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J Balsinde

Publications and source records attributed to J Balsinde.

At least 37 records · Page 2Linked to original sources
PubMed1997

Inflammatory activation of arachidonic acid signaling in murine P388D1 macrophages via sphingomyelin synthesis.

Ceramide has emerged as an important lipid messenger for many cellular processes triggered via surface receptors. In the present study, inflammatory activation of P388D1 macrophages with bacterial lipopolysaccharide (LPS) and platelet-activating factor (PAF) stimulated a transient accumulation of ceramide. Moreover, cell-permeable ceramide mimicked LPS/PAF in triggering arachidonate mobilization in these cells. LPS/PAF-induced ceramide synthesis did not result from sphingomyelinase activation but from increased de novo synthesis. Participation of this pathway in arachidonate signaling was detected since fumonisin B1, an inhibitor of de novo ceramide synthesis, was able to inhibit the LPS/PAF-induced response. These studies have uncovered a new role for sphingolipid metabolism in cellular signaling and constitute evidence that products of the sphingomyelin biosynthetic pathway may serve a specific role in signal transduction by influencing the activity of the novel Group V secretory phospholipase A2.

Animals↗
PubMed1997

Identity between the Ca2+-independent phospholipase A2 enzymes from P388D1 macrophages and Chinese hamster ovary cells.

A novel Ca2+-independent phospholipase A2 (iPLA2) has recently been purified and characterized from P388D1 macrophages (Ackermann, E. J., Kempner, E. S., and Dennis, E. A. (1994) J. Biol. Chem. 269, 9227-9233). This enzyme appears to play a key role in regulating basal phospholipid remodeling reactions. Also an iPLA2 from Chinese hamster ovary (CHO) cells has been purified, molecularly cloned, and expressed (Tang, J., Kriz, R., Wolfman, N., Shaffer, M., Seehra, J., and Jones, S. S. (1997) J. Biol. Chem. 272, 8567-8575). We report herein that the cloned CHO iPLA2 is equivalent to the mouse enzyme purified from P388D1 cells. Polymerase chain reaction amplification of cDNA fragments from P388D1 cells using primers based on the CHO iPLA2 sequence, revealed a high degree of homology between the mouse and hamster enzymes at both the nucleotide and amino acid levels (92 and 95%, respectively). Identity between the two proteins was further demonstrated by using immunochemical, pharmacological, and biochemical approaches. Thus, an antiserum generated against the CHO enzyme recognized the P388D1 cell enzyme and gave similar molecular masses (about 83 kDa) for the two enzymes under the same experimental conditions. Further, the CHO enzyme has exactly the same sensitivity to inhibition by a variety of compounds previously shown to inhibit the P388D1 enzyme, including bromoenol lactone, palmitoyl trifluoromethyl ketone, and methyl arachidonyl fluorophosphonate. Additionally, covalent modification of the CHO enzyme by [3H]bromoenol lactone is dependent on active enzyme as is the P388D1 iPLA2. Finally, both enzymes have the same specific activities under identical experimental conditions.

Amino Acid Sequence↗
PubMed1997

Differential regulation of phospholipase D and phospholipase A2 by protein kinase C in P388D1 macrophages.

Activation of P388D1 macrophages by phorbol myristate acetate (PMA) resulted in the translocation of the protein kinase C (PKC) isoforms alpha, delta, and epsilon from the cytosol to membranes. Furthermore, PMA activated phospholipase D (PLD) in these cells, and potentiated the effect of the inflammatory lipid mediator platelet-activating factor (PAF) on PLD activation. PAF also activated phospholipase A2 (PLA2) and enhanced arachidonic acid (AA) release in P388D1 macrophages, and bacterial lipopolysaccharide (LPS) increased the responsiveness of these cells to PAF. In contrast with PLD, PLA2 activation in P388D1 macrophages was found to take place independently of PKC. This was supported by the following evidence: (i) PMA neither induced AA release nor enhanced the PAF response; (ii) inclusion of PMA along with LPS during priming did not have any effect on PAF-stimulated AA release; (iii) down-regulation of PMA-activatable PKC isoforms by chronic treatment with the phorbol ester had no effect on the PAF response; and (iv) the PKC inhibitor staurosporine did not alter the PAF-induced AA release. The present study provides an example of cells in which the direct activation of PKC by phorbol esters does not lead to a primed and/or enhanced AA release. As a unique example in which PKC activation is neither necessary nor sufficient for AA release to occur, this now allows study of the separate and distinct roles for PLD and PLA2 in signal-transduction processes. This has hitherto been difficult to achieve because of the lack of specific inhibitors of these two phospholipases.

Animals↗
PubMed1996

Bromoenol lactone inhibits magnesium-dependent phosphatidate phosphohydrolase and blocks triacylglycerol biosynthesis in mouse P388D1 macrophages.

Bromoenol lactone (BEL) has previously been identified as a potent, irreversible, mechanism-based phospholipase A2 (PLA2) inhibitor that possesses greater than 1000-fold selectivity for inhibition of Ca2+-independent PLA2 (iPLA2) versus the Ca2+-dependent ones. Thus, this compound has been used as a selective tool for studies aimed at elucidating the role of iPLA2 in certain cellular functions. Herein we report that BEL also inhibits cellular phosphatidic acid phosphohydrolase (PAP) activity in intact P388D1 macrophages with an IC50 of about 8 microM, which is very similar to that previously found for inhibition of iPLA2 under the same experimental conditions. This results in the blockage of the incorporation of exogenous arachidonate and palmitate into diacylglycerol and triacylglycerol. Thus, inhibition of PAP by BEL blocks triacylglycerol biosynthesis in P388D1 cells due to decreased diacylglycerol availability. Because two forms of PAP activity exist in mammalian cells, differential assays were performed to identify which of these forms was inhibited by BEL. The results of these experiments revealed that BEL selectively inhibits the cytosolic, Mg2+-dependent enzyme. No apparent effect of BEL on the membrane-bound Mg2+-independent PAP form could be detected. Collectively, the results reported herein establish that BEL inhibits two cellular phospholipases, namely iPLA2 and Mg2+-dependent PAP, with similar potency. Therefore, the inhibitory effect of BEL on Mg2+-dependent PAP might explain several cellular functions previously attributed to iPLA2.

Animals↗
PubMed1996

Novel group V phospholipase A2 involved in arachidonic acid mobilization in murine P388D1 macrophages.

Four related genes encode four different secretory phospholipase A2 (sPLA2) enzymes in mammals, namely the well described Group I and IIA enzymes and the more recently described Groups IIC and V. A large body of research has putatively demonstrated that the Group IIA sPLA2 is involved in diverse pathologic processes, such as rheumatoid arthritis, septic shock, intestinal neoplasia, and epidermal hyperplasia, as well as in cellular signaling by regulating the formation of arachidonate-derived lipid messengers. However, we demonstrate herein the involvement of another sPLA2, i.e. the Group V sPLA2, in arachidonic acid release and prostaglandin production in the mouse macrophage-like cell line P388D1. Abundant message for Group V sPLA2 was detected in both resting and activated cells. In contrast, Group IIA sPLA2 message was undetectable as analyzed by Northern blot and reverse transcriptase-polymerase chain reaction. Moreover, blockage of Group V sPLA2 gene expression by antisense RNA oligonucleotides resulted in inhibition of prostaglandin E2 production as well as reduction of the amount of sPLA2 protein at the cellular surface. Collectively, these results uncover Group V sPLA2 as a novel effector involved in arachidonic acid-mediated signal transduction.

Animals↗
PubMed1996

Irreversible inhibition of Ca(2+)-independent phospholipase A2 by methyl arachidonyl fluorophosphonate.

Methyl arachidonyl fluorophosphonate (MAFP) has been recently reported to be a selective, active-site directed, irreversible inhibitor of the Group IV 85 kDa cytosolic phospholipase A2 (cPLA2). We have now shown that this compound also potently inhibits the Ca(2+)-independent cytosolic phospholipase A2 (iPLA2). MAFP inhibited iPLA2 in a concentration-dependent manner with half-maximal inhibition observed at 0.5 microM after a 5 min preincubation at 40 degrees C. This inhibition was not reversed upon extensive dilution of the enzyme into the assay mixture. Preincubation of iPLA2 with MAFP resulted in a linear, time-dependent inactivation of enzyme activity, and the enzyme was protected from inactivation by the reversible inhibitor PACOCF3. The ability of MAFP to inhibit the iPLA2 suggests that this enzyme proceeds through an acyl-enzyme intermediate as has been proposed for the cPLA2. Further testing indicated that MAFP did not inhibit the arachidonoyl-CoA synthetase, CoA-dependent acyltransferase, or CoA-independent transacylase activities from P388D1 cells. Thus, MAFP is not a general inhibitor for enzymes which act on arachidonoyl substrates. Instead, the inhibitor appears to show some selectivity for PLA2, although it does not discriminate between cPLA2 and iPLA2. Particular caution must be exercised to distinguish these activities if this inhibitor is used in intact cells.

Animals↗
PubMed1996

Distinct roles in signal transduction for each of the phospholipase A2 enzymes present in P388D1 macrophages.

Receptor-stimulated arachidonic acid (AA) mobilization in P388D1 macrophages consists of a transient phase in which AA accumulates in the cell and a sustained phase in which AA accumulates in the incubation medium. We have shown previously that a secretory group II phospholipase A2 (sPLA2) is the enzyme responsible for most of the AA released to the incubation medium. By using selective inhibitors for each of the PLA2s present in P388D1 macrophages, we demonstrate herein that the cytosolic group IV PLA2 (cPLA2) mediates accumulation of cell-associated AA during the early steps of P388D1 cell activation. The contribution of both cPLA2 and sPLA2 to AA release can be distinguished on the basis of the different spatial and temporal characteristics of activation and substrate preferences of the two phospholipase A2s (PLA2s). Furthermore, the results suggest the possibility that a functionally active cPLA2 may be necessary for sPLA2 to act. cPLA2 action precedes that of sPLA2, and overcoming cPLA2 inhibition by artificially increasing intracellular free AA levels restores extracellular AA release. Although this suggests cross-talk between cPLA2 and sPLA2, selective inhibition of one other PLA2 present in these cells, namely the Ca2+-independent PLA2, does not block, but instead enhances receptor-coupled AA release. These data indicate that Ca2+-independent PLA2 does not mediate AA mobilization in P388D1 macrophages. Collectively, the results of this work suggest that each of the PLA2s present in P388D1 macrophages serves a distinct role in cell activation and signal transduction.

Animals↗
PubMed1996

The incorporation of arachidonic acid into triacylglycerol in P388D1 macrophage-like cells.

When 388D1 cells are incubated in media containing 10 microM [3H]arachidonic acid (delta4Ach), the label is rapidly incorporated into phospholipids and triacylglycerol. However, incorporation of [3H]delta4Ach into phospholipids clearly precedes incorporation into triacylglycerol, indicating that the phospholipid pool constitutes the primary metabolic fate of the delta4Ach via a remodelling pathway. In contrast, [3H]delta4Ach is incorporated into triacylglycerol almost exclusively via de novo synthesis, as evidenced by studies using propranolol, a phosphatidate phosphohydrolase inhibitor. This compound induced a time-dependent and concentration-dependent increase in the levels of [3H]delta4Ach-containing phosphatidate that is directly correlated with a decrease in the levels of [3H]delta4Ach-containing triacylglycerol. In addition, no change in the levels of [3H]delta4Ach-containing diacylglycerol and monoacylglycerol were apparent along the time course of fatty acid incorporation into triacylglycerol. However, a sharp and transient accumulation of cell-associated free [3H]delta4Ach was detected shortly after exposure of the cells to the radioactive fatty acid. Collectively, the results reported herein suggest that free delta4Ach availability determines the patterns of incorporation and distribution of this fatty acid among the various lipid classes of P338D1 cells.

Animals↗
PubMed1995

Inhibition of calcium-independent phospholipase A2 prevents arachidonic acid incorporation and phospholipid remodeling in P388D1 macrophages.

Cellular levels of free arachidonic acid (AA) are controlled by a deacylation/reacylation cycle whereby the fatty acid is liberated by phospholipases and reincorporated by acyltransferases. We have found that the esterification of AA into membrane phospholipids is a Ca(2+)-independent process and that it is blocked up to 60-70% by a bromoenollactone (BEL) that is a selective inhibitor of a newly discovered Ca(2+)-independent phospholipase A2 (PLA2) in macrophages. The observed inhibition correlates with a decreased steady-state level of lysophospholipids as well as with the inhibition of the Ca(2+)-independent PLA2 activity in these cells. This inhibition is specific for the Ca(2+)-independent PLA2 in that neither group IV PLA2, group II PLA2, arachidonoyl-CoA synthetase, lysophospholipid:arachidonoyl-CoA acyltransferase, nor CoA-independent transacylase is affected by treatment with BEL. Moreover, two BEL analogs that are not inhibitors of the Ca(2+)-independent PLA2--namely a bromomethyl ketone and methyl-BEL--do not inhibit AA incorporation into phospholipids. Esterification of palmitic acid is only slightly affected by BEL, indicating that de novo synthetic pathways are not inhibited by BEL. Collectively, the data suggest that the Ca(2+)-independent PLA2 in P388D1 macrophages plays a major role in regulating the incorporation of AA into membrane phospholipids by providing the lysophospholipid acceptor employed in the acylation reaction.

Animals↗
PubMed1995

A phospholipase D-mediated pathway for generating diacylglycerol in nuclei from Madin-Darby canine kidney cells.

Many receptors, in response to their specific ligands, trigger activation of phospholipase D (PLD), resulting in the production of phosphatidic acid which, in turn, is acted upon by a specific phosphatase, phosphatidate phosphohydrolase, to produce diacylglycerol. We report here that isolated nuclei from Madin-Darby canine kidneys (MDCK)-D1 cells exhibit a PLD activity that is enhanced by the presence of ATP. PLD activity was measured in the presence of ethanol, by quantitating the production of phosphatidylethanol. Non-phosphorylating ATP analogs were unable to substitute for ATP in activating PLD, indicating that ATP acts as a phosphoryl group donor in a kinase-mediated phosphorylation reaction. The protein kinase C inhibitors chelerythrine and calphostin completely suppressed the ATP-induced nuclear PLD, implicating protein kinase C as the kinase involved in ATP-dependent PLD activity in nuclei from MDCK-D1 cells. In the absence of ethanol, phosphatidic acid was detected in ATP-treated nuclei. Accumulation of phosphatidic acid preceded or closely paralleled that of diacylglycerol, suggesting a precursor-product relationship. Consistent with those results, we detected phosphatidate phosphohydrolase activity in MDCK-D1 cell nuclei. Measurements of phosphatidic acid and diacylglycerol levels at increasing amounts of ethanol demonstrated that PLD and phosphatidate phosphohydrolase are responsible for generating the majority of the diacylglycerol accumulating in MDCK-D1 cell nuclei. The ability of nuclei to generate diacylglycerol from the concerted action of those two enzymes provides a means to regulate nuclear lipid synthesis as well as protein kinase C activity.

Adenosine Triphosphate↗
PubMed1994

Arachidonic acid mobilization in P388D1 macrophages is controlled by two distinct Ca(2+)-dependent phospholipase A2 enzymes.

Macrophage-like P388D1 cells mobilize arachidonic acid (AA) and produce prostaglandin E2 upon stimulation with bacterial lipopolysaccharide and platelet-activating factor. We have now demonstrated that AA mobilization in these cells is composed of two distinct events: a transient phase in which AA accumulates in the cell and a sustained phase in which the fatty acid accumulates in the incubation medium. Both phases are markedly dependent on the presence of Ca2+ in the extracellular medium. Treatment with an antisense oligonucleotide to group II phospholipase A2 inhibits the accumulation of AA in the incubation medium, but has no effect on the accumulation of this fatty acid in the cell. In addition, treatment with antisense oligonucleotide to group II phospholipase A2 has no effect on the uptake or the esterification of AA. Collectively, these results indicate that, in addition to the previously demonstrated role of group II phospholipase A2 in AA mobilization in activated P388D1 cells, another phospholipase A2, distinct from the group II enzyme, is implicated in raising the levels of intracellular AA during the early steps of P388D1 cell activation and in modulating deacylation/reacylation reactions involving AA. The data suggest that each of the different phospholipase A2 enzymes present in P388D1 cells serves a distinct role in cell function.

Animals↗
PubMed1994

Phosphatidate-induced arachidonic acid mobilization in mouse peritoneal macrophages.

Phosphatidate (PA) is synthesized by a variety of cells in response to physiological agonists. Addition of PA vesicles to [3H]arachidonic acid (AA)-labeled macrophages was found to induce the release of radiolabel in a dose- and time-dependent manner. This effect correlated with the uptake of PA by the macrophages and appeared to be attributable to PA itself and not to a PA metabolite. In parallel with AA release, PA induced a rapid increase in lysophosphatidylcholine in cells prelabeled with [14C]glycerol. Down-regulation of protein kinase C by long term exposure of the cells to phorbol myristate acetate or cell treatment with the protein kinase C inhibitor staurosporine did not affect the PA response. Also, removal of external calcium or cell treatment with the calmodulin antagonist trifluoperazine did not affect PA-induced AA release, while inhibiting the responses to zymosan, phorbol 12-myristate 13-acetate, and ionophore A23187. PA-induced AA release was not affected by intracellular calcium depletion by treatment with quin2/AM in the presence of EGTA. When assayed toward an AA-containing substrate, PA was able to enhance phospholipase A2 activity from cell homogenates in the absence of calcium. The dose dependence and magnitude of the PA effect correlated with those observed for PA-induced AA release in whole cells. Inclusion of ATP in the assay mixture did not affect the activity of the PA-stimulated phospholipase A2. These findings suggest a role for PA in the cascade of events leading to AA release in macrophages through Ca(2+)-independent stimulation of an AA-selective phospholipase A2.

Adenosine Triphosphate↗
PubMed1994

Increased incorporation of arachidonic acid into phospholipids in zymosan-stimulated mouse peritoneal macrophages.

Zymosan, a particle that can be phagocytosed, has been shown to stimulate the release of arachidonic acid (delta 4Ach) in macrophages via a phospholipase-A2-mediated mechanism, and to promote the incorporation of this fatty acid into cellular phospholipids [Balsinde, J., Fernández, B., Solís-Herruzo, J. A. & Diez, E. (1992) Biochim. Biophys. Acta 1136, 75-82]. This work was designed to better understand the regulation of and relationship between these two processes during cellular activation. Initial studies were conducted to examine the incorporation of exogenous [3H]delta 4Ach into the different phospholipid classes. Phosphatidylcholine and phosphatidylinositol initially accounted for most of the radioactivity incorporated into the cell. Prolonged incubation resulted in a decrease in radioactivity in phosphatidylcholine with a concomitant increase in phosphatidylethanolamine. Stimulation of the cells with zymosan led to a remarkable enhancement of the response without changing the pattern of phospholipid acylation by delta 4Ach. In the next series of experiments, the regulatory features of both delta 4Ach release and phospholipid acylation by delta 4Ach in zymosan-treated cells were comparatively investigated. Zymosan-stimulated [3H]delta 4Ach release from previously labeled cells was notably reduced when calcium was absent from the incubation medium and also when the cells were treated with pertussis toxin. Cell treatment with cholera toxin promoted a potentiation of the response. In contrast, neither of these treatments had appreciable effects on zymosan-stimulated [3H]delta 4Ach incorporation into phospholipids. Taken together, these data suggest that zymosan-stimulated delta 4Ach release and phospholipid acylation by delta 4Ach, although closely related, are independently regulated events.

Acylation↗
PubMed1994

Ethanol inhibits zymosan-stimulated eicosanoid production in mouse peritoneal macrophages.

Resident peritoneal macrophages synthesized and released eicosanoids when challenged by zymosan, a phagocytosable particle. Incubation of these cells with ethanol resulted in dose-dependent inhibition of arachidonic acid release and eicosanoid generation in response to zymosan. Ethanol affected the extent but not the ratio of eicosanoids released. When assayed in a cell-free system, endogenous phospholipase A2 activity was neither affected by the presence of ethanol in the incubation medium nor by preincubation of the cells with ethanol. Ethanol also inhibited arachidonic acid release in response to phorbol myristate acetate, a compound that, like zymosan, triggered a pertussis-toxin-sensitive response. When cells that had been previously treated with pertussis toxin were used, no further inhibitory effect of ethanol was seen in response to both zymosan and phorbol myristate acetate. On the other hand, ethanol had no effect on arachidonic acid release stimulated by ionophore A23187 or lipopolysaccharide, two compounds that triggered a pertussis-toxin-insensitive response. Moreover, ethanol was able to nearly abolish arachidonic acid release in response to fluoroaluminate, a direct activator of G-proteins. Altogether, the results of this study suggest that ethanol inhibits zymosan-stimulated eicosanoid production by interacting with a G-protein--or a G-protein-mediated process--that is critically involved in arachidonic acid mobilization.

Acyltransferases↗
PubMed1993

Mechanism of arachidonic acid liberation in ethanol-treated mouse peritoneal macrophages.

Ethanol has previously been found to induce the release of unmetabolized free arachidonic acid from resident peritoneal macrophages (Diez, E., Balsinde, J., Aracil, M. and Schüller, A. (1987) Biochim. Biophys. Acta 921, 82-89). The molecular mechanism by which ethanol promoted this effect has been investigated in this report. The results reported herein suggest that ethanol stimulated [3H]arachidonate liberation from prelabeled macrophages by inhibiting AA reesterification into phospholipids rather than by enhancing phospholipase A2 activity. Evidence supporting this view was 3-fold. First, ethanol-induced [3H]arachidonic acid release was neither affected by depletion of extracellular Ca2+ nor by cell treatment with the protein kinase C inhibitor staurosporine, two conditions which are known to prevent agonist-induced phospholipase A2 activation in macrophages. Second, ethanol appreciably inhibited the uptake and esterification of [3H]arachidonate into phospholipids of unlabeled cells. Third, combined treatment with ethanol and albumin resulted in an additive release of radiolabel. Altogether, the results of this study underscore the importance of the reacylation pathway in controlling free arachidonic acid levels in macrophages.

Alkaloids↗
PubMed1993

Calcium- and G-protein-dependent activation of arachidonic acid release by concanavalin-A-stimulated mouse macrophages.

In this work, signaling mechanisms put into function by concanavalin A in macrophages and its relationship to arachidonic acid release were investigated. After a lag period of approx. 3 min, concanavalin A induced the release of arachidonic acid from macrophages in a time- and dose-dependent manner. Removal of calcium from the extracellular medium led to a strong inhibition of the response. However, down-regulation of protein kinase C by prolonged treatment of macrophages with phorbol myristate acetate did not affect concanavalin-A-induced arachidonic acid release, suggesting that protein kinase C does not mediate the concanavalin A response. The role of G proteins in mediating the concanavalin A response was also investigated. Concanavalin-A-stimulated arachidonic acid release was inhibited by treatment with pertussis toxin but was enhanced by preincubation with cholera toxin. An increase of cAMP did not appear to mediate the stimulatory effect of cholera toxin since non-hydrolyzable cAMP derivatives or agents which raise cAMP levels, such as prostaglandin E2 and forskolin, were without effect on Con-A-stimulated arachidonate release. The direct G-protein activator fluoroaluminate was able to stimulate arachidonic acid release in a Ca(2+)-dependent manner. Combined treatment with fluoroaluminate and concanavalin A resulted in a greater than additive effect on arachidonic acid release. Altogether, these results suggest that concanavalin-A-induced arachidonic acid release in macrophages is co-ordinately regulated by Ca2+ and G proteins, but not by protein kinase C.

Aluminum↗