Toward a molecular and bioanalytical understanding of endogenous cannabinoid recycling and biosynthesis
Abstract
The endocannabinoids anandamide and 2-arachidonyl glycerol mimic the actions of Δ9-tetrahydrocannabinol, the active ingredient in marijuana. Recently the endocannabinoid system has been receiving much attention due to therapeutic potential. There have been many advances characterizing endocannabinoid biosynthesis, uptake and intracellular metabolism, however these processes are still not well understood. The aims of this work are (1) to investigate endocannabinoid recycling in addition to the role of calcium in endocannabinoid uptake and biosynthesis, (2) to explore endocannabinoid recycling and biosynthesis in a neuronal context, (3) to develop a sensitive and quantitative method for endocannabinoid detection using mass spectrometry, (4) to characterize the endocannabinoid lipid metabolite pool. I hypothesized that anandamide and 2-arachidonyl glycerol are synthesized in and released from lipid raft/caveolae membrane microdomains. Disruption of the lipid raft organization in RBL-2H3 cells attenuated endocannabinoid synthesis and release in response to the ionomycin treatment. I established that anandamide may be recycled by the cell to form new anandamide and 2-arachidonyl glycerol molecules. Furthermore, I determined that extracellular calcium is necessary for AEA and 2-arachidonyl glycerol biosynthesis because release of intracellular calcium stores alone does not promote endocannabinoid biosynthesis. I also discovered that neuronal cells CAD and MES 23.5 may utilize a different mechanism for endocannabinoid biosynthesis compared to RBL-2H3 cells. I examined the role of calcium as a “switch” to activate the synthesis of anandamide and simultaneously reduce uptake. Anandamide uptake was reduced in the presence of calcium, implying a mechanism indicative of calcium-modulated activation of synthesis and simultaneous termination of uptake. To enhance the ability to characterize endocannabinoid biosynthesis, I developed a liquid chromatography mass spectrometry method for endocannabinoid detection. By coordinating silver cations to the arachidonate backbone of anandamide and 2-arachidonyl glycerol, I was able to achieve an on-column detection limit of 15 fmoles for each species, a value consistent with published reports. I exploited the method to determine the lipid metabolite pool that contained the arachidonate backbone of anandamide. I detected a population of anandamide-derived molecules and discovered that anandamide-derived lipid pools were distinct from those formed from exogenous arachidonic acid. Furthermore, from the list of AEA metabolites, I selected and characterized 1- O-arachidonyl-sn-glycero-3-phosphocholine, a lipid that may serve as a precursor in the biosynthesis of anandamide.
Degree
Ph.D.
Advisors
Barker, Purdue University.
Subject Area
Biochemistry
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