Characterization of Substance P (SP) Aminoterminal SP (1-7) Binding in Brain Regions and Spinal Cord of the Male Rat Studies on the Interaction with Opioid Related Pathways

Abstract: Binding sites for substance P(1-7), SP(1-7) have been identified and characterized for the first time in crude membrane fraction from rat CNS using tritiated ([3H]) SP(1-7) as tracer. These putative receptors were investigated in relation to their affinity for tachykinins, opioid peptides and sigma receptor ligands. [3H]-SP(1-7) specifically binds to high affinity binding sites identified as receptor targets for the heptapeptide SP (1-7). Two distinct binding sites were observed in the spinal cord. One site is recognized by high affinity for SP(1-7) with a Kd of 0.5 nM, whereas the other site showed low affinity for the heptapeptide (Kd=12 nM). In the brain, the binding of SP(1-7) fitted a single site binding model with a Kd of 4.4 nM and a Ki of 4.2 nM. Further, using the spinal cord membranes the binding of [3H]-SP (1-7) was weakly displaced by SP and other N-terminal fragments thereof and no or negligible affinity was observed for ligands of the NK-1, NK-2 and NK-3 tachykinin receptors, C-terminal SP(5-11), Tyr-w-MIF-1 or the mu-opioid receptor antagonists naloxone and naloxonazine. On the other hand it was significantly displaced by endomorphin-2, DAMGO, and Try-MIF-1 and exhibit some affinity for MIF-1, ß-casomorphin and endomorphin-1. However, only endomorphin-2, DAMGO and Tyr-MIF-1 showed affinity in the close range of the native peptide SP(1-7). The affinity of endomorphin-2 for the spinal cord site was 10 times lower than that of SP(1-7) but more than 100 times higher than the affinity recorded for endomorphin-1. Tyr-MIF-1 but not Tyr-w-MIF-1 showed similar affinity as endomorphin-2 for SP(1-7) site. All peptides exhibiting high affinity at the SP(1-7) site, have a phenylalanine or a leucine residue in their C-terminal structure.Further, synthetic analogues of SP(1-7) were tested for their affinity for the SP(1-7) receptor in the rat spinal cord. An important finding here was that the receptor-ligand-interaction was favoured by the C-terminal region of SP(1-7). Residues at positions 5-7 appeared crucial for binding to the specific SP(1-7) site. The presence of the amidated Phe7 residue was extremely critical for binding to the SP(1-7) site.The analogue Gln5-Gln6-Phe7-NH2 was almost equipotent with the parent peptide in the SP (1-7) receptor binding assay.Furthermore, the SP(1-7)-amide potently and dose dependently reduced several signs of the reaction to morphine withdrawal and was significantly attenuated by the addition of the sigma agonist SK-10047.In conclusion, the work presented in this thesis has contributed the characterization of the properties of highly selective binding sites for SP(1-7) in the rat spinal cord and VTA. These sites appear to be distinct from the µ-opioid receptor or any of the known neurokinin receptors. The study further indicates that the SP(1-7)-amide mimics the effect of the nativ heptapeptide and that the mechanisms for its action involve a sigma receptor site.