Studies on the activation of cytosolic phospholipase A2 and 15-lipoxygenase-1

Abstract

Arachidonic acid is a polyunsaturated fatty acid, which has four double bonds and belongs to the omega-6 family of fatty acids. Even if it can be formed in humans from one essential fatty acid, the linoleic acid, most of the arachidonic acid in the body comes with the intake of food. In mammalian cells, arachidonic acid is found esterified in cellular membranes.

Phospholipases release arachidonic acid upon cellular stimulation. There are several mammalian lipases but the cPLA2-á has been shown to preferentially release arachidonic acid. Depending on which cell and what stimuli, arachidonic acid can he further metabolized into for example prostaglandins, thromboxan A2 or leukotrienes.

The first publication investigates the arachidonic acid release induced by cPLA2-á in human platelets after stimulation with polychlorinated biphenyls (PCBs). The release of arachidonic acid by PCBs was shown to be cPLA2-á dependent since it was completely blocked by the cPLA2-á inhibitors AACOCF3 or pyrrolidine-1. Two anti-estrogens, nafoxidin and tamoxifen, but not 17â-estradiol inhibited PCB-induced arachidonic acid release. Interestingly, platelets incubated with PCBs did not aggregate despite the fact that a robust release of arachidonic acid was observed.

The second publication investigates the 15-LO-1, which catalyzes the metabolism of several polyunsaturated fatty acids of both the omega-6 and omega-3 families. A unique feature of 15-LO-1 is that it translocates to internal cellular membranes and oxygenates fatty acids also when they are esterified into lipids. The 15-LO-1 is expressed in lung epithelial cells, eosinophils, reticulocytes and IL-4 stimulated monocytes and dendritic cells. In the second publication, 15-LO-1 was found to translocate to the plasma membrane in IL-4 stimulated human dendritic cells upon calcium stimulation. The 15-LO-1 was shown to bind the membrane even when calcium was chanted.

In a lipid-overlay assay, 15-LO-1 bound to certain phospholipids and especially phosphatidylinositol s The vesicle assay model was set up to investigate if phosphalidylinositols also influenced the enzyme activity of 15-LO-1. In the presence of calcium, the addition of phosphatidylinositol-4.5-bisphosphate (PI(4.5)P2) or PI(3.4)P2 to the vesicles increased the 15-LO-1 activity on the substrate arachidonic acid. Kinetic assays were performed and the Vmax was shown to be unchanged but the apparent Km of 15-LO-1 towards arachidonic acid was significantly lower in the presence of PI(4.5)P2 or PI(3.4)P2 in the vesicles.

Taken together, this thesis demonstrates that 15-LO-1 and cPLA2-á share several regulatory properties even though they catalyze different reactions in the arachidonic acid metabolism.