MODULAÇÃO DA VIA DA PI3K/AKT EM CULTURA DE CÉLULAS DE RETINA: PAPEL DO ÓXIDO NÍTRICO E VIAS DE SINALIZAÇÃO MEDIADAS POR CÁLCIOCALMODULINA

• Autor(a) principal: García, Telmo Agustín Mejía
• Data de Publicação: 2009
• Tipo de documento: Tese
• Idioma: por
• Título da fonte: Repositório Institucional da Universidade Federal Fluminense (RIUFF)
• Texto Completo: https://app.uff.br/riuff/handle/1/17256
• topic: Óxido Nítrico; AKT/PKB; Retina animal; L-ARG; CHX; ANIS; Translocação nuclear; Cálciocalmodulina; Nitric oxide; AKT / PKB; Animal retina, L-ARG; CHX, ANIS, nuclear translocation; Calciumcalmodulin; CNPQ::CIENCIAS BIOLOGICAS::IMUNOLOGIA

Previous studies have shown that nitric oxide (NO), produced from the NO donor Snitroso- Acetyl-D-L-Penicillamine (SNAP), inhibits apoptosis of purified retinal neurons in cultured through a cGMP/PKG-dependent mechanism also involving multiple kinase pathways including PI3K, MEK, PKA and CaMKs. The PI3K-AKT pathway and NO are important molecules with neuronal survival-promoting properties and which have shown to play an important role in plasticity associated with CNS development. The purpose of this study was to explore whether endogenous NO produced from L-ARG or exogenous NO from NO donor SNAP exerts its vital effects by activating the AKT protein kinase in cultures of chick retinal neurons. Cultures prepared from 8 day-oldchick embryo retinas were treated or not with SNAP or L-ARG and processed for the detection of AKT phosphorylation levels on Ser-473 and Thr-308 sites. Our results demonstrate that either SNAP or L-ARG lead to enhanced AKT phosphorylation in a concentration and time-dependent manner through the activation of soluble guanylyl cyclase. The effect was blocked by the enzyme inhibitors ODQ or LY83583, and mimicked by 8-Bromo cyclic GMP, a PKG activator or YC-1, a guanylyl cyclase activator. In addition, the effect of NO was also suppressed by LY294002, Wortmannin, KN93 or KN62, respectively indicating the involvement of phosphatidylinositol 3 kinase and calcium calmodulin-dependent protein kinase signaling pathways. Interestingly, pre-treatment by EGTA, a calcium chelator, or BAPTA-AM or Tapsigargin, intracellular calcium chelators, or Triflorperazine or W-7, calmodulin inhibitors, also abrogated the effects of SNAP or L-ARG. This revealed that the phosphorylation of AKT/PKB is mediated by calcium influx from extracellular medium and a calmodulin-dependent mechanism. NO from L-ARG or SNAP promoted Ca2+-influx through L-type Ca2+ channels. Furthermore, we found that NMDA receptor activation stimulates AKT/PKB and can be mimicked by cycloheximide or anisomycin (CHX, ANIS), two inhibitors of protein synthesis, and a consequent increase of an intracellular L-ARG pool available for NO synthesis. An additional result shows that activated AKT/PKB was translocated from cytoplasm to nucleus upon stimulation with SNAP or L-ARG. We then conclude that generation of endogenous or exogenous NO participates in the activation of AKT protein kinase in retinal neurons.