BINDING CHARACTERISTICS OF [I-125] BOLTON-HUNTER [SAR9,MET(O2)11]SUBSTANCE-P, A NEW SELECTIVE RADIOLIGAND FOR THE NK1 RECEPTOR

The selective tachykinin agonist [Sar9,Met(O2)11]substance P (Sar-SP) was radioiodinated with [125I]Bolton-Hunter reagent and the product [125I]Bolton-Hunter-[Sar9,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 (KD 261 pM) and rat submandibular gland (KD 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 3.7×109 M-1 min -1) was faster than to membranes from salivary gland (k+1 5.6 × 108 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)≫[Nle10]NKA-4(4-10)>[MePhe7]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 [MePhe7]NKB-(4-10) and [Nle10]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. © 1990.