Abstract
The role of adenosine A1 and A3 receptors in mediating cardioprotection has been studied predominantly in rabbits, yet the pharmacological characteristics of rabbit adenosine A1 and A3 receptor subtypes are unknown. Thus, the rabbit adenosine A3 receptor was cloned and expressed, and its pharmacology was compared with that of cloned adenosine A1 receptors. Stable transfection of rabbit A1or A3 cDNAs in Chinese hamster ovary-K1 cells resulted in high levels of expression of each of the receptors, as demonstrated by high-affinity binding of the A1/A3 adenosine receptor agonistN 6-(4-amino-3-[125I]iodobenzyl)adenosine (125I-ABA). For both receptors, binding of125I-ABA was inhibited by the GTP analog 5′-guanylimidodiphosphate, and forskolin-stimulated cyclic AMP accumulation was inhibited by the adenosine receptor agonist (R)-phenylisopropyladenosine. The rank orders of potency of adenosine receptor agonists for inhibition of 125I-ABA binding were as follows: rabbit A1,N 6-cyclopentyladenosine = (R)-phenylisopropyladenosine >N-ethylcarboxamidoadenosine ≥ I-ABA ≥N 6-2-(4-aminophenyl)ethyladenosine ≫N 6-(3-iodobenzyl)adenosine-5′-N-methyluronamide > N 6-(4-amino-3-benzyl)adenosine; rabbit A3,N 6-(3-iodobenzyl)adenosine-5′-N-methyluronamide ≥ I-ABA ≫ N-ethylcarboxamidoadenosine >N 6-2-(4-aminophenyl)ethyladenosine =N 6-cyclopentyladenosine = (R)-phenylisopropyladenosine >N 6-(4-amino-3-benzyl)adenosine. The adenosine receptor antagonist rank orders were as follow: rabbit A1, 8-cyclopentyl-1,3-dipropylxanthine > 1,3-dipropyl-8-(4-acrylate)phenylxanthine ≥ xanthine amine congener ≫ 8-(p-sulfophenyl)theophylline; rabbit A3, xanthine amine congener > 1,3-dipropyl-8-(4-acrylate)phenylxanthine ≥ 8-cyclopentyl-1,3-dipropylxanthine ≫ 8-(p-sulfophenyl)theophylline. These observations confirm the identity of the expressed proteins as A1 and A3 receptors. The results will facilitate further in-depth studies of the roles of A1 and A3 receptors in adenosine-mediated cardioprotection in rabbits, which can now be based on the appropriate recombinant rabbit A1 and A3receptor pharmacology.
Footnotes
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Send reprint requests to: Dr. Roger J. Hill, Department of Cardiovascular and Metabolic Diseases, Pfizer Inc., Eastern Point Road, Groton, CT 06340.
- Abbreviations:
- ABA
- N 6-(4-amino-3-benzyl)adenosine
- ADA
- adenosine deaminase
- cAMP
- adenosine 3′,5′-cyclic monophosphate
- CHO
- Chinese hamster ovary
- CPA
- N 6-cyclopentyladenosine
- DMEM
- Dulbecco’s modified Eagle’s medium
- DPCPX
- 8-cyclopentyl-1,3-dipropylxanthine
- Gpp(NH)p
- 5′-guanylimidodiphosphate
- I-ABA
- N 6-(4-amino-3-iodobenzyl)adenosine
- IB-MECA
- N 6-(3-iodobenzyl)adenosine-5′-N-methyluronamide
- PBS
- phosphate-buffered saline
- PCR
- polymerase chain reaction
- (R)-PIA
- (R)-phenylisopropyladenosine
- Received July 24, 1996.
- Accepted September 30, 1996.
- The American Society for Pharmacology and Experimental Therapeutics
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