Interaction of Clonidine and Clonidine Analogues with α‐Adrenergic Receptors of Neuroblastoma × Glioma Hybrid Cells and Rat Brain: Comparison of Ligand Binding with Inhibition of Adenylate Cyclase

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.