CB1 receptor-independent actions of SR141716 on G-protein signaling: Coapplication with the μ-opioid agonist tyr-D-ala-gly-(NMe)phe- gly-ol unmasks novel, pertussis toxin-insensitive opioid signaling in μ-opioid receptor-Chinese hamster ovary cells

Resat Cinar, M. Szücs

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28 Citations (Scopus)

Abstract

The CB1 cannabinoid receptor antagonist N-(piperidin-1-yl)-5-(4- chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride (SR141716) has been shown by many investigators to inhibit basal G-protein activity, i.e., to display inverse agonism at high concentrations. However, it is not clear whether this effect is cannabinoid CB1 receptor-mediated. Using the ligand-stimulated [35S]guanosine 5′-3-O-(thio)triphosphate (GTPγS) assay, we have found that 10 μM SR141716 slightly but significantly decreases the basal [35S] GTPγS binding in membranes of the wild-type and CB1 receptor knockout mouse cortex, parental Chinese hamster ovary (CHO) cells, and CHO cells stably transfected with μ-opioid receptors, MOR-CHO. Accordingly, we conclude that the inverse agonism of SR141716 is CB1 receptor-independent. Although the specific MOR agonist Tyr-D-Ala-Gly-(NMe)Phe- Gly-ol (DAMGO) saturably and concentration-dependently stimulated [ 35S]GTPγS binding, SR141716 (10 μM) inhibited the basal by 25% and competitively inhibited DAMGO stimulation in the mouse cortex. In MOR-CHO membranes, DAMGO caused a 501 ± 29% stimulation of the basal activity, which was inhibited to 456 ± 22% by 10 μM SR141716. The inverse agonism of SR141716 was abolished, and DAMGO alone displayed weak, naloxone-insensitive stimulation, whereas the combination of DAMGO and SR141716 (10 μM each) resulted in a 169 ± 22% stimulation of the basal activity (that was completely inhibited by the prototypic opioid antagonist naloxone) because of pertussis toxin (PTX) treatment to uncouple MORs from G i/Go proteins. SR141716 proved to bind directly to MORs with low affinity (IC50 = 5.7 μM). These results suggest the emergence of novel, PTX-insensitive G-protein signaling that is blocked by naloxone when MORs are activated by the combination of DAMGO and SR141716.

Original languageEnglish
Pages (from-to)567-574
Number of pages8
JournalJournal of Pharmacology and Experimental Therapeutics
Volume330
Issue number2
DOIs
Publication statusPublished - Aug 2009

Fingerprint

rimonabant
Ala(2)-MePhe(4)-Gly(5)-enkephalin
Cannabinoid Receptor CB1
Pertussis Toxin
Opioid Receptors
Cricetulus
GTP-Binding Proteins
Opioid Analgesics
Ovary
Naloxone
Gi-Go GTP-Binding Protein alpha Subunits
tyrosyl-alanyl-glycyl-phenylalanine
Cannabinoid Receptor Antagonists
Guanosine 5'-O-(3-Thiotriphosphate)

ASJC Scopus subject areas

  • Pharmacology
  • Molecular Medicine
  • Medicine(all)

Cite this

@article{e846b9cc694a4148898308824ac49309,
title = "CB1 receptor-independent actions of SR141716 on G-protein signaling: Coapplication with the μ-opioid agonist tyr-D-ala-gly-(NMe)phe- gly-ol unmasks novel, pertussis toxin-insensitive opioid signaling in μ-opioid receptor-Chinese hamster ovary cells",
abstract = "The CB1 cannabinoid receptor antagonist N-(piperidin-1-yl)-5-(4- chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride (SR141716) has been shown by many investigators to inhibit basal G-protein activity, i.e., to display inverse agonism at high concentrations. However, it is not clear whether this effect is cannabinoid CB1 receptor-mediated. Using the ligand-stimulated [35S]guanosine 5′-3-O-(thio)triphosphate (GTPγS) assay, we have found that 10 μM SR141716 slightly but significantly decreases the basal [35S] GTPγS binding in membranes of the wild-type and CB1 receptor knockout mouse cortex, parental Chinese hamster ovary (CHO) cells, and CHO cells stably transfected with μ-opioid receptors, MOR-CHO. Accordingly, we conclude that the inverse agonism of SR141716 is CB1 receptor-independent. Although the specific MOR agonist Tyr-D-Ala-Gly-(NMe)Phe- Gly-ol (DAMGO) saturably and concentration-dependently stimulated [ 35S]GTPγS binding, SR141716 (10 μM) inhibited the basal by 25{\%} and competitively inhibited DAMGO stimulation in the mouse cortex. In MOR-CHO membranes, DAMGO caused a 501 ± 29{\%} stimulation of the basal activity, which was inhibited to 456 ± 22{\%} by 10 μM SR141716. The inverse agonism of SR141716 was abolished, and DAMGO alone displayed weak, naloxone-insensitive stimulation, whereas the combination of DAMGO and SR141716 (10 μM each) resulted in a 169 ± 22{\%} stimulation of the basal activity (that was completely inhibited by the prototypic opioid antagonist naloxone) because of pertussis toxin (PTX) treatment to uncouple MORs from G i/Go proteins. SR141716 proved to bind directly to MORs with low affinity (IC50 = 5.7 μM). These results suggest the emergence of novel, PTX-insensitive G-protein signaling that is blocked by naloxone when MORs are activated by the combination of DAMGO and SR141716.",
author = "Resat Cinar and M. Sz{\"u}cs",
year = "2009",
month = "8",
doi = "10.1124/jpet.109.152710",
language = "English",
volume = "330",
pages = "567--574",
journal = "Journal of Pharmacology and Experimental Therapeutics",
issn = "0022-3565",
publisher = "American Society for Pharmacology and Experimental Therapeutics",
number = "2",

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TY - JOUR

T1 - CB1 receptor-independent actions of SR141716 on G-protein signaling

T2 - Coapplication with the μ-opioid agonist tyr-D-ala-gly-(NMe)phe- gly-ol unmasks novel, pertussis toxin-insensitive opioid signaling in μ-opioid receptor-Chinese hamster ovary cells

AU - Cinar, Resat

AU - Szücs, M.

PY - 2009/8

Y1 - 2009/8

N2 - The CB1 cannabinoid receptor antagonist N-(piperidin-1-yl)-5-(4- chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride (SR141716) has been shown by many investigators to inhibit basal G-protein activity, i.e., to display inverse agonism at high concentrations. However, it is not clear whether this effect is cannabinoid CB1 receptor-mediated. Using the ligand-stimulated [35S]guanosine 5′-3-O-(thio)triphosphate (GTPγS) assay, we have found that 10 μM SR141716 slightly but significantly decreases the basal [35S] GTPγS binding in membranes of the wild-type and CB1 receptor knockout mouse cortex, parental Chinese hamster ovary (CHO) cells, and CHO cells stably transfected with μ-opioid receptors, MOR-CHO. Accordingly, we conclude that the inverse agonism of SR141716 is CB1 receptor-independent. Although the specific MOR agonist Tyr-D-Ala-Gly-(NMe)Phe- Gly-ol (DAMGO) saturably and concentration-dependently stimulated [ 35S]GTPγS binding, SR141716 (10 μM) inhibited the basal by 25% and competitively inhibited DAMGO stimulation in the mouse cortex. In MOR-CHO membranes, DAMGO caused a 501 ± 29% stimulation of the basal activity, which was inhibited to 456 ± 22% by 10 μM SR141716. The inverse agonism of SR141716 was abolished, and DAMGO alone displayed weak, naloxone-insensitive stimulation, whereas the combination of DAMGO and SR141716 (10 μM each) resulted in a 169 ± 22% stimulation of the basal activity (that was completely inhibited by the prototypic opioid antagonist naloxone) because of pertussis toxin (PTX) treatment to uncouple MORs from G i/Go proteins. SR141716 proved to bind directly to MORs with low affinity (IC50 = 5.7 μM). These results suggest the emergence of novel, PTX-insensitive G-protein signaling that is blocked by naloxone when MORs are activated by the combination of DAMGO and SR141716.

AB - The CB1 cannabinoid receptor antagonist N-(piperidin-1-yl)-5-(4- chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride (SR141716) has been shown by many investigators to inhibit basal G-protein activity, i.e., to display inverse agonism at high concentrations. However, it is not clear whether this effect is cannabinoid CB1 receptor-mediated. Using the ligand-stimulated [35S]guanosine 5′-3-O-(thio)triphosphate (GTPγS) assay, we have found that 10 μM SR141716 slightly but significantly decreases the basal [35S] GTPγS binding in membranes of the wild-type and CB1 receptor knockout mouse cortex, parental Chinese hamster ovary (CHO) cells, and CHO cells stably transfected with μ-opioid receptors, MOR-CHO. Accordingly, we conclude that the inverse agonism of SR141716 is CB1 receptor-independent. Although the specific MOR agonist Tyr-D-Ala-Gly-(NMe)Phe- Gly-ol (DAMGO) saturably and concentration-dependently stimulated [ 35S]GTPγS binding, SR141716 (10 μM) inhibited the basal by 25% and competitively inhibited DAMGO stimulation in the mouse cortex. In MOR-CHO membranes, DAMGO caused a 501 ± 29% stimulation of the basal activity, which was inhibited to 456 ± 22% by 10 μM SR141716. The inverse agonism of SR141716 was abolished, and DAMGO alone displayed weak, naloxone-insensitive stimulation, whereas the combination of DAMGO and SR141716 (10 μM each) resulted in a 169 ± 22% stimulation of the basal activity (that was completely inhibited by the prototypic opioid antagonist naloxone) because of pertussis toxin (PTX) treatment to uncouple MORs from G i/Go proteins. SR141716 proved to bind directly to MORs with low affinity (IC50 = 5.7 μM). These results suggest the emergence of novel, PTX-insensitive G-protein signaling that is blocked by naloxone when MORs are activated by the combination of DAMGO and SR141716.

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