Endocannabinoid-like Molecules Research Articles

Host- and Helminth-Derived Endocannabinoids That Have Effects on Host Immunity Are Generated during Infection.

Helminths have coevolved with their hosts, resulting in the development of specialized host immune mechanisms and parasite-specific regulatory products. Identification of new pathways that regulate helminth infection could provide a better understanding of host-helminth interaction and may identify new therapeutic targets for helminth infection. Here we identify the endocannabinoid system as a new mechanism that influences host immunity to helminths. Endocannabinoids are lipid-derived signaling molecules that control important physiologic processes, such as feeding behavior and metabolism. Following murine infection with Nippostrongylus brasiliensis, an intestinal nematode with a life cycle similar to that of hookworms, we observed increased levels of endocannabinoids (2-arachidonoylglycerol [2-AG] or anandamide [AEA]) and the endocannabinoid-like molecule oleoylethanolamine (OEA) in infected lung and intestine. To investigate endocannabinoid function in helminth infection, we employed pharmacological inhibitors of cannabinoid subtype receptors 1 and 2 (CB1R and CB2R). Compared to findings for vehicle-treated mice, inhibition of CB1R but not CB2R resulted in increased N. brasiliensis worm burden and egg output, associated with significantly decreased expression of the T helper type 2 cytokine interleukin 5 (IL-5) in intestinal tissue and splenocyte cultures. Strikingly, bioinformatic analysis of genomic and transcriptome sequencing (RNA-seq) data sets identified putative genes encoding endocannabinoid biosynthetic and degradative enzymes in many parasitic nematodes. To test the novel hypothesis that helminth parasites produce their own endocannabinoids, we measured endocannabinoid levels in N. brasiliensis by mass spectrometry and quantitative PCR and found that N. brasiliensis parasites produced endocannabinoids, especially at the infectious larval stage. To our knowledge, this is the first report of helminth- and host-derived endocannabinoids that promote host immune responses and reduce parasite burden.

Oleoylethanolamide, an endogenous PPAR-α ligand, attenuates liver fibrosis targeting hepatic stellate cells.

Oleoylethanolamide (OEA), an endocannabinoid-like molecule, was revealed to modulate lipid metabolism through a peroxisome proliferator-activated receptor-α (PPAR-α) mediated mechanism. In present study, we further investigated the activities and mechanisms of OEA in ameliorating hepatic fibrosis in Sv/129 mice induced by a methionine choline-deficient (MCD) diet or thioacetamide (TAA) treatment. Liver fibrosis development was assessed by Hematoxylin-eosin and Sirius red staining. Treatment with OEA (5 mg/kg/day, intraperitoneal injection, i.p.) significantly attenuated the progress of liver fibrosis in both two experimental animal models by blocking the activation of hepatic stellate cells (HSCs). Gene expression analysis of hepatic tissues indicated that OEA inhibited the expression of α-smooth muscle action (α-SMA) and collagen matrix, fibrosis markers, and genes involved in inflammation and extracellular matrix remodeling. In vitro studies showed that OEA inhibited transforming growth factor β1-stimulated HSCs activation through suppressing Smad2/3 phosphorylation, α-SMA expression and myofibroblast transformation. These improvements could not be observed in PPAR-α knockout mice models with OEA administration, which suggested all the anti-fibrotic effects of OEA in vivo and in vitro were mediated by PPAR-α activation. Collectively, our results suggested that OEA exerted a pharmacological effect on modulating hepatic fibrosis development through the inhibition of HSCs activation in liver and therefore may be a potential therapeutic agent for liver fibrosis.

Anandamide drives cell cycle progression through CB1 receptors in a rat model of synchronized liver regeneration.

The endocannabinoid system, through cannabinoid receptor signaling by endocannabinoids, is involved in a wide range of functions and physiopathological conditions. To date, very little is known concerning the role of the endocannabinoids in the control and regulation of cell proliferation. An anti-proliferative action of CB1 signaling blockade in neurogenesis and angiogenesis argues in favor of proliferation-promoting functions of endocannabinoids through CB1 receptors when pro-growth signals are present. Furthermore, liver regeneration, a useful in vivo model of synchronized cell proliferation, is characterized by a peak of anandamide that elicits through CB1 receptor, the expression of critical mitosis genes. The aim of this study was to focus on the timing of endocannabinoid signaling changes during the different phases of the cell cycle, exploiting the rat liver regeneration model following partial hepatectomy, the most useful to study synchronized cell cycle in vivo. Hepatic regeneration led to increased levels of anandamide and endocannabinoid-like molecules oleoylethanolamide (OEA) and palmitoylethanolamide (PEA) in the G1 phase of the cell cycle, with a concomitant increase in CB1 mRNA levels, whose protein expression peaked later during the S phase. Blocking of CB1 receptor with a low dose of the selective antagonist/inverse agonist SR141716 (0.7 mg/kg/dose) affected cell cycle progression reducing the expression of PCNA, and through the inhibition of pERK and pSTAT3 pathways. These results support the notion that the signaling mediated by anandamide through CB1 receptor may be important for the entry and progression of cells into the cell cycle and hence for their proliferation under mitogenic signals.

Troubleshooting in LC-MS/MS method for determining endocannabinoid and endocannabinoid-like molecules in rat brain structures applied to assessing the brain endocannabinoid/endovanilloid system significance

Introduction: In recent years, a potential participation of endocannabinoids (eCBs) and related endocannabinoid-like molecules, including N-acylethanolamines (NAEs), in the physiological and pathophysiological processes has been highlighted, whereas measurement of their levels still remains difficult. The aim of this study was to develop a bioanalytical method that would enable researchers to simultaneously determine quantitatively eCBs (anandamide – AEA and 2-arachidonoylglycerol – 2-AG) and NAEs (oleoylethanolamide or oleoylethanolamine – OEA, palmitoylethanolamide or palmitoylethanolamine – PEA and linoleoylethanolamide or linoleoylethanolamine – LEA) in the rat brain. The analytical problems with analysis and possible solutions have been also shown.Methods: The methodology for quantifying eCBs/NAEs by means of a sensitive and selective liquid chromatography tandem mass spectrometry (LC-MS/MS) with electrospray positive ionization and multiple reaction monitoring (MRM) mode was developed and validated. Analytical problems with analyzed compounds were estimated.Results: Reasonably high precision and accuracy of the method were demonstrated in the validation process. The method is linear up to 200 ng/g for AEA, OEA, PEA and LEA and up to 100 μg/g for 2-AG, while the quantification limit reaches 0.2 ng/g and 0.8 μg/g, respectively.Discussion: Simplicity and rapidity of the assay allows analyzing many samples on a routine basis. This article presents the new procedure applied to the analysis of brain tissues.

Changes in the level of endocannabinoids and endocannabinoidlike molecules after acute and repeated administration of escitalopram or FAAH inhibitor in rat brain structures

Changes in the level of endocannabinoids and endocannabinoidlike molecules after acute and repeated administration of escitalopram or FAAH inhibitor in rat brain structures

Conference Report (42nd Annual Meeting of the Italian Society of Neurology, 22-25 October 2011, Turin, Italy)

Conference Report (42nd Annual Meeting of the Italian Society of Neurology, 22-25 October 2011, Turin, Italy)

Endocannabinoids and Human Sperm Cells

N-acylethanolamides (NAEs) are naturally occurring signaling lipids consisting of amides and esters of long-chain polyunsaturated fatty acids. Usually they are present in a very small amounts in many mammalian tissues and cells, including human reproductive tracts and fluids. Recently, the presence of N-arachidonoylethanolamide (anandamide, AEA), the most characterised member of endocannabinoids, and its congeners palmitoylethanolamide (PEA) and oleylethanolamide (OEA) in seminal plasma, oviductal fluid, and follicular fluids was demonstrated. AEA has been shown to bind not only type-1 (CB1) and type-2 (CB2) cannabinoid receptors, but also type-1 vanilloid receptor (TRPV1), while PEA and OEA are inactive with respect to classical cannabinoid CB1 and CB2 but activate TRPV1 or peroxisome proliferator activate receptors (PPARs). This review concerns the most recent experimental data on PEA and OEA, endocannabinoid-like molecules which appear to exert their action exclusively on sperm cells with altered features, such as membrane characteristics and kinematic parameters. Their beneficial effects on these cells could suggest a possible pharmacological use of PEA and OEA on patients affected by some forms of idiopathic infertility.

Vasorelaxation to N‐oleoylethanolamine in rat isolated arteries: mechanisms of action and modulation via cyclooxygenase activity

The endocannabinoid-like molecule N-oleoylethanolamine (OEA) is found in the small intestine and regulates food intake and promotes weight loss. The principal aim of the present study was to evaluate the vascular effects of OEA. Perfused isolated mesenteric arterial beds were pre-contracted with methoxamine or high potassium buffers and concentration-response curves to OEA were constructed. Combinations of inhibitors to block nitric oxide production, sensory nerve activity, cyclooxygenase activity, potassium channels, chloride channels and gap junctions, and a cannabinoid CB(1) receptor antagonist, were used during these experiments. The effects of OEA on caffeine-induced contractions in calcium-free buffer were also assessed. Isolated thoracic aortic rings were used as a comparison. OEA caused concentration-dependent vasorelaxation in rat isolated mesenteric arterial beds and thoracic aortic rings, with a greater maximal response in mesenteric vessels. This relaxation was sensitive to inhibition of sensory nerve activity and endothelial removal in both preparations. The cyclooxygenase inhibitor indomethacin reversed the effects of capsaicin pre-treatment in perfused mesenteric arterial beds and indomethacin alone enhanced vasorelaxation to OEA. The OEA-induced vasorelaxation was inhibited by a CB(1) receptor antagonist only in aortic rings. In mesenteric arteries, OEA suppressed caffeine-induced contractions in calcium-free buffer. The vasorelaxant effects of OEA are partly dependent on sensory nerve activity and a functional endothelium in the vasculature. In addition, vasorelaxation to OEA is enhanced following cyclooxygenase inhibition. OEA may also interfere with the release of intracellular calcium in arterial preparations.

From Surface to Nuclear Receptors: The Endocannabinoid Family Extends its Assets

Peroxisome proliferator-activated receptors (PPARs) have long been known as mediators of several physiological functions, among which the best characterized are lipid metabolism, energy balance and anti-inflammation. Their rather large and promiscuous ligand binding site has been recently discovered to accommodate, among a plethora of lipid molecules and metabolic intermediates, endocannabinoids and their cognate compounds, specifically belonging to the Nacylethanolamine group. In fact, oleoylethanolamide, palmitoylethanolamide and probably anandamide bind with relatively high affinity to PPARs and have now been included among their endogenous ligands. Through activation of PPARs these molecules exert a variety of physiological processes. Particularly, both long-term effects via genomic mechanisms and rapid non-genomic actions have been described, which in several instances are opposite to those evoked by activation of "classical" surface cannabinoid receptors. In this review, we describe how these effects are relevant under diverse physiological and pathophysiological circumstances, such as lipid metabolism and feeding behaviour, neuroprotection and epilepsy, circadian rhythms, addiction and cognition. A picture is emerging where nuclear receptors are involved in anorexiant, anti-inflammatory, neuroprotective, anti-epileptic, wakefulness- and cognitive-enhancing, and anti-addicting properties of endocannabinoid-like molecules. Further studies are necessary to fully understand cellular mechanisms underlying the interactions between endocannabinoids and PPARs, but also between their surface and nuclear receptors, and to exploit their potential therapeutic applications.

Lipidomic methodologies applicable to the study of endocannabinoids and related compounds: Endocannabinoidomics

AbstractThe identification of the endogenous ligands of the cannabinoid CB1 and CB2 receptors (endocannabinoids) as lipids nominally derived from arachidonic acid was a further demonstration that lipid molecules can be endowed with important signaling functions. This discovery was paralleled by the development of ever more sophisticated technologies for the profiling of small molecules, and the subsequent birth and exponential growth of the discipline of “metabolomics” and, in the case of analytical techniques for lipids, of its subdiscipline known as “lipidomics”. The application of lipidomics to the analysis of endocannabinoids, and of endocannabinoid‐like molecules with activities also at non‐cannabinoid receptors, is now culminating in the identification of more families of fatty acid derivatives, thus revealing what has been defined as the “endocannabinoid metabolome”, and in the development of specific techniques for the lipidomic profiling of these molecules. These new analytical advances are discussed in this article.

The complications of promiscuity: endocannabinoid action and metabolism.

In this review, we present our understanding of the action and metabolism of endocannabinoids and related endogenous molecules. It is clear that the interactions between the multiple endocannabinoid-like molecules (ECLs) are highly complex, both at the level of signal transduction and metabolism. Thus, ECLs are a group of ligands active at 7-transmembrane and nuclear receptors, as well as transmitter-gated and ion channels. ECLs and their metabolites can converge on common endpoints (either metabolic or signalling) through contradictory or reinforcing pathways. We highlight the complexity of the endocannabinoid system, based on the promiscuous nature of ECLs and their metabolites, as well as the synthetic modulators of the endocannabinoid system.