The Bicarbonate Transporter SLC4A7 Plays a Key Role in Macrophage Phagosome Acidification.

Macrophages represent the first line of immune defense against pathogens, and phagosome acidification is a necessary step in pathogen clearance. Here, we identified the bicarbonate transporter SLC4A7, which is strongly induced upon macrophage differentiation, as critical for phagosome acidification. Loss of SLC4A7 reduced acidification of phagocytosed beads or bacteria and impaired the intracellular microbicidal capacity in human macrophage cell lines. The phenotype was rescued by wild-type SLC4A7, but not by SLC4A7 mutants, affecting transport capacity or cell surface localization. Loss of SLC4A7 resulted in increased cytoplasmic acidification during phagocytosis, suggesting that SLC4A7-mediated, bicarbonate-driven maintenance of cytoplasmic pH is necessary for phagosome acidification. Altogether, we identify SLC4A7 and bicarbonate-driven cytoplasmic pH homeostasis as an important element of phagocytosis and the associated microbicidal functions in macrophages.

Deficient acid handling with distal RTA in the NBCe2 knockout mouse.

In many circumstances, the pathogenesis of distal renal tubular acidosis (dRTA) is not understood. In the present study, we report that a mouse model lacking the electrogenic Na(+)-HCO3 (-) cotransporter [NBCe2/Slc4a5; NBCe2 knockout (KO) mice] developed dRTA after an oral acid challenge. NBCe2 expression was identified in the connecting tubule (CNT) of wild-type mice, and its expression was significantly increased after acid loading. NBCe2 KO mice did not have dRTA when on a standard mouse diet. However, after acid loading, NBCe2 KO mice exhibited complete features of dRTA, characterized by insufficient urinary acidification, hyperchloremic hypokalemic metabolic acidosis, and hypercalciuria. Additional experiments showed that NBCe2 KO mice had decreased luminal transepithelial potential in the CNT, as revealed by micropuncture. Further immunofluorescence and Western blot experiments found that NBCe2 KO mice had increased expression of H(+)-ATPase B1 in the plasma membrane. These results showed that NBCe2 KO mice with acid loading developed increased urinary K(+) and Ca(2+) wasting due to decreased luminal transepithelial potential in the CNT. NBCe2 KO mice compensated to maintain systemic pH by increasing H(+)-ATPase in the plasma membrane. Therefore, defects in NBCe2 can cause dRTA, and NBCe2 has an important role to regulate urinary acidification and the transport of K(+) and Ca(2+) in the distal nephron.

Modulation of sodium-bicarbonate co-transporter (SLC4A4/NBCe1) protein and mRNA expression in rat's uteri by sex-steroids and at different phases of the oestrous cycle.

UNLABELLED: Oestrogen-induced uterine fluid sodium (Na(+)) and bicarbonate (HCO3(-)) secretion may involve SLC4A4. We hypothesized that uterine SLC4A4 expression changes under different sex-steroid influence, therefore may account for the fluctuation in uterine fluid Na(+) and HCO3(-) content throughout the oestrous cycle. The aim of this study is to investigate the differential effects of sex-steroids and oestrous cycle phases on uterine SLC4A4 expression. METHODS: Adult female WKY rats were ovariectomised and treated with different doses of 17ß-oestradiol (E2) (0.2, 2, 20 and 50 µg/ml/day) or progesterone (P4) (4 mg/ml/day) for three consecutive days and 3 days treatment with 0.2 µg/ml/day E2 followed by another 3 days with P4 to mimic the hormonal changes in early pregnancy. Oestrous cycle phases in intact, non-ovariectomised rats were determined by vaginal smear. The animals were then sacrificed and uteri were removed for protein and mRNA expression analyses by Western blotting and Real Time PCR, respectively. SLC4A4 distribution was observed by immunohistochemistry. RESULTS: Treatment with increasing E2 doses resulted in a dose-dependent increase in SLC4A4 protein expression. High SLC4A4 protein and mRNA expression can be seen at estrus. SLC4A4 is distributed mainly at the apical as well as basolateral membranes of the luminal and glandular epithelia following E2 treatment and at Es. Meanwhile, SLC4A4 expression was reduced following P4 treatment and was low at diestrus. CONCLUSION: High SLC4A4 expression under estrogen dominance may contribute to the increase in uterine fluid Na(+) and HCO3(-) content, while its low expression under P4 dominance may result in vice versa.

Contribution of Na+,HCO3(-)-cotransport to cellular pH control in human breast cancer: a role for the breast cancer susceptibility locus NBCn1 (SLC4A7).

Genome-wide association studies recently linked the locus for Na(+),HCO(3)(-)-cotransporter NBCn1 (SLC4A7) to breast cancer susceptibility, yet functional insights have been lacking. To determine whether NBCn1, by transporting HCO(3)(-) into cells, may dispose of acid produced during high metabolic activity, we studied the expression of NBCn1 and the functional impact of Na(+),HCO(3)(-)-cotransport in human breast cancer. We found that the plasmalemmal density of NBCn1 was 20-30% higher in primary breast carcinomas and metastases compared to matched normal breast tissue. The increase in NBCn1 density was similar in magnitude to that observed for Na(+)/H(+)-exchanger NHE1 (SLC9A1), a transporter previously implicated in cell migration, proliferation and malignancy. In primary breast carcinomas, the apparent molecular weight for NBCn1 was increased compared to normal tissue. Using pH-sensitive fluorophores, we showed that Na(+),HCO(3)(-)-cotransport is the predominant mechanism of acid extrusion and is inhibited 34 ± 9% by 200 µM 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid in human primary breast carcinomas. At intracellular pH (pH(i) ) levels >6.6, CO(2)/HCO(3)(-)-dependent mechanisms accounted for >90% of total net acid extrusion. Na(+)/H(+)-exchange activity was prominent only at lower pH(i) -values. Furthermore, steady-state pH(i) was 0.35 ± 0.06 units lower in the absence than in the presence of CO(2)/HCO(3)(-). In conclusion, expression of NBCn1 is upregulated in human primary breast carcinomas and metastases compared to normal breast tissue. Na(+),HCO(3)(-)-cotransport is a major determinant of pH(i) in breast cancer and the modest DIDS-sensitivity is consistent with NBCn1 being predominantly responsible. Hence, our results suggest a major pathophysiological role for NBCn1 that may be clinically relevant.

Electrogenic Na⁺/HCO3⁻ co-transporter-1 is essential for the parathyroid hormone-stimulated intestinal HCO3⁻ secretion.

Parathyroid hormone (PTH) was recently demonstrated to enhance the HCO(3)(-) secretion through the apical anion channel, cystic fibrosis transmembrane conductance regulator (CFTR), but how the HCO(3)(-) entered the epithelial cells was not well understood, in part, due to the lack of specific inhibitors of the basolateral HCO(3)(-) transporters. Moreover, the function of the PTH-stimulated HCO(3)(-) secretion has never been investigated in vivo. Here, we designed three specific pairs of small interfering RNA sequences to simultaneously knockdown three variants of the electrogenic Na(+)/HCO(3)(-) co-transporter (NBCe)-1 in the intestinal epithelium-like Caco-2 monolayer. The results showed that NBCe1 mRNA levels were markedly reduced, and the PTH-induced transepithelial current and voltage changes were diminished after triple knockdown as determined by quantitative real-time PCR and Ussing chamber technique, respectively. An in vivo ligated intestinal loop study further showed that there was an increased fluid secretion, presumably driven by HCO(3)(-) transport, in the ileum, but not in jejunum or colon, of rats administered intravenously with 2 µg/kg body weight of rat PTH 1-34. Therefore, the present results suggested that PTH stimulated intestinal HCO(3)(-) secretion, particularly in the ileum, by inducing the basolateral HCO(3)(-) uptake via NBCe1.

Astrocytes in the retrotrapezoid nucleus sense H+ by inhibition of a Kir4.1-Kir5.1-like current and may contribute to chemoreception by a purinergic mechanism.

Central chemoreception is the mechanism by which CO(2)/pH sensors regulate breathing in response to tissue pH changes. There is compelling evidence that pH-sensitive neurons in the retrotrapezoid nucleus (RTN) are important chemoreceptors. Evidence also indicates that CO(2)/H(+)-evoked adenosine 5'-triphosphate (ATP) release in the RTN, from pH-sensitive astrocytes, contributes to chemoreception. However, mechanism(s) by which RTN astrocytes sense pH is unknown and their contribution to chemoreception remains controversial. Here, we use the brain slice preparation and a combination of patch-clamp electrophysiology and immunohistochemistry to confirm that RTN astrocytes are pH sensitive and to determine mechanisms by which they sense pH. We show that pH-sensitive RTN glia are immunoreactive for aldehyde dehydrogenase 1L1, a marker of astrocytes. In HEPES buffer the pH-sensitive current expressed by RTN astrocytes reversed near E(K(+)) (the equilibrium potential for K(+)) and was inhibited by Ba(2+) and desipramine (blocker of Kir4.1-containing channels), characteristics most consistent with heteromeric Kir4.1-Kir5.1 channels. In bicarbonate buffer, the sodium/bicarbonate cotransporter also contributed to the CO(2)/H(+)-sensitive current in RTN astrocytes. To test the hypothesis that RTN astrocytes contribute to chemoreception by a purinergic mechanism, we used fluorocitrate to selectively depolarize astrocytes while measuring neuronal activity. We found that fluorocitrate increased baseline activity and pH sensitivity of RTN neurons by a P2-receptor-dependent mechanism, suggesting that astrocytes may release ATP to activate RTN chemoreceptors. We also found in bicarbonate but not HEPES buffer that P2-receptor antagonists decreased CO(2) sensitivity of RTN neurons. We conclude that RTN astrocytes sense CO(2)/H(+) in part by inhibition of a Kir4.1-Kir5.1-like current and may provide an excitatory purinergic drive to pH-sensitive neurons.

NBCn1 and NHE1 expression and activity in DeltaNErbB2 receptor-expressing MCF-7 breast cancer cells: contributions to pHi regulation and chemotherapy resistance.

Altered pH-regulatory ion transport is characteristic of many cancers; however, the mechanisms and consequences are poorly understood. Here, we investigate how a truncated, constitutively active ErbB2 receptor (DeltaNErbB2) common in breast cancer impacts on the Na(+)/H(+)-exchanger NHE1 and the Na(+),HCO(3)(-)-cotransporter NBCn1 in MCF-7 human breast cancer cells and address the roles of these transporters in chemotherapy resistance. Upon DeltaNErbB2 expression, mRNA and protein levels of NBCn1, yet not of NHE1, increased several-fold, and the localization of both transporters was altered paralleling extensive morphological changes. The rate of pH(i) recovery after acid loading increased by 50% upon DeltaNErbB2 expression. Knockdown and pharmacological inhibition confirmed the involvement of both NHE1 and NBCn1 in acid extrusion. NHE1 inhibition or knockdown sensitized DeltaNErbB2-expressing cells to cisplatin-induced programmed cell death (PCD) in a caspase-, cathepsin-, and reactive oxygen species-dependent manner. NHE1 inhibition augmented cisplatin-induced caspase activity and lysosomal membrane permeability followed by cysteine cathepsin release. In contrast, NBCn1 inhibition attenuated cathepsin release and had no net effect on viability. These findings warrant studies of NHE1 as a potential target in breast cancer and demonstrate that in spite of their similar transport functions, NHE1 and NBCn1 serve different functions in MCF-7 cells.

Secretagogues stimulate electrogenic HCO3- secretion in the ileum of the brushtail possum, Trichosurus vulpecula: evidence for the role of a Na+/HCO3- cotransporter.

Fluid secretion is essential for intestinal function and, in eutherian mammals, is driven by electrogenic Cl(-) transport, which is dependent upon a bumetanide-sensitive, basolateral Na(+)/K(+)/2 Cl(-) cotransporter, NKCC1. However, ileal secretion in the brushtail possum, a marsupial, involves a fundamentally different process, since NKCC1 expression is low in this tissue and the secretagogue-induced short circuit current (I(sc)) is insensitive to bumetanide. In view of these differences we have investigated the basis of the secretory response of the possum ileum. In the Ussing chamber the secretory I(sc) is independent of Cl(-) but dependent upon Na(+) and serosal HCO(3)(-)/CO(2), suggesting that secretagogues stimulate electrogenic HCO(3)(-) secretion. In agreement with this, serosal DIDS (4,4'-diisothiocyano-stilbene-2,2'-disulfonate; 1 mmol l(-1)) inhibited the secretory response. However, acetazolamide (1 mmol l(-1)) and serosal amiloride (1 mmol l(-1)) had little effect, indicating that HCO(3)(-) secretion is driven by HCO(3)(-) transport from the serosal solution into the cell, rather than hydration of CO(2) by carbonic anhydrase. Consistent with this the pancreatic variant of the electrogenic Na(+)/HCO(3)(-) cotransporter (pNBC) is highly expressed in the ileal epithelium and is located in the basolateral membrane of the epithelial cells, predominantly in the mid region of the villi, with lower levels of expression in the crypts and no expression in the villous tips. We conclude that the secretory response of the possum ileum involves electrogenic HCO(3)(-) secretion driven by a basolateral pNBC and that the ileal HCO(3)(-) secretion is associated with a specialised function of the possum ileum, most probably related to hindgut fermentation.

Inhibition of rat Na+(-)HCO3(-) cotransporter (NBCn1) function and expression by the alternative splice domain.

The Na(+)-HCO(3)(-) cotransporter NBCn1 (SLC4A7) has multiple variants depending upon splice domains in the cytoplasmic amino- and carboxy-termini of the protein. In this study, we examined the role of the amino-terminal splice domain containing 123 amino acids (cassette II) in the regulation of NBCn1 function and expression. Polymerase chain reaction detected NBCn1 mRNAs containing cassette II in a variety of tissues. Two variants, NBCn1-B containing cassette II and NBCn1-E lacking cassette II, were expressed in Xenopus oocytes and assessed by two-electrode voltage clamp to measure the ionic current mediated by the transporters. The two variants showed similar current-voltage (I-V) relations when measured 3-4 days after RNA injection. Replacment of Cl() with gluconate did not affect the I-V relations. When exposed to solutions containing 20-50 mm Na(+), the current produced by NBCn1-B was slightly more positive than that produced by NBCn1-E. The two currents were similar at 100 mm Na(+). The slope conductances for the two variants were progressively increased at higher Na(+) levels, and the increases were parallel and superimposed. Measured at different time points after RNA injection, NBCn1-B produced lower conductance than NBCn1-E at 24-48 h. Protein expression of NBCn1-B was also low at these time points as determined by immunoblot of oocyte membrane preparation. Expressed in opossum kidney (OK) cells, NBCn1-E caused a 1.5-fold increase in ouabain-sensitive production of p-nitrophenol from p-phenyl phosphate compared with control preparations, whereas NBCn1-B had negligible effect. We conclude that the primary function of cassette II is to reduce NBCn1 protein expression.

Studies on bicarbonate transporters and carbonic anhydrase in porcine nonpigmented ciliary epithelium.

PURPOSE: Bicarbonate transport plays a role in aqueous humor (AH) secretion. The authors examined bicarbonate transport mechanisms and carbonic anhydrase (CA) in porcine nonpigmented ciliary epithelium (NPE). METHODS: Cytoplasmic pH (pH(i)) was measured in cultured porcine NPE loaded with BCECF. Anion exchanger (AE), sodium bicarbonate cotransporter (NBC), and CA were examined by RT-PCR and immunolocalization. AH secretion was measured in the intact porcine eye using a fluorescein dilution technique. RESULTS: Anion exchanger AE2, CAII, and CAIV were abundant in the NPE layer. In cultured NPE superfused with a CO(2)/HCO(3)(-)-free HEPES buffer, exposure to a CO(2)/HCO(3)(-)-containing buffer caused rapid acidification followed by a gradual increase in pH(i). Subsequent removal of CO(2)/HCO(3)(-) with HEPES buffer caused rapid alkalinization followed by a gradual decrease in pH(i). The rate of gradual alkalinization after the addition of HCO(3)(-)/CO(2) was inhibited by sodium-free conditions, DIDS, and the CA inhibitors acetazolamide and methazolamide but not by the Na-H exchange inhibitor dimethylamiloride or low-chloride buffer. The phase of gradual acidification after removal of HCO(3)(-)/CO(2) was inhibited by DIDS, acetazolamide, methazolamide, and low-chloride buffer. DIDS reduced baseline pH(i). In the intact eye, DIDS and acetazolamide reduced AH secretion by 25% and 44%, respectively. CONCLUSIONS: The results suggest the NPE uses a Na(+)-HCO(3)(-) cotransporter to import bicarbonate and a Cl(-)/HCO(3)(-) exchanger to export bicarbonate. CA influences the rate of bicarbonate transport. AE2, CAII, and CAIV are enriched in the NPE layer of the ciliary body, and their coordinated function may contribute to AH secretion by effecting bicarbonate transport into the eye.

Sodium ion transporters as new therapeutic targets in heart failure.

Sodium ion transporters in sarcolemma are involved in numerous vital cell functions, such as excitability, excitation-contraction coupling, energy metabolism, pH and volume regulation, development and growth. In a number of cardiac pathologies, the intracellular sodium concentration ([Na+]i) is elevated. Since [Na+]i and intracellular Ca2+ concentration ([Ca2+]i are coupled through the Na+/Ca(2+)-exchanger, these cardiac pathologies display disturbed calcium handling. For instance, [Na+]i is increased in heart failure (HF) leading to Na+/Ca(2+)-exchanger mediated increase in [Ca2+]i, reduced contractility and increased propensity to arrhythmias. Several studies support the contention that an increase in [Na+]i and [Ca2+]i transduces a signal the nucleus, that triggers development of cardiac remodelling and hypertrophy. Pharmacological intervention, which favourably interferes with [Na+]i and [Ca2+]i homeostasis, might prevent hypertrophy, cardiac remodelling, arrhythmias and HF. The most important sodium transport mechanisms that may underlie increased [Na+]i are: Na+/H(+)-exchanger (NHE-1), Na+-HCO(3)(-) co-transporter (NBC), Na(+)-K(+)-Cl(-) co-transporter (NKCC), Na(+)-channel, Na+/K(+)-ATPase and Na+/Ca(2+)-exchanger (NCX). Preclinical studies showed that pharmacological interventions, targeted against sarcolemmal sodium ion transporters, proved effective in ameliorating heart failure. In this respect: 1) NHE-1 inhibition reduces cardiac remodelling, hypertrophy and HF, although, in the patients following coronary artery bypass graft surgery, it was associated with an increase of stroke. 2) The activity of NBC is up-regulated, during the development of hypertrophy and may be a therapeutic strategy to prevent the development of hypertrophy and HF. 3) NKCC is increased in post-infarction HF, and the inhibition of NKCC attenuated post-infarction remodelling. 4) Inactivation of sodium channels is impaired in HF, which may result, in increased Na+ influx and prolongation of the action potential. 5) Blockade of NCX may be useful as a part of a combined therapeutic approach. Inhibition of reversed mode, or activation of forward mode NCX reduce Ca2+ overload. 6) Inhibition of Na+/K(+)-ATPase (digoxin), is used to increase contractility, however, it enhances progression of HF. Oppositely, new drugs which increase activity of Na+/K(+)-ATPase may prevent the development of cardiac remodelling hypertrophy and HF.

Isothiocyanate inhibits restitution and wound repair after injury in the stomach: ex vivo and in vitro studies.

The role of isothiocyanate (ITC) in blocking epithelial restitution after injury and in the recovery of round wounds was examined in the ex vivo guinea pig stomach and in rat gastric mucosal-1 (RGM1) cells, respectively. For this, recovery of transepithelial electrical resistance and morphology after injury or the closure of round wounds was evaluated in the presence of 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) or 4,4-diisothiocyanatodihydrostilbene-2,2'-disulfonic acid (H2DIDS) (two ITC groups), 4-acetamido-4-isothiocyanatostilbene-2,2'-disulfonic acid (SITS) (one ITC group), or 4,4-diinitrostilbene-2,2'-disulfonic acid (DNDS) (no ITC groups). Wounded RGM1 cells were also incubated with bicarbonate-free buffer, ATP, barium, or phloretin to determine the mechanism of ITC inhibition. At 300 microM, DIDS or H2DIDS blocked restitution and wound repair by 100%, SITS blocked wound repair by 50%, and DNDS blocked wound repair by 2%. These results demonstrate the dependence of restitution and wound repair on ITC. ITC-binding purino (ATP) receptors and KATP channels were investigated as potential sites of inhibition, but they were found not to be the target of ITC in wound repair. Phloretin, blocking the monocarboxylate transporter (MCT), dose-dependently inhibited wound repair, and this result was exacerbated when the sodium bicarbonate cotransporter (NBC) was also blocked by bicarbonate-free conditions, resulting in 100% inhibition of wound repair with no reduction in viability when both transporters were blocked simultaneously. ITC potently inhibits both MCT and NBC, which may account for the inhibitory action of DIDS during restitution and wound repair. Reverse transcription-polymerase chain reaction data verified that MCT-1 is expressed in RGM1 cells. In conclusion, our results suggest that bicarbonate and monocarboxylate transport may work cooperatively to facilitate restitution of the gastric mucosa after injury.

Interactions between Na+ channels and Na+-HCO3- cotransporters in the freshwater fish gill MR cell: a model for transepithelial Na+ uptake.

Isolated mitochondria-rich (MR) cells from the rainbow trout gill epithelium were subjected to intracellular pH (pH(i)) imaging with the pH-sensitive dye BCECF-AM. MR cells were categorized into two distinct functional subtypes based on their ability to recover pH(i) from an NH(4)Cl-induced acidification in the absence of Na(+). An apparent link between resting pH(i) and Na(+)-independent pH(i) recovery was made. We observed a unique pH(i) acidification event that was induced by extracellular Na(+) addition. This further classified the mixed MR cell population into two functional subtypes: the majority of cells (77%) demonstrated the Na(+)-induced pH(i) acidification, whereas the minority (23%) demonstrated an alkalinization of pH(i) under the same circumstances. The focus of this study was placed on the Na(+)-induced acidification and pharmacological analysis via the use of amiloride and phenamil, which revealed that Na(+) uptake was responsible for the intracellular acidification. Further experiments revealed that pH(i) acidification could be abolished when Na(+) was allowed entry into the cell, but the activity of an electrogenic Na(+)-HCO(3)(-) cotransporter (NBC) was inhibited by DIDS. The electrogenic NBC activity was supported by a DIDS-sensitive, Na(+)-induced membrane potential depolarization as observed via imaging of the voltage-sensitive dye bis-oxonol. We also demonstrated NBC immunoreactivity via Western blotting and immunohistochemistry in gill tissue. We propose a model for transepithelial Na(+) uptake occurring via an apical Na(+) channel linked to a basolateral, electrogenic NBC in one subpopulation of MR cells.

Identification of a novel signal in the cytoplasmic tail of the Na+:HCO3- cotransporter NBC1 that mediates basolateral targeting.

The Na(+):HCO(3)(-) cotransporter 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. Furthermore, 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.

Colonic anion secretory defects and metabolic acidosis in mice lacking the NBC1 Na+/HCO3- cotransporter.

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 (pH(i)) was not altered in cAMP-stimulated epithelial cells of NBC1-/- cecum, but pH(i) 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- and HCO3-, 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.

Transporters involved in regulation of intracellular pH in primary cultured rat brain endothelial cells.

Fluid secretion across the blood-brain barrier, critical for maintaining the correct fluid balance in the brain, entails net secretion of HCO(3)(-), which is brought about by the combined activities of ion transporters situated in brain microvessels. These same transporters will concomitantly influence intracellular pH (pH(i)). To analyse the transporters that may be involved in the maintenance of pH(i) and hence secretion of HCO(3)(-), we have loaded primary cultured endothelial cells derived from rat brain microvessels with the pH indicator BCECF and suspended them in standard NaCl solutions buffered with Hepes or Hepes plus 5% CO(2)/HCO(3)(-). pH(i) in the standard solutions showed a slow acidification over at least 30 min, the rate being less in the presence of HCO(3)(-) than in its absence. However, after accounting for the difference in buffering, the net rates of acid loading with and without HCO(3)(-) were similar. In the nominal absence of HCO(3)(-) the rate of acid loading was increased equally by removal of external Na(+) or by inhibition of Na(+)/H(+) exchange by ethylisopropylamiloride (EIPA). By contrast, in the presence of HCO(3)(-) the increase in the rate of acid loading when Na(+) was removed was much larger and the rate was then also significantly greater than the rate observed in the absence of both Na(+) and HCO(3)(-). Removal of Cl(-) in the presence of HCO(3)(-) produced an alkalinization followed by a resumption of the slow acid gain. Removal of Na(+) following removal of Cl(-) increased the rate of acid gain. In the presence of HCO(3)(-) and initial presence of Na(+) and Cl(-), DIDS inhibited the changes in pH(i) produced by removal of either Na(+) or Cl(-). These are the expected results if these cells possess an AE-like Cl(-)/HCO(3)(-) exchanger, a 'channel-like' permeability allowing slow influx of acid (or efflux of HCO(3)(-)), a NBC-like Cl(-)-independent Na(+)-HCO(3)(-) cotransporter, and a NHE-like Na(+)/H(+) exchanger. The in vitro rates of HCO(3)(-) loading via the Na(+)-HCO(3)(-) cotransporter could, if the transporter is located on the apical, blood-facing side of the cells, account for the net secretion of HCO(3)(-) into the brain.

A cysteine-scanning mutagenesis study of transmembrane domain 8 of the electrogenic sodium/bicarbonate cotransporter NBCe1.

Na/HCO(3) cotransporters (NBCs) such as NBCe1 are members of a superfamily of bicarbonate transporters that includes anion exchangers. Residues within putative transmembrane domain 8 (TMD8) of anion exchanger 1 are involved in ion translocation (Tang, X. B., Kovacs, M., Sterling, D., and Casey, J. R. (1999) J. Biol. Chem. 274, 3557-3564), and the corresponding domain in NBCe1 variants is highly homologous. We performed cysteine-scanning mutagenesis to examine the role of TMD8 residues in ion translocation by rat NBCe1-A. We accessed function and/or sulfhydryl sensitivity and p-chloromercuribenzene sulfonate (pCMBS) accessibility of 21 cysteine-substituted NBC mutants expressed in Xenopus oocytes using the two-electrode, voltage clamp technique. Five NBC mutants displayed <10% wild-type activity: P743C, A744C, L746C, D754C, and T758C. For the remaining 16 mutants, we compared transporter-mediated inward currents elicited by removing external Na(+) before and after exposing oocytes to either 2-aminoethylmethane thiosulfonate (MTSEA) or pCMBS. MTSEA inhibited NBC mutants T748C, I749C, I751C, F752C, M753C, and Q756C by 9-19% and stimulated mutants A739C, A741C, L745C, V747C, Q755C, and I757C by 11-21%. pCMBS mildly inhibited mutants A739C, A740, V747C, and Q756C by 5 or 8%, and stimulated I749C by 10%. However, both sulfhydryl reagents strongly inhibited the L750C mutant by > or =85%. Using the substituted cysteine accessibility method, we examined the accessibility of the NBC mutant L750C under different transporter conditions. pCMBS accessibility is (i) reduced when the transporter is active in the presence of both Na(+) and HCO(3)(-), likely due to substrate competition with pCMBS; (ii) reduced in the presence of a stilbene inhibitor; and (iii) stimulated at more positive membrane potentials. In summary, TMD8 residues of NBCe1, particularly L750, are involved in ion translocation, and accessibility is influenced by the state of transporter activity.

Functional and molecular properties of Na+:HCO-3 cotransporters (NBC).

The sodium bicarbonate cotransporter (NBC) is located on the basolateral membrane of the kidney proximal tubule and is responsible for the reabsorption of the majority of filtered bicarbonate. In secretory epithelia (such as pancreatic duct) NBC is responsible for the transport of bicarbonate from the blood to the cell for the eventual secretion at the apical membrane. As such and due to its versatility, NBC can function in 2 distinct modes of bicarbonate reabsorption or secretion depending on the epithelia. NBC is also present in a wide variety of non-epithelial tissues where it is mainly involved with cell pH regulation. Recent molecular cloning experiments have identified the existence of 4 NBC isoforms (NBC1, 2, 3 and 4) and 2 NBC-related proteins AE4 and NCBE (anion exchanger 4 and sodium-dependent chloride-bicarbonate exchanger). Except for AE4 which does not transport Na, all are presumed to mediate the cotransport of Na+ and HCO(3)-. NBC isoforms show significant homology to each other and to the AE (anion exchange) family. NBC1 shows a limited tissue expression pattern, is electrogenic and plays an important role in bicarbonate reabsorption in kidney proximal tubule. A variant of NBC1 is expressed in pancreatic duct and gastrointestinal tract, and plays an important role in HCO(3)- secretion. NBC2 and NBC3 have a wider tissue distribution than NBC1, are electroneutral, and are involved with cell pH regulation. The electrogenecity of NBC4 may be variant-specific. An NBC related protein called NCBE mediates Na-dependent, Cl-/bicarbonate ex-change. The purpose of this review is to summarize functional and molecular properties of NBC isoforms and related proteins, and discuss their role and regulation in pathophysiologic states.