Specific Binding of Vasoactive Intestinal Peptide to Brain Membranes from the Guinea Pig

Abstract

1 The interaction of the vasoactive intestinal peptide family of hormones with synaptic membranes from guinea pig brain was examined using 125I-labeled vasoactive intestinal peptide as a tracer molecule. Binding of this peptide was rapid and temperature-dependent, as well as reversible and saturable. Scatchard plots were compatible with the existence of two classes of binding sites, the first class with an apparent Kd of 36 nM and a low binding capacity (4 pmol vasoactive intestinal peptide/mg membrane protein) and a second class of binding sites with a lower affinity (apparent Kd of 285 nM) and a higher capacity (20 pmol vasoactive intestinal peptide/mg membrane protein). 2 The specificity of binding sites for vasoactive intestinal peptide was established from the fact that binding of the 125I-labeled peptide was inhibited by the unlabeled peptide and by higher concentrations of unlabeled secretin but not by the parent hormone glucagon or by the neuropeptides somatiostatin, bombesin, C-terminal octapeptide of pancreozymin, Leu-enkephalin, and substance P. 3 Na+, K+, Ca2+, Mg2+ and EDTA reduced the amount of 125I-labeled vasoactive intestinal peptide bound at equilibrium. The monovalent and divalent cations decreased the binding rate constant, whereas EDTA increased the dissociation rate constant of binding. 4 Nucleoside triphosphates and diphosphates decreased binding of the 125I-labeled peptide at equilibrium and increased the rate constant of dissociation of membrane-bound vasoactive intestinal peptide. Guanyl nucleotides were the most potent and in this class of nucleotides GTP was 6-fold more potent than guanosine 5′-[β,γ-imido]triphosphate.