RESUMO
A previous study demonstrated that glutathione (GSH) produces specific antidepressant-like effect in the forced swimming test (FST), a predictive test of antidepressant activity. The present study investigated the involvement of multiple cellular targets implicated in the antidepressant-like effect of GSH in the FST. The antidepressant-like effect of GSH (300 nmol/site, icv) lasted up to 3 h when mice were submitted to FST. The central administration of oxidized GSH (GSSG, 3-300 nmol/site) did not alter the behavior of mice submitted to the FST. Furthermore, the combined treatment of sub-effective doses of GSH (100 nmol/site, icv) with a sub-effective dose of classical antidepressants (fluoxetine 10 mg/kg, and imipramine 5 mg/kg, ip) presented synergistic effect by decreasing the immobility time in the FST. The antidepressant-like effect of GSH was abolished by prazosin (1 mg/kg, ip, α1-adrenoceptor antagonist), baclofen (1 mg/kg, ip, GABAB receptor agonist), bicuculline (1 mg/kg, ip, GABAA receptor antagonist), l-arginine (750 mg/kg, ip, NO precursor), SNAP (25 µg/site, icv, NO donor), but not by yohimbine (1 mg/kg, ip, α2-adrenoceptor antagonist). The NMDA receptor antagonists, MK-801(0.001 mg/kg, ip) or GMP (0.5 mg/kg, ip), potentiated the effect of a sub-effective dose of GSH in the FST. These results suggest that the antidepressant-like effect induced by GSH is connected to the activation of α1 adrenergic and GABAA receptors, as well as the inhibition of GABAB and NMDA receptors and NO biosyntesis. We speculate that redox-mediated signaling on the extracelular portion of cell membrane receptors would be a common mechanism of action of GSH.
Assuntos
Antidepressivos/farmacologia , Glutationa/farmacologia , Terapia de Alvo Molecular , Antagonistas Adrenérgicos/farmacologia , Animais , Arginina/farmacologia , Sinergismo Farmacológico , Feminino , Glutationa/administração & dosagem , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Imobilização , Masculino , Camundongos , Receptores Adrenérgicos/metabolismo , Receptores de GABA/metabolismo , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Receptores de N-Metil-D-Aspartato/metabolismo , S-Nitroso-N-Acetilpenicilamina/farmacologia , NataçãoRESUMO
This study aimed to examine the effects of nitric oxide (NO) and different phosphodiesterase (PDE) families on meiosis resumption, nucleotides levels and embryo production. Experiment I, COCs were matured in vitro with the NO donor S-nitroso-N-acetylpenicillamine (SNAP) associated or not with the soluble guanylate cyclase (sGC) inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), meiotic resumption and nucleotides levels were assessed. SNAP delayed germinal vesicle breakdown (GVBD) (53.4 ± 1.2 versus 78.4 ± 2.4% for controls, P 0.05). Cyclic GMP levels were higher in SNAP (3.94 ± 0.18, P 0.05). Embryo development did not differ from the control for SNAP and cilostamide groups (38.7 ± 5.8, 37.9 ± 6.2 and 40.5 ± 5.8%, P > 0.05), but SNAP + cilostamide decreased embryo production (25.7 ± 6.9%, P < 0.05). In conclusion, SNAP was confirmed to delay meiosis resumption by the NO/sGC/cGMP pathway, by increasing cGMP, but not cAMP. Inhibiting different PDEs to further increase nucleotides in association with SNAP did not show any additive effects on meiosis resumption, indicating that other pathways are involved. Moreover, SNAP + cilostamide affected the meiosis progression and decreased embryo development.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Blastocisto/fisiologia , Óxido Nítrico/metabolismo , Oócitos/efeitos dos fármacos , Oócitos/crescimento & desenvolvimento , 3',5'-AMP Cíclico Fosfodiesterases/antagonistas & inibidores , Animais , Bovinos , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Dipiridamol/metabolismo , Feminino , Fertilização in vitro , Técnicas de Maturação in Vitro de Oócitos/métodos , Masculino , Meiose/efeitos dos fármacos , Doadores de Óxido Nítrico/farmacologia , Oócitos/metabolismo , Inibidores de Fosfodiesterase/farmacologia , Quinolonas/farmacologia , S-Nitroso-N-Acetilpenicilamina/farmacologia , Citrato de Sildenafila/farmacologiaRESUMO
Nitric oxide (NO) is a chemical messenger involved in the control of oocyte maturation. It stimulates guanylate cyclase to produce cyclic guanosine monophosphate (cGMP), which in turn activates cGMP-dependent protein kinase (PKG) and some phosphodiesterases that may interfere with cAMP levels, a nucleotide also involved in meiosis resumption. The aim of this study was to determine the role played by NO on the cGMP/cAMP pathway during meiosis resumption in bovine oocytes. The effects of increasing NO generated by S-nitroso-N-acetylpenicillamine (SNAP; 10(-7)-10(-3) mol/L) and of other drugs that may affect the NO/cGMP pathway (proptoporfirin IX and 8-Br-cGMP) on meiosis resumption were investigated in bovine cumulus-oocyte complexes (COCs) matured for 9 hours in a semidefined medium (TCM199 + 3 mg/mL BSA). The COCs matured with 10(-7) mol/L SNAP associated or not with 100 µmol/L oxadiazole-one quinoxaline, a guanylate cyclase inhibitor, also had their cGMP and cAMP levels measured during the first hours of maturation (1, 3, and 6 hours). Quantitative polymerase chain reaction was performed by real-time polymerase chain reaction to determine the effects of NO on expression of genes encoding for enzymes of the NO/guanylate cyclase/cGMP and cAMP pathways during the first 9 hours of oocyte maturation. Increasing NO levels using 10(-7) mol/L SNAP resulted in lower rate of germinal vesicle breakdown (36% germinal vesicle breakdown; P < 0.05) at 9 hours IVM, whereas control group and the treatments with 10(-9) and 10(-8) mol/L SNAP showed about 70% germinal vesicle breakdown (P > 0.05). A temporary increase in cGMP levels was also observed with the same treatment (4.51 pmol/COC) at 1 hour IVM, which was superior to the control group (2.97 pmol/COC; P < 0.05) and was reversed by inhibiting guanylate cyclase activity with 100 µmol/L oxadiazole-one quinoxaline. Neither cAMP levels nor gene expression were affected by NO. These results suggest that NO acts via guanylate cyclase/cGMP and that even a temporary increase in cGMP levels leads to a delay in meiosis resumption, even when cAMP levels have declined. Nitric oxide does not act on oocyte maturation by affecting cAMP levels or the expression of genes related to the NO/guanylate cyclase/cGMP and cAMP pathways. Also, to our knowledge this is the first report to detect PKG1, PKG2, phosphodiesterase-5A, ADCY3, ADCY6, and ADCY9 transcripts in bovine oocytes.
Assuntos
Bovinos/fisiologia , Guanilato Ciclase/fisiologia , Meiose/fisiologia , Óxido Nítrico/fisiologia , Oócitos/fisiologia , Transdução de Sinais/fisiologia , Animais , AMP Cíclico/genética , AMP Cíclico/fisiologia , GMP Cíclico/genética , GMP Cíclico/fisiologia , Feminino , Oxidiazóis/farmacologia , Proteínas Quinases/genética , Proteínas Quinases/fisiologia , RNA/química , RNA/genética , Distribuição Aleatória , Reação em Cadeia da Polimerase em Tempo Real/veterinária , S-Nitroso-N-Acetilpenicilamina/farmacologia , Transdução de Sinais/efeitos dos fármacosRESUMO
Nitric oxide (NO) plays an important role as a leishmanicidal agent in murine macrophages. NO resistant Escherichia coli and Mycobacterium tuberculosis have been associated with poor outcomes of their resulting diseases. NO resistant Leishmania braziliensis has also been identified and exacerbates the clinical course of human leishmaniasis. We report, for the first time, natural resistance of Leishmania chagasi promastigotes to NO. These parasites were isolated from humans and dogs with visceral leishmaniasis. We also demonstrate that this resistance profile was associated with a greater survival capacity and a greater parasite burden in murine macrophages, independent of activation and after activation by IFN-γ and LPS.
Assuntos
Leishmania/efeitos dos fármacos , Leishmaniose Visceral/parasitologia , Óxido Nítrico/farmacologia , Animais , Brasil , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cães , Resistência a Medicamentos , Humanos , Concentração Inibidora 50 , Leishmaniose Visceral/tratamento farmacológico , Leishmaniose Visceral/veterinária , Estágios do Ciclo de Vida , Macrófagos/parasitologia , Camundongos , Doadores de Óxido Nítrico/farmacologia , Carga Parasitária , S-Nitroso-N-Acetilpenicilamina/farmacologiaRESUMO
NO (nitric oxide) donating drugs have been investigated for their important role in the sensitization of neoplastic cells to chemotherapy drugs. The goal of this work was to investigate the involvement of NO in the resistance of K562 cells to DNR (daunorubicin). Only simultaneous addition of DNR and SNAP (S-nitroso-N-acetyl-dl-penicillamine) caused significant cell death by apoptosis. Combination of the compounds decreased Bcl-2 and survivin, and increased Bax and active-caspase 3 expression. Fluorescence microscope and cytometric analysis showed that DNR and SNAP together caused DNR intracellular accumulation in K562 cells. RT-PCR (reverse transcription-PCR) analysis showed that DNR and SNAP, alone or in association, produced significant decreases in lrp expression. abcc1 gene expression was unaffected by the presence of SNAP, but when treated with DNR there was a small reduction that was intensified by DNR and SNAP in combination. The transport mechanism involved in the resistance to DNR in K562 cells involves ABCC1 and LRP (lung resistance protein) resistance proteins. DNR and SNAP inhibition of the expression of these proteins occurs by distinct mechanisms, and this disrupts the K562 resistance to DNR.
Assuntos
Antibióticos Antineoplásicos/metabolismo , Daunorrubicina/metabolismo , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico/farmacologia , S-Nitroso-N-Acetilpenicilamina/farmacologia , Antibióticos Antineoplásicos/farmacologia , Proteínas Reguladoras de Apoptose/metabolismo , Caspase 3/metabolismo , Linhagem Celular Tumoral , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Daunorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Humanos , Proteínas Inibidoras de Apoptose/metabolismo , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Survivina , Proteína X Associada a bcl-2/metabolismoRESUMO
Stem cell therapy has been considered a promise for damaged myocardial tissue. We have previously shown that S-nitroso-N-acetyl-D,L-penicillamine (SNAP) increases the expression of several muscular markers and VEGF in mesenchymal stem cells, indicating that transplantation of SNAP-treated cells could provide better functional outcomes. Here, we transplanted SNAP-treated adipose tissue-derived stem cells (ADSCs) in rat infarcted myocardium. After 30days, we observed a significant improvement of the ejection fraction in rats that received SNAP-treated ADSCs, compared with those that received untreated cells (p=0.008). Immunohistochemical reactions showed an increased expression of troponin T-C and von Willebrand factor, and organized vascular units in the infarcted area of tissue transplanted with treated ADSCs. SNAP exposure induced intracellular S-nitrosation, a decreased GSH/GSSG ratio, but did not increase cGMP levels. Collectively, these results indicate that SNAP alters the redox environment of ADSCs, possibly associated with a pre-differentiation state, which may improve cardiac function after transplantation.
Assuntos
Tecido Adiposo/citologia , Coração/fisiopatologia , Infarto do Miocárdio/terapia , Neovascularização Fisiológica/efeitos dos fármacos , S-Nitroso-N-Acetilpenicilamina/farmacologia , Transplante de Células-Tronco , Células-Tronco/citologia , Animais , Glutationa/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Coração/efeitos dos fármacos , Testes de Função Cardíaca/efeitos dos fármacos , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Masculino , Infarto do Miocárdio/fisiopatologia , Nitrosação/efeitos dos fármacos , Ratos , Ratos Wistar , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Volume Sistólico/efeitos dos fármacos , Troponina/metabolismo , Fator de von Willebrand/metabolismoRESUMO
Mitochondria generated nitric oxide (NO) regulates several cell functions including energy metabolism, cell cycling, and cell death. Here we report that the NO synthase inhibitors (L-NAME, L-NNA and L-NMMA) administered either in vitro or in vivo induce Ca2+-dependent mitochondrial permeability transition (MPT) in rat liver mitochondria via a mechanism independent on changes in the energy state of the organelle. MPT was determined by the occurrence of cyclosporin A sensitive mitochondrial membrane potential disruption followed by mitochondrial swelling and Ca2+ release. In in vitro experiments, the effect of NOS inhibitors was dose-dependent (1 to 50 microM). In addition to cyclosporin A, L-NAME-induced MPT was sensitive to Mg2+ plus ATP, EGTA, and to a lower degree, to catalase and dithiothreitol. In contrast to L-NAME, its isomer D-NAME did not induce MPT. L-NAME-induced MPT was associated with a significant decrease in both the rate of NO generation and the content of mitochondrial S-nitrosothiol. Acute and chronic in vivo treatment with L-NAME also promoted MPT and decreased the content of mitochondrial S-nitrosothiol. SNAP (a NO donor) prevented L-NAME mediated MPT and reversed the decrease in the rate of NO generation and in the content of S-nitrosothiol. We propose that S-nitrosylation of critical membrane protein thiols by NO protects against MPT.
Assuntos
Mitocôndrias Hepáticas/metabolismo , Óxido Nítrico/metabolismo , S-Nitrosotióis/metabolismo , Animais , Inibidores Enzimáticos/farmacologia , Feminino , Técnicas In Vitro , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias Hepáticas/efeitos dos fármacos , Proteínas de Transporte da Membrana Mitocondrial/efeitos dos fármacos , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Poro de Transição de Permeabilidade Mitocondrial , Dilatação Mitocondrial/efeitos dos fármacos , NG-Nitroarginina Metil Éster/farmacologia , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Ratos , Ratos Wistar , S-Nitroso-N-Acetilpenicilamina/farmacologiaRESUMO
The role of NO in regulating the focal adhesion proteins, Src, FAK, p130 Cas, and PTP-alpha, was investigated. Fibroblasts expressing PTP-alpha (PTP-alpha(WT) cells), fibroblasts "knockout" for PTP-alpha (PTP-alpha(-/-) cells), and "rescued" "knockout" fibroblasts (PTP-alpha A5/3 cells) were stimulated with either S-nitroso-N-acetylpenicillamine (SNAP) or fetal bovine serum (FBS). FBS increased inducible NO synthase in both cell lines. Activation of Src mediated either by SNAP or by FBS occurred independent of dephosphorylation of Tyr527 in PTP-alpha(-/-) cells. Both stimuli promoted dephosphorylation of Tyr527 and activation of Src kinase in PTP-alpha(WT) cells. NO-mediated activation of Src kinase affected the activities of FAK and p130Cas and was dependent on the expression of PTP-alpha. Analogous to tyrosine phosphorylation, SNAP and FBS stimulated differential generation of NO and S-nitrosylation of Src kinase in both cell lines. Incubation with SNAP resulted in higher levels of NO and S-nitrosylation of immunoprecipitated Src in PTP-alpha(-/-) cells (oxidizing redox environment) as compared with the levels of NO and S-nitrosylated Src in PTP-alpha(WT) cells (reducing redox environment). SNAP differentially stimulated cell proliferation of both cell lines is dependent on the intracellular redox environment, Src activity, and PTP-alpha expression. This dependence also is observed with FBS-stimulated cell migration.
Assuntos
Proteína Substrato Associada a Crk/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Óxido Nítrico/metabolismo , Proteínas Tirosina Fosfatases Classe 4 Semelhantes a Receptores/metabolismo , Quinases da Família src/metabolismo , Animais , Bovinos , Movimento Celular , Proliferação de Células , Células Cultivadas , Proteína Substrato Associada a Crk/genética , Fibroblastos/citologia , Fibroblastos/fisiologia , Proteína-Tirosina Quinases de Adesão Focal/genética , Camundongos , Camundongos Knockout , Óxido Nítrico/genética , Doadores de Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Oxirredução , Proteínas Tirosina Fosfatases Classe 4 Semelhantes a Receptores/genética , S-Nitroso-N-Acetilpenicilamina/metabolismo , Quinases da Família src/genéticaRESUMO
In contrast to other metal-dithiocarbamate [DEDTC] complexes, the copper-DEDTC complex is highly cytotoxic, inducing oxidative stress, preferentially in tumor cells. Because nitric oxide (NO) forms adducts with Cu[DEDTC](2), we investigated whether NO donors like S-nitroso-N-acetyl penicillamine (SNAP) or sodium nitroprusside (SNP), and nitrite, a NO decomposition product, modulate Cu[DEDTC](2) cytotoxicity against human tumor cells. We show that apoptosis-associated PARP cleavage and inducible nitric oxide synthase (iNOS) down-regulation induced by nanomolar Cu[DEDTC](2), are counteracted by 50 muM SNAP, SNP, or CoCl(2), an inducer of hypoxia and NO signaling. Nitrite was stochiometrically effective in antagonizing Cu[DEDTC](2) cytotoxicity and inducing shifts in the absorption spectrum of the binary complex in the 280 and 450 nm regions. Subtoxic concentrations of Cu[DEDTC](2) became lethal when tumor cells were pretreated with c-PTIO, a membrane-impermeable scavenger for extracellular NO. Our results suggest that: (a) reactive oxygen species induced by Cu[DEDTC](2) are scavenged by nitrite released from NO, (b) the extent of lethality of Cu[DEDTC](2) is dependent on the reciprocal formation of an inactive ternary Cu[DEDTC](2)NO copper-nitrosyl complex.
Assuntos
Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico Sintase Tipo II/biossíntese , Nitritos/metabolismo , Compostos Organometálicos/metabolismo , Compostos Organometálicos/farmacologia , Morte Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cobalto/farmacologia , Óxidos N-Cíclicos/farmacologia , Regulação para Baixo , Sequestradores de Radicais Livres/farmacologia , Humanos , Imidazóis/farmacologia , Melanoma , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Óxido Nítrico/biossíntese , Nitroprussiato/farmacologia , Compostos Nitrosos/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo , S-Nitroso-N-Acetilpenicilamina/farmacologia , Nitrito de Sódio/farmacologiaRESUMO
Antidepressant-like activity of folic acid in forced swimming test and in the tail suspension test was demonstrated previously by our group. In this study we investigated the involvement of N-methyl-d-aspartate (NMDA) receptors and l-arginine-nitric oxide (NO)-cyclic guanosine monophosphate pathway in its antidepressant-like effect in the forced swimming test in mice. The antidepressant-like effect of folic acid (10 nmol/site, i.c.v.) was prevented by the pretreatment of mice with NMDA (0.1 pmol/site, i.c.v.), l-arginine (750 mg/kg, i.p., substrate for nitric oxide synthase), S-nitroso-N-acetyl-penicillamine (SNAP, 25 microg/site, i.c.v, a NO donor) or sildenafil (5 mg/kg, i.p., phosphodiesterase 5 inhibitor). The administration of 7-nitroindazole (25 and 50 mg/kg, i.p., a specific neuronal nitric oxide synthase (nNOS) inhibitor) or methylene blue (20 mg/kg, i.p., direct inhibitor of both nitric oxide synthase and soluble guanylate cyclase) in combination with a sub-effective dose of folic acid (1 nmol/site, i.c.v.) reduced the immobility time in the FST as compared with either drug alone. Together the results suggest that the antidepressant-like effect of folic acid in the forced swimming test is dependent on an inhibition of either NMDA receptors or NO and cGMP synthesis.