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

The present work characterizes, from a pharmacological and biochemical point of view, adenosine receptors in the human malignant melanoma A375 cell line. Adenosine receptors were detected by RT – PCR experiments. A 1 receptors were characterized using [ 3 H]‐DPCPX binding with a K D of 1.9±0.2 n M and B max of 23±7 fmol mg −1 of protein. A 2A receptors were studied with [ 3 H]‐SCH 58261 binding and revealed a K D of 5.1±0.2 n M and a B max of 220±7 fmol mg −1 of protein. A 3 receptors were studied with the new A 3 adenosine receptor antagonist [ 3 H]‐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 n M and B max of 291±50 fmol mg −1 of protein. The pharmacological profile of radioligand binding on A375 cells was established using typical adenosine ligands which displayed a rank order of potency typical of the different adenosine receptor subtype. Thermodynamic data indicated that radioligand binding to adenosine receptor subtypes in A375 cells was entropy‐ and enthalpy‐driven. 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 Ca 2+ mobilization. In conclusion, all these data indicate, for the first time, that adenosine 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 cell line.British Journal of Pharmacology (2001) 134 , 1215–1226; doi: 10.1038/sj.bjp.0704352