Roberto Paes-de-Carvalho

Long-term activation of adenosine A2a receptors blocks glutamate excitotoxicity in cultures of avian retinal neurons

Previous work showed the presence of adenosine receptors as well as adenosine uptake and release mechanisms in developing chick retinal neurons in culture. In the present work we show that exogenous glutamate or kainate promotes extensive cell death in these cultures which is blocked when the cultures are previously incubated with adenosine. Addition of glutamate or kainate to purified cultures of retinal neurons and photoreceptors induced massive death of cultured cells which was inhibited in both cases by preincubation with MK801, an NMDA antagonist, or DNQX, an AMPA/kainate antagonist. Cell death was also greatly attenuated by preincubation with adenosine plus EHNA, an adenosine deaminase inhibitor, NBI, an adenosine uptake blocker, the permeable cAMP analogs 8-Br cAMP and Sp cAMP and the A(2a) agonists CGS 21680 and DPMA, but not with the A(1) receptor agonist CHA. Kinetic studies performed determining the intracellular LDH activity showed that maximal death was observed after 8 h and in concentrations of glutamate as low as 50 microM. We also observed a time-dependent protective effect of adenosine beginning after 1 h of preincubation and reaching a maximal effect after 24 h, indicating its association with changes in cellular metabolism induced by long-term exposure of cells to the nucleoside. The results show that adenosine inhibits glutamate toxicity in retinal neurons through a long-term activation of A(2a) receptors and elevation of intracellular cyclic AMP levels.

Nitric oxide regulates cell survival in purified cultures of avian retinal neurons: involvement of multiple transduction pathways.

Nitric oxide (NO) is an important signaling molecule in the CNS, regulating neuronal survival, proliferation and differentiation. Here, we explored the mechanism by which NO, produced from the NO donor S‐nitroso‐acetyl‐d‐l‐penicillamine (SNAP), exerts its neuroprotective effect in purified cultures of chick retinal neurons. Cultures prepared from 8‐day‐old chick embryo retinas and incubated for 24 h (1 day in culture, C1) were treated or not with SNAP, incubated for a further 72 h (up to 4 days in culture, C4), fixed, and the number of cells estimated, or processed for cell death estimation, by measuring the reduction of the metabolic dye 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT). Experimental cultures were run in parallel but were re‐fed with fresh medium in the absence or presence of SNAP at culture day 3 (C3), incubated for a further 24 h up to C4, then fixed or processed for the MTT assay. Previous studies showed that the re‐feeding procedure promotes extensive cell death. SNAP prevented this death in a concentration‐ and time‐dependent manner through the activation of soluble guanylate cyclase; this protection was significantly reversed by the enzyme inhibitors 1H‐[1,2,4]oxadiazolo‐[4,3‐a]quinoxalin‐1‐one (ODQ) or LY83583, and mimicked by 8‐bromo cyclic guanosine 5′‐phosphate (8Br‐cGMP) (GMP) or 3‐(5′‐hydroxymethyl‐2′‐furyl)‐1‐benzyl indazole (YC‐1), guanylate cyclase activators. The effect was blocked by the NO scavenger 2‐phenyl‐4,4,5,5‐tetramethylimidazoline‐1‐oxyl‐3‐oxide (PTIO). The effect of NO was also suppressed by LY294002, Wortmannin, PD98059, KN93 or H89, indicating the involvement, respectively, of phosphatidylinositol‐3 kinase, extracellular‐regulated kinases, calmodulin‐dependent kinases and protein kinase A signaling pathways. NO also induced a significant increase of neurite outgrowth, indicative of neuronal differentiation, and blocked cell death induced by hydrogen peroxide. Cyclosporin A, an inhibitor of the mitochondrial permeability transition pore considered an important mediator of apoptosis and necrosis, as well as boc‐aspartyl (OMe) fluoromethylketone (BAF), a caspase inhibitor, also blocked cell death induced by re‐feeding the cultures. These findings demonstrate that NO inhibits apoptosis of retinal neurons in a cGMP/protein kinase G (PKG)‐dependent way, and strengthens the notion that NO plays an important role during CNS development.

Glutamate receptors modulate sodium-dependent and calcium-independent vitamin C bidirectional transport in cultured avian retinal cells.

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 [14C] 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. [14C] 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‐β‐threo‐benzylaspartate, indicating that vitamin C release was not mediated by glutamate uptake. Also, ascorbate had no effect on [3H] 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.

Adenosine as a signaling molecule in the retina: biochemical and developmental aspects

The nucleoside adenosine plays an important role as a neurotransmitter or neuromodulator in the central nervous system, including the retina. In the present paper we review compelling evidence showing that adenosine is a signaling molecule in the developing retina. In the chick retina, adenosine transporters are present since early stages of development before the appearance of adenosine A1 receptors modulating dopamine-dependent adenylate cyclase activity or A2 receptors that directly activate the enzyme. Experiments using retinal cell cultures revealed that adenosine is taken up by specific cell populations that when stimulated by depolarization or neurotransmitters such as dopamine or glutamate, release the nucleoside through calcium-dependent transporter-mediated mechanisms. The presence of adenosine in the extracellular medium and the long-term activation of adenosine receptors is able to regulate the survival of retinal neurons and blocks glutamate excitoxicity. Thus, adenosine besides working as a neurotransmitter or neuromodulator in the mature retina, is considered as an important signaling molecule during retinal development having important functions such as regulation of neuronal survival and differentiation.

Nitric Oxide Modulates Sodium Vitamin C Transporter 2 (SVCT-2) Protein Expression via Protein Kinase G (PKG) and Nuclear Factor-κB (NF-κB)

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. 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.

Nitric oxide regulates the proliferation of chick embryo retina cells by a cyclic GMP-independent mechanism

Nitric oxide (NO) is an intercellular messenger involved in many physiological and pathological processes of vertebrate and invertebrate animal tissues. In the embryonic chick retina, nitric oxide synthase (NOS) activity and a system for l‐arginine transport between neurons and glial cells were described, supporting the idea that nitric oxide is a critical molecule during retinal development. In the present work we show that nitric oxide is a modulator of cell proliferation in chick embryo retina. Mixed cultures of retinal neurons and glial cells were submitted to [3H]‐thymidine incorporation after drug treatment. Incubation for 24 h with the NO donors S‐nitroso‐N‐acetyl‐penicillamine (SNAP) or Spermine nitric oxide (SpNO) complex promoted a decrease of approximately 70% of [3H]‐thymidine incorporation in a dose‐dependent manner. SNAP did not increase Lactate dehydrogenase release and its effect was not mimicked by 8‐bromo cyclic GMP, or blocked by the guanylate cyclase inhibitor 1H‐[1,2,4]oxadiazole[4,3‐a]quinoxalin‐1‐one (ODQ), indicating that the effect was not due to cell death or mediated by increases of cyclic GMP levels. The inhibition was completely prevented by dithiotreitol (DTT), strongly indicating the participation of an S‐nitrosylation mechanism. SNAP blocked the increase of [3H]‐thymidine incorporation induced by ATP. Using purified cultures of glial cells we showed that the NO donor SNAP produced an inhibition of 50% in cell proliferation and did stimulate ERK1/2 phosphorylation, indicating that the inhibition of this pathway was not involved in its cytostatic effect. [3H]‐Thymidine autoradiography of mixed cultures showed labeling of oval nuclei of glial flat cells. The injection of eggs with SNAP also did promote an intense inhibition of [3H]‐thymidine incorporation in retinas from 9‐day‐old embryos. These data suggest that nitric oxide affects the proliferation of chick embryo retina glial cells in culture or “in vivo” through cyclic GMP and ERK‐independent pathways.

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.

Developmental regulation of group iii metabotropic glutamate receptors modulating adenylate cyclase activity in the avian retina

The regulation of adenylate cyclase by neurotransmitters is observed in early development of the chick retina. In the present work we show that L-2-amine-4-phosphonobutyric acid (L-AP4), the major agonist of group III metabotropic glutamate receptors (mGluRs), inhibits the accumulation of cyclic AMP induced by forskolin in the chick retina. This effect is observed after 8 days of development (E8), is maximal from E12-E17 and decreases at the post-hatching period (PH). The inhibition is also observed in cultures of retinal cells incubated for 2-8 days. We have also investigated the interaction between group III mGluRs and other receptors coupled to adenylate cyclase in the developing retina. The inhibition by L-AP4 is partially additive with that induced by the A1 adenosine agonist Cyclohexyladenosine and is not observed when cyclic AMP levels are increased with 2-chloroadenosine or dopamine. The group II mGluR agonist trans-(1S,3R)-1-amino-cyclopentanedicarboxylic acid has an inhibitory effect only on PH retinas, indicating that group II and group III mGluRs have a differential ontogenesis in this tissue. The results show that Group III mGluRs are expressed early during chick retina development and do not interact with other receptors known to be coupled to adenylate cyclase in the developing retina.

IL-6 treatment increases the survival of retinal ganglion cells in vitro: the role of adenosine A1 receptor.

IL-6 is a pleiotropic cytokine classically denominated pro-inflammatory. It has been already demonstrated that IL-6 can increase the survival of retinal ganglion cells (RGC) in culture. In this work, we show that the trophic effect of IL-6 is mediated by adenosine receptor (A1R) activation. The neutralization of extracellular BDNF abolished the IL-6 effect and the treatment with IL-6 and CHA (an agonist of A1R) modulated BDNF expression as well as pCREB and pTrkB levels.

Glutamate and nitric oxide modulate ERK and CREB phosphorylation in the avian retina: evidence for direct signaling from neurons to Müller glial cells

Glutamate signaling in the mature retinal tissue is very important for accurate sensory decoding by retinal neurons and orchestrates the fine‐tuned output from the retina to higher‐order centers at the cerebral cortex. In this study, we show that glutamate induces a rapid extracellular‐regulated kinase and cAMP‐responsive element binding protein (CREB) phosphorylation in cultured developing retinal neurons. This process is reliant on α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate receptors and nitric oxide (NO) signaling and independent of NMDA receptors activation, as it is blocked by α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate/kainate antagonists as well as inhibiting NO synthase with NG‐nitro‐l‐arginine methyl ester but not by the NMDA channel blocker dizocilpine maleate. The effect of NO on extracellular‐regulated kinase and CREB is mediated by the classical NO/soluble guanylyl cyclase/protein kinase G pathways as it is inhibited by the soluble guanylyl cyclase blocker 1H‐[1,2,4]oxadiazole[4,3‐a]quinoxalin‐1‐one and the protein kinase G inhibitor KT5823, respectively. Immunocytochemical data suggest that increased CREB phosphorylation in response to glutamate occurs in glial cell nuclei. We also have supporting evidence suggesting that neuronally produced NO directly reaches the glial cells and stimulates CREB phosphorylation. Hence, the results indicate the importance of neuronal–glial communication and glutamate/NO/CREB linkage during retinal development.