Regulatory aspects of rat liver mitochondrial phospholipase A2: effects of calcium ions and calmodulin

A comparative study was made of the metal ion requirement of rat liver mitochondrial phospholipase A2 in purified and membrane-associated forms. Membrane-bound enzyme was assayed using either exogenous or endogenous phosphatidylethanolamine. Although several divalent metal ions caused increased activity of the membrane-associated enzyme, only Ca2+ and Sr2+ activated the purified phospholipase A2. The activity in the presence of Sr2+ amounted to about 25% of that found with Ca2+. When the Ca2+ concentration was varied two activity plateaus were observed. The corresponding dissociation constants varied from 6 to 20 microM Ca2+ and from 1.4 to 12 mM Ca2+ for the high- and low-affinity binding sites, respectively, depending on the assay conditions and whether purified or membrane-bound enzyme was used. A kSr2+ of 60 microM was found for the high-affinity binding site. The effect of calmodulin and its antagonist trifluoperazine was also investigated using purified and membrane-associated enzyme. When membrane-bound enzyme was measured with exogenous phosphatidylethanolamine, small stimulations by calmodulin were found. However, these were not believed to indicate a specific role for calmodulin in the Ca2+ dependency of the phospholipase A2, since trifluoperazine did not lower the activity of the membrane-bound enzyme to levels below those found in the presence of Ca2+ alone. Membrane-bound enzyme in its action toward endogenous phosphatidylethanolamine was neither stimulated by calmodulin nor inhibited by trifluoperazine. Purified enzyme was also not stimulated by calmodulin, while trifluoperazine caused small stimulations, presumably due to interactions at the substrate level. These results indicate that calmodulin involvement in phospholipase A2 activation should not be generalized.     

Regulation of cytosolic phospholipase A2 activation and cyclooxygenase 2 expression in macrophages by the beta-glucan receptor

Phagocytosis of non-opsonized microorganisms by macrophages initiates innate immune responses for host defense against infection. Cytosolic phospholipase A(2) is activated during phagocytosis, releasing arachidonic acid for production of eicosanoids, which initiate acute inflammation. Our objective was to identify pattern recognition receptors that stimulate arachidonic acid release and cyclooxygenase 2 (COX2) expression in macrophages by pathogenic yeast and yeast cell walls. Zymosan- and Candida albicans-stimulated arachidonic acid release from resident mouse peritoneal macrophages was blocked by soluble glucan phosphate. In RAW264.7 cells arachidonic acid release, COX2 expression, and prostaglandin production were enhanced by overexpressing the beta-glucan receptor, dectin-1, but not dectin-1 lacking the cytoplasmic tail. Pure particulate (1, 3)-beta-D-glucan stimulated arachidonic acid release and COX2 expression, which were augmented in a Toll-like receptor 2 (TLR2)-dependent manner by macrophage-activating lipopeptide-2. However, arachidonic acid release and leukotriene C(4) production stimulated by zymosan and C. albicans were TLR2-independent, whereas COX2 expression and prostaglandin production were partially blunted in TLR2(-/-) macrophages. Inhibition of Syk tyrosine kinase blocked arachidonic acid release and COX2 expression in response to zymosan, C. albicans, and particulate (1, 3)-beta-D-glucan. The results suggest that cytosolic phospholipase A(2) activation triggered by the beta-glucan component of yeast is dependent on the immunoreceptor tyrosine-based activation motif-like domain of dectin-1 and activation of Syk kinase, whereas both TLR2 and Syk kinase regulate COX2 expression.     

Regulatory aspects of mitochondrial phospholipase A2 from rat liver: effects of proteins, phospholipids and calcium ions

This paper deals with the search for specific inhibitors or activators of the mitochondrial phospholipase A2. Convincing evidence for the existence of proteins in the mitochondrial or cytosolic fraction that function as specific regulators of this enzyme was not obtained. The enzymatic activity appeared to be inhibited at low substrate concentrations by lipocortin isolated from human monocytes. However, at higher substrate concentrations, the inhibition disappeared, suggesting either that lipocortin sequestered the phospholipid substrate or that the putative inactive complex of enzyme and lipocortin dissociated in the presence of excess phospholipids. The hydrolysis of the neutral phospholipid phosphatidylethanolamine was stimulated by the presence of cardiolipin and phosphatidylglycerol. It is unlikely that this is caused merely by the negative charge of these phospholipids, since other negatively charged phospholipids did not show this effect. Using a phospholipid extract from mitochondria as substrate, the enzymatic activity as a function of the Ca2+ concentration was determined. Only one enzyme activity plateau was observed. The calculated KCa2+ value of 0.05 mM suggests that the mitochondrial phospholipase A2 could be regulated strictly by the modulation of the free Ca2+ concentration in vivo. The two activity plateaus observed previously upon variation of the Ca2+ concentration using phosphatidylethanolamine as substrate could be explained by a Ca2+-induced transition of the phospholipid structure.     

Mechanisms of rat liver thiamine pyrophosphokinase activation by magnesium ions]

The role of Mg2+ in the activation of thiamine pyrophosphokinase from rat liver was studied. The dependence of the thiamine pyrophosphokinase reaction rate on total and free ATP concentrations suggests that the role of the metal comes down to optimization of conditions of the active enzyme-substrate complex formation due to incorporation of the reactions of Mg2+ and ATP-4 consecutive acception, the affinity of the latter for the enzyme being higher than that of Mg-ATP-2. The kinetic parameters of the reaction were determined. In the absence of ATP-4 free Mg2+ ions were shown to compete with the Mg-ATP-2 complex (Ki = 18 . 10(-3) M). Thiamine pyrophosphokinase is also inhibited by free ATP-4 with Ki = 3 . 10(-3) M.     

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.     

Prostaglandin levels in stimulated macrophages are controlled by phospholipase A2-activating protein and by activation of phospholipase C and D

Prostaglandins (PG), which are responsible for a large array of biological functions in eukaryotic cells, are produced from arachidonic acid by phospholipases and cyclooxygenase enzymes COX-1 and COX-2. We demonstrated that PG levels in cells were partly controlled by a regulatory protein, phospholipase A2 (PLA2)-activating protein (PLAA). Treatment of murine macrophages with lipopolysaccharide, interleukin-1beta, and tumor necrosis factor-alpha increased PLAA levels at early time points (2-30 min), which correlated with an up-regulation in cytosolic PLA2 and PGE2 levels. Both COX-2 and secretory PLA2 were also increased in lipopolysaccharide-stimulated macrophages, however, at later time points of 4-24 h. The role of PLAA in eicosanoid formation in macrophages was confirmed by the use of an antisense plaa oligonucleotide. Within amino acid residues 503-538, PLAA exhibited homology with melittin, and increased PGE(2) production was noted in macrophages stimulated with melittin. In addition to PLA2, we demonstrated that activation of phospholipase C and D significantly controlled PGE2 production. Finally, increased antigen levels of PLAA, COX-2, and phospholipases were demonstrated in biopsy specimens from patients with varying amounts of intestinal mucosal inflammation, which corresponded to increased levels of phospholipase activity. These results could provide a basis for the development of new therapeutic tools to control inflammation.     

Toll-like receptor 4 signaling regulates cytosolic phospholipase A2 activation and lipid generation in lipopolysaccharide-stimulated macrophages

Inflammatory lipid mediators such as prostaglandins and leukotrienes play crucial roles in the pathogenesis of bacterial lipopolysaccharide (LPS)-induced inflammation. Cytosolic phospholipase A(2) (cPLA(2)) is a key enzyme in the generation of pro-inflammatory lipid mediators. Here, we found that Toll-like receptor 4 (TLR4) is essential for LPS-induced cPLA(2) activation and lipid release. Inhibition of TLR4 protein expression by TLR4 small interfering RNA or neutralization of TLR4 by the specific antibody against TLR4/MD2 blocked cPLA(2) phosphorylation and cPLA(2)-hydrolyzed arachidonic acid release. Furthermore, activation of the TLR4 signaling pathway by LPS regulated cPLA(2) activation and lipid release. cPLA(2) phosphorylation and cPLA(2)-hydrolyzed lipid release were significantly impaired when TLR4 adaptor protein, either MyD88 or TRIF, was knocked down in LPS-stimulated macrophages. Similarly, LPS-induced arachidonate release was inhibited in cells transfected with a dominant-negative MyD88 or TRIF construct. Subsequently, cPLA(2) activation could be suppressed by inhibition of the TLR4 adaptor protein-directed p38 and ERK MAPK pathways. These findings suggest that, in LPS-induced inflammation, the TLR4-mediated MyD88- and TRIF-dependent MAPK pathways result in cPLA(2) activation and production of pro-inflammatory lipid mediators.     

Anionic phospholipids stimulate an arachidonoyl-hydrolyzing phospholipase A2 from macrophages and reduce the calcium requirement for activity

Mechanisms involved in regulating the activity of intracellular phospholipase A2 enzymes that function in eicosanoid and platelet-activating factor production are poorly understood. The properties of the substrate in the membrane may play a role in modulating phospholipase A2 activity. In this study, the effect of anionic phospholipids, diacylglycerol (DAG) and phosphatidylethanolamine (PE) on the activity of a partially purified, intracellular, arachidonoyl-hydrolyzing phospholipase A2 from the macrophage cell line, RAW 264.7 was studied. For these experiments phospholipase A2 activity was assayed in the presence of 1 microM calcium by measuring the hydrolysis of [3H]arachidonic acid from sonicated dispersions of the ether-linked substrate, 1-O-hexadecyl-2[3H]arachidonoylglycerophosphocholine. All the anionic phospholipids tested, including phosphatidylserine (PS), phosphatidic acid (PA), phosphatidylinositol (PI) and phosphatidylinositol-4,5-bisphosphate (PIP2), stimulated phospholipase A2 activity. At the lowest concentration of anionic phospholipids tested. PIP2 was the most stimulatory, resulting in a 7-fold increase in phospholipase A2 activity at 1 mol%. Co-dispersion of either DAG or PE with the substrate also induced a dose-dependent increase in phospholipase A2 activity, whereas sphingomyelin was inhibitory suggesting that the phospholipase A2 more readily hydrolyzed the ether-linked substrate when there was a decrease in the packing density of the bilayer. PIP2, together with either DAG or PE, synergistically stimulated phospholipase A2 activity by about 20-fold, and dramatically decreased the calcium concentration (from mM to nM) required for full activity of the enzyme. The results of this study demonstrate that the presence of anionic phospholipids and the packing characteristics of the bilayer can have pronounced effects on the activity and calcium requirement of an intracellular, arachidonoyl-hydrolyzing phospholipase A2 from macrophages.     

Role of phospholipase A2 activation and calcium in CYP2E1-dependent toxicity in HepG2 cells

Previous studies suggested a role for calcium in CYP2E1-dependent toxicity. The possible role of phospholipase A2 (PLA2) activation in this toxicity was investigated. HepG2 cells that overexpress CYP2E1 (E47 cells) exposed to arachidonic acid (AA) +Fe-NTA showed higher toxicity than control HepG2 cells not expressing CYP2E1 (C34 cells). This toxicity was inhibited by the PLA2 inhibitors aristolochic acid, quinacrine, and PTK. PLA2 activity assessed by release of preloaded [3H]AA after treatment with AA+Fe was higher in the CYP2E1 expressing HepG2 cells. This [3H]AA release was inhibited by PLA2 inhibitors, alpha-tocopherol, and by depleting Ca2+ from the cells (intracellular + extracellular sources), but not by removal of extracellular calcium alone. Toxicity was preceded by an increase in intracellular calcium caused by influx from the extracellular space, and this was prevented by PLA2 inhibitors. PLA2 inhibitors also blocked mitochondrial damage in the CYP2E1-expressing HepG2 cells exposed to AA+Fe. Ca2+ depletion and removal of extracellular calcium inhibited toxicity at early time periods, although a delayed toxicity was evident at later times in Ca2+-free medium. This later toxicity was also inhibited by PLA2 inhibitors. Analogous to PLA2 activity, Ca2+ depletion but not removal of extracellular calcium alone prevented the activation of calpain activity by AA+Fe. These results suggest that release of stored calcium by AA+Fe, induced by lipid peroxidation, can initially activate calpain and PLA2 activity, that PLA2 activation is critical for a subsequent increased influx of extracellular Ca2+, and that the combination of increased PLA2 and calpain activity, increased calcium and oxidative stress cause mitochondrial damage, that ultimately produces the rapid toxicity of AA+Fe in CYP2E1-expressing HepG2 cells.     

A novel phosphatidylglycerol-selective phospholipase A2 from macrophages

In our recent studies on the synthesis of bis(monoacylglycero)phosphate (BMP), we postulated that the first step involved a PLA2 that cleaved the 2-acyl group from phosphatidylglycerol (PG). In the present study, a novel lysosomal PLA2 was partially purified and characterized from RAW 264.7, macrophage like cells. Cells were homogenized and delipidated, and the PLA2 activity in the soluble fraction was purified by Sephacryl S100 and DEAE Sephacel. Further purification was performed using Con-A Sepharose, Phenyl Sepharose, DEAE Sephacel, and Superdex 75 FPLC. The enzyme at this stage of purification showed a dominant band around 45 kDa plus several minor bands on SDS-PAGE. The molecular mass determined by Superdex 75 column FPLC was about 45 kDa. The highly purified fraction hydrolyzed at the sn-1 position, implying that this PLA2 also has some intrinsic PLA1 activity. This enzyme preferentially hydrolyzed PG, has an acidic pH optima, and does not require divalent metal ions. Comparison using PG with various acyl chains on the sn-2 position showed that oleate and linoleate were preferred relative to arachidonate. MAFP, a known cytosolic PLA2 inhibitor, strongly inhibited this PLA2 activity. MJ33, AACOCF3, DENP, and Amiodarone also gave moderate inhibition. The characteristics of this enzyme showed this to be a new type of PLA, and the overwhelming preference for PG as substrate suggests its physiological role is in the biosynthesis of BMP.     

GTP-binding protein mediated phospholipase A2 activation in rat liver during the progression of sepsis

Effects of GTP-binding proteins on the activation of secretory phospholipase A2 (sPLA2) and cytosolic phospholipase A2 (cPLA2) in rat liver during two different phases of sepsis were studied. Sepsis was induced by cecal ligation and puncture (CLP). Experiments were divided into three groups: control, early sepsis, and late sepsis. Early and late sepsis refers to those animals sacrificed at 9 and 18 h, respectively, after CLP. The results show that in the absence of G-protein modulator, hepatic sPLA2 and cPLA2 activities were activated by 40.8-46 and 91.6-105.8%, respectively, during early and late phases of sepsis. GTPS and fluoroaluminate (A1F4-) stimulated sPLA2 and cPLA2 activities within each experimental group, i.e., control, early sepsis, and late sepsis. The GTPS and A1F4--stimulated sPLA2 and cPLA2 activities remained significantly elevated during early phase (22.3-65.6% increase) and late phase (32.5-109.1% increase) of sepsis. Further analyses demonstrate that cholera toxin significantly stimulated sPLA2 and cPLA2 activities within each experimental group, and that the cholera toxin stimulated sPLA2 and cPLA2 activities remained significantly higher during early phase (23.5-37%increase) and late phase (56.7-70% increase) of sepsis. In contrast, pertussis toxin significantly inhibited sPLA2 and cPLA2 activities within each experimental group, and that the pertussis toxin-inhibited sPLA2 and cPLA2 activities remained significantly higher in early septic (57-68.5% increase) and late septic (34.6-45.5% increase) experiments. These data demonstrate that cholera toxin-sensitive Gs and pertussis toxin-sensitive Gi were both involved in the activation of sPLA2 and cPLA2 activities in rat liver during the progression of sepsis.     

Translocation of phospholipase A2 from cytosol to membranes in rat brain induced by calcium ions

Phospholipase A2 (PLA2) activities were found in the cytosolic fractions of rat brain. Using the gel filtration chromatography, two major peaks of PLA2 activities were demonstrated: PLA2-H (200-500 kDa) and PLA2-L (100 kDa). PLA2-L was active at both neutral and alkaline pH and absolutely required Ca2+ for the activity, while the activity of PLA2-H was detected only at alkaline pH and independent of Ca2+. The activation of PLA2-L by Ca2+ was biphasic; the first observed at 1-100 microM Ca2+ and the second at 10 mM Ca2+. In the reconstitution system of partially purified PLA2-L and synaptosomal membranes from rat brain, PLA2-L associated with the membranes in a Ca2(+)-dependent manner. The association was completed within 5-10 min at 25 degrees C both at 10 microM and 1 mM Ca2+, though amount of PLA2-L translocated was dependent on Ca2+ concentrations. These results suggest that Ca2+ promotes the translocation of the cytosolic PLA2-L to membranes where phospholipids, substrate of PLA2, are present.     

Effect of parathyroidectomy on calcium and phospholipase A2 in the liver of carbon tetrachloride-treated rats.

The present study shows that carbon tetrachloride fails to cause hypercalcemia in the serum and liver of rats after parathyroidectomy. Secondly, a diminution in the activity of calcium-dependent proteolytic enzyme, phospholipase A2 occurs in parathyroidectomized rats, suggesting a plausible protective mechanism against carbon tetrachloride toxicity.     

On the importance of calcium and magnesium ions in yeast sporulation

Irrespective of the nutritional conditions, the sporulation frequency of wild and industrially used yeasts on agar or agarose plates has been found to vary from one experiment to another. An analysis of agar- and agarose-extracts by ion-exchange column chromatography proved that the amount of calcium and/or magnesium ions contained in the agar was a factor in the fluctuation of sporulation frequency. Furthermore, these two cations enhanced the formation of four-spored asci. When calcium or magnesium ions were added to a nutrition-deprived medium solidified with agarose containing no detectable calcium and magnesium ions, wild and industrially used sake yeasts efficiently sporulated with a frequency of 10–40%. A strictly controlled sporulation condition suitable for the analysis of meiosis and sporulation of yeast cells was constructed by using calcium and/or magnesium ions and highly purified agarose.     

Bradykinin-induced activation of phospholipase A2 is independent of the activation of polyphosphoinositide-hydrolyzing phospholipase C

This study evaluates the role of phosphatidylinositol 4,5-bisphosphate (PIP2) and its metabolites as possible mediators in the activation of phospholipases A2 in porcine aortic endothelial cells. We compared the time courses of bradykinin-induced turnover of phosphoinositides and the appearance of unesterified arachidonic acid (uAA) and eicosanoids. The metabolism of phosphoinositides was examined in cells prelabeled with [3H]inositol, which has a similar distribution as the endogenous inositol lipids. At 37 degrees C, bradykinin induced a rapid rise in lysophosphatidylinositol (lyso-PI) and inositol 1,4,5-trisphosphate (IP3) as well as a decrease in PIP2. Lyso-PI formation was detected at 10 s, as early as PIP2 degradation and IP3 formation. This suggests that the activation of PIP2-hydrolyzing phospholipase C and PI-hydrolyzing phospholipase A2 are simultaneous. However, at 30 degrees C, lyso-PI formation was detected in the absence of an increase in IP3 indicating that the activation of phospholipase A2 does not require the accumulation of IP3. The time course of formation of uAA and eicosanoids were examined in [3H]arachidonic acid-prelabeled cells. The 3H radioactivity was distributed among the phospholipid classes and subclasses the same as the endogenous phospholipids. Bradykinin stimulated the intracellular accumulation of uAA, detectable at 5 s, earlier than that of 1,2-diacylglycerol and phosphatidic acid. Such immediate formation of uAA further supports the notion that activation of phospholipase A2 is a very early event during the interaction of bradykinin with porcine endothelial cells, and that PIP2 hydrolysis is not prerequisite for the initial activation of phospholipase A2.     

A phospholipase A2 hydrolyzing arachidonoyl-phospholipids in mouse peritoneal macrophages

A calcium-dependent phospholipase A2 with half-maximal activity at approx. 0.7 microM free Ca2+ has been identified in the cytosolic fraction from macrophages. The enzyme eluted as a 70 kDa protein upon gel chromatography and showed increased activity after 10 min pretreatment of the cells with 10 nM phorbol myristate acetate. No significant activity could be detected in the membrane fraction. The enzyme hydrolyzed arachidonic acid-containing phosphatidylcholine and -ethanolamine as well as phosphatidylinositol. The release of arachidonic acid in the in vitro assay was inhibited in a dose-dependent manner by nordihydroguaiaretic acid and quercetin that are also potent inhibitors of the mobilization of arachidonic acid in intact macrophages.