RESUMO
Hypoxic-ischemic encephalopathy (HIE) causes mortality and long-term neurologic morbidities in newborns, affecting pathways related to energy failure, excitotoxicity and oxidative stress that often lead to cell death. The whole process of HIE injury is coupled to changes in the expression of a great array of proteins. A nanoliposomal preparation of the flavonoid quercetin has been shown to exert neuroprotective effects in perinatal asphyxia models. This study aimed to identify neonatal HIE markers and explore the effect of quercetin administration in two perinatal asphyxia models: newborn rats and piglets. In the rat model, nanoliposomal quercetin administration reduced mortality after asphyxia. In the piglet model, quercetin partially overrode the reduction of HIF-1α mRNA levels in the cortex induced by asphyxia. Quercetin administration also reduced increased level of HO-1 mRNA in asphyctic piglets. These results suggest that quercetin neuroprotection might be involved in the regulation of HIF-1α, HO-1 and their targets. A proteomic approach revealed that the glycolytic pathway is strongly regulated by quercetin in both species. We also identified a set of proteins differentially expressed that could be further considered as markers. In piglets, this set includes Acidic Leucine-rich nuclear phosphoprotein 32 (ANP32A), associated with nervous system differentiation, proteins related with death pathways and alpha-enolase which can be converted to neuron-specific enolase, a glycolytic enzyme that may promote neuroprotection. In newborn rats, other promising proteins associated with neurogenesis and neuroprotection emerged, such as dihydropyrimidinase-related proteins, catalytic and regulatory subunits of phosphatases and heterogeneous nuclear ribonucleoprotein K (hnRNPK). Our results show that a nanoliposomal preparation of quercetin, with protective effect in two HIE mammal models, modulates the expression of proteins involved in energy metabolism and other putative neuroprotective signals in the cortex. Identification of these signals could reveal potential molecular pathways involved in disease onset and the novel quercetin neuroprotective strategy.
Assuntos
Asfixia/tratamento farmacológico , Hipóxia-Isquemia Encefálica/tratamento farmacológico , Neuroproteção/efeitos dos fármacos , Quercetina/farmacologia , Animais , Animais Recém-Nascidos , Asfixia/metabolismo , Modelos Animais de Doenças , Humanos , Hipóxia-Isquemia Encefálica/metabolismo , Recém-Nascido , Fármacos Neuroprotetores/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Ratos , SuínosRESUMO
Epidemiological studies have shown that flavonoid-rich plants induce beneficial health effects that are likely beyond their potent antioxidant capacity. Thus, the mechanisms by which Achyrocline satureioides (AS), a popular South American medicinal plant, protects cells and neurons in culture, are still unclear. In this sense, a recently described trophic capacity for flavonoids, similar to that evoked by growth factors, could be one of the mechanisms involved in AS cellular protection. Since this trophic activity causes differentiation of PC12 cells, the cell differentiation capacity of AS and some of its flavonoids were evaluated. PC12 cells were treated with AS infusion (10 or 20 microg/mL of total polyphenols), quercetin (Q) (12.5 or 25 microm), luteolin (L) (25 microm), Q + L (12.5 microm each one) or nerve growth factor (NGF) for 3 days. Four morphological parameters (percentage of cells with neurites longer than one cell body diameter, percentage of cells with neurites, average number of neurites per cell and percentage of fusiform cells) were explored. The AS infusion showed differentiation capacity on all parameters with similar potency when compared with NGF. Besides, AS was more potent than some of its constituent flavonoids: Q, L or their combination.
Assuntos
Achyrocline/química , Diferenciação Celular/efeitos dos fármacos , Flavonoides/farmacologia , Neuritos/efeitos dos fármacos , Animais , Fatores de Crescimento Neural/farmacologia , Células PC12 , Quercetina/farmacologia , RatosRESUMO
Achyrocline satureioides (Lam.) DC (Marcela) is known to possess a broad spectrum of pharmacological, medicinal and therapeutic properties. Previous studies have demonstrated various protective abilities of the marcela extracts against various pathological conditions. However, no extensive safety studies have been conducted on these extracts to date. In this paper, we evaluated the acute toxicity (dose levels of 30-300 mg/kg) of an aqueous extract of marcela, administered intraperitoneally and orally in mice and rats. The acute oral maximun tolerable dose in repeated administration during 4 h (1, 3 until 5 g/kg) was also studied in rats. The extract had low acute toxicity when administered intraperitoneally and no toxicity upon oral administration. The LD(50) of aqueous extracts of marcela was found to be greater than 5 g/kg when administered once via gastric intubation to rats. Weight gain, toxicity signs, enzymatic studies (transaminases and phosphatases) and histological evaluation of several organs indicated that the extract was devoid of acute toxicity. These acute studies demonstrated that an aqueous extract of marcela obtained after a 2% infusion is safe and did not cause any detrimental effects in vivo under the conditions investigated in this study.
Assuntos
Achyrocline/toxicidade , Testes de Toxicidade Aguda/métodos , Administração Oral , Animais , Relação Dose-Resposta a Droga , Injeções Intraperitoneais , Masculino , Camundongos , Componentes Aéreos da Planta , Extratos Vegetais/isolamento & purificação , Extratos Vegetais/toxicidade , Ratos , Ratos Sprague-Dawley , Testes de Toxicidade Aguda/estatística & dados numéricos , Água/farmacologiaRESUMO
Epidemiological studies indicate that dietary antioxidants can influence the incidence of neurodegenerative diseases. Among them flavonoids have been proposed to be effective cytoprotectors. Consequently, herbs with a high concentration of these compounds such as Achyrocline satureioides, Ginkgo biloba and Epilobium parviflorum are of special interest. In this context a comparative study of the cytoprotective capacity of infusions from the three plants against an oxidative insult was performed. Hence, the cytoprotective activity of each infusion against H2O2 injury to PC12 cells was tested and the antioxidant capacity was assessed by the ABTS*+ radical bleaching assay. Free and glycosylated flavonoids contained in the infusions were identified by HPLC and the cytoprotective effect of some of these individual flavonoids was tested. The analysis of the flavonoid content of the infusions revealed different profiles. Epilobium parviflorum infusion showed the highest antioxidant capacity but only Achyrocline satureioides infusion proved to be cytoprotective. Moreover, the free flavonoids quercetin and luteolin contained in this infusion were also cytoprotective. In conclusion, the free radical scavenger capacity did not correlate with the cytoprotective profile of the infusions. The special mixture of unglycosylated Achyrocline satureioides flavonoids could be a clue to explain the unique effect of this plant.
Assuntos
Achyrocline , Antioxidantes/uso terapêutico , Epilobium , Flavonoides/uso terapêutico , Ginkgo biloba , Estresse Oxidativo/efeitos dos fármacos , Fitoterapia , Extratos Vegetais/uso terapêutico , Animais , Antioxidantes/isolamento & purificação , Células Cultivadas , Flavonoides/isolamento & purificação , Células PC12 , RatosRESUMO
En el complejo y multicausal proceso que lleva a la hipertensión esencial, los radicales libres parecen jugar un papel clave, particularmente en la regulación local del lecho capilar. Además de la producción de ion superóxido en la célula endotelial, existen otros factores de producción de radicales libres, especialmente el radical hidroxilo, aún cuando la hipertensión este medicada y clínicamente controlada. Si bien se han estudiado las defensas antioxidantes no se ha abordado, todavía, la generación de radicales libres en sangre en pacientes hipertensos, en el presente trabajo se estudió la producción del radical hidroxilo en sangre total en pacientes hipertensos controlados, mayores de 65 años, sin otra enfermedad y se compararon los niveles obtenidos con individuos de las mismas características, algunos de ellos hipertensos, que participaban en un programa de ejercicio y controlaban su dieta desde el punto de vista calórico. La producción del radical hidroxilo se determinó por la hidroxilación del salicilato, determinando la concentración del derivado 2,3 di-hidroxibenzoico (2,3-DHBA) por técnicas de cromatografía líquida de alta performance con detección electroquímica. Las concentraciones de 2,3-DHBA luego de la interacción de la sangre con la molécula de salicilato en tubo fueron significativamente mayores en pacientes hipertensos que en los individuos controles. En los individuos hipertensos que realizaban ejercicios se observó una tendencia a una menor producción de radicales hidroxilo. De acuerdo con estos datos, la hipertensión esencial, aún medicada, se acompañaría de una producción elevada de radicales hidroxilo por los elementos formes de la sangre, que el ejercicio y la dieta tienden a disminuir. Aunque no podemos hablar de estrés oxidativo ya que no se determinó el estado de las defensas antioxidantes, es probable que estos radicales, si no son neutralizados, contribuyan a la enfermedad vascular que se observa en la hipertensión esencial. Estos resultados se ubicarían en la línea de investigación que propende a un control activo de la producción radicalaria aumentada en la hipertensión, además de la medicación antihipertensiva.
Assuntos
Humanos , Idoso , Idoso , Radical Hidroxila , Hipertensão/complicaçõesRESUMO
En el complejo y multicausal proceso que lleva a la hipertensión esencial, los radicales libres parecen jugar un papel clave, particularmente en la regulación local del lecho capilar. Además de la producción de ion superóxido en la célula endotelial, existen otros factores de producción de radicales libres, especialmente el radical hidroxilo, aún cuando la hipertensión esté medicada y clínicamente controlada. Si bien se han estudiado las defensas antioxidantes no se ha abordado, todavía, la generación de radicales libres en sangre en pacientes hipertensos, en el presente trabajo se estudió la producción del radical hidroxilo en sangre total en pacientes hipertensos controlados, mayores de 65 años, sin otra enfermedad y se compararon los niveles obtenidos con individuos de las mismas características, algunos de ellos hipertensos, que participaban en un programa de ejercicio y controlaban su dieta desde el punto de vista calórico. La producción del radical hidroxilo se determinó por la hidroxilación del salicilato, determinando la concentración del derivado 2,3 di-hidroxibenzoico (2,3-DHBA) por técnicas de cromatografía líquida de alta performance con detección electroquímica. Las concentraciones de 2,3-DHBA luego de la interacción de la sangre con la molécula de salicilato en tubo fueron significativamente mayores en pacientes hipertensos que en los individuos controles. En los individuos hipertensos que realizaban ejercicios se observó una tendencia a una menor producción de radicales hidroxilo. De acuerdo con estos datos, la hipertensión esencial, aún medicada, se acompañaría de una producción elevada de radicales hidroxilo por los elementos formes de la sangre, que el ejercicio y la dieta tienden a disminuir. Aunque no podemos hablar de estrés oxidativo ya que no se determinó el estado de las defensas antioxidantes, es probable que estos radicales, si no son neutralizados, contribuyan a la enfermedad vascular que se observa en la hipertensión esencial. Estos resultados se ubicarían en la línea de investigación que propende a un control activo de la producción radicalaria aumentada en la hipertensión, además de la medicación antihipertensiva. (AU)
Assuntos
Humanos , Idoso , Radical Hidroxila/sangue , Hipertensão/complicações , IdosoRESUMO
The high morbidity, high socioeconomic costs and lack of specific treatments are key factors that define the relevance of brain pathology for human health and the importance of research on neuronal protective agents. Epidemiological studies have shown beneficial effects of flavonoids on arteriosclerosis-related pathology in general and neurodegeneration in particular. Flavonoids can protect the brain by their ability to modulate intracellular signals promoting cellular survival. Quercetin and structurally related flavonoids (myricetin, fisetin, luteolin) showed a marked cytoprotective capacity in in vitro experimental conditions in models of predominantly apoptotic death such as that induced by medium concentrations (200 M) of H2O2 added to PC12 cells in culture. Nevertheless, quercetin did not protect substantia nigra neurons in vivo from an oxidative insult (6-hydroxydopamine), probably due to difficulties in crossing the blood-brain barrier. On the other hand, treatment of permanent focal ischemia with a lecithin/quercetin preparation decreased lesion volume, showing that preparations that help to cross the blood-brain barrier may be critical for the expression of the effects of flavonoids on the brain. The hypothesis is advanced that a group of quercetin-related flavonoids could become lead molecules for the development of neuroprotective compounds with multitarget anti-ischemic effects.
Assuntos
Arteriosclerose/tratamento farmacológico , Flavonoides/uso terapêutico , Fármacos Neuroprotetores/uso terapêutico , Doenças do Sistema Nervoso Central/tratamento farmacológico , Doenças do Sistema Nervoso Central/metabolismo , Humanos , Estresse OxidativoRESUMO
The high morbidity, high socioeconomic costs and lack of specific treatments are key factors that define the relevance of brain pathology for human health and the importance of research on neuronal protective agents. Epidemiological studies have shown beneficial effects of flavonoids on arteriosclerosis-related pathology in general and neurodegeneration in particular. Flavonoids can protect the brain by their ability to modulate intracellular signals promoting cellular survival. Quercetin and structurally related flavonoids (myricetin, fisetin, luteolin) showed a marked cytoprotective capacity in in vitro experimental conditions in models of predominantly apoptotic death such as that induced by medium concentrations (200 æM) of H2O2 added to PC12 cells in culture. Nevertheless, quercetin did not protect substantia nigra neurons in vivo from an oxidative insult (6-hydroxydopamine), probably due to difficulties in crossing the blood-brain barrier. On the other hand, treatment of permanent focal ischemia with a lecithin/quercetin preparation decreased lesion volume, showing that preparations that help to cross the blood-brain barrier may be critical for the expression of the effects of flavonoids on the brain. The hypothesis is advanced that a group of quercetin-related flavonoids could become lead molecules for the development of neuroprotective compounds with multitarget anti-ischemic effects.
Assuntos
Humanos , Arteriosclerose , Flavonas , Fármacos NeuroprotetoresRESUMO
Although, the mechanism of 2,4-dichlorophenoxyacetic acid (2,4-D) neurotoxicity remains unknown, the monoaminergic system appears to mediate some of its effects in rats as we previously reported. In this study; we examined the 2,4-D effects on locomotor activity, circling behavior and monoamine levels after the injection into the basal ganglia of male adult rats. These effects were compared with those induced after selective lesions of dopaminergic neurons with 6-hydroxydopamine (6-OHDA). 2,4-D-injected into one striatum (100 microg/rat) produced a marked depression in locomotor activity and elicited a moderate circling towards the ipsilateral side at 6 and 24 h postinjection. These behavioral changes were accompanied by a decrease and an increase of serotonin (5-HT) and homovanillic acid (HVA) levels, respectively. 2,4-D administration (100 microg/rat) into the nucleus accumbens, induced similar behavioral and neurochemical patterns to the intrastriatal 2,4-D injection, although rats did not present notorious turning. When 2,4-D was injected into one medial forebrain bundle (MFB, 50 microg/rat), animals presented ipsilateral circling, while locomotor activity was unchanged at 3 and 7 days post-injection. These last rats also exhibited diminished levels of striatal 5-HT, dopamine (DA) and their metabolites without changes in the substantia nigra (SN). Animals sacrificed 3 and 7 days after a 6-OHDA injection into one of the MFB, presented progressive depletion of dopamine in striatum and SN. 2,4-D as well as 6-OHDA-treated rats into one of the MFB were challenged with low dose (0.05 mg/kg s.c.) of apomorphine (only at 7 days post-injection) to evaluate a possible DA-receptor supersensitivity. Only 6-OHDA treated rats showing a vigorous contralateral rotation activity. These results indicate that 2,4-D induced a regionally-specific neurotoxicity in the basal ganglia of rats. The neurotoxic effects of 2,4-D on basal ganglia by interacting with the monoaminergic system depended not only on the exact location of the 2,4-D injection, but also on the dose and time period of post-injection. Toxicity produced by 2,4-D appears to be different in monoaminergic terminals, axonal fibers, and cell bodies.
Assuntos
Ácido 2,4-Diclorofenoxiacético/toxicidade , Comportamento Animal/efeitos dos fármacos , Química Encefálica/efeitos dos fármacos , Herbicidas/toxicidade , Ácido 2,4-Diclorofenoxiacético/administração & dosagem , Animais , Monoaminas Biogênicas/metabolismo , Herbicidas/administração & dosagem , Masculino , Feixe Prosencefálico Mediano/efeitos dos fármacos , Feixe Prosencefálico Mediano/metabolismo , Atividade Motora/efeitos dos fármacos , Neostriado , Núcleo Accumbens , Oxidopamina , Ratos , Ratos Wistar , Comportamento Estereotipado/efeitos dos fármacosRESUMO
While the work of several groups has shown the neuroprotective effects of nicotine in vitro, evidences for the same effects in vivo are controversial, mainly regarding neuroprotection in experimental models of Parkinson's disease. In this context, we investigated the capability of various systemic administration schedules of nicotine to prevent the loss of striatal dopamine levels produced by partial or extensive 6-hydroxydopamine (6-OHDA) lesion of rat substantia nigra (SN). Eight days after 6- and 10-microg injections of 6-OHDA in the SN there was a significant decrease of dopamine concentrations in the corpus striatum (CS) and a concomitant increase in dopamine turnover. While 10 microg 6-OHDA produced an almost complete depletion of dopamine in the SN, 6 microg decreased dopamine levels by 50%. Subcutaneous nicotine (1 mg/kg) administered 4 h before and 20, 44 and 68 h after 6 microg 6-OHDA, prevented significantly the striatal dopamine loss. Administered only 18 or 4 h before or only 20, 44 and 68 h after, nicotine failed to counteract the loss of dopamine or the increase in dopamine turnover observed in the CS. Nicotine also failed to prevent significantly the decrease of striatal dopamine levels produced by the 10-microg 6-OHDA intranigral dose. Chlorisondamine, a long-lasting nicotinic acetylcholine receptor antagonist, reverted significantly the nicotinic protective effects on dopamine concentrations. These results are showing that putative neuroprotective effects of nicotine in vivo depend on an acute intermittent administration schedule and on the extent of the brain lesion.
Assuntos
Dopamina/metabolismo , Nicotina/administração & dosagem , Agonistas Nicotínicos/administração & dosagem , Doença de Parkinson Secundária/metabolismo , Substância Negra/efeitos dos fármacos , Ácido 3,4-Di-Hidroxifenilacético/metabolismo , Animais , Clorisondamina/administração & dosagem , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Modelos Animais de Doenças , Dopamina/deficiência , Relação Dose-Resposta a Droga , Esquema de Medicação , Injeções Subcutâneas , Masculino , Fármacos Neuroprotetores/administração & dosagem , Antagonistas Nicotínicos/administração & dosagem , Oxidopamina , Doença de Parkinson Secundária/induzido quimicamente , Ratos , Ratos Sprague-Dawley , Substância Negra/metabolismoAssuntos
Citoproteção/fisiologia , Dopamina/fisiologia , Terminações Nervosas/fisiopatologia , Transtornos Parkinsonianos/fisiopatologia , Receptores Nicotínicos/fisiologia , Animais , Técnicas In Vitro , Fármacos Neuroprotetores/farmacologia , Neurotoxinas , Nicotina/farmacologia , Oxidopamina , Transtornos Parkinsonianos/induzido quimicamenteRESUMO
Fasciculin 2 (FAS), an acetylcholinesterase (AChE) peripheral site ligand that inhibits mammalian AChE in the picomolar range and chicken AChE only at micromolar concentrations, was used in chick retinal cell cultures to evaluate the influence of AChE on neuronal development. The effects of other AChE inhibitors that bind the active and/or the peripheral site of the enzyme [paraoxon, eserine, or 1,5-bis(4-allyldimethylammoniumphenyl) pentan-3-one dibromide (BW284c51)] were also studied. Morphological changes of cultured neurons were observed with the drugs used and in the different cell culture systems studied. Cell aggregates size decreased by more than 35% in diameter after 9 days of FAS treatment, mainly due to reduction in the presumptive plexiform area of the aggregates. Eserine showed no effect on the morphology of the aggregates, although it fully inhibited the activity of AChE. In dense stationary cell culture, cluster formation increased after 3 days and 6 days of FAS treatment. However, FAS, at concentrations in which changes of morphological parameters were observed, did not inhibit the AChE activity as measured histochemically. In contrast, paraoxon treatment produced a slight morphological alteration of the cultures, while a strong inhibition of enzyme activity caused by this agent was observed. BW284c51 showed a harmful, probably toxic effect, also causing a slight AChE inhibition. It is suggested that the effect of an anticholinesterase agent on the morphological modifications of cultured neurons is not necessarily associated with the intensity of the AChE inhibition, especially in the case of FAS. Moreover, most of the effects of AChE on culture morphology appear to be independent of the cholinolytic activity of the enzyme. The results obtained demonstrate that FAS is not toxic for the cells and suggest that regions of the AChE molecule related to the enzyme peripheral site are likely to be involved with the nonclassical role of AChE.
Assuntos
Acetilcolinesterase/fisiologia , Inibidores da Colinesterase/farmacologia , Venenos Elapídicos/farmacologia , Proteínas do Olho/fisiologia , Retina/embriologia , Animais , Benzenamina, 4,4'-(3-oxo-1,5-pentanodi-il)bis(N,N-dimetil-N-2-propenil-), Dibrometo/farmacologia , Sítios de Ligação/efeitos dos fármacos , Domínio Catalítico/efeitos dos fármacos , Agregação Celular , Técnicas de Cultura de Células/métodos , Células Cultivadas/efeitos dos fármacos , Embrião de Galinha , Proteínas do Olho/antagonistas & inibidores , Neurônios/efeitos dos fármacos , Neurônios/enzimologia , Paraoxon/farmacologia , Fisostigmina/farmacologia , Retina/citologia , Retina/enzimologiaRESUMO
The dopaminergic and antioxidant properties of pukateine [(R)-11-hydroxy-1,2-methylenedioxyaporphine, PUK], a natural aporphine derivative, were analyzed in the rat central nervous system. At dopamine (DA) D1 ([3H]-SCH 23390) and D2 ([3H]-raclopride) binding sites, PUK showed IC50 values in the submicromolar range (0.4 and 0.6 microM, respectively). When the uptake of tritiated dopamine was assayed by using a synaptosomal preparation, PUK showed an IC50 = 46 microM. In 6-hydroxydopamine unilaterally denervated rats, PUK (8 mg/kg but not 4 mg/kg) elicited a significant contralateral circling, a behavior classically associated with a dopaminergic agonist action. When perfused through a microdialysis probe inserted into the striatum, PUK (340 microM) induced a significant increase in dopamine levels. In vitro experiments with a crude rat brain mitochondrial suspension showed that PUK did not affect monoamine oxidase activities, at concentrations as high as 100 microM. PUK potently (IC50 = 15 microM) and dose-dependently inhibited the basal lipid peroxidation of a rat brain membrane preparation. As a whole, PUK showed a unique profile of action, comprising an increase in extracellular DA, an agonist-like interaction with DA receptors, and antioxidant activity. Thus, PUK may be taken as a lead compound for the development of novel therapeutic strategies for Parkinson disease.
Assuntos
Antioxidantes/farmacologia , Aporfinas/farmacologia , Dopaminérgicos/farmacologia , Dopamina/metabolismo , Transmissão Sináptica/efeitos dos fármacos , Animais , Antioxidantes/uso terapêutico , Aporfinas/uso terapêutico , Sistema Nervoso Central/efeitos dos fármacos , Sistema Nervoso Central/metabolismo , Dopaminérgicos/uso terapêutico , Peroxidação de Lipídeos , Masculino , Microdiálise , Monoaminoxidase/metabolismo , Inibidores da Monoaminoxidase/farmacologia , Doença de Parkinson/tratamento farmacológico , Desempenho Psicomotor/efeitos dos fármacos , Ensaio Radioligante , Ratos , Ratos Sprague-DawleyRESUMO
Snake neurotoxins (NTX) have proven to be valuable tools for the characterisation of muscular nicotinic acetylcholine receptor structure and function. It is very likely that they could also be utilised to identify subtypes of neuronal nicotinic receptors controlling specific functions within the central nervous system. In this study we examined the effects of long alpha NTX (alpha-bungarotoxin, alpha-Bgt, and alpha-cobratoxin, alpha-Cbt) and short alpha NTX (alpha-erabutoxin a, alpha-Ebt) as well as the anticholinesterase toxin fasciculin-2 (FAS), on the nicotine-evoked release of dopamine (DA) in the striatum, using the in vivo push-pull technique. The short toxins alpha-Ebt and FAS blocked the extracellular increase of DA evoked by nicotine at 4.2 microM concentrations and alpha-Ebt was more potent, as reflected by the blockade at the lower dose of 0.42 microM. In contrast, the long toxins showed a different profile of action. Alpha-Cbt did not show any blockade of the nicotine-evoked release of DA at the doses studied while alpha-Bgt did block it only at the higher dose (4.2 microM) These results indicate that short neurotoxins show a stronger interaction with striatal nicotinic receptors subtypes controlling DA release when compared to the long ones. This interaction of short neurotoxin polypeptides and presynaptic receptors may permit the further elucidation of the particular nicotinic receptor populations responsible for the modulation of striatal DA release.
Assuntos
Corpo Estriado/metabolismo , Dopamina/metabolismo , Neurotoxinas/farmacologia , Nicotina/farmacologia , Serpentes/metabolismo , Animais , Bungarotoxinas/farmacologia , Proteínas Neurotóxicas de Elapídeos/farmacologia , Corpo Estriado/efeitos dos fármacos , Relação Dose-Resposta a Droga , Venenos Elapídicos/farmacologia , Erabutoxinas/farmacologia , Masculino , Neurotoxinas/metabolismo , Concentração Osmolar , Ratos , Ratos EndogâmicosRESUMO
To study the involvement of oxidative stress in 6-OHDA neurotoxicity, we investigated the production of the hydroxyl free radical (OH.) in the substantia nigra (SN) and the striatum (CS) several moments after intranigral injection of the neurotoxin, with or without an added episode of hypoxia (30 min, 95% N2, 5% O2). We utilized the hydroxylation of salicylate to 2,3 dihydroxybenzoic acid (2,3 DHBA) as indication of OH. production. When 2.3 DHBA levels were not modified, the levels of 2,5 DHBA were taken as an indication of cytochrome P450 (CYP 450) metabolism. 6-OHDA alone did not increase the production of 2,3 DHBA in the SN. 2,5 DHBA increased significantly after 120 min and was high up to 24 h. An episode of hypoxia (60 min after 6-OHDA injection) significantly worsened the decrease of dopamine (DA) in the striatum assessed 8 days after injection of 6-OHDA in the SN. Hypoxia performed 60 min and 24 h before or 24 h after 6-OHDA did not show any additional effect on striatal DA levels. Contrary to results obtained after 6-OHDA alone, 2,3 DHBA increased significantly 120 min after the injection, when the hypoxia-reoxygenation was added to the 6-OHDA treatment. Our data are showing a relationship between the increase in OH. production and a concomitant worsening of neuronal degeneration. As a whole, the results support the idea that neurons undergoing 6-OHDA neurotoxicity have their antioxidant defences affected and that oxidative stress is actually an important eliciting factor in 6-OHDA dependant neurodegeneration. However, OH. may not be the main radical species involved in this process. Additionally, 6-OHDA also appeared to provoke a long-term increase in CYP 450 activity.
Assuntos
Radical Hidroxila/metabolismo , Hipóxia Encefálica/fisiopatologia , Neurotoxinas/toxicidade , Oxidopamina/toxicidade , Oxigênio/farmacologia , Substância Negra/metabolismo , Ácido 3,4-Di-Hidroxifenilacético/metabolismo , Animais , Dopamina/metabolismo , Hidroxibenzoatos/metabolismo , Hidroxilação , Hipóxia Encefálica/induzido quimicamente , Masculino , Pressão Parcial , Ratos , Ratos Sprague-Dawley , Ácido Salicílico/metabolismoRESUMO
Acetylcholinesterase (AChE) molecular forms were studied during mouse brain development. Mouse embryos expressed a monomeric (G1) and a tetrameric (G4) AChE form. Our results indicate that G4 AChE expressed at embryonic day (ED) 9 and ED15 could be purified by acridinium-Sepharose chromatography and shared similar biochemical and kinetic properties with the adult form. However, the G1 form expressed at either embryonic stage did not bind to acridinium, was not inhibited by excess substrate, and possessed higher K(m) and lower Vmax values than the adult G1 form. Two peripheral anionic binding site inhibitors, fasciculin and propidium, had a significantly lower affinity for the monomeric form at ED9. Results are discussed in terms of the biological significance of the embryonic G1 form, and its resemblance to the AChE activity found, associated with the senile plaques present in the brains of Alzheimer's patients.
Assuntos
Acetilcolinesterase/metabolismo , Encéfalo/enzimologia , Encéfalo/crescimento & desenvolvimento , Acetilcolinesterase/análise , Acetilcolinesterase/isolamento & purificação , Animais , Encéfalo/embriologia , Inibidores da Colinesterase/farmacologia , Cromatografia de Afinidade , Edrofônio/farmacologia , Feminino , Cinética , Camundongos , Gravidez , Propídio/farmacologia , Especificidade por SubstratoRESUMO
Brain acetylcholinesterase (AChE) forms stable complexes with amyloid-beta peptide (Abeta) during its assembly into filaments, in agreement with its colocalization with the Abeta deposits of Alzheimer's brain. The association of the enzyme with nascent Abeta aggregates occurs as early as after 30 min of incubation. Analysis of the catalytic activity of the AChE incorporated into these complexes shows an anomalous behavior reminiscent of the AChE associated with senile plaques, which includes a resistance to low pH, high substrate concentrations, and lower sensitivity to AChE inhibitors. Furthermore, the toxicity of the AChE-amyloid complexes is higher than that of the Abeta aggregates alone. Thus, in addition to its possible role as a heterogeneous nucleator during amyloid formation, AChE, by forming such stable complexes, may increase the neurotoxicity of Abeta fibrils and thus may determine the selective neuronal loss observed in Alzheimer's brain.
Assuntos
Acetilcolinesterase/química , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/química , Proteínas do Tecido Nervoso/química , Neurônios/patologia , Doença de Alzheimer/metabolismo , Animais , Morte Celular , Células Cultivadas , Embrião de Galinha , Estabilidade Enzimática , Modelos Logísticos , Células PC12 , Ratos , SolubilidadeRESUMO
In the rat striatum, acetylcholine (ACh) increases dopamine (DA) release. The role of increased cholinergic activity provoked by acetylcholinesterase inhibitors (AChEi) on DA release is currently under revision after recent papers have shown a blockade of nicotinic transmission by AChEi in vitro. To study the effects of AChEi in vivo, Fasciculin2 (FAS), a peptidergic AChEi, and physostigmine (PHY), a classical carbamate AChEi, were applied through push-pull or microdialysis cannulae respectively, to the striatum of rats, alone or with ACh. Extracellular concentrations of DA were assessed by HPLC with electrochemical detection. Alone, the AChEi studied did not provoke changes in basal extracellular levels of DA, in the different doses studied. ACh (100 microM, 1 and 5 mM) applied through the push-pull cannulae in basal conditions provoked a dose-dependent increase of extracellular DA. This effect was not observed with ACh in concentrations of 100 microM and 1 mM if FAS (0.4 and 4.2 microM) was applied first. Higher concentrations of ACh (5 mM) evoked a partial response after FAS 0.42 microM, an effect still blocked by FAS at 4.2 microM. PHY 50 microM applied through microdialysis completely blocked the increase in DA release provoked by ACh 10, 20 mM, while at ACh 30 mM, PHY 50 microM only partially blocked the evoked increase. A partial blockade was also observed with PHY 20 microM, on the three different concentrations of ACh. On the other hand PHY 10 microM did not block any of the ACh doses perfused. These results showed that AChEi like FAS and PHY interfere with the ACh-evoked DA release in the striatum.
Assuntos
Acetilcolina/farmacologia , Inibidores da Colinesterase/farmacologia , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Dopamina/metabolismo , Venenos Elapídicos/farmacologia , Animais , Espaço Extracelular/metabolismo , Masculino , Microdiálise , Concentração Osmolar , Perfusão/métodos , Fisostigmina/farmacologia , Ratos , Ratos EndogâmicosRESUMO
Fasciculin 2 (FAS), a potent acetylcholinesterase (AChE, EC 3.1.1.7) inhibitory peptide with affinity for the enzyme in the nanomolar range was utilized together with two other AChE inhibitors (Paroxon and BW284c51) to study the role of AChE in central nervous system development. When drugs were intracisternally injected at postnatal days 3 and 5, only FAS showed a significant inhibition of hippocampus and striatum AChE (39% and 77% inhibition, respectively). After FAS treatment, animals showed convulsive behaviour which was blocked by subcutaneous pretreatment with atropine sulfate (10 mg/kg). An assessment of developmental indices showed no alteration in neurological reflex maturation, motor behaviour or cell morphology. Body weight gain was significantly lower only in FAS-treated animals compared to controls during the preweaning period. To investigate the specificity of this effect a synthetic loop of FAS (showing no activity in vitro or in vivo) and oxidized FAS (showing a weak inhibition in vitro and no activity in vivo) were also intracisternally injected. Animals injected with the loop showed normal body weight development while those treated with oxidized FAS showed impairment in body weight. In conclusion, FAS was the most potent drug at inhibiting neonatal AChE in vivo without nonspecific brain damage. Impairment in body weight seems to be dependent on AChE involvement, although the possibility of a direct FAS effect is discussed. These results point to FAS intracisternal treatment as a useful in vivo model to study the role of AChE in the critical period of early postnatal central nervous system development.
Assuntos
Encéfalo/efeitos dos fármacos , Inibidores da Colinesterase/toxicidade , Venenos Elapídicos/toxicidade , Animais , Animais Recém-Nascidos/crescimento & desenvolvimento , Benzenamina, 4,4'-(3-oxo-1,5-pentanodi-il)bis(N,N-dimetil-N-2-propenil-), Dibrometo/toxicidade , Encéfalo/enzimologia , Encéfalo/crescimento & desenvolvimento , Injeções Intraventriculares , Masculino , Paraoxon/toxicidade , Ratos , Ratos EndogâmicosRESUMO
The dried flower heads of Matricaria recutita L. (Asteraceae) are used in folk medicine to prepare a spasmolytic and sedative tea. Our fractionation of the aqueous extract of this plant led to the detection of several fractions with significant affinity for the central benzodiazepine receptor and to the isolation and identification of 5,7,4'-trihydroxyflavone (apigenin) in one of them. Apigenin competitively inhibited the binding of flunitrazepam with a Ki of 4 microM and had no effect on muscarinic receptors, alpha 1-adrenoceptors, and on the binding of muscimol to GABAA receptors. Apigenin had a clear anxiolytic activity in mice in the elevated plusmaze without evidencing sedation or muscle relaxant effects at doses similar to those used for classical benzodiazepines and no anticonvulsant action was detected. However, a 10-fold increase in dosage produced a mild sedative effect since a 26% reduction in ambulatory locomotor activity and a 35% decrement in hole-board parameters were evident. The results reported in this paper demonstrate that apigenin is a ligand for the central benzodiazepine receptors exerting anxiolytic and slight sedative effects but not being anticonvulsant or myorelaxant.