JWH018, a common constituent of 'Spice' herbal blends, is a potent and efficacious cannabinoid CB receptor agonist

Abstract

Background and purpose: 'Spice' is an herbal blend primarily marketed in Europe as a mild hallucinogen with prominent cannabis-like effects and as a legal alternative to cannabis. However, a recent report identified a number of synthetic additives in samples of 'Spice'. One of these, the indole derivative JWH018, is a ligand for the cannabinoid receptor 1 (CB(1)) cannabinoid receptor and inhibits cAMP production in CB(1) receptor-expressing CHO cells. Other effects of JWH018 on CB(1) receptor-mediated signalling are not known, particularly in neurons. Here we have evaluated the signalling pathways activated by JWH018 at CB(1) receptors.

Experimental approach: We investigated the effects of JWH018 on neurotransmission in cultured autaptic hippocampal neurons. We further analysed its activation of ERK1/2 mitogen activated protein kinase (MAPK) and internalization of CB(1) receptors in HEK293 cells stably expressing this receptor.

Key results: In cultured autaptic hippocampal neurons, JWH018 potently inhibited excitatory postsynaptic currents (IC(50)= 14.9 nM) in a concentration- and CB(1) receptor-dependent manner. Furthermore, it increased ERK1/2 MAPK phosphorylation (EC(50)= 4.4 nM). We also found that JWH018 potently induced rapid and robust CB(1) receptor internalization (EC(50)= 2.8 nM; t(1/2)= 17.3 min).

Conclusions and implications: JWH018, a prominent component of several herbal preparations marketed for their psychoactivity, is a potent and effective CB(1) receptor agonist that activates multiple CB(1) receptor signalling pathways. Thus, it is likely that the subjective effects of 'Spice' are due to activation of cannabinoid CB(1) receptors by JWH018, added to this herbal preparation.

Figure 1

JWH018 decreases neurotransmitter release by activating presynaptic CB₁ receptors. (A) Chemical structure of JWH018. (B) JWH018 concentration-dependently decreased EPSC charge (n= 8 to 14 for each concentration tested). This inhibitory effect was reversed by 1 µM rimonabant (n= 12) and was absent in CB₁−/− neurons (n= 5). (C) A representative experiment showing a time course of EPSC inhibition by 1 nM JWH018 followed by 100 nM JWH018. 1 µM rimonabant reversed JWH018 inhibition. Inset shows representative traces for three indicated time points. JWH018 (100 nM) significantly increases the paired-pulse ratio (right: representative traces of paired pulses, numbers indicate order of pulses) (D) and decreases miniature EPSC frequency (E) without affecting miniature EPSC amplitude (F), indicative of decreasing the probability of neurotransmitter release by acting at a presynaptic site (n= 5 to 8 for each). Inset scale bars: 1 nA and 10 ms. (G) Coefficient of variation analysis demonstrating that r < π < 1, which is consistent with a presynaptic site for JWH018's action resulting in synaptic depression. Values are presented as mean ± SEM where applicable. *P < 0.05, **P < 0.01, ***P < 0.0001. CB₁, cannabinoid receptor 1; EPSC, excitatory postsynaptic current.

Figure 2

JWH018 activation of CB₁ receptors stimulated ERK1/2 MAPK phosphorylation. (A) 100 nM JWH018 treatment of CB₁ expressing HEK293 cells, transiently increased ERK1/2 phosphorylation, similar to the time course of 100 nM WIN55,212 activation (4–6 replicate samples from 7–15 independent experiments). (B) CB₁ receptor expressing HEK293 cells were treated for 7.5 min with increasing concentrations of JWH018 and WIN55,212. JWH018 activated ERK1/2 in a concentration-dependent manner and was more potent than WIN55,212 (4–6 replicate samples from 8–15 independent experiments). 1 µM rimonabant reversed the effect of JWH018 and WIN55,212 on ERK1/2 activation. Values are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.0001: significantly different from WIN55,212 treatment. ###P < 0.0001: significantly different from rimonabant treatment. CB₁, cannabinoid receptor 1; MAPK, mitogen activated protein kinase.

Figure 3

JWH018 induced CB₁ receptor internalization. (A) In CB₁ receptor expressing HEK293 cells, 100 nM JWH018 treatment resulted in robust receptor internalization that was quicker than that induced by 100 nM WIN55,212 (4–6 replicate samples from 5–10 independent experiments). (B) Following 2 h of exposure to increasing concentrations of each drug, CB₁ receptors were internalized in a concentration-dependent manner with JWH018 being more potent than WIN55,212 (4–6 replicate samples from 5–10 independent experiments). 1 µM rimonabant, a CB₁ receptor antagonist, prevented receptor internalization due to JWH018 or WIN55,212 treatment. Values are presented as mean ± SEM. *P < 0.05, **P < 0.01: significantly different from WIN55,212 treatment. ###P < 0.0001: significantly different from rimonabant treatment. CB₁, cannabinoid receptor 1.