[3H]Neurokinin B and 125I‐Bolton Hunter Eledoisin Label Identical Tachykinin Binding Sites in the Rat Brain

Abstract

[³H]Neurokinin B ([³H]NKB) of high specific activity (75 Ci/mmol) was synthesized for study of its binding to crude synaptosomes from the rat cerebral cortex. The specific binding of [³H]NKB (75% of total binding) was temperature dependent, saturable, and reversible. Scatchard analyses and Hill plots showed the existence of a single population of noninteracting binding sites (K_D= 4.3 nM; B_max= 123 fmol/mg of protein). Competition studies indicated the following rank order of potencies among tachykinins: NKB > eledoisin (E) > kassinin > physalaemin > neurokinin A (NKA) > substance P (SP), a result suggesting that NKB might be the endogenous ligand for [³H]NKB binding sites. It is of interest that ¹²⁷I-Bolton Hunter (BH) NKA (¹²⁷I-BHNKA) was much more potent than NKA in inhibiting the specific binding of [³H]NKB, which raises certain questions concerning the use of ¹²⁵I-BHNKA as a Iigand for NKA binding sites in the brain. These results, as well as those obtained with different SP analogues, show a close similarity to those obtained previously with ¹²⁵I-BHE binding to cortical synaptosomes. This suggested that the two ligands labeled identical binding sites. In addition, using either [³H]NKB or ¹²⁵I-BHE as ligands, similar displacement curves were obtained with increasing concentrations of NKB and ¹²⁷I-BHE. The similarity of the [³H]NKB and ¹²⁵I-BHE binding sites was further confirmed by comparison of their localization on rat brain sections by autoradiography. The distribution of binding sites for [³H]NKB and ¹²⁵I-BHE was identical throughout the brain, and the highest density of binding sites for the two ligands was found in layers IV and V of the cerebral cortex, the paraventricular nucleus of the hypothalamus (magnocellular part), and the ventral tegmental area.