Cell surface lipids and adhesion. I. The effects of lysophosphatidyl compounds, phospholipase A2 and aggregation-inhibiting protein.

Aggregation-inhibiting protein (AIP: Curtis & Greaves, 1965), which diminishes the adhesiveness of cells, particularly at low temperatures, is identified in the present paper as phospholipase A2 (EC. 3.1.1.4). Our reasons for this identification are because phospholipase activity parallels AIP activity on cell adhesion, and because various inhibitors and sera act in a parallel manner on adhesion in the presence of AIP or phospholipase. We suggest that the enzyme acts on adhesion by producing lysolecithin and other lysolipids in the plasmalemma. Addition of lysolipids diminishes cell adhesion in a manner similar to phospholipase A. Incubation of cells in Hanks' medium at 37 degrees C has a parallel effect. Conditions which would be expected to stimulate reacylation of lysolipids in the plasmalemma, i.e. incubation of cells in the external presence of CoA, ATP and a fatty acid, lead to a recovery or maintenance of adhesion after or during Hanks' incubation at 37 degrees C. All these results suggest that lipid components of the cell, probably in the plasmalemma, are of importance in adhesion. The results are discussed in relation to the long-standing controversy about the effects of low temperatures and trypsinization on cell adhesion, for phospholipase treatment of cells affects adhesion in a manner similar to trypsinization.     

Cell surface lipids and adhesion. III. The effects on cell adhesion of changes in plasmalemmal lipids.

The two preceding papers of this series suggest that the state of the plasmalemmal lipids affects cell adhesion. Plasmalemmal composition was altered by the experimental incorporation of fatty acids into R1 and R2 positions in the phosphatidyl components of the cell surface. In this paper we report that: (1) If the incorporation is of long chain length fatty acids (saturated) cell adhesion rises. (2) If the incorporation is of unsaturated fatty acids cell adhesion falls as the unsaturation increases. (3) Incorporation has to be extensive to produce a large change in adhesion. (4) Changes in adhesion parallel the plasmalemmal incorporation but do not follow the total cell incorporation. Item (4) argues that it is plasmalemmal and not other membrane lipids that are involved in cell adhesion. Item (3) suggests that bulk membrane properties and not some very specific grouping are involved in the effects of lipids on adhesion. The similar extents of incorporation of the various different fatty acids and the negligible amounts of lysophospholipids in the membranes of cells that have incorporated fatty acids argue that the effects are not due to differential accumulations of these lysolipids when incubations are done with different fatty acids. The changes in adhesion cannot be accounted for by changes in surface charge density since the electrophoretic mobility of the cells is unchanged by these incubations. It is suggested that these effects on adhesion due to changes in plasmalemmal lipids can be explained either in terms of the action of intermembrane van der Waals--London (electrodynamic) forces in cell adhesion or of changes in surface fluidity. These alternatives are discussed.     

Evidence for the Involvement of Carnitine-Dependent Long-Chain Acyltransferases in Neuronal Triglyceride and Phospholipid Fatty Acid Turnover

Abstract: This study focuses on the potential involvement of carnitine palmitoyltransferase (CRT) on the phospholipid and triglyceride fatty acid turnover in neurons. This category of enzymes, which has been identified in several rat brain tissues, is well known for its role in modulating cellular fatty acid oxidation. Neuronal cell cultures from rat brain cortex incorporated radioactive palmitate or oleate into phospholipids and triglycerides. The largest fraction of radioactive fatty acids was recovered in phosphatidyl- choline followed by triglycerides and, to a lesser extent, phosphatidylethanolamine. CPT activity measured in neuronal lysates obtained from neurons treated with 40 μ M 2-tetradecylglycidic acid (TDGA) was almost completely abolished. Furthermore, between 2 and 10 μ M TDGA CPT activity dropped more rapidly than between 10 and 40 μ M. When the cells were pretreated with TDGA, the incorporation process of either radioactive fatty acid into triglycerides was dose-dependently suppressed. Radioactive fatty acid incorporation into phosphatidylcholine was significantly decreased in cells treated with TDGA. In contrast, phosphatidylethanolamine reacylation was essentially not affected by the CpT inhibitor. Similar results on the fatty acid incorporation into triglycerides and phospholipids were observed with neurons treated with palmitoyl- dl - aminocarnitine (PAC), a reversible CPT inhibitor, which does not consume free CoA. These effects do not seem to be the result of an inhibitory activity toward one of the steps involved in the acylation-deacylation process of triglycerides or phospholipids, as cellular lysates from TDGA-treated cells or lysates containing PAC incorporated radioactive fatty acids at rates comparable to controls. Our results suggest that CRT may be an important partner in the pathway of phospholipid and triglyceride fatty acid turnover in neurons.     

Phospholipid remodeling in human neutrophils. Parallel activation of a deacylation/reacylation cycle and platelet-activating factor synthesis

A23187 stimulated two enzymatic activities of human neutrophils (polymorphonuclear leukocytes), phospholipase A2 and fatty acyl-CoA acyltransferase, which resulted in a stimulated deacylation/reacylation cycle. The incorporation of fatty acids, other than arachidonic or eicosapentaenoic acid, into diacyl and alkylacyl species of choline phosphoglycerides was stimulated by 10-fold by A23187. These fatty acids were exclusively incorporated into the sn-2 position, and [3H]glycerol labeling showed there was no stimulation of de novo synthesis. A23187 also stimulated fatty acid incorporation into other phospholipids, but de novo synthesis accounted for a portion of this uptake. Inhibitors of protein kinase C prevented the stimulated recycling of phosphatidylcholine, and the simultaneous induction of platelet-activating factor synthesis, by inhibiting phospholipase A2 activation. They inhibited [3H]arachidonate release from prelabeled polymorphonuclear leukocytes, but had no effect on in vitro fatty acyl-CoA acyltransferase or acetyl-CoA acetyltransferase activity. Extracts from A23187-treated cells contained a fatty acyl-CoA acyltransferase, which did not utilize arachidonoyl-CoA, that was 2.3-fold more active than that of control extracts. Phosphatase treatment decreased this stimulated activity by 66%. Thus, A23187 stimulated a phospholipase A2 activity that generated both 1-alkyl and 1-acyl lysophosphatidylcholines. A stimulated acetyltransferase used a portion of the alkyl species for platelet-activating factor synthesis, while the acyl species and residual alkyl species were rapidly reacylated to phosphatidylcholine by a stimulated acyl-transferase. Arachidonate, an eicosanoid precursor, was spared by this process.     

IL-1 alpha increases arachidonyl-CoA: lysophospholipid acyltransferase activity and stimulates [3H]arachidonate incorporation into phospholipids in rat mesangial cells.

The proinflammatory cytokine interleukin-1 alpha is a potent stimulus of prostaglandin synthesis. We have previously shown that IL-1 amplifies mesangial cell prostaglandin synthesis by inducing synthesis of a non-pancreatic phospholipase A2. Phospholipase A2 activation results in the formation of lysophospholipids and free fatty acids. We now investigate the effects of IL-1 alpha on reacylation of lysophospholipids. Incubations with IL-1 alpha for 24 hours significantly stimulated mesangial cell [3H]arachidonic acid incorporation but not [3H]oleic acid incorporation into phosphatidylinositol and phosphatidylethanolamine. Lysophospholipid acyltransferase activity was measured in vitro. Cytokine treatment increased enzyme activity when lysophosphatidylcholine, lysophosphatidylethanolamine and lysophosphatidylinositol were used as exogenous substrates. We conclude that IL-1 promotes cellular phospholipid remodeling by stimulating the deacylation and reacylation of phospholipids.     

Fatty acid remodelling of phosphatidylinositol under conditions of de novo synthesis in rat liver microsomes

Phosphatidylinositol (PI) is initially synthesized in mammalian cells with a fatty acid composition similar to that of its precursor, primarily monounsaturated forms of cytidine diphosphodiglyceride (CDP-DAG). However, at the steady state, over 80% of PI exists in the 1-stearoyl, 2-arachidonoyl form. The fatty acid remodelling of PI is due to a number of deacylation/reacylation mechanisms. In the preceding paper we demonstrated that de novo synthesized PI is rapidly deacylated and subsequently reacylated. In this report we present further evidence that cycles of deacylation and reacylation are involved in the remodelling of PI. Incubation of microsomes with CDP-DAG of different fatty acid composition results in quantitative and qualitative differences in lysoPI formation. Additionally, analyses of the resulting lysoPI and PI species reveal that multiple species of fatty acids are incorporated into the 1-position of both PI and lysoPI. Addition of acylation cofactors (fatty acyl CoAs or ATP plus CoA) potentiate reacylation in this system. The addition of stearoyl or myristoyl CoA during de novo synthesis of PI results in the incorporation of these added fatty acids into the I-positive of PI. In addition, some evidence is presented that multiple mechanisms for remodelling of the 1-position of PI may be active in the microsomes, including ATP- and CoA-dependent acylation, ATP-independent, CoA-dependent acylation and CoA-independent mechanisms. Finally, the disappearance of only a subset of lysoPI species upon the addition of acylation cofactors suggests that the reacylation step exhibits some substrate specificity.     

The signal molecule lysophosphatidylcholine in Eschscholzia californica is rapidly metabolized by reacylation

In cultured cells of California poppy (Eschscholzia californica), lysophosphatidylcholine (LPC) triggers a signal path that finally induces alkaloid biosynthesis. LPC is transiently generated by elicitor-activated phospholipase A(2) of the plasma membrane. Externally added LPC is rapidly acylated by a membrane-bound enzyme that shows the highest specific activity in the purified plasma membrane. The fatty acid incorporated into the sn-2 position of LPC is preferentially linoleic (18:2), which is the most abundant acyl component in the PC species of Eschscholzia cells, but a minor component of the pool of free fatty acids. The fatty acid at the sn-1 position of LPC is less important for substrate specificity. The capacity of LPC acylation by intact cells or isolated plasma membranes by far exceeds the rate of LPC generation by activated phospholipase A(2) and is not limited by the availability of acyl donors. Metabolites other than phosphatidylcholine (PC) were not significantly produced from labeled LPC within 20 min, indicating that lysophospholipases are not significantly contributing to the short-time metabolism of LPC. It is concluded that reacylation to PC is the dominating process in the detoxication of LPC and ensures the transient character of its steady state concentrations, even at maximum phospholipase A(2) activities.     

t-Butyl hydroperoxide alters fatty acid incorporation into erythrocyte membrane phospholipid

Because the ability of cells to replace oxidized fatty acids in membrane phospholipids via deacylation and reacylation in situ may be an important determinant of the ability of cells to tolerate oxidative stress, incorporation of exogenous fatty acid into phospholipid by human erythrocytes has been examined following exposure of the cells to t-butyl hydroperoxide. Exposure of human erythrocytes to t-butyl hydroperoxide (0.5-1.0 mM) results in oxidation of glutathione, formation of malonyldialdehyde, and oxidation of hemoglobin to methemoglobin. Under these conditions, incorporation of exogenous [9,10-3H]oleic acid into phosphatidylethanolamine is enhanced while incorporation of [9,10-3H]oleic acid into phosphatidylcholine is decreased. These effects of t-butyl hydroperoxide on [9,10-3H]oleic acid incorporation are not affected by dissipating transmembrane gradients for calcium and potassium. When malonyldialdehyde production is inhibited by addition of ascorbic acid, t-butyl hydroperoxide still decreases [9,10-3H]oleic acid incorporation into phosphatidylcholine but no stimulation of [9,10-3H]oleic acid incorporation into phosphatidylethanolamine occurs. In cells pre-treated with NaNO2 to convert hemoglobin to methemoglobin, t-butyl hydroperoxide reduces [9,10-3H]oleic acid incorporation into phosphatidylcholine by erythrocytes but does not stimulate [9,10-3H]oleic acid incorporation into phosphatidylethanolamine. Under these conditions oxidation of erythrocyte glutathione and formation of malonyldialdehyde still occur. These results indicate that membrane phospholipid fatty acid turnover is altered under conditions where peroxidation of membrane phospholipid fatty acids occurs and suggest that the oxidation state of hemoglobin influences this response.     

Turnover of fatty acids in the 1-position of phosphatidylethanolamine in Escherichia coli

Phosphatidylethanolamine is the major membrane phospholipid of Escherichia coli, and two experimental approaches were used to investigate the metabolic activity of the fatty acids occupying the 1-position of this phospholipid. [3H]Acetate pulse-chase experiments with logarithmically growing cells indicated that 3-5% of the acyl groups were removed from the phosphatidylethanolamine pool/generation. The reacylation aspect of the turnover cycle was demonstrated by the incorporation of fatty acids into the 1-position of pre-existing phosphatidylethanolamine when de novo phospholipid biosynthesis was inhibited using the plsB acyltransferase mutant. 2- Acylglycerophosphoethanolamine would be the intermediate in a 1-position turnover cycle, and this lysophospholipid was identified as a membrane component that could re-esterified by a membrane-bound acyltransferase. The acyltransferase either utilized acyl-acyl carrier protein directly as an acyl donor or activated fatty acids for acyl transfer in the presence of ATP and Mg2+. Acyl-acyl carrier protein was also indicated as an intermediate in the latter reacylation reaction by the complete inhibition of phosphatidylethanolamine formation from fatty acids by acyl carrier protein-specific antibodies and by the observation that the inhibition of the acyltransferase by LiCl was reversed by the addition of acyl carrier protein. Coenzyme A thioesters were not substrates for this acyltransferase. These results suggest the existence of a metabolic cycle for the utilization of 1-position acyl moieties of phosphatidylethanolamine followed by the resynthesis of this membrane phospholipid from 2- acylglycerophosphoethanolamine by an acyl carrier protein-dependent 1-position acyltransferase.     

Control of baculovirus gp64-induced syncytium formation by membrane lipid composition.

We have investigated the effects of membrane lipid composition on biological membrane fusion triggered by low pH and mediated by the baculovirus envelope glycoprotein gp64. Lysolipids, either added exogenously or produced in situ by phospholipase A2 treatment of cell membranes, reversibly inhibited syncytium formation. Lysolipids also decreased the baculovirus infection rate. In contrast, oleic and arachidonic acids and monoolein promoted cell-cell fusion. Membrane lipid composition affected pH-independent processes which followed the low-pH-induced change in fusion protein conformation. Inhibition and promotion of membrane fusion by a number of lipids could not be explained by mere binding or incorporation into membranes, but rather was correlated with the effective molecular shape of exogenous lipids. Our data are consistent with the hypothesis that membrane fusion proceeds through highly bent membrane intermediates (stalks) having a net negative curvature. Consequently, inverted cone-shaped lysolipids inhibit and cone-shaped cis-unsaturated fatty acids promote stalk formation and, ultimately, membrane fusion.     

Control of arachidonic acid accumulation in bone marrow-derived macrophages by acyltransferases

The turnover of phospholipid fatty acid moieties of bone marrow-derived macrophages was analyzed by separate determination of degrading and acylating activities. Acylating activities were followed in intact cells by incubation with excess arachidonic acid and degradation of phospholipids was followed in cells prelabeled with fatty acids. Significant phospholipase A2 activity was detectable only if the reutilization of liberated fatty acid was inhibited , e.g. by p-chloromercuribenzoate. It was of interest that the divalent cation ionophore A 23187 and various antiphlogistic drugs like indomethacin, diclofenac, and acetylsalicylic acid were found to inhibit the acylation reaction. These compounds led to increased levels of free arachidonic acid in stimulated, as well as in unstimulated cells. Increased activities of phospholipase A2 were achieved by treatment with the bivalent cation ionophore A 23187 and with zymosan. The effect of zymosan obtained from various sources was found to be exclusively due to contamination of tee zymosan particles with phospholipase A2 activity. Even when the cellular phospholipase activity was increased by the addition of exogenous phospholipase activity contained in the zymosan particles, degradation of cellular phospholipids was not measurable unless the reacylation was inhibited. These results suggest that in the cells studied, the level of free arachidonic acid is mainly controlled by the activity of the lysophosphatide acyltransferase.     

Membranes of animal cells. II. The metabolism and turnover of the surface membrane

Turnover studies of the surface membrane and of cell particulate matter of L cells in tissue culture in logarithmic and plateau phase of growth have been made. The rate of incorporation of isotope into these fractions and the rate of fall of specific activities of labeled L-cell fractions have been observed. The following interpretation of the data appears most likely although other interpretations are possible. Growing and nongrowing cells synthesize approximately similar amounts of surface membrane and particulate material. In the growing cell the material is incorporated with net increases in substance. There is relatively little turnover. In the nongrowing cell newly synthesized material is incorporated, but a corresponding amount of material is eliminated so that there is turnover without net increase of substance. Our results suggest that there is no gross differential turnover between the protein, lipid, and carbohydrate of the surface membrane under the conditions of our experiments. Metabolic inhibitors or omission of amino acids in the culture medium lead to a decrease in synthesis of surface membrane and cell particulates and cause an equivalent decrease in the rate of degradation of surface membrane and of particulates; therefore the synthetic and degradative aspects of turnover appear to be coupled. As cultures of nongrowing cells in suspension or on a glass surface age, their synthetic and turnover capacities diminish. Our results suggest that the cell may exist in a nongrowing state with a level of synthesis similar to that of a growing cell. It can exist in this state with a high level of turnover.     

Spontaneous and plasma factor-mediated transfer of pyrenyl cerebrosides between model and native lipoproteins

A series of pyrenyl glucocerebrosides was synthesized by reacylation of psychosine with pyrene-labeled fatty acids having 3-11 methylene units. When incorporated into model high-density lipoproteins consisting of dimyristoylphosphatidylcholine-apolipoprotein A-II complexes and incubated with unlabeled complexes, these lipids exhibited spontaneous transfer. Half times of transfer varied from 1.5 min to 365 min at 37 degrees C. The logarithm of the rate of transfer was linearly related to the number of fatty acyl methylene units and HPLC retention time. Transfer occurred by passage of lipid monomers through the aqueous phase. Spontaneous transfer of the glycolipids also occurred when they were incorporated into native high-density lipoproteins. Rates of transfer between native high-density lipoprotein particles were higher than those observed between model high-density lipoprotein particles. A partially purified lipid exchange protein from plasma, as well as unfractionated lipoprotein-deficient serum, stimulated the transfer of fluorescent glycolipid between model high-density lipoprotein or native high-density lipoprotein and low-density lipoprotein 2-24 fold. The protein also stimulated the transfer of tritiated ganglioside GM3 between native low-density lipoprotein and high-density lipoprotein. This protein may play a role in glycolipid exchange in vivo.     

Phospholipase B activity enhances adhesion of Cryptococcus neoformans to a human lung epithelial cell line

Secreted phospholipase B (PLB1), which contains three enzyme activities in the one protein, is necessary for the initiation of pulmonary infection by Cryptococcus neoformans and for dissemination from the lung via the lymphatics and blood. Adhesion to lung epithelium is the first step in this process, therefore we investigated the role of PLB1 in adhesion to a human lung epithelial cell line, A549, using C. neoformans var. grubii wild-type strain H99, a PLB1 deletion mutant (deltaplb1), and a reconstituted strain (deltaplb1rec). Adhesion of H99 and deltaplb1rec was approximately 69% greater than deltaplb1 at 4 h. Adhesion of deltaplb1 significantly increased after killing by chemicals or heat, and Fourier-transformed analysis by FTIR spectroscopy indicated this was due to changes in capsular and/or cell wall polysaccharides and proteins. Inhibition by specific PLB1 antibodies, or inhibitors of phospholipase B (PLB), but not lysophospholipase (LPL) or lysophospholipase transacylase (LPTA) activities decreased the adhesion of H99 and deltaplb1rec by 33-58%. Growth under conditions of osmotic stress and high glucose concentration increased both PLB secretion and subsequent cryptococcal adhesion. Dose-dependent increases (to 67%) in adhesion of live deltaplb1 were observed in the presence of 0.1-2 mM palmitic acid. We conclude that PLB1 plays a role in the binding of C. neoformans to host lung epithelial cells, possibly due to production of fatty acids from plasma membranes and/or surfactant by PLB activity.     

Lipids, curvature stress, and the action of lipid prodrugs: free fatty acids and lysolipid enhancement of drug transport across liposomal membranes

Molecular shape and its impact on bilayer curvature stress are powerful concepts for describing the effects of lipids and fatty acids on fundamental membrane properties, such as passive permeability and derived properties like drug transport across liposomal membranes. We illustrate these relationships by studying the effects of fatty acids and lysolipids on the permeation of a potent anti-cancer drug, doxorubicin, across the bilayer of a liposome in which the drug is encapsulated. Using a simple fluorescence assay, we have systematically studied the passive permeation of doxorubicin across liposomal membranes in different lipid phases: the solid-ordered phase (DPPC bilayers), the liquid-disordered phase (POPC lipid bilayers), and the liquid-ordered phase induced by high levels of cholesterol (DOPC + cholesterol lipid bilayers). The effect of different free fatty acids (FA) and lysolipids (LL), separately and in combination, on permeability was assessed to elucidate the possible mechanism of phospholipase A₂-triggered release in cancer tissue of liposomal doxorubicin formulations. In all cases, FAs applied separately lead to significant enhancement of permeability, most pronounced in liquid-disordered bilayers and less pronounced in solid and solid-ordered bilayers. LLs applied separately had only a marginal effect on permeability. FA and LL applied in combination lead to a synergistic enhancement of permeability in solid bilayers, whereas in liquid-disordered bilayers, the combined effect suppressed the otherwise strong permeability enhancement due to the FAs.     

A soluble phospholipase of Toxoplasma gondii associated with host cell penetration

We previously reported that phospholipase increases host cell penetration by Toxoplasma gondii. Here we show that calcium-dependent phospholipase A (PLA) activity is found in the supernatant of sonically disrupted T. gondii. When fractions of disrupted T. gondii were incubated with host cells, the release of fatty acids and lysolipids was detected. Fractions of sonically disrupted T. gondii with PLA activity increased T. gondii host cell penetration in a bioassay. In addition, a protein of approximately 20 kDa was detected by immunoblot of T. gondii antigens with horse antiserum to snake venom, the major antibody of which recognizes PLA2. Incubation of T. gondii with exogenous PLA2 resulted in increased solubility of a rhoptry protein. This protein, which we previously characterized as involved with enhanced parasite invasion of host cells and which is recognized by monoclonal antibody Tg49, was detected in increased amounts in supernatant fractions of extracellular parasites treated with PLA2. Whereas without PLA2 treatment, it is only slightly soluble under physiological conditions. This raises the possibility that PLA may be implicated in the release of rhoptry proteins.     

Blockade of arachidonic acid incorporation into phospholipids induces apoptosis in U937 promonocytic cells

Arachidonic acid (AA) participates in a reacylation/deacylation cycle of membrane phospholipids, the so-called Lands cycle, that serves to keep the concentration of this free fatty acid in cells at a very low level. To manipulate the intracellular AA level in U937 phagocytes, we have used several pharmacological strategies to interfere with the Lands cycle. We used inhibitors of the AA reacylation pathway, namely thimerosal and triacsin C, which block the conversion of AA into arachidonoyl-CoA, and a CoA-independent transacylase inhibitor that blocks the movement of AA within phospholipids. In addition, we used cells overexpressing group VIA phospholipase A(2), an enzyme with key roles in controlling basal fatty acid deacylation reactions in phagocytic cells. All of these different strategies resulted in the expected increase of cellular free AA but also in the induction of cell death by apoptosis. Moreover, when used in combination with any of the aforementioned drugs, AA itself was able to induce apoptosis at doses as low as 10 muM. Blocking cyclooxygenase or lipoxygenases had no effect on the induction of apoptosis by AA. Collectively, these results indicate that free AA levels within the cells may provide an important cellular signal for the onset of apoptosis and that perturbations of the mechanisms controlling AA reacylation, and hence free AA availability, may decisively affect cell survival.     

Role of triglycerides in endothelial cell arachidonic acid metabolism

Arachidonic acid was incorporated into triglycerides by cultured bovine endothelial cells in a time- and concentration-dependent manner. At 75 microM or higher, more arachidonic acid was incorporated into triglycerides than into phospholipids. The triglyceride content of the cells increased as much as 5.5-fold, cytoplasmic inclusions appeared, and arachidonic acid comprised 22% of the triglyceride fatty acids. Triglyceride turnover occurred during subsequent maintenance culture; there was a 60% decrease in the radioactive arachidonic acid contained in triglycerides and a 40% decrease in triglyceride content in 6 hr. Most of the radioactivity was released into the medium as free fatty acid. The turnover of arachidonic acid, but not oleic acid in cellular triglycerides, decreased when supplemental fatty acid was added to the maintenance medium. Incorporation and turnover of radioactive arachidonic acid in triglycerides also was observed in human skin fibroblasts, 3T3-L1 cells, and MDCK cells. Other fatty acids were incorporated into triglycerides by the endothelial cells; the amounts after a 16-hr incubation with 50 microM fatty acid were 20:3 greater than 20:4 greater than 18:1 greater than 18:2 greater than 22:6 greater than 16:0 greater than 20:5. These findings indicate that triglyceride formation and turnover can play a role in the fatty acid metabolism of endothelial cells and that arachidonic acid can be stored in endothelial cell triglycerides.     

Differential turnover of phospholipid acyl groups in mouse peritoneal macrophages

Phospholipid acyl turnover was assessed in mouse peritoneal exudate cells which consisted primarily of macrophages. The cells were incubated for up to 5 h in media containing 40% H218O, and uptake of 18O into ester carbonyls of phospholipids was determined by gas chromatography-mass spectrometry of hydrogenated methyl esters. The uptake was highest in choline phospholipids and phosphatidylinositol, less in ethanolamine phospholipids, and much less in phosphatidylserine. Acyl groups at the sn-1 and sn-2 positions of diacyl glycerophospholipids, including arachidonic and other long-chain polyunsaturated fatty acids, acquired 18O at about the same rate. Acyl groups of alkylacyl glycerophosphocholine exhibited lower rates of 18O uptake, and acyl groups of ethanolamine plasmalogens (alkenylacyl glycerophosphoethanolamines) acquired only minimal amounts of 18O within 5 h, indicating a low average acyl turnover via free fatty acids. Pulse experiments with exogenous 3H-labeled arachidonic acid supported the concept that acylation of alkenyl glycerophosphoethanolamine occurs by acyl transfer from other phospholipids rather than via free fatty acids and acyl-CoA. The 18O content of intracellular free fatty acids increased gradually over a 5-h period, whereas in extracellular free fatty acids it reached maximal 18O levels within the first hour. Arachidonate and other long-chain polyunsaturated fatty acids were found to participate readily in deacylation-reacylation reactions but were present only in trace amounts in the free fatty acid pools inside and outside the cells. We conclude that acyl turnover of macrophage phospholipids through hydrolysis and reacylation is rapid but tightly controlled so that appreciable concentrations of free arachidonic acid do not occur.     

Modulation of cell-mediated cytotoxicity function after alteration of fatty acid composition in vitro

Fatty acids incorporated into cytotoxic T lymphocytes (CTL) during in vitro stimulation can enhance or inhibit the subsequent expression of cell-mediated cytotoxicity, depending on the class of fatty acid. Unsaturated fatty acids enhance cytolysis, whereas saturated fatty acids inhibit it. The effects of fatty acids on cytolysis can be mediated in the absence of cell division, thus eliminating relative clonal amplification or contraction as a basis for the observed effects. Nevertheless, the net result of fatty acid alteration is an increase (unsaturated fatty acids) or decrease (saturated fatty acids) in the frequency, in the treated immune population, of CTL capable of lysing target cells. These observations are best explained by a model in which fatty acid incorporated into cell membrane phospholipids results in a direct recruitment into or out of the pool of CTL within the immune population capable of lysing target cells under the conditions employed to assay cytotoxicity.