Serra M, Mei L, Roeske W R, Lui G K, Watson M, Yamamura H I
Department of Pharmacology, College of Medicine, University of Arizona, Tucson 85724.
J Neurochem. 1988 May;50(5):1513-21. doi: 10.1111/j.1471-4159.1988.tb03038.x.
The binding of [3H]pirenzepine to a human neuroblastoma cell line (SH-SY5Y) and its correlation with hydrolysis of phosphatidylinositols were characterized. Specific [3H]pirenzepine binding to intact cells was rapid, reversible, saturable, and of high affinity. Kinetic studies yielded association (k+1) and dissociation (k-1) rate constants of 5.2 +/- 1.4 X 10(6) M-1 min-1 and 1.1 +/- 0.06 X 10(-1) min-1, respectively. Saturation experiments revealed a single class of binding sites (nH = 1.1) for the radioligand with a total binding capacity of 160 +/- 33 fmol/mg protein and an apparent dissociation constant of 13 nM. The specific [3H]pirenzepine binding was inhibited by the presence of selected muscarinic drugs. The order of antagonist potency was atropine sulfate greater than pirenzepine greater than AF-DX 116, with K0.5 of 0.53 nM, 2.2 nM, and 190 nM, respectively. The binding properties of 3H-quinuclidinyl benzilate and its quaternary derivative 3H-methylquinuclidinyl benzilate were also investigated. The muscarinic agonist carbachol stimulated formation of inositol phosphates which could be inhibited by muscarinic antagonists. The inhibition constants of pirenzepine and AF-DX 116 were 11 nM and 190 nM, respectively. In conclusion, we show that the nonclassical muscarinic receptor antagonist [3H]pirenzepine identifies a high-affinity population of muscarinic sites which is associated with hydrolysis of phosphatidylinositols in this human neuroblastoma cell line.
对[3H]哌仑西平与人神经母细胞瘤细胞系（SH-SY5Y）的结合及其与磷脂酰肌醇水解的相关性进行了表征。[3H]哌仑西平与完整细胞的特异性结合迅速、可逆、可饱和且具有高亲和力。动力学研究得出结合速率常数（k+1）和解离速率常数（k-1）分别为5.2±1.4×10(6) M-1 min-1和1.1±0.06×10(-1) min-1。饱和实验揭示了放射性配体的单一结合位点类别（nH = 1.1），总结合容量为160±33 fmol/mg蛋白质，表观解离常数为13 nM。特定的[3H]哌仑西平结合受到所选毒蕈碱药物的抑制。拮抗剂效力顺序为硫酸阿托品大于哌仑西平大于AF-DX 116，K0.5分别为0.53 nM、2.2 nM和190 nM。还研究了3H-奎宁环基苯甲酸酯及其季铵衍生物3H-甲基奎宁环基苯甲酸酯的结合特性。毒蕈碱激动剂卡巴胆碱刺激肌醇磷酸的形成，这可被毒蕈碱拮抗剂抑制。哌仑西平和AF-DX 116的抑制常数分别为11 nM和190 nM。总之，我们表明非经典毒蕈碱受体拮抗剂[3H]哌仑西平识别出与该人神经母细胞瘤细胞系中磷脂酰肌醇水解相关的高亲和力毒蕈碱位点群体。
