Sera of patients suffering from inflammatory diseases contain group IIA but not group V phospholipase A(2).

During recent years, the high phospholipase A(2) (PLA(2)) concentrations at sites of inflammation and in circulation in several life-threatening diseases, such as sepsis, multi-organ dysfunction and acute respiratory distress syndrome, has generally been ascribed to the non-pancreatic group IIA PLA(2). Recently the family of secreted low molecular mass PLA(2) enzymes has rapidly expanded. In some cases, a newly described enzyme appeared to be cross-reactive with antibodies against the group IIA enzyme. For this reason, reports describing the expression of group IIA PLA(2) during inflammatory conditions need to be reevaluated. Here we describe the identification of the PLA(2) activity in sera of acute chest syndrome patients and in sera of trauma victims. In both cases, the PLA(2) activity was identified as group IIA. This classification was based upon cross-reactivity with monoclonal antibodies against group IIA PLA(2) which do not recognize the recombinant human group V enzyme. Moreover, purification of the enzymatic activity from the two sera followed by N-terminal amino acid sequence analyses revealed only the presence of group IIA enzyme.

Regulation of the expression of group IIA and group V secretory phospholipases A(2) in rat mesangial cells.

Rat mesangial cells synthesize and secrete a secretory phospholipase A(2) upon stimulation of the cells with cytokines, like IL-1beta and TNF and with cAMP elevating agents like forskolin. This enzyme was previously characterized to belong to group IIA sPLA(2). The discovery of several other low molecular weight phospholipases, like group IIC in murine testis and group V in human and rat heart, prompted investigations on the presence of group IIC and group V sPLA(2) in rat mesangial cells. This was done by isolating the RNA from stimulated cells and performing RT-PCR, using primers specific for group IIC and V sPLA(2). The results indicate that rat mesangial cells upon stimulation express next to group IIA also group V sPLA(2). No indications were obtained for the expression of group IIC sPLA(2). The regulation of the expression of group V sPLA(2) at the mRNA level was further investigated by examining the time-dependent expression, the influence of dexamethasone and the signaling route of the IL-1beta stimulation. The results show that the IL-1beta induced expression of group V sPLA(2) mRNA was time dependent and, similar to that of group IIA sPLA(2) mRNA, involves activation of NF-kappaB. However, in contrast to the group IIA sPLA(2), the expression of group V sPLA(2) was not influenced by the presence of dexamethasone. The expression of both phospholipases was also examined at the protein level in stimulated mesangial cells. Western blot analysis shows that stimulated mesangial cells synthesize both group IIA and group V sPLA(2) protein but the expression of group V is lower compared to that of group IIA sPLA(2). In addition, the extent of secretion into the medium appears to be considerably higher for group IIA than for group V sPLA(2).

Half-life of interleukin-1 beta-induced group II phospholipase A2 in rat mesangial cells.

Group II phospholipase A2 (sPLA2) has been implicated as an important agent involved in a number of inflammatory processes. Potent pro-inflammatory cytokines, such as interleukin-1 beta (IL-1 beta) and tumor necrosis factor (TNF) have been found to induce sPLA2 synthesis and release from many cell types among which mesangial cells. Although considerable research has been devoted to unravelling the mechanisms underlying the induction of sPLA2 not much is known about the time scale at which the cytokine elicited signals for sPLA2 induction persist in target cells. In this study we addressed that question by using rat renal mesangial cells as a model target cell. We found that after removal of IL-1 beta from the culture medium, the induced-sPLA2 synthesis continues at gradually decreasing rates for approximately 8 h. This is accompanied by a decrease in sPLA2 mRNA levels. Furthermore, with pulse-chase experiments we investigated the half-life of sPLA2 disappearance from the cells. This disappearance was found to be biphasic. A rapidly disappearing pool, constituting approx. 74% of the total, exhibited a half-life of 1.6 +/- 0.2 h. The remaining pool of the induced enzyme was much more stable and its level remained constant for at least 24 h. Analysis of the appearance of newly synthesized enzyme in the culture medium indicated this process to be completed in an hour.

Purification and characterization of Ca(2+)-dependent phospholipases A2 from rat kidney.

Three phospholipase A2 (PLA2) activities were identified in rat kidney. In the particulate fraction a PLA2 activity was present which was cross-reactive with polyclonal antibodies against the 14-kDa group II PLA2. This PLA2 was partially solubilized and purified to near homogeneity. The amino acid sequence at the N-terminus of the purified enzyme was identical to that of the 14-kDa rat group II PLA2 from rat liver mitochondria, platelet, and spleen. The cytosolic fraction of rat kidney contained at least two PLA2 activities which could be separated on a Mono Q column. Upon gel filtration the activity that eluted from the anion-exchange column in the salt gradient behaved as a high molecular mass PLA2, exhibited a preference for arachidonic acid at the sn-2 position of glycerophospholipids, and was already optimally active at submillimolar Ca2+ concentrations. The cytosolic PLA2 activity that did not bind to the anion-exchange column was purified by gel filtration, immunoaffinity chromatography using immobilized polyclonal antibodies to group I PLA2, and C18 reversed-phase chromatography. Immunological properties and N-terminal sequence analysis identified this enzyme as rat group I PLA2. Rat glomerular mesangial cells contained only group II and high molecular mass PLA2 enzymes.

Catabolism of platelet-activating factor and its acyl analog. Differentiation of the activities of lysophospholipase and platelet-activating-factor acetylhydrolase.

Recent investigations have shown the presence of 1-acyl-2-acetyl-sn-glycero-3-phosphocholine, i.e. the acyl analog of platelet-activating factor (PAF), in unstimulated tissues as well as its formation along with platelet-activating factor upon stimulation of a variety of cells. We demonstrate here that this acyl analog of PAF can be catabolized by purified lysophospholipases I and II from bovine liver with near stoichiometric formation of 2-acetyl-sn-glycero-3-phosphocholine. Lysophospholipase II also deacetylated PAF to lysoPAF and evidence is presented to show that this is an intrinsic activity of this enzyme. This suggested that some lysophospholipases may contribute to intracellular inactivation of PAF by deacetylation. Anion-exchange chromatography of rat liver cytosol confirmed this possibility. However, similar experiments with rat kidney cytosol and rat and human platelet cytosol clearly separated lysophospholipase activities without PAF acetylhydrolase activity from specific PAF acetylhydrolases not having lysophospholipase activity. Thus, lysophospholipases are clearly involved in the metabolism of the acyl analog of PAF and in some tissues, such as liver, may even contribute to abolishing the biological activity of PAF through deacetylation.

Immunoaffinity purification, partial sequence, and subcellular localization of rat liver phospholipase A2.

Monoclonal antibodies against rat liver mitochondrial phospholipase A2 were used to develop a rapid immunoaffinity chromatography for enzyme purification. The purified enzyme showed a single band upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The sequence of the N-terminal 24 amino acids was determined. This part of the sequence showed only 25% homology with that of rat pancreatic phospholipase A2 but was 96% identical to that of rat platelet and rat spleen membrane-associated phospholipase A2. These enzymes are distinguished from pancreatic phospholipases A2 by the absence of Cys-11. In rat liver phospholipase A2 activity has been reported in various subcellular fractions. All of these require Ca2+ and have a pH optimum in the alkaline region, but little is known about the structural relationship and quantitative distribution of these enzymes. We have investigated these points after solubilization of the phospholipase A2 activity from total homogenates and crude subcellular fractions by extraction with 1 M potassium chloride. Essentially all of the homogenate activity could be solubilized by this procedure indicating that the enzymes occurred in soluble or peripherally membrane-associated form. Gel filtration and immunological cross-reactivity studies indicated that phospholipases A2 solubilized from membrane fractions shared a common epitope with the mitochondrial enzyme. The quantitative distribution of the immunopurified enzyme activity among subcellular fractions followed closely that of the mitochondrial marker cytochrome c oxidase. Rat liver cytosol contained additional Ca2+-dependent and -independent phospholipase activities.

Phospholipase A2 activity in platelets. Immuno-purification and localization of the enzyme in rat platelets.

A comparative study on phospholipase A2 activity in platelet lysates from various species was carried out using identical assay conditions with phosphatidylethanolamine as substrate. Platelet phospholipase A2, both when expressed as activity per ml blood and as specific activity in KCl extracts, was low in human, cow, pig and goat. Moderate activities, in increasing order, were found in sheep, horse and rabbit, while rats showed by far the highest activity. In the latter four species total lysate activity was recovered in 1 M KCl extracts, suggesting that the enzyme occurs either in soluble form or as a peripheral membrane-associated protein. Immune cross-reactivity with monoclonal antibodies against rat liver mitochondrial phospholipase A2 was studied in dot-blot and monoclonal antibody-Sepharose binding experiments. Only sheep and rat platelet extracts contained cross-reactive phospholipase(s) A2. Immuno-affinity chromatography of rat platelet extracts indicated virtually complete binding of total phospholipase A2 activity and yielded pure enzyme in a single purification step. Enzyme visualization by immunogold electron microscopy showed a predominant localization in the matrix of alpha-granules.

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.

Hydrolysis of membrane-associated phosphoglycerides by mitochondrial phospholipase A2.

Conversion of membrane-bound substrates by membrane-associated enzymes can proceed in principle via intramembrane and intermembrane action. By using rat-liver mitochondria containing labeled phosphatidylethanolamine and inactivated phospholipase A2 as substrate source, and mitochondria containing unlabeled substrate and active enzyme, it is shown that hydrolysis of phosphatidylethanolamine by mitochondrial phospholipase A2 proceeds nearly entirely via intramembrane enzyme action. A study of the characteristics of this mode of enzyme action showed that all mitochondrial phosphoglycerides were hydrolyzed. Plots of approximate initial velocities of hydrolysis against the remaining amounts of each individual phospholipid, indicated that phosphatidylethanolamine was hydrolyzed fastest, with a rate about twice that for phosphatidylcholine and about 10-fold that for cardiolipin. The initial rates remained nearly constant in the initial phase of the hydrolysis, suggesting that the enzyme is surrounded by excess substrate.

Hydrolysis of exogenous substrates by mitochondrial phospholipase A2.

Evidence is provided in this paper to indicate that hydrolysis of exogenously added phosphatidylethanolamine and phosphatidylcholine by the membrane-bound phospholipase A2 from rat-liver mitochondria is preceded by association of the substrates with the membranes. Hydrolysis of phosphatidylethanolamine after preincubation of mitochondria and substrate is nearly independent of incubation volume, indicating that substrate and mitochondria are not independently diluted. The association is greatly enhanced in the presence of Ca2+, especially for phosphatidylethanolamine. Association can be measured after sucrose-gradient centrifugation of mitochondria preincubated with phosphatidylethanolamine and can be visualized by freeze-fracture electronmicroscopy, showing substrate clusters fused with mitochondria. The association provides an explanation for the hydrolysis of exogenous substrates by a membrane-associated phospholipase A2 as well as for the high preference for phosphatidylethanolamine degradation often observed in studies on membrane-bound phospholipases A. This preference is likely to result in part from the tendency of unsaturated phosphatidylethanolamines to adopt non-bilayer lipid phases allowing a more extensive association with biomembranes in the presence of Ca2+, and does not reflect enzyme specificity per se. This phenomenon should be kept in mind when determining the substrate specificity of membrane-bound phospholipases A by the use of exogenous substrates.