Evidence has been produced that macrophages can actively generate endocannabinoids (eCBs) in response to inflammatory stimuli. As eCBs are involved in the control of several physiological processes, including reproduction, here, we explored whether seminal levels of the eCBs, N-arachidonoylethanolamine (AEA), and 2-arachidonoylglycerol (2-AG), were higher in the presence of leukocytospermia, and were correlated with semen concentration of macrophages. The content of AEA and 2-AG was measured by high-performance liquid chromatography/mass spectrometry in seminal plasma of ejaculates from 18 leukocytospermic patients (>1 × 106 leukocytes/mL) and 21 normozoospermic controls. In the same ejaculates, round cells were phenotyped by flow-cytometry as leukocytes (CD45+), macrophages (CD14+), and activated macrophages (CD14+, HLA-DR+). The levels of 2-AG, but not of AEA, were significantly higher in ejaculates from leukocytospermic patients than in controls and exhibited a significant correlation with semen concentration of macrophages and activated macrophages. Significant associations of 2-AG with macrophages and activated macrophages persisted after adjustment for semen volume and sperm concentration. In conclusion, here we provide evidence that seminal plasma levels of 2-AG are higher in the presence of leukocytospermia, as a marker of macrophages activation. Further studies are warranted to elucidate possible clinical implications.

<AbstractText>To determine if plasma concentrations of the <em>N</em>-acylethanolamines (<em>N</em>AEs) <em>N</em>-<em>arachidonoylethanolamine</em> (AEA), <em>N</em>-oleoylethanolamide (OEA) and <em>N</em>-palmitoylethanolamide (PEA) increase in women at high risk for preterm birth (PTB) and whether these could be used to predict preterm delivery and if so, how they compare with current methods.</AbstractText><AbstractText>Prospective cohort study.</AbstractText><AbstractText>A large UK teaching hospital.</AbstractText><AbstractText>217 pregnant women were recruited between 24 and 34 gestational weeks at 'high-risk' for PTB, recruited from a prematurity prevention clinic or antenatal wards.</AbstractText><AbstractText>Plasma AEA, OEA, and PEA concentrations were measured using ultra-high performance liquid chromatography-tandem mass spectrometry whilst FAAH enzyme activity was measured by fluorometric radiometric assay and CL by ultrasound scan. The clinical usefulness of these measurements were determined by ROC and multivariate analyses.</AbstractText><AbstractText>AEA and PEA concentrations were significantly higher in women who delivered prematurely. An AEA concentration >1.095 nM predicted PTB, the gestational age at delivery and the recruitment to delivery interval (RTDI). A PEA concentration >17.50 nM only predicted PTB; FAAH enzyme activity was not related to these changes. Multivariate analysis (all variables) generated an equation to accurately predict the RTDI.</AbstractText><AbstractText>A single plasma AEA or PEA measurement can predict PTB. A single AEA measurement predicts the gestational age of delivery and the remaining period of pregnancy with reasonable accuracy and better than existing conventional tests thus offering a better window for primary prevention of PTB.</AbstractText>

Neurons are especially susceptible to oxidative damage, which is increasingly implicated in neurodegenerative disease. Certain N-acylethanolamines (NAEs) have been shown to protect neurons from oxidative stress. Since glaucoma may be considered a neurodegenerative disorder and the survival of retinal neurons could also be influenced by N-acylethanolamines, our goal was to quantify changes in certain N-acylethanolamine species and their oxylipin derivatives in the retina of a mouse model for glaucoma. We also sought to identify relationships between these and parameters of glaucoma disease development, specifically intraocular pressure, visual acuity, and contrast sensitivity. Five N-acylethanolamine species and three NAE oxylipin derivatives were quantified in retina from young and aged DBA/2Crl mice. N-Acylethanolamines and NAE-oxylipins in retinal extracts were quantified against deuterated standards by isotope dilution gas chromatography-mass spectrometry. Levels (nmol/g dry weight) of N-arachidonoylethanolamine (anandamide; NAE 20:4) were significantly (p = 0.008) decreased in aged (2.875 ± 0.6702) compared to young animals (5.175 ± 0.971). Conversely, the anandamide oxylipin, 15(S)-HETE ethanolamide (15(S)-HETE EA), was significantly (p = 0.042) increased in aged (0.063 ± 0.009) compared to young animals (0.039 ± 0.011). Enzymatic depletion of the anandamide pool by 15-lipoxygenase and consequent accumulation of 15(S)-HETE ethanolamine may contribute to decreased visual function in glaucomatous mice. Since N-acylethanolamines effectively attenuate glaucoma pathogenesis and associated visual impairment, our data provides additional rationale and novel targets for glaucoma therapies.

The use of cannabinoids to treat fibrotic skin diseases is an emergent issue. Therefore, we aimed to evaluate systemic and skin endocannabinoid responses in the wound-healing process in humans. A prospective study was performed in 50 patients who underwent body-contouring surgery. Anandamide (N-arachidonoylethanolamine, AEA), 2-arachidonoylglycerol (2-AG), palmitoylethanolamide (PEA) and oleoylethanolamide (OEA) were quantified using LC-MS/MS. Ten (20%) patients developed hypertrophic (HT) scars. No significant changes were observed between the normal (N) scar and HT scar groups in terms of plasma and skin endocannabinoids. Nevertheless, a positive correlation between plasma and skin AEA concentrations was found in the N group (r = 0.38, p = 0.015), which was absent in the HT group. Moreover, the AEA concentration was significantly lower in HT scar tissue than in normal scar tissue (0.77 ± 0.12 ng/g vs 1.15 ± 0.15 ng/g, p < 0.001). Interestingly, in all patients, the surgical intervention produced a time-dependent effect with a U shape for AEA, PEA and OEA plasma concentrations. In contrast, 2-AG plasma concentrations increased 5 days after surgery and were reduced and stabilized 3 months later. These results suggest crosstalk between systemic and local skin endocannabinoid systems during human wound healing. AEA appears to be the most likely candidate for this link, which is deficient in patients with HT scars.

An improved understanding of the endocannabinoid system has provided new avenues of drug discovery and development toward the management of pain and other behavioral maladies. Exogenous cannabinoid type 1 (CB1) receptor agonists such as Δ9-tetrahydrocannabinol are increasingly used for their medicinal actions; however, their utility is constrained by concern regarding abuse-related subjective effects. This has led to growing interest in the clinical benefit of indirectly enhancing the activity of the highly labile endocannabinoids N-arachidonoylethanolamine [AEA (or anandamide)] and/or 2-arachidonoylglycerol (2-AG) via catabolic enzyme inhibition. The present studies were conducted to determine whether such actions can lead to CB1 agonist-like subjective effects, as reflected in CB1-related discriminative stimulus effects in laboratory subjects. Squirrel monkeys (n = 8) that discriminated the CB1 full agonist AM4054 (0.01 mg/kg) from vehicle were used to study, first, the inhibitors of fatty acid amide hydrolase (FAAH) or monoacylglycerol lipase (MGL) alone or in combination [FAAH (URB597, AM4303); MGL (AM4301); FAAH/MGL (JZL195, AM4302)] and, second, the ability of the endocannabinoids AEA and 2-AG to produce CB1 agonist-like effects when administered alone or after enzyme inhibition. Results indicate that CB1-related discriminative stimulus effects were produced by combined, but not selective, inhibition of FAAH and MGL, and that these effects were nonsurmountably antagonized by low doses of rimonabant. Additionally, FAAH or MGL inhibition revealed CB1-like subjective effects produced by AEA but not by 2-AG. Taken together, the present data suggest that therapeutic effects of combined, but not selective, enhancement of AEA or 2-AG activity via enzyme inhibition may be accompanied by CB1 receptor-mediated subjective effects.

N-Acylethanolamines are lipid signaling molecules found throughout the plant and animal kingdoms. The best-known mammalian compound of this class is anandamide, N-arachidonoylethanolamine, one of the endogenous ligands of cannabinoid CB1 and CB2 receptors. Signaling by N-acylethanolamines is terminated by release of the ethanolamine moiety by hydrolyzing enzymes such as fatty acid amide hydrolase (FAAH) and N-acylethanolamine-hydrolyzing amidase (NAAA). Herein, we report the design and synthesis of N-(16-(18)F-fluorohexadecanoyl)ethanolamine ((18)F-FHEA) as a positron emission tomography (PET) probe for imaging the activity of N-acylethanolamine hydrolyzing enzymes in the brain. Following intravenous administration of (18)F-FHEA in Swiss Webster mice, (18)F-FHEA was extracted from blood by the brain and underwent hydrolysis at the amide bond and incorporation of the resultant (18)F-fluorofatty acid into complex lipid pools. Pretreatment of mice with the FAAH inhibitor URB-597 (1 mg/kg IP) resulted in significantly slower (18)F-FHEA incorporation into lipid pools, but overall (18)F concentrations in brain regions were not altered. Likewise, pretreatment with a NAAA inhibitor, (S)-N-(2-oxo-3-oxytanyl)biphenyl-4-carboxamide (30 mg/kg IV), did not significantly affect the uptake of (18)F-FHEA in the brain. Although evidence was found that (18)F-FHEA behaves as a substrate of FAAH in the brain, the lack of sensitivity of brain uptake kinetics to FAAH inhibition discourages its use as a metabolically trapped PET probe of N-acylethanolamine hydrolyzing enzyme activity.

Vesicants including sulfur mustard (SM) and nitrogen mustard (NM) are bifunctional alkylating agents that cause skin inflammation, edema and blistering. This is associated with alterations in keratinocyte growth and differentiation. Endogenous cannabinoids, including N-arachidonoylethanolamine (anandamide, AEA) and 2-arachidonoyl glycerol (2-AG), are important in regulating inflammation, keratinocyte proliferation and wound healing. Their activity is mediated by binding to cannabinoid receptors 1 and 2 (CB1 and CB2), as well as peroxisome proliferator-activated receptor alpha (PPARα). Levels of endocannabinoids are regulated by fatty acid amide hydrolase (FAAH). We found that CB1, CB2, PPARα and FAAH were all constitutively expressed in mouse epidermis and dermal appendages. Topical administration of NM or SM, at concentrations that induce tissue injury, resulted in upregulation of FAAH, CB1, CB2 and PPARα, a response that persisted throughout the wound healing process. Inhibitors of FAAH including a novel class of vanillyl alcohol carbamates were found to be highly effective in suppressing vesicant-induced inflammation in mouse skin. Taken together, these data indicate that the endocannabinoid system is important in regulating skin homeostasis and that inhibitors of FAAH may be useful as medical countermeasures against vesicants.

Marijuana has been used as a traditional medicine and a pleasure-inducing drug for thousands of years around the world, especially in Asia. Delta(9)-tetrahydrocannabinol, major psychoactive component of marijuana, has been shown to interact with specific cannabinoid receptors, thereby eliciting a variety of pharmacological responses in experimental animals and human. In 1990, the gene encoding a cannabinoid receptor (CB1) was cloned. This prompted the search for endogenous ligands. In 1992, N-arachidonoylethanolamine (anandamide) was isolated from pig brain as an endogenous ligand, and in 1995, 2-arachidonoylglycerol was isolated from rat brain and canine gut as another endogenous ligand. Both anandamide and 2-arachidonoylglycerol exhibit various cannabimimetic activities. The results of structure-activity relationship experiments, however, revealed that 2-arachidonoylglycerol, but not anandamide, is the intrinsic natural ligand for the cannabinoid receptor. 2-arachidonoylglycerol is a degradation product of inositol phospholipids that links the function of the cannabinoid receptors with the enhanced inositol phospholipid turnover in stimulated tissues and cells. The possible physiological roles of cannabinoid receptors and 2-arachidonoylglycerol in various mammalian tissues such as those of the nervous and inflammatory cells are demonstrated. Furthermore, the future development of therapeutic drugs coming from this endocannabinoid system are discussed.

Long-chain N-acylethanolamines (NAEs) have been found to uncouple oxidative phosphorylation and to inhibit uncoupled respiration of rat heart mitochondria [Wasilewski, M., Wieckowski, M.R., Dymkowska, D. and Wojtczak, L. (2004) Biochim. Biophys. Acta 1657, 151-163]. The aim of the present work was to investigate in more detail the mechanism of the inhibitory effects of NAEs on the respiratory chain. In connection with this, we also investigated a possible action of NAEs on the generation of reactive oxygen species (ROS) by respiring rat heart mitochondria. It was found that unsaturated NAEs, N-oleoylethanolamine (N-Ole) and, to a greater extent, N-arachidonoylethanolamine (N-Ara), inhibited predominantly complex I of the respiratory chain, with a much weaker effect on complexes II and III, and no effect on complex IV. Saturated N-palmitoylethanolamine had a much smaller effect compared to unsaturated NAEs. N-Ara and N-Ole were found to decrease ROS formation, apparently due to their uncoupling action. However, under specific conditions, N-Ara slightly but significantly stimulated ROS generation in uncoupled conditions, probably due to its inhibitory effect on complex I. These results may contribute to our better understanding of physiological roles of NAEs in protection against ischemia and in induction of programmed cell death.

Liver fatty-acid-binding protein (FABP1, L-FABP) is the major cytosolic binding/chaperone protein for both precursor arachidonic acid (ARA) and the endocannabinoid (EC) products N-arachidonoylethanolamine (AEA) and 2-arachidonoylglycerol (2-AG). Although FABP1 regulates hepatic uptake and metabolism of ARA, almost nothing is known regarding FABP1's impact on AEA and 2-AG uptake, intracellular distribution, and targeting of AEA and 2-AG to degradative hepatic enzymes. In vitro assays revealed that FABP1 considerably enhanced monoacylglycerol lipase hydrolysis of 2-AG but only modestly enhanced AEA hydrolysis by fatty-acid amide hydrolase. Conversely, liquid chromatography-mass spectrometry of lipids from Fabp1 gene-ablated (LKO) hepatocytes confirmed that loss of FABP1 markedly diminished hydrolysis of 2-AG. Furthermore, the real-time imaging of novel fluorescent NBD-labeled probes (NBD-AEA, NBD-2-AG, and NBD-ARA) resolved FABP1's impact on uptake vs intracellular targeting/hydrolysis. FABP1 bound NBD-ARA with 2:1 stoichiometry analogous to ARA, but bound NBD-2-AG and NBD-AEA with 1:1 stoichiometry-apparently at different sites in FABP1's binding cavity. All three probes were taken up, but NBD-2-AG and NBD-AEA were targeted to lipid droplets. LKO reduced the uptake of NBD-ARA as expected, significantly enhanced that of NBD-AEA, but had little effect on NBD-2-AG. These data indicated that FABP1 impacts hepatocyte EC levels by binding EC and differentially impacts their intracellular hydrolysis (2-AG) and uptake (AEA).

The opioid-induced rise of extracellular dopamine, endocannabinoid anandamide and γ-aminobutyric acid (GABA) concentrations triggered by opioids in the nucleus accumbens shell (NACSh) most likely participate in opioid reward. We have previously demonstrated that systemic administration of ghrelin antagonist (JMV2959) significantly decreased morphine-induced dopamine and anandamide (N-arachidonoylethanolamine, AEA) increase in the NACSh. Fentanyl is considered as a µ-receptor-selective agonist. The aim of this study was to test whether JMV2959, a growth hormone secretagogue receptor (GHS-R1A) antagonist, can influence the fentanyl-induced effects on anandamide, 2-arachidonoylglycerol (2-AG) and GABA in the NACSh and specify the involvement of GHS-R1A located in the ventral tegmental area (VTA) and nucleus accumbens (NAC). Using in vivo microdialysis in rats, we have found that pre-treatment with JMV2959 reversed dose dependently fentanyl-induced anandamide increases in the NACSh, resulting in a significant AEA decrease and intensified fentanyl-induced decreases in accumbens 2-AG levels, with both JMV2959 effects more expressed when administered into the NACSh in comparison to the VTA. JMV2959 pre-treatment significantly decreased the fentanyl-evoked accumbens GABA efflux and reduced concurrently monitored fentanyl-induced behavioural stimulation. Our current data encourage further investigation to assess if substances affecting GABA or endocannabinoid concentrations and action, such as GHS-R1A antagonists, can be used to prevent opioid-seeking behaviour.

The three amide oxyhomologues of the endolipids N-arachidonoylethanolamine (anandamide, AEA, 1a), N-oleoylethanolamine (OEA, 2a), and N-palmitoylethanolamine (PEA, 3a) have been prepared in a chemoselective way, capitalizing on the easy availability of O-[2-(triisopropylsilyoxy)ethyl]hydroxylamine (6) and the surprising complementary selectivity observed in the acylation of N-[2-(tert-butyldiphenylsilyloxy)ethyl]hydroxylamine (7) with the PPAA and the DCC/HOBT protocols. Reversal of the cannabinoid CB(1)/CB(2) receptor affinity ratio was observed for the first time in a derivative of anandamide (the O-alkyl-N-acyl hydroxylamine 1b), while the other oxyhomologues (1c and 1d) showed only marginal cannabimimetic activity. Compounds with unsaturated acyl chains generally retained vanilloid activity and showed an increased stability toward FAAH compared to their corresponding ethanolamides. Taken together, these observation show that oxyhomologation has a pronounced effect on both the pharmacodynamic and the pharmacokinetic properties of endogenous ethanolamides, suggesting a general relevance of this maneuver for the modification of amide pharmacophores.

The endocannabinoids 2-arachidonoylglycerol (2-AG) and anandamide (N-arachidonoylethanolamine, AEA) are produced by neurons and other cells, including platelets, in a stimulus-dependent manner and act as signaling molecules; they are then inactivated through transport into cells followed by enzymatic degradation. A number of studies showed that monoacylglycerol lipase (MAGL) plays an important role in the degradation of 2-AG. In this study we investigated the enzymatic degradation of 2-acylglycerols in rabbit platelets and we characterized the responsible enzyme(s). [(3)H]2-AG and [(3)H]2-oleoylglycerol (2-OG) were both metabolized to [(3)H]glycerol and the respective fatty acid in a time and protein concentration-dependent manner, apparently by the action of MAGL activity. In the presence of the specific fatty acid amide hydrolase (FAAH) inhibitors URB597 and AM374, though, 2-OG hydrolysis was inhibited up to 55% in a concentration-dependent manner (IC(50) = 129.8 nM and 20.9 nM respectively). These results indicate the involvement of both MAGL and FAAH on 2-acylglycerol hydrolysis. MAGL was further characterized in the presence of URB597 and it was found that 2-monoacylglycerols were hydrolyzed in a time, pH and protein concentration-dependent manner and hydrolysis followed Michaelis-Menten kinetics, with an apparent K(M) of 0.11 microM and V(max) of 1.32 nmol/min*mg protein. Subcellular fractionation of platelet homogenate showed that MAGL activity was present in both the cytosolic and membrane fractions. In conclusion, the endocannabinoid 2-AG, as well as other 2-acylglycerols, are substrates of both FAAH and MAGL; the latter was characterized for the first time in platelets. In human platelets, under the same experimental conditions, the hydrolysis of 2-acylglycerols was higher and MAGL activity showed a different sensitivity against the inhibitors mentioned above. Finally, immunoblot analysis revealed the presence of MAGL, both in rabbit and human platelets, with a molecular mass of approximately 33 kDa.

In 1929 Burr and Burr discovered the essential fatty acids omega-6 and omega-3. Since then, researchers have shown a growing interest in polyunsaturated fatty acids (PUFA) as precursors of "lipid mediator" molecules, often with opposing effects, prostaglandins, prostacyclins, thromboxanes, leukotrienes, lipossines, resolvines, protectines, maresins that regulate immunity, platelet aggregation, inflammation, etc. They showed that the balance between omega-3 and omega-6 acids has a profound influence on all the body's inflammatory responses and a raised level of PUFA omega-3 in tissue correlate with a reduced incidence of degenerative cardiovascular disease, some mental illnesses such as depression, and neuro-degenerative diseases such as Alzheimer's. The CYP-catalyzed epoxidation and hydroxylation of arachidonic acid (AA) were established recently as the so-called third branch of AGE cascade. Cytochrome P450 (CYP) epoxygenases convert AA to four epoxyeicosatrienoic acid (EET) regioisomers, that produce vascular relaxation anti-inflammatory effects on blood vessels and in the kidney, promote angiogenesis, and protect ischemic myocardium and brain. Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are accessible to CYP enzymes in the same way as AA. Metabolites derived from EPA include epoxye-icosatetraenoic acids (EETR) and hydroxyeicosapentaenoic acids (19- and 20-HEPE), whereas DHA include epoxydocosapentaenoic acids (EDPs) hydroxydocosahexaenoic acids (21- and 22-HDoHE). For many of the CYP isoforms, the n-3 PUFAs are the preferred substrates and the available data suggest that some of the vasculo- and cardioprotective effects attributed to dietary n-3 PUFAs may be mediated by CYP-dependent metabolites of EPA and DHA. From AA derives also endocannabinoids like anandamide (N-arachidonoylethanolamine) and 2-arachidonoylglycerol, capable of mimicking the pharmacological actions of the active principle of Cannabis sativa preparations such as hashish and marijuana (-)-Delta9-tetrahydrocannabinol. They act as true 'endogenous cannabinoids' by binding and functionally activating one or both cannabinoid receptor present on nervous and peripheral cell membranes. Enzymes that carry out anandamide oxidation are the same fatty acid oxygenases that are known to act on endogenous arachidonic acid namely, the members of the COX, LOX, and P450 families of enzymes. Recent advances in the biochemistry and pharmacology of the endocannabinoid system, also for its central and peripheral roles in regulating food intake, will offer the development of novel therapeutic agents.

Nearly 30 years after the discovery in 1964 of the psychoactive ingredient of cannabis (Cannabis sativa), Δ(9)-tetrahydrocannabinol, its endogenous counterparts were discovered and collectively termed endocannabinoids (eCBs): N-arachidonoylethanolamine (anandamide) in 1992 and 2-arachidonoylglycerol in 1995. Since then, intense research has identified additional eCBs and an ensemble of proteins that bind, synthesize and degrade them, the so-called eCB system. Altogether, these new compounds have been recognized as key mediators of several aspects of human pathophysiology, and in particular of female fertility. Here, the main features of the eCB system are presented, in order to put in a better perspective the relevance of eCB signaling in virtually all steps of human reproduction and to highlight emerging hopes that elements of this system might indeed become novel targets to combat fertility problems.

Background: Modulation of the endocannabinoid system has been shown to alleviate neuropathic pain. The aim of this study was to evaluate if treatment with paclitaxel, a chemotherapeutic agent that induces neuropathic pain, affects endocannabinoid levels at a time when mice develop paclitaxel-induced mechanical allodynia. We also evaluated the peripheral antiallodynic activity of the endocannabinoid 2-arachidonoyl glycerol (2-AG) and an inhibitor of monoacylglycerol lipase (MAGL), an enzyme responsible for 2-AG hydrolysis.

Methods: Female BALB/c mice were treated intraperitoneally with paclitaxel to induce mechanical allodynia. Levels of the endocannabinoids, N-arachidonoylethanolamine (anandamide, AEA), 2-AG, and the N-acylethanolamines (NAEs), N-palmitoylethanolamide (PEA) and N-oleoylethanolamide (OEA), which are structurally-related to AEA, in the brain, spinal cord and paw skin were measured using LC-MS/MS. Protein expression of MAGL in the paw skin was measured using Wes™. The effects of subcutaneous (s.c.) injection of 2-AG and JZL184 (a MAGL inhibitor) into the right hind paw of mice with paclitaxel-induced mechanical allodynia were assessed using the dynamic plantar aesthesiometer. The effects of pretreatment, s.c., into the right hind paw, with cannabinoid type 1 (CB₁) receptor antagonist AM251 and CB₂ receptor antagonist AM630 on the antiallodynic effects of 2-AG were also evaluated.

Results: The levels of 2-AG were reduced only in the paw skin of paclitaxel-treated mice, whilst the levels of AEA, PEA and OEA were not significantly altered. There was no change in the expression of MAGL in the paw skin. Administration of 2-AG and JZL184 produced antiallodynic effects against paclitaxel-induced mechanical allodynia in the injected right paw, but did not affect the uninjected left paw. The antiallodynic activity of 2-AG was antagonized by both AM251 and AM630.

Conclusion: These results indicate that during paclitaxel-induced mechanical allodynia there is a deficiency of 2-AG in the periphery, but not in the CNS. Increasing 2-AG in the paw by local administration of 2-AG or a MAGL inhibitor, alleviates mechanical allodynia in a CB₁ and CB₂ receptor-dependent manner.

Keywords: 2-Arachidonoyl glycerol; Allodynia; Cannabinoid receptors; Chemotherapy-induced neuropathic pain; Endocannabinoid; Monoacylglycerol lipase.

Traumatic brain injury (TBI) affects approximately 3 million Americans yearly and increases vulnerability to developing psychiatric comorbidities. Alcohol use disorder (AUD) is the most prevalent psychiatric diagnosis preceding injury and TBI may increase subsequent alcohol use. The basolateral amygdala (BLA) is a limbic structure commonly affected by TBI that is implicated in anxiety and AUD. Endocannabinoids (eCBs) regulate synaptic activity in the BLA, and BLA eCB modulation alters anxiety-like behavior and stress reactivity. Previous work from our laboratories showed that systemic eCB degradation inhibition ameliorates TBI-induced increases in anxiety-like behavior and motivation to respond for alcohol in male rats. Here, we used a lateral fluid percussion model to test moderate TBI effects on anxiety-like behavior, alcohol drinking, and eCB levels and cell signaling in BLA, as well as the effect of alcohol drinking on anxiety-like behavior and the BLA eCB system, in female rats. Our results show that TBI does not promote escalation of operant alcohol self-administration or increase anxiety-like behavior in female rats. In the BLA, TBI and alcohol drinking alter tissue amounts of 2-arachidonoylglycerol (2-AG) and N-arachidonoylethanolamine (anandamide; AEA) one hour post-injury, and 2-AG levels remain low eleven days post-injury. Eleven days after injury, BLA pyramidal neurons were hyperexcitable, but measures of synaptic transmission and eCB signaling were unchanged. These data show that TBI impacts BLA 2-AG tissue levels, that this effect is modified by alcohol drinking, and also that TBI increases BLA cell excitability.

Keywords: ALCOHOL AND DRUG ABUSE; BEHAVIORAL ASSESSMENTS; MODELS OF INJURY; Rat; TRAUMATIC BRAIN INJURY.

Anandamide (N-arachidonoylethanolamine, AEA) is an endogenous lipid that binds to cannabinoid receptors in the central nervous system and in peripheral cells. Quantitative analysis of AEA is generally based on the normalization to the fresh weight of the samples. Here, we show that the normalization procedure of AEA content is such a critical factor, that it might introduce per se significant discrepancies in the quantification of AEA even in the same sample. We suggest that a rapid, accurate and most reliable reference to quantify AEA and congeners from different sources is the protein content, a common parameter to cells and tissues.

The endocannabinoids (eCBs), N-arachidonoylethanolamine (anandamide, AEA) and 2-ararchidonylglycerol (2-AG) have been identified as main endogenous ligands for cannabinoid receptors. Developing a sensitive and robust method to determine AEA and 2-AG has been shown to be essential to understand their effects in stress regulation and the pathogenesis of affective disorders. Since eCBs are endogenous molecules, there is no true blank matrix available to construct calibration curves, thus, it has been a challenge to determine eCBs in plasma and brain matrix. A liquid chromatography tandem mass spectrometry (LC-MS/MS) method is developed to determine the concentrations of AEA and 2-AG in murine plasma and different brain substructures (prefrontal cortex, hippocampus and hypothalamus). To overcome the endogenous interference, a "surrogate analyte" approach was adopted using stable isotope-labeled standards as surrogates of authentic analytes to generate calibration curves in biological matrix. The mobile phase, composed of formic acid 0.1% in water-acetonitrile (40:60, v/v), was optimized to separate 2-AG and its non-bioactive isomer 1-AG. The analytes were extracted with ethyl acetate/n-hexane (9:1, v/v) and separated on an Xbridge C18 (2.1 × 100 mm, 3.5 μm) column using N-Oleoylethanolamine-d2 (OEA-d2) as the internal standard. Detection was performed in multiple reaction monitoring (MRM) mode with an electrospray ionization source operated in positive ion mode. The method was applied to assess plasma and brain eCBs in tumor-bearing mice.

Endocannabinoids (eCBs), among which N-arachidonoylethanolamine (AEA) and 2-arachidonoylglycerol (2-AG) are the most biologically active members, are polyunsaturated lipids able to bind cannabinoid, vanilloid and peroxisome proliferator-activated receptors. Depending on the target engaged, these bioactive mediators can regulate different signalling pathways, at both central and peripheral levels. The biological action of eCBs is tightly controlled by a plethora of metabolic enzymes which, together with the molecular targets of these substances, form the so-called "endocannabinoid system". The ability of eCBs to control manifold peripheral functions has received a great deal of attention, especially in the light of their widespread distribution in the body. In particular, eCBs are important regulators in blood, where they modulate haematopoiesis, platelet aggregation and apoptosis, as well as chemokine release and migration of immunocompetent cells. Here, we shall review the current knowledge on the pathophysiological roles of eCBs in blood. We shall also discuss the involvement of eCBs in those disorders affecting the haematological system, including cancer and inflammation. Knowledge gained to date underlines a fundamental role of the eCB system in blood, thus suggesting that it may represent a therapeutic promise for a broad range of diseases involving impaired hematopoietic cell functions.

OBJECTIVE

Cannabinoids are effective antiemetics and the "endogenous cannabinoids" (endocannabinoids) are thought to modulate emesis in both humans and animal models. Endocannabinoids, their receptors and their metabolising enzymes are present in peripheral blood and a reduction in blood endocannabinoid concentration has been observed in individuals with excessive nausea and vomiting following parabolic flight manoeuvres. We tested the hypothesis that plasma endocannabinoid levels are similarly perturbed in women with hyperemesis gravidarum (HG), a condition where the aetiopathogenesis is still unknown, compared to normal pregnant controls.

METHODS

Plasma N-arachidonoylethanolamine (anandamide), N-oleoylethanolamide and N-palmitoylethanolamide were quantified in women with HG (n = 15) and matched normal pregnant controls (n = 30) using UHPLC-ESI-MS/MS utilising an isotope dilution method and selective ion monitoring.

RESULTS

No significant differences in anandamide, oleoylethanolamide and palmitoylethanolamide levels were observed between the two groups. There were no significant correlations between these endocannabinoids and plasma haematocrit and serum urea or sodium concentrations.

CONCLUSIONS

These results would suggest that either the circulating endocannabinoids quantified may not be key modulating factors in HG or that the expected endocannabinoid system response to the stress induced by nausea and vomiting of early pregnancy remain unchanged in women with HG.

Purpose: Hypertensive lesions induce alterations at hemodynamic, peripheral, and central levels. Anandamide (N-arachidonoylethanolamine; AEA) protects neurons from inflammatory damage, but its free administration may cause central adverse effects. AEA controlled release by nanoformulations could reduce/eliminate its side effects. The present study aimed to evaluate the effects of nanoformulated AEA (nf-AEA) on systolic blood pressure (SBP), behavior, and central/peripheral inflammatory, oxidative, and apoptotic state in spontaneously hypertensive rats (SHR).

Materials/methods: Male rats were used, both Wistar Kyoto (WKY) and SHR (n ​= ​10 per group), with/without treatment with nf-AEA (obtained by electrospraying) at a weekly dose of 5 ​mg/kg IP for 4 weeks. SBP was measured and behavioral tests were performed. Inflammatory/oxidative markers were quantified at the central (brain cortex) and peripheral (serum) level.

Results: SHR showed hyperactivity, low anxiety, and high concentrations of central/peripheral inflammatory/oxidative markers, also higher apoptosis of brain cortical cells compared to WKY. As opposed to this group, treatment with nf-AEA in SHR significantly reduced SBP, peripheral/central inflammatory/oxidative makers, and central apoptosis. Nf-AEA also increased neuroprotective mechanisms mediated by intracellular heat shock protein 70 (Hsp70), which were attenuated in untreated SHR. Additionally, nf-AEA reversed the abnormal behaviors observed in SHR without producing central adverse effects.

Conclusions: Our results suggest protective properties of nf-AEA, both peripherally and centrally, through a signaling pathway that would involve the type I angiotensin II receptor, Wilms tumor transcription factor 1, Hsp70, and iNOS. Considering non-nf-AEA limitations, this nanoformulation could contribute to the development of new antihypertensive and behavioral disorder treatments associated with neuroinflammation.

Keywords: Anandamide; Hypertension; Inflammation; Nanoformulation; Oxidative stress.

BACKGROUND

The endocannabinoid system plays a substantial role in analgesia.

OBJECTIVE

To analyze N-arachidonoylethanolamine (AEA), N-oleoylethanolamine (OEA), linoleoyl ethanolamide (LEA), α-linoleoyl ethanolamine (α-LNEA), N-palmitoylethanolamine (PEA) and N-stearoyl ethanolamine (SEA) in two groups of patients having chronic pancreatic diseases.

METHODS

Twenty-six patients with chronic pancreatitis, 26 patients with pancreatic ductal adenocarcinoma and 36 healthy subjects were studied. The visual analogic scale (VAS) was used for assessing pain immediately before the venipuncture to obtain blood in all subjects. Six endocannabinoids were measured in serum of the patients enrolled.

RESULTS

Only OEA, LEA and PEA serum levels were significantly higher in patients with pain as compared to those without. Using the cutoff values, the sensitivity and specificity of the various endocannabinoids in evaluating pain in patients with chronic pancreatitis and in those with pancreatic ductal adenocarcinoma were: 44.2% and 95.6% for AEA, 83.7% and 73.3% for LEA, 88.4% and 91.1% for LNEA, 81.4% and 82.2% for OEA, 81.4% and 88.9% for PEA, 86.0% and 88.9% for SEA, respectively.

CONCLUSIONS

Endocannabinoids are not useful in assessing pain in patients with chronic pancreatic diseases and they cannot replace a simple method such as VAS for assessing the pain and its intensity.

Aim: We aimed to investigate whether maternal malnutrition during gestation/lactation induces long-lasting changes on inflammation, lipid metabolism and endocannabinoid signaling in the adult offspring hypothalamus and the role of hypothalamic astrocytes in these changes. Methods: We analyzed the effects of a free-choice hypercaloric palatable diet (P) during (pre)gestation, lactation and/or post-weaning on inflammation, lipid metabolism and endogenous cannabinoid signaling in the adult offspring hypothalamus. We also evaluated the response of primary hypothalamic astrocytes to palmitic acid and anandamide. Results: Postnatal exposure to a P diet induced factors involved in hypothalamic inflammation (Tnfa and Il6) and gliosis (Gfap, vimentin and Iba1) in adult offspring, being more significant in females. In contrast, maternal P diet reduced factors involved in astrogliosis (vimentin), fatty acid oxidation (Cpt1a) and monounsaturated fatty acid synthesis (Scd1). These changes were accompanied by an increase in the expression of the genes for the cannabinoid receptor (Cnr1) and Nape-pld, an enzyme involved in endocannabinoid synthesis, in females and a decrease in the endocannabinoid degradation enzyme Faah in males. These changes suggest that the maternal P diet results in sex-specific alterations in hypothalamic endocannabinoid signaling and lipid metabolism. This hypothesis was tested in hypothalamic astrocyte cultures, where palmitic acid (PA) and the polyunsaturated fatty acid N-arachidonoylethanolamine (anandamide or AEA) were found to induce similar changes in the endocannabinoid system (ECS) and lipid metabolism. Conclusion: These results stress the importance of both maternal diet and sex in long term metabolic programming and suggest a possible role of hypothalamic astrocytes in this process.

Keywords: Astrocytes; endocannabinoid system; hypothalamus; inflammation; lipid metabolism; maternal diet; perinatal diet; perinatal programing.