Receptor Binding Affinities of Synthetic Cannabinoids Determined by Non-Isotopic Receptor Binding Assay

Jin, Hongzhe; Song, Yun; Lee, Da Eun; Kim, Young-Hoon; Shin, Jisoon; Jang, Choon‐Gon; Suh, Soo Jhi; Kim, Sung Jin; Yun, Jaesuk

doi:10.5487/tr.2019.35.1.037

Cited by 2 publications

(2 citation statements)

References 29 publications

(37 reference statements)

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“…Employing an in vitro receptor-binding assay, we previously reported that JWH-210 binds to CB1 [ 33 ]. However, to the best of our knowledge, there are no studies showing the interaction of JWH-210 and CB1 in vivo [ 34 ].…”

Section: Resultsmentioning

confidence: 99%

Cannabinoid Receptor Type 1 Regulates Drug Reward Behavior via Glutamate Decarboxylase 67 Transcription

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Park

et al. 2021

IJMS

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Interaction of cannabinoid receptor type 1 (CB1) and GABAergic neuronal activity is involved in drug abuse-related behavior. However, its role in drug-dependent Pavlovian conditioning is not well understood. In this study, we aimed to evaluate the effects of a CB1 agonist, JWH-210, on the development of conditioned place preference (CPP)-induced by methamphetamine (METH). Pretreatment with a synthetic cannabinoid, JWH-210 (CB1 agonist), increased METH-induced CPP score and METH-induced dopamine release in acute striatal slices. Interestingly, CB1 was expressed in glutamate decarboxylase 67 (GAD67) positive cells, and overexpression of CB1 increased GAD67 expression, while CB1 knockdown reduced GAD67 expression in vivo and in vitro. GAD67 is known as an enzyme involved in the synthesis of GABA. CB1 knockdown in the mice striatum increased METH-induced CPP. When GAD67 decreased in the mice striatum, mRNA level of CB1 did not change, suggesting that CB1 can regulate GAD67 expression. GAD67 knockdown in the mouse striatum augmented apomorphine (dopamine receptor D2 agonist)–induced climbing behavior and METH-induced CPP score. Moreover, in the human brain, mRNA level of GAD67 was found to be decreased in drug users. Therefore, we suggest that CB1 potentiates METH-induced CPP through inhibitory GABAergic regulation of dopaminergic neuronal activity.

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Section: Resultsmentioning

confidence: 99%

Cannabinoid Receptor Type 1 Regulates Drug Reward Behavior via Glutamate Decarboxylase 67 Transcription

Seo

Park

et al. 2021

IJMS

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“…During the last decades, there is a growing trend to replace radioligand-based techniques with more costeffective and environmentally sustainable alternative procedures for the study of different aspects of G protein-coupled receptors (GPCR), including the cannabinoid CB 1 receptor [41]. Thus, immunohistochemistry has largely displaced autoradiography for the study of CB 1 receptor distribution in histological sections [42][43][44], with the advantage of providing more accurate information on receptor distribution [45].…”

Section: Discussionmentioning

confidence: 99%

Design and validation of recombinant protein standards for quantitative Western blot analysis of cannabinoid CB1 receptor density in cell membranes: an alternative to radioligand binding methods

et al. 2022

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Background: Replacement of radioligand binding assays with antibody-antigen interaction-based approaches for quantitative analysis of G protein-coupled receptor (GPCR) levels requires the use of purified protein standards containing the antigen. GPCRs in general and cannabinoid CB 1 receptor in particular show a progressive tendency to aggregate and precipitate in aqueous solution outside of their biological context due to the low solubility that the hydrophobic nature imprinted by their seven transmembrane domains. This renders full-length recombinant GPCRs useless for analytical purposes, a problem that can be overcome by engineering soluble recombinant fragments of the receptor containing the antigen.Results: Here we generated highly soluble and stable recombinant protein constructs GST-CB1 414-472 and GST-CB1 414-442 containing much of the human CB 1 receptor C-terminal tail for use as standard and negative control, respectively, in quantitative Western blot analysis of CB 1 receptor expression on crude synaptosomes of the adult rat brain cortex. To this end we used three different antibodies, all raised against a peptide comprising the C-terminal residues 443-473 of the mouse CB 1 receptor that corresponds to residues 442-472 in the human homolog. Estimated values of CB 1 receptor density obtained by quantitative Western blot were of the same order of magnitude but slightly higher than values obtained by the radioligand saturation binding assay. Conclusions:Collectively, here we provide a suitable Western blot-based design as a simple, cost-effective and radioactivity-free alternative for the quantitative analysis of CB 1 receptor expression, and potentially of any GPCR, in a variety of biological samples. The discrepancies between the results obtained by quantitative Western blot and radioligand saturation binding techniques are discussed in the context of their particular theoretical bases and methodological constraints.

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Receptor Binding Affinities of Synthetic Cannabinoids Determined by Non-Isotopic Receptor Binding Assay

Cited by 2 publications

References 29 publications

Cannabinoid Receptor Type 1 Regulates Drug Reward Behavior via Glutamate Decarboxylase 67 Transcription

Cannabinoid Receptor Type 1 Regulates Drug Reward Behavior via Glutamate Decarboxylase 67 Transcription

Design and validation of recombinant protein standards for quantitative Western blot analysis of cannabinoid CB1 receptor density in cell membranes: an alternative to radioligand binding methods

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