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OBJECTIVE: To evaluate the role of induced nitric oxide synthase (iNOS) in nociception/orofacial discomfort in rats submitted to tooth whitening with hydrogen peroxide (H2O2). DESIGN: Wistar rats were divided into three groups (n = 24/group): a sham group not submitted to whitening treatment, a saline group submitted to whitening treatment, and a test group submitted to whitening treatment and blockade of iNOS with aminoguanidine 50 mg/kg/day. After 24 and 48 h, and 7 days, the animals were euthanized to collect trigeminal ganglia and maxillae to histomorphometric analysis (size of neuronal bodies and percentage of pulp area filled by vessels) and behavior/nociception (Grimace scales, scratching and biting counting, weight loss and nociception assay). ANOVA-1- or - 2-way tests were used (p < 0.05, GraphPadPrism 5.0). RESULTS: The aminoguanidine-treated group showed a reduction in nociceptive threshold in the masseteric region (p < 0.001), Grimace scale scores (p < 0.001), number of scratching (p = 0.011) and body mass loss (p = 0.007). After 24 and 48 h of tooth bleaching, the saline group showed a significant increase in the mean area of the blood vessels (p = 0.020) and iNOS immunostaining in odontoblasts (p = 0.002) and non-odontoblasts cells (p = 0.025). Aminoguanidine reversed both increases. Tooth bleaching reduced the mean area of neuronal bodies, and aminoguanidine significantly reversed it (p = 0.019), but an increase in GFAP immunostaining in neuronal bodies did not reduce after seven-days or after aminoguanidine treatment (p = 0.003). CONCLUSION: iNOS blockage by aminoguanidine plays an important role in nociception and orofacial discomfort by control of inflammation in dental pulp after tooth bleaching with hydrogen peroxide (H2O2) 35%.
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Guanidinas , Clareadores Dentários , Clareamento Dental , Ratos , Animais , Peróxido de Hidrogênio/farmacologia , Nociceptividade , Óxido Nítrico , Ratos Wistar , Óxido Nítrico SintaseRESUMO
BACKGROUND: Leishmaniasis is a worldwide health problem, highly endemic in developing countries. Among the four main clinical forms of the disease, visceral leishmaniasis is the most severe, fatal in 95% of cases. The undesired side-effects from first-line chemotherapy and the reported drug resistance search for effective drugs that can replace or supplement those currently used in an urgent need. Aminoguanidine hydrazones (AGH's) have been explored for exhibiting a diverse spectrum of biological activities, in particular the antileishmanial activity of MGBG. The bioisosteres thiosemicarbazones (TSC's) offer a similar biological activity diversity, including antiprotozoal effects against Leishmania species and Trypanosoma cruzi. OBJECTIVES: Considering the impact of leishmaniasis worldwide, this work aimed to design, synthesize, and perform a screening upon L. chagasi amastigotes and for the cytotoxicity of the small "inhouse" library of both AGH and TSC derivatives and their structurally-related compounds. METHODS: A set of AGH's (3-7), TSC's (9, 10), and semicarbazones (11) were initially synthesized. Subsequently, different semi-constrained analogs were designed and also prepared, including thiazolidines (12), dihydrothiazines (13), imidazolines (15), pyrimidines (16, 18) azines (19, 20), and benzotriazepinones (23-25). All intermediates and target compounds were obtained with satisfactory yields and exhibited spectral data consistent with their structures. All final compounds were evaluated against L. chagasi amastigotes and J774.A1 cell line. Molecular docking was performed towards trypanothione reductase using GOLD® software. RESULTS: The AGH's 3i, 4a, and 5d, and the TSC's 9i, 9k, and 9o were selected as valuable hits. These compounds presented antileishmanial activity compared with pentamidine, showing IC50 values ranged from 0.6 to 7.27 µM, maximal effects up to 55.3%, and satisfactory SI values (ranged from 11 to 87). On the other hand, most of the resulting semi-constrained analogs were found cytotoxic or presented reduced antileishmanial activity. In general, TSC class is more promising than its isosteric AGH analogs, and the beneficial aromatic substituent effects are not similar in both series. In silico studies have suggested that these hits are capable of inhibiting the trypanothione reductase from the amastigote forms. CONCLUSION: The promising antileishmanial activity of three AGH's and three TSC's was characterized. These compounds presented antileishmanial activity compared with PTD, showing IC50 values ranged from 0.6 to 7.27 µM, and satisfactory SI values. Further pharmacological assays involving other Leishmania strains are in progress, which will help choose the best hits for in vivo experiments.
Assuntos
Leishmania infantum , Tiossemicarbazonas , Guanidinas , Hidrazonas/farmacologia , Simulação de Acoplamento Molecular , Relação Estrutura-Atividade , Tiossemicarbazonas/farmacologiaRESUMO
Oxidative stress produces macromolecules dysfunction and cellular damage. Renal ischemia-reperfusion injury (IRI) induces oxidative stress, inflammation, epithelium and endothelium damage, and cessation of renal function. The IRI is an inevitable process during kidney transplantation. Preliminary studies suggest that aminoguanidine (AG) is an antioxidant compound. In this study, we investigated the antioxidant effects of AG (50 mg/kg, intraperitoneal) and its association with molecular pathways activated by IRI (30 min/48 h) in the kidney. The antioxidant effect of AG was studied measuring GSSH/GSSG ratio, GST activity, lipoperoxidation, iNOS, and Hsp27 levels. In addition, we examined the effect of AG on elements associated with cell survival, inflammation, endothelium, and mesenchymal transition during IRI. AG prevented lipid peroxidation, increased GSH levels, and recovered the GST activity impaired by IRI. AG was associated with inhibition of iNOS, Hsp27, endothelial activation (VE-cadherin, PECAM), mesenchymal markers (vimentin, fascin, and HSP47), and inflammation (IL-1ß, IL-6, Foxp3, and IL-10) upregulation. In addition, AG reduced kidney injury (NGAL, clusterin, Arg-2, and TFG-ß1) and improved kidney function (glomerular filtration rate) during IRI. In conclusion, we found new evidence of the antioxidant properties of AG as a renoprotective compound during IRI. Therefore, AG is a promising compound to treat the deleterious effect of renal IRI.
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We sought to investigate whether RVLM iNOS activity and oxidative profile may participate in the reduction of sympathetic responsiveness in swimming trained normotensive rats. Sedentary (S) and swimming trained (T) Wistar male rats chronically instrumented with an arterial catheter and guide cannula into the RVLM were submitted to continuous pressure and heart rate (HR) recordings and determination of autonomic control (power spectral analysis) before and after unilateral RVLM iNOS inhibition (aminoguanidine, 250 pmol/100 nL). Other S and T rats received local l-glutamate microinjection (5 nmol/100 nL). In separate S and T groups not submitted to brainstem cannulation, fresh bilateral RVLM punchs were collected for iNOS gene expression (qPCR); reduced glutathione and lipid peroxidation quantification (spectrophotometry); iron-reducing antioxidant (FRAP) and 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) radical cation (ABTSË+) scavenger assays. iNOS gene expression was confirmed in fixed RVLM slices (immunofluorescence). T rats exhibited resting bradycardia, lower sympathovagal balance, reduced RVLM iNOS gene/protein expression and higher antioxidant capacity. Decreased iNOS expression was positively correlated with reduced HR. Pressor and tachycardic response to l-Glutamate were smaller in T rats. Aminoguanidine microinjection reduced sympathetic activity in S rats but did not change it in T rats expressing reduced RVLM iNOS content. Our data indicate that iNOS, expressed in the RVLM of normotensive male rats, has tonic effects on sympathetic activity and that swimming training is an efficient tool to reduce iNOS expression and augment the antioxidant defense, thus reducing glutamatergic responsiveness and sympathetic drive to cardiovascular effectors.
Assuntos
Sistema Nervoso Autônomo/metabolismo , Pressão Sanguínea/fisiologia , Frequência Cardíaca/fisiologia , Bulbo/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Estresse Oxidativo/fisiologia , Condicionamento Físico Animal/fisiologia , Animais , Sistema Nervoso Autônomo/efeitos dos fármacos , Pressão Sanguínea/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Guanidinas/farmacologia , Frequência Cardíaca/efeitos dos fármacos , Peroxidação de Lipídeos/fisiologia , Masculino , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Estresse Oxidativo/efeitos dos fármacos , Ratos , NataçãoRESUMO
Objectives: The study aimed to determine the effect of coffee intake on AGEs formation and platelet aggregation in diabetic Wistar rats. Methods: Coffee powder samples were used to prepare a 10% beverage. Diabetes mellitus was induced in the animals by administering 2% alloxan. All animal experiments were approved by the ethics committee for animal experiments under N°. 420/2012 and 536/2013. Diabetic and non-diabetic rats were divided into 6 groups treated and untreated with coffee (7.2 mL/Kg body weight) and aminoguanidine (AGE inhibiting agent) (100 mg/Kg body weight) for 50 days. After 50 days, the animals were fasted for 12 h and anesthetized (40 mg/Kg sodium pentobarbital) intraperitoneally. Blood samples were collected from the abdominal artery puncture. Hematological parameters (red cells, hemoglobin, hematocrit and leukocyte) and glycemic and HbA1c levels were measured. AGEs quantification (spectrofluorometric method) and the platelet aggregation test (aggregation of cuvettes in a four-channel platelet aggregometer) were also conducted. The rats' renal function was evaluated by measuring serum urea and creatinine. Results: Data showed that coffee intake had no effect on the hematological parameters. Fasting glucose and HbA1c dosage were significantly higher in diabetic animals compared to non-diabetic animals (confirmed the effectiveness of inducing and maintaining diabetic status). Results showed that coffee reduced AGE formation and platelet aggregation in our animal model, not altering the animals' renal function. Conclusions: These results suggest beneficial effects on vasculopathy, a common complication in diabetic patients.
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One of the promising fields for improving the effectiveness of antimicrobial agents is their combination with efflux pump inhibitors (EPIs), which besides expanding the use of existing antibiotics. The goal of this research was to evaluate a series of aminoguanidine hydrazones (AGH's, 1-19) as antibacterial agents and NorA efflux pump inhibitors in Staphylococcus aureus strain SA-1199B. Molecular modeling and docking studies were also performed in order to explain at the molecular level the interactions of the compounds with the generated NorA efflux pump model. The MICs of the antibiotic and ethidium bromide were determined by microdilution assay in absence or presence of a subinhibitory concentration of aminoguanidine hydrazones and macrophages viability was determined through MTT assay. Bioinformatic software Swiss-Model and AutoDock 4.2 were used to perform modeling and docking studies, respectively. As results, all AGH's were able to potentiate the action for the antibiotic norfloxacin, causing MIC's reduction of 16-fold and 32-fold to ethidium bromide. In the cell viability test, the concentration of 10 µg/mL showed better results than 90% and the concentration of 1000 µg/mL showed the lowest viability, reaching a maximum of 50% for the analyzed aminoguanidine hydrazones. Molecular docking studies showed that both norfloxacin and derivative 13 were recognized by the same binding site of NorA pump, suggesting a competitive mechanism. The present work demonstrated for the first time that AGH derivatives have potential to be putative inhibitors of NorA efflux pump, showing a promising activity as an antibacterial drug development.
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Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Farmacorresistência Bacteriana/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Hidrazonas/química , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Staphylococcus aureus/metabolismo , Animais , Proteínas de Bactérias/antagonistas & inibidores , Sítios de Ligação , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/toxicidade , Guanidinas/química , Hidrazonas/síntese química , Hidrazonas/farmacologia , Camundongos , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Proteínas Associadas à Resistência a Múltiplos Medicamentos/antagonistas & inibidores , Norfloxacino/farmacologia , Estrutura Terciária de Proteína , Staphylococcus aureus/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
In this paper we present the convenient syntheses of six new guanylhydrazone and aminoguanidine tetrahydropyran derivatives 2-7. The guanylhydrazone 2, 3 and 4 were prepared in 100% yield, starting from corresponding aromatic ketones 8a-c and aminoguanidine hydrochloride accessed by microwave irradiation. The aminoguanidine 5, 6 and 7 were prepared by reduction of guanylhydrazone 2-4 with sodium cyanoborohydride (94% yield of 5, and 100% yield of 6 and 7). The aromatic ketones 8a-c were prepared from the Barbier reaction followed by the Prins cyclization reaction (two steps, 63%-65% and 95%-98%). Cytotoxicity studies have demonstrated the effects of compounds 2-7 in various cancer and normal cell lines. That way, we showed that these compounds decreased cell viabilities in a micromolar range, and from all the compounds tested we can state that, at least, compound 3 can be considered a promising molecule for target-directed drug design.
Assuntos
Guanidinas/síntese química , Hidrazonas/síntese química , Neoplasias/tratamento farmacológico , Piranos/síntese química , Boroidretos/síntese química , Boroidretos/química , Linhagem Celular Tumoral , Ciclização , Guanidinas/administração & dosagem , Guanidinas/química , Humanos , Hidrazonas/administração & dosagem , Hidrazonas/química , Cetonas/síntese química , Cetonas/química , Estrutura Molecular , Piranos/administração & dosagem , Piranos/químicaRESUMO
In this study, we show that aminoguanidine (AMG), an inhibitor of protein glycation, increases the NOX2 (phagocyte NADPH oxidase) response and microbicidal activity by neutrophils, regardless of diabetic status. The non-enzymatic glycation of proteins, yielding irreversible advanced glycation end products (AGEs), is involved in the development of diabetes complications, including alterations of signaling pathways and the generation of reactive oxygen species by phagocytes. The phagocytes produce ROS (reactive oxygen species) through activation of the NOX2 complex, which generates superoxide. The purpose of this study was to evaluate the effect of hyperglycemia and the glycation of proteins on the NOX2 activity of neutrophils and its implications for cellular physiology, with a focus on the microbicidal activity of these cells. We treated diabetic rats with AMG and evaluated neutrophil ROS generation and Candida albicans killing ability. We observed a large increase in the microbicidal activity of peritoneal neutrophils from AMG-treated rats. The increase was independent of diabetic status and myeloperoxidase activity. Collectively, our results suggest that AMG has an immunomodulator role that triggers an increase in the microbicidal response of neutrophils mainly related to reactive oxygen species production by NOX2.
Assuntos
Candida albicans , Diabetes Mellitus Experimental/enzimologia , Guanidinas/farmacologia , Glicoproteínas de Membrana/metabolismo , NADPH Oxidases/metabolismo , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Fagocitose/efeitos dos fármacos , Animais , Diabetes Mellitus Experimental/imunologia , Diabetes Mellitus Experimental/microbiologia , Hemoglobinas Glicadas/metabolismo , Produtos Finais de Glicação Avançada/metabolismo , Fatores Imunológicos/farmacologia , Rim/efeitos dos fármacos , Rim/patologia , Masculino , NADPH Oxidase 2 , Neutrófilos/metabolismo , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismoRESUMO
BACKGROUND: This study evaluated the effect of aminoguanidine on liver of diabetic rats subject to physical exercises using histological and histochemical techniques. METHODS: THE RATS USED IN THIS STUDY WERE DIVIDED INTO FIVE GROUPS: sedentary control, sedentary diabetic, trained diabetic, sedentary diabetic and treated with aminoguanidine, trained diabetic and treated with aminoguanidine. RESULTS: The results showed no effect of aminoguanidine on the liver tissue, although there was improvement with exercise training showing cytological, morpho-histological and histochemical alterations in liver cells of animals from groups trained diabetic and/or treated diabetic compared to those individuals in the sedentary control and sedentary diabetic. These changes included: hepatocytes hypertrophy, presence and distribution of polysaccharides in the hepatocytes cytoplasm and, especially, congestion of the liver blood vessels. CONCLUSION: Our results suggest that aminoguanidine is not hepatotoxic, when used at dosage of 1 g/L for the treatment of diabetes complications, and confirmed that the practice of moderate physical exercise assuaged the damage caused by diabetes without the use of insulin.
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OBJECTIVE: The objective of this study was to evaluate the involvement of peripheral nitric oxide (NO) in vagotomy-induced pulmonary edema by verifying whether the nitric oxide synthases (NOS), constitutive (cNOS) and inducible (iNOS), participate in this mechanism. INTRODUCTION: It has been proposed that vagotomy induces neurogenic pulmonary edema or intensifies the edema of other etiologies. METHODS: Control and vagotomized rats were pretreated with 0.3 mg/kg, 3.0 mg/kg or 39.0 mg/kg of L-NAME, or with 5.0 mg/kg, 10.0 mg/kg or 20.0 mg/kg of aminoguanidine. All animals were observed for 120 minutes. After the animals' death, the trachea was catheterized in order to observe tracheal fluid and to classify the severity of pulmonary edema. The lungs were removed and weighed to evaluate pulmonary weight gain and edema index. RESULTS: Vagotomy promoted pulmonary edema as edema was significantly higher than in the control. This effect was modified by treatment with L-NAME. The highest dose, 39.0 mg/kg, reduced the edema and prolonged the survival of the animals, while at the lowest dose, 0.3 mg/kg, the edema and reduced survival rates were maintained. Aminoguanidine, regardless of the dose inhibited the development of the edema. Its effect was similar to that observed when the highest dose of L-NAME was administered. It may be that the non-selective blockade of cNOS by the highest dose of L-NAME also inhibited the iNOS pathway. CONCLUSION: Our data suggest that iNOS could be directly involved in pulmonary edema induced by vagotomy and cNOS appears to participate as a protector mechanism.
Assuntos
Animais , Masculino , Ratos , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Óxido Nítrico/metabolismo , Edema Pulmonar/metabolismo , Vagotomia/efeitos adversos , Inibidores Enzimáticos/uso terapêutico , Guanidinas/uso terapêutico , NG-Nitroarginina Metil Éster/uso terapêutico , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , Edema Pulmonar/tratamento farmacológico , Edema Pulmonar/etiologia , Edema Pulmonar/prevenção & controle , Ratos Wistar , Índice de Gravidade de Doença , Fatores de TempoRESUMO
Avaliaram-se o papel do óxido nítrico (NO) por meio da inibição da enzima óxido nítrico sintase induzível (iNOS), após a adição da aminoguanidina (AG), na motilidade, no vigor e na integridade da membrana plasmática nos tempos de 15, 60, 120, 180, 240 e 300min e a atividade mitocondrial e a capacitação de espermatozoides bovinos após 300min de cultivo. Adicionaram-se diferentes concentrações (0,001, 0,01 e 0,1M) de AG durante a capacitação induzida pela heparina e 500μM de nitroprussiato de sódio (SNP, doador de NO) à concentração deletéria. A adição de 0,1M de AG diminuiu a motilidade e o vigor espermático e a integridade da membrana (P<0,05). A adição de SNP ao meio de cultivo com 0,1M de AG somente reverteu a integridade da membrana após 300min. A inibição da síntese de NO pela adição de AG não alterou a atividade mitocondrial. A percentagem de oócitos penetrados com espermatozoides tratados com 0,01 e 0,1M de AG diminuiu 20,3 e 100 por cento, respectivamente, em relação aos não tratados (controle) (P<0,05), contudo houve aumento de 15 por cento na percentagem de oócitos desnudados penetrados com espermatozoides capacitados em presença de 0,1M de AG. Conclui-se que a inibição da síntese de NO pela AG diminuiu a qualidade espermática durante a capacitação de espermatozoides bovinos in vitro, exceto a atividade mitocondrial. Somente a integridade da membrana foi revertida após adição de NO, sugerindo diferentes vias de ação do NO na qualidade espermática ao longo da capacitação in vitro de espermatozoides bovinos.
The role of nitric oxide (NO) was evaluated by inhibition of inducible nitric oxide synthase (iNOS), with aminoguanidine (AG) on motility, vigor, and plasmatic membrane integrity of bovine spermatozoa culture after 15, 60, 120, 180, 240, and 300min and on mitochondrial activity and capacitation after 300min, respectively. Different concentrations, 0.001, 0.01, and 0.1M of AG were added during the heparin induced capacitation and sodium nitroprusside (SNP, NO donor-500μM) to the deleterious concentration. The addition of 0.1M of AG diminished progressive motility, spermatic vigor, and membrane integrity (P<0.05). SNP addition to the 0.1M of AG did revert only plasmatic membrane integrity after 300min. Mitochondrial activity was not influenced by addition of AG. Percentage of penetrated oocytes after addition of 0.01 and 0.1M of AG diminished, 20.3 and 100 percent, respectively, in relation to the control oocytes (P<0.05). However, an increase of 15 percent was observed when denuded oocytes were used with 0.1M AG treated sperm (P<0.05). It was concluded that the inhibition of NO synthesis with aminoguanidine diminished sperm quality during in vitro capacitation of bovine spermatozoa, except the mitochondrial activity. Only membrane integrity was reverted with the addition of NO to culture medium, suggesting different pathways of NO action on bovine sperm quality during in vitro capacitation.
Assuntos
Animais , Masculino , Bovinos , Capacitação Espermática , Óxido Nítrico Sintase/antagonistas & inibidores , Bovinos , EspermatozoidesRESUMO
Avaliaram-se o papel do óxido nítrico (NO) por meio da inibição da enzima óxido nítrico sintase induzível (iNOS), após a adição da aminoguanidina (AG), na motilidade, no vigor e na integridade da membrana plasmática nos tempos de 15, 60, 120, 180, 240 e 300min e a atividade mitocondrial e a capacitação de espermatozoides bovinos após 300min de cultivo. Adicionaram-se diferentes concentrações (0,001, 0,01 e 0,1M) de AG durante a capacitação induzida pela heparina e 500μM de nitroprussiato de sódio (SNP, doador de NO) à concentração deletéria. A adição de 0,1M de AG diminuiu a motilidade e o vigor espermático e a integridade da membrana (P<0,05). A adição de SNP ao meio de cultivo com 0,1M de AG somente reverteu a integridade da membrana após 300min. A inibição da síntese de NO pela adição de AG não alterou a atividade mitocondrial. A percentagem de oócitos penetrados com espermatozoides tratados com 0,01 e 0,1M de AG diminuiu 20,3 e 100 por cento, respectivamente, em relação aos não tratados (controle) (P<0,05), contudo houve aumento de 15 por cento na percentagem de oócitos desnudados penetrados com espermatozoides capacitados em presença de 0,1M de AG. Conclui-se que a inibição da síntese de NO pela AG diminuiu a qualidade espermática durante a capacitação de espermatozoides bovinos in vitro, exceto a atividade mitocondrial. Somente a integridade da membrana foi revertida após adição de NO, sugerindo diferentes vias de ação do NO na qualidade espermática ao longo da capacitação in vitro de espermatozoides bovinos.(AU)
The role of nitric oxide (NO) was evaluated by inhibition of inducible nitric oxide synthase (iNOS), with aminoguanidine (AG) on motility, vigor, and plasmatic membrane integrity of bovine spermatozoa culture after 15, 60, 120, 180, 240, and 300min and on mitochondrial activity and capacitation after 300min, respectively. Different concentrations, 0.001, 0.01, and 0.1M of AG were added during the heparin induced capacitation and sodium nitroprusside (SNP, NO donor-500μM) to the deleterious concentration. The addition of 0.1M of AG diminished progressive motility, spermatic vigor, and membrane integrity (P<0.05). SNP addition to the 0.1M of AG did revert only plasmatic membrane integrity after 300min. Mitochondrial activity was not influenced by addition of AG. Percentage of penetrated oocytes after addition of 0.01 and 0.1M of AG diminished, 20.3 and 100 percent, respectively, in relation to the control oocytes (P<0.05). However, an increase of 15 percent was observed when denuded oocytes were used with 0.1M AG treated sperm (P<0.05). It was concluded that the inhibition of NO synthesis with aminoguanidine diminished sperm quality during in vitro capacitation of bovine spermatozoa, except the mitochondrial activity. Only membrane integrity was reverted with the addition of NO to culture medium, suggesting different pathways of NO action on bovine sperm quality during in vitro capacitation.(AU)