Abstract
This study examined the ability of the endocannabinoids 2-arachidonoyl glycerol (2-AG) and noladin ether as well as the synthetic cannabinoid CP-55,940 [(-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl) cyclohexanol] to regulate three intracellular effectors via CB2 receptors in transfected Chinese hamster ovary cells. Although the three agonists regulate all effectors with equivalent efficacy, the rank order of potencies differs depending on which effector is evaluated. Noladin ether and CP-55,940 most potently inhibit adenylyl cyclase, requiring higher concentrations to stimulate the extracellular signal-regulated kinase subgroup of the mitogen-activated protein kinases (extracellular signal-regulated kinase-mitogen-activated protein kinase; ERK-MAPK) and Ca2+-transients. In contrast, 2-AG most potently activates ERK-MAPK, necessitating greater concentrations to inhibit adenylyl cyclase and even higher amounts to stimulate Ca2+-transients. Endocannabinoids also seem to be more “efficient” agonists at CB2 receptors relative to synthetic agonists. 2-AG and noladin ether require occupancy of less than one-half the number of receptors to produce comparable regulation of adenylyl cyclase and ERK-MAPK, relative to the synthetic cannabinoid CP-55,940. The CB2 antagonist 6-iodo-2-methyl-1-[2-(4-morpholinyl)-ethyl]-1H-indol-3-yl](4-methoxyphenyl)-methanone (AM630) reverses the actions of all agonists except Ca2+-transient stimulation by 2-AG. However, the effect of 2-AG on Ca2+-transients is attenuated by a second CB2 antagonist N-[(1S)-endo-1,3,3-trimethylbicyclo[2.2.1]heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-1-pyrazole-3-carboxamide (SR144528). This suggests that 2-AG stimulates Ca2+-transients by binding to sites on CB2 receptors distinct from those occupied by AM630 and the other cannabinoids examined. Agonists produce no effects in pertussis toxin-treated cells. In summary, cannabinoid agonists distinctly bind to CB2 receptors and display different rank order of potencies and fractional receptor occupancies for regulation of intracellular effectors. These data provide direct evidence for agonist-directed trafficking of response by endocannabinoids acting at CB2 receptors.
Footnotes
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This work was supported in part by National Institute of on Drug Abuse Grant DA13660 (to P.L.P.).
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.105.089474.
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ABBREVIATIONS: CB1, cannabinoid receptor 1; CB2, cannabinoid receptor 2; PTX, pertussis toxin; ERK-MAPK, extracellular signal-regulated kinase subgroup of the mitogen-activated protein kinases; PLCβ, phospholipase-Cβ; 2-AG, 2-arachidonoyl glycerol [(5Z,8Z,11Z,14Z)-5,8,11,14-eicosatetraenoic acid, 2-hydroxy-1-(hydroxymethyl) ethyl ester]; CP-55,940, (-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl) cyclohexanol; GPCR, G protein-coupled receptor; CHO, Chinese hamster ovary; DMEM, Dulbecco's modified Eagle's medium; AM630 [6-iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl](4-methoxyphenyl)-methanone; SR144528, N-[(1S)-endo-1,3,3-trimethylbicyclo[2.2.1]heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-1-pyrazole-3-carboxamide; PBS, phosphate-buffered saline; ECL, enhanced chemiluminescence; FACE, fast-activated cell-based enzyme-linked immunosorbent assay; TBS, Tris-buffered saline; ANOVA, analysis of variance; WIN 55,212-2, (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone; HU-210, (6aR)-trans-3-(1,1-dimethylheptyl)-6a,7,10,10a-tetrahydro-1-hydroxy-6,6-dimethyl-6H-dibenzo[b,d]pyran-9-methanol; DOR, δ opioid receptor; DPDPE, [d-Pen2,d-Pen5]-enkephalin; U73122, 1-[6-[[17-β-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione; U73343, 1-[6-[[17-β-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-2,5-pyrrolidinedione; FRO, fractional receptor occupancy; AF102B, (±)-cis-2-methyl-spiro(1,3-oxathiolane-5,3′)quinuclidine; AC, adenylyl cyclase; ADTR, agonist-directed trafficking of response; 5-HT, 5-hydroxytryptamine.
- Received May 12, 2005.
- Accepted July 26, 2005.
- The American Society for Pharmacology and Experimental Therapeutics
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