Fatty acid amide hydrolase (FAAH) is a widely conserved amidase in eukaryotes, best known for inactivating the signal of N-acylethanolamine (NAE) lipid mediators. In the plant Arabidopsis thaliana, FAAH-mediated hydrolysis of NAEs has been associated with numerous biological processes. Recently, the phylogenetic distribution of FAAH into two major branches (group I and II FAAHs) across angiosperms outside of Arabidopsis (and in other Brassicaceae), suggests a previously unrecognized complexity of this enzyme. Although A. thaliana has long been used to assess biological questions for plants, in this case it will fall short in understanding the significance of multiple FAAHs in other plant systems. Thus, in this study, I examined the role (s) of six FAAH isoforms in upland cotton (Gossypium hirsutum L.) and two FAAHs in the legume Medicago truncatula.

In the early nineties cannabinoid receptors, the main target for Δ9-tetrahydrocannabinol (THC), the psychoactive component of marijuana were identified. Shortly after their endogenous ligands, N-arachidonoylethanolamine (anandamide, AEA) and 2-diacylglycerol (2-AG) were characterized. The enzymes primarily responsible for catalysing the degradation of AEA and 2-AG are fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MGL) respectively. AEA is a member of the N-acylethanolamine (NAE) class of lipids, which depending on the acyl chain length and number of double bonds can act as ligands for a variety of biological targets. Exogenous cannabinoids have long been reported to have analgesic effects, however the clinical usefulness of such substances is limited by their psychoactive effects. Inhibition of endocannabinoid degradation would mean enhancing the therapeutic effects without producing these unwanted side effects. In order to succeed in developing such compounds the pharmacology of the enzymes responsible for the degradation of endocannabinoids has to be thoroughly understood. When the preclinical part of this thesis was planned, FAAH had been well characterized whereas little was known as to the pharmacology of MGL. A series of compounds were tested in this first study aiming to find MGL-selective compounds. Although no compounds showed selectivity for MGL over FAAH, several interesting agents affecting both enzymes were identified. In order to increase the knowledge concerning which patient group would benefit from such treatment strategies it is important to investigate in which pain states the endocannabinoids/NAEs are altered. Thus the general aim of the clinical part of this thesis was to investigate the levels of endocannabinoids/NAEs in the interstitium of the trapezius muscle in women suffering from chronic neck/shoulder pain (CNSP) and chronic wide spread pain (CWP) and in healthy pain-free controls. Furthermore for the CNSP the effect of training, which is a commonly recommended treatment for these patients, on the levels of endocannabinoids/NAEs was also investigated. Microdialysis technique in the trapezius muscle was used for sampling and masspectrometry was used for analysing. Two NAEs, N-palmitoylethanolamine (PEA) and N-stearoylethanolamine (SEA), could be repeatedly measured. The levels of these two lipids were significantly higher in CNSP compared to CON. The result showed also that PEA and SEA mobilize differently in CWP compared to both CNSP and CON. Taken together the results presented in thesis represent an early characterization of the pharmacology of MGL and provides novel information on NAEs in chronic muscle pain.

N-acyl ethanolamines (NAEs) are a class of lipids recently recognized as signaling molecules which are controlled, in part, by their degradation by fatty acid amide hydrolase (FAAH). On the basis of previous studies indicating increased NAE levels in a tobacco cell suspension-xylanase elicitor exposure system and the availability of FAAH mutants, overexpressor and knockout (OE and KO) genotypes in Arabidopsis thaliana, further roles of NAEs in A. thaliana plant defense was investigated. The commonly occurring urban antimicrobial contaminant triclosan (TCS) has been shown to suppress lipid signaling associated with plant defense responses. Thus, a second objective of this study was to determine if TCS exposure specifically interferes with NAE levels. No changes in steady state NAE profiles in A. thaliana-Pseudomonas syringae pv. syringae and A. thaliana-flagellin (bacterial peptide, flg22) challenge systems were seen despite evidence that defense responses were activated in these systems. There was a significant drop in enoyl-ACP reductase (ENR) enzyme activity, which catalyzes the last step in the fatty acid biosynthesis pathway in plants, on exposure of the seedlings to TCS at 10 ppm for 24 h and decreased reactive oxygen species (ROS) production due to flg22 in long term exposure of 0.1 ppm and short term exposure of 5 ppm. However, these responses were not accompanied by significant changes in steady state NAE profiles.

The lipoxygenase (LOX) pathway plays an important role in the oxidative metabolism of polyunsaturated N-acylethanolamines (PU-NAEs). The LOX pathway functions in conjugation with hydrolysis by fatty acid amide hydrolase (FAAH) and to produce oxidized NAEs during seed germination and early seedling development. When Arabidopsis seedlings were grown in low micromolar concentrations of lauroylethanolamide (NAE 12:0), growth retardation and elevated endogenous PU-NAE levels were observed due to the competitive inhibition of LOX by NAE 12:0. The elevated levels of endogenous PU-NAEs were more pronounced in genotypes with reduced NAE hydrolase capacity (faah knockouts), and less evident with overexpression of FAAH. Alterations in PU-NAE metabolism were studied in seedlings of various lox and FAAH mutants. The partitioning of PU-NAEs into oxylipin metabolites was exaggerated in the presence of exogenous linolenoylethanolamide (NAE18:3) and resulted in bleaching of cotyledons. The bleaching phenotype was restricted to a narrow developmental window (3-to-5 days after sowing), and was attributed to a reversible disruption of thylakoid membranes in chloroplasts. Biochemical and genetic evidence suggested that 9-hydro(pero)xy and 13-hydro(pero)xy octadecatrienoylethanolamides (9- and 13-NAE-H(P)OT), but not their corresponding hydro(pero)xy free fatty acids, induced cotyledon bleaching. The LOX-mediated metabolites of NAE18:3 shared some overlapping effects on seedling development with those of linoleoylethanolamide (NAE18:2) such as a reduction in seedling root growth. On the other hand, NAE18:3 oxylipin metabolites also exhibited distinct effects during seedling development such as the inhibition of photomorphogenesis. Biochemical and genetic evidence indicated that a LOX-mediated metabolite of NAE18:2, 9-hydro(pero)xy octadecadienoylethanolamide (9-NAE-H(P)OD), acted as a potent negative regulator of seedling root development, and this depended on an intact abscisic acid (ABA) signaling pathway. Synergistic inhibition of root elongation between 9-NAE-H(P)OD and ABA was restricted to a narrow developmental window (3-to-5 d after sowing) of seedling development. Genetic evidence with Arabidopsis mutants in ABA synthesis (aba1, aba2), perception (pyr1, pyl2, pyl4, pyl5, pyl8) and transcriptional regulation (abi3-1) suggested that negative regulation of growth by 9-NAE-H(P)OD likely was mediated through an increase in ABA synthesis, and this was confirmed biochemically. Induction of a secondary dormancy program in Arabidopsis seedlings by environmental stresses also requires an intact ABA signaling cascade, and our study has shown that this regulatory seedling program is dependent, in large part, on NAE oxylipin formation. Together, results presented here indicated that LOX-mediated metabolites of NAE18:3 and NAE18:2 in Arabidopsis represent a newly-discovered group of bioactive metabolites, and their accumulation during the embryo-to-seedling transition of plant development may act to synchronize seedling establishment with environmental cues.

N-Acylethanolamines (NAEs) are fatty acid derivatives in plants that negatively influence seedling growth. N-Lauroylethanolamine (NAE 12:0), one type of NAE, inhibits root length, increases radial swelling of root tips and reduces root hair numbers in a dose dependent manner in Arabidopis thaliana L. (ecotype Columbia). A forward genetics approach was employed by screening a population of T-DNA “activation-tagged” developed by the Salk Institute lines for NAE resistance to identify potential genes involved in NAE signaling events in Arabidopsis thaliana L. (ecotype Columbia). Seeds of the activation tagged lines were grown at 0, 25, 30, 50, 75 and 100 µM N-lauroylethanolamime (NAE 12:0). Ten plants which displayed NAE tolerance (NRA) seedling phenotypes, compared with wildtype (Columbia, Col-0) seedlings were identified. I focused on one mutant line, identified as NRA 25, where the tolerance to NAE 12:0 appears to be mediated by a single dominant, nuclear gene. Thermal asymmetric interlaced (TAIL) PCR identified the location of the T-DNA insert as 3.86 kbp upstream of the locus At1g68510. Quantitative PCR indicated that the transcript level corresponding to At1g68510 is upregulated approximately 20 fold in the mutant relative to wildtype. To determine whether the NAE tolerance in NRA 25 is associated with overexpression of At1g68510 I created overexpressing lines of At1g68510 with and without GFP fusions behind the 2X35S CaMV promoter. As predicted, results with overexpressing lines of At1g68510 also exhibited enhanced resistance to NAE when compared with the wildtype. Confocal images of the fusion proteins suggest that GFP-At1g68510 is concentrated in the nucleus and this was confirmed by counterstaining with 4', 6-Diamidino-2-phenylindol (DAPI). Futhermore, At1g68510 overexpressing lines and NRA 25 line also exhibited tolerance to abscisic acid (ABA) during seedling germination. The findings suggests that At1g68510 overexpression mediates seedling tolerance to both ABA and NAE, a mechanism independent of fatty acid amide hydrolase in which its overexpression leads to NAE resistance but hypersensitivity to ABA. The next steps are to identify the association of At1g68510 with specific genomic regions or interacting proteins that may be additional components of NAE signaling in plants.

N-Acylethanolamines (NAEs), although are ubiquitous in plants and animals the occurrence of endocannabinoid ligands and the corresponding cannabinoid receptors was limited to mammals. Interestingly, bryophytes, unlike seed plants possess arachidonic acid (AA, 20:4) and eicosapentaenoic acid (EPA, 20:5), which are fatty acid precursors for endocannabinoid ligands. Here, we show that the moss Physcomitrella patens contains ~24 and 7 % of AA and EPA, respectively. Using selective lipidomic profiling, we identified polyunsaturated NAEs, including N-arachidonoyl ethanolamide (anandamide/AEA/NAE 20:4) and N-eicosapentaenoyl ethanolamide (EPEA) and also their corresponding N-acyl-phosphatidylethanolamine (NAPE) precursors in various developmental stages of Physcomitrella. Quantification of various NAPE and NAE species indicated the abundance of unsaturated species over saturated. In all haploid developmental stages analyzed, NAE 20:4 levels contributed to ~ 30 % (~ 26 ng mg-1 lipid) of the total NAE while NAE 20:5 remained as a minor component (~ 5 %; ~ 4.5 ng mg-1 lipid). Exogenous application of AEA, EPEA and their corresponding fatty acid precursors (AA and EPA, respectively) inhibited the growth of gametophytes and protonemata in a dose-dependent manner. AEA has shown the exclusive effect on the F-actin dynamics at the apex of protonemal cells, which was similar to the effect of abscisic acid (ABA) on protonemal growth inhibition. Additionally, we identified moss ortholog for NAPE-hydrolyzing phospholipase D (NAPE-PLD) enzyme that was responsive to exogenous ABA. Putative PpNAPE-PLD was expressed in E. coli for further characterization. Our data demonstrate the occurrence of evolutionarily conserved NAE metabolic pathway in the moss, with the occurrence of AEA and EPEA.

Endocannabinoids are endogenous lipid mediators involved in various biological processes, and have immunomodulatory and anti-inflammatory activities. Anandamide (arachidonoyl ethanolamine, AEA) and 2-arachidonoyl glycerol (2-AG) are the main representatives of this group. The endocannabinoid receptors CB1 and CB2 with AEA have been found in human HaCaT keratinocytes and fibroblasts, but the metabolic pathway leading to endocannabinoid production in the skin has not been fully elucidated. This study aimed to investigate the profile of endocannabinoids and their main metabolizing enzymes in human skin cells and assess whether omega-3 polyunsaturated fatty acids (n-3 PUFA) altered these profiles. In addition, an investigation was carried out to check whether UV radiation could stimulate the production of endocannabinoids and N-acylethanolamines (NAE) in human skin cells. For this purpose HaCaT keratinocytes and 46RB.1N fibroblast cells were treated with 10 and 50µM of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) or oleic acid (OA) in the presence or absence of UVR (15mJ/cm2). Data suggest that n-3 PUFA may both directly (by up-regulating NAPE-PLD levels) and indirectly (by decreasing FAAH levels) increased endocannabinoid and NAE levels in HaCaT keratinocytes and 46BR.IN fibroblasts. DHA treatment significantly decreased COX-2 expression in the absence of UVR and inhibited UVR-induced COX-2 overexpression in 46BR.IN fibroblasts. In contrast, DHA appeared to induce COX-2 up-regulation in the absence of UVR and did not prevent UVR induced COX-2 up-regulation in HaCaT keratinocytes. EPA appeared to induce COX-2 down-regulation in the absence of UVR and did not prevent UVR induced COX-2 up-regulation in both HaCaT keratinocytes and 46BR.IN fibroblasts. UVR did not have any significant effect on endocannabinoid and NAE biosynthesis. However, UVR induced endocannabinoid production in some experiments of this study. A clinical study was carried on 16 volunteers from two different ethnic groups and two different skin types. The purpose was to assess the effect of UVR on the serum endocannabinoids and NAE, therefore, the volunteers were subjected to multiple doses (1.3, SED/ 6 min) of UVR for 6 weeks. Data showed that UVR did not have major effect on human serum NAE in both skin phototypes II and V but increased 2-AG in human serum in both skin types but the more pronounced effect was evident in skin phototypes V rather than in skin phototypes II. Human serum docosahxaenoylethanolamide levels were found to be higher in White Caucasians group (skin phototypes II). Based on these it can be concluded that n-3 PUFA and UVR alter the endocannabinoids and NAE profile in HaCaT keratinocytes and 46BR.IN fibroblasts. In addition, results of the clinical study indicated that UVR has no major effects on serum endocannabinoids or NAE therefore, further studies are required to address this question in vivo.