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Techniques for Determining the Metabolic Pathways of Eicosanoids and for Evaluating the Rate-Controlling Enzymes

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Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 186))

Abstract

The term eicosanoid is derived from the chemical name for arachidonic acid, eicosatetraenoic acid. The term eicosanoid includes the prostaglandins (PG), thromboxanes, and leukotrienes (1). Multiple biosynthetic pathways exist for eicosanoid formation (2) and specific eicosanoids seem to be produced by specific cell types in response to various inflammatory stimuli (3). The multiple synthetic pathways suggest that multiple sites of inhibition may exist. Phospholipase A2 (PLA2) acts on cell membranes to release arachidonic acid (4). While numerous enzymes have demonstrated PLA2 activity, two groups of enzymes are primarily responsible for the arachidonic acid generated during inflammatory responses. One enzyme with extensive involvement in arachidonic acid formation in response to inflammatory stimuli in many cell types is 14 kDa secretory PLA2 (sPLA2). sPLA2 requires millimolar concentrations of calcium ion for activity and binds to cell-surface proteoglycans to catalyze the release of fatty acids from the sn-2 position of cell-membrane phospholipids (5). Group IV cytosolic 85 kDa PLA2 (cPLA2) is also an important enzyme associated with the production of arachidonic acid and subsequent eicosanoid formation during inflammation (6). cPLA2 is also calcium-dependent and selectively hydrolyzes phospholipids with arachidonyl residues at the sn-2 position (7).

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References

  1. Eberhart, C. E. and DuBois, R. N. (1995) Eicosanoids and the gastrointestinal tract. Gastroenterology 109, 285–301.

    Article  CAS  PubMed  Google Scholar 

  2. Fu, J. Y., Masferrer, J. L., Seibert, K., Raz, A., and Needleman, P. (1990) The induction and suppression of prostaglandin H2 synthase (cyclooxygenase) in human monocytes. J. Biol. Chem. 265, 16,737–16,740.

    CAS  PubMed  Google Scholar 

  3. Longo, W. E., Panesar, N., Mazuski, J., and Kaminski, D. L. (1998) Contribution of cyclooxygenase-1 and cyclooxygenase-2 to prostanoid formation by human enterocytes stimulated by calcium ionophore and inflammatory agents. Pros. Other Lipid Med. 56, 325–339.

    Article  CAS  Google Scholar 

  4. Dennis, E. A. (1994) Diversity of group types, regulation and function of phospholipase A2. J. Biol. Chem. 269, 13,057–13,060.

    CAS  PubMed  Google Scholar 

  5. Hara, S., Kudo, I., Chang, H. W., Matsuta, K., Miyamoto, T., and Inoue, K. (1989) Purification and characterization of extracellular phospholipase A2 from human synovial fluid in rheumatoid arthritis. J. Biochem. 105, 395–399.

    CAS  PubMed  Google Scholar 

  6. Huang, Z., Payette, P., Abdullah, K., Cromlish, W. A., and Kennedy, B. P. (1996) Functional identification of the active site nucleophile of the human 85-kDa cytosolic phospholipase A2. Biochem. 35, 3712–3721.

    Article  CAS  Google Scholar 

  7. Takayama, K., Kudo, I., Kim, D. K., Nagata, K., Nozawa, Y., and Inoue, K. (1991) Purification and characterization of human platelet phospholipase A2 which preferentially hydrolyzes an arachidonyl residue. FEBS 282, 326–330.

    Article  CAS  Google Scholar 

  8. Capdevila, J. H., Falck, J. R., and Eastbrook, R. W. (1992) Cytochrome P450 and the arachidonic acid cascade. FASEB J. 6, 731–736.

    CAS  PubMed  Google Scholar 

  9. Peplov, P. V. (1996) Actions of cytokines in relation to arachidonic acid metabolism and eicosanoid production. Pros. Leuk. Ess. Fatty Acids 54, 303–317.

    Article  Google Scholar 

  10. Xie, W. L., Chipman, J. G., Robertson, D. L., Erikson, R. L., and Simmons, D. L. (1991) Expression of a mitogen responsive gene encoding prostaglandin synthase is regulated by mRNA splicing. Proc. Natl. Acad. Sci. USA 88, 2692–2696.

    Article  CAS  PubMed  Google Scholar 

  11. Miyamoto, T. N., Ogino, N., Yamamoto, S., and Hayaishi, O. (1976) Purification of prostaglandin endoperoxide synthase from bovine vesicular gland microsomes. J. Biol. Chem. 251, 2629–2636.

    CAS  PubMed  Google Scholar 

  12. Williams, C. S. and DuBois, R. B. (1996) Prostaglandin endoperoxide synthase: why two isoforms? Am. J. Physiol. 270, (Gastrointest. Liver Physiol., 33), G393–G400.

    CAS  Google Scholar 

  13. Seibert, K. and Masferrer, J. L. (1994) Role of inducible cyclooxygenase (COX-2) in inflammation,Receptor 4, 17–23.

    CAS  PubMed  Google Scholar 

  14. Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram qauntities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254.

    Article  CAS  PubMed  Google Scholar 

  15. Sharp, J. D., White, D. L., Chiou, G., Goodson, T., Gamboa, G. C., McClure, D., et al. (1991) Molecular cloning and expression of human Ca2-sensitive cytosolic phospholipase A2. J. Biol. Chem. 266, 14,850–14,853.

    CAS  PubMed  Google Scholar 

  16. Grossman, E. M., Longo, W. E., Mazuski, J. E., Panesar, N., and Kaminski, D. L. (2000) Role of cytosolic phospholipase A2 in cytokine stimulated prostaglandin release by human gallbladder cells. J. Gastrointest. Surg. in press.

    Google Scholar 

  17. Thommesen, L., Sjursen, W., Gasvik, K., Hanssen, W., Brekke, O. L., Skattebol, L., et al. (1998) Selective inhibitors of cytosolic or secretory phospholipase A2 block TNF-induced activation of transcription factor nuclear factor-κB and expression of ICAM-1. J. Immunol. 161, 3421–3430.

    CAS  PubMed  Google Scholar 

  18. Belley, A. and Chadee, K. (1999) Prostaglandin E2 stimulates rat and human colonic mucin exocytosis via the EP4 receptor. Gastroenterology 117, 1352–1362.

    Article  CAS  PubMed  Google Scholar 

  19. McCool, D. J., Marcon, M. A., Forstner, J. F., and Forstner, G. G. (1990) The T84 human colonic adenocarcinoma cell line produced mucin in culture and releases it in response to various secretagogues. Biochem. J. 267, 491–500.

    CAS  PubMed  Google Scholar 

  20. German, D., Barcia, J., Brems, J., Merenda, G., and Kaminski, D. L. (1989) Effects of bradykinin on feline gallbladder water transport and prostanoid formation. Dig. Dis. Sci. 34, 1770–1776.

    Article  CAS  PubMed  Google Scholar 

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Panesar, N., Deshpande, Y.G., Kaminski, D.L. (2002). Techniques for Determining the Metabolic Pathways of Eicosanoids and for Evaluating the Rate-Controlling Enzymes. In: Armstrong, D. (eds) Oxidative Stress Biomarkers and Antioxidant Protocols. Methods in Molecular Biology™, vol 186. Humana Press. https://doi.org/10.1385/1-59259-173-6:45

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  • DOI: https://doi.org/10.1385/1-59259-173-6:45

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-850-9

  • Online ISBN: 978-1-59259-173-2

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