Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Comparative Study
. 2012 Oct;63(5):905-15.
doi: 10.1016/j.neuropharm.2012.06.046. Epub 2012 Jul 4.

AM-251 and rimonabant act as direct antagonists at mu-opioid receptors: implications for opioid/cannabinoid interaction studies

Kathryn A Seely  1 , Lisa K Brents, Lirit N Franks, Maheswari Rajasekaran, Sarah M Zimmerman, William E Fantegrossi, Paul L Prather
Affiliations
Comparative Study

AM-251 and rimonabant act as direct antagonists at mu-opioid receptors: implications for opioid/cannabinoid interaction studies

Kathryn A Seely et al. Neuropharmacology. 2012 Oct.

Abstract

Mu-opioid and CB1-cannabinoid agonists produce analgesia; however, adverse effects limit use of drugs in both classes. Additive or synergistic effects resulting from concurrent administration of low doses of mu- and CB1-agonists may produce analgesia with fewer side effects. Synergism potentially results from interaction between mu-opioid receptors (MORs) and CB1 receptors (CB1Rs). AM-251 and rimonabant are CB1R antagonist/inverse agonists employed to validate opioid-cannabinoid interactions, presumed to act selectively at CB1Rs. Therefore, the potential for direct action of these antagonists at MORs is rarely considered. This study determined if AM-251 and/or rimonabant directly bind and modulate the function of MORs. Surprisingly, AM-251 and rimonabant, but not a third CB1R inverse agonist AM-281, bind with mid-nanomolar affinity to human MORs with a rank order of affinity (K(i)) of AM-251 (251 nM) > rimonabant (652 nM) > AM281 (2135 nM). AM-251 and rimonabant, but not AM-281, also competitively antagonize morphine induced G-protein activation in CHO-hMOR cell homogenates (K(b) = 719 or 1310 nM, respectively). AM-251 and rimonabant block morphine inhibition of cAMP production, while only AM-251 elicits cAMP rebound in CHO-hMOR cells chronically exposed to morphine. AM-251 and rimonabant (10 mg/kg) attenuate morphine analgesia, whereas the same dose of AM-281 produces little effect. Therefore, in addition to high CB1R affinity, AM-251 and rimonabant bind to MORs with mid-nanomolar affinity and at higher doses may affect morphine analgesia via direct antagonism at MORs. Such CB1-independent of these antagonists effects may contribute to reported inconsistencies when CB1/MOR interactions are examined via pharmacological methods in CB1-knockout versus wild-type mice.

PubMed Disclaimer

Figures

Figure 1
Figure 1. Chemical structures of test compounds used in this study
The colored boxes highlight structural differences between the drugs examined.
Figure 2
Figure 2. Determination of the affinity of test compounds for mMORs, mCB1Rs and hCB2s by competition receptor binding performed with membrane homogenates
Specific binding was determined as described in the Materials and Methods by incubating 0.1 nM of [3H]CP 55,950 or 1 nM of [3H]DAMGO with increasing concentrations of A) morphine, B) AM-251 or C) AM-281 and 100 μg of membranes prepared from mouse brains or CHO-hCB2 cells. The Cheng-Prusoff equation (Cheng and Prusoff, 1973) was used to convert the experimental IC50 values obtained from competition receptor binding experiments to Ki values, a quantitative measure of receptor affinity (presented in Table 1).
Figure 3
Figure 3. Modulation of basal and morphine-stimulated G-protein activity by test compounds utilizing [35S]GTPγS binding in mouse brain homogenates
A) Mouse brain membranes (10 μg) were incubated in the presence of 0.1 nM [35S]GTPγS with increasing concentrations of morphine (filled squares), AM-251 (open triangles) or AM-281 (open squares) as described in the Materials and Methods. B) The effect of AM-251 (1 μM) alone or in the presence of 1 μM of the neutral MOR antagonist naloxone (NX), 1 μM of the neutral CB1R antagonist O-2050 or both naloxone and O-2050 in combination on basal G-protein activity in mouse brain membranes was determined. The reduction in basal G-protein activity produced by AM-251 alone was not significantly attenuated by co-incubation with any drug or drug combination. Values designated with different letters above the error bars are significantly different (P<0.05, one-way ANOVA followed by a Newman-Keuls post-hoc test, mean ± SEM). C) Morphine produced a concentration-related activation of G-proteins in mouse membrane homogenates that was significantly shifted-to-the-right by co-incubation with AM-251 (10 μM), but not AM-281 (10 μM). Data are expressed as percent specific [35S]GTPγS binding normalized to the average maximal response produced by morphine and individual ED50 values are discussed in the Results section.
Figure 4
Figure 4. Determination of the affinity of test compounds for hMORs stably expressed in CHO cells by competition receptor binding performed with membrane homogenates
Specific binding was determined as described in the Materials and Methods by incubating 1 nM of [3H]DAMGO with increasing concentrations of morphine (filled squares), AM-251 (open triangles), rimonabant (open circles) or AM-281 (open squares) and 100 μg of membranes prepared from CHO-hMOR cells. The Cheng-Prusoff equation (Cheng and Prusoff, 1973) was used to convert the experimental IC50 values obtained from competition receptor binding experiments to Ki values, a quantitative measure of receptor affinity (presented in Table 1).
Figure 5
Figure 5. Modulation of basal G-protein activity by test compounds utilizing [35S]GTPγS binding in CHO-hMOR cell homogenates
A) CHO-hMOR membranes (10 μg) were incubated in the presence of 0.1 nM [35S]GTPγS with increasing concentrations of morphine (filled squares), AM-251 (open triangles), rimonabant (open circles) or AM-281 (open squares) as described in the Materials and Methods. B) The effect of morphine (100 nM), AM-251 (1 μM), rimonabant (1 μM) or AM-281 (1 μM) alone or in the presence of 1 μM of the neutral MOR antagonist naloxone (NX) on basal G-protein activity in membranes prepared from CHO-hMOR cells was determined. G-protein stimulation by morphine was significantly antagonized by naloxone; however, the reduction in basal G-protein activity produced by AM-251, rimonabant or AM-281 alone was not significantly attenuated by co-incubation with naloxone. An asterisk (*) above the histogram denotes a significant difference between the mean value of the indicate drug alone and the drug in the presence of naloxone (P<0.05, Student’s t-test, mean ± SEM). C) Morphine (10 μM) and naloxone (10 μM) alone produce no effect on basal G-protein activity, but AM-251 (10 μM), rimonabant (10 μM) and AM-281 (10 μM) all produce approximately 20% inhibition of GTPγS binding in membranes prepared from CHO-WT cells. Values designated with different letters above the error bars are significantly different (P<0.05, one-way ANOVA followed by a Newman-Keuls post-hoc test, mean ± SEM).
Figure 6
Figure 6. Modulation of morphine-stimulated G-protein activity by test compounds utilizing [35S]GTPγS binding in CHO-hMOR cell homogenates
Morphine produced a concentration-related activation of G-proteins in CHO-hMOR membrane homogenates that was significantly shifted-to-the-right by co-incubation with rimonabant (10 μM; Panel B) and AM-251 (1 and 10 μM; Panel C), but not by AM-281 (10 μM; Panel A). AM-251 competitively antagonizes G-protein activation by morphine with a Schild slope of 1.02 and a Kb value of 719 nM (Panel C; inset). Data are expressed as percent specific [35S]GTPγS binding normalized to the average maximal response produced by morphine and individual ED50 values are discussed in the Results section.
Figure 7
Figure 7. Modulation of forskolin-stimulated adenylyl cyclase activity by acute administration of test compounds in intact CHO-hMOR cells
A) Intact CHO-hMOR cells, pre-loaded with [3H]adenine, were incubated with forskolin (10 μM) and increasing concentrations of morphine (filled squares) as described in the Materials and Methods. B) Naloxone (1 μM), AM-251 (10 μM), rimonabant (10 μM) or AM-281 (10 μM) administered alone had no significant effect on forkolin-stimulated cAMP levels. C) Inhibition of adenylyl cyclase activity produced by 10 nM morphine was significantly attenuated by co-incubation with either the neutral MOR antagonist naloxone (1 μM), AM-251 (10 μM), rimonabant (10 μM) or AM-281 (10 μM). Values designated with different letters above the error bars are significantly different (P<0.05, one-way ANOVA followed by a Newman-Keuls post-hoc test, mean ± SEM).
Figure 8
Figure 8. Modulation of forskolin-stimulated adenylyl cyclase activity by test compounds following chronic morphine exposure
Following overnight exposure to 1 μM morphine, adenylyl cyclase inhibition by morphine (100 nM) was reduced (e.g., desensitization), while levels of cAMP were elevated above baseline by naloxone (1 μM) and AM-251 (10 μM), but not by rimonabant (10 μM) or AM281 (10 μM) (e.g., cAMP rebound). Data are expressed as the percent control of forskolin-stimulated cAMP production. Values designated with different letters above the error bars are significantly different (P<0.05, one-way ANOVA followed by a Newman-Keuls post-hoc test, mean ± SEM).
Figure 9
Figure 9. Antagonism of in vivo morphine analgesia in two different strains of mice by test compounds utilizing the tail-flick procedure
A) Morphine was administered subcutaneously (s.c.) to B6/SJL mice in doses ranging from 0.5 to 10 mg/kg to determine an ED50 for analgesia of 4.2 ± 0.61 mg/kg, using the tail-flick technique. Thirty-min pre-treatment of B) B6SJL or C) C57BL/6J mice by intra-peritoneal (i.p.) injection of naloxone (4 mg/kg) or AM-251 (10 mg/kg), but not AM-281 (10 mg/kg) significantly reduced analgesia produced by 5 mg/kg morphine. The test doses of naloxone, AM-251, rimonabant or AM-281 had no effect on basal tail-flick latencies when administered alone (data not shown). All data are expressed as the percent of maximum possible effect (% MPE). Values designated with different letters above the error bars are significantly different (P<0.05, one-way ANOVA followed by a Newman-Keuls post-hoc test, mean ± SEM).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Atwood BK, Straiker A, Mackie K. CB(2) cannabinoid receptors inhibit synaptic transmission when expressed in cultured autaptic neurons. Neuropharmacol. 2012 Epub ahead of print. - PMC - PubMed
    1. Avidor-Reiss T, Bayewitch M, Levy R, Matus-Leibovitch N, Nevo I, Vogel Z. Adenylyl cyclase supersensitization in mu-opioid receptor-transfected Chinese hamster ovary cells following chronic opioid treatment. J Biol Chem. 1995;270:29732–29738. - PubMed
    1. Bouaboula M, Poinot-Chazel C, Bourrie B, Canat X, Calandra B, Rinaldi-Carmona M, Le Fur G, Casellas P. Activation of mitogen-activated protein kinases by stimulation of the central cannabinoid receptor CB1. Biochem J. 1995;312 (Pt 2):637–641. - PMC - PubMed
    1. Bouaboula M, Poinot-Chazel C, Marchand J, Canat X, Bourrie B, Rinaldi-Carmona M, Calandra B, Le Fur G, Casellas P. Signaling pathway associated with stimulation of CB2 peripheral cannabinoid receptor. Involvement of both mitogen-activated protein kinase and induction of Krox-24 expression. Eur J Biochem. 1996;237:704–711. - PubMed
    1. Brents LK, Reichard EE, Zimmerman SM, Moran JH, Fantegrossi WE, Prather PL. Phase I hydroxylated metabolites of the K2 synthetic cannabinoid JWH-018 retain in vitro and in vivo cannabinoid 1 receptor affinity and activity. PLoS One. 2011;6:c21917. - PMC - PubMed

Publication types

MeSH terms