12-O-tetradecanoyl phorbol 13-acetate stimulates the myristylation of an approximately 82 kDa protein in HL-60 cells

We have studied protein acylation using [3H]myristate in the two leukemia cell lines HL-60 and HL-60 Blast II. The latter is a variant which does not differentiate after treatment with 12-O-tetradecanoyl phorbol 13-acetate (TPA). The acylation profiles of the two cell lines as examined by SDS-PAGE differed. TPA induced the myristylation of an approximately 82 kDa protein in the sensitive cells, but not in the resistant cells. Myristic acid was shown to be covalently linked to these proteins. Analysis of the cell lipids labelled with [3H]myristate showed that in contrast to observations with the proteins, the changes induced by TPA were observed in both TPA-sensitive and TPA-resistant cells. We conclude that the induction of myristylation may be an important step in the mechanism of differentiation.     

Changes in sterol metabolism in the skin of developing chick embryo and alterations in the presence of an anticholesterolemic agent and a chemical carcinogen

Changes in sterol metabolism in the skin of chick embryo during its development were studied with embryonal chick skin and with the cultured skin tissues. Changes in sterol metabolism of the skin of chick embryo began to appear at day 17, as observed by the accumulation of dihydrolanosterol, and the ratio of dihydrolanostrol:cholesterol increased thereafter until hatching. A similar change in sterol metabolism was also observed with the cultured skin tissue of chick embryo, although the stages of development seem to have been delayed by 3 days. The active sterol metabolism of the cultured skin tissue was also confirmed by studies of incorporation of [2-14C]acetate into sterols. 20,25-Diazacholesterol almost completely inhibited the incorporation of [2-14C]acetate into C27 sterols, whereas a chemical carcinogen, 4-hydroxyaminoquinoline 1-oxide, inhibited the incorporation of [2-14C]acetate into lathosterol but not that into cholesterol.     

Reversal of cerulenin-induced inhibition of phospholipids and sterol synthesis by exogenous fatty acids/sterols in Epidermophyton floccosum

Cerulenin, a specific inhibitor of fatty acids and sterol biosynthesis inhibited the growth of Epidermophyton floccosum, which was reversed when growth medium was supplemented with palmitic acid and sterols. Unsaturated fatty acids partially restored the growth. Cerulenin inhibited both phospholipid and sterol biosynthesis (60-70%) at the minimum inhibitory concentration (0.5 microgram/ml) as demonstrated by [32P]orthophosphoric acid and [14C]acetate incorporation into the respective lipids. Cerulenin-induced inhibition of phospholipid and sterol synthesis was dose dependent up to 0.5 microgram/ml. Exogenously supplied fatty acids and sterols restored the biosynthesis of phospholipids in cerulenin-treated cultures, while that of sterols was enhanced. The biosynthesis of both saturated and unsaturated fatty acids was inhibited by cerulenin.     

The effect of carbon monoxide on the nature of the accumulated 4,4-dimethyl sterol precursors of cholesterol during its biosynthesis from [2-14C]mevalonic acid in vitro

Cholesterol biosynthesis was studied in rat liver subcellular fractions incubated with dl-[2-(14)C]mevalonic acid under gas phases consisting of either N(2)+O(2) (90:10) or CO+O(2) (90:10). CO inhibits cholesterol biosynthesis from [2-(14)C]mevalonic acid and results in a large accumulation of radioactive 4,4-dimethyl sterols. Separation of the components of the 4,4-dimethyl sterol fraction showed that lanosterol and dihydrolanosterol are the major components that accumulate during cholesterol biosynthesis in an atmosphere containing CO, whereas 14-demethyl-lanosterol and 14-demethyldihydrolanosterol are the major components of the much less intensely radioactive 4,4-dimethyl sterol fraction isolated from incubations with N(2)+O(2) as the gas phase. The identities of lanosterol, dihydrolanosterol and 14-demethyldihydrolanosterol were confirmed by both radiochemical and physicochemical methods, including g.l.c. and combined g.l.c.-mass spectrometry. CO therefore results in a qualitative as well as a quantitative difference in the 4,4-dimethyl sterol fraction which arises during cholesterol biosynthesis from mevalonic acid. The specific radioactivity of the [(14)C]lanosterol biosynthesized in the presence of CO was lower than that of its companion, [(14)C]dihydrolanosterol. The relative amounts of 4,4-dimethyl-Delta(24)-sterols and 4,4-dimethyl-24,25-dihydrosterols present in each type of incubation suggest that enzymic reduction of the sterol side chain occurs predominantly at a stage after that of lanosterol.     

Inhibition by the fungicide fenpropimorph of cholesterol biosynthesis in 3T3 fibroblasts.

Fenpropimorph (N-[3-(p-t-butylphenyl)-2-methylpropyl]-cis-2,6-dimethylmorpholine), a morpholine fungicide known to be an inhibitor of sterol biosynthesis in fungi and in higher plants, was demonstrated to be an efficient inhibitor of cholesterol biosynthesis in cultured Swiss 3T3 fibroblasts. Treatment of the mammalian cells with fenpropimorph resulted in a dose-dependent inhibition of [14C]acetate incorporation into the C27 sterols [IC50 (concentration causing half-maximal inhibition) = 0.5 microM], which was accompanied by an accumulation of polar sterols and a decrease in cellular hydroxymethylglutaryl-CoA reductase activity. Exposure of the cells to the drug affected cell growth. Analysis of the sterols in the growth-arrested and in the pulse-labelled cells indicate that fenpropimorph has, in the sterol-biosynthetic pathway, target enzymes in mammalian cells different from those in the other phyla. Whereas in plants and fungi fenpropimorph mainly affects sterol isomerases and reductases, in the fibroblasts its main target seems to be the demethylation of lanosterol.     

Chloroquine inhibits cyclization of squalene oxide to lanosterol in mammalian cells

Chloroquine inhibits the incorporation of [14C]acetate into sterols at a concentration of 10 microM or more in mouse L cells but has no effect on fatty acid synthesis and CO2 production from the same substrate even at a 10-fold higher concentration of the drug. The site of inhibition is distal to the formation of mevalonate since chloroquine also inhibits [14C]mevalonate metabolism to sterols and does not decrease the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.34) or the incorporation of [14C]acetate into the total nonsaponifiable lipids. Analyses by thin layer and high pressure liquid chromatography of the nonsaponifiable lipid fraction from cultures incubated with chloroquine show an accumulation of radioactivity in the region of squalene oxide. Identification of the radiolabeled lipid as squalene oxide has been established by: (a) its co-migration with the authentic squalene oxide standard; (b) its conversion into squalene glycol by acid hydrolysis; and (c) its further metabolism to desmosterol when chloroquine is removed from the medium. Addition of chloroquine (12.5-50 microM) to 20,000 X g supernatant fractions of mouse liver homogenates inhibits the incorporation of [14C]mevalonolactone into cholesterol and lanosterol, with corresponding increases of [14C]squalene oxides, in a concentration-dependent manner. It appears, therefore, that chloroquine inhibits the enzymatic step catalyzed by 2,3-oxidosqualene-lanosterol cyclase (EC 5.4.99.7). Incubation of cell cultures with chloroquine (50 microM) arrests cell growth and causes cell death after 1-3 days. However, simultaneous incubation of chloroquine with either cholesterol or lanosterol prevents cell death and permits cell growth. Uptake of chloroquine is not affected by exogenous sterols since intracellular chloroquine concentrations are the same in cells grown with or without added sterols. The cytotoxicity of chloroquine, under our experimental conditions, must, therefore, be due primarily to its inhibition of sterol synthesis. In addition to its well known effect on protein catabolism, chloroquine has been found to inhibit protein synthesis. The significance of these findings concerning the use of chloroquine in studying the regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity is discussed.     

Sterol biosynthesis in the echinoderm Asterias rubens.

1. [2(-14)C]Mevalonic acid injected into the echinoderm Asterias rubens (Class Asteroidea) was effectively incorporated into the non-saponifiable lipid. 2. The most extensively labelled compounds were squalene and the 4,4-dimethyl sterols with much lower incorporations into the 4alpha-monomethyl and 4-demethyl sterol fractions. 3. Labelled compounds identified were squalene, lanosterol, 4,4-dimethyl-5alpha-cholesta-8,24-dien-3beta-ol and 4alpha-methyl-5alpha-cholest-7-en-3beta-ol; these are all intermediates in sterol biosynthesis. 4. The major sterol in A. rubens, 5alpha-cholest-7-en-3beta-ol, was also labelled showing that this echinoderm is capable of sterol biosynthesis de novo. 5. No evidence was obtained for the incorporation of [2(-14)C]mevalonic acid into the C28 and C29 components of the 4-demethyl sterols or 9beta,19-cyclopropane sterols found in A. rubens and it is assumed that these sterols are of dietary origin. 6. Another starfish Henricia sanguinolenta also incorporated [2(-14)C]mevalonic acid into squalene and lanosterol. 7. Various isolated tissues of A. rubens were all capable of incorporation of [2(-14)C]mevalonic acid into the nonsaponifiable lipid. With the body-wall and stomach tissues radioactivity accumulated in squalene and the 4,4-dimethyl sterols, but with the gonads and pyloric caecae there was a more efficient incorporation of radioactivity into the 4-demethyl sterols, principally 5alpha-cholest-7-en-3beta-ol.     

Cell-free transfer of sterols from dictyosome-like structures to plasma membrane vesicles of guinea pig testes

Summary Transfer of radiolabeled lipids from dictyosome-like structures (DLS) from testis tubules of the guinea pig as donor to unlabeled plasma membrane from testis tubules immobilized on nitrocellulose as acceptor was studied in a completely cell-free system. As a general label for lipids of the donor DLS, isolated testis tubules were incubated with [¹⁴C]acetate. Time- and temperature-dependent transfer of [¹⁴C]acetate labeled constituents was observed in the cellfree system. However, despite the fact that phospholipids and other constituents were highly labeled in the donor fraction, primarily radioactive sterols were transferred to the plasma membrane acceptor vesicles. Transfer at 37°C represented 0.4 to 0.7% of the total radiolabeled cholesterol at 37°C but little or no transfer occurred at 4°C. The sterols transferred exhibited Chromatographic mobilities corresponding to those of cholesterol and lanosterol. Similar results were obtained with [¹⁴C]mevalonic acid. In subsequent experiments, cholesterol transfer from DLS to plasma membrane was demonstrated by incubation of DLS with [³H]squalene which was converted into sterol or with [¹⁴C]cholesterol. Transfer of sterols required ATP, but not cytosol, and was both time- and temperature-dependent. DLS were more effective than either endoplasmic reticulum or plasma membrane as the donor fraction. The results from the cell-free analysis suggest a possible functional role of the DLS in sterol biogenesis and transfer to the plasma membrane during spermatid development.Abbreviations DLS dictyosome-like structure(s) - PBS phosphatebuffered saline - HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - BSA bovine serum albumin     

Lipid synthesis in inositol-starved Saccharomyces cerevisiae

Lipid synthesis was analyzed in an inositol-requiring mutant of Saccharomyces cerevisiae (MC13). Both rates and cellular amounts of [U-14C]acetate incorporation into phospholipids, triacylglycerols, free sterols and steryl esters were elevated in an inositol-starved culture compared to the supplemented control at a time when the deprived culture was losing viability (inositol-less death). The rates at a later time were greatly reduced. During the period when de novo lipid synthesis was high in the starved culture, phospholipid turnover and presumed conversion to triacylglycerols was also accelerated; no differences were apparent in the turnover of the sterol fractions between the two cultures. No change in the fractional percent of ergosterol or of the sterol precursors could be attributed to inositol starvation. The synthesis and maintenance of membrane lipids (phospholipids and free sterols) and their coupling in cellular metabolism are discussed in light of these results.     

Lanosterol 14 alpha-demethylase (P45014DM): effects of P45014DM inhibitors on sterol biosynthesis downstream of lanosterol

Lanosterol 14 alpha-demethylase (P45014DM) is the cytochrome P450 enzyme complex responsible for an early step in cholesterol biosynthesis, namely the 14 alpha-demethylation of lanosterol. We have synthesized a novel series of steroidal substrate analogues, designed to be specific and potent inhibitors of P45014DM. We describe here the effects of these compounds on sterol biosynthesis downstream from lanosterol, focusing ultimately on their efficacy as inhibitors of cholesterol biosynthesis. Results using a radio-high performance liquid chromatography (HPLC) assay show that in rat liver microsomal preparations, with [24,25-3H]dihydrolanosterol as substrate, the compounds do indeed inhibit the biosynthesis of sterols downstream from lanosterol. A range of inhibitory potencies was observed, and the key enzyme being inhibited was believed to be P45014DM. Inhibitor efficacy was readily correlated with non-metabolized [24,25-3H]dihydrolanosterol, formation of 4,4-dimethyl-cholest-8-en-3 beta-ol, and formation of lathosterol, a sterol believed to be an excellent indicator of whole body cholesterol biosynthesis in humans.     

Ketone body utilization for energy production and lipid synthesis in isolated rat brain capillaries

Isolated brain capillaries from 2-month-old rats were incubated for 2 h in the presence of [3-14C]acetoacetate, D-3-hydroxy[3-14C]butyrate, [U-14C]glucose, [1-14C]acetate or [1-14C]butyrate. Labelled CO2 was collected as an index of oxidative metabolism and incorporation of label precursors into lipids was determined. The rate of CO2 production from glucose was slightly higher than from the other substrates. Interestingly, acetoacetate was oxidized at nearly the same rate as glucose. This shows that ketone bodies could be used as a source of energy by brain capillaries. Radiolabelled substrates were also used for the synthesis of lipids, which was suppressed by the addition of albumin. The incorporation of [U-14C]glucose in total lipids was 10-times higher than that from other precursors. However, glucose labelled almost exclusively the glycerol backbone of phospholipids, especially of phosphatidylcholine. Ketone bodies as well as glucose were incorporated mainly into phospholipids, whereas acetate and butyrate were mainly incorporated into neutral lipids. The contribution to fatty acid synthesis of various substrates was in the following order: butyrate greater than or equal to acetate greater than ketone bodies greater than or equal to glucose. All precursors except glucose were used for sterol synthesis. Glucose produced almost exclusively the glycerol backbone of phospholipids.     

Sterol biosynthesis via cycloartenol and other biochemical features related to photosynthetic phyla in the amoeba Naegleria lovaniensis and Naegleria gruberi

The sterols and sterol precursors of two amoebae of the genus Naegleria, Naegleria lovaniensis and Naegleria gruberi were investigated. Cycloartenol, the sterol precursor in photosynthetic organisms, is present in both amoebae. In N. lovaniesis, it is accompanied by lanosterol and parkeol, as well as by the 24,25-dihydro derivatives of these triterpenes. One of the most striking features of these amoebae is the accumulation of 4 alpha-methylsterols which are present in similar amounts as those of 4,4-desmethylsterols (3-5 mg/g, dry weight). 4 alpha-Methylergosta-7,22-dienol was identified as a new compound. Ergosterol was the major 4,4-desmethylsterol, accompanied by small amounts of C27 and other C28 sterols. Treatment of N. lovaniensis with fenpropimorph modified the sterol pattern of this amoeba and inhibited its growth. This fungicide, known to inhibit steps of sterol biosynthesis in fungi and plants, induced the disappearance of 4 alpha-methyl-delta 7-sterols and the appearance of the unusual delta 6,8,22-ergostatrienol as in A. polyphaga. These results might be explained by a partial inhibition of the delta 8----delta 7 isomerase, the small amounts of delta 7-sterols formed being converted into ergosterol which is still present in fenpropimorph-exposed cells. De novo sterol biosynthesis in N. lovaniensis was shown by incorporation of [1-14C]acetate into sterols and sterol precursors, especially cycloartenol. Lanosterol and parkeol were not significantly labelled. Furthermore, [3-3H]squalene epoxide was efficiently cyclized by a cell-free system of this amoeba into cycloartenol, and again no significant radioactivity was detected in lanosterol and parkeol. This shows that cycloartenol, the sterol precursor in plants and algae, is also the sterol precursor in Naegleria species, and that these amoebae, like A. polyphaga, are related by some biosynthetic pathways to photosynthetic phyla. Lanosterol, the sterol precursor in non-photosynthetic phyla (animal and fungi) and parkeol are more likely dead-ends of this biosynthetic pathway. The peculiar phylogenetic position of these protozoa was further emphasized by the action of indole acetic acid and other auxine-like compounds on their growth. Indeed amoebic growth was enhanced in the presence of these higher plant growth hormones. The differences in the sterol composition of the protozoa we have hitherto examined is related to their sensitivity toward polyene macrolide antibiotics.(ABSTRACT TRUNCATED AT 400 WORDS)     

The effects of temperature acclimation on membrane sterols and phospholipids of Neurospora crassa

Experiments were conducted to examine the effects of temperature acclimation on sterol and phospholipid biosynthesis in Neurospora crassa. Cultures grown at high (37 degrees C) and low (15 degrees C) temperatures show significant differences in free and total sterol content, sterol/phospholipid ratios and distribution of major phospholipid species in total lipids and two functionally distinct membrane fractions. The ratio of free sterols to phospholipids in total cellular lipids from 15 degrees C cultures was found to be about one-half that found at 37 degrees C, whereas sterol/phospholipid ratios of mitochondrial and microsomal membranes were found to be higher at the low growth temperature. Total sterol and phospholipid biosynthetic rates showed parallel reductions in cultures acclimating to a shift from 37 to 15 degrees C growth conditions. Distribution of [14C]acetate label into free sterols was significantly lower under these conditions, however; indicating an increase in the conversion rate of sterols to sterol esters at the lower temperature. Mitochondrial and microsomal membrane fractions showed distinct phospholipid distributions which also differed from total lipid distributions at the two growth temperatures. In each case there was a consistent decrease in phosphatidylcholine and a corresponding increase in phosphatidylethanolamine as growth temperatures were lowered.     

Plasma membrane phospholipid and sterol synthesis in soybean hypocotyl segments undergoing auxin-induced elongation

Summary Auxin-induced cell elongation necessitates plasma membrane enlargement. The effect of auxin (10 M 2,4-dichlorophenoxyacetic acid) treatment on amount, composition, and rate of synthesis of plasma membrane lipids was examined. Auxin-treated and control soybean (Glycine max L.) hypocotyl segments were incubated with [¹⁴C]acetate for times ranging from 0.5 to 18 h, prior to isolation of plasma membrane by aqueous two-phase partitioning. The composition of individual plasma membrane lipids in elongating segments did not differ from the composition in treatment time-matched control segments, except that after longer auxin treatments, phospholipids had more unsaturated fatty acids. Plasma membrane phospholipid and free sterol content both increased in elongating segments. The relative proportion of sterols and phospholipids in the plasma membrane primarily depended on time after segment excision, for both auxin-treated and control segments. Auxin enhanced the rate of lipid incorporation into the plasma membrane by 6 h, and stimulated the synthesis of some phospholipids and sterols.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - ER endoplasmic reticulum - GC gas chromatography - IAA indole-3-acetic acid - PA phosphatidic acid - PC phosphatidylcholine - PE phosphatidylethanolamine - PG phosphatidylglycerol - PI phosphatidylinositol - PM plasma membrane - PS phosphatidylserine     

Effects of the calcium ionophore A 23187 on low-density lipoprotein processing and lipid metabolism in cultured human fibroblasts

Pretreatment of cultured human fibroblasts with the calcium ionophore A 23187 resulted in a decrease in low-density lipoprotein internalization. This effect was dose-dependent and did not occur in a medium devoid of calcium. About 2-fold reduction was observed with 10(-5)M A 23187. In contrast, the low-density lipoprotein binding was only slightly affected. The incorporation of [14C]acetate and [14C]oleate into all classes of lipids (sterol, triacylglycerols and phospholipids) was strikingly reduced by ionophore pretreatment.     

Absolute rates of cholesterol synthesis in extrahepatic tissues measured with 3H-labeled water and 14C-labeled substrates.

This study was undertaken to develop techniques for measuring absolute rates of sterol synthesis in extrahepatic tissues in vitro and to estimate the magnitude of the errors inherent in the use of various 14C-labeled substrates for such measurements. Initial studies showed that significant errors were introduced when rates of synthesis were estimated using [3H]water since about 20 nmol of water were bound to each mg of tissue cholesterol isolated as the digitonide. This source of error could be eliminated by subtracting apparent incorporation rates obtained at 0 degrees C from those obtained at 37 degrees C or by regenerating and drying the free sterol. In a second set of experiments, the H/C incorporation ratio in cholesterol was determined in the liver by measuring the absolute rates of hydrogen and acetyl CoA flux into sterols. The ratio of 0.69 +/- 0.03 was found to be independent of the rate of hepatic cholesterol synthesis, the rate of hepatic acetyl CoA generation, or the source of the acetyl CoA. In a third set of studies, rates of incorporation of [3H]water or 14C-labeled acetate, octanoate, and glucose into digitonin-precipitable sterols were simultaneously measured in nine different extrahepatic tissues. Assuming that the H/C ratio measured in the liver also applied to these tissues, the [3H]water incorporation rates were multipled by the reciprocal of the H/C ratio to give the absolute rates of sterol synthesis in each tissue. When these were compared to the incorporation rates determined with the 14C-labeled substrates the magnitude of the errors in the rates of sterol synthesis obtained with these substrates in each tissue could be assessed. Only [14C]octanoate gave synthesis rates approaching 100% of those obtained with [3H]water and this occurred only in the intestine and kidney; in the other extrahepatic tissues this substrate gave rates of 6--66+ of the absolute rates. Rates of [14C]acetate incorporation in sterols varied from 4 to 62% of the [3H]water incorporation rates while those obtained with [14C]glucose were only 2--88% of the true rates. These studies document the large and highly variable errors inherent in estimating rates of sterol synthesis in extrahepatic tissues using 14C-labeled substrates under in vitro conditions.     

A mammalian mutant cell lacking detectable lanosterol 14 alpha-methyl demethylase activity

A Chinese hamster ovary cell mutant, AR45, was selected for amphotericin B resistance after treatment with the mutagen ethyl methanesulfonate. The mutant is a cholesterol auxotroph with a deficiency in cholesterol biosynthesis. Whole cell experiments demonstrate that the mutant accumulates the C30 sterols, lanosterol and dihydrolanosterol, under culture conditions which promote active sterol biosynthesis. Metabolic studies show that the C29 sterol demethylation product of lanosterol, but not lanosterol itself, is actively converted to end product cholesterol by whole cells as well as by microsomal preparations derived from the mutant. Detectable amounts of several cytochromes can be observed spectrally in the AR45 demonstrating that it is not a general heme-deficient mutant. Collectively, these results characterize the AR45 mutant cells as being lanosterol 14 alpha-methyl demethylase-deficient. The cell line should prove useful in studying regulation of the demethylase enzyme and the putative endogenous regulatory oxysterol. It should also be a useful tool in the molecular cloning and elucidation of genetic properties of the demethylase.     

The effects of retinoic acid on [14C]acetate incorporation into lipids of normal and transformed hamster fibroblasts

This study examined the effects of retinoic acid (RA) on [14C]acetate incorporation and fatty acid composition of hamster embryo fibroblasts (HEF) and two cell lines derived from the same inbred strain but transformed by herpes simplex-2 virus (HSV) or polyoma virus (HFT). Cells were exposed to all trans RA, or dimethylsulfoxide (DMSO), the vehicle for RA, and the lipids labeled with [14C]acetate. Lipids were extracted from the cells, separated by paper chromatography, located by autoradiography, and acetate incorporation determined by liquid scintillation spectrometry. The distribution of fatty acids in total cell lipids was examined by gas chromatography. HEF cells incorporated more acetate into cholesterol than either transformed cell type. The HFT line incorporated more acetate into triglycerides and less into total phospholipids than either the HSV line or the HEF line. RA caused a significant decrease in incorporation of acetate into cholesterol and sphingomyelin in all three cell lines. HEF and HSV cells had decreased incorporation into phosphatidyl inositol-phosphatidyl serine and increased incorporation into triglycerides, changes not evident in the HFT cell. The control fatty acid profiles of the HEF and HSV cells were similar, while the HFT cells had a larger proportion of C16:0 and 18:1 fatty acids. Following treatment with RA all three cell types showed an increase in palmitic and a decrease in oleic acids. The three related cell types showed different [14C]acetate labeling patterns which did not respond uniformly to RA. On the other hand, exposure elicited some like responses in all cell types.     

Metabolic fate of [14C]-ethanol into endothelial cell phospholipids including platelet-activating factor, sphingomyelin and phosphatidylethanol

The metabolic fate of ethanol into the phospholipid pool of calf pulmonary artery endothelial cells was studied. [14C]-ethanol was incorporated into various endothelial cell phospholipids including phosphatidylethanol (PEth), which may represent a substantial fraction in microdomains of membrane phospholipids. The incorporation into phospholipids was reduced in the presence of pyrazole and cyanamide, inhibitors of ethanol metabolism. Wortmannin, the phosphatidylinositol 3-kinase inhibitor, increased [14C]-PEth formation. [3H]-acetate was also incorporated into endothelial cell phospholipids but in a different pattern. Distribution of [3H]-acetate and [14C]-ethanol into the fatty acyl moiety versus the glycerophosphoryl backbone of the phospholipids was also different. Stimulation of the endothelial cells with ATP increased [3H]-acetate incorporation into platelet-activating factor (PAF) and ethanol decreased it. Ethanol exposure increased ATP-stimulated [3H]-acetate incorporation into sphingomyelin. However, ATP had no effect on the incorporation of [14C]-ethanol into any phospholipids. The results suggest that the two precursors contribute to a separate acetate pool and that the sphingomyelin cycle may be sensitized in ethanol-treated cells. Thus, metabolic conversions of ethanol into lipids and the effect of ethanol on specific lipid mediators, e.g PAF, PEth and sphingomyelin, may be critical determinants in the altered responses of the endothelium in alcoholism.