Specific binding of 3H-substance P to rat brain membranes

Abstract

The undecapeptide substance P is a putative neurotransmitter in the mammalian central nervous system (CNS), and may be associated with pain fibres in the spinal cord^1,2. Radiolabelled derivatives of other neuropeptides havd been used to demonstrate specific interactions with receptor sites on brain membranes^3–6, and this approach has now been explored with substance P. We have now prepared [4-³H-Phe⁸]-substance P and we find that it binds reversibly to a saturable population of sites in rat brain particulate fractions. Scatchard analysis of concentration-dependent saturation of binding indicates a single population of non-interacting sites with a high affinity (K_d = 0.38 nM) and a low density (B_max = 27.2 fmol per mg protein). Kinetic analyses indicate an apparent dissociation equilibrium constant of 0.46 nM. A variety of neurotransmitter amines and amino acids, and other peptides do not compete at the substance P sites, but structurally related peptides or shorter C-terininal fragments of substance P are active. The rank order of potency of these substance P-related peptides agrees with that reported for their effects in depolarizing spinal cord neurones⁷. The regional distribution of the specific binding sites for ³H-substance P parallels that of substance P immunoreactivity, being high in the hypothalamus and low in the cerebellum and cerebral cortex. The characteristics of the ³H-substance P binding sites are consistent with those expected for substance P receptors.