Cloning and pharmacological characterization of the equine adenosine A2A receptor: a potential therapeutic target for the treatment of equine endotoxemia.
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 eA(2A)-R expression construct was generated by ligation of the eA(2A) cDNA into the pcDNA3.1 expression vector, and stably transfected into human embryonic kidney cells (HEK). Binding assays identified those clones expressing the eA(2A)-R, and equilibrium saturation isotherm experiments were utilized to determine dissociation constants (K(D)), and receptor densities (B(max)) 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 eA(2A)-R functionally coupled to Galpha(s) as indicated by an increase in intracellular [(3)H]cAMP upon receptor activation. Finally, NF-kappaB reporter gene assays revealed a CGS21680 concentration-dependent inhibition of NF-kappaB activity. These results indicate that the heterologously expressed eA(2A)-R has a pharmacological profile similar to that of other mammalian A(2A) receptors and thus can be utilized for further characterization of the eA(2A)-R to ascertain whether it can serve as a suitable pharmacological target for equine inflammatory disease.
Publication Date: 2006-07-19 PubMed ID: 16846461DOI: 10.1111/j.1365-2885.2006.00746.xGoogle Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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This study aimed to clone the horse-specific variant of the adenosine A2A receptor to study its pharmacological properties and determine if it’s a potential therapeutic target for inflammatory conditions in horses, such as endotoxemia.
About the Study
- The study’s objective was to clone the gene of the equine adenosine A2A receptor, create an eA2A-R expression assembly, and transfer it into human embryonic kidney cells (HEKs). The purpose of this was to establish a system for examining the pharmacological properties of this receptor.
- Moreover, the researchers wanted to examine the profile of the receptor’s interaction with different drug compounds using radioligand binding and functional assays.
Methods and Results
- The researchers managed to create the eA2A-R expression construct by inserting the eA2A cDNA into the pcDNA3.1 expression vector. This construct, carrying the receptor gene, was then successfully integrated into HEK cells.
- Through binding assays, the team identified the clones having the equine adenosine A2A receptor (eA2A-R). They used equilibrium saturation isotherm experiments to calculate dissociation constants and receptor densities for the chosen clones.
- The results of the equilibrium competition binding experiments indicated a rank-ordered potency of various agonists and antagonists when interacting with the eA2A receptor.
- Followed by this, adenylate cyclase assays were performed, which showed that the eA2A-R functionally linked to the Galpha(s) protein as revealed by an increase in intracellular cAMP with receptor activation, indicating the receptor’s role in the cell signaling process.
- They also conducted NF-kappaB reporter gene assays that exhibited a concentration-dependent decrease in NF-kappaB activity with the drug CGS21680.
Conclusion
- The findings suggest that the pharmacological profile of the heterologously expressed eA2A-R is similar to that of other mammalian A2A receptors. Hence, it could provide potential insights for treating inflammatory conditions like endotoxemia in horses, as the eA2A-R may serve as a valuable pharmacological target.
Cite This Article
APA
Brandon CI, Vandenplas M, Dookwah H, Linden J, Murray TF.
(2006).
Cloning and pharmacological characterization of the equine adenosine A2A receptor: a potential therapeutic target for the treatment of equine endotoxemia.
J Vet Pharmacol Ther, 29(4), 243-253.
https://doi.org/10.1111/j.1365-2885.2006.00746.x Publication
Researcher Affiliations
- Department of Physiology, College of Veterinary Medicine, University of Georgia, Athens, GA 30602, USA.
MeSH Terms
- Adenosine / agonists
- Adenosine / pharmacology
- Amino Acid Sequence
- Animals
- Binding Sites / drug effects
- Cloning, Molecular
- DNA, Complementary / genetics
- DNA, Complementary / metabolism
- Endotoxemia / drug therapy
- Endotoxemia / veterinary
- Horse Diseases / drug therapy
- Horses
- Humans
- Molecular Sequence Data
- Receptor, Adenosine A2A / genetics
- Receptor, Adenosine A2A / metabolism
- Transfection
Citations
This article has been cited 1 times.- Nishat S, Khan LA, Ansari ZM, Basir SF. Adenosine A3 Receptor: A promising therapeutic target in cardiovascular disease. Curr Cardiol Rev 2016;12(1):18-26.
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