BioMed Research International / 2015 / Article / Fig 3

Research Article

The Combined Inhibitory Effect of the Adenosine A1 and Cannabinoid CB1 Receptors on cAMP Accumulation in the Hippocampus Is Additive and Independent of A1 Receptor Desensitization

Figure 3

Combined effect of WIN55212-2 and CPA on forskolin-stimulated cAMP accumulation in rat hippocampal slices; influence of the preincubation period with CPA. (a) and (c) Combined effect of WIN55212-2 and CPA. In experiments where CPA was added 95 min before forskolin (c), slices were incubated in the absence (control) or in the presence of CPA (100 nM) for 50 min. After this period incubation continued for 45 min in the presence of rolipram (50 M) and in the absence (control) or in the presence of WIN55212-2 (30 M). Then incubation proceeded in the presence of forskolin (10 M) for a further 35 min period. In experiments where CPA was added 15 min before forskolin (a) CPA (100 nM final concentration) or vehicle (control) was added 30 min after rolipram. In each experiment four parallel assays were performed, corresponding, respectively, to incubation with WIN55212-2, CPA, WIN55212-2 + CPA and incubation in the absence of WIN55212-2 and CPA (control). Solid bars represent the % inhibition of control cAMP accumulation produced by (from left to right) WIN55212-2, CPA, and WIN55212-2 plus CPA; the dashed bar represents the arithmetical sum (calculated for each experiment) of the % inhibition produced by WIN55212-2 and CPA alone. (b) Time-dependent attenuation of the CPA effect. In experiments where CPA was added 95 min before forskolin (open bar), slices were incubated in the absence (control) or in the presence of CPA (100 nM) for 50 min. After this period incubation continued for 45 min in the presence of rolipram (50 M). Then incubation proceeded in the presence of forskolin (10 M) for a further 35 min period. In experiments where CPA was added 15 min before forskolin (solid bar) CPA (100 nM final concentration) or vehicle (control) was added 30 min after rolipram. Bars represent the % inhibition produced by CPA of control cAMP accumulation. Statistically different from CPA added 15 min before forskolin (, Student’s -test). In the bottom of (a) and (c) are presented the corresponding time lines of addition of drugs. In (b), for CPA 15 min applies the time line presented in (a) and for CPA 95 min applies the timeline presented in (c), but without WIN55212-2. R: rolipram, F: forskolin, W: WIN55212-2, and PCA: perchloric acid. Data are mean ± SEM from 4 independent experiments run at least in triplicate. Statistically different from zero (). Statistically significant () when comparing the cAMP accumulation obtained in the presence of CPA, WIN55212-2, or WIN55212-2 plus CPA, with control cAMP accumulation (One-way ANOVA, followed by LSD test). Statistically different from the effect of WIN55212-2 (A) or CPA (B) alone (; One-way ANOVA, followed by LSD test). NS: the WIN55212-2 plus CPA effect was not statistically different from the sum of the inhibitory effects of WIN55212-2 and CPA alone (A + B, dashed line; , when compared within the same experiment, paired Student’s -test) in (a) and (c), or from WIN55212-2 alone (A; , One-way ANOVA, followed by LSD test) in (c).
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