Alpha-1 and alpha-2 adrenoceptor binding in cerebral cortex: role of disulfide and sulfhydryl groups

The tritiated adrenergic antagonists Prazosin ([3H]PRZ) and Idazoxan ([3H]IDA, or RX-781094) bind specifically and with high affinity to alpha 1 and alpha 2-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 [3H]PRZ and [3H]IDA to the alpha 1 and alpha 2 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 [3H]IDA binding (alpha 2-sites) was more sensitive to both DTT and NEM than the [3H]PRZ sites (alpha 1-adrenoceptors), and the initial changes induced by alkylation of the alpha 2-site were due to an important decrease in the affinity for [3H]IDA, as judged by the increase in the Kd. 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 alpha 2-site. The results provide evidence for the participation of -SS- and -SH groups in the binding site of alpha 1- and alpha 2-adrenoceptors in the cerebral cortex.