An isolated growth cone-enriched fraction from developing rat brain has substance P binding sites

Abstract

A fraction enriched in neuronal growth cones isolated from developing rat forebrain was shown to possess binding sites for the substance P analog, Bolton-Hunter substance P ([ 125I]BHSP). Specific binding of this ligand reached an equilibrium after 10 min at 20 °C, and was reversible and temperature-dependent. Removal of extracellular Na + did not block but rather augmented [ 125I]BHSP binding suggesting that the labeled analog was not transported into the growth cone fraction. Scatchard analysis of the binding indicated a single class of non-interacting binding sites in the growth cone fraction ( K d: 257 pM; B max: 56 fmol/mg protein). From competition studies using substance P and other tachykinins, their rank order of potency for inhibiting [ 125I]BHSP binding was SP > physalaemin >> eledoisin > kassinin > NKB ⩾ NKA. Such order is consistent with the presence of an SP receptor (Neurokinin-1) in the growth cone fraction. The N-terminal fragments of substance P, SP 1–7 and SP 1–11 free acids, and the C-terminal fragment, SP 7–11, were devoid of affinity for the [ 125I]BHSP binding site. However SP 6–11 and SP 1–11 methyl esters showed more potency.