Background:Ascorbate is an important antioxidant that is carried into and out of cells by its high-affinity transporter sodium vitamin C transporter-2 (SVCT-2). Results: Nitric oxide increases SVCT-2 protein levels and ascorbate uptake. Conclusion: Nitric oxide exerts a fine-tuned control of ascorbate availability to retinal cells. Significance: Neuroprotective role of nitric oxide may be achieved by regulating SVCT-2 expression.

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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 D- and D-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 DR 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 DR-induced ascorbate release. Confirming the physiological relevance of this pathway, activation of DR 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 DR/adenylyl cyclase/cAMP/EPAC2, thereby suggesting that vitamin C might fine-tune dopaminergic neurotransmission in the retina.

Dopamine and glutamate are critical neurotransmitters involved in light-induced synaptic activity in the retina. In brain neurons, dopamine D1 receptors (D1Rs) and the cytosolic protein tyrosine kinase Src can, independently, modulate the behavior of NMDA-type glutamate receptors (NMDARs). Here we studied the interplay between D1Rs, Src and NMDARs in retinal neurons. We reveal that dopamine-mediated D1R stimulation provoked NMDAR hypofunction in retinal neurons by attenuating NMDA-gated currents, by preventing NMDA-elicited calcium mobilization and by decreasing the phosphorylation of NMDAR subunit GluN2B. This dopamine effect was dependent on upregulation of the canonical D1R/adenylyl cyclase/cAMP/PKA pathway, of PKA-induced activation of C-terminal Src kinase (Csk) and of Src inhibition. Accordingly, knocking down Csk or overexpressing a Csk phosphoresistant Src mutant abrogated the dopamine-induced NMDAR hypofunction. Overall, the interplay between dopamine and NMDAR hypofunction, through the D1R/Csk/Src/GluN2B pathway, might impact on light-regulated synaptic activity in retinal neurons.

Ascorbate, the reduced form of Vitamin C, is one of the most abundant and important low-molecular weight antioxidants in living tissues. Most animals synthesize vitamin C, but some primates, including humans, have lost this capacity due to disruption in L-gulono-gamma-lactone oxidase gene. Because of this incapacity, those animals must obtain Vitamin C from the diet. Ascorbate is highly concentrated in the central nervous system (CNS), including the retina, and plays essential roles in neuronal physiology. Ascorbate transport into cells is controlled by Sodium Vitamin C Co-Transporters (SVCTs). There are four SVCT isoforms and SVCT2 is the major isoform controlling ascorbate transport in the CNS. Regarding ascorbate release from retinal neurons, Glutamate, by activating its ionotropic receptors leads to ascorbate release via the reversion of SVCT2. Moreover, dopamine, via activation of D 1 receptor/cyclic AMP/EPAC2 pathway, also induces ascorbate release via SVCT2 reversion. Because the dopaminergic and glutamatergic systems are interconnected in the CNS, we hypothesized that dopamine could regulate ascorbate release indirectly, via the glutamatergic system. Here we reveal that dopamine increases the release of D-Aspartate from retinal neurons in a way independent on calcium ions and dependent on excitatory amino acid transporters. In addition, dopamine-dependent SVCT2 reversion leading to ascorbate release occurs by activation of AMPA/Kainate receptors and downstream ERK/AKT pathways. Overall, our data reveal a dopamine-to-glutamate signaling that regulates the bioavailability of ascorbate in neuronal cells.

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