Characterization of the [I-125]-neurokinin A binding site in the circular muscle of human colon

1 Neurokinin A (NKA) is a potent contractile agonist of human colon circula r muscle. These responses are mediated predominantly through tachykinin NK2 receptors. In the present study, the NK2 receptor radioligand [I-125]-NKA has been used to characterize binding sites in this tissue, using tachykini n agonists and antagonists. 2 (125)INKA labelled a single, high affinity binding site. Specific binding (95% of total binding) of [I-125]-NKA was saturable (K-D 0.47 +/- 0.05 nM) , of high capacity (B-max 2.1 +/- 0.1 fmol mg(-1) wet weight tissue) and re versible (kinetically derived K-D 0.36 +/- 0.07 nM). 3 The rank order of agonists competing for the [I-125]-NKA binding site was neuropeptide gamma (NP gamma) greater than or equal to NKA greater than or equal to [Lys(5),MeLeu(9),Nle(10)] NKA (4 - 10) (NK2 agonist) > > substanc e P (SP) > neurokinin B (NKB) greater than or equal to [Pro(9)]SP (NK1 agon ist) > > senktide (NK3 agonist), indicating binding to an NK2 site. 4 The nonpeptide selective NK2 antagonist SR48968 showed higher affinity fo r the [I-125]-NKA Site than selective peptide NK2 antagonists. The rank ord er of potency for NK2 antagonists was SR48968 greater than or equal to MEN1 1420 > GR94800 greater than or equal to MEN10627 > MEN10376 g R396. The NK1 antagonist SR140333 was a weak competitor. 5 The competition curve for SP could be resolved into two sites. When exper iments were repeated in the presence of SR140333 (0.1 mu M), the curve for SP became monophasic and showed a significant shift to the right, whereas c urves to NKA and NKB were unaffected. 6 In conclusion, binding of the radioligand [I-125]-NKA to membranes from c ircular muscle is predominantly to the NK2 receptor. There may be a small c omponent of binding to the NK1 receptor. The NK2 receptor mediates circular muscle contraction, whereas the role of the NK1 receptor in circular muscl e is unclear.