Concurrent stimulation of cannabinoid CB1 and dopamine D2 receptors augments cAMP accumulation in striatal neurons: evidence for a Gs linkage to the CB1 receptor

Abstract

Cannabinoids act at the CB1 receptor to inhibit adenylate cyclase activity via a pertussis toxin-sensitive G-protein. Within the striatum, CB1 receptors have been shown to be localized on the same neurons as Gi-coupled dopamine D2 receptors. In this study we have examined the interactions of CB1 and D2 receptors on adenylate cyclase. In striatal neurons in primary culture, both the CB1 receptor agonist [3-(1, 1-dimethylheptyl)-11-hydroxy-Delta8tetrahydrocannabinol] (HU210) and the D2 receptor agonist quinpirole inhibited forskolin-stimulated cAMP accumulation when applied separately. In contrast, HU210 and quinpirole in combination augmented cAMP accumulation. This augmentation was blocked by the CB1 receptor antagonist SR141716A or the D2 antagonist sulpride. Pertussis toxin treatment of striatal neurons prevented the inhibition of cAMP accumulation by D2 receptors but unmasked a cannabinoid receptor-mediated stimulatory effect on cAMP accumulation. The cannabinoid receptor-stimulated accumulation of cAMP was blocked in a concentration-dependent manner by SR141716A, suggesting that the response was regulated through the CB1 receptor. Similar augmentation of cAMP accumulation after pertussis toxin treatment was observed in Chinese hamster ovary (CHO) cells transfected with, and stably expressing, the CB1 receptor. This stimulation of cAMP was not Ca2+-sensitive and was unaffected by a range of protein kinase inhibitors. Treatment of the pertussis toxin-treated cells with cholera toxin before CB1 receptor activation amplified the stimulatory pathway, suggesting that this response was mediated through a Gs-type G-protein. Stimulation of cAMP accumulation was not observed after pertussis toxin treatment of CHO cells expressing the human CB2 receptor, suggesting that this novel signaling pathway is unique to the cannabinoid CB1 receptor.

Fig. 1.

Effect of CB1 and D2 agonists on forskolin-stimulated cAMP accumulation in primary striatal cultured neurons. Forskolin-stimulated (50 nm) cAMP accumulation was measured in primary culture of the striatum in the presence or absence of quinpirole (100 nm) and HU210 (1 μm) as described in Materials and Methods. The inhibition of cAMP accumulation by these compounds was blocked by the D2 receptor antagonist sulpride (1 μm) or the CB1 antagonist SR141716A (1 μm), respectively. Data are the mean ± SEM of at least three experiments performed in duplicate. One hundred percent accumulation is equivalent to 39 ± 8.5 pmol/mg cAMP.

Fig. 2.

Effect of concurrent activation of D2 and CB1 receptors on cAMP accumulation in primary striatal culture. Forskolin-stimulated (50 nm) cAMP accumulation was measured in primary striatal culture as described in Materials and Methods in combination with 100 nm quinpirole and increasing concentrations of HU210. HU210 reversed the quinpirole-mediated inhibition of forskolin-stimulated cAMP accumulation in a concentration-dependent manner to levels above that demonstrated with forskolin alone (EC₅₀ = 6.1 ± 1.2 nm). The stimulation of cAMP accumulation in the presence of forskolin and quinpirole at 1 μm HU210 was blocked by 1 μm SR141716A. Data are the mean ± SEM of at least three experiments performed in duplicate.

Fig. 3.

Effect of pertussis toxin on HU210-mediated inhibition of forskolin-stimulated cAMP accumulation in primary striatal culture (A) and CHO-hCB1 cells (B). Forskolin-stimulated (50 nm) cAMP accumulation was measured in primary striatal culture and CHO-hCB1 as described in Materials and Methods. Pertussis toxin (+PTX;5 ng/ml) was added ∼18 hr before the measurement of cAMP accumulation. In the absence of pertussis toxin, HU210 inhibited forskolin-stimulated cAMP (A, EC50 = 4.6 ± 1.2 nm; B, EC₅₀ = 0.17 ± 0.01 nm). After pertussis toxin treatment, HU210 resulted in an increase in forskolin-stimulated cAMP (A, EC50 = 1.2 ± 1.1 nm;B, EC50 = 1.6 ± 0.04 nm). Both the inhibition and the stimulation of cAMP were prevented by concurrent exposure to 1 μm SR141716A (open symbols). Data are the mean ± SEM of at least three experiments performed in triplicate. One hundred percent accumulation is equivalent toA, 39 ± 8.5 pmol/ml; B, 39.4 ± 3 pmol/ml.

Fig. 4.

Evidence suggesting a G_s linkage for the CB1 receptor. A, CHO-hCB1 cells were treated overnight with pertussis toxin. HU210 did not affect basal levels of cAMP accumulation in the absence of forskolin. Increasing concentrations of forskolin resulted in an amplification of HU210-induced cAMP accumulation above forskolin levels. Data are mean ± SEM from a representative experiment. B, cAMP levels after pretreatment with cholera toxin. Cholera toxin results in an increase in basal cAMP accumulation. HU210 stimulation in the absence of forskolin resulted in a U-shaped concentration response curve, with inhibition at lower doses but stimulation at high doses. Data are mean ± SEM from a representative experiment.C, Cells were treated overnight with pertussis toxin before treatment with cholera toxin. An amplified signal was observed after cholera toxin treatment (+PTX/CTX), compared with pertussis treatment (+PTX) alone. All experiments were performed in the absence of forskolin. Data are mean ± SEM from a representative experiment.