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¹ Canadian Institutes of Health Research Membrane Protein Research Group, Department of Physiology and Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7
² Department of Physiology and Pharmacology, School of Biomedical Sciences, University of Queensland, Brisbane, Queensland 4072, Australia

	Abstract

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Bicarbonate facilitate more than 50% of pH recovery in the acidotic myocardium, and have roles in cardiac hypertrophy and steady-state pH regulation. To determine which bicarbonate transporters are responsible for this activity, we measured the expression levels of all known HCO3^––anion exchange proteins in mouse heart, by quantitative real time RT-PCR. Bicarbonate–anion exchangers are members of either the SLC4A or the SLC26A gene families. In neonatal and adult myocardium, AE1 (Slc4a1), AE2 (Slc4a2), AE3 (Slc4a3) (AE3fl and AE3c variants), Slc26a3 and Slc26a6 were expressed. Adult hearts expressed Slc26a3 and Slc4a1–3 mRNAs at similar levels, while Slc26a6 mRNA was about seven-fold higher than AE3, which was more abundant than any other. Immunohistochemistry revealed that Slc26a6 and AE3 are present in the plasma membrane of ventricular myocytes. Slc26a6 expression levels were higher in ventricle than atrium, whereas AE3 was detected only in ventricle. Cl^––HCO₃^– and Cl^––OH^– exchange activity of SLC26A6 and AE3 were investigated in transfected HEK293 cells, using intracellular fluorescence measurements of 2',7'-bis (2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF), to monitor intracellular pH (pH_i). Rates of pH_i change were measured under HCO3^–-containing (Cl^––HCO₃^–) or nominally HCO3^–-free (Cl^––OH^–) conditions. HCO3^– fluxes were similar for cells expressing AE3fl, SLC26A6 or Slc26a3, suggesting that they have similar transport activity. However, only SLC26A6 and Slc26a3 functioned as Cl^––OH^– exchangers. Activation of -adrenergic receptors, which stimulates protein kinase C, inhibited SLC26A6 Cl^––HCO₃^– exchange activity. We conclude that Slc26a6 is the predominant Cl^––HCO₃^– and Cl^––OH^– exchanger of the myocardium and that Slc26a6 is negatively regulated upon -adrenergic stimulation.

(Received 12 October 2004; accepted after revision 18 October 2004; first published online 21 October 2004)
Corresponding author J. R. Casey: CIHR Membrane Protein Research Group, Department of Physiology and Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7. Email: joe.casey{at}ualberta.ca

	Introduction

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Myocardial intracellular pH (pH_i) is tightly controlled, as cardiac contractility is directly affected by changes in pH_i (Fabiato & Fabiato, 1978). Intracellular acidification reduces Ca²⁺ binding to troponin-C, consequently attenuating the force of cardiac contraction (Orchard & Kentish, 1990). Conversely, a rise in pH_i induces a positive inotropic effect on cardiac muscle, resulting from sensitization of cardiac myofilaments to intracellular Ca²⁺ (Kramer et al. 1991).

Several membrane ion transporters regulate pH_i in the myocardium, including members of the SLC4a bicarbonate transport family of electroneutral Cl^––HCO₃^– exchangers (AEs) (Alvarez et al. 2001) and the Na⁺–HCO₃^– symporters (NBCs) (Dart & Vaughan-Jones, 1992; Sterling & Casey, 2002). Two others transporters contribute to pH_i regulation, the electroneutral Na⁺–H⁺ exchanger (NHE1) (Lazdunski et al. 1985) and the H⁺-coupled lactate transporters (MCT) (Vandenberg et al. 1993).

The contribution of an unidentified chloride-dependent acid loader, CDAL, to ventricular myocyte pH_i regulation has been postulated (Sun et al. 1996). This novel anion exchanger was proposed to mediate Cl^– influx in exchange for OH^– efflux at the plasma membrane of cardiomyocytes. However, this transporter has been characterized only at the functional level (Hun Leem & Vaughan-Jones, 1997); the protein(s) responsible has not been identified.

The first Cl^––HCO₃^– exchangers to be identified were members of the Slc4a family (AE1, AE2 and AE3) (Kopito, 1990). More recently members of the SLC26 gene family, now comprising 11 genes (SLC26A1–A11), have been found to facilitate exchange of a variety of anions, including Cl^–, HCO₃^–, SO₄^2–, OH^–, iodide, formate and oxalate anions, with differing ion preferences (Hastbacka et al. 1994; Markovich et al. 1994; Melvin et al. 1999; Soleimani et al. 2001; Xie et al. 2002; Mount & Romero, 2004). Some members of the SLC26 anion transport gene family mediate Cl^––HCO₃^– exchange at the plasma membrane of mammalian cells (Everett & Green, 1999). The SLC26A6 gene has recently been cloned from both humans (SLC26A6) (Lohi et al. 2000; Waldegger et al. 2001) and mice (Slc26a6, also known as CFEX) (Knauf et al. 2001). Note that mouse and human Slc26a members will be referred to as Slc26a and SLC26A, respectively, by convention. Both mouse Slc26a6 (Knauf et al. 2001) and its human orthologue SLC26A6 (Waldegger et al. 2001) have been identified on Northern blots of heart tissues. SLC26A3, the causative gene for congenital chloride diarrhoea, was originally cloned as a gene down-regulated in adenoma (DRA) (Schweinfest et al. 1993; Hoglund et al. 1996). Slc26a3 functions as both a Cl^––HCO₃^– and Cl^––OH^– exchanger (Melvin et al. 1999). SLC26A4, the gene that causes Pendred syndrome (Everett et al. 1997), is responsible for Cl^––HCO₃^– exchange at the apical surface of renal ß-intercalated cells (Petrovic et al. 2003b) and also acts as a Cl^––OH^– exchanger (Xie et al. 2002). However, SLC26A4 expression was not detected in the heart, even on over-exposed Northern blots (Everett et al. 1997). The final human Cl^––HCO₃^– exchanger, SLC26A7, has Cl^––HCO₃^– exchange activity but its expression is not detectable in the heart (Vincourt et al. 2002; Petrovic et al. 2003a). Taken together AE1, AE2, AE3, Slc26a3 and Slc26a6 are the only reported cardiac proteins with Cl^––HCO₃^– exchange activity.

To understand the contribution of individual Cl^––HCO₃^– exchangers to myocardial pH_i regulation, we have characterized the expression of all known Cl^––HCO₃^– exchangers at the mRNA level by quantitative real time RT-PCR. These data complement previous characterization of transporter expression on Northern blots. This is the first study to compare absolute mRNA levels of members of the Slc4a (AE1, AE2 and AE3) and the Slc26 (Slc26a6 and Slc26a3) anion exchanger and anion transporter families, respectively, in both neonatal and adult mouse heart. Real time RT-PCR data demonstrate that Slc26a6 mRNA is the predominant anion exchanger in the adult heart. The surprising discovery of the high abundance of Slc26a6 in the heart led us to examine the expression pattern of the protein on immunoblots and by immunohistochemistry of heart sections. In addition, because adrenergic signalling is important to cardiac function, we examined the regulation of SLC26A6 in response to stimulation of the _1a adrenergic receptor. Slc26a6 is also highly expressed at the protein level in isolated adult mouse cardiomyocytes. On the basis of the ability to transport HCO3^– and OH^– ions, we conclude that Slc26a6 is a dual Cl^––HCO₃^–, Cl^––OH^– exchanger with particular implications for myocardial intracellular pH regulation.

	Methods

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RNA isolation

Total RNA was isolated from adult mouse heart ventricles and atria and from neonatal mouse heart ventricles using Trizol reagent (Gibco BRL, Life Technologies, CA, USA), according to the manufacturer's instructions. Samples of isolated RNA were incubated with DNaseI (0.5 U (10 µg)^–1 of RNA, Gibco BRL) at 37°C for 5 min, to remove DNA. DNase-treated RNA was re-extracted with TRIZOL reagent. RNA integrity was confirmed by denaturing agarose gel electrophoresis, and RNA was quantified spectrophotometrically at 260 nm.

cDNA synthesis

For RT-PCR single strand cDNA synthesis was carried out using SuperScript First-Strand Synthesis for RT-PCR (Invitrogen, Life Technologies, Canada), according to the manufacturer's instructions. In each PCR reaction, 2 µl of cDNA from each synthesis was added to 50 µl of ‘master mix’ containing 5 µl of 10x PCR buffer, 1 µl of 10 mM dNTP mix, 1.5 µl 50 mM MgCl₂, 1 µl 1 M KCl, 0.4 µl (5 U µl^–1) of Taq DNA polymerase (New England Biolabs Ltd, CA, USA) and 37.1 µl H₂O. To each reaction was added 1 µl of 10 µM of each specific flank primers.

PCR primers

cDNA sequences were obtained from the public GenBank sequence database of the National Centre for Biotechnology Information (http://www.ncbi.nlm.nih.gov), and primers were designed with the Oligo software of the DNA Star program (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3.cgi). Mouse Slc26a10 has not yet been reported. To prepare primers the Slc26a10 mouse gene was identified by homology search of the mouse genome (Blast Search, http://www.ncbi.nlm.nih.gov/genome/seq/), using the human SLC26A10 sequence (Mount & Romero, 2004). The accession number used for mouse Slc26a10 was NT_081856. In conventional RT-PCR, all primers generated only one amplification band visualized by agarose gel electrophoresis on 1% agarose gels stained with ethidium bromide, demonstrating specificity. Sequences for all PCR primers are shown in Table 1.

Real time RT-PCR was performed in an ABI Prism 7900H sequence detection system (Applied Biosystems). Each real time RT-PCR reaction contained: 50 mM KCl, 3 mM MgCl₂, 0.08% (v/v) glycerol, 0.001% (v/v) Tween 20, 0.02% (v/v) DMSO, 1/40000 dilution SYBR Green (Molecular Probes), 0.03 U µl^–1 Jumpstart Taq (Sigma), 3.2 µM of each primer, 5 µl of template diluted (0, 1–4, 1–16 and 1–64), and 1 mM Tris; pH 8.3. In each case the template was reverse transcription reaction prepared from 2 µg total RNA and in a total of 20 µl. Replicate samples were pipetted into Axygen 384 well reaction plates using a Biomek Fx Pipetting Robot (Beckman Coulter). Results were presented as cycle threshold (CT). That is, the PCR cycle number at which exponential PCR-generated fluorescence is first detected; the lower the CT value, the higher the expression. To quantify the number of DNA molecules present in the mouse ventricle samples, real time RT-PCR was performed using standard amounts of mouse AE1, AE2, AE3 and Slc26a3, and human SLC26A6 plasmid DNA, generating standard curves of CT versus copy number. The CT values obtained for each anion exchanger in adult mouse hearts were individually calibrated according to the plasmid DNA curves and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression. DNA copy number was then obtained from CT values. Data are presented as DNA copy number on a logarithmic scale. Real time RT-PCR was also used to quantify the amount of mRNA present in samples of adult mouse atria and ventricles, for all Slc26 family members (Slc261–11, see Table 1). The CT values obtained for each Slc26 family member were individually normalized to GAPDH expression.

Isolation of mouse cardiomyocytes

Adult mice were killed with pentobarbital (150 mg kg^–1, I.P.) according to the University of Alberta Animal Policy and Welfare Committee and Canadian Council on Animal Care (CCAC) guidelines. The hearts were removed and ventricular myocytes were then obtained by enzymatic dissociation using standard protocols, which have been previously described (Bouchard et al. 1993; Light et al. 1998).

Preparation of mouse heart membranes

Freshly isolated mouse heart atria and ventricles were separately homogenized in a polytron (Kinematica GMBH, Switzerland) in either 0.5 ml, or 1.5 ml, respectively, of ice-cold solution containing 0.32 M sucrose, 1 mM EGTA, 0.1 mM EDTA, 10 mM Hepes (pH 7.5) and protease inhibitors (MiniComplete tablets, Roche). Homogenates were centrifuged at 1440 g for 5 min in a Beckman G5-6K centrifuge. Supernatants were removed and centrifuged at 66 700 g for 30 min at 4°C in a Beckman TLA 100.4 rotor. The resulting membrane fraction was resuspended in either 100 µl (atria) or 300 µl (ventricles) of PBS containing (mM): NaCl 140, KCl 3, Na₂HPO₄ 6.5, KH₂PO₄ 1.5; pH 7.5. Protein was quantified by Bradford assay, and 50 µg of protein used for immunoblots.

Protein expression

Expression constructs for human SLC26A6 (Lohi et al. 2000), mouse Slc26a3 (Melvin et al. 1999), rat AE3fl (Sterling et al. 2001) and human _1a receptor (Stanasila et al. 2003) have been previously described. SLC26A6, Slc26a3, AE3fl and _1a receptor proteins were expressed by transient transfection of HEK293 cells (Sterling & Casey, 1999), using the calcium phosphate method (Ruetz et al. 1993). Cells were grown at 37°C in an air–CO₂ (19:1) environment in Dulbecco's modified Eagle's medium (DMEM), supplemented with 5% (v/v) fetal bovine serum and 5% (v/v) calf serum.

Immunodetection

HEK293 cells were transfected with human SLC26A6 cDNA (Ko et al. 2002), or cotransfected with SLC26A6 and haemagglutinin-epitope tagged _1a receptor (_1a-HA) (Stanasila et al. 2003). Two days post-transfection, cells were washed in PBS containing (mM): NaCl 140, KCl 3, Na₂HPO₄ 6.5 and KH₂PO₄ 1.5 at pH 7.5, and lysates of the whole tissue culture cells were prepared by addition of 150 µl SDS-PAGE sample buffer to a 60-mm Petri dish. In other experiments, isolated cardiomyocytes from mouse hearts were sedimented by centrifugation and prepared by addition of 150 µl SDS-PAGE sample buffer. Samples (20 µg protein for cell lysates, 50 µg protein for isolated cardiomyocytes, 50 µg protein for mouse atria membranes, and 50 µg protein for mouse ventricle membranes) were resolved by SDS-PAGE on 8% acrylamide gels (Laemmli, 1970). Proteins were transferred to polyvinylidene fluoride membranes, and then incubated with rabbit anti-human SLC26A6 (N-terminus antibody raised against peptide corresponding to the last 20 amino acids of human SLC26A6) (Lohi et al. 2000), rabbit anti-AE3 (AP3) (Sterling & Casey, 1999), or rabbit anti-HA probe (Y11, Santa Cruz, CA, USA) antibody. Immunoblots were then incubated with donkey anti-rabbit IgG conjugated to horseradish peroxidase (Sterling et al. 2002). Blots were visualized and quantified using enhanced chemoluminescence reagent and a Kodak Image Station.

Immunohistochemistry of SLC26A6

Antibodies raised in rabbits against to the C-terminus of all AE3 variants, and against the amino-terminal amino acid sequence of the SLC26A6 cDNA sequence (accession no. AF279265), have been previously described (Sterling & Casey, 1999; Lohi et al. 2000). Sections (4 µm thick) from formalin-fixed, paraffin embedded longitudinal specimens of normal adult mouse heart were used for immunohistochemistry. Serial sections of deparaffinized slides were pretreated by heating in a microwave oven for 8 min in 10 mM citrate buffer (pH 6.0), and endogenous peroxidase activity was blocked by 0.3% hydrogen peroxide–methanol for 30 min at 25°C. Anti-SLC26A6 and anti-AE3 anti-serum was diluted to 1:200, and immunostaining was performed with the Vectastain Elite ABC Kit (Vector Laboratories, Inc., Burlingame, CA, USA). Diaminobenzidine (DAB) was used as the chromogenic substrate. Control slides were stained with the standard haematoxylin–eosin method to observe heart morphology. Pre-immune serum was used as negative control for parallel sections.

Measurement of SLC26A6 chloride–bicarbonate exchange activity

HEK293 cells, grown on 7.5 mm x 11 mm glass poly L-lysine-coated coverslips (Erie Scientific Co, NH, USA) in 60-mm dishes, were transfected with cDNA encoding human SLC26A6, rat AE3fl, mouse Slc26a3 or pcDNA3.1 (Invitrogen; empty vector), or cotransfected with SLC26A6 and _1a receptor. Two days post-transfection, coverslips were rinsed in serum-free DMEM and incubated in serum-free DMEM, containing 2 M the acetoxymethyl ester form of BCECF (BCECF-AM; Molecular Probes) at 37°C for 20 min. Coverslips were mounted in a fluorescence cuvette and perfused at 3.5 ml min^–1 alternately with bicarbonate-buffered Tyrode solution containing (mM): NaCl 128, glucose 11, CaCl₂ 1.35, MgSO₄ 1.05, NaHCO₃^– 20.23 and KCl 4.5 mM, or Cl^– -free, bicarbonate-buffered Tyrode solution containing (mM): sodium gluconate 140, glucose 11, calcium gluconate (hemicalcium salt) 4, MgSO₄ 1.05 and potassium gluconate 4.5. Both buffers were continuously bubbled with air–5% CO₂, and adjusted to pH 7.4 at 25°C with NaOH. In other experiments, coverslips were alternately perfused with Hepes-buffered Tyrode solution conaining (mM): NaCl 140, glucose 11, CaCl₂ 1.35, MgSO₄ 1.05, KCl 4.5 and Hepes 20; pH 7.4, or Cl^–-free, Hepes-buffered Tyrode solution containing (mM): sodium gluconate 140, glucose 11, calcium gluconate (hemicalcium salt) 4, MgSO₄ 1.05, potassium gluconate 4.5 and Hepes 20; pH 7.4. Both buffers were continuously bubbled with 100% O₂, and adjusted to pH 7.4 at 25°C with NaOH. All solutions contained 1 mM amiloride (Sigma) to block Na⁺–H⁺ exchanger activity. In other experiments, the lactate–H⁺ symport inhibitor, -cyano-4-hydroxycynnamate (Sigma; 3 mM), was added to the solutions. Fluorescence changes were monitored in a Photon Technologies International RCR fluorometer at excitation wavelengths 440 and 502 nm and emission wavelength 528 nm. After each experiment, fluorescence data were converted to pH_i by calibration using the nigericin/high potassium method (Thomas et al. 1979). The initial rate of change of pH_i determined during the removal and re-addition of Cl^–, was then fitted to a straight line by linear least squares fit, using Kaleidagraph software. All transport data have been corrected for background activity of HEK293 cells transfected with pcDNA3 vector alone.

Measurement of intrinsic buffer capacity and proton fluxes

Intracellular buffer capacity measurements were made by the ammonium pulse method (Boron & De Weer, 1976). HEK293 cells grown on glass coverslips were transfected with rat AE3fl, human SLC26A6, or mouse Slc26a3 cDNAs, as previously described. Two days post-transfection, cells were loaded with BCECF-AM as described above. Coverslips were mounted in a fluorescence cuvette and allowed to equilibrate in Hepes-buffered Tyrode solution, containing 1 mM amiloride, bubbled with 100% O₂ to ensure nominally bicarbonate-free conditions. Cells were then perfused consecutively for 300 s or until pH_i was steady with Hepes-buffered Tyrode solution, containing varying concentrations of NH₄Cl (0, 1, 5, 10, 20 and 30 mM). [NH₄⁺]_i was calculated from the Henderson-Hasselbalch equation, and the intrinsic buffer capacity (ß_i) was then calculated as [NH₄⁺]_i/pH_i. The total buffering capacity of the system (ß_tot) was then determined as ß_tot = ß_i + ß_CO2, where ß _CO2 = 2.3 x [HCO₃^–]_i. Total flux of proton equivalents was then calculated as: J_H⁺ = ß_tot x pH_i/t (Roos & Boron, 1981).

Statistical analysis

Statistical significance was evaluated using one-way ANOVA (followed by Bonferroni's test) or paired t test as indicated, with P < 0.05 considered significant. Error bars show standard error of the mean.

	Results

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Expression of Slc26a6 in mouse heart

The chloride bicarbonate anion exchange activity in the heart has, in the past been attributed to members of the SLC4A1 (AE) family (Xu & Spitzer, 1994; Linn et al. 1995; Richards et al. 1999). More recently members of the SLC26A family have been recognized as facilitating chloride–bicarbonate exchange (Mount & Romero, 2004). To determine the role of SLC26 transporters in cardiac chloride–bicarbonate exchange, we examined the expression of the 11 members of the SLC26 family in the heart. Expression of anion exchanger transcripts from ventricular and atrial samples was quantified by real time RT-PCR (Fig. 1), by using straight CT values normalized to expression of GAPDH. CT denotes the threshold cycle of PCR amplification at which product is first detected by fluorescence. The CT value is dependent on the quantity of the target molecule in the sample. The higher the CT value, the lower the expression of the transcript. CT values of 32 or higher were considered as representing samples with no expression of the transcripts (Fig. 1, dashed line). Expression of Slc26a3 and Slc26a6 Cl^––HCO₃^– exchanger mRNA showed comparable levels in both atria and ventricles. Slc26a2, a Cl^––SO₄^2–, transporter involved in sulphate uptake in fibroblasts and chondrocytes, was detected at very low levels, consistent with reported Northern blot hybridization experiments (Haila et al. 2001). Slc26a10, suggested to be an expressed pseudogene with no uninterrupted open reading frame (Mount & Romero, 2004), was also detected in the mouse heart atria and ventricles. However, Slc26a1, Slc26a4, Slc26a5, Slc26a7, Slc26a8, Slc26a9 and Slc26a11 transcripts were not expressed in the mouse heart, as assessed by RT-PCR. We conclude that Slc26a3 and Slc26a6 are the only Cl^––HCO₃^– exchangers of the Slc26 family expressed in the heart.

View larger version (32K): [in this window] [in a new window]   Figure 1.  Expression of Slc26 transcripts in adult mouse heart atria and ventricles mRNA expression was compared in samples from atria and ventricles of adult mice using real time quantitative RT-PCR. Data are corrected for individual variation using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) standard curves and results are expressed as cycle threshold. The cycle threshold is dependent on the quantity of the target molecule in the sample. The higher the cycle threshold value, the lower the expression of the transcript. Dashed line marked limit of expressed transcripts. Cycle threshold values of 32 or higher means no expression of the transcript. Error bars represent S.E.M. (n = 3 separate adult mouse hearts supplying atrial and ventricular samples).

View larger version (32K): [in this window] [in a new window]   Figure 2.  Expression of anion exchanger transcripts in mouse heart and quantification by real time quantitative RT-PCR A, RT-PCR analysis of various anion exchangers in adult mouse ventricle. First strand cDNA was produced using First Strand RT-PCR Kit. Amplicons were analysed on a 1% agarose–ethidium bromide gel. Lane 1, AE1; lane 2, AE2; lane 3, AE3c; lane 4, AE3fl; lane 5, Slc26a6; lane 6, Slc26a3. B and C, mRNA expression was compared from ventricles of adult (filled bars) or neonatal (grey bars) mice using real time quantitative RT-PCR. Data are corrected for individual variation using plasmid DNA standards and results are expressed as copy number (logarithmic scale). Two different pairs of forward and reverse primers (1 and 2), which amplified different regions of Slc26a6 cDNA, and which had slightly different amplicon size and Tm (see Table 1), were used. *Significant difference from the expression level of Slc26a6 (primer set 1) in adult ventricle, with P < 0.05, one-way ANOVA. #P < 0.05 (paired t test) compared to Slc26a6 (primer set 2) in adult heart samples. Neonatal samples different from the expression level of Slc26a6 (primer set 1) with P < 0.05, one-way ANOVA. Error bars represent S.E.M. (n = 4 separate hearts for adults and 3 for neonates).

Quantification of specific genes by real time RT-PCR was performed with the use of the SYBR Green reagent. SYBR Green binds any double-strand DNA generated by PCR. Therefore, undesired products could generate a signal. To verify the specificity of the real time RT-PCR data, we performed RT-PCR using each of the primer pairs and ran the products on agarose gels. A single band was found for each product (Fig. 2A). We also calibrated the real time data by performing real time RT-PCR on a dilution series of plasmid encoding each anion exchanger cDNA. These standard curves facilitated conversion of the threshold cycle number data, for each amplified PCR product, to log₁₀ number of template molecules.

The finding that Slc26a6 is about two orders of magnitude more highly expressed than other anion exchangers was surprising because Cl^––HCO₃^– exchange has been extensively studied in the heart, yet the expression and role of Slc26a6 in the heart have not been examined. To examine if the observation was particular to the set of primers used, we examined expression level of Slc26a6 using another pair of forward and reverse primers (Slc26a6 (2); Table 1), which amplified a different region of the cDNA and which had slightly different amplicon size and melting temperature (T_m). Quantification of Slc26a6 expression with a second set of primers was compared to AE3fl in adult mouse heart. Expression was significantly higher for Slc26a6 (9116 ± 1040 copies) compared to AE3fl (195 ± 54; Fig. 2C; n = 4, P < 0.05 paired t test). Looking at the expression levels on a linear scale, Slc26a6 is about seven-fold more abundant than AE3fl. The two different primer sets used to quantify Slc26a6 expression (Fig. 2B and C) gave similar values for the level of the transcript (9970 ± 192 and 9116 ± 1040 copies). This result confirmed that the higher expression of Slc26a6 detected in mouse heart did not result from peculiarities of the primer pair used.

Expression of AE1, AE2, AE3fl and AE3c proteins, has been observed in rat neonatal cardiomyocytes (Richards et al. 1999). However, there are no data on Slc26a6 protein expression in heart cells. An antibody raised against a peptide corresponding to the C-terminal 20 amino acids of human SLC26A6 was used to detect Slc26a6 protein (Lohi et al. 2000). Amino acid sequences of human SLC26A6 and its mouse orthologue, Slc26a6, are virtually identical in this region, so that cross reactivity of the antibody for different species was anticipated. Immunoblots of mouse heart atrial and ventricular membranes showed a different expression pattern for Slc26a6 compared to AE3 anion exchanger (Fig. 3A and B). Slc26a6 was expressed in both atria and ventricles, although at considerably higher levels in ventricles than atria. In contrast, AE3 protein was detected only in ventricular samples. Expression of Slc26a6 protein in isolated mouse cardiomyocytes was also investigated on immunoblots. Immunoreactivity was found for human embryonic cells (HEK293) transfected with SLC26A6 cDNA, and cardiomyocytes isolated from adult mouse heart (Fig. 3C, lanes 2 and 3, respectively). The apparent molecular mass of Slc26a6 was 85 kDa for both HEK cells expressing SLC26A6 and cardiomyocytes. However, no immunoreactivity was found in non-transfected cells (Fig. 3C, lane 1), or on parallel blots incubated with non-immune rabbit serum (Fig. 3D).

View larger version (53K): [in this window] [in a new window]   Figure 3.  Expression of Slc26a6 and AE3 proteins in adult mouse heart atria and ventricles, and expression of Slc26a6 protein in isolated adult mouse cardiomyocytes A and B, heart membranes were isolated from adult mouse atria and ventricles. Membrane protein (50 µg) was subjected to SDS-PAGE analysis, transferred to PVDF membrane, and probed with specific anti-SLC26A6 (A), or anti-AE3 (B) antibodies. C and D, lysates of HEK293 cells (20 µg protein) transfected with either empty vector (lane 1), or transfected with human SLC26A6 cDNAs (lane 2), or lysates of freshly isolated mouse cardiomyocytes (50 µg, lane 3), were prepared. Samples were analysed by SDS-PAGE, transferred to PVDF membranes, and probed with anti-SLC26A6 antibody (C) or serum from nonimmune rabbits (D). Filled and open arrows indicate positions of the SLC26A6–Slc26a6 and AE3 proteins, respectively.

Expression of Slc26a6 and AE3 proteins was further investigated by immunohistochemistry of parallel longitudinal sections of adult mouse heart (Fig. 4A). Haematoxylin–eosin staining of the heart ventricular muscle shows normal cardiac muscle morphology. Immunostaining of heart muscle specimens with anti-Slc26a6 antibodies showed intense cell surface-associated expression of Slc26a6 protein in cardiac ventricular muscle (Fig. 4B). Anti-AE3 antibody, which recognizes both AE3fl and AE3c variants of AE3, also demonstrated the presence of AE3 protein in adult heart ventricular muscle (Fig. 4C). In addition some intracellular staining, either intracellular AE3 or non-specific staining, was evident. No immunoreactivity was seen on sections of ventricle treated with pre-immune rabbit serum (Fig. 4D). These results further suggest an important functional role for Slc26a6, as the predominant anion exchanger of the heart.

View larger version (172K): [in this window] [in a new window]   Figure 4.  Immunocytochemistry of Slc26a6 and AE3 proteins in adult mouse cardiac ventricle A, haematoxylin–eosin staining of ventricular muscle sections. Immunostaining of Slc26a6 protein (B) and AE3 protein (C) was detected on the surface of cardiomyocytes, in ventricular muscle sections. No immunoreactivity was seen on cardiac muscle sections processed with pre-immune rabbit serum (D). Scale bar represents 25 µm.

The physiological role of a transporter depends on substrate specificity and expression pattern. SLC26A6 mediates Cl^––OH^– and Cl^––HCO₃^– exchange activities, when expressed in Xenopus laevis oocytes (Xie et al. 2002). We thus examined the Cl^––OH^– and Cl^––HCO₃^– exchange activity of SLC26A6, Slc26a3 and AE3fl. The complexity of converging expression of various anion exchangers/transporters at the plasma membrane makes it difficult to study the anion exchange activity in myocardial preparations. Moreover, members of SLC4 and SLC26 families are equally sensitive to the stilbene derivatives such as DIDS and SITS, making pharmacological dissection of the transporters impossible (McConnell & Aronson, 1994; Papageorgiou et al. 2001; Xie et al. 2002). To compare transport activity of SLC4 and SLC26 transporters, AE3fl, SLC26A6 and Slc26a3 were individually expressed in HEK293 cells. Under these conditions, AE3fl is highly expressed (Alvarez et al. 2001). As seen in Fig. 3A, SLC26A6 is also readily detectable. Intrinsic buffer capacity (ß_i) was measured in HEK293 cells individually expressing AE3fl, SLC26A6 and Slc26a3 proteins. ß_i values for HEK293 transfected with SLC26A6, Slc26a3 and AE3fl were 11.64 ± 2.18 mM, 14.64 ± 1.14 mM and 14.91 ± 1.43 mM, respectively. ß_i values were estimated at similar pH_i, 6.83 ± 0.23, 6.90 ± 0.23 and 6.96 ± 0.26 for SLC26A6, Slc26a3 and AE3fl, respectively. The total buffer capacity of the system (ß_tot) was then determined as ß_tot = ß_i + ß_CO2, where ß_CO2 = 2.3 x [HCO₃^–]_i. ß_tot was 26.46 ± 4.12 mM, 32.05 ± 4.31 mM and 34.90 ± 4.63 mM for HEK293 cells expressing SLC26A6, Slc26a3 and AE3fl, respectively. We conclude that total and intrinsic buffering power did not differ in HEK293 expressing different anion exchangers/transporters.

Anion exchange activity was measured in the presence (bicarbonate-buffered Tyrode solution) or absence (Hepes-buffered Tyrode solution, nominally bicarbonate free) of HCO₃^–. Under these conditions, after stabilization in normal Tyrode solution, removal of extracellular Cl^– induced a rise in the pH_i due to the influx of HCO₃^– (Cl^–-free bicarbonate-buffered Tyrode solution) or OH^– (Cl^–-free Hepes-buffered Tyrode solution). After Cl^– re-addition, pH_i reached steady-state conditions, as previously described (Sterling & Casey, 1999). Hence, Cl^–-dependent HCO3^– or OH^– fluxes were measured in HEK293 cells individually expressing SLC26A6, Slc26a3 or AE3fl.

Figure 5A shows typical transport activity of HEK293 cells expressing human SLC26A6 anion transporter. Human SLC26A6 mediates Cl^––HCO₃^– and Cl^––OH^– exchange when subjected to a Cl^– removal/Cl^– re-addition protocol, as previously shown in Xenopus laevis oocytes (Xie et al. 2002). All experiments were conducted in the presence of 1 mM amiloride to block Na⁺–H⁺ exchanger activity. In other experiments, the lactate–H⁺ inhibitor, -cyano-4-hydroxycynnamate (3 mM), showed no effect on observed pH_i changes associated with changes of [Cl^–], with both HCO3^– buffer and Hepes buffer (data not shown), indicating that monocarboxylate–H⁺ cotransport is not a confounding factor in the study. Both Cl^––HCO₃^– and Cl^––OH^– transport activities were inhibited by 1 mM DIDS (73 ± 3% and 85 ± 2%, respectively; n = 4, P < 0.05; Fig. 5B). The ability of SLC26A6 to mediate changes of pH_i upon change of medium (Cl^–), in both HCO3^–-containing and HCO₃^–-free media is consistent with the action of SLC26A6 functioning as a Cl^––HCO₃^– and Cl^––OH^– exchanger. Note that the rate of pH_i change in SLC26A6-transfected cells is similar in bicarbonate-containing and nominally bicarbonate-free media (Hepes) (Fig. 5A). Yet, the calculated transport rates are much lower for Cl^––OH^–than Cl^––HCO₃^– exchange (Fig. 5B). This results from the larger buffer capacity in bicarbonate media than under bicarbonate-free conditions.

View larger version (27K): [in this window] [in a new window]   Figure 5.  Chloride–base exchange activity of cardiac anion exchange proteins A, HEK293 cells were transfected with SLC26A6 cDNA. Two days after transfection, cells were loaded with pH-sensitive dye, BCECF. Cells were perfused with either Cl–-free (filled bar), or Cl–-containing (open bar) buffer. Dark and light traces show experiments performed using bicarbonate- and Hepes-buffered Tyrode solutions, respectively. B, mean values of transport rates expressed as mmol l–1 min–1, of HEK293 cell transiently transfected with SLC26A6 cDNA. Cells expressing human SLC26A6 were exposed to DIDS (1 mM). Filled and grey bars represent experiments performed in bicarbonate- and Hepes-buffered Tyrode solutions, respectively. *P < 0.05, paired t test. C, Cl––HCO3– (bicarbonate) and Cl––OH– (Hepes) exchange activity was measured in HEK293 cells transiently transfected with rat AE3fl (rAE3fl), human SLC26A6 (hSLC26A6), mouse Slc26a3 (mSlc26a3) cDNAs, or empty vector (pCDNA3). Transport rates are expressed as flux (mmol l–1 min–1); (number of experiments). *P < 0.05 compared to pCDNA3, P < 0.05 compared to hSLC26A6; P < 0.05 compared to rAE3fl; one-way ANOVA.

In contrast with the Cl^––HCO₃^– transport results, Cl^––OH^– exchange fluxes were higher for SLC26A6 compared to Slc26a3 (Fig. 5C). However, Cl^––OH^– exchange fluxes by the AE3fl anion exchanger were not significantly different from the background activity of sham-transfected cells. On the basis of these results, we conclude that AE3fl is not a Cl^––OH^– exchanger, but that SLC26A6 and Slc26a3 both mediate significant Cl^––OH^– exchange.

_1a-adrenoceptor stimulation decreased Cl^––HCO₃^– exchange activity of Slc26a6

The identification of Slc26a6 as the predominant Cl^––HCO₃^– exchanger of the heart led us to examine the role of adrenergic signalling in the regulation of Slc26a6. ₁-adrenergic stimulation exerts diverse physiological actions in the mammalian myocardium, such us positive inotropic effects (Layland & Kentish, 2000), protein synthesis (Taimor et al. 2004) and arrhythmogenesis (Kurz et al. 1991). ₁-adrenoceptors are coupled to phospholipase C activation, which generates inositol-1,4,5-trisphosphate (IP₃) and diacylglycerol (DAG). In turn, DAG is a potent activator of protein kinase C (PKC). To assess the role of ₁-adrenergic stimulation on Slc26a6, HEK293 cells were cotransfected with HA-epitope tagged _1a-adrenergic receptor (_1a-HA) and SLC26A6 cDNAs. Cells expressing _1a-HA receptor (Fig. 6, lane 2) and SLC26A6 anion transporter were subjected to Cl^––HCO₃^– exchange assays, in the presence or absence of the ₁-adrenergic agonist phenylephrine (PE, 10 µM). PE elicited a 60 ± 2% decreased in the Cl^––HCO₃^– exchange activity of SLC26A6 (n = 4). When HEK293 cells expressing SLC26A6 alone where treated with the PKC activator, PMA-phorbol esters (200 nM), the Cl^––HCO₃^– exchange activity by SLC26A6 was reduced by 42 ± 3% (n = 4). The decreased Cl^––HCO₃^– exchange activity of SLC26A6 induced by PKC, was completely blocked (activity relative to control was 102 ± 14% (n = 4)) by the broadly specific PKC inhibitor, chelerythrine (CHE, 10 µM; Fig. 6B). We conclude that unlike AE3fl anion exchanger, which is activated by PKC (Alvarez et al. 2001), stimulation of PKC decreases Slc26a6-mediated HCO₃^– transport.

View larger version (15K): [in this window] [in a new window]   Figure 6.  Negative regulation of Slc26a6 Cl––HCO3– exchange activity upon 1a-adrenergic receptor stimulation A, expression of haemaglutinin epitope-tagged 1a receptors (1a-HA), in HEK293 cells. Lysates of HEK293 cells (20 µg protein) transfected with either empty vector (lane 1), or transfected with 1a-HA cDNA (lane 2), were prepared. Samples were analysed by SDS-PAGE, transferred to PVDF membranes, and probed with anti-HA antibody. Open arrows indicate position of different glycosylated forms of the 1a receptor. B, mean values of Cl––HCO3– exchange activity relative to SLC26A6 transport activity, of HEK293 cell transiently transfected with SLC26A6, or cotransfected with SLC26A6 and 1a-HA, cDNAs, as indicated at the bottom. Cells expressing human SLC26A6 and 1a receptors were incubated with phenylephrine (PE, 10 µM), or with PMA-phorbol esters (200 nM) to stimulate protein kinase C (PKC). Chelerythrine (CHE, 10 µM) was used to block PKC activity in cells expressing SLC26A6. *P < 0.05, paired t test.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Plasma membrane Cl^––HCO₃^– exchange, which is well documented in cardiomyocytes (Xu & Spitzer, 1994; Leem et al. 1999), is important during normal pH regulation (Leem et al. 1999), recovery from ischaemic acidosis (Vandenberg et al. 1993) and in the development of cardiac hypertrophy (Perez et al. 1995; Ennis et al. 1998). However, the molecular identity of the transporters that mediate this exchange is not fully characterized. Here we have revealed that Slc26a3 and Slc26a6 are the only Cl^––HCO₃ exchangers of the Slc26a family that are expressed in the heart (Fig. 1). These transporters join AE1, AE2 and AE3 as the only established Cl^––HCO₃^– exchangers of the heart and together are responsible for all forms of Cl^––HCO₃^– exchange activity observed in myocardium. The observation that both Slc26a3 and Slc26a6 are Cl^––OH^– exchangers leads to the conclusion that these isoforms are together responsible for cardiac Cl^––OH^– exchange activity (CDAL).

Slc26a6: the predominant Cl^––HCO₃^– exchanger in the mouse heart

This paper reports for the first time the quantification in parallel of all anion exchange proteins known to be expressed in the myocardium. Real time RT-PCR revealed that Slc26a6 is the predominant Cl^––HCO₃^– exchanger in adult hearts. The abundance of Slc26a6 was verified by the ability to detect protein at levels comparable to that found in transfected HEK293 cells. The ability of SLC26A6 and Slc26a3 transporters to act as acid loading Cl^––OH^– exchangers in mammalian cells underscores the relevance of the findings to cardiac physiology.

The primary anion exchanger isoforms studied so far in mammalian myocardium are the two splicing variants of the AE3 gene, AE3fl and AE3c (Yannoukakos et al. 1994; Cingolani et al. 2001). The existence of a novel AE1 (AE1_n) splicing variant, which represents a truncated version of the erythroid AE1 (AE1_e) has also been proposed (Richards et al. 1999). The finding suggests the existence in adult and neonatal cardiomyocytes of AE1 mRNAs that are spliced in one of introns 4–6 and 10–13, respectively. The authors concluded that AE1_n was the predominant AE of cardiac myocytes, but the novel isoform remains to be identified. Furthermore, even though the blots were overexposed, only one band corresponding to the AE1_e was identified by Northern blots in rat heart tissue (Alvarez et al. 2001).

In real time RT-PCR experiments primers amplified the region corresponding to exon 5 of the mouse AE1 mRNA (Table 1). These primers will recognize the kAE1-splicing variant, if present in mouse heart, because from the beginning of exon 4 to the polyadenylation site in exon 20, both AE1_e and kAE1 mRNAs are identical (Kudrycki & Shull, 1993). However, AE1_n is apparently spliced from a sequence downstream of exon 5 (Richards et al. 1999). Therefore, the primers used here would not recognize the AE1_n splicing variant.

Quantification of anion exchangers by real time RT-PCR showed that mouse heart expresses comparable levels of AE1 and Slc26a6 mRNA at the neonatal stage. Conversely, AE1 mRNA expression was down-regulated with mouse development; AE3c and AE3fl are the predominant AEs in the adult mouse heart (Fig. 2B). Slc26a6 mRNA was highly expressed in both neonatal and adult mouse heart. The mRNA expression levels of Slc26a3 in adult heart were comparable to AE3fl (Fig. 2B).

Expression of Slc26a6 protein in mouse cardiac cells and in transfected cells was verified using an antibody against the N-terminus of human SLC26A6. The antibody recognizes both human SLC26A6 protein, and its mouse orthologue Slc26a6 protein. Expression of Slc26a6 in isolated cardiomyocytes at comparable protein levels with SLC26A6 over-expressed in HEK293 cells, suggests a very high expression for the transporter in the myocardium. Taken together, the real time RT-PCR and immunoblot data show that Slc26a6 is the most abundant anion exchanger of the adult mouse heart. Transfected HEK293 cells manifest similar Cl^––HCO₃^– exchange activity whether transfected with AE3fl or SLC26A6. This suggests that, to a first approximation, SLC26A6 and AE3fl have similar transport rates. Thus, the total ionic flux carried by SLC26A6 is about seven-fold higher than AE3fl, reflecting their relative expression levels. On this basis SLC26A6 may be the most important Cl^––HCO₃^– exchanger of the myocardium. However, differential regulation of Cl^––HCO₃^– exchanger isoforms could alter their relative contributions in the context of the heart.

The expression pattern for Slc26a6 and AE3 was revealed on immunoblots and by immunohistochemistry of ventricular sections. The presence of both proteins in myocyte membranes is consistent with a role in normal myocyte function. The greater abundance of both proteins in ventricle than atrium (as seen on immunoblots and in quantitative real time RT-PCR analysis of mRNA levels) suggests that expression of both anion exchangers correlates with the level of metabolic load. That is, ventricular muscle works against a greater load than atrial muscle, and the ventricle is thus more metabolically active. The role of Slc26a6 and AE3 may be either to contribute to regulation of myocyte pH or handling of metabolic bicarbonate load.

In summary, we have examined the cardiac expression of mRNA for all known chloride–bicarbonate exchangers. In addition we examined the expression of all Slc26a family members, some of which (Slc26a3, a4, a6, a7) have been shown to function as chloride–bicarbonate exchangers. Among all these transporters, we found that only AE1, AE2, AE3, Slc26a2, Slc26a3, Slc26a6 and Slc26a10 are expressed at significant levels in the heart, as assessed by a cut-off threshold in real time quantitative RT-PCR experiments. Slc26a10 has been proposed to be an expressed pseudogene and is unlikely to eventuate as a chloride–bicarbonate exchanger (Mount & Romero, 2004). Slc26a2 has not been shown to transport bicarbonate, but rather like other Slc26a family members, to act as a sulphate transporter (Mount & Romero, 2004).

Identity of the CDAL previously described in ventricular myocytes

Previously only anion exchangers of the SLC4 family (AE) were considered as acid loaders, regulating pH_i in the heart (Vaughan-Jones, 1981, 1986; Xu & Spitzer, 1994; Richards et al. 1999). However, a Cl^––OH^– exchanger was functionally characterized in ventricular myocytes, as a part of a dual acid-loading mechanism along with the AEs (Sun et al. 1996). Unlike AE, the CDAL-mediated acid influx was proposed as functional in the absence of HCO₃^–. The molecular identity of the cardiac CHE remained unclear until the present report. Previously there has been speculation that CHE might be a novel member of the AE family, or a member of a different family of anion exchangers (Hun Leem & Vaughan-Jones, 1997). The authors concluded that CDAL is a Cl^–-dependent, DIDS-insensitive, but 4,4'-dibenzoylstilbene-2,2'-disulphonate (DBDS)-inhibitable transporter. Further, a CDAL was suggested to operate in the absence of residual HCO3^–, in a Cl^––OH^– exchange, or a H⁺–Cl^– symport mode. In CO₂–HCO₃^–-buffered solutions, the CHE accounts for 50% of acid loading of the cardiac cell during extracellular acidosis, considered by the authors as the major acid-transport mechanism of cardiomyocytes.

More recently, Cingolani and collaborators demonstrated that 50% of the recovery after intracellular alkaline loads in myocardial tissues, is attributable to AE3 anion exchangers (Cingolani et al. 2003). The authors used a functionally inhibitory antibody approach to block AE3-dependent acid loading. The antibody was specifically designed to recognize the third extracellular loop of the AE3. Therefore, the two splicing variants of the AE3 gene, AE3fl and AE3 cardiac, were both inhibited. Clearly, a residual acid-loading activity in the myocardium, independent from the AE3, was observed. Again, a large part of the acid loading was attributable to a Cl^––HCO₃^– exchanger, and most probably to a different AE than AE3.

Members of the Slc4a (AE) family of transporter are not likely to contribute to cardiac Cl^––OH^– exchange activity. In the present report we found that AE3 did not support Cl^––OH^– exchange. Similarly, tumour cells that expressed AE1, AE2 and AE3 exhibited Cl^––HCO₃^– exchange activity, but not Cl^––OH^– exchange activity (Papageorgiou et al. 2001), suggesting that none of the AE family members is a Cl^––OH^– exchanger. However, the conclusion that Slc4a transporters cannot act as Cl^––OH^– exchangers requires some caution, since one report found Cl^––OH^– associated with AE1 expressed in HEK cells (Ko et al. 2002).

The data presented here identified only two transporters that are expressed in the adult myocardium and which facilitate Cl^––OH^– exchange: Slc26a3 and Slc26a6. On the basis of the expression pattern and transport data we speculate that these transporters are the missing mechanism in the control of pH_i regulation in cardiac cells. Nonetheless, there are some discrepancies between our data and the work of Vaughan-Jones' group. CDAL was described as a Cl^–-dependent, but HCO3^–-independent acid-loading mechanism. CDAL was also reported to be sensitive to the stilbene-derivative DBDS, but DIDS-insensitive (Sun et al. 1996). Consistent with this characterization, Slc26a3, which we found to be present in adult mouse heart at the mRNA level (Fig. 2B), is subject to only 24% inhibition by high concentrations (1 mM) of DIDS (Melvin et al. 1999). Inconsistent with the previous report of CDAL in heart, SLC26A6 Cl^––HCO₃^– exchange activity was found here to be 73% inhibited by 1 mM DIDS. If SLC26A6 contributes to cardiac CHE, the discrepancy with regard to DIDS sensitivity may reflect differences in protein behaviour dependent on context: isolated tissue culture cells versus cardiomyocytes. It is also possible that species differences account for the discrepancy; mouse heart expression and human SLC26As were studied here, while guinea-pig ventricular myocytes were the focus of Vaughan-Jones and coworkers. Alternately, as Xie and coauthors proposed (Xie et al. 2002), the DIDS sensitivity of Slc26a6 and other members of the family is likely to be highly dependent on both the concentration and the identity of the transported anion (Karniski & Aronson, 1987).

On balance we conclude that cardiac CDAL results from the activity of Slc26a6 and Slc26a3. The greater abundance of Slc26a6 and its higher Cl^––OH^– exchange activity lead to the conclusion that Slc26a6 is the major Cl^––OH^– exchanger of the mouse heart.

This is the first report on Slc26a6 and Slc26a3 in the myocardium. Both transporters may be important for normal cardiac electrical activity, as they are reported to function with electrogenic mechanisms (Ko et al. 2002; Mount & Romero, 2004). The transport stoichiometry Slc26a6 is 2 HCO₃^–: 1 Cl^–, while Slc26a3 has a transport stoichiometry of 1 HCO₃^–: 2 Cl^– (Ko et al. 2002). This opposite stoichiometry of the transporters means that their relative transport activity will vary with the membrane potential; Slc26a6 will act to cause efflux of HCO₃^– at resting membrane potential (–80 mV), while Slc26a3 will become active in HCO₃^– efflux as the cell moves to more positive potential during the action potential.

Cardiac anion exchange is subject to hormonal regulation (Camilion de Hurtado et al. 1998, 2000; Alvarez et al. 2001). Angiotensin II and endothelin-1, both of which stimulate PKC, activate AE3fl Cl^––HCO₃^– exchange activity. In addition, hyperactivity of AE3 Cl^––HCO₃^– exchange activity in the hypertrophic myocardium of spontaneously hypertensive rats (SHRs) was demonstrated (Perez et al. 1995). The angiotensin-converting enzyme inhibitor, enalapril, reverted cardiac hypertrophy, and normalized the increased transport activity of AE3 Cl^––HCO₃^– exchanger in SHRs (Ennis et al. 1998). Here we found that 1-adrenergic-stimulation decreased SLC26A6-mediated HCO₃^– transport. Similarly, activation of PKC also decreased Cl^––HCO₃^– exchange activity by SLC26A6. SLC26A6 thus responds in manner counter to AE3fl in response to PKC stimulation and may therefore compensate for hypertrophic stimulation.

We have identified Slc26a6 as a Cl^––OH^–, Cl^––HCO₃^– transporter that is highly expressed in cardiac tissues. The high abundance and functional activity of Slc26a6 lead us to conclude that it is the predominant Cl^––HCO₃^– exchanger of the heart. Characterization of the Cl^––OH^– exchange activity of anion exchange proteins expressed in the adult heart, leads to the conclusion that Slc26a3 and Slc26a6 are the only cardiac Cl^––OH^– exchangers. On the basis of expression level and transport activity, Slc26a6 contributes the most CDAL activity to the mouse heart. HCO₃^– transport mediated by these transporters is relevant to the cardiac pH_i regulation, but the physiological relevance of the Cl^––OH^– exchange mediated by the Slc26a6 and Slc26a3 in the heart is not clear yet. However, the reciprocal responses of Slc26a6 and AE3fl to cardiac hypertrophic pathways coupled to PKC activation suggest that SLC26A6 may have a role in limiting hypertrophic processes. Identification of Slc26a6 as the predominant anion exchanger of the heart will need to be taken into account in all models of cardiac pH regulation.

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