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The Endocannabinoids Anandamide and Virodhamine Modulate the Activity of the Candidate Cannabinoid Receptor GPR55

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Abstract

The role of cannabinoid receptors in inflammation has been the topic of many research endeavors. Despite this effort, to date the involvement of the endocannabinoid system (ECS) in inflammation remains obscure. The ambiguity of cannabinoid involvement may be explained by the existence of cannabinoid receptors, other than CB1 and CB2, or a consequence of interaction of endocannabinoids with other signaling systems. GPR55 has been proposed to be a cannabinoid receptor; however the interaction of the endocannabinoid system with GPR55 remains elusive. Consequently this study set about to examine the effects of the endocannabinoids, anandamide (AEA) and virodhamine, on GPR55 mediated signaling. Specifically, we assessed changes in β-arrestin2 (βarr2) distribution and GPR55 receptor internalization following activation by lysophosphatidylinositol (LPI), the synthetic cannabinoid ligand SR141716A, and new selective synthetic GPR55 agonists. Data obtained from the experiments presented herein demonstrate that AEA and virodhamine modulate agonist-mediated recruitment of βarr2. AEA and virodhamine act as partial agonists; enhancing the agonist effect at low concentrations and inhibiting it at high concentrations. Furthermore, both virodhamine and AEA significantly attenuated agonist-induced internalization of GPR55. These effects are attributed to the expression of GPR55, and not CB1 and CB2 receptors, as we have established negligible expression of CB1 and CB2 in these GPR55-transfected U2OS cells. The identification of select endocannabinoids as GPR55 modulators will aide in elucidating the function of GPR55 in the ECS.

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Abbreviations

AEA:

Anandamide; N-arachidonyl ethanolamine

2-AG:

2-arachidonyl glycerol

βarr2:

β-arrestin2

BV2:

murine microglial cell line

BSA:

bovine serum albumin

CB1 :

cannabinoid receptor type 1

CB2 :

cannabinoid receptor type 2

CHO:

Chinese hamster ovary cell line

DMEM:

Dulbecco’s modified Eagle’s medium

ECS:

endocannabinoid system

FBS:

fetal bovine serum

FCA:

Freund’s complete adjuvant

GFP:

Green Fluorescent Protein

GPCR:

G-protein coupled receptor

HBSS:

Hanks’ balanced salt solution

HA:

hemagglutinin

HEK293:

human embryonic kidney cell line

IFN-γ:

interferon gamma

LDH:

lactate dehydrogenase

LPI:

lysophosphatidylinositol

LPS:

lipopolysaccharide

ML184 (CID2440433):

3-[4-(2,3-dimethylphenyl)piperazine-1-carbonyl]-N,N-dimethyl-4-pyrrolidin-1-ylbenzenesulfonamide

ML185 (CID1374043):

N-[4-(4-methoxyphenyl)-1,3-thiazol-2-yl]-2-[(4-methyl-[1,2,4]triazolo[4,3-a]quinolin-1-yl)sulfanyl]acetamide

ML186 (CID15945391):

N-(2-methoxy-5-morpholin-4-ylsulfonylphenyl)-4,5,6,7-tetrahydro-1-benzothiophene-2-carboxamide

PBS:

phosphate-buffered saline

qRT-PCR:

quantitative real time PCR

RT-PCR:

reverse transcriptase-polymerase chain reaction

SR141716A:

N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide

U2OS:

osteosarcoma cell line

Virodhamine:

O-arachidonoyl ethanolamine

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Acknowledgments

This research was supported by the National Institutes of Health National Institute on Drug Abuse [Grants DA023204, DA005274, T32DA07237, DA13429]. We would like to thank Dr. Pingwei Zhao for providing the plug-in for data quantification, Drs. Lawrence Barak and Marc Caron for providing the GPR55/βarr2-GFP U2OS cells and express gratitude to Mrs. Genevieve V. Regan for her valuable assistance with the confocal imaging.

Conflict of interest

The authors declare no conflict of interest.

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Authors and Affiliations

  1. Department of Anatomy and Cell Biology, Temple University School of Medicine, 3420 North Broad St, Philadelphia, PA, 19140, USA

    Haleli Sharir, Linda Console-Bram, Christina Mundy, Steven N. Popoff, Ankur Kapur & Mary E. Abood

  2. Center for Substance Abuse Research, Temple University School of Medicine, 3420 North Broad St, Philadelphia, PA, 19140, USA

    Haleli Sharir, Linda Console-Bram, Ankur Kapur & Mary E. Abood

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  1. Haleli Sharir
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  2. Linda Console-Bram
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  6. Mary E. Abood
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Correspondence to Mary E. Abood.

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Sharir, H., Console-Bram, L., Mundy, C. et al. The Endocannabinoids Anandamide and Virodhamine Modulate the Activity of the Candidate Cannabinoid Receptor GPR55. J Neuroimmune Pharmacol 7, 856–865 (2012). https://doi.org/10.1007/s11481-012-9351-6

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