RESUMEN
We have previously demonstrated that inhibition of extracellularly oriented carbonic anhydrase (CA) isoforms protects the myocardium against ischemia-reperfusion injury. In this study, our aim was to assess the possible further contribution of CA intracellular isoforms examining the actions of the highly diffusible cell membrane permeant inhibitor of CA, ethoxzolamide (ETZ). Isolated rat hearts, after 20 min of stabilization, were assigned to the following groups: (1) Nonischemic control: 90 min of perfusion; (2) Ischemic control: 30 min of global ischemia and 60 min of reperfusion (R); and (3) ETZ: ETZ at a concentration of 100 µM was administered for 10 min before the onset of ischemia and then during the first 10 min of reperfusion. In additional groups, ETZ was administered in the presence of SB202190 (SB, a p38MAPK inhibitor) or chelerythrine (Chel, a protein kinase C [PKC] inhibitor). Infarct size, myocardial function, and the expression of phosphorylated forms of p38MAPK, PKCε, HSP27, and Drp1, and calcineurin Aß content were assessed. In isolated mitochondria, the Ca2+ response, Ca2+ retention capacity, and membrane potential were measured. ETZ decreased infarct size by 60%, improved postischemic recovery of myocardial contractile and diastolic relaxation increased P-p38MAPK, P-PKCε, P-HSP27, and P-Drp1 expression, decreased calcineurin content, and normalized calcium and membrane potential parameters measured in isolated mitochondria. These effects were significantly attenuated when ETZ was administered in the presence of SB or Chel. These data show that ETZ protects the myocardium and mitochondria against ischemia-reperfusion injury through p38MAPK- and PKCε-dependent pathways and reinforces the role of CA as a possible target in the management of acute cardiac ischemic diseases.
Asunto(s)
Inhibidores de Anhidrasa Carbónica/farmacología , Etoxzolamida/farmacología , Corazón/efectos de los fármacos , Mitocondrias Cardíacas/efectos de los fármacos , Miocardio/metabolismo , Animales , Benzofenantridinas/farmacología , Calcio/metabolismo , Inhibidores Enzimáticos/farmacología , Imidazoles/farmacología , Preparación de Corazón Aislado , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias Cardíacas/metabolismo , Poro de Transición de la Permeabilidad Mitocondrial/metabolismo , Daño por Reperfusión Miocárdica , Proteína Quinasa C/antagonistas & inhibidores , Piridinas/farmacología , Ratas , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidoresRESUMEN
Heart failure is the leading cause of death among diabetic people. Cellular and molecular entities leading to diabetic cardiomyopathy are, however, poorly understood. Coupling of cardiac carbonic anhydrase II (CAII) and Na+/H+ exchanger 1 (NHE1) to form a transport metabolon was analyzed in obese type 2 diabetic mice (ob-/-) and control heterozygous littermates (ob+/-). Echocardiography showed elevated systolic interventricular septum thickness and systolic posterior wall thickness in ob-/- mice at 9 and 16â¯weeks. ob-/- mice showed increased left ventricular (LV) weight/tibia length ratio and increased cardiomyocyte cross sectional area as compared to controls, indicating cardiac hypertrophy. Immunoblot analysis showed increased CAII expression in LV samples of ob-/-vs. ob+/- mice, and augmented Ser703 phosphorylation on NHE1 in ob-/- hearts. Reciprocal co-immunoprecipitation analysis showed strong association of CAII and NHE1 in LV samples of ob-/- mice. NHE1-dependent rate of intracellular pH (pHi) normalization after transient acid loading of isolated cardiomyocytes was higher in ob-/- mice vs. ob+/-. NHE transport activity was also augmented in cultured H9C2 rat cardiomyoblasts treated with high glucose/high palmitate, and it was normalized after CA inhibition. We conclude that the NHE1/CAII metabolon complex is exacerbated in diabetic cardiomyopathy of ob-/- mice, which may lead to perturbation of pHi and [Na+] and [Ca2+] handling in these diseased hearts.
Asunto(s)
Anhidrasa Carbónica II/metabolismo , Cardiomegalia/patología , Diabetes Mellitus Tipo 2/complicaciones , Intercambiador 1 de Sodio-Hidrógeno/metabolismo , Animales , Anhidrasa Carbónica II/antagonistas & inhibidores , Inhibidores de Anhidrasa Carbónica/farmacología , Cardiomegalia/diagnóstico por imagen , Cardiomegalia/metabolismo , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/patología , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patología , Electrocardiografía , Etoxzolamida/farmacología , Femenino , Ventrículos Cardíacos/patología , Concentración de Iones de Hidrógeno , Ratones Mutantes , Ratones Transgénicos , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Fosforilación , Procesamiento Proteico-Postraduccional , Ratas , Serina/metabolismoRESUMEN
BACKGROUND: Two potent carbonic anhydrase (CA) inhibitors with widely differing membrane permeability, poorly diffusible benzolamide (BZ), and highly diffusible ethoxzolamide (ETZ) were assessed to determine whether they can reduce cardiac dysfunction in rats subjected to coronary artery ligation (CAL)-induced myocardial infarction. METHODS AND RESULTS: Rats with evidence of heart failure (HF) at 32 weeks following a permanent left anterior coronary artery occlusion were treated with placebo, BZ, or ETZ (4 mg kgday-1) for 4 weeks at which time left ventricular function and structure were evaluated. Lung weight/body weight (LW/BW) ratio increased in CAL rats by 17±1% vs. control, suggesting pulmonary edema. There was a trend for BZ and ETZ to ameliorate the increase in LW/BW by almost 50% (9±5% and 9±8%, respectively, versus CAL) (P=.16, NS). Echocardiographic assessment showed decreased left ventricular midwall shortening in HF rats, 21±1% vs. control 32±1%, which was improved by BZ to 29±1% and ETZ to 27±1%, and reduced endocardial shortening in HF rats 38±3% vs. control 62±1%, partially restored by BZ and ETZ to ~50%. Expression of the hypoxia-inducible membrane-associated CAIX isoform increased by ~60% in HF rat hearts, and this effect was blocked by ETZ. CONCLUSIONS: We conclude that CAL-induced myocardial interstitial fibrosis and associated decline in left ventricular function were diminished with BZ or ETZ treatment. The reductions in cardiac remodeling in HF with both ETZ and BZ CA inhibitors suggest that inhibition of a membrane-bound CA appears to be the critical site for this protection.
Asunto(s)
Benzolamida/farmacología , Inhibidores de Anhidrasa Carbónica/farmacología , Etoxzolamida/farmacología , Corazón/efectos de los fármacos , Infarto del Miocardio/patología , Animales , Vasos Coronarios/cirugía , Modelos Animales de Enfermedad , Immunoblotting , Ligadura , Masculino , Ratas , Ratas Wistar , Función Ventricular Izquierda/efectos de los fármacos , Remodelación Ventricular/efectos de los fármacosRESUMEN
Myocardial stretch is an established signal that leads to hypertrophy. Myocardial stretch induces a first immediate force increase followed by a slow force response (SFR), which is a consequence of an increased Ca(2+) transient that follows the NHE1 Na(+)/H(+) exchanger activation. Carbonic anhydrase II (CAII) binds to the extreme COOH terminus of NHE1 and regulates its transport activity. We aimed to test the role of CAII bound to NHE1 in the SFR. The SFR and changes in intracellular pH (pHi) were evaluated in rat papillary muscle bathed with CO2/HCO3(-) buffer and stretched from 92% to 98% of the muscle maximal force development length for 10 min in the presence of the CA inhibitor 6-ethoxzolamide (ETZ, 100 µM). SFR control was 120 ± 3% (n = 8) of the rapid initial phase and was fully blocked by ETZ (99 ± 4%, n = 6). The SFR corresponded to a maximal increase in pHi of 0.18 ± 0.02 pH units (n = 4), and pHi changes were blocked by ETZ (0.04 ± 0.04, n = 6), as monitored by epifluorescence. NHE1/CAII physical association was examined in the SFR by coimmunoprecipitation, using muscle lysates. CAII immunoprecipitated with an anti-NHE1 antibody and the CAII immunoprecipitated protein levels increased 58 ± 9% (n = 6) upon stretch of muscles, assessed by immunoblots. The p90(RSK) kinase inhibitor SL0101-1 (10 µM) blocked the SFR of heart muscles after stretch 102 ± 2% (n = 4) and reduced the binding of CAII to NHE1, suggesting that the stretch-induced phosphorylation of NHE1 increases its binding to CAII. CAII/NHE1 interaction constitutes a component of the SFR to heart muscle stretch, which potentiates NHE1-mediated H(+) transport in the myocardium.
Asunto(s)
Anhidrasa Carbónica II/antagonistas & inhibidores , Inhibidores de Anhidrasa Carbónica/farmacología , Etoxzolamida/farmacología , Husos Musculares/metabolismo , Músculos Papilares/efectos de los fármacos , Intercambiadores de Sodio-Hidrógeno/metabolismo , Animales , Anhidrasa Carbónica II/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Concentración de Iones de Hidrógeno , Inmunoprecipitación , Mediciones Luminiscentes , Masculino , Músculos Papilares/enzimología , Fosforilación , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Mapeo de Interacción de Proteínas/métodos , Inhibidores de Proteínas Quinasas/farmacología , Ratas , Ratas Wistar , Proteínas Quinasas S6 Ribosómicas 90-kDa/antagonistas & inhibidores , Proteínas Quinasas S6 Ribosómicas 90-kDa/metabolismo , Intercambiador 1 de Sodio-Hidrógeno , Factores de TiempoRESUMEN
The metabolism of gas gland cells of the swimbladder epithelium is specialized for the production of acidic metabolites that are released into the blood stream and provoke an increase in gas partial pressure by reducing the effective gas-carrying capacity of the blood. In a subsequent step this initial increase in gas partial pressure is multiplied by back-diffusion of gas molecules from the venous to the arterial side in the countercurrent system, the rete mirabile. Thus, gas partial pressures of up to several hundred atmospheres can be generated in the swimbladder. Measurements of metabolic end products and analysis of the formation of 14C02 from [1-14(superscription) C] glucose and [6-14(superscription) C] glucose revealed that the acidic metabolises are lactic acid, produced in the glycolytic pathway, and also C02, formed in the pentose phosphate shunt. C02 easily enters the blood stream by diffusion. The release of protons from isolated gas gland cells, however, is highly dependent on the extracellular sodium concentration. This sodium dependence can in part be blocked by addition of amiloride, indicating that a Na+/ H+ exchanger is involved in the release of protons. A significant decrease in the rate of proton secretion in the presence of the carbonic anhydrase inhibitor ethoxzolamide indicates that the second major route for the release of protons includes carbonic anhydrase activity and the diffusion of C02.
Asunto(s)
Humanos , beta-Galactosidasa/biosíntesis , Dióxido de Carbono/sangre , Metabolismo Energético , Glucosa/metabolismo , Sacos Aéreos/metabolismo , Ácido Oxámico/metabolismo , Cianuro de Sodio/metabolismo , Etoxzolamida/farmacología , Fluoruro de Sodio/metabolismo , Concentración de Iones de Hidrógeno , Sacos Aéreos/irrigación sanguíneaRESUMEN
The metabolism of gas gland cells of the swimbladder epithelium is specialized for the production of acidic metabolites that are released into the blood stream and provoke an increase in gas partial pressure by reducing the effective gas-carrying capacity of the blood. In a subsequent step this initial increase in gas partial pressure is multiplied by back-diffusion of gas molecules from the venous to the arterial side in the countercurrent system, the rete mirabile. Thus, gas partial pressures of up to several hundred atmospheres can be generated in the swimbladder. Measurements of metabolic end products and analysis of the formation of 14CO2 from [1-14C]glucose and [6-14C]glucose revealed that the acidic metabolites are lactic acid, produced in the glycolytic pathway, and also CO2, formed in the pentose phosphate shunt. CO2 easily enters the blood stream by diffusion. The release of protons from isolated gas gland cells, however, is highly dependent on the extracellular sodium concentration. This sodium dependence can in part be blocked by addition of amiloride, indicating that a Na+/H+ exchanger is involved in the release of protons. A significant decrease in the rate of proton secretion in the presence of the carbonic anhydrase inhibitor ethoxzolamide indicates that the second major route for the release of protons includes carbonic anhydrase activity and the diffusion of CO2.