Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 277, Issue 13, Pages 11058-11063Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M110985200
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Funding
- NCRR NIH HHS [P20 RR15592] Funding Source: Medline
- NHLBI NIH HHS [HL64056, HL58475, HL62844] Funding Source: Medline
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Recently it has been demonstrated that high density lipoprotein (HDL) binding to scavenger receptors, class B, type I (SR-BI) stimulates endothelial nitric-oxide synthase (eNOS) activity. In the present studies we used a Chinese hamster ovary cell system and a human microvascular endothelial cell line to confirm that HDL stimulates eNOS activity in a SR-BI-dependent manner. Importantly, we have extended these studies to examine the mechanism whereby HDL binding to SR-BI stimulates eNOS. eNOS can be stimulated by an increase in intracellular calcium, by phosphorylation by Akt kinase, or by an increase in intracellular ceramide. Calcium imagining studies and experiments with the calcium chelator, 1,2-bis(o-aminophenoxy)ethane-NN, N',N'-tetraacetic acid tetra (acetoxymethyl) ester demonstrated that HDL binding to SR-BI does not induce an increase in intracellular calcium. Antibodies specific for activated Akt kinase demonstrated that HDL binding to SR-BI does not induce Akt kinase activation. However, HDL binding to SR-BI caused a reversible increase in intracellular ceramide levels from 97 +/- 14 pmol/mg of protein to 501 +/- 21 pmol/mg of protein. In addition, C-2-ceramide stimulated eNOS to the same extent as HDL, whereas C-2-dihydroceramide did not stimulate eNOS. We conclude that EO:)L binding to SR-BI stimulates eNOS by increasing intracellular ceramide levels and is independent of an increase in intracellular calcium or Akt kinase phosphorylation.
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