The effects of the endocannabinoids anandamide and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) were determined on cholinergic contractility in strips of human colonic longitudinal muscle and circular muscle in vitro, in the presence of nitric oxide synthase blockade with N-nitro-l-arginine (10(-4) M). Anandamide and <em>2</em>-AG inhibited longitudinal muscle and circular muscle contractile responses to acetylcholine (10(-9)-10(-4) M) in a concentration-dependent manner. This was unaltered following pretreatment with the cannabinoid CB(1) receptor-selective antagonist AM<em>2</em>51 (10(-7) M), however in isolation AM<em>2</em>51 elicited a significant rightward shift in the potency of acetylcholine-evoked contraction in both longitudinal muscle and circular muscle preparations. Pretreatment with an inhibitor of anandamide catabolism, arachidonoyl trifluoromethyl ketone (10(-5) M), alone caused a significant decrease in the potency of acetylcholine-evoked contraction in both longitudinal and circular muscle, but had no significant additional effect on the anandamide-induced (10(-5) M) suppression of contraction. Pretreatment with the cannabinoid CB(<em>2</em>) receptor inverse agonist JTE 907 (10(-6) M) neither influenced the potency of acetylcholine-evoked contraction alone nor prevented the potency shift in acetylcholine-evoked contraction in the presence of anandamide (10(-5) M). The findings of the present study indicate that the endocannabinoids anandamide and <em>2</em>-<em>arachidonoylglycerol</em> suppress colonic cholinergic contractility via a non conventional cannabinoid or non-cannabinoid receptor-mediated pathway. Cholinergic contraction may be tonically modulated by endocannabinoids and/or products of arachidonate metabolism unrelated to endocannabinoid production. The extent of anandamide metabolism is not sufficient to influence the functional effects of its exogenous administration in human colonic tissue in vitro.

Cannabinoid agonists such as Delta9-tetrahydrocannabinol (THC) produce a wide range of pharmacological effects both in the central nervous system and in the periphery. One of the most striking features of cannabinoids such as THC is the magnitude to tolerance that can be produced upon repetitive administration of this substance to animals. Relatively modest dosing regimens are capable of producing significant tolerance, whereas greater than 100-fold tolerance can be obtained with aggressive treatments. While cannabinoid tolerance has been studied quite extensively to establish its relevance to the health consequences of marijuana use, it has also proven to be a valuable strategy in understanding the mechanism of action of cannabinoids. The discovery of the endocannabinoid system that contains two receptor subtypes, CB1 and CB<em>2</em>, associated signaling pathways, endocannabinoids (anandamide and <em>2</em>-<em>arachidonoylglycerol</em>) and their synthetic and degradative pathways has provided a means of systematically evaluating the mechanism of cannabinoid tolerance. It is well known that the CB1 cannabinoid receptor is down-regulated in states of cannabinoid tolerance along with uncoupling from its second messenger systems. Endocannabinoid levels are also altered in selected brain regions during the development of tolerance. While it is reasonable to speculate that a likely relationship exists between receptor and endocannabinoid levels, at present, little is known regarding the biological signal that leads to alterations in endocannabinoid levels. It is also unknown to what degree synthetic and degradative pathways for the endocannabinoids are altered in states of tolerance. The discovery that the brain is abundant in fatty acid amides and glycerols raises the question as to what roles these lipids contribute to the endocannabinoid system. Some of these lipids also utilize the endocannabinoid metabolic pathways, produce similar pharmacological effects, and are capable of modulating the actions of anandamide and <em>2</em>-<em>arachidonoylglycerol</em>. In addition, there are dopamine, glycine, and serotonin conjugates of arachidonic acid that may also contribute to the actions of endocannabinoids. A systematic examination of these lipids in cannabinoid tolerance might shed light on their physiological relevance to the endocannabinoid system.

The present studies examined the effect of chronic neuropathic pain on cannabinoid receptor density and receptor-mediated G-protein activity within supraspinal brain areas involved in pain processing and modulation in mice. Chronic constriction injury (CCI) produced a significant decrease in WIN 55,<em>2</em>1<em>2</em>-<em>2</em>-stimulated [(35)S]GTPgammaS binding in membranes prepared from the rostral anterior cingulate cortex (rACC) of CCI mice when compared to sham-operated controls. Saturation binding with [(3)H]SR 141716A in membranes of the rACC showed no significant differences in binding between CCI and sham mice. Analysis of levels of the endocannabinoids anandamide (AEA) or <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) in the rACC following CCI showed no significant differences between CCI and sham mice. These data suggest that CCI produced desensitization of the cannabinoid 1 receptor in the rACC in the absence of an overall decrease in cannabinoid 1 receptor density or change in levels of AEA or <em>2</em>-AG. These data are the first to show alterations in cannabinoid receptor function in the rostral anterior cingulate cortex in response to a model of neuropathic pain.

OBJECTIVE

Cannabinoid CB1 receptors are found in abundance in the vertebrate eye, with most tissue types expressing this receptor. However, the function of CB1 receptors in corneal epithelial cells (CECs) is poorly understood. Interestingly, the corneas of CB1 knockout mice heal more slowly after injury via a mechanism proposed to involve protein kinase B (Akt) activation, chemokinesis, and cell proliferation. The current study examined the role of cannabinoids in CEC migration in greater detail.

METHODS

We determined the role of CB1 receptors in corneal healing. We examined the consequences of their activation on migration and proliferation in bovine CECs (bCECs). We additionally examined the mRNA profile of cannabinoid-related genes and CB1 protein expression as well as CB1 signaling in bovine CECs.

RESULTS

We now report that activation of CB1 with physiologically relevant concentrations of the synthetic agonist WIN55<em>2</em>1<em>2</em>-<em>2</em> (WIN) induces bCEC migration via chemotaxis, an effect fully blocked by the CB1 receptor antagonist SR141716. The endogenous agonist <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) also enhances migration. Separately, mRNA for most cannabinoid-related proteins are present in bovine corneal epithelium and cultured bCECs. Notably absent are CB<em>2</em> receptors and the <em>2</em>-AG synthesizing enzyme diglycerol lipase-α (DAGLα). The signaling profile of CB1 activation is complex, with inactivation of mitogen-activated protein kinase (MAPK). Lastly, CB1 activation does not induce bCEC proliferation, but may instead antagonize EGF-induced proliferation.

CONCLUSIONS

In summary, we find that CB1-based signaling machinery is present in bovine cornea and that activation of this system induces chemotaxis.

The role of spinal cannabinoid systems in neuropathic pain of streptozotocin (STZ)-induced diabetic mice was studied. In normal mice, injection of the cannabinoid receptor agonist WIN-55,<em>2</em>1<em>2</em>-<em>2</em> (1 and 3μg, i.t.) dose-dependently prolonged the tail-flick latency, whereas there were no changes with the injection of either cannabinoid CB1 (AM <em>2</em>51, 1 μg, i.t.) or CB<em>2</em> (AM 630, 4 μg, i.t.) receptor antagonists. AM <em>2</em>51 (1 μg, i.t.), but not AM 630 (4 μg, i.t.), significantly inhibited the prolongation of the tail-flick latency induced by WIN-55,<em>2</em>1<em>2</em>-<em>2</em> (3 μg, i.t.). In STZ-induced diabetic mice, the tail-flick latency was significantly shorter than that in normal mice. A low dose of WIN-55,<em>2</em>1<em>2</em>-<em>2</em> (1 μg, i.t.) significantly recovered the tail-flick latency in STZ-induced diabetic mice. The effect of WIN-55,<em>2</em>1<em>2</em>-<em>2</em> (1 μg, i.t.) in STZ-induced diabetic mice was significantly inhibited by AM 630 (4 μg, i.t.), but not AM <em>2</em>51 (1 μg). The selective cannabinoid CB<em>2</em> receptor agonist L-759,656 (19 and 38 μg, i.t.) also dose-dependently recovered the tail-flick latency in STZ-induced diabetic mice, and this recovery was inhibited by AM 630 (4 μg, i.t.). The protein levels of cannabinoid CB1 receptors, CB<em>2</em> receptors and diacylglycerol lipase α (DGL-α), the enzyme that synthesizes endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em>, in the spinal cord were examined using Western blotting. The protein levels of both cannabinoid CB1 and CB<em>2</em> receptors were increased in STZ-induced diabetic mice, whereas the protein level of DGL-α was significantly decreased. These results indicate that spinal cannabinoid systems are changed in diabetic mice and suggest that cannabinoid CB<em>2</em> receptor agonists might have an ability to recover diabetic neuropathic pain.

The biological activity of endocannabinoids like anandamide (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) is subjected in vivo to a "metabolic control", exerted mainly by catabolic enzymes. AEA is inactivated by fatty acid amide hydrolase (FAAH), that is inhibited competitively by hydroxyanandamides (HAEAs) generated from AEA by lipoxygenase activity. Among these derivatives, 15-HAEA has been shown to be an effective (K(i) approximately 0.6 muM) FAAH inhibitor, that blocks also type-1 cannabinoid receptor (CB1R) but not other components of the "endocannabinoid system (ECS)", like the AEA transporter (AMT) or CB<em>2</em>R. Here, we extended the study of the effect of 15-HAEA on the AEA synthetase (NAPE-PLD) and the AEA-binding vanilloid receptor (TRPV1), showing that 15-HAEA activates the former (up to approximately 140% of controls) and inhibits the latter protein (down to approximately 70%). We also show that 15-HAEA halves the synthesis of <em>2</em>-AG and almost doubles the transport of this compound across the membrane. In addition, we synthesized methyl and acetyl derivatives of 15-HAEA (15-MeOAEA and 15-AcOAEA, respectively), in order to check their ability to modulate FAAH and the other ECS elements. In fact, methylation and acetylation are common biochemical reactions in the cellular environment. We show that 15-MeOAEA, unlike 15-AcOAEA, is still a powerful competitive inhibitor of FAAH (K(i) approximately 0.7 muM), and that both derivatives have negligible interactions with the other proteins of ECS. Therefore, 15-MeOAEA is a FAAH inhibitor more selective than 15-HAEA. Further molecular dynamics analysis gave clues to the molecular requirements for the interaction of 15-HAEA and 15-MeOAEA with FAAH.

In this study, we have ascertained the presence and functionality in mouse embryonic stem cells (ESCs) of members of the endocannabinoid system that have been proposed as possible modulators of the survival and differentiation of various type of stem cells. We show that mouse ESCs, in addition to classical CB(1) and CB(<em>2</em>) cannabinoid receptors, express the transient receptor potential vanilloid receptor, at mRNA, protein, and binding levels. Remarkably, we demonstrate that ESCs have the mRNA, protein, and enzyme activity to synthesize and degrade the prominent endocannabinoids anandamide (through N-acyl-phosphatidylethanolamine-specific phospholipase D and fatty acid amide hydrolase) and <em>2</em>-<em>arachidonoylglycerol</em> (through diacylglycerol lipase and monoacylglycerol lipase). In addition, both endocannabinoids were detected in ESCs that were also shown to constitutively release a fatty acid amide hydrolase-activating compound. Finally, we document that the stimulation of ESCs by methanandamide, a nonhydrolysable analog of anandamide, does not lead to overt alteration of the expression of Oct3/4, Nanog, and Cdx<em>2</em>, genes that are involved in early cell fate in the preimplantation embryo and stemness, or of the expression patterns of Brachyury and Hnf4, genes that are used as late markers of lineage differentiation capability of ESC-derived embryoid bodies. Similarly ineffective on the expression of the tested stemness genes was <em>2</em>-<em>arachidonoylglycerol</em>. Taken together, these results confirm and extend the notion that ESCs express several functional members of the endocannabinoid system, but they leave open the question about their role in stem cells as modulators of stemness and differentiation potential.

Novel monocyclic analogues of <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) were designed in order to explore the pharmacophoric conformations of this endocannabinoid ligand at the key cannabinergic proteins. All <em>2</em>-arachidonoyl esters of 1,<em>2</em>,3-cyclohexanetriol [meso-7 (AM5504), (+/-)-8 (AM5503), and meso-9 (AM5505)] were synthesized by regioselective acylation of <em>2</em>,3-dihydroxycyclohexanone followed by selective reductions. The optically active isomers (+)-8 (AM4434) and (-)-8 (AM4435) were synthesized from (<em>2</em>S,3S)- and (<em>2</em>R,3R)-<em>2</em>,3-dihydroxycyclohexanone, respectively, via a chemoenzymatic route. These head group constrained and conformationally restricted analogues of <em>2</em>-AG as well as the 1-keto precursors were evaluated as substrates for the endocannabinoid deactivating hydrolytic enzymes monoacylglycerol lipase (MGL) and fatty acid amide hydrolase (FAAH), and also were tested for their affinities for CB1 and CB<em>2</em> cannabinoid receptors. The observed biochemical differences between these ligands can help define the conformational requirements for <em>2</em>-AG activity at each of the above endocannabinoid protein targets.

Endocannabinoid system is reported to be activated during myocardial ischemia-reperfusion (IR) injury and protects against heart injury. We, therefore, observed changes in endocannabinoids levels during acute myocardial infarction (AMI) and myocardial IR injury and evaluated the role of cannabinoid-<em>2</em> (CB<em>2</em>) receptor in infarct and IR heart injury. In contrast to 16 control patients with normal coronary artery angiogram, the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> level in the infarct-side coronary artery of <em>2</em>3 AMI patients increased significantly, with increased reactive oxygen species and tumor necrosis factor-α levels in both infarct-side coronary artery and radial artery. Then, 35 C57BL/6J mice were made into SHAM, AMI, or IR models. AMI and IR groups were treated with CB<em>2</em>-selective agonist HU308 ((+)-(1aH,3H,5aH)-4-[<em>2</em>,6-dimethoxy-4-(1,1-dimethylheptyl)phenyl]-6,6-dimethylbicyclo[3.1.1]hept-<em>2</em>-ene-<em>2</em>-carbinol), with or without CB<em>2</em>-selective antagonist AM630 [6-iodo-<em>2</em>-methyl-1-[<em>2</em>-(4-morpholinyl)ethyl]-1H-indol-3-yl](4-methoxyphenyl)methanone through intraperitoneal injection. Compared with the SHAM, expressions of cannabinoid CB1/CB<em>2</em> receptor proteins in AMI/IR animals were upregulated; production of <em>2</em>-<em>arachidonoylglycerol</em> and anandamide and release of reactive oxygen species and tumor necrosis factor-α also increased. HU308 significantly decreased the infarct size and the levels of reactive oxygen species and tumor necrosis factor-α in AMI/IR animals. However, these effects were blocked by AM630. In conclusion, the endocannabinoid system was activated during AMI and IR, and CB<em>2</em> receptor activation produces a protective role, thus offering a novel pharmaceutical target for treating these diseases.

Cannabinoid signaling by CB1 receptors drives fibrogenesis and fat accumulation in liver. A report in this issue of Cell Metabolism (Jeong et al., <em>2</em>008) now links hepatic stellate cells, a resident liver fibrogenic cell type, to the generation of steatosis through production of the endocannabinoid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) after ethanol feeding, leading to paracrine stimulation of hepatocyte CB1 receptors.

<em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) is an endogenous ligand for the cannabinoid receptors with a variety of potent biological activities. In this study, we first examined the effects of potassium-induced depolarization on the level of <em>2</em>-AG in rat brain synaptosomes. We found that a significant amount of <em>2</em>-AG was generated in the synaptosomes following depolarization. Notably, depolarization did not affect the levels of other molecular species of monoacylglycerols. Furthermore, the level of anandamide was very low and did not change markedly following depolarization. It thus appeared that the depolarization-induced accelerated generation is a unique feature of <em>2</em>-AG. We obtained evidence that phospholipase C is involved in the generation of <em>2</em>-AG in depolarized synaptosomes: U731<em>2</em><em>2</em>, a phospholipase C inhibitor, markedly reduced the depolarization-induced generation of <em>2</em>-AG, and the level of diacylglycerol was rapidly elevated following depolarization. A significant amount of <em>2</em>-AG was released from synaptosomes upon depolarization. Interestingly, treatment of the synaptosomes with SR141716A, a CB1 receptor antagonist, augmented the release of glutamate from depolarized synaptosomes. These results strongly suggest that the endogenous ligand for the cannabinoid receptors, i.e. <em>2</em>-AG, generated through increased phospholipid metabolism upon depolarization, plays an important role in attenuating glutamate release from the synaptic terminals by acting on the CB1 receptor.

This chapter briefly describes the physiological neural mechanisms by which diverse neurotransmitter receptor systems control several aspects of gastrointestinal functions such as motility, secretion, feeding, and emesis. The current techniques used to study the effects of cannabinoids on these gastrointestinal functions are then sequentially described, starting with isolated gastrointestinal muscle preparations and ultimately evolving to whole animal models. Both delta9-tetrahydrocannibinol (delta9-THC) and well-studied representatives of other classes of exogenous cannabinoid CB1/CB<em>2</em> receptor agonists inhibit gastrointestinal motility, peristalsis, defecation, and secretions via cannabinoid CB1 receptors since the CB1 (SR141716A)- and not the CB<em>2</em> (SR1445<em>2</em>8)-receptor antagonist reverses these effects in a dose-dependent manner. In addition, exogenous cannabinoids inhibit vomiting produced by diverse emetic stimuli in a SR141716A-sensitive manner in different animal models of emesis. Often these cannabinoids produce hyperphagic effects under laboratory conditions in most human and animal models of feeding. Administration of SR141716A by itself can produce effects opposite to cannabinoid agonists (e.g., increases in gastrointestinal motility and secretions, hyperphagia and vomiting), which suggests an important role for endocannabinoids in these gastrointestinal functions. Indeed, the presence of cannabinoid CB1 receptor markers, endocannabinoids such as anandamide and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), their metabolic enzymes, and an endocannabinoid reuptake system have been confirmed in the gastrointestinal tract (GIT). The well-studied endocannabinoid anandamide also seems to reduce both gastrointestinal motility and secretion while producing hyperphagia. On the other hand, while the less well-investigated endocannabinoid <em>2</em>-AG is a potent emetogen, anandamide may possess weak antiemetic activity.

Fatty acid amide hydrolase (FAAH) and monoglyceride lipase (MGL) are the main enzymes responsible for the hydrolysis of endogenous cannabinoids N-arachidonoylethanolamide (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), respectively. Phenyl alkylcarbamates are FAAH inhibitors with anxiolytic and analgesic activities in vivo. Herein we present for the first time the synthesis and biological evaluation of a series of chiral 3-(<em>2</em>-oxazoline)-phenyl N-alkylcarbamates as FAAH inhibitors. Furthermore, the structural background of chirality on the FAAH inhibition is explored by analyzing the protein-ligand interactions. Remarkably, 10-fold difference in potency was observed for (R)- and (S)-derivatives of 3-(5-methyl-4,5-dihydrooxazol-<em>2</em>-yl)phenyl cyclohexylcarbamate (6a vs. 6b). Molecular modelling indicated an important interaction between the oxazoline nitrogen and FAAH active site.

Cannabinoids suppress nocifensive behaviors in rodents. We presently investigated peripheral endocannabinoid modulation of itch- and pain-related behaviors elicited from facial vs. spinally-innervated skin of rats. Intradermal (id) injection of the pruritogen serotonin (5-HT) elicited significantly more hindlimb scratch bouts, and longer cumulative time scratching, when injected in the rostral back compared to the cheek. Pretreatment of skin with inhibitors of degrading enzymes for the endocannabinoids anandamide (URB597) or <em>2</em>-<em>arachidonoylglycerol</em> (JZL184) significantly reduced scratching elicited by 5-HT in the rostral back. These effects were prevented by co-treatment with antagonists of the CB₁ (AM<em>2</em>51) or CB₂ receptor (AM630), implicating both receptor subtypes in endocannabinoid suppression of scratching in spinally-innervated skin. Conversely, pretreatment with either enzyme inhibitor, or with AM630 alone, increased the number of scratch bouts elicited by id 5-HT injection in the cheek. Moreover, pretreatment with JZL184 also significantly increased pain-related forelimb wipes directed to the cheek following id injection of the algogen, allyl isothiocyanate (AITC; mustard oil). Thus, peripheral endocannabinoids have opposite effects on itch-related scratching behaviors in trigeminally- vs. spinally-innervated skin. These results suggest that increasing peripheral endocannabinoid levels represents a promising therapeutic approach to treat itch arising from the lower body, but caution that such treatment may not relieve, and may even exacerbate, itch and pain arising from trigeminally-innervated skin of the face or scalp.

Cytosolic phospholipase A<em>2</em> alpha (cPLA<em>2</em>α) responds to micromolar intracellular Ca(<em>2</em>+) and produces arachidonic acid, which regulates cellular homeostasis, neurotoxicity, and inflammation. Endocannabinoids are the derivates of arachidonic acid and widely distributed in the cerebellum. However, the role of cPLA<em>2</em>α/arachidonic acid pathway in cerebellar synaptic transmission and plasticity is unknown. We utilized cPLA<em>2</em>α knockout mice and slice whole-cell patch clamp to study the action of cPLA<em>2</em>α/arachidonic acid signaling on the depolarization-induced suppression of excitation (DSE) and long-term potentiation at parallel fiber-Purkinje cell synapses. Our data showed that DSE was significantly inhibited but rescued by arachidonic acid in cPLA<em>2</em>α knockout mice. The degradation enzyme of <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), monoacylglycerol lipase, blocked DSE, while another catabolism enzyme for N-arachidonoylethanolamine, fatty acid amide hydrolase, did not, suggesting that <em>2</em>-AG is responsible for DSE in Purkinje cells. Co-application of paxilline reversed the blockade of DSE by internal K(+), indicating that large-conductance Ca(<em>2</em>+)-activated potassium channel is sufficient to inhibit cPLA<em>2</em>α/arachidonic acid-mediated DSE. On the other hand, we found that 1 Hz parallel fiber stimuli-triggered long-term potentiation (LTP) was deficient in cPLA<em>2</em>α knockout mice. LTP was also inhibited when AACOCF3, an inhibitor of cPLA<em>2</em>α, was given. Arachidonic acid was necessary for the LTP induction. Therefore, these data showed that cPLA<em>2</em>α/arachidonic acid/<em>2</em>-AG signaling pathway mediates DSE and LTP at parallel fiber-Purkinje cell synapse.

This study aimed to investigate potential new target(s)/mechanism(s) for the palmitoylethanolamide (PEA) analogue, adelmidrol, and its role in an in vitro model of contact allergic dermatitis. Freshly isolated canine keratinocytes, human keratinocyte (HaCaT) cells and human embryonic kidney (HEK)-<em>2</em>93 cells, wild-type or transfected with cDNA encoding for N-acylethanolamine-hydrolysing acid amidase (NAAA), were treated with adelmidrol or azelaic acid, and the concentrations of endocannabinoids (anandamide and <em>2</em>-<em>arachidonoylglycerol</em>) and related mediators (PEA and oleoylethanolamide) were measured. The mRNA expression of PEA catabolic enzymes (NAAA and fatty acid amide hydrolase, FAAH), and biosynthetic enzymes (N-acyl phosphatidylethanolamine-specific phospholipase D, NAPE-PLD) and glycerophosphodiester phosphodiesterase 1, was also measured. Brain or HEK-<em>2</em>93 cell membrane fractions were used to assess the ability of adelmidrol to inhibit FAAH and NAAA activity, respectively. HaCaT cells were stimulated with polyinosinic-polycytidylic acid and the release of the pro-inflammatory chemokine, monocyte chemotactic protein-<em>2</em> (MCP-<em>2</em>), was measured in the presence of adelmidrol. Adelmidrol increased PEA concentrations in canine keratinocytes and in the other cellular systems studied. It did not inhibit the activity of PEA catabolic enzymes, although it reduced their mRNA expression in some cell types. Adelmidrol modulated the expression of PEA biosynthetic enzyme, NAPE-PLD, in HaCaT cells, and inhibited the release of the pro-inflammatory chemokine MCP-<em>2</em> from stimulated HaCaT cells. This study demonstrates for the first time an 'entourage effect' of adelmidrol on PEA concentrations in keratinocytes and suggests that this effect might mediate, at least in part, the anti-inflammatory effects of this compound in veterinary practice.

BACKGROUND

Endogenous corticosteroids and endocannabinoids are both known to be involved in stress adaption and anti-inflammatory and immuneregulatory effects. The application of hair as retrospective specimen for long-term recording of corticosteroids and its association with stress-induced biochemical alterations was intensively examined.

METHODS

To evaluate the stability and correlation of various parameters of the endocannabinoid and corticosteroid systems, a prospective study was carried out. Hair samples were collected monthly over a pregnancy cycle (sixth week of pregnancy to 9 weeks postpartum). By comparison of hair concentrations in particular segments (ie, grown in the same time span but collected at different times), an examination of analyte stability in hair was achieved. Additionally, the comparison of proximal segments provided on biochemical information that is independent of alteration due to physical instability. The detection limits of a validated procedure using solid-phase extraction cleanup and liquid chromatography-mass spectrometry proved to be suitable to identify the endogenous levels of cortisol (limits of detection = 1.6 pg/mg), cortisone (<em>2</em>.1 pg/mg), anandamide (AEA, 0.3 pg/mg), and <em>2</em>-<em>arachidonoylglycerol</em> (15 pg/mg).

RESULTS

Corticosteroid concentrations in corresponding hair segments were found to be reduced with increasing hair age; an average decline of cortisol and cortisone by 50% in 4 months was estimated. Independently, an increase of cortisol and cortisone in proximal segments collected during pregnancy was confirmed, which is assumed to be stress related. Endocannabinoids proved to be by far more stable, as demonstrated by subsequent monthly collection of corresponding segments and there was hardly any washout of AEA detectable. Elevated hair concentrations of AEA and <em>2</em>-<em>arachidonoylglycerol</em> were detected in the first-second trimester of pregnancy, which corresponds to negative correlations between AEA, cortisol, and cortisone.

The metabolic intermediate and endocannabinoid signaling lipid <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) has not been readily labeled, primarily because of its instability toward rearrangement. We now detail a synthetic method that easily gives tritiated <em>2</em>-AG from [5,6,8,9,11,1<em>2</em>,14,15-(3)H(N)]arachidonic acid in two steps. We utilized a short chain 1,3-diacylglycerol and proceeded through the "structured lipid" [5'',6'',8'',9'',11'',1<em>2</em>'',14'',15''-(3)H(N)]<em>2</em>-arachidonoyl-1,3-dibutyrylglycerol, a triacylglycerol that was conveniently deprotected in ethanol with acrylic beads containing Candida antarctica lipase B to give [5'',6'',8'',9'',11'',1<em>2</em>'',14'',15''-(3)H(N)]<em>2</em>-<em>arachidonoylglycerol</em> ([(3)H]<em>2</em>-AG). The flash chromatographic separation necessary to isolate the labeled <em>2</em>-acylglycerol [(3)H]<em>2</em>-AG resulted in only 4% of the rearrangement byproducts that have been a particular problem with previous methodologies. This reliable "kit" method to prepare the radiolabeled endocannabinoid as needed gave tritiated <em>2</em>-<em>arachidonoylglycerol</em> [(3)H]<em>2</em>-AG with a specific activity of <em>2</em>00 Ci/mmol for enzyme assays, metabolic studies, and tissue imaging. It has been run on unlabeled materials on over 10 mg scales and should be generally applicable to other <em>2</em>-acylglycerols.

We represented the endocannabinoid system (ECS) as a biological network, where ECS molecules are the nodes (1<em>2</em>3) and their interactions the links (189). ECS network follows a scale-free topology, which confers robustness against random damage, easy navigability, and controllability. Network topological parameters, such as clustering coefficient (i.e., how the nodes form clusters) of 0.0009, network diameter (the longest shortest path among all pairs of nodes) of 1<em>2</em>, averaged number of neighbors (the mean number of connections per node) of 3.073, and characteristic path length (the expected distance between two connected nodes) of 4.715, suggested that molecular messages are transferred through the ECS network quickly and specifically. Interestingly, ∼75% of nodes are located on, or are active at the level of, the cell membrane. The hubs of ECS network are anandamide (AEA) and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), which have also the highest value of betweeness centrality, and their removal causes network collapse into multiple disconnected components. Importantly, AEA is a ubiquitous player while <em>2</em>-AG plays more restricted actions. Instead, the product of their degradation, arachidonic acid, and their hydrolyzing enzyme, fatty acid amide hydrolase, FAAH, have a marginal impact on ECS network, indeed their removal did not significantly affect its topology.

α/β-Hydrolase domain-containing 6 (ABHD6) represents a potentially attractive therapeutic target for indirectly potentiating <em>2</em>-<em>arachidonoylglycerol</em> signaling; however, the enzyme is currently largely uncharacterized. Here, we describe a five element, ligand-based pharmacophore model along with a refined homology model of ABHD6. Following a virtual screen of a modest database, both the pharmacophore and homology models were found to be highly predictive, preferentially identifying ABHD6 inhibitors over drug-like non-inhibitors. The models yield insight into the features required for optimal ligand binding to ABHD6 and the atomic structure of the binding site. In combination, the two models should be very helpful not only in high-throughput virtual screening, but also in lead optimization, and will facilitate the development of novel, selective ABHD6 inhibitors as potential drugs.

Cyclooxygenase-<em>2</em> (COX-<em>2</em>) oxygenates arachidonic acid (AA) and its ester analog, <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), to prostaglandins (PGs) and prostaglandin glyceryl esters (PG-Gs), respectively. Although the efficiency of oxygenation of these substrates by COX-<em>2</em> in vitro is similar, cellular biosynthesis of PGs far exceeds that of PG-Gs. Evidence that the COX enzymes are functional heterodimers suggests that competitive interaction of AA and <em>2</em>-AG at the allosteric site of COX-<em>2</em> might result in differential regulation of the oxygenation of the two substrates when both are present. Modulation of AA levels in RAW<em>2</em>64.7 macrophages uncovered an inverse correlation between cellular AA levels and PG-G biosynthesis. In vitro kinetic analysis using purified protein demonstrated that the inhibition of <em>2</em>-AG oxygenation by high concentrations of AA far exceeded the inhibition of AA oxygenation by high concentrations of <em>2</em>-AG. An unbiased systems-based mechanistic model of the kinetic data revealed that binding of AA or <em>2</em>-AG at the allosteric site of COX-<em>2</em> results in a decreased catalytic efficiency of the enzyme toward <em>2</em>-AG, whereas <em>2</em>-AG binding at the allosteric site increases COX-<em>2</em>'s efficiency toward AA. The results suggest that substrates interact with COX-<em>2</em> via multiple potential complexes involving binding to both the catalytic and allosteric sites. Competition between AA and <em>2</em>-AG for these sites, combined with differential allosteric modulation, gives rise to a complex interplay between the substrates, leading to preferential oxygenation of AA.

OBJECTIVE

Changes in tissue levels of <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), an endocannabinoid, during the evolution of bile duct ligation (BDL) may indicate that endocannabinoids have a role in the hemodynamic changes that occur in this condition.

METHODS

<em>2</em>-AG levels, in various organs and vascular beds of BDL rats, <em>2</em> and 4 weeks post surgery, were determined. Untouched and sham-operated (SO) rats were used as controls.

RESULTS

<em>2</em>-AG content of a specific organ was not a static finding and depended on the rat's age, the time from the surgical procedure and the type of procedure. The most pronounced changes were observed in BDL rats 4 weeks post surgery. In these rats, hepatic, pulmonary, cardiac and renal medullary and papillary <em>2</em>-AG levels were highest observed. No changes in splenic, aortic and renal cortical <em>2</em>-AG levels were observed. In addition a stepwise increase in <em>2</em>-AG levels from the cortex to the papilla was detected and was followed by a decrease in creatinine clearance.

CONCLUSIONS

<em>2</em>-AG probably has a role in the pathophysiologic changes in the liver, heart, lung and kidney that follows BDL.

BACKGROUND

Increased endocannabinoid tonus by dual-action fatty acid amide hydrolase (FAAH) and substrate selective cyclooxygenase (COX-<em>2</em>) inhibitors is a promising approach for pain-relief. One such compound with this profile is <em>2</em>-(<em>2</em>-fluorobiphenyl-4-yl)-N-(3-methylpyridin-<em>2</em>-yl)propanamide (Flu-AM1). These activities are shown by Flu-AM1 racemate, but it is not known whether its two single enantiomers behave differently, as is the case towards COX-<em>2</em> for the parent flurbiprofen enantiomers. Further, the effects of the compound upon COX-<em>2</em>-derived lipids in intact cells are not known.

RESULTS

COX inhibition was determined using an oxygraphic method with arachidonic acid and <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG) as substrates. FAAH was assayed in mouse brain homogenates using anandamide (AEA) as substrate. Lipidomic analysis was conducted in unstimulated and lipopolysaccharide + interferon γ- stimulated RAW <em>2</em>64.7 macrophage cells. Both enantiomers inhibited COX-<em>2</em> in a substrate-selective and time-dependent manner, with IC50 values in the absence of a preincubation phase of: (R)-Flu-AM1, COX-1 (arachidonic acid) 6 μM; COX-<em>2</em> (arachidonic acid) <em>2</em>0 μM; COX-<em>2</em> (<em>2</em>-AG) 1 μM; (S)-Flu-AM1, COX-1 (arachidonic acid) 3 μM; COX-<em>2</em> (arachidonic acid) 10 μM; COX-<em>2</em> (<em>2</em>-AG) 0.7 μM. The compounds showed no enantiomeric selectivity in their FAAH inhibitory properties. (R)-Flu-AM1 (10 μM) greatly inhibited the production of prostaglandin D<em>2</em> and E<em>2</em> in both unstimulated and lipopolysaccharide + interferon γ- stimulated RAW <em>2</em>64.7 macrophage cells. Levels of <em>2</em>-AG were not affected either by (R)-Flu-AM1 or by 10 μM flurbiprofen, either alone or in combination with the FAAH inhibitor URB597 (1 μM).

CONCLUSIONS

Both enantiomers of Flu-AM1 are more potent inhibitors of <em>2</em>-AG compared to arachidonic acid oxygenation by COX-<em>2</em>. Inhibition of COX in lipopolysaccharide + interferon γ- stimulated RAW <em>2</em>64.7 cells is insufficient to affect <em>2</em>-AG levels despite the large induction of COX-<em>2</em> produced by this treatment.

We have previously reported that maternal separation (MS) for 3 h daily during the first two postnatal weeks increases wakefulness, whereas it reduces sleep in rats. Oleamide, an agonist of the cannabinoid receptor type 1, increases sleep in MS rats to such a level that we cannot differentiate their sleep patterns from those of their non-MS (NMS) siblings. However, <em>2</em>-<em>arachidonoylglycerol</em> (<em>2</em>-AG), an endocannabinoid, infused into the lateral hypothalamus of NMS rats at the beginning of the dark phase of the cycle increases rapid eye movement sleep and the expression of c-Fos on the rapid eye movement sleep promoting melanin-concentrating hormone neurons. We recorded the sleep-wake cycle of adult rats subjected to MS for 3 h daily from postnatal days <em>2</em> to 16, as well as in their NMS siblings. Besides the electrodes for recording the sleep-wake cycle, a couple of cannulae aimed bilaterally to the lateral hypothalamus were implanted to infuse <em>2</em>-AG. We found that administration of <em>2</em>-AG into the lateral hypothalamus of MS rats at the beginning of the light phase of the cycle restores sleep, whereas sleep and wakefulness of NMS rats under <em>2</em>-AG infusion do not show any significant change.