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1.
Am J Respir Cell Mol Biol ; 62(6): 732-746, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32048876

RESUMO

Pulmonary vasoconstriction resulting from intermittent hypoxia (IH) contributes to pulmonary hypertension (pHTN) in patients with sleep apnea (SA), although the mechanisms involved remain poorly understood. Based on prior studies in patients with SA and animal models of SA, the objective of this study was to evaluate the role of PKCß and mitochondrial reactive oxygen species (mitoROS) in mediating enhanced pulmonary vasoconstrictor reactivity after IH. We hypothesized that PKCß mediates vasoconstriction through interaction with the scaffolding protein PICK1 (protein interacting with C kinase 1), activation of mitochondrial ATP-sensitive potassium channels (mitoKATP), and stimulated production of mitoROS. We further hypothesized that this signaling axis mediates enhanced vasoconstriction and pHTN after IH. Rats were exposed to IH or sham conditions (7 h/d, 4 wk). Chronic oral administration of the antioxidant Tempol or the PKCß inhibitor LY-333531 abolished IH-induced increases in right ventricular systolic pressure and right ventricular hypertrophy. Furthermore, scavengers of O2- or mitoROS prevented enhanced PKCß-dependent vasoconstrictor reactivity to endothelin-1 in pulmonary arteries from IH rats. In addition, this PKCß/mitoROS signaling pathway could be stimulated by the PKC activator PMA in pulmonary arteries from control rats, and in both rat and human pulmonary arterial smooth muscle cells. These responses to PMA were attenuated by inhibition of mitoKATP or PICK1. Subcellular fractionation and proximity ligation assays further demonstrated that PKCß acutely translocates to mitochondria upon stimulation and associates with PICK1. We conclude that a PKCß/mitoROS signaling axis contributes to enhanced vasoconstriction and pHTN after IH. Furthermore, PKCß mediates pulmonary vasoconstriction through interaction with PICK1, activation of mitoKATP, and subsequent mitoROS generation.


Assuntos
Hipertensão Pulmonar/fisiopatologia , Hipóxia/fisiopatologia , Mitocôndrias/fisiologia , Proteína Quinase C beta/fisiologia , Artéria Pulmonar/fisiopatologia , Vasoconstrição/fisiologia , Animais , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/metabolismo , Células Cultivadas , Óxidos N-Cíclicos/farmacologia , Proteínas do Citoesqueleto/antagonistas & inibidores , Proteínas do Citoesqueleto/metabolismo , Sequestradores de Radicais Livres/farmacologia , Humanos , Hipertensão Pulmonar/etiologia , Hipóxia/complicações , Hipóxia/enzimologia , Indóis/farmacologia , Masculino , Maleimidas/farmacologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Artérias Mesentéricas/efeitos dos fármacos , Artérias Mesentéricas/fisiopatologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/enzimologia , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/metabolismo , Canais de Potássio/metabolismo , Mapeamento de Interação de Proteínas , Artéria Pulmonar/enzimologia , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Síndromes da Apneia do Sono/fisiopatologia , Marcadores de Spin , Acetato de Tetradecanoilforbol/farmacologia
2.
Am J Physiol Heart Circ Physiol ; 314(5): H1011-H1021, 2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29373038

RESUMO

Chronic hypoxia (CH) augments basal and endothelin-1 (ET-1)-induced pulmonary vasoconstrictor reactivity through reactive oxygen species (ROS) generation and RhoA/Rho kinase (ROCK)-dependent myofilament Ca2+ sensitization. Because ROCK promotes actin polymerization and the actin cytoskeleton regulates smooth muscle tension, we hypothesized that actin polymerization is required for enhanced basal and ET-1-dependent vasoconstriction after CH. To test this hypothesis, both end points were monitored in pressurized, endothelium-disrupted pulmonary arteries (fourth-fifth order) from control and CH (4 wk at 0.5 atm) rats. The actin polymerization inhibitors cytochalasin and latrunculin attenuated both basal and ET-1-induced vasoconstriction only in CH vessels. To test whether CH directly alters the arterial actin profile, we measured filamentous actin (F-actin)-to-globular actin (G-actin) ratios by fluorescent labeling of F-actin and G-actin in fixed pulmonary arteries and actin sedimentation assays using homogenized pulmonary artery lysates. We observed no difference in actin polymerization between groups under baseline conditions, but ET-1 enhanced actin polymerization in pulmonary arteries from CH rats. This response was blunted by the ROS scavenger tiron, the ROCK inhibitor fasudil, and the mDia (RhoA effector) inhibitor small-molecule inhibitor of formin homology domain 2. Immunoblot analysis revealed an effect of CH to increase both phosphorylated (inactive) and total levels of the actin disassembly factor cofilin but not phosphorylated cofilin-to-total cofilin ratios. We conclude that actin polymerization contributes to increased basal pulmonary arterial constriction and ET-1-induced vasoconstrictor reactivity after CH in a ROS- and ROCK-dependent manner. Our results further suggest that enhanced ET-1-mediated actin polymerization after CH is dependent on mDia but independent of changes in the phosphorylated cofilin-to-total cofilin ratio. NEW & NOTEWORTHY This research is the first to demonstrate a role for actin polymerization in chronic hypoxia-induced basal pulmonary arterial constriction and enhanced agonist-induced vasoconstrictor activity. These results suggest that a reactive oxygen species-Rho kinase-actin polymerization signaling pathway mediates this response and may provide a mechanistic basis for the vasoconstrictor component of pulmonary hypertension.


Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Hipertensão Pulmonar/etiologia , Hipóxia/complicações , Artéria Pulmonar/metabolismo , Remodelação Vascular , Vasoconstrição , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/patologia , Fatores de Despolimerização de Actina/metabolismo , Animais , Doença Crônica , Modelos Animais de Doenças , Endotelina-1/farmacologia , Hipertensão Pulmonar/metabolismo , Hipertensão Pulmonar/patologia , Hipertensão Pulmonar/fisiopatologia , Hipóxia/metabolismo , Hipóxia/patologia , Hipóxia/fisiopatologia , Masculino , Estresse Oxidativo , Fosforilação , Polimerização , Artéria Pulmonar/efeitos dos fármacos , Artéria Pulmonar/patologia , Artéria Pulmonar/fisiopatologia , Ratos Sprague-Dawley , Remodelação Vascular/efeitos dos fármacos , Vasoconstrição/efeitos dos fármacos , Vasoconstritores/farmacologia , Proteínas rho de Ligação ao GTP/metabolismo , Quinases Associadas a rho/metabolismo
3.
Bioorg Med Chem Lett ; 16(3): 731-6, 2006 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16263283

RESUMO

A series of conformationally constrained bicyclic derivatives derived from SR141716 was prepared and evaluated as hCB(1)-R antagonists and inverse agonists. Optimization of the structure-activity relationships around the 2,6-dihydro-pyrazolo[4,3-d]pyrimidin-7-one derivative 2a led to the identification of two compounds with oral activity in rodent feeding models (2h and 4a). Replacement of the PP group in 2h with other bicyclic groups resulted in a loss of binding affinity.


Assuntos
Analgésicos/síntese química , Analgésicos/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/química , Comportamento Alimentar/efeitos dos fármacos , Receptor CB1 de Canabinoide/antagonistas & inibidores , Animais , Sítios de Ligação , Comportamento Alimentar/fisiologia , Modelos Biológicos , Piperidinas/química , Pirazóis/química , Pirazolonas/química , Pirimidinonas/química , Receptor CB1 de Canabinoide/agonistas , Rimonabanto , Roedores , Relação Estrutura-Atividade
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