelia. NBC1 mutations cause proximal renal tubular acidosis in humans, consistent with its role in HCO3- absorption in the kidney. In intestinal and colonic epithelia, NBC1 localizes to basolateral membranes and is thought to function in anion secretion. To test the hypothesis that NBC1 plays a role in transepithelial HCO3- secretion in the intestinal tract, null mutant (NBC1-/-) mice were prepared by targeted disruption of its gene (Slc4a4). NBC1-/- mice exhibited severe metabolic acidosis, growth retardation, reduced plasma Na+, hyperal-dosteronism, splenomegaly, abnormal dentition, intestinal obstructions, and death before weaning. Intracellular pH (pHi) was not altered in cAMP-stimulated epithelial cells of NBC1-/- cecum, but pHi regulation during sodium removal and readdition was impaired. Bioelectric measurements of NBC1-/- colons revealed increased amiloride-sensitive Na+ absorption. In Ringer solution containing both Cl- andHCO3-, the magnitude of cAMP-stimulated anion secretion was normal in NBC1-/- distal colon but increased in proximal colon, with the increase largely supported by enhanced activity of the basolateral NKCC1 Na+-K+-2Cl- cotransporter. Anion substitution studies in which carbonic anhydrase was inhibited and transepithelial anion conductance was limited to HCO3- revealed a sharp decrease in both cAMP-stimulated HCO3- secretion and SITS-sensitive current in NBC1-/- proximal colon. These results are consistent with the known function of NBC1 in HCO3- absorption in the kidney and demonstrate that NBC1 activity is a component of the basolateral mechanisms for HCO3- uptake during cAMP-stimulated anion secretion in the proximal colon. Na+/HCO3- cotransporter (NBC) 2The abbreviations used are: NBC, Na+/HCO3- cotransporter (the number following NBC refers to the specific isoform); NBC1-/-, NBC1+/-, and NBC1+/+, NBC1 homozygous mutant, heterozygous mutant, and wild type, respectively; pRTA, proximal renal tubular acidosis; ES, embryonic stem; BCECF-AM, 2′,7′-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein tetraacetoxy methylester; EIPA, 5-(N-ethyl-N-isopropyl)-amiloride; SITS, 4-acetamide-4′-isothiocyanato-2,2′-stilbene disulfonic acid; CFTR, cystic fibrosis transmembrane conductance regulator; NKCC1, Na+-K+-2Cl- cotransporter; pHi, intracellular pH; ENaC, epithelial sodium channel; TES, N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid; Pipes, 1,4-piperazinediethanesulfonic acid. isoform 1 is a member of the Slc4a gene family, which includes both C1-/HCO3- exchangers andNa+/HCO3- cotransporters (1Alper S.L. Annu. Rev. Physiol. 2002; 64: 899-923Crossref PubMed Scopus (167) Google Scholar, 2Soleimani M. Burnham C.E. J. Membr. Biol. 2001; 183: 71-84Crossref PubMed Scopus (85) Google Scholar, 3Romero M.F. Fulton C.M. Boron W.F. Pflugers Arch. 2004; 447: 495-509Crossref PubMed Scopus (383) Google Scholar). NBC1 has two protein variants, which localize to basolateral membranes (4Roussa E. Nastainczyk W. Thevenod F. Biochem. Biophys. Res. Commun. 2004; 314: 382-389Crossref PubMed Scopus (45) Google Scholar) and mediate electrogenic Na+/HCO3- cotransport (2Soleimani M. Burnham C.E. J. Membr. Biol. 2001; 183: 71-84Crossref PubMed Scopus (85) Google Scholar, 3Romero M.F. Fulton C.M. Boron W.F. Pflugers Arch. 2004; 447: 495-509Crossref PubMed Scopus (383) Google Scholar). The kNBC1 variant is expressed in kidney epithelia and eye (4Roussa E. Nastainczyk W. Thevenod F. Biochem. Biophys. Res. Commun. 2004; 314: 382-389Crossref PubMed Scopus (45) Google Scholar, 5Bok D. Schibler M.J. Pushkin A. Sassani P. Abuladze N. Naser Z. Kurtz I. Am. J. Physiol. 2001; 281: F920-F935Crossref PubMed Google Scholar), and the pNBC1 variant is expressed in pancreas, duodenum, colon, and several other tissues (4Roussa E. Nastainczyk W. Thevenod F. Biochem. Biophys. Res. Commun. 2004; 314: 382-389Crossref PubMed Scopus (45) Google Scholar – 8Jacob P. Christiani S. Rossmann H. Lamprecht G. Viellard-Baron D. Muller R. Gregor M. Seidler U. Gastroenterology. 2000; 119: 406-418Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). The stoichiometry of the transporter can be altered from 1Na+/2HCO3- to 1Na+/3HCO3- by phosphorylation of a residue near the carboxyl terminus (9Gross E. Kurtz I. Am. J. Physiol. 2002; 283: F876-F887PubMed Google Scholar). In the kidney, the ion stoichiometry and electrochemical driving forces for NBC1 result in Na+ and HCO3- extrusion across the basolateral membrane (2Soleimani M. Burnham C.E. J. Membr. Biol. 2001; 183: 71-84Crossref PubMed Scopus (85) Google Scholar, 3Romero M.F. Fulton C.M. Boron W.F. Pflugers Arch. 2004; 447: 495-509Crossref PubMed Scopus (383) Google Scholar, 9Gross E. Kurtz I. Am. J. Physiol. 2002; 283: F876-F887PubMed Google Scholar, 10Gross E. Hopfer U. J. Membr. Biol. 1996; 152: 245-252Crossref PubMed Scopus (33) Google Scholar); thus, in the kidney, NBC1 functions in HCO3- reabsorption in the proximal tubule (2Soleimani M. Burnham C.E. J. Membr. Biol. 2001; 183: 71-84Crossref PubMed Scopus (85) Google Scholar, 3Romero M.F. Fulton C.M. Boron W.F. Pflugers Arch. 2004; 447: 495-509Crossref PubMed Scopus (383) Google Scholar). In pancreas and the intestinal tract, the ion stoichiometry and driving forces for NBC1 appear to result in Na+ and HCO3- entry into the cell (2Soleimani M. Burnham C.E. J. Membr. Biol. 2001; 183: 71-84Crossref PubMed Scopus (85) Google Scholar, 8Jacob P. Christiani S. Rossmann H. Lamprecht G. Viellard-Baron D. Muller R. Gregor M. Seidler U. Gastroenterology. 2000; 119: 406-418Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar); thus, in intestine and colon, NBC1 has been proposed to mediate HCO3- uptake across the basolateral membrane to support transepithelial anion secretion (8Jacob P. Christiani S. Rossmann H. Lamprecht G. Viellard-Baron D. Muller R. Gregor M. Seidler U. Gastroenterology. 2000; 119: 406-418Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 11Bachmann O. Reichelt D. Tuo B. Manns M.P. Seidler U. Am. J. Physiol. 2006; 291: G650-G657Crossref PubMed Scopus (34) Google Scholar). Human patients with proximal renal tubular acidosis resulting from mutations in NBC1 have been reported (12Igarashi T. Inatomi J. Sekine T. Cha S.H. Kanai Y. Kunimi M. Tsukamoto K. Satoh H. Shimaszu M. Tozawa F. Mori T. Shiobara M. Seki G. Endou H. Nat. Genet. 1999; 23: 264-266Crossref PubMed Scopus (246) Google Scholar, 13Dinour D. Chang M.H. Satoh J. Smith B.L. Angle N. Knecht A. Serban I. Holtzman E.J. Romero M.F. J. Biol. Chem. 2004; 279: 52238-52246Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 14Horita S. Yamada H. Inatomi J. Moriyama N. Sekine T. Igarashi T. Endo Y. Dasouki M. Ekim M. Al-Gazali L. Shimadzu M. Seki G. Fujita T. J. Am. Soc. Nephrol. 2005; 16: 2270-2278Crossref PubMed Scopus (99) Google Scholar, 15Igarashi T. Inatomi J. Sekine T. Seki G. Shimadzu M. Tozawa F. Takeshima Y. Takumi T. Takahashi T. Yoshikawa N. Nakamura H. Endou H. J. Am. Soc. Nephrol. 2001; 12: 713-718Crossref PubMed Google Scholar, 16Inatomi J. Horita H. Braverman N. Sekine T. Yamada H. Suzuki Y. Kawahara K. Moriyama N. Kudo A. kawakami H. Shimadzu M. Endou H. Fujita T. Seki G. Igarashi T. Pfluegers Arch. 2004; 448: 438-444Crossref PubMed Scopus (79) Google Scholar), thereby confirming a bicarbonate-absorptive role for NBC1 in kidney. The primary mutations were single amino acid substitutions (R298S, T485S, R510H, A799V, R881C, and S427L), which appeared to cause decreased function of the cotransporter rather than loss of function (12Igarashi T. Inatomi J. Sekine T. Cha S.H. Kanai Y. Kunimi M. Tsukamoto K. Satoh H. Shimaszu M. Tozawa F. Mori T. Shiobara M. Seki G. Endou H. Nat. Genet. 1999; 23: 264-266Crossref PubMed Scopus (246) Google Scholar, 13Dinour D. Chang M.H. Satoh J. Smith B.L. Angle N. Knecht A. Serban I. Holtzman E.J. Romero M.F. J. Biol. Chem. 2004; 279: 52238-52246Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 14Horita S. Yamada H. Inatomi J. Moriyama N. Sekine T. Igarashi T. Endo Y. Dasouki M. Ekim M. Al-Gazali L. Shimadzu M. Seki G. Fujita T. J. Am. Soc. Nephrol. 2005; 16: 2270-2278Crossref PubMed Scopus (99) Google Scholar). One patient had an inactivating mutation in the unique N terminus of the kidney NBC1 variant (Q29X), but the pancreatic variant, which is expressed in many other tissues and at low levels in kidney (4Roussa E. Nastainczyk W. Thevenod F. Biochem. Biophys. Res. Commun. 2004; 314: 382-389Crossref PubMed Scopus (45) Google Scholar), was intact (15Igarashi T. Inatomi J. Sekine T. Seki G. Shimadzu M. Tozawa F. Takeshima Y. Takumi T. Takahashi T. Yoshikawa N. Nakamura H. Endou H. J. Am. Soc. Nephrol. 2001; 12: 713-718Crossref PubMed Google Scholar). Only a single patient has been identified with a complete inactivating mutation, a nucleotide deletion that causes a frameshift at codon 721 (16Inatomi J. Horita H. Braverman N. Sekine T. Yamada H. Suzuki Y. Kawahara K. Moriyama N. Kudo A. kawakami H. Shimadzu M. Endou H. Fujita T. Seki G. Igarashi T. Pfluegers Arch. 2004; 448: 438-444Crossref PubMed Scopus (79) Google Scholar). The pRTA resulting from NBC1 mutations clearly shows that this transporter is essential for renal HCO3- absorption; however, clinically significant intestinal disease has not been reported. NBC1 has been localized to the basolateral membrane of epithelial cells lining both the small and large intestine (8Jacob P. Christiani S. Rossmann H. Lamprecht G. Viellard-Baron D. Muller R. Gregor M. Seidler U. Gastroenterology. 2000; 119: 406-418Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 17Praetorius J. Hager H. Nielson S. Aalkjaer C. Friis U.G. Ainsworth M.A. Johansen T. Am. J. Physiol. 2001; 280: G332-G343PubMed Google Scholar, 18Seidler U. Bachmann O. Jacob P. Christiani S. Blumenstein I. Rossmann H. J. Pancreas. 2001; 2: 247-256Google Scholar). In the colon, its expression was greatest in crypt cells, consistent with a role in transepithelial anion secretion (19Bachmann O. Rossmann H. Berger U. Colledge W.H. Ratcliff R. Evans M.J. Gregor M. Seidler U. Am. J. Physiol. 2003; 284: G37-G45Google Scholar). Previous studies investigatingNa+/HCO3- cotransport activity in intestinal epithelia using relatively nonspecific inhibitors of HCO3- transport support the hypothesis that NBC1 is a component of the basolateral anion uptake mechanisms that facilitate transepithelial anion secretion and regulate pHi (18Seidler U. Bachmann O. Jacob P. Christiani S. Blumenstein I. Rossmann H. J. Pancreas. 2001; 2: 247-256Google Scholar, 19Bachmann O. Rossmann H. Berger U. Colledge W.H. Ratcliff R. Evans M.J. Gregor M. Seidler U. Am. J. Physiol. 2003; 284: G37-G45Google Scholar, 20Romero M.F. Boron W.F. Annu. Rev. Physiol. 1999; 61: 699-723Crossref PubMed Scopus (183) Google Scholar); however, at least two additional Na+/HCO3- cotransporters, the electroneutral NBCn1 and the electrogenic NBC4, are also expressed in intestinal epithelia (17Praetorius J. Hager H. Nielson S. Aalkjaer C. Friis U.G. Ainsworth M.A. Johansen T. Am. J. Physiol. 2001; 280: G332-G343PubMed Google Scholar, 21Pushkin A. Abuladze N. Newman D. Lee I. Xu G. Kurtz I. Biochim. Biophys. Acta. 2000; 1493: 215-218Crossref PubMed Scopus (80) Google Scholar). NBCn1 is present on the basolateral membrane of small intestinal enterocytes (17Praetorius J. Hager H. Nielson S. Aalkjaer C. Friis U.G. Ainsworth M.A. Johansen T. Am. J. Physiol. 2001; 280: G332-G343PubMed Google Scholar) and is co-expressed with NBC1 in colonic crypts (18Seidler U. Bachmann O. Jacob P. Christiani S. Blumenstein I. Rossmann H. J. Pancreas. 2001; 2: 247-256Google Scholar), whereas the membrane localization of NBC4 has not been determined in intestinal epithelia (21Pushkin A. Abuladze N. Newman D. Lee I. Xu G. Kurtz I. Biochim. Biophys. Acta. 2000; 1493: 215-218Crossref PubMed Scopus (80) Google Scholar). To understand the role of NBC1 in transepithelial anion secretion in the intestinal tract and also to assess its importance in kidney and other tissues, we developed a mouse carrying a targeted disruption of the Slc4a4 gene. Our data show that the loss of NBC1 in mice causes severe metabolic acidosis and impaired transepithelial HCO3- secretion in the colon. These results are consistent with the known function of NBC1 in bicarbonate absorption in the kidneys and demonstrate that it also serves a major secretory function in the intestinal tract. Preparation of Targeting Construct and Generation of Mutant Mice—PCR generated fragments of the mouse Slc4a4 gene and the pMJKO vector (22Schultheis P.J. Clarke L.L. Meneton P. Harline M. Boivin G.P. Stemmermann G. Duffy J.J. Doetschman T. Miller M.L. Shull G.E. J. Clin. Invest. 1998; 101: 1243-1253Crossref PubMed Scopus (222) Google Scholar) were used for the targeting construct. A 3.2-kb genomic fragment starting in intron 7 and ending in exon 9 was inserted into a cloning site 5′ to the promoter of the neomycin resistance gene, and a 2.3-kb fragment corresponding to sequences in intron 9 was inserted between the 3′ end of the neomycin resistance gene and the herpes simplex virus thymidine kinase gene. Targeting of ES cells (cells derived from 129S6/SvEv Tac (Taconic, Germantown, NY) mice) was performed as previously described (22Schultheis P.J. Clarke L.L. Meneton P. Harline M. Boivin G.P. Stemmermann G. Duffy J.J. Doetschman T. Miller M.L. Shull G.E. J. Clin. Invest. 1998; 101: 1243-1253Crossref PubMed Scopus (222) Google Scholar). ES cells that were positive for homologous recombination were identified by Southern blot analysis of EcoRV-digested DNA using a 5′ probe corresponding to genomic sequences from intron 6. Targeted ES cells were used to generate chimeric mice that were bred with wild-type Black Swiss females (Taconic). All studies were performed using mice of the mixed 129S6/SvEv and Black Swiss background with wild-type age-matched littermates serving as controls. At ∼5 days of age, offspring were genotyped by PCR analysis of tail DNA using the following primers: a forward primer from the deleted region of intron 9 (5′-TCACAAACCTTTCAGCAAAAGAGTGC-3′) that identifies only the wild-type allele; a reverse primer from intron 9 (5′-CAAAGAGCAACAGTCAGACAGC-3′) that identifies both wild-type and mutant alleles; and a primer from the neomycin resistance gene (5′-GACAATAGCAGGCATGCTGG-3′) that identifies only the mutant allele. Amplification using DNA from a tail biopsy and all three primers in the same reaction yields 269- and 241-bp products for the wild-type and mutant alleles, respectively. Northern Blot Analysis—Total RNA (30 μg) isolated from tissues of 14-day-old mice using Tri Reagent (Molecular Research Center, Inc., Cincinnati, OH) was denatured with glyoxal, fractionated by electrophoresis in 1% agarose, transferred to a nylon membrane, and hybridized with a 32P-labeled NBC1 cDNA probe. Histology—Tissue samples (brain, eye, kidney, spleen, pancreas, small intestine, cecum, proximal colon, distal colon, lung, and heart) were fixed in 10% neutral buffered formalin, dehydrated, and embedded in paraffin. Samples were stained with hematoxylin and eosin and examined for histological changes by light microscopy. Analysis of Blood and Blood Counts—At 14–15 days of age, NBC1+/+, NBC1+/-, and NBC1-/- pups were sacrificed by decapitation, and trunk blood was collected into heparinized tubes for analysis on a blood gas analyzer (Chiron Diagnostics model 248, Norwood, MA). Plasma Na+ and K+ concentrations were determined using flame photometry (model 480; Corning Glass). For peripheral blood counts, blood smears were obtained from the tail vein and allowed to dry. Samples were then stained for 2 min with Giemsa stain, washed in distilled water, and coverslipped using CytoSeal. Smears were analyzed for the percentage of nucleated red blood cells, myelocytes (includes myelocytes, neutrophils, eosinophils, and basophils), lymphocytes, and mononuclear white blood cells. Analysis of Serum Aldosterone—Mice were euthanized with CO2, and blood was drawn by cardiac puncture. Serum was separated from whole blood via centrifugation and was stored at -80 °C. Serum from two mice was pooled together and diluted 1:4 in phosphate-buffered saline; a total of 14 mice were used for each genotype. Aldosterone concentrations were determined using a commercially available radioimmunoassay kit (Diagnostic Products, Los Angeles, CA) according to the manufacturer's directions. Samples were counted using a γ counter (Packard Instrument Co.). BCECF Microfluorimetry—The method used for imaging intact intestinal epithelium of the cecum was based on a previously described technique for imaging epithelial cells in intact duodenum (23Gawenis L.R. Franklin C.L. Simpson J.E. Palmer B.A. Walker N.M. Wiggins T.M. Clarke L.L. Gastroenterology. 2003; 125: 1148-1163Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Following asphyxiation in 100% CO2 and bilateral pneumothorax, the cecum of 14–18-day-old animals was removed and placed immediately in an oxygenated, ice-cold Ringer solution. The ceca were opened along the mesenteric border, and the serosa and muscularis externa were removed from the underlying mucosa as previously described (24Simpson J.E. Gawenis L.R. Walker N.M. Boyle K.T. Clarke L.L. Am. J. Physiol. 2005; 288: G1241-G1251Crossref PubMed Scopus (59) Google Scholar). The muscle-stripped preparations were mounted basolateral (serosal) side up on a horizontal bilateral perfusion chamber (24Simpson J.E. Gawenis L.R. Walker N.M. Boyle K.T. Clarke L.L. Am. J. Physiol. 2005; 288: G1241-G1251Crossref PubMed Scopus (59) Google Scholar). The luminal (mucosal) and serosal surfaces were independently bathed with Ringer solution containing 1 μm indomethacin to minimize the effect of endogenous prostaglandins and 10 μm forskolin (to stimulate anion secretion and basolateral anion uptake) (25Bukhave K. Rask-Madsen J. Gastroenterology. 1980; 78: 32-42Abstract Full Text PDF PubMed Scopus (103) Google Scholar, 26Sheldon R.J. Malarchik M.E. Fox D.A. Burks T.F. Porreca F. J. Pharmacol. Exp. Ther. 1989; 249: 572-582PubMed Google Scholar). In addition, the serosal bathing medium contained 0.1 μm tetrodotoxin to minimize neural tone and 1 μm EIPA to inhibit activity of the basolateral Na+/H+ exchanger. The cecal segments were incubated with 16 μm BCECF-AM for 10 min on the luminal side in a modified Krebs bicarbonate Ringer solution containing TES and 140.0 mm Na+, 114.8 mm Cl-, 5.0 mm TES, 25.0 mm HCO3-, 5.2 mm K+, 2.8 mm PO42-, 1.2 mm Ca2+, 1.2 mm Mg2+, and 16.8 mm glucose that was gassed with 95% O2, 5% CO2 at 37 °C (pH 7.4). As described previously (24Simpson J.E. Gawenis L.R. Walker N.M. Boyle K.T. Clarke L.L. Am. J. Physiol. 2005; 288: G1241-G1251Crossref PubMed Scopus (59) Google Scholar), ∼10 epithelial cells were selected for ratiometric analysis of BCECF fluorescence. During Na+ removal, the serosal surface of the tissue was bathed in a Na+-free Ringer solution containing 140.0 mm N-methyl-d-glucamine, 114.8 mm Cl-, 5.0 mm TES, 25.0 mm choline, 25.0 mm HCO3-, 5.2 mm K+, 2.8 mm PO42-, 1.2 mm Ca2+, 1.2 mm Mg2+, and 16.8 mm glucose (gassed with 95% O2, 5% CO2 at 37 °C, pH 7.4). Ussing Chamber Analysis—Following asphyxiation of 14–18-day-old animals in 100% CO2 and bilateral thoracotomy, proximal, and distal colon samples were removed by a midline incision and placed in oxygenated, ice-cold Ringer solution (with 1 μm indomethacin). Tissues were opened along the mesenteric border and the muscle layers underlying the mucosa of the proximal colon segments removed by sharp dissection. Distal colon segments were used with the muscle layer intact. Colons were mounted in standard Ussing chambers (0.1-cm2 exposed surface area), and the mucosal and serosal surfaces were independently bathed in 4 ml of Krebs-bicarbonate Ringer solution (37 °C, gassed with 95% O2 and 5% CO2)as described previously (27Gawenis L.R. Boyle K.T. Palmer B.A. Walker N.M. Clarke L.L. Am. J. Physiol. 2004; 286: G1015-G1023Google Scholar, 28Gawenis L.R. Stien X. Shull G.E. Schultheis P.J. Woo A.L. Walker N. Clarke L.L. Am. J. Physiol. 2002; 282: G766-G784Google Scholar). For Cl- substitution experiments, gluconate was substituted on an equimolar basis. To minimize the variations in neural tone and the generation of endogenous prostanoids, tetrodotoxin (0.1 μm serosal) and indomethacin (1 μm, mucosal and serosal) were added to the bathing solutions (25Bukhave K. Rask-Madsen J. Gastroenterology. 1980; 78: 32-42Abstract Full Text PDF PubMed Scopus (103) Google Scholar, 26Sheldon R.J. Malarchik M.E. Fox D.A. Burks T.F. Porreca F. J. Pharmacol. Exp. Ther. 1989; 249: 572-582PubMed Google Scholar). Forskolin (10 μm) and 3-isobutyl-1-methylxanthine (100 μm) were added to the mucosal and serosal baths of amiloride-pretreated (10 μm) tissues to stimulate intracellular cAMP prior to the sequential addition of bumetanide (100 μm) and SITS (1 mm) to the serosal side. For all experiments, the final concentration of dimethyl sulfoxide in the bath Ringer solution was maintained at or below 0.1%. Transepithelial short circuit current (Isc; reported as μA/cm2 tissue surface area) was measured using an automatic voltage clamp (VCC-600; Physiologic Instruments, San Diego, CA) as previously described (29Clarke L.L. Harline M.C. Am. J. Physiol. 1998; 274: G718-G726PubMed Google Scholar). Transepithelial conductance (Gt, reported in mS/cm2 tissue surface area) was determined at 5-min intervals during the experiment by measuring the magnitude of the current deflection from application of a 5-mV pulse across each tissue and applying Ohm's law. All experiments were performed under short circuited conditions with the serosal bath serving as ground. Immunoblot Analysis—Mice were euthanized, and the colons were removed, cleaned in ice-cold 1× phosphate-buffered saline, flash-frozen in liquid nitrogen, and stored at -70 °C until further processing. Frozen tissues were pulverized in liquid nitrogen using a tissue grinder, and the tissue powder was suspended in prechilled 1× homogenization buffer (10 mm NaCl, 20 mm Pipes (pH 7.0), 5 mm EDTA, 0.5% Nonidet P-40, 2 mm dithiothreitol plus protease inhibitors and phosphatase inhibitors). The samples were then homogenized using a Polytron 3000 homogenizer, and the proteins were allowed to solubilize over ice for 2 h. Protein concentration was estimated by the Bradford method. The presence of NKCC1 protein and phosphorylated NKCC1 was determined by immunoblotting after separation of lysate proteins by electrophoresis on a discontinuous, 7% reducing SDS-polyacrylamide gel. The antibodies (described in Refs. 30Lytle C. Xu J.C. Biemesderfer D. Forbush 3rd, B. Am. J. Physiol. 1995; 269: C1496-C1505Crossref PubMed Google Scholar and 31Flemmer A.W. Gimenez I. Dowd B.F.X. Darman R.B. Forbush B. J. Biol. Chem. 2002; 277: 37551-37558Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar) used were T4 for NKCC1 (contributed to the Developmental Studies Hybridoma Bank, University of Iowa (Iowa City, IA) by C. Lytle and B. Forbush), R5 for NKCC1 that was phosphorylated on two regulatory threonine residues (a gift from Dr. Biff Forbush, Yale University), and antiactin (Sigma A4700) for the loading control. Statistics—A two-tailed unpaired Student's t test assuming equal variances was used to compare data from two treatment groups. A one-way analysis of variance with a post hoc Tukey's t test was used for comparisons among more than two treatment groups. A probability value of p < 0.05 was considered statistically significant. All values are reported as the mean ± S.E. Materials—All reagents were obtained from Sigma. Tetrodotoxin was dissolved at a stock concentration of 100 μm in 0.2% acetic acid. Forskolin and indomethacin were dissolved at stock concentrations of 10 mm in dimethyl sulfoxide. 3-Isobutyl-1-methylxanthine and amiloride were dissolved at stock concentrations of 10 mm in sterile water. EIPA was dissolved at a stock concentration of 1 mm in dimethyl sulfoxide. Acetazolamide and SITS were dissolved at stock concentrations of 10 and 100 mm in the perfusion Ringer solution for the experiment in which they were used (i.e. Krebs bicarbonate Ringer or Cl--free Ringer solution). Bumetanide was dissolved at a stock concentration of 100 mm in ethanol. Generation of NBC1 Null Mutant Mice—The targeting procedure replaced sequences that include part of exon 9 (including the 3′ splice site), which is present in the mRNAs for both NBC1 variants, and part of intron 9 with the neomycin resistance gene (Fig. 1A). Chimeric male mice generated using the targeted ES cells were bred to wild-type females to produce NBC1+/- mice, and breeding of heterozygous mutant mice resulted in live offspring of all three genotypes (Fig. 1, B and C). Northern blot analysis of kidney, small intestine, and colon demonstrated that the NBC1 mRNA was ablated in NBC1-/- mice (Fig. 1D). Gross Phenotype—Genotype frequencies of pups obtained from heterozygous matings exhibited a normal 1:2:1 Mendelian ratio (25.9% wild type, 48.6% heterozygous, and 25.5% null mutant among more than 1000 pups), with no alteration in the percentage of male or female null mutant mice. At birth, ∼25% of NBC1-/- mice were notably smaller than their wild-type and heterozygous littermates. As they developed during the postnatal period, all of the knockouts were emaciated, exhibited severe growth retardation (Fig. 2, A and B), and their teeth had a chalky white appearance and were easily chipped. NBC1 null mutants had poor survival, with death beginning about 5 days after birth and no survival beyond 24 days (Fig. 2C). Of the knock-out pups that survived to at least 20 days of age, ∼80% exhibited mild to severe intestinal impactions in the terminal ileum, cecum, and colon; however, the intestinal tracts of mice that died at much earlier ages were not obstructed. Regardless of whether they had intestinal impactions, 12–21-day-old NBC1-null mice had small, corkscrew ceca, a phenotype that has been observed in mice lacking either the CFTR Cl- channel (32Snouwaert J.N. Brigman K.K. Latour A.M. Malouf N.N. Boucher R.C. Smithies O. Koller B.H. Science. 1992; 257: 1083-1088Crossref PubMed Scopus (769) Google Scholar, 33Clarke L.L. Gawenis L.R. Franklin C.L. Harline M.C. Lab. Anim. Sci. 1996; 46: 612-618PubMed Google Scholar) or the NKCC1 Na+-K+-2Cl- cotransporter (34Flagella M. Clarke L.L. Miller M.L. Erway L.C. Giannella R.A. Andringa A. Gawenis L.R. Kramer J. Duffy J.J. 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Satoh H. Shimaszu M. Tozawa F. Mori T. Shiobara M. Seki G. Endou H. Nat. Genet. 1999; 23: 264-266Crossref PubMed Scopus (246) Google Scholar, 13Dinour D. Chang M.H. Satoh J. Smith B.L. Angle N. Knecht A. Serban I. Holtzman E.J. Romero M.F. J. Biol. Chem. 2004; 279: 52238-52246Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 14Horita S. Yamada H. Inatomi J. Moriyama N. Sekine T. Igarashi T. Endo Y. Dasouki M. Ekim M. Al-Gazali L. Shimadzu M. Seki G. Fujita T. J. Am. Soc. Nephrol. 2005; 16:

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