Cannabinoid-induced enhanced interaction and protein levels of serotonin 5-HT2A and dopamine D2 receptors in rat prefrontal cortex

Drugs

(-)-cis-3-[2-Hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol (CP55,940), a CB1 and CB2 receptor agonist; N-(Piperidin-1-yl)- 1-(2,4-dichlorophenyl)-1, 4-dihydro-6-methylindeno [1,2-c]pyrazole-3-carboxamide (GP 1a), a highly selective CB2 receptor agonist; 3-(1,1-Dimethylbutyl) -1-deoxy-Δ⁸- tetrahydrocannabinol (JWH 133), a selective CB2 receptor agonist; [6-iodo-2-methyl-1- [2-(4-morpholinyl)ethyl]- 1H-indol-3-yl](4-methoxyphenyl)-methanone (AM 630), a selective CB2 receptor antagonist; Pertussis Toxin (PTX); N,N- Dimethyl-(3R, 4aR,5S, 6aS, 10S,10aR,10bS )-5-(acetyloxy)- 3-ethenyldodecahydro-10, 10b-dihydroxy-3,4a,7,7, 10a-pentamethyl-1-oxo-1 H-naphtho [2,1-b]pyran-6- yl ester β -alanine hydrochloride (NKH 477), a potent activator of adenyl cyclase; 2-[1-(3-Dimethylaminopropyl) indol-3-yl]-3-(indol-3-yl) maleimide (GF 109203X), a very potent and selective inhibitor of PKC; 5,6,7,13-Tetrahydro-13- methyl-5-oxo-12H-indolo[2,3-a]pyrrolo [3,4-c]carbazole-12-propanenitrile (Go 6967), a potent PKC inhibitor; (1aR,1bS,4aR,7aS,7bS,8R,9R,9aS)-1a,1b,4,4a,5, 7a,7b,8,9,9a-Decahydro-4a,7b-dihydroxy- 3-(hydroxymethyl)-1,1,6,8- tetramethyl-5-oxo-1 H-cyclopropa[3,4] benz[1,2-e]azulen-9,9a- diyl butanoic acid ester (Phorbol 12,13-dibutylrate, PDBu), a PKC activator; (E,E,Z,E)-3-Methyl-7-(4- methylphenyl)-9-(2,6,6- trimethyl-1-cyclohexen-1- yl)-2,4,6,8-nonatetraenoic acid (SR 11302), an inhibitor of activating protein-1 transcription factor activity and N-(2-Chloroethyl)-5Z,8Z,11Z,14Z-eicosatetraenamide (ACEA), a highly selective CB1 receptor agonist, were purchased from Tocris (Ellisville, MO, USA). Naphthol AS-E phosphate, a CREB inhibitor, was purchased from Sigma-Aldrich Inc. (St. Louis, MO, USA).

Animal experimental protocol

Male Sprague-Dawley rats (225–275 g; Harlan Laboratories, Indianapolis, IN, USA) were housed two per cage in a temperature-, humidity-, and light-controlled room (12 hr light/dark cycle, lights on 7:00 am–19:00 pm). Food and water were available ad libitum. All procedures were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals as approved by the University of Kansas Institutional Animal Care and Use Committee (IACUC).

After arrival, the rats were allowed to acclimate to their environment for at least 4 days prior to the start of the treatment period. Eight rats were randomly assigned to each group; cage mates were assigned to the same treatment group. The body weight of each rat was recorded every other day. All solutions were made fresh before administration and rats were injected with either vehicle (Tween-80/ethanol/saline (1:1:18); 1 ml/kg, i.p.) or CP55,940 (0.05 mg/kg, i.p.) once a day for 7 days. Rats were sacrificed by decapitation 48 h after the last CP55,940 injection. The brains were immediately removed and the PFCx was dissected and frozen in dry ice.

Co-immunoprecipitation

Co-immunoprecipitation (co-IP) was carried out using the Thermo Scientific Pierce co-IP kit following manufacturer’s protocol. 5-HT_2A receptor antibody was a generous gift from Dr Nancy A Muma, and the D₂ receptor antibody was purchased from Santa Cruz, CA, USA. Briefly, 5-HT_2A receptor antibody or D₂ receptor antibody was first immobilized for 2 h using AminoLink Plus coupling resin. The resin was washed and incubated with pre-cleared prefrontal cortex lysate (300 µg) from vehicle and CP55,940-treated rats overnight. A negative control in this assay included a non-reactive resin that was also incubated with either 5-HT_2A or D₂ receptor antibodies. In this control, the coupling resin is not amine reactive, preventing covalent immobilization of the primary antibody onto the resin. This inactive resin was provided with the IP kit to assess non-specific binding in samples that received the same treatment as the co-IP samples, including 5-HT_2A receptor antibody or D₂ receptor antibody. After the overnight incubation of all the prefrontal cortex lysates from vehicle- and CP55,940-treated samples with either active or inactive resins, the resins were washed (3×) and the protein eluted using elution buffer. Samples were analyzed by Western blot using 5-HT_2A receptor antibody or D₂ receptor antibody (Figure 1). The specificity of the 5-HT_2A and D₂ receptor antibody has been verified in the literature (Montezinho et al., 2006; Nam and Kim, 2008; Singh et al., 2007).

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Figure 1.

CP55,940-induced enhanced co-immunoprecipitation of 5-HT_2A and D₂ receptors in rat PFCx.

Western blot

Membrane-associated proteins were isolated using the ProteoExtract^TM Native Membrane Protein Extraction kit (Calbiochem, La Jolla, CA, USA). Nuclear-associated proteins were isolated using NE-PER® Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific, IL, USA). Samples containing 5 µg of protein were separated by sodium dodecyl-polyacrylamide gel electrophoresis containing 0.1% SDS, 12.5% acrylamide/bisacrylamide (30:0.2), 4.6 M urea, and 275 mM Tris, pH 8.7. Gels were transferred electrophoretically by semi-dry blot to nitrocellulose membranes. After incubation with a blocking buffer (phosphate buffered saline containing 0.2% casein and 0.1% Tween 20), immunodetection was performed at 4°C overnight using primary antibody. c-Fos antibody was purchased from Santa Cruz, CA, USA. The anti-dopamine D₂ receptor, cytoplasmic domain, long form antibody was purchased from Millipore (Billerica, MA, USA) and the dopamine D₂ receptor (Short Isoform 239-246) antibody was purchased from Acris Antibodies GmbH (Germany). The specificity of the antibodies has been verified in the literature (Baltzis et al., 2004; Boundy et al., 1993; Montezinho et al., 2006; Nam and Kim, 2008; Singh et al., 2007). Antibodies were used at the following dilutions: c-Fos (1:1000), D₂L (1:1000), D₂S (1:1000), 5-HT_2A (1:5000) and D₂ (1:1000). The overnight incubation was followed by incubation with peroxidase-labeled secondary antibody for 1 h at room temperature. The membranes were incubated with enhanced chemiluminescence substrate solution (Amersham Biosciences Inc., Piscataway, NJ, USA). Protein loading for each lane was verified using an anti-actin antibody (Santa Cruz Biotechnology, Inc.). Negative controls included either the omission of primary antibody or addition of preimmune rabbit immunoglobulins.

Film analysis

Films were analyzed densitometrically with values calculated from the integrated optical density (IOD) of each band using Scion Image software (Scion Corporation, Frederick, MD, USA). The grayscale density readings were calibrated using a transmission step-wedge standard. The IOD of each band was calculated as the sum of the optical densities of all the pixels within the area of the band outlined. An adjacent area was used to calculate the background optical density of the film. The IOD for the film background was subtracted from the IOD for each band. The resulting IOD for each protein was then divided by the amount of protein loaded on the corresponding lane, and each sample was expressed as IOD per microgram of protein. Each sample was measured on three independent gels. All samples were standardized to controls and normalized to their respective actin levels.

Quantitative real-time PCR

Total RNA was isolated from either cell culture or prefrontal cortex tissue using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA) protocol as described by the manufacturer. Total mRNA was reverse transcribed to generate cDNA. Quantitative real-time PCR reactions were prepared using QuantiFast SYBR Green PCR Kit (Qiagen, Valencia, CA, USA), a 4% (v/v) concentration of cDNA product, and forward and reverse primers at a final concentration of 0.35 mM. All reactions were performed in triplicate using the ABI 7500 fast real-time PCR system (Applied Biosystems, Foster City, CA, USA). A negative control lacking cDNA or any known DNA template was included for each primer pair. The primers used in this manuscript were: 5-HT_2A (F:5’-AACGGTCCATCCACAGAG-3’ and R:5’-AACAGGAAGAACACGATGC-3’), D₂ (F:5’-CACCAC-GGCCTA CATAGCAA-3’ and R:5’-GGCGTGCCCATTCTTCTCT-3’), and GAPDH (F: 5’- TGGAGTCTACTGGCGTCTTCAC-3’ and R:5’-GGCATGGACTGTGGTCATGA-3’). These primers have been previously validated in the literature (Kindlundh-Hogberg et al., 2006; Roessner et al., 2010; Singh et al., 2010; Zhang et al., 2008).

In all real-time PCR experiments, measurements were made from the number of cycles required to reach the threshold fluorescence intensity (cycle threshold (Ct)). Ct values for each reaction were subtracted from Ct values for GADPH and then subtracted from Ct values for vehicle-treated animals that served as a baseline, and the result was referred to as ΔΔCt. Fold changes in gene expression were calculated as 2-^ΔΔCt to reflect the fact that, under optimal conditions, the amount of PCR product doubles with each amplification cycle. Results were normalized to those obtained for amplifications of the same cDNA samples using primers designed against GADPH, which acts as an internal standard, and averaged for each treatment group.

Cell culture protocol

We purchased CLU213 cells from Cedarlane Laboratories (Burlington, NC, USA). We selected this neuronal cell line because (1) it coexpresses 5-HT_2A, D₂, CB1 and CB2 receptors; and (2) the preliminary results in our lab showed that it reproduces the effect of sustained cannabinoid exposure in vivo experiments. This was confirmed in experiments reported in this paper (see Figure 2E and 2F). Although many in vitro cannabinoids studies use transformed cells that overexpress neurotransmitter receptors, we chose this neuronal cell line because it endogenously expresses 5-HT_2A, D₂, CB1 and CB2 receptors. Therefore, we anticipate that the results reported here could be a good model of the mechanisms underlying 5-HT_2A upregulation in vivo.

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Figure 2.

CP55,940-induced increased membrane-associated expression of 5-HT_2A and D₂ receptors in rat PFCx.

CLU213 cells were grown on 100-mm² plates treated with polystyrene (Corning Incorporated, Corning, NY, USA) and maintained in 5% CO₂ at 37°C, in Dulbecco’s modified eagle medium (DMEM) (Mediatech Inc, Manassas, VA, USA) containing 10% fetal bovine serum (FBS) (Thermo Scientific, Logan, UT, USA).

Effect of non-selective and selective CB1 and CB2 receptor agonists on 5-HT_2A and D₂ receptor mRNA

CLU213 cells were incubated with either vehicle (ethanol 0.01% final concentration), CP55,940 (CB1 and CB2 agonist, 1 nM) (Thomas et al., 1998; Wiley et al., 1995); ACEA (CB1 agonist, 15 nM) (Hillard et al., 1999; Rutkowska and Jachimczuk, 2004); or GP 1a (CB2 agonist, 1 nM) (Gorantla et al., 2010; Murineddu et al., 2006) for 24 h. mRNA was isolated and qRT-PCR for 5-HT_2A and D₂ mRNA were performed as described above.

Effect of highly selective CB2 receptor agonists on 5-HT_2A receptor mRNA in cultured cells

CLU213 cells were pretreated with either vehicle (ethanol 0.01% final concentration) or 1 µM AM 630 (Barrio et al., 2011), a highly selective CB2 receptor antagonist. After 20 min cells were treated with either vehicle or one of the following highly selective CB2 agonists: 30 nM JWH 133 (Barrio et al., 2011; Zarruk et al., 2012) or 1 nM GP 1a (Gorantla et al., 2010; Murineddu et al., 2006); 24 h later mRNA was isolated and qRT-PCR for 5-HT_2A mRNA was performed as previously described.

Effect of PTX on GP 1a-induced increases in 5-HT_2A receptor mRNA and protein levels

CLU213 cells were treated with either vehicle (PBS) or PTX (100 ng/ml) (Bokoch et al., 1983; Casey et al., 1989); 20 min later, cells were treated with either vehicle (ethanol 0.01% final concentration) or GP 1a (1 nM) for 24 h. mRNA was isolated and qRT-PCR for 5-HT_2A was performed as described above.

In a different experiment, CLU213 cells were treated with either vehicle (PBS) or PTX (100 ng/ml) for 20 min. Cells were then incubated with either vehicle (ethanol 0.01% final concentration) or GP 1a (1 nM) for 72 h. Cells were washed (3×) with PBS every 24 h and fresh vehicle or GP 1a was added. Expression of membrane-associated 5-HT_2A receptors was determined by Western blot as previously described.

Effect of a selective ERK1/2 inhibitor (PD 198306) or adenylyl cyclase activator (NKH 477) on GP 1a-induced increases in 5-HT_2A receptor mRNA

CLU213 cells were treated with either vehicle (ethanol 0.01% final concentration), NKH 477 (20 µM) (Sobolewski et al., 2004; Toya et al., 1998) or PD 198306 (200 nM) (Ciruela et al., 2003; Pelletier et al., 2003); 20 min later, cells were incubated with either vehicle (ethanol 0.01% final concentration) or GP 1a (1 nM) for 24 h. mRNA was isolated and qRT-PCR for 5-HT_2A was performed as described.

Effect of PKC inhibitors on GP 1a-induced increases in 5-HT_2A receptor mRNA

CLU213 cells were treated with either vehicle (ethanol 0.01% final concentration), GF 109203X (5 µM) (Jacobson et al., 1995; Toullec et al., 1991), or Go 6967 (10 nM) (Martiny-Baron et al., 1993) for 20 min. Cells were then treated with either vehicle (ethanol 0.01% final concentration) or GP 1a (1 nM) for 24 h. mRNA was isolated and qRT-PCR for 5-HT_2A was performed as described above.

Effect of PKC activator on GP 1a-induced increases in 5-HT_2A receptor mRNA

CLU213 cells were treated with either vehicle (DMSO 0.01% final concentration), PDBu (1 µM), or PDBu (30 nM) (Burns et al., 1990; Kim et al., 2005) for 20 min. Cells were then treated with either vehicle (ethanol 0.01% final concentration) or GP 1a (1 nM) for 24 h. mRNA was isolated and qRT-PCR for 5-HT_2A was performed as described above.

Effect of transcription factor inhibitors on GP 1a-induced upregulation of 5-HT_2A receptors

CLU213 cells were treated with either vehicle (ethanol 0.01%), Naphthol AS-E phosphate (10 µM) (Best et al., 2004) or SR 11302 (1 µM) (Fanjul et al., 1994; Huang et al., 1997) for 20 min. Cells were then treated with either vehicle (ethanol 0.01%) or GP 1a (1 nM) for 24 h. mRNA was isolated and qRT-PCR for 5-HT_2A was performed as previously described.

Effect of a selective ERK1/2 inhibitor on GP 1a-induced increases in nuclear levels of c-Fos protein

CLU213 cells were treated with either vehicle (ethanol 0.01% final concentration) or PD 198306 (200 nM) (Ciruela et al., 2003; Pelletier et al., 2003) for 20 min. Cells were then incubated with either vehicle (ethanol 0.01% final concentration) or GP 1a (1 nM) for 15 min. After 15 min of incubation, cells were collected and nuclear-associated proteins were isolated. Expression of nuclear-associated c-Fos was determined by Western blot as previously described.

Statistics

All data are expressed as the mean ± S.E.M., where n indicates the number of replicates per group. Data were analyzed by an unpaired Student’s t-test or analysis of variance (ANOVA) (Newman-Keuls post-hoc test). GB-STAT software (Dynamic Microsystems, Inc., Silver Spring, MD, USA) was used for all statistical analyses.

Effect of CP55,940 treatment on the co-immunoprecipitation of 5-HT_2A and D₂ receptors in rat PFCx

We used co-immunoprecipitation protocols to study the effect of CP55,940 on the physical interaction between 5-HT_2A and D₂ receptors in rat PFCx (Figure 1). PFCx lysate of rats treated with either vehicle or CP55,940 (a non-selective CB1/CB2 receptor agonist) for 7 days was used in this experiment as described in the methods section. We used either D₂ or 5-HT_2A receptor antibodies as baits in two different co-immunoprecipitation experiments. In the first experiment, we used active columns to precipitate 5-HT_2A receptors using D₂ receptors as bait (Figure 1A, lanes 1 and 2). We also used inactive columns, unable to bind D₂ receptor antibody as controls (Figure 1A, lanes 3 and 4), as described in methods. We found that 5-HT_2A receptors co-precipitate with D₂ receptors when we used D₂ receptors as bait. Indeed, we found an enhanced co-immunoprecipitation of 5-HT_2A and D₂ receptors in PFCx of CP55,940-treated rats compared with vehicle controls (approx. 200% increase, Figure 1A lanes 1 and 2 for vehicle or CP55,940 samples, respectively). No co-precipitation of 5-HT_2A and D₂ receptors was detected when using inactive columns (Figure 1A, lanes 3 and 4). Similarly, we found an approximate two-fold increased co-precipitation of D₂ receptors with 5-HT_2A receptors in PFCx lysate of CP55,940-treated rats compared with controls when we used 5-HT_2A receptor as a bait (Figure 1B, lanes 5 and 6 for vehicle of CP55,940 samples, respectively). No co-precipitation of 5-HT_2A and D₂ receptors was detected when using inactive columns (Figure 1B, lanes 7 and 8). This evidence suggests that CP55,940 treatment enhances formation of a 5-HT_2A–D₂ receptor heteromer in rat PFCx.

Effect of chronic CP55,940 treatment on the protein expression of D₂ and 5-HT_2A receptors in rat PFCx

CP55,940 enhanced expression of post-synaptically located D₂ and 5-HT_2A receptors could underlie the enhanced co-immunoprecipitation of these receptors detected in Figure 1. In our next experiments, we studied the effect of CP55,940 exposure on the membrane-associated protein levels of 5-HT_2A and D₂ receptors. There are two alternatively spliced isoforms of the D₂ receptor that are codified for the same gene (Doly et al., 2004; Khan et al., 1998; Usiello et al., 2000). These are the dopamine D₂ receptor long (D₂L) and short (D₂S) isoforms that differ by a 29 amino acid insert in the third cytoplasmic loop (Dal Toso et al., 1989). The D₂S receptor (molecular weight 48 kDa) is mainly presynaptically localized, while the D₂L receptor (molecular weight 50 kDa) and the 5-HT_2A receptor (molecular weight 42 kDa) are mainly located post-synaptically (Doly et al., 2004; Khan et al., 1998; Usiello et al., 2000).

Chronic administration of CP55,940 produced significant increases in membrane-associated levels of D₂S receptors (Figure 2A), D₂L receptors (Figure 2B), and 5-HT_2A receptors (Figure 2C) in rat PFCx. Membrane-associated levels of D₂L and 5-HT_2A receptors increased between 60% and 100% compared with vehicle-treated animals (p<0.01, t 3.264, df 10 and p<0.05, t 2.55, df 10, respectively), while D₂S receptor levels increased almost three-fold compared with vehicle-treated controls (p<0.05, t 2.299, df 10). Actin was used as a control for protein loading in all these Western blots. We also determined the effect of chronic CP55,940 treatment on 5-HT_2A and D₂ mRNA levels in rat PFCx. 5-HT_2A receptor mRNA was significantly (p<0.05) increased (an approximate 90% increase) in PFCx of CP55,940-treated rats compared with vehicle-treated controls (Figure 2D). Interestingly, D₂ receptor mRNA was significantly (p<0.05) reduced (an approximate 45% reduction) in PFCx of CP55,940-treated rats compared with vehicle-treated controls.

Effect of non-selective and selective cannabinoid agonists on the 5-HT_2A and D₂ mRNA levels in a neuronal cell line

We used a neuronal cell line, CLU213 cells, in our next experiments to better examine the mechanisms involved in the cannabinoid-induced upregulation of 5-HT_2A receptors. CLU213 cells express 5-HT_2A, D₂, CB1 and CB2 receptors. In these experiments we tested the effect of either a non-selective cannabinoid agonist (CP55,940) (Thomas et al., 1998; Wiley et al., 1995); a selective CB1 receptor agonist (ACEA) (Hillard et al., 1999; Rutkowska and Jachimczuk, 2004); or a selective CB2 receptor agonist (GP 1a) (Gorantla et al., 2010; Murineddu et al., 2006).

We found that either CP55,940 or GP 1a produced a significant (p<0.01) upregulation of 5-HT_2A receptor mRNA levels in CLU213 cells (Figure 2E). Cells treated with either CP55,940 or GP 1a exhibited an approximate two-fold increase in 5-HT_2A receptor mRNA levels compared with controls. No significant differences (p>0.05) in the 5-HT_2A receptor mRNA levels were detected between cells treated with either CP55,940 or GP 1a. The CB1 agonist ACEA did not have significant effects on 5-HT_2A receptor mRNA levels (Figure 2E). On the other hand, cells treated with either CP55,940 or ACEA exhibited a significant (p<0.05) downregulation of D₂ mRNA levels in CLU213 cells. Cells treated with CP55,940 exhibited an approximate 60% reduction (p<0.05) in D₂ mRNA levels, while cells treated with ACEA exhibited an approximate 52% reduction (p<0.05) in D₂ mRNA levels. No significant differences (p>0.05) in D₂ mRNA levels were detected between cells treated with either CP55,940 or ACEA.

Since we detected a very strong regulation of 5-HT_2A receptor mRNA induced by GP 1a, a highly selective CB2 receptor agonist, we also studied the effect of other selective CB2 agonist and antagonist on 5-HT_2A upregulation. In this experiment cells were pretreated with either vehicle or AM 630, a selective CB2 antagonist; 20 min later the cells were incubated with either vehicle, JWH 133 or GP 1a as described in the methods section. We found that both JWH 133 and GP 1a produced a significant (p<0.01) upregulation of 5-HT_2A receptor mRNA in CLU213 cells (Figure 2F). There were no significant (p>0.05) differences between the 5-HT_2A upregulation induced by JWH 133 or GP 1a. This strong 5-HT_2A mRNA upregulation induced by these CB2 receptor agonists was significantly (p<0.01) inhibited in cells pretreated with a selective CB2 antagonist, AM 630 (Figure 2F). No significant (p>0.05) differences in 5-HT_2A mRNA were found between vehicle-treated cells and cells pretreated with AM 630 and later treated with either vehicle, JWH 133, or GP 1a (Figure 2F). The two-way ANOVA for 5-HT_2A mRNA showed a significant main effect of AM 630 pretreatment (F_1,134.75, p<0.0001) and CB2 agonists treatment (F_1,65.98, p<0.0001). There was also a significant interaction between AM 630 pretreatment and CB2 agonists treatment (F_1,40.03, p<0.0001).

Effect of G-protein and ERK1/2 signaling inhibitors on the GP 1a-induced upregulation of 5-HT_2A receptors in CLU213 cells

Our next experiments were designed to identify some signaling components that would mediate the upregulation of 5-HT_2A receptors by the CB2 receptor agonist GP 1a. Previous reports suggested that CB1 and CB2 cannabinoid receptors couple to Gα_i/o G-proteins receptors to inhibit adenylyl cyclase activation and to induce the activation of the ERK1/2 signaling cascade (Bouaboula et al., 1996). Here we used PTX to prevent the GP 1a-induced activation of Gα_i/o G-proteins (Bouaboula et al., 1996). PTX-induced ADP ribosylation of Gα_i/o subunits mediates the inactivation of their signaling by interfering with Gα/receptor coupling (Bokoch et al., 1983; Casey et al., 1989).

Figure 3 illustrates the effect of PTX pretreatment on GP 1a-induced upregulation of 5-HT_2A receptors in CLU213 cells. CLU213 cells were pretreated with either vehicle or PTX (100 ng/ml) for 20 min, then vehicle or GP 1a (1 nM) was added to the media. We found that in vehicle-pretreated cells, GP 1a significantly (p<0.01) increased 5-HT_2A mRNA levels (two-fold increase) over controls (Figure 3A). This effect of GP 1a was prevented (p<0.01) in cells pretreated with PTX. No significant (p>0.05) effect of PTX was found on basal 5-HT_2A receptor mRNA levels. The two-way ANOVA for 5-HT_2A mRNA showed significant main effects of PTX pretreatment (F_1,23.52, p<0.0004) and GP 1a treatment (F_1,34.11, p<0.0001). There was a significant interaction between PTX pretreatment and GP 1a treatment (F_1,47.74, p<0.0001).

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Figure 3.

GP 1a, a selective CB2 receptor agonist, upregulated 5-HT_2A receptors via Ga_i G-protein in CLU213 cells.

In Figure 3B, CLU213 cells were treated with either vehicle or PTX (100 ng/ml), then vehicle or GP 1a (1 nM) was added to the media 20 min later. Membrane-associated 5-HT_2A receptor protein expression was measured in these cells after 3 days of incubation with GP 1a, as described in the methods section. We found that in vehicle-pretreated cells, GP 1a significantly (p<0.01) increased 5-HT_2A receptor protein levels (an approximate 60% increase) over controls (Figure 3B). The effect of GP 1a on 5-HT_2A receptor protein levels was prevented (p<0.01) in cells pretreated with PTX. No significant (p>0.05) effect of PTX was found on basal 5-HT_2A receptor protein levels. The two-way ANOVA for 5-HT_2A receptor protein levels showed significant main effects of PTX pretreatment (F_1,23.18, p<0.0001) and GP 1a treatment (F_1,19.34, p<0.0003). There was a significant interaction between PTX pretreatment and GP 1a treatment (F_1,7.14, p<0.0151). These data suggest that the GP 1a-induced upregulation of 5-HT_2A receptors is mediated by a Gα_i/o G-protein mechanism.

Coupling of CB2 cannabinoid receptors to Gα_i/o G-proteins mediates the increases in ERK signaling and also the inhibition adenylate cyclase that results in reduced cAMP levels (Felder et al., 2006). In our next experiment, we studied the effect of a ERK1/2 inhibitor (PD 198306) (Pelletier et al., 2003) and an adenylyl cyclase activator (NKH 477) (Sobolewski et al., 2004) on the GP 1a-induced upregulation of 5-HT_2A receptor mRNA. CLU213 cells were treated with either vehicle, PD 198306 (200 nM) or NKH 477 (20 µM); 20 min later cells were treated with either vehicle or GP 1a (1 nM) for 24 h. Consistent with our previous findings, GP 1a significantly (p<0.05) increased 5-HT_2A mRNA levels (an approximate two-fold increase) over controls (Figure 3C). The effect of GP 1a was prevented (p<0.05) in cells pretreated with either PD 198306 or NKH 477. No significant (p>0.05) effect of PD 198306 or NKH 477 was found on basal 5-HT_2A receptor mRNA levels (Figure 3C). These results suggest that the GP 1a-induced upregulation of 5-HT_2A receptors is dependent on ERK1/2 activation and prevented by activation of adenylyl cyclase.

Effect of PKC on GP 1a-induced upregulation of 5-HT_2A receptor mRNA

Figures 4A and 4B illustrate the effect of PKC inhibition on GP 1a-induced 5-HT_2A receptor upregulation. Bouaboula et al. (1996) proposed that cannabinoid receptors activate the ERK1/2 signaling cascade through PKC activation. In addition, they reported evidence to suggest that Ca²⁺-dependent PKC isoforms could be involved in CB2 signal transduction which are not involved in CB1 signal transduction (Bouaboula et al., 1996). Here we studied the effect of two different PKC inhibitors (GF 109203X and Go 6967) on GP 1a-induced increases in 5-HT_2A receptor mRNA levels. GF 109203X does not discriminate between Ca²⁺-dependent and -independent isoforms of PKC (IC₅₀ values are 0.0084, 0.0180, 0.210, 0.132, and 5.8 µM for α, β1, δ, ϵ and ζ isoforms, respectively) (Toullec et al., 1991), while Go 6967 selectively inhibits Ca²⁺-dependent isoforms PKCα and PKCβ1 (IC₅₀ values are 2.3 and 6.2 nM, respectively) (Martiny-Baron et al., 1993).

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Figure 4.

Ca²⁺-independent and -dependent isoforms of PKC regulate 5-HT_2A receptor mRNA levels in CLU213 cells.

In Figure 4A, CLU213 cells were pretreated with either vehicle or GF 109203X (5 µM) for 20 min and then treated with vehicle or GP 1a (1 nm). In this experiment, 5 µM GF 109203X should produce a substantial inhibition of most PKC isoforms. We found that GP 1a significantly (p<0.05) increased 5-HT_2A receptor mRNA levels (two-fold increase) in vehicle-pretreated cells compared with vehicle-treated controls (Figure 4A). Furthermore, GF 109203X pretreatment significantly (p<0.05) increased basal 5-HT_2A receptor mRNA levels (two-fold increase) over vehicle-treated controls and had no significant effect (p>0.05) on GP 1a-induced increases in 5-HT_2A receptor mRNA levels. The two-way ANOVA for 5-HT_2A mRNA showed significant main effects of GF 109203X pretreatment (F_1,6.68, p<0.0324) and GP 1a treatment (F_1,10.82, p<0.011). There was no significant interaction between GF 109203X pretreatment and GP 1a treatment (F_1,2.17, p>0.05).

In order to address the role of PKC Ca²⁺-dependent isoforms on the regulation of 5-HT_2A receptor mRNA, CLU213 cells were pretreated with either vehicle or Go 6967 (10 nm) for 20 min then treated with either vehicle or GP 1a (1 nm) for 24 h. GP 1a significantly (p<0.01) increased 5-HT_2A receptor mRNA levels (two-fold increase) compared with vehicle-pretreated controls (Figure 4B). Pretreatment with Go 6967 significantly (p<0.01) increased basal 5-HT_2A receptor mRNA levels (58% increase) over vehicle-pretreated controls, while Go 6967 pretreatment did not have a significant effect (p>0.05) on GP 1a-induced upregulation of 5-HT_2A receptors mRNA. The two-way ANOVA for 5-HT_2A receptor mRNA showed a main effect of Go 6967 pretreatment (F_{1, 5.85}, p<0.0418) and a main effect of GP 1a treatment (F_1,16.15, p<0.0038). There was no significant interaction between Go 6967 pretreatment and GP 1a treatment (F_1,2.03, p>0.05).

Next we examined the effect a PKC activator, PDBu, (K_d values are 1 µM, 0.98 µM, 26 nM, 11 nM, and 9 nM for ϵ, δ, β1, α, and ζ isoforms, respectively; Burns et al., 1990) has on GP 1a-induced increases in 5-HT_2A receptor mRNA. We used two doses of PDBu in our experiments, 1 µM and 30 nM. We expect to activate all the different isoforms with the 1 µM dose and selectively activate the β1, α, and ζ isoforms (Ca²⁺-dependent isoforms) with the lowest dose (30 nM) (Burns et al., 1990).

CLU213 cells were pretreated with either vehicle or PDBu (1 µM) for 20 min. Cells were then treated with either vehicle or GP 1a (1 nm). GP 1a significantly (p<0.05) increased 5-HT_2A receptor mRNA levels (two-fold increase, Figure 4C). This effect of GP 1a was prevented (p<0.05) in cells pretreated with PDBu. No significant (p>0.05) effect of PDBu was found on basal 5-HT_2A receptor mRNA levels. The two-way ANOVA for 5-HT_2A receptor mRNA showed significant main effects of PDBu pretreatment (F_1,6.12, p<0.0385) and GP 1a treatment (F_1,6.10, p<0.0375). There was a significant interaction between PDBu pretreatment and GP 1a treatment (F_1,5.38, p<0.0489).

In order to examine the effect Ca²⁺-dependent PKC isoforms on GP 1a-induced increases in 5-HT_2A receptor mRNA, we used a concentration of PDBu (30 nm) that activated the Ca²⁺-dependent isoforms (Burns et al., 1990). GP 1a significantly (p<0.01) increased 5-HT_2A receptor mRNA levels (two-fold increase, Figure 4D). There was no significant (p>0.05) effect of PDBu 30 nm found on basal 5-HT_2A receptor mRNA levels, and PDBu pretreatment significantly reduced (an approximate 20% decrease, p<0.05) the GP 1a-induced upregulation of 5-HT_2A receptors. The two-way ANOVA for 5-HT_2A receptor mRNA showed significant main effects of PDBu pretreatment (F_1,6.26, p<0.0368) and GP 1a treatment (F_1,79.39, p<0.0001). There was no significant interaction between PDBu pretreatment and GP 1a treatment (F_1,1.62, p<0.2385). The use of PKC activators seems to suggest that both Ca²⁺-dependent and Ca²⁺-independent PKC isoforms play a role preventing the GP 1a-induced upregulation of 5-HT_2A receptor mRNA.

Effect of CREB and AP-1 transcription factors inhibitors on the GP 1a-induced upregulation of 5-HT_2A receptor mRNA

Figure 5 illustrates the effect of CREB or AP-1 transcription factor inhibitor pretreatment on GP 1a-induced upregulation of 5-HT_2A receptor mRNA in CLU213 cells. Here we wanted to identify possible transcription factor(s) that would contribute to GP 1a-induced increases of 5-HT_2A receptor mRNA. In our previous experiments we showed that the GP 1a-induced upregulation of 5-HT_2A receptors is prevented by PD 198306, an inhibitor of ERK1/2 activation. Activation of ERK involves the phosphorylation of this protein in the cytoplasm and its translocation to the nucleus (Campbell et al., 1995; Chang et al., 2003; Seger and Krebs, 1995). In the nucleus, phosphorylated ERK (pERK) can activate several transcription factors such as CREB, c-Fos, ELK-1, SP-1, and EGR-1 (Campbell et al., 1995; Chang et al., 2003; Seger and Krebs, 1995). The transcription factors CREB and AP-1 have consensus sequences within the promoter region of the rat 5-HT_2A receptor gene (Chalecka-Franaszek et al., 1999; Du et al., 1994; Ferry and Molinoff, 1996). Therefore, we decided to test the effects of inhibitors of these transcription factors on the GP 1a-induced upregulation of 5-HT_2A receptor mRNA.

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Figure 5.

CB2 receptor-induced upregulation of 5-HT_2A receptor involves AP-1 and c-Fos, but not CREB, activation.

CREB is a transcription factor that binds to certain DNA sequences called cAMP response elements (CRE), thereby increasing or decreasing transcription of downstream genes (Chrivia et al., 1993; Kwok et al., 1994). c-Fos belongs to the immediate early gene family of transcription factors. Members of the Fos family dimerize with c-Jun to form the AP-1 transcription factors, which can upregulate transcription of various genes (Karin, 1995; Karin et al., 1997). In our first experiment, we studied the effect of CREB inhibitor pretreatment on the GP 1a-induced upregulation of 5-HT_2A receptors.

CLU213 cells were treated with either vehicle or Naphthol AS-E phosphate (10 μM) for 20 min and were then treated with vehicle or GP 1a (1 nm). Naphthol AS-E phosphate blocks cAMP-induction of CREB-dependent gene transcription (K_i 10 μM) (Best et al., 2004). We found that Naphthol AS-E phosphate did not inhibit or decrease GP 1a-induced increases in 5-HT_2A receptor mRNA (Figure 5A). No significant (p>0.05) effect of Naphthol AS-E phosphate was found on basal 5-HT_2A mRNA levels. The two-way ANOVA for 5-HT_2A receptor mRNA showed no significant main effect of Naphthol AS-E phosphate pretreatment (F_1,0.006, p>0.9384) and a significant main effect of GP 1a treatment (F_1,28.91, p<0.0007). There was no significant interaction between Naphthol AS-E pretreatment and GP 1a treatment (F_1,0.23, p>0.6453). These data indicate that CREB is not involved in GP 1a-induced 5-HT_2A receptor upregulation.

We then studied the effect of AP-1 inhibition on GP 1a-induced increases in 5-HT_2A receptor mRNA. CLU213 cells were treated with either vehicle or SR 11302 (1 μM) for 20 min, then vehicle or GP 1a (1 nM) was added to the incubation media. SR 11302 is a retinoid that transrepresses AP-1 without transactivating the retinoic acid response element (E_max 1 µM) (Fanjul et al., 1994). As expected, GP 1a induced a significant (p<0.05) increase in 5-HT_2A mRNA levels (an approximate two-fold increase in 5-HT_2A mRNA) (Figure 5B). SR 11302 pretreatment significantly reduced (approximately 55% decrease, p<0.05) the GP 1a-induced upregulation of the 5-HT_2A receptor mRNA (Figure 5B). No significant (p>0.05) effect of SR 11302 was found in basal 5-HT_2A mRNA levels. The two-way ANOVA for 5-HT_2A mRNA did not show a significant main effects of SR 11302 pretreatment (F_1,2.89, p>0.1271) and did show a significant effect of GP 1a treatment (F_1,32.80, p<0.0004). There was a significant interaction between SR 11302 pretreatment and GP 1a treatment (F_1,7.48, p<0.0256).

Our data seem to indicate that GP 1a-induced upregulation of 5-HT2A receptors would be mediated, at least in part, by ERK1/2 and AP-1 activation. Here we examined whether inhibition of ERK1/2 can prevent the GP 1a-induced increases in the nuclear-associated protein levels of c-Fos. CLU213 cells were treated with either vehicle or PD 198306 (200 nm) for 20 min and then treated with either vehicle or GP 1a (1 nm) for 15 min. As mentioned above, PD 198306 is a potent inhibitor of ERK1/2 (IC₅₀ 100 nM) (Pelletier et al., 2003). We found that in vehicle-pretreated cells, GP 1a significantly (p<0.05) increased c-Fos levels over controls (Figure 5C). Indeed, GP 1a induced an approximate 40% increase in the nuclear-associated protein levels of c-Fos. This effect of GP 1a was prevented (p<0.01) in cells pretreated with PD 198306 (Figure 5C). No significant (p>0.05) effect of PD 198306 was found on basal 5-HT_2A mRNA levels. The two-way ANOVA for 5-HT_2A mRNA showed significant main effects of PD 198306 pretreatment (F_1,15.74, p<0.0008) and GP 1a treatment (F_1,6.09, p<0.0147). There was a significant interaction between PD 198306 pretreatment and GP 1a treatment (F_1,5.95, p<0.0241).