Navia-Paldanius Dina, Aaltonen Niina, Lehtonen Marko, Savinainen Juha R, Taschler Ulrike, Radner Franz P W, Zimmermann Robert, Laitinen Jarmo T
School of Medicine, Institute of Biomedicine/Physiology, University of Eastern Finland, P.O. Box 1627, 70211 Kuopio, Finland; School of Pharmacy, University of Eastern Finland, P.O. Box 1627, 70211 Kuopio, Finland.
School of Pharmacy, University of Eastern Finland, P.O. Box 1627, 70211 Kuopio, Finland.
Eur J Pharm Sci. 2015 Sep 18;77:180-8. doi: 10.1016/j.ejps.2015.06.005. Epub 2015 Jun 9.
In mammalian brain, monoacylglycerol lipase (MAGL) is the primary enzyme responsible for terminating signaling function of the endocannabinoid 2-arachidonoylglycerol (2-AG). Previous in vivo studies with mice indicate that both genetic and chronic pharmacological inactivation of MAGL result in 8-30-fold increase of 2-AG concentration in the brain, causing desensitization and downregulation of cannabinoid CB1 receptor (CB1R) activity, leading to functional and behavioral tolerance. However, direct evidence for reduced CB1R activity in the brain is lacking. In this study, we used functional autoradiography to assess basal and agonist-stimulated CB1R-dependent Gi/o protein activity in multiple brain regions of MAGL-KO mice in comparison to their wild-type (WT) littermates. In addition, the role of endogenous cannabinoids in basal CB1R signaling was assessed after comprehensive pharmacological blockade of 2-AG hydrolysis by determining the contents of endocannabinoids (eCBs) in WT and MAGL-KO brain tissues by LC/MS/MS technology. To show whether lack of MAGL cause compensatory alterations in the serine hydrolase activity, we compared serine hydrolase pattern of WT and MAGL-KO using activity-based protein profiling. Consistent with studies using chronic pharmacological MAGL inactivation in vivo, we observed a statistically significant decrease of CB1R-Gi/o signaling in most of the studied brain regions. In MAGL-KO brain sections, elevated 2-AG levels were mirrored to heightened basal CB1R-dependent Gi/o-activity, as well as, dampened agonist-evoked responses in several brain regions. The non-selective serine hydrolase inhibitor methylarachidonoylfluorophosphonate (MAFP) was able to significantly elevate 2-AG levels in brain sections of MAGL-KO mice, indicating that additional serine hydrolases possess 2-AG hydrolytic activity in MAGL-KO brain sections.
在哺乳动物大脑中，单酰甘油脂肪酶（MAGL）是负责终止内源性大麻素2-花生四烯酸甘油酯（2-AG）信号功能的主要酶。先前对小鼠的体内研究表明，MAGL的基因失活和慢性药理失活都会导致大脑中2-AG浓度增加8至30倍，引起大麻素CB1受体（CB1R）活性的脱敏和下调，导致功能和行为耐受性。然而，大脑中CB1R活性降低的直接证据仍然缺乏。在本研究中，我们使用功能放射自显影术来评估MAGL基因敲除（KO）小鼠与其野生型（WT）同窝小鼠相比，多个脑区中基础和激动剂刺激的CB1R依赖性Gi/o蛋白活性。此外，通过LC/MS/MS技术测定WT和MAGL-KO脑组织中内源性大麻素（eCBs）的含量，在全面药理学阻断2-AG水解后，评估内源性大麻素在基础CB1R信号传导中的作用。为了显示MAGL的缺失是否会导致丝氨酸水解酶活性的代偿性改变，我们使用基于活性的蛋白质谱分析比较了WT和MAGL-KO的丝氨酸水解酶模式。与体内使用慢性药理学MAGL失活的研究一致，我们观察到在大多数研究的脑区中，CB1R-Gi/o信号有统计学意义的下降。在MAGL-KO脑切片中，升高的2-AG水平反映为基础CB1R依赖性Gi/o活性增强，以及几个脑区中激动剂诱发反应减弱。非选择性丝氨酸水解酶抑制剂甲基花生四烯酰氟磷酸酯（MAFP）能够显著提高MAGL-KO小鼠脑切片中的2-AG水平，表明在MAGL-KO脑切片中，其他丝氨酸水解酶具有2-AG水解活性。
