Biomedical subjects

J Balsinde

Publications and source records attributed to J Balsinde.

At least 19 recordsLinked to original sources

PubMed2001

Inflammatory activation of prostaglandin production by microglial cells antagonized by amyloid peptide.

The murine cell line MMGT-16 is of microglial origin and capable of releasing immunoinflammatory cytokines. When stimulated by the proinflammatory stimulus lipopolysaccharide (LPS), MMGT-16 cells secrete large amounts of prostaglandin E(2) (PGE(2)). This PGE(2) production is nearly abolished if amyloid beta-peptide (Abeta (1-40)) is present in the incubation medium. In addition, Abeta (1-40) inhibits cyclooxygenase-2 (COX-2) induction by LPS. Since these effects are not reproduced by the reverse control Abeta (40-1), these results suggest a novel, intriguing modulatory role for amyloid beta peptide in the inflammatory response of microglial cells.

Amyloid beta-Peptides↗

PubMed2000

Calcium-independent phospholipase A(2): structure and function.

The classical Ca(2+)-independent phospholipase A(2) enzyme, now known as Group VIA PLA(2), was initially purified and characterized from the P388D(1) macrophage-like cell line. The corresponding cDNA was subsequently cloned from a variety of sources, and it is now known that multiple splice variants of the enzyme are expressed, some of which may act as negative regulators of the active enzyme. Group VIA PLA(2) has a consensus lipase motif (GTSTG) containing the catalytic serine, is 85-88 kDa, and exists in an aggregated form. The enzyme contains multiple ankyrin repeats, which may play a role in oligomerization. The Group VIA enzyme exhibits lysophospholipase activity as well as phospholipase A(2) activity, and it is capable of hydrolyzing a wide variety of phospholipid substrates. A major function of Group VIA PLA(2) is to mediate phospholipid remodeling, but the enzyme may play other roles as well. Other Ca(2+)-independent PLA(2) enzymes have more recently been identified, and it may be possible to discriminate between the various Ca(2+)-independent PLA(2) enzymes based on sequence or inhibitor-sensitivity. However, the physiological functions of the newly identified enzymes have yet to be elucidated.

Adenosine Triphosphate↗

PubMed2000

Identification of a third pathway for arachidonic acid mobilization and prostaglandin production in activated P388D1 macrophage-like cells.

Previous studies have demonstrated that P388D(1) macrophages are able to mobilize arachidonic acid (AA) and synthesize prostaglandins in two temporally distinct phases. The first phase is triggered by platelet-activating factor within minutes, but needs the cells to be previously exposed to bacterial lipopolysaccharide (LPS) for periods up to 1 h. It is thus a primed immediate phase. The second, delayed phase occurs in response to LPS alone over long incubation periods spanning several hours. Strikingly, the effector enzymes involved in both of these phases are the same, namely the cytosolic group IV phospholipase A(2) (cPLA(2)), the secretory group V phospholipase A(2), and cyclooxygenase-2, although the regulatory mechanisms differ. Here we report that P388D(1) macrophages mobilize AA and produce prostaglandins in response to zymosan particles in a manner that is clearly different from the two described above. Zymosan triggers an immediate AA mobilization response from the macrophages that neither involves the group v phospholipase A(2) nor requires the cells to be primed by LPS. The group VI Ca(2+)-independent phospholipase A(2) is also not involved. Zymosan appears to signal exclusively through activation of the cPLA(2), which is coupled to the cyclooxygenase-2. These results define a secretory PLA(2)-independent pathway for AA mobilization in the P388D(1) macrophages, and demonstrate that, under certain experimental settings, stimulation of the cPLA(2) is sufficient to generate a prostaglandin biosynthetic response in the P388D(1) macrophages.

Animals↗

PubMed2000

Cellular regulation of cytosolic group IV phospholipase A2 by phosphatidylinositol bisphosphate levels.

Cytosolic group IV phospholipase A2 (cPLA2) is a ubiquitously expressed enzyme with key roles in intracellular signaling. The current paradigm for activation of cPLA2 by stimuli proposes that both an increase in intracellular calcium and mitogen-activated protein kinase-mediated phosphorylation occur together to fully activate the enzyme. Calcium is currently thought to be needed for translocation of the cPLA2 to the membrane via a C2 domain, whereas the role of cPLA2 phosphorylation is less clearly defined. Herein, we report that brief exposure of P388D1 macrophages to UV radiation results in a rapid, cPLA2-mediated arachidonic acid mobilization, without increases in intracellular calcium. Thus, increased Ca2+ availability is a dispensable signal for cPLA2 activation, which suggests the existence of alternative mechanisms for the enzyme to efficiently interact with membranes. Our previous in vitro data suggested the importance of phosphatidylinositol 4,5-bisphosphate (PtdInsP2) in the association of cPLA2 to model membranes and hence in the regulation of cPLA2 activity. Experiments described herein show that PtdInsP2 also serves a similar role in vivo. Moreover, inhibition of PtdInsP2 formation during activation conditions leads to inhibition of the cPLA2-mediated arachidonic acid mobilization. These results suggest that cellular PtdInsP2 levels are involved in the regulation of group IV cPLA2 activation.

Animals↗

PubMed2000

Group V phospholipase A(2)-mediated oleic acid mobilization in lipopolysaccharide-stimulated P388D(1) macrophages.

P388D(1) macrophages prelabeled with [(3)H]arachidonic acid (AA) respond to bacterial lipopolysaccharide (LPS) by mobilizing AA in a process that takes several hours and is mediated by the concerted actions of the group IV cytosolic phospholipase A(2) and the group V secretory phospholipase A(2) (sPLA(2)). Here we show that when the LPS-activated cells are prelabeled with [(3)H]oleic acid (OA), they also mobilize and release OA to the extracellular medium. The time and concentration dependence of the LPS effect on OA release fully resemble those of the AA release. Experiments in which both AA and OA release are measured simultaneously indicate that AA is released 3 times more efficiently than OA. Importantly, LPS-stimulated OA release is strongly inhibited by the selective sPLA(2) inhibitors 3-(3-acetamide-1-benzyl-2-ethylindolyl-5-oxy)propane sulfonic acid and carboxymethylcellulose-linked phosphatidylethanolamine. The addition of exogenous recombinant sPLA(2) to the cells also triggers OA release. These data implicate a functionally active sPLA(2) as being essential for the cells to release OA upon stimulation with LPS. OA release is also inhibited by methyl arachidonyl fluorophosphonate but not by bromoenol lactone, indicating that the group IV cytosolic phospholipase A(2) is also involved in the process. Together, these data reveal that OA release occurs during stimulation of the P388D(1) macrophages by LPS and that the regulatory features of the OA release are strikingly similar to those previously found for the AA release.

Animals↗

PubMed2000

Chitosan-induced phospholipase A2 activation and arachidonic acid mobilization in P388D1 macrophages.

We have found that chitosan, a polysaccharide present in fungal cell walls, is able to activate macrophages for enhanced mobilization of arachidonic acid in a dose- and time-dependent manner. Studies aimed at identifying the intracellular effector(s) implicated in chitosan-induced arachidonate release revealed the involvement of the cytosolic Group IV phospholipase A2 (PLA2), as judged by the inhibitory effect of methyl arachidonoyl fluorophosphonate but not of bromoenol lactone. Interestingly, priming of the macrophages with lipopolysaccharide renders the cells more sensitive to a subsequent stimulation with chitosan, and this enhancement is totally blocked by the secretory PLA2 inhibitor 3-(3-acetamide)-1-benzyl-2-ethylindolyl-5-oxy-propanesulfonic acid (LY311727). Collectively, the results of this work establish chitosan as a novel macrophage-activating factor that elicits AA mobilization in P388D1 macrophages by a mechanism involving the participation of two distinct phospholipases A2.

Animals↗

PubMed2000

Phosphorylation of cytosolic group IV phospholipase A(2) is necessary but not sufficient for Arachidonic acid release in P388D(1) macrophages.

Activation of the cytosolic Group IV phospholipase A(2) (cPLA(2)) by agonists has been correlated with the direct phosphorylation of the enzyme by members of the mitogen-activated protein kinase (MAPK) cascade. Phosphorylation of the cPLA(2) increases the specific activity of the enzyme, thereby stimulating the arachidonic acid release. We show here, however, that conditions that lead to full phosphorylation of the cPLA(2) do not lead to enhanced AA release. As the above observations were made under both Ca(2+)-dependent and Ca(2+)-independent conditions, they emphasize that the current paradigm for activation of the cPLA(2) in cells involving both phosphorylation and Ca(2+) is incomplete and that other factors should be taken into account.

Animals↗

PubMed2000

Group IV cytosolic phospholipase A2 activation by diacylglycerol pyrophosphate in murine P388D1 macrophages.

Diacylglycerol pyrophosphate (DGPP) is a novel phospholipid identified in yeast, bacteria, and plants, but not yet in mammalian cells. Given its structural resemblance to other phospholipid-activating molecules, such as lysophosphatidate, phosphatidate, and diacylglycerol, it was questioned whether DGPP was capable of activating macrophages to release arachidonic acid (AA) metabolites such as the prostaglandins. It has been found that DGPP is able to potently stimulate prostaglandin production in the murine cell line P388D1 by a mechanism that involves activation of the cytosolic Group IV phospholipase A2 (cPLA2). Our results demonstrate that DGPP possesses macrophage-activating-factor properties and suggest a role for this novel compound in the inflammatory response.

Animals↗

PubMed1999

Regulation of arachidonic acid mobilization in lipopolysaccharide-activated P388D(1) macrophages by adenosine triphosphate.

Murine P388D(1) macrophages exhibit a delayed prostaglandin biosynthetic response when exposed to bacterial lipopolysaccharide (LPS) for prolonged periods of time that is dependent on induction of the genes coding for Group V secretory phospholipase A(2) and cyclooxygenase-2. We herein report that LPS-induced arachidonic acid (AA) metabolite release in P388D(1) macrophages is strongly attenuated by the P2X(7) purinergic receptor antagonists periodate-oxidized ATP and pyridoxal-phosphate-6-azophenyl-2', 4'-disulfonic acid, and this is accompanied by suppression of the expression of both Group V secretory phospholipase A(2) and cyclooxygenase-2. The effect appears to be specific for LPS, because the P2 purinergic receptor antagonists do not affect P388D(1) cell stimulation by other stimuli such as platelet-activating factor or the Ca(2+) ionophore A23187. Moreover, extracellular nucleotides are found to stimulate macrophage AA mobilization with a pharmacological profile that implicates the participation of the P2X(7) receptor and that is inhibited by periodate-oxidized ATP. Collectively these results demonstrate coupling of the P2X(7) receptor to the AA cascade in P388D(1) macrophages and implicate the participation of this type of receptor in LPS-induced AA mobilization.

Adenosine Triphosphate↗

PubMed1999

Regulation of cyclooxygenase-2 expression by phosphatidate phosphohydrolase in human amnionic WISH cells.

Prostaglandins are known to play a key role in the initiation of labor in humans, but the mechanisms governing their synthesis in amnion are largely unknown. In this study, we have examined the regulatory pathways for prostaglandin E(2) (PGE(2)) production during protein kinase C-dependent activation of human WISH cells. In these cells, PGE(2) synthesis appears to be limited not by free arachidonic acid availability but by the expression levels of cyclooxygenase-2 (COX-2). Concomitant with the cells being able to synthesize and secrete PGE(2), we detected significant elevations of both COX-2 protein and mRNA levels. Specific inhibition of COX-2 by NS-398 totally ablated PGE(2) synthesis. All of these responses were found to be strikingly dependent on an active phosphatidate phosphohydrolase 1 (PAP-1). Inhibition of PAP-1 activity by three different strategies (i.e. use of bromoenol lactone, propranolol, and ethanol) resulted in inhibition of COX-2 expression and hence of PGE(2) production. These data unveil a novel signaling mechanism for the regulation of PGE(2) production via regulation of COX-2 expression and implicate phosphatidate phosphohydrolase 1 as a key regulatory component of eicosanoid metabolic pathways in the amnion.

Amnion↗

PubMed1999

Group V phospholipase A(2)-dependent induction of cyclooxygenase-2 in macrophages.

When exposed for prolonged periods of time (up to 20 h) to bacterial lipopolysaccharide (LPS) murine P388D(1) macrophages exhibit a delayed prostaglandin biosynthetic response that is entirely mediated by cyclooxygenase-2 (COX-2). Both the constitutive Group IV cytosolic phospholipase A(2) (cPLA(2)) and the inducible Group V secretory phospholipase A(2) (sPLA(2)) are involved in the cyclooxygenase-2-dependent generation of prostaglandins in response to LPS. Using the selective sPLA(2) inhibitor 3-(3-acetamide-1-benzyl-2-ethylindolyl-5-oxy)propane sulfonic acid (LY311727) and an antisense oligonucleotide specific for Group V sPLA(2), we found that induction of COX-2 expression is strikingly dependent on Group V sPLA(2), which was further confirmed by experiments in which exogenous Group V sPLA(2) was added to the cells. Exogenous Group V sPLA(2) was able to induce significant arachidonate mobilization on its own and to induce expression of the COX-2. None of these effects was observed if inactive Group V sPLA(2) was utilized, implying that enzyme activity is crucial for these effects to take place. Therefore, not only delayed prostaglandin production but also COX-2 gene induction are dependent on a catalytically active Group V sPLA(2). COX-2 expression was also found to be blunted by the Group IV cPLA(2) inhibitor methyl arachidonyl fluorophosphonate, which we have previously found to block Group V sPLA(2) induction as well. Collectively, the results support a model whereby Group IV cPLA(2) activation regulates the expression of Group V sPLA(2), which in turn is responsible for delayed prostaglandin production by regulating COX-2 expression.

Animals↗

PubMed1999

Trifluoromethyl ketones and methyl fluorophosphonates as inhibitors of group IV and VI phospholipases A(2): structure-function studies with vesicle, micelle, and membrane assays.

A series of fatty alkyl trifluoromethyl ketones and methyl fluorophosphonates have been prepared and tested as inhibitors and inactivators of human groups IV and VI phospholipases A(2) (cPLA(2) and iPLA(2)). Compounds were analyzed with phospholipid vesicle-, detergent-phospholipid mixed-micelle-, and natural membrane-based assays, and, with few exceptions, the relative inhibitor potencies measured with the three assays were similar. Ph(CH(2))(4)COCF(3) and Ph(CH(2))(4)PO(OMe)F emerged as a potent inhibitor and inactivator, respectively, of iPLA(2), and both are poorly effective against cPLA(2). Of all 13 fatty alkyl trifluoromethyl ketones tested, the trifluoromethyl ketone analog of arachidonic acid is the most potent cPLA(2) inhibitor, and structurally similar compounds including the trifluoromethyl ketone analog of docosahexenoic acid are much poorer cPLA(2) inhibitors. Inactivation of cPLA(2) by fatty alkyl fluoromethylphosphonates is greatly promoted by binding of enzyme to the interface. The use of both vesicles and mixed micelles to assay phospholipase A(2) inhibitors and inactivators present at low mol fraction in the interface provides reliable rank order potencies of a series of compounds that correlate with their behavior in a natural membrane assay.

Enzyme Inhibitors↗

PubMed1999

Regulation of delayed prostaglandin production in activated P388D1 macrophages by group IV cytosolic and group V secretory phospholipase A2s.

Group V secretory phospholipase A2 (sPLA2) rather than Group IIA sPLA2 is involved in short term, immediate arachidonic acid mobilization and prostaglandin E2 (PGE2) production in the macrophage-like cell line P388D1. When a new clone of these cells, P388D1/MAB, selected on the basis of high responsivity to lipopolysaccharide plus platelet-activating factor, was studied, delayed PGE2 production (6-24 h) in response to lipopolysaccharide alone occurred in parallel with the induction of Group V sPLA2 and cyclooxygenase-2 (COX-2). No changes in the level of cytosolic phospholipase A2 (cPLA2) or COX-1 were observed, and Group IIA sPLA2 was not detectable. Use of a potent and selective sPLA2 inhibitor, 3-(3-acetamide 1-benzyl-2-ethylindolyl-5-oxy)propanesulfonic acid (LY311727), and an antisense oligonucleotide specific for Group V sPLA2 revealed that delayed PGE2 was largely dependent on the induction of Group V sPLA2. Also, COX-2, not COX-1, was found to mediate delayed PGE2 production because the response was completely blocked by the specific COX-2 inhibitor NS-398. Delayed PGE2 production and Group V sPLA2 expression were also found to be blunted by the inhibitor methylarachidonyl fluorophosphonate. Because inhibition of Ca2+-independent PLA2 by an antisense technique did not have any effect on the arachidonic acid release, the data using methylarachidonyl fluorophosphonate suggest a key role for the cPLA2 in the response as well. Collectively, the results suggest a model whereby cPLA2 activation regulates Group V sPLA2 expression, which in turn is responsible for delayed PGE2 production via COX-2.

Animals↗

PubMed1999

Proinflammatory macrophage-activating properties of the novel phospholipid diacylglycerol pyrophosphate.

We have found that the novel phospholipid diacylglycerol pyrophosphate (DGPP), identified in bacteria, yeast, and plants, but not in mammalian cells, is able to potently activate macrophages for enhanced secretion of arachidonate metabolites, a key event in the immunoinflammatory response of leukocytes. Macrophage responses to DGPP are specific and are not mediated by its conversion into other putative lipid mediators such as phosphatidic acid, lysophosphatidic acid, or diacylglycerol. The responses to DGPP are compatible with a receptor-recognition event because they are blocked by suramin. Intracellular signaling initiated by DGPP includes phosphorylation and activation of the Group IV cytosolic phospholipase A2 and of the extracellular-signal regulated p42 mitogen-activated protein kinase (MAPK) and p44 MAPK, and membrane translocation of the protein kinase C isoenzymes alpha, epsilon, delta. These results establish DGPP as a novel macrophage-activating factor and suggest a potential role for this compound in triggering homeostatic cellular responses.

Arachidonic Acid↗

PubMed1999

Regulation and inhibition of phospholipase A2.

In recent years, there has been great interest in the study of phospholipid metabolism in intact cell systems. Such an interest arises mainly from the discovery that cellular membrane phospholipids serve not only in structural roles, but are also reservoirs of preformed second messenger molecules with key roles in cellular signaling. These second messenger molecules are generated by agonist-induced activation and secretion of intracellular and extracellular phospholipases, respectively, i.e. enzymes that cleave ester bonds within phospholipids. Prominent members of the large collection of signal-activated phospholipases are the phospholipase A2s. These enzymes hydrolyze the sn-2 ester bond of phospholipids, releasing a free fatty acid and a lysophospholipid, both of which may alter cell function. In addition to its role in cellular signaling, phospholipase A2 has recently been recognized to be involved in a wide number of pathophysiological situations, ranging from systemic and acute inflammatory conditions to cancer. A growing number of pharmacologic inhibitors will help define the role of particular phospholipase A2s in signaling cascades.

Animals↗

PubMed1998

Functional coupling between secretory phospholipase A2 and cyclooxygenase-2 and its regulation by cytosolic group IV phospholipase A2.

Secretory phospholipase A2 (sPLA2) is the major effector involved in arachidonic acid (AA) mobilization and prostaglandin E2 (PGE2) production during stimulation of P388D1 macrophages with the inflammatory stimuli bacterial lipopolysaccharide and platelet-activating factor. We herein demonstrate that PGE2 in stimulated P388D1 cells is accounted for by the inducible cyclooxygenase (COX)-2. COX-1, though present, appears not to participate significantly in stimulus-induced PGE2 production in P388D1 macrophages. Reconstitution experiments utilizing exogenous recombinant sPLA2 demonstrate that activation of the sPLA2 at the plasma membrane is highly dependent on previous activation of the cytosolic phospholipase A2 (cPLA2). Collectively these results demonstrate (i) that functional coupling exists between sPLA2 and COX-2 in activated cells, (ii) the critical role that cPLA2 plays in lipid mediator production, and (iii) that there is crosstalk between cPLA2 and sPLA2 in the cell.

Animals↗

PubMed1998

Involvement of phosphatidate phosphohydrolase in arachidonic acid mobilization in human amnionic WISH cells.

Prostaglandins are known to play a central role in the initiation of labor in humans, and amnionic cells constitute a major source of these compounds. Prostaglandin synthesis and release by amnion cells in response to hormones and ligands takes place after a characteristic 4-5 h lag. However, we report herein that free arachidonic acid (AA), the metabolic precursor of prostaglandins, can be induced at much shorter times (1 h) in human amnionic WISH cells by phorbol 12-myristate 13-acetate (PMA) through activation of protein kinase Calpha (PKCalpha). WISH cells were found to possess both cytosolic group IV phospholipase A2 (cPLA2) and Group VI Ca2+-independent phospholipase A2 (iPLA2). Of these, the cPLA2 was found to be the likely mediator of AA mobilization in PMA-activated WISH cells. PMA also activates phospholipase D (PLD) in these cells and ethanol, a compound that inhibits PLD-mediated phosphatidic acid (PA) formation, blocked AA release. Moreover, prevention of PA dephosphorylation by the PA phosphohydrolase inhibitors propranolol and bromoenol lactone, resulted in inhibition of AA release by PMA-treated WISH cells. Collectively, these data suggest that activation of cPLA2 and attendant AA release by phorbol esters in WISH cells requires prior generation of DAG by phosphatidate phosphohydrolase.

Amnion↗

PubMed1997

Antisense inhibition of group VI Ca2+-independent phospholipase A2 blocks phospholipid fatty acid remodeling in murine P388D1 macrophages.

A major issue in lipid signaling relates to the role of particular phospholipase A2 isoforms in mediating receptor-triggered responses. This has been difficult to study because of the lack of isoform-specific inhibitors. Based on the use of the Group VI Ca2+-independent phospholipase A2 (iPLA2) inhibitor bromoenol lactone (BEL), we previously suggested a role for the iPLA2 in mediating phospholipid fatty acid turnover (Balsinde, J., Bianco, I. D., Ackermann, E. J., Conde-Frieboes, K., and Dennis, E. A. (1995) Proc. Natl. Acad. Sci. U. S. A. 92: 8527-8531). We have now further evaluated the role of the iPLA2 in phospholipid remodeling by using antisense RNA technology. We show herein that inhibition of iPLA2 expression by a specific antisense oligonucleotide decreases both the steady-state levels of lysophosphatidylcholine and the capacity of the cell to incorporate arachidonic acid into membrane phospholipids. These effects correlate with a decrease in both iPLA2 activity and protein in the antisense-treated cells. Collectively these data provide further evidence that the iPLA2 plays a major role in regulating phospholipid fatty acyl turnover in P388D1 macrophages. In stark contrast, experiments with activated cells confirmed that the iPLA2 does not play a significant role in receptor-coupled arachidonate mobilization in these cells, as manifested by the lack of an effect of the iPLA2 antisense oligonucleotide on PAF-stimulated arachidonate release.

Animals↗