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Background: Expression and activity of spermidine/spermine N1-acetyltransferase (SSAT) increases in kidneys subjected to ischemia/reperfusion (I/R) injury, while its ablation reduces the severity of such injuries. These results suggest that increased SSAT levels contribute to organ injury; however, the role of SSAT specifically expressed in proximal tubule epithelial cells, which are the primary targets of I/R injury, in the mediation of renal damage remains unresolved., Methods: Severity of I/R injury in wt and renal proximal tubule specific SSAT-ko mice (PT-SSAT-Cko) subjected to bilateral renal I/R injury was assessed using cellular and molecular biological approaches., Results: Severity of the loss of kidney function and tubular damage are reduced in PT-SSAT-Cko- compared to wt-mice after I/R injury. In addition, animals treated with MDL72527, an inhibitor of polyamine oxidases, had less severe renal damage than their vehicle treated counter-parts. The renal expression of HMGB 1 and Toll like receptors (TLR) 2 and 4 were also reduced in PT-SSAT-Cko- compared to wt mice after I/R injury. Furthermore, infiltration of neutrophils, as well as expression of tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1) and interleukin-6 (IL-6) transcripts were lower in the kidneys of PT-SSAT-Cko compared to wt mice after I/R injury. Finally, the activation of caspase3 was more pronounced in the wt compared to PT-SSAT-Cko animals., Conclusions: Enhanced SSAT expression by proximal tubule epithelial cells leads to tubular damage, and its deficiency reduces the severity of renal I/R injury through reduction of cellular damage and modulation of the innate immune response.

Several members of the SLC26 gene family have highly-restricted expression patterns in the auditory and vestibular periphery and mutations in mice of at least two of these (SLC26A4 and SLC26A5) lead to deficits in hearing and/or balance. A previous report pointed to SLC26A7 as a candidate gene important for cochlear function. In the present study, inner ears were assayed by immunostaining for Slc26a7 in neonatal and adult mice. Slc26a7 was detected in the basolateral membrane of Reissner's membrane epithelial cells but not neighboring cells, with an onset of expression at P5; gene knockout resulted in the absence of protein expression in Reissner's membrane. Whole-cell patch clamp recordings revealed anion currents and conductances that were elevated for NO3- over Cl- and inhibited by I- and NPPB. Elevated NO3- currents were absent in Slc26a7 knockout mice. There were, however, no major changes to hearing (auditory brainstem response) of knockout mice during early adult life under constitutive and noise exposure conditions. The lack of Slc26a7 protein expression found in the wild-type vestibular labyrinth was consistent with the observation of normal balance. We conclude that SLC26A7 participates in Cl- transport in Reissner's membrane epithelial cells, but that either other anion pathways, such as ClC-2, possibly substitute satisfactorily under the conditions tested or that Cl- conductance in these cells is not critical to cochlear function. The involvement of SLC26A7 in cellular pH regulation in other epithelial cells leaves open the possibility that SLC26A7 is needed in Reissner's membrane cells during local perturbations of pH.

The NH(2) terminus of the sodium-bicarbonate cotransporter 1 (NBCe1) plays an important role in its targeting to the plasma membrane. To identify the amino acid residues that contribute to the targeting of NBCe1 to the plasma membrane, polarized MDCK cells were transfected with expression constructs coding for green fluorescent protein (GFP)-tagged NBCe1 NH(2)-terminal deletion mutants, and the localization of GFP-tagged proteins was analyzed by confocal microscopy. Our results indicate that the amino acids between residues 399 and 424 of NBCe1A contain important sequences that contribute to its localization to the plasma membrane. Site-directed mutagenesis studies showed that GFP-NBCe1A mutants D405A and D416A are retained in the cytoplasm of the polarized MDCK epithelial cells. Examination of functional activities of D405A and D416A reveals that their activities are reduced compared with the wild-type NBCe1A. Similarly, aspartic acid residues 449 and 460 of pancreatic NBCe1 (NBCe1B), which correspond to residues 405 and 416 of NBCe1A, are also required for its full functional activity and accurate targeting to the plasma membrane. In addition, while replacement of D416 with glutamic acid did not affect the targeting or functional activity of NBCe1A, substitution of D405 with glutamic acid led to the retention of the mutated protein in the intracellular compartment and impaired functional activity. These studies demonstrate that aspartic acid residues 405 and 416 in the NH(2) terminus of NBCe1A are important in its accurate targeting to the plasma membrane.

Expression of spermidine/spermine N(1)-acetyltransferase (SSAT) increases in kidneys subjected to ischemia-reperfusion injury (IRI). Increased expression of SSAT in vitro leads to alterations in cellular polyamine content, depletion of cofactors and precursors of polyamine synthesis, and reduced cell proliferation. In our model system, a >28-fold increase in SSAT levels in HEK-293 cells leads to depletion of polyamines and elevation in the enzymatic activities of ornithine decarboxylase and S-adenosylmethionine decarboxylase, suggestive of a compensatory reaction to increased polyamine catabolism. Increased expression of SSAT also led to DNA damage and G(2) arrest. The increased DNA damage was primarily due to the depletion of polyamines. Other factors such as increased production of H(2)O(2) due to polyamine oxidase activity may play a secondary role in the induction of DNA lesions. In response to DNA damage the ATM/ATR --> Chk1/2 DNA repair and cell cycle checkpoint pathways were activated, mediating the G(2) arrest in SSAT-expressing cells. In addition, the activation of ERK1 and ERK2, which play integral roles in the G(2)/M transition, is impaired in cells expressing SSAT. These results indicate that the disruption of polyamine homeostasis due to enhanced SSAT activity leads to DNA damage and reduced cell proliferation via activation of DNA repair and cell cycle checkpoint and disruption of Raf --> MEK --> ERK pathways. We propose that in kidneys subjected to IRI, one mechanism through which increased expression of SSAT may cause cellular injury and organ damage is through induction of DNA damage and the disruption of cell cycle.

HCO3- secretion by gastric mucous cells is essential for protection against acidic injury and peptic ulcer. Herein we report the identification of an apical HCO3- transporter in gastric surface epithelial cells. Northern hybridization and RT-PCR demonstrate the expression of this transporter, also known as SLC26A9, in mouse and rat stomach and trachea (but not kidney). In situ hybridization in mouse stomach showed abundant expression of SLC26A9 in surface epithelial cells with apical localization on immunofluorescence labeling. Functional studies in HEK-293 cells demonstrated that SLC26A9 mediates Cl-/HCO3- exchange and is also capable of Cl--independent HCO3- extrusion. Unlike other anion exchangers or transport proteins reported to date, SLC26A9 activity is inhibited by ammonium (NH4+). The inhibitory effect of NH4+ on gastric HCO3- secretion was also indicated by reduced gastric juxtamucosal pH (pHjm) in rat stomach in vivo. This report is the first to describe the inhibition of HCO3- transport in vitro and the reduction of pHjm in stomach in vivo by NH4+. Given its critical localization on the apical membrane of surface epithelial cells, its ability to transport HCO3-, and its inhibition by NH4+, we propose that SLC26A9 mediates HCO3- secretion in surface epithelial cells and is essential for protection against acidic injury in the stomach. Disease states that are associated with increased ammonia (NH3)/NH4+ generation (e.g., Helicobacter pylori) may impair gastric HCO3- secretion and therefore predispose patients to peptic ulcer by inhibiting SLC26A9.

The endolymph in the endolymphatic sac (ES) is acidic (pH 6.6-7). Maintaining this acidic lumen is believed to be important for the normal function of the ES. The acid-base regulation mechanisms of the ES are unknown. Here we investigated the expression patterns of acid-base regulators, including vacuolar (v)H+-ATPase (proton pump), carbonic anhydrase (CA) II, and pendrin in the murine ES epithelium by immunohistochemistry (IHC) and compared their expression patterns by double immunostaining. We found that pendrin and vH+-ATPase were co-localized in the apical membrane of a specific type of ES epithelial cell. Pendrin- and vH+-ATPase-positive cells also expressed cytoplasmic CA II. Co-expression of pendrin, vH+-ATPase, and CA II in the same subgroup of ES cells suggests that this specific type of ES cell is responsible for the acid-base balance processes in the ES and pendrin, vH+-ATPase, and CA II are involved in these processes., (Copyright The Histochemical Society, Inc.)

Background: The absence of NCC does not cause significant salt wasting in NCC deficient mice under basal conditions. We hypothesized that ENaC and pendrin play important roles in compensatory salt absorption in the setting of NCC inactivation, and their inhibition and/or downregulation can cause significant salt wasting in NCC KO mice. Methods: WT and NCC KO mice were treated with a daily injection of either amiloride, an inhibitor of ENaC, or acetazolamide (ACTZ), a blocker of salt and bicarbonate reabsorption in the proximal tubule and an inhibitor of carbonic anhydrases in proximal tubule and intercalated cells, or a combination of acetazolamide plus amiloride for defined durations. Animals were subjected to daily balance studies. At the end of treatment, kidneys were harvested and examined. Blood samples were collected for electrolytes and acid base analysis. Results: Amiloride injection significantly increased the urine output (UO) in NCC KO mice (from 1.3 ml/day before to 2.5 ml/day after amiloride, p<0.03, n = 4) but caused only a slight change in UO in WT mice (p>0.05). The increase in UO in NCC KO mice was associated with a significant increase in sodium excretion (from 0.25 mmol/24 hrs at baseline to 0.35 mmol/24 hrs after amiloride injection, p<0.05, n = 4). Daily treatment with ACTZ for 6 days resulted in >80% reduction of kidney pendrin expression in both WT and NCC KO mice. However, ACTZ treatment noticeably increased urine output and salt excretion only in NCC KO mice (with urine output increasing from a baseline of 1.1 ml/day to 2.3 ml/day and sodium excretion increasing from 0.22 mmole/day before to 0.31 mmole/day after ACTZ) in NCC KO mice; both parameters were significantly higher than in WT mice. Western blot analysis demonstrated significant enhancement in ENaC expression in medulla and cortex of NCC KO and WT mice in response to ACTZ injection for 6 days, and treatment with amiloride in ACTZ-pretreated mice caused a robust increase in salt excretion in both NCC KO and WT mice. Pendrin KO mice did not display a significant increase in urine output or salt excretion after treatment with amiloride or ACTZ. Conclusion: 1. ENaC plays an important role in salt reabsorption in NCC KO mice. 2. NCC contributes to compensatory salt reabsorption in the setting of carbonic anhydrase inhibition, which is associated with increased delivery of salt from the proximal tubule and the down regulation of pendrin. 3. ENaC is upregulated by ACTZ treatment and its inhibition by amiloride causes significant diuresis in NCC KO and WT mice. Despite being considered mild agents individually, we propose that the combination of acetazolamide and amiloride in the setting of NCC inhibition (i.e., hydrochlorothiazide) will be a powerful diuretic regimen. [ABSTRACT FROM AUTHOR]

Background: Expression and activity of spermidine/spermine N1-acetyltransferase (SSAT) increases in kidneys subjected to ischemia/reperfusion (I/R) injury, while its ablation reduces the severity of such injuries. These results suggest that increased SSAT levels contribute to organ injury; however, the role of SSAT specifically expressed in proximal tubule epithelial cells, which are the primary targets of I/R injury, in the mediation of renal damage remains unresolved. Methods: Severity of I/R injury in wt and renal proximal tubule specific SSAT-ko mice (PT-SSAT-Cko) subjected to bilateral renal I/R injury was assessed using cellular and molecular biological approaches. Results: Severity of the loss of kidney function and tubular damage are reduced in PT-SSAT-Cko- compared to wt-mice after I/R injury. In addition, animals treated with MDL72527, an inhibitor of polyamine oxidases, had less severe renal damage than their vehicle treated counter-parts. The renal expression of HMGB 1 and Toll like receptors (TLR) 2 and 4 were also reduced in PT-SSAT-Cko- compared to wt mice after I/R injury. Furthermore, infiltration of neutrophils, as well as expression of tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1) and interleukin-6 (IL-6) transcripts were lower in the kidneys of PT-SSAT-Cko compared to wt mice after I/R injury. Finally, the activation of caspase3 was more pronounced in the wt compared to PT-SSAT-Cko animals. Conclusions: Enhanced SSAT expression by proximal tubule epithelial cells leads to tubular damage, and its deficiency reduces the severity of renal I/R injury through reduction of cellular damage and modulation of the innate immune response. [ABSTRACT FROM AUTHOR]

Background. The epithelial calcium channel (ECaC) (TRPV5) and the Cl−/HCO3− exchanger pendrin (SLC26A4) are expressed on the apical membrane of tubular cells in the distal nephron and play essential roles in calcium re-absorption and bicarbonate secretion, respectively, in the kidney. Methods. A combination of functional and molecular biology techniques were employed to examine the role of pendrin deletion in calcium excretion. Results. Here, we demonstrate that deletion of pendrin causes acidic urine [urine pH 4.9 in knockout (KO) versus 5.9 in wild-type (WT) mice, P < 0.03)] and downregulates the calcium-absorbing molecules ECaC and Na/Ca exchanger in the kidney, as shown by northern hybridization, immunoblot analysis and/or immunofluorescent labeling. These changes were associated with a ∼100% increase in 24-h urine calcium excretion in pendrin null mice. Subjecting the pendrin WT and KO mice to oral bicarbonate loading for 12 days increased the urine pH to ∼8 in both genotypes, normalized the expression of ECaC and Na/Ca exchanger and reduced the urine calcium excretion in pendrin-null mice to levels comparable to WT mice. Conclusions. We suggest that pendrin dysfunction should be suspected and investigated in humans with an otherwise unexplained acidic urine and hypercalciuria. [ABSTRACT FROM PUBLISHER]

The majority of dietary amino acids are absorbed via the H+-di-/tripeptide transporter Pepti of the small intestine. Proton influx via Pept1 requires maintenance of intracellular pH (pHi) to sustain the driving force for peptide absorption. The apical membrane Na+/H+ exchanger Nhe3 plays a major role in minimizing epithelial acidification during H+-di-/tripeptide absorption. However, the contributions of HCO3--dependent transporters to this process have not been elucidated. In this study, we investigate the role of putative anion transporter-1 (Pat-1), an apical membrane anion exchanger, in epithelial pH, regulation during H+-peptide absorption. Using wild-type (WT) and Pat-1(-) mice, Ussing chambers were employed to measure the short-circuit current (Isc) associated with Pept1-mediated glycyl-sarcosine (Gly-Sar) absorption. Microfluorometry was used to measure pHi and Cl-/HCO3- exchange in the upper villous epithelium. In CO2/HCO3--buffered Ringers, WT small intestine showed significant Gly-Sar-induced Isc and efficient pHi regulation during pharmacological inhibition of Nhe3 activity. In contrast, epithelial acidification and reduced Isc response to Gly-Sar exposure occurred during pharmacological inhibition of Cl-/HCO3- exchange and in the Pat-1(-) intestine. Pat-1 interacts with carbonic anhydrase II (CAll), and studies using CAII(-) intestine or the pharmacological inhibitor methazolamide on WT intestine resulted in increased epithelial acidification during Gly-Sar exposure. Increased epithelial acidification during Gly-Sar exposure also occurred in WT intestine during inhibition of luminal extracellular CA activity. Measurement of Cl-/HCO3- exchange in the presence of Gly-Sar revealed an increased rate of Cl-OUT/HCO3-lN exchange that was both Pat-1 dependent and CA dependent. In conclusion, Pat-1 CI-/HCO3- exchange contributes to pHi regulation in the villous epithelium during H+-dipeptide absorption, possibly by providing a HCO3- import pathway. [ABSTRACT FROM AUTHOR]

Slc26a9 is a recently identified anion transporter that is abundantly expressed in gastric epithelial cells. To study its role in stomach physiology, gene targeting was used to prepare mice lacking Slc26a9. Homozygous mutant (Slc26a9-/-) mice appeared healthy and displayed normal growth. Slc26a9 deletion resulted in the loss of gastric acid secretion and a moderate reduction in the number of parietal cells in mutant mice at 5 weeks of age. lmmunofluorescence labeling detected the H-K-ATPase exclusively on the apical pole of gastric parietal cells in Slc26a9+/+ mice, in contrast to the predominant cytoplasmic localization in Slc26a94-/- mice. Light microscopy indicated that gastric glands were dilated, and electron micrographs displayed a distinct and striking absence of tubulovesicles in parietal cells and reductions in the numbers of parietal and zymogen cells in Slc26a9-/- stomach. Expression studies indicated that Slc26a9 can function as a chloride conductive pathway in oocytes as well as a Cl-/HCO3- exchanger in cultured cells, and localization studies in parietal cells detected its presence in tubulovesicles. We propose that Slc26a9 plays an essential role in gastric acid secretion via effects on the viability of tubulovesicles/ secretory canaliculi and by regulating chloride secretion in parietal cells. [ABSTRACT FROM AUTHOR]

The Na+:HCO3- co-transporter NBC1 (SLC4A4, variant A, kidney specific) is located exclusively on the basolateral membrane of epithelial cells, implying that this molecule has acquired specific signals for targeting to the basolateral membrane. A motif with the sequence QQPFLS (positions 1010-1015) in the cytoplasmic tail of NBC1 was recently demonstrated to mediate targeting of NBC1 to the basolateral membrane. Here, we demonstrate that mutating the amino acid F (phenylalanine) or L (leucine) at positions 1013 or 1014 to alanine, respectively, resulted in the retargeting of NBC1 to the apical membrane. Further- more, mutation of the FL motif to FF showed similar properties as the wild-type; however, mutation of the FL motif to LL showed significant intracellular retention of NBC1. Mutating the amino acids Q-Q-P and S (positions 1010-1011-1012 and 1015) to A-A-A and A, respectively, did not affect the membrane targeting of NBC1. Functional studies in oocytes with microelectrode demonstrated that the apically targeted mutants, as well as basolaterally targeted mutants, are all functional. We propose that the FL motif in the COOH-terminal tail of NBC1 is essential for the targeting of NBC1 to the basolateral membrane but is distinct from the membrane-targeting di-leucine motif identified in other membrane proteins. [ABSTRACT FROM AUTHOR]

Basal HCO3- secretion across the duodenum has been shown in several species to principally involve the activity of apical membrane Cl-/HCO3- exchanger(s). To investigate the identity of relevant anion exchanger(s), experiments were performed using wild-type (WT) mice and mice with gene-targeted deletion of the following Cl-/HCO3- exchangers localized to the apical membrane of murine duodenal villi: Slc26a3 [down-regulated in adenoma (DRA)], Slc26a6 [putative anion transporter 1 (PAT-1)], and Slc4a9 [anion exchanger 4 (AE4)]. RT-PCR of the isolated villous epithelium demonstrated PAT-1, DRA, and AE4 mRNA expression. Using the pH-sensitive dye BCECF, anion exchange rates were measured across the apical membrane of epithelial cells in the upper villus of the intact duodenal mucosa. Under basal conditions, Cl-/HCO3- exchange activity was reduced by 65- 80% in the PAT-1(-) duodenum, 30-40% in the DRA(-) duodenum, and <5% in the AE4(-) duodenum compared with the WT duodenum. SO42-/HCO3- exchange was eliminated in the PAT-1(-) duodenum but was not affected in the DRA(-) and AE4(-) duodenum relative to the WT duodenum. Intracellular pH (pHi) was reduced in the PAT-1(-) villous epithelium but increased to WT levels in the absence of CO2/HCO3- or during methazolamide treatment. Further experiments under physiological conditions indicated active pHi compensation in the PAT-1(-) villous epithelium by combined activities of Na+/H+ exchanger 1 and Cl--dependent transport processes at the basolateral membrane. We conclude that 1) PAT-1 is the major contributor to basal Cl-/HCO3- and SO42-/HCO3- exchange across the apical membrane and 2) PAT-1 plays a role in pHi regulation in the upper villous epithelium of the murine duodenum. [ABSTRACT FROM AUTHOR]

Na-H exchanger NHE3 and Cl-anion exchanger CFEX (SLC26A6, PAT1) play principal roles in the reabsorption of Na and Cl in the proximal tubule of the mammalian kidney. The mechanisms by which NHE3 and CFEX are localized to and maintained in the brush border of the proximal tubule are largely unknown. To investigate the possible interaction of NHE3 and CFEX with the PDZ-domain-containing scaffolding protein PDZK1, we performed a series of in vitro interaction assays with GST-fusion proteins and native brush border membrane proteins. These studies demonstrated that, not only were NHE3 and CFEX capable of directly interacting with PDZK1, but that this interaction was mediated through their C-terminal PDZ-interaction sites. To determine whether PDZK1 interaction is essential for brush border localization of NHE3 and CFEX in vivo, we examined the expression of NHE3 and CFEX in kidneys of wild-type and PDZK1-null mutant mice by both Western analysis and immunocytochemistry. These studies indicated that although brush border expression of NHE3 was unaffected by the loss of PDZK1, the expression of CFEX was markedly reduced. Finally, we assayed CFEX functional activity as Cl-oxalate exchange in brush border membrane vesicles and oxalate-stimulated volume absorption in microperfused proximal tubules. Consistent with the observed decrease in CFEX protein expression, both measures of CFEX functional activity were dramatically reduced in PDZK1-null animals. In conclusion, the scaffolding protein PDZK1 is essential for the normal expression and function of Cl-anion exchanger CFEX in the proximal tubule of the mammalian kidney. [ABSTRACT FROM AUTHOR]

HCO3- secretion by gastric mucous cells is essential for protection against acidic injury and peptic ulcer. Herein we report the identification of an apical HCO3- transporter in gastric surface epithelial cells. Northern hybridization and RT-PCR demonstrate the expression of this transporter, also known as SLC26A9, in mouse and rat stomach and trachea (but not kidney). In situ hybridization in mouse stomach showed abundant expression of SLC26A9 in surface epithelial cells with apical localization on immunofluorescence labeling. Functional studies in HEK-293 cells demonstrated that SLC26A9 mediates Cl-/HCO3- exchange and is also capable of Cl--independent HCO3- extrusion. Unlike other anion exchangers or transport proteins reported to date, SLC26A9 activity is inhibited by ammonium (NH4+). The inhibitory effect of NH4+ on gastric HCO3- secretion was also indicated by reduced gastric juxtamucosal pH (pHjm) in rat stomach in vivo. This report is the first to describe the inhibition of HCO3- transport in vitro and the reduction of pHjm in stomach in vivo by NH4+. Given its critical localization on the apical membrane of surface epithelial cells, its ability to transport HCO3-, and its inhibition by NH4+, we propose that SLC26A9 mediates HCO3- secretion in surface epithelial cells and is essential for protection against acidic injury in the stomach. Disease states that are associated with increased ammonia (NH3)/NH4+ generation (e.g., Helicobacter pylon) may impair gastric HCO3- secretion and therefore predispose patients to peptic ulcer by inhibiting SLC26A9. [ABSTRACT FROM AUTHOR]

Examines the effect of long term hyperosmolality on the exchanger isoform NHE-3 in cultured renal epithelial cells. Role of plasma membrane exchange in a variety of cell functions; Inhibition of NHE-3 activity in renal tubule cells; Impairment of urinary acidification in patients with diabetes mellitus.

Abstract: The mouse Slc39a8 gene encodes the ZIP8 transporter, which has been shown to be a divalent cation/ symporter. Using ZIP8 cRNA-injected Xenopus oocyte cultures, we show herein that: [a] ZIP8-mediated cadmium (Cd2+) and zinc (Zn2+) uptake have V max values of 1.8±0.08 and 1.0±0.08pmol/oocyte/h, and K m values of 0.48±0.08 and 0.26±0.09μM, respectively; [b] ZIP8-mediated Cd2+ uptake is most inhibited by Zn2+, second-best inhibited by Cu2+, Pb2+ and Hg2+, and not inhibited by Mn2+ or Fe2+; and [c] electrogenicity studies demonstrate an influx of two anions per one Cd2+ (or one Zn2+) cation, i.e. electroneutral complexes. Using Madin–Darby canine kidney (MDCK) polarized epithelial cells retrovirally infected with ZIP8 cDNA and tagged with hemagglutinin at the C-terminus, we show that—similar to ZIP4—the ZIP8 eight-transmembrane protein is largely internalized during Zn2+ homeostasis, but moves predominantly to the cell surface membrane (trafficking) under conditions of Zn2+ depletion. [Copyright &y& Elsevier]

Mutations in the SLC26A3 (DIM (down-regulated in adenoma)) gene constitute the molecular etiology of congenital chloride-losing diarrhea in humans. To ascertain its role in intestinal physiology, gene targeting was used to prepare mice lacking slc26ɑ3. slc26ɑ3-deficient animals displayed postpartum lethality at low penetrance. Surviving dra-deficient mice exhibited high chloride content diarrhea, volume depletion, and growth retardation. In addition, the large intestinal loops were distended, with colonic mucosa exhibiting an aberrant growth pattern and the colonic crypt proliferative zone being greatly expanded in slc26ɑ3-null mice. Apical membrane chloride/base exchange activity was sharply reduced, and luminal content was more acidic in slc26ɑ3-null mouse colon. The epithelial cells in the colon displayed unique adaptive regulation of ion transporters; NHE3 expression was enhanced in the proximal and distal colon, whereas colonic H,K-ATPase and the epithelial sodium channel showed massive up-regulation in the distal colon. Plasma aldosterone was increased in slc26ɑ3-null mice. We conclude that slc26ɑ3 is the major apical chloride/base exchanger and is essential for the absorption of chloride in the colon. In addition, slc26ɑ3 regulates colonic crypt proliferation. Deletion of slc26ɑ3 results in chloride-rich diarrhea and is associated with compensatory adaptive up-regulation of ion-absorbing transporters. [ABSTRACT FROM AUTHOR]

HCO3- secretion by gastric mucous cells is essential for protection against acidic injury and peptic ulcer. Herein we report the identification of an apical HCO3- transporter in gastric surface epithelial cells. Northern hybridization and RT-PCR demonstrate the expression of this transporter, also known as SLC26A9, in mouse and rat stomach and trachea (but not kidney). In situ hybridization in mouse stomach showed abundant expression of SLC26A9 in surface epithelial cells with apical localization on immunofluorescence labeling. Functional studies in HEK-293 cells demonstrated that SLC26A9 mediates Cl-/HCO3- exchange and is also capable of Cl--independent HCO3- extrusion. Unlike other anion exchangers or transport proteins reported to date, SLC26A9 activity is inhibited by ammonium (NH4+). The inhibitory effect of NH4+ on gastric HCO3- secretion was also indicated by reduced gastric juxtamucosal pH (pHjm) in rat stomach in vivo. This report is the first to describe the inhibition of HCO3- transport in vitro and the reduction of pHjm in stomach in vivo by NH4+. Given its critical localization on the apical membrane of surface epithelial cells, its ability to transport HCO3-, and its inhibition by NH4+, we propose that SLC26A9 mediates HCO3- secretion in surface epithelial cells and is essential for protection against acidic injury in the stomach. Disease states that are associated with increased ammonia (NH3)/NH4+ generation (e.g., Helicobacter pylon) may impair gastric HCO3- secretion and therefore predispose patients to peptic ulcer by inhibiting SLC26A9. [ABSTRACT FROM AUTHOR]

The Na+-HCO3- cotransporter NBC1 is located exclusively on the basolateral membrane and mediates vectorial transport of bicarbonate in a number of epithelia, including kidney and pancreas. To identify the motifs that direct the targeting of kidney NBC1 to basolateral membrane, wild type and various carboxyl-terminally truncated kidney NBC1 mutants were generated, fused translationally in-frame to GFP, and transiently expressed in kidney epithelial cells. GFP was linked to the NH2 terminus of NBC1, and labeling was examined by confocal microscopy. Full-length (1035 aa) and mutants with the deletion of 3 or 20 amino acids from the COOHterminal end of NBC1 (lengths 1032 and 1015 aa, respectively) showed strong and exclusive targeting on the basolateral membrane. However, the deletion of 26 amino acid residues from the COOH-terminal end (length 1010 aa) resulted in retargeting of NBC1 to the apical membrane. Expression studies in oocytes demonstrated that the NBC1 mutant with the deletion of 26 amino acid residues from the COOH-terminal end is functional. Additionally, the deletion of the last 23 amino acids or mutation in the conserved residue Phe at position 1013 on the COOH-terminal end demonstrated retargeting to the apical membrane. We propose that a carboxyl-terminal motif with the sequence QQPFLS, which spans amino acid residues 1010-1015, and specifically the amino acid residue Phe (position 1013) are essential for the exclusive targeting of NBC1 to the basolateral membrane. [ABSTRACT FROM AUTHOR]