Hypertension is a critical public health issue worldwide, and in the United States, it is the leading cause of heart disease, stroke, and kidney failure, contributing to more than 1,100 deaths per day. It is proposed that the renal medulla combats increased blood pressure by releasing a neutral lipid from the lipid droplets of medullary interstitial cells, termed medullipin, which induces diuresis- natriuresis and vasodepression. The renal medulla is enriched with fatty acid lipid ethanolamides including the endocannabinoid anandamide (AEA), palmitoylethanolamide (PEA), and oleoylethanolamide (OEA), along with their primary hydrolyzing enzyme fatty acid amide hydrolase (FAAH). Our lab is investigating the relationship of these lipid ethanolamides and their metabolites to medullipin. We have shown that intramedullary infusion of AEA stimulated diuresis-natriuresis without changing mean arterial pressure (MAP) in an acute surgical model using anesthetized normotensive C57BL/6J mice. The hypothesis that infusion of a FAAH-selective inhibitor, PF-3845, would produce similar responses as exogenous AEA was tested. Intramedullary infusion of PF-3845 stimulated diuresis-natriuresis, decreased MAP, and increased lipid ethanolamide concentrations in kidney tissue in C57BL/6J mice. Since the decrease in MAP observed with PF-3845 was not consistent with the results of exogenous AEA, this study hypothesized that increased PEA concentrations in the renal medulla observed with PF-3845 produced the decrease in MAP. Therefore, the effects of PEA administration into the renal medulla were investigated. Intramedullary infusion of PEA stimulated diuresis and natriuresis without changing MAP in normotensive C57BL/6J mice. However, intramedullary PEA administration to mice made hypertensive using L-NAME, an inhibitor of nitric oxide synthase, was assessed. Intramedullary infusion of PEA stimulated diuresis, but also decreased MAP in L-NAME-induced hypertensive mice. The mechanism of PEA-induced diuresis was evaluated for the contributions of its FAAH-mediated hydrolysis and the CB1 receptor. Intramedullary infusion of PEA stimulated diuresis in FAAH knockout mice and CB1 knockout mice. The possible source of PEA in the renal medulla was investigated using renal medullary interstitial cells cultured from mice. In cultured mouse medullary interstitial cells (MMICs), treatment with PF-3845 increased cytoplasmic lipid droplets detected by Sudan Black B (SBB) staining and increased PEA in the culture medium. Physiologic stimuli that may regulate PEA production and release from MMICs were also evaluated. Increased osmolarity increased NAPE-PLD protein levels, increased SBB stained droplets in MMICs, and increased PEA concentrations in the culture medium. Overall, it is concluded that the PEA-induced diuretic and natriuretic effect is independent of FAAH-mediated hydrolysis and the CB1 receptor, and that PEA can serve as an antihypertensive regulator in the renal medulla that may be regulated by medullary interstitial cells.

Anandamide (AEA) is an endogenous fatty acid which activates the same cannabinoid receptors as ∆9-tetrahydrocannabinol, the psychoactive substance in marijuana. In vivo, anandamide exerts a number of actions including effects upon pain and inflammation. However, AEA has a short duration of action since it is rapidly metabolised, primarily by the intracellular enzyme fatty acid amide hydrolase (FAAH). The general aim of this thesis has been to identify and characterize compounds capable of preventing the metabolism of anandamide. The chemical approach was based on the endogenous anti-inflammatory compound palmitoylethanolamide (PEA), a compound related to anandamide with the ability to inhibit anandamide degradation by substrate competition, but without the ability to directly activate cannabinoid receptors. A number of compounds were identified as inhibitors of rat brain FAAH in the initial in vitro studies, without having major affinity for the cannabinoid receptors. In particular, palmitoylisopropylamide (PIA) was found to reduce the metabolism of AEA in intact C6 glioma cells with potency similar to the prototypical AEA reuptake inhibitor AM404. This compound was in addition found to exert less effect upon C6 glioma cell proliferation than either AM404 or the closely related uptake inhibitor VDM11. To evaluate if PIA was effective in vivo, a model of mast cell dependent inflammation, oedema of the ear following local injection of compound 48/80, was set up using anaesthetised mice. Initially, a CB2 cannabinoid receptor selective agonist was used to probe the model and demonstrated to produce an anti-oedema effect. In contrast, the compound was inactive in vitro in skin slice preparations. PIA showed a similar pattern, although there was a large variation in responses which affected the significance of the result obtained, as did the vehicle used to dissolve the compound. Taken together, the present data would suggest that PIA can inhibit the degradation of AEA without having deleterious effects upon cell proliferation or affinity for the cannabinoid receptors. Further experimentation is necessary to elucidate the usefulness of this compound in vivo.

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.