Nicolas C, Carter C
Department of Preclinical Research, Synthélabo Recherche (LERS), Rueil Malmaison, France.
J Neurochem. 1994 Dec;63(6):2248-58. doi: 10.1046/j.1471-4159.1994.63062248.x.
We have studied the regional distribution and characteristics of polyamine-sensitive [3H]ifenprodil binding sites by quantitative autoradiography in the rat brain. In forebrain areas ifenprodil displaced [3H]ifenprodil (40 nM) in a biphasic manner with IC50 values ranging from 42 to 352 nM and 401 to 974 microM. In hindbrain regions, including the cerebellum, ifenprodil displacement curves were monophasic with IC50 values in the high micromolar range. Wiping studies using forebrain slices (containing both high- and low-affinity sites) or cerebellar slices (containing only the low-affinity site) showed that high- and low-affinity ifenprodil sites are sensitive to spermine and spermidine, to the aminoglycoside antibiotics neomycin, gentamicin, and kanamycin, and to zinc. Two calmodulin antagonists, W7 and calmidazolium, also displaced [3H]ifenprodil from both sites. Other calmodulin antagonists, including trifluoperazine, prenylamine, and chlorpromazine, selectively displaced [3H]ifenprodil from its low-affinity site in hindbrain and forebrain regions. High-affinity [3H]ifenprodil sites, defined either by ifenprodil displacement curves or by [3H]ifenprodil binding in the presence of 1 mM trifluoperazine, were concentrated in the cortex, hippocampus, striatum, and thalamus with little or no labeling of hindbrain or cerebellar regions. This distribution matches that of NMDAR2B mRNA, supporting data showing that ifenprodil has a preferential action at NMDA receptors containing this subunit. Low-affinity [3H]ifenprodil sites have a more ubiquitous distribution but are especially concentrated in the molecular layer of the cerebellum. [3H]ifenprodil was found to bind to calmodulin-agarose with very low affinity (IC50 of ifenprodil = 516 microM). This binding was displaced by calmodulin antagonists and by polyamines, with a potency that matched their displacement of [3H]ifenprodil from its low-affinity site in brain sections. However, the localization of the low-affinity [3H]ifenprodil site does not strictly correspond to that of calmodulin, and its identity remains to be further characterized. The restricted localization of high-affinity [3H]ifenprodil binding sites to regions rich in NMDAR2B subunit mRNA may explain the atypical nature of this NMDA antagonist.
我们通过定量放射自显影术研究了大鼠脑中多胺敏感的[3H]ifenprodil结合位点的区域分布和特征。在前脑区域，ifenprodil以双相方式取代[3H]ifenprodil（40 nM），IC50值范围为42至352 nM和401至974 microM。在后脑区域，包括小脑，ifenprodil取代曲线为单相，IC50值在高微摩尔范围内。使用前脑切片（包含高亲和力和低亲和力位点）或小脑切片（仅包含低亲和力位点）的擦拭研究表明，高亲和力和低亲和力的ifenprodil位点对精胺和亚精胺、氨基糖苷类抗生素新霉素、庆大霉素和卡那霉素以及锌敏感。两种钙调蛋白拮抗剂W7和卡米达唑也能从这两个位点取代[3H]ifenprodil。其他钙调蛋白拮抗剂，包括三氟拉嗪、普尼拉明和氯丙嗪，在脑后部和前脑区域能选择性地从其低亲和力位点取代[3H]ifenprodil。通过ifenprodil取代曲线或在1 mM三氟拉嗪存在下的[3H]ifenprodil结合定义的高亲和力[3H]ifenprodil位点集中在皮质、海马体、纹状体和丘脑，后脑或小脑区域几乎没有标记。这种分布与NMDAR2B mRNA的分布相匹配，支持了ifenprodil对含有该亚基的NMDA受体具有优先作用的数据。低亲和力的[3H]ifenprodil位点分布更广泛，但特别集中在小脑分子层。发现[3H]ifenprodil以非常低的亲和力与钙调蛋白琼脂糖结合（ifenprodil的IC50 = 516 microM）。这种结合被钙调蛋白拮抗剂和多胺取代，其效力与其从脑切片中的低亲和力位点取代[3H]ifenprodil的效力相匹配。然而，低亲和力[3H]ifenprodil位点的定位并不严格对应于钙调蛋白的定位，其身份仍有待进一步表征。高亲和力[3H]ifenprodil结合位点在富含NMDAR2B亚基mRNA的区域的受限定位可能解释了这种NMDA拮抗剂的非典型性质。
