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Journal of Basic and Clinical Physiology and Pharmacology

Editor-in-Chief: Horowitz, Michal

Editorial Board: Das, Kusal K. / Epstein, Yoram / S. Gershon MD, Elliot / Kodesh , Einat / Kohen, Ron / Lichtstein, David / Maloyan, Alina / Mechoulam, Raphael / Roth, Joachim / Schneider, Suzanne / Shohami, Esther / Sohmer, Haim / Yoshikawa, Toshikazu / Tam, Joseph


CiteScore 2016: 1.01

SCImago Journal Rank (SJR) 2016: 0.349
Source Normalized Impact per Paper (SNIP) 2016: 0.495

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2191-0286
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Volume 27, Issue 3

Issues

CB1 cannabinoid receptor-mediated increases in cyclic AMP accumulation are correlated with reduced Gi/o function

Khalil Eldeeb
  • Department of Physiology and Pharmacology, Wake Forest School of Medicine, Winston-Salem, NC, USA
  • ALAzhar Faculty of Medicine, New Damietta, Egypt
  • Other articles by this author:
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/ Sandra Leone-Kabler
  • Department of Physiology and Pharmacology, Wake Forest School of Medicine, Winston-Salem, NC, USA
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/ Allyn C. Howlett
  • Corresponding author
  • Department of Physiology and Pharmacology, Wake Forest School of Medicine, One Medical Center Blvd., Winston-Salem, NC 27157, USA, Phone: +1-336-716-8545
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Published Online: 2016-04-18 | DOI: https://doi.org/10.1515/jbcpp-2015-0096

Abstract

Background: CB1 cannabinoid receptors (CB1Rs) stimulate Gi/o-dependent signaling pathways. CB1R-mediated cAMP increases were proposed to result from Gs activation, but CB1R-stimulated GTPγS binding to Gs has not heretofore been investigated.

Methods: Three models of CB1R-stimulated cAMP production were tested: pertussis toxin disruption of Gi/o in N18TG2 cells; L341A/A342L-CB1R expressed in Chinese hamster ovary (CHO) cells; and CB1 and D2 dopamine receptors endogenously co-expressed in MN9D cells. cAMP was assayed by [3H]cAMP binding competition. G protein activation was assayed by the antibody-targeted scintillation proximity assay.

Results: In L341A/A342L-CB1-CHO cells, cannabinoid agonists significantly stimulated cAMP accumulation over vehicle; (–)-3-[2-hydroxyl-4-(1,1-dimethylheptyl)phenyl]-4-[3-hydroxyl propyl] cyclohexan-1-ol (CP55940)-stimulated [35S]GTPγS binding to Gi1/2/3 was reversed, whereas binding to Gs was not different from CB1R. In MN9D cells, CB1 agonist HU210 or D2 agonist quinpirole alone inhibited forskolin-activated cAMP accumulation, whereas HU210 plus quinpirole increased cAMP accumulation above basal. HU210 alone stimulated [35S]GTPγS binding to Gi1/2/3, whereas co-stimulation with quinpirole reversed HU210-stimulated [35S]GTPγS binding to Gi1/2/3.

Conclusions: CB1R couples to Gs but with low efficacy compared to Gi/o. The L341A/A342L mutation in CB1R reversed CP55940 activation of Gi to an inhibition, but had no effect on Gs. Combined CB1 plus D2 agonists in MN9D cells converted the CB1 agonist-mediated activation of Gi to inhibition of Gi. In these models, the CB1 agonist response was converted to an inverse agonist response at Gi activation. Cannabinoid agonist-stimulated cAMP accumulation can be best explained as reduced activation of Gi, thereby attenuating the tonic inhibitory influence of Gi on the major isoforms of adenylyl cyclase.

Keywords: adenylyl cyclase; biased signaling; cannabinoids; D2 dopaminergic receptors; G protein coupled receptor (GPCR); inverse agonism

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About the article

aCurrent address: Department of Pharmacology, Campbell University School of Osteopathic Medicine, Buies Creek, NC 27506, USA


Received: 2015-08-04

Accepted: 2016-03-10

Published Online: 2016-04-18

Published in Print: 2016-05-01


Funding Source: National Institutes of Health

Award identifier / Grant number: R01-DA03690

Funding Source: National Institutes of Health

Award identifier / Grant number: K12-GM102773

This work was supported by National Institutes of Health (Grant/Award Number: R01-DA03690 and K12-GM102773).


Citation Information: Journal of Basic and Clinical Physiology and Pharmacology, Volume 27, Issue 3, Pages 311–322, ISSN (Online) 2191-0286, ISSN (Print) 0792-6855, DOI: https://doi.org/10.1515/jbcpp-2015-0096.

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