Altered arachidonate distribution in macrophages from caveolin-1 null mice leading to reduced eicosanoid synthesis

In this work we have studied the effect of caveolin-1 deficiency on the mechanisms that regulate free arachidonic acid (AA) availability. The results presented here demonstrate that macrophages from caveolin-1-deficient mice exhibit elevated fatty acid incorporation and remodeling and a constitutively increased CoA-independent transacylase activity. Mass spectrometry-based lipidomic analyses reveal stable alterations in the profile of AA distribution among phospholipids, manifested by reduced levels of AA in choline glycerophospholipids but elevated levels in ethanolamine glycerophospholipids and phosphatidylinositol. Furthermore, macrophages from caveolin-1 null mice show decreased AA mobilization and prostaglandin E(2) and LTB(4) production upon cell stimulation. Collectively, these results provide insight into the role of caveolin-1 in AA homeostasis and suggest an important role for this protein in the eicosanoid biosynthetic response.     

Acyltransferases and transacylases that determine the fatty acid composition of glycerolipids and the metabolism of bioactive lipid mediators in mammalian cells and model organisms

Over one hundred different phospholipid molecular species are known to be present in mammalian cells and tissues. Fatty acid remodeling systems for phospholipids including acyl-CoA:lysophospholipid acyltransferases, CoA-dependent and CoA-independent transacylation systems, are involved in the biosynthesis of these molecular species. Acyl-CoA:lysophospholipid acyltransferase system is involved in the synthesis of phospholipid molecular species containing sn-1 saturated and sn-2 unsaturated fatty acids. The CoA-dependent transacylation system catalyzes the transfer of fatty acids esterified in phospholipids to lysophospholipids in the presence of CoA without the generation of free fatty acids. The CoA-dependent transacylation reaction in the rat liver exhibits strict fatty acid specificity, i.e., three types of fatty acids (20:4, 18:2 and 18:0) are transferred. On the other hand, CoA-independent transacylase catalyzes the transfer of C20 and C22 polyunsaturated fatty acids from diacyl phospholipids to various lysophospholipids, especially ether-containing lysophospholipids, in the absence of any cofactors. CoA-independent transacylase is assumed to be involved in the accumulation of PUFA in ether-containing phospholipids. These enzymes are involved in not only the remodeling of fatty acids, but also the synthesis and degradation of some bioactive lipids and their precursors. In this review, recent progresses in acyltransferase research including the identification of the enzyme’s genes are described.     

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.     

Anti-CD3 and concanavalin A-induced human T cell proliferation is associated with an increased rate of arachidonate-phospholipid remodeling. Lack of involvement of group IV and group VI phospholipase A2 in remodeling and increased susceptibility of proliferating T cells to CoA-independent transacyclase inhibitor-induced apoptosis

In this study arachidonate-phospholipid remodeling was investigated in resting and proliferating human T lymphocytes. Lymphocytes induced to proliferate with either the mitogen concanavalin A or with anti-CD3 (OKT3) in combination with interleukin 2 (OKT3/IL-2) showed a greatly accelerated rate of [3H]arachidonate-phospholipid remodeling compared with resting lymphocytes or with lymphocytes stimulated with OKT3 or IL-2 alone. The concanavalin A-stimulated cells showed a 2-fold increase in the specific activity of CoA-independent transacylase compared with unstimulated cells, indicating that this enzyme is inducible. Stimulation with OKT3 resulted in greatly increased quantities of the group VI calcium-independent phospholipase A2 but not of the quantity of group IV cytosolic phospholipase A2. However, group IV phospholipase A2 became phosphorylated in OKT3-stimulated cells, as determined by decreased electrophoretic mobility. Incubation of cells with the group VI phospholipase A2 inhibitor, bromoenol lactone, or the dual group IV/group VI phospholipase A2 inhibitor, methyl arachidonyl fluorophosphonate, did not block arachidonate-phospholipid remodeling resting or proliferating T cells, suggesting that these phospholipases A2 were not involved in arachidonate-phospholipid remodeling. The incubation of nonproliferating human lymphocytes with inhibitors of CoA-independent transacylase had little impact on cell survival. In contrast, OKT3/IL-2-stimulated T lymphocytes were very sensitive to apoptosis induced by CoA-independent transacylase inhibitors. Altogether these results indicate that increased arachidonate-phospholipid remodeling is associated with T cell proliferation and that CoA-independent transacylase may be a novel therapeutic target for proliferative disorders.     

A decrease in remodeling accounts for the accumulation of arachidonic acid in murine mast cells undergoing apoptosis

The goal of this study was to examine arachidonic acid (AA) metabolism by murine bone marrow-derived mast cells (BMMC) during apoptosis induced by cytokine depletion. BMMC deprived of cytokines for 12-48 h displayed apoptotic characteristics. During apoptosis, levels of AA, but not other unsaturated fatty acids, correlated with the percentage of apoptotic cells. A decrease in both cytosolic phospholipase A(2) expression and activity indicated that cytosolic phospholipase A(2) did not account for AA mobilization during apoptosis. Free AA accumulation is also unlikely to be due to decreases in 5-lipoxygenase and/or cyclooxygenase activities, since BMMC undergoing apoptosis produced similar amounts of leukotriene B(4) and significantly greater amounts of PGD(2) than control cells. Arachidonoyl-CoA synthetase and CoA-dependent transferase activities responsible for incorporating AA into phospholipids were not altered during apoptosis. However, there was an increase in arachidonate in phosphatidylcholine (PC) and neutral lipids concomitant with a 40.7 +/- 8.1% decrease in arachidonate content in phosphatidylethanolamine (PE), suggesting a diminished capacity of mast cells to remodel arachidonate from PC to PE pools. Further evidence of a decrease in AA remodeling was shown by a significant decrease in microsomal CoA-independent transacylase activity. Levels of lyso-PC and lyso-PE were not altered in cells undergoing apoptosis, suggesting that the accumulation of lysophospholipids did not account for the decrease in CoA-independent transacylase activity or the induction of apoptosis. Together, these data suggest that the mole quantities of free AA closely correlated with apoptosis and that the accumulation of AA in BMMC during apoptosis was mediated by a decreased capacity of these cells to remodel AA from PC to PE.     

Incorporation and effect of arachidonic acid on the growth of human myeloma cell lines

The objectives of this work are to investigate the incorporation of arachidonic acid (AA) in the human myeloma cell lines OPM2, U266 and IM9, and to assess the effect of AA and lipoxygenase products of AA on their growth. The kinetics of acylation of [3H]AA indicates that myeloma cells incorporate AA into their membrane phospholipids and triglycerides. PLA2-treatment and base hydrolysis experiments confirm that [3H]AA is incorporated unmodified in U266, IM9 and OPM2 phospholipids, and is linked by an ester bond. Prelabeling-chase experiments indicate no trafficking of labeled AA among the various phospholipid species. Addition of AA and lipoxygenase products of AA (leukotriene B4 and C4, lipoxin A4 and B4, 12- and 15-hydroxyeicosatetraenoic acid) have no effect on U266, IM9 and OPM2 proliferation assessed by [3H]thymidine incorporation into DNA. In conclusion, while human myeloma cells readily incorporate AA in their membrane phospholipids and triglycerides, AA and lipoxygenase products are not important modulators of their proliferation.     

LPS-stimulated release of prostaglandin E and thromboxane B2 from the U937 cell line

Human monocytes are known to metabolize arachidonic acid (AA) and to release prostaglandins upon stimulation. Previous data indicate that in vitro maturation and differentiation of monocytes result in alteration of this property with greatly diminished response to stimulators of release of prostaglandin E (PGE) and thromboxane B2 (TxB2) occurring after cells have been cultured. To further study the effects of differentiation on human monocyte AA metabolism, a model system was established based upon the human histiocytic cell line U937. Among tested stimulants, which included opsonized zymosan, complement fragment C3b, phorbol myristate acetate (PMA), calcium ionophore A23187, and concanavalin A, it was found that Escherichia coli lipopolysaccharide (LPS) was unique in that it stimulated increased release of TxB2 from U937 cells. The effect of the phorbol ester PMA, a compound commonly used to induce differentiation of U937, on the ability of U937 to respond to LPS was examined. Following 48 hr of treatment with PMA, U937 became capable of releasing both PGE and TxB2 in response to small doses of LPS. As previously observed for human monocytes, the release of PGE was delayed for several hours following stimulation and failed to reach maximal cumulative levels in culture until 24-48 hr following stimulation. In contrast to human monocytes, PMA-induced U937 were capable of maintaining their responsiveness to LPS for several days. Thus, the U937 cell line provides a useful model for study of the effects of differentiation of human mononuclear phagocytes on their ability to metabolize AA, and for the effects of LPS on histiocytic tumor cell prostaglandin release.     

Calcium dependency of arachidonic acid incorporation into cellular phospholipids of different cell types.

Ca2+ -independent phospholipase A2 (iPLA2) is involved in the incorporation of arachidonic acid (AA) into resting macrophages by the generation of the lysophospholipid acceptor. The role of iPLA2 in AA remodeling in different cells was evaluated by studying the Ca2+ dependency of AA uptake from the medium, the incorporation into cellular phospholipids, and the effect of the iPLA2 inhibitor bromoenol lactone on these events. Uptake and esterification of AA into phospholipids were not affected by Ca2+ depletion in human polymorphonuclear neutrophils and rat fibroblasts. The uptake was Ca2+ independent in chick embryo glial cells, but the incorporation into phospholipids was partially dependent on extracellular Ca2+. Both events were fully dependent on extra and intracellular Ca2+ in human platelets. In human polymorphonuclear neutrophils, the kinetics of incorporation in several isospecies of phospholipids was not affected by the absence of Ca2+ at short times (<30 min). The involvement of iPLA2 in the incorporation of AA from the medium was confirmed by the selective inhibition of this enzyme with bromoenol lactone, which reduced < or =50% of the incorporation of AA into phospholipids of human neutrophils. These data provide evidence that suggests iPLA2 plays a major role in regulating AA turnover in different cell types.     

Regulation of arachidonic acid availability for eicosanoid production.

Mammalian cells have developed specific pathways for the incorporation, remodeling, and release of arachidonic acid. Acyltransferase and transacylase pathways function to regulate the levels of esterified arachidonic acid in specific phospholipid pools. There are several distinct, differentially regulated phospholipases A2 in cells that mediate agonist-induced release of arachidonic acid. These pathways are important in controlling cellular levels of free arachidonic acid. Both arachidonic acid and its oxygenated metabolites are potent bioactive mediators that regulate a myriad of physiological and pathophysiological processes.     

Lipidomic approaches to the study of phospholipase A2-regulated phospholipid fatty acid incorporation and remodeling

The distribution of fatty acids among cellular glycerophospholipids is finely regulated by the CoA-dependent acylation of lysophospholipids followed by transacylation reactions. Arachidonic acid is the fatty acid precursor of a wide family of bioactive compounds called the eicosanoids, with key roles in innate immunity and inflammation. Because availability of free AA constitutes a rate-limiting step in the generation of eicosanoids by mammalian cells, many studies have been devoted to characterize the processes of arachidonate liberation from phospholipids by phospholipase A₂s and its re-incorporation and further remodeling back into phospholipids by acyltransferases and transacylases. These studies have traditionally been conducted by using radioactive precursors which do not allow the identification of the phospholipid molecular species involved in these processes. Nowadays, lipidomic approaches utilizing mass spectrometry provide a new frame for the analysis of unique phospholipid species involved in fatty acid release and phospholipid incorporation and remodeling. This review focuses on the mass spectrometry techniques applied to the study of phospholipid fatty acid trafficking and the recent advances that have been achieved by the use of this technique.     

Differential effects of arachidonoyl trifluoromethyl ketone on arachidonic acid release and lipid mediator biosynthesis by human neutrophils. Evidence for different arachidonate pools.

The goal of this study was to determine the effects of a putative specific cytosolic phospholipase A2 inhibitor, arachidonyl trifluoromethyl ketone (AACOCF3), on arachidonic acid (AA) release and lipid mediator biosynthesis by ionophore-stimulated human neutrophils. Initial studies indicated that AACOCF3 at concentrations 0-10 micro m did not affect AA release from neutrophils. In contrast, AACOCF3 potently inhibited leukotriene B4 formation by ionophore-stimulated neutrophils (IC50 approximately 2.5 micro m). Likewise, AACOCF3 significantly inhibited the biosynthesis of platelet activating factor. In cell-free assay systems, 10 micro m AACOCF3 inhibited 5-lipoxygenase and CoA-independent transacylase activities. [3H]AA labeling studies indicated that the specific activities of cell-associated AA mimicked that of leukotriene B4 and PtdCho/PtdIns, while the specific activities of AA released into the supernatant fluid closely mimicked that of PtdEtn. Taken together, these data argue for the existence of segregated pools of arachidonate in human neutrophils. One pool of AA is linked to lipid mediator biosynthesis while another pool provides free AA that is released from cells. Additionally, the data suggest that AACOCF3 is also an inhibitor of CoA-independent transacylase and 5-lipoxygenase. Thus, caution should be exercised in using AACOCF3 as an inhibitor of cytosolic phospholipase A2 in whole cell assays because of the complexity of AA metabolism.     

Human monocyte differentiation stage affects response to arachidonic acid

AA-induced cell death mechanisms acting on human monocytes and monocyte-derived macrophages (MDM), U937 promonocytes and PMA-differentiated U937 cells were studied. Arachidonic acid induced apoptosis and necrosis in monocytes and U937 cells but only apoptosis in MDM and U937D cells. AA increased both types of death in Mycobacterium tuberculosis-infected cells and increased the percentage of TNFα+ cells and reduced IL-10+ cells. Experiments blocking these cytokines indicated that AA-mediated death was TNFα- and IL-10-independent. The differences in AA-mediated cell death could be explained by high ROS, calpain and sPLA-2 production and activity in monocytes. Blocking sPLA-2 in monocytes and treatment with antioxidants favored M. tuberculosis control whereas AA enhanced M. tuberculosis growth in MDM. Such evidence suggested that AA-modulated effector mechanisms depend on mononuclear phagocytes’ differentiation stage.     

Scavenger receptor BI transfers major lipoprotein-associated phospholipids into the cells

The phospholipids of lipoproteins can be transferred to cells by an endocytosis-independent uptake pathway. We analyzed the role of scavenger receptor BI (SR-BI) for the selective cellular phospholipid import. Human monocytes rapidly acquired the pyrene (py)-labeled phospholipids sphingomyelin (SM), phosphatidylcholine, and phosphatidylethanolamine from different donors (low and high density lipoproteins (LDL, HDL), lipid vesicles). The anti-SR-BI antibody directed against the extracellular loop of the membrane protein lowered the cellular import of the phospholipids by 40-80%. The phospholipid transfer from the lipid vesicles into the monocytes was suppressed by LDL, HDL, and apoprotein AI. Transfection of BHK cells with the cDNA for human SR-BI enhanced the cellular import of the vesicle-derived py-phospholipids by 5-6-fold. In the case of the LDL donors, transfer of py-SM to the transfected cells was stimulated to a greater extent than the uptake of the other py-phospholipids. Similar differences were not observed when the vesicles and HDL were used as phospholipid donors. The concentration of LDL required for the half-maximal phospholipid import was close to the previously reported apparent dissociation constant for LDL binding to SR-BI. The low activation energy of the SR-BI-mediated py-phospholipid import indicated that the transfer occurs entirely in a hydrophobic environment. Disruption of cell membrane caveolae by cyclodextrin treatment reduced the SR-BI-catalyzed incorporation of py-SM, suggesting that intact caveolae are necessary for the phospholipid uptake. In conclusion, SR-BI mediates the selective import of the major lipoprotein-associated phospholipids into the cells, the transfer efficiency being dependent on the structure of the donor lipoprotein.     

Biosynthesis of paf-acether. XVII. Regulation by the CoA-independent transacylase in human neutrophils

Treatment of intact human polymorphonuclear neutrophils (PMN) with low concentrations of phorbol myristate acetate (PMA, 1-10 ng/ml) induced paf-acether (paf) and lyso paf formation, arachidonate release, and simultaneous inhibition of CoA-independent lyso paf: transacylase as assayed in a cell-free system. Inhibition of [3H]lyso paf reacylation was also observed when it was exogenously added to the PMA-treated intact PMN. When higher concentrations of PMA (40-100 ng/ml) were used, paf biosynthesis was severely impaired and the level of the CoA-independent transacylase activity returned to basal level. Since lyso paf appears to be the substrate for PMA-activated paf formation (remodeling pathway), we showed that [14C]acetate was incorporated into the paf molecule. By contrast, labeling with [3H]choline was not appropriate in this model. The presented results are against the involvement of a de novo route in paf synthesis initiated by PMA and open a new possibility of an important role for the CoA-independent transacylase in controlling the level of lyso paf availability for paf formation.     

alpha-Tocopherol, but not gamma-tocopherol inhibits 7 beta-hydroxycholesterol-induced apoptosis in human U937 cells.

Oxysterols, particularly those oxidised at position 7, are toxic to cells in culture and have been shown to induce apoptosis in cell types such as vascular endothelial cells, smooth muscle cells and monocytes. The precise mechanism by which oxysterols induce apoptosis is unknown but may involve the generation of oxidative stress. In the present study we examined the ability of alpha-TOC, alpha-TOC acetate (alpha-TOCA) and gamma-TOC to protect against 7 beta-hydroxycholesterol (7 beta-OHC)-induced apoptosis of human monocytic U937 cells. 7 beta-OHC is one of the most commonly detected oxysterols in foods and its level in plasma has been positively associated with an increased risk of atherosclerosis. The present study demonstrates a significant decrease in cell membrane integrity and cellular glutathione levels when U937 cells were treated with 30 microM 7 beta-OHC. DNA fragmentation also occurred, as measured by agarose gel electrophoresis, and the number of apoptotic cells increased as assessed by nuclear morphology. Analysis by HPLC showed that there was a greater incorporation of gamma-TOC into U937 cells after a 48 h incubation, than either alpha-TOC or alpha-TOCA. However, despite the increased uptake of gamma-TOC, only alpha-TOC, and not gamma-TOC or alpha-TOCA was effective at inhibiting 7 beta-OHC-induced apoptosis in U937 cells.     

Endothelial cell PAF synthesis following thrombin stimulation utilizes Ca(2+)-independent phospholipase A(2).

Platelet activating factor (PAF) is a potent lipid autocoid that is rapidly synthesized and presented on the surface of endothelial cells following thrombin stimulation. PAF production may occur via de novo synthesis or by the combined direct action of phospholipase A(2) (PLA(2)) and acetyl-CoA:lyso-PAF acetyltransferase or via the remodeling pathway. This study was undertaken to define the role of PLA(2) and plasmalogen phospholipid hydrolysis in PAF synthesis in thrombin-treated human umbilical artery endothelial cells (HUAEC). Basal PLA(2) activity in HUAEC was primarily found to be Ca(2+)-independent (iPLA(2)), membrane-associated, and selective for arachidonylated plasmenylcholine substrate. Thrombin stimulation of HUAEC resulted in a preferential 3-fold increase in membrane-associated iPLA(2) activity utilizing plasmenylcholine substrates with a minimal increase in activity with alkylacyl glycerophospholipids. No change in cystolic iPLA(2) activity in thrombin-stimulated HUAEC was observed. The thrombin-stimulated activation of iPLA(2) and associated hydrolysis of plasmalogen phospholipids was accompanied by increased levels of arachidonic acid (from 1.1 +/- 0.1 to 2.8 +/- 0.1%) and prostacyclin release (from 38 +/- 12 to 512 +/- 24%) as well as an increased level of production of lysoplasmenylcholine (from 0.6 +/- 0.1 to 2.1 +/- 0.3 nmol/mg of protein), lysophosphatidylcholine (from 0.3 +/- 0.1 to 0.6 +/- 0.1 nmol/mg of protein), and PAF (from 790 +/- 108 to 3380 +/- 306 dpm). Inhibition of iPLA(2) with bromoenol lactone resulted in inhibition of iPLA(2) activity, plasmalogen phospholipid hydrolysis, production of choline lysophospholipids, and PAF synthesis. These data indicate that PAF production requires iPLA(2) activation in thrombin-stimulated HUAEC and may occur through the CoA-independent transacylase remodeling pathway rather than as a direct result of the PLA(2)-catalyzed hydrolysis of membrane alkylacyl glycerophosphocholine.     

Role of group VIA calcium-independent phospholipase A2 in arachidonic acid release, phospholipid fatty acid incorporation, and apoptosis in U937 cells responding to hydrogen peroxide

Group VIA calcium-independent phospholipase A2 (iPLA2) has been shown to play a major role in regulating basal phospholipid deacylation reactions in certain cell types. More recently, roles for this enzyme have also been suggested in the destruction of membrane phospholipid during apoptosis and after oxidant injury. Proposed iPLA2 roles have rested heavily on the use of bromoenol lactone as an iPLA2-specific inhibitor, but this compound actually inhibits other enzymes and lipid pathways unrelated to PLA2, which makes it difficult to define the contribution of iPLA2 to specific functions. In previous work, we pioneered the use of antisense technology to decrease cellular iPLA2 activity as an alternative approach to study iPLA2 functions. In the present study, we followed the opposite strategy and prepared U937 cells that exhibited enhanced iPLA activity by stably expressing a plasmid containing iPLA2 cDNA. Compared with control cells, the iPLA2 -overexpressing U937 cells showed elevated responses to hydrogen peroxide with regard to both arachidonic acid mobilization and incorporation of the fatty acid into phospholipids, thus providing additional evidence for the key role that iPLA2 plays in these events. Long-term exposure of the cells to hydrogen peroxide resulted in cell death by apoptosis, and this process was accelerated in the iPLA2-overexpressing cells. Increased phospholipid hydrolysis and fatty acid release also occurred in these cells. Unexpectedly, however, abrogation of U937 cell iPLA2 activity by either methyl arachidonyl fluorophosphonate or an antisense oligonucleotide did not delay or decrease the extent of apoptosis induced by hydrogen peroxide. These results indicate that, although iPLA2-mediated phospholipid hydrolysis occurs during apoptosis, iPLA2 may actually be dispensable for the apoptotic process to occur. Thus, beyond a mere destructive role, iPLA2 may play other roles during apoptosis.     

The arachidonate-dependent cytoprotective signaling evoked by peroxynitrite is a general response of the monocyte/macrophage lineage

U937, THP-1, and J774 cells or human monocytes and macrophages display similar levels of sensitivity to peroxynitrite and exposure to an otherwise non-toxic concentration of the oxidant in the presence of a phospholipase A(2) inhibitor was invariably associated with the onset of mitochondrial permeability transition (MPT)-dependent toxicity. These events were prevented by exogenous arachidonic acid (AA). In general, the protective concentrations of AA were greater in those cell types releasing more AA. Thus, non-toxic concentrations of peroxynitrite commit cells belonging to the monocyte/macrophage lineage to MPT-dependent toxicity that is however prevented by endogenous AA.     

Corticosteroid effect on Caco-2 cell lipids depends on cell differentiation

Previous studies from our laboratory have indicated that secondary hyperaldosteronism affects phospholipids of rat colonic enterocytes. To assess whether this represents a direct effect of mineralocorticoids on enterocytes, the role of aldosterone and dexamethasone in the regulation of lipid metabolism was examined in Caco-2 cells during development of their enterocyte phenotype. Differentiation of Caco-2 cells was associated with increased levels of triglycerides (TG) and cholesteryl esters (CE), a decreased content of cholesterol and phospholipids and changes in individual phospholipid classes. The phospholipids of differentiated cells had a higher content of n-6 polyunsaturated fatty acids (PUFA) and lower amounts of monounsaturated (MUFA) and saturated fatty acids than subconfluent undifferentiated cells. Differentiated cells exhibited a higher ability to incorporate [3H]arachidonic acid (AA) into cellular phospholipids and a lower ability for incorporation into TG and CE. Incubation of subconfluent undifferentiated cells with aldosterone or dexamethasone was without effect on the content of lipids, their fatty acids and [3H]AA incorporation. In contrast, aldosterone treatment of differentiated cells diminished the content of TG, increased the content of phospholipids and modulated their fatty acid composition. The percentage of n-6 and n-3 PUFA in phospholipids was increased and that of MUFA decreased, whereas no changes in TG were observed. The incorporation of [3H]AA into phospholipids was increased and into TG decreased and these changes were blocked by spironolactone. Treatment of differentiated cells with dexamethasone increased their CE content but no effect was identified upon other lipids, their fatty acid composition and on the incorporation of [3H]AA. As expected for the involvement of corticosteroid hormones the mineralocorticoid and glucocorticoid receptors were identified in Caco-2 cells by RT-PCR. The results suggest that aldosterone had a profound influence on lipid metabolism in enterocytes and that its effect depends on the stage of differentiation. The aldosterone-dependent changes occurring in phospholipids and their fatty acid composition may reflect a physiologically important phenomenon with long-term consequences for membrane structure and function.