Regulation of endothelial nitric oxide synthase activation in endothelial cells by S1P1 and S1P3

Autor: Mirjam Schuchardt, Markus Tölle, Annette Wiedon, M. van der Giet, L Klöckl, Walter Zidek
Rok vydání: 2016
Předmět:
0301 basic medicine
Agonist
medicine.medical_specialty
Nitric Oxide Synthase Type III
medicine.drug_class
Biophysics
Organophosphonates
Thiophenes
Nitric Oxide
Biochemistry
Nitric oxide
03 medical and health sciences
chemistry.chemical_compound
Phosphoserine
Enos
Sphingosine
Internal medicine
medicine
Human Umbilical Vein Endothelial Cells
Humans
Anilides
Sphingosine-1-phosphate
Phosphorylation
RNA
Small Interfering
Receptor
Molecular Biology
Sphingosine-1-Phosphate Receptors
biology
Chemistry
Endothelial Cells
Cell Biology
biology.organism_classification
Organophosphates
Cell biology
Enzyme Activation
Receptors
Lysosphingolipid
030104 developmental biology
Endocrinology
Gene Knockdown Techniques
beta-Alanine
Thiazolidines
Signal transduction
Proto-Oncogene Proteins c-akt
Signal Transduction
Zdroj: Biochemical and biophysical research communications. 476(4)
ISSN: 1090-2104
Popis: Endothelial nitric oxide synthase (eNOS) plays a crucial role in vascular homeostasis. Lysophospholipid interaction with sphingosine 1-phosphat (S1P) receptors results in eNOS activation in different cells. In endothelial cells, eNOS activation via S1P1 or S1P3 was shown controversially. The aim of this study is to investigate the meaning of both S1P receptors for eNOS activation in human endothelial cells. Therefore, several S1P1 and S1P3 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™ multiplex, and Western Blot were used. In HUVEC, both the S1P1 agonist AUY954 as well as the S1P1,3 agonist FTY720P induced eNOS activation in a time- and dose-dependent manner. Other S1P1 agonists activated eNOS to a lesser extent. The AUY954-induced eNOS activation was blocked by the S1P1 antagonist W146, the combination of W146 and the S1P3 antagonist CAY10444 and the S1P1,3 antagonist VPC23019, but not by CAY10444 indicating the meaning of S1P1 for the AUY954-induced eNOS activation. The FTY720P-induced eNOS activation was blocked only by the combination of W146 and CAY10444 and the combined S1P1,3 antagonist VPC23019, but not by W146 or CAY10444 indicating the importance of both S1P1 and S1P3 for FTY720-induced eNOS activation. These results were confirmed using specific siRNA against S1P1 and S1P3. The S1P1,3 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 S1P receptor activation these data show potent selective S1P1 activation by using AUY954 and with selective S1P receptor inhibition evidence was provided that both S1P1 and S1P3 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.
Databáze: OpenAIRE
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