RESUMO
ADP activates a family of cell surface receptors that modulate signaling pathways in a broad range of cells. ADP receptor antagonists are widely used to treat cardiovascular disease states. These studies identify a critical role for the stable reactive oxygen species hydrogen peroxide (H2O2) in mediating cellular responses activated by the G protein-coupled P2Y1 receptor for ADP. We found that ADP-dependent phosphorylation of key endothelial signaling proteins--including endothelial nitric oxide synthase, AMP-activated protein kinase, and the actin-binding MARCKS protein--was blocked by preincubation with PEG-catalase, which degrades H2O2. ADP treatment promoted the H2O2-dependent phosphorylation of c-Abl, a nonreceptor tyrosine kinase that modulates the actin cytoskeleton. Cellular imaging experiments using fluorescence resonance energy transfer-based biosensors revealed that ADP-stimulated activation of the cytoskeleton-associated small GTPase Rac1 was independent of H2O2. However, Rac1-dependent activation of AMP-activated protein kinase, the signaling phospholipid phosphatidylinositol-(4, 5)-bisphosphate, and the c-Abl-interacting protein CrkII are mediated by H2O2. We transfected endothelial cells with differentially targeted HyPer2 H2O2 biosensors and found that ADP promoted a marked increase in H2O2 levels in the cytosol and caveolae, and a smaller increase in mitochondria. We performed a screen for P2Y1 receptor-mediated receptor tyrosine kinase transactivation and discovered that ADP transactivates Fms-like tyrosine kinase 3 (Flt3), a receptor tyrosine kinase expressed in these cells. Our observation that P2Y1 receptor-mediated responses involve Flt3 transactivation may identify a unique mechanism whereby cancer chemotherapy with receptor tyrosine kinase inhibitors promotes vascular dysfunction. Taken together, these findings establish a critical role for endogenous H2O2 in control of ADP-mediated signaling responses in the vascular wall.
Assuntos
Difosfato de Adenosina/metabolismo , Células Endoteliais/metabolismo , Ativação Enzimática/fisiologia , Peróxido de Hidrogênio/metabolismo , Receptores Purinérgicos P2Y1/metabolismo , Transdução de Sinais/fisiologia , Tirosina Quinase 3 Semelhante a fms/metabolismo , Animais , Bovinos , Linhagem Celular , Impedância Elétrica , Células Endoteliais/fisiologia , Ativação Enzimática/genética , Transferência Ressonante de Energia de Fluorescência , Humanos , Immunoblotting , Microscopia de FluorescênciaRESUMO
Elevated levels of serum retinol-binding protein 4 (RBP4) contribute to insulin resistance and correlate with increased prevalence of hypertension and myocardial infarction. We sought to determine whether lowering RBP4 would improve blood pressure (BP) and protect against obesity- or angiotensin (Ang)-II-induced hypertension. Systolic and diastolic BP were lower in the RBP4-knockout (RBP4-KO) mice and higher in the RBP4-overexpressing (RBP4-Tg) mice compared with BP in the wild-type (WT) littermates. Carbachol-induced vasodilatation was increased in arteries from the RBP4-KO compared with the WT mice and was impaired in the RBP4-Tg mice. Aortic eNOS(Ser1177) phosphorylation was enhanced â¼50% in the RBP4-KO mice, with no change in total eNOS protein. Feeding a high-fat diet increased BP in the RBP4-KO mice only to the level in the WT mice fed chow and had no effect on aortic eNOS(Ser1177) phosphorylation. Ang-II infusion resulted in 22 mmHg lower systolic BP in the RBP4-KO than in the WT mice, although the relative BP increase over saline infusion was â¼30% in both. Ang-II treatment decreased aortic eNOS(Ser1177) phosphorylation in the WT and RBP4-KO mice, but phosphorylation remained higher in the RBP4-KO mice. Cardiac hypertrophy with Ang-II treatment was diminished by 56% in the RBP4-KO mice. Thus, elevated serum RBP4 raises BP and lack of RBP4 reduces it, with commensurate changes in aortic eNOS(Ser1177) phosphorylation. Lowering RBP4 may reduce BP through enhanced eNOS-mediated vasodilatation and may be a novel therapeutic approach for hypertension.
Assuntos
Pressão Sanguínea/fisiologia , Hipertensão/metabolismo , Proteínas Plasmáticas de Ligação ao Retinol/metabolismo , Angiotensina II/metabolismo , Animais , Aorta/metabolismo , Cardiomegalia/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico Sintase Tipo III/metabolismo , Obesidade/metabolismo , Fosforilação/fisiologia , Vasodilatação/fisiologiaRESUMO
Nitric oxide (NO) and hydrogen peroxide (H(2)O(2)) are synthesized within cardiac myocytes and play key roles in modulating cardiovascular signaling. Cardiac myocytes contain both the endothelial (eNOS) and neuronal (nNOS) NO synthases, but the differential roles of these NOS isoforms and the interplay of reactive oxygen species and reactive nitrogen species in cardiac signaling pathways are poorly understood. Using a recently developed NO chemical sensor [Cu(2)(FL2E)] to study adult cardiac myocytes from wild-type, eNOS(null), and nNOS(null) mice, we discovered that physiological concentrations of H(2)O(2) activate eNOS but not nNOS. H(2)O(2)-stimulated eNOS activation depends on phosphorylation of both the AMP-activated protein kinase and kinase Akt, and leads to the robust phosphorylation of eNOS. Cardiac myocytes isolated from mice infected with lentivirus expressing the recently developed H(2)O(2) biosensor HyPer2 show marked H(2)O(2) synthesis when stimulated by angiotensin II, but not following ß-adrenergic receptor activation. We discovered that the angiotensin-II-promoted increase in cardiac myocyte contractility is dependent on H(2)O(2), whereas ß-adrenergic contractile responses occur independently of H(2)O(2) signaling. These studies establish differential roles for H(2)O(2) in control of cardiac contractility and receptor-dependent NOS activation in the heart, and they identify new points for modulation of NO signaling responses by oxidant stress.
Assuntos
Peróxido de Hidrogênio/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Óxido Nítrico Sintase Tipo III/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Proteínas Quinases Ativadas por AMP/metabolismo , Agonistas Adrenérgicos beta/farmacologia , Angiotensina II/farmacologia , Animais , Tamanho Celular/efeitos dos fármacos , Células Cultivadas , Cobre/química , Corantes Fluorescentes/química , Peróxido de Hidrogênio/metabolismo , Immunoblotting , Isoproterenol/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia de Fluorescência , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/enzimologia , Óxido Nítrico/química , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo I/genética , Óxido Nítrico Sintase Tipo III/genética , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pirazóis/farmacologia , Pirimidinas/farmacologia , Vasoconstritores/farmacologiaRESUMO
These studies explore the effects of statins on cyclic AMP-modulated signaling pathways in vascular endothelial cells. We previously observed (Kou, R., Sartoretto, J., and Michel, T. (2009) J. Biol. Chem. 284, 14734-14743) that simvastatin treatment of endothelial cells leads to a marked decrease in PKA-modulated phosphorylation of the protein VASP. Here we show that long-term treatment of mice with simvastatin attenuates the vasorelaxation response to the ß-adrenergic agonist isoproterenol, without affecting endothelin-induced vasoconstriction or carbachol-induced vasorelaxation. We found that statin treatment of endothelial cells dose-dependently inhibits PKA activation as assessed by analyses of serine 157 VASP phosphorylation as well as Epac-mediated Rap1 activation. These effects of simvastatin are completely reversed by mevalonate and by geranylgeranyl pyrophosphate, implicating geranylgeranylation as a critical determinant of the stain response. We used biochemical approaches as well as fluorescence resonance energy transfer (FRET) methods with a cAMP biosensor to show that simvastatin treatment of endothelial cells markedly inhibits cAMP accumulation in response to epinephrine. Importantly, simvastatin treatment significantly decreases Gα(s) abundance, without affecting other Gα subunits. Simvastatin treatment does not influence Gα(s) protein stability, and paradoxically increases the abundance of Gα(s) mRNA. Finally, we found that simvastatin treatment inhibits Gα(s) translation mediated by Akt/mTOR/eIF4/4EBP. Taken together, these findings establish a novel mechanism by which simvastatin modulates ß-adrenergic signaling in vascular wall, and may have implications for cardiovascular therapeutics.
Assuntos
Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Transdução de Sinais/efeitos dos fármacos , Sinvastatina/farmacologia , Animais , Bovinos , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Células Endoteliais/citologia , Endotélio Vascular/citologia , Estabilidade Enzimática , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Camundongos , Fosforilação/efeitos dos fármacos , Transdução de Sinais/fisiologia , Serina-Treonina Quinases TOR/metabolismoRESUMO
MARCKS is an actin-binding protein that modulates vascular endothelial cell migration and cytoskeleton signaling (Kalwa, H., and Michel, T. (2011) J. Biol. Chem. 286, 2320-2330). Angiotensin-II is a vasoactive peptide implicated in vascular physiology as well as pathophysiology; the pathways connecting angiotensin-II and cytoskeletal remodeling are incompletely understood. Here we show that MARCKS is expressed in intact arterial preparations, with prominent staining of the endothelium. In endothelial cells, angiotensin-II-promoted MARCKS phosphorylation is abrogated by PEG-catalase, implicating endogenous H(2)O(2) in the angiotensin-II response. Studies using the H(2)O(2) biosensor HyPer2 reveal that angiotensin-II promotes increases in intracellular H(2)O(2). We used a Rac1 FRET biosensor to show that angiotensin-II promotes Rac1 activation that is attenuated by PEG-catalase. siRNA-mediated Rac1 knockdown blocks angiotensin-II-stimulated MARCKS phosphorylation. Cell imaging studies using a phosphoinositide 4,5-bisphosphate (PIP(2)) biosensor revealed that angiotensin-II PIP(2) regulation depends on MARCKS and H(2)O(2). siRNA-mediated knockdown of MARCKS or Rac1 attenuates receptor-mediated activation of the tyrosine kinase c-Abl and disrupts actin fiber formation. These studies establish a critical role for H(2)O(2) in angiotensin-II signaling to the endothelial cytoskeleton in a novel pathway that is critically dependent on MARCKS, Rac1, and c-Abl.
Assuntos
Angiotensina II/metabolismo , Endotélio Vascular/metabolismo , Peróxido de Hidrogênio/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Neuropeptídeos/metabolismo , Transdução de Sinais/fisiologia , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Angiotensina II/genética , Animais , Técnicas Biossensoriais/métodos , Catalase/farmacologia , Bovinos , Citoesqueleto/genética , Citoesqueleto/metabolismo , Endotélio Vascular/citologia , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Substrato Quinase C Rico em Alanina Miristoilada , Neuropeptídeos/genética , Fosfatidilinositol 4,5-Difosfato/genética , Fosfatidilinositol 4,5-Difosfato/metabolismo , Polietilenoglicóis/farmacologia , Proteínas Proto-Oncogênicas c-abl/genética , Proteínas Proto-Oncogênicas c-abl/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas rac de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/genéticaRESUMO
Hydrogen peroxide and other reactive oxygen species are intimately involved in endothelial cell signaling. In many cell types, the AMP-activated protein kinase (AMPK) has been implicated in the control of metabolic responses, but the role of endothelial cell redox signaling in the modulation of AMPK remains to be completely defined. We used RNA interference and pharmacological methods to establish that H(2)O(2) is a critical activator of AMPK in cultured bovine aortic endothelial cells (BAECs). H(2)O(2) treatment of BAECs rapidly and significantly increases the phosphorylation of AMPK. The EC(50) for H(2)O(2)-promoted phosphorylation of AMPK is 65 + or - 15 microM, within the physiological range of cellular H(2)O(2) concentrations. The Ca(2+)/calmodulin-dependent protein kinase kinase-beta (CaMKKbeta) inhibitor STO-609 abolishes H(2)O(2)-dependent AMPK activation, whereas eNOS inhibitors enhance AMPK activation. Similarly, siRNA-mediated knockdown of CaMKKbeta abrogates AMPK activation, whereas siRNA-mediated knockdown of eNOS leads to a striking increase in AMPK phosphorylation. Cellular imaging studies using the H(2)O(2) biosensor HyPer show that siRNA-mediated eNOS knockdown leads to a marked increase in intracellular H(2)O(2) generation, which is blocked by PEG-catalase. eNOS(-/-) mice show a marked increase in AMPK phosphorylation in liver and lung compared to wild-type mice. Lung endothelial cells from eNOS(-/-) mice also show a significant increase in AMPK phosphorylation. Taken together, these results establish that CaMKKbeta is critically involved in mediating the phosphorylation of AMPK promoted by H(2)O(2) in endothelial cells, and document that eNOS is an important negative regulator of AMPK phosphorylation and intracellular H(2)O(2) generation in endothelial cells.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Endotélio Vascular/enzimologia , Peróxido de Hidrogênio/farmacologia , Óxido Nítrico Sintase Tipo III/metabolismo , Animais , Aorta/enzimologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Bovinos , Homeostase , Cinética , Fígado/enzimologia , Pulmão/enzimologia , Camundongos , Camundongos Knockout , Óxido Nítrico Sintase Tipo III/deficiência , Óxido Nítrico Sintase Tipo III/genética , Fosforilação , RNA Interferente Pequeno/genéticaRESUMO
Vasodilator-stimulated phosphoprotein (VASP) is a major substrate for cyclic nucleotide-dependent kinases that has been implicated in cardiac pathology, yet many aspects of VASP's molecular regulation in cardiomyocytes are incompletely understood. In these studies, we explored the role of VASP, both in signaling pathways in isolated murine myocytes, as well as in a model of cardiac hypertrophy in VASP(null) mice. We found that the beta-adrenergic agonist isoproterenol promotes the rapid and reversible phosphorylation of VASP at Ser157 and Ser239. Forskolin and the cAMP analog 8-(4-chlorophenylthio)-cAMP promote a similar pattern of VASP phosphorylation at both sites. The effects of isoproterenol are blocked by atenolol and by compound H-89, an inhibitor of the cAMP-dependent protein kinase. By contrast, phosphorylation of VASP only at Ser239 is seen following activation of particulate guanylate cyclase by atrial natriuretic peptide, or following activation of soluble guanylate cyclase by sodium nitroprusside, or following treatment of myocytes with cGMP analog. We found that basal and isoproterenol-induced VASP phosphorylation is entirely unchanged in cardiomyocytes isolated from either endothelial or neuronal nitric oxide synthase knockout mice. In cardiomyocytes isolated from diabetic mice, only basal VASP phosphorylation is increased, whereas, in cells isolated from mice subjected to ascending aortic constriction (AAC), we found a significant increase in basal VASP expression, along with an increase in VASP phosphorylation, compared with cardiac myocytes isolated from sham-operated mice. Moreover, there is further increase in VASP phosphorylation in cells isolated from hypertrophic hearts following isoproterenol treatment. Finally, we found that VASP(null) mice subjected to transverse aortic constriction develop cardiac hypertrophy with a pattern similar to VASP(+/+) mice. Our findings establish differential receptor-modulated regulation of VASP phosphorylation in cardiomyocytes by cyclic nucleotides. Furthermore, these studies demonstrate for the first time that VASP expression is upregulated in hypertrophied heart.
Assuntos
Cardiomegalia/metabolismo , Moléculas de Adesão Celular/metabolismo , Diabetes Mellitus/metabolismo , Proteínas dos Microfilamentos/metabolismo , Miócitos Cardíacos/metabolismo , Nucleotídeos Cíclicos/metabolismo , Fosfoproteínas/metabolismo , Agonistas Adrenérgicos beta/farmacologia , Antagonistas Adrenérgicos beta/farmacologia , Animais , Pressão Sanguínea , Cardiomegalia/genética , Cardiomegalia/fisiopatologia , Moléculas de Adesão Celular/deficiência , Moléculas de Adesão Celular/genética , Células Cultivadas , AMP Cíclico/análogos & derivados , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Diabetes Mellitus/genética , Diabetes Mellitus/fisiopatologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Ativação Enzimática , Ativadores de Enzimas/farmacologia , Guanilato Ciclase/metabolismo , Frequência Cardíaca , Masculino , Camundongos , Camundongos Knockout , Proteínas dos Microfilamentos/deficiência , Proteínas dos Microfilamentos/genética , Miócitos Cardíacos/efeitos dos fármacos , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo I/genética , Óxido Nítrico Sintase Tipo I/metabolismo , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Fosfoproteínas/deficiência , Fosfoproteínas/genética , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Receptores Adrenérgicos beta/efeitos dos fármacos , Receptores Adrenérgicos beta/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Serina , Guanilil Ciclase Solúvel , Tionucleotídeos/metabolismo , Fatores de Tempo , Regulação para CimaRESUMO
RATIONALE: Hydrogen peroxide (H2O2) is a stable reactive oxygen species (ROS) that has long been implicated in insulin signal transduction in adipocytes. However, H2O2's role in mediating insulin's effects on the heart are unknown. OBJECTIVE: We investigated the role of H2O2 in activating insulin-dependent changes in cardiac myocyte metabolic and inotropic pathways. The sources of insulin-dependent H2O2 generation were also studied. METHODS AND RESULTS: In addition to the canonical role of insulin in modulating cardiac metabolic pathways, we found that insulin also inhibited beta adrenergic-induced increases in cardiac contractility. Catalase and NADPH oxidase (NOX) inhibitors blunted activation of insulin-responsive kinases Akt and mTOR and attenuated beta adrenergic receptor-mediated responses. These insulin responses were lost in a mouse model of type 2 diabetes, suggesting a role for these H2O2-dependent pathways in the diabetic heart. The H2O2-sensitive fluorescent biosensor HyPer revealed rapid increases in cytosolic and caveolar H2O2 concentrations in response to insulin treatment, which were blocked by NOX inhibitors and attenuated in NOX2 KO and NOX4 KO mice. In NOX2 KO cardiac myocytes, insulin-mediated phosphorylation of Akt and mTOR was blocked, while these responses were unaffected in cardiac myocytes from NOX4 KO mice. In contrast, insulin's effects on contractility were lost in cardiac myocytes from NOX4 KO animals but were retained in NOX2 KO mice. CONCLUSIONS: These studies identify a proximal point of bifurcation in cardiac insulin signaling through the simultaneous activation of both NOX2 and NOX4. Each NOX isoform generates H2O2 in cardiac myocytes with distinct time courses, with H2O2 derived from NOX2 augmenting Akt-dependent metabolic effects of insulin, while H2O2 from NOX4 blocks beta adrenergic increases in inotropy. These findings suggest that insulin resistance in the diabetic heart may lead to potentially deleterious potentiation of beta adrenergic responses.
Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Insulina/metabolismo , Miócitos Cardíacos/metabolismo , NADPH Oxidase 2/metabolismo , NADPH Oxidase 4/metabolismo , Transdução de Sinais , Animais , Diabetes Mellitus Tipo 2/enzimologia , Modelos Animais de Doenças , Peróxido de Hidrogênio/metabolismo , Camundongos , Miócitos Cardíacos/enzimologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Serina-Treonina Quinases TOR/metabolismoRESUMO
Caveolin-1 is a scaffolding/regulatory protein that interacts with diverse signaling molecules. Caveolin-1(null) mice have marked metabolic abnormalities, yet the underlying molecular mechanisms are incompletely understood. We found the redox stress plasma biomarker plasma 8-isoprostane was elevated in caveolin-1(null) mice, and discovered that siRNA-mediated caveolin-1 knockdown in endothelial cells promoted significant increases in intracellular H2O2. Mitochondrial ROS production was increased in endothelial cells after caveolin-1 knockdown; 2-deoxy-D-glucose attenuated this increase, implicating caveolin-1 in control of glycolytic pathways. We performed unbiased metabolomic characterizations of endothelial cell lysates following caveolin-1 knockdown, and discovered strikingly increased levels (up to 30-fold) of cellular dipeptides, consistent with autophagy activation. Metabolomic analyses revealed that caveolin-1 knockdown led to a decrease in glycolytic intermediates, accompanied by an increase in fatty acids, suggesting a metabolic switch. Taken together, these results establish that caveolin-1 plays a central role in regulation of oxidative stress, metabolic switching, and autophagy in the endothelium, and may represent a critical target in cardiovascular diseases.
Assuntos
Aorta/patologia , Autofagia , Caveolina 1/fisiologia , Endotélio Vascular/patologia , Metaboloma , Estresse Oxidativo , Animais , Aorta/metabolismo , Catalase/metabolismo , Bovinos , Caveolina 1/antagonistas & inibidores , Células Cultivadas , Endotélio Vascular/metabolismo , Glutationa/metabolismo , Peróxido de Hidrogênio/metabolismo , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismo , Mitocôndrias/patologia , RNA Interferente Pequeno/genética , Espécies Reativas de Oxigênio/metabolismo , Transdução de SinaisRESUMO
Endothelial dysfunction is a central hallmark of diabetes. The transcriptional coactivator PGC-1α is a powerful regulator of metabolism, but its role in endothelial cells remains poorly understood. We show here that endothelial PGC-1α expression is high in diabetic rodents and humans and that PGC-1α powerfully blocks endothelial migration in cell culture and vasculogenesis in vivo. Mechanistically, PGC-1α induces Notch signaling, blunts activation of Rac/Akt/eNOS signaling, and renders endothelial cells unresponsive to established angiogenic factors. Transgenic overexpression of PGC-1α in the endothelium mimics multiple diabetic phenotypes, including aberrant re-endothelialization after carotid injury, blunted wound healing, and reduced blood flow recovery after hindlimb ischemia. Conversely, deletion of endothelial PGC-1α rescues the blunted wound healing and recovery from hindlimb ischemia seen in type 1 and type 2 diabetes. Endothelial PGC-1α thus potently inhibits endothelial function and angiogenesis, and induction of endothelial PGC-1α contributes to multiple aspects of vascular dysfunction in diabetes.
Assuntos
Diabetes Mellitus/metabolismo , Células Endoteliais/metabolismo , Fatores de Transcrição/metabolismo , Animais , Movimento Celular/fisiologia , Células Cultivadas , Células Endoteliais/citologia , Regulação da Expressão Gênica , Membro Posterior/patologia , Humanos , Camundongos , Camundongos Knockout , Coativador 1-alfa do Receptor gama Ativado por Proliferador de PeroxissomoRESUMO
Nitric oxide (NO) and hydrogen peroxide (H2O2) are synthesized within cardiac myocytes, and both molecules play key roles in modulating cardiovascular responses. However, the interconnections between NO and H2O2 in cardiac myocyte signaling have not been properly understood. Adult mouse cardiac myocytes represent an informative model for the study of receptor-modulated signaling pathways involving reactive oxygen species and reactive nitrogen species. However, these cells typically survive for only 1-2 days in culture, and the limited abundance of cellular protein undermines many biochemical analyses. We have exploited chemical sensors and biosensors for use in in vivo imaging studies of H2O2 and NO in adult cardiac myocytes. Here we describe detailed methods for the isolation of cardiac myocytes suitable for imaging studies. We also present our methods for the generation of recombinant lentiviral preparations encoding the H2O2 biosensor HyPer2 that permit analysis of intracellular H2O2 levels using fluorescence microscopy in living cardiac myocytes following tail vein injection and in cultured endothelial cells following infection. We also describe our protocols for using the NO chemical sensor Cu2(FL2E) in living adult mouse cardiac myocytes to study the effects of agonist-modulated H2O2 production on NO synthesis. Using these techniques, we have demonstrated that receptor-stimulated increases in intracellular H2O2 modulate NO levels in living cardiac myocytes. These and similar approaches may facilitate a broad range of studies in other terminally differentiated cells that involve the interaction of NO- and H2O2-regulated signaling responses.
Assuntos
Ventrículos do Coração/metabolismo , Peróxido de Hidrogênio/metabolismo , Imagem Molecular/métodos , Miócitos Cardíacos/metabolismo , Óxido Nítrico/metabolismo , Animais , Técnicas Biossensoriais , Células Endoteliais/metabolismo , Células Endoteliais/ultraestrutura , Corantes Fluorescentes , Vetores Genéticos , Células HEK293 , Ventrículos do Coração/ultraestrutura , Humanos , Peróxido de Hidrogênio/análise , Lentivirus/genética , Masculino , Camundongos , Miócitos Cardíacos/ultraestrutura , Óxido Nítrico/análise , Cultura Primária de Células , Transdução de Sinais , Replicação ViralRESUMO
Obesity places major demands on the protein folding capacity of the endoplasmic reticulum (ER), resulting in ER stress, a condition that promotes hepatic insulin resistance and steatosis. Here we identify the transcription factor, Kruppel-like factor 15 (KLF15), as an essential mediator of ER stress-induced insulin resistance in the liver. Mice with a targeted deletion of KLF15 exhibit increased hepatic ER stress, inflammation, and JNK activation compared to WT mice; however, KLF15 (-/-) mice are protected against hepatic insulin resistance and fatty liver under high-fat feeding conditions and in response to pharmacological induction of ER stress. The mammalian target of rapamycin complex 1 (mTORC1), a key regulator of cellular energy homeostasis, has been shown to cooperate with ER stress signaling pathways to promote hepatic insulin resistance and lipid accumulation. We find that the uncoupling of ER stress and insulin resistance in KLF15 (-/-) liver is associated with the maintenance of a low energy state characterized by decreased mTORC1 activity, increased AMPK phosphorylation and PGC-1α expression and activation of autophagy, an intracellular degradation process that enhances hepatic insulin sensitivity. Furthermore, in primary hepatocytes, KLF15 deficiency markedly inhibits activation of mTORC1 by amino acids and insulin, suggesting a mechanism by which KLF15 controls mTORC1-mediated insulin resistance. This study establishes KLF15 as an important molecular link between ER stress and insulin action.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Estresse do Retículo Endoplasmático , Resistência à Insulina , Fígado/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Proteínas de Ligação a DNA/genética , Gorduras na Dieta/efeitos adversos , Gorduras na Dieta/farmacologia , Fígado Gorduroso/induzido quimicamente , Fígado Gorduroso/genética , Fígado Gorduroso/metabolismo , Fígado Gorduroso/patologia , Humanos , Fatores de Transcrição Kruppel-Like , Fígado/patologia , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Camundongos Knockout , Complexos Multiproteicos , Obesidade/induzido quimicamente , Obesidade/genética , Obesidade/metabolismo , Obesidade/patologia , Fosforilação , Serina-Treonina Quinases TOR , Fatores de Transcrição/genéticaRESUMO
Nitric oxide (NO) and hydrogen peroxide (H(2)O(2)) play key roles in physiological and pathological responses in cardiac myocytes. The mechanisms whereby H(2)O(2)-modulated phosphorylation pathways regulate the endothelial isoform of nitric oxide synthase (eNOS) in these cells are incompletely understood. We show here that H(2)O(2) treatment of adult mouse cardiac myocytes leads to increases in intracellular Ca(2+) ([Ca(2+)](i)), and document that activity of the L-type Ca(2+) channel is necessary for the H(2)O(2)-promoted increase in sarcomere shortening and of [Ca(2+)](i). Using the chemical NO sensor Cu(2)(FL2E), we discovered that the H(2)O(2)-promoted increase in cardiac myocyte NO synthesis requires activation of the L-type Ca(2+) channel, as well as phosphorylation of the AMP-activated protein kinase (AMPK), and mitogen-activated protein kinase kinase 1/2 (MEK1/2). Moreover, H(2)O(2)-stimulated phosphorylations of eNOS, AMPK, MEK1/2, and ERK1/2 all depend on both an increase in [Ca(2+)](i) as well as the activation of protein kinase C (PKC). We also found that H(2)O(2)-promoted cardiac myocyte eNOS translocation from peripheral membranes to internal sites is abrogated by the L-type Ca(2+) channel blocker nifedipine. We have previously shown that kinase Akt is also involved in H(2)O(2)-promoted eNOS phosphorylation. Here we present evidence documenting that H(2)O(2)-promoted Akt phosphorylation is dependent on activation of the L-type Ca(2+) channel, but is independent of PKC. These studies establish key roles for Ca(2+)- and PKC-dependent signaling pathways in the modulation of cardiac myocyte eNOS activation by H(2)O(2).