The Endocannabinoid Anandamide : Metabolism & Neuroprotection

Abstract

Marijuana is an extract of the Cannabis sativa and is the most used illegal drug in the world. Public debate centres upon the possible legalization of marijuana for recreational and therapeutic uses. DELTA-exp.9-Tetrahydrocannabinol (THC), the main psychoactive compound in marijuana, exerts its action by binding to G-protein-coupled membrane receptors, i.e. the cannabinoid CB1 and CB2 receptors. The biochemical and pharmacological actions of THC are mimicked by an endogenous lipid termed anandamide [N-arachidonoylethanolamine,5Z,8Z,11Z,14Z-eicosatetraenoyl-N-(2-hydroxyethyl)-amine]. Together with 2-arachidonoylglycerol, anandamide represents a class of endogenous ligands for CB receptors, i.e. the endocannabinoids. The CB receptors, endocannabinoids and proteins for theid biosynthesis and inactivation constitute the endocannabinoid system. The am of the research described in this thesis was to study a) the metabolism of anandamide in human cells and brain tissue, b) the interaction of its oxygenated metabolites with the proteins of the endocannabinoid system, and c) the role of the endocannabinoid system in in vivo neurodegeneration. nchapter 2 of this thesis, we have shown that the life span of anandamide in human brain and cells in culture is limited by a rapid inactivation process consisting of two steps, i.e. a translocation into the cell via a transporter protein, followed by a cleavage by the enzyme fatty acid amide hydrolase [FAAH] (Maccarrone et al., J. Biol. Chem., 1998, 273, 32332-32339). Endocannabinoids are not only inactivated via FAAH, but are also oxygenated by other enzymes such as lipoxygenases. In chapter 3, we have described the synthesis of various oxygenated metabolites of anandamide and 2-AG and characterized their cannabinoid properties (Van der Stelt et al., J. Med. Chem., 2002, 45, 3709-3720) The oxygenated metabolites were shown to competitively inhibit FAAH. Furthermore, depending on the position of the hydroxyl group in the metabolites, they were able to selectively bind to the CB1 or CB2 receptor. The metabolites did not interfere with anandamide uptake into the cell. These results indicate that small changes in the chemical structure of anandamide can lead to the generation of novel compounds which can specifically interact with each of the proteins of the endogenous cannabinoid system. With Molecular Dynamics simulations we have tried to identify the essential structural elements of anandamide, which are necessary for the interaction with the CB1 receptor. The endocannabinoid system is implicated in the regulation of various processes, such as learning, memory, pain and appetite. Its involvement in pathophysiological conditions is less clear, but we have shown in chapters 4, 5 and 6 that THC and anandamide can protect rat brain against neurodegenerative insults in vivo (Van der Stelt et al., J. of Neurosci., 2001, 21, 8765-8771; Van der Stelt et al., J. of Neurosci., 2001, 21, 6475-6479; Van der Stelt et al., Mol. Neurobiol., 2002, in press). We demonstrated that THC and anandamide could significantly reduce the volume of cytotoxic edema in neonatal rats in the acute phase after the intrastriatal injection with the Na+/K+-ATPase inhibitor ouabain. The effect of THC was antagonized by blocking the CB1 receptor with SR141716A, whereas the reduction in cellular swelling by anandamide was insensitive to this CB1 receptor antagonist. After seven days the infarct volume in THC- and anandamide-treated rats was ~40% and ~64%, respectively, smaller compared to control animals. When assessed at this time point, the neuroprotective actions of both anandamide and THC were abolished by SR141716A. A CB1-receptor mediated reduction in calcium influx and a reduced glutamate release are thought to be responsible for the neuroprotection in the hippocampus, striatum and cortex. In summary, the results reported in this thesis indicate that agonists of CB1 receptors might be useful to enhance the neurological outcome after acute brain damage. If the extent of neuronal damage is not too large, inhibitors of endocannabinoid clearance may prove to be suitable leads for drug development, because they have probably limited side effects, e.g. psychotropic activity