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.

Introduction

Ascorbate is a six carbon-composed sugar that displays many functions in neuronal cells, such as maturation of the glutamatergic system in cultured hippocampal cells (1) and modulation of NMDA receptor function (2). Vitamin C is a widespread term that defines its oxidized (dehydroascorbate) and its reduced forms (ascorbate). Ascorbate is transported by a high-affinity protein, named sodium vitamin C transporter (SVCT)3 (3–5), which transports ascorbate in a sodium-dependent manner, using two Na+ ions for each ascorbate molecule transported (3, 5). There are two human cloned isoforms of SVCT (SVCT-1 and -2), which are encoded by the genes SLC23A1 and SLC23A2, respectively (6, 7). The two isoforms display a high percentage homology in their primary transcripts and serve different functions among distinct tissues (6). Consensus motifs for phosphorylation by PKA and PKC have also been described within the SVCT (3–5). In the nervous system, ascorbate contributes to the formation of the myelin shaft (8, 9) and negatively modulates Na+/K+-ATPase activity (10, 11). Ascorbate has also been reported to be present in chicken embryo eyes since early stages of development (12). Previous studies have demonstrated the presence of SVCT-2 transcripts in the inner nuclear layer of the rat retina (3). Moreover, presence of the SVCT-2 protein has been described in the inner nuclear and ganglion cell layers of the chicken retina (13). In cultured chicken retinal cells it has been demonstrated that ascorbate release is modulated by ionotropic glutamate receptors and proposed that ion flux through AMPA/kainate receptors promotes SVCT-2 reversal (13).

The gaseous signaling molecule NO is produced from the amino acid l-arginine in a reaction catalyzed by nitric-oxide synthase (NOS) enzymes (14). The reaction also requires molecular oxygen with the concomitant production of NO and l-citrulline (15). NO can mediate its effects by two mechanisms: first, the classical activation of soluble guanylyl cyclase (sGC, also defined as a NO-receptor) with production of cGMP and activation of PKG, known as the sGC/cGMP/PKG pathway (16–19); second, NO can covalently attach to cysteine residues within the amino acid sequence of a specific sets of proteins, a mechanism known as S-nitrosylation (19, 20). Hitherto, the involvement of NO and its related downstream signaling pathways in several developmental aspects of the CNS has been demonstrated, for instance, neuronal proliferation, survival, and differentiation (21–25). Previous studies have demonstrated the anti-proliferative role of NO in the developing chick retina (25) and its neuroprotective role upon cultured retinal cells in relationship to culture-refeeding stress (21). Moreover, a high-affinity transport system for l-arginine as well as the neuronal NOS have been shown to be expressed in cultured retinal neurons (26). NO has also been shown to modulate the phosphorylation of ERK MAP kinase and cAMP responsive element-binding protein (CREB) transcription factor in cultured retinal cells (27).

The NF-κB is a dimeric transcription factor involved in cell survival and differentiation (28). It modulates a large number of genes in response to infection, inflammation, and other stressful situations that require rapid reprogramming of gene expression (29). When NF-κB is attached to IκB (NF-κB inhibitor), it remains in its inactive state at the cytoplasm (28). The IκB kinase (IKK) is a regulatory complex composed of two catalytic subunits (IKKα/1 and IKKβ/2) and a structural subunit known as NEMO/IKKγ (30). When active, this complex phosphorylates IκB, which is degraded by the ubiquitin system (29, 31, 32). As a result, free NF-κB can be translocated to the nucleus and modulates gene transcription. NF-κB is also a redox-sensitive transcription factor and has already been shown to modulate SVCT2 mRNA expression in response to redox-state unsteadiness in C2C12 myotubes (7). Furthermore, it has already been suggested that LPS-modulated iNOS activity may regulate NF-κB activation in non-neuronal cells (33). Moreover, the control of NO production in inflammatory processes, such as LPS-induced macrophage activation, is achieved by transcriptional regulation of the iNOS gene by the NF-κB pathway (34).

As ascorbate and NO seem to play critical roles in neuronal cells, we are interested in studying the effects of NO and its related signaling pathways on the ascorbate uptake in cultured retinal cells. Here we show for the first time that NO strictly modulates SVCT-2 expression and ascorbate uptake in retinal cells in culture, an effect mediated by the sGC/cGMP/PKG signaling pathway and NF-κB system.