Development of new chromatographic tools based on A2A adenosine receptor subtype for ligand characterization and...

A liquid chromatographic stationary phase containing immobilized membranes from cells expressing A(2A) adenosine receptor (A(2A)AR) is firstly described. Cellular membranes from CHO cells stably transfected with human A(2A)AR vector (A(2A)(+)) and from the same cell line transfected with the corresponding empty vector (A(2A)(-)) were entrapped on immobilized artificial membrane (IAM) support and packed into 6.6 mm I.D. glass columns to create A(2A)(+)-IAM and A(2A)(-)-IAM stationary phases. Frontal chromatography experiments on both A(2A)(+)-IAM and A(2A)(-)-IAM columns demonstrated the presence of a low specific interaction with the receptor. However, immobilized A(2A) retained its ability to specifically bind known ligands as demonstrated by the agreement of the calculated K(d) values with two different chromatographic protocols in comparison to previously reported data. In order to maximize the specific interaction, the same cellular membranes were immobilized on the inner surface of a silica capillary (40 cm × 100 μm I.D.) by non-covalent interactions using the avidin-biotin coupling system to create two open tubular columns A(2A)(+)-OT and A(2A)(-)-OT. The open tubular system was characterized by ranking experiments for affinity studies in mixture useful for the selection of new potential candidates.