RESUMEN
In the systemic circulation, 11,12-epoxyeicosatrienoic acid (11,12-EET) elicits nitric oxide (NO)- and prostacyclin-independent vascular relaxation, partially through the activation of large conductance Ca(2+)-activated potassium (BK) channels. However, in the lung 11,12-EET contributes to hypoxia-induced pulmonary vasoconstriction. Since pulmonary artery smooth muscle cells also express BK channels, we assessed the consequences of BKß(1) subunit deletion on pulmonary responsiveness to 11,12-EET as well as to acute hypoxia. In buffer-perfused mouse lungs, hypoxia increased pulmonary artery pressure and this was significantly enhanced in the presence of NO synthase (NOS) and cyclooxygenase (COX) inhibitors. Under these conditions the elevation of tissue EET levels using an inhibitor of the soluble epoxide hydrolase (sEH-I), further increased the hypoxic contraction. Direct administration of 11,12-EET also increased pulmonary artery pressure, and both the sEH-I and 11,12-EET effects were prevented by iberiotoxin and absent in BKß(1)(-/-) mice. In pulmonary artery smooth muscle cells treated with NOS and COX inhibitors and loaded with the potentiometric dye, di-8-ANEPPS, 11,12-EET induced depolarization while the BK channel opener NS1619 elicited hyperpolarization indicating there was no effect of the EET on classical plasma membrane BK channels. In pulmonary artery smooth muscle cells a subpopulation of BK channels is localized in mitochondria. In these cells, 11,12-EET elicited an iberiotoxin-sensitive loss of mitochondrial membrane potential (JC-1 fluorescence) leading to plasma membrane depolarization, an effect not observed in BKß(1)(-/-) cells. Mechanistically, stimulation with 11,12-EET time-dependently induced the association of the BK α and ß(1) subunits. Our data indicate that in the absence of NO and prostacyclin 11,12-EET contributes to pulmonary vasoconstriction by stimulating the association of the α and ß(1) subunits of mitochondrial BK channels. The 11,12-EET-induced activation of BK channels results in loss of the mitochondrial membrane potential and depolarization of the pulmonary artery smooth muscle cells.
Asunto(s)
Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/metabolismo , Subunidades beta de los Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismo , Pulmón/irrigación sanguínea , Mitocondrias/metabolismo , Vasoconstricción , Ácido 8,11,14-Eicosatrienoico/metabolismo , Animales , Presión Arterial , Epóxido Hidrolasas/antagonistas & inhibidores , Epóxido Hidrolasas/metabolismo , Eliminación de Gen , Células HEK293 , Humanos , Hipoxia/genética , Hipoxia/fisiopatología , Subunidades beta de los Canales de Potasio de Gran Conductancia Activados por el Calcio/genética , Pulmón/metabolismo , Pulmón/fisiopatología , Potencial de la Membrana Mitocondrial , Potenciales de la Membrana , Ratones , Ratones Endogámicos C57BL , Arteria Pulmonar/citologíaRESUMEN
AIMS: The C-terminal domain of the soluble epoxide hydrolase (sEH) metabolizes epoxyeicosatrienoic acids (EETs) to their less active diols, while the N-terminal domain demonstrates lipid phosphatase activity. As EETs are potent vasoconstrictors in the pulmonary circulation, we assessed the development of pulmonary hypertension induced by exposure to hypoxia (10% O(2)) for 21 days in wild-type (WT) and sEH(-/-) mice and compared the effects with chronic (4 months) sEH inhibition. METHODS AND RESULTS: In isolated lungs from WT mice, acute hypoxic vasoconstriction (HPV) was potentiated by sEH inhibition and attenuated by an EET antagonist. After prolonged hypoxia, the acute HPV and sensitivity to the EET antagonist were increased, but potentiation of vasoconstriction following sEH inhibition was not evident. Chronic hypoxia also stimulated the muscularization of pulmonary arteries and decreased sEH expression in WT mice. In normoxic sEH(-/-) mice, acute HPV and small artery muscularization were greater than that in WT lungs and enhanced muscularization was accompanied with decreased voluntary exercise capacity. Acute HPV in sEH(-/-) mice was insensitive to sEH inhibition but inhibited by the EET antagonist and chronic hypoxia induced an exaggerated pulmonary vascular remodelling. In WT mice, chronic sEH inhibition increased serum EET levels but failed to affect acute HPV, right ventricle weight, pulmonary artery muscularization, or voluntary running distance. In human donor lungs, the sEH was expressed in the wall of pulmonary arteries, however, sEH expression was absent in samples from patients with pulmonary hypertension. CONCLUSION: These data suggest that a decrease in sEH expression is intimately linked to pathophysiology of hypoxia-induced pulmonary remodelling and hypertension. However, as sEH inhibitors do not promote the development of pulmonary hypertension it seems likely that the N-terminal lipid phosphatase may play a role in the development of this disease.
Asunto(s)
Epóxido Hidrolasas/fisiología , Hipertensión Pulmonar/etiología , Hipoxia/complicaciones , Animales , Cardiomegalia/etiología , Epóxido Hidrolasas/antagonistas & inhibidores , Epóxido Hidrolasas/genética , Humanos , Hipertensión Pulmonar/enzimología , Hipoxia/enzimología , Hipoxia/fisiopatología , Ratones , Ratones Endogámicos C57BL , Condicionamiento Físico Animal , Regiones Promotoras Genéticas , VasoconstricciónRESUMEN
Inhibition of the soluble epoxide hydrolase (sEH) has beneficial effects on vascular inflammation and hypertension indicating that the enzyme may be a promising target for drug development. As the enzymatic core of the hydrolase domain of the human sEH contains two tyrosine residues (Tyr(383) and Tyr(466)) that are theoretically crucial for enzymatic activity, we addressed the hypothesis that the activity of the sEH may be affected by nitrosative stress. Epoxide hydrolase activity was detected in human and murine endothelial cells as well in HEK293 cells and could be inhibited by either authentic peroxynitrite (ONOO(-)) or the ONOO(-) generator 3-morpholino-sydnonimine (SIN-1). Protection of the enzymatic core with 1-adamantyl-3-cyclohexylurea in vitro decreased sensitivity to SIN-1. Both ONOO(-) and SIN-1 elicited the tyrosine nitration of the sEH protein and mass spectrometry analysis of tryptic fragments revealed nitration on several tyrosine residues including Tyr(383) and Tyr(466). Mutation of the latter residues to phenylalanine was sufficient to abrogate epoxide hydrolase activity. In vivo, streptozotocin-induced diabetes resulted in the tyrosine nitration of the sEH in murine lungs and a significant decrease in its activity. Taken together, these data indicate that the activity of the sEH can be regulated by the tyrosine nitration of the protein. Moreover, nitrosative stress would be expected to potentiate the physiological actions of arachidonic acid epoxides by preventing their metabolism to the corresponding diols.
Asunto(s)
Epóxido Hidrolasas/antagonistas & inhibidores , Epóxido Hidrolasas/metabolismo , Ácido Peroxinitroso/metabolismo , Tirosina/metabolismo , Animales , Células Cultivadas , Células Endoteliales/citología , Células Endoteliales/enzimología , Inhibidores Enzimáticos/metabolismo , Epóxido Hidrolasas/genética , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , Molsidomina/análogos & derivados , Molsidomina/metabolismo , Mutagénesis Sitio-Dirigida , Estrés OxidativoRESUMEN
Recent findings have indicated a role for cytochrome P-450 (CYP) epoxygenase-derived epoxyeicosatrienoic acids (EETs) in acute hypoxic pulmonary vasoconstriction (HPV). Given that the intracellular concentration of EETs is determined by the soluble epoxide hydrolase (sEH), we assessed the influence of the sEH and 11,12-EET on pulmonary artery pressure and HPV in the isolated mouse lung. In lungs from wild-type mice, HPV was significantly increased by sEH inhibition, an effect abolished by pretreatment with CYP epoxygenase inhibitors and the EET antagonist 14,15-EEZE. HPV and EET production were greater in lungs from sEH(-/-) mice than from wild-type mice and sEH inhibition had no further effect on HPV, while MSPPOH and 14,15-EEZE decreased the response. 11,12-EET increased pulmonary artery pressure in a concentration-dependent manner and enhanced HPV via a Rho-dependent mechanism. Both 11,12-EET and hypoxia elicited the membrane translocation of a transient receptor potential (TRP) C6-V5 fusion protein, the latter effect was sensitive to 14,15-EEZE. Moreover, while acute hypoxia and 11,12-EET increased pulmonary pressure in lungs from TRPC6(+/-) mice, lungs from TRPC6(-/-) mice did not respond to either stimuli. These data demonstrate that CYP-derived EETs are involved in HPV and that EET-induced pulmonary contraction under normoxic and hypoxic conditions involves a TRPC6-dependent pathway.
Asunto(s)
Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Ácido 8,11,14-Eicosatrienoico/farmacología , Eicosanoides/fisiología , Epóxido Hidrolasas/fisiología , Hipoxia/fisiopatología , Arteria Pulmonar/fisiología , Circulación Pulmonar/fisiología , Vasoconstricción/fisiología , Animales , Presión Sanguínea , Inhibidores Enzimáticos del Citocromo P-450 , Sistema Enzimático del Citocromo P-450/metabolismo , Técnicas In Vitro , Ratones , Circulación Pulmonar/efectos de los fármacos , Canales Catiónicos TRPC/fisiología , Canal Catiónico TRPC6 , Vasoconstrictores/farmacología , Quinasas Asociadas a rho/metabolismoRESUMEN
The rate-limiting enzyme for cholesterol synthesis, the hydroxy-methylglutaryl coenzyme A reductase (HCR), is phosphorylated by the AMP-activated protein kinase (AMPK). As shear stress activates the AMPK in endothelial cells, we determined whether it affects HCR activity and subsequent HCR-dependent signaling. Shear stress (12 dynes cm(-2)) rapidly increased the phosphorylation and activity (6.5- and 4-fold, respectively) of the AMPK in cultured endothelial cells and the activated AMPK phosphorylated the HCR in vitro. Moreover, shear stress and the AMPK activator 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) attenuated endothelial HCR activity by 37% and 33%, respectively. Inhibition of NO production attenuated the acute shear stress-induced phosphorylation of the AMPK and the decrease in HCR activity. Prolonged shear stress (18 hours) led to a significant (50%) decrease in HCR mRNA expression that was dependent on NO, AMPK, and the subsequent phosphorylation and degradation of FoxO1a. Correspondingly, the downregulation of FoxO (small interfering RNA) decreased HCR expression. Prolonged shear stress also attenuated the bradykinin-induced activation of Ras and extracellular signal-regulated kinase 1/2, a phenomenon that was comparable to the effects of cerivastatin and that was reversed by mevalonate and thus attributed to HCR inhibition. A decrease (35%) in HCR expression was also detected in femoral arteries from mice following voluntary exercise, and the bradykinin-induced vasodilatation of the mouse hindlimb was attenuated by both exercise and the HCR inhibitor cerivastatin. These data indicate that fluid shear stress regulates the activity and expression of the HCR in endothelial cells and determines responsiveness to stimuli, such as bradykinin via a mechanism involving NO, AMPK, FoxO1a, and p21Ras.
Asunto(s)
Células Endoteliales/enzimología , Endotelio Vascular/enzimología , Factores de Transcripción Forkhead/fisiología , Hidroximetilglutaril-CoA Reductasas/metabolismo , Inhibidores de Hidroximetilglutaril-CoA Reductasas/metabolismo , Complejos Multienzimáticos/antagonistas & inhibidores , Óxido Nítrico/fisiología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Quinasas Activadas por AMP , Secuencia de Aminoácidos , Animales , Células Cultivadas , Células Endoteliales/metabolismo , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Activación Enzimática/fisiología , Proteína Forkhead Box O1 , Factores de Transcripción Forkhead/antagonistas & inhibidores , Factores de Transcripción Forkhead/genética , Humanos , Hidroximetilglutaril-CoA Reductasas/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Datos de Secuencia Molecular , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/metabolismo , Óxido Nítrico/antagonistas & inhibidores , Óxido Nítrico/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Resistencia al Corte , Estrés Mecánico , PorcinosRESUMEN
Resveratrol and its analogs are promising cancer chemoprevention agents, currently under investigation in clinical trials. However, patients administered other plant polyphenols experienced severe diarrhea, likely due to an increase in intracellular cyclic AMP (cAMP). Resveratrol itself raises intracellular cAMP levels in breast cancer cells in vitro. Its future use as a cancer chemopreventive agent could therefore be compromised by its severe side effects. The aim of the study was (a) to define the influence of resveratrol on intestinal Cl(-) secretion and (b) to elucidate possible intracellular transduction pathways involved. Resveratrol caused a dose- and time-dependent increase in DeltaIsc in T(84) cells. The specificity of resveratrol was confirmed by using piceatannol 100 mumol/L, the hydroxylated resveratrol analog, which did not alter DeltaIsc. A significant elevation of [cAMP](i) by resveratrol was assessed in T(84) cells. In mouse jejunum, resveratrol induced a time- and dose-dependent increase in DeltaIsc as well. In bilateral Cl(-)-free medium, as well as after inhibition of protein kinase A, resveratrol-induced DeltaIsc was reduced significantly. Preincubation of T(84) cells with butyrate 2 mmol/L (24 and 48 hours) significantly inhibited resveratrol as well as forskolin-induced Cl(-) secretion. In summary, the main mechanism of action of resveratrol in intestinal epithelia is cAMP-induced chloride secretion which can be suppressed by butyrate. It can therefore be suggested that in cancer chemoprevention, both agents should be combined to reduce an undesired side effect such as diarrhea and to benefit from the known agonistic effect of both agents on differentiation of colon cancer cells.
Asunto(s)
Anticarcinógenos/farmacología , Cloruros/metabolismo , Estilbenos/farmacología , Animales , Butiratos/farmacología , Diferenciación Celular , Línea Celular Tumoral , Cloruros/farmacología , Colforsina/farmacología , AMP Cíclico/química , AMP Cíclico/metabolismo , AMP Cíclico/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Electrofisiología , Humanos , Técnicas In Vitro , Yeyuno/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos BALB C , Resveratrol , Transducción de Señal , Factores de TiempoRESUMEN
An intrauterine environment may play a role in predisposing a developing fetus to metabolic diseases during adulthood. We investigated the hypothesis that a maternal diet high in omega-6 polyunsaturated fat can modify the programming of an offspring's glucose tolerance, insulin sensitivity, body composition, lipid metabolism, and insulin signaling. High omega-6 polyunsaturated fat diets were fed to female rats 4 weeks before mating and throughout the gestation period. The offspring were maintained on chow diet. At 3 months of age, indirect calorimetry, oral glucose tolerance tests, and dual x-ray absorptiometry measurements were performed. Triglyceride content and beta-hydroxyacyl coenzyme A dehydrogenase activity were determined in the liver and quadriceps muscle. Expression levels of key insulin signaling pathway proteins were measured in the liver and quadriceps muscle of the 3-month-old offspring. Offspring from the fat-fed dams had significantly increased proportions of both total body fat and abdominal fat. All offspring displayed normal insulin sensitivity and glucose tolerance, although the offspring from the fat-fed dams were significantly more hyperinsulinemic 15 minutes after an oral glucose challenge. Whole body fuel oxidation was not altered. The offspring of fat-fed dams had significantly elevated liver triglyceride content. Insulin signaling protein expression levels in the offspring of fat-fed dams were consistent with reduced hepatic insulin sensitivity but increased quadriceps insulin sensitivity. A maternal diet high in omega-6 polyunsaturated fat evokes programming within the metabolic processes of the offspring that may predispose the offspring to the development of metabolic diseases.