Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1997 Mar 1;322(Pt 2):671–677. doi: 10.1042/bj3220671

Biosynthesis, release and degradation of the novel endogenous cannabimimetic metabolite 2-arachidonoylglycerol in mouse neuroblastoma cells.

T Bisogno 1, N Sepe 1, D Melck 1, S Maurelli 1, L De Petrocellis 1, V Di Marzo 1
PMCID: PMC1218241  PMID: 9065792

Abstract

The monoacylglycerol 2-arachidonoylglycerol (2-AG) has been recently suggested as a possible endogenous agonist at cannabinoid receptors both in brain and peripheral tissues. Here we report that a widely used model for neuronal cells, mouse N18TG2 neuroblastoma cells, which contain the CB1 cannabinoid receptor, also biosynthesize, release and degrade 2-AG. Stimulation with ionomycin (1-5 microM) of intact cells prelabelled with [3H]arachidonic acid ([3H]AA) led to the formation of high levels of a radioactive component with the same chromatographic behaviour as synthetic standards of 2-AG in TLC and HPLC analyses. The amounts of this metabolite were negligible in unstimulated cells, and greatly decreased in cells stimulated in the presence of the Ca2+-chelating agent EGTA. The purified component was further characterized as 2-AG by: (1) digestion with Rhizopus arrhizus lipase, which yielded radiolabelled AA; (2) gas chromatographic-MS analyses; and (3) TLC analyses on borate-impregnated plates. Approx. 20% of the 2-AG produced by stimulated cells was found to be released into the incubation medium when this contained 0.1% BSA. Subcellular fractions of N18TG2 cells were shown to contain enzymic activity or activities catalysing the hydrolysis of synthetic [3H]2-AG to [3H]AA. Cell homogenates were also found to convert synthetic [3H]sn-1-acyl-2-arachidonoylglycerols (AcAGs) into [3H]2-AG, suggesting that 2-AG might be derived from AcAG hydrolysis. When compared with ionomycin stimulation, treatment of cells with exogenous phospholipase C, but not with phospholipase D or A2, led to a much higher formation of 2-AG and AcAGs. However, treatment of cells with phospholipase A2 10 min before ionomycin stimulation caused a 2.5-3-fold potentiation of 2-AG and AcAG levels with respect to ionomycin alone, whereas preincubation with the phospholipase C inhibitor neomycin sulphate did not inhibit the effect of ionomycin on 2-AG and AcAG levels. These results suggest that the Ca2+-induced formation of 2-AG proceeds through the intermediacy of AcAGs but not necessarily through phospholipase C activation. By showing for the first time the existence of molecular mechanisms for the inactivation and the Ca2+-dependent biosynthesis and release of 2-AG in neuronal cells, the present paper supports the hypothesis that this cannabimimetic monoacylglycerol might be a physiological neuromodulator.

Full Text

The Full Text of this article is available as a PDF (452.8 KB).

Selected References

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

  1. Allen A. C., Gammon C. M., Ousley A. H., McCarthy K. D., Morell P. Bradykinin stimulates arachidonic acid release through the sequential actions of an sn-1 diacylglycerol lipase and a monoacylglycerol lipase. J Neurochem. 1992 Mar;58(3):1130–1139. doi: 10.1111/j.1471-4159.1992.tb09372.x. [DOI] [PubMed] [Google Scholar]
  2. Askari A., Xie Z. J., Wang Y. H., Periyasamy S., Huang W. H. A second messenger role for monoacylglycerols is suggested by their activating effects on the sodium pump. Biochim Biophys Acta. 1991 Oct 14;1069(1):127–130. doi: 10.1016/0005-2736(91)90113-m. [DOI] [PubMed] [Google Scholar]
  3. De Petrocellis L., Orlando P., Di Marzo V. Anandamide, an endogenous cannabinomimetic substance, modulates rat brain protein kinase C in vitro. Biochem Mol Biol Int. 1995 Aug;36(6):1127–1133. [PubMed] [Google Scholar]
  4. Desarnaud F., Cadas H., Piomelli D. Anandamide amidohydrolase activity in rat brain microsomes. Identification and partial characterization. J Biol Chem. 1995 Mar 17;270(11):6030–6035. doi: 10.1074/jbc.270.11.6030. [DOI] [PubMed] [Google Scholar]
  5. Deutsch D. G., Chin S. A. Enzymatic synthesis and degradation of anandamide, a cannabinoid receptor agonist. Biochem Pharmacol. 1993 Sep 1;46(5):791–796. doi: 10.1016/0006-2952(93)90486-g. [DOI] [PubMed] [Google Scholar]
  6. Devane W. A., Axelrod J. Enzymatic synthesis of anandamide, an endogenous ligand for the cannabinoid receptor, by brain membranes. Proc Natl Acad Sci U S A. 1994 Jul 5;91(14):6698–6701. doi: 10.1073/pnas.91.14.6698. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Devane W. A., Hanus L., Breuer A., Pertwee R. G., Stevenson L. A., Griffin G., Gibson D., Mandelbaum A., Etinger A., Mechoulam R. Isolation and structure of a brain constituent that binds to the cannabinoid receptor. Science. 1992 Dec 18;258(5090):1946–1949. doi: 10.1126/science.1470919. [DOI] [PubMed] [Google Scholar]
  8. Devane W. A. New dawn of cannabinoid pharmacology. Trends Pharmacol Sci. 1994 Feb;15(2):40–41. doi: 10.1016/0165-6147(94)90106-6. [DOI] [PubMed] [Google Scholar]
  9. Di Marzo V., De Petrocellis L., Sepe N., Buono A. Biosynthesis of anandamide and related acylethanolamides in mouse J774 macrophages and N18 neuroblastoma cells. Biochem J. 1996 Jun 15;316(Pt 3):977–984. doi: 10.1042/bj3160977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Di Marzo V., De Petrocellis L., Sugiura T., Waku K. Potential biosynthetic connections between the two cannabimimetic eicosanoids, anandamide and 2-arachidonoyl-glycerol, in mouse neuroblastoma cells. Biochem Biophys Res Commun. 1996 Oct 3;227(1):281–288. doi: 10.1006/bbrc.1996.1501. [DOI] [PubMed] [Google Scholar]
  11. Di Marzo V., Fontana A. Anandamide, an endogenous cannabinomimetic eicosanoid: 'killing two birds with one stone'. Prostaglandins Leukot Essent Fatty Acids. 1995 Jul;53(1):1–11. doi: 10.1016/0952-3278(95)90077-2. [DOI] [PubMed] [Google Scholar]
  12. Di Marzo V., Fontana A., Cadas H., Schinelli S., Cimino G., Schwartz J. C., Piomelli D. Formation and inactivation of endogenous cannabinoid anandamide in central neurons. Nature. 1994 Dec 15;372(6507):686–691. doi: 10.1038/372686a0. [DOI] [PubMed] [Google Scholar]
  13. Facci L., Dal Toso R., Romanello S., Buriani A., Skaper S. D., Leon A. Mast cells express a peripheral cannabinoid receptor with differential sensitivity to anandamide and palmitoylethanolamide. Proc Natl Acad Sci U S A. 1995 Apr 11;92(8):3376–3380. doi: 10.1073/pnas.92.8.3376. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Galadari S. H., Morris H. R., Di Marzo V. The effect of a cAMP analogue on Ca2+ ionophore-, antigen- and agonist-induced inositol phosphate release in rat basophilic leukaemia (RBL-1) cells. Biochim Biophys Acta. 1992 Jan 13;1133(2):218–222. doi: 10.1016/0167-4889(92)90072-j. [DOI] [PubMed] [Google Scholar]
  15. Hansen H. S., Lauritzen L., Strand A. M., Moesgaard B., Frandsen A. Glutamate stimulates the formation of N-acylphosphatidylethanolamine and N-acylethanolamine in cortical neurons in culture. Biochim Biophys Acta. 1995 Oct 5;1258(3):303–308. doi: 10.1016/0005-2760(95)00134-x. [DOI] [PubMed] [Google Scholar]
  16. Hanus L., Gopher A., Almog S., Mechoulam R. Two new unsaturated fatty acid ethanolamides in brain that bind to the cannabinoid receptor. J Med Chem. 1993 Oct 1;36(20):3032–3034. doi: 10.1021/jm00072a026. [DOI] [PubMed] [Google Scholar]
  17. Hasegawa-Sasaki H. Early changes in inositol lipids and their metabolites induced by platelet-derived growth factor in quiescent Swiss mouse 3T3 cells. Biochem J. 1985 Nov 15;232(1):99–109. doi: 10.1042/bj2320099. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hillard C. J., Wilkison D. M., Edgemond W. S., Campbell W. B. Characterization of the kinetics and distribution of N-arachidonylethanolamine (anandamide) hydrolysis by rat brain. Biochim Biophys Acta. 1995 Aug 3;1257(3):249–256. doi: 10.1016/0005-2760(95)00087-s. [DOI] [PubMed] [Google Scholar]
  19. Kruszka K. K., Gross R. W. The ATP- and CoA-independent synthesis of arachidonoylethanolamide. A novel mechanism underlying the synthesis of the endogenous ligand of the cannabinoid receptor. J Biol Chem. 1994 May 20;269(20):14345–14348. [PubMed] [Google Scholar]
  20. Lee M., Yang K. H., Kaminski N. E. Effects of putative cannabinoid receptor ligands, anandamide and 2-arachidonyl-glycerol, on immune function in B6C3F1 mouse splenocytes. J Pharmacol Exp Ther. 1995 Nov;275(2):529–536. [PubMed] [Google Scholar]
  21. Matsuda L. A., Lolait S. J., Brownstein M. J., Young A. C., Bonner T. I. Structure of a cannabinoid receptor and functional expression of the cloned cDNA. Nature. 1990 Aug 9;346(6284):561–564. doi: 10.1038/346561a0. [DOI] [PubMed] [Google Scholar]
  22. Maurelli S., Bisogno T., De Petrocellis L., Di Luccia A., Marino G., Di Marzo V. Two novel classes of neuroactive fatty acid amides are substrates for mouse neuroblastoma 'anandamide amidohydrolase'. FEBS Lett. 1995 Dec 11;377(1):82–86. doi: 10.1016/0014-5793(95)01311-3. [DOI] [PubMed] [Google Scholar]
  23. Mechoulam R., Ben-Shabat S., Hanus L., Ligumsky M., Kaminski N. E., Schatz A. R., Gopher A., Almog S., Martin B. R., Compton D. R. Identification of an endogenous 2-monoglyceride, present in canine gut, that binds to cannabinoid receptors. Biochem Pharmacol. 1995 Jun 29;50(1):83–90. doi: 10.1016/0006-2952(95)00109-d. [DOI] [PubMed] [Google Scholar]
  24. Mechoulam R., Hanus L., Martin B. R. Search for endogenous ligands of the cannabinoid receptor. Biochem Pharmacol. 1994 Oct 18;48(8):1537–1544. doi: 10.1016/0006-2952(94)90197-x. [DOI] [PubMed] [Google Scholar]
  25. Munro S., Thomas K. L., Abu-Shaar M. Molecular characterization of a peripheral receptor for cannabinoids. Nature. 1993 Sep 2;365(6441):61–65. doi: 10.1038/365061a0. [DOI] [PubMed] [Google Scholar]
  26. Sugiura T., Kondo S., Sukagawa A., Nakane S., Shinoda A., Itoh K., Yamashita A., Waku K. 2-Arachidonoylglycerol: a possible endogenous cannabinoid receptor ligand in brain. Biochem Biophys Res Commun. 1995 Oct 4;215(1):89–97. doi: 10.1006/bbrc.1995.2437. [DOI] [PubMed] [Google Scholar]
  27. Thomas B. F., Adams I. B., Mascarella S. W., Martin B. R., Razdan R. K. Structure-activity analysis of anandamide analogs: relationship to a cannabinoid pharmacophore. J Med Chem. 1996 Jan 19;39(2):471–479. doi: 10.1021/jm9505167. [DOI] [PubMed] [Google Scholar]
  28. Ueda H., Kobayashi T., Kishimoto M., Tsutsumi T., Okuyama H. A possible pathway of phosphoinositide metabolism through EDTA-insensitive phospholipase A1 followed by lysophosphoinositide-specific phospholipase C in rat brain. J Neurochem. 1993 Nov;61(5):1874–1881. doi: 10.1111/j.1471-4159.1993.tb09829.x. [DOI] [PubMed] [Google Scholar]
  29. Ueda N., Kurahashi Y., Yamamoto S., Tokunaga T. Partial purification and characterization of the porcine brain enzyme hydrolyzing and synthesizing anandamide. J Biol Chem. 1995 Oct 6;270(40):23823–23827. doi: 10.1074/jbc.270.40.23823. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES