Cloning and pharmacological characterization of the equine adenosine A3 receptor.
The aim of this study was to establish a heterologous expression system for the equine adenosine A(3) receptor (eA(3)-R) in an effort to ascertain its pharmacologic profile. Initially, radioligand binding assays identified clones expressing the eA(3)-R in human embryonic kidney cells (HEK) based on the specific binding of [(125)I]AB-MECA. Subsequently, adenylate cyclase assays were utilized to demonstrate functional coupling of the eA(3)-R to the G-protein/adenylate cyclase system. Equilibrium competition binding assays were then performed using selective and non-selective A(3) agonists and antagonists. Results from these experiments revealed a rank order of agonist potency to be IB-MECA > NECA > CGS21680, and an antagonist potency of MRS1220 > ZM241385 > 8-p-sulphophenyltheophylline; these rank orders were in agreement with that of other mammalian A(3)-R's. Lastly, NF-kappaB reporter gene assays revealed an IB-MECA concentration-dependent inhibition of TNFalpha-stimulated NF-kappaB activity. These results indicate that the heterologously expressed eA(3)-R is functional, has a pharmacological profile similar to that of other mammalian A(3) receptors, and its activation has an inhibitory effect on a key regulatory pathway in the inflammatory response. Thus, the eA(3)-R may serve as a pharmacological target in the treatment of equine inflammatory disease.
Brandon
C I
CI
Department of Physiology and Pharmacology, College of Veterinary Medicine, University of Georgia, Athens, GA 30602, USA.
Vandenplas
M
M
Dookwah
H
H
Murray
T F
TF
D000241:Adenosine / Q000819:agonists / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism*; D019446:Endotoxemia / Q000188:drug therapy / Q000662:veterinary*; D006734:Horse Diseases / Q000188:drug therapy*; D006736:Horses; D006801:Humans; D008969:Molecular Sequence Data; D043684:Receptor, Adenosine A3 / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D018076:DNA, Complementary; D043684:Receptor, Adenosine A3; D000241:Adenosine