Binding characteristics of [3H] guanfacine to rat brain α-adrenoceptors: Comparison with [3H]clonidine

The tritium-labeled α-adrenoceptor agonist and antihypertensive drug guanfacine, N-amidino-2-(2,6-dichlorophenyl)-acetamide (sp. act. 24.2 $\text{Ci}\text{mmole}$) was employed for a direct identification and characterization of α-adrenoceptors in rat brain membranes. Its usefulness as a radioligand was studied in comparison with [³H]clonidine (sp. act. 26.7 $\text{Ci}\text{mmole}$). The nonspecific binding of [³H]guanfacine to rat cerebral membranes was considerably more pronounced than that observed for [³H]clonidine. The specific binding of [³H] guanfacine (0.1–20 nM) and [³H]clonidine (0.1–20 nM) as defined as the excess over blanks containing (?)-norepinephrine (10μM) was saturable. Scatchard analyses of these binding data indicated single populations of binding sites for both ligands. K_D values of 3.9 ([³H]guanfacine) and 3.7 nM ([³H]clonidine) were calculated. Maximal number of specific binding sites amounted to 220 and 195 $\text{fmole}\text{mg}$ protein for [³H]guanfacine and [³H]clonidine, respectively. In case unlabeled guanfacine (1 μM) was used to characterize the specific binding of [³H] guanfacine, K_d value and maximal number of binding sites were about twice as high as determined in the presence of excess (?)-norepinephrine. The rate of association of both radioligands was rapid. Binding reached equilibrium by about 10–15 min of incubation. Half-maximal binding was attained at approximately 1–2 min. The rates of dissociation were biphasic. A rapid and a slow component were identified. The specific binding sites of [³H] guanfacine in rat brain possess the general characteristics of α₂-adrenoceptors. Selective antagonists of α₂-adrenoceptors, like yohimbine and rauwolscine strongly interfered with this binding. However, preferential blocking agents of α₁-adrenoceptors, such as prazosin and corynanthine, were weak competitors. The relative potency of agonists and antagonists in displacing [³H]guanfacine was identical to their effectiveness in competing for [³H]clonidine specific binding sites. It is concluded that [³H]guanfacine labels the same α₂-adrenoceptor population in rat brain as [³H]clonidine. However, [³H]guanfacine seems not as suitable as [³H]clonidine for routine use in the direct identification of α₂-adrenoceptors in view of its relatively high nonspecific binding.