RESUMEN
Slc4a genes encode various types of transporters, including Na+-HCO3- cotransporters, Cl-/HCO3- exchangers, or Na+-driven Cl-/HCO3- exchangers. Previous research has revealed that Slc4a9 (Ae4) functions as a Cl-/HCO3- exchanger, which can be driven by either Na+ or K+, prompting investigation into whether other Slc4a members facilitate cation-dependent anion transport. In the present study, we show that either Na+ or K+ drive Cl-/HCO3- exchanger activity in cells overexpressing Slc4a8 or Slc4a10. Further characterization of cation-driven Cl-/HCO3- exchange demonstrated that Slc4a8 and Slc4a10 also mediate Cl- and HCO3--dependent K+ transport. Full-atom molecular dynamics simulation on the recently solved structure of Slc4a8 supports the coordination of K+ at the Na+ binding site in S1. Sequence analysis shows that the critical residues coordinating monovalent cations are conserved among mouse Slc4a8 and Slc4a10 proteins. Together, our results suggest that Slc4a8 and Slc4a10 might transport K+ in the same direction as HCO3- ions in a similar fashion to that described for Na+ transport in the rat Slc4a8 structure.
Asunto(s)
Potasio , Simportadores de Sodio-Bicarbonato , Animales , Ratones , Bicarbonatos/metabolismo , Sitios de Unión , Antiportadores de Cloruro-Bicarbonato/metabolismo , Antiportadores de Cloruro-Bicarbonato/genética , Cloruros/metabolismo , Transporte Iónico , Simulación de Dinámica Molecular , Potasio/metabolismo , Sodio/metabolismo , Simportadores de Sodio-Bicarbonato/metabolismo , Simportadores de Sodio-Bicarbonato/genéticaRESUMEN
Two alkalinizing mechanisms coexist in cardiac myocytes to maintain intracellular pH: sodium/bicarbonate cotransporter (electroneutral isoform NBCn1 and electrogenic isoform NBCe1) and sodium/proton exchanger (NHE1). Dysfunction of these transporters has previously been reported to be responsible for the development of cardiovascular diseases. The aim of this study was to evaluate the contribution of the downregulation of the NBCe1 to the development of cardiac hypertrophy. To specifically reduce NBCe1 expression, we cloned shRNA into a cardiotropic adeno-associated vector (AAV9-shNBCe1). After 28 days of being injected with AAV9-shNBCe1, the expression and the activity of NBCe1 in the rat heart were reduced. Strikingly, downregulation of NBCe1 causes significant hypertrophic heart growth, lengthening of the action potential in isolated myocytes, an increase in the duration of the QT interval and an increase in the frequency of Ca2+ waves without any significant changes in Ca2+ transients. An increased compensatory expression of NBCn1 and NHE1 was also observed. We conclude that reduction of NBCe1 is sufficient to induce cardiac hypertrophy and modify the electrical features of the rat heart.
Asunto(s)
Bicarbonatos , Simportadores de Sodio-Bicarbonato , Ratas , Animales , Simportadores de Sodio-Bicarbonato/genética , Simportadores de Sodio-Bicarbonato/metabolismo , Bicarbonatos/metabolismo , Cardiomegalia/genética , Cardiomegalia/metabolismo , Sodio/metabolismo , Isoformas de Proteínas/metabolismo , Concentración de Iones de HidrógenoRESUMEN
Myocardial infarction (MI) is one of the leading causes of death worldwide. Prognosis and mortality rate are directly related to infarct size and post-infarction pathological heart remodeling, which can lead to heart failure. Hypoxic MI-affected areas increase the expression of hypoxia-inducible factor (HIF-1), inducing infarct size reduction and improving cardiac function. Hypoxia translocates HIF-1 to the nucleus, activating carbonic anhydrase IX (CAIX) transcription. CAIX regulates myocardial intracellular pH, critical for heart performance. Our objective was to investigate CAIX participation and relation with sodium bicarbonate transporters 1 (NBC1) and HIF-1 in cardiac remodeling after MI. We analyzed this pathway in an "in vivo" rat coronary artery ligation model and isolated cardiomyocytes maintained under hypoxia. Immunohistochemical studies revealed an increase in HIF-1 levels after 2 h of infarction. Similar results were observed in 2-h infarcted cardiac tissue (immunoblotting) and in hypoxic cardiomyocytes with a nuclear distribution (confocal microscopy). Immunohistochemical studies showed an increase CAIX in the infarcted area at 2 h, mainly distributed throughout the cell and localized in the plasma membrane at 24 h. Similar results were observed in 2 h in infarcted cardiac tissue (immunoblotting) and in hypoxic cardiomyocytes (confocal microscopy). NBC1 expression increased in cardiac tissue after 2 h of infarction (immunoblotting). CAIX and NBC1 interaction increases in cardiac tissue subjected to MI for 2h when CAIX is present (immunoprecipitation). These results suggest that CAIX interacts with NBC1 in our infarct model as a mechanism to prevent acidic damage in hypoxic tissue, making it a promising therapeutic target.
Asunto(s)
Anhidrasa Carbónica IX/metabolismo , Factor 1 Inducible por Hipoxia/metabolismo , Hipoxia/enzimología , Infarto del Miocardio/enzimología , Simportadores de Sodio-Bicarbonato/metabolismo , Animales , Masculino , Cultivo Primario de Células , Ratas Wistar , Remodelación VentricularRESUMEN
Physical training stimulates the development of physiologic cardiac hypertrophy (CH), being a key event in this process the inhibition of the Na+/H+ exchanger. However, the role of the sodium bicarbonate cotransporter (NBC) has not been explored yet under this circumstance. C57/Bl6 mice were allowed to voluntary exercise (wheel running) for five weeks. Cardiac mass was evaluated by echocardiography and histomorphometry detecting that training promoted the development of physiological CH (heart weight/tibia length ratio, mg/mm: 6.54 ± 0.20 vs 8.81 ± 0.24; interstitial collagen content, %: 3.14 ± 0.63 vs. 1.57 ± 0.27; and cross-sectional area of cardiomyocytes, µm2: 200.6 ± 8.92 vs. 281.9 ± 24.05; sedentary (Sed) and exercised (Ex) mice, respectively). The activity of the electrogenic isoform of the cardiac NBC (NBCe1) was estimated by recording intracellular pH under high potassium concentration and by measuring action potential duration (APD). NBCe1 activity was significantly increased in isolated cardiomyocytes of trained mice. Additionally, the APD was shorter and the alkalization due to high extracellular potassium-induced depolarization was greater in this group, indicating that the NBCe1 was hyperactive. These results are online with the observed myocardial up-regulation of the NBCe1 (Western Blot, %: 100 ± 13.86 vs. 202 ± 29.98; Sed vs. Ex, n = 6 each group). In addition, we detected a reduction in H2O2 production in the myocardium of trained mice. These results support that voluntary training induces the development of physiologic CH with up-regulation of the cardiac NBCe1 in mice. Furthermore, the improvement in the antioxidant capacity contributes to the beneficial cardiovascular consequences of physical training.
Asunto(s)
Miocardio/metabolismo , Condicionamiento Físico Animal , Simportadores de Sodio-Bicarbonato/metabolismo , Animales , Cardiomegalia Inducida por el Ejercicio/fisiología , Peróxido de Hidrógeno/farmacología , Masculino , Ratones Endogámicos C57BL , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Isoformas de Proteínas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Regulación hacia ArribaRESUMEN
The soluble adenylyl cyclase (sAC) was identified in the heart as another source of cyclic AMP (cAMP). However, its cardiac physiological function is unknown. On the other hand, the cardiac Na+/HCO3- cotransporter (NBC) promotes the cellular co-influx of HCO3- and Na+. Since sAC activity is regulated by HCO3-, our purpose was to investigate the potential functional relationship between NBC and sAC in the cardiomyocyte. Rat ventricular myocytes were loaded with Fura-2, Fluo-3, or BCECF to measure Ca2+ transient (Ca2+i) by epifluorescence, Ca2+ sparks frequency (CaSF) by confocal microscopy, or intracellular pH (pHi) by epifluorescence, respectively. Sarcomere or cell shortening was measured with a video camera as an index of contractility. The NBC blocker S0859 (10 µM), the selective inhibitor of sAC KH7 (1 µM), and the PKA inhibitor H89 (0.1 µM) induced a negative inotropic effect which was associated with a decrease in Ca2+i. Since PKA increases Ca2+ release through sarcoplasmic reticulum RyR channels, CaSF was measured as an index of RyR open probability. The generation of CaSF was prevented by KH7. Finally, we investigated the potential role of sAC activation on NBC activity. NBC-mediated recovery from acidosis was faster in the presence of KH7 or H89, suggesting that the pathway sAC-PKA is negatively regulating NBC function, consistent with a negative feedback modulation of the HCO3- influx that activates sAC. In summary, the results demonstrated that the complex NBC-sAC-PKA plays a relevant role in Ca2+ handling and basal cardiac contractility.
Asunto(s)
Adenilil Ciclasas/metabolismo , Contracción Miocárdica , Miocitos Cardíacos/metabolismo , Simportadores de Sodio-Bicarbonato/metabolismo , Inhibidores de Adenilato Ciclasa/farmacología , Animales , Benzamidas/farmacología , Señalización del Calcio , Células Cultivadas , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Ventrículos Cardíacos/citología , Isoquinolinas/farmacología , Masculino , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/fisiología , Ratas , Ratas Wistar , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Simportadores de Sodio-Bicarbonato/antagonistas & inhibidores , Sulfonamidas/farmacologíaRESUMEN
The electrogenic sodium bicarbonate co-transporter isoform 1 (NBCe1) plays an important role in ischemia-reperfusion injury. The cardioprotective action of an antibody directed to the extracellular loop 3 (a-L3) of NBCe1 was previously demonstrated by us. However, the role of a-L3 on mitochondrial post-ischemic alterations has not yet been determined. In this study, we aimed to elucidate the effects of a-L3 on post-ischemic mitochondrial state and dynamics analysing the involved mechanisms. Isolated rat hearts were assigned to the following groups: 1) Non-ischemic control (NIC): 110â¯min of perfusion; 2) Ischemic control (IC): 30â¯min of global ischemia and 60â¯min of reperfusion (R); 3) a-L3: a-L3 was administered during the initial 10â¯min of R; 4) SBâ¯+â¯a-L3: SB202190 (p38MAPK inhibitor) plus a-L3. Infarct size (IS) was measured by TTC staining. Developed pressure (LVDP), maximal velocities of rise and decay of LVP (+dP/dt max, -dP/dt max) and end-diastolic pressure (LVEDP) of the left ventricle were used to assess systolic and diastolic function. Mitochondrial Ca2+ response (CaR), Ca2+ retention capacity (CRC), membrane potential (ΔΨm) and MnSOD levels were measured. The expression of P-p38MAPK, calcineurin, P-HSP27, P-Drp1, Drp1, and OPA1 were determined. a-L3 decreased IS, improved post-ischemic recovery of myocardial function, increased P-p38MAPK, P-HSP27, P-Drp1, cytosolic Drp1, and OPA1 expression and decreased calcineurin. These effects were abolished by p38MAPK inhibition with SB. These data show that NBCe1 inhibition by a-L3 limits the cell death, improves myocardial post-ischemic contractility and mitochondrial state and dynamic through calcium decrease/calcineurin inhibition-mediated p38MAPK activation and p38MAPK/HSP27-dependent pathways. Thus, we demonstrated that a-L3 is a potential therapeutic strategy in post-ischemic alterations.
Asunto(s)
Calcineurina/metabolismo , Proteínas de Choque Térmico HSP27/metabolismo , Daño por Reperfusión Miocárdica/metabolismo , Simportadores de Sodio-Bicarbonato/antagonistas & inhibidores , Simportadores de Sodio-Bicarbonato/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Anticuerpos/farmacología , Preparación de Corazón Aislado/métodos , Masculino , Daño por Reperfusión Miocárdica/prevención & control , Ratas , Ratas Wistar , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
Pseudohypoaldosteronism type II (PHAII) is a genetic disease characterized by association of hyperkalemia, hyperchloremic metabolic acidosis, hypertension, low renin, and high sensitivity to thiazide diuretics. It is caused by mutations in the WNK1, WNK4, KLHL3 or CUL3 gene. There is strong evidence that excessive sodium chloride reabsorption by the sodium chloride cotransporter NCC in the distal convoluted tubule is involved. WNK4 is expressed not only in distal convoluted tubule cells but also in ß-intercalated cells of the cortical collecting duct. These latter cells exchange intracellular bicarbonate for external chloride through pendrin, and therefore, account for renal base excretion. However, these cells can also mediate thiazide-sensitive sodium chloride absorption when the pendrin-dependent apical chloride influx is coupled to apical sodium influx by the sodium-driven chloride/bicarbonate exchanger. Here we determine whether this system is involved in the pathogenesis of PHAII. Renal pendrin activity was markedly increased in a mouse model carrying a WNK4 missense mutation (Q562E) previously identified in patients with PHAII. The upregulation of pendrin led to an increase in thiazide-sensitive sodium chloride absorption by the cortical collecting duct, and it caused metabolic acidosis. The function of apical potassium channels was altered in this model, and hyperkalemia was fully corrected by pendrin genetic ablation. Thus, we demonstrate an important contribution of pendrin in renal regulation of sodium chloride, potassium and acid-base homeostasis and in the pathophysiology of PHAII. Furthermore, we identify renal distal bicarbonate secretion as a novel mechanism of renal tubular acidosis.
Asunto(s)
Acidosis Tubular Renal/fisiopatología , Túbulos Renales Colectores/fisiopatología , Proteínas Serina-Treonina Quinasas/genética , Seudohipoaldosteronismo/complicaciones , Transportadores de Sulfato/metabolismo , Acidosis Tubular Renal/sangre , Acidosis Tubular Renal/etiología , Animales , Modelos Animales de Enfermedad , Técnicas de Inactivación de Genes , Humanos , Túbulos Renales Colectores/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación Missense , Potasio/sangre , Potasio/metabolismo , Seudohipoaldosteronismo/genética , Seudohipoaldosteronismo/fisiopatología , Eliminación Renal , Cloruro de Sodio/metabolismo , Simportadores de Sodio-Bicarbonato/metabolismo , Transportadores de Sulfato/genética , Regulación hacia ArribaRESUMEN
To fertilize an egg, sperm must reside in the female reproductive tract to undergo several maturational changes that are collectively referred to as capacitation. From a molecular point of view, the HCO3--dependent activation of the atypical soluble adenylyl cyclase (ADCY10) is one of the first events that occurs during capacitation and leads to the subsequent cAMP-dependent activation of protein kinase A (PKA). Capacitation is also accompanied by hyperpolarization of the sperm plasma membrane. We previously reported that PKA activation is necessary for CFTR (cystic fibrosis transmembrane conductance regulator channel) activity and for the modulation of membrane potential (Em). However, the main HCO3- transporters involved in the initial transport and the PKA-dependent Em changes are not well known nor characterized. Here, we analyzed how the activity of CFTR regulates Em during capacitation and examined its relationship with an electrogenic Na+/HCO3- cotransporter (NBC) and epithelial Na+ channels (ENaCs). We observed that inhibition of both CFTR and NBC decreased HCO3- influx, resulting in lower PKA activity, and that events downstream of the cAMP activation of PKA are essential for the regulation of Em. Addition of a permeable cAMP analog partially rescued the inhibitory effects caused by these inhibitors. HCO3- also produced a rapid membrane hyperpolarization mediated by ENaC channels, which contribute to the regulation of Em during capacitation. Altogether, we demonstrate for the first time, that NBC cotransporters and ENaC channels are essential in the CFTR-dependent activation of the cAMP/PKA signaling pathway and Em regulation during human sperm capacitation.
Asunto(s)
Bicarbonatos/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Canales Epiteliales de Sodio/metabolismo , Potenciales de la Membrana , Capacitación Espermática , Espermatozoides/fisiología , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Humanos , Masculino , Fosforilación , Transducción de Señal , Sodio/metabolismo , Simportadores de Sodio-Bicarbonato/metabolismoRESUMEN
NBC Na+/HCO3- cotransporter (NBCn1) and NHE1 Na+/H+ exchanger have been associated with cardiac disorders and recently located in coronary endothelial cells (CEC) and cardiomyocytes mitochondria, respectively. Mitochondrial NHE1 blockade delays permeability transition pore (MPTP) opening and reduces superoxide levels, two critical events exacerbated in cells of diseased hearts. Conversely, activation of NBCn1 prevented apoptosis in CEC subjected to ischemic stress. We characterized the role of the NHE1 and NBCn1 transporters in heart mitochondria from hypertrophic (SHR) and control (Wistar) rats. Expression of NHE1 was analyzed in left ventricular mitochondrial lysates (LVML), by immunoblots. NHE1 expression increased by ~40% in SHR compared to control (P < 0.05, n = 4). To examine NHE1-mediated Na+/H+ exchange activity in cardiac hypertrophy, mitochondria were loaded with BCECF-AM dye and the maximal rate of pHm change measured after the addition of 50 mM NaCl. SHR mitochondria had greater changes in pHm compared to Wistar, 0.10 ± 0.01 vs. 0.06 ± 0.01, respectively (P < 0.05, n = 5). In addition, mitochondrial suspensions from SHR and control myocardium were exposed to 200 µM CaCl2 to induce MPTP opening (light-scattering decrease, LSD) and swelling. Surprisingly, SHR rats showed smaller LSD and a reduction in mitochondrial swelling, 67 ± 10% (n = 15), compared to control, 100 ± 8% (n = 13). NBC inhibition with S0859 (1 µM) significantly increased swelling in both control 139 ± 10% (n = 8) and SHR 115 ± 10% (n = 4). Finally, NBCn1 Na+/HCO3- cotransporter increased by twofold its expression in SHR LVML, compared to normal (P < 0.05, n = 5). We conclude that increased NBCn1 activity may play a compensatory role in hypertrophic hearts, protecting mitochondria from Ca2+-induced MPTP opening and swelling.
Asunto(s)
Cardiomegalia/metabolismo , Mitocondrias/patología , Dilatación Mitocondrial , Simportadores de Sodio-Bicarbonato/metabolismo , Animales , Cardiomegalia/patología , Modelos Animales de Enfermedad , Células HEK293 , Humanos , Immunoblotting , Inmunohistoquímica , Microscopía Confocal , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Poro de Transición de la Permeabilidad Mitocondrial , Miocitos Cardíacos , Ratas , Ratas Endogámicas SHR , Ratas WistarRESUMEN
Some cardiac non-genomic effects of aldosterone (Ald) are reported to be mediated through activation of the classic mineralocorticoid receptor (MR). However, in the last years, it was proposed that activation of the novel G protein-coupled receptor GPR30 mediates certain non-genomic effects of Ald. The aim of this study was to elucidate if the sodium/bicarbonate cotransporter (NBC) is stimulated by Ald and if the activation of GPR30 mediates this effect. NBC activity was evaluated in rat cardiomyocytes perfused with HCO3(-)/CO2 solution in the continuous presence of HOE642 (sodium/hydrogen exchanger blocker) during recovery from acidosis using intracellular fluorescence measurements. Ald enhanced NBC activity (% of ΔJHCO3(-); control: 100±5.82%, n=7 vs Ald: 151.88±11.02%, n=5; P<0.05), which was prevented by G15 (GPR30 blocker, 90.53±7.81%, n=7). Further evidence for the involvement of GPR30 was provided by G1 (GPR30 agonist), which stimulated NBC (185.13±18.28%, n=6; P<0.05) and this effect was abrogated by G15 (124.19±10.96%, n=5). Ald- and G1-induced NBC stimulation was abolished by the reactive oxygen species (ROS) scavenger MPG and by the NADPH oxidase inhibitor apocynin. In addition, G15 prevented Ald- and G1-induced ROS production. Pre-incubation of myocytes with wortmannin (PI3K-AKT pathway blocker) prevented Ald- or G1-induced NBC stimulation. In summary, Ald stimulates NBC by GPR30 activation, ROS production and AKT stimulation.
Asunto(s)
Aldosterona/farmacología , Miocardio/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Simportadores de Sodio-Bicarbonato/metabolismo , Animales , Factor de Crecimiento Epidérmico/farmacología , Receptores ErbB/metabolismo , Concentración de Iones de Hidrógeno , Espacio Intracelular/metabolismo , Masculino , Modelos Biológicos , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismo , Receptores de Mineralocorticoides/metabolismo , Activación Transcripcional/efectos de los fármacosRESUMEN
BACKGROUND: Bicarbonate transport has crucial roles in regulating intracellular pH (pHi) in a variety of cells. The purpose of this study was to evaluate its participation in the regulation of pHi in resting and stimulated human neutrophils. METHODS: Freshly isolated human neutrophils acidified by an ammonium prepulse were used in this study. RESULTS: We demonstrated that resting neutrophils have a bicarbonate transport mechanism that prevents acidification when the Na(+)/H(+) exchanger is blocked by EIPA. Neutrophils acidified by an ammonium prepulse showed an EIPA-resistant recovery of pHi that was inhibited by the blocker of the anionic transporters SITS or the Na(+)/HCO3(-) cotransporter (NBC) selective inhibitor S0859, and abolished when sodium was removed from the extracellular medium. In western blot and RT-PCR analysis the expression of NBCe2 but not NBCe1 or NBCn1 was detected in neutrophils Acidified neutrophils increased the EIPA-insensitive pHi recovery rate when its activity was stimulated with fMLF/ cytochalasin B. This increase in the removal of acid equivalents was insensitive to the blockade of the NADPH oxidase with DPI. CONCLUSION: It is concluded that neutrophils have an NBC that regulates basal pHi and is modulated by chemotactic agents.
Asunto(s)
Neutrófilos/metabolismo , Simportadores de Sodio-Bicarbonato/metabolismo , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-disulfónico/farmacología , Amilorida/análogos & derivados , Amilorida/farmacología , Cloruro de Amonio/farmacología , Benzamidas/farmacología , Bicarbonatos/farmacología , Citocalasina B/farmacología , Células HEK293 , Humanos , Concentración de Iones de Hidrógeno , Transporte Iónico/efectos de los fármacos , N-Formilmetionina Leucil-Fenilalanina/farmacología , NADPH Oxidasas/antagonistas & inhibidores , NADPH Oxidasas/metabolismo , Neutrófilos/efectos de los fármacos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/metabolismo , Simportadores de Sodio-Bicarbonato/genética , Intercambiadores de Sodio-Hidrógeno/metabolismo , Sulfonamidas/farmacologíaRESUMEN
BACKGROUND: Although the participation of the electrogenic sodium/bicarbonate cotransporter (NBCe1) in the recovery from an intracellular acid load is recognized, its role in ischemia-reperfusion is still unclear. METHODS AND RESULTS: Our objective was to assess the role of NBCe1 in reperfusion injury. We use selective functional antibodies against extracellular loop 3 (a-L3) and loop 4 (a-L4) of NBCe1. a-L3 inhibits and a-L4 stimulates NBCe1 activity. Isolated rat hearts were submitted to 40 min of coronary occlusion and 1 h of reperfusion. a-L3, a-L4 or S0859--selective Na(+)-HCO3(-) co-transport inhibitor--were administered during the initial 10 min of reperfusion. The infarct size (IS) was measured by triphenyltetrazolium chloride staining technique. Postischemic systolic and diastolic functions were also assessed. a-L3 and S0859 treatments decreased significantly (P < .05) the IS (16 ± 3% for a-L3 vs. 32 ± 5% in hearts treated with control nonimmune serum and 19 ± 3% for S0859 vs. 39 ± 2% in untreated hearts). Myocardial function during reperfusion improved after a-L3 treatment, but it was not modified by S0859. The infusion of a-L4 did not modify neither the IS nor myocardial function. CONCLUSIONS: The NBCe1 hyperactivity during reperfusion leads to Na(+) and Ca(2+) loading, conducing to Ca(2+) overload and myocardial damage. Consistently, we have shown herein that the selective NBCe1 blockade with a-L3 exerted cardioprotection. This beneficial action strongly suggests that NBCe1 could be a potential target for the treatment of coronary disease.
Asunto(s)
Daño por Reperfusión Miocárdica/etiología , Daño por Reperfusión Miocárdica/fisiopatología , Miocardio/metabolismo , Simportadores de Sodio-Bicarbonato/metabolismo , Animales , Benzamidas/farmacología , Cardiotónicos/farmacología , Contracción Miocárdica , Infarto del Miocardio/tratamiento farmacológico , Infarto del Miocardio/metabolismo , Infarto del Miocardio/patología , Daño por Reperfusión Miocárdica/prevención & control , Miocardio/patología , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Ratas , Ratas Wistar , Simportadores de Sodio-Bicarbonato/agonistas , Simportadores de Sodio-Bicarbonato/antagonistas & inhibidores , Sulfonamidas/farmacología , Función Ventricular IzquierdaRESUMEN
AIMS: Electroneutral (NBCn1) and electrogenic (NBCe1) isoforms of the Na(+)-HCO3(-) cotransporter (NBC) coexist in the heart. We studied the expression and function of these isoforms in hearts of Wistar and spontaneously hypertensive rats (SHR), elucidating the direct implication of the renin-angiotensin system in the NBC regulation. METHODS AND RESULTS: We used myocytes from Wistar, SHR, losartan-treated SHR (Los-SHR), and Angiotensin II (Ang II)-induced cardiac hypertrophy. We found an overexpression of NBCe1 and NBCn1 proteins in SHR that was prevented in Los-SHR. Hyperkalaemic-induced pHi alkalization was used to study selective activation of NBCe1. Despite the increase in NBCe1 expression, its activity was lower in SHR than in Wistar or Los-SHR. Similar results were found in Ang II-induced hypertrophy. A specific inhibitory antibody against NBCe1 allowed the discrimination between NBCe1 and NBCn1 activity. Whereas in SHR most of the pHi recovery was due to NBCn1 stimulation, in Wistar and Los-SHR the activity of both isoforms was equitable, suggesting that the deteriorated cardiac NBCe1 function observed in SHR is compensated by an enhanced activity of NBCn1. Using the biotin method, we observed greater level of internalized NBCe1 protein in SHR than in the non-hypertophic groups, while with immunofluorescence we localized the protein in endosomes near the nucleus only in SHR. CONCLUSIONS: We conclude that Ang II is responsible for the impairment of the NBCe1 in hypertrophied hearts. This is due to retained transporter protein units in early endosomes. Moreover, NBCn1 activity seems to be increased in the hypertrophic myocardium of SHR, compensating impaired function of NBCe1.
Asunto(s)
Bicarbonatos/metabolismo , Cardiomegalia/metabolismo , Hipertensión/metabolismo , Miocitos Cardíacos/metabolismo , Sistema Renina-Angiotensina , Sarcolema/metabolismo , Simportadores de Sodio-Bicarbonato/metabolismo , Compuestos de Amonio/metabolismo , Angiotensina II , Bloqueadores del Receptor Tipo 1 de Angiotensina II/farmacología , Animales , Antihipertensivos/farmacología , Cardiomegalia/inducido químicamente , Cardiomegalia/patología , Modelos Animales de Enfermedad , Regulación hacia Abajo , Endosomas/metabolismo , Concentración de Iones de Hidrógeno , Hiperpotasemia/metabolismo , Hipertensión/tratamiento farmacológico , Hipertensión/patología , Losartán/farmacología , Masculino , Miocitos Cardíacos/patología , Potasio/metabolismo , Transporte de Proteínas , Ratas , Ratas Endogámicas SHR , Ratas Wistar , Sistema Renina-Angiotensina/efectos de los fármacos , Sarcolema/patología , Factores de TiempoRESUMEN
The NBCn1 Na(+)/HCO3(-) cotransporter catalyzes the electroneutral movement of 1 Na(+):1 HCO3(-) into kidney cells. We characterized the intracellular pH (pHi) regulation in human embryonic kidney cells (HEK) subjected to NH4Cl prepulse acid loading, and we examined the NBCn1 expression and function in HEK cells subjected to 24-h elevated Pco2 (10-15%). After acid loading, in the presence of HCO3(-), â¼50% of the pHi recovery phase was blocked by the Na(+)/H(+) exchanger inhibitors EIPA (10-50 µM) and amiloride (1 mM) and was fully cancelled by 30 µM EIPA under nominally HCO3(-)-free conditions. In addition, in the presence of HCO3(-), pHi recovery after acid loading was completely blocked when Na(+) was omitted in the buffer. pHi recovery after acidification in HEK cells was repeated in the presence of the NBC inhibitor S0859, and the pHi recovery was inhibited by S0859 in a dose-dependent manner (Ki = 30 µM, full inhibition at 60 µM), which confirmed NBC Na(+)/HCO3(-) cotransporter activation. NBCn1 expression increased threefold after 24-h exposure of cultured HEK cells to 10% CO2 and sevenfold after exposure to 15% CO2, examined by immunoblots. Finally, exposure of HEK cells to high CO2 significantly increased the HCO3(-)-dependent recovery of pHi after acid loading. We conclude that HEK cells expressed the NBCn1 Na(+)/HCO3(-) cotransporter as the only HCO3(-)-dependent mechanism responsible for cellular alkaline loading. NBCn1, which expresses in different kidney cell types, was upregulated by 24-h high-Pco2 exposure of HEK cells, and this upregulation was accompanied by increased NBCn1-mediated HCO3(-) transport.
Asunto(s)
Dióxido de Carbono/farmacología , Células HEK293/efectos de los fármacos , Células HEK293/metabolismo , Simportadores de Sodio-Bicarbonato/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Amilorida/análogos & derivados , Amilorida/farmacología , Bicarbonatos/metabolismo , Relación Dosis-Respuesta a Droga , Células HEK293/citología , Humanos , Concentración de Iones de Hidrógeno , Factores de TiempoRESUMEN
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.
Asunto(s)
Angiotensina II/fisiología , Corazón/fisiología , Simportadores de Sodio-Bicarbonato/metabolismo , Remodelación Ventricular/fisiología , Calcio/metabolismo , Cardiomegalia/metabolismo , Fenómenos Electrofisiológicos/fisiología , Cardiopatías/metabolismo , Humanos , Sodio/metabolismoRESUMEN
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.
Asunto(s)
Anhidrasas Carbónicas/metabolismo , Miocardio/metabolismo , Simportadores de Sodio-Bicarbonato/metabolismo , Animales , Bicarbonatos/metabolismo , Transporte Biológico Activo , Anhidrasa Carbónica IX , Línea Celular , Células HEK293 , Humanos , Concentración de Iones de Hidrógeno , Ratas , Simportadores de Sodio-Bicarbonato/químicaAsunto(s)
Proteínas Bacterianas/genética , Etanol/farmacología , Regulación Bacteriana de la Expresión Génica , Genes Reguladores , Proteoma/genética , Synechocystis/efectos de los fármacos , Adaptación Fisiológica , Secuencias de Aminoácidos , Biocombustibles , Proteínas Bacterianas/metabolismo , Etanol/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Anotación de Secuencia Molecular , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Unión Proteica , Fotosíntesis/efectos de los fármacos , Proteoma/metabolismo , Simportadores de Sodio-Bicarbonato/genética , Simportadores de Sodio-Bicarbonato/metabolismo , Synechocystis/genética , Synechocystis/crecimiento & desarrollo , Synechocystis/metabolismo , Transcripción GenéticaRESUMEN
Amelogenesis imperfecta is a group of genetic conditions that affect the structure and clinical appearance of tooth enamel. The types (hypoplastic, hypocalcified, and hypomature) are correlated with defects in different stages of the process of enamel synthesis. Autosomal dominant, recessive, and X-linked types have been previously described. These disorders are considered clinically and genetically heterogeneous in etiology, involving a variety of genes, such as AMELX, ENAM, DLX3, FAM83H, MMP-20, KLK4, and WDR72. The mutations identified within these causal genes explain less than half of all cases of amelogenesis imperfecta. Most of the candidate and causal genes currently identified encode proteins involved in enamel synthesis. We think it is necessary to refocus the search for candidate genes using biochemical processes. This review provides theoretical evidence that the human SLC4A4 gene (sodium bicarbonate cotransporter) may be a new candidate gene.
Asunto(s)
Amelogénesis Imperfecta/genética , Amelogénesis Imperfecta/metabolismo , Bicarbonatos/metabolismo , Esmalte Dental/metabolismo , Simportadores de Sodio-Bicarbonato , Ameloblastos/metabolismo , Amelogénesis/genética , Amelogénesis Imperfecta/clasificación , Amelogénesis Imperfecta/fisiopatología , Animales , Esmalte Dental/fisiopatología , Estudios de Asociación Genética , Heterogeneidad Genética , Humanos , Concentración de Iones de Hidrógeno , Transporte Iónico/genética , Ratones , Mutación/genética , Ratas , Simportadores de Sodio-Bicarbonato/genética , Simportadores de Sodio-Bicarbonato/metabolismo , Diente/metabolismo , Diente/fisiopatologíaRESUMEN
The Na(+)/HCO(3)(-) cotransporter (NBC) plays an important role in intracellular pH (pH(i)) 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 45 mM (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 pH(i) units after 14 min 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 100 nM Ang II (-0.008 ± 0.018*; n=5). This inhibitory effect was prevented when the myocytes were incubated with losartan (AT(1) 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 AT(1) receptors, exerts an inhibitory effect on eNBC activity in a p38 kinase-dependent manner.
Asunto(s)
Angiotensina II/farmacología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Simportadores de Sodio-Bicarbonato/antagonistas & inhibidores , Ácidos/metabolismo , Potenciales de Acción/efectos de los fármacos , Animales , Gatos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Ventrículos Cardíacos/citología , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/metabolismo , Modelos Biológicos , Miocitos Cardíacos/enzimología , Potasio/metabolismo , Simportadores de Sodio-Bicarbonato/metabolismo , Factores de Tiempo , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
The sarcolemmal Na+/HCO3- cotransporter (NBC) plays an important role in intracellular pH (pH(i)) regulation in the heart. In the present work we studied, in isolated cat ventricular myocytes, the role of Angiotensin II (Ang II) and reactive oxygen species (ROS) production as potential activators of the NBC. pH(i) was measured in single cells in a medium with HCO3- using the fluorescent pH indicator BCECF. The NH4+ pulse method was used to induce an intracellular acid load and the acid efflux (JH) in the presence of the Na+/H+ exchanger blocker HOE642 (10 microM) was calculated as indicator of NBC activity. The following JH data are presented at pH(i) of 6.8 (* and # indicate p<0.05 after ANOVA vs. control and Ang II, respectively). The basal JH (1.03+/-0.12 mM/min, n=11) was significantly increased in the presence of 100 nM Ang II (1.70+/-0.15 mM/min, n=8*). This effect of Ang II was abolished when we added to the extracellular solution 2 mM MPG (ROS scavenger; 0.80+/-0.08 mM/min, n=11#), 300 microM apocynin (NADPH oxidase blocker; 0.80+/-0.13 mM/min, n=6#), 500 microM 5-hydroxidecanoate (mitochondrial ATP dependent K+ channel, mK(ATP), blocker; 0.97+/-0.21 mM/min, n=9#), or the inhibitor of the MAP kinase ERK pathway U0126 (10 microM; 0.56+/-0.18 mM/min, n=6#). We also determined the phosphorylation of ERK during the first min of acidosis and we detected that Ang II significantly enhanced the ERK phosphorylation levels, an effect that was cancelled by scavenging ROS with MPG. In conclusion, we propose that Ang II enhances the production of ROS through the activation of the NADPH oxidase, which in turn triggers mK(ATP) opening and mitochondrial ROS production ("ROS-induced ROS-release mechanism"). Finally, these mitochondrial ROS stimulate the ERK pathway, leading to the activation of the NBC.