Identification and characterization of the leech CNS cannabinoid receptor: coupling to nitric oxide release

The present study demonstrates that stereoselective binding sites for anandamide, a naturally occurring cannabinoid substance, can be found in leech (Theromyzon tessulatum and Hirudo medicinalis) central nervous system. The anandamide binding site is monophasic and of high affinity exhibiting a Kd of approximately 32 nM with a Bmax of 550 fmol/mg protein in both animals. These sites are highly select as demonstrated by the inability of other types of signaling molecules to displace [3H]anandamide. Furthermore, this binding site is coupled to nitric oxide release. A deduced amino acid sequence (153 residues) analysis from a 480 pb amplified RT-PCR fragment cDNA exhibits a 49.3% and 47.2% sequence identity with human and rat cannabinoid receptors (CB1R), respectively. Thus, the leech cannabinoid receptor may be a G-protein coupled receptor with seven transmembrane domains as in CB1R. Moreover, this sequence exhibits highly conserved regions, particularly in the putative transmembrane domains 1 and 2. The presence of a cannabinoid receptor in these organisms indicates that this signaling system has been conserved during evolution.