Nitric oxide inhibition of free radical-mediated cholesterol peroxidation in liposomal membranes

The ability of nitric oxide ((*)NO) to inhibit propagative lipid peroxidation was investigated using unilamellar liposomes (LUVs) constituted with egg phosphatidylcholine (PC) or 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), [(14)C]cholesterol (Ch), and a nonregenerable singlet oxygen-derived primer, 5alpha-hydroperoxycholesterol (5alpha-OOH). Exposing LUVs to ascorbate and a lipophilic iron chelate at 37 degrees C resulted in an exponential decay of 5alpha-OOH and accumulation of free radical-derived 7alpha- and 7beta-hydroperoxycholesterol (7alphabeta-OOH), as detected by high-performance liquid chromatography with electrochemical detection. Thiobarbituric acid-reactive species (TBARS) were generated concurrently in egg PC-containing LUVs. Including the (*)NO donor spermine NONOate (SPNO, 5-50 microM) or S-nitroso-N-acetyl-D,L-penicillamine (SNAP, 50-100 microM) in the reaction mixture had no effect on 5alpha-OOH decay (suggesting that iron was not redox-inhibited) but slowed TBARS and 7alphabeta-OOH accumulation in a strongly dose-dependent fashion. Decomposed SPNO or SNAP had no such effects, implying that (*)NO was the responsible agent. Accumulation of several [(14)C]Ch oxidation products, detected by high-performance thin-layer chromatography with phosphorimaging, was similarly diminished by active SPNO or SNAP. Concomitantly, a new band referred to as RCh.4 appeared, the radioactivity of which increased as a function of incubation time and (*)NO donor concentration. RCh.4 material was also generated via direct iron/ascorbate reduction of 7alpha-OOH in the presence of (*)NO, consistent with 7alpha-nitrite (7alpha-ONO) identity. However, various other lines of evidence suggest that RCh.4 is not 7alpha-ONO, but rather 5alpha-hydroxycholesterol (5alpha-OH) generated by reduction of 5alpha-ONO arising from 7alpha-ONO rearrangement. 5alpha-OH was only detected when (*)NO was present in the reaction system, thus providing indirect evidence for the existence of nitrosated Ch intermediates arising from (*)NO chain-breaking activity.     

Histamine Stimulation of Cyclic AMP Accumulation in Astrocyte-Enriched and Neuronal Primary Cultures from Rat Brain

Histamine stimulates cyclic AMP accumulation in astrocyte-enriched and neuronal primary cultures from rat brain in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine. The response in the astrocyte cultures (Emax = 304 +/- 44% over basal, EC50 = 43 +/- 5 microM) was much higher than in neuronal cultures (Emax = 24 +/- 2%, EC50 = 14 +/- 7 microM). The histamine effect in astrocytes was competitively inhibited by the H2 antagonists cimetidine (Ki = 1.1 +/- 0.2 microM) and ranitidine (Ki = 46 +/- 10 nM) but was insensitive to the H1 antagonist mepyramine (1 microM). The two selective H2 agonists impromidine and dimaprit behaved as partial agonists and showed relative potencies (139 and 0.5, respectively) consistent with an interaction with H2 receptors. The more selective H1 agonist 2-thiazolylethylamine (0.01-1 mM) did not potentiate the response to impromidine (10 microM). Thus, in contrast to what is generally observed in intact cell preparations from brain, the histamine-induced cyclic AMP accumulation in astroglial cells is mediated solely by H2 receptors. The small effect shown in neuronal cultures also appears to be mediated by H2 receptors.     

Calmodulin antagonist W-7 inhibits de novo synthesis of cholesterol and suppresses secretion of de novo synthesized and preformed lipids from cultured hepatocytes

The effects of a calmodulin antagonist W-7 were studied on the synthesis and secretion of lipids in primary rat hepatocytes and McArdle-RH7777 cells. In time course experiments, W-7 (20 microM) inhibited secretion of newly synthesized triacyl[(3)H]glycerol by 35%. When the cells were pre-treated overnight with W-7 (20 microM), followed by incubation with [(3)H]oleate, a significant decrease in the secretion of triacylglycerol (TG) and cholesteryl ester (CE) was observed. De novo synthesis of cholesterol from acetate or mevalonolactone was inhibited by W-7, but not glycerolipid synthesis from glycerol and oleic acid precursors. Concentration-response curves for the effects of overnight pre-incubation with W-7 followed labeling with [(3)H]glycerol and [(14)C]mevalonolactone revealed that: (1). the inhibitory effect of W-7 was concentration-dependent and appeared even at the lowest concentration examined (1 microM). W-7 at a concentration of 20 microM suppressed secretion of TG by 60% (P相似文献   

Cytotoxic effects of oxysterols produced during ozonolysis of cholesterol in murine GT1-7 hypothalamic neurons

Ozone present in the photochemical smog or generated at the inflammatory sites is known to oxidize cholesterol and its 3-acyl esters. The oxidation results in the formation of multiple "ozone-specific" oxysterols, some of which are known to cause abnormalities in the metabolism of cholesterol and exert cytotoxicity. The ozone-specific oxysterols have been shown to favor the formation of atherosclerotic plaques and amyloid fibrils involving pro-oxidant processes. In the present communication, cultured murine GT1-7 hypothalamic neurons were studied in the context of cholesterol metabolism, formation of reactive oxygen species, intracellular Ca2 + levels and cytotoxicity using two most commonly occurring cholesterol ozonolysis products, 3beta- hydroxy-5-oxo-5,6-secocholestan-6-al (ChSeco) and 5beta, 6beta-epoxy-cholesterol (ChEpo). It was found that ChSeco elicited cytotoxicity at lower concentration (IC50 = 21 +/- 2.4 microM) than did ChEpo (IC50 = 43 +/- 3.7 microM). When tested at their IC50 concentrations in GT1-7 cells, both ChSeco and ChEpo resulted in the generation of ROS, the magnitude of which was comparable. N-acetyl-l-cysteine and Trolox attenuated the cytotoxic effects of ChSeco and ChEpo. The intracellular Ca2 + levels were not altered by either ChSeco or ChEpo. Methyl-beta-cyclodextrins, which cause depletion of cellular cholesterol, prevented ChSeco- but not ChEpo-induced cytotoxicity. The cell death caused by ChEpo, but not ChSeco, was prevented by exogenous cholesterol. Although oxidative stress plays a significant role, the results of the present study indicate differences in the pathways of cell death induced by ChSeco and ChEpo in murine GT1-7 hypothalamic neurons.     

Effect of esterastin, an acid lipase inhibitor, on the free and esterified cholesterol contents of cultured aortic smooth muscle cells treated with LDL and cholesterol ester liquid crystals

The effects of esterastin, an acid lipase inhibitor, on the free and esterified cholesterol contents of cultured smooth muscle cells from pig aorta were examined. The post-nuclear supernatant fraction of the cell homogenate showed maximum acid cholesterol esterase activity at pH 4.5, and 50% of this activity was inhibited by 0.31 microM esterastin. During a 48 h incubation with esterastin, the esterified cholesterol content of the cells increased to about 13 times that of control cells in the presence of low density lipoprotein and to 7 times that of control cells in the presence of cholesterol oleate liquid crystals. The ratio of esterified to free cholesterol also increased to about 5 times the control value in both conditions.     

Lethal damage to murine L1210 cells by exogenous lipid hydroperoxides: protective role of glutathione-dependent selenoperoxidases

The effect of selenium deprivation on the viability of murine L1210 cells exposed to various exogenous lipid hydroperoxides has been investigated. Selenoperoxidase activities of cells grown for longer than 1 week in 1% serum with no added selenium [Se(-) cells] were less than 10% of the activities of selenium-satisfied controls [Se(+) cells] or selenium-repleted counterparts [Se(-/+) cells]. The enzymes measured were classical glutathione peroxidase (GPX) and phospholipid hydroperoxide glutathione peroxidase (PHGPX). Se(-) cells exhibited a compensatory increase in catalase activity. Dye exclusion and clonal survival assays indicated that Se(-) and Se(+) cells were relatively insensitive to photochemically generated phospholipid hydroperoxides in liposomal form. However, both cell types were sensitive to liposomal cholesterol hydroperoxides, e.g., 7-hydroperoxycholesterol (7-OOH), Se(-) being much more so (LD50 approximately 10 microM) than Se(+) (LD50 approximately 75 microM). By contrast, 7-hydroxycholesterol over a comparable concentration range was minimally toxic to Se(-) and Se(+) cells. Cell killing by 7-OOH was inhibited by desferrioxamine and by butylated hydroxytoluene, suggesting that iron-mediated free radical reactions are involved. The involvement of glutathione in cytoprotection was confirmed by showing that Se(+) cells were more sensitive to 7-OOH after treating with buthionine sulfoximine, an inhibitor of GSH synthesis. Cellular detoxification of 7-OOH is provisionally attributed to PHGPX rather than GPX, since 7-OOH and other cholesterol hydroperoxides were found to be good substrates for PHGPX in a cell free system, but were unreactive with GPX.     

Synthesis and antitumor activity of s-tetrazine derivatives

Fifty-five compounds of s-tetrazine derivative including hexahydro-, 1,6-dihydro, 1,4-dihydro-, 1,2-dihydro- and aromatic s-tetrazine were prepared. Their antitumor activities were evaluated in vitro by MTT method for P-388 cell and SRB method for A-549 cell. The results show that there are 9 compounds which in 10(-6) microM have more than 50% inhibition rate to A-549 cancer cell growth, and 7 compounds in 10(-6) microM have more than 50% inhibition rate to P-388 cancer cell growth. The IC(50) of compound 3q for P-388, Bel-7402, MCF-7 and A-549 are 0.6 microM, 0.6 microM, 0.5 microM and 0.7 microM, respectively. So s-tetrazine derivative is a kind of compound which possesses potential antitumor activities and is worth to research further.     

Effect of cholesta-3,5-dien-7-one on human liver aldehyde dehydrogenase

A recently isolated cholesterol oxidation product, cholesta-3,5-dien-7-one, which was present at high concentrations in fatty/cirrhotic alcoholic liver was identified as a potent endogenous inhibitor of the cytosolic, E1, isozyme of aldehyde dehydrogenase (EC 1.2.1.3). The oxysterol was a less potent inhibitor of mitochondrial, E2, isozyme. The inhibition of the E1 isozyme was irreversible on the IEF gels, upon dilution and with 33 microM 2-mercaptoethanol during activity assay. The calculated 1-50% values from the inhibition curves for the E1 isozyme were 5-10 microM and approx. 180 microM for the E2 isozyme. The E3 isozyme was not sensitive to the oxysterol. Judging from the Lineweaver-Burk plot, the inhibition of the E1 isozyme with a constant concentration of cholesta-3,5-dien-7-one (52 microM) appeared to be noncompetitive.     

The mevalonate/isoprenoid pathway inhibitor apomine (SR-45023A) is antiproliferative and induces apoptosis similar to farnesol

Apomine (SR-45023A) is a new antineoplastic compound which is currently in clinical trials and representative of the family of cholesterol synthesis inhibitors 1,1-bisphosphonate esters. Apomine inhibits growth of a wide variety of tumor cell lines with IC(50) values ranging from 5 to 14 microM. The antiproliferative activity of apomine was studied in comparison with that of other inhibitors of the mevalonate/isoprenoid pathway of cholesterol synthesis, simvastatin, farnesol, and 25-hydroxycholesterol. All these compounds inhibit 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity. Apomine (IC(50) = 14 microM), simvastatin (IC(50) = 3 microM), farnesol (IC(50) = 60 microM), and 25-hydroxycholesterol (IC(50) = 2 microM) inhibited HL60 cell growth. Growth inhibition due to simvastatin was reverted by mevalonate, whereas the antiproliferative activity of apomine, farnesol, and 25-hydroxycholesterol was not. Apomine triggered apoptosis in HL60 cells in less than 2 h. Apomine and farnesol induced caspase-3 activity at concentrations similar to their IC(50) values for cell proliferation, whereas a 10-fold excess of simvastatin was necessary to trigger apoptosis compared to its potency on proliferation. Caspase-3 activity was not induced by 25-hydroxycholesterol. The overall similar profile on mevalonate synthesis inhibition, cell growth inhibition, and apoptosis suggests that apomine acts as a synthetic mimetic of farnesol.     

Evoked effects of cholesterol binding on integral proteins and lipid fluidity of dog brain synaptosomal plasma membranes

Binding of cholesterol into dog brain synaptosomal plasma membranes (SPM) within the limits of concentration used (0.5-5 microM) follows an exponential curve described by the general formula y = a.ebx. This curve, which represents the total binding (specific and nonspecific), acquires sigmoid character in the presence of 100 microM cholesterol glucoside, with a Hill coefficient of h = 2.98 +/- 0.18. The specific activity of the Na+/K+-transporting ATPase and Ca2+-transporting ATPase rose after a 2-h preincubation of SPM with cholesterol (up to 5 microM) or its glucoside (up to 50 microM) to at least 50% above their original values. Fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) increased with cholesterol glucoside (50 microM) incorporation. Cholesterol (5 microM) had no effect on the DPH fluorescence polarization. Arrhenius plots of Na+/K+-transporting ATPase activity exhibited a break point at 23.2 +/- 1.1 degrees C in control SPM, which was elevated to 29.5 +/- 1.4 degrees C in SPM treated with cholesterol glucoside (50 microM) and abolished in SPM treated with cholesterol (5 microM). The allosteric properties of SPM-bound Na+/K+-transporting ATPase inhibited by F- and Ca2+-transporting ATPase inhibited by Na+ (as reflected by changes in the Hill coefficient) were modulated by cholesterol. It could be stated that cholesterol glucoside (50 microM) produced an increased packing of the bulk lipids, while cholesterol (5 microM) increased the fluidity of the lipid microenvironment of both Na+/K+-transporting ATPase and Ca2+-transporting ATPase.     

Assay of cholesterol 7 alpha-hydroxylase activity in rat hepatocytes in primary monolayer culture

A sensitive and precise method is described to assay cholesterol 7 alpha-hydroxylase activity in homogenates of rat hepatocytes cultured in monolayers for up to 76 h. The assay is based on measurement of the amount of radioactive cholesterol converted into 7 alpha-[14C]-hydroxycholesterol. Since no subcellular fractionation was applied to measure enzyme activity, this method is rapid and can be performed with cell protein, corresponding to as little as 1 to 2 million hepatocytes. Optimal assay conditions were determined and the reproducibility of this cholesterol 7 alpha-hydroxylase determination was established. Exogenous cholesterol (105 microM), solubilized in Tween 80, was added to saturate the enzyme, giving an apparent Km of 56 microM. Under these conditions, 70% of the cholesterol present in the homogenates is directly accessible to the cholesterol 7 alpha-hydroxylase. The detection limit of the assay was found to be about 10 pmol per incubation. A time course of the cholesterol 7 alpha-hydroxylase activity in cultured hepatocytes revealed that after an initial loss of approximately 60% of the activity as compared with 287 pmol/h/mg for freshly isolated cells, the enzyme activity was increased to the initial level in hepatocytes cultured for 52 h. This result and the finding that the cholesterol 7 alpha-hydroxylase activity was diminished by 94% after a 24-h incubation with 5 microM cycloheximide suggest that the enzyme activity is associated with de novo protein synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of bile acid synthesis. V. Inhibition of conversion of 7-dehydrocholesterol to cholesterol is associated with down-regulation of cholesterol 7 alpha-hydroxylase activity and inhibition of bile acid synthesis.

In the chronic bile fistula rat, the administration of a bolus dose of mevinolinic acid, an inhibitor of HMG-CoA reductase, was followed by rapid down-regulation of cholesterol 7 alpha-hydroxylase activity and a decrease in bile acid synthesis. These observations suggested that either newly synthesized cholesterol or some other metabolite of mevalonate may be involved in the regulation of bile acid synthesis. In order to distinguish between these two alternatives, we carried out experiments in which cholesterol synthesis was blocked by AY9944, a compound that inhibits the conversion of 7-dehydrocholesterol to cholesterol, a last step in the cholesterol biosynthesis pathway. Rats underwent biliary diversion for 72 h at which time they were given intravenously either a bolus dose of AY9944 (1 mg/kg) or control vehicle. At 0 (pre-treatment control), 0.5, 1.5, and 3 h post bolus, livers were harvested and specific activities of cholesterol 7 alpha-hydroxylase were determined. At 1.5, 3, and 6 h post bolus, AY9944 inhibited bile acid synthesis by 19 +/- 6%, 40 +/- 4%, and 41 +/- 6%, respectively, as compared to pretreatment baseline. Cholesterol 7 alpha-hydroxylase activity determined at 0.5, 1.5, and 3 h was decreased by 44 +/- 6%, 44 +/- 2%, and 36 +/- 2%, respectively, as compared to the control value. In in vitro experiments using microsomes from livers of control bile fistula rats, the addition of AY9944 (up to 100 microM) failed to inhibit cholesterol 7 alpha-hydroxylase activity. The results of this study demonstrate that, in the chronic bile fistula rat, acute inhibition of cholesterol synthesis at either early or late steps leads to a rapid down-regulation of cholesterol 7 alpha-hydroxylase activity and decrease in bile acid synthesis.     

Inhibition of protein synthesis by antagonists of calmodulin in Ehrlich ascites tumor cells

Several recent publications indicate that Ca2+ is required for protein synthesis in mammalian cells, including the Ehrlich ascites tumor cell. The present communication examines whether the effects of Ca2+ might be mediated through calmodulin or a related protein. Four calmodulin antagonists belonging to different chemical categories were used to provide evidence of calmodulin involvement. Three of the antagonists inhibited protein synthesis in intact cells; 50% inhibitory concentrations were 10 microM calmidazolium, 12 microM N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W7) and 17.5 microM trifluoperazine (TFP). Initiation was preferentially inhibited as indicated by an increase in the 80S monomers accompanied by a significant disaggregation of polyribosomes. All the antagonists also inhibited protein synthesis initiation in the cell-free protein-synthesizing system; 50% inhibitory concentrations for compound 48/80, calmidazolium, TFP, and W7 were 10 microM, 125 microM, 300 microM and 500 microM, respectively. A weak analogue of W7 inhibited only 20% at 1000 microM. Inhibition in the cell-free system was reversed by the addition of exogenous calmodulin in all four cases. The levels of 43S complexes were significantly elevated with all four antagonists, indicating a block in the utilization of 43S complexes. The similarity of the effects of four distinct classes of antagonists and their ready reversal by exogenous calmodulin leads us to suggest that there may be a role for calmodulin or a very similar calcium-binding protein in protein synthesis.     

Zinc-induced neuronal death in cortical neurons.

Although Zn2+ is normally stored and released in the brain, excessive exposure to extracellular Zn2+ can be neurotoxic. The purpose of the present study was to determine the type of neuronal cell death, necrosis versus apoptosis, induced by Zn2+ exposure. Addition of 10-50 microM ZnCl2 to the bathing medium of murine neuronal and glial cell cultures induced, over the next 24 hrs., Zn2+-concentration-dependent neuronal death; some glial death also occurred with Zn2+ concentrations above 30 microM. The neuronal death induced by 20 microM Zn2+ was characterized by coarse chromatin condensation, the formation of apoptotic bodies, and internucleosomal DNA fragmentation. It was attenuated in cortical cell cultures prepared from mice null for the bax gene, and by the caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-CH2F (ZVAD, 100 microM), but not by the NMDA receptor antagonist, D-2-amino-5-phosphonovalerate (D-APV, 200 microM ). In contrast, the neuronal death induced by 50 microM Zn2+ was characterized by plasma membrane disruption and random DNA fragmentation; this death was attenuated by D-APV, but exhibited little sensitivity to ZVAD or deletion of bax. These results suggest that Zn2+ can induce cell death with characteristics of either apoptosis or necrosis, depending on the intensity of the Zn2+ exposure.     

Adrenergic and calcium-mediated subcellular redistribution of protein kinase C in primary neuronal cultures

Incubation of primary neuronal cultures prepared from the brains of neonatal rats with 50 microM epinephrine resulted in the transient redistribution of protein kinase C from the cytosol to the particulate fraction. This effect occurred after 1 and 5 min of incubation and resulted in a decrease in cytosolic protein kinase C activity with a corresponding increase in particulate protein kinase C of approximately 30% and 15%, respectively. The epinephrine-stimulated translocation of protein kinase C was blocked by 1 microM prazosin indicating the involvement of alpha 1-adrenergic receptors. Further, inclusion of 0.1 microM Ca2+ in the homogenization buffer was found to significantly enhance the binding of protein kinase C to cellular membranes prepared from neuronal cultures. These results indicate that alpha 1-adrenergic receptors in neuronal brain cell cultures are linked to the activation of protein kinase C and that the mobilization of Ca2+ may enhance this effect.     

The hypolipidemic action of antibiotic 86/88 (enniatin B) in a hepatoblastoma G2 cell culture]

A cyclodepsipeptide antibiotic 86/88 (enniatin B) with strong hypolipidemic action was isolated from the culture liquid of the fungus INA F-86/88 identified as Fusarium lateritium Nees var. stilboides (Wr.) Bilai. In the Hep G2 cell culture the antibiotic suppressed 14C-acetate incorporation into cholesterol (IC50 1.75 microM), cholesterol ethers (IC50 1 microM), triglycerides (IC50 1.3 microM) and free fatty acids (IC50 2.2 microM). The most pronounced effect of the drugs, i.e. the suppression of the cholesterol ethers synthesis is likely due not only to the ACAT inhibition but also to the inhibition of the triglyceride synthesis and the diminishing of the free fatty acids pool in the cells.     

The growth inhibitory effect of conjugated linoleic acid on a human hepatoma cell line, HepG2, is induced by a change in fatty acid metabolism, but not the facilitation of lipid peroxidation in the cells

We investigated the growth inhibitory effect of conjugated linoleic acid (CLA) on HepG2 (human hepatoma cell line), exploring whether the inhibitory action occurs via lipid peroxidation in the cells. When the cells were incubated up to 72 h with 5-40 microM of CLA (a mixture of 9c,11t-18:2 and 10t,12c-18:2), cell proliferation was clearly inhibited in a dose and time dependent manner but such an inhibition was not confirmed with linoleic acid (LA). In order to evaluate the possible contribution of lipid peroxidation exerted by CLA to cell growth inhibition, alpha-tocopherol (5-20 microM) and BHT (1-10 microM) as potent antioxidants were added to the medium with CLA (20 microM), which did not restore cell growth at all. Furthermore, after 72 h incubation, the membranous phospholipid hydroperoxide formation in the CLA-supplemented cells was suppressed respectively to 25% and 50% of that in LA-supplemented cells and control cells. No difference was observed by a conventional lipid peroxide assay, the TBA test, between CLA-supplemented cells and LA-supplemented cells. Although the cellular lipid peroxidation was not stimulated, lipid contents (triacylglycerol, total cholesterol and free cholesterol) and fatty acid contents (palmitic acid, palmitoleic acid and stearic acid) markedly increased in CLA-supplemented cells compared with LA-supplemented and control cells. Moreover, supplementation with 20 microM LA and 20 microM arachidonic acid profoundly interfered with the inhibitory effect of CLA in HepG2. These results suggest that the growth inhibitory effect of CLA on HepG2 is due to changes in fatty acid metabolism but not to lipid peroxidation.     

Quantitative determination of cholesterol, sitosterol, and sitostanol in cultured Caco-2 cells by liquid chromatography-atmospheric pressure chemical ionization mass spectrometry

In this study, we describe a simple liquid extraction (methanol/choloroform, 1:1, v/v) method for endogenous free cholesterol and administered sterols extracted from cultured Caco-2 cells. To quantify sterol contents in Caco-2 cells, a new HPLC-APCI-MS method was developed. All the sterols were baseline separated using reversed-phase column (C8, 2.1 mm x 150 mm, 3.5 microm) and isocratic conditions (90%, v/v, methanol-water mixture containing 0.2 mM ammonium acetate). The full scan mass spectra of sterols were measured by an ion trap mass spectrometer equipped with an APCI ion source. The intense fragment ions resulting from the loss of water [M+H-H2O]+ (m/z 369, 395, 397 and 399 for cholesterol, stigmasterol, sitosterol, and sitostanol, respectively) were used for determinations. The absolute extraction recovery of sterols from the spiked cell samples were 109.7+/-26.2, 105.7+/-5.1, 109.8+/-5.0 and 99.0+/-7.0% for cholesterol, stigmasterol, sitosterol, and sitostanol, respectively. Furthermore, no significant matrix effect was observed for the sterols in the cell samples. The sample assay was based on the internal standard method using stigmasterol as an internal standard. The method was linear over the concentration ranges of 0.45-9.0 microM (cholesterol) and 0.225-7.2 microM (sitosterol and sitostanol). The within- and between-day precision was less than 7% and accuracy ranged from 93.51 to 101.77%. The lowest limit of quantitation (LLOQ) was 0.225 microM for sitosterol and sitostanol, and 0.45 microM for cholesterol. The accuracy range was 95-106% and precision was lower than 9% for all LLOQ values.     

Cellular cholesterol efflux is modulated by phospholipid-derived signaling molecules in familial HDL deficiency/Tangier disease fibroblasts

Familial HDL deficiency (FHD) is the heterozygous form of Tangier disease (TD). Mutations of the ABCA1 gene cause FHD and TD. FHD/TD cells are unable to normally efflux cholesterol onto nascent HDL particles, which are rapidly catabolized. TD fibroblasts have an abnormal pattern of PLC and PLD activation following cell stimulation with HDL(3) or apolipoprotein A-I (apoA-I). We examined cellular cholesterol efflux in FHD and TD fibroblasts by phospholipid-derived-molecules, compared with that of normal cells. We used the PKC agonist 1,2-dioctanoylglycerol (DOG) and phorbol myristate acetate (PMA) to activate PKC, calphostin C, and GO 6976, as inhibitors of PKC; phosphatidic acid (PA), which is the product of PLD, and lysophosphatidic acid (LPA), phosphatidylcholine, sphingomyelin, and beta-cyclodextrin to investigate their potential effect in modulating cellular cholesterol efflux in [(3)H]cholesterol-labeled and cholesterol-loaded fibroblasts. Phosphatidylcholine, sphingomyelin, and beta-cyclodextrin promoted cholesterol efflux in an identical fashion in control, FHD, or TD fibroblasts. In a dose-dependent fashion, DOG (0-200 microM) increased apoA-I-mediated cellular cholesterol efflux by 40% in controls, 71% in FHD, and 242% in TD cells. PMA similarly increased cholesterol efflux to a maximum of 256% in controls, 182% in FHD, and 191% in TD cells. This effect was inhibited by calphostin C. PA (100 microM) also increased cholesterol efflux by 25% in control, 44% in FHD, and 100% in TD cells. Conversely, LPA reduced cholesterol efflux in a dose-dependent fashion in control and FHD cells (-50%, 200 microM) but not in TD cells, where efflux was increased by 140%. Propranolol (100 microM) significantly increased cholesterol efflux at 24 h in all three cell lines. n-Butanol partially decreased the DOG-mediated increase in cholesterol efflux. The inhibitory effect of calphostin C on DOG-stimulated cholesterol efflux could be partially overcome by propranolol, suggesting that PA is a downstream mediator of PKC-stimulated cholesterol efflux.We conclude that PLC and PLD activities are required for apoA-I-mediated cellular cholesterol efflux, and modulating cellular PA concentration can correct, at least partially, the cholesterol efflux defect in FHD and TD.