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The solubility of Eu(III) was investigated under undersaturated conditions in acidic, dilute to concentrated MgSO4 and Na2SO4 solutions at T = (22 ± 2) °C. After attaining equilibrium conditions, solid phases were characterized by a multi-method approach, including X-ray diffraction (XRD), Raman and infrared (IR) spectroscopy, quantitative chemical analysis (ICP-OES) and thermogravimetric analysis (TG-DTA). A total of 45 solubility samples were investigated for the systems Eu2(SO4)3-MgSO4-H2O (19 samples) and Eu2(SO4)3-Na2SO4-H2O (26 samples). Eu2(SO4)3·8H2O(cr) was found to control the solubility of Eu(III) in all investigated MgSO4 solutions, as well as in dilute Na2SO4 systems. The transformation of Eu2(SO4)3·8H2O(cr) into the double salt Na2Eu2(SO4)4·2H2O(cr) was observed at mNa2SO4 > 0.01 mol kg-1. The latter phase is characterized by significantly lower solubility. Based on these experimental solubility measurements, thermodynamic and activity models were proposed based on the Pitzer equations considering the full dissociation of the Eu(III) species in MgSO4 and Na2SO4 aqueous solutions, i.e. deliberately excluding Eu(III)-sulfate complex formation. A combination of the geochemical calculation code PhreeSCALE and the parameter estimation code PEST was used to determine the values of solubility products and binary and ternary specific interaction parameters (ß(0)ij, ß(1)ij, CÏij, θik, Ψijk).
RESUMO
A time-resolved laser fluorescence spectroscopy (TRLFS) study was carried out to investigate the Eu(III)-SO4 complexation at room temperature over a wide range of Na2SO4 concentrations (0-2 mol kg-1). Spectroscopic observations confirm the step-wise formation of the aqueous complexes Eu(SO4)+, Eu(SO4)2- and Eu(SO4)33- over the investigated Na2SO4 concentrations. Combining TRLFS data obtained in this study and solubility data reported in Part I of this work for the Eu2(SO4)3-Na2SO4-H2O and Eu2(SO4)3-MgSO4-H2O systems, thermodynamic and activity models were derived based on the SIT and Pitzer formalisms. A combination of the geochemical calculation codes PhreeqC (SIT), PhreeSCALE (Pitzer) and the parameter estimation code PEST was used to determine the solubility products of Eu2(SO4)3·8H2O(cr) and Na2Eu2(SO4)4·2H2O(cr), stability constants of the Eu(III)-SO4 complexes (ß0i), and the specific binary and ternary interaction parameters (εij, ß(0)ij, ß(1)ij, CÏij, θik, Ψijk) for both activity models. The thermodynamic constants determined in this work are discussed with reference to values available in the literature.
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Echinacea purpurea (L.) Moench is widely known for its medicinal properties, being one of the most used medicinal plants for its immunostimulant properties. Nevertheless, there is still scarce information on its cytotoxic activity. Thus, this study aims at evaluating the cytotoxicity and antimicrobial activity of several aqueous and organic extracts of the aerial parts of this plant and chemically characterizing the obtained extracts. The analysis was performed by HPLC-DAD-ESI/MS. Fifteen compounds were identified; of these, seven were phenolic acids and eight were flavonoids. Non-polar compounds were evaluated by GC/MS, with a total of sixty-four compounds identified, and the most abundant groups were the sterols, fatty acids and long-chain hydrocarbons. The highest antimicrobial activity was exhibited by the dichloromethane, ethyl acetate, and acetone extracts. Dichloromethane and n-hexane extracts showed the highest cytotoxic activity. Therefore, they were fractionated, and the obtained fractions were also assessed for their cytotoxicity. Notwithstanding, the cytotoxicity of the extracts was superior to that of the obtained fractions, evidencing a possible synergistic effect of different compounds in the whole extracts.
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The inflorescences of Calluna vulgaris were nutritionally and chemically characterized. Furthermore, different organic and aqueous extracts were prepared for the evaluation of their bioactive properties. From the obtained results, carbohydrates were the major compounds, followed by protein, lipid and ashes. It was possible to identify the sugars fructose and glucose, five organic acids, 26 individual fatty acids and the four tocopherol isoforms. Concerning the extract composition, 12 phenolic compounds were identified, with myricetin-3-O-glucoside and myricetin-O-rhamnoside predominating. Concerning the bioactive effects, the more polar extracts showed not only the highest amount in phenolic compounds, but also the strongest antioxidant and antibacterial activities. In contrast, for the anti-inflammatory and cytotoxic potential, the most effective extracts were the n-hexane and the ethyl acetate extracts, respectively. C. vulgaris presented a wide range of biological effects, highlighting their capacity to inhibit pathogenic bacteria without affecting beneficial microflora, corroborating their use in traditional medicine.
Assuntos
Bactérias/efeitos dos fármacos , Calluna/química , Microbiota/efeitos dos fármacos , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Vagina/microbiologia , Antibacterianos/farmacologia , Antioxidantes/química , Antioxidantes/farmacologia , Bactérias/classificação , Bactérias/genética , Bactérias/isolamento & purificação , Ácidos Graxos/química , Ácidos Graxos/farmacologia , Feminino , Humanos , Testes de Sensibilidade Microbiana , Fenóis/química , Fenóis/farmacologia , Tocoferóis/química , Tocoferóis/farmacologiaRESUMO
Geranium molle L., commonly known as Dove's-foot Crane's-bill or Dovesfoot Geranium, is an herbaceous plant belonging to the Geraniaceae family. Contrary to many other Geranium species, the bioactivity and the phytochemical composition of G. molle seem not to have attracted attention until a recent study from our group regarding the bioactivity of several aqueous and organic extracts of the plant. In particular, we assessed the cytotoxic activity of these extracts against several human tumor cell lines (breast, lung, cervical and hepatocellular carcinomas) and a non-tumor porcine liver primary cell line, inspired by an ethnopharmacological report describing the traditional use of this medicinal plant in some regions of Northeast Portugal for the treatment of cancer. Following this preliminary evaluation, the most active extracts (acetone and methanol) were fractionated by column chromatography and the resulting fractions were evaluated for their antioxidant activity and cytotoxicity against the same cell lines. The bio-guided fractionation of the extracts resulted in several fractions exhibiting improved bioactivity in comparison with the corresponding crude extracts. The fractions obtained from the acetone extract consistently displayed the lowest EC50 and GI50 values and presented the highest content of total phenolic compounds. The phytochemical composition of the most bioactive fractions of the acetone and methanol extracts was also determined and about thirty compounds, mainly flavonoids and phenolic acids, could be identified for the first time in G. molle.
Assuntos
Antineoplásicos Fitogênicos/isolamento & purificação , Antioxidantes/isolamento & purificação , Descoberta de Drogas , Flavonoides/isolamento & purificação , Geranium/química , Fenol/isolamento & purificação , Extratos Vegetais/química , Acetona/química , Animais , Antineoplásicos Fitogênicos/efeitos adversos , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/farmacologia , Antioxidantes/efeitos adversos , Antioxidantes/química , Antioxidantes/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Etnofarmacologia , Flavonoides/efeitos adversos , Flavonoides/química , Flavonoides/farmacologia , Geranium/crescimento & desenvolvimento , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Humanos , Metanol/química , Estrutura Molecular , Neoplasias/tratamento farmacológico , Concentração Osmolar , Fenol/efeitos adversos , Fenol/química , Fenol/farmacologia , Extratos Vegetais/efeitos adversos , Extratos Vegetais/isolamento & purificação , Extratos Vegetais/farmacologia , Portugal , Solventes/química , Sus scrofaRESUMO
In this study, diabetes mellitus (DM) was induced in Wistar rats during pregnancy and maintained in the postpartum period (PP) and we evaluated systolic blood pressure (SBP), glomerular filtration rate (GFR) and renal immunohistochemical and morphometric studies from different groups: G1 (non-pregnant control rats), G2 (non-pregnant diabetic rats), G3 (control mothers) and G4 (diabetic mothers). We found that there were no differences in relation to SBP, but there was a tendency for reduction in GFR from G4 compared with the other groups (G). There was increased total kidney weight/body weight ratio of G4 compared with other G. There were increase in glomerular tuft area in G3 and G4 compared with G1 and G2. G2 and G4 showed even higher percentage of cortical collagen. G3 showed increased glomerular proliferating cells compared with G1 and G2, while in G4 this number was smaller than G3. Cell proliferation was higher in the tubulointerstitial (TBI) compartment from G4. Glomerular and TBI α-smooth muscle actin expression was increased in G4 compared with other G. The glomerular p-p38 expression showed a pattern similar to proliferation cell nuclear antigen, with a reduction of p-p38 in G4 relative to other G. The immunoreactivity of p-JNK was higher in both the glomeruli and TBI compartment in G4 compared with G1, G2 and G3. The DM induced during pregnancy and maintained in the PP resulted in renal structural and functional changes to mothers. In addition, altered mitogen-activated protein kinase expression in association with these changes may play an important role in renal damage observed in the present investigation.
Assuntos
Diabetes Mellitus Experimental/fisiopatologia , Nefropatias Diabéticas/fisiopatologia , Período Pós-Parto/fisiologia , Complicações na Gravidez/fisiopatologia , Aloxano/toxicidade , Animais , Pressão Sanguínea/fisiologia , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/patologia , Nefropatias Diabéticas/sangue , Nefropatias Diabéticas/patologia , Modelos Animais de Doenças , Feminino , Taxa de Filtração Glomerular/fisiologia , Humanos , Rim/patologia , Rim/fisiopatologia , Masculino , Mães , Gravidez , Complicações na Gravidez/sangue , Complicações na Gravidez/induzido quimicamente , Complicações na Gravidez/patologia , Ratos , Ratos WistarRESUMO
Geranium robertianum L. has been used in folk medicine and herbalism practice for the treatment of various conditions, but the study of its bioactivity has been barely addressed. Although its phytochemical composition has received some attention, contributions to the nutritional composition are practically unknown. Herein, G. robertianum gathered in Trás-os-Montes, Northeastern Portugal, was chemically characterized regarding nutritional parameters, and the antioxidant activity and cytotoxicity against several human tumor cell lines and non-tumor porcine liver primary cells of several aqueous and organic extracts were evaluated. G. robertianum showed to be an equilibrated valuable herb, rich in carbohydrates and proteins, and poor in fat, providing sugars, tocopherols, organic and essential fatty acids. Amongst the extracts, the acetone one showed the highest total phenol and total flavonoid contents, as well as the greatest antioxidant and cytotoxic activities. This extract showed to contain hydrolysable tannins (e.g. geraniin and castalagin/vescalagin), as the main phenolic compounds.
Assuntos
Geranium/química , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Acetona , Antineoplásicos Fitogênicos , Antioxidantes , Linhagem Celular Tumoral , Flavonoides/análise , Glucosídeos/análise , Humanos , Taninos Hidrolisáveis/análise , Valor Nutritivo , Fenóis/análise , Fitoterapia , Portugal , ÁguaRESUMO
The neurodegenerative and inflammatory environment that is prevalent in the diabetic eye is a key player in the development and progression of diabetic retinopathy. The adenosinergic system is widely regarded as a significant modulator of neurotransmission and the inflammatory response, through the actions of the four types of adenosine receptors (A1R, A2AR, A2BR, and A3R), and thus could be revealed as a potential player in the events unfolding in the early stages of diabetic retinopathy. Herein, we review the studies that explore the impact of diabetic conditions on the retinal adenosinergic system, as well as the role of the said system in ameliorating or exacerbating those conditions. The experimental results described suggest that this system is heavily affected by diabetic conditions and that the modulation of its components could reveal potential therapeutic targets for the treatment of diabetic retinopathy, particularly in the early stages of the disease.
Assuntos
Adenosina/metabolismo , Retinopatia Diabética/metabolismo , Receptores Purinérgicos P1/metabolismo , Retina/metabolismo , Transdução de Sinais , Animais , Microambiente Celular , Retinopatia Diabética/tratamento farmacológico , Retinopatia Diabética/patologia , Retinopatia Diabética/fisiopatologia , Humanos , Antagonistas de Receptores Purinérgicos P1/uso terapêutico , Receptores Purinérgicos P1/efeitos dos fármacos , Retina/efeitos dos fármacos , Retina/patologia , Retina/fisiopatologia , Transdução de Sinais/efeitos dos fármacosRESUMO
After a period of indifference, in which synthetic compounds were favored, there is an increasing interest in the study of the biological properties of plants and the active principles responsible for their therapeutic properties. Geranium molle L. has been used in the Portuguese folk medicine for the treatment of various ailments including cancer but, unlike many of the species from the Geranium genus, its phytochemical characterization and biological activity are virtually unexplored. In this study a G. molle sample from Trás-os-Montes, north-eastern Portugal, was chemically characterized regarding nutritional value, free sugars, organic acids, fatty acids and tocopherols, and several aqueous (decoction, infusion) and organic (n-hexane, dichloromethane, ethyl acetate, acetone, methanol) extracts of the plant were assessed for their bioactive properties. The antioxidant activity was evaluated by means of the free radicals scavenging activity, reducing power and inhibition of lipid peroxidation. The cytotoxicity of the different extracts was assessed in vitro against several human cancer cell lines (breast, lung, cervical and hepatocellular carcinomas) and, additionally, their hepatotoxicity was evaluated using a porcine liver primary cell culture. G. molle was shown to be rich in carbohydrates and proteins, providing tocopherols and essential fatty acids. Amongst the various extracts, the acetone extract was found to have the highest content of phenolic compounds (mainly ellagitannins, but also some flavone and flavonol glycosides) as well as the highest antioxidant and cytotoxic activities. To the best of our knowledge, this is the first report on the chemical composition and bioactive properties of G. molle.
Assuntos
Geranium/química , Compostos Fitoquímicos/química , Compostos Fitoquímicos/farmacologia , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Animais , Antioxidantes/química , Antioxidantes/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Linhagem Celular Tumoral , Doença Hepática Induzida por Substâncias e Drogas , Ácidos Graxos Essenciais/química , Flavonas/química , Flavonoides/análise , Flavonóis , Sequestradores de Radicais Livres/farmacologia , Glicosídeos , Humanos , Taninos Hidrolisáveis/química , Peroxidação de Lipídeos/efeitos dos fármacos , Valor Nutritivo , Portugal , Cultura Primária de Células , Suínos , Tocoferóis/químicaRESUMO
This study presents several new squarylium dyes derived from benzothiazole and benzoselenazole with several structural variations, namely the nature of the heteroaromatic ring and the length of the N,N'-dialkyl groups. Before being investigated in connection with their effect on living cells and/or tissues, these novel compounds were characterized, namely with respect to the determination of their main photophysical parameters. Therefore, a study of the ground state absorption, fluorescence emission (quantum yields and lifetimes) and singlet oxygen generation quantum yields was performed for all the compounds synthesized in order to evaluate their efficiency as photosensitizers. An increase of the alkyl chain length from ethyl to hexyl did not produce a clear change in the fluorescence quantum yields, showing no influence on the photoisomerization process. Heavy atom inclusion (Se instead of S) enhanced the singlet oxygen generation efficiency and decreased the intensity of the fluorescence emission. The external heavy atom effect (I(-) as a counterion instead of CF3SO3(-)) produced a significant increase in the singlet oxygen formation quantum yield (about 20%). Transient absorption studies in aerated and oxygen free samples revealed that the photoisomerization process, which could compete with the triplet state formation for all dyes in solution, is a negligible pathway for the excited state deactivation, in accordance with the rigidity introduced by the squaric ring into the polymethine chain of the dye, both in chloroform and ethanol. However, in the case of the chloroform solution a new transient was detected in air equilibrated solutions, resulting from a reaction of the excited squarylium dye in the singlet state with CHCl3Ë, and assigned to the radical cation (SQ(+)Ë) of the dye.
RESUMO
The most selective purification method for proteins and other biomolecules is affinity chromatography. This method is based on the unique biological-based specificity of the biomolecule-ligand interaction and commonly uses biological ligands. However, these ligands may present some drawbacks, mainly because of their cost and lability. Dye-affinity chromatography overcomes the limitations of biological ligands and is widely used owing to the low cost of synthetic dyes and to their resistance to biological and chemical degradation. In this work, immobilized aminosquarylium cyanine dyes are used in order to exploit affinity interactions with standard proteins such as lysozyme, α-chymotrypsin and trypsin. These studies evaluate the affinity interactions occurring between the immobilized ligand and the different proteins, as a reflection of the sum of several molecular interactions, namely ionic, hydrophobic and van der Waals, spread throughout the structure, in a defined spatial manner. The results show the possibility of using an aminosquarylium cyanine dye bearing a N-hexyl pendant chain, with a ligand density of 1.8 × 10(-2) mmol of dye/g of chromatographic support, to isolate lysozyme, α-chymotrypsin and trypsin from a mixture. The application of a decreasing ammonium sulfate gradient resulted in the recovery of lysozyme in the flowthrough. On the other hand, α-chymotrypsin and trypsin were retained, involving different interactions with the ligand. In conclusion, this study demonstrates the potential applicability of ligands such as aminosquarylium cyanine dyes for the separation and purification of proteins by affinity chromatography.
Assuntos
Carbocianinas/química , Cromatografia de Afinidade/métodos , Corantes Fluorescentes/química , Proteínas/isolamento & purificação , Carbocianinas/síntese química , Carbocianinas/metabolismo , Cromatografia de Afinidade/instrumentação , Eletroforese em Gel de Poliacrilamida , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/metabolismo , Ligantes , Proteínas/química , Proteínas/metabolismoRESUMO
Diabetic retinopathy (DR), a leading cause of vision loss and blindness among working-age adults, holds several hallmarks of an inflammatory disease. The increase in cell death in neural retina is an early event in the diabetic retina, preceding the loss of microvascular cells. Since tumor necrosis factor-α (TNF-α) has been shown to trigger the death of perycites and endothelial cells as well as the breakdown of the blood-retinal barrier, we set out to investigate whether TNF-α acting through tumor necrosis factor receptor 1 (TNFR1), the major receptor responsible for mediating TNF-induced cell death, could also be responsible for the early neuronal cell death observed in DR. We used retinal neural cell cultures exposed to high glucose conditions, to mimic hyperglycaemia, and evaluated the contribution of TNFR1 in neural cell death. TNFR1 was found to be present to a great extent in retinal neurons and the levels of this receptor were found to be altered in cells cultured in high glucose conditions. High glucose induced an early decrease in cell viability, an increase in apoptosis and a higher immunoreactivity for the cleaved caspase-3, indicating a high glucose-induced caspase-dependent cell death. These observations were correlated with an increase in TNF-α expression. Nonetheless, inhibiting the activation of TNFR1 was sufficient to prevent the decrease in cell viability and the increase in retinal cell death by apoptosis. In conclusion, our data indicate that TNF-α acting through TNFR1 is responsible for the high glucose-induced cell death and that blocking the activity of this receptor is an adequate strategy to avoid cell loss in such conditions.
Assuntos
Apoptose/fisiologia , Glucose/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Neurônios Retinianos/metabolismo , Animais , Animais Recém-Nascidos , Caspase 3/metabolismo , Sobrevivência Celular , Células Cultivadas , Retinopatia Diabética/metabolismo , Ratos , Ratos Wistar , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Diabetic retinopathy (DR) is the leading cause of blindness in adults. In diabetes, there is activation of microglial cells and a concomitant release of inflammatory mediators. However, it remains unclear how diabetes triggers an inflammatory response in the retina. Activation of P2 purinergic receptors by adenosine triphosphate (ATP) may contribute to the inflammatory response in the retina, insofar as it has been shown to be associated with microglial activation and cytokine release. In this work, we evaluated how high glucose, used as a model of hyperglycemia, considered the main factor in the development of DR, affects the extracellular levels of ATP in retinal cell cultures. We found that basal extracellular ATP levels were not affected by high glucose or mannitol, but the extracellular elevation of ATP, after a depolarizing stimulus, was significantly higher in retinal cells cultured in high glucose compared with control or mannitol-treated cells. The increase in the extracellular ATP was prevented by application of botulinum neurotoxin A or by removal of extracellular calcium. In addition, degradation of exogenously added ATP was significantly lower in high-glucose-treated cells. It was also observed that, in retinal cells cultured under high-glucose conditions, the changes in the intracellular calcium concentrations were greater than those in control or mannitol-treated cells. In conclusion, in this work we have shown that high glucose alters the purinergic signaling system in the retina, by increasing the exocytotic release of ATP and decreasing its extracellular degradation. The resulting high levels of extracellular ATP may lead to inflammation involved in the pathogenesis of DR.
Assuntos
Trifosfato de Adenosina/metabolismo , Glucose/metabolismo , Retina/metabolismo , Análise de Variância , Animais , Toxinas Botulínicas Tipo A/administração & dosagem , Cálcio/metabolismo , Células Cultivadas , Retinopatia Diabética/etiologia , Modelos Animais de Doenças , Espaço Extracelular/metabolismo , Hiperglicemia/metabolismo , Manitol/metabolismo , Neurotoxinas/administração & dosagem , Ratos , Ratos WistarRESUMO
Proteolytic cleavage of the Na(+)/Ca(2+) exchanger (NCX) by calpains impairs calcium homeostasis, leading to a delayed calcium overload and excitotoxic cell death. However, it is not known whether reversal of the exchanger contributes to activate calpains and trigger neuronal death. We investigated the role of the reversal of the NCX in Ca(2+) dynamics, calpain activation and cell viability, in alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor-stimulated hippocampal neurons. Selective overactivation of AMPA receptors caused the reversal of the NCX, which accounted for approximately 30% of the rise in intracellular free calcium concentration ([Ca(2+)](i)). The NCX reverse-mode inhibitor, 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea (KB-R7943), partially inhibited the initial increase in [Ca(2+)](i), and prevented a delayed increase in [Ca(2+)](i). In parallel, overactivation of AMPA receptors strongly activated calpains and led to the proteolysis of NCX3. KB-R7943 prevented calpain activation, cleavage of NCX3 and was neuroprotective. Silencing of NCX3 reduced Ca(2+) uptake, calpain activation and was neuroprotective. Our data show for the first time that NCX reversal is an early event following AMPA receptor stimulation and is linked to the activation of calpains. Since calpain activation subsequently inactivates NCX, causing a secondary Ca(2+) entry, NCX may be viewed as a new suicide substrate operating in a Ca(2+)-dependent loop that triggers cell death and as a target for neuroprotection.
Assuntos
Cálcio/metabolismo , Calpaína/metabolismo , Hipocampo/citologia , Neurônios/metabolismo , Receptores de AMPA/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Animais , Células Cultivadas/citologia , Células Cultivadas/metabolismo , Inativação Gênica , Homeostase , Degeneração Neural , Neurônios/citologia , Ratos , Trocador de Sódio e Cálcio/antagonistas & inibidores , Trocador de Sódio e Cálcio/genética , Tioureia/análogos & derivados , Tioureia/farmacologiaRESUMO
Diabetic retinopathy is a leading cause of blindness among adults in the western countries. It has been reported that neurodegeneration may occur in diabetic retinas, but the mechanisms underlying retinal cell death are poorly understood. We found that high glucose increased the number of cells with condensed nuclei and the number of TUNEL-positive cells, and caused an increase in the translocation of phosphatidylserine to the outer leaflet of the plasma membrane, indicating that high glucose induces apoptosis in cultured retinal neural cells. The activity of caspases did not increase in high glucose-treated cells, but apoptosis-inducing factor (AIF) levels decreased in the mitochondria and increased in the nucleus, indicating a translocation to the nucleus where it may cause DNA fragmentation. These results demonstrate that elevated glucose induces apoptosis in cultured retinal neural cells. The increase in apoptosis is not dependent on caspase activation, but is mediated through AIF release from the mitochondria.
Assuntos
Apoptose/efeitos dos fármacos , Glucose/farmacologia , Neurônios/citologia , Neurônios/efeitos dos fármacos , Retina/citologia , Trifosfato de Adenosina/metabolismo , Animais , Antropologia Cultural , Apoptose/fisiologia , Fator de Indução de Apoptose/metabolismo , Caspases/metabolismo , Núcleo Celular/metabolismo , Retinopatia Diabética/patologia , Mitocôndrias/metabolismo , Neurônios/metabolismo , Ratos , Ratos WistarRESUMO
We investigated the effect of adenosine A1 receptors on the release of acetylcholine (ACh) and GABA, and on the intracellular calcium concentration ([Ca2+]i) response in cultured chick amacrine-like neurons, stimulated by KCl depolarization. The KCl-induced release of [3H]ACh, but not the release of [14C]GABA, was potentiated when adenosine A1 receptor activation was prevented by perfusing the cells with adenosine deaminase (ADA) or with 1,3-dipropyl-8-cycloentylxanthine (DPCPX). The changes in the [Ca2+]i induced by KCl depolarization, measured in neurite segments of single cultured cells, were also modulated by endogenous adenosine, acting on adenosine A1 receptors. Our results show that adenosine A1 receptors inhibit Ca2+ entry coupled to ACh release, but not to the release of GABA, suggesting that the synaptic vesicles containing each neurotransmitter are located in different zones of the neurites, containing different VSCC and/or different densities of adenosine A1 receptors.
Assuntos
Acetilcolina/metabolismo , Canais de Cálcio/metabolismo , Receptores Purinérgicos P1/fisiologia , Retina/metabolismo , Ácido gama-Aminobutírico/metabolismo , Animais , Cálcio/metabolismo , Canais de Cálcio Tipo N/metabolismo , Células Cultivadas , Embrião de Galinha , Retina/citologia , Retina/embriologiaRESUMO
The Dale's law postulates that a neuron releases the same neurotransmitter from all its branches. In the case of multiple neurotransmitters it would require all transmitters to be released from all branches. The retinal cholinergic amacrine cells contain and release gamma-aminobutyric (GABA) and, therefore, if GABA and acetylcholine (ACh) are released at the same sites, this could mean that amacrine cells simultaneously excite and inhibit postsynaptic cells. Conversely, if the two neurotransmitters are released at different synapses, or if their release is regulated in a distinct manner, they may play different physiological roles. Recent studies carried out in cultured cholinergic amacrine-like neurons showed that Ca(2+)-dependent release of ACh and GABA have a different sensitivity to membrane depolarization, to the effect of blockers of voltage gated Ca(2+) channels (VGCC) and to the effect of presynaptic A(1) adenosine receptors. Therefore, it is proposed that in retinal amacrine cells the Ca(2+)-dependent release of ACh and GABA occurs at distinct cellular locations. The possible nature of these release sites and the physiological significance of this model are discussed in this review.
Assuntos
Neurotransmissores/metabolismo , Retina/metabolismo , Animais , Humanos , Neurotransmissores/fisiologia , Receptores de Neurotransmissores/metabolismo , Retina/citologia , Retina/fisiologiaRESUMO
Retinal amacrine cells express metabotropic glutamate receptors (mGluRs), but their physiological role is unknown. We investigated the effect of mGluR on [(3)H]acetylcholine release ([(3)H]ACh) from cultured chick amacrine-like neurons. Activation of group III mGluR with the agonist L(+)-2-amino-4-phosphonobutyric acid (L-AP4) inhibited [(3)H]ACh release evoked by 25 mM KCl in a dose-dependent manner, and this effect was sensitive to pertussis toxin. In contrast, activation of group I or II mGluR with (S)-3, 5-dihydroxyphenylglycine (DHPG) and (2S,2'R,3'R)-2-(2', 3'-dicarboxycyclopropyl)glycine (DCG-IV), respectively, did not affect significantly [(3)H]ACh release. The effect of L-AP4 on [(3)H]ACh release was sensitive to nitrendipine, suggesting that it is, at least in part, due to inhibition of L-type Ca(2+) channels. Activation of group III mGluR also partly inhibited omega-conotoxin GVIA-sensitive Ca(2+) channels, coupled to [(3)H]ACh release. The L-AP4 did not affect the cAMP levels measured in amacrine-like neurons depolarized with 25 mM KCl or stimulated with forskolin, indicating that the effect of group III mGluR on [(3)H]ACh release is not due to inhibition of adenylyl cyclase activity. Inhibition of protein kinase A with KT-5720 was without effect on [(3)H]ACh release evoked by 25 mM KCl, further indicating that the effect of group III mGluR on [(3)H]ACh release cannot be attributed to the inhibition of the kinase. The effect of L-AP4 on [(3)H]ACh release was reversed by DHPG or by DCG-IV, and activation of group II mGluR also partially inhibited cAMP production stimulated by forskolin. Taken together, our results show that the effect of group III mGluR on [(3)H]ACh release may be due to a direct inhibition of L- and N-type Ca(2+) channels and is modulated by group I and group II mGluR.
Assuntos
Acetilcolina/metabolismo , Neurônios/metabolismo , Receptores de Glutamato Metabotrópico/fisiologia , Retina/metabolismo , 4-Aminopiridina/farmacologia , Inibidores de Adenilil Ciclases , Adenilil Ciclases/metabolismo , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo L/metabolismo , Canais de Cálcio Tipo N/metabolismo , Células Cultivadas , Embrião de Galinha , Colina/metabolismo , Colforsina/farmacologia , AMP Cíclico/metabolismo , Receptor Cross-Talk/efeitos dos fármacos , Receptor Cross-Talk/fisiologia , Retina/citologiaRESUMO
Adenosine triphosphate (ATP) has been proposed to play a role as a neurotransmitter in the retina, but not much attention has been given to the regulation of ATP release from retinal neurons. In this work, we investigated the release of ATP from cultures enriched in amacrine-like neurons. Depolarization of the cells with KCl, or activation of alpha-amino-3-hydroxy- 5-methyl-4-isoxazole-propionate (AMPA) receptors, evoked the release of ATP, as determined by the luciferin/luciferase luminescent method. The ATP release was found to be largely Ca(2+) dependent and sensitive to the botulinum neurotoxin A, which indicates that the ATP released by cultured retinal neurons originated from an exocytotic pool. Nitrendipine and omega-Agatoxin IVA, but not by omega-Conotoxin GVIA, partially blocked the release of ATP, indicating that in these cells, the Ca(2+) influx necessary to trigger the release of ATP occurs in part through the L- and the P/Q types of voltage-sensitive Ca(2+) channels (VSCC), but not through N-type VSCC. The release of ATP increased in the presence of adenosine deaminase, or in the presence of 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), an adenosine A(1) receptor antagonist, showing that the release is tonically inhibited by the adenosine A(1) receptors. To our knowledge, this is the first report showing the release of endogenous ATP from a retinal preparation.