Endothelial nitric oxide synthase (eNOS) plays a crucial role in vascular homeostasis. Lysophospholipid interaction with sphingosine 1-phosphat (SIP) receptors results in eNOS activation in different cells. In endothelial cells, eNOS activation via S1P(1) or S1P(3) was shown controversially. The aim of this study is to investigate the meaning of both SIP receptors for eNOS activation in human endothelial cells. Therefore, several S1P(1) and S1P(3) agonists in combination with antagonists and specific RNAi approach were used. eNOS activation was measured in human umbilical vein endothelial cells (HUVEC) via DAF2-DA-based fluorescence microscopy. For investigation of the signaling pathway, agonists/antagonist studies, RNAi approach, Luminex (TM) multiplex, and Western Blot were used. In HUVEC, both the S1P(1) agonist AUY954 as well as the S1P(1,3) agonist FTY720P induced eNOS activation in a time- and dose-dependent manner. Other S1P(1) agonists activated eNOS to a lesser extent. The AUY954-induced eNOS activation was blocked by the S1P(1) antagonist W146, the combination of W146 and the S1P(3) antagonist CAY10444 and the S1P(1,3) antagonist VPC23019, but not by CAY10444 indicating the meaning of S1P(1) for the AUY954-induced eNOS activation. The FTY720P-induced eNOS activation was blociced only by the combination of W146 and CAY10444 and the combined S1P(1,3) antagonist VPC23019, but not by W146 or CAY10444 indicating the importance of both S1P(1) and S1P(3) for FTY720-induced eNOS activation. These results were confirmed using specific siRNA against S1P(1) and S1P(3). The S1P(1,3) activation results in Akt phosphorylation and subsequent activation of eNOS via phosphorylation at serine(1177) and dephosphorylation at threonine(495). Beside former investigations with rather unspecific SIP receptor activation these data show potent selective S1P(1) activation by using AUY954 and with selective SW receptor inhibition evidence was provided that both S1P(1) and S1P(3) lead to downstream activation of eNOS in HUVEC in the same experimental setting. Inhibition or knockdown of one of these receptor subtypes did not abolish the eNOS activation and subsequent NO production. (C) 2016 Elsevier Inc. All rights reserved.