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

Abstract

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.

The nitric oxide-cGKII system relays death and survival signals during embryonic retinal development via AKT-induced CREB1 activation

“…Considering the capacity of microenvironmental molecules to modulate/determine retinal neuronal cells development, and taking into account the neuromodulatory role played by NO in our culture system, 11,14,15,23 we tested whether prolonged NO treatment of cultured neurons would modulate the viability of those cells. S-nitroso-N-acetyl-D,L-penicillamine (SNAP; NO donor) effect was only detected when it was added 2 h after seeding E6 and E8 neurons, designed here as E6C0 or E8C0 (Figure 1).…”

“…47 We previously demonstrated that neuronal-derived NO increased phospho-CREB in cultured retinal cells in an ERK-dependent manner. 23 On the other hand, nNOS activity has been associated with both glutamate-induced excitotoxicity in cortical neurons 48 and AMPA-induced apoptosis in the retina. 15 We showed that calcium influx, nNOS activity and NO production were high in E6 when compared with E8 retinas.…”

“…Antibodies. Primary antibodies: mouse anti-CREB (#9197), mouse antiphospho-CREB Ser133 (#9196), rabbit anti-phospho-CREB Ser133 (#9191) were from Cell Signaling (Danvers, MA, USA) and their specificity for chick CREB has been described previously 23,27 ; rabbit anti-cGKII (ab110124) was from Abcam (Cambridge, MA, USA), rabbit anti-phospho-AKT Ser473 (#9271) was from Cell Signaling and the specificity of both antibodies in the chick was previously determined 22 ; rabbit anti-phospho-nNOS Ser1417 (07-544) was from Millipore (Merck/Millipore) and its specificity was previously determined. 15 Antibody labeling in chick retinal cells was re-validated (Supplementary Figure 1).…”

“…15,23,27 E6 or E8 chick embryo retinas were suspended in calcium and magnesium-free HBSS and digested with 0.1% trypsin (w/v) for 17 min at 37 1C. Cells were suspended in MEM (supplemented with 3% FBS (v/v), 100 U/ml penicillin, 100 mg/ml streptomycin and 2 mM glutamine), dissociated using a Pasteur pipette, and seeded onto 24-or 12-well culture plastic dishes in a density of 2 Â 10 5 cells/mm 2 .…”

“…22 In addition, cyclic AMPresponsive element-binding protein (CREB) phosphorylation in retinal cells is dependent on a cGK-mediated extracellular regulated kinase (ERK) activation, which directly correlates glutamate and NO pathway to nuclear CREB activity in the retina. 23 Therefore, NO-mediated cGK activation also regulates physiological events in developing retinal cells.…”