Pharmacological and biochemical characterization of adenosine receptors inthe human malignant melanoma A375 cell line

1 The present work characterizes, from a pharmacological and biochemical po int of view, adenosine receptors in the human malignant melanoma A375 cell line. 2 Adenosine receptors were detected by RT-PCR experiments. A, receptors wer e characterized using [H-3]-DPCPX binding with a K-D of 1.9 +/-0.2 nm and B -max of 23 +/-7 fmol mg(-1) of protein. A(2A) receptors were studied with [ H-3]-SCH 58261 binding and revealed a K-D of 5.1 +/-0.2 nm and a B-max of 2 20 +/-7 fmol mg(-1) of protein. A(3) receptors were studied with the new A( 3) adenosine receptor antagonist [H-3]-MRE 3008F20, the only A(3) selective radioligand currently available. Saturation experiments revealed a single high affinity binding site with K-D of 3.3 +/-0.7 nm and B-max of 291 +/- 5 0 fmol mg(-1) of protein. 3 The pharmacological profile of radioligand binding on A375 cells was esta blished using typical adenosine ligands which displayed a rank order,of pot ency typical of the different adenosine receptor subtype. 4 Thermodynamic data indicated that radioligand binding to adenosine recept or subtypes in A375 cells was entropy- and enthalpy-driven. 5 In functional assays the high affinity A(2A) agonists HE-NECA, CGS 21680 and A(2A)-A(2B) agonist NECA were able to increase cyclic AMP accumulation in A375 cells whereas A(3) agonists Cl-IB-MECA, IB-MECA and NECA were able to stimulate Ca2+ mobilization. 6 In conclusion, all these data indicate, for the first time, that adenosin e receptors with a pharmacological and biochemical profile typical of the A (1), A(2A), A(2B) and A(3) receptor subtype are present on A375 melanoma ce ll line.