Release of anandamide from blood cells

Background: Endogenous ligands of cannabinoid receptors ( endocannabinoids), in particular anandamide ( arachidonylethanolamide), have been recognized as being of crucial importance in a variety of physiological functions. Plasma concentrations of anandamide have been measured in a number of investigations; however, discrepant data on "normal'' anandamide plasma concentrations were reported. Since this might be caused by pre-analytical variables, we investigated the impact of different sample handling conditions on measured plasma anandamide concentrations. Methods: Blood samples were taken from healthy volunteers in EDTA- or heparin-containing tubes; whole blood samples were kept at +4 degrees C, room temperature, or 37 degrees C, respectively, for up to 120 min before obtaining plasma by centrifugation. Plasma anandamide concentrations were measured by an isotope-dilution liquid chromatography tandem mass spectrometry ( LC-MS/MS) method. Results: A marked time- and temperature-dependent increase in plasma anandamide concentrations ex vivo was observed in both EDTA- and heparin-containing tubes. Mean anandamide concentrations approximately doubled when EDTA samples were kept at 4 degrees C for 60 min before centrifugation {[}immediately centrifuged, 1.3 mg/L ( SD 0.3 mg/L); 2.8 mg/L ( SD 0.5 mg/L) after storage for 60 min; n=12). After storage of heparinized whole-blood samples for 120 min at 37 degrees C, a mean plasma anandamide concentration of 11.9 mg/L ( SD 1.8 mg/L) was found. In cell-free plasma, no increase in anandamide concentrations was found. Conclusion: Anandamide is released from blood cells ex vivo at a very high rate; therefore, strictly standardized pre-analytical protocols have to be applied for plasma anandamide determination

Interaction between anandamide and sphingosine-1-phosphate in mediating vasorelaxation in rat coronary artery

<b>BACKGROUND AND PURPOSE</b> Anandamide and sphingosine-1-phosphate (S1P) both regulate vascular tone in a variety of vessels. This study aimed to examine the mechanisms involved in the regulation of coronary vascular tone by anandamide and S1P, and to determine whether any functional interaction occurs between these receptor systems. <br></br> <b>EXPERIMENTAL APPROACH</b> Mechanisms used by anandamide and S1P to regulate rat coronary artery (CA) reactivity were investigated using wire myography. Interactions between S1P and the cannabinoid (CB)2 receptor were determined using human embryonic kidney 293 (HEK293) cells that stably over-express recombinant CB2 receptor. <br></br> <b>KEY RESULTS</b> Anandamide and S1P induced relaxation of the rat CA. CB2 receptor antagonists attenuated anandamide-induced relaxation, while S1P-mediated relaxation was dependent on the vascular endothelium and S1P3. Anandamide treatment resulted in an increase in the phosphorylation of sphingosine kinase-1 within the CA. Conversely, anandamide-mediated relaxation was attenuated by inhibition of sphingosine kinase. Moreover, S1P3, specifically within the vascular endothelium, was required for anandamide-mediated vasorelaxation. In addition to this, S1P-mediated relaxation was also reduced by CB2 receptor antagonists and sphingosine kinase inhibition. Further evidence that S1P functionally interacts with the CB2 receptor was also observed in HEK293 cells over-expressing the CB2 receptor. <br></br> <b>CONCLUSIONS AND IMPLICATIONS</b> In the vascular endothelium of rat CA, anandamide induces relaxation via a mechanism requiring sphingosine kinase-1 and S1P/S1P3. In addition, we report that S1P may exert some of its effects via a CB2 receptor- and sphingosine kinase-dependent mechanism, where subsequently formed S1P may have privileged access to S1P3 to induce vascular relaxation

Requirement for sphingosine kinase 1 in mediating phase 1 of the hypotensive response to anandamide in the anaesthetised mouse

In the isolated rat carotid artery, the endocannabinoid anandamide induces endothelium-dependent relaxation via activation of the enzyme sphingosine kinase (SK). This generates sphingosine-1-phosphate (S1P) which can be released from the cell and activates S1P receptors on the endothelium. In anaesthetised mice, anandamide has a well-characterised triphasic effect on blood pressure but the contribution of SK and S1P receptors in mediating changes in blood pressure has never been studied. Therefore, we assessed this in the current study. The peak hypotensive response to 1 and 10 mg/kg anandamide was measured in control C57BL/6 mice and in mice pretreated with selective inhibitors of SK1 (BML-258, also known as SK1-I) or SK2 ((R)-FTY720 methylether (ROMe), a dual SK1/2 inhibitor (SKi) or an S1P1 receptor antagonist (W146). Vasodilator responses to S1P were also studied in isolated mouse aortic rings. The hypotensive response to anandamide was significantly attenuated by BML-258 but not by ROMe. Antagonising S1P1 receptors with W146 completely blocked the fall in systolic but not diastolic blood pressure in response to anandamide. S1P induced vasodilation in denuded aortic rings was blocked by W146 but caused no vasodilation in endothelium-intact rings. This study provides evidence that the SK1/S1P regulatory-axis is necessary for the rapid hypotension induced by anandamide. Generation of S1P in response to anandamide likely activates S1P1 to reduce total peripheral resistance and lower mean arterial pressure. These findings have important implications in our understanding of the hypotensive and cardiovascular actions of cannabinoids

Enhanced Anandamide Plasma Levels in Patients with Complex Regional Pain Syndrome following Traumatic Injury: A Preliminary Report

The complex regional pain syndrome (CRPS) is a disabling neuropathic pain condition that may develop following injuries of the extremities. The pathogenesis of this syndrome is not clear; however, it includes complex interactions between the nervous and the immune system resulting in chronic inflammation, pain and trophic changes. This interaction may be mediated by chronic stress which is thought to activate the endogenous cannabinoid (endocannabinoid) system (ECS). We conducted an open, prospective, comparative clinical study to determine plasma level of the endocannabinoid anandamide by high-performance liquid chromatography and a tandem mass spectrometry system in 10 patients with CRPS type I versus 10 age- and sex-matched healthy controls. As compared to healthy controls, CRPS patients showed significantly higher plasma concentrations of anandamide. These results indicate that the peripheral ECS is activated in CRPS. Further studies are warranted to evaluate the role of the ECS in the limitation of inflammation and pain. Copyright (C) 2009 S. Karger AG, Base

The evolution and comparative neurobiology of endocannabinoid signalling

CB(1)- and CB(2)-type cannabinoid receptors mediate effects of the endocannabinoids 2-arachidonoylglycerol (2-AG) and anandamide in mammals. In canonical endocannabinoid-mediated synaptic plasticity, 2-AG is generated postsynaptically by diacylglycerol lipase alpha and acts via presynaptic CB(1)-type cannabinoid receptors to inhibit neurotransmitter release. Electrophysiological studies on lampreys indicate that this retrograde signalling mechanism occurs throughout the vertebrates, whereas system-level studies point to conserved roles for endocannabinoid signalling in neural mechanisms of learning and control of locomotor activity and feeding. CB(1)/CB(2)-type receptors originated in a common ancestor of extant chordates, and in the sea squirt Ciona intestinalis a CB(1)/CB(2)-type receptor is targeted to axons, indicative of an ancient role for cannabinoid receptors as axonal regulators of neuronal signalling. Although CB(1)/CB(2)-type receptors are unique to chordates, enzymes involved in biosynthesis/inactivation of endocannabinoids occur throughout the animal kingdom. Accordingly, non-CB(1)/CB(2)-mediated mechanisms of endocannabinoid signalling have been postulated. For example, there is evidence that 2-AG mediates retrograde signalling at synapses in the nervous system of the leech Hirudo medicinalis by activating presynaptic transient receptor potential vanilloid-type ion channels. Thus, postsynaptic synthesis of 2-AG or anandamide may be a phylogenetically widespread phenomenon, and a variety of proteins may have evolved as presynaptic (or postsynaptic) receptors for endocannabinoids

Glucocorticoid-endocannabinoid interaction in cardiac surgical patients: relationship to early cognitive dysfunction and late depression

Background: Endocannabinoids (ECs) are rapidly acting immune-modulatory lipid-signaling molecules that are important for adaptation to stressful and aversive situations. They are known to interact with glucocorticoids and other stress-responsive systems. Maladaptation to acute or chronic stress represents a major risk factor for the development of psychiatric disorders. In the present study, we administered stress doses of hydrocortisone in a prospective, randomized, placebo-controlled double-blind study in patients undergoing cardiac surgery (CS) to examine the relationship between the use of glucocorticoids, plasma EC levels, and the occurrence of early postoperative cognitive dysfunction (delirium) and of later development of depression. Methods: We determined plasma levels of the ECs anandamide and 2-arachidonoylglycerol (2-AG) in CS patients of the hydrocortisone (n=56) and the placebo group (n=55) preoperatively, at postoperative day (POD) 1, at intensive care unit discharge, and at 6 months after CS (n=68). Postoperative delirium was diagnosed according to Diagnostic and Statistical Manual of the American Psychiatric Association IVth Edition (DSM-IV) criteria, and depression was determined by validated questionnaires and a standardized psychological interview (Structured Clinical Interview for DSM-IV). Results: Stress doses of hydrocortisone did not affect plasma EC levels and the occurrence of delirium or depression. However, patients who developed delirium on POD 1 had significantly lower preoperative 2-AG levels of the neuroprotective EC 2-AG (median values, 3.8 vs. 11.3 ng/ml; p=0.03). Preoperative 2-AG concentrations were predictive of postoperative delirium (sensitivity=0.70; specificity=0.69; cutoff value=4.9 ng/ml; receiver operating characteristic curve area=0.70; 95% confidence interval=0.54-0.85). Patients with depression at 6 months after CS (n=16) had significantly lower anandamide and 2-AG levels during the perioperative period. Conclusions: A low perioperative EC response may indicate an increased risk for early cognitive dysfunction and long-term depression in patients after CS. Glucocorticoids do not seem to influence this relationship

New Synthetic Endocannabinoid as Anti-Inflammaging Cosmetic Active: an In Vitro Study on a Reconstructed Skin Model

Endocannabinoids have been recently appointed as interesting cosmetic actives in regulating inflammaging, a state of chronic low-grade inflammation, known for being involved in many senescence\u2019s manifestations, included skin aging. The aim of this study was to assess the anti-inflammaging activity of a new synthetic endocannabinoid, Isopalmide\uae, on a reconstructed skin model, on which inflammaging has been reproduced through UVA radiation and light mechanical stress. We tested Isopalmide\uae both as a single active and conveyed in a cosmetic product, in comparison with Anandamide, a well-known natural endocannabinoid with anti-inflammatory action. The anti-inflammaging activity of topically applied products has been assessed, after 6 hours of treatment post-irradiation, through the transcriptional modification of genes involved in the NF-\u3baB pathway and the epigenetic pathway targeting miRs as potential biomarkers of inflammaging: miR-21, miR-126 and miR-146a. The results confirmed the anti-inflammatory action of Anandamide which inhibits NF-\u3baB, while Isopalmide\uae showed its anti-inflammaging activity through the establishment of an inflammatory/anti-inflammatory balance by maintaining NF-\u3baB inactive in the cytoplasm and active in the nucleus. The anti-inflammaging activity was shown also by the cosmetic product containing Isopalmide

Inactivation of anandamide signaling : A continuing Debate

Copyright 2011 Elsevier B.V., All rights reserved.Peer reviewedPublisher PD

Interaction between Cannabinoid System and Toll-Like Receptors Controls Inflammation

Since the discovery of the endocannabinoid system consisting of cannabinoid receptors, endogenous ligands, and biosynthetic and metabolizing enzymes, interest has been renewed in investigating the promise of cannabinoids as therapeutic agents. Abundant evidence indicates that cannabinoids modulate immune responses. An inflammatory response is triggered when innate immune cells receive a danger signal provided by pathogen- or damage-associated molecular patterns engaging pattern-recognition receptors. Toll-like receptor family members are prominent pattern-recognition receptors expressed on innate immune cells. Cannabinoids suppress Toll-like receptor-mediated inflammatory responses. However, the relationship between the endocannabinoid system and innate immune system may not be one-sided. Innate immune cells express cannabinoid receptors and produce endogenous cannabinoids. Hence, innate immune cells may play a role in regulating endocannabinoid homeostasis, and, in turn, the endocannabinoid system modulates local inflammatory responses. Studies designed to probe the interaction between the innate immune system and the endocannabinoid system may identify new potential molecular targets in developing therapeutic strategies for chronic inflammatory diseases. This review discusses the endocannabinoid system and Toll-like receptor family and evaluates the interaction between them