title: Nitric oxide regulates AKT phosphorylation and nuclear translocation in cultured retinal cells.
abstract: Previous studies have shown that nitric oxide (NO) inhibits apoptosis of retinal neurons in culture through the canonical cyclic GMP/protein kinase G (PKG)-dependent pathway, but also involving multiple kinase pathways, such as phosphatidylinositol 3' kinase (PI3k) and AKT. NO and AKT exhibit survival-promoting properties and display important roles in both CNS development and plasticity. The purpose of this study was to evaluate the effects of exogenous NO, derived from the NO donor S-nitroso-N-acetylpenicillamin (SNAP), or endogenous NO, produced from l-arginine, on AKT phosphorylation in cultured chick retinal neurons. Our results demonstrate that SNAP or l-arginine enhances AKT phosphorylation on both serine-473 and threonine-308 residues in a concentration and time-dependent manner. This effect was mediated by the activation of soluble guanylyl cyclase and PKG, since it was blocked by the respective enzyme inhibitors ODQ or LY83583 and KT5823, as well as by transduction with shRNA lentiviruses coding PKGII shRNA, and mimicked by the respective enzyme activators YC-1 and 8-Bromo cyclic GMP, and also by the cyclic GMP phosphodiesterase inhibitor zaprinast. In addition, LY294002 or wortmannin suppressed the SNAP effect, indicating the involvement of phosphoinositide 3' kinase. Moreover, the mTOR inhibitor KU0063794 blocked SNAP-induced AKT phosphorylation at both residues, suggesting the participation of the mTORC2 complex in the process. Glutamate and NMDA also promoted AKT phosphorylation and a nitric oxide synthase inhibitor abrogated these effects, revealing a mechanism involving the activation of NMDA receptors and NO production. We have also found that SNAP and l-arginine induced AKT translocation into the nucleus of retinal neurons as well as other neuronal cell lines. SNAP also protects retinal cells from death induced by hydrogen peroxide and this effect was blocked by the phosphoinositide 3' kinase inhibitor LY294002. We therefore conclude that NO produced from endogenous or exogenous sources promotes AKT activation and its shuttling to the nucleus, probably participating in neuronal survival pathways important during CNS development.
authors: Program of Neurosciences and Department of Neurobiology, Institute of Biology, Fluminense Federal University, Niterói, RJ, Brazil. Electronic address: tmejiagarcia@hotmail.com.
authors: Telmo A
authors: TA
authors: Mejía-García
authors: Camila C
authors: CC
authors: Portugal
authors: Thaísa G
authors: TG
authors: Encarnação
authors: Marco Antônio M
authors: MA
authors: Prado
authors: Roberto
authors: R
authors: Paes-de-Carvalho
mesh_terms: D021581:Active Transport, Cell Nucleus / Q000187:drug effects; D000818:Animals; D001120:Arginine / Q000378:metabolism; D002478:Cells, Cultured; D002645:Chickens; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism*; D020030:Nitric Oxide Donors / Q000494:pharmacology*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D016194:Receptors, N-Methyl-D-Aspartate / Q000378:metabolism; D055351:Retinal Neurons / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D026423:S-Nitroso-N-Acetylpenicillamine / Q000494:pharmacology*; D015398:Signal Transduction / Q000187:drug effects; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism
chemical_list: D001120:Arginine; D017869:Cyclic GMP-Dependent Protein Kinases; D009569:Nitric Oxide; D020030:Nitric Oxide Donors; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; D016194:Receptors, N-Methyl-D-Aspartate; D026423:S-Nitroso-N-Acetylpenicillamine; D058570:TOR Serine-Threonine Kinases
keywords: (5R,2S)-(1)5methyl-10,11-dihydro-5H-dibenzo [a,d] cyclohepten-5,10-imine hydrogen maleate; 1H-[1,2,4]oxadiazolo[4,3-aquinoxalin-1-one]; 3-(5′hydroxymethyl-2′-furyl)-1-1benzyl indazole; 5-(2-Propoxyphenyl)-1H-[1,2,3]triazolo[4,5-d]pyrimidin-7(4H)-one; AKT; AMPA; CMF; CREB; Chick retina; Cyclic GMP; EDTA; ERK; KT5823; KU0063794; L-Arg; L-NAME; MAPK; MEM; MK-801; N-acetylcysteine; N-methyl-d-aspartate; N-nitro-l-arginine methyl ester; NAC; NMDA; NMDA receptor; NO; NOS; ODQ; PD; PD98058; PI3K; PKG; Protein kinase G; S-nitroso-N-acetylpenicillamin; SDS-PAGE; SNAP; Wor; YC-1; [1-oxo-9.12-epoxy-1H-diidolo[1,2,3-fg:3′,2′,1′kl]pyrrolo[3,4][1,6]benzodiazocine-10carboxylic acid methyl ester; a-amino-3-hydroxy-5-methylisoxazole-4-propionate; cGMP; calcium and magnesium-free balanced salt solution; cyclic guanosine monophosphate; cyclic nucleotide responsive element binding protein; ethylene diamine tetraacetic acid; extracellular signal-regulated kinase; l-Arginine; l-arginine; mTOR; minimum essential medium; mitogen-associated protein kinase; nitric oxide; nitric-oxide synthase; phosphatidylinositol 3′ kinase; protein kinase B; protein kinase G; rel-5-[2-[(2R,6S)-2,6-dimethyl-4-morpholinyl]-4-(4-morpholinyl)pyrido[2,3-d]pyrimidin-7-yl]-2-methoxybenzenemethanol; sGC; sodium-dodecyl sulfate polyacrylamide gel electrophoresis; soluble guanylate cyclase; wortmannin, (1S,6bR,9aS,11R,11bR) 11-(Acetyloxy)-1,6b,7,8,9a,10,11,11b-octahydro-1-(methoxymethyl)-9a,11b-dimethyl-3H-furo[4,3,2-de]indeno[4,5,-h]-2-h]-2-benzopyran-3,6,9-trione; zaprinast