Guanine Nucleotides Regulates Both Agonist and Antagonist Binding to Cod Brain α1-Adrenoceptors

Abstract

The effect of guanine nucleotides on the binding of ³H‐prazosin and its displacement by various agonists and antagonists were studied in membranes prepared from the cod brain. When cod brain membranes were maintained in a buffer containing 8 mM Mg²⁺, GTP (1 mM) was found to increase the specific binding of ³H‐prazosin Computer modelling ³H‐prazosin saturation curves, suggested that the number of binding sites for ³H‐prazosin was increased by 18 ± 5% by GTP without affecting the affinity for ³H‐prazosin (dissociation constant, K_d, 56±3 pM). Displacement of ³H‐prazosin by adrenaline produced shallow displacement curves. Computer modelling these curves suggested that adrenaline bound to two sites ‐ one high and one low affinity site ‐ the K_d's being 0.14±0.03 (KH) and 7.6±0.5 μM (K_L), respectively. When I mM GTP was present the displacement curves were shifted to the right and became steeper. Computer modelling these curves suggested that adrenaline now bound to only one low affinity site with a K_d of 7.6±0.5 μM. When unlabelled prazosin was used to displace ³H‐prazosin the displacement curves were uniphasic and steep, irrespective of whether GTP was present or not. Computer modelling these curves suggested that prazosin bound to only one site with a K_d of 68±11 pM. In the absence of Mg²⁺ and in the presence of EDTA (0.8 mM) the displacement curve of adrenaline became steeper and the effect of GTP was almost abolished. During these conditions the ability of GTP to enhance ³H‐prazosin binding was also abolished. Substitution with 0.08–8 mM Mg²⁺ concentration‐dependently restored the effects of GTP. The data is compatible with a ternary receptor model for the α₁‐adrenoceptor in which the receptor may become associated with another membrane component, which presumably is a guanine nucleotide regulatory protein.