Role of spinal phospholipase A(2) in inflammatory hyperalgesia and characterization of the human group IVA, IVB and IVC phospholipase A(2) paralogs

Free arachidonic acid (AA) is released from phospholipids via the activity of a variety of phospholipase A₂'s (PLA₂) and is further processed by downstream enzymes to produce a number of potent lipid mediators named eicosanoids. Eicosanoids initiate a wide variety of physiological functions including inflammation, partuition, bronchoconstriction, and neutrophil chemotaxis and are indicated as potential targets of therapy in a number of diseases. This thesis focuses on two main aspects of phospholipase A₂ biochemistry.; First, in collaboration with Camilla Svensson and Tony Yaksh, the role of spinal PLA₂ in inflammatory hyperalgesia was investigated. AA is known to facilitate spinally mediated hyperalgesia following conversion to eicosanoids and may also act directly through potentiation of N-methyl-D-aspartate (NMDA) receptors. Our studies indicate that intrathecal administration of the Group IV cPLA₂ and Group VI iPLA₂ inhibitors methyl arachidonyl fluorophosphonate (MAFP) and arachidonyl trifluoromethylketone (ATK) as well as the sPLA₂ indole inhibitor (LY311727) dose dependently prevent hyperalgesia in two established rodent models. Intrathecal injection of ATK decreased the NMDA evoked release of prostaglandin E₂ (PGE ₂) into the spinal dialysate. The presence of constitutive mRNA in spinal cord for PLA₂ Groups IB, IIA, IIC, IVA, V and VI was determined by Q-PCR and sequencing. Activity assays specific for Group IV cPLA₂ , Group VI iPLA₂, and the sPLA₂'s were optimized and used to verify that these enzymes are present and active in spinal cord homogenates and that ATK, MAFP, BEL, and LY311727 inhibit PLA₂ activity in spinal preparations.; In a separate line of investigation an enzymatic characterization and comparison of the three known human Group IV PLA₂ paralogs, Group IVA, IVB and IVC Phospholipase A₂'s was carried out. All three enzymes were over-expressed in Sf9 insect cells using the baculovirus expression system and characterized from cell lysates. Like the IVA paralog, Group IVC was found to have significant lysophospholipase activity while Group IVB had none. Of the three paralogs, only the Group IVA PLA₂ shows enhanced activity in the presence of PIP₂. Laborious efforts to purify the Group IVB and IVC PLA₂ for further characterization met with limited success.