Bergström L, Beaujouan J C, Torrens Y, Saffroy M, Glowinski J, Lavielle S, Chassaing G, Marquet A, D'Orleans-Juste P, Dion S
Chaire de Neuropharmacologie, INSERM U.114, Collège de France.
Mol Pharmacol. 1987 Dec;32(6):764-71.
3H-Neurokinin A (3H-NKA) with high specific activity (75 Ci/mmol) was synthesized to study NKA (NK-2)-binding sites on membrane preparations of various tissues in the rat, including brain, spinal cord, duodenum, vas deferens, and ileum. The binding capacity of 3H-NKA (0.9 nM) was very low in membrane preparations of different central nervous system regions and the ileum smooth muscle (0.2-2 fmol/mg of protein). In contrast, relatively high specific binding was found in membrane suspensions of the rat duodenal smooth muscle (18 fmol/mg of protein) and the vas deferens (8 fmol/mg of protein). 3H-NKA-binding sites were further characterized on the rat duodenal smooth muscle. The specific binding of 3H-NKA was shown to be temperature dependent, saturable, reversible, and increased in parallel with the protein concentration. Scatchard analyses and Hill plots of equilibrium binding studies in the concentration range of 0.40-30 nM revealed that 3H-NKA bound to a single class of noninteracting binding sites (Bmax = 270 fmol/mg of protein, KD = 13.3 nM). Displacement of 3H-NKA with different tachykinin-related peptides gave the following rank order of potencies: NKA greater than NKA (4-10) greater than kassinin greater than eledoisin greater than NKB much greater than substance P greater than physalaemin, which suggests that the binding site labeled by 3H-NKA is different from substance P (NK-1)-and NKB (NK-3)-binding sites. The biological activities of tachykinins and related peptides were tested by measuring their contractile effects on the rat duodenum and rabbit pulmonary artery, two tissues known to be sensitive for NKA. Ki values were correlated with the EC50 obtained in biological assays. The results revealed a significant correlation (r = 0.86, p less than 0.01) between Ki and EC50 values obtained in the isolated rabbit pulmonary artery, whereas there was no significant correlation between binding affinities and biological responses on the rat duodenum (r = 0.62, p greater than 0.05).
合成了具有高比活性（75 Ci/mmol）的3H-神经激肽A（3H-NKA），以研究大鼠各种组织（包括脑、脊髓、十二指肠、输精管和回肠）膜制剂上的NKA（NK-2）结合位点。在不同中枢神经系统区域和回肠平滑肌的膜制剂中，3H-NKA（0.9 nM）的结合能力非常低（0.2 - 2 fmol/mg蛋白质）。相比之下，在大鼠十二指肠平滑肌（18 fmol/mg蛋白质）和输精管（8 fmol/mg蛋白质）的膜悬液中发现了相对较高的特异性结合。对大鼠十二指肠平滑肌上的3H-NKA结合位点进行了进一步表征。结果表明，3H-NKA的特异性结合具有温度依赖性、可饱和性、可逆性，且与蛋白质浓度呈平行增加。在0.40 - 30 nM浓度范围内进行的平衡结合研究的Scatchard分析和Hill图显示，3H-NKA与一类非相互作用的结合位点结合（Bmax = 270 fmol/mg蛋白质，KD = 13.3 nM）。用不同的速激肽相关肽置换3H-NKA得到以下效力顺序：NKA大于NKA（4 - 10）大于蛙皮素大于eledoisin大于神经激肽B远大于P物质大于physalaemin，这表明3H-NKA标记的结合位点与P物质（NK-1）和神经激肽B（NK-3）结合位点不同。通过测量速激肽和相关肽对大鼠十二指肠和兔肺动脉的收缩作用来测试它们的生物学活性，已知这两种组织对NKA敏感。Ki值与生物学测定中获得的EC50相关。结果显示，在分离的兔肺动脉中获得的Ki和EC50值之间存在显著相关性（r = 0.86，p小于0.01），而在大鼠十二指肠上，结合亲和力与生物学反应之间没有显著相关性（r = 0.62，p大于0.05）。
