Effects of the cannabinoid HU210 on proliferation of cultured hippocampal NS/PCs. (A) In the WST-8 assay, incubation of NS/PCs with 10 nM to 1 μM of HU210 for 48 hours significantly promoted NS/PC proliferation, which was blocked by the CB1 receptor antagonist AM281. AM281 alone significantly decreased NS/PC proliferation only with 10 μM, but this concentration of AM281 was not able to block the lethal effects of 10 μM of HU210 on NS/PCs. (B) BrdU incorporation assay confirmed the results obtained with the WST-8 assay shown in A. (C) Incubation of NS/PCs with 1 μM to 10 μM of AEA for 48 hours significantly promoted NS/PC proliferation in the WST-8 assay. (D) Application of 10 nM to 1 μM of HU210 significantly promoted NS/PC proliferation in both the presence and absence of the growth factors bFGF and EGF in the culture medium. (E) Pertussis (PTX; 100 ng/ml), a selective blocker for G_i/o protein activation, prevented the effects of 10 nM to 1 μM of HU210 on promoting NS/PC proliferation. (F) Incubation of NS/PCs with 1 mg/ml of cholera toxin, a selective G_s activator, stimulated a profound increase in cAMP accumulation in NS/PCs 0.5, 1, 2, and 24 hours after the addition of cholera toxin. (G) Incubation of NS/PCs with 1 mg/ml of cholera toxin for 0.5, 1, 2, 24, or 48 hours did not induce significant change in NS/PC proliferation. Error bars represent SEM. *P < 0.05 and **P < 0.01 by Tukey post-hoc tests after 1-way ANOVA.