The effects of cannabinoid receptor antagonists on the synergistic effects of pentobarbital with ∆9-THC are shown in Fig. 2. ∆9-THC [vehicle (Veh) + ∆9-THC 10 mg/kg, i.v.] significantly prolonged pentobarbital-induced sleep by 3.3-folds compared with the vehicle-pretreated group (Veh + Veh). The result supports our previous finding that ∆9-THC prolonged pentobarbital-induced sleeping time in mice by various routes of administrations [22]. At this time, once an abused drug, such as pentobarbital, has entered the body, a drug interaction between ∆9-THC and pentobarbital may occur. Moreover, this should be treated as a serious problem, rather than simply a case of single administration of cannabis or barbiturates alone.

Fig. 2 Effects of CB 1 receptor antagonists on ∆9-THC-induced pentobarbital potentiation in mice. Mice were pretreated with 2, 5, or 10 mg/kg of CB 1 receptor antagonists by i.v. injection 10 min before ∆9-THC administration (10 mg/kg, i.v.), and then administered 40 mg/kg of pentobarbital by i.p. injection (n = 8). Sleeping time was expressed as the mean % of control (Veh + Veh + pentobarbital) sleeping time. Concentration of CB 1 receptor antagonist: 2 mg/kg, i.v., 5 mg/kg, i.v., 10 mg/kg, i.v., Control sleeping time was 37 ± 5 min. Significant difference from control group (Veh + Veh; *p < 0.05, **p < 0.01). Significant difference from ∆9-THC alone treated group (Veh + ∆9-THC; ##p < 0.01) Full size image

The cannabinoid receptor is subdivided into CB 1 and CB 2 receptors, with each specific ligand (agonist and antagonist) previously determined [37]. In the present study, SR141716A and AM251 were used as CB 1 receptor antagonists to investigate pharmacological effects and perform the receptor binding affinity experiment. Compton et al. [14] reported antagonistic response of SR141716A for pharmacological and behavioral experiments such as measurements of spontaneous locomotor activity, tail-flick responsiveness, or rectal temperature at the range of 1–10 mg/kg, i.v. to mice. We also used an equivalent dose of SR141716A for the effect of ∆9-THC-induced pentobarbital potentiation. When SR141716A or AM251 (2–10 mg/kg) was pre-administered by i.v. injection 10 min before ∆9-THC treatment, the potentiation of pentobarbital-induced sleep by ∆9-THC decreased dose-dependently. In particular, a low dose of the CB 1 receptor antagonists (2 mg/kg, i.v.) significantly suppressed the prolonging effect of ∆9-THC. Cannabinoid receptor antagonists themselves (AM251 + Veh or SR141716A + Veh) then caused no change in pentobarbital-induced sleeping time. The attenuated synergistic effect of ∆9-THC on pentobarbital-induced sleep was thought to be due to blockade of CB 1 receptor binding by specific CB 1 receptor antagonists.

Since a low dose of CB 1 receptor antagonists attenuated the synergistic effects of pentobarbital with Δ9-THC and CB 1 receptor affinity of SR141716A is much higher than the affinity of AM251, doses of 2 or 5 mg/kg, i.v. of SR141716A were used for the synergistic experiment of CBD and pentobarbital. CBD (Veh + CBD 10 mg/kg, i.v.) also significantly prolonged pentobarbital-induced sleep 1.8-fold compared with the control (Veh + Veh), while pretreatment with SR141716A (2 or 5 mg/kg, i.v.) failed to inhibit CBD-enhanced pentobarbital-induced sleep (Fig. 3). CBD is well known as a major constituent of marijuana, causing pharmacological effects, such as barbiturate synergism [8,9,10, 24]. Watanabe et al. [30] previously reported the potentiation mechanism of CBD on pentobarbital-induced sleep, which is caused by inhibition of the hepatic drug-metabolizing enzymes by CBD. CBD inhibits the metabolism of pentobarbital, leading to increased levels of the drug in the body and enhances its action. Namely, the synergistic effect of CBD with pentobarbital is different from ∆9-THC, which reflects how the CB 1 receptor influences CNS pharmacological effects. Therefore, this failure of SR141716A to produce antagonistic effects on CBD-induced pentobarbital potentiation suggests that CBD does not bind to the CB 1 receptor in CNS at the supplied dosage of 10 mg/kg i.p. of CBD in mice.

Fig. 3 Effects of CB 1 receptor antagonist on CBD-induced pentobarbital potentiation in mice. Mice were pretreated with 2 or 5 mg/kg SR141716A by i.v. injection 10 min before CBD administration (10 mg/kg, i.v.) and then administered pentobarbital at 40 mg/kg by i.p. injection. Sleeping time was expressed as the mean % of control (Veh + Veh + pentobarbital) sleeping time (n = 8). Control sleeping time was 35 ± 3 min. Significant difference from control group (**p < 0.01) Full size image

As shown in Fig. 4, the affinities of antagonists for CB 1 receptor binding were evaluated using a radio receptor assay measuring specific [3H]CP55940 binding to the mouse brain synaptic membrane. All compounds concentration-dependently inhibited the specific [3H]CP55940 binding. ∆9-THC and CB 1 receptor antagonists, except for CBD, possessed the same potency for the CB 1 receptor binding. The Ki values of cannabinoid ligands are summarized in Table 1. The Ki values of ∆9-THC and CBD were 6.62 and 2010 nM, respectively, showing the high affinity of ∆9-THC and the low affinity of CBD for the CB 1 receptor. The Ki values of CB 1 receptor antagonists, SR141716A and AM251, were 9.54 and 2.58 nM, respectively, indicating the high affinities of these antagonists to the CB 1 receptor binding site, as well as ∆9-THC. Yamamoto et al. [38] also reported that active metabolites of ∆9-THC bound to the cannabinoid CB 1 receptor in the brain. The results confirm that the potentiation mechanism of pentobarbital-induced sleep by CBD is not mediated through the CB 1 receptor, but rather by other mechanisms, such as inhibition of barbiturate metabolism (Table 2).

Fig. 4 Displacement effects of ∆9-THC (circle), CBD (square), AM251 (triangle), and SR141716A (diamond) on specific [3H]CP55940 binding to the synaptic membrane from ddY mice brains. Displacement curves were fitted by ORIGIN ver. 7.5 and calculated as IC 50 Full size image

Table 1 Displacement of specific [3H]CP-55940 binding to the synaptic membrane from ddY mice brains by cannabinoid receptor ligands Full size table

The involvement of pentobarbital in CB 1 receptor function was also examined. We added 1 mM pentobarbital to the CB 1 receptor binding assay mixture. However, pentobarbital did not affect specific [3H]CP55940 binding to the mice brain synaptic membrane (Table 2). In other words, pentobarbital did not directly affect the CB 1 receptor binding site. The cannabinoid CB 1 receptor antagonists, SR141716A and AM251, are effective in antagonizing in vivo effects of ∆9-THC, such as hypothermia and antinociception, as well as decreasing locomotor activity [14, 37]. To date, several lines of evidence indicate that cannabinoids potentiate barbiturate potency as a pharmacological property of marijuana [5, 7]. We also revealed that marijuana constituents, their active metabolites, and synthetic analogues prolonged pentobarbital-induced sleep in mice [18, 23, 24]. In the structure–activity relationship of cannabinoids showing CNS pharmacological effects, the cannabinoid-induced barbiturate synergism was similar to other CNS indices, such as catalepsy and hypothermia [21], suggesting a close interaction between cannabinoid and barbiturate action sites.

Table 2 Effect of pentobarbital on specific [3H]CP55940 binding to the synaptic membrane from mice brains Full size table

Many reports explore the barbiturate active site, but a precise action mechanism of barbiturates is not fully elucidated. In general, barbiturates act on the GABA A supramolecule receptor complex, which couples with the GABA and benzodiazepine binding sites, as well as the picrotoxinin and chloride ion channel [39]. We previously reported the effects of cannabinoids on the benzodiazepine receptor of the synaptic membrane in the bovine brain [40]. ∆9-THC and its metabolites competitively bound to the benzodiazepine receptor, indicating a slight co-modification of benzodiazepine receptor binding by these cannabinoids. Despite this, the receptor binding affinities of the cannabinoids to the benzodiazepine receptor were low. For the synergy of barbiturate with ∆9-THC, the direct interaction of pentobarbital with the CB 1 receptor was ruled out, since adding high concentrations of pentobarbital (1 mM) to the receptor binding mixture did not change the specific [3H]CP55940 binding to the membrane. In addition, treatment of CB 1 receptor antagonists without Δ9-THC resulted in no change in sleeping time compared with vehicle treatment, indicating pentobarbital did not compete with the CB 1 receptor binding site. Regarding the mechanism explaining the synergy between cannabinoids and barbiturate, the barbiturate action site differed from the CB 1 receptor, suggesting the CB 1 receptor signal may activate the barbiturate site downstream to the CB 1 receptor site. CB 1 receptors are known to be coupled through G protein-dependent and G protein-independent (Gi/o) receptors to certain ion channels [41]. The present results suggest that positive cross-talk is present between CB 1 receptor signaling and pentobarbital-induced chloride channels. However, concerning the action mechanism downstream of the CB 1 receptor binding site is still unknown. It may be related to a certain signal transduction between the cannabinoid receptor and barbiturate active sites, including the GABA A receptor and G protein. Since the CB 1 receptor is known to couple Gi/o protein, measurement of adenylate cyclase activity of CB 1 receptor in the presence of pentobarbital may be evidence of this interaction. Moreover, barbiturates are known to affect to GABA A receptor. Study of involvement of GABA A receptor to CB 1 receptor is future work.