Molecular cloning and characterization of the human A3 adenosine receptor.

Abstract

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.

Inhibition of fMLP-triggered respiratory burst of human monocytes by adenosine: involvement of A3 adenosine receptor

“…In addition, we observed that the methylxanthine theophylline, a nonspecific A1 and A2 Ado receptor antagonist, decreased the inhibitory effect of Ado. Unlike A3 receptor in rat [30], the human A3 receptor can bind certain xanthines that display little binding at this receptor because 10 µM concentrations of traditional antagonists produced approximately 10-30% displacement of agonist binding [31]. Moreover, we showed that A3 Ado receptor mRNA was expressed by freshly isolated monocytes.…”

Activation and desensitization of rat A3-adenosine receptors by selective adenosine derivatives and xanthine-7-ribosides

“…The classical adenosine antagonists, the xanthines, display much weaker affinity at rat A 3 receptors than at other adenosine receptor subtypes Salvatore et al, 1993;Ji et al, 1994]. Numerous xanthines that are potent antagonists at rat, rabbit, and human A 1 and A 2 receptors only weakly displaced the binding of the radioligand [ 125 I]APNEA (N 6 -[2-(4-amino-3-iodophenyl)ethyl]adenosine) from cloned rat A 3 receptors even at concentrations in the 10 −4 M range.…”

“…The anionic xanthine BWA522 (5) (IABOPX, Fig. 2), which contains nonequivalent substitution at the xanthine N1 and N3 positions, was reported to have K i values of 3 and 18 nM at sheep and human A 3 receptors, respectively Salvatore et al, 1993], and to antagonize A 3 receptor activation in the rat [Fozard and Hannon, 1994]. Ji et al [1994] found Compound 5 to be much weaker at rat A 3 receptors (K i 1.2 μM), and thus it is not a generally useful antagonist at this subtype.…”

Adenosine A3 receptor stimulation and cerebral ischemia

“…Very recently a novel adenosine receptor subtype (A 3 ) has been identified in the rat (Meyerhof et al, 1991;Zhou et al, 1992), and similar receptors have been cloned from both human (Salvatore et al, 1993) and sheep brain cDNA libraries . It has been shown that activation of adenosine A 3 receptors causes inhibition of adenylate cyclase and stimulation of phospholipase C (Zhou et al, 1992;Ramkumar et al, 1993).…”