Ativação da nNOS pela via PKA/PP1: implicações na captação de LDL oxidada via Toll Like Receptor 4

Abstract

nNOS has been described as atheroprotective and its activity is compromised in hyperlipidemia. However, there is no known relationship of receptors recognizing modified LDL in macrophages and nNOS activity. in this work we evaluated the role of nNOS-derived NO in the uptake of oxLDL and its relation to the activation of TLR4 receptors. Therefore, peritoneal macrophages of C57BL/6J and TLR4−/− mice were stimulated for 1h with FITC-conjugated oxLDL in the presence or absence of nNOS inhibitor, amitriptyline (AM, TLR2/4 receptor antagonist), SNAP (NO donor) or H2O2. FITC-oxLDL was visualized by fluorescence microscopy and cytometry. production of NO and superoxide and calcium influx were evaluated by DAF-FM, DHE, Fluo-4 and Fluo-5N probes. Activation of endothelial nNOS was evaluated in acetylcholine-stimulated endothelial cells and aortic rings. it was observed that inhibition of nNOS increased the uptake of oxLDL in macrophages and that NO donor decreased it. these effects were not observed when polymerization of actin was inhibited. In addition, increased uptake by nNOS inhibition was abolished in TLR4−/− animals and AM-treated cells. this treatment, as well as inhibition of nNOS, decreased NO production. The phosphorylation levels of nNOS (Ser852-inactivation) decreased throughout oxLDL exposure and increased in TLR4−/− cells and AM-treated cells. On the other hand, oxLDL stimulation led to the release of calcium from the reticulum and calcium influx dependent on TLR4, PLC and PKA. inhibition of PKA increased LDLox uptake and decreased NO production. In addition, it increased the phosphorylation of nNOS-Ser852 and prevented the translocation of PP1 into the cytoplasm. in endothelial cells and aortic rings dephosphorylation of the ser 852 residue by Ach was observed. silenced aorta rings for eNOS showed inhibition of the acetylcholine response when PP1 was inhibited. In addition, PP1 deattachment of the endogenous NIPP1 inhibitor and subsequent cytoplasmic translocation were evidenced. cAMP analogue stimulation mimicked Ach activation, and inhibition of PKA prevented the dephosphorylation of nNOS-Ser852 and prevented PP1 translocation. Consequently, we conclude that the activation of nNOS occurs by a mechanism dependent on PKA and PP1. in macrophages, this pathway is induced by TLR4 stimulation in response to oxLDL resulting in NO release that possibly regulates macropinocytosis, preventing further oxLDL uptake.