Selective blockade of 2-arachidonoylglycerol hydrolysis produces cannabinoid behavioral effects

Abstract

2-Arachidonoylglycerol (2-AG) and anandamide are endocannabinoids that activate the cannabinoid receptors CB1 and CB2. Endocannabinoid signaling is terminated by enzymatic hydrolysis, a process that for anandamide is mediated by fatty acid amide hydrolase (FAAH), and for 2-AG is thought to involve monoacylglycerol lipase (MAGL). FAAH inhibitors produce a select subset of the behavioral effects observed with CB1 agonists, which suggests a functional segregation of endocannabinoid signaling pathways in vivo. Testing this hypothesis, however, requires specific tools to independently block anandamide and 2-AG metabolism. Here, we report a potent and selective inhibitor of MAGL called JZL184 that, upon administration to mice, raises brain 2-AG by eight-fold without altering anandamide. JZL184-treated mice exhibited a broad array of CB1-dependent behavioral effects, including analgesia, hypothermia and hypomotility. These data indicate that 2-AG endogenously modulates several behavioral processes classically associated with the pharmacology of cannabinoids and point to overlapping and unique functions for 2-AG and anandamide in vivo.

Fig. 1

Structures and competitive ABPP profiles of MAGL inhibitors. a, Structures of MAGL inhibitors. b, Competitive ABPP showing the effect of MAGL inhibitors on serine hydrolase activities in the mouse brain membrane proteome. Shown for comparison are the profiles of the selective FAAH and ABHD6 inhibitors, URB597 and WWL70 (32) respectively. Inhibitors were incubated with brain membranes for 30 min, followed by treatment with the serine hydrolase-directed ABPP probe FP-rhodamine (2 μM, 30 min), and the proteomes were then analyzed by SDS-PAGE and in-gel fluorescence scanning to detect inhibited enzymes. Control proteomes were treated with DMSO alone. Fluorescent gel is shown in grayscale. Note that brain MAGL migrates as a 35 kDa doublet by SDS-PAGE, as reported previously,.

Fig. 2

In vitro characterization of JZL184. a, Concentration-dependent effects of JZL184 on mouse brain membrane serine hydrolases as determined by competitive ABPP. b, Blockade of brain membrane MAGL and FAAH activity by JZL184 as determined with substrate assays (2-AG and oleamide, respectively). c, Blockade of recombinant MAGL and FAAH activity by JZL184 as determined with substrate assays (2-AG and anandamide, respectively). Enzymes were recombinantly expressed in COS7 cells. Note that JZL184 produced a near-complete blockade of recombinant MAGL activity (> 95%), but ∼15% residual 2-AG hydrolysis activity was observed in brain membranes, likely reflecting the activity of other enzymes. For a-c, samples were treated with JZL184 for 30 min prior to analysis. For b and c, data are presented as means ± SEM for three independent experiments.

Fig. 3

In vivo characterization of JZL184. a and b, Serine hydrolase activity profiles (a) and MAGL and FAAH activities (b) of brain membranes prepared from mice treated with JZL184 at the indicated doses (4-40 mg kg^-1, i.p.) for 4 h. c, ABPP-MudPIT analysis of serine hydrolase activities in brain membranes prepared from mice treated with JZL184 (16 mg kg^-1, i.p., 4 h). MAGL and FAAH control signals are shown in red and blue bars, respectively. d-f, Brain levels of 2-AG (d), arachidonic acid (e), and NAEs (f) from mice treated with JZL184 at the indicated doses (4-40 mg kg^-1, i.p.) for 4 h. For f, data from mice treated with URB597 (10 mg kg^-1, i.p.) are also shown, confirming the elevations of NAEs induced by this FAAH inhibitor. For b-f, *, p < 0.05; **, p < 0.01 for inhibitor-treated versus vehicle-treated animals. Data are presented as means ± SEM. n = 3-5 mice per group.

Fig. 4

JZL184 raises interstitial levels of 2-AG following neuronal depolarization. Effects of JZL184 (10 mg kg^-1, i.p.) on interstitial levels of 2-AG and AEA were measured by in vivo microdialysis sampling from the nucleus accumbens of C57Bl/6 mice. Endocannabinoid release was stimulated by neuronal depolarization during perfusion with a high potassium & calcium artificial CSF solution (t = 0-90 min; shaded bar). Depolarization significantly increased dialysate 2-AG levels in both vehicle-(F(10,50) = 2.12, p < 0.05) and JZL184-treated (F(10,70) = 5.567, p < 0.0001) mice and this effect was substantially more robust in JZL184 treated animals as demonstrated by analysis of both the temporal profile (pretreatment x time interaction (F(10,120) = 3.355, *, p < 0.001; a) and area under the curve (AUC) measures (AUC t = 0-150 min; F(1,12) = 8.737; *, p < 0.05; b). There was no significant alteration in dialysate AEA levels following JZL184 administration and no significant effect of the high potassium/calcium solution on dialysate AEA levels in either group of mice as determined by analysis of both temporal profile and AUC measures (c and d). Data are the mean ± SEM of the percent change from baseline levels. Baseline dialysate 2-AG levels were 4.6 ± 0.7 nM and 4.2 ± 0.4 nM and dialysate AEA levels were 0.54 ± 0.1 nM vs. 0.58 ± 0.08 nM for the JZL184 (n = 8) and vehicle (n = 6) groups, respectively. Pretreatments with JZL184 were administered at t = -60 min (denoted by arrow).

Fig. 5

Time course analysis of inhibitory activity of JZL184 in vivo. a and b, Serine hydrolase activity profiles (a) and MAGL and FAAH activities (b) of brain membranes prepared from mice treated with JZL184 (16 mg kg^-1, i.p.) for the indicated times. c-e, Brain levels of 2-AG (c), arachidonic acid (d), and AEA (e) from mice treated with JZL184 (16 mg kg^-1, i.p.) for the indicated times. For b-e, *, p < 0.05; **, p < 0.01 for inhibitor-treated versus vehicle-treated control animals. Data are presented as means ± SEM. n = 3-5 mice per group.

Fig. 6

Behavioral effects of JZL184. a-d, JZL184 produced antinociceptive effects in the tail immersion assay of thermal pain sensation (a; 16 mg kg^-1, i.p.), acetic acid abdominal stretching assay of noxious chemical pain sensation (b; 16 mg kg^-1, i.p.), and both phase 1 (c; 40 mg kg^-1, p.o.) and phase 2 (d; 40 mg kg^-1, p.o.) of the formalin test. These effects were blocked by pre-treatment with the CB1 antagonist rimonabant (Rim., 3 mg kg^-1). JZL184 (16 mg kg^-1 i.p.) also produced significant hypothermia (e) and hypomotility (f) that were significantly attenuated by rimonabant. The baseline tail immersion latency and rectal temperature was 0.82 ± 0.02 s and 37.4 ± 0.1°C, respectively. *, p < 0.05; **, p < 0.01, for vehicle-vehicle versus vehicle-JZL184-treated mice. #, p < 0.05; ##, p < 0.01, for vehicle-JZL184 versus rimonabant-JZL184-treated mice. Data are presented as means ± SEM. n = 6-14 mice per group.

Fig. 7

Fig. 8