Native low-density lipoprotein induces endothelial nitric oxide synthase dysfunction: role of heat shock protein 90 and caveolin-1.
Although native LDL (n-LDL) is well recognized for inducing endothelial cell (EC) dysfunction, the mechanisms remain unclear. One hypothesis is n-LDL increases caveolin-1 (Cav-1), which decreases nitric oxide (*NO) production by binding endothelial nitric oxide synthase (eNOS) in an inactive state. Another is n-LDL increases superoxide anion (O(2)(*-)), which inactivates *NO. To test these hypotheses, EC were incubated with n-LDL and then analyzed for *NO, O(2)(*-), phospho-eNOS (S1179), eNOS, Cav-1, calmodulin (CaM), and heat shock protein 90 (hsp90). n-LDL increased NOx by more than 4-fold while having little effect on A23187-stimulated nitrite production. In contrast, n-LDL decreased cGMP under basal and A23187-stimulated conditions and increased O(2)(*-) by a mechanism that could be inhibited by L-nitroargininemethylester (L-NAME) and BAPTA/AM. n-LDL increased phospho-eNOS by 149%, eNOS by approximately 34%, and Cav-1 by 28%, and decreased the association of hsp90 with eNOS by 49%. n-LDL did not appear to alter eNOS distribution between membrane fractions (approximately 85%) and cytosol (approximately 15%). Only 3-6% of eNOS in membrane fractions was associated with Cav-1. These data support the hypothesis that n-LDL increases O(2)(*-), which scavenges *NO, and suggest that n-LDL uncouples eNOS activity by decreasing the association of hsp90 as an initial step in signaling eNOS to generate O(2)(*-).
Pritchard
Kirkwood A
KA
Department of Surgery, Division of Pediatric Surgery, Pharmacology and Toxicology, and Cardiovascular Center, Medical College of Wisconsin, Milwaukee, WI 53226, USA. kpritch@mcw.edu
Ackerman
Allan W
AW
Ou
Jingsong
J
Curtis
Michelle
M
Smalley
David M
DM
Fontana
Jason T
JT
Stemerman
Michel B
MB
Sessa
William C
WC
D000818:Animals; D001692:Biological Transport; D015153:Blotting, Western; D002147:Calmodulin / Q000378:metabolism; D002417:Cattle; D051242:Caveolin 1; D022461:Caveolins / Q000378:metabolism*; D002478:Cells, Cultured; D002614:Chelating Agents / Q000494:pharmacology; D006152:Cyclic GMP / Q000378:metabolism; D004533:Egtazic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000201:enzymology; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D008077:Lipoproteins, LDL / Q000494:pharmacology*; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D009573:Nitrites / Q000378:metabolism; D010766:Phosphorylation; D013481:Superoxides / Q000378:metabolism
C495540:CAV1 protein, human; D002147:Calmodulin; D051242:Caveolin 1; D022461:Caveolins; D002614:Chelating Agents; D004791:Enzyme Inhibitors; D018841:HSP90 Heat-Shock Proteins; D008077:Lipoproteins, LDL; D009573:Nitrites; D013481:Superoxides; D009569:Nitric Oxide; D004533:Egtazic Acid; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D006152:Cyclic GMP; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D019331:NG-Nitroarginine Methyl Ester