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

Department of Clinical and Experimental Medicine, Pharmacology Unit (S.G., K.V., S.M., E.C., A.A., P.A.B.) and Department of Biochemistry and Molecular Biology (R.G., C.F.), University of Ferrara, Ferrara, Italy; "Centro Nazionale di Eccellenza per lo Sviluppo di Metodologie Innovative per lo Studio ed il Trattamento delle Patologie Infiammatorie" Ferrara, Ferrara, Italy (S.G., K.V., S.M., E.C., A.A., P.A.B.); and King Pharmaceuticals, Cary, North Carolina (E.L., S.M).

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 B_max values of 125 ± 15 and 225 ± 23 fmol/mg of protein and K_D 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.

Received July 14, 2003; accepted December 3, 2003.

Address correspondence to: Prof. Dr. Pier Andrea Borea, Chair of Pharmacology, Faculty of Medicine, University of Ferrara, Department of Clinical and Experimental Medicine, Pharmacology Unit, Via Fossato di Mortara 17-19, 44100 Ferrara, Italy. E-mail: bpa{at}dns.unife.it

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