IMMUNOLOGICAL CHARACTERIZATION OF ADENOSINE A(2A) RECEPTORS IN HUMAN AND PORCINE CARDIOVASCULAR TISSUES

Antipeptide antibody was raised in rabbit against the sequence (361-39 0) of RDC-8, the presumed adenosine A(2A) receptor cDNA from canine. T he antibody titer was estimated by solid phase radioimmunoassay. Weste rn blot analysis under reducing conditions identified a major 45 +/- 1 kDa protein in bovine striatal membranes. This immunoreactive band wa s competed in the presence of excess peptide. Furthermore, the antibod y recognized a single 45-kDa immunoreactive band in membranes from cel ls transfected with the recombinant human adenosine A(2A) receptors, w hereas, fail to cross-react with membranes from cells transfected with recombinant rat A(1) and human A(3) receptors. Membranes from human a nd porcine coronary artery, ventricle, atria and platelets (human only ) showed a major immunoreactive band at 45 +/- 1 kDa size. Under nonre ducing conditions, the migration patterns of the immunoreactive bands were not altered indicating the absence of interchain disulfide bond. The 45-kDa immunoreactive band co-migrated with )phenyl]ethylamino-5'- N-ethylcarboxyamidoadenosine photoaffinity labeled A(2A) adenosine rec eptor using SANPAH as the photoaffinity cross-linker. We provide immun ological evidence for the presence of A(2A) adenosine receptor in huma n cardiovascular tissues that exists as a 45-kDa monomeric protein. Th is study also presents evidence for the presence of A(2A) adenosine re ceptor in ventricle and atria in both human and porcine.