Guanyl nucleotide influences on ³H-ligand binding to α-noradrenergic receptors in calf brain membranes

Guanyl triphosphate and diphosphate nucleotides decrease the binding of the agonist ligands (±)-[³H]epinephrine and (-)-[ ³H]norepinephrine to α-noradrenergic receptors in calf frontal cerebral cortex membranes, with IC₅₀ values (concentrations which reduced specific binding by 50%) of 1 to 10 μM and the potency order GTP = guanyl-5'-yl imidodiphosphate > GDP. Corresponding adenyl nucleotides have a similar effect, but are more than 100 times weaker. Guanosine, 5'-GMP, adenosine, and 5'-AMP have no effect on (±)-[³H]epinephrine binding at concentrations up to 0.1 mM. Guanyl and adenyl nucleotides do not decrease the binding of the α-receptor antagonist ligand [³H]WB-4101 or the mixed agonist-antagonist ligand [³H]dihydroergokryptine at concentrations up to 1.0 mM. Ten micromolar GTP has no effect on the affinities of α-receptor agonists and antagonists at [³H]WB-4101 and [³H]dihydroergokryptine binding sites or on the affinities of antagonists at (±)-[³H]epinephrine binding sites, but selectively lowers the potencies of agonist inhibitors of (±)-[³H]epinephrine binding by 4- to 5-fold. In (±)-[³H]epinephrine saturation experiments, 1.0 μM GTP and 10 μM GTP lower the affinity of the ligand 2 and 6 times, respectively, without altering the Bmax. GTP increases the rate of association at 25°C of (±)-[³H]epinephrine and (-)-[³H]norepinephrine binding to α-receptors and also greatly increases the rate of dissociation at 25°C of the ³H-catecholamine ligands, both when added during initial labeling of the receptors and when added at the onset of dissociation. Guanyl-5'-yl imidodiphosphate and GTP are equipotent in accelerating (±)-[³H]epinephrine dissociation. The effect of GTP on (±)-[³H]epinephrine dissociation seemed more striking than its effect on (±)-[³H]epinephrine association and may cause the observed decrease in (±)-[³H]epinephrine affinity at α-receptors. Both NaC1 and GTP lower ³H-catecholamine α-receptor binding, but the effectiveness of one agent is unaltered by the presence of the other. Thus, the effects of monovalent cations and guanyl nucleotides on α-receptor agonist ligand binding appear to be mutually independent. Dissociation of ³H-catecholamines from α-receptors in calf cerebellum is more rapid at 25°C than in frontal cortex and is greatly accelerated by 10 μM GTP. However, GTP and guanyl-5'-yl imidodiphosphate have no effect on (±)-[³H]epinephrine binding at 4°C to β₂-noradrenergic receptors in calf cerebellar membranes, and these nucleotides also have no effect on the potency of β-agonists in competing for the binding of (-)-[³H]dihydroalprenolol at 25°C to these cerebellar β₂-receptors.