RESUMEN
Preeclampsia is a serious complication of pregnancy characterized by the development of vasospasm, hypertension and often associated with proteinuria after the 20th week of gestation. Because termination of pregnancy results in the most efficacious resolution of preeclampsia, it is a leading cause of premature delivery worldwide. In pregnancy, 14,15-epoxyeicosatrienoic acids (EETs) have been shown to facilitate uterine blood flow during preeclampsia, in which the classic vasodilator agents such as nitric oxide and prostacyclin are reduced. EETs are converted to dihydroxyeicosatrienoic acids (DHETs) by the activity of soluble epoxide hydrolase (sEH). We tested the hypothesis that sEH activity is increased in preeclampsia by measuring urinary 14,15-DHET in healthy and preeclamptic pregnant women. Urine samples were collected and incubated with or without ß-glucuronidase to enable the measurement of both the glucuronidated and free forms of 14,15-DHET, which were quantified using a 14,15-DHET ELISA. Levels of total (free+glucuronidated) 14,15-DHET, which is a measurement of EET-dependent sEH activity, were higher in urine samples obtained from preeclamptic women compared to healthy pregnant women. Considering the fact that free+glucuronidated 14,15-DHET levels are increased in urine of preeclamptic women, we hypothesize that sEH expression or activity is augmented in these patients, reducing EET and increasing blood pressure. Moreover we suggest that novel anti-hypertensive agents that target sEH might be developed as therapeutics to control high blood pressure in women with preeclampsia.
Asunto(s)
Epóxido Hidrolasas/sangre , Preeclampsia/sangre , Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Ácido 8,11,14-Eicosatrienoico/sangre , Ácido 8,11,14-Eicosatrienoico/orina , Adulto , Antihipertensivos/farmacología , Presión Sanguínea , Epoprostenol/sangre , Femenino , Glucuronidasa/sangre , Humanos , Hipertensión/tratamiento farmacológico , Edad Materna , Óxido Nítrico/sangre , Embarazo , Complicaciones del Embarazo/sangre , Vasoconstricción , Vasodilatadores/farmacología , Adulto JovenRESUMEN
Cytochrome P-450 (CYP) epoxygenases metabolize arachidonic acid into epoxyeicosatrienoic acids (EETs), which play important roles in regulating cardiovascular functions. The anti-inflammatory, antiapoptotic, proangiogenic, and antihypertensive properties of EETs suggest a beneficial role for EETs in diabetic nephropathy. Endogenous EET levels are maintained by a balance between synthesis by CYP epoxygenases and hydrolysis by epoxide hydrolases into physiologically less active dihydroxyeicosatrienoic acids. Genetic disruption of soluble epoxide hydrolase (sEH/EPHX2) results in increased EET levels through decreased hydrolysis. This study investigated the effects of sEH gene disruption on diabetic nephropathy in streptozotocin-induced diabetic mice. Streptozotocin-induced diabetic manifestations were attenuated in sEH-deficient mice relative to wild-type controls, with significantly decreased levels of Hb A(1c), creatinine, and blood urea nitrogen and urinary microalbumin excretion. The sEH-deficient diabetic mice also had decreased renal tubular apoptosis that coincided with increased levels of antiapoptotic Bcl-2 and Bcl-xl, and decreased levels of the proapoptotic Bax. These effects were associated with activation of the PI3K-Akt-NOS3 and AMPK signaling cascades. sEH gene inhibition and exogenous EETs significantly protected HK-2 cells from TNFα-induced apoptosis in vitro. These findings highlight the beneficial role of the CYP epoxygenase-EETs-sEH system in the pathogenesis of diabetic nephropathy and suggest that the sEH inhibitors available may be potential therapeutic agents for this condition.
Asunto(s)
Citoplasma/enzimología , Nefropatías Diabéticas/metabolismo , Epóxido Hidrolasas/metabolismo , Túbulos Renales Proximales/metabolismo , Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Ácido 8,11,14-Eicosatrienoico/metabolismo , Ácido 8,11,14-Eicosatrienoico/farmacología , Ácido 8,11,14-Eicosatrienoico/orina , Albuminuria/prevención & control , Animales , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/metabolismo , Línea Celular Transformada , Citoplasma/efectos de los fármacos , Citoplasma/metabolismo , Nefropatías Diabéticas/sangre , Nefropatías Diabéticas/tratamiento farmacológico , Nefropatías Diabéticas/orina , Modelos Animales de Enfermedad , Epóxido Hidrolasas/antagonistas & inhibidores , Epóxido Hidrolasas/genética , Silenciador del Gen , Humanos , Hiperglucemia/prevención & control , Corteza Renal/efectos de los fármacos , Corteza Renal/metabolismo , Corteza Renal/patología , Túbulos Renales Proximales/efectos de los fármacos , Túbulos Renales Proximales/patología , Ratones , Terapia Molecular Dirigida , ARN Interferente Pequeño , Transducción de Señal/efectos de los fármacos , Estreptozocina , Factor de Necrosis Tumoral alfaRESUMEN
We have previously established a cytochrome P450 4F2 (CYP4F2) transgenic mouse model. The present study elucidated the molecular foundation of hypertension by androgen-induction in this model. The renal expression of CYP4F2 in transgenic mice was highly expressed and strongly induced with 5α-dihydrotestosterone (DHT) treatment determined by Western blot. DHT also increased the renal arachidonic acid ω-hydroxylation and urinary 20-hydroxyeicosatetraenoic acid (20-HETE) excretion (P<0.01), and furthermore elevated the systolic blood pressure by 10 and 22 mm Hg (P<0.05) in female and castrated male transgenic mice, respectively. HET0016 completely eliminated the androgen-induced effects (P<0.01). Endogenous Cyp4a ω-hydroxylases, evaluated by real-time quantitative PCR, were significantly suppressed in transgenic mice (P<0.05). Importantly, transgenic mice with increased 20-HETE showed decreased epoxyeicosatrienoic acids (EETs) and increased dihydroxyeicosatetraenoic acids determined by liquid chromatography-tandem mass spectrometry, contributing to significantly raised ratio of 20-HETE/EETs in the urine and kidney homogenate (P<0.01). These data demonstrate that the androgen aggravated hypertension possibly through an altered ratio of 20-HETE/EETs in CYP4F2 transgenic mice.
Asunto(s)
Ácido 8,11,14-Eicosatrienoico/metabolismo , Andrógenos/efectos adversos , Sistema Enzimático del Citocromo P-450/metabolismo , Ácidos Hidroxieicosatetraenoicos/metabolismo , Hipertensión/inducido químicamente , Riñón/metabolismo , Ácido 8,11,14-Eicosatrienoico/orina , Animales , Citocromo P-450 CYP4A/metabolismo , Sistema Enzimático del Citocromo P-450/genética , Femenino , Ácidos Hidroxieicosatetraenoicos/orina , Masculino , Ratones , Ratones TransgénicosRESUMEN
Epoxyeicosatrienoic acids (EET), the primary arachidonic acid metabolites of cytochrome P450 2J (CYP2J) epoxygenases, possess potent vasodilatory, anti-inflammatory, antiapoptotic, and mitogenic effects. To date, little is known about the role of CYP2J2 and EETs in tumor necrosis factor (TNF)-α-induced cardiac injury. We utilized cell culture and in vivo models to examine the effects of exogenously applied EETs or CYP2J2 overexpression on TNF-α-induced cardiac apoptosis and cardiac dysfunction. In neonatal rat cardiomyocytes, TNF-α-induced apoptosis was markedly attenuated by EETs or CYP2J2 overexpression, leading to significantly improved cell survival. Further studies showed that TNF-α decreased expression of the antiapoptotic proteins Bcl-2 and Bcl-xL, decreased IκBα and PPARγ, and also inhibited PI3K-dependent Akt and EGFR signaling. Both EETs and CYP2J2 overexpression reversed the effects of TNF-α on these pathways. Furthermore, overexpression of CYP2J2 in rats prevented the decline in cardiac function that is normally observed in TNF-α-challenged animals. These results demonstrate that EETs or CYP2J2 overexpression can prevent TNF-α-induced cardiac cell injury and cardiac dysfunction by inhibiting apoptosis, reducing inflammation, and enhancing PPARγ expression. Targeting the CYP2J2 epoxygenase pathway may represent a novel approach to mitigate cardiac injury in diseases such as heart failure, where increased TNF-α levels are known to occur.
Asunto(s)
Apoptosis/efectos de los fármacos , Corazón/efectos de los fármacos , Factor de Necrosis Tumoral alfa/toxicidad , Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Ácido 8,11,14-Eicosatrienoico/orina , Animales , Animales Modificados Genéticamente , Animales Recién Nacidos , Línea Celular , Células Cultivadas , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Eicosanoides , Citometría de Flujo , Hemodinámica/efectos de los fármacos , Masculino , Receptores Activados del Proliferador del Peroxisoma/genética , Receptores Activados del Proliferador del Peroxisoma/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
The cytochrome P450 epoxygenase, CYP2J2, converts arachidonic acid to four regioisomeric epoxyeicosatrienoic acids (EETs), which are highly abundant in the kidney and considered renoprotective. Accumulating evidence suggests that EETs are important in regulating renal and cardiovascular function. Further, EETs have been confirmed to exert diverse biological activities including potent vasodilation; fibrinolytic properties; and antiinflammatory, antiapoptotic, and mitogenic effects. In the current study, we investigated the effects of overexpression of CYP2J2 via recombinant adeno-associated virus (rAAV) in protection against renal damage in a rat 5/6 nephrectomy (5/6-Nx) model of chronic renal failure. The rAAV-CYP2J2 gene delivery in vivo increased EET generation; attenuated the rise in blood pressure; and reduced the levels of proteinuria, serum creatinine, and blood urea nitrogen. Morphological analysis indicated that rAAV-CYP2J2 gene delivery reduced 5/6 nephrectomy-induced glomerular sclerosis, tubular dilatation, luminal protein cast formation, and tubulointerstitial fibrosis. rAAV-CYP2J2 gene delivery also significantly lowered collagen I and IV deposition, as well as renal cell apoptosis detected by TUNEL staining, caspase-3 activity, and the loss of mitochondrial membrane potential (ΔΨ(m)). Furthermore, rAAV-CYP2J2 gene delivery regulated the level of protein expression including transforming growth factor (TGF)-ß(1)/SMADs; matrix metalloproteinases (MMPs); mitogen-activated protein kinases (MAPKs); and apoptosis-related proteins Bax, Bcl-2, and Bcl-x(L). Together, these findings demonstrated that rAAV-CYP2J2 gene delivery can protect remnant kidney against renal injury in 5/6-Nx rats by inhibiting apoptosis and fibrosis via regulation of protein expression including TGF-ß(1)/SMADs, MMPs, MAPKs, and apoptosis-related proteins.
Asunto(s)
Apoptosis , Sistema Enzimático del Citocromo P-450/genética , Dependovirus/genética , Terapia Genética , Riñón/patología , Insuficiencia Renal Crónica/terapia , Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Ácido 8,11,14-Eicosatrienoico/orina , Animales , Presión Sanguínea , Colágeno/metabolismo , Citocromo P-450 CYP2J2 , Sistema Enzimático del Citocromo P-450/biosíntesis , Fibrosis , Técnicas de Transferencia de Gen , Riñón/metabolismo , Riñón/fisiopatología , Masculino , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Proteínas Quinasas Activadas por Mitógenos , Nefrectomía , Ratas , Ratas Wistar , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Insuficiencia Renal Crónica/patología , Insuficiencia Renal Crónica/orina , Proteínas Smad/metabolismoRESUMEN
Cytochrome P450 (CYP) epoxygenases metabolize arachidonic acid into epoxyeicosatrienoic acids (EETs), which play important and diverse roles in the cardiovascular system. The anti-inflammatory, anti-apoptotic, pro-angiogenic, and anti-hypertensive properties of EETs in the cardiovascular system suggest a beneficial role for EETs in diabetic nephropathy. This study investigated the effects of endothelial specific overexpression of CYP2J2 epoxygenase on diabetic nephropathy in streptozotocin-induced diabetic mice. Endothelial CYP2J2 overexpression attenuated renal damage as measured by urinary microalbumin and glomerulosclerosis. These effects were associated with inhibition of TGF-ß/Smad signaling in the kidney. Indeed, overexpression of CYP2J2 prevented TGF-ß1-induced renal tubular epithelial-mesenchymal transition in vitro. These findings highlight the beneficial roles of the CYP epoxygenase-EET system in the pathogenesis of diabetic nephropathy.
Asunto(s)
Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Sistema Enzimático del Citocromo P-450/metabolismo , Diabetes Mellitus Experimental/sangre , Nefropatías Diabéticas/sangre , Riñón/metabolismo , Transducción de Señal , Factor de Crecimiento Transformador beta1/farmacología , Ácido 8,11,14-Eicosatrienoico/orina , Animales , Ácido Araquidónico/sangre , Ácido Araquidónico/orina , Línea Celular , Citocromo P-450 CYP2J2 , Sistema Enzimático del Citocromo P-450/sangre , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/orina , Diabetes Mellitus Experimental/inducido químicamente , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/fisiopatología , Diabetes Mellitus Experimental/orina , Nefropatías Diabéticas/inducido químicamente , Nefropatías Diabéticas/complicaciones , Nefropatías Diabéticas/fisiopatología , Nefropatías Diabéticas/orina , Transición Epitelial-Mesenquimal/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Riñón/efectos de los fármacos , Riñón/fisiopatología , Pruebas de Función Renal , Ratones , Ratones Transgénicos , Proteínas Smad/sangre , Proteínas Smad/genética , Proteínas Smad/orina , Estreptozocina/administración & dosificación , Estreptozocina/efectos adversosRESUMEN
Cytochrome P450 (P450)-derived epoxyeicosatrienoic acids (EETs) exert well recognized vasodilatory, diuretic, and tubular fluid-electrolyte transport actions that are predictive of a hypotensive effect. The study sought to determine the improvement of hypertension and cardiac function by overexpressing P450 epoxygenases in vivo. Long-term expression of CYP102 F87V or CYP2J2 in spontaneously hypertensive rats (SHR) was mediated by using a type 8 recombinant adeno-associated virus (rAAV8) vector. Hemodynamics was measured by a Millar Instruments, Inc. (Houston, TX) microtransducer catheter, and atrial natriuretic peptide (ANP) mRNA levels were tested by real-time polymerase chain reaction. Results showed that urinary excretion of 14,15-EET was increased at 2 and 6 months after injection with rAAV-CYP102 F87V and rAAV-CYP2J2 compared with controls (p < 0.05). During the course of the 6-month study, systolic blood pressure significantly decreased in P450 epoxygenase-treated rats, but the CYP2J2-specific inhibitor C26 blocked rAAV-CYP2J2-induced hypotension and the increase in EET production. Cardiac output was improved by P450 epoxygenase expression at 6 months (p < 0.05). Furthermore, cardiac collagen content was reduced in P450 epoxygenase-treated rats. ANP mRNA levels were up-regulated 6- to 14-fold in the myocardium, and ANP expression was significantly increased in both myocardium and plasma in P450 epoxygenase-treated rats. However, epidermal growth factor (EGF) receptor antagonist 4-(3'-chloroanilino)-6,7-dimethoxy-quinazoline (AG-1478) significantly attenuated the increase in the EET-induced expression of ANP in vitro. These data indicate that overexpression of P450 epoxygenases attenuates the development of hypertension and improves cardiac function in SHR, and that these effects may be mediated, at least in part, by ANP via activating EGF receptor.
Asunto(s)
Factor Natriurético Atrial/fisiología , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/fisiología , Hipertensión/genética , Hipertensión/fisiopatología , Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Ácido 8,11,14-Eicosatrienoico/metabolismo , Ácido 8,11,14-Eicosatrienoico/orina , Adenoviridae/genética , Animales , Aorta Torácica/efectos de los fármacos , Factor Natriurético Atrial/genética , Presión Sanguínea/genética , Presión Sanguínea/fisiología , Western Blotting , Citocromo P-450 CYP2J2 , Sistema Enzimático del Citocromo P-450/biosíntesis , Vectores Genéticos , Pruebas de Función Cardíaca , Hemodinámica/genética , Hemodinámica/fisiología , Inmunohistoquímica , Técnicas In Vitro , Masculino , Relajación Muscular/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Ratas , Ratas Endogámicas SHR , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Pulmonary arterial hypertension (PAH) is a life-threatening disease that leads to progressive pulmonary hypertension, right heart failure, and death. Endothelial dysfunction and inflammation were implicated in the pathogenesis of PAH. Epoxyeicosatrienoic acids (EETs), products of the cytochrome P450 epoxygenase metabolism of arachidonic acid, are potent vasodilators that possess anti-inflammatory and other protective properties in endothelial cells. We investigated whether gene delivery with the human cytochrome P450 epoxygenase 2J2 (CYP2J2) ameliorates monocrotaline (MCT)-induced pulmonary hypertension in rats. Significant pulmonary hypertension developed 3 weeks after the administration of MCT, but gene therapy with CYP2J2 significantly attenuated the development of pulmonary hypertension and pulmonary vascular remodeling, without causing changes in systemic arterial pressure or heart rate. These effects were associated with increased pulmonary endothelial NO synthase (eNOS) expression and its activity, inhibition of inflammation in the lungs, and transforming growth factor (TGF)-ß/type II bone morphogenetic protein receptor (BMPRII)-drosophila mothers against decapentaplegic proteins (Smads) signaling. Collectively, these data suggest that gene therapy with CYP2J2 may have potential as a novel therapeutic approach to this progressive and oftentimes lethal disorder.
Asunto(s)
Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/uso terapéutico , Técnicas de Transferencia de Gen , Terapia Genética , Hipertensión Pulmonar/terapia , Arteria Pulmonar/patología , Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Ácido 8,11,14-Eicosatrienoico/metabolismo , Ácido 8,11,14-Eicosatrienoico/orina , Animales , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/metabolismo , Citocromo P-450 CYP2J2 , Células Endoteliales/efectos de los fármacos , Células Endoteliales/enzimología , Hemodinámica/efectos de los fármacos , Humanos , Hipertensión Pulmonar/sangre , Hipertensión Pulmonar/complicaciones , Hipertensión Pulmonar/fisiopatología , Hipertrofia Ventricular Derecha/sangre , Hipertrofia Ventricular Derecha/complicaciones , Hipertrofia Ventricular Derecha/fisiopatología , Hipertrofia Ventricular Derecha/orina , Interleucina-10/sangre , Interleucina-6/sangre , Monocrotalina , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico Sintasa de Tipo III/metabolismo , Arteria Pulmonar/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Receptores del Factor de Crecimiento Derivado de Plaquetas/metabolismo , Transducción de Señal/efectos de los fármacos , Análisis de Supervivencia , Extractos de Tejidos/metabolismo , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
OBJECTIVE: Accumulating evidence suggests that cytochrome P450 (CYP) epoxygenases metabolize arachidonic acid into epoxyeicosatrienoic acids (EETs), which play crucial and diverse roles in cardiovascular homeostasis. The anti-inflammatory, antihypertensive, and pro-proliferative effects of EETs suggest a possible beneficial role for EETs on insulin resistance and diabetes. RESEARCH DESIGN AND METHODS: This study investigated the effects of CYP2J3 epoxygenase gene therapy on insulin resistance and blood pressure in diabetic db/db mice and in a model of fructose-induced hypertension and insulin resistance in rats. RESULTS: CYP2J3 gene delivery in vivo increased EET generation, reduced blood pressure, and reversed insulin resistance as determined by plasma glucose levels, homeostasis model assessment insulin resistance index, and glucose tolerance test. Furthermore, CYP2J3 treatment prevented fructose-induced decreases in insulin receptor signaling and phosphorylation of AMP-activated protein kinases (AMPKs) in liver, muscle, heart, kidney, and aorta. Thus, overexpression of CYP2J3 protected against diabetes and insulin resistance in peripheral tissues through activation of insulin receptor and AMPK pathways. CONCLUSIONS: These results highlight the beneficial roles of the CYP epoxygenase-EET system in diabetes and insulin resistance.
Asunto(s)
Sistema Enzimático del Citocromo P-450/genética , Fructosa/uso terapéutico , Síndrome Metabólico/prevención & control , Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Ácido 8,11,14-Eicosatrienoico/orina , Animales , Presión Sanguínea , Cartilla de ADN , Endotelina-1/genética , Ensayo de Inmunoadsorción Enzimática , Regulación de la Expresión Génica , Prueba de Tolerancia a la Glucosa , Hipertensión/genética , Hipertensión/prevención & control , Resistencia a la Insulina/genética , Resistencia a la Insulina/fisiología , Síndrome Metabólico/genética , Ratones , Óxido Nítrico Sintasa de Tipo III/genética , ARN Mensajero/genética , Ratas , Receptor de Endotelina A/genéticaRESUMEN
The cytochrome P450-derived epoxyeicosatrienoic acids (EETs) have potent effects on renal vascular reactivity and tubular sodium and water transport; however, the role of these eicosanoids in the pathogenesis of hypertension is controversial. The current study examined the hydrolysis of the EETs to the corresponding dihydroxyeicosatrienoic acids (DHETs) as a mechanism for regulation of EET activity and blood pressure. EET hydrolysis was increased 5- to 54-fold in renal cortical S9 fractions from the spontaneously hypertensive rat (SHR) relative to the normotensive Wistar-Kyoto (WKY) rat. This increase was most significant for the 14,15-EET regioisomer, and there was a clear preference for hydrolysis of 14, 15-EET over the 8,9- and 11,12-EETs. Increased EET hydrolysis was consistent with increased expression of soluble epoxide hydrolase (sEH) in the SHR renal microsomes and cytosol relative to the WKY samples. The urinary excretion of 14,15-DHET was 2.6-fold higher in the SHR than in the WKY rat, confirming increased EET hydrolysis in the SHR in vivo. Blood pressure was decreased 22+/-4 mm Hg (P:<0.01) 6 hours after treatment of SHRs with the selective sEH inhibitor N:, N:'-dicyclohexylurea; this treatment had no effect on blood pressure in the WKY rat. These studies identify sEH as a novel therapeutic target for control of blood pressure. The identification of a potent and selective inhibitor of EET hydrolysis will be invaluable in separating the vascular effects of the EET and DHET eicosanoids.
Asunto(s)
Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Ácidos Araquidónicos/metabolismo , Epóxido Hidrolasas/metabolismo , Compuestos Epoxi/metabolismo , Hipertensión/metabolismo , Ácido 8,11,14-Eicosatrienoico/metabolismo , Ácido 8,11,14-Eicosatrienoico/orina , Animales , Ácidos Araquidónicos/farmacología , Presión Sanguínea/efectos de los fármacos , Citosol/metabolismo , Eicosanoides/metabolismo , Eicosanoides/farmacología , Inhibidores Enzimáticos/farmacología , Epóxido Hidrolasas/antagonistas & inhibidores , Epóxido Hidrolasas/farmacología , Hidrólisis/efectos de los fármacos , Hipertensión/etiología , Corteza Renal/enzimología , Masculino , Microsomas/enzimología , Microsomas Hepáticos/enzimología , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Ratas Sprague-Dawley , Especificidad de la Especie , Urea/análogos & derivados , Urea/farmacologíaRESUMEN
Epoxyeicosatrienoic acids (EETs) are major products of cytochrome P450 (CYP)-catalyzed metabolism of arachidonic acid in the kidney. The potent effect of EETs on renal vascular tone and tubular ion and water transport implicates their role in the regulation of renal function and blood pressure. The present study was designed to test the hypothesis that CYP-catalyzed EET formation was altered in the spontaneously hypertensive rat (SHR) kidney. The formation of 14,15- and 11,12-EET was approximately 2-fold higher in incubations of arachidonic acid with SHR renal cortical microsomes relative to microsomes from normotensive Wistar-Kyoto (WKY) rats. This was consistent with increased expression of a CYP2J2 immunoreactive protein in the SHR cortex and outer medulla. In contrast, there was no significant difference in the levels of the CYP2E and CYP2C epoxygenases in SHR and WKY kidneys. Protein and RNA analysis suggests that the CYP2J2 immunoreactive protein that is overexpressed in the SHR kidney is distinct from the known rat CYP2J isoforms. EET formation also was documented in vivo from measurements of urinary EET excretion. Importantly, the excretion rates of 14,15-, and 11,12-EETs were 2.5- and 1.8-fold higher, respectively, in SHR than WKY kidney. These studies provide both in vitro and in vivo evidence for increased EET formation in the SHR kidney and identify a novel CYP2J2 immunoreactive protein that is differentially expressed in the hypertensive kidney. In light of the known biological properties of the EETs, these findings may be important in elucidating the mechanisms that control renal vascular tone and tubular ion transport in the SHR.
Asunto(s)
Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Sistema Enzimático del Citocromo P-450/biosíntesis , Hipertensión/enzimología , Riñón/enzimología , Oxigenasas/biosíntesis , Ácido 8,11,14-Eicosatrienoico/orina , Animales , Citocromo P-450 CYP2J2 , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Hipertensión/metabolismo , Riñón/metabolismo , Hígado/enzimología , Hígado/metabolismo , Masculino , Oxigenasas/genética , Oxigenasas/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKYRESUMEN
The rat kidney microsomal epoxygenase catalyzed the asymmetric epoxidation of arachidonic acid to generate as major products: 8(R),9(S)-, 11(R),12(S)- and 14(S),15(R)-epoxyeicosatrienoic acids with optical purities of 97, 88, and 70%, respectively. Inhibition studies utilizing a panel of polyclonal antibodies to several rat liver cytochrome P-450 isoforms, indicated that the renal epoxygenase(s) belongs to the cytochrome P-450 2C gene family. Dietary salt, administered either as a 2-2.5% (w/v) solution in the drinking water or as a modified solid diet containing 8% NaCl (w/w), resulted in marked and selective increases in the renal microsomal epoxygenase activity (416 and 260% of controls, for the liquid and solid forms of NaCl, respectively) with no significant changes in the microsomal omega/omega-1 oxygenase or in the hepatic arachidonic acid monooxygenase reaction. Immunoblotting studies demonstrated that dietary salt induced marked increases in the concentration of a cytochrome P-450 isoform(s) recognized by polyclonal antibodies raised against human liver cytochrome P-450 2C10 or rat liver cytochrome P-450 2C11. Comparisons of the stereochemical selectivity of the induced and non-induced microsomal epoxygenase(s) with that of purified rat liver cytochrome P-450 2C11 suggest that the salt-induced protein(s) is catalytically and structurally different from liver cytochrome P-450 2C11. The in vivo significance of dietary salt in regulating the activities of the kidney endogenous arachidonic acid epoxygenase was established by the demonstration of a salt-induced 10-20-fold increase in the urinary output of epoxygenase metabolites. These results, in conjunction with published evidence demonstrating the potent biological activities of its metabolites, suggest a role for the epoxygenase in the renal response to dietary salt.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Dieta , Riñón/enzimología , Oxigenasas/metabolismo , Cloruro de Sodio/farmacología , Ácido 8,11,14-Eicosatrienoico/orina , Animales , Cromatografía Líquida de Alta Presión , Citocromo P-450 CYP2J2 , Sistema Enzimático del Citocromo P-450/genética , Cromatografía de Gases y Espectrometría de Masas , Ácidos Hidroxieicosatetraenoicos/orina , Inmunoelectroforesis , Masculino , Espectrometría de Masas , Microsomas/enzimología , Familia de Multigenes , Oxigenasas/genética , Ratas , Ratas Sprague-DawleyAsunto(s)
Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Angina Inestable/metabolismo , Ácidos Araquidónicos/metabolismo , Preeclampsia/metabolismo , 1-Metil-3-Isobutilxantina/farmacología , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico , Ácido 8,11,14-Eicosatrienoico/metabolismo , Ácido 8,11,14-Eicosatrienoico/farmacología , Ácido 8,11,14-Eicosatrienoico/orina , Angioplastia Coronaria con Balón , Ácido Araquidónico , Colforsina/farmacología , AMP Cíclico/farmacología , Citocromo P-450 CYP2J2 , Sistema Enzimático del Citocromo P-450/metabolismo , Femenino , Humanos , Oxigenasas/metabolismo , Activación Plaquetaria/efectos de los fármacos , Embarazo/metabolismo , Endoperóxidos de Prostaglandinas Sintéticos/farmacología , Trombina/farmacologíaRESUMEN
Arachidonic acid is metabolized by means of P450 isoenzyme(s) to form epoxyeicosatrienoic acids (EETs) and their corresponding dihydroxy derivatives (DHETs). In the present study, we established the presence in human urine of 8,9-, 11,12-, and 14,15-EETs and their corresponding DHETs by developing quantitative assays and using negative ion, chemical ionization GC/MS and octadeuterated internal standards. Urinary excretion of 8,9- and 11,12-DHET increased in healthy pregnant women compared with nonpregnant female volunteers. By contrast, excretion of 11,12-DHET and 14,15-DHET, but not the 8,9-DHET regioisomer, increased even further in patients with pregnancy-induced hypertension. Intravenous administration of [3H]14,15-EET to three dogs markedly increased its DHET in plasma. The terminal half-life ranged from 7.9-12.3 min and the volume of distribution (3.5-5.3 liters) suggested limited distribution outside the plasma compartment. Negligible radioactivity was detected in urine; this fact infers that under physiological circumstances, urinary DHETs largely derive from the kidney. That P450 metabolites of arachidonic acid are formed in humans supports the hypothesis that these metabolites contribute to the physiological response to normal pregnancy and the pathophysiology of pregnancy-induced hypertension.
Asunto(s)
Ácido 8,11,14-Eicosatrienoico/orina , Ácidos Grasos Insaturados/orina , Hipertensión/metabolismo , Preeclampsia/metabolismo , Complicaciones Cardiovasculares del Embarazo/metabolismo , Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Ácido 8,11,14-Eicosatrienoico/farmacocinética , Animales , Perros , Femenino , Cromatografía de Gases y Espectrometría de Masas , Humanos , Hipertensión/orina , Embarazo , Técnica de Dilución de Radioisótopos , Valores de Referencia , TritioRESUMEN
Epoxyeicosatrienoic acids, metabolites of the cytochrome P-450-mediated epoxygenase reaction, were detected in human urine by gas chromatographic-mass spectroscopic techniques after conversion to their hydrogenated and non-hydrogenated methyl and pentafluorobenzyl esters. Initial analysis of the regioisomeric composition utilizing the corresponding hydrogenated pentafluorobenzyl esters revealed the presence of the 8,9- and 14,15-isomers.