The overall hypothesis of this proposal is that anandamide (an endogenous ligand that binds to cannabinoid and vanilloid receptors) is taken up into cells by a mechanism of simple diffusion rather than, as currently believed, by an anandamide transporter. Examining approximately a dozen immortalized and primary cell lines reported in the literature to have an anandamide transporter, the specific aims of the proposal are: 1) to determine if anandamide uptake occurs by simple diffusion (non-saturable) or facilitated diffusion (saturable). These experiments will differ from other published reports in that uptake of anandamide will be studied at initial times (25 seconds). These initial rates will distinguish actual uptake from downstream events such as metabolism or intracellular sequestration, which may give the appearance of saturation and an anandamide carrier. 2) to investigate the role of the """"""""anandamide transport inhibitors"""""""" described in the literature. These """"""""anandamide transport inhibitors"""""""" will be tested as uptake competitors using initial uptake rates as opposed to the prevailing studies in the literature that employed times greater than one minute that measures net uptake. If our hypothesis is correct, none of these will inhibit transport but will, in fact, be shown to inhibitor FAAH. Therefore the approach being used by other investigators in the field to synthesize """"""""anandamide transport inhibitors"""""""" may be flawed from a theoretical viewpoint. 3) to elucidate the cellular distribution of FAAH with particular emphasis on its intracellular localization. If immunofluorescence indicates that FAAH staining resides mainly on intracellular membranes of neuroblastoma cells, it suggests that anandamide hydrolysis will appear as a downstream event that will be confirmed by the observed kinetics of anandamide hydrolysis in Specific Aims 1 and 2. In contrast to prevailing studies, this proposal will unequivocally determine if anandamide uptake is a process of simple diffusion. The clarification of the mechanism of anandamide inactivation is important for the development of therapeutics in areas such as drug addiction, mood, pain, fertility, and appetite regulation. ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute on Drug Abuse (NIDA)
Type
Research Project (R01)
Project #
1R01DA016419-01A1
Application #
6725067
Study Section
Molecular, Cellular and Developmental Neurosciences 2 (MDCN)
Program Officer
Hillery, Paul
Project Start
2004-04-01
Project End
2007-03-31
Budget Start
2004-04-01
Budget End
2005-03-31
Support Year
1
Fiscal Year
2004
Total Cost
$188,125
Indirect Cost
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
Elmes, Matthew W; Kaczocha, Martin; Berger, William T et al. (2015) Fatty acid-binding proteins (FABPs) are intracellular carriers for ?9-tetrahydrocannabinol (THC) and cannabidiol (CBD). J Biol Chem 290:8711-21
Sanson, BenoƮt; Wang, Tao; Sun, Jing et al. (2014) Crystallographic study of FABP5 as an intracellular endocannabinoid transporter. Acta Crystallogr D Biol Crystallogr 70:290-8
Kaczocha, Martin; Rebecchi, Mario J; Ralph, Brian P et al. (2014) Inhibition of fatty acid binding proteins elevates brain anandamide levels and produces analgesia. PLoS One 9:e94200
Leung, Kwannok; Elmes, Matthew W; Glaser, Sherrye T et al. (2013) Role of FAAH-like anandamide transporter in anandamide inactivation. PLoS One 8:e79355
Kaczocha, Martin; Vivieca, Stephanie; Sun, Jing et al. (2012) Fatty acid-binding proteins transport N-acylethanolamines to nuclear receptors and are targets of endocannabinoid transport inhibitors. J Biol Chem 287:3415-24
Kaczocha, Martin; Lin, Qingqing; Nelson, Lindsay D et al. (2012) Anandamide externally added to lipid vesicles containing trapped fatty acid amide hydrolase (FAAH) is readily hydrolyzed in a sterol-modulated fashion. ACS Chem Neurosci 3:364-8
Tsuboi, Kazuhito; Okamoto, Yasuo; Ikematsu, Natsuki et al. (2011) Enzymatic formation of N-acylethanolamines from N-acylethanolamine plasmalogen through N-acylphosphatidylethanolamine-hydrolyzing phospholipase D-dependent and -independent pathways. Biochim Biophys Acta 1811:565-77
Howlett, Allyn C; Reggio, Patricia H; Childers, Steven R et al. (2011) Endocannabinoid tone versus constitutive activity of cannabinoid receptors. Br J Pharmacol 163:1329-43
Kaczocha, Martin; Glaser, Sherrye T; Chae, Janiper et al. (2010) Lipid droplets are novel sites of N-acylethanolamine inactivation by fatty acid amide hydrolase-2. J Biol Chem 285:2796-806
Ulloa, Nadine M; Deutsch, Dale G (2010) Assessment of a spectrophotometric assay for monoacylglycerol lipase activity. AAPS J 12:197-201

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