RESUMO
Chronic hypoxia contributes to pulmonary hypertension through complex mechanisms that include enhanced NADPH oxidase expression and reactive oxygen species (ROS) generation in the lung. Stimulation of peroxisome proliferator-activated receptor gamma (PPARgamma) reduces the expression and activity of NADPH oxidase. Therefore, we hypothesized that activating PPARgamma with rosiglitazone would attenuate chronic hypoxia-induced pulmonary hypertension, in part, through suppressing NADPH oxidase-derived ROS that stimulate proliferative signaling pathways. Male C57Bl/6 mice were exposed to chronic hypoxia (CH, Fi(O2) 10%) or room air for 3 or 5 weeks. During the last 10 days of exposure, each animal was treated daily by gavage with either the PPARgamma ligand, rosiglitazone (10 mg/kg/d) or with an equal volume of vehicle. CH increased: (1) right ventricular systolic pressure (RVSP), (2) right ventricle weight, (3) thickness of the walls of small pulmonary vessels, (4) superoxide production and Nox4 expression in the lung, and (5) platelet-derived growth factor receptor beta (PDGFRbeta) expression and activity and reduced phosphatase and tensin homolog deleted on chromosome 10 (PTEN) expression. Treatment with rosiglitazone prevented the development of pulmonary hypertension at 3 weeks; reversed established pulmonary hypertension at 5 weeks; and attenuated CH-stimulated Nox4 expression and superoxide production, PDGFRbeta activation, and reductions in PTEN expression. Rosiglitazone also attenuated hypoxia-induced increases in Nox4 expression in pulmonary endothelial cells in vitro despite hypoxia-induced reductions in PPARgamma expression. Collectively, these findings indicate that PPARgamma ligands attenuated hypoxia-induced pulmonary vascular remodeling and hypertension by suppressing oxidative and proliferative signals providing novel insights for mechanisms underlying therapeutic effects of PPARgamma activation in pulmonary hypertension.
Assuntos
Hipertensão Pulmonar/metabolismo , Hipóxia/metabolismo , PPAR gama/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tiazolidinedionas/farmacologia , Vasodilatadores/farmacologia , Animais , Pressão Sanguínea/efeitos dos fármacos , Proliferação de Células , Doença Crônica , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Ventrículos do Coração/metabolismo , Ventrículos do Coração/patologia , Humanos , Hipertensão Pulmonar/tratamento farmacológico , Hipertensão Pulmonar/etiologia , Hipertensão Pulmonar/patologia , Hipóxia/tratamento farmacológico , Hipóxia/etiologia , Hipóxia/patologia , Ligantes , Masculino , Camundongos , NADPH Oxidase 4 , NADPH Oxidases/metabolismo , Tamanho do Órgão , PPAR gama/agonistas , PTEN Fosfo-Hidrolase/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Rosiglitazona , Superóxidos/metabolismoRESUMO
Vascular endothelial cells express the ligand-activated transcription factor, peroxisome proliferator-activated receptor-gamma (PPARgamma), which participates in the regulation of metabolism, cell proliferation, and inflammation. PPARgamma ligands attenuate, whereas the loss of function mutations in PPARgamma stimulate, endothelial dysfunction, suggesting that PPARgamma may regulate vascular endothelial nitric oxide production. To explore the role of endothelial PPARgamma in the regulation of vascular nitric oxide production in vivo, mice expressing Cre recombinase driven by an endothelial-specific promoter were crossed with mice carrying a floxed PPARgamma gene to produce endothelial PPARgamma null mice (ePPARgamma(-/-)). When compared with littermate controls, ePPARgamma(-/-) animals were hypertensive at baseline and demonstrated comparable increases in systolic blood pressure in response to angiotensin II infusion. When compared with those of control animals, aortic ring relaxation responses to acetylcholine were impaired, whereas relaxation responses to sodium nitroprusside were unaffected in ePPARgamma(-/-) mice. Similarly, intact aortic segments from ePPARgamma(-/-) mice released less nitric oxide than those from controls, whereas endothelial nitric oxide synthase expression was similar in control and ePPARgamma(-/-) aortas. Reduced nitric oxide production in ePPARgamma(-/-) aortas was associated with an increase in the parameters of oxidative stress in the blood and the activation of nuclear factor-kappaB in aortic homogenates. These findings demonstrate that endothelial PPARgamma regulates vascular nitric oxide production and that the disruption of endothelial PPARgamma contributes to endothelial dysfunction in vivo.
Assuntos
Endotélio Vascular/metabolismo , Hipertensão/metabolismo , Óxido Nítrico/metabolismo , PPAR gama/metabolismo , Vasodilatação , Acetilcolina/farmacologia , Animais , Biomarcadores/sangue , Pressão Sanguínea , Relação Dose-Resposta a Droga , Regulação para Baixo , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/fisiopatologia , Hipertensão/genética , Hipertensão/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Nitroprussiato/farmacologia , Estresse Oxidativo , PPAR gama/deficiência , PPAR gama/genética , Fatores de Tempo , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologiaRESUMO
Nucleoside reverse transcriptase inhibitors (NRTIs), such as zidovudine (AZT) and stavudine (d4T), cause toxicities to numerous tissues, including the liver and vasculature. While much is known about hepatic NRTI toxicity, the mechanism of toxicity in endothelial cells is incompletely understood. Human aortic endothelial and HepG2 liver cells were exposed to 1 muM AZT or d4T for up to 5 weeks. Markers of oxidative stress, mitochondrial function, NRTI phosphorylation, mitochondrial DNA (mtDNA) levels, and cytotoxicity were monitored over time. In endothelial cells, AZT significantly oxidized glutathione redox potential, increased total cellular and mitochondrial-specific superoxide, decreased mitochondrial membrane potential, increased lactate release, and caused cell death from weeks 3 through 5. Toxicity occurred in the absence of di- and tri-phosphorylated AZT and mtDNA depletion. These data show that oxidative stress and mitochondrial dysfunction in endothelial cells occur with a physiologically relevant concentration of AZT, and require long-term exposure to develop. In contrast, d4T did not induce endothelial oxidative stress, mitochondrial dysfunction, or cytotoxicity despite the presence of d4T-triphosphate. Both drugs depleted mtDNA in HepG2 cells without causing cell death. Endothelial cells are more susceptible to AZT-induced toxicity than HepG2 cells, and AZT caused greater endothelial dysfunction than d4T because of its pro-oxidative effects.
Assuntos
Células Endoteliais/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Inibidores da Transcriptase Reversa/toxicidade , Estavudina/toxicidade , Zidovudina/toxicidade , Biomarcadores/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Células Cultivadas , DNA Mitocondrial/metabolismo , Células Endoteliais/metabolismo , Glutationa/metabolismo , Humanos , Ácido Láctico/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Fosforilação , Inibidores da Transcriptase Reversa/metabolismo , Estavudina/metabolismo , Superóxidos/metabolismo , Fatores de Tempo , Zidovudina/metabolismoRESUMO
Obstructive sleep apnea, characterized by intermittent periods of hypoxemia, is an independent risk factor for the development of pulmonary hypertension. However, the exact mechanisms of this disorder remain to be defined. Enhanced NADPH oxidase expression and superoxide (O2(-).) generation in the pulmonary vasculature play a critical role in hypoxia-induced pulmonary hypertension. Therefore, the current study explores the hypothesis that chronic intermittent hypoxia (CIH) causes pulmonary hypertension, in part, by increasing NADPH oxidase-derived reactive oxygen species (ROS) that contribute to pulmonary vascular remodeling and hypertension. To test this hypothesis, male C57Bl/6 mice and gp91phox knockout mice were exposed to CIH for 8 hours per day, 5 days per week for 8 weeks. CIH mice were placed in a chamber where the oxygen concentration was cycled between 21% and 10% O2 45 times per hour. Exposure to CIH for 8 weeks increased right ventricular systolic pressure (RVSP), right ventricle (RV):left ventricle (LV) + septum (S) weight ratio, an index of RV hypertrophy, and thickness of the right ventricular anterior wall as measured by echocardiography. CIH exposure also caused pulmonary vascular remodeling as demonstrated by increased muscularization of the distal pulmonary vasculature. CIH-induced pulmonary hypertension was associated with increased lung levels of the NADPH oxidase subunits, Nox4 and p22phox, as well as increased activity of platelet-derived growth factor receptor beta and its associated downstream effector, Akt kinase. These CIH-induced derangements were attenuated in similarly treated gp91phox knockout mice. These findings demonstrate that NADPH oxidase-derived ROS contribute to the development of pulmonary vascular remodeling and hypertension caused by CIH.
Assuntos
Hipertensão Pulmonar/enzimologia , Hipertensão Pulmonar/etiologia , Hipóxia/complicações , Hipóxia/enzimologia , NADPH Oxidases/metabolismo , Animais , Disponibilidade Biológica , Doença Crônica , Hemoglobinas/metabolismo , Pulmão/irrigação sanguínea , Pulmão/enzimologia , Pulmão/patologia , Masculino , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NADPH Oxidase 2 , NADPH Oxidase 4 , Óxido Nítrico/metabolismo , Subunidades Proteicas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de Sinais , Superóxidos/metabolismoRESUMO
Human immunodeficiency virus (HIV)-infected patients have a higher incidence of oxidative stress, endothelial dysfunction, and cardiovascular disease than uninfected individuals. Recent reports have demonstrated that viral proteins upregulate reactive oxygen species, which may contribute to elevated cardiovascular risk in HIV-1 patients. In this study we employed an HIV-1 transgenic rat model to investigate the physiological effects of viral protein expression on the vasculature. Markers of oxidative stress in wild-type and HIV-1 transgenic rats were measured using electron spin resonance, fluorescence microscopy, and various molecular techniques. Relaxation studies were completed on isolated aortic rings, and mRNA and protein were collected to measure changes in expression of nitric oxide (NO) and superoxide sources. HIV-1 transgenic rats displayed significantly less NO-hemoglobin, serum nitrite, serum S-nitrosothiols, aortic tissue NO, and impaired endothelium-dependent vasorelaxation than wild-type rats. NO reduction was not attributed to differences in endothelial NO synthase (eNOS) protein expression, eNOS-Ser1177 phosphorylation, or tetrahydrobiopterin availability. Aortas from HIV-1 transgenic rats had higher levels of superoxide and 3-nitrotyrosine but did not differ in expression of superoxide-generating sources NADPH oxidase or xanthine oxidase. However, transgenic aortas displayed decreased superoxide dismutase and glutathione. Administering the glutathione precursor procysteine decreased superoxide, restored aortic NO levels and NO-hemoglobin, and improved endothelium-dependent relaxation in HIV-1 transgenic rats. These results show that HIV-1 protein expression decreases NO and causes endothelial dysfunction. Diminished antioxidant capacity increases vascular superoxide levels, which reduce NO bioavailability and promote peroxynitrite generation. Restoring glutathione levels reverses HIV-1 protein-mediated effects on superoxide, NO, and vasorelaxation.
Assuntos
Aorta/metabolismo , Glutationa/metabolismo , Infecções por HIV/metabolismo , HIV-1/metabolismo , Proteínas do Vírus da Imunodeficiência Humana/metabolismo , Óxido Nítrico/metabolismo , Estresse Oxidativo , Provírus/metabolismo , Acetilcolina/farmacologia , Animais , Animais Geneticamente Modificados , Antioxidantes/farmacologia , Aorta/efeitos dos fármacos , Aorta/enzimologia , Aorta/fisiopatologia , Aorta/virologia , Biopterinas/análogos & derivados , Biopterinas/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Regulação para Baixo , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Endotélio Vascular/virologia , Infecções por HIV/fisiopatologia , Infecções por HIV/virologia , HIV-1/genética , Proteínas do Vírus da Imunodeficiência Humana/genética , Masculino , NADPH Oxidases/metabolismo , Óxido Nítrico/sangue , Óxido Nítrico Sintase Tipo III/metabolismo , Nitroprussiato/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Provírus/genética , Ácido Pirrolidonocarboxílico/farmacologia , Ratos , Ratos Endogâmicos F344 , Superóxidos/metabolismo , Tiazolidinas/farmacologia , Tirosina/análogos & derivados , Tirosina/metabolismo , Vasodilatação , Vasodilatadores/farmacologia , Xantina Oxidase/metabolismoRESUMO
BACKGROUND: Chronic alcohol consumption perturbs cellular function in a variety of organ systems. Previous studies have suggested that moderate alcohol consumption reduces vascular disease, whereas heavier alcohol consumption may worsen it. The mechanisms for these vascular effects of chronic alcohol ingestion continue to be defined and constitute the focus of this study. METHODS: Male Sprague Dawley rats were fed an isocaloric, Lieber-Decarli liquid diet containing either ethanol (36% calories) or Maltose-Dextrin (substituted for ethanol) for 6 weeks. Telemetric blood pressure measurements were taken before and after ethanol feeding. After the rats were killed, the aortas were analyzed for endothelial nitric oxide (NO) synthase expression and NO production. RESULTS: Chronic ethanol ingestion decreased mean arterial pressure and increased aortic NO production as demonstrated by direct ex vivo measurements using iron diethyldithio-carbamic acid as well as analysis of nitrosyl-hemoglobin (NO-Hb) levels. Consistent with these assays of vascular NO production, endothelium-dependent relaxation responses to acetycholine (Ach) were enhanced in ethanol-fed animals. Aortic endothelial nitric oxide synthase expression was also increased by chronic ethanol ingestion. CONCLUSIONS: These findings demonstrate that a regimen of chronic alcohol ingestion in the rat produced generally salutary effects in the systemic vasculature following a 6-week treatment regimen. These findings extend previous in vitro studies to demonstrate that alcohol has potent effects on vascular endothelial nitric oxide synthase expression, NO production, and vascular function. Consistent with previous reports, these findings confirm that alcohol-induced alterations in the production of reactive nitrogen species play an important role in the pathogenesis of alcohol-mediated tissue effects.
Assuntos
Aorta/efeitos dos fármacos , Pressão Sanguínea/efeitos dos fármacos , Depressores do Sistema Nervoso Central/farmacologia , Etanol/farmacologia , Óxido Nítrico/metabolismo , Animais , Aorta/metabolismo , Células Endoteliais/enzimologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/enzimologia , Endotélio Vascular/metabolismo , Masculino , Óxido Nítrico Sintase Tipo III/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Acute leptin exposure stimulates endothelial nitric oxide (NO) production in vitro. In contrast, chronic elevations in circulating leptin levels in patients with obesity are associated with endothelial dysfunction and impaired endothelial NO production. Therefore, the goal of the current study was to examine the direct effects of acute and more sustained leptin stimulation on endothelial nitric oxide synthase (eNOS) and NO production in human aortic endothelial cells (HAECs). HAECs were treated with vehicle or with leptin (5 or 60 ng/mL) acutely (30-60 minutes) or for 72 hours. HAEC NO release into culture media was measured with a chemiluminescence technique, and superoxide (O(2)(-.)) production was measured with electron spin resonance (ESR) spectroscopy. HAEC eNOS activity was measured as the conversion of (3)H-arginine to (3)H-citrulline, and protein levels of eNOS, phospho-eNOS (serine 1177), Erk, phospho-Erk, suppressor of cytokine signaling (SOCS3), xanthine oxidase (XO), and the reduced nicotinamide adenine dinucleotide (NADPH) oxidase components p22phox, p67phox, Nox-4, and gp91phox were examined by Western blotting or immunoprecipitation. Acute leptin exposure increased eNOS serine 1177 phosphorylation and caused Erk activation. In contrast, prolonged leptin stimulation was not cytotoxic and failed to alter eNOS expression, phosphorylation, or HAEC NO release. Furthermore, prolonged leptin stimulation did not alter O(2)(-.) production or NADPH oxidase or XO expression but increased SOCS3 expression. In contrast to acute stimulation, prolonged (72 hours) stimulation does not alter endothelial cell NO or O(2)(-.) production. We postulate that chronic leptin stimulation, through increased SOCS3 expression, may attenuate the effects of leptin on vascular endothelial function.
Assuntos
Aorta/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/fisiologia , Leptina/farmacologia , Óxido Nítrico/metabolismo , Transdução de Sinais/efeitos dos fármacos , Aorta/citologia , Técnicas de Cultura de Células , HumanosRESUMO
Oxidative stress plays an important role in diabetic vascular dysfunction. The sources and regulation of reactive oxygen species production in diabetic vasculature continue to be defined. Because peroxisome proliferator-activated receptor gamma (PPARgamma) ligands reduced superoxide anion (O(2)(-.)) generation in vascular endothelial cells in vitro by reducing NADPH oxidase and increasing Cu/Zn superoxide dismutase (SOD) expression, the current study examined the effect of PPARgamma ligands on vascular NADPH oxidase and O(2)(-.) generation in vivo. Lean control (db(+)/db(-)) and obese, diabetic, leptin receptor-deficient (db(-)/db(-)) mice were treated with either vehicle or rosiglitazone (3 mg/kg/day) by gavage for 7-days. Compared to controls, db(-)/db(-) mice weighed more and had metabolic derangements that were not corrected by treatment with rosiglitazone for 1-week. Aortic O(2)(-.) generation and mRNA levels of the NADPH oxidase subunits, Nox-1, Nox-2, and Nox-4 as well as Nox-4 protein expression were elevated in db(-)/db(-) compared to db(+)/db(-) mice, whereas aortic Cu/Zn SOD protein and PPARgamma mRNA levels were reduced in db(-)/db(-) mice. Treatment with rosiglitazone for 1-week significantly reduced aortic O(2)(-.) production and the expression of Nox-1, 2, and 4 but failed to increase Cu/Zn SOD or PPARgamma in aortic tissue from db(-)/db(-) mice. These data demonstrate that the vascular expression of Nox-1, 2, and 4 subunits of NADPH oxidase is increased in db(-)/db(-) mice and that short-term treatment with the PPARgamma agonist, rosiglitazone, has the potential to rapidly suppress vascular NADPH oxidase expression and O(2)(-.) production through mechanisms that do not appear to depend on correction of diabetic metabolic derangements.
Assuntos
Aorta/efeitos dos fármacos , Diabetes Mellitus Tipo 2/tratamento farmacológico , Hipoglicemiantes/farmacologia , NADPH Oxidases/metabolismo , Estresse Oxidativo/efeitos dos fármacos , PPAR gama/agonistas , Tiazolidinedionas/farmacologia , Animais , Aorta/enzimologia , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Regulação para Baixo , Hipoglicemiantes/uso terapêutico , Masculino , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Knockout , NADH NADPH Oxirredutases/metabolismo , NADPH Oxidase 1 , NADPH Oxidase 2 , NADPH Oxidase 4 , NADPH Oxidases/genética , PPAR gama/genética , PPAR gama/metabolismo , RNA Mensageiro/biossíntese , Receptores de Superfície Celular/deficiência , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Receptores para Leptina , Rosiglitazona , Superóxidos/metabolismo , Tiazolidinedionas/uso terapêuticoRESUMO
Hepatic insulin gene therapy (HIGT) ameliorates hyperglycemia in diabetic rodents, suggesting that similar approaches may eventually provide a means to improve treatment of diabetes mellitus. However, whether the metabolic and hormonal changes produced by HIGT benefit vascular function remains unclear. The impact of HIGT on endothelium-dependent vasodilation, nitrosyl-hemoglobin content (NO-Hb), and insulin sensitivity were studied using aortic ring preparations, electron spin resonance spectroscopy (ESR), homeostasis assessment of insulin resistance (HOMA-IR) calculations, and insulin tolerance testing (ITT). Data were correlated with selected hormone and adipocytokine concentrations. Rats made diabetic with streptozotocin were treated with subcutaneous insulin pellets dosed to sustain body weights and hyperglycemia or with HIGT; nondiabetic rats served as controls. Hyperglycemic rats demonstrated impaired endothelium-dependent vasodilation, reduced levels of NO-Hb, and diminished insulin, leptin, and adiponectin concentrations compared with controls. In contrast, HIGT treatment significantly reduced blood sugars and sustained both endothelium-mediated vasodilation and NO-Hb at control levels. HOMA-IR calculations and ITT indicated enhanced insulin sensitivity among HIGT-treated rats. HIGT partially restored suppressed leptin levels in hyperglycemic rats and increased adiponectin concentrations to supranormal levels, consistent with indicators of insulin sensitivity. Our findings indicate that the metabolic milieu produced by HIGT is sufficient to preserve vascular function in diabetic rodents. These data suggest that improved glycemia, induction of a beneficial adipocytokine profile, and enhanced insulin sensitivity combine to preserve endothelium-dependent vascular function in HIGT-treated diabetic rats. Consequently, HIGT may represent a novel and efficacious approach to reduce diabetes-associated vascular dysfunction.
Assuntos
Diabetes Mellitus Experimental/terapia , Terapia Genética/métodos , Insulina/genética , Fígado/metabolismo , Acetilcolina/farmacologia , Adenoviridae/genética , Adiponectina/sangue , Animais , Aorta/efeitos dos fármacos , Aorta/fisiopatologia , Glicemia/metabolismo , Peso Corporal/efeitos dos fármacos , Peso Corporal/fisiologia , Fenômenos Fisiológicos Cardiovasculares , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/fisiopatologia , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Glucagon/sangue , Hemoglobinas/metabolismo , Humanos , Técnicas In Vitro , Injeções Intravenosas , Insulina/sangue , Insulina/farmacologia , Resistência à Insulina/fisiologia , Leptina/sangue , Lipídeos/sangue , Masculino , Óxido Nítrico Sintase/metabolismo , Nitroprussiato/farmacologia , Hormônios Peptídicos/metabolismo , Ratos , Ratos Sprague-Dawley , Vasodilatação/efeitos dos fármacosRESUMO
Nitric oxide (NO) produced by vascular endothelial cells (ECs) plays a critical role in normal vascular physiology. Important insights into mechanisms regulating the production of endothelial NO have been derived from in vitro studies employing cultured ECs. Although many techniques for the detection of NO have been described, many of these methods lack adequate sensitivity to detect the small amount of NO produced by cultured ECs. In this chapter, we describe three protocols that employ chemiluminescence, electron spin resonance, or electrochemical techniques to permit the reliable detection of EC NO production.
Assuntos
Endotélio Vascular/metabolismo , Óxido Nítrico/biossíntese , Calibragem , Células Cultivadas , Eletroquímica , Espectroscopia de Ressonância de Spin Eletrônica , Endotélio Vascular/citologia , Medições LuminescentesRESUMO
Several studies have shown that nitric oxide (NO) inhibits Na(+) transport in renal and alveolar monolayers. However, the mechanisms by which NO alters epithelial Na(+) channel (ENaC) activity is unclear. Therefore, we examined the effect of applying the NO donor drug l-propanamine 3,2-hydroxy-2-nitroso-1-propylhidrazino (PAPA-NONOate) to cultured renal epithelial cells. A6 and M1 cells were maintained on permeable supports in medium containing 1.5 microM dexamethasone and 10% bovine serum. After 1.5 microM PAPA-NONOate was applied, amiloride-sensitive short-circuit current measurements decreased 29% in A6 cells and 44% in M1 cells. This differed significantly from the 3% and 19% decreases in A6 and M1 cells, respectively, treated with control donor compound (P < 0.0005). Subsequent application of PAPA-NONOate to amiloride-treated control (no NONOate) A6 and M1 cells did not further decrease transepithelial current. In single-channel patch-clamp studies, NONOate significantly decreased ENaC open probability (P(o)) from 0.186 +/- 0.043 to 0.045 +/- 0.009 (n = 7; P < 0.05) without changing the unitary current. We also showed that aldosterone significantly decreased NO production in primary cultures of alveolar type II (ATII) epithelial cells. Because inducible nitric oxide synthase (iNOS) coimmunoprecipitated with the serum- and glucocorticoid-inducible kinase (SGK1) and both proteins colocalized in the cytoplasm (as shown in our studies in mouse ATII cells), SGK1 may also be important in regulating NO production in the alveolar epithelium. Our study also identified iNOS as a novel SGK1 phosphorylated protein (at S733 and S903 residues in miNOS) suggesting that one way in which SGK1 could increase Na(+) transport is by altering iNOS production of NO.
Assuntos
Óxido Nítrico Sintase/metabolismo , Óxido Nítrico/antagonistas & inibidores , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Canais de Sódio/metabolismo , Aldosterona/farmacologia , Sequência de Aminoácidos , Animais , Células Cultivadas , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Canais Epiteliais de Sódio , Hidrazinas/farmacologia , Proteínas Imediatamente Precoces , Ativação do Canal Iônico/fisiologia , Camundongos , Dados de Sequência Molecular , Néfrons/citologia , Óxido Nítrico/metabolismo , Óxido Nítrico/farmacologia , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase Tipo II , Fosforilação , Alvéolos Pulmonares/citologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Sódio/metabolismo , XenopusRESUMO
Recently, we demonstrated that the peroxisome proliferator-activated receptor-gamma (PPAR-gamma) ligands, either 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) or ciglitazone, increased endothelial nitric oxide (.NO) release without altering endothelial nitric oxide synthase (eNOS) expression (4). However, the precise molecular mechanisms of PPAR-gamma-stimulated endothelial.NO release remain to be defined. Superoxide anion radical (O2-.) combines with .NO to decrease.NO bioavailability. NADPH oxidase, which produces O2-., and Cu/Zn-superoxide dismutase (Cu/Zn-SOD), which degrades O2-., thereby contribute to regulation of endothelial cell.NO metabolism. Therefore, we examined the ability of PPAR-gamma ligands to modulate endothelial O2-. metabolism through alterations in the expression and activity of NADPH oxidase or Cu/Zn-SOD. Treatment with 10 microM 15d-PGJ2 or ciglitazone for 24 h decreased human umbilical vein endothelial cell (HUVEC) membrane NADPH-dependent O2-. production detected with electron spin resonance spectroscopy. Treatment with 15d-PGJ2 or ciglitazone also reduced relative mRNA levels of the NADPH oxidase subunits, nox-1, gp91phox (nox-2), and nox-4, as measured using real-time PCR analysis. Concordantly, Western blot analysis demonstrated that 15d-PGJ2 or ciglitazone decreased nox-2 and nox-4 protein expression. PPAR-gamma ligands also stimulated both activity and expression of Cu/Zn-SOD in HUVEC. These data suggest that in addition to any direct effects on endothelial.NO production, PPAR-gamma ligands enhance endothelial.NO bioavailability, in part by altering endothelial O2-. metabolism through suppression of NADPH oxidase and induction of Cu/Zn-SOD. These findings further elucidate the molecular mechanisms by which PPAR-gamma ligands directly alter vascular endothelial function.
Assuntos
Membrana Celular/metabolismo , Células Endoteliais/metabolismo , PPAR gama/metabolismo , Prostaglandina D2/análogos & derivados , Prostaglandina D2/farmacologia , Superóxidos/metabolismo , Tiazolidinedionas/farmacologia , Western Blotting , Membrana Celular/efeitos dos fármacos , Células Cultivadas , Espectroscopia de Ressonância de Spin Eletrônica , Células Endoteliais/efeitos dos fármacos , Humanos , Hipoglicemiantes/farmacologia , Ligantes , NADPH Oxidases/efeitos dos fármacos , NADPH Oxidases/metabolismo , Óxido Nítrico/metabolismo , PPAR gama/efeitos dos fármacos , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Superóxido Dismutase/biossíntese , Superóxido Dismutase/efeitos dos fármacos , Veias Umbilicais/efeitos dos fármacos , Veias Umbilicais/metabolismoRESUMO
BACKGROUND: Insulin increases endothelial nitric oxide (NO) production by activating endothelial nitric oxide synthase (eNOS) through protein kinase B (Akt)-mediated phosphorylation of serine residue 1179 (p-eNOS serine 1179). Because fatty acids modulate insulin-stimulated Akt signaling cascades in smooth muscle cells, we hypothesized that fatty acids would differentially regulate endothelial Akt signaling, eNOS phosphorylation, and NO production. METHODS: Porcine pulmonary artery endothelial cells (PAECs) were treated for 3 hours with 100 microM oleic (18:1) or eicosapentaenoic (20:5) acids or with an equivalent volume of ethanol vehicle (0.1%). PAECs were then treated with graded concentrations (10(9)-10(-5) M) of insulin or incubated overnight (24 hours) in culture medium without fatty acids before insulin treatment. Activation and phosphorylation of Akt and eNOS were determined by immunoblotting. NO production was measured with a chemiluminescence NO analyzer or with a NO-selective carbon fiber microelectrode. RESULTS: Insulin-stimulated Akt phosphorylation, eNOS phosphorylation, and NO production. The phosphatidylinositol-3 kinase inhibitor wortmannin attenuated insulin-stimulated Akt activation and NO production. Treatment with the omega-3 fatty acid 20:5, but not 18:1, enhanced insulin-stimulated NO production but failed to alter insulin-stimulated Akt activation or eNOS serine 1179 phosphorylation. CONCLUSION: Individual fatty acyl species have distinct effects on insulin-stimulated endothelial NO production. Although fatty acids alter Akt signaling in muscle cells, the current results indicate that fatty acids do not modulate endothelial NO production through alterations in insulin-stimulated, Akt-mediated eNOS phosphorylation.
Assuntos
Endotélio Vascular/efeitos dos fármacos , Ácidos Graxos/farmacologia , Insulina/farmacologia , Óxido Nítrico/metabolismo , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas/metabolismo , Androstadienos/farmacologia , Animais , Western Blotting , Células Cultivadas , Relação Dose-Resposta a Droga , Combinação de Medicamentos , Ácido Eicosapentaenoico , Endotélio Vascular/metabolismo , Ácidos Graxos Ômega-3/farmacologia , Ácidos Graxos Insaturados/farmacologia , Ácido Oleico/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas c-akt , Artéria Pulmonar , Suínos , WortmaninaRESUMO
The reliable measurement of nitric oxide (NO) production by endothelial cells in vitro has become an important tool for investigating mechanisms of endothelial dysfunction. This study evaluates measuring NO production by cultured porcine pulmonary artery endothelial cells (PAEC) using the assay based on the fluorometric detection of 1-(H)-naphthotriazole, the fluorescent product of the reaction between nitrite (NO2-) and 2,3-diaminonapthalene (DAN). To stimulate NO production, PAEC were treated for 60 min with agonists known to stimulate endothelial NO production. The DAN assay was unable to detect NO production from agonist-stimulated PAEC. In contrast, chemiluminescence analysis, which detects NO, NO2-, and nitrate (NO3-) (collectively referred to as NO(x)), detected significant increases in NO(x) from stimulated PAEC. Nitrate reductase-mediated reduction of NO3-to NO2- in media from stimulated PAEC enhanced the ability of the DAN assay to detect NO release from PAEC. These results provide the first direct comparison of the sensitivity of these two commonly employed assays. Our findings emphasize that NO3-reduction may be required to enable the DAN assay to detect small amounts of NO produced by cultured endothelial cells.