Effects of Atorvastatin on the Intracellular Stability and Secretion of Apolipoprotein B in HepG2 Cells

From the Department of Chemistry and Biochemistry, University of Windsor, Windsor, Ontario, Canada (A.M., J.M., T.R., D.D., K.A.); and Parke-Davis Pharmaceutical Research, Warner-Lambert Co, Ann Arbor, Mich (R.N.).

Correspondence to Khosrow Adeli, Department of Chemistry and Biochemistry, University of Windsor, 401 Sunset Ave, Windsor, Ontario, Canada N9B 3P4. E-mail adeli{at}uwindsor.ca

	Abstract

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Abstract—We investigated the effects of atorvastatin, a new 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, on the biogenesis of apolipoprotein B (apoB) in intact and permeabilized HepG2 cells. Intact cells were pretreated either with single or multiple doses of atorvastatin (0.1 to 20 µmol/L) for periods of 6 to 20 hours and pulsed with [³⁵S]methionine. In some cases the cells were permeabilized with digitonin. Experiments were performed to investigate the effects of atorvastatin on (1) the rates of lipid synthesis and secretion, (2) the synthesis and accumulation of apoB, (3) the intracellular stability of apoB, (4) the amount of apoB-containing lipoprotein particles assembled in HepG2 microsomes, and (5) the secretion and accumulation of apoB into the culture medium. ApoB synthesis, degradation, and secretion were measured by pulse-chase experiments with [³⁵S]methionine in both intact and permeabilized HepG2 cells. Lipid synthesis was assessed by pulse-labeling experiments with [³H]acetate or [³H]oleate bound to bovine serum albumin. Comparisons were made under basal conditions and in the presence of oleate (0.36 µmol/L). Atorvastatin acutely inhibited the synthesis of cholesterol and cholesterol ester but did not have a significant effect on triglyceride or phospholipid synthesis. Atorvastatin did not affect the uptake of [³⁵S]methionine by the cells nor did it influence the synthesis of apoB or a control protein, albumin. However, atorvastatin reduced the secretion of apoB into the culture medium, apparently by enhancing the degradation of apoB in the cell under basal and induced conditions with oleate. The stability of apoB associated with the lipoprotein particles was also significantly lowered by atorvastatin. The stimulated degradation of apoB in atorvastatin-treated cells was sensitive to MG132, a proteasome inhibitor. The net effect of atorvastatin was a reduction in the number of apoB-containing lipoprotein particles of different sizes isolated from microsomes and a reduction in apoB secretion into the culture medium. The data suggest that atorvastatin may impair the translocation of apoB into the lumen of the endoplasmic reticulum, thus increasing the amount of apoB degraded intracellularly. It is hypothesized that atorvastatin alters these parameters primarily as a result of inhibiting cholesterol synthesis and limiting the availability of cholesterol and/or cholesterol ester for the normal assembly of apoB-containing lipoprotein particles.

Key Words: apolipoprotein B • atorvastatin • HMG-CoA reductase inhibitor • degradation • translocation • secretion

	Introduction

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Competitive inhibitors of HMG-CoA reductase can dramatically reduce plasma total and LDL cholesterol as well as LDL-apoB in subjects with either familial hypercholesterolemia^{1 2} or nonfamilial hypercholesterolemia.³ It is well accepted that these inhibitors act at least in part by upregulating LDL receptors in the liver, thereby facilitating the clearance of atherogenic apoB-containing lipoproteins. Recent evidence suggests that HMG-CoA reductase inhibitors may also reduce apoB secretion by limiting the availability of cholesterol derived from de novo synthesis.⁴ Because free cholesterol is necessary as a substrate for ACAT-derived cholesteryl esters in the hydrophobic core of lipoproteins, it has been hypothesized that either triglycerides or cholesteryl esters are obligatory for protecting apoB from intracellular proteolytic degradation.^{5 6 7 8} If certain inhibitors chronically inhibit cholesterol biosynthesis, there exists the potential for this pharmacological action to decrease apoB secretion. Indeed, apoB turnover studies in patients with combined hyperlipidemia and moderate hyperlipidemia have shown that HMG-CoA reductase inhibitors significantly reduce the in vivo production rate of apoB-containing lipoproteins by influencing both VLDL apoB secretion as well as direct production of LDL apoB.^{9 10}

Human apoB₁₀₀ is expressed exclusively in the liver. ApoB₁₀₀ is essential for the hepatic assembly and secretion of the triglyceride-rich lipoprotein particle VLDL. The formation of VLDL-apoB particles is a very complex process that requires the coordinate synthesis and assembly of apoB, triglycerides, cholesterol esters, phospholipids, and other components. Ample evidence suggests that acute modulation of apoB secretion is posttranscriptionally regulated.^{11 12 13 14 15} Control at the level of apoB translocation across the ER membrane has been suggested to be the key regulatory motif controlling the secretion of apoB-containing lipoproteins. Translocation efficiency and the rate of transport out of the ER may determine whether apoB is secreted or shunted into degradative pathways.^{16 17} Any apoB that is not translocated across the ER membrane and assembled into a lipoprotein particle is subsequently diverted for intracellular degradation.^{18 19} ApoB translocation is blocked in Chinese hamster ovary cells, suggesting that apoB requires a unique process for complete translocation that is not expressed in nonhepatic cells.²⁰ More recently, Rusiñol and coworkers^{21 22} showed that apoB translocation into the lumen of microsomes of rat hepatocytes is disrupted in membranes enriched in phosphatidyl-monomethylethanolamine and demonstrated that impaired translocation leads to increased degradation of the protein.²² Protease protection studies in permeabilized HepG2 cells²³ recently provided direct evidence that apoB translocation can be altered by oleate treatment of HepG2 cells as well as by agents such as DTT and cyclosporin A that affect the correct conformation of the apoB molecule.

Inefficient translocation of apoB appears to lead to its intracellular degradation. A significant proportion of newly synthesized apoB is rapidly degraded in rat hepatocytes^{17 18 24} and HepG2 cells.²⁵ ApoB degradation may occur early in the ER,¹⁸ and degradation of freshly translated apoB may regulate the proportion of apoB that enters the secretory pathway.^{17 26 27 28} In permeabilized HepG2 cells, degradation of apoB occurs by a pH- and temperature-dependent, calcium-independent, and ALLN-sensitive process.⁸ Degradation of apoB generates a distinct 70-kD fragment that is N-terminal in nature and is detectable in the lumen of the ER.^{8 29} The site of apoB degradation is generally thought to be the ER or a closely associated compartment,^{8 17 18 28} although three recent studies have reported post-ER degradation of apoB in insulin-treated rat hepatocytes,³⁰ choline-deficient rat hepatocytes,³¹ and glucagon-stimulated rat hepatocytes.³² Few data are available on the turnover of different intracellular pools of apoB₁₀₀ and the mechanisms involved. Studies by Boren et al^{33 34} and Cartwright et al³⁵ suggest that the membrane-bound apoB, as well as a fraction of the luminal apoB, may be sorted to degradation. Recent data from our laboratory confirm that both membrane-bound apoB and lipoprotein-associated apoB are subject to intracellular degradation, possibly involving distinct degradative mechanisms.³⁶ Yeung and coworkers³⁷ have recently demonstrated the involvement of the ubiquitin-dependent proteasome system in degradation of apoB. Recent data from two other laboratories provide further evidence for the role of the proteasome in apoB degradation.^{38 39}

In the present study, intact and permeabilized HepG2 cells have been used to investigate the effect of atorvastatin, a new HMG-CoA reductase inhibitor, on the intracellular degradation of apoB as well as the formation of apoB-containing lipoprotein particles in the lumen of the ER. Measurements of lipid synthetic rates, apoB degradation, the amount of luminal apoB particles, and secretion into the media provide evidence to support the possibility that atorvastatin may act to limit the hepatic production of apoB-containing lipoproteins, possibly by reducing the intracellular availability of cholesterol or cholesterol esters.

	Methods

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Materials
HepG2 cells (ATCC HB 8065) were obtained from American Type Culture Collection. Cell culture media, reagents, and fetal bovine serum (certified grade) were from Life Technologies. Culture dishes and flasks were obtained from Corning or Falcon. Digitonin (50% purity), leupeptin, pepstatin, ALLN, and other common laboratory reagents were from Sigma Chemical Co. Digitonin of a higher purity (100%) was obtained from Calbiochem. Ready Safe was from Beckman. Trasylol (aprotinin) was from Bayer. Ultrapure electrophoresis reagents were from Bio-Rad. L-[³⁵S]methionine (specific activity >1000 Ci/mmol), [³⁵S]protein labeling mixture (specific activity of >1000 Ci/mmol), and prestained protein standards (rainbow markers) were purchased from Dupont Canada. Monospecific apoB antibody was obtained from Medix-Biotech. Rabbit anti-goat IgG was from Sigma. Immunoprecipitin was obtained from Life Technologies. Enhance (fluorographic reagent) was from Dupont Canada and Amplify was from Amersham International.

Cell Culture
Monolayer cell cultures were maintained in -MEM in culture flasks or multiwell dishes containing 10% fetal calf serum.⁴⁰ Cells were grown in 35-, 60-, or 100-mm dishes at 37°C, 5% CO₂ in complete medium (-MEM, 10% fetal bovine serum). Cultures were allowed to reach 80% to 100% confluence before any experiment was performed.

Determination of Synthesis and Secretion of Cellular and Secreted Lipids
HepG2 cells were pulsed for 3 or 18 hours with 5 µCi/mL [³H]acetate to assess the rates of synthesis and secretion of cholesterol, cholesterol ester, and phospholipids. Triglyceride synthesis and secretion were monitored by labeling HepG2 cells for 3 to 5 hours with 5 µCi/mL [³H]oleate bound to BSA. After labeling was performed, cells were extracted with hexane:isopropanol (3:2), and the total lipid extract was dried, suspended in hexane, and applied to a thin-layer chromatogram. The TLC plates were developed using a two-solvent system to separate polar lipids with chloroform:methanol:acetic acid:formic acid:H₂O (70:30:12:4:2) and neutral lipids with petroleum ether:ethyl ether:acetic acid (90:10:1). The lipids were stained with iodine vapor and identified based on the use of a set of known lipid standards (Sigma). The spots identified on the TLC plates were cut and counted using a scintillation counter.

Note that the doses of atorvastatin used in this study (0.1 to 10 µmmol/L) relate closely to blood levels of atorvastatin in patients treated with 10 to 80 mg of the drug. Assuming complete absorption of the drug, 10 to 80 mg of atorvastatin (calcium salt) is estimated to result in blood levels of 1.8 to 18 µmol/L (in a 70-kg man with a blood volume of 5 L). Although the blood levels would decrease gradually after the absorption phase, it is likely that the concentration of the drug would remain high in hepatic tissue considering the liver selective nature of the drug.

Metabolic Labeling and Permeabilization of HepG2 Cells
Confluent HepG2 cultures grown in 100-mm dishes were depleted of methionine by incubation in methionine-free MEM for 60 minutes at 37°C under 5% CO₂. HepG2 cells were pulse-chased and made semipermeable as described below. Cells were incubated with 50 to 100 µCi/mL of [³⁵S]methionine, washed in Earle's balanced salt solution three times, and chased in complete medium containing 5 to 10 mmol/L methionine. After a washing with MEM, the cells were incubated in cytoskeletal (CSK) buffer (0.3 mol/L sucrose, 0.1 mol/L KCl, 2.5 mmol/L MgCl₂, 1 mmol/L Na-free EDTA, and 10 mmol/L PIPES, pH 6.8) containing 50 µg/mL of digitonin for 10 minutes. Digitonized cells were washed three times in CSK buffer and were used for the degradation studies.

Determination of ApoB Translocation Efficiency
To determine the efficiency of apoB translocation across the membrane of the ER, we used a protocol recently published by our laboratory.²³ Briefly, HepG2 cells were pulsed for 5 minutes with [³⁵S]methionine and then chased for various periods of time. At each time point, cells were permeabilized with digitonin (75 µg/mL) for 5 minutes, washed, and then treated with trypsin (200 µg/mL). After trypsinization for 10 minutes at room temperature, proteolysis was inhibited by the addition of a 10-fold excess of soybean trypsin inhibitor and the cells were solubilized for immunoprecipitation, which was performed as below. The protease inhibitor cocktail was included in all steps to prevent any residual trypsin activity.

Isolation and Analysis of ApoB-Containing Lipoproteins
Subcellular fractionation was performed as described.^{33 34 36 41 42} Briefly, permeabilized cells incubated for 0 to 2 hours were washed once with 250 mmol/L sucrose and 3 mmol/L imidazole, pH 7.4, and once with 50 mmol/L sucrose and 3 mmol/L imidazole, pH 7.4. The cells were then scraped in 0.5 mL of 50-mmol/L sucrose solution supplemented with a cocktail of protease inhibitors (0.1 mmol/L leupeptin, 1 mmol/L PMSF, 100 KIU/mL Trasylol (aprotinin), 1 µmol/L pepstatin A, and 5 µmol/L ALLN) and homogenized with a glass Dounce homogenizer as described.^{36 41 42} The homogenate was centrifuged for 10 minutes at 2200g, and the supernatant containing the crude microsomes was subjected to carbonate extraction followed by fractionation of membrane and luminal fractions by ultracentrifugation, as described.³² In some experiments, luminal contents isolated from microsomes were supplemented with protease inhibitors (0.1 mmol/L leupeptin, 1 mmol/L PMSF, 100 KIU/mL Trasylol, 1 µmol/L pepstatin A, and 5 µmol/L ALLN) and then subjected to ultracentrifugation on a step sucrose gradient (1.5 mL 49%/3.0 mL 25%/2.0 mL 20%/3.5 mL sample/1.9 mL 5%/0.9 mL 0% sucrose) at 35 000 rpm in a SW41 rotor for 65 hours at 12°C. All solutions contained the protease inhibitor cocktail as above. Gradients were fractionated into 1-mL fractions, and the density of the fractions was determined to ensure the linearity of the sucrose gradient.

Immunoprecipitation
Cell lysates or fractions collected from sucrose gradients were diluted with 800 µL of the solubilization buffer containing 360 µL 5XC buffer (250 mmol/L Tris/HCl [pH 7.4], 750 mmol/L NaCl, 25 mmol/L EDTA, 5 mmol/L PMSF, and 5% Triton X-100), 410 µL PBS, 20 µL Trasylol, and 10 µL PMSF and were preimmunoprecipitated by the addition of 2 µL preimmune serum. After 2 hours of incubation at room temperature, 60 µL immunoprecipitin was added, and samples were further incubated for 1 hour. Samples were cleared by centrifuging in a microfuge for 2 minutes, and the supernatant was subjected to specific immunoprecipitation with antibodies to apoB or albumin. Immunoprecipitation was performed by adding 10 µL of antibody or antiserum to each sample and incubating overnight at 4°C. The amount of antibody required was optimized to ensure quantitative immunoprecipitation of apoB from the HepG2 lysates. By testing increasing concentrations of the primary antibody, we found that 10 µL of apoB antiserum was sufficient to precipitate all of the radiolabeled apoB in the HepG2 lysate from a 35-mm culture dish. Immunoprecipitin (100 µL) was then added to each sample and further incubated at room temperature for 1 hour. Samples were centrifuged for 2 minutes at 14 000 rpm to pellet the immunoprecipitates. Immunoprecipitates were washed three times with the washing buffer (10 mmol/L Tris/HCl [pH 7.4], 2 mmol/L EDTA, 0.1% SDS, and 1% Triton X-100). Finally, the immunoprecipitates were prepared for SDS-PAGE by suspending and boiling in 100 µL of electrophoresis sample buffer (see below).

SDS-PAGE and Fluorography
SDS-PAGE was performed essentially as described.⁴³ Gels were composed of 5% (wt/vol) stacking and 6% (wt/vol) resolving gels or 3% to 15% gradient gels with a 5% stacking layer. Electrophoresis was at 66 V for 16 hours. The gels were fixed, stained, and fluorographed by incubation in Enhance or Amplify. The gels were dried and exposed to Kodak X-Omat AR5 film at -80°C for 1 to 4 days. Molecular weight markers (Sigma) were carbonic anhydrase (29 000), egg albumin (45 000), BSA (66 000), phosphorylase b (97 400), ß-galactosidase (116 000), and myosin (205 000).

To determine the radioactivity in apoB₁₀₀, the bands corresponding to the proteins visualized by fluorography were cut out of the gel and digested⁴⁴ and radioactivity was counted. In some cases, the apoB bands were scanned using a Bio-Rad imaging densitometer.

	Results

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Atorvastatin Acutely Inhibits Synthesis and Secretion of Cholesterol and Cholesterol Ester in HepG2 Cells
The effects of atorvastatin on the intracellular synthesis and extracellular secretion of lipids were determined by [³H]acetate- or [³H]oleate-labeling of HepG2 cells pretreated for 18 hours with various concentrations of atorvastatin. The radioactivity in various lipid fractions was determined by solvent extraction, TLC analysis, and scintillation counting. Fig 1 shows the level of newly synthesized lipids at various concentrations of atorvastatin. There was a dramatic decrease in the intracellular levels of cholesterol (Fig 1a) and cholesterol esters (Fig 1b) with increasing doses of atorvastatin (significantly lower at 1 µmol/L compared with 0 control, P<.05, n=3). Maximal inhibition of both free cholesterol and cholesterol ester levels was achieved at 1 µmol/L atorvastatin, and further increases in the dose did not have a further lowering effect on these lipid fractions. To assess the synthesis and secretion of triglycerides, cells were labeled with [³H]oleate bound to BSA. Interestingly, atorvastatin did not have an inhibitory effect on triglyceride accumulation in the cell (triglyceride levels at different doses not significantly different from 0 control, P>.05, n=3) (Fig 1c). Furthermore, atorvastatin at doses up to 5 µmol/L had no significant effect on the intracellular levels of phosphotidylethanolamine (P>.05, n=3), suggesting that the drug does not induce any significant change in phospholipid accumulation in HepG2 cells (Fig 1d). Note that although there was an apparent increase at 10 µmol/L, this change was not significant when compared with the level at 2.5 µmol/L (P>.05). Similarly, no significant changes were observed in phosphotidylcholine (data not shown).

View larger version (37K): [in this window] [in a new window]   Figure 1. Effect of atorvastatin on intracellular lipid synthesis in HepG2 cells. Near-confluent HepG2 cells were treated with 0.1 to 10 µmol/L atorvastatin for 18 hours and labeled for 3 hours with [3H]acetate or [3H]oleate bound to BSA. Lipids were extracted from cells and subjected to TLC analysis. The amount of radioactivity in various lipid fractions was estimated by scintillation counting of lipid spots identified on TLC plates (mean±SD, n=3). a, Cholesterol (CH); b, cholesterol ester (CE); c, triglyceride (TG); and d, phosphatidylethanolamine (PE).

Analysis of lipids in the culture media of HepG2 cells (Fig 2) showed that pretreatment with atorvastatin caused a similar pattern of changes as observed in the cells. Atorvastatin at a concentration of 1 µmol/L significantly (P<.05, n=3) inhibited the secretion of cholesterol and cholesterol ester (Fig 2a and 2b). However, atorvastatin had no appreciable effects on secretion of triglyceride (Fig 2c), phosphotidylethanolamine (Fig 2d), or phosphatidylcholine (Fig 2e). The data suggest that the atorvastatin-induced inhibition of intracellular cholesterol and cholesterol ester levels also results in a significant inhibition of their extracellular secretion into the culture medium.

View larger version (48K): [in this window] [in a new window]   Figure 2. Effect of atorvastatin on extracellular lipid secretion in HepG2 cells. Near-confluent HepG2 cells were treated with 1 µmol/L atorvastatin for 18 hours. Lipid secretion was monitored by labeling for either 18 hours with [3H]acetate or 5 hours with [3H]oleate bound to BSA. Lipids were extracted from media and subjected to TLC analysis. The amount of radioactivity in various lipid fractions was estimated by scintillation counting of lipid spots identified on TLC plates (mean±SD, n=3). a, Cholesterol (CH); b, cholesterol ester (CE); c, triglyceride (TG); d, phosphatidylethanolamine (PE); and e, phosphatidylcholine (PC). *Significantly different from control (P<.05, n=3).

Atorvastatin Does Not Alter Intracellular Synthesis of ApoB or Control Protein Albumin
To investigate the effect of atorvastatin treatment on the synthesis of apoB, a series of pulse-chase labeling experiments were performed using [³⁵S]methionine. First, the uptake of [³⁵S]methionine was compared between control cells and cells pretreated with atorvastatin for 18 hours. Fig 3a shows the total trichloroacetic acid–precipitable radioactivity recovered from HepG2 cells after a 10-minute pulse. There was no significant difference (P>.05, n=6) between the total amount of radiolabeled protein accumulated in control versus drug-treated cells, suggesting that atorvastatin treatment did not significantly influence the uptake of [³⁵S]methionine by HepG2 cells. Similarly, there was no appreciable effect on albumin synthesis, as shown in Fig 3b and 3c. The amount of radiolabeled albumin immunoprecipitated from the cells (quantified by cutting and scintillation counting of the bands) was similar between control cells and those treated with atorvastatin (Fig 3c) (P>.05, n=6). This again confirmed the notion that atorvastatin treatment of HepG2 cells does not alter total protein synthesis or the specific synthesis of individual proteins such as albumin. In addition, pulse-labeling of control and atorvastatin-treated HepG2 cells and immunoprecipitation of apoB also showed that the synthesis of apoB was not significantly affected with atorvastatin treatment (Fig 3b and 3d). There was no significant change (P>.05, n=6) in cellular apoB (quantified by cutting and scintillation counting of the bands), suggesting that atorvastatin does not alter the level of apoB synthesis under these conditions.

View larger version (40K): [in this window] [in a new window]   Figure 3. Atorvastatin does not alter the synthesis of apoB or of the control protein albumin. Near-confluent HepG2 cells were treated with 10 µmol/L atorvastatin for 18 hours and then labeled for 15 minutes with [35S]methionine. a, Total amount of radiolabeled proteins was assessed by solubilizing the cells and determining the amount of trichloroacetic acid–precipitable radioactivity. b, Cell extracts were also subjected to immunoprecipitation with a monospecific antibody against albumin or apoB, and the immunoprecipitates were analyzed by SDS-PAGE and fluorography. c and d, The albumin or apoB bands were cut out of the gel, solubilized, and counted using a scintillation counter. The quantification of albumin and apoB bands (mean±SD, n=6) is shown in c and d, respectively.

Atorvastatin Inhibits Secretion of ApoB and Reduces Its Intracellular Stability
We initially determined the effect of atorvastatin on the secretion of apoB in intact HepG2 cells that were treated overnight with a single dose of the drug. Fig 4a shows the amount of apoB secreted over 2 hours in the presence and absence of atorvastatin. There was a small but significant decrease in the amount of apoB secreted in drug-treated cells (P<.05, n=3). A similar pulse-chase labeling experiment was performed to assess the stability of apoB in the presence of atorvastatin. ApoB stability was determined by comparing the amount of apoB recovered from control and atorvastatin-treated cells after a 2-hour chase as a percentage of the amount recovered at the beginning of the chase (percentage of the 0 time for control and atorvastatin-treated cells, respectively). As shown in Fig 4b, there was a substantial decrease (significantly different, P<.05, n=3) in the amount of apoB recovered in atorvastatin-treated cells, suggesting a higher level of intracellular degradation in the presence of the drug.

View larger version (41K): [in this window] [in a new window]   Figure 4. Effect of atorvastatin on apoB secretion and stability in intact HepG2 cells. Near-confluent HepG2 cells were treated with 10 µmol/L atorvastatin for 18 hours in complete MEM. Cells were pulsed for 15 minutes with [35S]methionine and were chased for 2 hours with excess cold methionine. Media and cells were collected, and apoB was immunoprecipitated with a specific anti-apoB antibody followed by SDS-PAGE and fluorography. Quantification of apoB was performed either by scintillation counting of the apoB100 band or by densitometric scanning. a, Immunoprecipitable apoB recovered in media of control and atorvastatin-treated HepG2 cells. b, ApoB stability in HepG2 cells expressed as the percentage of apoB remaining after a 2-hour chase in control and atorvastatin-treated cells (mean±SD, n=3). *Significantly different from control (P<.05).

Atorvastatin Partially Prevents the Stimulatory Effect of Oleate on ApoB
We also determined the effect of atorvastatin on the secretion of apoB in oleate-treated HepG2 cells that had been treated overnight with a single dose of atorvastatin. Fig 5a and 5b demonstrate the amount of apoB secreted over 2 hours in the presence and absence of atorvastatin. ApoB secretion and degradation were assessed in the presence of media alone, media+BSA, media+BSA/oleate, and media+BSA/oleate+atorvastatin (a representative autoradiograph is shown in Fig 5a, and quantification is shown in Fig 5b). Treatment with BSA did not significantly affect the secretion of apoB compared with control cells. However, there was a 2.3-fold stimulation of apoB secretion in cells incubated with BSA/oleate (13.19±0.87, n=4) compared with BSA-treated cells (5.79±0.37, n=4). Incubation with both oleate/BSA and atorvastatin resulted in a lower stimulation of apoB secretion (10.46±0.43, n=4). There was a significant decrease (P<.005) in the amount of immunoprecipitable apoB in cells treated with both the drug and oleate/BSA when compared with that in cells treated with oleate/BSA alone. Treatment with the inhibitor therefore appeared to partially abolish the stimulatory effect of oleate on apoB secretion.

View larger version (27K): [in this window] [in a new window]   Figure 5. Effect of atorvastatin on apoB secretion and degradation in oleate-treated HepG2 cells. Near-confluent HepG2 cells were treated with 10 µmol/L atorvastatin for 18 hours in the presence or absence of BSA-bound oleate (360 µmol/L). Cells were pulsed for 15 minutes with [35S]methionine and chased for 2 hours with excess cold methionine. Media and cells were collected, and apoB was immunoprecipitated with a specific anti-apoB antibody followed by SDS-PAGE and fluorography. a, Representative fluorograph showing the apoB immunoprecipitated from cells treated with media, media+BSA, media+BSA/oleate, and media+BSA/oleate+atorvastatin in duplicate. Quantification of apoB was performed either by scintillation counting of the apoB band or by densitometric scanning. b, The amount of apoB secreted in cells treated with media, media+BSA, media+BSA/oleate, and media+BSA/oleate+atorvastatin. c, Percentage of apoB remaining after a 2-hour chase in intact cells treated with media, media+BSA, media+BSA/oleate, and media+BSA/oleate+atorvastatin (mean±SD, n=4). *Significantly different from cells treated with BSA/oleate but not atorvastatin (P<.05).

To determine whether the effect of atorvastatin was partially at the level of apoB degradation, we performed a pulse-chase experiment in intact HepG2 cells (Fig 5c). Cells were pretreated with oleate (with or without a single dose of the inhibitor), pulsed for 10 minutes, and then chased for 0 or 2 hours. ApoB was immunoprecipitated at both 0 time and 2 hours, and the extent of apoB degradation was estimated by determining the percentage of apoB remaining after the 2-hour chase. As expected, oleate treatment resulted in a significant elevation (4.1-fold) in the percentage of apoB remaining in cells chased for 2 hours versus that in control cells. This suggests oleate-triggered inhibition of apoB degradation. However, the percentage of apoB remaining was significantly lower in cells treated with BSA/oleate+atorvastatin compared with BSA/oleate-treated cells (P<.05, n=3). This suggests that atorvastatin destabilizes the newly synthesized apoB in oleate-treated cells, resulting in a higher rate of degradation in the cell and a decreased rate of secretion.

Effect of Atorvastatin on ApoB Stability in Permeabilized HepG2 Cells
To further investigate the effect of atorvastatin on apoB degradation, we performed several degradation experiments in cells permeabilized with digitonin. We previously used permeabilized HepG2 cells to investigate apoB degradation and showed the usefulness of this system for studying the degradation process. It is important to note that the necessary control experiments showing the integrity of microsomal membranes in digitonin-permeabilized HepG2 cells previously have been published.^{8 23 36} Intact HepG2 cells were treated with atorvastatin, pulsed with [³⁵S]methionine, briefly chased, and permeabilized with digitonin. Permeabilized cells were incubated in buffer, and degradation was monitored by immunoprecipitation of apoB before and after a 2-hour chase. Atorvastatin-treated cells contained significantly lower amounts of immunoprecipitable apoB at the end of the 2-hour chase, suggesting that atorvastatin reduced stability and increased intracellular degradation of apoB. There was a 42% decrease (average of two experiments) in immunoprecipitable apoB remaining in atorvastatin-treated cells (apoB recovered in control cells was 2250±76 cpm per dish versus 1226±84 cpm per dish in atorvastatin-treated cells.

To assess whether the effect of atorvastatin on apoB degradation in permeabilized cells is dose dependent, HepG2 cells were treated with multiple doses of atorvastatin at two different concentrations (10 and 20 µmol/L). Fig 6a and 6b show the effect of different doses of atorvastatin on the immunoprecipitable apoB recovered in permeabilized cells (Fig 6a, representative fluorograph; Fig 6b, quantification of apoB degradation expressed as percentage of apoB remaining). Drug treatment at 20 µmol/L every 3 hours (4 doses) decreased the percentage of apoB remaining by 47%, compared with a 24% decrease at a multiple dose of 10 µmol/L (significantly different, P<.05, n=3). Atorvastatin-mediated stimulation of apoB degradation was therefore concentration dependent.

View larger version (47K): [in this window] [in a new window]   Figure 6. Effect of atorvastatin on apoB degradation in permeabilized HepG2 cells. Near-confluent HepG2 cells were treated with 10 or 20 µmol/L atorvastatin for 6 or 18 hours. Cells were pulsed for 15 minutes with [35S]methionine and chased for 10 minutes with excess cold methionine. Cells were then permeabilized with digitonin (50 µg/mL) for 10 minutes, and the permeabilized cells were incubated in a cytoskeletal buffer for 1 and 2 hours before immunoprecipitation of the cells with a specific anti-apoB antibody. Immunoprecipitates were analyzed by SDS-PAGE and fluorography. a and c, Representative fluorographs of apoB recovered in cells treated with atorvastatin at different doses (a) or different times (c). ApoB radioactivity was quantified by cutting and scintillation counting of the bands, and apoB degradation was assessed by calculating the percentage of apoB remaining in cells under various conditions. b, Percentage of apoB remaining after a 2-hour chase in control cells and cells treated with atorvastatin at 2 different doses (10 and 20 µmol/L) (mean±SD, n=3). d, Percentage of apoB remaining in cells treated with 10 µmol/L atorvastatin for either 6 or 20 hours (mean±SD, n=3). *Significantly different from control (P<.05).

The effect of atorvastatin on apoB degradation was also determined in cells treated for 6 versus 20 hours. Cells pretreated for either 6 or 20 hours were pulsed, briefly chased, and permeabilized with digitonin. Permeabilized cells were incubated in buffer to monitor apoB degradation. Fig 6c and 6d show the effect of atorvastatin at different times of drug pretreatment (Fig 6c, representative fluorograph; Fig 6d, quantification of apoB degradation expressed as the percentage of apoB remaining). Treatment at either 6 or 20 hours resulted in a significant decrease (P<.05, n=3) in the percentage of apoB remaining (expressed as the apoB radioactivity remaining at 2 hours in control and atorvastatin-treated cells as a percentage of the 0 time radioactivity for control and atorvastatin-treated cells, respectively). The data suggest an enhancement of apoB degradation with atorvastatin. Drug treatment for both time periods appeared to have similar effects on apoB stability, indicating that atorvastatin elicited its effect within 6 hours of preincubation.

The rate of apoB degradation in permeabilized HepG2 cells was also monitored in both control and atorvastatin-treated cells. Treatment with the inhibitor appeared to accelerate the degradation rate of apoB. Over the first hour of chase, the percentage of apoB radioactivity remaining was 72.75±3.93 (n=3) in control cells and 49.61±1.96 (n=3) in drug-treated cells. The rate of degradation during the first hour of chase was approximately twofold greater in atorvastatin-treated cells (based on regression analysis, the slope of apoB loss in control cells was –27.2 compared with –50.4 for drug-treated cells). The rise in apoB degradation rate also correlated well with the data presented above, indicating a 24.7% decline in the total apoB radioactivity after a 2-hour chase period.

Effect of MG132, a Proteasome Inhibitor, on Degradation of ApoB in Atorvastatin-Treated Cells
Interestingly, the degradation of apoB in atorvastatin-treated cells was sensitive to MG132, a potent inhibitor of the proteasome (Fig 7). Treatment of HepG2 cells with MG132 abolished the stimulated degradation of apoB observed in atorvastatin-treated cells. To study the sensitivity to MG132, HepG2 cells were pretreated with atorvastatin or atorvastatin+MG132, pulsed, and chased for 2 hours in the absence or presence of the protease inhibitor (Fig 7). The percentage of apoB remaining was then calculated from immunoprecipitable apoB recovered at 0 and 2 hours (46.5±1.3% in control cells, 69.6±2.6% in MG132-treated cells, 36.5±2.0% in atorvastatin-treated cells, and 72.8±4.0% in atorvastatin+MG132-treated cells; n=3). As expected, atorvastatin treatment induced a significant decrease in the percentage of apoB remaining. However, there was no significant difference in the percentage of apoB remaining in MG132- versus atorvastatin+MG132-treated cells. Thus, the presence of MG132 inhibited apoB degradation and appeared to abolish the stimulated degradation of apoB in atorvastatin-treated cells. The data suggest that apoB degradation in atorvastatin-treated cells is mediated by a MG132-sensitive pathway, most likely the proteasome.

View larger version (49K): [in this window] [in a new window]   Figure 7. Effect of MG132 on apoB degradation in control and atorvastatin-treated cells. Near-confluent HepG2 cells were treated with 10 µmol/L atorvastatin for 18 hours. One hour before the pulse, cells were incubated with MG132 (25 µmol/L). Cells were then pulsed for 15 minutes with [35S]methionine and chased for 2 hours with excess cold methionine in the presence or absence of MG132. Media and cells were collected, and apoB was immunoprecipitated with a specific anti-apoB antibody followed by SDS-PAGE and fluorography. ApoB stability was compared among the various conditions by calculating the percentage of apoB remaining (cells+media) at 2 hours as a percentage of the radioactivity in cells at 0 hours. *Significantly different from control cells (P<.05). **Not significantly different from MG132-treated cells.

Atorvastatin Inhibits Translocation of ApoB into the Secretory Pathway
We also attempted to determine the effect of atorvastatin on the translocational efficiency of apoB across microsomal membranes. Using a permeabilized cell translocation assay,²³ we determined the level of trypsin-resistant apoB and used it as a measure of the efficiency of protein translocation across the membrane of the ER. In this assay, intact HepG2 cells were briefly pulsed, chased for various periods of time, and then permeabilized with digitonin. Permeabilized cells were then treated with exogenous trypsin to hydrolyze apoB nascent chains that were not translocated. Immunoprecipitation of apoB from control and trypsin-treated permeabilized cells enabled us to determine the amount of full-length apoB fully translocated into the ER lumen. The assay is therefore capable of measuring the efficiency of accumulation of fully translocated apoB, which is known to be secretion-competent and a key determinant of the rate of apoB secretion from the cell. Results of all the appropriate control experiments for the translocation protocol have recently been published²³ and include evidence showing the intactness of microsomal membranes in digitonin-permeabilized HepG2 cells. In addition, treatment with atorvastatin does not alter the integrity of microsomal membranes in permeabilized HepG2 cells, as determined by monitoring the activity of NADH–cytochrome c reductase, as described.⁸ Trypsin digestion of apoB in permeabilized cells is also specific, since other control secretory proteins were not susceptible to trypsin digestion.²³ Fig 8a shows the rate of apoB translocation in HepG2 cells pretreated with oleate and ALLN to maximize the translocation efficiency. In this assay, an increasing amount of apoB becomes protected from trypsin digestion as a function of time, suggesting a gradual translocation of the protein into the luminal compartment of the ER, as reported previously.²³

View larger version (80K): [in this window] [in a new window]   Figure 8. Determination of the efficiency of translocation of apoB in control and atorvastatin-treated cells. a, Basal rate of apoB translocation across the ER membrane. Near-confluent HepG2 cells were pulsed for 5 minutes with [35S]methionine and chased for various time periods with excess cold methionine. Cells were then permeabilized with digitonin (75 µg/mL) for 5 minutes, and the permeabilized cells were incubated with trypsin to digest any untranslocated apoB chains. ApoB was immunoprecipitated, the immunoprecipitates were analyzed by SDS-PAGE and fluorography, and apoB radioactivity was quantified by cutting and scintillation counting of the apoB100 band. b, Near-confluent HepG2 cells were treated with or without 10 µmol/L atorvastatin for 24 hours and were pulsed for 5 minutes, chased for 30 minutes, and permeabilized as in panel a. Cells were trypsinized, and apoB was immunoprecipitated and analyzed by SDS-PAGE and fluorography. ApoB radioactivity was quantified by cutting and scintillation counting of the apoB100 band. *Significantly different from control cells treated with trypsin but not atorvastatin (P<.05) (mean±SD, n=4). c, Representative fluorograph of the apoB translocation experiment in control and atorvastatin-treated cells. Arrowhead indicates the position of the 550-kD apoB100 band.

Pretreatment of HepG2 cells with the HMG-CoA reductase inhibitor before the translocation assay was performed resulted in a significant reduction in the accumulation of trypsin-resistant apoB. Fig 8b shows the translocational efficiency of apoB in control and drug-treated HepG2 cells expressed as the percentage of apoB protected, and Fig 8c shows a representative fluorograph. There was a significant decline in the amount of trypsin-resistant or protected apoB₁₀₀ in cells pretreated with atorvastatin. In translocation experiments, there was an average of 61.7% decrease in trypsin-resistant apoB after a 30-minute chase (n=4, P<.05). It is also important to note that there was a reduction in the amount of trypsin-protected fragments in atorvastatin-treated cells in addition to a reduced level of intact apoB₁₀₀ (Fig 8c). Together, the above data suggest that apoB translocation across microsomal membrane and the accumulation of luminal apoB are significantly hampered in atorvastatin-treated HepG2 cells.

Atorvastatin Inhibits Intracellular Formation of ApoB-Containing Lipoprotein Particles
The effect of atorvastatin on the assembly of lipoprotein particles in HepG2 cells was also investigated. HepG2 cells were pulsed, briefly chased, and permeabilized with digitonin. Permeabilized cells were incubated for various times and then subjected to subcellular fractionation. Luminal lipoprotein particles were isolated from total microsomes and then analyzed by sucrose gradient ultracentrifugation and immunoprecipitation. As depicted in Fig 8, there was a considerably lower amount of apoB-containing lipoprotein particles in the lumen of microsomes isolated from atorvastatin-treated cells both at 0 time (Fig 9a) and after a 2-hour chase (Fig 9b), suggesting that (1) fewer apoB lipoprotein particles accumulated in the microsomal lumen and (2) luminal apoB lipoprotein particles were less stable in drug-treated cells. Fractions 2 through 5 represent high-density apoB lipoprotein particles (apoB lipoproteins with density similar to that of HDL, peak density 1.065 to 1.170 g/mL), and fractions 6 through 12 represent the lower-density apoB lipoprotein particles (LDL/VLDL-apoB, peak density 1.011 to 1.045 g/mL) (see References 33 and 34^{33 34} ). Therefore, we also observed a reduction in the accumulation of apoB-containing lipoprotein particles in the lumen of the ER associated with the observed increase in apoB degradation and a decrease in apoB translocation.

View larger version (29K): [in this window] [in a new window]   Figure 9. Distribution of luminal apoB-containing lipoproteins in control and atorvastatin-treated cells. HepG2 cells were preincubated with or without atorvastatin (10 µmol/L) for 18 hours. Cells were then pulsed for 15 minutes with [35S]methionine, briefly chased with excess cold methionine, and permeabilized with digitonin (50 µg/mL) for 10 minutes. Permeabilized cells were incubated in a cytoskeletal buffer for 0 (a) or 2 hours (b) before homogenization and fractionation of microsomes. Luminal lipoproteins were extracted from microsomes by carbonate treatment and separated from the membrane fraction by centrifugation (SW55 rotor; 35 000 rpm, 93 minutes). Fractionation of luminal lipoproteins was performed by sucrose gradient centrifugation (SW41 rotor; 35 000 rpm, 65 hours). After centrifugation, gradient fractions were collected and immunoprecipitated with a monospecific anti-apoB antibody. Immunoprecipitates were analyzed by SDS-PAGE and fluorography, and apoB radioactivity was quantified by cutting and scintillation counting of the apoB100 band. a, Luminal lipoproteins at 0 hours; b, luminal lipoproteins after 2-hour chase. Both a and b show the results of a representative experiment (two others performed).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Numerous evidence suggests that, once synthesized, apoB is subjected to posttranslational regulation, a process that is closely linked to the lipid status of the cell. The supply of one or more of the core lipids (triglycerides and cholesterol ester) in apoB-containing lipoproteins plays an important role in regulating the assembly and secretion of these lipoproteins. Human liver can apparently "vary the type and quantity of apoB particles secreted in response to the load and type of lipid it has received."⁴⁵ Increased delivery of either fatty acids or sterols, or both, to the liver may result in overproduction of apoB particles.⁴⁶ The amount of newly synthesized apoB used for secretion may be modulated by the available supply of triglycerides,^{26 46 47 48} cholesterol ester,^{4 5 6} or possibly specific pools of phospholipids.⁴⁹ Some studies have suggested that increased synthesis of triglyceride may upregulate apoB secretion by increasing the recruitment of the ER-translocated apoB to form mature lipoproteins,^{47 48} while in other studies increased cholesterol ester synthesis rather than triglyceride synthesis has been suggested as the immediate regulator of apoB secretion.^{4 5 25} Overall, these studies suggest that apoB may be made in surplus and its secretion rate may be decided by the availability of the intracellular lipid supply.

Intracellular lipid pools most likely regulate apoB production via posttranslational mechanisms that may involve facilitating the translocation of newly synthesized apoB across the ER membrane and reducing apoB degradation or enhancing its assembly into secretion-competent lipoprotein particles. In the present study, we used atorvastatin, a new HMG-CoA reductase inhibitor with potent inhibitory effects on the intracellular rate of cholesterol synthesis, to investigate the effect of inhibition of cholesterol synthesis on apoB translocation into the ER and its intracellular degradation. Atorvastatin has been shown to decrease cholesterol synthesis to a considerable extent in vitro, ex vivo, and in vivo.^{50 51 52 53} Results obtained in our laboratory also demonstrated that treatment of HepG2 cells with atorvastatin under basal or lipid-rich conditions results in a significant reduction in cholesterol, cholesterol ester, and apoB secretion. Several previous in vitro studies have been performed to investigate the effect of HMG-CoA reductase inhibitors on apoB secretion and have yielded conflicting results. Ribeiro et al⁵⁴ studied the effect of simvastatin on primary cultures of rat hepatocytes and reported stimulation of apoB secretion. On the other hand, Sato et al²⁵ found that treatment of HepG2 cells by CS-514 did not influence the synthesis and secretion of apoB. Our observations that atorvastatin inhibits apoB secretion in HepG2 cells compare with the results of two recent in vivo studies in miniature pigs and guinea pigs. Huff and coworkers⁵⁵ recently used miniature pigs to demonstrate that atorvastatin reduced plasma cholesterol by inhibiting the hepatic secretion of VLDL apoB.⁵⁵ Similarly, studies by Fernandez and coworkers⁵⁶ also showed a significant reduction in hepatic VLDL apoB secretion rates in guinea pigs treated with atorvastatin. These in vivo studies support our in vitro evidence showing a reduction in apoB secretion with atorvastatin treatment.

Previous in vitro studies of statins have mostly focused on the effect of these inhibitors on the secretion of apoB and have not explored the intracellular mechanisms by which these drugs may exert their effects on the production of apoB-containing lipoproteins. To further investigate the mechanism of atorvastatin action on apoB secretion, we used both intact cells as well as a semipermeable HepG2 system.^{8 23 29 36} Results from these experiments appeared to suggest that atorvastatin decreased apoB secretion by stimulating its degradation in HepG2 cells. Further investigation showed that atorvastatin may stimulate apoB secretion by inhibiting apoB translocation across the ER membrane, which in turn may result from depletion of cholesterol or cholesterol ester. Atorvastatin showed its stimulatory effect on apoB degradation under basal as well as oleate-treated conditions. However, the drug was clearly more effective in reducing apoB secretion when HepG2 cells were enriched in lipids by pretreatment with oleate. This is not unexpected because apoB secretion, which is very low under basal conditions in HepG2 cells, is significantly increased with oleate treatment. Atorvastatin appears to interfere more effectively with lipoprotein secretion under such stimulated conditions.

Our results regarding the effect of atorvastatin on the translocation of apoB are consistent with current research, which suggests that translocation may be a key regulatory point in apoB secretion.^{17 18 19 20 22 57} Evidence that the actual process of apoB translocation across the ER may be a crucial regulatory point in the production of lipoproteins has recently been suggested by Rusiñol and Vance.²² They have shown that the supplementation of primary rat hepatocytes with phosphatidyl-monomethylethanolamine decreased the secretion of apoB. This decrease was attributed to a decrease in the translocation of apoB and was independent of lipid availability. Furthermore, Bonnardel and Davis⁵⁷ recently used HepG2 cells to demonstrate that apoB translocation rather than degradation may be the primary mechanism that regulated apoB secretion. Recent data from our laboratory²³ have also shown that the efficiency of apoB translocation can be modulated by lipid availability as well as by altering the conformation of the nascent protein with agents such as DTT and cyclosporin. There is considerable evidence to suggest the existence of two distinct pools of apoB in the ER: a luminal (trypsin-resistant) apoB pool, which is used for the assembly of lipoproteins, and a membrane-bound (trypsin-susceptible) apoB pool, which is thought to be shunted to a degradative pathway.^{17 26 55} The membrane-bound apoB pool may also act as a precursor for the formation of dense apoB particles in the ER lumen.³⁶ Our data in the present report demonstrate that atorvastatin reduces the amount of apoB that is translocated into the lumen of the ER. An alternative explanation for the apparent effect on apoB translocation is that atorvastatin induces intraluminal degradation of apoB and therefore reduces the amount of luminal apoB. This possibility is unlikely given that the translocation experiment was performed in the presence of ALLN, which appears to inhibit both proteasome-mediated apoB degradation³⁷ as well as degradation of apoB in the ER lumen.^{8 36} We suggest that the reduced translocation may result from the depletion of cholesterol or cholesterol ester from HepG2 cells. This depletion of lipid may reduce the number of apoB-containing lipoprotein particles and increase the amount of superfluous apoB. Several studies have demonstrated the importance of lipid availability for the translocation of apoB.^{23 33 34} The presence of oleate has been suggested to facilitate the translocation of apoB into the lumen and thereby reduce the percentage of apoB that becomes prone to degradation.^{26 36}

The data demonstrating that HepG2 cells incubated in the presence of atorvastatin contained a significantly lower amount of luminal apoB-containing lipoproteins further support the suggestion that atorvastatin may hamper apoB translocation across the ER membrane and reduce the amount of apoB molecules available for lipoprotein assembly. Fractionation of luminal apoB into dense (HDL-like) and light (LDL/VLDL-like) particles^{33 34} showed that both fractions undergo a higher level of degradation in the presence of atorvastatin. However, the HDL-like particles showed a more profound susceptibility to degradation under conditions of atorvastatin treatment. This effect could possibly be exerted through inhibition of cholesterol synthesis and reduction in the level of cholesterol ester pool available for lipoprotein assembly. The exact mechanism(s) for the effect of atorvastatin on apoB degradation remains to be elucidated through further investigations. In preliminary experiments, we found that apoB degradation in atorvastatin-treated cells was sensitive to MG132, a potent inhibitor of the proteasome. This protease inhibitor appeared to normalize the stability of apoB in atorvastatin-treated cells. The data suggest that the stimulated degradation of apoB on atorvastatin treatment can be prevented by inhibition of the proteasome. This finding is in agreement with the hypothesis that atorvastatin treatment may result in inhibition of apoB translocation leading to an increased proportion of membrane-associated apoB chains and their degradation by the cytosolic proteasome.

Overall, the results of our study further support the clinical evidence that atorvastatin decreases the production of LDL-apoB. This effect may be occurring at several levels. Atorvastatin has been shown to decrease the translocation of apoB into the lumen and increase its intracellular degradation. This increased degradation may be the result of the impaired translocation. Taken together, the data suggest that atorvastatin may decrease plasma LDL-apoB not only through upregulation of LDL receptors but also through a decrease in the hepatic production of apoB-containing lipoproteins.

	Selected Abbreviations and Acronyms

 

-MEM = modification of Eagle's minimal essential medium ACAT = acyl CoA:cholesterol acyltransferase ALLN = N-acetylleucylleucylnorleucinal ER = endoplasmic reticulum HMG-CoA = 3-hydroxy-3-methylglutaryl coenzyme A TLC = thin-layer chromatography

	Acknowledgments

 
This study was supported by grants from the Heart and Stroke Foundation of Ontario and Parke-Davis (to K.A.). We gratefully acknowledge the excellent technical assistance of Debbie Rudy and Andrea Aiton.

Received June 10, 1997; accepted December 12, 1997.

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