Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 40
Filtrar
Mais filtros










Intervalo de ano de publicação
1.
Front Pharmacol ; 14: 1223132, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37637427

RESUMO

The increase of intracellular Ca2+ concentration, produced principally by its influx through the L-type Ca2+ channels, is one of the major contributors to the ischemia-reperfusion injury. The inhibition of those channels in different experimental models was effective to ameliorate the post-ischemic damage. However, at a clinical level, the results were contradictory. Recent results of our group obtained in an ¨ex vivo¨ heart model demonstrated that a chemical derived from acetazolamide, the N-methylacetazolamide (NMA) protected the heart against ischemia-reperfusion injury, diminishing the infarct size and improving the post-ischemic recovery of myocardial function and mitochondrial dynamic. A significant inhibitory action on L-type Ca2+ channels was also detected after NMA treatment, suggesting this action as responsible for the beneficial effects on myocardium exerted by this compound. Although these results were promising, the effectiveness of NMA in the treatment of ischemic heart disease in humans as well as the advantages or disadvantages in comparison to the classic calcium antagonists needs to be investigated.

3.
Front Cardiovasc Med ; 9: 862118, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35548416

RESUMO

Cardiac cells depend on specific sarcolemmal ion transporters to assure the correct intracellular pH regulation. The sodium/bicarbonate cotransporter (NBC) is one of the major alkalinizing mechanisms. In the heart two different NBC isoforms have been described: the electroneutral NBCn1 (1Na+:1 HCO 3 - ) and the electrogenic NBCe1 (1Na+:2 HCO 3 - ). NBCe1 generates an anionic repolarizing current that modulates the action potential duration (APD). In addition to regulating the pH, the NBC is a source of sodium influx. It has been postulated that NBC could play a role in the development of hypertrophy. The aim of this research was to study the contribution of NBCe1 in heart electrophysiology and in the development of heart hypertrophy in an in vivo mouse model with overexpression of NBCe1. Heart NBCe1 overexpression was achieved by a recombinant cardiotropic adeno-associated virus (AAV9) and was evidenced by western-blot and qPCR. AAV9-mCherry was used as a transduction control. NBCe1 overexpression fails to increase heart growth. Patch clamp and electrocardiogram were performed. We observed a reduction on both, ventricular myocytes APD and electrocardiogram QT interval corrected by cardiac rate, emphasizing for the first time NBCe1 relevance for the electrical activity of the heart.

4.
Biochim Biophys Acta Gen Subj ; 1866(5): 130098, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35104623

RESUMO

Our objective was to examine the effects of N-methylacetazolamide (NMA), a non­carbonic anhydrase inhibitor, on ischemia-reperfusion injury. Isolated rat hearts were assigned to the following groups: 1) Non-ischemic control (NIC):110 min of perfusion and 2) Ischemic control (IC): 30 min of global ischemia and 60 min of reperfusion (R). Both groups were repeated in presence of NMA (5 µM), administered during the first 10 min of R. Infarct size (IS) was measured by TTC staining. Developed pressure (LVDP) and end-diastolic pressure (LVEDP) of the left ventricle were used to assess systolic and diastolic function, respectively. The content of P-Akt, P-PKCε, P-Drp1 and calcineurin Aß were measured. In cardiomyocytes the L-type Ca2+ current (ICaL) was recorded with the whole-cell configuration of patch-clamp technique. The addition of NMA to non-ischemic hearts decreased 15% the contractility. In ischemic hearts (IC group), NMA decreased IS (22 ± 2% vs 32 ± 2%, p < 0.05) and improved the post-ischemic recovery of myocardial function. At the end of R, LVDP was 54 ± 7% vs 18 ± 3% and LVEDP was 23 ± 8 vs. 55 ± 7 mmHg ¨p < 0.05¨. The level of P-Akt, P-PKCε and P-Drp1 increased and the expression of calcineurin Aß decreased in NMA treated hearts. Peak ICaL density recorded at 0 mV was smaller in myocytes treated with NMA than in non-treated cells (-1.91 ± 0.15 pA/pF vs -2.32 ± 0.17 pA/pF, p < 0.05). These data suggest that NMA protects the myocardium against ischemia-reperfusion injury through an attenuation of mitochondrial fission by calcineurin/Akt/PKCε-dependent pathways associated to the decrease of ICaL current.


Assuntos
Bloqueadores dos Canais de Cálcio , Cardiotônicos , Metazolamida , Traumatismo por Reperfusão Miocárdica , Animais , Calcineurina , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo L/metabolismo , Cardiotônicos/farmacologia , Metazolamida/farmacologia , Traumatismo por Reperfusão Miocárdica/metabolismo , Miócitos Cardíacos/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos
5.
Arch Biochem Biophys ; 694: 108600, 2020 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-33007282

RESUMO

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.


Assuntos
Miocárdio/metabolismo , Condicionamento Físico Animal , Simportadores de Sódio-Bicarbonato/metabolismo , Animais , Cardiomegalia Induzida por Exercícios/fisiologia , Peróxido de Hidrogênio/farmacologia , Masculino , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Isoformas de Proteínas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Regulação para Cima
6.
J Gen Physiol ; 152(11)2020 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-32986800

RESUMO

Each heartbeat is followed by a refractory period. Recovery from refractoriness is known as Ca2+ release restitution (CRR), and its alterations are potential triggers of Ca2+ arrhythmias. Although the control of CRR has been associated with SR Ca2+ load and RYR2 Ca2+ sensitivity, the relative role of some of the determinants of CRR remains largely undefined. An intriguing point, difficult to dissect and previously neglected, is the possible independent effect of SR Ca2+ content versus the velocity of SR Ca2+ refilling on CRR. To assess these interrogations, we used isolated myocytes with phospholamban (PLN) ablation (PLNKO), knock-in mice with pseudoconstitutive CaMKII phosphorylation of RYR2 S2814 (S2814D), S2814D crossed with PLNKO mice (SDKO), and a previously validated human cardiac myocyte model. Restitution of cytosolic Ca2+ (Fura-2 AM) and L-type calcium current (ICaL; patch-clamp) was evaluated with a two-pulse (S1/S2) protocol. CRR and ICaL restitution increased as a function of the (S2-S1) coupling interval, following an exponential curve. When SR Ca2+ load was increased by increasing extracellular [Ca2+] from 2.0 to 4.0 mM, CRR and ICaL restitution were enhanced, suggesting that ICaL restitution may contribute to the faster CRR observed at 4.0 mM [Ca2+]. In contrast, ICaL restitution did not differ among the different mouse models. For a given SR Ca2+ load, CRR was accelerated in S2814D myocytes versus WT, but not in PLNKO and SDKO myocytes versus WT and S2814D, respectively. The model mimics all experimental data. Moreover, when the PLN ablation-induced decrease in RYR2 expression was corrected, the model revealed that CRR was accelerated in PLNKO and SDKO versus WT and S2814D myocytes, consistent with the enhanced velocity of refilling, SR [Ca2+] recovery, and CRR. We speculate that refilling rate might enhance CRR independently of SR Ca2+ load.


Assuntos
Cálcio , Retículo Sarcoplasmático , Animais , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Células Cultivadas , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Teóricos , Miócitos Cardíacos/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina , Retículo Sarcoplasmático/metabolismo
7.
Life Sci ; 242: 117211, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31891720

RESUMO

Ventricular hypertrophy is a risk factors for arrhythmias, ischemia and sudden death. It involves cellular modifications leading to a pathological remodeling and is associated with heart failure. The activation of the G protein-coupled estrogen receptor (GPER) mediates beneficial actions in the cardiovascular system. Our goal was to prevent and regress the hypertrophy by the activation of GPER in neonatal cardiac myocytes (NRCM) and SHR male rats. Aldosterone increased the neonatal cardiomyocytes cell surface area after 48 h of incubation. The aldo-induced hypertrophy was blocked by the mineralocorticoid receptor (MR) inhibitor Eplererone or the reduction of MR expression by siRNA. The activation of GPER by the agonist G-1 totally prevented the increase surface area by Ald. The transfection of neonatal rat cardiac myocytes with a siRNA against GPER or the incubation with GPER blockers G-15 and G-36 inhibited the protection of G-1. The significant increase of cell surface area after 48 h of incubation with Ald was totally regressed in 24 h by the presence of G-1, indicating that the activation of GPER not only prevent the hypertrophy but also regress the hypertrophy when it is already established. In the in vivo model, G-1 or Vehicle was constantly infused via the minipump to SHR. The reduction of the hypertrophy by G-1 was evident by the cross-sectional area, BNP and ANP markers and by echocardiography. In this studied we demonstrated that the activation of GPER prevented and regressed the hypertrophy induced by Ald in NRCM and regressed hypertrophy in SHR rats.


Assuntos
Cardiomegalia/prevenção & controle , Receptores Acoplados a Proteínas G/metabolismo , Animais , Animais Recém-Nascidos , Western Blotting , Cardiomegalia/diagnóstico por imagem , Células Cultivadas , Ciclopentanos/farmacologia , Ecocardiografia , Eplerenona/farmacologia , Masculino , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Quinolinas/farmacologia , Ratos , Ratos Endogâmicos SHR , Ratos Wistar , Reação em Cadeia da Polimerase em Tempo Real , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/fisiologia
8.
Pflugers Arch ; 472(1): 103-115, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31754830

RESUMO

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.


Assuntos
Adenilil Ciclases/metabolismo , Contração Miocárdica , Miócitos Cardíacos/metabolismo , Simportadores de Sódio-Bicarbonato/metabolismo , Inibidores de Adenilil Ciclases/farmacologia , Animais , Benzamidas/farmacologia , Sinalização do Cálcio , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Ventrículos do Coração/citologia , Isoquinolinas/farmacologia , Masculino , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/fisiologia , Ratos , Ratos Wistar , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Simportadores de Sódio-Bicarbonato/antagonistas & inibidores , Sulfonamidas/farmacologia
9.
Biochem Pharmacol ; 161: 26-36, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30615862

RESUMO

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): 110 min of perfusion; 2) Ischemic control (IC): 30 min of global ischemia and 60 min of reperfusion (R); 3) a-L3: a-L3 was administered during the initial 10 min of R; 4) SB + 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.


Assuntos
Calcineurina/metabolismo , Proteínas de Choque Térmico HSP27/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Simportadores de Sódio-Bicarbonato/antagonistas & inibidores , Simportadores de Sódio-Bicarbonato/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Anticorpos/farmacologia , Preparação de Coração Isolado/métodos , Masculino , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia
11.
Pflugers Arch ; 469(12): 1663-1673, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28836001

RESUMO

The force-frequency relationship (FFR) is an important intrinsic regulatory mechanism of cardiac contractility. However, a decrease (negative FFR) or no effect (flat FFR) on contractile force in response to an elevation of heart rate is present in the normal rat or in human heart failure. Reactive oxygen species (ROS) can act as intracellular signaling molecules activating diverse kinases as calcium-calmodulin-dependent protein kinase II (CaMKII) and p-38 MAP kinase (p-38K). Our aim was to elucidate the intracellular molecules implicated in the FFR of isolated rat ventricular myocytes. The myocytes were field-stimulated via two-platinum electrodes. Sarcomere length was recorded with a video camera. Ca2+ transients and intracellular pHi were recorded by epifluorescence. Increasing frequency from 0.5 to 3 Hz decreased cell shortening without changes in pHi. This negative FFR was changed to positive FFR when the myocytes were pre-incubated with the ROS scavenger MPG, the NADPH oxidase blocker apocynin, or by inhibiting mitochondrial ROS production with 5-HD. Similar results were obtained when the cells were pre-incubated with the CaMKII blocker, KN-93, or the p-38K inhibitor, SB-202190. Consistently, the levels of phosphorylation of p-38K and the oxidation of CaMKII were significantly higher at 2 Hz than at 0.5 Hz. Despite the presence of positive inotropic effect during stimulation frequency enhancement, Ca2+ transient amplitudes were reduced in MPG- and SB-202190-treated myocytes. In conclusion, our results indicate that the activation of the intracellular pathway involving ROS-CaMKII-p-38K contributes to the negative FFR of rat cardiomyocytes, likely by desensitizing the response of contractile myofilaments to Ca2+.


Assuntos
Contração Miocárdica/fisiologia , Miócitos Cardíacos/metabolismo , Miofibrilas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Ventrículos do Coração/metabolismo , Masculino , Ratos , Ratos Wistar
12.
Pflugers Arch ; 469(2): 251-261, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-28013412

RESUMO

Cellular energetic deregulation is widely known to produce an overproduction of acidic species in cancer cells. This acid overload must be counterbalanced with a high rate of H+ extrusion to maintain cell viability. In this sense, many H+ transporters have been reported to be crucial for cell survival and proposed as antineoplastic target. By the way, voltage-gated proton channels (Hv1) mediate highly selective H+ outward currents, capable to compensate acid burden in brief periods of time. This structure is canonically described acting as NADPH oxidase counterbalance in reactive oxygen species production. In this work, we show, for the first time in a oncohematologic cell line, that inhibition of Hv1 channels by Zn2+ and the more selective blocker 2-(6-chloro-1H-benzimidazol-2-yl)guanidine (ClGBI) progressively decreases intracellular pH in resting conditions. This acidification is evident minutes after blockade and progresses under prolonged exposure (2, 17, and 48 h), and we firstly demonstrate that this is followed by cell death through apoptosis (annexin V binding). Altogether, these results contribute strong evidence that this channel might be a new therapeutic target in cancer.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Canais Iônicos/antagonistas & inibidores , Linfócitos T/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Humanos , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Ativação do Canal Iônico/efeitos dos fármacos , Canais Iônicos/metabolismo , Células Jurkat , NADPH Oxidases/metabolismo , Prótons , Espécies Reativas de Oxigênio/metabolismo , Linfócitos T/metabolismo , Zinco/farmacologia
13.
Channels (Austin) ; 10(5): 428-434, 2016 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-27249584

RESUMO

The sodium/bicarbonate cotransporter (NBC) transports extracellular Na+ and HCO3- into the cytoplasm upon intracellular acidosis, restoring the acidic pHi to near neutral values. Two different NBC isoforms have been described in the heart, the electroneutral NBCn1 (1Na+:1HCO3-) and the electrogenic NBCe1 (1Na+:2HCO3-). Certain non-genomic effects of aldosterone (Ald) were due to an orphan G protein-couple receptor 30 (GPR30). We have recently demonstrated that Ald activates GPR30 in adult rat ventricular myocytes, which transactivates the epidermal growth factor receptor (EGFR) and in turn triggers a reactive oxygen species (ROS)- and PI3K/AKT-dependent pathway, leading to the stimulation of NBC. The aim of this study was to investigate the NBC isoform involved in the Ald/GPR30-induced NBC activation. Using specific NBCe1 inhibitory antibodies (a-L3) we demonstrated that Ald does not affect NBCn1 activity. Ald was able to increase NBCe1 activity recorded in isolation. Using immunofluorescence and confocal microscopy analysis we showed in this work that both NBCe1 and GPR30 are localized in t-tubules. In conclusion, we have demonstrated that NBCe1 is the NBC isoform activated by Ald in the heart.


Assuntos
Aldosterona/fisiologia , Miócitos Cardíacos/fisiologia , Receptores Acoplados a Proteínas G/fisiologia , Simportadores de Sódio-Bicarbonato/fisiologia , Animais , Masculino , Ratos
14.
J Mol Cell Cardiol ; 89(Pt B): 260-7, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26497404

RESUMO

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.


Assuntos
Aldosterona/farmacologia , Miocárdio/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Simportadores de Sódio-Bicarbonato/metabolismo , Animais , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/metabolismo , Concentração de Íons de Hidrogênio , Espaço Intracelular/metabolismo , Masculino , Modelos Biológicos , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Receptores de Mineralocorticoides/metabolismo , Ativação Transcricional/efeitos dos fármacos
15.
Cardiovasc Pathol ; 24(4): 236-40, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25670255

RESUMO

BACKGROUND: Reactive oxygen species, such as superoxide, are being increasingly recognized as key components of a vast array of signaling pathways. Angiotensin II is a well-recognized stimulus for superoxide production through NADPH oxidase activation and opening of the mitochondrial ATP-sensitive potassium channels (mKATP). A role for this mechanism has been proposed to explain several physiological effects of the peptide. The aim of this study was to evaluate the involvement of this mechanism in the inotropic response to 100nmol/L angiotensin II. METHODS: Sarcomere shortening and intracellular pH (BCECF-epifluorescence technique) were evaluated in isolated cat ventricular myocytes placed in a perfusion chamber on the stage of an inverted microscope. Myocardial superoxide production was evaluated by the lucigenin quimioluminiscence method. RESULTS: Angiotensin II (100nmol/L) increased~70% sarcomere shortening, effect that was only partially prevented by NADPH oxidase inhibition, mKATP channel blockade or inhibition of the cardiac Na(+)/H(+) exchanger (NHE-1). Moreover, angiotensin II stimulates NHE-1 activity by a NADPH oxidase-dependent mechanism. Myocardial superoxide production was also increased by angiotensin II, and this action was completely prevented either by NADPH oxidase inhibition or mKATP channel blockade. CONCLUSIONS: The positive inotropic response to 100nmol/L angiotensin II is due to both ROS/NHE-1 dependent and independent pathways, this being a point of divergence with the signaling previously described to be triggered by lower concentrations of angiotensin II (i.e.: 1nmol/L).


Assuntos
Angiotensina II/farmacologia , Cardiotônicos/farmacologia , Ventrículos do Coração/efeitos dos fármacos , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Superóxidos/metabolismo , Animais , Gatos , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Ventrículos do Coração/metabolismo , Concentração de Íons de Hidrogênio , Miócitos Cardíacos/metabolismo , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/metabolismo , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio/efeitos dos fármacos , Canais de Potássio/metabolismo , Sarcômeros/efeitos dos fármacos , Sarcômeros/metabolismo , Transdução de Sinais/efeitos dos fármacos , Trocadores de Sódio-Hidrogênio/antagonistas & inibidores , Trocadores de Sódio-Hidrogênio/metabolismo
16.
J Mol Cell Cardiol ; 74: 274-83, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24949568

RESUMO

Ca(2+)-calmodulin kinase II (CaMKII) activation is deleterious in cardiac ischemia/reperfusion (I/R). Moreover, inhibition of CaMKII-dependent phosphorylations at the sarcoplasmic reticulum (SR) prevents CaMKII-induced I/R damage. However, the downstream targets of CaMKII at the SR level, responsible for this detrimental effect, remain unclear. In the present study we aimed to dissect the role of the two main substrates of CaMKII at the SR level, phospholamban (PLN) and ryanodine receptors (RyR2), in CaMKII-dependent I/R injury. In mouse hearts subjected to global I/R (45/120min), phosphorylation of the primary CaMKII sites, S2814 on cardiac RyR2 and of T17 on PLN, significantly increased at the onset of reperfusion whereas PKA-dependent phosphorylation of RyR2 and PLN did not change. Similar results were obtained in vivo, in mice subjected to regional myocardial I/R (1/24h). Knock-in mice with an inactivated serine 2814 phosphorylation site on RyR2 (S2814A) significantly improved post-ischemic mechanical recovery, reduced infarct size and decreased apoptosis. Conversely, knock-in mice, in which CaMKII site of RyR2 is constitutively activated (S2814D), significantly increased infarct size and exacerbated apoptosis. In S2814A and S2814D mice subjected to regional myocardial ischemia, infarct size was also decreased and increased respectively. Transgenic mice with double-mutant non-phosphorylatable PLN (S16A/T17A) in the PLN knockout background (PLNDM) also showed significantly increased post-ischemic cardiac damage. This effect cannot be attributed to PKA-dependent PLN phosphorylation and was not due to the enhanced L-type Ca(2+) current, present in these mice. Our results reveal a major role for the phosphorylation of S2814 site on RyR2 in CaMKII-dependent I/R cardiac damage. In contrast, they showed that CaMKII-dependent increase in PLN phosphorylation during reperfusion opposes rather than contributes to I/R damage.


Assuntos
Proteínas de Ligação ao Cálcio/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Cálcio/metabolismo , Traumatismo por Reperfusão Miocárdica/genética , Miócitos Cardíacos/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Animais , Sinalização do Cálcio , Proteínas de Ligação ao Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Morte Celular , Regulação da Expressão Gênica , Técnicas de Introdução de Genes , Ventrículos do Coração/citologia , Ventrículos do Coração/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Mutação , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Miócitos Cardíacos/citologia , Técnicas de Cultura de Órgãos , Fosforilação , Cultura Primária de Células , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo
17.
Cardiovasc Pathol ; 23(4): 224-30, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24721237

RESUMO

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.


Assuntos
Traumatismo por Reperfusão Miocárdica/etiologia , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Miocárdio/metabolismo , Simportadores de Sódio-Bicarbonato/metabolismo , Animais , Benzamidas/farmacologia , Cardiotônicos/farmacologia , Contração Miocárdica , Infarto do Miocárdio/tratamento farmacológico , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Ratos , Ratos Wistar , Simportadores de Sódio-Bicarbonato/agonistas , Simportadores de Sódio-Bicarbonato/antagonistas & inibidores , Sulfonamidas/farmacologia , Função Ventricular Esquerda
18.
Cell Physiol Biochem ; 33(4): 982-90, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24714077

RESUMO

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.


Assuntos
Neutrófilos/metabolismo , Simportadores de Sódio-Bicarbonato/metabolismo , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-dissulfônico/farmacologia , Amilorida/análogos & derivados , Amilorida/farmacologia , Cloreto de Amônio/farmacologia , Benzamidas/farmacologia , Bicarbonatos/farmacologia , Citocalasina B/farmacologia , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Transporte de Íons/efeitos dos fármacos , N-Formilmetionina Leucil-Fenilalanina/farmacologia , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/metabolismo , Neutrófilos/efeitos dos fármacos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/metabolismo , Simportadores de Sódio-Bicarbonato/genética , Trocadores de Sódio-Hidrogênio/metabolismo , Sulfonamidas/farmacologia
19.
Cardiovasc Res ; 101(2): 211-9, 2014 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-24253522

RESUMO

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.


Assuntos
Bicarbonatos/metabolismo , Cardiomegalia/metabolismo , Hipertensão/metabolismo , Miócitos Cardíacos/metabolismo , Sistema Renina-Angiotensina , Sarcolema/metabolismo , Simportadores de Sódio-Bicarbonato/metabolismo , Compostos de Amônio/metabolismo , Angiotensina II , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Animais , Anti-Hipertensivos/farmacologia , Cardiomegalia/induzido quimicamente , Cardiomegalia/patologia , Modelos Animais de Doenças , Regulação para Baixo , Endossomos/metabolismo , Concentração de Íons de Hidrogênio , Hiperpotassemia/metabolismo , Hipertensão/tratamento farmacológico , Hipertensão/patologia , Losartan/farmacologia , Masculino , Miócitos Cardíacos/patologia , Potássio/metabolismo , Transporte Proteico , Ratos , Ratos Endogâmicos SHR , Ratos Wistar , Sistema Renina-Angiotensina/efeitos dos fármacos , Sarcolema/patologia , Fatores de Tempo
20.
Am J Physiol Renal Physiol ; 305(12): F1765-74, 2013 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-24005470

RESUMO

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(-), ∼50% 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.


Assuntos
Dióxido de Carbono/farmacologia , Células HEK293/efeitos dos fármacos , Células HEK293/metabolismo , Simportadores de Sódio-Bicarbonato/metabolismo , Regulação para Cima/efeitos dos fármacos , Amilorida/análogos & derivados , Amilorida/farmacologia , Bicarbonatos/metabolismo , Relação Dose-Resposta a Droga , Células HEK293/citologia , Humanos , Concentração de Íons de Hidrogênio , Fatores de Tempo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...