High density lipoprotein binding to scavenger receptor, Class B, type I activates endothelial nitric-oxide synthase in a ceramide-dependent manner.
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-N,N,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 HDL binding to SR-BI stimulates eNOS by increasing intracellular ceramide levels and is independent of an increase in intracellular calcium or Akt kinase phosphorylation.
Li
Xiang-An
XA
Department of Physiology, University of Kentucky Medical School, Lexington, Kentucky 40536-0230.
Titlow
William B
WB
Jackson
Brian A
BA
Giltiay
Nathalia
N
Nikolova-Karakashian
Mariana
M
Uittenbogaard
Annette
A
Smart
Eric J
EJ
D000818:Animals; D018955:CD36 Antigens; D016466:CHO Cells; D002518:Ceramides / Q000378:metabolism*; D006224:Cricetinae; D004789:Enzyme Activation; D006801:Humans; D008077:Lipoproteins, LDL / Q000378:metabolism*; D008565:Membrane Proteins; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D010766:Phosphorylation; D011485:Protein Binding; D011971:Receptors, Immunologic / Q000378:metabolism*; D018110:Receptors, Lipoprotein; D051116:Receptors, Scavenger; D051122:Scavenger Receptors, Class B
D018955:CD36 Antigens; D002518:Ceramides; D008077:Lipoproteins, LDL; D008565:Membrane Proteins; D011971:Receptors, Immunologic; D018110:Receptors, Lipoprotein; D051116:Receptors, Scavenger; C495124:SCARB1 protein, human; C454739:Scarb1 protein, mouse; D051122:Scavenger Receptors, Class B; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III