Specific Binding of a ¹²⁵I‐Substance P Derivative to Rat Brain Synaptosomes

Abstract

Binding characteristics of 125I‐labeled Bolton‐Hunter substance P (125I‐BHSP), a radioactive analogue of substance P, were studied with a crude syn‐aptosomal fraction (P2) of the rat brain. A specific binding of 125I‐BHSP that was saturable, reversible, and temperature dependent was demonstrated. Under the conditions of the binding assay (20°C, 5 min), nonspecific binding represented no more than 25% of total binding, and in contrast to [3H]dopamine 125I‐BHSP was not taken up within synaptosomes by a ouabain‐sensitive process. Scatchard analysis indicated the existence of a single population of noninteracting sites with a high affinity (KD, 470 pM) and a low‐density (Bmax, 13 fmol/mg protein). Substance P and different substance P analogues were tested for their competitive potencies in inhibiting 125I‐BHSP binding to synaptosomes. (Tyr8) substance P, substance P, and BHSP strongly inhibited 125I‐BHSP specific binding. Among various tachykinins, physalaemin was the most potent (one fourth the potency of substance P). When substance P C‐terminal fragments were tested for their ability to compete with 125I‐BHSP binding, a good relationship was found between competitive activity and peptide length. N‐terminal fragments of substance P were ineffective. However, (nor‐Leu11)substance P and the methyl ester of substance P1‐11 slightly reduced 125I‐BHSP binding. (D‐Pro2, D‐Phe7, D‐Trp9) substance P and (D‐Pro2, D‐Trp7,9)substance P, two substance P antagonists, were also tested. (D‐Pro2, D‐Trp7,9) substance P was the only one to inhibit 125I‐BHSP binding even slightly. Results were compared with those previously obtained concerning 125I‐BHSP binding to mesencephalic embryonic cells in primary cultures using similar conditions for the binding assay.

Substance P receptors in primary cultures of cortical astrocytes from the mouse.

“…Substance P (SP) receptors have been characterized in the central nervous system of adult mammals in binding studies performed on synaptosomes or membranes using [3H]SP (1,2) or the 125I-labeled Bolton-Hunter (125I-BH) derivative of SP (125I-BHSP) (3)(4)(5). The regional localization of these binding sites has been shown by microdissection (1,6) or by autoradiography (7)(8)(9), and a correlation has been observed between the distribution of [3H]SP binding sites and the ability of SP to stimulate phosphatidylinositol turnover (10).…”

Pharmacological specificity of novel, synthetic, cyclic peptides as antagonists at tachykinin receptors

“…(Viger et al, 1983;Cascieri et al, 1981;Lee et al, 1986;Iversen et al, 1988; branes from hamster urinary bladder (Lee et al, 1986), and we have found that the correlation between potency for agonists to displace the binding of [125I-BH]-Ele in rat brain cortex (NK3 site) and to contract the portal vein of the rat is highly significant (McKnight & Maguire, 1987).…”

Interaction of tachykinins with their receptors studied with cyclic analogues of substance P and neurokinin B.

“…The most stable predicted conformations exhibit a turn around either the Gly-9 and Phe-8 or Phe-7 and Phe-8 residues leading to a spatial proximity of the C-terminal methionine and of the glutamine side chains (2)(3)(4)(5)(6)(7). However, we have demonstrated the existence in the rat brain (8,9) and spinal cord (10) of a specific binding site for SP that requires the whole sequence of SP for full binding potency. This result was further confirmed by several groups (11)(12)(13).…”

“…Starting from 335 mg of crude peptide, 103 mg was recovered from filtration on Sephadex G-25F (25% acetic acid), which was purified to homogeneity on were used, respectively. Synaptosomes were prepared as described previously (9,20), the final pellet being'resuspended in a Krebs-Ringer phosphate buffer containing bovine serum albumin (0.4 mg/ml), bacitracin (30 ,ug/ml), and glucose (1 mg/ml). Finally, synaptosomes were incubated in Eppendorf tubes at 20°C in 200 gl of buffer containing 40 pM of 125I-BHSP (5 min) or 125I-BHELE (15 min) and increasing concentrations of peptides (9,20).…”

“…The selectivities of the analogues were then determined using binding and pharmacological tests specific for the SP, neurokinin B (NKB), and neurokinin A (NKA) receptor types. Binding studies were done with appropriate ligands and tissue preparations that allow for the discrimination of the three different tachykinin binding sites discovered in mammals-i.e., 125I-labeled Bolton and Hunter SP (125I-BHSP)-and 125I-labeled Bolton and Hunter eledoisin (125I-BHELE)-specific bindings on rat brain synaptosomes (9,20) and 3H-labeled NKA-specific binding on membranes from the rat duodenum (41). Indeed, pharmacological investigations have established that SP, NKB, and NKA are the preferred endogenous ligands for these respective binding sites (19,21).…”