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

Br J Pharmacol. 2001 Nov;134(6):1215-26. doi: 10.1038/sj.bjp.0704352.


1. The present work characterizes, from a pharmacological and biochemical point of view, adenosine receptors in the human malignant melanoma A375 cell line. 2. Adenosine receptors were detected by RT - PCR experiments. A1 receptors were characterized using [3H]-DPCPX binding with a KD of 1.9+/-0.2 nM and Bmax of 23+/-7 fmol x mg(-1) of protein. A2A receptors were studied with [3H]-SCH 58261 binding and revealed a KD of 5.1+/-0.2 nM and a Bmax of 220+/-7 fmol x mg(-1) of protein. A3 receptors were studied with the new A3 adenosine receptor antagonist [3H]-MRE 3008F20, the only A3 selective radioligand currently available. Saturation experiments revealed a single high affinity binding site with KD of 3.3+/-0.7 nM and Bmax of 291+/-50 fmol x mg(-1) of protein. 3. 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. 4. Thermodynamic data indicated that radioligand binding to adenosine receptor subtypes in A375 cells was entropy- and enthalpy-driven. 5. In functional assays the high affinity A2A agonists HE-NECA, CGS 21680 and A2A - A2B agonist NECA were able to increase cyclic AMP accumulation in A375 cells whereas A3 agonists Cl-IB-MECA, IB-MECA and NECA were able to stimulate Ca2+ mobilization. In conclusion, all these data indicate, for the first time, that adenosine receptors with a pharmacological and biochemical profile typical of the A1, A2A, A2B and A3 receptor subtype are present on A375 melanoma cell line.

MeSH terms

  • Adenosine Deaminase / metabolism
  • Binding, Competitive
  • Calcium / metabolism
  • Cell Membrane / metabolism
  • Cyclic AMP / metabolism
  • Humans
  • Melanoma, Experimental / metabolism*
  • Phenylurea Compounds / chemistry
  • Phenylurea Compounds / pharmacology*
  • Purinergic P1 Receptor Antagonists*
  • Pyrimidines / chemistry
  • Pyrimidines / pharmacology*
  • Radioligand Assay
  • Receptors, Purinergic P1 / chemistry
  • Reverse Transcriptase Polymerase Chain Reaction
  • Skin Neoplasms / metabolism*
  • Triazoles / chemistry
  • Triazoles / pharmacology*
  • Tritium
  • Tumor Cells, Cultured
  • Xanthines / chemistry
  • Xanthines / pharmacology*


  • 5-amino-7-(2-phenylethyl)-2-(2-furyl)pyrazolo(4,3-e)-1,2,4-triazolo(1,5-c)pyrimidine
  • MRE 3008-F20
  • Phenylurea Compounds
  • Purinergic P1 Receptor Antagonists
  • Pyrimidines
  • Receptors, Purinergic P1
  • Triazoles
  • Xanthines
  • Tritium
  • 1,3-dipropyl-8-cyclopentylxanthine
  • Cyclic AMP
  • Adenosine Deaminase
  • Calcium