Miyayasu K, Mak J C, Nishikawa M, Barnes P J
Department of Thoracic Medicine, National Heart and Lung Institute, London, UK.
Mol Pharmacol. 1993 Sep;44(3):539-44.
We have characterized the binding of a novel radioligand, [3H] FK888, to neurokinin (NK)1 receptors in guinea pig lung membranes and localized its binding in guinea pig lung sections by autoradiography. Lung membranes were incubated with [3H] FK888 at 25 degrees and the assays were terminated by rapid filtration; nonspecific binding was defined as binding in the presence of 1 microM concentrations of the nonpeptide NK1-selective antagonist CP-96,345. Kinetic analysis showed that specific binding of [3H] FK888 (approximately 70% of total binding) was rapid, reaching a plateau by 20 min, and that binding was reversed by addition of 1 microM CP-96,345, giving a kinetic Kd of 0.46 nM. Binding of [3H] FK888 was saturable at approximately 1 nM, and equilibrium binding analysis gave a Kd of 0.32 +/- 0.03 nM and a Bmax of 46.9 +/- 7.1 fmol/mg of protein (four experiments). In competition studies, substance P, CP-96,345, and FK888 competed for [3H] FK888 binding, but NKA, NKB, and NK2-selective antagonists such as SR48968 and L-659,877 did not. Guanosine-5'-O-(3-thio)triphosphate significantly shifted the competition curve for substance P competition against [3H]FK888 binding to a lower affinity state, confirming that NK1 receptors are coupled to a G protein. Autoradiographic mapping in cryostat sections of lung showed that [3H]FK888 binding was dense over smooth muscle of all airways, with moderate binding over epithelium of bronchi and bronchioles as well as submucosal glands of trachea. No significant labeling of blood vessels was observed. [3H]FK888 binds to NK1 receptors in guinea pig lung and may be a useful tool for studying the expression and regulation of NK1 receptors.
我们已对一种新型放射性配体[3H]FK888与豚鼠肺膜中神经激肽（NK）1受体的结合特性进行了表征，并通过放射自显影术在豚鼠肺切片中定位了其结合位点。将肺膜与[3H]FK888在25摄氏度下孵育，通过快速过滤终止实验；非特异性结合定义为在1 microM浓度的非肽类NK1选择性拮抗剂CP - 96,345存在下的结合。动力学分析表明，[3H]FK888的特异性结合（约占总结合的70%）迅速，在20分钟时达到平台期，并且通过添加1 microM CP - 96,345可使结合逆转，动力学解离常数（Kd）为0.4 nM。[3H]FK888的结合在约1 nM时达到饱和，平衡结合分析得出Kd为0.32±0.0 nM，最大结合容量（Bmax）为46.9±7.1 fmol/mg蛋白质（四个实验）。在竞争研究中，P物质、CP - 96,345和FK888竞争[3H]FK888的结合，但神经激肽A（NKA）、神经激肽B（NKB）以及NK2选择性拮抗剂如SR48968和L - 659,877则不竞争。鸟苷 - 5'-O -（3 - 硫代）三磷酸显著将P物质竞争[3H]FK888结合的竞争曲线向较低亲和力状态移动，证实NK1受体与G蛋白偶联。肺冷冻切片的放射自显影图谱显示，[3H]FK888结合在所有气道的平滑肌上密集，在支气管和细支气管的上皮以及气管的黏膜下腺上有中等程度的结合。未观察到血管有明显标记。[3H]FK888与豚鼠肺中的NK1受体结合，可能是研究NK1受体表达和调节的有用工具。
