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Comparative pharmacology of human adenosine receptor subtypes - characterization of stably transfected receptors in CHO cells

Klotz, K N; Hessling, J; Hegler, J; Owman, Christer LU ; Kull, B; Fredholm, B B and Lohse, M J (1998) In Naunyn-Schmiedeberg's Archives of Pharmacology 357(1). p.1-9
Abstract
Four adenosine receptor subtypes of the family of G protein-coupled receptors, designated A1, A2A, A2B and A3 are currently known. In this study all human subtypes were stably transfected into Chinese hamster ovary (CHO) cells in order to be able to study their pharmacological profile in an identical cellular background utilizing radioligand binding studies (A1, A2A, A3) or adenylyl cyclase activity assays (A2B). The A1 subtype showed the typical pharmacological profile with 2-chloro-N6-cyclopentyladenosine (CCPA) as the agonist with the highest affinity and a marked stereoselectivity for the N6-phenylisopropyladenosine (PIA) diastereomers. In competition with antagonist radioligand biphasic curves were observed for agonists. In the... (More)
Four adenosine receptor subtypes of the family of G protein-coupled receptors, designated A1, A2A, A2B and A3 are currently known. In this study all human subtypes were stably transfected into Chinese hamster ovary (CHO) cells in order to be able to study their pharmacological profile in an identical cellular background utilizing radioligand binding studies (A1, A2A, A3) or adenylyl cyclase activity assays (A2B). The A1 subtype showed the typical pharmacological profile with 2-chloro-N6-cyclopentyladenosine (CCPA) as the agonist with the highest affinity and a marked stereoselectivity for the N6-phenylisopropyladenosine (PIA) diastereomers. In competition with antagonist radioligand biphasic curves were observed for agonists. In the presence of GTP all receptors were converted to a single low affinity state indicating functional coupling to endogenous G proteins. For A2A adenosine receptors CGS 21680 (2-[p-(2-carboxyethyl)phenylethylamino]-5'-N-ethylcarboxamidoadeno sine) and N-ethylcarboxamidoadenosine (NECA) were found to be the most potent agonists followed by R- and S-PIA with minor stereoselectivity. The relative potencies of agonists for the A2B adenosine receptor could only be tested by measurement of receptor-stimulated adenylyl cyclase activity. NECA was the most potent agonist with an EC50-value of 2.3 microM whereas all other compounds tested were active at concentrations in the high micromolar range. Inhibition of NECA-stimulated adenylyl cyclase identified xanthine amino congener (XAC; 8-[4-[[[[(2-aminoethyl)amino]-carbonyl]methyl]oxy]phenyl]-1,3-dipropylxa nthine) as the most potent antagonist at this receptor subtype. The A3 receptor was characterized utilizing the nonselective agonist [3H]NECA. The N6-benzyl substituted derivatives of adenosine-5'-N-methyluronamide (MECA) turned out to be the most potent agonists. The notion of xanthine-insensitivity of the A3 receptor should be dropped at least for the human receptor as xanthines with submicromolar affinity were found. Overall, the pharmacological characteristics of the human receptors are similar to other species with some species-specific characteristics. In this study we present for the first time the comparative pharmacology of all known human adenosine receptor subtypes. The CHO cells with stably transfected adenosine receptors provide an identical cellular background for such a pharmacological characterization. These cells are valuable systems for further characterization of specific receptor subtypes and for the development of new ligands. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Stable transfection, G protein-coupled receptors, Signal transduction, Adenosine receptors, A1, A2A, A2B, A3, ligand
in
Naunyn-Schmiedeberg's Archives of Pharmacology
volume
357
issue
1
pages
1 - 9
publisher
Springer
external identifiers
  • pmid:9459566
  • scopus:0031931065
ISSN
0028-1298
DOI
10.1007/PL00005131
language
English
LU publication?
yes
id
fe3b221c-835c-4df5-901e-bca1b8d43265 (old id 1113454)
date added to LUP
2008-07-15 10:20:11
date last changed
2017-11-19 03:25:22
@article{fe3b221c-835c-4df5-901e-bca1b8d43265,
  abstract     = {Four adenosine receptor subtypes of the family of G protein-coupled receptors, designated A1, A2A, A2B and A3 are currently known. In this study all human subtypes were stably transfected into Chinese hamster ovary (CHO) cells in order to be able to study their pharmacological profile in an identical cellular background utilizing radioligand binding studies (A1, A2A, A3) or adenylyl cyclase activity assays (A2B). The A1 subtype showed the typical pharmacological profile with 2-chloro-N6-cyclopentyladenosine (CCPA) as the agonist with the highest affinity and a marked stereoselectivity for the N6-phenylisopropyladenosine (PIA) diastereomers. In competition with antagonist radioligand biphasic curves were observed for agonists. In the presence of GTP all receptors were converted to a single low affinity state indicating functional coupling to endogenous G proteins. For A2A adenosine receptors CGS 21680 (2-[p-(2-carboxyethyl)phenylethylamino]-5'-N-ethylcarboxamidoadeno sine) and N-ethylcarboxamidoadenosine (NECA) were found to be the most potent agonists followed by R- and S-PIA with minor stereoselectivity. The relative potencies of agonists for the A2B adenosine receptor could only be tested by measurement of receptor-stimulated adenylyl cyclase activity. NECA was the most potent agonist with an EC50-value of 2.3 microM whereas all other compounds tested were active at concentrations in the high micromolar range. Inhibition of NECA-stimulated adenylyl cyclase identified xanthine amino congener (XAC; 8-[4-[[[[(2-aminoethyl)amino]-carbonyl]methyl]oxy]phenyl]-1,3-dipropylxa nthine) as the most potent antagonist at this receptor subtype. The A3 receptor was characterized utilizing the nonselective agonist [3H]NECA. The N6-benzyl substituted derivatives of adenosine-5'-N-methyluronamide (MECA) turned out to be the most potent agonists. The notion of xanthine-insensitivity of the A3 receptor should be dropped at least for the human receptor as xanthines with submicromolar affinity were found. Overall, the pharmacological characteristics of the human receptors are similar to other species with some species-specific characteristics. In this study we present for the first time the comparative pharmacology of all known human adenosine receptor subtypes. The CHO cells with stably transfected adenosine receptors provide an identical cellular background for such a pharmacological characterization. These cells are valuable systems for further characterization of specific receptor subtypes and for the development of new ligands.},
  author       = {Klotz, K N and Hessling, J and Hegler, J and Owman, Christer and Kull, B and Fredholm, B B and Lohse, M J},
  issn         = {0028-1298},
  keyword      = {Stable transfection,G protein-coupled
receptors,Signal transduction,Adenosine receptors,A1,A2A,A2B,A3,ligand},
  language     = {eng},
  number       = {1},
  pages        = {1--9},
  publisher    = {Springer},
  series       = {Naunyn-Schmiedeberg's Archives of Pharmacology},
  title        = {Comparative pharmacology of human adenosine receptor subtypes - characterization of stably transfected receptors in CHO cells},
  url          = {http://dx.doi.org/10.1007/PL00005131},
  volume       = {357},
  year         = {1998},
}