Expression of A3 adenosine receptors in human lymphocytes:: Up-regulation in T cell activation

The present study investigates mRNA and protein levels of A(3) adenosine receptors in resting (R) and activated (A) human lymphocytes. The receptors were evaluated by the antagonist radioligand [H-3] 5-N-(4-methoxyphenyl-carbamoyl) amino-8-propyl-2(2furyl)-pyrazolo-[4,3e]-1,2,4-triazolo-[1,5-c]-pyrimidine ([H-3] 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 A(3) 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 A(3) mRNA after T cell activation. Competition of radioligand binding by adenosine ligands displayed a rank order of potency typical of the A(3) 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 A(3)-related binding mechanisms. Functionally, the up-regulation of A(3) adenosine receptors in A versus R cells corresponded to a potency increase of the A(3) agonist N-6-(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 A(3) receptors in human lymphocytes and demonstrate that, under activating conditions, they are up-regulated and may contribute to the effects triggered by adenosine.