THE S1P1 AGONIST AUY954 ACTIVATES ENOS VIA AKT-MEDIATED PATHWAY: 1D.02

Abstract

Objective: In the mammalian cardiovascular system, nitric oxide (NO) plays a pivotal role in the maintenance of vascular homeostasis. The endothelial isoform (eNOS) is a key determinant of blood pressure or other fundamental response in the vascular wall. Sphingosin-1-phosphate (S1P) is a bioactive sphingolipid which regulates diverse physiological processes by serving as ligand to G-protein coupled S1P receptors and recently identified as eNOS activator, whereas the S1P1 receptor play a key role. A newly developed mono-selective S1P1 modulator – AUY954 – should be investigated in this study on its potential to active eNOS. Methods: Human umbicial vein endothelial cells (HUVEC) were used. S1P receptor mRNA was detected via RT-PCR. Phosphorylation of eNOS was detected by Western Blot technique. Akt phosphorylation was measured using Luminex™ technology. The production of NO was measured in DAF2-DA labeled cells. Results: AUY954 [0.1 – 10 μmol/L] led to a significant and dose-dependent increase in NO liberation in HUVEC detected by fluorescence increase in DAF2-DA labelled cells. This effect could be blocked by L-NAME [100 μmol/L], an inhibitor of NO synthases. AUY954 significantly increase Ser1177 phosphorylation time- and dose-dependently. S1P1 and S1P3 receptor are the most prominent S1P receptors in HUVECs and only a weak expression of S1P2 was detectable. So, we next investigated a specific, non-selective inhibitor of S1P1/3 receptor, VPC23019 which significantly block the AUY954-induced NO liberation. Cay10444, a selective S1P3 antagonist doesn't have any effect. This indicates a S1P1 specific effect. Furthermore, AUY954 is able to stimulate Akt phosphorylation in a significant and time-dependent manner. The co-stimulation of AUY954 with a specific Akt inhibitor blocked the NO production in DAF2-DA labeled HUVECs. Conclusions: Here, we showed that the selective S1P1 receptor agonist AUY954 is a very potent activator of endothelial NO synthase via a Akt-mediated process which results in increased NO liberation of human endothelial cells. Therefore, eNOS/NO activation by AUY954 might play a role in cardiovascular response by promoting endothelial cell survival, proliferation and migration.