The Use of Ligand Binding for the Characterisation of α-Adrenoceptors

Binding sites with characteristics of α2- and α1-adrenoceptors were found on rat brain membranes with the help of labelled clonidine and prazosin, respectively. Prazosin also identified α1-adrenoceptors in rat myocardium. Two classes of modulators, mono- and divalent cations, are shared by the α2-and α1-adrenoceptors. Divalent cations (Ni2+, Mn2+, Mg2+) potentiate clonidine (α2-receptor) binding in a temperature-dependent manner. The potentiation by Mg2+ is due to increased availability of sites and an increase in the association constant. The effects of Mg2+ can be reversed by chelation or by lowering the temperature from 30 to 2°C; guanosine triphosphate (half-maximal effect at 2.5 μM) prevents the potentiation. Mg2+ also decreases the affinity of rat brain α2-adrenoceptors for the α1-antagonist prazosin. α2-Adrenoceptors decrease their affinity for certain agonists by addition of sodium ions. The “sodium shift” is pronounced for adrenaline, α-methylnoradrenaline, and noradrenaline relative to clonidine. There is little or no sodium shift for phenylephrine, yohimbine, and prazosin. In the presence of Na+, the Kd values of certain agonists may be similar as found for the α1-adrenoceptors in the presence of Mg2+. It is suggested that α2-adrenoceptors in vivo are under constraint, most likely by sodium ions. Characterisation of these receptors in the absence of sodium ions in vitro reveals a conformation through which the receptor may cycle during excitation. It is suggested that a thermodynamic approach should be taken to explain the driving forces for coupling initiated by agonists. α1-Adrenoceptors in rat brain and rat heart membranes are influenced with respect to agonist binding by magnesium and sodium ions. Mg2+ increases the affinity of adrenaline, noradrenaline, and phenylephrine for the rat heart α1-adrenoceptors. If sodium is added together with magnesium, noradrenaline and α-methylnoradrenaline lose, whereas naphazoline and clonidine gain, affinity. The rank order of potencies (measured by the respective KD values) was reversed for α-methylnoradrenaline and phenylephrine when sodium was added. In addition to monovalent and divalent cations, α2-adrenoceptors are modulated in vitro by guanyl nucleotides. A heat-labile factor confers relatively high affinity for the agonist clonidine to α2-adrenoceptors in rat brain membranes.