125I]-Bolton-Hunter scyliorhinin II: A novel, selective radioligand for the tachykinin NK3 receptor in rat brain

Abstract

The cyclic tachykinin scyliorhinin II (SCYII) has high affinity for the [neurokinin B (NKB)-preferring] NK3 receptor. SCYII was iodinated using [ 125I]-Bolton-Hunter reagent and the product BHSCYII purified using reverse phase HPLC. In rat brain membranes, binding of BHSCYII and of the relatively unselective radioligand [ 125I]-Bolton-Hunter eledoisin (BHELE) was saturable, reversible and to an NK3 site. In competition studies, the rank order of potency in inhibiting binding of BHSCYII and BHELE was: SCYII ≥ [MePhe 7]-NKB ≈ senktide > NKB ≥ kassinin ≥ eledoisin > [Pro 7]-NKB > neurokinin A > neuropeptide K ≥ substance P > [Sar 9,Met(O 2) 11]-substance P. In “cold” saturation experiments, binding of BHELE occurred to a single class of high affinity sites (K D, 18.6±0.91 nM). Binding of BHSCYII was of greater affinity than for BHELE and could be resolved into a high (K D, 1.33±0.98 nM; 27% of sites) and low affinity (K D, 9.84±2.75; 73% of sites) component. The total number of binding sites was similar for both radioligands (BHSCYII, 8.27±0.98; BHELE, 7.94±0.32 fmol/mg wet weight). In vitro autoradiography in slide-mounted sections of rat brain showed identical binding patterns for both radioligands (100 pM), with dense binding localized predominantly to the cortex, Ammon's horn field 1, premammillary nuclei and interpeduncular nucleus. In membranes from several peripheral tissues, specific binding of BHELE was much higher than for BHSCYII, suggesting few NK3 receptors in the periphery. In comparison with BHELE, BHSCYII exhibits lower nonspecific binding (15%), better selectivity and higher affinity for the tachykinin NK3 receptor in rat brain.