AM630 behaves as a protean ligand at the human cannabinoid CB2 receptor

Daniele Bolognini, Maria Grazia Cascio, Daniela Parolaro, Roger G Pertwee

Research output: Contribution to journalArticle

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Abstract

BACKGROUND AND PURPOSE We have investigated how pre-incubating hCB(2) CHO cells with the CB(2) receptor antagonists/inverse agonists, AM630 and SR144528, affects how these and other ligands target hCB(2) receptors in these cells or their membranes. EXPERIMENTAL APPROACH We tested the ability of AM630, SR144528 and of the CB(1) /CB(2) receptor agonists, CP55940 and R-(+)-WIN55212, to modulate forskolin-stimulated cAMP production in hCB(2) CHO cells or [(35) S]-GTP¿S binding to membranes prepared from these cells, or to displace [(3) H]-CP55940 from whole cells and membranes. Assays were also performed with the CB(2) receptor partial agonist, ¿(9) -tetrahydrocannabivarin. Some cells were pre-incubated with AM630 or SR144528 and then washed extensively. KEY RESULTS AM630 behaved as a low-potency neutral competitive antagonist in AM630-pre-incubated cells, a low-potency agonist in SR144528-pre-incubated cells, and a much higher-potency inverse agonist/antagonist in vehicle-pre-incubated cells. AM630 pre-incubation (i) reduced the inverse efficacy of SR144528 without abolishing it; (ii) increased the efficacy of ¿(9) -tetrahydrocannabivarin; and (iii) did not affect the potency with which AM630 displaced [(3) H]-CP55940 from whole cells or its inverse agonist potency and efficacy in the [(35) S]-GTP¿S membrane assay. CONCLUSIONS AND IMPLICATIONS These results suggest that AM630 is a protean ligand that can target a constitutively active form of the hCB(2) receptor (R*) with low affinity to produce agonism or neutral antagonism and a constitutively inactive form of this receptor (R) with much higher affinity to produce inverse agonism, and that the constitutive activity of whole cells is decreased less by pre-incubation with AM630 than with the higher-efficacy inverse agonist, SR144528. LINKED ARTICLES This article is part of a themed section on Cannabinoids in Biology and Medicine. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.165.issue-8. To view Part I of Cannabinoids in Biology and Medicine visit http://dx.doi.org/10.1111/bph.2011.163.issue-7.
Original languageEnglish
Pages (from-to)2561-2574
Number of pages14
JournalBritish Journal of Pharmacology
Volume165
Issue number8
Early online date23 Mar 2012
DOIs
Publication statusPublished - Apr 2012

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Cannabinoid Receptor CB2
Ligands
Cannabinoids
CHO Cells
Cell Membrane
Medicine
iodopravadoline
Aptitude
Membranes
Colforsin
SR 144528

Keywords

  • cannabinoid CB2 receptor
  • AM630
  • SR144528
  • CP55940
  • ¿9-tetrahydrocannabivarin
  • R-(+)-WIN55212
  • inverse agonism
  • neutral antagonism
  • two-state model of agonism and inverse agonism
  • protean ligand

Cite this

AM630 behaves as a protean ligand at the human cannabinoid CB2 receptor. / Bolognini, Daniele; Cascio, Maria Grazia; Parolaro, Daniela; Pertwee, Roger G.

In: British Journal of Pharmacology, Vol. 165, No. 8, 04.2012, p. 2561-2574.

Research output: Contribution to journalArticle

Bolognini, Daniele ; Cascio, Maria Grazia ; Parolaro, Daniela ; Pertwee, Roger G. / AM630 behaves as a protean ligand at the human cannabinoid CB2 receptor. In: British Journal of Pharmacology. 2012 ; Vol. 165, No. 8. pp. 2561-2574.
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T1 - AM630 behaves as a protean ligand at the human cannabinoid CB2 receptor

AU - Bolognini, Daniele

AU - Cascio, Maria Grazia

AU - Parolaro, Daniela

AU - Pertwee, Roger G

N1 - © 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.

PY - 2012/4

Y1 - 2012/4

N2 - BACKGROUND AND PURPOSE We have investigated how pre-incubating hCB(2) CHO cells with the CB(2) receptor antagonists/inverse agonists, AM630 and SR144528, affects how these and other ligands target hCB(2) receptors in these cells or their membranes. EXPERIMENTAL APPROACH We tested the ability of AM630, SR144528 and of the CB(1) /CB(2) receptor agonists, CP55940 and R-(+)-WIN55212, to modulate forskolin-stimulated cAMP production in hCB(2) CHO cells or [(35) S]-GTP¿S binding to membranes prepared from these cells, or to displace [(3) H]-CP55940 from whole cells and membranes. Assays were also performed with the CB(2) receptor partial agonist, ¿(9) -tetrahydrocannabivarin. Some cells were pre-incubated with AM630 or SR144528 and then washed extensively. KEY RESULTS AM630 behaved as a low-potency neutral competitive antagonist in AM630-pre-incubated cells, a low-potency agonist in SR144528-pre-incubated cells, and a much higher-potency inverse agonist/antagonist in vehicle-pre-incubated cells. AM630 pre-incubation (i) reduced the inverse efficacy of SR144528 without abolishing it; (ii) increased the efficacy of ¿(9) -tetrahydrocannabivarin; and (iii) did not affect the potency with which AM630 displaced [(3) H]-CP55940 from whole cells or its inverse agonist potency and efficacy in the [(35) S]-GTP¿S membrane assay. CONCLUSIONS AND IMPLICATIONS These results suggest that AM630 is a protean ligand that can target a constitutively active form of the hCB(2) receptor (R*) with low affinity to produce agonism or neutral antagonism and a constitutively inactive form of this receptor (R) with much higher affinity to produce inverse agonism, and that the constitutive activity of whole cells is decreased less by pre-incubation with AM630 than with the higher-efficacy inverse agonist, SR144528. LINKED ARTICLES This article is part of a themed section on Cannabinoids in Biology and Medicine. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.165.issue-8. To view Part I of Cannabinoids in Biology and Medicine visit http://dx.doi.org/10.1111/bph.2011.163.issue-7.

AB - BACKGROUND AND PURPOSE We have investigated how pre-incubating hCB(2) CHO cells with the CB(2) receptor antagonists/inverse agonists, AM630 and SR144528, affects how these and other ligands target hCB(2) receptors in these cells or their membranes. EXPERIMENTAL APPROACH We tested the ability of AM630, SR144528 and of the CB(1) /CB(2) receptor agonists, CP55940 and R-(+)-WIN55212, to modulate forskolin-stimulated cAMP production in hCB(2) CHO cells or [(35) S]-GTP¿S binding to membranes prepared from these cells, or to displace [(3) H]-CP55940 from whole cells and membranes. Assays were also performed with the CB(2) receptor partial agonist, ¿(9) -tetrahydrocannabivarin. Some cells were pre-incubated with AM630 or SR144528 and then washed extensively. KEY RESULTS AM630 behaved as a low-potency neutral competitive antagonist in AM630-pre-incubated cells, a low-potency agonist in SR144528-pre-incubated cells, and a much higher-potency inverse agonist/antagonist in vehicle-pre-incubated cells. AM630 pre-incubation (i) reduced the inverse efficacy of SR144528 without abolishing it; (ii) increased the efficacy of ¿(9) -tetrahydrocannabivarin; and (iii) did not affect the potency with which AM630 displaced [(3) H]-CP55940 from whole cells or its inverse agonist potency and efficacy in the [(35) S]-GTP¿S membrane assay. CONCLUSIONS AND IMPLICATIONS These results suggest that AM630 is a protean ligand that can target a constitutively active form of the hCB(2) receptor (R*) with low affinity to produce agonism or neutral antagonism and a constitutively inactive form of this receptor (R) with much higher affinity to produce inverse agonism, and that the constitutive activity of whole cells is decreased less by pre-incubation with AM630 than with the higher-efficacy inverse agonist, SR144528. LINKED ARTICLES This article is part of a themed section on Cannabinoids in Biology and Medicine. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.165.issue-8. To view Part I of Cannabinoids in Biology and Medicine visit http://dx.doi.org/10.1111/bph.2011.163.issue-7.

KW - cannabinoid CB2 receptor

KW - AM630

KW - SR144528

KW - CP55940

KW - ¿9-tetrahydrocannabivarin

KW - R-(+)-WIN55212

KW - inverse agonism

KW - neutral antagonism

KW - two-state model of agonism and inverse agonism

KW - protean ligand

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DO - 10.1111/j.1476-5381.2011.01503.x

M3 - Article

VL - 165

SP - 2561

EP - 2574

JO - British Journal of Pharmacology

JF - British Journal of Pharmacology

SN - 0007-1188

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ER -