The study of the naturally occurring """"""""cannabinoid"""""""" system in our bodies will help us elucidate any harmful and beneficial effects of marijuana use. The long term goal of this research is to understand how the actions of the endogenous cannabinomimetic (anandamide) are enzymatically terminated (by an amidase) in the nervous system and peripheral tissues. In this proposal, we plan to build upon our progress and capitalize upon recent advances in the area by others (cloning of the enzyme) to answer questions of fundamental importance.
The specific aims are to: 1) Identify the active-site and the transmembrane region (s) of anandamide amidase. The amino acids crucial for the active site of the enzyme (a putative serine) will be identified as well as those regions of the enzyme which may be responsible for insertion or association of the enzyme into the membrane. These studies will employ site-directed mutagenesis, radiolabeling of the active site with an irreversible inhibitor, and deletion mutagenesis. 2) Characterize the enzymatic properties of the amidase. We will answer the fundamental questions: (a) Does the enzyme catalyze the reverse reaction; i.e., the synthesis of anandamide from the condensation of ethanolamine and arachidonic acid; (b) What are the effects of some novel irreversible inhibitors upon the enzyme; and How many molecules of anandamide can the enzyme degrade per unit time (turnover number). These experiments will utilize purified amidase preparations from tissue or from the cloned amidase. 3) Expression of the amidase in tissue. These experiments are designed to determine the levels of the enzyme and its mRNA in organs where the cannabinoid receptors (CB1 and CB2) have been shown to occur. In addition, any variants of the mRNA that are detected will be analyzed by sequencing. In situ hybridization experiments will be undertaken to determine the cell specific expression of the amidase mRNA in some selected organs including uterus, kidney and brain.
|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|
|Kaczocha, Martin; Glaser, Sherrye T; Deutsch, Dale G (2009) Identification of intracellular carriers for the endocannabinoid anandamide. Proc Natl Acad Sci U S A 106:6375-80|
|Hermann, Anita; Kaczocha, Martin; Deutsch, Dale G (2006) 2-Arachidonoylglycerol (2-AG) membrane transport: history and outlook. AAPS J 8:E409-12|
|Kaczocha, Martin; Hermann, Anita; Glaser, Sherrye T et al. (2006) Anandamide uptake is consistent with rate-limited diffusion and is regulated by the degree of its hydrolysis by fatty acid amide hydrolase. J Biol Chem 281:9066-75|
|Glaser, Sherrye T; Deutsch, Dale G; Studholme, Keith M et al. (2005) Endocannabinoids in the intact retina: 3 H-anandamide uptake, fatty acid amide hydrolase immunoreactivity and hydrolysis of anandamide. Vis Neurosci 22:693-705|
|Glaser, Sherrye T; Abumrad, Nada A; Fatade, Folayan et al. (2003) Evidence against the presence of an anandamide transporter. Proc Natl Acad Sci U S A 100:4269-74|
|Deutsch, D G; Glaser, S T; Howell, J M et al. (2001) The cellular uptake of anandamide is coupled to its breakdown by fatty-acid amide hydrolase. J Biol Chem 276:6967-73|
|Puffenbarger, R A; Kapulina, O; Howell, J M et al. (2001) Characterization of the 5'-sequence of the mouse fatty acid amide hydrolase. Neurosci Lett 314:21-4|
|Yazulla, S; Studholme, K M; McIntosh, H H et al. (1999) Immunocytochemical localization of cannabinoid CB1 receptor and fatty acid amide hydrolase in rat retina. J Comp Neurol 415:80-90|
|Omeir, R L; Arreaza, G; Deutsch, D G (1999) Identification of two serine residues involved in catalysis by fatty acid amide hydrolase. Biochem Biophys Res Commun 264:316-20|
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