Computational model of electrode-induced microenvironmental effects on pH measurements near a cell membrane.

The mechanism of gas transport across cell membranes remains a topic of considerable interest, particularly regarding the extent to which lipids vs. specific membrane proteins provide conduction pathways. Studies of transmembrane (CO2) transport often rely on data collected under controlled conditions, using pH-sensitive microelectrodes at the extracellular surface to record changes due to extracellular CO2 diffusion and reactions. Although recent detailed computational models can predict a qualitatively correct behavior, a mismatch between the dynamical ranges of the predicted and observed pH curves raises the question whether the discrepancy may be due to a bias introduced by the pH electrode itself. More specifically, it is reasonable to ask whether bringing the electrode tip near or in contact with the membrane creates a local microenvironment between the electrode tip and the membrane, so that the measured data refer to the microenvironment rather than to the free surface. Here, we introduce a detailed computational model, designed to address this question. We find that, as long as a zone of free diffusion exists between the tip and the membrane, the microenvironment behaves effectively as the free membrane. However, according to our model, when the tip contacts the membrane, partial quenching of extracellular diffusion by the electrode rim leads to a significant increase in the pH dynamics under the electrode, matching values measured in physiological experiments. The computational schemes for the model predictions are based on semi-discretization by a finite-element method, and an implicit-explicit time integration scheme to capture the different time scales of the system.

NBCe1 (SLC4A4) a potential pH regulator in enamel organ cells during enamel development in the mouse.

During the formation of dental enamel, maturation-stage ameloblasts express ion-transporting transmembrane proteins. The SLC4 family of ion-transporters regulates intra- and extracellular pH in eukaryotic cells by cotransporting HCO3 (-) with Na(+). Mutation in SLC4A4 (coding for the sodium-bicarbonate cotransporter NBCe1) induces developmental defects in human and murine enamel. We have hypothesized that NBCe1 in dental epithelium is engaged in neutralizing protons released during crystal formation in the enamel space. We immunolocalized NBCe1 protein in wild-type dental epithelium and examined the effect of the NBCe1-null mutation on enamel formation in mice. Ameloblasts expressed gene transcripts for NBCe1 isoforms B/D/C/E. In wild-type mice, weak to moderate immunostaining for NBCe1 with antibodies that recognized isoforms A/B/D/E and isoform C was seen in ameloblasts at the secretory stage, with no or low staining in the early maturation stage but moderate to high staining in the late maturation stage. The papillary layer showed the opposite pattern being immunostained prominently at the early maturation stage but with gradually less staining at the mid- and late maturation stages. In NBCe1 (-/-) mice, the ameloblasts were disorganized, the enamel being thin and severely hypomineralized. Enamel organs of CFTR (-/-) and AE2a,b (-/-) mice (CFTR and AE2 are believed to be pH regulators in ameloblasts) contained higher levels of NBCe1 protein than wild-type mice. Thus, the expression of NBCe1 in ameloblasts and the papillary layer cell depends on the developmental stage and possibly responds to pH changes.

Immunocytochemical identification of electroneutral Na⁺-coupled HCO3⁻ transporters in freshly dissociated mouse medullary raphé neurons.

The medullary raphé (MR) of the medulla oblongata contains chemosensitive neurons that respond to increases in arterial [CO2], by altering firing rate, with increases being associated with serotonergic (5-hydroxytryptamine [5HT]) neurons and decreases, with GABAergic neurons. Both types of neurons contribute to increased alveolar ventilation. Decreases in intracellular pH are thought to link the rise in [CO2] to increased ventilation. Because electroneutral Na(+)-coupled HCO3(-) transporters (nNCBTs), which help protect cells from intracellular acidosis, are expressed robustly in the neurons of the central nervous system, a key question is whether these transporters are present in chemosensitive neurons. Therefore, we used an immunocytochemistry approach to identify neurons (using a microtubule associated protein-2 monoclonal antibody) and specifically 5HT neurons (TPH monoclonal antibody) or GABAergic neurons (GAD2 monoclonal antibody) in freshly dissociated cells from the mouse MR. We also co-labeled with polyclonal antibodies against the three nNCBTs: NBCn1, NDCBE, and NBCn2. We exploited ePet-EYFP (enhanced yellow fluorescent protein) mice (with EYFP-labeled 5HT neurons) as well as mice genetically deficient in each of the three nNCBTs. Quantitative image analysis distinguished positively stained cells from background signals. We found that >80% of GAD2(+) cells also were positive for NDCBE, and >90% of the TPH(+) and GAD2(+) cells were positive for the other nNCBTs. Assuming that the transporters are independently distributed among neurons, we can conclude that virtually all chemosensitive MR neurons contain at least one nNCBT.

Distribution of NBCn2 (SLC4A10) splice variants in mouse brain.

The five known Na-coupled HCO(3)(-) transporters (NCBTs) of the solute carrier 4 (SLC4) family play important roles in pH regulation and transepithelial HCO(3)(-) transport. Nearly all of the NCBTs have multiple splice variants. One particular NCBT, the electroneutral Na/HCO(3)(-) cotransporter NBCn2 (SLC4A10), which is predominantly expressed in brain, has three known splice variants-NBCn2-A, -B, and -C-as well as a potential variant-D. It is important to know the tissue-specific expression of the splice variants for understanding the physiological roles of NBCn2 in central nervous system. In the present study, we developed three novel rabbit polyclonal antibodies against NBCn2: (1) anti-ABCD, which recognizes all four variants; (2) anti-BD, which recognizes NBCn2-B and -D; (3) anti-CD, which recognizes NBCn2-C and -D. By western blotting, we examined the expression and distribution of NBCn2 splice variants in five brain regions: cerebral cortex, subcortex, cerebellum, hippocampus, and medulla. The expression pattern revealed with anti-ABCD is distinct from those revealed with anti-BD and anti-CD. Moreover, by using immunoprecipitation in combination with western blotting, we demonstrate that NBCn2-D does indeed exist and that it is predominantly expressed in subcortex, to a lesser extent in medulla, but at very low levels in cortex, cerebellum, and hippocampus. NBCn2-A may be the dominant variant in mouse brain as a whole, and may also dominate in cerebral cortex, cerebellum, and hippocampus. Immunohistochemistry with anti-ABCD shows that NBCn2 is highly expressed in choroid plexus, cortex, molecular layer of cerebellum, hippocampus, and some specific regions of the brainstem.

Localization of electrogenic Na/bicarbonate cotransporter NBCe1 variants in rat brain.

The activity of HCO(3)(-) transporters contributes to the acid-base environment of the nervous system. In the present study, we used in situ hybridization, immunoblotting, immunohistochemistry, and immunogold electron microscopy to localize electrogenic Na/bicarbonate cotransporter NBCe1 splice variants (-A, -B, and -C) in rat brain. The in situ hybridization data are consistent with NBCe1-B and -C, but not -A, being the predominant NBCe1 variants in brain, particularly in the cerebellum, hippocampus, piriform cortex, and olfactory bulb. An antisense probe to the B and C variants strongly labeled granule neurons in the dentate gyrus of the hippocampus, and cells in the granule layer and Purkinje layer (e.g. Bergmann glia) of the cerebellum. Weaker labeling was observed in the pyramidal layer of the hippocampus and in astrocytes throughout the brain. Similar, but weaker labeling was obtained with an antisense probe to the A and B variants. In immunoblot studies, antibodies to the A and B variants (alphaA/B) and C variant (alphaC) labeled approximately 130-kDa proteins in various brain regions. From immunohistochemistry data, both alphaA/B and alphaC exhibited diffuse labeling throughout brain, but alphaA/B labeling was more intracellular and punctate. Based on co-localization studies with antibodies to neuronal or astrocytic markers, alphaA/B labeled neurons in the pyramidal layer and dentate gyrus of the hippocampus, as well as cortex. alphaC labeled glia surrounding neurons (and possibly neurons) in the neuropil of the Purkinje cell layer of the cerebellum, the pyramidal cell layer and dentate gyrus of the hippocampus, and the cortex. According to electron microscopy data from the cerebellum, alphaA/B primarily labeled neurons intracellularly and alphaC labeled astrocytes at the plasma membrane. In summary, the B and C variants are the predominant NBCe1 variants in rat brain and exhibit different localization profiles.

Expression and localization of Na-driven Cl-HCO(3)(-) exchanger (SLC4A8) in rodent CNS.

The Na(+)-driven Cl-HCO(3) exchanger (NDCBE or SLC4A8) is a member of the solute carrier 4 (SLC4) family of HCO(3)(-) transporters, which includes products of 10 genes with similar sequences. Most SLC4 members play important roles in regulating intracellular pH (pH(i)). Physiological studies suggest that NDCBE is a major pH(i) regulator in at least hippocampal (HC) pyramidal neurons. We generated a polyclonal rabbit antibody directed against the first 18 residues of the cytoplasmic N terminus (Nt) of human NDCBE. By Western blotting, the antibody distinguishes NDCBE-as a purified Nt peptide or a full-length transporter (expressed in Xenopus oocytes)-from other Na(+)-coupled HCO(3)(-) transporters. By Western blotting, the antiserum recognizes an approximately 135-kDa band in several brain regions of adult mice: the cerebral cortex (CX), subcortex (SCX), cerebellum (CB), and HC. In CX, PNGase F treatment reduces the molecular weight to approximately 116 kDa. By immunocytochemistry, affinity-purified (AP) NDCBE antibody stains the plasma membrane of neuron cell bodies and processes of rat HC neurons in primary culture as well as freshly dissociated mouse HC neurons. The AP antibody does not detect substantial NDCBE levels in freshly dissociated HC astrocytes, or astrocytes in HC or CB sections. By immunohistochemistry, the AP antibody recognizes high levels of NDCBE in neurons of CX, HC (including pyramidal neurons in Cornu Ammonis (CA)1-3 and dentate gyrus), substantial nigra, medulla, cerebellum (especially Purkinje and granular cells), and the basolateral membrane of fetal choroid plexus. Thus, NDCBE is in a position to contribute substantially to pH(i) regulation in multiple CNS neurons.

Use of a new polyclonal antibody to study the distribution and glycosylation of the sodium-coupled bicarbonate transporter NCBE in rodent brain.

NCBE (SLC4A10) is a member of the SLC4 family of bicarbonate transporters, several of which play important roles in intracellular-pH regulation and transepithelial HCO(3)(-) transport. Here we characterize a new antibody that was generated in rabbit against a fusion protein consisting of maltose-binding protein and the first 135 amino acids (aa) of the N-terminus of human NCBE. Western blotting--both of purified peptides representing the initial approximately 120 aa of the transporters and of full-length transporters expressed in Xenopus oocytes--demonstrated that the antibody is specific for NCBE versus the two most closely related proteins, NDCBE (SLC4A8) and NBCn1 (SLC4A7). Western blotting of tissue in four regions of adult mouse brain indicates that NCBE is expressed most abundantly in cerebral cortex (CX), cerebellum (CB) and hippocampus (HC), and less so in subcortex (SCX). NCBE protein was present in CX, CB, and HC microdissected to avoid choroid plexus. Immunocytochemistry shows that NCBE is present at the basolateral membrane of embryonic day 18 (E18) fetal and adult choroid plexus. NCBE protein is present by Western blot and immunocytochemistry in cultured and freshly dissociated HC neurons but not astrocytes. By Western blot, nearly all NCBE in mouse and rat brain is highly N-glycosylated (approximately 150 kDa). PNGase F reduces the molecular weight (MW) of natural NCBE in mouse brain or human NCBE expressed in oocytes to approximately the predicted MW of the unglycosylated protein. In oocytes, mutating any one of the three consensus N-glycosylation sites reduces glycosylation of the other two, and the triple mutant exhibits negligible functional expression.

Evidence that aquaporin 1 is a major pathway for CO2 transport across the human erythrocyte membrane.

We report here the application of a previously described method to directly determine the CO2 permeability (P(CO2)) of the cell membranes of normal human red blood cells (RBCs) vs. those deficient in aquaporin 1 (AQP1), as well as AQP1-expressing Xenopus laevis oocytes. This method measures the exchange of (18)O between CO2, HCO3(-), and H2O in cell suspensions. In addition, we measure the alkaline surface pH (pH(S)) transients caused by the dominant effect of entry of CO2 vs. HCO3(-) into oocytes exposed to step increases in [CO2]. We report that 1) AQP1 constitutes the major pathway for molecular CO2 in human RBCs; lack of AQP1 reduces P(CO2) from the normal value of 0.15 +/- 0.08 (SD; n=85) cm/s by 60% to 0.06 cm/s. Expression of AQP1 in oocytes increases P(CO2) 2-fold and doubles the alkaline pH(S) gradient. 2) pCMBS, an inhibitor of the AQP1 water channel, reduces P(CO2) of RBCs solely by action on AQP1 as it has no effect in AQP1-deficient RBCs. 3) P(CO2) determinations of RBCs and pH(S) measurements of oocytes indicate that DIDS inhibits the CO2 pathway of AQP1 by half. 4) RBCs have at least one other DIDS-sensitive pathway for CO2. We conclude that AQP1 is responsible for 60% of the high P(CO2) of red cells and that another, so far unidentified, CO2 pathway is present in this membrane that may account for at least 30% of total P(CO2).

Expression of Na+/H+ and HCO3- -dependent transporters in Na+/H+ exchanger isoform 1 null mutant mouse brain.

Acid-base transporters, such as the sodium-hydrogen exchangers (NHEs) and bicarbonate-dependent transporters, play an important role in the regulation of intracellular pH (pH(i)) in the CNS. Previous studies from our laboratory have shown that the absence of the major NHE isoform 1 (NHE1) reduced the steady-state pH(i) and recovery rate from an acid load in the hippocampal neurons not only in HEPES but also in HCO(3)(-) solutions (Yao et al., 1999). The purpose of the current study was to determine whether the NHE1 null mutation affects the expression of pH-regulatory transporters in the mouse CNS. Immunoblotting and semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) were performed to examine the protein and mRNA levels of NHE1-4, electrogenic sodium-bicarbonate cotransporter 1 variants (NBCe1), and brain-specific anion exchanger 3 (AE3) in four brain regions (cerebral cortex, hippocampus, cerebellum and brainstem-diencephalon). NHE1 null mutant mice were compared with their wild type controls at the average age of approximately 4 weeks. Our results revealed that the NHE1 null mutation caused a significant increase in NHE3 in the cerebellum (84% for protein, 105% for mRNA), an increase in NBCe1 expression in the brainstem-diencephalon (approximately 40-50% for protein, 9-15% for mRNA), as well as a decrease in AE3 in the hippocampus (approximately 60% for protein, 24% for mRNA). We conclude that the NHE1 null mutation does alter the expression of other membrane transporters at both protein and mRNA levels. The alteration is region-specific. An increase in acid extruders (e.g. NHE3) and a decrease in acid loaders (e.g. AE3) suggest that there are some compensatory mechanisms that occur in NHE1 null mutant mice.

Inhibition of the cardiac electrogenic sodium bicarbonate cotransporter reduces ischemic injury.

OBJECTIVE: Although it is believed that sodium-driven acid-base transport plays a central role in the development of the reperfusion injury that follows cardiac ischemia, research to date has demonstrated only a role for Na(+)/H(+) exchange (NHE). However, Na(+)-driven HCO(-)(3) transport, which is quantitatively as important as NHE in cardiac cells, has not been examined. METHODS AND RESULTS: Here the results show that a neutralizing antibody raised against the human heart electrogenic Na(+)/HCO(3)(-) cotransporter (hhNBC) blocked the recovery of pH after acidic pulse both in HEK-293 cells expressing hhNBC and in rat cardiac myocytes demonstrating the presence of an electrogenic NBC in rat cardiac myocytes similar to hhNBC. Administration of anti-NBC antibody to ischemic-reperfused rat hearts markedly protects systolic and diastolic functions of the heart during reperfusion. Furthermore, using a quantitative real-time RT-PCR (TaqMan) and Western blot analysis we demonstrated that in human cardiomyopathic hearts, mRNA and protein levels of hhNBC increase, whereas mRNA levels of the electroneutral Na(+)/HCO(3)(-) cotransporter (NBCn1) remain unchanged. CONCLUSION: Our data provide evidence that inhibition of hhNBC, whose role in cardiac pathologies could be amplified by overexpression, represents a novel therapeutic approach for ischemic heart disease.

Cloning and functional expression of an MIP (AQP0) homolog from killifish (Fundulus heteroclitus) lens.

The major intrinsic protein (MIP) of lens fiber cells is a member of the aquaporin (AQP) water channel family. The protein is expressed at very high levels in lens fiber cells, but its physiological function is unclear. By homology to known AQPs, we have cloned a full-length cDNA encoding an MIP from the lens of killifish (Fundulus heteroclitus). The predicted protein (263 amino acids; GenBank accession no. AF191906) shows 77% identity to amphibian MIPs, 70% identity to mammalian MIPs, and 46% identity to mammalian AQP1. Expression of MIPfun in Xenopus laevis oocytes causes an approximately 40-fold increase in oocyte water permeability. This stimulation is comparable to that seen with AQP1 and substantially larger than that seen with other MIPs. The mercurials HgCl(2) and p-chloromercuribenzenesulfonate inhibit the water permeability of MIPfun by approximately 25%. MIPfun is not permeable to glycerol, urea, or formic acid but is weakly permeable to CO(2).

Inhibition of K/HCO(3) cotransport in squid axons by quaternary ammonium ions.

Previous squid-axon studies identified a novel K/HCO3 cotransporter that is insensitive to disulfonic stilbene derivatives. This cotransporter presumably responds to intracellular alkali loads by moving K(+) and HCO(3)(-) out of the cell, tending to lower intracellular pH (pH(i)). With an inwardly directed K/HCO(3) gradient, the cotransporter mediates a net uptake of alkali (i.e., K(+) and HCO(3)(-) influx). Here we test the hypothesis that intracellular quaternary ammonium ions (QA(+)) inhibit the inwardly directed cotransporter by interacting at the intracellular K(+) site. We computed the equivalent HCO(3)(-) influx (J(HCO3)) mediated by the cotransporter from the rate of pH(i) increase, as measured with pH-sensitive microelectrodes. We dialyzed axons to pH(i) 8.0, using a dialysis fluid (DF) free of K(+), Na(+) and Cl(-). Our standard artificial seawater (ASW) also lacked Na(+), K(+) and Cl(-). After halting dialysis, we introduced an ASW containing 437 mm K(+) and 0.5% CO(2)/12 mm HCO(3)(-), which (i) caused membrane potential to become transiently very positive, and (ii) caused a rapid pHi decrease, due to CO(2) influx, followed by a slower plateau-phase pH(i) increase, due to inward cotransport of K(+) and HCO(3)(-). With no QA(+) in the DF, J(HCO3) was approximately 58 pmole cm(-2) sec(-1). With 400 mm tetraethylammonium (TEA(+)) in the DF, J(HCO3) was virtually zero. The apparent K(i) for intracellular TEA(+) was approximately 78 mm, more than two orders of magnitude greater than that obtained by others for inhibition of K(+) channels. Introducing 100 mm inhibitor into the DF reduced J(HCO3) to approximately 20 pmole cm(-2) sec(-1) for tetramethylammonium (TMA(+)), approximately 24 for TEA(+), approximately 10 for tetrapropylammonium (TPA(+)), and virtually zero for tetrabutylammonium (TBA(+)). The apparent K(i) value for TBA(+) is approximately 0.86 mm. The most potent inhibitor was phenyl-propyltetraethylammonium (PPTEA(+)), with an apparent K(i) of approximately 91 microm. Thus, trans-side quaternary ammonium ions inhibit K/HCO(3) influx in the potency sequence PPTEA(+) > TBA(+) > TPA(+) > TEA(+) congruent with TMA(+). The identification of inhibitors of the K/HCO(3) cotransporter, for which no inhibitors previously existed, will facilitate the study of this transporter.

Sodium-hydrogen exchangers and sodium-bicarbonate co-transporters: ontogeny of protein expression in the rat brain.

We used western blotting to examine the developmental profiles (at embryonic day 16 and postnatal days 1, 13, 23, 33 and 105) of protein expression for three sodium-hydrogen exchanger isoforms (1, 2 and 4) and for a sodium-bicarbonate co-transporter in three CNS regions (cortex, cerebellum and brainstem-diencephalon). In microsomal preparations, sodium-hydrogen exchanger isoform 1 and sodium-bicarbonate co-transporter protein expression in the CNS increases gradually from embryonic day 16 (25-40% of the adult level) to postnatal day 105. In contrast, sodium-hydrogen exchanger isoform 2 and 4 expression reaches a maximum (three to 20 times the adult level) at around three to four weeks of age. There is significant regional heterogeneity in the expression of sodium-hydrogen exchanger and sodium-bicarbonate co-transporter proteins in the rat CNS. Sodium-hydrogen exchanger isoform 1 was highly expressed in the brainstem-diencephalon, whereas the sodium-bicarbonate co-transporter was robustly expressed in the cerebellum and brainstem-diencephalon. These data indicate that the expression of sodium-hydrogen exchanger and sodium-bicarbonate co-transporter proteins varies as a function of both development and specific brain region.

Cloning, characterization, and chromosomal mapping of a human electroneutral Na(+)-driven Cl-HCO3 exchanger.

The electroneutral Na(+)-driven Cl-HCO3 exchanger is a key mechanism for regulating intracellular pH (pH(i)) in neurons, glia, and other cells. Here we report the cloning, tissue distribution, chromosomal location, and functional characterization of the cDNA of such a transporter (NDCBE1) from human brain (GenBank accession number AF069512). NDCBE1, which encodes 1044 amino acids, is 34% identical to the mammalian anion exchanger (AE2); approximately 50% to the electrogenic Na/HCO3 cotransporter (NBCe1) from salamander, rat, and humans; approximately 73% to mammalian electroneutral Na/HCO3 cotransporters (NBCn1); 71% to mouse NCBE; and 47% to a Na(+)-driven anion exchanger (NDAE1) from Drosophila. Northern blot analysis of NDCBE1 shows a robust approximately 12-kilobase signal in all major regions of human brain and in testis, and weaker signals in kidney and ovary. This human gene (SLC4A8) maps to chromosome 12q13. When expressed in Xenopus oocytes and running in the forward direction, NDCBE1 is electroneutral and mediates increases in both pH(i) and [Na(+)](i) (monitored with microelectrodes) that require HCO3(-) and are blocked by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). The pH(i) increase also requires extracellular Na(+). The Na(+):HCO3(-) stoichiometry is 1:2. Forward-running NDCBE1 mediates a 36Cl efflux that requires extracellular Na(+) and HCO3(-) and is blocked by DIDS. Running in reverse, NDCBE1 requires extracellular Cl(-). Thus, NDCBE1 encodes a human, electroneutral Na(+)-driven Cl-HCO3 exchanger.

Sodium-coupled bicarbonate transporters.

Together, the Na(+)-coupled HCO(3)(-) transporters and the AE family of anion exchangers (i.e., Cl-HCO3 exchangers) comprise the bicarbonate transporter (BT) superfamily. Virtually all BTs are important for the regulation of intracellular pH (pH(i)) in cells throughout the body. Specific BTs also play roles in cell-volume regulation, as well as for the transport of salt and/or acid-base equivalents across many epithelia. Electrogenic Na/HCO3 cotransporters (NBCe's) play key roles in HCO(3)(-) reabsorption by the renal proximal tubule, and HCO(3)(-) secretion by the pancreatic duct. Electroneutral NBC's (NBCn's) regulate pH(i) in vascular smooth muscle and are present in/near axons in the brain. Finally, the Na(+)-driven Cl-HCO3 exchanger (NDCBE's) appear to be the major pH(i) regulators in CNS neurons. A characteristic of most, but not all, BT's is that they are inhibited rather effectively by 4,4'-diisothiocyanostilbene-4,4'-disulfonate (DIDS).

Immunolocalization of electroneutral Na-HCO(3)(-) cotransporter in rat kidney.

An electroneutral Na-HCO(3)(-) cotransporter (NBC(N)1) was recently cloned, and Northern blot analyses indicated its expression in rat kidney. In this study, we determined the cellular and subcellular localization of NBC(N)1 in the rat kidney at the light and electron microscopic level. A peptide-derived antibody was raised against the COOH-terminal amino acids of NBC(N)1. The affinity-purified antibody specifically recognized one band, approximately 180 kDa, in rat kidney membranes. Peptide-N-glycosidase F deglycosylation reduced the band to approximately 140 kDa. Immunoblotting of membrane fractions from different kidney regions demonstrated strong signals in the inner stripe of the outer medulla (ISOM), weaker signals in the outer stripe of the outer medulla and inner medulla, and no labeling in cortex. Immunocytochemistry demonstrated that NBC(N)1 immunolabeling was exclusively observed in the basolateral domains of thick ascending limb (TAL) cells in the outer medulla (strongest in ISOM) but not in the cortex. In addition, collecting duct intercalated cells in the ISOM and in the inner medulla also exhibited NBC(N)1 immunolabeling. Immunoelectron microscopy demonstrated that NBC(N)1 labeling was confined to the basolateral plasma membranes of TAL and collecting duct type A intercalated cells. Immunolabeling controls were negative. By using 2, 7-bis-carboxyethyl-5,6-caboxyfluorescein, intracellular pH transients were measured in kidney slices from ISOM and from mid-inner medulla. The results revealed DIDS-sensitive, Na- and HCO(3)(-)-dependent net acid extrusion only in the ISOM but not in mid-inner medulla, which is consistent with the immunolocalization of NBC(N)1. The localization of NBC(N)1 in medullary TAL cells and medullary collecting duct intercalated cells suggests that NBC(N)1 may be important for electroneutral basolateral HCO(3)(-) transport in these cells.

Na/HCO3 cotransporters in rat brain: expression in glia, neurons, and choroid plexus.

We studied the expression and distribution of Na/HCO(3) cotransporters in rat brain using polynucleotide probes and polyclonal antibodies derived from the electrogenic rat kidney Na/HCO(3) cotransporter (rkNBC). In whole brain, we observed a single mRNA ( approximately 7.5 kb) by Northern hybridization and a major approximately 130 kDa protein by immunoblotting with a polyclonal antiserum directed against the C terminus of rkNBC. NBC mRNA and protein were present in cortex, brainstem-diencephalon, and cerebellum. In situ hybridization revealed NBC mRNA expression throughout the CNS, with particularly high levels in olfactory bulb, hippocampal dentate gyrus, and cerebellum. NBC mRNA was present in glial cells (e.g., Bergmann glia of cerebellum and hippocampal astrocytes) and neurons (e.g., granule cells of dentate gyrus and neurons of cortex or striatum). Double hybridization of mRNA encoding NBC and glutamate transporter 1 (glial marker) confirmed that both glia and neurons express NBC. Indirect immunofluorescence microscopy demonstrated NBC protein throughout the CNS, particularly in hippocampus and cerebellum. Although NBC mRNA was restricted to cell bodies, NBC protein was distributed diffusely, compatible with a localization in cell processes and perhaps cell bodies. Double labeling with glial fibrillary acidic protein (astrocytic marker), microtubule-associated protein 2 (neuronal marker), or 2',3'-cyclic mononucleotide 3'-phosphodiesterase (oligodendrocytic marker) demonstrated expression of NBC protein in specific subpopulations of both glia and neurons. Moreover, NBC protein was present in both cultured hippocampal astrocytes and cortical neurons. NBC mRNA and protein were also present in epithelial cells of choroid plexus, ependyma, and meninges. Our results are thus consistent with multiple novel roles for Na/HCO(3) cotransport in CNS physiology.

An electroneutral sodium/bicarbonate cotransporter NBCn1 and associated sodium channel.

Two electroneutral, Na+-driven HCO3- transporters, the Na+-driven Cl-/HCO3- exchanger and the electroneutral Na+/HCO3- cotransporter, have crucial roles in regulating intracellular pH in a variety of cells, including cardiac myocytes, vascular smooth-muscle, neurons and fibroblasts; however, it is difficult to distinguish their Cl- dependence in mammalian cells. Here we report the cloning of three variants of an electroneutral Na+/HCO3- cotransporter, NBCn1, from rat smooth muscle. They are 89-92% identical to a human skeletal muscle clone, 55-57% identical to the electrogenic NBCs and 33-43% identical to the anion exchangers. When expressed in Xenopus oocytes, NBCn1-B (which encodes 1,218 amino acids) is electroneutral, Na+-dependent and HCO3(-)-dependent, but not Cl(-)-dependent. Oocytes injected with low levels of NBCn1-B complementary RNA exhibit a Na+ conductance that 4,4-diisothiocyanatostilbene-2,2'-disulphonate stimulates slowly and irreversibly.

An electrogenic Na(+)-HCO(-)(3) cotransporter (NBC) with a novel COOH-terminus, cloned from rat brain.

We screened rat brain cDNA libraries and used 5' rapid amplification of cDNA ends to clone two electrogenic Na(+)-HCO(-)(3) cotransporter (NBC) isoforms from rat brain (rb1NBC and rb2NBC). At the amino acid level, one clone (rb1NBC) is 96% identical to human pancreas NBC. The other clone (rb2NBC) is identical to rb1NBC except for 61 unique COOH-terminal amino acids, the result of a 97-bp deletion near the 3' end of the open-reading frame. Using RT-PCR, we confirmed that mRNA from rat brain contains this 97-bp deletion. Furthermore, we generated rabbit polyclonal antibodies that distinguish between the unique COOH-termini of rb1NBC (alpharb1NBC) and rb2NBC (alpharb2NBC). alpharb1NBC labels an approximately 130-kDa protein predominantly from kidney, and alpharb2NBC labels an approximately 130-kDa protein predominantly from brain. alpharb2NBC labels a protein that is more highly expressed in cortical neurons than astrocytes cultured from rat brain; alpharb1NBC exhibits the opposite pattern. In expression studies, applying 1.5% CO(2)/10 mM HCO(-)(3) to Xenopus oocytes injected with rb2NBC cRNA causes 1) pH(i) to recover from the initial CO(2)-induced acidification and 2) the cell to hyperpolarize. Subsequently, removing external Na(+) reverses the pH(i) increase and elicits a rapid depolarization. In the presence of 450 microM DIDS, removing external Na(+) has no effect on pH(i) and elicits a small hyperpolarization. The rate of the pH(i) decrease elicited by removing Na(+) is insensitive to removing external Cl(-). Thus rb2NBC is a DIDS-sensitive, electrogenic NBC that is predominantly expressed in brain of at least rat.

Extracellular HCO(3)(-) dependence of electrogenic Na/HCO(3) cotransporters cloned from salamander and rat kidney.

We studied the extracellular [HCOabstract (3) (-)] dependence of two renal clones of the electrogenic Na/HCO(3) cotransporter (NBC) heterologously expressed in Xenopus oocytes. We used microelectrodes to measure the change in membrane potential (DeltaV(m)) elicited by the NBC cloned from the kidney of the salamander Ambystoma tigrinum (akNBC) and by the NBC cloned from the kidney of rat (rkNBC). We used a two-electrode voltage clamp to measure the change in current (DeltaI) elicited by rkNBC. Briefly exposing an NBC-expressing oocyte to HCOabstract (3 )(-)/CO(2) (0.33-99 mM HCOabstract (3)(-), pH(o) 7.5) elicited an immediate, DIDS (4, 4-diisothiocyanatostilbene-2,2-disulfonic acid)-sensitive and Na(+)-dependent hyperpolarization (or outward current). In DeltaV(m) experiments, the apparent K(m ) for HCOabstract (3)(-) of akNBC (10. 6 mM) and rkNBC (10.8 mM) were similar. However, under voltage-clamp conditions, the apparent K(m) for HCOabstract (3)(-) of rkNBC was less (6.5 mM). Because it has been reported that SOabstract (3)(=)/HSO abstract (3)(-) stimulates Na/HCO(3 ) cotransport in renal membrane vesicles (a result that supports the existence of a COabstract (3)(=) binding site with which SOabstract (3)(=) interacts), we examined the effect of SOabstract (3)(=)/HSO abstract (3)(-) on rkNBC. In voltage-clamp studies, we found that neither 33 mM SOabstract (4)(=) nor 33 mM SOabstract (3) (=)/HSOabstract (3)(-) substantially affects the apparent K(m) for HCO abstract (3)(-). We also used microelectrodes to monitor intracellular pH (pH(i)) while exposing rkNBC-expressing oocytes to 3.3 mM HCOabstract (3 )(-)/0.5% CO(2). We found that SO abstract (3)(=)/HSOabstract (3 )(-) did not significantly affect the DIDS-sensitive component of the pH(i) recovery from the initial CO(2 )-induced acidification. We also monitored the rkNBC current while simultaneously varying [CO(2)](o), pH(o), and [COabstract (3)(=)](o) at a fixed [HCOabstract (3)(-)](o) of 33 mM. A Michaelis-Menten equation poorly fitted the data expressed as current versus [COabstract (3)(=)](o ). However, a pH titration curve nicely fitted the data expressed as current versus pH(o). Thus, rkNBC expressed in Xenopus oocytes does not appear to interact with SOabstract (3 )(=), HSOabstract (3)(-), or COabstract (3)(=).