Cognitive Impairment Induced by Delta9-tetrahydrocannabinol Occurs through Heteromers between Cannabinoid CB1 and Serotonin 5-HT2A Receptors
Fig 5
A profile of CB1R-5-HT2AR heteromer signaling.
In (A and E), cAMP production was determined in HEK-293Tcells expressing CB1R and 5-HT2AR after stimulation with 100 nM DOI, 100 nM WIN 55,212–2 (WIN), or both in the absence or in the presence of 0.5 μM forskolin. In (E), cells were first preincubated either with the CB1R antagonist rimonabant (1 μM, RIM) or the 5-HT2AR antagonist MDL 100,907 (300 nM) for 20 min prior to being stimulated. Values (cAMP produced in each condition minus basal stimulation in the absence of forskolin or agonists) represent mean ± SEM of n = 3–4 and are expressed as the percentage of the forskolin-treated cells in each condition (120–150 pmols cAMP/106 cells). One-way ANOVA followed by a Bonferroni post hoc tests showed a significant effect over the forskolin-alone effect in each condition (** p < 0.01, *** p <0.001) or of the antagonist plus agonist treatment over the agonist treatment (# p < 0.05, ## p < 0.01, ### p < 0.001). In (B and F), β arrestin II recruitment was measured by BRET experiments in cells transfected with 2 μg of CB1R cDNA, 0.2 μg of β-arrestin II-Rluc cDNA, and 1 μg of 5-H2AR-YFP cDNA. Cells were not preincubated with antagonist (B) or were preincubated (F) for 20 min with vehicle, rimonabant (1 μM, RIM), or MDL 100,907 (300 nM, MDL). Cells were not treated (BRET < 10) or were treated for 7 min with WIN 55,212–2 (100 nM, WIN) or DOI (100 nM) before BRET determination. Values represent mean ± SEM of n = 4–6. One-way ANOVA followed by a Bonferroni post hoc tests showed a significant effect over not-treated cells (* p < 0.05, ** p < 0.01, *** p <0.001) or of the antagonist plus agonist treatment over the agonist treatment (## p < 0.01, ### p < 0.001). In (C, D, G, and H), cells expressing CB1R and 5-HT2AR were not preincubated with antagonist (C and D) or were preincubated for 15 min with rimonabant (1 μM, RIM) or MDL 100,907 (300 nM, MDL) (G and H) and stimulated for 5 min with WIN 55,212–2 (100 nM,WIN) or DOI (100 nM). Quantification of phosphorylated ERK 1/2 (C and G) or Akt (D and H) was determined by western blot. Values, expressed as percentage of basal (nontreated cells), were mean ± SEM of n = 3–10. One-way ANOVA followed by a Bonferroni post hoc tests showed a significant effect over basal (* p < 0.05, ** p < 0.01, ** p < 0.001) or of the antagonist plus agonist treatment over the agonist treatment (# p < 0.05, ## p < 0.01, ### p < 0.001).