NBC Isoforms Research Articles

In vivo Overexpression of Electrogenic Sodium/Bicarbonate Cotransporter (NBCe1) by AAV9 Modifies the Cardiac Action Potential and the QT Interval in Mice.

Cardiac cells depend on specific sarcolemmal ion transporters to assure the correct intracellular pH regulation. The sodium/bicarbonate cotransporter (NBC) is one of the major alkalinizing mechanisms. In the heart two different NBC isoforms have been described: the electroneutral NBCn1 (1Na+:1) and the electrogenic NBCe1 (1Na+:2). NBCe1 generates an anionic repolarizing current that modulates the action potential duration (APD). In addition to regulating the pH, the NBC is a source of sodium influx. It has been postulated that NBC could play a role in the development of hypertrophy. The aim of this research was to study the contribution of NBCe1 in heart electrophysiology and in the development of heart hypertrophy in an in vivo mouse model with overexpression of NBCe1. Heart NBCe1 overexpression was achieved by a recombinant cardiotropic adeno-associated virus (AAV9) and was evidenced by western-blot and qPCR. AAV9-mCherry was used as a transduction control. NBCe1 overexpression fails to increase heart growth. Patch clamp and electrocardiogram were performed. We observed a reduction on both, ventricular myocytes APD and electrocardiogram QT interval corrected by cardiac rate, emphasizing for the first time NBCe1 relevance for the electrical activity of the heart.

Sodium bicarbonate transporter NBCe1 regulates proliferation and viability of human prostate cancer cells LNCaP and PC3.

Studies on cultured cancer cells or cell lines have revealed multiple acid extrusion mechanisms and their involvement in cancer cell growth and progression. In the present study, the role of the sodium bicarbonate transporters (NBCs) in prostate cancer cell proliferation and viability was examined. qPCR revealed heterogeneous expression of five NBC isoforms in human prostate cancer cell lines LNCaP, PC3, 22RV1, C4-2, DU145, and the prostate cell line RWPE-1. In fluorescence pH measurement of LNCaP cells, which predominantly express NBCe1, Na+ and HCO3–-mediated acid extrusion was identified by bath ion replacement and sensitivity to the NBC inhibitor S0859. NBCe1 knockdown using siRNA oligonucleotides decreased the number of viable cells, and pharmacological inhibition with S0859 (50 µM) resulted in a similar decrease. NBCe1 knockdown and inhibition also increased cell death, but this effect was small and slow. In PC3 cells, which express all NBC isoforms, NBCe1 knockdown decreased viable cell number and increased cell death. The effects of NBCe1 knockdown were comparable to those by S0859, indicating that NBCe1 among NBCs primarily contributes to PC3 cell proliferation and viability. S0859 inhibition also decreased the formation of cell spheres in 3D cultures. Immunohistochemistry of human prostate cancer tissue microarrays revealed NBCe1 localization to the glandular epithelial cells in prostate tissue and robust expression in acinar and duct adenocarcinoma. In conclusion, our study demonstrates that NBCe1 regulates acid extrusion in prostate cancer cells and inhibiting or abolishing this transporter decreases cancer cell proliferation.

Regulation of Intracellular pH is Altered in Cardiac Myocytes of Ovariectomized Rats.

BackgroundIt is well known that after menopause women are exposed to a greater cardiovascular risk, but the intracellular modifications are not properly described. The sodium/proton exchanger (NHE) and the sodium/bicarbonate cotransporter (NBC) regulate the intracellular pH and, indirectly, the intracellular sodium concentration ([Na+]). There are 2 isoforms of NBC in the heart: the electrogenic (1Na+/2HCO3−; NBCe1) and the electroneutral (1Na+/1HCO3−; NBCn1). Because NHE and NBCn1 hyperactivity as well as the NBCe1 decreased activity have been associated with several cardiovascular pathologies, the aim of this study was to investigate the potential alterations of the alkalinizing transporters during the postmenopausal period.Methods and ResultsThree‐month ovariectomized rats (OVX) were used. The NHE activity and protein expression are significantly increased in OVX. The NBCe1 activity is diminished, and the NBCn1 activity becomes predominant in OVX rats. p‐Akt levels showed a significant diminution in OVX. Finally, NHE activity in platelets from OVX rats is also higher in comparison to sham rats, resulting in a potential biomarker of cardiovascular diseases.ConclusionsOur results demonstrated for the first time that in the cardiac ventricular myocytes of OVX rats NHE and NBC isoforms are altered, probably because of the decreased level of p‐Akt, compromising the ionic intracellular homeostasis.

Expressional and Functional Characterization of Intracellular pH Regulators and Effects of Ethanol in Human Oral Epidermoid Carcinoma Cells

Background/Aims: To functionally characterize intracellular pH (pH_i) regulating mechanisms, such as Na⁺-H⁺ exchanger (NHE) and Na⁺-HCO₃^- co-transporter (NBC), and further examine effects of ethanol on the pH_i regulating mechanism in human oral epidermoid carcinoma (OEC-M1) cells. Methods: OEC-M1 cells were a gift from Tri-Service General Hospital. Changes of pH_i were detected by microspectrofluroimetry with a pH-sensitive fluorescent dye, BCECF. Isoforms of transporters were examined by Western blot technique. Results: i) the steady-state pH_i value shifted from alkaline (7.35∼7.49) to acidic (7.0∼7.03) following acid/base impacts; ii) in HEPES-buffer system, pH_i recovery following induced-acidification was totally blocked by either removing [Na]_o⁺ or adding HOE 694 (a NHE1 specific inhibitor), which demonstrates existence of NHE1; iii) in HCO₃^-/CO₂-buffer system, the pH_i recovery following induced-acidification was entirely blocked by either removing [Na]_o⁺ or adding HOE 694 plus DIDS (a NBC specific inhibitor), which suggests existence of Na⁺- and HCO₃^–dependent acid-extruder, i.e. NBC; iv) the isoforms of the two acid extruders were NHE1, NBCn1, NBCe1 and NDCBE; v) ethanol (10-1000 mM) showed a biphasic and concentration-dependent effect on resting pH_i (i.e. increase then decrease) by changing the activity of NHE1 and NBC accordingly; vi) treatment with ethanol for 24 hr (<unterline>></unterline> 300 mM) significantly inhibited the expression of NHE1, NBCn1 and NDCBE, while up-regulated NBCe1. Conclusions: Ethanol affects pH_i in a concentration-dependent manner by changing function and expression of NHE1 and NBC isoforms in OEC-M1 cells.

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

ABSTRACTThe sodium/bicarbonate cotransporter (NBC) transports extracellular Na+ and HCO3− into the cytoplasm upon intracellular acidosis, restoring the acidic pHi 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.

Physiological and pharmacological characterization of transmembrane acid extruders in cultured human umbilical artery smooth muscle cells

Background: Intracellular pH (pH_i) is a pivotal factor for cellular functions and homeostasis. Apart from passive intracellular buffering capacity, active transmembrane transporters responsible for kinetic changes of pH_i impacts. Acid extrusion transporters such as Na^+/H^+ exchanger (NHE) and Na^+/HCO_3^− cotransporter (NBC) have been found to be activated when cells are in an acidic condition in different cell types. However, such far, the pH_i regulators have not been characterized in human umbilical artery smooth muscle cells (HUASMCs).Materials and Methods: We, therefore, investigated the mechanism of pH_i recovery from intracellular acidosis, induced by NH_4Cl-prepulse, using pH-sensitive fluorescence dye: 2',7'-bis(2-carboxethyl)-5(6)-carboxy-fluorescein in HUASMCs. Cultured HUASMCs were derived from the segments of the human umbilical artery that were obtained from women undergoing children delivery. Results: The resting pH_i is 7.23 ± 0.03 when cells in HEPES (nominally HCO_3^−-free) buffered solution. The resting pH_i is higher as 7.27 ± 0.03 when cells in CO_2/HCO_3^−-buffered solution. In HEPES-buffered solution, a pH_i recovery following induced intracellular acidosis could be inhibited completely by 30 μM HOE 694 (a specific NHE inhibitor) or by removing [Na^+]_o. In 5% CO_2/HCO_3^−-buffered solution, 30 μM HOE 694 slowed the pH_i recovery from the induced intracellular acidosis only. On the contrary, HOE 694 adding together with 0.2 mM 4, 4'-diisothiocyanatostilbene-2, 2'-disulphonic acid (a specific NBC inhibitor) or removal of [Na^+]_o entirely blocked the acid extrusion. By using Western blot technique, we demonstrated that four different isoforms of NBC, that is, SLC4A8 (NBCBE), SLC4A7 (NBCn1), SLC4A5 (NBCe2) and SLC4A4 (NBCe1), co-exist in the HUASMCs. Conclusions: We demonstrate, for the 1^(st) time, that apart from the housekeeping NHE1, another Na^+ couple HCO_3^−-transporter, that is, NBC, functionally coexists to responsible for acid-extruding in HUASMCs.

Intracellular Acid-Extruding Regulators and the Effect of Lipopolysaccharide in Cultured Human Renal Artery Smooth Muscle Cells

Homeostasis of the intracellular pH (pHi) in mammalian cells plays a pivotal role in maintaining cell function. Thus far, the housekeeping Na+-H+ exchanger (NHE) and the Na+-HCO3 − co-transporter (NBC) have been confirmed in many mammalian cells as major acid extruders. However, the role of acid-extruding regulators in human renal artery smooth muscle cells (HRASMCs) remains unclear. It has been demonstrated that lipopolysaccharide (LPS)-induced vascular occlusion is associated with the apoptosis, activating calpain and increased [Ca2+]i that are related to NHE1 activity in endothelia cells. This study determines the acid-extruding mechanisms and the effect of LPS on the resting pHi and active acid extruders in cultured HRASMCs. The mechanism of pHi recovery from intracellular acidosis (induced by NH4Cl-prepulse) is determined using BCECF-fluorescence in cultured HRASMCs. It is seen that (a) the resting pHi is 7.19±0.03 and 7.10±0.02 for HEPES- and CO2/HCO3 −- buffered solution, respectively; (b) apart from the housekeeping NHE1, another Na+-coupled HCO3 − transporter i.e. NBC, functionally co-exists to achieve acid-equivalent extrusion; (c) three different isoforms of NBC: NBCn1 (SLC4A7; electroneutral), NBCe1 (SLC4A4; electrogenic) and NBCe2 (SLC4A5), are detected in protein/mRNA level; and (d) pHi and NHE protein expression/activity are significantly increased by LPS, in both a dose- and time- dependent manner, but NBCs protein expression is not. In conclusion, it is demonstrated, for the first time, that four pHi acid-extruding regulators: NHE1, NBCn1, NBCe1 and NBCe2, co-exist in cultured HRASMCs. LPS also increases cellular growth, pHi and NHE in a dose- and time-dependent manner.

Modulation of the cardiac sodium/bicarbonate cotransporter by the renin angiotensin aldosterone system: pathophysiological consequences

The sodium/bicarbonate cotransporter (NBC) is one of the major alkalinizing mechanisms in the cardiomyocytes. It has been demonstrated the existence of at least two functional isoforms, one that promotes the co-influx of 1 molecule of Na+ per 1 molecule of HCO−3 (electroneutral isoform; NBCn1) and the other one that generates the co-influx of 1 molecule of Na+ per 2 molecules of HCO−3 (electrogenic isoform; NBCe1). Both isoforms are important to maintain intracellular pH (pHi) and sodium concentration ([Na+]i). In addition, NBCe1 generates an anionic repolarizing current that modulates the action potential duration (APD). The renin-angiotensin-aldosterone system (RAAS) is implicated in the modulation of almost all physiological cardiac functions and is also involved in the development and progression of cardiac diseases. It was reported that angiotensin II (Ang II) exhibits an opposite effect on NBC isoforms: it activates NBCn1 and inhibits NBCe1. The activation of NBCn1 leads to an increase in pHi and [Na+]i, which indirectly, due to the stimulation of reverse mode of the Na+/Ca2+ exchanger (NCX), conduces to an increase in the intracellular Ca2+ concentration. On the other hand, the inhibition of NBCe1 generates an APD prolongation, potentially representing a risk of arrhythmias. In the last years, the potentially altered NBC function in pathological scenarios, as cardiac hypertrophy and ischemia-reperfusion, has raised increasing interest among investigators. This review attempts to draw the attention on the relevant regulation of NBC activity by RAAS, since it modulates pHi and [Na+]i, which are involved in the development of cardiac hypertrophy, the damage produced by ischemia-reperfusion and the generation of arrhythmic events, suggesting a potential role of NBC in cardiac diseases.

Identification of Functionally Distinct Na-HCO3 Co-Transporters in Colon

Na-HCO3 cotransport (NBC) regulates intracellular pH (pHi) and HCO3 secretion in rat colon. NBC has been characterized as a 5,5′-diisothiocyanato-2-2′-stilbene (DIDS)-sensitive transporter in several tissues, while the colonic NBC is sensitive to both amiloride and DIDS. In addition, the colonic NBC has been identified as critical for pHi regulation as it is activated by intravesicular acid pH. Molecular studies have identified several characteristically distinct NBC isoforms [i.e. electrogenic (NBCe) and electroneutral (NBCn)] that exhibit tissue specific expression. This study was initiated to establish the molecular identity and specific function of NBC isoforms in rat colon. Northern blot and reverse transcriptase PCR (RT-PCR) analyses revealed that electrogenic NBCe1B or NBCe1C (NBCe1B/C) isoform is predominantly expressed in proximal colon, while electroneutral NBCn1C or NBCn1D (NBCn1C/D) is expressed in both proximal and distal colon. Functional analyses revealed that amiloride-insensitive, electrogenic, pH gradient-dependent NBC activity is present only in basolateral membranes of proximal colon. In contrast, amiloride-sensitive, electroneutral, [H+]-dependent NBC activity is present in both proximal and distal colon. Both electrogenic and electroneutral NBC activities are saturable processes with an apparent Km for Na of 7.3 and 4.3 mM, respectively; and are DIDS-sensitive with apparent Ki of 8.9 and 263.8 µM, respectively. In addition to Na-H exchanger isoform-1 (NHE1), pHi acidification is regulated by a HCO3-dependent mechanism that is HOE694-insensitive in colonic crypt glands. We conclude from these data that electroneutral, amiloride-sensitive NBC is encoded by NBCn1C/D and is present in both proximal and distal colon, while NBCe1B/C encodes electrogenic, amiloride-insensitive Na-HCO3 cotransport in proximal colon. We also conclude that NBCn1C/D regulates HCO3-dependent HOE694-insensitive Na-HCO3 cotransport and plays a critical role in pHi regulation in colonic epithelial cells.

Sarcolemmal localisation of Na+/H+ exchange and Na+–HCO3− co‐transport influences the spatial regulation of intracellular pH in rat ventricular myocytes

Membrane acid extrusion by Na(+)/H(+) exchange (NHE1) and Na(+)-HCO3(-) co-transport (NBC) is essential for maintaining a low cytoplasmic [H(+)] (∼60 nm, equivalent to an intracellular pH (pHi) of 7.2). This protects myocardial function from the high chemical reactivity of H(+) ions, universal end-products of metabolism. We show here that, in rat ventricular myocytes, fluorescent antibodies map the NBC isoforms NBCe1 and NBCn1 to lateral sarcolemma, intercalated discs and transverse tubules (t-tubules), while NHE1 is absent from t-tubules. This unexpected difference matches functional measurements of pHi regulation (using AM-loaded SNARF-1, a pH fluorophore). Thus, myocyte detubulation (by transient exposure to 1.5 m formamide) reduces global acid extrusion on NBC by 40%, without affecting NHE1. Similarly, confocal pHi imaging reveals that NBC stimulation induces spatially uniform pHi recovery from acidosis, whereas NHE1 stimulation induces pHi non-uniformity during recovery (of ∼0.1 units, for 2-3 min), particularly at the ends of the cell where intercalated discs are commonly located, and where NHE1 immunostaining is prominent. Mathematical modelling shows that this induction of local pHi microdomains is favoured by low cytoplasmic H(+) mobility and long H(+) diffusion distances, particularly to surface NHE1 transporters mediating high membrane flux. Our results provide the first evidence for a spatial localisation of [H(+)]i regulation in ventricular myocytes, suggesting that, by guarding pHi, NHE1 preferentially protects gap junctional communication at intercalated discs, while NBC locally protects t-tubular excitation-contraction coupling.

Regulation of the Cardiac Sodium/Bicarbonate Cotransporter by Angiotensin II: Potential Contribution to Structural, Ionic and Electrophysiological Myocardial Remodelling

The sodium/ bicarbonate cotransporter (NBC) is, with the Na+/H+ exchanger (NHE), an important alkalinizing mechanism that maintains cellular intracellular pH (pHi). In the heart exists at least three isoforms of NBC, one that promotes the co-influx of 1 molecule of Na+ per 1molecule of HCO3-(electroneutral isoform; nNBC) and two others that generates the co-influx of 1 molecule of Na+ per 2 molecules of HCO3- (electrogenic isoforms; eNBC). In addition, the eNBC generates an anionic repolarizing current that modulate the cardiac action potential (CAP), adding to such isoforms the relevance to modulate the electrophysiological function of the heart. Angiotensin II (Ang II) is one of the main hormones that regulate cardiac physiology. The alkalinizing mechanisms (NHE and NBC) are stimulated by Ang II, increasing pHi and intracellular Na+ concentration, which indirectly, due to the stimulation of the Na+/Ca2+ exchanger (NCX) operating in the reverse form, leads to an increase in the intracellular Ca2+ concentration. Interestingly, it has been shown that Ang II exhibits an opposite effect on NBC isoforms: it activates the nNBC and inhibits the eNBC. This inhibition generates a CAP prolongation, which could directly increase the intracellular Ca2+ concentration. The regulation of the intracellular Na+ and Ca2+ concentrations is crucial for the cardiac cellular physiology, but these ions are also involved in the development of cardiac hypertrophy and the damage produced by ischemia-reperfusion, suggesting a potential role of NBC in cardiac diseases.

Regulation of the Cardiac Sodium/Bicarbonate Cotransporter by Angiotensin II: Potential Contribution to Structural, Ionic and Electrophysiological Myocardial Remodelling

The sodium/ bicarbonate cotransporter (NBC) is, with the Na+/H+ exchanger (NHE), an important alkalinizing mechanism that maintains cellular intracellular pH (pHi). In the heart exists at least three isoforms of NBC, one that promotes the co-influx of 1 molecule of Na+ per 1molecule of HCO3 -(electroneutral isoform; nNBC) and two others that generates the co-influx of 1 molecule of Na+ per 2 molecules of HCO3 - (electrogenic isoforms; eNBC). In addition, the eNBC generates an anionic repolarizing current that modulate the cardiac action potential (CAP), adding to such isoforms the relevance to modulate the electrophysiological function of the heart. Angiotensin II (Ang II) is one of the main hormones that regulate cardiac physiology. The alkalinizing mechanisms (NHE and NBC) are stimulated by Ang II, increasing pHi and intracellular Na+ concentration, which indirectly, due to the stimulation of the Na+/Ca2+ exchanger (NCX) operating in the reverse form, leads to an increase in the intracellular Ca2+ concentration. Interestingly, it has been shown that Ang II exhibits an opposite effect on NBC isoforms: it activates the nNBC and inhibits the eNBC. This inhibition generates a CAP prolongation, which could directly increase the intracellular Ca2+ concentration. The regulation of the intracellular Na+ and Ca2+ concentrations is crucial for the cardiac cellular physiology, but these ions are also involved in the development of cardiac hypertrophy and the damage produced by ischemia-reperfusion, suggesting a potential role of NBC in cardiac diseases. Keywords: Angiotensin II, calcium overload, cardiac arrhythmias, cardiac hypertrophy, electrogenic sodium/bicarbonate cotransporter, electroneutral sodium/bicarbonate cotransporter, sodium overload

Angiotensin II inhibits the electrogenic Na+/HCO3− cotransport of cat cardiac myocytes

The Na+/HCO3− cotransporter (NBC) plays an important role in intracellular pH (pHi) regulation in the heart. In the myocardium co-exist the electrogenic (eNBC) and electroneutral (nNBC) isoforms of NBC. We have recently reported that angiotensin II (Ang II) stimulated total NBC activity during the recovery from intracellular acidosis through a reactive oxygen species (ROS) and ERK-dependent pathway. In the present work we focus our attention on eNBC. In order to study the activity of the eNBC in isolation, we induced a membrane potential depolarization by increasing extracellular K+ [K+]o from 4.5 to 45mM (K+ pulse). This experimental protocol enhanced eNBC driving force leading to intracellular alkalization (0.19±0.008, n=6; data expressed as an increase of pHi units after 14min of applying the K+ pulse). This alkalization was completely abrogated by the NBC blocker S0859 (−0.004±0.016*, n=5; * indicates p<0.05 vs control) but not by the Na+/H+ exchanger blocker HOE642 (0.185±0.04, n=4), indicating that we are exclusively measuring eNBC. The K+ pulse induced alkalization was canceled by 100nM Ang II (−0.008±0.018*; n=5). This inhibitory effect was prevented when the myocytes were incubated with losartan (AT1 receptor blocker, 0.18±0.02; n=4) or SB202190 (p38 MAP kinase inhibitor, 0.25±0.06; n=5). Neither chelerythrine (PKC inhibitor, −0.06±0.04*; n=4), nor U0126 (ERK inhibitor, −0.07±0.04*; n=4) nor MPG (ROS scavenger, −0.02±0.05*; n=8) affected the Ang II-induced inhibition of eNBC. The inhibitory action of Ang II on eNBC was corroborated with perforated patch-clamp experiments, since no impact of the current produced by eNBC on action potential repolarization was observed in the presence of Ang II. In conclusion, we propose that Ang II, binding to AT1 receptors, exerts an inhibitory effect on eNBC activity in a p38 kinase-dependent manner.

Na+:HCO(3-) cotransporters (NBC): cloning and characterization.

The sodium bicarbonate cotransporter (NBC1) is essential for bicarbonate transport across plasma membranes in epithelial and nonepithelial cells. The direction of the NaHCO3 movement in secretory epithelia is opposite to that in reabsorptive epithelia. In secretory epithelia (such as pancreatic duct cells) NBC is responsible for the transport of bicarbonate from blood to the cell for eventual secretion at the apical membrane. In reabsorptive epithelia (such as kidney proximal tubule cells) NBC is responsible for the reabsorption of bicarbonate from cell to the blood. In nonepithelial cells this transporter is mainly involved with cell pH regulation. Recent molecular cloning experiments have identified the existence of four NBC isoforms (NBC1, 2, 3 and 4) and two NBC-related proteins AE4 and NCBE (Anion Exchanger 4 and Na-dependent Chloride-Bicarbonate Exchanger). All but AE4 are presumed to mediate the cotransport of Na+ and HCO(3-) under normal conditions and may be functionally altered in certain pathologic states. NBC1 shows a limited tissue expression pattern, is electrogenic and plays an important role in bicarbonate reabsorption in kidney proximal tubule. In addition to the kidney, NBC1 is expressed in pancreatic duct cells, is activated by cystic fibrosis transmembrane conductance regulator (CFTR) and plays an important role in HCO3- secretion. NBC2 and NBC3 have a wider tissue distribution than NBC1, are electroneutral, and are involved with cell pH regulation. The characterization of NBC4 is incomplete. The NBC-related protein called NCBE mediates Na-dependent, Cl-/Bicarbonate Exchange. The purpose of this review is to summarize recent advances on the cloning of NBC isoforms and related proteins and their role and regulation in physiologic and pathologic states.

Expression and localization of Na(+)-HCO(3)(-) cotransporter in bovine corneal endothelium.

Functional studies support the presence of the Na(+)-HCO(3)(-) cotransporter (NBC) in corneal endothelium and possibly corneal epithelium; however, molecular identification and membrane localization have not been reported. To test whether NBC is expressed in bovine cornea, Western blotting was performed, which showed a single band at approximately 130 kDa for freshly isolated and cultured endothelial cells, but no band for epithelium. Two isoforms of NBC have recently been cloned in kidney (kNBC) and pancreas (pNBC). RT-PCR was run using cultured and fresh bovine corneal endothelial and fresh corneal epithelial total RNA and specific primers for kNBC and pNBC. RT-PCR analysis for pNBC was positive in endothelium and weak in epithelium. The RT-PCR product was subcloned and confirmed as pNBC by sequencing. No specific bands for kNBC were obtained from corneal cells. Indirect immunofluorescence and confocal microscopy indicated that NBC locates predominantly to the basolateral membrane in corneal endothelial cells. Furthermore, Na(+)-dependent HCO(3)(-) fluxes and HCO(3)(-)-dependent cotransport with Na(+) were elicited only from the basolateral side of corneal endothelial cells. Therefore, we conclude that pNBC is present in the basolateral membrane of both fresh and cultured bovine corneal endothelium and weakly expressed in the corneal epithelium.

Physiologic and molecular aspects of the Na+:HCO3- cotransporter in health and disease processes

Approximately 80% of the filtered load of HCO3- is reabsorbed in the proximal tubule via a process of active acid secretion by the luminal membrane. The major mechanism for the transport of HCO3- across the basolateral membrane is via the electrogenic Na+:3HCO3- cotransporter (NBC). Recent molecular cloning experiments have identified the existence of three NBC isoforms (NBC-1, NBC-2, and NBC-3).1 Functional and molecular studies indicate the presence of all three NBC isoforms in the kidney. All are presumed to mediate the cotransport of Na+ and HCO3- under normal conditions and may be functionally altered in certain pathophysiologic states. Specifically, NBC-1 may be up-regulated in metabolic acidosis and potassium depletion and in response to glucocorticoid excess and may be down-regulated in response to HCO3- loading or alkalosis. Recent studies provide molecular evidence indicating the expression of NBC-1 in pancreatic duct cells. NBC is activated by cystic fibrosis transmembrane conductance regulator (CFTR) and plays an important role in HCO3- secretion in the agonist-stimulated state in pancreatic duct cells. The purpose of this review is to summarize recent functional and molecular studies on the regulation of NBCs in physiologic and pathophysiologic states. Possible signals responsible for the regulation of NBCs in these conditions are examined. Furthermore, the possible role of this transporter in acid-base disorders (such as proximal renal tubular acidosis) is discussed.

Cloning and Immunolocalization of a Rat Pancreatic Na+ Bicarbonate Cotransporter

In the rat, pancreatic HCO−3 secretion is believed to be mediated by duct cells with an apical Cl−/HCO−3 exchanger acting in parallel with a cAMP-activated Cl− channel and protons being extruded through a basolateral Na+/H+ exchanger. However, this may not be the only mechanism for HCO−3 secretion by the rat pancreas. Recently, several members of electrogenic Na+/HCO−3 cotransporters (NBC) have been cloned. Here we report the cloning of a NBC from rat pancreas (rpNBC). This rpNBC is 99% identical to the longer, more common form of NBC [pNBC; 1079 amino acids (aa); 122 kDa in human heart, pancreas, prostate, and a minor clone in kidney]. The longer NBC isoforms are identical to the rat and human kidney-specific forms (kNBC; 1035 aa; 116 kDa) at the ∼980 C-terminal aa's and are unique (with different lengths) at the initial N-terminus. Using polyclonal antibodies to the common N- and C-termini of rat kidney NBC, a ∼130-kDa protein band was labeled by immunoblotting of rat pancreas homogenate and was enriched in the plasma membrane fraction. Immunofluorescence and immunoperoxidase light microscopy of rat pancreatic tissue with both antibodies revealed basolateral labeling of acinar cells. Labeling of both apical and basolateral membranes was found in centroacinar cells, intra- and extralobular duct, and main duct cells. The specificity of the antibody labeling was confirmed by antibody preabsorption experiments with the fusion protein used for immunization. The data suggest that rpNBC likely plays a more important role in the transport of HCO−3 by rat pancreatic acinar and duct cells than previously believed.