There is growing evidence that eicosanoid generation is regulated by the temporal and spatial localization of the individual enzymes of their biosynthesis, most likely at the nuclear envelope. This is achieved through their localization in the resting cell, their induced expression at specific subcellular locations, and their translocation to a source of substrate when a cell is activated. The mouse bone marrow derived mast cell (mBMMC) may be activated for three phases of eicosanoid generation. In the constitutive-immediate phase, mBMMC are activated by the ligand for c-kit ((KL) or by cross-linking of FepsilonRI by IgE and antigen for the rapid generation of leukotriene (LT) C4 and prostaglandin (PG) D2 that is complete within 10 minutes. In the delayed phase KL and interleukin (IL)10, with either IL-1beta or IgE and antigen, elicits PGD2 generation from 2 to 10 hours. In the third phase, which begins after 12 hours and is termed prime-immediate, KL with accessory cytokines primes mBMMC for augmented PGD2 generation. Immediate (whether constitutive or primed) and delayed PGD2 generation re dependent upon prostaglandin endoperoxide synthase (PGHS)-1 and PGHS-2, respectively. The initial step in eicosanoid biosynthesis is the release of free arachidonic acid from cell membrane phospholipids by the action of phospholipase A2 (PLA2), a growing family of enzymes. The segregation of the supply of arachidonic acid to PGHS-1 and PGHS-2 is likely mediated by the participation of different PLA2 enzymes in each phase. The group IV and group V enzymes likely act in a co-operative fashion in the constitutive-immediate phase, and a unidentified heparin- sensitive PLA2 acts in the delayed phase. We have used rabbit anti-peptide antibodies with specificity for the group V enzyme to show its translocation to the nuclear envelope during the immediate phase of mBMMC activation. We have also identified a cysteine-rich PLA2, termed group XI PLA2, preferentially expressed in T cells of the T helper (Th) 2 subset. We propose to extend these novel observations to a study of the expression, subcellular localization, and translocation of individual PLA2 species during each phase of eicosanoid generation. We will confirm their participation through inhibition by antisense DNA, pharmacological agents, and gene disruption. We will use site-directed mutagenesis to determine the motifs critical to the resting location and translocation of the group V enzyme. We will further characterize the group XI PLA2, examin3e its expression in developing and activated mouse mast cells, and test the hypothesis that it has a role in the differentiation and/or effector function of Th2 cells.
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