Biology subjects

Sarroza, D.

Publications and source records attributed to Sarroza, D..

3 recordsLinked to original sources

bioRxiv2023

Pharmacological characterization of the endocannabinoid sensor GRABeCB2.0

IntroductionThe 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, GRABeCB2.0, 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. MethodsGRABeCB2.0 was expressed in cultured HEK293 cells. Live cell confocal microscopy and high-throughput fluorescent signal measurements. Results2-AG increased GRABeCB2.0 fluorescent signal (EC50 = 85 nM), and the cannabinoid 1 receptor (CB1R) antagonist, SR141617, decreased GRABeCB2.0 signal (SR1, IC50 = 3.3 nM), responses that mirror their known potencies at cannabinoid 1 receptors (CB1R). GRABeCB2.0 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 M, respectively), {Delta}9-tetrahydrocannabinol ({Delta}9-THC) and{Delta} 8-THC (EC50s = 1.6 and 2.0 M, 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 GRABeCB2.0 fluorescent signal and yet reduced the 2-AG stimulated GRABeCB2.0 responses (IC50 = 8.8 nM). Conclusions2-AG and SR1 modulate the GRABeCB2.0 fluorescent signal with EC50s that mirror their potencies at CB1R whereas AEA, eCB analogues, THC and CP increase GRABeCB2.0 fluorescent signal with EC50s significantly lower than their potencies at CB1R. CBD reduces the 2-AG response without affecting basal signal, suggesting that GRABeCB2.0 retains the negative allosteric modulator (NAM) property of CBD at CB1R. This study describes the pharmacological profile of GRABeCB2.0 to improve interpretation of changes in fluorescent signal in response to a series of known eCBs and CB1R ligands.

pharmacology and toxicology↗

bioRxiv2022

A preclinical model of THC edibles that produces high-dose cannabimimetic responses

No preclinical experimental approach enables the study of voluntary oral consumption of high-concentration {Delta}9-tetrahydrocannabinol (THC) and its intoxicating effects, mainly owing to the aversive response of rodents to THC that limits intake. Here we developed a palatable THC formulation and an optimized access paradigm in mice to drive voluntary consumption. THC was formulated in chocolate gelatin (THC-E-gel). Adult male and female mice were allowed ad libitum access for 1 and 2 h. Cannabimimetic responses (hypolocomotion, analgesia, and hypothermia) were measured following access. Levels of THC and its metabolites were measured in blood and brain tissue. Acute acoustic startle responses were measured to investigate THC-induced psychotomimetic behavior. When allowed access for 2 h to THC-E-gel on the second day of a three-day exposure paradigm, adult mice consumed up to {approx}30 mg/kg over 2 h which resulted in robust cannabimimetic behavioral responses (hypolocomotion, analgesia and hypothermia). Consumption of the same gelatin decreased on the following 3rd day of exposure. Pharmacokinetic analysis show that THC-E-gel consumption led to parallel accumulation of THC and its psychoactive metabolite, 11-OH-THC, in brain, a profile that contrasts with the known rapid decline in brain 11-OH-THC levels following THC intraperitoneal (i.p.) injections. THC-E-gel consumption increased the acoustic startle response in males but not in females, demonstrating a sex-dependent effect of consumption. Thus, while voluntary consumption of THC-E-gel triggered equivalent cannabimimetic responses in male and female mice, it potentiated acoustic startle responses preferentially in males. We build a dose-prediction model that included cannabimimetic behavioral responses elicited by i.p. versus THC-E-gel to test the accuracy and generalizability of this experimental approach and found that it closely predicted the measured acoustic startle results in males and females. In summary, THC-E-gel offers a robust preclinical experimental approach to study cannabimimetic responses triggered by voluntary consumption in mice, including sex-dependent psychotomimetic responses.

animal behavior and cognition↗

bioRxiv2022

ABHD6 selectively controls Gq-dependent increases in 2-AG production

The most abundant endocannabinoid (eCB) in the brain, 2-arachidonoyl glycerol (2-AG), is hydrolyzed by /{beta}-hydrolase domain containing 6 (ABHD6); yet how ABHD6 controls stimuli-dependent increases in 2-AG production is unknown. To explore this question, we leveraged the recently developed 2-AG sensor, GRABeCB2.0, and found that stimulation of Neuro2a cells in culture with bradykinin (BK) acting at metabotropic B2K receptors and ATP acting at ionotropic P2X7 receptors led to differential increases in 2-AG levels. B2K triggered increases in 2-AG levels via diacylglycerol lipase (DAGL), and this mechanism was potentiated by increases in intracellular calcium and ABHD6 inhibition. By contrast, P2X7-triggered increases in 2-AG levels were dependent on DAGL and extracellular calcium but unaffected by ABHD6 inhibition. Thus, ABHD6 preferentially regulates metabotropic-dependent increases in 2-AG levels over ionotropic-dependent increases in 2-AG levels. Our study indicates that ABHD6 selectively controls stimuli-dependent increases in 2-AG production and emphasizes its specific role in eCB signaling.

cell biology↗