Guineensine is a novel inhibitor of endocannabinoid uptake showing cannabimimetic behavioral effects in BALB/c mice

From Phytocannabinoids to Cannabinoid Receptors and Endocannabinoids: Pleiotropic Physiological and Pathological Roles Through Complex Pharmacology

“…The primary characteristic of such a mechanism is that chemical probes can block it. Indeed, several inhibitors of AEA cellular uptake were developed and provided indirect evidence of the existence of the putative membrane transporter (188, 226,271,585,626,627,642). In one case, nanotechnologies and the AEA cytosolic binding site on TRPV1 channels were exploited to detect AEA entry into cells.…”

Endocannabinoid Transport Revisited

“…Any inhibitor of FAAH will thus also inhibit AEA uptake. The existence of cell lines that do not express FAAH but still show AEA uptake that can be inhibited by small organic molecules is fortunate and may help to discover more selective probes Nicolussi, Chicca, et al, 2014;Nicolussi, Viveros-Paredes, et al, 2014). More recently, different cytoplasmic AEA-binding proteins have been proposed to regulate or to be involved in AEA transport and to be the target of some of the reported AEA uptake inhibitors (Kaczocha, Vivieca, Sun, Glaser, & Deutsch, 2012;Maccarrone, Dainese, & Oddi, 2010).…”

Assay of Endocannabinoid Uptake

“…2 . This assay allows to characterize differential EC cellular uptake inhibition in U937 cells [ 7 ], to investigate the profi les of FAAH and MAGL inhibitors [ 7 ]], suitable and to characterize new selective AEA uptake inhibitors like guineensine [ 20 ], or highly potent inhibitors of FAAH [ 4 ]. …”

“…However, each process can be inhibited independently, thus leading to EtNH 2 curve shifts that reveal an apparent selectivity for AEA cellular uptake (e.g., with OMDM-2 ( Fig. 2a ) or guineensine [ 20 ], but see also [ 4 , 7 ] in comparison to FAAH inhibitors). After identifi cation of a new AEA uptake inhibitor, further potential targets such as intracellular shuttles AEA hydrolysis assay in U937 cell homogenates, performed as previously described [ 4 , 19 ].…”

“…A bottom phase with CHCl 3 and lipophilic substances such as the arachidonic acid, phospholipids, 2-AG, or AEA; a precipitated phase of proteins in the middle; and an upper aqueous phase with MeOH, H 2 O, and either free glycerol or ethanolamine, depending on the assay. After centrifugation at maximal speed , and guineensine in separate assays, as previously described [ 20 ]. 0.5 × 10 6 U937 cells or cell homogenates, respectively, were used per sample.…”