Anandamide Research Articles

Effects of N-Arachidonoylethanolamine on the Quality of Storage Platelets

Objective To evaluate the impact of N-Arachidonoylethanolamine(ANA)on the quality of platelets(PLT)stored at(22±2)℃.Methods From November 7th to 25th 2011,a total of 20 volunteers who donated PLT in Blood Center of Shandong Province were included into this study.Apheresis PLTs were collected through the Amicus instrument.Different working concentrations of ANA(0.1,0.5,1,5,10 and 50 μmol/L)were added to the PLTs samples,respectively,as the experimental group,another corresponding parts with no ANA as the controls,respectively.All the PLT samples were stored at(22±2)℃ for 7 days,then were evaluated the viability by measuring the mitochondrial reduction with methyl thiazolyl tetrazolium colorimetric for detecting the most effective concentration of ANA.Samples(50 mL/each)were taken and split into two storage parts(25 mL/each).The most effective concentration of ANA was added one part as the experimental group(n=20),the other with no ANA as the control group(n=20).These PLT samples were stored on a flat-bed shaker at(22±2)℃.PLT count,mean PLT volume (MPV),PLT distribution width(PDW),phosphatidyl serine(PS)and soluble P-selectin of the two groups were detected on the 1st,3rd,5th,7th,9th and 11th day of storage.Results The most effective final concentration of ANA was 0.5 μmol/L,which showed significant increasing PLTs survival(about 1.7 fold more than controls)(t=14.32,P＜0.05).During 11 days of storage,the PLT counts were not changed significantly until on the 11th day of storage between the experimental group and control group.Moreover,the MPV and PDW were decreased significantly in the experimental group than those in the control group on the 11th day of storage(P＜0.05),respectively.The rates of PLT PS positive were on the rise during the storage period:the experimental group were from(8.29 ±1.44)％ to(21.18 ± 1.78)％ while the control group were from(14.24±2.47)％ to(40.6± 1.95)％,with significant differences between the two groups (P＜0.05)on the 7th,9th and 11th day of storage,respectively.Soluble P-selectin contents of the experimental group on the 7th,9th and 11th day of storage were(75.08± 6.35)ng/mL,(81.58±6.09)ng/mL and(103.55±7.91)ng/mL,while those in the control group were(90.37±8.91)ng/mL,(117.56 ± 9.98)ng/mL and(144.7 ± 7.76)ng/mL,respectively,with significant differences between the two groups(P＜0.05).Conclusions 0.5 μmol/L ANA had a good protective effect on PLTs,and could potentially alleviate PLT storage lesions to some extent. Key words: endocannabinoids; blood platelet; blood preservation

Take an NSAID for Anxiety

Acting through cannabinoid receptors, endogenous endocannabinoids (eCBs), such as anandamide, modulate pain and anxiety, among other things; substances that inhibit enzymes that break down eCBs may have antianxiety properties. Because cyclooxygenase-2 (COX-2), which catalyzes synthesis of prostaglandins from arachidonic acid, also inactivates anandamide, researchers explored the …

The effect of anandamide on inhibiting the abilities of adhesion, chemotactic motility and invasion of U251 glioma cell line

Objective To investigate the effects of the cell adhesion,chemotactic motility and invasion in U251 glioma cell by anandamide (AEA) treatment.Methods Human U251 glioma cell line were cultured with DMEM's medium in vitro.Cultured cells were divided into control group,and 5 μmol/L AEA groups for 12 h and 24 h.The cell adhesion rate of U251 cell was investigated by methyl thiazol tetrazolium (MTT) assay at different times of AEA treatment.The abilities of chemotactic motility and invasion were detected by Transwell.Results MTF showed that AEA inhibited cell adhesion and the rate increased with increasing treatment time of 5 μmol/L AEA.The inhibitive rates were respectively 45.14％ for 12 h (P ＜ 0.05) and 66.53％ for 24 h (P ＜ 0.01).The abilities of chemotactic motility and invasion also were depressed respectively 45.24％ and 41.86％ for 12 h (P ＜ 0.05).Those rates were 81.08％ and 83.85％ for 24h (P ＜0.01).Conclusion AEA could inhibit the abilities of cell adhesion,chemotactic motility and invasion on human U251 glioma cell line,and this effect has the time dependence. Key words: Anandamide ; Adhesion ; Chemotactic motility ; Cell invasion; Glioma cell

Abstract 501: Enhanced Expression of the Endocannabinoid 2-AG and CB-1 Receptor in the Kidney of the Diabetic Hypertensive Female Rat

The cannabinoids arachidonoylethanolamide (AEA) and 2-arachidonoylglycerol (2-AG) are endogenous ligands for the cannabinoid receptors including CB1 and CB2 subtypes. The endocannabinoid system (ECS) is typically associated with regulation of metabolic balance and appetite, although CB1 receptors (CB1R) may contribute to diabetic renal injury while CB2R are protective. The hypertensive mRen2.Lewis (mRen2) females have less renal injury than the males; however, diabetic female mRen2 lack this protection and show comparable indices of injury to the diabetic males. The present study addressed whether female and male diabetic mRen2 exhibit similar alterations in the renal ECS. Diabetes was induced with a single dose of streptozotocin (STZ at 65 mg/kg; ip). Cortical content of AEA and 2-AG was quantified by UPLC MS/MS. Receptor levels and the metabolizing enzymes for AEA (FAAH) and 2-AG (MAGL) were quantified by protein immunoblot. Basal 2-AG content was higher in male versus female mRen2, and significantly increased in both diabetic females and males (figure, n=6) while AEA was unchanged. Cortical Ang II correlated to 2-AG [p<0.01; r = 0.89]. CB1R protein expression was higher in female diabetic mRen2 but unchanged in the males (figure, n=4). CB2R expression was lower in the male diabetics [p<0.01; n=4], but not changed in females. MAGL expression was >70% lower in female diabetics compared to controls [p<0.05; n=4] while FAAH was not changed. Elements of the renal ECS are differentially regulated in the diabetic mRen2. Higher expression of both 2-AG and CB1R, but reduced MAGL (females) and CB2R (males) may contribute to the development and progression of renal injury in this model.

Plasma Concentrations of Endocannabinoids and Related Primary Fatty Acid Amides in Patients with Post-Traumatic Stress Disorder

BackgroundEndocannabinoids (ECs) and related N-acyl-ethanolamides (NAEs) play important roles in stress response regulation, anxiety and traumatic memories. In view of the evidence that circulating EC levels are elevated under acute mild stressful conditions in humans, we hypothesized that individuals with traumatic stress exposure and post-traumatic stress disorder (PTSD), an anxiety disorder characterized by the inappropriate persistence and uncontrolled retrieval of traumatic memories, show measurable alterations in plasma EC and NAE concentrations.MethodsWe determined plasma concentrations of the ECs anandamide (ANA) and 2-arachidonoylglycerol (2-AG) and the NAEs palmitoylethanolamide (PEA), oleoylethanolamide (OEA), stearoylethanolamine (SEA), and N-oleoyldopamine (OLDA) by HPLC-MS-MS in patients with PTSD (n = 10), trauma-exposed individuals without evidence of PTSD (n = 9) and in healthy control subjects (n = 29). PTSD was diagnosed according to DSM-IV criteria by administering the Clinician Administered PTSD Scale (CAPS), which also assesses traumatic events.ResultsIndividuals with PTSD showed significantly higher plasma concentrations of ANA (0.48±0.11 vs. 0.36±0.14 ng/ml, p = 0.01), 2-AG (8.93±3.20 vs. 6.26±2.10 ng/ml, p<0.01), OEA (5.90±2.10 vs. 3.88±1.85 ng/ml, p<0.01), SEA (2.70±3.37 vs. 0.83±0.47, ng/ml, p<0.05) and significantly lower plasma levels of OLDA (0.12±0.05 vs. 0.45±0.59 ng/ml, p<0.05) than healthy controls. Moreover, PTSD patients had higher 2-AG plasma levels (8.93±3.20 vs. 6.01±1.32 ng/ml, p = 0.03) and also higher plasma concentrations of PEA (4.06±1.87 vs. 2.63±1.34 ng/ml, p<0.05) than trauma-exposed individuals without evidence of PTSD. CAPS scores in trauma-exposed individuals with and without PTSD (n = 19) correlated positively with PEA (r = 0.55, p = 0.02) and negatively with OLDA plasma levels (r = −0.68, p<0.01). CAPS subscores for intrusions (r = −0.65, p<0.01), avoidance (r = −0.60, p<0.01) and hyperarousal (r = −0.66, p<0.01) were all negatively related to OLDA plasma concentrations.ConclusionsPTSD appears to be associated with changes in plasma EC/NAE concentrations. This may have pathophysiological and diagnostic consequences but will need to be reproduced in larger cohorts.

Enrichment of Arachidonic Acid for the Enzymatic Synthesis of Arachidonoyl Ethanolamide

AbstractArachidonoyl ethanolamide is an endogenous cannabinoid neurotransmitter that potentially has therapeutic properties. In this study, we report an enzymatic method to synthesize this bioactive ethanolamide. First, free fatty acids were obtained from arachidonic acid‐rich oil and then arachidonic acid was enriched by urea inclusion and AgNO3 solution fractionation. Arachidonic acid content was increased from 40.2 to 78.4 % with 27.6 ± 1.8 % total fatty acid mass yield (w/w, relative to total free fatty acid) after urea inclusion. Purification of arachidonic acid by AgNO3 solution fractionation was optimized, and under the optimal conditions, a product with 90.7 % arachidonic acid was obtained with 80.4 % mass yield (w/w, relative to total free fatty acid). Finally, arachidonoyl ethanolamide was synthesized by reacting the purified arachidonic acid with ethanolamine in hexane using Novozym 435 lipase of Novozymes America (Blair, NE), which resulted in the formation of 88.4 ± 0.6 % arachidonoyl ethanolamide with 72.7 ± 2.2 % mass yield. The main novelties of this study are the enrichment of polyunsaturated fatty acids by AgNO3 solution fractionation which has received little attention in recent years, and this is the first time the synthesis of arachidonoyl ethanolamide is reported.

Detection of the endocannabinoid metabolome in human plasma and breast milk

The endocannabinoid metabolome (ENDOMET), endogenous cannabinoids that are lipid messengers, is critical for early development, including fertilization, implantation and pre‐ and postpartum (PP). The regulation underlying these physiological effects remains to be completely elucidated. We evaluated the ENDOMET in plasma of i) non‐pregnant (n=6) and pregnant women (20–22weeks‐ delivery, n=68) and ii) breast milk (BM) at 2 and 10 weeks PP (n= 20). The plasma and BM ENDOMETs [arachidonoyl ethanolamide (ET) (AEA), palmitoyl ET (PEA), oleoyl ET (OEA), docosahexaenoyl ET (DHEA), 2‐arachidonol glycerol (GLY) (2‐AG), 2‐palmitoyl GLY (PEA), and 2‐oleoyl GLY (2‐OG)] were measured using liquid chromatography‐mass spectrometry. ANOVA determined differences in the ENDOMET across time.Resultsplasma ETs increase with pregnancy and peak at delivery (AEA, PEA, OEA, DHEA, p&lt;0.01); GLYs decrease during pregnancy and are stable (2‐AG, p=0.14; 2‐PG, p=0.07, 2‐OG, p=0.02). While BM ETs are considerably lower than plasma ET in pregnancy, BM levels remain stable during PP. BM GLYs are higher than plasma GLYs in late pregnancy. 2‐OG and 2‐AG decrease during PP (p=0.02; 0.05). Plasma and BM ETs are much lower in concentrations than GLYs. Our data point to a reciprocal relationship between ETs and GLYs during pregnancy and PP. Work is needed to better understand how maternal plasma and BM ENDOs regulate pre‐and postnatal outcomes.

Characterisation of the prostaglandin E2-ethanolamide suppression of tumour necrosis factor-α production in human monocytic cells

Background and purposeProstaglandin ethanolamides or prostamides are naturally occurring neutral lipid derivatives of prostaglandins that have been shown to be synthesised in vivo following COX-facilitated oxygenation of arachidonoyl ethanolamine (anandamide). Although the actions of prostaglandins have been extensively studied, little is known about the physiological or pathophysiological effects of prostamides. Since prostaglandin E2 has potent immunosuppressive/immunomodulating actions, the aim of the present study was to determine whether the derivative, prostaglandin E2 ethanolamide (PGE2-EA), could modulate the production of the pro-inflammatory cytokine tumour necrosis factor-α in human blood and human monocytic cells and indicate whether this action involved the same receptor systems/signals as PGE2. Experimental approachWhole human blood, monocytes isolated from the blood or the human monocytic cell line THP-1 was incubated with LPS and the level of TNF-α produced was measured by ELISA assay. The actions of PGE2-EA were assessed on the LPS-induced TNF-α release. In addition, in order to ascertain the receptors involved, the levels of cyclic AMP in cells were measured in monocytes and THP-1 cells in response to PGE2-EA and directly compared to those of PGE2. The effect of PGE2-EA on the binding of radiolabelled PGE2 to cells was also measured. Cells were incubated with radiolabelled arachidonic acid and ethanolamine to estimate the production of PGE2-EA. Key resultsPGE2-EA potently suppressed TNF-α production in blood, monocytes and the cell line THP-1 in a concentration-dependent manner. This occurred via cyclic AMP pathways as indicated by agents which interfere with these pathways and also direct ligand binding experiments. It was also shown that the cells were able to endogenously produce PGE2-EA. Conclusions and implicationsThis study reports that PGE2-EA can downregulate the production of TNF-α by human mononuclear cells in response to an immune stimulus, i.e. LPS-activated TLR4, and that this appears to occur via a cAMP-dependent mechanism that most likely involves binding to the EP2 receptor.

The Contractile Effect of Anandamide in the Guinea‐Pig Small Intestine is Mediated by Prostanoids but not TRPV1 Receptors or Capsaicin‐Sensitive Nerves

Although exogenous and endogenous cannabinoid receptor agonists have well-documented inhibitory effects on gastrointestinal motility, a TRPV1 receptor-mediated excitatory action of anandamide (arachidonoyl ethanolamide, AEA) in the guinea-pig ileum strip has also been described. We used in vitro capsaicin desensitization for assessing the possible participation of sensory neurons in the contractile effect of anandamide on the guinea-pig whole ileum, as well as autonomic drugs and a cyclooxygenase inhibitor for characterizing this response. Isolated organ experiments were used with isotonic recording. Contractions induced by anandamide (1 or 10 μM) were strongly inhibited by tetrodotoxin, indomethacin or atropine plus a tachykinin NK(1) receptor antagonist, but weakly to moderately reduced by atropine alone and partly diminished by the fatty acid amide hydrolase inhibitor URB 597. Neither capsaicin pre-treatment nor the TRPV1 receptor antagonist BCTC, the ganglionic blocking drug hexamethonium or cannabinoid (CB1 or CB2 ) receptor antagonists, influenced the effect of anandamide. It is concluded that the capsaicin-insensitive, neuronal excitatory effect of anandamide in the intestine is most probably mediated by cyclooxygenase products. Such a mechanism may also play a role at other sites in the mammalian body.

Liquid chromatography–tandem mass spectrometry analysis of free and esterified fatty acid N-acyl ethanolamines in plasma and blood cells

The origin of N-acyl ethanolamides (NAEs) in plasma is not well understood, and it is possible that NAEs are present in plasma in esterified form. To test this hypothesis, a new and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for the quantification of arachidonoyl ethanolamide, 2-arachidonoyl glycerol, docosahexaenoyl ethanolamide, dihomo-γ-linolenoyl ethanolamide, oleoyl ethanolamide, palmitoyl ethanolamide, and stearoyl ethanolamide in 100μl of human plasma using a simple acetonitrile extraction step. Using this method, we determined (i) free and esterified NAE levels in human plasma, (ii) free and esterified NAE levels in plasma of mice fed with diets with different amounts of n-3 fatty acids, and (iii) esterified NAE levels in blood cells. Murine and human plasma extracts contained 20- to 60-fold higher levels of esterified NAEs than free NAEs. Moreover, the effect of dietary n-3 fatty acids on murine free plasma NAE profiles was similar for esterified NAEs. Finally, esterified NAEs were also present in murine blood cells, and their pattern followed the same diet effect as observed for free and esterified NAEs in plasma. Together, these data point to the presence of previously ignored pools of esterified NAEs in plasma and blood cells that correlated well with free NAE levels in plasma.

Early Endogenous Activation of CB1 and CB2 Receptors after Spinal Cord Injury Is a Protective Response Involved in Spontaneous Recovery

Spinal cord injury (SCI) induces a cascade of processes that may further expand the damage (secondary injury) or, alternatively, may be part of a safeguard response. Here we show that after a moderate-severe contusive SCI in rats there is a significant and very early increase in the spinal cord content of the endocannabinoids 2-arachidonoylglycerol (2-AG) and arachidonoyl ethanolamide (anandamide, AEA). Since 2-AG and AEA act through CB1 and CB2 cannabinoid receptors, we administered at 20 minutes after lesion a single injection of their respective antagonists AM281 and AM630 alone or in combination to block the effects of this early endocannabinoid accumulation. We observed that AM281, AM630 or AM281 plus AM630 administration impairs the spontaneous motor recovery of rats according to the Basso-Beattie-Bresnahan (BBB) locomotor scale. However, blockade of CB1, CB2 or both receptors produced different effects at the histopathological level. Thus, AM630 administration results at 90 days after lesion in increased MHC-II expression by spinal cord microglia/monocytes and reduced number of serotoninergic fibres in lumbar spinal cord (below the lesion). AM281 exerted the same effects but also increased oedema volume estimated by MRI. Co-administration of AM281 and AM630 produced the effects observed with the administration of either AM281 or AM630 and also reduced white matter and myelin preservation and enhanced microgliosis in the epicentre. Overall, our results suggest that the endocannabinoids acting through CB1 and CB2 receptors are part of an early neuroprotective response triggered after SCI that is involved in the spontaneous recovery after an incomplete lesion.

Quantitative analysis of multiple fatty acid ethanolamides using ultra-performance liquid chromatography–tandem mass spectrometry

Fatty acid ethanolamides (FAE) represent a group of lipid signaling molecules associated with many physiological and pharmacological actions; however, low FAE tissue levels pose challenges in terms of analytical characterization. The objective was to develop a competent ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method for analysis of multiple FAE in animal and human tissue samples. Analytes were extracted using lipid-phase and solid-phase extraction procedures. Chromatographic separation was achieved using a gradient elution in 8min. FAE were quantified by MS/MS in positive electrospray ionization mode. Linearity was shown in lower and higher FAE concentration ranges, with a limit of quantification (LOQ) ≤0.2ng/ml for FAE including alpha-linolenoylethanolamide (ALEA), arachidonoylethanolamide (AEA), docosahexaenoylethanolamide (DHEA), linoleoylethanolamide (LEA), oleoylethanolamide (OEA) and palmitoylethanolamide (PEA). Accuracy was shown to be between 92.4% and 108.8%, and precision was <10% for all FAE species. In sum, this sensitive and reproducible method can be used to simultaneously determine multiple FAE at low concentrations in order to facilitate further study of the role of FAE on physiological state.

Subventricular zone neural progenitors protect striatal neurons from glutamatergic excitotoxicity

The functional significance of adult neural stem and progenitor cells in hippocampal-dependent learning and memory has been well documented. Although adult neural stem and progenitor cells in the subventricular zone are known to migrate to, maintain and reorganize the olfactory bulb, it is less clear whether they are functionally required for other processes. Using a conditional transgenic mouse model, selective ablation of adult neural stem and progenitor cells in the subventricular zone induced a dramatic increase in morbidity and mortality of central nervous system disorders characterized by excitotoxicity-induced cell death accompanied by reactive inflammation, such as 4-aminopyridine-induced epilepsy and ischaemic stroke. To test the role of subventricular zone adult neural stem and progenitor cells in protecting central nervous system tissue from glutamatergic excitotoxicity, neurophysiological recordings of spontaneous excitatory postsynaptic currents from single medium spiny striatal neurons were measured on acute brain slices. Indeed, lipopolysaccharide-stimulated, but not unstimulated, subventricular zone adult neural stem and progenitor cells reverted the increased frequency and duration of spontaneous excitatory postsynaptic currents by secreting the endocannabinod arachidonoyl ethanolamide, a molecule that regulates glutamatergic tone through type 1 cannabinoid receptor (CB(1)) binding. In vivo restoration of cannabinoid levels, either by administration of the type 1 cannabinoid receptor agonist HU210 or the inhibitor of the principal catabolic enzyme fatty acid amide hydrolase, URB597, completely reverted the increased morbidity and mortality of adult neural stem and progenitor cell-ablated mice suffering from epilepsy and ischaemic stroke. Our results provide the first evidence that adult neural stem and progenitor cells located within the subventricular zone exert an 'innate' homeostatic regulatory role by protecting striatal neurons from glutamate-mediated excitotoxicity.

Lack of effect of chronic pre-treatment with the FAAH inhibitor URB597 on inflammatory pain behaviour: evidence for plastic changes in the endocannabinoid system.

Elevating levels of endocannabinoids with inhibitors of fatty acid amide hydrolase (FAAH) is a major focus of pain research, purported to be a safer approach devoid of cannabinoid receptor-mediated side effects. Here, we have determined the effects of sustained pharmacological inhibition of FAAH on inflammatory pain behaviour and if pharmacological inhibition of FAAH was as effective as genetic deletion of FAAH on pain behaviour. Effects of pre-treatment with a single dose, versus 4 day repeated dosing with the selective FAAH inhibitor, URB597 (i.p. 0.3 mg·kg⁻¹), on carrageenan-induced inflammatory pain behaviour and spinal pro-inflammatory gene induction were determined in rats. Effects of pain induction and of the drug treatments on levels of arachidonoyl ethanolamide (AEA), palmitoyl ethanolamide (PEA) and oleolyl ethanolamide (OEA) in the spinal cord were determined. Single, but not repeated, URB597 treatment significantly attenuated the development of inflammatory hyperalgesia (P < 0.001, vs. vehicle-treated animals). Neither mode of URB597 treatment altered levels of AEA, PEA and OEA in the hind paw, or carrageenan-induced paw oedema. Single URB597 treatment produced larger increases in AEA, PEA and OEA in the spinal cord, compared with those after repeated administration. Single and repeated URB597 treatment decreased levels of immunoreactive N-acylphosphatidylethanolamine phospholipase D (NAPE-PLD) in the spinal cord and attenuated carrageenan-induced spinal pro-inflammatory gene induction. Changes in the endocannabinoid system may contribute to the loss of analgesic effects following repeated administration of low dose URB597 in this model of inflammatory pain.

Endocannabinoid system and periodontal diseases: a histological preliminary study

Periodontal disease is a chronic condition characterized by an opportunistic infection of the periodontal tissues by Gram- anaerobic bacteria (1). Tissue destruction can be limited by the production of anti-inflammatory substances and its final entity depends on the equilibrium between pro- and anti-inflammatory mechanisms. The endocannabinoid system (EC) is supposed to have an anti-inflammatory action. The two most extensively studied molecules of the EC are anandamide and 2-AG with their specific receptors, CB1 and CB2. Nakajima et al (2) showed the presence of CB1 and CB2 receptors in human gingival fibroblasts from healthy patients and subjects affected by periodontal inflammation and found that AEA significantly reduced the production of cytokines (IL-6, IL-8 and MCP-1) by gingival fibroblasts after bacteria stimulation, through CB1 and CB2 receptors. The aim of this project was to quantitatively and qualitatively evaluate the presence of CB1 and CB2 receptors in the gingival tissue from patients affected by periodontitis compared to healthy subjects. Ten patients were recruited, six healthy subjects (H) and four with chronic periodontitis (P). In each patient, a gingival biopsy was harvested during periodontal treatment and processed for immunohistochemistry to reveal CB1 and CB2 expression. Every sample was mapped at 200X total magnification and the presence of CB1 and CB2 was quantified using a standardized method with Adobe Photoshop PS5. The percentage of marked tissue in the connective tissue and in the epithelium was calculated. In P the mean CB1 percentage on the connective tissue (CB1-c) was 2.27% + 0.14% and CB2 (CN2-c) was 2.12% + 0.43%, while in H the values were 0.13% + 0.31% and 0.06% + 0.11% respectively. In P the mean CB1 value on the epithelium (CB1-e) was 5.08 + 2.27% and CB2 (CB2-e) was 4.07% + 1.64%, while in H the corresponding values were respectively 0.16% + 0.36% and 0.03% + 0.02%. CB1 and CB2 expression resulted significantly higher in P than in H samples in both epithelial and connective tissue (Wilcoxon sum rank test, p<0.01). Such data confirm that EC are involved in periodontal disease.

Substrate-Selective Inhibition of Cyclooxygenase-2: Development and Evaluation of Achiral Profen Probes

Cyclooxygenase-2 (COX-2) oxygenates arachidonic acid and the endocannabinoids 2-arachidonoylglycerol (2-AG) and arachidonoylethanolamide (AEA). We recently reported that (R)-profens selectively inhibit endocannabinoid oxygenation but not arachidonic acid oxygenation. In this work, we synthesized achiral derivatives of five profen scaffolds and evaluated them for substrate-selective inhibition using in vitro and cellular assays. The size of the substituents dictated the inhibitory strength of the analogs, with smaller substituents enabling greater potency but less selectivity. Inhibitors based on the flurbiprofen scaffold possessed the greatest potency and selectivity, with desmethylflurbiprofen (3a) exhibiting an IC50 of 0.11 μM for inhibition of 2-AG oxygenation. The crystal structure of desmethylflurbiprofen complexed to mCOX-2 demonstrated a similar binding mode to other profens. Desmethylflurbiprofen exhibited a half-life in mice comparable to that of ibuprofen. The data presented suggest that achiral profens can act as lead molecules toward in vivo probes of substrate-selective COX-2 inhibition.

Identification and recombinant expression of anandamide hydrolyzing enzyme from Dictyostelium discoideum

BackgroundAnandamide (Arachidonoyl ethanolamide) is a potent bioactive lipid studied extensively in humans, which regulates several neurobehavioral processes including pain, feeding and memory. Bioactivity is terminated when hydrolyzed into free arachidonic acid and ethanolamine by the enzyme fatty acid amide hydrolase (FAAH). In this study we report the identification of a FAAH homolog from Dictyostelium discoideum and its function to hydrolyze anandamide.ResultsA putative FAAH DNA sequence coding for a conserved amidase signature motif was identified in the Dictyostelium genome database and the corresponding cDNA was isolated and expressed as an epitope tagged fusion protein in either E.coli or Dictyostelium. Wild type Dictyostelium cells express FAAH throughout their development life cycle and the protein was found to be predominantly membrane associated. Production of recombinant HIS tagged FAAH protein was not supported in E.coli host, but homologous Dictyostelium host was able to produce the same successfully. Recombinant FAAH protein isolated from Dictyostelium was shown to hydrolyze anandamide and related synthetic fatty acid amide substrates.ConclusionsThis study describes the first identification and characterisation of an anandamide hydrolyzing enzyme from Dictyostelium discoideum, suggesting the potential of Dictyostelium as a simple eukaryotic model system for studying mechanisms of action of any FAAH inhibitors as drug targets.

Highly Selective Inhibitors of Monoacylglycerol Lipase Bearing a Reactive Group that Is Bioisosteric with Endocannabinoid Substrates

The endocannabinoids 2-arachidonoyl glycerol (2-AG) and N-arachidonoyl ethanolamine (anandamide) are principally degraded by monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH), respectively. The recent discovery of O-aryl carbamates such as JZL184 as selective MAGL inhibitors has enabled functional investigation of 2-AG signaling pathways in vivo. Nonetheless, JZL184 and other reported MAGL inhibitors still display low-level cross-reactivity with FAAH and peripheral carboxylesterases, which can complicate their use in certain biological studies. Here, we report a distinct class of O-hexafluoroisopropyl (HFIP) carbamates that inhibits MAGL in vitro and in vivo with excellent potency and greatly improved selectivity, including showing no detectable cross-reactivity with FAAH. These findings designate HFIP carbamates as a versatile chemotype for inhibiting MAGL and should encourage the pursuit of other serine hydrolase inhibitors that bear reactive groups resembling the structures of natural substrates.

Role of arachidonoylethanolamine in blood pressure regulation in volume-resistant patients on peritoneal dialysis

In this study we explored the possible role of arachidonoylethanolamine (AEA) in regulating blood pressure in patients on continuous ambulatory peritoneal dialysis (CAPD). One hundred and five patients on CAPD were enrolled. Volume status was evaluated by the overhydration (OH) value obtained by bioimpedance analysis. OH<2.0 kg was defined as normal volume (NV) and OH≥2.0 kg as high volume (HV). Home mean systolic blood pressure<130 mmHg was defined as controlled hypertension (CHT) and ≥130 mmHg as uncontrolled hypertension (UHT). The patients were divided into four subgroups: (1) controlled hypertension with normal volume (CHT-NV), (2) controlled hypertension with high volume (CHT-HV), (3) uncontrolled hypertension with normal volume (UHT-NV), and (4) uncontrolled hypertension with high volume (UHT-HV). AEA was measured by ultra performance liquid chromatography-tandem mass spectrometry. AEA was significantly higher in the HV group as compared with the NV group (P<0.05). In addition, AEA was also significantly higher in the CHT-HV group as compared with the UHT-NV group (P<0.05). These results may suggest a compensatory function of AEA and TRPV1 pathway to lower blood pressure during volume expansion in CAPD patients.

Δ9‐Tetrahydrocannabinol and N‐arachidonyl glycine are full agonists at GPR18 receptors and induce migration in human endometrial HEC‐1B cells

Endometriosis is a disorder in which the endometrium forms growths outside the uterus and is associated with chronic pain. Recent evidence suggests that endometrial motility plays a role in the aetiology of endometriosis. The endocannabinoid system regulates cellular migration. Given the growing involvement of the endocannabinoids in reproduction, we investigated the role of the endocannabinoid system in migration of endometrial cells. Migration of the human endometrial HEC-1B cells was assayed. Standard PCR techniques were used to determine the presence of the GPCR, GPR18, in HEC-1B cells, and p44/42 MAPK was assayed in stably transfected HEK293-GPR18 cells to determine receptor specificity for known cannabinoid agonists and antagonists. N-arachidonoyl ethanolamine (AEA) metabolism was measured, using HPLC/MS/MS for lipid analysis. AEA, Δ(9) -tetrahydrocannabinol (Δ(9) -THC) and N-arachidonoyl glycine (NAGly) induce migration of HEC-1B cells through cannabinoid CB(1) receptor-independent mechanisms. MAPK activation in HEK293-GPR18 cells revealed novel pharmacology for known CB(1) and CB(2) receptor ligands at GPR18 receptors, including Δ(9) -THC, which activates MAPK at nanomolar concentrations, whereas WIN 55212-2, CP55940, JWH-133 and JWH-015, and arachidonyl-1-hydroxy-2-propylamide (R1-methanandamide) had no effect. Moreover, HEC-1B migration and MAPK activation by NAGly and Δ(9) -THC were antagonized by Pertussis toxin, AM251 and cannabidiol. An understanding of the function and regulation of GPR18 and its molecular interactions with endogenous ligands, and how phytocannabinoids play a role with GPR18 signalling is vital if we are to comprehensively assess the function of the cannabinoid signalling system in human health and disease. This article is commented on by Alexander, pp. 2411-2413 of this issue and is part of a themed section on Cannabinoids in Biology and Medicine. To view Alexander visit http://dx.doi.org/10.1111/j.1476-5381.2011.01731.x. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.165.issue-8. To view Part I of Cannabinoids in Biology and Medicine visit http://dx.doi.org/10.1111/bph.2011.163.issue-7.