Dual Effects of Anandamide on NMDA Receptor-Mediated Responses and Neurotransmission

Abstract: Anandamide is an endogenous ligand of cannabinoid receptors that induces pharmacological responses in animals similar to those of cannabinoids such as Δ⁹-tetrahydrocannabinol (THC). Typical pharmacological effects of cannabinoids include disruption of pain, memory formation, and motor coordination, systems that all depend on NMDA receptor mediated neurotransmission. We investigated whether anandamide can influence NMDA receptor activity by examining NMDA-induced calcium flux (ΔCa²⁺_NMDA) in rat brain slices. The presence of anandamide reduced ΔCa²⁺_NMDA and the inhibition was disrupted by cannabinoid receptor antagonist, pertussis toxin treatment, and agatoxin (a calcium channel inhibitor). Whereas these treatments prevented anandamide inhibiting ΔCa²⁺_NMDA, they also revealed another, underlying mechanism by which anandamide influences ΔCa²⁺_NMDA. In the presence of cannabinoid receptor antagonist, anandamide potentiated ΔCa²⁺_NMDA in cortical, cerebellar, and hippocampal slices. Anandamide (but not THC) also augmented NMDA-stimulated currents in Xenopus oocytes expressing cloned NMDA receptors, suggesting a capacity to directly modulate NMDA receptor activity. In a similar manner, anandamide enhanced neurotransmission across NMDA receptor-dependent synapses in hippocampus in a manner that was not mimicked by THC and was unaffected by cannabinoid receptor antagonist. These data demonstrate that anandamide can modulate NMDA receptor activity in addition to its role as a cannabinoid receptor ligand.     

GC/MS analysis of anandamide and quantification of N-arachidonoylphosphatidylethanolamides in various brain regions, spinal cord, testis, and spleen of the rat

Anandamide [N-arachidonoylethanolamide (NAE)] was initially isolated from porcine brain and proposed as an endogenous ligand for cannabinoid receptors in 1992. Accumulating evidence has now suggested that, in the tissue, NAE is generated from N-arachidonoylphosphatidylethanolamides (N-ArPEs) by phosphodiesterase. In this study a sensitive and specific procedure was developed to quantify NAE and N-ArPE, including organic solvent extraction, reverse-phase C-18 cartridge separation, derivatization, and gas chromatography/mass spectrometry (GC/MS) analysis. NAE is converted by a two-step derivatization procedure to a pentafluorobenzoyl ester followed by pentafluoropropionyl acylation. Quantification was performed by isotope dilution GC/MS using deuterium-labeled NAE (NAE-2H8) as an internal standard. The same chemical derivatization was applicable to N-ArPE quantification. The separated N-ArPE fractions were converted by a two-step cleavage/derivatization procedure into the pentafluorobenzoyl ester of NAE and then to its pentafluoropropionyl amide. The derivative was quantified by GC/MS using deuterium-labeled 1,2-[2H8]dioleoyl-sn-glycero-3-phospho(arachidonoyl)ethanolamid e as an internal standard. Using these methods, we have found that endogenous NAE levels in rat brain, spleen, testis, liver, lung, and heart were below the level of quantification achievable (0.1 pmol/mg of protein) but that N-ArPE is readily quantifiable and is widely distributed in the rat CNS with the highest level in the spinal cord. The striatum, hippocampus, and accumbens contain intermediate concentrations of N-ArPE, whereas the value is lowest in the cerebellum.     

New tetrazole-based selective anandamide uptake inhibitors

A new series of 1,5- and 2,5-disubstituted tetrazoles have been synthesized and evaluated as inhibitors of anandamide cellular uptake. Some of them inhibit the uptake process with a relatively high potency (IC₅₀ = 2.3–5.1 μM) and selectively over other proteins involved in endocannabinoid action and metabolism.     

Rapid High-Energy Microwave Fixation is Required to Determine the Anandamide (<Emphasis Type="BoldItalic">N</Emphasis>-arachidonoylethanolamine) Concentration of Rat Brain

Anandamide (N-arachidonoylethanolamine, AEA) is the putative endogenous ligand for the CB₁ receptor. Despite being regulated enzymatically, brain AEA concentrations are quite variable and have been reported to increase in response to ischemia and post-mortem delay. Because these observations are similar to the effects of decapitation on brain concentrations of unesterified arachidonic acid and several of its metabolites, we propose that brain AEA concentrations also increase with decapitation and that immediate head-focused microwave irradiation is necessary to quantify basal brain AEA levels correctly. To test this hypothesis, we measured brain AEA levels in rats that were subjected to head-focused microwave irradiation 5 min. following decapitation (5.5 kW, 3.4 s) (ischemic) and prior to decapitation (controls). Brain AEA concentrations were quantified by LC/MS/MS. AEA concentrations from ischemic animals (10.01 ± 4.41 pmol/g, mean ± SD) were significantly higher and more variable than control concentrations (2.45 ± 0.39 pmol/g). Thus, the basal concentration of AEA in the brain is lower than previously thought and future studies attempting to quantify brain AEA should consider using head-focused microwave fixation to prevent anomalous results.     

Effects of anandamide on embryo implantation in the mouse

Anandamide (N-arachidonoylethanolamine), an arachidonic acid derivative, is an endogenous ligand for both the brain-type (CB1-R) and spleen-type (CB2-R) cannabinoid receptors. To investigate the possible effects of anandamide on embryo implantation in the mouse, we used a co-culture system in which mouse embryos are cultured with a monolayer of uterine epithelial cells. Our results indicate that 14 nM anandamide significantly promotes the attachment and outgrowth of the blastocysts on the monolayer of uterine epithelial cells, and those effects could be blocked by CB1-R antagonists SR141716A, but not by SR144528, a CB2-R antagonist. It suggests that the effects of anandamide on embryo attachment and outgrowth are mediated by CB1-R. However, 56 nM anandamide is capable of inhibiting the blastocyst attachment and outgrowth, we, therefore, conclude that anandamide may play an essential role at the outset of implantation.     

Simultaneous determination of endocannabinoids (arachidonylethanolamide and 2-arachidonylglycerol) and isoprostane (8-epiprostaglandin F2alpha) by gas chromatography-mass spectrometry-selected ion monitoring for medical samples

This article describes the overall procedure for the simultaneous determination of endocannabinoids (arachidonylethanolamide and 2-arachidonyglycerol) and isoprostane by gas chromatography-mass spectrometry in the selected-ion monitoring SIM mode (GC-MS-SIM) for medical samples. It also describes the general points of this method which a scientist who wants to assay a new, unidentified prostanoids and related compounds in medical samples would need to be clarified. The similar structures of prostaglandins, thromboxane, their metabolites, isoprostane, and arachidonyl compounds, allow them to be assayed after the simultaneous preparation of a single sample. The dimethyl isopropylsilyl ether forms of derivatized compounds are suitable for multiple GC-MS-SIM assay because of their molecular stability, and because they produce positive, strong, and large fragments on MS.     

Anandamide is an endogenous inhibitor for the migration of tumor cells and T lymphocytes

Cell migration is of paramount importance in physiological processes such as immune surveillance, but also in the pathological processes of tumor cell migration and metastasis development. The factors that regulate this tumor cell migration, most prominently neurotransmitters, have thus been the focus of intense investigation. While the majority of neurotransmitters have a stimulatory effect on cell migration, we herein report the inhibitory effect of the endogenous substance anandamide on both tumor cell and lymphocyte migration. Using a collagen-based three-dimensional migration assay and time-lapse videomicroscopy, we have observed that the anandamide-mediated signals for CD8⁺ T lymphocytes and SW 480 colon carcinoma cells are each mediated by distinct cannabinoid receptors (CB-Rs). Using the specific agonist docosatetraenoylethanolamide (DEA), we have observed that the norepinephrine-induced migration of colon carcinoma cells is inhibited by the CB₁-R. The SDF-1–induced migration of CD8⁺ T lymphocytes was, however, inhibited via the CB₂-R, as shown by using the specific agonist JWH 133. Therefore, specific inhibition of tumor cell migration via CB₁-R engagement might be a selective tool to prevent metastasis formation without depreciatory effects on the immune system of cancer patients.     

Levels, metabolism, and pharmacological activity of anandamide in CB(1) cannabinoid receptor knockout mice: evidence for non-CB(1), non-CB(2) receptor-mediated actions of anandamide in mouse brain

Anandamide [arachidonylethanolamide (AEA)] appears to be an endogenous agonist of brain cannabinoid receptors (CB(1)), yet some of the neurobehavioral effects of this compound in mice are unaffected by a selective CB(1) antagonist. We studied the levels, pharmacological actions, and degradation of AEA in transgenic mice lacking the CB(1) gene. We quantified AEA and the other endocannabinoid, 2-arachidonoyl glycerol, in six brain regions and the spinal cord by isotope-dilution liquid chromatography-mass spectrometry. The distribution of endocannabinoids and their inactivating enzyme, fatty acid amide hydrolase, were found to overlap with CB(1) distribution only in part. In CB(1) knockout homozygotes (CB(1)-/-), the hippocampus and, to a lesser extent, the striatum exhibited lower AEA levels as compared with wild-type (CB(1)+/+) controls. These data suggest a ligand/receptor relationship between AEA and CB(1) in these two brain regions, where tonic activation of the receptor may tightly regulate the biosynthesis of its endogenous ligand. 2-Arachidonoyl glycerol levels and fatty acid amide hydrolase activity were unchanged in CB(1)-/- with respect to CB(1)+/+ mice in all regions. AEA and Delta(9)-tetrahydrocannabinol (THC) were tested in CB(1)-/- mice for their capability of inducing analgesia and catalepsy and decreasing spontaneous activity. The effects of AEA, unlike THC, were not decreased in CB(1)-/- mice. AEA, but not THC, stimulated GTPgammaS binding in brain membranes from CB(1)-/- mice, and this stimulation was insensitive to CB(1) and CB(2) antagonists. We suggest that non-CB(1), non-CB(2) G protein-coupled receptors might mediate in mice some of the neuro-behavioral actions of AEA.     

Mechanisms for the coupling of cannabinoid receptors to intracellular calcium mobilization in rat insulinoma beta-cells

In RIN m5F rat insulinoma beta-cells, agonists at cannabinoid CB(1) receptors modulate insulin release. Here we investigated in these cells the effect of the activation of cannabinoid CB(1) and CB(2) receptors on intracellular Ca(2+) ([Ca(2+)](i)). The CB(1) agonist arachidonoyl-chloro-ethanolamide (ACEA), and the CB(2) agonist JWH133, elevated [Ca(2+)](i) in a way sensitive to the inhibitor of phosphoinositide-specific phospholipase C (PI-PLC), U73122 (but not to pertussis toxin and forskolin), and independently from extracellular Ca(2+). PI-PLC-dependent Ca(2+) mobilization by ACEA was entirely accounted for by activation of inositol-1,3,4-phosphate (IP(3)) receptors on the endoplasmic reticulum (ER), whereas the effect of JWH133 was not sensitive to all tested inhibitors of IP(3) and ryanodine receptors. ACEA, but not JWH133, significantly inhibited the effect on [Ca(2+)](i) of bombesin, which acts via G(q/11)- and PI-PLC-coupled receptors in insulinoma cells. The endogenous CB(1) agonists, anandamide and N-arachidonoyldopamine, which also activate transient receptor potential vanilloid type 1 (TRPV1) receptors expressed in RIN m5F cells, elevated [Ca(2+)](i) in the presence of extracellular Ca(2+) in a way sensitive to both CB(1) and TRPV1 antagonists. These results suggest that, in RIN m5F cells, CB(1) receptors are coupled to PI-PLC-mediated mobilization of [Ca(2+)](i) and might inhibit bombesin signaling.