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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
2R01AI017287-12
Application #
3127106
Study Section
Physiological Chemistry Study Section (PC)
Project Start
1981-09-30
Project End
1998-06-30
Budget Start
1993-07-01
Budget End
1994-06-30
Support Year
12
Fiscal Year
1993
Total Cost
Indirect Cost
Name
Wake Forest University Health Sciences
Department
Type
Schools of Medicine
DUNS #
041418799
City
Winston-Salem
State
NC
Country
United States
Zip Code
27106
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Nixon, A B; O'Flaherty, J T; Salyer, J K et al. (1999) Acetyl-CoA:1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine acetyltransferase is directly activated by p38 kinase. J Biol Chem 274:5469-73
Nixon, A B; Seeds, M C; Bass, D A et al. (1997) Comparison of alkylacylglycerol vs. diacylglycerol as activators of mitogen-activated protein kinase and cytosolic phospholipase A2 in human neutrophil priming. Biochim Biophys Acta 1347:219-30
O'Flaherty, J T; Kuroki, M; Nixon, A B et al. (1996) 5-Oxo-eicosanoids and hematopoietic cytokines cooperate in stimulating neutrophil function and the mitogen-activated protein kinase pathway. J Biol Chem 271:17821-8
O'Flaherty, J T; Kuroki, M; Nixon, A B et al. (1996) 5-Oxo-eicosatetraenoate is a broadly active, eosinophil-selective stimulus for human granulocytes. J Immunol 157:336-42
Nixon, A B; Greene, D G; Wykle, R L (1996) Comparison of acceptor and donor substrates in the CoA-independent transacylase reaction in human neutrophils. Biochim Biophys Acta 1300:187-96
Huang, C; Wykle, R L; Daniel, L W (1995) Phospholipase D hydrolyzes ether- and ester-linked glycerophospholipids by different pathways in MDCK cells. Biochem Biophys Res Commun 213:950-7
Wijkander, J; O'Flaherty, J T; Nixon, A B et al. (1995) 5-Lipoxygenase products modulate the activity of the 85-kDa phospholipase A2 in human neutrophils. J Biol Chem 270:26543-9
Huang, C; Wykle, R L; Cabot, M C (1993) Comparison of phospholipase D activity in vasopressin- and phorbol ester-stimulated fibroblasts. FEBS Lett 319:141-4
Venable, M E; Olson, S C; Nieto, M L et al. (1993) Enzymatic studies of lyso platelet-activating factor acylation in human neutrophils and changes upon stimulation. J Biol Chem 268:7965-75

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