Expression of A3 Adenosine Receptors in Human Lymphocytes: Up-Regulation in T Cell Activation

• Published in: Molecular pharmacology 2004-03, Vol.65 (3), p.711-719
• Main Authors: Gessi, Stefania, Varani, Katia, Merighi, Stefania, Cattabriga, Elena, Avitabile, Arianna, Gavioli, Riccardo, Fortini, Cinzia, Leung, Edward, Lennan, Stephen Mac, Borea, Pier Andrea
• Format: Article
• Language: English
• Subjects: Binding Sites; Binding, Competitive; Blotting, Western; Cyclic AMP - metabolism; Humans; Kinetics; Lymphocyte Activation - physiology; Radioligand Assay; Receptor, Adenosine A3 - metabolism; Reverse Transcriptase Polymerase Chain Reaction; T-Lymphocytes - metabolism; Thermodynamics; Up-Regulation
• ISSN: 0026-895X; 1521-0111
• DOI: 10.1124/mol.65.3.711

The present study investigates mRNA and protein levels of A3 adenosine receptors in resting (R) and activated (A) human lymphocytes. The receptors were evaluated by the antagonist radioligand [3H]5-N-(4-methoxyphenyl-carbamoyl)amino-8-propyl-2(2furyl)-pyrazolo-[4,3e]-1,2,4-triazolo-[1,5-c]-pyrimidine ([3H]MRE 3008F20), which yielded Bmax values of 125 ± 15 and 225 ± 23 fmol/mg of protein and KD values of 1.79 ± 0.30 and 1.85 ± 0.25 nM in R and A cells, respectively. The protein seems to be induced with remarkable rapidity starting at 15 min and reaches a plateau at 30 min. Western blot assays revealed that the up-regulation of the A3 subtype after lymphocyte activation was caused by an increase in an enriched CD4+ cell fraction. Real-time reverse transcription-polymerase chain reaction experiments confirmed the rapid increase of A3 mRNA after T cell activation. Competition of radioligand binding by adenosine ligands displayed a rank order of potency typical of the A3 subtype. Thermodynamic data indicated that the binding is enthalpy- and entropy-driven in both R and A cells, suggesting that the activation process does not involve, at a molecular level, receptor alterations leading to modifications in the A3-related binding mechanisms. Functionally, the up-regulation of A3 adenosine receptors in A versus R cells corresponded to a potency increase of the A3 agonist N6-(3-iodo-benzyl)-2-chloro-adenosine-5′-N-methyluronamide in inhibiting cAMP accumulation (IC50 = 1.5 ± 0.4 and 2.7 ± 0.3 nM, respectively); this effect was antagonized by MRE 3008F20 (IC50 = 5.0 ± 0.3 nM). In conclusion, our results provide, for the first time, an in-depth investigation of A3 receptors in human lymphocytes and demonstrate that, under activating conditions, they are up-regulated and may contribute to the effects triggered by adenosine.