PubMed: Pharmacological characterization of the endocannabinoid sensor GRAB <sub>eCB2.0</sub>

PubMed: Pharmacological characterization of the endocannabinoid sensor GRAB <sub>eCB2.0</sub>

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


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–a3-xbLzPoB9xM&fc=20220928170152&ff=20230322191001&v=2.17.9.post6+86293ac March 22, 2023 10:00 am