Electrogenic Na/HCO3 cotransporter (NBCe1) variants expressed in Xenopus oocytes: functional comparison and roles of the amino and carboxy termini

Using pH- and voltage-sensitive microelectrodes, as well as the two-electrode voltage-clamp and macropatch techniques, we compared the functional properties of the three NBCe1 variants (NBCe1-A, -B, and -C) with different amino and/or carboxy termini expressed in Xenopus laevis oocytes. Oocytes expressing rat brain NBCe1-B and exposed to a CO(2)/HCO(3)(-) solution displayed all the hallmarks of an electrogenic Na(+)/HCO(3)(-) cotransporter: (a) a DIDS-sensitive pH(i) recovery following the initial CO(2)-induced acidification, (b) an instantaneous hyperpolarization, and (c) an instantaneous Na(+)-dependent outward current under voltage-clamp conditions (-60 mV). All three variants had similar external HCO(3)(-) dependencies (apparent K(M) of 4-6 mM) and external Na(+) dependencies (apparent K(M) of 21-36 mM), as well as similar voltage dependencies. However, voltage-clamped oocytes (-60 mV) expressing NBCe1-A exhibited peak HCO(3)(-)-stimulated NBC currents that were 4.3-fold larger than the currents seen in oocytes expressing the most dissimilar C variant. Larger NBCe1-A currents were also observed in current-voltage relationships. Plasma membrane expression levels as assessed by single oocyte chemiluminescence with hemagglutinin-tagged NBCs were similar for the three variants. In whole-cell experiments (V(m) = -60 mV), removing the unique amino terminus of NBCe1-A reduced the mean HCO(3)(-)-induced NBC current 55%, whereas removing the different amino terminus of NBCe1-C increased the mean NBC current 2.7-fold. A similar pattern was observed in macropatch experiments. Thus, the unique amino terminus of NBCe1-A stimulates transporter activity, whereas the different amino terminus of the B and C variants inhibits activity. One or more cytosolic factors may also contribute to NBCe1 activity based on discrepancies between macropatch and whole-cell currents. While the amino termini influence transporter function, the carboxy termini influence plasma membrane expression. Removing the entire cytosolic carboxy terminus of NBCe1-C, or the different carboxy terminus of the A/B variants, causes a loss of NBC activity due to low expression at the plasma membrane.     

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.     

Inhibition of the Na/Bicarbonate Cotransporter NBCe1-A by diBAC Oxonol Dyes Relative to Niflumic Acid and a Stilbene

Na/HCO(3) cotransporters (NBCs) are important regulators of intracellular pH (pH(i) in a variety of organ systems where acid-base status is critical for tissue function. To characterize the pharmacology of NBCs in more detail, we used the two-electrode voltage-clamp technique to examine the effect of previously identified inhibitors of anion exchanger 1 (AE1) on the activity of rat NBCe1-A expressed in Xenopus laevis oocytes. NBC-expressing oocytes voltage-clamped at -60 mV and exposed to a 5% CO(2)/33 mM HCO(3)(-) solution displayed NBC-mediated outward currents that were inhibited by either niflumic acid or one of the two bis-oxonol dyes diBA(3)C4 and diBA(5)C4. NBCe1-A was less sensitive to niflumic acid (apparent K(i) of 100 microM) than 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS, apparent K(i) of 36 microM) but more sensitive to the diBAC dyes (apparent K(i) of approximately 10 microM). Based on current-voltage relationships, the diBAC dyes inhibited HCO(3)(-) -induced NBCe1-mediated inward currents more so than outward currents. NBCe1 sensitivity to the dyes was (1) lower in the presence of 40 microM DIDS, (2) unaffected by changes in external HCO(3)(-) concentration and (3) only modestly higher at an external Na(+) concentration of 5, but not 15 or 33, mM. Therefore, the diBAC dyes compete with DIDS but not appreciably with Na(+) or HCO(3)(-) for binding. The mechanism of diBAC inhibition of NBCe1 appears similar to that previously reported for AE1.     

Binding of carbonic anhydrase IX to extracellular loop 4 of the NBCe1 Na+/HCO3- cotransporter enhances NBCe1-mediated HCO3- influx in the rat heart

Na(+)/HCO(3)(-) cotransporter (NBC)e1 catalyze the electrogenic movement of 1 Na(+):2 HCO(3)(-) into cardiomyocytes cytosol. NBC proteins associate with carbonic anhydrases (CA), CAII, and CAIV, forming a HCO(3)(-) transport metabolon. Herein, we examined the physical/functional interaction of NBCe1 and transmembrane CAIX in cardiac muscle. NBCe1 and CAIX physical association was examined by coimmunoprecipitation, using rat ventricular lysates. NBCe1 coimmunoprecipitated with anti-CAIX antibody, indicating NBCe1 and CAIX interaction in the myocardium. Glutathione-S-transferase (GST) pull-down assays with predicted extracellular loops (EC) of NBCe1 revealed that NBCe1-EC4 mediated interaction with CAIX. Functional NBCe1/CAIX interaction was examined using fluorescence measurements of BCECF in rat cardiomyocytes to monitor cytosolic pH. NBCe1 transport activity was evaluated after membrane depolarization with high extracellular K(+) in the presence or absence of the CA inhibitors, benzolamide (BZ; 100 μM) or 6-ethoxyzolamide (ETZ; 100 μM) (*P < 0.05). This depolarization protocol produced an intracellular pH (pH(i)) increase of 0.17 ± 0.01 (n = 11), which was inhibited by BZ (0.11 ± 0.02; n = 7) or ETZ (0.06 ± 0.01; n = 6). NBCe1 activity was also measured by changes of pH(i) in NBCe1-transfected human embryonic kidney 293 cells subjected to acid loads. Cotransfection of CAIX with NBCe1 increased the rate of pH(i) recovery (in mM/min) by about fourfold (12.1 ± 0.8; n = 9) compared with cells expressing NBCe1 alone (3.1 ± 0.5; n = 7), which was inhibited by BZ (7.5 ± 0.3; n = 9). We demonstrated that CAIX forms a complex with EC4 of NBCe1, which activates NBCe1-mediated HCO(3)(-) influx in the myocardium. CAIX and NBCe1 have been linked to tumorigenesis and cardiac cell growth, respectively. Thus inhibition of CA activity might be useful to prevent activation of NBCe1 under these pathological conditions.     

Euryhaline pufferfish NBCe1 differs from nonmarine species NBCe1 physiology

Marine fish drink seawater and eliminate excess salt by active salt transport across gill and gut epithelia. Euryhaline pufferfish (Takifugu obscurus, mefugu) forms a CaCO(3) precipitate on the luminal gut surface after transitioning to seawater. NBCe1 (Slc4a4) at the basolateral membrane of intestinal epithelial cell plays a major role in transepithelial intestinal HCO(3)(-) secretion and is critical for mefugu acclimation to seawater. We assayed fugu-NBCe1 (fNBCe1) activity in the Xenopus oocyte expression system. Similar to NBCe1 found in other species, fNBCe1 is an electrogenic Na(+)/HCO(3)(-) cotransporter and sensitive to the stilbene inhibitor DIDS. However, our experiments revealed several unique and distinguishable fNBCe1 transport characteristics not found in mammalian or other teleost NBCe1-orthologs: electrogenic Li(+)/nHCO(3)(-) cotransport; HCO(3)(-) independent, DIDS-insensitive transport; and increased basal intracellular Na(+) accumulation. fNBCe1 is a voltage-dependent Na(+)/nHCO(3)(-) cotransporter that rectifies, independently from the extracellular Na(+) or HCO(3)(-) concentration, around -60 mV. Na(+) removal (0Na(+) prepulse) is necessary to produce the true HCO(3)(-)-elicited current. HCO(3)(-) addition results in huge outward currents with quick current decay. Kinetic analysis of HCO(3)(-) currents reveals that fNBCe1 has a much higher transport capacity (higher maximum current) and lower affinity (higher K(m)) than human kidney NBCe1 (hkNBCe1) does in the physiological range (membrane potential = -80 mV; [HCO(3)(-)] = 10 mM). In this state, fNBCe1 is in favor of operating as transepithelial HCO(3)(-) secretion, opposite of hkNBCe1, from blood to the luminal side. Thus, fugu-NBCe1 represents the first ortholog-based tool to study amino acid substitutions in NBCe1 and how those change ion and voltage dependence.     

Carbonic anhydrase II increases the activity of the human electrogenic Na+/HCO3- cotransporter

Several acid/base-coupled membrane transporters, such as the electrogenic sodium-bicarbonate cotransporter (NBCe1), have been shown to bind to different carbonic anhydrase isoforms to create a "transport metabolon." We have expressed NBCe1 derived from human kidney in oocytes of Xenopus leavis and determined its transport activity by recording the membrane current in voltage clamp, and the cytosolic H(+) and Na(+) concentrations using ion-selective microelectrodes. When carbonic anhydrase isoform II (CAII) had been injected into oocytes, the membrane current and the rate of cytosolic Na(+) rise, indicative for NBCe1 activity, increased significantly with the amount of injected CAII (2-200 ng). The CAII inhibitor ethoxyzolamide reversed the effects of CAII on the NBCe1 activity. Co-expressing wild-type CAII or NH(2)-terminal mutant CAII together with NBCe1 provided similar results, whereas co-expressing the catalytically inactive CAII mutant V143Y had no effect on NBCe1 activity. Mass spectrometric analysis and the rate of cytosolic H(+) change following addition of CO(2)/HCO(3)(-) confirmed the catalytic activity of injected and expressed CAII in oocytes. Our results show that the transport capacity of NBCe1 is enhanced by the catalytic activity of CAII, in line with the notion that CAII forms a transport metabolon with NBCe1.     

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.     

Ion transport mechanisms linked to bicarbonate secretion in the esophageal submucosal glands

The esophageal submucosal glands (SMG) secrete HCO(3)(-) and mucus into the esophageal lumen, where they contribute to acid clearance and epithelial protection. This study characterized the ion transport mechanisms linked to HCO(3)(-) secretion in SMG. We localized ion transporters using immunofluorescence, and we examined their expression by RT-PCR and in situ hybridization. We measured HCO(3)(-) secretion by using pH stat and the isolated perfused esophagus. Using double labeling with Na(+)-K(+)-ATPase as a marker, we localized Na(+)-coupled bicarbonate transporter (NBCe1) and Cl(-)-HCO(3)(-) exchanger (SLC4A2/AE2) to the basolateral membrane of duct cells. Expression of cystic fibrosis transmembrane regulator channel (CFTR) was confirmed by immunofluorescence, RT-PCR, and in situ hybridization. We identified anion exchanger SLC26A6 at the ducts' luminal membrane and Na(+)-K(+)-2Cl(-) (NKCC1) at the basolateral membrane of mucous and duct cells. pH stat experiments showed that elevations in cAMP induced by forskolin or IBMX increased HCO(3)(-) secretion. Genistein, an activator of CFTR, which does not increase intracellular cAMP, also stimulated HCO(3)(-) secretion, whereas glibenclamide, a Cl(-) channel blocker, and bumetanide, a Na(+)-K(+)-2Cl(-) blocker, decreased it. CFTR(inh)-172, a specific CFTR channel blocker, inhibited basal HCO(3)(-) secretion as well as stimulation of HCO(3)(-) secretion by IBMX. This is the first report on the presence of CFTR channels in the esophagus. The role of CFTR in manifestations of esophageal disease in cystic fibrosis patients remains to be determined.     

Entry to "formula tunnel" revealed by SLC4A4 human mutation and structural model

Glaucoma, cataracts, and proximal renal tubular acidosis are diseases caused by point mutations in the human electrogenic Na(+) bicarbonate cotransporter (NBCe1/SLC4A4) (1, 2). One such mutation, R298S, is located in the cytoplasmic N-terminal domain of NBCe1 and has only moderate (75%) function. As SLC transporters have high similarity in their membrane and N-terminal primary sequences, we homology-modeled NBCe1 onto the crystal structure coordinates of Band 3(AE1) (3). Arg-298 is predicted to be located in a solvent-inaccessible subsurface pocket and to associate with Glu-91 or Glu-295 via H-bonding and charge-charge interactions. We perturbed these putative interactions between Glu-91 and Arg-298 by site-directed mutagenesis and used expression in Xenopus oocyte to test our structural model. Mutagenesis of either residue resulted in reduced transport function. Function was "repaired" by charge reversal (E91R/R298E), implying that these two residues are interchangeable and interdependent. These results contrast the current understanding of the AE1 N terminus as protein-binding sites and propose that hkNBCe1 (and other SLC4) cytoplasmic N termini play roles in controlling HCO(3)(-) permeation.     

Cloning of a Na+-driven Cl/HCO3 exchanger from squid giant fiber lobe

We extracted RNA from the giant fiber lobe (GFL) of the squid Loligo pealei and performed PCR with degenerate primers that were based on highly conserved regions of Na+-coupled HCO3- transporters. This approach yielded a novel, 290-bp sequence related to the bicarbonate transporter superfamily. Using an L. opalescens library, we extended the initial fragment in the 3' and 5' directions by a combination of library screening and PCR and obtained the full-length clone (1,198 amino acids) by PCR from L. pealei GFL. The amino acid sequence is 46% identical to mammalian electrogenic and electroneutral Na-HCO3 cotransporters and 33% identical to the anion exchanger AE1. Northern blot analysis showed strong signals in L. pealei GFL, optic lobe, and heart and weaker signals in gill and stellate ganglion. To assess function, we injected in vitro-transcribed cRNA into Xenopus oocytes and subsequently used microelectrodes to monitor intracellular pH (pHi) and membrane voltage (Vm). Superfusing these oocytes with 5% CO2-33 mM HCO3- caused a CO2-induced fall in pHi, followed by a slow recovery. The absence of a rapid HCO3- -induced hyperpolarization indicates that the pHi recovery mechanism is electroneutral. Ion substitutions showed that Na+ and Cl- are required on opposite sides of the membrane. Transport was blocked by 50 microM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). The characteristics of our novel clone fit those of a Na+-driven Cl/HCO3 exchanger (NDCBE).     

Enhanced activity of ventricular Na+-HCO3- cotransport in pressure overload hypertrophy

The Na(+)-HCO(3)(-) cotransporter (NBC) plays a key role in intracellular pH (pH(i)) regulation in normal ventricular muscle. However, the state of NBC in nonischemic hypertrophied hearts is unresolved. In this study, we examined functional and molecular properties of NBC in adult rat ventricular myocytes. The cells were enzymatically isolated from both normal and hypertrophied hearts. Ventricular hypertrophy was induced by pressure overload created by suprarenal abdominal aortic constriction of 50% for 7 wk. pH(i) was measured in single cells using the fluorescent pH indicator 2',7'-bis(2-carboxyethyl)5-(6)carboxyfluorescein. Real-time PCR analysis was used to quantitatively assess expression of NBC-encoding mRNA, including SLC4A4 (encoding electrogenic NBC, NBCe1) and SLC4A7 (electroneutral NBC, NBCn1). Our results demonstrate that: 1) mRNA levels of both the electrogenic NBCe1 (SLC4A4) and electroneutral NBCn1 (SLC4A7) forms of NBC were increased by aortic constriction, 2) the onset of NBC upregulation occurred within 3 days after constriction, 3) normal and hypertrophied ventricles displayed regional differences in NBC expression, 4) acid extrusion via NBC (J(NBC)) was increased significantly in hypertrophied myocytes, 5) although acid extrusion via Na(+)/H(+) exchange was also increased in hypertrophied myocytes, the relative enhancement of J(NBC) was larger, 6) membrane depolarization markedly increased J(NBC) in hypertrophied myocytes, and 7) losartan, an ANG II AT(1) receptor antagonist, significantly attenuated the upregulation of both NBCs induced by 3 wk of aortic constriction. Enhanced NBC activity during hypertrophic development provides a mechanism for intracellular Na(+) overload, which may render the ventricles more vulnerable to Ca(2+) overload during ischemia-reperfusion.     

A novel missense mutation in the sodium bicarbonate cotransporter (NBCe1/SLC4A4) causes proximal tubular acidosis and glaucoma through ion transport defects

In humans and terrestrial vertebrates, the kidney controls systemic pH in part by absorbing filtered bicarbonate in the proximal tubule via an electrogenic Na+/HCO3- cotransporter (NBCe1/SLC4A4). Recently, human genetics revealed that NBCe1 is the major renal contributor to this process. Homozygous point mutations in NBCe1 cause proximal renal tubular acidosis (pRTA), glaucoma, and cataracts (Igarashi, T., Inatomi, J., Sekine, T., Cha, S. H., Kanai, Y., Kunimi, M., Tsukamoto, K., Satoh, H., Shimadzu, M., Tozawa, F., Mori, T., Shiobara, M., Seki, G., and Endou, H. (1999) Nat. Genet. 23, 264-266). We have identified and functionally characterized a novel, homozygous, missense mutation (S427L) in NBCe1, also resulting in pRTA and similar eye defects without mental retardation. To understand the pathophysiology of the syndrome, we expressed wild-type (WT) NBCe1 and S427L-NBCe1 in Xenopus oocytes. Function was evaluated by measuring intracellular pH (HCO3- transport) and membrane currents using microelectrodes. HCO3- -elicited currents for S427L were approximately 10% of WT NBCe1, and CO2-induced acidification was approximately 4-fold faster. Na+ -dependent HCO3- transport (currents and acidification) was also approximately 10% of WT. Current-voltage (I-V) analysis reveals that S427L has no reversal potential in HCO3-, indicating that under physiological ion gradient conditions, NaHCO3 could not move out of cells as is needed for renal HCO3- absorption and ocular pressure homeostasis. I-V analysis without Na+ further shows that the S427L-mediated NaHCO3 efflux mode is depressed or absent. These experiments reveal that voltage- and Na+ -dependent transport by S427L-hkNBCe1 is unfavorably altered, thereby causing both insufficient HCO3- absorption by the kidney (proximal RTA) and inappropriate anterior chamber fluid transport (glaucoma).     

Functional characterization of human NBC4 as an electrogenic Na+-HCO cotransporter (NBCe2)

We have functionally characterized Na+-driven bicarbonate transporter (NBC)4, originally cloned from human heart by Pushkin et al. (Pushkin A, Abuladze N, Newman D, Lee I, Xu G, and Kurtz I. Biochem Biophys Acta 1493: 215-218, 2000). Of the four NBC4 variants currently present in GenBank, our own cloning efforts yielded only variant c. We expressed NBC4c (GenBank accession no. AF293337) in Xenopus laevis oocytes and assayed membrane potential (Vm) and pH regulatory function with microelectrodes. Exposing an NBC4c-expressing oocyte to a solution containing 5% CO2 and 33 mM HCO elicited a large hyperpolarization, indicating that the transporter is electrogenic. The initial CO2-induced decrease in intracellular pH (pH(i)) was followed by a slow recovery that was reversed by removing external Na+. Two-electrode voltage clamp of NBC4c-expressing oocytes revealed large HCO- and Na+-dependent currents. When we voltage clamped V(m) far from NBC4c's estimated reversal potential (E(rev)), the pH(i) recovery rate increased substantially. Both the currents and pH(i) recovery were blocked by 200 microM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). We estimated the transporter's HCO:Na+ stoichiometry by measuring E(rev) at different extracellular Na+ concentration ([Na+]o) values. A plot of E(rev) against log[Na+]o was linear, with a slope of 54.8 mV/log[Na+]o. This observation, as well as the absolute E(rev) values, are consistent with a 2:1 stoichiometry. In conclusion, the behavior of NBC4c, which we propose to call NBCe2-c, is similar to that of NBCe1, the first electrogenic NBC.     

Intracellular pH regulation in colonocytes of rat proximal colon

The regulation of intracellular pH (pH(i)) in colonocytes of the rat proximal colon has been investigated using the pH-sensitive dye BCECF and compared with the regulation of pH(i) in the colonocytes of the distal colon. The proximal colonocytes in a HEPES-buffered solution had pH(i)=7.24+/-0.04 and removal of extracellular Na(+) lowered pH(i) by 0.24 pH units. Acid-loaded colonocytes by an NH(3)/NH(4)(+) prepulse exhibited a spontaneous recovery that was partially Na(+)-dependent and could be inhibited by ethylisopropylamiloride (EIPA). The Na(+)-dependent recovery rate was enhanced by increasing the extracellular Na(+) concentration and was further stimulated by aldosterone. In an Na(+)- and K(+)-free HEPES-buffered solution, the recovery rate from the acid load was significantly stimulated by addition of K(+) and this K(+)-dependent recovery was partially blocked by ouabain. The intrinsic buffer capacity of proximal colonocytes at physiological pH(i) exhibited a nearly 2-fold higher value than in distal colonocytes. Butyrate induced immediate colonocyte acidification that was smaller in proximal than in distal colonocytes. This acidification was followed by a recovery phase that was both EIPA-sensitive and -insensitive and was similar in both groups of colonocytes. In a HCO(3)(-)/CO(2)-containing solution, pH(i) of the proximal colonocytes was 7.20+/-0.04. Removal of external Cl(-) caused alkalinization that was inhibited by DIDS. The recovery from an alkaline load induced by removal of HCO(3)(-)/CO(2) from the medium was Cl(-)-dependent, Na(+)-independent and blocked by DIDS. Recovery from an acid load in EIPA-containing Na(+)-free HCO(3)(-)/CO(2)-containing solution was accelerated by addition of Na(+). Removal of Cl(-) inhibited the effect of Na(+). In summary, the freshly isolated proximal colonocytes of rats express Na(+)/H(+) exchanger, H(+)/K(+) exchanger ((H(+)-K(+))-ATPase) and Na(+)-dependent Cl(-)/HCO(3)(-) exchanger that contribute to acid extrusion and Na(+)-independent Cl(-)/HCO(3)(-) exchanger contributing to alkali extrusion. All of these are likely involved in the regulation of pH(i) in vivo. Proximal colonocytes are able to maintain a more stable pH(i) than distal cells, which seems to be facilitated by their higher intrinsic buffer capacity.     

Molecular and functional evidence for electrogenic and electroneutral Na(+)-HCO(3)(-) cotransporters in murine duodenum

Inward Na(+)-HCO(3)(-) cotransport has previously been demonstrated in acidified duodenal epithelial cells, but the identity and localization of the mRNAs and proteins involved have not been determined. The molecular expression and localization of Na(+)-HCO(3)(-) cotransporters (NBCs) were studied by RT-PCR, sequence analysis, and immunohistochemistry. By fluorescence spectroscopy, the intracellular pH (pH(i)) was recorded in suspensions of isolated murine duodenal epithelial cells loaded with 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. Proximal duodenal epithelial cells expressed mRNA encoding two electrogenic NBC1 isoforms and the electroneutral NBCn1. Both NBC1 and NBCn1 were localized to the basolateral membrane of proximal duodenal villus cells, whereas the crypt cells did not label with the anti-NBC antibodies. DIDS or removal of extracellular Cl(-) increased pH(i), whereas an acidification was observed on removal of Na(+) or both Na(+) and Cl(-). The effects of inhibitors and ionic dependence of acid/base transporters were consistent with both inward and outward Na(+)-HCO(3)(-) cotransport. Hence, we propose that NBCs are involved in both basolateral electroneutral HCO(3)(-) transport as well as basolateral electrogenic HCO(3)(-) transport in proximal duodenal villus cells.     

Muscarinic activation of Na+-dependent ion transporters and modulation by bicarbonate in rat submandibular gland acinus

To investigate the interaction between the ion channels and transporters in the salivary fluid secretion, we measured the membrane voltage (V(m)) and intracellular concentrations of Ca(2+), Na(+) ([Na(+)](c)), Cl(-), and H(+) (pH(i)) in rat submandibular gland acini (RSMGA). After a transient depolarization induced by a short application of acetylcholine (ACh; 5 muM, 20 s), RSMGA showed strong delayed hyperpolarization (V(h,ACh); -95 +/- 1.8 mV) that was abolished by ouabain. In the HCO(3)(-)-free condition, the V(h,ACh) was also blocked by bumetanide, a blocker of Na(+)-K(+)-2Cl(-) cotransporter (NKCC). In the presence of HCO(3)(-) (24 meq, bubbled with 5% CO(2)), however, the V(h,ACh) was not blocked by bumetanide, but it was suppressed by ethylisopropylamiloride (EIPA), a Na(+)/H(+) exchanger (NHE) inhibitor. Similarly, the ACh-induced increase in [Na(+)](c) was totally blocked by bumetanide in the absence of HCO(3)(-), but only by one-half in the presence of HCO(3)(-). ACh induced a prominent acidification of pH(i) in the presence of HCO(3)(-), and the acidification was further increased by EIPA treatment. Without HCO(3)(-), an application of ACh strongly accelerated the NKCC activity that was measured from the decay of pH(i) during the application of NH(4)(+) (20 mM). Notably, the ACh-induced activation of NKCC was largely suppressed in the presence of HCO(3)(-). In summary, the ACh-induced anion secretion in RSMGA is followed by the activation of NKCC and NHE, resulting an increase in [Na(+)](c). The intracellular Na(+)-induced activation of electrogenic Na(+)/K(+)-ATPase causes V(h,ACh). The regulation of NKCC and NHE by ACh is strongly affected by the physiological level of HCO(3)(-).     

The electrogenic Na+-HCO3- cotransporter NBCe1-B is regulated by intracellular Mg2+

NBCe1-B, a major splice variant of the electrogenic Na+--HCO3- cotransporter (NBCe1) fulfills basic cellular functions including regulation of intracellular pH and epithelial HCO3- secretion. However, its cellular regulatory mechanism still remains elusive. Here, we provide evidence for the first time that NBCe1-B activity can be controlled by intracellular Mg2+ (Mg2+(i)), the physiologically most abundant intracellular divalent cation. Using the whole-cell patch-clamp technique, we found that recombinant NBCe1-B currents expressed in HEK293 and NIH3T3 cells were inhibited voltage-independently by Mg2+(i) in a concentration-dependent manner (K(i) approximately 0.01 mM). The Mg2+(i) inhibition was partially relieved by truncation of the NBCe1-B specific N-terminal region (K(i) approximately 0.3 mM), and was also observed for native electrogenic Na+--HCO3- cotransporter current in bovine parotid acinar cells that endogenously express NBCe1-B (K(i) approximately 1 mM). These results suggest that Mg2+ may be a cytosolic factor that limits intrinsic cotransport activity of NBCe1-B in mammalian cells.     

Granulosa cells regulate intracellular pH of the murine growing oocyte via gap junctions: development of independent homeostasis during oocyte growth

Oocytes grow within ovarian follicles in which the oocyte is coupled to the surrounding granulosa cells by gap junctions. It was previously found that small growing oocytes isolated from juvenile mice and freed of their surrounding granulosa cells (denuded) lacked the ability to regulate their intracellular pH (pH(i)), did not exhibit the pH(i)-regulatory HCO(3)(-)/Cl(-) and Na(+)/H(+) exchange activities found in fully-grown oocytes, and had low pH(i). However, both exchangers became active as oocytes grew near to full size, and, simultaneously, oocyte pH(i) increased by approximately 0.25 pH units. Here, we show that, in the more physiological setting of the intact follicle, oocyte pH(i) is instead maintained at approximately 7.2 throughout oocyte development, and the growing oocyte exhibits HCO(3)(-)/Cl(-) exchange, which it lacks when denuded. This activity in the oocyte requires functional gap junctions, as gap junction inhibitors eliminated HCO(3)(-)/Cl(-) exchange activity from follicle-enclosed growing oocytes and substantially impeded the recovery of the oocyte from an induced alkalosis, implying that oocyte pH(i) may be regulated by pH-regulatory exchangers in granulosa cells via gap junctions. This would require robust HCO(3)(-)/Cl(-) exchange activity in the granulosa cells, which was confirmed using oocytectomized (OOX) cumulus-oocyte complexes. Moreover, in cumulus-oocyte complexes with granulosa cells coupled to fully-grown oocytes, HCO(3)(-)/Cl(-) exchange activity was identical in both compartments and faster than in denuded oocytes. Taken together, these results indicate that growing oocyte pH(i) is controlled by pH-regulatory mechanisms residing in the granulosa cells until the oocyte reaches a developmental stage where it becomes capable of carrying out its own homeostasis.     

Mechanisms contributing to intracellular pH homeostasis in an immortalised human chondrocyte cell line

The maintenance of chondrocyte pH is an important parameter controlling cartilage matrix turnover rates. Previous studies have shown that, to varying degrees, chondrocytes rely on Na(+)/H(+) exchange to regulate pH. HCO(3)(-)-dependent buffering and HCO(3)(-)-dependent acid-extrusion systems seem to play relatively minor roles. This situation may reflect minimal carbonic anhydrase activity in cartilage cells. In the present study, the pH regulation of the human chondrocyte cell line, C-20/A4 has been characterised. Intracellular pH (pH(i)) was measured using the H(+)-sensitive fluoroprobe BCECF. In solutions lacking HCO(3)(-)/CO(2), pH(i) was approximately 7.5, and the recovery from intracellular acidification was predominantly mediated by a Na(+)-dependent, amiloride- and HOE 694-sensitive process. A small additional component which was sensitive to chloro-7-nitrobenz-2-oxa-1,3-diazole, an inhibitor of the V-type H(+)-ATPase, was also apparent. In solutions containing HCO(3)(-)/CO(2), pH(i) was approximately 7.2. Comparison of buffering capacity in the two conditions showed that this variable was not significantly augmented in HCO(3)(-)/CO(2)-containing media. The recovery from intracellular acidification was more rapid in the presence of HCO(3)(-)/CO(2), although under these conditions it was again largely dependent on Na(+) ions and inhibited by amiloride and HOE 694. A small component was inhibited by SITS, although this effect did not reach the level of statistical significance. These findings indicate that HCO(3)(-)-dependent processes play only a minimal role in pH regulation in C-20/A4 chondrocytes. pH regulation instead relies heavily on the Na(+)/H(+) exchanger together with a H(+)-ATPase. The absence of extrinsic (HCO(3)(-)/CO(2)) buffering is likely to reflect the low levels of carbonic anhydrase in these cells. In addition to providing fundamental information about a widely-used cell line, these findings support the contention that the unusual nature of pH regulation in chondrocytes reflects the paucity of carbonic anhydrase activity in these cells.