Phospholipase A2 (PLA2) enzymes are critical regulators of prostaglandins and leukotriene synthesis, play important roles in the generation of PAF and reactive oxygen species, and can directly modify cellular membranes. Prostaglandins have been implicated in glutamate release from astrocytes. This grant has supported the creation of cytosolic PLA2 knockout (cPLA2-/-) mice to evaluate the physiological importance of this enzyme. CPLA2-/- mice are protected against ischemic injury to the brain. In the first specific aim the mechanisms of protection afforded by the absence of cPLA2 will be explored in vivo in the """"""""knockout"""""""" mouse. The effects of deletion of cPLA2 on eicosanoid, lyphospholipid, PAF, hydroxyl ion production, glutamate and sPLA2 release in response to ischemia will be examined. Mouse brain will be evaluated for the presence of Group V PLA2. Absolute measurements of cerebral blood flow will be determined. In the second specific aim the role of cPLA2 on excitotoxicity and glutamate release in vitro will be evaluated using neurons and astrocytes from Cpla2-/- and +/+ mice. Glutamate enhances cPLA2 enzymatic activity in neuronal cultures. Neurons from cPLA2-/- and +/+ mice will be prepared and the effects of the absence of cPLA2 on excitotoxicity evaluated. Agonist-stimulated glutamate release will be studied in cortical astrocytes from cPLA2-/- and +/+ cells. In the third specific aim the relationships between ERK and p38 MAP kinase pathways, cPLA2 activation, and excitotoxicity and glutamate release in vivo and in vitro will be explored. The roles of the ERK and p38 MAP kinase cascades in neuronal cell injury and astrocyte glutamate release in vitro will be studied using adenoviral constructs encoding dominant negative constitutively active forms of upstream activating kinases of ERK and p38. In the fourth specific aim the role of PLIP, a cPLA-2 interacting protein, in neuronal injury and glutamate release from astrocytes will be explored. PLIP expression and intracellular localization will be determined and compared to cPLA2 localization in the post- ischemic brain. The effects of expression of this protein on glutamate toxicity and glutamate release in cultured cPLA2-?- and +/+ neurons and astrocytes will be evaluated.
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