Evidence against the presence of an anandamide transporter.
On the basis of temperature dependency, saturability, selective inhibition, and substrate specificity, it has been proposed that an anandamide transporter exists. However, all of these studies have examined anandamide accumulation at long time points when downstream effects such as metabolism and intracellular sequestration are operative. In the current study, we have investigated the initial rates (<1 min) of anandamide accumulation in neuroblastoma and astrocytoma cells in culture and have determined that uptake is not saturable with increasing concentrations of anandamide. However, anandamide hydrolysis, after uptake in neuroblastoma cells, was saturable at steady-state time points (5 min), suggesting that fatty acid amide hydrolase (FAAH) may be responsible for observed saturation of uptake at long time points. In general, arvanil, olvanil, and N-(4-hydroxyphenyl)arachidonylamide (AM404) have been characterized as transport inhibitors in studies using long incubations. However, we found these "transport inhibitors" did not inhibit anandamide uptake in neuroblastoma and astrocytoma cells at short time points (40 sec or less). Furthermore, we confirmed that these inhibitors in vitro were actually inhibitors of FAAH. Therefore, the likely mechanism by which the transport inhibitors raise anandamide levels to exert pharmacological effects is by inhibiting FAAH, and they should be reevaluated in this context. Immunofluorescence has indicated that FAAH staining resides mainly on intracellular membranes of neuroblastoma cells, and this finding is consistent with our observed kinetics of anandamide hydrolysis. In summary, these data suggest that anandamide uptake is a process of simple diffusion. This process is driven by metabolism and other downstream events, rather than by a specific membrane-associated anandamide carrier.
Glaser
Sherrye T
ST
Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY 11794, USA.
Abumrad
Nada A
NA
Fatade
Folayan
F
Kaczocha
Martin
M
Studholme
Keith M
KM
Deutsch
Dale G
DG
D001095:Arachidonic Acids / Q000493:pharmacokinetics*; D001254:Astrocytoma; D001692:Biological Transport / Q000187:drug effects; D002186:Cannabinoids / Q000493:pharmacokinetics; D002211:Capsaicin / Q000031:analogs & derivatives* / Q000494:pharmacology; D002352:Carrier Proteins / Q000378:metabolism*; D063388:Endocannabinoids; D006801:Humans; D007150:Immunohistochemistry; D007700:Kinetics; D009447:Neuroblastoma; D053284:Polyunsaturated Alkamides; D014407:Tumor Cells, Cultured
D001095:Arachidonic Acids; D002186:Cannabinoids; D002352:Carrier Proteins; D063388:Endocannabinoids; D053284:Polyunsaturated Alkamides; C121153:arvanil; C054256:olvanil; D002211:Capsaicin; C078814:anandamide