Protein synthesis inhibition promotes nitric oxide generation and activation of CGKII-dependent downstream signaling pathways in the retina.

Nitric oxide is an important neuromodulator in the CNS, and its production within neurons is modulated by NMDA receptors and requires a fine-tuned availability of L-arginine. We have previously shown that globally inhibiting protein synthesis mobilizes intracellular L-arginine "pools" in retinal neurons, which concomitantly enhances neuronal nitric oxide synthase-mediated nitric oxide production. Activation of NMDA receptors also induces local inhibition of protein synthesis and L-arginine intracellular accumulation through calcium influx and stimulation of eucariotic elongation factor type 2 kinase. We hypothesized that protein synthesis inhibition might also increase intracellular L-arginine availability to induce nitric oxide-dependent activation of downstream signaling pathways. Here we show that nitric oxide produced by inhibiting protein synthesis (using cycloheximide or anisomycin) is readily coupled to AKT activation in a soluble guanylyl cyclase and cGKII-dependent manner. Knockdown of cGKII prevents cycloheximide or anisomycin-induced AKT activation and its nuclear accumulation. Moreover, in retinas from cGKII knockout mice, cycloheximide was unable to enhance AKT phosphorylation. Indeed, cycloheximide also produces an increase of ERK phosphorylation which is abrogated by a nitric oxide synthase inhibitor. In summary, we show that inhibition of protein synthesis is a previously unanticipated driving force for nitric oxide generation and activation of downstream signaling pathways including AKT and ERK in cultured retinal cells. These results may be important for the regulation of synaptic signaling and neuronal development by NMDA receptors as well as for solving conflicting data observed when using protein synthesis inhibitors for studying neuronal survival during development as well in behavior and memory studies.

Chlorogenic acids inhibit glutamate dehydrogenase and decrease intracellular ATP levels in cultures of chick embryo retina cells.

Chlorogenic acids (CGAs) are a group of phenolic compounds found in worldwide consumed beverages such as coffee and green tea. They are synthesized from an esterification reaction between cinnamic acids, including caffeic (CFA), ferulic and p-coumaric acids with quinic acid (QA), forming several mono- and di-esterified isomers. The most prevalent and studied compounds are 3-O-caffeoylquinic acid (3-CQA), 4-O-caffeoylquinic acid (4-CQA) and 5-O-caffeoylquinic acid (5-CQA), widely described as having antioxidant and cell protection effects. CGAs can also modulate glutamate release from microglia by a mechanism involving a decrease of reactive oxygen species (ROS). Increased energy metabolism is highly associated with enhancement of ROS production and cellular damage. Glutamate can also be used as an energy source by glutamate dehydrogenase (GDH) enzyme, providing α-ketoglutarate to the tricarboxylic acid (TCA) cycle for ATP synthesis. High GDH activity is associated with some disorders, such as schizophrenia and hyperinsulinemia/hyperammonemia syndrome. In line with this, our objective was to investigate the effect of CGAs on GDH activity. We show that CGAs and CFA inhibits GDH activity in dose-dependent manner, reaching complete inhibition at high concentration with IC50 of 52 µM for 3-CQA and 158.2 µM for CFA. Using live imaging confocal microscopy and microplate reader, we observed that 3-CQA and CFA can be transported into neuronal cells by an Na+-dependent mechanism. Moreover, neuronal cells treated with CGAs presented lower intracellular ATP levels. Overall, these data suggest that CGAs have therapeutic potential for treatment of disorders associated with high GDH activity.

Vitamin C modulates glutamate transport and NMDA receptor function in the retina.

Vitamin C (in the reduced form ascorbate or in the oxidized form dehydroascorbate) is implicated in signaling events throughout the central nervous system (CNS). In the retina, a high-affinity transport system for ascorbate has been described and glutamatergic signaling has been reported to control ascorbate release. Here, we investigated the modulatory role played by vitamin C upon glutamate uptake and N-methyl-d-aspartate (NMDA) receptor activation in cultured retinal cells or in intact retinal tissue using biochemical and imaging techniques. We show that both forms of vitamin C, ascorbate or dehydroascorbate, promote an accumulation of extracellular glutamate by a mechanism involving the inhibition of glutamate uptake. This inhibition correlates with the finding that ascorbate promotes a decrease in cell surface levels of the neuronal glutamate transporter excitatory amino acid transporter 3 in retinal neuronal cultures. Interestingly, vitamin C is prone to increase the activity of NMDA receptors but also promotes a decrease in glutamate-stimulated [3 H] MK801 binding and decreases cell membrane content of NMDA receptor glutamate ionotropic receptor subunit 1 (GluN1) subunits. Both compounds were also able to increase cAMP response element-binding protein phosphorylation in neuronal nuclei in a glutamate receptor and calcium/calmodulin kinase-dependent manner. Moreover, the effect of ascorbate is not blocked by sulfinpyrazone and then does not depend on its uptake by retinal cells. Overall, these data indicate a novel molecular and functional target for vitamin C impacting on glutamate signaling in retinal neurons.