The human A3 adenosine receptor was cloned from a striatal cDNA library using a probe derived from the homologous rat sequence. The cDNA encodes a protein of 318 amino acids and exhibits 72% and 85% overall identity with the rat and sheep A3 adenosine receptor sequences, respectively. Specific and saturable binding of the adenosine receptor agonist N6-(4-amino-3-[125I]iodobenzyl)adenosine [125I]ABA was measured on the human A3 receptor stably expressed in Chinese hamster ovary cells with a Kd = 10 nM. The potency order for adenosine receptor agonists was N-ethylcarboxamidoadenosine (NECA) > or = (R)-N6-phenyl-2-propyladenosine [(R)-PIA] > N6-cyclopentyladenosine (CPA) > (S)-N6-phenyl-2-propyladenosine [(S)-PIA]. The human receptor was blocked by xanthine antagonists, most potently by 3-(3-iodo-4-aminobenzyl)-8-(4-oxyacetate)phenyl-1-propylxanthine (I-ABOPX) with a potency order of I-ABOPX > 1,3-dipropyl-8-(4-acrylate)phenylxanthine > or = xanthine amino congener >> 1,3-dipropyl-8-cyclopentylxanthine. Adenosine, NECA, (R)- and (S)-PIA, and CPA inhibited forskolin-stimulated cAMP accumulation by 30-40% in stably transfected cells; I-ABA is a partial agonist. When measured in the presence of antagonists, the dose-response curves of NECA-induced inhibition of forskolin-stimulated cAMP accumulation were right-shifted. Antagonist potencies determined by Schild analyses correlated well with those established by competition for radioligand binding. The A3 adenosine receptor transcript is widespread and, in contrast to the A1, A2a, and A2b transcripts, the most abundant expression is found in the lung and liver. The tissue distribution of A3 mRNA is more similar to the widespread profile found in sheep than to the restricted profile found in the rat. This raises the possibility that numerous physiological effects of adenosine may be mediated by A3 adenosine receptors.

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Adenosine functions as a counterregulatory hormone in the myocardium by decreasing work and thereby protecting the myocardium against ischemia. Functional adenosine A₁ receptors could serve as an important regulatory system in the developing preinnervated heart by balancing the humoral sympathetic input to the heart. The aims of this study were to determine if A₁ adenosine receptors were functionally coupled to their G_i protein in the immature preinnervated heart and to determine if A₁ adenosine receptors were present in greater numbers in the immature heart. One- to 3-day-old rat ventricular cardiomyocyte cultures were exposed to (1) control conditions; (2) isoproterenol, a β-receptor agonist; (3) R-PIA, an A₁ agonist, or (4) isoproterenol and R-PIA. cAMP levels were determined by RIA in each group. Adenosine A₁ receptor density and the equilibrium dissociation constant were determined by binding of an adenosine At receptor antagonist in newborn, 1-week-old, 2-week-old, and adult rat hearts. A₁ stimulation decreased the isoproterenol-induced increase in cAMP by 30%, demonstrating functional A₁ receptors in immature preinnervated myocytes. The A₁ receptor density in the newborn age group was twice the adult and 2-week-old level. We conclude that A₁ receptors in the immature heart are functionally coupled to their effector and that A₁ receptors are present in greater numbers in the immature heart.