Gehlert D R, Schober D A, Gackenheimer S L, Mais D E, Ladouceur G, Robertson D W
Central Nervous System and Cardiovascular Research, Lilly Research Eli Lilly and Company, Indianapolis, IN 46285.
Neurochem Int. 1993 Oct;23(4):373-83. doi: 10.1016/0197-0186(93)90081-f.
We have developed a high specific activity radioiodinated ligand for the biochemical evaluation and autoradiographic localization of 5HT3 receptors in the brain. [125I]-(S)-iodozacopride was synthesized by radioiodination of deschloro-(S)-zacopride using chloramine-T, and the product was purified by HPLC. The equilibrium kinetics and pharmacology of the binding of this radioligand were studied in homogenates of rat cerebral cortex, while the distribution of binding was examined by quantitative autoradiography. [125I]-(S)-iodozacopride bound to a single, saturable, specific binding site (Kd = 192 +/- 9 pM, Bmax = 1.2 +/- 0.2 fmol/mg protein). The binding had the pharmacological properties of a 5HT3 receptor, being potently inhibited by a variety of 5HT3 agonists and antagonists including (S)-zacopride (Ki = 0.032 nM), Quipazine (Ki = 0.45 nM), LY278584 (Ki = 0.5 nM), (1-m-chlorophenyl)-biguanide (Ki = 0.6 nM) and ICS 205-930 (Ki = 1.0 nM). Autoradiographic studies were undertaken by incubating sections with 400 pM [125I]-(S)-iodozacopride and exposing them to film for 3-7 days to obtain suitable autoradiograms. Specific binding of [125I]-(S)-iodozacopride was found at various amounts in a variety of brain regions. The highest levels of binding were found in the brainstem, principally the nucleus of the solitary tract with somewhat lower levels in the area postrema, substantia gelatinosa of the trigeminal nucleus and dorsal motor nucleus of the vagus. In the rat forebrain, moderate levels of specific binding were found in the glomerular layer of the olfactory bulb, anterior olfactory nucleus and various subnuclei of the amygdala. Lower levels of binding were seen in the superficial laminae of the parietal cerebral cortex and diffusely distributed throughout the hippocampal formation. In conclusion, [125I]-(S)-iodozacopride binds to a receptor site with the pharmacological properties and distribution that is consistent with the 5HT3 receptor. [125I]-(S)-iodozacopride represents a significant improvement in autoradiographic studies of the 5HT3 receptor by reducing the required exposure time for producing autoradiograms from the 3-6 months required for [3H]-labeled ligands to 3-7 days.
我们已经开发出一种高比活度的放射性碘标记配体，用于脑内5-羟色胺3（5HT3）受体的生化评估和放射自显影定位。[125I]-(S)-碘扎考必利通过用氯胺-T对去氯-(S)-扎考必利进行放射性碘化合成，产物通过高效液相色谱法纯化。在大鼠大脑皮层匀浆中研究了这种放射性配体结合的平衡动力学和药理学，同时通过定量放射自显影检查结合分布。[125I]-(S)-碘扎考必利与单一、可饱和、特异性结合位点结合（解离常数Kd = 192±9皮摩尔，最大结合容量Bmax = 1.2±0.2飞摩尔/毫克蛋白）。这种结合具有5HT3受体的药理学特性，受到多种5HT3激动剂和拮抗剂的强烈抑制，包括(S)-扎考必利（抑制常数Ki = 0.032纳摩尔）、喹哌嗪（Ki = 0.45纳摩尔）、LY278584（Ki = 0.5纳摩尔）、(1-间氯苯基)-双胍（Ki = 0.6纳摩尔）和ICS 205-930（Ki = 1.0纳摩尔）。放射自显影研究通过用400皮摩尔的[125I]-(S)-碘扎考必利孵育切片并将其在胶片上曝光3至7天以获得合适的放射自显影片进行。在各种脑区发现了不同量的[125I]-(S)-碘扎考必利特异性结合。结合水平最高的是脑干，主要是孤束核，最后区、三叉神经核胶状质和迷走神经背运动核中的水平略低。在大鼠前脑，嗅球的小球层、前嗅核和杏仁核的各个亚核中发现中等水平的特异性结合。在顶叶大脑皮层的浅层结合水平较低，并在整个海马结构中广泛分布。总之，[125I]-(S)-碘扎考必利与一个受体位点结合，其药理学特性和分布与5HT3受体一致。[125I]-(S)-碘扎考必利在5HT3受体的放射自显影研究中代表了一项重大改进，通过将产生放射自显影片所需的曝光时间从[3H]标记配体所需的3至6个月减少到3至7天。
