In vivo metabolism of labeled oleic and linoleic acids by the laying hen.

Radioactive oleic and linoleic acids, labeled with 3H in the chain and 14C in the carbonyl group, were administered to white leghorn laying hens. Mixtures fed in separate experiments included: (1) 3H- and 14C-labeled oleic acid, (2) 3H- and 14C-labeled linoleic acid and (3) [3H]oleic aicd and [14C] linoleic acid. The 3H/14C ratios of both the neutral lipid and phospholipid fractions from the egg yolk and of the isolated acids from these lipid fractions were compared to that in the administered mixture. Agreement in the 3H/14C ratios for the neutral lipid fraction from each of the feeding experiments indicated that neither the 3H- and 14C labeled acids nor the oleic or linoleic acids were distinguishable during synthesis of the neutral lipid. Analysis of the phospholipid fractions showed that when dual-labeled mixtures of oleic acid were administered, 3H/14C ratios were elevated and, therefore, there was selective elimination of the 14C label. When dual-labeled mixtures of linoleic acid were administered, the 3H/14C ratios were in agreement; and when the two acids were administered simultaneously as a dual-labeled mixture, there was selective incorporation of linoleic acid. These findings indicate separate metabolic pathways for synthesis of neutral lipid and phospholipid in egg yolk as expected, as well as preferential use of the essential fatty acid in the phospholipid by the hen.     

Characterization and metabolism of ovine foetal lipids

1. Total phospholipid concentrations in liver, kidney and brain of the 140-day ovine foetus were only half of those in comparable maternal tissues. 2. Phosphatidylcholine was the predominant phospholipid in all foetal tissues examined. The most striking difference between foetal and maternal tissues in individual phospholipids was in the heart; foetal heart contained more ethanolamine plasmalogen than choline plasmalogen, whereas in adult tissue the concentration of these was reversed. Sphingomyelin content of foetal brain was only one-sixth of that of maternal brain tissue. 3. Oleic acid (18:1) was the predominant acid in the phospholipid extracted from foetal tissues, except in brain where palmitic acid (16:0) was slightly higher. In phospholipids from adult tissues there was a higher proportion of unsaturated fatty acids (linoleic acid, 18:2, and linolenic acid, 18:3) and a correspondingly lower proportion of oleic acid (18:1). The distribution of fatty acids in the neutral lipid fraction of foetal and maternal tissues was very similar; oleic acid (18:1) was generally the principal component. 4. (14)C derived from [U-(14)C]-glucose and [U-(14)C]fructose infused into the foetal circulation in utero was incorporated into the neutral lipids and phospholipids of heart, liver, kidney, brain and adipose tissue. 5. Phospholipid analysis revealed that the specific activity of phosphatidic acid was higher in liver than in other tissues. The specific activity of phosphatidylethanolamine was less than that of phosphatidylcholine in heart, but in other tissues they were about the same. The specific activities of phosphatidylinositol and phosphatidic acid in brain were very similar and were higher than the other components. The specific activity of phosphatidylserine was highest in liver and brown fat. 6. The pattern of incorporation of (14)C derived from [(14)C]glucose and [(14)C]fructose into foetal neutral lipids was similar. Diglyceride accounted for most of the radioactivity in brain, whereas triglyceride had more label in heart, liver, kidney and fat.     

The role of holotrichs in the metabolism of dietary linoleic acid in the rumen

The uptake and metabolism of linoleic acid by rumen holotrichs (mainly Isotricha prostoma and I. intestinalis) has been examined in in vitro infusion experiments. Maximum absorption and metabolism of [1-14C]linoleate by 2 . 10(6) Isotricha suspended in 100 ml buffer was obtained using an infusion rate of 1.6 mg linoleate/h. After 90 min, 84% of the added substrate was recovered within the cells, mainly as free fatty acid or phospholipid. There was a rapid incorporation of radioactivity into phospholipid, mainly phosphatidylcholine, at the commencement of linoleate infusion but no further incorporation after about 40 min. The presence of bacteria during incubations, in approximately the same Isotricha/bacteria ratio as found in the rumen, reduced the uptake of linoleate and the accumulation of free fatty acid by holotrichs but the incorporation into phospholipid remained similar to that obtained in the absence of bacteria. Very little biohydrogenation of linoleic acid occurred in incubations with holotrichs alone. Bacterial suspensions converted linoleic acid to mainly trans monoene and a small amount of stearic acid, but in incubations containing both bacteria and holotrichs, both stearic acid and trans monoene were major products. Using the latter mixed culture, about 20% of the added [1-14C]linoleic acid was present in holotrich phospholipid of which 62% remained as octadecadienoic acid. The Isotricha population was 3 . 10(3)--2 . 10(4)/ml rumen fluid and it contributed about 23% of the linoleic acid in the rumen of a cow on a hay diet.     

Characterization and separation of the arachidonic acid 5-lipoxygenase and linoleic acid omega-6 lipoxygenase (arachidonic acid 15-lipoxygenase) of human polymorphonuclear leukocytes

The cytosolic fraction of human polymorphonuclear leukocytes precipitated with 60% ammonium sulfate produced 5-lipoxygenase products from [14C]arachidonic acid and omega-6 lipoxygenase products from both [14C]linoleic acid and, to a lesser extent, [14C]- and [3H]arachidonic acid. The arachidonyl 5-lipoxygenase products 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid (5-HPETE) and 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) derived from [14C]arachidonic acid, and the omega-6 lipoxygenase products 13-hydroperoxy-9,11-octadecadienoic acid (13-OOH linoleic acid) and 13-hydroxy-9,11-octadecadienoic acid (13-OH linoleic acid) derived from [14C]linoleic acid and 15-hydroxyperoxy-5,8,11,13-eicosatetraenoic acid (15-HPETE), and 15-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE) derived from [14C]- and [3H]arachidonic acid were identified by TLC-autoradiography and by reverse-phase high-performance liquid chromatography (RP-HPLC). Products were quantitated by counting samples that had been scraped from replicate TLC plates and by determination of the integrated optical density during RP-HPLC. The arachidonyl 5-lipoxygenase had a pH optimum of 7.5 and was 50% maximally active at a Ca2+ concentration of 0.05 mM; the Km for production of 5-HPETE/5-HETE from arachidonic acid was 12.2 +/- 4.5 microM (mean +/- S.D., n = 3), and the Vmax was 2.8 +/- 0.9 nmol/min X mg protein (mean +/- S.D., n = 3). The omega-6 linoleic lipoxygenase had a pH optimum of 6.5 and was 50% maximally active at a Ca2+ concentration of 0.1 mM in the presence of 5 mM EGTA. When the arachidonyl 5-lipoxygenase and the omega-6 lipoxygenase were separated by DEAE-Sephadex ion exchange chromatography, the omega-6 lipoxygenase exhibited a Km of 77.2 microM and a Vmax of 9.5 nmol/min X mg protein (mean, n = 2) for conversion of linoleic acid to 13-OOH/13-OH linoleic acid and a Km of 63.1 microM and a Vmax of 5.3 nmol/min X mg protein (mean, n = 2) for formation of 15-HPETE/15-HETE from arachidonic acid.     

Stimulation of prostaglandin synthesis in WI-38 human lung fibroblasts following inhibition of phospholipid acylation by p-hydroxymercuribenzoate

The release of arachidonic acid and its metabolites, prostaglandin E2 and thromboxane A2, from WI-38 human lung fibroblasts was modulated by p-hydroxymercuribenzoate. Exposure to the inhibitor resulted in a dose-dependent decrease in [1-14C]arachidonic acid uptake and incorporation into phospholipids and neutral lipid pools. Activities of lung fibroblast arachidonyl-CoA synthetase and lysolecithin acyltransferase were inhibited by 100 microM p-hydroxymercuribenzoate. [14C]Arachidonic acid labelled fibroblasts exhibited an increased release of [14C]arachidonate and [14C]prostaglandin E2 of 54% and 112%, respectively, when exposed to 100 microM of inhibitor. The stimulatory effects of 8.0 microM delta 1-tetrahydrocannabinol on arachidonate release and prostaglandin E synthesis (Burstein, S., Hunter, S.A., Sedor, C. and Shulman, S. (1982) Biochem. Pharmacol. 31, 2361-2365) were modified by the inclusion of inhibiting agent, resulting in a 608% stimulation in arachidonic acid release, while prostaglandin E2 and thromboxane A2 synthesis increased 894% and 390%, respectively, over levels obtained by untreated cells. The levels of arachidonate metabolites were altered by inhibitor when compared to cells treated with cannabinoid alone. No significant inhibition by delta 1-tetrahydrocannabinol was found on arachidonic uptake in these cells. In unlabelled studies, p-hydroxymercuribenzoate resulted in a profound, dose-dependent stimulation of prostaglandin E synthesis of 1490% at 150 microM inhibitor concentration. These results provide evidence that free arachidonate is reincorporated via acylation, thereby implicating this pathway as a possible control mechanism for the synthesis of arachidonic acid metabolites.     

Alterations of phospholipid metabolism by phorbol esters and fatty acids occur by different intracellular mechanisms in cultured glioma, neuroblastoma, and hybrid cells

Differences between the influences of phorbol esters (such as 4 beta-12-O-tetradecanoylphorbol 13-acetate) and of fatty acids (such as oleic acid) on the synthesis and turnover of phosphatidylcholine (PtdCho) and other phospholipids have been studied in glioma (C6), neuroblastoma (N1E-115), and hybrid (NG108-15) cells in culture using [methyl-3H]choline, [32P]Pi, [1,2-14C]ethanolamine, or 1-14C-labeled fatty acids as lipid precursors. 100-500 microM oleic acid stimulated PtdCho synthesis 3- to 5-fold in all three cell lines, but had little influence on chase of choline label following a 24-h pulse. Phorbol ester (50-200 nM) stimulated PtdCho synthesis 1.5- to 3-fold in C6 cells, was without effect in N1E-115 cells, and had intermediate effects on NG108-15 cells. Phorbol ester stimulated both uptake of extracellular choline and synthesis of PtdCho, whereas fatty acid stimulated only synthesis. Release of radioactivity from 24-h pulse-labeled PtdCho to the medium was enhanced by phorbol ester in C6 cells. Incorporation of [32P]Pi, primarily into PtdCho, was stimulated, whereas utilization of [1,2-14C]ethanolamine or 1-14C-fatty acid was little altered by phorbol ester. C6 cells "down-regulated" with phorbol ester lost the stimulatory response of subsequent treatment with phorbol esters on PtdCho synthesis, but the response to fatty acid was enhanced. Fatty acid had little influence on the relative binding of phorbol ester or "translocation" of phorbol ester binding sites. Accordingly, metabolism of phospholipids in these cultured cells of neural origin is markedly influenced by cell type, phospholipid class, condition of incubation medium, and nature of stimulator. Phorbol esters and fatty acids appear to enhance phospholipid synthesis and turnover by distinct intracellular mechanisms.     

Phospholipid metabolism, calcium flux, and the receptor-mediated induction of chemotaxis in rabbit neutrophils

Rabbit neutrophils were stimulated with the chemotactic peptide fMet-Leu-Phe in the presence of the methyltransferase inhibitors homocysteine (HCYS) and 3-deazaadenosine (3-DZA). HCYS and 3-DZA inhibited chemotaxis, phospholipid methylation, and protein carboxymethylation in a dose-dependent manner. The chemotactic peptide-stimulated release of [14C]arachidonic acid previously incorporated into phospholipid was also partially blocked by the methyltransferase inhibitors. Stimulation by fMet-Leu-Phe or the calcium ionophore A23187 caused release of arachidonic acid but not of previously incorporated [14C]-labeled linoleic, oleic, or stearic acids. Unlike the arachidonic acid release caused by fMet-Leu-Phe, release stimulated by the ionophore could not be inhibited by HCYS and 3-DZA, suggesting that the release was caused by a different mechanism or by stimulating a step after methylation in the pathway from receptor activation to arachidonic acid release. Extracellular calcium was required for arachidonic acid release, and methyltransferase inhibitors were found to partially inhibit chemotactic peptide-stimulated calcium influx. These results suggest that methylation pathways may be associated with the chemotactic peptide receptor stimulation of calcium influx and activation of a phospholipase A2 specific for cleaving arachidonic acid from phospholipids.     

Phospholipid synthesis in isolated alveolar type II cells exposed in vitro to paraquat and hyperoxia.

Isolated alveolar epithelial type II cells were exposed to paraquat and to hyperoxia by gas diffusion through the thin Teflon bottom of culture dishes. After exposure, type II cells were further incubated in the presence of labelled substrates to assess their capacity to synthesize lipids. Hyperoxia alone (90% O2; 5 h) had minor effects on lipid metabolism in the type II cells. At low paraquat concentrations (5 and 10 microM), hyperoxia enhanced the paraquat-induced decrease of [Me-14C]choline incorporation into phosphatidylcholines. The incorporation rates of [Me-14C]choline, [1-14C]palmitate, [1-14C]glucose and [1,3-3H]glycerol into various phospholipid classes and neutral lipids were decreased by paraquat, depending on the concentration and duration of the exposure. The incorporation of [1-14C]acetate into phosphatidylcholines, phosphatidylglycerols and neutral lipids appeared to be very sensitive to inactivation by paraquat. At 5 microM-paraquat the rate of [1-14C]acetate incorporation was decreased to 50% of the control values. The rate of [1-14C]palmitate incorporation into lipids was much less sensitive; it even increased at low paraquat concentrations. At 10 microM-paraquat both NADPH and ATP were significantly decreased. It is concluded that lipid synthesis in isolated alveolar type II cells is extremely sensitive to paraquat. At low concentrations of this herbicide, lipid synthesis, and particularly fatty acid synthesis, is decreased. The effects on lipid metabolism may be partly related to altered NADPH and ATP concentrations.     

Incorporation into liver microsomal lipids of linoleic and stearic acids and of their respective products of delta 6 and delta 9 desaturation, gamma-linolenic and oleic acids: effect of age and of blackcurrant seed oil.

The incorporation of [1-14C]linoleic and [1-14C]stearic acid and of their delta 6 and delta 9 desaturation products (gamma-linolenic and oleic acids, respectively) into different classes of lipids was studied in liver microsomes of rats in function of the diet (blackcurrant seed oil diet, containing gamma-linolenic acid, versus control diet) and in function of age (3, 6 and 9 months). After delta 6 desaturation, total radioactivity was distributed between phospholipids, especially phosphatidylcholine, and neutral lipids. The desaturation product, gamma-linolenic acid, was totally recovered in the phospholipid fraction. Blackcurrant seed oil, which decreased the rate of delta 6 desaturation in 6- and 9-month-old rats, also decreased the incorporation of radioactivity in total phospholipids, especially in phosphatidylcholine. At 6 months of age, after delta 9 desaturation, the majority of radioactivity was recovered in neutral lipids principally as oleic acid, the desaturation product. The precursor, stearic acid, was highly incorporated into phospholipids, especially in rats on a diet of blackcurrant seed oil.     

Metabolism of Linoleic Acid or Mevalonate and 6-Pentyl-α-Pyrone Biosynthesis by Trichoderma Species

The understanding of the biosynthetic pathway of 6-pentyl-α-pyrone in Trichoderma species was achieved by using labelled linoleic acid or mevalonate as a tracer. Incubation of growing cultures of Trichoderma harzianum and T. viride with [U-¹⁴C]linoleic acid or [5-¹⁴C]sodium mevalonate revealed that both fungal strains were able to incorporate these labelled compounds (50 and 15%, respectively). Most intracellular radioactivity was found in the neutral lipid fraction. At the initial time of incubation, the radioactivity from [¹⁴C]linoleic acid was incorporated into 6-pentyl-α-pyrone more rapidly than that from [¹⁴C]mevalonate. No radioactivity incorporation was detected in 6-pentyl-α-pyrone when fungal cultures were incubated with [1-¹⁴C]linoleic acid. These results suggested that β-oxidation of linoleic acid was a probable main step in the biosynthetic pathway of 6-pentyl-α-pyrone in Trichoderma species.     

Linoleate and linolenate desaturation by rat hepatoma cells

Morris 7777 rat hepatoma cells in culture possess high delta 6 and delta 5 desaturase activities over linolenic acid added to the medium as albumin or alpha-fetoprotein complexes. After 2 hours incubation with [1-14C] linolenic acid (7 microM), around 40% of the radioactivity was recovered in other polyene fatty acids, mainly pentaenes. After 24 hours incubation with this substrate the polyene derivatives raised to more than 60%. However, [1-14C] linoleic acid was poorly converted to other polyene fatty acids. Linoleic acid up to 58 microM concentration in the medium do not inhibited linolenic acid desaturation. Long-term supplementation with 50 microM linoleic or linolenic acid, which modified the fatty acid profile of hepatoma lipids, enhanced the desaturase activities against linoleic acid. Desaturase activities were not affected by the fatty acid protein carrier, alpha-fetoprotein or albumin.     

Ionophore-induced metabolism of phospholipids and eicosanoid production in porcine aortic endothelial cells: selective release of arachidonic acid from diacyl and ether phospholipids

Confluent cultures of porcine aortic endothelial cells were prelabeled with 1 microM [14C]arachidonic acid complexed to 1 microM bovine serum albumin. After washing, the cells were stimulated with 1 microM A23187 for time intervals between 30 s and 30 min. Cellular lipids were extracted and separated into major lipid classes and phospholipid subclasses. The external medium was analyzed for released radioactive eicosanoids. The time-course of total release of 14C radioactivity demonstrated a biphasic nature of A23187-induced changes in endothelial cell lipids. Early, from 30 s to 5 min, substantial losses of [14C]arachidonic acid from diacylphosphatidylethanolamine and phosphatidylinositol, as well as an abrupt increase in diacylphosphatidylcholine-associated radioactivity were observed. These initial changes coincided with the release of 14C-labeled cyclooxygenase products. Later changes (5-30 min) included a sustained progressive loss of 14C radioactivity from alkenyl (alk-1-enyl) acylphosphatidylethanolamine and diacylphosphatidylcholine. These later changes coincided with the elaboration of 14C-labeled lipoxygenase products. Although unequivocal assignments cannot be made, the data suggest that specific pools of arachidonic acid provide precursors for individual classes of eicosanoids.     

IgE-mediated histamine release in rat basophilic leukemia cells: receptor activation, phospholipid methylation, Ca2+ flux, and release of arachidonic acid

Stimulation of IgE receptors on rat basophilic leukemia cells causes a transient rise and fall of methylated phopholipids, Ca²⁺ influx, and release of arachidonic acid previously incorporated into phosphatidylcholine and liberation of histamine. Inhibition of phospholipid methylation by methyltransferase inhibitors, 3-deazaadenosine and homocysteine thiolactone, almost completely blocks the influx of Ca²⁺, and release of arachidonic acid and histamine. Stimulation of immunoglobulin E receptors by antigen releases only [¹⁴C]arachidonic acid but not [¹⁴C]linoleic acid, [¹⁴C]oleic acid and [¹⁴C]stearic acid, all of which were previously incorporated into phospholipids. [¹⁴C]Arachidonate was found to be incorporated mainly into phosphatidylcholine. The phosphatidycholine rich in arachidonate appeared to be synthesized to a considerable extent by the transmethylation pathway. These findings suggest that in rat basophilic leukemia cells, immunoglobulin E receptors, phospholipid methyltransferases, Ca²⁺ ion channel, and phospholipase(s) that cause release of arachidonic acid and the discharge of histamine are associated.     

Are blastocyst prostaglandins produced endogenously?

This study was undertaken to determine whether preimplantation mouse and rabbit blastocysts possess cyclooxygenase activity and therefore are able to metabolize arachidonic acid (AA) to prostaglandins (PGs) and thromboxane B2. Single rabbit blastocysts or groups of 100 mouse blastocysts were incubated for 4 h in the presence of 5 muCi/ml [3H] AA (sp. ac. 61 Ci/mmol) or for 24 h and 18 h with 0.2 and 0.1 muCi/ml [14C] AA (sp. ac. 56.5 Ci/mmol), respectively. Incubated blastocysts were subsequently exposed for 10 min to 24 h to the Ca2+ specific ionophore, X-537A (10 microM), to stimulate release of radiolabeled AA from the phospholipid pool. After incubation, incorporation of AA into the phospholipid and neutral lipid pools, and metabolism of the fatty acid to PGs and thromboxane B2, were determined using thin-layer chromatographic (TLC) and liquid scintillation spectroscopic techniques. In addition, spent incubation media were analyzed for radiolabeled PG content. In blastocysts of both species, incorporation of AA into phospholipids was greater than that into neutral lipids (mouse: 1.0 vs. 0.7 pmol/blastocyst; rabbit: 159.7 vs. 56.9 pmol/blastocyst). The ionophore stimulated the release of AA from the phospholipid, and to a lesser extent, from the neutral lipid pool, of both blastocysts. No newly synthesized PGs were detected in either mouse blastocysts or their spent incubation media after stimulation with X-537A. Radiolabeled PGs (PGE2, PGF2 alpha, PGD2, PGA2) and thromboxane B2 were present in the media of rabbit blastocyst incubations, however, but were undetectable in the tissue extracts. Increases in metabolism of AA into each compound were observed with an increase in time of exposure to ionophore, and meclofenamic acid (2 microM) partially inhibited the synthesis of all compounds during a 24-h incubation. The results are discussed with regard to the role of blastocyst PGs in the events of implantation.     

Studies in Tetrahymena membranes substrates for desaturation of fatty acyl chains in Tetrahymena pyriformis microsomes

[1-14-C]Palmitoyl-Co A was incubated with Tetrahymena microsomes containing the complete enzyme system for desaturation during various time periods. The level of [1-14C]palmitoleoyl-CoA increased to a maximum during the 1--3 min incubation time, while [1-14C]palmitoleic acid in the phospholipid reached a maximum level during 6--7 min incubation time. The radioactivity of [1-14C]palmitoleic acid in free fatty acid and the triglyceride fraction was not significantly observed upon 3 min incubation. Incubation of [1-14C]palmitoyl-CoA with microsomes in the absence of NADH produced [1-14C]palmitoyl lipid without desaturation. Radioactive palmitic acids in the microsomal lipids were not converted to palmitoleic acids after addition of NADH by the complete enzyme system. When microsomes prepared from cells labeled with [1-14C]palmitic acid or [1-14C]stearic acid were incubated alone in the presence of O2 and NADH, no significant increase in [1-14C]palmitoleic acid in the phospholipid was observed, wherease an increase in [1-14C]linoleic acid and gamma-[1-14C]linolenic acid did occur at the expense of [1-14C]oleic acid in the phospholipid. From these results it can be concluded that the enzyme involving desaturation of palmitic acid to palmitoleic acid requires palmitoyl-CoA as the substrate. However, the possibility of oleoyl and linoleoyl phospholipids being substrates in the desaturation of Tetrahymena microsomes was suggested.     

Metabolism of polyunsaturated fatty acids by rabbit reticulocytes

Rabbit reticulocytes obtained by repeated bleeding metabolize exogenous [1-14C]linoleic acid and [1-14C]arachidonic acid by three different pathways. 1. Incorporation into cellular lipids: 50% of the fatty acids metabolized are incorporated into phospholipids, mainly phosphatidylcholine (32.8%) but also into phosphatidylethanolamine (12%), whereas about 10% of the radioactivity was found in the neutral lipids (mono- di- and triacylglycerols, but not cholesterol esters). 2. Formation of lipoxygenase products: 30% of the fatty acids metabolized are converted via the lipoxygenase pathway mainly to hydroxy fatty acids. Their formation is strongly inhibited by lipoxygenase inhibitors such as 5,8,11,14-eicosatetraynoic acid or nordihydroguaiaretic acid. Inhibition of the lipoxygenase pathway results in an increase of the incorporation of the fatty acids into cellular lipids. 15-Hydroxy-5,8,11,13(Z,Z,Z,E)eicosatetraenoic acid and 13-hydroxy-9,11(Z,E)-octadecadienoic acid are incorporated by reticulocytes into cellular lipids and also are metabolized via beta-oxidation. The metabolism of arachidonic acid and linoleic acid is very similar except for a higher incorporation of linoleic acid into neutral lipids. 3. beta-Oxidation of the exogenous fatty acids: about 10% of the polyenoic fatty acids are metabolized via beta-oxidation to 14CO2. Addition of 5,8,11,14-eicosatetraynoic acid strongly increased the 14CO2 formation from the polyenoic fatty acids whereas antimycin A completely abolished beta-oxidation. Erythrocytes show very little incorporation of unsaturated fatty acids into phospholipids and neutral lipids. Without addition of calcium and ionophore A23187 lipoxygenase metabolites could not be detected.     

The lipid composition and permeability to the triazole antifungal antibiotic ICI 153066 of serum-grown mycelial cultures of Candida albicans

The total lipid content of Candida albicans (serotype A: NCPF 3153) exponential-phase mycelial cultures grown in tissue-culture medium 199 (containing 10%, v/v, foetal calf serum) was 29.8 +/- 8 mg (g dry weight)-1 (mean +/- SD). The weight ratios of phospholipid to neutral lipid and phospholipid to non-esterified sterol were 2.6 +/- 0.4 and 24.9 +/- 0.5, respectively. The major phospholipid was phosphatidylcholine with smaller amounts of phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidylglycerol and diphosphatidylglycerol; the most abundant fatty acids were palmitic, palmitoleic, oleic and linoleic acids. The major neutral lipids comprised esterified sterol, triacylglycerol and non-esterified fatty acid with a smaller amount of non-esterified sterol. The fatty acid compositions of the three fatty-acid-containing neutral lipids were distinct from each other and the phospholipids. Comparison with previous data on yeast cultures of C. albicans A grown in glucose broth shows that mycelial cultures have a larger lipid content, lower phospholipid to neutral lipid ratio and higher phospholipid to non-esterified sterol ratio. We now show that mycelial cultures were more permeable to a [14C]triazole antifungal antibiotic compared with exponentially growing yeast cultures of several azole-sensitive strains. Taken together these data are consistent with there being a relationship between the phospholipid/non-esterified sterol ratio of a culture and its ability to accumulate a triazole.     

Arachidonate released upon agonist stimulation preferentially originates from arachidonate most recently incorporated into nuclear membrane phospholipids

When icosanoid-producing cells are stimulated by an agonist, 2-10% of total cellular arachidonate is released from phospholipids, and a variable percentage of the released arachidonate is subsequently converted into icosanoids. We used a mouse fibrosarcoma cell line (HSDM1C1) which synthesizes prostaglandin E2 in response to bradykinin stimulation to address the following questions: 1) upon cell stimulation is newly incorporated arachidonate preferentially released from phospholipids over previously incorporated arachidonate and 2) is there a corresponding change in phospholipid or membrane compartmentation of arachidonate to explain preferential release of newly incorporated arachidonate? To study changes in the availability of arachidonate for release from phospholipids, we incubated HSDM1C1 cells with 0.67 microM [14C]arachidonate for 15 min and chased the pulse of radiolabeled arachidonate with normal serum fatty acids. We found that of the [14C]arachidonate incorporated into phospholipids during the 15-min pulse, the percent released upon stimulation decreased nearly 3-fold from 8.9 +/- 0.5% at 5 min of chase to 3.6 +/- 0.2% (mean +/- S.E., n = 6, P less than 0.001) after only 60 min of chase. Percent release of arachidonate from nonpulsed controls was 3-4%. Although arachidonate release from phospholipids decreased significantly after 60 min of chase, the arachidonate which was released always originated predominantly from phosphatidylinositol. There was no decrease in the activities of enzymes required for arachidonate release during this time period. We also observed that throughout the period of the chase, the radiolabeled arachidonate remained esterified to the same phospholipid class into which it was initially incorporated (approximately 40% of [14C]arachidonate in diacyl phosphatidylcholine, 40% in phosphatidylinositol, and 15% in diacyl phosphatidylethanolamine. In cell fractionation experiments, we found that after 1-3 h of chase, [14C]arachidonate decreased in subcellular fractions containing nuclei, as it became progressively unavailable for release from phospholipids. Thus, our results indicate that 1) upon cell stimulation, the most recently incorporated pool of arachidonate, which is in high concentration in the nuclear membrane, is preferentially released and that 2) arachidonate rapidly moves out of the nuclear membrane into a less releasable pool while remaining esterified to the phospholipid moiety into which it was initially incorporated. This study indicates that the subcellular compartmentation of arachidonate has a marked influence on the cellular metabolism of arachidonate.     

Effect of dietary fats on linoleic acid metabolism. A radiolabel study in rats

Effects on the linoleic acid metabolism in vivo of three dietary fats, rich in either oleic acid, trans fatty acids or alpha-linolenic acid, and all with the same linoleic acid content, were investigated in male Wistar rats. After 6 weeks of feeding, the rats were intubated with [1-14C]linoleic acid and [3H]oleic acid. The incorporation of these radiolabels into liver, heart and serum was investigated 2, 4, 8, 24 and 48 h after intubation. The amount of 14C-labelled arachidonic acid incorporated into the liver phospholipid of the group fed the oleic acid-rich diet was significantly higher than that of the other groups. However, compared to the trans fatty acids-containing diet, the oleic acid-rich diet induced only a slightly higher arachidonic acid level in the phospholipid fraction of the tissues as determined by GLC. Dietary alpha-linolenic acid more than halved the arachidonic acid levels. Our results do not support the hypothesis that the delta 6-desaturase system actually determines the polyunsaturated fatty acid levels in tissue lipids by regulating the amount of polyunsaturated fatty acids (e.g., arachidonic acid) synthesized. The biosynthesis of polyunsaturated fatty acids only is not sufficient to explain the complicated changes in fatty acid compositions as observed after feeding different dietary fats.     

Stimulation of deacylation in Madin-Darby canine kidney cells. Specificity of deacylation and prostaglandin production in 12-O-tetradecanoylphorbol-13-acetate-treated cells

Madin-Darby canine kidney cells deacylate arachidonic acid from cellular phospholipid in response to 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and convert the free arachidonic acid to prostaglandins. We have used this system to characterize the acyl specificity of deacylation. Cells were labeled with either [14C]linoleic, [14C]eicosatrienoic (delta 8,11,14 or delta 5,8,11), or [14C]arachidonic acid and stimulated with 10 nM TPA. We found that TPA stimulated the deacylation of all four acids, primarily from phosphatidylethanolamine and phosphatidylcholine.l Only products from linoleic (presumably through chain elongation and desaturation), eicosatrienoic (delta 8,11,14), and arachidonic acids produced prostaglandins. Those produced from linoleic and eicosatrienoic acid (delta 8,11,14)-labeled cells were determined to be primarily of the 1-series, while arachidonic acid-labeled cells produced prostaglandins of the 2-series. Together these results indicate that the stimulated deacylation of phospholipids is not specific for arachidonic acid and that the membrane acyl composition controls the particular series of prostaglandin which is produced.