Fowler Christopher J, Jonsson Kent-Olov, Andersson Anna, Juntunen Juha, Järvinen Tomi, Vandevoorde Séverine, Lambert Didier M, Jerman Jeffrey C, Smart Darren
Department of Pharmacology and Clinical Neuroscience, Umeå University, SE-90187 Umeå, Sweden.
Biochem Pharmacol. 2003 Sep 1;66(5):757-67. doi: 10.1016/s0006-2952(03)00392-7.
It has previously been shown that the endocannabinoids anandamide and 2-arachidonoylglycerol (2-AG) inhibit the proliferation of C6 glioma cells in a manner that can be prevented by a combination of capsazepine (Caps) and cannabinoid (CB) receptor antagonists. It is not clear whether the effect of 2-AG is due to the compound itself, due to the rearrangement to form 1-arachidonoylglycerol (1-AG) or due to a metabolite. Here, it was found that the effects of 2-AG can be mimicked with 1-AG, both in terms of its potency and sensitivity to antagonism by Caps and CB receptor antagonists. In order to determine whether the effect of Caps could be ascribed to actions upon vanilloid receptors, the effect of a more selective vanilloid receptor antagonist, SB366791 was investigated. This compound inhibited capsaicin-induced Ca(2+) influx into rVR1-HEK293 cells with a pK(B) value of 6.8+/-0.3. The combination of SB366791 and CB receptor antagonists reduced the antiproliferative effect of 1-AG, confirming a vanilloid receptor component in its action. 1-AG, however, showed no direct effect on Ca(2+) influx into rVR1-HEK293 cells indicative of an indirect effect upon vanilloid receptors. Identification of the mechanism involved was hampered by a large inter-experimental variation in the sensitivity of the cells to the antiproliferative effects of 1-AG. A variation was also seen with anandamide, which was not a solubility issue, since its water soluble phosphate ester showed the same variability. In contrast, the sensitivity to methanandamide, which was not sensitive to antagonism by the combination of Caps and CB receptor antagonists, but has similar physicochemical properties to anandamide, did not vary between experiments. This variation greatly reduces the utility of these cells as a model system for the study of the antiproliferative effects of anandamide. Nevertheless, it was possible to conclude that the antiproliferative effects of anandamide were not solely mediated by either its hydrolysis to produce arachidonic acid or its CB receptor-mediated activation of phospholipase A(2) since palmitoyltrifluoromethyl ketone did not prevent the response to anandamide. The same result was seen with the fatty acid amide hydrolase inhibitor palmitoylethylamide. Increasing intracellular arachidonic acid by administration of arachidonic acid methyl ester did not affect cell proliferation, and the modest antiproliferative effect of umbelliferyl arachidonate was not prevented by a combination of Caps and CB receptor antagonists.
先前的研究表明，内源性大麻素花生四烯酸乙醇胺和2-花生四烯酸甘油酯（2-AG）以一种可被辣椒素（Caps）和大麻素（CB）受体拮抗剂联合阻断的方式抑制C6胶质瘤细胞的增殖。目前尚不清楚2-AG的作用是由于该化合物本身、重排形成1-花生四烯酸甘油酯（1-AG）还是由于一种代谢产物。在此，研究发现1-AG在效力及其对Caps和CB受体拮抗剂拮抗作用的敏感性方面都能模拟2-AG的作用。为了确定Caps的作用是否可归因于对香草酸受体的作用，研究了一种更具选择性的香草酸受体拮抗剂SB366791的作用。该化合物抑制辣椒素诱导的Ca(2+)流入rVR1-HEK293细胞，其pK(B)值为6.8±0.3。SB366791和CB受体拮抗剂的联合使用降低了1-AG的抗增殖作用，证实了其作用中存在香草酸受体成分。然而，1-AG对Ca(2+)流入rVR1-HEK293细胞没有直接影响，表明其对香草酸受体有间接作用。由于细胞对1-AG抗增殖作用敏感性的实验间差异较大，阻碍了相关作用机制的确定。花生四烯酸乙醇胺也存在差异，这不是溶解度问题，因为其水溶性磷酸酯也表现出相同的变异性。相比之下，对甲氧基花生四烯酸乙醇胺的敏感性在不同实验之间没有变化，甲氧基花生四烯酸乙醇胺对Caps和CB受体拮抗剂的联合拮抗不敏感，但具有与花生四烯酸乙醇胺相似的理化性质。这种差异极大地降低了这些细胞作为研究花生四烯酸乙醇胺抗增殖作用模型系统的实用性。然而，可以得出结论，花生四烯酸乙醇胺的抗增殖作用并非仅由其水解产生花生四烯酸或其CB受体介导的磷脂酶A(2)激活所介导，因为棕榈酰三氟甲基酮并不能阻止对花生四烯酸乙醇胺的反应。脂肪酸酰胺水解酶抑制剂棕榈酰乙酰胺也得到了相同的结果。通过给予花生四烯酸甲酯增加细胞内花生四烯酸并不影响细胞增殖，并且辣椒素和CB受体拮抗剂的联合使用并不能阻止伞形酮花生四烯酸酯的适度抗增殖作用。
