Monohydroxylated fatty acid content in peripheral blood mononuclear cells and immune status of people at long times after the Chernobyl accident.

The monohydroxylated fatty acid content of peripheral blood mononuclear cells from 23 cleanup workers and 16 unexposed individuals was studied in relation to their immune status after the Chernobyl accident. Men with absorbed doses below 0.32 Gy showed higher levels of free and esterified 12-hydroxyeicosatetraenoic acid (12-HETE) than unexposed men, whereas 15-HETE and the 17-hydroxy derivative of C22 fatty acid (17-OH 22), either free or esterified in phospholipids, were increased in a dose-dependent manner. The percentage of CD4-positive cells was also increased significantly in heavily irradiated men, whereas the percentage of CD8-positive cells tended to decrease with dose. Furthermore, the absolute count of CD4-positive cells was correlated positively with the amount of esterified 15-HETE in the phospholipid fraction of the mononuclear cells and with the total 15-HETE. These results show for the first time that the accumulation of autoxidized/lipoxygenase products of polyunsaturated fatty acids in the mononuclear cells of irradiated individuals was associated with immune imbalance. This may be the basis for certain late effects of radiation such as autoimmune disorders, somatic and neoplastic diseases, and early aging.

Endothelial cell prostacyclin synthesis induced by lymphocytes is independent of the membrane fatty acid composition of both cell types and of E-selectin, VCAM-1 or ICAM-1-mediated adhesion.

Prostacyclin (PGI2), the main prostanoid in most vascular tissues regulates haemostasis and vascular tone, as well as the proliferation of smooth muscle cells. We have previously reported that lymphocyte contact with endothelium enhances endothelial cell PGI2 output. Here, we demonstrate the specificity of lymphocytes for switching on this response. Co-incubation of human umbilical vein endothelial cells (HUVEC) in serum-free medium with allogeneic peripheral blood lymphocytes (PBL), at a PBL:HUVEC ratio of 9:1, enhanced the basal (HUVEC alone) PGI2 output by 2.5-fold under static conditions, and was not altered in conditions mimicking shear stress. It occurred without previous activation of either cell type and was dependent upon specific interactions with PBL. Indeed, the PGI2 output induced by the co-incubation with resting neutrophils, non-activated platelets or latex beads was significantly lower than that induced by PBL. Blocking endothelial cell adhesion molecules (ECAM) E-selectin, vascular cell adhesion molecule-1 (VCAM-1) or intracellular adhesion molecule-1(ICAM-1) did not modify the PBL-induced PGI2 output, although 51Cr-labelled PBL adhesion was significantly decreased with anti-ICAM-1 antibody. Changes in the fatty acid composition of membrane phospholipids induced by incubation with eicosapentaenoic (EPA) or docosahexaenoic acids (DHA) resulted in diminished basal PGI2 output and adhesion of 51Cr-labelled PBL, whereas the PBL-stimulated PGI2 output was not modified. This specific cell-cell interaction represents a new stimulus for PGI2 synthesis that does not primarily involve the ECAM pathway, is independent of cell membrane fatty acid composition and shear stress. This switch-on for PGI2 synthesis, which is induced by lymphocytes, might serve as a protection against atherogenesis.

Human lymphocytes stimulate prostacyclin synthesis in human umbilical vein endothelial cells. Involvement of endothelial cPLA2.

Prostacyclin (PGI2) contributes to the maintenance of a nonadhesive luminal surface in blood vessels due to its anti-platelet and vasodilatory properties. Here, we sought to determine whether peripheral blood lymphocytes (PBL) may regulate the PGI2 production of human umbilical vein endothelial cells (HUVEC). Cell-cell contact between HUVEC and lymphocytes markedly enhanced PGI2 synthesis as a function of the number of lymphocytes added. This stimulated synthesis was totally suppressed when lymphocytes and HUVEC were separated by a microporous insert. It was not due to prostaglandin H synthase up-regulation. The pretreatment of lymphocytes with the PGI2 synthase inhibitor tranylcypromine partially inhibited PGI2 synthesis (47%), suggesting a transcellular metabolism of the endothelial prostaglandin endoperoxide PGH2 by the lymphocyte PGI2 synthase. Experiments using [14C]arachidonate-labeled lymphocytes coincubated with unlabeled HUVEC, and [14C]arachidonate-labeled HUVEC coincubated with unlabeled lymphocytes showed that the arachidonic acid used for PGI2 synthesis was totally of endothelial origin. Furthermore, the PGI2 synthesis was strongly inhibited by the cytosolic phospholipase A2 inhibitor, MAFP and totally suppressed by the combination of the calcium chelators, BAPTA and EGTA. Collectively, these results suggest that lymphocytes trigger an outside-in signaling in endothelial cells involving cPLA2 activation. Overall, the switch-on for PGI2 synthesis induced by lymphocytes might serve as a protection against atherothrombogenesis.

The cAMP-specific phosphodiesterase PDE4D3 is regulated by phosphatidic acid binding. Consequences for cAMP signaling pathway and characterization of a phosphatidic acid binding site.

Hormones and growth factors induce in many cell types the production of phosphatidic acid (PA), which has been proposed to play a role as a second messenger. We have previously shown in an acellular system that PA selectively stimulates certain isoforms of type 4 cAMP-phosphodiesterases (PDE4). Here we studied the effect of endogenous PA on PDE activity of transiently transfected MA10 cells overexpressing the PA-sensitive isoform PDE4D3. Cell treatment with inhibitors of PA degradation, including propranolol, induced an accumulation of endogenous PA accompanied by a stimulation of PDE activity and a significant decrease in both cAMP levels and protein kinase A activity. Furthermore, in FRTL5 cells, which natively express PDE4D3, pretreatment with compounds inducing PA accumulation prevented both cAMP increase and cAMP-responsive element-binding protein phosphorylation triggered by thyroid-stimulating hormone. To determine the mechanism of PDE stimulation by PA, endogenous phospholipids were labeled by preincubating MA10 cells overexpressing PDE4D3 with [(32)P]orthophosphate. Immuno- precipitation experiments showed that PA was specifically bound to PDE4D3, supporting the hypothesis that PDE4D3 activation occurs through direct binding of PA to the protein. PA binding site on PDE4D3 was characterized by engineering deletions of selected regions in the N-terminal regulatory domain of the enzyme. Deletion of amino acid residues 31-59 suppressed both PA-activating effect and PA binding, suggesting that this region rich in basic and hydrophobic residues contains the PA binding site. These observations strongly suggest that endogenous PA can modulate cAMP levels in intact cells, through a direct activation of PDE4D3.

Anthracyclines trigger apoptosis of both G0-G1 and cycling peripheral blood lymphocytes and induce massive deletion of mature T and B cells.

The anthracyclines daunorubicin and doxorubicin were shown to induce apoptosis of hematopoietic cell lines. Here we report that they induce apoptosis of both nonactivated and phytohemagglutinin-activated human peripheral blood lymphocytes. Apoptosis demonstrated by surface expression of phosphatidylserine and typical nuclear alterations reached a maximum after 48 h of incubation with these agents. In contrast to topoisomerase inhibitors (etoposide and camptothecin) and antimetabolites (methotrexate and 5-fluorouracil) that induced apoptosis of activated cells only, daunorubicin and doxorubicin triggered apoptosis of cells in the G0-G1 phases of the cell cycle. In agreement with in vitro data, a single i.p. injection of daunorubicin or doxorubicin in BALB/c mice induced T- and B-cell depletion in spleen, lymph nodes, and to a lesser extent in the thymus. Soluble Fas-Fc, CD95 antagonistic antibodies, as well as the p55 tumor necrosis factor receptor-immunoglobulin fusion protein, did not inhibit drug-induced apoptosis. The level of reactive oxygen species was significantly increased in the presence of daunorubicin or doxorubicin only in nonactivated lymphocytes. However, antioxidants such as N-acetyl-L-cysteine or glutathione did not prevent apoptosis. Activation of caspase-3 after daunorubicin or doxorubicin treatment of either nonactivated or activated lymphocytes was demonstrated by the cleavage of poly(ADP-ribose) polymerase, which was, as apoptosis, inhibited by the peptide benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone. Finally, daunorubicin and doxorubicin induced a rapid production of ceramides. These data indicate that anthracyclines may induce major peripheral T-cell deletion, a property not shared by many cytotoxic agents.

The topoisomerase inhibitors camptothecin and etoposide induce a CD95-independent apoptosis of activated peripheral lymphocytes.

The effect of etoposide and camptothecin, two topoisomerase inhibitors directed against topoisomerases II and I, respectively, was evaluated on human peripheral blood lymphocytes. Etoposide and camptothecin induced apoptosis of mitogen-activated but not resting CD4+ and CD8+ T lymphocytes. Cell sensitivity to these agents required G1 to S-phase transition of the cell cycle. Conversely, daunorubicin, an intercalating agent and topoisomerase II inhibitor, induced apoptosis of both resting and activated lymphocytes. Although etoposide and camptothecin induced CD95-ligand mRNA expression, drug-induced apoptosis of activated human lymphocytes was not inhibited by CD95 antagonists. Drug-induced cell death was also not inhibited by p55 TNFR-Ig fusion protein. Activation of the caspases cascade was suggested by the partial inhibitory effect of the tripeptide zVAD-fmk and documented by activation of caspase 3. Finally etoposide and camptothecin induced a rapid production of ceramide in activated but not resting peripheral blood lymphocytes, suggesting that ceramide might initiate the signaling apoptotic cascade in sensitive cells.

Brief preconditioning ischemia alters diacylglycerol content and composition in rabbit heart.

In order to give further insight into the potential role of PKC in the beneficial effects of ischemic preconditioning, we have characterized the production of diacylglycerol, the endogenous activator of PKC, and its molecular species composition in ischemic control and preconditioned hearts. Preconditioning was induced by 1 cycle of 5 min of ischemia followed by 5 min of reperfusion. In control and preconditioned groups, hearts were harvested under deep anesthesia at baseline (preischemia) and at 2, 5 and 10 min into the sustained coronary artery occlusion, i.e., preceding myocyte death. Diacylglycerol content and fatty acid composition were analyzed by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC), respectively. Myocardial diacylglycerol content was increased at 2 min into the sustained ischemia in the control group (481 +/- 34 vs 292 +/- 64 ng x mg(-1) at baseline; p < 0.05), but was comparable to the baseline value at 5 and 10 min. In the preconditioned group, diacylglycerol production remained unchanged throughout the 10-min test ischemia (317 +/- 17 at 2 min vs 312 +/- 38 ng x mg(-1) at baseline; p = NS). A detailed analysis of the molecular species composition at the time of 2 min revealed a reduced contribution of phosphatidylinositol to diacylglycerol production in preconditioned myocardium (global correlation coefficient 0.57 vs 0.66 in control myocardium) with a trend toward an enrichment of diacylglycerol composition with some species originating from phosphatidylcholine. Thus, our study revealed that brief preconditioning ischemia: (1) prevents the increase of diacylglycerol content in the early minutes of the sustained ischemia, and (2) emphasizes the contribution of phosphatidylcholine in diacylglycerol formation to the detriment of that of phosphatidylinositol.

Chernobyl clean-up workers erythrocyte membrane lipid content at the remote period.

Chernobyl Nuclear Power Plant accident clean-up workers were exposed to different radiation doses. Realization of remote effects depends on cellular membrane structure and function. We studied lipid composition of erythrocyte ghost and blood plasma in clean-up workers on the 12-th year after the accident. Groups of Chernobyl accident clean-up workers, non-irradiated patients with the same diseases and healthy controls were studied. Phospholipids were analyzed by two dimentional thin layer chromatography on silica gel. To analyse fatty acids and cholesterol gas chromatography was used. Total cholesterol and total phospholipids amounts in clean-up workers erythrocyte ghost were increased. The level of sphingomyelin and phosphatidylethanolamine raised. In some of the studied irradiated patients lysophosphatidylethanolamine appeared. The distribution of (diacyl)- and plasmalogen forms of phosphatidylcholine and phosphatidylethanolamine was not affected. The fatty acid composition changed slightly except 22:5 n-3, which level significantly increased and 22:5 n-6, which quantity lowered. The unsaturation index remains unchanged. No change in lipid composition of blood plasma was detected. All the changes in lipid composition of erythrocyte ghost of irradiated and unexposed patients were very similar. The obtained results confirm the suggestion about non-specific remote effects after exposure to low doses of ionizing irradiation.

Possible interactions between the NS-1 protein and tumor necrosis factor alpha pathways in erythroid cell apoptosis induced by human parvovirus B19.

Human erythroid progenitor cells are the main target cells of the human parvovirus B19 (B19), and B19 infection induces a transient erythroid aplastic crisis. Several authors have reported that the nonstructural protein 1 (NS-1) encoded by this virus has a cytotoxic effect, but the underlying mechanism of NS-1-induced primary erythroid cell death is still not clear. In human erythroid progenitor cells, we investigated the molecular mechanisms leading to apoptosis after natural infection of these cells by the B19 virus. The cytotoxicity of NS-1 was concomitantly evaluated in transfected erythroid cells. B19 infection and NS-1 expression induced DNA fragmentation characteristic of apoptosis, and the commitment of erythroid cells to undergo apoptosis was combined with their accumulation in the G(2) phase of the cell cycle. Since B19- and NS-1-induced apoptosis was inhibited by caspase 3, 6, and 8 inhibitors, and substantial caspase 3, 6, and 8 activities were induced by NS-1 expression, there may have been interactions between NS-1 and the apoptotic pathways of the death receptors tumor necrosis factor receptor 1 and Fas. Our results suggest that Fas-FasL interaction was not involved in NS-1- or B19-induced apoptosis in erythroid cells. In contrast, these cells were sensitized to tumor necrosis factor alpha (TNF-alpha)-induced apoptosis. Moreover, the ceramide level was enhanced by B19 infection and NS-1 expression. Therefore, our results suggest that there may be a connection between the respective apoptotic pathways activated by TNF-alpha and NS-1 in human erythroid cells.

The priming effect of 12(S)-hydroxyeicosatetraenoic acid on lymphocyte phospholipase D involves specific binding sites.

We have previously shown that 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE)-enrichment primed human peripheral blood mononuclear cells for phospholipase D activation by mitogens. Given that 12(S)-HETE-enriched cells stimulated with concanavalin A released free 12(S)-HETE in the extracellular medium, and that the priming effect of 12(S)-HETE on phospholipase D was suppressed by the non-permeant drug, suramin, we hypothesized an extracellular mechanism for 12(S)-HETE-induced PLD activation. Using [3H]12(S)-HETE as a ligand and a rapid filtration technique, we have pointed out the presence of specific low-affinity 12(S)-HETE binding sites on intact human mononuclear cells and lymphocytes. [3H]12(S)-HETE binding was efficiently displaced by other monohydroxylated and n-3 fatty acids but not by oleate and arachidonate, and was also significantly inhibited by suramin and pertussis toxin. Furthermore, 12(S)-HETE-induced PLD activation was strongly inhibited by pertussis toxin and genistein, but was not PKC-dependent. In addition, 12(S)-HETE also potentiated the ConA-induced tyrosine phosphorylation of a 46-50 kDa protein, which was inhibited by genistein. Collectively, these results suggest that 12(S)-HETE binding sites on human lymphocytes may be coupled to phospholipase D through pertussis toxin sensitive G-proteins and tyrosine kinases.

Relationships between phosphatidic acid and cyclic nucleotide phosphodiesterases in activated human blood mononuclear cells.

We have previously shown that mitogenic activation of human PBMC rapidly increases both the intracellular phosphatidic acid (PA) level and cyclic nucleotide phosphodiesterase (PDE) activity, with time-course responses, suggesting a causative relationship between the two events. PA also directly stimulated cAMP-PDE activity in acellular systems. Thus the mitogenic properties of PA night be due to its ability to lower the level of cAMP, a negative effector of lymphocyte activation, through PDE activation. In this study, human PBMC were stimulated either with the mitogenic lectin ConA, the anti-CD3 mAb OKT3, or the phorbol ester TPA. All three agonists increased the radiolabeled PA level and the PA mass in treated cells and simultaneously increased cytosolic and particulate cAMP- and cGMP-PDE activities, with significant positive correlations between PA accumulation and PDE activities. Furthermore, the ConA-induced PDE activation was dose-dependently reduced by treatment of PBMC with the diacylglycerol-kinase inhibitor R59022. This compound also dose-dependently lowered the PA level and inhibited the proliferative response to ConA. In addition, TPA-induced PDE activation was totally abolished by ethanol, which strongly reduced PA accumulation in response to the phorbol ester. These data suggest that PA increase may be linked to mitogen-induced PDE activation. Experiments performed in the presence of rolipram indicated that ConA and TPA stimulated both the rolipram-sensitive PDE4 and the rolipram-insensitive PDE activities, OKT3 being more active on PDE4. All three agonists stimulated the cGMP-specific PDE5. These results suggest that PA is an important component of the mechanisms that maintain a low level of cyclic nucleotides, which is a prerequisite for an optimal lymphoproliferative response.

Protective effect of docosahexaenoic acid against hydrogen peroxide-induced oxidative stress in human lymphocytes.

Oxidatively stressed lymphocytes exhibit decreased proliferative response to mitogenic stimulation. Although several sensitive targets involved in lymphocyte suppression have already been identified, little is known about the influence of oxidative stress on cyclic nucleotide phosphodiesterases (PDE) (EC 3.1.4.17), thought to play a major role in the control of cyclic AMP (cAMP) level, a well-recognized negative effector of lymphoproliferation. Although the polyunsaturated fatty acid content of membrane phospholipids is thought to be directly related to the extent of oxidant-induced lipid peroxidation, some n-3 fatty acids also seem to have antioxidant effects, depending on the concentration used and the overall redox status of the cells in question. Results of the present study showed that human peripheral blood mononuclear cells (PBMC) as well as rat thymocytes were relatively resistant to a short-term exposure (10 min) to hydrogen peroxide (H2O2). Indeed, H2O2-induced lipid peroxidation, estimated by malondialdehyde (MDA) production, was only 2-fold increased by H2O2 concentrations lower than 2 mM, whereas a larger increase (10-fold) could be observed in PBMC at the highest dose (5 mM). Previous enrichment of PBMC with 5 microM docosahexaenoic acid (22:6n-3), brought to the cells as a fatty acid-albumin complex (ratio 1), significantly reduced MDA production induced by low doses of H2O2, the protective effect no longer being observed at the highest doses. In contrast, eicosapentaenoic acid (20:5n-3) did not have any protective effect. Cytosolic PDE activities of both human PBMC and rat thymocytes were significantly inhibited (40-50%) after H2O2 treatment of the cells, whereas particulate PDE activities were not modified. Different responses of PDE activities to H2O2 treatment were observed when PBMC were first enriched with 22:6n-3 prior to H2O2 addition. In 22:6n-3-treated cells, the H2O2-induced inhibition of both cAMP- and cGMP-PDE cytosolic activities was abolished, whereas the particulate activities were increased by the highest H2O2 concentration used (5 mM). At the same time, the glutathione peroxidase (glutathione: oxidoreductase, EC 1.11.1.9) (GSH-Px) activity of PBMC and thymocytes was only marginally inhibited by H2O2 addition (20%), and pretreatment of the cells with 22:6n-3 did not modify the slight inhibitory effect of H2O2. Collectively, these results suggest that lymphocytes are relatively resistant to H2O2-induced lipid peroxidation due to their high GSH-Px content, and that low doses of 22:6n-3 are able to prevent some of the H2O2-induced alterations such as lipid peroxidation and PDE inhibition. Docosahexaenoic acid might thus offer some protection against oxidant-induced lymphocyte suppression.

Regulation of PDE-4 cAMP phosphodiesterases by phosphatidic acid.

Phosphatidic acid (PA) has been previously shown to activate specifically some of the isoforms of type 4 cylic nucleotide phosphodiesterases (PDE-4) in an acellular system. In the present work, we have investigated the mechanism of PA-activating effect by using a recombinant PA-sensitive isoform, PDE-4D3. The enzyme was specifically activated by acidic phospholipids, but not by zwitterionic phospholipids or anionic detergents. The importance of the role of PA acidic groups in the activation process was confirmed by studying the influence of pH and ionic strength on activation. Crosslinking experiments suggested that PA might influence the ability of PDE-4D3 to form dimers. Binding studies performed with radiolabeled PA showed that PA binds to a PDE-4D3 preparation in a saturable manner. Specifically bound PA was displaced by anionic, but not by zwitterionic phospholipids. With a preparation of PDE-4B2, a PDE-4 isoform insensitive to PA activation, PA binding was only displaced by high concentrations of unlabeled PA, suggesting that high-affinity PA binding sites are only present on PDE-4D3. These data support the hypothesis that PA-activating effect depends on direct binding of the effector on specific sites carried by the PDE-4D3 protein.

Docosahexaenoic acid lowers phosphatidate level in human activated lymphocytes despite phospholipase D activation.

N-3 polyunsaturated fatty acids from marine oil have been shown to decrease T cell-mediated immune function both in animals and humans, and to inhibit the mitogen-induced lymphoproliferative response when added to lymphocyte culture medium. As phosphatidic acid (PA) is a key mediator of the mitogenic process, the present study aims to investigate whether docosahexaenoic (DHA) and eicosapentaenoic (EPA) acids, the main n-3 fatty acids from fish oil, are able to alter the mitogen-induced synthesis of PA, when added to the culture medium of human peripheral blood mononuclear cells (PBMC). Incubation of PBMC in a medium containing 5 microM DHA bound to 5 microM human delipidated serum albumin induced a 2-fold increase in the basal PA mass whereas incubation with EPA, in the same conditions, had no effect. In contrast, both fatty acids markedly reduced the concanavalin A (ConA)-induced production of PA as compared with untreated cells. Paradoxically, phospholipase D (PLD) activity, evidenced by the synthesis of phosphatidylbutanol, was only detected in DHA-treated cells further stimulated by ConA, indicating that both DHA and ConA are required for PLD activation. Similarly, an increased diacylglycerol (DAG) mass was only observed in DHA-treated cells stimulated by ConA, whereas no modification occurred in control or EPA-treated cells stimulated or not by ConA. Furthermore, 1-butanol suppressed the ConA-induced increase of DAG mass observed in DHA-treated cells, indicating that phosphatidate was the source of the newly synthesized diacylglycerol. Altogether, these results show that, in concanavalin A-activated human peripheral blood mononuclear cells, docosahexaenoate stimulates both phospholipase D and phosphatidate phosphohydrolase activities, which ultimately results in an increased diacylglycerol production at the expense of phosphatidate.

Caspase-dependent ceramide production in Fas- and HLA class I-mediated peripheral T cell apoptosis.

We recently demonstrated that the engagement of HLA class I alpha1 domain induced Fas-independent apoptosis in human T and B lymphocytes. We analyzed the signaling pathway involved in HLA class I-mediated apoptosis in comparison with Fas (APO-1, CD95)-dependent apoptosis. The mouse mAb90 or the rat YTH862 monoclonal antibodies which bind the human HLA class I alpha1 domain induced the production of ceramide which was blocked by addition of the phosphatidylcholine-dependent phospholipase C inhibitor, D609. Furthermore, HLA class I-mediated apoptosis involved at least two different caspases, an interleukin-1 converting enzyme-like protease and another protease inhibited by the CPP32-like protease inhibitor Ac-DEVD-CHO. Despite similarity between Fas and HLA class I signaling pathways, we failed to demonstrate any physical association between these two molecules. We also report that the pan-caspase inhibitory peptide zVAD-fmk, but not Ac-DEVD-CHO and Ac-YVAD-CHO, inhibited decrease of mitochondrial transmembrane potential and generation of ceramide induced by anti-HLA class I and anti-Fas monoclonal antibodies, whereas all three peptides efficiently inhibited apoptosis. Altogether these results suggest that signaling through Fas and HLA class I involve caspase(s), targeted by zVAD-fmk, which act upstream of ceramide generation and mitochondrial events, whereas interleukin-1 converting enzyme-like and CPP32-like proteases act downstream of the mitochondria.

Selective stimulation of a cAMP-specific phosphodiesterase (PDE4A5) isoform by phosphatidic acid molecular species endogenously formed in rat thymocytes.

We have previously reported that concanavalin A (ConA) stimulation of rat thymocytes induces an increase in the cellular phosphatidic acid mass as well as a change in its fatty acid composition. An increase in phosphodiesterase (PDE) activity, mostly due to cAMP-specific (PDE4) isoforms, has also been observed in thymocytes stimulated by ConA. Furthermore, phosphatidic acid was able to stimulate PDE4 activity in vitro. In the present study, cAMP levels have been shown to decrease upon ConA stimulation of thymocytes. Decreasing phosphatidic acid level using diacylglycerol kinase inhibitors induced a parallel decrease of the ConA-stimulated cAMP-specific PDE activity in these cells. Analyses of phosphatidic acid molecular species in cells stimulated for 5 min by ConA revealed a significant increase in 1-stearoyl-2-arachidonoyl-sn-glycerol-3-phosphate and a relative decrease in the other molecular species of phosphatidic acid, mainly species containing palmitate. On the other hand, phosphatidic acid extracted from ConA-stimulated cells activated more efficiently the recombinant PDE4A5 isoform in vitro, as compared to phosphatidic acid extracted from unstimulated cells. In addition, phosphatidic acid species containing unsaturated fatty acids were stimulatory, while those containing two saturated fatty acids had only a marginal effect on the enzyme activity. Taken together, these data suggest that the mitogenic stimulation of thymocytes is accompanied by the synthesis of peculiar phosphatidic acid molecular species able to activate a PDE4 isoform. This activation might be of physiological relevance since cAMP is a major negative effector of the mitogenic response.