Pharmacological Characterization of the Endocannabinoid Sensor GRABeCB2.0

Abstract

Introduction: The endocannabinoids (eCBs), 2-arachidonoylglycerol (2-AG) and arachidonoyl ethanolamine (AEA), are produced by separate enzymatic pathways, activate cannabinoid (CB) receptors with distinct pharmacological profiles, 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-CBs 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-CB action. Materials and 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, SR141716 (SR1), decreased GRAB_eCB2.0 signal (IC₅₀=3.3 nM), responses that mirror their known potencies at the CB₁R. GRAB_eCB2.0 fluorescent signal also increased in response to AEA (EC₅₀=815 nM), the eCB analogues 2-linoleoylglycerol and 2-oleoylglycerol (EC₅₀=632 and 868 nM, respectively), Δ⁹-tetrahydrocannabinol (Δ⁹-THC), and Δ⁸-THC (EC₅₀=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=9.7 nM). Conclusions: 2-AG and SR1 modulate the GRAB_eCB2.0 fluorescent signal with EC₅₀ values that mirror their potencies at CB₁R, whereas AEA, eCB analogues, THC, and CP increase GRAB_eCB2.0 fluorescent signal with EC₅₀ values 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.

Keywords: CB1; endocannabinoids; pharmacology; phytocannabinoids.