Staphylococcal enterotoxin-B (SEB) alters [14C]-choline transport and phosphatidylcholine metabolism in cultured human kidney proximal tubular cells

We studied the effects of SEB on [¹⁴C]-choline transport and metabolism of choline containing phospholipids in cultured human kidney proximal tubular (PT) cells. SEB increased the uptake of [¹⁴C]-choline in PT cells as a function of toxin concentration, incubation time, and pH. The maximum increase in uptake (3.5–5-fold compared to control) was observed at a toxin concentration of 10 ug/10⁴ cells, at 4 h and at pH 7.4. Two toxins structurally related to SEB, Staphylococcal enterotoxin-A and toxic shock toxin (TST-1) failed to alter [¹⁴C]-choline uptake in PT cells, a finding which indicates that SEB-mediated alteration in choline uptake in PT cells has high specificity.We found that SEB markedly and significantly increased the incorporation of [¹⁴C]-choline into phosphatidylcholine, Iysophosphatidylcholine and sphingomyelin, but not into phosphatidylethanolamine. Maximum increase in the incorporation of [¹⁴C]-choline into phosphatidlycholine (3-fold compared to control) was observed at 4 h after incubation with toxin. In contrast, SEB did not alter the incorporation of [¹⁴C]-choline in phosphatidylethanolamine. The cellular level of phosphatidylcholine was also increased (2-fold compared to control) in PT cells incubated with SEB. This was accompanied by a 3-to-4-fold increase in CTP: phosphocholine, cytidyltransferase activity.In sum, SEB specifically stimulates phosphatidylcholine synthesis in PT cells by increasing choline uptake or by activating CTP: phosphocholine, cytidyltransferase, or both. We believe this is the first-ever report indicating that a toxin can increase phosphatidylcholine synthesis. This high order of specificity may be in part due to the presence of a glycosphingolipid receptor in PT cells that specifically binds SEB but not SEA or TST-1. Accordingly, it is tempting to speculate that the receptor may somehow be involved in the SEB-mediated regulation of phosphatidylcholine synthesis.Abbreviations SEB Staphylococcal entertoxin-B - SEA Staphylococcal enterotoxin-A - TST-1 Toxic shock syndrome toxin-1 - PT Proximal tubular - PC Phosphatidylcholine - SM Sphingomyelin - LPC Lysophosphatidyl-choline - CT Cytidyltransferase     

Synthesis and transport of different sphingomyelin species in rat Sertoli cells

Cellular phospholipids of Sertoli cells from immature rats were labeled with [¹⁴C]-choline. Two sphingomyelin bands (SM1 and SM2) were identified by TLC. The incorporation of [¹⁴C]-choline over a 45 h period of incubation demonstrated that there are differences in labeling kinetics between SM1 and SM2. The subcellular location of SM1 and SM2 was investigated by accessibility to bacterial sphingomyelinase. The results showed the existence of two SM pools in Sertoli cells, but an equal cellular distribution of SM1 and SM2. SM2 is characterized by a relatively high content of unsaturated fatty acids. The inhibition of vesicular flow by monensin determines a decrease of about 60–70% in incorporation into SM1 and SM2, suggesting the existence of at least two sites of sphingomyelin synthesis. Pulse-chase and time-course experiments indicated a phosphatidylcholine SM precursor product relationship and differences in kinetic properties between SM1 and SM2. Resynthesis experiments showed that monensin had only a partial inhibitory effect on SM1 resynthesis, and a second sphingomyelinase treatment demonstrated that the resynthesized fraction reached the outer leaflet of the plasma membrane. The 60–70% inhibition of SM synthesis by monensin showed that the trans-Golgi cisternae and the trans-Golgi network are the most likely sites of bulk SM synthesis, and that about 15% of SM was synthesized in the cis/medial Golgi apparatus. Additionally the results indicated that plasma membrane SM synthase activity could be the site of about 15% of SM synthesis in Sertoli cells.     

Phospholipid metabolism in boar spermatozoa and role of diacylglycerol species in the De Novo formation of phosphatidylcholine

We have investigated pathways of lipid metabolism in boar spermatozoa sperm cells incubated for up to 3 days with [¹⁴C]palmitic acid, [¹⁴C]glycerol, [¹⁴C]choline, or [¹⁴C]arachidonic acid or incorporated these precursors into diglycerides and/or phospholipids. When spermatozoa were incubated with [¹⁴C]palmitic acid or [¹⁴C]glycerol, there was first an incorporation into phosphatidic acid, followed by labelling of 1,2-diacylglycerol (DAG) and then phosphatidyl-choline (PC). This indicates that the de novo pathway of phospholipid synthesis is active in these cells. However, not all DAG was converted to PC. A pool of di-saturated DAG, which represented a considerable proportion of the high basal levels of DAG, accumulated the majority of label. Another DAG pool, containing saturated fatty acids in position 1 and unsaturated fatty acids in position 2 and representing the remaining basal DAG, was in equilibrium with PC. When spermatozoa were incubated with [¹⁴C]arachidonic acid, there was a considerable incorporation of label into PC, which indicates the presence of an active deacylation/reacylation cycle. The behaviour of certain lipid pools varied depending on the temperature at which spermatozoa were incubated. For example, in the presence of [¹⁴C]palmitic acid or [¹⁴C]arachidonic acid, there was more incorporation of label into PC when spermatozoa were incubated at 25°C than when incubated at 17°C. Taken together, these results indicate that spermatozoa have an active lipid synthetic capacity. It may therefore be possible to design methods to evaluate the metabolic activity of boar spermatozoa based on the incorporation of lipid precursors under standardized conditions. Mol. Reprod. Dev. 47:105–112, 1997. © 1997 Wiley-Liss, Inc.     

Ethanol inhibits phosphatidylcholine and phosphatidylethanolamine biosynthesis in human leukemic monocyte-like U937 cells

The effect of ethanol (ETOH) on the incorporation of [¹⁴C]oleic acid (18:1) into lipid in human monocyte-like U937 cells was investigated. With increasing time of exposure to ETOH, the percentage of the label distributed into neutral lipid (NL) declined from 35 per cent (3 h) to 10 per cent (24 h) accompanied by increased incorporation into phospholipid (PL). [¹⁴C] 18 : 1 was preferentially incorporated into triglyceride (TG) and phosphatidylcholine (PC), comprising over 65 per cent and 50 per cent of the label associated with NL and PL, respectively. Low concentrations of ETOH (⩽ 1·0 per cent; v/v) had no effect. At concentrations greater than 1·5 per cent, there was enhanced incorporation into TG and diacylglycerol (DAG) in a 24-h incubation period, while at 16 h the label in phosphatidylethanolamine (PE) was decreased. The effect of ETOH on the CDP-choline or ethanolamine pathway was examined by monitoring the incorporation of [³H]choline or [¹⁴C]ethanolamine into PC or PE, respectively. At low concentrations ETOH had no effect on either choline uptake or the incorporation into PC. Higher concentrations (≥ 1·5 per cent) for 3 and 6 h resulted in a slightly decreased choline uptake, and the reduction (40–50 per cent) of incorporation into PC suggests that the CDP-choline pathway was inhibited. There was a similar inhibition of the incorporation of [¹⁴C]ethanolamine into PE. When the cells were incubated for 3 h in the presence of 2 per cent ETOH and with labelled 18 : 1 and PL-base, the ratios of incorporation (base/18 : 1) into PC and PE fractions decreased, indicating that the major inhibition lay in blockage of the availability of the base moiety for PL formation. Analysis of the distribution of the label into metabolites revealed that ETOH inhibited the conversion of [¹⁴C] ethanolamine into [¹⁴C]phosphorylethanolamine. The reduction in incorporation was not due to the enhanced breakdown of base-labelled PL. Our results indicate that ETOH has an inhibitory effect on the CDP-choline or ethanolamine pathway.     

Rapid Methylation of Cell Proteins and Lipids in Trypanosoma brucei

ABSTRACT. The fate of the [methyl-¹⁴C] group of S-adenosylmethionine (AdoMet) in bloodstream forms of Trypanosoma brucei brucei, was studied. Trypanosomes were incubated with either [methyl-¹⁴C]methionine, [U-¹⁴C]methionine, S-[methyl-¹⁴C]AdoMet or [³⁵S]methionine and incorporation into the total TCA precipitable fractions was followed. Incorporation of label into protein through methylation was estimated by comparing molar incorporation of [methyl-¹⁴C] and [U-¹⁴C]methionine to [³⁵S]methionine. After 4-h incubation with [U-¹⁴C]methionine, [methyl-¹⁴C]methionine or [³⁵S]methionine, cells incorporated label at mean rates of 2,880 pmol, 1,305 pmol and 296 pmol per mg total cellular protein, respectively. Cells incubated with [U-¹⁴C] or [methyl-¹⁴C]methionine in the presence of cycloheximide (50 μg/ml) for four hours incorporated label eight- and twofold more rapidly, respectively, than cells incubated with [³⁵S]methionine and cycloheximide. [Methyl-¹⁴C] and [U-¹⁴C]methionine incorporation were > 85% decreased by co-incubation with unlabeled AdoMet (1 mM). The level of protein methylation remaining after 4-h treatment with cycloheximide was also inhibited with unlabeled AdoMet. The acid precipitable label from [U-¹⁴C]methionine incorporation was not appreciably hydrolyzed by DNAse or RNAse treatment but was 95% solubilized by proteinase K. [U-¹⁴C]methionine incorporated into the TCA precipitable fraction was susceptible to alkaline borate treatment, indicating that much of this label (55%) was incorporated as carboxymethyl groups. The rate of total lipid methylation was found to be 1.5 times that of protein methylation by incubating cells with [U-¹⁴C]methionine for six hours and differential extraction of the TCA lysate. These studies show T. b. brucei maintains rapid lipid and protein methylation, confirming previous studies demonstrating rapid conversion of methionine to AdoMet and subsequent production of post-methylation products of AdoMet in African trypanosomes.     

Differential effects of unsaturated fatty acids on phospholipid synthesis in human leukemia monocytic U937 cells

The biosynthesis of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in monocyte-like leukemia U937 cells was monitored by adding [³H]choline, [¹⁴C]ethanolamine or [¹⁴C]glycerol to the culture media; incorporation into phospholipid (PL) increased with time. The effect of unsaturated fatty acids (UFA) on PC and PE synthesis was investigated by pretreating U937 cells for 72h with 10 μM 18:1 (n –9), 18:2 (n –6), 18:3 (n –3), 20:4 (n –6) and 20:5 (n –3). The UFA caused no alteration in cell growth, as evidenced by light microscopy and the incorporation of [³H]thymidine and [³H]leucine. Total cellular uptake of radioactive precursors remained unaffected by all the treatments. Pretreatment with 20:5 resulted in approximately 25 per cent reduction in the incorporation of [³H]choline into PL, while no significant effect was detected with the other UFAs. 18:3, 20:4 and 20:5 depressed the incorporation of [¹⁴C]ethanolamine into PL by 34 per cent, 28 per cent and 49 per cent respectively. However, there was no redistribution of label with any of the treatments. 18:3, 20:4 and 20:5 also antagonized the stimulatory effect of endotoxin (LPS) on PC and PE synthesis. In addition, the incorporation from [¹⁴C]glycerol into PC and PE was reduced by 18:3, 20:4 and 20:5. Although the PL composition of the cells remained essentially unaffected, our study shows that chronic treatment of U937 cells with n –3 PUFA (20:5) depressed PC and PE synthesis, and 18:3 and 20:4 also caused inhibition of PE synthesis.     

Impact of endothelial lipase on cellular lipid composition

Using mass spectrometry (MS), we examined the impact of endothelial lipase (EL) overexpression on the cellular phospholipid (PL) and triglyceride (TG) content of human aortic endothelial cells (HAEC) and of mouse plasma and liver tissue. In HAEC incubated with the major EL substrate, HDL, adenovirus (Ad)-mediated EL overexpression resulted in the generation of various lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE) species in cell culture supernatants. While the cellular phosphatidylethanolamine (PE) content remained unaltered, cellular phosphatidylcholine (PC)-, LPC- and TG-contents were significantly increased upon EL overexpression. Importantly, cellular lipid composition was not altered when EL was overexpressed in the absence of HDL. [¹⁴C]-LPC accumulated in EL overexpressing, but not LacZ-control cells, incubated with [¹⁴C]-PC labeled HDL, indicating EL-mediated LPC supply. Exogenously added [¹⁴C]-LPC accumulated in HAEC as well. Its conversion to [¹⁴C]-PC was sensitive to a lysophospholipid acyltransferase (LPLAT) inhibitor, thimerosal. Incorporation of [³H]-Choline into cellular PC was 56% lower in EL compared with LacZ cells, indicating decreased endogenous PC synthesis. In mice, adenovirus mediated EL overexpression decreased plasma PC, PE and LPC and increased liver LPC, LPE and TG content. Based on our results, we conclude that EL not only supplies cells with FFA as found previously, but also with HDL-derived LPC and LPE species resulting in increased cellular TG and PC content as well as decreased endogenous PC synthesis.     

Distribution and metabolism of arachidonic and docosahexaenoic acids in rat pineal cells. Effect of norepinephrine

The time-course incorporation of 10 μM [¹⁴C]arachidonic (AA) and docosahexaenoic (DHA) acids into glycerolipids was studied in rat pineal cells. The incorporation of both labeled fatty acids into total lipids was approximately equal, but their distribution profiles among the various cell lipids showed marked differences. The esterification of [¹⁴C]DHA in the neutral lipids, triacylglycerols (TAG) and cholesterol esters (CE), was 2-fold higher than that of [¹⁴C]AA whereas the opposite could be observed in total phospholipids (PL). The order of incorporation into PL was phosphatidylcholine (PC) > phosphatidylinositol (PI) = phosphatidylethanolamine (PE) for [¹⁴C]AA and PC = PE for [¹⁴C]DHA, the incorporation of both fatty acids being not detected in phosphatidylserine (PS) and that of DHA not in PI. When using 0.5 μM [³H] fatty acids, the respective distribution patterns resembled that of fatty acids at 10 μM, except for a lower proportion in TAG. The stimulation of ³H-labeled cells by 100 μM norepinephrine induced a 170% increase of basal release of [³H]AA into the medium, while [³H]DHA was virtually not released. However, the analysis of cell labeling revealed that both [³H] fatty acid levels were decreased in PL and increased in TAG. These findings suggest different involvement for AA and DHA in the pineal function. The preferential incorporation of DHA in TAG suggests that TAG might play an important role in the pineal enrichment with DHA. The absence of DHA release after NE stimulation, which however cannot be ascertained, may raise the question of the role of DHA in NE transduction.     

De novo synthesis and recycling pathways of sphingomyelin in rat Sertoli cells

Sertoli cells from 19-day-old rats have two molecular species of sphingomyelin (SM1 and SM2) with different kinetic characteristics and fatty acid composition. Here, we have studied the incorporation of [14C]-choline and [14C]-palmitic acid into SM in presence or absence of fumonisin B1, an inhibitor of ceramide synthesis, and beta-chloroalanine, an inhibitor of sphinganine synthesis. The contributions of de novo synthesis and recycling pathways were estimated by analysis of the inhibition caused by these drugs. SM1 was synthesized more by sphingosine recycling, and SM2 was synthesized principally by ceramide recycling than SM1. De novo synthesis seems to be important for the two SM types, but our results showed that this pathway is more extensively utilized by SM2. In conclusion, using Sertoli cell cultures, we have shown for the first time that in the same cell different molecular species of SM are synthesized by different pathways.     

The use of exogenous δ-aminolaevulinic acid for the studies of the regulation of haem synthesis in rabbit reticulocytes

δ-Amino [4-¹⁴C]laevulinate added to reticulocytes incubated in vitro is incorporated into haem. Exogenous δ-aminolaevulinate restores the incorporation of ⁵⁹Fe into haem in reticulocytes which had been treated with isonicotinic acid hydrazide (INH) or penicillamine and were hence unable to synthesize δ-aminolaevulinate. On the other hand, the addition of δ-aminolaevulinate does not restore the incorporation of Fe into reticulocytes incubated with haemin. The inhibition of the incorporation of iron is neither restored by δ-aminolaevulinate in reticulocytes incubated with cycloheximide (which inhibits globin synthesis and thus elevates the free intracellular haem pool). These results suggest that in intact reticulocytes haemin does not inhibit δ-aminolaevulinate synthetase. This conclusion is further supported by the finding that the pattern of incorporation of [2-¹⁴C]glycine and δ-amino[4-¹⁴C]-laevulinate into haem differs in reticulocytes incubated with an inhibitor of δ-aminolaevulinate synthetase (INH) and in reticulocytes incubated with haemin and cycloheximide.     

Regulation of hormone biosynthesis in cultured islet cells from anglerfish

Summary The effects of glucose and arginine on islet hormone biosynthesis were investigated using primary cell cultures prepared from islets of the anglerfish (Lophius americanus). After dispersion under sterile conditions, islet cells were maintained at 23° C in medium containing RPMI 1640 with Hanks' buffer, pH 7.5, modified by the adjustment of glucose (to 0.56 or 5.6 mM) and arginine (to 0.1, 1.15, or 10 mM) with the addition of 10% fetal bovine serum (dialyzed, heat inactivated) and penicillin/streptomycin. After 48 h, media were replaced by incorporation media containing [¹⁴C]isoleucine and [³H]tryptophan and incubated for an additional 8 h under otherwise identical conditions. Culture samples (cells plus media) were extracted, desalted, and gel filtered to identify and quantitate [¹⁴C]insulin, [³H]glucagon(s) plus [³H]somatostatin-28, and [³H]somatostatin-14 were In some experiments, [¹⁴C]insulin, [³H]glucagon(s), [³H]somatostatin-28, and [³H]somatostatin-14 were separated by high performance liquid chromatography. Raising the medium glucose from 0.56 (control) to 5.6 mM resulted in an augmentation in incorporation of [¹⁴C]isoleucine into insulin and an augmentation of [³H]tryptophan into glucagon(s) and somatostatin-14, but no change in incorporation of [³H]tryptophan into somatostatin-28. Raising the concentration of arginine from 0.1 to 1.15 or 10 mM resulted in a dose-dependent inhibition of labeled amino acid incorporation into all hormones except somatostatin-28. The results demonstrate the usefulness of the culture system for studying the modulation of hormone biosynthesis in anglerfish islet cells. This work was supported by Grants AM 16921 and AM 26378 from the National Institutes of Health, Bethesda, MD.     

The regulation of ubiquinone synthesis in fibroblasts: The effect of modulators of β-hydroxy-β-methylglutaryl-coenzyme A reductase activity

The effect of inhibitors of β-hydroxy-β-methylglutaryl-coenzyme A (HMG-CoA) reductase such as low-density lipoprotein (LDL) and compactin were tested for their effects on the biosynthesis of ubiquinone in fibroblasts using [2-¹⁴C]acetic acid as a labeled precursor. LDL added to fibroblasts incubated in lipoprotein-deficient serum inhibited acetate incorporation into ubiquinone by 35%. Compactin, 2.5 μm, inhibited acetate incorporation by 60%. Further increases in compactin concentration up to 20 μm gradually increased the extent of inhibition but leveled off between 70 and 80%. The incorporation of ³H]mevalonic acid and 4-[U-¹⁴C]hydroxybenzoic acid into ubiquinone were determined with a range of compactin concentrations. Whereas the incorporation of [³H]mevalonate showed an apparent increase in response to compactin, the incorporation of 4-[U-¹⁴C]hydroxybenzoate into ubiquinone decreased. Both curves leveled off at concentrations of 5 μm did not significantly change with further increases in compactin concentration approaching 20 μm. Thus, the inhibition of acetate and 4-hydroxybenzoate incorporation into ubiquinone by compactin showed similar patterns. Cells incubated in lipoprotein-deficient serum compared to whole human serum showed inhibition of acetate incorporation similar to that observed previously for 4-hydroxybenzoate (9), thereby suggesting the presence of a stimulatory factor for ubiquinone biosynthesis in whole human serum. These data confirm and extend our earlier conclusions that inhibition of HMG-CoA reductase greatly affects ubiquinone synthesis in fibroblasts.     

Substrate utilization for energy production and lipid synthesis in oligodendrocyte-enriched cultures prepared from rat brain

The metabolism of oligodendrocytes has been studied using cultures of oligodendrocyte-enriched glial cells isolated from cerebra of 5–8-day old rats. Cultures containing 60–80% oligodendrocytes were incubated for 16h with [3-¹⁴C]acetoacetate, d-[3-¹⁴C]3-hydroxybutyrate, [U-¹⁴C]glucose, l-[U-¹⁴C]glutamine and [1-¹⁴C]pyruvate or [2-¹⁴C]pyruvate in the presence or absence of other oxidizable substrates. Labelled CO₂ was collected as an index of oxidative metabolism and the incorporation of label into total lipids, fatty acids and cholesterol was used as an index of the de novo synthesis of lipids. Glucose, acetoacetate, D-3-hydroxybutyrate, pyruvate and l-lactate were measured to determine substrate utilization and product formation under various conditions. Our results indicate that glucose is rapidly converted to lactate and is a relatively poor substrate for oxidative metabolism and lipid synthesis. Ketone bodies were used as an energy source and as precursors for the synthesis of fatty acids and cholesterol. Preferential incorporation of acetoacetate into cholesterol was not observed. Exogenous pyruvate was incorporated into both the glycerol skeleton of complex lipids and into cholesterol and fatty acids. l-Glutamine appeared to be an important substrate for the energy metabolism of these cells.     

Incorporation of [14C]Methanol and [14C]Bicarbonate by Hyphomicrobium sp. 53-49 under Various Growth Conditions: Aerobic,Denitrifying and Autotrophic

A study was made of the incorporation of methanol and bicarbonate into the cell constituents of denitrifying or aerobic methanol grown and autotrophic H₂–O₂–CO₂ grown Hyphomicrobium sp. 53-49. Cells were incubated with [¹⁴C]methanol or [¹⁴C]bicarbonate, and the distribution of the radioactivity in the nonvolatile constituents of ethanol extracts of cells was examined. When denitrifying grown cells were incubated with [¹⁴C]methanol, the major part of the radioactivity was fixed to serine as the first stable compound. Aerobic methanol grown cells also fixed [¹⁴C]methanol mainly to serine. These results suggest that methanol grown cells assimilate methanol by the serine pathway. When denitrifying or aerobic methanol grown cells were incubated with [¹⁴C]bicarbonate, malate was mainly observed as a nonvolatile compound in the initial period of the incubation. Autotrophic grown cells also fixed the major part of [¹⁴C]bicarbonate to malate. In this case, phosphoglyceric acid was found in the phosphorylated compounds area.     

The fate of labelled glucose molecules in the rat adipocyte: Dependence on glucose concentration

Isolated rat adipocytes were incubated with 15 nM [3-³H]glucose or 100 nM [U-¹⁴C]glucose with or without insulin and in the absence or presence of unlabelled glucose. Following a 2 h incubation with 15 nM [3-³H]glucose, about two thirds of the cell-associated ³H-labelled metabolic products were hydrophilic largely anionic intermediates and about one third was lipids. The equivalent values were 40 and 60%, respectively, when using 100 nM [U-¹⁴C]glucose. The only ¹⁴C-labelled metabolite escaping to the incubation medium was ¹⁴CO₂, which accounted for about 15% of the rate of metabolism. Therefore, the rate of incorporation of 100 nM [U-¹⁴C]glucose into the cell-associated metabolites was quite a good measure of its net influx rate. The conversion of the two tracers to the sum of the metabolic products in cells treated with a maximally stimulating insulin concentration remained constant with glucose concentrations up to about 100 μM and then decreased progressively. The incorporation of radioactivity into the different metabolites varied markedly over the glucose concentration range 0–100 μM, presumably due to the saturation of different metabolic pools at different glucose concentrations. This variation was much less in cells not stimulated with insulin. Consequently, the maximal effect of insulin on the incorporation of the tracers into a given metabolite (e.g., labelled lipids) varied over the entire glucose concentration range. In addition, the apparent sensitivity (ED₅₀) with respect to the incorporation into a given metabolite was also dependent on the glucose concentration.     

Cholesterol biosynthesis in human lymphocytes,monocytes, and granulocytes

Lymphocytes, monocytes and granulocytes were separated by counter-flow centrifugation from the blood of normal individuals and were incubated in full serum medium or lipid-depleted medium. The monocytes incorporated about five times more [2-¹⁴C]acetate into sterols than did the lymphocytes in full serum medium and approximately twenty times more than the lymphocytes in lipid-depleted medium. The granulocytes were unable to synthesize sterols from either [2-¹⁴C]acetate or [2-¹⁴C]mevalonate, but they were able to use these substrates for the synthesis of squalene and demonstrated approximately a two fold increase in the incorporation of [2-¹⁴C]acetate (but not [2-¹⁴C]mevalonate) into squalene when incubated in the lipid-depleted medium as compared to the full serum medium.     

Differential distribution and metabolism of arachidonic acid and docosahexaenoic acid by human placental choriocarcinoma (BeWo) cells

The time course of incorporation of [¹⁴C]arachidonic acid and [³H]docosahexaenoic acid into various lipid fractions in placental choriocarcinoma (BeWo) cells was investigated. BeWo cells were found to rapidly incorporate exogenous [¹⁴C]arachidonic acid and [³H] docosahexaenoic acid into the total cellular lipid pool. The extent of docosahexaenoic acid esterification was more rapid than for arachidonic acid, although this difference abated with time to leave only a small percentage of the fatty acids in their unesterified form. Furthermore, uptake was found to be saturable. In the cellular lipids these fatty acids were mainly esterified into the phospholipid (PL) and the triacyglycerol (TAG) fractions. Smaller amounts were also detected in the diacylglycerol and cholesterol ester fractions. Almost 60% of the total amount of [3H]Docosahexaenoic acid taken up by the cells was esterified into TAG whereas 37% was in PL fractions. For arachidonic acid the reverse was true, 60% of the total uptake was incorporated into PL fractions whereas less than 35% was in TAG. Marked differences were also found in the distribution of the fatty acids into individual phospholipid classes. The higher incorporation of docosahexaenoic acid and arachidonic acid was found in PC and PE, respectively. The greater cellular uptake of docosahexaenoic acid and its preferential incorporation in TAG suggests that both uptake and transport modes of this fatty acid by the placenta to fetus is different from that of arachidonic acid.     

Lecithin biosynthesis in liver mitochondrial fractions

The pretreatment of rat liver mitochondrial fractions with phospholipase C preparations enhanced the incorporation of cytidine diphospho-[¹⁴C]-choline into phospholipids several-fold. Similar pretreatment of the microsomal fraction produced a similar stimulation. When the extent of microsomal contamination in the mitochondria was determined, and increments of pretreated microsomes were added to the mitochondria, the incorporation values extrapolated to zero for zero microsomal contamination. It was concluded that lecithin biosynthesis from endogenous diglycerides in the mitochondrial fractions could be ascribed to contaminating microsomes.     

Effect of various aminoacid analogues on chick tendon procollagen synthesis and secretion: Selective inhibition by S-2-aminoethyl cysteine

Matrix-free chick embryo tendon cells were incubated with [¹⁴C]-proline in the presence of various aminoacid analogues and the effects of the analogues on [¹⁴C]-proline incorporation, [¹⁴C]-hydroxyproline synthesis and secretion of labeled molecules were examined. It was found that the structural lysine analogue S-2-aminoethylcysteine was a potent inhibitor of procollagen synthesis and secretion. At a concentration of 1 mM it produced a 75% decrease in [¹⁴C]-hydroxyproline synthesis and a 90% decrease in the secretion of [¹⁴C]-hydroxyproline-containing macromolecules.     

Arachidonic acid turnover in peritoneal macrophages is altered in endotoxin-tolerant rats

Peritoneal macrophages from endotoxin-tolerant rats have been found to exhibit depressed metabolism of arachidonic acid (AA) to prostaglandins and thromboxane in response to endotoxin. The effect of endotoxin tolerance on AA turnover in peritoneal macrophages was investigated by measuring [14C]AA incorporation and release from membrane phospholipids. Endotoxin tolerance did not affect the amount of [14C]AA incorporated into macrophages (30 min-24 h). However, the temporal incorporation of [14C]AA into individual phospholipid pools (15 min-24 h) was altered. In endotoxin-tolerant macrophages, [14C]AA incorporation into phosphatidylcholine (PC) (2, 4, 24 h) and phosphatidylethanolamine (PE) (8 h) was increased, while the incorporation into phosphatidylserine (PS) (2-24 h) was reduced (P less than 0.005) compared to control macrophages. There was no change in [14C]AA incorporation into phosphatidylinositol (PI). Following 2 or 24 h of incorporation of [14C]AA, macrophages were incubated (3 h) with endotoxin (50 micrograms/ml) or A23187 (1 microM), and [14C]AA release was measured. Endotoxin-tolerant macrophages released decreased (P less than 0.05) amounts of [14C]AA in response to both endotoxin and the calcium ionophore A23187 compared to controls. Control macrophages in response to endotoxin released [14C]AA from PC, PI and PE. In contrast, tolerant cells released [14C]AA only from PC (P less than 0.05). A23187 released [14C]AA from all four pools in the control cells, but only from PC and PE in the tolerant cells. These data demonstrate that endotoxin tolerance alters the uptake and release of AA from specific macrophage phospholipid pools. These results suggest that changes in AA turnover and/or storage are associated with endotoxin tolerance.