Lipid metabolites including prostaglandins, leukotrienes, other eicosanoids, and platelet activating factor are produced in a wide variety of cells. Their production is a key factor in the inflammatory response.
The aim of this project is to determine the basic mechanisms of prostaglandin regulation in inflammation by studying the prostaglandin pathways in the munne P388D1 macrophage-like cell line. During the course of previous grants, we have characterized the pathways for prostaglandin production and their control mechanisms in the P388D1 macrophages. We have identified five separate mechanisms that modulate prostaglandin production. The point of control appears to be focused on the first two steps in the prostaglandin pathway, i.e. the liberation of arachidonic acid (AA) by phospholipase A2s (PLA2) and oxidation of this AA by cyclooxygenases. We have identified the two PLA2s that participate in this pathway in P388D1 cells, i.e. the Group IV Ca2+-dependent cytosolic PLA2 and the Group V Ca2+-dependent secreted PLA2, and have shown that modulating their activity is the key to controlling prostaglandin production. We have expanded these studies in the current proposal to explore in depth how the activities of these two PLA2s are controlled. We will use confocal microscopy to follow changes in the subcellular location of the GIV PLA2 in response to changes in the levels of Ca2+, PIP2, and vimentin and ascertain how this affects prostaglandin production. We have also shown that phosphorylation processes play an important role in regulating the production of prostaglandins. We have now proposed a set of experiments that will determine how phosphorylation controls these processes. We will also explore how the extracellular GV PLA2 is controlled, i.e. via up-regulation, subcellular translocation, or activation. We will determine the factors that control the expression of GV PLA2. We will use the confocal procedures to follow the subcellular migration of this enzyme through out the course of the cellular activation.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM064611-01A1
Application #
6547065
Study Section
Physiological Chemistry Study Section (PC)
Program Officer
Chin, Jean
Project Start
2002-09-17
Project End
2006-08-31
Budget Start
2002-09-17
Budget End
2003-08-31
Support Year
1
Fiscal Year
2002
Total Cost
$299,373
Indirect Cost
Name
University of California San Diego
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
077758407
City
La Jolla
State
CA
Country
United States
Zip Code
92093
Gregus, Ann M; Buczynski, Matthew W; Dumlao, Darren S et al. (2018) Inhibition of spinal 15-LOX-1 attenuates TLR4-dependent, nonsteroidal anti-inflammatory drug-unresponsive hyperalgesia in male rats. Pain 159:2620-2629
Norris, Paul C; Dennis, Edward A (2014) A lipidomic perspective on inflammatory macrophage eicosanoid signaling. Adv Biol Regul 54:99-110
Gregus, Ann M; Dumlao, Darren S; Wei, Spencer C et al. (2013) Systematic analysis of rat 12/15-lipoxygenase enzymes reveals critical role for spinal eLOX3 hepoxilin synthase activity in inflammatory hyperalgesia. FASEB J 27:1939-49
Norris, Paul C; Dennis, Edward A (2012) Omega-3 fatty acids cause dramatic changes in TLR4 and purinergic eicosanoid signaling. Proc Natl Acad Sci U S A 109:8517-22
Gregus, Ann M; Doolen, Suzanne; Dumlao, Darren S et al. (2012) Spinal 12-lipoxygenase-derived hepoxilin A3 contributes to inflammatory hyperalgesia via activation of TRPV1 and TRPA1 receptors. Proc Natl Acad Sci U S A 109:6721-6
Dumlao, Darren S; Cunningham, Anna M; Wax, Laura E et al. (2012) Dietary fish oil substitution alters the eicosanoid profile in ankle joints of mice during Lyme infection. J Nutr 142:1582-9
Norris, Paul C; Reichart, Donna; Dumlao, Darren S et al. (2011) Specificity of eicosanoid production depends on the TLR-4-stimulated macrophage phenotype. J Leukoc Biol 90:563-74
Quehenberger, Oswald; Armando, Aaron M; Dennis, Edward A (2011) High sensitivity quantitative lipidomics analysis of fatty acids in biological samples by gas chromatography-mass spectrometry. Biochim Biophys Acta 1811:648-56
Dumlao, Darren S; Buczynski, Matthew W; Norris, Paul C et al. (2011) High-throughput lipidomic analysis of fatty acid derived eicosanoids and N-acylethanolamines. Biochim Biophys Acta 1811:724-36
Harkewicz, Richard; Dennis, Edward A (2011) Applications of mass spectrometry to lipids and membranes. Annu Rev Biochem 80:301-25

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