Changes in cultured arterial smooth muscle cells isolated from chicks upon cholesterol feeding

We have developed cultures of smooth muscle cells (SMC) isolated from arterial hypercholesterolemic chicks (cholesterol-SMC). These cultures are suitable for the study at the molecular level of the changes in arterial SMC induced by a cholesterol diet. By using a strong dose of cholesterol (5%) for 10 d, we obtained very proliferative SMC which became foam cells after 30 d in culture. On the other hand, SMC cultures isolated from control-fed chicks has a lower growth rate than the SMC ones under the same culture conditions. DNA synthesis was fourfold greater in cholesterol-SMC than in control-SMC cultures. Intracellular cholesterol concentrations were the same in both cholesterol and control SMC during the first 14 d of culture but afterward increased in differing ways: after 20 d of culture the cholesterol-SMC increased their cholesterol content to double the control. We give here the results obtained from transmission electron microscopy, lipid analysis, proliferation studies, DNA, RNA and protein synthesis, and then discuss their implications.     

Purification and properties of aortic cholesteryl ester hydrolase

The enzyme(s) present in acetonedried powder of rat and rabbit aortas, which catalyzes the synthesis and hydrolysis of cholesteryl ester, was purified partially by acid precipitation, acetone fractionation, 0-(diethylaminoethyl) cellulose chromatography, and Sephadex G-100 filtration. The synthetic activity was purified by 120-fold (rat) and 140-fold (rabbit). Purification of hydrolytic activity was 90-fold (rat) and 103-fold (rabbit). Cholesteryl ester hydrolase activity was separated from nonspecific, esterase by column chromatography. Both synthetic and the hydrolytic activities are apprently the functions of one enzyme. The mol wt of the enzyme was estimated to be 140,000 dalton as determined by Sephadex G-200 gel filtration. The extracts of the acetone-dired powders of aortas of both species contained an inhibitor of synthetic activity. The inhibitor was nondialyzable and was precipitated at pH 5.7 Both activities were found to be fairly nonspecific with regard to sterol and fatty acids. With oleic acid, the relative rates of sterol ester synthesis were: cholesterol, 100; cholestanol, 94; desmosterol, 35; corprostanol, 24; ergosterol, 20; and β-sitosterol, 19. Epicholesterol was not esterified. Oleic acid was most active in cholesteryl ester synthesis, the relative rates being: oleic>linoleic>arachidonic>palmitic>stearic>butyric. The rate of hydrolysis was maximum with cholesteryl linoleate followed by oleate, linolenate, palmitate, stearate and laurate in decreasing order.     

Effects of purified eicosapentaenoic acid on arachidonic acid metabolism in cultured murine aortic smooth muscle cells,vessel walls and platelets

The effects of highly purified eicosapentaenoic acid (97% pure) on the arachidonic acid cascade in isolated murine vascular cells and platelets were studied. The incorporation of eicosapentaenoic acid was not as active as that of arachidonic acid in platelets. The ratio of incorporation of eicosapentaenoic acid to arachidonic acid into platelet phospholipids was about 0.7. Analysis of the phospholipid fractions of platelets after labeling with¹⁴C-eicosapentaenoic acid and¹⁴C-arachidonic acid revealed that the incorporation of¹⁴C-eicosapentaenoic acid into the phosphatidylinositol fraction is significantly less than that of¹⁴C-arachidonic acid, while the incorporation of both fatty acids into other phospholipid fractions was almost the same. On the other hand, no significant difference between either fatty acid in incorporation rate, kinetics or distribution in cellular phospholipids was found in cultured aortic smooth muscle cells. Following treatment with eicosapentaenoic acid, cells produced less prostacyclin from endogenous arachidonic acid than did control cells. This was not due to the decrease in fatty acid cyclooxygenase activity, but rather, due to the decrease in arachidonic acid content in cellular phospholipids. In addition, eicosapentaenoic acid was neither converted to prostaglandin I₃ by the vascular cells nor to thromboxane A₃ by platelets. Furthermore, similar results were also obtained by in vivo experiments in which rats were fed with eicosapentaenoic acid enriched diet.     

Fatty acid and sterol specificity of cholesterol esterifying enzyme in developing rat brain

The properties and fatty acid and sterol specificity of cholesterol-esterifying enzyme (EC 3.1.1.13) in rat brain were studied. The enzyme utilized free fatty acid for esterification, and activity was maximal at pH 5.6. Exogenous ATP and CoA did not stimulate the incorporation of free fatty acids into sterol esters. Substrates dispersed in Tween 20 or Triton X-100 were just as effective as the substrates dissolved in acetone solution, while dispersion in propylene glycol or sodium taurocholate was not as effective. Snake venom phospholipase A₂ (EC 3.1.1.4) increased the esterification of cholesterol in the absence of added fatty acid. The fatty acid specificity data indicated that oleic and palmitic acids were the preferred fatty acids. Little or no esterification occurred in the presence of long chain fatty acids (C₂₀–C₂₄). Esterification of cholesterol with palmitate or stearate was not affected by the presence of oleic acid in the mixture. Thus, the nonrequirement of the brain-esterifying enzyme for a bile acid or for an amphiphile such as an unsaturated fatty acid suggests that micellar solubilization of the substrate is not essential for activity. Although the brain enzyme catalyzed the esterification of desmosterol, cholesterol was the preferred substrate. Neither lanosterol (C₂₉ sterols) nor Δ7-dehydrocholesterol was esterified to any significant extent. The presence of low concentrations of desmosterol increased cholesterol esterification slightly, while there was a concentration-dependent inhibition of demosterol esterification by cholesterol. These data on fatty acid and sterol specificity of the esterifying enzyme correlate well with the composition of sterol esters present in developing rat brain.     

Effects of passive smoking on the regulation of rat aortic cholesteryl ester hydrolases by signal transduction

The effects of exogenous oxidative stress due to passive smoking on cholesteryl ester (CE)-metabolizing enzymes and their regulatory kinases were examined by exposing rats to cigarette smoke (CS) for a 1-h period twice a day for 8, 12, or 20 wk. An oxidatively modified low density lipoprotein (Ox-LDL) with a high lipid peroxide was identified in three CS groups after all three exposure periods. The rat aortic acid and neutral CE hydrolases (ACEH and NCEH) were activated to similar extents by both cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) in the presence of their respective cofactors. The aortic PKC activity in the three CS groups exhibited significant reductions of 72, 84, and 75% as compared with the respective controls, which coincided with the reductions in the ACEH activities (86, 71, and 80%, respectively), whereas the PKA activities increased to 121, 197, and 252% in the three CS groups, respectively. Reflecting the increase of the PKA activity, the NCEH activity exhibited increases of 112% at 8 wk and 140% until 12 wk of exposure and decreased by 50% of the control value at 20 wk of exposure, suggesting inactivation of NCEH itself. The activation of acyl-CoA:cholesterol O-acyl-transferase activity was associated with an increase of free cholesterol in aorta. The vitamin E diet prevented the formation of Ox-LDL and the oxidative inactivation of most enzymes, especially PKC, until 12 wk, but was less effective by 20 wk. The oxidative inactivation of PKC, particularly its activated form that translocated to the membrane fraction, was confirmed in the in vitro exposure to active oxygen generators at an optimal concentration; this inactivation was prevented by catalase and superoxide dismutase. These results suggested that the formation of Ox-LDL and alterations in CE-metabolizing enzymes caused by passive smoking could contribute to a twofold increase in the aortic CE content, thereby contributing to one of the mechanisms for atherosclerosis associated with smoking.     

Oxysterol mediated changes in fatty acid distribution and lipid synthesis in cultured bovine aortic smooth muscle cells

We studied the actions of oxysterols on fatty acid distribution and lipid synthesis in cultured bovine aortic smooth muscle cells. Cultures were labeled with [1-¹⁴C] arachidonate or [1-¹⁴C]oleate. During a 24-hr incubation, 25-or 22R-hydroxycholesterol enhanced the incorporation of label into triglycerides, concomitant with a reduction in the labeling of phospholipids. Cholestantriol or 20-hydroxycholesterol had the opposite effects. They caused a higher incorporation of radiolabel into phospholipids and a reduction of labeling of triacylglycerols. Similar changes were seen in cells labeled with [1-¹⁴C]acetate. Therefore, we conclude that oxysterols can promote changes in the distribution of fatty acids between neutral lipids and phospholipids through mechanisms that still need to be clarified.     

Effects of cholesterol and 25-hydroxycholesterol on smooth muscle cell and endothelial cell growth

Auto-oxidation products of cholesterol may play a role in atherogenesis. In order to determine whether cholesterol or 25-hydroxycholesterol, a cholesterol auto-oxidation product, affected growth of vessel wall cells, sparse and confluent cultures of rabbit thoracic aorta smooth muscle cells and human umbilical vein endothelial cells were exposed to these compounds for 88 hr. The compounds were administered at 10⁻⁴, 10⁻⁵, 10⁻⁶ or 10⁻⁷ M in either ethanol or fetal bovine serum (FBS) vehicle. Cells were counted electronically, and the results were expressed as the percent growth in experimental vs control wells. Cholesterol did not inhibit cell growth under any experimental condition. 25-Hydroxycholesterol had the following effects: inhibited confluent smooth muscle cell growth at 10⁻⁴ M in ethanol vehicle only; inhibited sparse smooth muscle cell growth in a dose-related manner at 10⁻⁴, 10⁻⁵ and 10⁻⁶ M in ethanol vehicle, but in FBS vehicle inhibited at only 10⁻⁴ and 10⁻⁵ M; inhibited confluent human umbilical vein endothelial cells at 10⁻⁴ M in ethanol vehicle only; and inhibited sparse human umbilical vein endothelial cell growth at 10⁻⁴ and 10⁻⁵ M in ethanol vehicle only. Thus, rabbit aortic smooth muscle cell growth was more sensitive to inhibition by 25-hydroxycholesterol than human umbilical vein endothelial cell growth was. Part of this work was presented at the 10th Annual Hugh Lofland Conference on Arterial Wall Metabolism, Boston, MA, in 1985 and at the 70th Annual FASEB Meeting, St. Louis, MO, in 1986. It is published in abstract form (Fed. Proc.45;684, 1986).     

The effect of oral contraceptives on mononuclear cell cholesteryl ester hydrolase activity

The influence of sex steroids on mononuclear cell cholesteryl ester hydrolase (CEH) activity in premenopausal women and women on combined estrogen-progestin oral contraceptives has been studied. In addition, plasma and mononuclear cell cholesterol and esters were measured along with plasma estrogen and progesterone levels. Mononuclear cell CEH activity in control women is highest on Day 20 of their menstrual cycle. The control women had significantly higher CEH activities than women on oral contraceptives. Plasma esters were higher in the oral contraceptive group. However, in mononuclear cells free cholesterol but not cholesteryl esters were higher in women on oral contraceptives.     

Esterification of cholesterol and hydrolysis of cholesteryl ester in alcohol induced fatty liver of rats

Repeated oral administrations of ethanol to rats induced accumulation of cholesteryl ester, as well as triglyceride in the livers. The contents of free cholesterol and phospholipid in the livers were not changed significantly in the present experiment in which ethanol ingestions were repeated four times. Although in vitro esterification of cholesterol by particle fractions of the alcoholic fatty liver was not affected, hydrolysis of cholesteryl palmitate by the supernatant fraction of the liver homogenate was reduced when compared with those of the control group which was given water or isocaloric glucose. The results of in vitro esterification of cholesterol and hydrolysis of cholesteryl palmitate in the liver of the rats which were ingested with glucose were larger than those of the control rats which were given water.     

p-Chlorophenoxyisobutyrate enhanced retention of homologous lipoproteins by human aortic smooth muscle cells

Human aortic smooth muscle cells (SMC) specifically bind and take up indiscriminately both the lipid and protein moietics of homologous²⁵I-very low density lipoproteins (VLDL) and¹²⁵I-low density lipoproteins LDL). Sixty-five to 80% of absorbed lipids are incorporated into the cell lipids, preferentially into the phospholipid fraction. Twenty to 35% of the lipid bound and the protein moiety are eliminated from the cells. Half of the eliminated protein label is recovered as TCA soluble products. Five mM of p-chlorophenoxyisobutyrate (CPIB) raise the level of intracellular radioactivity derived from the lipid moieties of VLDL and LDL by about 40% via a reduced elimination. The processing of the protein moiety and lipoprotein binding to the cell surface are not affected by 5.0 mM of CPIB. CPIB lowers the incorporation of¹⁴C-acetate,¹⁴C-pyruvate, and³²phosphate radioactivity into fatty acids and phospholipids of aortic SMC. Five mM of CPIB reduce the overall palmitic acid synthesis by shifting from de novo synthesis to the mechanism of chain elongation, although the further elongation to saturated C₁₈–C₂₄ fatty acids is also depressed. The CPIB-enhanced retention of the lipid-derived lipoprotein radio-activity is interpreted as a compensatory mechanism providing cellular fatty acids which are deficient as a result of the CPIB inhibited synthetic processes.     

Effect of 25-hydroxycholesterol on cholesteryl ester formation in Caco-2 cells

Incubation of Caco-2 cells, a human intestinal cell line, with 25-hydroxycholesterol (25-HOC) markedly enhanced cellular cholesteryl ester formation determined by incorporation of [¹⁴C]oleic acid into intracellular cholesteryl [¹⁴C]oleate. The stimulation by 25-HOC of cholesteryl ester formation was suppressed by staurosporine, a kinase inhibitor, but not by cycloheximide or actinomycin D. The specific activity of microsomal acyl-coenzyme A:cholesterol acyltransferase (ACAT) increased two-fold in cells treated with 10 μM 25-HOC for 5 h. ACAT activity decreased when microsomes were incubated without sodium fluoride, a phosphatase inhibitor, but the decrease in ACAT activity in cells stimulated with 25-HOC was more pronounced. The results suggest that protein phosphorylation may be involved in the stimulation of cholesteryl ester formation by 25-HOC in Caco-2 cells.     

Polyunsaturated fatty acids,vitamine E,and the proliferation of aortic smooth muscle cells

Smooth muscle cell cultures were obtained from the aortas of prepubertal guinea pigs. Cell proliferation in these cultures was inhibited by 8,11,14-eicosatrienoic acid, 5,8,11,14-eicosatetraenoic acid, and their prostaglandin E derivatives, PGE₁ and PGE₂. Prostaglandin F derivatives, PGF_1α and PGF_2α, stimulated cell proliferation. Cell proliferation was also inhibited by 5,8,11-eicosatrienoic acid and 11,14,17-eicosatrienoic acid. The monoene and diene precursors of the triene acids, 9-octadecenoic acid and 9,12-octadecadienoic acid, did not inhibit cell, proliferation. Indomethacin alone had no effect on cell proliferation, and indomethacin did not suppress the inhibition of cell proliferation with a triene acid. The antioxidant α-naphthol alone stimulated cell proliferation and suppressed prostaglandin E formation. α-Naphthol in the presence of either triene or tetraene acids also stimulated cell proliferation and suppressed prostaglandin E formation. The antioxidants butylated hydroxy toluene and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid either alone or in the presence of triene and tetraene acids stimulated cell proliferation and had no effect on prostaglandin E formation. Vitamin E either alone or in the presence of triene or tetraene acids stimulated cell proliferation and had no effect on prostaglandin E formation. More prostaglandin E was formed from 8,11,14-eicosatrienoic acid than from 5,8,11,14-eicosatetraenoic acid in the presence of antioxidants. Vitamin E suppressed the inhibitory effects of both PGE₂ and palmitic acid on cell proliferation. The cyclic nucleotide phosphodiesterase inhibitors, caffeine and papaverine, suppressed the stimulatory effect of vitamin E on cell proliferation and enhanced the inhibitory effect of a triene acid on cell proliferation. Substrate and inhibitor specificities are consistent with the oxidative regulation of cell proliferation through the formation of hydroperoxy fatty acids. We propose that hydroperoxy fatty acids may regulate both cyclase and cyclic nucleotide phosphodiesterase enzymes through sulfhydryl-disulfide interconversions. We suggest that this regulatory mechanism may help to explain the acculation of 5,8,11-eicosatrienoic acid in essential fatty acid deficiency, the effects of antioxidants on cell proliferation, and one of the several effects of polyunsaturated fatty acids in proliferative disorders such as cancer and atherosclerosis.     

Effects of high glucose on vascular endothelial growth factor synthesis and secretion in aortic vascular smooth muscle cells from obese and lean zucker rats

Type 1 diabetes is characterized by insulin deficiency, type 2 by both insulin deficiency and insulin resistance: in both conditions, hyperglycaemia is accompanied by an increased cardiovascular risk, due to increased atherosclerotic plaque formation/instabilization and impaired collateral vessel formation. An important factor in these phenomena is the Vascular Endothelial Growth Factor (VEGF), a molecule produced also by Vascular Smooth Muscle Cells (VSMC). We aimed at evaluating the role of high glucose on VEGF-A(164) synthesis and secretion in VSMC from lean insulin-sensitive and obese insulin-resistant Zucker rats (LZR and OZR). In cultured aortic VSMC from LZR and OZR incubated for 24 h with d-glucose (5.5, 15 and 25 mM) or with the osmotic controls l-glucose and mannitol, we measured VEGF-A(164) synthesis (western, blotting) and secretion (western blotting and ELISA). We observed that: (i) d-glucose dose-dependently increases VEGF-A(164) synthesis and secretion in VSMC from LZR and OZR (n = 6, ANOVA p = 0.002-0.0001); (ii) all the effects of 15 and 25 mM d-glucose are attenuated in VSMC from OZR vs. LZR (p = 0.0001); (iii) l-glucose and mannitol reproduce the VEGF-A(164) modulation induced by d-glucose in VSMC from both LZR and OZR. Thus, glucose increases via an osmotic mechanism VEGF synthesis and secretion in VSMC, an effect attenuated in the presence of insulin resistance.     

Effects of perturbations in hepatic free and esterified cholesterol pools on bile acid synthesis,cholesterol 7α-hydroxylase,HMG-CoA reductase,acyl-CoA:Cholesterol acyltransferase and cytosolic cholesteryl ester hydrolase

Effects of expansion of the hepatic free cholesterol pool on bile acid and cholesterol metabolism and homeostasis were examined in rats fed cholesterol in high-fat diets or treated with oleyl-p-(n-decyl)-benzenesulfonate (ODS) or progesterone. Cholesterol feeding for 10–16 days, which increased free (33%) and esterified (6-fold) cholesterol, had no effect on cholate synthesis, total bile acid synthesis, or cholate turnover, whereas these activities were increased 60–80% by ODS and progesterone, which produced only small increases (19%) in free cholesterol. Cholesterol feeding reduced β-hydroxy-β-methylglutaryl (HMG)-CoA reductase (72%) and cholesteryl ester hydrolase (48%) and increased acyl-CoA:cholesterol acyltransferase (184%), whereas ODS and progesterone reversed these compensatory responses in cholesterol-fed rats. Cholesterol 7α-hydroxylase was changed no more than 22% by any treatment. A bolus of ODS elevated biliary cholesterol output 41% and shifted biliary bile acid synthesis and composition toward 12-deoxy bile acids. These effects were not seen in ODS-fed or progesterone-treated rats, in which cholesteryl ester stores were depleted. It is concluded that effects of free cholesterol on bile acid synthesis and biliary cholesterol are probably mediated by specific precursor or regulatory pools which can be independently regulated and which represent a relatively small fraction of hepatic free cholesterol.     

Cholesterol metabolism in human monocyte-derived macrophages: Stimulation of cholesteryl ester formation and cholesterol excretion by serum lipoproteins

The role of lipoproteins and serum in the formation and accumulation of cholesteryl esters in human monocyte-derived macrophages (HMD macrophages) was investigated; studies were also carried out with IC21 cells (a cell line derived from mouse peritoneal macrophages). Following preincubation of HMD macrophages with lipoprotein-depleted serum (LPDS), both native and acetylated low density lipoprotein (LDL and AcLDL, respectively) stimulated the formation of cholesteryl esters with a resultant increase in cellular cholesteryl ester content. Cholesteryl ester formation and accumulation was also stimulated in macrophages exposed continuously to 25-hydroxycholesterol. However, the stimulation of cholesterol esterification by either lipoproteins or 25-hydroxycholesterol was not inhibited by progesterone in HMD macrophages, but was in the IC21 cells. Cholesterol efflux and the hydrolysis of cellular cholesterol ester, promoted by serum components, were studied in HMD macrophages preloaded with cholesteryl ester by incubation with 25-hydroxy cholesterol. Replacement of the medium with one devoid of 25-hydroxycholesterol resulted within 24 hr in at least a 30% decrease in the cholesteryl ester content of the HMD macrophages; replacement with a medium high in cholesterol acceptor content (LPDS or high density lipoprotein) and incubation for three days led to the most marked decreases in cellular cholesterol content. Thus, hydrolysis of the cholesteryl esters by HMD macrophages was not dependent on the presence of cholesterol acceptors in the medium, but cellular cholesterol content was.     

Inhibition of cholesterol esterification in macrophages and vascular smooth muscle foam cells: Evaluation of E5324, an Acyl-CoA cholesterol acyltransferase inhibitor

Cholesteryl esters (CE) comprise the principal lipid class that accumulates within macrophages and smooth muscle cells of the atherosclerotic lesion. Acyl-CoA cholesterol acyl-transferase (ACAT) is the major enzyme responsible for esterification of intracellular cholesterol. We evaluated the ability of E5324 (n-butyl-N″-[-2-[3-(5-ethyl-4-phenyl-1H-imidazol-1-yl)propoxyl]-6-methyl-phenyllurea), a novel, orally absorbable ACAT inhibitor, to inhibit esterification of fatty acids to cholesterol and CE accumulation in macrophages and in smooth muscle cells. E5324 significantly inhibited cholesterol esterification in rat aortic smooth muscle cells and in macrophages. In addition, E5324 reduced the cellular mass of CE, the significant measure of the efficacy of drugs designed to modulate cholesterol metabolism. E5324 treatment of macrophages exposed to acetylated low-density lipoprotein reduced CE mass by 97%, and treatment of lipid-loaded smooth muscle cells reduced CE mass by 29%. Although free cholesterol increased approximately twofold, this free cholesterol would presumably be accessible to the membrane for effluxin vivo (reverse cholesterol transport). These results demonstrate that E5324 can inhibit cholesterol esterification and CE mass in atherosclerotic foam cells, derived from either macrophages or arterial smooth muscle cells.     

Oxysterols in cultured bovine aortic smoth muscle cells and in the monocyte-like cell line U937

Oxysterols in cultured bovine aortic smooth muscle cells and in the monocyte-like cell line U937 were analyzed by gas-liquid chromatography. The following products were detected: 7α- and 7β-hydroxycholesterol, 7-ketocholesterol, cholesterol-α-epoxide, cholesterol-β-epoxide, cholestanetriol, and 20- and 25-hydroxycholesterol. The proportions of oxidized cholesterol varied from 1 to 3% of total cholesterol. The concentrations of oxysterols were lower when the smooth muscle cells were maintained in a vitamin E-enriched medium, and were higher in U937 cells when the cells were activated with phorbol myristate acetate. The cell oxysterol content appears to be regulated by factors that inhibit or enhanced free radical generation. The concentrations of oxysterols found in cells may serve as an indicator of the extent of lipid peroxidation.     

Dual action of neutral sphingomyelinase on rat hepatocytes: Activation of cholesteryl ester metabolism and biliary cholesterol secretion and inhibition of VLDL secretion

To address the role of cell membrane neutral sphingomyelinase (EC 3.1.4.12; SMase) in the regulation of cholesterol metabolism in the liver parenchymal cell, we examined the effect of exogenous neutral SMase on the metabolism of cholesteryl esters and the secretion of VLDL and biliary lipids in isolated rat hepatocytes. We show that treatment of hepatocytes with SMase (20 mU/mL) resulted in the intracellular buildup of cholesteryl esters, increased ACAT (EC 2.3.1.26) activity without affecting the ACAT2 mRNA level, and increased cytosolic and microsomal cholesteryl ester hydrolase (EC 3.1.1.13) activity. This was accompanied by increases in the secretion of biliary. bile acid, phospholipid, and cholesterol and in increased cholesterol 7α-hydroxylase (EC 1.14.13.17) activity and levels of mRNA, as well as decreased levels of apoB mRNA and a decreased secretion of VLDL apoB (apoB-48, ∼45%; apoB-100, ∼32%) and lipids (∼55%). Moreover, the VLDL particles secreted had an abnormal size and lipid composition; they were larger than controls, were relatively enriched in cholesteryl ester, and depleted in TG and cholesterol. Cell-permeable ceramides did not replicate any of the reported effects. These findings demonstrate that the increased cholesteryl ester turnover, oversecretion of biliary cholesterol and bile acids, and undersecretion of VLDL cholesterol and particles are concerted responses of the primary hepatocytes to exogenous neutral SMase brought about by regulation at several levels. We suggest that plasma membrane neutral SMase may have a specific, ceramide-independent effect in the regulation of cholesterol out-put pathways in hepatocytes.