Marcelo Cossenza

L-arginine uptake and release by cultured avian retinal cells: differential cellular localization in relation to nitric oxide synthase.

Abstract: The availability of L‐arginine is of pivotal importance for the synthesis of nitric oxide, a signaling molecule in the CNS. Here we show the presence of a high‐affinity L‐arginine uptake system (Km of 4.4 ± 0.5 μM and a Vmax of 26.0 ± 0.9 fmol/well/min) in cultured chick retinal cells. Different compounds, such as NG‐mono‐methyl‐L‐arginine and L‐lysine, were able to inhibit the uptake that was also inhibited 60‐70% in the absence of sodium and/or calcium ions. No trans stimulation was observed when cells were preloaded with L‐lysine. The data indicate that the L‐arginine uptake in cultured retinal cells is partially mediated by the y+ system, but has a great contribution of the B0,+ system. Autoradiographic studies revealed that the uptake is predominant in glial cells and can also be detected in neurons, whereas immunocytochemistry of nitric oxide synthase and L‐citrulline showed that the enzyme is present in neurons and photoreceptors, but not in glial cells. L‐[3H]Arginine is released from purified glial cultures incubated with high concentrations of potassium in the extracellular medium. Moreover, the amino acid released from preloaded glial cells was taken up by purified neuronal cultures. These results indicate that L‐arginine released from glial cells is taken up by neurons and used as substrate for the synthesis of nitric oxide.

Inhibition of protein synthesis by activation of NMDA receptors in cultured retinal cells: a new mechanism for the regulation of nitric oxide production

The synthesis of nitric oxide (NO) is limited by the intracellular availability of l‐arginine. Here we show that stimulation of NMDA receptors promotes an increase of intracellular l‐arginine which supports an increase in the production of NO. Although l‐[3H]arginine uptake measured in cultured chick retina cells incubated in the presence of cycloheximide (CHX, a protein synthesis inhibitor) was inhibited approximately 75% at equilibrium, quantitative thin‐layer chromatography analysis showed that free intracellular l‐[3H]arginine was six times higher in CHX‐treated than in control cultures. Extracellular l‐[3H]citrulline levels increased threefold in CHX‐treated groups, an effect blocked by NG‐nitro‐l‐arginine, a NO synthase (NOS) inhibitor. NMDA promoted a 40% increase of free intracellular l‐[3H]arginine in control cultures, an effect blocked by the NMDA antagonist 2‐amino 5‐phosphonovaleric acid. In parallel, NMDA promoted a reduction of 40–50% in the incorporation of 35[S]methionine or l‐[3H]arginine into proteins. Western blot analysis revealed that NMDA stimulates the phosphorylation of eukaryotic elongation factor 2 (eEF2, a factor involved in protein translation), an effect inhibited by (+)‐5‐methyl‐10,11‐dihydro‐5H‐dibenzo[a,d]cyclohepten‐5,10‐imine maleate (MK801). In conclusion, we have shown that the stimulation of NMDA receptors promotes an inhibition of protein synthesis and a consequent increase of an intracellular l‐arginine pool available for the synthesis of NO. This effect seems to be mediated by activation of eEF2 kinase, a calcium/calmodulin‐dependent enzyme which specifically phosphorylates and blocks eEF2. The results raise the possibility that NMDA receptor activation stimulates two different calmodulin‐dependent enzymes (eEF2 kinase and NOS) reinforcing local NO production by increasing precursor availability together with NOS catalytic activity.

c-Src deactivation by the polyphenol 3-O-caffeoylquinic acid abrogates reactive oxygen species-mediated glutamate release from microglia and neuronal excitotoxicity

3-O-caffeoylquinic acid (3-CQA) is an isomer of chlorogenic acid, which has been shown to regulate lipopolysaccharide-induced tumor necrosis factor production in microglia. Whereas overactivation of microglia is associated with neuronal loss in brain diseases via reactive oxygen species (ROS) production and glutamate excitotoxicity, naïve (nonactivated) microglia are believed to generate little ROS under basal conditions, contributing to the modulation of synaptic activity and nerve tissue repair. However, the signaling pathways controlling basal ROS homeostasis in microglial cells are still poorly understood. Here we used time-lapse microscopy coupled with highly sensitive FRET biosensors (for detecting c-Src activation, ROS generation, and glutamate release) and lentivirus-mediated shRNA delivery to study the pathways involved in antioxidant-regulated ROS generation and how this associates with microglia-induced neuronal cell death. We report that 3-CQA abrogates the acquisition of an amoeboid morphology in microglia triggered by Aβ oligomers or the HIV Tat peptide. Moreover, 3-CQA deactivates c-Src tyrosine kinase and abrogates c-Src activation during proinflammatory microglia stimulation, which shuts off ROS production in these cells. Moreover, forced increment of c-Src catalytic activity by overexpressing an inducible c-Src heteromerization construct in microglia increases ROS production, abrogating the 3-CQA effects. Whereas oxidant (hydrogen peroxide) stimulation dramatically enhances glutamate release from microglia, such release is diminished by the 3-CQA inhibition of c-Src/ROS generation, significantly alleviating cell death in cultures from embryonic neurons. Overall, we provide further mechanistic insight into the modulation of ROS production in cortical microglia, indicating antioxidant-regulated c-Src function as a pathway for controlling microglia-triggered oxidative damage.

Dopamine promotes NMDA receptor hypofunction in the retina through D1 receptor-mediated Csk activation, Src inhibition and decrease of GluN2B phosphorylation.

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.

Determination of ascorbic acid in the retina during chicken embryo development using high performance liquid chromatography and UV detection

The retina is a specialized tissue of the central nervous system (CNS) and it is the only part of the CNS that can be visualized non-invasively. During vertebrate development, the retina originates together with the optic nerve as outgrowths of the developing brain, and in this respect, the avian retina is a very convenient model for neurochemical studies of the CNS. In this study, a HPLC-UV method was developed and validated for the determination of ascorbic acid (AA) in the chicken embryo retina. AA has an important role in the retina because of its antioxidant properties. The developed method showed very good figures of merit (recovery = 91 ± 2%; repeatability and intermediate precision better than 1.67% and 2.53% and a limit of quantification of 0.03 mg L−1). Retinas of two embryo ages (12 days and 18 days) showed AA concentrations of 0.0107 ± 0.0010 and 0.0055 ± 0.0005 μg of AA per μg of protein, respectively, and the statistical comparison of results confirmed the decrease of the AA level. These results seem to correlate well with oxidative stress protection, but this fact is still under investigation. As far as we are aware, this is the first study that demonstrates the HPLC-UV determination of ascorbic acid in the chicken embryo retina and its variation along embryo development.

Combination Therapy with Sulfasalazine and Valproic Acid Promotes Human Glioblastoma Cell Death Through Imbalance of the Intracellular Oxidative Response

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.

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

Graphical abstract Figure. No caption available. &NA; 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 &agr;‐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 &mgr;M for 3‐CQA and 158.2 &mgr;M 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.

Ascorbate Transport in Retinal Cells and Its Relationship with the Nitric Oxide System

Abstract Retinal tissue derives from the anterior neural tube during early embryogenesis and thus belongs to the central nervous system. Classical neurotransmitter and second messenger systems are all expressed early during retinal tissue development and may be implicated in retinal maturation and differentiation. Vitamin C and nitric oxide are bioactive cellular messengers that regulate retinal functions. Vitamin C is concentrated in the retina by a mechanism employing glucose transporters, expressed in the blood–retinal barrier. Within the retina, vitamin C may cycle between retinal neurons in either its reduced (ascorbate) or oxidized (dehydroascorbate) form. Neuronal nitric oxide synthase is the major nitric oxide-producing enzyme in the retina. Recent evidence strongly suggests that endogenous nitric oxide actively participates in ascorbate transport by modulating the expression of its high-affinity transporter, the sodium-dependent vitamin C transporter (SVCT-2). In this chapter recent topics relating acutely produced nitric oxide with ascorbate transport in retinal neurons are discussed.