Manipulation of fatty acid composition of membrane phospholipid and its effects on cell growth in mouse LM cells

Fatty acid composition of the phospholipids of mouse LM cells grown in suspension culture in serum-free chemically defined medium was modified by supplementing the medium with various fatty acids bound to bovine serum albumin.Following supplementation with saturated fatty acids of longer than 15 carbons (100 μM) profound inhibition of cell growth occurred; this inhibitory effect was completely abolished when unsaturated fatty acids were added at the same concentration. Supplementing with unsaturated fatty acids such as linoleic acid, linolenic acid or arachidonic acid had no effect on the cell growth.Fatty acid composition of membrane phospholipids could be manipulated by addition of different fatty acids. The normal percentage of unsaturated fatty acids in LM cell membrane phospholipids (63%) was reduced to 35–41% following incorporation of saturated fatty acids longer than 15 carbon atoms and increased to 72–82% after addition of unsaturated fatty acids.A good correlation was found between the unsaturated fatty acid content of membrane phospholipids and cell growth. When incorporated saturated fatty acids reduced the percentage of unsaturated fatty acids in membrane phospholipids to less than 50%, severe inhibition of the cell growth was found. Simultaneous addition of an unsaturated fatty acid completely abolished this effect of saturated fatty acids.     

Modification of membrane phospholipid fatty acyl composition in a leukemic T cell line: effects on receptor mediated intracellular Ca2+ increase.

The effect of modifying fatty acyl composition of cellular membrane phospholipids on receptor-mediated intracellular free Ca2+ concentration ([Ca2+]i) increase was investigated in a leukemic T cell line (JURKAT). After growing for 72 h in medium supplemented with unsaturated fatty acids (UFAs) and alpha-tocopherol, the fatty acyl composition of membrane phospholipids in JURKAT cells was extensively modified. Each respective fatty acid supplemented in the culture medium was readily incorporated into phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine and phosphatidylcholine in the JURKAT cells. The total n-6 fatty acyl content was markedly reduced in phosphatidylinositol and phosphatidylcholine of cells grown in the presence of n-3 fatty acids (alpha-linolenic acid, eicosapentaenoic acid and docosahexaenoic acid). Conversely, in the presence of n-6 fatty acids (linoleic acid and arachidonic acid), the total n-3 fatty acyl content was reduced in all the phospholipids examined. In n-3 and n-6 polyunsaturated fatty acid (PUFA) modified JURKAT cells, the total n-9 monounsaturated fatty acyl content in the phospholipids were markedly reduced. Changing the fatty acyl composition of membrane phospholipids in the JURKAT cells appears to have no affect on the presentation of the T cell receptor/CD3 complex or the binding of anti-CD3 antibodies (OKT3) to the CD3 complex. However, the peak increase in [Ca2+]i and the prolonged sustained phase elicited by OKT3 activation were suppressed in n-3 and n-6 PUFA but not in n-9 monounsaturated fatty acid modified cells. In Ca2+ free medium, OKT3-induced transient increase in [Ca2+]i representing Ca2+ release from the inositol 1,4,5-trisphosphate-sensitive Ca2+ stores, were similar in control and UFA modified cells. Using Mn2+ entry as an index of plasma membrane Ca2+ permeability, the rate of fura-2 fluorescence quenching as a result of Mn2+ influx stimulated by OKT3 in n-9 monounsaturated fatty acid modified cells was similar to control cells, but the rates in n-3 and n-6 PUFA modified cells were significantly lower. These results suggest that receptor-mediated Ca2+ influx in JURKAT cells is sensitive to changes in the fatty acyl composition of membrane phospholipids and monounsaturated fatty acids appears to be important for the maintenance of a functional Ca2+ influx mechanism.     

Effect of phospholipid fatty acid composition of endothelial cells on cholesterol efflux rates.

Human endothelial cells (EA.hy 926 line) were loaded with cationized low density lipoprotein (LDL) and subsequently incubated with fatty acid/bovine serum albumin complexes. The fatty acids were palmitic, oleic, linoleic, arachidonic, and eicosapentaenoic acids. The preincubations resulted in extensively modified fatty acid profiles in cell membrane phospholipids and in cellular cholesteryl esters. The cholesterol efflux from these fatty acid-modified cells was measured using 0.2 mg high density lipoprotein3 (HDL3)/ml medium. The efflux was significantly higher for the palmitic acid-treated cells, compared to all other fatty acid treatments. These differences in efflux rates were not caused by changes in the binding of HDL3 to high affinity receptors on the EA.hy 926 cells. Efflux mediated by dimethyl suberimidate-treated HDL3, which does not interact with high affinity HDL receptors, was similar to efflux induced by native HDL3 after all fatty acid treatments. Our results indicate that high affinity HDL receptors are not important for HDL-mediated efflux of cell cholesterol. The fatty acid composition of the cell membrane phospholipids may be an important determinant.     

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.     

Altered membrane free unsaturated fatty acid composition in human colorectal cancer tissue

Polyunsaturated free fatty acids (PUFAs) participate in normal functioning of the cell, particularly in control intracellular cell signalling. As nutritional components they compose a human diet with an indirect promoting influence on tumourogenesis. The PUFAs level depends on the functional state of the membrane. This work is focused on changes only of free unsaturated fatty acids amount (AA – arachidonic acid, LA – linoleic acid, ALA – α-linolenic acid, palmitoleic acid (PA) and oleic acid) in cell membranes of colorectal cancer of pT3 stage, G2 grade without metastasis. Qualitative and quantitative composition of free unsaturated fatty acids in the membrane was determined by high-performance liquid chromatography. It was shown that the malignant transformation was accompanied by a decrease in amount of LA and ALA while arachidonic and oleic acids increased. It is of interest that free AA levels are elevated in colon cancer, as AA is the precursor to biologically active eicosanoids.     

The role of arachidonic acid in vasogenic brain edema

Arachidonic acid is released rapidly from cellular membrane phospholipids after pathological insults associated with the delayed development of brain edema. Intracerebral injection of arachidonic acid caused significant increases in brain water and sodium content with decreases in potassium content and Na+,K+-ATPase activity. The 125I-labeled bovine serum albumin spaces in brain (a measure of blood-brain barrier permeability) rose threefold 24 h after arachidonic acid injection. There was gross and microscopic evidence of edema. Saturated fatty acids and monounsaturated fatty acids were not effective. These data indicate that the endothelial cells of the blood-brain barrier are target sites for the action of arachidonic acid. It is hypothesized that the increased permeability of endothelial cells to macromolecules and water results from alterations of membrane phospholipids and increased vesicular transport, changes that are responsible for the delayed development of vasogenic edema.     

Alterations in lipid composition and fluidity of liver plasma membranes in copper-deficient rats

In view of the importance of membrane fluidity on cell functions, the influence of phospholipid acyl groups on membrane fluidity, and the changes in lipid metabolism induced by copper (Cu) deficiency, this study was designed to examine the influence of dietary Cu on the lipid composition and fluidity of liver plasma membranes. Male Sprague-Dawley rats were divided into two dietary treatments, namely Cu deficient and Cu adequate. After 8 weeks of treatment, liver plasma membranes were isolated by sucrose density gradient centrifugation. The lipid fluidity of plasma membranes, as assessed by the intramolecular eximer fluorescence of 1,3-di(1-pyrenyl) propane, was significantly depressed by Cu deficiency. In addition, Cu deficiency significantly reduced the content of arachidonic and palmitoleic acids but increased the docosatetraenoic and docosahexaenoic acids of membrane phospholipids. This alteration in unsaturated phospholipid fatty acid composition, especially the large reduction in arachidonic acid, may have contributed to the depressed membrane fluidity. Furthermore, Cu deficiency also markedly altered the fatty acid composition of the triacylglycerols associated with the plasma membranes. Thus, the lipid composition and fluidity of liver plasma membranes are responsive to the animal's Cu status.     

Fatty acid composition in native and cultured human endothelial cells

Endothelial cells from human umbilical veins were isolated by collagenase treatment. Cells were cultured in the presence of either 20% fetal bovine serum (FBS) or 20% human serum (HS). At confluency, endothelial cell lipids were labeled with tracer concentrations of tritiated arachidonic acid, then extracted and separated into lipid subclasses by thin layer chromatography. The fatty acid composition of each lipid class was determined by glass capillary gas-liquid chromatography analysis and compared to that of cells freshly isolated from the cord (NC cells). The fatty acid compositions differed only in phospholipids. Polyunsaturated fatty acids (PFAs), arachidonic, and linoleic acids were depleted in FBS cell phospholipids and replaced by both stearic and oleic acids. No significant difference could be observed between NC cell and HS cell phospholipids. We conclude that PFAs might be decreased in FBS cells because of the relative paucity of PFAs in FBS as compared to HS. It seems therefore more convenient to cultivate endothelial cells in the presence of HS, especially in respect to their phospholipid content of arachidonic acid, which is the physiological reservoir for prostacyclin synthesis.     

Alanine transport by Chinese hamster ovary cells with altered phospholipid acyl chain composition

The Na+-dependent transport of alanine has been examined in Chinese hamster ovary (CHO) cells as a function of the fatty acid composition of their membrane lipids. Significant changes in the fatty acid composition of the CHO cell phospholipids were achieved by supplementation of the growth medium with specific saturated (palmitate) or monoenoic (oleate) free fatty acids. Arrhenius plots of the temperature-dependent uptake of alanine were constructed for cells of altered fatty acid composition. Alanine uptake was characterized by a single discontinuity in the Arrhenius plot. The temperature of this break was observed to be dependent upon the fatty acid composition of the cell phospholipids, ranging from 16 degrees C for cells enriched with oleate to 32 degrees C for cells enriched in palmitate. Calculation of the Km value for the uptake process showed no significant change with temperature or fatty acid supplementation. Correlations are made between the physical state of the membrane lipids and the temperature-dependence for alanine transport. The results are discussed in terms of membrane fatty acid composition, ordered in equilibrium fluid phase transitions and amino acid transport.     

The relationship between the fatty acid composition of immune cells and their function

The immune system, including its inflammatory components, is fundamental to host defence against pathogenic invaders. It is a complex system involving interactions amongst many different cell types dispersed throughout the body. Central to its actions are phagocytosis of bacteria, processing of antigens derived from intracellular and extracellular pathogens, activation of T cells with clonal expansion (proliferation) and production of cytokines that elicit effector cell functions such as antibody production and killing cell activity. Inappropriate immunologic activity, including inflammation, is a characteristic of many common human disorders. Eicosanoids produced from arachidonic acid have roles in inflammation and regulation of T and B lymphocyte functions. Eicosapentaenoic acid (EPA) also gives rise to eicosanoids and these may have differing properties from those of arachidonic acid-derived eicosanoids. EPA and docosahexaenoic acid (DHA) give rise to newly discovered resolvins which are anti-inflammatory and inflammation resolving. Human immune cells are typically rich in arachidonic acid, but arachidonic acid, EPA and DHA contents can be altered through oral administration of EPA and DHA. This results in a changed pattern of production of eicosanoids and probably also of resolvins, although the latter are not well examined in the human context. Changing the fatty acid composition of immune cells also affects phagocytosis, T cell signaling and antigen presentation capability. These effects appear to mediated at the membrane level suggesting important roles of fatty acids in membrane order, lipid raft structure and function, and membrane trafficking. Thus, the fatty acid composition of human immune cells influences their function and the cell membrane contents of arachidonic acid, EPA and DHA are important. Fatty acids influence immune cell function through a variety of complex mechanisms and these mechanisms are now beginning to be unraveled.     

Long chain‐polyunsaturated fatty acids modulate membrane phospholipid composition and protein localization in lipid rafts of neural stem cell cultures

Rat neural stem cells/neural progenitors (NSC/NP) are generally grown in serum‐free medium. In this study, NSC/NP were supplemented with the main long‐chain polyunsaturated fatty acids (PUFAs) present in the brain, arachidonic acid (AA), or docosahexaenoic acid (DHA), and were monitored for their growth. Lipid and fatty acid contents of the cells were also determined. Under standard conditions, the cells were characterized by phospholipids displaying a highly saturated profile, and very low levels of PUFAs. When cultured in the presence of PUFAs, the cells easily incorporated them into the phospholipid fraction. We also compared the presence of three membrane proteins in the lipid raft fractions: GFR and connexin 43 contents in the rafts were increased by DHA supplementation, whereas Gβ subunit content was not significantly modified. The restoration of DHA levels in the phospholipids could profoundly affect protein localization and, consequently, their functionalities. J. Cell. Biochem. 110: 1356–1364, 2010. © 2010 Wiley‐Liss, Inc.     

Modification of membrane phospholipid fatty acyl composition in a leukemic T cell line: effects on receptor mediated intracellular Ca2+ increase

The effect of modifying fatty acyl composition of cellular membrane phospholipids on receptor-mediated intracellular free Ca²⁺ concentration ([Ca²⁺]_i) increase was investigated in a leukemic T cell line (JURKAT). After growing for 72 h in medium supplemented with unsaturated fatty acids (UFAs) and α-tocopherol, the fatty acyl composition of membrane phospholipids in JURKAT cells was extensively modified. Each respective fatty acid supplemented in the culture medium was readily incorporated into phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine and phosphatidylcholine in the JURKAT cells. The total n ? 6 fatty acyl content was markedly reduced in phosphatidylinositol and phosphatidylcholine of cells grown in the presence of n ? 3 fatty acids (α-linolenic acid, eicosapentaenoic acid and docosahexaenoic acid). Conversely, in the presence of n ? 6 fatty acids (linoleic acid and arachidonic acid), the total n ? 3 fatty acyl content was reduced in all the phospholipids examined. In n ? 3 and n ? 6 polyunsaturated fatty acid (PUFA) modified JURKAT cells, the total n ? 9 monounsaturated fatty acyl content in the phospholipids were markedly reduced. Changing the fatty acyl composition of membrane phospholipids in the JURKAT cells appear to have no affect on the presentation of the T cell receptor/CD3 complex or the binding of anti-CD3 antibodies (OKT3) to the CD3 complex. However, the peak increase in [Ca²⁺]_i and the prolonged sustained phase elicited by OKT3 activation were suppressed in n ? 3 and n ? 6 PUFA but not in n ? 9 monounsaturated fatty acid modified cells. In Ca²⁺ free medium, OKT3-induced transient increase in [Ca²⁺]_i, representing Ca²⁺ release from the inositol 1,4,5-triphosphate-sensitive Ca²⁺ stores, were similar in control and UFA modified cells. Using Mn²⁺ entry as an index of plasma membrane Ca²⁺ permeability, the rate of fura-2 fluorescence quenching as a result of Mn²⁺ influx stimulated by OKT3 in n ? 9 monounsaturated fatty acid modified cells was similar to control cells, but the rates in n ? 3 and n ? 6 PUFA modified cells were significantly lower. These results suggest that receptor-mediated Ca²⁺ influx in JURKAT cells is sensitive to changes in the fatty acyl composition of membrane phospholipids and n ? 9 monounsaturated fatty acids appears to be important for the maintenance of a functional Ca²⁺ influx mechanism.     

Omegacoeur, a Mediterranean nutritional complement, stimulates Na,K-ATPase activity in human endothelial cells.

Fatty acids are known as modulators of the vasoactive properties of the vessel wall and can influence the physical and functional properties of cell membrane. The membrane-bound enzyme Na,K-ATPase plays a central role in endothelial function such as vasoconstriction. In a previous study, we have shown that omega3 fatty acids inhibited Na,K-ATPase activity in human endothelial cells. As Mediterranean diet is known to protect from cardiovascular diseases, we have investigated the effects of Omegacoeur, a Mediterranean nutritional complement consisting of omega3, omega6, omega9 fatty acids, garlic and basil, on Na,K-ATPase activity in human endothelial cells (HUVECs). Cells were incubated for 18 hr with pure lecithin liposomes or Omegacoeur-enriched emulsions (4 mg lecithin/ml). Na,K-ATPase and 5'-nucleotidase activities were determined using coupled assay methods on microsomal fractions obtained from HUVECs. Cell fatty acid composition was evaluated by gas chromatography after extraction of lipids and fatty acids methylation. The results showed that Omegacoeur (0.1 mM) increased Na,K-ATPase activity by 40% without changes in 5'-nucleotidase activity. Cells incubated with Omegacoeur preferentially incorporated linoleic acid. Therefore, linoleic acid or others constituents of Omegacoeur could be responsible of the stimulation of the Na,K-ATPase activity that might be related to changes in endothelial membrane fluidity.     

The Relationship Between Lipid Composition of Red Blood Cells and Their Susceptibility to Lipid Peroxidation

Red blood cells from 31 healthy donors were examined for the cholesterol content, the fatty acid composition. and the susceptibility to lipid peroxidation induced by either hydrogen peroxide or phenylhy-drazine. Lipid peroxidation was monitored by the release of pentane and ethane. In addition, plasma fatty acids were measured in order to find out, whether plasma and red cell fatty acids were correlated. In experiments with hydrogen peroxide, a significant positive correlation was found between the proportion of arachidonic acid (C 20:4n – 6; r = 0.57, p < 0.01) and docosahexaenoic acid (C 22:6; - 3; r = +0.71, p < 0.01), and the release of pentane and ethane, respectively. A significant negative correlation was found between the membrane cholesterol content and the pentane release (r -0.44, p< 0.05). In experiments performed with phenylhydrazine, red cell membrane lipid composition did not influence the susceptibility of red cells to lipid peroxidation. A close correlation was found between plasma and red cell fatty acids (palmitic acid, r = +0.46, p < 0.01; linoleic acid, r = +0.41, p < 0.05; arachidonic acid, r = +0.59, p < 0.01; docosahexaenoic acid, r = +0.67, p < 0.01). The results demonstrated that the degree of peroxide-induced oxidation of erythrocyte lipids depends on the content of polyunsaturated fatty acids in the membrane, which on the other hand, is determined by plasma fatty acids. It is suggested that dietary variations may influence the susceptibility of red cells to lipid peroxidation.     

4-Hydroxynonenal induces dysfunction and apoptosis of cultured endothelial cells.

Lipolytic products of triglyceride-rich lipoproteins, i.e., free fatty acids, may cause activation and dysfunction of the vascular endothelium. Mechanisms of these effects may include lipid peroxidation. One of the major and biologically active products of peroxidation of n-6 fatty acids, such as linoleic acid or arachidonic acid, is the aldehyde 4-hydroxynonenal (HNE). To study the hypothesis that HNE may be a critical factor in endothelial cell dysfunction caused by free fatty acids, human umbilical endothelial cells (HUVEC) were treated with up to160 microM of linoleic or arachidonic acid. HNE formation was detected by immunocytochemistry in cells treated for 24 h with either fatty acid, but more markedly with arachidonic acid. To study the cellulareffects of HNE, HUVEC were treated with different concentrations of this aldehyde, and several markers of endothelial cell dysfunction were determined. Exposure to HNE for 6 and 9 h resulted in increased cellular oxidative stress. However, short time treatment with HNE did not cause activation of nuclear factor-kappaB (NF-kappaB). In addition, HUVEC exposure to HNE caused a dose-dependent decrease in production of both interleukin-8 (IL-8) and intercellular adhesion molecule-1 (ICAM-1). On the other hand, HNE exerted prominent cytotoxic effects in cultured HUVEC, manifested by morphological changes, diminished cellular viability, and impaired endothelial barrier function. Furthermore, HNE treatment induced apoptosis of HUVEC. These data provide evidence that HNE does not contribute to NF-kappaB-related mechanisms of the inflammatory response in HUVEC, but rather to endothelial dysfunction, cytotoxicity, and apoptotic cell death.     

Alterations of Na,K-ATPase Isoenzymes in the Rat Diabetic Neuropathy: Protective Effect of Dietary Supplementation with n-3 Fatty Acids

Abstract: Diabetic neuropathy is a degenerative complication of diabetes accompanied by an alteration of nerve conduction velocity (NCV) and Na,K-ATPase activity. The present study in rats was designed first to measure diabetes-induced abnormalities in Na,K-ATPase activity, isoenzyme expression, fatty acid content in sciatic nerve membranes, and NCV and second to assess the preventive ability of a fish oil-rich diet (rich in n-3 fatty acids) on these abnormalities. Diabetes was induced by intravenous streptozotocin injection. Diabetic animals (D) and nondiabetic control animals (C) were fed the standard rat chow either without supplementation or supplemented with either fish oil (DM, CM) or olive oil (DO, CO) at a daily dose of 0.5 g/kg by gavage during 8 weeks. Analysis of the fatty acid composition of purified sciatic nerve membranes from diabetic animals showed a decreased incorporation of C16:1(n-7) fatty acids and arachidonic acids. Fish oil supplementation changed the fatty acid content of sciatic nerve membranes, decreasing C18:2(n-6) fatty acids and preventing the decreases of arachidonic acids and C18:1(n-9) fatty acids. Protein expression of Na,K-ATPase α subunits, Na,K-ATPase activity, and ouabain affinity were assayed in purified sciatic nerve membranes from CO, DO, and DM. Na,K-ATPase activity was significantly lower in sciatic nerve membranes of diabetic rats and significantly restored in diabetic animals that received fish oil supplementation. Diabetes induced a specific decrease of α1- and α3-isoform activity and protein expression in sciatic nerve membranes. Fish oil supplementation restored partial activity and expression to varying degrees depending on the isoenzyme. These effects were associated with a significant beneficial effect on NCV. This study indicates that fish oil has beneficial effects on diabetes-induced alterations in sciatic nerve Na,K-ATPase activity and function.     

Polyunsaturated fatty acid supplements modulate mast cell membrane microdomain composition

In the present study, the lipid raft composition of a canine mastocytoma cell line (C2) was analyzed. Lipid rafts were well separated from non-raft plasma membranes using a detergent-free isolation technique. To study the influence of n-3 and n-6 polyunsaturated fatty acids (PUFA) on raft fatty acid composition in comparison to non-raft cell membrane, C2 were supplemented with one of the following: α-linolenic acid, eicosapentaenoic acid, docosahexaenoic acid, linoleic acid or arachidonic acid. Enrichment of the culture medium with a specific PUFA resulted in an increase in the content of this fatty acid both in rafts and non-raft membranes. Contents of cholesterol and protein were found not to be affected by the changes in the fatty acid profiles. In conclusion, our data provide strong evidence that PUFA modulate lipid composition and physiological properties of membrane micro domains of mast cells which in turn may have effects on mast cell function.     

Inhibition of platelet aggregation by unsaturated fatty acids through interference with a thromboxane-mediated process

cis- and trans-unsaturated fatty acids with 18 carbon atoms (oleic, linoleic, elaidic and linolelaidic acid) inhibited aggregation of washed rabbit platelets stimulated with collagen, arachidonic acid and U46619 when in the same concentration ranges. Thrombin-induced aggregation was not affected by any of them. Saturated fatty acid (stearic acid) had no effect on this response. The inhibition is independent of the induced change in membrane fluidity, since trans-isomers could not induce the change in fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene. Unsaturated fatty acids, except linoleic acid, did not interfere with the formation of thromboxane B2 from exogenously added arachidonic acid. All the unsaturated fatty acids only slightly inhibited the arachidonic acid liberation by phospholipase A2 in platelet lysate. This indicates that the unsaturated fatty acids may block a process after formation of thromboxane A2 in response to collagen and arachidonic acid. The increase in phosphatidic acid formation stimulated with U46619 was inhibited dose dependently by each of the unsaturated fatty acids but that stimulated with thrombin was not affected by any of them. Phospholipase C activity measured by diacylglycerol formation in unstimulated platelet lysate was not inhibited by the fatty acids. The elevation of cytosolic free Ca2+ induced by arachidonic acid or U46619 and Ca2+ influx by collagen were inhibited almost completely at the same concentration as that which inhibited their aggregation. These data suggest that the unsaturated fatty acids were intercalated into the membrane and inhibited collagen- and arachidonic acid-induced platelet aggregation by causing a significant suppression of the thromboxane A2-mediated increase in cytosolic free Ca2+, probably due to interference with the receptor-operated Ca2+ channel.     

Fatty acid composition in native and cultured human endothelial cells

Summary Endothelial cells from human umbilical veins were isolated by collagenase treatment. Cells were cultured in the presence of either 20% fetal bovine serum (FBS) or 20% human serum (HS). At confluency, endothelial cell lipids were labeled with tracer concentrations of tritiated arachidonic acid, then extracted and separated into lipid subclasses by thin layer chromatography. The fatty acid composition of each lipid class was determined by glass capillary gas-liquid chromatography analysis and compared to that of cells freshly isolated from the cord (NC cells). The fatty acid compositions differed only in phospholipids. Polyunsaturated fatty acids (PFAs), arachidonic, and linoleic acids were depleted in FBS cell phospholipids and replaced by both stearic and oleic acids. No significant difference could be observed between NC cell and HS cell phospholipids. We conclude that PFAs might be decreased in FBS cells because of the relative paucity of PFAs in FBS as compared to HS. It seems therefore more convenient to cultivate endothelial cells in the presence of HS, especially in respect to their phospholipid content of arachidonic acid, which is the physiological reservoir for prostacyclin synthesis. This work was supported by a grant from the Délégation Générale à la Recherche Scientifique et Technique, Paris, France (79.7.0091).