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

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.

Phosphatidic Acid as the Biosynthetic Precursor of the Endocannabinoid 2‐Arachidonoylglycerol in Intact Mouse Neuroblastoma Cells Stimulated with Ionomycin

“…Anandamide and 2-AG were synthesized as described previously (Bisogno et al, 1997;Di Marzo et al, 1998a). Phosphatidylethanol (PPE), methylarachidonoylfluorophosphonate (MAFP), and arachidonoyl trifluoromethyl ketone (ATFMK) were purchased from Biomol, U.S.A. sn-l-Oleoylglycerol-3-phosphate [lysophosphatidic acid (LPA) J was obtained from Fluka, Switzerland.…”

“…The [3H]AA-containing medium was then removed gently to avoid the detachment of cells from dishes and replaced with serum-free medium containing 2.5 p M ionomycin and 10 pA4 ATFMK [as an inhibitor of 2-AG hydrolysis (Di Marzo et al, 1998a)J. Cell stimulation was carried out for 20 min at 37"C, after which lipids were extracted from cells plus incubation medium and fractionated as described previously (Bisogno et al, 1997) by means of sequential silica gel column chromatography, TLC, and normal-phase HPLC.…”

“…The chemical nature of ['H]2-AG was confirmed by reverse-phase HPLC and gas chromatographic/mass spectrometric analyses of the tnmethylsilyl-ether derivative, both performed as described (Bisogno et al, 1997).…”

“…Apart from its several cannabimimetic actions, a possible role for 2-AG as an extracellular neuromodulator was strongly supported by the finding that the monoglyceride can be biosynthesized and released by intact neuronal cells in response to depolarizing stimuli, such as ionomycin stimulation, in a fashion dependent on extracellular CaZf (Bisogno et al, 1997;Stella et al, 1997). The Ca*+-dependent formation of 2-AG was also shown to occur in rat brain homogenates (Kondo et a]., 1998).…”

“…As a model for neuronal cells we have used the N18TG2 mouse neuroblastoma cell line, widely used for studies on endocannabinoid action and metabolism. When stimulated with ionomycin, intact N18TG2 cells produce and release 2-AG in a Ca2+-dependent manner and in levels more than fivefold higher than basal levels (Bisogno et al, 1997). Therefore, these cells can be used as an experimental model for the membrane depolarization-induced biosynthesis of extracellular, cannabimimetic 2-AG.…”