The triated adrenergic antagonists Prazosin (3HPRZ) and Idazoxan (3HIDA, or RX-781094) bind specifically and with high affinity to α1and α2-adrenoceptors respectively, in membrane preparations from cerebral cortex. Saturation experiments performed to determine the density of receptors and the dissociation constant (Kd) were analyzed by the methods of Eadie Hofstee, iterative modelling, and the procedure of Hill, while the specificity of the labelling was verified by displacement experiments. Since receptors are proteins, we examined the role of disulfide (−SS−) bridges and sulfhydryl (−SH) groups in the specific combination of 3HPRZ and 3HIDA to the α1and α2-adrenoceptors. Pretreatment of the membranes with the −SS− reactive DL-dithiothreitol (DTT) or the alkylating agent N-ethylmaleimide (NEM), alone or in combination, decreased specific binding of both ligands, with only minor changes in the non-specific counts. The 3HIDA binding (α2-sites) was more sensitive to both DTT and NEM than the 3HPRZ sites (α2-adrenoceptors), and the initial changes induced by alkylation of the α2-site were due to an important decrease in the affinity for 3HIDA, as judged by the increase in theKd. This modulation in the affinity caused by alkylation of a thiol group could explain the higher potency of the blocking agent tetramine disulfide benextramine at the α2-site. The results provide evidence for the participation of −SS− and −SH groups in the binding site of α1and α2-adrenocepto
展开▼
