The effects of endocannabinoids on human, mouse and rabbit bone cells were investigated.  At high concentrations anandamide and 2-arachidonyl glycerol (2-AG) inhibited human osteoclast formation with no effects at lower concentrations. The inhibition was not attenuated by antagonists for the CB₁, CB₂ or TRPV1 receptors, indicating a non-receptor mediated effect.  Conversely, anandamide and 2-AG increased mouse osteoclast formation.  The effect of anandamide was enhanced in cells from fatty acid amide hydrolase (FAAH)-null mice and abolished in cells from CB_1/2 knockout mice.  The effect of 2-AG was not eliminated in CB_1/2 knockout cells, indicating a non-CB₁/CB₂ action.  The CB₁ antagonist, AM251, and the CB₂ antagonist, AM630, both inhibited mouse osteoclast formation.  These effects were not rescued in the CB_1/2-knockout mouse cells.  Both anandamide and 2-AG stimulated actin ring formation and osteoclast activity in human and rabbit osteoclast.  This was prevented in the presence of AM630 but not AM251, indicating a CB₂-mediated response.  The endocannabinoids and the cannabinoid receptor antagonists do not have a regulatory action on osteoblast activity. The effects of the novel cyclooxygenase-2 (COX-2) metabolite of 2-AG, prostaglandin E₂-glycerol ester (PGE₂-G), on human osteoclasts were examined.  Treatment with PGE₂-G inhibited formation and ERK phosphorylation of human osteoclasts.  These effects were attenuated by a selective EP₄ antagonist and mimicked by PGE₂ alone, indicating that PGF₂-G is rapidly metabolised into PGE₂ in human osteoclast cultures.  However, PGE₂-G treatment elevated intracellular calcium levels in human osteoclasts, through a phospholipase C (PLC)- and IP₃- dependent mechanism, indicative of a G-protein coupled receptor effect.  This was not mimicked by PGE₂, or prevented by the EP₄ antagonist, but blocked by a putative PGE₂-G receptor antagonist, PDA-94 indicating that PDA-94 may be a PGE₂-G receptor antagonist.

Bioactive compounds from Cannabis sativa have been used for millennia to alleviate the symptoms of a range of diseases. The physiological basis of effects such as analgesia, stimulation of hunger and reduction of inflammation was established in the late 20th century with the discovery of cannabinoid receptors but efforts to synthesise safe and potent drugs targeting these proteins have so far failed. The major barrier to research in this area is the instability of the receptors outside of biological settings, rendering elucidation of the binding sites by traditional means difficult. Certain small molecules can interact with the cannabinoid type 1 receptor (CB1) at locations distinct to the primary ligand docking site. Such allosteric modulation of the endocannabinoid system offers significant advantages over using orthosteric drugs and in this research a range of indole based structures were synthesised and tested in an attempt to improve the activity and drug-like nature of a lead compound. A partial structure-activity relationship was established, including the description of the most potent allosteric enhancer of CB1 so far reported. Efforts were also undertaken to investigate the allosteric binding environments using photoactivatable ligands based on a CB1 inhibitor. In combination with mutation studies and computer modelling this technique could allow the rational design of allosteric modulators, a task which is not trivial at present. Two photoactivatable compounds were synthesised and shown to interact with the receptor, with a method for isolating covalently labelled peptide fragments from other biomolecules demonstrated using “click chemistry” and a modified Wang resin. This work may find application in future investigations aiming to produce allosteric pharmaceuticals targeting CB1. Furthermore, the techniques described may be applied to study the binding site of a recently described allosteric endocannabinoid or could potentially be adapted to look at secondary binding domains in other G protein-coupled receptors.

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 93-112).

Thesis (Ph.D.)--Aberdeen University, 2008. / Title from web page (viewed on July 20, 2009). Includes bibliographical references.

Thesis (Ph.D.)--Aberdeen University, 2009. / Title from web page (viewed on Oct. 8, 2009). Includes bibliographical references.

Endocannabinoids are derivatives of arachidonate-based lipids that activate a network of signaling pathways in eukaryotes. Specifically, in mammals, a C20 N-acylethanolamine (NAE) or anandamide is a ligand for cannabinoid receptor (a G protein-coupled receptor) and acts as neuromodulator for a variety of physiological processes, including pain sensation and protection against stroke. Interestingly, while only C12-C18 NAEs occur in seed plants, anandamide and its membrane lipid precursor, N-arachidonylphosphatidylethanolamine were identified in a nonseed early land plant, Physcomitrella patens. Selective lipidomics also revealed other 20C-derived NAEs including eicosapentaenoyl ethanolamide (EPEA or NAE20:5) that were previously unidentified in angiosperms. Furthermore, putative genes that likely encode for enzymes that participate in NAE metabolism were also identified in P. patens and were heterologously expressed. Fatty acid amide hydrolase (FAAH), an enzyme that catabolizes NAEs showed preferential activity towards NAE20:4 at pH 8.0 and 30 C. While biological implications of anandamide and its metabolic pathway in mosses are still under investigation, we show that both AEA and EPEA and their corresponding fatty acids act as potent inhibitors of protonema and gametophore growth in a dose-dependent manner and similar to ABA. Furthermore, short-term exposure to AEA or ABA inhibited tip growth that was associated with depolymerization of F-actin but not microtubule organization, which remained unaffected. While these data indicate that the occurrence of NAEs and their metabolism is evolutionarily conserved and composition in early land plants is unique, if the functions of AEA are mediated by a G-protein coupled receptor, similar to ABA, and endocannabinoid signaling in mammals, remains to be elucidated.

No description available.

The endocannabinoid system is composed of endocannabinoids (eCBs), their cognate receptors, and their biosynthetic and catabolic enzymes. Inhibition of serine hydrolases (catabolic enzymes), such as carboxylesterases (CES), might result in the accumulation of eCBs. eCBs, such as 2-arachidonoylglycerol (2-AG), have been shown to increase or reduce inflammation via engagement with cannabinoid receptors on immune cells. This research focuses on exploring the ability of eCBs and their metabolizing enzymes to regulate inflammation. First, a negative feedback mechanism between inflammation and the eCB system was examined by identifying serine hydrolases inhibited by lipopolysaccharide (LPS) stimulation in mice. Ces2g activity was inhibited and Il6 levels were induced in the murine spleen, suggesting a role for this enzyme in an inflammatory response, possibly to limit inflammation. IL-6 did not influence 2-AG hydrolytic activity in human peripheral blood mononuclear cells (PBMCs), but monocytic MAGL was shown to be the predominant 2-AG hydrolytic enzyme in these cells. To investigate a separate mechanism by which serine hydrolases and eCBs may regulate immune responses, mice were treated with chlorpyrifos (CPF), a pesticide known to inhibit serine hydrolases, at doses that do not inhibit acetylcholinesterase in the nervous system. This research is focused on lung tissue since epidemiologic studies have linked pesticide exposures to respiratory diseases. At low doses, Ces1c (adult and neonatal mice) and Ces1d (neonatal mice) were markedly inhibited by CPF (2.5 mg/kg, 7 d, PO). Stimulation with LPS (1.25 mg/kg, IP) following the final CPF dose produced minimal differences in lung immune responses to LPS. In follow up experiments utilizing wild-type and Ces1d-/- mice, a downregulation of Ces1c mRNA in adult Ces1d-/- mice corresponded to an upregulation of Tnfa mRNA in response to LPS in CPF-treated mice. Additionally, Ces1d was found to be expressed in murine alveolar macrophages, suggesting these cells could be used to study the role of CES1 in immunity. Overall, Ces enzymes appear to play a role in immune homeostasis either through a protective mechanism or a negative feedback mechanism to control inflammation.

Here we demonstrate that both exogenous and endogenous cannabinoids affect different aspects of learning and memory in the rat.  For example, the potent CB₁ receptor agonist, WIN-2 was able to delay-dependently impair short-term memory (STM) sparing reference memory (RM).  This demonstrates that it is the STM but  not RM processes that are more sensitive to the effects of cannabinoids. In addition, given that cannabinoids were able to hinder the recruitment of hippocampal firing correlates that are crucial for correct performance of a STM task, suppress hippocampal principal cell firing during the encoding phase of a STM task, reduce spontaneous bursting and disrupt synchronous firing of hippocampal principal cells respectively, confirm that they do alter the neurophysiology of the hippocampus.  These cannabinoid induced alterations in hippocampal neuronal activity may well explain the observed deficits across numerous other working memory (WM) and STM tasks. The results also revealed that cannabinoid-induced deficits in learning and memory are brought about due to an interaction between cannabinoid and cholinergic systems.  Although endocannabinoids failed to produce impairments in STM under normal physiological conditions, STM deficits were observed when anadamide levels were pharmacologically elevated beyond normal physiological levels.  Moreover, results demonstrate that the endocannabinoid system is involved in behavioural flexibility (i.e. reversal learning) and modulation of acquisition and/or consolidation of certain spatial elements that are necessary to perform an operant conditioning risk. Overall, the results in this thesis show that cannabinoid induced deficits in learning and memory are produced as a result of their direct effects on hippocampal processing.  The exact mechanisms that mediate these cannabinoid-induced deficits in memory are yet unclear and remain to be determined.

The elucidation of the binding of marijuana’s psychoactive compound, (-)-D9- tetrahydrocannabinol (THC), to specific membrane receptors, in the early 1990s, led to the identification of endogenous arachidonate-based lipids that activate cannabinoid receptors in mammals. While the metabolic and signaling pathway for these 20 carbon N-acylethanolamines (NAE) and their derivatives has been well characterized in mammals, thus far, only 12-18 carbon NAEs have been identified in plants and their metabolic pathway has been partly characterized. In plants, NAEs have been shown to modulate a number of physiological processes, including seed and seedling development and ability to respond to stress; however, the mechanisms by which they function remain to be elucidated. Our recent identification of a 20C NAE (arachidonylethanolamide) in moss provided us with an exciting possibility to identify receptor-mediated endocannabinoid signaling responses in plants that is akin to mammals. In this seminar, I will provide insights into the past, present and future aspects of plant endocannabinoid research.