Matias I, Bisogno T, Melck D, Vandenbulcke F, Verger-Bocquet M, De Petrocellis L, Sergheraert C, Breton C, Di Marzo V, Salzet M
Laboratoire d'Endocrinologie et immunité des Annélides UPRES-A CNRS 8017, SN3-USTL, 59655 Villeneuve d'Ascq, France.
Brain Res Mol Brain Res. 2001 Mar 5;87(2):145-59. doi: 10.1016/s0169-328x(00)00290-4.
In invertebrates, like Hydra and sea urchins, evidence for a functional cannabinoid system was described. The partial characterization of a putative CB1 cannabinoid receptor in the leech Hirudo medicinalis led us to investigate the presence of a complete endogenous cannabinoid system in this organism. By using gas chromatography-mass spectrometry, we demonstrate the presence of the endocannabinoids anandamide (N-arachidonoylethanolamine, 21.5+/-0.7 pmol/g) and 2-arachidonoyl-glycerol (147.4+/-42.7 pmol/g), and of the biosynthetic precursor of anandamide, N-arachidonylphosphatidyl-ethanolamine (16.5+/-3.3 pmol/g), in the leech central nervous system (CNS). Anandamide-related molecules such as N-palmitoylethanolamine (32.4+/-1.6 pmol/g) and N-linolenoylethanolamine (5.8 pmol/g) were also detected. We also found an anandamide amidase activity in the leech CNS cytosolic fraction with a maximal activity at pH 7 and little sensitivity to typical fatty acid amide hydrolase (FAAH) inhibitors. Using an antiserum directed against the amidase signature sequence, we focused on the identification and the localization of the leech amidase. Firstly, leech nervous system protein extract was subjected to Western blot analysis, which showed three immunoreactive bands at ca. approximately 42, approximately 46 and approximately 66 kDa. The former and latter bands were very faint and were also detected in whole homogenates from the coelenterate Hydra vulgaris, where the presence of CB1-like receptors, endocannabinoids and a FAAH-like activity was reported previously. Secondly, amidase immunocytochemical detection revealed numerous immunoreactive neurons in the CNS of three species of leeches. In addition, we observed that leech amidase-like immunoreactivity matches to a certain extent with CB1-like immunoreactivity. Finally, we also found that stimulation by anandamide of this receptor leads, as in mammals, to inhibition of cAMP formation, although this effect appeared to be occurring through the previously described anandamide-induced and CB1-mediated activation of nitric oxide release. Taken together, these results suggest the existence of a complete and functional cannabinoid system in leeches.
在无脊椎动物，如水螅和海胆中，已报道了功能性大麻素系统的证据。对医用水蛭中一种假定的CB1大麻素受体的部分特性分析，促使我们研究该生物体内完整内源性大麻素系统的存在情况。通过气相色谱 - 质谱联用技术，我们证明了水蛭中枢神经系统（CNS）中存在内源性大麻素花生四烯酸乙醇胺（N - 花生四烯酰乙醇胺，21.5±0.7 pmol/g）和2 - 花生四烯酰甘油（147.4±42.7 pmol/g），以及花生四烯酸乙醇胺的生物合成前体N - 花生四烯酰磷脂酰乙醇胺（16.5±3.3 pmol/g）。还检测到了与花生四烯酸乙醇胺相关的分子，如N - 棕榈酰乙醇胺（32.4±1.6 pmol/g）和N - 亚麻酸乙醇胺（5.8 pmol/g）。我们还在水蛭CNS胞质组分中发现了一种花生四烯酸乙醇胺酰胺酶活性，其在pH 7时活性最高，且对典型的脂肪酸酰胺水解酶（FAAH）抑制剂不敏感。使用针对酰胺酶特征序列的抗血清，我们专注于水蛭酰胺酶的鉴定和定位。首先，对水蛭神经系统蛋白提取物进行蛋白质印迹分析，结果显示在约42 kDa、约46 kDa和约66 kDa处有三条免疫反应带。前一条带和后一条带非常微弱，在腔肠动物普通水螅的全匀浆中也检测到了，此前已报道普通水螅中存在CB1样受体、内源性大麻素和FAAH样活性。其次，酰胺酶免疫细胞化学检测显示三种水蛭的CNS中有大量免疫反应性神经元。此外，我们观察到水蛭酰胺酶样免疫反应性在一定程度上与CB1样免疫反应性相匹配。最后，我们还发现，与哺乳动物一样，花生四烯酸乙醇胺对该受体的刺激会导致cAMP形成的抑制，尽管这种效应似乎是通过先前描述的花生四烯酸乙醇胺诱导的、CB1介导的一氧化氮释放激活而发生的。综上所述，这些结果表明水蛭中存在完整且功能性的大麻素系统。
