RESUMO
T2 toxin synthetized by Fusarium spp. negatively affects various internal organs and systems, including the digestive tract and the immune, endocrine, and nervous systems. However, knowledge about the effects of T2 on the enteric nervous system (ENS) is still incomplete. Therefore, during the present experiment, the influence of T2 toxin with a dose of 12 µg/kg body weight (b.w.)/per day on the number of enteric nervous structures immunoreactive to neuronal isoform nitric oxide synthase (nNOS-used here as a marker of nitrergic neurons) in the porcine duodenum was studied using the double immunofluorescence method. Under physiological conditions, nNOS-positive neurons amounted to 38.28 ± 1.147%, 38.39 ± 1.244%, and 35.34 ± 1.151 of all enteric neurons in the myenteric (MP), outer submucous (OSP), and inner submucous (ISP) plexuses, respectively. After administration of T2 toxin, an increase in the number of these neurons was observed in all types of the enteric plexuses and nNOS-positive cells reached 46.20 ± 1.453% in the MP, 45.39 ± 0.488% in the OSP, and 44.07 ± 0.308% in the ISP. However, in the present study, the influence of T2 toxin on the intramucosal and intramuscular nNOS-positive nerves was not observed. The results obtained in the present study indicate that even low doses of T2 toxin are not neutral for living organisms because they may change the neurochemical characterization of the enteric neurons.
Assuntos
Duodeno/inervação , Fusarium/fisiologia , Óxido Nítrico Sintase Tipo I/metabolismo , Suínos/fisiologia , Toxina T-2/metabolismo , Animais , Duodeno/enzimologia , Feminino , Fusariose/metabolismo , Fusariose/microbiologia , Fusariose/veterinária , Neurônios Nitrérgicos/enzimologia , Óxido Nítrico Sintase Tipo I/análise , Dados Preliminares , Suínos/microbiologia , Doenças dos Suínos/metabolismo , Doenças dos Suínos/microbiologiaRESUMO
We aimed to determine the influence of nitrergic innervation function on the decreased mesenteric arterial tone induced by high levels of triiodothyronine (T3), as a model of acute thyroiditis, as well as the mechanism/s implicated. We analysed in mesenteric segments from male Wistar rats the effect of 10â¯nmol/L T3 (2â¯h) on the vasomotor response to electrical field stimulation (EFS) in the presence/absence of specific neuronal NOS (nNOS) inhibitor L-NPA, or superoxide anion scavenger tempol. Nitric oxide (NO) release was measured in the presence/absence of tempol or PI3K inhibitor LY294002. Superoxide anion and peroxynitrite releases, nNOS, PI3K, AKT and superoxide dismutase (SOD) 1 and 2 expressions, nNOS and AKT phosphorylation, and SOD activity were analysed. T3 decreased EFS-induced vasoconstriction. L-NPA increased EFS-induced vasoconstriction more markedly in T3-incubated segments. T3 increased NO release. Tempol decreased EFS-induced vasoconstriction and augmented NO release only in segments without T3. LY294002 decreased NO release in T3-incubated segments. T3 diminished superoxide anion and peroxynitrite formation, enhanced SOD-2 expression, nNOS and AKT phosphorylations and SOD activity, and did not modify nNOS, PI3K, AKT and SOD-1 expressions. In conclusion, these results show a compensatory mechanism aimed at reducing the enhanced blood pressure that appears during acute thyroiditis.
Assuntos
Artérias Mesentéricas/inervação , Neurônios Nitrérgicos/efeitos dos fármacos , Óxido Nítrico/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Tri-Iodotironina/farmacologia , Vasoconstrição/efeitos dos fármacos , Vasodilatação/efeitos dos fármacos , Animais , Relação Dose-Resposta a Droga , Estimulação Elétrica , Inibidores Enzimáticos/farmacologia , Sequestradores de Radicais Livres/farmacologia , Técnicas In Vitro , Masculino , Neurônios Nitrérgicos/enzimologia , Óxido Nítrico Sintase Tipo I/antagonistas & inibidores , Óxido Nítrico Sintase Tipo I/metabolismo , Estresse Nitrosativo/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Ácido Peroxinitroso/metabolismo , Fosforilação , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Superóxido Dismutase/metabolismo , Superóxidos/metabolismo , Fatores de TempoRESUMO
Increase in hyperglycaemia-induced oxidative stress and decreased effectiveness of endogenous defense mechanisms plays an essential role in the initiation of diabetes-related neuropathy. We demonstrated that nitrergic myenteric neurons display different susceptibilities to diabetic damage in different gut segments. Therefore, we aim to reveal the gut segment-specific differences in the expression of heme oxygenase (HO) isoforms and the colocalization of these antioxidants with neuronal nitric oxide synthase (nNOS) in myenteric neurons. After ten weeks, samples from the duodenum, ileum, and colon of control and streptozotocin-induced diabetic rats were processed for double-labelling fluorescent immunohistochemistry and ELISA. The number of both HO-immunoreactive and nNOS/HO-immunoreactive myenteric neurons was the lowest in the ileal and the highest in the colonic ganglia of controls; it increased the most extensively in the ileum and was also elevated in the colon of diabetics. Although the total number of nitrergic neurons decreased in all segments, the proportion of nNOS-immunoreactive neurons colocalizing with HOs was enhanced robustly in the ileum and colon of diabetics. We presume that those nitrergic neurons which do not colocalize with HOs are the most seriously affected by diabetic damage. Therefore, the regional induction of the HO system is strongly correlated with diabetes-related region-specific nitrergic neuropathy.
Assuntos
Diabetes Mellitus Experimental/enzimologia , Heme Oxigenase (Desciclizante)/metabolismo , Neurônios Nitrérgicos/enzimologia , Óxido Nítrico Sintase Tipo I/metabolismo , Animais , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/patologia , Ensaio de Imunoadsorção Enzimática , Heme Oxigenase (Desciclizante)/sangue , Imuno-Histoquímica , Intestinos/enzimologia , Intestinos/patologia , Masculino , Plexo Mientérico/enzimologia , Plexo Mientérico/patologia , Neurônios Nitrérgicos/patologia , Óxido Nítrico Sintase Tipo I/sangue , Ratos , Ratos WistarRESUMO
The diverse signaling events following mitochondrial depolarization in neurons are not clear. We examined for the first time the effects of mitochondrial depolarization on mitochondrial function, intracellular calcium, neuronal nitric oxide synthase (nNOS) activation, and nitric oxide (NO) production in cultured neurons and perivascular nerves. Cultured rat primary cortical neurons were studied on 7-10 days in vitro, and endothelium-denuded cerebral arteries of adult Sprague-Dawley rats were studied ex vivo. Diazoxide and BMS-191095 (BMS), activators of mitochondrial KATP channels, depolarized mitochondria in cultured neurons and increased cytosolic calcium levels. However, the mitochondrial oxygen consumption rate was unaffected by mitochondrial depolarization. In addition, diazoxide and BMS not only increased the nNOS phosphorylation at positive regulatory serine 1417 but also decreased nNOS phosphorylation at negative regulatory serine 847. Furthermore, diazoxide and BMS increased NO production in cultured neurons measured with both fluorescence microscopy and electron spin resonance spectroscopy, which was sensitive to inhibition by the selective nNOS inhibitor 7-nitroindazole (7-NI). Diazoxide also protected cultured neurons against oxygen-glucose deprivation, which was blocked by NOS inhibition and rescued by NO donors. Finally, BMS induced vasodilation of endothelium denuded, freshly isolated cerebral arteries that was diminished by 7-NI and tetrodotoxin. Thus pharmacological depolarization of mitochondria promotes activation of nNOS leading to generation of NO in cultured neurons and endothelium-denuded arteries. Mitochondrial-induced NO production leads to increased cellular resistance to lethal stress by cultured neurons and to vasodilation of denuded cerebral arteries.
Assuntos
Artérias Cerebrais/metabolismo , Potencial da Membrana Mitocondrial , Mitocôndrias/enzimologia , Neurônios Nitrérgicos/enzimologia , Óxido Nítrico Sintase Tipo I/metabolismo , Óxido Nítrico/metabolismo , Comunicação Parácrina , Vasodilatação , Animais , Benzopiranos/farmacologia , Células Cultivadas , Artérias Cerebrais/efeitos dos fármacos , Artérias Cerebrais/inervação , Diazóxido/farmacologia , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Imidazóis/farmacologia , Indazóis/farmacologia , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Neurônios Nitrérgicos/efeitos dos fármacos , Óxido Nítrico Sintase Tipo I/antagonistas & inibidores , Comunicação Parácrina/efeitos dos fármacos , Fosforilação , Canais de Potássio/agonistas , Canais de Potássio/metabolismo , Cultura Primária de Células , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Serina , Transdução de Sinais , Vasodilatação/efeitos dos fármacosRESUMO
Immunohistochemical methods for the demonstration of tyrosine hydrolase (TH) and neuronal form of nitric oxide synthase (nNOS) were used to study the distribution of catecholaminergic and nitroxidergic vasomotor neurons respectively, in the nuclei of the medulla oblongata and the pons of 12 Wistar rats. Most often the expression of TG was found in neurons located in the nucleus and several reticular nuclei (gigantocellular, paragigantocellular, caudal pons nucleus), but the proportion of immunoreactive neurons did not exceed 8-14%. In the other nuclei (reticular parvocellular nucleus and oral pons nucleus, spinal nucleus of the trigeminal nerve) the value of this parameter ranged from 1 to 3%. In a large group of nuclei with proven vasomotor function such neurons were constantly not detected. In the structures with high content of catecholaminergic neurons, nNOS-positive cells were found, as a rule, in fewer numbers than in the nuclei with a limited number of TH-positive neurons.
Assuntos
Neurônios Nitrérgicos , Óxido Nítrico Sintase Tipo I/metabolismo , Núcleo Inferior Caudal do Nervo Trigêmeo , Tirosina 3-Mono-Oxigenase/metabolismo , Sistema Vasomotor , Animais , Neurônios Nitrérgicos/citologia , Neurônios Nitrérgicos/enzimologia , Ratos , Ratos Wistar , Núcleo Inferior Caudal do Nervo Trigêmeo/citologia , Núcleo Inferior Caudal do Nervo Trigêmeo/enzimologia , Sistema Vasomotor/citologia , Sistema Vasomotor/enzimologiaRESUMO
The aim of this study was to examine the distribution of cholinergic and nitroxidergic neurons in the spinal cord (SC) of adult and newborn rats. Using immunohistochemical demonstration of choline acetyltransferase (ChAT) and nitric oxide synthase (NOS), cervical portions of SC were studied in newborn (n=5) and adult (n=5) Wistar rats. It was found that ChAT-positive neurons were localized in the anterior horns of the SC, while individual cells were located in of SC posterior horns, in the central gray matter and at the boundary of VI-VII Rexed laminae. Nitroxidergic neurons were located in the superficial layers of SC posterior horns of grey matter, in the central gray matter and in the area of VI-VII Rexed laminae. It is found that SC of newborn and adult rats contained cholinergic neurons expressing NOS. Detection of cells containing both enzymes already at postnatal Day 1, suggests that they were formed in rat SC during prenatal ontogenesis
Assuntos
Neurônios Colinérgicos , Neurônios Nitrérgicos , Óxido Nítrico Sintase Tipo I/metabolismo , Corno Dorsal da Medula Espinal , Corno Lateral da Medula Espinal , Animais , Animais Recém-Nascidos , Neurônios Colinérgicos/citologia , Neurônios Colinérgicos/enzimologia , Neurônios Nitrérgicos/citologia , Neurônios Nitrérgicos/enzimologia , Ratos , Ratos Wistar , Corno Dorsal da Medula Espinal/citologia , Corno Dorsal da Medula Espinal/enzimologia , Corno Lateral da Medula Espinal/citologia , Corno Lateral da Medula Espinal/enzimologiaRESUMO
CONTEXT: The rectal distension in dogs increases the rate of transitory lower esophageal sphincter relaxation considered the main factor causing gastroesophageal reflux. OBJECTIVES: The aim of this study was evaluate the participation of the nitrergic pathway in the increased transitory lower esophageal sphincter relaxation rate induced by rectal distension in anesthetized dogs. METHODS: Male mongrel dogs (n = 21), weighing 10-15 kg, were fasted for 12 hours, with water ad libitum. Thereafter, they were anesthetized (ketamine 10 mg.Kg-1 + xylazine 20 mg.Kg-1), so as to carry out the esophageal motility evaluation protocol during 120 min. After a 30-minute basal period, the animals were randomly intravenous treated whith: saline solution 0.15M (1ml.Kg-1), L-NAME (3 mg.Kg-1), L-NAME (3 mg.Kg-1) + L-Arginine (200 mg.Kg-1), glibenclamide (1 mg.Kg-1) or methylene blue (3 mg.Kg-1). Forty-five min after these pre-treatments, the rectum was distended (rectal distension, 5 mL.Kg-1) or not (control) with a latex balloon, with changes in the esophageal motility recorded over 45 min. Data were analyzed using ANOVA followed by Student Newman-Keuls test. RESULTS: In comparison to the respective control group, rectal distension induces an increase in transitory lower esophageal sphincter relaxation. Pre-treatment with L-NAME or methylene blue prevents (P<0.05) this phenomenon, which is reversible by L-Arginine plus L-NAME. However, pretreating with glibenclamide failed to abolish this process. CONCLUSIONS: Therefore, these experiments suggested, that rectal distension increases transitory lower esophageal sphincter relaxation in dogs via through nitrergic pathways.
Assuntos
Esfíncter Esofágico Inferior/fisiologia , Junção Esofagogástrica/fisiologia , Neurônios Nitrérgicos/metabolismo , Nitroarginina/farmacologia , Peristaltismo/fisiologia , Reto/fisiologia , Animais , Cães , Motilidade Gastrointestinal/fisiologia , Masculino , Manometria , Neurônios Nitrérgicos/efeitos dos fármacos , Neurônios Nitrérgicos/enzimologia , Reflexo/fisiologiaRESUMO
AIM: To investigate the relationship between neuronal nitric oxide synthase (nNOS) expression and the natriuretic peptide signaling pathway in the gastric fundus of streptozotocin (STZ)-induced diabetic mice. METHODS: Diabetic mice were induced by injection of STZ solution. Immunofluorescence labeling of HuC/D, nNOS and natriuretic peptide receptor-A, B, C (NPRs) in the gastric fundus (GF) was used to observe nNOS expression and whether NPRs exist on enteric neurons. The expression levels of nNOS and NPRs in the diabetic GF were examined by western blotting. An isometric force transducer recorded the electric field stimulation (EFS)-induced relaxation and contraction in the diabetic GF. An intracellular recording method assessed EFS-induced inhibitory junction potentials (IJP) on the GF. GF smooth muscles acquired from normal mice were incubated with different concentrations of the NPRs agonist C-type natriuretic peptide (CNP) for 24 h, after which their nNOS expressions were detected by western blotting. RESULTS: Eight weeks after injection, 43 diabetic mice were obtained from mouse models injected with STZ. Immunofluorescence indicated that the number of NOS neurons was significantly decreased and that nNOS expression was significantly downregulated in the diabetic GF. The results of physiological and electrophysiological assays showed that the EFS-induced relaxation that mainly caused by NO was significantly reduced, while the contraction was enhanced in the diabetic GF. EFS-induced IJP showed that L-NAME sensitive IJP in the diabetic GF was significantly reduced compared with control mice. However, both NPR-A and NPR-B were detected on enteric neurons, and their expression levels were upregulated in the diabetic GF. The nNOS expression level was downregulated dose-dependently in GF smooth muscle tissues exposed to CNP. CONCLUSION: These findings suggested that upregulation of the NPs signaling pathway may be involved in GF neuropathy caused by diabetes by decreasing nNOS expression.
Assuntos
Diabetes Mellitus Experimental/enzimologia , Sistema Nervoso Entérico/enzimologia , Fundo Gástrico/inervação , Músculo Liso/inervação , Peptídeos Natriuréticos/metabolismo , Neurônios Nitrérgicos/enzimologia , Óxido Nítrico Sintase Tipo I/metabolismo , Animais , Glicemia/metabolismo , Peso Corporal , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/fisiopatologia , Regulação para Baixo , Estimulação Elétrica , Sistema Nervoso Entérico/fisiopatologia , Masculino , Camundongos Endogâmicos ICR , Contração Muscular , Óxido Nítrico/metabolismo , Receptores do Fator Natriurético Atrial/metabolismo , Transdução de Sinais , Estreptozocina , Técnicas de Cultura de Tecidos , Regulação para CimaRESUMO
Context The rectal distension in dogs increases the rate of transitory lower esophageal sphincter relaxation considered the main factor causing gastroesophageal reflux. Objectives The aim of this study was evaluate the participation of the nitrergic pathway in the increased transitory lower esophageal sphincter relaxation rate induced by rectal distension in anesthetized dogs. Methods Male mongrel dogs (n = 21), weighing 10-15 kg, were fasted for 12 hours, with water ad libitum. Thereafter, they were anesthetized (ketamine 10 mg.Kg-1 + xylazine 20 mg.Kg-1), so as to carry out the esophageal motility evaluation protocol during 120 min. After a 30-minute basal period, the animals were randomly intravenous treated whith: saline solution 0.15M (1ml.Kg-1), L-NAME (3 mg.Kg-1), L-NAME (3 mg.Kg-1) + L-Arginine (200 mg.Kg-1), glibenclamide (1 mg.Kg-1) or methylene blue (3 mg.Kg-1). Forty-five min after these pre-treatments, the rectum was distended (rectal distension, 5 mL.Kg-1) or not (control) with a latex balloon, with changes in the esophageal motility recorded over 45 min. Data were analyzed using ANOVA followed by Student Newman-Keuls test. Results In comparison to the respective control group, rectal distension induces an increase in transitory lower esophageal sphincter relaxation. Pre-treatment with L-NAME or methylene blue prevents (P<0.05) this phenomenon, which is reversible by L-Arginine plus L-NAME. However, pretreating with glibenclamide failed to abolish this process. Conclusions Therefore, these experiments suggested, that rectal distension increases transitory lower esophageal sphincter relaxation in dogs via through nitrergic pathways. .
Contexto A distensão retal aumenta a taxa de relaxamento transitório do esfíncter esofágico inferior em cães, sendo o relaxamento transitório do esfíncter esofágico inferior considerado o principal fator responsável pelo refluxo gastroesofágico. Objetivos Avaliar a participação da via nitrérgica no aumento da taxa relaxamento transitório do esfíncter esofágico inferior induzida por distensão retal em cães anestesiados. Métodos Cães sem raça definida, machos (n = 21), pesando entre 10-15 kg, foram mantidos em jejum durante 12 horas, no entanto, com água ad libitum. Depois disso, eles foram anestesiados (cetamina 10 mg.Kg-1 + xilazina 20 mg.Kg-1), para a realização do protocolo de avaliação da motilidade esofágica durante 120 minutos. Após um período basal de 30 minutos, os animais foram aleatoriamente tratados intravenosa com: solução salina 0,15 (1 ml.Kg-1), L-NAME (3 mg.Kg-1), L-NAME (3 mg.Kg-1) + L-arginina (200 mg.Kg-1), glibenclamida (1 mg.Kg-1) e azul de metileno (3 mg.Kg-1). Quarenta e cinco minutos após os pré-tratamentos, o reto foi distendido com um balão de látex (DR, 5 mg.Kg-1) ou não (grupo controle), e as variações da motilidade esofágica foram registradas e gravadas ao longo dos 45 minutos seguintes. Os dados foram analisados utilizando-se ANOVA seguido pelo teste de Student Newman-Keuls. Resultados Em comparação com o respectivo grupo controle, a distensão retal demonstrou induzir um aumento na taxa de relaxamento transitório do esfíncter esofágico inferior. O pré-tratamento com L -NAME ou azul de metileno impediu (P<0,05) este fenômeno, que foi reversível após a administração de L-Arginina + L-NAME. No entanto, o pré-tratamento com a glibenclamida não ...
Assuntos
Animais , Cães , Masculino , Esfíncter Esofágico Inferior/fisiologia , Junção Esofagogástrica/fisiologia , Neurônios Nitrérgicos/metabolismo , Nitroarginina/farmacologia , Peristaltismo/fisiologia , Reto/fisiologia , Motilidade Gastrointestinal/fisiologia , Manometria , Neurônios Nitrérgicos/efeitos dos fármacos , Neurônios Nitrérgicos/enzimologia , Reflexo/fisiologiaRESUMO
The presence of nitrergic cells in the prefrontal cortex has been confirmed, however little is known about the postnatal development of these cells. Nitrergic neurons were studied histochemically by using NADPH-diaphorase staining in the prefrontal cortex of male Wistar rats from postnatal day 7-21 (P7-21). Neuronal NADPH-diaphorase is a nitric oxide synthase that provides a specific histochemical marker for neurons producing nitric oxide (NO). NO acts as a neurotransmitter and intracellular signaling molecule in the nervous system. We observed in 7 day old rats NADPH-d containing neurons that were intensely stained. These neurons were bipolar with a short dendrite with average length of 23 µm. During the second postnatal week, the neurons were mainly bipolar and were rarely multipolar. By P14 the cells were located primarily in cortical layers III-VI. Nitrergic neurons of the 21 day old rats were histochemically identified as multipolar cells with long radial extending dendrites. Dendrites of neurons in 14 and 21 day old rats were a similar length with an average of 57 µm. These results suggest that nitrergic neurons differentiate during a relatively short period of time and reach their structural maturity by the end of the second week of postnatal development.
Assuntos
Neurônios Nitrérgicos/citologia , Córtex Pré-Frontal/citologia , Córtex Pré-Frontal/crescimento & desenvolvimento , Animais , Animais Recém-Nascidos , Diferenciação Celular , Histocitoquímica , Masculino , NADPH Desidrogenase/metabolismo , Neurônios Nitrérgicos/enzimologia , Córtex Pré-Frontal/enzimologia , Ratos , Ratos WistarRESUMO
The ionic basis of nitrergic "slow'" inhibitory junction potential (sIJP) is not fully understood. The purpose of the present study was to determine the nature and the role of calmodulin-dependent protein kinase II (CaMKII)-dependent ion conductance in nitrergic neurotransmission at the intestinal smooth muscle neuromuscular junction. Studies were performed in guinea pig ileum. The modified Tomita bath technique was used to induce passive hyperpolarizing electrotonic potentials (ETP) and membrane potential change due to sIJP or drug treatment in the same cell. Changes in membrane potential and ETP were recorded in the same smooth muscle cell, using sharp microelectrode. Nitrergic IJP was elicited by electrical field stimulation in nonadrenergic, noncholinergic conditions and chemical block of purinergic IJP. Modification of ETP during hyperpolarization reflected active conductance change in the smooth muscle. Nitrergic IJP was associated with decreased membrane conductance. The CAMKII inhibitor KN93 but not KN92, the Cl(-) channel blocker niflumic acid (NFA), and the K(ATP)-channel opener cromakalim hyperpolarized the membrane. However, KN93 and NFA were associated with decreased and cromakalim was associated with increased membrane conductance. After maximal NFA-induced hyperpolarization, hyperpolarization associated with KN93 or sIJP was not seen, suggesting a saturation block of the Cl(-) channel signaling. These studies suggest that inhibition of CaMKII-dependent Cl(-) conductance mediates nitrergic sIJP by causing maximal closure of the Cl(-) conductance.
Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/fisiologia , Canais de Cloreto/fisiologia , Íleo/enzimologia , Potenciais da Membrana/fisiologia , Músculo Liso/enzimologia , Neurônios Nitrérgicos/enzimologia , Animais , Benzilaminas/farmacologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Canais de Cloreto/efeitos dos fármacos , Cromakalim/farmacologia , Feminino , Cobaias , Íleo/efeitos dos fármacos , Masculino , Potenciais da Membrana/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Ácido Niflúmico/farmacologia , Neurônios Nitrérgicos/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Sulfonamidas/farmacologiaRESUMO
Spinal cord ischemia belongs to serious and relatively frequent diseases of CNS. The aim of the present study was to find out the vulnerability of nitrergic neurons to 15 min transient spinal cord ischemia followed by 1 and 2 weeks of reperfusion. We studied neuronal nitric oxide synthase (nNOS) and nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) in structural elements of lumbosacral spinal cord along its rostrocaudal axis. In addition, a neurological deficit of experimental animals was evaluated. Spinal cord ischemia, performed on the rabbit, was induced by abdominal aorta occlusion using Fogarty catheter introduced into the right femoral artery for a period of 15 min. After surgical intervention the animals survived for 7 and 14 days. nNOS-immunoreactivity (nNOS-IR) was measured by immunohistochemical and NADPHd-positivity by histochemical method, and both immunohistochemical and histochemical stainings were quantified by densitometric analyses. Neurological deficit was evaluated according Zivin's criteria. The number of nNOS-IR and/or NADPH-d positive neurons and the density of neuropil were markedly increased in superficial dorsal horn (laminae I-III) after 15 min ischemia and 7 days of reperfusion. However, ischemia followed by longer time of survival (14 days) returned the number of nNOS-IR and NADPH-d positive neurons to control. In the pericentral region (lamina X) containing interneurons and crossing fibers of spinal tracts, than in lamina VII and in dorsomedial part of the ventral horn (lamina VIII) we recorded a decreased number of nNOS-IR and NADPH-d positive neurons after both ischemia/reperfusion periods. In the medial portion of lamina VII and dorsomedial part of the ventral horn (lamina VIII) we observed many necrotic loci. This area was the most sensitive to ischemia/reperfusion injury. Fifteen minute ischemia caused a marked deterioration of neurological function of hind limbs, often developing into paraplegia. A quantitative immunohistochemical and histochemical study have shown a strong vulnerability of nitrergic neurons in intermediate zone to transient spinal cord ischemia.
Assuntos
Neurônios Nitrérgicos/patologia , Paraplegia/patologia , Traumatismo por Reperfusão/patologia , Isquemia do Cordão Espinal/patologia , Medula Espinal/patologia , Animais , Aorta Abdominal/patologia , Aorta Abdominal/cirurgia , Cateterismo , Contagem de Células , Feminino , Membro Posterior , Imuno-Histoquímica , NADPH Desidrogenase/metabolismo , Neurônios Nitrérgicos/enzimologia , Óxido Nítrico Sintase Tipo I/metabolismo , Paraplegia/complicações , Paraplegia/enzimologia , Coelhos , Traumatismo por Reperfusão/complicações , Traumatismo por Reperfusão/enzimologia , Medula Espinal/enzimologia , Isquemia do Cordão Espinal/complicações , Isquemia do Cordão Espinal/enzimologiaRESUMO
The distribution of neuronal nitric oxide synthase (nNOS) in the process of normal mouse brain growth from embryonic (E) Day 11 to postnatal (P) Day 1 was investigated by means of immunohistochemical and immunofluorescent methods. Our results demonstrated that nNOS positive neurons appeared early in superficial cortex at E11. At E13, nNOS positive neurons were located in lateral hypothalamus and amygdala, and temporarily in medullar and ventral hypothalamic neuroepithelia. From E15 to P0, nNOS positive neurons were distributed in superior and inferior colliculi, positive staining could also be seen in superior and inferior tectal neuroepithelium at E15. From E17 to birth, the medial geniculate nucleus had a high density of nNOS labeling. The distribution of nNOS gradually increased and extended laterally in embryo brain, which in turn implies that NO might be involved in the development of mouse brain.
Assuntos
Encéfalo/embriologia , Encéfalo/enzimologia , Neurônios Nitrérgicos/enzimologia , Óxido Nítrico Sintase Tipo I/biossíntese , Óxido Nítrico Sintase Tipo I/genética , Animais , Animais Recém-Nascidos , Encéfalo/citologia , Diferenciação Celular/fisiologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos ICR , Neurogênese/fisiologia , Neurônios Nitrérgicos/citologia , Óxido Nítrico/fisiologia , Óxido Nítrico Sintase Tipo I/metabolismo , GravidezRESUMO
The results of the application of a "pixel method" for the automated measurement of the intensity of histochemical reaction in the neurons are presented. The method is based on measuring principle, which includes the automatic counting the sum of brightnesses of all the pixels forming the image with the help of standard computer programs. The potential of this method is demonstrated on the example of NADPH-diaphorase activity study in the nitroxidergic neurons of sensory and motor nuclei of the medulla of healthy rats.
Assuntos
Histocitoquímica/métodos , Neurônios Motores , NADPH Desidrogenase/isolamento & purificação , Neurônios Nitrérgicos , Animais , Processamento de Imagem Assistida por Computador , Masculino , Modelos Teóricos , Neurônios Motores/citologia , Neurônios Motores/enzimologia , NADPH Desidrogenase/metabolismo , Neurônios Nitrérgicos/citologia , Neurônios Nitrérgicos/enzimologia , RatosRESUMO
We have studied the development of NADPH-diaphorase activity in the retinorecipient layers of the superior colliculus (SC) in rats from embryonic day 17 to adulthood, during aging, and following neonatal tetrodotoxin injection or unilateral eye removal in the neonatal or in the adult animal. In the superficial SC, NADPH-d activity is first seen in neurons on postnatal day (P) 4; over the next two weeks, enzyme expression increases gradually, in cells as well as in the neuropil. By P12-14, around the time of eye opening, NADPH-d reactivity increases dramatically. In parallel, the dendrites of many NADPH-d-positive neurons in the superficial gray layer, more or less randomly distributed at first, gradually align their orientation relative to the dorsoventral axis. The pattern of NADPH-d activity in the superficial layers of the SC (i.e. stratum griseum superficiale and stratum opticum) is adult-like by the fourth week of age. Deafferentation of the superficial SC, both in the neonatal and adult rat, and block of retinal activity lead to reduction in the size of the SC and changes in NADPH-d-positive neurons, including dendrite misorientation, decreased cell size and reduced number. Some of these changes are seen also in the aging animal. These results document a protracted and progressive increase in the development of NADPH-d expression in the SC. Our results suggest a strong influence of retinal afferents and activity on the development and maintenance of NAPHD-positive neurons in the retinorecipient layers of the SC, where NO can act as a retrograde signal to carve the terminal arbors of retinal axons.
Assuntos
Senescência Celular/fisiologia , NADPH Desidrogenase/metabolismo , Neurônios Nitrérgicos/enzimologia , Colículos Superiores/enzimologia , Colículos Superiores/crescimento & desenvolvimento , Animais , Animais Recém-Nascidos , Axônios/fisiologia , Feminino , Masculino , Neurogênese/fisiologia , Neurônios Nitrérgicos/citologia , Óxido Nítrico/fisiologia , Ratos , Retina/citologia , Retina/fisiologia , Colículos Superiores/citologiaRESUMO
In this study, we investigated the expression of neuronal nitric oxide synthase (nNOS) and nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d), two specific enzymes for nitric oxide (NO) synthesis, in the development of liver fibrosis induced by chronic bile duct ligation (BDL) in the rabbit. We specifically studied the liver-innervated nitroxidergic neurons that originate in the nodose ganglion (NG), nucleus of the solitary tract (NTS) and dorsal motor vagal nucleus (DMV). Our data showed that BDL resulted in overexpression of NADPH-d/nNOS in the NG, NTS and DMV neurons. Using densitometric analysis, we found a significant increase in NADPH-d expression as a result of BDL in the NG, NTS and DMV (72.6, 79.4 and 57.4% increase, respectively). These findings were corroborated by serum biochemistry and hepatic histopathological examination, which were influenced by NADPH-d/nNOS-generated NO in the liver following BDL. Upregulation of NADPH-d/nNOS expression may have important implications, including (1) facilitation of extrahepatic biliary parasympathetic tone that promotes gallbladder emptying of excess stagnant bile; (2) relaxation of smooth muscles of bile canaliculi thus participating in the pathogenesis of cholestasis; (3) dilation of hepatic sinusoids to counter BDL-induced intrahepatic portal hypertension in which endothelia may be damaged, and (4) alterations in hepatic metabolism, such as glycogenesis, bile formation and secretion, and bilirubin clearance.
Assuntos
Sistema Biliar/fisiologia , Icterícia Obstrutiva/patologia , NADPH Desidrogenase/metabolismo , Neurônios Nitrérgicos/patologia , Óxido Nítrico Sintase Tipo I/metabolismo , Nervo Vago/patologia , Animais , Icterícia Obstrutiva/metabolismo , Neurônios Nitrérgicos/enzimologia , Gânglio Nodoso/enzimologia , Gânglio Nodoso/patologia , Coelhos , Nervo Vago/enzimologiaRESUMO
This study focussed on the development of the corpus striatum in the fetus, using silver impregnation and immunohistochemistry. For the latter, we looked for nNOS positive cells and 5-HT(2A) receptors positive cells in the corpus striatum during development. During the initial formation of the corpus striatum, there was migration cells of the ganglionic eminence toward the putamen by 15-17 weeks of gestation. Process formation in the neurons started by week 17 and became very complex before term (31/32 weeks of gestation). By 25-27 gestational weeks, the globus pallidus already had two parts and the corpus striatum was similar to the adult in configuration. The nNOS positive cells appeared early (21-23 weeks in gestation) while 5-HT(2A) receptors positive cells were not observed until 31/32 weeks gestation. The number of positive cells in both groups was relatively small. It is anticipated that further developmental changes would occur in the postnatal/neonatal phases.
Assuntos
Corpo Estriado/embriologia , Corpo Estriado/metabolismo , Neurogênese/fisiologia , Neurônios Nitrérgicos/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Receptor 5-HT2A de Serotonina/metabolismo , Padronização Corporal/fisiologia , Movimento Celular/fisiologia , Corpo Estriado/enzimologia , Feminino , Feto , Humanos , Masculino , Neurônios Nitrérgicos/citologia , Neurônios Nitrérgicos/enzimologia , Óxido Nítrico Sintase Tipo I/biossíntese , Gravidez , Receptor 5-HT2A de Serotonina/biossínteseRESUMO
Dorsal root ganglia (DRGs) contain the cell bodies of primary afferent neurons that transmit sensory information from the periphery into the spinal cord. Distinct populations of DRG neurons have been characterized by a variety of different immunohistochemical markers. A subpopulation of ganglionic neurons containing neuronal nitric oxide synthase (nNOS), an enzyme known to generate nitric oxide, has been detected in a number of mammalian species. Despite previous studies, no information is known on the presence and exact distribution of nNOS-immunoreactive neurons in the DRGs of the bottlenose dolphin. In this investigation, immunoperoxidase for nNOS was used to determine the distribution and the perikaryal size of nitrergic neurons in the DRGs of this species. Double immunofluorescence protocol was used to determine the percentage of nNOS-immunoreactive (IR) neurons over the total primary afferent neurons. In addition, double immunostaining was used to verify whether there was colocalization of nNOS with substance P (SP). In all DRGs, a subpopulation of small- and medium-sized neurons (about 9%) exhibited nNOS immunoreactivity. Data analysis revealed that the majority of nNOS-IR neurons (81.3%) expressed SP. The density of nNOS-immunoreactive and nNOS/SP-double immunopositive cells was relatively constant throughout the ganglia. However, as observed in others mammals, the number of nitrergic neurons decreased in the caudalmost DRGs. Our results, in conjunction with previous observations, suggest that nNOS-IR neurons may be involved in the afferent transmission of visceral and nociceptive information as well as in the regulation of the vascular tone.
Assuntos
Golfinho Nariz-de-Garrafa , Gânglios Espinais/química , Neurônios Nitrérgicos/química , Óxido Nítrico Sintase Tipo I/metabolismo , Óxido Nítrico/análise , Animais , Golfinho Nariz-de-Garrafa/metabolismo , Gânglios Espinais/enzimologia , Neurônios Nitrérgicos/enzimologia , Óxido Nítrico/metabolismoRESUMO
Nitric oxide (NO)-containing neurons are widely distributed within the central nervous system, including regions involved in the control of reproduction and sexual behavior. Nitrergic neurons may co-localize with gonadal hormone receptors and gonadal hormones may influence neuronal NO synthase expression in adulthood as well as during development. In rodents, the female, in physiological conditions, is exposed to short-term changes of gonadal hormones levels (estrous cycle). Our studies, performed in mouse hypothalamic and limbic systems, reveal that the expression of neuronal NO synthase may vary according to the rapid variations of hormonal levels that take place during the estrous cycle. This is in accordance with the hypothesis that gonadal hormone activation of NO-cGMP pathway is important for mating behavior. NO-producing system appears particularly sensitive to alterations of endocrine balance during development, as demonstrated by our experiments utilizing perinatal exposure to bisphenol A, an endocrine disrupting chemical. In fact, significant effects were detected in adulthood in the medial preoptic nucleus and in the ventromedial subdivision of the bed nucleus of the stria terminalis. Therefore, alteration of the neuronal NO synthase expression may be one of the causes of the important behavioral alterations observed in bisphenol-exposed animals.
Assuntos
Comportamento Animal/fisiologia , Disruptores Endócrinos/toxicidade , Ciclo Estral/fisiologia , Hipotálamo/metabolismo , Neurônios Nitrérgicos/metabolismo , Óxido Nítrico/biossíntese , Animais , Comportamento Animal/efeitos dos fármacos , Compostos Benzidrílicos , Disruptores Endócrinos/metabolismo , Estrogênios não Esteroides/metabolismo , Estrogênios não Esteroides/toxicidade , Ciclo Estral/efeitos dos fármacos , Feminino , Hipotálamo/efeitos dos fármacos , Hipotálamo/enzimologia , Masculino , Camundongos , Neurônios Nitrérgicos/efeitos dos fármacos , Neurônios Nitrérgicos/enzimologia , Óxido Nítrico Sintase Tipo I/biossíntese , Fenóis/metabolismo , Fenóis/toxicidadeRESUMO
1. Nitrergic neurons regulate gastrointestinal (GI) activity and their dysfunction has been associated with various GI diseases. Nitric oxide (NO) typically relaxes GI smooth muscle, but nitrergic contractions also occur. Although guanylate cyclase is well established as mediating nitrergic GI relaxation, its role in contraction remains uncertain. 2. We used electrical field stimulation (EFS; 0.3 msec pulses, three trains of 1.2 s width, 2 Hz, at 30 s intervals) to evoke biphasic contraction-relaxation responses in rat ileum strips (longitudinal muscle-myenteric plexus preparations), mediated by the endogenous nitrergic transmitter, under non-adrenergic, non-cholinergic (NANC) conditions (1 micromol/L atropine and 4 micromol/L guanethidine). 3. All EFS responses were abolished by tetrodotoxin (1 micromol/L). Inhibition of NO synthase with N(omega)-nitro-L-arginine-methyl-ester (l-NAME; 100 and 300 micromol/L) prevented both EFS-evoked contractions and relaxations. L-Arginine (3 mmol/L) reversed l-NAME inhibition, primarily restoring contractions and suggesting that these require lower nitrergic transmitter levels than relaxations. 4. Pretreatment of preparations with subrelaxant concentrations of sodium nitroprusside (1 micromol/L) selectively desensitized EFS-evoked contractions without affecting relaxations, suggesting different downstream mechanisms. Nevertheless, the selective guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (3 and 10 micromol/L) inhibited both nitrergic contractions and relaxations, indicating that guanylate cyclase activation is required for both responses. 5. The results of the present study support the hypothesis that the endogenous nitrergic transmitter differentially regulates guanylate cyclase, leading to either contractions or relaxations depending on its concentrations, thus providing additional insight into the regulation of ileum contractility by nitrergic activity.