Back to Search
Start Over
Biosynthesis, release and degradation of the novel endogenous cannabimimetic metabolite 2-arachidonoylglycerol in mouse neuroblastoma cells.
- Source :
-
The Biochemical journal [Biochem J] 1997 Mar 01; Vol. 322 ( Pt 2), pp. 671-7. - Publication Year :
- 1997
-
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.
- Subjects :
- Animals
Arachidonic Acids pharmacology
Calcium pharmacology
Calcium Channel Blockers pharmacology
Cannabinoids pharmacology
Endocannabinoids
Enzyme Inhibitors pharmacology
Hydrolysis drug effects
Ionomycin pharmacology
Ionophores pharmacology
Mice
Neuroblastoma
Neurons drug effects
Polyunsaturated Alkamides
Receptors, Cannabinoid
Subcellular Fractions metabolism
Tumor Cells, Cultured
Glycerides metabolism
Neurons metabolism
Receptors, Drug agonists
Subjects
Details
- Language :
- English
- ISSN :
- 0264-6021
- Volume :
- 322 ( Pt 2)
- Database :
- MEDLINE
- Journal :
- The Biochemical journal
- Publication Type :
- Academic Journal
- Accession number :
- 9065792
- Full Text :
- https://doi.org/10.1042/bj3220671