Skip to main content
NCBI home page
The Journal of Experimental Medicine logoLink to The Journal of Experimental Medicine
. 1984 Jan 1;159(1):89–102. doi: 10.1084/jem.159.1.89

Subcellular localization of the PGE2 synthesis activity in mouse resident peritoneal macrophages

PMCID: PMC2187202  PMID: 6420497

Abstract

The aim of this work was to establish, on a quantitative basis, the subcellular distribution of the enzyme system that converts arachidonic acid into prostaglandin (PG) E2 in mouse resident peritoneal (MRP) macrophages. Kinetic studies were conducted on cell-free extracts derived from cells cultivated for 1 d, using [1-14C]arachidonic acid as substrate and measuring the label in PGE2 after extraction and thin layer chromatography. The activity was synergistically enhanced by L- adrenaline and reduced glutathione, inhibited by indomethacin, and linearly related to the concentration of the cell-free extract. It was labile at 0 degrees C in the medium used for homogenization and fractionation of the cells (half-life less than 2 h). Addition of catalase (0.15 mg/ml) to the suspension medium increased the initial activity (by congruent to 70%) and the stability (half-life congruent to 6 h) of the enzyme in cytoplasmic extracts. It enabled us to establish the density distribution after isopycnic centrifugation in a linear gradient of sucrose. The sample centrifuged consisted of untreated cytoplasmic extracts, or cytoplasmic extracts treated with digitonin and Na pyrophosphate. Comparison of the centrifugation behavior of PGE2 synthesis activity with that of various enzymes used as reference for the major subcellular entities has revealed that PGE2 synthesis fairly fits the density profile of sulfatase C in each case. The conclusion is that at least the rate-limiting reaction in the conversion of arachidonic acid into PGE2 is catalyzed by an enzyme associated with the endoplasmic reticulum.

Full Text

The Full Text of this article is available as a PDF (1.0 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bonney R. J., Naruns P., Davies P., Humes J. L. Antigen-antibody complexes stimulate the synthesis and release of prostaglandins by mouse peritoneal macrophages. Prostaglandins. 1979 Oct;18(4):605–616. doi: 10.1016/0090-6980(79)90027-3. [DOI] [PubMed] [Google Scholar]
  2. Brune K., Glatt M., Kälin H., Peskar B. A. Pharmacological control of prostaglandin and thromboxane release from macrophages. Nature. 1978 Jul 20;274(5668):261–263. doi: 10.1038/274261a0. [DOI] [PubMed] [Google Scholar]
  3. Böhlen P., Stein S., Dairman W., Udenfriend S. Fluorometric assay of proteins in the nanogram range. Arch Biochem Biophys. 1973 Mar;155(1):213–220. doi: 10.1016/s0003-9861(73)80023-2. [DOI] [PubMed] [Google Scholar]
  4. Canonico P. G., Beaufay H., Nyssens-Jadin M. Analytical fractionation of mouse peritoneal macrophages: physical and biochemical properties of subcellular organelles from resident (unstimulated) and cultivated cells. J Reticuloendothel Soc. 1978 Aug;24(2):115–138. [PubMed] [Google Scholar]
  5. Cantarow W. D., Cheung H. T., Sundharadas G. Effects of prostaglandins on the spreading, adhesion and migration of mouse peritoneal macrophages. Prostaglandins. 1978 Jul;16(1):39–46. doi: 10.1016/0090-6980(78)90200-9. [DOI] [PubMed] [Google Scholar]
  6. Darte C., Beaufay H. Analytical subcellular fractionation of cultivated mouse resident peritoneal macrophages. J Exp Med. 1983 Apr 1;157(4):1208–1228. doi: 10.1084/jem.157.4.1208. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Doig M. V., Ford-Hutchinson A. W. The production and characterisation of products of the lipoxygenase enzyme system released by rat peritoneal macrophages. Prostaglandins. 1980 Dec;20(6):1007–1019. doi: 10.1016/0090-6980(80)90055-6. [DOI] [PubMed] [Google Scholar]
  8. Egan R. W., Paxton J., Kuehl F. A., Jr Mechanism for irreversible self-deactivation of prostaglandin synthetase. J Biol Chem. 1976 Dec 10;251(23):7329–7335. [PubMed] [Google Scholar]
  9. Gordon D., Bray M. A., Morley J. Control of lymphokine secretion by prostaglandins. Nature. 1976 Jul 29;262(5567):401–402. doi: 10.1038/262401a0. [DOI] [PubMed] [Google Scholar]
  10. Hamberg M., Samuelsson B. Prostaglandins in human seminal plasma. Prostaglandins and related factors 46. J Biol Chem. 1966 Jan 25;241(2):257–263. [PubMed] [Google Scholar]
  11. Hemler M. E., Crawford C. G., Lands W. E. Lipoxygenation activity of purified prostaglandin-forming cyclooxygenase. Biochemistry. 1978 May 2;17(9):1772–1779. doi: 10.1021/bi00602a031. [DOI] [PubMed] [Google Scholar]
  12. Hemler M. E., Lands W. E. Evidence for a peroxide-initiated free radical mechanism of prostaglandin biosynthesis. J Biol Chem. 1980 Jul 10;255(13):6253–6261. [PubMed] [Google Scholar]
  13. Hemler M., Lands W. E. Purification of the cyclooxygenase that forms prostaglandins. Demonstration of two forms of iron in the holoenzyme. J Biol Chem. 1976 Sep 25;251(18):5575–5579. [PubMed] [Google Scholar]
  14. Humes J. L., Bonney R. J., Pelus L., Dahlgren M. E., Sadowski S. J., Kuehl F. A., Jr, Davies P. Macrophages synthesis and release prostaglandins in response to inflammatory stimuli. Nature. 1977 Sep 8;269(5624):149–151. doi: 10.1038/269149a0. [DOI] [PubMed] [Google Scholar]
  15. Kurland J. I., Bockman R. Prostaglandin E production by human blood monocytes and mouse peritoneal macrophages. J Exp Med. 1978 Mar 1;147(3):952–957. doi: 10.1084/jem.147.3.952. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Leung K. H., Mihich E. Prostaglandin modulation of development of cell-mediated immunity in culture. Nature. 1980 Dec 11;288(5791):597–600. doi: 10.1038/288597a0. [DOI] [PubMed] [Google Scholar]
  17. MARGOLIASH E., NOVOGRODSKY A. A study of the inhibition of catalase by 3-amino-1:2:4:-triazole. Biochem J. 1958 Mar;68(3):468–475. doi: 10.1042/bj0680468. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Mertin J., Stackpoole A. Anti-PGE antibodies inhibit in vivo development of cell-mediated immunity. Nature. 1981 Dec 3;294(5840):456–458. doi: 10.1038/294456a0. [DOI] [PubMed] [Google Scholar]
  19. Miyamoto T., Ogino N., Yamamoto S., Hayaishi O. Purification of prostaglandin endoperoxide synthetase from bovine vesicular gland microsomes. J Biol Chem. 1976 May 10;251(9):2629–2636. [PubMed] [Google Scholar]
  20. Miyamoto T., Yamamoto S., Hayaishi O. Prostaglandin synthetase system--resolution into oxygenase and isomerase components. Proc Natl Acad Sci U S A. 1974 Sep;71(9):3645–3648. doi: 10.1073/pnas.71.9.3645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Nitta T., Suzuki T. Fc gamma 2b receptor-mediated prostaglandin synthesis by a murine macrophage cell line (P388D1). J Immunol. 1982 Jun;128(6):2527–2532. [PubMed] [Google Scholar]
  22. Ogino N., Miyamoto T., Yamamoto S., Hayaishi O. Prostaglandin endoperoxide E isomerase from bovine vesicular gland microsomes, a glutathione-requiring enzyme. J Biol Chem. 1977 Feb 10;252(3):890–895. [PubMed] [Google Scholar]
  23. Roth G. J., Siok C. J., Ozols J. Structural characteristics of prostaglandin synthetase from sheep vesicular gland. J Biol Chem. 1980 Feb 25;255(4):1301–1304. [PubMed] [Google Scholar]
  24. Rouzer C. A., Scott W. A., Cohn Z. A., Blackburn P., Manning J. M. Mouse peritoneal macrophages release leukotriene C in response to a phagocytic stimulus. Proc Natl Acad Sci U S A. 1980 Aug;77(8):4928–4932. doi: 10.1073/pnas.77.8.4928. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Rouzer C. A., Scott W. A., Griffith O. W., Hamill A. L., Cohn Z. A. Arachidonic acid metabolism in glutathione-deficient macrophages. Proc Natl Acad Sci U S A. 1982 Mar;79(5):1621–1625. doi: 10.1073/pnas.79.5.1621. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Rouzer C. A., Scott W. A., Kempe J., Cohn Z. A. Prostaglandin synthesis by macrophages requires a specific receptor-ligand interaction. Proc Natl Acad Sci U S A. 1980 Jul;77(7):4279–4282. doi: 10.1073/pnas.77.7.4279. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Samuelsson B., Goldyne M., Granström E., Hamberg M., Hammarström S., Malmsten C. Prostaglandins and thromboxanes. Annu Rev Biochem. 1978;47:997–1029. doi: 10.1146/annurev.bi.47.070178.005025. [DOI] [PubMed] [Google Scholar]
  28. Schultz R. M., Pavlidis N. A., Stylos W. A., Chirigos M. A. Regulation of macrophage tumoricidal function: a role for prostaglandins of the E series. Science. 1978 Oct 20;202(4365):320–321. doi: 10.1126/science.694537. [DOI] [PubMed] [Google Scholar]
  29. Scott W. A., Pawlowski N. A., Andreach M., Cohn Z. A. Resting macrophages produce distinct metabolites from exogenous arachidonic acid. J Exp Med. 1982 Feb 1;155(2):535–547. doi: 10.1084/jem.155.2.535. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Scott W. A., Zrike J. M., Hamill A. L., Kempe J., Cohn Z. A. Regulation of arachidonic acid metabolites in macrophages. J Exp Med. 1980 Aug 1;152(2):324–335. doi: 10.1084/jem.152.2.324. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Snyder D. S., Beller D. I., Unanue E. R. Prostaglandins modulate macrophage Ia expression. Nature. 1982 Sep 9;299(5879):163–165. doi: 10.1038/299163a0. [DOI] [PubMed] [Google Scholar]
  32. Suzuki T., Saito-Taki T., Sadasivan R., Nitta T. Biochemical signal transmitted by Fc gamma receptors: phospholipase A2 activity of Fc gamma 2b receptor of murine macrophage cell line P388D1. Proc Natl Acad Sci U S A. 1982 Jan;79(2):591–595. doi: 10.1073/pnas.79.2.591. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Takeguchi C., Kono E., Sih C. J. Mechanism of prostaglandin biosynthesis. I. Characterization and assay of bovine prostaglandin synthetase. Biochemistry. 1971 Jun 8;10(12):2372–2376. doi: 10.1021/bi00788a030. [DOI] [PubMed] [Google Scholar]
  34. Ueno R., Shimizu T., Kondo K., Hayaishi O. Activation mechanism of prostaglandin endoperoxide synthetase by hemoproteins. J Biol Chem. 1982 May 25;257(10):5584–5588. [PubMed] [Google Scholar]
  35. Valone F. H., Franklin M., Sun F. F., Goetzl E. J. Alveolar macrophage lipoxygenase products of arachidonic acid: isolation and recognition as the predominant constituents of the neutrophil chemotactic activity elaborated by alveolar macrophages. Cell Immunol. 1980 Sep 1;54(2):390–401. doi: 10.1016/0008-8749(80)90219-1. [DOI] [PubMed] [Google Scholar]
  36. Vane J. R. Inhibition of prostaglandin synthesis as a mechanism of action for aspirin-like drugs. Nat New Biol. 1971 Jun 23;231(25):232–235. doi: 10.1038/newbio231232a0. [DOI] [PubMed] [Google Scholar]
  37. Vane J. R. Prostaglandins as mediators of inflammation. Adv Prostaglandin Thromboxane Res. 1976;2:791–801. [PubMed] [Google Scholar]
  38. WILSON H. Chromogenic values of various ketosteroids in a micro modification of the Zimmermann reaction: comparison with the macro procedure. Arch Biochem Biophys. 1954 Sep;52(1):217–235. doi: 10.1016/0003-9861(54)90105-8. [DOI] [PubMed] [Google Scholar]
  39. Wedmore C. V., Williams T. J. Control of vascular permeability by polymorphonuclear leukocytes in inflammation. Nature. 1981 Feb 19;289(5799):646–650. doi: 10.1038/289646a0. [DOI] [PubMed] [Google Scholar]
  40. Zimecki M., Webb D. R. The regulation of the immune response to T-independent antigens by prostaglandins and B cells. J Immunol. 1976 Dec;117(6):2158–2164. [PubMed] [Google Scholar]

Articles from The Journal of Experimental Medicine are provided here courtesy of The Rockefeller University Press

RESOURCES