A novel probe for the cannabinoid receptor

Devane, William A.; Breuer, Aviva; Sheskin, Tzviel; Järbe, Torbjörn U. C.; Eisen, Moris S.; Mechoulam, Raphael

doi:10.1021/jm00089a018

Cited by 184 publications

(119 citation statements)

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“…Synaptosomal membranes were prepared from the brains of Sabra male rats (250 g) after removal of the brainstem. The [ 3 H]HU-243 probe, with a dissociation constant of 45 pM (11), was incubated with synaptosomal membranes (3-4 g) for 90 min at 30°C with the indicated concentrations of noladine ether or the vehicle alone (fatty-acid-free BSA at a final concentration of 0.5 mg͞ml). Bound and free radioligand were separated by centrifugation (see ref.…”

Section: Methodsmentioning

confidence: 99%

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2-Arachidonyl glyceryl ether, an endogenous agonist of the cannabinoid CB ₁ receptor

Hanŭs

Abu‐Lafi

Fride

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

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694

379

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Two types of endogenous cannabinoid-receptor agonists have been identified thus far. They are the ethanolamides of polyunsaturated fatty acids-arachidonoyl ethanolamide (anandamide) is the best known compound in the amide series-and 2-arachidonoyl glycerol, the only known endocannabinoid in the ester series. We report now an example of a third, ether-type endocannabinoid, 2-arachidonyl glyceryl ether (noladin ether), isolated from porcine brain. The structure of noladin ether was determined by mass spectrometry and nuclear magnetic resonance spectroscopy and was confirmed by comparison with a synthetic sample. It binds to the CB1 cannabinoid receptor (Ki ‫؍‬ 21.2 ؎ 0.5 nM) and causes sedation, hypothermia, intestinal immobility, and mild antinociception in mice. It binds weakly to the CB2 receptor (Ki > 3 M).W e have reported the isolation and identification of two types of endogenous cannabinoids that bind and activate the known cannabinoid receptors CB 1 and CB 2 . Arachidonoyl ethanolamide (anandamide; ref. 1) and later two more polyunsaturated fatty acid ethanol amides (2) were found in porcine brain. An ester, 2-arachidonoyl glycerol (2-AG) was isolated by us from canine gut (3) and by Sugiura et al. from brain (4). Anandamide and 2-AG have been the objects of numerous investigations in various areas of biology and have been found to affect processes in the nervous, cardiovascular, immune, and reproductive systems (5-8). They interact with many neurotransmitters and affect hormone levels (9-10). This ubiquity of effects led us to look for additional endocannabinoids.We report now that we have isolated from porcine brain a third endocannabinoid, 2-arachidonyl glyceryl ether, which we have named noladin ether ( Fig. 1). Materials and MethodsIsolation of Noladin Ether. Porcine brain (100 g, approximately a single brain) was added to a mixture of chloroform (200 ml) and methanol (200 ml) and mixed in a blender for 2 min. Water (100 ml) was added, and the mixing process was continued for another minute. The mixture was filtered. Two layers were formed. The water-methanol layer was separated and evaporated under reduced pressure. The residue obtained was extracted with methanol. The above isolation was repeated numerous times (from a total of 2.4 kg porcine brain). The extract obtained was chromatographed on a gravity column (i.d. 2.5 cm, height 28 cm, 82 g ICN Silica TSC, 60 A) with hexane͞acetone initially in a ratio of 10:1 (vol͞vol) (400 ml), then 9:1 (100 ml), and finally 4:1 (100 ml). The fractions eluted were monitored for binding to the CB 1 receptor from rat brain synaptosomes (prepared as described below; ref. 11) on the basis of displacement of the potent labeled agonist [ 3 H]HU-243 purchased from Tocris (Bristol, U.K.). The material eluted in fractions 45-54 (10-ml each) was found to bind to the receptor. These fractions were combined and purified further by HPLC (see below). A polar-active compound developed on a TLC plate (silica gel 60 F 254 , Merck) in a hexane͞ acetone (4:1) solvent system gave ...

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Section: Methodsmentioning

confidence: 99%

“…Bound and free radioligand were separated by centrifugation (see ref. 11 for full details of the assay). The data were normalized to 100% of specific binding, which was determined with 50 nM unlabeled HU-243.…”

Section: Methodsmentioning

confidence: 99%

2-Arachidonyl glyceryl ether, an endogenous agonist of the cannabinoid CB ₁ receptor

Hanŭs

Abu‐Lafi

Fride

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

694

379

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“…The cannabinoids HU-243, HU-210, HU-211, Ag-tetrahydrocannabinol (A9-THC), and anandamide have been previously described [5,[16][17][18]. The synthesis of the cannabinoids HU-293 and HU-293a will be described separately.…”

Section: Methodsmentioning

confidence: 99%

The peripheral cannabinoid receptor: adenylate cyclase inhibition and G protein coupling

et al. 1995

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Two cannabinoid receptors, designated neuronal (or CB1) and peripheral (or CB2), have recently been cloned. Activation of CB1 receptors leads to inhibition of adenylate cyclase and N-type voltage-dependent Ca 2+ channels. Here we show, using a CB2 transfected Chinese hamster ovary cell line, that this receptor binds a variety of tricyclic cannabinoid ligands as well as the endogenous ligand anandamide. Activation of the CB2 receptor by various tricydic cannabinoids inhibits adenylate cyclase activity and this inhibition is pertussis toxin sensitive indicating that this receptor is coupled to the GilGo GTP-binding proteins. Interestingly, contrary to results with CB1, anandamide did not inhibit the CB2 coupled adenylate cyclase activity and A9-tetrahydrocannabinol had only marginal effects. These results characterize the CB2 receptor as a functional and distinctive member of the cannabinoid receptor family.

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“…The a nity of CBD and (+)-CBD for human VR1 receptors was assessed by means of displacement assays carried out with membranes (50 mg tube 71 ) from HEKhVR1 cells, prepared as described previously (Ross et al, 2001), and the high a nity VR1 ligand [ 3 H]-resiniferatoxin (48 Ci mmol 71 , NEN-Dupont), using the incubation conditions described previously (Ross et al, 2001 Cannabinoid CB 1 and CB 2 receptor binding assays These methods have been described previously by Devane et al (1992a) for CB 1 , and Bayewitch et al (1996) for CB 2 . For CB 1 receptor binding assays synaptosomal membranes from rat brains were used.…”

Section: Vr1 Receptor Binding Assaysmentioning

confidence: 99%

Molecular targets for cannabidiol and its synthetic analogues: effect on vanilloid VR1 receptors and on the cellular uptake and enzymatic hydrolysis of anandamide

Bisogno¹,

Hanŭs

Petrocellis

et al. 2001

British J Pharmacology

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1 (7)-Cannabidiol (CBD) is a non-psychotropic component of Cannabis with possible therapeutic use as an anti-in¯ammatory drug. Little is known on the possible molecular targets of this compound. We investigated whether CBD and some of its derivatives interact with vanilloid receptor type 1 (VR1), the receptor for capsaicin, or with proteins that inactivate the endogenous cannabinoid, anandamide (AEA). 2 CBD and its enantiomer, (+)-CBD, together with seven analogues, obtained by exchanging the C-7 methyl group of CBD with a hydroxy-methyl or a carboxyl function and/or the C-5' pentyl group with a di-methyl-heptyl (DMH) group, were tested on: (a) VR1-mediated increase in cytosolic Ca 2+ concentrations in cells over-expressing human VR1; (b) [ 14 C]-AEA uptake by RBL-2H3 cells, which is facilitated by a selective membrane transporter; and (c) [ 14 C]-AEA hydrolysis by rat brain membranes, which is catalysed by the fatty acid amide hydrolase. 3 Both CBD and (+)-CBD, but not the other analogues, stimulated VR1 with EC 50 =3.2 ± 3.5 mM, and with a maximal e ect similar in e cacy to that of capsaicin, i.e. 67 ± 70% of the e ect obtained with ionomycin (4 mM). CBD (10 mM) desensitized VR1 to the action of capsaicin. The e ects of maximal doses of the two compounds were not additive. 4 (+)-5'-DMH-CBD and (+)-7-hydroxy-5'-DMH-CBD inhibited [ 14 C]-AEA uptake (IC 50 =10.0 and 7.0 mM); the (7)-enantiomers were slightly less active (IC 50 =14.0 and 12.5 mM). CBD and (+)-CBD were also active (IC 50 =22.0 and 17.0 mM). 5 CBD (IC 50 =27.5 mM), (+)-CBD (IC 50 =63.5 mM) and (7)-7-hydroxy-CBD (IC 50 =34 mM), but not the other analogues (IC 50 4100 mM), weakly inhibited [ 14 C]-AEA hydrolysis. 6 Only the (+)-isomers exhibited high a nity for CB 1 and/or CB 2 cannabinoid receptors. 7 These ®ndings suggest that VR1 receptors, or increased levels of endogenous AEA, might mediate some of the pharmacological e ects of CBD and its analogues. In view of the facile high yield synthesis, and the weak a nity for CB 1 and CB 2 receptors, (7)-5'-DMH-CBD represents a valuable candidate for further investigation as inhibitor of AEA uptake and a possible new therapeutic agent.

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A novel probe for the cannabinoid receptor

Cited by 184 publications

References 2 publications

2-Arachidonyl glyceryl ether, an endogenous agonist of the cannabinoid CB ₁ receptor

2-Arachidonyl glyceryl ether, an endogenous agonist of the cannabinoid CB ₁ receptor

The peripheral cannabinoid receptor: adenylate cyclase inhibition and G protein coupling

Molecular targets for cannabidiol and its synthetic analogues: effect on vanilloid VR1 receptors and on the cellular uptake and enzymatic hydrolysis of anandamide

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A novel probe for the cannabinoid receptor

Cited by 184 publications

References 2 publications

2-Arachidonyl glyceryl ether, an endogenous agonist of the cannabinoid CB 1 receptor

2-Arachidonyl glyceryl ether, an endogenous agonist of the cannabinoid CB 1 receptor

The peripheral cannabinoid receptor: adenylate cyclase inhibition and G protein coupling

Molecular targets for cannabidiol and its synthetic analogues: effect on vanilloid VR1 receptors and on the cellular uptake and enzymatic hydrolysis of anandamide

Contact Info

Product

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About

2-Arachidonyl glyceryl ether, an endogenous agonist of the cannabinoid CB ₁ receptor

2-Arachidonyl glyceryl ether, an endogenous agonist of the cannabinoid CB ₁ receptor