Biosynthesis, release and degradation of the novel endogenous cannabimimetic metabolite 2-arachidonoylglycerol in mouse neuroblastoma cells
Biosynthesis, release and degradation of the novel endogenous cannabimimetic metabolite 2-arachidonoylglycerol in mouse neuroblastoma cells
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DOI：
10.1042/bj3220671
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发表时间：
1997-03-01
影响因子：
4.1
通讯作者：
DiMarzo, V
中科院分区：
文献类型：
--
作者：
Bisogno, T;Sepe, N;DiMarzo, V
单酰基甘油2-花生四烯酰甘油（2-AG）最近已被认为是一种可能的内源性激动剂在大麻素受体在脑和外周组织。在这里，我们报告了一个广泛使用的神经元细胞模型，小鼠N（18）TG（2）神经母细胞瘤细胞，其中含有CB 1大麻素受体，也生物合成，释放和降解2-AG。用离子霉素（1-5 μ M）刺激用[H-3]花生四烯酸（[H-3]AA）预标记的完整细胞，导致形成高水平的放射性组分，其在TLC和HPLC分析中具有与2-AG合成标准品相同的色谱行为。这种代谢产物的量在未刺激的细胞中可以忽略不计，并且在Ca 2+螯合剂EGTA存在下刺激的细胞中大大降低。纯化的组分通过以下方法进一步表征为2-AG：（1）用少根根霉脂肪酶消化，产生放射性标记的AA;（2）气相色谱-MS分析;和（3）在Berate浸渍板上的TLC分析。约当含有0.1%BSA时，发现由刺激的细胞产生的20%的2-AG被释放到孵育培养基中。N1，TG，细胞的亚细胞级分显示含有催化合成[H-3]2-AG水解为[H-3]AA的酶活性或活性。还发现细胞匀浆将合成的[H-3]sn-1-酰基-2-花生四烯酰甘油（AcAGs）转化为[H-3]2-AG，表明2-AG可能来自AcAG水解。与离子霉素刺激相比，用外源性磷脂酶C处理细胞，而不是用磷脂酶D或A（2），导致更高的2-AG和AcAG形成。然而，在离子霉素刺激前10分钟用磷脂酶A（2）处理细胞，导致2-AG和AcAG水平比单独使用离子霉素增强2.5-3倍，而用磷脂酶C抑制剂硫酸新霉素预孵育不能抑制离子霉素对2-AG和AcAG水平的影响。这些结果表明，Ca 2+诱导的2-AG的形成通过AcAGs的中介作用进行，但不一定通过磷脂酶C激活。通过首次显示神经元细胞中2-AG的失活和Ca 2+依赖性生物合成和释放的分子机制的存在，本文支持了这一假设，即大麻拟单酰甘油可能是一种生理神经模块。
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 N(18)TG(2) neuroblastoma cells, which contain the CB1 cannabinoid receptor, also biosynthesize, release and degrade 2-AG. Stimulation with ionomycin (1-5 mu M) of intact cells prelabelled with [H-3]arachidonic acid ([H-3]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 berate-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 N,,TG, cells were shown to contain enzymic activity or activities catalysing the hydrolysis of synthetic [H-3]2-AG to [H-3]AA. Cell homogenates were also found to convert synthetic [H-3]sn-1-acyl-2-arachidonoylglycerols (AcAGs) into [H-3]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 A(2), led to a much higher formation of 2-AG and AcAGs. However, treatment of cells with phospholipase A(2) 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 neuromodular.