Binding characteristics of [ 125I]Bolton-Hunter [Sar 9, Met(O 2) 11]substance P, a new selective radioligand for the NK1 receptor

Abstract

The selective tachykinin agonist [Sar 9,Met(O 2) 11]substance P (Sar-SP) was radioiodinated with [ 125I]Bolton-Hunter reagent and the product [ 125I]Bolton-Hunter-[Sar 9,Met(O) 2) 11]SP (BHSar-SP) purified using reverse phase HPLC. Autoradiographic studies showed dense specific binding of BHSar-SP over the rat submandibular gland and over several regions in rat brain, with very low nonspecific binding, identical with the pattern of binding sites seen in a parallel study with [ 125I]Bolton-Hunter SP (BHSP). In homogenate binding experiments, BHSar-SP bound with high affinity to a single site in membranes from rat brain (K D 261 pM) and rat submandibular gland (K D 105 pM). Comparative values for BHSP were 495 and 456 pM, i.e. of two and four fold lower affinity than BHSar-SP. Association of BHSar-SP to membranes from brain (k +1 3.7×10 9 M −1 min −1) was faster than to membranes from salivary gland (k +1 5.6 × 10 8 M −1 min −1). In competition studies, BHSar-SP was displaced from salivary gland membranes by substance P (SP)≈physalaemin≥Sar-SP≈SP-(3–11)>SP-(5–11)⪢neurokinin A (NKA)≈eledoisin =kassinin=SP-methyl ester≥neurokinin B (NKB)⪢[Nle 10]NKA-4(4–10)>[MePhe 7]NKB-(4–10). In brain membranes, the rank potency order was SP>Sar-SP≥physalaemin>SP-(3–11)>SP-(5–11)>NKA≥eledoisin⪢NKB >kassinin>SP-methyl ester; however [MePhe 7]NKB-(4–10) and [Nle 10]NKA-(4–10) were ineffective competitors at concentrations up to 1 μM. Both binding patterns are consistent with BHSar-SP binding to an NK1 site. With the exception of SP, Sar-SP, SP-(3–11) and physalaemin, all competitors were 5 to 54 times less potent at BHSar-SP binding sites in brain than in salivary gland. These data reveal some differences in characteristics of NK1 binding sites in brain and submandibular gland. Although of higher affinity, BHSar-SP does not appear greatly more selective than BHSP in its ability to define NK1 binding sites.