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Research Activities

Overview

    
     

Lipid Second Messengers and Phospholipases

Characterization of Phospholipase A2
 

Inhibitors and Cellular Studies

The inhibitor studies provide the knowledge and tools to study the novel enzymes in intact cells [Review: 11]. We have developed assays to distinguish each of these three types of enzymes in cell extracts. Our laboratory was the first to develop antisense oligonucleotides to block sPLA2 (12) in intact cells and we have used this technology to distinguish between the PLA2’s present in cells (13) and to inhibit the iPLA2 (14). More importantly, we have designed and developed numerous chemical inhibitors of the phospholipases and studied their mechanism of inhibition (15). We recently designed a new and novel class of cPLA2 inhibitors, the 2-oxo amides (16) and are exploring their effects in vitro, ex vivo, and in vivo animal models of hyperalgesia and pain. These inhibitors have been and will continue to be developed both in vitro and in cellular studies where the function of each PLA2 subtype is to be assessed. We have been able to differentiate the role of each of these enzymes and have developed a model for signal transduction in macrophages where cellular activation leads to the translocation of the cPLA2 from the cytosol to membranes and release of arachidonic acid followed by secretion of sPLA2 outside the cell where arachidonic acid is released and rapidly taken up by the cells to be converted to prostaglandins (17).
We discovered cross-talk between not only the sPLA2 and cPLA2 but also a coupling to one specific cyclooxygenase, COX-2 (18). Unsaturated fatty acids such as arachidonic acid when ingested or when added to cells are rapidly taken up and incorporated into phospholipids. We have shown that the iPLA2 plays a central role in membrane remodeling generating the lysophospholipid precursor that combines with the unsaturated fatty acid (19). This essential enzyme is necessary to provide the storage form of unsaturated fatty acids in phospholipids for release upon signal transduction.
We have more recently expanded our understanding of the novel regulatory cascade we discovered for cPLA2/sPLA2/COX-2 which is induced by LPS (20,21,22) and have now identified a number of distinct activation systems for macrophages including purinergic (23), zymosan (24) and UV light (25). The latter provides a Ca2+-independent mode of activating cPLA2 which may involve PIP2. We are now determining the intracellular localization of these enzymes using confocal microscopy and enhancing our understanding of the subtle levels of PLA2 regulation in macrophages.
 

Membrane Phenomenon and Surface Dilution Kinetics

Other Novel Enzymes

Oxidized Lipids
 
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Significant References

  1. Balsinde,J., Balboa,M.A., Insel,P.A., and Dennis,E.A., Regulation and Inhibition of Phospholipase A2., Ann.Reviews of Pharmacology and Toxicology, 39, 175-189 (1999).
  2. Barbour, S. and Dennis, E.A., Antisense Inhibition of Group II Phospholipase A2 Expression Blocks the Production of Prostaglandin E2 by P388D1 Cells, J.Biol.Chem., 268, 21875-21882 (1993).
  3. Balsinde, J., Barbour, S.E., Bianco, I.D., and Dennis, E.A., Arachidonic Acid Mobilization in P388D1 Macrophages is Controlled by Two Distinct Ca2+-Dependent Phospholipase A2 Enzymes, Proc.Natl.Acad.Sci.U.S.A., 91, 11060-11064 (1994).
  4. Balsinde, J., Balboa, M.A., and Dennis, E.A., Antisense Inhibition of Group VI Ca2+-Independent Phospholipase A2 Blocks Phospholipid Fatty Acid Remodelling in Murine P388D1 Macrophages, J.Biol.Chem., 272, 29317-29321 (1997).
  5. Conde-Frieboes, K., Reynolds, L.J., Lio, Y., Hale, M., Wasserman, H.H., and Dennis, E.A., Activated Ketones as Inhibitors of Intracellular Ca2+-Dependent and Ca2+-Independent Phospholipase A2, J.Am.Chem.Soc., 118, 5519-5525 (1996).
  6. Kokotos,G., Kotsovolou,S., Six,D.A., Constantinou-Kokotou,V., Beltzner,C.C., and Dennis,E.A., Novel 2-Oxo Amide Inhibitors of Human Group IVA Phospholipase A2 , J.Med.Chem, 45, 2891-2893 (2002).
  7. Balsinde, J. and Dennis, E.A., Distinct Roles in Signal Transduction for Each of the Phospholipase A2 Enzymes Present in P388D1 Macrophages, J.Biol.Chem., 271, 6758-6765 (1996).
  8. Balsinde, J., Balboa, M.A., and Dennis, E.A., Functional Coupling Between Secretory Phospholipase A2 and Cyclooxygenase-2 and Its Regulation by Cytosolic Group IV Phospholipase A2, Proc.Natl.Acad.Sci.U.S.A., 95, 7951-7956 (1998).
  9. Balsinde, J., Bianco, I.D., Ackermann, E.J., Conde-Frieboes, K., and Dennis, E.A., Inhibition of Calcium-Independent Phospholipase A2 Prevents Arachidonic Acid Incorporation and Phospholipid Remodeling in P388D1 Macrophages., Proc.Natl.Acad.Sci.U.S.A., 92, 8527-8531 (1995).
  10. Shinohara,H., Balboa,M.A., Johnson,C.A., Balsinde,J., and Dennis,E.A., Regulation of Delayed Prostaglandin Production in Activated P388D1 Macrophages by Group IV Cytosolic and Group V Secretory Phospholipase A2s, J.Biol.Chem., 274, 12263-12268 (1999).
  11. Shinohara,H., Lefkowitz,L.J., Johnson,C.A., Balboa,M.A., and Dennis,E.A., Group V Phospholipase A2 Dependent Induction of Cyclooxygenase-2 in Macrophages, J.Biol.Chem., 274, 25967-25970 (1999).
  12. Balsinde,J., Balboa,M.A., Yedgar,S., and Dennis,E.A., Group V Phospholipase A2-Mediated Oleic Acid Mobilization in Lipopolysaccharide-Stimulated P388D1 Macrophages, J.Biol.Chem., 275, 4783-4786 (2000).
  13. Balboa,M.A., Balsinde,J., Johnson,C.A., and Dennis,E.A., Regulation of Arachidonic Acid Mobilization in Lipopolysaccharide-activated P388D1 Macrophages by Adenosine Triphosphate, J.Biol.Chem., 274, 36764-36768 (1999).
  14. Balsinde,J., Balboa,M.A., and Dennis,E.A., Identification of a Third Pathway for Arachidonic Acid Mobilization and Prostaglandin Production in Activated P388D1 Cells, J.Biol.Chem., 275, 22544-22549 (2000).
  15. Balsinde,J., Balboa,M.A., Li,W.-H. , Llopis,L., and Dennis,E.A., Cellular Regulation of Cytosolic Group IV Phospholipase A2 by Phosphatidylinositol Bisphosphate Levels, J.Immunology, 164, 5398-5402 (2000).
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