We also determined what effect CP55940, JWH-133, and GP1a treatment had on -Arrestin 2 protein levels

We also determined what effect CP55940, JWH-133, and GP1a treatment had on -Arrestin 2 protein levels. -Arrestin 2. Our results suggest that sustained activation of CB2 receptors would enhance -Arrestin 2 expression possibly contributing to its increased conversation with ERK1/2 thereby driving the upregulation of 5-HT2A receptors. The CB2 receptor-mediated upregulation of -Arrestin 2 would be mediated, at least in part, by an ERK1/2-dependent activation of AP-1. These data could provide the rationale for some of the adverse effects associated with repeated cannabinoid exposure and shed light on some CB2 receptor agonists that could symbolize an alternative therapeutic because of their minimal effect on serotonergic neurotransmission. and [9;10]. Cannabinoid agonists can produce their physiological effects through the activation of two G-protein coupled cannabinoid receptors in the brain, CB1 and CB2 receptors [11;12]. CB1 and CB2 receptors bind endocannabinoids, synthetic cannabinoids, and cannabinoids found in nature (such as 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 utilized for all statistical analyses. 3. Results 3.1 Chronic CP55940 treatment induces enhanced -Arrestin 2 and ERK1/2 interaction in PFCx Our previous work has shown that some cannabinoid agonists can enhance 5-HT2A receptor expression by means of a mechanism that involves CB2 receptor regulation of ERK1/2 activation. PHA690509 [9;10]. Cannabinoid receptors could produce a long-term ERK1/2 activation by a mechanism that may involve a -Arrestin-ERK1/2 scaffolding complex [17C19]. Specifically, CB2 receptors that are a class A GPCR would preferentially interact with -Arrestin 2, which may facilitate and enhance the conversation between -Arrestin and ERK1/2 resulting in long-term ERK1/2 activation [20]. Here, we used co-immunoprecipitation protocols to study the effect of CP55940 treatment around the physical conversation between -Arrestin 2 and ERK1/2 in rat PFCx (Fig. 1. A). We used -Arrestin 2 antibody as bait and ERK1/2 antibody as prey. Inactive columns which are unable to bind -Arrestin 2 antibody were used as a control as explained in methods. We found that ERK1/2 co-precipitates with -Arrestin 2 when we used -Arrestin 2 as bait (Fig. 1. A, lanes 3 & 4). Interestingly, we detected a significant (p 0.05) two-fold increase in the conversation between -Arrestin 2 and ERK1/2 in PFCx of CP55940-treated rats compared to vehicle treated controls (Fig. 1. A, lane 3 and 4, vehicle- and CP55940-treated animals, respectively). No co-precipitation of -Arrestin 2 and ERK1/2 was detected using the inactive columns (Fig. 1. A, lanes 5 & 6). Open in a separate window Physique 1 CP55940-induced enhanced co-immunoprecipitation of -Arrestin 2 and ERK1/2 and increased -Arrestin 2 protein expression in rat PFCx(A) Enhanced immunoprecipitation of the ERK1/2 (Lane 4) compared to PHA690509 vehicle-treated controls (Lane 3). Negative controls (Lanes 5 and 6) received the same concentration of -Arrestin 2 antibody except that this coupling resin was replaced with control agarose resin that is not amine reactive. All columns were incubated with prefrontal cortex lysate (300 g) from vehicle (Lanes 3 and 5 ) or CP55940 (Lanes 4 and 6) treated rats. Prefrontal cortex PHA690509 lysate (30 g of protein) was used as an Rabbit Polyclonal to COX41 input control (Lane 1 and 2). (B) Increased pERK protein levels in CP55940 treated rats compared to vehicle treated rats. **p 0.01, significant effect of CP55940 treatment compared to vehicle-treated controls. (C) Increased membrane associated -Arrestin 2 protein levels in PFCx of CP55940 treated rats. **p 0.01 significant PHA690509 effect of CP55940 treatment compared to vehicle-treated controls. (D) CP55940 treatment does not impact total ERK1/2 expression in the PFCx. (E) Increased -Arrestin 2 mRNA levels in PFCx of CP55940.