RESUMO
During development, neurons switch among growth states, such as initial axon outgrowth, axon pruning, and regrowth. By studying the stereotypic remodeling of the Drosophila mushroom body (MB), we found that the heme-binding nuclear receptor E75 is dispensable for initial axon outgrowth of MB γ neurons but is required for their developmental regrowth. Genetic experiments and pharmacological manipulations on ex-vivo-cultured brains indicate that neuronally generated nitric oxide (NO) promotes pruning but inhibits regrowth. We found that high NO levels inhibit the physical interaction between the E75 and UNF nuclear receptors, likely accounting for its repression of regrowth. Additionally, NO synthase (NOS) activity is downregulated at the onset of regrowth, at least partially, by short inhibitory NOS isoforms encoded within the NOS locus, indicating how NO production could be developmentally regulated. Taken together, these results suggest that NO signaling provides a switching mechanism between the degenerative and regenerative states of neuronal remodeling.
Assuntos
Axônios/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Plasticidade Neuronal , Óxido Nítrico/metabolismo , Fatores de Transcrição/metabolismo , Animais , Encéfalo/metabolismo , Drosophila melanogaster/enzimologia , Corpos Pedunculados , Neurônios/metabolismo , Óxido Nítrico Sintase/metabolismo , Isoformas de Proteínas/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismoRESUMO
Maintenance of renal function and fluid transport are essential for vertebrates and invertebrates to adapt to physiological and pathological challenges. Human patients with malignant tumours frequently develop detrimental renal dysfunction and oliguria, and previous studies suggest the involvement of chemotherapeutic toxicity and tumour-associated inflammation1,2. However, how tumours might directly modulate renal functions remains largely unclear. Here, using conserved tumour models in Drosophila melanogaster3, we characterized isoform F of ion transport peptide (ITPF) as a fly antidiuretic hormone that is secreted by a subset of yki3SA gut tumour cells, impairs renal function and causes severe abdomen bloating and fluid accumulation. Mechanistically, tumour-derived ITPF targets the G-protein-coupled receptor TkR99D in stellate cells of Malpighian tubules-an excretory organ that is equivalent to renal tubules4-to activate nitric oxide synthase-cGMP signalling and inhibit fluid excretion. We further uncovered antidiuretic functions of mammalian neurokinin 3 receptor (NK3R), the homologue of fly TkR99D, as pharmaceutical blockade of NK3R efficiently alleviates renal tubular dysfunction in mice bearing different malignant tumours. Together, our results demonstrate a novel antidiuretic pathway mediating tumour-renal crosstalk across species and offer therapeutic opportunities for the treatment of cancer-associated renal dysfunction.
Assuntos
Antidiuréticos , Nefropatias , Neoplasias , Neuropeptídeos , Receptores da Neurocinina-3 , Animais , Humanos , Camundongos , Antidiuréticos/metabolismo , GMP Cíclico/metabolismo , Modelos Animais de Doenças , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Nefropatias/complicações , Nefropatias/tratamento farmacológico , Nefropatias/metabolismo , Túbulos de Malpighi/citologia , Túbulos de Malpighi/metabolismo , Neoplasias/complicações , Neoplasias/metabolismo , Óxido Nítrico Sintase/metabolismo , Receptores da Neurocinina-3/antagonistas & inibidores , Receptores da Neurocinina-3/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Arginina Vasopressina/metabolismo , Proteínas de Drosophila/metabolismo , Neuropeptídeos/metabolismoRESUMO
Type 1 CD8α+ conventional dendritic cells (cDC1s) are required for CD8+ T cell priming but, paradoxically, promote splenic Listeria monocytogenes infection. Using mice with impaired cDC2 function, we ruled out a role for cDC2s in this process and instead discovered an interleukin-10 (IL-10)-dependent cellular crosstalk in the marginal zone (MZ) that promoted bacterial infection. Mice lacking the guanine nucleotide exchange factor DOCK8 or CD19 lost IL-10-producing MZ B cells and were resistant to Listeria. IL-10 increased intracellular Listeria in cDC1s indirectly by reducing inducible nitric oxide synthase expression early after infection and increasing intracellular Listeria in MZ metallophilic macrophages (MMMs). These MMMs trans-infected cDC1s, which, in turn, transported Listeria into the white pulp to prime CD8+ T cells. However, this also facilitated bacterial expansion. Therefore, IL-10-mediated crosstalk between B cells, macrophages, and cDC1s in the MZ promotes both Listeria infection and CD8+ T cell activation.
Assuntos
Linfócitos B/imunologia , Células Dendríticas/imunologia , Interleucina-10/metabolismo , Listeria monocytogenes/fisiologia , Listeriose/imunologia , Macrófagos/imunologia , Baço/imunologia , Animais , Antígenos CD19/metabolismo , Antígenos CD8/metabolismo , Linhagem Celular Tumoral , Regulação da Expressão Gênica , Fatores de Troca do Nucleotídeo Guanina/genética , Interleucina-10/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase/metabolismo , Comunicação Parácrina , Baço/microbiologiaRESUMO
GTP cyclohydrolase I (GTPCH), 6-pyruvoyltetrahydropterin synthase (PTPS), and sepiapterin reductase (SR) are sequentially responsible for de novo synthesis of tetrahydrobiopterin (BH4), a known co-factor for nitric oxide synthase (NOS). The implication of BH4-biosynthesis process in tumorigenesis remains to be investigated. Here, we show that PTPS, which is highly expressed in early-stage colorectal cancer, is phosphorylated at Thr 58 by AMPK under hypoxia; this phosphorylation promotes PTPS binding to LTBP1 and subsequently drives iNOS-mediated LTBP1 S-nitrosylation through proximal-coupling BH4 production within the PTPS/iNOS/LTBP1 complex. In turn, LTBP1 S-nitrosylation results in proteasome-dependent LTBP1 protein degradation, revealing an inverse relationship between PTPS pT58 and LTBP1 stability. Physiologically, the repressive effect of PTPS on LTBP1 leads to impaired transforming growth factor ß (TGF-ß) secretion and thereby maintains tumor cell growth under hypoxia. Our findings illustrate a molecular mechanism underlying the regulation of LTBP1-TGF-ß signaling by the BH4-biosynthesis pathway and highlight the specific requirement of PTPS for tumor growth.
Assuntos
Proliferação de Células/fisiologia , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Hipóxia/metabolismo , Proteínas de Ligação a TGF-beta Latente/metabolismo , Fósforo-Oxigênio Liases/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Células HCT116 , Células HEK293 , Células HT29 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Óxido Nítrico Sintase/metabolismo , Fosforilação/fisiologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta/metabolismoRESUMO
Nitric oxide (NO) from endothelial NO synthase importantly contributes to vascular homeostasis. Reduced NO production or increased scavenging during disease conditions with oxidative stress contribute to endothelial dysfunction and NO deficiency. In addition to the classical enzymatic NO synthases (NOS) system, NO can also be generated via the nitrate-nitrite-NO pathway. Dietary and pharmacological approaches aimed at increasing NO bioactivity, especially in the cardiovascular system, have been the focus of much research since the discovery of this small gaseous signaling molecule. Despite wide appreciation of the biological role of NOS/NO signaling, questions still remain about the chemical nature of NOS-derived bioactivity. Recent studies show that NO-like bioactivity can be efficiently transduced by mobile NO-ferroheme species, which can transfer between proteins, partition into a hydrophobic phase, and directly activate the soluble guanylyl cyclase-cGMP-protein kinase G pathway without intermediacy of free NO. Moreover, interaction between red blood cells and the endothelium in the regulation of vascular NO homeostasis have gained much attention, especially in conditions with cardiometabolic disease. In this review we discuss both classical and nonclassical pathways for NO generation in the cardiovascular system and how these can be modulated for therapeutic purposes. SIGNIFICANCE STATEMENT: After four decades of intensive research, questions persist about the transduction and control of nitric oxide (NO) synthase bioactivity. Here we discuss NO signaling in cardiovascular health and disease, highlighting new findings, such as the important role of red blood cells in cardiovascular NO homeostasis. Nonclassical signaling modes, like the nitrate-nitrite-NO pathway, and therapeutic opportunities related to the NO system are discussed. Existing and potential pharmacological treatments/strategies, as well as dietary components influencing NO generation and signaling are covered.
Assuntos
Sistema Cardiovascular , Óxido Nítrico , Transdução de Sinais , Humanos , Óxido Nítrico/metabolismo , Animais , Sistema Cardiovascular/metabolismo , Doenças Cardiovasculares/metabolismo , Doenças Cardiovasculares/tratamento farmacológico , Doenças Cardiovasculares/fisiopatologia , Óxido Nítrico Sintase/metabolismoRESUMO
Nitric oxide synthases (NOSs) are multidomain metalloproteins first identified in mammals as being responsible for the synthesis of the wide-spread signaling and protective agent nitric oxide (NO). Over the past 10 years, prokaryotic proteins that are homologous to animal NOSs have been identified and characterized, both in terms of enzymology and biological function. Despite some interesting differences in cofactor utilization and redox partners, the bacterial enzymes are in many ways similar to their mammalian NOS (mNOS) counterparts and, as such, have provided insight into the structural and catalytic properties of the NOS family. In particular, spectroscopic studies of thermostable bacterial NOSs have revealed key oxyheme intermediates involved in the oxidation of substrate L-arginine (Arg) to product NO. The biological functions of some bacterial NOSs have only more recently come to light. These studies disclose new roles for NO in biology, such as taking part in toxin biosynthesis, protection against oxidative stress, and regulation of recovery from radiation damage.
Assuntos
Bactérias/metabolismo , Óxido Nítrico Sintase/química , Óxido Nítrico Sintase/metabolismo , Animais , Arginina/metabolismo , Bactérias/enzimologia , Óxidos de Nitrogênio/metabolismoRESUMO
Binge drinking (BD) is an especially pro-oxidant pattern of alcohol consumption, particularly widespread in the adolescent population. In the kidneys, it affects the glomerular filtration rate (GFR), leading to high blood pressure. BD exposure also disrupts folic acid (FA) homeostasis and its antioxidant properties. The aim of this study is to test a FA supplementation as an effective therapy against the oxidative, nitrosative, and apoptotic damage as well as the renal function alteration occurred after BD in adolescence. Four groups of adolescent rats were used: control, BD (exposed to intraperitoneal alcohol), control FA-supplemented group and BD FA-supplemented group. Dietary FA content in control groups was 2 ppm, and 8 ppm in supplemented groups. BD provoked an oxidative imbalance in the kidneys by dysregulating antioxidant enzymes and increasing the enzyme NADPH oxidase 4 (NOX4), which led to an increase in caspase-9. BD also altered the renal nitrosative status affecting the expression of the three nitric oxide (NO) synthase (NOS) isoforms, leading to a decrease in NO levels. Functionally, BD produced a hydric-electrolytic imbalance, a low GFR and an increase in blood pressure. FA supplementation to BD adolescent rats improved the oxidative, nitrosative, and apoptotic balance, recovering the hydric-electrolytic equilibrium and blood pressure. However, neither NO levels nor GFR were recovered, showing in this study for the first time that NO availability in the kidneys plays a crucial role in GFR regulation that the antioxidant effects of FA cannot repair.
Assuntos
Antioxidantes , Consumo Excessivo de Bebidas Alcoólicas , Ratos , Animais , Antioxidantes/farmacologia , Antioxidantes/metabolismo , Óxido Nítrico/metabolismo , Pressão Sanguínea , Consumo Excessivo de Bebidas Alcoólicas/metabolismo , Taxa de Filtração Glomerular , Rim/metabolismo , Suplementos Nutricionais , Etanol/farmacologia , Ácido Fólico/farmacologia , Ácido Fólico/metabolismo , Óxido Nítrico Sintase/metabolismo , Estresse OxidativoRESUMO
The exchange protein directly activated by cAMP (EPAC) has been implicated in cardiac proarrhythmic signaling pathways including spontaneous diastolic Ca2+ leak from sarcoplasmic reticulum and increased action potential duration (APD) in isolated ventricular cardiomyocytes. The action potential (AP) lengthening following acute EPAC activation is mainly due to a decrease of repolarizing steady-state K+ current (IKSS) but the mechanisms involved remain unknown. This study aimed to assess the role of EPAC1 and EPAC2 in the decrease of IKSS and to investigate the underlying signaling pathways. AP and K+ currents were recorded with the whole cell configuration of the patch-clamp technique in freshly isolated rat ventricular myocytes. EPAC1 and EPAC2 were pharmacologically activated with 8-(4-chlorophenylthio)-2'-O-methyl-cAMP acetoxymethyl ester (8-CPTAM, 10 µmol/L) and inhibited with R-Ce3F4 and ESI-05, respectively. Inhibition of EPAC1 and EPAC2 significantly decreased the effect of 8-CPTAM on APD and IKSS showing that both EPAC isoforms are involved in these effects. Unexpectedly, calmodulin-dependent protein kinase II (CaMKII) inhibition by AIP or KN-93, and Ca2+ chelation by intracellular BAPTA, did not impact the response to 8-CPTAM. However, inhibition of PLC/PKC and nitric oxide synthase (NOS)/PKG pathways partially prevents the 8-CPTAM-dependent decrease of IKSS. Finally, the cumulative inhibition of PKC and PKG blocked the 8-CPTAM effect, suggesting that these two actors work along parallel pathways to regulate IKSS upon EPAC activation. On the basis of such findings, we propose that EPAC1 and EPAC2 are involved in APD lengthening by inhibiting a K+ current via both PLC/PKC and NOS/PKG pathways. This may have pathological implications since EPAC is upregulated in diseases such as cardiac hypertrophy.NEW & NOTEWORHTY Exchange protein directly activated by cAMP (EPAC) proteins modulate ventricular electrophysiology at the cellular level. Both EPAC1 and EPAC2 isoforms participate in this effect. Mechanistically, PLC/PKC and nitric oxide synthase (NO)/PKG pathways are involved in regulating K+ repolarizing current whereas the well-known downstream effector of EPAC, calmodulin-dependent protein kinase II (CaMKII), does not participate. This may have pathological implications since EPAC is upregulated in diseases such as cardiac hypertrophy. Thus, EPAC inhibition may be a new approach to prevent arrhythmias under pathological conditions.
Assuntos
Potenciais de Ação , Fatores de Troca do Nucleotídeo Guanina , Ventrículos do Coração , Miócitos Cardíacos , Proteína Quinase C , Transdução de Sinais , Animais , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/enzimologia , Proteína Quinase C/metabolismo , Ratos , Potenciais de Ação/efeitos dos fármacos , Ventrículos do Coração/metabolismo , Ventrículos do Coração/citologia , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase/antagonistas & inibidores , Fosfolipases Tipo C/metabolismo , Fosfolipases Tipo C/antagonistas & inibidores , Masculino , Ratos Wistar , Potássio/metabolismo , AMP Cíclico/metabolismoRESUMO
The bacterial metabolic enzyme 1-deoxy-d-xylulose-5-phosphate synthase (DXPS) catalyzes the thiamin diphosphate (ThDP)-dependent formation of DXP from pyruvate and d-glyceraldehyde-3-phosphate (d-GAP). DXP is an essential bacteria-specific metabolite that feeds into the biosynthesis of isoprenoids, pyridoxal phosphate (PLP), and ThDP. DXPS catalyzes the activation of pyruvate to give the C2α-lactylThDP (LThDP) adduct that is long-lived on DXPS in a closed state in the absence of the cosubstrate. Binding of d-GAP shifts the DXPS-LThDP complex to an open state which coincides with LThDP decarboxylation. This gated mechanism distinguishes DXPS in ThDP enzymology. How LThDP persists on DXPS in the absence of cosubstrate, while other pyruvate decarboxylases readily activate LThDP for decarboxylation, is a long-standing question in the field. We propose that an active site network functions to prevent LThDP activation on DXPS until the cosubstrate binds. Binding of d-GAP coincides with a conformational shift and disrupts the network causing changes in the active site that promote LThDP activation. Here, we show that the substitution of putative network residues, as well as nearby residues believed to contribute to network charge distribution, predictably affects LThDP reactivity. Substitutions predicted to disrupt the network have the effect to activate LThDP for decarboxylation, resulting in CO2 and acetate production. In contrast, a substitution predicted to strengthen the network fails to activate LThDP and has the effect to shift DXPS toward the closed state. Network-disrupting substitutions near the carboxylate of LThDP also have a pronounced effect to shift DXPS to an open state. These results offer initial insights to explain the long-lived LThDP intermediate and its activation through disruption of an active site network, which is unique to DXPS. These findings have important implications for DXPS function in bacteria and its development as an antibacterial target.
Assuntos
Difosfatos , Tiamina Pirofosfato , Domínio Catalítico , Tiamina Pirofosfato/metabolismo , Transferases/metabolismo , Ácido Pirúvico , Bactérias/metabolismo , Óxido Nítrico Sintase/metabolismo , AntibacterianosRESUMO
In our efforts to develop inhibitors selective for neuronal nitric oxide synthase (nNOS) over endothelial nitric oxide synthase (eNOS), we found that nNOS can undergo conformational changes in response to inhibitor binding that does not readily occur in eNOS. One change involves movement of a conserved tyrosine, which hydrogen bonds to one of the heme propionates, but in the presence of an inhibitor, changes conformation, enabling part of the inhibitor to hydrogen bond with the heme propionate. This movement does not occur as readily in eNOS and may account for the reason why these inhibitors bind more tightly to nNOS. A second structural change occurs upon the binding of a second inhibitor molecule to nNOS, displacing the pterin cofactor. Binding of this second site inhibitor requires structural changes at the dimer interface, which also occurs more readily in nNOS than in eNOS. Here, we used a combination of crystallography, mutagenesis, and computational methods to better understand the structural basis for these differences in NOS inhibitor binding. Computational results show that a conserved tyrosine near the primary inhibitor binding site is anchored more tightly in eNOS than in nNOS, allowing for less flexibility of this residue. We also find that the inefficiency of eNOS to bind a second inhibitor molecule is likely due to the tighter dimer interface in eNOS compared with nNOS. This study provides a better understanding of how subtle structural differences in NOS isoforms can result in substantial dynamic differences that can be exploited in the development of isoform-selective inhibitors.
Assuntos
Óxido Nítrico Sintase Tipo III , Óxido Nítrico Sintase , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/química , Óxido Nítrico Sintase Tipo I , Isoformas de Proteínas/química , Cristalografia por Raios X , Inibidores Enzimáticos/farmacologia , Heme/química , Tirosina , Óxido NítricoRESUMO
Heat shock protein 90 (Hsp90) is known to mediate heme insertion and activation of heme-deficient neuronal nitric oxide (NO) synthase (apo-nNOS) in cells by a highly dynamic interaction that has been extremely difficult to study mechanistically with the use of subcellular systems. In that the heme content of many critical hemeproteins is regulated by Hsp90 and the heme chaperone GAPDH, the development of an in vitro system for the study of this chaperone-mediated heme regulation would be extremely useful. Here, we show that use of an antibody-immobilized apo-nNOS led not only to successful assembly of chaperone complexes but the ability to show a clear dependence on Hsp90 and GAPDH for heme-mediated activation of apo-nNOS. The kinetics of binding for Hsp70 and Hsp90, the ATP and K+ dependence, and the absolute requirement for Hsp70 in assembly of Hsp90â¢apo-nNOS heterocomplexes all point to a similar chaperone machinery to the well-established canonical machine regulating steroid hormone receptors. However, unlike steroid receptors, the use of a purified protein system containing Hsp90, Hsp70, Hsp40, Hop, and p23 is unable to activate apo-nNOS. Thus, heme insertion requires a unique Hsp90-chaperone complex. With this newly developed in vitro system, which recapitulates the cellular process requiring GAPDH as well as Hsp90, further mechanistic studies are now possible to better understand the components of the Hsp90-based chaperone system as well as how this heterocomplex works with GAPDH to regulate nNOS and possibly other hemeproteins.
Assuntos
Gliceraldeído-3-Fosfato Desidrogenases , Proteínas de Choque Térmico HSP70 , Proteínas de Choque Térmico HSP90 , Heme , Hemeproteínas , Chaperonas Moleculares , Óxido Nítrico Sintase , Heme/química , Hemeproteínas/química , Hemeproteínas/metabolismo , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/química , Proteínas de Choque Térmico HSP90/metabolismo , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Ligação Proteica , Óxido Nítrico Sintase/química , Óxido Nítrico Sintase/metabolismo , Enzimas Imobilizadas , Gliceraldeído-3-Fosfato Desidrogenases/química , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Ativação EnzimáticaRESUMO
The anti-tuberculosis therapeutic bedaquiline (BDQ) is used against Mycobacterium abscessus. In M. abscessus BDQ is only bacteriostatic and less potent compared to M. tuberculosis or M. smegmatis. Here we demonstrate its reduced ATP synthesis inhibition against M. abscessus inside-out vesicles, including the F1FO-ATP synthase. Molecular dynamics simulations and binding free energy calculations highlight the differences in drug-binding of the M. abscessus and M. smegmatis FO-domain at the lagging site, where the drug deploys its mechanistic action, inhibiting ATP synthesis. These data pave the way for improved anti-M. abscessus BDQ analogs.
Assuntos
Mycobacterium abscessus , Mycobacterium tuberculosis , Antituberculosos/farmacologia , Diarilquinolinas/farmacologia , Diarilquinolinas/metabolismo , Mycobacterium tuberculosis/metabolismo , Óxido Nítrico Sintase/metabolismo , Trifosfato de Adenosina/metabolismo , Testes de Sensibilidade MicrobianaRESUMO
Nitric oxide (NO) is a gaseous molecule that regulates various reproductive functions. It is a well-recognized regulator of GnRH-FSH/LH-sex steroid secretion in vertebrates including fish. Kisspeptin is a recently discovered neuropeptide which also regulates GnRH secretion. Nitrergic and kisspeptin neurons are reported in close physical contact in the mammalian brain suggesting their interactive role in the release of GnRH. The existence of kisspeptin and NOS is also demonstrated in vertebrate gonads, but information on their reciprocal relation in gonads, if any, is obscure. Therefore, attempts were made to evaluate the functional reciprocal relation between nitric oxide and kisspeptin in the catfish gonads, if any, by administering the nitric oxide synthase (NOS) inhibitor, L-NAME {N(G)-nitro-L-arginine methyl ester}, which reduces NO production, and kisspeptin agonist (KP-10) and assessing their impacts on the expressions of kisspeptin1, different NOS isoforms, NO and steroid production in the gonadal tissue. The results revealed that L-NAME suppressed the expression of kiss1 in gonads of the catfish establishing the role of NO in kisspeptin expression. However, KP-10 increased the expression of all the isoforms of NOSs (iNOS, eNOS, nNOS) and concurrently NO and steroids in the ovary and testis. In vitro studies also indicate that kisspeptin stimulates the production of NO and estradiol and testosterone levels in the gonadal explants and medium. Thus, in vivo results clearly suggest a reciprocal interaction between kisspeptin and NO to regulate the gonadal activity of the catfish. The in vitro findings further substantiate our contention regarding the interactive role of kisspeptin and NO in gonadal steroidogenesis.
Assuntos
Peixes-Gato , Gametogênese , Kisspeptinas , NG-Nitroarginina Metil Éster , Óxido Nítrico , Animais , Óxido Nítrico/metabolismo , Peixes-Gato/metabolismo , Kisspeptinas/metabolismo , Masculino , NG-Nitroarginina Metil Éster/farmacologia , Feminino , Gametogênese/efeitos dos fármacos , Esteroides/biossíntese , Óxido Nítrico Sintase/metabolismo , Testículo/metabolismo , Testículo/efeitos dos fármacos , Gônadas/metabolismo , Gônadas/efeitos dos fármacos , Ovário/metabolismoRESUMO
Parasitoids are important components of the natural enemy guild in the biological control of insect pests. They depend on host resources to complete the development of a specific stage or whole life cycle and thus have evolved towards optimal host exploitation strategies. In the present study, we report a specific survival strategy of a fly parasitoid Exorista sorbillans (Diptera: Tachinidae), which is a potential biological control agent for agricultural pests and a pest in sericulture. We found that the expression levels of nitric oxide synthase (NOS) and nitric oxide (NO) production in host Bombyx mori (Lepidoptera: Bombycidae) were increased after E. sorbillans infection. Reducing NOS expression and NO production with an NOS inhibitor (NG-nitro-L-arginine methyl ester hydrochloride) in infected B. mori significantly impeded the growth of E. sorbillans larvae. Moreover, the biosynthesis of 20-hydroxyecdysone (20E) in infected hosts was elevated with increasing NO production, and inhibiting NOS expression lowered 20E biosynthesis. More importantly, induced NO synthesis was required to eliminate intracellular bacterial pathogens that presumably competed for shared host resources. Inhibiting NOS expression down-regulated the transcription of antimicrobial peptide genes and increased the number of bacteria in parasitized hosts. Collectively, this study revealed a new perspective on the role of NO in host-parasitoid interactions and a novel mechanism for parasitoid regulation of host physiology to support its development.
Assuntos
Bombyx , Dípteros , Ecdisterona , Interações Hospedeiro-Parasita , Óxido Nítrico , Animais , Bombyx/genética , Bombyx/microbiologia , Bombyx/parasitologia , Dípteros/fisiologia , Ecdisterona/metabolismo , Larva/crescimento & desenvolvimento , Larva/parasitologia , Larva/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase/genéticaRESUMO
BACKGROUND: It has been previously demonstrated that the maintenance of ischemic acidic pH or the delay of intracellular pH recovery at the onset of reperfusion decreases ischemic-induced cardiomyocyte death. OBJECTIVE: To examine the role played by nitric oxide synthase (NOS)/NO-dependent pathways in the effects of acidic reperfusion in a regional ischemia model. METHODS: Isolated rat hearts perfused by Langendorff technique were submitted to 40 min of left coronary artery occlusion followed by 60 min of reperfusion (IC). A group of hearts received an acid solution (pH = 6.4) during the first 2 min of reperfusion (AR) in absence or in presence of l-NAME (NOS inhibitor). Infarct size (IS) and myocardial function were determined. In cardiac homogenates, the expression of P-Akt, P-endothelial and inducible isoforms of NOS (P-eNOS and iNOS) and the level of 3-nitrotyrosine were measured. In isolated cardiomyocytes, the intracellular NO production was assessed by confocal microscopy, under control and acidic conditions. Mitochondrial swelling after Ca2+ addition and mitochondrial membrane potential (Δψ) were also determined under control and acidosis. RESULTS: AR decreased IS, improved postischemic myocardial function recovery, increased P-Akt and P-eNOS, and decreased iNOS and 3-nitrotyrosine. NO production increased while mitochondrial swelling and Δψ decreased in acidic conditions. l-NAME prevented the beneficial effects of AR. CONCLUSIONS: Our data strongly supports that a brief acidic reperfusion protects the myocardium against the ischemia-reperfusion injury through eNOS/NO-dependent pathways.
Assuntos
Óxido Nítrico , Animais , Concentração de Íons de Hidrogênio , Óxido Nítrico/metabolismo , Masculino , Ratos , Ratos Wistar , Óxido Nítrico Sintase Tipo III/metabolismo , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , NG-Nitroarginina Metil Éster/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/patologia , Óxido Nítrico Sintase/metabolismoRESUMO
BACKGROUND: Applications of nonthermal plasma have expanded beyond the biomedical field to include antibacterial, anti-inflammatory, wound healing, and tissue regeneration. Plasma enhances epithelial cell repair; however, the potential damage to deep tissues and vascular structures remains under investigation. RESULT: This study assessed whether liquid plasma (LP) increased nitric oxide (NO) production in human umbilical vein endothelial cells by modulating endothelial NO synthase (eNOS) phosphorylation and potential signaling pathways. First, we developed a liquid plasma product and confirmed the angiogenic effect of LP using the Matrigel plug assay. We found that the NO content increased in plasma-treated water. NO in plasma-treated water promoted cell migration and angiogenesis in scratch and tube formation assays via vascular endothelial growth factor mRNA expression. In addition to endothelial cell proliferation and migration, LP influenced extracellular matrix metabolism and matrix metalloproteinase activity. These effects were abolished by treatment with NG-L-monomethyl arginine, a specific inhibitor of NO synthase. Furthermore, we investigated the signaling pathways mediating the phosphorylation and activation of eNOS in LP-treated cells and the role of LKB1-adenosine monophosphate-activated protein kinase in signaling. Downregulation of adenosine monophosphate-activated protein kinase by siRNA partially inhibited LP-induced eNOS phosphorylation, angiogenesis, and migration. CONCLUSION: The present study suggests that LP treatment may be a novel strategy for promoting angiogenesis in vascular damage. Video Abstract.
Assuntos
Matriz Extracelular , Óxido Nítrico Sintase Tipo III , Plasma , Lesões do Sistema Vascular , Humanos , Monofosfato de Adenosina/metabolismo , Monofosfato de Adenosina/farmacologia , Angiogênese , Matriz Extracelular/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Neovascularização Fisiológica , Óxido Nítrico/metabolismo , Óxido Nítrico/farmacologia , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase/farmacologia , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Fosforilação , Proteínas Quinases/metabolismo , Regulação para Cima , Fator A de Crescimento do Endotélio Vascular/metabolismo , Lesões do Sistema Vascular/metabolismo , Lesões do Sistema Vascular/terapia , Plasma/metabolismoRESUMO
BACKGROUND: Migraine research has highlighted the pivotal role of nitric oxide (NO) in migraine pathophysiology. Nitric oxide donors such as glyceryl trinitrate (GTN) induce migraine attacks in humans, whereas spontaneous migraine attacks can be aborted by inhibiting NO production. The present study aimed to investigate how GTN triggers migraine through its three nitric oxide synthase (NOS) isoforms (neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS)) via a suspected feed-forward phenomenon. METHODS: Migraine-relevant hypersensitivity was induced by repeated injection of GTN in an in vivo mouse model. Cutaneous tactile sensitivity was assessed using von Frey filaments. Signaling pathways involved in this model were dissected using non-selective and selective NOS inhibitors, knockout mice lacking eNOS or nNOS and their wild-type control mice. Also, we tested a soluble guanylate cyclase inhibitor and a peroxynitrite decomposition catalyst (Ntotal = 312). RESULTS: Non-selective NOS inhibition blocked GTN-induced hypersensitivity. This response was partially associated with iNOS, and potentially nNOS and eNOS conjointly. Furthermore, we found that the GTN response was largely dependent on the generation of peroxynitrite and partly soluble guanylate cyclase. CONCLUSIONS: Migraine-relevant hypersensitivity induced by GTN is mediated by a possible feed-forward phenomenon of NO driven mainly by iNOS but with contributions from other isoforms. The involvement of peroxynitrite adds to the notion that oxidative stress reactions are also involved.
Assuntos
Modelos Animais de Doenças , Camundongos Knockout , Transtornos de Enxaqueca , Nitroglicerina , Ácido Peroxinitroso , Animais , Transtornos de Enxaqueca/metabolismo , Transtornos de Enxaqueca/induzido quimicamente , Nitroglicerina/toxicidade , Nitroglicerina/farmacologia , Camundongos , Ácido Peroxinitroso/metabolismo , Masculino , Guanilil Ciclase Solúvel/metabolismo , Camundongos Endogâmicos C57BL , Óxido Nítrico Sintase Tipo I/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico Sintase Tipo II/antagonistas & inibidoresRESUMO
Insulin resistance (IR) being the major cause behind different metabolic disorders, has attracted a lot of attention. Epidemiological data shows marked rise in the cases over a period of time. Nitric oxide (NO), produced from nitric oxide synthases (NOS), is involved in a variety of biological functions, alteration in which causes various disorders like hypertension, atherosclerosis, and angiogenesis-associated disorders. IR has been found to be a contributing factor, which is associated with abnormal NO signalling. Skeletal muscle is essential for metabolism, both for its role in glucose uptake and its importance in metabolic disease. In this article, we give an overview of the significance of NO in oxidative stress (OS) mediated IR, describing its role in different conditions that are associated with skeletal muscle IR. NO is found to be involved in the activation of insulin receptor downstream pathway, which suggests absence of NO could lead to reduced glucose uptake, and may ultimately result in IR.
Assuntos
Resistência à Insulina , Músculo Esquelético , Óxido Nítrico , Estresse Oxidativo , Transdução de Sinais , Óxido Nítrico/metabolismo , Humanos , Músculo Esquelético/metabolismo , Animais , Receptor de Insulina/metabolismo , Glucose/metabolismo , Óxido Nítrico Sintase/metabolismo , Insulina/metabolismoRESUMO
Neural circuits for appetites are regulated by both homeostatic perturbations and ingestive behaviour. However, the circuit organization that integrates these internal and external stimuli is unclear. Here we show in mice that excitatory neural populations in the lamina terminalis form a hierarchical circuit architecture to regulate thirst. Among them, nitric oxide synthase-expressing neurons in the median preoptic nucleus (MnPO) are essential for the integration of signals from the thirst-driving neurons of the subfornical organ (SFO). Conversely, a distinct inhibitory circuit, involving MnPO GABAergic neurons that express glucagon-like peptide 1 receptor (GLP1R), is activated immediately upon drinking and monosynaptically inhibits SFO thirst neurons. These responses are induced by the ingestion of fluids but not solids, and are time-locked to the onset and offset of drinking. Furthermore, loss-of-function manipulations of GLP1R-expressing MnPO neurons lead to a polydipsic, overdrinking phenotype. These neurons therefore facilitate rapid satiety of thirst by monitoring real-time fluid ingestion. Our study reveals dynamic thirst circuits that integrate the homeostatic-instinctive requirement for fluids and the consequent drinking behaviour to maintain internal water balance.
Assuntos
Ingestão de Líquidos/fisiologia , Vias Neurais , Área Pré-Óptica/citologia , Área Pré-Óptica/fisiologia , Órgão Subfornical/citologia , Órgão Subfornical/fisiologia , Sede/fisiologia , Animais , Apetite/fisiologia , Feminino , Neurônios GABAérgicos/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Homeostase , Instinto , Masculino , Camundongos , Óxido Nítrico Sintase/metabolismo , Resposta de Saciedade/fisiologia , Equilíbrio HidroeletrolíticoRESUMO
Ceria nanoparticles (CeO2NPs) exhibit great potential in cardiovascular disease and nonalcoholic fatty liver disease due to its excellent antioxidant capacity. However, the profitable effect of CeO2NPs on many diseases is almost all attributed to the regulation of ROS. Apart from the general antioxidant function, there seems to be no more distinct mechanism to reflect its unique multi-disease improvement effect. Here, we for the first time reveal a new discovery of CeO2NPs in mimicking nitric oxide synthase (NOS) by catalyzing L-arginine (L-Arg) to produce nitric oxide (NO) or the derivatives. NOS-like activity of CeO2NPs is original and associated with multiple factors like substrate concentration, pH, temperature and time, etc. where oxygen vacancy ratio plays a more critical role. Meanwhile, NOS-like activity of CeO2NPs successfully elevates NO secretion in endothelial cells and macrophages without expanding eNOS/iNOS expression. Importantly, NOS-like activity of CeO2NPs and the responsive endogenous NO promote the re-distribution of blood lipids and stabilize eNOS expression but suppress iNOS, thus collectively alleviate the accumulation of vascular plaque. Altogether, we provide a new angle of view to survey the outstanding potential of CeO2NPs, apart from the inevitable antioxidant capacity, the covert but possible and more critical NOS-like enzymatic activity is more noteworthy.