RESUMEN
BACKGROUND: Previous studies suggested that Cg1 area of the cingulate cortex of rats controls glutamate-mediated fear-induced defensive behaviour and antinociception organised at the posterior hypothalamus. In turn, microinjection of the nitric oxide donor SIN-1 into the anterior hypothalamus of mice produced defensive behaviours and fear-induced antinociception. However, it remains unknown whether Cg1 also modulates the latter mechanisms in mice. AIMS: The present study examined the influence of Cg1 on SIN1-evoked fear-induced defensive behaviour and antinociception organised at the anterior hypothalamus of mice. METHODS: The fear-like behavioural and antinociceptive responses to the microinjection of SIN-1 (300 nmol) into the anterior hypothalamus were evaluated after the microinjection of either N-methyl-D-aspartic acid receptor agonist (0.1, 1 and 10 nmol) or physiological saline into the cingulate cortex of C57BL/6 male mice. In addition, neurotracing and immunohistochemistry were used to characterise Cg1-anterior hypothalamus glutamatergic pathways. RESULTS: The data showed that activation of Cg1 N-methyl-D-aspartic acid receptors increased escape while reducing freezing and antinociceptive responses to SIN-1 microinjections into the anterior hypothalamus. Anterograde neural tract tracer co-localised with VGLUT2-labelled fibres suggests these responses are mediated by glutamatergic synapses at the anterior hypothalamus. CONCLUSIONS: In contrast with previous studies showing that Cg1 facilitates both escape and antinociception to chemical stimulation of the posterior hypothalamus in rats, the present data suggest that Cg1 facilitates escape while inhibiting defensive antinociception produced by the microinjection of SIN-1 in the anterior hypothalamus of mice. Accordingly, Cg1 may have opposite effects on antinociceptive responses organised in the anterior and posterior hypothalamus of mice and rats, respectively.
Asunto(s)
Miedo , Giro del Cíngulo , Hipotálamo Anterior , Donantes de Óxido Nítrico/metabolismo , Óxido Nítrico/metabolismo , Percepción del Dolor/fisiología , Receptores de N-Metil-D-Aspartato/agonistas , Analgesia/psicología , Animales , Conducta Animal/efectos de los fármacos , Miedo/efectos de los fármacos , Miedo/fisiología , Giro del Cíngulo/efectos de los fármacos , Giro del Cíngulo/metabolismo , Hipotálamo Anterior/efectos de los fármacos , Hipotálamo Anterior/fisiología , Ratones , Ratones Endogámicos C57BL , Microinyecciones/métodos , Molsidomina/análogos & derivados , Molsidomina/farmacología , Vías Nerviosas , Neurotransmisores/farmacologíaRESUMEN
Molsidomine is currently used as a vasodilator drug for the treatment of myocardial ischemic syndrome and congestive heart failure, although still presenting some mitochondrial-targeted side effects in many human cells. As a model of molsidomine mitotoxicity, the reaction of cytochrome c with phosphatidylserine (PS)- and cardiolipin (CL)-containing liposomes was investigated in oxidative/nitrosative conditions imposed by SIN-1 decomposition, which renders peroxynitrite (ONOO-) as a main reactive product. In these conditions, the production of thiobarbituric acid-reactive substance (TBARs) and LOOH was affected by the lipid composition and the oxidative/nitrative conditions used. The oxidative/nitrative conditions were the exposure of lipids to SIN-1 decomposition, native cytochrome c after previous exposure to SIN-1, concomitantly to SIN-1 and native cytochrome c, native cytochrome c, and cytochrome c modified by SIN-1 that presents a less-rhombic heme iron (L-R cytc). TBARs and LOOH production by lipids and cytochrome c exposed concomitantly to SIN-1 differed from that obtained using L-R cytc and featured similar effects of SIN-1 alone. This result suggests that lipids rather than cytochrome c are the main targets for oxidation and nitration during SIN-1 decomposition. PS- and CL-containing liposomes challenged by SIN-1 were analyzed by Fourier transform infrared spectroscopy that revealed oxidation, trans-isomerization, and nitration. These products are consistent with reaction routes involving lipids and NOx formed via peroxynitrite or direct reaction of NO⢠with molecular oxygen that attacks LOOH and leads to the formation of substances that are not reactive with thiobarbituric acid.
Asunto(s)
Citocromos c/antagonistas & inhibidores , Membranas Mitocondriales/efectos de los fármacos , Modelos Biológicos , Molsidomina/análogos & derivados , Molsidomina/farmacología , Citocromos c/metabolismo , Humanos , Estructura Molecular , Molsidomina/química , Molsidomina/metabolismo , Oxidación-ReducciónRESUMEN
BACKGROUND: An excitatory imbalance in the hypothalamus of rodents caused by local chemical stimulation elicits fear-related defensive reactions such as escape and freezing. In addition, these panic attack-like defensive reactions induced by hypothalamic neurons may cause antinociception. However, there is a shortage of studies showing the participation of the anterior hypothalamic nucleus in these adaptive defensive mechanisms. Nitric oxide (NO) donors have been shown to evoke fear-related defensive responses when microinjected into paralimbic and limbic structures, and this excitatory neuromodulation can recruit the glutamatergic system. AIMS: The aim of this work was to investigate the influence of the glutamatergic system in the nitrergic effects on fear-related defensive responses organised by anterior hypothalamic neurons. METHODS: The present study evaluates the effects of the molsidomine active metabolite SIN-1 NO donor administered into the anterior hypothalamus (AH) of mice at different concentrations (75, 150 and 300 nmol/0.1 µL). Then, we investigated the effects of pre-treatment of the AH with AP-7 (an N-methyl-d-aspartate (NMDA) receptor-selective antagonist; 0.02, 0.2 and 2 nmol/0.1 µL) on the behavioural and antinociceptive effects provoked by AH chemical stimulation with SIN-1 microinjections. RESULTS: The 300 nmol dose of SIN-1 was the most effective at causing panic-like defensive behaviours followed by a significant antinociceptive response. In addition, both of these effects were attenuated or inhibited by AH pre-treatment with AP-7. CONCLUSIONS: These findings suggest that the panicogenic and antinociceptive effects evoked by intra-AH microinjections of SIN-1 depend on NMDA receptor activation.
Asunto(s)
Miedo/efectos de los fármacos , Molsidomina/análogos & derivados , Donantes de Óxido Nítrico/administración & dosificación , Pánico/efectos de los fármacos , 2-Amino-5-fosfonovalerato/administración & dosificación , 2-Amino-5-fosfonovalerato/análogos & derivados , 2-Amino-5-fosfonovalerato/farmacología , Animales , Conducta Animal/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Hipotálamo Anterior/efectos de los fármacos , Hipotálamo Anterior/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Microinyecciones , Molsidomina/administración & dosificación , Molsidomina/farmacología , Donantes de Óxido Nítrico/farmacología , Receptores de N-Metil-D-Aspartato/efectos de los fármacos , Receptores de N-Metil-D-Aspartato/metabolismoRESUMEN
The most toxic species in live systems include reactive nitrogen species such as peroxynitrite, which at high levels induces nitrosative stress. In human spermatozoa, the negative effect of peroxynitrite on motility and mitochondrial membrane potential was recently demonstrated, and the hypothesis of this work is that impairment of ATP production could be one cause of the effect on motility. Therefore, the aim here was to evaluate ATP production by both glycolysis and oxidative phosphorylation (OXPHOS) in spermatozoa exposed to peroxynitrite in vitro. Human spermatozoa were incubated with SIN-1, a molecule which generates peroxynitrite, and the ATP level was evaluated. Then, to inactivate glycolysis or OXPHOS, spermatozoa were incubated with pharmacological inhibitors of these pathways. Spermatozoa treated for inactivating one or the other pathway were exposed to SIN-1, and the ATP level was compared to the control without SIN-1 in each condition. The ATP level fell after peroxynitrite exposure. The ATP in spermatozoa treated for inactivating one or the other metabolic pathway and subsequently exposed to peroxynitrite was reduced compared with the control. These results show for the first time that an important mechanism by which peroxynitrite reduces sperm function is the inhibition of ATP production, affecting both glycolysis and OXPHOS.
Asunto(s)
Adenosina Trifosfato/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ácido Peroxinitroso/toxicidad , Motilidad Espermática/efectos de los fármacos , Espermatozoides/efectos de los fármacos , Antimetabolitos/toxicidad , Desoxiglucosa/toxicidad , Glucólisis/efectos de los fármacos , Humanos , Masculino , Mitocondrias/efectos de los fármacos , Molsidomina/análogos & derivados , Molsidomina/metabolismo , Fosforilación Oxidativa/efectos de los fármacos , Estrés Oxidativo , Rotenona/toxicidad , Espermatozoides/metabolismo , Desacopladores/toxicidadRESUMEN
Short-term exposure of gametes to different types of stress might induce stress tolerance in mammalian embryos. The aim of this study was to evaluate the effect of short-term exposure of bovine mature cumulus-oocyte complex (COC) to 3-morpholinosydnonimine (SIN-1) on subsequent in vitro embryo development, embryo quality and relative gene expression. Matured COCs were incubated with SIN-1 (0, 0.1, 1, 10 and 100 µM SIN-1) for 1 hr before in vitro fertilization and zygotes were cultured until Day 7. The cleavage rate at 72 hr did not show any differences among groups. However, the blastocyst rate on Day 7 decreased with all treatments evaluated, with the embryos generated with 10 µM SIN-1 showing the lowest embryo production rate. Embryo quality analysis did not show any differences in total cell number (TCN) or inner cell mass (ICM) among groups. Relative gene expression analysis showed a downregulation of eNOS expression and an upregulation of nNOS expression in all treatments evaluated compared to the control group. Also, a downregulation was observed in some treatments: SOD2 at 0.1 µM; SOD1 at 0.1 and 100 µM; PRDX5 at 0.1, 10 and 100 µM; and NANOG at 10 and 100 µM; and an upregulation of CDX2 expression was observed at 100 µM. The other genes (OCT4, HIF1A, HSPA1A, BCL2A and iNOS) did not show any differences in the relative gene expression. These results suggest that the short-term exposure of mature bovine COCs to SIN-1 does not induce stress tolerance and has no beneficial effect on bovine in vitro embryo production.
Asunto(s)
Bovinos/embriología , Células del Cúmulo/fisiología , Técnicas de Cultivo de Embriones/veterinaria , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Molsidomina/análogos & derivados , Oocitos/fisiología , Animales , Femenino , Regulación del Desarrollo de la Expresión Génica/fisiología , Molsidomina/farmacologíaRESUMEN
Peroxynitrite is a highly reactive nitrogen species and when it is generated at high levels it causes nitrosative stress, an important cause of impaired sperm function. High levels of peroxynitrite have been shown to correlate with decreased semen quality in infertile men. Thiol groups in sperm are mainly found in enzymes, antioxidant molecules, and structural proteins in the axoneme. Peroxynitrite primarily reacts with thiol groups of cysteine-containing proteins. Although it is well known that peroxynitrite oxidizes sulfhydryl groups in sperm, the subcellular localization of this oxidation remains unknown. The main objective of this study was to establish the subcellular localization of peroxynitrite-induced nitrosative stress in thiol groups and its relation to sperm motility in human spermatozoa. For this purpose, spermatozoa from healthy donors were exposed in vitro to 3-morpholinosydnonimine (SIN-1), a compound which generates peroxynitrite. In order to detect peroxynitrite and reduced thiol groups, the fluorescent probes, dihydrorhodamine 123 and monobromobimane (mBBr), were used respectively. Sperm viability was analyzed by propidium iodide staining. Peroxynitrite generation and thiol redox state were monitored by confocal microscopy whereas sperm viability was evaluated by flow cytometry. Sperm motility was analyzed by CASA using the ISAS(®) system. The results showed that exposure of human spermatozoa to peroxynitrite results in increased thiol oxidation which is mainly localized in the sperm head and principal piece regions. Thiol oxidation was associated with motility loss. The high susceptibility of thiol groups to peroxynitrite-induced oxidation could explain, at least in part, the negative effect of reactive nitrogen species on sperm motility. ABBREVIATIONS: DHR: dihydrorhodamine 123; mBBr: monobromobimane ONOO(-): peroxynitrite RNS: reactive nitrogen species RFI: relative fluorescence intensity SIN-1: 3-morpholinosydnonimine CASA: Computer-Aided Sperm Analysis PARP: poli ADP ribose polimerasa VCL: curvilinear velocity VSL: straight-line velocity VAP: average path velocity PRDXs: peroxiredoxins ODF: outer dense fiber ODF1: outer dense fiber 1 PI: propidium iodide DMSO: dimethyl sulfoxide SD: standard deviation ANOVA: analysis of variance.
Asunto(s)
Ácido Peroxinitroso/metabolismo , Espermatozoides/metabolismo , Compuestos de Sulfhidrilo/metabolismo , Colorantes Fluorescentes , Humanos , Masculino , Molsidomina/análogos & derivados , Molsidomina/metabolismo , Oxidación-Reducción , Análisis de Semen , Motilidad Espermática , Espermatozoides/citologíaRESUMEN
Nitrosative stress is produced by high levels of reactive nitrogen species (RNS). The RNS include peroxynitrite, a highly reactive free radical produced from a diffusion-controlled reaction between nitric oxide and superoxide anion. Peroxynitrite causes nitration and oxidation of lipids, proteins and DNA, and is thus considered an important pathogenic mechanism in various diseases. Although high levels of peroxynitrite are associated with astenozoospermia, few reports exist regarding the in vitro effect of high levels of this RNS on human sperm. The aim of this study was to evaluate the in vitro effect of nitrosative stress caused by peroxynitrite on the viability, motility and mitochondrial membrane potential of human spermatozoa. To do this, human spermatozoa from healthy donors were exposed in vitro to 3-morpholinosydnonimine (SIN-1), a molecule that generates peroxynitrite. Incubations were done at 37°C for up to 4 h with SIN-1 concentrations between 0.2 and 1.0 mmol/l. Generation of peroxynitrite was confirmed using dihydrorhodamine 123 (DHR) by spectrophotometry and flow cytometry. Sperm viability was assessed by propidium iodide staining; sperm motility was analyzed by CASA, and the state of mitochondrial membrane potential (ΔΨm) by JC-1 staining. Viability and ΔΨm were measured by flow cytometry. The results showed an increase in DHR oxidation, demonstrating the generation of peroxynitrite through SIN-1. Peroxynitrite decreased progressive and total motility, as well as some sperm kinetic parameters. Mitochondrial membrane potential also decreased. These alterations occurred with no decrease in sperm viability. In conclusion, peroxynitrite-induced nitrosative stress impairs vital functions in the male gamete, possibly contributing to male infertility.
Asunto(s)
Molsidomina/análogos & derivados , Ácido Peroxinitroso/farmacología , Motilidad Espermática/efectos de los fármacos , Espermatozoides/efectos de los fármacos , Adulto , Bencimidazoles , Carbocianinas , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Colorantes Fluorescentes , Humanos , Masculino , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Potencial de la Membrana Mitocondrial/fisiología , Molsidomina/química , Molsidomina/farmacología , Estrés Oxidativo , Ácido Peroxinitroso/química , Rodaminas , Análisis de Semen , Espectrometría de Fluorescencia , Motilidad Espermática/fisiología , Espermatozoides/citología , Espermatozoides/fisiologíaRESUMEN
Increased production of reactive nitrogen (RNS) and oxygen (ROS) species and its detrimental effect to mitochondria are associated with endothelial dysfunction. This study was designed to determine the effect of a peroxynitrite flux, promoted by 1,3-morpholinosydnonimine (SIN-1), in mitochondrial function and some redox homeostasis parameters in bovine aortic endothelial cells (BAEC). Moreover, the effect of diphenyl diselenide (PhSe)2, a simple organic selenium compound, in preventing peroxynitrite-mediated cytotoxicity was also investigated. Our results showed that overnight exposure to SIN-1 (250 µM) caused a profound impairment of oxygen consumption, energy generation and reserve capacity in mitochondria of BAEC. Mitochondrial dysfunction resulted in an additional intracellular production of peroxynitrite, amplifying the phenomenon and leading to changes in redox homeostasis. Moreover, we observed an extensive decline in mitochondrial membrane potential (ΔΨm) induced by peroxynitrite and this event was associated with apoptotic-type cell death. Alternatively, the pretreatment of BAEC with (PhSe)2, hindered peroxynitrite-mediated cell damage by preserving mitochondrial and endothelial function and consequently preventing apoptosis. The protective effect of (PhSe)2 was related to its ability to improve the intracellular redox state by increasing the expression of different isoforms of peroxiredoxins (Prx-1-3), efficient enzymes in peroxynitrite detoxification.
Asunto(s)
Derivados del Benceno/farmacología , Células Endoteliales/efectos de los fármacos , Metabolismo Energético/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Molsidomina/análogos & derivados , Compuestos de Organoselenio/farmacología , Peroxirredoxinas/metabolismo , Ácido Peroxinitroso/metabolismo , Animales , Aorta/citología , Bovinos , Células Endoteliales/enzimología , Homeostasis/efectos de los fármacos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/enzimología , Molsidomina/química , Oxidación-Reducción , Ácido Peroxinitroso/química , Ácido Peroxinitroso/toxicidadRESUMEN
The dorsal raphe nucleus (DRN) is the origin of ascending serotonergic projections and is considered to be an important component of the brain circuit that mediates anxiety- and depression-related behaviors. A large fraction of DRN serotonin-positive neurons contain nitric oxide (NO). Disruption of NO-mediated neurotransmission in the DRN by NO synthase inhibitors produces anxiolytic- and antidepressant-like effects in rats and also induces nonspecific interference with locomotor activity. We investigated the involvement of the 5-HT1A autoreceptor in the locomotor effects induced by NO in the DRN of male Wistar rats (280-310 g, N = 9-10 per group). The NO donor 3-morpholinosylnomine hydrochloride (SIN-1, 150, and 300 nmol) and the NO scavenger S-3-carboxy-4-hydroxyphenylglycine (carboxy-PTIO, 0.1-3.0 nmol) were injected into the DRN of rats immediately before they were exposed to the open field for 10 min. To evaluate the involvement of the 5-HT1A receptor and the N-methyl-D-aspartate (NMDA) glutamate receptor in the locomotor effects of NO, animals were pretreated with the 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT, 8 nmol), the 5-HT1A receptor antagonist N-(2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl)-N-2-pyridinyl-cyclohexanecarboxamide maleate (WAY-100635, 0.37 nmol), and the NMDA receptor antagonist DL-2-amino-7-phosphonoheptanoic acid (AP7, 1 nmol), followed by microinjection of SIN-1 into the DRN. SIN-1 increased the distance traveled (mean ± SEM) in the open-field test (4431 ± 306.1 cm; F7,63 = 2.44, P = 0.028) and this effect was blocked by previous 8-OH-DPAT (2885 ± 490.4 cm) or AP7 (3335 ± 283.5 cm) administration (P < 0.05, Duncan test). These results indicate that 5-HT1A receptor activation and/or facilitation of glutamate neurotransmission can modulate the locomotor effects induced by NO in the DRN.
Asunto(s)
Animales , Masculino , Ratas , Molsidomina/análogos & derivados , Actividad Motora/efectos de los fármacos , Óxido Nítrico/farmacología , Núcleos del Rafe/efectos de los fármacos , /efectos de los fármacos , /farmacología , Relación Dosis-Respuesta a Droga , Glicina/análogos & derivados , Glicina/farmacología , Molsidomina/farmacología , Actividad Motora/fisiología , Ratas WistarRESUMEN
The dorsal raphe nucleus (DRN) is the origin of ascending serotonergic projections and is considered to be an important component of the brain circuit that mediates anxiety- and depression-related behaviors. A large fraction of DRN serotonin-positive neurons contain nitric oxide (NO). Disruption of NO-mediated neurotransmission in the DRN by NO synthase inhibitors produces anxiolytic- and antidepressant-like effects in rats and also induces nonspecific interference with locomotor activity. We investigated the involvement of the 5-HT(1A) autoreceptor in the locomotor effects induced by NO in the DRN of male Wistar rats (280-310 g, N = 9-10 per group). The NO donor 3-morpholinosylnomine hydrochloride (SIN-1, 150, and 300 nmol) and the NO scavenger S-3-carboxy-4-hydroxyphenylglycine (carboxy-PTIO, 0.1-3.0 nmol) were injected into the DRN of rats immediately before they were exposed to the open field for 10 min. To evaluate the involvement of the 5-HT(1A) receptor and the N-methyl-D-aspartate (NMDA) glutamate receptor in the locomotor effects of NO, animals were pretreated with the 5-HT(1A) receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT, 8 nmol), the 5-HT(1A) receptor antagonist N-(2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl)-N-2-pyridinyl-cyclohexanecarboxamide maleate (WAY-100635, 0.37 nmol), and the NMDA receptor antagonist DL-2-amino-7-phosphonoheptanoic acid (AP7, 1 nmol), followed by microinjection of SIN-1 into the DRN. SIN-1 increased the distance traveled (mean ± SEM) in the open-field test (4431 ± 306.1 cm; F(7,63) = 2.44, P = 0.028) and this effect was blocked by previous 8-OH-DPAT (2885 ± 490.4 cm) or AP7 (3335 ± 283.5 cm) administration (P < 0.05, Duncan test). These results indicate that 5-HT(1A) receptor activation and/or facilitation of glutamate neurotransmission can modulate the locomotor effects induced by NO in the DRN.
Asunto(s)
Molsidomina/análogos & derivados , Actividad Motora/efectos de los fármacos , Óxido Nítrico/farmacología , Núcleos del Rafe/efectos de los fármacos , Receptor de Serotonina 5-HT1A/efectos de los fármacos , Antagonistas del Receptor de Serotonina 5-HT1/farmacología , Animales , Relación Dosis-Respuesta a Droga , Glicina/análogos & derivados , Glicina/farmacología , Masculino , Molsidomina/farmacología , Actividad Motora/fisiología , Ratas , Ratas WistarRESUMEN
The aim of this work was to study the effect of molsidomine (MOLS), a nitric oxide (NO) donor, on the nitrergic system changes in an experimental model of cholinergic damage induced by 192 IgG saporin (SAP). Male rats were injured by intraseptal administration of SAP (0.22 µg), after seven days, rats were administered with MOLS (4 mg/kg, i.p.) 60 min before sacrifice. Prefrontal cortex (PC), striatum (S) and hippocampus (HC) were dissected out. Results showed significant recovery of the constitutive NOS activity (cNOS) in PC and S regions by MOLS but not in HC compared against controls. SAP reduced the cellular population in the lesion site and MOLS was able to avoid the progression of damage in this area. NO donor is able to modulate the nitrergic status in an experimental model induced by SAP.
Asunto(s)
Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Molsidomina/farmacología , Donantes de Óxido Nítrico/farmacología , Óxido Nítrico Sintasa/metabolismo , Animales , Anticuerpos Monoclonales/toxicidad , Colinérgicos/toxicidad , Masculino , Ratas , Ratas Wistar , Proteínas Inactivadoras de Ribosomas Tipo 1/toxicidad , SaporinasRESUMEN
The purpose of this study was to verify the occurrence of pigment dispersion in retinal pigment cells exposed to UVA and UVB radiation, and to investigate the possible participation of a nitric oxide (NO) pathway. Retinal pigment cells from Neohelice granulata were obtained by cellular dissociation. Cells were analyzed for 30 min in the dark (control) and then exposed to 1.1 and 3.3 J cm(-2) UVA, 0.07 and 0.9 J cm(-2) UVB, 20 nmß-PDH (pigment dispersing hormone) or 10 µm SIN-1 (NO donor). Histological analyses were performed to verify the UV effect in vivo. Cultured cells were exposed to 250 µm L-NAME (NO synthase blocker) and afterwards were treated with UVA, UVB or ß-PDH. The retinal cells in culture displayed significant pigment dispersion in response to UVA, UVB and ß-PDH. The same responses to UVA and UVB were observed in vivo. SIN-1 did not induce pigment dispersion in the cell cultures. L-NAME significantly decreased the pigment dispersion induced by UVA and UVB but not by ß-PDH. All retinal cells showed an immunopositive reaction against neuronal nitric oxide synthases. Therefore, UVA and UVB radiation are capable of inducing pigment dispersion in retinal pigment cells of Neohelice granulata and this dispersion may be nitric oxide synthase dependent.
Asunto(s)
Braquiuros/metabolismo , Braquiuros/efectos de la radiación , Pigmentos Retinianos/metabolismo , Pigmentos Retinianos/efectos de la radiación , Animales , Braquiuros/efectos de los fármacos , Técnicas In Vitro , Molsidomina/análogos & derivados , Molsidomina/farmacología , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico/metabolismo , Donantes de Óxido Nítrico/farmacología , Péptidos/farmacología , Células Fotorreceptoras de Invertebrados/efectos de los fármacos , Células Fotorreceptoras de Invertebrados/metabolismo , Células Fotorreceptoras de Invertebrados/efectos de la radiación , Rayos UltravioletaRESUMEN
Electrical or chemical stimulation of the dorsal periaqueductal gray matter (DPAG) evokes escape, a defensive behavior that has been related to panic attacks. Injection of 5-HT(1A) or 5-HT(2A) receptor agonists into this midbrain area inhibits this response. It has been proposed that the impairment of 5-HT mechanisms controlling escape at the level of the DPAG may underlie the susceptibility to panic attacks that characterizes the panic disorder. In this study we evaluated the effects of the pharmacological manipulation of the dorsal raphe nucleus (DRN), which are the main source of 5-HT input to the DPAG, on the escape response evoked in rats by the intra-DPAG injection of the nitric oxide donor SIN-1. The results showed that DRN administration of the 5-HT(1A) receptor agonist 8-OH-DPAT which inhibits the activity of 5-HT neurons favored the expression of escape induced by SIN-1. Intra-DRN injection of the excitatory amino acid kainic acid or the 5-HT(1A) receptor antagonist WAY-100635 did not change escape expression. However, both compounds fully blocked the escape reaction generated by intra-DPAG injection of the excitatory amino acid d,l-homocysteic acid (DLH). Overall, the results indicate that 5-HT neurons in the DRN exert a bidirectional control upon escape behavior generated by the DPAG. Taking into account the effect of WAY-100635 on DLH-induced escape, they also strengthen the view that DRN 5-HT(1A) autoreceptors are under tonic inhibitory influence by 5-HT.
Asunto(s)
Reacción de Fuga/fisiología , Homocisteína/análogos & derivados , Molsidomina/análogos & derivados , Trastorno de Pánico/fisiopatología , Sustancia Gris Periacueductal/fisiología , Núcleos del Rafe/fisiología , 8-Hidroxi-2-(di-n-propilamino)tetralin/farmacología , Animales , Modelos Animales de Enfermedad , Reacción de Fuga/efectos de los fármacos , Homocisteína/antagonistas & inhibidores , Homocisteína/farmacología , Ácido Kaínico/farmacología , Masculino , Molsidomina/farmacología , Piperazinas/farmacología , Piridinas/farmacología , Núcleos del Rafe/efectos de los fármacos , Ratas , Ratas Wistar , Agonistas del Receptor de Serotonina 5-HT1 , Antagonistas del Receptor de Serotonina 5-HT1RESUMEN
Reactive oxygen species have an important role in the control of platelet activity. Superoxide anion (O(2)(-)) is a free radical that can be converted into other reactive oxygen species such as peroxynitrite (ONOO(-)) that is formed from the reaction between O(2)(-) and nitric oxide (NO). There are conflicting data on ONOO(-) effects in platelets because it presents pro- or anti-aggregatory actions. 3-morpholinosydnonimine (SIN-1) co-generates NO and O(2)(-), yielding ONOO(-). Therefore, the present study aimed to investigate the mechanisms involved in the inhibition of human platelet adhesion by SIN-1. Microtiter plates were coated with human fibrinogen, after which washed platelets (6 x 10(8)platelets/ml) were added to adhere. Exposure of non-activated and thrombin-activated platelets to SIN-1 (0.001-100 microM) concentration-dependently inhibited adhesion, which was accompanied by marked increases in the cyclic GMP levels. In non-activated platelets, the soluble guanylate cyclase inhibitor ODQ prevented the SIN-1-induced cGMP elevations and adhesion inhibition. In thrombin-activated platelets, ODQ fully prevented the SIN-1-induced cGMP elevations, but only partly prevented the adhesion inhibition. The O(2)(-) and ONOO(-) scavengers superoxide dismutase (SOD) and -(-)epigallocatechin gallate, respectively, had minimal effects in non-activated platelets. The inhibition of activated platelets by SIN-1 was reversed by SOD and partly reduced by ECG. Western blot analysis of SIN-1-treated platelets showed a single 105 kDa-nitrated band. Nanospray LC-MS-MS identified the protein containing 3-nitrotyrosine residues as human alpha-actinin-1-cytoskeletal isoform. Our data show that platelet adhesion inhibition by SIN-1 in activated platelets involves cGMP-independent mechanism through O(2)(-) generation. Superoxide anion signaling pathway includes ONOO(-) formation and alpha-actinin nitration.
Asunto(s)
Plaquetas/citología , Plaquetas/efectos de los fármacos , Molsidomina/análogos & derivados , Superóxidos/farmacología , Trombina/farmacología , Plaquetas/metabolismo , Proteínas Sanguíneas/metabolismo , Adhesión Celular/efectos de los fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Fibrinógeno/metabolismo , Depuradores de Radicales Libres/farmacología , Guanilato Ciclasa/antagonistas & inhibidores , Humanos , Molsidomina/farmacología , Nitratos/metabolismo , Ácido Peroxinitroso/farmacología , Solubilidad , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMEN
Previous studies have showed that SIN-1, a nitric oxide (NO) donor, injected into the dorsolateral column of the periaqueductal gray (dlPAG) induces flight reactions. This drug, however, can also produce peroxynitrite, which may interfere in this effect. In addition, it is also unknown if this effect is mediated by local activation of soluble guanylate cyclase (sGC). The aims of this study, therefore, were (1) to investigate if NOC-9 (6-(2-Hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-hexanamine), a NO donor that does not produce peroxynitrite, would produce flight reactions after intra-dlPAG administration similar to those induced by SIN-1; (2) to verify if these responses could be prevented by local injection of a selective guanylate cyclase inhibitor (ODQ). Male Wistar rats (n=5-12) with cannulae aimed at the dlPAG received injections of TRIS (pH 10.0, 0.5 microl), NOC-9 (75 and 150 nmol), saline or SIN-1 (200 nmol) and were placed in an open arena for 10 min. In a subsequent experiment animals (n=7-8) were pretreated with ODQ (1 nmol/0.5 microl) before receiving NOC-9 150 nmol. NOC-9 induced a significant dose-dependent increase in flight reactions in the first minute after injection (% of animals displaying flight: vehicle=0%, NOC 75=67%, NOC 150=75%). SIN-1 had a similar effect (100% of animals showing flight) but the effects lasted longer (10 min) than those of NOC-9. The effect of NOC-9 (150 nmol) was prevented by pretreatment with ODQ (% of animals displaying flight: vehicle+NOC 150=71%, ODQ+NOC 150=37%). The results suggest that NO donors injected into the dlPAG induce defensive responses that are not mediated by secondary peroxynitrite production. Moreover, they also indicate that these defensive responses depend on activation of local sGC. The data strengthen the proposal that NO can modulate defensive reactions in the dlPAG.
Asunto(s)
Reacción de Fuga/efectos de los fármacos , Guanilato Ciclasa/metabolismo , Donantes de Óxido Nítrico/farmacología , Sustancia Gris Periacueductal/efectos de los fármacos , Triazenos/farmacología , Animales , Cateterismo , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/administración & dosificación , Guanilato Ciclasa/antagonistas & inhibidores , Masculino , Microinyecciones , Molsidomina/administración & dosificación , Molsidomina/análogos & derivados , Actividad Motora/efectos de los fármacos , Análisis Multivariante , Oxadiazoles/administración & dosificación , Quinoxalinas/administración & dosificación , Distribución Aleatoria , Ratas , Ratas Wistar , Factores de TiempoRESUMEN
The cell signaling cascades that mediate pigment movements in crustacean chromatophores are not yet well established, although Ca(2+) and cyclic nucleotide second messengers are involved. Here, we examine the participation of cyclic guanosine monophosphate (cGMP) in pigment aggregation triggered by red pigment concentrating hormone (RPCH) in the red ovarian chromatophores of freshwater shrimp. In Ca(2+)-containing (5.5 mmol l(-1)) saline, 10 micromol l(-1) dibutyryl cGMP alone produced complete pigment aggregation with the same time course ( approximately 20 min) and peak velocity ( approximately 17 microm/min) as 10(-8) mol l(-1) RPCH; however, in Ca(2+)-free saline (9 x 10(-11) mol l(-1) Ca(2+)), db-cGMP was without effect. The soluble guanylyl cyclase (GC-S) activators sodium nitroprusside (SNP, 0.5 micromol l(-1)) and 3-morpholinosydnonimine (SIN-1, 100 micromol l(-1)) induced moderate aggregation by themselves ( approximately 35%-40%) but did not affect RPCH-triggered aggregation. The GC-S inhibitors zinc protoporphyrin IX (ZnPP-XI, 30 micromol l(-1)) and 6-anilino-5,8-quinolinedione (LY83583, 10 micromol l(-1)) partially inhibited RPCH-triggered aggregation by approximately 35%. Escherichia coli heat-stable enterotoxin (STa, 1 micromol l(-1)), a membrane-receptor guanylyl cyclase stimulator, did not induce or affect RPCH-triggered aggregation. We propose that the binding of RPCH to an unknown membrane-receptor type activates a Ca(2+)-dependent signaling cascade coupled via cytosolic guanylyl cyclase and cGMP to protein kinase G-phosphorylated proteins that regulate aggregation-associated, cytoskeletal molecular motor activity. This is a further example of a cGMP signaling cascade mediating the effect of a crustacean X-organ neurosecretory peptide.
Asunto(s)
Cromatóforos/metabolismo , GMP Cíclico/metabolismo , Palaemonidae/metabolismo , Pigmentos Biológicos/metabolismo , Transducción de Señal/fisiología , Aminoquinolinas/farmacología , Animales , Brasil , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Agua Dulce , Guanilato Ciclasa/antagonistas & inhibidores , Guanilato Ciclasa/metabolismo , Molsidomina/análogos & derivados , Molsidomina/farmacología , Nitroprusiato/farmacología , Palaemonidae/fisiología , Protoporfirinas/farmacologíaRESUMEN
Yerba maté (Ilex paraguariensis) is a native South America plant widely consumed as different beverages. Yerba maté leaves contains high concentrations of polyphenols that are responsible for its high in vitro and in vivo antioxidant activity. The in vivo antioxidant properties vis a vis LDL particles has not yet been studied for maté tea, the roasted yerba maté product. The aim of this study was to evaluate the antioxidant activity of maté tea ingestion ex vivo on human LDL. Fasting peripheral venous blood samples of healthy women were taken in three different times: before drinking the tea, one hour later and after one week (7 days) of daily consumption of maté tea. The isolated LDL was oxidized by three different pathways [copper (CuSO4), lipoxygenase and peroxynitrite (SIN-1)]. Conjugated dienes and structural modifications on LDL were evaluated. Ingestion of maté tea increased LDL resistance towards ex vivo copper oxidation, but did not alter the peroxidation pattern when SIN-1 or lipoxygenase were used as oxidants.
Asunto(s)
Bebidas , LDL-Colesterol/sangre , Ilex paraguariensis/química , Hojas de la Planta/química , Preparaciones de Plantas/farmacología , Antioxidantes/farmacología , Sulfato de Cobre/farmacología , Femenino , Humanos , Lipooxigenasa/farmacología , Molsidomina/análogos & derivados , Molsidomina/farmacología , Ácido Peroxinitroso/farmacologíaRESUMEN
The effect of peroxynitrite (ONOO(-)) on the capacitation rates of cryopreserved bull spermatozoa and the participation of protein kinases in the capacitation process were evaluated. A pool of spermatozoa from five bulls was incubated in Tyrode's albumin lactate pyruvate (TALP) medium in the presence of heparin (10 IU/ml), sodium nitroprusside (SNP, 50 nM), a nitric oxide donor or 3-morpholinosydnonimine (SIN-1, 1-20 microM), a ONOO(-) donor. The participation of ONOO(-) was evaluated at 15, 30 and 45 min and confirmed by using a specific scavenger, uric acid (2-20 mM). Spermatozoa capacitated with SIN-1 were incubated with ovarian follicular fluid of cattle to evaluate their ability to undergo acrosome reaction. The role of ONOO(-) during capacitation induced by heparin or nitric oxide was evaluated by the addition of uric acid. The participation of protein kinase A (PKA), protein kinase C (PKC) and protein tyrosine kinase (PTK) in capacitation induced by ONOO(-) was evaluated by incubation with specific inhibitors (50 microM H-89, 0.1 microM bisindolylmaleimide I, and 3 microM genistein, respectively). Capacitation percentages were determined by the fluorescence technique with chlortetracycline (CTC) and true acrosome reaction was determined by trypan blue and Differential-Interferential Contrast (DIC). SIN-1 concentrations employed had no effect on progressive motility or sperm viability. Capacitation values of 10 microM SIN-1 treatment (23+/-2%) were significantly greater with respect to the control (4.6+/-1.62%). At 15 min of incubation the greatest capacitation was observed (P<0.05), reaching a plateau between 15 and 45 min. Follicular fluid induced acrosome reaction in spermatozoa previously capacitated with 10 microM SIN-1 (P<0.05). Uric acid prevented SIN-1-induced capacitation and significantly diminished capacitation induced by heparin or SNP. The addition of PKA and PKC inhibitors failed to modify the capacitation induced by SIN-1 (27.4+/-3.85 and 24.8+/-4.75, respectively). Genistein, a PTK inhibitor, produced a significant capacitation decrease (8.6+/-5.5%). These results indicate that endogenous ONOO(-) may be generated during heparin- or SNP-induced capacitation. Exogenous ONOO(-) acts as a capacitation inducer and involves the participation of PTK, as part of the intracellular mechanisms that lead to capacitation in cryopreserved bovine spermatozoa.
Asunto(s)
Bovinos/fisiología , Criopreservación/veterinaria , Heparina/farmacología , Ácido Peroxinitroso/metabolismo , Preservación de Semen/veterinaria , Capacitación Espermática/efectos de los fármacos , Espermatozoides/efectos de los fármacos , Reacción Acrosómica/efectos de los fármacos , Animales , Criopreservación/métodos , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Femenino , Líquido Folicular/metabolismo , Genisteína/farmacología , Indoles/farmacología , Masculino , Maleimidas/farmacología , Molsidomina/análogos & derivados , Molsidomina/farmacología , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/metabolismo , Preservación de Semen/métodos , Espermatozoides/enzimologíaRESUMEN
The ability of UV radiation to stimulate color change in vertebrates is well known; however, the signaling pathway involved is not fully explained. Since nitric oxide (NO) is among the candidates for this role, in this study the participation of NO signaling in the pigment migration induced by UV radiation in melanophores of the crab Chasmagnathus granulatus was investigated. When the NO donor, SIN-1, was incubated with pieces of epidermis, there was an induction of a dose-dependent pigment dispersion (in vitro assays). When male adults were exposed to different doses of UVA and UVB, N(G)-nitro-l-arginine-methyl-ester, an NO synthase (NOS) blocker produced a decrease of the pigment dispersion induced by UV (in vivo assays). However, in similar assays, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, an NO scavenger, decreased only the pigment dispersion induced by UVA. Interestingly, buthionine sulfoximine did not produce any change in pigment dispersion induced by UVA (in vivo assays) and SIN-1 (in vitro assays). Our results using NADPH-diaphorase histochemistry and immunocytochemistry against nNOS indicated the production of NO by epidermal cells. In conclusion, we suggest that NO is a key molecule for the induction of pigment dispersion in the melanophores of Chasmagnthus granulatus, and also that NOS activation is a fundamental step for this process.