Eur J Biochem. these effects were prevented by selective CB2, but not selective CB1, receptor antagonists. CB2 receptors couple to the extracellular kinase (ERK) signaling pathway by Gi/o class of G-proteins. Noteworthy, GP 1a (selective CB2 receptor agonist) produced a strong upregulation of 5-HT2A receptor mRNA and protein, an effect that was prevented by selective CB2 receptor antagonists and by an ERK1/2 inhibitor, PD 198306. In summary, our results identified a strong cannabinoid-induced upregulation of 5-HT2A receptor signaling in rat PFCx. Our cultured cell studies suggest that selective CB2 receptor agonists upregulate 5-HT2A receptor signaling by activation of the ERK1/2 signaling pathway. Activity of cortical 5-HT2A receptors has been associated with several physiological functions and neuropsychiatric disorders such as stress response, stress & depressive disorder and schizophrenia. Therefore, these results might provide a molecular mechanism by which activation of cannabinoid receptors might be relevant to the pathophysiology of some cognitive and mood disorders in humans. as this behavior is usually prevented by pretreatment CIQ with selective 5-HT2A receptor antagonists and is absent in 5-HT2A receptor knock out animals (Cheer et al., 1999; Darmani, 2001; Gorzalka et al., 2005). The detailed molecular mechanism by which cannabinoid receptor agonists regulate 5-HT2A receptor signaling in brain remains unknown; however, we have recently reported that selective cannabinoid agonists can upregulate 5-HT2A receptors (Franklin et al., 2012; Franklin and Carrasco, 2012). Nevertheless in those manuscripts we did not assess the effect of cannabinoid agonists on the activity of 5-HT2A receptors or indicates the number of rats or cell culture plates per group. Data was analyzed by an unpaired Students t-test or ANOVA (Newman-Keuls post-hoc test). GB-STAT software (Dynamic Microsystems, Inc., Silver Spring, MD, USA) was used for all statistical analyses. RESULTS CP 55,940 exposure enhances 5-HT2A CIQ receptor signaling and expression in rat PFCx We first examined the effect of chronic administration of CP 55,940 (50g/kg for 7 days), a CB1/CB2 receptor agonist (Bouaboula et al., 1996), on the activity and expression of CIQ 5-HT2A receptors in rat PFCx. Previously we have found that chronic CP 55,940 treatment increases 5-HT2A receptor expression but the effect on 5-HT2A receptor activity is usually unknown (Franklin and Carrasco, 2012). Initially, we measured activity of 5-HT2A receptors in PFCx because 5-HT-stimulated phosphoinositol hydrolysis in this brain area has been reported to be mediated primarily by activation of 5-HT2A receptors (Carrasco and Battaglia, 2007; Wolf and Schutz, 1997). 5-HT2A receptor stimulated PLC activity was significantly (p 0.01) greater in CP 55,940-treated rats compared with controls (578 44 and 397 34 pmoles/mg protein/min for CP 55,940 and vehicle-treated rats, respectively) (Fig. 1A). The two-way ANOVA detected a main effect of treatment with CP 55,940 (F1,58.18, p 0.0001) and 5-HT (F1,1000.48, p 0.0001) around the PLC activity and a main interaction between these two factors (F1,58,45, p 0.0001). Noteworthy, this CP 55,940-induced enhanced PLC activity was associated with a significant (p 0.05) two-fold increase in the membrane-associated levels of 5-HT2A receptors in PFCx compared to controls (Fig. 1B). Here, 5-HT2A receptors were identified as a single and prominent band with a molecular mass of approximately 42C43 kDa as previously described (Singh et al., 2007). Open in a separate window Physique 1 CP 55,940-induced enhanced activity and upregulation of 5-HT2A receptors in PFCx. (A) 5-HT2A receptor stimulated PLC activity in PFCx of rats treated with either vehicle or CP 55,940 (50g/kg, i.p.) for 7 days and withdrawn for 48 hours. Rabbit Polyclonal to CNOT7 We detected an increased 5-HT-stimulated PLC activity in rats treated with CP 55,940 compared to control rats (** 0.01, significant effect compared to vehicle-treated rats; ## 0.01, significant effect of 5-HT-stimulated PLC activity compared to vehicle-treated rats). (B) Increased membrane-associated 5-HT2A receptor protein levels in PFCx of CP 55,940-treated rats. -actin was used as a loading control. Representative Western blots are shown in this physique and IOD was calculated as described in Experimental Procedures (* 0.05, significant effect compared to vehicle-treated rats). The data represent mean SEM (n=4C6). 5-HT2A receptor mRNA was significantly (p 0.05) increased in PFCx of CP 55,940-treated rats compared to vehicle-treated controls (approx. 90% increase, Fig. 1C). No significant changes in the levels of 5-HT1A receptor or 5-HT2A receptor coupled-Gq G-protein mRNAs were detected in PFCx of CP CIQ 55,940-treated animals. This highlights the specificity of the effect of CP 55,940 on 5-HT2A receptor signaling. Also, we found a significant (p 0.05) downregulation of CB1 and CB2 receptor mRNA in PFCx of CP 55,940-treated rats compared to vehicle controls (Fig. 1C). CB1 and CB2 mRNA levels were reduced.