RESUMO
The roles of endothelial nitric oxide synthase (eNOS) in the ventilatory responses during and after a hypercapnic gas challenge (HCC, 5% CO2, 21% O2, 74% N2) were assessed in freely-moving female and male wild-type (WT) C57BL6 mice and eNOS knock-out (eNOS-/-) mice of C57BL6 background using whole body plethysmography. HCC elicited an array of ventilatory responses that were similar in male and female WT mice, such as increases in breathing frequency (with falls in inspiratory and expiratory times), and increases in tidal volume, minute ventilation, peak inspiratory and expiratory flows, and inspiratory and expiratory drives. eNOS-/- male mice had smaller increases in minute ventilation, peak inspiratory flow and inspiratory drive, and smaller decreases in inspiratory time than WT males. Ventilatory responses in female eNOS-/- mice were similar to those in female WT mice. The ventilatory excitatory phase upon return to room-air was similar in both male and female WT mice. However, the post-HCC increases in frequency of breathing (with decreases in inspiratory times), and increases in tidal volume, minute ventilation, inspiratory drive (i.e., tidal volume/inspiratory time) and expiratory drive (i.e., tidal volume/expiratory time), and peak inspiratory and expiratory flows in male eNOS-/- mice were smaller than in male WT mice. In contrast, the post-HCC responses in female eNOS-/- mice were equal to those of the female WT mice. These findings provide the first evidence that the loss of eNOS affects the ventilatory responses during and after HCC in male C57BL6 mice, whereas female C57BL6 mice can compensate for the loss of eNOS, at least in respect to triggering ventilatory responses to HCC.
Assuntos
Óxido Nítrico Sintase Tipo III/metabolismo , Ventilação Pulmonar/genética , Ventilação Pulmonar/fisiologia , Animais , Feminino , Hipercapnia/fisiopatologia , Hipóxia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico Sintase Tipo III/fisiologia , Respiração , Insuficiência Respiratória/fisiopatologia , Volume de Ventilação PulmonarRESUMO
NADPH diaphorase is used as a histochemical marker of nitric oxide synthase (NOS) in aldehyde-treated tissues. It is thought that the catalytic activity of NOS promotes NADPH-dependent reduction of nitro-blue tetrazolium (NBT) to diformazan. However, it has been argued that a proteinaceous factor other than NOS is responsible for producing diformazan in aldehyde-treated tissues. We propose this is a NO-containing factor such as an S-nitrosothiol and/or a dinitrosyl-iron (II) cysteine complex or nitrosated proteins including NOS. We now report that (1) S-nitrosothiols covalently modify both NBT and TNBT, but only change the reduction potential of NBT after modification, (2) addition of S-nitrosothiols or ß- or α-NADPH to solutions of NBT did not elicit diformazan, (3) addition of S-nitrosothiols to solutions of NBT plus ß- or α-NADPH elicited rapid formation of diformazan in the absence or presence of paraformaldehyde, (4) addition of S-nitrosothiols to solutions of NBT plus ß- or α-NADP did not produce diformazan, (5) S-nitrosothiols did not promote NADPH-dependent reduction of tetra-nitro-blue tetrazolium (TNBT) in which all four phenolic rings are nitrated, (6) cytoplasmic vesicles in vascular endothelial cells known to stain for NADPH diaphorase were rich in S-nitrosothiols, and (7) procedures that accelerate decomposition of S-nitrosothiols, markedly reduced NADPH diaphorase staining in tissue sections subsequently subjected to paraformaldehyde fixation. Our results suggest that NADPH diaphorase in aldehyde-fixed tissues is not enzymatic but is due to the presence of NO-containing factors (free SNOs or nitrosated proteins such as NOS), which promote NADPH-dependent reduction of NBT to diformazan.
Assuntos
NADPH Desidrogenase/metabolismo , Óxido Nítrico Sintase/metabolismo , S-Nitrosotióis/metabolismo , Animais , Compostos Azo/metabolismo , Tronco Encefálico/química , Tronco Encefálico/efeitos dos fármacos , Tronco Encefálico/metabolismo , Cerebelo/química , Cerebelo/efeitos dos fármacos , Cerebelo/metabolismo , Formaldeído/farmacologia , Masculino , Nitroazul de Tetrazólio/metabolismo , Oxirredução , Polímeros/farmacologia , Ratos , Ratos Sprague-Dawley , Coloração e Rotulagem/métodos , Coloração e Rotulagem/normasRESUMO
S-Nitrosoglutathione is an endogenous airway smooth muscle relaxant. Increased airway S-nitrosoglutathione breakdown occurs in some asthma patients. We asked whether patients with increased airway catabolism of this molecule had clinical features that distinguished them from other asthma patients. We measured S-nitrosoglutathione reductase expression and activity in bronchoscopy samples taken from 66 subjects in the Severe Asthma Research Program. We also analysed phenotype and genotype data taken from the program as a whole. Airway S-nitrosoglutathione reductase activity was increased in asthma patients (p=0.032). However, only a subpopulation was affected and this subpopulation was not defined by a "severe asthma" diagnosis. Subjects with increased activity were younger, had higher IgE and an earlier onset of symptoms. Consistent with a link between S-nitrosoglutathione biochemistry and atopy: 1) interleukin 13 increased S-nitrosoglutathione reductase expression and 2) subjects with an S-nitrosoglutathione reductase single nucleotide polymorphism previously associated with asthma had higher IgE than those without this single nucleotide polymorphism. Expression was higher in airway epithelium than in smooth muscle and was increased in regions of the asthmatic lung with decreased airflow. An early-onset, allergic phenotype characterises the asthma population with increased S-nitrosoglutathione reductase activity.
Assuntos
Aldeído Oxirredutases/metabolismo , Asma/enzimologia , Brônquios/enzimologia , Regulação Enzimológica da Expressão Gênica , Adolescente , Adulto , Biópsia , Lavagem Broncoalveolar , Broncoscopia , Estudos de Casos e Controles , Células Cultivadas , Criança , Feminino , Genótipo , Humanos , Imunoglobulina E/sangue , Imuno-Histoquímica , Interleucina-13/metabolismo , Pulmão/enzimologia , Imageamento por Ressonância Magnética , Masculino , Metabolismo , Pessoa de Meia-Idade , Músculo Liso/enzimologia , Fenótipo , Polimorfismo de Nucleotídeo Único , Adulto JovemRESUMO
C57BL6 mice display non-eupneic breathing and spontaneous apneas during wakefulness and sleep as well as markedly disordered breathing following cessation of a hypoxic challenge. We examined whether (1) C57BL6 mice display marked non-eupneic breathing following hypercapnic or hypoxic-hypercapnic challenges, and (2) compared the post-hypoxia changes in non-eupneic breathing of C57BL6 mice to those of B6AF1 (57BL6 dam × A/J sire) and Swiss-Webster mice, which display different ventilatory responses than C57BL6 mice. C57BL6 mice displayed marked increases in respiratory frequency and non-eupneic breathing upon return to room-air after hypoxic (10% O2, 90% N2), hypercapnic (5% CO2, 21% O2 and 74% N2) and hypoxic-hypercapnic (10% O2, 5% CO2 and 85% N2) challenges. B6AF1 mice displayed less tachypnea and reduced non-eupneic breathing post-hypoxia, whereas Swiss-Webster mice displayed robust tachypnea with minimal increases in non-eupneic breathing post-hypoxia. These studies demonstrate that non-eupneic breathing increases after physiologically-relevant hypoxic-hypercapnic challenge in C57BL6 mice and suggest that further studies with these and B6AF1 and Swiss-Webster mice will help define the genetics of non-eupneic breathing.
Assuntos
Hipercapnia/fisiopatologia , Hipóxia/fisiopatologia , Respiração , Taquipneia/fisiopatologia , Animais , Estado de Consciência , Hipercapnia/genética , Hipóxia/complicações , Hipóxia/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pletismografia Total , Especificidade da Espécie , Taquipneia/etiologia , Taquipneia/genéticaRESUMO
When erythrocyte hemoglobin (Hb) is fully saturated with O2, nitric oxide (NO) covalently binds to the cysteine 93 residue of the Hb ß-chain (B93-CYS), forming S-nitrosohemoglobin. Binding of NO is allosterically coupled to the O2 saturation of Hb. As saturation falls, the NO group on B93-CYS is transferred to thiols in the erythrocyte, and in the plasma, forming circulating S-nitrosothiols. Here, we studied whether the changes in ventilation during and following exposure to a hypoxic challenge were dependent on erythrocytic B93-CYS. Studies were performed in conscious mice in which native murine Hb was replaced with human Hb (hB93-CYS mice) and in mice in which murine Hb was replaced with human Hb containing an alanine rather than cysteine at position 93 on the Bchain (hB93-ALA). Both strains expressed human γ-chain Hb, likely allowing a residual element of S-nitrosothiol-dependent signaling. While resting parameters and initial hypoxic (10% O2, 90% N2) ventilatory responses were similar in hB93-CYS mice and hB93-ALA mice, the excitatory ventilatory responses (short-term potentiation) that occurred once the mice were returned to room air were markedly diminished in hB93-ALA mice. Further, short-term potentiation responses were virtually absent in mice with bilateral transection of the carotid sinus nerves. These data demonstrate that hB93-CYS plays an essential role in mediating carotid sinus nerve-dependent short-term potentiation, an important mechanism for recovery from acute hypoxia.
Assuntos
Seio Carotídeo/fisiopatologia , Cisteína/metabolismo , Hemoglobinas/metabolismo , Hipóxia/fisiopatologia , Ventilação Pulmonar , Mecânica Respiratória , Animais , Estado de Consciência , Cisteína/química , Hemoglobinas/química , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Relação Estrutura-AtividadeRESUMO
S-nitrosothiols have been implicated in the etiology of various pulmonary diseases. Many of these diseases display gender preferences in presentation or altered severity that occurs with puberty, the mechanism by which is unknown. Estrogen has been shown to influence the expression and activity of endothelial nitric oxide synthase (eNOS) which is associated with increased S-nitrosothiol production. The effects of gender hormones on the expression and activity of the de-nitrosylating enzyme S-nitrosoglutathione reductase (GSNO-R) are undefined. This report evaluates the effects of gender hormones on the activity and expression of GSNO-R and its relationship to N-acetyl cysteine (NAC)-induced pulmonary hypertension (PH). GSNO-R activity was elevated in lung homogenates from female compared to male mice. Increased activity was not due to changes in GSNO-R expression, but correlated with GSNO-R S-nitrosylation: females were greater than males. The ability of GSNO-R to be activated by S-nitrosylation was confirmed by: 1) the ability of S-nitrosoglutathione (GSNO) to increase the activity of GSNO-R in murine pulmonary endothelial cells and 2) reduced activity of GSNO-R in lung homogenates from eNOS(-/-) mice. Gender differences in GSNO-R activity appear to explain the difference in the ability of NAC to induce PH: female and castrated male animals are protected from NAC-induced PH. Castration results in elevated GSNO-R activity that is similar to that seen in female animals. The data suggest that GSNO-R activity is modulated by both estrogens and androgens in conjunction with hormonal regulation of eNOS to maintain S-nitrosothiol homeostasis. Moreover, disruption of this eNOS-GSNO-R axis contributes to the development of PH.
Assuntos
Aldeído Oxirredutases/metabolismo , Endotélio Vascular/enzimologia , Pulmão/enzimologia , Acetilcisteína/farmacologia , Animais , Broncodilatadores/farmacologia , Células Cultivadas , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Estrogênios/farmacologia , Feminino , Sequestradores de Radicais Livres/farmacologia , Hipóxia , Pulmão/citologia , Pulmão/efeitos dos fármacos , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Orquiectomia , S-Nitrosoglutationa/farmacologia , Fatores SexuaisRESUMO
Allogeneic bone marrow transplantation (BMT) is an effective therapy for hematologic malignancies. However graft-versus-host disease (GVHD) is a major limiting factor for a successful patient outcome. GVHD is a result of alloimmune responses of donor T lymphocytes attacking the recipient's cells and tissues. Chemokine receptor CCR5 plays a role in solid organ allograft rejection and mediates murine GVHD pathogenesis. Herein, we report that infiltrating lymphocytes in the skin of human acute GVHD (aGVHD) samples are predominantly CCR5(+) T cells. In addition, we characterized the features of the CCR5 expression on alloreactive T lymphocytes. We found that the CCR5(+) population exhibits the characteristics of the activated effector T cell phenotype. CCR5 expression is upregulated upon allogenic stimulation, and CCR5(+) cells are proliferating with coexpression of T cell activation markers. Furthermore, the activated T cells producing inflammatory cytokine tumor necrosis factor (TNF)alpha, interleukin (IL)-2, or interferon (IFN)-gamma, are positive for CCR5. Thus, CCR5 is a marker for GVHD effector cells and CCR5(+) T cells are active participants in the pathogenesis of human aGVHD.
Assuntos
Doença Enxerto-Hospedeiro/imunologia , Receptores CCR5/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Imunologia de Transplantes/imunologia , Antígenos CD1/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/patologia , Proliferação de Células , Células Dendríticas/imunologia , Doença Enxerto-Hospedeiro/patologia , Humanos , Interferon gama/metabolismo , Interleucina-2/metabolismo , Células de Langerhans/metabolismo , Células de Langerhans/patologia , Leucócitos Mononucleares/imunologia , Lábio/patologia , Ativação Linfocitária/imunologia , Teste de Cultura Mista de Linfócitos , Pele/patologia , Linfócitos T/patologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
NO transfer reactions between protein and peptide cysteines have been proposed to represent regulated signaling processes. We used the pharmaceutical antioxidant N-acetylcysteine (NAC) as a bait reactant to measure NO transfer reactions in blood and to study the vascular effects of these reactions in vivo. NAC was converted to S-nitroso-N-acetylcysteine (SNOAC), decreasing erythrocytic S-nitrosothiol content, both during whole-blood deoxygenation ex vivo and during a 3-week protocol in which mice received high-dose NAC in vivo. Strikingly, the NAC-treated mice developed pulmonary arterial hypertension (PAH) that mimicked the effects of chronic hypoxia. Moreover, systemic SNOAC administration recapitulated effects of both NAC and hypoxia. eNOS-deficient mice were protected from the effects of NAC but not SNOAC, suggesting that conversion of NAC to SNOAC was necessary for the development of PAH. These data reveal an unanticipated adverse effect of chronic NAC administration and introduce a new animal model of PAH. Moreover, evidence that conversion of NAC to SNOAC during blood deoxygenation is necessary for the development of PAH in this model challenges conventional views of oxygen sensing and of NO signaling.
Assuntos
Hipóxia/metabolismo , Hipóxia/patologia , Artéria Pulmonar/patologia , S-Nitrosotióis/metabolismo , Transdução de Sinais , Acetilcisteína/análogos & derivados , Acetilcisteína/farmacologia , Animais , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Glutationa/análogos & derivados , Glutationa/farmacologia , Hipertensão/fisiopatologia , Hipóxia/induzido quimicamente , Camundongos , Camundongos Knockout , Óxido Nítrico Sintase Tipo III/deficiência , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Nitrocompostos/farmacologia , Compostos Nitrosos/química , Compostos Nitrosos/farmacologia , Oxigênio/metabolismo , Artéria Pulmonar/fisiopatologiaRESUMO
S-nitrosoglutathione (GSNO) stabilizes the alpha-subunit of hypoxia inducible factor-1 (HIF-1) in normoxic cells, but not in the presence of PI3K inhibitors. In this report, the biochemical pathway by which GSNO alters PI3K/Akt activity to modify HIF-1 expression was characterized in Cos cells and primary pulmonary vascular endothelial cells. GSNO increased Akt kinase activity--and downstream HIF-1alpha protein accumulation and DNA-binding activity--in a dose- and time-dependent manner. The PI3K inhibitors, wortmannin and LY294002, blocked these responses. Neither glutathione nor 8-bromo-cyclic GMP mimicked the GSNO-induced increases in Akt kinase activity. GSNO-induced Akt kinase activity and downstream HIF-1alpha stabilization were blocked by acivicin, an inhibitor of gamma-glutamyl transpeptidase (gammaGT), a transmembrane protein that can translate extracellular GSNO to intracellular S-nitrosocysteinylglycine. Dithiothreitol blocked GSNO-induced Akt kinase activity and HIF-1alpha stabilization. Moreover, the 3'-phosphatase of phosphoinositides, PTEN (phosphatase and tensin homolog deleted on chromosome ten) was S-nitrosylated by GSNO in pulmonary arterial endothelial cells, which was reversed by dithiothreitol and ultraviolet light. Interestingly, the abundance of S-nitrosylated PTEN also correlated inversely with PTEN activity. Taken together, these results suggest that GSNO induction of Akt appears to be mediated by S-nitrosylation chemistry rather than classic NO signaling through guanylate cyclase/cGMP. We speculate that gammaGT-dependent activation of Akt and subsequent activation of HIF-1 in vascular beds may be relevant to the regulation of HIF-1-dependent gene expression in conditions associated with oxyhemoglobin deoxygenation, as opposed to profoundly low Po(2), in the pulmonary vasculature.
Assuntos
Células Endoteliais/metabolismo , Fator 1 Induzível por Hipóxia/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Artéria Pulmonar/metabolismo , S-Nitrosoglutationa/farmacologia , Animais , Células COS , Bovinos , Células Cultivadas , Chlorocebus aethiops , Células Endoteliais/efeitos dos fármacos , Camundongos , Modelos Biológicos , PTEN Fosfo-Hidrolase/química , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Artéria Pulmonar/citologia , Artéria Pulmonar/efeitos dos fármacos , Proteínas Recombinantes/metabolismo , S-Nitrosoglutationa/metabolismoRESUMO
The endogenous bronchodilator, S-nitrosoglutathione (GSNO), increases expression, maturation, and function of both the wild-type and the DeltaF508 mutant of the cystic fibrosis transmembrane conductance regulatory protein (CFTR). Though transcriptional mechanisms of action have been identified, GSNO seems also to have post-transcriptional effects on CFTR maturation. Here, we report that 1) GSNO is only one of a class of S-nitrosylating agents that, at low micromolar concentrations, increase DeltaF508 and wild-type CFTR expression and maturation; 2) NO itself (at these concentrations) and 8-bromocyclic GMP are minimally active on CFTR; 3) a novel agent, S-nitrosoglutathione diethyl ester, bypasses the need for GSNO bioactivation by gamma-glutamyl transpeptidase to increase CFTR maturation; 4) surprisingly, expression-but not S-nitrosylation-of cysteine string proteins (Csp) 1 and 2 is increased by GSNO; 5) the effect of GSNO to increase full maturation of wild-type CFTR is inhibited by Csp silencing (si)RNA; 6) proteins relevant to CFTR trafficking are SNO-modified, and SNO proteins traffic through the endoplasmic reticulum (ER) and Golgi after GSNO exposure; and 7) GSNO alters the interactions of DeltaF508 CFTR with Csp and Hsc70 in the ER and Golgi. These data suggest that GSNO is one of a class of S-nitrosylating agents that act independently of the classic NO radical/cyclic GMP pathway to increase CFTR expression and maturation. They also suggest that the effect of GSNO is dependent on Csp and on intracellular SNO trafficking. We speculate that these data will be of relevance to the development of NO donor-based therapies for CF.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Células Epiteliais/fisiologia , Regulação da Expressão Gênica , Processamento de Proteína Pós-Traducional , S-Nitrosoglutationa/farmacologia , Diferenciação Celular , Linhagem Celular , Retículo Endoplasmático/metabolismo , Proteínas de Choque Térmico HSP40/fisiologia , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Proteínas de Membrana/fisiologia , Estrutura Molecular , S-Nitrosotióis/farmacologiaRESUMO
Although Jun upregulation and activation have been established as critical to oncogenesis, the relevant downstream pathways remain incompletely characterized. In this study, we found that c-Jun blocks erythroid differentiation in primary human hematopoietic progenitors and, correspondingly, that Jun factors block transcriptional activation by GATA-1, the central regulator of erythroid differentiation. Mutagenesis of c-Jun suggested that its repression of GATA-1 occurs through a transcriptional mechanism involving activation of downstream genes. We identified the hairy-enhancer-of-split-related factor HERP2 as a novel gene upregulated by c-Jun. HERP2 showed physical interaction with GATA-1 and repressed GATA-1 transcriptional activation. Furthermore, transduction of HERP2 into primary human hematopoietic progenitors inhibited erythroid differentiation. These results thus define a novel regulatory pathway linking the transcription factors c-Jun, HERP2, and GATA-1. Furthermore, these results establish a connection between the Notch signaling pathway, of which the HERP factors are a critical component, and the GATA family, which participates in programming of cellular differentiation.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Eritropoese/fisiologia , Regulação da Expressão Gênica , Células-Tronco Hematopoéticas/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Antígenos CD34 , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Proteínas de Ciclo Celular/genética , Diferenciação Celular , Fatores de Ligação de DNA Eritroide Específicos , Fator de Transcrição GATA1 , Sequências Hélice-Alça-Hélice , Células-Tronco Hematopoéticas/citologia , Humanos , Células K562 , Proteínas Proto-Oncogênicas c-jun/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/genética , Transcrição GênicaRESUMO
The activities of certain nuclear regulatory proteins are modified by high concentrations of S-nitrosothiols associated with nitrosative stress. In the present study, we have studied the effect of physiological (low microM) concentrations of the endogenous S-nitrosothiol, GSNO (S-nitrosoglutathione), on the activities of nuclear regulatory proteins Sp3 and Sp1 (specificity proteins 3 and 1). Low concentrations of GSNO increased Sp3 binding, as well as Sp3-dependent transcription of the cystic fibrosis transmembrane conductance regulatory gene, cftr. However, higher GSNO levels prevented Sp3 binding, augmented Sp1 binding and prevented both cftr transcription and CFTR (cystic fibrosis transmembrane conductance regulator) expression. We conclude that low concentrations of GSNO favour Sp3 binding to 'housekeeping' genes such as cftr, whereas nitrosative stress-associated GSNO concentrations shut off Sp3-dependent transcription, possibly to redirect cellular resources. Since low micromolar concentrations of GSNO also increase the maturation and activity of a clinically common CFTR mutant, whereas higher concentrations have the opposite effect, these observations may have implications for dosing of S-nitrosylating agents used in cystic fibrosis clinical trials.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/biossíntese , Proteínas de Ligação a DNA/fisiologia , Regulação da Expressão Gênica/efeitos dos fármacos , S-Nitrosoglutationa/farmacologia , Fator de Transcrição Sp1/fisiologia , Fatores de Transcrição/fisiologia , Animais , Linhagem Celular , Linhagem Celular Tumoral/efeitos dos fármacos , Meios de Cultura Livres de Soro , Cicloeximida/farmacologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Dactinomicina/farmacologia , Relação Dose-Resposta a Droga , Drosophila melanogaster/citologia , Humanos , Isoxazóis/farmacologia , Neoplasias Pulmonares/patologia , Plicamicina/farmacologia , Ligação Proteica/efeitos dos fármacos , Alvéolos Pulmonares/patologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Proteínas Recombinantes de Fusão/fisiologia , S-Nitrosoglutationa/administração & dosagem , Transdução de Sinais/fisiologia , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp3 , Transcrição Gênica , TransfecçãoRESUMO
S-Nitrosylated proteins form when a cysteine thiol reacts with nitric oxide (NO) in the presence of an electron acceptor to form an S-NO bond. Under physiological conditions, this posttranslational modification affects the function a wide array of cell proteins, ranging from ion channels to nuclear regulatory proteins. Recent evidence suggests that 1) S-nitrosylated proteins can be synthesized by exposure of specific redox-active motifs to NO, through transnitrosation/transfer reactions, or through metalloprotein-catalyzed reactions; 2) S-nitrosothiols can be sequestered in membranes, lipophilic protein folds, or in vesicles to preserve their activity; and 3) S-nitrosothiols can be degraded by a number of enzymes systems. These recent insights regarding the bioactivities, molecular signaling pathways, and metabolism of endogenous S-nitrosothiols have suggested several new therapies for disease ranging from cystic fibrosis to pulmonary hypertension.
Assuntos
S-Nitrosotióis/metabolismo , Proteínas de Ligação a DNA/metabolismo , Inibidores Enzimáticos/metabolismo , Humanos , Fator 1 Induzível por Hipóxia , Subunidade alfa do Fator 1 Induzível por Hipóxia , Isoxazóis/metabolismo , Óxido Nítrico/metabolismo , Doadores de Óxido Nítrico/metabolismo , Proteínas Nucleares/metabolismo , Oxirredução , S-Nitrosoglutationa/metabolismo , Transdução de Sinais/fisiologia , Superóxido Dismutase/metabolismo , Fatores de Transcrição/metabolismoRESUMO
BACKGROUND: Anesthetics are protective during ischemic-reperfusion injury and associated inflammation; therefore, the authors hypothesized that anesthetic pretreatment may provide protection in culture from cytokine-induced cell death. METHODS: Rat vascular smooth muscle (VSM) cell and human umbilical vascular endothelial cell (HUVEC) cultures were used to determine whether pretreatment with 30 min of isoflurane decreases cell death from tumor necrosis factor alpha (TNF-alpha), interleukin 1 (IL-1 beta), and interferon (IFN-gamma) alone or in combination. Cell survival and viability were determined by trypan blue staining and cell proliferation assay, as well as by DNA fragmentation assays. The roles of protein kinase C (PKC) and adenosine triphosphate-sensitive potassium (K(ATP)) channels in mediating isoflurane (and halothane) protection were evaluated with the antagonists staurosporine or glibenclamide in cytokine- and also hydrogen peroxide (H(2)O(2))-induced cell death. RESULTS: Pretreatment with 1.5% isoflurane immediately prior to cytokine exposure increased cell survival and viability from cytokines by 10-60% for 24, 48, 72, and 96 h in VSMs and up to 72 h in HUVECs. DNA fragmentation (TUNEL) was also attenuated by isoflurane. Isoflurane was equally effective in VSMs at 0.75, 1.5, and 2.5%, whereas in HUVECs, 1.5 and 2.5% were more effective than 0.75%. In VSMs, isoflurane administered 1 h prior to or simultaneously with cytokines was also effective, whereas isoflurane 2 h prior to cytokines was less effective, and either 4 h prior to or 30 min after cytokines was not effective. In both cytokine- and H(2)O(2)-induced cell death, isoflurane and halothane pretreatment were equally protective, and staurosporine and glibenclamide attenuated the protective effect. CONCLUSIONS: Thirty minutes of isoflurane attenuates cytokine-induced cell death and increases cell viability in VSMs for 96 h and in HUVECs for 72 h. Isoflurane must be administered less than 2 h prior to or simultaneously with the cytokines to be protective. These initial inhibitor studies suggest involvement of PKC and K(ATP) channels in isoflurane and halothane protection against both cytokine- and H(2)O(2)-induced cell death of VSMs and HUVECs.