Cysteine substitutions in apolipoprotein A-I primary structure modulate paraoxonase activity.

Paraoxonase (PON) is transported primarily on apolipoprotein A-I (apoA-I) -containing high-density lipoprotein (HDL) and is thought to protect against early atherogenic events including low-density lipoprotein (LDL) oxidation and monocyte migration. It has been proposed that apoA-I may be necessary for PON's association with plasma HDL. On the basis of this, we examined the effect of apoA-I on PON's enzymatic activity and its ability to associate with HDL. Additionally, we examined whether changes in apoA-I primary structure (cysteine substitution mutations) could modulate these effects. Chinese hamster ovary cells stably transfected with human PON1A cDNA were incubated in the presence and absence of recombinant wild-type apoA-I (apoA-I(WT)) and specific Cys substitution mutations. Extracellular accumulation of PON activity in the presence of apoA-I(WT) was 0.095 +/- 0.013 unit/mg of cell protein (n = 7) compared to 0.034 +/- 0.010 unit/mg of cell protein in the absence of apoA-I (n = 7), a 2.79-fold increase in activity when apoA-I was incubated with the cells. Lipid-free apoA-I did not increase PON activity, while preformed nascent HDL increased PON activity only 30%, suggesting that maximal PON activity is lipid-dependent and requires coassembly of PON and apoA-I on nascent HDL. The cysteine mutations R10C, R27C, and R61C significantly increased (p < 0.01) PON activity 32.6% +/- 14.7%, 31.6% +/- 18.9%, and 27.4% +/- 20%, respectively, over that of wild type (WT). No changes in PON activity were observed with apoA-I cysteine substitution mutations in the C-terminal portion of the protein. The data suggest that, for optimal PON activity, coassembly of the enzyme onto nascent HDL is required and that the N-terminal region of apoA-I may be important in the assembly process.

Baculovirus-mediated expression and purification of human serum paraoxonase 1A.

Human paraoxonase 1 (hPON1) is a lipid-associated enzyme transported on HDL. There is considerable interest in hPON1 because of its putative antioxidative/antiatherogenic properties. We have created a recombinant baculovirus (BV) to generate hPON1A in large quantities for structure-function studies and here describe the method for production and isolation of the enzyme. A high level of recombinant hPON1 type A (rPON1A) was produced by Hi-5 insect cells (40 mg/l); a fraction ( approximately 10 mg/l) was secreted into the cell culture medium, but the majority ( approximately 30 mg/l) remained associated with the host insect cells. Cell-associated rPON1A was purified by detergent extraction (Tergitol NP-10) followed by three simple chromatography steps (DEAE-Sepharose, Sephacryl S-200, and concanavalin A). The purified enzyme bound to concanavalin A and was converted to a lower molecular mass by endoglycosidase H digestion, suggesting that rPON1A contained high-mannose N-glycan chains. There was a significant decrease in arylesterase activity (>99%) concomitant with enzymatic deglycosylation. rPON1A was dependent on Ca(2+) for arylesterase activity, exhibiting kinetic parameters similar to native hPON1A (K(m) = 3.8 +/- 2.1 vs. 3.7 +/- 2.0 mM and V(max) = 1,305 +/- 668 vs. 1,361 +/- 591 U/mg protein, rPON1A and hPON1A, respectively). Both rPON1A and hPON1A efficiently inhibited lipoxygenase-mediated peroxidation of phospholipid. In contrast to the arylesterase activity, which was sensitive to endoglycosidase H treatment, enzymatic deglycosylation did not inhibit the antioxidant activity of rPON1A. In conclusion, our BV-mediated PON1A expression system appears ideally suited for the production of relatively large quantities of rPON1A for structure-function studies.

Regulation and activity of the human ABCA1 gene in transgenic mice.

The ABCA1 transporter is one of the limiting steps in cellular cholesterol efflux. To study the expression and activity of the human ABCA1 gene in vivo we have examined mice containing two human BAC transgenes with different 5' ends. Mice containing a 255-kilobase (kb) BAC transgene, including 70 kb upstream of the previously defined exon 1, demonstrated a pattern of tissue-specific expression mimicking that of the endogenous mouse gene. Compared with macrophages from control mice, macrophages from these transgenics had increases in apoA-I cholesterol efflux heightened in response to increases in cell cholesterol content. The observed increase in macrophage apoA-I-mediated cholesterol efflux was not accompanied by alterations in plasma high density lipoprotein in the transgenics. Although mice containing a smaller 171-kb human BAC transgene, lacking the previously described exon 1 and ABCA1 promoter, did not express human ABCA1 in macrophages, they did express the human transgene in liver at levels comparable with those of the orthologous mouse gene. Analysis by 5' rapid amplification of cDNA ends of liver mRNA from these animals revealed a new ABCA1 exon 1 (exon 1A) and a previously unrecognized promoter. Analysis of human tissue revealed that exon 1A containing transcripts accounted for a high proportion of the ABCA1 mRNAs present in human liver. This analysis of ABCA1 transgenics showed that the expression of human ABCA1 transgenes can result in increased cholesterol efflux from macrophages, unaccompanied by changes in plasma high density lipoprotein, and identified a new ABCA1 promoter in humans.

Relative sensitivities of plasma lecithin:cholesterol acyltransferase, platelet-activating factor acetylhydrolase, and paraoxonase to in vitro gas-phase cigarette smoke exposure.

In order to identify potential atherogenic properties of gas-phase cigarette smoke, we utilized an in vitro exposure model to determine whether the activities of several putative anti-atherogenic enzymes associated with plasma lipoproteins were compromised. Exposure of heparinized human plasma to gas-phase cigarette smoke produced a dose-dependent reduction in the activity of platelet-activating factor acetylhydrolase (PAF-AH). Reductions of nearly 50% in PAF-AH activity were observed following exposure to gas-phase smoke from four cigarettes over an 8-h period. During this time of exposure, lecithin:cholesterol acyltransferase (LCAT) was rendered almost completely inactive (>80%). In contrast, paraoxonase was totally unaffected by cigarette smoke. Supplementation of plasma with 1 mM reduced glutathione was found to protect both PAF-AH and LCAT from cigarette smoke, suggesting that cysteine modifications may have contributed to the inhibition of these two enzymes. To evaluate this possibility, we blocked the free cysteine residues of these enzymes with the reversible thiol-modifying reagent dithiobisnitrobenzoic acid (DTNB). Reversal of the DTNB-cysteine adducts following cigarette smoke exposures revealed that LCAT, but not PAF-AH, was protected. Moreover, high doses (1.0-10 mM) of acrolein and 4-hydroxynonenal, reactive aldehydic species associated with cigarette smoke, completely inhibited plasma LCAT activity, whereas PAF-AH was resistant to such exposures. Taken together, these results indicate a divergence regarding the underlying mechanism of PAF-AH and LCAT inhibition upon exposure to gas-phase cigarette smoke. While LCAT was sensitive to exposure to volatile aldehydic products involving, in part, cysteine and/or active site modifications, the enzyme PAF-AH exhibited an apparent resistance. The latter suggests that the active site of PAF-AH is in a microenvironment that lacks free cysteine residues and/or is shielded from volatile aldehydic combustion products. Based on these results, we propose that cigarette smoke may contribute to atherogenesis by inhibiting the activities of plasma PAF-AH and LCAT, but the nature of this inhibition differs for the enzymes.

Evidence that lipid hydroperoxides inhibit plasma lecithin:cholesterol acyltransferase activity.

The oxidation of low density lipoproteins (LDL) has been implicated in the development of atherosclerosis. Recently, we found that polar lipids isolated from minimally oxidized LDL produced a dramatic inhibition of lecithin: cholesterol acyltransferase (LCAT) activity, suggesting that HDL-cholesterol transport may be impaired during early atherogenesis. In this study, we have identified molecular species of oxidized lipids that are potent inhibitors of LCAT activity. Treatment of LDL with soybean lipoxygenase generated small quantities of lipid hydroperoxides (20 +/- 4 nmol/mg LDL protein, n = 3); but when lipoxygenase-treated LDL (1 mg protein/ml) was recombined with the d > 1.063 g/ml fraction of human plasma, LCAT activity was rapidly inhibited (25 +/- 4 and 65 +/- 16% reductions by 1 and 3 h, respectively). As phospholipid hydroperoxides (PL-OOH) are the principal oxidation products associated with lipoxygenase-treated LDL, we directly tested whether PL-OOH inhibited plasma LCAT activity. Detailed dose-response curves revealed that as little as 0.2 and 1.0 mole % enrichment of plasma with PL-OOH produced 20 and 50% reductions in LCAT activity by 2 h, respectively. To gain insight into the mechanism of LCAT impairment, the enzyme's free cysteines (Cys31 and Cys184) and active site residues were "capped" with the reversible sulfhydryl compound, DTNB, during exposure to either minimally oxidized LDL or PL-OOH. Reversal of the DTNB "cap" after such exposures revealed that the enzyme was completely protected from both sources of peroxidized phospholipids. We, therefore, conclude that PL-OOH inhibited plasma LCAT activity by modifying the enzyme's free cysteine and/or catalytic residues. These studies are the first to suggest that PL-OOH may accelerate the atherogenic process by impairing LCAT activity.

Apolipoprotein A-I promotes cholesterol release and apolipoprotein E recruitment from THP-1 macrophage-like foam cells.

Apolipoprotein E (apoE) is synthesized and secreted by arterial macrophages while apolipoprotein A-I (apoA-I) is present in surrounding interstitial fluids. Both apolipoproteins play important roles in macrophage cholesterol homeostasis by forming lipid complexes (nascent-HDL) with cellular phospholipids (PL) and cholesterol (UC) thereby promoting cholesterol efflux. In this study, we evaluated the relative contributions of apoA-I and endogenously produced apoE in mediating the recruitment of cellular cholesterol. THP-1 human monocytes were differentiated (300 nm phorbol dibutyrate) into macrophages and macrophage-foam cells were generated by cholesterol loading with acetylated LDL (50 microg protein/ml). ApoA-I (10 microg/ml) depleted macrophage-foam cell cholesteryl esters by 50% in 24 h. This reduction was accompanied by a significant increase in the UC/PL mole ratio of nascent HDL (UC/PL = 0.80 +/- 0.15) in the medium compared to complexes isolated from macrophages (UC/PL = 0.59 +/- 0.08). Significantly more (70%) nascent-HDL were formed in incubations of apoA-I with macrophage-foam cells than with macrophages. Medium apoE accumulation paralleled the assembly of apoA-I containing nascent HDL where 2- and 4-fold increases were observed with macrophages and macrophage-foam cells, respectively, compared to incubations in the absence of apoA-I. Despite the increase in medium apoE accumulation, a majority (85%) of particles (11, 9, and 7.4 nm in diameter) from macrophages and macrophage-foam cells possessed apoA-I without apoE. ApoA-I plus apoE particles (13-16 nm) were also formed along with a small quantity of apoE-only particles (19-20 nm). The predominance of apoA-I only particles indicates, however, that the assembly of apoA-I-containing nascent-HDL represents a major metabolic pathway of cellular cholesterol recruitment compared to the endogenous production of apoE.

Deletion of amino acids Glu146-->Arg160 in human apolipoprotein A-I (ApoA-ISeattle) alters lecithin:cholesterol acyltransferase activity and recruitment of cell phospholipid.

Human apolipoprotein A-I (apoA-I) has an important role in the efflux of cholesterol from peripheral cells, the first step in reverse cholesterol transport. Deletion of amino acids Glu146-->Arg160 in apoA-I (apoA-ISeattle) removes a large section of a lipid binding helix and is associated in vivo with an atherogenic lipoprotein profile characterized by a deficiency in high-density lipoproteins (HDL). In the present study, we asked whether apoA-ISeattle had normal ability to recruit lipids from cells and to form nascent high-density lipoprotein (HDL) particles and whether the altered secondary structure affected lecithin:cholesterol acyltransferase (LCAT) activity. Wild-type apoA-I and apoA-ISeattle expressed in transfected Chinese hamster ovary cells formed nascent HDL particles with similar density distribution and protein-to-lipid ratio. Phospholipid subclass distribution of apoA-ISeattle nascent HDL demonstrated a significant increase in sphingomyelin and phosphatidylethanolamine compared to wild type. ApoA-ISeattle nascent HDL had a unique size distribution compared to wild-type nascent HDL; large (9-20 nm) particles predominated while there were virtually no small (7.5 nm) particles. LCAT reactivity was impaired by apoA-ISeattle nascent HDL where cholesterol esterification was only half that of wild-type complexes. The apoA-ISeattle conformation on nascent HDL was studied with a panel of monoclonal antibodies (Mabs) specific for apoA-I. Mabs that recognize the putative LCAT activation site, residues 95-122, had normal reactivity. As expected, the Mabs that recognized residues 141-164 were unreactive because of the 146-160 deletion; in addition, there was low reactivity with a Mab that recognizes residues 220-242. The data suggest that apoA-I residues 146-160 and/or 220-242 partake in normal LCAT activation and that cooperative interactions between helices may be important for maximal cholesterol esterification.

Elevated triglycerides and low HDL cholesterol in transgenic mice expressing human apolipoprotein A-I(Milano).

In general, plasma concentrations of high density lipoproteins (HDL) are inversely related to the incidence of coronary artery disease. One exception to this trend is individuals with apolipoprotein A-I(Milano) (apo A-IM), a molecular variant of apo A-I, which results in very low plasma apo A-I and HDL-cholesterol levels. Despite these low levels, and other lipoprotein defects, individuals with this mutation have no increased risk for cardiovascular disease. As a first step in proving why apo A-IM carriers appear to be protected from the pro-atherogenic effect of a low HDL, transgenic mice expressing apo A-IM were generated. Mice expressing either wild-type human apo A-I or apo A-IM, together with human apo A-II, were crossed into mice lacking murine apo A-I. Apo A-IM/A-II mice had lower cholesterol and HDL plasma levels compared to apo A-I/A-II mice. Moreover, as in human carriers, apo A-IM mice were characterized by elevated triglyceride plasma levels and by the presence of a population of very small HDL particles. These results indicate that the expression of apo A-IM in a mouse model reproduces the major lipid/lipoprotein abnormalities observed in human carriers. Thus, apo A-IM transgenic mice appear to be a suitable model in which to assess whether the mutation has an anti-atherogenic effect.

High density lipoprotein particle size restriction in apolipoprotein A-I(Milano) transgenic mice.

Human carriers of apolipoprotein A-I(Milano) (Arg173 --> Cys substitution in apolipoprotein A-I) are characterized by an HDL deficiency in which small, dense HDL accumulate in plasma. Because affected individuals are heterozygous for this mutation, the full impact of apolipoprotein A-I(Milano) (apoA-I(Milano)) on HDL-cholesterol metabolism is unknown. In this study, apoA-I(Milano) transgenic mice were used to evaluate the extent of apoA-I(Milano) dimerization and HDL particle size restriction in the absence of wild-type apoA-I. Murine apoA-I knockout mice were utilized to express apoA-I(Milano) and human apoA-II in the presence of wild-type, human apoA-I (apoA-IMilano/A-Iwt/A-II) and in its absence (apoA-IMilano/A-II). Plasma HDL-cholesterol concentrations were similar (30 mg/dl) in both lines of apoA-I(Milano) transgenic mice. In the apoA-IMilano/A-Iwt/A-II phenotype, 14% of the apoA-I(Milano) formed homodimers and 33% formed heterodimers with apoA-II. ApoA-I(Milano) homodimers increased by 71% in the apoA-IMilano/A-II transgenics and was associated with an abundance of small, 7.6-nm HDL3-sized particles compared to the 9.5, 8.3, and 7.6-nm-sized particles in apoA-IMilano/A-Iwt/A-II mice. The unesterified cholesterol/cholesteryl ester mole ratio of HDL was elevated by 45% in apoA-IMilano/A-Iwt/A-II mice and by 90% in apoA-IMilano/A-II transgenics compared to wild-type (human apoA-I/A-II). Both apoA-I(Milano) transgenics possessed normal levels of plasma LCAT activity, but endogenous cholesterol esterification rates were reduced by 50% compared to controls. Thus, HDL particle size restriction was not the result of impaired LCAT activation; rather, dimerization of apoA-I(Milano) limited the esterification of cholesterol on endogenous HDL. In the absence of wild-type apoA-I, the more extensive dimerization of apoA-I(Milano) severely limited cholesteryl ester accumulation on plasma HDL accounting for the abundance of small, 7.6-nm HDL3 particles in apoA-IMilano/A-II mice.

Evidence that apolipoprotein A-IMilano has reduced capacity, compared with wild-type apolipoprotein A-I, to recruit membrane cholesterol.

Human carriers of apolipoprotein (apo) A-IMilano are heterozygous for an Arg173-->Cys substitution in the apoA-I primary sequence; despite severe reductions in HDL cholesterol concentrations, affected individuals do not develop coronary heart disease, suggesting that apoA-IMilano may possess antiatherogenic properties. As the beneficial effects of wild-type apoA-I are linked to its role in HDL cholesterol transport, we examined the capacity of apoA-IMilano to recruit cell cholesterol and activate lecithin:cholesterol acyltransferase (LCAT) (two key events in the antiatherogenic reverse cholesterol transport pathway). ApoA-IMilano and wild-type apoA-I were expressed in Chinese hamster ovary cells, and their ability to recruit membrane phospholipid and cholesterol for the assembly of nascent HDL was compared. Both clonal cell lines exhibited similar levels of apolipoprotein accumulation in serum-free medium (approximately 2 micrograms/mg cell protein per 24 hours), and 15% of each apolipoprotein was associated with membrane lipids to form nascent HDL (d = 1.063 to 1.21 g/mL). SDS-PAGE showed that a majority (66 +/- 12%) of the lipidated apoA-IMilano was in the homodimer form. Compositional analyses revealed that apoA-IMilano nascent HDL had a significantly lower (P < .001) unesterified cholesterol/phospholipid mole ratio (0.47 +/- 0.10) than wild-type apoA-I complexes (1.29 +/- 0.14), indicating that apoA-IMilano had a reduced capacity to recruit cell cholesterol. In addition to the reduced unesterified cholesterol/phospholipid ratio, apoA-IMilano nascent HDL consisted mostly of small 7.4-nm particles compared with wild-type apoA-I, in which 11- and 9-nm particles predominated. Despite these changes in nascent HDL particle size and composition, apoA-IMilano activated LCAT normally. We conclude that, even though apoA-IMilano is a normal activator of LCAT, it is less efficient that wild-type apoA-I in recruiting cell cholesterol, suggesting that the putative antiatherogenic properties attributed to apoA-IMilano may be unrelated to the initial stages of reverse cholesterol transport.

Minimally oxidized LDL is a potent inhibitor of lecithin:cholesterol acyltransferase activity.

The oxidation of low density lipoproteins (LDL) has been implicated in the development of atherosclerosis. As a variety of highly reactive lipid peroxidation products can transfer from oxidized LDL to HDL, we evaluated the potential deleterious effects of LDL oxidation on HDL-cholesterol metabolism. To address this issue, we exposed the HDL-containing d > 1.063 g/ml fraction of human plasma to copperoxidized LDL and assessed lecithin:cholesterol acyltransferase (LCAT) activity and apolipoproteinA-I (apoA-I) structure. To determine whether LCAT was directly affected by oxidized LDL, independent of crosslinking of apoA-I, we used an exogenous, [14C]cholesterol-labeled proteoliposome substrate to measure plasma LCAT activity. We observed an inhibition of LCAT activity where copper-oxidized LDL possessing only 2.3 +/- 0.1 and 7.3 +/- 1.4 TBARS produced 24 +/- 3% and 47 +/- 10% reductions in [14C]cholesterol esterification by 1 h, respectively. Copper-oxidized LDL that had been passed through a GF-5 desalting column, while retaining only one-third of its original TBARS, possessed nearly all of its LCAT inhibitory capacity suggesting that the LCAT inhibitory factor(s) was a lipophilic oxidation product. Analysis of polarlipids isolated from copper-oxidized LDL indicated that phospholipid and sterol fractions effectively inhibited LCAT. Copper-oxidized LDL, with as little as 6.3 TBARS, also produced intermolecular crosslinking of apoA-I molecules. Taken together, these data suggest that products of LDL oxidation may adversely affect HDL-cholesterol metabolism by two separate mechanisms: 1) a direct inhibitory effect on LCAT activity and 2) through crosslinking of apoA-I. If occurring in vivo, minimally oxidized LDL may impair cholesteryl ester formation on HDL thereby limiting the ability of HDL to function efficiently in the putative antiatherogenic reverse cholesterol transport pathway.

Structural relationships between nascent apoA-I-containing particles that are extracellularly assembled in cell culture.

Apolipoprotein A-I (apoA-I) incubated with CHO cells assembles three major nascent lipid complexes with diameters of 7.3, 9, and 11 nm. Previous studies suggested that the smaller nascent particles were precursors for the larger nascent ones. To test this hypothesis, the 7.3, 9, and 11 nm apoA-I-lipid complexes formed by incubating CHO cells with lipid-free apoA-I were isolated and subsequently each subpopulation was re-incubated with cells in the absence of other subpopulations. The physical-chemical characteristics of each subpopulation were examined before and after re-incubation in an effort to understand relationships. if any, between the different nascent complexes. The 7.3, 9, and 11 nm complexes were unique in that each of the particles had pre-alpha mobility on agarose gels: this rapid migration was not altered by re-incubation with cells. Protein crosslinking studies indicated that the 7.3, 9, and 11 nm complexes possessed 2, 3, and 4 apoA-I molecules per complex, respectively; it is unlikely that the size of the particle and number of apoA-I molecules per particle played a role in the increased negative charge of the particles. The present study shows that smaller particles did not give rise to larger ones upon re-incubation with cells. Rather, the 11 and 9 nm particles both generated smaller discs (the 11 nm giving rise primarily to 9 nm discs and the 9 nm complex giving rise to 7.3 nm discs) suggesting that, during incubation with cells, the complexes are destabilized and remodeled into smaller, not larger, complexes. Surprisingly, the 7.3 nm complexes during re-incubation with cells were extremely stable and did not undergo size alteration. When the 7.3 nm particles were incubated with additional small quantities of lipid-free apoA-I (1-2 microgram/ml), larger discoidal complexes were generated suggesting that the formation of larger particles may be driven by the availability of lipid-free apoA-I.

Inhibition of lecithin-cholesterol acyltransferase and modification of HDL apolipoproteins by aldehydes.

Experimental evidence suggests that aldehydes generated as a consequence of lipid peroxidation may be involved in the pathogenesis of atherosclerosis. It is well documented that aldehydes modify LDL: however, less is known concerning the effects of aldehydes on other plasma and interstitial fluid components. In the present study, we investigated the effects of five physiologically relevant aldehydes (acetaldehyde, acrolein, hexanal, 4-hydroxynonenal [HNE], and malondialdehyde [MDA]) on two key constituents of the antiatherogenic reverse cholesterol transport pathway, lecithin-cholesterol acyltransferase (LCAT) and HDL. Human plasma was incubated for 3 hours at 37 degrees C with each one of the five aldehydes at concentrations ranging from 0.16 to 84 mmol/L. Dose-dependent decreases in LCAT activity were observed. The short-chain (acrolein) and long-chain (HNE) alpha,beta-unsaturated aldehydes were the most effective LCAT inhibitors. Micromolar concentrations of these unsaturated aldehydes resulted in significant reductions in plasma LCAT activity. The short- and longer-chain saturated aldehydes acetaldehyde and hexanal and the dialdehyde MDA were considerably less effective at inhibiting LCAT than were acrolein and HNE. In addition to inhibiting LCAT, aldehydes increased HDL electrophoretic mobility and cross-linked HDL apolipoproteins. Cross-linking of apolipoproteins A-I and A-II required higher aldehyde concentrations than inhibition of LCAT. The alpha,beta-unsaturated aldehydes acrolein and HNE were fourfold to eightfold more effective cross-linkers of apolipoproteins A-I and A-II than the other aldehydes studied. These data suggest that products of lipid peroxidation, especially unsaturated aldehydes, may interfere with normal HDL cholesterol transport by inhibiting LCAT and modifying HDL apolipoproteins.

Gas-phase cigarette smoke inhibits plasma lecithin-cholesterol acyltransferase activity by modification of the enzyme's free thiols.

Cigarette smoking is associated with an increased risk of premature atherosclerosis. The underlying mechanisms responsible for this association are unknown. Recent work from this laboratory has shown that ex vivo exposure to plasma to gas-phase cigarette smoke (CS) produces a rapid inhibition of lecithin-cholesterol acyltransferase (LCAT) activity and crosslinking of HDL-apolipoproteins. The goal of the present study was to investigate the mechanism(s) by which CS inhibited LCAT and modified HDL. When dialyzed human plasma (12 ml) was exposed to the gas-phase of an equivalent of 1/8 of a cigarette (one 'puff') at 15 min intervals for 3 h, LCAT activity was reduced by 76 +/- 1% compared to controls; supplementation of plasma with glutathione produced a dose-dependent protection of LCAT activity where at the highest concentration (1 mM) 78% protection was observed. A similar protection was obtained with N-acetyl cysteine (1 mM). In addition to LCAT inhibition, HDL-apolipoproteins were crosslinked after 3 h exposure of plasma to CS; crosslinking was reduced by the addition of either glutathione or N-acetyl cysteine to plasma. The amino compounds N-acetyl lysine, N-acetyl arginine, and aminoguanidine failed to protect LCAT and HDL indicating a specificity with regard to the ability of free thiols to buffer the deleterious components of CS which inhibited LCAT and crosslinked HDL-apolipoproteins. Since LCAT contains two free cysteine residues (Cys-31 and -184) near the active site of the enzyme, we tested whether pretreatment of plasma with the reversible sulfhydryl modifying compound, 5,5'-dithiobis-2-nitrobenzoic acid (DTNB), could protect LCAT from CS-induced inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)

Efflux of cellular cholesterol and phospholipid to lipid-free apolipoproteins and class A amphipathic peptides.

The mechanism(s) by which lipid-free apolipoprotein (apo) AI is able to stimulate efflux of cholesterol and phospholipid from cells in cultures has (have) been examined. This process was found to be enhanced when macrophages were enriched with cholesterol. There were 12- and 4-fold increases in cholesterol and phospholipid efflux, respectively, from cholesterol-enriched mouse macrophages when compared to cells not loaded with cholesterol. This enhancement in cholesterol efflux to lipid-free apo AI from macrophages enriched with cholesterol was found to be controlled by the level of free cholesterol in the cells. When cholesterol-enriched mouse macrophages were exposed to lipid-free apo AI at 20 micrograms/mL (706 nM), there was significant efflux of [14C]cholesterol and [3H]phospholipid (20% +/- 0.5%/24 h and 6% +/- 0.3%/24 h, respectively). In comparison, HDL at equivalent protein concentrations only stimulated 11% and 4% efflux of cholesterol and phospholipid, respectively. Synthetic peptides containing amphipathic helical segments that mimic those present in apo AI were used to examine the structural features of the apoprotein which stimulate lipid efflux. Peptides containing only one (18A) or two (37pA) amphipathic helical segments stimulated as much cholesterol efflux from both mouse macrophages and L-cells as apo AI. The order of efficiency, as assessed by the mass concentration at which half-maximal efflux was reached (EC50), was apo AI > 37pA > 18A, indicating that acceptor efficiency was dependent on the number of amphipathic helical segments per molecule. When the helical content of 18A was increased by neutralizing the charges at the ends of the peptide (Ac-18A-NH2), there was a substantial increase in the efficiency for cholesterol efflux (EC50 18A = 17 micrograms/mL vs Ac-18A-NH2 = 6 micrograms/mL). In contrast, when the amphipathicity of the helix in 18A was decreased by scrambling the amino acid sequence, thereby reducing its lipid affinity, cholesterol and phospholipid efflux were not stimulated. The efficiency with which the peptides stimulated cholesterol efflux was in order of their lipid affinity (37pA > Ac-18A-NH2 > 18A), and this order was similar for phospholipid efflux. The time course of lipid release from mouse macrophages and L-cells indicated that phospholipid appeared in the extracellular medium before cholesterol. These results suggest that the apo AI or peptides first interacted with the cell to form protein/phospholipid complexes, that could then accept cholesterol.

Copper and gas-phase cigarette smoke inhibit plasma lecithin:cholesterol acyltransferase activity by different mechanisms.

Cigarette smokers have reduced levels of plasma high density lipoprotein (HDL) compared to nonsmokers and are at risk of premature cardiovascular disease. Previous work from this laboratory has shown that exposure of human plasma to gas-phase cigarette smoke (CS) inhibited the activity of lecithin:cholesterol acyltransferase (LCAT), the enzyme that catalyzes the formation of cholesteryl ester in HDL and thereby promotes HDL maturation. As CS contains free radicals that could potentially oxidize plasma lipoproteins, we examined the involvement of lipid peroxidation in LCAT inhibition. Results obtained with CS were compared with those obtained by initiating lipid peroxidation with copper ions. Exposure of dialyzed human plasma to an equivalent of one-eighth of a cigarette at 15-min intervals resulted in a progressive loss of LCAT activity (50 and 90% reductions by 1 and 6 h, respectively). A similar pattern of LCAT inhibition was produced with copper (0.5 mM) where 50 and 97% reductions were observed at 1 and 6 h, respectively. To determine whether LCAT inhibition was related to lipid peroxidation, lipoprotein fractions corresponding to VLDL-IDL, LDL, and HDL were isolated from plasma exposed to CS or copper and analyzed for changes in TBARS, the polyunsaturated fatty acid arachidonate relative to palmitate (20:4/16:0 ratio), and vitamin E concentrations. Exposure of plasma for 6 h to CS had no effect on the levels of TBARS and 20:4/16:0 ratio; however, 6 h copper treatment (0.5 mM) caused a 3.0-, 4.0-, and 1.4-fold increase in TBARS and a 17, 25, and 13% reduction in the 20:4/16:0 ratio in VLDL-IDL, LDL, and HDL fractions, respectively. In addition, a complete depletion of lipoprotein vitamin E was observed with CS, whereas copper decreased vitamin E levels by approximately 50% in each fraction. Supplementation of plasma with either vitamin C (85 microM) or butylated hydroxytoluene (BHT, 0.45 mM) was unable to protect LCAT from CS. In contrast, BHT completely protected LCAT activity from inhibition by copper. We conclude that unlike copper, CS-induced inhibition of plasma LCAT activity was unrelated to free radical-induced lipid peroxidation. The inhibition of LCAT activity by cigarette smoke may contribute to the development of atherosclerosis by impairing HDL metabolism and the reverse cholesterol transport process.

Recruitment of cell phospholipids and cholesterol by apolipoproteins A-II and A-I: formation of nascent apolipoprotein-specific HDL that differ in size, phospholipid composition, and reactivity with LCAT.

Studies were carried out to determine whether apolipoprotein (apo) A-II, like apoA-I, can recruit phospholipid and cholesterol from cell membranes, thereby forming nascent apoA-II-specific HDL. ApoA-II and apoA-I were purified from plasma and each was incubated with CHO cells at a concentration of 10 micrograms/ml. Lipid-containing complexes were isolated from the medium in both cases; the composition of the apoA-II- and apoA-I-specific complexes were similar where percent protein, phospholipid, and cholesterol were 35 +/- 3, 38 +/- 2, and 25 +/- 1 for apoA-II, respectively, and 40 +/- 2, 35 +/- 1, and 24 +/- 2 for apoA-I, respectively. On a per mole of apolipoprotein basis, apoA-I recruited significantly more phospholipid and cholesterol than dimeric apoA-II suggesting that apoA-I with its greater number of alpha helices binds more lipid. By electron microscopy, nascent apoA-II- and apoA-I-specific particles were predominantly discoidal in morphology. ApoA-II complexes were unique in their nondenaturing polyacrylamide gradient gel size distribution as six distinct populations of particles with diameters of 8.1, 9.3, 10.4, 11.8, 13.1, and 14.6 nm were routinely noted, compared with apoA-I which formed only three major populations with diameters of 7.3, 9.2, and 11.0 nm. Nascent apoA-I complexes incubated with purified lecithin:cholesterol acyltransferase (LCAT) were transformed into predominantly 8.4 nm particles. The latter is similar in size to plasma HDL3a, LpA-I particles, suggesting that extracellularly assembled apoA-I-lipid complexes can directly give rise to a major plasma LpA-I subpopulation upon interaction with LCAT. Unlike apoA-I, apoA-II-lipid complexes could not serve as substrates for LCAT and did not undergo transformation. This study also demonstrates, for the first time, that apoA-II and apoA-I show a preference in phospholipid recruitment from membranes. Although phosphatidylcholine is the major phospholipid removed by both apolipoproteins, apoA-II preferentially recruits phosphatidylethanolamine (PE) as its second most abundant phospholipid while apoA-I recruits sphingomyelin. As PE is usually associated with the inner leaflet of the membrane, it is likely that dimeric apoA-II, compared with apoA-I, can penetrate farther into the membrane and extract PE. This ability of apoA-II to insert more deeply into the lipid milieu may explain the known ability of apoA-II to resist dissociation from the mature HDL particle.

Efflux of lipid from fibroblasts to apolipoproteins: dependence on elevated levels of cellular unesterified cholesterol.

Earlier work from this laboratory showed that enrichment of cells with free cholesterol enhanced the efflux of phospholipid to lipoprotein acceptors, suggesting that cellular phospholipid may contribute to high density lipoprotein (HDL) structure and the removal of sterol from cells. To test this hypothesis, we examined the efflux of [3H]cholesterol (FC) and [32P]phospholipid (PL) from control and cholesterol-enriched fibroblasts to delipidated apolipoproteins. The percentages of [3H]cholesterol and [32P]phospholipid released from control cells to human apolipoprotein A-I were 2.2 +/- 0.5%/24 h and 0.8 +/- 0.1%/24 h, respectively. When the cellular cholesterol content was doubled, efflux of both lipids increased substantially ([3H]FC efflux = 14.6 +/- 3.6%/24 h and [32P]PL efflux = 4.1 +/- 0.3%/24 h). Phosphatidylcholine accounted for 70% of the radiolabeled phospholipid released from cholesterol-enriched cells. The cholesterol to phospholipid molar ratio of the lipid released from cholesterol-enriched cells was approximately 1. This ratio remained constant throughout an incubation time of 3 to 48 h, suggesting that there was a coordinate release of both lipids. The concentrations of apoA-I, A-II, A-IV, E, and Cs that promoted half-maximal efflux of phospholipid from cholesterol-enriched fibroblasts were 53, 30, 68, 137, and 594 nM, respectively. With apoA-I and A-IV, these values for half-maximal efflux of phospholipid were identical to the concentrations that resulted in half-maximal efflux of cholesterol. Agarose gel electrophoresis of medium containing apoA-I that had been incubated with cholesterol-enriched fibroblasts revealed a particle with alpha to pre-beta mobility. We conclude that the cholesterol content of cellular membranes is an important determinant in the ability of apolipoproteins to promote lipid removal from cells. We speculate that apolipoproteins access cholesterol-phosphatidylcholine domains within the plasma membrane of cholesterol-enriched cells, whereupon HDL is generated in the extracellular compartment. The release of cellular lipid to apolipoproteins may serve as a protective mechanism against the potentially damaging effects of excess membrane cholesterol.

Assessment of [h]thymidine incorporation into DNA as a method to determine bacterial productivity in stream bed sediments.

We performed several checks on the underlying assumptions and procedures of the thymidine technique applied to stream bed sediments. Bacterial production rates were not altered when sediments were mixed to form a slurry. Incubation temperature did affect production rates. Controls fixed and washed with formaldehyde had lower backgrounds than trichloroacetic acid controls. DNA extraction by base hydrolysis was incomplete and variable at 25 degrees C, but hydrolysis at 120 degrees C extracted 100% of the DNA, of which 84% was recovered upon precipitation. Production rates increased as thymidine concentrations were increased over 3 orders of magnitude (30 nM to 53 muM thymidine). However, over narrower concentration ranges, thymidine incorporation into DNA was independent of thymidine concentration. Elevated exogenous thymidine concentrations did not eliminate de novo synthesis. Transport of thymidine into bacterial cells occurred at least 5 to 20 times faster than incorporation of label into DNA. We found good agreement between production rates of bacterial cultures based upon increases in cell numbers and estimates based upon thymidine incorporation and amount of DNA per cell. Those comparisons emphasized the importance of isotopic dilution measurements and validated the use of the reciprocal plot technique for estimating isotopic dilution. Nevertheless, the thymidine technique cannot be considered a routine assay and the inability to measure the cellular DNA content in benthic communities restricts the accuracy of the method in those habitats.

Efflux of phospholipid from fibroblasts with normal and elevated levels of cholesterol.

To address the hypothesis that phospholipid efflux from cells contributes to lipoprotein structure, we have examined the efflux of biosynthetically labeled [32P]phospholipid from cells to lipoproteins. With normal human skin fibroblasts in monolayer culture, high density lipoprotein (HDL3) promoted the efflux of phospholipid in a concentration-dependent manner. As analyzed by TLC, the major phospholipids released from fibroblasts were phosphatidylcholine, sphingomyelin, lysophosphatidylcholine and phosphatidylethanolamine. At identical concentrations, HDL3 and dimethylsuberimidate treated-HDL3 promoted similar efflux, suggesting that efflux did not depend on specific binding of HDL3 to the cell surface. When the content of cholesterol in fibroblasts was doubled by pre-incubation with LDL and cholesterol-rich liposomes, the fractional efflux of phospholipid to HDL3 and other acceptors was stimulated about 2-fold. Most of this stimulation was due to enhanced release of phosphatidylcholine. Similar effects of enrichment were found with Fu5AH rat hepatoma cells, but not with J774 mouse macrophages. The results support the hypothesis that the efflux of phospholipid from cells contributes to the phospholipid content of HDL. This may enhance the ability of HDL to remove cholesterol from cells, the initial step in reverse cholesterol transport.