Direct and indirect effects of sulfhydryl blocking agents on agonist and antagonist binding to central alpha 1- and alpha 2-adrenoceptors.

The effects of p-chloromercuribenzoate and N-ethylmaleimide were evaluated on the binding of (3H)-p-aminoclonidine, (3H)-rauwolscine and (3H)-prazosin on rat brain alpha-adrenergic receptors. Pretreatment of the particulate fraction with increasing concentrations of p-chloromercuribenzoate indicated that the binding of all three radioligands was similarly inhibited with an IC50 of about 30 microM. This effect was then reduced when agonist [(-)-norepinephrine] or antagonist (phentolamine) were present during the pretreatment. Pretreatment of the particulate fraction at N-ethylmaleimide concentrations less than 100 microM specifically decreased the (3H)-p-aminoclonidine binding while binding of antagonist was unchanged. N-ethylmaleimide produced binding changes similar to those induced by GTP in control membranes, i.e. interconversion of the alpha 2-adrenoceptors states from a high affinity to a low affinity for agonists. Norepinephrine but not phentolamine reduced the effects of N-ethylmaleimide when present during the pretreatment. Taken together, these results suggest that the alpha 1- and alpha 2-adrenoceptors possess, within or close by the recognition site, an --SH group which can be blocked at low concentrations by p-chloromercuribenzoate but not by N-ethylmaleimide. In contrast, the group alkylated by the latter does not seem to be located in the recognition site domain but rather at a site important for the coupling between the alpha 2-receptor and the GTP-binding protein.