Chemical biology of calcium-independent phospholipase A2 mediating signaling in the vasculature

The biologic roles of intracellular phospholipases are complex and reflect their pleiotropic roles in cell signaling and energy metabolism. The phospholipases A2 (PLA2) catalyze the rate determining step in the hydrolysis of phospholipids thereby facilitating the generation of eicosanoid lipid second messengers. Two types of intracellular phospholipases have been described, the cPLA2 and the iPLA2 family. Although most work has focused on cPLA2alpha as the mediator of cellular activation, this thesis examines the role of iPLA2beta in mediating arachidomc acid (AA) release through biochemical and genetic approaches. Chiral mechanism-based inhibition of the iPLA2 enzymes in conjunction with mice null for the iPLA2beta, have revealed a more detailed understanding of the complexity of vascular cell signaling and the communication between endothelial and smooth muscle cells. Pharmacologic inhibition of iPLA 2 previously demonstrated that iPLA2 activity is an important constituent in the capacitative calcium entry pathway and in acetylcholine-induced nitric oxide (NO)-mediated vasodilation. In this thesis, we utilize the iPLA 2 inhibitor (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2 H-tetrahydropyran-2-one (BEL) to investigate the contributions of iPLA2 on vascular cell function. Herein, we demonstrate that BEL-inhibited iPLA2 activity attenuated agonist-stimulated intracellular calcium signaling and NO production through modulation of protein phosphorylation and calcium homeostasis in EA.hy 926 cells. The products of iPLA2 reaction, AA and lysophospholipids (and their metabolites), were two distinguishable components contributing to calcium release from internal stores and calcium influx across the plasma membrane. Additionally, we demonstrate that AA-facilitated phosphorylation of endothelial NO synthase leads to enhance NO production. These findings were unambiguously clarified by a genetic approach utilizing iPLA2beta knockout. Aortic smooth muscle cells from iPLA 2beta-deficient mice possessed impairment of AA release and calcium entry after application of calcium mobilizing agents when compared to wild-type cells. These results imply that iPLA2beta activity may modulate AA-induced vascular smooth muscle cell relaxation through alterations in calcium signaling and NO production. Furthermore, knock-out of iPLA2beta significantly impeded mesenteric artery smooth muscle cell migration and growth, which was rescued by extracellular provision of lysophosphatidic acid. Collectively, this thesis demonstrates that iPLA2beta activity is implicated in AA (and eicosanoid)-regulated vascular tone, cellular calcium signaling and cell growth-related processes.