Inhibitors of neutral cholesteryl ester hydrolase

p-Nitrophenyl N-butyl, N-octyl, and N-dodecyl carbamates and a newly synthesized diethyl phosphate compound were studied as potential inhibitors of the cholesteryl ester hydrolases of Fu5AH rat hepatoma cells. Whole homogenates of Fu5AH cells were used as an enzyme source for the assay of cholesteryl ester hydrolase activity. All four compounds led to marked inhibition (70-80%) of neutral cholesteryl ester hydrolase activity (assayed at pH 7) at concentrations where the activity of acid cholesteryl ester hydrolase (assayed at pH 4) was unaffected. Cholesteryl ester hydrolysis was also evaluated in intact cultured cells induced to accumulate cholesteryl esters in cytoplasmic lipid droplets by exposure to cholesterol-rich phospholipid dispersions. Hydrolysis was then assessed during subsequent incubations in the presence of an inhibitor of cholesterol esterification. All compounds caused significant inhibition of cholesterol ester hydrolysis with the diethyl phosphate being the most effective. At a concentration that caused greater than 90% inhibition of the hydrolysis of cytoplasmic cholesteryl esters, the compound had only a minimal effect on lysosomal hydrolysis of cholesteryl esters. These results suggest that diethyl phosphates and N-alkylcarbamates may be of value in future studies on the substrate specificities, regulation, and physiological role(s) of cholesteryl ester hydrolases.     

Effect of substrate physical state on the activity of acid cholesteryl ester hydrolase

The effects of the physicochemical properties of the substrate vehicle on the activity of acid cholesteryl ester hydrolase (ACEH; EC 3.1.1.13) isolated from rat liver lysosomes have been studied. In particular, the influence of the physical state of the neutral lipid core of substrate emulsion particles on the enzymatic activity has been probed in the light of previous studies on the clearance of cholesteryl esters (CE) from lipid-loaded cells which indicated that inclusions that are in the isotropic (liquid) state can be hydrolyzed faster than those in the anisotropic (liquid-crystalline) state. In the present study, such lipid inclusions were isolated from cultured cells and used as substrates for the hydrolase. No appreciable difference between the hydrolysis rates of isotropic and anisotropic inclusions was observed; the Vmax values were 93.0 +/- 6.7 and 84.0 +/- 3.3 nmol CE/mg.h, respectively. To elucidate the factors which affect the activity of ACEH, model inclusions were prepared by sonication and used as substrates. The physical state of these models was varied in a systematic way by changes of droplet composition and incubation temperature. The rate of hydrolysis was found to be insensitive to the physical state of the core of the model inclusions in good agreement with the results obtained with cellular inclusions. However, the activity of ACEH is sensitive to such interfacial properties of the lipid droplets as surface area available to the enzyme, net surface charge and surface solubility of the substrate CE molecules. The enzymatic activity is also sensitive to the amount of free cholesterol present in the emulsion droplets. The interfacial concentration and molecular packing of substrate CE molecules in the droplet surface significantly affect the hydrolytic activity of ACEH.     

Identification of neutral cholesterol ester hydrolase, a key enzyme removing cholesterol from macrophages

Unstable lipid-rich plaques in atherosclerosis are characterized by the accumulation of macrophage foam cells loaded with cholesterol ester (CE). Although hormone-sensitive lipase and cholesteryl ester hydrolase (CEH) have been proposed to mediate the hydrolysis of CE in macrophages, circumstantial evidence suggests the presence of other enzymes with neutral cholesterol ester hydrolase (nCEH) activity. Here we show that the murine orthologue of KIAA1363, designated as neutral cholesterol ester hydrolase (NCEH), is a microsomal nCEH with high expression in murine and human macrophages. The effect of various concentrations of NaCl on its nCEH activity resembles that on endogenous nCEH activity of macrophages. RNA silencing of NCEH decreases nCEH activity at least by 50%; conversely, its overexpression inhibits the CE formation in macrophages. Immunohistochemistry reveals that NCEH is expressed in macrophage foam cells in atherosclerotic lesions. These data indicate that NCEH is responsible for a major part of nCEH activity in macrophages and may be a potential therapeutic target for the prevention of atherosclerosis.     

Hormone-sensitive lipase is not required for cholesteryl ester hydrolysis in macrophages

Storage of cholesteryl esters in the cytoplasm of macrophages is one of the earliest and most ubiquitous event observed in the development of arteriosclerosis. Macrophages have an enormous capacity to uptake and store cholesterol in the form of cytosolic cholesteryl ester droplets. These stores are mobilized by the action of a neutral cholesteryl ester hydrolase (NCEH), producing free cholesterol that is either secreted to extracellular acceptors or reesterified. It has been proposed that hormone-sensitive lipase (HSL) is responsible for the NCEH activity in macrophages. The present work shows, however, that peritoneal macrophages from HSL null mice hydrolyze cytosolic stores of cholesteryl esters at a comparable rate to that of peritoneal macrophages from wild-type mice, therefore demonstrating that HSL is not the main NCEH in macrophages.     

Temperature-dependent molecular motions and phase behavior of cholesteryl ester analogues

The phase behavior and temperature-dependent molecular motions of three cholesteryl ethers (caproyl, myristyl, oleyl) and a cholesteryl carbonate (oleyl) were characterized. The properties of each ether were qualitatively similar to, but quantitatively different from, those of the corresponding cholesteryl ester. For example, cholesteryl oleyl ether exhibited the same phase transitions as cholesteryl oleate, but at much lower temperatures (e.g., the ether isotropic liquid to cholesteric transition is at 29 degrees C). 13C NMR spectra of ethers in the isotropic liquid and liquid crystalline phases were similar to those of the ester analogue. However, near the liquid to liquid crystalline transition, the steroid ring C3 and C6 linewidths, the C3/C6 linewidth ratio, and the steroid ring rotational correlation times tau rx and tau rz calculated from the linewidths were larger for the ether than the ester analogue. The oleyl carbonate had qualitatively different properties from its analogues (e.g., stable vs. metastable cholesteric and smectic phases). Quantitative results (e.g., relatively long tau rx and tau rz in the isotropic liquid phase) for the carbonate were also distinct from those of both the ester and ether analogues. A comparison of analogues in which the polar linkage is the only structural variable yielded insights into the intermolecular interactions which influence phase behavior.     

Characterization of multiple forms of cholesteryl ester hydrolase in the rat testis

Cholesterol ester hydrolase (EC 3.1.1.13) activity from the 104,000 X g supernatant of rat testis was fractionated into 28-kDa, 72-kDa, and 420-kDa molecular mass forms by high performance size exclusion chromatography. The 72-kDa and 420-kDa forms (temperature-labile) were completely inactivated by elevation of temperature from 32 to 37 degrees C. Apparent disaggregation of the 420-kDa form suggested that the 72-kDa and 420-kDa enzymes are monomeric and multimeric forms of the same enzyme. The 28-kDa form was shown to be a different enzyme (temperature-stable) which retained activity at 37 degrees C. In contrast, cholesteryl ester hydrolase activities from 104,000 X g supernatants of liver or adrenal gland were unaffected and increased 4-fold, respectively, by elevation of temperature from 32 to 37 degrees C. Both testicular enzymes exhibited pH optima at about 7.3, and were activated by sodium cholate at concentrations near the critical micellar concentration (0.03-0.07%), but inhibited by higher concentrations. The temperature-labile cholesteryl ester hydrolase exhibited a high specificity for cholesteryl esters of monoenoic fatty acids of 18-24 carbons, especially nervonate (24:1), whereas the temperature-stable cholesteryl ester hydrolase exhibited highest specificity for cholesteryl oleate and arachidonate. Neither enzyme hydrolyzed cholesteryl acetate, myristate, palmitate, linoleate, or docosahexaenoate . Both enzymes reached maximum rates of hydrolysis at 150 microM substrates, with each substrate and at both reaction temperatures. Substrate inhibition was observed at higher concentrations (200 microM). The temperature-labile cholesteryl ester hydrolase was induced 20-fold in hypophysectomized rats by injection of follicle-stimulating hormone (FSH) and was localized in Sertoli cells, the target cells for FSH, but was not induced by luteinizing hormone. The temperature-stable cholesteryl ester hydrolase was induced by both FSH and LH and was found in both Sertoli cells and Leydig cells, the respective target cells for FSH and luteinizing hormone. Neither form of the enzyme was present at detectable levels in the germinal cells. The unique properties, localization, and hormonal regulation of both temperature-labile and temperature-stable cholesterol ester hydrolases suggest important roles for these enzymes in the testis.     

Stimulation of a neutral cholesteryl ester hydrolase by cAMP system in P388D1 macrophages

Cholesteryl ester laden foam cells in atherosclerotic lesions derive, in part, from macrophages. Mobilization of stored cholesteryl esters involves hydrolysis by a neutral cholesteryl ester hydrolase. Incubation of intact P388D1 macrophages with dibutyryl cAMP in the presence of 1-methyl-3-isobutylxanthine resulted in a dose-dependent increase in neutral cholesteryl ester hydrolase activity of up to 50% (ED50 = 0.1 mM). Incubation with prostaglandin E1 in the presence of 1-methyl-3-isobutylxanthine also increased neutral cholesterol ester hydrolase activity by about 50%. In cell-free preparation, cAMP-dependent protein kinase caused about a 2-fold activation of the neutral cholesteryl ester hydrolase. Activation was blocked by protein kinase inhibitor. These data suggest that the P388D1 macrophage may be a useful model for studying the hormonal regulation of cholesteryl ester mobilization in macrophage-derived foam cells.     

Cholesteryl ester storage disease and Wolman disease: phenotypic variants of lysosomal acid cholesteryl ester hydrolase deficiency

The lysosomal enzyme responsible for cholesteryl ester hydrolysis, acid cholesteryl ester hydrolase, or acid lipase (E.C.3.1.1.13) plays an important role in cellular cholesterol metabolism. Loss of the activity of this enzyme in tissues of individuals with both Wolman disease and cholesteryl ester storage disease is believed to play a causal role in these conditions. The objectives of our studies were not only to directly compare and contrast the clinical features of Wolman disease and cholesteryl ester storage disease but also to determine the reasons(s) for the varied phenotype expression of acid cholesteryl ester hydrolase deficiency. Although both diseases manifest a type II hyperlipoproteinemic phenotype and hepatomegaly secondary to lipid accumulation, a more malignant clinical course with more significant hepatic and adrenal manifestations was observed in the patient with Wolman disease. However, the acid cholesteryl ester hydrolase activity in cultured fibroblasts in both diseases was virtually absent. In addition, fibroblasts from both Wolman disease and cholesteryl ester storage disease were able to utilize exogenously supplied enzyme, suggesting that neither disease was due to defective enzyme delivery by the mannose-6-phosphate receptor pathway. Coculture and cell fusion of fibroblasts from Wolman disease and cholesteryl ester storage disease subjects did not lead to correction of the enzyme deficiency, indicating that these disorders are allelic. However, the activities of the hepatic acid and neutral lipase in these two clinical variants were quite different. Hepatic acid lipase activity was only 4% normal in Wolman disease, but the activity was 23% normal in cholesteryl ester storage disease. The hepatic neutral lipase activity was normal in Wolman disease but increased more than twofold in cholesteryl ester storage disease. These combined results indicate that the clinical heterogeneity in acid cholesteryl ester hydrolase deficiency can be explained by a varied hepatic metabolic response to an allelic mutation.     

Effect of estrogens on the acitivities of cholesteryl ester synthetase and cholesteryl ester hydrolases in pigeon aorta.

This study is the first to report the effect of conjugated equine estrogens on the acitivity of cholesteryl ester synthetase and cholesteryl ester hydrolases in the aorta. In spontaneously atherosclerosis-susceptible White Carneau pigeons, estrogens significantly decreased (P less 0.01) the activity of cholesteryl ester synthetase and increased (P less than 0.01) the cholesteryl ester hydrolase activity in the microsomal fraction of the aorta. There was no effect on the cholesteryl ester hydrolase activity in the supernatant fraction. The inhibition of cholesteryl ester synthesis and the stimulation of cholesteryl ester hydrolase might be responsible for the decreased content of cholesteryl esters noted in pigeon aorta after estrogen treatment.     

Neutral cholesteryl ester hydrolase in the rat lactating mammary gland: regulation by phosphorylation-dephosphorylation

The characteristics of neutral cholesteryl ester hydrolase activities found in the microsomal and cytosolic subcellular fractions of rat lactating mammary tissue were investigated. The enzymes were assayed using cholesteryl oleate dispersed as a mixed micelle with phosphatidylcholine and sodium taurocholate (molar ratio 1:4:2) as substrate. This method gave activities approx. 20-fold higher than those seen when cholesteryl oleate was added in ethanol. Addition of phosphatidylcholine and sodium taurocholate to the assays using the ethanol-dissolved substrate did not increase the activities observed. When the cholesteryl oleate was dispersed with phosphatidylcholine only (molar ratio, 1:4) the activity of the two neutral cholesteryl ester hydrolases was also decreased considerably compared to that found with mixed micelles. In this case, however, approx. 60% of the cytosolic, but only 10% of the microsomal activity, was restored by separate addition of sodium taurocholate. The activities of both the microsomal and the cytosolic neutral cholesteryl ester hydrolases were inhibited by MgCl2, and this inhibition was almost completely reversed by the addition of an equimolar concentration of ATP. At a fixed concentration of MgCl2 increasing concentrations of ATP increased the enzyme activities in a dose-dependent way. The activity of the microsomal, but not the cytosolic enzyme was enhanced by a cyclic AMP-dependent protein kinase and both activities were inhibited by alkaline phosphatase (bovine milk). These results provide evidence for the regulation of neutral cholesteryl ester hydrolases in the rat lactating mammary gland by mechanisms involving phosphorylation-dephosphorylation and therefore suggest that these enzymes may be under hormonal control.     

Putative role of cholesteryl ester transfer protein in removal of cholesteryl ester from vascular interstitium, studied in a model system in cell culture

A model system to study the putative role of cholesteryl ester transfer protein in the egress of interstitial cholesteryl ester is described. Confluent cultures of bovine aortic smooth muscle cells were labeled for 24 h with [3H]cholesteryl linoleyl ether and [14C]cholesteryl linoleate by incubation with bovine milk lipoprotein lipase. This method of labeling results in the transfer of cholesteryl linoleyl ether and cholesteryl ester to three compartments: a trypsin-releasable, trypsin-resistant and catabolic compartment (Stein, O., Halperin, G., Leitersdorf, E., Olivecrona, T. and Stein, Y. (1984) Biochim. Biophys. Acta 795, 47-59). The efflux of labeled cholesteryl linoleyl ether and cholesteryl ester from the extracellular and cell-surface related compartments into a serum-free culture medium containing 1% bovine serum albumin was studied during 24 h of postincubation. The efflux was expressed as a percentage of pulse value, i.e., radioactivity retained by the cell culture at the end of the labeling period. The efflux of [3H]cholesteryl linoleyl ether, [14C]cholesteryl ester and 14C-labeled free cholesterol (formed by cellular hydrolysis of cholesterol ester) into the culture medium with 1% bovine serum albumin was about 5% of the pulse value. Addition of human lipoprotein-deficient serum resulted in a 3-10-fold increase in the efflux of [3H]cholesteryl linoleyl ether and [14C]cholesteryl ester, but did not change markedly the efflux of 14C-labeled free cholesterol. Rat lipoprotein-deficient serum which does not contain cholesteryl ester transfer protein did not increase the efflux of [3H]cholesteryl linoleyl ether or [14C]cholesteryl ester. The rate of cholesteryl ester efflux in the presence of human lipoprotein-deficient serum was linear for about 6 h and increased further up to 24 h. Addition of Intralipid to medium containing human lipoprotein-deficient serum further enhanced the efflux of [3H]cholesteryl linoleyl ether and, to a lesser extent, that of cholesteryl ester. A similar effect was observed also by addition of rat VLDL to medium containing human lipoprotein-deficient serum. Inhibition of cholesteryl linoleyl ether and cholesteryl ester efflux and marked enhancement of free cholesterol efflux occurred when rat HDL was added to medium containing human lipoprotein-deficient serum, while human HDL was only slightly inhibitory. The results obtained with human lipoprotein-deficient serum were reproduced with partially purified cholesteryl ester transfer protein. Using the partially purified cholesteryl ester transfer protein, the efflux of cholesteryl linoleate was compared to that of cholesteryl oleate and was found to be the same.     

Critical role of neutral cholesteryl ester hydrolase 1 in cholesteryl ester hydrolysis in murine macrophages

Hydrolysis of intracellular cholesteryl ester (CE) is the rate-limiting step in the efflux of cholesterol from macrophage foam cells. In mouse peritoneal macrophages (MPMs), this process is thought to involve several enzymes: hormone-sensitive lipase (Lipe), carboxylesterase 3 (Ces3), neutral CE hydrolase 1 (Nceh1). However, there is some disagreement over the relative contributions of these enzymes. To solve this problem, we first compared the abilities of several compounds to inhibit the hydrolysis of CE in cells overexpressing Lipe, Ces3, or Nceh1. Cells overexpressing Ces3 had negligible neutral CE hydrolase activity. We next examined the effects of these inhibitors on the hydrolysis of CE and subsequent cholesterol trafficking in MPMs. CE accumulation was increased by a selective inhibitor of Nceh1, paraoxon, and two nonselective inhibitors of Nceh1, (+)-AS115 and (−)-AS115, but not by two Lipe-selective inhibitors, orlistat and 76-0079. Paraoxon inhibited cholesterol efflux to apoA-I or HDL, while 76-0079 did not. These results suggest that Nceh1 plays a dominant role over Lipe in the hydrolysis of CE and subsequent cholesterol efflux in MPMs.     

High-density-lipoprotein-induced cholesterol efflux from arterial smooth muscle cell derived foam cells: functional relationship of the cholesteryl ester cycle and eicosanoid biosynthesis

Eicosanoids have been implicated in the regulation of arterial smooth muscle cell (SMC) cholesteryl ester (CE) metabolism. These eicosanoids, which include prostacyclin (PGI2), stimulate CE hydrolytic activities. High-density lipoproteins (HDL), which promote cholesterol efflux, also stimulate PGI2 production, suggesting that HDL-induced cholesterol efflux is modulated by eicosanoid biosynthesis. To ascertain the role of endogenously synthesized eicosanoids produced by arterial smooth muscle cells in the regulation of CE metabolism, we examined the effects of cyclooxygenase inhibition on CE hydrolytic enzyme activities, cholesterol efflux, and cholesterol content in normal SMC and SMC-derived foam cells following exposure to HDL and another cholesterol acceptor protein, serum albumin. Alterations of these activities were correlated with cholesterol efflux in response to HDL or bovine serum albumin (BSA) in the presence or absence of aspirin. HDL stimulated PGI2 synthesis and CE hydrolases in a dose-dependent manner. Eicosanoid dependency was established by demonstrating that HDL-induced acid cholesteryl ester hydrolase (ACEH) activity was blocked by aspirin. CE enrichment essentially abrogated HDL-induced PGI2 production in cells which also exhibited decreased lysosomal and cytoplasmic CE hydrolase activities. In CE-enriched cells whose cytoplasmic CE pool was metabolically labeled with [3H]oleate or cLDL containing [3H]cholesteryl linoleate, aspirin did not alter HDL- or BSA-induced net CE hydrolysis or efflux, respectively. Finally, aspirin treatment did not alter the mass of either free or esterified cholesterol content of untreated or CE-enriched SMC following exposure to acceptor proteins. These data demonstrated that CE enrichment significantly reduced HDL-induced activation of CE hydrolytic activity via inhibition of endogenous PGI2 production.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rat liver retinyl palmitate hydrolase activity. Relationship to cholesteryl oleate and triolein hydrolase activities

Studies were conducted to explore relationships in rat liver between retinyl palmitate hydrolase activity and the hydrolytic activities against cholesteryl oleate and triolein. Previous studies have shown positive correlations between these three lipid ester hydrolase activities. In order to extend this work, the hydrolase activities were further purified and characterized. The activities against cholesteryl oleate and triolein resembled retinyl palmitate hydrolase activity in showing great variability from rat to rat as assayed in vitro. The relative levels of the three activities were highly correlated with each other over a 50-fold range of activity in a series of 66 liver homogenates. Partial purification (approx. 200-fold) in the absence of detergents was achieved by sequential chromatography of an acetone powder extract of liver on columns of phenyl-Sepharose, DEAE-Sepharose and heparin-Sepharose. The three hydrolase activities copurified during each of these chromatographic steps. The properties of the three copurifying activities were similar with regard to stimulation of activity by trihydroxy bile salts, pH optimum (near 8.0), and observance of Michaelis-Menten-type saturation kinetics. The three activities were different in their sensitivity towards the serine esterase inhibitors diisopropylfluorophosphate and phenylmethanesulfonyl fluoride, and in their solubility properties in 10 mM sodium acetate, pH 5.0. Thus, triolein hydrolase activity was much less sensitive than the other two activities to the two inhibitors. In addition, the activity against cholesteryl oleate could be separated from the other two activities by extraction of an acetone powder with acetate buffer, pH 5.0. These results indicate that the three lipid hydrolase activities are due to at least three different catalytically active centers, and at least two distinct and separable enzymes. It is likely that three separate but similar enzymes, that appear to be coordinately regulated, are involved.     

Neutral and acid retinyl ester hydrolases associated with rat liver microsomes: relationships to microsomal cholesteryl ester hydrolases

We recently reported the presence of a neutral, bile salt-independent retinyl ester hydrolase (REH) activity in rat liver microsomes and showed that it was distinct from the previously studied bile salt-dependent REH and from nonspecific carboxylesterases (Harrison, E. H., and M. Z. Gad. 1989. J. Biol. Chem. 264: 17142-17147). We have now further characterized the hydrolysis of retinyl esters by liver microsomes and have compared the observed activities with those catalyzing the hydrolysis of cholesteryl esters. Microsomes and microsomal subfractions enriched in plasma membranes and endosomes catalyze the hydrolysis of retinyl esters at both neutral and acid pH. The acid and neutral REH enzyme activities can be distinguished from one another on the basis of selective inhibition by metal ions and by irreversible, active site-directed serine esterase inhibitors. The same preparations also catalyze the hydrolysis of cholesteryl esters at both acid and neutral pH. However, the enzyme(s) responsible for the neutral REH activity can be clearly responsible for the neutral REH activity can be clearly differentiated from the neutral cholesteryl ester hydrolase(s) on the basis of differential stability, sensitivity to proteolysis, and sensitivity to active site-directed reagents. These results suggest that the neutral, bile salt-independent REH is relatively specific for the hydrolysis of retinyl esters and thus may play an important physiological role in hepatic vitamin A metabolism. In contrast to the neutral hydrolases, the activities responsible for hydrolysis of retinyl esters and cholesterol esters at acid pH are similar in their responses to the treatments mentioned above. Thus, a single microsomal acid hydrolase may catalyze the hydrolysis of both types of ester.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transfer of cholesteryl ester into high density lipoprotein by cholesteryl ester transfer protein: effect of HDL lipid and apoprotein content

Recombinant high density lipoprotein (rHDL) particles were prepared by cosonication of purified lipids and human apoproteins and incubated with partly purified cholesteryl ester transfer protein (CETP) and low density lipoprotein (LDL) containing [3H]cholesteryl ester. Increasing the triglyceride content relative to cholesteryl ester in rHDL significantly decreased the ability of the particles to accept cholesteryl esters transferred by CETP. Kinetic analysis of the data was performed to numerically define the maximum velocity of lipid transfer, Tmax, and the HDL concentration required for half maximal velocity, KH. Increases in rHDL-triglyceride content were shown to result in a significant reduction in the Tmax without a major change in KH. When the free cholesterol content was increased relative to phospholipid, the ability of the particles to accept cholesteryl esters was also decreased in a similar manner. Conversely, rHDL prepared from purified apoprotein A-I, A-II, or mixtures of both, had significantly elevated Tmax and KH values for their interaction with CETP. The results suggest that increases in triglyceride or free cholesterol content of an rHDL particle decrease the catalytic ability of CETP by noncompetitive inhibition. In addition, some component(s) of HDL apoproteins, other than A-I or A-II, were shown to uncompetitively inhibit the activity of CETP, by modifying both Tmax and the KH for the reaction. This study has shown that altered HDL composition may have marked effects on the transfer and equilibration of cholesteryl esters within the HDL pool.     

Immunological characterization of neutral cholesteryl ester hydrolase from rat liver cytosol.

Rabbit polyclonal antibodies were raised against rat liver bile salt-independent neutral cholesteryl ester hydrolase (CEH) and used for subcellular localization and immunological comparison with isoforms from other tissues. Antibodies exhibited a high degree of specificity for the liver CEH through all stages of purification and neutralized 70-80% of the activity of liver cytosolic CEH. They exhibited various levels of cross-reactivity with cytosolic proteins from other tissues, but reacted weakly with pancreatic and intestinal proteins and did not inhibit pancreatic CEH. Cytosol contained 78% of total cellular CEH activity and 75% of CEH immunoreactive protein. Washed microsomes contained 3% of CEH activity and 5% of CEH protein.     

Regulation of hepatic cholesterol ester hydrolase and acyl-coenzyme A:cholesterol acyltransferase in the rat

Cholesterol exists within the hepatocyte as free cholesterol and cholesteryl ester. The proportion of intrahepatic cholesterol in the free or ester forms is governed in part by the rate of cholesteryl ester formation by acyl-coenzyme A:cholesterol acyltransferase (ACAT) and cholesteryl ester hydrolysis by neutral cholesterol ester (CE) hydrolase. In other cell types both ACAT and CE hydrolase activities are regulated in response to changes in the need for cellular free cholesterol. In rats, we performed a variety of experimental manipulations in order to vary the need for hepatic free cholesterol and to examine what effect, if any, this had on the enzymes that govern cholesteryl ester metabolism. Administration of a 20-mg bolus of lipoprotein cholesterol or a diet supplemented with 2% cholesterol resulted in an increase in microsomal cholesteryl ester content with little change in microsomal free cholesterol. This was accomplished by an increase in cholesteryl esterification as measured by ACAT but no change in CE hydrolase activity. An increased need for hepatic free cholesterol was experimentally induced by intravenous bile salt infusion or cholestyramine (3%) added to the diet. ACAT activity was decreased with both experimental manipulations compared to controls, while CE hydrolase activity did not change. Microsomal cholesteryl ester content decreased significantly with little change in microsomal free cholesterol content. Addition of exogenous liposomal cholesterol to liver microsomes from cholestyramine-fed and control rats resulted in a 784 +/- 38% increase in ACAT activity. Nevertheless, the decrease in ACAT activity with cholestyramine feeding was maintained. These studies allowed us to conclude that changes in hepatic free cholesterol needs are met in part by regulation of the rate of cholesterol esterification by ACAT without a change in the rate of cholesteryl ester hydrolysis by CE hydrolase.     

Metabolic activity of cholesteryl esters in aortic smooth muscle cells is altered by prostaglandins I2 and E2

Among the biochemical processes associated with the atherogenic process are increased aortic cholesteryl ester (CE) accumulation and altered prostaglandin (PG) production. The precise physiological role of PG, particularly prostacyclin (PGI2), in the control of CE metabolism in intact aortic smooth muscle cells remains to be fully elucidated. We report here that cytosolic neutral cholesteryl ester hydrolytic activity (NCEH) in intact cultured aortic smooth muscle cells is significantly increased by 75-250 nM PGI2 at the end of a 2-hr incubation period. The effect was mediated by increased intracellular cAMP levels since the effect of PGI2 on NCEH activity was abolished in the presence of an inhibitor of adenylate cyclase activity, viz., dideoxyadenosine (DDA0. Although the addition of 20-100 microM dibutyryl cAMP (Bt2cAMP) and 50-100 microM sodium arachidonate also increased NCEH activity twofold, 6-keto PGF1 alpha, PGE1, and PGE2 did not increase the activity of this enzyme. In contrast to these findings, 75-250 nM PGE2 significantly inhibited CE synthetic activity (ACAT) approximately 60%. Arachidonate or Bt2cAMP did not affect ACAT activity. This decrease in ACAT activity induced by PGE2 does not appear to be mediated by cAMP. Taken together, these findings suggest that PGI2, a well known potent vasodilator and inhibitor of platelet aggregation, and PGE2 may have an important regulatory role in aortic CE metabolism.