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1.
Mol Pharmacol ; 98(3): 243-249, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32591478

RESUMO

Heat shock protein (Hsp) 70 modulators are being developed to enhance the removal of toxic proteins in a variety of protein misfolding diseases. In the course of our studies on neuronal nitric oxide synthase (nNOS), a client of the Hsp90 and Hsp70 chaperone system, we have established that inactivation of nNOS by heme or tetrahydrobiopterin (BH4) alteration and loss triggers ubiquitination by the Hsp70-associated E3 ligase c-terminus of Hsp70-interacting protein (CHIP) and subsequent degradation in cells. Although in cells Hsp90 and Hsp70 work together to maintain protein quality control, in this study, we specifically developed an assay to assess the selectivity of the Hsp70:CHIP complex for inactivated nNOS. We developed a highly sensitive ELISA to measure Hsp70:CHIP-dependent nNOS ubiquitination without interference from direct ubiquitination by CHIP, as evidenced by Bcl-2 associated athanogene 1-M completely abolishing ubiquitination. To further validate the assay we demonstrated, JG-98, a rhodocyanin compound that acts on Hsp70 but not its inactive structural analog JG-258, enhances the ubiquitination of nNOS 3-fold. Utilizing this assay, we have shown that the Hsp70:CHIP complex preferentially ubiquitinates heme-deficient nNOS (apo-nNOS) over heme-containing nNOS (holo-nNOS). Moreover, depletion of nNOS-bound BH4 triggers ubiquitination of holo-nNOS by the Hsp70:CHIP complex. Most importantly, JG-98 was shown to enhance the ubiquitination of only dysfunctional nNOS while leaving the native functional nNOS untouched. Thus, the finding that enhancing Hsp70:CHIP-mediated ubiquitination does not affect native proteins has important pharmacological implications. Moreover, development of a facile in vitro method for Hsp70:CHIP-mediated ubiquitination will be beneficial for testing other Hsp70 modulators. SIGNIFICANCE STATEMENT: The heat shock protein 70 (Hsp70):c-terminus of Hsp70-interacting protein (CHIP) complex facilitates the ubiquitination and subsequent degradation of several hundred-client proteins, and activation of Hsp70 has been suggested as a therapeutic strategy to enhance the degradation of disease-causing proteins. The current study shows that the pharmacological activation of Hsp70 enhances the ubiquitination of dysfunctional but not native nNOS, and it suggests that this therapeutic strategy will likely be highly selective.


Assuntos
Proteínas de Choque Térmico HSP70/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Proteínas de Ligação a DNA/metabolismo , Ensaio de Imunoadsorção Enzimática , Humanos , Células Sf9 , Fatores de Transcrição/metabolismo , Ubiquitinação
2.
Am J Physiol Regul Integr Comp Physiol ; 319(2): R233-R242, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32579854

RESUMO

Continuous infusion of prostaglandin E1 (PGE1) is used to maintain ductus arteriosus patency in infants with critical congenital heart disease, but it can also cause central apnea suggesting an effect on respiratory neural control. In this study, we investigated whether 1) PGE1 inhibits the various phases of the acute hypoxic ventilatory response (HVR; an index of respiratory control dysfunction) and increases apnea incidence in neonatal rats; and 2) whether these changes would be reversible with caffeine pretreatment. Whole body plethysmography was used to assess the HVR and apnea incidence in neonatal rats 2 h following a single bolus intraperitoneal injection of PGE1 with and without prior caffeine treatment. Untreated rats exhibited a biphasic HVR characterized by an initial increase in minute ventilation followed by a ventilatory decline of the late phase (~5th minute) of the HVR. PGE1 had a dose-dependent effect on the HVR. Contrary to our hypothesis, the lowest dose (1 µg/kg) of PGE1 prevented the ventilatory decline of the late phase of the HVR. However, PGE1 tended to increase postsigh apnea incidence and the coefficient of variability (CV) of breathing frequency, suggesting increased respiratory instability. PGE1 also decreased brainstem microglia mRNA and increased neuronal nitric oxide synthase (nNOS) and platelet-derived growth factor-ß (PDGF-ß) gene expression. Caffeine pretreatment prevented these effects of PGE1, and the adenosine A2A receptor inhibitor MSX-3 had similar preventative effects. Prostaglandin appears to have deleterious effects on brainstem respiratory control regions, possibly involving a microglial-dependent mechanism. The compensatory effects of caffeine or MSX-3 treatment raises the question of whether prostaglandin may also operate on an adenosine-dependent pathway.


Assuntos
Alprostadil/farmacologia , Tronco Encefálico/efeitos dos fármacos , Cafeína/farmacologia , Ventilação Pulmonar/efeitos dos fármacos , Respiração/efeitos dos fármacos , Animais , Tronco Encefálico/metabolismo , Microglia/efeitos dos fármacos , Microglia/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Pletismografia Total , Proteínas Proto-Oncogênicas c-sis/genética , Proteínas Proto-Oncogênicas c-sis/metabolismo , Antagonistas de Receptores Purinérgicos P1/farmacologia , Ratos , Ratos Sprague-Dawley
3.
Biochim Biophys Acta Mol Cell Res ; 1867(8): 118732, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32360667

RESUMO

Nitric oxide is an important neuromodulator in the CNS, and its production within neurons is modulated by NMDA receptors and requires a fine-tuned availability of L-arginine. We have previously shown that globally inhibiting protein synthesis mobilizes intracellular L-arginine "pools" in retinal neurons, which concomitantly enhances neuronal nitric oxide synthase-mediated nitric oxide production. Activation of NMDA receptors also induces local inhibition of protein synthesis and L-arginine intracellular accumulation through calcium influx and stimulation of eucariotic elongation factor type 2 kinase. We hypothesized that protein synthesis inhibition might also increase intracellular L-arginine availability to induce nitric oxide-dependent activation of downstream signaling pathways. Here we show that nitric oxide produced by inhibiting protein synthesis (using cycloheximide or anisomycin) is readily coupled to AKT activation in a soluble guanylyl cyclase and cGKII-dependent manner. Knockdown of cGKII prevents cycloheximide or anisomycin-induced AKT activation and its nuclear accumulation. Moreover, in retinas from cGKII knockout mice, cycloheximide was unable to enhance AKT phosphorylation. Indeed, cycloheximide also produces an increase of ERK phosphorylation which is abrogated by a nitric oxide synthase inhibitor. In summary, we show that inhibition of protein synthesis is a previously unanticipated driving force for nitric oxide generation and activation of downstream signaling pathways including AKT and ERK in cultured retinal cells. These results may be important for the regulation of synaptic signaling and neuronal development by NMDA receptors as well as for solving conflicting data observed when using protein synthesis inhibitors for studying neuronal survival during development as well in behavior and memory studies.


Assuntos
Proteína Quinase Dependente de GMP Cíclico Tipo II/metabolismo , Óxido Nítrico/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , Retina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Arginina/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Embrião de Galinha , Galinhas , Proteína Quinase Dependente de GMP Cíclico Tipo II/genética , Quinase do Fator 2 de Elongação/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/metabolismo , Nitratos/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Nitritos , Fosforilação
4.
Nat Commun ; 11(1): 2501, 2020 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-32427844

RESUMO

Anxiety is common in patients suffering from chronic pain. Here, we report anxiety-like behaviors in mouse models of chronic pain and reveal that nNOS-expressing neurons in ventromedial prefrontal cortex (vmPFC) are essential for pain-induced anxiety but not algesia, using optogenetic and chemogenetic strategies. Additionally, we determined that excitatory projections from the posterior subregion of paraventricular thalamic nucleus (pPVT) provide a neuronal input that drives the activation of vmPFC nNOS-expressing neurons in our chronic pain models. Our results suggest that the pain signal becomes an anxiety signal after activation of vmPFC nNOS-expressing neurons, which causes subsequent release of nitric oxide (NO). Finally, we show that the downstream molecular mechanisms of NO likely involve enhanced glutamate transmission in vmPFC CaMKIIα-expressing neurons through S-nitrosylation-induced AMPAR trafficking. Overall, our data suggest that pPVT excitatory neurons drive chronic pain-induced anxiety through activation of vmPFC nNOS-expressing neurons, resulting in NO-mediated AMPAR trafficking in vmPFC pyramidal neurons.


Assuntos
Dor Crônica/enzimologia , Dor Crônica/psicologia , Núcleos da Linha Média do Tálamo/enzimologia , Neurônios/enzimologia , Óxido Nítrico Sintase Tipo I/metabolismo , Córtex Pré-Frontal/enzimologia , Animais , Ansiedade , Comportamento Animal , Dor Crônica/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Núcleos da Linha Média do Tálamo/citologia , Neurônios/citologia , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo I/genética , Córtex Pré-Frontal/citologia
5.
J Med Chem ; 63(9): 4528-4554, 2020 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-32302123

RESUMO

Inhibition of neuronal nitric oxide synthase (nNOS), an enzyme implicated in neurodegenerative disorders, is an attractive strategy for treating or preventing these diseases. We previously developed several classes of 2-aminoquinoline-based nNOS inhibitors, but these compounds had drawbacks including off-target promiscuity, low activity against human nNOS, and only modest selectivity for nNOS over related enzymes. In this study, we synthesized new nNOS inhibitors based on 7-phenyl-2-aminoquinoline and assayed them against rat and human nNOS, human eNOS, and murine and (in some cases) human iNOS. Compounds with a meta-relationship between the aminoquinoline and a positively charged tail moiety were potent and had up to nearly 900-fold selectivity for human nNOS over human eNOS. X-ray crystallography indicates that the amino groups of some compounds occupy a water-filled pocket surrounding an nNOS-specific aspartate residue (absent in eNOS). This interaction was confirmed by mutagenesis studies, making 7-phenyl-2-aminoquinolines the first aminoquinolines to interact with this residue.


Assuntos
Aminoquinolinas/farmacologia , Ácido Aspártico/química , Inibidores Enzimáticos/farmacologia , Óxido Nítrico Sintase Tipo I/antagonistas & inibidores , Aminoquinolinas/síntese química , Aminoquinolinas/metabolismo , Aminoquinolinas/farmacocinética , Animais , Barreira Hematoencefálica/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Ensaios Enzimáticos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacocinética , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Microssomos Hepáticos/metabolismo , Estrutura Molecular , Mutagênese Sítio-Dirigida , Mutação , Óxido Nítrico Sintase Tipo I/química , Óxido Nítrico Sintase Tipo I/genética , Óxido Nítrico Sintase Tipo I/metabolismo , Permeabilidade , Ligação Proteica , Ratos , Relação Estrutura-Atividade
6.
Biochem Biophys Res Commun ; 525(2): 520-527, 2020 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-32113678

RESUMO

Extremely high relapse rate is the dramatic challenge of drug abuse at present. Environmental cues play an important role in relapse of drug abuse. However, the specific mechanism underlying relapse remains unclear. Using morphine conditioned place preference (CPP) model, we show that association of neuronal nitric oxide synthase (nNOS) with postsynaptic density-95 (PSD-95) plays a significant role in morphine priming-induced reinstatement. The nNOS-PSD-95 coupling and c-Fos expression in the medial prefrontal cortex (mPFC) was significantly increased after extinction of morphine CPP. Dissociation of nNOS-PSD-95 in the mPFC by ZL006 inhibited the reinstatement of morphine CPP induced by a priming dose of morphine. Significantly reduced phosphorylation of cAMP-response element binding protein (CREB) in the mPFC was observed in the mice exposed to morphine after the extinction training. Uncoupling nNOS-PSD-95 reversed the morphine-induced CREB dysfunction. Moreover, effects of ZL006 on the reinstatement of morphine CPP and CREB activation depended on nNOS-PSD-95 target. Together, our findings suggest that nNOS-PSD-95 in the mPFC contributes to reinstatement of morphine CPP, possibly through CREB dysfunction, offering a potential target to prevent relapse of drug abuse.


Assuntos
Proteína 4 Homóloga a Disks-Large/metabolismo , Morfina/farmacologia , Entorpecentes/farmacologia , Óxido Nítrico Sintase Tipo I/metabolismo , Córtex Pré-Frontal/efeitos dos fármacos , Animais , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Extinção Psicológica/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Córtex Pré-Frontal/metabolismo , Mapas de Interação de Proteínas/efeitos dos fármacos
7.
Endocrinology ; 161(4)2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32166324

RESUMO

Genetic research has revealed pro-opiomelanocortin (POMC) to be a fundamental regulator of energy balance and body weight in mammals. Within the brain, POMC is primarily expressed in the arcuate nucleus of the hypothalamus (ARC), while a smaller population exists in the brainstem nucleus of the solitary tract (POMCNTS). We performed a neurochemical characterization of this understudied population of POMC cells using transgenic mice expressing green fluorescent protein (eGFP) under the control of a POMC promoter/enhancer (PomceGFP). Expression of endogenous Pomc mRNA in the nucleus of the solitary tract (NTS) PomceGFP cells was confirmed using fluorescence-activating cell sorting (FACS) followed by quantitative PCR. In situ hybridization histochemistry of endogenous Pomc mRNA and immunohistochemical analysis of eGFP revealed that POMC is primarily localized within the caudal NTS. Neurochemical analysis indicated that POMCNTS is not co-expressed with tyrosine hydroxylase (TH), glucagon-like peptide 1 (GLP-1), cholecystokinin (CCK), brain-derived neurotrophic factor (BDNF), nesfatin, nitric oxide synthase 1 (nNOS), seipin, or choline acetyltransferase (ChAT) cells, whereas 100% of POMCNTS is co-expressed with transcription factor paired-like homeobox2b (Phox2b). We observed that 20% of POMCNTS cells express receptors for adipocyte hormone leptin (LepRbs) using a PomceGFP:LepRbCre:tdTOM double-reporter line. Elevations in endogenous or exogenous leptin levels increased the in vivo activity (c-FOS) of a small subset of POMCNTS cells. Using ex vivo slice electrophysiology, we observed that this effect of leptin on POMCNTS cell activity is postsynaptic. These findings reveal that a subset of POMCNTS cells are responsive to both changes in energy status and the adipocyte hormone leptin, findings of relevance to the neurobiology of obesity.


Assuntos
Tronco Encefálico/metabolismo , Neurônios/metabolismo , Pró-Opiomelanocortina/metabolismo , Receptores para Leptina/metabolismo , Animais , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Colecistocinina/metabolismo , Colina O-Acetiltransferase/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Camundongos Transgênicos , Óxido Nítrico Sintase Tipo I/metabolismo , Nucleobindinas/metabolismo , Regiões Promotoras Genéticas , Receptores para Leptina/genética , Tirosina 3-Mono-Oxigenase/metabolismo
8.
Neurourol Urodyn ; 39(3): 916-925, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32040866

RESUMO

AIMS: The urethral dysfunction produced by a rat model of peripheral neurogenic detrusor underactivity (DU) using pelvic nerve crush (PNC) injury was characterized and then tested with the administration of tadalafil, a phosphodiesterase type 5 (PDE 5) inhibitor. METHODS: Ten days after producing PNC rats, awake cystometrograms (CMGs) and isovolumetric cystometrograms with urethral perfusion pressure (IC-UPP) measurements were performed. Also, in control rats, IC-UPP was recorded before and after intravenous atropine administration to determine if the reduction of bladder contraction pressure affects urethral relaxation during voiding. Then, CMG and IC-UPP measurements in PNC rats were recorded after intravenous administration of tadalafil. Lastly, real-time polymerase chain reaction was used to measure transcript levels of neuronal nitric oxide synthases (nNOS), endothelial nitric oxide synthases, and PDE 5 in urethral specimens from PNC and control rats. RESULTS: PNC rats demonstrated the characteristics of DU in CMG. Also, PNC rats exhibited significant decreases in isovolumetric bladder contraction amplitudes and urethral relaxation. Atropine attenuated the amplitude of isovolumetric bladder contractions; however, atropine did not affect urethral relaxation in control rats. Tadalafil decreased postvoid residual and increased voiding efficiency without changing bladder contraction amplitude in PNC rats. Also, tadalafil improved the amplitude of urethral relaxation during bladder contraction in PNC rats. Urethral nNOS transcript levels were upregulated in PNC rats compared to control rats. CONCLUSIONS: PNC rats revealed both DU and impaired urethral relaxation. PDE 5 inhibition in PNC rats enhanced urethral relaxation during voiding, resulting in improved voiding efficiency. Thus, urethral dysfunction could be a potential target for the treatment of inefficient voiding associated with neurogenic DU.


Assuntos
Traumatismos dos Nervos Periféricos/fisiopatologia , Inibidores da Fosfodiesterase 5/farmacologia , Tadalafila/farmacologia , Uretra/efeitos dos fármacos , Bexiga Urinaria Neurogênica/fisiopatologia , Bexiga Inativa/fisiopatologia , Bexiga Urinária/efeitos dos fármacos , Micção/efeitos dos fármacos , Animais , Lesões por Esmagamento/fisiopatologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5 , Feminino , Óxido Nítrico Sintase Tipo I/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Pelve , Ratos , Ratos Sprague-Dawley , Uretra/inervação , Uretra/metabolismo , Uretra/fisiopatologia , Bexiga Urinária/inervação , Bexiga Urinária/fisiopatologia , Micção/fisiologia
9.
Endocrinology ; 161(3)2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-32067028

RESUMO

Elevated and sustained estradiol concentrations cause a gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) surge that is necessary for ovulation. In sheep, several different neural systems have been implicated in this stimulatory action of estradiol and this study focused on somatostatin (SST) neurons in the ventral lateral region of the ventral medial nucleus (vlVMN) which express c-Fos during the surge. First, we determined if increased activity of SST neurons could be related to elevated GnRH secretion by assessing SST synapses onto GnRH neurons and neurons coexpressing kisspeptin, neurokinin B, dynorphin (KNDy). We found that the percentage of preoptic area GnRH neurons that receive SST input increased during the surge compared with other phases of the cycle. However, since SST is generally inhibitory, and pharmacological manipulation of SST signaling did not alter the LH surge in sheep, we hypothesized that nitric oxide (NO) was also produced by these neurons to account for their activation during the surge. In support of this hypothesis we found that (1) the majority of SST cells in the vlVMN (>80%) contained neuronal nitric oxide synthase (nNOS); (2) the expression of c-Fos in dual-labeled SST-nNOS cells, but not in single-labeled cells, increased during the surge compared with other phases of the cycle; and (3) intracerebroventricular (ICV) infusion of the nitric oxide synthase inhibitor, N(G)-nitro-L-arginine methyl ester, completely blocked the estrogen-induced LH surge. These data support the hypothesis that the population of SST-nNOS cells in the vlVMN are a source of NO that is critical for the LH surge, and we propose that they are an important site of estradiol positive feedback in sheep.


Assuntos
Hormônio Luteinizante/sangue , Óxido Nítrico/metabolismo , Ovulação , Ovinos/sangue , Núcleo Hipotalâmico Ventromedial/enzimologia , Animais , Feminino , Óxido Nítrico Sintase Tipo I/metabolismo , Somatostatina/metabolismo
10.
J Cardiovasc Pharmacol ; 75(1): 31-40, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31895878

RESUMO

Endothelium-dependent hyperpolarization (EDH) factor is one of endothelium-derived relaxing factors and plays important roles especially in microvessels. We have previously demonstrated that endothelium-derived hydrogen peroxide (H2O2) is an EDH factor produced by all types of nitric oxide synthases (NOSs), including endothelial NOS (eNOS), neuronal NOS (nNOS), and inducible NOS. Recent studies have suggested the association between coronary microvascular dysfunction and cardiac diastolic dysfunction. However, the role of EDH in this issue remains to be fully elucidated. We thus examined whether EDH plays an important role in coronary microcirculation and if so, whether endothelial dysfunction, especially impaired EDH, is involved in the pathogenesis of cardiac diastolic dysfunction in mice. Using a Langendorff-perfused heart experiment, we examined the increase in coronary flow in response to bradykinin in the presence of indomethacin and N-nitro-L-arginine (EDH condition) in wild-type, eNOS-knockout (KO), and nNOS/eNOS-double-KO mice. Compared with wild-type mice, EDH-mediated relaxations were increased in eNOS-KO mice but were significantly reduced in n/eNOS-KO mice. Catalase, a specific H2O2 scavenger, markedly inhibited EDH-mediated relaxations in all 3 genotypes, indicating compensatory roles of nNOS-derived H2O2 as an EDH factor in coronary microcirculation. Although both eNOS-KO and n/eNOS-KO mice exhibited similar extents of cardiac morphological changes, only n/eNOS-KO mice exhibited cardiac diastolic dysfunction. The expression of oxidized protein kinase G I-α (PKGIα) in the heart was significantly increased in eNOS-KO mice compared with n/eNOS-KO mice. These results indicate that EDH/H2O2 plays important roles in maintaining coronary microcirculation and cardiac diastolic function through oxidative PKGIα activation.


Assuntos
Fatores Biológicos/metabolismo , Circulação Coronária , Vasos Coronários/metabolismo , Endotélio Vascular/metabolismo , Hipertrofia Ventricular Esquerda/metabolismo , Microcirculação , Microvasos/metabolismo , Vasodilatação , Disfunção Ventricular Esquerda/metabolismo , Função Ventricular Esquerda , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Vasos Coronários/fisiopatologia , Proteína Quinase Dependente de GMP Cíclico Tipo I/metabolismo , Diástole , Endotélio Vascular/fisiopatologia , Peróxido de Hidrogênio/metabolismo , Hipertrofia Ventricular Esquerda/genética , Hipertrofia Ventricular Esquerda/fisiopatologia , Preparação de Coração Isolado , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microvasos/fisiopatologia , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo I/genética , Óxido Nítrico Sintase Tipo I/metabolismo , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Oxirredução , Fosforilação , Transdução de Sinais , Disfunção Ventricular Esquerda/genética , Disfunção Ventricular Esquerda/fisiopatologia
11.
J Stroke Cerebrovasc Dis ; 29(3): 104531, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31882337

RESUMO

BACKGROUND: The purpose of this study was to investigate the effects of edaravone on nitric oxide (NO) production, hydroxyl radical (OH-) metabolism, and neuronal nitric oxide synthase (nNOS) expression during cerebral ischemia and reperfusion. METHODS: Edaravone (3 mg/kg) was administered intravenously to 14 C57BL/6 mice just before reperfusion. Eleven additional mice received saline (controls). NO production and OH- metabolism were continuously monitored using bilateral striatal in vivo microdialysis. OH- formation was monitored using the salicylate trapping method. Forebrain ischemia was produced in all mice by bilateral occlusion of the common carotid artery for 10 minutes. Levels of NO metabolites, nitrite (NO2-) and nitrate (NO3-), were determined using the Griess reaction. Brain sections were immunostained with an anti-nNOS antibody and the fractional area density of nNOS-immunoreactive pixels to total pixels determined. RESULTS: Blood pressure and regional cerebral blood flow were not significantly different between the edaravone and control groups. The levels of NO2- did not differ significantly between the 2 groups. The level of NO3- was significantly higher in the edaravone group compared with the control group after reperfusion. 2,3-dihydroxybenzoic acid levels were lower in the edaravone group compared with those in the control group after reperfusion. Immunohistochemistry showed nNOS expression in the edaravone group to be significantly lower than that in the control group 96 hours after reperfusion. CONCLUSIONS: These in vivo data indicate that edaravone may have a neuroprotective effect by reducing levels of OH- metabolites, increasing NO production and decreasing nNOS expression in brain cells.


Assuntos
Isquemia Encefálica/tratamento farmacológico , Encéfalo/efeitos dos fármacos , Edaravone/farmacologia , Depuradores de Radicais Livres/farmacologia , Radical Hidroxila/metabolismo , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Óxido Nítrico Sintase Tipo I/metabolismo , Óxido Nítrico/metabolismo , Traumatismo por Reperfusão/prevenção & controle , Animais , Encéfalo/enzimologia , Encéfalo/patologia , Isquemia Encefálica/enzimologia , Isquemia Encefálica/patologia , Modelos Animais de Doenças , Camundongos Endogâmicos C57BL , Neurônios/enzimologia , Neurônios/patologia , Traumatismo por Reperfusão/enzimologia , Traumatismo por Reperfusão/patologia , Fatores de Tempo
12.
Chemosphere ; 238: 124602, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31545211

RESUMO

Polybrominated diphenyl ethers (PBDEs) have been known to exhibit neurotoxicity in rats; however, the underlying mechanism remains unknown and there is no available intervention. In this study, we aimed to investigate the role of oxidative and nitrosative stress in the neurotoxicity in the cerebral cortex and primary neurons in rats following the BDE-153 treatment. Compared to the untreated group, BDE-153 treatment significantly induced the neurotoxic effects in rats, as manifested by the increased lactate dehydrogenase (LDH) activities and cell apoptosis rates, and the decreased neurotrophic factor contents and cholinergic enzyme activities in rats' cerebral cortices and primary neurons. When compared to the untreated group, the oxidative and nitrosative stress had occurred in the cerebral cortex or primary neurons in rats following the BDE-153 treatment, as manifested by the increments in levels of reactive oxygenspecies (ROS), malondialdehyde (MDA), nitric oxide (NO), and neuronal nitric oxide synthase (nNOS) mRNA and protein expressions, along with the decline in levels of superoxide dismutase (SOD) activity, glutathione (GSH) content, and peroxiredoxin I (Prx I) and Prx II mRNA and protein expressions. In addition, the ROS scavenger N-acetyl-l-cysteine (NAC) or NO scavenger NG-Nitro-l-arginine (L-NNA) significantly rescued the LDH leakage and cell survival, reversed the neurotrophin contents and cholinergic enzymes, mainly via regaining balance between oxidation/nitrosation and antioxidation. Overall, our findings suggested that oxidative and nitrosative stresses are involved in the neurotoxicity induced by BDE-153, and that the antioxidation is a potential targeted intervention.


Assuntos
Córtex Cerebral/patologia , Éteres Difenil Halogenados/toxicidade , Síndromes Neurotóxicas/patologia , Estresse Nitrosativo/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Bifenil Polibromatos/toxicidade , Acetilcisteína/farmacologia , Animais , Antioxidantes/metabolismo , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Córtex Cerebral/efeitos dos fármacos , Glutationa/metabolismo , Éteres Difenil Halogenados/metabolismo , Masculino , Malondialdeído/metabolismo , Fatores de Crescimento Neural/metabolismo , Neurônios/efeitos dos fármacos , Neurotrofina 3/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Estresse Nitrosativo/fisiologia , Estresse Oxidativo/fisiologia , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos
13.
Biochem Biophys Res Commun ; 523(2): 299-306, 2020 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-31864709

RESUMO

Anxiety is recognized as primary clinical phenotype of psychiatric disorders. However, many patients with anxiety have not yet received effective treatment. Our previous study demonstrated that hippocampal nNOS-CAPON interaction is implicated in anxiety-related behaviors, and blocking nNOS-CAPON interaction in the hippocampus produces anxiolytic-like effects. Here, ZLc-002, a small molecule inhibitor of nNOS-CAPON coupling, was evaluated for anxiolytic-like properties after systemic administered using anxiety behavioral tests, including open-field (OF), elevated plus maze (EPM), novelty-suppressed feeding (NSF) and light-dark (LD) tests. We reported that ZLc-002 when administered intraperitoneally at the dose of 40 or 80 mg/kg/d for 14 days produces anxiolytic-like effects. Furthermore, the similar effects of ZLc-002 were observed when administered intravenously at the dose of 10, 20 or 40 mg/kg/d for 7 days. More importantly, the mice dosing with 80 mg/kg/d ZLc-002 intraperitoneally or 40 mg/kg/d ZLc-002 intravenously for 3 days exerted significant behavioral effects. However, intragastric administration with ZLc-002 was devoid of effect on anxiety behaviors, even at high doses. Furthermore, intraperitoneal or intravenous treatment of ZLc-002 significantly disrupted the interaction between nNOS and CAPON in the hippocampus of adult mice, and there was a significant anxiolytic-like effect of ZLc-002 at day 3 after intrahippocampal microinjection. Our results verified that systemic administration of putative small molecule inhibitor of nNOS-CAPON can be used for the treatment of anxiety disorders.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Ansiolíticos/administração & dosagem , Óxido Nítrico Sintase Tipo I/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Transtornos de Ansiedade/tratamento farmacológico , Transtornos de Ansiedade/metabolismo , Comportamento Animal/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/administração & dosagem , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Injeções Intraperitoneais , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos ICR , Óxido Nítrico Sintase Tipo I/metabolismo
14.
Antioxid Redox Signal ; 32(1): 18-34, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31642335

RESUMO

Aims: Neuronal nitric oxide synthase (nNOS) and nitric oxide (NO) signaling have been implicated in learning, memory, and underlying long-lasting synaptic plasticity. In this study, we aimed at detecting whether nNOS is a target protein of SUMOylation in the hippocampus and its contributions to hippocampal long-term potentiation (LTP) of synaptic transmission. Results: We showed that N-methyl-d-aspartate receptor-dependent neuronal activity enhancement induced the attachment of small ubiquitin-like modifier 1 (SUMO1) to nNOS. Protein inhibitor of activated STAT3 (PIAS3) promoted SUMO1 conjugation at K725 and K739 on nNOS, which upregulated NO production and nNOS S1412 phosphorylation (activation). In addition, the N-terminus (amino acids 43-86) of PIAS3 bound nNOS directly. Tat-tagged PIAS3 segment representing amino acids 43-86, a cell-permeable peptide containing PIAS3 residues 43-86, suppressed activity-induced nNOS SUMOylation by disrupting PIAS3-nNOS association. It also decreased LTP-related expression of Arc and brain-derived neurotrophic factor and blocked signaling via extracellular signal-regulated kinase (ERK) 1/2 and Elk-1 in the hippocampus. More importantly, PIAS3-mediated nNOS SUMOylation was required for activity-regulated ERK1/2 activation in nNOS-positive neurons and hippocampal LTP induction. Innovation and Conclusion: These findings indicated that network activity-regulated nNOS SUMOylation underlies excitatory synaptic LTP by facilitating nNOS-NO-ERK1/2 signal cascades.


Assuntos
Hipocampo/citologia , Chaperonas Moleculares/metabolismo , Óxido Nítrico Sintase Tipo I/química , Óxido Nítrico Sintase Tipo I/metabolismo , Proteínas Inibidoras de STAT Ativados/metabolismo , Proteína SUMO-1/metabolismo , Animais , Sítios de Ligação , Células Cultivadas , Hipocampo/metabolismo , Potenciação de Longa Duração , Sistema de Sinalização das MAP Quinases , Masculino , Modelos Moleculares , Chaperonas Moleculares/química , Óxido Nítrico/metabolismo , Ligação Proteica , Proteínas Inibidoras de STAT Ativados/química , Ratos , Sumoilação , Transmissão Sináptica
15.
Biochem Pharmacol ; 176: 113779, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-31881190

RESUMO

Previous studies from this lab and others have demonstrated that nitric oxide (NO) in a concentration dependent manner, modulated neutrophil and leukemic cell survival. Subsequent studies delineated importance of iNOS in neutrophil differentiation and leukemic cell death. On the contrary, role of nNOS in survival of these cells remains least understood. Present study was therefore undertaken to assess and compare the role of iNOS and nNOS in the survival of NOS overexpressing myelocytic K562 cells. Cells with almost similar iNOS and nNOS activities displayed comparable cell cycle perturbation, Annexin V positivity, mitochondrial dysfunction, augmented DCF fluorescence, and also attenuated expression of antioxidants. Moreover, induction in cell death was also accompanied by the activation of pJNK/p38MAPK/Erk1/2 and reduction in PI3K/Akt/mTOR signaling. Treatment of NOS isoform overexpressing K562 cells with NAC, a potent free radical scavenger prevented cell death and also the modulations in the signaling proteins. In addition, enhanced expression of CASP1 and CASP4 genes, along with increased Caspase-1 cleavage and increased IL-1ß release were significantly more in K562iNOS cells, which indicate priming of these cells for pyroptotic cell death. On the other hand, K562nNOS cells, displayed much enhanced CASP3 gene expression, Caspase-3 cleavage and Caspase-3 activity. Results obtained indicate that similar level of iNOS or nNOS activation in K562 cells, preferred pyroptotic and apoptotic cell death respectively.


Assuntos
Apoptose/fisiologia , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Piroptose/fisiologia , Acetilcisteína/farmacologia , Apoptose/genética , Caspase 1/genética , Caspase 1/metabolismo , Caspase 3/genética , Caspase 3/metabolismo , Ciclo Celular/genética , Ciclo Celular/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , Depuradores de Radicais Livres/farmacologia , Humanos , Células K562 , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo I/genética , Óxido Nítrico Sintase Tipo II/genética , Piroptose/genética , Espécies Reativas de Oxigênio/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética
16.
Folia Histochem Cytobiol ; 57(4): 179-187, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31840794

RESUMO

INTRODUCTION: Stomach hyperacidity leads to damage of the mucus/bicarbonate barrier, ulcerations and the development of stomach cancer. Key regulators of the mucosal barrier/luminal acid balance are neurotransmitters secreted by intramural neurons. The aim of the current study was to determine the expression of gastric neuropeptides and nNOS in the porcine stomach following hydrochloric acid instillation. We report on increased expression of enteric neurotransmitters involved in adaptive reaction to an experimentally-induced hyperacidity state. MATERIAL AND METHODS: The investigation was conducted on eight 12-18 kg pigs. The influence of intragastric infusion of hydrochloric acid on the expression of cocaine- and amphetamine-regulated transcript peptide (CART), neuronal nitric oxide synthase (nNOS), vasoactive intestinal polypeptide (VIP), pituitary adenylate cyclase-activating peptide (PACAP), substance P (SP) and galanin (GAL) in the submucous and myenteric gastric neurons of the pig has been studied with double immunofluorescence. RESULTS: A mimicked hyperacidity state significantly increased the proportion of enteric neurons immunoreactive to CART, nNOS, VIP, PACAP, SP and GAL in the submucous gastric neurons. In the myenteric plexus, a significant increase of the number of VIP-, CART- and GAL-immunoreactive (IR) neurons was found. Similarly, the percentage of myenteric nNOS-IR and PACAP-IR neurons tended to increase, while the fraction of SP-IR cells did not change. CONCLUSIONS: Stomach hyperacidity modifies the expression of the studied neurotransmitters in a specific way depending on the location of the neurons in particular plexuses of the stomach. Increased numbers of neurons expressing CART, nNOS, VIP, PACAP, SP and GAL clearly indicate their regulatory engagement in the restoration of the physiological gastric balance following hyperacidity.


Assuntos
Ácido Clorídrico/farmacologia , Plexo Mientérico/metabolismo , Neurônios/metabolismo , Neuropeptídeos/metabolismo , Estômago/inervação , Plexo Submucoso/metabolismo , Animais , Feminino , Ácido Clorídrico/administração & dosagem , Infusões Parenterais , Plexo Mientérico/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Óxido Nítrico Sintase Tipo I/metabolismo , Estômago/efeitos dos fármacos , Plexo Submucoso/efeitos dos fármacos , Suínos
17.
Life Sci ; 238: 116922, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31634463

RESUMO

AIMS: Nitric oxide (NO) has a critical, but not well understood, influence in the physiology of the lower urinary tract. We evaluated the effect of NO/phosphodiesterase (PDE)5 signaling in voiding dysfunction in the sickle cell disease (SCD) mouse, characterized by low NO bioavailability. MAIN METHODS: Adult SCD (Sickle) and wild-type (WT) male mice were treated daily with sodium nitrate (10 mM) or vehicle. After 18 days, blood was obtained for nitrite measurement, urethra was collected for organ bath study, and bladder and urethra were collected for Western blot analysis of PDE5 phosphorylation (Ser-92) (activated form). Non-anesthetized mice underwent evaluation of urine volume by void spot assay. eNOS phosphorylation (Ser-1177) and nNOS phosphorylation (Ser-1412) (positive regulatory sites) were evaluated in the bladder and urethra of untreated mice. KEY FINDINGS: Sickle mice exhibited decreased eNOS, nNOS, and PDE5 phosphorylation in the bladder and urethra, decreased plasma nitrite levels, increased relaxation of phenylephrine-contracted urethral tissue to an NO donor sodium nitroprusside, and increased total urine volume, compared with WT mice. Nitrate treatment normalized plasma nitrite levels, relaxation of urethra to sodium nitroprusside, PDE5 phosphorylation in the urethra and bladder, and urine volume in Sickle mice. SIGNIFICANCE: Derangement in PDE5 activity associated with basally low NO bioavailability in the bladder and urethra contributes to the molecular basis for voiding abnormalities in Sickle mice. Inorganic nitrate supplementation normalized voiding in Sickle mice through mechanisms likely involving upregulation of PDE5 activity. These findings suggest that interventions targeting dysregulatory NO/PDE5 signaling may ameliorate overactive bladder in SCD.


Assuntos
Anemia Falciforme/fisiopatologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/metabolismo , Nitratos/administração & dosagem , Óxido Nítrico/metabolismo , Uretra/efeitos dos fármacos , Bexiga Urinária/efeitos dos fármacos , Administração Oral , Animais , Masculino , Camundongos , Óxido Nítrico Sintase Tipo I/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Fosforilação , Uretra/metabolismo , Uretra/patologia , Bexiga Urinária/metabolismo , Bexiga Urinária/patologia
18.
Life Sci ; 239: 116912, 2019 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-31634465

RESUMO

AIMS: Cardiac dysfunction is a major cause of multi-organ dysfunction in critical care units following severe burns. The purpose of this study was to investigate the role of inducible nitric oxide synthase (iNOS) in cardiac dysfunction in burned mice. MATERIALS AND METHODS: Wild-type and iNOS-knockout mice were subjected to 30% total body surface area burns. Next, the expression of iNOS was measured at 1, 3 and 7 days post-burn. Cardiac function, insulin sensitivity, inflammation, oxidative stress, and apoptosis in the hearts of the mice were assessed at 3 days post-burn. KEY FINDINGS: Compared to control mice, iNOS expression was increased and reached a maximum in the heart of burned mice at 3 days post-burn. iNOS deficiency significantly alleviated the cardiac dysfunction and insulin resistance in burned mice. In addition, burn-induced inflammation, oxidative stress, and apoptosis in the heart were markedly reduced in iNOS-knockout burned mice when compared to corresponding values in wild-type burned mice. SIGNIFICANCE: Our study demonstrates that iNOS contributes to insulin resistance in the hearts of mice following burn injury, and iNOS deficiency protects cardiac function against burn injury in mice, suggesting iNOS as a potential therapeutic target to treat burn injuries.


Assuntos
Queimaduras/fisiopatologia , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico Sintase Tipo II/fisiologia , Animais , Apoptose , Queimaduras/complicações , Coração , Cardiopatias/metabolismo , Inflamação/metabolismo , Resistência à Insulina/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Esquelético/metabolismo , Miocárdio/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Estresse Oxidativo/fisiologia , Ratos , Ratos Sprague-Dawley
19.
PLoS One ; 14(10): e0223551, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31596883

RESUMO

Although there is good evidence to indicate a major role of intrinsic impairment of the contractile apparatus in muscle weakness seen in several pathophysiological conditions, the factors responsible for control of myofibrillar function are not fully understood. To investigate the role of mechanical load in myofibrillar function, we compared the skinned fiber force between denervated (DEN) and dexamethasone-treated (DEX) rat skeletal muscles with or without neuromuscular electrical stimulation (ES) training. DEN and DEX were induced by cutting the sciatic nerve and daily injection of dexamethasone (5 mg/kg/day) for 7 days, respectively. For ES training, plantarflexor muscles were electrically stimulated to produce four sets of five isometric contractions each day. In situ maximum torque was markedly depressed in the DEN muscles compared to the DEX muscles (-74% vs. -10%), whereas there was not much difference in the degree of atrophy in gastrocnemius muscles between DEN and DEX groups (-24% vs. -17%). Similar results were obtained in the skinned fiber preparation, with a greater reduction in maximum Ca2+-activated force in the DEN than in the DEX group (-53% vs. -16%). Moreover, there was a parallel decline in myosin heavy chain (MyHC) and actin content per muscle volume in DEN muscles, but not in DEX muscles, which was associated with upregulation of NADPH oxidase (NOX) 2, neuronal nitric oxide synthase (nNOS), and endothelial NOS expression, translocation of nNOS from the membrane to the cytosol, and augmentation of mRNA levels of muscle RING finger protein 1 (MuRF-1) and atrogin-1. Importantly, mechanical load evoked by ES protects against DEN- and DEX-induced myofibrillar dysfunction and these molecular alterations. Our findings provide novel insights regarding the difference in intrinsic contractile properties between DEN and DEX and suggest an important role of mechanical load in preserving myofibrillar function in skeletal muscle.


Assuntos
Dexametasona/farmacologia , Contração Muscular , Miofibrilas/fisiologia , Actinas/metabolismo , Animais , Cálcio/metabolismo , Células Cultivadas , Masculino , Proteínas Musculares/metabolismo , Miofibrilas/efeitos dos fármacos , Miofibrilas/metabolismo , Miosinas/metabolismo , NADPH Oxidase 2/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Nervos Periféricos/fisiologia , Ratos , Ratos Wistar , Estresse Mecânico , Torque , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina-Proteína Ligases/metabolismo
20.
Andrologia ; 51(9): e13349, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31482616

RESUMO

The nitric oxide (NO) pathway plays a role in maintaining the function of the prostate. An impairment in the activity of the NO system may have an impact in the manifestation of lower urinary tract symptomatology and benign prostatic hyperplasia. Arginase enzymes (Arg) counteract the generation of NO by depleting the intracellular pool of L-arginine, known to be the substrate of the NO synthases. This study investigated the expression of arginase type I and II in the human prostate. Nondiseased prostate tissue was obtained during pelvic surgeries (prostatectomy, cystoprostatectomy). Tissue sections were exposed to antibodies directed against Arg I and II, cGMP, the phosphodiesterase 5 and nNOS. The expression of mRNA transcripts encoding for Arg I and Arg II was investigated using molecular biology. Reverse transcriptase polymerase chain reaction (RT-PCR) revealed the presence of mRNA encoding for Arg I and II, immunofluorescence specific for Arg I was seen in the stromal smooth musculature, and labelling for PDE5 and cyclic GMP was also observed. Nerve fibres containing nNOS were identified running across the smooth musculature. Immunostainings for Arg II did not yield signals. These findings are in support of the notion that, in the prostate, Arg is involved in the modulation of the activity of the NO system.


Assuntos
Arginase/metabolismo , Óxido Nítrico/metabolismo , Próstata/metabolismo , Arginase/análise , Arginase/genética , Arginina/metabolismo , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Músculo Liso/metabolismo , Fibras Nervosas/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Próstata/inervação , Próstata/cirurgia , Prostatectomia , RNA Mensageiro/metabolismo , Transdução de Sinais
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