Evidence for an endocannabinoid system in the central nervous system of the leech Hirudo medicinalis

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.