MOLECULAR-CLONING AND FUNCTIONAL EXPRESSION OF A SHEEP-A3 ADENOSINE RECEPTOR WITH WIDESPREAD TISSUE DISTRIBUTION

Using the polymerase chain reaction, an A3 adenosine receptor has been cloned from the hypophysial par tuberalis of sheep. The clone encodes a 317-amino acid protein that is 72% identical to the rat A3 adenosin e receptor. In contrast to rat, where abundant A3 mRNA transcript is f ound primarily in testis, the sheep transcript is most abundant in lun g, spleen, and pineal gland and is present in moderate levels in brain , kidney, and testis. The agonist N6-amino[I-125]iodobenzyladenosine b inds with high affinity (K(d) congruent-to 6 nm) and specificity to re combinant A3 adenosine receptors expressed transiently in COS-1 cells or stably in CHO K1 cells. The potency order of agonists is N6-aminoio dobenzyladenosine > N-ethylcarboxamidoadenosine greater-than-or-equal- to (R)-phenylisopropyladenosine much greater than cyclopentyladenosine . Little or no binding of purine nucleotides was detected. The potency order of antagonists is nobenzyl)-8-(4-oxyace-tate)phenyl-1-propylxan thine (I-ABOPX) (K(i) = 3 nm) > 1,3-dipropyl-8-(4-acrylate)phenylxanth ine (BW-A1433) > 1,3-dipropyl-8-sulfophenylxanthine = xanthine amine c ogener much greater than 8-cyclopentyl-1,3-dipropylxanthine. Enprofyll ine does not bind. These data indicate that, in contrast to A1 adenosi ne receptors, A3 adenosine receptors preferentially bind ligands with aryl rings in the N6-position of adenine and in the C-8-position of xa nthine. Among antagonists, the A3 adenosine receptor preferentially bi nds 8-phenylxanthines with acidic versus basic para-substituents (I-AB OPX > BW-A1433 > 1,3-dipropyl-8-sulfophenylxanthine = xanthine amine c ogener). Agonists reduce forskolin-stimulated cAMP accumulation in Chi nese hamster ovary cells stably transfected with recombinant sheep A3 adenosine receptors; the reduction is blocked by BW-A1433 but not by 8 -cyclopentyl-1,3-dipropylxanthine. These data suggest that (i) A3 aden osine receptors display unusual structural diversity for species homol ogs, (ii) in contrast to rat, sheep A3 adenosine receptors have a broa d tissue distribution, and (iii) some xanthines with acidic side chain s bind with high affinity to A3 adenosine receptors.