RESUMO
The endocannabinoid system (ECS) refers to a complex cell-signaling system highly conserved among species formed by numerous receptors, lipid mediators (endocannabinoids) and synthetic and degradative enzymes. It is widely distributed throughout the body including the CNS, where it participates in synaptic signaling, plasticity and neurodevelopment. Besides, the olfactory ensheathing glia (OEG) present in the olfactory system is also known to play an important role in the promotion of axonal growth and/or myelination. Therefore, both OEG and the ECS promote neurogenesis and oligodendrogenesis in the CNS. Here, we investigated if the ECS is expressed in cultured OEG, by assessing the main markers of the ECS through immunofluorescence, western blotting and qRT-PCR and quantifying the content of endocannabinoids in the conditioned medium of these cells. After that, we investigated whether the production and release of endocannabinoids regulate the differentiation of oligodendrocytes co-cultured with hippocampal neurons, through Sholl analysis in oligodendrocytes expressing O4 and MBP markers. Additionally, we evaluated through western blotting the modulation of downstream pathways such as PI3K/Akt/mTOR and ERK/MAPK, being known to be involved in the proliferation and differentiation of oligodendrocytes and activated by CB1, which is the major endocannabinoid responsive receptor in the brain. Our data show that OEG expresses key genes of the ECS, including the CB1 receptor, FAAH and MAGL. Besides, we were able to identify AEA, 2-AG and AEA related mediators palmitoylethanolamide (PEA) and oleoylethanolamide (OEA), in the conditioned medium of OEG cultures. These cultures were also treated with URB597 10-9 M, a FAAH selective inhibitor, or JZL184 10-9 M, a MAGL selective inhibitor, which led to the increase in the concentrations of OEA and 2-AG in the conditioned medium. Moreover, we found that the addition of OEG conditioned medium (OEGCM) enhanced the complexity of oligodendrocyte process branching in hippocampal mixed cell cultures and that this effect was inhibited by AM251 10-6 M, a CB1 receptor antagonist. However, treatment with the conditioned medium enriched with OEA or 2-AG did not alter the process branching complexity of premyelinating oligodendrocytes, while decreased the branching complexity in mature oligodendrocytes. We also observed no change in the phosphorylation of Akt and ERK 44/42 in any of the conditions used. In conclusion, our data show that the ECS modulates the number and maturation of oligodendrocytes in hippocampal mixed cell cultures.
RESUMO
The avian retina has been used as a model to study signaling by different neuro- and gliotransmitters. It is unclear how dopaminergic and cannabinoid systems are related in the retina. Here we studied the expression of type 1 and 2 cannabinoid receptors (CB1 and CB2), as well as monoacylglycerol lipase (MAGL), the enzyme that degrades 2-arachidonoylglycerol (2-AG), during retina development. Our data show that CB1 receptor is highly expressed from embryonic day 5 (E5) until post hatched day 7 (PE7), decreasing its levels throughout development. CB1 is densely found in the ganglion cell layer (GCL) and inner plexiform layer (IPL). CB2 receptor was also found from E5 until PE7 with a decrease in its contents from E9 afterwards. CB2 was mainly present in the lamination of the IPL at PE7. MAGL is expressed in all retinal layers, mainly in the IPL and OPL from E9 to PE7 retina. CB1 and CB2 were found both in neurons and glia cells, but MAGL was only expressed in Müller glia. Older retinas (PE7) show CB1 positive cells mainly in the INL and co-expression of CB1 and tyrosine hydroxylase (TH) are shown in a few cells when both systems are mature. CB1 co-localized with TH and was heavily associated to D1 receptor labeling in primary cell cultures. Finally, cyclic AMP (cAMP) was activated by the selective D1 agonist SKF38393, and inhibited when cultures were treated with WIN55, 212-2 (WIN) in a CB1 dependent manner. The results suggest a correlation between the endocannabinoid and dopaminergic systems (DSs) during the avian retina development. Activation of CB1 limits cAMP accumulation via D1 receptor activation and may influence embryological parameters during avian retina differentiation.
RESUMO
Endocannabinoids are endogenous lipids that activate selective G protein coupled receptors (CB1 and CB2), mostly found at neuronal presynaptic sites in the nervous system. One of the main consequences of the activation of CB receptors is a decrease in GABA or glutamate release, controlling cell excitability. Here we studied the expression of CB1 and CB2 receptors in E8C8 cultured retina cells (embryonic day 8 and 8 days in vitro) using immunocytochemistry and western blot analysis. We also evaluated their functions in terms of cyclic AMP (cAMP) production, single cell calcium imaging (SCCI) and GABA release induced in basal conditions or activated by l-Aspartate (L-ASP) in cell cultures or under ischemia in young chick retina. We show that both cannabinoid receptors are expressed in retinal neurons and glial cells. WIN 55,212-2 (WIN, a CB1/CB2 agonist) decreased cAMP production in cultured avian embryonic retinal cells in basal conditions. WIN also led to a decrease in the number of glial cells that increased Ca2+ levels evoked by ATP, but had no effect in Ca2+ shifts in neuronal cells activated by KCl. Finally, WIN inhibited [3H]-GABA release induced by KCl or L-ASP, accumulated in amacrine cells, but had no effect in the amount of GABA released in an oxygen glucose deprivation (OGD) condition. Altogether, our data indicate that cannabinoid receptors function as regulators of avian retina signaling at critical embryonic stages during synapse formation.
Assuntos
Neuroglia/metabolismo , Neurônios/metabolismo , Receptor CB1 de Canabinoide/fisiologia , Receptor CB2 de Canabinoide/fisiologia , Retina/embriologia , Retina/metabolismo , Analgésicos/farmacologia , Animais , Benzoxazinas/farmacologia , Embrião de Galinha , Técnicas de Cocultura , Morfolinas/farmacologia , Naftalenos/farmacologia , Neuroglia/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Retina/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
Aging represents a major risk factor for numerous illnesses that are of increasing importance to society, including two of the most prevalent: diabetes and Alzheimer's disease. Studies have shown that diabetes is a risk factor for spontaneous Alzheimer's disease. While these studies suggest that diabetes can contribute to Alzheimer's disease, the implications of AD on diabetes are practically unexplored. The major mediator of the pathophysiological effects, the Aß42 peptide, has been shown to enter neurons and lead to an alteration of the intracellular distribution of the molecular motor myosin Vb. Myosin Vb functions in memory and learning by participating in the strengthening of the long-term potentiation (LTP) of synaptic transmissions. It has also been implicated in the translocation of the glucose transporter, GLUT4, to the plasma membrane in response to insulin, a process that is defective in diabetes. Here, the effect on GLUT4 upon entry of the Aß42 peptide into cultured chick retinal neurons was explored. The results suggest an alteration in distribution and a reduced level at the cell surface, as well as an increased colocalization with myosin Vb, which can partially explain the changes in glucose metabolism associated with AD. It is also shown that the presence of the Aß40 peptide inhibits the internalization of the Aß42 peptide in cultured cells. Together, the results provide additional targets for the development of therapeutics against the progression and effects of Alzheimer's disease.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Transportador de Glucose Tipo 4/metabolismo , Miosina Tipo V/metabolismo , Neurônios/metabolismo , Doença de Alzheimer/etiologia , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/farmacologia , Animais , Células Cultivadas , Embrião de Galinha , Galinhas , Retina/citologia , Retina/metabolismoRESUMO
GABA (γ-amino butyric acid) is the major inhibitory transmitter in the central nervous system and its action is terminated by specific transporters (GAT), found in neurons and glial cells. We have previously described that GAT-3 is responsible for GABA uptake activity in cultured avian Müller cells and that it operates in a Na(+) and Cl(-) dependent manner. Here we show that glutamate decreases [(3)H] GABA uptake in purified cultured glial cells up to 50%, without causing cell death. This effect is mediated by ionotropic glutamatergic receptors. Glutamate inhibition on GABA uptake is not reverted by inhibitors of protein kinase C or modified by agents that modulate cyclic AMP/PKA. Biotinylation experiments demonstrate that this reduction in GABA uptake correlates with a decrease in GAT-3 plasma membrane levels. Interestingly, both GAT-1 and GAT-3 mRNA levels are also decreased by glutamate. Conditioned media (CM) prepared from retinal neurons could also decrease GABA influx, and glutamate receptor antagonists (MK-801 + CNQX) were able to prevent this effect. However, glutamate levels in CM were not different from those found in fresh media, indicating that a glutamatergic co-agonist or modulator could be regulating GABA uptake by Müller cells in this scenario. In the whole avian retina, GAT-3 is present from embryonic day 5 (E5) increasing up to the end of embryonic development and post-hatch period exclusively in neuronal layers. However, this pattern may change in pathological conditions, which drive GAT-3 expression in Müller cells. Our data suggest that in purified cultures and upon extensive neuronal lesion in vivo, shown as a Brn3a reduced neuronal cells and an GFAP increased gliosis, Müller glia may change its capacity to take up GABA due to GAT-3 up regulation and suggests a regulatory interplay mediated by glutamate between neurons and glial cells in this process.
Assuntos
Células Ependimogliais/fisiologia , Proteínas da Membrana Plasmática de Transporte de GABA/fisiologia , Ácido Glutâmico/fisiologia , Ácido gama-Aminobutírico/metabolismo , Animais , Transporte Biológico Ativo , Biotinilação , Cálcio/análise , Membrana Celular/metabolismo , Células Cultivadas , Embrião de Galinha , Galinhas , Meios de Cultivo Condicionados , Células Ependimogliais/efeitos dos fármacos , Proteínas da Membrana Plasmática de Transporte de GABA/genética , Perfilação da Expressão Gênica , Ácido Glutâmico/farmacologia , Ácido Caínico/farmacologia , N-Metilaspartato/administração & dosagem , N-Metilaspartato/farmacologia , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/fisiologia , Inibidores de Proteínas Quinases/farmacologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Retina/crescimento & desenvolvimento , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Müller cells constitute the main glial cell type in the retina where it interacts with virtually all cells displaying relevant functions to retinal physiology. Under appropriate stimuli, Müller cells may undergo dedifferentiation, being able to generate other neural cell types. Here, we show that purified mouse Müller cells in culture express a group of proteins related to the dopaminergic phenotype, including the nuclear receptor-related 1 protein, required for dopaminergic differentiation, as well the enzyme tyrosine hydroxylase. These dopaminergic components are active, since Müller cells are able to synthesize and release dopamine to the extracellular medium. Moreover, Müller-derived tyrosine hydroxylase can be regulated, increasing its activity because of phosphorylation of serine residues in response to agents that increase intracellular cAMP levels. These observations were extended to glial cells obtained from adult monkey retinas with essentially the same results. To address the potential use of dopaminergic Müller cells as a source of dopamine in cell therapy procedures, we used a mouse model of Parkinson's disease, in which mouse Müller cells with the dopaminergic phenotype were transplanted into the striatum of hemi-parkinsonian mice generated by unilateral injection of 6-hydroxydopamine. These cells fully decreased the apomorphine-induced rotational behavior and restored motor functions in these animals, as measured by the rotarod and the forelimb-use asymmetry (cylinder) tests. The data indicate local restoration of dopaminergic signaling in hemi-parkinsonian mice confirmed by measurement of striatal dopamine after Müller cell grafting.
Assuntos
Neurônios Dopaminérgicos/transplante , Células Ependimogliais/transplante , Transtornos Parkinsonianos/patologia , Transtornos Parkinsonianos/terapia , Animais , Cebus , Diferenciação Celular/fisiologia , Células Cultivadas , Corpo Estriado/citologia , Corpo Estriado/fisiologia , Modelos Animais de Doenças , Dopamina/metabolismo , Neurônios Dopaminérgicos/citologia , Neurônios Dopaminérgicos/metabolismo , Células Ependimogliais/citologia , Células Ependimogliais/metabolismo , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Atividade Motora/fisiologia , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Transtornos Parkinsonianos/metabolismo , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Recuperação de Função Fisiológica/fisiologia , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
Pituitary Adenylyl Cyclase-Activating Polypeptide (PACAP) is a neuroactive peptide present in the avian retina where it activates adenylyl cyclase (AC) since early in development via PACAP receptors. The synthesis of cAMP in response to PACAP is observed since embryonic day 8/9 (E8/9). After E12, signaling via PACAP receptors desensitizes, reaching very low levels in the mature tissue. We show here that chronic administration of PACAP in vitro desensitizes PACAP-induced cAMP accumulation, while the administration of the PACAP antagonist (PACAP 6-38) re-sensitizes PACAP receptor/cyclase system in vitro and in vivo. Moreover, a twofold increase in the number of tyrosine hydroxylase positive (THâº) cells is observed after in vivo injection of PACAP6-38. NURR1, a transcription factor associated with the differentiation of dopaminergic cells in the CNS, is present in the chick retina in all developmental stages studied. The presence of NURR1 positive cells in the mature tissue far exceeds the number of TH⺠cells, suggesting that these NURR1-positive cells might have the potential to express the dopaminergic phenotype. Our data show that if PACAP signaling is increased in mature retinas, plastic changes in dopaminergic phenotype can be achieved.
Assuntos
Plasticidade Neuronal/fisiologia , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Retina/metabolismo , Animais , Western Blotting , Galinhas , AMP Cíclico , Dopamina , Imuno-Histoquímica , Fenótipo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Dysregulated cholinergic signaling is an early hallmark of Alzheimer disease (AD), usually ascribed to degeneration of cholinergic neurons induced by the amyloid-ß peptide (Aß). It is now generally accepted that neuronal dysfunction and memory deficits in the early stages of AD are caused by the neuronal impact of soluble Aß oligomers (AßOs). AßOs build up in AD brain and specifically attach to excitatory synapses, leading to synapse dysfunction. Here, we have investigated the possibility that AßOs could impact cholinergic signaling. The activity of choline acetyltransferase (ChAT, the enzyme that carries out ACh production) was inhibited by ~50% in cultured cholinergic neurons exposed to low nanomolar concentrations of AßOs. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction, lactate dehydrogenase release, and [(3)H]choline uptake assays showed no evidence of neuronal damage or loss of viability that could account for reduced ChAT activity under these conditions. Glutamate receptor antagonists fully blocked ChAT inhibition and oxidative stress induced by AßOs. Antioxidant polyunsaturated fatty acids had similar effects, indicating that oxidative damage may be involved in ChAT inhibition. Treatment with insulin, previously shown to down-regulate neuronal AßO binding sites, fully prevented AßO-induced inhibition of ChAT. Interestingly, we found that AßOs selectively bind to ~50% of cultured cholinergic neurons, suggesting that ChAT is fully inhibited in AßO-targeted neurons. Reduction in ChAT activity instigated by AßOs may thus be a relevant event in early stage AD pathology, preceding the loss of cholinergic neurons commonly observed in AD brains.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Proteínas Aviárias/metabolismo , Colina O-Acetiltransferase/metabolismo , Neurônios/metabolismo , Estresse Oxidativo , Doença de Alzheimer/patologia , Animais , Antioxidantes/metabolismo , Proteínas Aviárias/genética , Técnicas de Cultura de Células , Células Cultivadas , Galinhas , Antagonistas de Aminoácidos Excitatórios/farmacologia , Neurônios/patologiaRESUMO
The most common neurodegenerative disorder afflicting the aging human population is Alzheimer's disease (AD). A major hallmark of AD is dementia from a loss of neuronal function, attributed to the presence and accumulation of ß-amyloid (Aß) peptide into senile plaques. Preceding senile plaque formation, abnormalities in axons can be observed as changes in morphologies and intracellular trafficking. Recently, it has been recognized that Aß also accumulates within neurons and this intraneuronal Aß accumulation has been reported to be critical in the disruption of synapses and cognitive function. Here, we report on the internalization of a fluorescently labeled Aß peptide into cultured chick retinal neurons. The pattern of Aß distribution during the time course of incubation is reminiscent of the endocytic pathway. Furthermore, the distribution of the internalized Aß peptide converges with that of myosin Vb and both relocalize from the axon to cell body. These observations are consistent with the hypothesis that AD proceeds as a result of an imbalance between Aß production and Aß clearance, suggesting a role for myosin Vb in this process.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Axônios/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Miosina Tipo V/metabolismo , Neurônios/metabolismo , Retina/metabolismo , Sinapses/metabolismo , Animais , Sobrevivência Celular , Células Cultivadas , Embrião de Galinha , Cromatografia Líquida de Alta Pressão , Humanos , Técnicas Imunoenzimáticas , Neurônios/citologia , Retina/citologiaRESUMO
The amyloid precursor protein (APP) is well known for giving rise to the amyloid-ß peptide and for its role in Alzheimer's disease. Much less is known, however, on the physiological roles of APP in the development and plasticity of the central nervous system. We have used phage display of a peptide library to identify high-affinity ligands of purified recombinant human sAPPα(695) (the soluble, secreted ectodomain from the main neuronal APP isoform). Two peptides thus selected exhibited significant homologies with the conserved extracellular domain of several members of the semaphorin (Sema) family of axon guidance proteins. We show that sAPPα(695) binds both purified recombinant Sema3A and Sema3A secreted by transfected HEK293 cells. Interestingly, sAPPα(695) inhibited the collapse of embryonic chicken (Gallus gallus domesticus) dorsal root ganglia growth cones promoted by Sema3A (K(d)≤8·10(-9) M). Two Sema3A-derived peptides homologous to the peptides isolated by phage display blocked sAPPα binding and its inhibitory action on Sema3A function. These two peptides are comprised within a domain previously shown to be involved in binding of Sema3A to its cellular receptor, suggesting a competitive mechanism by which sAPPα modulates the biological action of semaphorins.
Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Gânglios Espinais/metabolismo , Cones de Crescimento/fisiologia , Fragmentos de Peptídeos/metabolismo , Semaforina-3A/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animais , Células Cultivadas , Embrião de Galinha , Meios de Cultivo Condicionados/farmacologia , Gânglios Espinais/citologia , Humanos , Imunoprecipitação , Rim/citologia , Rim/efeitos dos fármacos , Rim/metabolismo , Biblioteca de Peptídeos , Conformação ProteicaRESUMO
D-aspartate is present in significant concentrations throughout the nervous tissue but its physiological role is still under discussion. Here, we report the process of d-aspartate release in retinal cells. [(3)H]-d-aspartate release occurs through a glutamate/aspartate exchange mechanism using excitatory amino acid transporters. This process is sodium-dependent and it is not prevented by glutamate receptor antagonists such as MK-801, DNQX or AIDA nor mimicked by glutamatergic agonists like kainate, NMDA or trans-ACPD. In vitro experiments indicate that the great majority of d-aspartate release is performed by neuronal cells and to a much lower extent by glial cells. This glutamate-mediated release process is mimicked by the competitive glutamate transporter antagonist l-trans-PDC and inhibited by the non-competitive transporter antagonist TBOA. Instead of the classical calcium-dependent exocytosis or transporter-reversal mediated neuronal release, d-aspartate efflux in the retina occurs mostly, if not exclusively, via an exchange of external l-glutamate by d-aspartate predominantly present in the cytoplasmatic compartment of neurons. These data also suggest that this process narrows down the specificity of excitatory signaling in the microenvironment of the synapses, reinforcing NMDA receptor activation by d-aspartate at the cost of reduction in the overall activation of excitatory amino acid receptors promoted by l-glutamate.
Assuntos
Ácido D-Aspártico/metabolismo , Ácido Glutâmico/metabolismo , Retina/metabolismo , Animais , Galinhas , Cromatografia Líquida de Alta PressãoRESUMO
The neurotoxicity of the amyloid-ß peptide (Aß) appears to be, at least in part, related to pathological activation of glutamate receptors by Aß aggregates. However, the downstream signaling pathways leading to neurodegeneration are still incompletely understood. Hyperactivation of nitric oxide synthase (NOS) and increased nitric oxide (NO) production have been implicated in excitotoxic neuronal damage caused by overactivation of glutamate receptors, and it has been suggested that increased NO levels might also play a role in neurotoxicity in Alzheimer's disease. We have examined the effect of blockade of NO production on the neurotoxicity instigated by Aß42 and by elevated concentrations of glutamate in chick embryo retinal neurons in culture. Results showed that L-nitroarginine methyl ester, a potent inhibitor of all NOS isoforms, had no protective effect against neuronal death induced by either Aß42 (20 µM) or glutamate (1 mM). Surprisingly, at short incubation times both Aß and glutamate decreased NO production in retinal neuronal cultures in the absence of neuronal death. Thus, excitotoxic insults induced by Aß and glutamate cause inhibition rather than activation of NO synthase in retinal neurons, suggesting that cell death induced by Aß or glutamate is not related to increased NO production. On the other hand, considering the role of NO in long term potentiation and synaptic plasticity, the decrease in NO levels instigated by Aß and glutamate suggests a possible mechanism leading to synaptic failure in AD.
Assuntos
Doença de Alzheimer/fisiopatologia , Peptídeos beta-Amiloides/farmacologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Óxido Nítrico/metabolismo , Fragmentos de Peptídeos/farmacologia , Retina/citologia , Doença de Alzheimer/metabolismo , Animais , Células Cultivadas , Embrião de Galinha , Inibidores Enzimáticos/farmacologia , Ácido Glutâmico/farmacologia , NG-Nitroarginina Metil Éster/farmacologia , Neurônios/citologia , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/metabolismo , Nitritos/metabolismo , Sinapses/fisiologiaRESUMO
The expression of A1 and A2a adenosine receptors is developmentally regulated in the chick retina, but little is known about the factors important for this regulation. Here, we show that cell aggregation and cAMP analogs promote a dramatic increase in A1 receptor expression. Importantly, a long-term stimulation of A2a receptors also promotes an increase of A1 receptor expression accompanied by a down-regulation of A2a receptors. Chick embryo retina cultures grown in the form of aggregates or dispersed cells accumulate cAMP when stimulated with dopamine or the adenosine agonist 2-chloroadenosine. However, inhibition of dopamine-dependent cAMP accumulation by 2-chloroadenosine was observed in aggregate cultures but not in dispersed cell cultures. Accordingly, A1 receptor binding sites were detected in aggregate cultures, but were low or absent from dispersed cell cultures. Interestingly, an increase of A1 binding sites was detected when dispersed cell cultures were treated for 5 days with permeable cAMP analogs, the adenylyl cyclase activator forskolin or A2a receptor agonists. Although a significant amount of A1 receptor protein was detected in dispersed cell cultures by western blot or immunocytochemistry, the long-term stimulation of A2a receptors also promoted an increase of the A1 receptor protein and mRNA, indicating that A2a receptors and cAMP were regulating transcription and/or translation of A1 receptors. We also found an increase of A1 receptors in locations in or near the membrane after treatment with A2a agonist. The long-term stimulation of retinal explants with A2a agonist also promoted an increase of A1 receptor protein. The results indicate that A2a receptors and the cAMP-dependent protein kinase pathway are involved in the regulation of A1 receptor expression during retinal development.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , AMP Cíclico/fisiologia , Receptor A1 de Adenosina/biossíntese , Receptor A2A de Adenosina/biossíntese , Retina/metabolismo , 2-Cloroadenosina/farmacologia , Animais , Western Blotting , Agregação Celular , Células Cultivadas , Embrião de Galinha , Dopamina/farmacologia , Dopamina/fisiologia , Expressão Gênica/fisiologia , Imuno-Histoquímica , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Retina/citologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Xantinas/metabolismoRESUMO
Polyamines are important regulators of growth and differentiation in a variety of cells, including parasitic protozoa. Promastigotes of Leishmania species have high levels of putrescine and spermidine and their growth can be inhibited by polyamine biosynthesis antagonists. The putrescine analogue 1,4-diamino-2-butanone (DAB) is microbicidal against Tritrichomonas foetus and Trypanosoma cruzi, so we tested its effects on Leishmania amazonensis proliferation, viability, organization, putrescine transport and synthesis as well as in vitro infectivity. DAB impaired promastigote proliferation dose-dependently (IC(50) 144 microM) and the parasite putrescine concentration was reduced by nearly 50 %. This analogue markedly inhibited both ornithine decarboxylase activity and [H(3)]putrescine uptake by promastigotes. Pre-treatment with DAB for 24 h led to compensatory enhancement of putrescine uptake, indicating an adaptive mechanism in DAB-treated parasites. Remarkably, DAB caused mitochondrial damage, assessed by transmission electron microscopy, and 3 h treatment with 1 mM DAB enhanced lipid peroxidation, whereas incubation with 10 mM DAB or for 24 h resulted in decreased peroxidation levels in the parasites. This effect was probably due to the loss of mitochondrial function, demonstrated by the diminished reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), not observed in macrophages. Murine macrophages infected with L. amazonensis amastigotes treated with DAB had parasite loads significantly (P<0.05) lower than controls, presumably due to interference with putrescine uptake and/or synthesis. These results suggest that putrescine may be involved in leishmanial survival, possibly by maintaining the parasite's mitochondrial function. The use of analogues to interfere with polyamine/diamine synthesis and transport may shed light on its function in intracellular parasite survival and lead to identification of new targets for leishmaniasis chemotherapy.
Assuntos
Antiprotozoários/farmacologia , Leishmania/metabolismo , Poliaminas/metabolismo , Putrescina/análogos & derivados , Animais , Antiprotozoários/metabolismo , Proliferação de Células , Células Cultivadas , Leishmania/efeitos dos fármacos , Leishmania/crescimento & desenvolvimento , Leishmania/ultraestrutura , Peroxidação de Lipídeos , Macrófagos/parasitologia , Camundongos , Microscopia Eletrônica de Transmissão , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Ornitina Descarboxilase/metabolismo , Putrescina/metabolismo , Putrescina/farmacologia , Sais de Tetrazólio/farmacologia , Tiazóis/farmacologiaRESUMO
Purified retina glial Müller cells can express the machinery for dopamine synthesis and release when maintained in culture. Dopamine is detected in cell extracts of cultures exposed to its precursor, L-DOPA. A large portion of synthesized dopamine is recovered in the superfusing medium showing the tendency of the accumulated dopamine to be released. Müller cells purified from developing chick and mouse retinas express L-DOPA decarboxylase (DDC; aromatic-L-amino-acid decarboxylase; EC 4.1.1.28) and the dopamine transporter DAT. The synthesis of dopamine from L-DOPA supplied to Müller cultures is inhibited by m-hydroxybenzylhydrazine, a DDC inhibitor. Dopamine release occurs via a transporter-mediated process and can activate dopaminergic D(1) receptors expressed by the glia population. The synthesis and release of dopamine were also observed in Müller cell cultures from mouse retina. Finally, cultured avian Müller cells display increased expression of tyrosine hydroxylase, under the influence of agents that increase cAMP levels, which results in higher levels of dopamine synthesized from tyrosine. A large proportion of glial cells in culture do express Nurr1 transcription factor, consistent with the dopaminergic characteristics displayed by these cells in culture. The results show that Müller cells, deprived of neuron influence, differentiate dopaminergic properties thought to be exclusive to neurons.
Assuntos
Diferenciação Celular/fisiologia , Dopamina/metabolismo , Neuroglia/metabolismo , Neurônios/metabolismo , Retina/metabolismo , Animais , Inibidores das Descarboxilases de Aminoácidos Aromáticos , Biomarcadores/metabolismo , Células Cultivadas , Embrião de Galinha , AMP Cíclico/metabolismo , Proteínas de Ligação a DNA/metabolismo , Dopa Descarboxilase/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Inibidores Enzimáticos/farmacologia , Corantes Fluorescentes , Camundongos , Neuroglia/citologia , Neurônios/citologia , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares , Fenótipo , Receptores de Dopamina D1/metabolismo , Retina/citologia , Fatores de Transcrição/metabolismo , Tirosina/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
The role of dopamine in the retina has been studied for the last 30 years and there is now increasing evidence that dopamine is used as a developmental signal in the embryonic retina. Dopamine is the main catecholamine found in the retina of most species, being synthesized from the L-amino acid tyrosine. Its effects are mediated by G protein coupled receptors constituting the D(1) (D(1) and D(5)) and D(2) (D(2), D(3) and D(4)) receptor subfamilies that can be coupled to adenylyl cyclase in opposite manners. Dopamine-mediated cyclic AMP (cAMP) accumulation, via D(1)-like receptors, is observed very early during retina ontogeny, before synaptogenesis and, in some species, before the expression of tyrosine hydroxylase (TH), the enzyme that characterizes the neuronal dopaminergic phenotype. D(2)-like receptors appear in the tissue days after D(1)-like activity is detected. In the embryonic avian retina, before the tissue is capable of synthesizing its own dopamine via TH, dopamine synthesis is observed from L-DOPA supplied to the neuroretina from retina pigmented epithelium which results in dopaminergic communication in the embryonic tissue before TH expression. Müller cells, the main glia type found in the retina, seem to actively contribute to dopaminergic activity in the retinal tissue. Understanding the dopaminergic role during retina development may contribute to novel strategies approaching certain visual dysfunctions such as those found in ocular albinism.
Assuntos
Dopamina/biossíntese , Neurônios/metabolismo , Retina/embriologia , Retina/crescimento & desenvolvimento , Transdução de Sinais/fisiologia , Animais , AMP Cíclico/metabolismo , Vias Neurais/embriologia , Vias Neurais/crescimento & desenvolvimento , Vias Neurais/metabolismo , Neuroglia/metabolismo , Receptores Dopaminérgicos/metabolismo , Retina/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
PURPOSE: Toxoplasma gondii, the most common cause of retinochoroiditis in humans, is an obligate intracellular protozoan parasite that depends on te host cell's microenvironment to proliferate. Because congenital infection is associated with a higher risk of ocular involvement than a postnatally acquired infection, this study was conducted to investigate the ability of Toxoplasma gondii to infect retinal tissue during development, when cellular environmental changes normally occur. METHODS: Retinas from 5- to 9-day-old chick embryos were used. Stationary cultures were prepared in 24-well cell culture dishes and maintained at 37 degrees C in DMEM plus 5% fetal bovine serum for 2 to 6 days. Then the wells were infected with 4 x 10(5) tachyzoites. Retina explants and aggregate cell cultures were maintained in DMEM under rotation at 37 degrees C. T. gondii proliferation was measured using [(3)H]-thymidine incorporation after 72 hours. Ornithine and arginine decarboxylase (ODC and ADC) activities were determined by measuring CO(2) production from [1-(14)C]-ornithine and [1-(14)C]-arginine, respectively. RESULTS: The proliferation of tachyzoites was high in dense, stationary cultures expressing elevated ODC and ADC activity. The addition of ODC or ADC inhibitors reduced T. gondii proliferation by approximately 20% to 40%. As for cultured retina cells, retina explants also allowed T. gondii proliferation whenever ODC activity was high. CONCLUSIONS: The data suggest a direct correlation between retinal polyamine biosynthesis and the proliferation of T. gondii, in agreement with the observation that individuals infected congenitally have a greater risk of development of toxoplasmic retinochoroiditis.
Assuntos
Retina/embriologia , Toxoplasma/fisiologia , Toxoplasmose Animal/parasitologia , Toxoplasmose Congênita/parasitologia , Toxoplasmose Ocular/parasitologia , Animais , Carboxiliases/metabolismo , Células Cultivadas , Embrião de Galinha , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Técnicas Imunoenzimáticas , Camundongos , Ornitina Descarboxilase/metabolismo , Poliaminas/metabolismo , Retina/parasitologia , Retina/ultraestrutura , Toxoplasmose Animal/enzimologia , Toxoplasmose Congênita/enzimologia , Toxoplasmose Ocular/enzimologiaRESUMO
Alzheimer's disease (AD) and several other neurological disorders have been linked to the overactivation of glutamatergic transmission and excitotoxicity as a common pathway of neuronal injury. The beta-amyloid peptide (Abeta) is centrally related to the pathogenesis of AD, and previous reports have demonstrated that the blockade of glutamate receptors prevents Abeta-induced neuronal death. We show that taurine, a beta-amino acid found at high concentrations in the brain, protects chick retinal neurons in culture against the neurotoxicity of Abeta and glutamate receptor agonists. The protective effect of taurine is not mediated by interaction with glutamate receptors, as demonstrated by binding studies using radiolabeled glutamate receptor ligands. The neuroprotective action of taurine is blocked by picrotoxin, an antagonist of GABA(A) receptors. GABA and the GABA(A) receptor agonists phenobarbital and melatonin also protect neurons against Abeta-induced neurotoxicity. These results suggest that activation of GABA receptors decreases neuronal vulnerability to excitotoxic damage and that pharmacological manipulation of the excitatory and inhibitory neurotransmitter tonus may protect neurons against a variety of insults. GABAergic transmission may represent a promising target for the treatment of AD and other neurological disorders in which excitotoxicity plays a relevant role.
Assuntos
Precursor de Proteína beta-Amiloide/toxicidade , Proteínas do Olho/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Receptores de GABA/efeitos dos fármacos , Receptores de Glutamato/efeitos dos fármacos , Taurina/farmacologia , Doença de Alzheimer/tratamento farmacológico , Animais , Células Cultivadas , Senescência Celular , Embrião de Galinha , Maleato de Dizocilpina/farmacologia , Agonistas de Aminoácidos Excitatórios/farmacologia , Proteínas do Olho/fisiologia , Ácido Glutâmico/farmacologia , Ácido Caínico/toxicidade , N-Metilaspartato/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Picrotoxina/farmacologia , Receptores de AMPA/efeitos dos fármacos , Receptores de GABA/fisiologia , Receptores de GABA-A/efeitos dos fármacos , Receptores de GABA-A/fisiologia , Receptores de Glutamato/fisiologia , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Retina/citologia , Retina/embriologia , Taurina/uso terapêutico , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiônico/farmacologiaRESUMO
DOPA decarboxylase (DDC; aromatic-l-amino acid decarboxylase; EC 4.1.1.28) is absent in retinas from 6-day-old chicken embryos (E6) but is expressed in retina of E8 embryos, in the presumptive outer plexiform layer. Thereafter, DDC appears in cell bodies of presumptive amacrine cells. The dopamine (DA) content of E9/10 and E15/16 retinas, pre-incubated with l-DOPA for 1 h, increased 250- and 600-fold, respectively, showing that DDC is active since early in development. Intercellular communication, measured by endogenous cyclic AMP accumulation, was observed when retinas from E9/10 to E15/16 were pre-incubated for 1 h with 1 mm l-DOPA, washed and followed by incubation in the presence of 0.5 mm 3-isobutyl-1-methylxanthine, a phosphodiesterase inhibitor. Cyclic AMP accumulation was prevented when pre-incubation with l-DOPA was carried out in the presence of carbidopa. Moreover, the accumulation of cyclic AMP was inhibited by SCH 23390 (2 micro m). The incubation of retinas in medium previously conditioned by retina-pigmented epithelium (RPE) also increased its cyclic AMP content with the characteristics described for l-DOPA. Our results show that dopaminergic communication takes place in the embryonic retina, before tyrosine hydroxylase expression, provided l-DOPA is supplied to the tissue. It also shows that RPE is a potential source of l-DOPA early in development.