Liu L, Warner F J, Conlon J M, Burcher E
School of Physiology and Pharmacology, University of New South Wales, Sydney, Australia.
Naunyn Schmiedebergs Arch Pharmacol. 1999 Aug;360(2):187-95. doi: 10.1007/s002109900069.
This is the first report of the development of a new radioligand [125I]Bolton-Hunter bufokinin ([125I]BH-bufokinin) and its use in the characterisation of tachykinin receptors in the small intestine of the cane toad, Bufo matrinus. The binding of [125I]BH-bufokinin to toad intestinal membranes was rapid, saturable, of high affinity and to a single population of binding sites with KD 0.57 nM and Bmax 3.1 fmol mg wet weight tissue(-1). The rank order of affinity of tachykinins to compete for [125I]-BH bufokinin binding revealed similarities with that of the mammalian NK1 receptor, being bufokinin (IC50, 1.7 nM)>physalaemin (6.7 nM)>substance P (SP, 10.7 nM)> or =neuropeptide gamma (NPgamma, 12.4 nM)> or =kassinin (17.8 nM)>scyliorhinin I (35.3 nM)> or =eledoisin (40.6 nM)> or =carassin (43.2 nM)> or =neurokinin A (NKA, 57.8 nM)> or =neurokinin B (NKB, 77.5 nM)>scyliorhinin II (338 nM). The mammalian NK3-selective agonist senktide was a very weak competitor. The radioligand [125I]neurokinin A showed no specific binding to toad intestinal membranes. In the toad isolated small intestine, the maximum contractile response to bufokinin was over 150% greater than that to acetylcholine in longitudinal muscle, whereas responses to bufokinin and acetylcholine were similar in circular muscle. Bufokinin was the most potent agonist (EC501 0.34 nM) and produced a long-lasting contraction. Other tachykinins such as physalaemin, SP and kassinin were also potent contractile agents. The potency values of mammalian and amphibian tachykinins derived from functional studies (pD2) correlated significantly with those from binding assays (pKi). The data for fish and molluscan tachykinins, however, showed poor correlation. Contractions to bufokinin and SP were unaffected by atropine, indomethacin and tetrodotoxin. The highly selective NK1 receptor antagonists CP 99994, GR 82334 and RP 67580 were ineffective in both binding and functional studies. Bufokinin increased inositol monophosphate formation in a concentration-dependent manner with an EC50 value of 10.7 nM, suggesting that the tachykinin receptor may be coupled to phosphoinositol hydrolysis. In summary, this study provides evidence for a high-affinity, bufokinin-preferring, NK1-like tachykinin receptor in the toad small intestine. This is probably not the receptor which mediates contraction to carassin, scyliorhinin II and eledoisin. The study also provides evidence that bufokinin and its receptor play an important physiological role in regulating intestinal motility.
这是关于新型放射性配体[125I]博尔顿 - 亨特蟾蜍激肽（[125I]BH - 蟾蜍激肽）的开发及其在表征海蟾蜍（Bufo matrinus）小肠中速激肽受体方面应用的首次报告。[125I]BH - 蟾蜍激肽与蟾蜍肠膜的结合迅速、可饱和、具有高亲和力，且针对单一结合位点群体，解离常数（KD）为0.57 nM，最大结合容量（Bmax）为3.1 fmol mg湿重组织（-1）。速激肽竞争[125I]-BH蟾蜍激肽结合的亲和力排序显示与哺乳动物NK1受体相似，即蟾蜍激肽（半数抑制浓度[IC50]，1.7 nM）> 雨蛙素（6.7 nM）> P物质（SP，10.7 nM）≥神经肽γ（NPγ，12.4 nM）≥ 卡辛肽（17.8 nM）> 鲨肌肽I（35.3 nM）≥ 伊里多辛（40.6 nM）≥ 卡雷辛（43.2 nM）≥ 神经激肽A（NKA，57.8 nM）≥ 神经激肽B（NKB，77.5 nM）> 鲨肌肽II（338 nM）。哺乳动物NK3选择性激动剂森克肽是非常弱的竞争者。放射性配体[125I]神经激肽A对蟾蜍肠膜无特异性结合。在蟾蜍离体小肠中，蟾蜍激肽在纵肌中引起的最大收缩反应比乙酰胆碱引起的大150%以上，而在环肌中，蟾蜍激肽和乙酰胆碱引起的反应相似。蟾蜍激肽是最有效的激动剂（半数有效浓度[EC501] 0.34 nM），并产生持久收缩。其他速激肽如雨蛙素、SP和卡辛肽也是有效的收缩剂。功能研究（pD2）得出的哺乳动物和两栖动物速激肽的效价与结合试验（pKi）得出的效价显著相关。然而，鱼类和软体动物速激肽的数据显示相关性较差。蟾蜍激肽和SP引起的收缩不受阿托品、吲哚美辛和河豚毒素影响。高度选择性的NK1受体拮抗剂CP 99994、GR 82334和RP 67580在结合和功能研究中均无效。蟾蜍激肽以浓度依赖方式增加肌醇单磷酸的形成，半数有效浓度（EC50）值为10.7 nM，表明速激肽受体可能与磷酸肌醇水解偶联。总之，本研究为蟾蜍小肠中存在高亲和力、偏好蟾蜍激肽的NK1样速激肽受体提供了证据。这可能不是介导对卡雷辛、鲨肌肽II和伊里多辛收缩反应的受体。该研究还提供了证据表明蟾蜍激肽及其受体在调节肠道运动中起重要生理作用。
