Radioactive Phospholipids Research Articles

Phospholipase D Regulates Myogenic Differentiation through the Activation of Both mTORC1 and mTORC2 Complexes

How phospholipase D (PLD) is involved in myogenesis remains unclear. At the onset of myogenic differentiation of L6 cells induced by the PLD agonist vasopressin in the absence of serum, mTORC1 complex was rapidly activated, as reflected by phosphorylation of S6 kinase1 (S6K1). Both the long (p85) and short (p70) S6K1 isoforms were phosphorylated in a PLD1-dependent way. Short rapamycin treatment specifically inhibiting mTORC1 suppressed p70 but not p85 phosphorylation, suggesting that p85 might be directly activated by phosphatidic acid. Vasopressin stimulation also induced phosphorylation of Akt on Ser-473 through PLD1-dependent activation of mTORC2 complex. In this model of myogenesis, mTORC2 had a positive role mostly unrelated to Akt activation, whereas mTORC1 had a negative role, associated with S6K1-induced Rictor phosphorylation. The PLD requirement for differentiation can thus be attributed to its ability to trigger via mTORC2 activation the phosphorylation of an effector that could be PKCα. Moreover, PLD is involved in a counter-regulation loop expected to limit the response. This study thus brings new insights in the intricate way PLD and mTOR cooperate to control myogenesis.

Rapid de-esterification and loss of eicosapentaenoic acid from rat brain phospholipids: an intracerebroventricular study

Eicosapentaenoic acid (EPA, 20:5n-3) is being explored as a therapy in neurological diseases and disorders. Although it is known that palmitate is the most abundant fatty acid in the brain while EPA is one of the lowest, the mechanism by which the brain maintains this balance is unclear. Therefore, to trace the metabolism of these fatty acids in the brain, (14) C-palmitate or (14) C-EPA was administered via intracerebroventricular infusion to rats. From 4 to 128 days post-infusion, brains were collected after head-focused, high-energy microwave irradiation for biochemical analysis. At day 4 post-infusion, 57% (82 ± 26 nCi) of the total phospholipid radioactivity in (14) C-palmitate-infused brains was intact palmitate; whereas in (14) C-EPA-infused brains, 9% (2 ± 0.9 nCi) of the radioactivity was intact EPA. The half-life of esterified (14) C-palmitate and (14) C-EPA was 32 ± 4 (2% loss per day) and 5 ± 0.2 days (14% loss per day), respectively. Radioactivity was also detected in other saturates, monounsaturates, and cholesterol, suggesting that the infused radiolabeled fatty acids were β-oxidized. In conclusion, the low concentration of EPA in brain phospholipids may be the result of extensive metabolism of EPA, in part by β-oxidation, upon entry into the brain and upon de-esterification from phospholipids.

Expression and Biological Activity of ABCA1 in Alveolar Epithelial Cells

The mechanisms used by alveolar type I pneumocytes for maintenance of the lipid homeostasis necessary to sustain these large squamous cells are unknown. The processes may involve the ATP-binding cassette transporter A1 (ABCA1), a transport protein shown to be crucial in apolipoprotein A-I (apoA-I)-mediated mobilization of cellular cholesterol and phospholipid. Immunohistochemical data demonstrated the presence of ABCA1 in lung type I and type II cells and in cultured pneumocytes. Type II cells isolated from rat lungs and cultured for 5 days in 10% serum trans-differentiated toward cells with a type I-like phenotype which reacted with the type I cell-specific monoclonal antibody VIIIB2. Upon incubation of the type I-like pneumocytes with agents that up-regulate the ABCA1 gene (9-cis-retinoic acid [9cRA] and 22-hydroxycholesterol [22-OH, 9cRA/22-OH]), ABCA1 protein levels were enhanced to maximum levels after 8 to 16 hours and remained elevated for 24 hours. In the presence of apoA-I and 9cRA/22-OH, efflux of radioactive phospholipid and cholesterol from pneumocytes was stimulated 3- to 20-fold, respectively, over controls. Lipid efflux was inhibited by Probucol. Sucrose density gradient analysis of the media from stimulated cells incubated with apoA-I identified heterogeneous lipid particles that isolated at a density between 1.063 and 1.210 g/ml, with low or high apoA-I content. Thus, pneumocytes with markers for the type I phenotype contained functional ABCA1 protein, released lipid to apoA-I protein, and were capable of producing particles resembling nascent high-density lipoprotein, indicating an important role for ABCA1 in the maintenance of lung lipid homeostasis.

Upregulated liver conversion of α-linolenic acid to docosahexaenoic acid in rats on a 15 week n-3 PUFA-deficient diet

We quantified incorporation rates of plasma-derived alpha-linolenic acid (alpha-LNA, 18:3n-3) into "stable" liver lipids and the conversion rate of alpha-LNA to docosahexaenoic acid (DHA, 22:6n-3) in male rats fed, after weaning, an n-3 PUFA-adequate diet (4.6% alpha-LNA, no DHA) or an n-3 PUFA-deficient diet (0.2% alpha-LNA, no DHA) for 15 weeks. Unanesthetized rats were infused intravenously with [1-14C]alpha-LNA, and arterial plasma was sampled until the liver was microwaved at 5 min. Unlabeled alpha-LNA and DHA concentrations in arterial plasma and liver were reduced >90% by deprivation, whereas unlabeled arachidonic acid (20:4n-6) and docosapentaenoic acid (22:5n-6) concentrations were increased. Deprivation did not change alpha-LNA incorporation coefficients into stable liver lipids but increased synthesis-incorporation coefficients of DHA from alpha-LNA by 6.6-, 8.4-, and 2.3-fold in triacylglycerol, phospholipid, and cholesteryl ester, respectively. Assuming that synthesized-incorporated DHA eventually would be secreted within lipoproteins, calculated liver DHA secretion rates equaled 2.19 and 0.82 micromol/day in the n-3 PUFA-adequate and -deprived rats, respectively. These rates exceed the published rates of brain DHA consumption by 6- and 10-fold, respectively, and should be sufficient to maintain normal and reduced brain DHA concentrations, respectively, in the two dietary conditions.

Identification and characterization of rodent ABCA1 in isolated type II pneumocytes.

ATP-binding cassette transporter A1 (ABCA1) promotes transfer of cholesterol and phospholipid from cells to lipid-free serum apolipoproteins. ABCA1 mRNA and protein expression in primary cultures of rodent type II cells was sensitive to upregulation with 5 microM 9-cis-retinoic acid (9cRA) and 6.2 microM 22-hydroxycholesterol (22-OH). The increase in ABCA1 protein levels was time dependent and was maximal after 16 h of exposure to 9cRA + 22-OH. Inducible ABCA1 was also found in transformed cell lines of lung origin: WI38/VA13, A549, and NIH-H441 cells. Stimulation of ABCA1 in rat type II cells by 9cRA + 22-OH resulted in a four- or fivefold enhancement of efflux of radioactive phospholipid or cholesterol, respectively, from the pneumocytes to apolipoprotein AI (apo AI), whereas cAMP (0.3 mM) had no effect. ABCA1-mediated lipid efflux to apo AI was independent of the surfactant secretion pathway, inasmuch as upregulation of ABCA1 resulted in a reduction of secretagogue-stimulated surfactant phospholipid release. These studies demonstrate the presence of functional ABCA1 in type II cells from the lung.

Synthesis of lipids in isolated nuclei from rat thymus and liver cells.

Synthesis of lipids was studied in isolated nuclei from rat thymus and liver cells. On incubation of the isolated nuclei with [2-14C]acetate and [1-14C]glycerol, the label was intensively incorporated into phospholipids and with a significantly lower intensity into fatty acids and cholesterol. Only trace amounts of radioactivity were detected in the lipids of chromatin prepared from isolated thymus nuclei after their incubation, and this suggested that lipids were mainly synthesized on the nuclear membrane. On the preincubation of thymus tissue homogenate with [2-14C]acetate and the subsequent isolation of the nuclei and chromatin, the radioactivity of chromatin lipids was comparable to the radioactivity of nuclear lipids. The findings suggested that in the isolated nuclei the newly synthesized lipids were not transported into chromatin from the nuclear membrane. The specific radioactivities of individual phospholipids and fatty acids were different in the isolated nuclei and in nuclei obtained from preincubated homogenate. Mechanisms of lipid synthesis in isolated nuclei and causes of the different radioactivities of lipids in the isolated nuclei and in the nuclei obtained from the preincubated homogenate are discussed.

Resistance of HepG2 cells against the adverse effects of ethanol related to neutral lipid and phospholipid metabolism

The influence of both short- and long-term ethanol exposure on the lipid metabolism was determined in the human hepatoma cell line HepG2. Ethanol did not cause any cytotoxicity or lipid peroxidation even after 7 days of 100 mM ethanol treatment of HepG2 cells. Incubation of cells in the presence of [1- 14 C ]ethanol demonstrated that these cells actively metabolize ethanol to acetyl CoA, incorporating the radioactive label into neutral lipids and phospholipids. [1,2,3- 3 H ]glycerol was efficiently used in phospholipid and neutral lipid biosynthesis, showing higher radioactivity in phosphatidylcholine, phosphatidylethanolamine and triacylglycerols. Exposure of HepG2 cells to 100 mM ethanol for 24 hr did not significantly modify the incorporation of glycerol into newly synthesized phospholipids and neutral lipids, nor was lipid degradation affected by the presence of ethanol. When the alcohol treatment was prolonged for 7 days, incorporation of [1,2,3- 3 H ]glycerol into triacylglycerols and diacylglycerols showed a slight increase concomitantly with decreased radioactivity in the major phospholipids, phosphatidylcholine and phosphatidylethanolamine. In addition, these changes were associated with a greater release of radiolabeled triacylglycerols into the culture medium. These results indicate that ethanol does not cause in HepG2 cells the marked lipogenic stimulation widely shown in hepatocytes, and demonstrate that HepG2 cells strongly resist the adverse effects of ethanol. Since these cells lack the isoenzymatic form of cytochrome P 450 mainly involved in the ethanol metabolism (namely cytochrome P 4502E1) and also are devoid of alcohol dehydrogenase activity, we propose that the toxic actions of ethanol on liver must be linked to the activity of one or both of these systems.

Choline supplementation inhibits diethanolamine-induced morphological transformation in syrian hamster embryo cells: evidence for a carcinogenic mechanism.

DEA, an amino alcohol, and its fatty acid condensates are widely used in commerce. DEA is hepatocarcinogenic in mice, but shows no evidence of mutagenicity or clastogenicity in a standard testing battery. However, it increased the number of morphologically transformed colonies in the Syrian hamster embryo (SHE) cell morphologic transformation assay. The goal of this work was to test the hypothesis that DEA treatment causes morphologic transformation by a mechanism involving altered cellular choline homeostasis. As a first step, the ability of DEA to disrupt the uptake and intracellular utilization of choline was characterized. SHE cells were cultured in medium containing DEA (500 microg/ml), and (33)P-phosphorus or (14)C-choline was used to label phospholipid pools. After 48 h, SHE cells were harvested, lipids were extracted, and radioactive phospholipids were quantified by autoradiography after thin layer chromatographic separation. In control cells, phosphatidylcholine (PC) was the major phospholipid, accounting for 43 +/- 1% of total phospholipid synthesis. However, with DEA treatment, PC was reduced to 14 +/- 2% of total radioactive phospholipids. DEA inhibited choline uptake into SHE cells at concentrations > or = 50 microg /ml, reaching a maximum 80% inhibition at 250-500 microg/ml. The concentration dependence of the inhibition of PC synthesis by DEA (0, 10, 50, 100, 250, and 500 microg/ml) was determined in SHE cells cultured over a 7-day period under the conditions of the transformation assay and in the presence or absence of excess choline (30 mM). DEA treatment decreased PC synthesis at concentrations > or = 100 microg/ml, reaching a maximum 60% reduction at 500 microg/ml. However, PC synthesis was unaffected when DEA-treated cells were cultured with excess choline. Under 7-day culture conditions, (14)C-DEA was incorporated into SHE lipids, and this perturbation was also inhibited by choline supplementation. Finally, DEA (10-500 microg/ml) transformed SHE cells in a concentration-dependent manner, whereas with choline supplementation, no morphologic transformation was observed. Thus, DEA disrupts intracellular choline homeostasis by inhibiting choline uptake and altering phospholipid synthesis. However, excess choline blocks these biochemical effects and inhibits cell transformation, suggesting a relationship between the two responses. Overall, the results provide a plausible mechanism to explain the morphologic transformation observed with DEA and suggest that the carcinogenic effects of DEA may be caused by intracellular choline deficiency.

Lipid Metabolism in Primary Cultured Hepatocytes from Daunomycin-Nephrotic Rats

We investigated the lipid metabolism in primary cultured hepatocytes to elucidate the causes of hyperlipidemia, increased cholesteryl esters, and decreased triglyceride levels in the livers of daunomycin-nephrotic rats. The incorporation of ¹⁴C-palmitate into phospholipids and triglycerides in primary cultured hepatocytes and medium was similar in daunomycin-nephrotic and control rats. The incorporation of ¹⁴C-acetate into phospholipids, triglycerides, cholesterol, cholesteryl esters, and total fatty acids in primary cultured hepatocytes was increased in daunomycin-nephrotic rats. The radioactivity of phospholipids, triglycerides, cholesterol, cholesteryl esters, and very-low-density lipoprotein lipids in medium was increased in the hepatocytes of daunomycin-nephrotic rats using ¹⁴C-acetate as a precursor. The increased cholesterogenesis and the increased secretion of triglycerides synthesized from acetate by hepatocytes may be due to an increased cholesteryl ester content and a decreased triglyceride content in the livers of daunomycin-nephrotic rats. The increased secretion of lipids synthesized from acetate by hepatocytes may be due to increased accumulation of lipids in serum and very-low-density lipoprotein in daunomycin-nephrotic rats.

Iron Deficiency Reduces the Hydrolysis of Cell Membrane Phosphatidyl Inositol-4,5-Bisphosphate during Splenic Lymphocyte Activation in C57BL/6 Mice

Iron deficiency impairs lymphocyte proliferation in humans and laboratory animals by unknown mechanisms. In this study, we investigated whether this alteration can be attributed in part to impaired hydrolysis of cell membrane phosphatidyl inositol-4,5-bisphosphate (PIP2), a required early event of T-lymphocyte activation. The study involved 46 iron-deficient (ID), 26 control (C) and 23 pair-fed (PF) mice, and ID mice that were repleted for 3 (n = 16), 7 (n = 17) or 14 d (n = 18). Mice were killed after 40–63 d (mean, 48 d) of consuming the test diet (0.09 mmol/kg iron) or the control diet (0.9 mmol/kg). The mean (±SEM) hemoglobin concentrations were 57 ± 16.7, 176 ± 2.6 and 181 ± 9.7 g/L for ID, C and PF groups, respectively. After splenic lymphocytes were labeled in vitro with 3H-myoinositol for 3 h, PIP2 hydrolysis was estimated by measuring the radioactivity recovered as a mixture of inositol mono-, di- and triphosphate (IP) from concanavalin A (0, 1, 2.5, 5 and 10 mg/L) activated cells. Although cells from ID mice and those from mice repleted for 3 d incorporated slightly more radioactivity in cellular phospholipids than did cells from C or PF mice, less (P < 0.005) was recovered as IP than in controls, suggesting impaired conversion of the precursor to PIP2. At almost all incubation periods (10–120 min) and mitogen concentrations, the rate of PIP2 hydrolysis expressed as the ratio of radioactivity obtained in Con A–treated to untreated cells was significantly (P < 0.05) reduced in cells from ID mice compared with those obtained from C and PF mice. For cells that were activated for 60 min or less, iron repletion for 14 d significantly (P < 0.05) improved the rate of PIP2 hydrolysis. PIP2 hydrolysis positively and significantly (P < 0.05) correlated (r = 0.27–0.56) with indicators of iron status. Mitogenic response was also significantly (P < 0.05) reduced in ID but not PF mice, and it was corrected by iron repletion for 3, 7 or 14 d. Lymphocyte proliferation positively (r = 0.27–0.37, P < 0.01) correlated with indices of iron status and IP ratios. The data suggest that reduced PIP2 hydrolysis contributes to impaired blastogenesis in iron deficiency.

The Acid Activation ofHelicobacter pyloriToxin VacA: Structural and Membrane Binding Studies

The cell vacuolating activity of the protein toxin VacA, released byHelicobacter pylori,is strongly increasedin vitroby exposure to acidic pH followed by neutralization. This short acid exposure does not increase significantly the binding of VacA to cell or to lipid membranes. However, membrane photolabeling with photoactivatable radioactive phospholipids and ANS binding studies show that VacA transiently exposed to pH equal or lower than 5 changes conformation and exposes on its surface hydrophobic segments. Both the 32 and the 58 kDa subunits of the toxin insert in the lipid bilayer and interact with the fatty acid chains of phospholipids. Membrane binding and penetration are enhanced by incubating target cells or liposomes with the toxin at mild acidic pH values, similar to those present aroundH. pylorion the stomach mucosa. These findings are discussed with respect to the critical step in cell intoxication consisting in the translocation of the active toxin domain into the cell cytosol. We suggest that membrane translocation takes place at the plasma membrane level.

No evidence for direct incorporation of esterified palmitic acid from plasma into brain lipids of awake adult rat

Awake adult rats were given a solution of [9,10-3H]palmitate ([3H]PAM) by gavage. The appearance of radiolabel in plasma lipid fractions was monitored by thin-layer chromatography at fixed intervals thereafter. At 2 h, the rats were killed by microwave irradiation and radioactivity in whole brain and individual brain phospholipids was determined. In plasma, esterified [3H]PAM was mainly associated with triglyceride, phospholipid, and cholesterol ester. Radioactivity appeared to a larger extent in triglyceride than in unesterified fatty acid, suggesting that unesterified [3H]PAM in plasma was largely due to release from esterified [3H]PAM by lipoprotein lipase hydrolysis. Brain radioactivity could be accounted for entirely by incorporation of unesterified plasma [3H]PAM. Esterified [3H]PAM in chylomicrons or lipoproteins was calculated to make no measurable contribution using a published value for the incorporation coefficient of [3H]PAM into brain in the evaluation. These results suggest that ingested palmitic acid (PAM) in adult rats enters blood as esterified triglyceride within chylomicrons and lipoproteins and, in part, eventually is converted to circulating unesterified PAM. It is the circulating unesterified PAM that is incorporated into brain from blood, whereas esterified PAM within plasma chlomicrons and lipoproteins makes no measurable direct contribution.

Exogenously added alkylmethylglycerophosphocholine and alkylmethylcarbamylglycerophosphocholine accumulate in plasma membranes more than in intracellular membranes of rabbit platelets

We found that extracellular addition of 2% bovine serum albumin (BSA) to a suspension of rabbit platelets after stimulation with platelet-activating factor resulted in a biphasic extraction of [ 3 H]1-O- alkyl-2-O- methyl (or 2- O-methylcarbamyl)- sn-glycero-3-phosphocholine. A fast phase of extraction of the phospholipid probe by BSA was found to be mainly due to removal of the probe remaining in an outer layer of platelet plasma membrane, whereas a second phase of extraction of the probe by BSA was mostly attributed to redistribution of the probe which had been flipped across the plasma membrane. On the basis of analysis of the biphasic extraction by BSA of 1- O-alkyl-2- O-methyl(or methylcarbamyl)- sn-glycero-3-phosphocholine at various times after its addition, we suggested that the radioactive phospholipid accumulated in plasma membrane more than in intracellular membranes of rabbit platelets. In similar experiments with guinea-pig polymorphonuclear leukocytes, we observed a monophasic extraction of 1- O-alkyl-2- O-methyl, (or methylcarbamyl)- sn-glycero-3-phosphocholine by BSA, indicating its unidirectional movement across the plasma membrane.

Translocation of cytosolic phospholipase A2 to the nuclear envelope elicits topographically localized phospholipid hydrolysis.

Cytosolic phospholipase A2 (cPLA2) is a good candidate for mediating the agonist-stimulated release of arachidonic acid (AA) from membrane phospholipids. This enzyme undergoes a Ca(2+)-dependent translocation from the cytosol to a membrane site in a variety of cell types, and this site has recently been identified as the nuclear envelope in leucocytes. The functional correlate of this finding has not yet been established. The present study was therefore undertaken to determine whether translocation of cPLA2 to the nuclear envelope was associated with localized phospholipid hydrolysis at this site. Rat alveolar epithelial cells, previously shown to contain cPLA2, were prelabelled with [3H]AA and stimulated with the model agonist, ionophore A23187. Ionophore-induced AA release exhibited characteristics typical of a cPLA2-mediated response, in that it was Ca(2+)-dependent, sn-2 AA-selective, and inhibited by arachidonyl trifluoromethyl ketone. As determined by indirect immunofluorescence microscopic analysis as well as subcellular fractionation with immunoblotting, ionophore treatment resulted in a translocation of cPLA2 protein from the cytoplasm to the nuclear envelope. To determine whether the nuclear membrane was indeed the source of released AA, prelabelled cells were incubated in the presence or absence of A23187, after which the phospholipid radioactivity was quantified in nuclear and non-nuclear membrane fractions. [3H]AA was distributed in both nuclear and non-nuclear membrane phospholipids. Following A23187 stimulation, the loss of [3H]AA from nuclear membrane phospholipids accounted for 88.1 +/- 5.8% of the total loss from phospholipids and for 92.9 +/- 2.3% of the total [3H]AA released into the medium. These results demonstrate for the first time that agonist-stimulated translocation of cPLA2 to the nuclear envelope is associated with phospholipid hydrolysis which is preferentially localized to that site.

The influence of a fish oil‐enriched diet on the phospholipid fatty acid turnover in the rabbit red cell membranein vivo

The influence of the polyunsaturated fatty acid (PUFA) composition of the diet on the rate of fatty acid turnover of individual phospholipids in the erythrocyte membrane in vivo was studied. Following modification of the fatty acid composition of the membrane phospholipids by the use of a fish oil or a linoleic acid enriched diet, phospholipids--labelled in the unsaturated fatty acid at the 2-position of the glycerol moiety--were introduced into the membrane of freshly isolated rabbit erythrocytes. Thereafter, the labelled erythrocytes were reinjected into the bloodstream of the animal. It appears that, with the exception of 1-palmitoyl,2-linoleoyl phosphatidylcholine, all other phosphatidylcholines disappear faster from the erythrocytes of fish oil-fed rabbits than from the red cells of linoleic acid-fed rabbits. Another parameter, which possibly influences the turnover rates of PUFA containing phospholipids, can be peroxidation. An attempt was made to measure peroxidative damage of lipids in vivo by the introduction of 1-palmitoyl,2-cis-parinaroyl phosphatidylcholine (PnPC)--a probe to measure oxidative stress--into the membrane of freshly isolated erythrocytes, in the same way as is described for the radioactive phospholipids. The data demonstrate that the fluorescent signal from the PnPC decreases at a fast rate which is independent of the dietary conditions.

Quantitative chromatographic analysis of inositol phospholipids and related compounds.

The metabolism of phospholipids and the mobilization of second messengers such as inositol-1,4,5-trisphosphate, 1,2-diacylglycerol (DAG) and arachidonic acid (AA) from phospholipids is commonly studied by radiolabelling phospholipids with [3H]myo-inositol or [32P]ATP and measuring the incorporation of radioactivity in different phospholipids or their hydrolysis products. However, for the radiolabelling method to accurately reflect changes in the compound's mass, it is essential that the tissue is labelled to isotopic equilibrium which is difficult to achieve. To circumvent the disadvantages of the radiolabelling method, several analytical procedures have been developed for the mass analysis of phospholipids and inositolphosphates (IPs). Quantitation of the mass or the radiolabelling of phospholipids is a complex multi-step procedure that involves quantitative isolation of phospholipids, fractionation of individual phospholipids and either determination of radioactivity in each component or the measurement of their mass. Phospholipids, DAG and AA are extracted from tissue sample with organic solvents such as chloroform-methanol (2:1) containing HCl or formic acid. The extract is separated by TLC, cartridge-column chromatography or HPLC on a reversed-phase column. Phospholipids are quantitated by measuring inorganic phosphate, absorption at 200 nm or mass spectrometry. Inositol phosphates are extracted with perchloric acid or trichloroacetic acid and separated by ion-exchange cartridge-column or HPLC with an ion-exchange column. IPs are quantitated by measuring inorganic phosphate or by using enzymatic reaction, metal-dye coupling, NMR or mass spectrometry.

Hexachlorocyclohexanes affect the arachidonic acid release from phosphatidylinositol but not from other phospholipid classes in tubular cell cultures.

Gamma- and delta-isomers of hexachlorocyclohexane caused marked decreases in the levels of radioactive phospholipids, and increases in the levels of [3H]arachidonate incorporated into free fatty acids in rat renal tubular cells. The increased radioactivity of free fatty acids arises from the decrease of [3H]arachidonate incorporated into phosphatidylinositol, but not into phosphatidylcholine, phosphatidylserine or phosphatidylethanolamine. This fact suggests that phosphatidylinositol can be broken down to the fatty acid from the sn-2 position and lysophospholipid by a phospholipase activity increased by hexachlorocyclohexanes. The observed specific toxicant action could be achieved in two ways: (a) operating upon a specific phospholipase A2 that acts on phosphatidylinositol, but not on other phospholipids as substrates and/or (b) involving substrate-phospholipase A2 interactions. Interestingly, the observed effect of the delta-isomer was more pronounced than that of the gamma-one.

A phospholipase D-mediated pathway for generating diacylglycerol in nuclei from Madin-Darby canine kidney cells.

Many receptors, in response to their specific ligands, trigger activation of phospholipase D (PLD), resulting in the production of phosphatidic acid which, in turn, is acted upon by a specific phosphatase, phosphatidate phosphohydrolase, to produce diacylglycerol. We report here that isolated nuclei from Madin-Darby canine kidneys (MDCK)-D1 cells exhibit a PLD activity that is enhanced by the presence of ATP. PLD activity was measured in the presence of ethanol, by quantitating the production of phosphatidylethanol. Non-phosphorylating ATP analogs were unable to substitute for ATP in activating PLD, indicating that ATP acts as a phosphoryl group donor in a kinase-mediated phosphorylation reaction. The protein kinase C inhibitors chelerythrine and calphostin completely suppressed the ATP-induced nuclear PLD, implicating protein kinase C as the kinase involved in ATP-dependent PLD activity in nuclei from MDCK-D1 cells. In the absence of ethanol, phosphatidic acid was detected in ATP-treated nuclei. Accumulation of phosphatidic acid preceded or closely paralleled that of diacylglycerol, suggesting a precursor-product relationship. Consistent with those results, we detected phosphatidate phosphohydrolase activity in MDCK-D1 cell nuclei. Measurements of phosphatidic acid and diacylglycerol levels at increasing amounts of ethanol demonstrated that PLD and phosphatidate phosphohydrolase are responsible for generating the majority of the diacylglycerol accumulating in MDCK-D1 cell nuclei. The ability of nuclei to generate diacylglycerol from the concerted action of those two enzymes provides a means to regulate nuclear lipid synthesis as well as protein kinase C activity.

Phospholipid Biosynthesis in Growing and Non-growing Conditions in Candida albicans.

The major membrane phospholipids of Candida albicans (C. albicans) are phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidylserine (PS). In Saccharomyces cerevisiae (S. cerevisiae), the most abundant component PC is considered to be synthesized mainly by two pathways; stepwise methylation pathway of PE produced from PS by decarboxylation (de novo pathway) and diacylglycerol (DG)/CDP-choline pathway. However, relatively little information is available regarding the phospholipid biosynthesis in C. albicans. In order to clarify the phospholipid metabolism of this organism, we have investigated the 32Pi incorporation into different phospholipids by continuous and pulse-labeling in cells grown in the synthetic medium (SM) and yeast extractproteose peptone-dextrose (YPD) medium.The continuous labeling with 32Pi in YPD medium showed that the decreases in radioactivity in acidic phospholipids (PA, PI and PS) were followed by the great increase in PC, suggesting the existence of de novo pathway. In SM cultured cells showing no growth, PS and PI were heavily labeled, while PC was much less labeled. By the switch from SM to YPD, the rapid increase in 32P-labeled PC occurred at the expense of PI and PS, suggesting that the principal PC synthesis for membrane biogenesis is via the de novo pathway in C. albicans.

Effect of inhibition of β-oxidation on incorporation of [ U- 14C]palmitate and [1- 14C]arachidonate into brain lipids

The purpose of the present study was to determine the effect of inhibiting the mitochondrial β-oxidation of free fatty acids on the incorporation of radiolabeled free fatty acids into brain lipids. To this end, methyl 2-tetradecylglycidate (MEP), an irreversible inhibitor of carnitine palmitoyltransferase I, was given orally to male rats 2, 4, and 6 h prior to an intravenous infusion of the saturated fatty acid [ U- 14C]palmitic acid (PA) or the polyunsaturated fatty acid [1- 14C]arachidonate (AA). With [U- 14C]PA, MEP (10–25 mg/kg) increased brain organic radioactivity 2-fold and decreased brain aqueous radioactivity 3- to 5-fold relative to control values at all pretreatment times. The effect was due to prolongation of the plasma integral of [ U- 14C]PA due to peripheral inhibition of β-oxidation, and to direct inhibition of β-oxidation of the tracer within the brain. MEP had no effect on brain organic radioactivity after infusion of [1- 14C]AA. Increasing the interval between MEP administration and [ U- 14C]PA infusion from 2 to 6 h resulted in a dramatic redistribution of [ U- 14C]PA within brain lipids. The percentage of radioactivity in phospholipids decreased from 65 to 33%, whereas that in the free fatty acid fraction increased from 10 to 47% and that in triglycerides was elevated 2–3 fold over control levels. These results indicate that MEP may facilitate the use of radiolabeled PA as an in vivo probe of brain lipid metabolism using quantitative autoradiography or position emission tomography.