The endocannabinoids, anandamide (AEA) and 2-arachidonoylglycerol (2-AG), are the major signaling molecules for the cannabinoid receptors CB1 and CB2. All neurotransmitters require an efficient mechanism to terminate their action at the receptor. Water-soluble neurotransmitters are removed from the synapse by specific plasma transmembane transporters and they then diffuse unassisted within the cell to an enzyme for breakdown. The hypothesis of this proposal is that, owing to their neutral lipid structure the inactivation of AEA and 2-AG occurs by a unique pathway. It is proposed that this inactivation occurs by two steps: 1) Passive diffusion through the plasma membrane, independently of a transmembrane transporter, and 2) intracellular trafficking with chaperone proteins to their inactivating enzymes. To examine whether AEA and 2-AG can passively diffuse across the membrane, synthetic lipid vesicles (liposomes) will be employed with internalized FAAH (fatty acid amide hydrolase) or MAGL (monoacylglycerol lipase). These inactivating enzymes maintain the outward/inward gradient that drives the uptake. This model system will also be valuable in determining if simple diffusion demonstrates saturation kinetics, owing to an unstirred water layer, and if membrane composition regulates uptake. The latter will be addressed by comparing uptake in liposomes with and without lipid rafts. Of the possible mechanisms for the movement of AEA and 2-AG from the plasma membrane to their inactivating enzymes in the cell: unassisted simple diffusion, endocytosis, or trafficking by a chaperone protein, the latter appears most plausible. Accordingly, these proposed studies will examine endogenous levels of endocannabinoids trafficking proteins in cells and will elucidate the role of these proteins by comparing endocannabinoid metabolism rates and cellular uptake when the proteins are up-regulated by transfection and down-regulated by RNA interference or specific inhibitors. From a health related viewpoint, understanding the mechanism of endocannabinoid inactivation may lead to drug targets for addiction, mood disorders, pain and inflammation, cognition, and appetite regulation.

Public Health Relevance

Maintenance of the levels of the two endocannabinoid neurotransmitters, anandamide and 2-AG, is required for normal physiological functioning in humans. The steps by which these compounds are inactivated in the body are the focus of this proposal and will lead to new targets for pharmaceutical drugs. Since anandamide and 2-AG signaling molecules are involved in such diverse functions as reinforcement, mood, memory, appetite, pain and movement;drugs that prevent their inactivation may be good for treating drug addiction, depression, compulsive behaviors, neuropathic pain and movement disorders.

Agency
National Institute of Health (NIH)
Institute
National Institute on Drug Abuse (NIDA)
Type
Research Project (R01)
Project #
5R01DA016419-07
Application #
8247052
Study Section
Molecular Neuropharmacology and Signaling Study Section (MNPS)
Program Officer
Hillery, Paul
Project Start
2003-01-01
Project End
2014-03-31
Budget Start
2012-04-01
Budget End
2014-03-31
Support Year
7
Fiscal Year
2012
Total Cost
$260,538
Indirect Cost
$92,485
Name
State University New York Stony Brook
Department
Biochemistry
Type
Schools of Medicine
DUNS #
804878247
City
Stony Brook
State
NY
Country
United States
Zip Code
11794
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