Modulation of extracellular signal-regulated kinase (ERK) by opioid and cannabinoid receptors that are expressed in the same cell

BR A IN RE S E A RCH 1 1 89 ( 20 0 8 ) 2 3 –3 2

a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s

Research Report

Modulation of extracellular signal-regulated kinase (ERK) by opioid and cannabinoid receptors that are expressed in the same cell Alexander Korzh, Ora Keren, Mikhal Gafni, Hilla Bar-Josef, Yosef Sarne⁎ The Mauerberger Chair in Neuropharmacology, Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv 69978, Israel

A R T I C LE I N FO

AB S T R A C T

Article history:

In the present study we investigated the signal transduction pathways leading to the

Accepted 28 October 2007

activation of extracellular signal-regulated kinase (ERK) by opioid or cannabinoid drugs,

Available online 4 November 2007

when their receptors are coexpressed in the same cell-type. In N18TG2 neuroblastoma cells, the opioid agonist etorphine and the cannabinoid agonist CP-55940 induced the

Keywords:

phosphorylation of ERK by a similar mechanism that involved activation of δ-opioid

Opioid

receptors or CB1 cannabinoid receptors coupled to Gi/Go proteins, matrix metalloproteases,

Cannabinoid

vascular endothelial growth factor (VEGF) receptors and MAPK/ERK kinase (MEK). In HEK-

Extracellular signal-regulated kinase

293 cells, these two drugs induced the phosphorylation of ERK by separate mechanisms.

Vascular endothelial growth

While CP-55940 activated ERK by transactivation of VEGFRs, similar to its effect in N18TG2

factor receptors

cells, the opioid agonist etorphine activated ERK by a mechanism that did not involve

Receptor tyrosine Kinase

transactivation of a receptor tyrosine kinase. Interestingly, the activation of ERK by etorphine was resistant to the inhibition of MEK, suggesting the possible existence of a novel, undescribed yet mechanism for the activation of ERK by opioids. This mechanism was found to be specific to etorphine, as activation of ERK by the μ-opioid receptor (MOR) agonist DAMGO ([D-Ala2, N-Me-Phe4, Gly5-ol] enkephalin) was mediated by MEK in these cells, suggesting that etorphine and DAMGO activate distinct, ligand-specific, conformations of MOR. The characterization of cannabinoid- and opioid-induced ERK activation in these two cell-lines enables future studies into possible interactions between these two groups of drugs at the level of MAPK signaling. © 2007 Elsevier B.V. All rights reserved.

⁎ Corresponding author. Fax: +972 3 6409113. E-mail address: [email protected] (Y. Sarne). Abbreviations: DAMGO, [D-Ala2, N-Me-Phe4, Gly5-ol] enkephalin; DOR, δ-opioid receptor; EGF, Epidermal growth factor; ERK, Extracellular signal-regulated kinase; FGF, Fibroblast growth factor; GPCR, G-protein-coupled receptor; GRK, G-protein receptor kinase; HEK, Human embryonic kidney cells; MAPK, Mitogen-activated protein-kinase; MEK, MAPK/ERK kinase; MOR, μ-opioid receptor; PDGF, Platelet-derived growth factor; I3K, Phosphatidyl inositol-3 kinase; PKC, Protein kinase C; PP2A, protein phosphatase 2A; PTX, pretussis toxin; 7TMR, Seven-transmembrane receptor; VEGF, Vascular endothelial growth factor 0006-8993/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2007.10.070

24

1.

BR A IN RE S EA RCH 1 1 89 ( 20 0 8 ) 2 3 –32

Introduction

Opioids and cannabinoids are two separate groups of psychoactive drugs that have a similar pharmacological profile (the induction of analgesia, hypothermia, hypotension, sedation and motor depression), although they activate different receptors: μ (MOR), δ (DOR) and κ (KOR) in the case of opioids and CB1 and CB2 in the case of cannabinoids (Loh and Smith, 1990; Pertwee, 1997; Reisine and Brownstein, 1994). Both opioid and cannabinoid receptors belong to the family of seventransmembrane (7TM) receptors that act through heterotrimeric G proteins to modulate various intracellular systems. Opioid and cannabinoid agonists were shown to inhibit adenylate cyclase activity, block voltage-dependent calcium channels and activate inward rectifying potassium channels (Howlett, 1995; Law et al., 2000). Similar to other 7TM receptors, opioid and cannabinoid receptors were also shown to mediate the activation of mitogen-activated protein kinase (MAPK) pathways, including the Ras-Raf-ERK (extracellular signalregulated kinase) cascade, in a variety of cells in-vitro (Ai et al., 1999; Belcheva et al., 2002; Davis et al., 2003; Derkinderen et al., 2003; Galve-Roperh et al., 2002; Kramer et al., 2002; Sanchez et al., 2003) and in-vivo (Derkinderen et al., 2003; Eitan et al., 2003; Rubino et al., 2004). Through the activation of MAPK cascades, opioid and cannabinoid agonists can modulate, in addition to their well recognized pharmacological effects (see above), diverse biological processes such as growth, differentiation, cell migration, survival and death (Guzman et al., 2001; Persson et al., 2003; Tegeder and Geisslinger, 2004). Furthermore, the involvement of ERK was also demonstrated in desensitization and internalization of opioid (Schmidt et al., 2000) and cannabinoid (Rubino et al., 2005) receptors and in adaptive processes resulting from chronic administration of either opioid (Eitan et al., 2003) or cannabinoid drugs (Rubino et al., 2004). ERK1/2 proteins are classically activated through the sequential mobilization of Ras G proteins, Raf kinases and MAPK/ERK kinase 1 and 2 (MEK1 and 2). MEKs phosphorylate and activate their only known substrates, ERK 1 and 2, which are themselves serine/threonine kinases that phosphorylate a large number of substrates throughout the cell, including various transcription factors and immediate early genes products. The linear pathway that leads to ERK phosphorylation can be regulated by a variety of signals such as G-protein subunits, protein kinase A (PKA), protein kinase C (PKC) and phosphatidyl inositol-3 kinase (PI3K). It is now evident that 7TM receptors can activate ERKs by multiple mechanisms including transactivation of cell-surface receptor tyrosine kinases (RTKs) that leads to ERK phosphorylation; integrinbased focal adhesion complexes containing focal adhesion kinases (FAKs) that provide scaffolds upon which the receptoractivated Ras complex can assemble (Della Rocca et al., 1999); β-arrestin scaffolding directly on the 7TMR (Lefkowitz and Shenoy, 2005); and the modulation of the Ras-Raf-MEK-ERK pathway through a wide range of intracellular intermediates (reviewed in (Belcheva and Cosia, 2002; Caunt et al., 2006; Gavi et al., 2006)). Opioid and cannabinoid agonists were shown to activate ERK by various mechanisms. Opioids elevated ERK phosphor-

ylation by transactivation of epidermal growth factor receptors (EGFRs) In human embryonic kidney cells (HEK-293) and in astrocytes (Belcheva et al., 2001, 2003; Eisinger and Schulz, 2004), and by transactivation of fibroblast growth factor receptors (FGFRs) in rat C6 glioma cells (Belcheva et al., 2002). Similarly, we have previously shown that cannabinoids modulate ERK activation by transactivation of vascular endothelial growth factor receptors (VEGFRs) in N18TG2 neuroblastoma cells (Rubovitch et al., 2004). Both opioids and cannabinoids were also shown to utilize other cellular mechanisms for the modulation of ERK phosphorylation in other experimental systems. For instance, PKC was found to mediate opioids activation of ERK in transfected Chinese hamster ovary (CHO) cells (Ai et al., 1999), Jurkat leukemia Tcells (Shahabi et al., 1999) and adult hippocampal progenitor cells (Persson et al., 2003), and PI3K was involved in opioidinduced ERK phosphorylation in HEK-293 (Eisinger and Schulz, 2004) and in CHO cells (Ai et al., 1999). Furthermore, cannabinoids were shown to modulate ERK phosphorylation either through PKA in NIE-115 neuroblastoma cells (Davis et al., 2003), through cAMP and the Src tyrosine kinase FYN in hippocampal slices (Derkinderen et al., 2003) or through PI3K in astrocytoma and prostate PC-3 cells (Galve-Roperh et al., 2002; Sanchez et al., 2003). In view of the heterogeneity and complexity of the pathways leading from 7TM receptors to ERK activation, the present study was designed to characterize the mechanisms underlying ERK1/2 activation via opioid and cannabinoid receptors that are expressed in the same cell. Since colocalization of opioid and cannabinoid receptors in neurons of the spinal cord and the brain was reported (Childers, 1991; Pickel et al., 2004; Rodriguez et al., 2001; Salio et al., 2001), and as interactions between opioid and cannabinoid classic signal transduction pathways at the cellular level were previously found by ourselves (Shapira et al., 1998, 2003) as well as by others (reviewed in (Vigano et al., 2005)), the outcome of the present study will provide information on new possible points of interaction between opioid- and cannabinoid-signaling along the MAPK cascade. The study was carried out using two different cell lines: N18TG2 neuroblastoma cells that naturally express both δopioid receptors (DORs) and CB1 cannabinoid receptors, and HEK-293 cells which were transfected with μ-opioid receptors (MORs) or CB1 receptors. We found that in N18TG2 cells both the opioid agonist etorphine and the cannabinoid agonist CP55940 induced ERK phosphorylation by transactivation of VEGFR. On the other hand, separate intracellular mechanisms were activated by these two drugs in HEK-293 cells: CP-55940 induced ERK phosphorylation by transactivation of VEGFR, similar to its effect in N18TG2 cells, while the opioid agonist etorphine induced ERK phosphorylation by a different mechanism, which did not involve transactivation of a TRK receptor. Interestingly, the effect of etorphine in HEK-293 cells was resistant to the inhibition of MEK, the only kinase that is known, so far, to phosphorylate ERK, suggesting the existence of a novel, unidentified yet mechanism for opioid-induced activation of ERK (see Discussion). This novel mechanism for the activation of ERK was specific to etorphine, since the effect of the MOR selective ligand [D-Ala2, N-Me-Phe4, Gly5-ol] enkephalin (DAMGO) was mediated by MEK, suggesting that

BR A IN RE S E A RCH 1 1 89 ( 20 0 8 ) 2 3 –3 2

25

different agonists of MOR can activate, in the same cell, distinct signaling pathways leading to ERK1/2 phosphorylation.

2.

Results

2.1. Cannabinoid-induced ERK1/2 phosphorylation in N18TG2 neuroblastoma and HEK-293 cells We have previously shown that the cannabinoid agonist desacetyllevonantradol (DALN) induces the phosphorylation of ERK1/2 in N18TG2 neuroblastoma cells via the CB1 cannabinoid receptor. This activation of ERK by DALN was mediated by Gi/Go proteins, PKC, matrix metalloproteases, VEGFRs and MEK (Rubovitch et al., 2004). In the present study we tested the effect of the cannabinoid agonist CP-55940 on ERK phosphorylation. Similar to our previous results with DALN, exposure of the cells to CP-55940 (1 μM, 5 min) increased ERK1/2 phosphorylation compared to control, vehicletreated cells. As expected, this effect was abolished by 20 μM of the MEK inhibitor PD98059 (n = 9), similar to what we have found before for DALN (Fig. 3 in (Rubovitch et al., 2004)). Since the transactivation of VEGFR by CB1 agonists is a novel mechanism by which cannabinoids can activate ERK1/2, we next examined whether this is unique to the N18TG2 cellline, or whether a similar mechanism is used by these drugs in other cell types as well. In order to examine this question we chose to use the HEK-293 cell-line in which various signaling pathways that lead from GPCRs activation to ERK phosphorylation were described, including transactivation of RTKs and the modulation of downstream elements along the ERK cascade (Lefkowitz et al., 2002). The cannabinoid agonist CP-55940 induced a concentration-dependent increase in ERK1/2 phosphorylation in HEK293 cells that had been transfected with a mouse CB1 receptor. The elevation in pERK1/2 was transient, peaking at 3 min and then declining (Fig. 1). The activation of ERK1/2 by CP-55940 was mediated by the CB1 receptor as it was completely blocked by the CB1 receptor antagonist SR141716A (10 μM,

Fig. 1 – Phosphorylation of ERK1/2 by the cannabinoid agonist CP-55940 (CP) in HEK-293 cells. Representative experiments illustrating the time-dependency (A) and the blocking effect of PTX (B) and o-phenanthroline (OPh) (C). A quantitative analysis of the effects of PTX and o-phenanthroline is presented in Fig. 2.

Fig. 2 – The involvement of CB1 receptors, Gi/Go binding proteins and metalloproteases in CP-55940-induced ERK activation in HEK-293 cells. The phosphorylation of ERK2 by CP-55940 (CP, 0.1 μM) was completely blocked by 10 μM SR-141716A (SR, n = 8) (A). Pretreatment with PTX (100 ng/ml, 18 h, n = 8) (B) or with o-phenanthroline (OPh, 200 μM, 30 min, n = 9) (C) completely inhibited the effect of 1 μM CP-55940. (*) denotes p < 0.01.

n = 8, p < 0.01) (Fig. 2), and involved Gi/Go proteins, as it was abolished by pretreatment with PTX (100 ng/ml, 18 h, n = 8, p < 0.01) (Figs. 1 and 2). Transactivation of TRKs by G-protein coupled receptors (GPCRs) involves membrane-bound metalloproteases that cleave and release growth factor-like substances to the extracellular medium (Belcheva and Cosia, 2002). To examine whether CP-55940 modulates ERK in HEK293 cells by transactivation of a RTK via metalloproteases, the cells were pretreated with the metalloprotease inhibitor 1, 10phenanthroline (o-phenanthroline) (200 μM, 30 min), that was found to abolish the effect of CP-55940 (n = 9, p < 0.01) (Figs. 1 and 2). Further experiments showed that the effect of CP55940 was completely blocked by the non-selective RTK inhibitor SU 5416 (50 μM, n = 4) that blocks VEGFR, FGFR and PDGFR, and by the selective inhibitor of VEGFR oxindole-1 (3 μM, 10 min) (n = 8, p < 0.01), but not by AG1478, a specific inhibitor of EGFR (1 μM, n = 4) or by AG1296, a specific inhibitor of PDGFR (10 μM, n = 4) (Fig. 3). These results indicate that cannabinoids activate ERK in HEK-293 cells by

26

BR A IN RE S EA RCH 1 1 89 ( 20 0 8 ) 2 3 –32

vation of ERK by CP-55940 was inhibited by the MEK inhibitors PD-98059 (100 μM, 30 min, n = 8, p < 0.01) (Fig. 4) and UO126 (10 μM, 30 min, n = 10, p < 0.01). It should be noted that pretreatment with either of the MEK inhibitors considerably reduced basal pERK levels, indicating a significant constitutive activity along this pathway (see Discussion).

2.2. Opioid-induced ERK1/2 phosphorylation in N18TG2 neuroblastoma and in HEK-293 cells

Fig. 3 – The effect of various RTK blockers on cannabinoid-induced phosphorylation of ERK in HEK-293 cells. A–D depict representative experiments with the various blockers. (E) shows a quantitative analysis of the effects of the various blockers. Results are presented as percent of the matched control (either vehicle or blocker alone) in each experiment, with control values standardized to 100%. For the concentrations and the specificity of the blockers see text. (CP = CP-55940; OXIN = oxindole-1). n = 4–8. (*) denotes p < 0.01.

So far we have established that cannabinoids, through CB1 receptors, activate ERK1/2 in two different cell types by a similar mechanism, namely, transactivation of VEGFRs. The next question was whether activation of opioid receptors, which are coexpressed within the same cell, will also lead to transactivation of VEGFRs followed by ERK phosphorylation. We first examined the effect of the opioid agonist etorphine in N18TG2 neuroblastoma cells. Exposure of N18TG2 cells to etorphine induced a transient dose-dependent increase in ERK1/2 phosphorylation. This effect was carried out by the opioid receptor, since it was blocked by the opioid antagonist naloxone (10 μM, n = 5), was mediated by Gi/Go GTP-binding proteins, as it was completely abolished by pretreatment of the cells with pertussis toxin (PTX) (100 ng/ml, 18 h, n = 4) and involved PKC, since it was abolished by its selective inhibitor chelerytrine (10 μM, n = 3) (Fig. 5). In order to determine whether ERK1/2 activation by opioids involves metalloproteases, we tested the effect of o-phenanthroline, the inhibitor of metalloproteases, on ERK1/2 phosphorylation by etorphine. Pretreatment with o-phenanthroline (100 μM, 30 min, n = 4) completely eliminated the effect of 1 μM etorphine (Fig. 5), thus suggesting that activation of the opioid receptor resulted in transactivation of a RTK. As cannabinoids were previously found by us to transactivate VEGFR in this cellline (Rubovitch et al., 2004), we next tested the effect of oxindole1, a selective inhibitor of VEGFR, on etorphine-induced ERK1/2 phosphorylation. Oxindole-1 (0.9–3 μM, 30 min, n = 5) completely blocked the phosphorylation of ERK1/2 by etorphiμne (Fig. 5). These findings demonstrate that in N18TG2 neuroblastoma cells cannabinoids and opioids utilize a similar pathway to activate ERK1/2, namely, transactivation of VEGF receptors.

the transactivation of VEGF receptors, similar to what we previously found in N18TG2 cells (Rubovitch et al., 2004). To further characterize the pathway leading from the CB1 receptor to ERK1/2 phosphorylation, we tested the involvement of several mediators that were previously shown to regulate the activation of ERK by various GPCRs. Pretreatment with wortmannin, a specific PI3K inhibitor (1 μM, 30 min) did not modify the effect of CP-55940. On the other hand, the PKC inhibitor chelerythrine (10 μM, 30 min) completely abolished the activation of ERK by CP-55940 (n = 12, p < 0.001). Similarly, pretreatment of the cells with the nonreceptor tyrosine kinase Src family inhibitor PP2 (10 μM, 30 min, n = 8) abolished almost completely the effect of CP-55940 (p < 0.03). These findings indicate that the pathway leading from the CB1 receptor to ERK activation is modulated by PKC and by Src, but not by PI3K. Finally, we tested the involvement of MEK, the only kinase that is known to phosphorylate ERK. As expected, the acti-

Fig. 4 – Inhibition of CP-55940-induced ERK phosphorylation by the MEK blocker PD-98059 (100 μM) in HEK-293 cells. A representative experiment (A) and a quantitative analysis of 8 quadruplets drawn from 4 experiments (B). (*) denotes p < 0.01.

BR A IN RE S E A RCH 1 1 89 ( 20 0 8 ) 2 3 –3 2

27

indicating that VEGFRs were not involved in etorphineinduced ERK activation. The results with the metalloprotease and the VEGFR inhibitors demonstrate that etorphine activates ERK in HEK-293 cells by a different pathway than the cannabinoid agonist CP-55940. We next examined the involvement of other possible mediators in the opioid effect. Neither wortmannin (1 μM), nor PP2 (10 μM), nor chelerythrine (10 μM) blocked the effect of etorphine on ERK phosphorylation, indicating that the activation of ERK by etorphine did not involve PI3K, neither Src nor

Fig. 5 – Phosphorylation of ERK1/2 by the opioid agonist etorphine (Et; 1 μM) in N18TG2 neuroblastoma cells. Representative experiments illustrating the blocking effect of PTX (100 ng/ml, 18 h) (A), chelerytrine (Chel, 10 μM) (B), o-phenanthroline (OPh, 200 μM) (C) and oxindole-1 (Oxin, 1 μM) (D), indicating the involvement of Gi/Go binding proteins, PKC, metalloproteases and VEGFR, respectively. Each experiment was repeated 3–5 times with similar results.

The next step was to examine whether the transactivation of the same RTK (namely, VEGFR) by both cannabinoids and opioids is unique to the N18TG2 cell-line, or whether these drugs utilize a similar common mechanism in HEK-293 cells as-well. The opioid agonist etorphine induced a transient dose-dependent increase in ERK1/2 phosphorylation in HEK293 cells that had been transfected with a rat μ-opioid receptor (MOR). The elevation in pERK peaked at 5 min and then declined (Fig. 6). The effect of etorphine (0.1 μM, 5 min) was mediated by the opioid receptor as it was completely blocked by the opioid antagonist naloxone (10 μM, n = 8, p < 0.001) (Fig. 6). Pretreatment of the cells with PTX (100 ng/ml, 18 h) abolished the opioid effect (1 μM, n = 22, p < 0.005) (Fig. 6) indicating the involvement of Gi/Go proteins. To examine whether etorphine, similar to the cannabinoid agonist CP-55940, activated ERK in HEK-293 cells through metalloproteases and transactivation of VEGFR, we next tested the effects of o-phenanthroline and oxindole-1. When the effect of the metalloprotease inhibitor o-phenanthroline (100–200 μM, 30 min) was tested, we found that in 12 out of 19 sample quadruplets drawn from 5 separate experiments, o-phenanthroline did not affect the activation of ERK by etorphine, while in the rest of the samples only a partial inhibition was observed. Analysis of the results showed a significant effect of etorphine (p < 0.001) in the cells that were pretreated with ophenanthroline, although a small decrease (about 25%) was noted, compared to the effect of etorphine in vehicle-treated cells. When the effect of oxindol-1, the selective VEGFR inhibitor (3 μM, 10 min) was examined, we found that in 9 out of 12 comparisons oxindole-1 did not affect the activation of ERK by etorphine, and in the remaining samples only a partial inhibition of ERK phosphorylation was detected,

Fig. 6 – Phosphorylation of ERK1/2 by the opioid agonist etorphine (Et) in HEK-293 cells. Representative experiments illustrating the time-dependency of the effect of 1 μM etorphine (A), inhibition of the effect of 0.1 μM etorphine by the opioid antagonist naloxone (Nal, 10 μM), (B) and the elimination of the effect of 1 μM etorphine by PTX (100 ng/ml, 18 h). Histograms D (n = 8) and E (n = 22) present a quantitative analysis of these experiments. (*) denotes p < 0.01.

28

BR A IN RE S EA RCH 1 1 89 ( 20 0 8 ) 2 3 –32

PKC. We then tested the effect of the MEK inhibitor PD98059 (100 μM, 30 min, n = 11) on the activation of ERK by etorphine. To our surprise, in 7 out of 11 sample quadruplets drawn from 5 separate experiments, PD98059 failed to block the effect of etorphine, and in the other 4 samples only a partial inhibition was observed (Fig. 7). A statistical analysis of all 11 quadruplets failed to show a significant effect of PD98059 on the activation of ERK by etorphine. Although PD98059 is considered a specific inhibitor of MEK, there is evidence that this drug may sometimes enhance ERK1/2 phosphorylation (Cerioni et al., 2003). Therefore, in order to confirm the unexpected finding that the inhibition of MEK does not affect the activation of ERK by etorphine, another inhibitor of MEK, UO126 was tested. In 7 out of 10 comparisons the effect of etorphine was not affected by UO126 (10 μM, 30 min) while in the other 3 samples only partial inhibition was observed. Since we have shown that these two MEK inhibitors could antagonize the activation of ERK by the cannabinoid agonist in this cell-line (see above, and Fig. 4), the fact that these inhibitors failed to affect the activation of ERK by etorphine suggests that etorphine activates ERK by a different mechanism, which does not involve MEK. It should be noticed that the application of PD98059 or of UO126 resulted in a pronounced reduction in pERK (see Figs. 4 and 7). Similarly, the application of the tyrosine-phosphatase inhibitor vanadate (either 1 mM or 3 mM for 30 min) considerably elevated the level of pERK (by 300% or 500%, respectively) in these cells. These findings point to the ongoing phosphorylation and dephosphorylation that regulate the level of pERK in the cells under basal conditions (see Discussion). It is well accepted now that GPCRs can exist in multiple active conformations, and that different agonists can stabilize different active states of the receptor that lead to distinct signaling pathways (reviewed in (Kenakin, 2003; Reiter and Lefkowitz, 2006)). Hence, our next step was to test whether this

Fig. 7 – Phosphorylation of ERK1/2 in HEK-293 cells in the presence and absence of PD98059: the MEK blocker PD98059 reduces the ongoing phosphorylation of ERK, but failes to block the stimulation of ERK by etorphine (Et). A representative experiment is shown in (A), and a quantitative analysis of 11 quadruplets drawn from 5 experiments is presented in (B). PD98059 (PD, 100 μM) was introduced 30 min before the addition of 1 μM etorphine. (*) denotes p < 0.01.

unexpected MEK-independent ERK activation is unique to the opioid agonist etorphine, or whether it is also induced by another MOR agonist. DAMGO (0.1–1 μM), a selective agonist of MOR, increased ERK phosphorylation in HEK-293 cells. However, unlike the effect of etorphine, the effect of DAMGO was completely inhibited by the MEK inhibitor PD98059 (100 μM, 30 min, n = 6). It is therefore evident that activation of MOR in HEK-293 cells can result in the induction of the classic pathway for ERK phosphorylation via MEK, as the effect of DAMGO was abolished by the MEK inhibitor. The activation of ERK by etorphine, however, is mainly mediated by another, unknown yet, process (see Discussion). The next question was whether the effect of etorphine on ERK phosphorylation, which was not mediated by MEK, is unique to the μ-opioid receptor in HEK-293 cells, or whether in this cell-line, etorphine will activate another opioid receptor in a similar manner. To test this question, we used HEK-293 cells that were transfected with a rat δ-opioid receptor (DOR). Etorphine (1 μM, 5 min) induced the phosphorylation of ERK1/2 in these cells similar to its effect in MOR transfected cells. The MEK inhibitor PD98059 (100 μM, 30 min, n = 6) abolished the effect of etorphine, suggesting that etorphine induces ERK phosphorylation via DOR in HEK-293 cells by the classic pathway that involves MEK activation. The unexpected finding that etorphine activates ERK in this cell-line by a pathway that does not involve MEK is therefore unique to MOR.

3.

Discussion

The similarity between opioid and cannabinoid effects, both in-vivo and in-vitro, raises the possibility of interactions between these two groups of drugs. Indeed, several in-vivo studies described acute as well as chronic interactions. Opioid antagonists were found to block the effect of cannabinoids (Reche et al., 1996; Smith et al., 1998; Welch, 1994), and synergism between opioids and cannabinoids was reported (Cichewicz et al., 1999; Smith et al., 1998). Interactions at the level of the whole organism can result either from the sequential activation of opioid and endogenous cannabinoid systems or from interactions that take place at the cellular level. Indeed, we (Shapira et al., 1998, 2003) and others (Di Toro et al., 1998) have demonstrated cross-desensitization and cross- downregulation between opioid and cannabinoid agonists in cells that express opioid and cannabinoid receptors. As both opioid and cannabinoid agonists have been shown to regulate the ERK signaling pathway (see Introduction), it is possible that interactions between these two groups of drugs can also occur along the ERK cascade. Actually, it was recently suggested that opioid and cannabinoid agonists reciprocally inhibited ERK1/2 phosphorylation in HEK-293 cells (Rios et al., 2006). The goal of our study was to characterize and compare the signaling pathways leading from cannabinoid and opioid receptors to ERK1/2 phosphorylation, when both receptors are expressed in the same cell, in order to be able to identify new possible points of interactions between cannabinoid and opioid drugs at the cellular level. We have already shown that activation of the CB1 receptors in N18TG2 neuroblastoma cells resulted in ERK1/2 phosphorylation that was mediated by Gi/Go proteins and PKC and

BR A IN RE S E A RCH 1 1 89 ( 20 0 8 ) 2 3 –3 2

involved transactivation of VEGF receptors through matrix metalloproteases (Rubovitch et al., 2004). In the present study we show that the activation of opioid receptors by etorphine in the same cells induces ERK1/2 phosphorylation by a similar mechanism. The common pathway provides the mechanistic basis for interactions between cannabinoid and opioid drugs in relation to ERK activation in these cells. We further show in the present study that cannabinoids utilize VEGF receptors to activate ERK1/2 not only in N18TG2 cells but also in HEK-293 cells, thus pointing to VEGFRs as a common target of GPCRs activation. In recently published papers (Chen et al., 2006; Singleton et al., 2006) it was shown that MOR agonists induced VEGF receptors phosphorylation that resulted in increased microvascular endothelial cells proliferation and migration. Since opioids are widely used in cancer patients for their pain reducing properties and cannabinoids are used as antiemetic drugs in chemotherapy, this possible angiogenic effect should therefore be taken into consideration when prescribing these drugs. On the other hand, opioid and cannabinoid antagonists may prove useful therapeutically as inhibitors of angiogenesis. While the cannabinoid agonist CP55940 and the opioid agonist etorphine induced ERK1/2 phosphorylation by a similar mechanism in N18TG2 cells, which endogenously express cannabinoid and opioid receptors, these drugs were found to activate ERK by different signaling pathways in HEK293 cells transfected with either CB1 receptor or MOR. CP55940-induced ERK activation in HEK-293 cells occurred, as in N18TG2 cells, via Gi/Go mediated transactivation of VEGFR. Etorphine-induced ERK activation in HEK-293 cells was also mediated by Gi/Go proteins but was largely unaffected by the VEGFR antagonist oxindole-1 and the metalloprotease inhibitor o-phenanthroline, a fact that implied that the main pathway leading from the activation of MOR by etorphine to ERK phosphorylation does not involve transactivation of a RTK. We have further shown that etorphine-induced ERK phosphorylation did not involved PKC neither Src nor PI3K. A surprising result was the finding that the phosphorylation of ERK by etorphine was not abolished by the MEK inhibitors PD98059 and UO126, although MEK is the only kinase that is known, so far, to phosphorylate ERK. Hence, an alternative explanation for the phosphorylation of ERK by etorphine should be considered. The state of phosphorylation of a protein is regulated by both protein kinases and protein phosphatases. We have found that the basal levels of phosphorylated ERK in HEK-293 cells were greatly reduced by MEK inhibitors (Figs. 4 and 7), and therefore concluded that this pathway was constitutively active. Assuming that both kinases and phosphatases must be constitutively active in order to maintain a steady level of basal pERK, one can predict that inhibition of a phosphatase by etorphine will result in increased levels of pERK. Our finding that vanadate, the specific inhibitor of tyrosine- phosphatases, dramatically increased the phosphorylation of ERK1/2 in HEK-293 cells supports the possibility that such a mechanism can take place in these cells. Recently, Mao et al. (Mao et al., 2005) described a novel metabotrophic glutamate receptor 5-triggered signaling mechanism that involved inactivation of protein phosphatase 2A (PP2A) and resulted in the activation of ERK1/2. The authors found, however, that in their case the effect on ERK was indirect, and was probably mediated by decreased depho-

29

sphorylation of the upstream kinase MEK1/2, and thus was inhibited by the MEK inhibitors PD98059 and UO126. Since in our experiments the two MEK inhibitors did not affect etorphine-induced ERK phosphorylation, we must assume a mechanism in which etorphine activates a pathway that involves inhibition of dephosphorylation of ERK1/2 itself. The existence of such a signaling pathway was not investigated within the framework of the present study. There are reports in the literature, however, that link opioids to phosphatase inhibition (Liu et al., 2002; Sood and Mohanakumar, 1985). Interestingly, McLaughlin and Chavkin (McLaughlin and Chavkin, 2001) suggested that the conserved amino acid sequence surrounding tyrosine 166 in MOR has similarities to the key amino acids conferring protein tyrosine phosphatase substrate specificity, and may thus allow the binding of the phosphatase to the second cytoplasmic loop of MOR. It is therefore tempting to suggest that activation of MOR by etorphine may trigger a signaling pathway that involves inhibition of a phosphatase that is associated with the receptor. In contrast to etorphine that induced ERK1/2 phosphorylation in HEK-MOR cells by a process that was independent of MEK, the MOR-selective agonist DAMGO was found to activate ERK in the same cells by the classic process that involved MEK, as it was abolished by pretreatment with either PD98059 or UO126. These data indicate that distinct signaling pathways leading to ERK phosphorylation can be activated by different agonists of MOR in HEK-293 cells. The traditional two-state model of receptor activation, that assumes that the receptor exists in equilibrium between an active and an inactive state and that agonists stabilize the active conformation and promote G proteins activation, has been challenged in recent years. Theoretical considerations, supported by experimental data, led to the suggestion that multiple discrete active conformations of the receptor exist, and that different ligands will direct signaling through distinct transduction pathways by stabilizing one of these active conformations (Kenakin, 2003; Reiter and Lefkowitz, 2006), thus leading to agonistspecific effects (Berg et al., 1998; Gesty-Palmer et al., 2006; Johnson et al., 2006). Our results support the suggestion that MOR has more than one active state, as etorphine and DAMGO induced ERK1/2 phosphorylation by different cellular mechanisms. The signaling pathway that leads from the activation of MOR by etorphine to ERK phosphorylation seems to be unique to the μ-opioid receptor type, since in HEK-293 cells that were transfected with the δ-opioid receptor, etorphine induced the activation of ERK via MEK. The finding that MOR may have distinct ligand-specific active states should be further explored as it has important implications for future drug design. It may thus be possible to select for drugs that will activate only specific pathways, with more beneficial and less harmful properties.

4.

Experimental procedures

4.1.

Cell culture

N18TG2 neuroblastoma cells (passage number 25–40) were grown in 75 cm2 culture flasks in Dulbeco's modified Eagle's

30

BR A IN RE S EA RCH 1 1 89 ( 20 0 8 ) 2 3 –32

medium (DMEM) supplemented with 10% fetal calf serum, 2 mM L-glutamine, 40 U/ml penicillin and 40 μg/ml streptomycin, at 37 °C, in a humidified atmosphere of 5% CO2/95% air. For the experiments, confluent cultures were harvested with DMEM medium and the cells were seeded into 24-well plates. 24 h before the experiments the medium was changed to serum-free DMEM. HEK-293 cells were transfected with mouse CB1 receptor cDNA, or co-transfected with mouse CB1 receptor cDNA and either rat MOR cDNA or rat DOR cDNA as previously described (Shapira et al., 2001). Cells (5 × 10− 4 cells/ml) were seeded in 30 mm Petri dishes and were transfected 24 h later with 2 μg/ ml mouse CB1 receptor cDNA in pcDNA3 (Abood et al., 1997) alone or in combination with 2 ìg/ml of either rat MOR cDNA in pCMV-neo vector (Thompson et al., 1993) or with rat DOR cDNA in pcDNA3 vector (Abood et al., 1994). Selection was done in the presence of 1 mg/ml G418. The selected clones were grown in DMEM supplemented with 10% fetal calf serum, 2 mM L-glutamine, 40 U/ml penicillin, 40 μg/ml streptomycin and 0.25 mg/ml G418 (to prevent the growing of cells without the transfected vector) in 100 mm Petri dishes. At confluence, the cells were harvested with trypsin/EDTA and seeded for the experiments into polylysine-coated 24-well plates, in the same medium. 24 h before the experiments the medium was changed to serum-free medium.

4.2.

Phosphorylation of ERK1/2

Following drug treatment, cells were lysed with an ice cold solubilization buffer solution containing 50 mM HEPES, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 1.5 mM MgCl2, 2 mM phenylmethylsulfonylfluoride (PMSF), 1 μg/ml leupeptin and 100 μM sodium orthovanadate. Solubilized cell extracts were then centrifuged at 12,000×g for 10 min, and protein concentrations were determined with the Bradford Reagent (Sigma). 33 μl of the supernatants, that contained about 20 μg protein, were added to 10 μl of “sample buffer” (200 mM Tris, pH 6.8, 40% glycerol, 20% mercaptoethanol, 8% sodium dodecyl sulphate (SDS) and bromophenol blue) and subjected to Western blot analysis. Following separation on 8% SDS-polyacrylamide gels, the proteins were electrophoretically transferred to nitrocellulose membranes for immunoblotting. Phosphorylated ERK1/2 was detected by immunoblotting with mouse monoclonal anti-phospho-p44/42 MAPK (Santa Cruz, 1:1000). Subsequently, the membranes were incubated with horseradish peroxidase (HRP)-conjugated anti-mouse IgGs for anti pERK. Antibody binding was visualized by enhanced chemiluminescence (ECL) substrate for detecting HRP. In some experiments total ERK1/2 levels were determined after stripping by 10 min incubation in 0.1 M NaOH containing 0.2% SDS and then reprobing with a rabbit polyclonal antibody raised against total ERK (1:1000; Santa Cruz Biotechnology Inc.) (Rubovitch et al., 2004). Quantification of pERK2 levels was carried out by scanning the films with an optical scanner (BioImaging Systems, Rhenium, Israel) and then analyzing band intensities with TINA 2.07 software. The results were statistically analyzed by paired t-test and the p-value refers to the comparison between the effect of an agonist (opioid or cannabinoid) and its matched control (either vehicle or

blocker) that was run within the same experiment. The inhibitory effect of a blocker was determined by comparing the relative effect of the drug in the presence and absence of the blocker. When the effects of various blockers that were tested in different experiments were presented together, standardization of control in each experiment to 100% was carried out and the effect of the agonist was analyzed again using the non-parametric Wilcoxon test. Each experiment was repeated at least 3 times, and 1–4 samples of each treatment were drawn from each experiment. Comparison was carried out between matched pairs of samples (agonist vs. vehicle) or quadruplets of samples (agonist vs. vehicle in the presence and absence of a blocker) that were grown in the same plate and separated on the same gel, with “n” denoting number of comparisons (pairs or quadruplets, as specified).

4.3.

Materials

Etorphine, CP-55,940 and SR141716A were gifts from NIDA (USA). Naloxone hydrochloride was a gift from DuPont (USA). Pertussis toxin (PTX) was purchased from List Biological Laboratories (USA). Wortmannin, PD98059, chelerythrin, PP2, G418, oxindole-1, AG1296, AG1478, okadaic acid and sodium orthovanadate were obtained from Calbiochem (USA). 1,10phenanthroline, UO126, Poly-D-Lysine, DMEM and SDS were purchased from Sigma (USA). Leupeptin and PMSF were from Roche (Germany). Fetal calf serum, penicillin and streptomycin were acquired from Beit-Haemek Biological Industries (Israel). Mouse monoclonal antibodies against phospoERK1/2 were from Santa Cruz Biotechnology, CA (USA). Secondary antibodies were from Jackson ImmunoResearch Laboratories (USA). ECL, Supersignal West Pico Chemiluminescent Substrate was obtained from Pierce (USA). Films and nitrocellulose membranes were from Amersham (UK). Tissue culture flasks (75 cm2) and Petri dishes were from Corning (USA) and 24-well plates were purchased from Greiner (Germany).

Acknowledgments This research was supported by The Israel Science Foundation (grant no. 180/04) and by the Israel Anti-drug Authority.

REFERENCES

Abood, M.E., Noel, M.A., Farnsworth, J.S., Tao, Q., 1994. Molecular cloning and expression of a delta-opioid receptor from rat brain. J. Neurosci. Res. 37, 714–719. Abood, M.E., Ditto, K.E., Noel, M.A., Showalter, V.M., Tao, Q., 1997. Isolation and expression of a mouse CB1 cannabinoid receptor gene. Comparison of binding properties with those of native CB1 receptors in mouse brain and N18TG2 neuroblastoma cells. Biochem. Pharmacol. 53, 207–214. Ai, W., Gong, J., Yu, L., 1999. MAP kinase activation by mu opioid receptor involves phosphatidylinositol 3-kinase but not the cAMP/PKA pathway. FEBS Lett. 456, 196–200. Belcheva, M.M., Cosia, C.J., 2002. Diversity of G protein-coupled receptor signaling pathways to ERK/MAP kinase. NeuroSignals 11, 34–44.

BR A IN RE S E A RCH 1 1 89 ( 20 0 8 ) 2 3 –3 2

Belcheva, M.M., Szucs, M., Wang, D., Sadee, W., Coscia, C.J., 2001. mu-Opioid receptor-mediated ERK activation involves calmodulin-dependent epidermal growth factor receptor transactivation. J. Biol. Chem. 276, 33847–33853. Belcheva, M.M., Haas, P.D., Tan, Y., Heaton, V.M., Coscia, C.J., 2002. The fibroblast growth factor receptor is at the site of convergence between mu-opioid receptor and growth factor signaling pathways in rat C6 glioma cells. J. Pharmacol. Exp. Ther. 303, 909–918. Belcheva, M.M., Tan, Y., Heaton, V.M., Clark, A.L., Coscia, C.J., 2003. Mu opioid transactivation and down-regulation of the epidermal growth factor receptor in astrocytes: implications for mitogen-activated protein kinase signaling. Mol. Pharmacol. 64, 1391–1401. Berg, K.A., Maayani, S., Goldfarb, J., Scaramellini, C., Leff, P., Clarke, W.P., 1998. Effector pathway-dependent relative efficacy at serotonin type 2A and 2C receptors: evidence for agonistdirected trafficking of receptor stimulus. Mol. Pharmacol. 54, 94–104. Caunt, C.J., Finch, A.R., Sedgley, K.R., McArdle, C.A., 2006. Seven-transmembrane receptor signalling and ERK compartmentalization. Trends Endocrinol. Metab. 371, 1–8. Cerioni, L., Palomba, L., Cantoni, O., 2003. The Raf/MEK inhibitor PD98059 enhances ERK1/2 phosphorylation mediated by peroxynitrite via enforced mitochondrial formation of reactive oxygen species. FEBS Lett. 547, 92–96. Chen, C., Farooqui, M., Gupta, K., 2006. Morphine stimulates vascular endothelial growth factor-like signaling in mouse retinal endothelial cells. Curr. Neurovascular Res. 3, 171–180. Childers, R., 1991. Opioid receptor coupled second messenger systems. Life Sci. 48, 1991–2003. Cichewicz, D.L., Martin, Z.L., Smith, F.L., Welch, S.P., 1999. Enhancement mu opioid antinociception by oral delta9-tetrahydrocannabinol: dose–response analysis and receptor identification. J. Pharmacol. Exp. Ther. 289, 859–867. Davis, M.I., Ronesi, J., Lovinger, D.M., 2003. A predominant role for inhibition of the adenylate cyclase/protein kinase A pathway in ERK activation by cannabinoid receptor 1 in N1E-115 neuroblastoma cells. J. Biol. Chem. 278, 48973–48980. Della Rocca, G.J., Maudsley, S., Daaka, Y., Lefkowitz, R.J., Luttrell, L.M., 1999. Pleiotropic coupling of G protein-coupled receptors to the mitogen-activated protein kinase cascade. Role of focal adhesions and receptor tyrosine kinases. J. Biol. Chem. 274, 13978–13984. Derkinderen, P., Valjent, E., Toutant, M., Corvol, J.C., Enslen, H., Ledent, C., Trzaskos, J., Caboche, J., Girault, J.A., 2003. Regulation of extracellular signal-regulated kinase by cannabinoids in hippocampus. J. Neurosci. 23, 2371–2382. Di Toro, R., Campana, G., Sciarretta, V., Murari, G., Spampinato, S., 1998. Regulation of delta opioid receptors by delta9-tetrahydrocannabinol in NG108-15 hybrid cells. Life Sci. 63, PL197–PL204. Eisinger, D.A., Schulz, R., 2004. Extracellular signal-regulated kinase/mitogen-activated protein kinases block internalization of delta-opioid receptors. J. Pharmacol. Exp. Ther. 309, 776–785. Eitan, S., Bryant, C.D., Saliminejad, N., Yang, Y.C., Vojdani, E., Keith Jr, D., Polakiewicz, R., Evans, C.J., 2003. Brain region-specific mechanisms for acute morphine-induced mitogen-activated protein kinase modulation and distinct patterns of activation during analgesic tolerance and locomotor sensitization. J. Neurosci. 23, 8360–8369. Galve-Roperh, I., Rueda, D., Gomez del Pulgar, T., Velasco, G., Guzman, M., 2002. Mechanism of extracellular signal-regulated kinase activation by the CB(1) cannabinoid receptor. Mol. Pharmacol. 62, 1385–1392. Gavi, S., Shumay, E., Wang, H.Y., Malbon, C.C., 2006. G-protein-coupled receptors and tyrosine kinases: crossroads in cell signaling and regulation. Trends Endocrinol. Metab. 17, 48–54.

31

Gesty-Palmer, D., Chen, M., Reiter, E., Ahn, S., Nelson, C.D., Wang, S., Eckhardt, A.E., Cowan, C.L., Spurney, R.F., Luttrell, L.M., Lefkowitz, R.J., 2006. Distinct beta-arrestin- and G protein-dependent pathways for parathyroid hormone receptor-stimulated ERK1/2 activation. J. Biol. Chem. 281, 10856–10864. Guzman, M., Sanchez, C., Galve-Roperh, I., 2001. Control of the cell survival/death decision by cannabinoids. J. Mol. Med. 78, 613–625. Howlett, A.C., 1995. Cannabinoiod compounds and signal transduction mechanisms. In: Pertwee, R.G. (Ed.), Cannabinoid receptors. Academic Press, pp. 169–204. Johnson, E.A., Oldfield, S., Braksator, E., Gonzalez-Cuello, A., Couch, D., Hall, K.J., Mundell, S.J., Bailey, C.P., Kelly, E., Henderson, G., 2006. Agonist-selective mechanisms of mu-opioid receptor desensitization in human embryonic kidney 293 cells. Mol. Pharmacol. 70, 676–685. Kenakin, T., 2003. Ligand-selective receptor conformations revisited: the promise and the problem. Trends Pharmacol. Sci. 24, 346–354. Kramer, H.K., Onoprishvili, I., Andria, M.L., Hanna, K., Sheinkman, K., Haddad, L.B., Simon, E.J., 2002. Delta opioid activation of the mitogen-activated protein kinase cascade does not require transphosphorylation of receptor tyrosine kinases. BMC Pharmacol. 2, 5. Law, P.Y., Wong, Y.H., Loh, H.H., 2000. Molecular mechanisms and regulation of opioid receptor signaling. Annu. Rev. Pharmacol. Toxicol. 40, 389–430. Lefkowitz, R.J., Shenoy, S.K, 2005. Transduction of receptor signals by beta-arrestins. Science 308, 512–517. Lefkowitz, R.J., Pierce, K.L., Luttrell, L.M., 2002. Dancing with different partners: protein kinase a phosphorylation of seven membrane-spanning receptors regulates their G protein-coupling specificity. Mol. Pharmacol. 62, 971–974. Liu, Q.R., Zhang, P.W., Zhen, Q., Walther, D., Wang, X.B., Uhl, G.R., 2002. KEPI, a PKC-dependent protein phosphatase 1 inhibitor regulated by morphine. J. Biol. Chem. 277, 13312–13320. Loh, H.H., Smith, A.P., 1990. Molecular characterization of opioid receptors. Annu. Rev. Pharmacol. Toxicol. 30, 123–147. Mao, L., Yang, L., Arora, A., Choe, E.S., Zhang, G., Liu, Z., Fibuch, E.E., Wang, J.Q., 2005. Role of protein phosphatase 2A in mGluR5-regulated MEK/ERK phosphorylation in neurons. J. Biol. Chem. 280, 12602–12610. McLaughlin, J.P., Chavkin, C., 2001. Tyrosine phosphorylation of the mu-opioid receptor regulates agonist intrinsic efficacy. Mol. Pharmacol. 59, 1360–1368. Persson, A.I., Thorlin, T., Bull, C., Eriksson, P.S., 2003. Opioid-induced proliferation through the MAPK pathway in cultures of adult hippocampal progenitors. Mol. Cell. Neurosci. 23, 360–372. Pertwee, R.G., 1997. Pharmacology of cannabinoid CB1 and CB2 receptors. Pharmacol. Ther. 74, 129–180. Pickel, V.M., Chan, J., Kash, T.L., Rodriguez, J.J., MacKie, K., 2004. Compartment-specific localization of cannabinoid 1 (CB1) and mu-opioid receptors in rat nucleus accumbens. Neuroscience 127, 101–112. Reche, I., Fuentes, J.A., Ruiz-Gayo, M., 1996. A role for central cannabinoid and opioid systems in peripheral delta 9-tetrahydrocannabinol-induced analgesia in mice. [erratum appears in Eur. J. Pharmacol. 1996 Oct 3;312(3):391] Eur. J. Pharmacol. 301, 75–81. Reisine, T., Brownstein, M.J., 1994. Opioid and cannabinoid receptors. Curr. Opin. Neurobiol. 4, 406–412. Reiter, E., Lefkowitz, R.J., 2006. GRKs and beta-arrestins: roles in receptor silencing, trafficking and signaling. Trends Endocrinol. Metab. 17, 159–165. Rios, C., Gomes, I., Devi, L.A., 2006. mu opioid and CB1 cannabinoid receptor interactions: reciprocal inhibition of receptor signaling and neuritogenesis.[see comment]. Br. J. Pharmacol. 148, 387–395.

32

BR A IN RE S EA RCH 1 1 89 ( 20 0 8 ) 2 3 –32

Rodriguez, J.J., Mackie, K., Pickel, V.M., 2001. Ultrastructural localization of the CB1 cannabinoid receptor in mu-opioid receptor patches of the rat caudate putamen nucleus. J. Neurosci. 21, 823–833. Rubino, T., Forlani, G., Vigano, D., Zippel, R., Parolaro, D., 2004. Modulation of extracellular signal-regulated kinases cascade by chronic delta 9-tetrahydrocannabinol treatment. Mol. Cell. Neurosci. 25, 355–362. Rubino, T., Forlani, G., Vigano, D., Zippel, R., Parolaro, D., 2005. Ras/ERK signalling in cannabinoid tolerance: from behaviour to cellular aspects. J. Neurochem. 93, 984–991. Rubovitch, V., Gafni, M., Sarne, Y., 2004. The involvement of VEGF receptors and MAPK in the cannabinoid potentiation of Ca2+ flux into N18TG2 neuroblastoma cells. Brain Res. Mol. Brain Res. 120, 138–144. Salio, C., Fischer, J., Franzoni, M.F., Mackie, K., Kaneko, T., Conrath, M., 2001. CB1-cannabinoid and mu-opioid receptor co-localization on postsynaptic target in the rat dorsal horn. NeuroReport 12, 3689–3692. Sanchez, M.G., Ruiz-Llorente, L., Sanchez, A.M., Diaz-Laviada, I., 2003. Activation of phosphoinositide 3-kinase/PKB pathway by CB(1) and CB(2) cannabinoid receptors expressed in prostate PC-3 cells. Involvement in Raf-1 stimulation and NGF induction. Cell Signal 15, 851–859. Schmidt, H., Schulz, S., Klutzny, M., Koch, T., Handel, M., Hollt, V., 2000. Involvement of mitogen-activated protein kinase in agonist-induced phosphorylation of the mu-opioid receptor in HEK 293 cells. J. Neurochem. 74, 414–422. Shahabi, N.A., Daaka, Y., McAllen, K., Sharp, B.M., 1999. Delta opioid receptors expressed by stably transfected Jurkat cells signal through the map kinase pathway in a ras-independent manner. J. Neuroimmunol. 94, 48–57. Shapira, M., Gafni, M., Sarne, Y., 1998. Independence of, and interactions between, cannabinoid and opioid signal

transduction pathways in N18TG2 cells. Brain Res. 806, 26–35. Shapira, M., Keren, O., Gafni, M., Sarne, Y., 2001. Divers pathways mediate delta-opioid receptor down regulation within the same cell. Brain Res. Mol. Brain Res. 96, 142–150. Shapira, M., Gafni, M., Sarne, Y., 2003. Long-term interactions between opioid and cannabinoid agonists at the cellular level: cross-desensitization and downregulation. Brain Res. 960, 190–200. Singleton, P.A., Lingen, M.W., Fekete, M.J., Garcia, J.G., Moss, J., 2006. Methylnaltrexone inhibits opiate and VEGF-induced angiogenesis: role of receptor transactivation. Microvasc. Res. 72, 3–11. Smith, F.L., Cichewicz, D., Martin, Z.L., Welch, S.P., 1998. The enhancement of morphine antinociception in mice by delta9-tetrahydrocannabinol. Pharmacol. Biochem. Behav. 60, 559–566. Sood, P.P., Mohanakumar, K.P., 1985. Inhibition of acid and alkaline phosphatases in the central nervous system of mice during morphine dependence development, withdrawal and naloxone administration. Cell. Mol. Biol. 31, 469–473. Tegeder, I., Geisslinger, G., 2004. Opioids as modulators of cell death and survival–unraveling mechanisms and revealing new indications. Pharmacol. Rev. 56, 351–369. Thompson, R.C., Mansour, A., Akil, H., Watson, S.J., 1993. Cloning and pharmacological characterization of a rat mu opioid receptor. Neuron 11, 903–913. Vigano, D., Rubino, T., Parolaro, D., 2005. Molecular and cellular basis of cannabinoid and opioid interactions. Pharmacol. Biochem. Behav. 81, 360–368. Welch, S.P., 1994. Blockade of cannabinoid-induced antinociception by naloxone benzoylhydrazone (NalBZH). Pharmacol. Biochem. Behav. 49, 929–934.