RESUMO
Ascorbate, the reduced form of vitamin C, is highly concentrated in the central nervous system (CNS), including the retina, where it plays important physiological functions. In the CNS, the plasma membrane transporter sodium vitamin C co-transporter 2 (SVCT2) is responsible for ascorbate transport in neurons. The neurotransmitter dopamine (DA), acting through D1- and D2-like receptor subfamilies and classically coupled to adenylyl cyclase, is known to modulate synaptic transmission in the retina. Here, we reveal that DA controls the release of ascorbate from retinal neurons. Using primary retinal cultures, we show that this DA effect is dose-dependent, occurring by the reversal of the SVCT2, and could be elicited by brief and repetitive pulses of DA. The DA effect in inducing ascorbate release occurs by the activation of D1R and is independent of PKA. Moreover, the exchange protein directly activated by cAMP type 2 (EPAC2) is present in retinal neurons and its specific knockdown using shRNAs abrogates the D1R-induced ascorbate release. Confirming the physiological relevance of this pathway, activation of D1R or EPAC2 also triggered ascorbate release ex vivo in acute preparations of the intact retina. Overall, DA plays pivotal roles in regulating ascorbate homeostasis through an unanticipated signaling pathway involving D1R/adenylyl cyclase/cAMP/EPAC2, thereby suggesting that vitamin C might fine-tune dopaminergic neurotransmission in the retina.
Assuntos
Ácido Ascórbico/metabolismo , Dopamina/farmacologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Receptores de Dopamina D1/metabolismo , Neurônios Retinianos/metabolismo , Adenilil Ciclases/metabolismo , Animais , Células Cultivadas , Embrião de Galinha , Neurônios Retinianos/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
Glioblastoma (GBM) is the most common and aggressive malignant primary brain tumor and still lacks effective therapeutic strategies. It has already been shown that old drugs like sulfasalazine (SAS) and valproic acid (VPA) present antitumoral activities in glioma cell lines. SAS has also been associated with a decrease of intracellular glutathione (GSH) levels through a potent inhibition of xc- glutamate/cystine exchanger leading to an antioxidant deprotection. In the same way, VPA was recently identified as a histone deacetylase (HDAT) inhibitor capable of activating tumor suppression genes. As both drugs are widely used in clinical practice and their profile of adverse effects is well known, the aim of our study was to investigate the effects of the combined treatment with SAS and VPA in GBM cell lines. We observed that both drugs were able to reduce cell viability in a dose-dependent manner and the combined treatment potentiated these effects. Combined treatment also increased cell death and inhibited proliferation of GBM cells, while having no effect on human and rat cultured astrocytes. Also, we observed high protein expression of the catalytic subunit of xc- in all the examined GBM cell lines, and treatment with SAS blocked its activity and decreased intracellular GSH levels. Noteworthy, SAS but not VPA was also able to reduce the [14C]-ascorbate uptake. Together, these data indicate that SAS and VPA exhibit a substantial effect on GBM cell's death related to an intracellular oxidative response imbalance, making this combination of drugs a promising therapeutic strategy.
Assuntos
Neoplasias Encefálicas/patologia , Glioblastoma/patologia , Espaço Intracelular/metabolismo , Sulfassalazina/farmacologia , Ácido Valproico/farmacologia , Sistema y+ de Transporte de Aminoácidos/metabolismo , Animais , Ácido Ascórbico/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Quimioterapia Combinada , Glutationa/metabolismo , Humanos , Mesoderma/efeitos dos fármacos , Mesoderma/patologia , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , Neuroglia/patologia , Oxirredução , Ratos , Fatores de TempoRESUMO
Ascorbate is an important antioxidant, which also displays important functions in neuronal tissues, including the retina. The retina is responsible for the initial steps of visual processing, which is further refined in cerebral high-order centers. The retina is also a prototypical model for studying physiologic aspects of cells that comprise the nervous system. Of major importance also is the cellular messenger nitric oxide (NO). Previous studies have demonstrated the significance of NO for both survival and proliferation of cultured embryonic retinal cells. Cultured retinal cells express a high-affinity ascorbate transporter, and the release of ascorbate is delicately regulated by ionotropic glutamate receptors. Therefore, we proposed whether there is interplay between the ascorbate transport system and NO signaling pathway in retinal cells. Here we show compelling evidence that ascorbate uptake is tightly controlled by NO and its downstream signaling pathway in culture. NO also modulates the expression of SVCT-2, an effect mediated by cGMP and PKG. Kinetic studies suggest that NO increases the transport capacity for ascorbate, but not the affinity of SVCT-2 for its substrate. Interestingly, NO utilizes the NF-κB pathway, in a PKG-dependent manner, to modulate both SVCT-2 expression and ascorbate uptake. These results demonstrate that NO exerts a fine-tuned control of the availability of ascorbate to cultured retinal cells and strongly reinforces ascorbate as an important bioactive molecule in neuronal tissues.
Assuntos
Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Regulação da Expressão Gênica/fisiologia , NF-kappa B/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Óxido Nítrico/metabolismo , Retina/metabolismo , Transportadores de Sódio Acoplados à Vitamina C/metabolismo , Animais , Ácido Ascórbico/genética , Ácido Ascórbico/metabolismo , Transporte Biológico Ativo/fisiologia , Proliferação de Células , Embrião de Galinha , Galinhas , Proteínas Quinases Dependentes de GMP Cíclico/genética , NF-kappa B/genética , Proteínas do Tecido Nervoso/genética , Óxido Nítrico/genética , Retina/citologia , Retina/embriologia , Transdução de Sinais/fisiologia , Transportadores de Sódio Acoplados à Vitamina C/genéticaRESUMO
Vitamin C is transported in the brain by sodium vitamin C co-transporter 2 (SVCT-2) for ascorbate and glucose transporters for dehydroascorbate. Here we have studied the expression of SVCT-2 and the uptake and release of [(14)C] ascorbate in chick retinal cells. SVCT-2 immunoreactivity was detected in rat and chick retina, specially in amacrine cells and in cells in the ganglion cell layer. Accordingly, SVCT-2 was expressed in cultured retinal neurons, but not in glial cells. [(14)C] ascorbate uptake was saturable and inhibited by sulfinpyrazone or sodium-free medium, but not by treatments that inhibit dehydroascorbate transport. Glutamate-stimulated vitamin C release was not inhibited by the glutamate transport inhibitor l-beta-threo-benzylaspartate, indicating that vitamin C release was not mediated by glutamate uptake. Also, ascorbate had no effect on [(3)H] D-aspartate release, ruling out a glutamate/ascorbate exchange mechanism. 2-Carboxy-3-carboxymethyl-4-isopropenylpyrrolidine (Kainate) or NMDA stimulated the release, effects blocked by their respective antagonists 6,7-initroquinoxaline-2,3-dione (DNQX) or (5R,2S)-(1)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801). However, DNQX, but not MK-801 or 2-amino-5-phosphonopentanoic acid (APV), blocked the stimulation by glutamate. Interestingly, DNQX prevented the stimulation by NMDA, suggesting that the effect of NMDA was mediated by glutamate release and stimulation of non-NMDA receptors. The effect of glutamate was neither dependent on external calcium nor inhibited by 1,2-bis (2-aminophenoxy) ethane-N',N',N',N',-tetraacetic acid tetrakis (acetoxy-methyl ester) (BAPTA-AM), an internal calcium chelator, but was inhibited by sulfinpyrazone or by the absence of sodium. In conclusion, retinal cells take up and release vitamin C, probably through SVCT-2, and the release can be stimulated by NMDA or non-NMDA glutamate receptors.