The cardiac electrogenic sodium/bicarbonate cotransporter (NBCe1) is activated by aldosterone through the G protein-coupled receptor 30 (GPR 30)

The sodium/bicarbonate cotransporter (NBC) transports extracellular Na⁺ and HCO₃^? into the cytoplasm upon intracellular acidosis, restoring the acidic pH_i to near neutral values. Two different NBC isoforms have been described in the heart, the electroneutral NBCn1 (1Na⁺:1HCO₃^?) and the electrogenic NBCe1 (1Na⁺:2HCO₃^?). Certain non-genomic effects of aldosterone (Ald) were due to an orphan G protein-couple receptor 30 (GPR30). We have recently demonstrated that Ald activates GPR30 in adult rat ventricular myocytes, which transactivates the epidermal growth factor receptor (EGFR) and in turn triggers a reactive oxygen species (ROS)- and PI3K/AKT-dependent pathway, leading to the stimulation of NBC. The aim of this study was to investigate the NBC isoform involved in the Ald/GPR30-induced NBC activation. Using specific NBCe1 inhibitory antibodies (a-L3) we demonstrated that Ald does not affect NBCn1 activity. Ald was able to increase NBCe1 activity recorded in isolation. Using immunofluorescence and confocal microscopy analysis we showed in this work that both NBCe1 and GPR30 are localized in t-tubules. In conclusion, we have demonstrated that NBCe1 is the NBC isoform activated by Ald in the heart.     

Cytosolic sodium regulation in mouse cortical astrocytes and its dependence on potassium and bicarbonate

Sodium plays a major role in different astrocytic functions, including maintenance of ion homeostasis and uptake of neurotransmitters and metabolites, which are mediated by different Na⁺-coupled transporters. In the current study, the role of an electrogenic sodium-bicarbonate cotransporter (NBCe1), a sodium-potassium-chloride transporter 1 (NKCC1) and sodium-potassium ATPase (Na⁺-K⁺-ATPase) for the maintenance of [Na⁺]_i was investigated in cultured astrocytes of wild-type (WT) and of NBCe1-deficient (NBCe1-KO) mice using the Na⁺-sensitive dye, asante sodium green-2. Our results suggest that cytosolic Na⁺ was higher in the presence of CO₂/HCO₃⁻ (15 mM) than CO₂/HCO₃⁻-free, HEPES-buffered solution in WT, but not in NBCe1-KO astrocytes (12 mM). Surprisingly, there was a strong dependence of cytosolic [Na⁺] on the extracellular [HCO₃⁻] attributable to NBCe1 activity. Pharmacological blockage of NKCC1 with bumetanide led to a robust drop in cytosolic Na⁺ in both WT and NBCe1-KO astrocytes by up to 6 mM. There was a strong dependence of the cytosolic [Na⁺] on the extracellular [K⁺]. Inhibition of the Na⁺-K⁺-ATPase led to larger increase in cytosolic Na⁺, both in the absence of K⁺ as compared with the presence of ouabain and in NBCe1-KO astrocytes as compared with WT astrocytes. Our results show that cytosolic Na⁺ in mouse cortical astrocytes can vary considerably and depends greatly on the concentrations of HCO₃⁻ and K⁺, attributable to the activity of the Na⁺-K⁺-ATPase, of NBCe1 and NKCC1.     

Na+-dependent HCO3- uptake into the rat choroid plexus epithelium is partially DIDS sensitive

Several studies suggest the involvement of Na⁺ and HCO₃^– transport in the formation of cerebrospinal fluid. Two Na⁺-dependent HCO₃^– transporters were recently localized to the epithelial cells of the rat choroid plexus (NBCn1 and NCBE), and the mRNA for a third protein was also detected (NBCe2) (Praetorius J, Nejsum LN, and Nielsen S. Am J Physiol Cell Physiol 286: C601–C610, 2004). Our goal was to immunolocalize the NBCe2 to the choroid plexus by immunohistochemistry and immunogold electronmicroscopy and to functionally characterize the bicarbonate transport in the isolated rat choroid plexus by measurements of intracellular pH (pH_i) using a dual-excitation wavelength pH-sensitive dye (BCECF). Both antisera derived from COOH-terminal and NH₂-terminal NBCe2 peptides localized NBCe2 to the brush-border membrane domain of choroid plexus epithelial cells. Steady-state pH_i in choroidal cells increased from 7.03 ± 0.02 to 7.38 ± 0.02 (n = 41) after addition of CO₂/HCO₃^– into the bath solution. This increase was Na⁺ dependent and inhibited by the Cl^– and HCO₃^– transport inhibitor DIDS (200 µM). This suggests the presence of Na⁺-dependent, partially DIDS-sensitive HCO₃^– uptake. The pH_i recovery after acid loading revealed an initial Na⁺ and HCO₃^–-dependent net base flux of 0.828 ± 0.116 mM/s (n = 8). The initial flux in the presence of CO₂/HCO₃^– was unaffected by DIDS. Our data support the existence of both DIDS-sensitive and -insensitive Na⁺- and HCO₃^–-dependent base loader uptake into the rat choroid plexus epithelial cells. This is consistent with the localization of the three base transporters NBCn1, Na⁺-driven Cl^– bicarbonate exchanger, and NBCe2 in this tissue. bicarbonate metabolism; BCECF; cerebrospinal fluid; acid/base transport; ammonium prepulse     

Mutation of Aspartate 555 of the Sodium/Bicarbonate Transporter SLC4A4/NBCe1 Induces Chloride Transport

To understand the mechanism for ion transport through the sodium/bicarbonate transporter SLC4A4 (NBCe1), we examined amino acid residues, within transmembrane domains, that are conserved among electrogenic Na/HCO₃ transporters but are substituted with residues at the corresponding site of all electroneutral Na/HCO₃ transporters. Point mutants were constructed and expressed in Xenopus oocytes to assess function using two-electrode voltage clamp. Among the mutants, D555E (charge-conserved substitution of the aspartate at position 555 with a glutamate) produced decreasing HCO₃⁻ currents at more positive membrane voltages. Immunohistochemistry showed D555E protein expression in oocyte membranes. D555E induced Na/HCO₃-dependent pH recovery from a CO₂-induced acidification. Current-voltage relationships revealed that D555E produced an outwardly rectifying current in the nominally CO₂/HCO₃⁻-free solution that was abolished by Cl⁻ removal from the bath. In the presence of CO₂/HCO₃⁻, however, the outward current produced by D555E decreased only slightly after Cl⁻ removal. Starting from a Cl⁻-free condition, D555E produced dose-dependent outward currents in response to a series of chloride additions. The D555E-mediated chloride current decreased by 70% in the presence of CO₂/HCO₃⁻. The substitution of Asp⁵⁵⁵ with an asparagine also produced a Cl⁻ current. Anion selectivity experiments revealed that D555E was broadly permissive to other anions including NO₃⁻. Fluorescence measurements of chloride transport were done with human embryonic kidney HEK 293 cells expressing NBCe1 and D555E. A marked increase in chloride transport was detected in cells expressing D555E. We conclude that Asp⁵⁵⁵ plays a role in HCO₃⁻ selectivity.The electrogenic Na/HCO₃ cotransporter NBCe1 (SLC4A4) is one of the SLC4A gene family members transporting HCO₃⁻ across the plasma membrane (1–3). NBCe1 plays a role in transepithelial HCO₃⁻ movement and pH_i regulation in many tissues (4–6). NBCe1 is responsible for HCO₃⁻ reabsorption in the proximal tubules of the kidney (7). The proximal tubule cells reclaim HCO₃⁻ from the lumen through a series of reactions involving titration of HCO₃⁻ by H⁺ secretion via the apical Na/H exchanger, production of CO₂, and regeneration of HCO₃⁻ and H⁺ in the tubule cells. HCO₃⁻ then moves to the interstitium via the basolateral NBCe1. The essential feature driving this basolateral Na⁺/HCO₃⁻ exit is the stoichiometry of 1:3 Na⁺:HCO₃⁻, which makes the equilibrium potential for NBCe1 more positive than the resting membrane potential of the proximal tubule cells (8). The stoichiometry of 1Na⁺:1HCO₃⁻ or 1Na⁺:2HCO₃⁻ causes both ions to move into cells in other tissues such as pancreas, brain, and cardiovascular tissues (9, 10).Despite the importance of NBCe1 for basolateral HCO₃⁻ reabsorption in the proximal tubules, the mechanism of electrogenic Na/HCO₃ transport via the transporter is not well understood. Ion movement depends on loading ions at their translocation or binding sites that likely reside within the membrane field at some distance from the bath solution (11). This implies that the transmembrane domains (TMs)2 of NBCe1 and amino acid residues within TMs play critical roles in ion transport.Sequence analysis of different SLC4A proteins shows similar hydropathy plots, predicting that these proteins share structural elements of transport function (12). Such similarities have facilitated structure/function studies to define molecular domains or motifs responsible for conferring Na/HCO₃ transport of NBCe1. Abuladze et al. (13) performed a large scale mutagenesis on acidic and basic amino acids in non-TMs and found many residues affecting Na⁺-dependent base flux. McAlear et al. (14) identified amino acids in TM8 involving ion translocation. By a systematic approach of chimeric transporters between NBCe1 and the electroneutral Na/HCO₃ cotransporter NBCn1 (SLC4A7) (15), we and our colleagues (16) demonstrated that electrogenic Na/HCO₃ transport of NBCe1 requires interactions between the regions TM1–5 and TM6–13 of the protein. Zhu et al. (17) recently proposed TM1 as a domain lining the ion translocation pathway. On the other hand, Chang et al. (18) reported that the cytoplasmic N-terminal domain might contribute to HCO₃⁻ permeation.In the present study, we searched amino acid residues that are highly conserved among electrogenic Na/HCO₃ transporters but not among electroneutral Na/HCO₃ transporters and examined their role in electrogenic Na/HCO₃ transport. Nine candidate residues in human renal NBCe1-A (5, 19) were selected and mutated by replacement with the amino acids at the corresponding sites of NBCn1. Mutant transporters were expressed in Xenopus oocytes and assessed via two-electrode voltage clamp. Our data show that Asp⁵⁵⁵ of NBCe1 plays an important role in HCO₃⁻ selectivity.     

The acid-base transport proteins NHE1 and NBCn1 regulate cell cycle progression in human breast cancer cells

Precise acid-base homeostasis is essential for maintaining normal cell proliferation and growth. Conversely, dysregulated acid-base homeostasis, with increased acid extrusion and marked extracellular acidification, is an enabling feature of solid tumors, yet the mechanisms through which intra- and extracellular pH (pH_i, pH_e) impact proliferation and growth are incompletely understood. The aim of this study was to determine the impact of pH, and specifically of the Na⁺/H⁺ exchanger NHE1 and Na⁺, HCO₃^? transporter NBCn1, on cell cycle progression and its regulators in human breast cancer cells. Reduction of pH_e to 6.5, a common condition in tumors, significantly delayed cell cycle progression in MCF-7 human breast cancer cells. The NHE1 protein level peaked in S phase and that of NBCn1 in G2/M. Steady state pH_i changed through the cell cycle, from 7.1 in early S phase to 6.8 in G2, recovering again in M phase. This pattern, as well as net acid extrusion capacity, was dependent on NHE1 and NBCn1. Accordingly, knockdown of either NHE1 or NBCn1 reduced proliferation, prolonged cell cycle progression in a manner involving S phase prolongation and delayed G2/M transition, and altered the expression pattern and phosphorylation of cell cycle regulatory proteins. Our work demonstrates, for the first time, that both NHE1 and NBCn1 regulate cell cycle progression in breast cancer cells, and we propose that this involves cell cycle phase-specific pH_i regulation by the two transporters.     

Splice Cassette II of Na+,HCO3? Cotransporter NBCn1 (slc4a7) Interacts with Calcineurin A: IMPLICATIONS FOR TRANSPORTER ACTIVITY AND INTRACELLULAR pH CONTROL DURING RAT ARTERY CONTRACTIONS*

Activation of Na⁺,HCO₃⁻ cotransport in vascular smooth muscle cells (VSMCs) contributes to intracellular pH (pH_i) control during artery contraction, but the signaling pathways involved have been unknown. We investigated whether physical and functional interactions between the Na⁺,HCO₃⁻ cotransporter NBCn1 (slc4a7) and the Ca²⁺/calmodulin-activated serine/threonine phosphatase calcineurin exist and play a role for pH_i control in VSMCs. Using a yeast two-hybrid screen, we found that splice cassette II from the N terminus of NBCn1 interacts with calcineurin Aβ. When cassette II was truncated or mutated to disrupt the putative calcineurin binding motif PTVVIH, the interaction was abolished. Native NBCn1 and calcineurin Aβ co-immunoprecipitated from A7r5 rat VSMCs. A peptide (acetyl-DDIPTVVIH-amide), which mimics the putative calcineurin binding motif, inhibited the co-immunoprecipitation whereas a mutated peptide (acetyl-DDIATAVAA-amide) did not. Na⁺,HCO₃⁻ cotransport activity was investigated in VSMCs of mesenteric arteries after an NH₄⁺ prepulse. During depolarization with 50 mm extracellular K⁺ to raise intracellular [Ca²⁺], Na⁺,HCO₃⁻ cotransport activity was inhibited 20–30% by calcineurin inhibitors (FK506 and cyclosporine A). FK506 did not affect Na⁺,HCO₃⁻ cotransport activity in VSMCs when cytosolic [Ca²⁺] was lowered by buffering, nor did it disrupt binding between NBCn1 and calcineurin Aβ. FK506 augmented the intracellular acidification of VSMCs during norepinephrine-induced artery contractions. No physical or functional interactions between calcineurin Aβ and the Na⁺/H⁺ exchanger NHE1 were observed in VSMCs. In conclusion, we demonstrate a physical interaction between calcineurin Aβ and cassette II of NBCn1. Intracellular Ca²⁺ activates Na⁺,HCO₃⁻ cotransport activity in VSMCs in a calcineurin-dependent manner which is important for protection against intracellular acidification.     

Regulation of intracellular pH by electrogenic Na+/HCO3– co-transporters in embryonic neural stem cell-derived radial glia-like cells

A stroke causes a hypoxic brain microenvironment that alters neural cell metabolism resulting in cell membrane hyperpolarization and intracellular acidosis. We studied how intracellular pH (pH_i) is regulated in differentiated mouse neural progenitor cells during hyperpolarizing conditions, induced by prompt reduction of the extracellular K⁺ concentration. We found that the radial glia-like population in differentiating embryonic neural progenitor cells, but not neuronal cells, was rapidly acidified under these conditions. However, when extracellular calcium was removed, an instant depolarization and recovery of the pH_i, back to normal levels, took place. The rapid recovery phase seen in the absence of calcium, was dependent on extracellular bicarbonate and could be inhibited by S0859, a potent Na/HCO3 cotransporter inhibitor. Immunostaining and PCR data, showed that NBCe1 (SLC4A4) and NBCn1 (SLC4A7) were expressed in the cell population and that the pH_i recovery in the radial glial-like cells after calcium removal was mediated mainly by the electrogenic sodium bicarbonate transporter NBCe1 (SLC4A4). Our results indicate that extracellular calcium might hamper pH_i regulation and Na/HCO3 cotransporter activity in a brain injury microenvironment. Our findings show that the NBC-type transporters are the main pH_i regulating systems prevailing in glia-like progenitor cells and that these calcium sensitive transporters are important for neuronal progenitor cell proliferation, survival and neural stem cell differentiation.     

Bicarbonate-Independent Sodium Conductance of Na/HCO3 Cotransporter NBCn1 Decreases NMDA Receptor Function

The sodium bicarbonate cotransporter NBCn1 is an electroneutral transporter with a channel activity that conducts Na⁺ in a HCO₃^–-independent manner. This channel activity was suggested to functionally affect other membrane proteins which permeate Na⁺ influx. We previously reported that NBCn1 is associated with the NMDA receptors (NMDARs) at the molecular and physiological levels. In this study, we examined whether NBCn1 channel activity affects NMDAR currents and whether this effect involves the interaction between the two proteins. NBCn1 and the NMDAR subunits GluN1A/GluN2A were expressed in Xenopus oocytes, and glutamate currents produced by the receptors were measured using two-electrode voltage clamp. In the absence of CO₂/HCO₃^–, NBCn1 channel activity decreased glutamate currents mediated by GluN1A/GluN2A. NBCn1 also decreased the slope of the current–voltage relationships for the glutamate current. Similar effects on the glutamate current were observed with and without PSD95, which can cluster NBCn1 and NMDARs. The channel activity was also observed in the presence of CO₂/HCO₃^–. We conclude that NBCn1 channel activity decreases NMDAR function. Given that NBCn1 knockout mice develop a downregulation of NMDARs, our results are unexpected and suggest that NBCn1 has dual effects on NMDARs. It stabilizes NMDAR expression but decreases receptor function by its Na⁺ channel activity. The dual effects may play an important role in fine-tuning the regulation of NMDARs in the brain.     

Molecular expression of SLC4-derived Na+-dependent anion transporters in selected human tissues

NaHCO(3) transporters are involved in maintenance of intracellular pH and transepithelial HCO(3)(-) movement in many rodent tissues. To establish the human relevance of the many investigations on rodents, this study aimed to map these transporters and a related polypeptide, NaBC1 [solute carrier 4 (SLC4)A11], to several human tissues by using PCR on reverse transcribed human mRNA and immunoperoxidase histochemistry. The mRNA encoding the electroneutral Na(+):HCO(3)(-) cotransporter (NBCe1; SLC4A4), was expressed in renal cortex, renal medulla, stomach, duodenum, jejunum, ileum, colon, pancreas, choroid plexus, cerebellum, cerebrum, and hippocampus. NBCe2 (SLC4A5) and NBCn1 (SLC4A7) mRNAs were mainly found in kidney and brain tissues, as was mRNA encoding the Na(+)-dependent anion exchangers NCBE (SLC4A10) and NDCBE1 (SLC4A8). In addition to previous findings, NBCn1 protein was localized to human renal medullary thick ascending limbs and duodenal epithelial villus cells and NBCe2 protein to renal collecting ducts. Finally, the message encoding NaBC1 was found in kidney, stomach, duodenum, pancreas, and brain, and the corresponding protein in the anterior and posterior corneal epithelia, renal corpuscules, proximal tubules, collecting ducts, pancreatic ducts, and the choroid plexus epithelium. In conclusion, the selected human tissues display distinct expression patterns of HCO(3)(-) transporters, which closely resemble that of rodent tissues.     

Effect of Simultaneously Replacing Putative TM6 and TM12 of Human NBCe1-A with Those from NBCn1 on Surface Abundance in <Emphasis Type="Italic">Xenopus</Emphasis> Oocytes

HCO₃ ⁻ translocation across the plasma membrane via the electrogenic Na/HCO₃ ⁻ cotransporter NBCe1 plays an important role in intracellular pH regulation and transepithelial HCO₃ ⁻ transport. However, the structural determinants of transporter function remain largely unknown. A previous study showed that the putative fourth extracellular loop (EL4) plays an essential role in determining the electrogenicity of NBCe1. In the present study, we generated eight new chimeras of human NBCe1-A and NBCn1-A. All possess the putative NBCe1 EL4 and are electrogenic. Chimera O, in which the putative sixth transmembrane segment (TM6) and EL5 through the C terminus (Ct) of NBCe1 was replaced by corresponding NBCn1 sequence, produces the smallest hyperpolarization (1–2 mV) when CO₂/HCO₃ ⁻ is added to the extracellular solution. Biotinylation experiments show that O has a very low abundance at the plasma membrane. However, chimeras in which we simultaneously replaced the putative TM6 and smaller subdomains of the EL5-Ct region for the NBCn1 sequence were strongly electrogenic except for chimera T, in which we replaced TM6 and TM12 of NBCe1 with the corresponding regions of NBCn1. T exhibited greatly reduced transporter surface expression compared to wild-type NBCe1-A, while retaining at least some electrogenic character. We hypothesize that putative TM6 and TM12 are part of a functional unit and that if the two TMs are replaced by those of the same transporter type, high surface expression would require that the surrounding TMs are also from the same transporter type.     

NBCn1 and NHE1 expression and activity in ΔNErbB2 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 (ΔNErbB2) common in breast cancer impacts on the Na⁺/H⁺-exchanger NHE1 and the Na⁺,HCO₃⁻-cotransporter NBCn1 in MCF-7 human breast cancer cells and address the roles of these transporters in chemotherapy resistance. Upon ΔNErbB2 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 ΔNErbB2 expression. Knockdown and pharmacological inhibition confirmed the involvement of both NHE1 and NBCn1 in acid extrusion. NHE1 inhibition or knockdown sensitized ΔNErbB2-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.     

CFTR-mediated anion secretion in parathyroid hormone-treated Caco-2 cells is associated with PKA and PI3K phosphorylation but not intracellular pH changes or Na+/K+-ATPase abundance

Parathyroid hormone (PTH) has previously been shown to enhance the transepithelial secretion of Cl^? and HCO₃^? across the intestinal epithelia including Caco-2 monolayer, but the underlying cellular mechanisms are not completely understood. Herein, we identified the major signaling pathways that possibly mediated the PTH action to its known target anion channel, i.e., cystic fibrosis transmembrane conductance regulator anion channel (CFTR). Specifically, PTH was able to induce phosphorylation of protein kinase A and phosphoinositide 3-kinase. Since the apical HCO₃^? efflux through CFTR often required the intracellular H⁺/HCO₃^? production and/or the Na⁺-dependent basolateral HCO₃^? uptake, the intracellular pH (pH_i) balance might be disturbed, especially as a consequence of increased endogenous H⁺ and HCO₃^? production. However, measurement of pH_i by a pH-sensitive dye suggested that the PTH-exposed Caco-2 cells were able to maintain normal pH despite robust HCO₃^? transport. In addition, although the plasma membrane Na⁺/K⁺-ATPase (NKA) is normally essential for basolateral HCO₃^? uptake and other transporters (e.g., NHE1), PTH did not induce insertion of new NKA molecules into the basolateral membrane as determined by membrane protein biotinylation technique. Thus, together with our previous data, we concluded that the PTH action on Caco-2 cells is dependent on PKA and PI3K with no detectable change in pH_i or NKA abundance on cell membrane.     

AHCYL2 (long-IRBIT) as a potential regulator of the electrogenic Na-HCO3 cotransporter NBCe1-B

Although AHCYL2 (long-IRBIT) is highly homologous to IRBIT, which regulates ion-transporting proteins including the electrogenic Na⁺-HCO₃⁻ cotransporter NBCe1-B, its functions are poorly understood. Here, we found that AHCYL2 interacts with NBCe1-B in bovine parotid acinar cells using yeast two-hybrid, immunofluorescence confocal microscopy and co-immunoprecipitation analyses. Whole-cell patch-clamp experiments revealed that co-expression of AHCYL2 reduces the apparent affinity for intracellular Mg²⁺ in inhibition of NBCe1-B currents specifically in a HCO₃⁻-deficient cellular condition. Our data unveil AHCYL2 as a potential regulator of NBCe1-B in mammalian cells. We propose that cytosolic ionic condition appropriate for AHCYL2 to function might be different from IRBIT.     

Role of Sodium Bicarbonate Cotransporters in Intracellular pH Regulation and Their Regulatory Mechanisms in Human Submandibular Glands

Sodium bicarbonate cotransporters (NBCs) are involved in the pH regulation of salivary glands. However, the roles and regulatory mechanisms among different NBC isotypes have not been rigorously evaluated. We investigated the roles of two different types of NBCs, electroneutral (NBCn1) and electrogenic NBC (NBCe1), with respect to pH regulation and regulatory mechanisms using human submandibular glands (hSMGs) and HSG cells. Intracellular pH (pH_i) was measured and the pH_i recovery rate from cell acidification induced by an NH₄Cl pulse was recorded. Subcellular localization and protein phosphorylation were determined using immunohistochemistry and co-immunoprecipitation techniques. We determined that NBCn1 is expressed on the basolateral side of acinar cells and the apical side of duct cells, while NBCe1 is exclusively expressed on the apical membrane of duct cells. The pH_i recovery rate in hSMG acinar cells, which only express NBCn1, was not affected by pre-incubation with 5 μM PP2, an Src tyrosine kinase inhibitor. However, in HSG cells, which express both NBCe1 and NBCn1, the pH_i recovery rate was inhibited by PP2. The apparent difference in regulatory mechanisms for NBCn1 and NBCe1 was evaluated by artificial overexpression of NBCn1 or NBCe1 in HSG cells, which revealed that the pH_i recovery rate was only inhibited by PP2 in cells overexpressing NBCe1. Furthermore, only NBCe1 was significantly phosphorylated and translocated by NH₄Cl, which was inhibited by PP2. Our results suggest that both NBCn1 and NBCe1 play a role in pH_i regulation in hSMG acinar cells, and also that Src kinase does not regulate the activity of NBCn1.     

Intracellular pH Regulation in Cultured Astrocytes from Rat Hippocampus : II. Electrogenic Na/HCO3 Cotransport

In the preceding paper (Bevensee, M.O., R.A. Weed, and W.F. Boron. 1997. J. Gen. Physiol. 110: 453–465.), we showed that a Na⁺-driven influx of HCO₃ ⁻ causes the increase in intracellular pH (pH_i) observed when astrocytes cultured from rat hippocampus are exposed to 5% CO₂/17 mM HCO₃ ⁻. In the present study, we used the pH-sensitive fluorescent indicator 2′,7′-biscarboxyethyl-5,6-carboxyfluorescein (BCECF) and the perforated patch-clamp technique to determine whether this transporter is a Na⁺-driven Cl-HCO₃ exchanger, an electrogenic Na/HCO₃ cotransporter, or an electroneutral Na/HCO₃ cotransporter. To determine if the transporter is a Na⁺-driven Cl-HCO₃ exchanger, we depleted the cells of intracellular Cl⁻ by incubating them in a Cl⁻-free solution for an average of ∼11 min. We verified the depletion with the Cl⁻-sensitive dye N-(6-methoxyquinolyl)acetoethyl ester (MQAE). In Cl⁻-depleted cells, the pH_i still increases after one or more exposures to CO₂/HCO₃ ⁻. Furthermore, the pH_i decrease elicited by external Na⁺ removal does not require external Cl⁻. Therefore, the transporter cannot be a Na⁺-driven Cl-HCO₃ exchanger. To determine if the transporter is an electrogenic Na/ HCO₃ cotransporter, we measured pH_i and plasma membrane voltage (V_m) while removing external Na⁺, in the presence/absence of CO₂/HCO₃ ⁻ and in the presence/absence of 400 μM 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid (DIDS). The CO₂/HCO₃ ⁻ solutions contained 20% CO₂ and 68 mM HCO₃ ⁻, pH 7.3, to maximize the HCO₃ ⁻ flux. In pH_i experiments, removing external Na⁺ in the presence of CO₂/HCO₃ ⁻ elicited an equivalent HCO₃ ⁻ efflux of 281 μM s⁻¹. The HCO₃ ⁻ influx elicited by returning external Na⁺ was inhibited 63% by DIDS, so that the predicted DIDS-sensitive V_m change was 3.3 mV. Indeed, we found that removing external Na⁺ elicited a DIDS-sensitive depolarization that was 2.6 mV larger in the presence than in the absence of CO₂/ HCO₃ ⁻. Thus, the Na/HCO₃ cotransporter is electrogenic. Because a cotransporter with a Na⁺:HCO₃ ⁻ stoichiometry of 1:3 or higher would predict a net HCO₃ ⁻ efflux, rather than the required influx, we conclude that rat hippocampal astrocytes have an electrogenic Na/HCO₃ cotransporter with a stoichiometry of 1:2.     

Renal cortical basolateral Na+/HCO 3 − cotransporter III. Evidence for a regulatory protein in the inhibitory effect of protein kinase A

The activity of the Na-H antiporter is inhibited by cyclic AMP-dependent protein kinase A (cAMP.PKA). The inhibitory effect of PKA on the Na-H antiporter is mediated through a regulatory protein that can be dissociated from the antiporter by limited protein digestion. PKA also inhibits the activity of the Na⁺/ HCO ₃ ^? cotransporter. We investigated whether the activity of Na⁺/HCO ₃ ^? cotransporter and the effect of PKA on this transporter may also be regulated by limited protein digestion. In rabbit renal cortical basolateral membranes (BLM) and in solubilized BLM reconstituted in liposomes (proteoliposomes), trypsin (100 μg) increased ²²Na uptake in the presence of HCO₃ but not in the presence of gluconate, indicating that trypsin does not alter diffusive ²²Na uptake but directly stimulates the Na⁺/HCO ₃ ^? cotransporter activity. In proteoliposomes phosphorylated with ATP, the catalytic subunit (CSU) of cAMP-PKA decreased the activity of the Na⁺/HCO ₃ ^? cotransporter (expressed as nanomoles/mg protein/3s) from 23 ± 10 to 14 ± 6 (P < 0.01). In the presence of trypsin, the inhibitory effect of CSU of cAMP-PKA on the activity of Na⁺/HCO ₃ ^? cotransporter was blunted. To identify a fraction that was responsible for the inhibitory effect of the CSU on the Na⁺/HCO ₃ ^? cotransporter activity, solubilized proteins were separated by size exclusion chromatography. The effect of CSU of cAMP-PKA on the Na⁺/HCO ₃ ^? cotransporter activity was assayed in proteoliposomes digested with trypsin with the addition of a fraction containing the 42 kDa protein (fraction S+) or without the 42 kDa protein (fraction S?). With the addition of fraction S?, the CSU of cAMP-PKA failed to inhibit the Na⁺/HCO ₃ ^? cotransporter activity (control 27 ± 6, CSU 27 ± 3) while the addition of fraction S+ restored the inhibitory effect of CSU (27 ± 6 to 3 ± 0.3 P < 0.01). The CSU of cAMP-PKA phosphorylated several proteins in solubilized protein including a 42 kDa protein. Fluorescein isothiocyanate (FITC) labels components of the Na⁺/HCO ₃ ^? cotransporter including the 56 kDa and 42 kDa proteins. In trypsin-treated solubilized protein the 42 kDa protein was not identified with FITC labeling. The results demonstrate that the activity of the Na⁺/HCO ₃ ^? cotransporter is regulated by protein(s) which mediates the inhibitory effect of PKA. Limited protein digestion can dissociate this protein from the cotransporter.     

Distribution of sodium transporters and aquaporin-1 in the human choroid plexus

The choroid plexus epithelium secretes electrolytes and fluid in the brain ventricular lumen at high rates. Several channels and ion carriers have been identified as likely mediators of this transport in rodent choroid plexus. This study aimed to map several of these proteins to the human choroid plexus. Immunoperoxidase-histochemistry was employed to determine the cellular and subcellular localization of the proteins. The water channel, aquaporin (AQP) 1, was predominantly situated in the apical plasma membrane domain, although distinct basolateral and endothelial immunoreactivity was also observed. The Na⁺-K⁺-ATPase ₁-subunit was exclusively localized apically in the human choroid plexus epithelial cells. Immunoreactivity for the Na⁺-K⁺-2Cl^– cotransporter, NKCC1, was likewise confined to the apical plasma membrane domain of the epithelium. The Cl^–/HCO₃^– exchanger, AE2, was localized basolaterally, as was the Na⁺-dependent Cl^–/HCO₃^– exchanger, NCBE, and the electroneutral Na⁺-HCO₃^– cotransporter, NBCn1. No immunoreactivity was found toward the Na⁺-dependent acid/base transporters NHE1 or NBCe2. Hence, the human choroid plexus epithelium displays an almost identical distribution pattern of water channels and Na⁺ transporters as the rat and mouse choroid plexus. This general cross species pattern suggests central roles for these transporters in choroid plexus functions such as cerebrospinal fluid production. immunohistochemistry; metabolism; cerebrospinal fluid secretion