Structure-activity relationships among N-arachidonylethanolamine (Anandamide) head group analogues for the anandamide transporter.

Two putative endocannabinoids, N-arachidonylethanolamine (AEA) and 2-arachidonylglycerol, are inactivated by removal from the extracellular environment by a process that has the features of protein-mediated facilitated diffusion. We have synthesized and studied 22 N-linked analogues of arachidonylamide for the purpose of increasing our understanding of the structural requirements for the binding of ligands to the AEA transporter. We have also determined the affinities of these analogues for both the CB(1) cannabinoid receptor and fatty acid amide hydrolase (FAAH). We have identified several structural features that enhance binding to the AEA transporter in cerebellar granule cells. We have confirmed the findings of others that replacing the ethanolamine head group with 4-hydroxybenzyl results in a high-affinity ligand for the transporter. However, we find that the same molecule is also a competitive inhibitor of FAAH. Similarly, replacement of the ethanolamine of AEA with 3-pyridinyl also results in a high-affinity inhibitor of both the transporter and FAAH. We conclude that the structural requirements for ligand binding to the CB(1) receptor and binding to the transporter are very different; however, the transporter and FAAH share most, but not all, structural requirements.

Selective effects of the endogenous cannabinoid arachidonylethanolamide (anandamide) on regional cerebral blood flow in the rat.

Recent biochemical data suggest that arachidonylethanolamide (AEA; anandamide) may be an endogenous ligand for brain cannabinoid receptors. The functional neuronal consequences of AEA binding to cannabinoid receptors are only poorly understood. Using regional cerebral blood flow (rCBF) as an indirect marker of neuronal activity, acute AEA administration dose-dependently depressed rCBF in unanesthetized rats. Although 3.0 mg/kg was ineffective in altering rCBF, 10 mg/kg led to a decrease in rCBF in seven brain areas including the amygdala, cingulate, frontal, prepyriform, sensorimotor, and claustrocortex. An additional 16 areas responded in a similar manner to AEA, but only after 30 mg/kg, including the CA1 and CA3 regions of the hippocampus, the rostral core portion of the nucleus accumbens, and rostral caudate nucleus. Most of these rCBF effects dissipated between 15 and 20 min after drug administration, with only 4 regions, the basomedial and lateral amygdala, CA3 hippocampus and claustrocortex still depressed 60 min after an acute drug injection. No significant changes in heart rate, blood pressure, or blood gases were seen at the time of rCBF measurement, suggesting that the observed drug effects were neuronally mediated. Taken together with existing behavioral data, these data support the hypothesis that an endogenous cannabinoid neural system exists in mammalian brain and may help to explain the unique behavioral profile seen after cannabinoid administration.

Identification of the 11,14,15- and 11,12, 15-trihydroxyeicosatrienoic acids as endothelium-derived relaxing factors of rabbit aorta.

A number of endothelium-derived relaxing factors have been identified including nitric oxide, prostacyclin, and the epoxyeicosatrienoic acids. Previous work showed that in rabbit aortic endothelial cells, arachidonic acid was metabolized by a lipoxygenase to vasodilatory eicosanoids. The identity was determined by the present study. Aortic homogenates were incubated in the presence of [U-14C]arachidonic acid, [U-14C]arachidonic acid plus 15-lipoxygenase (soybean lipoxidase), or [U-14C]15-hydroxyeicosatetraenoic acid (15-HPETE) and analyzed by reverse phase high pressure liquid chromatography (RP-HPLC). Under both experimental conditions, there was a radioactive metabolite that migrated at 17.5-18.5 min on RP-HPLC. When the metabolite was isolated from aortic homogenates, it relaxed precontracted aortas in a concentration-dependent manner. Gas chromatography/mass spectrometry (GC/MS) of the derivatized metabolite indicated the presence of two products; 11,12,15-trihydroxyeicosatrienoic acid (THETA) and 11,14,15-THETA. A variety of chemical modifications of the metabolite supported these structures and confirmed the presence of a carboxyl group, double bonds, and hydroxyl groups. With the combination of 15-lipoxygenase, arachidonic acid, and aortic homogenate, an additional major radioactive peak was observed. This fraction was analyzed by GC/MS. The mass spectrum was consistent with this peak, containing both the 11-hydroxy-14, 15-epoxyeicosatrienoic acid (11-H-14,15-EETA) and 15-H-11,12-EETA. The hydroxyepoxyeicosatrienoic acid (HEETA) fraction also relaxed precontracted rabbit aorta. Microsomes derived from rabbit aortas also synthesized 11,12,15- and 11,14,15-THETAs from 15-HPETE, and pretreatment with the cyctochrome P450 inhibitor, miconazole, blocked the formation of these products. The present studies suggest that arachidonic acid is metabolized by 15-lipoxygenase to 15-HPETE, which undergoes an enzymatic rearrangement to 11-H-14,15-EETA and 15-H-11,12-EETA. Hydrolysis of the epoxy group results in the formation of 11,14,15- and 11,12,15-THETA, which relaxed rabbit aorta. Thus, the 15-series THETAs join prostacyclin, nitric oxide, and epoxyeicosatrienoic acids as new members of the family of endothelium-derived relaxing factors.

Human platelets and polymorphonuclear leukocytes synthesize oxygenated derivatives of arachidonylethanolamide (anandamide): their affinities for cannabinoid receptors and pathways of inactivation.

Arachidonylethanolamide (AEA), the putative endogenous ligand of the cannabinoid receptor, has been shown to be a substrate for lipoxygenase enzymes in vitro. One goal of this study was to determine whether lipoxygenase-rich cells metabolize AEA. [14C]AEA was converted by human polymorphonuclear leukocytes (PMNs) to two major metabolites that comigrated with synthetic 12(S)- and 15(S)-hydroxy-arachidonylethanolamide (HAEA). Human platelets convert [14C]AEA to 12(S)-HAEA. 12(S)-HAEA binds to both CB1 and CB2 receptors with approximately the same affinity as AEA. 12(R)-HAEA, which is not produced by PMNs, has 2-fold lower affinity for the CB1 receptor and 10-fold lower affinity for the CB2 receptor than 12(S)-HAEA. 15-HAEA has a lower affinity than AEA for both receptors, with Ki values of 738 and >1000 nM for CB1 and CB2 receptors, respectively. The addition of a hydroxyl group at C20 of AEA resulted in a ligand with the same affinity for the CB1 receptor but a 4-fold lower affinity for the CB2 receptor than AEA. 12(S)-HAEA and 15-HAEA are poor substrates for AEA amidohydrolase and do not bind to the AEA uptake carrier. In conclusion, the addition of a hydroxyl group at C12 of the arachidonate backbone of AEA does not affect binding to CB receptors but is likely to increase its half-life. The addition of hydroxyl groups at other positions affects ligand affinity for CB receptors; both the position of the hydroxyl group and the configuration of the remaining double bonds are determinants of affinity.

Synthesis and characterization of diazomethylarachidonyl ketone: an irreversible inhibitor of N-arachidonylethanolamine amidohydrolase.

N-Arachidonylethanolamine (AEA), a putative endogenous agonist of neuronal (CB1) cannabinoid receptors, is a substrate for N-arachidonylethanolamine amidohydrolase (AEA amidohydrolase), a serine amidase present in cell membranes. Following a strategy that has been used to develop inhibitors that covalently bind to the active site of serine peptidases, diazomethyl arachidonyl ketone (DAK) was synthesized and its effects on AEA amidohydrolase were determined. DAK inhibits the hydrolysis of AEA by rat brain membranes with an IC50 value of 0.5 microM. At low concentrations, DAK reduces the Vmax and increases the K(m) of the enzyme for its substrate AEA, which suggests that it is both a competitive and noncompetitive inhibitor. At higher concentrations, DAK inhibition is completely noncompetitive. DAK inhibition of membrane-associated AEA amidohydrolase is irreversible because hydrolytic activity is not restored with extensive washing or dialysis of the membranes. Furthermore, DAK inhibition is not reversible by anion exchange chromatography of the subsequently solubilized enzyme. In contrast, DAK inhibition of detergent-solubilized enzyme exhibits competitive kinetics and is reversible upon ion exchange chromatography. Exposure of C6 glioma cells to DAK results in concentration-related inhibition of AEA amidohydrolase activity in cellular membranes with an IC50 value of 0.3 microM. In summary, these studies demonstrate that DAK is an irreversible inhibitor of AEA amidohydrolase in its native membrane and provides a useful tool with which to study the role of AEA amidohydrolase in the termination of action of AEA.

N-arachidonylethanolamide relaxation of bovine coronary artery is not mediated by CB1 cannabinoid receptor.

It has been reported that the endogenous cannabinoid N-arachidonylethanolamide (AEA), commonly referred to as anandamide, has the characteristics of an endothelium-derived hyperpolarizing factor in rat mesenteric artery. We have carried out studies to determine whether AEA affects coronary vascular tone. The vasorelaxant effects of AEA were determined in isolated bovine coronary artery rings precontracted with U-46619 (3 x 10(-9) M). AEA decreased isometric tension, producing a maximal relaxation of 51 +/- 9% at a concentration of 10(-5) M. Endothelium-denuded coronary arteries were not significantly affected by AEA. The CB1 receptor antagonist SR-141716A (10(-6)M) failed to reduce the vasodilatory effects of AEA, suggesting that the CB1 receptor is not involved in this action of AEA. Because AEA is rapidly converted to arachidonic acid and ethanolamine in brain and liver by a fatty acid amide hydrolase (FAAH), we hypothesized that the vasodilatory effect of AEA results from its hydrolysis to arachidonic acid followed by enzymatic conversion to vasodilatory eicosanoids. In support of this hypothesis, bovine coronary arteries incubated with [3H]AEA for 30 min hydrolyzed 15% of added substrate; approximately 9% of the radiolabeled product was free arachidonic acid, and 6% comigrated with the prostaglandins (PGs) and epoxyeicosatrienoic acids (EETs). A similar result was obtained in cultured bovine coronary endothelial cells. Inhibition of the FAAH with diazomethylarachidonyl ketone blocked both the metabolism of [3H]AEA and the relaxations to AEA. Whole vessel and cultured endothelial cells prelabeled with [3H]arachidonic acid synthesized [3H]PGs and [3H]EETs, but not [3H]AEA, in response to A-23187. Furthermore, SR-141716A attenuated A-23187-stimulated release of [3H]arachidonic acid, suggesting that it may have actions other than inhibition of CB1 receptor. These experiments suggest that AEA produces endothelium-dependent vasorelaxation as a result of its catabolism to arachidonic acid followed by conversion to vasodilatory eicosanoids such as prostacyclin or the EETs.

Accumulation of N-arachidonoylethanolamine (anandamide) into cerebellar granule cells occurs via facilitated diffusion.

N-Arachidonoylethanolamine (anandamide, AEA) is a putative endogenous ligand of the cannabinoid receptor. Intact cerebellar granule neurons in primary culture rapidly accumulate AEA. [3H]AEA accumulation by cerebellar granule cells is dependent on incubation time (t(1/2) of 2.6 +/- 0.8 min at 37 degrees C) and temperature. The accumulation of AEA is saturable and has an apparent Km of 41 +/- 15 microM and a Vmax of 0.61 +/- 0.04 nmol/min/10(6) cells. [3H]AEA accumulation by cerebellar granule cells is significantly reduced by 200 microM phloretin (57.4 +/- 4% of control) in a noncompetitive manner. [3H]AEA accumulation is not inhibited by either ouabain or removal of extracellular sodium. [3H]AEA accumulation is fairly selective for AEA among other naturally occurring N-acylethanolamines; only N-oleoylethanolamine significantly inhibited [3H]AEA accumulation at a concentration of 10 microM. The ethanolamides of palmitic acid and linolenic acid were inactive at 10 microM. N-Arachidonoylbenzylamine and N-arachidonoylpropylamine, but not arachidonic acid, 15-hydroxy-AEA, or 12-hydroxy-AEA, compete for AEA accumulation. When cells are preloaded with [3H]AEA, temperature-dependent efflux occurs with a half-life of 1.9 +/- 1.0 min. Phloretin does not inhibit [3H]AEA efflux from cells. These results suggest that AEA is accumulated by cerebellar granule cells by a protein-mediated transport process that has the characteristics of facilitated diffusion.

Nonclassical and endogenous cannabinoids: effects on the ordering of brain membranes.

The effects of several nonclassical cannabinoids and the endogenous cannabinoid ligand, anandamide on the lipid ordering of rat brain synaptic plasma membranes (SPM) were examined and compared to delta 9-tetrahydrocannabinol (delta 9-THC). SPM order was determined using fluorescence polarization. All compounds tested affected membrane ordering. delta 9-THC, CP-55,940, CP-55,244 and WIN-55212 decreased lipid ordering in SPM. Some stereospecificity was observed with delta 9-THC and WIN-55212, but not other compounds. Anandamide also decreased lipid order as did its putative precursor, arachidonic acid. In contrast to these compounds, levonantradol increased SPM lipid order. Although all pharmacologically active cannabinoids affect SPM lipid order, potency on this measure does not correlate well with their pharmacological potency. The results of this study suggest that membrane perturbation (either increases or decreases in lipid order) may be a necessary characteristic for cannabinoid pharmacological activity, but it is not a primary or sufficient determinate of action for this class of drugs.

Physiological and behavioural effects of the endogenous cannabinoid, arachidonylethanolamide (anandamide), in the rat.

1. Arachidonylethanolamide (AEA; anandamide) has been isolated from mammalian brain and found to bind to, and is thought to be, an endogenous ligand for the cannabinoid receptor. In order to understand better its behavioural and physiological properties, we have examined its acute effects in unanaesthetized freely behaving rats. 2. Intravenous AEA caused dose-related decreases in locomotor behaviour, a pronounced hyperreflexia, and a moderate antinociceptive state. At doses between 3 and 30 mg kg-1, a dose-dependent hypothermia and profound, time-dependent cardiovascular changes were also observed. 3. An immediate bradycardia exceeding 50% was seen within 10-15 s of administration and lasted up to 11 min following the highest dose of the drug. In contrast, the change in mean arterial pressure was biphasic: an immediate 20% decrease in mean arterial pressure followed by a significant increase in blood pressure that lasted about 13 min after the highest dose. 4. These data demonstrate that AEA in the unanaesthetized rat exerts behavioural and physiological effects generally similar to those seen following natural cannabinoids and synthetic cannabimimetic agents and suggests a role for AEA in regulation of various physiological processes.

Vasorelaxation by an endothelium-derived metabolite of arachidonic acid.

Arachidonic acid elicited relaxation responses in normal rabbit aorta precontracted with norepinephrine. The relaxation response was enhanced by the cyclooxygenase inhibitor indomethacin and inhibited by lipoxygenase inhibitors, including nordihydroguaiaretic acid and cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate. The cytochrome P-450 epoxygenase inhibitor metyrapone had no effect on arachidonic acid-induced relaxations. The present study hypothesized that a lipoxygenase metabolite of arachidonic acid mediated the response. Incubation of rabbit aorta with [14C]arachidonic acid resulted in the synthesis of a previously unidentified 14C-labeled metabolite and was called the unknown factor. Production of the unknown factor was not inhibited by indomethacin and decreased by lipoxygenase inhibitors. Production of the unknown factor and arachidonic acid-induced relaxations were dependent on an intact endothelium, indicating that the cellular source of the unknown relaxant factor was the endothelial cell. This was confirmed by demonstrating the ability of cultured rabbit aortic endothelial cells to produce the unknown factor from [14C]arachidonic acid. Feeding rabbits a 2% cholesterol diet for 2 wk induced hypercholesterolemia without causing atherosclerosis. In the cholesterol-fed rabbits, indomethacin enhanced arachidonic acid-induced relaxations in norepinephrine-precontracted aortas (maximal relaxation 49.0 +/- 2.5 vs. 35.5 +/- 1.7%, cholesterol-fed vs. normal) and increased production of the unknown factor compared with normal rabbits. The partially purified unknown factor elicited an approximately 26% inhibition of the vasoconstrictor response to norepinephrine in intact rabbit aorta. Further purification of the unknown factor by reverse-phase high-pressure liquid chromatography system resulted in isolation of a radioactive product that relaxed precontracted rabbit aorta. Therefore these data suggest that in normal and hypercholesterolemic rabbit aorta the endothelium produces an unknown metabolite of arachidonic acid that causes vasorelaxation and may regulate vascular tone.

Characterization of the kinetics and distribution of N-arachidonylethanolamine (anandamide) hydrolysis by rat brain.

Arachidonoylethanolamide or 'anandamide' is a naturally occurring derivative of arachidonic acid that has been shown to activate cannabinoid receptors in the brain. Its metabolic inactivation by brain tissue has been investigated. Anandamide is hydrolyzed by the membrane fraction of rat brain homogenate to arachidonic acid and ethanolamine. The hydrolysis is temperature and pH- dependent (pH maximum at 8.5) and abolished by boiling. Anandamide hydrolysis is protein dependent in the range of 25-100 micrograms protein/ml; does not require calcium and is inhibited by phenylmethylsulfonylfluoride, diisopropylfluorophosphate, thimerosal and arachidonic acid. Hydrolysis of 10 microM anandamide by brain membranes follows first order kinetics; at 30 degrees C, the rate constant for anandamide catabolism is 0.34 min-1 mg protein-1. The Km for anandamide hydrolysis is 3.4 microM, and the Vmax is 2.2 nmol/min per mg protein. Hydrolysis occurs in all subcellular fractions except cytosol with the highest specific activity in myelin and microsomes. The distribution of anandamide hydrolytic activity correlates with the distribution of cannabinoid receptor-binding sites; the hippocampus, cerebellum and cerebral cortex exhibit the highest metabolic activity, while activity is lowest in the striatum, brain stem and white matter.

Characterization of ligand binding to the cannabinoid receptor of rat brain membranes using a novel method: application to anandamide.

Ligand binding to the cannabinoid receptor of brain membranes has been characterized using [3H]CP 55,940 and the Multiscreen Filtration System. Binding of [3H]CP 55,940 is saturable and reaches equilibrium by 45 min at room temperature. At a concentration of 10 micrograms of membrane protein/well, the KD for [3H]CP 55,940 is 461 pM and the Bmax is 860 fmol/mg of protein. The apparent KD of [3H]CP 55,940 is dependent upon tissue protein concentration, increasing to 2,450 pM at 100 micrograms of membrane protein. Binding of [3H]CP 55,940 is dependent upon the concentration of bovine serum albumin in the buffer; the highest ratio of specific to nonspecific binding occurs between 0.5 and 1.0 mg/ml. The Ki of anandamide, a putative endogenous ligand of the cannabinoid receptor, is 1.3 microM in buffer alone and 143 nM in the presence of 0.15 mM phenylmethylsulfonyl fluoride. When [14C]anandamide is incubated with rat forebrain membranes at room temperature, it is degraded to arachidonic acid; the hydrolysis is inhibited by 0.15 mM phenylmethylsulfonyl fluoride. These results support the hypothesis that anandamide is a high-affinity ligand of the cannabinoid receptor and that it is rapidly degraded by membrane fractions.

The binding of novel phenolic derivatives of anandamide to brain cannabinoid receptors.

Arachidonylethanolamide (N-2-hydroxyethyl-arachidonamide) or 'anandamide' is a naturally occurring derivative of arachidonic acid that has been shown to bind and activate cannabinoid receptors in the brain. Since other potent ligands for the cannabinoid receptor have an aromatic hydroxyl group, we investigated the hypothesis that replacement of the ethanolamine hydroxyl with an aromatic hydroxyl will increase the binding affinity for the cannabinoid receptor. Two novel congeners of anandamide containing aromatic hydroxyl groups were synthesized: N-2-(4-hydroxyphenyl)ethyl arachidonamide (HEA) and N-2-hydroxyphenyl arachidonamide (HPA). The affinity of these congeners for the brain cannabinoid receptor was determined by competition with [3H]CP55940. HEA competed for [3H]CP55940 binding with a Ki of 600 nM; HPA had a Ki of 2200 nM. These results indicate that increased size in the amide portion of anandamide decreases affinity for the receptor. Phenylmethylsulfonyl fluoride (PMSF), an inhibitor of anandamide catabolism by brain membranes, had no effect on the binding of either HEA or HPA. We conclude that these congeners are not substrates for the amidase that catabolizes anandamide.