Caveolin is a 22-kDa protein that appears to play a critical role in regulating the cholesterol concentration of caveolae. Even though caveolin is thought to be a membrane protein, several reports suggest that this peculiar protein can traffic independently of membrane vesicles. We now present evidence that a cytosolic pool of caveolin is part of a heat-shock protein-immunophilin chaperone complex consisting of caveolin, heat-shock protein 56, cyclophilin 40, cyclophilin A, and cholesterol. Treatment of NIH 3T3 cells with 1 M cyclosporin A or 100 nM rapamycin disrupted the putative transport complex and prevented rapid (10 -20 min) transport of cholesterol to caveolae. The lymphoid cell line, L1210-JF, does not express caveolin, does not form an immunophilin-caveolin complex, and does not transport newly synthesized cholesterol to caveolae. Transfection of caveolin cDNA into L1210-JF cells allowed the assembly of a transport complex identical to that found in NIH 3T3 cells. In addition, newly synthesized cholesterol in transfected cells was rapidly (10 -20 min) and specifically transported to caveolae. These data strongly suggest that a caveolin-chaperone complex is a mechanism by which newly synthesized cholesterol is transported from the endoplasmic reticulum through the cytoplasm to caveolae.

Oxidized LDL (oxLDL) depletes caveolae of cholesterol, resulting in the displacement of endothelial nitric-oxide synthase (eNOS) from caveolae and impaired eNOS activation. In the present study, we determined if the class B scavenger receptors, CD36 and SR-BI, are involved in regulating nitric-oxide synthase localization and function. We demonstrate that CD36 and SR-BI are expressed in endothelial cells, co-fractionate with caveolae, and co-immunoprecipitate with caveolin-1. Co-incubation of cells with 10 g/ml high density lipoprotein (HDL) prevented oxLDLinduced translocation of eNOS from caveolae and restored acetylcholine-induced nitric-oxide synthase stimulation. Acetylcholine caused eNOS activation in cells incubated with 10 g/ml oxLDL (10-15 thiobarbituric acid-reactive substances) and blocking antibodies to CD36, whereas cells treated with only oxLDL were unresponsive. Furthermore, CD36-blocking antibodies prevented oxLDL-induced redistribution of eNOS. SR-BI-blocking antibodies were used to demonstrate that the effects of HDL are mediate by SR-BI. HDL binding to SR-BI maintained the concentration of caveola-associated cholesterol by promoting the uptake of cholesterol esters, thereby preventing oxLDL-induced depletion of caveola cholesterol. We conclude that CD36 mediates the effects of oxLDL on caveola composition and eNOS activation. Furthermore, HDL prevents oxLDL from decreasing the capacity for eNOS activation by preserving the cholesterol concentration in caveolae and, thereby maintaining the subcellular location of eNOS.Hypercholesterolemia-induced vascular disease and atherosclerosis are characterized by an early, selective decrease in the bioavailability of endothelium-derived nitric oxide (NO) (1). Responsiveness to receptor-dependent stimuli, such as acetylcholine, is decreased in the initial phase of the disease process, whereas responsiveness to receptor-independent stimuli such as the calcium ionophore A23187 is not altered. Therefore, the early pathogenesis is characterized by attenuated endothelial NO production in response to extracellular stimuli, even though the capacity for maximal enzyme activation and the breakdown of NO are not affected. As the disease progresses, nonspecific inhibition of NO bioavailability occurs, which is at least partly due to enhanced inactivation of NO by superoxide anions (2-4). The chronic inhibition of NO synthesis in rabbit models of hypercholesterolemia accelerates the development of vascular dysfunction and intimal lesions, providing additional evidence that the impairment of NO synthesis promotes atherogenesis (5). In vitro investigations have further shown that oxLDL 1 inhibits NO-mediated responses (6). Numerous studies have demonstrated that the endothelial isoform of NO synthase (eNOS) is localized in plasmalemmal caveolae and that caveolin is a negative regulator of eNOS enzymatic activity (7). Caveolae are lipid domains that typically represent about 1-4% of the total plasma membrane surface area (8). The structure and function of caveolae is dep...

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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. 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.

Cardiovascular diseases remain the leading cause of death in the United States. Two factors associated with a decreased risk of developing cardiovascular disease are elevated HDL levels and sexspecifically, a decreased risk is found in premenopausal women. HDL and estrogen stimulate eNOS and the production of nitric oxide, which has numerous protective effects in the vascular system including vasodilation, antiadhesion, and anti-inflammatory effects. We tested the hypothesis that HDL binds to its receptor, scavenger receptor class B type I (SR-BI), and delivers estrogen to eNOS, thereby stimulating the enzyme. HDL isolated from women stimulated eNOS, whereas HDL isolated from men had minimal activity. Studies with ovariectomized and ovariectomized/estrogen replacement mouse models demonstrated that HDL-associated estradiol stimulation of eNOS is SR-BI dependent. Furthermore, female HDL, but not male HDL, promoted the relaxation of muscle strips isolated from C57BL/6 mice but not SR-BI null mice. Finally, HDL isolated from premenopausal women or postmenopausal women receiving estradiol replacement therapy stimulated eNOS, whereas HDL isolated from postmenopausal women did not stimulate eNOS. We conclude that HDL-associated estrodial is capable of the stimulating eNOS. These studies establish a new paradigm for examining the cardiovascular effects of HDL and estrogen.