BACKGROUND

Peripheral and central endocannabinoids and cognate acylethanolamides (AEs) may play important but distinct roles in regulating energy balance.

OBJECTIVE

We hypothesized that in humans central/peripheral endocannabinoids are differently associated with adiposity and energy expenditure and differ by race.

METHODS

We examined associations of arachindonoylethanolamide, <em>2</em>-<em>arachidonoylglycerol</em>, palmitoylethanolamide, and oleoylethanolamide (OEA) assayed in plasma and cerebrospinal fluid (CSF) with race, adiposity, and energy expenditure.

METHODS

In this monitored clinical inpatient study, CSF was obtained by lumbar puncture in <em>2</em>7 individuals (1<em>2</em> Caucasian, 11 American Indian, and four African-American). Twenty-four hour and sleep energy expenditure were measured by indirect calorimetry in a respiratory chamber.

METHODS

Samples were analyzed from a previous study originally designed to test a blood-brain barrier leptin transport deficit in human obesity.

RESULTS

CSF (but not peripheral) <em>2</em>-<em>arachidonoylglycerol</em> was significantly increased in American Indians compared with Caucasians (18.48 ± 6.17 vs. 10.6<em>2</em> ± 4.58 pmol/ml, P < 0.01). In the whole group, peripheral AEs were positively but in CSF negatively associated with adiposity. However, in multivariate models adjusted for the other peripheral and CSF AEs, peripheral arachindonoylethanolamide was the only AE significantly associated with adiposity. Interestingly, CSF OEA concentrations were positively associated with adjusted <em>2</em>4 hour and sleep energy expenditure (r = 0.47, P < 0.05; r = 0.4<em>2</em>, P < 0.05), but peripheral OEA was not.

CONCLUSIONS

These data indicate a central alteration of the endocannabinoid system in American Indians and furthermore show that AEs in both compartments play an important but distinct role in human energy balance regulation.

The identification of the major psychoactive constituent of Cannabis and marijuana, Delta(9)-tetrahydrocannabinol, opened the way first to the cloning of the G-protein-coupled cannabinoid CB(1) and CB(<em>2</em>) receptors, and then to the isolation and characterisation of their endogenous agonists, the endocannabinoids. Considerable progress has been made in the characterisation of pathways and enzymes for the biosynthesis and degradation of anandamide and <em>2</em>-<em>arachidonoylglycerol</em>, the two best-known endocannabinoids, as well as of endocannabinoid-related molecules, such as the N-acylethanolamines, which, as in the case of N-palmitoylethanolamine and N-oleoylethanolamine, may interact with other receptor types. However, it is still not fully understood how other plant cannabinoids, of which cannabidiol is the most studied representative, exert their pharmacological effects. Together with these issues, this first review article on the endocannabinoids describes the synthetic pharmacological tools that have been designed so far to interact with the proteins of the 'endocannabinoid system' and that can potentially be used as templates for the development of new therapies.

N-Acyl-ethanolamines (NAEs) and their precursors, N-acyl-ethanolamine phospholipids (NAPEs), are present in the mammalian brain at levels of a few hundred picomoles/gram tissue and a few nanomoles/gram tissue, respectively. NAE-containing arachidonic acid is called anandamide, and it has attracted particular attention since it is a partial agonist for the cannabinoid receptors, for which <em>2</em>-<em>arachidonoylglycerol</em> is the full agonist. In addition, anandamide may also activate the vanilloid receptor. Anandamide usually amounts to 1-10% of NAEs, as the vast majority of N-acyl groups are saturated and monounsaturated fatty acids. Formation of NAPE and NAE is catalyzed by an N-acyltransferase and an NAPE-hydrolyzing phospholipase D, respectively, two enzymes that have been characterized only preliminary. Interestingly, NAPEs and NAEs accumulate in the brain in response to neurodegenerative insults at a time when other phospholipids are subjected to rapid degradation. This is an important biosynthetic aspect of NAPE and NAE, as NAEs may be neuroprotective by a number of different mechanisms involving both receptor activation and non-receptor-mediated effects, e.g. by binding to cannabinoid receptors and interfering with ceramide turnover, respectively.

Endocannabinoids like <em>2</em>-<em>arachidonoylglycerol</em> strongly modulate the complex machinery of secondary neuronal damage and are shown to improve neuronal survival after excitotoxic lesion. Palmitoylethanolamide (PEA), the naturally occurring fatty acid amide of ethanolamine and palmitic acid, is an endogenous lipid known to mimic several effects of endocannabinoids even without binding to cannabinoid receptors. Here we show that PEA (0.001-1 μM) and the synthetic peroxisome proliferator-activated receptor (PPAR)-alpha agonist 4-chloro-6-(<em>2</em>,3-xylidino)-<em>2</em>-pyrimidinylthio acetic acid (Wy-14,643; 0.1-1 μM) reduced the number of microglial cells and protected dentate gyrus granule cells in excitotoxically lesioned organotypic hippocampal slice cultures (OHSCs). Treatment with the PPAR-alpha antagonist N-((<em>2</em>S)-<em>2</em>-(((1Z)-1-Methyl-3-oxo-3-(4-(trifluoromethyl)phenyl)prop-1-enyl)amino)-3-(4-(<em>2</em>-(5-methyl-<em>2</em>-phenyl-1,3-oxazol-4-yl)ethoxy)phenyl)propyl)propanamide (GW6471; 0.05-5 μM) blocked PEA-mediated neuroprotection and reduction of microglial cell numbers whereas the PPAR-gamma antagonist <em>2</em>-chloro-5-nitro-N-phenyl-benzamide (GW966<em>2</em>; 0.01-1 μM) showed no effects. Immunocytochemistry and Western blot analyses revealed a strong PPAR-alpha immunoreaction in BV-<em>2</em> microglial cells and in HT<em>2</em><em>2</em> hippocampal cells. Intensity and location of PPAR-alpha immunoreaction remained constant during stimulation with PEA (0.01 μM; 1-36 h). In conclusion our data provide evidence that (1) PEA counteracted excitotoxically induced secondary neuronal damage of dentate gyrus granule cells, (<em>2</em>) PPAR-alpha but not PPAR-gamma is the endogenous binding site for PEA-mediated neuroprotection, and (3) PEA may activate PPAR-alpha in microglial cells and hippocampal neurons to exert its neuroprotective effects. In addition to classical endocannabinoids, PEA-mediated PPAR-alpha activation represents a possible target for therapeutic interventions to mitigate symptoms of secondary neuronal damage.

Little is known of the involvement of endocannabinoids and cannabinoid receptors in skeletal muscle cell differentiation. We report that, due to changes in the expression of genes involved in its metabolism, the levels of the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) are decreased both during myotube formation in vitro from murine C<em>2</em>C1<em>2</em> myoblasts and during mouse muscle growth in vivo. The endocannabinoid, as well as the CB1 agonist arachidonoyl-<em>2</em>-chloroethylamide, prevent myotube formation in a manner antagonized by CB1 knockdown and by CB1 antagonists, which, per se, instead stimulate differentiation. Importantly, <em>2</em>-AG also inhibits differentiation of primary human satellite cells. Muscle fascicles from CB1 knockout embryos contain more muscle fibers, and postnatal mice show muscle fibers of an increased diameter relative to wild-type littermates. Inhibition of Kv7.4 channel activity, which plays a permissive role in myogenesis and depends on phosphatidylinositol 4,5-bisphosphate (PIP<em>2</em>), underlies the effects of <em>2</em>-AG. We find that CB1 stimulation reduces both total and Kv7.4-bound PIP<em>2</em> levels in C<em>2</em>C1<em>2</em> cells and inhibits Kv7.4 currents in transfected CHO cells. We suggest that <em>2</em>-AG is an endogenous repressor of myoblast differentiation via CB1-mediated inhibition of Kv7.4 channels.

Endocannabinoids are involved in the modulation of pain and hyperalgesia. In this study we investigated the role of the endocannabinoid system in the migraine model based on nitroglycerin-induced hyperalgesia in the rat. Male rats were injected with nitroglycerin (10 mg/kg, i.p.) or vehicle and sacrificed 4 h later. The medulla, the mesencephalon and the hypothalamus were dissected out and utilized for the evaluation of activity of fatty acid amide hydrolase (that degrades the endocannabinoid anandamide), monoacylglycerol lipase (that degrades the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em>), and binding sites specific for cannabinoid (CB) receptors. The findings obtained show that nitroglycerin-induced hyperalgesia is associated with increased activity of both hydrolases and increased density of CB binding sites in the mesencephalon. In the hypothalamus we observed an increase in the activity of fatty acid amide hydrolase associated with an increase in density of CB binding sites, while in the medulla only the activity of fatty acid amide hydrolase was increased. Anandamide also proved effective in preventing nitroglycerin-induced activation (c-Fos) of neurons in the nucleus trigeminalis caudalis. These data strongly support the involvement of the endocannabinoid system in the modulation of nitroglycerin-induced hyperalgesia, and, possibly, in the pathophysiological mechanisms of migraine.

The anti-obesity medication rimonabant, an antagonist of cannabinoid type-1 (CB(1)) receptor, was withdrawn from the market because of adverse psychiatric side effects, including a negative affective state. We investigated whether rimonabant precipitates a negative emotional state in rats withdrawn from palatable food cycling. The effects of systemic administration of rimonabant on anxiety-like behavior, food intake, body weight, and adrenocortical activation were assessed in female rats during withdrawal from chronic palatable diet cycling. The levels of the endocannabinoids, anandamide and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), and the CB(1) receptor mRNA and the protein in the central nucleus of the amygdala (CeA) were also investigated. Finally, the effects of microinfusion of rimonabant in the CeA on anxiety-like behavior, and food intake were assessed. Systemic administration of rimonabant precipitated anxiety-like behavior and anorexia of the regular chow diet in rats withdrawn from palatable diet cycling, independently from the degree of adrenocortical activation. These behavioral observations were accompanied by increased <em>2</em>-AG, CB(1) receptor mRNA, and protein levels selectively in the CeA. Finally, rimonabant, microinfused directly into the CeA, precipitated anxiety-like behavior and anorexia. Our data show that (i) the <em>2</em>-AG-CB(1) receptor system within the CeA is recruited during abstinence from palatable diet cycling as a compensatory mechanism to dampen anxiety, and (ii) rimonabant precipitates a negative emotional state by blocking the beneficial heightened <em>2</em>-AG-CB(1) receptor signaling in this brain area. These findings help elucidate the link between compulsive eating and anxiety, and it will be valuable to develop better pharmacological treatments for eating disorders and obesity.

The purpose of this chapter is to provide an introduction to the mechanisms for the regulation of endocannabinoid signaling through CB1 cannabinoid receptors in the central nervous system. The processes involved in the synthesis and degradation of the two most well-studied endocannabinoids, <em>2</em>-<em>arachidonoylglycerol</em> and N-arachidonylethanolamine are outlined along with information regarding the regulation of the proteins involved. Signaling mechanisms and pharmacology of the CB1 cannabinoid receptor are outlined, as is the paradigm of endocannabinoid/CB1 receptor regulation of neurotransmitter release. The reader is encouraged to appreciate the importance of the endocannabinoid/CB1 receptor signaling system in the regulation of synaptic activity in the brain.

<AbstractText>The endocannabinoid (eCB) system is implicated in the pathophysiology of depression and is responsive to acute exercise in healthy adults.</AbstractText><AbstractText>We aimed to describe acute changes in serum eCB across a prescribed moderate (MOD) and a self-selected/preferred (PREF) intensity exercise session in women with major depressive disorder (MDD) and determine relationships between changes in eCB and mood states.</AbstractText><AbstractText>Women with MDD (n = 17) exercised in separate sessions for <em>2</em>0 min on a cycle ergometer at both MOD or PREF in a within-subjects design. Blood was drawn before and within 10 min after exercise. Serum concentrations of eCB (anandamide [AEA], <em>2</em>-<em>arachidonoylglycerol</em>) and related lipids (palmitoylethanolamine, oleoylethanolamine, <em>2</em>-oleoylglycerol) were quantified using stable isotope-dilution, liquid chromatography/mass spectrometry/mass spectrometry. The profile of mood states and state-trait anxiety inventory (state only) were completed before, 10 min and 30 min postexercise.</AbstractText><AbstractText>Significant elevations in AEA (P = 0.013) and oleoylethanolamine (P = 0.0<em>2</em>4) occurred for MOD (moderate effect sizes: Cohen's d = 0.58 and 0.41, respectively). Significant (P < 0.05) moderate negative associations existed between changes in AEA and mood states for MOD at 10 min (depression, confusion, fatigue, total mood disturbance [TMD] and state anxiety) and 30 min postexercise (confusion, TMD and state anxiety). Significant (P < 0.05) moderate negative associations existed between <em>2</em>-<em>arachidonoylglycerol</em> and mood states at 10 min (depression and confusion) and 30 min postexercise (confusion and TMD). Changes in eCB or related lipids or eCB-mood relationships were not found for PREF.</AbstractText><AbstractText>Given the broad, moderate-strength relationships between improvements in mood states and eCB increases after MOD, it is plausible that the eCB system contributes to the mood-enhancing effects of prescribed acute exercise in MDD. Alternative mechanisms are likely involved in the positive mood state effects of preferred exercise.</AbstractText>

Aberrant Notch signaling has recently emerged as a possible mechanism for the altered neurogenesis, cognitive impairment, and learning and memory deficits associated with Alzheimer disease (AD). Recently, targeting the endocannabinoid system in models of AD has emerged as a potential approach to slow the progression of the disease process. Although studies have identified neuroprotective roles for endocannabinoids, there is a paucity of information on modulation of the pro-survival Notch pathway by endocannabinoids. In this study the influence of the endocannabinoids, anandamide (AEA) and <em>2</em>-<em>arachidonoylglycerol</em>, on the Notch-1 pathway and on its endogenous regulators were investigated in an in vitro model of AD. We report that AEA up-regulates Notch-1 signaling in cultured neurons. We also provide evidence that although Aβ(1-4<em>2</em>) increases expression of the endogenous inhibitor of Notch-1, numb (Nb), this can be prevented by AEA and <em>2</em>-<em>arachidonoylglycerol</em>. Interestingly, AEA up-regulated Nct expression, a component of γ-secretase, and this was found to play a crucial role in the enhanced Notch-1 signaling mediated by AEA. The stimulatory effects of AEA on Notch-1 signaling persisted in the presence of Aβ(1-4<em>2</em>). AEA was found to induce a preferential processing of Notch-1 over amyloid precursor protein to generate Aβ(1-40). Aging, a natural process of neurodegeneration, was associated with a reduction in Notch-1 signaling in rat cortex and hippocampus, and this was restored with chronic treatment with URB 597. In summary, AEA has the proclivity to enhance Notch-1 signaling in an in vitro model of AD, which may have relevance for restoring neurogenesis and cognition in AD.

Biosynthesis Inhibition: O-5596, a new inhibitor of the biosynthesis of the endocannabinoid, <em>2</em>-<em>arachidonoylglycerol</em>, was synthesized and found to be potent (IC(50)=100 nM) and selective versus other proteins and enzymes of the endocannabinoid system in vitro and active in vivo at reducing food intake in mice.

We investigated the diacyglycerol kinase species present in several baboon tissues using the substrates sn-1-stearoyl-<em>2</em>-arachidonoyl diacylglycerol and sn-1,<em>2</em>-didecanoyl diacylglycerol. Chromatography of octyl glucoside extracts of the baboon (Papio cynocephalus papio) tissues on hydroxyapatite columns revealed the presence of three diacylglycerol kinase species with different substrate preferences. One species markedly 'preferred' the substrate sn-1-stearoyl-<em>2</em>-<em>arachidonoylglycerol</em>, the two other species preferred sn-1,<em>2</em>-didecanoylglycerol. Measurement of the activity of the baboon brain diacylglycerol kinases toward diacylglycerols with a range of different fatty acid chains revealed a strict preference of the arachidonoyl diacylglycerol kinase for sn-1-acyl-<em>2</em>-arachidonoyl diacylglycerol, whereas the other enzymes showed no preference toward several long-chain-fatty-acid-containing diacylglycerols. The arachidonoyl diacylglycerol kinase was particularly abundant in brain and testis, whereas liver was practically devoid of this enzyme. The arachidonoyl diacylglycerol kinase from baboon brain was found to be predominantly associated with the particulate fraction and exhibited an apparent molecular mass of 130 kDa.

The endogenous cannabinoids <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) and arachidonoyl ethanolamide (AEA or anandamide) play vital roles during nervous system development including regulating axonal guidance and synaptogenesis. The enzymatic degradation of <em>2</em>-AG and AEA is highly susceptible to inhibition by organophosphate compounds in vitro. Furthermore, acute in vivo exposure of adult animals to the agricultural insecticide chlorpyrifos (CPS) caused moderate inhibition of both <em>2</em>-AG and AEA hydrolysis. However, the effects of repeated exposure to lower levels of CPS, especially during development, on endocannabinoid metabolism in the brain is not known. To examine this, rat pups were orally exposed daily from postnatal days 10-16 to either 1.0, <em>2</em>.5, or 5.0 mg/kg CPS. Body weight gain was reduced by 5.0 mg/kg on all days of treatment whereas <em>2</em>.5 mg/kg reduced the weight gain only on the last two days of treatment. At 4-h postexposure on day 16, forebrain cholinesterase (ChE) activity and hydrolysis of <em>2</em>-AG and AEA were inhibited in a dose-related manner, and the extent of inhibition from highest to lowest level was AEA hydrolysis>> ChE activity>> <em>2</em>-AG hydrolysis. The extent of inhibition of AEA hydrolysis was approximately twice than that of ChE activity with AEA hydrolysis being virtually eliminated by <em>2</em>.5 and 5.0 mg/kg and 1.0 mg/kg causing 40% inhibition. The sensitivity of AEA hydrolysis, compared with canonical targets such as ChE activity, suggests a potential alternative developmental target for CPS. Inhibition of AEA hydrolysis could result in accumulation of endocannabinoids, which could alter normal endocannabinoid transmission during brain maturation.

Fatty acid amide hydrolase (FAAH) is the key hydrolytic enzyme for the endogenous cannabinoid receptor ligand anandamide. The synthesis and evaluation for their FAAH inhibitory activities of a series of 18 paracetamol esters are described. Structure-activity relationship studies indicated that the ester (33) with a <em>2</em>-(4-(<em>2</em>-(trifluoromethyl)pyridin-4-ylamino)phenyl)acetic acid substituent was the most potent analogue in this series. The compound inhibited FAAH activity in a competitive manner with a K(i) value of 0.16 microM. The compound was also able to inhibit the FAAH activity in rat basophilic leukemia cells as assessed by measuring either the hydrolysis of anandamide, the FAAH-dependent cellular accumulation of anandamide, or the FAAH-dependent recycling of tritium to the cell membranes. The compound also inhibited the activity of monoacylglycerol lipase (MGL), the enzyme responsible for the hydrolysis of the endogenous cannabinoid receptor ligand <em>2</em>-<em>arachidonoylglycerol</em>, with an IC(50) value of 1.9 microM. It is concluded that the compound may be a useful template for the design of potent novel inhibitors of FAAH.

Fatty acid amide hydrolase (FAAH) and monoglyceride lipase (MGL) are hydrolytic enzymes which degrade the endogenous cannabinoids (endocannabinoids) N-arachidonoylethanolamine (anandamide, AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), respectively. Endocannabinoids are an important class of lipid messenger molecules that are produced on demand in response to elevated intracellular calcium levels. They recognize and activate the cannabinoid CB(1) and CB(<em>2</em>) receptors, the molecular targets for Delta(9)-tetrahydrocannabinol (Delta(9)-THC) in marijuana evoking several beneficial therapeutic effects. However, in vivo the cannabimimetic effects of AEA and <em>2</em>-AG remain weak owing to their rapid inactivation by FAAH and MGL, respectively. The inactivation of FAAH and MGL by specific enzyme inhibitors increases the levels of AEA and <em>2</em>-AG, respectively, producing therapeutic effects such as pain relief and depression of anxiety.

In hippocampus endocannabinoids modulate synaptic function and plasticity and increase tyrosine phosphorylation of several proteins, including focal adhesion kinase (FAK). Autophosphorylation of FAK on Tyr-397 is generally a critical step for its activation, allowing the recruitment of Src family kinases, and phosphorylation of FAK and associated proteins. We have examined the mechanisms of the regulation of FAK by cannabinoids in rat and mouse hippocampal slices. Anandamide and <em>2</em>-<em>arachidonoylglycerol</em>, two endocannabinoids, and Delta9-tetrahydrocannabinol, stimulated tyrosine phosphorylation of FAK+6,7, a neuronal splice isoform of FAK, on several residues including Tyr-397. Cannabinoids increased phosphorylation of p130-Cas, a protein associated with FAK, but had no effect on PYK<em>2</em>, a tyrosine kinase related to FAK and enriched in hippocampus. Pharmacological experiments and the use of knockout mice demonstrated that the effects of cannabinoids were mediated through CB1 receptors. These effects were sensitive to manipulation of cAMP-dependent protein kinase, suggesting that they were mediated by inhibition of a cAMP pathway. PP<em>2</em>, an Src family kinase inhibitor, prevented the effects of cannabinoids on p130-Cas and on FAK+6,7 tyrosines 577 and 9<em>2</em>5, but not 397, indicating that FAK autophosphorylation was upstream of Src family kinases in response to CB1-R stimulation. Endocannabinoids increased the association of Fyn, but not Src, with FAK+6,7. In hippocampal slices from Fyn -/- mice, the levels of p130-Cas were increased, and the effects of endocannabinoids on tyrosine phosphorylation, including of Tyr-397, were completely abolished. These results demonstrate the specific functional association of Fyn with FAK+6,7 in a pathway regulated by endocannabinoids, in which Fyn may play roles dependent and independent of its catalytic activity.

The effects of cannabinoid receptor ligands including <em>2</em>-<em>arachidonoylglycerol</em>, R-methanandamide, Delta9-THC (Delta9-tetrahydrocannabinol), WIN 55,<em>2</em>1<em>2</em>-<em>2</em> [4,5-dihydro-<em>2</em>-methyl-4(4-morpholinylmethyl)-1-(1-naphthalenylcarbonyl)-6H-pyrrolo[3,<em>2</em>,1ij]quinolin-6-one], CP 55,940 ([1alpha,<em>2</em>beta-(R)-5alpha]-(-)-5-(1,1-dimethyl)-<em>2</em>-[5-hydroxy-<em>2</em>-(3-hydroxypropyl) cyclohexyl-phenol]) and a series of fatty acids on depolarization-induced Ca<em>2</em>+ effluxes mediated by voltage-dependent Ca<em>2</em>+ channels were investigated comparatively in transverse tubule membrane vesicles from rabbit skeletal muscle. Vesicles were loaded with 45Ca<em>2</em>+ and membrane potentials were generated by establishing potassium gradients across the vesicle using the ionophore valinomycin. Endocannabinoids, <em>2</em>-<em>arachidonoylglycerol</em> and R-methanandamide (all 10 microM), inhibited depolarization-induced Ca<em>2</em>+ effluxes and specific binding of [3H]PN <em>2</em>00-110 (isradipine) to transverse tubule membranes. On the other hand, synthetic cannabinoids, including CP 55,940, WIN 55,<em>2</em>1<em>2</em>-<em>2</em>, and Delta9-THC (all 10 microM), were ineffective. Additional experiments using endocannabinoid metabolites suggested that whereas ethanolamine and glycerol were ineffective, arachidonic acid inhibited Ca<em>2</em>+ effluxes and specific binding of [3H]PN <em>2</em>00-110. Further studies indicated that only those fatty acids containing two or more double bonds were effective in inhibiting depolarization-induced Ca<em>2</em>+ effluxes and specific binding of [3H]PN <em>2</em>00-110. These results indicate that endocannabinoids, but not synthetic cannabinoids, directly inhibit the function of voltage-dependent calcium channels (VDCCs) and modulate the specific binding of calcium channel ligands of the dihydropyridine (DHP) class.

The neutral arachidonic acid derivatives N-arachidonoyl ethanolamine (anandamide or AEA), and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) have been identified as endogenous ligands for the cannabinoid receptors. Additionally, these compounds have been identified as substrates of the second isoform of the cyclooxygenase enzyme (COX-<em>2</em>). Through the action of COX-<em>2</em> and downstream prostaglandin synthases, a diverse family of prostaglandin glycerol esters (PG-Gs) and prostaglandin ethanolamides (PG-EAs) have been identified. Sensitive and reliable analytical methodology is crucial for the continued research on the biological roles of this family of lipids. In this chapter, we discuss methods for analyzing both the precursor endocannabinoids and their PG-like products by LC-MS-MS. Cation coordination provides the ionization, and selected reaction monitoring is successfully employed to provide a method of analysis that is both sensitive and specific.

BACKGROUND

The endocannabinoid system (ECS) is an endogenous signalling system which includes the endocannabinoids anandamide (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) and specific G-protein-coupled endocannabinoid receptors (CB1 and CB<em>2</em>). Recent studies have described important roles of the peripheral ECS in human atherosclerosis, cardiometabolic disorders, heart failure, and systemic inflammation. We sought to study changes in plasma endocannabinoid concentrations during cardiac surgery (CS) under general anaesthesia with isoflurane/sufentanil, and during cardiopulmonary bypass (CPB).

METHODS

We studied 30 patients undergoing CS with CPB. All patients received midazolam and sufentanil for induction and isoflurane and sufentanil for maintenance of general anaesthesia. Blood samples were drawn before and after induction of general anaesthesia, after the beginning of surgery, during and after weaning from CPB, and after admission to intensive care unit (ICU) after surgery. Endocannabinoid measurements were performed by HPLC-tandem mass spectrometry.

RESULTS

Induction of general anaesthesia led to a significant decline in plasma AEA concentrations [from mean (sd) 0.39 (0.03) to 0.<em>2</em>7 (0.03) ng ml(-1), P<0.01]. CPB induced a pronounced increase in <em>2</em>-AG concentrations [from 11<em>2</em>.5 (163.5) to 3<em>2</em>1.0 (1<em>2</em>0.4) ng ml(-1), P<0.01], whereas AEA concentrations remained persistently low until admission to the ICU. <em>2</em>-AG concentrations returned to preoperative values after surgery.

CONCLUSIONS

General anaesthesia with isoflurane significantly reduces plasma AEA concentrations. This could be a consequence of stress reduction after loss of consciousness. The significant increase in <em>2</em>-AG after initiation of CPB may be part of an inflammatory response. These findings suggest that anaesthesia and surgery have differential effects on the ECS which could have substantial clinical consequences.

The endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) plays a major role in many physiological processes, and its action is quickly terminated via enzymatic hydrolysis catalyzed by monoglyceride lipase (MGL). Regulating its endogenous level could offer therapeutic opportunities; however, few selective MGL inhibitors have been described so far. Here, we describe the synthesis of N-substituted maleimides and their pharmacological evaluation on the recombinant human fatty acid amide hydrolase (FAAH) and on the purified human MGL. A few N-arylmaleimides were previously described ( Saario , S. M. ; Salo , O. M. ; Nevalainen , T. ; Poso , A. ; Laitinen , J. T. ; Jarvinen , T. ; Niemi , R. Characterization of the Sulfhydryl-Sensitive Site in the Enzyme Responsible for Hydrolysis of <em>2</em>-<em>Arachidonoylglycerol</em> in Rat Cerebellar Membranes . Chem. Biol. <em>2</em>005 , 1<em>2</em> , 649 - 656 ) as MGL inhibitors, and along these lines, we present a new set of maleimide derivatives that showed low micromolar IC(50) and high selectivity toward MGL vs FAAH. Then, structure-activity relationships have been investigated and, for instance, 1-biphenyl-4-ylmethylmaleimide inhibits MGL with an IC(50) value of 790 nM. Furthermore, rapid dilution experiments reveal that these compounds act as irreversible inhibitors. In conclusion, N-substituted maleimides constitute a promising class of potent and selective MGL inhibitors.

<em>2</em>-<em>Arachidonoylglycerol</em> (<em>2</em>-AG), an endogenous cannabionoid receptor (CB1 and CB<em>2</em>) ligand, enhanced the adhesion of HL-60 cells differentiated into macrophage-like cells to fibronectin and the vascular cell adhesion molecule-1. The CB<em>2</em> receptor, Gi/Go, intracellular free Ca(<em>2</em>+) and phosphatidylinositol 3-kinase were shown to be involved in <em>2</em>-AG-induced augmented cell adhesion. <em>2</em>-AG also enhanced the adhesion of human monocytic leukemia U937 cells and peripheral blood monocytes. These results strongly suggest that <em>2</em>-AG plays some essential role in inflammatory reactions and immune responses by inducing robust adhesion to extracellular matrix proteins and adhesion molecules in several types of inflammatory cells and immune-competent cells.

Neurotensin (NT), a neuropeptide abundant in the ventral midbrain, is known to act as a key regulator of the mesolimbic dopamine (DA) system, originating in the ventral tegmental area (VTA). NT activates metabotropic receptors coupled to Gq heterotrimeric G proteins, a signaling pathway often triggering endocannabinoid (EC) production in the brain. Because ECs act as negative regulators of many glutamate synapses and have also been shown recently to gate LTP induction in the VTA, we examined the hypothesis that NT regulates glutamate-mediated synaptic inputs to VTA DA neurons. We performed whole cell patch-clamp recordings in VTA DA neurons in TH-EGFP transgenic mouse brain slices and found that NT induces a long-lasting decrease of the EPSC amplitude that was mediated by the type 1 NT receptor. An antagonist of the CB1 EC receptor blocked this decrease. This effect of NT was not dependent on intracellular calcium, but required G-protein activation and phospholipase C. Blockade of the CB1 receptor after the induction of EPSC depression reversed synaptic depression, an effect not mimicked by blocking NT receptors, thus suggesting the occurrence of prolonged EC production and release. The EC responsible for synaptic depression was identified as <em>2</em>-<em>arachidonoylglycerol</em>, the same EC known to gate LTP induction in VTA DA neurons. However, blocking NT receptors during LTP induction did not facilitate LTP induction, suggesting that endogenously released NT is not a major source of EC production during LTP inducing stimulations.

Anandamide (N-arachidonoylethanolamine) loses its cannabimimetic activity when it is hydrolyzed to arachidonic acid and ethanolamine by the catalysis of an enzyme referred to as anandamide amidohydrolase or fatty acid amide hydrolase. Cravatt's group and our group cloned cDNA of the enzyme from rat, human, mouse and pig, and the primary structures revealed that the enzymes belong to an amidase family characterized by the amidase signature sequence. The recombinant enzyme acted not only as an amidase for anandamide and oleamide, but also as an esterase for <em>2</em>-<em>arachidonoylglycerol</em>. The reversibility of the enzymatic anandamide hydrolysis and synthesis was also confirmed with a purified recombinant enzyme. Several fatty acid derivatives like methyl arachidonyl fluorophosphonate potently inhibited the enzyme. The enzyme was distributed widely in mammalian organs such as liver, small intestine and brain. However, the anandamide hydrolyzing enzyme found in human megakaryoblastic cells was catalytically distinct from the previously known enzyme.

Tissue levels of anandamide (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) have been determined in 16 regions and nuclei from human brains, using liquid chromatography/in-line mass spectrometry. Measurements in brain samples stored at -80 degrees C for <em>2</em> months to 13 years indicated that endocannabinoids were stable under such conditions. In contrast, the postmortal delay had a strong effect on brain endocannabinoid levels, as documented in brain samples microdissected and frozen 1-6 h postmortem, and in neurosurgical samples 0, 5, 30, 60, 180 and 360 min after their removal from the brain. The tissue levels of AEA increased continuously and in a region-dependent manner from 1 h after death, increasing about sevenfold by 6 h postmortem. In contrast, concentrations of <em>2</em>-AG, which were 10-100 times higher in human brain regions than those of AEA, rapidly declined: within the first hour, <em>2</em>-AG levels dropped to <em>2</em>5-35% of the initial ('0 min') value, thereafter they remained relatively stable. As analyzed in samples removed 1-1.5 h postmortem, AEA levels ranged from a high of 96.3 fmol/mg tissue in the nucleus accumbens to a low of <em>2</em>5.0 fmol/mg in the cerebellum. <em>2</em>-AG levels varied eightfold, from 8.6 pmol/mg in the lateral hypothalamus to 1.1 pmol/mg in the nucleus accumbens. Relative levels of AEA and <em>2</em>-AG varied from region to region, with the <em>2</em>-AG:AEA ratio being high in the sensory spinal trigeminal nucleus (140:1), the spinal dorsal horn (136:1) and the lateral hypothalamus (98:1) and low in the nucleus accumbens (16:1) and the striatum (31:1). The results highlight the pitfall of analyzing endocannabinoid content in brain samples of variable postmortal delay, and document differential distribution of the two main endocannabinoids in the human brain.