title: Pharmacological and biochemical characterization of adenosine receptors in the human malignant melanoma A375 cell line.
abstract: 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.
authors.affiliation: Department of Clinical and Experimental Medicine, Pharmacology Unit, University of Ferrara, Centro Nazionale Di Eccellenza Per Lo Sviluppo Di Metodologie Innovative Per Lo Studio Ed Il Trattamento Delle Patologie Infiammatorie, Italy.
authors.forename: S
authors.initials: S
authors.lastname: Merighi
authors.forename: K
authors.initials: K
authors.lastname: Varani
authors.forename: S
authors.initials: S
authors.lastname: Gessi
authors.forename: E
authors.initials: E
authors.lastname: Cattabriga
authors.forename: V
authors.initials: V
authors.lastname: Iannotta
authors.forename: C
authors.initials: C
authors.lastname: Ulouglu
authors.forename: E
authors.initials: E
authors.lastname: Leung
authors.forename: P A
authors.initials: PA
authors.lastname: Borea
mesh_terms: D000243:Adenosine Deaminase / Q000378:metabolism; D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D006801:Humans; D008546:Melanoma, Experimental / Q000378:metabolism*; D010671:Phenylurea Compounds / Q000737:chemistry / Q000494:pharmacology*; D058915:Purinergic P1 Receptor Antagonists; D011743:Pyrimidines / Q000737:chemistry / Q000494:pharmacology*; D011869:Radioligand Assay; D018047:Receptors, Purinergic P1 / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012878:Skin Neoplasms / Q000378:metabolism*; D014230:Triazoles / Q000737:chemistry / Q000494:pharmacology*; D014316:Tritium; D014407:Tumor Cells, Cultured; D014970:Xanthines / Q000737:chemistry / Q000494:pharmacology*
chemical_list: C098657:5-amino-7-(2-phenylethyl)-2-(2-furyl)pyrazolo(4,3-e)-1,2,4-triazolo(1,5-c)pyrimidine; C405605:MRE 3008-F20; D010671:Phenylurea Compounds; D058915:Purinergic P1 Receptor Antagonists; D011743:Pyrimidines; D018047:Receptors, Purinergic P1; D014230:Triazoles; D014970:Xanthines; D014316:Tritium; C051360:1,3-dipropyl-8-cyclopentylxanthine; D000242:Cyclic AMP; D000243:Adenosine Deaminase; D002118:Calcium