CLONING, EXPRESSION AND PHARMACOLOGICAL CHARACTERIZATION OF RABBIT ADENOSINE A(1) AND A(3) RECEPTORS

The role of adenosine A(1) and A(3) receptors in mediating cardioprote ction has been studied predominantly in rabbits, yet the pharmacologic al characteristics of rabbit adenosine A(1) and A(3) receptor subtypes are unknown. Thus, the rabbit adenosine A(3) receptor was cloned and expressed, and its pharmacology was compared with that of cloned adeno sine A(1) receptors. Stable transfection of rabbit A(1) or A(3) cDNAs in Chinese hamster ovary-K1 cells resulted in high levels of expressio n of each of the receptors, as demonstrated by high-affinity binding o f the A(1)/A(3) adenosine receptor agonist N-6-(4-amino-3-[I-125]iodob enzyl)adenosine (I-125-ABA). For both receptors, binding of I-125-ABA was inhibited by the GTP analog 5'-guanylimidodiphosphate, and forskol in-stimulated cyclic AMP accumulation was inhibited by the adenosine r eceptor agonist (R)-phenylisopropyladenosine. The rank orders of poten cy of adenosine receptor agonists for inhibition of I-125-ABA binding were as follows: rabbit A(1), N-6-cyclopentyladenosine = (R)-phenyliso propyladenosine > N-ethylcarboxamidoadenosine greater than or equal to 1-ABA greater than or equal to N-6-2-(4-aminophenyl)ethyladenosine >> N-6-(3-iodobenzyl)adenosine-5'-N-methyluronamide > N-6-(4-amino-3-ben zyl)adenosine; rabbit A(3), N-6-(3-iodobenzyl)adenosine-5'-N-methyluro namide greater than or equal to I-ABA >> N-ethylcarboxamidoadenosine > N-6-2-(4-aminophenyl)ethyladenosine = N-6-cyclopentyladenosine = (R)- phenylisopropyladenosine > N-6-(4-amino-3-benzyl)adenosine. The adenos ine receptor antagonist rank orders were as follows: rabbit A(1), 8-cy clopentyl-1,3-dipropylxanthine > 1,3-dipropyl-8-(4-acrylate)phenylxant hine greater than or equal to xanthine amine congener >> 8-(p-sulfophe nyl)theophylline; rabbit A(3), xanthine amine congener > 1,3-dipropyl- 8-(4-acrylate)phenylxanthine greater than or equal to 8-cyclopentyl-1, 3-dipropylxanthine >> 8-(p-sulfophenyl)theophylline. These observation s confirm the identity of the expressed proteins as A(1) and A(3) rece ptors. The results will facilitate further in-depth studies of the rol es of A(1) and A(3) receptors in adenosine-mediated cardioprotection i n rabbits, which can now be based on the appropriate recombinant rabbi t A(1) and A(3) receptor pharmacology.