Abstract
The aim of the current study was to clone the equine adenosine A 2A receptor gene and to establish a heterologous expression system to ascertain its pharmacologic profile via radioligand binding and functional assays. An eA2A-R expression construct was generated by ligation of the eA2A cDNA into the pcDNA3.1 expression vector, and stably transfected into human embryonic kidney cells (HEK). Binding assays identified those clones expressing the eA2A-R, and equilibrium saturation isotherm experiments were utilized to determine dissociation constants (K D), and receptor densities (Bmax) of selected clones. Equilibrium competition binding revealed a rank order of agonist potency of ATL > CV-1808 > NECA > 2-CADO > CGS21680, and a rank order of antagonist potency as ZM241385 > 8-phenyltheophylline > p-sulfophenyltheophylline > caffeine. Furthermore, adenylate cyclase assays using selective A 2A-R agonists revealed that the eA2A-R functionally coupled to Gαs as indicated by an increase in intracellular [3H]cAMP upon receptor activation. Finally, NF-κB reporter gene assays revealed a CGS21680 concentration-dependent inhibition of NF-κB activity. These results indicate that the heterologously expressed eA 2A-R has a pharmacological profile similar to that of other mammalian A2A receptors and thus can be utilized for further characterization of the eA2A-R to ascertain whether it can serve as a suitable pharmacological target for equine inflammatory disease.
| Original language | English |
|---|---|
| Pages (from-to) | 243-253 |
| Number of pages | 11 |
| Journal | Journal of Veterinary Pharmacology and Therapeutics |
| Volume | 29 |
| Issue number | 4 |
| DOIs | |
| State | Published - Aug 2006 |
| Externally published | Yes |
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All Science Journal Classification (ASJC) codes
- Pharmacology
- veterinary(all)
Cite this
Cloning and pharmacological characterization of the equine adenosine A 2A receptor : A potential therapeutic target for the treatment of equine endotoxemia. / Brandon, C. I.; Vandenplas, M.; Dookwah, H.; Linden, J.; Murray, Thomas F.
In: Journal of Veterinary Pharmacology and Therapeutics, Vol. 29, No. 4, 08.2006, p. 243-253.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Cloning and pharmacological characterization of the equine adenosine A 2A receptor
T2 - A potential therapeutic target for the treatment of equine endotoxemia
AU - Brandon, C. I.
AU - Vandenplas, M.
AU - Dookwah, H.
AU - Linden, J.
AU - Murray, Thomas F.
PY - 2006/8
Y1 - 2006/8
N2 - The aim of the current study was to clone the equine adenosine A 2A receptor gene and to establish a heterologous expression system to ascertain its pharmacologic profile via radioligand binding and functional assays. An eA2A-R expression construct was generated by ligation of the eA2A cDNA into the pcDNA3.1 expression vector, and stably transfected into human embryonic kidney cells (HEK). Binding assays identified those clones expressing the eA2A-R, and equilibrium saturation isotherm experiments were utilized to determine dissociation constants (K D), and receptor densities (Bmax) of selected clones. Equilibrium competition binding revealed a rank order of agonist potency of ATL > CV-1808 > NECA > 2-CADO > CGS21680, and a rank order of antagonist potency as ZM241385 > 8-phenyltheophylline > p-sulfophenyltheophylline > caffeine. Furthermore, adenylate cyclase assays using selective A 2A-R agonists revealed that the eA2A-R functionally coupled to Gαs as indicated by an increase in intracellular [3H]cAMP upon receptor activation. Finally, NF-κB reporter gene assays revealed a CGS21680 concentration-dependent inhibition of NF-κB activity. These results indicate that the heterologously expressed eA 2A-R has a pharmacological profile similar to that of other mammalian A2A receptors and thus can be utilized for further characterization of the eA2A-R to ascertain whether it can serve as a suitable pharmacological target for equine inflammatory disease.
AB - The aim of the current study was to clone the equine adenosine A 2A receptor gene and to establish a heterologous expression system to ascertain its pharmacologic profile via radioligand binding and functional assays. An eA2A-R expression construct was generated by ligation of the eA2A cDNA into the pcDNA3.1 expression vector, and stably transfected into human embryonic kidney cells (HEK). Binding assays identified those clones expressing the eA2A-R, and equilibrium saturation isotherm experiments were utilized to determine dissociation constants (K D), and receptor densities (Bmax) of selected clones. Equilibrium competition binding revealed a rank order of agonist potency of ATL > CV-1808 > NECA > 2-CADO > CGS21680, and a rank order of antagonist potency as ZM241385 > 8-phenyltheophylline > p-sulfophenyltheophylline > caffeine. Furthermore, adenylate cyclase assays using selective A 2A-R agonists revealed that the eA2A-R functionally coupled to Gαs as indicated by an increase in intracellular [3H]cAMP upon receptor activation. Finally, NF-κB reporter gene assays revealed a CGS21680 concentration-dependent inhibition of NF-κB activity. These results indicate that the heterologously expressed eA 2A-R has a pharmacological profile similar to that of other mammalian A2A receptors and thus can be utilized for further characterization of the eA2A-R to ascertain whether it can serve as a suitable pharmacological target for equine inflammatory disease.
UR - http://www.scopus.com/inward/record.url?scp=33745635024&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=33745635024&partnerID=8YFLogxK
U2 - 10.1111/j.1365-2885.2006.00746.x
DO - 10.1111/j.1365-2885.2006.00746.x
M3 - Article
C2 - 16846461
AN - SCOPUS:33745635024
VL - 29
SP - 243
EP - 253
JO - Journal of Veterinary Pharmacology and Therapeutics
JF - Journal of Veterinary Pharmacology and Therapeutics
SN - 0140-7783
IS - 4
ER -