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

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. Radiolabelled derivatives of other neuropeptides have been used to demonstrate specific interactions with receptor sites on brain membranes, 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 (Kd=0.38 nM) and a low density (Bmax=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-terminal 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 neurones. 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.