Witting Anke, Walter Lisa, Wacker Jennifer, Möller Thomas, Stella Nephi
Department of Pharmacology, University of Washington, Seattle, WA 98195-7280, USA.
Proc Natl Acad Sci U S A. 2004 Mar 2;101(9):3214-9. doi: 10.1073/pnas.0306707101. Epub 2004 Feb 19.
Endogenous cannabinoid ligands (endocannabinoids) produced by neurons, astrocytes, and microglial cells activate cannabinoid receptors, the molecular target for marijuana's bioactive ingredient Delta(9)-tetrahydrocannabinol. The molecular mechanism underlying the production of the most abundant endocannabinoid, 2-arachidonoylglycerol (2-AG), is unclear. A prevalent hypothesis proposes that activation of metabotropic receptors coupled to the phosphatidylinositol-specific phospholipase C and diacylglycerol (DG) lipase pathway will systematically lead to increases in 2-AG production. Here, we show that ATP increases 2-AG production by cultured microglial cells in a phosphatidylinositol-specific phospholipase C and DG lipase-dependent manner. However, efficacious activation of metabotropic P2Y purinergic receptors coupled to phosphatidylinositol-specific phospholipase C does not increase 2-AG production. This suggests that ionotropic, and not metabotropic, purinergic receptors control 2-AG production at an unexpected enzymatic step of its metabolic pathway. We show that activation of P2X(7) ionotropic receptors, which are highly permeable to calcium, is necessary and sufficient to increase 2-AG production in microglial cells. We also show that the sustained rise in intracellular calcium induced by activation of P2X(7) receptors directly increases DG lipase activity while inhibiting the activity of monoacylglycerol lipase, the enzyme that degrades 2-AG. This inverse sensitivity of DG lipase and monoacylglycerol lipase to calcium constitutes an original and efficient modality for sustained accumulation of 2-AG. Because prolonged increases in 2-AG amounts in brain parenchyma are thought to orchestrate neuroinflammation, the enzymatic steps involved in 2-AG synthesis and degradation by microglial cells constitute appealing targets for therapy aimed at controlling exacerbated neuroinflammation.
由神经元、星形胶质细胞和小胶质细胞产生的内源性大麻素配体（内源性大麻素）激活大麻素受体，大麻素受体是大麻生物活性成分Δ⁹-四氢大麻酚的分子靶点。最丰富的内源性大麻素2-花生四烯酸甘油酯（2-AG）产生的分子机制尚不清楚。一个普遍的假说是，与磷脂酰肌醇特异性磷脂酶C和二酰基甘油（DG）脂肪酶途径偶联的代谢型受体的激活将系统性地导致2-AG产量增加。在此，我们表明ATP以磷脂酰肌醇特异性磷脂酶C和DG脂肪酶依赖性方式增加培养的小胶质细胞中2-AG的产生。然而，与磷脂酰肌醇特异性磷脂酶C偶联的代谢型P2Y嘌呤能受体的有效激活并不会增加2-AG的产生。这表明离子型而非代谢型嘌呤能受体在其代谢途径的一个意想不到的酶促步骤中控制2-AG的产生。我们表明，对钙具有高度通透性的P2X₇离子型受体的激活对于增加小胶质细胞中2-AG的产生是必要且充分的。我们还表明，由P2X₇受体激活诱导的细胞内钙的持续升高直接增加DG脂肪酶活性，同时抑制降解2-AG的单酰基甘油脂肪酶的活性。DG脂肪酶和单酰基甘油脂肪酶对钙的这种反向敏感性构成了2-AG持续积累的一种原始而有效的方式。由于脑实质中2-AG量的长期增加被认为会引发神经炎症，小胶质细胞中参与2-AG合成和降解的酶促步骤构成了旨在控制加剧神经炎症的治疗的有吸引力的靶点。
