RESUMEN
BACKGROUND: Endothelium-derived relaxing factors play an important role in cardiovascular homeostasis. Among them, endothelium-derived hyperpolarizing factor (EDHF) is important especially in microcirculation. It has previously been demonstrated that endothelium-derived hydrogen peroxide (H(2)O(2)) is an EDHF in animals and humans and that endothelial nitric oxide synthase (eNOS) plays diverse roles as a nitric oxide (NO) generating system in conduit arteries and as an EDHF/H(2)O(2) generating system in microvessels. As compared with NO-mediated responses, those by EDHF are resistant to atherosclerosis, contributing to the maintenance of cardiovascular homeostasis. The aim of this study is to elucidate the molecular mechanisms for enhanced EDHF-mediated responses in microvessels. METHODS AND RESULTS: This study used male wild-type mice and caveolin-1-deficient mice (caveolin-1(-/-) mice). In the endothelium, eNOS was functionally suppressed in mesenteric arteries (microvessels) compared with the aorta (conduit arteries), for which Ca(2+)/calmodulin-dependent protein kinase kinase ß (CaMKKß) and caveolin-1 are involved, as EDHF-mediated responses were inhibited by STO-609 (an inhibitor of CaMKKß) and in caveolin-1(-/-) mice, respectively. In vascular smooth muscle, relaxation responses to H(2)O(2) were enhanced through a protein kinase G1α (PKG1α)-mediated mechanism in mesenteric arteries compared with the aorta, as they were inhibited by Rp-8-Br-cGMPS (an inhibitor of PKG1α). CONCLUSIONS: These results indicate that CaMKKß, caveolin-1, and PKG1α are substantially involved in the mechanisms for the enhanced EDHF-mediated responses in microvessels in mice.
Asunto(s)
Factores Biológicos/metabolismo , Endotelio Vascular/metabolismo , Mesenterio/irrigación sanguínea , Músculo Liso Vascular/metabolismo , Vasodilatación , Animales , Quinasa de la Proteína Quinasa Dependiente de Calcio-Calmodulina/antagonistas & inhibidores , Quinasa de la Proteína Quinasa Dependiente de Calcio-Calmodulina/metabolismo , Caveolina 1/deficiencia , Caveolina 1/genética , Proteína Quinasa Dependiente de GMP Cíclico Tipo I , Proteínas Quinasas Dependientes de GMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Endotelio Vascular/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microvasos/efectos de los fármacos , Microvasos/metabolismo , Músculo Liso Vascular/efectos de los fármacos , Óxido Nítrico Sintasa de Tipo III/metabolismo , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Transducción de Señal , Vasodilatación/efectos de los fármacos , Vasodilatadores/farmacologíaRESUMEN
We have previously demonstrated that endothelium-derived hydrogen peroxide (H(2)O(2)) plays an important role in the canine coronary microcirculation as an endothelium-derived hyperpolarizing factor in vivo. However, it remains to be examined whether endogenous H(2)O(2) is involved in the dilatation of coronary collaterals during myocardial ischemia in vivo and, if so, whether erythropoietin (EPO) enhances the responses. Canine subepicardial native collateral small arteries (CSA; ≥ 100 µm) and arterioles (CA; <100 µm) were observed using an intravital microscope. Experiments were performed after left anterior descending coronary artery ischemia (90 min) under the following eight conditions (n = 5 each): control, EPO, EPO+catalase, EPO+N-monomethyl-l-arginine (l-NMMA), EPO+l-NMMA+catalase, EPO+l-NMMA+iberiotoxin [Ca(2+)-activated K(+) (K(Ca)) channel blocker], EPO+l-NMMA+apamin+charybdotoxin (K(Ca) channel blocker), and EPO+wortmannin (phosphatidylinositol 3-kinase inhibitor). Myocardial ischemia caused significant vasodilatation in CA but not in CSA under control conditions, which was significantly decreased by catalase in CA. After EPO, the vasodilatation was significantly increased in both sizes of arteries and was significantly decreased by catalase. The enhancing effect of EPO was reduced by l-NMMA but not by catalase in CSA and was reduced by l-NMMA+catalase in CA, where the greater inhibitory effects were noted with l-NMMA+catalase, l-NMMA+iberiotoxin, L-NMMA+apamin+charybdotoxin, or wortmannin. EPO significantly ameliorated ischemia-induced impairment of myocardial Akt phosphorylation, which was abolished by l-NMMA+catalase or wortmannin. EPO also ameliorated oxidative stress and myocardial injury, as assessed by plasma 8-hydroxydeoxyguanosine and troponin-T, respectively. These results indicate that EPO enhances H(2)O(2)-mediated dilatation of coronary collateral arterioles during myocardial ischemia in dogs in vivo.
Asunto(s)
Circulación Colateral/efectos de los fármacos , Circulación Coronaria/efectos de los fármacos , Vasos Coronarios/efectos de los fármacos , Eritropoyetina/farmacología , Peróxido de Hidrógeno/metabolismo , Isquemia Miocárdica/fisiopatología , Vasodilatación/efectos de los fármacos , Vasodilatadores/farmacología , 8-Hidroxi-2'-Desoxicoguanosina , Animales , Antioxidantes/farmacología , Arteriolas/efectos de los fármacos , Arteriolas/metabolismo , Arteriolas/fisiopatología , Dióxido de Carbono/sangre , Vasos Coronarios/metabolismo , Vasos Coronarios/fisiopatología , Desoxiguanosina/análogos & derivados , Desoxiguanosina/sangre , Modelos Animales de Enfermedad , Perros , Inhibidores Enzimáticos/farmacología , Femenino , Masculino , Isquemia Miocárdica/metabolismo , Óxido Nítrico Sintasa de Tipo III/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo III/metabolismo , Oxígeno/sangre , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación , Bloqueadores de los Canales de Potasio/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Troponina T/sangreRESUMEN
BACKGROUND: The endothelium modulates vascular tone by synthesizing and releasing several vasodilating factors, including vasodilator prostaglandins, nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF). In the present study, we examined whether an angiotensin-receptor blocker, a calcium-channel blocker or their combination improved EDHF-mediated responses in diabetic apolipoprotein E-deficient (ApoE(-/-)) mice. METHODS AND RESULTS: We used male C57BL/6N (control) and streptozocin-induced diabetic ApoE(-/-) mice. The diabetic ApoE(-/-) mice were administered oral vehicle (untreated), olmesartan (OLM, 30 mgxkg(-1)xday(-1)), azelnidipine (AZL, 10 mgxkg(-1)xday(-1)), their combination (OLM + AZL), or hydralazine (HYD 5 mgxkg(-1)xday(-1)) for 5 weeks. In the untreated group, systolic blood pressure was significantly higher and both EDHF-mediated relaxation and endothelium-dependent hyperpolarization were markedly reduced as compared with the control group. Although EDHF-mediated relaxation was not significantly improved in the HYD, OLM and AZL groups, it was significantly improved in the OLM + AZL group, as was also the case with phosphorylation of Akt and endothelial NO synthase (eNOS). In contrast, the endothelium-independent relaxation response to sodium nitroprusside or NS-1619 (a direct opener of K(Ca) channels) was unaltered in any group. CONCLUSIONS: OLM + AZL may improve the severely impaired EDHF-mediated responses in diabetic ApoE(-/-) mice, in which activation of the endothelial Akt - eNOS pathway may be involved.
Asunto(s)
Apolipoproteínas E/deficiencia , Ácido Azetidinocarboxílico/análogos & derivados , Factores Biológicos/metabolismo , Diabetes Mellitus Experimental/metabolismo , Dihidropiridinas/uso terapéutico , Endotelio Vascular/metabolismo , Imidazoles/uso terapéutico , Tetrazoles/uso terapéutico , Bloqueadores del Receptor Tipo 1 de Angiotensina II/farmacología , Bloqueadores del Receptor Tipo 1 de Angiotensina II/uso terapéutico , Animales , Apolipoproteínas E/genética , Ácido Azetidinocarboxílico/farmacología , Ácido Azetidinocarboxílico/uso terapéutico , Bloqueadores de los Canales de Calcio/farmacología , Bloqueadores de los Canales de Calcio/uso terapéutico , Diabetes Mellitus Experimental/fisiopatología , Dihidropiridinas/farmacología , Modelos Animales de Enfermedad , Quimioterapia Combinada , Endotelio Vascular/efectos de los fármacos , Imidazoles/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Óxido Nítrico Sintasa de Tipo III/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/fisiología , Estreptozocina , Tetrazoles/farmacología , Vasodilatación/efectos de los fármacos , Vasodilatación/fisiologíaRESUMEN
BACKGROUND: Direct evidence for Rho-kinase activation in patients with pulmonary hypertension (PH) is still lacking. METHODS AND RESULTS: Rho-kinase activity in circulating neutrophils was examined by determining the ratio of phosphorylated/total forms of myosin-binding subunit, a substrate of Rho-kinase, in 40 consecutive PH patients and 40 healthy controls. Next, Rho-kinase expression and activity was examined in isolated human lung tissues (5 patients with idiopathic pulmonary arterial hypertension [IPAH], 5 controls) and vascular reactivity of isolated small human pulmonary arteries in vitro (4 IPAH, 4 controls). Rho-kinase activity in circulating neutrophils was significantly increased in the PH patients overall compared with controls (P<0.0001). Significant correlations were noted between Rho-kinase activity and the severity and duration of PAH (all P<0.05). Rho-kinase expression and activity in isolated lung tissues also were significantly increased in the IPAH patients compared with the controls (both P<0.0001). Endothelium-dependent relaxation was markedly impaired and serotonin-induced contraction (in the absence of the endothelium) markedly enhanced in the PAH patients compared with the controls, and the hypercontraction to serotonin was abolished by hydroxyfasudil, a specific Rho-kinase inhibitor. CONCLUSIONS: These results provide the first direct evidence for Rho-kinase activation in patients with PAH, suggesting the therapeutic importance of Rho-kinase in the disorder.
Asunto(s)
Hipertensión Pulmonar/enzimología , Pulmón/irrigación sanguínea , Neutrófilos/enzimología , Arteria Pulmonar/enzimología , Quinasas Asociadas a rho/metabolismo , Adulto , Estudios de Casos y Controles , Relación Dosis-Respuesta a Droga , Activación Enzimática , Femenino , Humanos , Hipertensión Pulmonar/fisiopatología , Masculino , Persona de Mediana Edad , Fosfatasa de Miosina de Cadena Ligera/metabolismo , Fosforilación , Estudios Prospectivos , Proteína Quinasa C-alfa/metabolismo , Proteína Quinasa C-delta/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Arteria Pulmonar/efectos de los fármacos , Arteria Pulmonar/fisiopatología , Índice de Severidad de la Enfermedad , Factores de Tiempo , Vasoconstricción , Vasoconstrictores/farmacología , Vasodilatación , Vasodilatadores/farmacología , Quinasas Asociadas a rho/antagonistas & inhibidoresRESUMEN
The endothelium synthesizes and releases several vasodilator substances, including prostacyclin, nitric oxide (NO), and endothelium-derived hyperpolarizing factor (EDHF). We have demonstrated that endothelium-derived hydrogen peroxide (H2O2) is an EDHF in animals and humans and that superoxide anions derived from endothelial nitric oxide synthases (NOSs) system are an important precursor for EDHF/H2O2 in mice. There are several intracellular sources of superoxide anions other than NOSs, including NAD(P)H oxidase, xanthine oxidase, lipoxygenase, and mitochondrial electron transport chain. In this study, we examined the possible role of endothelial oxidases other than NOSs in the EDHF-mediated responses. In angiotensin II-infused mice, both EDHF-mediated relaxations and hyperpolarizations to acetylcholine were significantly reduced, nitric oxide-mediated relaxations were rather enhanced, and vascular smooth muscle responses were preserved. Antihypertensive treatment normalized blood pressure but failed to improve EDHF-mediated responses in those mice. Acute inhibition of endothelial oxidases other than NOSs, including NAD(P)H oxidase, xanthine oxidase, lipoxygenase, or mitochondrial electron transport chain, had no inhibitory effects on EDHF-mediated responses. Furthermore, in p47phox-knockout mice, EDHF-mediated responses were unaltered. These results suggest that endothelial oxidases other than NOSs are not involved in EDHF/H2O2 responses in mice, suggesting a specific link between endothelial NOSs system and EDHF responses under physiological conditions.
Asunto(s)
Factores Biológicos/metabolismo , Endotelio Vascular/enzimología , Hipertensión/enzimología , Oxidorreductasas/metabolismo , Superóxidos/metabolismo , Vasodilatación , Angiotensina II , Animales , Antihipertensivos/farmacología , Presión Sanguínea , Catalasa/metabolismo , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/fisiopatología , Inhibidores Enzimáticos/farmacología , Glutatión Peroxidasa/metabolismo , Hipertensión/inducido químicamente , Hipertensión/tratamiento farmacológico , Hipertensión/fisiopatología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , NADPH Oxidasas/genética , NADPH Oxidasas/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Oxidorreductasas/antagonistas & inhibidores , Oxidorreductasas/genética , Superóxido Dismutasa/metabolismo , Vasodilatación/efectos de los fármacos , Vasodilatadores/farmacologíaRESUMEN
The endothelium plays an important role in maintaining vascular homeostasis by synthesizing and releasing several relaxing factors, such as prostacyclin, nitric oxide (NO), and endothelium-derived hyperpolarizing factor (EDHF). We have previously demonstrated in animals and humans that endothelium-derived hydrogen peroxide (H(2)O(2)) is an EDHF that is produced in part by endothelial NO synthase (eNOS). In this study, we show that genetic disruption of all three NOS isoforms (neuronal [nNOS], inducible [iNOS], and endothelial [eNOS]) abolishes EDHF responses in mice. The contribution of the NOS system to EDHF-mediated responses was examined in eNOS(-/-), n/eNOS(-/-), and n/i/eNOS(-/-) mice. EDHF-mediated relaxation and hyperpolarization in response to acetylcholine of mesenteric arteries were progressively reduced as the number of disrupted NOS genes increased, whereas vascular smooth muscle function was preserved. Loss of eNOS expression alone was compensated for by other NOS genes, and endothelial cell production of H(2)O(2) and EDHF-mediated responses were completely absent in n/i/eNOS(-/-) mice, even after antihypertensive treatment with hydralazine. NOS uncoupling was not involved, as modulation of tetrahydrobiopterin (BH(4)) synthesis had no effect on EDHF-mediated relaxation, and the BH(4)/dihydrobiopterin (BH(2)) ratio was comparable in mesenteric arteries and the aorta. These results provide the first evidence that EDHF-mediated responses are dependent on the NOSs system in mouse mesenteric arteries.
Asunto(s)
Endotelio Vascular/enzimología , Óxido Nítrico Sintasa/fisiología , Acetilcolina/metabolismo , Animales , Biopterinas/análogos & derivados , Biopterinas/farmacología , Electrofisiología/métodos , Femenino , Peróxido de Hidrógeno/farmacología , Masculino , Arterias Mesentéricas/metabolismo , Ratones , Ratones Transgénicos , Modelos Biológicos , Neuronas/metabolismo , Óxido Nítrico Sintasa/metabolismo , Isoformas de ProteínasRESUMEN
We have recently demonstrated that endothelium-derived hydrogen peroxide (H2O2) is an endothelium-derived hyperpolarizing factor and that endothelial Cu/Zn-superoxide dismutase (SOD) plays an important role in the synthesis of endogenous H2O2 in both animals and humans. We examined whether SOD plays a role in the synthesis of endogenous H2O2 during in vivo reactive hyperemia (RH), an important regulatory mechanism. Mesenteric arterioles from wild-type and Cu,Zn-SOD(-/-) mice were continuously observed by a pencil-type charge-coupled device (CCD) intravital microscope during RH (reperfusion after 20 and 60 s of mesenteric artery occlusion) in the cyclooxygenase blockade under the following four conditions: control, catalase alone, N(G)-monomethyl-L-arginine (L-NMMA) alone, and L-NMMA + catalase. Vasodilatation during RH was significantly decreased by catalase or L-NMMA alone and was almost completely inhibited by L-NMMA + catalase in wild-type mice, whereas it was inhibited by L-NMMA and L-NMMA + catalase in the Cu,Zn-SOD(-/-) mice. RH-induced increase in blood flow after L-NMMA was significantly increased in the wild-type mice, whereas it was significantly reduced in the Cu,Zn-SOD(-/-) mice. In mesenteric arterioles of the Cu,Zn-SOD(-/-) mice, Tempol, an SOD mimetic, significantly increased the ACh-induced vasodilatation, and the enhancing effect of Tempol was decreased by catalase. Vascular H(2)O(2) production by fluorescent microscopy in mesenteric arterioles after RH was significantly increased in response to ACh in wild-type mice but markedly impaired in Cu,Zn-SOD(-/-) mice. Endothelial Cu,Zn-SOD plays an important role in the synthesis of endogenous H(2)O(2) that contributes to RH in mouse mesenteric smaller arterioles.
Asunto(s)
Peróxido de Hidrógeno/metabolismo , Hiperemia/metabolismo , Mesenterio/irrigación sanguínea , Circulación Esplácnica , Superóxido Dismutasa/metabolismo , Vasodilatación , Vasodilatadores/metabolismo , Acetilcolina/farmacología , Animales , Arteriolas/metabolismo , Arteriolas/fisiopatología , Presión Sanguínea , Dióxido de Carbono/sangre , Catalasa/metabolismo , Óxidos N-Cíclicos/farmacología , Inhibidores de la Ciclooxigenasa/farmacología , Relación Dosis-Respuesta a Droga , Endotelio Vascular/metabolismo , Endotelio Vascular/fisiopatología , Inhibidores Enzimáticos/farmacología , Frecuencia Cardíaca , Hiperemia/enzimología , Hiperemia/fisiopatología , Indometacina/farmacología , Masculino , Ratones , Ratones Noqueados , Microscopía Fluorescente , Microscopía por Video , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico Sintasa/metabolismo , Oxígeno/sangre , Marcadores de Spin , Circulación Esplácnica/efectos de los fármacos , Superóxido Dismutasa/deficiencia , Superóxido Dismutasa/genética , Factores de Tiempo , Vasodilatación/efectos de los fármacos , Vasodilatadores/farmacología , omega-N-Metilarginina/farmacologíaRESUMEN
Pulmonary arterial hypertension is a progressive and fatal disease for which Rho-kinase may be substantially involved. In this study, we examined the acute vasodilator effects of fasudil, a Rho-kinase inhibitor, in monocrotaline (MCT)-induced pulmonary hypertension (PH) in rats. Three weeks after a single subcutaneous injection of MCT (60 mg/kg), hemodynamic variables were measured under conscious and free-moving conditions before and after oral administration of fasudil. MCT caused a significant elevation of mean pulmonary arterial pressure (mPAP). Although a low dose of fasudil (3 mg/kg) had no effect on mPAP, a middle dose (10 mg/kg) caused a significant reduction in mPAP without change in mean systemic arterial pressure (mSAP), and a high dose (30 mg/kg) significantly reduced both mPAP and mSAP. Rho-kinase activity was significantly increased by MCT injection in pulmonary arteries but not in the aorta. Fasudil (10 mg/kg) inhibited only the Rho-kinase activity in pulmonary arteries without any effect in the aorta. Plasma concentration of hydroxyfasudil, a metabolite of fasudil, was within its clinical range in humans. These results demonstrate that fasudil exerts effective and selective vasodilatation of pulmonary arteries in rats with MCT-induced PH at a given dose, suggesting its usefulness for the treatment of the fatal disorder.
Asunto(s)
1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , Inhibidores Enzimáticos/uso terapéutico , Hipertensión Pulmonar/tratamiento farmacológico , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Monocrotalina , Venenos , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacocinética , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/uso terapéutico , Animales , Presión Sanguínea/efectos de los fármacos , Western Blotting , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacocinética , Frecuencia Cardíaca/efectos de los fármacos , Hipertensión Pulmonar/inducido químicamente , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Masculino , Proteínas Serina-Treonina Quinasas/metabolismo , Arteria Pulmonar/efectos de los fármacos , Circulación Pulmonar/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Vasodilatación/efectos de los fármacos , Quinasas Asociadas a rhoRESUMEN
Vasospastic angina is induced by stress, for which cortisol secreted by activated hypothalamic/pituitary/adrenal axis may play an important role. However, direct evidence for this notion is still lacking. In this study, we examined whether sustained elevation of serum cortisol level sensitizes coronary vasoconstricting responses in pigs in vivo and, if so, whether Rho-kinase, which we found is a key molecule of coronary vasospasm, is involved. Oral administration of cortisol (20 mg/kg per day) increased its serum level to that seen in restraint stress in pigs. Thus, we examined coronary vasomotor responses in the following 4 groups: (1) control (without cortisol); (2) cortisol (20 mg/kg per day, p.o.) for 9 days; (3) cortisol plus RU38486 (a glucocorticoids receptor antagonist, 10 mg/kg per day, p.o.) for 9 days; and (4) cortisol for 9 days followed by 6-week withdrawal. Coronary angiography showed that intracoronary serotonin caused coronary hyperconstriction and reduction in coronary blood flow associated with ischemic ECG changes (coronary vasospasm) in only the cortisol group. All of these responses were abolished by hydroxyfasudil, a specific Rho-kinase inhibitor, in vivo. Organ chamber experiments demonstrated that serotonin concentration-dependently caused hypercontractions of coronary vascular smooth muscle associated with Rho-kinase activation (as evidenced by the enhanced phosphorylation of myosin binding subunit, a substrate of Rho-kinase) in only the cortisol group. All of these responses were again inhibited by hydroxyfasudil in vitro. These results indicate that sustained elevation of serum cortisol level sensitizes coronary vasoconstricting responses through Rho-kinase activation, suggesting the link between stress and coronary vasospasm.
Asunto(s)
Vasos Coronarios/fisiopatología , Hidrocortisona/sangre , Estrés Fisiológico/fisiopatología , Vasoconstricción , Administración Oral , Animales , Western Blotting , Vasoespasmo Coronario/etiología , Vasos Coronarios/efectos de los fármacos , Esquema de Medicación , Endotelio Vascular/fisiopatología , Hidrocortisona/administración & dosificación , Hidrocortisona/farmacología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Masculino , Proteínas Serina-Treonina Quinasas/metabolismo , Restricción Física , Serotonina/farmacología , Serotoninérgicos/farmacología , Estrés Fisiológico/sangre , Estrés Fisiológico/complicaciones , Estrés Fisiológico/etiología , Porcinos , Quinasas Asociadas a rhoAsunto(s)
Factores Biológicos/fisiología , Endotelio Vascular/fisiopatología , Animales , Arteriosclerosis/etiología , Diabetes Mellitus/etiología , Diabetes Mellitus/fisiopatología , Humanos , Peróxido de Hidrógeno , Hipertensión/etiología , Hipertensión/fisiopatología , Óxido Nítrico/fisiología , Factores de RiesgoRESUMEN
Long-term cadmium exposure leads to mitochondrial dysfunction in the proximal tubular epithelial cells. Mitochondrial DNA deletion may contribute to the pathogenesis of cadmium-induced nephropathy. The aim of our study is to clarify the accumulation of mitochondrial DNA deletion and mitochondrial dysfunction in the renal cortex of rats injected three times/week with 1 ml of 1 mM CdCl2 or saline for 80 weeks. After 40-week cadmium injection, mitochondrial number diminished, and cadmium in the renal cortex reached a saturation level. At this time interval, nearly 30% of cadmium in the whole cell fraction was found in the mitochondria. Cytochrome c oxidase (COX) activity in the proximal tubular epithelial cells decreased after 40-week exposure of cadmium. Oxidized phosphatidylcholine (oxPC) started to accumulate in the cytochrome c-positive mitochondria in some tubular epithelial cells after 80-week exposure. After 40 weeks, accumulation of the 4834-bp deletion in mitochondrial DNA was evident in both control and cadmium-treated groups. However, the amount of accumulated mitochondrial DNA deletion tended to increase after 40-week exposure, and was significantly greater after 80 weeks of exposure, compared to the control. Our results indicate that long-term cadmium exposure in rats accelerates accumulation of 4834-bp mitochondrial DNA deletions and impairment of mitochondrial function associated with accumulation of oxidized product.
Asunto(s)
Envejecimiento/efectos de los fármacos , Intoxicación por Cadmio/patología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/patología , Mitocondrias/efectos de los fármacos , Mitocondrias/patología , Adenosina Trifosfato/metabolismo , Animales , ADN Mitocondrial/biosíntesis , ADN Mitocondrial/metabolismo , Complejo IV de Transporte de Electrones/metabolismo , Femenino , Inmunohistoquímica , Corteza Renal/efectos de los fármacos , Corteza Renal/metabolismo , Túbulos Renales Proximales/efectos de los fármacos , Túbulos Renales Proximales/metabolismo , Oxidación-Reducción , Fosfatidilcolinas/metabolismo , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Cadmium (Cd) and arsenic (As) are known toxic metals in humans. As trioxide (As(2)0(3)) has been recently used as a mitochondria-targeting drug in acute promyelocytic leukemia. In the present study, we examined the intracellular action of these metals using rat kidney tubular cells and cells tolerant to the metals. The cells were cultured with CdCl(2) (1-10 micro M) or As(2)O(3) (1-2.5 micro M). Cells tolerant to Cd and As (Cd-T and As-T, respectively) were defined as cells that survived at toxic concentrations of each metal. Both Cd and As induced cell toxicity in a dose-dependent fashion, which was accompanied by fragmented DNA and decreased mitochondrial membrane potential. Intracellular glutathione (GSH) increased with the increase of Cd and As concentration. In Cd-T and As-T cells, GSH levels were twice those observed in normal cells. When each metal-tolerant culture was exposed to the other different metal, i.e., As or Cd, the protective property was maintained. However, when buthionine sulfoximine (BSO) was added to the metal-tolerant cultures, apoptosis was restored in both Cd-T and As-T. Our results indicate that (1) although GSH is increased in NRK52E by the addition of Cd and As, mitochondria-mediated apoptosis can be still induced, (2) the protective property against metal-induced cytotoxicity is identical in Cd-T and As-T cultures, and (3) although GSH was higher in the metal-tolerant cell lines, depression of GSH by BSO induced apoptosis. We conclude that Cd- and As-induced apoptosis is mediated by an identical mechanism involving intracellular GSH reactive oxidation.
Asunto(s)
Arsénico/toxicidad , Cadmio/toxicidad , Muerte Celular/efectos de los fármacos , Animales , Línea Celular , Fragmentación del ADN , Relación Dosis-Respuesta a Droga , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Glutatión/antagonistas & inhibidores , Glutatión/metabolismo , Humanos , Túbulos Renales/citología , Peroxidación de Lípido , Potenciales de la Membrana/fisiología , Metalotioneína/metabolismo , Mitocondrias/metabolismo , Oxidación-Reducción , RatasRESUMEN
Long-term exposure to cadmium (Cd) induces perturbation of kidney proximal tubular epithelial cells. Mitochondrial dysfunction in renal cortical cells may contribute to the pathogenesis of Cd-induced nephropathy. In this study, we examined the accumulation of mitochondrial DNA (mtDNA) with a large deletion and cellular senescence in the renal cortex. Wistar rats at 8 weeks of age were intraperitoneally injected with 1 mL of 1 mM CdCl(2) or saline, 3 times/week for 5, 20, 40, or 80 weeks. Mitochondrial Cd content in the renal cortex was quantified by atomic absorption analysis. Cytochrome c oxidase (CCO) and senescence-associated beta-galactosidase (SA-beta-gal) activity were determined in renal cortex by enzyme-histochemistry. mtDNA in total DNA extracted from the renal cortex was amplified by PCR, and mtDNA deletions, including 4,834-bp (nt8118-nt12937) deletion, were determined and semiquantified. After 40 weeks of Cd injection, Cd levels in the renal cortex reached a saturation level, and 30% of the level of the whole-cell fraction was found in the mitochondria. CCO activity in the renal cortex, which was predominantly found in proximal tubular cells, decreased after 40 weeks of Cd exposure. Expression of SA-beta-gal was detected primarily in the proximal tubular cells and significantly increased after 80 weeks of Cd exposure. After 40 weeks of study, accumulation of 4,834-bp deletion in mtDNA was evident in both groups of rats; however, the amount of the deletion was significantly greater in Cd-treated rats than in control rats. Our results indicate that long-term Cd exposure induced a post-regenerative state of proximal tubular cells, which accelerated accumulation of 4,834-bp mtDNA deletions in the renal cortex, suggesting that Cd may be a senescence acceleration factor for kidney proximal tubular epithelial cells, which results in Cd-induced nephropathy.
Asunto(s)
Cadmio/toxicidad , Senescencia Celular/fisiología , ADN Mitocondrial/análisis , ADN Mitocondrial/genética , Túbulos Renales Proximales/efectos de los fármacos , Túbulos Renales Proximales/metabolismo , Eliminación de Secuencia , beta-Galactosidasa/metabolismo , Animales , Complejo IV de Transporte de Electrones/metabolismo , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Femenino , Humanos , Corteza Renal/química , Corteza Renal/citología , Túbulos Renales Proximales/citología , Ratas , Ratas Wistar , Estadística como AsuntoRESUMEN
BACKGROUND: The pathogenesis of itai-itai disease continues to be controversial, although cadmium (Cd) poisoning which arises via polluted water and rice in Japan is likely involved. Until recently, however, a well-defined animal model for Cd intoxication was not available. An animal model for itai-itai disease was produced in rats by low-dose Cd treatment, intraperitoneally for a period of 70-80 weeks. Osteomalacia followed the renal damage. RESULTS: A gene deletion in the mitochondrial DNA was found in the mitochondria of the proximal tubule cells of rats with chronic Cd intoxication, as was shown by the increased smaller PCR product seen by gel electrophoresis in one DNA region, where ATPase and cytochrome oxidase genes are located. However, the PCR product was different from that seen with a gene deletion associated with aging: del4834bp. Renal damage from Cd intoxication initially caused mitochondrial dysfunction indicated by the disturbance in reabsorption in the proximal tubules and decreased amounts of ATP, ATPase, and cytochrome oxidase with gradually progressing tubular proteinuria, and, finally, chronic renal failure with tubulointerstitial damage throughout the renal cortex. These gave rise to osteomalacia, subsequently. CONCLUSION: We concluded that in Cd poisoning, a mitochondrial gene deletion in the mitochondria of the proximal tubule cells was the primary event for the pathogenesis of osteomalacia in itai-itai disease.