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83 Structures of human nNOS, 55 structures of human eNOS, 13 structures of iNOS, and about 126 reported NOS-bound compounds are summarized and analyzed. Structural and statistical analysis show that, at least one copy of each analyzed compound binds to the active site (the substrate arginine binding site) of human NOS. And binding features of the three isoforms show differences, but the binding preference of compounds is not in the way helpful for inhibitor design targeting nNOS and iNOS, or for activator design targeting eNOS. This research shows that there is a strong structural and functional similarity between oxygenase domains of human NOS isoforms, especially the architecture, residue composition, size, shape, and distribution profile of hydrophobicity, polarity and charge of the active site. The selectivity and efficacy of inhibitors over the rest of isoforms rely a lot on chance and randomness. Further increase of selectivity via rational improvement is uncertain, unpredictable and unreliable, therefore, to achieve high selectivity through targeting this site is complicated and requires combinative investigation. After analysis on the current two targeting sites in NOS, the highly conserved arginine binding pocket and H4B binding pocket, new potential drug-targeting sites are proposed based on structure and sequence profiling. This comprehensive analysis on the structure and interaction profiles of human NOS and bound compounds provides fresh insights for drug discovery and pharmacological research, and the new discovery here is practically applied to guide protein-structure based drug discovery.
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Nitric oxide (NO) plays an important and diverse signalling role in the cardiovascular system, contributing to the regulation of vascular tone, endothelial function, myocardial function, haemostasis, and thrombosis, amongst many other roles. NO is synthesised through the nitric oxide synthase (NOS)-dependent L-arginine-NO pathway, as well as the nitrate-nitrite-NO pathway. The three isoforms of NOS, namely neuronal (NOS1), inducible (NOS2), and endothelial (NOS3), have different localisation and functions in the human body, and are consequently thought to have differing pathophysiological roles. Furthermore, as we continue to develop a deepened understanding of the different roles of NOS isoforms in disease, the possibility of therapeutically modulating NOS activity has emerged. Indeed, impaired (or dysfunctional), as well as overactive (or dysregulated) NOS activity are attractive therapeutic targets in cardiovascular disease. This review aims to describe recent advances in elucidating the physiological role of NOS isoforms within the cardiovascular system, as well as mechanisms of dysfunctional and dysregulated NOS in cardiovascular disease. We then discuss the modulation of NO and NOS activity as a target in the development of novel cardiovascular therapeutics.
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Enfermedades Cardiovasculares , Humanos , Enfermedades Cardiovasculares/terapia , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Óxido Nítrico Sintasa/metabolismo , Miocardio/metabolismo , Isoformas de Proteínas/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Óxido Nítrico Sintasa de Tipo I/metabolismoRESUMEN
OBJECTIVES: Cerebral infarction is a subtype of stroke with high incidence and disability rate. Ischemia reperfusion injury (IRI) is the key point of cerebral infarction treatment. UbiA prenyltransferase domain containing 1 (UBIAD1) is a kind of enzyme with various biological functions including electron transport in mitochondrial respiratory chain, lipid metabolism, and oxidative stress which are related to IRI. The purpose of this study aims to determine the neuroprotective effects and the underlying mechanisms of UBIAD1 in cerebral IRI. METHODS: We employed oxygen-glucose deprivation/reoxygenation (OGD/R) model in mouse neuroblastoma Neuro2a (N2a) cells to mimic cerebral IRI. Lentivirus vector over-expressed UBIAD1 was transfacted into N2a cells to maintain high and stable expression of UBIAD1. In the first part of the experiment, N2a cells were divided into 5 groups: A non-OGD (N2a cells without exposure to OGD) group, groups of reoxygenation 0, 4, 12 and 24 h after 4 h of OGD, respectively. In the second part of the experiment, N2a cells were divided into 6 groups: A Con (normal cell)+non-OGD group, an EV (cell transfected with empty vector)+non-OGD group, an OE (over-expressed UBIAD1)+non-OGD group, a Con+OGD/R group, an EV+OGD/R group, and an OE+OGD/R group. In the third part, the N2a cells were divided into 8 groups: A Con+non-OGD group, an OE+non-OGD group, a Con+non-OGD+nNOS inhibitior 7-nitroindazole (7-NI) group, an OE+non-OGD+7-NI group, a Con+OGD/R group, an OE+OGD/R group, a Con+OGD/R+7-NI group, and an OE+OGD/R+7-NI group. The morphological changes of Golgi apparatus were observed under the confocal laser scanning microscope. The mRNA and protein levels of UBIAD1, secretory pathway Ca2+-ATPase isoform 1 (SPCA1), and NOS were determined by real-time PCR and Western blotting, respectively. Cell apoptosis rate was detected with flow cytometry; cell viability was detected with MTT assay, and NO release was determined with Griess assay. RESULTS: Compared with the non-OGD group, the expression levels of UBIAD1 mRNA and protein in N2a cells in the groups of 0, 4, 12 and 24 h reoxygenation after OGD 4 h decreased significantly (P<0.05 or P<0.01), and the longer the reoxygenation time, the more significant the reduction of UBIAD1 expression. Compared with the Con+OGD/R group and the EV+OGD/R group, mRNA and protein levels of UBIAD1 and SPCA1 were increased (P<0.05 or P<0.01), the apoptosis rate was decreased (all P<0.01), and the cell viability was increased (all P<0.01) in the OE+OGD/R group. The Golgi fragmentation was less in the OE+OGD/R group than that in the Con+ OGD/R group and the EV+OGD/R group. The mRNA and protein levels of endothelial NOS (eNOS) and neuronal NOS (nNOS) were decreased (P<0.05 or P<0.01), and the level of NO was decreased (all P<0.01) in the groups over-expressed UBIAD1 (OE+non-OGD group vs Con+non-OGD group, OE+OGD/R group vs Con+OGD/R group). The level of NO and apoptosis rate of N2a cells were decreased (all P<0.01) in the the groups pretreated with 7-NI (Con+OGD/R+7-NI group vs Con+OGD/R group, OE+OGD/R+7-NI group vs OE+OGD/R group). CONCLUSIONS: UBIAD1 may exerts protective effects on OGD/R induced N2a cells by ameliorating Golgi apparatus dysfunction via the nNOS/NO pathway.
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Dimetilaliltranstransferasa , Oxígeno , Animales , Ratones , Supervivencia Celular , Infarto Cerebral , Glucosa , Metabolismo de los Lípidos , Óxido Nítrico/metabolismoRESUMEN
Nitric oxide (NO) is a small molecule produced by nitric oxide synthase (NOS) with various physio-pathological functions in the body. There are three main NOS isoforms, including the endothelial (eNOS), inducible (iNOS), and neuronal NOS (nNOS), that exist in the peripheral organs and nervous systems of humans and rodents. Moreover, NOS includes other identified NOS isoforms, such as retinal Muller glial cells (mNOS), mitochondrial (mtNOS), penile (PnNOS), testis-specific (TnNOS), and invertebrate Drosophila NOS (dNOS), which are the lesser-known types. It is proposed that the versatile functions of NOS isoforms depend on various NOS splice variant subtypes and their expression in the neural (e.g., brain, and spinal cord) and non-neuronal tissues (e.g., lung, kidney, liver, and GI tract). Therefore, this review summarizes the NOS subtypes, splice variants, targeted splicing expression in the body, and their proposed physio-pathological functions. At last, alternative NOS subtypes and isoforms, which have previously received scant attention, will be addressed in this article.
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Óxido Nítrico Sintasa de Tipo III , Óxido Nítrico Sintasa , Humanos , Masculino , Óxido Nítrico Sintasa de Tipo II/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico/metabolismo , Isoformas de Proteínas/metabolismo , Encéfalo/metabolismoRESUMEN
Fish have colonized nearly all aquatic niches, making them an invaluable resource to understand vertebrate adaptation and gene family evolution, including the evolution of complex neural networks and modulatory neurotransmitter pathways. Among ancient regulatory molecules, the gaseous messenger nitric oxide (NO) is involved in a wide range of biological processes. Because of its short half-life, the modulatory capability of NO is strictly related to the local activity of nitric oxide synthases (Nos), enzymes that synthesize NO from L-arginine, making the localization of Nos mRNAs a reliable indirect proxy for the location of NO action domains, targets, and effectors. Within the diversified actinopterygian nos paralogs, nos1 (alias nnos) is ubiquitously present as a single copy gene across the gnathostome lineage, making it an ideal candidate for comparative studies. To investigate variations in the NO system across ray-finned fish phylogeny, we compared nos1 expression patterns during the development of two well-established experimental teleosts (zebrafish and medaka) with an early branching holostean (spotted gar), an important evolutionary bridge between teleosts and tetrapods. Data reported here highlight both conserved expression domains and species-specific nos1 territories, confirming the ancestry of this signaling system and expanding the number of biological processes implicated in NO activities.
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Evolución Molecular , Pez Cebra , Animales , Sistema Nervioso , Óxido Nítrico , FilogeniaRESUMEN
Neuroanatomic and functional studies show the paraventricular (PVN) of the hypothalamus to have a central role in the autonomic control that supports cardiovascular regulation. Direct and indirect projections from the PVN preautonomic neurons to the sympathetic preganglionic neurons in the spinal cord modulate sympathetic activity. The preautonomic neurons of the PVN adjust their level of activation in response to afferent signals arising from peripheral viscerosensory receptors relayed through the nucleus tractus solitarius. The prevailing sympathetic tone is a balance between excitatory and inhibitory influences that arises from the preautonomic PVN neurons. Under physiologic conditions, tonic sympathetic inhibition driven by a nitric oxide-γ-aminobutyric acid-mediated mechanism is dominant, but in pathologic situation such as heart failure there is a switch from inhibition to sympathoexcitation driven by glutamate and angiotensin II. Angiotensin II, reactive oxygen species, and hypoxia as a result of myocardial infarction/ischemia alter the tightly regulated posttranslational protein-protein interaction of CAPON (carboxy-terminal postsynaptic density protein ligand of neuronal nitric oxide synthase (NOS1)) and PIN (protein inhibitor of NOS1) signaling mechanism. Within the preautonomic neurons of the PVN, the disruption of CAPON and PIN signaling leads to a downregulation of NOS1 expression and reduced NO bioavailability. These data support the notion that CAPON-PIN dysregulation of NO bioavailability is a major contributor to the pathogenesis of sympathoexcitation in heart failure.
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Hipotálamo , Núcleo Hipotalámico Paraventricular , Ácido Glutámico , Humanos , Neuronas , Sistema Nervioso SimpáticoRESUMEN
NO (nitric oxide) is an important regulator of neutrophil functions and has a key role in diverse pathophysiological conditions. NO production by nitric oxide synthases (NOS) is under tight control at transcriptional, translational, and post-translational levels including interactions with heterologous proteins owing to its potent chemical reactivity and high diffusibility; this limits toxicity to other cellular components and promotes signaling specificity. The protein-protein interactions govern the activity and spatial distribution of NOS isoform to regulatory proteins and to their intended targets. In comparison with the vast literature available for endothelial, macrophages, and neuronal cells, demonstrating neuronal NOS (nNOS) interaction with other proteins through the PDZ domain, neutrophil nNOS, however, remains unexplored. Neutrophil's key role in both physiological and pathological conditions necessitates the need for further studies in delineating the NOS mediated NO modulations in signaling pathways operational in them. nNOS has been linked to depression, schizophrenia, and Parkinson's disease, suggesting the importance of exploring nNOS/NO-mediated neutrophil physiology in relation to such neuronal disorders. The review thus presents the scenario of neutrophil nNOS from the genetics to the functional level, including protein-protein interactions governing its intracellular sequestration in diverse cell types, besides speculating possible regulation in neutrophils and also addressing their clinical implications.
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Neutrófilos , Óxido Nítrico Sintasa de Tipo I/metabolismo , Humanos , Neuronas , Óxido NítricoRESUMEN
Epilepsy is identified as a brain disorder and characterized by unpredictable disruption of normal brain function. Due to adverse side effect associated with antiepileptic drugs and also resistance profile, improvement of antiepileptic medications with more beneficial anticonvulsant activity is essential. Natural products have demonstrated their therapeutic properties such as anxiolytic, antidepressant and anticonvulsant activities and a source for identification of novel lead compounds. Therefore, the purpose of this study was to evaluate the effects of Onopordon acanthium secondary metabolite, onopordia, on pentylenetetrazole (PTZ)-induced seizure in male mice and investigate the possible role of nitric oxide pathway. Different doses of onopordia (0.1, 1 and 10 mg/kg) and phenobarbital (20 mg/kg) were administered intraperitoneally (i.p., 30, 60 and 120 min) prior to induction of epileptic seizure and compared to control groups. Onopordia demonstrated anticonvulsant effects when administrated at dose of 10 mg/kg, i.p. and optimum time 60 min prior to induction of seizure. Anticonvulsant effect of onopordia was blocked by applying a single dose of a non-selective nitric oxide synthase (NOS) inhibitor, Nω-nitro-l-arginine methyl ester hydrochloride (l-NAME; 10 mg/kg, i.p.), and also a single dose of a selective neuronal NOS (nNOS) inhibitor, 7-nitroindazole (7-NI; 30 mg/kg, i.p.). Administration of ketamine as a N-Methyl-d-aspartic acid (NMDA) receptor antagonist (0.5 mg/kg; i.p.) with onopordia did not change the anticonvulsant effect of onopordia. The results of the present study demonstrated the anticonvulsant effect of onopordia as a new lead compound and also contribution of NO/nNOS pathway on PTZ-induced seizure in mice.
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Transient congenital hypothyroidism (TCH) has long-lasting consequences on the cardiovascular system during adulthood. The aim of this study was to determine whether nitric oxide (NO) and NO-producing enzymes are involved in impaired cardiac function as well as decreased tolerance to ischemia-reperfusion (IR) injury in adult male rats with TCH. Pregnant rats were divided into control and hypothyroid groups. Male offspring rats were categorized in control and hypothyroid (TCH) groups at week 16. Levels of NOx (nitrate+nitrite) and neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS) were measured in hearts of rats and isolated perfused hearts from both groups were subjected to IR. Levels of NOx and NOSs were also measured in both groups after ischemia. Compared with controls, heart NOx levels were higher at baseline (48.0 ± 4.9 vs. 35.0 ± 2.6 µmol/L; P = 0.034) and following IR (103.6 ± 4.2 vs. 70.2 ± 2.7 µmol/L; P < 0.001) in rat with TCH. At baseline, compared with controls, heart iNOS and nNOS levels were significantly higher in rats with TCH (6.12 ± 0.34 vs. 4.78 ± 0.27 ng/mg protein; P = 0.008 for iNOS and 4.87 ± 0.28 vs. 3.55 ± 0.23 ng/mg protein; P = 0.003 for nNOS). Following IR, in rats with TCH, heart iNOS levels increased (11.75 ± 2.02 vs. 6.12 ± 0.34, ng/mg protein; P = 0.015) whereas nNOS level decreased (4.10 ± 0.25 vs. 4.87 ± 0.28 ng/mg protein; P = 0.063). Adverse effects of TCH on cardiac function are associated with increased ratio of iNOS/eNOS; in addition, increased heart nNOS levels are involved in impaired cardiac function while its decrease is associated with decreased tolerance to IR injury.
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Hipotiroidismo Congénito/fisiopatología , Corazón/fisiopatología , Daño por Reperfusión Miocárdica/fisiopatología , Óxido Nítrico Sintasa/fisiología , Animales , Femenino , Hemodinámica , Masculino , Óxido Nítrico Sintasa/análisis , Ratas , Ratas Wistar , Hormonas Tiroideas/sangreRESUMEN
Structural changes affecting cardiomyocyte function may contribute to the pathophysiological remodeling underlying cardiac function impairment. Recent reports have shown that endogenous nitric oxide (NO) plays an important role in this process. In order to examine the role of NO in cardiomyocyte remodeling, male rats were acclimated to room temperature (22 ± 1 °C) or cold (4 ± 1 °C) and treated with 2.25% l-arginine·HCl or 0.01% l-NAME (Nω-nitro-l-arginine methyl ester)·HCl for 45 days. Untreated groups served as controls. Right heart ventricles were routinely prepared for light microscopic examination. Stereological estimations of volume densities of cardiomyocytes, surrounding blood vessels and connective tissue, as well as the morphometric measurements of cardiomyocyte diameters were performed. Tissue sections were also analyzed for structural alterations. We observed that both l-arginine and l-NAME supplementation induced cardiomyocyte hypertrophy, regardless of ambient temperature. However, cardiomyocyte hypertrophy was associated with fibrosis and extra collagen deposition only in the l-NAME treated group. Taken together, our results suggest that NO has a modulatory role in right heart ventricle remodeling by coordinating hypertrophy of cardiomyocytes and fibrous tissue preventing cardiac fibrosis.
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BACKGROUND/PURPOSE: During the early phase of sepsis, hypotension is accompanied by increase of plasma vasopressin hormone (AVP) levels, which decline during the late phase. This hypotension is due in part to increase of nitric oxide (NO) synthesis by nitric oxide synthase (NOS) enzyme. Neuronal isoform of this enzyme (nNOS) is present in vasopressinergics neurons of hypothalamus, but its role in vasopressin secretion during sepsis is unknown. METHODS: We evaluated the role of nNOS in NO production and vasopressin secretion during sepsis. Wistar rats received 7-nitroindazole (50 mg/kg, i.p.), an inhibitor of nNOS activity, or vehicle and were submitted to septic stimulus by cecal ligation and puncture (CLP). At the time points 0, 4, 6, 18 and 24 h after sepsis induction the animals were decapitated and neurohypophysis and hypothalamus were removed for analysis of vasopressin content and NOS activity, respectively. Hematocrit, serum sodium, osmolality, proteins and plasmatic AVP were quantified. RESULTS: Mortality was not affected by 7-nitroindazole (7-NI). Sodium and plasma proteins levels decreased after CLP and the treatment anticipated the protein loss, and delayed serum sodium decrease. Septic animals treated with 7-NI showed decrease of osmolality 4 h after CLP. Nitric oxide synthase activity in hypothalamus increased at 4 and 24 h after CLP and was reduced with 7-NI. Neurohypophysis content of AVP diminished after CLP and 7-NI did not alter this parameter. Plasma AVP levels increased at 6 h and decreased 18 and 24 h after CLP. Treatment with 7-NI did not alter plasma vasopressin levels. CONCLUSION: We concluded that nNOS does not have a substantial role in vasopressin secretion during experimental sepsis.
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Arginina Vasopresina/metabolismo , Óxido Nítrico Sintasa de Tipo I/metabolismo , Sepsis/metabolismo , Vasopresinas/metabolismo , Animales , Masculino , Óxido Nítrico/metabolismo , Radioinmunoensayo , Ratas , Ratas WistarRESUMEN
The nitric oxide synthases (NOS) catalyze a two-step oxidation of l-arginine (Arg) to generate NO. In the first step, O2 activation involves one electron being provided to the heme by an enzyme-bound 6R-tetrahydro-l-biopterin cofactor (H4 B), and the H4 B radical must be reduced back to H4 B in order for NOS to continue catalysis. Although an NADPH-derived electron is used to reduce the H4 B radical, how this occurs is unknown. We hypothesized that the NOS flavoprotein domain might reduce the H4 B radical by utilizing the NOS heme porphyrin as a conduit to deliver the electron. This model predicts that factors influencing NOS heme reduction should also influence the extent and rate of H4 B radical reduction in kind. To test this, we utilized single catalytic turnover and stop-freeze methods, along with electron paramagnetic resonance spectroscopy, to measure the rate and extent of reduction of the 5-methyl-H4 B radical formed in neuronal NOS (nNOS) during Arg hydroxylation. We used several nNOS variants that supported either a slower or faster than normal rate of ferric heme reduction. We found that the rates and extents of nNOS heme reduction correlated well with the rates and extents of 5-methyl-H4 B radical reduction among the various nNOS enzymes. This supports a model where the heme porphyrin transfers an electron from the NOS flavoprotein to the H4 B radical formed during catalysis, revealing that the heme plays a dual role in catalyzing O2 activation or electron transfer at distinct points in the reaction cycle.
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Biopterinas/análogos & derivados , Electrones , Hemo/metabolismo , Óxido Nítrico Sintasa de Tipo I/metabolismo , Animales , Arginina/química , Arginina/metabolismo , Biocatálisis , Biopterinas/química , Biopterinas/metabolismo , Espectroscopía de Resonancia por Spin del Electrón , Transporte de Electrón , Radicales Libres/química , Radicales Libres/metabolismo , Hemo/química , Cinética , Modelos Moleculares , Estructura Molecular , Mutación , Óxido Nítrico Sintasa de Tipo I/química , Óxido Nítrico Sintasa de Tipo I/genética , Oxidación-Reducción , Unión Proteica , Dominios Proteicos , RatasRESUMEN
The development of multiple sclerosis, a major neurodegenerative disease, is due to both genetic and environmental factors that might trigger aberrant epigenetic changes of the genome. In this study, we analysed global DNA methylation in the brain of mice upon induction of experimental autoimmune encephalomyelitis (EAE), and the effect of environmental enrichment (EE). We demonstrate that global DNA methylation decreased in the striatum, but not in the cortex, of EAE mice compared to healthy controls, in particular in neuronal nitric oxide synthase (nNOS)-positive interneurons of this brain area. Also, in the striatum but again not in the cortex, decreased DNA methylation of the nNOS downstream effector, dexamethasone-induced Ras protein 1 (Dexras 1), was observed in EAE mice, and was paralleled by an increase in its mRNA. Interestingly, EE was able to revert EAE effects on mRNA expression and DNA methylation levels of Dexras 1 and reduced gene expression of nNOS and 5-lipoxygenase (Alox5). Conversely, interleukin-1ß (IL-1ß) gene expression was found up-regulated in EAE mice compared to controls and was not affected by EE. Taken together, these data demonstrate an unprecedented epigenetic modulation of nNOS-signaling in the pathogenesis of multiple sclerosis, and show that EE can specifically revert EAE effects on Dexras 1 along this pathway.
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Encéfalo/metabolismo , Encefalomielitis Autoinmune Experimental/patología , Epigénesis Genética/fisiología , Óxido Nítrico Sintasa de Tipo I/metabolismo , Transducción de Señal/fisiología , Proteínas ras/metabolismo , 5-Metilcitosina/metabolismo , Animales , Antiinflamatorios/farmacología , Araquidonato 5-Lipooxigenasa/metabolismo , Encéfalo/efectos de los fármacos , Encéfalo/patología , Citocinas/genética , Citocinas/metabolismo , Dexametasona/farmacología , Modelos Animales de Enfermedad , Fosfoproteína 32 Regulada por Dopamina y AMPc/metabolismo , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/metabolismo , Epigénesis Genética/efectos de los fármacos , Femenino , Ratones , Ratones Endogámicos C57BL , Glicoproteína Mielina-Oligodendrócito/inmunología , Neuronas/metabolismo , Fragmentos de Péptidos/inmunología , Transducción de Señal/efectos de los fármacos , Proteínas ras/genéticaRESUMEN
Testosterone (TES) and other androgens exert a direct vasorelaxing action on the vasculature in vitro that is structurally specific and independent of cytosolic androgen receptor (AR). The effects of intravenous androgen infusions on mean arterial blood pressure (BP) and heart rate (HR) were determined in conscious, unrestrained, chronically catheterized, ganglionically blocked (hexamethonium, HEX; 30 mg/kg ip) male Sprague-Dawley (SD) and testicular-feminized male (Tfm; AR-deficient) rats, 16-20 wk of age. BP and HR were recorded at baseline and with increasing doses of androgens (0.375-6.00 µmol·kg(-1)·min(-1) iv; 10 min/dose). Data are expressed as means ± SE (n = 5-8 rats/group). In SD rats, baseline BP and HR averaged 103 ± 4 mmHg and 353 ± 12 beats/min (bpm). TES produced a dose-dependent reduction in BP to a low of 87 ± 4 mmHg (Δ16%), while HR was unchanged (354 ± 14 bpm). Neither BP (109 ± 3 mmHg) nor HR (395 ± 13 bpm) were altered by vehicle (10% EtOH in 0.9% saline; 0.15 ml·kg(-1)·min(-1), iv). In Tfm, TES produced a similar reduction in BP (99 ± 3 to 86 ± 3 mmHg, Δ13%); HR was unchanged (369 ± 18 bpm). In SD, 5ß-dihydrotestosterone (genomically inactive metabolite) produced a greater reduction in BP than TES (102 ± 2 to 79 ± 2 mmHg, Δ23%); HR was unchanged (361 ± 9). A 20-µg iv bolus of sodium nitroprusside in both SD and Tfm rats reduced BP 30-40 mmHg, while HR was unchanged, confirming blockade by HEX. Pretreatment of SD rats with neuronal nitric oxide synthase (nNOS) inhibitor (S-methyl-thiocitrulline, SMTC; 20 µg·kg(-1)·min(-1) × 30 min) abolished the hypotensive effects of TES infusion on BP (104 ± 2 vs. 101 ± 2 mmHg) and HR (326 ± 11 vs. 324 ± 8 bpm). These data suggest the systemic hypotensive effect of TES and other androgens involves a direct vasodilatory action on the peripheral vasculature which, like the effect observed in isolated arteries, is structurally specific and AR-independent, and involves activation of nNOS.
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Andrógenos/administración & dosificación , Presión Arterial/efectos de los fármacos , Arterias/efectos de los fármacos , Hipotensión/inducido químicamente , Óxido Nítrico Sintasa de Tipo I/metabolismo , Testosterona/administración & dosificación , Síndrome de Resistencia Androgénica/genética , Síndrome de Resistencia Androgénica/metabolismo , Síndrome de Resistencia Androgénica/fisiopatología , Andrógenos/química , Animales , Arterias/enzimología , Arterias/fisiopatología , Dihidrotestosterona/administración & dosificación , Relación Dosis-Respuesta a Droga , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Frecuencia Cardíaca/efectos de los fármacos , Hipotensión/enzimología , Hipotensión/fisiopatología , Infusiones Intravenosas , Masculino , Estructura Molecular , Óxido Nítrico Sintasa de Tipo I/antagonistas & inhibidores , Ratas , Ratas Sprague-Dawley , Receptores Androgénicos/genética , Receptores Androgénicos/metabolismo , Relación Estructura-Actividad , Testosterona/análogos & derivados , Testosterona/química , Factores de Tiempo , Vasodilatación/efectos de los fármacosRESUMEN
Neuronal nitric oxide synthase (nNOS) catalyzes production of nitric oxide in vertebrate brains. Recent findings indicate that endothelial NOS and reactive oxygen species (ROS) are significantly increased during hypoxic stress and are modulated by antioxidants. However, the influence of antioxidants and steroids on nNOS upregulation by hypoxia is largely unknown. In this study, we characterized nNOS cDNA and examined the effects of hypoxia and antioxidant and steroid treatments on nNOS expression in Atlantic croaker hypothalamus. Hypoxia exposure (dissolved oxygen, DO: 1.7 mg/L for 2 and/or 4weeks) caused significant increases in hypothalamic nNOS mRNA, protein and its neuronal expression. Hypothalamic nNOS expression and superoxide radical (O2(·-), an index of ROS) production were increased by pharmacological treatment of fish exposed to normoxic conditions with N-ethylmaleimide, an alkene drug which covalently modifies sulfhydryl groups and inhibits aromatase activity. In contrast, treatments with Nω-nitro-L-arginine methyl ester, a competitive NOS-inhibitor, or vitamin E, an antioxidant, prevented the upregulation of O2(·-) production and nNOS expression in hypoxia-exposed (DO: 1.7 mg/L for 4 weeks) fish. Moreover, treatment with 1,4,6-androstatrien-3,17-dione, an aromatase inhibitor, increased hypothalamic O2(·-) production and nNOS expression in normoxic control fish; whereas estradiol-17ß treatment significantly reduced O2(·-) production and nNOS expression in hypoxia-exposed fish. Double-labeled immunohistochemical results showed that nNOS and aromatase proteins are co-expressed in the hypothalamus. Taken together, the results suggest that upregulation of nNOS and ROS in the croaker hypothalamus in response to hypoxia is influenced by antioxidant and overall estrogen status.
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Antioxidantes/farmacología , Proteínas de Peces/metabolismo , Hipotálamo/enzimología , Óxido Nítrico Sintasa de Tipo I/metabolismo , Perciformes/metabolismo , Secuencia de Aminoácidos , Animales , Hipoxia de la Célula , Secuencia Conservada , Inducción Enzimática/efectos de los fármacos , Estradiol/farmacología , Estrógenos/fisiología , Femenino , Proteínas de Peces/genética , Expresión Génica , Hipotálamo/efectos de los fármacos , Masculino , Datos de Secuencia Molecular , Óxido Nítrico Sintasa de Tipo I/genética , Especificidad de Órganos , Transporte de ProteínasRESUMEN
BACKGROUND: An investigation was made to explore the possibility of anxiolytic activity of piperine in unstressed and stressed mice along with the underlying role of nitriergic and GABAergic modulation for the noted activity of piperine. METHODS: Piperine (5, 10 and 20mg/kg, ip) was administered to unstressed mice. In another groups of animals, piperine was administered 30 min before subjecting them to immobilization stress for 6h. Antianxiety activity was evaluated by employing elevated plus maze, light-dark box and social interaction test. Diazepam was employed as standard anxiolytic drug. RESULTS: Piperine produced significant antianxiety-like activity in unstressed and stressed mice. The anxiolytic-like activity of piperine was comparable to diazepam. In unstressed mice, piperine significantly increased brain GABA levels, but could not produce any change in plasma nitrite levels. Meanwhile, in stressed mice, piperine did not produce any significant change in GABA levels, but significantly decreased nitrite levels. Pre-treatment with aminoguanidine (50mg/kg, ip), an inducible nitric oxide synthase (NOS) inhibitor, significantly potentiated the anxiolytic-like activity of piperine, as compared to piperine and aminoguanidine alone in stressed mice. On the other hand, pretreatment with 7-nitroindazole (20mg/kg, ip), a neuronal NOS inhibitor significantly potentiated the antianxiety-like activity of piperine, as compared to piperine and 7-nitroindazole alone in unstressed mice. CONCLUSION: These data suggest that the piperine produced significant anxiolytic activity in unstressed mice possibly through increase in GABA levels and inhibition of neuronal NOS. On the other hand, antianxiety activity in stressed mice might be through inhibition of inducible NOS.
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Alcaloides/farmacología , Ansiolíticos/farmacología , Ansiedad/tratamiento farmacológico , Benzodioxoles/farmacología , Piperidinas/farmacología , Alcamidas Poliinsaturadas/farmacología , Estrés Psicológico/tratamiento farmacológico , Alcaloides/administración & dosificación , Animales , Ansiolíticos/administración & dosificación , Benzodioxoles/administración & dosificación , Diazepam/farmacología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Guanidinas/farmacología , Indazoles/farmacología , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Ratones , Óxido Nítrico Sintasa de Tipo I/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo I/metabolismo , Óxido Nítrico Sintasa de Tipo II/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo II/metabolismo , Nitritos/sangre , Piperidinas/administración & dosificación , Alcamidas Poliinsaturadas/administración & dosificación , Ácido gamma-Aminobutírico/efectos de los fármacos , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Thalidomide is an old glutamic acid derivative which was initially used as a sedative medication but withdrawn from the market due to the high incidence of teratogenicity. Recently, it has reemerged because of its potential for counteracting number of diseases, including neurodegenerative disorders. Other than the antiemetic and hypnotic aspects, thalidomide exerts some anticonvulsant properties in experimental settings. However, the underlying mechanisms of thalidomide actions are not fully realized yet. Some investigations revealed that thalidomide could elicit immunomodulatory or neuromodulatory properties by affecting different targets, including cytokines (such as TNF α), neurotransmitters, and nitric oxide (NO). In this regard, we used a model of clonic seizure induced by pentylenetetrazole (PTZ) in male NMRI mice to investigate whether the anticonvulsant effect of thalidomide is affected through modulation of the l-arginine-nitric oxide pathway or not. Injection of a single effective dose of thalidomide (10 mg/kg, i.p. or higher) significantly increased the seizure threshold (P<0.05). On the one hand, pretreatment with low and per se noneffective dose of l-arginine [NO precursor] (10, 30 and 60 mg/kg) prevented the anticonvulsant effect of thalidomide. On the other hand, NOS inhibitors [l-NAME and 7-NI] augmented the anticonvulsant effect of a subeffective dose of thalidomide (1 and 5 mg/kg, i.p.) at relatively low doses. Meanwhile, several doses of aminoguanidine [an inducible NOS inhibitor] (20, 50 and 100 mg/kg) failed to alter the anticonvulsant effect of thalidomide significantly. In summary, our findings demonstrated that the l-arginine-nitric oxide pathway can be involved in the anticonvulsant properties of thalidomide, and the role of constitutive nNOS is prominent in the reported neuroprotective feature.
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Anticonvulsivantes/uso terapéutico , Óxido Nítrico/metabolismo , Convulsiones/tratamiento farmacológico , Talidomida/uso terapéutico , Animales , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Masculino , Ratones , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Pentilenotetrazol , Convulsiones/inducido químicamente , Convulsiones/metabolismoRESUMEN
Behavioral sensitization to cocaine is associated with increased AMPA receptor (AMPAR) surface expression in the nucleus accumbens (NAc). This upregulation is withdrawal-dependent, as it is not detected on withdrawal day (WD) 1, but is observed on WD7-21. Its underlying mechanisms have not been clearly established. Nitric oxide (NO) regulates AMPAR trafficking in the brain by S-nitrosylation of the AMPAR auxiliary subunit, stargazin, leading to increased AMPAR surface expression. Our goal was to determine if stargazin S-nitrosylation contributes to AMPAR upregulation during sensitization. First, we measured stargazin S-nitrosylation in NAc core and shell subregions on WD14 after 8 daily injections of saline or 15 mg/kg cocaine. Stargazin S-nitrosylation was markedly increased in NAc shell but not core. To determine if this is associated with AMPAR upregulation, rats received 8 cocaine or saline injections followed by twice-daily treatments with vehicle or the nitric oxide synthase inhibitor l-NAME (50 mg/kg) on WD1-6, the time when AMPAR upregulation is developing in cocaine-exposed rats. Cocaine/vehicle rats showed elevated stargazin and GluA1 surface expression on WD7 compared to saline/vehicle rats; the GluA1 increase was more robust in core, while stargazin increased more robustly in shell. These effects of cocaine were attenuated in shell but not core when cocaine injections were followed by l-NAME treatment on WD1-6. Together, these results indicate that elevated S-nitrosylation of stargazin contributes to AMPAR upregulation during sensitization selectively in the NAc shell. It is possible that AMPAR upregulation in core involves a different TARP, γ4, which also upregulates in the NAc of sensitized rats.
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Canales de Calcio/metabolismo , Trastornos Relacionados con Cocaína/metabolismo , Cocaína/administración & dosificación , Núcleo Accumbens/metabolismo , Receptores AMPA/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Animales , Trastornos Relacionados con Cocaína/genética , Masculino , Actividad Motora/efectos de los fármacos , Núcleo Accumbens/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Receptores AMPA/genética , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/metabolismoRESUMEN
The muscle-specific variant of neuronal nitric oxide (NO) synthase (NOS-I), is developmentally regulated in mouse suggesting a role of NO during myogenesis. In chick embryo, a good model of development, we found that the expression of NOS-I is up-regulated, but only in the early phase of development. Through a pharmacological intervention in ovo we found that NO signalling plays a relevant role during embryonic development. The inhibition of NOS-I decreased the growth of embryo, in particular of muscle tissue, while the restoring of physiological NO levels, via administration of a NO donor, reversed this effect. We found a selective action of NO, produced by NOS-I, on regulatory factors involved in myogenic differentiation in the early phase of chick embryo development: inhibition of NO generation leads to a decreased expression of the Myocyte enhancer factor 2a (Mef2a), Mef2c, Myogenin and Myosin, which was reversed by the administration of a NO donor. NO had no effects on Myf5 and MyoD, the myogenic regulatory factors necessary for myogenic determination. The action of NO on the myogenic regulatory factors was mediated via generation of cyclic GMP (cGMP) and activation of the cGMP-dependent protein kinase G (PKG). Finally we found in myoblasts in vitro that the activation of Mef2c was the key event mediating the NO-induced modulation of myogenesis. Our results identify NO produced by NOS-I as a key messenger in the early phase of embryonic development of chicken, acting as a critical determinant of myogenesis through its physiological cGMP/PKG pathway.
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Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Desarrollo de Músculos/efectos de los fármacos , Desarrollo de Músculos/genética , Factores Reguladores Miogénicos/genética , Óxido Nítrico/farmacología , Animales , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Células Cultivadas , Embrión de Pollo , Pollos/genética , Pollos/metabolismo , Humanos , Ratones , Factores Reguladores Miogénicos/metabolismo , Óxido Nítrico Sintasa de Tipo I/genética , Óxido Nítrico Sintasa de Tipo I/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Neuronal nitric oxide synthase µ (nNOSµ) contains 34 additional residues in an autoregulatory element compared to nNOSα. Cytochrome c and flavin reductions in the absence of calmodulin (CaM) were faster in nNOSµ than nNOSα, while rates in the presence of CaM were smaller. The magnitude of stimulation by CaM is thus notably lower in nNOSµ. No difference in NO production was observed, while electron transfer between the FMN and heme moieties and formation of an inhibitory ferrous-nitrosyl complex were slower in nNOSµ. Thus, the insert affects electron transfer rates, modulation of electron flow by CaM, and heme-nitrosyl complex formation.