Pharmacological characterization of the endocannabinoid sensor GRABeCB2.0

Simar Singh; Dennis Sarroza; Anthony English; Maya McGrory; Ao Dong; Larry Zweifel; Benjamin B Land; Yulong Li; Michael R Bruchas; Nephi Stella

doi:10.1101/2023.03.03.531053

Pharmacological characterization of the endocannabinoid sensor GRAB_eCB2.0

bioRxiv [Preprint]. 2023 Mar 6:2023.03.03.531053. doi: 10.1101/2023.03.03.531053.

Authors

Simar Singh^{1

2

3}, Dennis Sarroza¹, Anthony English^{1

2

3}, Maya McGrory¹, Ao Dong⁴, Larry Zweifel^{1

2

3

5}, Benjamin B Land^{1

2

3}, Yulong Li⁴, Michael R Bruchas^{1

2

3

6}, Nephi Stella^{1

2

3

5}

Affiliations

¹ Department of Pharmacology, University of Washington, Seattle, USA.
² Center for Cannabis Research, University of Washington, Seattle, USA.
³ Center for the Neurobiology of Addiction, Pain, and Emotion, University of Washington, Seattle, USA.
⁴ Peking University School of Life Sciences, PKU-IDG/McGovern Institute for Brain Research, Peking-Tsinghua Center for Life Sciences, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China.
⁵ Department of Psychiatry and Behavioral Sciences, University of Washington, Seattle, USA.
⁶ Department of Anesthesiology, School of Medicine, University of Washington, Seattle, USA.

Abstract

Introduction: The endocannabinoids (eCBs), 2-arachidonoylglycerol (2-AG) and arachidonoyl ethanolamine (AEA), are produced by separate enzymatic pathways, activate cannabinoid receptors with distinct pharmacology, and differentially regulate pathophysiological processes. The genetically encoded sensor, GRAB_eCB2.0, detects real-time changes in eCB levels in cells in culture and preclinical model systems; however, its activation by eCB analogues produced by cells and by phyto-cannabinoids remains uncharacterized, a current limitation when interpreting changes in its response. This information could provide additional utility for the tool in in vivo pharmacology studies of phyto-cannabinoid action.

Methods: GRAB_eCB2.0 was expressed in cultured HEK293 cells. Live cell confocal microscopy and high-throughput fluorescent signal measurements.

Results: 2-AG increased GRAB_eCB2.0 fluorescent signal (EC₅₀ = 85 nM), and the cannabinoid 1 receptor (CB₁R) antagonist, SR141617, decreased GRAB_eCB2.0 signal (SR1, IC₅₀ = 3.3 nM), responses that mirror their known potencies at cannabinoid 1 receptors (CB₁R). GRAB_eCB2.0 fluorescent signal also increased in response to AEA (EC₅₀ = 815 nM), the eCB analogues 2-linoleoylglycerol and 2-oleoylglycerol (2-LG and 2-OG, EC₅₀s = 1.5 and 1.0 μM, respectively), Δ⁹-tetrahydrocannabinol (Δ⁹-THC) and Δ⁸-THC (EC₅₀s = 1.6 and 2.0 μM, respectively), and the artificial CB₁R agonist, CP55,940 (CP, EC₅₀ = 82 nM); however their potencies were less than what has been described at CB₁R. Cannabidiol (CBD) did not affect basal GRAB_eCB2.0 fluorescent signal and yet reduced the 2-AG stimulated GRAB_eCB2.0 responses (IC₅₀ = 8.8 nM).

Conclusions: 2-AG and SR1 modulate the GRAB_eCB2.0 fluorescent signal with EC₅₀s that mirror their potencies at CB₁R whereas AEA, eCB analogues, THC and CP increase GRAB_eCB2.0 fluorescent signal with EC₅₀s significantly lower than their potencies at CB₁R. CBD reduces the 2-AG response without affecting basal signal, suggesting that GRAB_eCB2.0 retains the negative allosteric modulator (NAM) property of CBD at CB₁R. This study describes the pharmacological profile of GRAB_eCB2.0 to improve interpretation of changes in fluorescent signal in response to a series of known eCBs and CB₁R ligands.

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Abstract

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