Specific binding of substance P aminoterminal heptapeptide [SP(1-7)] to mouse brain and spinal cord membranes

Abstract

Aminoterminal fragments of substance P (SP) have been previously shown to produce effects distinct, and often opposite, from those produced by the C-terminal of SP. The present investigation was initiated to determine whether N-terminal fragments interact at binding sites distinct from the neurokinin-1 (NK-1) receptor where the C-terminal sequence of SP binds with high affinity, and distinct from mu-opiate receptors, where we have previously shown the N-terminal sequence of SP to interact. A tritium-labeled aminoterminal heptapeptide of SP, 3H-SP(1-7), was synthesized, purified, and used to characterize the binding of a variety of fragments of SP and opioids in the mouse brain and spinal cord membranes. Using the reduction of SP-induced caudally directed biting and scratching behaviors as an index of biological activity, 3H-SP(1-7) was shown to be equipotent to unlabeled SP(1-7). 3H-SP(1-7) was found to bind reversibly to a saturable population of sites. Scatchard analyses of concentration-dependent saturation of binding in the brain indicated a single population of noninteracting sites with a high affinity (Kd = 2.5 nM) and a low capacity (Bmax = 29.2 fmol/mg protein). Kinetic analyses indicated an apparent dissociation equilibrium constant of 2.1 nM. Two populations of binding sites were observed in the spinal cord, one with a very high affinity (Kd = 0.03 nM) and low capacity (Bmax = 0.87 fmol/mg protein), and the other with lower affinity (Kd = 5.4 nM) and intermediate capacity (Bmax = 19.6 fmol/mg protein). Specific agonists for NK-1, NK-2, and NK-3 and delta opioid receptors, carboxyterminal fragments of SP, and a variety of other peptides did not compete at the 3H-SP(1-7) binding sites, but structurally related N-terminal peptides and (D-Ala2, NMe-Phe4, Gly-ol)-enkephalin (DAMGO) were active in displacing the ligand. The binding site for 3H-SP(1-7) appeared to be a membrane-bound complex whose specific binding was dependent on the integrity of both proteins and phospholipids. These studies are the first to characterize the binding sites for the SP N-terminal partial sequence of SP that can be generated by metabolism in vivo. The expanding body of evidence for distinct biological activities of N-terminal metabolites of SP, together with the current characterization of N-terminal binding, strongly support the existence of an N-terminal-directed SP receptor. The characteristics of SP(1-7) binding sites are consistent with those expected for an SP N-terminal receptor.

Nociceptin/Orphanin FQ Peptide Receptors: Pharmacology and Clinical Implications

“…This observation indicated that the SP(1-7) binding in the spinal cord was due to G-protein coupled receptors specific for the heptapeptide. The presence of specific binding sites for SP (1)(2)(3)(4)(5)(6)(7) in mice brain and spinal cord has also been demonstrated [71]. …”

Synergistic effect with Phe‐Gly‐Leu‐Met‐NH2 of the C‐terminal of substance P and insulin‐like growth factor‐1 on epithelial wound healing of rabbit cornea

“…Furthermore, unlike substance P or Phe‐Gly‐Leu‐Met‐NH 2 , Gly‐Leu‐Met‐NH 2 , the tachykinin C‐terminal sequence of 3 amino‐acids, did not demonstrate any synergistic effect with IGF‐1. On the other hand, most of the biological effects of substance P have been thought to be mediated by the C‐terminal of substance P, but the N‐terminal of substance P, such as substance P (1–7), also has been shown to produce biological effects and often has produced effects opposite from those produced by the C‐terminal of substance P ( Hall & Stewart, 1984 ; Igwe et al ., 1990 ; Larson & Sun, 1992 ; Yukhanaov & Larson, 1994 ). Substance P (1–7) did not, however, demonstrate any synergistic effect with IGF‐1.…”

Differences in genetic predisposition to high anxiety in two inbred rat strains: role of substance P, diazepam binding inhibitor fragment and neuropeptide Y

“…This may indicate that decreased anxiety-like behavior after administration of both SP, an endogenous agonist at NK-1 receptors, and NK-1 receptor antagonists may be a function of the different dose ranges (e.g., 1-7 nmol SP versus 100-500 nmol of the NK-1 antagonists FK 888 and SR 48968; Teixeira et al 1996) or different routes of administration (e.g., oral, File 1997) in the different studies. Alternatively, anxiolytic effects of ICV administration of low doses of SP in the present experiments may have been mediated by the amino-terminus metabolite fragment of SP, SP(1-7), which may act on different receptor sites (e.g., Igwe et al 1990;Kreeger and Larson 1996).…”