Bernardo V. Alvarez

Metabolon disruption: a mechanism that regulates bicarbonate transport

Carbonic anhydrases (CA) catalyze the reversible conversion of CO2 to HCO3−. Some bicarbonate transporters bind CA, forming a complex called a transport metabolon, to maximize the coupled catalytic/transport flux. SLC26A6, a plasma membrane Cl−/HCO3− exchanger with a suggested role in pancreatic HCO3− secretion, was found to bind the cytoplasmic enzyme CAII. Mutation of the identified CAII binding (CAB) site greatly reduced SLC26A6 activity, demonstrating the importance of the interaction. Regulation of SLC26A6 bicarbonate transport by protein kinase C (PKC) was investigated. Angiotensin II (AngII), which activates PKC, decreased Cl−/HCO3− exchange in cells coexpressing SLC26A6 and AT1a‐AngII receptor. Activation of PKC reduced SLC26A6/CAII association in immunoprecipitates. Similarly, PKC activation displaced CAII from the plasma membrane, as monitored by immunofluorescence. Finally, mutation of a PKC site adjacent to the SLC26A6 CAB site rendered the transporter unresponsive to PKC. PKC therefore reduces CAII/SLC26A6 interaction, reducing bicarbonate transport rate. Taken together, our data support a mechanism for acute regulation of membrane transport: metabolon disruption.

Carbonic anhydrase inhibition prevents and reverts cardiomyocyte hypertrophy

Hypertrophic cardiomyocyte growth contributes substantially to the progression of heart failure. Activation of the plasma membrane Na+–H+ exchanger (NHE1) and Cl−–HCO3− exchanger (AE3) has emerged as a central point in the hypertrophic cascade. Both NHE1 and AE3 bind carbonic anhydrase (CA), which activates their transport flux, by providing H+ and HCO3−, their respective transport substrates. We examined the contribution of CA activity to the hypertrophic response of cultured neonatal and adult rodent cardiomyocytes. Phenylephrine (PE) increased cell size by 37 ± 2% and increased expression of the hypertrophic marker, atrial natriuretic factor mRNA, twofold in cultured neonatal rat cardiomyocytes. Cell size was also increased in adult cardiomyocytes subjected to angiotensin II or PE treatment. These effects were associated with increased expression of cytosolic CAII protein and the membrane‐anchored isoform, CAIV. The membrane‐permeant CA inhibitor, 6‐ethoxyzolamide (ETZ), both prevented and reversed PE‐induced hypertrophy in a concentration‐dependent manner in neonate cardiomyocytes (IC50= 18 μm). ETZ and the related CA inhibitor methazolamide prevented hypertrophy in adult cardiomyocytes. In addition, ETZ inhibited transport activity of NHE1 and the AE isoform, AE3, with respective EC50 values of 1.2 ± 0.3 μm and 2.7 ± 0.3 μm. PE significantly increased neonatal cardiomyocyte Ca2+ transient frequency from 0.33 ± 0.4 Hz to 0.77 ± 0.04 Hz following 24 h treatment; these Ca2+‐handling abnormalities were completely prevented by ETZ (0.28 ± 0.07 Hz). Our study demonstrates a novel role for CA in mediating the hypertrophic response of cardiac myocytes to PE and suggests that CA inhibition represents an effective therapeutic approach towards mitigation of the hypertrophic phenotype.

Blindness caused by deficiency in AE3 chloride/bicarbonate exchanger.

Background Vision is initiated by phototransduction in the outer retina by photoreceptors, whose high metabolic rate generates large CO2 loads. Inner retina cells then process the visual signal and CO2. The anion exchanger 3 gene (AE3/Slc4a3) encodes full-length AE3 (AE3fl) and cardiac AE3 (AE3c) isoforms, catalyzing plasma membrane Cl−/HCO3 − exchange in Müller (AE3fl) and horizontal (AE3c) cells. AE3 thus maintains acid-balance by removing photoreceptor-generated CO2 waste. Methodology/Principal Findings We report that Slc4a3−/− null mice have inner retina defects (electroretinogram b-wave reduction, optic nerve and retinal vessel anomalies). These pathologic features are common to most human vitreoretinal degenerations. Immunobloting analysis revealed that Na+/HCO3 − co-transporter (NBC1), and carbonic anhydrase II and CAXIV, protein expression were elevated in Slc4a3 −/− mouse retinas, suggesting compensation for loss of AE3. TUNEL staining showed increased numbers of apoptotic nuclei from 4–6 months of age, in Slc4a3 −/− mice, indicating late onset photoreceptor death. Conclusions/Significance Identification of Slc4a3 as underlying a previously unrecognized cause of blindness suggests this gene as a new candidate for a subset of hereditary vitreoretinal retinal degeneration.

Bicarbonate homeostasis in excitable tissues: role of AE3 Cl−/HCO3− exchanger and carbonic anhydrase XIV interaction

Bicarbonate transport and metabolism are key elements of normal cellular function. Two alternate transcripts of anion exchanger 3 (AE3), full-length (AE3fl) and cardiac (AE3c), are expressed in central nervous system (CNS), where AE3 catalyzes electroneutral Cl(-)/HCO(3)(-) exchange across the plasma membrane of neuronal and glial cells of CNS. Anion exchanger isoforms, AE3fl and AE3c, associate with the carbonic anhydrases (CA) CAII and CAIV, forming a HCO(3)(-) transport metabolon, to maximize HCO(3)(-) flux across the plasma membrane. CAXIV, with catalytic domain anchored to the extracellular surface, is also expressed in CNS. Here physical association of AE3 and CAXIV was examined by coimmunoprecipitation experiments, using mouse brain and retinal lysates. CAXIV immunoprecipitated with anti-AE3 antibody, and both AE3 isoforms were immunoprecipitated using anti-CAXIV antibody, indicating CAXIV and AE3 interaction in the CNS. Confocal images revealed colocalization of CAXIV and AE3 in Müller and horizontal cells, in the mouse retina. Cl(-)/HCO(3)(-) exchange activity of AE3fl was investigated in transiently transfected human embryonic kidney 293 cells, using intracellular fluorescence measurements of BCECF, to monitor intracellular pH. CAXIV increased the rate of AE3fl-mediated HCO(3)(-) transport by up to 120%, which was suppressed by the CA inhibitor acetazolamide. Association of AE3 and CAXIV may represent a mechanism to enhance disposal of waste CO(2) and to balance pH in excitable tissues.

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.

Influence of Na+-Independent Cl−-HCO3− Exchange on the Slow Force Response to Myocardial Stretch

Abstract— Previous work demonstrated that the slow force response (SFR) to stretch is due to the increase in calcium transients (Ca2+T) produced by an autocrine-paracrine mechanism of locally produced angiotensin II/endothelin activating Na+-H+ exchange. Although a rise in pHi is presumed to follow stretch, it was observed only in the absence of extracellular bicarbonate, suggesting pHi compensation through the Na+-independent Cl−-HCO3− exchange (AE) mechanism. Because available AE inhibitors do not distinguish between different bicarbonate-dependent mechanisms or even between AE isoforms, we developed a functional inhibitory antibody against both the AE3c and AE3fl isoforms (anti-AE3Loop III) that was used to explore if pHi would rise in stretched cat papillary muscles superfused with bicarbonate after AE3 inhibition. In addition, the influence of this potential increase in pHi on the SFR was analyzed. In this study, we present evidence that cancellation of AE3 isoforms activity (either by superfusion with bicarbonate-free buffer or with anti-AE3Loop III) results in pHi increase after stretch and the magnitude of the SFR was larger than when AE was operative, despite of similar increases in [Na+]i and Ca2+T under both conditions. Inhibition of reverse mode Na+-Ca2+ exchange reduced the SFR to the half when the AE was inactive and totally suppressed it when AE3 was active. The difference in the SFR magnitude and response to inhibition of reverse mode Na+-Ca2+ exchange can be ascribed to a pHi-induced increase in myofilament Ca2+ responsiveness.

Cl − /HCO 3− Exchanger slc26a6 : A pH Regulator Shapes the Cardiac Action Potential

See Article by Sirish et al Regulation of intracellular pH (pHi) is often considered a housekeeping function, contributing little to cardiac contractile activity. With the study published by Sirish et al1 in this issue of Circulation: Arrhythmia and Electrophysiology , a Cl−/HCO3− exchanger is revealed to have a more central role in the heart. pHiis an important modulator of cardiac excitation and contraction2 and can adversely contribute to electric arrhythmia.3 Correspondingly, cardiac myocytes express a complex apparatus to regulate pHi. Cardiac muscle cytosolic pH (≈7.2) is maintained by sarcolemmal ion transport proteins that move H+, OH−, or HCO3− ions across the membrane.4 Along with the acid extruders, Na+/H+ exchanger 1 and Na+/HCO3− cotransporter (NBC, electrogenic NBCe1/e2 and electroneutral NBCn1) myocytes possess Cl−/HCO3− exchangers (SLC4 family members AE1, AE2, and AE3) and Cl−/OH− exchanger (with no molecular identity) alkali extruders.4 SLC26 gene family members were identified as (mouse slc26a65 and its human orthologue SLC26A6,6 and slc26a37) responsible for Cl−/HCO3− and Cl−/OH− exchange at plasma membrane of heart ventricles.7 The work of Sirish et al1 in this issue revealed that ablation of slc26a6 , encoding a plasma membrane Cl−/HCO3− exchange protein, results in cardiac action potential (AP) shortening, cardiomyocyte Ca2+ transient and sarcoplasmic reticulum …