Arachidonic acid (AA) is the precursor of an array of potent oxidation products eicosanoids) whose role in inflammation and hypersensitivity is widely recognized. Cellular AA is bound in the 2-position of membrane phospholipids and must be released before conversion to eicosanoids. In neutrophils (PMN), phospholipase A2 (PLA2) appears to be the major enzyme responsible for the release of free AA, but the specific PLA2 isozyme involved has not been established. In human PMN approximately half the phospholipids are ether-linked and these subclasses are highly enriched in AA. It has recently been demonstrated that the ethanolamine- containing phosphoglycerides (PE) are a major source of AA in stimulated PMN. On the other hand, alkyl-2-AA-sn-glycero-3-phosphocholine (alkyl-2- AA-GPC) serves as a source of both eicosanoids and platelet activating factor (PAF), another powerful mediator of PMN function. It has been assumed that PLA2 acts directly on alkyl-2-AA-GPC to initiate this conversion. However, the PI's laboratory recently found evidence that hydrolysis of AA from PE results in an accumulation of 1-0-alkenyl-2- lyso-sn-glycero-3-phosphoethanolamine (alkenyl-2-lyso-GPE) which can trigger transfer of AA from alkyl-2-AA-GPC to alkenyl-2-lyso-GPE via a CoA-independent transacylase reaction, thus regenerating alkenyl-2-AA-GPE and forming alkyl-2-lyso-GPC (1yso-PAF). The studies outlined are designed to assess the importance of this indirect route of AA release and PAF synthesis relative to that catalyzed by the direct action of PLA2 on alkyl-2-AA-GPC. The transacylase will be characterized both in membrane preparations and as the purified enzyme, if attempts to isolate the enzyme are successful. The substrate specificity, kinetic parameters and protein properties of the transacylase will be examined. In the postulated transacylase-dependent route of AA release and PAF synthesis, the action of PLA2 on alkenyl-2-AA-GPE plays a central role in triggering the pathway. Studies will be carried out to examine the hydrolysis of exogenous and membrane-bound alkenyl-AA-GPE by PLA2 in both isolated subcellular fractions and in permeabilized cells. These studies will provide information on the nature of the PLA2 responsible for the release of AA and synthesis of PAF and will elucidate the control mechanisms responsible for activating the enzyme upon stimulation. Preliminary evidence suggests that the alkyl-2-AA-GPC and alkenyl-2-AA-GPE of the specific granules may play an important role as a source of eicosanoids and PAF in stimulated neutrophils. The possible role of fusion of specific granules and plasma membrane in mobilization of these lipids will be examined.

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National Institute of Allergy and Infectious Diseases (NIAID)
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Wake Forest University Health Sciences
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Baker, Paul R S; Owen, John S; Nixon, Andrew B et al. (2002) Regulation of platelet-activating factor synthesis in human neutrophils by MAP kinases. Biochim Biophys Acta 1592:175-84
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