PubMed: Pharmacological characterization of the endocannabinoid sensor GRAB _eCB2.0

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

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 (EC ₅₀ = 85 nM), and the cannabinoid 1 receptor ( CB ₁ R ) antagonist, SR141617, decreased GRAB _eCB2.0 signal ( SR1 , IC ₅₀ = 3.3 nM), responses that mirror their known potencies at cannabinoid 1 receptors ( CB ₁ R ). GRAB _eCB2.0 fluorescent signal also increased in response to AEA (EC ₅₀ = 815 nM), the eCB analogues 2-linoleoylglycerol and 2-oleoylglycerol ( 2-LG and 2-OG , EC ₅₀ s = 1.5 and 1.0 μM, respectively), Δ ⁹ -tetrahydrocannabinol ( Δ ⁹ -THC ) and Δ ⁸ -THC (EC ₅₀ s = 1.6 and 2.0 μM, respectively), and the artificial CB ₁ R agonist, CP55,940 ( CP , EC ₅₀ = 82 nM); however their potencies were less than what has been described at CB ₁ R. Cannabidiol ( CBD ) did not affect basal GRAB _eCB2.0 fluorescent signal and yet reduced the 2-AG stimulated GRAB _eCB2.0 responses (IC ₅₀ = 8.8 nM).

CONCLUSIONS: 2-AG and SR1 modulate the GRAB _eCB2.0 fluorescent signal with EC ₅₀ s that mirror their potencies at CB ₁ R whereas AEA, eCB analogues, THC and CP increase GRAB _eCB2.0 fluorescent signal with EC ₅₀ s significantly lower than their potencies at CB ₁ R. 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 CB ₁ R. 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 CB ₁ R ligands.

PMID:36945533 | PMC:PMC10028790 | DOI:10.1101/2023.03.03.531053