Fred W. Neys

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).

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.

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 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.

Sera of patients suffering from inflammatory diseases contain group IIA but not group V phospholipase A2

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.

A simple and versatile affinity column for phospholipase A2

An affinity adsorbent for phospholipase A2 (EC 3.1.1.4) was prepared by reacting 10-O-p-toluenesulfonyldecane-1-O-phosphocholine with AH-Sepharose 4B. Phospholipases A2 bind to the immobilized ligand in the presence of Ca2+ and can be eluted with buffers containing EDTA. This principle held not only for soluble phospholipase A2 from porcine pancreas and Crotalus adamanteus, but proved also effective in the purification of phospholipases A2 solubilized from the membranes of rat liver mitochondria and rat platelets.

Half-life of interleukin-1β-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.

Some Properties of Membrane-Bound Phospholipases A2

Phospholipase A2 (EC 3.1.1.4) is found in high concentrations in venoms of bees, snakes and scorpions (1), and also in pancreatic juice (2). These soluble enzymes are nowadays well characterized (1,3). Much less is known about the cellular phospholipases A2, although these enzymes are thought to be involved in many important cellular processes like phospholipid turnover and generation of free arachidonic acid. This polyunsaturated fatty acid is the rate-limiting precursor in prostaglandins, leukotrienes, thromboxane and prostacyclin formation.