Aharony D, Conner G E, Woodhouse D P
Pulmonary Sct., ICI Americas Inc., Wilmington, Delaware.
Neuropeptides. 1992 Oct;23(2):121-30. doi: 10.1016/0143-4179(92)90089-f.
We synthesized a novel ligand [4,5-3H-Leu9]-Neurokinin A (3H-NKA, S.A 117-144 Ci/mmol), and evaluated its binding to hamster urinary bladder membranes (HUBM). The ligand bound to HUBM in a highly-specific (94 +/- 4%) and protein-dependent manner. Binding was rapid (k1 = 0.037 nM-1*min-1) and saturable (Bmax = 1210 +/- 177 fmol/mg protein), to a single population of high-affinity sites (KD = 2.41 +/- 0.15 nM, nH = 0.99 +/- 0.02). Binding was inhibited by non-hydrolyzable GTP analogs. Competition experiments with HUBM demonstrated the following rank order of potency: NKA > Kassinin > [beta-Ala8]-NKA(4-10) > [Nle10]-NKA(4-10) = Eledoisin = NKB > Physaelamin > Substance P. The selective NK-1 and NK-3 ligands, [Sar9-Met (O2)11]-SP, (+/-) CP96,345 and Senktide respectively, did not inhibit binding at 10 microM, whereas, the selective NK-2 antagonists: (+/-) SR-48,968 >> L-659,877 > R396 >> MEN-10,207 > MEN-10,376, inhibited binding in a competitive manner. In contrast, the low specific binding (< 30%) detected in guinea pig lung membranes, was not inhibited by selective NK-2 ligands. Over 30 ligands (0.1-10 microM) from other receptor classes, were not inhibitory. The data suggest that this new ligand binds with high-affinity and selectivity to homogeneous population of NK-2 receptors on HUBM but not on lung membranes, and is a suitable ligand to study NK-2 receptors.
我们合成了一种新型配体[4,5-³H-亮氨酸⁹]-神经激肽A（³H-NKA，比活117 - 144 Ci/mmol），并评估了其与仓鼠膀胱膜（HUBM）的结合情况。该配体以高度特异性（94±4%）和蛋白质依赖性方式与HUBM结合。结合迅速（k1 = 0.037 nM⁻¹·min⁻¹）且具有饱和性（Bmax = 1210±177 fmol/mg蛋白质），结合到单一的高亲和力位点群体（KD = 2.41±0.15 nM，nH = 0.99±0.02）。结合受到不可水解的GTP类似物的抑制。与HUBM的竞争实验表明以下效力顺序：神经激肽A＞速激肽＞[β-丙氨酸⁸]-神经激肽A(4 - 10)＞[异亮氨酸¹⁰]-神经激肽A(4 - 10)=伊列替丁=神经激肽B＞海参肽＞P物质。选择性NK-1和NK-3配体，即[Sar⁹-甲硫氨酸（O2）¹¹]-P物质、（±）CP96,345和森克肽，在10 μM时不抑制结合，而选择性NK-2拮抗剂：（±）SR-48,968＞＞L-659,877＞R396＞＞MEN-10,207＞MEN-10,376，以竞争性方式抑制结合。相反，在豚鼠肺膜中检测到的低特异性结合（＜30%）不受选择性NK-2配体的抑制。超过30种来自其他受体类别的配体（0.1 - 10 μM）没有抑制作用。数据表明这种新配体以高亲和力和选择性与HUBM上的NK-2受体同质群体结合，但不与肺膜上的NK-2受体结合，是研究NK-2受体的合适配体。
