NO propagates a number of antiatherogenic effects in the endothelium, and diminished availability has been associated with vascular disease. Recently it has been reported that phosphorylation of endothelial NO synthase (eNOS) at Ser-1179 is required to increase activity in response to stimuli, including high-density lipoprotein (HDL). The current study was undertaken to further examine the mechanism by which HDL activates eNOS and to specifically determine the role of the major apolipoprotein of HDL, apolipoprotein AI (ApoAI). Phosphorylation of eNOS residues Ser-116, Ser-617, Ser-635, Ser-1179, and Thr-497 after incubation with ApoAI and HDL was examined. There were significant increases in phosphorylation at Ser-116 in response to both HDL and ApoAI and similar magnitudes of dephosphorylation at Thr-497. Ser-1179 phosphorylation increased transiently but returned to basal level after 2.5 min. Data demonstrating activation of AMP activated protein kinase (AMPK) during HDL and ApoAI incubation suggests that AMPK may play a role in activation of eNOS. NO release in response to HDL and ApoAI stimulation in endothelial cells paralleled the time frames of phosphorylation, suggesting a causal relationship. Furthermore, ApoAI was found to associate with eNOS in endothelial cells and bind transfected eNOS in Chinese hamster ovary cells, whereas confocal data demonstrates colocalization of ApoAI and eNOS in the perinuclear region, suggesting a protein-protein interaction. Collectively, the results indicate that HDL and ApoAI increase eNOS activity by multisite phosphorylation changes, involving AMPK activation after protein association between ApoAI and eNOS. D ecreased bioavailability of endothelium-derived NO is an important antecedent to atherosclerosis (1). NO inhibits events that promote atherosclerotic progression, including vasoconstriction, monocyte adhesion, and smooth muscle cell proliferation (2). The bulk of endothelium-derived NO is generated from L-arginine conversion by endothelial NO synthase (eNOS, NOS III) (3). Activity of eNOS is modulated by complex mechanisms including phosphorylation, protein-protein interactions, substrate availability, and intracellular Ca 2ϩ flux. Numerous biological agents have been associated with changes in eNOS activity, including caveolin (4), Ca 2ϩ calmodulin (5), HSP90 (6), Dynamin-2 (7), bradykinin (8), and more recently high-density lipoprotein (HDL) (9). HDL plays a major role in reversing and preventing progression of vascular disease through its role in reverse cholesterol transport and its involvement in signaling͞receptor pathways of cholesterol metabolism (10). Possibly, some cardiovascular protective effects of HDL are mediated via activation of eNOS, although the precise nature of this interaction remains unclear. The current study was undertaken to examine the mechanism by which HDL activates eNOS and to determine whether the major apolipoprotein of HDL, apolipoprotein AI (ApoAI), mediates the response.Endothelial cells incubated with HDL exhibit an increase in...

Since accelerated atherosclerosis may be induced by excess circulating remnants of triglyceride-rich lipoprotein catabolism, we looked for evidence of remnant particle accumulation in the lipoproteins of 11 patients on long-term dialysis. We found several abnormalities in lipoprotein protein and lipids: enrichment of intermediate-density lipoproteins (ILD) and low-density lipoproteins (LDL) with triglyceride; the presence of apoprotein B48 (a "marker" for intestinal lipoproteins) in very-low-density lipoproteins (VLDL); an increased concentration of apoprotein AIV (a protein related to chylomicron transport); the presence of AIV in VLDL, IDL, and LDL; and the presence in LDL of apoproteins C and E (proteins not normally found in LDL). These findings strongly suggest accumulation of remnants of triglyceride-rich lipoproteins in patients with chronic renal failure who are undergoing peritoneal dialysis or hemodialysis, and may explain in part the increased incidence of coronary deaths among these patients.

Human plasma apolipoprotein A-I (apoA-I) and recombinant full-length proapoA-I (apoA-I-(؊6 -243)) as well as four truncated forms of proapoA-I were used as acceptors to study cholesterol and phospholipid efflux from HepG2 cells. Efflux of both cholesterol and phospholipid to the lipid-free plasma apoA-I was twice that of apoA-I-(؊6 -243). When apoA-I was incorporated into reconstituted high density lipoprotein, cholesterol efflux increased, phospholipid efflux decreased and the difference between plasma apoA-I and apoA-I-(؊6 -243) disappeared. Truncation of recombinant apoA-I to residues 222 (apoA-I-(؊6 -222)) and 210 (apoA-I-(؊6 -210)) resulted in a 70 -95% decrease in their ability to promote the efflux of both intracellular and plasma membrane cholesterol. Further truncation to residues 150 (apoA-I-(؊6 -150)) and 135 (apoA-I-(؊6 -135)) fully restored the ability of apoA-I to promote cholesterol efflux. Phospholipid efflux closely paralleled the efflux of cholesterol. Interaction of 125 I-labeled apoA-I with the cells was similar for apoA-I-(؊6 -243), apoA-I-(؊6 -222), and apoA-I-(؊6 -210), but slightly higher for apoA-I-(؊6 -150) and apoA-I-(؊6 -135).When complexed with phospholipid, all forms except apoA-I-(؊6 -210) formed discoidal reconstituted high density lipoprotein particles. When the same amounts of free or lipid-associated apoA-I were compared, association of apoA-I with phospholipid increased cholesterol efflux and decreased phospholipid efflux, and the difference in the ability of different mutants to promote cholesterol and phospholipid efflux disappeared. We conclude that the capacity of lipid-free apoA-I to promote cholesterol efflux is related to its ability to mobilize cellular phospholipid, which apparently involves a region around residues 222-243. A second lipid-binding region is exposed when the carboxylterminal half of apoA-I is absent.

A B S T R A C a The turnover and the catabolic fate of the B apoprotein of very low density lipoprotein (VLDL-B) was studied in 15 normal and hyperlipidemic subjects using reinjected autologous VLDL labeled with radioiodine. The specific radioactivity-time curve ofthe B apoprotein in total VLDL (Sf 20-400) was multiexponential but conformed to a two-pool model during the first 48 h of catabolism. The flux was highest in several hypertriglyceridemic subjects. The mass of pool A exceeded the intravascular content of VLDL-B by 30% on average, indicating extravascular metabolism of VLDL. The two-pool model might reflect the input of several populations of particles or heterogeneity of catabolic processes or pools. The flux of B apoprotein was also measured in several subclasses of VLDL, in smailler intermediate density lipoproteins, and in low density lipoproteins (LDL). In three subjects the flux was similar in Sf 60-400 and in Sf 12-60 lipoproteins, suggesting that VLDL was catabolized at least to a particle in the density range Sf 12-60. Subsequent catabolism appeared to proceed by two pathways: in normotriglyceridemiic subjects, B apoprotein flux in the Sf 20-400 and in Sf 12-20 lipoproteins was similar, whereas in hypertriglyceridemic subjects flux through Sf 12-20 accounted for only part of the VLDL-B flux.The flux of low density' lipoprotein B apoprotein (LDL-B), which is believed to be derived from VLDL catabolism, was calculated f'rom the area between the specific activity time curves of' VLDL-B and LDL-B. In subjects with normal plasmla triglyceride concentration, LDL-B flux was froml 91% to 113% of that of VLDL-B; but in three hy'pertriglyceridemic subjects showing high rates of VLDL-B transport, LDL-B flux was only one-third that of VLDL-B. This suggests that