Chu J, Wilczynski W, Wilcox R E
Institute for Neuroscience, Department of Psychology, University of Texas, Austin, Tex., USA.
Brain Behav Evol. 2001 Jun;57(6):328-42. doi: 10.1159/000047251.
The pharmacological profiles of D1- and D2-like dopamine receptors were investigated for native brain receptors in the leopard frog, Rana pipiens, using direct binding assays, which characterize functional receptors rather than assess total receptor protein. We used homogenate assays of R. pipiens fore- and midbrains to determine, via saturation isotherms, that the dissociation constant, Kd, for (3)H-SCH-23390 binding to the D1-like receptors was 0.29 nM, and the maximal receptor density, Bmax, was 40 fmoles/mg protein. This compares with the more than 10-fold higher density of D1 sites in rat striatum. Specific binding for the D2-like receptors was measurable using these methods with (3)H-spiperone as the ligand. However, saturation of binding was not achieved. This contrasts with the > 400 fmoles/mg protein Bmax in rat striatum. Pharmacological profiles (rank order of potency of displacing drugs) for each receptor type were determined. We used non-radioactive SCH-23390, SKF-38393, sulpiride, and spiperone to displace (3)H-SCH-23390 and (3)H-spiperone at D1 and D2 receptors, respectively. Parallel displacement assays were performed with rat striatal controls. Results indicated that the relative rank order displacements in anuran dopamine receptors were characteristic of D1- and D2-like receptors. However, the rank orders were not identical to those in mammals. The rank order for affinity at D1-like receptors in both rats and frogs was SCH-23390 > SKF-38393 > spiperone > sulpiride. The rank order for affinity at D2-like receptors was spiperone > SCH-23390 > sulpiride > SKF-38393 in frogs, and spiperone > sulpiride > SCH-23390 > SKF-38393 in rats. SKF-38393 and spiperone had similar affinities for the 'D1' receptors in both species. SCH-23390 had a slightly lower affinity for the D1-like receptors in Rana, whereas sulpiride had a significantly lower affinity for Rana D1-like receptors compared to rat D1 receptors. In Rana D2-like receptors, spiperone and sulpiride were significantly less potent compared to rat. However, SCH-23390 and SKF-38393 were equally potent for the D2-like receptors in both species. The results indicate that amphibian brain dopamine receptors fall into two classes similar to the mammalian D1 and D2 subfamilies, but with binding characteristics slightly different from those typically described in mammals. This work represents the first pharmacological characterization of native brain dopaminergic receptors in an anuran amphibian. Because direct binding assays measure the initial aspect of the functional interaction between transmitter and receptor, these data provide an important complement to studies using cell expression systems.
利用直接结合试验研究了豹蛙（Rana pipiens）大脑中天然的D1样和D2样多巴胺受体的药理学特征，该试验表征的是功能性受体，而非评估总受体蛋白。我们采用豹蛙前脑和中脑的匀浆试验，通过饱和等温线来确定，(3)H-SCH-23390与D1样受体结合的解离常数Kd为0.29 nM，最大受体密度Bmax为40 fmol/mg蛋白。这与大鼠纹状体中密度高出10倍以上的D1位点形成对比。使用(3)H-螺哌隆作为配体，通过这些方法可检测到D2样受体的特异性结合。然而，未实现结合饱和。这与大鼠纹状体中>400 fmol/mg蛋白的Bmax形成对比。确定了每种受体类型的药理学特征（取代药物效力的排序）。我们分别使用非放射性的SCH-23390、SKF-38393、舒必利和螺哌隆来取代D1和D2受体上的(3)H-SCH-23390和(3)H-螺哌隆。对大鼠纹状体对照进行了平行取代试验。结果表明，无尾两栖类多巴胺受体的相对取代排序具有D1样和D2样受体的特征。然而，排序与哺乳动物中的不同。大鼠和青蛙中D1样受体的亲和力排序均为SCH-23390>SKF-38393>螺哌隆>舒必利。青蛙中D2样受体的亲和力排序为螺哌隆>SCH-23390>舒必利>SKF-38393，大鼠中为螺哌隆>舒必利>SCH-23390>SKF-38393。SKF-38393和螺哌隆对两个物种的“D1”受体具有相似的亲和力。SCH-23390对豹蛙中D1样受体的亲和力略低，而舒必利对豹蛙D1样受体的亲和力相比大鼠D1受体显著更低。在豹蛙D2样受体中，螺哌隆和舒必利的效力相比大鼠显著更低。然而，SCH-23390和SKF-38393对两个物种的D2样受体效力相同。结果表明，两栖动物大脑多巴胺受体分为两类，类似于哺乳动物的D1和D2亚家族，但结合特征与哺乳动物中通常描述的略有不同。这项工作代表了对无尾两栖动物大脑中天然多巴胺能受体的首次药理学表征。由于直接结合试验测量的是递质与受体之间功能相互作用的初始方面，这些数据为使用细胞表达系统的研究提供了重要补充。
