RESUMEN
The use of antagonists of the mineralocorticoid receptor in the treatment of myocardial hypertrophy and heart failure has gained increasing importance in the last years. The cardiac Na(+)/H(+) exchanger (NHE-1) upregulation induced by aldosterone could account for the genesis of these pathologies. We tested whether aldosterone-induced NHE-1 stimulation involves the transactivation of the epidermal growth factor receptor (EGFR). Rat ventricular myocytes were used to measure intracellular pH with epifluorescence. Aldosterone enhanced the NHE-1 activity. This effect was canceled by spironolactone or eplerenone (mineralocorticoid receptor antagonists), but not by mifepristone (glucocorticoid receptor antagonist) or cycloheximide (protein synthesis inhibitor), indicating that the mechanism is mediated by the mineralocorticoid receptor triggering nongenomic pathways. Aldosterone-induced NHE-1 stimulation was abolished by the EGFR kinase inhibitor AG1478, suggesting that is mediated by transactivation of EGFR. The increase in the phosphorylation level of the kinase p90(RSK) and NHE-1 serine703 induced by aldosterone was also blocked by AG1478. Exogenous epidermal growth factor mimicked the effects of aldosterone on NHE-1 activity. Epidermal growth factor was also able to increase reactive oxygen species production, and the epidermal growth factor-induced activation of the NHE-1 was abrogated by the reactive oxygen species scavenger N-2-mercaptopropionyl glycine, indicating that reactive oxygen species are participating as signaling molecules in this mechanism. Aldosterone enhances the NHE-1 activity via transactivation of the EGFR, formation of reactive oxygen species, and phosphorylation of the exchanger. These results call attention to the consideration of the EGFR as a new potential therapeutic target of the cardiovascular pathologies involving the participation of aldosterone.
Asunto(s)
Aldosterona/farmacología , Receptores ErbB/metabolismo , Miocitos Cardíacos/efectos de los fármacos , Intercambiadores de Sodio-Hidrógeno/efectos de los fármacos , Animales , Células Cultivadas , Receptores ErbB/genética , Modelos Animales , Miocitos Cardíacos/metabolismo , Fosforilación/fisiología , Distribución Aleatoria , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismo , Sensibilidad y Especificidad , Transducción de Señal/efectos de los fármacos , Intercambiadores de Sodio-Hidrógeno/metabolismo , Superóxidos/metabolismo , Activación TranscripcionalRESUMEN
Cardiac Na(+)/H(+) exchanger (NHE1) hyperactivity is a central factor in cardiac remodeling following hypertension, myocardial infarction, ischemia-reperfusion injury, and heart failure. Treatment of these pathologies by inhibiting NHE1 is challenging because specific drugs that have been beneficial in experimental models were associated with undesired side effects in clinical practice. In the present work, small interference RNA (siRNA) produced in vitro to specifically silence NHE1 (siRNA(NHE1)) was injected once in vivo into the apex of the left ventricular wall of mouse myocardium. After 48 h, left ventricular NHE1 protein expression was reduced in siRNA(NHE1)-injected mice compared with scrambled siRNA by 33.2 ± 3.4% (n = 5; P < 0.05). Similarly, NHE1 mRNA levels were reduced by 20 ± 2.0% (n = 4). At 72 h, siRNA(NHE1) spreading was evident from the decrease in NHE1 expression in three portions of the myocardium (apex, medium, base). NHE1 function was assessed based on maximal velocity of intracellular pH (pH(i)) recovery (dpH(i)/dt) after an ammonium prepulse-induced acidic load. Maximal dpH(i)/dt was reduced to 14% in siRNA(NHE1)-isolated left ventricular papillary muscles compared with scrambled siRNA. In conclusion, only one injection of naked siRNA(NHE1) successfully reduced NHE1 expression and activity in the left ventricle. As has been previously suggested, extensive NHE1 expression reduction may indicate myocardial spread of siRNA molecules from the injection site through gap junctions, providing a valid technique not only for further research into NHE1 function, but also for consideration as a potential therapeutic strategy.
Asunto(s)
Proteínas de Transporte de Catión/genética , Silenciador del Gen/efectos de los fármacos , Corazón/efectos de los fármacos , Corazón/fisiología , ARN Interferente Pequeño/farmacología , Intercambiadores de Sodio-Hidrógeno/genética , Animales , Tampones (Química) , Proteínas de Transporte de Catión/efectos de los fármacos , Células HEK293 , Humanos , Concentración de Iones de Hidrógeno , Inmunoquímica , Inyecciones , Masculino , Ratones , Ratones Endogámicos BALB C , Miocardio/metabolismo , Músculos Papilares/efectos de los fármacos , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Intercambiador 1 de Sodio-Hidrógeno , Intercambiadores de Sodio-Hidrógeno/efectos de los fármacos , Técnicas de Cultivo de Tejidos , Función Ventricular Izquierda/genética , Función Ventricular Izquierda/fisiologíaRESUMEN
The effects of aldosterone on the intracellular pH recovery rate (pHirr) via Na+/H+ exchanger and on the [Ca2+]i were investigated in isolated rat S3 segment. Aldosterone [10(-12), 10(-10), or 10(-8) M with 1-h, 15- or 2-min preincubation (pi)] caused a dose-dependent increase in the pHirr, but aldosterone (10(-6) M with 1-h, 15- or 2-min pi) decreased it (these effects were prevented by HOE694 but not by S3226). After 1 min of aldosterone pi, there was a transient and dose-dependent increase of the [Ca2+]i and after 6-min pi there was a new increase of [Ca2+]i that persisted after 1 h. Spironolactone, actinomycin D, or cycloheximide did not affect the effects of aldosterone (15- or 2-min pi) but inhibited the effects of aldosterone (1-h pi) on pHirr and on [Ca2+]i. RU 486 prevented the stimulatory effect of aldosterone (10(-12) M, 15- or 2-min pi) on both parameters and maintained the inhibitory effect of aldosterone (10(-6) M, 15- or 2-min pi) on the pHirr but reversed its stimulatory effect on the [Ca2+]i to an inhibitory effect. The data indicate a genomic (1 h, via MR) and a nongenomic action (15 or 2 min, probably via GR) on [Ca2+]i and on the basolateral NHE1 and are compatible with stimulation of the NHE1 by increases in [Ca2+]i in the lower range (at 10(-12) M aldosterone) and inhibition by increases at high levels (at 10(-6) M aldosterone) or decreases in [Ca2+]i (at 10(-6) M aldosterone plus RU 486).
Asunto(s)
Aldosterona/farmacología , Señalización del Calcio/fisiología , Túbulos Renales Proximales/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Cloruro de Amonio/farmacología , Animales , Calcio/metabolismo , Cicloheximida/farmacología , Citosol/metabolismo , Dactinomicina/farmacología , Expresión Génica/efectos de los fármacos , Guanidinas/farmacología , Concentración de Iones de Hidrógeno/efectos de los fármacos , Técnicas In Vitro , Túbulos Renales Proximales/efectos de los fármacos , Masculino , Metacrilatos/farmacología , Mifepristona/farmacología , Ratas , Ratas Wistar , Receptores de Glucocorticoides/genética , Receptores de Mineralocorticoides/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sodio/farmacología , Intercambiador 1 de Sodio-Hidrógeno , Intercambiadores de Sodio-Hidrógeno/antagonistas & inhibidores , Intercambiadores de Sodio-Hidrógeno/efectos de los fármacos , Espironolactona/farmacología , Sulfonas/farmacologíaRESUMEN
Tubular dysfunction is a hallmark of severe leptospirosis. Antimicrobial therapy is thought to interfere on renal involvement. We evaluated the expression of a proximal tubule type-3 Na+/H+ exchanger (NHE3) and a thick ascending limb Na+-K+-2Cl(-) cotransporter (NKCC2) in controls and treated hamsters. Animals infected by a serovar Copenhageni isolate, were treated or not with ampicillin (AMP) and/or N-acetylcysteine (NAC). Leptospiral antigen(s) and expression of renal transporters were evaluated by immunohistochemistry, and serum thiobarbituric acid (TBARS) was quantified. Infected hamsters had high amounts of detectable leptospiral antigen(s) in target tissues while renal expression of NHE3 and NKCC2 decreased. Ampicillin treatment was associated with minimal or no detection of leptospiral antigens, normal expression of NHE3 and NKCC2 transporters, and reduced levels of TBARS. NAC effect was restricted to lowering TBARS. Early and late AMP treatment rescued tubular defects in severe leptospirosis disease, and there was no evidence of benefit from antioxidant therapy.
Asunto(s)
Ampicilina/farmacología , Antibacterianos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Intercambiadores de Sodio-Hidrógeno/biosíntesis , Simportadores de Cloruro de Sodio-Potasio/biosíntesis , Enfermedad de Weil/tratamiento farmacológico , Acetilcisteína/administración & dosificación , Acetilcisteína/farmacología , Ampicilina/administración & dosificación , Animales , Antibacterianos/administración & dosificación , Antígenos Bacterianos/análisis , Cricetinae , Regulación hacia Abajo , Femenino , Depuradores de Radicales Libres/administración & dosificación , Depuradores de Radicales Libres/farmacología , Perfilación de la Expresión Génica , Riñón/patología , Riñón/fisiopatología , Hígado/patología , Mesocricetus , Intercambiador 3 de Sodio-Hidrógeno , Intercambiadores de Sodio-Hidrógeno/análisis , Intercambiadores de Sodio-Hidrógeno/efectos de los fármacos , Simportadores de Cloruro de Sodio-Potasio/análisis , Simportadores de Cloruro de Sodio-Potasio/efectos de los fármacos , Miembro 1 de la Familia de Transportadores de Soluto 12 , Tiobarbitúricos/sangre , Enfermedad de Weil/patología , Enfermedad de Weil/fisiopatologíaRESUMEN
BACKGROUND: Sodium/hydrogen ion exchange is hyperactive in hypertension. Myocardial sodium/hydrogen ion exchange hyperactivity accompanies the regression of cardiac hypertrophy in spontaneously hypertensive rats (SHR) after long term control of blood pressure with enalapril. OBJECTIVES: To explore whether this effect is shared by other antihypertensive agents or is specific to angiotensin-converting enzyme inhibition. ANIMALS AND METHODS: SHR and normotensive Wistar Kyoto (WKY) rats were treated for five weeks with enalapril (20 mg/kg/day), nifedipine (10 mg/kg/day) or losartan (40 mg/kg/day). Sodium/hydrogen ion exchange activity was estimated in terms of both steady intracellular pH in HEPES buffer and the rate of intracellular pH recovery from intracellular acid loads in isolated superfused 2'-7'-bis(2-carboxyethyl)-5,-(and-6)-carboxyfluorescein, acetoxymethyl ester form-loaded papillary muscles. RESULTS: Enalapril, nifedipine and losartan decreased systolic blood pressure in SHR to about the same value (140 3, 140 2 and 146 3 mmHg, respectively, at the end the treatment). However, the index of cardiac hypertrophy (heart weight to body weight ratio) was decreased to a smaller value with losartan than with nifedipine or enalapril (2.66 0.09, 3.06 0.05 and 2.86 0.04 mg/g respectively; P<0.05 ANOVA). For the untreated SHR, the index of cardiac hypertrophy was 3.30 0.04 mg/g. Myocardial sodium/hydrogen ion exchange hyperactivity in SHR was normalized by all treatments. CONCLUSIONS: The three treatments regressed cardiac hypertrophy and normalized sodium/hydrogen ion exchange exchange activity in SHR, and losartan was the most effective treatment for reversing cardiac hypertrophy, despite producing effects on blood pressure and sodium/hydrogen exchange activity similar to that of other antihypertensive drugs.
Asunto(s)
Antihipertensivos/farmacología , Hipertensión/tratamiento farmacológico , Intercambiadores de Sodio-Hidrógeno/efectos de los fármacos , Animales , Antihipertensivos/uso terapéutico , Presión Sanguínea/efectos de los fármacos , Bloqueadores de los Canales de Calcio/farmacología , Bloqueadores de los Canales de Calcio/uso terapéutico , Cardiomegalia/metabolismo , Modelos Animales de Enfermedad , Enalapril/farmacología , Enalapril/uso terapéutico , Losartán/farmacología , Losartán/uso terapéutico , Masculino , Miocardio/metabolismo , Nifedipino/farmacología , Nifedipino/uso terapéutico , Ratas , Ratas WistarAsunto(s)
Miocardio/metabolismo , Intercambiadores de Sodio-Hidrógeno/fisiología , Acidosis/metabolismo , Angiotensina II/farmacología , Animales , Bicarbonatos/metabolismo , Calcio/metabolismo , Dióxido de Carbono/metabolismo , Permeabilidad de la Membrana Celular , Diabetes Mellitus/metabolismo , Diabetes Mellitus/fisiopatología , Endotelinas/farmacología , HEPES/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Hipertensión/fisiopatología , Hipertrofia Ventricular Izquierda/metabolismo , Hipertrofia Ventricular Izquierda/fisiopatología , Contracción Miocárdica/fisiología , Reperfusión Miocárdica , Protones , Canales de Sodio/metabolismo , Intercambiadores de Sodio-Hidrógeno/efectos de los fármacosAsunto(s)
Humanos , Animales , Intercambiadores de Sodio-Hidrógeno/fisiología , Miocardio/metabolismo , Protones , Acidosis/metabolismo , Bicarbonatos/metabolismo , Angiotensina II/farmacología , Dióxido de Carbono/metabolismo , Reperfusión Miocárdica , Canales de Sodio/metabolismo , Permeabilidad de la Membrana Celular , Calcio/metabolismo , Endotelinas/farmacología , Hipertrofia Ventricular Izquierda/fisiopatología , Hipertrofia Ventricular Izquierda/metabolismo , Intercambiadores de Sodio-Hidrógeno/efectos de los fármacos , Diabetes Mellitus/fisiopatología , Diabetes Mellitus/metabolismo , HEPES/metabolismo , Concentración de Iones de Hidrógeno , Hipertensión/fisiopatología , Contracción Miocárdica/fisiologíaRESUMEN
Intracellular pH is under strict control in myocardium; H+ are extruded from the cells by sodium-dependent mechanisms, mainly Na+/H+ exchanger and Na+/HCO3- symport, whereas Na+-independent Cl-/HCO3- exchanger extrudes bases on intracellular alkalinization. Hypertrophic myocardium from spontaneously hypertensive rats (SHR) exhibits increased Na+/H+ exchange activity that is accompanied by enhanced extrusion of bases through Na+-independent Cl-/HCO3- exchange. The present experiments were designed to investigate the effect of enalapril-induced regression of cardiac hypertrophy on the activity of these exchangers. Male SHR and normotensive Wistar-Kyoto rats (WKY) received enalapril maleate (20 mg/kg per day) in the drinking water for 5 weeks. Gender- and age-matched SHR and WKY were used as untreated controls. Enalapril treatment significantly reduced systolic blood pressure in SHR and completely regressed cardiac hypertrophy. Na+/H+ activity was estimated in terms of both steady pHi value in HEPES buffer and the rate of pHi recovery from CO2-induced acid load. Na+-independent Cl-/HCO3- activity was assessed by measuring the rate of pHi recovery from intracellular alkalinization produced by trimethylamine exposure. Regression of cardiac hypertrophy was accompanied by normalization of Na+/H+ and Na+-independent Cl-/HCO3- exchange activities. Inhibition of protein kinase C (PKC) activity with chelerythrine (10 mmol/L) or calphostin C (50 nmol/L) returned both exchange activities to normal values. These results show that angiotensin-converting enzyme inhibition normalizes the enhanced activity of both exchangers while regressing cardiac hypertrophy. Because normalization of exchange activities could be also achieved by PKC inhibition, the data would suggest that PKC-dependent mechanisms play a significant role in the increased ion exchange activities of hypertrophic myocardium and in their normalization by angiotensin-converting enzyme inhibition.
Asunto(s)
Antihipertensivos/uso terapéutico , Cardiomegalia/tratamiento farmacológico , Enalapril/uso terapéutico , Concentración de Iones de Hidrógeno/efectos de los fármacos , Intercambiadores de Sodio-Hidrógeno/efectos de los fármacos , Análisis de Varianza , Animales , Presión Sanguínea/efectos de los fármacos , Cardiomegalia/etiología , Cardiomegalia/metabolismo , Hipertensión/complicaciones , Transporte Iónico/efectos de los fármacos , Masculino , Miocardio/metabolismo , Proteína Quinasa C/efectos de los fármacos , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Intercambiadores de Sodio-Hidrógeno/metabolismoRESUMEN
Hypertension has been associated with increased activity of the Na(+)-H+ exchanger. To study the role played by protein kinase C in this process, we used chelerythrine, a potent and specific inhibitor of the kinase. After an acid load by ammonium chloride preincubation, platelets isolated from spontaneously hypertensive rats showed a faster and larger increase in intracellular pH than platelets from Wistar-Kyoto rats. The initial rate of intracellular pH recovery was 2.46 +/- 0.26 pH units per minute in spontaneously hypertensive rats and 1.74 +/- 0.19 in Wistar-Kyoto rats. For protein kinase C inhibition, platelets were incubated for 30 minutes with 10 mumol/L chelerythrine. This treatment induced a significant reduction in the recovery rate only in spontaneously hypertensive rat platelets, indicating that a pathway involving protein kinase C participates in the prestimulation of the exchanger in cells from this rat strain. Addition of chelerythrine reduced the baseline intracellular pH of platelets. No significant difference was found between the decrease of steady-state intracellular pH induced by chelerythrine in either rat strain. These findings indicate that this model of hypertension is characterized by increased Na(+)-H+ activity mediated by protein kinase C stimulation.
Asunto(s)
Sangre/efectos de los fármacos , Fenantridinas/farmacología , Proteína Quinasa C/antagonistas & inhibidores , Intercambiadores de Sodio-Hidrógeno/efectos de los fármacos , Alcaloides , Animales , Benzofenantridinas , Sangre/metabolismo , Inhibidores Enzimáticos , Concentración de Iones de Hidrógeno , Masculino , Proteína Quinasa C/sangre , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Especificidad de la Especie , Trombina/farmacologíaRESUMEN
The effect of protein kinase C (PKC) activation on fluid and bicarbonate transport in renal tubules has been discussed controversially. Stimulation and inhibition have been shown to depend on factors such as experimental model and exposure time to the mediator of enzyme activation. We studied the role of PKC activation by phorbol-12-myristate-13-acetate (PMA) and by 1,2-dioctanoyl glycerol (DOG) in proximal bicarbonate reabsorption (JHCO3-) by 'in vivo' stationary microperfusion and ion-exchange resin microelectrode determination of luminal pH. Both PMA (10(-8) mol/l) and DOG (10(-3) mol/l) added to lumen or to peritubular capillaries reduced the net JHCO3- significantly. When added to lumen, the inhibition was 44 and 32%, respectively. This reduction did not involve changes in lumen stationary pH, but was mediated by a marked increase in the halftime of luminal bicarbonate disappearance; from 4.22 +/- 0.23 to 6.27 +/- 0.51 s with PMA and from 3.90 +/- 0.25 to 6.33 +/- 0.48 s with DOG. This effect was intensified by 10(-6) mol/l okadaic acid, a phosphatase inhibitor (inhibition of JHCO3- increased to 61%), and reduced by 30% by 10(-6) mol/l H7, an inhibitor of PKC. H89, a protein kinase A inhibitor, did not affect the inhibitory action of PMA. Our data suggest that PKC activation reduces the rate of H ion secretion (bicarbonate reabsorption) in convoluted segments of rat renal proximal tubules and that phosphorylation of the Na+/H+ exchanger by this kinase is the cause of the reduction in net secretion of H ions.