Durieux C, Ruiz-Gayo M, Corringer P J, Bergeron F, Ducos B, Roques B P
Département de Chimie Organique, U266 INSERM, URA 1500 CNRS, Paris, France.
Mol Pharmacol. 1992 Jun;41(6):1089-95.
[3H]Propionyl-Tyr-(SO3H)-gNle-mGly-Trp-(NMe)Nle-Asp-Phe-NH2 ([3H]pBC 264) (98-100 Ci/mmol), a new peptidase-resistant cholecystokinin (CCK) agonist that is 1000-fold more potent for CCK-B than for CCK-A receptors, interacts, with a similar subnanomolar affinity, with a single class of binding sites (Kd, 0.15-0.2 nM) in brain membranes of mouse, rat, guinea pig, and cat, in Tris and Krebs buffers. The concentration of CCK-A receptors in rodent brain was estimated to be 8-10 fmol/mg of protein, by measurement of the Bmax values of the nonselective agonist [3H] propionyl-CCK8, with or without 10 nM pBC 264 to saturate CCK-B sites. In guinea pig and mouse brain, specific [3H]pBC 264 binding was not affected by NaCl and/or guanyl-5'-yl-imidodiphosphate. In contrast, in rat brain the affinity of [3H]pBC 264 was decreased and the maximal number of binding sites was increased by NaCl and the guanyl nucleotide or by alkaline treatment, suggesting that a proportion of CCK-B receptors are linked to guanine nucleotide-binding proteins. The Bmax of a CCK8 analog, [3H]SNF 8702, was higher (57 fmol/mg of protein) than that of [3H]pBC 264 (40 fmol/mg of protein) in guinea pig brain cortex but not in mouse brain. The relative potencies of various analogs differed among species. The CCK-B antagonist L365,260 was 18-, 30-, and 64-fold less potent than [3H]pBC 264 in guinea pig, mouse, and rat, respectively. PD 134308, a CCK-B antagonist, was 20-fold less potent in rat brain than in guinea pig brain. Likewise, the cyclic analog BC 254 displayed a 30- and 60-fold lower affinity for mouse and rat than for guinea pig brain preparations. Together, these results suggest the presence of CCK-B receptor subtypes. In all tissues, the specific binding of [3H]pBC 264 at its Kd values was very high (75-90%) and higher than that of the hydrophobic CCK-B probe [3H]SNF 8702 (approximately 50%). Therefore, unlike [3H]SNF 8702, [3H]pBC 264 can be used to study preparations with low receptor concentrations, such as rat brain, making this radiolabeled molecule the most appropriate ligand available to date for CCK-B receptor studies.
[3H]丙酰基 - 酪氨酸 -（磺酸基）-γ-鸟氨酸 - 甲基甘氨酸 - 色氨酸 -（N-甲基）鸟氨酸 - 天冬氨酸 - 苯丙氨酸 - 酰胺（[3H]pBC 264）（98 - 100居里/毫摩尔）是一种新型的抗肽酶胆囊收缩素（CCK）激动剂，对CCK - B受体的效力比对CCK - A受体强1000倍。在 Tris 和 Krebs 缓冲液中，它以类似的亚纳摩尔亲和力与小鼠、大鼠、豚鼠和猫的脑膜中的单一类结合位点（解离常数Kd，0.15 - 0.2纳摩尔）相互作用。通过测量非选择性激动剂[3H]丙酰基 - CCK8的最大结合量（Bmax）值，估计啮齿动物脑中CCK - A受体的浓度为8 - 10飞摩尔/毫克蛋白质，测量时有无10纳摩尔pBC 264以饱和CCK - B位点。在豚鼠和小鼠脑中，特异性[3H]pBC 264结合不受氯化钠和/或鸟苷 - 5'-基 - 亚氨基二磷酸的影响。相反，在大鼠脑中，氯化钠、鸟苷酸或碱性处理会降低[3H]pBC 264的亲和力并增加结合位点的最大数量，这表明一部分CCK - B受体与鸟嘌呤核苷酸结合蛋白相连。在豚鼠脑皮层中，CCK8类似物[3H]SNF 8702的Bmax（57飞摩尔/毫克蛋白质）高于[3H]pBC 264（40飞摩尔/毫克蛋白质），但在小鼠脑中并非如此。各种类似物的相对效力在不同物种间有所不同。CCK - B拮抗剂L365,260在豚鼠、小鼠和大鼠中分别比[3H]pBC 264弱18倍、30倍和64倍。CCK - B拮抗剂PD 134308在大鼠脑中的效力比在豚鼠脑中低20倍。同样，环状类似物BC 254对小鼠和大鼠脑制剂的亲和力比对豚鼠脑制剂低30倍和60倍。这些结果共同表明存在CCK - B受体亚型。在所有组织中，[3H]pBC 264在其Kd值下的特异性结合非常高（75 - 90%），高于疏水性CCK - B探针[3H]SNF 8702（约50%）。因此，与[3H]SNF 8702不同，[3H]pBC 264可用于研究受体浓度低的制剂，如大鼠脑，这使得这种放射性标记分子成为迄今为止用于CCK - B受体研究的最合适配体。
