Interaction of Clonidine and Clonidine Analogues with α‐Adrenergic Receptors of Neuroblastoma × Glioma Hybrid Cells and Rat Brain

Abstract

Clonidine and several analogues of clonidine are shown to be useful probes for α₂-adrenergic receptors in a comparative study of ligand binding and inhibition of adenylate cyclase. The α-adrenergic properties of a new potential probe, N-(4-hydroxyphenacetyl)-4-aminoclonidine hydrochloride, are described. [³H]Clonidine binds to α-receptors of NG108-15 neuroblastoma × glioma hybrid cell membranes with K_d values of 1.7 and 33 nM for putative high-affinity and low-affinity sites, respectively. p-Aminoclonidine and hydroxyphenacetyl aminoclonidine displace [³H]clonidine from the high-affinity sites with K_d values of 2.3 and 5.8 nM, respectively. Rat brain α₂-receptors also exhibit high affinity toward clonidine, p-aminoclonidine, and hydroxyphenacetyl aminoclonidine, as determined by displacement of specifically bound [³H]clonidine. Clonidine, p-aminoclonidine, and hydroxyphenacetylaminoclonidine elicit modest inhibition (up to 24%) of NG108-15 adenylate cyclase by interaction with α₂-receptors (K_d,app 300, 50, and 130 nM, respectively); these compounds also partially reverse the inhibition elicited by (–)-norepinephrine. Components of the adenylate cyclase assay mixture, particularly ATP, GTP, sodium ions, and a nucleoside-triphosphate-regenerating system, decrease the high-affinity [³H)clonidine binding to NG108-15 membranes; in the presence of these components, α-receptors possess only low affinity (K^d 43 nM) for [³H]clonidine. These results are consistent with the concept that certain components required for the receptor-mediated inhibition of adenylate cyclase convert α₂-receptors from a high-affinity inactive state to a low-affinity active state.