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Development and preliminary validation of a plate-based CB1/CB2 receptor functional assay.

By April 17, 2013No Comments

Pub Med

Development and preliminary validation of a plate-based CB1/CB2 receptor functional assay.


 [Epub ahead of print]

Development and preliminary validation of a plate-based CB1/CB2 receptor functional assay.


Biomedical Research Center, National Institute on Aging, National Institutes of Health, 251 Bayview Boulevard, Suite 100, Baltimore, MD 21224, USA.


Cannabinoids receptors (CB) are being targeted therapeutically for the treatment of anxiety, obesity, movement disorders, glaucoma, and pain. More recently, cannabinoid agonists have displayed anti-proliferative activity against breast cancer and prostate cancer in animal models. In order to studycannabinoid receptor ligands, we have developed a novel plate-based assay that measures internalization of CB1/CB2 receptors by determining the change in the intracellular levels of the radiolabeled agonists: [3H]-Win 55-212-2 for CB1 and [3H]-CP 55-940 for CB2. The developed plate-based assay was validated by determining IC50 values for known antagonists: AM251, AM281, AM630 and AM6545. The data obtained was consistent with previously reported values, therefore confirming that the assay can be used to determine the functional binding activities (IC50) of antagonists for the CB1 and CB2 receptors. In addition, we demonstrated that the plate-based assay may be used for screening against complex matrices. Specifically, we demonstrated that the plate-based assay was able to identify which extracts of several species of the genus Zanthoxylum had activity at the CB1/CB2 receptors.

Copyright © 2013. Published by Elsevier Inc.



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Figures and tables from this article:

Full-size image (52 K)
Fig.1. Chemical structures of tested compounds.
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Fig.2. Dose–response curve of the selective CB1 cannabinoid receptor antagonist AM281 on the inhibition of uptake of [3H]Win55,212,2 for CB1 receptors (A) or of [3H]CP55,940 for CB2 receptors (B). Each experiment was run using 0.5-fold serial dilutions, starting at 20 μM for CB1 and 40 μM for CB2.
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Fig.3. Effects of 10 and 0.1 μ/ml Z. bungeanum A (a), Z. bungeanum B (b), Z. schinifolium (c), Z. piperitum (d), and Z. armatum (e) on the uptake of [3H]Win55,212,2 for the CHO–CB1 cell line (A) or of [3H]CP55,940 for the CHO–CB2 cell line (B).
Table 1. IC50 values of tested ligands for inhibition of internalization of uptake of 10 nM [3H]Win55,212-2 and 2 nM [3H]CP55,940 for CB1 and CB2 receptors, respectively.
Note: Literature values are in parentheses. SE, standard error.
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Corresponding author. Fax: +1 410 558 8409.

Published by Elsevier Inc.

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